Practical Manual of Experimental

and Clinical Pharmacology

Practical Manual of Experimental
and Clinical Pharmacology

Bikash Medhi
MBBS, MD(AIIMS), MAMS, FIMSA
Associate Professor
Department of Pharmacology
Research Block B, 4th Floor
Postgraduate Institute of Medical Education and Research
Chandigarh, 160012, India
E-mail: drbikashus@yahoo.com

Ajay Prakash
MSc (Pharmacology), DPPM, DPMM
Ex. Jr. Demonstrator
Department of Pharmacology
Research Block B, 4th Floor
Postgraduate Institute of Medical Education and Research
Chandigarh, 160012, India
E-mail: ajayprakashpgi@gmail.com

JAYPEE BROTHERS MEDICAL PUBLISHERS (P) LTD

New Delhi • St Louis (USA) • Panama City (Panama) • London (UK) • Ahmedabad
Bengaluru • Chennai • Hyderabad • Kochi • Kolkata • Lucknow • Mumbai • Nagpur
Published by
Jitendar P Vij
Jaypee Brothers Medical Publishers (P) Ltd
Corporate Office
4838/24 Ansari Road, Daryaganj, New Delhi - 110002, India, Phone: +91-11-43574357, Fax: +91-11-43574314
Registered Office
B-3 EMCA House, 23/23B Ansari Road, Daryaganj, New Delhi - 110 002, India
Phones: +91-11-23272143, +91-11-23272703, +91-11-23282021
+91-11-23245672, Rel: +91-11-32558559, Fax: +91-11-23276490, +91-11-23245683
e-mail: jaypee@jaypeebrothers.com, Website: www.jaypeebrothers.com

Offices in India
• Ahmedabad, Phone: Rel: +91-79-32988717, e-mail: ahmedabad@jaypeebrothers.com
• Bengaluru, Phone: Rel: +91-80-32714073, e-mail: bangalore@jaypeebrothers.com
• Chennai, Phone: Rel: +91-44-32972089, e-mail: chennai@jaypeebrothers.com
• Hyderabad, Phone: Rel:+91-40-32940929, e-mail: hyderabad@jaypeebrothers.com
• Kochi, Phone: +91-484-2395740, e-mail: kochi@jaypeebrothers.com
• Kolkata, Phone: +91-33-22276415, e-mail: kolkata@jaypeebrothers.com
• Lucknow, Phone: +91-522-3040554, e-mail: lucknow@jaypeebrothers.com
• Mumbai, Phone: Rel: +91-22-32926896, e-mail: mumbai@jaypeebrothers.com
• Nagpur, Phone: Rel: +91-712-3245220, e-mail: nagpur@jaypeebrothers.com

Overseas Offices
• North America Office, USA, Ph: 001-636-6279734, e-mail: jaypee@jaypeebrothers.com,
anjulav@jaypeebrothers.com
• Central America Office, Panama City, Panama, Ph: 001-507-317-0160, e-mail: cservice@jphmedical.com
Website: www.jphmedical.com
• Europe Office, UK, Ph: +44 (0) 2031708910, e-mail: dholman@jpmedical.biz

Practical Manual of Experimental and Clinical Pharmacology

© 2010, Jaypee Brothers Medical Publishers (P) Ltd.
The authors of this book have used their best scientific knowledge and skills to provide correct information of their best
capacity, to provide accurate and authentic information with recent update as far as possible based on published data
available at national and international level. However, readers are requested to take their own decision in case of any
differences of opinion based on published data.

All rights reserved. No part of this publication should be reproduced, stored in a retrieval system, or transmitted in any form
or by any means: electronic, mechanical, photocopying, recording, or otherwise, without the prior written permission of the
authors and the publisher.

This book has been published in good faith that the material provided by authors is original. Every effort is made to ensure
accuracy of material, but the publisher, printer and authors will not be held responsible for any inadvertent error (s). In
case of any dispute, all legal matters are to be settled under Delhi jurisdiction only.

First Edition: 2010

ISBN 978-81-8448-953-8
Typeset at JPBMP typesetting unit

Printed at Ajanta Offset

 Dedicated to
My parents, wife (Dr Sujata Upadhyay), son (Debayan Medhi) and all my students for
their constant encouragement during my teaching career
Bikash Medhi

Dedicated to
Mentor “Dr Bikash Medhi” who always encouraged and
motivated me to do well
My grandma and parents for their emotional caring support and entire family for their
constant support whenever I needed
To all “True Friends” who are always with me and encouraged me to excel in life
Ajay Prakash
 PREFACE
The purpose of the present book is to provide fundamental knowledge of practical aspects of the
subject ranging from laboratory animals to clinical aspects and practical implications of various
important recent advances. Learning pharmacology without animal experiment is not practically
suitable though various computer assistance learning models are available for teaching experimental
pharmacology as an integral part. The postgraduates perform animal experiments to learn and
conduct research studies, finally to establish scientific facts and to make their career in the research
field. Fundamental principles of pharmacology deal with essential points of pharmacology, animal
experimentation methodology, and interpretation of results. Most important is to impart skill to
budding pharmacologists, which is an essential area of teaching. In this book, reader could find
some of the useful aspects, e.g. number of worked out examples which will help to translate theory
into practice. Authors made a sincere attempt to include as much relevant information as possible
with illustrated points and suitable examples to make this book comprehensive. Topics covered in
this book have been carefully selected based on most of the recent improvised problems as per
curriculum designed for pharmacology. We are hopeful that the present book will be helpful for all
the postgraduates related to pharmacology, trainees, research workers during their day-to-day
activities including allied health discipline and scientists in industrial drug discovery set-up and
CRO. Several simple and newer experimental models have been incorporated which will help the
students to engage in drug discovery in future. Besides this, several important points have been
discussed in this book, e.g. ethics of animal experimentation, care of animals, preparation of solutions.
Established technologies have been used in different experiments including cell culture in drug
discovery. Clinical pharmacology and pharmacokinetics are special features of this book. Several
clinical pharmacology topics including pharmacokinetics related to various aspects have been
incorporated systematically which will provide exposure to pharmacology residents.
Lastly suggestions and criticism are most welcome.

Bikash Medhi
Ajay Prakash
 ACKNOWLEDGMENTS
We would like to thank: Dr Monika Singla and Dr Sathish Kumar V (Department of Neurology),
Dr Bikash Naredi (Pediatric Surgery), Dr Basanta Hazarika (Department of Pulmonary Medicine),
Dr Pranab Bhattacharyya (Department of Cardiology), Dr Ajay Meena and Dr Neeraj (Department
of General Surgery), Mr Subodh Kumar (Department of Biophysics), Dr YS Bansal and Mr Sunil
Dutt Attrey (Department of Forensic Medicine), Dr Deonis Xess (Apollo Hospital), Mr Devinder
Toor (School of Public Health), Dr Prasad Byrav DS, Dr Harjot Kaur and Ms Sazal Patyar (Department
of Pharmacology), Postgraduate Institute of Medical Education and Research, Chandigarh for their
help in scrutinizing the book. We wish to thank and express gratitude for those books and
bibliography, we have consulted for preparing the manuscript of this book. We would like to thank
Mr Tarun Duneja (Director–Publishing) of Jaypee Brothers Medical Publishers (P) Ltd for his
continuous support and excellent coordination and also to staff of Jaypee Brothers for their hard
work and efforts in handling the manuscript with accurate professional skills.
 CONTENTS
PART 1: GENERAL CONSIDERATIONS IN EXPERIMENTAL PHARMACOLOGY
1. Introduction to Experimental Pharmacology ............................................................................... 3
A. Experimental background .......................................................................................................... 3
B. Drug development and use of animals: An overview ........................................................... 5
C. Commonly used experimental animals ................................................................................... 6
D. Animal behavior and terminology ......................................................................................... 16
E. Animal care, handling and sex determination ...................................................................... 17
F. Diet and experimental animals ............................................................................................... 21
G. Dose calculation for experimental animals............................................................................ 23
H. Routes of drug administration in experimental animals ..................................................... 25
I. Blood collection from the experimental animals .................................................................. 30
J. Variability of drug responses in experimental animals ....................................................... 33
K. Diseases caused by animals (zoonotic diseases) ................................................................... 34
L. Euthanasia method used in the experimental study ............................................................ 37
M. Anesthesia and experimental animals ................................................................................... 37
N. Ethical considerations of animal use in indian scenario ...................................................... 39
2. Bioassay ............................................................................................................................................. 45
A. Introduction ................................................................................................................................ 45
B. Principles of bioassay ................................................................................................................ 45
C. Error in bioassay ........................................................................................................................ 46
D. Applications of bioassay ........................................................................................................... 47
E. Methodology .............................................................................................................................. 47
F. Physiological salt solution (PSS) .............................................................................................. 51
G. Lever and magnification ........................................................................................................... 53
H. Dose cycle and response ........................................................................................................... 56
I. Type of tissue ............................................................................................................................. 57
J. Classification of bioassay .......................................................................................................... 57
i. Direct end point assay (depa) ............................................................................................ 57
ii. Quantal assay (all or none assay) ...................................................................................... 57
iii. Graded response assay (GRA) ........................................................................................... 58
1. Bracketing assay........................................................................................................... 59
2. Matching assay ............................................................................................................. 59
3. Interpolation assay ...................................................................................................... 59
4. Multiple point assay .................................................................................................... 59
K. Bioassay of antagonist ............................................................................................................... 63
L. Human tissue bioassay ............................................................................................................. 66
M. Bioassay of cytokines ................................................................................................................ 67
N. Example of performing a set of bioassay ............................................................................... 69
3. Commonly Used Instruments in Pharmacology Laboratory ................................................... 76
4. Sophisticated Instruments and Techniques Used in Pharmacology Laboratory ................ 82
5. Pyrogen Test (In Vivo and In Vitro Methods).............................................................................. 91
xii  Practical Manual of Experimental and Clinical Pharmacology

6. Practical Aspects of Cell Culture .................................................................................................. 95

7. Preclinical to Clinical Drug Dose Calculation ......................................................................... 100
8. Protocol and Thesis Writing for Postgraduate Students ........................................................ 105
9. Toxicology Study ........................................................................................................................... 115
10. Biomedical Waste Disposal ......................................................................................................... 120
11. Biostatistics in Pharmacology ...................................................................................................... 123

PART 2: EXPERIMENTAL (IN VITRO STUDIES: ISOLATED TISSUE PREPARATION)

12. General Considerations and Collection of the Tissue/Muscle ............................................. 137

13. Identification and Collection of Tissue/Muscle ...................................................................... 140
14. Principle of Muscle Contraction ................................................................................................. 150
15. Fast Contracting Smooth Muscle Preparation .......................................................................... 152
A. To determine unknown concentration of histamine by using guinea pig ileum ........... 152
B. To determine unknown concentration of acetylcholine (ACh) using
rat ascending/descending colon ........................................................................................... 154
C. To determine unknown concentration of acetylcholine (ACh) using rat uterus ........... 155
D. To determine unknown concentration of adrenaline using guinea pig atria ................. 157
E. To determine unknown concentration of acetylcholine (ACh) using rat
anococcygeus muscle preparation ........................................................................................ 159
F. To determine unknown concentration of acetylcholine (ACh) using
rat vas deferens ........................................................................................................................ 160
G. To determine unknown concentration of antagonist (atropine) using acetylcholine (ACh)
as an agonist employing guinea pig ileum preparation by pA2 method ........................ 161
16. Slow Contracting Muscle ............................................................................................................. 164
A. To determine unknown concentration of serotonin (5-HT) using rat
stomach (fundus) .................................................................................................................... 164
B. To determine unknown concentration of acetylcholine (ACh) using frog
rectus abdominis muscle ........................................................................................................ 166
C. To determine unknown concentration of acetylcholine (ACh) using
guinea pig trachea .................................................................................................................. 167
D. To determine unknown concentration of acetylcholine (ACh) using rat
phrenic nerve diaphragm ....................................................................................................... 169
E. To determine neuromuscular blocking drugs using innervated
biventer cervicis preparation of the chick ............................................................................ 171
17. Cardiac Muscle Preparation ......................................................................................................... 173
A. To observe the effect of various drugs on the isolated heart
(Langendorff’s preparation) ................................................................................................... 173
B. To determine the effect of different drugs on the normal and hypodynamic
rabbit heart ............................................................................................................................... 176
C. To demonstrate the effect of the inotropic and chronotropic effects of various drugs on
frog heart normal/hypodynamic) ........................................................................................ 177
i. Isolated preparation ............................................................................................................ 177
ii. In situ preparation ............................................................................................................... 177
 Contents  xiii

PART 3: EXPERIMENTAL (IN VIVO STUDIES)

18. Animal Experiment on Central Nervous System (CNS) ........................................................ 183

A. To demonstrate the effect of pentobarbital on righting reflex (Hypnosis) in mouse .... 183
B. To demonstrate muscle relaxant property of diazepam in mouse using rotarod
apparatus .................................................................................................................................. 184
C. To demonstrate muscle relaxant property of diazepam in mouse using
chimney test .............................................................................................................................. 186
D. To demonstrate anti-anxiety effect of diazepam in rat using
elevated plus maze apparatus ............................................................................................... 187
E. To demonstrate amnesic effect of diazepam in rat using Morris water
maze apparatus ........................................................................................................................ 190
F. To demonstrate the anticonvulsant property of diazepam against pentylenetetrazole
(PTZ) induced convulsions in mice ...................................................................................... 191
G. To demonstrate the anticonvulsant property of diazepam against pentylenetetrazole
(PTZ) induced kindling in rats .............................................................................................. 194
H. To demonstrate the anti-convulsant activity of phenytoin against maximal electroshock
(MES) induced convulsions in rats ....................................................................................... 195
I. To demonstrate effect of phenothiazine (haloperidol) induced catatonia in rat ........... 197
J. To demonstrate the Straub tail reaction/phenomenon induced by morphine .............. 199
K. To demonstrate the analgesic effect of morphine in mouse using hot plate/tail
flick method .............................................................................................................................. 201
L. To demonstrate partial global cerebral ischemia in mice .................................................. 203
19. Animal Experiment on Cardiovascular System ....................................................................... 207
A. To record blood pressure (BP) in rodents (Rat BP). ............................................................ 207
B. To record ECG in rodents (rat and mouse) .......................................................................... 211
C. To demonstrate isoproterenol induced myocardial infarction in rats ............................. 213
D. To demonstrate deoxycorticosterone acetate (DOCA) salt induced
hypertension in rats ................................................................................................................. 214
E. To demonstrate Ferric Chloride (FeCl3) -induced thrombosis in rat model ................... 216
20. Animal Experiment on Gastrointestinal Tract (GIT) .............................................................. 217
A. To demonstrate gastric ulcer induction/formation by different methods ..................... 217
B. To demonstrate cerulein induced acute pancreatitis in rat ............................................... 219
C. To demonstrate Tri Nitro Benzene Sulphonic acid (TNBS) induced colitis in rat ......... 220
21. Animal Experiment on Respiratory System ............................................................................. 222
A. To measure respiratory volume in guinea pig using body plethysmograph ................. 222
B. To collect the Broncho Alveolar Lavage (BAL) fluid for analysis .................................... 223
22. Anti-inflammatory ........................................................................................................................ 224
A. To demonstrate the anti-inflammatory property of indomethacin against carrageenan
induced paw edema ................................................................................................................ 224
B. To demonstrate analgesic effect of morphine against acetic acid induced
writhing in rat .......................................................................................................................... 225
23. Local Anesthetics (LA) .................................................................................................................. 228
A. To demonstrate the effect of any given local anesthetic (LA) using guinea pig (GP) ... 228
B. To demonstrate the effect of the local anesthetic property of procaine HCl using foot
withdrawal reflex of frog ........................................................................................................ 229
xiv  Practical Manual of Experimental and Clinical Pharmacology

24. Experiment on Rabbit Eye ............................................................................................................ 230

A. To study the effect of different drugs on the rabbit eye ..................................................... 230
25. Experimental Pharmacokinetics ................................................................................................. 233
A. To study the pharmacokinetics of phenytoin following oral single dose administration
for 7 days ................................................................................................................................... 233
B. To study the pharmacokinetic interaction of phenytoin with etoricoxib after single oral
dose for 7 days ......................................................................................................................... 234

PART 4: CLINICAL EXPERIMENTS

26. Cardiovascular System (CVS) ..................................................................................................... 239
Blood pressure measurement and validation of sphygmomanometer
A. Introduction .............................................................................................................................. 239
B. Chronobiology of blood pressure .......................................................................................... 241
C. To prepare standard operating procedure (SOP) for blood pressure measurement ..... 241
D. Regulation of blood pressure ................................................................................................. 243
E. Exercise and blood pressure (BP) .......................................................................................... 245
F. Blood pressure guidelines ...................................................................................................... 247
CVS Exp. 1. To measure blood pressure in healthy volunteers .............................................. 250
CVS Exp. 2. To evaluate chronobiology of blood pressure in healthy volunteers ............... 252
CVS Exp. 3. To evaluate the effect of body posture and arm position on arterial blood
pressure and heart rate ........................................................................................... 254
CVS Exp. 4. To evaluate the effect of propranolol on blood pressure, heart rate and cardiac
workload following different submaximal exercises (Tread mill test [TMT],
Master’s 2 step test, Bicycle ergo meter and Hand Dynamometer) in healthy
volunteers .................................................................................................................. 256
A. Treadmill test [TMT] ...................................................................................... 257
B. Master’s 2 step test .......................................................................................... 258
C. Bicycle ergometer ............................................................................................ 258
D. Hand dynamometer ....................................................................................... 259
CVS Exp. 5. To evaluate the effect propranolol on mental stress induced rise in blood
pressure and heart rate in healthy volunteer ...................................................... 261
CVS Exp. 6. To evaluate the postural hypotension in the 60-year old male volunteers ..... 263
CVS Exp. 7. A. To evaluate the effect of glyceryl trinitrate (GTN) on blood pressure,
heart rate in healthy volunteers ................................................................... 264
B. To evaluate the effect of glyceryl trinitrate (GTN) transdermal
patches on blood pressure, heart rate arterial vasodilatation in
healthy volunteers .......................................................................................... 266
C. Recording of an electrocardiogram (ECG) .................................................. 269
Practical Exercise for Cardiovascular System ................................................................................... 275
27. Respiratory System ........................................................................................................................ 280
RESP. Exp. 9. To compare the effect of salbutamol with placebo on peak expiratory flow
rate (PEFR) in healthy volunteers .................................................................... 280
RESP. Exp. 10. To evaluate the effect of salbutamol inhalation in pulmonary function test
in healthy male volunteers ............................................................................... 283
RESP. Exp. 11. To evaluate pulmonary function test following Stair climbing exercise
tolerance test ....................................................................................................... 285
Exercise .............................................................................................................................................. 288
 Contents  xv

28. Central Nervous System (CNS) ................................................................................................... 291

CNS Exp. 12. To demonstrate the effect of various drugs on psychomotor function of
healthy volunteers. .................................................................................................. 291
Exercise .............................................................................................................................................. 297
29. Kidney .............................................................................................................................................. 303
Kid Exp. 13. To evaluate the effect of frusemide on urine volume and Na+ and K+ excretion
in healthy volunteers ............................................................................................... 303
Kid Exp. 14. To evaluate saluretic, natriuretic and carbonic anhydrase inhibitory effect of
various diuretics in healthy volunteers ............................................................... 305
Exercise .............................................................................................................................................. 307
30. Ophthalmology .............................................................................................................................. 309
Ophtha Exp. 15. To evaluate the effect of the hyoscine on pupillary diameter,
salivary secretion and heart rate ..................................................................... 309
Ophtha Exp. 16. To evaluate the effect of Tropicamide (1%) on pupillary
diameter and accommodation reflex ............................................................. 312
Ophtha Exp. 17. To evaluate the effect of topical pilocarpine (2%) on pupillary
diameter in healthy volunteers ....................................................................... 314
Ophtha Exp. 18. To demonstrate water induced ocular hypertension in
healthy volunteers ............................................................................................ 316
31. Clinical Pharmacokinetics............................................................................................................ 319
Exp. 19. To study the pharmacokinetics of Aceclofenac tablet following
single oral dose. ............................................................................................................... 319
32. Miscellaneous Practicals ............................................................................................................... 322
Exp. 20. To evaluate the analgesic activity of NSAIDs on different human pain models ... 322
A. Cold water stress ....................................................................................................... 323
B. Radiant heat ................................................................................................................ 323
C. BP cuff inflation ......................................................................................................... 324
D. Hand dynamometer .................................................................................................. 324
Exp. 21. To evaluate plasma salicylate level by fluorometric methods in
healthy volunteer............................................................................................................. 325
Exp. 22. To evaluate acetylator status by isoniazid (INH) estimation in
healthy volunteers ........................................................................................................... 328
Exp. 23. To evaluate anticholinergic effect of oxybutynin (30 mg tablet) on salivary secretion
in healthy volunteers ...................................................................................................... 330
Exp. 24. To demonstrate histamine induced wheal and flare in healthy volunteers ........... 331
33. Laboratory Experiments (Assay) ................................................................................................. 333
Exp. 25. Therapeutic drug monitoring in pharmacology
(antiepileptic/lithium/digoxin) .................................................................................... 333
34. Impact Factor ................................................................................................................................... 336
35. Computational Pharmacology ..................................................................................................... 338
36. Pharmacokinetics/Pharmacodynamics ...................................................................................... 340
37. Promotional Product Literature ................................................................................................... 342
38. Analytical Toxicology ................................................................................................................... 346
 xvi  Practical Manual of Experimental and Clinical Pharmacology

39. Recent Advances in Pharmacology ............................................................................................ 350

A. Translational medicine ........................................................................................................... 350
B. Reverse pharmacology ............................................................................................................ 350
C. Microdosing (Phase 0) ............................................................................................................. 351
Appendices
I. Abbreviations ..................................................................................................................................... 353
II Drug and solubility ........................................................................................................................... 355
III. List of drugs in clinical pharmacology practicals .............................................................................. 357
IV. Equipment required in clinical pharmacology laboratory ................................................................. 363
V. Analytical and molecular mass .......................................................................................................... 364
VI. Log conversion table........................................................................................................................... 365
VII. SI unit conversion .............................................................................................................................. 368
VIII. Practical examination question paper ................................................................................................ 369
Index ............................................................................................................................................................ 371
Part 1

General Considerations in
Experimental Pharmacology
 Introduction to Experimental
1 Pharmacology
EXPERIMENTAL BACKGROUND
Drugs and their use were well started in pre-
historic era and even the beneficial or toxic effects
of many plants and animal sources were recog-
nized. In ancient time, India and China enlist
many types of remedies, out of which a few are
recognized till today as useful drugs. Before 1693,
“Pharmacology” word was not coined but in the
year 1693 “Samuel Dale” published the 1st edition
of his book entitled as ‘Pharmacologia sen
Manuduction ad Materiam Medicam’. 2500BC,
there were tremendous attempts to introduce
rational methods of experimentation into drug
discovery and research, but the limitation of
thought; which proposed to explain disease
without the need for experimentation and obser-
vation was dominated. The oldest belief was that,
India and China dominated in the ancient
therapeutics. Indian contribution is with
“Rigveda” (2500-3000 BC) which is one of the
ancient preparation compendium, followed by
“Charaka Samhita” written by physician
“Charaka” and renowned surgeon “Sushruta”
described various preparations of different
sources. “Pan Tsao” a Materia Medica of China
has contributed of many preparations from the
animal, plant or mineral origins.
The concept of pharmacology began in
17thcentury, when observation and experi-
mentation began to replace traditional drug use.
Many physicians from Great Britain and of this
continent understood the values of experimen-
tation when they applied them to the effects of
traditional drugs used in their own practices in
the treatment of several diseases. Thus, ‘materia
medica’ the science of drug preparation and
therapeutic use of drugs began to develop as the
part of pharmacology. But, limitation in use was
lack of methods for purifying active agents from
the crude materials and methods for testing
hypotheses.
In the late 18th and early 19th centuries, François
Magendie and later his student Claude Bernard
began to develop the methods of experimental
animal physiology and pharmacology and was
recognized as father of physiology and prince of
vivisectors. Antoine Lavoisier proved that
respiration was a form of combustion by using a
guinea pig and Stephen Hales measured blood
pressure in the horse in the same era. In the 1880s,
Louis Pasteur demonstrated the germ theory of
medicine by giving anthrax to sheep. Furthermore,
advances in chemistry and development of
physiology in the 18th, 19th, and early 20th
centuries contributed to the foundation needed
for basic understanding, how drug works at the
organ and tissue levels?
Real advances in the basic pharmacology
during this time were mainly promoted by the
private manufacturers and marketers. But, the
concept of rational therapeutics was started with
the randomized controlled clinical trial (compa-
rative study of drug action in human) which were
reintroduced into drug development about 50
years ago that it became possible to accurately
evaluate therapeutic drug. At the same time major
expansion of research was taken into all the
4  Practical Manual of Experimental and Clinical Pharmacology
biological fields. As new concepts and new
techniques were introduced, information
accumulated about drug action and their target
on receptor at more molecular level. So, during
the last half-century, many fundamental new
drug groups and new members of old groups were
introduced.
The studies of the molecular basis for drug
action have been seen in last three decades due to
rapid growth of information and understanding
in the concerned area. The molecular mechanisms
of action of many drugs have now been identified;
numerous receptors have been isolated, struc-
turally characterized, and cloned. In fact, the use
of receptor identification methods has led to the
discovery of many orphan receptors for which no
ligand has been discovered.
Genetic studies such as, decoding of the
genomes of many species from bacteria to humans
have led to an emerging area of unsuspected
relationships between receptor families. In the
present scenario, pharmacogenomics reveals the
relation of the individual’s genetic makeup to his
or her response to specific drugs. It helps to
understand individual’s inherited abnormality in
their DNA, so it is now possible in the case of
some inherited diseases to define exactly which
DNA base pairs are abnormal and in which
chromosome they appear thus making it easy to
target the disease progression.
One of the most powerful new genetic tech-
nique is the ability to produce transgenic animals
(e.g. mice) in which the gene for the receptor or its
endogenous ligand has been “knocked out,” i.e.
mutated so that the gene product is absent or
nonfunctional. Homozygous “knock out” mice
have complete suppression of that function, while
heterozygous animals have partial suppression.
On the other hand “knock in” mice have been bred
that can over express certain receptors of interest.
In last two decades, many nanomedicines like
polymeric nanoparticles, biochips, nanosensors,
bioreactors, neural stem cells, immune nano-
particles, nanotubes, micelles, liposomes,
quantum dots, dendrimers, fullerenes, and
hydrogels have been developed for the more
targeted disease therapy in CVS, CNS, GIT,
chemotherapy, etc.
So, based on previous studies and experience,
pharmacological products are broadly governed
by two principles;
1. All therapies for health should meet the same
standards of evidence of efficacy and safety in
preclinical and clinical studies and
2. Substances used as drugs can be toxic under
certain conditions
Pioneers of Pharmacology
• Father of Modern Experimental Medicine
Claude Bernard (1813-1878)
• Father of American Pharmacology
John Jacob Abel (1857-1938)
• Father of Indian Pharmacology
Ram Nath Chopra (1882-1973)
• Father of Clinical Pharmacology
Lou Lasagna (1923-2003)
• Father of Modern Medicine
Hippocrates (460 BC-370 BC)
• Father of Modern Pharmacology
Oswald Schmiedeberg (1838-1921)
• Father of Modern Chemotherapy
Paul Ehrlich (1854-1915)
Claude Bernard
(July 12, 1813 – February 10, 1878)
A French physiologist recognized for his three
major works:
1. He worked on the functions of the pancreas
gland, and postulated that the pancreatic juice
has great significance in digestion. For this
work, he won a prize for experimental
physiology from the French Academy of
Sciences.
2. He investigated glycogenic function of the liver.
3. He established the existence of vasomotor
system.
Note: Claude Bernard is known as the father of
physiology and prince of vivisectors.

John Jacob Abel
(May 19, 1857 – May 26, 1938)
1883 : Ph.D from the University of Michigan.
1891 : Founded and chaired the first depart-
ment of pharmacology in the United
States at the University of Michigan.
1893 : Chaired the pharmacology department
at Johns Hopkins University
1897 : Second to isolate epinephrine (First was
Napoleon Cybulski in 1895)
1914 : Isolated amino acids from the blood
1926 : Reported the isolation and crystal-
lization of insulin
Note: Formulated the idea of the artificial kidney
Ram Nath Chopra
(August 17, 1882 - June 13, 1973)
1908 : Obtained MD degree from Cambridge
University
1921 : Appointed as the first Professor of
Pharmacology in the newly established
Calcutta School of Tropical Medicine
and parallely headed the Department of
Pharmacology at the Calcutta Medical
College
1941-1957
: Appointed as Director of the Drug
Research Laboratory at Srinagar 1958
onwards; Honorary Scientific Advisor to
the Regional Research Laboratory,
Jammu
He first introduced and done systematic study
of Rauwolfia serpentina and had a major
contribution in establishing the first National Drug
Research Institute of India, Lucknow (Presently,
known as Central Drug Research Institute (CDRI)).
He pioneered research on herbal drugs in India.
Note: Indian Posts and Telegraphs Department
has issued a commemorative stamp in his honor.
Paul Ehrlich
(March 14, 1854 – August 20, 1915)
1878 : Obtained his Doctor of Medicine (MD);
Dissertation on the theory and practice
of staining animal tissues and in the
Introduction to Experimental Pharmacology  5
same year appointed as assistant to
Professor Frerichs at the Berlin Medical
Clinic.
1882 : Ehrlich published his method of staining
the tubercle bacillus that Koch had
discovered and also derived the Gram
method of staining bacteria, so much used
by modern bacteriologists. He has
become Professor.
1890 : Appointed as one of assistants of Robert
Koch, Director of the newly established
Institute for Infectious Diseases where
Ehrlich began the immunological studies
1896 : Appointed as Director of Institute for the
control of therapeutic sera, Steglitz, Berlin
1897 : Appointed as Public Health Officer at
Frankfurt-am-Main
1899 : Became Director of the Royal Institute of
Experimental Therapy, Frankfurt, where
he devoted himself to chemotherapy
1904 : Ehrlich became honorary Professor of
the University of Göttingen, Germany
1906 : Received Prize of Honor at the XVth
International Congress of Medicine at
Lisbon and discovered the structural
formula of atoxyl, a chemical compound
able to treat sleeping sickness
1908 : Shared Nobel Prize with Metchnikoff the
highest scientific distinction
1909 : He and his student Sahachiro Hata
developed Salvarsan, a treatment
effective against syphilis
1911 : Liebig Medal of the German Chemical
Society
1914 : The Cameron Prize of Edinburgh
Note: He coined the term “chemotherapy” and
popularized the concept of “magic bullet”. He is
credited with the first empirical observation of the
blood-brain barrier and the development of the
first antibacterial drug in modern medicine.
DRUG DEVELOPMENT AND USE OF
ANIMALS: AN OVERVIEW
Drug development mainly deals with three stages:
Stage I: Hit and lead compound development
phase (Identification of lead compound amongst
6  Practical Manual of Experimental and Clinical Pharmacology
the million compounds and selection for further
study)
Stage II: Preclinical studies (Done in form of
in vitro and in vivo animal experiments)
Stage III: Clinical studies (Experiment in human:
Clinical trial Phase 0, I, II, III, IV and V)
The process of finding a new drug for a
particular disease, usually involves several steps
beforehand. The discovery of drugs, mainly
involves in vitro as well as in vivo studies in
different species. In late 18th century, pharma-
ceutical companies played an important role in
research and development (R & D) and due to
their focused and planned activity in the drug
discovery R & D grew rapidly.
In the first stage of drug development, target
identification is the most important step. It saves
lots of time. Targeted chemical moiety identification
also makes some sense. After the target
identification, next step is to find lead compounds.
Lead compound is identified by the cloning of the
target protein. Amongst several species sequences,
human sequence is preferred due to the species to
species variation. There is specific lengthy protocol
for the development of lead compound, which is beyond
the limit of this chapter. Briefly, cloning of the target
protein, identification of the functional activity of
target protein, combinatorial chemistry study and
high throughput screening (HTS) or ultra high-
throughput screening (UHTS) are mainly involved
in the identification of hit compound and then lead
compound for the further stage. After the lead
compound identification, the next step is lead
compound optimization, which mainly involves
increasing the potency of the compound with
respect to selectivity, metabolic stability,
pharmacokinetics and toxicological effects on its
selected target.
Note: High-throughput screening (HTS) involves assays
performed in a parallel fashion using multi-well assay plates
of 96, 384 and above, whereas ultra high-throughput
screening (UHTS) is performed using single-digit microliter
to nanoliter scale or 1536 well plate and more. UHTS is
considered to be more sensitive and can screen more
compounds as compared to the HTS.
The objective of the lead optimization phase
is to identify one or more drug candidates suitable
for further development. Thereafter, the drug
enters into the stage II (preclinical studies),
which involves pharmacodynamics, pharma-
cokinetics and toxicity studies (acute toxicity,
chronic toxicity, genotoxicity, etc.) in animals
as well as receptor identification and
characterization studies. The aim of the
preclinical study is to find out the maximum
recommended starting dose (MRSD) for the
human. This is done by several in vitro (cell line,
enzyme inhibition, etc.) and in vivo (animal
model) studies. In stage III, after getting the
MRSD, dose can be extrapolated and calculated
for the safe starting dose (SSD) with application
of safety factor. After the selection of SSD,
clinical trial begins with the healthy volunteers
(Phase I), But, there are few exceptions for the
Phase I study, in which patients are preferred
instead of healthy volunteers such as cancer,
HIV, cystic fibrosis, etc. The complete steps of
drug development are summarized in the Table
1.1 and Figure 1.1.
COMMONLY USED EXPERIMENTAL
ANIMALS
Selection of an animal model is one of the most
important steps in any of the experimental
pharmacological study. Animal model preferred
for the study must be producing similar disease
profile as in the human. Hence, suitable animal
model should be selected which follows three main
objectives:
1. Use of an animal phylogenetically closer to
man or
2. Use of an animal in which the process under
investigation is as close as possible to that in
man,
3. The Anatomy, Physiology and Biochemistry
are considered to be similar
 Introduction to Experimental Pharmacology  7

Table 1.1: Drug discovery timeframe and compounds screened

Duration Events during the period Compounds Screened *
(years)*
0-1 Identify targeted disease, budget, research team, drug design,
devise assays and select ‘hits’
1-2 Establish utility of ‘hits’ in animals
2-5 Identify promising ‘hits’; then identify lead compound and optimize
it with (literature, potency, acute toxicity, ADME, synthesis,
feasibility, etc.)
4-5 Patent protection
4-9 IND application, Phase 0, I to III clinical trials
8-11 Regulatory review
10-15 NDA application, Marketing and phase IV and V
15-20 Patent protection expires

* Years and compounds screened are approximate values which may vary with drug to drug
Roughly about $850 Million are required to develop a drug
Note: In the present scenario due to use of high throughput or ultra high throughput screening drug development
timeframe is significantly reduced with better output.

Fig. 1.1: Summary of developmental stages of a drug. Divided into the three stages of drug development includes lead
compound identification and optimization, preclinical studies and clinical studies. (IND= Investigational New Drug; NDA=
New Drug Application; ANDA= Abbreviated New Drug Application)
8  Practical Manual of Experimental and Clinical Pharmacology
Broadly experimental animals are divided into
three categories.
Mouse as an Experimental Animal
(Mus musculus)
According to paleontological data, men and mice
have been in contact since the early “Pleistocene”
(From two million to 11 thousand years ago). In
the biomedical research mice are preferred which
are the smallest rodents used in the laboratory.
They have several advantages over other species,
e.g. they are easy to keep, handle and require small
place for housing.
Mice proved as an invaluable resource in
identifying the several alleles which further
develop in the research of mutagenesis. Mice are
the only known species in which it is possible to
grow totipotent embryonic stem (ES) cells in vitro,
and can form germ line once re-injected into a
developing embryo.
Mice (subgenus Mus) comprise several species
that are similar in size and shape but never
hybridize in wild. Region to region, its species
varies which is described in the box below. Mus
musculus has Indian subcontinent origin but now
have spreaded to several countries. DBA/2 is the
most ancient of all in bred strains, established by
CC Little in 1909 whereas C57BL/6 was
established by Miss Lathrop after 10 years. Other
commonly used strains C3H, CBA and A were
discovered by LC Strong.
The ability to add and selectively alter the mouse
genome is the potential tool for understanding the
genetic basis of human health and disease. Due to
the large similarity in mice and human genome
(>99% conserved), it provides good model for
research not only on mammalian biology but also
on a wide variety of human diseases like, cancer,
diabetes, ageing, atherosclerosis, immunological
disease, autoimmune disorders, neurological
dysfunction and other endocrine diseases and
several other diseases. Inbred and mutant mice are
ubiquitously accepted as the preferred models for
identifying and understanding inherited human
diseases. Most importantly, its selection as the first
model animal to has its genome sequenced in the Human
Genome.
Knockout and Knock in mice have been
developed for the selective assessment at the
genetic level. In the Knockout mice, selective gene
is taken out whereas in Knock in mice gene of
interest is introduced into the mice. The first
knockout mouse was created by Mario R Capecchi,
Martin Evans and Oliver Smithies in 1989, for
which they were awarded the Nobel Prize for
Medicine in 2007. In 2006, Nobel Prize was
awarded to Andrew Z. Fire and Craig C Mello for
RNA interference, in which genes are silenced or
“knocked down” by short pieces of double-
stranded RNA.
Region and Species Found
• Asian species: Mus cervicolar, Mus cookee, Mus
caroli, Mus famulus (India) and Mus fragilicauda
(Thailand-latest strain)
• Western Mediterranean region: Mus spretus
• Eastern Mediterranean and Central Europe: Mus
spicilegus, Mus macedonicus
• Western Europe and Africa: Mus domesticus
• India, Eastern Europe, Japan, Russia, Northern
China: Mus musculus

 Important Points to Remember
• Smallest laboratory animal
• Common strain Swiss albino mice
• Nude mice: Hairless genetic mutant which lacks
thymus gland
• Biege mice: Lack natural killer cells and are susceptible
to cancer
• Mice use their tail to help in thermoregulation
Rat as an Experimental Animal
(Rattus norvegicus)
Rat is the most commonly used animal in the
biomedical research. The randomly bred strains
are used almost exclusively and are derived from
the Norway rat (Rattus norvegicus) which is
thought to have originated in the area between
the Caspian Sea and Tobolsk. Among these,
“Wistar rat” and the “Sprague Dawley rat” are
preferred because of easy handling, sensitivity and
low cost. The Albino rat (officially known as the
Pink-Eyed White or PEW) is most likely the very
first mutation to be discovered and purposely bred.
Albino rats were introduced to Great Britain by a
travelling entertainer around the year 1800.
Nude rats resemble nude mice in their lack of a
normal thymus and functionally mature T cells
and are phenotypically hairless with possible
fine-sparse hair growth and most preferred model
in immunological research.
Rat is preferably used in the research of
behavior, pharmacology, physiology, neuro-
sciences, immunogenetics, transplantation,
cancer risk assessment, cardiovascular diseases
and aging. Further, development and genetic
characterization of inbred, congenic, and
recombinant strains took place in the United States,
Japan and other European countries.
Introduction to Experimental Pharmacology  9
 Important Points to Remember
• Albino (subgroup, Wistar and Sprague Dawley (SD)
rat)
• Wistar rat: wide head and the ear is long whereas tail
length is less than the body length
• SD rats: longer and narrow head. Tail is longer than the
body length
• Do not vomit (due to strong sphincter between the
stomach and the esophagus and lack vomiting center)
• Do not have tonsil and gallbladder
• Diffuse pancreas, so not a good model for type I diabetes
• Coprophagy (eating their own stool)
• Tail of rat helps in thermoregulation of body
Note: Some important phenotypic differences
between baby rats and mice
1. Baby rat has blunt and broad large head
relative to body whereas mice have triangular,
small head relative to body
2. Baby rat has small ears relative to the head
whereas mice have large ears
3. In the baby rat hind paw and body ratio is
larger as compared to mice
4. Tail is thick and shorter than body length in
baby rat while mice have thin and larger or
same length tail as compared to body
Guinea Pig (GP) as an Experimental Animal
(Cavia porcellus)
About 400 years ago, Guinea pig (Cavia porcellus)
was introduced into Europe from South America.
There are 3 major varieties of strains used in the
experimental studies and is the member of rodents
suborder “Hystricomorpha”. Guinea pig is
herbivorous and eats green foods, seeds and roots,
but now in many laboratories feed is provided
with a readymade chow diet which fulfills its daily
10  Practical Manual of Experimental and Clinical Pharmacology

dietary requirement. But, it is essential to add

vitamin C (Ascorbic acid) in the chow, while it is
important to note that GP are not able to synthesize
the required vitamin C daily. It is recommended
that when GP is provided with the greens,
then ascorbic acid should be given at the rate of
1gm/L of drinking water on a weekly basis. If
they are completely dependent on the chow
then vitamin C should be added in the dose of
200 mg/L daily.
Newborn GP can eat solid food by the fifth
day and may be weaned by 2 weeks. Young GP
are best mated after 3 months of age. The maturing
is slow in male as compared with the female sibs.
Coitus generally occurs at night and that followed
by polygamous group mating. Duration of estrous
cycle will range from 13-20 days an average of 16
days can be further divided into the other stages
such as proestrus, estrus, metestrus and diestrus.
Anatomically, guinea pig coronary arteries
have duality concerning the origin and branching
which are represented by 4 instead of 2 aortic
branches compared to other species. It is sensitive
to various diseases and infections which makes it
suitable for the diagnostic tests. It is an ideal model
for the enteric amoebiasis and widely used in the
hypersensitivity, immune response, anaphylactic
shock, encephalomyelitis, tuberculosis and
ascorbic acid metabolism. GP is used widely in
the screening of local anesthetics and is good
proposed model for bronchial asthma, COPD, etc.
 Important Points to Remember
• Highly sensitive to histamine (1000 times more
sensitive)
• Serum contains an enzyme asparaginase, which shows
antileukemic action
• Very susceptible to tuberculosis and anaphylactic
shock
• Highly sensitive to penicillin (100-1000 times more than
rat)
phenotypically related to rat and also known as
“jirds” or “sand rat”. The original habitat of wild
species is Mongolia and adjacent parts of Southern
Siberia and Northern China, Sinkiang, and
Manchuria. This animal is preferred in the
laboratory because of ease in handling, mild and
quiet nature. Housing guidelines varies from
country to country.
Housing space of the gerbils are different at
different places such as in USA, it is said that 5
gallons (1 gallon = 231 cubic inches) are required
for each gerbil, i.e. 10 gallons for 2 gerbil, 15
gallons for 3 gerbil, 20 gallons for 4 gerbil, etc.
whereas in UK, for a pair of Gerbils a 15” by 12”
by 10” tank is ideal.
Gerbil is widely used as a research animal in the
field of stroke, epilepsy, auditory studies (hearing
curve similar to man), parasite and bacterial
infections and lipid metabolism and heart disease
studies (high serum cholesterol levels). This animal
is one of the few species which were originally
developed in Japan as laboratory animals.
Hamster as an Experimental Animal
(Mesocricetus auratus)

Gerbil as an Experimental Animal

(Meriones unguiculatus)
The Mongolian gerbil is a small laboratory rodent,
having length in between rat and mice. It is
Among small rodents, hamster a brown to gold
color animal has become the third most commonly
used laboratory experimental animal in the
biomedical research. They have different strains

namely Syrian hamsters (Golden), Chinese hamster
(striped back), European hamster and Armenian
hamster (gray).
Syrian hamster is the most commonly used
in biomedical research because of availability and
ease of reproduction. They are relatively free from
spontaneous disease and susceptible to many
introduced pathogenic agents. Their anatomical
and physiological features are unique for the
experimental study, and have rapid development
with shorte life cycles. European hamster (quite
larger than other hamster species (300-400 gm))
is a more suitable model for highly concentrated
and prolonged smoke inhalation studies than
the Syrian hamster. Armenian hamster is more
specific for the research to mutagenic and
carcinogenic agents and for studying meiosis
due to its susceptibility. Chinese hamster has the
lowest number of chromosomes compared to any
other placental nurtured laboratory animal and
it is useful for cytogenesis research.
This is a spontaneous model of human
diseases such as diabetes mellitus (similar to the
juvenile type in man), Syrian hamster dystrophy
(Autosomal recessive skeletal muscle degene-
ration, cardiomyopathy, cardiohypertrophy, and
congestive heart failure), cholesterol cholelithiasis
or gall stones, polycystic diseases, dental caries
etc. It is a good model for physiology and
pathogenesis of Duchenne’s dystrophy, variable
sizes of muscle, fiber, centrally located nuclei,
with fatty infiltration and fibrous connective tissue
replacement.
Hamsters are used extensively in slow virus
(Scrapie, chronic measles, etc.) type C, Onco virus,
influenza virus, respiratory syncytial virus (RSV)
studies and vaccine production (Foot and Mouth).
Due to anatomical advantages, cheek pouches do
not have intact lymphatic drainage and hence,
they are an ideal site for tissue transplants, such
as, tumors and grafts. Hamsters are used for in
vivo and in vitro diagnostic techniques for
numerous infectious agents (i.e. Clostridium spp;
Leptospirosis spp).
Introduction to Experimental Pharmacology  11
Note: Cheek pouch: They have characteristic cheek
pouch which is used for collection or transport of food
materials or any nesting materials.
Whiskers: It is found on the face and the side of the body
to navigate the surroundings and object around them,
especially at night. It is also known as “vibrissae”.
Non-rodents (Mammalians)
Rabbit as an Experimental Animal
(Oryctolagus cuniculus)
The most common strain in use is New Zealand
white rabbit, followed by the Dutch, the Flemish
Giant and other minor strains of the domestic
rabbit. In 1916, WS Preshaw bred the first litter of
New Zealand white rabbits and it is considered
to be 100% American bred.
New Zealand white rabbits have been used
in the screening of different drugs for diseases
like diabetes, diphtheria, tuberculosis, cancer,
and heart diseases. Biomedical research
studies in which rabbits is commonly used are
genetics, nutrition, toxicology, physiology,
immunology and reproduction. Classically
the rabbit has been utilized in human medicine
to determine pregnancy in women by injecting
the serum from the patient into the rabbit
and thereby inducing ovulation in the doe.
Apart from the drugs, effects of skin
creams, cosmetics, special diets, and food
additives have also been tested on New Zealand
white rabbits.
Other important uses are:
1. Standard animal for pyrogen testing of all
solutions for human medical use
12  Practical Manual of Experimental and Clinical Pharmacology
2. To test toxic effects of cosmetics and
pharmaceuticals
3. Good model for the production of antibodies
and antiserums.
New Zealand white rabbits have a genetic
deviation called albinism. Albinism is caused by
lack of melanin, which is a vital pigment that gives
their skin/ fur/ hair/ eye color in all creatures,
including humans. Rabbit is homologous to
humans to react similarly to diseases and
medications. A female rabbit (doe) is fertile all year
long. The gestation period is around 28 to 31 days.
Mainly, chemical method is preferred for
euthanasia in rabbits. Cervical dislocation which
is generally preferred for the rodent is not suitable
for the rabbit because of short neck. It has very
simple cardiac conductive tissue, free of
connective tissue and is an animal of choice for
many cardiac studies.
 Important Points to Remember
• Very sensitive to histamine
• Cannot vomit (like the rat and the horse)
• Ideal animal for pharmacokinetic studies
• Cytochrome 3A4 is absent which is corresponding to
cytochrome 3A6
• Enzyme atropinesterase present in blood which
degrade atropine
• Has an ability to taste water, a characteristic absent in
humans or rats
• Very important for pyrogen testing in parenteral
preparation
• Only known mammal from which tubules of the kidney
can be dissected with basement membrane intact
• Lacks vasomotor reversal phenomenon (Absence of
adrenergic vasodilator nerve)
Monkey as an Experimental Animal
(Macaca mulatta)
Mammalians are used widely in the biomedical
research due to the anatomical and physiological
resemblance to humans. Monkeys are one of the most
commonly used mammalian in the experimental
studies others in the cue are dogs and cats. The
rhesus monkey (Macaca mulatta) commonly used is
found in the South Asia. Phenotypic property in the
male and female are almost same except face is
slightly elongated, low brow ridges are continuous
more in male. Adult body weight is 10-12 kg for
male and 8-10 Kg for female.
Husbandry is based with the objective to keep
animal in a hygienic and physical environment
to fulfill their optimum requirement. Standard
room temperature is kept at 22º- 25ºC with the
standard deviation of ±5ºC. Humidity is
maintained between 55-60% and 12 hours of light
cycle (photo cycle) to maintain their breeding and
physiological rhythm.
During the study animal can be maintained in
the gang cages (single or in group). The determined
dimension for the cage is 24" × 30" × 30" for single
or individual housing and 28 ft × 10 ft for group
housing (8 female and 1 male may be maintained).
Monkeys eat a variety of foods such as fruits,
insects, buds, bark, etc. but in the laboratory set-
up, it is important to keep animal healthy and
disease free hence a standard food composition is
used to fulfill their daily requirement. Diet consists
of 18-20% protein, 65-70% carbohydrates, 4-6%
fat, 4-5% crude fiber, minerals, and vitamins.
Monkey infants depend on the breast milk of
mother and continue clinging till the 6 months of
age, thereafter caged separately in a group of 2-4
and are fed with milk, fruits or cereal food.
Monkeys are widely used as primate model to
study drug metabolism because they generally
show a metabolic pattern similar to humans.
 Important Points to Remember
• Uterus resembles humans and exhibiting regular
menstrual periods
• Metabolism approximately same as human
• Ideal model for pharmacokinetic study
• Best for studying drugs acting on CNS (memory, anxiety,
antidepressants, etc.), CVS (antihypertensive,
antianginal, etc.), GIT and fertility
• Require regular check up for rabies, tuberculosis and
timely immunization
 Introduction to Experimental Pharmacology  13

Dog as an Experimental Animal

(Canis familiaris)

or rodent, hence they have been extensively used

in cardiovascular (CVS), behavioral and biomedical
research.
Cats are useful models in studying the
transmission of vitamins and minerals to the fetus
After monkey, dog is the most preferred large
and newborn. The collection of blood samples is
experimental animal. The advantages being small
relatively easy. Cat has distinct nictiating
alimentary tract and easily get trained. Mongrel
membrane hence commonly used in the screening
and Beagles are the most preferred for the
of ganglion blocking drugs. Cats are mainly
experiment purpose due to manageable size,
carnivorous and sedentary except when hunting.
moderate length of hair coat, docile nature and
Smell is less developed than dog. It is not a good
ease to handle.
model in the experiment on the loss of righting
Cardiovascular research is preferred in the
reflex, because it regain its righting reflex even fall
dogs. Drugs acting on blood pressure and from a high altitude.
vascular system are preferably screened. It is also Cats are mainly used in the field of behavioral
a good model for diabetes mellitus and studies, cardiovascular studies, nerve impulse
reproduction. The dog is frequently used as a transmission, e.g. reflexes of the respiratory system
model for many human conditions in areas such and spinal system, reflexes associated with
as cardiovascular research, diabetes mellitus, nociception, light perception, sound perception
ulcerative colitis, open heart surgery, organ and body reaction to exposure to chemical stimuli.
transplantation, central nervous system (CNS), Additionally, it is also used in the neuropharma-
safety pharmacology and toxicology. cology (particularly the testing of psychotropic
drugs), toxicology, oncology and chromosomal
 Important Points to Remember
abnormalities studies.
• Stomach and intestinal tract resemble human
• Distinct structure of pancreas, allowing it as good model Pig as an Experimental Animal
for the research on diabetes (Sus scrofa domestica)
• It may develop spontaneous hypertension resembling
human
• Cervical sympathetic and vagal nerves are run together
hence stimulation of nictitating membrane through
preganglionic sympathetic is complicated by central
vagal stimulation causing reflex variations in blood
pressure

Cat as an Experimental Animal

(Felis catus)
Anatomical and physiological similarities
Cat has similar physiological features which are between man and pig makes this animal a good
common with humans than the laboratory rabbit model in many research areas including
14  Practical Manual of Experimental and Clinical Pharmacology
pharmacological and toxicological research.
Several isolated organ models, investigation of
skin permeation and for digestive systems, etc.
are few important areas of research with pig as an
experimental animal. This is preferred because, it
is small sized at maturation as well as is selecting
for less hair contains on the body. In Europe, pigs
are used in pharmaceutical R & D studies in place
of dogs and primates.
Dissimilarities are in the vascularization (rich
in man, poor in the pig) and in the sebaceous
glands. Humans have mostly eccrine sweat
glands over the body surface, whereas the pig has
only apocrine glands. Similar findings also
observed while studying the skin of other domestic
mammals. Pig has small lungs in relation to body
size and is susceptible to bronchitis and
pneumonia.
 Important Points to Remember
• Alimentary tract resembles human
• Important animal model in cardiovascular research
such as atherosclerosis, MI, etc.
Zebra Fish (Danio rerio)
Dr George Streisinger at the University of Oregon
observed that the Zebra fish is a suitable model
for studying vertebrate development and genetics
in early 1970’s and is considered to be the “Father
of Zebra fish research”. The zebra fish is native to
the freshwater streams of the southeastern
Himalayan region such as Eastern India and
Pakistan, Bangladesh, Nepal, and Myanmar.
Zebra fish are vertebrates and have a backbone
like humans which gives more close relation to
humans than commonly used invertebrate
models, such as insects and worms (Drosophila-
fruit flies and Caenorhabditis elegans: nematodes)
which do not have backbones. Nowadays,
studying embryo development and genetics make
it useful animal model as in genetic techniques. It
is getting popularity into the biomedical research
due to its clear eggs which can be developed
outside the mother’s body and allow watching a
zebra fish egg grow into a newly formed fish under
a microscope in 2-4 days. The lifespan of zebra
fish is considered to be approximately 5 years and
length of the adult fish is about 6 cm.
The advantages of this model is that it can be
kept at fairly high densities in a small tank, so it is
cheaper to maintain than other experimental
animals, a single spawning produces 100-200 eggs
which are easily collectible, development of eggs is
clear and easily observed and manipulated
whereas drawbacks are that it requires an
aquarium to maintain, not closely related to humans
as a mouse or other animal model (not mammalian)
and genetic modification has not been possible as
it is in the mouse (Knock out/Knock in).
Frog (Rana tigrina)
Frog is the only tail less experimental amphibian
which is used very commonly in the biomedical
research. They are mainly characterized by long
hind legs, a short oval body, webbed fingers or
toes and projected eyes. Generally, frogs and toads
are two characterized amphibians and have no
taxonomic basis to make difference, but can be
differentiated by their appearance. (Toads are dry
whereas frog is wet). Anatomically their heart
contains three chambers which is different from the
other mammals used in the experiment. Barbouruta
kalimantanensis (Flat headed Frog) is the first
species of frog which is found without lungs. Some
frog species like Xenopus laevis was first widely

used in laboratories in pregnancy assays. The
relationship between electricity and the nervous
system were well studied in frogs by biologist
Luigi Galvani. Frogs are generally used in the field
of CVS and CNS research, also used in cloning
research and other branches of embryology
because frogs are among the closest living relatives
of man who lack egg shells. Some hybridized frogs
are also used in biomedical research such as
Edible Frog (Rana esculenta), which is a hybrid of
the Pool Frog (R.lessonae) and the Marsh Frog (R.
ridibunda).
There are several important substances like
epibatidine (an alkaloid), a painkiller 200 times
more potent than morphine and other toxins like
irritants, hallucinogens, convulsants, nerve
poisons, and vasoconstrictors which are obtained
from different species of frog. Other chemicals
isolated from the skin of frogs may offer resistance
to HIV infection which is considered to be a good
target for research.
 Important Points to Remember
• Oxygen can pass through their highly permeable skin
and hence “breathe” largely through their skin
• Camouflage is a common defensive mechanism in frogs
(hiding or color change)
• Very commonly used in the CVS related experiments
or bioassays
Chicken (Gallus domesticus)
In the experimental pharmacology, chicken are
widely in use due to the ease of availability and
its organs. The maintenance is quite economical.
The room temperature should be around 23°C
which is achieved by reducing the temperature
from 32-33°C (1°-2°C per week) until a temperature
of 23°C is obtained. It is thought that chickens
Introduction to Experimental Pharmacology  15
were domesticated in India about 2000 BC and
then introduced to Japan via Korea about 300 BC.
Chickens can be easily bred and housed. So, they
are being increasingly used as experimental
animals. The digestive tract of the chicken is
simple, relatively short and highly efficient.
The use of chicken as an experimental animal
was started since the 1960s. They are being tested
in many areas of biomedical research such as
breeding and genetics, growth, performance
testing, embryology, incubation, fertility, artificial
insemination, hatchability, anatomy, toxicology
and pharmacology, behavior and welfare,
physiology, biochemistry, endocrinology and
neurobiology. Some of the important models
developed in the chicken are chick comb method,
chicken blood pressure measurement, heart rate,
EEG and activity through telemetric system,
Scleroderma model in chicken, measurement of
contractile force of isolated cardiac myocytes, and
vasodilating activity of chicken. Other few system
involved in the research of chick are angiogenesis
(chick chorioallantoic membrane assay: to test
angiogenesis and inhibition of angiogenesis),
catalepsy antagonism in chicken (white Leghorn),
in learning and memory (Aversive discrimination
in chickens) and model for spontaneous
autoimmune thyroiditis (Obese strain chicken: OS
chicken, e.g. UCD-200 chickens). At the finale, the
only model for studying Avian diseases.
Pigeon (Columbia livia)
Pigeons constitute the family Columbidae within
the order Columbiformes, which include some 300
species. In general, the terms “dove” and “pigeon”
are used interchangeably. Pigeon is survived on
seeds, fruit and plants. Pigeons have strong wing
muscles comprises 31-44% of their total body
16  Practical Manual of Experimental and Clinical Pharmacology

weight and are amongst the strongest fliers of all
birds.
Pigeons are mainly preferred in screening
antiemetic activity, cardiovascular diseases such
as spontaneous arteriosclerosis in pigeons and
standardization of cardiac glycosides, CNS such
as anxiety pigeon method, apomorphine induces
stereotypic behavior in pigeons and screening of
intravenous anesthetics in pigeon. Bioassay of
prolactin through the pigeon crop method is one
of the important methods in prolactin assessment.
Note: General information of laboratory animals
and their vital parameters are explained in Tables
1.2 and 1.3.
ANIMAL BEHAVIOR AND TERMINOLOGY
Animal behavior: It is defined as the behavioral
reaction of an animal to any internal or external
stimuli.
Abnormal appearance: It involves the postures like
head down, tucked abdomen, hunched back, facial
distortion, or pallor of an animal.
Allogroom: Rapid little nibbles like activity of one
animal to another, it is head or neck grooming or
Table 1.2: General information of laboratory animals
Animal Weight(g) Lifespan Floor space*/
(Year) animal
(cm2 × h (cm)
whole body grooming. For example: rat, mice,
guinea pig, gerbil, hamster, dog, etc.
Barber: This is the extent of grooming in which
the fur is nibbled off. Animal may barber
themselves or another animal. For example: rat,
mice, hamster, dog, etc.
Behavioral estrus: It is one type of reproductive
behavior in which female entreats male to grab
her, occurs mainly in the vaginal proestrus (12
hour period before ovulation).
Box behavior: One type of play or defense behavior
in which two rats stands on their hind legs/paws
facing each other nearly nose to nose, and pushes
or keep paw at each other with their front legs/
paws.
Chase or pursuit: Animals pursue behavior in
which an animal runs after another animal
Chatter: Repetitive grinding of the incisors against
each other
Cannibalism: The act of killing and eating of its
own species.
Coprophagy: Reingestion of feces by the animal
Ear wiggle: Female vibrates her ears rapidly in
behavioral estrus (about every 4-5 days) to solicit
and maintain mounting behavior by the male.
Food intake Water intake Gestation Animal House
(g/day) (ml/day) period (Days) Temperature Humidity
(°C) (%)

Mouse 18-40 1.5-3 77.4 × 12 3-5 6-7 19-21 19-23 40-70

Rat 150-400 2-4 187.0 × 14 5-10/100g 10/100g 21-23 19-23 40-70
Hamster 85-150 1-3 103.2 × 12 5-7 10/100g 15.5-16 19-23 40-60
Gerbil 55-100 2-4 103.2 × 12 5-8 4-7 24-26 19-23 30-50
Guinea pig 600-1200 4-8 651.4 × 18 6/100 g 10 /100 g 59-72 18-26 40-70
Rabbit 1000-3500 6-12 270 × 35.5 5/100g 10 /100 g 31-32 16-20 40-60
Chicken 1000-3500 3-5 30.48 × 60.96 125-250 200-300 20-22 18-26 40-70
* Floor space × height (h) is given as per adult animal requirement for housing in centimeter of the cage.

Table 1.3: Vital parameters of different experimental animals

Animal Rectal Heart Blood pressure (mmHg) Blood volume Respiratory Tidal
temperature (°C) rate (min) Systole Diastole (ml/Kg) rate (min) volume (ml)
Mouse 38-39 310-840 133-160 90-110 58.5 60-220 0.18
Rat 36-40 250-450 84-134 60 54-70 70-115 0.6-2
Hamster 37-38 250-500 150 100 78 35-135 0.6-1.4
Gerbil 37-38.5 360 — — 66-78 90 —
Guinea pig 37.2-40 230-380 80-94 55-58 69-75 42-104 2.3-5.3
Rabbit 38.5-40 130-325 90-130 60-90 57-65 30-60 4-6

Flank mark: It is a scent marking olfactory behavior
which is left on the objects in the surroundings by
rubbing their flanks. For example: rat, mice, dog, etc.
Gallop: Movement of both limbs (left and right)
nearly in synchrony. The gallop is fast and
asymmetric movement at the time of free-flight
(all four limbs off the ground) whereas trot is
the movement in which diagonal pairs of legs
move in synchrony (left front and right hind
paw)
Infanticide: Killing the young one’s of their own
species. For example: rat, mice, guinea pig, etc
Lordosis: Female mating posture which is a
reflexive behavior that is triggered by a touch
on the lower back, flanks, or genital region.
Vulva, which normally faces the floor, rotates
almost 90° to the vertical, i.e. to backward facing
position.
Mount: This is the male copulatory position, and
is seen when a male mounts a female prior to
mating.
Mutilation: Symptom of pain including, licking,
biting, scratching, shaking, or rubbing.
Nibble: Rats may nibble their own skin or that of
other rats with their teeth, appear to be combing
the fur with their teeth and nibbling the skin
underneath.
Nip: Light pinching activity which may elicit a
squeak with the teeth whereas skin remains
unbroken.
Peep: Brief high pitched sound heard during head
and body allogrooming.
Pica: The nausea response of rat called “Pica”
but rat cannot vomit (lacks vomiting center).
Piloerection: This is phenomenon in cold, or stress
in which body hair rises or erect on end.
Popcorning: Animal show this behavior during
play or happy moments, they run-around and
jump into the air and land on all fours paws. For
example: Baby guinea pig, dog, cat, etc.
Poofing: see Piloerection
Pup-killing: See infanticide
Introduction to Experimental Pharmacology  17
Pup retrieval: Carrying behavior of mother animal.
They keep rambling pups in her mouth with its back
to targeted place. For example: rat, cat, dog, etc.
Recumbency: Unusual length of time for pain as
symptom.
Sniff: It is an exploratory behavior which involves
cluster of movement sequences with snout and
head.
Solicitation: One of the sexual behaviors of female
in which female skims towards male and runs a
short distance, then wait for a while. “Full
solicitation” in which she repeats the same activity,
whereas “partial solicitation” in which she does it
once
Squeak: Vocalization response of rodents in pain
or any head grooming and mild social interactions
Vocalization: Crying out sound, when palpated
or given some painful stimuli
ANIMAL CARE, HANDLING AND SEX
DETERMINATION
Handling and restraining of experimental animals
is an important aspect in practical pharmacology,
which includes transferring the cage, feeding,
treatment (gavages or parenteral) or any other
procedure with the animals. In India CPCSEA has
given complete guideline for animal caring,
housing and handling requirements. As per GLP,
handling the commonly used rodents, there are
several points to be keep in mind which will limit
the unnecessary stress or strain to the animals.
Precaution to be taken before
handling the animals
• Before restrain, first pet or soothe the animal
by slow deliberate movements on their body
• Overcrowding near the animal cage should
be avoided
• Noise should be kept to a minimum as much
as possible
• Don’t hold animal too hard, it may face
difficulty in breathing and may die too
• Never agitate the animal, it may become violent
for self protection.
18  Practical Manual of Experimental and Clinical Pharmacology

Mouse Way 3: Hold the complete body by grabbing back

of the neck by using all fingers. (Forceful gripping
This is the smallest animal in the laboratory and
should be avoided) (Fig. 1.4).
used very commonly.
Way 1: One can handle it with the help of blunt
forceps by grasping the skin behind the neck/
body. This technique is often used to transfer mice
from one cage to another. (Should not hold too
hard, it may injure the tissue) (Fig. 1.2).

Fig. 1.4: Mouse handling technique (way 3)

Rat
Way 1: Lift rat out of the cage by grasping the base
Fig. 1.2: Mouse handling technique (way 1)
of the tail and place on a soft surface. (Hard smooth
surfaces can make the rat tense) (Fig. 1.5).
Way 2: Grasp the base of the tail with one hand
and with the other grasp the loose skin behind its
neck (Fig. 1.3)

Fig. 1.3: Mouse handling technique (way 2) Fig. 1.5: Rat handling technique (way 1)
 Introduction to Experimental Pharmacology  19

Way 2 (A and B): Place your index and middle

fingers (way 2A) alongside the rat’s head and your
thumb and ring fingers under its forelegs (way 2B).
Use your index and middle fingers to secure its
head and the remaining fingers to support the body
(Figs 1.6A and B).

Fig. 1.7: Rat handling technique (way 3)

(For color version see Plate 1)

Figs 1.6A and B: Rat handling technique (way 2)

(For color version of Figure 1.6A, see Plate 1)

Way 3: Hold the complete body by grabbing the

back by using complete palm (Fig. 1.7).

Guinea Pig
These are very humble rodents and can be easily
handled because of their docile nature
Way 1(A and B): By using both hands, calmly
grasp it with one hand under the chest and use B
your other hand to support its hindquarters (Figs Figs 1.8A and B: Guinea pig handling technique
1.8A and B) (way 1A and B)
20  Practical Manual of Experimental and Clinical Pharmacology

Way 2: Handle guinea pig with one hand, by

holding its hind quarter (Fig. 1.9).

Fig. 1.9: Guinea pig handling technique (way 2)

(For color version see Plate 1)
Hamster
Way 1: Hold the complete body by grabbing at the
nap of the neck by thumb and index finger and Fig. 1.11: Hamster handling technique (way 2)
grasp the complete body by using rest of fingers
Way 3: (Same as way 2; Position of fingers) Hold
(Fig. 1.10).
the complete body by grabbing back by using
complete palm (Fig. 1.12).

Fig. 1.10: Hamster handling technique (way 1)

(For color version see Plate 1)

Way 2: Hold the complete body by grabbing back

by using complete palm (Fig. 1.11). Fig. 1.12: Hamster handling technique (way 3)

Rabbit
Way 1: By using single hand, hold the pelvic
region. This technique is mainly used to transfer
rabbits from one cage to another (Fig. 1.13)
Fig. 1.13: Rabbit handling technique (way 1)
Way 2: By using both the hands, hold the complete
hindquarter (Fig. 1.14).
Fig. 1.14: Rabbit handling technique (way 2)
Way 3: By using the both hands, calmly grasp it
with one hand supporting back of neck and the
other hand supporting its hindquarters (Fig. 1.15).
Introduction to Experimental Pharmacology  21
Fig. 1.15: Rabbit handling technique (way 3)
Sex Determination in Rodents
Mouse/ Rat
Direction 1: Restrain the mouse/rat and lift the base
of the tail. Sex is most easily determined by anogenital
distance. Males normally have a greater distance
between the anus and urogenital openings. Male mice
also have a larger genital papilla (Fig. 1.16).
Direction 2: Scrotum can be easily palpable in male
Note: In winter scrotum shrinks inside the body,
hence direction 2 should be followed carefully in
winter.
Guinea Pig
Both male and female guinea pigs display similar
anogenital distances.
Direction 1: The female has detectable pink nipple
at either side, a separate urethal orifice, a vaginal
membrane, a perineal sac, and an anus whereas
male has a penis, a larger perineal sac, and an
anus (Fig. 1.17).
Direction 2: The penis lies just under the skin and
can be inverted with gentle pressure. The testes
and penis are palpable in adults.
DIET AND EXPERIMENTAL ANIMALS
The influence of the diet is directly on the health
of any animal or human. Diet is important in order
22  Practical Manual of Experimental and Clinical Pharmacology

Fig. 1.16: Gender identification in male/female Rat

Fig. 1.17: Gender identification in Guniea pig

to maintain health and energy. Main constituents
of the diet remain the same as for human, i.e.
proteins, carbohydrates, lipids, fibers, vitamins
and some ions and elements. Blood, urine
concentration, pH and extent of ionization of
compounds all depend on the diet of the
individual. In the experimental setting, one can
develop the different models of the animals by
restricting or enhancing the constituents of diet.
For example: atherosclerosis model, hypertension
model, diabetic model, pancreatitis model, etc.
Some species are very susceptible to the specific
type of diet, hence it can be used in the develop-
ment of certain disease model such as rabbits are
 Introduction to Experimental Pharmacology  23

susceptible to hypercholesterolemia and So, 0.25 mg is to be administered as per 10 gm

arteriosclerosis after excessive cholesterol feeding, of mice
pigeons develop spontaneous arteriosclerosis Suppose this has to be given in the volume of 0.1 ml
whereas hypercholesterolemia can be induced in Then, (1) will be written as
rats by daily administration of 1 ml/100 g body 0.25 mg/10 gm/0.1 ml (2)
weight of a cocktail containing in 1 ml peanut oil, Step 3: Now, weighing and dilution of the drug,
100 g cholesterol, 30 g propylthiouracil, and 100 From (2),
g cholic acid by gavages over a period of 7 days. = 0.25 mg/0.1 ml (multiply by 10)
All animal diets are tested in the government = 2.5 mg/1 ml
laboratory for their constituents (Proteins, = 25 mg/10 ml
carbohydrates, fibers, lipids, etc.) before supply to Hence, the solution should be prepared by
any institute or research center. adding 2.5 mg in 1ml or 25 mg in 10 ml of solvent,
which will give the dose of 0.25 mg/10 gm or
DOSE CALCULATION FOR 25 mg/kg of body weight.
EXPERIMENTAL ANIMALS
For Injectables
Sometimes pure powder of the drug is not
available, so student have to use the injectable
form or ampoule of the drug
Step 1: So, in this case two important things should
be kept in mind
Strength of the injection and its solubility in
the solvent
Suppose, morphine is available in the strength
of 15 mg/ml
Calculation 1 Students have to use this strength in rat at a
dose of 5 mg/kg, to show the analgesic activity of
For Powder Drugs
the drug.
One should follow the following steps given So, drug dose = 5 mg/kg
below, for calculating the doses and volume of = 5 mg/1000 gm
injection of any given drug, to be administered in = 0.5 mg/100 gm (for rat) (1)
the animal. That means, 0.5 mg of drug should be
administered in the 100 gm of body weight rat.
Step 1: Think of “What you have?” Suppose this is given in 0.1ml volume
Drug dose, weight of the animal and volume to be Then, (1) can be written as
injected 0.5 mg/100 gm/0.1 ml (2)
Suppose drug dose is 25 mg/kg for mice (Calculate Step 2:
the drug dose in the multiple of 10 gm in case of Dilution of injection, strength 15 mg/ml
mice or 100 gm in case of rat, guinea pig or rabbit) Put 1ml of drug in test tube and then add 2 ml
of solvent in the test tube (solution A)
Step2: Now, test tube contains 15 mg of drug in 3 ml
Hence, 25 mg/kg = 25 mg/1000g Hence, = 15 mg/3 ml
= 0.25 mg/10 gm (mice) (1) = 5 mg/1 ml (3)
= 2.5 mg/100 gm (rat and = 0.5 mg/0.1 ml
higher weight animal) [equivalent to equation (2)]
24  Practical Manual of Experimental and Clinical Pharmacology

So, student can use directly 0.1 ml of the
“solution A”, to provide 0.5 mg of drug in 100 gm
of body weight rat.
Calculation 2
This formula is applicable in most of the cases
and used very commonly,
If concentration of solution 1 is given in
mg/ml and the concentration of solution 2 is
given in µg/ml, then, make both units same
(Annexure-VII).
Calculation 3
By applying the conversion factor

C1 × V1 = C2 × V2 Sometime, doses of a drug is not known for the

Where,
C1 is the concentration of solution 1,
C2 is the concentration of solution 2,
V1 is the volume of solution 1,
V2 is the volume of solution 2.
animals, then it may be converted to the respective
animal drug dose by the help of conversion factors
developed according to the body surface area.
The steps involved are:

For example
Suppose, you are provided with the 50 ml solution 1. From human drug dose
of a 5 mg/ml concentration and the instructor Suppose, human dose of drug A = 100 mg/
wants you to make it, 10 ml of 3.5 mg/ml. kg/day [for the calculation, dose will be
So, you have calculated either for 20 gm mice or 200 gm rat
C1 = 5 mg/ml V1 = ? = A or 400 gm guinea pig or 1500 gm rabbit (as per
C2 = 3.5 mg/ml V2 = 10 ml animal selection)]
Step I: Dose given = 100 mg/kg
Apply the formula Step II: Convert into absolute dose*
Human dose given = 100 mg/kg = 70 × 100 =
C1 × V 1 = C2 × V2
7000 mg/70 kg
So, 5 mg/ml × A = 3.5 mg/ml × 10 ml
(Absolute dose is converted for 70 kg adult).
3.5 mg/ml × 10 ml * If, a adult dose is taken, then avoid step II.
Hence, A =
5 mg/ml For example, drug dose is 50 mg/day, then it
is assumed that, it is for 50 mg/70 kg of adult.
3.5 × 10 ml Note: Conversion of absolute dose is
= 7 ml
5 important because it keep the dose as per the
Therefore, take 7 ml of the solution 1 in a test body weight of animal/human to make easy
tube and make it volume to the 10 ml by the solvent. calculation such as convet dose mg/kg into
The resultant solution will be 10 ml of 3.5 mg/ml. the mg/70 kg for human, mg/1.5 kg for rabbit,
Note: The units of the solution concentration must mg/400 gm for guinea pig, mg/200 mg for rat
be same such as if it is in mg/ml then both solution and mg/20 mg for mice.
concentrations should be in mg/ml. Step III: Multiply by conversion factor
 Introduction to Experimental Pharmacology  25

Step IV: Then, convert into per kg dose according ROUTES OF DRUG ADMINISTRATION IN
to animal EXPERIMENTAL ANIMALS

Mice: 18.2 mg/20 mg = 910 mg/kg/day
Rat: 126 mg/200 gm = 630 mg/kg/day
Guinea pig: 217 mg/400 gm = 542.5 mg/kg/
day
Rabbit: 490 mg/1500 gm = 326.66 mg/kg/day
2. From animal to animal drug dose
Mice dose of drug A is 5 mg/kg, then, calculate
the drug dose for the other animals
The same as example 1
Step I: Dose given -= 5 mg/kg
Step II: Convert into absolute dose
For mice (20 mg) = 5 mg/kg = 0.1 mg/20 mg
Step III: Multiply by conversion factor
Feeding or Oral Gavage (Figs 1.18A to E)
Step I: Hold the rodent in a hand carefully
Step II: Measure the tube length from the nose to
the last rib of the rodent and mark it
Step III: Give a gentle tight grip at the back of
neck, so that it opens its mouth widely (if possible,
use any hard wooden or plastic gag)
Step IV: Push the rodent head slightly upward
and back to straighten the esophagus and then
either from right or left side of teeth, insert the tube
by gentle rotation to avoid the resistance (do not
force the tube in esophagus, this may injure
mucous membrane).

Step IV: Then, convert into per kg dose according

to animal
From Step III
Rat: 0.7 mg/200 gm = 3.5 mg/kg/day
Guinea pig: 1.22 mg/400 gm = 1.83 mg/kg/day
A
Rabbit: 2.78 mg/1500 gm = 1.85 mg/kg/day
In the same pattern, if the rat dose is known then
other animal dose can be calculated by using the
above mentioned steps with conversion factor
given below:
• Rat (200 gm) to mice (20 gm) conversion factor
is 0.14
• Rat (200 gm) to guinea pig (400 gm) conversion
factor is 1.74
• Rat (200 gm) to rabbit (1500 gm) conversion
factor is 3.9
Note: After converting the drug dose to
corresponding animals, do as per Calculation 1 B
or Calculation 2 for the volume to be injected. Figs 1.18A and B
26  Practical Manual of Experimental and Clinical Pharmacology

C
Figs 1.18A to C: Feeding or oral gavage to the rodent (A) Mice, (B) Rat, (C) Guinea pig

D E

Figs 1.18D and E: Feeding or oral gavage to the (D) Hamster, (E) Rabbit: with wooden gag

Step V: Slowly pass the tube observing for the
swallowing reflex and when desired length of tube
has been inserted, inject solution with the help of
syringe
Note: Recommended in mice and rats but is not
preferable in guinea pig because they have a small
palatal ostium which gets easily damaged.
Injection Site and Techniques
Following precautions should be taken before
injection:
• Injection sites should be cleaned with a
suitable disinfectant/antiseptic (isopropyl
alcohol, ethanol, spirit, etc.)
• Sterile syringes and needles must be used for
any type of injections
• Always select the smallest possible gauge (G)
needle to limit tissue trauma and injection
discomfort. For example: 25-27G needle is pre-
ferred in adult mice for the injection in tail vein.
• Aspiration technique is always an important
aspect before pushing the injection solution at
the site.
Note: Aspiration is a technique of creating vacuum
at the site of injection by pulling piston back to
check the right placement of the needle.
 Introduction to Experimental Pharmacology  27

Intraperitoneal (i.p) Injection (Figs 1.19 to 1.22)

Fig. 1.19: Mouse Fig. 1.21: Hamster

Fig. 1.20: Rat Fig. 1.22: Rabbit (But not preferred)

28  Practical Manual of Experimental and Clinical Pharmacology

Intravenous (i.v) injection (Figs 1.23 and 1.24)

Fig. 1.23: Cross-section of tail vein and procedure of injection or blood withdrawal

Fig. 1.24: Rabbit marginal vein (Prefered route)

Intramuscular (i.m) Injection (Figs 1.25 and 1.26)

Fig. 1.25: Thigh (Mouse/Rat) Fig. 1.26: Gluteus (Rabbit)

 Introduction to Experimental Pharmacology  29

Subcutaneous (s.c) Injection Nap of the Neck (Figs 1.27 to 1.31)

Fig. 1.27. Mouse (Nap of the neck) Fig. 1.28: Rat (Nap of the neck)

Fig. 1.29: Hamster (Nap of the neck) Fig. 1.30: Hamster (Flank)

Fig. 1.31: Rat (Flank)

30  Practical Manual of Experimental and Clinical Pharmacology
Intracardiac Injection (Figs 1.32 and 1.33)
Fig. 1.32: Rat
Note: Other less preferred routes are Intradermal
or Intrathecal.
BLOOD COLLECTION FROM THE
EXPERIMENTAL ANIMALS
Blood/plasma/serum of animals is required for a
variety of analytical purposes such as to measure
the drug concentration in the pharmacokinetics
study or for the estimation of different biochemical
or hematological parameters of a study. So the
experimenter should know the proper technique
to withdraw the blood sample from the animals.
Guideline says that always use the technique
which is causing less distress, pain and discomfort
to the animal. The collection technique of blood depends
on the three major objectives,
The principle for blood collection from veins
and arteries is almost the same, whereas arteries
are preferred only when a large sample has to be
withdrawn. Moreover, this is rapid and relatively
easy. There are some basic principles for collecting
blood from an experimental animal which
Fig. 1.33: Hamster
includes handling and restraint, needle size, site and
location of the vein and dilation of the vein. Animal
should be restrained which make procedure easy
and comfortable for both animal and
experimenter. Sometimes use of anesthesia is
preferred in small animals compared to the larger
animals. But, some reports suggest that use of
anesthetics may interfere with the hematological
and biochemical parameters of the animal still in
some situations handling with anesthetic is
preferred at the time of blood withdrawal because
it determines the quality and accuracy of the blood
sample. For example: In stress catecholamine’s
level are increased in the blood which may be
reduced by the use of anesthetics. The general
principle for selection of needle size (i.e. length
and bore) is determined by the diameter of the
vein, i.e. bore size selected should have less
diameter than the vein (needle diameter < vein
diameter). In the experimental studies needles
between 10-50 mm in length and 17-27G bore is
preferred for most of species. Meanwhile, dilation
of the vein can be facilitated by several means such
as by anesthetic or warming or use of any irritant.
In conscious animals, however, blood can be more
easily obtained if the animal is warmed first. Some
laboratories also preferred thermostatically warmed
box (e.g. made from perspex). Animals are kept at
30° C for 10-15 min. (kept under constant
 Introduction to Experimental Pharmacology  31

observation in order to prevent hyperthermia). then this requires little more blood. The amount of
Then, selection of the appropriate site is important blood withdrawn is mainly correlated to their body
because it makes the procedure easy and the weight and the total blood volume. Cannulation is done
amount of the blood required is easily obtained. for the multiple sampling which reduces the stress
and trauma to animals. The general information
Agents Used for Vasodilatation during for animal blood withdrawal is explained briefly
Blood Withdrawal in Table 1.4 and procedure for collection of blood
• Anesthetics sample is briefly given in Flow Chart 1.1.
• Dipping the tail of rodents into warm water (temperature
of around 45°C)
• Xylene (use with caution because it has carcinogenic
potency or may causes skin rashes ) and
• Fentanyl/fluanisone and acetyl promazine (mainly in
rabbits)

Volume of blood withdrawn depends on the
analytical process to be used. For example: if it is
to be used in the latest instruments like, HPLC,
PCR, spectrophotometer, ELISA kit, etc. then it is
required only in micro liters (μl) and if it is to be
used in the biochemical parameters assessment
Adverse Effects Experienced during Blood
Withdrawal
Blood withdrawal is a technique which requires
skill and experienced hand. Inexperienced hand
and improper technique may induce thrombosis
(clotting) and phlebitis (inflammation of the vein),

Flow Chart 1.1: Procedure for collection of blood sample

Caution: Sample taken too quickly or abrupt withdrawal of needle may collapse vein
Note: As a general rule, blood may be withdrawn up to 10% of total blood volume of animal (single
withdrawal) and 1% of total blood volume/24 hr for repeated withdrawal
32  Practical Manual of Experimental and Clinical Pharmacology

Table 1.4: General information on blood withdrawal in animals

Animals Site for blood Needle No. of Sample Unwanted effects
withdrawal sample/ 24 hr Volume
Mouse/Rat Tail vein 25-27G (M) 1or 2 (M) 50 μl-0.2 ml (M) Infection and hemorrhage
(M/R) 21-23G (R) < 8(R) 0.1-2 ml (R)
Tail snipping Sterile scalpel ≤ 4 (M) 10 μl (M) Bruising, hemorrhage and
blade (M) Infection
Saphenous vein 27G or 25G (M) ≤ 4 (M/R) Up to 0.15 ml (M) Infection, hemorrhage and
23G (R) Up to 0.2 ml (R) Bruising
Sublingual vein 23G (R) 1(R) Up to 0.2 ml (R) Infection, hemorrhage and
Bruising
Retro-orbital Pasteur pipette Only 1(M/R) 0.2 ml with May produce blindness,
or Glass capillary recovery 0.5 ml Infection and hemorrhage
tube (M/R) without recovery
(M);Up to 4 ml
recovery, 4-10 ml
non-recovery (R)
Jugular vein 23G (R) < 8 (R) 0.1-2 ml (R) Infection, hemorrhage and
bruising
Cardiac puncture 23-25 G (M) 1 Up to 1 ml (M) ———————
19-21G (R) Up to 15 ml (R)
Abdominal/ 25G (M) 1 (M/R) Up to 1 ml (M); ———————
thoracic blood 19 -21G (R) 10 ml Hepatic Portal
vessel (ATBV) vein/15 ml from
other ATBV
Hamster Saphenous vein 25G Up to 4 0.5% of BW/ Bruising, hemorrhage,
sample infection and temporary
favoring of the limb
Cardiac puncture Insulin syringe 1 0.5 ml Bruising, hemorrhage,
with recovery (0.5-2 ml) Infection and temporary
favoring of the limb
Cardiac puncture 23G 1 Up to 5 ml ———————
without recovery
Retro-orbital Pasteur pipette or 1 0.1-0.5 ml May produce blindness,
Glass capillary Histological changes, abnormal
tube clinical signs and Tissue
damage
Gerbil Cardiac puncture Insulin syringe 1 0.5 ml Bruising, hemorrhage and
with recovery (0.5-2 ml) or 5/8" Infection
25 G, 1" 22 G
Guinea pig Cannulation 23G - 25G cannula Up to 6 0.1-0.5 ml Infection, hemorrhage,
sample/2 hr Blocked cannula, Swelling
around the jacket, Skin sores
from the jacket
Tarsal vein 23G Up to 6 0.1-0.3 ml Skin abrasion from the
samples shaving, bruising, Infection
and hemorrhage
Saphenous vein 23G Up to 4 0.5% of BW/ Bruising, hemorrhage,
samples sample infection

(Contd...)
 Introduction to Experimental Pharmacology  33
(Contd...)

Animals Site for blood Needle No. of Sample Unwanted effects

withdrawal sample/ 24hr Volume

Abdominal/thoracic 19-21G 1 Up to 15 ml Bruising, hemorrhage,

infection
Cardiac puncture 20-21G 1 1-25 ml ———————

Decapitation — 1 10-20 ml ———————

Rabbit Marginal ear vein/ 19-23G Up to 8 0.5-10 ml Bruising/hemorrhage,
artery infection
Cardiac puncture 19-21G 1 60-200 ml ———————

Cat Cephalic vein 21 or more G Up to 8 (each 2-5 ml Hemorrhage, infection,

leg 4 sample) swelling

Jugular vein 21G (1" long) Up to 8 2-20 ml Infection, hemorrhage and

Bruising

Cardiac puncture 16G (1.5"long) 1 Up to 900 ml ———————

Dog Cephalic vein 21 or more G Up to 8 (each 2-5 ml Hemorrhage, infection,
leg 4 sample) swelling
Jugular vein 21G (1" long) Up to 8 2-20 ml Bruising/hemorrhage,
infection
Cardiac puncture 16G(1.5"long) 1 Up to 900 ml ——————

Pig Cranial vena cava 19-21G- pig 1 in 7 days 5-30 ml Bruising/hemorrhage,

20-21G-minipig infection

Ear vein 21-23G Up to 8/24 hr 1-3 ml Bruising/hemorrhage/

infection

Femoral vein 25G Up to 7 0.1-3 ml Hematoma

Monkey Abdominal vena 19G 1 Up to 10 ml ——————
(Marmoset) cava (Terminal)
Cephalic vein, 1" 22 G Up to 8 (each 5 - 10 ml Bruising/hemorrhage,
leg 4 sample) Infection
Saphenous vein 22 G Up to 8 10% of BW Bruising/hemorrhage,
Infection
Femoral vein 22 G Up to 8 5 - 10 ml Infection, Bruising/
hemorrhage
Jugular vein 22 G Up to 8 5 - 10 ml Bruising/hemorrhage,
Infection

etc. Other adverse effects are hemorrhage, bruising,
thrombosis and infection at the site of needle entry.
If any of the side effects occurs, then treatment
should be given from the assigned veterinary
surgeon. Hemorrhage is not common and can be
associated with the animal which have clotting
defect. Bruising is mainly due to subcutaneous
bleeding at the time of venous puncture.
VARIABILITY OF DRUG RESPONSES IN
EXPERIMENTAL ANIMALS
Species differences in drug sensitivity can often
be explained by differences in their pharma-
cokinetic, including quantitative and qualitative
differences in the ability to detoxify drugs. Sex and
age can also modulate the rates of absorption,
34  Practical Manual of Experimental and Clinical Pharmacology

Table 1.5: Variability of drug responses

Drug Variable responses
Adrenaline Cat: Stimulates pregnant uterus but contracts non-pregnant uterus; Rabbit: stimulates uterus; Rat:
relax uterus
Alloxan Produces diabetes in most animals but not in guinea pig
Atropine Mice > cat > human > dog= rabbit (100 times of human dose) (sensitivity in order)
ATP Relaxes rabbit anococcygeus muscle whereas contracts rat anococcygeus muscle
Cardiac Rat heart is resistant
glycosides
Dopamine Shows fall in BP of dog (instead rise seen in man and other animal)
Histamine Cat: Produces Bronchodilation and pulmonary vasoconstriction (increases blood pressure); Guinea
pig and human: contract uterus (500 times more sensitive than mice and rat);Hamster stomach
strip is not sensitive to 5HT and histamine, but suitable for the bioassay of prostaglandins
(PGE2 > PGF2)
Insulin Mice susceptibility to insulin induced epilepsy is highly reduced in summer
Morphine Produces depression in rabbit, rat, dog, monkey and man whereas stimulate mice, cat, pig or horses
Pethidine Dog is resistant to pethidine

ATP: Adenosine triphosphate; 5HT: 5-hydroxytryptamine; PGE2: Prostaglandins E2; PGF2: Prostaglandins F2;

metabolism, distribution, and elimination of DISEASES CAUSED BY ANIMALS

drugs. The distribution and storage of drugs are
reasonably consistent among mammalian
species, including humans, although plasma
binding tends to be more extensive in humans than
in small mammals. Diet intake can also be
influenced by the urinary excretion in different
animal species because diet influences urinary
pH and thus the extent of ionization of
compounds. After oral administration, absorption
in laboratory animals is generally considered to
be similar to that in humans, although there are
quantitative differences for some compounds. For
example, species differences in the absorption and
action of some compounds are related to
differences in the bacterial flora of the
gastrointestinal tract. Biliary excretion is quite
variable from species to species and is apparently
more extensive in mice and rabbits than in rats
and humans. Species differences in response to
drugs appear to be related mainly to rates of
biotransformation, which are generally more
rapid in small laboratory animals than in humans.
Recent studies shown that the genetic makeup
can influence the drug transporter, enzyme or even
receptors in animal or humans. Table 1.5 explains
the drug variability in different species.
(ZOONOTIC DISEASES)
Transmissible or communicable diseases
which are not too new in this context but the
important thing is that these diseases can be
also being spread through the laboratory
animals. Hence, it is very important to take
precautions while experimenting with
laboratory animals.
Zoonotic diseases or animal transmitted
diseases are caused directly or indirectly between
vertebrates, animals like rodents, rabbits, cat or
dogs and human through bacteria, viruses,
Chlamydia, fungi, parasites or animal bite (Fig.
1.34). The complete list of the zoonoses related to
experimental animals in research, teaching or
testing is quite long. But, some of the important
zoonotic diseases are mentioned in the Table 1.6.
Control of Zoonotic Disease
Several programs are run by CPCSEA to control
these diseases and to produce disease-free
animals or by taking proper health care of animal
in the animal house. Veterinary monitoring and
care programs by the qualified veterinary
practitioner are one of the important aspects of
the program. The risk of exposure to zoonotic
 Introduction to Experimental Pharmacology  35

Table 1.6: Zoonotic disease it management

Zoonotic disease Animal (organism involved) Mode of transmission Treatment

Rat fever Rat (Streptobacillus Animal bite, direct contact Penicillin or erythromycin or
moniliformis ) with secretions of the mouth, tetracyclines for 7-14 days
nose, eye of an infected animal
Ring worm Mice, Rat, guinea pig, Direct or indirect contact with Fluconazole, Itraconazole,
hamster, gerbil etc. skin lesions or infected hair, Ketoconazole and Terbinafine
(Microsporum spp., or fomites
Trichophyton spp., fungal
organisms)
Rabies Dog, cat, rat, mice Animal bite Prophylactic Rabies vaccine at 0, 3,
(Rhabdovirus, Rabies, 7, 14 and 28 days. Booster at 90 days
Hydrophobia)
Herpes B virus Monkey Herpesvirus Bite or direct or indirect Acyclovir
Infection simiae, a DNA herpesvirus contact with infected saliva
or tissues
Hantavirus Mice (Peromyscus spp.) Inhalation of the virus in the Better take precaution and prevention
Infection dust of mice excreta (urine from rodents
and feces) Ribavirin (some benefit)
Tetanus Nearly every laboratory Animal bite Immune globine im injection and
animal penicillin to eradicate the bacteria
Salmonella Dog, cat, ferrets etc Bite or direct or indirect Ciprofloxacin, ceftriaxone and
contact with infected saliva amoxicillin
or tissues
Campylobacter Dog, non-human Bite or direct or indirect Erythromycin, azithromycin and
primates contact with infected saliva Doxycycline
or tissues
Psittacosis Birds Nasal secretions and in the Doxycycline, Azithromycin,
stool from infected birds Erythromycin, Rifampin and
Tetracycline
Cutaneous Amphibians and Fish Direct or indirect contact Amikacin
mycobacteriosis with urine or feces

diseases is greater for those who work with
experimental animals. Hence, proper vaccination
program should be run in the institute.
To prevent zoonotic disease following
precautions should be taken while handling the
animals
• Regular health testing of animals should be
done and use respiratory mask during the
experiment
• Prophylactic vaccination of experimenters,
post-bite treatment of victim and animal
quarantine should be maintained.
• Avoid direct contact with animal urine and
feces, and wear protective clothing, e.g: gloves
or apron, etc.
• Test serum to assess prior exposure
• Post-exposure treatment and assessment
36  Practical Manual of Experimental and Clinical Pharmacology

Fig. 1.34: Mode of transfer of zoonoses

Allergy due to Animal Exposure be done to identify those with pre-existing

allergies or IgE antibodies whereas pulmonary
Allergy onset to the experimenter while handling
function testing (PFT) to identify occupational
animal is mainly depending on the duration of
asthma and to evaluate and monitor symptoms
exposure, and allergen exposure concentration.
is advisable.
The common mode allergy is animal fur, skin urine
or faces or saliva (Table 1.7). Allergies develop Prevention
immediately or within 2 years of exposure through
• Ventilation exhaust should be proper
direct contact or inhalation. Immediate allergic
which minimizes the generation of micro-
reaction (Type 1), mediated through release of IgE
organisms
antibody and less commonly, includes IgG-
• Proper cleanup of hazardous dust, fumes, etc.
mediated allergy, which may occur 1-12 h after
which limit the area of contamination by
exposure. Non-IgE-mediated allergy involving
reducing allergens
other immunoglobulin and giving rise to specific
• Avoid crowding of animals per area
pathology, such as extrinsic allergic alveolitis.
• Regular cleaning and disinfection of contami-
Symptoms: Rhinitis, Conjunctivitis, skin effects nated walls, surfaces, etc.
(Urticaria, Wheals and Eczema) • Animal bed should be change at regular basis
Diagnosis of allergy: Skin tests, RAST or enzyme- to avoid the accumulation of urine and faeces
linked immunosorbent assay (ELISA) may in the animal house (act as reservoir of allergen)
 Introduction to Experimental Pharmacology  37

• Storage and disposal of animal waste should ANESTHESIA AND EXPERIMENTAL ANIMALS
be proper
• Eating, drinking, smoking, etc should be strictly Aim of the anesthesia used in an experimental
prohibited in contaminated areas. study is to ensure analgesia, amnesia and
immobilization of animal for the ease of handling
Table 1.7: Medium of allergens spread from or any surgical procedure. While anesthetizing the
experimental animal animal one should maintain the three vitals such
as “blood circulation”, “oxygenation” (to ensure
Animal Allergen present
adequate oxygen concentration in the animal’s
Rat Proteins in urine and saliva arterial blood) and “respiration” (to ensure that
Mice Urinary proteins the animal’s ventilation is adequately maintained).
Guinea pigs Urinary proteins (penetrate low into the Anesthetic is commonly used in the experimental
respiratory tract) study so as to make the procedure more simple,
Rabbits Glycoprotein found in the fur (major).
reliable, and reproducible. Generally, all types of
Proteins in urine and saliva (minor)
anesthetics are used in the study (Table 1.8).
Cats Proteins from the sebaceous glands (hair
shafts and saliva) Ether must be used in a fume hood and stored
Dog Proteins found in saliva, hair and skin appropriately, otherwise use of ether as an
anesthetic agent is prohibited.

INHALATIONAL INJECTION LOCAL

• Difficulty in use, requires special
apparatus
• Thermoregulation required, cold
gas may reduce temperature
• Short lasting
• Required maintenance dose
• Special arrangement required
such as Drop System, gas
scavenging system, etc.
• Example: Ether, Halothane,
Chloroform, Nitrous oxide, Isoflu-
rane, etc.
• Bypass first pass metabolism
• Depth of anesthesia that cannot
be easily altered
• Safe and effective to use
• High first pass metabolism
• Usually, animals under injectable
anesthesia are not intubated
• Requires experienced person to
use (identifying location and time
to use)
• Onset within 15 minutes of
application and may last from 45
minutes to several hours
• No special arrangement required
• No effect on the physiological
property of animal
• No first pass metabolism

EUTHANASIA METHOD USED IN THE “Euthanasia means the humane killing

EXPERIMENTAL STUDY
Animals are used in the experimental
pharmacological studies and to assess the drug
activity, it requires analyzing the organ (brain,
kidney, lung, etc.), blood (plasma or serum), and
body fluid (CSF, urine, etc.) to conclude the result.
Hence for the purpose, animals have to be
sacrificed to obtain the said part of the body.
Therefore, according to the animal care guidelines
they should be euthanized.
(sacrifice) of an animal which produces rapid
unconsciousness and subsequent death without
or minimal pain or distress to animal.”
The choice of a method depends on
species, age, and availability of restraint and
skill of the individuals performing euthanasia.
It is very important that, in an experimental
setting, the method of euthanasia must
be primarily consistent with the experimental
goals.
38  Practical Manual of Experimental and Clinical Pharmacology

Table 1.8: Anesthetic agents used in experimental animals

Anesthetic agent Dose (mg/kg), Route of administration
Mouse Rat Hamster Guinea pig Rabbit Frog* Zebra fish*

α-Chloralose ————— ————— ————— ————— 80 – 100, iv ————— —————

Halothane, 3-4% induction; 3-4% induction; 3-4% induction; 3-4% induction; ————— 1-5% 1-5%
Isoflurane, 1-2% 1-2% 1-2% 1-2%
Enflurane maintenance maintenance maintenance maintenance

Hexobarbital 60, iv or ip 60, iv or ip ————— ————— ————— ————— —————

Ketamine HCl 22-24, im 22-24, im ————— 22-24, im 22-24, im/iv 50-150 50-150
Pentobarbitone 35-50, iv or ip 25-50, iv or ip 35, iv 30-40, iv or ip 30-40, iv or ip 60, dorsal —————
sodium lymph sac

Thiopentone 25-50, iv or ip 20-40, iv or ip 20-40, iv or ip 20-55, iv or ip 20, iv ————— —————

sodium
Urethane# ————— 0.75-1.5g/kg, ip ————— 1.25 -1.5 g/kg, ip 1g/kg, iv or ip ————— —————
Tricaine and ————— ————— ————— ————— ————— 25-100 mg/L 25-100 mg/L
benzocaine

Ketamine (K) + 100 (K) + (60-90) (K) + (80-100) (K) + 40 (K) + 5 (25-50) (K) + ————— —————
Xylazine (5-10)(X) i.p. (6-9) (X), i.p. (7-10) (X), i.p. (X), i.p. (6-10) (X), i.m.

Ketamine (K) + 75 (K) + 1 75 (K) + 100 (K) + 40 (K) + 25 (K) + ————— —————
Medetomidine (M), i.p. 0.5 (M), i.p., 0.25 (M), i.p. 0.5 (M), s.c. 0.5 (M), i.m.
(M) s.c.

# Carcinogen! Use with caution and have prolonged anesthesia

* Add anesthetic in the water as per the depth of anesthesia required
Note: Chloral hydrate, is also used as an anesthetic agent, subject to availability (Dose = 400 mg/kg, i.p. for mice)

Euthanasia Objectives hypoxia which further leads to depression of vital

• Reliability and irreversibility
• Minimum pain, distress, anxiety or
apprehension
• Minimum delay until unconsciousness
• Safety and emotional effect on personnel
• Compatibility with requirement and purpose,
including subsequent use of tissue and
• Compatibility with species, age and health
status
Euthanasia methods are broadly classified as;
Chemical methods (Inhalants/Non-inhalants)
and physical methods.
Inhalant Anesthetics
Halothane, enflurane, sevoflurane, methoxy-
flurane, isoflurane and desflurane are preferred
for euthanasia in animals.
(CO2): Carbon dioxide is an effective and widely
used agent to euthanize rodents due to rapid
centers. (Maintain 20% -70% of the chamber volume
per minute) this method is preferred in several
animal but it is dangerous to use, so precaution
should be taken while using it.
Nitrous oxide (N2O) is not preferred due to lack in
fast onset of anesthesia, but may produce
hypoxemia and cardiac or respiratory arrest.
However, it may be used in combination with
other anesthetics to speed anesthesia onset.
Ether was formerly used extensively, but is now
only acceptable conditionally. The reason is being
irritant to mucous membranes and risk of fire and
explosion. The use of ether is prohibited in many
countries.
Non-inhalant Anesthetics
Barbiturates: Sodium pentobarbital is the most
rapid and reliable method of euthanasia for most
experimental animals. In non-rodent species,

barbiturates are given intravenously to be most
effective. Intraperitoneal injection of barbiturates
is acceptable for euthanasia in small mammals.
Potassium chloride (KCl): KCl induces immediate
cardiac arrest without any significant depression
of the central nervous system. Hence, it must only
be used after the animal is deeply anesthetized.
Magnesium sulphate (MgSO4): MgSO4 produces
its action through cardiac arrhythmia,
neuromuscular blockade and deep anesthesia,
hence ultimately animal gets euthanized due to
cardiac arrest and neuromuscular blockade.
Neuromuscular Blocking Agents (Succinycholine,
Curare, etc.): These agents induce muscular
paralysis and death because of suffocation. Distress
onset is more, hence less preferred for euthanasia.
Physical Methods
Physical methods are performed by skilled and
experienced personnel with appropriate, well-
maintained equipment.
Cervical dislocation: Humane technique for
euthanasia which is frequently used for mice, rats,
guinea pig, rabbits (weighing less than 1 kg) and
other rodents.
Decapitation: Decapitation may be used to
euthanize rodents and small rabbits. Except in
neonatal animals, a guillotine is generally used.
Microwave irradiation: Special instruments
designed (appropriate power and microwave
distribution) for this purpose, which is used when
an experiment requires fixation of mouse or rat
brain metabolites in vivo without losing anatomic
integrity of the brain.
Penetrating captive bolt: This method is
conditionally acceptable and made for ruminants,
horses, and swine when chemical agents are
scientifically contraindicated. This method is not
employed in the laboratory animals.
Euthanasia of Poikilothermic (Cold-blooded)
Animals
The euthanasia of poikilothermic animals is
different due to differences in the pharmacokinetic,
Introduction to Experimental Pharmacology  39
respiration and tolerance to cerebral hypoxia
between these species and homeothermic animals.
Chemical agents: Pentobarbital, Tricainemethane
sulfonate or benzocaine HCl.
Intraperitoneal administration of pento-
barbital is an effective method of euthanasia in
amphibians. Tricaine methane sulfonate or
benzocaine hydrochloride may be placed in the
water of amphibians and fish to produce
anesthesia and prolonged contact may produce
death. Inhalant anesthetics may be used for
amphibians and reptiles but due to the low oxygen
requirements for amphibian, the onset of
unconsciousness and death will be significantly
lengthened.
Physical methods: Poikilotherms may be
euthanized by stunning followed by decapitation
or pithing to ensure death. In frogs and toads,
pithing the brain (single pithing) and spinal cord
(double pithing) are effective and acceptable
methods.
Preferred euthanasia Animal
Cervical dislocation Mice, rat, guinea pig and gerbil
Decapitation Mice, rat, guinea pig, gerbil,
hamster
70-80% CO 2 Mice, rat, guinea pig, gerbil,
hamster, rabbit, cat, dog
Sodium Mice, rat and guinea pig (150mg/
pentobarbitone kg; i.p.); Hamster (300 mg/kg;
i.p.); rabbit (120mg/kg; i.v)
Inhalation Nearly all laboratory animals
including amphibian
Pithing (single or Amphibian
double)
ETHICAL CONSIDERATIONS OF ANIMAL
USE IN INDIAN SCENARIO
Ethics are based on that “Each animal has the right
to life and humans should not take such a right away
from them.”
In clinical setting, pain or suffering in a patient
is considered unethical unless it is for the direct
benefit of that patient, so does it applies with the
use of animals in experiments. Animal use in the
biomedical research is common to understand
fundamental aspects of molecular and cellular
level that in turn facilitate the development of
40  Practical Manual of Experimental and Clinical Pharmacology
therapeutic measures for both animals and
humans. Those who oppose the use of animals in
research may object to the means by which
scientists attempt to achieve their goals, further in
the research animal use can be minimized but not
possibly be stopped.
Researchers are frequently faced with
questions about the use of animals in research.
Medical researchers in particular face the
challenge of allegations that the use of animals
for scientific research is not necessary and that it
is possible to develop new drugs in the test-tube or
even by computer (In silico) or microdosing directly
to the healthy volunteers (to assess pharma-
cokinetics/pharmacodynamic/toxicological
aspect).
The understanding of the human body has come
from more than 200 years of research on the function
of normal cells, tissues and organs, and on disease
processes. Animal model or species are used to test
possibilities that would be difficult or impossible to test
using the target species. In general, one species may be
used as a model for another when, despite other
differences between them, the two species strongly
resemble each other in particular ways. Research
that involves low suffering to the animals and was
likely to be highly beneficial would generally be
regarded as acceptable. Animals can instead provide
a vital contribution to fundamental scientific
understanding that may provide benefit in the future.
The use of animals is not permitted where a
replacement alternative is available. The place
where no replacement alternative is available,
then experimental protocols should be refined in
such a way that it reduces any pain or suffering to
animal or is minimized by using analgesics or
anesthetics.
Finally, the number of animals used should
be reduced to the minimum consistent with
achieving the scientific objectives of the study. At
least 5 animal per group should be taken, to
achieve the hypothesis or to find a significance
difference between the compared grops throughs
a statistical analysis. This principle can be
achieved by the following 3R’s such as Replacement
(if possible replace the method used such as in
vitro or in silico), Refinement (to minimize potential
pain, suffering or distress to the animal use in
experiments) and Reduction (if not possible to select
any other in vitro procedure then minimize the
animal use as much as possible to achieve the
result).
Note: 4th ‘R’ coming up as ‘Rehabilitation’ of used
animals.
Animal use in Indian Scenario
The use of animals in the institutions is
supervised by the Committee for the Purpose of
Control and Supervision of Experimentation on
Animals (CPCSEA) guidelines which are
controlled by Ministry of Environment and Forests
(Animal Welfare Division), Government of India.
CPCSEA mainly controls the Institutional Animal
Ethics Committee (IAEC) and Institute Biosafety
Committee (IBSC/IBC).
Hierarchy of the CPCSEA
The objective of guidelines is to promote the
humane care of animals used in biomedical
research and provide the legal aspect for the
experimentation in the animals.
Basic Principles of CPCSEA
• Advancement by the new discovery in the
experimental animals to improve the well
being of the society
• Minimize the animal use by proper design to
give the result under 95% of confidence interval
 Introduction to Experimental Pharmacology  41
Fig. 1.35: Experimental animals which come under the CPCSEA regulation
(Order of animals coming under regulation; Amphibian are not explained under regulation)
• Minimize the pain, stress and discomfort in
experimental animals
• Investigators are responsible for the well-being
of the animals and the euthanasia is permitted
during the study in some special circums-
tances such as:
– Animal is paralyzed or incapable of
locomotion
– Extreme or recurring pain and distress
– Situation at which lack of euthanasia may
become life-threatening to human or other
animals
• Animal house should follow the GLP
guidelines for their housing, feeding, care and
disposal.
Institutional Animal Ethics Committee (IAEC)
As per Rule 13 of the Breeding of and Experiments
on Animals (Control and supervision) Rules,
1998,
Members of IAEC are:
• A biological scientist
• Two scientists from different biological
disciplines
• A veterinarian involved in the care of animals
• The scientist in charge of animals facility of
the establishment concerned
• A scientist from outside the institute
• A non-scientific socially aware member and a
representative or nominee of the CPCSEA
Role of the IAEC is to approve experiments on
the phylogenetic level of rodents (e.g. mice, rats
and rabbits) and for approval level higher than
rodents, application must be forwarded to the
CPCSEA (Fig. 1.35).
Institutional Biosafety Committee (IBSC/IBC)
Institutional Biosafety Committee (IBSC) is
engaged in hazardous chemical use, genetic
engineering research and production activities.
Members of IBSC are:
• Head of the institution or his nominee
• 3 or more scientists engaged in DNA work or
molecular biology with an outside expert in
the relevant discipline
• A member with medical qualification-
Biosafety officer (in case of work with
pathogenic agents/large scale used).
• One member nominated by Department of
Biotechnology (DBT), Govt. of India
Role of IBSC is mainly review and clearance of
project proposals falling under restricted category,
training of personnel on biosafety, instituting
health monitoring program for laboratory
personnel and adopting emergency plans if any
mishap happens in the Institution.
SUGGESTED READING
History and Drug Development
1. Benjamini Y, Hochberg Y. Controlling the false
discovery rate: A practical and powerful approach
to multiple testing. J Roy Stat Soc B 1995;57:289-
300.
2. Bernard E Rollin. “The Regulation of Animal Research
and the Emergence of Animal Ethics: A Conceptual
History” Theoretical Medicine and Bioethics 2006;
27(4):285-304.
42  Practical Manual of Experimental and Clinical Pharmacology
3. Caron PR, Mullican MD, Mashal RD, Wilson KP, Su
MS, Murcko MA. Chemogenomic approaches to drug
discovery. Curr Opin Chem Biol 2001;5:464-70.
4. Chang C, Ekins S, Bahadduri P, Swaan PW.
Pharmacophorebased discovery of ligands for drug
transporters. Adv Drug Del Rev 2006b;58:1431-50.
5. de Graaf C, Vermeulen NP, Feenstra KA. Cytochrome
P450 in silico: An integrative modeling approach. J
Med Chem 2005;48:2725-55.
6. Fraser D, McDonald M. “Expanding the three R’s to
meet new challenges in humane animal experi-
mentation”. Altern Lab Anim 2004;32(5):525-32.
7. Hann MM, Oprea TI. Pursuing the lead likeness
concept in pharmaceutical research. Current Opinion
in Chemical Biology 2004;8:255-63.
8. LaFollette H, Shanks N. Animal Experimentation:
the Legacy of Claude Bernard, International Studies
in the Philosophy of Science 1994:195-210.
9. Langer T, Eder M, Hoffmann RD, Chiba P, Ecker GF.
Lead identification for modulators of multidrug
resistance based on in silico screening with a
pharmacophoric feature model. Arch Pharm
(Weinheim) 2004;337:317-27.
10. Liu B, Li S, Hu J. Technological advances in high-
throughput screening. Am J Pharmacogenomics
2004;4(4):263-76.
11. Mattsson JL, Spencer PJ and Albee RR. A performance
standard for clinical and Functional Observational
Battery examinations of rats. J Am Coll Toxicol
1996;15:239.
12. Paget G E, Barnes J M. In Evaluation Of Drug Activities:
Pharmacometrics, Eds. Laurence D R, Bacharach A L,
vol 1,Academic Press, New York and London, 1964.
13. Smith LL. “Key challenges for toxicologists in the
21st century”. Trends Pharmacol Sci 2001;22(6):
281-85.
Experimental Animals
1. Adams CE. The rabbit. In: The UFAW Handbook on
the Care and Management of Laboratory Animals (6th
ed. 1987) T. Poole, ed., Longman Scientific and
Technical: Harlow.
2. Burgess SC. Proteomics in the Chicken: Tools for
Understanding Immune Responses to Avian
Diseases. Poultry Science 2004;83:552-73.
3. Bustad LK. Pigs in the laboratory. Sci Am 1966;214:
94-100.
4. Clarkson TB, Lofland HB. Therapeutic studies on
spontaneous arteriosclerosis in pigeons. In: Garattini
S, Paoletti R (Eds) Drugs affecting lipid metabolism.
Elsevier Publ Comp. Amsterdam, 1961; 314-17.
5. Dahm, Ralf. “The Zebrafish Exposed”, American
Scientist 2006; 94(5):446-53.
6. Feldman DB, McConnel EE, Knapka JJ. Growth,
Kidney Disease, and Longevity of Syrian Hamsters
Fed Varying Levels of Protein. Lab Anim Sci 1982;
32(6):613-18.
7. Fillios LC, Andrus StB, Mann GV, Stare FJ.
Experimental production of gross atherosclerosis in
the rat. J Exper Med 1956;104:539-52.
8. Gibbs ME, Barnett JM. Drug effects on successive
discrimination learning in young chickens. Brain Res
Bull 1976;1:295-99.
9. Griesemer R A. Laboratory animal management cats,
ILAR News, 1978;21(3),C3-C20.
10. Guide for the Care and Use of Laboratory Animals-
Institute of Laboratory Animal Resources
Commission on Life Sciences - National Research
Council
11. Hans J Hedrich, Gillian Bullock. The laboratory
Mouse. Elsevier Academic Press, Londan, 2004.
12. Hanzlik JP. New method of estimating the potency
of digitalis in pigeons: Pigeon emesis. J Pharmacol
Exp Ther 1929;35:363-91.
13. Hoffman RA. The Golden Hamster. Iowa State
University Press 1968.
14. Hurni H, Rossbach W. The laboratory cat, UFAW
Handbook on the Care and Management of
Laboratory Animals, 6th ed, (ed. TB Poole), Longman
Scientific and Technical, London 1987;476-92.
15. Jackson F, Scott PP. Laboratory Animals 1970; 4:135-
37.
16. Jilge B. The rabbit: A diurnal or a nocturnal animal?
Journal of Experimental Animal Science 1991; 34(5-
6):170-83.
17. Lustalot P, Schuler W, Albrecht W. Comparison of
drug actions in a spectrum of experimental anti-
atherosclerotic test systems. In: Garattini S, Paoletti
R (eds) Drugs affecting lipid metabolism. Elsevier
Publ Comp., Amsterdam 1961;271-76.
18. MacArthur JA. The dog. In: Poole T (ed). The UFAW
Handbook on the Care and Management of
Laboratory A n i m a l s. 6th ed. Longman Scientific
and Technical, London 1987:456-75.
19. Mandsager RE, Raffe MR. Chemical restraint
techniques in dogs and cats. In: Kirk, R.W. (ed).
Current Veterinary Therapy X. Small Animal Practice.
W.B. Saunders, Philadelphia 1989:63-70.
20. Masahiko Nishimura, Masatoshi Inoue, Toru
Nakano, Tetsu Nishikawa, Megumu Miya-moto,
Takaaki Kobayashi, Yukihiko Kitamura. Beige Rat:

A New Animal Model of Chediak-Higashi Syndrome.
Blood 1989;74(1):270-73.
21. McGrath P, Li CQ. Zebrafish: A predictive model for
assessing drug-induced toxicity. Drug Discovery
Today 2008;13(9-10):394-401.
22. NHMRC Animal Welfare Committee. Ways of
minimising pain and distress in Animals in Research.
Australian Government Publishing Service, Canberra,
1994.
23. Onderdonk AB, Brodasky TF, Bannister B.
Comparative Effects of Clindamycin and Metabolites
in the Hamster Model of Antibiotic Associated
Colitis. J Antimicrob Chemother 1981;8(5):383-94.
24. Panepinto LM, Phillips RW, Will DH. The Yucatan
miniature pig as a laboratory animal. Lab Anim Sci
1978;28:308-313.
25. Parng C, Anderson N, Ton C, McGrath P. Zebrafish
apoptosis assays for drug discovery. Methods Cell
Biol 2004;76:75-85.
26. Parng C, Seng WL, Semino C, McGrath P. Zebrafish:
A preclinical model for drug screening. Assay Drug
Dev Technol 2002; 1(1 Pt 1):41-48.
27. Phillipe G, Angenot L. “Recent developments in the
field of arrow and dart poisons”. J Ethnopharmacol
2005;100(1-2):85-91.
28. Rolstad B. The athymic nude rat: An animal
experimental model to reveal novel aspects of innate
immune responses? Immunological Reviews 2001;
184(1):136-44.
29. Schwentker V. “The Gerbil. A new laboratory animal.”
Ill Vet 1963;6:5-9.
30. Simon GA. The pig as an experimental animal in
biomedical research. Israel J Vet Med 1993;48:
161-67.
31. Suzuki M, Harada Y, Hirakawa H, Hirakawa K,
Omura R. An experimental study demonstrating the
physiological polarity of the frog’s utricle. Eur Arch
torhinolaryngol 1987;244(4):215-17.
32. Thomas J, Gill Ill, Garry J Smith, Robbery W, Wissler,
et al. The rat as an experimental animal. Science
1989;245(4915):269-76.
33. VanCompernolle Scott E, Taylor R J, Oswald-Richter
K, Jiang J, Youree BE, Bowie JH, et al. “Antimicrobial
peptides from amphibian skin potently inhibit
Human Immunodeficiency Virus infection and
transfer of virus from dendritic cells to T cells”.
Journal of Virology 2005;79:11598-606.
34. Vicentini CA, Orsi AM, Dias SM. Anatomical
observations of the coronary artery vascularization
in the guinea pigs (Cavia porcellus, L). Anat Anz
1991;172(3):209-12.
Introduction to Experimental Pharmacology  43
35. Wuttke W, Kelleher RT. Effects of some
benzodiazepines on punished and unpunished
behavior in the pigeon. J Pharmacol Exper Ther 1970;
172:397-405.
Blood Collection
1. Besch EL, Chou BJ. Physiological responses to blood
collection methods in rats. Proceedings of the Society
of Experimental Biology and Medicine 1971;
138:1019-21.
2. Flecknell PA, Liles JH, Williamson HA. The use of
lignocaine-prilocaine local anaesthetic cream for pain-
free venepuncture in laboratory animals. Laboratory
Animals 1990;24,142-46.
3. Harms PG, Ojeda SR. A rapid and simple procedure
for chronic cannulation of the rat jugular veins.
Journal of Applied Physiology 1974;36:391-92.
4. Jackson RK, Kieffer VA, Sauber JJ, King GL. A
tethered-restraint system for blood collection from
ferrets. Lab Anim Sci. 1988;38(5):625-28.
5. Ladewig J, Stribrny K. A simplified method for stress
free continuous blood collection in large animals.
Laboratory Animal Science 1988;38:333-35.
6. MacLeod JN, Shapiro BH. Repetitive blood sampling
in unrestrained and unstressed mice using a chronic
indwelling right atrial catheterization apparatus.
Laboratory Animal Science 1988;38:603-08.
7. McGuill MW, Rowan AN. Biological effects of blood
loss: Implications for sampling volumes and
techniques. ILAR News 1989;31:5-18.
8. Sarlis NJ. Chronic blood sampling techniques in stress
experiments in the rat - mini-review. Animal
Technology 1991;42,51-59.
9. Stuhlman RA, Packer JT, Rose SD. Repeated blood
sampling of Mystromys albicaudatus. Laboratory
Animal Science 1972;22:268-70.
10. Timm KI. Orbital venous anatomy of the Mongolian
Gerbil with comparison to the mouse, hamster and
the rat. Laboratory Animal Science 1989;39:262-64.
Zoonoses and Animal Allergy
1. Duncan CJ, Scott S. What caused the Black Death?
Postgraduate Medical Journal 2006;81;315-20.
2. Heeney JL. Zoonotic viral diseases and the frontier of
early diagnosis, control and prevention. Journal of
Internal Medicine 2006;260:399-408.
3. Kallio-Kokko H, Uzcategui N, Vapalahti I and Vaheri
A. Viral zoonosis in Europe. Fems Microbiology
Reviews 2005;29;1051-77.
44  Practical Manual of Experimental and Clinical Pharmacology
4. Kibby T, Power G, Croner J. Allergy to laboratory
animals: A prospective sectional study. Journal of
Occupational Medicine 1989;31:842-46.
5. Pal M. Importance of zoonoses in public health.
Indian Journal of Animal Sciences 2005;75;586-91.
6. Reynolds D. Policy making in areas of uncertainty,
conference “The Prevention and Control of Zoonoses
– from Science to Policy”, (2005) Health Protection
Agency, UK. Slides on www.hpazoonoses
conference.org.uk/programme.htm
7. Wolfe N D, Panosian D C, Diamond J. Origins of
major human infectious diseases. Nature 2007; 447:
279-83.
Anesthesia and Euthanasia
1. Defalque RJ, Stoelting VK. Latency and duration of
action of common local anesthetics. Anesth Analg
1967; 46: 311-14.
2. Defalque RJ, Stoelting VK. Latency and duration of
action of some local anesthetic mixtures. Anesth
Analg 1966; 45: 106-16.
3. Feldman HS, Covino BG. Comparative motor-
blocking effects of bupivacaine and ropivacaine, a
new amino amide local anesthetic, in the rat and
dog. Anesth Analg 1988;67:1047-52.
4. Grant GJ, Vermeulen K, Zakowski MI, Sutin KM,
Ramanathan S, Langerman L, Weisman TE, Turndorf
H. A rat sciatic nerve model for independent
assessment of sensory and motor block induced by
local anesthetics. Anesth Analg 1992;75:889-94.
5. Markham A, Faulds D. Ropivacaine: A review of its
pharmacology and therapeutic use in regional
anaesthesia. Drugs 1996;52:429-49.
6. Miller EV, Ben M, Cass JS. Comparative Anesthesia
in Laboratory Animals. Fed Proc 1969; 28: 1369-
1586.
Ethical Consideration and Others
1. Broadhead CL, Bottrill K. Strategies for replacing
animals in biomedical research. Mol Med Today 1997;
3(11):483-87.
2. Kurosawa TM. Alternatives to animal experi-
mentation vs animal rights terrorism. Yakugaku
Zasshi 2008; 128(5): 741-46.
3. Manciocco A, Chiarotti F, Vitale A, Calamandrei G,
Laviola G, Alleva E. The application of Russell and
Burch 3R principle in rodent models of
neurodegenerative disease: the case of Parkinson’s
disease. Neurosci Biobehav Rev 2009;33(1):18-32.
4. Ormandy EH, Schuppli CA, Weary DM. Worldwide
trends in the use of animals in research: The
contribution of genetically-modified animal models.
Altern Lab Anim 2009;37(1):63-68.
5. Principles and methods for evaluating the toxicity of
chemicals, part 1. Environmental health criteria 6,
world health organization, Geneva, 1978;35-36.

2
INTRODUCTION
Bioassay was started in late 18th century, when
standardization of Diphtheria antitoxin was done
by Paul Ehrlich. Thereafter, it was a common
practice to standardize any substance through
biological assay. Bioassay is a most important
basic step towards the drug discovery and can
determine the effect of any natural source of
unknown substances without affecting complete
system. So, it is the method for the estimation of
the potency of a material on living system.
Bioassay comprises of “bio” means living
material and “assay” means assessment at
laboratory, i.e. assessment of unknown substance
on any living tissue. Hence, bioassay is defined as
comparative assessment of relative potency of a test
compound (T) to a standard compound (S) on any living
animal or biological tissue. This procedure is for
determining the quantitative relationship between
the concentrations (dose) and magnitude of
response. A bioassay experiment may be quantal
or quantitative, direct or indirect. Qualitative
bioassays are used for assessing the physical
effects of a substance that may not be quantified,
such as abnormal development or deformity,
whereas quantitative bioassay assessed at the
laboratory level and the concentration or drug dose
can be evaluated.
Physical, chemical or biological methods are
few important quantitative methods for
assessment of any drug. The popularity of
bioassay among the other methods is due to some
important criteria such as improved reliability,
Bioassay
specificity, accuracy, sensitivity and probability which
are required for any assay to reduce the
variability. If, a relationship is established
between the bioassay and chemical assay, then
bioassay has the superiority due to greater
specificity while chemical assay has the greater
precision.
Substances Used in Bioassay
Substances derived from plants and animal
sources are mainly assessed by biological assay.
Chemical and synthetic drugs are ideally not
required for bioassay because of known structure.
Moreover, their own chemical standardization
methods are available.
Objective or Purpose of Bioassay
Bioassays have mainly three main constituents
namely, stimulus, subject and response. Through
these constituents, one can make
• Identification of various compounds
• Quantify the screening procedure and
• Commercial production of drugs like anti-
biotics
PRINCIPLES OF BIOASSAY
A bioassay is a quantitative procedure using a
functional response in a living system, either in
vivo or in vitro. The basic principle of bioassay is
to compare the potencies of the different
treatments instead of their respective responses.
Its main purpose is the determination (including
46  Practical Manual of Experimental and Clinical Pharmacology
assessment of errors) of the amount of a
substance in relation to the functional response
of a standard of the same substance. A bioassay
measures the ‘relative activity’ or ‘potency’ of a
substance and its specific capacity to achieve
an intended biological effect. In other words,
potency is the quantitative measure of biological
activity. It is usually measured as an observable
or objectively measurable parameter at a set
interval from the bioassay starting point (at time
zero). Normally, as potency increases with
increasing concentration of a substance (in
solution), the biological activity is directly linked
to subdivisions or dilutions of the original
concentration. The most useful bioassays are those
in which the range of functional responsiveness is
the scale of readings for the measurable parameter
between the minimum and maximum response which
is relatively large and falls between an effective,
practical range of concentrations/dilutions of a
substance to generate analyzable ‘dose-response’
data.
Principles in Short
• To compare a test drug potency with a standard drug,
quantitatively
• To evaluate both test and standard drugs, as identical
to each other
• To estimate the biological variation of the drug to
species and other groups of animals or living beings
• Methods for comparing therapeutic potency of unknown
and standard drug
ERROR IN BIOASSAY
While performing bioassay, sometime responses
are varied, i.e. sometimes it may increase and
sometimes it may decrease, which is calculated
as error of the procedure. Broadly error is divided
into two categories, i.e. due to biological variation
or due to methodological error.
Biological variation: It is known that animal to
animal or individual biological variation occurs
in response to a drug action. The effect of drug
dose to the tissue response may vary and will fall
in a range. So, when we plot the response on the
Y-axis and drug dose on the X-axis, the graph
obtained is the symmetric frequency distribution
curve (Fig. 2.1). So, the response taken is the mean
of all responses observed.
Fig. 2.1: Symmetric frequency distribution curve, plot of
response versus drug dose
Reason and its correction for biological
variation:
• Downregulation of receptors (repeated
washing of tissue; avoid over washing)
• Loss of tissue sensitivity (change the tissue)
• Animal use of different species, sex, age, weight
and health status (must use same species)
• Laboratory condition may be variable (must be
kept constant)
• Housing and handling of animals (must be
handled by a qualified experienced staff)
Methodological error: Usually this error occurs
due to the faulty method selection or due to the
incorrect procedure, i.e human error and
experimental error.

Human error is one which is done by the
experimenter during the experiment. It may
happen from the time of tissue selection or
physiological salt solution (PSS) preparation or
during the response recording. In every step, there
are chances of error, so to maintain the standard
conditions for the isolated tissue, be sure regarding
the accuracy of the procedure.
Experimental error may happen due to the
faulty procedure selection or due to the calibration
error of the instrument. This may be corrected by
properly balancing the lever and by maintaining
the temperature or pH of the PSS.
Reasons for Methodological error and its correction
• Lack of standardization of procedure (standar-
dize your procedure beforehand)
• Set-up of apparatus (Check every instrument
used in the experiment for their proper
working)
• Tissue isolation/extraction and preparation
for the experiment (minimize handling and
excess cleaning of the tissue while mounting
in the organ bath)
• Preparation of physiological salt solution
(PSS) and maintenance of its pH (Follow the
standard procedure to prepare and maintain
the pH of PSS)
• Drug preparation or it dilution (while making
serial dilution of the drug, mix it slowly; Avoid
the vigorous mixing).
APPLICATIONS OF BIOASSAY
According to several pharmacopeias, assay used
for the drug may be varied. For example:
1. Estimate potency of natural drug, for which
chemical method is not known or established
2. Standardization of drugs of natural origin
(plant and animal origin) whose structure or
origin is unpredictable
3. Screening of new compound for biological
activity
4. Estimation of biologically active substance like
histamine, acetylcholine, 5-hydroxytrypta-
mine, adrenaline, bradykinin, substance P,
prostaglandins, etc.
Bioassay  47
5. Estimation of ED50/TD50 and LD50. (Presently
instead of LD50, NOAEL or LD10 is preferred).
METHODOLOGY (FLOW CHART 2.1)
In academic set-up, traditional organ bath is
commonly in use, which contains inner organ
bath, water bath with coiled inlet for physiological
salt solution (PSS), stirrer, heating coil, kymograph
with revolving motor, etc. (Fig. 2.2). But in industry
more recent sophisticated equipments are
available for drug discovery such as multiple
organ baths (may be up to 8 inner organ bath).
Organ bath assembly was first used by Rudolph
Magnus in early 19th century. He standardized
the set-up with intestinal strips. More or less set is
still same with some modification.
Essential Parts and Uses of Organ Bath
Two types of organ baths are designed which got
access in most of the laboratories such as,
1. Single unit organ bath: This set-up was
designed and developed by Rudolph Magnus.
It has only one inner organ bath (Fig. 2.3).
2. Double or multiple unit organ baths: The
concept of these organ baths were developed
by Gaddum. He was developed a double unit
organ bath. It has two inner tissue organ baths,
but it is not able to replace the conventional
organ bath. Nowadays, in many industries
automated multiple unit organ baths are used
for more efficient and fast drug discovery
(Fig. 2.4).
Essential Parts of Organ Bath which is
Required to Record the Response of a
Tissue/Muscle
Rotating drum and kymograph: Rotating drum is
also known as Sherrington rotating drum. Its speed
can be adjusted by the attached gear and lever.
For the slow contracting tissue, the drum is kept
on low speed and for the fast contracting tissue
the drum speed should be kept on comparable
high speed. The standard speed of the drum is 1
revolution/96 min.
48  Practical Manual of Experimental and Clinical Pharmacology
Flow Chart 2.1: Overview of complete steps for bioassay

Kymogram is the paper used to record the
response of the tissue/muscle against the known/
unknown concentration of the drug. Paper has
two sides, one side is glossy or smooth and other
side is rough. While mounting the paper on the
drum, one should make sure that the smooth
surface is outside (helps or don’t produce
resistance in the movement of the lever on
kymogram) and the rough surface is inside (help
in sticking of paper to the drum).
Paper is smoked with the help of burning cotton soaked in
benzene or kerosene. The drum is kept on the height
where it gets dark and densely smoked (At center and
adequate height). Precaution should be taken while smoking
drum and should maintain sufficient distance from fire. For
better writing on the kymograph paper, smoking should
be uniform and dense.
Presently in most of the pharmacology laboratories, ink
kymograph paper is also in use where there is no need to
smoke the paper. Stylus works as a writer on the kymograph.
 Bioassay  49

1. Power input 12. Stirrer

2. Switch board 13. Reservoir for PSS
3. Heating rod 14. Clip to control the flow of PSS to inner bath
4. Thermostat 15. Stylus or writing point
5. Coiled PSS inlet to inner bath 16. Motor
6. Outer bath water outlet 17. Gear to adjust the speed
7. Inner bath PSS outlet 18. Clutch lever
8. Inner bath with air supply (aerator) and tissue holder 19. Screw to adjust the level of drum
9. Thermometer 20. Drum rotation indicator
10. Recording lever 21. Sherrington rotating drum
11. Fulcrum 22. Lift screw (to move drum up or down)

Fig. 2.2: Schematic presentation of commonly used organ bath set-up

Fig. 2.3: Single unit organ bath Fig. 2.4: Double unit organ bath (For color version see Plate 1)
50  Practical Manual of Experimental and Clinical Pharmacology
Outer bath/Outer water jacket: Mainly used to
store water outside the inner organ bath to
maintain the temperature for the experiment. It is
made up of perspex glass, glass or steel. Perspex
is more widely used due to its durability.
Inner organ bath: It is transparent and made up of
glass to observe the tissue during experiment.
Inner bath has varying capacity from 5 to 50 ml.
Proper marking is done on the tube to fill the PSS at
fixed level every time.
Tissue holder and oxygen supply: Tissue is attached
inside the inner organ bath with the help of tissue
holder and it also supports the air or oxygen
supply through its motor.
Fulcrum: Writing lever is attached to this and help
in free vertical movement to record the response
of tissue against the drug.
Heating iron coil and thermostat: Heating coil is
attached to the outer bath and is made up of iron.
It maintains the temperature outside the inner
bath as well as of PSS which moves in the inner
bath through coiled inlet. Whereas “thermostat”
as name suggests maintains static or constant
temperature throughout the experiment and
avoids wide variation in temperature.
Stirrer: It gives circulating water to maintain the
similar temperature throughout the outer organ
bath.
Fig. 2.5: Fixing of kymogram; fixing solution is made by dissolving the shellac in methanol and saturate the solution for
5-7 days to settle down the particles at bottom and then filter it and store at cool and dark place)
Fixing of the recording: Tracing on kymogram is
fixed with help of fixing solution at the end of
the experiment. Fixing solution is made up of
shellac and colophony saturated in the alcohol.
Sufficient powdered shellac is mixed in the
alcohol (methanol) and dissolved till it gets
saturated and precipitated then keep solution for
5-7 days to settle down the particles and decant
the remnants to get non-precipitated solution.
Store the prepared fixing solution in the cool and
dark place (Fig. 2.5).
Drug Response Relationship
Potency comparison of standard and test
preparation always depends on the response
produced by the particular concentration of
standard/test. The dose response is always directly
co-related to the concentration of the standard/
test drugs. Students always prefer to make a dose
response curve (DRC) or concentration response
curve (CRC) in an ascending order (Fig. 2.6A) (It is
less common practice of making in the descending
order due to the lack of known high concentration).
But, sometimes it shows, reduced sensitivity of the
tissue. Hence, to retain the tissue sensitivity for a
longer duration, one should vary the dose order
intermittently (neither ascending nor descending)
(Fig. 2.6B).
 Bioassay  51

Fig. 2.6A: Ascending dose response plot

Fig. 2.6B: Intermittent order dose response plot

The dose response curve depends on the linear
regression relationship on log dose of potency of
standard and test preparation. The dose response
curve of standard and test preparation should be
parallel. (Figs 2.7A to C) This is not only useful as
to estimation of potency (two parallel lines) but it
also estimates the errors in the method applied. If
there is no regression of potency, there is no use of
dose response relationship and it is thought that
if the mean square for regression is relatively large
then there is good co-relation with potency of the
two preparations.
PHYSIOLOGICAL SALT SOLUTION (PSS)
Preparation of PSS depends on tissue selection
for the experiment (Table 2.1). PSS is very
important to maintain tissue outside the animal
body which fulfills their internal environment of
ions and nutrition. This is often called as Ringer
solution.
52  Practical Manual of Experimental and Clinical Pharmacology

Figs 2.7A to C: (A) Correct linear

regression relationship of standard and
test (log dose versus percentage
response plot); (B) Incorrect linear
regression relationship of standard and
test; (C) Guide to select the dose of
standard and test during the experiment
and showing the several effects of drug
on a tissue (supramaximal effect is
commonly known as ceiling effect of
the drug)

Table 2.1: Composition of different physiological salt solution (PSS) [salts in g/10 liters]
Ingredients Tyrode Krebs De Jalon Frog Ringer Ringer Locke McEwens
NaCl 80.0 69 90.0 65.0 90.0 76.0
NaHCO3 10.0 21 5.0 2.0 2.0 21.0
D Glucose 10.0 20 5.0 20.0 10.0 20.0
KH2PO4 – 1.6 – – – –
NaH2PO4 0.50 – – – – 1.44
KCl 2.0 3.6 4.2 1.4 4.2 4.2
MgSO4.7H2O – 2.90 – – – –
MgCl2 1.0 – – – – –
Sucrose – – – – – 5
CaCl2 2.64 3.70 0.8 1.58 3.2 3.0
Aeration Air 95%O2 + 95%O2 + Air O2 95%O2 +
5% CO2 5% CO2 5% CO2

Solution prepared with the help of distilled or
double distilled or deionized water. Main
components of PSS are sodium (Na+), chloride
(Cl–), potassium (K+), magnesium (Mg+), calcium
(Ca+) and glucose.
It is always important to select PSS in which tissue
last longest. Aeration is important for solution with
oxygen (O2) or 95% O2 + 5% CO2 (carbogen). This
process helps to provide O2 to tissue and mix PSS
thoroughly in organ bath. Additionally, carbogen
is important because, pure O2 may interact with
bicarbonate (HCO3–) buffer in PSS which will
cause CO2 loss and PSS become alkaline.
Certain precautions must be taken, while
addition of ingredients into the distilled water
(DW)/double distilled water (DDW) or deionized
water (DIW). Calcium chloride should be added
at last, to prevent precipitation or chelation of the
bicarbonate which make solution turbid or
opaque. This may interfere with internal property
of solution and may reduce visibility of tissue in
inner organ bath. Thumb rule for selection of PSS
is that, Tyrode may be used for experiment with
non-innervated muscle while Krebs is used for
nerve muscle preparation. The pH is important to
maintain PSS property and should be at range
7.3-7.4.
The preparation of PSS is important and the
error in making should not exceed 1%. The
physical properties of ingredients should also be
taken into consideration, such as calcium chloride
and magnesium chloride are very hygroscopic,
so it is best to add them from stock solution. MgSO4
is not hygroscopic in nature and hence used instead of
MgCl2. The reason being exchange of chloride with
sulphate (SO4–) does not make any markedly
difference, because most part of chloride ion is
fulfilled by NaCl and sulphate (SO4–) ion have no
harmful effect.
Preparation of various PSS is given in Table 2.1.
Fresh solution is always prepared while it can be
stored for 24 hr in fridge, but storage can’t be preferred
for longer time because of microbial growth (due to
presence of glucose). If preparation of solution is
needed and requires storage longer than 24 hr then
Bioassay  53
avoid addition of calcium and glucose which should
be added at the time of experiment.
Role of Each Ingredient
• Sodium (Na+): One of the major extracellular cations
which maintain the electrolyte level in the tissue. It
makes solution isotonic by maintaining the osmolarity.
• Potassium (K +): It is the major intracellular cation
having important role in nerve conduction, muscle
contraction, etc. Its role is remarkable in maintaining
heart rate and rhythm.
• Calcium chloride (CaCl 2 ): It controls excitability of
muscle, nerve and glands. Among contraction-
relaxation coupling it also helps in maintaining the
integrity and permeability of the cell. The major role
remains excitation.
• Magnesium chloride (MgCl 2): Being second most
common intracellular cation, its main action to reduce
the spontaneous activity of tissue, also play an
important role in neurotransmission in muscle
contraction.
• Bicarbonate (HCO3–) and Sodium hydrogen phos-
phate (NaHPo 4): Acts as a buffer.
• Glucose: Major nutrients for the tissue in ‘in vitro’ set-up.
Important Points to Remember
• McEwen solution contains sucrose in addition to
glucose.
• DeJalon, Frog-Ringer and Ringer-Locke do not contain
magnesium (Mg 2+) and phosphate (PO4–)
• Krebs, DeJalon and McEwen are aerated with 95% O 2
+ 5% CO 2 and used for mammalian isolated organ
(nerve associates) and avian skeletal muscle
• Tyrode specially used for the mammalian smooth
muscle
• For amphibian tissue, use Frog-Ringer solution
• For heart muscle preparation, use Ringer-Locke
solution
• Krebs solution may be used for any tissue
LEVER AND MAGNIFICATION
It is the mechanical instrument based on the
principle of static equilibrium which is derived
from Newton’s laws of motion and statics. Hence,
working of the lever is denoted by the
“Load force × length of load arm = Effort force
× length of effort arm.”
Lever has three basic parts (1) effort arm (point
where you apply the force) (2) load arm (effect
54  Practical Manual of Experimental and Clinical Pharmacology

observed due to application of force) and (3)
fulcrum. Both load arm and the effort arm are measured
from the fulcrum to the load and effort, respectively
(Figs 2.8 to 2.10).
The lever used in the in vitro bioassays is of the
“type 1 lever”. In most of the experiments, the change
in the tissue length is measured and recorded by the
lever on a kymogram (Fig. 2.11). Stylus is the point of the
lever which touches the kymograph for recording the response
(writing point). It is made of stainless steel, aluminum
or wood. Lever is attached to fulcrum and mainly used
to magnify the response of the isolated tissue
preparation. So, lever records and amplifies the
response obtained during the drug effect on the isolated
tissue. Magnification can vary for tissue to tissue from
5 to 15 times. The standard rule is that, if tissue
preparation is slow contracting magnification is large
either 10-15 times but when tissue is fast contracting
the magnification may reduce to 5-10 times.
Magnification (Mx) and its Adjustment
The transducer which amplifies contraction are
mainly divided into two types, namely, when there
is change in the length of tissue recorded after
contraction but no change in tone or tension
recorded, then it is called as isotonic transducer
such as simple writing, frontal writing, etc. whereas
in isometric transducer there is no change in the
length but change in tone or tension of tissue is
recorded. Such as in the muscle twitching, cramp,
etc. this type of recording needs special set-up.
In isotonic set-up magnification of the
recording lever plays important role for a
magnified response. Usually, magnification is
kept low in the case of fast contracting tissues
whereas it is kept high in the case of slow
contracting tissues, so as to record the proper
response. The magnification of the lever is
calculated as followed (Fig. 2.12):

Fig. 2.8: Fulcrum (F) is located between the input effort (E)
and the output load (L). For example: Bioassay, seesaw,
triceps brachii muscle, Bicycle hand brakes, scissors
(double lever), etc.

Fig. 2.9: Fulcrum (F) is located at the one end and effort
input (E) at the other. The load input (L) is at the middle of ‘E’
and ‘F’. For exmple: Dental elevator, Bottle opener, puss-up,
etc.

Fig. 2.10: Fulcrum (F) is located at the one end and at load
input (L) the other. The effort input (E) is at the middle of ‘L’
and ‘F’. For example: forceps, etc.
 Bioassay  55

Simple lever: Made of stainless steel, aluminum or wood
with stylus* attached to it.
Attachment of simple lever should be tangential to the
smoked drum
Frontal writing lever (FWL): Made of stainless steel,
and aluminum with stylus* (two arm) attached to it.
Attachment of FWL should be perpendicular to the smoked
drum. It magnified a small contraction of tissue/muscle on
the kymograph

Gimbal lever (GL): Made of stainless steel, or aluminum
with stylus* attached to it. A roller is fitted in between for
free movement by the gravity force. Attachment of GL
should be tangential to the smoked drum.
Sprung lever: Made of stainless steel, or aluminum with
stylus* attached to it with help of return spring. Tension of
the lever is adjusted with the screw attached at the opposite
end of stylus. Attachment of simple lever should be
tangential to the smoked drum

A 20 cm
5
B 4 cm
Auxotonic lever (AL): Made of stainless steel, aluminum Torsion lever (TL): Made of stainless steel, aluminum or
or wood with stylus* attached to it. wood with stylus* attached to it.
Attachment of AL should be perpendicular to the smoked Attachment of TL should be tangential to the smoked
drum. drum

Starling heart lever (SHL): Made of stainless steel or

aluminum with detachable stylus attached to it, Attachment
of SHL is perpendicular to the smoked drum.
* Stylus (Writing point): Ideally it should be made up of aluminum or stainless steel but in laboratory, it can be made
by the thin steady film of X-ray or photography film or any hard paper (for temporary use).

Fig. 2.11: Different types of levers, commonly used in experimental pharmacology

Distance from fulcrum to writing point (A) Answer:

Mx = A = 20 cm
Distance from fulcrum to tied tissue (B)
B = 4 cm
Mx = ?
Example: Calculate the magnification of a lever
attached to a fulcrum, if the distance of writing point Mx =
from the fulcrum is 20 cm and the tissue attached to the
Hence, the magnification of attached lever is 5x.
lever is 4 cm apart from the fulcrum?
56  Practical Manual of Experimental and Clinical Pharmacology

Fig. 2.12: Measurement of magnification of lever

DOSE CYCLE (DC) AND RESPONSE
Principles of the experiment depend on the dose
response relationship of test drug as well as
standard drug. So, for measuring responses, it is
necessary to first fulfill the basic requirements of
the experiment, i.e. tissue should properly relax
and proper load should be given for the specified
time. Baseline recording is initial step and
important to record, which helps to take a uniform
response of drug. (it can indicate the shift of
baseline; if any) It is not necessary that all tissue
show the steady straight line responses but few
tissues show the spontaneous tissue variability
which can be observed in the response recording
when there is any baseline shift found. Variability
(biological) of about ±10% is acceptable in the tissue
response recording. Tissue may show the
“tachyphylaxis”, in which there are decreased
responses to drugs after repeated dosing.
While taking response, dose cycle has three
important phases, baseline recording, dose
response and washing of tissue. Dose cycle is defined
as the time gap between drug dose additions whereas
contact time is defined as time duration at which
tissue come in contact with drug. Dose cycle is
commonly of 3 minutes for fast contracting
tissues or 5 minutes for slow contracting tissues.
(This includes at least two tissue wash)
(Fig. 2.13)

Fig. 2.13: Measurement of dose cycle and contact time in slow contracting and fast contracting tissues

TYPE OF TISSUE
Preparation of tissues used in the isolated tissue
experiments are broadly classified in two ways;
1. According to tissue/muscle obtained:
– Smooth muscle preparation, e.g.: Uterus,
tracheal smooth muscle, vas deferens,
aorta, ileum, ascending or descending
colon, etc.
– Skeletal muscle, e.g.: Rectus abdominus
muscle, etc.
– Cardiac muscle, e.g.: Frog heart etc.
2. According to response obtained by tissue/
muscle:
– Slow contracting tissue/muscle: For example:
Frog rectus abdominus muscle, stomach
fundus, biventer cervicis muscle of chick,
guinea pig tracheal smooth muscle, etc.
– Fast contracting tissue/muscle: For example:
Ileum, uterus, ascending and descending
colon, etc.
CLASSIFICATION OF BIOASSAY
Broadly bioassay is classified into three groups
namely,
1. Direct end point assay (DEPA)
2. Quantal assay (all or none assay)
3. Graded assay
a. Bracketing assay
b. Matching assay
c. Interpolation assay
d. Multiple point assay (3-point, 4-point,
6-point and 8-point)
Direct End-point Assay
In a direct assay, the threshold dose required for
response is determined for each experimental unit.
The principle of direct assay is to measure direct
response of dose of standard and test preparation.
The ratio between these doses estimates the
potency of the test preparation relative to the
standard.
The response should be clear and easily
recognized and the dose given to the experimental
animal should be in such a way that is easily
Bioassay  57
measured. Thus, the observed data are dose units,
e.g.: assay of digitalis in cat. Unlike, the indirect
assay the experimental unit receives one or more
specified doses of the preparation and the
observed data may be either quantal or
quantitative responses. Depending on the
experimental design, several dose levels of T and
S are given to the same or different experimental
units and the mean dose level is selected.
Mainly two experimental designs are followed,
one is crossover, and another is parallel group or
completely randomized design.
In the procedure, the test or standard
preparation is infused at a fixed rate into the
circulation of animal until a direct effect is
observed in the animal. For example: stopping of
heart beating after the continuous administration
of digitalis at the constant rate in the assay of
digitalis in cats.
The threshold dose of standard = Total period
of infusion × Rate of drug administration
TDS
Concentration of test = ×CSD
TDT
Where,
TDS = Threshold dose of standard
TDT = Threshold dose of test
CSD = Concentration of standard drug
Advantages
• Drug effects appear rapidly and are easily
recognized
• Drug effect is directly proportional to drug
dose
• Rapid end-point detection.
Disadvantages
• Only toxicity study or high dose study is
possible
• Dose ranging study cannot be done.
Quantal Assay (All or None Assays)
The unknown is compared with the standard
with respect to potency which produces the
58  Practical Manual of Experimental and Clinical Pharmacology
quantal affect, i.e. changes is easily recognized
sign or often death. These responses are recorded;
reason being effect of some drug or stimulus to
any targeted system is not able to record response
quantitatively or the reaction of subject is so
minimal which cannot be quantified or recorded.
In a quantal assay there is use of dose response
relationship, however assay are more closely
related to direct assay. As a procedure quantal
response to a drug is obtained and percentage of
positive response at each dose is calculated. Most
popular example in the drug discovery is
determination of LD50 (Fig. 2.14) and other is assay
of insulin by mice convulsion method. The
response in the quantal assay is varying, i.e. some
responses are irreversible and hence the animal
Fig. 2.14: Determination of LD50
Fig. 2.15: Serial dilution (descending order)
used is once but some responses have no
permanent effect and can be used in a design of
several test.
Graded Response Assay (GRA)
In this type of biological assays, the extent of the
reaction is a function of the dose of drug. Ideally,
the quantitative relation between dosage and
response would be expressed in terms of an
equation describing the mode of drug action. Most
graded reactions are consistent with the sigmoid
form, approaching asymptotically to a “floor” and
a “ceiling.” However, unlike the all-or-none
response, where the number exposed to treatment
is known or can be estimated readily from control
groups, the amount of reaction representing
100 % of a graded response would need to be deter-
mined experimentally to solve either formula.
By GRA, potency of a test agonist is determined
by comparing its mean response to standard mean
response. This process is known as ‘analytical
dilution assay’. (Serial dilution of standard/test
drug) (Fig. 2.15).
This assay simply depends on the several
graded responses by exponential increase in the
test dose and which is compared with the
standard graded dose response. GRA is simplest
way of determining potency of a test drug because it
does not require statistical analysis.

Bracketing Assay (Fig. 2.16)
This assay is preferred when test sample volume
is too small. It is the simplest way of GRA, in
which single or few response (s) is taken by using
any test drug concentration. Consequently, this
response is bracketed between two responses (one
higher and one lower) of the standard drug. Then
the potency of the test drug is directly calculated
from concentration of standard drug or by
interpolation through dose response curve.
Limitation of the assay is poor precision and
reliability and also unable to calculate error.
Matching Assay (Fig. 2.17)
Comparison of potency between the unknown and
standard drug is done by trial and error method.
Important part of this method is that response is
matched at only one dose, so it does not needs
dose response curve of test compound. It requires
very small sample volume, whereas meanwhile
having several disadvantages such as it is purely
Concentration of T =
Fig. 2.16: Bracketing assay; Test compound dose response (T) is bracketed between the two dose responses of
standard compound (S1 and S2), one response more and another is lower than test compound
Bioassay  59
subjective, experimental error is not excluded out
and there is no sign of parallelism as it lacks dose
response relationship. It requires most sensitive
tissue, so tissue selection is the most important
aspect in this assay.
Interpolation Assay (Fig. 2.18)
This method depends on the assumption of dose
response curve. Concentration of unknown is
interpolated from the dose response curve graph.
At the first step DRC of the standard drug is plotted
then single or few responses of the test drug are
plotted. The dose of the test drug which comes at
the linear log dose-response relationship is
interpolated from the dose response plot.
Multiple Point Assay
The above mentioned methods are not ideal
because of lack in sensitivity, accuracy and might
Dose of ‘S’
Conc. of ‘S’
Dose of ‘T’
60  Practical Manual of Experimental and Clinical Pharmacology

Fig. 2.17: Matching assay; the test compound response is matched with the selected response of standard compound.
Initially DRC of standard compound is obtained then take a response of fixed volume of test then either higher response
or lower response of standard is selected. In the above figure, matching assay is performed by selecting a higher response
of standard than the test response, hence the test volume is increased slowly to meet the response of standard response,
then concentration of test is find by formula given in the column. (T/T*/T** = Showing increasing dose of test compound to
match the standard response)

doses must be in the linear portion of the dose-

Fig. 2.18: Interpolation bioassay
involve many methodological errors such as tissue
sensitivity error, variable temperature error,
dilution error, etc. So, to correct the above
mentioned limitations, graded response assays
are preferred. Repeated response recording in
graded response assays minimize the tissue
sensitivity error and improve the methodological
errors. These assays are performed by the selection
of 1 or more dose responses of test compound and
these responses are compared with 2 or more
responses of standards. The selection of the test
response plot of standard compound, i.e. between
25 to 75% (Dose discrimination is better at these
portions) (Fig. 2.19).
Three-point assay (Fig. 2.20): Method depends on
the latin square randomization of total three
responses selected from DRC prepared for
standard as well as test (2 response from standard
and 1 response from test: response selection is made
between 25-75% of response). For many bioassay,
dose response data can be transformed to generate log
dose transformed response lines to yield as extended a
linear range as feasible. Estimates of relative potency
are then obtained as the displacement of parallel
log dose response lines of standard and test
compound.
S1 S2 T
Latin square randomization S2 T S1
T S1 S2
Calculation for 3’point assay:
T – S1 s
Relative potency (M) = log 2
S2 – S1 s1
 Bioassay  61

Fig. 2.19: Dose should be selected in the linear portion of the plot (25-75%)

s1
antilog M
t

Fig. 2.20: Three-point assay

Concentration of unknown compound s1 and s2 = Standard drug dose which came

= the response ‘S1’ and ‘S2’
= “x” times more concentrated than standard
compound
Where,
S1 and S2 = Length of standard dose
response selected between 25-
75%
T = Length of test dose response
selected in between of two
standard response
in contact with tissue and given
respectively
t = Test drug dose which came in
contact with tissue and given the
response ‘T’
Note: There are several factors plays role during
the experiments such as biological environmental
or methodological factors. So, there may chances
of some error present during the bioassay. Hence,
62  Practical Manual of Experimental and Clinical Pharmacology

the percentage of error is calculated by the formula T1 and T2 = Length of test dose response
given below: selected
s1 and s2 = Doses which produces mean
ACT – OCT
Percentage error (%) = 100 response of S1 and S2
ACT respectively
where, t1 and t2 = Doses which produces mean
ACT = Actual concentration of test response of T1 and T2 res-
OCT = Observed concentration of test pectively
Note: In bioassay, permiciable limit of percentage Concentration of unknown
error is < 10%.
Four-point assays (Fig. 2.21): Method is same as =
3-point assay, only difference is that in this
experiment responses are selected; 2 responses of = ‘x’
standard and 2 of test from the DRC for the So, the concentration of unknow is ‘x’ times
consecutive 16 response of Latin square more strengther than standard
randomization as shown below in figure. This
procedure is more sensitive than 3-point assay Note: There are several factors plays role during
and reduces the error or variability the experiments such as biological environmental
or methodological factors. So, there may chances
S1 S2 T1 T2 of some error present during the bioassay. Hence,
S2 T1 T2 S1 the percentage of error is calculated by the formula
Latin square randomization T1 T2 S1 S2 given below:
T2 S1 S2 T1 ACT – OCT
Percentage error (%) = 100
ACT
where,
M=
ACT = Actual concentration of test
OCT = Observed concentration of test
Where,
S1 and S2 = Length of standard dose Note: In bioassay, permiciable limit of percentage
response selected error is < 10%.

Fig. 2.21: Four-point assay


6-point and 8-point Assay
These methods of bioassays are generally not
adopted for the experiment purpose because of
the time consuming lengthy procedure. The
responses obtained for the 6-point is ‘36’ and ‘64’
for 8-point. But, the advantage being reduced error
and variability of the procedure over other
methods due to the large number of responses and
hence have greater specificity.
BIOASSAY OF ANTAGONIST
The term antagonist refers to any drug which can
completely or partially block a response of an
agonist. So, an antagonist is the opposite of an
agonist which stimulates an action and these two
are considered as a prime agent in pharmacology.
Antagonist activity may be reversible or
irreversible depending on antagonist–receptor
complex which in turn depends on the nature of
antagonist receptor binding. When assessing an
antagonist, the following points should kept in
mind such as,
a. To check whether the antagonism is surmoun-
table by increasing the concentration of agonist
or not and
b. To check whether the antagonism is reversible
or not (does agonist regain response, after
washing of antagonist)
This is due to identification of the type of
antagonism. Such as, if an antagonist is sur-
mountable or reversible, it is likely to be compe-
titive.
Experimentally antagonist is divided into the
two groups,
1. Preventive
• When the antagonist used before addition
of agonist, it is called the preventive
antagonist
2. Curative
• In this process of antagonism, first agonist
and then antagonist is added
Bioassay  63
In pharmacology, several different types of
antagonist are described such as
Competitive antagonist (Antagonists bind to the
receptor of agonist but show no efficacy, e.g.
propranolol, naloxone, etc.) (Figs 2.22A and C).
Non-competitive antagonist (Antagonists bind to
the receptor at sites which is not related to the agonist
binding site for example Ca2+ blockers) (Fig. 2.22B).
Physiological (functional) antagonist (Antagonist
has the opposite biological action of the agonist,
by action on a different receptor, e.g.: salbutamol
a β2-adrenoceptor agonist given in acute asthmatic
attack antagonizes the bronchoconstrictor action.
Pharmacokinetic antagonist (Antagonist which
reduces the free blood concentration of drug at its
target site either by reducing drug absorption or
accelerating renal or hepatic elimination)
Chemical antagonist (Particular drug may
antagonize the action of a second drug by binding
and inactivating the second drug. For example
protamine binds to heparin and makes it
unavailable for interactions with proteins
involved in the formation of blood clots)
But, in the practical pharmacology, the identification
of antagonist is limited to the competitive or non-
competitive or physiological antagonists only. For
analyzing the antagonist, a dose-response curve
of agonist is made in the absence and presence of a
fixed concentration of antagonist, which shifts the
DRC to the right (parallel shift), with the same
maximum response and the same shape; for the
competative antagonist.
Antagonist has no effect of its own, so, it is
determined experimentally by the intensity to
block the agonist activity. The process of dose
determination is same as it is with the agonist.
First, the agonist response is obtained thereafter
percentage inhibition is determined by using the
different doses of antagonist. Sometimes,
percentage inhibition is converted into the probit
scale against the log dose to make graph more linear
(Fig. 2.23).
64  Practical Manual of Experimental and Clinical Pharmacology

Fig. 2.22: (A) Cumulative plot showing competitive antagonism (parallel shift) in the presence of antagonism; (B) Cumulative
plot of non-competitive antagonism in the presence of antagonist, (C) Competitive antagonism response plot, showing a
partial response block of an agonist which is regained by the repeated tissue washing

experiment will be 1 X 10–10 M. The exposure time of

Fig. 2.23: Probit plot
As a general rule antagonist takes a long-time
to block an action of agonist. In the experimental
purpose, the use of antagonist concentration is around
10 times less than the agonist concentration, i.e. if
agonist used in the concentration of 1 X 10-9 M then
the concentration of antagonist used in the same
antagonist to tissue should be around 15min. but in
the routine experiment it is also seen that 3-5 min
contact time of antagonist with tissue can also elicited
the result.
Hence, various methods are employed for the
identification of competitive antagonist,
1. Schild plot method and pA2, pA10 values
2. Parallel shift of DRC
3. Double reciprocal plot Lineweaver and Burk
Schild Plot Method and pA2, pA10 Values
Generally, potencies of competitive antagonist are
expressed as pA2 values or pAx values. Hence
the term, pA2 is defined as the negative logarithm
to base 10 of the antagonist concentration in molar
units corresponding to a dose-ratio of 2, i.e. the
concentration that produces a 2-fold shift in the
 Bioassay  65

Figs 2.24A and B: (A) Schild plot; (B) Determination of IC50

response of agonist concentration-response curve. experimental units compared to the Schild method
Kd (the dissociation constant of the antagonist for to plot a graph.
the receptor) is the other form of expressing the Once the experiments are completed a series
potencies of competitive antagonists. Procedures of dose ratios (DR) are calculated for observed
(both design and model) for estimating Kd and responses. For example, the ratio of the dose of
pA2 have also been developed from the results of agonist (A’) to produce a specific effect (e.g. half
an antagonist inhibition curve in the presence of maximal effect) in the presence of the antagonist
a fixed concentration of the agonist (Figs 2.24A (B) to the dose required in the absence of the
and B). antagonist (A) is calculated.
Suppose, the antagonist is of competitive type,
then the dose ratio will be expressed as, Parallel Shift Plot (Fig. 2.25)
Dose ratio (d) = 1+ (antagonist)/(Kd) It is the simplest form of experimental approach
d-1 = (antagonist)/(Kd) of identifying an antagonist in which the shift of
log (d-1)= log [(antagonist)/(Kd)] DRC is parallel towards right after addition of
log (d-1)= log(antagonist)- log(Kd) competitive antagonist is seen.
If the antagonist is competitive, then slope will
be 1.0 and the X-intercept and Y-intercept will
both be equal to the Kd of the antagonist. The other
formula to identify the competitive inhibition is
by the difference of pA2 and pA10 (pA2 – pA10). If
the difference (pA2 – pA10) is 0.95 or in the ranges
of 0.8 - 1.20, the inhibition is competitive. (pA2
and pA10, where 2 and 10 is the dose ratio)
The limitation of the above mentioned methods
is that no one applies to evaluate antagonist
potencies for compounds that are not pure
antagonist, i.e. partial and inverse agonists and
antagonists lacking intrinsic activity. So, in these
cases Waud Model is applied for the estimation of
pA2, Kd, and IC50. Waud Method requires fewer Fig. 2.25: Parallel shift plot
66  Practical Manual of Experimental and Clinical Pharmacology
Lineweaver-Burk Plot or Woolf-Lineweaver-
Burk Plot/graph (Fig. 2.26)
This method was developed by Hans Lineweaver
and Dean Burk in 1934 for the assessment of
enzyme kinetics. Lineweaver-Burk plot is inverse
of response plotted against the inverse of dose
concentration of agonist, which was invented to
analyze how fast a drug can produce its response,
against a present antagonist. This method applied
to distinguish between competitive, non-
competitive and uncompetitive antagonists.
Findings of Different Antagonist
• Competitive inhibition: Have the same y-
intercept as uninhibited (since V max is
unaffected by competitive inhibitors the
inverse of Vmax also doesn’t change) but there
are different slopes and x-intercepts between
the two data sets.
• Noncompetitive inhibition: Produces plots
with the same x-intercept as uninhibited (Km
is unaffected) but different slopes and y-
intercepts.
• Uncompetitive inhibition: Causes different
intercepts on both the y- and x-axes but the
same slope.
Fig. 2.26: Woolf-Lineweaver Burk plot
HUMAN TISSUE BIOASSAY
There is a common practice of using animal tissues
in bioassay to ensure the activity of an unknown
compound in institute/industries which provides
a more convenient tool than using human tissues.
But, limitation of animal tissue use in the research
is that it can’t predict accurate outcome in relation
to the human. So, new insight includes bioassay
of human tissue and cytokines in the cell lines
which have very close precision to human.
Human tissue is used in areas such as in vitro/
in vivo, efficacy and toxicological pharmacology. But,
major difficulty is that, human tissue is difficult
to obtain, because of many ethical limitations and
difficult in standardization. Advantages of
human tissue over animal tissue are that, it can
provide a reliable and sensitive alternative. The
most commonly studied human tissue is vascular
tissue such as veins (commonly available from
removal of varicosities), cardiac blood vessels
(available after transplant program), large blood
vessels (available following major surgery, e.g.:
limb amputations or organ removal and small
cardiac vessels (can be obtained from the atrial
tip, removed to permit access to cannula during
cardiac surgery which requires bypass).
Recently, testing on human tissue validated many of the
pathways and proteins involved in the body functions such
as discovery of vascular endothelium as a major modulator
of vascular function and the discovery of the nitric oxide
pathway, the endothelin pathway, vascular endothelial
growth factor, and endothelium-derived hyperpolarizing
factor
Note: Tissue >2 mm exceeds the limits of diffusion and
which is maintained by continuous gassing, perfusion or
cooling at 4ºC.
Collection and storage of human tissue samples:
There are several problems in the collection of fresh
human tissue, namely cost, time and ethical issues
or even a conflict of interest between the surgeon,
pathologist and the researcher. The use of tissue or
storage varies with the quality of tissue and
experimental time, e.g. cardiac and brain tissues
have the shortest ‘experimental window’ and hence
it is difficult to assay.

Brain tissue: Being most important part of body, it
is most difficult tissue to obtain. The only possible
way out is the tumor tissue removal, which is
preferably used fresh due to shortest experimental
window.
Cardiac tissue: Vital part of body, hence one of the
difficult human tissue to obtain and only obtained
after some surgical procedure like cardiac resection,
transplant or postmortem, whereas, atrial tissues are
freely available from atrial appendage removal
during cardiac catheterization. Cardiac tissue
must be used freshly or may be stored in an
arrested state (4°C), to reduce metabolic activity
and avoid degradation. Preservation up to two
weeks (remain functional) can be achieved by
submerging tissue into physiological salt solution
(PSS) and oxygenating at 37.8°C. Experiments may
be carried out for maximum of 8-12 hours.
Lung tissues (bronchial rings or thin strips of
parenchymal tissue): Respiratory tissue is soft and
delicate hence, it gets easily damaged, so requires
skilled handling. Lung tissue is most commonly
obtained from tissue removed for the treatment of
lung cancer. Thin strips of parenchyma are used as
an isolated tissue preparation in an organ bath
for the measurement of contraction using a
modified strain gauge technique. Isolated bronchial
rings dissected within 24 hours of surgery retain
the ability to respond to nerve stimulation and
can generate an IgE-mediated sensitization of the
responses to bronchoconstrictors.
Vascular tissue (arterial tissue, intestine, stomach):
Arterial tissues may be obtained from small
arteries of skin, gut and skeletal muscle after
biopsy, whereas readily available from surgical
removal procedures such as tumor.
Objective
Technique should be sensitive and reliable
because it mimic in vivo environment equivalent
to human being.
Method
The choice of method depends on the level of
sensitivity required for the procedure.
Bioassay  67
Method of field stimulation: A measure of
sympathetic drive is assessed by using field-
stimulation. The strain gauge is preferred because
cannulation of small vessels is technically difficult
and therefore limits the number of arteries that
can be studied simultaneously. Blood vessel is
placed between two platinum or silver plates,
through which a small voltage is applied. The
resulting electrical field stimulates the
sympathetic nerves on the blood vessel to release
their catecholamine stores, producing a
measurable vasoconstriction response.
Method of pressure-flow system: A pressure-flow
system is an alternative methodology, where the
vascular tube is cannulated at each end, enabling
fluid to be perfused at physiological levels. The
pressure-flow systems offer up to ten times more
sensitivity than the strain gauge. In this assay, the
transducers measure pressure and flow rather
than force. The technique also uses imaging
microscopy to measure changes in the lumen
diameter that correlate directly with vaso-
constriction or relaxation.
Strain gauges also alter the natural orien-
tation and environment of the tested tissue,
whereas pressure-flow systems are much less
disruptive.
For example, cardiac tissue is cut into strips
that are placed in an organ bath and stimulated
using platinum or silver electrodes. Test drugs
are added, and the resulting changes in cardiac
muscle contractility are recorded.
Note: In experiment, students may use isolated arterial
strip or ring segment, uterus smooth muscle, lung slices
or tracheal rings, colon (mucosal layer) etc. in the human
tissues bioassay experiment.
Temperature condition: 37°-38°C
Effect observed: Contractility (In few tissues like isolated
lung slices or colon mucosal layer release of cytokines
and other signaling factors may study)
BIOASSAY OF CYTOKINES
Bioassay must generate a quantitatively
measurable parameter that reproducibly increases
with increasing cytokine concentration. Biological
assay (bioassay) of cytokines is important for
68  Practical Manual of Experimental and Clinical Pharmacology
biological characterization and potency
determination due to clinic as biotherapeutic
agents for malignant, infectious or autoimmune
diseases. The main reason of bioassay is that these
are biologically active proteins and cannot be
completely characterized by physiochemical
methods alone.
Objective
The parameters most often measured in cytokine
bioassays include increase/decrease in (live) cell
number, inhibition of virus replication, increase
in (cell) colony number, and increase in cell surface
antigen expression.
Cytokines for bioassay: Interleukins (ILs),
interferons (IFNs), hematopoietic (colony)
stimulating factors (CSFs), tumor necrosis factors
(TNFs), and certain (polypeptide) growth factors
(GFs) and many more.
Principle of Cytokines Bioassay
Principle of assaying cytokines depends on the
quantitative comparison of test sample to a
standard sample. Most proteins are relatively large
Flow Chart 2.2: An example of estimation of IL 6 cytokine by the ELISA method
Note: Washing is an important procedure during the assay, hence protocol of washing should be followed properly
molecules with complex structure and molecular
composition, which cannot be completely deter-
mined by physical and chemical methods alone.
Principle of bioassay is also applied when two
or more preparations of an individual protein are
being compared, they should be identical in the
bioassay. Repeatability, intermediate precision,
and reproducibility are the main advantages in
conducting cytokine bioassay. It helps greatly in
that a bioassay has the appropriate specificity,
i.e. the measured parameter is exclusively related
to the concentration of the biologically active
protein.
Note:
• Reproducibility is always essential for
analytical procedure. This is best achieved by
identifying and minimizing possible known
variables.
• Presence of other substances such as active
impurities/contaminants may interfere with
results.
Procedure for Cytokines Bioassay
(Flow Chart 2.2)
Currently, there is large number of cytokines,
mostly rDNA-derived, which are available
 Bioassay  69

commercially for several therapeutic purposes. Log dose conversion

A major problem with clinical-grade cytokine s 1 = 0.2 × 10–7= log (0.2 × 10–7)
products from an assay point of view is that they = log (0.2) + (–7) log 10 = –0.69897 + (–7) x 1
are very highly potent. Their potencies often = –7.69897
exceed 1.0 million IU (MIU) or greater and s 2 = 0.4 × 10–7 = log (0.4 × 10–7)
therefore they require considerable dilution = log (0.4) + (–7) log 10 = –0.39794 + (–7) × 1
before titration in bioassays. ‘Off-plate’ dilutions = –7.39794
are very important step which often involve t1 = 0.2 × 10-3 = log (0.2 x 10–3)
several 10-fold dilution steps. Undiluted = log (0.2) + (–3) log 10 = –0.69897 + (–3) × 1
constituents of the samples may interfere with = –3.69897
bioassay performance.
t2 = 0.4 × 10–3= log (0.4 × 10–3)
Note: Dilution at each stage carried out with = log (0.4) + (–3) log 10 = –0.39794 + (–3) × 1
micropipettes calibrated for precise delivery of the = –3.39794
dilution volume.
S1 S2 T1 T2
EXAMPLE OF PERFORMING A SET OF BIOASSAY S2 T1 T2 S1
Latin square design
T1 T2 S1 S2
Question: 1 T2 S1 S2 T1

Write the Protocol and undertake the 4-point

bioassay experiment to determine the
concentration of histamine in the given sample in
an in vitro tissue of your preference.
Answer:
Concentrate on the key words in the question
which is underlined.
Protocol: Means you have to write about the
principle, aim and objective, materials and
method, procedure, observation and result with
inference.
4 point bioassay: Write down the principle of
4- point bioassay, its study design and procedure.
Note: Dilution factor of the drug should be considered
according to the volume of inner organ bath such as
10 ml, 20 ml, 50 ml or 100 ml.
For example: If the drug concentration is (s1= 0.2 ×
10–7) and volume inner organ bath is 10 ml, then the
concentration of the drug to which tissue comes in
contact will be (s1= 0.2 × 10–8), so for the calculation
(s1 = 0.2 × 10–8) should be taken and all the drug
concentration changed accordingly.
M =
=

Histamine: The drug given in the experiment is

09 + 08
histamine, hence, as the rule in the experiment the = 0.30103
tissue selection will depend on the sensitivity of 18.50 17.50
the tissue which is most sensitive to histamine. 17
Therefore, your tissue of choice should be Guinea = 0.30103 = 0.142153
36
Pig ileum.
The response taken here is approx value (not Conc. of unknown
to scale; made for understanding purpose) s1
Responses (mm) Mean = antilog M
t1
S1 34 38 33 32 34.25 7
S2 52 57 49 53 52.75 0.2 10
= antilog (0.142153)
T1 26 29 23 27 26.25 0.2 10 3

T2 45 43 44 43 43.75
= “x”
70  Practical Manual of Experimental and Clinical Pharmacology

Hence, the given solution is the “x” times more
potent than standard.
Representation of plotted DRC is shown in
Figure 2.27 and representation of results is shown
in Figure 2.31.
Question: 2
To determine the unknown concentration of given
acetylcholine (ACh) by using suitable tissue.
Answer
Step 1: Start with aim then write down the
principle of bioassay, procedure and if possible
make the flow diagram for the procedure
(explained briefly, see text in Chapter; Flow Chart
2.1).
Step 2: Commonly all tissues are sensitive to ACh,
but select the tissue which is reliable, sensitive
and durable.
Tissue: Rat ileum (best tissue is leech dorsal
muscle or frog rectus abdominis muscle) (Subject
to availability in laboratory).
Step 3: Selection of PSS: Tyrode
Step 4: It is fast contracting hence, magnification
should be low
Magnification: 7x
Step 5: Adjust temperature between 35-37°C.
Step 6: Prepare the tyrode and collect the tissue.
Keep tissue in continuous slow oxygenation.
Step 7: Balance the lever and then tie the tissue
and adjust the weight by counterweight at other
side of fulcrum.
Step 8: Leave the tissue in bath for relaxation for
at least 30 - 45 min, meanwhile change the PSS 3-
4 times during the relaxation (chances of pH
change after prolonged oxygenation).

Aim of the experiment: To determine the unknown concentration of histamine in the given sample in an in vitro tissue
of your preference.
Tissue selected: Guinea pig ileum (Highly sensitive tissue for histamine)
PSS: Tyrode
Dose cycle: 3 min
Temp: 35-37°C
Level: Isotonic (Frontal writing lever)
Magnification: 7x
Inner organ bath volume: 10 ml
Dose cycle: 3 min

Fig. 2.27: Presentation of kymogram after the experiment is over. Write down every variables used in the experiment
 Bioassay  71

Step 9: Prepare the standard drug and select the
experimental design (3-point, 4-point or others).
Step 10: 3-point is selected due to less time
consumption than 4-point and reduced variability
than bracketing and matching assay.
Response recording (Arbitrary value;
not to scale)
For example: If the drug concentration is (S1= 0.8
× 10–7) and volume inner organ bath is 10 ml, then the
concentration of the drug to which tissue come in contact
will be (S1= 0.8 X 10-8), so for the calculation (S1= 0.8
X 10-8) should be taken and all the concentration will
be changed accordingly.

Responses (mm) Mean

= –0.31596 × 0.39794
S1 20 23 21 21.33
S2 27 27 29 27.66 = 0.12573
T 18 21 19 19.33 Concentration of unknown
s1 = 0.8 × 10–7 = log (0.8) + (–7)log 10
= –0.09691 – 7 = –7.09691 =
s2 = 0.2 × 10–6 = log (0.2) + (–6)log10
= –0.69897 – 6 = –6.69897
t = 0.4 × 10–3 = log (0.4) + (–3) log10 = Antilog (0.12573) = “y”
= –0.39794 – 3 = –3.39794
Concentration of the unknown compound is
Note: Dilution factor of the drug should be “y” times potent that of standard.
considered according to the volume of inner organ Representation of plotted DRC is shown in
bath such as 10 ml, 20 ml, 50 ml or 100 ml. Figure 2.28.

Fig. 2.28: Presentation of traced 3-point assay by using rat ileum

72  Practical Manual of Experimental and Clinical Pharmacology
Question 3
To determine unknown concentration of antagonist
atropine using ACh as an agonist using Rat ileum
preparation by pA2 method
Answer
Step 1: Write down the principle of bioassay,
procedure and if possible make the flow diagram
for the procedure (Flow Chart 2.1).
Step 2: Selection of tissue—Most of the tissue are
sensitive to ACh, but select the tissue which is
reliable, sensitive and durable.
Tissue: Rat ileum
Step 3: Selection of PSS: Tyrode
Step 4: Tissue is fast contracting hence,
magnification should be low
Magnification: 7x
Step 5: Temperature is maintained at 35-37°C
Step 6: Prepare the tyrode and collect the tissue.
Keep tissue in continuous slow oxygenation.
Step 7: Balance the lever and then tie the tissue
and adjust the lever by counterweight at other side
of fulcrum.
Step 8: Leave the tissue in bath for relaxation for
at least 30 - 45 min, meanwhile change the PSS 3-
Fig. 2.29: Response plot for pA2 determination
4 times during the relaxation (chances of pH
change after prolonged oxygenation).
Step 9: Select the dose ‘2R’ from the DRC of
standard drug.
Step 10: Then, take the response of ‘2R’; at least
three responses should be taken
(Aim of the experiment is to find out the dose of
antagonist which reduces the response of “2R” to its
half, i.e. “R”)
Step 11: Add the 10 times lower concentration of
antagonist and wait for 5-10 min, then add agonist
i.e. 2R; at least three responses should be taken
Step 12: Wash out the antagonist, and maintain
the dose cycle
Step 13: Repeat the step 11 until the response of
the ‘2R’ is about equal to ‘R’
Step 14: Plot the response in to the percentage
response verses -log (antagonist) dose curve
Representation of plotted DRC is shown in
Figure 2.29 and Calculation is shown in Figure
2.30.
Note: Fiducial limit (confedence limit) is
calculated for observe the variability instead of
relative potency among the response obtained
by test and standard compound.
 Bioassay  73

DRC response of agonist Antagonist concentration used is 10–10

Reponse in mm % Response of maximal response Antagonist logdose Response
12 44.44444 concentration
16 59.25926 0.2 × 10–10 log (0.2) + (–10) log 10 17
20 74.07407 = –0.69897 – 10 = –10.69897 20
24 88.88889 2R 23
27 100 0.4 × 10–10 log (0.4) – 10 = –0.39794 – 10 10
* If the 2R is selected as the 24 mm response, then = –10.39794 16
targeted ‘R’ response should be ‘12 mm 20
24
0.3 × 10–10 log (0.3) – 10 = –0.52288 – 8 12
–log (antagonist) Response % Response to 2R = –10.52288 16
10.70 17 41.66667 22
10.40 10 83.33333
10.52 12 70.83333

Fig. 2.30: Calculation and determination of pA2 ; Inference: Write down about the finding of the study [Value of –log (antagonist)]

Figs 2.31A to D: Representation of bioassay result by graphical methods. The student can represent their
findings by any of the graph given above
74  Practical Manual of Experimental and Clinical Pharmacology
SUGGESTED READING
Bioassay
1. Allen DD, Caviedes R, Cárdenas AM, Shimahara T,
Segura-Aguilar J, Caviedes PA. Cell lines as in vitro
models for drug screening and toxicity studies. Drug
Dev Ind Pharm 2005;31(8):757-68.
2. Arunlakshana O, Schild H O. Some quantitative uses
of drug antagonists. Brit J Pharmacol 1959;14:48-
50.
3. Bliss CI, Cattell McK. Biological assay. Annual review
of physiology 1943;5:479-539.
4. Bliss CI. The design of biological assays. Annals of
the New York Academy of Sciences 1950;52:877-88.
5. Box GEP, Hay WA. A statistical design for the
efficient removal of trends occurring in a comparative
experiment with an application in biological assay.
Biometrics 1953;9:304-19.
6. Burn JH. The errors of biological assay. Physiological
Reviews 1930;10:146-69.
7. Butcher E C, Berg E L, Kunkel E J. Systems biology in
drug discovery. Nature Biotechnol 2004;22:1253-59.
8. Cheng Hsien C. The power issue: Determination of
KB or Ki from IC50. A closer look at the Cheng-
Prusoff equation, the Schild plot and related power
equations. Journal of Pharmacological and
Toxicological Methods 2001;46:61-71.
9. DeBeer E J. The calculation of biological assay results
by the graphical methods. The all- or-none type of
response. J Pharmacol Exp Ther 1945;85:1-13.
10. Eugene M Laska, Morris J Meisner. Statistical
methods and applications of bioassay. Ann. Rev.
Pharmacol. Toxicol 1987;27:385-97.
11. Finney DJ. The principle of bioassay. Journal of the
Royal Statistical Society 1947a;S9:46-91.
12. Finney MJ, Karlsson JA, Persson CG. Effects of
bronchoconstrictors and bronchodilators on a novel
human small airway preparation. Br J Pharmacol
1985;85(1):29-36.
13. Ghelani AM, Holroyde MC, Sheard P. Response of
human isolated bronchial and lung parenchymal
strips to SRS-A and other mediators of asthmatic
bronchospasm. Br J Pharmacol 1980;71(1):107-12.
14. Ghosh K, Kowal D, Dawson L, A and Tasse R. Design
and models for estimating antagonist potency (pA2,
Kd and IC50) following the detection of antagonism
observed in the presence of intrinsic activity.
Neuropharmacology 1999;38(3):361-73.
15. Halpern W, Mulvany MJ, Warshaw DM. Mechanical
properties of smooth muscle cells in the walls of
arterial resistance vessels. J Physiol 1978;275:85-101.
16. Hulsmann AR, de Jongste JC. Studies of human
airways in vitro: A review of the methodology. J
Pharmacol Toxicol Methods 1993;30(3):117-32.
17. Jung SP, Siegrist B, Wang YZ, Wade MR, Anthony
CT, Hornick C, etc. Effect of human Angiostatin
protein on human angiogenesis in vitro. Angiogenesis
2003;6(3):233-40.
18. Lareau S, Moore R, Mainwood GW, Labow RS, Keon
WJ, Deslauriers R. An NMR probe to study function
and metabolism simultaneously in isolated human
cardiac tissue. Magn Reson Med 1999;20(2):312-18.
19. National Bioethics Advisory Commission, (2001)
Ethical and Policy Issues in Research Involving
Human Subjects. NBAC In: http://www.
bioethics.gov
20. Phung MW, Dass CR. In vitro and in vivo assays for
angiogenesis-modulating drug discovery and
development. J Pharm Pharmacol 2006;58(2):153-
60.
21. Schild H O. pAx a new scale for the measurement of
drug antagonisum. Br J Pharmac 1947;2:184-206.
22. Wadhwa M, Bird C, Dilger P, Mire-Sluis T, Thorpe
R, Quantitative biological assays for individual
cytokines, in: F. Balkwill (Ed.), Cytokine Cell Biology:
A Practical Approach, Oxford University Press,
Oxford, UK 2000;207-39.
23. WHO, Recommendations for the preparation,
characterization and establishment of International
biological reference materials. WHO/BS/04.1995.
www.who.int/biologicals.
Bioassay of Antagonism
1. Arunlakshana O, Schild H O. Some quantitative uses
of drug antagonists. Br J Pharmacol 1959;14:
48-50.
2. Gaddum JH. Theories of drug antagonism.
Pharmacol Rev 1957;9:211-18.
3. Ghosh K, Kowal D, Dawson L A, Tasse R. Design
and models for estimating antagonist potency (pA2,
Kd and IC50) following the detection of antagonism
observed in the presence of intrinsic activity.
Neuropharmacology 1999;38(30):361-73.
4. Jones RL, Wise1 H, Clark R, Whiting RL, Bley KR.
Investigation of the prostacyclin (IP) receptor
antagonist RO1138452 on isolated blood vessel and
platelet preparations. Br J Pharmacol 2006;149:
110-20

5. Kunchandy J, Kulkarni SK. Apparent pA2 estimation
of benzodiazepine receptor antagonists. Methods
Find Exp Clin Pharmacol 1986;8:553-55.
6. Lazareno S, Birdsall NJM. Estimation of antagonist
Kb from the inhibition curves in functional
experiments: Alternative to the Cheng-Prusoff
equation. Trends Pharmacol Sci 1993b;14:37-239.
7. Lazareno S, Birdsall NJM. Estimation of competitive
antagonist affinity from functional inhibition curves
using the Gaddum, Schild and Cheng-Prusoff
equations. Br J Pharmacol 1993a;109:1110-19.
8. Martin JR, Jenck F, Moreau JL. Comparison of
benzodiazepine receptor ligands with partial
agonistic, antagonistic or partial inverse agonistic
properties in precipitating withdrawal in squirrel
monkeys. J Pharmacol Exp Ther 1995;275:405-11.
9. Schild HO. Drug antagonism and pA2. Pharmacol
Rev 1957;9:242-46.
Bioassay  75
10. Schild HO. pA, a new scale for the measurement of
drug antagonisum. Br J Pharmacol 1947;2:184-206.
11. Shannon HE, Cone EJ, Gorodetsky CW. Morphine-
like discriminative stimulus effects of buprenorphine
and demethoxybuprenorphine in rats: Quantitative
antagonism by naloxone. J Pharmacol Exp Ther
1984a;229:768-74.
12. Spealman RD. Discriminative-stimulus effects of
midazolam in squirrel monkeys: Comparison with
other drugs and antagonism by Ro 15-1788. J
Pharmacol Exp Ther 1985;235:456-62.
13. Takemori AE, Kupferberg HJ, Miller JW. Quantitative
studies of the antagonism of morphine by nalorphine
and naloxone. J Pharmacol Exp Ther 1969;169(1):
39-45.
14. Waud DR. Analysis of dose-response curves. In: Daniel
EE, Paton, D.M. (Eds.), Methods in Pharmacology,
vol. 3. Plenum Press, New York, 1975, 471-506.
 Commonly Used Instruments
3 in Pharmacology Laboratory

Kymograph (Sherrington-Starling
Kymograph) (Fig. 3.1)
This is the basic instrument used to obtain a
graphical amplified measurable response of
a muscle or tissue (contraction/relaxation)
against a given concentration of drug or stimuli,
hence the resolution of the tissue response is
increased.
It consists of two important parts, i.e. a motor
box and drum. Motor operates drum in a variable
speed according to the need of the experiment or
the tissue attached to the recording lever.
Additionally, speed setting lever or variable speed
clutch lever, spindle and On/Off switch is
attached to motor box.
On/Off switch: This is the main power supply
board and it should remain ‘ON’ while doing the
practical.
Speed setting lever or Variable speed lever: This
lever is made to control the speed of the drum.
This ranges from 0.12-0.25 mm/sec to 320-640
mm/sec, i.e. slow or fast. The speed of the drum
mainly depends on the type of the tissue and dose Fig. 3.1: Kymograph and its different parts
cycle used.
Clutch lever: It disengages or engages the gear. to the electrically amplified impulse to record the
response. Its sensitivity is as high as 5 micro volts
Spindle and screw: Rod like structure which holds
per mm. The complete system is designed with
the drum in the vertical position, where stylus
couplers, matching transducers and pick-ups, and
touches the drum. Height of the drum can be adjusted
main console which includes the chart drive, pen
with lift screw attached at the top of the spindle.
recording system, the amplifier and coupler housing.
Student’s Physiograph (Fig. 3.2) Standard transducer for student physiograph: A
Physiograph is an electronic stimulator which is transducer is an electronic device which is
sensitive enough to convert the mechanical energy prompted by energy from one system (mechanical)
 Commonly Used Instruments in Pharmacology Laboratory  77
Fig. 3.2: Student’s physiograph
(For color version see Plate 1)
and converts energy to another. There are several
types of transducers such as pressure transducer,
respiration belt transducer, force transducer, pulse
transducer, isotonic transducer, etc. which are
very commonly used in the biomedical research.
Coupler is the main device which is attached
to the transducer, which should be compatible
with transducer and controls the input impulse.
There are several types of couplers such as strain
gauge coupler which is mainly involved in the
experiment of arterial and venous pulse, blood
pressure recording, BMR, spirometry
experiment, in vitro frog heart preparation,
isolated tissues, finger movement, etc. Second
is biopotential coupler used in the recording of
ECG, EEG, EMG, etc. and other less commonly
used are pulse/respiration coupler, temperature
coupler, etc.
So, the application of Students physiograph is in
several biomedical research fields such as
recording in cardiovascular physiology (arterial
and venous pulse, blood pressure in dog/rabbit,
ECG clinical, effect of vagal stimulation and drugs
on perfused isolated frog heart, etc.),
neuromuscular physiology (muscle twist, strength
of stimulus, fatigue, isometric contraction, etc.),
experimental respiratory analysis (respiratory
movement, lung volumes, tidal volume, IRV, ERV,
VC, IC, BMR, etc.), human experiment on ECG
clinical, EMG, EEG, etc. and to see the effect of
drugs on other in vitro experiments like, isolated
intestine, isolated uterus, sciatic nerve, etc.
Analgesiometer (Figs 3.3A and B)
The instrument works on the principle of
observing the pain threshold in the rodents before
and after the drug administration, so useful for
differentiating between centrally acting morphine-
like analgesics and non-opiate analgesics. This is
performed mainly to identify any pain stimulus
threshold in rodents against a radiant heat and to
screen analgesic drug by increasing pain
threshold. Hence, the radiant heat method is being
used for evaluation of systemic analgesic activity
of a drug. The end-point of the experiment is considered
as escape reaction which is considered to be controlled
by brain. There are two type of analgesiometer used
in the animal experiment namely tail flick
analgesiometer and hotplate analgesiometer.
Physiologically, tail flick is mainly mediated as a
spinal reflex whereas hotplate paw withdrawal
is mainly mediated by the brain. Therefore, the
observation of the escape reaction can be regarded
as a true assessment of the influence of the drug
acting on the brain.
Instrument and its Parts
A. Animal is placed into restrainer and leaving
the tail exposed outside the restrainer. Clean
the tail with the help of cotton soaked in water
or ethanol. Then leave the tail for drying and
also to familiarize with the restrainer. Then,
keep on the “tail flick analgesiometer”. 1/3rd
tail proximally left due to the thick and
keratinized skin and then keep tail on the place
made for tail above hot wire (made up of
nichrome wire) of the analgesiometer. The time
of tail flick is measured and recorded. The cut-
off time is set-up 15-20 sec for mice whereas in
the case of rat, cut off time is 20-30 sec to avoid
any further injury to the tail (Fig. 3.3A).
B. Instrument consists of an electrically heated
surface (made up of iron, aluminum or copper)
whose temperature is maintained by the
thermostat ‘Knob’ at 55° to 56 °C. After
maintaining the temperature mice are placed
on the hotplate and observed for either paw
licking or jumping reaction. The reaction time
78  Practical Manual of Experimental and Clinical Pharmacology

Figs 3.3A and B: (A) Tail flick analgesiometer: Showing on/off switch for power on/off, water inlet and outlet to maintain the
normal temperature surrounding the hot-wire and current control knob which control the hotness of nichrome wire.
(B) Eddy’s Hotplate analgesiometer: Showing on/off switch, Temperature control knob which can be adjusted on the
desired temperature (55-56°C), Thermometer is placed at the corner place for continuous observation of the temperature,
Hotplate on which the animal is kept for observation and lid cover of hotplate

is recorded by a stop-watch. Repeated reading
is taken at 20, 60, and 90 minutes after the
drug administration. The cut-off time is set-up
15-20 sec for mice whereas in the case of rat
cut-off time is 20-30 sec to avoid any further
injury to the paw (Fig. 3.3B).
Practical Application
Mainly used in the screening of centrally acting
analgesics. However, screening of local
anesthetics or screening of muscle relaxant may
also be performed.
Note: In screening of the analgesic drugs, result may
misinterpreted with false positive result due to muscle
relaxant or local anesthetic drugs
jerks as well as face and forelimb clonus that may
progress to rearing and falling down (such ictal
behavior is referred to as limbic seizures) whereas,
convulsions mediated by the brainstem involve
tonic-clonic convulsions. Brainstem seizures also
may occur independently of forebrain neural
substrates once generalized tonic-clonic
convulsions are induced by transauricular or
corneal electroshock (ES) in rats. The Seizure
severity score followed for screening the
anticonvulsant is evaluated by:
0 = no seizure; 1 = forelimb extension without
hind limb extension; 2 = complete forelimb
extension and partial hind limb extension,
3 = complete (parallel to the tail) tonic hind limb
extension (THLE), 4 = postictal depression.

Instrument and its Parts

Maximal Electroshock Seizure (MES)
Convulsiometer (Fig. 3.4)
This instrument is used to induce convulsions
experimentally which are hypothesized to
originate from the forebrain or brainstem.
Convulsions mediated by the forebrain involve
clonic spasms, behavioral immobility, myoclonic
Electroshock is applied through a pair of ear-clip
electrodes or corneal electrode, using a sinusoidal
maximal current of 150 mA, 50 Hz for 0.2 sec for
rat and 12 mA, 50 Hz for 0.2 sec for mice. Animal
is housed in acrylic cage, 1 min before the current
application, and observed for an additional 2 min
period. Recordings may be videotaped and
 Commonly Used Instruments in Pharmacology Laboratory  79
Fig. 3.4: Convulsiometer (For color version see Plate 1)
watched for all stages of seizure. Its only
application is the screening, if the antiepileptic
drug acting on tonic-clonic convulsion and animal
should be screened one week prior to the
experimental day.
Elevated Plus Maze (Fig. 3.5)
This instrument is made for a novel test for the
selective identification of ‘anxiogenic and
anxiolytic’ drug effects in rodents. It is also used
to evaluate the learning and memory in rodents
in presence or absence of a drug.
The plus maze apparatus consists of two open
(16 × 5 cm for mice and 50 × 10 cm for the rats) and
two closed arms (16 × 5 × 12 cm for mice and 50 ×
10 × 40 cm for rats), and an open roof with the
entire maze elevated (25 cm for mice and 50 cm for
rats) from the floor. The animals are placed
individually at the center of the elevated plus maze
with their head facing the open arm. During the 5
minutes test the preference of the animal for the
first entry, the numbers of entries into the open
Fig. 3.5: Plus Maze apparatus for the rat (50 × 10 × 40 cm).
The closed arm painted black inside to evoke the night like
situation. All the movements of rat are videotaped in dim
lighted calm room
and closed arms entries reflect the relative safety
of closed arms compared with the relative
fearfulness of open arms. Anxiolytics would be
expected to increase the frequency of entries and
time spent into open arms. It is also a good
screening method for learning and memory in
rodents.
Morris Water Maze (Fig. 3.6)
This instrument is designed to evaluate learning
and memory in rodents and to evaluate effect of
drugs. The test apparatus consists of a circular
fiberglass tank (130 cm in diameter, 50 cm in depth;
dimension for rat). The pool is filled to a height of
30 cm with water at room temperature, 21-22°C.
The pool is divided into four vertical quadrants
(Q1, Q2, Q3 and Q4) of equal surface area. A
transparent escape platform (10 cm in diameter, 29
cm in height) is placed in a fixed location in the
tank in the center of one of a quadrant, 1 cm below
the water surface. The platform is not visible from
just above water level. Many extra maze cues
surrounding the maze are available for the rats to
use in locating the escape platform. The maximum
cut-off time for learning and memory is 300s.
Precaution in instrument use: Animals that do not
float on the water and search for the platform
should be excluded from the study.
Fig. 3.6: Morris water maze for the rat
Passive Avoidance Test (Fig. 3.7)
Apparatus consist of Shuttle-box divided into an
illuminated compartment and a dark compartment
of the same size by a wall with a guillotine door.
80  Practical Manual of Experimental and Clinical Pharmacology

(TCP) method. This method is always preferred

Fig. 3.7: Shuttle-box for passive avoidance test
Adaptation, Training Trial and Retention Test
In the experimental session, each animal is trained
to adapt to the step through passive avoidance
apparatus. The animal is put into the illuminated
compartment. After 10 sec, the door between these
two boxes is opened and the animal is allowed to
move into the dark compartment freely. The latency
to leave the illuminated compartment is recorded.
Two hours after the adaptation trial, the animal is
again put into the illuminated compartment. The
learning trial is similar to the adaptation trial
except that the door is closed as soon as the animal
stepped into the dark compartment and an
inescapable foot-shock (0.2 mA, 2s for rat 0.15 mA,
2s for mice) is delivered through the grid floor.
The retention of passive avoidance response is
measured 1 and 7 days after the learning trial.
Each animal is again put into the illuminated
compartment and the latency of re-enter the dark
compartment is recorded. No foot-shock is
delivered while performing the retention test. The
maximum cut-off time for step-through latency is
300s.
BP Apparatus (Fig. 3.8)
In a cardiovascular experiment, measurement of
arterial pressure (AP) and heart rate (HR) in
rodents is done mainly by the tail cuff pressure
over the invasive arterial catheter method whereas
this method is indirect method of measuring
systolic blood pressure.
In this type of set-up, BP and HR recording
can be performed in anesthetized rodents or
heated on a heating pad to increase the blood flow
to the tail. Preferably, animals are placed in an
incubator or heating pad at 37°C for about 10 min
before the AP recording. Body warming increases
blood flow in the tail. A cuff with a pneumatic
pulse sensor is attached to the tail and allowed to
habituate to this procedure for 7 days before
experiment.
The systolic blood pressure (SBP) is recorded
for at least three consecutive cycles of inflation/
deflation on each rat. For calculating SBP, mean
of the last three recordings is taken but the
difference among reading should not be more than
10 mm Hg.
Instrument and its Parts
Fig. 3.8: Non-invasive BP apparatus
Electrolyte Analyzer (Fig. 3.9)
This medical analyzer is easy to use and provides
fast sample analysis. The instrument is for
performing electrolytic analysis from whole blood
as well as plasma, urine dialysate, serum or
aqueous standards. It has the capability to provide
accurate and consistent results within one minute.
This analyzer is used for the sodium, potassium,
chloride, ionized calcium and lithium.
 Commonly Used Instruments in Pharmacology Laboratory  81
Fig. 3.9: Electrolyte analyzer
This instrument is based on the enhanced
microprocessor-based technology and is easy to
use due to operational flexibility. Hence, it is based
on the principle that each electrolyte has an ion
selective membrane that undergoes a specific
reaction with the respective ions contained in the
sample being analyzed. Membrane used here is
basically an ion exchanger, reacting to the
electrical charges of ions being analyzed and
causing change in membrane potential or
measuring voltage. The difference between two
potential values on either side is determined by
the galvanic measuring chain within the electrode.
Ultimately, ion concentration in the unknown
sample is determined by the using a calibration
curve determined by measuring points of
standard solutions with the reference known
concentration.
Body Plethysmograph (Fig. 3.10)
Body plethysmograph is an instrument for
measuring changes in volume of whole body in
which signal represents the sum of two flows,
one is nasal flow (movement of air into and out
of the nose) and another is thoracic flow (rise
and fall of chest cavity). The animal makes
respiratory efforts against the closed shutter,
causing their chest volume to expand and
decompressing the air in their lungs. This is based
on the principle of changes of chest volume
Fig. 3.10: Body plethysmograph
which alter the internal box volume (increase in
chest volume slightly reduces the box volume)
and thus slightly increases the pressure in the
box.
The body plethysmograph is a two cham-
bered acrylic box. The animal chamber holds
the animal in position and is made up of four
parts: Posterior cylinder is designed to fit the
various sizes of rodents with slanted anterior
end. It house rodents comfortably for the experi-
ment and also fixes the animal for unwanted
movements. An anterior cylinder fits tightly over
the posterior cylinder. Its anterior end is
slanting downwards and has a conical opening
in the middle. When the anterior chamber is
pushed over the posterior chamber, the snout of
the animal enters the cone and the head gets
fixed. Neck plate has two plates with adjustable
hole to fit neck of the different rodents. Upper
plate is removable and adjusted with the screw
whereas the lower plate is fixed permanently.
A nares cap is screwed over the protruding end
of the anterior piece. This cap has a hole on front
to hold the pneumotachograph and another on
its top for air or aerosol. This animal chamber is
placed inside an outer box of about 15 liter
capacity (size may vary). This box contains six
openings, three for transmitting airflow and box
pressure signals, one for passing air or aerosol,
one for exhaust and one for pressure leakage.
 Sophisticated Instruments
and Techniques Used in
4 Pharmacology Laboratory

Centrifuge Machine whereas, effective mass of sedimenting particles

The most commonly used instrument in the
biomedical research to prepare or separate a
sample liquid/semiliquid for the further analysis.
Centrifuge is the instrument which separates solid
or large particles from liquid on the basis of
density, shape or size.
Centrifuge is based on the principle of laws of
inertia. The principle of the centrifuge is governed
by centrifugal force when it rotates at the high
speed towards the axis of the rotor (Figs 4.1A and
B). Hence, centrifugal force accelerates the particles
to separate and sediments the larger particles
(macroparticles) first at the bottom then the
smaller particles (microparticle) (Figs 4.2A and
B). Therefore, the centrifugal force (F) is defined
and denoted by the formula,
F = mω
ωr ω2
where, F = centrifugal force intensity
m = effective mass of sedimenting
particle
ω = angular velocity of rotation (rad/
sec)
r = distance of the migrating particle
from the central axis of rotation
(m) is defined by the actual mass (ma) minus the
correction factor for the weight of water displaced.
The other factor which influence the
centrifugation is gravitational force which
depends on the velocity of the rotation and the
distance of the particle from the axis of the rotation,
hence the relative centrifugal force (RCF) is
denoted by
RCF = 11.19 × 10–6 R2 (r)
where, “R” = revolution per minute (rpm) and
“r”= distance of the particle from the axis of the
rotation
Application of Centrifuge
Low-speed centrifuge is used for whole cells i.e.
separating red blood cells or platelets from whole
...(1)
blood samples
Other like protein precipitation, tissue culture,
subcellular isolation (i.e., ribosomes, Golgi bodies,
etc.), plasmid preparations, and DNA/RNA
separations are important.
Ultracentrifuge is used in the separation of
virus particles, DNA, protein, or RNA
fractionations, or lipoprotein flotation

Table 4.1: Different types of centrifuge and their properties

Type of centrifuge Speed (rpm) Refrigeration RCF (g) Centrifuge tube used
Density gradient

Material Size
Low speed 1-6000 Some 6000 Glass/Plastic 10-50 ml
High speed 1000-25,000 All (near 4°C) 50,000 Plastic 0.5-2 ml
Ultracentrifuge 20-80,000 All 6,00,000 Plastic 0.25-2 ml
 Sophisticated Instruments and Techniques Used in Pharmacology Laboratory  83

Components of Centrifuge

Figs 4.1A and B: (A) Showing the position of angle of rotation tube with axis of rotation axis. Its RCF value variation
with the increasing r/r’from axis of rotation (B) Showing the position of tube on the either side of axis of rotation

Figs 4.2A and B: (A) Showing the process of fixed angle rotor centrifuge and at time its separation in the two layers at
the rest; (B) Process of vertical rotor centrifuge and separation into two layer at the rest
84  Practical Manual of Experimental and Clinical Pharmacology
Fig. 4.3: Principle of taking the supernatant from the test
tube, after centrifugation micropipette should not touch the
precipitate at the bottom and make test tube slightly slanted,
so it make easy to remove the supernatant
In large scale may use for whole cell harvest
step in the processing of large volumes of cultures
from bioreactors.
Note: Cold centrifugation is done in high and ultrahigh
speed centrifuge to protect the proteins or other biological
products from the heat generated during the process.
Polymerase Chain Reaction (PCR)
Polymerase chain reaction (PCR) is a tool for
researchers which produces millions of copies of
a specific DNA sequence in short duration.
Polymerase is one of the important enzyme used
in the reaction and it is considered to be chain
reaction which processes exponentially.
PCR was developed by Kary Mullis in 1984
and won Nobel Prize in Chemistry in 1993. The
PCR works on the principle of amplification of a
targeted single strand DNA exponentially. The
amplification depends on the reaction cycle and
the cycle is nothing but the different heating cycle.
Further, total process is classified into three cycles
namely, (1) Denaturation of DNA template and
primer, (2) Annealing of single stranded DNA to
primer, and (3) Extension and Elongation of DNA
strand (Fig. 4.4).
The overview of experimental protocol to run
PCR is given in Flow Chart 4.1 and 4.2 as an
example.
Salient Feature of PCR
Primary application is in vitro amplification of
specific target DNA sequences and being highly
sensitivity. But, the limitation of the process is, it
requires highly skilled person, extremely liable to
contamination and it may not easy to set up a
quantitative assay.
High Performance Liquid Chromatography
(HPLC)
High Performance Liquid Chromatography
(HPLC) is one efficient chromatography which is
most widely used as a analytical technique. In
biomedical research, HPLC is a qualitative and
quantitative process. Russian botanist, Mikhail S
Tswett (1872-1920) described the liquid
chromatography. Chromatography is derived from
the Greek words chroma, meaning color, and
graph, meaning writing. HPLC is based on the
principle of partition differentiation between the
moving solvent (mobile phase), and the column
packed particles (stationary phase) to separate a
test substance. Mainly, two principal factors that
determine the overall separation power are
mechanical separation power (produced by the
column length, particle size, and packed-bed
uniformity) and chemical separation power
(produced by the packing material and the mobile
phase). Hence, degree of separation of two
compounds is known as chromatographic
resolution which depends on high flow pressure
through packed particle size and length of the
column. Smaller particle size for stationary phase
(<10 microns) is required to improve separation
power. However, smaller particles produce
greater resistance to flow, so need higher pressures
to create the desired solvent flow rate. The pressure
required to generate the flow in packed columns
ranges from 500 psi to 6,000 psi.
HPLC has the ability to separate, identify, and
quantitate the compounds that are present in any
sample that can be dissolved in a liquid. So, overall
performance of HPLC depends on the selection of
column and the detector.
 Sophisticated Instruments and Techniques Used in Pharmacology Laboratory  85

Flow Diagram of Experimental Procedure

Flow Chart 4.1: For PCR Run

Flow Chart 4.2: For nucleotide sequencing

Note: This is just a brief introduction to the PCR run and sequencing procedure for beginners. In each step, there are
several factors which have to be considered during the process such as temperature, concentration of PCR mixture,
agarose gel, primer designing, etc.
86  Practical Manual of Experimental and Clinical Pharmacology

Fig. 4.4: Steps of polymerase chain reaction (PCR);

Time of extension and elongation is depend on selection of DNA polymerase

Note: Buffer solution: Makes environment favorable to DNA polymerase (It may contains divalent like Mg2+ and Mn2+ and
monovalent like K+ cations)

Basis of Column Selection
Column selection is an important part in the
HPLC, which has whole influence on the process.
So, the selection of column depends on the several
factors which are molecular weight of the sample
(Exclusion limit molecular weight), Ability to
dissolve the sample (Applicable to packing
materials) and molecular weight distribution
(Range of calibration curve).
Column inner diameters and packing
material particle sizes play an important role in
the efficiency of particle. HPLC columns range
from 20 to 500 mm in length (L) and 1 to 100 mm
in internal diameter (id). Sample load is
proportional to the column cross-sectional area.
It is necessary to select a column inner diameter
suitable for the sample load. Smaller packing
particle sizes can provide higher column
efficiency, hence give higher resolution. In
addition, column length gives better separ-
ability.
Note: Smaller packing particle sizes and longer
column length may increase the column pressure;
hence column should be able to hold the pressure.
Basis of Detector Selection
The objective of selecting a detector is to sense the
presence of a sample and send its corresponding
electrical signal to a computer data system. The
selection is depending upon the characteristics
and concentrations of the compounds that need
to be separated and analyzed such as if,
 Sophisticated Instruments and Techniques Used in Pharmacology Laboratory  87

Fig. 4.5: Components of HPLC (Mobile phase reservoir: Solvent which moves through the column; High-pressure pump:
Used to generate and meter a specified flow rate of mobile phase, typically milliliters per minute; Injector or Autosampler:
Able to inject /introduce the sample into the continuously flowing mobile phase that carries the sample into the HPLC
column; Column: Contains the chromatographic packing material (stationary phase) needed to effect the separation;
Detector: Needed to see the separated compound bands as they elute from the HPLC column. Data processor: detector
connected to computer which records the electrical signal generated to form the chromatogram for a detected sample
which further identified and quantified; Waste mobile phase collection: Mobile phase exiting through detector is collected as
waste)

compound is fluorescent then selects a
fluorescence detector whereas if sample absorbs
ultraviolet light then use UV-absorbance detector.
Chromatogram
The curve obtained after the electrical signal is
generated and sent it to the computer data
system. It generates a graph on computer screen
commonly known as “chromatogram”. It has
several parts such as baseline i.e. graph
obtain by the mobile phase only, peaks, i.e
mainly obtained in the presence of the test
substance or even in presence of some impurities
(Fig. 4.6).
Important Point to Remember
Efficiency of HPLC is a measure of mechanical separation
power whereas selectivity of HPLC is a measure of
chemical separation power.
Ultra-performance Liquid Chromatography (UPLC)
HPLC is well known for its selectivity, efficiency and
high resolution due to the improved column technology.
But, if its resolution is increased by the columns
with smaller particles [1-1.7 micron] and instrumentation,
which is designed to deliver mobile phase at very high
pressure, 15,000 to 100,000 psi is known as ultra-
performance liquid chromatography(UPLC).
Separation method selection depends on
resolution (Selectivity of the packing material for
the compound of interest), load (Capacity of the
packing material) and speed (Isolation time).
Further, HPLC performs on the five important
separation mode namely (Table 4.2).
• Normal phase chromatography (NP),
• Reversed phase chromatography (RP),
• Size exclusion chromatography (SEC),
• Ion exchange chromatography(IEX) and
• Affinity chromatography (AC).
All these modes are summarized in the table.
Application
HPLC is widely used in biotechnological,
biomedical, and biochemical research, but very
88  Practical Manual of Experimental and Clinical Pharmacology

Fig. 4.6: HPLC chromatogram

commonly used to determine concentration of
drug in plasma/serum in the therapeutic drug
monitoring (TDM)
HPLC is also used in a variety of other fields
and industries including the cosmetics, energy,
food, and environmental studies
Liquid Chromatography and Mass-
Spectrometry (LC-MS) (Fig. 4.7)
In general, coupling of a mass spectrometer to an
HPLC system is called LC/MS. So, it is
combination of powerful analytical separation
technique with a powerful analysis and
detection of mass technique. There are two
common Atmospheric Pressure Ionization (API)
LC/MS processes; Electrospray Ionization (ESI)
and Atmospheric Pressure Chemical Ionization
(APCI). Both are soft ionization technique and
are compatible with most chromatographic
separations. APCI is regarded as a soft ionization
process which results in a mass spectrum typically
dominated by a single ion (either + or –) that
corresponds directly to the molecular weight of
the compound, which is protonated in the
positive ion mode (M+H)+ or deprotonated in
the negative ion mode (M-H)-.
 Sophisticated Instruments and Techniques Used in Pharmacology Laboratory  89

Table 4.2: Summary of different types of mode of HPLC, its requirement and features
NPC RPC SEC IEC AC
Stationary phase Silica gel (Polar) Silica-ODS Porous Polymer Ion exchange gel Packing with
(Silica-C18) Aqueous porous ligand
(Non-Polar) Polymer
Mobile phase Organic solvent MeOH/Water Organic solvent Buffer solution Buffer solution
(n-Hexane/IPE) (Polar) (THF)Buffer
(100% organic) solution
(Non-Polar)
Type of Adsorption Hydrophobic Gel permeation Ion exchange Affinity
Interaction
Feature Fat-soluble Most widely used Molecular weight Separation of Purification of
distribution ionic substances enzymes and
Protein Separation proteins
Normal phase chromatography (NPC); Reversed phase chromatography (RPC); Size exclusion chromatography (SEC);
Ion exchange chromatography (IEC); Affinity chromatography (AC)

Fig. 4.7: Component of LC-MS

Salient feature of LCMS is that it can detect the Telemetry

ions ranging from at least 2 to 3,000 mass-to-charge
ratio (m/e).
Schematic System Configuration
Application
LCMS is applied in qualitative and quantitative
analysis, impurities identification, metabolite
studies, pharmacokinetics, pharmaceutical and
biomedical applications in several industries and
research institutes.
Recent application is extended in deter-
mination of drugs and its metabolites concen-
tration in phase 0 or microdosing study during
the drug development process.
Telemetry is a highly automated communicational
technique for measurement and data transmission
through wireless or radio-signals or other means
from distant or remote sources. This skilled
technology was introduced in the beginning of the
20th century to monitor or supervise nature. Hence,
technique commonly uses wireless transmission.
Word is coined from Greek origin i.e. ‘tele’ means
remote or distance and ‘metron’ means measure.
Complete telemetry system basically consists of a
transducer as an input device, a transmission medium
in the form of wired lines if it is connected through
wire or radio waves if system is wireless, signal
processing devices and receiver or devices for
recording or monitoring data.
90  Practical Manual of Experimental and Clinical Pharmacology
Flow Chart 4.3: Procedure of application of telemetry

Application There are many more fields in which the

• Telemetry is used in the experimental as well
as clinical practices. For monitoring tempe-
rature
• Cardiovascular parameters (BP, HR, ECG, etc.)
• Respiratory parameters (FEV1 , VO 2 con-
sumption, etc.)
• CNS (EEG etc.)
• Others like locomotor activity, exploratory
behavior, etc.
telemetry is in use for the determination of natural
reading in experimental animal or human in
clinics. It is hypothesized that the biochemical or
physiological parameters may alter during the
anxiety or hormonal changes in the animal during
the handling or human in variable conditions.
Telemetry is also used clinically for patients who
are at risk of abnormal heart activity or CNS or
gastrointestinal activity.
 Pyrogen Test
5 (In Vivo and In Vitro Methods)
INTRODUCTION
Pyrogen is defined as an agent that causes rise
in temperature which can be polysaccharide or
protein in nature. The pyrogenic molecules can
be exogenous or endogenous whose presence
in injectables may be detrimental to the patients.
Effect of infusion of exogenous pyrogens in the
body may vary from mild pyrogenic effect to
shock. These manifestations underline the
importance of pyrogen testing in injectable
fluids.
A number of in vivo and in vitro methods are
available for pyrogen testing. In vivo methods
have the advantage of detecting any pyrogenic
molecule, which may be protein or poly-
saccharide, or some other unknown molecule
whereas disadvantage of being too sensitive
and may sometimes give erroneous results due
to biological variations. “In vitro” methods like
Limulus Amebocyte Lysate (LAL) test have also
been described but they are not cost effective
and may show variable results with new
molecules. Additionally, some protein
molecules like recombinant products may show
pyrogenic response just because of their
chemistry. Conventional method of in vivo
pyrogen testing is the method by using rabbits.
Principle: The test involves the measurement of
the rise in body temperature of rabbits following
the intravenous injection of a sterile solution of
the substance being examined. It is designed for
products that can be tolerated by the rabbit in a
dose not to exceed 10 ml per kg injected
intravenously within a period of not more than
ten minutes.
LABORATORY CONDITIONS
The test is performed in a quiet room maintained
at a temperature of 24-26°C. The temperature
should not differ for more than ± 5° C from the
animal house.
APPARATUS
1. Restrainer: Wooden restrainer is used. The
restrainer is of the size to allow normal sitting
position of the rabbit.
2. Glassware: The glassware used should be
sterilized by autoclave at 125°C for 8 minutes.
3. Syringes and needles: Sterile disposable syringes
and needles are used for the test.
4. Recording of temperature: An accurate
temperature-sensing device, a mercury
thermometer attached to a probe, calibrated to
assure an accuracy of ± 0.1oC is used. The
temperature-sensing probe is inserted into the
rectum of the test rabbit to a depth of not less
than 7.5 cm, and, after an interval of 5 minutes,
the rabbit’s body temperature is recorded.
TEST ANIMALS
Healthy, adult white New Zealand rabbits of
either sex, preferably of the same variety, weighing
not less than 1.5 kg, fed on a complete and
balanced diet and not showing loss of body weight
during the week preceding the test are used.
92  Practical Manual of Experimental and Clinical Pharmacology
METHOD
Preliminary Test (Sham Test)
In case animals are used for the first time in a
pyrogen test or have not been used during the two
previous weeks, conditioning for one to three days
before testing the substance is done. After an
overnight fast, the animals are examined, by
injecting intravenously into them 10 ml/kg of
body weight of a pyrogen free saline solution.
Water is withheld during the test. The temperature
of the animals is recorded beginning 90 minutes
before injection and continued for 3 hours after
injection of the solution being examined. Any
animal showing a temperature variation of 0.6oC
or more is excluded from the main test (Fig. 5.1).
Main Test
Main test is done by using a group of three rabbits.
Preparation of the Sample
The test substance is dissolved in a pyrogen free
saline solution. The liquids being examined is
warmed to approximately 38oC before injection.
The amount of the sample to be injected is varying
according to the preparation being examined. The
volume of injection is not being less than 0.5 ml
per kg and not more than 10 ml per kg of body
weight.
Fig. 5.1: In vivo Pyrogen test. Design and selection of rabbits for sham and main test
Procedure
The temperature of each animal is recorded at
intervals of thirty minutes, beginning ninety
minutes before the injection of the solution being
examined and is continued for three hours after
the injection. Thirty minutes immediately
preceding the injection of the test dose, the initial
temperature of each rabbit is recorded, which is
the mean of two temperature readings recorded
for that rabbit at an interval of fifteen minutes in
the thirty-minute period. In any one group of test
animals, only those animals whose initial
temperatures do not vary by more than +1oC from
each other is used.
Rabbits usually show a temperature
variation greater than ± 0.2oC between two
successive readings in the determination of
“initial temperature (IT)” should be excluded from
the test. The solution being examined is injected
slowly into the marginal vein of the ear of each
rabbit over a period not exceeding ten minutes,
unless otherwise prescribed in the monograph.
The temperature of each animal is recorded at
half-hourly intervals for three hours after the
injection. The highest temperature difference of
all the recorded different temperatures for a
rabbit is taken as its response. When this
difference is negative, the result is counted as a
zero response.
 Pyrogen Test (In Vivo and In Vitro Methods)  93
Interpretation of Results
If the sum of the responses of the group of three
rabbits does not exceed 1.4oC and if the response
of any individual rabbit is less than 0.6oC, the
preparation being examined passes the test. If the
response of any rabbit is 0.6oC or more, or if the
sum of the responses of the three rabbits exceeds
1.4oC, continue the test using five other rabbits. If
not more than three of the eight rabbits show
individual responses of 0.6oC or more, and if the
sum of the responses of the group of eight rabbits
does not exceed 3.7oC, the preparation being
examined passes the test.
Animals are not used for testing more
frequently than once every 48 hrs. After the animal
has shown positive response, at least two weeks
are allowed to recover the animal.
Pyrogen Testing by “in vitro” Method
Due to the expertise hand and long time required
for the experiment, in vivo method is not adopted
for the pyrogen testing in present scenario. So, in
vitro method was developed, which is equally
good and have been taking comparative less time.
It is also preferred due to its high precision,
sensitivity, reliability and reproducibility over the in
vivo method where chances for inter individual
variation is very high. There are parenteral drugs
which may contain endotoxin, augment in vivo
biological activities. Hence, such drugs require
not only quantifying endotoxin but also regulate
overall in vivo action of contaminated parenteral
drugs. Hence, in vitro method, not only establishes
the presence of pyrogen but also endotoxins in
contaminated parenteral drugs.
The performance standards consist of 1)
essential test method components, 2) reference
substances, and 3) the comparable accuracy and
reliability that should be achieved.
Principles
The principle of in vitro pyrogenicity test methods
is based predominantly on the human cell type
used, and its assessment for the pyrogen and
endotoxin. Specific human-derived cells used in
the in vitro test method and incubation of test
substance with the cells for a specified period of
time. Concentration of pro-inflammatory
cytokines (e.g., IL-1α, IL-6) is quantified via a
cytokine-specific enzyme-linked immunosorbent
assay (ELISA) by comparison to a standard curve.
Using an endotoxin standard curve, the endotoxin
content of the product is calculated.
A product “passes” if the endotoxin content
is < 0.5 endotoxin units (EU)/mL.
Laboratory condition: Cell culture grow at
specified conditions hence, the laboratory
condition is maintained at temperature (37°C ±
1°C), humidity (90% ± 10%), Air (5.0% O2 ± 1%
CO2 in ambient air).
Cell culture required are mammalian cell line,
primary cells, or heparinized whole blood or
cryopreserved cells/whole blood and fresh whole
blood (should be used within 4 hrs of collection)
may be also used.
Note: Cell cultures should be free of contamination
with bacteria, mycoplasma, or fungi.
Precaution during the Experiment
Instrument used in the test should be sterile,
pyrogen free (e.g., pipette tips, pipettes, culture
ware, etc.)
Test substances should be diluted in sterile,
pyrogen-free 0.9% NaCl.
Medical devices can be directly incubated with
the cells in suspension.
Test substances should be fully solubilized
(i.e., no visual observation of test substance in the
dosing solution).
Methods
During the experiment, it is advisable to add three
groups of control namely
“Negative control” = to assure the proper
functioning of the test
procedure (Vehicle group)
“Positive control” = to assure the enough
sensitivity to a pyrogenic
substances (international
reference standard endotoxin
such as E.coli) and
“Benchmark control” = to ensure the test method
is functioning properly.
94  Practical Manual of Experimental and Clinical Pharmacology

The reliability and relevance of each test SUGGESTED READING

method is evaluated with pyrogen-free parenteral
1. Akihiko Yamamoto, Masaki Ochiai, Kazunari
drugs spiked with different concentrations of an
Kamachi, Michiyo Kataoka, Hiromi Toyoizumi,
endotoxin standard. In vitro pyrogenicity test Yoshichika Arakawa, et al. A clinically relevant in
methods may be used to test parenteral drugs, vitro pyrogen test using a human cell line that
biological products and medical devices. have the similar responsiveness to various pyrogens
to that of human peripheral blood cells (hPBC). In:
Test Substance Application and Sample AATEX 14, Proc. 6th World Congress on Alternatives
Collection and Animal Use in the Life Sciences: Special Issue,
2007Aug 21-25; 2007;647-53.
2. Finney DJ. Statistical Methods in Biological Assay.
3rd edn. Charles Griffin and Co., Ltd., London 1978.
3. Indian pharmacopeia; 1996: A-28.
4. Moesby L, Jensen S, Hanse EW, Christensen JD. A
comparative study of Mono Mac 6 cells, isolated
mononuclar cells and Limulus amoebocyte lysate
assay in pyrogen testing, Int J Pharm 1999;191:
141-49.
5. Pool EJ, Johaar G, James S, Petersen I, Bouic P. The
detection of pyrogens in blood products using as ex
vivo whole blood culture assay. J Immunoassay
Interpretation of Result 1998:19;95-111.
6. Thomas H et al. Statement on the varidity of in vitro
Positive control samples curve is used to calculate
pyrogen tests 2006.
the level of relative release cytokine in the sample.
7. Yamamoto A, Ochiai M, Kataoka M, Toyoizumi H,
When, the level of cytokine release is greater than
Horiuchi Y. Development of a Highly Sensitive in
or equal to that induced by the 0.5 EU/mL vitro Assay Method for Biological Activity of
endotoxin standard, then the sample is considered Endotoxin Contamination in Biological Products.
positive for a pyrogen test. Biologicals 2002;30:85-92.
 Practical Aspects of
6 Cell Culture

This chapter includes the basic principles and
methodology of cell culture in the pharmacology.
The important advantages are that it facilitates
analysis of biological properties of the host
organism and effect of several drugs on the system
at the laboratory setting, which generally not
readily accessible at the level of the intact
organism. Historically, cell culture started with
the maintenance of medullary plate of an
embryonic chicken in warm saline by Wilhelm
Roux in 1885, whereas, methodology of tissue
culture was established by Ross Granville Harrison.
In the recent times, there is more ethical constraint
on the use of animal in the research or experiment,
when other alternative methods are available. So,
the use of cell culture is very efficient to produce
the reliable data.
In general, the cell culture is the cultivation or
growth of cells outside of the host organism that
may include eukaryotic or prokaryotic cells. The
advantage over other method of assessment is that,
it allows direct access to a population of cells
outside the parent organism and the effect is
directly seen. But, drawbacks are the architecture
of the original tissue is lost and the cells change
properties over time.
Two Types of Cell Culture (Fig. 6.1)
• Primary cell culture
• Cell line culture or subculture (secondary or
tertiary culture or more)

Organ culture: survival up to 3 weeks

Fig. 6.1: Classification of cell culture; primary culture is usually fibroblasts of the dermis or the basal epithelial layer of the
epidermis that gives rise to high density of the cells. These cells represent the largest compartment of proliferating, or
potentially proliferating, cells. Subculture is prepared from the primary culture by rinsing, dissection, and either mechanical
disaggregation or enzymatic digestion in trypsin and/or collagenase
96  Practical Manual of Experimental and Clinical Pharmacology
Primary Culture
These are the cell type which is explanted directly
from an organism, animals or human, which are
capable cell divisions in culture under the
standard maintained conditions. While main-
taining and developing in culture it retains
characteristics of normal cells as in the host organ.
Because of these reasons, it is the best experimental
in vitro models to assess the activity of any drug,
which gives rise to the comparable good result
than the other in vitro study.
But, the limitation being these are susceptible
to contamination and have limited period to grow
and eventually senesce and die. Senescence is
determined by a number of intrinsic factors
regulating cell cycle, such as Rb and p53 and is
accompanied by shortening of the telomeres on
the chromosomes. Once the telomeres reach a
critical minimum length, the cell can no longer
divide. Telomere length is maintained by
telomerase, which is down regulated in most
normal cells except germ cells.
Cell Line Culture or Secondary Cell Culture
As the name suggest, it is the second culture which
was originally explanted from a donor organism
and preserved. These cells are terminally
differentiated cells and harder to maintain than
less specialized cells. For example, animal (mouse
or rat) cells are easy to maintain than the human
cells. These cells do not divide indefinitely and
eventually their physical characteristics will keep
changing to mature and die. Therefore, limited
number of subcultivations from these cells is
possible. Cells always get to a certain density and
stop dividing even if nutrients are present in the
culture. These limits in the process is defined as
Hayflick’s limit (1961).
Whereas, other type of cells known as “infinite
cell line.” These cells are easy to maintain,
manipulate in culture and easy to obtain large
quantities. So, the other most important is that
these cell lines subcultured indefinitely, i.e. these
cells are immortalized.
Note: A cell line is the culture that is produced
from subculture of the primary cell culture and
further additional subculture. After this increases
the possibility of cross-contamination, so
verification of origin is important.
Principles of Cell Culture
There are four basic requirements for cell culture
namely, sterile condition, its medium and nutrition,
principles of cell growth and basic knowledge and
practice of techniques involved in the growth of a
particular cell type. The basic principles of cell
culture maintenance of a non-adherent cell line and
adherent cell line is almost similar.
Cryopreservation is other important principle for
the storage of cell lines for further subculture.
Principle of Sterile Condition is the most important
principle to get the good result. For this biological
safety cabinet should be turned on and allowed to
run for at least 15 min to remove the contaminated
air. All work surfaces within the cabinet should be
decontaminated with an appropriate solution
(either 70% ethanol or isopropanol). Antibiotics
are used sometimes to avoid any contamination in
the cell culture, e.g: Amphotericin B, Ampicillin,
Gentamicin, Kanamycin, Penicillin, Streptomycin,
Tetracycline and many more. Mainly antibiotics
are used during collection, transportation, and
dissection of biopsy samples because of the
intrinsic contamination risk involved in these
operations. The second principle is principle of
medium or nutrition used, which are required for
survival and growth of cell culture. Nutrient
medium should be sterile and cannot be autoclaved.
The compounds in nutrient medium are destroyed
by the heat of autoclaving. Medium must therefore
be sterilized by passing it through a sterile filter
small enough in pore size to hold back bacteria and
mycoplasmas, e.g: membrane filter. Then, the third
principle is principle of cell growth, majority of cell
culture carried out by researchers involves the use
of various nonadherent (i.e. P815, EL-4, etc.) or
adherent (i.e. FM3, STO, NIH 3T3, etc.) continuously
growing cell lines. The cell population can be
grown by selection of the correct medium (e.g.,
 Practical Aspects of Cell Culture  97

keratinocyte growth medium (KGM) or MCDB 153

for keratinocytes. Culture medium is a liquid or
gel designed to support the growth of cells. Cell
growth cycle explained in the Figure 6.2.

Common Requirements for

Culture Growth Media
(The amount and concentration varies according
to the cell selected for growth)

Ingredients
• Ions: Na + , K + , Mg 2+, Ca 2+, Cl - , CO 2 or
bicarbonate, P+ etc.
• Sugars: Glucose
• Amino acids: 13 essential
• Trace elements: Iron, selenium, zinc, etc.
• Vitamins
• Fetal calf serum: Usually 10%: Nutralizes the
trypsin, used to buffer toxic nutrients by
binding them and contains hormone like
growth factors or peptide hormones accelerate
the growth.
• Antibiotics: Amphotericin B, Ampicillin,
Gentamicin, Kanamycin, etc.: To prevent the
growth of bacterial or fungal contamination
Physiological Condition to Maintain the Culture
Temperature : 37°C
pH : 7.2-7.5
Humidity : 80-95%
Gas phase : CO2 (5-10%) or Bicarbonate
Visible light : Should be kept in dark
Cryopreservation is the process which
involved in the storage of primary or secondary
cell culture for further use. Cryopreservation is
done with a slow cooling, 1°C/min, down to –70°C
and thereafter, rapid transfer to a liquid nitrogen
freezer High cell density at freezing (1 × 106–1 ×
107 cells/ml). Preservative such as glycerol or
dimethyl sulfoxide (DMSO) at 5-10% may be
added to the cell culture to preserve for long
duration.
After cryopreservation if someone wants to use
the sample then rapid thawing and slow dilution,
<“20-fold, in medium is used to dilute the
preservative.
Fig. 6.2: Cell growth cycle during the cell growth in the
medium and showing the different procedure timeline and
its different phases of growth
Methodology
Tissue Collection and Transportation
The important step in beginning of the cell culture
is collection of samples in a labeled container of
culture medium containing antibiotics. The
samples should be labeled and stored in a
refrigerator, but freezing of sample should be
avoided. Transfer of sample should be protected,
preferably double wrapped in a sealed polythene
bag and transferred. (To avoid contamination).
The following precautions should be taken during
the experiment on the cell line
• Switch on the laminar flow and UV source of
safety cabinet for at least 15 min before
experiment
• Clean the area with disinfectant (70% ethanol
or isopropanol)
• Never uncover sterile articles like flask, bottle,
Petri dish, etc. in open air/place.
• Cover the cell line as soon as procedure is
finished (Never leave it open to the
environment)
• Sterile pipettes should be taken from the
wrapper at the time of use (Sterile pipettes don’t
need not to be flamed).
98  Practical Manual of Experimental and Clinical Pharmacology

• Cells may die with a hot pipette and always
use the different pipettes for different bottles.
(Do not draw from a different bottle with the
same pipette)
• After removing the cap from a bottle, flask, etc.
do not place the cap with the open end upright
on the laboratory bench (chances of contami-
nation)
• Do not hold the opened flask/bottle straight
up into the air. If possible, tilt the container so
that any falling microorganisms fall onto the
lip (Avoid hand contamination)
• Techniques should be performed as rapidly
as possible to minimize contamination.
Protocol for the Experiment (Flow Chart 6.1)
It is necessary to change the growth medium every
third day. Cells require subculturing according to
their doubling time. Generally, they should be
subcultured twice a week.
Adherent cells lines require trypsinization while
the non-adherent cell lines do not require trypsini-
zation (Figs. 6.3A and B). Generally cells should
be splitted 1: 3 to 1: 5 ratio depending upon the cell
doubling period.

Flow Chart 6.1: Procedure for secondary cell line development

Note: Cells should be grow into the culture flask (T-75 cm2 or T-25 cm2)

Figs 6.3A and B: (A) Adherent cell line growth
(B) Non-adherent cell line growth
In T-75 cm2 culture flask initially 10-15 lac cells
should be inoculated and the ideal capacity is 20-
25 ml while in T-25 cm2culture flask 2 lac cells
can be inoculated and can contain culture medium
up to 6-8 ml.
SUGGESTED READING
1. Beshay NM, Zordoky, Ayman OS. El-Kadi. H9c2
cell line is a valuable in vitro model to study the drug
metabolizing enzymes in the heart. Journal of
Pharmacological and Toxicological Methods 2007;
56:317-22.
2. Cerni C. Telomeres, telomerase, and myc. An update.
Mutat Res 2000;462:31-47.
Practical Aspects of Cell Culture  99
3. Das RC. Antibodies in biotech: year of the bear. Am
Biotechnol Lab 2003;20:4-6.
4. Finter NB, Garland AJM, Telling RC. Large-scale
mammalian cell culture: a perspective. Bioprocess
Technology 1990;10:1-14.
5. Freshney RI. Cell line provenance. Cytotechnology
2002;39:3-15.
6. Juliano RL. Signal transduction by cell adhesion
receptors and the cytoskeleton: functions of integrins,
cadherins, selectins, and immunoglobulin-
superfamily members. Annu Rev Pharmacol. Toxicol
2002;42:283-323.
7. Knazek RA, Gullin P, Kohler PO, Dedrick R. Cell
culture on artificial capillaries. An approach to tissue
growth in vitro. Science 1972;178:65-67.
8. Marcovic O, Marcovic N. Cell cross-contamination
in cell cultures: the silent and neglected danger. In
vitro Cell Dev Biol 1998;34:108.
9. Pereira RM, Marques CC, Baptista MC, Vasques MI
and Horta AEM. Embryos and culture cells: A model
for studying the effect of progesterone. Animal
Reproduction Science 2009;111:31-40.
10. Zhengshan Zhao, Dee Montgomery-Brock2, Cheng-
Sheng Lee and Yuanan Lu. Establishment,
characterization and viral susceptibility of 3 new cell
lines from snakehead, Channa striatus (Blooch).
Methods in Cell Science 2003;25:155-66.
 Preclinical to Clinical Drug
7 Dose Calculation
INTRODUCTION
The experiment on animals gives an overall idea
about the tested drug (pharmacodynamics,
pharmacokinetic and toxicology) through various
methods involved in the process. But, the ultimate
goal of any drug research is to use the drug in the
patients care. So, it is most important to perform a
planned scientific study in the humans before,
it’s clinical use. Preclinical study is primarily
designed to find a lead compound with desired
efficacy and safety for clinical study through
pharmacokinetics and pharmacodynamics data
obtained from in vitro and in vivo studies (Fig. 7.1).
Hence, the compound selected for the clinical
study is based on the bioavailability, volume of
distribution and tissue penetration, half-life and
its receptor binding study. Phase 0 or phase 1
clinical studies is end of preclinical study and
Fig. 7.1: Schematic diagram of dose conversion from the in vitro studies to in vivo (animal)
and finally to in vivo (human) studies
start of clinical study in drug discovery which
requires special permission through Investi-
gational New Drug Application (IND).
Derivation from the animal dose to the human
dose of a tested drug is important step due to
chances of toxicities. So, question arises that “How
much dose should be selected, which is safe for
the human?” The first dose used in human is
known as “maximum recommended starting
dose” (MRSD) or first human dose (FHD), entry
into human (EIH) or first time in man (FTIM) whose
derivation initiates clinical trials of new tested
drug in adult healthy volunteers/patients.
Requirements for Determination of the
First Human Dose
• Preclinical data, including
– Pharmacodynamic studies,
 Preclinical to Clinical Drug Dose Calculation  101
– Full toxicological studies of the drug, and
– Pharmacokinetics data (absorption, distri-
bution, metabolism, and excretion).
Procedure of Starting-dose Calculations
There are several approaches of extrapolation or
transformation of doses from preclinical to clinical
(human) study which mainly depend on the drugs
behavior across different species and its
physiochemical and pharmacological nature. It is
very important to find a starting dose in human but
the method should be economical and less time-
consuming. These approaches can vary from
scientist to scientist and their training and
experience but broadly extrapolation of the first
human dose is divided into two categories (Fig. 7.2).
1. Extrapolation of dose of cytotoxic drugs in
which patients are the target individuals and
2. Extrapolation of dose of non-cytotoxic drugs
in which healthy volunteers are the target
individuals
Cytotoxic Drugs
The main objective of extrapolation of doses for
cytotoxic drugs involves a therapeutic benefit and
Fig. 7.2: Different categories for extrapolation of safe starting dose
to minimize chances of patient exposure to sub-
therapeutic doses of drugs. So the approach
applied is usually a multiple ascending dose
(MAD) instead of a single ascending dose (SAD)
study. The principle in the treatment of cancer is
that more acceptance of toxicity to achieve
therapeutic benefit so the dose selected as starting-
dose calculation for anticancer is generally based
on a dose and dose schedule that have produced
some toxicity in animals rather than on a dose
that has been identified as safe in animals or
NOAEL.
Note: The dose selection should be appropriate
because cytotoxic compounds have a very low
therapeutic index and a steep concentration–
response curve for safety.
Procedure
The approach is based on the toxic dose low (TDL)
and the concept of lethal dose (LD10) (Fig. 7.3).
Toxic dose low (TDL): TDL is defined as the lowest
dose of a drug which does not produce any
lethality when its dose is doubled but able to
change pathology in hematological, chemical,
clinical, or morphological parameters.
102  Practical Manual of Experimental and Clinical Pharmacology
Fig. 7.3: Representation of therapeutic, toxic and
lethal dose
Animal preferred: Dog or monkey
Drug dosing: Single dose and dosing for 5 days
(Dose is expressed as mg/m2)
Drug dose selection: 1/3rd of TDL
Lethal Dose (LD10)
It is defined as the lowest dose of a drug which
produces lethality to 10% of normal (non-tumor)
mice (LD10) during a specific period of observation
under standard condition.
Animal preferred: Mice
Drug dosing : Single dose and dosing for 5 days
(Dose is expressed as mg/m2)
Drug dose selection : LD10 (1/10th of LD10)
Note: If the calculated 1/10th of LD10 in mice and
1/3rd TDL in large animal are different, then the
lower of the two doses be used as the starting dose.
Non-cytotoxic Drugs
There are four approaches which are preferred in
the extrapolation of first human dose for non-
cytotoxic drugs
• Approach 1: No observable adverse effect level
(NOAEL)
• Approach 2: Similar class of drug
• Approach 3: Pharmacokinetics
• Approach 4: Comparative
Approach 1: The Process of Calculating the
MRSD by using NOAEL Approach
Step 1: No observed adverse effect level
(NOAEL) determination
The NOAEL is a generally accepted benchmark
for safety which is derived from appropriate
animal models. This can serve as the starting
point for determining a reasonably safe starting
dose (SSD) of a new therapeutic in healthy human
volunteers. Hence, the NOAEL is defined as the
highest dose of a drug that does not produce significant
adverse effects.
Besides NOAEL, other more terms which may
confuse with is no observed effect level (NOEL)
which is any pharmacodynamic actions of the
drug and may not raise a safety concern, lowest
observed adverse effect level (LOAEL) is defined as
the dose of a drug at which it shows the lowest
adverse effect sometime also called as maximum
tolerated dose (MTD).
 Preclinical to Clinical Drug Dose Calculation  103
Three requisite findings of preclinical
toxicology studies that can be used to determine
the NOAEL are
1. Overt toxicity (e.g. clinical signs, macro- and
microscopic lesions);
2. Surrogate markers of toxicity (e.g. serum liver
enzyme levels); and
3. Exaggerated pharmacodynamic effects.
Step 2: Human equivalent dose (HED) calculation
Human equivalent dose (HED) is the dose which
is obtained from converting mg/kg dose for each
animal species to the mg/kg dose in human which is
equivalent to the animal’s NOAEL on a mg/m2
basis. This conversion process mainly depends
on three major factor based on the body Surface
area (mg/m2), body weight (mg/kg), route of
administration, etc. While extrapolating the
animal dose to the HED, most appropriate method
is used.
Step 3: Most appropriate species selection
After selection of HED, the MRSD is calculated by
using the most appropriate sensitive species
(defined as the species in which the minimum
HED can be obtained). While determining, 1st
HED, human ADME data is obtained by the in
vitro studies.
Step 4: Application of safety factor
Receiving 1st dose in the human after preclinical
study have many ethical issues, hence it is
important to give a marginally safe dose to the
volunteers/patients. So, safety factor is included
in the calculation of the human first dose.
Practically, for the calculation of MRSD, HED is
divided by safety factor 10. While a safety factor of
10 can generally be considered adequate for
protection of human subjects participating in
initial clinical trials, this safety factor may not be
appropriate for all cases. The safety factor should
be raised when there is favorable reason for
increase such as steep dose-response curve, severe
toxicities, non-monitorable toxicity, toxicities
without prodromal indicators, variable bio-
availability, irreversible toxicity, unexplained
mortality, large variability in doses or AUC levels
eliciting effect, novel therapeutic target and
lowered when there is design and conduct of
toxicological study is of good quality, therapeutics
should be administered by the same route,
schedule, and duration of administration or
similar toxicity profile.
According to Association of Food and Drug Officials
(AFDO) of the United States, the maximum starting dose
for the FHD study is the smallest of the following three
doses:
• 1/10 of the highest no-observed effect dose in rodents,
• 1/6 of the highest no-effect dose in dogs, or
• 1/3 of the highest no-effect dose in monkeys
Step 5: Consideration of the pharmacologically
active dose (PAD)
PAD is an in vivo study to compare the dose
decided by the HED and MRSD by the help of
pharmacodynamic model. HED can be derived
from a PAD estimate by using a body surface area
conversion factor (BSA-CF). This HED value
should be compared directly to the MRSD. If this
pharmacologic HED is lower than the MRSD, it may
be appropriate to decrease the clinical starting dose
for pragmatic or scientific reasons.
Drugs like vasodilators, anticoagulants,
monoclonal antibodies, or growth factors from
which toxicity may arise from magnified
pharmacologic effects. Hence, PAD in these cases
may be a more sensitive indicator of potential
toxicity than the NOAEL and might therefore
justify lowering the MRSD.
Other Approaches
Approach 2: Similar Class of Drug
These are the investigational drugs which have
same chemical class or related chemical structure
or having similar toxicological profiles. For these
similar drug profile, first human dose is calculated
as the ratio of an optimal starting dose of the
similar parent drug to its NOAEL dose.
FHDP/NDP = FHDi/NDi
104  Practical Manual of Experimental and Clinical Pharmacology
where,
FHDP : Optimal starting dose of parent
compound
NDP : No observed adverse effect level
of parent drug
FHDi : Starting dose of investigational
drug
NDi : No observed adverse effect level
of investigational drug
Approach 3: Pharmacokinetic
In this approach NOAEL and its corresponding
AUC are used. The lowest dose from more than
one animal species are calculated and selected.
The clearance of the drug in humans (CLh) is
predicted using allometric scaling. Then, the
starting dose is calculated as:
FHD = AUCp × CLh
where
FHD : First human dose
AUCp : AUC obtained at the NOAEL in
preclinical toxicology studies, and
CLh : Clearance in humans predicted by
allometric scaling
Approach 4: Comparative
This approach is comparative study of estimation
of the starting dose using at least two or more
methods and then compares the results to find an
optimal starting dose.
SUGGESTED READING
1. Bonate PL, Howard D. Critique of prospective
allometric scaling: Does the emperor have clothes? J
Clin Pharmacol 2000;40:335-40.
2. Boxenbaum H, DiLea C. “First-Time-in-Human
Dose Selection: Allometric Thoughts and Pers-
pectives,” Journal of Clinical Pharmacology 1995;
35:957-66.
3. Brodie BB, Reid WD. Some pharmacological
consequences of species variation in rates of
metabolism. Fed Proc 1967;26:1062-70.
4. Freireich EJ, Gehan EA, Rall DP, Schmidt LH, Skipper
HE. “Quantitative Comparison of Toxicity of
Anticancer Agents in Mouse, Rat, Hamster, Dog,
Monkey, and Man,”Cancer Chemotherapy Reports
1966;50:219-44.
5. Goldsmith MA, Slavik M, Carter SK. Quantitative
prediction of drug toxicity in humans from toxicology
in small and large animals. Cancer Res 1975;35:
1354-64.
6. Gomeni R, Bani M, D’Angeli C, Corsi M, Bye A.
Computer-Assisted Drug Development (CADD): An
emerging technology for designing first-time-in man
and proof-of concept studies from preclinical
experiments. Eur J Pharm Sci 2001;13:261-70.
7. Lave T, Coassolo P, Reigner B. Prediction of hepatic
metabolic clearance based on interspecies allometric
scaling techniques and in vitro–in vivo correlations.
Clin Pharmacokinet 1999;36:211-31.
8. Mahmood I, Balian JD. The pharmacokinetic
principles behind scaling from preclinical results to
phase I protocols. Clin Pharmacokinet 1999;36:
1-11.
9. Mahmood I. Allometric issues in drug development.
J Pharm Sci 1999;88:1101-06.
10. Penta JS, Rosner GL, Trump DL. Choice of starting
dose and escalation for phase I studies of antitumor
agents. Cancer Chemother Pharmacol 1992;31:
247-50.
11. Penta JS, Rozencweig M, Guarino AM, Muggia FM.
Mouse and large animal toxicology studies of twelve
antitumor agents: Relevance to starting dose for phase
I clinical trials. Cancer Chemother Pharmacol 1979;
3:97-101.
12. Posvar EL, Sedman AJ. New drugs: First time in
man. J Clin Pharmacol 1989;29:961-66.
13. Prieur DJ, Young DM, Davis RD, Cooney DA, Homan
ER, Dixon RL, Guarino AM. Procedures for
preclinical toxicologic evaluation of cancer
chemotherapeutic agents: Protocols of the laboratory
of toxicology. Cancer Chemother Rep 1973;4:1-6.
 Protocol and Thesis Writing
8 for Postgraduate Students

This chapter will provide a brief conceptual

framework for understanding the principles
involved in hypothesis of protocol design and
writing. Most of the PG students listen word
“Protocol”, “thesis” or “dissertation” first time at
the time of entry into the PG course and feel very
confused about, when to start planning to write a
protocol for a thesis or dissertation. Their mind is
full of questions which have to be cleared at each
step of protocol writing such as, why are you doing
the study?, what are the background information
available at the selected subject?, why you selected
the particular topic?, what you will get from the
study, etc.
Broadly if we see, the research protocol are
divided into the 2 types in postgraduate (PG) study
1. Experimental research protocol and
2. Clinical research protocol
Whatever be the nature of research protocol, the
first step in both the types is the selection of a research
topic, which requires a thorough understanding of Fig. 8.1: Stepwise approach from protocol to
prevailing operational realities along with the thesis writing
working limits (Flow Chart 8.1). While selecting a
research topic following factors should be consi- Experimental Research Protocol
dered: Experimental research protocol deals with the
i. Relevance to health in general community experimental studies done under controlled
ii. Likelihood of effective implications of the conditions on the animals, cell lines or in vitro
findings to the community setting, from which results are obtained whereas
iii. Feasibility of successfully carrying out the clinical research protocol deals with the studies
proposed study. which are of observational type, open trials or the
iv. Reproducibility of the findings to other randomized human trials. (Arm of the study is
settings depend on the intervention selected and its
v. Interest and experience of the research team control.)
106  Practical Manual of Experimental and Clinical Pharmacology

Different Components of Experimental and Clinical Protocol

Experimental Research Protocol Clinical Research Protocol

• Title page • Title page

• Abstract/Introduction • Abstract/Introduction
• Review of literature • Review of literature
• Materials and methods • Materials and methods
• Statistical analysis • Statistical analysis
• Ethical justification • References
• References • Ethical consideration
• Animal ethics form • Patients information sheet
• Case record form
• Informed consent form

Flow Chart 8.1: Common pathway to write a
research protocol
degree, name of the department and institution
where the work will conduct.
Note: Title should be properly designed which
should be self explanatory and should never
contain any abbreviation, chemical formula,
propriety names, etc.

Title Page
Composite question: What is the study title? Or who
will conduct the study and where the study will
be conducted?
Title page should contains the title of the study,
name of the candidate and academic degree for
which student is making the thesis protocol, name
of the supervisor with his/her highest academic
Introduction
Composite question: Why the study is needed? Or
what are the rational of doing the study?
The first part of introduction should summarize
the proposed study with the epidemiological
background or prevalence rate if available. (Which
shows the importance of the study?) Further, study
purpose should be described with the favorable
previous study. Next section should give the
background and rationale for initiating the study.
Findings from previous studies must be outlined
including appropriate detailed references to earlier
studies and brief data. If any pilot study conducted
in the same laboratory, then the results should be
included in the later part of introduction. This
section ends with a statement regarding aim and
objectives of study.
Review of Literature
Composite question: What are available data?
or How much background information available?
It is an essential part which involves a
collective review and critique in the candidates
own words of various viewpoints supported by
relevant data. The review should be properly
 Protocol and Thesis Writing for Postgraduate Students  107
referenced and the method of citing references in
the text and listing at the end of the text are depend
on the institution guidelines.
Aims and Objectives
This section of the protocol gives the ultimate goal
of the study and the objectives section should give
a concise statement to achieve research question.
Hence, the study design must be in such way that
it allow candidate to answer the research
objectives.
Materials and Methods
Composite question: Which materials, animals and
method should be used to answer the research
question?
Explain about the animal use and describe
about their species, numbers, breed (if applicable),
age, sex, and weight. Housing and laboratory
condition should be described during experiment
such as temperature, humidity, light/dark cycle,
etc. Properly explain the experimental groups of
the study such as control/normal group, negative
control group, treatment group and positive control
group, etc. Write brief paragraph on the disease
model development and the methods selected for
the estimation of various parameters to conclude
the study.
Statistical Analysis
Composite question: how the collected data will be
analyzed? Or which tests are more appropriate to
analyze the data?
In the first section, summarize the study design
and rationale. Thereafter, describe the statistical
plan for each objective described. While selecting
the statistical tests one should consider the end
point of the study
If the software will be used for the analysis of
data, then describe about “What software will be
used?”
Note: In clinical research protocol describe about
primary and secondary endpoints, sample size
and statistical power or precision associated with
the sample size, stratification factors and inter-
Types of Experimental Groups
• Control or normal group (Negative Control): To assess
the impact of external variables (environmental, genetic,
microorganism, etc.) or to recognize the possible effect
of unwanted variables without the treatment.
• Vehicle Control group: These groups are kept in the
experiment to assess whether vehicle alone causes
any effects or not. It is given in the same schedule as,
is in the other treatment groups. This group is compared
with the control or normal animals.
• Sham Control group: To assess any effect of the surgical
procedure or special treatment technique such as
implant on the animals. This group is compared with
the control or normal or vehicle group.
• Positive Control group: These are the group in which
effect is expected. These are either a disease model
group in which there are definite physiological changes
are occurred after the chemical or bacteria treatment
or it may be a standard treatment* group which also
show the definite reduction in the pathology of disease.
• Comparative Control group: It is the group of a standard
treatment which is directly compares the effect of a
test treatment.
*Standard treatment refers to clinically available prototype
drug for a disease. e.g. Phenytoin in the treatment of epilepsy
or best drug treatment available clinically for the same
disease.
vention allocation plan for randomized studies
(if any).
Ethical Justification
Composite question: Why study is important in the
animal? or is any alternative method is available?
If yes, then give the justification of not using the
alternative method.
This is a brief paragraph on the method used
and importance of the animal use in the study.
Justify the total number of animal use and also
give brief on the care and handling of the animal
during the study.
References
This section contains a list of references for the
study. Institutions have their own style of reference
writing hence always refer to your institution
guidelines for the style of references used.
“Vancouver and Harvard” referencing is very
commonly used worldwide.
108  Practical Manual of Experimental and Clinical Pharmacology

Example of Vancouver Style

Note: If the authors are more than six, then use the word “et al” (means ‘and others’) after six author and if it is less give
the name of all authors. Reference citation in the protocol is by no. of reference such as above mentioned reference no. is
‘1’, hence it is cited as Superscript.1

Example of Harvard Style

Note: Give the name of all authors. Reference citation in the text is not by the reference number (no reference number),
it is cited as Rawlin et al. (1977) or Rawlin (1977).
In both the referencing style, example given is for journal reference citation. Style for book, newsletter, conferences,
internet, etc. is different in both the styles. There is several other referencing styles which varies with the journal or
publishers

Animal Ethics Form use, surgical procedure adopted and disposal of

This is an ethical requirement for the approval of
the research protocol from the Institutional Animal
Ethics Committee (IAEC). It includes the all the
information regarding the study such as, title of
the study, investigator name and addresses or
additional investigators name, briefly procedure
of the study, justification of the study and animal
the animal if animal requires euthanasia and other
related information.
Clinical Research Protocol
Clinical research protocol is meant for obser-
vational population based research or any
randomized trial. The typical format and compo-
nents of a Clinical research protocol are:
 Protocol and Thesis Writing for Postgraduate Students  109
i. Title page
ii. Abstract/Introduction
iii. Review of literature
a. Rationale of the study
b. Previous studies on the subject either
experimental or clinical study
c. Study question
iv. Aim and objectives
a. Aim, hypotheses and objectives
v. Materials and method
a. Design and methods
(Study design, Study population,
Sample size and statistical power,
Subjects, Data collection methods, Data
management and Statistical analysis)
b. Project Management
(Personnel required, Duration of study
and Follow up procedures)
c. Strengths and limitations
vi. References
vii. Ethical considerations
viii. Patient information sheet
ix. Case Record Form (CRF)
x. Patients informed consent
xi. Budget
xii. Investigators.
Title Page
Same as the experimental research protocol, this
page contains the title of the study, name of the
candidate and academic degree for which student
is making the thesis protocol, name of the
supervisor with his/her highest academic degree,
name of the department and institution where the
work will conduct.
Abstract
Abstract is a concise summary which provides
information about the main purpose of the
study, the way in which it will be conducted
and its expected benefits. Although in a typical
protocol format, abstract is written on the top
after completion of the studies.
Review of Literature
It is one of the brief documentation of a collective
review and critique in the candidates own words
of various viewpoints supported by relevant data
either of experimental or clinical study. The review
should be properly referenced and cited with the
reference as per institute guidelines.
Study Description
Study description involves the outline of the
proposed study and its essential components
are:
Rationale and Previous Studies on the
Subject
An ideal protocol should provide the rationale
and the relevant background information
related of the proposed study. Rationale should
indicate in a progressive logical sequence that
how the study question has emerged. This is
done by reviewing the relevant literature and
current knowledge showing specifically the
ambiguity in knowledge which make the study
worth doing. Any preliminary work done in the
field of proposed study should be included in
the protocol. Rationale should clearly explain
how the proposed study will benefit the
community.
Study Question
This addresses the issue which one has to
pursue during the proposed study. The research
topic is generally formulated into a question,
known as study question. The study question
must clearly explain the objectives of the
proposed study. The necessary steps required
to answer the study question should also be
included in the protocol.
Aims and Objectives
Study question indicates the aim or the goal, which
is generally very vast. So, this is broken down into
smaller questions known as objectives.
110  Practical Manual of Experimental and Clinical Pharmacology
Hypotheses
Hypotheses are distinct and clearly defined
outcomes for any proposed study. Objectives
should be stated in the form of hypotheses.
Hypotheses can be written as statements to be
refuted i.e. no difference between study groups
and are referred to as Null hypotheses. Alternate
hypotheses are statements assuming the real
difference between the study groups. Thus the
research hypotheses provide information about
the comparisons which will be needed to answer
the study question and establish the format with
in which one will apply the statistical tests while
interpreting the results.
Materials and Methods
Design and Methods
Study design: Protocol should clearly indicate how
the proposed study will be performed and which
methods will be adopted. It should mention the
selected design of study along with the rationale
of choosing the proposed design.
Study population: Study population means the
target group in which the study will be carried
out.
Sample size and statistical power: Protocol must
indicate how big the target group has to be in order
to precisely answer the study question. This
should include the assumptions made for
calculation and a table of calculation of sample
size and power.
Subjects selection: This should explain the number
of subjects , criteria for subject selection i.e.
inclusion and exclusion criteria, various inclu-
sion and exclusion parameters, mechanism of
recruitment of subjects and feasibility of recruit-
ment.
Data collection methods: This section should
clearly mention how the data will be collected and
what are the determinants of the proposed study.
Quality control procedures should be involved
and if the procedure is a standard one, it should
be referenced.
Data management and Statistical analysis: This
section deals with data compilation and analysis.
This should indicate the procedures for coding
and entering the data into computer files. It must
mention how the results will be displayed, how
comparisons will be made and which tests will
be used to carry out the statistical analyses in order
to test the stated hypotheses.
Overall Project Management
This section describes the required personnel,
duration of study and follow-up procedures for
study participants where appropriate.
Strengths and Limitations
This section should address the strong points of the
proposed study making it worthy for financial
support. Along with the criticisms of the proposed
study and the compromises made in study design
while writing protocol should be included.
References
This section should include all the references
according to the order of their presentation in the
protocol. For the reference style refer to experi-
mental protocol section.
ETHICAL CONSIDERATIONS (ICMR GUIDELINE)
This section must state the ethical considerations
in accordance with the Helsinki Declaration. It
must indicate the expected hazards of the study,
expected benefits, safety of participants, responsi-
bility for liability for injury to participants and
voluntary consent to participate.
PATIENT INFORMATION SHEET (PIL)
As the name indicates, this information sheet
provides all the basic essential information of the
study to the patients to build up the confidence to
participate in the study. Such as what is the
purpose of the study?, what is the drug used and
its indication? What is the procedure to attend
the trial? Are there any alternatives of diagnosis
 Protocol and Thesis Writing for Postgraduate Students  111
Flow Chart 8.2: Ideal protocol should answer six basic questions stepwise

Why there is need of proposed study?

↓
How the study will perform and what are the rationales for choosing it?
↓
Who will be study population and what will be the sample size?
↓
What are the measurable determinants and outcomes?
↓
What is expected outcome/results and how it contributes to present knowledge?
↓
How these findings helps the community?

or treatment? What are the benefits to participate INVESTIGATORS

into it?, the possible side effect/risk involve, what
will happen if subject want to discontinue the trial?
or if adverse effect occur, then whom to report, etc.
CASE RECORD FORM (CRF)
It is an official data/information form, developed
to record all the relevant clinical data according
to the protocol such as medical history, physical
examinations, vital signs, continued medication,
etc. In the diagnostics, hematological, biochemical,
microbiological, pathological findings or radio-
logical findings should explain separately for
each patient. So, CRF is determined by the design
of the protocol.
PATIENTS INFORMED CONSENT
This is a legal document which ensures that
volunteers/patients participating into the clinical
study is understand the protocol in the simple
language (Hindi/their regional language/
English ) such as, purpose of the study, treatment/
alternative treatment offered and possible benefits
and side effect involved in the study. If volunteers/
patients are ready to participate in the study, they
have to give the consent by sign and countersigned
by the attendant or any other people present at
the site.
BUDGET
A written budget along with the justification
should be included to explain various expendi-
tures.
This section should describe the names of
investigators and role of each investigator in the
study along with their curriculum vitae.
THESIS WRITING
A research thesis is a formal document submitted
for fulfillment or partial fulfillment of a research
course/academic degree. The purpose of the
thesis is to prove that the chosen research
approach was carefully considered, ethically
applied and as a result, the research has made a
useful and original contribution to the current
research knowledge. A thesis aims to:
i. Report each stage of the research process
ii. Provide a comprehensive description of the
relevant background information
iii. Describe in detail methods used to
investigate the research question
iv. Present a discussion of problems arising
during the study
v. Outline the future prospects in the research
area selected
Thesis writing begins with:
i. Front page
ii. Certificate page
iii. Contents page
Front page is the first page of the thesis. It
should contain the title of the research, a statement
outlining the degree, where the degree was taken
and the academic institution with the month and
year. It should include the name of the researcher,
name of principal investigator and date of
112  Practical Manual of Experimental and Clinical Pharmacology
submission. Generally this page should be
designed as per the institute’s format. Then,
certificate page is includes the certification of the
student who have done the study. It includes name
of the institute, title of the study, name of the
candidate student, degree obtain and name of all
investigators with their original sign. Contents
page outlines the structure of the thesis and
chapter contents with relevant page numbers. The
list of tables, figures and appendices should also
be included.
The typical format and components of a
research thesis are:
i. Abstract
ii. Introduction
iii. Review of literature
iv. Project design
a. Study question
b. Aims and objectives
c. Hypotheses
v. Materials and methods
vi. Results
vii. Discussion
viii. References
ix. Appendices
x. Acknowledgments
Note: In a common process, thesis writing is, basically
expansion of the protocol with relevant recent findings in
several research paper and add the findings of the study
with proper results and discussion (in respect to other
author findings and conclusion).
Abstract
Abstract is a concise summary which provides
an overview of the entire thesis including the main
purpose of the study, the way in which it was
conducted and its major results. Although in a
typical protocol format, abstract is written on the
top yet it is written in last after completion of the
studies. It should be kept brief with words not
exceeding 300.
Introduction
A brief description regarding the relevant
background of the research topic being pursued
should be given. It should outline the motivation
and inspiration for doing the proposed study with
sufficient background information through
literatures.
Review of Literature
Thesis should provide the rationale and the
relevant background information related of the
proposed study. Rationale should indicate in a
progressive logical sequence that how the study
question has emerged and corrected. This is done
by reviewing the relevant literature and current
knowledge showing specifically the loopholes in
knowledge which make the study worth doing.
Any preliminary work done in the field of
proposed study should be also included.
Rationale should clearly explain how the
proposed study will contribute to current research
knowledge and will benefit the community with
recent relavant information.
Project Design
Project design involves the outline of the proposed
study and its essential components are:
Study Question
This addresses the issue which one has pursued
during the proposed study. The research topic is
generally formulated into a question, known as
study question. The study question must clearly
explain the objectives of the proposed study. The
necessary steps required to answer the study
question should also be included.
Aims and Objectives
Study question indicates the aim or the goal, which
is generally very vast. So this is broken down into
smaller questions known as objectives.
Hypotheses
Hypotheses are distinct and clearly defined
outcomes for any proposed study. Objectives
should be stated in the form of hypotheses.
Hypotheses can be written as statements to be
 Protocol and Thesis Writing for Postgraduate Students  113
refuted, i.e. no difference between experimental
groups and are referred to as null hypotheses.
Alternate hypotheses are statements assuming the
real difference between the experimental groups.
Thus the research hypotheses provide information
about the comparisons which will be needed to
answer the study question and establish the
format with in which one will apply the statistical
tests while interpreting the results.
MATERIALS AND METHODS
Materials/Chemicals
This section should describe the chemicals or
reagents used during the study.
Study Population
Study population means the target group in which
the study was carried out. This can be human
subjects, experimental animals or microorganisms.
Sample Size and Statistical Power
Thesis must indicate how big the target group has
to be in order to precisely answer the study
question. This should include the assumptions
made for calculation and a table of calculation of
sample size and power.
Methodology
Thesis should clearly indicate how the proposed
study was performed and which methods were
adopted. It should mention the selected design of
study along with the rationale of choosing the
proposed design. Ideally, this is the first step to start the
thesis writing. This section should clearly mention
how the data was collected and what were the
determinants of the proposed study. Quality control
procedures should be involved and if the procedure
is a standard one, it should be referenced.
Data Management and Statistical Analysis
This section deals with data compilation and
analysis. This should indicate the procedures for
coding and entering the data into computer files.
It must mention how comparisons will be made
and which tests were used to carry out the
statistical analyses in order to test the stated
hypotheses.
RESULTS
This section should clearly mention the research
findings supported with tables, figures and
graphs, etc. This should be totally convincing in
the sense that everything has been done to answer
the research question and supported with the
statistical test applied.
DISCUSSION
This mentions interpretations of the project
findings in detail. This should include the
variables influencing the research findings and
the newer insights which were revealed along
with the data supporting the same.
CONCLUSION
This should include very precise statements
encapsulating the project findings correlating
them to the study question posed in the beginning
of the thesis.
REFERENCES
This section should include all the references
according to the order of their presentation in
the protocol as per the style mentioned by the
institute.
APPENDICES
Material relevant to the development of the thesis
like preparation of solutions, buffers, samples of
questionnaires, etc. should be included in this
section.
ACKNOWLEDGMENTS
In this section, one should acknowledge the
people for their contribution in pursuing the
research project. This may be also given after the
front page at the starting of thesis.
114  Practical Manual of Experimental and Clinical Pharmacology

SUGGESTED READING
Note: Before writing the protocol, student/researcher
should perform a small sample study to check the
hypothesis made, is actually working. These type of study
is knwon as “Pilot study”. It is defined as “study conducted
on the small number of animal/human to check the
hypothesis made, procedure and technique used in the
study with relevant supported background information. If
the data collected is supportive, then start the protocol
writing and then actual experiment and finally thesis writing
and submission.
1. Barletta JF. Conducting a successful residency
research project. Am J Pharm Educ 2008;72(4):92.
2. Betkerur J. Guideline for writing a research project
synopsis or protocol. Indian J Dermatol Venereol
Leprol 2008;74(6):687-90.
3. Van Way CW. Writing a scientific paper. Nutr Clin
Pract 2007;22(6):636-40.
4. Betkerur J. Guideline for writing a research project
synopsis or protocol. Indian J Dermatol Venereol
Leprol 2008;74(6):687-90.
 Toxicology Study
9
Evaluation and development of a new drug
undergoes numerous procedures in order to get
the safe and efficacious drug. Apart from the other
studies like pharmacological and pharma-
cokinetic evaluation, toxicity study (also known
as hyperpharmacology) is also an important and
ethically necessary study during the drug
development process. There is species-to-species
variation in the pharmacological responses hence
during the toxicity study always two or more
species are preferred. Protocols must be reviewed
and approved by an Institutional Animal Ethics
Committee for the experiment.
Objective of the Toxicity Study
1. Identify any toxic substance prior to clinical
use
2. Qualitative* and quantitative# assessment of
drug use
3. Characterize the cumulative toxicity of the
drug under study (specially, in subchronic or
chronic study but not predicted by single dose
toxicity)
4. To allow a careful selection of doses for further
studies (including, carcinogenicity studies)
5. Different types of dose identification can be
done. For example maximum lethal dose (MLD),
Lethal dose (LD50), maximum tolerated dose
(MTD), minimum effective dose (ED50), etc.
6. Therapeutic index can be calculated (LD50/
ED50)
* Nature of effect of drug seen in the target organs
# Subacute/subchronic study: Repeated dose from
Drug level (plasma and tissue levels) at which,
drug effects definitely seen and not seen
General Principles
The principle of the toxicity study is to assess all
the relative toxic effects of the drug regarding its
dose, duration and its effect on the organs. Hence,
these studies not only provide adequate thera-
peutic margin of initial dose levels and duration
of dosing, but also give guidance for any special
measures to be taken in initial clinical trials.
In a large set-up, conducting toxicity studies
should comply the following important require-
ments,
1. Should follow the Good Laboratory Practice
(GLP)
2. Studies should be performed by suitably
trained and qualified staff
3. Instruments should be calibrated and
standardized properly and of adequate
capacity.
4. Standard operating procedures (SOPs) should
be followed.
Documentation part is proper and the most important to
save and anticipate should be the results. All documents
like approval of protocol, raw data, draft report, final report
and histology slides and paraffin tissue blocks should be
preserved for a minimum of 5 years after marketing of the
drug
Toxicological study involves toxicodynamics
and toxicokinetics. The toxicity studies are
conducted as per the duration of clinical use of
the drug. So, the study is mainly divided into the
three categories, i.e.
Acute toxicity study: Single dose toxicity study
2 weeks to 6 months
116  Practical Manual of Experimental and Clinical Pharmacology
Chronic toxicity study: Repeated dose study from
6 months to 12 months or even up to 2 years
Acute Toxicity Study
Single-dose Toxicity Studies
Animal: 2 rodent species (mice and rats); in each
group at least 5 animals of either sex
Route of administration*: Same as intended for
humans
*If the intended route of administration in humans is only
intravenous, at least one more route should be used in
one of the species to ensure systemic absorption of the
drug
Doses: At least three graded doses
Treatment: Given in a single bolus or several doses
or by continuous infusion within 24 hours
Animals should be observed for 14 days
Observation:
1. Signs of intoxication
2. Effect on body weight
3. Gross pathological changes
Note: It is desirable to include histopathology of
grossly affected organs. (if any)
Calculate the minimum lethal dose (MLD) and
maximum tolerated dose (MTD) and lethality dose
(LD50)
Selection of oral dose: Limit of 2 g/kg or 10 times
the normal dose that is intended in humans,
whichever is higher is recommended for oral
dosing.
Repeated Dose Toxicity Studies
Subacute/subchronic or chronic toxicity study
falls under these category and being use in the
drug which are intended for the repeated dose in
the clinical setting.
Subacute/Subchronic
Aim of the study is to identify target organ toxicity
and establishment of MTD for subsequent studies.
Animal: At least two mammalian species (1rodent
+ 1 non-rodent)
(Younger and still growing animals are
preferred at the initiation of a subchronic study)
Treatment and duration: Given in 14, 28, 90 or 180
days (duration depends on therapeutic indication)
• Group I; Control group—Normal or vehicle
group
• Group II; Treatment group—drug treatment
group
Route of administration*: Same as intended for
humans
Doses: At least three graded doses
Selection of Animals
• Rodents must be about six weeks and not more
than eight weeks.
• Non-rodent (e.g. dog) treatment should begin
at 4-6 months but no more than 9 months.
Selection of Dose
• Highest dose should produce observable
toxicity
• Intermediate dose should cause some symptoms,
but not gross toxicity or death, and should be
placed logarithmically between the other two
doses
• Lowest dose should not cause observable
toxicity.
Methods
• One rodent (6-10/sex/group; for 14 days) (15-
30/sex/group; for 90 and 180days)
• One non-rodent species (2-3/sex/group; for
14 days) (4-6/sex/group; for 90 and 180 days)
Observation
• Sites of injection should be subjected to gross
and microscopic examination if given
parenterally.
• Non-rodent species: Electrocardiogram and
fundus examination

Table 9.1: Analytical parameters which should be assessed
after the subscute/ subchronic/ chronic, toxicity test, listed
as follow:
• Liver function tests: Total bilirubin, direct or indirect
bilirubin, bile acids, ammonia
• Hepatocellular leakage enzymes: AST, ALT, SDH,
LDH
• Muscle parameters: Creatine kinase (CK), AST, ALT,
LDH
• Pancreatic parameters: Amylase, lipase
• Lipid parameters: Cholesterol, triglycerides Cholestatic
enzymes: AP, GGT
• Calcium, potassium: Often influenced by kidney
function, kidney parameters should be evaluated
concurrently
• Kidney parameters: Urea nitrogen, creatinine urine
specific gravity should be evaluated concurrently
when evaluating renal function
• Other, observation parameters should include
cage-side observations, body weight changes,
food/water intake, blood biochemistry,
hematology, and gross and microscopic
studies of all viscera and tissues (Table 9.1).
Chronic Toxicity Study
• Animal: 2 species (a rodent and a non-rodent).
• In rodents chronic studies are usually for 6
months to 2 years.
• In non-rodents chronic studies are usually for
1 year but may be longer.
Selection of Dose
• Highest dose should produce observable
toxicity
• Intermediate dose should cause some symptoms,
but not gross toxicity or death, and should be
placed logarithmically between the other two
doses
• Lowest dose should not cause observable toxicity
The length of drug treatment or exposure is
mainly dependent on the intended period of use
in humans.
Observation
• Sites of injection should be subjected to gross
and microscopic examination if given
parenterally.
Toxicology Study  117
• Non-rodent species: Electrocardiogram and
fundus examination
• Other, observation parameters should include
body weight changes, food/water intake,
blood biochemistry, hematology, and gross
and microscopic studies of all viscera and
tissues (Table 9.1).
Other specific toxicity studies are depend on
the nature of drug uses such as teratogenicity
study, male fertility or female fertility toxicity tests,
etc. and some depend on drugs site of use such as
local toxicity (dermal and photoallergy or dermal
phototoxicity, vaginal, ocular or inhalational
toxicity test, etc.) is done to evaluate the drug in
rodents or non-rodents.
Lethality Testing and Calculation
Lethality testing or determination is an essential
regulatory requirement for a drug or biological
products before clinical study. It is basically acute
toxicity testing in which death is considered as a
single end-point and other data or other
behavioral parameters are not taken under
consideration. Hence, lethal dose of drugs may be
defined as “dose of a given drug which produces
mortality in the treated animal, especially in most
sensitive species model.” The most commonly
calculated lethal dose is median lethal dose or
lethal dose 50 (LD50). LD50 is defined as the “dose
of a given drug which produces mortality in 50% of
total treated animal preferably in the most sensitive
species model”. LD50 was discovered by Trevan and
Behrens, who identified and read the midpoint
on the mortality dose response curve. Lethality
testing is preferred over subchronic or chronic
testing because of ease of end-point selection,
small procedure and still real and concerned
method. For a broader concern, lethality testing is
done mainly on the 3 doses of selection such as
one dose which has 100% lethality, second,
produces marginal lethality and last dose is taken
in between of first 2 doses. Route of administration
is same in all testing animal and preferably,
human intended route. Toxicity is varying
according to route of administration. There is a rule
of thumb, i.e. the i.p. LD50 is roughly 30% of oral
118  Practical Manual of Experimental and Clinical Pharmacology

and the i.v. is 10% of that of the oral. Duration of 4. Other factors like species, strain or substrain,
the lethality testing ranges from 7 days to 14 days. age, weight, and sex of the animals or even,
Note: Generally, LD 50 is calculated in the animals husbandry condition may interfere with result
(preferably rodents like mice or rat) whereas only one outcome.
direct human LD50 values was determined in “Nazi human There are two methods of calculating the LD50,
experimentation”; is reported.
one is mathematical method and second is
Precautions should be taken while performing graphical method.
the experiment because animals receive a single
calculated dose,
Mathematical Method
1. Animal must be dosed on the same day and
preferably at the same time. Karber’s method is very commonly used because
2. Great care must be taken while calculation, it is simple and does not need to plot the dose
preparation and delivery of the test drug. response curve. It is simplified in the example
3. So, keep laboratory conditions constant for given below,
each animal during the experiment (animal
model chosen must be most sensitive).
Example:
Exp. Dose Dose No.of No. of Mean mortality* DD x MM
Group (mg/kg) (difference (DD)) animals (n) dead animal (MM)
1 3 — 10 0 (A) - —
2 5 2 10 2 (B) 1 2
3 10 5 10 3 2.5 12.5
4 12 2 10 5 4 8
5 15 3 10 7 6.5 19.5
6 20 5 10 10 8.5 42.5
Σ(DD × MM) = 84.5

*Mean mortality = difference of two adjacent no. of dead animal/2

e.g. = (A +B)/2 = (0 + 2)/2 = 1
Σ(DD × MM) = 84.5; then value is divided by the no. of animal in the group (here, n = 10)
Hence, value obtained is 84.5/10 = 8.45
Finally, this value is sustracted from the minimum dose which produces the 100% mortality, i.e. 20 mg/kg
So, LD50 = 20 – 8.45 = 11.55 mg/kg

Graphical Method
This method is well described by the Miller
and Tainter (1944) as well as Litchfield and
Wilcoxon (1949). This is depending on the toxic
log dose response curve and the interpretation
of result is done by the curve. The preferred
method is, first convert the percentage response
into the probit, then plot the curve log dose
on ‘x’-axis and ‘y’-axis contains probit scale
(Fig. 9.1).
The LD50 is the antilog (ld) value which falls
on the ‘x’-axis. Fig. 9.1: Determination of LD50

Note: Probit analysis is a type of regression used to
analyze binomial response variables. It is most precise
but requires at least two groups of partial responses (i.e.
mortality greater than 0% but less than 100%). Initially,
Bliss (1939) developed the idea of probit through
transforming the sigmoid dose-response curve to a straight
line dose-response curve which was further refined by
Finney (1952)
SUGGESTED READING
1. Armitage P, Allen I. Methods of estimating the LD50
in quantal response data. J Hygiene 1950;48:398-422.
2. Bliss C. Some principles of bioassay. Am Scientist
1957;45:449-66.
3. Finney D. The median lethal dose and its estimation.
Arch Toxicol 1985;56:215-18.
Toxicology Study  119
4. Finney DJ, Ed. Probit Analysis. Cambridge, England,
Cambridge University Press 1952.
5. Leo T M van der Ven, Aart Verhoef, Ton van de Kuil,
Wout Slob, Pim E G Leonards, Theo J Visser, et al. A
28-day Oral Dose Toxicity Study Enhanced to Detect
Endocrine Effects of Hexabromocyclododecane in
Wistar Rats. Toxicol Sci 2006;94(2):281-92.
6. Lorke D. A new approach to practical acute toxicity
testing. Arch Toxicol 1983;54:275-87.
7. Phillips J, Gibson W, Yum J, Alden C, Hurd G. Survey
of the QSAR and in vitro approaches for developing
non-animal methods to supersede the in vivo. LD50
test. Fd. Chem. Toxicol 1990;28:375-94.
8. Ranjit Madhukar Bidhe, Sangita Ghosh. Acute and
Subchronic (28-day) Oral Toxicity Study in Rats Fed
with Novel Surfactants. AAPS PharmSci 2004; 6 (2)
Article 14.
 Biomedical Waste Disposal
10
It is important for the student in the laboratory to
note that work is always being associated with
the potentially toxic, flammable, irritable, painful
or the carcinogenic substances. So, the dangerous
situation can be developed unexpectedly.
Therefore, one should be aware about the
procedure, careful use of chemicals in the
laboratory and most importantly the other
important safety concern, facilities and emergency
action. Moreover, it all includes “Good Laboratory
Practice (GLP)” (Flow Chart 10.1).
In the pharmacology laboratory, students
mainly deal with the animals, chemicals and
different types of instruments. So, the students
should take care of the all the important points
while working into the laboratory such as:
With Respect to Drug/Chemicals
1. Never work alone/single in the laboratory.
2. Read the instructions, so, that student should
be familiar with the chemical and physical
properties such as reactivity, flammability,
toxicity, carcinogenicity and the disposal of
drugs or chemicals used in the experiment.
3. Use the apron, gloves, and cap while doing
the experiment.
4. Wear safety glasses with wide side shield to
protect the eye.
5. Avoid mouth suction to fill pipette or to start
siphons
6. Always be familiar with the safety equip-
ment like fire extinguisher, eye washes,
chemical spill kit, first aid supply, etc. in the
laboratory.
7. Avoid personal work like reading, eating,
drinking and smoking in the laboratory.
8. Always use the clean and dry glassware*
With Respect to Experimental Animals
1. Handle the animal with care (may get aggre-
ssive, if get agitated)
2. Avoid forceful hold to the animal
3. Laboratory should be dim lighted while
performing the experiment e.g.: CNS experi-
ment, etc.
4. For any surgical procedure, use the sterile
instruments (syringe/needle, forceps, scissor,
etc.) or nicely wiped with 70% alcohol
5. Waste material should be disposed according
to the labeled colored bags.
*Cleaning of glassware
• Most purposes: 0.5% of detergent in water followed by
6-10 water rinses whereas final rinse is with distilled or
deionised water.
• Glassware contaminated with metal ion: Rinse with
concentrated nitric acid, then extensively rinse with
the water
• When Glassware needs to get dried quickly: Rinse
with the acetone which quickly evaporates and dries
quickly
• Glass or quartz cuvettes: Carefully clean in 0.5%
detergent water in a cuvette washer or in a sonicator
bath (Never use ethanolic KOH or other strong
base which may cause etching)
The use of chromate solutions to clean glassware is
PROHIBITED due to extreme toxicity of chromates
including carcinogenicity.

Flow Chart 10.1: To minimize the risk in the laboratory
Waste Disposal Management
There are different types of material used in the
pharmacology laboratory such as from simple
chemicals to the animals. So, the minimization and
management of wastes such as chemicals, radio-
active materials, animal tissues/organs, cotton,
sharpen blades, etc. from the experimental procedure
should be maintained to ensure the significant
reduction in generation of hazardous, radioactive,
and mixed wastes in the environment. There are
guidelines to dispose the contaminated bio-
degradable or non-biodegradable wastes to reduce
the contamination and infections in the general
Biomedical Waste Disposal  121
environment. The wastes material is collected in the
respective “colored bags” in the laboratory and send
to the central disposal center to proper disposal of
biomedical wastes (Table: 10.1).
The other corrective measure to reduce the
biomedical wastes is improvements in laboratory
techniques, reduce use of toxic substance or check
for any alternative less/non-hazards chemicals/
materials.
The waste materials collected in the color
coded polythene bag (double bagged if required),
then send for incineration or other method of
destruction.
122  Practical Manual of Experimental and Clinical Pharmacology

Table 10.1: Different type of waste disposal bags and their contents
Bag type Contents Example of contents
Yellow Contains infectious and related waste. Cotton, dressing , plaster of Paris(POP), human or
Biodegradable wastes (decomposed by animal anatomical waste, human tissue, organs, body
bacteria or other living organisms) parts, microbiology and biotechnology waste, etc.
Blue Sharp things which can be responsible Glass syringes, needles, surgical blades, shaving blades ,
for infectious diseases etc.
Black Contains non-infectious and related Outdated medicines, contaminated medicines, discarded
waste medicines, packing cover of medicines, waste paper,
general non-infected waste, leftover food and peels of
fruits, etc.
Red Contains infectious and related waste Ryle’s tube, catheter, urinary catheter, suction catheter,
like surgical waste. chest drainage catheter, IV set, blood set and glucose
bottle and contains low level radioactive waste
Violet Cytotoxic drugs and related waste Cytotoxic drugs and related waste, contaminated sharps,
contaminated animal carcasses and cageLinings

SUGGESTED READING 3. National Research Council, Committee on

1. Lunn George, Eric B Sansone. Destruction of
Hazardous Chemicals in the Laboratory, 2nd
Edition. New York, NY: John Wiley and Sons. 1994.
2. Lunn George, Eric Sansone. Ethidium bromide:
Destruction and decontamination of solutions.
Analytical Biochemistry 1987;162:453-58.
Hazardous Substances in the Laboratory. Prudent
4. Practices in the Laboratory. Washington DC:
National Academy Press, 1995.
5. Washington State Department of Ecology. Dangerous
Waste Regulations, pp 173-303 WAC. Publication
No. 92-91 Olympia, WA: Department of Ecology
Publications, 2003.
 Biostatistics in
11 Pharmacology

INTRODUCTION grouped (Summarized into the Flow Chart (Fig.

11.2) and overall description in the Table 11.1)
This chapter deals with the brief introductory Generally, data show picture of the variability and
biostatistics which is considered to be an alien in central tendency.
the subject but logically important. Students may Data can be taken from the population or it
feel anxiety in many parts to understand and may be derived from the sample. Population is
implement it. So, the chapter deals with the basic nothing but the integral set of subjects (e.g.
understanding and implementation, briefly.
complete set of PGIMER or AIIMS patients or total
Before starting the chapter students should be
animals stored in animal house), whereas the
motivated enough to learn a subject that is
sample denotes the representation from the entire
perceived to be difficult and dry.
population which is selected randomly from the
Statistics is an art and science which mainly
population, but sample can never be a replication
deals with the ways to gather and analyze data
of population, e.g. patients coming in the OPD’s
and statistics which deals in the health and
of cardiology or neurology. Patients from the
medical sciences is commonly known as
cardiology and neurology represent the complete
Biostatistics. So, the biostatistics mainly differs
set of PGIMER and AIIMS patients. Hence, sample
from the core statistics in that it consists of steps
like, an assumption or hypothesis, then gathering is just a subset of population. The difference
data and thereafter statistical analysis is done to between the sample value and population value
make decision(s) or inference about the event. is defined as sampling error.
Hence, biostatistics is very important to design
an experimental study, its aim and objectives and
nature of the data obtained in the experiments. It
is thought that, French mathematician, “Pierre
Louis” developed and recommended his
“numerical method” for the assessment in the
experimental medicine.
Limiting to the basics, students should know
about the data obtained during the experiment and
its classification, distribution and most importantly
its analysis to conclude the experiment.

Data and its Types

Broadly, data is classified into two groups, namely
qualitative and quantitative which is further sub- Fig. 11.1: Population and sample
124  Practical Manual of Experimental and Clinical Pharmacology
Fig. 11.2: Classification of data
Primary data: Data collected directly from the
research experiment (first-hand experience)
Secondary data: Data from other sources like
published data, data collected by other
individual, or any other indirect sources
Non-numerical data: Data which shows the
quality but not quantity of given observation such
as smoker/non-smoker, normotensive/
hypertensive or mild asthma, moderate asthma
or severe asthma, etc.
Numerical data: Data which directly shows the
quantity of parameters assessed or observation
in numbers such as BP measurement (120/80),
biochemical levels, hematological estimation, etc.
Nominal data: Data which is countable but not
ordered or measure, e.g. male/female, yes/no,
old/new, alcoholic/non-alcoholic, etc.
Ordinal data: Data which observations can be
put in order or have a rating scale, e.g. mild/
moderate/sever, etc.
Discrete data: Data which show distinct and
separate observation such as blood group (O, A,
B, AB), no. of patients in doctors surgery, gender
(male, female), etc.
Continuous data: Data which have values within
a finite or infinite interval observation. For
example height, weight, temperature, Hb count,
RBS/WBC counts, creatinine level in urine, etc.
Interval data: Data provide a scale which shows
a range or distance between two adjacent units
of measurement or observation but the zero-point
is arbitrary, e.g. temperature, year, etc.
Ratio data: Data which is a continuous, ordered
and constant scale. Ratio data has a natural zero.
For example blood pressure, weight, height, Hb
count length, age, creatinine count, etc.
Errors in Data
Random error: Commonly, related to imprecision
of measurement done by the experimenter.
Different people may record different values of
same data, which are close to one another. This
type of error is usually on either side of mean and
close to the average or real value.
Systematic error: This error occurs due to
inaccurate or uncalibrated instruments, e.g. BP
apparatus, hematogram, balance, tape, etc. which
causes to persist recurrence. So, this type of error
can be avoided by the recalibration of all
measurement instruments, i.e. BP apparatus,
hematogram or other biochemical instruments,
etc.
 Biostatistics in Pharmacology  125

Table 11.1: Classification of data and brief introduction

Class Subclass Definition Examples Test applied Descriptive method
or representation
Qualitative Nominal Shows unordered Diabetic/non- Non-parametric Bar chart, Pie chart,
(Categorical) category diabetic; Smoker/ (Fisher exact test
non-smoker; Male/ or chi-square test)
female
Ordinal Shows ordered Mild/moderate/ Non-parametric Bar chart, Pie chart
category severe; Small/ (Fisher exact test
Data or Variables

smaller/smallest or chi-square test)

Quantitative Discrete Countable or BP reading, etc. Parametric test Box plot, Scatter
(Numerical) finite number (t-test,correlation plot (2 variables),
and Regression) Dot diagram
Histogram, Stem-
Leaf and Quantile-
Quantile Plot, etc.
Continuous Value lie in Age, height, etc. Parametric test Box plot, Scatter
(Interval/ continuous range (Mean,SD, plot (2 variables),
ratio) ANOVA, z- and Dot diagram
t-tests and Histogram, Stem-
Regression) Leaf and Quantile-
Quantile Plot, etc.

Different Measures of Data So, (7 + 8)/ 2 = “7.5” is median value for above
mentioned data
Mean: This is nothing but the arithmetic average
of any given data. Mean is denotes by the “x–” in Mode: It is very frequently occurring event in a
sample and “µ” in population. given data.
e.g.: 2, 2, 3, 3, 4, 2, 5, 6, 7, 7, 8, 4, 9, 3, 4, 8, 9, 3,
x
. Mean = In the given data ‘3’ is repeated 4 times and
n it is most frequently occurring number. So,
where, x = Observations; “3” is the mode of given data
n = Total number of observations
e.g.: 2, 2, 4, 7, 7, 8, 10, 11, 11,16; Mean of given Measure of Dispersion
data is calculated as:
(2+2+4+7+7+8+10+11+11+16)/10 = 7.8 Range: “Range” = Highest value – Lowest value
That means, range gives the value between the
Median: This is the middle value of any given data, minimum and maximum values of a variable.
when it is arranged in the ascending or descending Understanding this statistic is important in
order. understanding your data, especially for
e.g.: 1, 2, 2, 4, 7, 7, 8, 10, 11, 11, 16 (Odd value) management and diagnostic purposes.
Hence, in the odd numbers of samples, e.g.: 2, 2, 4, 7, 7, 8, 10, 11, 11, 16
middle value is treated as the median. So Highest value= 16 and Lowest value = 2
“7” is median in above data Hence, Range = 16- 2 = 14
1, 2, 4, 6, 7, 8, 10, 11, 12, 16 (Even value)
In the even number of sample, the mean of two Variance: It is a measure of an event that departs
middle values is considered as median value. from expectations, i.e. the dispersion of a group
126  Practical Manual of Experimental and Clinical Pharmacology
data around their mean value. It is mean of all
deviation score in the data, i.e. it is calculated as
where X = individual score
µ = mean of the total score (popu-
lation) [in sample it is denoted by
X]
N = number of total observation score
(sometime (N-1) is used instead of
N to eliminate bias
Standard deviation: It is the measure of dispersion
or variability of a data from its mean value, which
is calculated by the square root of variance. Hence,
it is calculated by the formula
where X = individual score
µ = mean of the total score (popu-
lation) [in sample it is denoted by
X]
N = number of total observation score
(sometime (N-1) is used instead of
N to elimnate bias
Standard Error of Mean (SEM): This is the measure
of variability, which is defined by the “S/√n”.
Difference between the standard Deviation (SD)
and Standard Error of Mean (SEM): Standard
deviation (SD) describes variation in values of
individuals which is denoted by ‘S’ whereas Standard
Error of Mean (SEM) describes variation in values
of a statistic from one conduct of study to the next
and it is the measure of S/√n.
Types of Data Distribution
Overall data distribution is divided into the two,
first continuous variable distribution (normal
distribution) and the discrete variable distribution
(Binomial and Poisson distribution).
Normal (Gaussian) Distribution
This distribution is also known as Gaussian
distribution. (Named after German mathematician,
Gauss (1777-1855)). In such distribution, variables
have the tendency to cluster around the central
value with a symmetric distribution on the either
side. (Positive and Negative)This type of
distribution is plotted as a normal distribution
curve. The mean, median and the mode
considered to be equal in such distribution. The
normal distribution curve has a central tendency
and a degree of dispersion. Various methods of
analysis are available to make assumptions about
normality, including‘t’-tests, analysis of variance
(ANOVA) and correlation and regression. Skew
is zero in normal distribution but when tail skew
is towards right it is known as positive skewed
and when it skew towards left it is known as
negative skewed and therefore measure of central
tendency differs in this context (Fig. 11.3).
Such distribution can be made to normal
distribution by a suitable transformation.
Logically, transformation which makes data
follow a normal distribution often makes the
variance uniform as well, and vice versa. Data
can be transformed by taking the logarithm, square
root, reciprocal, or some other function of the data.
In the normal distribution, 68% of data fall
between 1 SD, 95.4% of data fall between 2 SDs
and 99.7% of data fall between 3 SDs. Hence, 95%
of population falls within the 1.96 SD (Fig. 11.4).
Kurtosis of normal distribution found to be
zero. (Kurtosis means how distribution is selected)
Relation between the mean, median and mode is
well established in the normal distribution Figure 11.3.
Binomial Distribution
Binomial distribution is applicable to those data
where there are only two possibilities such as
smokers or non-smokers; diabetics or non-
diabetics; male or female; survived or not survived.
This type of data have properties like, only two
outcome possible, have fixed number of obser-
vation and each event have constant probability
to occur. Hence, the binomial distribution curve
has two peaks.
But, in the case of a large observation time
and corresponding small event, binomial
distribution calculation is very tedious and not

Fig. 11.3: Representation of data
Fig. 11.4: Data and standard deviation
Fig. 11.5: Binomial distribution curve
an appropriate method. Hence, describing and
analyzing such event requires “Poisson distri-
bution”. These two, i.e. Binomial and Poisson
distributions are most important discrete probability
distributions (Fig. 11.5).
Biostatistics in Pharmacology  127
Poisson Distribution
This is commonly used distribution in health
sciences defined the distribution of the number of
occurrences “x” of some random event in an
interval of time or space. For example, recruitment
of patients into the clinical trial Phase III studies:
Investigator recruits patients daily according to
inclusion/exclusion criteria. If investigator is
interested in looking the patients enrolled for the
study on particular time say, 1-2 months of study
begin, and then data obtained has the poisson
distribution. Hence, it calculates the probability
of event in a fixed interval. In this case event may
occur randomly and independently. This is
calculated by an exponential formula (Fig. 11.6).
Statistical Tests
Statistical test is a mathematical calculation or
analysis of observed and collected data from the
population or sampling. The result obtained, refers
the statistical power of a test. Therefore, statistical
power is defined as a measure of the probability
that a statistical test rejects a false null hypothesis,
or in other words, the probability of finding a
significant result, if there is any difference. The
higher the power of a statistical test, the more likely
128  Practical Manual of Experimental and Clinical Pharmacology
Fig. 11.6: Poisson distribution
one is to find statistical significance, if the null
hypothesis is false.
Note:
• Type I error (α error): When an experimenter concludes
that there is a difference between the analyzed groups
when, in fact, there is no difference (False positive)
• Type II error (β error): When an experimenter concludes
that there is not a difference between the analyzed
groups being studied when, in fact, there is a difference
(False negative)
As we have seen that there are different types
of data, and for analyzing such data, statistical
tests are mainly divided into two groups, one
which applies on the normally distributed data,
Fig. 11.7: Classification of biostatistical tests
i.e. quantitative numerical continuous data and the
second which applies on the ordinal or categorical
data or qualitative data (Fig. 11.7).
 Important Points to Remember
Equivalent Parametric and non-parametric tests
Parametric test Non-parametric tests
Unpaired student‘t’ test : Mann- Whitney U test
Paired ‘t’ test : Wilcoxon signed rank test
One way ANOVA : Kruskal- Wallis test
Repeated measures ANOVA : Friedman’s test
Pearson correlation coefficient : Spearman rank order
These non-parametric tests are the alternative to the
corresponding parametric tests in which data is not normally
distributed.
Note:
Assumption of the null hypothesis is tested by the p value
which represents the risk observed in the experiment due
to chance. The p value determines the significance or
non-significance of a result. (Measure of probability) If one
fixed the significance level at p value <0.05 that means
chances of 5 observation which may fall outside the range
i.e. uncertainty of 5%. Other p values which are commonly
set as significance levels are 0.01 or 0.001. Limitation of
the p value is that, it may influence by the study result or
do not show the magnitude of difference in the comparing
groups.
The alternative to the p value is 95% confidence interval
(CI) which represents that 95% chance of the cases to fall
into the range. Most commonly 95% and 99% of the
 Biostatistics in Pharmacology  129

confidence interval is in use. It has two values, lowest and distribution and one want it to do so,
highest value which is known as lower and upper transformation of data into logarithm or reciprocal
confidence limits. of all values may be done.
The important difference between the p value and
confidence interval is that confidence interval represents
clinical significance whereas p value indicates statistical Short Description of Commonly Used Tests
significance; therefore in many of the clinical study, CI is
preferred instead of p value.
The selection of the appropriate statistical test is
largely determined by the nature of the
Parametric tests: These are the tests which are experiment, the question being asked and the
mainly used to estimate numerical continuous types of variables being analyzed. In the text we
values which are based on normal sampling are providing a basic overview of several tests
distribution. So, there are few criteria for the which is widely used in statistical procedures to
parametric tests such as data should be at the analyze a data.
numerical scale, the distribution of the entire
population should be normal, the samples have Students‘t’ Test
the same variance, observation within the group Students‘t’ test mainly deals with the one or two
is independents and lastly the samples are
means comparison. Test tells about either
randomly drawn from the population. If the data
acceptance or rejection of the “null hypothesis”
is not distributed normally, i.e skewed data, then
between two means. In, 1908, ‘t’-test was
these can be transformed into the other form such
introduced by William Sealy Gosset, a statistician
as logarithm (log10). Sometime, data can be
working in Dublin. This test is divided into two
transformed into the natural logarithm (log 2) to
group according to the samples used for the
calculate the mean and standard deviation
analysis.
(antilogarithm of these estimated mean known as
geometric mean) and then analyzed by the
parametric tests. The advantages of the parametric
test is that it may be analyzed either way, inter
group or intra group and have more statistical
power than the non-parametric test.

Parametric tests can be applied to the data which

• Should be on numerical scale
• Should be normally distributed
• Observation should be independent in a group
Note: If the above mentioned criteria are not fulfilled, then
non-parametric tests can be applied
Requirement for the non-parametric test
• Data should be from the continuous distribution and on
at least ordinal scale
• Observations within a group are independent
• Sample have been drawn from the population
Non-parametric test: When the requirement of
parametric test is not fulfilled, then non-
parametric tests are the alternative. It does not
assume the normal distribution hence, it is also
explained as the “distribution free tests”. In some
situations when a data does not follows normal
In the ‘t’ test, sample size of less than 30 (n < 30)
is analyzed and the sample size n>30 is analyzed
by the ‘z’ test and the group variance may be
calculated by ‘F’ test which is the ratio of the
(variance 1/ variance2). ‘t’ distribution curve is
similar to that of the normal curve.
‘t’ tests are used to test mean differences
between two groups. In general, they require two
independent variables (e.g. an experimental and
a control group) and a single continuous
dependent variable.
For example, t tests can be used to test for mean
differences between experimental and control
130  Practical Manual of Experimental and Clinical Pharmacology

If you analyze the means of two groups by

ANOVA, you get the same results as doing it with
a t-test. Although, same data can be analyzed by
using a series of t-tests to examine the differences
between more than two groups, but this approach
would not only be less efficient, but it would add
Fig. 11.8: Data in normal distribution and ‘t’-distribution experimental error (Type I errors). Interestingly,
despite its name, the ANOVA works by comparing
groups in a randomized experiment, or to test for the differences between group means rather than
mean differences between two groups in a non- the differences between group variances.
experimental condition. “Analysis of Variance” comes from the procedure
which uses variances to decide whether the means
Unpaired ‘t’-test are different or not. There are numerous different
Group I Group II variations of the ANOVA procedure to choose
(Control) (Treatment) from, depending on the study hypothesis and
research design. For example, a one-way ANOVA
is used to compare the means of two or more levels
of a single independent variable. So, we may use
an ANOVA to examine the differential effects of
three types of treatment on ischemia.
Paired ‘t’-test One way ANOVA
Group I Group I Group II Group III
(Control) (Treatment 1) (Treatment 2)
Pretreatment Post-treatment

Treatment of Ischemia

Alternatively, multifactor ANOVAs can be used

Unpaired ‘t’-test Paired ‘t’-test
when a study involves two or more independent
1. Two independent group 1. Sample group is compared variables. For example, Factorial design of 2 × 3 to
are compared e.g.: before and after inter-
vention/treatment examine the effectiveness of the different
2. Intragroup variability is 2. No intragroup variability treatments (Factor 1) and high or low fat diet in
present reducing symptoms of hypertension (Factor-2).
3. More sample size require 3. Comparative lesser sample Another, type of the ANOVA is the multiple
to increase the power of size is required to achieve
the stury the same power of the
analysis of variance, or MANOVA. The MANOVA
study is used when there are two or more dependent
variables that are generally related in some way.
Using the previous example, let’s say that mea-
Analysis of Variance (ANOVA)
suring the effect of the different treatments, with
It is the extension of the t-test, major difference or without exercise, on hypertension measured in
between a t-test and an ANOVA is that the several different ways. Although we could
ANOVA can compare means across more than conduct separate ANOVAs for each of these
two groups or conditions. Therefore, a t-test is just outcomes, the MANOVA provides a more efficient
a special case of ANOVA. and more informative way of analyzing the data.
 Biostatistics in Pharmacology  131

Regression or ordinal because chi-square is a test of

proportions. Also, because it compares the
Linear regression is a method of estimating or
categorical responses between two or more
predicting a value on some dependent variable
groups, the chi-square statistic can be conducted
given the values of one or more independent
only on actual numbers and not on pre-calculated
variables. Like correlations, statistical regression
percentages or proportions. Chi-square
tells the association or relationship between
summarizes the discrepancy between observed
variables. Unlike with correlations, however, the
and expected frequencies. The smaller the overall
primary purpose of regression is prediction. For
discrepancy is between the observed and expected
example, health of a person is predicted by age,
scores, the smaller the value of the chi-square and
body weight, medical history, disease status,
so vice versa. The chi-square test calculates
marital status, and current behavioral patterns.
approximate p value, hence to make assumption
There are two basic types of regression analysis:
better Yates’ continuity correlation is applied.
simple regression and multiple regression. In simple
regression, we attempt to predict the dependent
variable with a single independent variable. In
multiple regression, one may use any number of
independent variables such as age, body weight,
medical history, etc. in the health of a person, to
predict the dependent variable.
Logistic regression, unlike its linear
This is the test of “2 × 2” table as that of chi-
counterpart, is unique in its ability to predict
square (χ2) test. It directly calculates the probability
dichotomous variables, such as the presence or
(give exact) p value of a data instead of calculating
absence of a specific outcome, based on a specific
statistics to a sampling distribution. Hence, it
set of independent or predictor variables. Like
preferred logically to use the exact probability
correlation, logistic regression provides
rather than approximate χ2 - test. Generally
information about the strength and direction of
this test performed when the sample size is too
the association between the variables. In addition,
low (n < 6). Other test may be use to evaluate the
logistic regression coefficients can be used to
significance of categorical data is Fischer’s exact
estimate odds ratio for each of the independent
test. This is applied to the less sample size (< 5
variables in the model. These odds ratio can tell us
samples) where, chi-square test shows inaccurate
how likely a dichotomous outcome is to occur
evaluation.
given a particular set of independent variables. A
common application of logistic regression is to Present Absent
determine whether and to what degree a set of Yes a b a+b
hypothesized risk factors might predict the onset No c d c+d
of a certain condition. a+c b+d a+b+c+d

χ2)
Chi-square (χ
The inferential statistics that we have discussed
so far (i.e., t-tests, ANOVA) are appropriate only
when the dependent variables being measured
are continuous (interval or ratio). Chi-square
analysis is often used to examine between-group
differences on categorical variables, such as
gender, marital status, or grade level. The main
thing to remember is that the data must be nominal
(a b) (c d) (a c) (b d)
p
(abcd)(a b c d)
The limitation of χ2- test and Fisher exact test
is that it does not indicate the strength of an
association whereas the experimental and clinical
practices, people more interested in how much
more likely an outcome will be, when a treatment
or intervention is given or how much is relative
risk (risk ratio) is present.
132  Practical Manual of Experimental and Clinical Pharmacology
Some other parameters* which depend on the “2 × 2”
table
a / (a b)
Relative risk or risk ratio :
c / (c d)
Absolute relative risk (ARR) : Collecting and screening data. Br Med J 1980;
Odds ratio : 5. Altman DG. Statistics and ethics in medical research.
Number need to treat (NNT) : 281(6249):1182-84.
*These are the measures of the adverse or hazards
effect of a drug
Mann-Whitney ‘U’ Test
Test is performed when data of two groups have
measured on an ordinal scale and this test may
applied to large sample size (n>100). It is very
useful test because it can have high power
approximately 95% compared to unpaired‘t’-test.
Wilcoxon-signed Ranks Test
It corresponds to the paired‘t’ test and have high
power approximately 95% compared to paired‘t’-
test. Test is performed by calculating difference
between pairs then the absolute difference is
ranked. The sum of positive ranks is compared
with the sum of the negative ranks.
Hence, the groups have no difference if,
Sum of positive ranks = sum of the
negative ranks
Note: (Software used in biostatistics)
Nowadays, there is increase in application of computer in
the biomedical research, so, as in the statistical tests.
Reason being, complicated and tedious job to do and thirdly,
very low interest of student to perform the statistical tests
manually.
There are several softwares, used in the biostatistics. But,
most commonly used softwares are SPSS, SigmaPlot, in-
stat, BioEstat, Dataplot, graph and prism Macanova, etc.
SUGGESTED READING
1. Altman DG, Bland JM. Presentation of numerical
data. Br Med J 1996;312(7030):572.
2. Altman DG, Bland JM. Statistics notes: The normal
distribution. Br Med J 1995;310(6975):298.
3. Altman DG, Whitehead J, Parmar MK, Stenning SP,
Fayers PM, Machin D. Randomised consent designs
in cancer clinical trials. Eur J Cancer. 1995;31A(12):
1934-44.
4. Altman DG. Statistics and ethics in medical research.
281(6252):1399-401.
Misuse of statistics is unethical. Br Med J 1980;
6. Altman DG. Statistics and ethics in medical research.
VII—Interpreting results. Br Med J 1980;281(6255):
1612-14.
7. Altman DG. Statistics and ethics in medical research.
VI—Presentation of results. Br Med J 1980; 281
(6254):1542-44.
8. Altman DG. Statistics and ethics in medical research:
III How large a sample? Br Med J 1980;281(6251):
1336-38.
9. Altman DG. Statistics and ethics in medical research:
Study design. Br Med J 1980;281(6250):1267-69.
10. Altman DG. Statistics and ethics in medical research:
V—Analysing data. Br Med J 1980;281(6253):1473-
75.
11. Altman DG. Statistics: Necessary and important. Br
J Obstet Gynaecol 1986;93(1):1-3.
12. Bland JM, Altman DG. Measurement error
proportional to the mean. Br Med J 1996;313(7049):
106.
13. Bland JM, Altman DG. Measurement error. Br Med J
1996;313(7059):744.
14. Bland JM, Altman DG. Multiple significance tests:
The Bonferroni method. Br Med J 1995;310(6973):
170.
15. Bland JM, Altman DG. Transformations, means, and
confidence intervals. Br Med J 1996;312(7038):1079.
16. Bland JM, Altman DG. Transforming data. Br Med J
1996;312(7033):770.
17. Gardner MJ, Altman DG. Confidence intervals rather
than P values: Estimation rather than hypothesis
testing. Br Med J (Clin Res Ed) 1986;292(6522):746-
50.
18. Glantz SA. Biostatistics: How to detect, correct and
prevent errors in the medical literature. Circulation
1980;61(1):1-7.
19. Godfrey K. Simple linear regression in medical
research. N Engl J Med 1985 ;313(26):1629-36.
20. Godfrey K. Statistics in practice. Comparing the
means of several groups. N Engl J Med 1985;313(23):
1450-56.

21. Ludbrook J, Dudley H. Issues in biomedical statistics:
Analysing 2 × 2 tables of frequencies. Aust N Z J
Surg 1994;64(11):780-87.
22. Ludbrook J, Dudley H. Issues in biomedical statistics:
Statistical inference. Aust NZJ Surg 1994;64(9):
630-36.
23. Ludbrook J. Advantages of permutation (rando-
mization) tests in clinical and experimental
pharmacology and physiology. Clin Exp Pharmacol
Physiol 1994;21(9):673-86.
24. Ludbrook J. Analysis of 2 x 2 tables of frequencies:
Matching test to experimental design. Int J Epidemiol
2008;37(6):1430-35.
Biostatistics in Pharmacology  133
25. Ludbrook J. Statistics in biomedical laboratory and
clinical science: Applications, issues and pitfalls. Med
Princ Pract 2008;17(1):1-13.
26. Ludbrook J. Statistics in physiology and pharma-
cology: A slow and erratic learning curve. Clin Exp
Pharmacol Physiol 2001;28(5-6):488-92.
27. Ludbrook J. The presentation of statistics in
Clinical and Experimental Pharmacology and
Physiology. Clin Exp Pharmacol Physiol 2008;
35(10):1271-74.
28. Moses LE, Emerson JD, Hosseini H. Analyzing data
from ordered categories. N Engl J Med. 1984;311(7):
442-48.
 Part 2

Experimental (In Vitro Studies:

Isolated Tissue Preparation)
 General Considerations and
Collection of the
12 Tissue/Muscle
General Considerations in Animal
Experiments
Exposure to animal study and different experi-
mental methodologies is one of the important part
of the MD (Pharmacology), M Pharm , MBBS and
B Pharm syllabus. Several new terminologies have
emerged in experimental pharmacology and
student should be familiar with these terms. So,
this part of the chapter deals with the termi-
nologies which are commonly used in the
experimental pharmacology.
General Terminology Used in the
Experimental Pharmacology
Acclimatization: Pre-exposure of an animal to the
particular procedures, the people and laboratory
involved in the experiment, before conduct of the
actual experiment (Fig. 12.1).
Accuracy: Degree of trueness which is measured
or calculated to its true value. It predominately
depends on the reproducibility or repeatability of
an experiment. Accuracy is closely related to
precision but not same (Figs 12.2A to D).
Fig. 12.1: Acclimatize the animal from animal house to the laboratory, person and procedure of the experiment
Exsanguinations: One of the methods to sacrifice
the experimental animal by excessive bleeding.
Decapitation: Process of sacrificing the experi-
mental animals by cutting off the head with the
guillotine.
Precision: Degree of an exactness of a calculated
value falling exactly within the range. It shows
the refinement of a process, by which experiment
is performed. An experimental data is considered
to be excellent, if it is precise and accurate (Figs
12.2A to D)
Reproducibility: It is the measure of a process or
result which indicates their closeness in the
independent set-up, that means repetitive and
same results are obtained with the same process,
method, materials but in distinct laboratory
under different conditions such as different
person, different apparatus, different environ-
ment, etc.
Sensitivity: It is a measure for the stimuli which
produces certain reaction or stimulation at
minimal strength.
138  Practical Manual of Experimental and Clinical Pharmacology

Figs 12.2A to D: Relation of accuracy and precision: An experiment, in which the accurate value is 20. In (A) all the four
measured value is 20, (B) value measured is not 20, (C) one value is 20 but other is closely related, and (D) all values are
closely related but not 20

Terminology of Experimental Procedure experiment whereas if experiments continue

In vitro: It is the experimental process which is
mainly done outside the living body. For example:
isolated tissue preparation, chemical analysis, etc
(Figs 12.3 and 12.4).
longer than 24 hours using living cells or tissue,
then it is considered to be “in vitro” experiment.
e.g.: cell line culture, etc (Fig. 12.4).
In vivo: It is the experimental process which is
done inside the living body. For example: Pharma-
codynamic or pharmacokinetic study of drugs
(Fig. 12.5).

Fig. 12.3: In vitro experiment

Ex vivo: It is the experimental process which is

performed outside the living body in an artificial
in vivo environment. The experiment usually
lasting up to 24 hour is called as “ex vivo” Fig. 12.5: In vivo experiment

In situ: It means to measure the function of an

Fig. 12.4: Ex vivo and in vitro
organ at the same anatomical position. (Mainly
done in anesthetized or sacrificed animal). For
example: Frog heart preparation, etc.
In silico: Experimental process which is “per-
formed on computer or via computer simulation”.
 General Considerations and Collection of the Tissue/Muscle  139

Terms Used as per the Treatment of Drug Duration to the Animal

Acute study: Short duration (day 0 to 1 week).

Subacute or Subchronic study: Short to medium duration (>1week to 12 weeks).

Chronic study: Long duration (>12 weeks to 24 weeks or even more up to 18 months in higher animals
like dog or monkey),
 Identification and Collection
13 of Tissue/Muscle
In vitro bioassay is performed in the excised tissue
or muscle. So, the proper identification and
collection of the tissue/muscle is the foremost
important procedure for successful bioassay.
Collection of the tissue always depends on the
animal selection as per requirement of the
experiment. The most sensitive tissue should be
Collection of Intestinal Tissue/Muscle
Ileum
selected to avoid errors in the experiment. The
procedure is almost same for the tissue collection
in all experimental rodents. Tissue/muscle are
first selected according to the drug sensitivity,
thereafter select the animal and continue with the
procedures. Maintain the aseptic condition
throughout the procedure.
 Identification and Collection of Tissue/Muscle  141

Note:
1. Minimize the tissue damage by keeping the tissue on the cotton soaked with PSS (do not hold it at
the middle part).
2. The procedure remains same in case of guinea pig, rat and rabbit ileum.

Rat Colon (Ascending/Descending)

Ascending Colon

Descending Colon
142  Practical Manual of Experimental and Clinical Pharmacology

Rat Uterus

Stomach Fundus
 Identification and Collection of Tissue/Muscle  143

Frog Rectus Abdominis Muscle

Guinea Pig Trachea

144  Practical Manual of Experimental and Clinical Pharmacology

Anococcygeus Muscle (Rat)

Vas Deferens (Rat)

 Identification and Collection of Tissue/Muscle  145

Phrenic Nerve Diaphragm (Rat)

Chick Biventer–Cervicis

Note: The animal sensitivity to the drugs used and the most appropriate tissue identification and collection is given briefly
in the Table 13.1.
146  Practical Manual of Experimental and Clinical Pharmacology

Figs 13.1A to D: (A) Surgical posture of rodent (rat), arrow denotes the straight head, body and paws, keep complete
body in stretched and straight leveled, (B) Step I to open up the abdominal cavity by midline incision; lift the skin gently to
make short horizontal cut, (C) Position of scissor and forceps to make short horizontal cut), (D) After short horizontal cut,
give the vertical (longitudinal) cut (For color version of Figures 13.1C and D see Plate 2)
 Identification and Collection of Tissue/Muscle  147

Figs 13.1E to J: (E) Vertical cut opens the abdomen (indicated by arrows), (F) Abdominal viscera and its natural positions
(indicated by arrows), (G) Cecum (indicated by arrows), (H) Ileum (indicated by arrows), (I) Ascending colon (indicated
by arrows), (J) Descending colon (indicated by arrows)
148  Practical Manual of Experimental and Clinical Pharmacology

M N

Figs 13.1K to N: (K) Position of; (1) Lung, (2) Diaphragm, (3) Fundus, (4) Pylorus, (5) Kidney and (6) Liver, (L) Anatomical
position; (1) Trachea and (2) Esophagus, (M) Localization of trachea (arrows indicate tracheal cartilage rings), (N) Uterus
(For color version of Figures 13.1K to N see Plate 2)
 Table 13.1: Different animal tissues/muscles; their identification points and drug sensitivity with predominant receptors present
Animal tissue Identification point of T/M
(T)/muscle (M)
Tracheal chain Present at the ventral neck region,
above esophagus and between
sternocleidomastoid muscles
Stomach fundus Upper grey part of the stomach
which attached to the thick and
red pylorus
Ileum Ileum connects cecum at the
middle part where as large
intestine begins at the distal part
Ascending colon (4-7 cm from the ileocaecum
junction)
Decending colon [5-7 cm above rectum, identify by
Muscle involved Circular Drug sensitivity Receptor predominantly
(C) or Longitudinal (L) present in (T/M)
C NA, A and ACh β2-receptor, M2/M3
L receptors
L 5-HT > ACh (10 times) > 5HT-D receptor
C Histamine (1000 times)
Bradykinin
L Histamine > ACh H1, muscarinic receptor
L NA > A β3-receptor, 5-HT2A,
ACh, 5-HT 5-HT4
L ACh Muscarinic receptor

Colon
the diagonal muscle strips on the
upper surface]
Anacoccygeus Thin muscle strip arises from the L NA, ACh, 5-HT, IsoP Adrenergic supply
sacral vertebrae and passes to No Histamine NANC
colon end
Vas deferens Attached to epididymis, should L NA and ACh α1-adrenergic receptors and
be distinguish from seminal vesicle muscarinic receptor
Uterus Clear two horn above rectum L Oxytocin, 5-HT, A, NA β, α (after estrogen
connected to ovary treatment*) receptors
ACh M2/M3 receptor

* 0.1 mg/kg stilbestrol, im 24 hr before the experiment with oxytocin/ACh and for 5-HT 0.25 mg/100 g of stilbestrol, i.p for three days before experiment
C- circular; L- longitudinal; alpha2- beta 2 receptor; 5-HT- 5- hydroxy tryptaminergic receptor; IsoP- Isoprenaline; ACh- Acetyl Chosline; H1- Histamine-
1 receptor; NANC- Non-adrenergic non-cholinergic receptor; NA- Noradrenaline; α-Alpha-adrenergic receptor; M2/M3- Muscarinic receptor M2/M3.
Identification and Collection of Tissue/Muscle  149
 Principle of Muscle
14 Contraction

Action potential generated due to chemical,
electrical or mechanical stimuli is responsible for
muscle contraction. In many studies, it is
confirmed that ability to generate force and
movement depends on the interactions of the two
contractile proteins, namely myosin (M) in the
thick filaments and actin (A) in the thin filaments,
where energy is provided by ATP. Shortening of
the contractile elements in muscle is brought about
by sliding of the thin filaments over the thick
filaments.
Each cycle consists of four steps (Fig. 14.1):
1. Attachment of the cross bridge to a thin (actin)
filament,
2. Movement of the cross bridge, producing
tension in the thin filament,
3. Detachment of the cross bridge from the thin
filament, and
4. Energizing the cross bridge so that it can again
attach to a thin filament and repeat the cycle.
Type of Contraction
The classification mainly depends on the role of
shortening of the contractile elements in muscle.
Depending on the property of a muscle either
reduction or no change in the length of muscle,
contraction is classified into the two groups;
1. Isotonic contraction: The length of muscle
against a constant load is reduced whereas
tone remains same. For example: Bioassay of
guinea pig ileum or other tissues, etc.

Fig. 14.1: Mechanism of muscle contraction

 Principle of Muscle Contraction  151

2. Isometric contraction: The length of muscle In the bioassay, contraction/relaxation of the

against a constant load does not change where- muscle takes place by circular or longitudinal
as tone of muscle varies, e.g. muscle twitch, etc. smooth or skeletal muscles (Fig. 14.2).

Fig. 14.2: Comparison of smooth and skeletal muscle contraction

 Fast Contracting Smooth
15 Muscle Preparation

EXPERIMENT NO: 15A reagent bottle may vary with

different salt compositions)
Aim
To determine unknown concentration of hista- Precautions before Experimentation
mine by using guinea pig ileum. • Clean the organ bath before starting the
Background experiment specially inner organ bath (chances
of presence of previous drug used)
Guinea pig ileum is most sensitive to histamine. • Balance the writing lever horizontally with the
Histamine, mainly act through the H1 and H2
help of load
receptor. Contractile response of histamine to the
• Prepare PSS for the experiment, while taking
ileum is caused by the H1 receptor found in the
exact quantity of chemicals (1% variability is
ileum, bronchi and capillaries. The assay of the
acceptable)
preparation is based on the magnus method
(1904). Guinea pig ileum has the spontaneous • Add the calcium chloride at the end of PSS
activity and its specificity is improved by using preparation (to avoid any precipitation: PSS
atropine or mepyramine in tyrode. In the assay of should be clear)
histamine, atropine (2 × 10–6 to 10–7 M) and for • Try to minimize the handling of tissue (espe-
acetylcholine (ACh) assay mepyramine (5 × 10–6 cially at the middle part)
to 2 × 10–7) is used for the enhancement of the • Always use the finger to hold the tissue instead
specificity. Some of the endogenous polypeptides of forceps
like angiotensin, bradykinin and substance P are • Maintain the dose cycle properly (tissue
also sensitive to guinea pig ileum. sensitivity depends on the cycle)
• Avoid contact of very high dose of the drug; tissue
Materials and Method may loss sensitivity due to “tachyphylaxis”
(tissue may remain in contractile stage, in
Animal/tissue : Guinea Pig/ileum presence of high drug concentration)
PSS : Tyrode/Ringer
Lever : Frontal writing Methods
Tension/load : upto 1 gm
Step I: Keep the animal (guinea pig) for fasting at
Magnification : 7-10x
least for 24-48 hr.
Aeration air : O2/Carbogen
Temperature : 32-37°C Step II: Sacrifice the guinea pig by stunning (a
Drug : Histamine (Mwt: 307.14) strong blow) on the head then, keep the animal on
(Always check M wt. on the the dissecting board (DB).
 Fast Contracting Smooth Muscle Preparation  153
Step III: Fix guinea pig on the DB by tying its legs
with the help of thread.
Step IV: Cut open the abdomen of guinea pig by a
vertical midline incision after a small horizontal
cut, and then expose the abdominal contents.
Step V: Identification of the ileum is done by two
ways; (1) identify the cecum then come back at least
of 10 cm, or (2) identify stomach, then go forward to
identify the ileum 10 cm before cecum (also see in
identification and collection of tissue section).
Step VI: Take out the 2 -3 cm or as desired length of
the ileum and remaining should be kept in
refrigerator for the future use.
Step VII: The ileum is trimmed away from
mesentery or attached tissues, then clean any
waste contains of the ileum by a gentle push of
PSS by the help of syringe (preferably PSS warmed
at 30-35°C.
Step VIII: Then, the ileum is attached to the one
end with the hook attached to the tissue holding
arm and the other end is tie to the lever.
(Attachment of the tissue should be in the direction
of the intestine in vivo as far as possible)
Step IX: The experimental design is selected 3-point,
4-point or any other design mentioned. Then, make
the DRC of standard and test drug then plan for
the selected experimental design accordingly such
as for 3-point select 2 response of standard and 1
response of test whereas for 4-point select 2
responses of standard and test each.
Step X: Prepare the complete graph and calculate
for determining the unknown concentration of
given drug.
Note: Rat or rabbit ileum bioassay procedure is
same as described in the Guinea pig ileum. But,
tissue shows variable spontaneous response
hence the tissue should be relaxed properly.
Calculation and Result
The calculation and graph depend on the
method/design of experimentation adopted either
3- point, 4-point or matching, etc. (see the example
given in bioassay chapter for calculation).
Inference
Write the findings of the study.
Discussion
Ileum is preferred for the experiment because of
less mesentery attached to ileum, and nearly all
receptors are present. But, 10 cm of ileum attached
to the cecum contains more of α-excitatory receptor
is present which should be excluded. Intestine is
supplied by both parasympathetic and sympa-
thetic nerves. Muscarinic receptors are predomi-
nant in parasympathetic action whereas inhi-
bitory activity is mediated by the sympathetic α
and β- receptors. It is reported that inhibitory α-
receptors is present located presynaptically
whereas β-receptor (especially β1) are present on
the smooth muscle fibers. Fasting leads to less
possibility of fecal matter present at the site. The
selection of tissue depends on the drug such as
guinea pig ileum is most sensitive for the histamine
or related compounds. Always select the tissue
which is most sensitive to the drug. For example: If
the drug is ACh, then select the dorsal leech muscle
(if available) or frog rectus abdominis muscle, etc.
For the intestinal preparation most commonly used
species are guinea pig and rabbit. Spontaneous
activity of the tissue is reduced by performing the
experiment 5-7°C lower than the body temperature.
SUGGESTED READING
1. Bian X, Burda JE, Carrasquillo M, Galligan JJ.
Postnatal down regulation of inhibitory
neuromuscular transmission to the longitudinal
muscle of the guinea pig ileum. Neurogastroenterol
Motil 2009 Apr 13.
2. Foong JP, Bornstein JC. 5-HT antagonists NAN-190
and SB 269970 block alpha2-adrenoceptors in the
guinea pig. Neuroreport 2009;20(3):325-30.
3. Guagnini F, Cogliati P, Mukenge S, Ferla G, Croci T.
Tolerance to cannabinoid response on the myenteric
plexus of guinea-pig ileum and human small
intestinal strips. Br J Pharmacol 2006;148(8):
1165-73.
154  Practical Manual of Experimental and Clinical Pharmacology
4. Hons IM, Burda JE, Grider JR, Mawe GM, Sharkey
KA. Alterations to enteric neural signaling underlie
secretory abnormalities of the ileum in experimental
colitis in the guinea pig. Am J Physiol Gastrointest
Liver Physiol 2009;296(4):G717-26.
5. Schulz R, Seidl E, Wüster M, Herz A. Opioid
dependence and cross-dependence in the isolated
guinea-pig ileum. Eur J Pharmacol 1982;84(1-2): 33-
40.
6. Takaki M, Mizutani M, Jin JG, Nakayama S. Slow
hyperpolarizing action of tryptamine on myenteric
neurons of the isolated guinea-pig ileum. Acta Med
Okayama 1990;44(2):87-91.
7. Yagasaki O, Sasaki N, Yanagiya I. Evidence of
ascending release of acetylcholine from the locally
distended guinea pig ileum. Jpn J Pharmacol. 1982;
32(5):938-40.
EXPERIMENT NO: 15B
Aim
To determine unknown concentration of acetyl-
choline (ACh) using rat ascending/descending
colon.
Background
Initial, few centimeters (cm) of rat colon are usually
used for the bioassay of noradrenaline (NA) and
adrenaline (Adr) like preparations. Colon is more
sensitive to the NA than Adr. ACh assay can be
also performed on the ascending or descending
colon. Its sensitivity may increase by keeping colon
at 4°C for 24 hr. Students should keep in mind
that after removing any tissue from the fridge, it
should be allowed to recover the room temperature,
before tying into the organ bath. It is also sensitive
to the substance P and little to prostaglandins and
angiotensin.
It is found that the calcium sensing receptor
(CaSR) is activated by extracellular calcium (Ca2+)
and mediates increases in inositol 1,4,5-
trisphosphate which is involved in the contraction
and relaxation of the colon.
Materials and Method
Animal/tissue : Rat/Ascending (AC) or des-
cending colon (DC)
PSS : Krebs (AC) /deJalon (DC)
Lever : Frontal writing
Magnification : 4-7x
Tension/load : 2g
Air : O2 or Carbogen
Temperature : 25°C (AC)/37°C (DC)
Drug : Acetylcholine chloride (ACh)
[M. wt: 181.68]
Precautions before Experimentation
• Clean the organ bath before starting the
experiment specially inner organ bath (chances
of presence of previous drug used or to avoid
any contamination)
• Balance the frontal writing lever horizontal
with the help of load
• Prepare PSS for the experiment, while taking
exact quantity of chemicals (1% variability is
acceptable)
• Add the calcium chloride at the end of PSS
preparation (to avoid any precipitation: PSS
should be clear)
• Try to minimize the handling of tissue (espe-
cially at the middle part)
• Maintain the dose cycle properly (tissue
sensitivity depend on this cycle)
• Identify the ascending or descending colon
properly, otherwise it may interfere with
response.
Method
(Rat ascending colon)
Step I: Keep the animal (rat) fasting for at least 24
hours
Step II: Sacrifice the rat by stunning (a strong blow)
on the head. After sacrifice, keep the animal on
the dissecting board (DB).
Step III: Rat should be fixed on the DB by tying its
legs with the help of thread.
Step IV: Cut open the abdomen of rat by a vertical
midline incision after a small horizontal cut, and
then expose the abdominal viscera.
Step V: Identify ascending colon through the
cecum. Identify the cecum and then go forward
towards the rectum at least 5-7cm.
 Fast Contracting Smooth Muscle Preparation  155
Step VI: Cut small piece (1.5-3 cm) as per the inner
organ bath volume and clean it with a lukewarm
water or PSS with the help of a syringe.
Step VII: Then tie both the ends with the help of
nylon or cotton thread and tie it to the balanced
horizontal lever.
Step VIII: Leave the tissue at least for 30 min for
relaxation.
Step IX: Then, take the response with the standard
and test drug (prepared by serial dilution).
Step X: Select the experimental design and take
the responses of the drug.
Step XI: Calculate recorded responses, according
to the selected experimental design.
(Rat descending colon)
Step I: Keep the animal (rat) fasting for at least 24
hours
Step II: Sacrifice the rat by stunning (a strong blow)
on the head. After sacrifice, keep the animal on
the dissecting board (DB).
Step III: Rat is fixed on the DB by tying it legs with
the help of thread.
Step IV: Cut open the abdomen of rat by a vertical
midline incision after a small horizontal cut, and
then expose the abdominal viscera.
Step V: Identify the rectum and then come back at
least 5-7 cm for the collection of, descending colon
Step VI: Cut small piece (1.5-3 cm) of DC as per the
inner organ bath volume and clean it with a
lukewarm water or PSS with the help of a syringe.
Step VII: Then, tie both the end with the help of
nylon or cotton threads and tie it to the balanced
horizontal lever.
Step VIII: Leave the tissue at least for 30 min for
relaxation.
Step IX: Then, take the response with the standard
and test drug (prepared by serial dilution).
Step X: Select the experimental design and take
the responses of the drug.
Step XI: Calculate and make graph of recorded
responses.
Calculation and Result
The calculation and graph are depend on the
method/design of experimentation adopted either
3- point, 4-point or matching, etc. (see the bioassay
section for calculation).
Inference
Write the findings of the study.
Discussion
For the ACh most sensitive tissue is dorsal leech
muscle and the frog rectus abdominis muscle. The
ascending/descending colon is not the suitable
tissue for the assay of ACh, where as it may give
erratic responses. Intestine preparations are very
commonly used in the isolated tissue experiment
due to the ease of isolation, more handling
resistant than other tissues which have variable
spontaneous responses.
SUGGESTED READING
1. Cheng SX, Okuda M, Hall AE, Geibel JP, Hebert SC.
Expression of calcium-sensing receptor in rat colonic
epithelium: Evidence for modulation of fluid
secretion. Am J Physiol Gastrointest Liver Physiol
2002; 283(1): G240-250.
EXPERIMENT NO: 15C
Aim
To determine unknown concentration of acetyl-
choline (ACh) using rat uterus.
Background
The response of uterus preparation mainly
depends on the animal age due to variation in the
estrus cycle. It is fast contracting tissue and shows
the spontaneous response. It responds to the
156  Practical Manual of Experimental and Clinical Pharmacology
minimal dose of ACh at 5 × 10–5M and carbachol
at 10–4M. The other drugs like adrenaline (3 ×
10–6 M), noradrenaline (3 × 10–5M), isoprenaline
(10 –6 M), ephedrine (10 –3 M) and tyramine
(10 –3 M) show the specificity to the uterus
preparation.
It is important to induce the estrus cycle into
the normal female animal before the experiment.
Artificially induced estrus cycle have the variable
sensitivity to tested drugs such as for bioassay of
‘oxytocin’ or ACh; rat is treated with stilbestrol
0.1 mg/kg (20 µg/ml), subcutaneous 24 hr before
the experiment whereas for assay of ‘5-HT ’ on
uterus is primed for 3 days at the dose of 0.25 mg/
100 ml. Other drugs like histamine (acting on H1),
adrenaline, and noradrenaline (acting on
β-receptor) are also sensitive to the uterus.
Materials and Method
Animal/tissue : Female rat/uterus
PSS : Krebs/deJalon/McEwen
Lever : Frontal writing
Magnification : 4-7x
Tension : 1-4g
Air : O2 or Carbogen
Temperature : 32-37°C (at 32°C spontaneous
responses are low)
Drug : Acetylcholine chloride (ACh)
[M. wt: 181.68] Oxytocin [M.
wt: 1007.19]
Precautions before Experimentation
• Clean the organ bath before starting the
experiment specially inner organ bath (chances
of presence of previous drug used)
• Balance the writing lever horizontal with the
help of load
• Prepare PSS for the experiment, while taking
exact quantity of chemicals (1% variability is
acceptable)
• Add the calcium chloride at the end of PSS
preparation (to avoid any precipitation: PSS
should be clear)
• Try to minimize the handling of tissue
(especially at the middle part)
• Maintain the dose cycle properly (tissue
sensitivity depend on this cycle)
• May give spontaneous response, hence relax
the tissue properly.
Method
Step I: Female rat (FR) weighing around 150-300 g
is taken, and 24 hr prior to the experiment, the rat
is primed with 0.1 mg/kg stilbestrol, IM.
Step II: Sacrifice the FR by stunning (a strong blow)
on the head. After sacrifice, keep the FR on the
dissecting board (DB).
Step III: FR is fixed on the DB by tying its leg with
the help of thread.
Step IV: Cut open the abdomen of FR by a vertical
midline incision after a small horizontal cut,
and then exposed the abdominal contains (Also
see in identification and collection of tissue/ muscle
section).
Step V: Identify the vaginal orifice.
Step VI: Trace the vaginal orifice interiorly to find
the two horns of the uterus.
Step VII: Cut the two horns and now, you have
two samples of the uterus.
Step VIII: Cut short the tissue according to the inner
organ bath (handle the tissue minimally at the
middle part which can reduce the sensitivity of
the tissue).
Step IX: Select the experimental design (matching,
bracketing, 3-point, or 4point assay).
Note: Rabbit uterus responses are approximately
homologous to human. It gives the spontaneous
pendulum like movement response at the kymo-
graph.
Calculation and Result
The calculation and graph depend on the
method/design of experimentation adopted either
3- point, 4-point or matching, etc. (see the bioassay
section for calculation).
 Fast Contracting Smooth Muscle Preparation  157

Inference Background
Write the findings of the study. The guinea pig or rabbit atria (2-4 cm) are generally
preferred for this assay. There are several advan-
Discussion tages of these tissues such as require very less
trimming or slicing of the tissue, thickness of tissue
Drug response to uterus in presence of different
gives easy way to the oxygen to pass through, tissue
estrous cycle differs due to the different receptors
thickness maintained throughout the preparation,
expression. α-adrenergic receptor is prominent have easy separation of right and left atria for
and it causes relaxation which is blocked by experiments using spontaneous beating or stimu-
α-receptor antagonists. Since, uterus has no lated preparation, and size and shape of the atria
inherent tone but the relaxation is observed by gives good contractile tension and stability over
physiological antagonism of the contractile the several hour of the experiment. Isoprenaline,
response of ACh or other drugs. In the estrous salbutamol and ACh shows the sensitivity
stage (uterus vascularity and the size is increased), (contraction) to the atrial tissue.
spontaneous contractility is increased. It is found
that after priming with estradiol, there is excess Materials and Method
expression of α-excitatory adrenoreceptors. Other Animal/tissue : Guinea pig/atria
drugs like 5-HT, noradrenaline, etc. can be PSS : Krebs /Ringer Locke
assayed with the uterus. Prostaglandins also Lever : Frontal writing/starling heart
show the contractile response via PGF2α in the Magnification : 6-8x
uterus smooth muscle of non-pregnant animals Tension : 1g
whereas PGE2 causes relaxation. Air : O2 or Carbogen
Temperature : 30-32°C
SUGGESTED READING Drug : Adrenaline (M. wt.: 183.21)
Isoprenaline HCL (M. wt:
1. Bossmar T, Osman N, Zilahi E, Haj MA, Nowotny 247.72)
N, Conlon JM. Expression of the oxytocin gene, but
Salbutamol (M. wt.: 239.31)
not the vasopressin gene, in the rat uterus during
pregnancy: influence of oestradiol and progesterone.
Precautions before Experimentation
J Endocrinol 2007;193(1):121-26.
2. Kumcu EK, Büyüknacar HS, Göçmen C, Evrüke IC, • Separate atria, ventricle safely, so that AV node
Onder S. Differential effect of neocuproine, a donot get disturbed
copper(I) chelator, on contractile activity in isolated • Clean the organ bath before starting the
ovariectomized non-pregnant rat, pregnant rat and
experiment specially inner organ bath (chances
pregnant human uterus. Eur J Pharmacol 2009 Jan
20.
of presence of previous drug used or to avoid
3. Orescanin-Dusiæ Z, Milovanoviæ S, Blagojeviæ D, any contamination)
Nikoliæ-Kokiæ A, Radojiciæ R, Spasojeviæ I, Spasiæ • Balance the frontal writing lever horizontal
M. Diethyldithiocarbamate potentiates the effects of with the help of load
protamine sulphate in the isolated rat uterus. Redox • Make PSS for the experiment, while taking
Rep 2009;14(2):48-54. exact quantity of chemicals (1% variability is
acceptable)
EXPERIMENT NO: 15D • Add the calcium chloride at the end of PSS
preparation (to avoid any precipitation: PSS
Aim
should be clear)
To determine unknown concentration of adrena- • Try to minimize the handling of tissue
line using guinea pig atria. (especially at the middle part)
158  Practical Manual of Experimental and Clinical Pharmacology
Figs15.1A to D: Isolation and separation of atria from the ventricles; (A) Showing heart holded with forcep and making cut
at the atrioventricular junction (shave the AV pacemaker); (B) Isolated atrium from ventricle, dissected into right (Rt) and
left (Lt) atrium; (C) Separate into right (Rt) and left (Lt) atrium, cut through the dotted line; (D) Two pieces (Rt & Lt) of atrium
for the experiment
• Maintain the dose cycle properly (tissue
sensitivity depend on this cycle)
• Carefully cut and separate ventricles
Method
Step I: Select the guinea pig weighing around 350-
450 g and euthanized by cervical dislocation.
Step II: Lateral thoracotomy is done and identify
the heart at the right bottom of thoracic.
Step III: Carefully cut the pericardium and remove
the heart then cut and clean the fat and other
tissue attached to the heart.
Step IV: Transfer the heart to the ice cold PSS so as
to reduce the metabolic activity and hence, it can
work longer.
Step V: Cut the ventricles carefully so that atria
pacemaker could not get disturbed.
Step VI: Then, tie the thread on the both side of the
atria and hang into the organ bath and attached
to the lever.
Step VII: Leave the tissue for relaxation for around
30-45 min.
Step VIII: Select the experimental design (matching,
bracketing, 3-point, or 4-point assay) and start the
response recording.
Calculation and Result
The calculation and graph are depend on the
method/design of experimentation adopted either
3- point, 4-point or matching, etc. (see the bioassay
section for calculation).
Inference
Write the findings of the study.
Discussion
Atria are highly supplied with the sympathetic and
slightly with parasympathetic nervous system. The
β2- receptor is widely present in the atria.
SUGGESTED READING
1. Chiao H, Caldwell RW. Local cardiac effects of
substance P: Roles of acetylcholine and noradrenaline.
Br J Pharmacol 1995;114(2):283-88.
2. Hussain M, Chorvatova A, Singh J. Physiological
effects and biochemical properties of a serum protein
that produces positive inotropic and chronotropic
effects on isolated guinea pig atria. Mol Cell Biochem.
2004;261(1-2):201-07.
3. Li L, Zhuang FE, Yang L, Zhang CL, Zhao GS, Zhao
DK. Effects of osthole on isolated guinea pig heart
atria. Zhongguo Yao Li Xue Bao 1995;16(3):251-54.
4. Pousti A, Bakhtiarian A, Najafi R, Deemyad T,
Brumand K, Hosseini MJ. Effect of sertraline on
ouabain-induced arrhythmia in isolated guinea-pig
atria. Depress Anxiety 2009 Feb 25.
5. Pousti A, Deemyad T, Malihi G, Brumand K. A
preliminary study on the interaction of fluvoxamine
and adenosine receptor on isolated Guinea-pig atria.
Int J Neurosci 2006;116(12):1491-99.
 Fast Contracting Smooth Muscle Preparation  159
6. Royse CF, Royse AG, Rohrlach R, Wright CE, Angus
JA. The cardiovascular effects of adrenaline,
dobutamine and milrinone in rabbits using
pressure-volume loops and guinea pig isolated
atrial tissue. Anaesth Intensive Care 2007;35(2):
180-88.
EXPERIMENT NO: 15E
Aim
To determine the unknown concentration of
acetylcholine (ACh) using rat anococcygeus
muscle preparation.
Background
It is a thin strip of smooth muscle (made of 2
anococcygeus muscle) which arises from sacral
vertebrae and reaches to terminal colon (near anus).
The sensitivity of the anococcygeus muscle is to
ACh, noradrenaline, isoprenaline, adenosine and
5-HT but insensitive to histamine. The atropine and
phentolamine block the contractile property of ACh
and noradrenaline respectively. This assay method
is previously described by the Gillespie (1972). The
receptor responsible for the contraction/relaxation
is considered to be prejunctional receptor which
results in enhancement of norepinephrine (NE)
release, which further induces contractile response
by activation of postjunctional α1-adrenoreceptors.
Other receptor found to be responsible is
5-HT1B/1D.
Materials and Method
Animal/tissue : Rat/anococcygeus muscle
PSS : Krebs
Lever : Frontal writing
Magnification : 7-10x
Tension : up to 1 gm
Air : O2 or Carbogen
Temperature : 35-37°C
Drug : Acetyl choline chloride (ACh)
[M. wt: 181.68]
Precautions before Experimentation
• Clean the organ bath before starting the
experiment specially inner organ bath (chances
of presence of previous drug used or to avoid
any contamination)
• Balance the frontal writing lever horizontal
with the help of load
• Make PSS for the experiment, while taking
exact quantity of chemicals (1% variability is
acceptable)
• Add the calcium chloride at the end of PSS
preparation (to avoid any precipitation: PSS
should be clear)
• Try to minimize the handling of tissue (espe-
cially at the middle part)
• Maintain the dose cycle properly (tissue
sensitivity depend on this cycle).
Method
Step I: Sacrifice the rat by stunning (a strong blow)
on the head and then transfer to the dissecting
board (DB).
Step II: Rat should be fixed on the DB by tying its
legs with the help of thread.
Step III: Cut open the abdomen of rat by a vertical
midline incision after a small horizontal cut, and
then exposed the abdominal viscera.
Step IV: Identify the root of rectum interiorly and
then identify the 2 thin strips of anococcygeus
muscle which arise from the vertebrae and meet
at the terminal part of colon (anus).
Step V: Cut the rectal colon 2-3 cm from the anus
and clear from the connective tissues and isolate
the 2 strips of anococcygeus muscle.
Step VI: Keep in PSS and supply continuous
constant air (O2 or carbogen).
Step VII: Prepare the standard and test drugs and
select the experimental design.
Calculation and Result
The calculation and graph are depend on the
method/design of experimentation adopted either
3- point, 4-point or matching, etc. (see the bioassay
section for calculation)
160  Practical Manual of Experimental and Clinical Pharmacology
Inference
Write the findings of the study.
Discussion
Practically, it is difficult to identify the anoco-
ccygeus muscle. It gets mixed with the surroun-
ding smooth muscles. The only differentiation is,
it has tendinous origin and do not appear soft.
These muscles have a dense adrenergic excitatory
innervation as well as inhibitory innervations in
the rabbit, rat and cat. In the studies, it is found
that adenosine triphosphate (ATP) have the
differential action on different animal such as, is
a powerful inhibitory agent in the rabbit anoco-
ccygeus, whereas in the rat it causes contraction
while in the cat, requires high concentrations to
produce relaxation. Other like Acetylcholine-
sterase-positive fibers and catecholaminergic
fibers are abundant in the anococcygeus. In an
interesting study, it was found that androgen and
estrogen play an important role in sexual dimor-
phism of the mouse anococcygeus muscle.
SUGGESTED READING
1. Creed K E, Gillespie J S. Some electrical properties
of the rabbit anococcygeus muscle and a
comparison of the effects of inhibitory nerve
stimulation in the rat and rabbit. J Physiol 1977;
273:137-53.
2. Emre S, Erdem SR, Tuncer M. Does serotonin relax
the rat anococcygeus muscle via 5-HT7 receptors?
Naunyn Schmiedebergs Arch Pharmacol 2000;
362(2):96-100.
3. Emre-Aydingöz S, Kocaefe C C, Tuncer M. Calcium-
antagonistic activity of sumatriptan in the rat
anococcygeus muscle. Pharmacology 2002;64(1): 43-
48.
4. Gillespie JS. The rat anococcygeus muscle and its
response to nerve stimulation and to some drugs. Br
J Pharmac 1972; 45: 404-16.
5. Kulkarni SK, Sharma A. Rat anococcygeus: A
dynamic smooth muscle preparation for experimental
pharmacology. Methods Find Exp Clin Pharmacol
1994;16(6):379-85.
6. Tirapelli CR, de Andrade CR, Cassano AO, De
Souza FA, Ambrosio SR, da Costa FB, de Oliveira
AM. Antispasmodic and relaxant effects of the
hidroalcoholic extract of Pimpinella anisum
(Apiaceae) on rat anococcygeus smooth muscle. J
Ethnopharmacol 2007;110(1):23-29.
EXPERIMENT NO: 15F
Aim
To determine unknown concentration of acetyl-
choline (ACh) using rat vas deferens
Background
Rat and guinea pig are suitable animals for the
preparation due to relatively large vas deferens
than other animals. For increasing the sensitivity
of preparation animal should be, fed with oats for
at least for 3 days. Adrenaline, noradrenaline and
phenylephrine (µ-sympathomimetic) contract the
vas deferens whose action is blocked by the
phentolamine. The method is previously described
by the Henderson et al (1972) and Hart et al (1979).
Materials and Method
Animal/tissue : Rat/vas deferens
PSS : Krebs/Ringer/Tyrode/
McEwen
Lever : Frontal writing
Magnification : 6-8x
Tension : 0.3-1g
Air : O2 or Carbogen
Temperature : 32-37°C
Drug : Acetyl choline chloride (ACh)
[M. wt: 181.68]
: Histamine [M. wt: 307.14]
Precautions before Experimentation
• Clean the organ bath before starting the
experiment specially inner organ bath (chances
of presence of previous drug used)
• Balance the writing lever horizontal with the
help of load
• Prepare PSS for the experiment, while taking
exact quantity of chemicals (1% variability is
acceptable)
 Fast Contracting Smooth Muscle Preparation  161
• Add the calcium chloride at the end of PSS
preparation (to avoid any precipitation: PSS
should be clear)
• Try to minimize the handling of tissue
(especially at the middle part)
• Maintain the dose cycle properly (tissue
sensitivity depend on this cycle)
• Carefully remove the connective tissues and
blood vessel attached to the vas deferens.
Method
Step I: Sacrifice the rat by stunning (a strong blow)
on the head and then transfer the animal on the
dissecting board (DB).
Step II: Rat should be fixed on the DB by tying its
legs with the help of thread.
Step III: Cut open the abdomen of rat by a vertical
midline incision after a small horizontal cut, and
then expose the abdominal viscera.
Step V: Identify the testes then pull it to one side
and vas deferens a white thin tubular structure is
identified easily.
Step VI: It is freed out from the epididymis which
can be distinguished clearly from where it joins
the urethra. Cut into desired length of tissue.
Step VI: Put into the Petri dish filled with PSS with
proper continuous distinguishable aeration.
Step VII: Put the tissue into the organ bath and
perform the experiment as per selected experi-
mental design.
Calculation and Result
The calculation and graph are depend on the
method/design of experimentation adopted either
3- point, 4-point or matching, etc. (see the bioassay
section for calculation).
Inference
Write the findings of the study.
Discussion
Contractile responses of rat vas deferens are due
to the presence of noradrenergic or adrenergic
receptor specifically β2-adrenoceptors. This effect
is found to be antagonized by yohimbine,
piperoxan, phentolamine and tolazoline.
Contractile effects of sympathomimetic agents
may either antagonize or potentiated by the
reversible µ-adrenoceptor antagonists according
to their doses and duration of contact of drug to
the tissue.
SUGGESTED READING
1. Berdysheva LV and Manukhin BN. Effect of
Activation of Muscarinic Cholinergic Receptors on
the Kinetics of α1-Adrenergic Contractile Response
of the Rat Vas Deferens. Doklady Biological Sciences
2001;381(1-6):522-25.
2. Minneman KP, Fox AW, Abel PW. Occupancy of
alpha 1-adrenergic receptors and contraction of rat
vas deferens. Mol Pharmacol 1983;23(2):359-68.
EXPERIMENT NO: 15G
Aim
To determine unknown concentration of anta-
gonist (atropine) using acetylcholine (ACh) as an
agonist employing guinea pig ileum preparation
by pA2 method.
Background
pA2 is the principle method for studying the
antagonist action for a selected agonist which is
defined as the negative logarithm to base 10 of
the antagonist concentration (molar units)
corresponding to a dose-ratio of 2 (i.e. the
concentration that produces a 2-fold shift in the
agonist concentration-response curve). This
method is developed by Schild in 1957. But, the
limitation being Schild design is that it requires
a large number of experimental units to obtain
even a minimal number of points for the Schild
plot.
Materials and Method
Animal/tissue : Guinea pig/ileum
PSS : Tyrode
Lever : Frontal writing
Magnification : 7-10x
162  Practical Manual of Experimental and Clinical Pharmacology
Tenin/load : Up to 1 gm
Air : O2/Carbogen
Temperature : 32-37°C
Drug : Acetylcholine chloride (ACh)
[M. wt: 181.68]
Atropine sulphate [M. wt:
694.84]
Precautions before Experimentation
• Clean the organ bath before starting the
experiment specially inner organ bath
(chances of presence of previous drug used)
• Balance the writing lever horizontal with the
help of load
• Prepare PSS for the experiment, while taking
exact quantity of chemicals (1% variability is
acceptable)
• Add the calcium chloride at the end of PSS
preparation (to avoid any precipitation: PSS
should be clear)
• Try to minimize the handling of tissue (espe-
cially at the middle part)
• Always use the finger to hold the tissue instead
of forceps
• Maintain the dose cycle properly (tissue
sensitivity depend on this cycle).
Example
Method
Step I: Keep the animal for fasting at least for 24
hours.
Step II: Standard drug DRC is plotted by the same
method as described in the guinea pig ileum
experiment no. 15A.
Step III: Select the dose ‘2R’ from the DRC of
standard drug (between 25-75% of response).
Step IV: Then, take at least three responses of ‘2R’.
Step V: Add the 10 times lesser concentration of
atropine (antagonist) and wait for 5-15 min, then
without washing add agonist i.e. 2R.
Step VI: Wash out the antagonist and take the
responses till the normal response of ‘2R’ is
achieved (maintain the dose cycle to preserve the
sensitivity of the tissue).
Step VII: Repeat the step V until the response of
the ‘2R’ is about equal to ‘R’(about the half
response of ‘2R’.
Step VIII: Plot the graph, of % response to 2R versus
-log (antagonist) dose (Figs 15.2A and B)
Calculation and Result
After plotting the %response versus –log (anta-
gonist), pA2 value is directly extrapolated through
the graph at which its % response remains 50%.
 Fast Contracting Smooth Muscle Preparation  163

Figs 15.2A and B: As an example, DRC plot by an agonist thereafter selection of ‘2R’ is shown. After selection of ‘2R’ plot
it at least for the 3 times, then add the antagonist at 10 times lower concentration of agonist and repeat the experiment
as shown in the plot; R1, R2, and R3 are the responses of ‘2R’ after addition of antagonist, (B) Represents the graph plot
of % response to 2R versus –log (antagonist) and determination of pA2 at the X-‘axis’
 Slow Contracting Muscle
16
EXPERIMENT NO: 16A
Aim
To determine unknown concentration of serotonin
(5-HT) using rat stomach (fundus).
Background
Stomach fundus is useful in the bioassay of 5-HT
which is considered to be most sensitive tissue
among the three parts of stomach namely, fundus,
corpus and pylorus. Fundus is identified by its gray
color and situated above the pink thick pyloric
region. Drugs like histamine are little or insensitive to
the rat fundus due to the lack of H1 receptors in the
stomach muscle. The other drugs which have
sensitivity against the fundus are ACh, bradykinin
and prostaglandin (PGE2).
The muscle preparation is the important step
in the bioassay using stomach fundus. Both
longitudinal and circular muscle may be used in
the experiment which depends on the transverse
cut made to prepare the tissue (Figs 16.1A to E).
Materials and Method
Animal/tissue : Rat/Stomach fundus
PSS : Tyrode/deJalon/Krebs

Figs 16.1A to E: (A) Showing three parts of stomach; (B) Stomach cut open through lesser curvature and spread on a
paper sheet wetted with PSS, (C) Divided into two parts, if want to preseve the longitudinal muscle, (D) vertical alternate
transverse cut to preserve the longitudinal tissue, (E) Horizontal alternate transverse cut for preserving circular muscle
(donot cut the stomach into two parts if want to see the effect of circular muscle)

Lever : Frontal writing/ simple lever
Magnification : 7-15x
Tension : 1-4g
Air : O2/Carbogen
Temperature : 35-37°C
Drug : Serotonin (5-HT) [M.wt: 176.
218]
Acetylcholine chloride (ACh)
[M. wt: 181.68]
Precautions before Experimentation
• Clean the organ bath before starting the
experiment specially inner organ bath (chances
of presence of previous drug used or to avoid
any contamination)
• Balance the simple writing lever, tangential to
the rotating drum
• Prepare PSS for the experiment, while taking
exact quantity of chemicals (1% variability is
acceptable)
• Add the calcium chloride at the end of PSS
preparation (to avoid any precipitation: PSS
should be clear)
• Try to minimize the handling of tissue (espe-
cially at the middle part)
• Maintain the dose cycle properly (tissue
sensitivity depend on this cycle)
• Animal should be fasted properly (any food
present in the stomach may interfere with the
assay)
• Carefully clean the fundus from the adjacent
tissues.
Method
Step I: Keep the animal (rat) fasting for at least
24hr, water is given ad libitum.
Step II: Sacrifice the rat by stunning (a strong blow)
on the head, and then keep on the dissecting board
(DB).
Step III: Rat is fixed on the DB by tying it legs with
the help of thread.
Step IV: Cut open the abdomen of rat by a vertical
long cut, and then expose the abdominal viscera.
Slow Contracting Muscle  165
Step V: Identify the stomach (just down the line of
peritoneal cavity touches the liver and spleen.
Step VI: Cut and separate the stomach attachment
at the end of esophagus and down at the
duodenum.
Step VII: Identify the fundus (grey part) which
discriminates from the pyloric region that is pink
in appearance.
Step VIII: Wash the stomach contents properly with
the PSS and then cut open the fundus by lesser
curvature.
Step IX: Then, cut the fundus at the midline into
two equal parts.
Step X: For preserving the longitudinal muscle the
alternate opposite vertical cut should be given (as
shown in the figure) and then tie the thread on the
either end of the muscle and stretch out the muscle.
Step XI: Then, attach into the organ bath and leave
for relaxation at least 30-45 min.
Step XII: Select the experimental design and get
the response with standard and test drug.
Calculation and Result
The calculation and graph depend on the method /
design of experimentation adopted either
3- point, 4-point or matching, etc. (see the bioassay
section for calculation).
Inference
Write the findings of the study.
Discussion
Fundus contains the swallowed air and has
functions mainly concerned with pressure
changes. Its preparation is somewhat tedious than
the other tissue preparations because it require
alternate longitudinal cutting of tissue in which
contraction of a circular muscle is measured in a
longitudinal fashion. It is a slow contracting
muscle hence, sometimes it needs a stretching
weight to bring it to the baseline. Bradykinin
166  Practical Manual of Experimental and Clinical Pharmacology

produces slow contraction of stomach fundus

whereas it is a strong stimulator of isolated
intestinal tissue. This tissue well responds to the
tryptamine and prostacycline (PGE2).

SUGGESTED READING
Frog rectus abdominis
1. Cohen ML, Fludzinski LA. Contractile serotonergic muscle (Typical silver grey
receptor in rat stomach fundus. J Pharmacol Exp color; differentiating from the
Ther 1987;243(1):264-69. surrounding muscle)
2. Komada T, Yano S. Pharmacological characterization
of 5-hydroxytryptamine-receptor subtypes in circular
muscle from the rat stomach. Biol Pharm Bull
2007;30(3):508-13.
3. Scarparo HC, Santos GC, Leal-Cardoso JH, Criddle
DN. Selective inhibitory effects of niflumic acid on 5- Fig. 16.2: Identification of frog rectus abdominis muscle
HT-induced contraction of the rat isolated stomach
Materials and Method
fundus. Br J Pharmacol 2000;130(3):678-84.
4. Vane JR. A sensitive method for the assay of 5- Animal/tissue : Frog/rectus abdominis muscle
hydroxytryptamine. 1957. Br J Pharmacol 1997; PSS : Ringer
120(4 Suppl):142-47.
Lever : Simple writing lever
5. Yu PL, Fujimura M, Hayashi N, Nakamura T,
Fujimiya M. Mechanisms in regulating the release of Magnification : 10-15x
serotonin from the perfused rat stomach. Am J Tension : 1-2 gm
Physiol Gastrointest Liver Physiol 2001;280(6): Air : O2/Carbogen
G1099-105. Temperature : Room temperature
Drug : Acetylcholine chloride (ACh)
EXPERIMENT NO: 16B [M. wt: 181.68]
Aim Precautions before Experimentation
To determine unknown concentration of ACh • Frog rectus abdominis muscle is skeletal
using frog rectus abdominis muscle. muscle and requires less precaution during
handling compared to other smooth isolated
Background tissues
• Clean the organ bath before starting the
Rectus abdominis muscle is a striated skeletal experiment specially inner organ bath (chances
muscle preparation which is sensitive to the ACh of presence of previous drug used)
and curare like substances. But, the most sensitive • Balance the simple writing lever tangentially
muscle for the ACh is considered to be the dorsal leech with smoked drum
muscle. The activity is considered due to presence • Prepare PSS for the experiment with sufficient
of nicotinic muscarinic (NM)-receptor. Mamma- quantity of chemicals (1% variability is
lians muscle fibers are of two types; single or focal acceptable)
-innervated fiber and multiple-innervated fibers. • Add the calcium chloride at the end of PSS
Among which single or focal –innervated fiber preparation (to avoid any precipitation: PSS
shows fast contractility whereas multiple- should be clear)
innervated fibers shows the slow contraction. Frog • Try to minimize the handling of tissue (espe-
rectus abdominis muscle is composed of both cially at the middle part)
fibers but the multiple-innervated fibers dominate • Maintain the dose cycle properly (tissue
and show the slow contraction (Fig. 16.2). sensitivity depend on this cycle)

Method
Step I: Pith the frog and lay it on its back on the
frog dissecting board. Pin the four limbs on the
dissecting board (ventral part facing above and
distal part facing downward).
Step II: Remove the abdominal skin and expose
the rectus abdominis muscle (silver grey, distinct
from surrounding tissue, at middle lining).
Step III: Dissect out the muscle on the PSS soaked
piece of paper and spread gently.
Step IV: Cut into two pieces (preserve the
longitudinal muscle).
Step V: Tie the thread on the one end and tie it to
the inner organ bath and the second end tied to
the simple lever in the upright position under the
tension of 1-2 gm.
Step VI: Leave tissue for relaxation for at least
45 min.
Step VII: Mean while prepare the standard and
test drug serial dilution.
Step VIII: Start the recording of response by using
the suitable experimental design.
Calculation and Result
The calculation and graph depend on the
method/design of experimentation adopted either
3- point, 4-point or matching, etc. (see the bioassay
section for calculation).
Inference
Write the findings of the study.
Discussion
Frog rectus abdominis muscle is the easiest
isolated tissue to handle, even safe with new
people. Being the amphibian, it responds under
room temperature and does not require
temperature maintenance. But, season variability
is noted in the response recording. It shows erratic
response in the winter but it is corrected by the
Slow Contracting Muscle  167
maintaining the room temperature and proper
oxygenation whereas, in summer it gives good
result in standard set-up.
SUGGESTED READING
1. Edge ND. The effect of antiadrenaline compounds
on acetylcholine responses of frog rectus abdominis
muscle. Br J Pharmacol 1970;38(2):386-93.
2. Erenmemisoglu A, Tekol Y, Gogusten B.
Neuro-muscular blocking effect of thiamphenicol on
frog rectus abdominis muscle. Gen Pharmacol 1 994;
25(7):1417-20.
3. Karataº Y, Ergün Y, Göçmen C, Seçilmiº A, Singirik
E, Dikmen A, Baysal F. Possible postsynaptic action
of aminoglycosides in the frog rectus abdominis. Acta
Med Okayama 2000;54(2):49-56.
4. Kounenis G, Koutsoviti-Papadopoulou M, Elezoglou
V. Effect of nizatidine and ranitidine on the D-
tubocurarine neuromuscular blockade in the toad
rectus abdominis muscle. Pharmacol Res 1994;
29(2):155-61.
5. Nagata M, Kadota K. Acetylcholine bioassay with
thin strip of frog rectus abdominis muscle. Nippon
Seirigaku Zasshi 1977;39(3):62-64.
6. Parle M, Kulkarni SK. Alpha 2 adrenoceptor-
mediated contraction of frog rectus abdominis
muscle. Arch Int Pharmacodyn Ther 1984;271(1):
122-26.
7. Premendran SJ, Khapre MD, Sharma ML. Effects of
calcium, strontium, and barium salts on frog rectus
abdominis. Indian J Exp Biol 1990;28(6):590-91.
8. Ramaswamy S, Geetha VS, Nazimudeen SK,
Kameswaran L. Carbachol-induced sensitivity
changes in skeletal muscle and their mechanism of
action. Eur J Pharmacol 1978;52(2):197-200.
9. Rao SS, Bhagwat AW, Parmanand VG. Interaction
of acetylcholine and caffeine on the isolated rectus
abdominis of frog (Rana tigrina). Indian J Physiol
Pharmacol 1978;22(2):155-57.
EXPERIMENT NO: 16C
Aim
To determine unknown concentration of acetyl-
choline (ACh) using guinea pig trachea.
Background
This method is based on the method of Castillo
and De Beer (1947). The response is taken by the
168  Practical Manual of Experimental and Clinical Pharmacology

extraction of at least 6 cm of trachea (containing B. Give the spiral cut to the trachea. Cut is
minimum of 6 rings). Tracheal ring is in “D” form, made at the mid part of cartilage then, tie
and smooth muscle is present in the straight line the thread on the either end and stretch the
of “D” shape. The ring is prepared for the assay tissue (Fig. 16.4C)
by the two means
This preparation is mainly to demonstrate the
1. Separate the ring and tie one another with the
help of thin thread (Fig. 16.3) respiratory dominant β2-adrenoreceptor which
Tie the ring with one another at cartilage, but causes bronchodilation by the adenylcyclase and
it is important to keep in mind that smooth cAMP. Limitation of the tissue preparation is that
muscle should be at longitudinal fashion. it is tedious to prepare and has the slow contrac-
2. A. Longitudinal cut along the mid dorsal tion and relaxation.
surface (cut at mid of cartilage part), then
separate each cut ring. Then, tie the each Materials and Method
cartilage end with one another to form loop
(Figs 16.4A and B) or Animal/tissue : Guinea pig/trachea
PSS : Krebs
Lever : Simple/frontal writing
Magnification : 10-20x
Tension : Up to 0.5 gm
Air : O2/Carbogen
Temperature : 35-37°C

Precautions before Experimentation

• Clean the organ bath before starting the

Fig. 16.3: Separation of tracheal rings and method to
make a chain
experiment specially inner organ bath (chances
of presence of previous drug used)
• Balance the writing lever horizontal with the
help of load
• Make PSS for the experiment, while taking
exact quantity of chemicals (1% variability is
acceptable)
• Add the calcium chloride at the end of PSS
preparation (to avoid any precipitation: PSS
should be clear)
• Try to minimize the handling of tissue (espe-
cially at the middle part)
• Maintain the dose cycle properly (tissue
sensitivity depend on this cycle).

Method

Figs 16.4A to C: Another method to prepare trachea for Step I: Guinea pig (GP) is sacrificed under
bioassay anesthesia.

Step II: Fix the GP on the dissecting board and
then shave the hair near the neck region.
Step III: The trachea is dissected out and put into
the petri dish containing Krebs solution (see the
identification and collection of tissue section for detail).
Step IV: Tracheal chain is then dissected out by
any of the above mentioned methods.
Step V: Mount the trachea in the inner organ bath
and give the 45-60 min of relaxation.
Step VI: Select the experimental design (bracketing,
matching, 3-point and 4-point).
Calculation and Result
Result and calculation depends on the experi-
mental design. After getting the complete response
recording, refer the calculation part in bioassay
chapter.
Discussion
The response of this tissue is slow to develop but
last for longer period. This preparation is utilized
to study the bronchodilators like theophylline,
adrenaline, etc. β-adrenoceptor subtypes which
induced relaxation is identified in smooth muscle
cells of the isolated guinea-pig trachea. Tracheal
preparation is an ideal model to study the
contractile drugs like acetylcholine, 5-HT and
histamine, additionally their antagonism is
studied by several drugs like adrenaline,
isoprenaline (ISO), aminophylline, theophylline,
etc. Noradrenaline lacks activity on β2- adrenergic
receptor, hence absence of relaxation in the
presence of the ACh, 5-HT or histamine. In the
recent study, role of nitric oxide (NO) as
bronchodilator is also well studied.
SUGGESTED READING
1. Dellabianca A, Faniglione M, De Angelis S, Tonini S,
Balestra B, Colucci M, Cervio M, Clavenzani P,
Chiocchetti R, De Giorgio R, Candura SM. Adenosine
A(1) and A(3) Receptor Agonists Inhibit
Nona-drenergic, Noncholinergic Relaxations in the
Guinea Pig Isolated Trachea. Respiration. 2008
Dec 11.
Slow Contracting Muscle  169
2. Gok S, Izanli-Paksoy A, Vural K. Contribution of
RhoA kinase and protein kinase C to weak relaxant
effect of pinacidil on carbachol-induced contractions
in sensitized guinea-pig trachealis. Arch Pharm Res
2009;32(2):243-50.
3. Larsson AK, Fumagalli F, DiGennaro A, Andersson
M, Lundberg J, Edenius C, Govoni M, Monopoli A,
Sala A, Dahlén SE, Folco GC. A new class of nitric
oxide-releasing derivatives of cetirizine; pharma-
cological profile in vascular and airway smooth
muscle preparations. Br J Pharmacol 2007; 151(1):
35-44.
4. Nascimento NR, Refosco RM, Vasconcelos EC,
Kerntopf MR, Santos CF, Batista FJ, De Sousa CM,
Fonteles MC. 1,8-Cineole induces relaxation in rat
and guinea-pig airway smooth muscle. J Pharm
Pharmacol 2009;61(3):361-66.
5. Schaafsma D, Gosens R, Bos IS, Meurs H, Zaagsma
J, Nelemans SA. Role of contractile prostaglandins
and Rho-kinase in growth factor-induced airway
smooth muscle contraction. Respir Res 2005;6:85.
6. Tanaka Y, Yamashita Y, Horinouchi T, Koike K.
Adrenaline produces the relaxation of guinea-pig
airway smooth muscle primarily through the
mediation of beta(2)-adrenoceptors. J Smooth Muscle
Res 2005;41(3):153-61.
EXPERIMENT NO: 16D
Aim
To determine unknown concentration of acetyl-
choline (ACh) using rat phrenic nerve dia-
phragm.
Background
This is primary motor nerve of the diaphragm
which arises mainly from the fourth cervical
nerve. Preparation is preferred in the nerve
mediated muscle contraction and its response in
presence of agonist and antagonists. This nerve
preparation performs well in Krebs or tyrode
solution at 32-37°C. However, it is seen that the
cholinesterase activity is absent at room temperature.
Cholinergic agonists, anticholinesterase,
sympathomimetics or neuromuscular blockers
may be used in the preparation. The response
observed by physostigmine and neostigmine is
different in the neuromuscular blockage
produced by the d-tubocurarine.
170  Practical Manual of Experimental and Clinical Pharmacology
Materials and Method
Animal/tissue : Rat/ phrenic nerve diaphragm
PSS : Krebs /tyrode
Lever : Preferably spring loaded lever
or simple lever
Magnification : 10-18x
Tension : 0.5-1g
Air : O2 or Carbogen
Temperature : 35-37°C
Drug : Acetylcholine chloride (ACh)
[M. wt: 181.68]
Precautions before Experimentation
• Clean the organ bath before starting the
experiment specially inner organ bath (chances
of presence of previous drug used)
• Balance the writing lever horizontal with the
help of load
• Prepare PSS for the experiment, while taking
exact quantity of chemicals (1% variability is
acceptable)
• Add the calcium chloride at the end of PSS
preparation (to avoid any precipitation: PSS
should be clear)
• Try to minimize the handling of tissue (espe-
cially at the middle part)
• Maintain the dose cycle properly (tissue
sensitivity depend on this cycle)
• Carefully remove the connective tissues and
blood vessel attached to the phrenic nerve.
Method
Step I: Sacrifice the rat by stunning (a strong blow)
on the head and then transfer the animal on the
dissecting board (DB).
Step II: Rat should be fixed on the DB by tying it
legs with the help of thread.
Step III: Cut open the thorax of rat by a vertical
midline incision, and then exposed the thoracic
cavity.
Step IV: Ribs are dissected from the base of the
sternum, and half thorax is cut and removed by
cutting through animal flanks.
Step V: Separate and clear the thoracic contents,
then identify the phrenic nerve, running from
diaphragm to the thymus gland.
Step VI: Two cuts are given, one at diaphragm and
second at the base of the thymus (avoid excessive
cleaning of the nerve from the muscle).
Step VII: Immediately transfer the nerve to the krebs
or tyrode, then attach to the organ bath.
Step VIII: Give proper relaxation for 45-60 min
and select the experimental design and record
the response by standard and test drugs
respectively.
Calculation and Result
The calculation and graph depend on the
method/design of experimentation adopted either
3- point, 4-point or matching, etc. (see the bioassay
section for calculation).
Inference
Write the findings of the study.
Discussion
Rat phrenic nerve–diaphragm preparation is
commonly used in the experimental
pharmacology for evaluation of neuromuscular
function. Studies showed 70% of ACh in the
diaphragm contain motor nerve terminal, 10%
intramuscular nerve fibers and 65% acetyl
transferase is in motor terminal with 35% in nerve
fiber. Studies showed atropine has variable
response with different concentration. In low
concentration, it enhanced neuromuscular trans-
mission, possibly via a presynaptic mechanism,
however in higher concentration, atropine causes
reduction or it may block transmission.
SUGGESTED READING
1. Alves-do-Prado W, Prado WA. Neuromuscular
facilitation induced by muscarinic antagonists in the
 Slow Contracting Muscle  171

rat isolated diaphragm. Gen Pharmacol 1993;24(6): Materials and Method

1501-14.
2. Correia-de-Sá P, Timóteo MA, Ribeiro JA. Pre- Animal/tissue : Chick/biventer-cervicis
synaptic A1 inhibitory/A2A facilitatory adenosine PSS : Krebs
receptor activation balance depends on motor Lever : Simple writing
nerve stimulation paradigm at the rat hemi- Magnification : 10-15x
diaphragm. J Neurophysiol 1996;76(6):3910-19. Tension : 1-2g
3. Snider RM, Gerald MC. Noradrenergic-mediated Air : Carbogen (95% O2 + 5% CO2)
potentiation of acetylcholine release from the phrenic
Temperature : 37°C
nerve: evidence for presynaptic alpha 1-
adrenoceptor involvement. Life Sci 1982;31(9):
Drug : Tubocurarine chloride [M.wt:
853-57. 771.72]
4. Wessler I, Anschütz S. Beta-adrenoceptor stimulation Physostigmine [M.wt: 275.
enhances transmitter output from the rat phrenic 346]
nerve. Br J Pharmacol 1988;94(3):669-74. Hexamethonium [M.wt: 362.
5. Wessler I, Ladwein E, Szrama E. Stimulation of alpha 188]
1-adrenoceptors increases electrically evoked Acetyl choline chloride (ACh)
[3H]acetylcholine release from the rat phrenic nerve. [M. wt: 181.68]
Eur J Pharmacol 1989;174(1):77-83.

Precautions before Experimentation

EXPERIMENT NO: 16E
Aim
To determine neuromuscular blocking drugs
using innervated biventer-cervicis preparation of
the chick.
Background
This preparation is for the study of neuromuscular
blocking agents such as suxamethonium, tubo-
curarine, etc. Muscles contain two types of slow or
twitch fibers which correspond to different
response at the different stimulus. When their nerve
supply is stimulated electrically, they give twitch
responses which is similar to the rat diaphragm
responses whereas its contraction responses are
similar to the frog rectus muscle. Decamethonium,
suxamethonium, etc. have good specificity for the
tissue and give good contractility. Tubocurarine
chloride is used in the higher concentration to
produce contraction as compared to other drugs.
Acetylcholine (ACh) is less contractile in this
muscle but produces marked effects in the presence
of physostigmine or neostigmine. In the presence
of suxamethonium and ACh, it shows contractile
and depolarization block which is increased by
neostigmine whereas tubocurarine has no activity
on slow muscle fiber and neostigmine reversed
neuromuscular block of twitch muscle.
• Clean the organ bath before starting the
experiment specially inner organ bath (chances
of presence of previous drug used)
• Balance the writing lever horizontal with the
help of load
• Prepare PSS for the experiment, while taking
exact quantity of chemicals (1% variability is
acceptable)
• Add the calcium chloride at the end of PSS
preparation (to avoid any precipitation: PSS
should be clear)
• Try to minimize the handling of tissue
(especially at the middle part)
• Maintain the dose cycle properly (tissue
sensitivity depend on this cycle).
Method
Step I: Select chick weighing about 400-800 g and
euthanize with sodium pentobarbitone (6 mg/
100g, IM) or chloroform.
Step II: Give the midline incision on the back of the
neck after removal of feathers.
Step III: Identify the biventer-cervicis on the both
side of the midline incision just under the skin.
Step IV: Tie the upper portion with thread and cut
the upper portion then cut the muscle from the
172  Practical Manual of Experimental and Clinical Pharmacology
bottom then attach the second thread at the bottom
to hang the tissue in inner organ bath.
Step V: Give the relaxation time about 45 min-1 hr.
Step VI: Make the standard solution and then make
serial dilution.
Step VII: Select the experimental design (matching,
bracketing, 3-point, or 4-point assay).
Calculation and Result
The calculation and graph depends on the
method/design of experimentation adopted either
3- point, 4-point or matching, etc. (see the bioassay
section for calculation).
Inference
Write the findings of the study.
Discussion
Biventer cervicis (BC) is an anatomically complex
tendinous muscle and respond differently
twitch/contraction to different stimuli.
Tubocurarine, suxamethonium, decamethonium,
carbachol, etc. may act on the chick biventer
cervicis muscle by releasing acetylcholine at the
synapse which then acts on the postjunctional
receptors to produce the response.
SUGGESTED READINGS
1. Barlow RB, Zoller A. Activity of analogues of de-
camethonium on the chick biventer cervicis preparation.
Br J Pharmacol Chemother 1962;19: 485-91.
2. Elliott RC. The role of acetylcholine in tetra-
ethylammonium induced contractures of the chick
biventer cervicis muscle in the presence of lidocaine.
Gen Pharmacol 1987;18(1):7-11.
3. Marshall I G. Actions of acetylcholine and carbachol
on the chick biventer cervicis muscle Br J Pharmac
1971;42:462-72.
4. Wali FA. Effect of lignocaine on chick biventer cervicis
skeletal muscle. Pharmacol Res Commun 1986;18(1):
31-48.
 Cardiac Muscle Preparation
17
EXPERIMENT NO: 17A
Aim
To observe the effect of various drugs on the
isolated heart (Langendorff’s preparation).
Background
In 1897, Oscar Langendorff established the isolated
perfused mammalian heart preparation which
was considered to be the breakthrough in
cardiovascular research. This is based on the
principle of retrograde flow in the aorta either at
constant flow or constant pressure. The entire
perfusate enters the coronary arteries via the
ostia at the aortic root (Fig. 17.1A). The physio-
logical salt solution (PSS) passes through the
coronary circulation and then, perfusate drains
into the right atrium via the coronary sinus.
Coronary flow rate is measured on volumetric
determination.
Animal Required
• Albino Rats (300 gm and at least of 1 yr of age),
or
• New Zealand Rabbits (1.5-3 kg and 3 years of
age) or
• Guinea pig (300-450 gm and 2-3 years of age)
Animal is housed at ambient temperature
(23°C ±2°C) under a 12:12 hr light and dark cycle
and with free access to tap water and food ad
libitum. The animals are acclimatized to the
laboratory conditions for at least 1 week prior to
experimentation.
Precautions
• Animal should be pretreated with heparin
(55-110 µg/kg, i.p or s.c [for rabbit] or heparin
(500-1000 IU/kg, i.v in rabbit and rat and i.p
in guinea pig), to avoid thrombosis in heart, if
mounting take > 2-3 min after the surgical
procedure) and then sacrificed after 20-30 mins
• Maintain the PSS flow rate adequately (Edema
may develop in cardiac tissues, if the perfusion
pressure is too high) (The expected flow rate
through the cannula differs depending on
species and it ranges from 7-9 ml/min for rat to
20 ml/min for rabbit)
• While attaching the heart to the cannula, there
should be already some flow through the aortic
cannula, which helps to avoid air bubbles
entering the coronary arteries
• Insert cannula firmly so that it should not
penetrate into the aortic valve (Fig. 17.1A to C)
• Maintain the sufficient hydrostatic pressure
by maintaining the distance between reservoir
and heart position.
Method
Animal is pretreated with the heparin (55-110 µg/
kg, i.p or s.c [for rabbit] or heparin (500–1000 IU/kg,
i.v in rabbit and rat and i.p in guinea pig). Animal
is stunned and the thorax is opened immediately
and the heart exposed. Heart is dissected out and
removed as rapidly as possible and transferred
immediately into cold Krebs solution (rat and guinea
pig heart) or McEwens solution (rabbit heart). The
ascending aorta is separated gently using forceps
from the pulmonary artery and dissect the heart
174  Practical Manual of Experimental and Clinical Pharmacology

Figs 17.1A to C: (A) Showing the coronary supply of the rat or rabbit heart; (B) Bilateral coronary supply in guinea pig,
different from the rat or rabbit. It has 2 right and 2 left coronary arteries and (C) Position of cannula in the ascending aorta
(above aortic valve)

with at least 1 cm aorta intact. (Usually the aorta is
cut just before it divides.) The heart is gently
squeezed several times to remove blood from the
heart and prevent formation of thrombus. Trim
away any excess connective and lung tissue from
the heart taking care not to damage the aorta. The
glass cannula (3 mm outer diameter for rats and
guinea pig; 4 mm outer diameter for rabbit) tip is
placed 0.5 cm into the aorta and firmly kept in place
using a thread. It is important to exclude the air
entrap into the system to prevent air emboli. Initially
side arm is flushed to remove air bubbles from the
apparatus before the experiment is started.
Langendorff’s preparation is a constant head
reservoir system, used for providing pressure, raise
this to about 18 inches above the heart or alter-
natively, if a peristaltic pump is used set the rate for
about 15-20 ml/min, reducing it to 3-5 ml/min
maintain perfusion chamber and aerated Krebs
solution in it at 37-38°C.
Attachment of Heart
Attach a heart clip complete with a length of thread
to the tip of the ventricles (another small clip may
be bound to the auricles). The thread is passed
around a pulley arm vertically below the heart.
Attach the thread to the transducer after passing
it over a second pulley horizontal to first and
about 20 cm apart and attach to the transducer

Fig. 17.2: Langendorff apparatus set-up for the assessment of activity of different drugs
which further attach to the coupler of student
physiograph. After the experiment animals are
disposed in the yellow polythene bags for the
incineration.
Instrument
The recording is done by attaching the thread to a
strain gauge transducer, an attachment with the
student physiograph. The paper speed can be
adjusted as required. The sensitivity of the
recording apparatus to the heart contractions can
be adjusted based on the sample tracings and the
baseline is adjusted so that the tracing is in middle
of the paper. Standardized speed is maintained.
Drugs
Drug additions by attaching a syringe to the
injection port (or into a rubber tubing) preferably
keeping the volumes < 0.2 ml.
Cardiac Muscle Preparation  175
Recording
Reading of the rate of heating and of the coronary
flow can most commonly be taken over a period of
30 seconds. If the movements are too fast to count,
the kymograph can be run at a high speed for 15-
30 seconds before the drug reaches the heart and
in most instances, the effects passes in a few
minutes as the drugs are washed through.
Recording of the rate of heating of flow should be
taken once every 2 minutes. Further, doses should
not be given until the preparation has recovered
completely and the rates are steady or until they
have settled down steadily at new control levels.
Note: Important to follow the step: Initially the
experiment is started with the stimulant drugs
(epinephrine, norepinephrine, isoprenaline and
calcium in increasing doses), followed with
depressant (acetylcholine, potassium) drugs and
finally antagonists (propranolol and atropine) are
added as the effects of the antagonists take a long
time to wear off.
176  Practical Manual of Experimental and Clinical Pharmacology
Observations: The preparation is observed for the
inotropic (by measuring the amplitude of contrac-
tions) effect, chronotropic (by measuring the rate of
heart beat) and effect on coronary vascular
resistance (by measuring the rate of collection of
drops from the preparation).
 Important Points to Remember
1. Based on principle of retrograde perfusion
2. Circulation of PSS in isolated heart: Directly delivered
to the coronary artery (left and right), collected at
coronary sinus then drained into right ventricle and
then pumped through the pulmonary artery
3. Rabbit heart can beat to 9 hour if not treated to the
toxic drugs
4. Transfer of isolated heart immediately to the ice cold
PSS leads to reduce the metabolic activity as well as
reduce beating, which results into low requirement of
glucose and oxygen
5. Isolated heart reduces work load, hence the cardiac
output in isolated heart cannot be directly correlated
as in in vivo condition
SUGGESTED READING
1. Beckett PR. The isolated perfused heart preparation:
Two suggested improvements. J Pharm Pharmac
1970;22:818.
2. Doring HJ, Dehnert H. The isolated perfused heart
according to Langendorff. BVM-Biomesstechnic
Verlag, 1987.
3. Fukunami M, Hearse DJ. The inotropic consequences
of cooling: Studies in the isolated rat heart. Heart
and Vessels 1989;5:1-9.
4. Hearse DJ, Sutherland FJ. Experimental models for
the study of cardiovascular function and disease.
Pharmacol Res 2000;41(6):597-603.
5. Hofer E, Stark U, Stark G, Tritthart HA. Detection
and continuous monitoring of intracardiac low-level
potentials from the surface of the Langendorff-
perfused heart. Basic Res Cardiol 1990;85:198-208.
6. Mattsson C, Hoylaerts M, Holmer E, Uthne T, Collen
D. Antithrombotic properties in rabbits of
heparin and heparin fragments covalently coupled
to human antithrombin III. J Clin Invest 1985;75(4):
1169-73.
7. Ravelli F, Allessie M. Effects of atrial dilatation on
refractory period and vulnerability to atrial fibrillation
in the isolated Langendorff-perfused rabbit heart.
Circulation 1997;96:1686-95.
8. Stark G, Stark U, Tritthart HA. Acute effects of
amiodarone on the pacemaker- and conduction
systems of the Langendorff perfused guinea pig heart.
Z Kardiol 1987;76(S2):67.
EXPERIMENT NO: 17B
Aim
To determine the effect of different drugs on the
normal and hypodynamic rabbit heart.
Background
Hypodynamic heart is defined as the heart
exhibiting subnormal power or force than the
normal one. Experimentally, it is developed by
the supply of the 1/4th of calcium chloride (CaCl2)
than the required one which reduces the heart
rate.
Requirements
• Animal: Rabbit (2-3 kg)
• Drugs: Digoxin, calcium chloride (CaCl2)
• Physiological salt solution (PSS): McEwens
solution (For normal heart), ¼th of CaCl2 in
McEwens solution (For the hypodynamic
heart)
• Instrument: Physiograph or Starling Heart lever
and drum.
Method
The entire primary set-up of instrument and the
attachment of the heart is same, as it is in the
Langendorff’s preparation.
Recording of the Responses
Step I: First record the effect of the drug on the
normal heart preparation with the (0.1, 0.2, 0.4
and 0.8 ml. (Digoxin, CaCl2)
Step II: Then, note the dose on the said response
accordingly.
Step III: McEwens solution is replaced by the
hypodynamic McEwens solution and leave for 5-
10 min for the acclimatization of the heart in the
hypodynamic solution.
Step IV: Record the response of the drug on the
same concentration as it is used previously in the
normal heart.
Step V: Note the response of drug after treating
with the hypodynamic solution and note the
 Cardiac Muscle Preparation  177

dose on the respective response. Then, fix the Step VI: Compare the response of the drug
tracing if used kymograph. on the normal and hypodynamic heart pre-
paration.

Observation and Result

Drug Dose (ml) Normal heart Hypodynamic heart Remarks
Rate Force Rhythm Rate Force Rhythm
Digoxin 0.1
0.2
0.4
0.8
Calcium chloride 0.1
0.2
0.4
0.8

SUGGESTED READING
1. Chapman RA, Niedergerke R. Effects of calcium on
the contraction of the hypodynamic frog heart. J
Physiol 1970;211(2):389-421.
2. Grupp IL, Subramaniam A, Hewett TE, Robbins J,
Grupp G. Comparison of normal, hypodynamic, and
hyperdynamic mouse hearts using isolated work-
performing heart preparations. Am J Physiol Heart
Circ Physiol 1993;265:H1401-10.

EXPERIMENT NO: 17C

Aim
To demonstrate the effect of the inotropic and Fig. 17.3: Three chambered frog heart
chronotropic effects of various drugs on frog heart
normal/hypodynamic)
i. Isolated preparation and mimetic drugs like adrenaline and noradrenaline.
ii. In situ preparation There are few drugs which decreases the rate and
force of contraction and hence they grouped as
Background “negative chronotropic” and “negative inotropic”
drugs respectively like parasympathetic drugs like
The heart is the most common site for the drug target acetylcholine (ACh).
and there are several drugs which influence the
rate, force or rhythm of the heart either as therapeutic REQUIREMENTS
effect or as side effects. Inotropic drugs are those
which increase the force of contraction and the Animal : Frog (50- 70 gm)
response is called as the “positive inotropic Drugs : Adrenaline
response” whereas drugs which increase the heart Noradrenaline
rate are chronotropic drugs and the response is Acetylcholine
known as “positive chronotropic response”. Calcium chloride
Broad classification of drugs into the positive Potassium chloride
inotropic and chronotropic involves sympatho- Atropine
178  Practical Manual of Experimental and Clinical Pharmacology

PSS : Frog ringer solution (FRS) and Step VI: The lever used is sterling-heart lever. Heart
hypodynamic solution is attached to the lever with the help of thin thread
(¼CaCl2 in FRS)
Step VII: Then, take the recording with different
Temperature : Room temperature
drugs
Instrument : Physiograph or kymograph

Method for Experiment No: 17(i)
Step I: Carefully hold the frog with its hind limb
and then anesthetize the frog by pithing or keep it
into the freezer (-4°C) for about 3-5 min
Step II: Give a little horizontal cut at the mid of
abdomen and then a long vertical incision at the
midline above sternum
Step III: Open the thoracic cage and locate the heart
Step IV: Carefully remove the pericardium and
remove the heart
Step V: Clear it from the surrounding tissue and
squeeze it gently to remove the blood from the heart
in cold FRS and then mount it in the organ bath.
Methods for Experiment No: 17(ii)
Step I: Carefully hold the frog with its hind limb
and then anesthetize the frog by pithing or keep it
into the freezer (-4°C) for about 3-5 min
Step II: Give a little horizontal cut at the mid of
abdomen and then give a long vertical incision at
the midline above sternum
Step III: Open the thoracic cage and locate the heart
Step IV: Cannulate the aorta, with a little incision
and tie the cannula with aorta in situ
Step V: Take the responses with different drugs
using 0.2 ml adrenaline, noradrenaline, ACh,
CaCl2, KCL and atropine.

Observation and Results

Drug Dose (ml) Normal heart Hypodynamic heart Remarks
Rate Force Rhythm Rate Force Rhythm
Adrenaline 0.1
0.2
0.4
0.8
Noradrenaline 0.1
0.2
0.4
0.8
Acetylcholine 0.1
(ACh)
0.2
0.4
0.8
CaCl2 0.1
0.2
0.4
0.8
KCl 0.1
0.2
0.4
0.8
Atropine 0.1
0.2
0.4
0.8

SUGGESTED READING
1. Broadley KJ. Negative inotropic responses of the
isolated heart of the rat to isoprenaline. Br J Pharmacol
1972;45(1):123-25.
2. Graham JA, Lamb JF. The effect of adrenaline on the
tension developed in contractures and twitches of
Cardiac Muscle Preparation  179
the ventricle of the frog. J Physiol 1968;197(2):
479-509.
3. Martin Morad, Chris Sanders, James Weiss. The
inotropic actions of adrenaline on frog ventricular
muscle: Relaxing versus potentiating effects. J Physiol
198;311:585-604.
 Part 3

Experimental (In Vivo Studies)

 Animal Experiment on
18 Central Nervous System (CNS)

EXPERIMENT NO.: 18A

Aim
To demonstrate the effect of pentobarbital on
righting reflex (Hypnosis) in mouse.

Background
Hypnosis is the process of natural sleep and the
drugs or agents inducing it are known as the
hypnotic agents. This concept is applicable to the
patient or human use, but in the animal
experiments, the term “hypnotic” means deeper
stage of central depression which induces Fig. 18.1: Loss of righting reflex in rat (unable to correct
unconsciousness, associated with loss of righting body posture)
reflexes and muscle tone. The “loss of righting
reflex” is the term mainly used to denote the ‘sleep’ Hexabarbital (60 mg/ kg,
of an animal and it is defined as the loss of postural i.p), Diazepam (5-20 mg/kg,
reaction which can’t be corrected when the animal i.p or s.c)
is kept on its back. In the righting reflex animal
turns body in such a way that, its paws or feet are Precautions before Experimentation
pointed at the ground. Righting reflex reaction is
• Laboratory should be dim lighted and noise
dependent on normal vestibular, visual and
free
proprioceptive functions (Fig. 18.1).
• Animal should be marked properly, to avoid
mixing in two groups
Materials and Methods
• Handle the animals with care (minimize the
Materials stress and pain to animal)
• Observe the animals in a plexi glass chamber
Animal/species : Mouse/Swiss albino
Sex/Body weight : Either sex/ 20-30 g
Methods
Syringe/needle : 1ml/ preferably 24G on-
wards Step 1
Drug : Pentobarbital (50 mg/kg, i.p) • Weigh the animals and mark them properly to
Other drugs : Barbital (180 mg/kg, i.p), distinguish from one another
184  Practical Manual of Experimental and Clinical Pharmacology
• Divide animals into two groups (n = 6 in each
group)
Step 2
• Group 1: Control group (n = 6); mice are given
saline at the equivalent dose of drug
• Group 2: Treatment group (n = 6); mice are
given Pentobarbital 50 mg/kg, i.p.
Observation Table
Sl. Righting reflex
No. Onset (min) (O) Recovery (min) (R)
Group 1 Group 2 Group 1
1.
2.
3.
4.
5.
6.
Note: Cat is not a suitable animal for the experiment on “loss of righting reflex”. This is because; they
have flexible backbone and no functional clavicle.
Discussion
Evaluation of the righting reflex is an ideal method
to evaluate drug acting as CNS depressants, since
the entire CNS depressant drugs cause loss of
righting reflex in animals at certain dose. e.g.:
Mouse and rat are the ideal models for studying
righting reflex whereas the cat is not a suitable
animal for the experiment “on righting reflex”.
This is because; they have flexible backbone and
no functional clavicle. There are several methods
of doing this experiment, generally putting animal
on its back (distal portion) to correct the position
on its four legs or dropping the animal from a
certain height and observe either animal touches
ground on its four paws or not, is very commonly
in use. Drugs used in such experiment have CNS
depressant properties such as anesthetics like
pentobarbital, thiopental etc. or long of short acting
hypnotics like diazepam, nitrazepam or zolpidem,
zopiclone, triazolam etc. respectively. Studies
suggest that when short or long acting hypnotics
are added with the anaesthetics, it significantly
increases the duration of the loss of righting reflex.
Step 3
• Observe the animals for 45 min to 1 hour
• Observe for the onset of loss of righting reflex
and duration of loss of righting reflex
Note: Observe the animals for any other behavior
changes during the observation time (sniffing,
rearing, exploratory behavior, etc.)
Duration of loss of righting
reflex (min) (D= (R) – (O))
Group 2 Group 1 Group 2
SUGGESTED READING
1. Liu X, Lee TL, Wong PT. Cyclooxygenase-1 Inhibition
Shortens the Duration of Diazepam-Induced Loss
of Righting Reflex in Mouse. Anesth Analg 2006;
102:135-40.
2. Simon P, Chermat R, Doaré L, Bourin M, Farinotti R.
Interactions imprévues de divers psychotropes avec
les effets du barbital et du pentobarbital chez la
souris. J Pharmacol (Paris) 1982:13;241-52.
EXPERIMENT NO: 18B
Aim
To demonstrate muscle relaxant property of
diazepam in mouse using rotarod apparatus
Background
Rota rod test is used to evaluate fore and hind
limb motor coordination of rodents. The apparatus
consists of a horizontal metal rod (coated with
rubber) of 3 cm diameter attached to a motor with
the speed adjusted to 2 rotations/minute to 6
 Animal Experiment on CNS  185

Fig. 18.2: Rota rod apparatus

rotation/min. The rod is 75 cm in length and is
divided into 6 sections by plastic discs, thereby
allowing the simultaneous testing of 6 mice. The
rod is at a height of about 50 cm above the tabletop
in order to discourage the animals to escape from
the instrument (Fig. 18.2). The cut off time for the
test is 2 min. The retention time (sec) for each
mouse/rat is recorded.
Materials and Methods
Materials
• Animal should be marked properly, to avoid
mixing in two groups
• Handle the animals with care (minimize the
stress and pain to animal)
• Precondition the mouse with the procedure
Methods
Step 1
• Weigh the animals and mark properly to
distinguish from one another
• Divide animals into two groups (n = 6 in each
group)

Animal/species : Mice/ Swiss albino Step 2

• Group 1: Control group (n = 6); mice are given
Sex/Body weight : Either sex/ 20-30 g
saline at the equivalent dose of drug
Syringe/needle : 1ml/ preferably 24G on- • Group 2: Treatment group (n = 6); mice are
wards given diazepam at the dose of 3 mg/kg, ip
Drug : Diazepam (3-5mg/kg, i.p)
Step 3
• Observe the animals for 2 min on the rota rod
Precautions before Experimentation
apparatus
• Laboratory should be dim lighted and noise • Observe the animals, to fall down and note
free the time of falling down
186  Practical Manual of Experimental and Clinical Pharmacology
Note: For mouse: Mouse is placed on a 2.5 cm
diameter rod rotating at 3 rev/min. The animal’s
muscle coordination is considered to be impaired,
if it falls from the rota rod within 120 sec
observation period.
Dimensions of Rota Rod
Mouse: Rod is 75 cm in length, 30 mm diameter
and is divided into 6 sections by plastic discs and
of about 50 cm above the table top.
Rat: Rod is 75 cm in length, 60 mm diameter and
is divided into 6 sections by plastic discs and of
about 50 cm above the table top.
Observation Table
Sl. No. Retention time on Rota rod (cut off time 120 sec)
Pre-drug Post-drug
1.
2.
3.
4.
5.
6.
Discussion: Motor coordination is a physiological
process to achieve movement which is an essential
interaction between neural processes involved in
moving a limb, and the actual limb in movement.
This is mainly processed through peripheral
nervous system (PNS). It is responsible for both
the transmission of the efferent to the CNS and
the movement of the limb. CNS also plays a vital
role in integrating the afferent feedback and
efferent signals. There are several experimental
models which are established to study the motor
co-ordination in the rodents such as rotarod
method, chimney test, grip strength, treadmill
performance etc. In the either tests, rodents (mouse
or rat) are tested for their ability to retain the muscle
co-ordination such as in rotarod test. Mouse/rat
should remain on the revolving rod or in chimney
test; it should climb backward to show the muscle
coordination. The drugs which can be screened
through these tests are anesthetics like Pheno-
barbital, etc. centrally active skeletal muscle
relaxants such as benzodiazepam, chlordia-
zepoxide or diazepam, zolpidem, zopiclone, etc.
SUGGESTED READING
1. Cartmell SM, Gelgor L, Mitchell D. A revised rotarod
procedure for measuring the effect of antinociceptive
drugs on motor function in the rat. J Pharmacol
Methods 1991;26(2):149-59.
2. Dunham NW, Miya T S. A note on a simple approach
for determining neurological deficits in rats and
mouse. J Am Pharmacol Assoc 1957;46:208.
3. Liu X, Lee TL, Wong PT. Cyclooxygenase-1 Inhibition
Shortens the Duration of Diazepam-Induced Loss
of Righting Reflex in Mouse. Anesth Analg 2006;
102:135-40.
4. Stanistaw J. Czuczwar, Kinga K. Borowicz, Zdzistaw
Kleinrok, Piotr Tutka, Tomasz Zarnowski,
Waldemar A. Turski. Influence of combined
treatment with NMDA and non-NMDA receptor
antagonists on electroconvulsions in mouse. Eur J
Pharmacol 1995;281:327-33.
EXPERIMENT NO: 18C
Aim
To demonstrate muscle relaxant property of
diazepam in mouse using chimney test.
Background
Mouse is made to climb backward up a plastic
Perspex tube (3 cm inner diameter and 25 cm
length). The animals usually unable to perform
the test within 60s are considered to be positive
for motor impairment. The internal diameter
varies with the animal’s weight such as for low
weight mouse, it requires less diameter and for
higher weight mouse, it require larger inner
diameter of tube (22-28 mm).
Materials and Methods
Materials
Animal/species : Mouse/Swiss albino
Sex/Body weight : Either sex/ 20-30g
Syringe/needle : 1ml/ preferably 24G on-
wards
Drug : Diazepam (3mg/kg, i.p)
Precautions before Experimentation
• Laboratory should be dim lighted and noise
free
 Animal Experiment on CNS  187

• Animals should be marked properly, to avoid 2. Stanistaw J Czuczwar, Kinga K. Borowicz, Zdzistaw
mixing in two groups Kleinrok, Piotr Tutka, Tomasz Zarnowski,
• Handle the animals with care (minimize the Waldemar A Turski. Influence of combined treatment
with NMDA and non-NMDA receptor antagonists
stress and pain to animal)
on electroconvulsions in mouse. Eur J Pharmacol
• Precondition the mouse with the procedure 1995;281:327-33.
one day prior to the experiment day.
EXPERIMENT NO.: 18D
Methods
Aim
Step 1
• Weigh the animals and mark properly to To demonstrate anti-anxiety effect of diazepam in
distinguish from one another rat using elevated plus maze apparatus
• Divide animals into two groups (n = 6 in each
group) Background
Step 2 It is a novel test for the selective identification of
• Group 1: Control group (n = 6); mice are given ‘anxiogenic and anxiolytic’ drug effects in rodents.
saline at the equivalent dose of drug The plus maze apparatus consists of two open
• Group 2: Treatment group (n = 6); mice are (16 × 5 cm for mouse and 50 × 10cm for the rats)
given diazepam at the dose of 3 mg/kg, ip and two closed arms (16 × 5 × 12 cm for mouse and
50 × 10 × 40 cm for rats), and an open roof of the
Step 3 entire maze elevated (25 cm for mouse and 50 cm
• Force mice backwards in a plastic perspex tube for rats) from the floor. The animals are placed
(3 cm inner dimeter, 25 cm length), and make individually at the centre of the elevated plus maze
tube slightly tilted. with their head facing towards the open arm during
• Observe the animal performance for 60 sec, the 90 sec-5 min test. The preference of the animal
(climbing backward into the perspex tube). for the first entry, the number of entries into the
open and closed arms reflect the relative safety of
Observations and Results closed arms as compared with the relative
fearfulness of open arms. Rat/mouse are rodents
Observation Table
and feel safe in dark, hence normal rodents prefer
Sl. Motor Co-ordination (cut off time 60s) dark arm first. Anxiolytics would be expected to
No. Group 1 Group 2 increase the proportion of entries into and time
Present Absent Present Absent spent in open arms (Figs 18.3 and 18.4).
1.
2.
Materials and Method
3. Materials
4.
5. Animal/species : Rat/Wistar
6. Sex/Body weight : Either sex/150-250g
Syringe/needle : 1ml/preferably 23G
Discussion Drug : Diazepam (0.5-1 mg/kg, i.p)
Refer to the exp 18b
Precautions before Experimentation
SUGGESTED READING • Laboratory should be dim lighted and noise
1. Boissier JR, J Tardy, JC Diverres. Une nouvelle method free
simple pour explorer l’action ‘tranquilisante’: le test • Animal should be marked properly, to avoid
de la chimin6e, Med Exp (Basel) 1960;3:81. mixing in two groups
188  Practical Manual of Experimental and Clinical Pharmacology

Fig. 18.3: Rat facing towards the open arm in an elevated plus maze (For color version see Plate 3)

Fig. 18.4: Diagram of rat elevated plus maze with dimensions of the arm and height
 Animal Experiment on CNS  189

• Handle the animals with care (minimize the Step 2

stress and pain to animal)
• Place the rat at the centre of the plus maze,
facing towards open field
• Expose rat to the experiment procedure 1-2
days prior to the experiment
• Video recorder is placed to record the experi-
ment in calm and dim lighted room
Methods
Step 1
• Weigh the animals and mark properly
• Divide animals into two groups (n = 6 in each
group)
• Group 1: Control group (n = 6); rats are given
saline at the equivalent dose of drug
• Group 2: Treatment group (n = 6); rats are given
diazepam at the dose of 1 mg/kg, ip
Step 3
• Observe the animals for 5 minutes (cut off time)
• Observation parameters: (1) First arm
preference, (2) No. of entries into the open and
closed arm, (3) Time spent in the open and
closed arm and (4) Total no. of entries in the arm
Note: Dimensions of mice elevated plus maze are
explained in introduction

Observations and Results

Observation Table
Group 1
Sl.no. No. of entries Time spent in the arm (sec) Total no. of entries 1st arm preference
Closed Open Closed Open Closed Open (open/closed)
1.
2.
3.
4.
5.
6.

Group 2
Sl.no. No. of entries Time spent in the arm (sec) Total no. of entries 1st Preference
Closed Open Closed Open Closed Open (open/closed)
1.
2.
3.
4.
5.
6.

Discussion whereas anxiogenic compounds increase time

There are several methods available for screening
the anxiolytic compounds such as elevated plus
maze, Z-maze, O-maze, Y-maze or radial maze
etc. Elevated plus maze is most popularly used in
the screening and evaluation of the drug by
decreasing anxiety. It is evaluated by increase in
the time spent and exploration time in open arm
spent in the closed arm. The judgement parameters
during the experiment are time spent in open arm,
latency to enter in open arm, total number of entries
in open arm, and first preference of arm by the
animal (open/closed). The principle is same for
all above mentioned mazes. Drugs like
benzodiazepine, alcohol, barbiturates etc. may be
screened through this method.
190  Practical Manual of Experimental and Clinical Pharmacology
SUGGESTED READING
1. Brett RR, Pratt JA. Chronic handling modifies the
anxiolytic effect of diazepam in the elevated plus-
maze. Eur J Pharmacol 1990;178:135-38.
2. Cao BJ, Rodgers RJ. Comparative effects of novel 5-
HT1A receptor ligands, LY293284, LY315712 and
LY297996 on plus-maze anxiety in mouse.
Psychopharmacology 1998;139:185-94.
3. Kauppila T, Tanila H, Carlson S, Taira T. Effects of
atipamezole, a novel α2-adrenoreceptor antagonist,
in open-field, plus-maze, two compartment
exploratory, and forced swimming tests in rats. Eur
J Pharmacol 1991;205:177-82.
4. Pellow S, Chopin P, File SE and Briley M. Validation
of open:closed arm entries in an elevated plus-maze
as a measure of anxiety in the rat. J Neurosci Methods
1985;14:149-67.
EXPERIMENT NO: 18E
Aim
To demonstrate amnesic effect of diazepam in rat
using Morris water maze apparatus.
Background
The test apparatus consists of a circular fiberglass
tank (130 cm in diameter, 50 cm in depth). The
pool is filled to a height of 30 cm with water at
room temperature, 21-22°C. The pool is divided
into four quadrants (Q1, Q2, Q3 and Q4) of equal
surface area. A transparent escape platform (10
cm in diameter, 29 cm in height) is placed in a
fixed location in the tank in the centre of one of the
quadrants, 1cm below the water surface. So, that
platform is not visible. Several clues arround the
maze are available for the rats to use in locating
the escape platform (Fig. 3.6).
Training of animals: The platform remains in a
constant location in the centre of one quadrant,
equidistant from the centre and the edge of the
pool. Each training trial involves placing the
animal into the pool facing the wall at one of the
four quadrants. A different starting point is
randomly used on each trial. The rats are allowed
to swim freely until they find the escape platform.
The latency to find the hidden platform is recorded
and used as a measure of acquisition of the task. If
a rat fails to locate the hidden platform within 60
sec, it is then manually guided to escape platform
by the experimenter. The rat remains on the
platform for at least 20 sec to orient itself to the
visual cues. Rats are then turned to their home
cage for a fixed interval (3-10 min), until the series
of trials are completed.
Note: The water maze test is ideal to measure
learning and memory rather than the anxiolytic
activity whereas elevated plus maze is ideal for
screening anxiolytic activity rather than learning
and memory.
Materials and Methods
Materials
Animal/species : Rat/ Wistar
Sex/Body weight : Either sex/ 150-250g
Syringe/needle : 1ml/ preferably 23G
Drug : Diazepam (1-2 mg/kg, i.p)
Precautions before Experimentation
• Laboratory should be dim lighted and noise
free
• Animal should be marked properly, to avoid
mixing in two groups
• Handle the animals with care (minimize the
stress and pain to animal)
• Proper training should be given to animals for
the swimming and for the location of the
platform for at least 4-5 days
• Animals who do not float on the water and
search for the platform should be excluded
from the study
• Check for the pregnancy if the female
rats are included into the experiment (Pre-
gnant animals should not be included in
 Animal Experiment on CNS  191

the study; true for every experiment until it Step 2

requires) • Group 1: Control group (n = 6); rats are given
saline at the equivalent dose of drug
• Group 2: Treatment group (n = 6); rats are given
Methods
diazepam at the dose of 1.5 mg/kg, ip
Step 1 Step 3
• Weigh the animals and mark properly • Observe the animal for 90 sec/5 min (cut off time)
• Divide animals into two groups (n = 6 in each • Time to reach at the platform is recorded and
group) the movement in the quadrant of the pool

Observations and Result

Sl. no. No. of entries/time spend (sec) Time to reach to
Group 1 Group 2 the platform
Q1 Q2 Q3 Q4 Q1 Q2 Q3 Q4 Group 1 Group 2
1.
2.
3.
4.
5.
6.

Discussion 2. D’Hooge Rudi, Deyn Peter P. De. Applications of

Morris water maze is mainly designed to evaluate
the drugs acting on learning and memory and
was devised by Prof Richard Morris. Morris water
maze has two advantages over other mazes, (1)
rat searches for the escape, so there is no waiting
time and (2) there are no local cues (e.g. olfactory
or auditory). Drugs like NMDA receptor
antagonists which improve the learning and
memory whereas benzodiazepines that impair the
memory, can also be evaluated in the experiment.
Study reported that it impairs acquisition but not
retrieval of spatial information and is not due to
the sedative, hypothermic or state-dependent
learning effects of diazepam.
the Morris water maze in the study of learning and
memory. Brain Research Reviews 2001;36:60-90.
3. Kant G J, Wylier R M, Vasilakis AA, Ghosh S. Effects
of triazolam and diazepam on learning and memory
as assessed using a water maze. Pharmacol Biochem
Behav 1996;53(2):317-22.
4. Kant GJ, Wylie RM, Vasilakis AA, Ghosh S. Effects
of triazolam and diazepam on learning and memory
as assessed using a water maze. Pharma-co-logy,
biochemistry and behavior 1996;53 (2):317-22.
5. McNamara RK, Skelton RW. Diazepam impairs
acquisition but not performance in the Morris water
maze. Pharmacol Biochem Behav 1991;38(3):
651-58.
EXPERIMENT NO: 18F

SUGGESTED READING Aim

1. Arolfo MP, Brioni JD. Diazepam impairs place To demonstrate the anticonvulsant property of
learning in the Morris water maze. Behav Neural Biol diazepam against pentylenetetrazole (PTZ)
1991;55(1):131-36. induced convulsions in mice.
192  Practical Manual of Experimental and Clinical Pharmacology
Background
Seizures are finite episodes because of abnormal
discharge of cerebral neurons and are broadly
divided into two groups (1) Epileptic seizure:
seizures which are periodic and unpredictable
associated with disorder of brain function and (2)
Non-epileptic: seizures which are evoked in a
normal brain by treatment with electroshock or
chemicals. That means animal model which is
developed to assess the effect of the drug is a non-
epileptic seizure model. Most animal models
cannot show all the pathophysiological,
behavioral, electrophysiological and neuro-
chemical alteration of the spontaneous human
epileptic syndrome. However, PTZ induced
seizures represent the absence seizures and
regarded as a good chemical model. The seizure
induced is characterized by generalized spike and
wave discharges on the EEG. PTZ mainly acts
through inhibition of GABAB coupled chloride
(Cl–) channel.
Materials and Methods
Materials
Animal/species : Mice/ albino Swiss
Sex/Body weight : Male/ 20-30 g
Syringe/needle : 1ml/ preferably 24G on-
wards
Drug : Pentylenetetrazole (PTZ; 80-
85mg/kg, i.p.)
Diazepam (4 mg/kg, i.p)
Precautions before Experimentation
• Laboratory should be dim lighted and noise
free
• Animals should be marked properly, to avoid
mixing in two groups
• Handle the animals with care (minimize the
stress and pain to animal)
• Observe the animals in a plexi glass chamber
• Female have been more sensitive to seizure,
hence should be avoided or if added, make
equal distribution in each group (Check for
non-pregnancy)
Methods
Step 1
• Weigh the animals and mark properly
• Divide animal into two groups (n = 6 in each
group)
Step 2
• Group 1: Control group (n = 6); mice are given
saline at the equivalent dose of drug
• Group 2: Treatment group (n = 6); mice are
given diazepam at the dose of 4 mg/kg, ip,
then after 15-30 min, give PTZ in a dose of 85
mg/kg, ip
Step 3
• Observe the animal for 1 hour
• Assessment: (1) onset of seizure (2) severity of
seizure (score) (3) No. of seizures in an hour
(4) percentage of positive responder (if seizures
score ≥3) and (5) total duration of seizure
Observations and Result
Seizures are recorded in a seven point score
according to the following scale.
Score
0 = no behavioral changes; 0.5 = atypical behavior
(e.g. intensive grooming, sniffing, and moving
arrests); 1 = isolated myoclonic jerks, ear and facial
twitching; 2 = atypical minimal seizures,
convulsive wave through the body; 3 = fully
developed minimal seizures, clonus of the head
muscles and forelimbs, righting reflex present;
4 = major seizures (generalized without the tonic
phase); 5 = generalized tonic-clonic seizures
beginning with running.
Followed by the loss of righting reflex, then
short tonic phase (flexion or extension of fore- and
hind limbs) progresses to the clonus of all four limbs
leading sometimes to the death of the animal.
 Animal Experiment on CNS  193

Observations and Result

Observation Table
Group 1
Sl. no. Onset of Severity of No. of seizure in % of positive Duration of
seizures (sec) seizure (score) 60 min responder* seizure

1.
2.
3.
4.
5.
6.
*Score ≥3 is considered as positive responder

Group 2
Sl. no. Onset of Severity of No. of seizure in % of positive Duration of
seizures (sec) seizure (score) 60 min responder* seizure

1.
2.
3.
4.
5.
6.

Discussion evaluated are phenytoin, valproate, pheno-

barbitone for the generalized seizures, but pheny-
There are several methods for induction of
toin is not be evaluated by the PTZ model (Model
seizures in animals which resembles human
for absence seizure).
seizures such as pentylenetetrazole (PTZ), a
chemical method which resembles human absence
SUGGESTED READING
seizure whereas other method like maximal
electroshock seizure (MES) which resembles 1. Fradley RL, Guscott MR, Bull S, Hallett DJ, Goodacre
human generalized tonic clonic seizure (GTCS). SC, Wafford KA, Garrett EM, Newman RJ, O’Meara
Diazepam shows protective effect on both the GF, Whiting PJ, Rosahl TW, Dawson GR, Reynolds
models and it is found to enhance GABAA binding DS, Atack JR. Differential contribution of GABA(A)
receptor subtypes to the anticonvulsant efficacy of
to receptors, increases chloride channel opening
benzodiazepine site ligands. J Psychopharmacol
and indirectly blocks T-type Ca ++ channels.
2007;21(4):384-91.
Whereas kindling model is induced by the 2. Scarlatelli-Lima AV, Magalhães LHM, Doretto MC,
continuous administration of sub-maximal Moraes MFD. Assessment of the seizure
chemical dose or electrical stimuli which evokes susceptibility of Wistar Audiogenic rat to
the GTCS, but its resembling condition in human electroshock, pentylenetetrazole and pilocarpine.
is still controversial. Other drugs which may be Brain Res 2003;960:184-89.
194  Practical Manual of Experimental and Clinical Pharmacology
3. Velisek L, Kubova H, Pohl M, Stankova L, Mares P,
Schickerova R. Pentylenetetrazol-induced seizures
in rats: an on to genetic study, Naunyn Schmiedebergs
Arch. Pharmacol 1992;346:588-91.
EXPERIMENT NO.: 18G
Aim
To demonstrate the anticonvulsant property of
diazepam against pentylenetetrazole (PTZ)
induced kindling in rats
Background
Kindling is another experimental model to
develop seizures which involves the delivery of
submaximal electrical or chemical stimuli. The
repeated administration of the stimuli lowers the
seizure threshold and produces behavior changes
in the animal. Its exact mechanism is not clear but
some studies demonstrate that brainstem, the
substantia nigra (SN), can regulate the kindled
seizure threshold. However, its applicability to
human epilepsy is still controversial.
Materials and Methods
Materials
Animal/species : Rat/ Wistar
Sex/Body weight : Male/ 150-250g
Syringe/needle : 1ml/ preferably 24G on-
wards
Drug : Pentylenetetrazole (PTZ; 30-
40mg/kg, i.p)
Diazepam (3 mg/kg, i.p)
Precautions before Experimentation
• Laboratory should be dim lighted and noise
free
• Animal should be marked properly, to avoid
mixing in two groups
• Handle the animals with care (minimize the
stress and pain to animal)
• Observe the animals in a plexiglass chamber
• Female have been more sensitive to seizure,
hence should be avoided
Methods
Step 1
• Weigh the animals and mark properly
• Divide animals into three groups (n = 6 in each
group)
Step 2
• Group 1(A): Control group (n = 6); rats are
given saline at the equivalent dose of drug
• Group 2(B): Vehicle group (n = 6); rats are
given vehicle at the equivalent dose of drug 40
min before PTZ (30-40 mg/kg, i.p.), 3 times/
week for 9 weeks.
• Group 3(C): Treatment group (n = 6); rats are
given Diazepam (3 mg/kg, i.p), 40 min before
PTZ (30-40 mg/kg, i.p. 3 times/week for 9
week.
Step 3
• Observe* the animals for 1 hour, after the PTZ
dose 3 times/week for 9 weeks.
• Assessment: (1) Onset of seizure (2) severity of
seizure (score) (3) No. of seizure in an hour (4)
percentage of positive responder (if seizures
score ≥3) and (5) total duration of seizure
Note: *Observe the animal for any other behavioral
changes such as sniffing, rearing, excitement,
aggressiveness etc.
Observation and Results
Kindling model scoring
0 – No response; 1 – Ear and facial twitching; 2 –
One to 20 myoclonic body jerks in 10 min; 3 –
More than 20 body jerks in 10 min; 4 – Clonic
forelimb convulsions; 5 – Generalized clonic
convulsions with rearing and falling down
episodes; 6 – Generalized convulsions with tonic
extension episodes (same score may be used to
score the PTZ induced seizure also. (Exp. 18F)
 Animal Experiment on CNS  195

Sl no. Onset of Severity of No. of seizure % of positive Duration of

seizure (sec) seizure (score) in 60 min responder seizure (sec)
A B C A B C A B C A B C A B C
1.
2.
3.
4.
5.
6.

Discussion EXPERIMENT NO: 18H

Same as Experiment 18f
SUGGESTED READING
1. Giorgi O, Orlandi M, Lecca D, Corda MG. MK-801
prevents chemical kindling induced by
pentylenetetrazol in rats. Eur J Pharmacol 1991;193:
363-65.
2. Goddard GV, McIntyre DC, Leech CK. A permanent
change in brain function resulting from daily electrical
stimulation. Exp Neurol 1969;25:295-330.
3. Kodama M, Yamada M, Sato K, Kitamura Y,
Koyama F, Sato T, Morimoto K, Kuroda S. Effects of
YM90K, a selective AMP receptor antagonist, on
amgdala-kindling and long-term hippocampal
potentiation in rats. Eur J Pharmacol 1999;374:
11-19.
4. Löscher W. Animal models of epilepsy for the
development of antiepileptic and disease-modifying
drugs. A comparison of the pharmacology of kindling
and poststatus epilepticus models of temporal
epilepsy. Epilepsy Res 2002b;50:105-23.
5. McNamara JO. Kindling model of epilepsy. Adv
Neurol 1986;44:303-18.
Aim
To demonstrate the anti-convulsant activity of
phenytoin against maximal electroshock (MES)
induced convulsions in rats
Background
The maximal electroshock (MES) model is a model
for grand mal epilepsy and the end point
considered as tonic hind limb extension (THLE)
which are evoked by electric stimuli. The agents
screened through this model considered an anti
epileptic drug if it corrects or suppresses THLE in
rat (Figs 18.5A and B). The maximal electroshock
is induced through corneal electrode and ear electrode.
Corneal electrode has the limited use due to the
risk of blindness and other harmful nerve effects
to the animal used. Ear electrode is commonly
used in the experiment which is easy to use and
less harmful compared to corneal electrodes.

Figs 18.5A and B: (A) Showing positive THLE (score 3) and (B) Postictal depression after the
THLE (score 4): it remains for few seconds then rat/mouse recovers
196  Practical Manual of Experimental and Clinical Pharmacology
Materials and Methods
Materials
Animal/species : Rat/ Wistar
Sex/Body weight : Male/ 150-250 g
Syringe/needle : 1ml/ preferably 23G
Drug : Phenytoin (20-25mg/kg, i.p
or po)
Instrument
Electro convulsiometer:
• (For rat: Intensity of stimulus: (150mA, 50Hz
for 0.2sec)
• (For mouse: Intensity of stimulus: (12mA, 50
Hz for 0.2 sec)
Other drugs: Diazepam (3-4 mg/kg, i.p. for mouse)
Precautions before Experimentation
• Laboratory should be dim lighted and noise
free
• Animals should be marked properly, to avoid
mixing in two groups
• Handle the animals with care (minimize the
stress and pain to animal)
• Animals should be screened 1 week prior to the
experiment
• Check the instrument and ear electrode for its
proper working before experiment
• Ear should be lubricated with gel or water to
enhance the conductivity of electricity.
Methods
Step 1
• Weigh the animals and mark properly
• Divide animals into two groups (n = 6 in each
group)
Step II
• Group 1: Control group (n = 6); rats are given
the saline as per body weight
• Group 2: Treatment group (n = 6); rats are given
phenytoin 20-25 mg/kg, i.p., thereafter 30 min
rats are given electro shock at the intensity of
150 mA, 50 Hz for 0.2 sec. (in case of oral test
drug, MES induced after 60 min)
Step 3
• Observe the animals after MES
• Record whether THLE “present or absent”
• Calculate percentage protection
Note:
1. All the procedure and scoring is the same for
mouse. The only difference is the selection of
electrical stimuli and duration of electrical
stimuli through ear/corneal electrodes
2. One can also videotape the whole seizure
events in rat/mouse and can use the scale to
score the seizure events in the slow motion
video run (Events takes place in the millisecond
of time, hence not possible to observe by the
naked eye; only THLE is seen as end point.
Seizure score
0 = no seizure; 1 = forelimb extension without
hind limb extension; 2 = complete forelimb
extension and partial hind limb extension, 3 =
complete tonic hind limb extension (THLE) (hind
limb become parallel to the tail) and 4 = post ictal
depression; See Figs 18.5A and B.
Observations and Results
Percentage protection (%) = No. of animals with
THLE absent / total no. of animal × 100
Observation Table
Sl. Tonic hind limb extension (THLE) % Pro-
no. Present (Yes) Absent (No) tection
Group 1 Group 2 Group 1 Group 2
1.
2.
3.
4.
5.
6.
Discussion
The end point of the experiment is considered as
the absence/presence of THLE following a drug
treatment which is a position during the GTCS in
rodents when tail and both hind limbs are parallel
 Animal Experiment on CNS  197

to each other. It is a subjective measure hence,
mouse/rat should be screened 1 week prior to the
experiment. The animal present with the positive
THLE is included in the study. One week time is
given to animal to recover from the excitatory
neuronal discharge in the brain.
(Also refer to discussion of experiment 18f)
and D1 to some extent. In the experiment, extra-
pyramidal side effects are identified by catalepsy.
It is an extreme tonus, muscular rigidity which is
characterized by a tendency to remain in a fixed
position for long period, hence unable to correct
an externally imposed, unusual posture over a pro-
longed period of time (Figs 18.7A to C).

SUGGESTED READING Materials and Methods

1. Blanco MM, Dos Santos Jr JG, Perez-Mendes P, Kohek Materials
SR, Cavarsan CF, Hummel M, Albuquerque C, Mello
LE. Assessment of seizure susceptibility in Animal/species : Rat/ Wistar
pilocarpine epileptic and nonepileptic Wistar rats Sex/Body weight : Male/ 150-250g
and of seizure reinduction with pentylenetetrazole Syringe/needle : 1ml/ preferably 23G
and electroshock models. Epilepsia 2008 Nov 19. Drug : Haloperidol (0.4-4 mg/kg,
2. Rastogi SA, Ticku MK. Involvement of a GABAergic i.p)
mechanism in the anticonvulsant effect of
Instrument : Two Wooden or cardboard
Phenobarbital against maximal electroshock-induced
seizures in rats. Pharmacol Biochem Behav pillar, 10cm apart with a rod
1985;222:141-46. and rod at the height of
3. Stanistaw J, Czuczwar, Kinga K, Borowicz, Zdzistaw 10-20 cm (for mice, height =
Kleinrok, Piotr Tutka, Tomasz Zarnowski, 7-10 cm) (Fig. 18.6)
Waldemar A. Turski. Influence of combined
treatment with NMDA and non-NMDA receptor Precautions before Experimentation
antagonists on electroconvulsions in mouse. Eur J
Pharmacol 1995;281:327-33. • Laboratory should be dim lighted and noise
4. Stephen H Koslow, Lloyd J Roth. Reserpine and free
acetazolamide in maximum electroshock seizure in
• Animal should be marked properly, to avoid
the rat. J Pharmacol Exp Therap 1971;176(3):711-17.
5. Woodbury LA, Davenport VO. Design and use of a mixing in two groups
new electroshock seizure apparatus and analysis of • Handle the animals with care (minimize the
factors altering seizure threshold and pattern. Arch stress and pain to animal)
Int Pharmacodyn 1952;92:97-107. • Condition the animal to the experimental box
for at least 2min, before the experiment (Fig.
EXPERIMENT NO.: 18I 18.6).
Aim
To demonstrate effect of phenothiazine (halo-
peridol) induced catatonia in rat.

Background
Anti-psychotics drugs are well known for their
Fig. 18.6: Dimensions of instrument used to evaluate
extrapyramidal side effects. Present experiment catalepsy in rat
demonstrates extra-pyramidal side effects like
tardive dyskinesia in animal followed by the Methods
phenothiazine (Haloperidol) treatment. Pheno- Step 1
thiazine and butyrophenone neuroleptics act • Weigh the animals and mark properly for
through the blockade of both β and D2 receptors identification
198  Practical Manual of Experimental and Clinical Pharmacology

Figs 18.7A to C: Different postures of catatonia produced in the rat

• Divide animals into two groups (n = 6 in each Observation Table

group)
Sl. Cataleptic (Abnormal) Posture (60s)
Step II % of
no. Present Absent
• Group 1: Control group (n = 6); rats are given cataleptic
Group 1 Group 2 Group 1 Group 2 animal
the saline as per body weight
• Group 2: Treatment group (n = 6); rats are given 1.
haloperidol 4 mg/kg, i.p., thereafter 10-15 min 2.
rats expose to the test 3.
4.
Step 3
5.
• Observe the animals behaviour and correction
6.
of its externally induced abnormal posture
(cutoff time 60s)
• Time of posture correction is noted down at Discussion
30, 60, 120 and 360 min Extrapyramidal adverse effects are commonly
• Calculate percentage of responders reported with the antipsychotic drugs like
Note: If rat remains on the bar for 60s, then chlorpromazine, flufenazine, haloperidol etc.
catalepsy is positive They are mediated through dopamine D 2 -
receptor blocked mainly in the nigrostriatal
Observations and Resulte pathway, which leads to dystonia, bradykinesia,
akathisia, dyskinesias etc. These behavioral
Calculation of percentage of cataleptic animals: changes are well studied in rodents by failure
No. of animals showing cataleptic posture to correct the externally exposed abnormal
100
Total number of animals position.

SUGGESTED READING
1. de Sousa Moreira LF, Pinheiro MC, Masur J.
Catatonic behavior induced by haloperidol, increased
by retesting and elicited without drug in rats.
Pharmacology 1982;25(1):1-5.
2. Cerbo R, Carchedi F, Casacchia M. Role of serotonin
in catatonia induced with haloperidol and reserpine.
Boll Soc Ital Biol Sper 1976;52(4):245-50.
3. Costall B, Naylor RJ. On catalepsy and catatonia
and the predictability of the catalepsy test for
neuroleptic activity. Psychopharmacologia 1974;
34(3):233-41.
4. Casey DE. Serotoninergic aspects of acute extra
pyramidal syndromes in nonhuman primates.
Psychopharmacol Bull 1989;25:457-59.
5. Casey DE. Serotonergic and dopaminergic aspects
of neuroleptic-induced extrapyramidal syndromes
in nonhuman primates. Psychopharmacology 1993;
112:S55-S59.
EXPERIMENT NO.: 18J
Aim
To demonstrate the Straub tail reaction/pheno-
menon induced by morphine
Background
Simply straub tail means erected tail. The
phenomenon was used to check doping i.e. opioid
level in the horses which took part in the race. A
subcutaneous injection of morphine hydro-
chloride (10-40 mg/kg, i.p./s.c) into the mouse
flank produces a curvature of the tail in the form
of an ‘S’ , following 2 to 15 minutes after the
injection, depending on the dose . Finally, the tail
curves back along the body of the animal, the tip
touching the center of the head (Figs 18.8A to C).
Straub tail phenomenon occurs as a result of
injection of the centrally acting analgesics such
as morphine. But, there are no such studies which
have supporting data for the peripheral analgesic
induced straub tail.
As straub tail reaction is thought to be due to
mechanical contraction of the dorsal sacro coccygeus
muscle and electrical stimulation of spinal cord. There
Animal Experiment on CNS  199
is the presence of the dorsal sacro coccygeus
muscle on the either side of tail originating at the
base of spinal cord (Fig. 18.8D). In the previous
study, it showed that either side cut of the muscle
deviate the tail towards the side of attached
muscle. The mode of action is not clear. But various
studies suggest that straub tail results because of
direct action on the opioids μ-receptor, most
importantly μ2–receptor. There are some studies
which suggest that, it also results through 5-HT
receptors, α 2-(alpha-2) receptors, dopamine
receptors and glucocorticoid receptors as well.
Materials and Methods
Materials
Animal/species : Mice/ albino Swiss
Sex/Body weight : Male/ 20-30 g
Syringe/needle : 1ml/ preferably 24G on-
wards
Drug : Morphine (10-40mg/kg, s.c
or ip)
Precautions before Experimentation
• Animals should be marked properly, to avoid
mixing in two groups
• Handle the animal with care (minimize the
stress and pain to animal)
• Observe the animal in a plexiglass chamber
• Laboratory should be noise free (noise may
delay the response)
Methods
Step 1
• Weigh the animals and mark properly to
distinghish from one another
• Divide animals into two groups (n = 6 in each
group)
Step II
• Group 1: Control group (n = 6); mice are given
the saline at the equvalent dose of drug
• Group 2: Treatment group (n = 6); mice are
given morphine at the dose of 40 mg/kg, s.c.
200  Practical Manual of Experimental and Clinical Pharmacology

Figs 18.8A to D: (A) Positive straub tail phenomenon, (B) “S” shaped positive straub tail phenomenon, (C) Extreme
positive straub tail phenomenon, sometimes tail touches the head of mouse and (D) Showing dorsal sacro coccygeus
muscle on the either side of tail root (pointed in the picture) (For color version of Figures 18.8A to C see Plate 3)

Step 3 Note: Straub tail is said to be positive, when the

• Observe the animals for 45 min in plexiglass
chamber
• Observe the animals behavior carefully, for (1)
onset of straub tail reaction/phenomenon (2)
duration of straub tail reaction and (3) any
additional behavior changes
tail rises more than 30° of angle with tail base
Numerical score for straub tail reaction/
phenomenon;
0 = 0 °, 0.5 = 1-30° ,1 = 31-45° , 1.5 = 46-60° , 2.0 =
61-90°, 2.5 = more than 90 °

Observations and Results

Observation Table
Sl. No. Straub Tail Reaction Straub tail
Group1 Group 2 reaction(Score)
Present Absent Present Absent Group 1 Group 2
1.
2.
3.
4.
5.
6.

Discussion α2-(alpha-2) receptors, dopamine receptors and

Straub tail reaction or phenomenon is the
characteristic contraction of the dorsal sacro-
coccygeus muscle and electrical stimulation of
spinal cord in which tail rises than its normal
position. The role of the μ2–receptor, 5-HT receptors,
glucocorticoid receptor are well studied in several
studies. The tail rise at an angle >30° is considered
to be the positive straub tail reaction. There is no
clinical condition resembling this condition hence
have only experimental implication.

SUGGESTED READING
1. Anna Capasso, Carmela Casciano and Alberto
Loizzo. Dexamethasone reduces morphine induced
straub reaction in mouse. J Pharmacy Pharmacol
2002;54:983-87.
2. Bilbey D LJ, Salem H, Grossman MH. The anatomical
basis of the straub phenomenon. Br J Pharmacol
1960;15:540-43.
3. Kameyama T, Ukai M, Nabeshima T. Effects of
catecholaminergic or tryptaminergie agents on the
morphine-induced Straub tail reaction. Jpn J
Pharmacol 1978:28;249-57.
4. Schwe Fang Pong, Janet Mary Sweetman, Amy Sue
Pong, John Franklin Carpenter. Evaluation of oral
skeletal muscle relaxants in the morphine-induced
straub tail test in mouse. Drug Development
Research 2004;11(1):53-57.
5. Tsutomu Kameyama, Makoto Ukai and Toshitaka
Nabeshima. Effect of catecholaminergic or trypta-
minergic agents on the morphine induced straub tail
reaction. Japan J Pharmacol 1978;28:249-57.
6. Zarrindast MR, Alaei-Nia K, Shafizadeh M. On the
mechanism of tolerance to morphine-induced Straub
tail reaction in mouse. Pharmacol Biochem Behav
2001;69(3-4):419-24.
EXPERIMENT NO: 18K
Aim
To demonstrate the analgesic effect of morphine
in mouse using hot plate/tail flick method
Background
The method deals on the principle of the thermal
radiation heat. This principle is used in the animal
experiment for the evaluation of the centrally
acting analgesics, and hence this method found
to be the differentiating between the centrally
acting opiates and non-opiates analgesics.
Hotplate Method
The instrument involved is known as “hotplate
analgesiometer”. Instrument consists of an
electrically heated surface (made up of iron,
aluminum or copper) whose temperature is
maintained by the thermostat ‘Knob’ at 55° to
56 °C. After maintaining the temperature mouse/
Animal Experiment on CNS  201
rat are placed on the hot plate and observed for
either paw licking or jumping reaction. The
reaction time is recorded by a stop-watch.
Repeated reading is taken at 20, 60, and 90
minutes after the drug administration. Cut off time
for rat is 20-30 sec and for mice it is 15-20 sec.
Tail Flick Method
Animal is placed into restrainer and leaving the
tail exposed outside the restrainer. Clean the tail
with the help of cotton soaked in water or ethanol.
Then, leave the tail for drying and also to settle
down the rat/mouse in the restrainer. When rat/
mouse setted, then keep restrainer on the “tail flick
analgesiometer”. 1/3rd tail proximally left due to
the thick and keratinized skin and then keep tail
on the place made for tail above hot wire (measure
the height of tail from wire) of the analgesiometer.
The time of tail flick is measured and recorded. The
cut off time is set up 15-20 sec in case of mouse
whereas in the case of rat, cut off time is 20-30 sec
to avoid any further injury to the tail (Fig. 18.9).
Materials and Methods
Materials
Animal/species : Mice/ albino Swiss
Sex/Body weight : Either sex/ 20-30 g
Syringe/needle : 1ml/ preferably 24G on-
wards
Drug : Morphine (5-7.5 mg/kg, i.p
or s.c.)
Fig. 18.9: Tail-flick test in rat
202  Practical Manual of Experimental and Clinical Pharmacology
Other drugs : Codeine hydrochloride (30
mg/kg s.c.), Pethidine hydro-
chloride (30 mg/kg s.c.) and
Phenazone (400 mg/kg s.c.)
Precautions before Experimentation
• Animals should be marked properly, to avoid
mixing in two groups
• Handle the animals with care (minimize the
stress and pain to animal)
• Check the instrument carefully for any error
and electricity connection
• Carefully check the temperature within the
range (chances of burning of paw or tail) [hot
plate]
• Clean the paw and hot plate for uniform
temperature distribution
• In tail flick method, clean the tail properly, to
avoid interference with result
• The time of wire getting red must be substracted
from the total time recorded [tail flick]
• Screening is must for the both tests before the
experiment, if mouse show reaction time more
than 6 sec, should be excluded from the study
(selection reaction time for rat is 10 sec).
Methods
Step 1
• Weigh the animals and mark properly
• Divide animals into two groups (n = 6 in each
group)
Step II
• Group 1: Control group (n = 6); mice are given
the saline at the equvalent dose of drug
• Group 2: Treatment group (n = 6); mice are
given morphine at the dose of 5 mg/kg, s.c.
Step 3
• Hot plate/tail flick: Observe the animal for 15-
20 seconds (cut off time for mice) 20-30 seconds
(cut off time for Rat)
• Observe for the licking of the paw or jumping
in case of hot plate or the tail flick in tail flick
test and record the time
Note:
1. Record the response at 20, 60 and 90 min after
the saline/drug treatment (Hot Plate test)
2. Record the response at 30, 60 and 120 min
after the saline/drug treatment (Tail flick
test)
Observations and Results
Centrally acting analgesics can be evaluated by
the hot plate method where as peripherally acting
analgesics are not effective. e.g: Aspirin showed
no effect in hot plate method even at very high
doses
False positive results: sedatives and muscle
relaxants (Woolfe and MacDonald, 1944) or
psychotomimetics (Knoll, 1967) may give the
increase reaction time.
Discussion
Hot-plate and tail-flick test mimic acute thermal
pain and persistent pain model by the formalin
test. Hot-plate and tail-flick test are two different
methods for evaluation of nociception. These
experimental models of pain commonly used to
tests for response thresholds to high intensity
stimuli (acute pain tests) or persistent pain
models. Tail-flick test is predominantly a spinal
response and Hot-plate is mostly at supraspinal
level. Studies have conducted to evaluate
participation of nitric oxide in the agmatine-
mediated potentiation of morphine-induced
analgesia in mice indicate that agmatine
potentiates morphine-induced spinal but not
supraspinal analgesia, and this effect is
not mediated by a nitric oxide-dependent
mechanism.
 Animal Experiment on CNS  203

Observation Table
For hot plate
Sl. Time of paw licking or jumping
No. 20 min 60 min 90 min
Group 1 Group 2 Group 1 Group 2 Group 1 Group 2
1.
2.
3.
4.
5.
6.

For tail flick

Sl. Time of tail flick
No. 30 min 60 min 120 min
Group 1 Group 2 Group 1 Group 2 Group 1 Group 2
1.
2.
3.
4.
5.
6.

SUGGESTED READING in Drug Evaluation. Yearbook Med Publ. Inc., Chicago

1. Aanonsen LM, Wilcox GL. Nociceptive action of
excitatory amino acids in the mouse: effects of
spinally administered opioids, phencyclidine and
sigma agonists. J Pharmacol Exp Ther 1987;243(1):9-
19.
2. Abbott FV, Melzack R, Leber BF. Morphine analgesia
and tolerance in the tail-flick and formalin tests: dose-
response relationships. Pharmacol Biochem Behav.
1982;17(6):1213-19.
3. Abbott FV, Melzack R, Samuel C. Morphine analgesia
in tail-flick and formalin pain tests is mediated by
different neural systems. Exp Neurol 1982;75(3):
644-51.
4. Kambur O, Männistö PT, Viljakka K, Reenilä I,
Lemberg K, Kontinen VK, Karayiorgou M, Gogos
JA, Kalso E. Stress-induced analgesia and morphine
responses are changed in catechol-O-methyl-
transferase-deficient male mouse. Basic Clin
Pharmacol Toxicol 2008;103(4):367-73.
5. Knoll J. Screening and grouping of psycho-
pharmacological agents. In: Siegler PE,Moyer HJ
(Eds) Animal and Clinical Pharmacologic Techniques
1967;305-21.
6. Luszczki JJ, Czuczwar SJ. Dose-response relationship
analysis of vigabatrin doses and their antinociceptive
effects in the hot-plate test in mouse. Pharmacol Rep
2008;60(3):409-14.
7. Ung D, Cowan A, Parkman HP, Nagar S. Lack of
interaction between metoclopramide and morphine
in vitro and in mouse. Xenobiotica 2008;38(11):1365-
76.
8. Woolfe G, MacDonald AD. The evaluation of the
analgesic action of pethidine hydrochloride (DE-
MEROL) J Pharmacol Exper Ther 1944;80:300-07.
9. Zimer PO, Wynn RL, Ford RD, Rudo FG. Effect of
hot plate temperature on the antinociceptive activity
of mixed opioid agonist antagonist compounds.
Drug Dev Res 1986;7:277-80.
EXPERIMENT NO.: 18L
Aim
To demonstrate partial global cerebral ischemia
in mice
204  Practical Manual of Experimental and Clinical Pharmacology
Background
Cerebral ischemia leads to a cascade of patho-
physiological processes which contribute to
ischemic cell damage, reduction in oxygen and
glucose availability to brain leading to cellular
energy crisis which interrupts the activity of
cellular ion pumps thus disturbing the ionic
gradients homeostasis resulting ultimately to an
increased release of neurotransmitters (mainly
glutamate) within 1-2 minutes of ischemia.
Glutamate release causes excitation and results
in early onset seizure. The contribution of over-
stimulation by excitatory amino acids leading to
neurotoxicity and cell death following cerebral
hypoxemia induced by ischemia is well estab-
lished. Activation of NMDA-glutamate receptors
results in an increase in free Ca2+ ion which
triggers a number of potential cytotoxic cascades
including activation of protein kinase-C (PKC),
release of platelet activating factor (PAF),
generation of free radicals and production of
nitric oxide.
Materials and Methods
Materials
Animal/species : Mice/albino Swiss
Sex/Body weight : Either sex/ 20-30 g
Syringe/needle : Aneurysm clip/bull dog clip
Precautions before Experimentation
• The area of surgery is thoroughly cleaned with
70% ethanol with the sterile cotton swab
• Use sterilized instruments during the surgery
• While performing the surgical procedure, the
animals are kept warm with the help of Infra-
red lamp
Method of Cerebral Ischemia Induction
Method
Refer Flow Chart 18.1.
Step 1
• After ischemia induction, divide animals into
two groups (n = 6 in each group)
Step 2
• Group 1: Control group (n = 6); mice are given
saline at the equivalent dose of drug
• Group 2: Treatment group (n = 6); mice are
given dizocilpine (MK-801) at the dose of 0.1
mg/kg, i.p. 1st dose is given 30 min before the
surgery thereafter, once every day at 24 hr, 48
hr and 72 hr
Step 3
• Brain is removed after 72 hr of cerebral
ischemia and then following observations are
carried out
• The infarct area
• Brain edema
Measurement of Brain Edema
Brain edema is measured with the wet-dry
method. After the animal is sacrificed by decapi-
tation under euthanasia, their brain is removed,
weighed immediately to yield wet weight. After
drying in a desiccating oven for 48 h at 70oC, the
tissue is reweighed to yield dry weight. The
percentage of water in the tissues is calculated
according to the formula:
Wet weight – dry weight
100
Wet weight
Measurement of Cerebral Infarct Size
After 72 hr, the animal is sacrificed by decapi-
tation, their brain is removed and infarct size is
calculated. The brain is kept overnight at (–4°C).
Frozen brain is sliced into uniform sections (7-8
in number per brain) of 1 mm thickness. The slices
are immersed in 1% triphenyltetrazolinium
chloride (TTC) at 37°C in 0.25 M phosphate buffer
(pH 8.5) for 5min; tissue sections are dipped in
10% formaldehyde solution for 5 min. Triphenyl-
tetrazolinium chloride (TTC) is converted to red
formazone pigment and therefore stained the
viable cells deep red. The infarcted cell have lost
the enzyme and cofactor and thus remained
unstained dull yellow. The brain slices are
placed in between two glass slides. A transparent
plastic grid with 100 squares per cm2 is placed
 Animal Experiment on CNS  205
Flow Chart 18.1: Procedure for producing cerebral ischemia

over slides. Number of squares falling over non- Observations and Results
stained dull yellow area and total number Sl. no. Cerebral infarct size Brain edema
of squares covered by each brain slice is counted.
Control Treatment Control Treatment
Infarcted area is expressed as a percentage
of total brain volume. For calculation of
infarcted area of a brain, all the brain sections
are studied with the help of magnifying glass
(40 X).
206  Practical Manual of Experimental and Clinical Pharmacology
Result is expressed in mean ± SD and is
compared by unpaired t-test (if treatment groups
are more than one and total mean is >2 then one
way ANOVA should be preferred)
Discussion
There are several other models are used to study
cerebral ischemia or stroke in the published
literature such as middle cerebral artery occlusion
model (MCAO) or unilateral carotid artery ligation
(UCAL). Four vessel occlusion models are also
used in the number of studies for the producing
global cerebral ischemia. The development of
ischemic brain damage depends on the reduction
of cerebral blood flow (CBF) below critical
threshold level. So, the decrease of CBF in ischemic
regions may result in an energy failure and further
lead to an activation of the toxic intracellular
pathway, additionally the infarct volume depend
on the duration of ischemia. Therefore, the severity
of ischemia has two components: degree of CBF
reduction and duration of the ischemic episode.
SUGGESTED READING
1. Aggarwal R, Medhi B, Pathak A, Dhawan V,
Chakrabarti A. Neuroprotective effect of progesterone
on acute phase changes induced by partial global
cerebral ischaemia in mice. J Pharm Pharmacol 2008;
60(6):731-37.
2. Bochelen, D., Rudin, M. and Saute, A. Calcineurin
inhibitors FK506 and SDZASM 981 alleviate the
outcome of focal cerebral ischemic/reperfusion
injury. J Pharmacol Exp Ther 1999;288:653-59.
3. Dempsey RJ, Baskaya, MK and Doglan, A. Attenu-
ation of brain edema, blood-brain barrier breakdown,
and injury volume by ifenprodil, a polyamine-site
NMDA receptor antagonist, after experimental
traumatic brain injury in rats. Neurosurgery
2000;47(2):399-406.
 Animal Experiment on
19 Cardiovascular System (CVS)
EXPERIMENT NO.: 19A
Aim
To record blood pressure (BP) in rodents (Rat BP).
Background
BP measurement in the animal is same as the human
measurement allowing readings when pulse/flow
disappears during cuff inflation and reappears
during deflation. Mainly, there are two methods of
BP measurement which are employed, one is
invasive or direct measurement and other is non-
invasive or indirect method. Direct or invasive
method follows the BP measurement through the
carotid artery. Major limitation, it is tedious work to
maintain the respiratory and surgical procedure.
(Must maintain the aseptic condition) Non- invasive
method includes tail-cuff method which is a
sensitive and accurate approach for the noninvasive
measurement of blood pressure in conscious or un-
conscious rat/mouse. It is simple and inexpensive
but can be misinterpreted due to stress-induced
changes in blood pressure during animal restraint
and heating. Systolic blood pressure (SBP) is still
preferably measured in rats by the tail-cuff method
(Figs 19.1A to D). Several methods of flow/pulse
detection have been described in addition to the
parent water plethysmograph or its modifications
mercury in-rubber or strain gauge detectors,
microphonic and piezoelectric pulse detectors,
doppler effect photoelectric and impedance.
Now days, telemetry is preferred method
employed for conscious rodents/animals blood
pressure measurement and overall cardiovascular
studies but major limitation lies with cost of the
telemetry apparatus and radio transmitter
implantation, whereas several advantages over
other methods are accurately obtain blood
pressure measurements at the lowest reco-
mmended temperatures and highly sensitive
photoelectric sensor for detection of blood pressure
pulses. Its software records real time systolic,
diastolic, mean and heart rate and allows complete
control of system.
Materials and Methods
Materials
Animal/species : Rat/Wistar
Sex/Body weight : Either sex/ 250-300 g
Syringe/needle : 1ml/ preferably 23 G
Drugs : Sodium pentobarbital (50
mg/kg i.p.)
Laboratory conditions: Temperature: 23°C±2°C
under a 12:12 hr light: dark cycle (The animals
are acclimatized to the laboratory conditions for
at least 1 week prior to experimentation.
Precautions before Experimentation
• Handle the animal with care (minimize the
stress and pain to animal)
• Proper training is given to animals to fami-
liarize with the procedure
• All procedure should be performed by the
same person
208  Practical Manual of Experimental and Clinical Pharmacology

Figs 19.1A to D: (A) Restrained rat showing attached BP cuff, (B) Close view tail-cuff attachment and
(C) and (D) Showing complete set of BP measurement with cardiogram (For color version of Figure 19.1C, see Plate 3)

Methods
Non-invasive method (Tail cuff method)
 Animal Experiment on CVS  209

Invasive method
Step I: For maintaining the ventilation

Note: Pentobarbitone has long duration of action which makes it, anesthesia of choice in the experiment

Step II: Procedure for BP measurement

1. Carotid artery
2. Femoral artery
Procedure for BP measurement through carotid artery
210  Practical Manual of Experimental and Clinical Pharmacology

BP measurement through Femoral artery

Recording recorder. Various cardiovascular drugs are added

Drugs are injected through the rubber tubing close to note the effect (inotropic/chronotropic etc.) of
to the cannula and a constant volume of saline is these drugs on the heart. After the experiment,
allowed to run each time after injection. The animals are sacrificed under euthanasia and then
recordings are taken on a smoked drum or electronic animal is disposed in a yellow polythene bags for

incineration. In vivo responses to the primary
action of the drug are the main advantage of this
preparation unlike the langendorf’s preparation
and hence, the net responses are measured.
Observations and Results
Sl. Weight of animal BP HR
No.
1.
2.
3.
Discussion
There are several methods described for
measuring BP in rodents. Among those, tail-cuff
method is very commonly used for measuring
systolic blood pressure (SBP) in rodents. Direct
methods have used tethers and indwelling
catheters connected to blood pressure measure-
ment devices, but limitations being are stressful
and give variable results. The inflation–
deflation cycle usually involved in the tail-cuff
methods and inflation is characterized by
disappearance of the pulse/flow signal during
cuff and reappearance during deflation.
Clinically, it is used for the diagnosis of pheo-
chromocytoma.
Telemetry is a modern technique which
includes an implantable telemetric probe, receiver
and monitoring system and gives very similar
results to the tail-cuff method. Telemetry allows
for a longer period of accurate measurement and
gives greater power to the study. So, telemetry is a
reliable method of direct measurement and has
been introduced to measure blood pressure (BP)
in the conscious unstressed animals. This system
simultaneously measures SBP, diastolic blood
pressure (DBP), mean arterial pressure (MAP) and
heart rate (HR).
SUGGESTED READING
1. Buñag R D. Pressor effects of the tail-cuff method in
awake normotensive and hypertensive rats. Journal
Animal Experiment on CVS  211
of Laboratory and Clinical Medicine 1971;78:
675-82.
2. Buñag RD, McCubbin JW, and Page IH. Lack of
correlation between direct and indirect measurements
of arterial pressure in unanesthetized rats. Cardio-
vascular Research 1971;5:24-31.
3. Byrom FB and Wilson C. A plethysmographic method
for measuring systolic blood pressure in the intact
rat. Journal of Physiology 1938;93:301-04.
4. Fritz M, Rinaldi G. Blood pressure measurement with
the tail-cuff method in Wistar and spon-taneously
hypertensive rats: Influence of adrenergic- and nitric
oxide-mediated vasomotion. Journal of Pharma-
cological and Toxicological Methods 2008; 58:215-
21.
5. Hermansen K. A new method for determination of
the systolic blood pressure in conscious rats. Life
Sciences 1970;9:1233-37.
6. Lucas J. A modified indirect method of blood pressure
measurement in the conscious and anaesthetized rat.
Journal of Physiology 1971;218:1-3.
7. Maistrello I and Matscher R. Measurement of systolic
blood pressure of rats: Comparison of intraarterial
and cuff values. Journal of Applied Physiology 1969;
26:188-93.
8. Van Vliet VN, Chafe LL, Antic V, Schnyder-Candrian
S and Montani JP. Direct and indirect methods used
to study arterial blood pressure. Journal of
Pharmacological and Toxicological Methods
2000;44:361-73.
9. Williams JR, Harrison TR and Wollmann AA. simple
method for determining the systolic blood pressure
of the unanesthetised rat. Journal of Clinical
Investigation 1939;18:373.
EXPERIMENT NO: 19B
Aim
To record ECG in rodents (rat and mouse).
Background
Recording electrocardiogram (ECG) in rodents (rat
and mouse) is commonly used parameter for
various pharmacological and toxicological
studies. It provides valuable information about
function and structure of heart. Recording of ECG
is similar to human ECG but some differences are
in smaller animals, such as the heart rate is very
high and ST segment is generally absent. Some of
212  Practical Manual of Experimental and Clinical Pharmacology
the important aspects of rat electrogram are
position of the animals, some of the investigators
prefer supine position and others recommend
prone as suitable posture. Mean electrical axis
(MEA) is varied from –22 to +120 in rat. Rat model
has become relatively invaluable for studying
mechanism of human disease whereas, mouse is
increasingly used in the recent times particularly
in drug development and to determine effect of
gene defect, disease and therapies. Though
conventional methods have several disadvan-
tages like requirement of anesthesia, insertion of
pin electrode in the limb, etc. So, new advance
technology has been used in the research like
telemetry. It was introduced in 1988, and latest
noninvasive foot plate technology is available to
Methods
Observations and Results
Attach the findings of the study (Electro-
cardiogram).
Discussion
In rat, ECG usually shows presence of more
prominent QRS complexes as compared to human
measure ECG in mouse. It has several advantages
such as it is without anesthesia, surgery and
implant.
Materials and Methods
Materials
Animal Species : Swiss mice/Wistar Rat
Sex/Body weight : Either sex/20-30 g mice or
150-200 g rat
Precautions before Experimentation
• Handle the animals with care (minimize the
stress and pain to animal)
• Keep laboratory calm while recording the ECG
beings because of difference in body shape
between rat and human chest and large ST
segment is probably because of early ventricular
repolarization. Selection of anesthetic agents, and
positioning of the animals are important while
recording ECG. It has been reported that ECG has
apparent absence of T wave in some of the smaller
animals and there is lack of isopotential segment

S and T wave (except in guinea pig) and comple-
xity of QRS wave.
SUGGESTED READING
1. Xing S, Tsaih SW, Yuan R, Svenson KL, Jorgenson L,
So M, Paigen B, Korstanje R. Genetic influence on
electrocardiogram time intervals and heart rate in
aging mice. Am J Physiol Heart Circ Physiol 2009 Apr
24.
EXPERIMENT NO.: 19C
Aim
To demonstrate isoproterenol induced myocardial
infarction in rats
Background
Myocardial infarction (MI) is caused due to non-
availability of oxygen to myocardium. Experi-
mental studies suggested that production of
catecholamine in cardiac tissue results in
increased oxygen requirement to myocardium by
increasing cardiac metabolism causing hypoxia.
Experimentally myocardial infarction can be
produced by injection of epinephrine and
isoproterenol (ISO), DIH (1-3-4 dihydroxyphenol-
Methods
Animal Experiment on CVS  213
2-isopropyl amino ethanol), but in massive dose
it produces myocardial necrosis. Role of
mineralocorticoids (aldosterone) have also been
observed. Studies also indicate increased urinary
excretion of aldosterone in experimentally
induced myocardial infarction and spontaneous
myocardial infarction in human beings. Experi-
mentally isoproterenol (a synthetic catecholamine
and β adrenergic agonist) induced myocardial
infarction is well established animal model and
resembles to those taking place in human MI.
Materials and Methods
Materials
Animal/species : Rat/ Wistar
Sex/Body weight : Either sex/ 100-150 g
Syringe/needle : 1 ml/ preferably 23G
Drug : Isoproterenol (8.5 mg/100
gm of rat)
Precautions before Experimentation
• Animals should be marked properly, to avoid
mixing in two groups
• Handle the animals with care (minimize the
stress and pain to animal)
214  Practical Manual of Experimental and Clinical Pharmacology
Histopathological Examination of Heart
Histopathological examination is done and heart
is washed immediately, after removal so that no
blood should be remaining in the cavity and it is
preserved in 10% formalin.
Histopathological grading is done according
to following criteria.
Grade 0 No change
Grade 1 Focal interstitial response
Grade 2 Focal lesions in many sections, con-
sisting of mottled staining and frag-
mentation of muscle fibers and sequis-
ting mucoid edema.
Grade 3 Confluent retrogressive lesion with
hyaline necrosis and fragmentation of
muscle fibers and sequisting mucoid
edema
Grade 4 Massive infarct with occasionally
acute aneurysm and mural thrombi
Observation and Results
Sl. Weight ECG HR Histological
No. score
1.
2.
3.
Discussion
Isoproterenol, synthetic sympathomimetics which
produces experimentally induced acute
myocardial infarction at the high dose by myocyte
necrosis. It has predominant action on β-1 receptor
which causes prolong systolic action, so diastolic
duration is reduced. Endocoronary filling occur
during diastole so, heart prone to develop MI.
Isoproterenol is light sensitive, hence solution
should be prepare in colored beaker or bottle
freshly, before use.
SUGGESTED READING
1. Tiwari R, Mohan M, Kasture S, Maxia A, Ballero M.
Cardioprotective potential of myricetin in isopro-
terenol-induced myocardial infarction in wistar rats.
Phytother Res 2009;23 (10):1361-66.
2. Krenek P, Kmecova J, Kucerova D, Bajuszova Z,
Musil P, Gazova A, Ochodnicky P, Klimas J,
Kyselovic J. Isoproterenol-induced heart failure in
the rat is associated with nitric oxide-dependent
functional alterations of cardiac function. Eur J Heart
Fail 2009;11(2):140-46.
EXPERIMENT NO.: 19D
Aim
To demonstrate deoxycorticosterone acetate
(DOCA) salt induced hypertension in rats
Background
DOCA-salt model is based on the principle of its
sodium and water retention properties in the body,
so it leads to increase in plasma and extracellular
volume. This model was first developed by the
Seyle et al, 1957. DOCA ultimately leads to
increase the sympathetic activity through the
renin-angiotensin-aldosterone system (RAAS)
system. Experimental studies suggest that the
female and young animals are more prone to the
DOCA induced hypertension.
Materials and Methods
Materials
Animal/species : Rat/ Wistar
Sex/Body weight : Either sex/ 100-150 g
Syringe/needle : 1ml/ preferably 23 G
Drug : DOCA (20mg/kg, s.c)
Methods
Step 1
• Weigh the animals and mark properly to
distinguish from one another
• Divide animals into two groups (n = 6 in each
group)
Step 2
• Group 1: Control group (n = 6); Rats are given
saline at the equvalent dose of NaCl 1%
solution or propylene glycol 0.5 ml for four
weeks
• Group 2: Treatment group (n = 6); Rats are
given DOCA 20 mg/kg, s.c. along with NaCl
1% sol. orally ad libitum for four weeks
Step 3
• Observe the animals behavior at baseline,
every week and after completion of 4 weeks
• Blood pressure, ECG is measured at the
beginning (baseline), 1, 2, 3, and 4 weeks
 Animal Experiment on CVS  215

Observations and Results

Group 1
Sl. no. BP ECG
0wk 1wk 2wk 3wk 4wk 0wk 1wk 2wk 3wk 4wk
1.
2.
3.
4.
5.
6.

Group 2
Sl. no. BP ECG
0wk 1wk 2wk 3wk 4wk 0wk 1wk 2wk 3wk 4wk
1.
2.
3.
4.
5.
6.

Discussion endopeptidase inhibitor, retrothiophan, on renal

In the DOCA-salt induced hypertension,
vasopressin plays an important role which is
observed elevated in plasma level and urinary
excretion in several studies. The others are nor-
epinephrine and angiotensin II, which induce the
DOCA –salt hypertension. These rats develop
hypertrophy, excessive collagen deposition,
perivascular and interstitial fibrosis, endothelial
dysfunction, and prolongation of the cardiac
action potential with hypertension within 4
weeks.
SUGGESTED READING
1. Pham I, el Amrani AI, Fournie-Zaluski MC, Corvol
P, Roques B, Michel JB. Effects of the selective
function and blood pressure in conscious
normotensive Wistar and hypertensive DOCA-salt
rats. J Cardiovasc Pharmacol 1992;20:847-57.
2. Badyal DK, Lata H, Dadhich AP. Animal models of
hypertension and effect of drugs. Ind J Pharmacol
2003;35:349-62.
3. Majima M, Katori M, Hanazuka M, Mizogami S,
Nakano T, Nakao Y,Mikami R, Uryu H, Okamura
R,Mohsin SSJ, Oh-Ishi S. Suppression of rat
desoxycorticosterone-salt hypertension by the
kallikrein-kinin system. Hypertension 1991;17:
806-13.
4. Schenk J, McNeill JH. The pathogenesis of DOCA-
salt hypertension. J Pharm Toxicol Meth 1992;27:
161-70.
5. Seyle H, Bois P. the hormonal production of
nephrosclerosis and periarteritis nodosa in the
primates. Br Med J 1957;1:183-86.
216  Practical Manual of Experimental and Clinical Pharmacology

EXPERIMENT NO: 19E Drug : FeCl3(25%) *

Aim *For mice 10% FeCl3 is used.

To demonstrate Ferric Chloride (FeCl3) -induced Methods

thrombosis in rat model.
Background
FeCl3 is commonly used as an oxidant and causes
endothelial injury, platelet aggregation, and a
rapid onset of thrombus formation when it is
directly applied to the blood vessel. The trans-
migration pathway of the ferric ion plays the main
role which shows that some ferric oxide aggregates
formed near the developing thrombus in the
vascular lumen whereas endothelial and smooth
muscle injuries are observed in segments of the
vessel in many studies which came in direct
contact with the FeCl3.
Materials and Methods
Materials
Animal/species : Rat/ Wistar
Sex/Body weight : Either sex/ 150-200 g
Syringe/needle : 1 ml/ preferably 23 G
Step 1
• Weigh the animals and anesthetise with
sodium pentobarbital (50 mg/kg, i.p.)
• Give the midline incision between mandible
of the neck and upper end of manubrium
sterni
• Sternocleidomastoid muscles are gently
retracted laterally and locate trachea
Step 2
• Identify the right or left carotid artery and
separate it from vagus and attached tissue
• Apply a weighed filter paper (2 × 5 mm) and
saturated with 25% FeCl3 solution to the artery
• Paper is allowed to remain at the vessel for 10
min and then removed and weighed
Step 3
• The experiment is continued for 60 min after
the induction of thrombosis. Then, thrombus
is removed and weighed
• Blood pressure, blood flow and ECG is
measured at the beginning (baseline), 1, 2, 3
and 4 week (To observe progression of
thrombosis).

Observation
Sl. no. BP Blood flow ECG
0wk 1wk 2wk 3wk 4wk 0wk 1wk 2wk 3wk 4wk 0wk 1wk 2wk 3wk 4wk
1.
2.
3.
4.
5.
6.

SUGGESTED READING
2. Michael T. Tseng, Alan Dozier, Bodduluri Haribabu
1. Kurz KD, Main BW, Sandusky GE. Rat model of and Uschi M. Graham. Transendothelial migration
arterial thrombosis induced by ferric chloride. Thromb of ferric ion in FeCl3 injured murine common carotid
Res 1990;60:269-80. arteryThrombosis Research 2006;118 (2):275-80.
 Animal Experiment on
20 Gastrointestinal Tract (GIT)

EXPERIMENT NO.: 20A Stomach is opened along the greater curvature

and stretched properly and pinned on a wax
Aim plate. The number of ulcers is noted and the
To demonstrate gastric ulcer induction/formation severity recorded with the following scores:
by different methods 0 = no ulcer; 1 = superficial ulcers; 2 = deep ulcers;
3 = perforation
Background
Pylorus ligation method (SHAY method): Animal Materials and Methods
is anesthetized and a midline abdominal incision Materials
is made. The stomach is identified and the pylorus
is ligated operatively. Then, leave the animal in Animal/species :Rat/ Wistar
the individual cages for 19-24 hr. The test Sex/Body weight :Either sex/ 150-250 g
compounds are given either orally (gavage) or Syringe/needle :1 ml/ preferably 23 G
injected subcutaneously 30 min before ligation. Drug :Indomethacin (20 mg/kg,
At specified time, animal is sacrificed under po)
euthanasia. The abdomen is opened and a first Other agent for ulcer : Aspirin (500 mg/kg, po)
ligature is placed around the esophagus close to
the diaphragm to stop the contents in the stomach. Precautions before Experimentation
Then, the stomach is removed, and the contents • Animals should be marked properly, to avoid
are drained in a centrifuge tube. (Volume of the mixing in two groups
gastric content is measured and then, after, • Handle the animals with care (minimize the
centrifugation, acidity is determined by titration stress and pain to animal)
with 0.1N NaOH). • Animals should be fasted for at least 24 hour
Indomethacin induced ulcer: The test drugs are in metabolic cage and water is given ad libitum
administered orally dissolved in 0.1% Tween 80 • Should be housed separately to avoid canni-
solution 10 min prior to oral administration of balism and coprophagy
indomethacin in a dose of 20 mg/kg, dissolved in • While ligation, care must be taken, so that
0.1% Tween 80 solution. Six hours later, the rats neither damage to the blood supply nor stress
are sacrificed by CO2 anesthesia and stomach is to the pylorus occurs.
removed.
Methods
Ethanol induced ulcer: The animals are adminis-
tered 1 ml absolute ethanol orally. Untreated or Step 1
vehicle treated animals are included as control. • Weigh the animals and mark properly
One hour after administration of ethanol, the • Divide animals into two groups (n = 6 in each
animals are euthanized with CO2. group)
218  Practical Manual of Experimental and Clinical Pharmacology

Step 2
• Group 1: Control group (n = 6); rat are given
saline at the equivalent dose of drug
• Group 2: Treatment group (n = 6); rat pylorus
is ligated for 19-24 hr or indomethacin 20 mg/
kg, po or aspirin 500 mg/kg, po
Step 3
• Sacrifice the animal after 19 hrs in case of
pylorus ligation or 6 hrs after indomethacin
or 1 hr after ethanol administration
• Observe for score
Note: Group: 3, may be added in the experimental Fig. 20.1: Arrows indicate gastric ulcer in rat stomach
group, if one wants to see the protective effect of (For color version see Plate 3)
the PPI’s such as omeprazole, rabeprazole etc. or
H-2 blockers such as Ranitidine, cimetidine, etc.
Test drug is administered 30-60 min prior to analgesics induced gastric ulcer, for example
induction of gastric ulcer. indomethacin, aspirin, alcohol induced, etc. or
Ulcer is observed by the following score: pylorus ligation method (SHAY method). All these
0 = no ulcer; 1 = superficial ulcers; 2 = deep ulcers; methods are based on the mechanism of the
3 = perforation. excessive retention or production of gastric acid
(HCl) prostaglandins, histamine, etc. which leads
Observation and Results to oxygen reactive radicals, and other infla-
An ulcer index (UI) is calculated: mmatory mediators, locally at the stomach; can
UI = AU + ASS+PU X 10–1 induce serve ulcer. Severity of ulcer may worsen
AU = average number of ulcers per animal due to the presence ulcer of H. Pylori infection
ASS = average of severity score which is difficult to treat.
PU = percentage of animals with ulcers
SUGGESTED READING
Observation Table
Sl. no. Gastric ulcer 1. Asad M, Shewade DG, Koumaravelou K, Abraham
Group 1 Group 2 BK, Vasu S, Ramaswamy S. Effect of centrally
administered oxytocin on gastric and duodenal ulcers
Score UI Score UI
in rats. Acta Pharmacol Sin 2001;22(6):488-92.
1. 2. Djahanguiri B. The production of acute gastric
2. ulceration by indomethacin in the rat. Scand J
Gastroenterol 1969;4:265-67.
3.
3. Fornai M, Natale G, Colucci R, Tuccori M, Carazzina
4. G, Antonioli L, Baldi S, Lubrano V, Abramo A,
5. Blandizzi C, Del Tacca M. Mechanisms of protection
6. by pantoprazole against NSAID-induced gastric
mucosal damage. Naunyn Schmiedebergs Arch
Discussion Pharmacol 2005;372(1):79-87.
4. Ishihara Masashi, ITO Mikio. Influence of aging on
There are several ideal models to induce the gastric gastric ulcer healing activities of cimetidine and
ulcer which resemble gastric ulcer in humans like omeprazole. Eur J Pharmacol 2002;444:209-15.
 Animal Experiment on GIT  219

EXPERIMENT NO: 20B Methods

Step 1
Aim • Weigh the animals and mark properly
To demonstrate cerulein induced acute pan- • Divide animals into two groups (n = 6 in each
creatitis in rat. group)
Step 2
Background • Group 1: Control group (n = 6); Rats are given
Inflammation of the pancreas is known as saline at the equivalent dose of drug
pancreatitis and it is proposed that the enzymes • Group 2: Treatment group (n = 6); Rats are
attack and damage their own tissues which given cerulein at the dose of 40 µg/kg, ip
produce them. Mainly pancreatitis is of two types Step 3
acute and chronic. Acute pancreatitis is infla- • Sacrifice the animals after 2 hr of cerulein
mmation of the pancreas that occurs suddenly administration and remove the pancreas
and usually resolves in a few days with treatment • Observe the following parameters: (1) pan-
creatic edema, (2) Pancreatic morphology
where as chronic pancreatitis is inflammation of
the pancreas that does not heal or improve and it Assessment of Intra-pancreatic Edema
gets worse over time leading to permanent damage. After sacrificing the animal, the pancreas is
It is easy to develop experimental model of the removed and weighed. Then, keep pancreas for
pancreatitis which is developed by the many the desiccation at 95° C for 24 hours.
means but the important are cerulein and L- The difference between the wet and dry tissue
arginine for the practical purpose. weights is calculated and expressed as percentage
of the tissue wet weight.
Materials and Methods (WT – DT)/WT × 100
Materials Where,
WT = weight of wet tissue
Animal/species : Rat/ Wistar DT = weight of dry tissue
Sex/Body weight : Either sex/ 150-250 g Morphological characters is checked for any
Syringe/needle : 1ml/ preferably 23G swelling or abnormal condition.
Drug : Cerulein (40µg/kg, i.p)
Other agent for Observations and Results
pancreatitis : L-arginine monohydro- Observation Table
chloride (5.0g/kg, in 0.9%
Group 1
NaCl, i.p)
Sl. no. Intra-pancreatic edema
Precautions before Experimentation WT DT WT - DT % of the tissue
wet weight
• Animals should be marked properly, to avoid
1.
mixing in two groups
2.
• Handle the animals with care (minimize the
3.
stress and pain to animal)
4.
• While locating pancreas one should be careful
because it is diffused and cover a large area in 5.
rats 6.
220  Practical Manual of Experimental and Clinical Pharmacology
Group 2
Sl. no. Intra-pancreatic edema
WT DT WT - DT % of the tissue
wet weight
1.
2.
3.
4.
5.
6.
Discussion
In the normal rat, pancreas is extensively diffuse
and difficult to identify in the peritoneum cavity.
But, in pancreatitis it appears as swollen trans-
parent structure beneath the stomach and
identified near the duodenum.
SUGGESTED READING
1. Frossard JL, Rubbia-Brandt L, Wallig MA, Benathan
M, Ott T, Morel P, Hadengue A, Suter S, Willecke K,
Chanson M. Severe acute pancreatitis and reduced
acinar cell apoptosis in the exocrine pancreas of
mouse deficient for the Cx32 gene. Gastroenterology
2003;124(2):481-93.
2. Schmidt J, Rattner DW, Lewandrowski K, Compton
CC, Mandavilli U, Knoefel WT, Warshaw AL. A
better model of acute pancreatitis for evaluating
therapy. Ann Surg 1992;215:44-56.
EXPERIMENT NO.: 20C
Aim
To demonstrate Tri Nitro Benzene Sulphonic acid
(TNBS) induced colitis in rat.
Background
Inflammatory bowel disease is an idiopathic,
chronic inflammatory condition, which affects the
gastrointestinal tract. The mechanism is through
cytokines like TNFα, IL-1 and IL-8 which are
secreted from macrophages. Due to the lack of
curative agent, screening of an effective drug is
always in pipeline. Experimentally colitis is
induced by a single intracolonic administration of
20 mg TNBS (volume to be administered is 0.25 ml)
which is dissolved in 35% ethanol into the
descending colon (8-10 cm from rectum). Rat is
placed under light ether anesthesia and a rubber
catheter lubricated with lignocaine jelly (outer
diameter = 2 mm) is inserted rectally into the colon
through anus such that tip is reached 8 cm inside
from anus, approximately at the splenic flexure.
TNBS is dissolved in 35% ethanol (v/v) and instilled
into the lumen of the colon through rubber catheter.
The total volume is expelled with additional air
and the catheter is removed. Rats are observed for
two weeks and after two weeks rats are sacrificed
under anesthesia for demonstration of colitis.
Materials and Methods
Materials
Animal/species : Rat/ Wistar
Sex/Body weight : Either sex/ 150-250 g
Syringe/needle : 1 ml/ preferably 23G
Drug : Tri Nitro Benzene Sulphonic
acid (TNBS; 20 mg in 0.25 ml
intracolonic)
Other agent for
colitis : Acetic acid (for 3 days), Dini-
trochlorobenzene (DNCB),
dextran sulfate, carrageenan,
acetic acid, etc
Precautions before Experimentation
• Animals should be marked properly, to avoid
mixing in two groups
• Handle the animals with care (minimize the
stress and pain to animal)
• TNBS is irritant and carcinogenic, so handle
with care and wear the gloves while handling
• Animals should be fasted for at least 24-48 hr
(colon should be cleaned of any fecal material).
Methods
Step 1
• Weigh the animals and mark properly
• Divide animals into two groups (n = 6 in each
group).
 Animal Experiment on GIT  221

Step 2 Discussion
• Group 1: Control group (n = 6); rats are given
Inflammatory bowel disease is a polygenic
35% ethanol at the equivalent dose of drug
• Group 2: Treatment group (n = 6); rats are given disorder that gives rise to multiple clinical
TNBS 20 mg in 0.25 ml dissolved into the 35% subgroups within ulcerative colitis and Crohn’s
ethanol disease. A series of cytokines, like TNF-α, IL-1 and
Step 3 IL-8 are thought to be involved in the process.
• Sacrifice the animals after 14 days after TNBS Among inflammatory cytokines, TNF-α has a
administration and remove 10 cm of descen- broad spectrum of biological effect which plays a
ding colon major role in inflammatory bowel disease. It can
• Cut it longitudinally and open and spread. activate resident macrophages and promote the
Then, observe the gross morphology of colon.
release of other pro-inflammatory mediators
Assessment of Colitis (Enteritis Gross including nitric oxide, prostacyclin and platelet
Morphology Score) activating factors. Among the several experimental
Gross inflammatory index (GII) will be visually models, none of them produced the particularly
assessed for inflammation according to the ulcerative colitis or Crohn’s disease like condi-
following scores: tions; hence it is generally expressed as experi-
0 No inflammatory sign in the whole of 10 cm of mental colitis model and TNBS is very widely used
intestine
to produce experimental colitis.
1 Slight inflammation, and redness, villi visible
less than 15x magnification
2 Intermediate inflammation, discontinuous SUGGESTED READING
hyperemia intermediate redness of villi
3 Intensive inflammation, hyperemia, intensive 1. Levine A, Kenet G, Bruk R, Avni Y, Avinoach I,Aeed
redness of villi H, et al. Effect of heparin on tissue binding activity
Histological examination (HP score) may be of fibroblast growth factor and heparin binding
done to confirm the finding according to the method epidermal growth factor in experimental colitis in
and score described in Levine A et al, 2002. rats. Pediatric Res 2002;51(5):635-40.
2. Medhi B, Prakash A, Avti PK, Saikia UN, Pandhi P,
Observations and Results
Khanduja KL. Effect of Manuka honey and
Observation Table sulfasalazine in combination to promote antioxidant
Sl. Colitis Score defense system in experimentally induced ulcerative
no. Group 1 Group 2 colitis model in rats. Indian J Exp Biol 2008;46(8):
583-90.
GII score HP score GII score HP score
3. Prakash A, Medhi B, Avti PK, Saikia UN, Pandhi P,
1. Khanduja KL. Effect of different doses of Manuka
2. honey in experimentally induced inflammatory
3. bowel disease in rats. Phytother Res 2008;22(11):
4. 1511-19.
5. 4. Vogel H Gerhard, Goethe J Wolfgang. Experimental
6. Colitis. In: Drug Discovery and Evaluation Pharma-
GII score: Gross inflammatory index (Score) and HP cological Assay, 2nd edition, Germany, Springer,
score: Histopathological score 2002;896-99.
 Animal Experiment on
21 Respiratory System
EXPERIMENT NO.: 21A
Aim
To measure respiratory volume in guinea pig
using body plethysmograph.
Background
Body plethysmograph measures the volume of air
present in the lung (while inhale and exhale of
the air), airway resistance, functional residual
capacity (FRC), etc. This test is based on the flow
and pressure measurements. Additionally,
specific airways conductance and airway/
bronchial reactivity to allergen like histamine
(inhaled) is carried out using the body plethys-
mographic technique which is well described in
Agrawal et al, 1977. An indigenously fabricated
Fig. 21.1: Body plethysmograph; for measuring lung volume
(for details of body plethysmograph, please refer to “Commonly
used instrument in pharmacology laboratory” section)
body plethysmograph is used for measurement of
lung volume which is attached to monitor for the
assessment of parameters (Fig. 21.1).
Materials and Methods
Materials
Animal/sex : Guinea pig/Male
Weight : 250-650 gm,
The following baseline investigations are carried
out in all the animals:
1. Measurement of specific airways conductance
in a body plethysmograph.
2. Measurement of airway response to inhaled
histamine to quantify baseline bronchial
reactivity.
Methods
Step 1: Weigh the animal; generally take the
healthy adult guinea pig
Step 2: Group 1*: Control group without any
intervention. 3 animals included in this group
Step 3: Lung volume is assessed
*This experiment just deals with the common way to
assess the lung volume. Other groups may be added
Group 2 may be included in the study if one wants to see
the effect of an allergen. Animals first sensitized to
ovalbumin followed by an inhalation challenge with histamine
(in vivo generation of reactive oxygen species associated
with airway inflammation)
Group 3 may be included if one want to see the effect of
therapeutic agents like anti-asthmatics (salbutamol
inhalation), etc. on the allergens induced hypersensitivity
reaction.
 Animal Experiment on Respiratory System  223

Observations and Result Methods

Write the findings of measurement.

Discussion
Lung volume is measured by the computer monitor
which is attached to the body plethysmograph
directly. Other assessment like bronchial reactivity
and histopathological score may be done for the
histamine treated or intervention after the allergen
induced hypersensitivity reaction.

SUGGESTED READING
1. Agrawal KP. Assessment of airway reactivity in
guinea pigs using a non-invasive body plethys-
mographic technique. Indian J Chest Dis Allied Sci
1977;19(1):3-7.

EXPERIMENT NO.: 21B

Aim
To collect the Broncho Alveolar Lavage (BAL) fluid
for analysis.
Background
BAL is an investigative tool to assess the patho-
logy of disease activity and stage of interstitial
lung diseases. Collection of fluid is squirted into
a small part of the lung and then recollected for
examination to diagnose several lung diseases in
lungs, pneumonia, tuberculosis, mycosis, allergic
alveolitis, etc.
Materials and Methods
Materials
Animal : Guinea Pig
Sex/Body weight : Either sex/ 400-800 gm
Syringe/needle : 1 ml/ preferably 24G on-
wards
Note: DLC analyzes the cellular infiltration into the
bronchoalveolar lumen
Same method may be performed in mouse/rat
for the collection of BAL.
SUGGESTED READING
1. Renz H, Smith HR, Henson JE, Ray BS, Irvin CG,
Gelfand EW. Aerosolized antigen exposure without
adjuvant causes increased IgE production and
increased airway responsiveness in the mouse.
J Allergy Clin Immunol 1992;89:1127-38.
2. Schmiedl A, Hoymann HG, Ochs M, Menke A,
Fehrenbach A, Krug N, Tschernig T, Höhlfeld JM.
Increase of inactive intra-alveolar surfactant subtypes
in lungs of asthmatic Brown Norway rats. Virchows
Arch 2003;442:56-65.
 Anti-inflammatory
22
EXPERIMENT NO: 22A plethysmograph which is used to measure the
changes in volume (Fig. 22.1). In the experiment,
Aim it measures the volume of the rat paw in the
To demonstrate the anti-inflammatory property presence and absence of irritant and after the
of indomethacin against carrageenan induced treatment of anti-inflammatory drug.
paw edema.
Materials and Methods
Background Materials
The principle of screening of anti-inflammatory Animal/species : Rat/Wistar
agents is based on the reduction of edema caused
Sex/body weight : Male/ 150-250 g
by any irritant or phlogistic agent (agent that may
Syringe/needle : 1 ml/ preferably 26G
induce inflammation or fever). The edema is
Drug : Indomethacin (8 mg/kg , po),
measured by an instrument known as the
carrageenan (0.05 ml of 1%
solution, s.c)
Instrument : Plethysmograph
Other irritants : Serotonin solution (0.05 ml of
0.02%), complete Freund’s
adjuvant (0.1 ml), dextran
solution (0.1 ml of 1 to 3%),
fresh egg white (undiluted
0.05 ml)

Precautions before Experimentation

• Animals should be marked properly, to avoid
mixing in two groups
• Clean the paw with wet cotton
• Mark the paw (the insertion part must be the
same every time).

Fig. 22.1: Plethysmograph; Rat paw is immersed into the Methods

tube’A’ and the reading is checked through the tube ‘B’ (some
set-up have digital display attached to it) Step 1: Weigh the animals and mark properly.
 Anti-inflammatory  225

Step 2 Alternative method for measuring paw edema

• Group 1: Control group (n = 6); choose right or
Glass cylinder with an internal diameter of 2 cm and height
left paw of rat as a control which is given saline of 5 cm is attached to the center of a 10 cm diameter Petri
at the equvalent dose of drug dish. Mercury is filled up to 4 centimeters of the cylinder.
• Group 2: Treatment group (n = 6); other paw is Then, the assembly is put on a digital balance with a suitable
given treatment of indomethacin (8 mg/kg, sensitivity.
p.o.), then after 30 min, carrageenan (0.05 ml Volume of paw edema= (W1 – W2)/ G
of 1% solution, s.c. is injected into the plantar Where, W1 = weight of the assembly with the paw
region. immersed
W2 = weight of the assembly without paw
Step 3
immersed
• Observe the animals at 0, 3, 6 and 24 hrs after G = gravity of fluid in the glass cylinder
the drug administration (mercury gravity=13.6)
• Observe the animals behavior carefully and
Note: (1) Take at least two readings, (2) Mark the paw at
calculate the percentage protection after the specific point to immerse every time at the same level
treatment.
Observations and Results
Observation Table
Group 1
Sl. Paw edema (0hr) Paw edema (3hr) Paw edema (6hr) Paw edema (24hr)
No. Right paw Left paw Right paw Left paw Right paw Left paw Right paw Left paw
1.
2.
3.
4.
5.
6.

SUGGESTED READING 4. Winter CA, Risley EA, Nuss GW (1963). Anti-

1. Fereidonia M, Ahmadiania A, Semnanianb S, Javan
M. An accurate and simple method for measurement
of paw edema. Journal of Pharmacological and
Toxicological Methods 2000;43:11-14.
2. Ferreira T, Camargo EA, Ribela MT, Damico DC,
Marangoni S, Antunes E, De Nucci G, Landucci
EC. Inflammatory edema induced by Lachesis muta
muta (Surucucu) venom and LmTX-I in
the rat paw and dorsal skin. Toxicon 2009;53(1):
69-77.
3. Ne•iæ L, Skrbiæ R, Dobriæ S, Stojiljkoviæ MP,
Jaæeviæ V, Satara SS, Milovanoviæ ZA, Stojakoviæ
N. Simvastatin and Indomethacin have similar anti-
inflammatory activity in a rat model of acute local
inflammation. Basic Clin Pharmacol Toxicol 2009
Jan 31.
inflammatory and antipyretic activities of
indomethacin, (1-(pchlorobenzoyl)-5-methoxy-2-
methyl-indole-3-acetic acid. J Pharmacol Exp Ther
1963;141:369-76.
EXPERIMENT NO: 22B
Aim
To demonstrate analgesic effect of morphine
against acetic acid induced writhing in rat.
Background
There are few methods to evaluate the peripheral
analgesic activity. The Randallselitto-Test in
rats and writhing tests in mouse are the
commonest. Many of the peripherally acting
226  Practical Manual of Experimental and Clinical Pharmacology

analgesics have the anti-inflammatory and • Divide animals into two groups (n = 6 in each
antipyretic effect. The method depends on the group)
principle that an irritant directly administered
Step 2
into the peritoneum cavity of the animal
• Group 1: Control group (n = 6); rats are given
produces severe pain and irritation in the
saline at the equivalent dose of drug
ventral part. The writhing is characterized by
• Group 2: Treatment group (n = 6); rats are given
typical stretching behavior of body and rodent
morphine at the dose of 3.5 mg/kg, ip,
tries to touch its ventral part to the ground.
thereafter 10-15 min, acetic acid 0.1 ml of a
0.6% solution, i.p. is given.
Materials and Methods
Step 3
Materials
• Observe the animals for 45 min in plexiglass
Animal/species : Rat/Wistar chamber
Sex/body weight : Male/150-280 gm • Observe the animals behavior carefully, for (1)
Syringe/needle : 1 ml/preferably 24G on- onset of writhing, (2) duration of writhing and
wards (3) number of writhing or any additional
Drug : Morphine (3.5-4 mg/kg, s.c.) behaviour changes.
Irritant : Acetic acid (0.1 ml of a 0.6%-
1.0% solution, i.p )
Other irritants : Phenylquinone [(0.02% in
1% suspension of carboxy-
methylcellulose (CMC): 0.25
ml, i.p], PGE1 and brady-
kinin may also be used
Other Drugs
screened : Aspirin (20-21 mg/kg, p.o)
Indomethacin (10-20 mg/kg,
i.p.); Amidopyrine 40 mg/kg
p.o.and Phenacetin 80 mg/
kg p.o.

Precautions before Experimentation

• Laboratory should be dim lighted and noise
free
• Animals should be marked properly, to avoid
mixing in two groups
• Handle the animals with care (minimize the
stress and pain to animal)
• Observe the animals in a plexi glass chamber.

Methods
Step 1 Figs 22.2A and B: (A) Showing the stretching of abdomen
• Weigh the animals and mark properly to and rat touches the ventral part to ground and (B) Showing
distinguish from one another the abdominal cramp or discomfort
 Anti-inflammatory  227

Observations and Results 2. Ferreira SH, Lorenzetti BB, Corrêa FMA. Central and
peripheral antialgesic action of aspirin-like drugs.
Observation Table Eur J Pharmacol 1978b;53:39-48.
3. Ferreira SH, Nakamura M, DeAbreu Castro MS. The
Writhing response
hyperalgesic effects of prostacyclin and prosta-
Onset Recovery No. of writhing Duration glandin E2. Prostaglandins 1978a;16:31-37.
4. Finck AD, Samaniego E, Ngai SH. Morphine tolerance
1.
decreases the analgesic effects of ketamine in mouse.
2. Anesthesiology 1988;68(3):397-400.
3. 5. Miranda HF, Puig MM, Dursteler C, Prieto JC, Pinardi
G. Dexketoprofen-induced antinociception in animal
4.
models of acute pain: synergy with morphine and
5. paracetamol. Neuropharmacology 2007;52(2):291-96.
6. 6. Rios L, Jacob JJC. Inhibition of inflammatory pain by
naloxone and its N-methyl quaternary analogue. Life
Sci 1982;31:1209-12.
SUGGESTED READING 7. Romer D. Pharmacological evaluation of mild
analgesics. Br J Clin Pharmacol 1980;10:247S-51S.
1. Chiba S, Nishiyama T, Yoshikawa M, Yamada Y. 8. Winter CW, Risley EA, Nuss GW. Carrageenen-
The antinociceptive effects of midazolam on three induced edema in hind paw of the rat as an assay for
different types of nociception in mouse. J Pharmacol anti-inflammatory drugs. Proc Soc Exp Biol Med
Sci 2009;109(1):71-77. 1962;111:544-47.
 Local Anesthetics (LA)
23
EXPERIMENT NO: 23 • Mark the control and treatment side properly
to avoid the confusion.
Aim
To demonstrate the effect of any given local Methods
anesthetic (LA) using guinea pig (GP).
Step 1
Background • Weigh the animals and mark properly to
Local anesthetics are agents which block con- distinguish from one another
duction of Na+ by decreasing or preventing the • Remove the hair on its flanks on the both sides.
large transient increase in the permeability of
excitable membranes and at higher concentration Step 2
they also block K+ channels. This is based on the • Control side: (Mark ‘C’): Saline or control jelly
principle of loss of the sensation, even after given is applied at ‘C’ site
an external stimuli. • Treatment side: (Mark ‘T’): Drug is applied
localy at the ‘T’ site
EXPERIMENT 23‘A’
• Anesthetic activity is checked by giving the
Materials and Methods small pinch with forcep on the control and
Materials treatment side and then check the animal
response (squeak response)
Animal/species : Guinea pig
Sex/Body weight : Either sex/250-350 gm
Step 3
Drug : Lignocaine (2% gel)
Instruments : Scissor, razor • Take the responses at the 0, 5, 10, 15, 20, 30, 45
and 60 min after application of gel
Precautions before Experimentation • The effect of the local anesthetic is expressed
• Carefully remove the hair from either side of as ‘Yes’ for the LA effect and ‘No’ for the
flanks with blade absence of the effect.
 Local Anesthetics (LA)  229

Observations and Result

Observation Table
Sl. No. Time after application of lignocaine gel (min) Remarks
0 5 10 15 20 30 45 60
‘C’ ‘T’ ‘C’ ‘T’ ‘C’ ‘T’ ‘C’ ‘T’ ‘C’ ‘T’ ‘C’ ‘T’ ‘C’ ‘T’ ‘C’ ‘T’
1.
2.
3.
4.
5.
6.

EXPERIMENT 23‘B’ • Fill the abdominal cavity by 10-12 ml of 1%

w/v of procaine HCl and wait for 5 min, then
Aim
reflex is checked by immersing the leg in
To demonstrate the effect of the local anesthetic normal saline and 0.1N HCl respectively
property of procaine HCl using foot withdrawal Step 3: Response is taken as reflex present and
reflex of frog. absent after the anesthetic treatment in left (test)
and right (control) leg.
Materials
Animal : Frog Observations and Result
Solution : Normal saline and 0.1 N HCl
Sl. No Reflex (Control) Reflex (Test)
Instruments : Beaker, scissor, dissection
Present Absent Present Absent
board
1.
Methods 2.
Step 1 3.
• Frog is desensitized by pithing 4.
• Give the midline abdominal incision. Open
5.
the abdomen and remove all abdominal
viscera and expose the sciatic nerve 6.

Step 2 SUGGESTED READING

• Expose the sciatic nerve in the abdomen cavity
• Fix two front limbs of frog on the dissecting
board, in such a way that both hind limbs of
the frog hang vertical by outside the board
• Place the leg into the either solution selectively,
i.e. right on normal saline (control) and left in
the 0.1 N HCl (test) (To check the response)
1. Arthur H Campbell, Jeanette A Stasse, Geoffrey H
Lord, John E Willson. In vivo evaluation of local
anesthetics applied topically. Journal of Pharma-
ceutical Sciences 2006;57(12):2045-48.
2. Ritchie JM, Greengard P. On the mode of action of
local anesthetics. Ann Rev Pharmacol 1966;6:
405-30.
 Experiment on Rabbit Eye
24
EXPERIMENT NO: 24A
Aim
To study the effect of different drugs on the rabbit
eye.
Background
The drugs like miotics (constrict the pupil) and
mydriatics (dilate the pupils) are generally screened
by this method. The role of circular muscle, radial
muscle and ciliary muscle is well-known (Fig. 24.1).
Effect should be noted on the various para-
meters
1. Effect on conjunctiva: Conjunctiva is usually thin
and clear when eyes are healthy which is used
in the experiment. Changes before and after the
drug administration are noted and compared.
2. Effect on pupil: The original size is measured
with the help of “pupilometer”( it is the scale
like structure with varying size of round holes,
i.e. 0.5 mm, 1 mm, 1.5 mm, 2 mm, 2.5 mm and
3 mm) (Fig. 24.2) and after the drug treatment
the change in pupil size is noted and then it is
compared with the initial size or the control
Fig. 24.1: Mechanism of miosis and mydriasis
Fig. 24.2: Pupilometer
eye size. It either increases in size (mydriasis)
or decreases the pupil size (miosis) according
to the drug used in the experiment.
3. Corneal reflex: When cornea is touched rabbit
closes the eyes. To check this reflex, a cotton
tip is prepared and touched at corneoscleral
junction by bringing the corneal tip from the
side of the eye. If the cornea is desensitized
with the local anesthetic, then there will be no
corneal reflex.
4. Light reflex: It is elicited by pointing the light
source on the pupil with the help of torch.
There is constriction of pupil when rabbit is
kept in a darker area or eye is covered with a
cardboard and the size of pupil is measured,
then light source is pointed on the pupil.
Thereafter the size of the pupil is noted and
then compared with the initial reading.
Pathway of Vision (Fig. 24.3)
The visual system is responsible for the vision and
is controlled by the central nervous system. It is
composed of many complex pathways through
optic nerve, optic chiasm, optic tract, lateral
geniculate nucleus (LGN) and visual cortex. Vision
is generated by retina (photoreceptors), a layer of
cells at the back of the eye and travel through optic
nerve to visual cortex. Importance of visual system

Fig. 24.3: Visual pathway; Visual information falls through
the cornea to the object left field to the right side of each eye
and right field to the left side of the eye; Optic nerve: Pass
the visual information to the optic chiasm; Optic chiasm:
Nerve or fibers of media half of either retina cross in the
optic chiasm; Optic tract: It carries the visual signal of
opposite side such as left visual field information pass
through right optic tract and right visual information pass
from left optic tract; LGN: Situated in thalamus and all nerve
synapse here. Then, neurons of the LGN, pass along the
visual image to the primary visual cortex; Visual cortex:
After receiving the image in primary cortex through LGN, it
passes to secondary visual cortex (extrastriate visual
cortex) where the visual perception take place about color,
height, place, motion, distance, etc.
is to do many complex tasks such as reception of
light, the identification and categorization of visual
objects by forming an image in the retina, assessing
characteristic colors, place, height, distances of an
objects and guiding body movements to visual clues.
This type of the psychological manifestation of
visual information is known as ‘visual perception’.
Interestingly visual system is the same in the higher
animals.
Materials and Methods
Materials
Animal : Rabbit (1.5-2.5 kg)
Drugs
miotics : Pilocarpine (1-4%),
Physostigmine (1%)
Experiment on Rabbit Eye  231
Mydriatics : Phenylephrine, Ephedrine
(3-5%)
Anesthetics : Lignocaine (4%)
Instruments : Corneal reflex meter, scissor,
dropper, torch.
Precautions before Experimentation
• Maintain the aseptic conditions while
instilling the drug
• Do not use the solution, if it is discolored (check
the expiry date of given drug)
• Experiment should be performed in the dim
lighted room
• Check for any eye disease such as conjunc-
tivitis, uvieitis, etc.
• Remove the eye lashes carefully.
Methods
Step 1
• Weigh and mark the rabbits. Select 3 rabbits
(one for pupil reflex, second for corneal reflex
and third for the light reflex)
• The alternate eye of a rabbit works as control
and test group (to avoid individual varia-
tion).
Step 2
• Restrain the rabbit in the suitable restrainer
and fix it for 5 min (to accommodate and
acclimatize rabbit into the restrainer)
• Select the control and test eye according to your
preference (right or left), and mark it with test
eye (TE) for test and control eye (CE) for control
• Eye lashes of both eyes is trimmed out with
scissor and normal saline is put in both the
eyes to make them clean and clear.
Step 3
• The drug is instilled* with the help of dropper
in the test eye (not more than 2-3 drops)
• Then, the response is noted and both test and
control eyes are compared.
*Note: Volume to be instilled in rabbit eye sac
should be 40-60 μl.
232  Practical Manual of Experimental and Clinical Pharmacology

Observations and Results

Observation Table
Time (min) Light reflex Corneal reflex Pupillary size Condition of
conjunctiva
TE CE TE CE TE CE TE CE
0
5
10
20
30
60

SUGGESTED READING 2. Strughold H. The mechanism threshold of the corneal

1. Green K, Downs SJ. Ocular penetration of pilocarpine reflex of the usual laboratory animals. Am J Physiol
in rabbits. Arch Ophthalmol 1975;93(11):1165-68. 1930;94:235-40.
 Experimental
25 Pharmacokinetics
EXPERIMENT NO: 25A
Aim
To study the pharmacokinetics of phenytoin
following oral single dose administration for 7
days.
Background
Pharmacokinetics is a process by which a drug is
absorbed, distributed, metabolized, and elimi-
nated through the body. So, during these processes
drug substances interact with different com-
pounds residing within an animal or system, such
as nutrients, metabolites, drugs, endogenous
hormones, toxins, etc. Hence, it is important to
assess the drug’s interaction which is preferred
for the chronic use such as antiepileptics, anti-
rheumatics, analgesics, etc. Pharmacokinetics in
the human and animals is not exactly the same.
Hence, animal model in this context is rabbit and
it is widely used in pharmacokinetic studies.
Special animal ethics permission is required from
CPCSEA for use of monkey, whereas rabbits are
easily available through the Institutional Animal
Ethics Committee (IAEC).
Materials and Methods
Animals : Male white New Zealand
rabbits (1.5 kg and 2.5 kg)
(n=6)
Drug : Phenytoin (30 mg/kg/day)
Laboratory
conditions : 12 hours day-night cycle at a
temperature of 25 ± 2°C. The
animal is allowed to take
water ad libitum and free
access to standard food.
Anesthesia : Topical lignocaine (4%) (To
minimize discomfort and
pain while withdrawing
blood)
Precautions before Experimentation
• Care and handling should be proper
• Route of blood withdrawal should be
convenient and easily approachable
• Selection of needle size should be proper to
minimize the pain and trauma to the marginal
vein or at site of blood withdrawal.
Methods
Step I: Rabbits are acclimatized to laboratory and
person, 1 week prior to the experimentation.
Step II: Rabbits (n = 6) are administered phenytoin
in a dose of 30 mg/kg/day per oral, at 08:00 hours
for seven consecutive days using an orogastric
tube.
Step III: On day 7, blood samples (1 ml) are collected
before administration of drug dose (7th day dose)
of phenytoin at 0 hr and then drug is administered.
Thereafter, blood sample is taken at 0.5, 1, 2, 3, 4,
5, 6, 9, 12 and 24 hours after drug administration
i.e. total of 11 samples.
Step IV: Blood sample is drawn from the marginal
ear vein after applying anesthesia with topical
lignocaine 2% gel. (See instructions for blood collection
in chapter 1).
234  Practical Manual of Experimental and Clinical Pharmacology
Step V: Each sample is centrifuged at 3000 rpm for
5 minutes and plasma separated and stored at
(–20°C) until HPLC analysis.
Step VI: Then, phenytoin concentration is
estimated by HPLC method.
Extraction procedure: To 0.2 ml plasma sample/
standard sample, add 0.2 ml of 1.0M of sodium
acetate buffer (pH 5.5) and 3.0 ml of chloroform.
Shake the tubes for 1 min and then centrifuge at
3000rpm for 10 min. Transfer 2.8 ml of
chloroform layer in another test tube and
evaporate the chloroform at 50°C on a water
bath. Reconstitute the residue in 0.2 ml of mobile
phase to be used for HPLC assay. Inject 100 µl
of this reconstituted solution to HPLC system
for assay.
HPLC conditions: Mobile phase: acetonitrile:
methanol: 4mM potassium phosphate buffer (pH
6.0) in ratio of 20:40:40(V/V/V)
Flow rate : 1.0 ml/min
Temperature : ambient
AUFS : 0.02
Detection : UV detector 215 nm
Chart speed : 4 mm/min
Injection volume : 100 µl
Sensitivity assay : 0.1 µg/ml
Recovery : 98% or more
Standard : phenytoin 0.5 to 32 µg/ml
Step VII: Record the reading and store chrom-
atogram.
Step VIII: The following pharmacokinetic para-
meters should be calculated for phenytoin.
• Peak plasma concentration(Cmax)
• Time to reach peak plasma concentration(Tmax)
• Absorption constant (ka)
• Absorption half-life (t½a)
• Elimination constant (kel)
• Elimination half-life(t½el )
• Area under the plasma-drug concentration
time curve (AUC0- 24) and (AUC0-∞).
Observation Table
Parameters Drug (Phenytoin)
Peak plasma concentration
(Cmax)
Time to reach peak plasma
concentration(Tmax-)
Absorption constant (ka)
Absorption half-life (t½a)
Elimination constant (kel)
Elimination half-life(t½el )
Area under the plasma-
drug concentration time
curve (AUC0-24)
(AUC0-∞)
SUGGESTED READING
1. Medhi B, Sukhija M, Prakash A, Gaikwad S, Bansal
V, Pandhi P. Effects of etoricoxib on the pharma-
cokinetics of phenytoin. Pharmacol Rep 2008;60(2):
233-37.
EXPERIMENT NO: 25B
Aim
To study the pharmacokinetic interaction of
phenytoin with etoricoxib after single oral dose
for 7days.
Background
Pharmacokinetic interactions result due to the
multiple use of drugs at a time or even with the
food. But, these interactions are more complicated
and difficult to predict because the interacting
drugs often have unrelated actions. For example:
Phenytoin is used as antiepileptic whereas
etoricoxib is used for the acute and chronic pain.
The interactions are mainly due to alteration of
absorption, distribution, metabolism, or excretion
which changes the amount and duration of a
drug’s availability at receptor sites. Some drugs
cause induction of hepatic mitochondrial
enzymes (P-450) such as phenytoin, barbiturates,
rifampin, digoxin, etc. and some drugs cause
inhibition of hepatic mitochondrial enzymes (P-
450) such as TCA, oral contraceptives, isoniazid,

cimetidine, allopurinol, disulfiram, etc. which can
influence the pharmacokinetics of several drugs
metabolized by the same enzyme system.
Materials and Methods
Animals : Male white New Zealand
rabbits (1.5 kg and 2.5 kg)
(n=6)
Drugs : Phenytoin (30 mg/kg/day)
Etoricoxib (5.6 mg/kg)
Laboratory
conditions : 12 hours day-night cycle at a
temperature of 25 ± 2°C. The
animal is allowed to take
water ad libitum and free
access to standard food.
Anesthesia : Topical lignocaine (4%) (To
minimize discomfort and
pain)
Methods
Step I: Total 12 rabbits are acclimatized to
laboratory and person, 1 week prior to the
experimentation.
Step II: On experimental day, rabbits are divided
into two groups control (phenytion alone) group
and treatment group (Phenytoin + etoricoxib).
In control group, rabbits (n=6) are adminis-
tered phenytoin in a dose of 30 mg/kg/day per
oral at 0900 hours for ten consecutive days using
an orogastric tube. But, in the treatment group
(n=6) phenytoin is administered in a dose of 30
mg/kg/day per oral at 0900 hours for 7 conse-
cutive days using an orogastric tube, then next
3days etoricoxib 5.6 mg/kg/day, po with same
dose of phenytoin is administered.
Step III: On day 10, blood samples (1 ml) are
collected before administration of next dose (10th
day dose) of phenytoin at 0 hr and then administer
Experimental Pharmacokinetics  235
the both drugs then, collect blood samples at 0.5,
1, 2, 3, 4, 5, 6, 9, 12 and 24 hours.
Step IV: Blood samples are drawn from the
marginal ear vein after applying anesthesia with
topical lignocaine 2% gel. (See instructions for
blood collection in chapter 1).
Step V: Samples are labelled properly, so that there
is no mixing of samples. Then, each sample is
centrifuged at 3000 rpm for 5 minutes and plasma
separated and stored at -20°C until HPLC
analysis.
Step VI: Then, phenytoin is estimated by HPLC
method.
Extraction procedure: To 0.2 ml plasma sample/
standard sample add 0.2 ml of 1.0M of sodium
acetate buffer (pH 5.5) and 3.0 ml of chloroform.
Shake the tubes for 1 min and then centrifuge at
3000 rpm for 10 min. Transfer 2.8 ml of chloroform
layer in another test tube and evaporate the
chloroform at 50°C on a water bath. Reconstitute
the residue in 0.2 ml of mobile phase to be used for
HPLC assay. Inject 100 µl of this reconstituted
solution to HPLC system for assay.
HPLC conditions: For HPLC conditions refer to
experiment no. 25A
Step VII: Record the readings and store chro-
matogram
Step VIII: The following pharmacokinetic para-
meters will be calculated for phenytoin
• Peak plasma concentration(Cmax)
• Time to reach peak plasma concentration(Tmax)
• Absorption constant (ka)
• Absorption half-life (t½a)
• Elimination constant (kel)
• Elimination half-life(t½el )
• Area under the plasma-drug concentration
time curve (AUC0- 24) and (AUC0-∞).
236  Practical Manual of Experimental and Clinical Pharmacology

Observation Table
Parameters Drug
Phenytoin Phenytoin + Etoricoxib
Peak plasma concentration(Cmax)
Time to reach peak plasma concentration(Tmax-)
Absorption constant (ka)
Absorption half-life (t½a)
Elimination constant (kel)
Elimination half-life(t½el )
Area under the plasma-drug concentration
time curve (AUC0- 24)
(AUC0-∞)

SUGGESTED READING 2. Sukhija M, Medhi B, Pandhi P. Effects of artemisinin,

1. Medhi B, Sukhija M, Prakash A, Gaikwad S, Bansal
V, Pandhi P. Effects of etoricoxib on the pharma-
cokinetics of phenytoin. Pharmacol Rep 2008;
60(2):233-37.
artemether, and arteether on the pharmacokinetics
of phenytoin. Methods Find Exp Clin Pharmacol 2006;
28(2):89-94.
3. Sukhija M, Medhi B, Pandhi P. Effects of artemisinin,
artemether, arteether on the pharmacokinetics of
carbamazepine. Pharmacology 2006;76(3):110-16.
Part 4

Clinical Experiments
 Cardiovascular System
26
BLOOD PRESSURE MEASUREMENT AND
VALIDATION OF SPHYGMOMANOMETER
Introduction
Blood pressure (BP) measurement is one of the
important tool for evaluation of cardiovascular
system, which helps in diagnosis and manage-
ment of hypertension and related end organ
damages. Hence, monitoring of BP is an essential
and basic step. So, briefly, blood pressure (BP) is
the force exerted against blood vessel walls due to
the blood flow in the vessels. As a result of
resistance of the blood vessels and the volume of
blood carried into the blood vessel. It is expressed
in millimeters of mercury (mm Hg). The blood
pressure is measured as two end-point, i.e. first
upper level is systolic pressure (the first sound
heard) and the second is diastolic pressure. (The
last sound heard) There are 5 phases of sound
heard (Korotkoff sound). The difference of systolic
and diastolic pressure is commonly denoted as
“pulse pressure”. (approximately 40 mm Hg).
In clinical practice, BP measurement may get
erroneous (high or low reading) due to the several
reasons such as cuff is too small or cuff too loose,
slow cuff deflation, arm is not at the level of heart,
column or dial not at eye level or it may be taken at
the obsolete time in case of anxiety, exercise, after
eating, etc.
Korotkoff sounds
Phase I : First sound, sharp tapping sound-systolic
pressure (lasts for 10-12 mm Hg) fall
Phase II : Soft swishing sound (lasts for 10-15 mm Hg fall)
Phase III : Rhythmic tapping sound (lasts for 12-14 mm Hg
fall)
Phase IV : Muffling/fading of tapping sound (lasts for 4-5
mm Hg fall)
Phase V : Point at which all sounds disappear-diastolic
pressure
Factors Affecting Blood Pressure
Several factors may interfere with blood pressure.
BP varies throughout the day according to the time,
season and even the daily routine of individual. It
can rise rapidly while doing physical exercise,
when having an emotional moment or even stress
and defense reaction of body to any stimuli.
Principle of BP Measurement
Principle of blood pressure measurement
depends on the compression length of artery
(brachial in arm, radial in wrist and femoral in
thigh) which blocks and reopens the artery blood
flow. The pressure is applied with the help of
bladder fixed in the cuff and rubber bulb. The
systolic and diastolic pressure is measured by the
palpatory, auscultatory or oscillometric method.
Instrument to Measure the BP
The gold standard instrument for blood pressure
measurement is considered to be mercury
sphygmomanometer. The term developed from the
Greek origin, “sphygmo” denotes pulse, “ manos”
denotes thin and “metron” denotes measure. It
consists of mercury manometer, graduated tube,
armlet (Riva Rocci cuff) and air pump.
240  Practical Manual of Experimental and Clinical Pharmacology

Fig. 26.1: Parts of sphygmomanometer and the size of the ideal cuff (Riva Rocci)

Cuff (Riva Rocci) (Table 26.1). These errors caused by the non-
calibrated blood pressure apparatus can not be
Selection of BP cuff should be very logical and
reduced by averaging the measurement. This is
scientific. Wrong selection can give false cuff
an error which is difficult to detect and correct.
hypertension or underestimated BP. The recom-
(Another error is the random error which is due to
mended cuff should have air bladder which
biological variability or any stress or emotional
covers at least 80% of the arm circumference and
condition, but it is reduced by averaging a number
width wise it should cover at least 40% of arm
of measurements.)
circumference (Fig. 26.1).
Arm circumference Class Cuff size Formal Calibration of the Pressure Indicator
22 to 26 cm “small adult” 12-22 cm
27 to 34 cm “adult” 16-30 cm
The validation of devices measuring blood
35 to 44 cm “large adult” 16-36 cm pressure is essential. It should be timely calibrated
45 to 52 cm “adult thigh” 16-42 cm and validated at the recommended time for
accurate measurement of blood pressure. The
Rubber bulb and tube: Inflation mode attached to
instrument should be regularly checked for the
the air bladder of the cuff which is at least of 70 cm
air leakage, the condition of cuff, tubes, bulb and
in length.
fittings, rapid exhaust time, scale visibility,
contamination of the glass tube or mercury, cuff
Mercury Reservoir and Manometer: Validation/
inflation and deflation control and security of
calibration of Sphygmomanometer
mercury containment (vapor of mercury is
The manometer provides a simple indication of poisonous).
difference of pressure, the most preferred one is The sphygmomanometer is calibrated at the
mercury manometer which is used to record or indicator from ‘0’ to the maximum pressure (280-
control difference of pressure or fluid flow. 300 mm Hg) on the sphygmomanometer scale at
Accurate measurement of blood pressure requires pressure increments not greater than 50 mm Hg.
the use of validated/calibrated sphygmo- The pressure indicator of all sphygmomanometers
manometer, which requires regular service and should be calibrated by an authorized laboratory
calibration. The calibration is done to avoid any at 0-300 mm Hg. The standard for instrument is
systemic error during the BP measurement set for least uncertainty of measurement and best
 Cardiovascular System  241

accuracy, i.e. a least uncertainty of measurement TO PREPARE STANDARD OPERATING

of 0.4 mm Hg or less. PROCEDURE (SOP) FOR BLOOD
PRESSURE MEASUREMENT
Table 26.1: Sphygmomanometer: Inspection and
calibration interval Precautions before BP Measurement
Sphygmomanometer Inspection Interval for • Sphygmomanometer should be kept properly
interval calibration while taking the readings
(Month) (Month)
• Always use same arm for readings
Mercury 6 12
Aneroid 1 6 • At the time of BP measurement, moving and
Electronic oscillometric 6 12 talking should be avoided
Electronic manual 6 12
• Air present in the bladder, affects the blood
pressure measurement so it should be emptied
CHRONOBIOLOGY OF BLOOD PRESSURE • Take two or three readings each about 2
minutes apart (Take the average of readings
The BP measurement at a single time may be either two or three and the reading range
erroneous due to the changes seen in almost every should not be more than 5 mm Hg)
biological variable like steroid and other hormones • Volunteers should avoid eating (should not
secretion under 24 hours synchronized condi- take any caffeinated drink or meal) or exercise
tions. These phases of circadian rhythms can be at least before 2 hr of the experiment
manipulated by changing the phase of the • Volunteer should be sitting quietly for about 5
environmental cycles. The systolic and diastolic minutes prior to recording blood pressure
BP variance of an adult is shown in Figure 26.2. • Upper right arm (dominated arm) should be
Hence, it is confirmed that the one time BP bare; cuff should not be placed over the
measurement may not give an accurate reading of clothes.
blood pressure. The BP measurement on the basis
of chronobiological measurement gives the exact Posture of Volunteer
interpretation. • Supine position (Fig. 26.3A and B)
• Sitting position (Figs 26.4A to C)

Fig. 26.2: Chronodesm of an adult Fig. 26.3A: Recording of BP in supine position

242  Practical Manual of Experimental and Clinical Pharmacology

– Normal (relaxed) (Figs 26.4A and B) Various other positions

– Crossed leg (Fig. 26.4C)
• Standing position
– Hand/arm position below heart level (Fig.
26.5A)
i. at the level of heart (26.5B)
ii. above heart level (Fig. 26.5C)

Fig. 26.3B: Supine position

Fig. 26.4C: Sitting with crossed leg

(For color version see Plate 4)

Fig. 26.4A: Recording of BP in sitting position (normal)

Fig. 26.4B: Sitting; cuff position should be at Fig. 26.5A: Standing with hand position below the level of heart
the level of heart (For color version see Plate 4) (cuff level is below heart) (For color version see Plate 4)

Fig. 26.5B: Standing with hand position at the heart level
(For color version see Plate 4)
Fig. 26.5C: Standing with hand position above heart level
(For color version see Plate 4)
Special Population and BP Measurement
Relationship between obesity and hypertension
is well established since 1930s. Hence, the BP
measurement in these populations is an essential
requirement to maintain the health. Caution,
should be taken while measuring the BP, selecting
arm circumference and taking bladder dimen-
sions. It may give false overestimation of BP (“cuff
hypertension”) if the cuff size is small and vice
versa. Variability in BP is more in the pediatric
population than in adults. Mercury sphygmo-
manometer is used in the BP measurement. For
the obese population, pediatric population
appropriate cuff size should be used. Korotkoff
sounds in child aged 1-5 years are not audible
and hence, murcury sphygmomanometer is not
used, so in these age groups doppler, ultrasound,
or oscillometry would be more sensitive and
reliable.
BP measurement in the geriatric population is
troublesome due to change in the physiological
Cardiovascular System  243
functions and rapid changes in the circadian
rhythm. In this population chronodiagnosis of BP
measurement is considered to be more reliable and
reproducible.
 Important Points to Remember
Finger Cuff Method (developed by Penaz). This is based
on the principle of the “unloaded arterial wall”. Arterial
pulsation in a finger is detected by a photoplethysmograph
under a pressure cuff. This method gives an accurate
estimate of the changes of systolic and diastolic pressure,
but less than brachial artery pressures. Mainly used for
the ambulatory BP measurement. Advantages being
monitoring of short change in BP, but cost, inconvenience,
and relative inaccuracy for measuring absolute levels of
blood pressure are major limitations.
REGULATION OF BLOOD PRESSURE
Blood pressure regulation (Fig. 26.6) involves
complex interaction of neural, renal and endocrine
mechanism. Arterial blood pressure is generated
as a result of blood flow and the resistance to blood
flow. Major factors affecting blood pressure are
cardiac output (CO) and total peripheral vascular
resistance (TPR) of blood vessels. So, blood
pressure (BP) = CO × TPR. CO is the major deter-
minant of SBP, whereas TPR mainly determines
DBP. Cardiac output and peripheral resistance
are regulated by the autonomic nervous system
(ANS). Blood pressure is regulated by alteration
in cardiac output and peripheral resistance
exerted at different target sites like arterioles,
postcapillar venules, kidneys and heart. Myo-
cardial contractility and heart rate are regulated
by both the sympathetic and parasympathetic
divisions of the ANS. In contrast, blood vessels
are not innervated by parasympathetic fibers,
therefore the parasympathetic nervous system has
minimal influence on vascular tone. The neural
control of blood pressure regulation is by
parasympathetic neurons that innervate the heart
and by three different action of sympathetic
cardiovascular efferents barosensitive, thermo-
sensitive and glucosensitive. The barosensitive
sympathetic efferents are controlled by arterial
baroreceptors which play a dominant role in
both short-term and long-term blood pressure
244  Practical Manual of Experimental and Clinical Pharmacology

Fig. 26.6: Regulation of blood pressure

regulation. The important regulatory systems in
controlling blood pressure are the sympathetic
nervous system and the renin-angiotensin-
aldosterone system. A rise in blood pressure
increases the impulses to the vasomotor center
resulting in decreased output from the center
resulting in compensatory decrease in the efferent
response of the sympathetic nervous system.
Similarly, a reduction in stretch due to fall in blood
pressure causes reduction in baroreceptor activity.
The sympathetic baroreflex mechanism is acti-
vated by stimulus of mechanoreceptors causes
distension of the arterial wall, an increase in blood
pressure activates baroreceptors there by causing
inhibition of cardiac renal vasomotor sympathetic
effect which help in normalization of blood
pressure.
Baroreflex resetting involves both neural and
humoral mechanisms.
Besides these various local hormones and
metabolites such as ANP, PGs, kinins, NO,
endothelins, etc. are involved in regulation of blood
pressure. Nitric oxide in vascular endothelial
cells, is primarily responsible for controlling
vascular tone and platelet aggregation. The recent
evidence suggest that nitric oxide may affect

vasodilation not only by activating guanyl cyclase
but also by activating calcium and potassium
channels in vascular smooth muscle cells, nitric
oxide appears to activate these potassium
channels directly via guanyl cyclase independent
mechanism leading to hyperpolarization of the
cells and subsequently causes vasodilatation.
Other factor like endothelin, which is a potent
endothelium derived vasoconstrictor, endothelin
has got positive inotrophic and chronotropic
action on the heart and also contributes to
remodeling in the cardiovascular system. Different
isoforms ET-1, ET-2, ET-3 have been identified.
The ET-1 is secreted from the endothelial cells
which is mainly involved in cardiovascular
actions as paracrine and autocrine locally. ET-1
concentration is very high in the vascular area
because it is secreted on the basal side of the
endothelial cells. Endothelins act through several
G protein couple receptors like ET-A and ET-B.
ET-A receptor on vascular smooth cells mediate
vasoconstriction whereas ET-B located pre-
dominantly on vascular endothelial cells where
they mediate vasodilatation via release of pro-
stacyclin mediate vasoconstriction. Thus endo-
thelins produce vasoconstriction by acting
through ET-A and ET-B receptors in the vascular
smooth muscle cells and vasodilation acting
through ET-B receptors in the endothelium. Other
mechanism involved are neurohormonal mecha-
nisms of adrenaline and NA influence vascular
tone alpha-1 and beta-2 receptors on vascular
smooth muscle cells.
Besides this, (ADH) secreted in response to
angiotensin-II affects the vascular tone by acting
on its 3 V types of receptors. This mechanism is
involved to maintain blood flow at constant level
and also to maintain vascular tone, so that blood
flow is alter by different metabolites like H+, CO2,
O2, adenosine, lactate, K+, etc. mostly in local
tissue. So, local mechanism of vascular tone
regulation is predominant in the vascular beds of
essential organs which help in maintenance of
blood flow and oxygen supply as per demand of
local metabolism in these organs.
Role of natriuretic peptides in regulation of
blood pressure: Atria and other tissue of mammals
Cardiovascular System  245
contain family of peptides with natriuretic diuretic,
vasorelaxant and other properties. These consist
of atrial natriuretic peptide (ANP), brain natriuretic
peptide (BNP), and C-type natriuretic peptide. ANP,
a 28 amino acid peptide is synthesized in cardiac
atrial cells but insignificant amount are also
synthesized in ventricular cells. It is also
synthesized in the CNS and peripheral nervous
system and in lungs. Several factors which increase
the release of ANP from the heart are atrial stretch
via mechanosensitive ion channels, blood volume
expansion, head out water emersion changing from
standing to supine position and exercise. Besides
this ANP participates in physiological regulation
of sodium excretion and blood pressure, e.g.
suppression of ANP production or blockade of its
action impairs the natriuretic response to volume
expansion and increase blood pressure.
Exercise and Blood Pressure
Exercise is a planned, structured and repetitive
bodily movement done to improve or maintain
one or more components of physical fitness.
Physical fitness includes several components:
cardiorespiratory, muscular strength, flexibility,
endurance and body composition. Exercise can
be divided into four types:
1. Acute and chronic
Acute exercise relates the physiological
responses that occur with a single bout of
exercise. Chronic exercise describes person
familiar with repeated bouts of physical
training.
2. Aerobic and anaerobic
Aerobic exercise consists of repetitive low-
resistance movements that last over a long
period of time, usually more than 10 minutes,
such as walking or cycling or it can be
described as exercise of muscle movement that
uses oxygen to burn both carbohydrates and
fats to produce energy. Anaerobic exercise, on
the other hand, consists of high-resistance,
low-repetitive movements that last only 1-3
minutes, interrupted by frequent periods of
rest between exercise bouts for example weight
lifting, push-ups and sit-ups, etc. or simply it
246  Practical Manual of Experimental and Clinical Pharmacology
is an exercise, where muscle movement does
not require oxygen and only burns carbo-
hydrates to produce energy. This builds up
muscles and gives physical strength through
short bursts of strenuous activity. So an ideal
exercise program should include both aerobic
and anaerobic exercises.
3. Isotonic and isometric
Isotonic exercise is explained as when a muscle
contracts and temporarily shortens with
movement of attached joint. And isometric
exercise can be described when the muscle
contracts against a fixed resistance. So, in
isometric exercise, there is no muscle shor-
tening or joint movement, e.g. pushing against
a wall, trying to a lift a weight, etc.
4. Maximal and submaximal
Maximal exercise testing is considered gold
standard but as compared to maximal exercise
testing submaximal exercise test has greater
applicability in clinical practice (Table 26.2).
These tests are used in order to predict
maximal aerobic capacity. Application of these
tests is selectively based on specific indica-
tions: like person with limited capacity
because of cardiopulmonary, musculoskeletal
and neuromuscular impairments with diffi-
culty during exertion, dyspnea, fatigue,
weakness and pain during daily activities. So,
Table 26.2: Examples of different Submaximal exercises
Predictive submaximal test
1. Modified Bruce treadmill test
2. Single-stage submaximal treadmill walking test
(SSTWT)
3. Astrand and Rhyming (A-R) cycle ergometer test
4. Canadian aerobic fitness test (CAFT) and modified
CAFT (MCAFT)
5. 1- Mile tract walking test (Rockport fitness test, 1-
MTW)
Performance submaximal test
1. Self paced walking test (SPWT)
2. Modified shuttle waking test (MSWT)
3. Bag and carry test (BCT)
4. Time up and go test (TUGT)
5. 12 minute walk test (12-MWT)
6. 6 minute walk test (6-MWT)
goal of submaximal testing is to produce
adequate level of exercise stress without
physiological and biological strain. Maximal
exercise is useful for VO 2 determination,
diagnosis and to test the treatment outcome,
whereas submaximal exercise is applicable to
predict VO2 max, to assess the effect of an
intervention in the exercise schedule, and to
observe the recovery strategies after the
exercise.
The American College of Sports Medicine
characterizes aerobic exercise intensity as low,
moderate or high based on elicited rise in heart
rate. Predicted Maximum Heart Rate (PMHR) is
calculated by the following formula, PMHR = 220
- Age in years. An exercise is categorized as low-
intensity if it elicits 35 to 59% of predicted
maximum heart rate and moderate-intensity,
which elicits 60 to 79% of PMHR. Exercise eliciting
a response greater than this is considered high-
intensity.
Metabolic equivalents (METs) units are usually
used to estimate the oxygen consumption,
following a specific physical activity. One MET
indicates energy cost of sitting quietly. Depending
on physical activity, it varies for example person
engaged in vigorous activity, his or her oxygen
demands will increase, and so do the METs
assigned to that activity. Several examples such as
activities like walking slowly, yoga and housework
use about 3 METs, fast walking and doubles tennis
use 3 to 6, and jogging, shoveling snow, and singles
tennis use more than 6. So, in day to day activity,
typical person uses about 3.5 milliliters of oxygen
per kg of body weight per minute. Aerobic exercise
intensity can also be categorized based on energy
equivalents, measured in METs. One MET is the
energy spent at rest to maintain basal bodily
functions. (1 MET = 3.5 ml O2/kg/min). An activity
or exercise requiring less than 3 METs is considered
light-intensity, that requires 3-6 METs is considered
as moderate intensity and those exercises which
demand more than 6 METs are called high-
intensity exercises.
Several studies have consistently reported that
regularly performed aerobic exercise of mild-to-

moderate intensity lowers blood pressure and it
is beneficial for patients with essential hyper-
tension. Regular aerobic exercise lowers blood
pressure as it involves various physiological
mechanisms like decreased sympathetic system
activity alone with potentiating of the baroreceptor
reflex and reduced arterial stiffness with enhanced
total systemic arterial compliance. It also helps in
increased release of endothelium-derived nitric
oxide which is probably related to lower plasma
cholesterol and increased insulin sensitivity.
Studies show that reduction in blood pressure
with regular aerobic exercise is approximately 8
to 10 mm Hg for systolic and 7 to 8 mm Hg for
diastolic blood pressure in hypertensive patients.
Several studies have consistently shown that
regularly performed aerobic exercise of mild-to-
moderate intensity lowers blood pressure in
patients with essential hypertension. The exercise
is recommended and it is convenient to remember
the anagram FITT: Frequency (F), the intensity (I),
the time (T) (or duration), and the type (T) or
exercise. Of the four components of the FITT
anagram, the intensity of exercise is by far the most
important. Most of the skill in prescribing exercise
is related to managing the intensity component.
BLOOD PRESSURE GUIDELINES
(TABLE 26.3)
Hypertension is the commonest cardiovascular
disease all over the world. Studies have reported
that about 25% of world population aged more
than 30 years suffer from hypertension. Presently,
Table 26.3: Blood pressure and different guidelines
Classification JNC* 7
<120/80 Normal
120-129/80-84 Pre-hypertensive
130-139/85-89
140-159/90-99 Stage I
160-179/100-109 Stage II
> 180/110
ISH$ G-1 SBP > 140
DBP < 90
ISH$ G-II
* JNC 7: Joint National Committee on Prevention, Detection, Evaluation, and Treatment of High Blood Pressure
#
BHS (IV): British Hypertensive Society, $ ISH International Society of Hypertension
Cardiovascular System  247
there are several guidelines available for manage-
ment of hypertension: WHO-International Society
of Hypertension (WHO-ISH), the Joint National
Committee (JNC VII) from USA, British Hyper-
tensive Society (BHS IV), Australian, Japanese and
Indian guidelines. All these guidelines provide
the guidance to practitioner based on statistical
data of particular country’s population. The
Indian guideline was released in 2000 and revised
in 2007. The Indian guideline is a joint guideline
of Hypertensive Society of India, Cardiological
Society of India, Indian Medical Association and
Association of Physicians of India (API). Most of
the international guideline updated every 4-5
years for example British Hypertensive Society
Guidelines were revised in 1989, 1993, 1999 and
2004. Though, there are several guidelines
available but there is no uniformity on cut off
levels of all the guidelines. Recently, optimal blood
pressure is considered less than 120/80 mm Hg.
So, normal blood pressure have been accepted in
most of the guidelines is systolic 120-129 and
diastolic is 80-84 mm Hg. There is also category
included in the guideline like high normal which
corresponds to systolic 130-139 and diastolic
85-89 mm Hg e.g. in British Hypertensive Society
and Indian Guideline. Similarly, JNC VII used
term as pre hypertension, mostly JNC VII catego-
rizes as stage I and stage II hypertension but
Indian guideline described as stage I, II, and III
hypertension which is similar to JNC VI guideline.
Most of the guidelines recommend use of mercury
BHS# IV Indian HTN Guidelines II
Optimal Optimal
Normal Normal
High normal High normal
Stage I Stage I
Stage II Stage II
Stage III Stage III
SBP 140-159 SBP 140-159
DBP < 90 DBP < 90
SBP > 160 SBP > 160
DBP < 90 DBP < 90
248  Practical Manual of Experimental and Clinical Pharmacology
sphygmomanometer as a gold standard. Besides,
this Indian guideline also stresses on daily intake
of salts, alcohol abstinence and described
metabolic syndromes as per our population.
SUGGESTED READING
1. Carney SL, Gillies AH, Green SL, Paterson O, Taylor
MS, Smith AJ. Hospital blood pressure measurement:
Staff and device assessment. J Qual Clin Pract 1999;
19(2):95-98.
2. Carney SL, Gillies AH, Smith AJ, Smitham S. Hospital
sphygmomanometer use: an audit. J Qual Clin Pract.
1995;15(1):17-22.
3. Coleman A, Freeman P, Steel S, Shennan A. Validation
of the Omron 705IT (HEM-759-E) oscillometric blood
pressure monitoring device according to the British
Hypertension Society protocol. Blood Press Monit
2006;11(1):27-32.
4. Coleman AJ, Steel SD, Ashworth M, Vowler SL,
Shennan A. Accuracy of the pressure scale of
sphygmomanometers in clinical use within primary
care. Blood Press Monit 2005;10(4):181-88.
5. Dahloff B, Lindholm LH, Hansson L, Schersten B,
Ekbom T,Wester PO. Morbidity and mortality in the
Swedish Trial in Old Patients with Hypertension
(STOP-Hypertension). Lancet 1991;394:405-12.
6. De Swiet M, Dillon MJ, Little W, O’Brien E, Padfield
PL, Petrie JC. Measurement of blood pressure in
children. Recommendations of a working party of
the British Hypertension Society. BMJ 1989;299:497.
7. De Swiet M, Dillon MJ, Little W, O’Brien E, Padfield
PL, Petrie JC. Measurement of blood pressure in
children. Recommendations of a working party of
the British Hypertension Society. BMJ 1989;299:497.
8. Joffres MR, Hamet P, Rabkin SW, Gelskey D, Hogan
K, Fodor G. Prevalence, control and awareness of
high blood pressure among Canadian adults.
Canadian Heart Health Surveys Research Group. Can
Med Assoc J 1992;146(11):1997-2005.
9. Marshall T, Rouse A. Blood pressure measurement.
Doctors who cannot calibrate sphygmomanometers
should stop taking blood pressures. BMJ 2001;
323(7316):806.
10. O’Brien E, Atkins N. A comparison of the British
Hypertension Society and Association for the
Advancement of Medical Instrumentation protocols
for validating blood pressure measuring devices: Can
the two be reconciled? J Hypertens 1994;12:1089-94.
11. O’Brien E, Pickering T, Asmar R, Myers M, Parati G,
Staessen J, Mengden T, Imai Y, Waeber B, Palatini P,
Gerin W. Working Group on Blood Pressure
Monitoring of the European Society of Hypertension
International Protocol for validation of blood pressure
measuring devices in adults. Blood Press Monit 2002;
7(1):3-17.
12. O’Brien E. A century of confusion: Which bladder
for accurate blood pressure measurement? J Hum
Hypertension 1996;10:565-72.
13. O’Brien E. A century of confusion: Which bladder
for accurate blood pressure measurement? J Hum
Hypertension 1996;10:565-72.
14. Penaz J. Photoelectric measurement of blood
pressure, volume and flow in the finger. Digest Tenth
International Conference Medical Biological
Engi-neering. Dresden; 1973:104.
15. Pickering TG, Hall JE, Appel LJ, Falkner BE, Graves
J, Hill MN, Jones DW, Kurtz T, Sheps SG, Roccella
EJ. Recommendations for blood pressure
measure-ment in humans and experimental animals:
part 1: blood pressure measurement in humans: A
statement for professionals from the Subcommittee
of Professional and Public Education of the American
Heart Association Council on High Blood Pressure
Research. Circulation 2005;111(5):697-16.
16. Pickering TG. Reflections in hypertension. How
should blood pressure be measured during
preg-nancy? J Clin Hypertens (Greenwich) 2005;7(1):
46-49.
17. Shah NC, Sibbritt DW, Heaney S, Sharples J.
Sphyg-momanometers—an audit in general practice.
Aust Fam Physician 2004;33(11):952-54.
18. Turner MJ, Baker AB, Kam PC. Effects of systematic
errors in blood pressure measurements on the
diagnosis of hypertension. Blood Press Monit 2004;
9(5):249-53.
19. Turner MJ, Irwig L, Bune AJ, Kam PC, Baker AB.
Lack of sphygmomanometer calibration causes over-
and under-detection of hypertension: A computer
simulation study. J Hypertens 2006;24(10):1931-38.
20. van Popele NM, Bos WJ, de Beer NA, van Der Kuip
DA, Hofman A, Grobbee DE, Witteman JC. Arterial
stiffness as underlying mechanism of disagreement
between an oscillometric blood pressure monitor and
a sphygmomanometer. Hypertension 2000;36(4):
484-88.
Regulation of Blood Pressure
21. Danpney RAL. Central mechanism underlying short
and long-term regulation of the cardiovascular
system. Clinical Exp Pharmacol Physiol 2002;29:
261-68.
22. Dempsey JA, Sheel AW, St Croix CM, Morgan BJ.
Respiratory influences on sympathetic vasomotor
outflow in humans. Respire Physiol Neurobiol 2002;
130:3-20.

23. Di bona GF, Kopp Uc. Neural control of renal
function. Physiol Rev 1997;77:75-197.
24. Fruchgott RF. Endothelium derived relaxing factor
discovery early studies and identification as NO.
Bioscci Rep 1999;19:235.
25. Galie N, Manes A, Branzi A. The endothelin system
in pulmonary arterial hypertension. Cardiovascular
Reseasch 2004;61:227.
26. Godfraind T, Kaba A. Role of calcium in the action
of drug on vascular smooth muscle. Arch Int
Pharmacodyn Ther 1972;196;S35-S49.
27. Guo ZL, Lai HC Lonhust HC. Medullar pathways
involved in cardiac symphato excitatory reflexes in
the cat. Brain Res 2002;925:55-66.
28. Guyenet PG. The sympathetic control of blood
pressure. Nat Rev Neurosci 2006;7:335-46.
29. Higashi Y, Yoshizumi M. Exercise and endothelial
function; role of endothelium derived nitric oxide
and oxidative stress in healthy subject and hyper-
tensive patients. Pharmacotherapy 2004; 102:87-96.
30. Liu KL. Regulation of renal medularry circulation by
rennin angiotensin system in genetically hypertensive
rats. Clin Exp Pharmacol Physiol 2009 Feb 10.
31. Lohmeier TE. The symphatetic nervous system and
long-term blood pressure regulation. Am J hyper-
tension 2001;14:147-54.
32. Pilowsky PM, Good child AK. Baroreceptor reflex
pathways and neurotransmitters 10-year on J on
hypertens 2006;20;1675-88.
33. Schreihofer AM, Guyenet PG. The baroreflex and
beyond control of symphatetic vasomotor tone by
GABAergic neurons in the venterolateral medulla.
Clin Exp Pharmacol Physiol 2002;29:514-21.
34. Vallbo AB, Harbarth KE, Wallin BG. micro-
neuerography; how the technique developed and its
role in the investigation of the fore the symphatetic
nervous system. J Appl Phsiol 2004: 96;1262-69.
Exercise and Blood Pressure
35. American College of Sports Medicine, Guidelines for
Exercise Testing and Prescriptions, Ed 6. Baltimore,
Lippincott Williams and Wilkins 2000;150.
36. American College of Sports Medicine. Exercise and
hypertension. Med Sci Sports Exerc 2004;34:533-53.
37. Bavikati VV, Sperling LS, Salmon RD, et al. Effect of
Comprehensive Therapeutic Lifestyle Changes in
Prehypertension. Am J Cardiol 2008;102:1677-80.
38. Church T, Earnest C, Skinner J, et al. Effects of
different doses of physical activity on cardio-
respiratory fitness among sedentary, overweight or
obese post-menopausal women with elevated blood
pressure: A randomized controlled trial. J Am Med
Cardiovascular System  249
Assoc 2007;297:2081-91.
39. Foster C, Cadwell K, Crenshaw B, et al. Physical
activity and exercise training prescriptions for
patients. Cardiol Clin 2001;19:447-57.
40. Fu Qi, Vongpatanasin W, Levine BD. Neural and
nonneural Mechanisms for Sex Differences in Elderly
Hypertension – Can Exercise Training Help?
Hypertension 2008;52:787-94.
41. Kokkinos PF, Narayan P, Papademetriou V. Exercise
as Hypertension Therapy: Cardiol Clin 2001;19:
507-16.
42. Labarthe D, Ayala C. Nondrug interventions in
hypertension prevention and control. Cardiol Clin
2002;20:249-63.
43. Vanessa N, Elizabeth D. Submaximal exercise
testing: Clinical application and interpreatation.
Physical Therapy 2000;80(8):782-807.
Blood Pressure Guideline
44. Guide to management of hypertension 2008. http://
www.heartfoundation.org.au/Professional_Information/
Clinical_Practice/Hypertension.htm
45. Guidelines for management of hypertension: Report
of the fourth working party of the British Hyper-
tension Society, 2004-BHS IV.B Williams, NR Poulter,
MJ Brown et al. Journal of Human Hypertension
2004;18:139-85.
46. Guidelines for the Management of Arterial
Hyper-tension. The Task Force for the Management
of Arterial Hypertension of the European Society of
Hypertension (ESH) and of the European. Society of
Cardiology (ESC). Authors/Task Force Members:
Giuseppe Mancia, Co-Chairperson (Italy), Guy de
Backer, Co- Chairperson(Belgium), Anna Domi-
niczak (UK), Renata Cifkova (Czech Republic),
Robert Fagard ( Belgium), Giuseppe Germano (Italy),
Anthony m. Heagerty (UK), Sverre E. Kjeldsen
(Norway), Stephane Laurent (France), Krzysztof
Narkiewicz (Poland), Luis Ruilope (Spain), Andrzej
Rynkiewicz (Poland), Roland E. Schmieder (Ger-
many), Harry A.J. Struijker Boudier (Nether-lands),
Alberto Zanchetti (Italy). Journal of Hypertension
2007;25:1105-1187.
47. Indian hypertension guidelines II. http://www.
apiindia.org/hypertensioguidelines/hyperhome.htm
48. The Seventh Report of the Joint National Committee
on Prevention, Detection, Evaluation and Treatment
of High Blood Pressure US Department of Health
and Human Services. National Institutes of Health.
National Heart, Lung and Blood Institute. http://
www.nhlbi.nih.gov/guidelines/hypertension/jnc7full.pdf
250  Practical Manual of Experimental and Clinical Pharmacology
49. World Health Organization, International Society of
Hypertension Writing Group. Journal of Hypertension
2003;21:1983-92.
CVS EXPERIMENT: 1
Aim
To measure blood pressure in healthy volunteers.
Background
Blood pressure is the force exerted by the blood
against a unit area of the vessel wall. For
example, if pressure is 100 mm Hg, this means
that the force, exerted is sufficient to push a
column of mercury up to a level of 100 mm
height. Occasionally, pressure is measured in
cm of water. One mm of Hg equals 1.36 cm of
water because the specific gravity of mercury is
13.6. The blood pressure in the large arteries
(Aorta, brachial and others) in the young adult
rises to a peak value (systolic pressure) of about
120 mm of Hg, during each cycle and falls to a
maximum value (diastolic) of about 70 mm of
Hg. The pulse pressure (PP), the difference
between SBP and DBP is normally about 40-50
mm Hg. The mean arterial pressure is the average
pressure throughout the cardiac cycle and is
calculated by
1 1
DBP + PP or (SBP + 2DBP)
3 3
The pressure in vessel below heart level is
increased (2 mm Hg at every inch) and that in
vessel above heart is decreased (2 mm Hg at every
inch) by the effect of gravity. The magnitude of the
gravitational effect, the product of the density of
blood, the acceleration due to gravity (980 cm/
sec2) and the vertical distance above and below
the heart is 0.77 mm Hg/cm at the density of
normal blood. The pressure in large artery in foot
(105 cm below the heart is 180 mm of Hg (100 +
{0.77 × 105}).
Materials and Methods
• Healthy adult volunteers (Willing to give
written informed consent)
• Stethoscope, Mercury sphygmomanometer,
Facilities for Clinical Pharmacology Labora-
tory (Appendix IV).
Initial Screening of Volunteers
Volunteer is screened for detailed evaluation of
any disease conditions.
Detailed history should be taken in respect to
smoking, alcohol consumption, drug therapy, or
any associated disease conditions.
Precautions during Experiment
• Subject should be advised for adequate sleep
over night and light breakfast in the morning
on the day of experiment (preferably 2 hr
before)
• Avoid caffeinated drink or smoking at least
2 hr before the experiment
• Take 2 or 3 readings, preferably take 2 readings
at the time of measurement
• Volunteer should have a comfortable room,
sitting in a chair or lying quietly for at least
5-10 min, with the arm unconstricted.
• Avoid (or if unavoidable, make a note of it)
extraneous factors, which may alter the BP.
For example: Recent smoking/eating, anxiety,
talking, exercise/exertion, cold medication
like, estrogen, corticosteroid, adernergics such
as nasal, drops, pentazocine, pupillary
dilators, phenylephrine, etc.
• Note the time of the day (preferably blood
pressure should be taken at the same time of
the day)
• Width of cuff should cover at least 40% of
upper arm
• Inflatable balloon should cover 80% of the arm
circumference
• Place the cuff 1.5-2 cm above the antecubital
fossa
• Record the exact blood pressure, do not round
off
 Cardiovascular System  251

Source of potential errors (Korotkoff V). However, in adults after exercise

and in children, the diastolic blood pressure
1. Improper equipment
a. Cuff too small/large for length of the arm correlates best with the pressure at which the
b. Inflatable balloon small for arm circumference sound becomes muffled (Korotkoff IV). The
c. Manometer is inaccurate sound of Korotkoff is produced by turbulent
2. Inaccurate reading flow in the brachial artery.
a. Improper placement of cuff
b. Error in value, missing an auscultatory gap or Note: It is always preferred practice to perform
confusion over muffling/disappearance of sound Palpatory method before Auscultatory method. It
c. Variation due to arrhythmia gives accurate blood pressure and heart rate
d. Position of arm not at level of heart
e. Arm held without support
monitoring and reduce variability.
Observer bias may occur with single reading.
Method of Measuring Blood Pressure
Observations and Result
1. Palpatory method: The systolic blood pressure
SBP DBP HR
can be determined by inflating an arm cuff and
letting the pressure fall and determining the 1st 2nd 3rd Mean 1st 2nd 3rd Mean
pressure at which the radial pulse first become
palpable. Pressure obtained by this method is
usually 2.5 mm Hg lower than those measured
by the auscultatory method. The importance
of this method is in avoiding the error in
measurement due to auscultatory gap seen in
some subjects.
2. Auscultatory method: Routinely used method
for blood pressure measurement. An inflatable Discussion
cuff (Riva-Rocci cuff) attached to a mercury The measurement of blood pressure is the
manometer (sphygmomanometer) is wrapped important clinical parameter which provides
around the arm and the stethoscope is placed
important information about cardiovascular
over the brachial artery at the elbow (cubital
system under normal and disease conditions.
fossa). The cuff is rapidly inflated until the
Though, there are several instruments available
pressure in it is well above the systolic pressure
but mercury sphygmomanometer is considered
in the brachial artery. The artery is occluded
as ideal one and palpatory method should be
by the cuff pressure and no sound is heard
practiced always before auscultatory mea-
with the stethoscope. The pressure in the cuff is
surement. Blood pressure should be measured in
then released slowly (2-3 mm Hg/sec). The point
both the arms and arm with highest reading
at which systolic pressure in the artery just
should be considered for recording. The normal
exceeds the cuff pressure, a spurt of blood
ratio of SBP, DBP and PP is 3:2:1. Blood pressure
passes through with each heart beat and
synchronically with each beat a tapping sound is usually lower in females by 8-10 mmHg, over
is heard below the cuff. The cuff pressure at weight person tend to have increased blood
which sounds are just heard is the SBP. As the pressure.
cuff pressure is lowered further, the sound
becomes louder dull and muffled, and finally SUGGESTED READING
in most individuals, it disappears. These are 1. De Swiet M, Dillon MJ, Little W, O’Brien E, Padfield
the sounds of Korotkoff. The diastolic blood PL, Petrie JC. Measurement of blood pressure in
pressure in resting adults correlates best with children. Recommendations of a working party of
the pressure at which the sounds disappear the British Hypertension Society. BMJ 1989;299:497.
252  Practical Manual of Experimental and Clinical Pharmacology
2. Marshall T, Rouse A. Blood pressure measurement.
Doctors who cannot calibrate sphygmomanometers
should stop taking blood pressures. BMJ 2001;
323(7316):806.
3. O’Brien E. A century of confusion: Which bladder
for accurate blood pressure measurement? J Hum
Hypertension 1996;10:565-72.
4. O’Brien E. A century of confusion: which bladder for
accurate blood pressure measurement? J Hum
Hypertension 1996;10:565-72.
5. Turner MJ, Baker AB, Kam PC. Effects of systematic
errors in blood pressure measurements on the
diagnosis of hypertension. Blood Press Monit 2004;
9(5):249-53.
CVS EXPERIMENT: 2
Aim
To evaluate chronobiology of blood pressure in
healthy volunteers.
Background
In any biological system, there is rhythm and
cycle of biological events. The blood pressure also
shows a circadian rhythm with release of
different hormones in the body. Molecular
studies suggest that genetic inheritance was
influenced from the environment during the
course of evolution. Hence, the blood pressure
and heart rate may vary with change in
the internal or external environment of the
body. The chronodiagnosis of a patient
provides the accurate measurement of the
HR and BP whereas one time measurement
may give the false blood pressure or heart rate
value. (Situations like, elder patients, stress,
anxiety, eating, exercise, etc.)
Materials and Method
• Healthy adult volunteers (willing to give
written informed consent)
• Stethoscope, mercury sphygmomanometer,
Facilities for Clinical Pharmacology Labo-
ratory (Appendix IV).
Screening of Volunteers
Volunteers should be screened for detailed
evaluation of any disease conditions, detailed
history should be taken in respect to smoking,
alcohol consumption, drug therapy, or any
associated disease condition.
Precautions during Experiment
Subject should be advised for adequate sleep
over night and light breakfast in the morning
on the day of experiment (preferably 2 hr before)
Any caffeinated drink or smoking at least 2 hr
before the experiment should be prohibited.
Method
Mercury sphygmomanometer is considered to be
the standard of all the other available methods.
Procedure
The blood pressure is monitored in a healthy
volunteer over a period of 24 hours at 2 hours
interval. The volunteer should have a normal sleep
over night and is allowed to do his daily routine
activities. At the time of each recording the subject
is rested in a supine position for 5 min, and then
readings are taken. Start the experiment at the
specified time (preferably at 8.00 am and then
record every 2 hr till 8.00 am, the next day).
 Cardiovascular System  253

Observations
Time (hr) Systolic blood pressure Diastolic blood pressure Heart rate (HR)
(SBP)(mm Hg) (DBP)(mm Hg)
0 (preferable
at 8:00 am)
2
4
6
8
10
12
14
16
18
20
22
24

Observe the time at which blood pressure
shows highest and lowest recordings of SBP, DBP
and monitor the heart rate.
Represent the result, as it given in Figure 26.7.
Statistical Analysis
The mean of the all 12 readings should be recorded
and data should be expressed as mean + SD.
Discussion
The Blood pressure is low under basal conditions
and it achieves peak in the late afternoon, mostly
systolic blood pressure and it is significantly low
while sleeping. As more frequent readings over
long-time have become available, the marked
individual variability of the blood pressure has
become apparent. The blood pressure taken with
an intra-arterial device continuously over 24 hours
in 20 untreated ambulant hypertensives showed
highest value in the mid morning, a progressive
fall during the day and much lower recording
during sleep. The readings varied as much as by
40 mm Hg throughout 24 hour. There is a marked
rise in the blood pressure upon awakening,
although plasma noradrenaline levels are lower
during sleep, but plasma cortisol and aldosterone
levels rise during the early morning. The rise in
systolic and diastolic blood pressure immediately
upon awakening may be as high as 20 mm Hg. A
further rise occurs upon arising and ambulation.
During period of REM sleep the blood pressure
tends to rise a bit and becomes variable.

SUGGESTED READING
1. Kawasaki T, Cugini P, Di Palma L. Chronobiology
approach to human hypertension. Ann Ist Super
Sanita 1993;29(4):679-92.
2. Cugini P, Kavasaki T, Palma LD, et al. Blood pressure
monitoring and chronobiometry: New reference
standard and definitions concerning norm tension
and hypertension. J Health Sci 1989;11:145-62.
3. Cugini P, Kavasaki T, Palma LD, et al. Innovative
Fig. 26.7: Chronobiology of volunteer blood pressure criteria for diagnosing arterial hypertension via blood
254  Practical Manual of Experimental and Clinical Pharmacology
pressure monitoring: The chronodiagnosis. J Health
Sci 1991;13:23-34.
4. Kudzhini P, Kavasaki T, Palma LD, Vatisti P,
Antonikoli S, Leone D, Uezono K, Sto-onev AG.
Circadian rhythm of arterial pressure: Chrono-
biological criteria for normotension and hypertension.
Fiziol Cheloveka 1991;17(4):73-79.
5. Cugini P, Lucia P, Di Palma L, Pozzilli P, Re M,
Canova R, Gasbarrone L, Cianetti A. Vasoactive
intestinal peptide: A chronoimmunomodulator?
Biochem Med Metab Biol 1991;46(2):274-76.
6. Cugini P, Di Palma L, Battisti P, Coppola A, Leone
G, Cresci G, Bruscagli G, Lembo T, Angeloni A,
Pelosio A. 24-hour blood pressure: Noninvasive
monitoring and biometric analysis in relation to age.
Recenti Prog Med 1991;82(9):463-74.
7. Cugini P, Cresci G, Di Palma L, Bruscagli G, Battisti
P, Lembo T, Angeloni A, Coppola A, Leone G, Pelosio
A. Recenti Prog Med 1991;82(9):452-62.
8. Cugini P, Lucia P, Di Palma L, Re M, Leone G, Battisti
P, Canova R, Gasbarrone L, Cianetti A. Vasoactive
intestinal peptide fluctuates in human blood with a
circadian rhythm. Regul Pept 1991;34(3):141-48.
9. Cugini P, Lucia P, Di Palma L, Pozzilli P, Re M,
Canova R, Gasbarrone L, Cianetti A. Temporal
interrelationships between circadian rhythms of
vasoactive intestinal peptide and T lymphocyte
subpopulations. J Clin Lab Immunol 1991;34(2):
49-54.
10. Kawasaki T, Cugini P, Uezono K, Sasaki H, Itoh K,
Nishiura M, Shinkawa K. Circadian variations of
total renin, active renin, plasma renin activity and
plasma aldosterone in clinically healthy young
subjects. Horm Metab Res 1990;22(12):636-39.
CVS EXPERIMENT: 3
Aim
To evaluate the effect of body posture and arm
position on arterial blood pressure and heart rate.
Background
Several studies suggest that the change in the body
posture can alter blood pressure to rise or fall.
Ideally, recording of the blood pressure is done in
supine or in normal relaxed sitting position.
However, diastolic pressure varies about 5 mm
Hg in sitting than the supine position whereas
systolic pressure remains same. When the arm
cuff position is at the level of the right atrium in
both positions, the systolic pressure has been
reported to be 8 mm Hg higher in the supine than
the upright position. Other posture like crossing
of leg may raise systolic pressure. The position of
the arm in respect to the heart level may also vary
the BP measurements. There is a progressive
increase in the pressure of about 5 mm Hg, when
arm position is higher than the heart position,
whereas about 5 mm Hg may fall when the arm
position is below the heart level.
Body Posture (Also see Figs 26.3 to 26.5)
Supine position
Sitting position
Other various positions:
• Sitting with crossed leg
• Standing with hand position below the heart
level.
• Standing with hand position at the heart level.
• Standing with hand position above the heart
level.
Materials and Method
• Healthy adult volunteers (willing to give
written informed consent)
• Stethoscope, Mercury sphygmomanometer,
Facilities for Clinical Pharmacology Labo-
ratory (Appendix IV).
Initial Screening of Volunteers
Subject is screened for detailed evaluation of any
disease conditions, detailed history should be
taken in respect to smoking, alcohol con-
sumption, drug therapy, or any associated
disease conditions.
Precautions during Experiment
Subject should be advised for adequate sleep over
night and light breakfast in the morning day of
experiments (preferably 2 hr before the experiment).
Any caffeinated drink, alcohol, cola drinks or
smoking should be avoided at least 2 hr before the
experiment.

Procedure
Volunteers with history of alcohol consumption
on previous day, smoking, drug addiction should
be excluded. Those taking any drug especially
affecting blood pressure are excluded. Preferred
age is 18-35 years. About 2 hr after a light breakfast
at morning, volunteer are advised to report to the
clinical pharmacology laboratory. Volunteer is
asked to lie comfortably for 15 min, after applying
the blood pressure cuff, BP should be recorded on
both upper arms and the arm which shows higher
blood pressure is selected for subsequent
measurement. Blood pressure should be recorded
Observation and Results
Body posture SBP
1st 2nd 3rd
Supine
Normal sitting
Sitting with cross leg
Standing with hand position
below the level of heart
Standing with hand position
at the level of heart
Standing with hand position
above the level of heart
Discussion
BP and HR usually increase when the volunteer
is standing by keeping hands at the heart level.
With hands upward BP is low but HR high. In the
sitting position, both BP and HR give intermediate
results. When the volunteers stand up, there is
pooling of blood in the dependant position, thus
there is decreased venous return. This leads to
lesser stretching of baroreceptors, sympathetic
activity increases and there is increase in blood
pressure and heart rate due to withdrawal of
parasympathetic baroreceptor activity.
Baroreceptor is located at the carotid sinus and
aortic arch. When the volunteer stands up, from
sitting position pooling of blood occurs in the
lower limb. While holding the arm hanging by
Cardiovascular System  255
twice or thrice and the average should be taken
for accuracy.
After 15 min of rest in supine position, BP
should be recorded. Then, volunteers are asked to
stand up immediately and BP at 1 and 3 min are
recorded for assessment of postural hypotension.
The volunteer is then asked to stand up for 5 min
with hand up and BP is recorded. It is again
measured with hand by the side and hand
hanging after 5 min each. Finally, volunteer is
asked to sit down and BP is recorded after 5 min.
If, there is fall in BP, it gives the evidence of postural
hypotension, and if present, volunteer should be
excluded.
DBP HR
Mean 1st 2nd 3rd Mean
the side, pooling occurs in the upper limb still
further. Hence, these situation show increase in
blood pressure and heart rate. When volunteer
stands up with arm raised, there is better venous
return, no pooling of blood in the upper limb and
thus less (lower) sympathetic activity manifesting
a lower blood pressure and heart rate.
SUGGESTED READING
1. Brar KS, Ramesh. Technique of blood pressure
measurement. MJAFI 2003;59:51-52.
2. Pickering TG, Hall JE, Appel LJ, Falkner BE, Graves
J, Hill MN, Jones DW, Kurtz T, Sheps SG, Roccella
EJ. Recommendations for blood pressure mea-
surement in humans and experimental animals: Part
1: blood pressure measurement in humans: A
statement for professionals from the Subcommittee
256  Practical Manual of Experimental and Clinical Pharmacology
of Professional and Public Education of the American
Heart Association Council on High Blood Pressure
Research. Hypertension 2005;45(1):142-61.
CVS EXPERIMENT: 4
Aim
To evaluate the effect of propranolol on blood
pressure, heart rate and cardiac workload
following different submaximal exercises (Tread
mill test [TMT], Master’s 2 step test, Bicycle ergo
meter and Hand Dynamometer) in healthy
volunteers.
Background
It depends on the validity, reliability, and
sensitivity of the instrument and on the objectives
of submaximal exercise, i.e. to induce a state of the
stress through exercise at which there is no
alteration of the biological and physiological
parameters. Clinically submaximal exercise
testing appears to have greater applicability to
identify the various diseases like arrhythmia,
angina, asthma, etc. There are two main categories
of submaximal tests, one is predictive, which
mainly identifies maximal aerobic capacity and
second is performance test which is done to identify
physical capability of volunteers or patients. Sub-
maximal exercise tests can be used to predict
VO2max, to make diagnosis and assess functional
limitations, to assess the outcome of interventions
such as exercise programs whereas maximal
exercise tests are mainly used to determine VO2max
which is used as diagnostic or treatment outcome
tools. At the laboratory basis, there are several
instruments through which experimenter can
determine the BP, HR, RPP and RR of a healthy
volunteer. Those are treadmill test, Master’s 2 test,
Bicycle ergometer and hand dynamometer. These
instruments were developed to meet the needs of
people with various functional limitations,
disabilities and requirements of older, obese
population. Wrong selection of protocol may
under stress or over stress the subject. In the
practical clinical pharmacology, the selection is not
so important because of the participation of
healthy volunteers (aged 21-50 years) but in the
clinical setting selection criteria is important and
limiting factor which depends on the several
factors include cognitive status, age, weight,
mobility, nutritional status, or use of walking aids.
Materials and Methods
Initial Screening of Volunteers
Volunteers are screened for any cardiovascular,
respiratory disorder or any conditions which may
contraindicate or interfere with exercise protocol.
Vertigo is one of the important criteria to screen
the volunteers. Volunteer should be screened for
postural hypotension also.
• Healthy adult volunteers (Willing to give
written informed consent)
• Stethoscope, mercury sphygmomanometer
Experiment: 4 A: (Tread mill exercise),
Experiment: 4 B: Master’s 2 steps,
Experiment: 4 C: Bicycle ergometer and
Experiment: 4 D: Hand-dynamometer
Drug: β-blocker ( Propranolol: 40 mg PO)
Precautions during Experiment
• Subject should be advised for adequate sleep
over night and light breakfast in the morning
on the day of experiments (preferably 2 hr
before)
• Avoid caffeinated drink or smoking, at least
2 hr before the experiment or any abuse sub-
stance
• Volunteer should get complete instructions of
the procedure and its rehearsal, so as to avoid
any conditional anxiety of volunteers
• Monitoring equipment should be calibrated
regularly
• Oxygen source and suction device should be
accessible
• Basic operating procedure of the selected
instrument should be well explained to the
volunteers
• Direction of movement on the master’s 2 step
should be clear to the volunteer
 Cardiovascular System  257

• Testing procedure and its consequences

should be discussed beforehand.

Procedure
These studies are performed after taking baseline
values for HR, SBP, DBP, RPP and RR at supine,
sitting and standing, and then subject is asked to
perform submaximal exercise protocol. After
normalization of all the baseline parameters one
tablet of propranolol (40 mg) is given with plain
water to the volunteers. Thereafter, the same
exercise protocol should be repeated following
2 hr post drug administration. The HR, SBP, DBP,
RPP and RR are recorded as described below and
volunteer asked to report any adverse effect.
Meanwhile, exertion, dyspnea, fatigue, weakness,
and pain during exercise should be observed. Fig. 26.8: Treadmill test (TMT)

EXPERIMENT NO: 4(A) grade (16%)-time (3 min); Stage 5: speed 5.0(mph)-

Treadmill test (TMT) (Fig. 26.8): TMT used in
cardiac stress testing and to test maximal
endurance testing. There are several protocols to
perform the TMT namely, Bruce, Modified Bruce,
Balke, Naughton, Wilson, Kattus, etc. whereas
Bruce or modified Bruce is most commonly used
protocol. Basically, Bruce protocol and modified
Bruce protocol are nearly same only difference is
that modified Bruce protocol have initial two
stages, i.e. stage 0 in which speed is 1.7 mph have
0% grade for 3 min following by the stage 0.5
which have same speed with 5% grade for 3 min.
thereafter, modified Bruce and Bruce protocol is
the same such as stage 1: speed (1.7 mph)- grade
(10%)-time (3 min); stage 2: speed (2.5mph)- grade
(12%)-Time (3 min); Stage 3: speed (3.4 mph) –
grade (14%)-time (3min); stage 4: speed (4.2 mph)-
grade (18%)- time (3 min) and stage 6: Speed (5.5
mph)-grade (20%)-time (3min). But, apart from
these stages modified Bruce also have stage 7 with
speed (6.0 mph)-grade (22%)- time (3 min).
Method
Mercury sphygmomanometer is considered to be
the standard of all the other sphygmomanometer
BP measurement.
Volunteer is asked to perform till submaximal
stage is attained or till his heart rate is 60-85% of
predicted maximum heart rate*. Then, monitor HR,
BP, RPP and ECG (if done) during each stage.
Note: *Predicted maximum heart rate (PMHR) is calculated
differently for adult male and female.
PMHR for male; 220- age
PMHR for female; 226- age

Observations and Results

HR BP RPP RR
Pre drug Post drug Pre drug Post drug Pre drug Post drug Pre drug Post drug

Supine

Standing

Measurement: HR, BP, respiratory rate (RR), and Rate Pressure Product (RPP)
258  Practical Manual of Experimental and Clinical Pharmacology

EXPERIMENT: 4(B)
Master’s 2 steps are designed to perform sub-
maximal exercise and are made of wood with the
specific dimensions of “23 cm × 25 cm” (Height ×
Width) of each step (Fig. 26.9).
The subjects are allowed to relax for 15 min in
supine position and all the vital parameters: HR, Fig. 26.9: Master’s 2 steps with its dimensions. Movement
SBP, DBP and RR should be recorded. The HR is of the volunteers should be such that it make “8” while
turning back on the each side of Master’s 2 steps; it prevents
recorded by palpatory method and BP to measure development of vertigo)
with sphygmomanometer. Then, repeat all
parameters readings in sitting and standing steps chart appropriate for volunteers weight and
positions. Now, volunteer has to undertake sub- age (Nomogram). After the completion of exercise
maximal exercise using master’s 2 steps exercise HR, SBP, DBP, RPP and RR are recorded every
protocol. The exercise is undertaken for 3 min and min till they reached to the baseline value.
the number of double steps to be carried out in 3 Similar, protocol is followed 2 hr after
min is calculated which is based on masters 2 administration of propranolol 40 mg.

Observations and Result

Values are as mean of 5 volunteers
HR BP RPP RR
Pre drug Post drug Pre drug Post drug Pre drug Post drug Pre drug Post drug
Supine

Sitting

Standing

EXPERIMENT: 4(C)
Bicycle Ergometer (Fig. 26.10)
Bicycle Ergometer test is one of the most frequently
used submaximal ergometer tests. This is are
mainly performed to assess the power or work
done by the muscle. This test is preferred due to
easily recordable HR and BP whereas limitations
of the test include the error margin in the predicted
VO2max values and discomfort at the lower part of
some volunteers.

Method
Precondition the volunteer regarding test
procedure, by instructing him/her to warm-up, Fig. 26.10: Bicycle ergometer

and familiarizing with the equipment. Written
informed consent should be obtained before the
experiment. Thereafter, select appropriate
protocol for the submaximal exercise. Volunteer
is asked to relax for 10 min and then his/her
baseline BP, HR and RR are noted. The volunteer
is then subjected to submaximal exercise as the
“Bruce protocol”.
Stage 1= 1kg x 150 meters x 2 min
Stage 2= 2kg x 150 meters x 2 min
Stage 3= 3kg x 150 meters x 2 min
Stage 4= 4kg x 150 meters x 2 min
(Reference: Bruce RA, Kusumi F, Hosmer D. Maximal
oxygen intake and nomographic assessment of
Observations and Result
HR BP
Pre drug Post drug Pre drug Post drug
Discussion
Myocardial oxygen consumption is determined by
HR, SBP, left ventricular diastolic volume and left
ventricular wall thickness. The RPP increases with
increasing walk/exercise and is a reliable indicator
of myocardial performance. β-blockers decrease the
RPP to allow optimal utilization of oxygen in states
of ischemia. Benzer, “2001”, reported that exercise
test is the primary mean used to evaluate the safety
of participating in an exercise program and to
formulate the exercise prescription. Because of, the
wide scatter of maximal heart rate when plotted
against age, it is much better to determine person’s
actual maximal heart rate by testing, by assigning
to a target heart rate for training rather than giving
Cardiovascular System  259
functional aerobic impairment in cardiovascular
disease. Am Heart J 1973; 85: 546 –562.)
At the end of all stages of submaximal exercise
BP, HR and RR are recorded at every 2 min until
recovery of baseline recordings. From the above
values “Rate Pressure Product” is calculated, for
the entire period of the study.
RPP= (HR x SBP)/100
All the above mentioned parameters are
recorded 2 hr after propranolol (40 mg) intake. If
volunteer complain of discomfort, exercise is
stopped quickly and volunteer is asked to relax in
sitting or supine position.
RPP RR
Pre drug Post drug Pre drug Post drug
a predicted value. Exercise test can also be used to
advance patients safely to a higher level of
performance; also the improvement in exercise
capacity demonstrated by an exercise test can be
effective incentive and can encourage risk factor
modification.
EXPERIMENT: 4(D)
Hand Dynamometer (Fig. 26.11)
Isometric exercise or static exercise generates force
with negligible muscle shortening. It also produces
alterations in cardiovascular parameters. Pressor
response is more with isometric exercise, it may
be hand grip exercise or weight lifting. β-blockers
260  Practical Manual of Experimental and Clinical Pharmacology

middle of four fingers. The subject presses the

dynamometer with maximum isometric effort.
Duration of effort is noted.
Note: Body part should be straight and should
not move forward.

Fig. 26.11: Hand dynamometer and technique to hold it
cause decrease in CV response to changes induced
by the exercise. Volunteer holds dynamometer in
the dominating hand with the arm at right angles
and the elbow by the side of the body. The base of
the dynamometer should rest on first metacarpal
(heel of palm), while the handle should rest on
Method
Volunteer is made to sit for 15 min BP, HR and
RPP are recorded until stable values are obtained.
It is recorded on the non-dominant arm. The
subject is asked to perform maximum exercise
using hand dynamometer. The maximum value
is noted down. The subject is then asked to grip
and performs exercise at 25% of maximum
voluntary effort for 90 sec the cuff is inflated at
70 sec and BP and HR at 90 sec is noted down,
while volunteer is still performing exercise with
the other hand. After stopping exercise BP, HR
and RPP are recorded for 10 min or till readings
reached back to baseline.

Observations and Result

HR/min BP (SBP/DBP) RPP RR
Time(min) Before drug After drug Before drug After drug Before drug After drug Before drug After drug
0
1
2
3
4
5
6
7
10
12
15
17

Experimenter may look for the other con-
sequences like, exertion, dyspnea, fatigue,
weakness, and pain or stress while conducting
test performance.
Discussion
The baseline heart rate is lower after adminis-
tration of propranolol 40 mg. The post exercise
rise in HR and SBP are slightly less as compared
to without drug post exercise values. The
recovery of HR and SBP are quicker after drug
administration. The rise in SBP after exercise is
attenuated by propranolol. There is little effect
on DBP. The rate pressure product (RPP) is
calculated as:
RPP= (HR × SBP)/100

There is large increase in RPP after exercise in
both the pre drug and post drug session but the
increase in RPP is less in post drug session after
exercise, also the recovery is quicker. The
reduction of RPP signifies that propranolol
reduces the cardiac work load and O2 demand for
the similar amount of exercise and thereby
improves exercise tolerance. Volunteers may
report giddiness, sedation and heaviness at 30
min of drug administration. Reduction in sub-
maximal exercise responses such as HR, RR, and
BP can be consistent with improved aerobic
conditioning and in clinical setting interpretation
based primarily on the type of test conducted, e.g.
assessment, diagnostic, exercise prescription for
specified outcomes.
SUGGESTED READING
1. Bruce RA, Kusumi F, Hosmer D. Maximal oxygen
intake and nomographic assessment of functional
aerobic impairment in cardiovascular disease. Am
Heart J 1973;85:546-62.
2. Bruce RA, Kusumi F, Hosmer D. Maximal oxygen
intake and nomographic assess-ment of functional
aerobic impairment in cardio-vascular disease. Am
Heart J 1973;85:546-62).
3. Bruce RA. Exercise testing of patients with coronary
heart disease: Principles and normal standards for
evaluation. Ann Clin Res 1971;3:323-32.
4. George JD, Vehrs PR, Allsen PE, Fellingham GW,
Fisher AG. Development of a submaximal treadmill
jogging test for fit college-aged individuals. Med Sci
Sports Exerc 1993;25(5):643-47.
5. Hartung GH, Krock LP, Crandall CG, Bisson RU,
Myhre LG. Prediction of maximal oxygen uptake
from submaximal exercise testing in aerobically fit
and nonfit men. Aviat Space Environ Med 1993;
64(8):735-40.
6. Legge BJ, Banister EW. The Astrand-Ryhming
nomogram revisited. J Appl Physiol 1986;61:
1203-09.
7. Marciniuk DD, Gallagher CG. Clinical exercise testing
in interstitial lung disease. Clin Chest Med 1994;15:
287-303.
8. Patterson JA, Naughton J, Pietras RJ, Gunnar RM.
Treadmill exercise in assessment of patients with
cardiac disease. Am J Cardiol 1972;30:757-62.
9. Schreuders TA, Roebroeck ME, Jaquet JB, Hovius
SE, Stam HJ. Long-term outcome of muscle strength
Cardiovascular System  261
in ulnar and median nerve injury: Comparing manual
muscle strength testing, grip and pinch strength
dynamometers and a new intrinsic muscle strength
dynamometer. J Rehabil Med 2004;36(6):273-78.
10. Wisen AGM, Wohlfart B. A comparison between two
exercise tests on cycle: A computerized test versus
the Astrand test. Clin Physiol 1995;15:91-102.
11. Wyndham CH. Submaximal tests for estimating
maximum oxygen intake. Can Med Assoc J 1967;
96:736-45.
12. Zeballos RJ, Weisman IM. Behind the scenes of
cardiopulmonary exercise testing. Clin Chest Med
1994;15:193-213.
CVS EXPERIMENT: 5
Aim
To evaluate the effect propranolol on mental stress
induced rise in blood pressure and heart rate in
healthy volunteer.
Background
Mental stress leads to marginal rise in systolic
blood pressure in both normal volunteers and
patients with hypertension. This is a result of
activation of central sympathetic pathways
leading to an increase in peripheral vascular
resistance. Earlier studies reported the efficacy of
β-adrenoceptor antagonism on the effects of
experimental stress in healthy volunteers; it
showed single oral dose of propranolol (40 mg)
can reduce the stress-induced increase in heart
rate and systolic blood pressure significantly
compared to placebo. However minimal effect is
documented in diastolic blood pressure following
β-adrenoceptor blockade like propranolol.
Materials and Methods
• Healthy adult volunteers (willing to give
written informed consent)
• Stethoscope, mercury sphygmomanometer,
Facilities for Clinical Pharmacology Labo-
ratory (Appendix IV).
Initial Screening of Volunteers
Volunteer is screened for detailed evaluation of
any disease conditions, detailed history should
262  Practical Manual of Experimental and Clinical Pharmacology
be taken in respect to smoking, alcohol con-
sumption, drug therapy, or any associated disease
conditions.
Precautions for Experiment
1. Volunteer should be advised for adequate
sleep over night and light breakfast in the
morning day of experiments (preferably
2 hr before)
2. Avoid any caffeinated drink or smoking at
least 2 hr before the experiment
3. Take 2 or 3 readings in every measurement
4. Volunteer should be sitting on a chair or
lying quietly for at least 5-10 min, with the
relaxed arm
5. Avoid (or if unavoidable, make a note of it)
extraneous factor, which may alter the BP,
e.g.: Recent smoking/eating, anxiety, talking,
exercise/exertion, medication that can
interfere the result of the experiment.
6. Note the time of the day (preferably blood
pressure should be taken at the same time of
the day)
7. Width of cuff should cover at least 40% of
upper arm.
Observations and Results
BP
Pre drug Post drug Pre drug
1st person
2nd person
3rd person
Discussion
Propranolol is a non-selective β-adrenergic
receptor blocker with no autonomic nervous
system activity. It is a competitive antagonist
which specifically competes with beta-adrenergic
receptor stimulating agents for available beta-
receptor sites. Peak plasma concentrations of
propranolol are attained 60 to 90 minutes
following oral administration and the apparent
8. Inflatable balloon should cover 80% of the
arm circumference
9. Place the cuff 2-3 cm above the antecubital
fossa
10. Read exact pressure, do not round off.
Methods
Healthy volunteers are included in the experiment
following adequate rest and baseline BP and HR
readings should be noted every 2 minutes till an
average of 3 recordings are made. The volunteers
are administered a mental task for 3 min. The
volunteers are asked to continuously go on
subtracting a digit from a 3 digit number in a
recurring fashion and to speak out the subtracted
number, that is noted by the observer. At the end
of 3 min, the BP and HR values are noted and no.
of correct and incorrect responses should be
recorded. The rise in BP and HR following mental
stress is observed and mean changes in arterial
BP are determined. Then one of the volunteers
receives propranolol 40 mg and other one placebo
in a double blind fashion and the same procedure
is repeated at 120 min after drug administration.
HR Correct response
Post drug Pre drug Post drug
plasma half-life has been reported to be between
10 and 12 hours. Mental stress results in increased
systolic blood pressure and propanolol have been
shown to reduce the stress-induced increase in
heart rate and systolic blood pressure to 49.9
percent and 8.3 percent respectively compared to
61.0% and 17.4% with placebo in a double blind
randomized study in 12 healthy volunteers.
Several other studies showed the effects of oral

treatment with atenolol and propranolol on blood
pressure, heart rate and plasma cyclic adenosine
3':5'-monophosphate (cAMP) in young borderline
hypertensives following psychological stress
testing. Studies showed there is a correlation
between heart rate and plasma cAMP at rest and
also following psychological stress test β-blockers
significantly reduce all the parameters and plasma
cAMP is mostly affected by propranolol. Another
study using a standardized form of mental stress
in 21 patients with idiopathic hypertension was
assessed at baseline and following treatment with
placebo, propranolol , or methyldopa. Studies have
shown satisfactory control of blood pressure with
both the drugs but propranolol treatment showed
modification of the pressor response to stress
testing ultimately provide more uniform control
of blood pressure in patients with essential
hypertension. Besides above mentioned effect, the
rise in temperature of the trunk skin is significantly
reduced by propranolol. The self-rating of anxiety,
alertness and concentration by the volunteers is
usually unaffected by propranolol.
SUGGESTED READING
1. Dunn FG, Lorimer AR, Lawrie TD. Objective
measurement of performance during acute stress in
patients with essential hypertension: Assessment of
the effects of propranolol and metoprolol. Clin Sci
(Lond) 1979;57 Suppl 5:413s-15s.
2. Dunn FG, Melville DI, Jones JV, Lorimer AR, Lawrie
TD. Standardized stress and hypertension: Com-
parison of effect of propranolol and methyldopa. Br
J Clin Pharmacol 1978;5(3):223-26.
3. González-Gómez A, Garcia-Barreto D, Cabrera R,
Toruncha A, Hernández-Cañero A. Effects of Oral
Atenolol and Propranolol on Blood Pressure, Heart
Rate and Plasma Cyclic Adenosine 3':5'-Mono-
phosphate in Borderline Hypertensive Patients.
Pharmacology 1982;25:33-38.
4. Pandhi P, Sharma PL. Comparative effects of beta,
alpha- and combined beta- plus alpha-adrenoceptor
blocking agents in stress-induced increase in arterial
blood pressure. Int J Clin Pharmacol Ther Toxicol
1987;25(6):297-300.
5. Taylor EA , Harrison J, Turner P. Propranolol in
experimentally induced stress. The British Journal of
Psychiatry 1981;139:545-49.
Cardiovascular System  263
CVS EXPERIMENT: 6
Aim
To evaluate the postural hypotension in the
60-year old male volunteers.
Background
Postural hypotension is defined as the sudden
drop of blood pressure because of change in body
posture position. Normally this change is
compensated by the body, but if the body’s
response to a change in vertical position is slow
or absent, the result is orthostatic hypotension
which indicates the low response of the body
compensation mechanism. If the change in
posture causes systolic BP fall below 20 mm Hg
or diastolic 10 mm Hg then, this shows that the
person is suffering from the postural hypotension.
It is clinically important to identify the postural
hypotensive person, so that drugs causing the
postural hypotension such as vasodilator
(glyceryl trinitrate, phenoxybenzamine or phento-
lamine), diuretics (furosemide, ethacrynic acid,
torsemide), etc. should be avoided in the pres-
cription.
Materials and Methods
• Healthy volunteer of about 60 years of age and
willing to give written informed consent
• Stethoscope, mercury sphygmomanometer,
Facilities for Clinical Pharmacology Labo-
ratory (Appendix IV).
Initial Screening of Volunteers
Volunteer is screened for detailed evaluation of any
disease conditions, detailed history should be taken
in respect to smoking, alcohol consumption, drug
therapy, or any associated disease conditioned.
Precaution
• Volunteer should be advised for adequate
sleep over night and light breakfast in the
morning day of experiments (preferably 2 hr
before)
• Avoid any caffeinated drink or smoking at
least 2 hr before the experiment
• Take 2 or 3 readings in each recording
264  Practical Manual of Experimental and Clinical Pharmacology
• Volunteer should be sitting on chair or ideally
in supine position quietly for at least 10-15
min, with the arms relaxed
• Avoid (or if unavoidable, make a note of it)
extraneous factors, which may alter the BP,
e.g.: Recent smoking/eating, anxiety, talking,
exercise/exertion, avoid medications which
can interfere with blood pressure.
• Note the time of the day (preferably blood
pressure should be taken at the same time of the
day).
Observations and Result
SBP
1st 2nd 3rd
Supine position
Standing Position
Parameters should be recorded
Detailed history should be taken regarding
volunteer experience with any dizziness, black-
outs, syncope, etc.
Statistical analysis: Data is expressed as mean ±
SD.
Discussion
Postural hypotension is associated with
increased morbidity and also mortality in elderly
people. The postural hypotension can be diag-
nosed if the systolic blood pressure in decrease
by 20 mm Hg or diastolic blood pressure decrease
10 mm Hg or more, with or without an increase
in heart or pulse rate, for 3 minutes after standing
from supine posture. The prevalence of postural
hypotension in older adults is 5-30%, and it is
reportedly higher among hospitalized patients
(52-69%) and patients receiving long-term
hospital care facilities (50%). This may be
because of autonomic insufficiency, in case of
prolonged illness, diabetic polyneuropathy and
patients treated with above mentioned
sympatholytic drugs. Studies have reported that
administration of a β-blocker is not usually
• Width of cuff should cover at least 40% of
upper arm
• Inflatable balloon should cover 80% of the arm
circumference
• Place the cuff 1.5- 2 cm above the antecubital
fossa
• Read exact pressure, do not round off
Method
For methodology please refer to CVS experiment
no. 2.
DBP
Mean 1st 2nd 3rd Mean
associated with postural hypotension, but with
α-blocker like prazosin showed significant risk
of first-dose postural effects, however with the
newer α1-blockers, for example doxazosin has
more gradual onset of action.
SUGGESTED READING
1. Christopher J Mathias. Postural hypotension: Causes,
Clinical Features, Investigation, and Management.
Annual Review of Medicine 1999;50:317-36.
2. Ismo Räihä, Sinikka Luutonen, Juhana Piha, Asko
Seppänen, Tuula Toikka, Leif Sourander. Prevalence,
Predisposing Factors, and Prognostic Importance of
Postural Hypotension. Arch Intern Med 1995;
155(9):930-35.
3. Peter A Meredith. Is Postural Hypotension a Real
Problem with Antihypertensive Medication?
Cardiology 2001;96:19-24.
CVS EXPERIMENT: 7(A)
Aims
To evaluate the effect of glyceryl trinitrate (GTN)
on blood pressure, heart rate in healthy volun-
teers.

Background
Glyceryl trinitrates (GTN), are drugs that release
nitric oxide (NO) and are widely used in the
treatment and prevention of angina. ‘NO’ activates
guanylyl cyclase by increasing cyclic GMP-
dephosphorylation of the myosin light chain and
the reduction of cystolic (Ca2+) and leads to the
relaxation of smooth muscle cells and additionally
causes inhibition of platelet aggregation. GTN
mainly acts in veins then the arterioles. It also
increases in subendocardial blood flow which
causes decreases oxygen consumption leads
ultimately favors subendocardial perfusion.
Decreasing peripheral arteriolar resistance
reduces after load and thus myocardial work and
oxygen consumption.
Materials and Methods
Initial Screening of Volunteers
Volunteers are screened for any cardiovascular,
respiratory disorder or any conditions which may
interfere with experimental protocol. Volunteers
with smoking habit should be excluded.
Healthy male adult volunteers (willing to give
written informed consent)
Stethoscope, mercury sphygmomanometer,
Facilities for Clinical Pharmacology Laboratory
(Appendix IV)
Drug: GTN tablet
Inclusion Criteria
Healthy volunteers between 18-45 years selected
for the study.
No history of nitrate allergy, cardiovascular
disease, psychiatric illness, intake of any drugs
especially that can affect BP and HR should be
confirmed. Intake of alcohol, smoking, drug abuse,
dehydration, postural hypotension should be
checked for exclusion. Normal ECG, LFT, RFT and
Cardiovascular System  265
other hemodynamic parameters should be carried
out.
Exclusion Criteria
1. Known hypersensitivity to GTN and related
organic nitrate compounds.
2. Acute circulatory failure associated with
marked hypotension (shock).
3. Conditions associated with elevated intra-
cranial pressure.
4. Myocardial insufficiency due to obstruction
(e.g. in the presence of aortic or mitral stenosis
or of constrictive pericarditis).
5. Concomitant use of GTN and phospho-
diesterase type 5 (PDE5) inhibitors such as
sildenafil is contraindicated, because PDE5
inhibitors may amplify the vasodilatory effects
of GTN resulting in severe hypotension.
Precautions
Patients should adequately and cautiously under
strict medical surveillance and/or hemodynamic
monitoring till completion of experiments and all
the supportive medical care should be in the
clinical pharmacology laboratory (Appendix IV).
Methods
After light breakfast volunteers are asked to report
to the clinical pharmacology laboratory in the
morning. Initially volunteers are advised to lie
down for 15 min and baseline HR, BP and RR
should be noted, the volunteers are given GTN as
mentioned above. The vital parameters are
recorded at baseline, every 15 min till 2 hrs.
Volunteers should be observed for any adverse
drug reaction like headache, giddiness, allergic
reaction till the completion of experiment. The
volunteers should be told that in case of any severe
headache; GTN tablet should be taken out.
266  Practical Manual of Experimental and Clinical Pharmacology

Observations and Result

Time (min) Systolic blood pressure Diastolic blood pressure Heart rate (HR) Respiratory rate
(SBP) (mm Hg) (DBP) (mm Hg)
Baseline (0)
15
30
45
60
75
90
105
120

Discussion active substance penetrates the skin and thus

Fall in SBP and DBP is observed with increase in
heart rate and no alteration of respiratory rate is
seen. GTN is a vasodilator which mainly dilates
the veins. Thus there is a systemic vascular
resistance reduction, though very mild vasodila-
tation lead to decreased end diastolic volume.
There is not much effect on arteries. Pulmonary
vascular resistance is decreased and cardiac
output falls. Heart rate tends to rise, so there may
be reflex tachycardia which can lead to para-
doxical anginal pain in patients with ischemic
heart disease.
CVS EXPERIMENT: 7(B)
Aim
To evaluate the effect of glyceryl trinitrate (GTN)
transdermal patches on blood pressure, heart rate
arterial vasodilatation in healthy volunteers.
Background
Glyceryl trinitrate (GTN) transdermal patches
available as 1,2,3-Propanetriol trinitrate trans-
dermal therapeutic system with dose ranging
from 25 to 75 mg. Nitroglycerin patch is an
organic nitrate derivative which is designed in
flat multilayer system to deliver nitroglycerin
continuously through a release membrane
following application to the skin. In cases where
the permeability of the skin is excessive, drug
release is limited by the release membrane. The
becomes directly bioavailable to the systemic
circulation at relatively constant concentrations
during the recommended application period. The
nominal rate of nitroglycerin release in vivo is
approximately 20-25 μg/cm2 h. It is commonly
indicated for Angina Pectoris as monotherapy
or in combination with other anti-anginal drugs
such as beta-blockers and/or calcium anta-
gonists. It is used in congestive heart failure as
supplementary medication in patients not
responding adequately to conventional therapy
with digitalis or other positive-inotropic agents
and diuretics. It is also used in prevention of
phlebitis and extravagation secondary to venous
cannulation for intravenous fluid and drug
administration when treatment is expected to last
for two days or longer.
Materials and Methods
Initial Screening of Volunteers
Volunteers are screened for any cardiovascular,
respiratory disorder or any conditions which may
interfere with experimental protocol. Volunteers
with smoking habit should be excluded.
Healthy male adult volunteers (willing to give
written informed consent)
Doppler flow meter (Fig. 26.12), Pulse oxymeter,
Facilities for Clinical Pharmacology Laboratory
(Appendix IV).
Stethoscope, mercury sphygmomanometer
Drug: Nitroglycerin skin patches

Dosage and Use
Volunteers should be given lowest effective dose
(25 mg). The application site should be cleaned
properly and observed for any local irritation.
Nitroglycerin skin patch is sealed in a separate
sachet with a tear-off edge to facilitate the
application. After removal of the white protective
backing, apply the Nitroglycerin skin patches to
a clean, non-hairy, dry area of intact skin on the
trunk or upper arm. Hold the patch in position for
10-20 seconds with the palm of the hand.
Inclusion Criteria
Healthy volunteers between 18-45 years are
selected for the study.
No history of nitrate allergy, cardiovascular
disease, psychiatric illness, intake of any drugs
especially that can effect BP and HR should be
confirmed. Intake of alcohol, smoking, drug abuse,
dehydration, postural hypotension should be
checked for exclusion. Normal ECG, LFT, RFT and
other haemodynamic parameters should be
carried out.
Exclusion Criteria
1. Known hypersensitivity to nitroglycerin and
related organic nitrate compounds.
2. Acute circulatory failure associated with
marked hypotension (shock).
3. Conditions associated with elevated intra-
cranial pressure.
4. Myocardial insufficiency due to obstruction.
5. Concomitant use of nitroglycerin skin patches
and phosphodiesterase type 5 (PDE5) inhi-
bitors such as sildenafil is contraindicated,
because PDE5 inhibitors may amplify the
vasodilatory effects of nitroglycerin skin
patches resulting in severe hypotension.
Precautions
1. Volunteers should be adequately and cau-
tiously under strict medical surveillance and/
or hemodynamic monitoring till completion
of experiments and all the supportive medical
Cardiovascular System  267
care should be in the clinical pharmacology
laboratory (Appendix IV).
2. The nitroglycerin skin patch contains an
aluminium layer. Therefore nitroglycerin skin
patches must be removed before applying
magnetic or electrical fields to the body during
procedures such as MRI, cardiversion or DC
defibrillation or diathermy treatment.
3. Precautions should be taken carefully as the
possibility of increased frequency of angina
during patch-off periods in that case use of
concomitant anti-anginal therapy is desirable.
4. Tolerance to sublingual nitroglycerin: As
tolerance to nitroglycerin patches develop, the
effect of sublingual nitroglycerin on exercise
tolerance may be partially diminished.
Methods
After light breakfast volunteers are asked to
report to the clinical pharmacology laboratory
in the morning. Initially volunteers are advised
to lie down for 15 min and baseline heart rate,
blood pressure and respiratory rate should be be
noted, the volunteers are given GTN as men-
tioned above. The vital parameters are recorded
at baseline, every 15 min till 2 hrs. Volunteers
should be observed for any adverse drug reaction
like headache, giddiness, allergic reaction till the
completion of experiment. The volunteers should
be told that if in case of severe headache,
Nitroglycerin skin patches should be removed.
Fig. 26.12: Doppler flow meter
268  Practical Manual of Experimental and Clinical Pharmacology

Ankle Brachial Index (ABI) ABI should be measure at baseline following

nitroglycerine skin patches at 2 and 4 hours. A
Doppler derived arterial segmental pressures
higher value signifies improvement in arterial
should be measured in the ankle and brachium
perfusion via collaterals, whereas a lower value
with a standard adult cuff. Normally, there is
indicates a decrease in perfusion either because
amplification of systolic pressure further down
of disease progression or as a result of problems
the limb, i.e. systolic pressure at the ankle level
with a reconstructive procedure. (This experiment
should be higher than that recorded from the
can also be done by using a suitable vasodilator,
upper arm. This means that the systolic pressures
as nitroglycerine patch has mostly venodilatory
recorded from both tibial arteries at the ankle
action).
should be at least equal to or higher than that
recorded from the arm. Oxygen saturation: Oxygen saturation should be
ABI = systolic ankle BP/ systolic arm BP assessed using pulse oxymeter (Fig. 26.13) at
baseline and following 2 and 4 hours of skin patch
ABI Interpretation/ Severity application.
>1.3 False high values
Adverse drug reactions: Volunteers should be
> 0.9 Normal
0.75 – 0.9 Normal evaluated for different ADRs like headache,
0.5 – 0.75 Medium PAD dizziness, tachycardia, postural hypotension,
<0.5 Severe PAD flushing, nausea, vomiting, dermatitis contact,
* PAD—Peripheral Artery Disease erythema, pruritus, burning, and irritation.

Observations and Result

Time (min) (SBP) (DBP) (HR) RR ABI Oxygen saturation
ADR
Baseline After Baseline After Baseline After Baseline After Baseline After Baseline After
drug drug drug drug drug drug

SBP= Systolic blood pressure; DBP= Diastolic blood pressure; HR= Heart rate; RR= Respiratory rate; ABI= Ankle
brachial index; ADR= Adverse drug reaction

Fig. 26.13: Pulse oxymeter
Discussion
Nitroglycerin relaxes smooth muscles throughout
the body. In the vascular system, it acts chiefly on
the systemic veins and the large coronary arteries.
Nitroglycerin dose-dependently dilates the
arteriolar vascular bed, thereby lowering systemic
vascular resistance (after load) and left-ventricular
systolic wall tension, further reducing myocardial
oxygen consumption. In addition, nitroglycerin
produces relaxation of vasospasm, whether
spontaneous or induced by ergonovine. Following
single application, the plasma concentrations of
nitroglycerin reach a plateau within 2 hours, which
is maintained over the recommended application
period. The height of this plateau is directly
proportional to the size of the system‘s drug
releasing area. The active substance is rapidly
metabolized by a glutathione-dependent organic
nitrate reductase in the liver. In addition, and
probably more important, studies with human
erythrocytes in vitro have shown that the erythro-
cyte is also a site of biotransformation of nitro-
glycerin by a sulphydryl-dependent enzymatic
process and by an interaction with reduced
hemoglobin. The amount of reduced hemoglobin
in human erythrocytes seem to play a major role in
their metabolic activity, and caution should
therefore be exercised in cases of anemia. Develop-
ment of tolerance or attenuation of therapeutic
effects commonly occurs with prolonged or
frequent administration of long-acting nitrates.
Concomitant treatment with other vasodilators,
PDE5 inhibitors, calcium antagonists, ACE
Cardiovascular System  269
inhibitors, beta-blockers, diuretics, antihyper-
tensives, tricyclic antidepressants, and major
tranquillizers, as well as the consumption of
alcohol, may potentiate the blood-pressure
lowering effect. Caution is required during
pregnancy, especially in the first 3 months,
lactation, while driving and machinery use. High
doses of nitroglycerin may lead to severe hypo-
tension and reflex tachycardia or to collapse and
syncope. This can be rapidly terminated simply by
removing the system(s). Hypotension or collapse
can be treated by elevation or, if necessary,
compression bandaging of the patient’s legs.
SUGGESTED READING
1. Agvald P, Hammar L, Gustafsson LE. Nitroglycerin-
patch induced tolerance is associated with reduced
ability of nitroglycerin to increase exhaled nitric oxide.
Vascul Pharmacol 2005;43(6):449-57.
2. Carter SA. Indirect systolic pressures and pulse
waves in arterial occlusive diseases of the lower
extremities. Circulation 1968;37:624-37.
3. Gori T, Harvey P, Floras JS, Parker JD. Continuous
therapy with nitroglycerin impairs endothelium-
dependent vasodilation but does not cause tolerance
in conductance arteries: A human in vivo study. J
Cardiovasc Pharmacol 2004;44(5):601-06.
4. Joyce WP, Walsh K, Gough DB, Gorey TF, Fitzpatrick
JM. Pulse oximetry: A new non-invasive assessment
of peripheral arterial occlusive disease. Br J Surg
1990;77(10):1115-17.
5. Kelly JJ, Tam SH, Williamson PM, Whitworth JA.
Decreased threshold for the nitric oxide donor
glyceryl trinitrate in cortisol-induced hypertension in
humans. Clin Exp Pharmacol Physiol 2007;
34(12):1317-18
6. Malcolm J Boyle. A dramatic drop in blood pressure
following prehospital GTN administration. Emer-
gency Medicine Journal 2007;24:225-26.
7. Schwemmer M, Bassenge E. New approaches to
overcome tolerance to nitrates. Cardiovasc Drugs
Ther 2003;17(2):159-73.
8. Sumimoto T, Hamada M, Kawakami H, Suzuki M,
Abe M, Matsuoka H, Shigematsu Y, Hiwada K.
Effects of glyceryl trinitrate on blood pressure and
arterial compliance. Angiology 1993;44(12):951-57.
CVS EXPERIMENT: 7(C)
Aim
Recording of an electrocardiogram (ECG).
270  Practical Manual of Experimental and Clinical Pharmacology

Introduction selves are too small to make a representation on

The electrocardiogram (ECG) is a representation
of action potential developed through the activity
of the cardiac muscles. The action potential is
generated through the every heart muscle cells
but the ECG measured at the surface of the body is
only formed when the activation (depolarization)
and the following relaxation (repolarization)
spread in the whole heart muscle and producing
a total cumulative electrical component, which is
recorded through the various leads attached to
the body (Fig. 26.14).
Electrocardiogram (ECG) helps to elucidate
cardiac arrhythmia and conduction defects. It is
used to diagnose and localize myocardial
hypertrophy, ischemia or infarction. It also gives
information about electrolyte imbalance and
toxicity of some drugs.
ECG is the recording of electrical current of
the heart which is produced by depolarization
and repolarization waves spreading across the
atrial an ventricular myocardium, while SA node,
AV node, His bundle and Purkinje fiber them-
the surface ECG. These just generate or conduct
the impulse.
ECG machine: It is a galvanometer with a
writing facility to record electrical activity through
a no. of leads. A lead is constituted by wires
connecting –ve and +ve poles.
ECG Machine: 2 types, mechanical and digital
Leads: ECG has the following Leads
(Figs 26.15 and 26.16)
1. Standard limb leads I, II, and III
2. Augmented unipolar leads aVR, aVL and aVF
3. Unipolar chest leads v1, v2, v3, v4, v5 and v6
12 lead-ECG is inadequate particularly in
patients with MI or CHD where V3R, and V4R
leads should be recorded routinely. So, a 14 lead
ECG is better than a 12 lead ECG.
Chest Leads
• V1 = 4th intercostals space (ICS) right of
sternum

Fig. 26.14: Electrocardiogram

 Cardiovascular System  271

• V2 = 4th intercostals space (ICS) left of sternum

• V3 = in between V2 and V4
• V4 = 5th ICS in midclavicular lime
• V5 = lateral to V4 in anterior axillary line
• V6 = lateral to V5 in mid axillary line
• V3R = Corresponding to V3 on right side
• V4R = Corresponding to V4 on right side
Calibration: For recording ECG some standard
calibration has to be used
• 1 mV = 10 mm deflection
• Standard calibration should be printed before
each ECG is taken
Paper speed should be 25 mm/sec
ECG paper: Thermolabile paper, can be wax or
chemical coated
ECG grid: A small square represents
1mm Height = 0.1 mV
1mm width = 0.04 second
The paper should be placed or stored in a cool
place (away from direct sun light).

Cardiac Contraction and ECG Development

Fig. 26.15: Standard limb leads is Well Explained in Figure 26.17

Normal ECG Complex

HR(beats/min) = 1500/no. of small square
between R-R wave
PR interval: Between beginning of P wave and Q
wave
Normal PR interval
Childhood = 0.1-0.12 sec
Adults = 0.12-0.25 sec
Elderly = 0.14-0.22 sec
QT interval: between onset of Q wave and end of
T wave. It varies with HR.
QTc (corrected QT interval) = QT/√RR
where RR = 60/HR
Fig. 26.16: Triaxial reference system (Polarity (+) and (–) Or QT + 1.75 X (vent.rate – 60).
272  Practical Manual of Experimental and Clinical Pharmacology

Fig. 26.17: Cardiac contraction and ECG development

(Arrows indicate movements of electrical current within cardiac muscle)

Fridericia formula= QTc= QT/ (R-Rint)0.33
FDA formula= QT/ (R-Rint)0.37
Central Terminal of Wilson (CTW)
The concept of electrocardiography is more than
100 years old. It was originally developed by
WILLEM EINTHOVEN for which he was
honored with “Nobel Prize”
CTW is the indifferent electrode, and the
exploring electrode is the active electrode.
Salient Features of CTW
• Homogeneity of the body volume conductor
• Asymmetry of the leads
• Single equivalent dipole at the center of the
volume conductor.
• Constructed by connecting RA, LA, and LL
electrodes through 5000 resistance (important
to cancel out the potential from 3-points)
The selection of the position for 6 unipolar
chest leads is based on the concept that the
proximity of the heart to the anterior chest wall
resulting in the unipolar chest lead functioning
as semi direct leads being influenced by the tissue
immediately beneath the electrode. The 6 standard
chest leads (V1 to V6) are recorded by positioning
the exploring chest electrodes.
Unipolar limb leads may be recorded by a
system in which CTW constitutes the indifferent
electrode and the exploring is on the three active
limb electrodes. These leads are referred to as VR,
VC, and VL. By disconnecting the input to CTW
from the extremity being explored an augmen-
tation of the voltage by 50% occurs. This modi-
fication is used inversely for clinical ECG and the
leads are labeled as aVR, aVL and aVF.
 Cardiovascular System  273

Figs 26.18A and B: (A) Central terminal of Wilson (CTW) and chest (C) electrode,
(B) Horizontal plane orientation of unipolar chest lead

Fig. 26.19: Augmented unipolar limb leads (aVR, aVL, aVF)

274  Practical Manual of Experimental and Clinical Pharmacology

Position of Chest Electrodes

Fig. 26.20: Position of chest electrodes

 Cardiovascular System  275

Practical Exercise for Cardiovascular System

Practical Exercise 1: A 63-year-old woman with 10 hours of chest pain and sweating, ECG of patient
showed changes in ST, leads V1 - 6, I and aVL, write the diagnosis from ECG and discuss the drug therapy.

Fig. 26.21
276  Practical Manual of Experimental and Clinical Pharmacology

Practical Exercise 2: A 55-year-old man with 4 hours of “crushing” chest pain, ECG of patient showed
changes in ST , leads II,III, aVF and in anterior lead , write the diagnosis from ECG and discuss the drug therapy.

Fig. 26.22
 Cardiovascular System  277

Practical Exercise 3: A 76-year-old man complains of breathlessness, ECG showed irregular ventricular
rhythm, sometimes on first look the rhythm may appear regular but on closer inspection it is clearly irregular ,
write the diagnosis from ECG and discuss the drug therapy.

Fig. 26.23
278  Practical Manual of Experimental and Clinical Pharmacology

Practical Exercise 4: A 45-year-old lady complains of palpitations and history of chronic renal failure, ECG
showed a wide QRS tachycardia, write the diagnosis from ECG and discuss the drug therapy.

Fig. 26.24
 Cardiovascular System  279

Practical Exercise 5: A male patient of 60 years, known hypertensive with recurrent episode of palpitation
since childhood, with increasing frequency and duration since last four years, ECG shows a regular rhythm of
150 beats per minute or higher, with a narrow QRS complex, write the diagnosis from ECG and discuss the drug
therapy.

Fig. 26.25
 Respiratory System
27
RESP. EXPERIMENT: 9
Aim
To compare the effect of salbutamol with placebo
on peak expiratory flow rate (PEFR) in healthy
volunteers.
Background
Presently, there is widespread use of peak flow
meters for the screening and follow-up of
patients with reversible airway obstruction
which has been advocated since the early 1970s
as a reliable method for evaluating airway
caliber. Peak Expiratory Flow Rate (PEFR) is a
simple bedside test of lung function, and it is
most commonly used by the patients with
asthma. It is popular because of quick and
simple bedside measurements. Basically, it is a
small tube with a slider device to measure how
fast air is driven through the tube by the
volunteer exhaling rapidly. Once subject takes
a deep breath in, and then breaths out as hard
and fast as possible, with a good airtight seal of
the lips around the tube. This is best done
standing up; the best of three attempts is usually
taken, with a short rest between attempts.
Several reports have shown the strong corre-
lation between FEV1 and PEFR. Studies of cross-
sectional analyses reported that subjects with
asthma, have correlation coefficients ranging
from 0.82 to 0.89. Besides this, PEFR monitoring
is relatively simple and inexpensive. Most of
guidelines for assessment and treatment of
asthma recommend modification of treatment
when PEFR or FEV, drop by 15% or more over
baseline suggesting that PEFR monitoring is as
reliable as FEV1. Periodic determination of
PEFR in asthmatics helps to find out the
severity. Long term daily monitoring is helpful
in the management of such patients for early
detection of change in airway obstruction that
meets changes in treatment, for evaluation of
responses to change the treatment and for
recognition of air way obstruction.
Materials and Method
Initial Screening of Volunteers
Volunteers are screened for any cardiovascular,
respiratory disorder or any condition which may
interfere with experimental protocol. Volunteers
with smoking habit should be excluded.
Healthy adult volunteer of either sex (willing
to give written informed consent)
Peak flow meter (PEFM) (Mini-Wright).
Stethoscope, mercury sphygmomanometer,
Facilities for Clinical Pharmacology Laboratory
(Appendix IV)
Drug: Salbutamol, Matching placebo
Inclusion criteria: Healthy volunteer aged 18-40
years of either sex
Exclusion criteria: Volunteer with history of
smoking, alcohol, and any other disease condi-
tions and drug therapy which can interfere with
experimental protocol
 Respiratory System  281

Precautions for Experiment

• Subject should be advised for adequate sleep
over night and light breakfast in the morning
on day of experiments (preferably 2 hr before)
• Avoid caffeinated drink or smoking at least
2 hr before the experiment or free of any
substance abuse
• Volunteer should get complete instructions
of the procedure and its rehearsal, so as to
avoid any conditional anxiety of volunteers
• Monitoring equipment should be maintained
and regularly calibrated
• Oxygen source and suction device should be
accessible
• Basic operating procedure of the selected
instrument should be well explained to the
volunteers
• Testing procedure and its consequences
should be discussed beforehand
• Training of volunteers for correct use of PEFM
should be explained properly. Volunteer
should be asked to take deep breath in, then
breath out as hard and as fast as possible, with
a good airtight seal of the lips around the tube,
usually done standing up; the best of three
attempts is usually taken, with a short rest
between attempts.

Peak Flow Meter (Mini-Wright)

Peak flow meter was introduced in 1959 and the
original Mini-Wright peak flow meter was
developed in the 1970s by Dr BM Wright, of the
Medical Research Council, and Clement Clarke Figs 27.1A and B: Peak flow meter
International, Harlow, England. Peak flow meter
is a simple device for quick measurement of lung
breakfast. Smoking and caffeine containing
function at bed sides or at home as mini version
beverages should be prohibited. Baseline BP, HR,
is available both for adult and children. Flow
RR and PEFR should be measured with the
meter has short cylinder which made of plastic
subject in the sitting position.
material with an indicator to move in a scale with
number usually express as liters/min. Instru-
ment has a handle in the mouth piece and Procedure for PEFR
opening for air exit from the instrument (Figs Volunteer is asked to hold the PEFM carefully
27.1A and B). so that fingers should not obstruct the scale, slot
and hole of apparatus. Then, volunteers are
Method
instructed to take a deep breath after putting
Volunteers should report to Clinical Pharma- peak flow meter between teeth and lips, then
cology Laboratory early morning with light blow rapidly; average of 3 flow meter readings
282  Practical Manual of Experimental and Clinical Pharmacology

should be taken; same procedure should be Discussion: Salbutamol is a β2-agonist, bron-

repeated at 30 min, 1 hr, 2 hr, and 4 hr after chodilator used in the treatment of bronchial
ingestion of blinded drug or placebo. Detailed asthma. Such short acting agents are used in
history should be taken to record adverse drug acute exacerbation of asthma and exercise
reaction (ADR). Measurement of PEFR is done induced asthma. They increase airway caliber.
using variable orifice flow meter. Calibration of PEFR is used to classify patients into various
the meter is expressed in “L/min”. Volunteers levels such as mild intermittent, mild persistent,
are advised to take a deep inspiration and from moderate persistent and severe persistent using
this stage expire fully and vigorously into the a parameter called PEFR variability and calcu-
orifice. Values are similar in standing and sitting lated as
position and nose clip is not essential for this
measurement. PEFR variability =
There are several sources of error for example:
(Evening PEFR – Morning PEFR)
(1) inadequate subject performance, either failure 100
of full inspiration or inadequate expiratory effort (Evening PEFR + Morning PEFR)
(2) error in the instrument, (3) failure to hold 2
instrument upright. Above mentioned minor Using PEFM, clinical pharmacological status of
errors may alter the readings. Hence, it is agents effective in asthma can be done. A 15%
advisable to take either last three of the total 5 change in airway caliber has been proposed as the
readings or the highest obtained reading, one of criteria for modifying treatment. Peak flow
the above methods should be practiced. monitoring is not recommended for children under
12 years.
Normal Range of PEFR
PEFR (L/min) SUGGESTED READING
Adults 350-600
1. Alessandro Brunelli, Francesco Xiume´, Majed
Observations and Result Refai, Michele Salati, Rita Marasco, Valeria Sciarra,
and Armando Sabbatini. Evaluation of Expiratory
Following parameters should be recorded at Volume, Diffusion Capacity, and Exercise Tolerance
different time intervals following administration Following Major Lung Resection A Prospective
of salbutamol and placebo. Follow-up Analysis. CHEST 2007; 131(1):141-147
5. Denyse Gautrin, Luis Carlos D’Aquino, Gilles
Placebo 0 hr 0.5 hr 1 hr 2 hr 4 hr Gagnon, Jean-Luc Malo and André Cartier. Com-
SBP (mm Hg) parison Between Peak Expiratory Flow Rates
DBP (mm Hg) (PEFR) and FEV1 in the Monitoring of Asthmatic
HR (per min) Subjects at an Outpatient Clinic. Chest
RR (per min) 1994;10615:1419-26.
PEFR (L/min) 1. Hansen JE, X-G Sun, Wasserman K. Should forced
Salbutamol 0 hr 0.5 hr 1 hr 2 hr 4 hr expiratory volume in six seconds replace forced vital
SBP (mm Hg) capacity to detect airway obstruction? Eur Respir J
DBP (mm Hg) 2006;27:1244-50.
HR (per min) 4. Mahhumad Inatullaya et al. Peak expiratory flow
RR (per min) rate (PEFR); normal values for people of Multan.
PEFR (L/min) The Professionals 2000;7(4):1-4.
2. Vandevoorde J, Verbanck S, Schuermans D,
Statistical analysis: All the data is expressed as Broekaert L, Devroey D, Kartounian J and incken
mean ±SD and comparison is done by using W. Forced vital capacity and forced expiratory
paired ‘t’ test. (if same volunteer is used pre and volume in six seconds as predictors of reduced total
post drug treatment) lung capacity. Eur Respir J 2008;31:391-95.

RESP. EXPERIMENT: 10
Aim
To evaluate the effect of salbutamol inhalation
in pulmonary function test in healthy male
volunteers.
Background
Presently, there are large number of pulmonary
tests available, from simple to sophisticated one,
which provide useful information regarding lung
functions like ventilation, perfusion and diffu-
sion of lungs. The PFT is used for different
reasons such as for diagnosis, to assess effective-
ness of treatment, to evaluate physical fitness
before cardiovascular surgery and in uncommon
situations like for legal opinion for hazardous
occupation.
Materials and Methods
Volunteers are screened for any cardiovascular,
respiratory disorder or any other conditions
which may interfere with experimental protocol.
Volunteers with smoking habit should be
excluded.
Healthy adult volunteers of either sex (willing
to give written informed consent)
Computerized Spirometer
Stethoscope, mercury sphygmomanometer
Drug: Salbutamol inhaler
Precautions for Experiment
• Subject should be advised for adequate sleep
over night and light breakfast in the morning
day of experiments (preferably 2 hr before)
• Avoid caffeinated drink or smoking or at least
2 hr before the experiment or free of any
substance abuse
• Volunteer should get complete instructions
of the procedure and its rehearsal, so to avoid
any conditional anxiety in the volunteers
• Monitoring equipment should be maintained
and regularly calibrated.
• Oxygen source and suction device should be
accessible.
Respiratory System  283
• Basic operating procedure of the selected
instrument should be well explained to the
volunteers
• Testing procedure and its consequences
should be discussed beforehand
• Training of volunteers for correct use of
spirometer should be explained properly
(Volunteer is asked to take deep breath in, then
breath out with maximum force and as hard
and as fast as possible, with a good airtight
seal of the lips around the tube, usually done
standing up; the best of three attempts is
usually taken, with a short rest between
attempts).
Methods
Volunteers are asked to report early morning before
breakfast. Smoking and caffeine containing
beverages are prohibited. Baseline BP, HR, RR and
PFT should be measured. After baseline measure-
ment volunteer is given salbutamol inhalation and
PFT and other evaluation should be repeated after
30 minutes.
Measurement of Pulmonary Function Test
At baseline and after treatment following para-
meters are recorded: Forced vital capacity (FVC),
Forced expiratory volume in half second (FEV0.5),
Forced expiratory volume in one second (FEV1),
Forced expiratory volume in three second (FEV3),
Peak expiratory flow rate (PEFM), Mean force
expiratory flow during middle half of the
FVC(FEF2-12), Mean forced expiratory flow rate
between 0.2 and 1.2 liters of volume change
(FEFR25-75), Forced expiratory flow after 25% of
FVC has been expired (FEF 25%), Forced
expiratory flow after 50% of FVC has been
expired (FEF 50%), Forced expiratory flow after
75% of FVC has been expired (FEF 75%), Forced
expiratory volume ratio, (FEV0.5/FVC), Forced
vital capacity ratio (FEV1/FVC), FEV3/FVC and
maximum voluntary ventilation (MVV). To
record above mentioned parameters instrument
is calibrated and volunteer’s data entry is done.
Before measuring, PFT procedure is explained
in detail to the volunteer and let him/her practice
284  Practical Manual of Experimental and Clinical Pharmacology
once or twice. Nasal clip is applied and clean
mouthpiece is attached to the breathing tube then
volunteer is asked to take maximum inspiration
after placing the mouthpiece appropriately
followed by maximum expiration for forced
expiratory phase.
There are several sources of error e.g.: (1)
inadequate subject performance, either failure of
full inspiration or inadequate expiratory effort
(2) error in the instrument, (3) failure to hold
mouth piece properly. Above mentioned minor
errors may alter the readings. Hence, it is
advisable to take either last three of the total 5
readings or the highest obtained reading, one of
the above methods should be practiced.
Procedure for Inhalation
The correct use of inhaler is very important and
volunteers should be explained in detail about
different steps of using inhaler. Inhaler has several
components like plastic body with mouth piece,
mouth piece cover and canister. Before using
inhaler, it should be shaken well and then test fire
shoule be done; means release one puff into air.
The following steps should be followed: After
removing mouth piece first clean the mouth piece,
shake adequately, place the inhaler upside down
in one or two finger on the top of the canister. Take
the respiration gently by mouth. Inhaler mouth-
piece should be placed between teeth and lips
without biting then ask volunteer to start breathing
slowly from mouth, as the volunteer breath in
canister should be pressed down to release the one
dose with continuously breath in deeply. After
removing the inhaler, hold the breath for at least
10 sec or as long as it is comfortable and breathing
should be slow.
Observations and Result
Following parameters should be recorded
Parameters 0 hr 0.5 hr
Placebo Salbutamol Placebo Sal-
butamol
SBP (mm Hg)
DBP (mm Hg)
HR (per min)
RR (per min)
FVC
FEV0.5
FEV1
FEV3
PEFR
FEF 2-12
FEF 25-75
FEF 25%
FEF 50%
FEF 75%
FEV0.5/FVC
FEV1/FVC
FEV3/FVC
MVV
Statistical analysis: All the data should be
expressed as mean ± SD, comparison of baseline
value and following salbutamol inhalation is
done by paired ‘t’ test (experiment on same
volunteer).
Discussion
Lung function depends on various factors such
as effect of posture on vital capacity, which is
higher in erect posture as compared to sitting and
lying posture. Since, more physical effort is
applied in erect posture and in this posture
diaphragm is decent it increase capacity of
thoracic cage with elimination of gravity. So,
blood flow to the lung is decreased because of
gravitational effect thus ultimately causing
increased vital capacity on standing. Studies
reported altered PFT in healthy volunteers with
complete trismus on pulmonary function using
objective and subjective parameters. In healthy
volunteers, it was showed that the significant
changes in test data stimulated mild to moderate
pulmonary impairment, whereas patients with
an already impaired pulmonary function
showed a marked deterioration of their initial
respiratory condition. Besides this, other sophisti-
cated methods like completely noninvasive
oxygen-enhanced pulmonary function test have
potential for clinical applications in the serial
diagnosis of lung diseases. Other study reported
that there is no difference on use of nasal clip on
PFT.

SUGGESTED READING
1. Agarwal D, Gupta PP, Sood S. Evaluation of
pulmonary function testing with and without nose
clip in healthy volunteers. Ind J Allergy Asthma
Immunol 2004;18(2):83-86.
2. Guleria R, Singh TR, Sinha S, Padhy AK, Gupta K,
Pande JN. Effect of inhalation of salbutamol,
beclomethasone dipropionate and ipratropium
bromide on mucociliary clearance in some patients
with chronic stable bronchial asthma. Indian J Med
Res 117, April 2003;158-63.
3. Jakob PM, Wang T, Schultz G, Hebestreit H,
Hebestreit A, Hahn D. Assessment of human
pulmonary function using oxygen-enhanced T(1)
imaging in patients with cystic fibrosis. Magn Reson
Med 2004;51(5):1009-16.
4. Spiekerkoetter E, Fabel H, Hoeper MM. Effects of
inhaled salbutamol in primary pulmonary hyper-
tension Eur Respir J 2002;20:524-28.
RESP. EXPERIMENT: 11
Aim
To evaluate pulmonary function test following
Stair climbing exercise tolerance test.
Background
Altered pulmonary function and exercise
capacity has been reported in various lung
diseases like bronchial asthma, chronic obstruc-
tive pulmonary disease or as a result of resection
of lung. Aim of present experiment is to evaluate
the changes of pulmonary function and exercise
tolerance using stair climbing test. Stair climbing
test is popular because it is simple, economical,
and can be performed in short period. This test
can be applied for multiple evaluations on a large
number of volunteers or in patients in clinic, since
it more closely resembles to normal daily activity
as compared to cycle ergometry, and cycle
ergometry is more stressful than any other form
of exercise test.
Materials and Method
Volunteers are screened for any cardiovascular,
respiratory disorder or any other conditions
which may interfere with experimental protocol.
Volunteers with smoking is excluded
Respiratory System  285
Healthy adult volunteers of either sex (willing
to give written informed consent)
Computerized spirometer
Stethoscope, mercury sphygmomanometer
Pulsoxymeter
Precautions
• Subject should be advised to have adequate
sleep over night and light breakfast in the
morning day of experiments (preferably 2 hr
before)
• Caffeinated drink or smoking should be
avoided at least 2 hr before the experiment
or free of any substance abuse
• Volunteer should get complete instruction of
the procedure and its rehearsal, so to avoid
any conditional anxiety in the volunteers
• Monitoring equipment should be maintained
and regularly calibrated.
• Oxygen source and suction device should be
accessible.
• Basic operating procedure of the selected
instrument should be well explained to the
volunteers
• Testing procedure and its consequences
should be discussed before hand
• Training of volunteers for correct use and
detailed explanation of pulmonary function
test for example there should be a good air
tight seal of the lips around the tube, usually
done standing up and stair climbing exercise
test.
Stair Climbing Exercise Test
The stair climbing test is a symptom-limited
exercise. In this exercise, volunteers are usually
advised to climb 16 flights of stairs, each flight
having 11 steps. Each step is 0.155 m in height.
The volunteers climb at a pace of their own
choice, the maximum number of steps and to stop
only on exhaustion, limiting dyspnea, leg fatigue,
or chest pain. During the exercise, pulse rate and
capillary oxygen saturation should be monitored
using a portable pulse-oxymeter. For every
volunteer, the number of steps climbed and the
time taken to complete the test should be
recorded to calculate different ergometric
variables: work and VO2 peak.
286  Practical Manual of Experimental and Clinical Pharmacology
Method
Volunteers are asked to report early morning
after light breakfast. Smoking and caffeine
containing beverages are prohibited. Baseline BP,
HR, RR, oxygen saturation and PFT are measured
with the subject in the sitting position. Same
procedure is repeated following exercise.
Measurement of Pulmonary Function Test
Following parameters are recorded: Forced vital
capacity (FEV), FEV 1 , FEV 1 /FVC, etc. as
mentioned in experiment number 10. To record
above mentioned parameters instrument should
be calibrated and volunteer’s data entry should
be done. Before measuring the PFT volunteers
should be informed in detail about the procedure
and volunteers should practice once or twice.
Nasal clip is applied and clean mouthpiece is
attached to the breathing tube then volunteer is
asked to take maximum inspiration after placing
the mouthpiece appropriately followed by
maximum expiration for force expiratory phase.
There are several sources of error for example:
(1) inadequate subject performance, either failure
of full inspiration or inadequate expiratory effort
(2) error in the instrument, (3) failure to hold
mouth piece properly. Above mentioned minor
errors may alter the readings. Hence, it is
advisable to take either last three of the total 5
readings or the highest obtained reading, one of
the above methods should be practiced.
Observations and Results
Following parameters should be recorded
Parameters Before exercise After exercise
(Baseline)
Age, yr
Body mass index, kg/m2
SBP (mm Hg)
DBP (mm Hg)
HR (per min)
RR (per min)
VO 2 peak, mL/kg/min
FVC
FVC 0.5
FVC1
FVC3
PEFR
FEF 2-12
FEF 25-75
FEF 25%
FEF 50%
FEF 75%
FEV 0.5/FVC
FEV 1/FVC
FEV3/FVC
MVV
Statistical Analysis
Volunteers are evaluated by using pulmonary
function testing (PFT) and stair climbing tests.
Pulmonary function test results are expressed
as percentage of predicted value according to
age, sex, and height as per standard guide-
line and all the data should be expressed as
mean ± SD.
Discussion
The objectives of the present experiment is to
evaluate the changes of pulmonary function
following exercise and to evaluate exercise
tolerance, the stair climbing test used as a form
of exercise testing. Several studies have already
shown a good correlation between stair clim-
bing and cycle ergometry test. It has been found
that higher values of VO2 peak measured during
stair climbing. Although the accuracy of the cal-
culation of VO2 peak during the stair climbing
test is not universally accepted, and a compa-
rison between the calculated VO2 peak at stair
climbing test and the VO2 peak measured at
cycle ergometry should be done. Stair climbing
is commonly used because it is simple, econo-
mical, brief and easier than cycle ergometry, so
in the clinic it can be widely used.

SUGGESTED READING
1. Holden DA, Rice TW, Stelmach K, et al. Exercise
testing, 6-min walk, and stair climb in the evaluation
of patients at high risk for pulmonary resection.
Chest 1992;102:1774-79.
2. Morice RC, Peters EJ, Ryan MB, et al. Exercise testing
in the evaluation of patients at high risk for lung
resection. Chest 1990;98:54S.
3. Nicholas Hart, Annabel H Nickol, Derek Cramer,
Simon P. Ward, Frédéric Lofaso, Neil B. Pride, John
Respiratory System  287
Moxham, Michael I. Polkey. Effect of Severe Isolated
Unilateral and Bilateral Diaphragm Weakness on
Exercise Performance. Am J Respir and Cri Car Med
2002;165:1265-70.
4. Pollock M, Roa J, Benditt J, et al. Estimation of
ventilator reserve by stair climbing: a study in
patients with chronic airflow obstruction. Chest
1993; 104:1378-83.
5. Swinburn CR, Wakefield JM, Jones PW. Performance,
ventilation, and oxygen consumption in three different
types of exercise tests in patients with chronic
obstructive lung disease. Thorax 1985;40:581-86.
288  Practical Manual of Experimental and Clinical Pharmacology

EXERCISE

Excercise No. 1: 39-year-old male presenting with history of occasional breathlessness since 6 months, has been
referred for pulmonary function test (PFT).
Interpret the findings of PFT and discuss the drug therapy?

Name: SR
ID
Diagnosis:
Age: 39 Height: 176 cm Weight: 92 kg Sex: Male

Spirometry Predicted Observed Observed Percent

Value Pre Pred Post Pred Change
FVC L 4.18 3.34 79 3.9 93 16
FEV0.5 L 2.64 1.66 62 1.92 72 15
FEV1 L 3.45 2.29 66 2.6 75 13
FEV3 L 3.94 3.03 76 3.43 87 13
FEV1/FVC % 82 69 84 67 81 –2
FEV3/FVC % 95 91 95 88 92 –3
FEF25-75 L/S 3.62 1.49 41 1.5 41
PEFR L/S 7.82 6.81 87 7.1 90 4
FEF25 L/D 7.24 3.98 54 4.57 63 14
FEF50 L/S 4.32 1.96 45 2.00 46 2
FEF75 L/S 1.77 0.6 33 0.54 30 –10
FET Sec 6.82 7.27 6
FTVC L 3.55 3.39 95 3.83 107 12
PIFR L/S 5.21 4.84 92 4.76 91 –1
FIF50 L/S 4.21 4.65 10
 Respiratory System  289

Exercise No. 2: 50-year-old female presenting with history of respiratory difficulty since 6 months, has been
referred for pulmonary function test (PFT).
Interpret the findings of PFT and discuss the drug therapy?

Name:
ID:
Diagnosis:
Age: 50 Height: 160 cm Weight: 63 kg Sex: F

Spirometry Predicted Observed Observed Percent

value Pre % pred Post % Pred Change
FVC L 2.61 1.97 75 2.03 77 3
FEV0.5 L 1.65 0.91 55 0.98 59 7
FEV1 L 2.16 1.28 59 1.3 60 1
FEV3 L 2.4 1.71 71 1.76 73 2
FEV1/FVC % 82 65 79 64 78 –1
FEV3/FVC % 95 87 91 87 91
FEF25-75 L/S 2.34 0.78 33 0.73 31 –6
PEFR L/S 4.98 2.73 54 3.91 78 43
FEF25 L/S 4.68 2.73 58 2.72 58
FEF50 L/S 1.91 1.04 54 0.94 49 –9
FEF75 L/S 1.16 0.25 21 0.26 22 4
FET Sec 6.89 6.97 1
FIVC L 2.61 2.06 78 2.05 78
PIFR L/S 3.32 2.45 73 2.97 89 21
FIF50 L/S 2.37 2.94 24

Diffusion Predicted Observed

Value Pre % Pred
Dlco corr 2326 12.62 54
Dlco Unc 23.26 12.76 54
Va obtps 5.5 2.33 42
DL/VA 5.14 5.41 105
290  Practical Manual of Experimental and Clinical Pharmacology

Excercise No. 3: 49 years old male presenting with history of respiratory difficulty since 6 months, has been
referred for pulmonary function test (PFT).
Interpret the findings of PFT and discuss the drug therapy?

Date:
Name:
Diagnosis:
Age: 49 Height: 170 cm Weight: 73 kg Sex: M
Physician: Consulting:

Spirometry Predicted Observed Observed Percent

Value Pre % Pred Post % Pred Change
FVC L 3.5 2.73 78
FEV0.5 L 2.31 0.86 37
FEV1 L 2.86 1.56 54
FEV3 L 3.36 2.66 79
FEV1/FVC % 81 57 70
FEV3/FVC % 94 97 103
FEF25-75 L/S 3.02 1.27 42
PEFR L/S 7.04 1.84 26
FEF25 L/S 6.49 1.73 26
FEF50 L/S 3.66 1.34 36
FEF75 L/S 1.41 0.99 70
FET Sec 3.94
FIVC L 2.98 2.4 80
PIFR L/S 4.69 1.34 28
 Central Nervous System
28
CNS EXERCISE NO: 12
Aim
To demonstrate the effect of various drugs on
psychomotor function of healthy volunteers.
Background
Psychomotor function tests are commonly used
to assess effect of environmental stressors with
wide range of clinical utility including evaluation
of various drugs. There are various psychomotor
assessment tests and these are aim at evaluation
of sensory, motor and central function
assessment (Figs 28.1 and 28.2). It is a procedure
for critical evaluation of subject’s ability to
perceive and follow instructions and perform
motor responses which also including psycho-
motor reaction time. Hence, it determines
psychomotor functions like physical responses,
intelligence, or ability to perform or solve several
psychological questionaires or tests. It confirms
the motor and psychological components of
mental process, for example as in depression
psychomotor function is altered. The test is
performed to assess the psychomotor the patients
with depression, Parkinson’s, Huntington’s or
even in seizure etc. and many more. Hence, it is
important to familiar with the methodology of
the procedures of the various kinds of tests.
Materials and Methods
• Healthy volunteers (willing to give written
informed consent)
• Visual analogue scale (VAS), Card, Critical
flicker fusion, Hand Stabilometer
• Drugs: Placebo, Chlorpheniramine (10 mg)
and Fexofenadine (120 mg)
(other drugs also can be used like diazepam,
lorazepam, clobazam, etc. for this practical’s)
Inclusion criteria: Healthy volunteers aged 18-
40 years with minimum education of 12
standards is advisable
Exclusion criteria: Incase of pregnancy, lactation
or planning pregnancy, volunteers with H/o
drug allergy, volunteers should be taking any
drugs which can affect psychomotor function,
renal and hepatic disease
Precautions
• Volunteers should be advised for adequate
sleep over night and light breakfast in the
morning on day of experiments (preferably
2 hr before)
• Caffeinated drink or smoking should be
avoided at least 2 hr before the experiment
or free of any substance abuse
• Volunteer should get complete instruction of
the procedure and its rehearsal, in order to
avoid any conditional anxiety of volunteers.
They should be explained that adequate
cooperation and concentration is essential.
Volunteers should be trained till the investi-
gator obtain consistent results
• Monitoring equipment should be maintained
and regularly calibrated
• Basic operating procedure of the selected
instrument and the test should be well
explained to the volunteers
292  Practical Manual of Experimental and Clinical Pharmacology

Fig. 28.1: Psychomotor function assessment test

Fig. 28.2: Psychomotor function tests


Methodology
Baseline psychomotor functions are assessed
prior to using the following tests. The drug is
administered and after that the following tests
are repeated at same sequences, and then mean
values before and after treatment are compared.
1. Card sorting: Volunteer should be given a pack
of cards and is asked to sort then into the
different groups of diamonds, hearts, spades
and clubs as quickly as possible. (fix the card
type among the four or all) Time, to do the task
is noted pre and post treatment.
2. Subjective Assessment: This assessment
done on the basis of VAS score. Volunteers
are assessed for their mood and well being
on a series of visual analogue scale (VAS)
with a range from 0-10 or 0-100. The
assessment is done for 6 different features.
Visual analogue scale (VAS: 0-100)
1. Sleepy—————————————Awake
2. Tense—————————————Relaxed
3. Lethargic———————————Energetic
4. Mentally dull———————————Alert
5. Inability to concentrate——————Ability
to concentrate
6. Anxious————————————Casual
3. Critical flicker fusion threshold: The CFF
apparatus consists of a flickering light source
against a dark background. A dial is provided
in front to adjust the number of flicker per
second. The volunteer is asked to see the
flickering light for a min at 5 Hz. The dial is
then slowly moved at the rate of 2 Hz/sec.
The volunteer is then asked when the light
appears fused. Volunteer is asked to look at
this frequency for a minute. Then the rate is
decreased at 2 Hz/sec till the light appears
as flickering. This frequency is then noted.
The average of 3 such reading is noted.
4. Finger Tapping: Volunteer is asked to tap a
particular key (computer/calculation/table
Central Nervous System  293
top etc.) with their index finger for 15 sec.
Then, total numbers of taps are noted.
5. Short term Memory: Volunteers are shown
10 unrelated objects or 10 words and left for
1min to memorize the objects. Then, 4hr later,
volunteers are given 1 min to recall and write
down the objects as many as possible.
6. Six letter cancellation: Volunteer is presented
with a sheet containing randomized letters
arranged in columns/row. Target six letters
and asked to cancel targeted letters as much
as possible in 2 min in any selected direction
(horizontal/vertical/random) and number of
letters correctly cancelled, attempted and
errors in selection are recorded.
7. Digit symbol substitution test: Test is applied
to evaluate cognitive function, attention and
psychomotor speed. Volunteers are given a
code table, displaying the correspondence
between pairs of digits (from 1 to 9) and
symbols. They have to fill in blank squares
with the symbol that is paired to the digit
displayed above the square. The subjects have
to fill in as many squares as possible in 90
seconds.
8. Digit span: It is a measure of short term
memory in which volunteers have to
memorize few digit spans from 1-9 and have
to recall in a span of time. The order of recall
the digit is important such as if the digit
sequences is 1-4-2-5-3 or 5-2-6-2-3 or 9-2-5-1-
4. Then the recall is checked in the order of
digits in any sequence. Volunteers are given
one digit/sec and volunteer have to repeat
the sequence in the correct order.
9. Immediate recall memory: This task is also
known as word list memory task. In which
volunteers are presented with a list of 10
related words at the interval of one word/2
sec (18 sec). Then, volunteers are asked to
294  Practical Manual of Experimental and Clinical Pharmacology

recall/write down all 10 words after 45 seconds.
The sequences of the word may be checked.
10. Hand Stabilometer: The volunteer is asked to
insert a probe into the different (descending
order of diameter) diameter hole. First, instruct
the volunteers and give a trial run, then start
the test from the largest diameter hole to the
lowest one. The moment probe touches the
margin of hole, a light indicate and experiment
is ended. The point and time taken to reach
the particular point is noted (Fig. 28.3).
11. Pegboard test: The test is used for the gross
movements of hands, fingers and fingertip
dexterity. Volunteers are demonstrated with
a board with the space to fix the pegs and
instructed (Fig. 28.4). Then, they are asked to
fix the entire peg in their correct places. The
time taken to fix the pegs and percentage of
correct performances are recorded.

Fig. 28.4: Pegboard test

Study design: Double blind placebo controlled
Fig. 28.3: Hand stabilomotor study

Observations and Results

Tests for Assessment Different groups of volunteers
Placebo Chlorpheniramine Fexofenadine
1. Card Sorting
2. Subjective assessment
3. Critical flicker fusion threshold
4. Finger tapping
5. Short term memory
6. Six letter cancellation
7. Digit symbol substitution test
8. Digit Span
9. Immediate recall memory
10. Hand Stabilometer
11. Pegboard test

Overall evaluation: Subjective evaluation Statistical Test

Objective evaluation Data should be express as mean ± SD and for
Percentage of impairment (%) comparison is made by unpaired ‘t’ test or
Conclusion: ANOVA, if mean is more than 2.

Discussion
Presently, various psychomotor function tests
have been used to measure the effects of
psychoactive compounds on human behavior
and every test has its own merits and demerits.
The psychopharmacologist should be familiar
with judgement in behavioural terms and
accurate assessment but at times the range of
behavioral activities presented is critical to
evaluate.
These tests help in evaluation various drugs
whether it is psychologically relevant, and it
should be evaluated in model of behavior. It is
very much clear that mode and level of activity of
the brain and central nervous system is dependent
upon personality, motivation and memory so it is
necessary to examine different factor interfering
with performance. Various studies have already
reported effect of different drug on psychomotor
functions for example, an early recovery time
propofol sedation is similar in elderly and younger
patients, but recovery of psychomotor function in
the elderly is delayed compared with younger
patients. Patients with epilepsy who require high
CBZ concentrations for optimal control of seizures
may be at risk of concurrent impairment of
psychomotor function. Study done to evaluate the
effects of various drugs like haloperidol (1 mg),
benzhexol (5 mg), diazepam (10 mg) and caffeine
(400 mg) on subjective and objective measures of
cognitive and psychomotor function compared
with placebo in 20 healthy volunteers showed
diazepam and benzhexol are associated with
significant impairments in subjective alertness,
critical flicker fusion threshold and choice reaction
time (CRT), however, haloperidol could not be
distinguished from placebo in psychomotor tests
but it is associated with an apparent improvement
in CRT (in males) and simple visual reaction time.
Finally, perceptual maze test detected impairment
by benzhexol on processing speed but was not
sensitive to any other drug effects. Another study
reported with efficiency of measurement of
variability which are generally not coated in the
Central Nervous System  295
study. So, efficiency was evaluated from five recent
studies to calculate the coefficients of variation for
different tests of psychomotor function, both during
and in study days. Study showed saccadic eye
movement analysis and critical flicker fusion
threshold is found to have low coefficients of
variation ( 2·7 to 7·5 %) and visual analogue rating
scales and the digit symbol substitution test are
reported to have much higher coefficients of
variation (7·9 to 17·6%). This indicates that
measures of test variability should be routinely
assessed during psychomotor assessment studies
to validate and also to check test methodology.
Note
Visual analogue assessment (VAS) can have variation
depending of volunteers/ patients right or left handed person
because right handed person have more tendency to mark
toward right side and same thing is applied for left handed
person also. So, it is advisable to place VAS in vertical
direction.
SUGGESTED READING
1. Adam JK, Rmaih WN. A double-blind placebo-
controlled investigation of the psychomotor
profile of clopidogrel in healthy volunteers. J
Cardiovasc Pharmacol 2008;52(6):507-09.
2. Aitken RCB. Measurement of feelings using
visual analogue scales. Proceedings of the Royal
Society of Medicine 1969;62:989-93 Freyd M.
The graphic rating scale. Journal of Educational
Psychology 1923;43: 83-102 Hayes, MHS, DG
Patterson. Experimental development of the
graphic rating method. Psychological Bulletin,
1921;18:98-99.
3. Clubley M, Bye CE, Henson TA, Peck AW,
Riddington CJ. Effects of caffeine and cyclizine
alone and in combination on human
performance, subjective effects and EEG
activity. Br J Clin Pharmacol 1979;7(2):U57-
63.
4. DJ King, G Henry. The effect of neuroleptics on
cognitive and psychomotor function. A
preliminary study in healthy volunteers. The
British Journal of Psychiatry 1992;160:647-53.
5. Effect of sodium valproate on carbamazepine
disposition and psychomotor profile in man.
Macphee GJ, Mitchell JR, Wiseman L, McLellan
AR, Park BK, McInnes GT, Brodie MJ. Br J Clin
Pharmacol 1988;25(1):59-66.
296  Practical Manual of Experimental and Clinical Pharmacology
6. Freyd M. The graphic rating scale. Journal of
Educational Psychology 1923;43:83-102.
7. Hart J, Hill HM, Bye CE, Wilkinson RT, Peck
AW. The effects of low doses of amylobarbitone
sodium and diazepam on human performance.
Br J Clin Pharmacol 1976; 3(2):289-98.
8. Hayes MHS, DG Patterson. Experimental
development of the graphic rating method.
Psychological Bulletin, 1921;18:98-99.
9. Ian Hindmarch. Psychomotor function and
psy-choactive drugs. ‘Br J Clin Pharmacol
1980;10: 189-209
10. Lynch G, King DJ, Green JF, Byth W, Wilson-
Davis K The effects of haloperidol on visual
search, eye movements and psychomotor
performance. Psycho-pharmacology (Berl)
1997;133(3):233-39.
11. MF Mannion1, G Lynch2, DJ King. Precision of
measures of saccadic eye movements and
conventional psychomotor function tests.
Human Psychopharmacology 2004;9(1):37-41.
12. Murphy SE, Downham C, Cowen PJ, Harmer
CJ. Direct effects of diazepam on emotional
processing in healthy volunteers. Psycho-
pharmacology 2008;199(4):503-13.
13. Peck AW, Bye CE, Clubley M, Henson T,
Riddington C. A comparison of bupropion
hydrochloride with dexamphetamine and
amitriptyline in healthy subjects. Br J Clin
Pharmacol 1979;7(5):469-78.
14. Toshimitsu Kitajima. Recovery of psychomotor
function after propofol sedation is prolonged in
the elderly. Can J Anesth 2002;49:9;927-31
15. Wechsler D. WAIS-R manual. New York, NY:
Psychological Corporation, 1981.
 Central Nervous System  297

EXERCISE

Exercise no. 1: 30-year-old female presenting with bilateral hand paresthesia, on examination patients could
lift, left hand more than right on radial three finger for 1st three years. More at night time with pain impairing
sleep also. Possibility of bilateral carpal tunnel syndrome was kept. Plan was to rule out, diabetes and
hypothyroidism. Nerve conduction study was planned and showing normal nerve conduction although latency
is prolonged at left side than right side. Describe your interpretation.

Fig. 28.5
298  Practical Manual of Experimental and Clinical Pharmacology

Fig. 28.6
 Central Nervous System  299

Exercise no. 2: 20-year old female presented with H/o seizure for 10 yr. GTCS with frequency once in every 3-
4 months. (Usually nocturnal seizure with myoclonic jerks. No h/o cognitive decline. Family history was
negative. Describe the EEG findings and discuss pharmacological therapy.

Fig. 28.7
300  Practical Manual of Experimental and Clinical Pharmacology

Fig. 28.8
 Central Nervous System  301

Exercise no. 3: 30-year-old female presented with seizure for past 15 yrs, GTCS at morning and night with
frequency of 1 in 2-3 months. h/o myoclonic jerk is present . Family h/o positive, no history of cognitive
decline. Describe the EEG findings and discuss pharmacological therapy.

Fig. 28.9
302  Practical Manual of Experimental and Clinical Pharmacology

Fig. 28.10

29
EXPERIMENT NO: 13
Aim
To evaluate the effect of frusemide on urine
volume and Na+ and K+ excretion in healthy
volunteers.
Background
Thiazide diuretics cause moderate diuresis and
natriuresis. Loop diuretics cause marked diuresis
and natriuresis, whereas K+ sparing diuretics
produce diuresis and preserve K+. Furosemide
(frusemide) is a loop diuretic commonly prescri-
bed for congestive cardiac failure and edema,
initially it was used in horse-race to prevent nose
bleeding. Presently, furosemide is included in the
world anti- doping banned drugs list. It acts by
inhibiting Na+-K+ -2Cl– sympoter in the thick
ascending limb of loop of Henle, action mostly
on distal tubules and has weak carbonic anhy-
drase-inhibiting activity. So, the experiment is
aimed to determine the diuretic and natriuretic
effects of frusemide.
Materials and Methods
Initial Screening of Volunteers
Volunteers are screened for any cardiovascular,
respiratory disorder or any other medical
unstable conditions which may interfere with
experimental protocol. Volunteers with smoking
are excluded.
Healthy adult volunteers of either sex (willing
to give written informed consent)
Kidney
Stethoscope, mercury sphygmomanometer,
Facilities for clinical pharmacology laboratory
(Appendix IV)
Drug: Tablet Frusemide (40 mg)
Precautions
• Subject is advised for adequate sleep over
night and light breakfast in the morning on
day of experiment (preferably 2 hr before)
• Avoid caffeinated drink or smoking or at least
12 hr before the experiment or free of any
substance abuse
• Volunteer should get complete instructions
of the procedure before the experiment
• Monitoring equipment is maintained and
regularly calibrated
• Basic operating procedure of the selected
instrument should be well explained to the
volunteers.
Methods
The study requires healthy volunteers. Following
preliminary physical examination, volunteer is
given breakfast and asked to refrain from
smoking tea, coffee or other caffeinated beve-
rages 12 hr before and during study. Fluid intake
is regulated and volunteers are adviced to rest
in a room with ambient temperature (25 ± 2ºC)
in clinical pharmacology laboratory.
Inclusion Criteria
1. Healthy volunteer aged 18-40 years
2. Willing to give written informed consent.
304  Practical Manual of Experimental and Clinical Pharmacology

Exclusion Criteria Detection limit of Na+= 0.0001ppm

1. h/o hypersensitivity (particularly allergy to
frusemide)
2. Electrolyte imbalance and hypertension.
Volunteers should be given frusemide 40 mg
with 200 ml of water. Voiding urine, collection
is done at every ½ hr, for 2 hrs and then hourly
for 6 hrs. Amount of water equal to volume of
urine each time is replaced. Samples are assessed
for Na+ and K+ by flame photometer or by auto-
analyzer. All the vital parameters BP and HR and
adverse drug reactions should be recorded.
Flame Photometer Method
Estimation of Na+, K+ routinely is done by flame
photometer method. Detection limit is as follows:
Detection limit of K+= 0.001ppm
Procedure
Stock solution of 1 gm/mEq/L of Na+ and K+ is
prepared. Then, solutions are diluted to make
different strength solution. Na+ std solution 110
mEq/L and 170 mEq/L and K+ std solution are
8, 6, 4 and 2 mEq/L. The reading of photometer
are adjusted and corresponding reading of
unknown samples are directly read from
photometer.
Discussion
In the flame photometer, the solution evaporates
and substance is first converted to atomic state.
The electrons in the higher energy orbit are in a

Observation and Results

Na+ conc. of unknown = ?
K+ conc. of unknown = ?

Volunteer No 1

Time Blood pressure Heart rate Urine volume Na+ K+ ADR

0.5 hr
1 hr
1.5 hr
2 hr
3 hr
4 hr
5 hr
6 hr

Volunteer No 2

Time Blood pressure Heart rate Urine volume Na+ K+ ADR

0.5 hr
1 hr
1.5 hr
2 hr
3 hr
4 hr
5 hr
6 hr

meta stable state and are prone to return to lower
energy orbit including ground state and energy
previously absorbed is released as quanta of light,
the wave length of which depends upon the
energy levels which the electrons can attain and
are characteristics of each substance commonly
called “emission spectrum”. In emission flame
photometry, the relatively low energy flame
excites a few elements mainly alkali metals. Part
of light which is emitted in all directions is
collected by a reflector and falls on a detector.
The light intensity and hence the detector output
is directly proportional to the concentration of
substance.
Frusemide causes marked diuresis in terms
of urinary volume. The onset of diuresis is within
1 hour. The peak effect occurs within the first
and second hour. The duration of action is 6-8
hours. The duration of diuretic effect is approxi-
mately 2 hours. It also causes an increase in Na+
and K+ excretion. Total urine output usually
occur within 6 hrs approximately 3500 ml. K+
and Na+ excretion increases peaking at 1 hr. BP
and HR is not usually affected. Volunteers may
complain of bad taste and weakness.
SUGGESTED READING
1. Arancibia A, Nella Gai M, Paulos C, Chávez J, Pinilla
E, Angel N, Ritschel WA. Effects of high altitude
exposure on the pharmacokinetics of furosemide
in healthy volunteers. Int J Clin Pharmacol Ther
2004; 42(6):314-20.
2. Klausner EA, Lavy E, Stepensky D, Cserepes E,
Barta M, Friedman M, Hoffman A. Furosemide
pharma-cokinetics and pharmacodynamics
following gastroretentive dosage form administra-
tion to healthy volunteers. J Clin Pharmacol
2003;43(7): 711-20.
3. Musso CG, Reynaldi J, Vilas M, De Miguel R,
Imperiali N, Algranati L. Fractional excretion of K,
Na and Cl following furosemide infusion in healthy,
young and very old people. Int Urol Nephrol 2009
Mar 10.
4. Noskov VB, Goncharov IV, Kovachevich IV,
Repenkova LG, Kodratenko SN, Starodubtsev AK.
The pharmacodynamics and pharmacokinetics of
furosemide during ordinary life activities and
during head-down tilt hypokinesia in a healthy
subject] Eksp Klin Farmakol 1998;61(4):29-33.
Kidney  305
5. Verma AK, da Silva JH, Kuhl DR. Diuretic effects of
subcutaneous furosemide in human volunteers: A
randomized pilot study. Ann Pharmacother 2004;
38(4):544-49.
KIDNEY EXPERIMENT: 14
Aim
To evaluate saluretic, natriuretic and carbonic
anhydrase inhibitory effect of various diuretics
in healthy volunteers.
Background
Excretion of salt and water is important for
treatment of condition like peripheral edema
following congestive heart failure and also used
for treatment of hypertension. Aim of this
experiment is to calculate saluretic activity by
calculating the sum of Na+ and Cl– excretion,
natriuretic activity by the ratio Na+ /K + and
carbonic anhydrase inhibition from the ratio
Cl-/(Na+) + (K+). Most commonly used drugs
are frusemide, thiazide diuretic in the clinical
practice.
Materials and Methods
Initial Screening of Volunteers
Volunteers are screened for any cardiovascular,
respiratory disorder or any other medical
unstable conditions which may interfere with
experimental protocol. Volunteers with smoking
habit and alcoholics should be excluded.
Healthy adult volunteers of either sex (willing
to give written informed consent)
Stethoscope, mercury sphygmomanometer
Drug: Frusemide, thiazide diuretic
Precautions
Refer to experiment no.: 13
Methods
The study requires healthy volunteers. After
preliminary physical examination, volunteer is
306  Practical Manual of Experimental and Clinical Pharmacology

given breakfast and asked to refrain from smo-
king tea, coffee or other caffeinated beverages 12
hr before and during study. Fluid intake is
regulated and volunteers are kept in a room with
ambient temperature (25±2ºC) in clinical phar-
macology laboratory.
Inclusion Criteria
1. Healthy volunteer aged 18-40 years
2. Willing to give written informed consent
Exclusion Criteria
1. h/o hypersensitivity,
2. Electrolyte imbalance and hypertension.
One volunteer advise to take frusemide 40 mg
with 200 ml of water and other one 25mg with
200ml of water. Voiding of urine is done at every
½ hr, for 2 hrs and then hourly for 6 hrs. Amount
of water equal to volume of urine each time is
replenished. Samples are assessed for Na+ and
K + on flame photometer and Cl - argento-
metrically by potentiometrical end point titration
method. All the vital parameters BP and HR are
recorded. Following parameters are assessed
saluretic activity from Na+ and Cl– excretion,
natriuretic activity by calculating the ratio, Na+/
K+ and calculation to estimate carbonic anhy-
drase inhibition from the ratio Cl-/(Na+) + (K+)
i.e. (ion quotient)
(For procedure refer experiment no.:13)

Observations and Results

Volunteer No 1

Time Blood Pressure Heart Rate Urine volume Na+ K+ Cl– ADR
0.5 hr
1 hr
1.5 hr
2 hr
3 hr
4 hr
5 hr
6 hr

Volunteer No 2

Time Blood Pressure Heart Rate Urine volume Na+ K+ Cl- ADR
0.5 hr
1 hr
1.5 hr
2 hr
3 hr
4 hr
5 hr
6 hr

Discussion
Above mentioned diuretics promote excretion of
salt in the urine as it has saluretic, natriuretic and
minimal effect on carbonic anhydrase inhibition.
The saluretic activity is calculated from excretion
of Na+, Cl-; natriuretic activity and potassium-
sparing effect from ratio of Na+/K+, while ratio
Cl-/(Na+) + (K+ ) is used to calculate carbonic
 Kidney  307

anhydrase inhibition effect. Recent study showed
thiazide and high ceiling diuretics inhibit all
mammalian isoforms of carbonic anhydrase.
SUGGESTED READING
1. Knauf H, Bailey MA, Hasenfuss G, Mutschler E. The
influence of cardiovascular and anti-inflammatory
drugs on thiazide-induced hemodynamic and
saluretic effects. Eur J Clin Pharmacol 2006;
62(11):885-92.
2. Lam NP, Kuk JM, Franson KL, Lau AH. Effect of
diuretic drugs on creatinine clearance determina-
tion. Ther Drug Monit 1995;17(2):142-44.
3. Musso CG, Reynaldi J, Vilas M, De Miguel R,
Imperiali N, Algranati L. Fractional excretion of K,
Na and Cl following furosemide infusion in healthy,
young and very old people. Int Urol Nephrol 2009
Mar 10.
4. Temperini C, Cecchi A, Scozzafava A, Supuran CT.
Carbonic anhydrase inhibitors. Comparison of
chlorthalidone, indapamide, trichloromethiazide,
and furosemide X-ray crystal structures in adducts
with isozyme II, when several water molecules
make the difference. Bioorg Med Chem 2009;
17(3):1214-21.

EXERCISE
Exercise 1: A 9 years old male child, who was operated at the age of 4 days for lumbar myelomeningocele.
Following operation parent notice urinary incontinence at the age of 2 years. All the investigations Hb, urea,
creatinine, USG,MCU,DMSA scan was within normal limit. Based on urodynamic study, a diagnosis of
neurogenic bladder was made and child was put on CIC with regular follow-up. Subsequently in urodynamic
high risk bladder was detected then oxybutynin 5 mg tds started along with CIC for 21 days. Child improved
symptomatically and urodynamically. Following are pre-drug and post oxybutynin urodynamic study. Pre-
drug maximum bladder volume = 87 ml, detrusor leak point pressure = 75 cm H2O, compliance = 1.5 ml/
cmH2O and post-drug maximum bladder volume = 130 ml, detrusor leak point pressure = 40 cmH2O,
compliance = 3.3 ml/ cmH2O. Describe your interpretation?
Pre-drug graph

Fig. 29.1
308  Practical Manual of Experimental and Clinical Pharmacology

Post-drug graph

Fig. 29.2
 Ophthalmology
30
OPHTHA. EXPERIMENT: 15
Aim
To evaluate the effect of the hyoscine on pupillary
diameter, salivary secretion and heart rate.
Background
Hyoscine (scopolamine) is an alkaloid which has
anticholinergic properties which leads to
decrease in salivary secretion resulting in dry
mouth increased pupillary diameter and increase
in basal heart rate. It is a competitive antagonist
of M1 receptor, primarily used for nausea,
motion sickness, intestinal cramps and ocular
purposes. The ophthalmic use is mostly for
pupillary dilatation, cycloplegia in patients
suffering from uveitis, iritis or iridocyclitis,
besides this it also provides beneficial effects as
preanesthetic, adjunct to narcotics, in nicotine
addiction, sea sickness and as a lie detector drug.
Materials and Method
Materials
Initial screening of volunteers: Volunteers are
screened for any cardiovascular, respiratory
disorder or any condition which may interfere
with experimental protocol
Healthy adult volunteers of either sex (willing
to give written informed consent)
Pupillometer, measuring cylinder
Stethoscope, mercury sphygmomanometer
Drug: Hyoscine tablet, matching placebo
Precautions for Experiment
• Subject is advised for adequate sleep over
night and light breakfast in the morning on
day of experiments (preferably 2 hr before)
• Avoid caffeinated drink or smoking or at least
2 hr before the experiment or any abuse
substance
• Volunteer should get complete instructions
of the procedure and its rehearsal, so it will
avoid any conditional anxiety of volunteers
• Monitoring equipment should be maintained
and regularly calibrated
• Basic operating procedure of the selected
instrument should be well explained to the
volunteers
Method
After initial screening volunteers are allowed to
participate in the study as per following inclusion
and exclusion criteria:
Inclusion Criteria
Healthy volunteers aged between 18-45 years of
either sex
Exclusion Criteria
1. History of Volunteers with: cardiovascular
disease , significant hepatic/renal dysfuction,
glaucoma, Benign prostatic hypertrophy
(BPH), psychiatric disorders
2. Anticholinergic drug taken within past 7 days
310  Practical Manual of Experimental and Clinical Pharmacology

3. History of substance abuse (narcotics) or any

other drugs likely to affect interpretation of
study parameters
4. Coffee, tea or smoking (past 24 hrs) and
alcohol intake (past 48 hrs)

Study Parameters
1. Pupillary diameter is measured with the help
of a pupillometer. It should be measured close
to the right eye with left eye closed with a
card and volunteers are asked to see a distant,
well illuminated object. The point at which 2
holes in the pupillometer just start overlap-
ping each other is taken as pupillary
diameter.
2. Heart rate: Estimated by palpating the radial
artery.
3. Salivary secretion: Assessed by using rock salt.
‘1g’ of the salt is given to keep under the
tongue and advised to keep there for 1 min,
then saliva is collected in a measuring
cylinder
Pupillometer (Fig. 30.1)
Pupillometer was developed for the assessment
of eye shape and condition, monitoring tiredness,
and for the detection of drugs or alcohol in
human beings. It is a portable instrument that
rapidly and simultaneously assesses records and
stores pupil size and reactivity to stimulus. It
provides a rapid diagnostic aid for neurological
assessment of unconscious patients, since pupil
response is known to be a vital aspect of the
diagnostic process. Regular assessment of the
size, reactivity to light and equality of pupils is
essential for early recognition of neurological
deterioration. The main features are to measure
pupil reaction, speed and size in patients with
suspected brain injury. The pupilometer mostly
detects and measures pupil diameter and pupil
response to a light stimulus. In the recent times,
so many models are available and these are with
software helping in the diagnosis of alcohol or
drug presence and use of this pupillometer
requires the active participation from the suspect.
Fig. 30.1: Pupillometer
Some of the updated models of pupillometer
require complex optometric components. The
current method of practice is to manually
measure these aspects using a bright light, which
stimulates reactivity of the pupil and make a note
of the dilation compared to the original size of
the pupil. Actual measurements compared with
a card having different pupil sizes matched
thereon. This method of assessment is time
consuming, and subjective.
Procedure for measurement of pupillary
diameter: Before using the pupillometer, instru-
ment is validated. Make sure batteries are
installed and are in operating order. Switch on
the pupillometer and then select the different size
option which can be viewed digitally with setting
of particular eye site or both. Volunteers should
be given detailed instruction to hold the
pupillometer up to their eyes with proper use of
nose rest. Then, volunteers stare through the
view windows and eyepiece, so that the pinpoint
of light is located inside the pupil to calculate
Pupillary Diameter.
Method
All the volunteers are asked to report in the clinical
pharmacology laboratory in the morning with
good overnight sleep and light breakfast. A
 Ophthalmology  311

through medical examination is done before the
experiment. All the procedure is explained in
detail. Above mentioned study parameters should
be recorded at baseline before giving the drug,
afterward volunteers are given 10 mg of hyoscine
or placebo in randomized single blinded fashion
with a glass of water. All the above mentioned
parameters should be recorded at frequent
intervals at 0, 0.5, 1, 1.5, 2, 3, 4, 5 and 6 hours
following drug intake.

Observations and Result

A. Hyoscine
Time (hr) BP (mmHg) H R (beats/min) RR (per min) PD (mm) SS (ml) ADR
0
0.5
1.0
1.5
2.0
3.0
4.0
5.0
6.0

B. Placebo
Time (hr) BP (mmHg) H R (beats/min) RR (per min) PD (mm) SS (ml) ADR
0
0.5
1.0
1.5
2.0
3.0
4.0
5.0
6.0
Blood Pressure (BP) mm Hg, Heart rate (H R) Rate/min, Respiratory rate (RR) Rate/min, Pupillary diameter (PD)
mm, salivary secretion (SS) ml.

Statistical analysis: All the data should be
expressed as mean± SD. Data is compared by the
unpaired ‘t’ test.
Discussion: Hyoscine causes decrease in salivary
secretion. Maximum effect is achieved approxi-
mately at 3 hrs which returns to baseline level
by 6 hrs which was found significantly different
from placebo group in several previous studies.
Similar results may be present with HR and
pupillary diameter in comparison to baseline and
when compared to placebo. Volunteers may
complain of dry mouth, throat, blurred vision,
photosensitivity, urination difficulty, flushing,
hallucinations etc. Special precaution should be
taken in patients with glaucoma because
hyoscine may precipitate glaucoma in borderline
patients. Studies reported oral scopolamine
hydrobromide decreases salivation by 70%,
following transdermal hyoscine the magnesium
secretion rate is unaltered, whereas sodium,
potassium, and calcium secretion rates are
significantly lowered and no significant modifi-
312  Practical Manual of Experimental and Clinical Pharmacology
cation is observed in the electroretinogram in
healthy volunteers following parenteral admini-
stration of hyoscine.
SUGGESTED READING
1. Ebert U, Grossmann M, Oertel R, Gramatté T, Kirch
W. Pharmacokinetic-pharmacodynamic modeling
of the electroencephalogram effects of scopolamine
in healthy volunteers. J Clin Pharmacol 2001;41(1):
51-60.
2. Gordon C, Ben-Aryeh H, Attias J, Szargel R, Gutman
D Effect of transdermal scopolamine on salivation.
J Clin Pharmacol 1985;25(6):407-12
3. Harding GF, Daniels R, Panchal S, Drasdo N,
Anderson SJ. Visual evoked potentials to flash and
pattern reversal stimulation after administration of
systemic or topical scopolamine. Doc Ophthalmol
1994;86(3):311-22.
4. Kochiadakis GE, Rombola AT, Kanoupakis EM,
Zuridakis EG, Skalidis EI, Vardas PE. Effect of
transdermal scopolamine on heart rate variability
in patients with severe coronary heart disease.
Pacing Clin Electrophysiol 1996;19(11 Pt 2):1867-71.
5. Markkanen YJ, Pihlajamäki K. Oral scopolamine
hydrobromide solution as an antisialagogic agent
in dentistry. Oral Surg Oral Med Oral Pathol 1987;
63(4):417-20
6. Renner UD, Oertel R, Kirch W. Pharmacokinetics
and pharmacodynamics in clinical use of scopo-
lamine. Ther Drug Monit 2005;27(5):655-65.
7. Sunahara FA, Farewell J, Mintz L, Johnson WH.
Pharmacological interventions for motion sickness:
cardiovascular effects. Aviat Space Environ Med
1987;58(9 Pt 2):A270-76.
OPHTHA. EXPERIMENT: 16
Aim
To evaluate the effect of Tropicamide (1%) on
pupillary diameter and accommodation reflex.
Background
Tropicamide, a muscarinic blocker acts as short
acting mydriatic and cycloplegic. The duration
of action is 4-8 hours so it is mostly preferred
over atropine for examination of lens, vitreous
humor and retina.
Materials and Method
Initial Screening of Volunteers
Volunteers are screened for any cardiovascular,
respiratory disorder or any conditions which
may interfere with experimental protocol.
Healthy adult volunteers of either sex (willing
to give written informed consent)
Pupillometer, Stethoscope, mercury sphy-
gmomanometer
Drug: Tropicamide (1%)
Precautions for Experiment
• Subject is advised for adequate sleep over
night and light breakfast in the morning on
day of experiments (preferably 2 hr before)
• Avoid caffeinated drink or smoking or at least
2 hr before the experiment or any abuse
substance
• Volunteers should get complete instruction
of the procedure and its rehearsal, so to avoid
any conditional anxiety of volunteers
• Basic operating procedure should be well
explained to the volunteers
• Advice the volunteers not to drive unless they
can see clearly
• Volunteers should not wear soft contact
lenses following use of tropicamide because
it contains a preservative which can be
absorbed by soft contact lenses and cause eye
irritation
Method
After initial screening, volunteers should partici-
pate in the study as per following inclusion and
exclusion criteria:
Inclusion Criteria
Healthy volunteers aged between 18-45 years of
either sex
Exclusion Criteria
• History of Volunteers with: cardiovascular
disease, significant hepatic/renal dysfuction,
glaucoma, Benign prostatic hypertrophy
(BPH), psychiatric disorders
 Ophthalmology  313

• Anticholinergic drug taken within past 7 days Assessment of Accommodation

• History of sensitivity or allergy to any
ingredient
• History of substance abuse (narcotics) or any
other drugs likely to affect interpretation of
study parameter.
• Coffee, tea or smoking (past 24 hrs) and alcohol
intake (past 48 hrs)
Pupillary Size Measurement
This is done with a simple pupillometer consis-
ting of a stiff piece of paper on which parallel
holes are made with a pin with increasing distance
between the 2 pin holes starting from 2 to 8 mm.
Between the one pair of holes from another pair is
kept constant to ½ inch. The volunteer looks at a
distant object which is illuminated and the
pupillometer is brought near the eye while the
other eye is closed. The volunteer looks through
the holes which appear as circles. The volunteer
looks through various holes and points out where
circles appear exactly adjacent to each other. This
gives an approximate idea of pupil size. (refer to
experiment no: 15)
This is examined by asking the volunteer to read
serial number of print of graded size ‘1’ as small
size then successively bigger prints as 2,3,4,5
accordingly. Then, the ability to see fine print is
tested. If there is less of accommodation then fine
prints are not readable and larger print can be
readable.
Procedure
All the volunteers should report to the clinical
pharmacology laboratory in the morning with
good overnight sleep and light breakfast. A
through medical examination is done before the
experiment. All the procedure should be
explained in detailed. In this experiment
volunteers act as self control. Tropicamide 1%
solution is instilled into right eye and left eye acts
as a control. Pupillary diameter and accommo-
dation are recorded before and after drug
administration at baseline 5, 10, 15, 30 min and
then 1, 2, 3, 4, 5, and 6 hr.

Observations and Result

Pupillary diameter (mm) Accommodation
Control (Lt eye) Test (Rt eye) Control(Lt eye) Test (Rt eye) ADR
Time
Baseline
5 min
10 min
15 min
30 min
1 hr
2 hr
3 hr
4 hr
5 hr
6 hr

Statistical analysis: All the data should be Discussion

expressed as mean ± SD. Data is analyzed by the Tropicamide is a short acting antimuscarinic drug.
unpaired‘t’ test. The receptors are present in pupillae and because
314  Practical Manual of Experimental and Clinical Pharmacology
continuous cholinergic input maintains pupil size.
Tropicamide blocks the action of sphincter pupillae
muscle of iris and ciliary muscle causing pupillary
dilatation and paralysis of ciliary body leads to
loss of accommodation reflex, particularly 1%
preparation paralyzes accommodation. The
increase is maximal at 1 hr baseline size sometimes
not reached even after 6hr. Cycloplegia with
decrease in near vision is noticed by inability to
read fine print. Usually it takes 15-30 minutes to
act and duration lasts for 3-8hrs. Deeply pigmen-
ted iris may require more doses as compared to
lightly pigmented iris. The commonest adverse
effect of Tropicamide reported are stinging,
irritation, dry mouth, blurring of vision, increased
intraocular pressure, etc.
SUGGESTED READING
1. Haddad DE, Rosenfield M, Portello JK, Krumholz
DM. Does prior instillation of a topical anaesthetic
alter the pupillary mydriasis produced by tropi-
camide (0.5%)? Ophthalmic Physiol Opt 2007;
27(3):311-14.
2. Hamasaki I, Hasebe S, Kimura S, Miyata M, Ohtsuki
H. Cycloplegic effect of 0.5% tropicamide and 0.5%
phenylephrine mixed eye drops: objective assess-
ment in Japanese schoolchildren with myopia. Jpn
J Ophthalmol 2007;51(2):111-15.
3. Inzelberge R, Feiler V, Korzcyn AD. The tropic-
amide eye drop test in Parkinson’s disease.
Parkinsonism Relat Disord 1997;3(4):215-18
4. Kawa P, Biaek M, Zagórski Z. Effect of mydriasis
and accommodation on intraocular pressure and
pigment release in patients with the pigment
dispersion syndrome. Klin Oczna 2007;109(4-6):198-
200.
5. Manny RE, Hussein M, Scheiman M, Kurtz D,
Niemann K, Zinzer K. Tropicamide (1%): an
effective cycloplegic agent for myopic children.
Investigative Ophthalmology and Visual Science
2001; 42 (8):1728-35.
6. Ratanapakorn T, Yospaiboon Y, Chaisrisawadsuk
N. Single dose of 1% tropicamide and 10% phenyle-
phrine for pupil dilation. J Med Assoc Thai.
2006;89(11):1934-39
OPHTHA. EXPERIMENT: 17
Aim
To evaluate the effect of topical pilocarpine (2%)
on pupillary diameter in healthy volunteers.
Background
Pilocarpine is a naturally occurring alkaloid
obtained from leaves of plant Pilocarpus. It acts
as parasympathetic stimulant (M3) to sphincter
pupillae and ciliary muscle thus producing
miosis, fixing of eyes for near vision, blurring
of distant vision, and lowering of intraocular
pressure. Besides this, it also increases salivary
secretion, urination and perspiration. Pilocar-
pine increases the outflow of aqueous humor
resulting in decreased intraocular pressure, so
it provides beneficial action in case of chronic
open angle and acute angle closer glaucoma. It
is also used as antidote for poisoning of
hyoscine, atropine, and in case of xerostomia
following radiotherapy.
Materials and Method
Initial Screening of Volunteers
Volunteers are screened for any systemic
disorder or any conditions which may interfere
with experimental protocol.
Healthy adult volunteers of either sex (willing
to give written informed consent)
Pupillometer, Stethoscope, mercury sphy-
gmomanometer
Drug: Pilocarpine 2%
Precautions for Experiment
• Subject is advised for adequate sleep over
night and light breakfast in the morning on
day of experiments (preferably 2 hr before)
• Avoid caffeinated drink or smoking or at least
2 hr before the experiment or free of any
substance abuse
• Volunteer should get complete instruction of
the procedure and its rehearsal, so to avoid
any conditional anxiety of volunteers
• Basic operating procedure should be well
explained to the volunteers
Method
After initial screening volunteers should parti-
cipate in the study as per following inclusion and
exclusion criteria:
 Ophthalmology  315

Inclusion Criteria as circles. The volunteer looks through various

Healthy volunteers aged between 18-45 years of
either sex.
Exclusion Criteria
• History of Volunteers with: cardiovascular
disease, significant hepatic/renal dysfuction,
glaucoma, Benign prostatic hypertrophy
(BPH), psychiatric disorders
• Anticholinergic drug taken within past 7 days
• History of (h/o) allergy to pilocarpine
• h/o substance abuse (narcotics) or any other
drugs likely to affect interpretation of study
parameter.
• Coffee, tea or smoking (past 24 hrs) and
alcohol intake (past 48 hrs).
Measurement of pupillary size: This is done with
a simple pupillometer consisting of a stiff piece
of paper on which parallel holes are made with
a pin with increasing distance between the 2 pin
holes starting from 2 to 8 mm. Between the one
pair of holes from another pair is kept constant
to ½ inch. The volunteer looks at a distant object
illuminated and the pupillometer is brought near
the eye while the other eye is closed. The
volunteer looks through the holes which appear
holes and points out where circles appear exactly
adjacent to each other. This gives an approximate
idea of pupil size. (Refer to exp. no: 15)
Procedure
All the volunteers should report to the clinical
pharmacology laboratory in the morning with
good over night sleep and light breakfast in the
morning. A through medical examination should
be done before the experiment. All the procedure
is explained in detail. In this experiment volun-
teers act as self control. One eye is used as test
and the other eye as control. One drop of
pilocarpine is instilled to the volunteer’s eyes.
Pupillary diameter, near and distant vision is
monitored for checking the effect on pupil size
and accommodation reflex changes. Pupillary
diameter and accommodation are recorded before
and after drug administration at baseline 0, 5, 10,
15, 20, 25, 30, 35, 40, 45, 50, 55 min then 1, 1.5, 2, 4,
and 8 hr. Along with pupillary diameters, accom-
modation, other vital parameters like blood
pressure, pulse rate are recorded in order to
evaluate systemic adverse drug reactions.
Volunteers should be asked in detail about any
adverse drug reaction and it should be recorded.

Observations and Result

Pupillary diameter (mm) Near Vision Distance Vision
Time HR BP Control (Lt eye) Test (Rt eye) Control (Lt eye) Test (Rt eye) Control (Lt eye) Test (Rt eye)
0
5
10
15
20
25
30
35
40
45
50
55
1 hr
1.5 hr
2 hr
4 hr
8 hr
In addition, observe for adverse drug reaction (ADR) or salivary secretion if any.
316  Practical Manual of Experimental and Clinical Pharmacology
Statistical analysis: All the data should be
expressed as mean ± SD. Data is analyzed by
unpaired’t’ test.
Discussion
Pilocarpine eyedrops produce pupillary contr-
action in 10 min and the effect is usually
observed up to 4 hrs. Near vision improves
whereas distant vision is blurred during the
same period. The drug produces burning pain
on instillation. Conjuctival hyperemia is
develops after 5 min, vague discomfort, pain
and blurring of distant vision is noted from 10
to 15 min onwards. Pilocarpine is well-known
for excessive sweating, salivation, broncho-
spasm, bradycardia, hypotension and diarrhea,
so it has limited clinical use.
SUGGESTED READING
1. Doughty MJ, Lyle WM. A review of the clinical
pharmacokinetics of pilocarpine, moxisylyte
(thymoxamine), and dapiprazole in the reversal of
diagnostic pupillary dilation. Optom Vis Sci. 1992;
69(5):358-68.
2. Druzgala P, Winwood D, Drewniak-Deyrup M,
Smith S, Bodor N, Kaminski JJ. New water-soluble
pilocarpine derivatives with enhanced and sus-
tained muscarinic activity. Pharm Res 1992; 9(3):
372-77.
3. Smith SE, Smith SA, Friedmann AI, Chaston
JM. Comparison of the pupillary, refractive,
and hypotensive effects of Ocusert-40 and pilo-
carpine eyedrops in the treatment of chronic
simple glaucoma. Br J Ophthalmol 1979;63(4):
228-32.
4. Yie T, Liang L, Chen X. Clinical observation on the
effect of 4% pilocarpine gel used one dose per day
in the treatment of glaucoma. Yan Ke Xue Bao 2000;
16(2):77-80.
5. Yuan J, Wei H. A clinical observation of the
therapeutic effects of pilocarpine gel for treatment
of glaucoma. Zhonghua Yan Ke Za Zhi 1998;
34(3):174-77.
OPHTHA. EXPERIMENT: 18
Aim
To demonstrate water induced ocular hyper-
tension in healthy volunteers.
Background
The normal intraocular pressure is maintained at
16-23 mm Hg as it is required for the maintenance
of the optical properties of refracting organs. The
pressure in the eyeball is mainly because of
elasticity of outer coats, volume of solid and liquid
contents which maintain the intraocular pressure.
The measurement of intraocular pressure can be
made by various methods like manometry, tono-
metry (digital tonometry, instrumental tonometry
like impression tonometry, applanation tono-
metry, etc.). These are used to measure the
intraocular pressure by flattening an area of
cornea. There are different factors responsible for
variations of intraocular pressure like respiration
and pulse which can change the pressure to 1-2
mm Hg. The intraocular pressure maintains
diurnal variations pressure which is usually high
in the morning then falls in the evening and at
night pressure rises again. There is different
provocative test used in clinical practice to
diagnose chronic simple glaucoma and also to find
out behavior of intraocular tension under stress.
Water drinking test is applied since it lowers the
osmotic tension of blood ultimately raising the
intraocular tension.
Materials and Method
Initial Screening of Volunteers
Volunteers are screened for any systemic
disorder or any conditions which may interfere
with experimental protocol.
Healthy adult volunteers of either sex (willing
to give written informed consent).
Schiotz Tonometry, 4% xylocaine, Stetho-
scope, mercury sphygmomanometer, Facilities
for Clinical Pharmacology Laboratory (Appendix
IV).
Precautions for Experiment
• Subject should be advised for adequate sleep
over night and light breakfast in the morning
on day of experiments (preferably 2 hr before)
• Avoid caffeinated drink or smoking or at least
2 hr before the experiment or any substance
abuse
 Ophthalmology  317

• Volunteer should get complete instructions of is advised to lie down in supine position without
the procedure, so to avoid any conditional any pillow under the head. Volunteer is asked
anxiety of volunteers to look vertically upward towards roof to relax
• Basic operating procedure should be well the accommodation. The eyelid is separated with
explained to the volunteers. finger without giving any pressure in the eye,
and then globe tonometer is vertically placed on
Method the cornea. The volunteers are advised not to
After initial screening volunteers should parti- move the eye during this time. The pressure is
cipate in the study as per following inclusion and recorded as the pointer reaches the steady state.
exclusion criteria: After recording the pressure, antibiotic should
be applied.
Inclusion Criteria
Observations and Result
Healthy volunteers aged between 18-45 years of
either sex Ocular pressure (mm Hg)
Time HR BP RR Control eye Test eye
Exclusion Criteria 0
0.5 hr
• History of volunteers with any systemic 1 hr
disease 2 hr
• Volunteers with glaucoma 3 hr
• Anticholinergic drug taken within past 7 days 4 hr
6 hr
• History of substance abuse (narcotics) or any
other drugs likely to affect interpretation of
study parameter. Graphical Representation
• Coffee, tea or smoking (past 24 hrs) and
alcohol intake (past 48 hrs).

Procedure
All the volunteers should report to the clinical
pharmacology laboratory in the morning with
good over night sleep and light breakfast. A
thorough medical examination is done before the
experiment. All the procedure should be explai-
ned in detail.
Water drinking test: The volunteers are asked to
drink “one liter” of water in 10 min before
breakfast and ocular pressure is measured at
baseline 0, 0.5, 1, 2, 3, 4 and 6 hr. Along with
ocular tension other parameters like blood
pressure, pulse rate, respiratory rate are also
recorded. Fig. 30.2: Representation of result

Measurement of Ocular Tension Statistical analysis: All the data is expressed

After taking written informed consent, cornea is as mean± SD. Data is analyzed by unpaired ‘t’
anesthetized with 4% xylocaine then volunteer test.
318  Practical Manual of Experimental and Clinical Pharmacology
Discussion
Provocative tests are commonly used in the
practice to diagnose chronic simple glaucoma
and to evaluate behavior of intraocular tension
under stress. There are different tests which are
used like water drinking test causes lowering
of osmotic tension of blood causing rise of intra-
ocular tension and if it is rise beyond 6 mm Hg,
this indicates glaucoma. Besides this other tests
are available like venous congestion test which
increases ocular pressure greater than 10 mm
Hg in case of glaucoma.
SUGGESTED READING
1. Vetrugno M, Sisto D, Trabucco T, Balducci F, Delle
Noci N, Sborgia C. Water-drinking test in patients
with primary open-angle glaucoma while treated
with different topical medications. J Ocul Pharmacol
Ther 2005;21(3):250-57.
 Clinical Pharmacokinetics
31
EXPERIMENT NO: 19
Aim
To study the pharmacokinetics of Aceclofenac
tablet following single oral dose.
Background
Pharmacokinetics is a composite discipline of
mathematics, physiology and pharmacology
involved in determination of absorption, distri-
bution, metabolism and elimination of a drug. Its
systemic analysis may predict overall progress of
a drug through the body and the interaction
between two or more drugs. Aceclofenac is taken
as an example and it is an orally administered
phenyl acetic acid derivatives and potent inhibitor
of the enzyme cyclooxygenase, which is involved
in the production of prostaglandins. It is commonly
prescribed for the acute and chronic conditions,
e.g. rheumatoid arthritis (RA) and osteoarthritis
(OA).
Materials and Methods
Adult male healthy volunteers, Facilities for
Clinical Pharmacology Laboratory (Appendix IV)
Drug: Aceclofenac (100 mg), HPLC, Centrifuge
Inclusion Criteria
a. Volunteer age group of l8-45 years
b. Be neither underweight nor overweight as per
chart provided by LIC, India
c. Have voluntarily given written informed
consent and comply with protocol requirement
d. Clinically normal profile of RBC, WBC and
DLC (differential leukocytes count)
e. Clinical normal profile of serum creatinine,
serum aspartate aminotransferase, serum
alanine aminotransferase.
f. No change in ECG
g. No abnormality detected in routine exam-
ination of urine.
Exclusion Criteria
Volunteers are excluded from the study on the
basis of following criteria:
a. History of hypersensitivity to any drug
particularly to NSAIDs
b. Volunteers who have received NSAIDs within
one week
c. Volunteers with cardiovascular, respiratory,
liver, psychiatric or any other systemic illness
d. Volunteers who are a regular smokers or
alcoholic
e. Volunteers who are HIV or Hepatitis B or C
positive.
Precautions
Following submission of written informed
consent, volunteers should undergo standardized
screening procedure before initiation of the study
(Physical and Laboratory examination) one week
prior to the period of drug administration.
320  Practical Manual of Experimental and Clinical Pharmacology
Methods
HPLC METHOD FOR PLASMA ANALYSIS
Sample Preparation
Samples are prepared by adding 0.5 ml sodium
floride solution (40 mg/mL), 1 ml of 1 mol/L
phosphoric acid and 0.1 mL internal standard
solution (0.05 mg ketoprofen) to 1 mL plasma
followed by the addition of 5ml n-hexane/diethyl
ether (50:50, v/v). The tubes are capped, shaken for
30 min and then centrifuged at 4000 rpm for 10 min.
The organic layer is transferred into a glass tube
and evaporated to dryness under a nitrogen stream
at room temperature. Prior to analysis, the residue is
dissolved in 120 mL of a solution consisting of 72%,
0.01 mol/L phosphate buffer, 15% acetonitrile, 10%
methanol, 3% tetrahydrofuran (final pH 2.5), which
is used for HPLC analysis.
HPLC VARIABLES USED FOR PLASMA
ESTIMATION
Column used in this method is : 250 × 4.6, 5 mm
spherisorb C18 and mobile phase is mixture of
solvent A (consisting of 20% (v/v) 0.005 mol/L
phosphate buffer, 80% (v/v) acetronitrile) and
solvent B ( 88% (v/v) 0.01 mol/L phosphate buffer
and 12% (v/v) acetonitrile, the flow rate should
be 1ml/min and injection volume should be 20
mL and read frequency at UV 275.
CHROMATOGRAM
Internal standard is ketoprofen and limit of
detection should be 10 ng/mL and the external
standard is bulk solution of aceclofenac.
DATA RECORDING
All data during the conduct of the study should
be directly entered in the data recording forms
and computer generated chromatograms should
be prepared. Data should be entered in software
program to calculate the pharmacokinetic para-
meters.
Results and calculation of pharmacokinetics
parameters
Volunteers Vitals: with different time intervals:
 Clinical Pharmacokinetics  321

Name: Age: in both young and elderly volunteers. Aceclofenac

Sex: is mainly metabolized by CYP2C9 pathway. The
Address: metabolism of aceclofenac differs in animals and
the human beings. Aceclofenac, additional side-
Time (hr) Plasma level (µg/ml)
chain (esterifies acetoxy) is compared with
0 structurally related diclofenac. 4’ OH aceclofenac
1 is the main metabolite in the plasma and urine
2 after the oral administration of aceclofenac. Other
3 minor metabolites are diclofenac and 4’ OH
4 diclofenac, 5’-OH diclofenac. Aceclofenac gets
5 converted to diclofenac after long time by
6 diclofenac hydroxylate or of hydrolysis of the
8 present compound.
10
Note: Presently, there are different softwares available
12
for pharmacokinetic parameters calculations for example:
16 WinNonlin, NONMEM, etc.
24
Pharmacokinetic parameters: SUGGESTED READING
1. Bioavailability : Rate and extent
1. Bhinge JR, Kumar RV, Sinha VR. A simple and
Concentration time curve sensitive stability-indicating RP-HPLC assay method
Cmax, Tmax for the determination of aceclofenac. J Chromatogr
2. Volume of distribution (Vd) Sci 2008;46(5):440-44.
3. Elimination half life (t1/2 el) 2. Bort R, Ponsonda X,Carrasco E, et al. Metabolism of
aceclofenac in humans. Drug Metab Dispos 1996;
4. Area under curve (AUC0-24 and AUC0-∝)
24(8):834-41.
5. Rate constants (Ka and Kel) 3. Brogden RN, Wiseman LR. Aceclofenac. A review of
a. Absorption rate constant (Ka) its pharmacodynamic properties and therapeutic
b. Elimination rate constant (Kel) potential in the treatment of rheumatic disorders
6. Clearance and in pain management. Drugs 1996;52(1):
Discussion: A newer NSAIDs Aceclofenac, with 113-24.
4. Dooley M, Spencer CM, Dunn CJ. Aceclofenac: A
anti-inflammatory and analgesic properties is reappraisal of its use in the management of pain and
most commonly prescribed in clinical practice. rheumatic disease. Drugs 2001;61(9):1351-78.
Following oral administration of a single 100 mg 5. Hinz B, Auge D, Rau T, Rietbrock S, Brune K, Werner
dose, mean maximum plasma aceclofenac U. Simultaneous determination of Aceclofenac and
concentration (Cmax) of 6.8 to 8.9 mg/L is reduced three of its metabolites in human plasma by high-
in about 1.4 to 2 hours. Cmax and area under the performance liquid chromatography. Biomed Chrom
2003, 17: 268-275.
plasma concentration time curve (AUC) increase
6. Rex N Brogden, Lynda R Wiseman. Aceclofenac: A
linearly after administration of single dose of review of the pharmacodynamic properties and
aceclofenac 50, 100 and 150 mg and the pharma- therapeutic potential in the treatment of Rheumatic
cokinetic properties of the drug are generally disorders and in pain management. Drugs 1996;
unchanged during multiple-dose administration 52(1):113-24.
 Miscellaneous
32 Practicals
EXPERIMENT NO: 20
Aim
To evaluate the analgesic activity of NSAIDs on
different human pain models.
Background
The evaluation of analgesics in volunteers has
got various difficulties and it is difficult to develop
an ideal model for example experimental stimulus
does not produce any emotional response which
is a part of any painful pathological condition.
This is because the subject can withdraw the
stimulus whenever the pain became intolerable.
Response is variable because of placebo response,
is very common in these type of screening. Pain
threshold is the time after the stimulus adminis-
tration the subject starts feeling the painful
sensation. Pain tolerance is the point at which the
subject starts feeling the pain which is unbearable
and subject withdraws hand away from the
painful stimulus. Non-steroidal anti-inflam-
matory drugs (NSAIDs) are most commonly
prescribed drug for acute or chronic pain and for
long-term anti-inflammatory therapy. NSAIDs are
believed to modulate inflammation and sensiti-
zation of nociceptors by the inhibition of COX 1
and COX 2 resulting in an inhibition of prosta-
glandins, particularly PGE 1 and PGE 2, which
mainly act locally by promoting inflammation and
other inflammatory agents. Detection of analgesic
effects of NSAIDs through the experimental pain
models seems to be controversial and less suited.
The hypothetical thought that most of the methods
used for the induction of pain do not produce
inflammation or substantial tissue damage and
hence the mechanism, by which NSAIDs mainly
act, i.e. by inhibiting prostaglandin synthesis, thus
might not come into play in such a short-time.
To develop an ideal pain model some of the
criteria should be fulfilled for example, it should
not cause tissue damage, or psychological injury,
it should be simple and reliable without any after
effect lastly volunteers should have controlled of
cessation during the experiment.
Precautions before the Experiment
• Procedure should be well explained to the
volunteers
• Eyes should be covered during the experiment
to avoid time clues and visual distraction
• Hypersensitivity to the cold or warm should
be checked (Complete allergic history should
be taken)
• Volunteers should be advised for adequate
sleep over night and light breakfast in the
morning day of experiments (preferably 2 hr
before)
• Avoid caffeinated drink, alcohol, cola drinks
or smoking at least 2 hr before the experiment.
Materials and Methods
• Healthy adult volunteers (willing to give
written informed consent)
• Cold/warm water reservoir, ice, and thermo-
meter (sensitivity ± 0.1°C)
Drug: Aceclofenac (100 mg/tablet)
 Miscellaneous Practicals  323

Experiment: A
Cold Water Stress
Step 1: Volunteers are asked to immerse their non-
dominant forearm (marked at the specific position)
into a container of warm water (34.5-35.5°C) for
2 min (Fig. 32.1).
Step 2: Then, immediate transfer the arm to
another container of cold water and ice (0.5-1.5°C)
(Fig. 32.1). Volunteers are instructed to leave the
arm immersed in the cold water/ice as long as the
pain could be tolerated (pain tolerance). Cold Fig. 32.1: Cold water stress test
pressor tolerance should be measured in seconds
from immersion in the cold water.
Time of onset of pain and tolerance is recorded
before and after the drug treatment.
Limitation of the Study
Observations: 0 min (baseline), 60 min, 120 min
and 180 min after the drug administration, tests Without inflammation and hyperalgesia, the
are repeated. result is inconsistent and unreliable.

Results
Time of hand withdrawal before drug (Sec) Time of hand withdrawal after drug (Sec)
Volunteer 1
0 min
60 min
120 min
180 min
Volunteer 2
0 min
60 min
120 min
180 min
Note: In this experiment additional parameters such as cardiac parameters (BP, HR, ECG, etc). can be evaluated

Experiment: B marked spots sequentially against a switch

Radiant Heat
The instrument for delivering radiant heat is kept
at a fixed distance from non-hairy marked skin
surface of forearm. The subject is told to indicate
the time of pain threshold and of maximum pain
tolerance.
Thermal stimulation radiant heat of a constant
intensity is used to induce pain. On the ventral
surface of the volunteer dominant forearm, eight
spots should be marked and numbered from one
to eight. The subjects are instructed to press the
mounted at an aperture, 6 × 6 mm in size, on the
stimulator. Without prior notice, a projection
filament lamp mounted within the stimulator is
then turned on by an experimenter. The volunteers
are instructed to pull their forearm away from the
apparatus as soon as they perceive the radiant
heat stimulus as painful and thereby, to indicate
their pain threshold. The time elapsing between
the turning on of the lamp and the withdrawal of
the arm from the apparatus, allowing the closure
of the switch is measured, in milliseconds, by a
digital clock and stored on magnetic disk. The
324  Practical Manual of Experimental and Clinical Pharmacology

intervals between the applications of the radiant
heat stimuli ranged randomly between 15 and 25
sec.
Observations: Total of seven measurements (each
lasting 8 min) are carried out at 0, 30, 60, 90, 120,
150, and 180 min after drug administration.
Measurements: Threshold and tolerance to
electrically induced pain, threshold to thermally
induced pain, reaction time to pain stimuli, fine
motor control, or side effects.
Precautions
• Specialized hairless forearm area should be
selected and marked properly
• Distance between the heat source and hand is
kept constant
• Hypersensitivity should be checked initially

Time of hand withdrawal before drug (Sec) Time of hand withdrawal after drug (Sec)
Volunteer 1
0 min
30 min
60 min
90 min
120 min
150 min
180 min
Volunteer 2
0 min
30 min
60 min
90 min
120 min
150 min
180 min
Note: In this experiment additional parameters such as cardiac parameters (BP, HR, ECG, etc). can be evaluated

Experiment: C its edges directly applied to the skin. The cuff is

BP Cuff Inflation
In this method, the BP cuff is tied to the arm with
the cap of a cold drink bottle under the cuff with
inflated at the mean arterial pressure and the time
at which the pain starts is noted and cuff pressure
is maintained and the time of maximal tolerance
developed is noted.

Time of hand withdrawal before drug (Sec) Time of hand withdrawal after drug (Sec)
Volunteer 1
0 min
60 min
120 min
180 min
Volunteer 2
0 min
60 min
120 min
180 min
Note: In this experiment additional parameters such as cardiac parameters (BP, HR, ECG, etc). can be evaluated

Experiment: D of hand at 50% of MVC. Time of onset of pain and

Hand Dynamometer time of maximal pain is noted.
The subject is asked to perform his maximal The same protocol is followed 2-3 hr after the
voluntary contraction (MVC) on Smodley’s hand NSAIDs administration (Depends on the half-life
dynamometer. The volunteer is asked to keep grip of drugs).

Time of hand withdrawal before drug (Sec)
Volunteer 1
0 min
60 min
120 min
180 min
Volunteer 2
0 min
60 min
120 min
180 min
Note: In this experiment additional parameters such as cardiac parameters (BP, HR, ECG, etc). can be evaluated
Other Methods
The submaximum effort tourniquet method (Smith
et al, 1966)
Discussion: Presently various human pain models
are available for example cold-induced pain
model, electrically induced pain model, dental
pain models inducing pain by removal of impac-
ted or semi-impacted third molars, laser-induced
pain model, mechanically induced pain model,
chemically induced pain model, freezing injury,
pain model, heat-burn injury pain model, and UV
pain models using UVA or UVB radiation, etc. in
most of the pain model with human healthy
volunteers established the comparative efficacy
but some models like in cold-induced pain model
various NSAIDs efficacy was failed to demons-
trated consistent analgesic effects. Several studies
reported inter individual variation, significant
influence of gender, etc.
SUGGESTED READING
1. Doverty M, White JM, Somogyi AA, Bochner F, Ali
R, Ling W. Hyperalgesic responses in methadone
maintenance patients. Pain 2001;90:91-96.
2. Garcia de Jalon PD, Harrison FJ, Johnson KI, Kozma
C, Schnelle K. A modified cold stimulation technique
for the evaluation of analgesic activity in human
volunteers. Pain 1985;22:183-89.
3. Jones SF, McQuay HJ, Moore RA, Hand CW.
Morphine and ibuprofen compared using the cold-
pressure test. Pain 1988;34:117-22.
4. Pandhi P, Sharma PL, Sharma BK, Wahi PL.
Comparative effect of propranolol and labetalol on
isometric exercise and cold stress induced increase
Miscellaneous Practicals  325
Time of hand withdrawal after drug (Sec)
in arterial blood pressure. Int J Clin Pharmacol Ther
Toxicol 1986;24(5):249-53.
5. SF La Vincente1, White1 JM , Somogyi1 AA, Bochner1
F, CB Chapleo. Enhanced Buprenorphine Analgesia
with the Addition of Ultra-low-dose Naloxone in
Healthy Subjects. Clinical pharmacology and
therapeutics 2008;83(1):144-52.
6. Stacher G, Bauer P, Schneider C, Winklehner S,
Schmierer G. Effects of a combination of oral
naproxen sodium and codeine on experimentally
induced pain. Eur J Clin Pharmac 1982a;21:485-90.
7. Stacher G, Steinringer H, Schmierer G, Schneider C,
Winklehner S, Mittelbach G, de Paolis C, Praga C.
Ceruletide increases dose dependently both jejunal
motor activity and threshold and tolerance to
experimentally induced pain in healthy man. Gut
1984;25:513-19.
8. Stacher G, Steinringer H, Schmierer G, Winklehner S,
Schneider C. Ceruletide increases threshold and
tolerance to experimentally induced pain in healthy
man. Peptides 1982b;3:955-62.
9. Stacher G, Steinringer H, Winklehner S, Mittelbach G,
Schneider C. Effects of graded oral doses of
meptazinol and pentazocine in comparison with
placebo on experimentally induced pain in healthy
humans. Br J Clin Pharrnac 1983;16:149-56.
10. Telekes A, Holland RL, Peck AW. Indomethacin:
Effects of cold-induced pain and the nervous system
in healthy volunteers. Pain 1987;30:321-28.
11. Wolff BB, Kantor TG, Jarvik ME, Laska E. Response
of experimental pain to analgesic drugs; III: codeine,
aspirine, secobarbital, and placebo. Clin Pharmacol
Ther 1969;10:217-28.
EXPERIMENT NO: 21
Aim
To evaluate plasma salicylate level by fluorometric
methods in healthy volunteer.
326  Practical Manual of Experimental and Clinical Pharmacology
Background
Aspirin, another name acetylsalicylic acid (ASA)
is commonly used as an analgesic for headache,
fever and as an anti-inflammatory agent. Felix
Hoffman of Friedrich Bayer and Co., developed a
stable and better-tolerated form of the drug
(acetylsalicylic acid) in 1897, though industrial
production of salicylic acid was started in 1859,
however early preparations are associated with
side effects, such as unpleasant taste and
dyspepsia. Aspirin acts by inhibiting the cyclo-
oxygenase (COX) activity of prostaglandin (PG),
which ultimately blocks the metabolism of
arachidonic acid to cyclic prostanoids such as
thromboxane A2 (TXA2). Aspirin acts as anti-
platelet because it acetylates serine 529 in the active
site of the cyclooxygenase-1 enzyme
(prostaglandin H2 synthase-1), permanently
deactivating it and preventing thromboxane A2
platelet activation. So in clinical practice it is most
commonly used for preventing thrombotic events,
such as ischemic strokes and acute myocardial
infarction. Aspirin is rapidly absorbed from the
gastrointestinal tract and peak plasma concen-
trations are achieved in 30 to 40 minutes, at 60
minutes following ingestion of 100 mg of aspirin
which inhibits TXA2 production. Though plasma
half-life is only 20 minutes but since platelet
inhibition is of irreversible nature so once daily
dose maintains the antithrombotic action, so it is
used for primary and secondary prevention of
vascular events.
Materials and Methods
Initial Screening of Volunteers
• Volunteers are screened for any systemic
disorder or any other disease conditions
which may interfere with experimental
protocol.
• Healthy adult volunteers of either sex (willing
to give written informed consent)
• Stethoscope, mercury sphygmomanometer
• Spectrofluorimeter
• Drug: Aspirin
Precautions for Experiment
• Subject should be advised for adequate sleep
over night and light breakfast in the morning
on day of experiments (preferably 2 hr before)
• Avoid caffeinated drink or smoking or at least
2 hr before the experiment or free of any
substance abuse
• Volunteer should get complete instruction of
the procedure
• Monitoring equipment should be maintained
with regular calibration
• Basic operating procedure of the selected
instrument should be well explained to the
volunteers.
Method: After initial screening volunteers should
participate in the study as per following inclusion
and exclusion criteria:
Inclusion Criteria
Healthy volunteers aged between 18-45 years of
either sex.
Exclusion Criteria
1. History of volunteers with: cardiovascular
disease , significant hepatic/renal dysfuction,
2. History of drug allergy particularly to sali-
cylate
3. History of substance abuse (narcotics) or any
other drugs likely to affect interpretation of
study parameter.
4. Coffee, tea or smoking (past 24 hours) and
alcohol intake (past 48 hours).
Methods
All the volunteers should report to the clinical
pharmacology laboratory in the morning with good
overnight sleep and light breakfast in the morning.
A thorough medical examination should be done
before the experiment. All the procedure should be
explained in detail. Above mentioned study
parameters should be recorded at baseline before
giving the drug, afterwards volunteers should be
given 325 mg of aspirin with a glass of water of 200
ml. All the above mentioned parameters should be

recorded at frequent intervals at 0, 0.5, 1, 1.5, 2, 3, 4,
5 and 6 hours following drug intake. Blood samples
are collected at various time interval as mentioned
above.
Laboratory procedure: Proteins are precipitated
from plasma with sulphuric acid. Salicylates
are analyzed by mixing a sample of the super-
natants fluids with NaOH and then measuring
the fluorescence. The intensity of fluorescence
is proportional to the amount of salicylate
present in the plasma. (Assay method: Jacobs JC,
Pesce M. Arthritis Rheumatism, 1978;21:129-31).
Results
Samples (μg/ml) Spectrofluorimetric reading
(Fluorescence unit)
Standard
Blank plasma
2.5
5 properties affecting platelet function. Aspirin
10
20
40
Test sample (T1)
Test sample (T2)
Calculation
FUT – FUB
Concentration of test sample = × CSS
FUS – FUB
Where,
FUT = Fluorescence unit of test
FUB = Fluorescence unit of blank
FUS = Fluorescence unit of standard
CSS = Concentration of standard solution
Discussion: Aspirin is a weak acid, following oral
administration little amount is ionized in the
stomach, approximately 50-80% aspirin is bound
to plasma protein and volume of distribution is
0.1-0.21 L/kg, 80% aspirin metabolized in the liver
by conjugation forms salicyluric acid and different
glucuronic acid form. It is excreted by kidney mainly
in the form of salicyluric acid, with elimination
half-life 2-4.5 hours in case of dose < 250 mg, while
with higher doses > 4 gm half-life increases up to
15-30 hours. Increased doses switch on from first
order to zero order because of saturation of
Miscellaneous Practicals  327
metabolic pathways. Aspirin has been in the
clinical practice since > 100 years but presently
new problem has been started as aspirin resistance
where aspirin fail to protect individuals from
thrombotic complications. Though there are no
formal diagnostic criteria but aspirin resistance
generally describes the failure of aspirin to produce
an expected biological response or the failure of
aspirin to prevent atherothrombotic events. The
incidence of aspirin resistance is 5 to 45% of the
general population but the exact prevalence of
aspirin resistance is unknown. Measurement of
platelet aggregation, platelet activation, and
bleeding time have all can confirmed variability in
patient’s antithrombotic responses to aspirin
therapy. Till now mechanism for aspirin resistance
remains uncertain but probably it is likely due to a
combination of clinical, biological, and genetic
resistance can improved by educating the patients
regarding importance of compliance or by elimi-
nating interfering substances like ibuprofen, and
avoiding increasing aspirin dose because increa-
sing the dose of aspirin does not enhance COX-1
inhibition. One can also use alternative drugs, e.g.
clopidogrel but scientific evidence are minimal that
switching to alternative treatment strategies
improves outcomes. Another problem is variability
in individual response to antiplatelet agents
emerging as a clinical problem: poor respon-
siveness has been associated with an increased
risk of ischemic events, including stent thrombosis.
SUGGESTED READING
1. Hankey GJ, Eikelboom JW. Aspirin resistance. BMJ
2004;328(7438):477-79.
2. Hankey GJ, Eikelboom JW. Aspirin resistance. Lancet
2006;367(9510):606-17.
3. Jacobs JC, Pesce M. Micromeasurement of plasma
salicylate in arthritic children. Arthritis Rheum 1978;
21(1):129-32.
4. Krasopoulos G, Brister SJ, Beattie WS, Buchanan
MR. Aspirin “resistance” and risk of cardiovascular
morbidity: Systematic review and meta-analysis. BMJ
2008;336(7637):195-98.
5. Tran HA, Anand SS, Hankey GJ, Eikelboom JW
Aspirin resistance. Thromb Res 2007;120(3):337-46.
328  Practical Manual of Experimental and Clinical Pharmacology
EXPERIMENT NO: 22
Aim
To evaluate acetylator status by isoniazid (INH)
estimation in healthy volunteers.
Background
Biotransformation of many drugs is genetically
determined. Different individuals have different
levels of metabolizing enzymes for a particular
drug. Rates of biotransformation of a drug in a
population follows normal distribution but may
be bimodal or trimodal also. INH undergoes
acetylation by N-acetyl transferase enzyme
present in the liver. Rate of metabolism follows
slow and fast acetylator pattern. In Europe, fast
acetylators constitute 40%, in Japan 85% and in
Asia 85-100%. In India, 30-40% considered as fast
acetylators (half-life 1 hr) and 60-70% as slow
acetylators (half-life 3 hr). Other drugs, the
metabolism of which can be affected by acetylator
status are, hydralazine, procainamide, dapsone
and some sulphonamides.
Materials and Methods
Initial Screening of Volunteers
• Volunteers are screened for any systemic
disorder or any conditions which may
interfere with experimental protocol.
• Healthy adult volunteers of either sex (willing
to give written informed consent)
• Stethoscope, mercury sphygmomanometer
• Spectrofluorimeter
• Drug: INH
Precautions for Experiment
• Subject should be advised for adequate sleep
over night and light breakfast in the morning
on day of experiments (preferably 2 hr before)
• Avoid caffeinated drink or smoking or at least
2 hr before the experiment or free of any
substance abuse
• Volunteer should get complete instruction of
the procedure
• Monitoring equipment should be maintained
with regular calibration
• Basic operating procedure of the selected
instrument should be well explained to the
volunteers.
Method: After initial screening, volunteers should
participate in the study as per following inclusion
and exclusion criteria:
Inclusion Criteria
Healthy volunteers aged between 18-45 years of
either sex.
Exclusion Criteria
• History of volunteers with: cardiovascular
disease, significant hepatic/renal dysfuction,
• History of drug allergy
• History of substance abuse (narcotics) or any
other drugs likely to affect interpretation of
study parameters.
• Coffee, tea or smoking (past 24 hours) and
alcohol intake (past 48 hours)
Methods
All the volunteers should report to the clinical
pharmacology laboratory in the morning with
good over night sleep and light breakfast in the
morning. A thorough medical examination
should be done before the experiment. All the
procedure should be explain in detailed.
Afterwards the volunteers are asked to take INH
600 mg orally and 2 ml blood sample should be
collected at 0 hr, 1 hr, 3 hr and 6 hr. Plasma INH
estimation should be done spectrofluorometri-
cally.

Results
Samples (μgm/ml) unit Spectrofluorimetric reading
Standard
Blank plasma
1.25 μg/ml
2.5
5
10
20
Test sample 0 hr
Spectrofluorimetric reading
(Fluorescence unit) Plasma level
of INH
Discussion
There are different methods for determination of
acetylator status of INH: (1) Urinary estimation of
metabolites: Urine sample is collected at 6 hr after
a dose of (600 mg) INH. Slow acetylators have
<70% metabolite excreted while fast acetylator
have >97% metabolite excreted. (2) Plasma levels:
INH is given orally 10 mg/kg (600 mg) and blood
sample is collected at 0 hr and 6 hr. If INH
>2.5 μg/ml at 6 hr = slow acetylator and if < 2.5
μg/ml at 6 hr = fast acetylator. (3) Acetyl INH/
INH, metabolite ratio: If ratio is <0.48 then it is
slow acetylator and if ratio is >0.77 then it is called
as fast acetylator.(4) t1/2 in INH: Slow acetyaltor:
140-200 min and Fast acetylator: 45-80 min, but
in this test repeated sampling is required.
SUGGESTED READING
1. Bozok Cetintaþ V, Erer OF, Kosova B, Ozdemir I,
Topçuoðlu N, Aktoðu S, Eroðlu Z. Determining the
relation between N-acetyltransferase-2 acetylator
phenotype and antituberculosis drug induced
hepatitis by molecular biologic tests. Tuberk Toraks
2008;56(1):81-86.
2. Furet Y, Bechtel Y, Le Guellec C, Bechtel PR, Autret-
Leca E, Paintaud G. Clinical relevance of N-
acetyltransferase type 2 (NAT 2) genetic poly-
morphism] Therapie 2002;57(5):427-31.
3. Huang YS, Chern HD, Su WJ, Wu JC, Chang SC,
Chiang CH, Chang FY, Lee SD. Cytochrome P450
Miscellaneous Practicals  329
1 hr 3 hr 6 hr
2E1 genotype and the suscepti-bility to antituber-
culosis drug-induced hepatitis. Hepatology 2003;
37(4):924-30
4. Hutchings AD, Routledge PA. A single sample
saliva test to determine acetylator phenotype. Br J
Clin Pharmacol 1996;42(5):635-37.
5. Matar KM, Mayet AY, Ayoola EA, Bawazir SA, Al-
Faleh FZ, Al-Wazzan A. Isoniazid acetylation
phenotyping in Saudi Arabs.J Clin Pharm Ther
2004;29(5):443-47.
6. Najim RA, Al-waizt M, Al-Razzuqi RA. Acetylator
phenotype in Iraqi patients with allergic contact
dermatitis. Ann Saudi Med 2005;25(6):473-76.
7. Pande JN, Pande A, Singh SP. Acetylator status,
drug metabolism and disease. Natl Med J India 2003;
16(1):24-26.
8. Scott EM, Wright RC. Fluorometric determination of
isonicotinic acid hydrazide in serum. J Lab Clin Med
1967;70(2):355-60.
9. Scott EM, Wright RC. Fluorometric determination of
isonicotinic acid hydrazide in serum. J Lab Clin Med
1967;70(2):355-60.
10. Singh SP, Pande JN, Khilnani GC, Kailash S.
Comparison between serum and urinary sulphadi-
midine acetylation as predictors of isoniazid
acetylator status in patients with pulmonary
tuberculosis. Indian J Chest Dis Allied Sci 1996;
38(1):5-11.
11. Sohrani AS, Ahmad B, Janbaz KH. Acetylation
percentage method for determination of acetylator
status in human volunteers and tuberculous patients.
Pak J Pharm Sci 1995;8(1):11-16.
330  Practical Manual of Experimental and Clinical Pharmacology
EXPERIMENT NO: 23
Aim
To evaluate anticholinergic effect of oxybutynin
(30 mg tablet) on salivary secretion in healthy
volunteers.
Background
Adverse effects of anticholinergic medications are
well documented. Drugs such as atropine and
scopolamine inhibit muscarinic receptors and can
produce both peripheral (constipation, dry mouth,
tachycardia, urinary retention, reduced sweating)
and central (cognitive and memory impairment,
confusion, delirium, headache, blurred vision,
dizziness, and drowsiness) effects. Other drugs
like oxybutynin and tolterodine are clinically used
to treat symptoms of overactive bladder, such as
incontinence, frequent or urgent urination and
increased night-time urination. Oxybutynin is
preferred in the experiment because it causes
severe dry mouth. In a study, it was reported that
¼th of patients who begain oxybutynin treatment
had to stop because of dry mouth.
Materials and Methods
• Healthy adult volunteers (Willing to give
written informed consent form)
• Cotton dental rolls and weighing balance
• Oxybutynin extended release (30 mg).
Initial Screening of Volunteers
Subject is screened for detailed evaluation of any
disease condition, detailed history should be taken
in respect to smoking, alcohol consumption, drug
therapy, or any associated disease conditioned.
Precautions for Experiment
Do not give medication to the volunteers who are
allergic to oxybutynin, or they have untreated or
uncontrolled glaucoma or are unable to urinate.
Volunteers are excluded, in cases of glaucoma,
any liver disease, kidney disease, an enlarged
prostate, ulcerative colitis, blockage in stomach
or intestines, or gastroesophageal reflux disease
(GERD) or slow digestion.
Methods
Method 1: The baseline salivation is measured by
placing the three dental rolls under the tongue for
2 min. Thereafter, interventional drug is adminis-
tered and wait for 1-2 hr (depending on half-life
of drug). Salivary secretion is again measured by
using three dental rolls which are inserted into
the buccal cavity under the tongue for 2 min.
The salivary secretion rate is observed at 0 hr
(baseline), 1, 2, 3, 6 and 12 hr and the evaluation
of the salivary flow is done by observing the
difference in weight of three dental rolls after and
before the intervention drug administration.
Rate of salivary secretion = (Weight of three
dental rolls after the treatment – Weight of
three dental rolls at baseline)
Method 2: Volunteer should rinse his/her mouth
with approximately 60-100 ml of tap water.
Volunteers are allowed to wait for 5-10 min and
then, an accurately weighed 1 × 1 inch square of
paraffin (pre-weight) is placed on each subject’s
tongue. The subject is then instructed to chew the
paraffin for 2 minutes, after which the accumu-
lated saliva and the chewed paraffin is expecto-
rated into a previously weighed clear dry container
(beaker, wide mouth glass tube, etc.).
The salivary secretion rate is observed at 0 hr
(baseline), 1, 2, 3, 6 and 12 hr and the evaluation
of the salivary flow is done by observing the
difference in weight of paraffin square after and
before the intervention drug administration.
Rate of salivary secretion = (Weight of paraffin
and sputum after the treatment - Weight of
paraffin square at baseline)
Observations and Result
Salivary secretion
Pre-drug(0 hr; Post-drug
baseline) 1 hr 2 hr 3 hr
1st person
2nd person

Discussion
Oxybutynin chloride exerts direct antispasmodic
effect on smooth muscles by competitively
antagonizing the M1, M2, and M3 subtypes of the
muscarinic acetylcholine receptors and inhibits
the muscarinic action of acetylcholine on smooth
muscles. As compared to atropine, it exhibits 1/
5th of the anticholinergic activity of atropine on
the rabbit detrusor muscle, but have 4 to 10 times
the antispasmodic activity. Transdermal patch is
also available for oxybutynin, but due to less
anticolinergic side effects, salivary secretion is
affected moderately.
SUGGESTED READING
1. Bye CE, Clubley M, Henson T, Peck AW, Smith SA,
Smith SE. Changes in the human light reflex as a
measure of anticholinergic effect of drugs. A
comparison with other measures. Eur J Clin
Pharmacol 1979;15:21-25.
2. Chancellor MB, Appell RA, Sathyan G, Gupta SK. A
comparison of the effects on saliva output of
oxybutynin chloride and tolterodine tartrate. Clin
Ther 2001;23:753-60.
3. Steiner JE, Birnbaum D, Karmeli F, Cohen S. Effect
of Diazepam on Human Salivary Secretion. Digestion
1970;3(5):262-68.
4. Tripathi SK, Griyappanavar CR, Lal A, Biswas K,
Biswas NR, Sankaranarayanan A, Sharma PL.
Evaluation of antimuscarinic activity in human
volunteers: A teaching aid in clinical pharmacology.
Indian J Physiol Pharmacol 1995;39(2):163-65.
EXPERIMENT NO: 24
Aim
To demonstrate histamine induced wheal and
flare in healthy volunteers.
Background
Quantitatively antihistaminic potency of drugs is
evaluated by the histamine-induced wheal and
flare response. It is an eruption of skin either by
injury or direct injection of an allergen in the skin.
It is typically characterized by three stages, begins
Miscellaneous Practicals  331
with the appearance of an erythema at the site of
injury/injection, followed by development of a
flare surrounding the site and finally due to fluid
leakage from capillaries under skin, a wheal forms
at the site. Hence, identified as redness or swelling
due to release of histamine. Drugs which can be
evaluated are levocetirizine, desloratadine,
fexofenadine, loratadine, etc.
Materials and Methods
• Healthy adult volunteers (willing to give
written informed consent form)
• Facilities for clinical pharmacology laboratory
(Appendix IV)
• Drugs: levocetirizine (5 mg), desloratadine 5
mg, fexofenadine HCl 180 mg, montelukast
sodium 10 mg, etc. and histamine.
Initial Screening of Volunteers
Subject is screen for detailed evaluation of any
disease condition; detailed history should be taken
in respect to smoking, alcohol consumption, drug
therapy, or any associated disease conditioned.
Inclusion Criteria
Volunteer aged 18-40 years, willing to give written
informed consent.
Exclusion Criteria
Volunteers should be excluded, if there is h/o any
concomitant chronic or acute illness; or history of
or current cardiovascular (including cardiac
arrhythmias), respiratory, hepatic, renal, gas-
trointestinal, endocrinological, neurological, or
psychiatric disease; anaphylactic shock as well
as disorders capable of altering the absorption,
metabolism or elimination of drugs, or constituting
a risk factor when taking the trial medication.
In case of concomitant drugs such as cortico-
steroids, antihistamines, cromoglycates, or
leukotriene antagonists.
332  Practical Manual of Experimental and Clinical Pharmacology
Volunteer with habit of heavy caffeine
drinkers (>five cups of coffee, tea, cola, etc. per
day).
Volunteers with exposure to skin irritants or
UV light in last 48 hours before each visit should
be excluded.
Precaution
Volunteers should be evaluated in detail about
allergy or intolerance to the study drugs-to-drugs
related to the study procedures, as well as any
medicine chemically related to the study drugs or
their excipients.
Observation and Result
Volunteers
Pre drug(15 minbefore drug intake; baseline)
Wheal Flare
0.1 0.4 1.6 0.1 0.4
1 WFB – WFT
2 100
Percentage (%) of wheel/flare inhibition is
calculated by,
where
WFB = Wheel/flare after histamine
WFT = Wheel/flare after drug treatment
Discussion
It is the direct method of evaluation of anti-
histaminic activity of a drug. Wheel and flare
method has been used traditionally as a challenge
in skin pharmacodynamic studies of the anti-
histaminic activity of new compounds. The subject
is sensitized with allergen, as normally used for
the diagnosis of allergy by skin prick testing and
so, the clinical efficacy may be strengthened by
studies using challenge with the specific aller-
gens. An advantage is that allergen-induced wheal
and flare studies recruit allergic subjects rather than
healthy volunteers. During the screening of a
Methods
The volunteers should be selected after proper
screening. Volunteers are injected histamine in
the increasing doses of 0.1, 0.4 and 1.6μg intra-
dermally at the back (at the middle part of both
shoulder) and after 3-4 hr, wheal and flare
formation are observed and assessed. Effect of
antihistaminic drug is analyzed by the same
method. The observation can be performed 15
minutes before and 90 minutes after antihista-
minic drugs such as levocetrizine (5 mg) intake.
Other drugs which may be used in the experiment
are desloratadine 5mg, fexofenadine HCl 180 mg,
montelukast sodium 10 mg, etc.
Histamine
Post-drug (90 min after drug intake)
Wheal Flare
1.6 0.1 0.4 1.6 0.1 0.4 1.6
WFB
antihistaminic drug one should keep in mind that
it may provide false negative result if the test is
not done in specified time.
SUGGESTED READING
1. Khosla PP, Saha N, Koul A, Chakrabarti A,
Sankaranarayanan A, Sharma PL. Effects of
ranitidine alone and in combination with chlor-
pheniramine on histamine-induced wheal and flare
and psychomotor performance. Indian J Physiol
Pharmacol 1993;37(2):132-34.
2. Nelly Frossard, Margherita Strolin-Benedetti, Ashok
Purohit, Gabrielle Pauli. Inhibition of allergen-
induced wheal and flare reactions by levocetirizine
and Desloratadin. Br J Clin Pharmacol 1965;2:179.
3. Peck AW, Fowle ASE, Bye C. A comparison of
triprolidine and clemastine on histamine antagonism
and performance tests in man: Implications for the
mechanism of drug induced drowsiness. Eur J clin
Pharmacol 1975;8:455-63.
4. Saha N, Sachdev A, Bhasin DK, Sankaranahyanan
A, Khosla PP, Singh K, Sharma PL. Clinical
evaluation of the effect of omeprazole, cimetidine,
famotidine and ranitidine on histamine induced
cutaneous wheal and flare response. Int J Clin
Pharmacol Ther Toxicol 1993;31(7):322-25.
 Laboratory Experiments
33 (Assay)

EXPERIMENT NO: 25 stationary phase in contained within a short,

small bored column through which the liquid
Aim mobile phase is pumped at high pressure.
Therapeutic drug monitoring in pharmacology In this chapter few examples are given, so that
(antiepileptic/lithium/digoxin). it will be convenient for students to know, how
TDM is done in the pharmacology laboratory.
Background
(A) Estimation of phenytoin, carbamazepine
Therapeutic drug monitoring (TDM) is an investi- and phenobarbitone by HPLC
gational procedure from last three decades and
found to be very beneficial in the management of Procedure
narrow index therapeutic agents such as anti- Sample preparation
epileptic drugs, digoxin, lithium, antipsychotics,
etc. during the therapy. It has an important place
in the patient’s management and managing their
effects in chronic therapy where clinical symptoms
and signs of toxicity can also be difficult to detect
and interpret. The main objective of the TDM is to
optimize drug therapy by individualizing dose
according to the serum/plasma concentration
and the clinical effects. It is also frequently used
to assess drug toxicity and patient’s compliance.
The most, laboratory procedure for TDM is
performed by the HPLC because of the high
sensitivity or some drug like digoxin is performed
by the chemical/ELISA methods. Hence, all
principle of HPLC separation is applied to the
same; briefly HPLC separation of solute molecule
depends on the distribution of molecules between Chromatographic Conditions
a stationary and a liquid mobile phase. The Mobile phase: Acetonitrile: Methanol : 4 mmK+
relative affinities of solute for two phases may phosphate buffer(pH 6.0) in ratio of:
involve one or more of adsorption, partition, ion 20:40:40 (v/v/v)
exchange or a salvation mechanism and these Flow rate : 1.0 ml/min
determine. The separation characteristics the Temperature : Ambient
334  Practical Manual of Experimental and Clinical Pharmacology
AUFS : 0.2
Detector : UV detector at 215 nm
Injection volume : 100 µl
Sensitivity : 0.1 µg/ml
Standards : 4-16 µg/ml
Results
Normal level
Phenytoin 10-20 mg/L, Carbamazepine 4-10
mg/L, phenobarbitone 15-40 mg/L
Calculate Concentration of Unknown?
Discussion
Several studies have already demonstrated
usefulness of TDM in epilepsy management.
Several tertiary centers reported that increasing
demand of TDM is because of increased awareness
of physicians and availability of facility in most
of the centers. So, in future physician will more
depend on TDM because of its usefulness,
however it increases the health care cost because
of limitation of methodology and most of the drug
required sophisticated instruments. In the recent
time various development has been taken place
for rationalizing the TDM request by compu-
terized program with educational program which
will definitely improve the quality of TDM. Most
of the TDM laboratories routinly conduct for older
antiepileptics like phenytoin, carbamazepine,
phenobarbitone, since it is still widely prescribed.
Studies reported frequent inter-individual varia-
tion in the plasma level/dose ratio for these drugs.
Though, there are lot of variations in Western and
Asian populations. However, TDM pattern is
similar in developed and developing countries.
Limitation is very few centers publish their TDM
pattern in the literature.
(B) Estimation of lithium
Background
Lithium remains the drug of choice for bipolar
disorder, though various other drugs are
available. Lithium has narrow therapeutic index
and the therapeutic level between toxic, thera-
peutic and subtherapeutic serum levels are liable
to change because of various factors. Individual
variation, poor compliance, renal disease,
seasonal variation, etc. are some reasons which
make plasma lithium level estimation essential
as a part of better management of patients. It is a
monovalent cation which shows a high degree of
interindividual variation and narrow therapeutic
index. The variability in lithium levels need to be
carefully reviewed with caution, since it can affect
the efficacy and safety in the patients. In case of
variation oral dose modification is needed for the
management toxicity as well as loss of efficacy.
So, the therapeutic drug monitoring (TDM) of
lithium is essential.
Method of Estimation
Estimation of plasma lithium is commonly done
by lithium autoanalyzer. The principle is potential
developed by the lithium ion selective electrode
with respect to the reference electrode. Most
sample salts dissociate to their ions and exchange
reaction occurs between the relevant electrode and
the ion which produces a potential between the
ion selective electrode and the reference one.
Lithium ion selective electrode consists of a
neutral carrier- based lithium sensor immobilized
in polyvinyl chloride. It has electrical connection
by silver or silver chloride wire. The reference
electrode usually contain shell filled with
saturated potassium chloride which is separated
from the sample by a membrane. (For other
methods refer to Brown and Legg et al, 1970. Ann
Clin Biochem 1970; 7: 13-18.)
Discussion: Therapeutic drug monitoring (TDM),
is considered useful in patients suffering from
mood disorders, though it increases the health
care cost. Several studies indicates a pattern of
increasing awareness and caution among
physicians while prescribing lithium. TDM
assisted better management of psychiatric
patients for early detection and prevention ADR
and to avoid lack of therapeutic response. Some

of recent studies suggested therapeutic drug
monitoring using salivary plasma lithium levels
however it was not found beneficial to find the
plasma correlation. The normal therapeutic range
of lithium is 0.6 – 1.2 meq/L. Studies showed,
patients with an early onset of MDP has greater
variation in the lithium levels. It has been reported
that variations observed in Italy 10% ,
Netherlands 5% and in a subtropical country
like India seasonal variations observed in plasma
lithium levels of up to 25%, with no significant
change in the oral lithium dosage. So, lithium
shows marked inter individual variation, genetic
variability in pharmacokinetics, drug-drug
interactions, and high levels can lead to adverse
effects require TDM in those patients who are on
prophylaxis.
Digoxin estimation: The clinical usefulness of the
measurement of serum digoxin is due to its narrow
therapeutic ratio. In addition, individuals may
present variable response to digoxin with an
apparent increase in susceptibility to toxicity with
age. ELISA is a sensitive method for digoxin
quantitation in serum. The activity of the enzyme
present on the surface of the well is quantities by
reaction with a suitable substrate to produce color.
The employment of several serum references of
known digoxin concentration permits cons-
truction of a graph of activity and concentration.
Laboratory Experiments (Assay)  335
Besides ELISA, other chemical methods are
available for quantitative estimation.
SUGGESTED READING
1. Manpreet Sukhija, Bikash Medhi, Pandhi P. Effects
of artemisinin, artemether, arteether on the pharma-
cokinetics of carbamazepine. Pharmacology,
2006;76:110-16.
2. Manpreet Sukhija, Bikash Medhi, Pandhi P. Effects
of artemisinin, artemether, arteether on the pharma-
cokinetics of phenytoin. Meth Find Clin Exp
Pharmacol,2006,28(3):89-94.
3. Medhi B, Prakash O, Jose VM, Pradhan B, Chakra-
barty S, Pandhi P. Seasonal variation in plasma levels
of lithium in the Indian population-is there need to
modify dose? Singapore Medical Journal 2008;
49(9):724-27.
4. Medhi B, Sukhija M, Prakash A, Gaikowad S, Bansal
V, Pandhi P. Effects of Etoricoxib on the Pharma-
cokinetics of Phenytoin. Pharmacological Reports
(Poland Journal of Pharmacology) 2008;60(2):
233-37.
5. Medhi B, Sukhija M, Sumedh G, Vinu M Jose,
Chakrabarty S, Pandhi P. Lithium Therapeutic drug
monitoring pattern at a tertiary care hospital in India.
JK Practotioner 2006,13(1):15-17.
6. Prakash A, Medhi B. TDM pattern of the antiepileptic
drugs in developed and developing countries: An
overview. Neurosciences 2009;14(3):447-49.
7. Vinu J, Medhi B, Pandhi P. Antiepileptic Therapeutic
Drug Monitoring pattern at a tertiary care hospital
in India. Nepal Med Coll J 2006;8(2):107-10.
 Impact Factor
34
It is the general belief that there is direct
correlation between impact factor (IF) and journal
quality ratings among the physicians and
researchers because it is directly related to the
journal citation frequency. Most importantly, IF
helps to guide the researcher to choose the best
journal according to their frequency of citation
in their field. The IF and citation varies with the
types of the journals such as journal cited by both
physicians and researchers have the high IF
(New England Medical journal, The Lancet etc.),
followed by the journals cited by either by
physician or researchers.
The main purpose of creation of impact
factor in the biomedical research field is to
measure the journal’s value by calculating the
average number of citations per article over a
period of time which was initially designed by
Eugene Garfield in 1950’s. It was introduced in
scientific community as an assessment tool to
evaluate the value of the scientific journal by
calculating the number of citations of an article
published in particular journal over a specific
time period.
The terminology of impact factor was first
used in 1961 after the publication in Science
Citation Index (SCI) in 1963. Presently, it is
popularly called Journal Citation Reports (JCR),
and burgeoning literature using bibliometric
measures. In general number of citations of a
particular article indicates only the mean interest
of scientists for that article thus the IF highlights
average interest of that article which got publish
in the journal.
Calculation of the IF of a journal for a selected
year depends on the average number of citations
of an article getting published in that journal
during the last two years from all the published
articles in that year, i.e. If the IF of a journal is
3.0 in 2008, it reflects that on an average the
articles published in 2006 and 2007 were cited
3 times in the collection of all ISI indexed
journals which was published in 2008.
Hence, the ratio obtained from dividing
citations received in one year (numerator) by paper
published during the two previous years (deno-
minator).
IF= CR/PP
Where,
CR = Citations received in one year
PP = Paper* published during the two pre-
vious years
*paper: original/research paper, review article,
peer review, proceedings, etc.
The recent trend in scientific world is that, if
the researchers or physicians want to be recog-
nized, they should have a good number of
publications with good citations. Thus, it has
become mandatory for a scientist to publish their
work in journals with high IF. And this criterion
has lead to the development of the long-time belief
that the number only counts. Therefore, a great
number of publications make scientists popular
and distinguished in their field. Major criteria in
present time among scientific community, for
evaluating the status of the scientific journals
and also the status of the scientist on the basis of
 Impact Factor  337

their publication output, to assess how they are List of leading scientific journals and their impact factor
actively engaged in the research. For example, In (IF)
some countries like South Korea, China, and Journals name Impact factor (2007)
Pakistan, their science ministries are offering
Nature Medicine 31.921
cash rewards to their scientists if they publish NEJM 52.589
papers in journals with high IFs such as Nature, The Lancet 29.887
Science, or Cell, etc. JAMA 25.547
Journal Citation Index (JCR) are used as the BJCP 2.681
JPET 4.003
only evaluation criteria rather than to quanti- Pharmacological Review 18.823
fication of scientific contribution itself. Because Molecular Pharmacology 4.088
original idea of citation analysis was developed Pharmacology Research 9.643
to protect against the uncritical citation of Trends in Pharmacological Sciences 10.4
fraudulent data or even disputed data. Some of Therapeutic Drug Monitoring 3.032 (2006)
study questioned the meaning of IFs, stating that NEJM: New England journal of medicine; BJCP:
they actually represent popularity rather than British journal of clinical pharmacology; JPET:
prestige. Journal of pharmacology and experimental
Eugene Garfield, the inventor of the IF, therapeutics; JAMA: Journal of the American
emphasized that its potential value is primarily medical association.
in the management of library journal collections
to determine their optimum makeup, providing SUGGESTED READING
solid basis for cost-benefit analysis of subscription 1. Garfield E. How can impact factor can be improved?
budgets. Infact inventor of IF never predicted that BMJ 1966;313:413-15.
in scientific community IF will be used as a criteria 2. Garfield E. Use and misuse of citation frequency.
for judging the scientist, quality and providing Curr Contents 1985;43:3-9.
3. Kumar V, Upadhyay S, Medhi B. Impact of Impact
research grant.
factor in Biomedical Research, its use and misuse:
People misuse the IF as there are no specifically an overview. Singapore Med J 2009;50(8):752-55.
defined principles governing its interpretation. 4. Linardi PM, Coelho PMZ, Coster HMA. The impact
The IF is used to measure the importance of factor as a criterion for the quality of scientific
journals as well as researcher potential, for which production is a relative, not absolute measure. Braz
it was never intended, and it is used to make faulty J Med and Biol Res 1996;29:555-61.
5. Moed HF. The impact factor debate the ISI’s uses
comparisons, including journals themselves. So
and limits. Nature 2002;45:731-32.
misuse of IF is a common problem in research field, 6. Rey-Rocha J, Martín-Sempere MJ, Martinez-Frías JY
as scholars have complained about the misuse of López-Vera F. “Some misuses of journal impact factor
the IF since long-time. in research evaluation” Cortex 2001;37(4): 595-97.
 Computational
35 Pharmacology
In the changing trend of education, need of
computer and its knowledge is now necessary in
each and every field of science, meanwhile it is
found very useful tool for explaining any unknown
or new topic to the students or any scientific body
in a attractive way such as making animated films
of drug action on a system or showing the
molecular action of a drug, side effects, transport
of drugs, etc. Additionally, computer helps in
demonstration of drug effects on whole animal or
on isolated tissues of practical pharmacology for
undergraduate students or postgraduate
medical/pharmacy students. So, computational
pharmacology or computational therapeutics is a
rapidly ubiquitously growing area in the field of
development of techniques for using software to
capture, analyze and integrate biological and
medical data from many diverse sources. The term
‘in silico’ is an experiment performed by computer
and is related to the more commonly known in
vivo and in vitro methods. Among the “3Rs” of
Russell and Burch (1959), developed to reduce
the animal use or use with caution in experiment
and research, the replacement with alternative
method like in vitro or in silico is the first choice,
whereas the other two are refinement of methods
to minimize any adverse effects to individual
animal and reduction of animal use to achieve
consistent scientific objectives. These methods are
increasingly used in discoveries or advances in
pharmacology and therapeutics. Computational
(in silico) methods have been developed and
applied to pharmacology in hypothesis deve-
lopment and testing. Additionally, computational
approaches are now being used to facilitate the
experimental determination of macromolecular
structures by aiding in structural refinement based
on either nuclear magnetic resonance (NMR) or
X-ray data. These can also be applied in situations
where experimentally determined structures are
not available. With the rapid advance in gene
technology, including the human genome project,
the ability of computational approaches to
accurately predict 3D structures based on primary
sequence represents an area that is expected to
have a significant impact.
Drug design and development is another area
of research in pharmacology and it is correlated
with computational biochemistry and biophysics
is having an ever-increasing impact. Compu-
tational approaches can be used to aid in the
refinement of drug candidates, systematically
changing a drug’s structure to improve its
pharmacological properties, as well as in the
identification of novel lead compounds. The latter
can be performed via the identification of
compounds with a high potential for activity from
available databases of chemical compounds or
via de novo drug design approaches, which build
totally novel ligands into the binding sites of target
molecules. At the molecular level, computational
genomics helps pharmacological studies, about
learning of the genomes of cells and sometime uses
DNA microarray to identify the genes expressed
in each cell types.
Other important related allied fields are
Computational biomodeling, a field concerned with
building computational models of biological

systems, Computational biochemistry and biophysics,
which make extensive use of structural modeling
and simulation methods such as molecular
dynamics to explain the kinetics and thermo-
dynamics of protein functions, whereas clinomics
is a bridge between basic biological data and its
effect in clinical setting. For example certain genes
such as BRCA1 are associated with a higher
probability of developing breast cancer.
Application of in silico Methods
1. Maintains databases, quantitative structure-
activity relationships, pharmacophores,
receptor modeling and other molecular
modeling such as data mining and data
analysis which requires a computer.
2. In silico methods are primarily used alongside
the generation of in vitro data both to create
the model and to test it in the discovery and
optimization of new drug with affinity to
receptor target, its pharmacokinetics and
toxicity properties in addition to physico-
chemical characterization.
3. Makes easy correlation of human genome
through computational and experimental data
which can be correlated with all data types.
4. Computational approaches can be used to
investigate the energetics associated with
changes in both conformation and chemical
structure of the drug.
SUGGESTED READING
1. Albert A. Relations between molecular structure and
biological activity: Stages in the evolution of current
concepts. Ann Rev Pharmacol 1971;11:13-36.
2. Aradi I, Erdi P. Computational neuropharmacology:
Dynamical approaches in drug discovery. Trends
Pharmacol Sci 2006;27:240-43.
3. Arý¨ens EJ. Receptors: From fiction to fact. Trends
Pharmacol Sci 1979;1:11-15.
Computational Pharmacology  339
4. Balakin KV, Ivanenkov YA, Savchuk NP, Ivaschenko
AA, Ekins S. Comprehensive computational
assessment of ADME properties using mapping
techniques. Curr Drug Disc Tech 2005;2:99-113.
5. Chen YZ, Zhi DG. Ligand–protein inverse docking
and its potential use in the computer search of protein
targets of a small molecule. Proteins 2001b;43:217-
26.
6. Dudek AZ, Arodz T, Galvez J. Computational
methods in developing quantitative structure–
activity relationships (QSAR): a review. Comb Chem
High Throughput Screen 2006;9:213-28.
7. Ekins S, Swaan PW. Development of computational
models for enzymes, transporters, channels and
receptors relevant to ADME/TOX. Rev Comp Chem
2004;20:333-415.
8. Grzybowski BA, Ishchenko AV, Kim C-K, Topalov
G, Chapman R, Christianson DW, et al. Combinatorial
computational method gives new picomolar ligands
for a known enzyme. Proc Natl Acad Sci USA 2002;
99:1270-73.
9. Kulkarni SA, Zhu J, Blechinger S. In silico techniques
for the study and prediction of xenobiotic meta-
bolism: a review. Xenobiotica 2005; 35: 955-73.
10. Langowski J, Long A. Computer systems for the
prediction of xenobiotic metabolism. Adv Drug Del
Rev 2002; 54: 407-15.
11. Lemmen C, Lengauer T. Computational methods
for the structural alignment of molecules. J Comput
Aided Mol Des 2000;14:215-32.
12. Lipinski CA, Lombardo F, Dominy BW, Feeney PJ.
Experimental and computational approaches to
estimate solubility and permeability in drug
discovery and development settings. Adv Drug Del
Rev 1997;23:3-25.
13. Mestres J. Computational chemogenomics approaches
to systematic knowledge-based drug discovery. Curr
Opin Drug Discov Dev 2004;7:304-13.
14. Rockey WM, Elcock AH. Rapid computational
identification of the targets of protein kinase
inhibitors. J Med Chem 2005;48:4138-52.
15. Sieburg HB. Physiological studies in silico. Studies
in the Sciences of Complexity 1990;12:321-42.
16. Sung M-H, Simon R. In silico simulation of inhibitor
drug effects on nuclear factor-kB pathway dynamics.
Mol Pharmacol 2004;66:70-75.
 Pharmacokinetics/
36 Pharmacodynamics

PHARMACOKINETICS/
PHARMACODYNAMICS (PK/PD)
Pharmacokinetic (PK) and pharmacodynamics
(PD) are the principles and separate part of
pharmacology which always play basic assess-
ment in the drug development. The new concept
of PK/PD model is one which bridges the
principles of both PK and PD. This is a measure of
response of a drug in a postulated time (Fig. 36.1).
In many condition, it is difficult to identify the
minimum effective dose through dose-response
data in a new drug such as antihypertensive,
anticancer drug, prostaglandins, H2-blockers,
respiratory drugs, etc. There are several clinical
factors which have influence on the dose – Fig. 36.1: Pharmacokinetics pharmacodynamics
response-time relationship such as demographics modeling
(age, sex, weight, body weight, body surface area, PK/PD modeling is varying with the com-
lean body mass, etc.) or any disease status. partmental model designed for the analysis. It is
The objectives of PK/PD model are: mainly of two type, follows the classification of
• Forecasting of response of a drug dose pharmacokinetic compartments, i.e. single com-
• Dose selection and dosing interval partment, time –independent PK/PD model and
• Duration of drug response complex PK/PD, time dependent models.
• Estimate therapeutic window The first approach is the simplest and the
• Identify mechanism of action drug concentration is distributed in the one com-
Hence, the pharmacological effect of drug is partment, i.e. blood and it correlates direct
varying accordingly in relation with the plasma pharmacological action, limiting its direct effect
concentration; either it is antihypertensive, anti- in one compartment only. So, in this context there
coagulant or diuretics. Animal study described is no time dependant event. Whereas, the second
the relationship between the concentration of drug approach complex PK/PD is the commonest in
in blood or plasma and drug receptor occupancy vivo approach, which is involving sequential
or functional response, provide clinically useful analysis of the concentration versus time and
test regarding potency, efficacy and duration of effect versus time. This model tells about the dose-
the effect. response-time relationship which gives a

biophase responses, i.e. compartment where drug
shows its effect. (Represents half-life, bioavai-
lability and potency). So, briefly complex PK/
PD model determine the drug effect in the
compartment in time course of drug concen-
tration.
PK/PD model approach limits, its use in in
vivo constants which can be applied to the other
in vivo data calculation but not link with in vitro
data conversion. The application of PK/PD
modeling is documented in cardiovascular (CVS),
central nervous system (CNS), oncology and
gastroenterology.
SUGGESTED READING
1. Derendorf H, Möllmann H, Hochhaus G, Meibohm
B, Barth J. Clinical PK/PD modelling as a tool in
drug development of corticosteroids. Int J Clin
Pharmacol Ther 1997;35(10):481-88.
2. Gibb IA, Anderson BJ. Paracetamol (acetaminophen)
pharma-codynamics: Interpreting the plasma
concentration. Arch Dis Child 2008;93(3):241-47.
Pharmacokinetics/Pharmacodynamics  341
3. Kristensen NR, Madsen H, Ingwersen SH. Using
stochas-tic di_erential equations for PK/PD model
development. J Pharmacokinet Pharmacodyn 2005;
32:109-41.
4. Lieberman R, Nelson R. Dose-response and
concentration-response relationships: Clinical and
regulatory perspectives. Ther Drug Monit 1993; 15(6):
498-502.
5. Meibohm B, Derendorf H. Basic concepts of
pharmacokinetic/pharmacodynamic (PK/PD)
modelling. Int J Clin Pharmacol Ther 1997;35(10):
401-13.
6. Schaefer HG, Heinig R, Ahr G, Adelmann H, Tetzloff
W, Kuhlmann J. Pharmacokinetic-pharmacodynamic
modelling as a tool to evaluate the clinical relevance
of a drug-food interaction for a nisoldipine controlled-
release dosage form. Eur J Clin Pharmacol 1997;
51(6):473-80.
7. Tornoe CW, Jacobsen J L, Pedersen O, Hansen T,
Madsen H. Grey-box modelling of pharmacokinetic/
pharmacodynamic systems. J Pharmacokinet
Pharmacodyn 2004b;31(5):401-17.
8. Wagner JG. Kinetics of pharmacologic response. I.
Proposed relationships between response and drug
concentration in the intact animal and man. J Theor
Biol 1968;20(2):173-201.
 Promotional Product
37 Literature
PROMOTIONAL PRODUCT LITERATURE
The commonest sources for providing information
from industry to practicing physician are verbal,
written and computerized, e.g. professional
meeting, advertising in journal, e-mail or from
medical representative, etc. Lot of money has been
utilized for effective communication to physician.
Most important aspect of promotional literature
is to look for sources of references which provide
comprehensive update to the physician from
leading medical national/international journals
or international organization data like WHO. So,
product literature is the needful source of
information regarding the drugs which provide
its complete knowledge about its nature, class,
pharmacological effects and the known side effect,
interaction and contraindication for the medical
practitioners as well as for patients. Hence,
promotional product literature can be defined as
graphic and/or written material prepared by/for
one party, which is made available to the public
for information and distribution, for the purpose
of promoting or marketing the particular product
or brand. Generally, the sources of product
literature are categorized into several categories,
but broadly they are divided into following
classes:
Primary, secondary and tertiary product
literatures are importantly informative literatures
for the researcher, clinicians and other health
professionals, whereas promotional product
literature is a form of advertising tool, provided to
the clinician, chemists and other related health
professional who directly/indirectly related to the
patients health. But, it is different from conven-
tional advertising in few modes:
1. Promotional product literature is more
selective. Since, it is distributed through
controlled means, rather than through general
media placement, the target audience can be
more sharply defined and the message can
be written with the targeted ‘consumer’ in
mind.
2. Initial readership is virtually 100%. When a
particular consumer group is targeted,
message can be tailored to group which is pre-
disposed to a particular message.
3. There is the opportunity for direct movement
into the objective. Because of the established
interest on the part of the recipient.
In a clinical set-up, a major marketing
technique used by pharmaceutical companies is
direct-to-physician marketing (DTP). This form of
marketing frequently employs promotional
marketing literature, based on clinical research,
which serves as an important source of drug
information and may influence the prescribing
behavior of a physician.
Role of Promotional Product Literature
It can be used in following different ways for the
best possible results:

1. Fulfilling requests for information: Many people
want detail information regading a product
before purchase. So, information can be
provided in the form of promotional literature
without visiting them personally.
2. Informational displays: This literature can serve
as a brochure display in a doctor’s office and
providing the information to both current
customers and prospects.
3. Leaving with prospects following sales meetings:
It is hard to close a sale with just one visit to a
prospect but making repeat visits is also not
cost-effective. Hence, leaving a piece of
promotional literature behind helps in provi-
ding additional information to clinician
without making the repeated sales meetings.
4. Use as direct mail pieces: Promotional literature
can be used proactively as direct mail pieces
and mailed to people in the target market.
5. Promotional literature as a sales tool: A well
written literature helps a salesperson in
remembering the benefits and features of a
product more easily and gives more effective
sales presentations by illustrating each and
every point and helps to take business to the
higher level of sales and profits.
Components of Promotional Product Literature
The Headline
The headline is an important part of a printed
piece. It sets the tone for the rest of the piece and is
responsible for sales effectiveness.
Body Copy
It is the main body of the literature. It includes the
text which conveys the advertiser’s message, name
and address. In case of a pharmaceutical product,
it should include the name of the product
(normally the brand name), the active ingredients,
using approved names where they exist, the name
and address of the pharmaceutical company or
its agent responsible for marketing the product,
date of production of the advertisement,
“abbreviated prescribing information” which
should include an approved indication or
Promotional Product Literature  343
indications for use together with the dosage and
method of use and a brief statement of the
contraindications, precautions and side effects.
Designing Promotional Literature
The need and production of the promotional
literature is a result of feedback from market
research of established need, its attractive
promotional designing and commercial need.
Establishing a need: First step requires the
establishment of the need of literature which can
be done by answering some questions like what
is the specific purpose of the proposed printed
piece.
• Is it absolutely necessary?
• What is the target audience? And
• How big is the “Universe” i.e. checking the
demographic information?
Deciding on a format: There are many formats
which include leaflets, brochures, master
brochures, fliers, scanable letters, sales letters, rack
cards, counter cards, postcards, pocket cards,
meaning and circulars, etc. The choice of one
which is best for the purpose is influenced by
various factors like the proposed use, number of
different printed pieces and the budget for
printing.
Selecting a printer: This should be done prior to
making any moves keeping in mind the quality
and budget.
Ideal Characteristics of Promotional Literature
Good promotional literature should inform,
educate, stimulate and direct the reader in the
simplest and most concise manner. It should be
focussed, attention-grabbing and benefits-driven.
It should have conviction value and memorizing
value. It should be suggestive and true. Promo-
tional literature should maintain high ethical
standards and comply with applicable legal,
regulatory and professional requirements.
To ensure the ethical promotional practices,
Pharmaceutical companies must comply with
IFPMA (International Federation of Pharma-
ceutical Manufacturers and Associations) Code.
344  Practical Manual of Experimental and Clinical Pharmacology
The IFPMA Code of Pharmaceutical Marketing
Practices (the “IFPMA Code”) sets out standards
for the ethical promotion of pharmaceutical
products to health care professionals to ensure
that member companies’ interactions with health
care professionals are appropriate and perceived
as such. Effective from January 1st, 2007, this Code
replaces the IFPMA Code of Pharmaceutical
Marketing Practices (Update 2000). In India,
standards of promotion are set by the Organization
of Pharmaceutical Producers of India (OPPI),
which is a premier organization of pharma-
ceutical, manufacturers in India.
Advantages of Promotional Product Literature
1. Introduces a new product in the market.
2. Increases prospects.
3. Increases sales.
4. Fights competition.
5. Enhances good will of concern.
6. Educates the target market.
7. Eliminates middlemen.
8. Supports the salesmanship.
9. Raises the standard of living.
Disadvantages of Promotional Product
Literature
1. May be inadequate, altogether inaccurate,
invalid, unethical, false, misleading, biased,
and deceptive or based on studies of poor
methodological quality.
2. Leads to monopoly in a particular brand of
product.
3. Creates artificial demand for the product.
4. Increases the price of the product as expenses
on it form the part of the total cost of the product.
5. May be harmful for the society, as the
pharmaceutical promotional activities have
powerful influences on prescribing behavior
of the clinicians.
6. Enhances the self medication of patients.
Current Recommendations
Physicians should be made aware of the limita-
tions of the current methods of medical industry
promotions and the influence of marketing on
prescribing behavior. More emphasis should be
laid on the evaluation of promotional and other
scientific literature while teaching under-
graduate students. Sessions of appraisal of
promotional literature should be conducted for
interns and resident doctors as they are the ones
who usually interact with pharmaceutical
representatives. Initiatives should be taken to
form a body for the review of promotional and
other scientific material before it reaches the
target, i.e. doctors or consumers as in direct-to-
consumer advertising. Government should
establish a national drug policy that is focused
on public health problems and is consistent
with general health policies, forming an
organization that will evaluate national medica-
tions and technology together with this policy,
and developing standard treatment guidelines.
One of the methods recommended is the creation
of an independent, reliable, easily accessible,
and current source of information for physicians
(like Internet web pages and drug bulletins). In
this process, it is important to create resources
to provide physicians an alternative source of
information that is evidence-based. Physicians
should equiped themselves with the skills of
critically appraising and assessing the lite-
rature. The new drug meant for promotion
should be preferred over the existing one, if it
offers clear advantages in terms of safety,
tolerability, efficacy and price, i.e. STEP criteria.
The new drug should be relevant to the
clinician’s practice in terms of population
studied, the disease and the need for new
treatment. Obtaining and assessing the quality
of references is important and should be
emphasized. Clinicians should work with
medical industry representatives to formulate
evidence-based literature. The methodology of
the study should be carefully judged to deter-
mine the authenticity of the evidence. By
critically appraising and assessing the litera-
ture, the ultimate goal of medical practice i.e. to
ensure the optimum care of the patients can be
achieved.

Check list for evolution of promotional product literature
Name (Active ingredients/ INN/ Generic
name/ brand name)
Active ingredient/ contents/ doses form/
regimen
Approved therapeutic use
Dosages from regimen
Adverse drug reaction: minor/ major
Precautions, contraindication and warnings
Introduction: Drugs food product
Name and address of manufacturer
Scientific references
Note:
FDA has given regulations which specify that
advertisments are false, lacking in fair balance, or
otherwise misleading if:
• Using headlines, sub-headlines, or pictorial
or other graphic material in way that is
misleading or erroneous.
• Use literature or references inappropriately to
support claims in the advertisement.
• If they make claims about relative safety and
efficacy or about the populations in which the
drug is useful that are not supported by the
current literatures.
Promotional Product Literature  345
SUGGESTED READING
Yes/No 1. http://www.p2pays.org/ref/16/15687.pdf.
2. Cardarelli R, Licciardone JC, Taylor LG. A cross-
Yes/No sectional evidence-based review of pharmaceutical
promotional marketing brochures and their under-
Yes/No lying studies: Is what they tell us important and
Yes/No true? BMC Fam Pract 2006;7:13.
Yes/No 3. http://www.hubpages.com/hub/Promotional_
Yes/No Literature.
4. Lal A. Pharmaceutical drug promotion: How it is
Yes/No being practiced in India? J Assoc Physicians India
Yes/No 2001;49:266-73.
Yes/No 5. IFPMA Code of Pharmaceutical Marketing Practices
2006 (Revision).
6. OPPI Code of Pharmaceutical Marketing Practices
2007.
7. Gupta RS, Sharma BD, Bhalla NS. Fundamentals of
commerce theory and functional management. 1st
Ed. Kalyani Publishers 1996;440-41.
8. Sharma RK, Gupta SK, Oberoi M, Sharma R. Principle
and practice of commerce theory and functional
management. 1st Ed. Kalyani Publishers 1996;
436-39.
9. Shetty VV, Karve AV. Promotional literature: How
do we critically appraise? J Postgrad Med 2008;
54:217-21.
10. Civaner M. A Proposal for the Prevention of Ethical
Problems Related to Drug Promotion: A National
Network for Drug Information. Turk J Psyc 2008;
19:310.
11. Wilkes MS, Doblin BH, Shapiro MF. Pharmaceutical
advertisements in leading medical journals: Experts’
assessments. Ann Int Med 1992;116:912-19.
 Analytical Toxicology
38
ANALYTICAL TOXICOLOGY
This chapter briefly emphasizes on principles and
practical information on the analysis of chemicals,
drugs and poisons in biological specimens
(serum, plasma, urine, etc.) particularly clinical
and forensic specimens. Toxicology is derived
from Greek word “Toxican” which was used for
the poisonous substance in which arrow head
were dipped. Poisonous substances are those
whose cumulative effect leads to the fatality either
in low or high dose. Several medicines like,
opioids, aspirin, barbiturates, antidepressants,
phenothiazine, benzodiazepines, warfarin,
paracetamol, etc. which is effective at therapeutic
dose but may produce the toxic effect in the high
doses (Table 38.1).
Broadly, toxicology is divided into the different
subspecialties such as analytical toxicology, clinical
toxicology, environmental toxicology, industrial
toxicology, etc. whereas for pharmacological
teaching purpose analytical toxicology and clinical
toxicology is important. The analytical toxicology
may be required to detect, identify, and in measure
a wide variety of compounds in samples from
almost any part of the body or in related materials
such as residues in syringes or in soil. Whereas,
according to the WHO, “clinical toxicology” is
defined as the detection, identification and the
measurement of drugs and other foreign compounds
(xenobiotics) in biological and related specimens to help
in the diagnosis, treatment, prognosis, and prevention
of poisoning.
Generally, acutely poisoned patients samples
are assessed for general clinical chemistry (blood
glucose, blood gases, etc.) and hematology.
Toxicological analyses can play a useful role if
the diagnosis is in doubt, the administration of
antidotes or protective agents is contemplated, or
the use of active elimination therapy is being
considered.
There are several advantages of analytical
toxicology, which have the influence on the
clinical toxicology, i.e. (1) it gives a correlation of
laboratory finding with the clinical diagnosis,
(2) it provide knowledge about the poison, (3) helps
in the selection of specific antidote, and (4) therapy
can be channelize based on the toxicology report.
Analytical techniques: Color tests and spectro-
photometry, chromatography and electrophoresis,
mass spectrometry, and immunoassay.
Methods in assessment of toxicology: In a
poisoning case, collection of tissues and the organ
in which the poison is suspected to be present
should be kept for the chemical examination. The
isolation and identification of unknown poison
are the very important step in the analysis of any
poisoning. Even small amount of poison present
may changes the physiology of body which often
requires the skilled hand and confirmatory results.
Complete analytical procedure is divided into
three phases, i.e pre-analytical, analytical, and post-
analytical phases (Fig. 38.1).
Pre-analytical phase is an important procedure
which involves appropriate sample collection (in

Fig. 38.1: Phases of analytical toxicology evaluation
appropriate sample tubes) and safe transport,
receipt, and storage of biological samples.
Transfer of samples to the laboratory and then
arranging it for the analysis is also a part of pre-
analytical phase. Analytical phase is one which
involves complete analytical procedure and
identification in which analysts used tried and
tested procedures to perform the requested or
appropriate analyses to the required degree of
accuracy and reliability in an appropriate,
clinically relevant time-scale. Post-analytical phase
includes reporting results by writing (proforma of
laboratory, telephone, fax, or other electronic
means.
Note: Typically full records of the analysis should be
kept for a minimum of 5 years (10 or more years for
medicolegal implications case). Remaining samples
must be disposed safely in an agreed time-frame.
Sample Preparation and Methods of Estimation
The method should be accurate, reliable and
reproducible. Uniform methodology is not
employed and the analytical methods used is
depend on local circumstances. There are two
types of sample/material referred to the toxi-
cological analysis, sample in survival cases and
sample in fatal cases.
Analytical Toxicology  347
The extraction procedure is the important step
in the analysis of toxic compound because further
steps and treatment is depends on the identified
toxins. Toxicological extraction method provides
the suitable concentration of sample to which the
detection technique may be applied. The design
of extraction method is depend on the analytical
aim, the type of the material analyzed and the
nature of the substance to be extracted.
The extraction method depends on the known
as well as the unknown nature of toxins. Whereas,
toxins are mainly classified according to the
isolation method involved,
1. Volatile poison, isolated by distillation or
diffusion,
2. Metallic poisons, isolated by the oxidation of
the organic matter,
3. Toxic anions, isolated by dialysis or ion
exchange methods,
4. Pesticides, direct solvent extraction method
5. Corrosives, e.g: Acids, alkali, etc.
6. Non-volatile organic substance, isolated by
solvent extraction and
7. Miscellaneous poisons like animal poisons,
e.g: Snake bite, cantharides, etc. or plants
poison like Papaver somniferum, Cannabis,
Belladona, etc. requiring special extraction
technique.
Reporting of Results
The results may be presented in mmol/L, ng/mL,
µg/L, and mg/L. But, to avoid any confusion in
units, it is important to generalize the results
report which should be clear and to maintain
consistency by the laboratory in a particular
institute or center.
Example: Lithium, Thyroxine, and Methotrexate:
Molar units - mmol/L whereas
In Scientific Papers and immunoassay results
may expressed as ng/mL, µg/L, mg/L, g/L, and
molar (mmol/L, etc.).
348  Practical Manual of Experimental and Clinical Pharmacology

Table 38.1: Drugs and chemicals concentrations in blood

Drugs Therapeutic dose (mg/L) Toxic dose (mg/L) Lethal dose (mg/L)
Acetaminophen 10-20 400 1500
Aminophylline 20-100
Barbiturate; Short acting 1 7 10
Intermediate acting 1-5 10-30 30
~
Phenobarbital 10 40-60 80-150
Barbital ~ 10 60-80 100
Carbamazepine 2 8-10
Chloral hydrate 10 100 250
Codeine 25 µg/L
DDT 13 µg/L
Diazepam 0.5-2.5 5-20 >50
Digitoxin 1.7-2.1 µg/L 320 µg/L
Digoxin 0.6-1.3 µg/L 2-9 µg/L
Diphenylhydantoin 5-22 50 100
Ethanol 1.5 g/L > 3.5 g/L
Halothane 200
Iron 500 6
Lead 0.05-1.3 0.7
LSD 1-4 µg/L
Lidocaine 2 6
Lithium 4.2-8.3 13.9 13.9-34.7
Methanol 200 > 890
Morphine 0.1 0.05-4
Nicotine 10 5-52
Propranolol 0.025-0.2 8-12
Quinidine 3-6 10 30-50
Quinine 12
Salicylate 20-100 150-300 500
Theophylline 20-100
Warfarin 1.0-10

SUGGESTED READING Technical Series No 5. Vienna: UN International Drug

1. Flanagan RJ. SI units - Common sense not dogma is
needed. British Journal of Clinical Pharmacology 1995;
39:589-94.
2. Flanagan RJ. Role of the laboratory in the diagnosis
and management of poisoning. In: Medical Toxicology,
Edition 3. Ed. Dart RC. Baltimore: Lippincott,
Williams and Wilkins, 2003: 337-58.
3. Flanagan RJ, Braithwaite RA, Brown SS, Widdop B,
de Wolff FA. Basic Analytical Toxicology. Geneva:
WHO, 1995.
4. French - http://www.intox.org/databank/
documents/supplem/supp/sup2f.htm.
5. Flanagan RJ, Streete PJ, Ramsey JD. Volatile substance
abuse - Practical guidelines for analytical investigation of
suspected cases and interpretation of results. UNDCP
Control Programme, 1997
6. http://www.undcp.org/odccp/
technical_series_1997-01-01_1.html.
7. Maurer HH. Systematic toxicological analysis
procedures for acidic drugs and/or metabolites
relevant to clinical and forensic toxicology and/or
doping control. Journal of Chromatography B 1999;
733:3-25.
8. SOFT/AAFS (Society of Forensic Toxicologists/
American Academy of Forensic Sciences). Forensic
toxicology laboratory guidelines 2002.
9. http://www.softtox.org/guidelines/default.asp
10. Stewart MJ, Watson ID. Analytical reviews in clinical
chemistry: methods for the estimation of salicylate
and paracetamol in serum, plasma and urine. Annals
of Clinical Biochemistry 1987;24:552-65.

11. Stewart MJ, Watson ID. Analytical reviews in clinical
chemistry: Methods for the estimation of salicylate
and paracetamol in serum, plasma and urine. Annals
of Clinical Biochemistry 1987;24:552-65.
12. Wilson J. External quality assessment schemes for
toxicology. Forensic Science International 2002;128:
98-103.
13. Wu AH, McKay C, Broussard LA, Hoffman RS,
Kwong TC, Moyer TP, Otten EM, Welch SL, Wax P.
National academy of clinical biochemistry laboratory
medicine practice guidelines: Recommendations for
Analytical Toxicology  349
the use of laboratory tests to support poisoned
patients who present to the emergency department.
Clinical Chemistry 2003; 49:357-79.
14. http://www.clinchem.org/cgi/content/full/49/3/
R50.
15. Duffus JH. Glossary for chemists of terms used in
toxicology. Pure and applied chemistry, 1993, 65: 2003-
2122.
16. Stead AH, et al. Standardised thin-layer chro-
matographic systems for the identification of drugs
and poisons. Analyst (London), 1982;107:1106-68.
 Recent Advances in
39 Pharmacology
TRANSLATIONAL MEDICINE
Simply, translational medicine is a concept of
translating the laboratory findings to the clinic
i.e. in the patients care. This concept is broadly
known as “bench to bedside”. So, this is the
cumulative support of physicians and scientists
to translate findings from the laboratory into better
treatment for patients. This is often named as
“Molecular Medicine” and “Personalized Medicine”,
which mainly refer to the process of molecular
research of drug in laboratory to clinical care.
Objective of translational medicine is to
discover the origin, pathway and mechanism of
diseases including the responsible biomarkers.
The main aim of such concept is to systematically
discover and develop new diagnostics and
therapeutic methods and drugs in short duration
of time.
The approach used in the translational
medicine is unidirectional either from the “bench
Fig. 39.1: Bench to bedside concept
to bed” such as the conventional targeted drug
discovery or “bed to bench” such as biological
samples like DNA, blood, or tissue fragments, etc.
from the patients and observed the disease cause
at molecular level and further helps in develop
drugs targeted at particular subgroups of disease.
REVERSE PHARMACOLOGY
Ayurvedic drugs are generally regarded as safe
and are commonly used in many acute and
chronic illness. Reverse pharmacology is actually
a rediscovery of drugs activity and its mechanism
of action. Hence, reverse pharmacology can be
defined as the integrating science of developing
candidate drugs from a clinical to experiential hits
and leads by transdisciplinary exploratory
studies and ultimately to understand the mecha-
nisms of action at different pathological stages of
biological organism. Then, confirm candidate
drugs experimental to clinical use on the basis of
safety, efficacy and acceptability on relevant
science.
The first stage experiential included clinical
trial 2 and 1 as an experience and data from
Fig. 39.2: Three stages of reverse pharmacology:
experiential, exploratory and experimental

previous uses. Whereas, exploratory studies, the
2nd stage would cover dose-activity in ambulant
patients and selected in vitro and in vivo experi-
mental models to evaluate the key target. The 3rd
stage experimental involves supporting pharma-
coepidemiological data, standardization of
formulation with HPLC pattern, exploratory
human/animal studies, human dose deter-
mination and other experimental parameters like
levels of biological parameters (biochemical,
hematological, tissue, etc.)
Common examples of reverse pharmacology
are Digitalis purpurea (Na + /K + ATPase),
Papaverum somniferum (Opioid receptors),
Cincohona (Antimalarial), Curcuma longa (Anti-
inflammatory, etc.), etc.
Fig. 39.3: Comparative flow diagram of pharmacological
research and the reverse pharmacology research
MICRODOSING (PHASE 0)
In the drug development, one of the reasons for
drug failures during late developmental phase is
suboptimal pharmacokinetics (clearance, volume
of distribution, t1/2 , etc.) or change in metabolic
status in human. Hence, the pharmacokinetic
studies before going for the larger trial have shown
to be beneficial and a new experimental approach
has been developed, known as microdosing or
Phase 0 in human.
“Microdosing” study designed, as a solution
of above mentioned problem. Therefore, the
objectives of the “Microdosing” are to reduce the
cost and resources spent on non-practicable drugs
Recent Advances in Pharmacology  351
Fig. 39.4: Procedure for microdosing
done on animals. “Microdosing” or “phase 0” is
defined as a microdose which is administered
100 µg of investigational drug or 1/100th of the
pharmacological active dose extrapolated from in
vitro or in vivo animal studies in most sensitive
species, whichever is the lesser.
Microdose is a very low dose compared to the
pharmacological active dose, so its analysis and
assessment depends on the availability of
ultrasensitive analytical methods to measure drug
and its metabolite. (May measure at the level of
picogram to femtogram range).
Analysis is done by the highly sensitive
analytical methods like “Accelerator Mass
Spectrometry” (AMS) and “Positron Emission
Tomography” (PET). The basic step is to label an
investigational drug using the radioisotope
carbon-14 (14C; t1/2 5740 yrs*) for AMS and carbon-
11 (11C: 20 min*) for PET. Both AMS and PET
quantify the total number of labeled atoms present
in a sample rather than distinguishing between
parent drug and metabolite(s).
Methodology may include the parallel study
groups between two and five molecules using
human subject. Each molecule might be admi-
nistered in a crossover design such as an
intravenous (i.v.) dose followed by an oral dose
after a suitable washout period. AMS is used
for determining PK data by taking body samples
over time, processing the samples in the
laboratory and then analyzing their drug
content. PET provides primarily PD data
352  Practical Manual of Experimental and Clinical Pharmacology
through real-time imaging and some limited PK
data. Thus, pharmacokinetic parameters like
absorption, Vd, CL, etc. can be obtained. Relative
proportion of drug and its metabolites can be
obtained through chromatographic separation
of an extract of blood or plasma followed by
analysis of collected chromatography fractions
such as liquid chromatography- mass spectro-
metry (LC-MS).
Note: *PK data can be obtained for only 2 hr after
drug administration by PET (i.e. 5-6 decay half-
life) while PK data can be obtained up to 100 days
after drug administration by using AMS.
SUGGESTED READING
1. Marincola FM. Translational Medicine: A two-way
road, J Transl Med 2003;1(1):1.
2. Stacey P Mankoff, Christian Brander, Soldano
Ferrone, Francesco M Marincola. Lost in Translation:
Obstacles to Translational Medicine, J Transl Med
2004;2:14.
3. Clinical and Translational Medicine —http://
www.ctsjournal.com
4. Pharmacology at a reverse-http://www. express-
pharmaonline.com/20060930/research03.shtml
5. Kutzleb Christian, Busmann Annette, Wendland
Martin, Maronde Erik. Discovery of Novel Regulatory
Peptides by Reverse Pharmacology: Spotlight on
Chemerin and the RF-amide Peptides Metastin and
QRFP. Current Protein and Peptide Science
2005;6(3):265-78.
6. Sakurai T. Reverse pharmacology of orexin: From an
orphan GPCR to integrative physiology. Regul Pept
2005;126(1-2):3-10.
7. Lappin G, Garner RC. Big physics, small doses: The
use of AMS and PET in human microdosing of
development drugs. Nature Rev Drug Discovery
2003;2:233-40.
8. Wilding I, Bell J. Improved early clinical development
through human microdosing studies. Drug Discovery
Today 2005;10(13):890-94.
9. Lappin G, Garner RC. Current perspectives of 14C-
isotope measurement in biomedical accelerator mass
spectrometry. Anal Bioanal Chem 2004;378:356-64.
10. Aboagye EO, Price PM, Jones T. In vivo pharma-
cokinetics and pharmacodynamics in drug develop-
ment using positron-emission tomography. Drug
Discovery Today 2001;6:293-302.
11. Bergström M, Grahnén A, Langström B. Positron
emission tomography microdosing: A new concept
with application in tracer and early clinical drug
development. Eur J Clin Pharmacol 2003;59:
357-66.
 Appendices

APPENDIX I: ABBREVIATIONS
AC Animal Care
AFDO Association of Food and Drug Officials
AMS Accelerator Mass Spectrometry
ANDA Abbreviated New Drug Application
ANOVA Analysis of Variance
AP Arterial Pressure
AVMA American Veterinary Medical Association
AWA Animal Welfare Act
AWIC Animal Welfare Information Center
CAAT Center for Alternatives to Animal Testing
CaSR Calcium Sensing Receptor
CDC Centers for Disease Control and Prevention
CFA Complete Freund’s Adjuvant
CFR Code of Federal Regulations
CIRA Center for Information on Research with Animals
CPCSEA Committee for the Purpose of Control and Supervision of Experimentation on Animals
CRC Concentration Response Curve
DEPA Direct endpoint assay
DMSO Dimethyl Sulfoxide
DRC Dose Response Curve
DTP Direct-To-Physician marketing
ED Effective Dose
ELISA Enzyme-Linked Immunosorbent Assay
EIH Entry Into Human
ESA Endangered Species Act
EU Endotoxin Units
FASEB Federation of American Societies of Experimental Biology
FBR Foundation for Biomedical Research
FDA Food and Drug Administration
FHD First Human Dose
FTIM First Time In Man
GLP Good Laboratory Practices
GCP Good Clinical Practices
GRA Graded Response Assay
HED Human Equivalent Dose
HEPA High-Efficiency Particulate Air Filter
HTS High-Throughput Screening
IACUC Institutional Animal Care and Use Committee
IAEC Institutional Animal Ethics Committee
354  Practical Manual of Experimental and Clinical Pharmacology
IBSC/IBC Institutional Biosafety Committee
IBRO International Brain Research organization
ICLAS International Council for Laboratory Animal Science
IFA Incomplete Freund’s Adjuvant
IF Impact Factor
ILAR Institute for Laboratory Animal Research
IND Investigational New Drug
JCR Journal Citation Reports
LAL Limulus Amebocyte Lysate
LAMA Laboratory Animal Management Association
LC-MS Liquid Chromatography and Mass-Spectrometry
LD Lethal Dose
LOAEL Lowest Observed Adverse Effect Level
mAb Monoclonal Antibody
MAD Multiple Ascending Dose
MANOVA Multiple Analysis of Variance
MES Maximal Electroshock Seizure
MLD Minimum Lethal Dose
MRSD Maximum Recommended Starting Dose
MTD Maximum Tolerated Dose
M.wt Molecular Weight
NABR National Association for Biomedical Research
NARRC National Advisory Research Resources Council
NAS National Academy of Sciences
NDA New Drug Application
NIH National Institutes of Health
NOAEL No Observable Adverse Effect Level
OLAW Office of Laboratory Animal Welfare
OHSP Occupational Health and Safety Program
OPPI Organization of Pharmaceutical Producers of India
OSHA Occupational Safety and Health Administration
PAD Pharmacologically Active Dose
PCR Polymerase Chain Reaction
PEFR Peak Expiratory Flow Rate
PEFM Peak Expiratory Flow Meter
PET Positron Emission Tomography
PK/PD Pharmacokinetics/Pharmacodynamics
PSS Physiological Salt Solution
RCF Relative Centrifugal Force
RSC Radiation Safety Committee
SAD Single Ascending Dose
SCAW Scientists Center for Animal Welfare
SCI Science Citation Index
SD Standard Deviation
SEM Standard Error of Mean
SOPs Standard Operating Procedures
SSD Safe Starting Dose
TCP Tail Cuff Pressure
TDL Toxic Dose Low
TDM Therapeutic Drug Monitoring
THLE Tonic Hind Limb Extension
UHTS Ultra High Throughput Screening
UPLC Ultra-performance Liquid Chromatography
US FDA United States Food and Drug Association
WHO World Health Organization
 Appendices  355

APPENDIX II: DRUG AND SOLUBILITY

Sl No. Drugs Molecular weight Solvents

a
1 Acetyl choline chloride 181.7 Hygroscopic, freely soluble in water
2 Adrenalineb 183.21 Slightly soluble in water; insoluble in ethanol (95%)
and in ether ; soluble in solutions of mineral acids,
of sodium hydroxide and of potassium hydroxide
3 Adrenaline bitartrate 333.29 Freely soluble in water; slightly soluble in ethanol
(95%); practically insoluble in chloroform and in
ether.
4 Aspirin 180.16 Slightly soluble in water, freely soluble in ethanol
(95%); soluble in chloroform and in ether.
5 Atropine methonitrate 366.42 Freely soluble in water; soluble in ethanol (95%);
practically insoluble in chloroform and in ether
6 Atropine sulphate 694.84 Very soluble in water; freely soluble in ethanol (95%)
and in glycerin; practically insoluble in chloroform
and in ether
7 Caffeine 194.19 Freely soluble in chloroform and in boiling water;
sparingly soluble in water and in ethanol (95%);
slightly soluble in ether
8 Codeine phosphate 406.37 Freely soluble in water; slightly soluble in ethanol
(95%); sparingly soluble in chloroform; practically
insoluble in ether
9 Diazepam 284.74 Freely soluble in chloroform; soluble in ethanol (95%);
very slightly soluble in water
10 Diclofenac sodium 318.13 Freely soluble in methanol; soluble in ethanol (95%);
sparingly soluble in water and in glacial acetic acid;
practically insoluble in ether, in chloroform and in
toluene
11 Diclofenac sodium 318.14 Water for Injection
injection
12 Digoxin 780.95 Practically insoluble in water, freely soluble in
pyridine and in a mixture of equal volumes of
dichloromethane and methanol; slightly soluble in
ethanol (95%).
13 Digoxin injection 780.95 Water for Injection and Ethanol or other suitable
solvents (base of injection)
14 Frusemide 330.74 Soluble in acetone; sparingly soluble in ethanol (95%);
slightly soluble in ether; practically insoluble in
water
15 Haloperidol 375.87 Practically insoluble in water, soluble in chloroform;
sparingly soluble in ethanol (95%).
16 Haloperidol 375.87 Lactic Acid diluted with Water for Injection.
injection
17 Indomethacin 357.79 Insoluble in water ; Soluble in chloroform; sparingly
soluble in ethanol (95%) and in ether

(Contd...)
356  Practical Manual of Experimental and Clinical Pharmacology
(Contd...)

Sl No. Drugs Molecular weight Solvents

18 Lithium carbonate 73.89 Soluble in water; practically insoluble in ethanol
(95%).
19 Morphine sulphate 758.83 Soluble in water; freely soluble in hot water; slightly
soluble in ethanol (95%) but more soluble in hot
ethanol (95%); practically insoluble in chloroform
and in ether.
20 Morphine sulphate Water for Injection
injection
21 Omeprazole 345.42 Freely soluble in chloroform; soluble in ethanol (95%)
and in methanol; very slightly soluble in water
22 Oxytocin 1007.20 Soluble in water
23 Oxytocin injection Water for Injection
24 Paracetamol 151.16 Freely soluble in ethanol (95%) and in acetone;
sparingly soluble in water
25. Pentobarbitone Sodium 248.26 Very soluble in water and ethanol
26 Phenobarbital sodium 252.22 Soluble in ethanol (95%) and in ether; very slightly
soluble in water
27 Phenobarbital Sodium 9:1 (Propylene Glycol : Water for Injection)
Injection
28 Phenytoin Sodium 274.25 Soluble in water and ethanol (95%); insoluble in
dichloromethane and in ether

a Should be stored in the air tight container at cool place and immediately close the cap of the bottle after transferring
the Ach from the bottle at the time of weighing.
b Store in tightly-closed and light-resistant containers (decomposes rapidly in presence of moisture and at higher
temperatures) and it is not stable in a neutral or alkaline solution which rapidly becomes red on exposure to air.

Other Solvents
Dimethyl sulfoxide (DMSO), Dichloromethane (DCM), 0.1N HCl or NaOH may use to dissolve the test drug
during the experiment.

Solubility Definitions
• Very soluble: less than 1 part
• Freely soluble: 1-30 parts
• Soluble: 10 – 30 parts
• Sparingly soluble: 30 – 100 parts;
• Slightly soluble: 100 – 1000 parts
• Very slightly soluble: 1000 – 10,000 parts
• Practically insoluble: more than 10,000 parts

Note: Drugs which are not freely soluble in water or warm water, those can be making soluble in DMSO.
The pharmacological activity of the solvent is also considered during the selection of the solvent such as ethanol or
ether have the CNS depressant property hence not preferred as a solvent in the experiment related to the CNS activity
assessment.
 Appendices  357

APPENDIX III: LIST OF DRUGS IN CLINICAL

PHARMACOLOGY PRACTICALS

ACECLOFENAC
• Chemically phenylacetic acid derivative
• Well absorbed from GIT
• Peak plasma time-1 – 3 hrs after oral dose
• 99% plasma protein bound
• Plasma elimination half life- 4 hr
• Two third dose excreted in urine as hydroxymetabolite
• Dose-100 mg twice daily p.o. 100 mg once daily in hepatic impairment
• 100 mg. 200 mg-SR. 100 mg BD
• ADR: Indigestion, heartburn, dyspepsia, diarrhea, nausea, abdominal pain, flatulence.
• Trade name- Dolodkind (mankind), Dolokind-SR (mankind), Zerodol etc.

ASPIRIN
• Acetyl salicylic acid
• Rapidly absorbed from GIT, skin. Non-ionised form absorbed in stomach and intestine
• During 1st 20 min after oral dose-aspirin is the dominant form in plasma
• Once absorbed, rapidly converted to salicylate
• 80-90% plasma protein bound, widely distributed
• Volume of distribution (Vd)-170 ml/kg in adults
• Salicylate crosses placental barrier, secreted in breast milk
• Mainly hepatic metabolism
• At 325 mg dose t 1/2 is 2-3 hr, at high dose 15-30 hr
• Excretion- unchanged in urine-30% in alkaline urine, 2% in acidic urine
• Removable by hemodialysis
• Dose- 300- 900 mg every 4-6 hrs for analgesic, anti-inflammatory, antipyretic
• 75-325 mg –as antiplatelet, 50mg, 75mg, 150mg
• ADR: Hyperventilation, bleeding, tinnitus, fluid retention acidosis
• Trade name: Aspicot, colsprin, ecosprin, ASA 50 (german remedies), Loprin -75, Delisprin-150 etc.

CARBAMAZEPINE
• Carbamazepine is related chemically to the tricyclic antidepressants. It is a derivative of iminostilbene with
a carbamyl group at the 5 position; this moiety is essential for potent antiseizure activity. The structural
formula of carbamazepine is slowly and irregularly absorbed from GIT, metabolized in liver – CYP3A4,
CYP2C8c-10,11 epoxide also active. Excreted mainly in urine, widely distributed, 75% plasma protein
bound
• Plasma t1/2 5-26 hr, induces its own metabolism.
• Plasma therapeutic range- 4-12 µg/ml
• Crosses placental barrier, present in breast milk.
• Therapeutic concentrations should be maintained at 6 to 12 µg/ml, although considerable variation occurs.
Side effects referable to the CNS are frequent at concentrations above 9 µg/ml.
• Dose 100-200 mg twice daily can increase 100-200 mg every week upto 0.8-1.2 g daily divided doses, 100,
200,400 mg TAB
• ADR: Stupor or coma, hyperirritability, convulsions, and respiratory depression. Long-term therapy
drowsiness, vertigo, ataxia, diplopia, and blurred vision. Other adverse effects include nausea, vomiting,
serious hematological toxicity (aplastic anemia, agranulocytosis), and hypersensitivity reactions (dermatitis,
358  Practical Manual of Experimental and Clinical Pharmacology

eosinophilia, lymphadenopathy, splenomegaly).transient elevation of hepatic transaminases in plasma in

5 to 10% of patients.
• Trade name: Mazetol, tegretol, carbetrol etc.

CHLORPHENIRAMINE
• Slowly absorbed, peak plasma time 2.5- 6 hr following oral administration. 25-50% bioavailability, undergoes
considerable first pass metabolism.
• Widely distributed, enter CNS, excreted in urine. Duration of action 4-6 hrs
• Dose- 4 mg orally 4-6 hourly up to 24 mg max
• I/V 10-20 mg slow
• Trade name: Avil etc.

DIGOXIN
• Absorption from GIT 70-90%
• Therapeutic plasma range- 0.5-2 ng/ml
• Large Vd, concentration in myocardium > plasma
• 20-30% plasma protein bound
• Crosses placenta, CSF, breast milk
• Elimination half life-1.5- 2 days
• Excreted unchanged in urine, not removed by dialysis
• Dose: effect within 2 hrs, max. Within 6 hrs loading dose needed. Steady state concentration (CSS) in sample
taken at least 6 hrs later- 0.5- 2 ng/ml
• Loading dose- 750-1500 µg as a single dose during the initial 24 hrs or in divided daily doses 6 hrly, 250ug
twice daily
• Trade name- Cardioxin, lanoxin etc.

ETORICOXIB
• COX-2 selective inhibitor (106.0 times more selective for COX-2 inhibition over COX-1)
• Bioavailability is 100%
• Plasma Protein binding is 92%
• Metabolism by CYP3A4
• t1/2 is 22 hours
• Drug is excreted through kidney 70% in stool 20%
• It is contraindicated in pregnancy
• Currently it is used in treatment of rheumatoid arthritis, psoriatic arthritis, osteoarthritis, ankylosing
spondylitis, chronic low back pain, acute pain and gout.
• Doses; 60, 90 mg/day for chronic pain and 120 mg/day for acute pain.
• Trade name: Arcoxia etc.

FEXOFENADINE
• Rapidly absorbed orally, peak plasma conc. 2-3 hrs, 60-70% plasma protein bound, elimination t-½ 14 hrs.
Excretion mainly in faeces.
• Dose- 120 mg daily or 60 mg b.d and 180 mg OD for chronic urticaria
• Trade name: Allegra etc.

FUROSEMIDE
• Rapid GI absorption, 60-70% bioavailability,
 Appendices  359

• Plasma t1/2 is 2 hr, increased in hepatic and renal failure.

• 99% albumin bound, excreted unchanged in urine, crosses placenta, excreted in breast milk.
• Hemodialysis does not clear it
• Increased free form in heart failure, renal and hepatic impairment.
• Effect of oral dose within 0.5–1 hr,
• Peak at 1-2 hr and last for 4-6 hrs. I/V within 5 min, lasts for 2 hrs
• Dose-oral 40 mg once daily
• I/V: 20-50 mg slowly at 4 mg/min
• Contraindications : Severe Na+ and volume depletion, hypersensitivity to sulfonamides and anuria
• ADR: Hyponatremia, circulatory collapse, thromboembolic episodes, hypochloremic alkalosis, hypokalemia,
hypomagnesemia, cardiac arrhythmias and hypocalcemia, rarely tetany, Ototoxicity, hyperuricemia ,
hyperglycemia , hypercholesteriamia, skin rashes, photosensitivity, paresthesias, bone marrow depression,
and gastrointestinal disturbances.
• Trade name: Lasix, frusenex, diucontin-K, Lasix,oral /iv etc.

GLYCERYL TRINITRATE
• Organic nitrates are polyol esters of nitric acid
• Rapidly absorbed from oral mucosa, GIT and skin
• <100% bioavailability due to pre-systemic clearance
• Sublingual or buccal tablet effect within 1-3 min, transdermal patch or ointment- within 30-60 min, I/V
within 1-2 min.
• Duration of action- sublingual-30-60 min, transdermal patch 24 hr, I/V 3-5 min
• Hydrolysis in plasma, metabolized in liver
• Dose-acute angina- 300-600 µg S/L tab, max 3 doses within 15 min.
• Buccal tablet 1-2 mg, ointment 2%
• Unstable angina-5-10 µg/min to up to 200 µg/min
• Hypertension control 5-25 µg/min
• Tablet for sublingual use-0.3-0.6 mg as needed
• Sublingual spray-0.4 mg as needed
• Chewable tablet 2.5-9 mg 2-4 times daily
• IV 10-20 µg/minute
• Contraindication in raised intracranial tension,tolerance etc.
• ADR: Orthostatic Hypotension, tachycardia, throbbing headache
• Trade name: Angised, nitrocontin, nitroderm, myovit, illisrol, Nitribid, nitrostat, nitrol, nitro- dur etc.

HYOSCINE
• Esters formed by combination of an aromatic acid, tropic acid, and complex organic bases, either tropine
(tropanol) or scopine. Scopine differs from tropine only in having an oxygen bridge between the carbon
atoms designated as 6 and 7
• Readily absorbed from GIT, entirely metabolized in liver, crosses blood brain barrier, placenta
• Absorbed through skin
• Dose: Tab.10 mg, 20 mg I/M OR I/V max. 100mg daily
• 20 mg orally-4 times daily
• 10-20 mg,3-5 times daily,
• IM/IV.20-40 mg,3-5 times daily
• ADR: Increased pulse rate, visual disturbance due to i.v administration
• Trade name: Buscopan etc.
360  Practical Manual of Experimental and Clinical Pharmacology

INH- ISONIAZID
• Readily absorbed from GIT
• Peak concentration of 3-7 µg/ ml appear in blood 1-2 hr after a fasting dose of 300 mg orally
• Plasma t ½ is 1-6 hrs
• Primary metabolic route- acetylation is genetically determined. However clinical effectiveness not influenced
by dosing 2 to 3 times per week
• Over 75% appears in urine within 24 hr, crosses BBB, placenta.
• Dose- 300 mg orally empty stomach
• ADR: Rash (2%), fever (1.2%), jaundice (0.6%), and peripheral neuritis (0.2%). Hypersensitivity reaction
manifest with fever, various skin eruptions, hepatitis, and morbilliform, maculopapular, purpuric, and
urticarial rashes. Other are agranulocytosis, eosinophilia, thrombocytopenia, anemia, arthralgia, peripheral
neuritis to isoniazid and occurs in about 2% of patients receiving 5 mg/kg of the drug daily. Precipitate
convulsions in patients with seizure disorders, Muscle twitching, dizziness, ataxia, paresthesias, stupor, and
toxic encephalopathy, euphoria, transient impairment of memory, dryness of the mouth, epigastric distress,
methemoglobinemia, tinnitus, and urinary retention.
• Trade name: ISOKIN, ISONEX, RifacomE-Z, Isokin.tab.100 mg, Isonex.tab. 100mg, Nydrazid etc.

LITHIUM
• Lithium is the lightest of the alkali metals (group Ia); the salts of this monovalent cation similar characteristics
with Na+ and K+.
• Readily absorbed from GIT.
• Peak concentration at 0.5-3 hr after oral dose in modified formulations peak at 2-12 hr after oral dose.
Completely distributed within 6-10 hr. Levels in bone, brain, thyroid more than in serum. Excreted in urine
mainly.
• Elimination half life-20-24 hr. Steady state conc. At 4-7 days. Blood sample to be taken 12 hrs after the last
dose following consistent dosing schedule of 4-7 days.
• Therapeutic level 0.4-1 mmol/liter
• Dose- 450-675 mg bid. or 0.4-1.2 mg in daily divided doses.
• 150, 300, 600 mg lithium carbonate
• ADR: Vomiting, profuse diarrhea, coarse tremor, ataxia, coma, and convulsions, mental confusion,
hyperreflexia, gross tremor, dysarthria, seizures, and cranial nerve and focal neurological signs, progressing
to coma and death. Other toxic effects are cardiac arrhythmias, hypotension, and albuminuria. Other
adverse effects common even in therapeutic dose ranges include nausea, diarrhea, daytime drowsiness,
polyuria, polydipsia, weight gain, fine hand tremor, and dermatological reactions including acne. The
prolonged use of Li+ causes a benign and reversible depression of the T wave of the ECG, an effect not
related to depletion of Na+ or K+. Seizures have been reported in Allergic reactions such as dermatitis and
vasculitis can occur with Li+ administration. mild alopecia. The use of Li+ in pregnancy has been associated
with neonatal goiter, CNS depression, hypotonia (“floppy baby” syndrome), and cardiac murmur, Ebstein’s
malformation.
• Trade name: Licab, eskalith etc.

PHENYTOIN
• It is 5-phenyl or other aromatic substituent appears essential for activity against generalized tonic-clonic
seizures. Alkyl substituent’s in position 5 contribute to sedation, a property absent in phenytoin
• Slowly but completely absorbed from upper intestine, metabolized in liver by cytochrome 2C 9 and 19.
Genetic polymorphism influences metabolism.
• Undergoes eneterohepatic recycling widely distributed. 90% plasma protein bound. Dose dependent halflife.
t1/2 22 hrs. Therapeutic levels- 10-20- ug /ml.
• Dose-3-4 mg/kg daily/150-300 mg daily up to 600 mg, 100 mg capsule
 Appendices  361

• Status epilepticus- 10-15 mg/kg slow iv at < 50 mg/min

• ADR: Cardiac arrhythmias, behavioral changes, gingival hyperplasia, osteomalacia, and megaloblastic
anemia. Hirsutism, neutropenia and leucopenia, red-cell aplasia, agranulocytosis, and mild thrombocytopenia,
lymphadenopathy, hypoprothrombinemia. Rarely Stevens-Johnson syndrome. Systemic lupus
erythematosus and potentially fatal hepatic necrosis.
• Trade names: Dilantin, epsolin, eptoin etc.

PHENOBARBITONE
• 5-phenyl-5-ethylbarbituric acid, it has less antiseizure potency than does of phenobarbital, but it is virtually
devoid of hypnotic activity. Rapidly absorbed from GIT, lipid insoluble, peak at 2 hrs after oral and at 4 hr
after IM
• Plasma protein binding-45-60%
• Metabolized in liver, plasma t-half 75-120 hrs, children 21-75 hrs, neonate- prolonged.
• Therapeutic range- 15-40 µg/ml
• Crosses placental barrier
• Dose 60-180 mg daily at night, children 8 mg/kg daily, status epilepticus-10 mg/kg
• ADR: Sedation, the most frequent undesired effect of phenobarbital, nystagmus and ataxia occur at excessive
dosage, irritability and hyperactivity in children, and agitation and confusion in the elderly. Scarlatiniform
or morbilliform rash, possibly with other manifestations of drug allergy, occurs in 1 to 2% of patients.
Hypoprothrombinemia, rarely exfoliative dermatitis.
• Trade name: Gardinal, luminal, luminettes, luminal, mysoline etc.

PILOCARPINE
• Mean elimination half-life-0.7- 1.3 hrs after 5-10 mg oral dose
• Inactivated in plasma and at neuronal synapse
• Glaucoma-0.5-4% eye drop daily 4 times, 1%, 2%, 3%, 4%
• ADR: Precautions, Toxicity, and Contraindications : It is contraindicated in asthma, hyperthyroidism,
coronary insufficiency, and acid-peptic disease and hyperthyroid patients. Other possible undesirable
effects are flushing, sweating, abdominal cramps, belching, a sensation of tightness in the urinary bladder,
difficulty in visual accommodation, headache, and salivation.
• Dry mouth- 5 mg tid
• Trade name: pilocar, carpomiotic, Pilocar/pilodrops, Salagen 5-7.5 mg oral etc.

PROPRANOLOL
• Completely absorbed from GIT, hepatic tissue binding, first pass metabolism
• Peak plasma concentration 1 -2 hr after oral dose. Plasma concentration vary greatly. High lipid solubility,
crosses BBB, placenta.
• 90% plasma protein bound
• Plasma t1/2 is 3-6 hrs, metabolised in liver
• Dose: Hypertension 40—80 mg twice daily
• Pheochromocytoma- 60 mg daily on 3 days before surgery always with alpha blockade angina- 40 mg 2-
3 times daily
• ADR: Bradycardia, asthma, PVD. diabetes
• Trade name: Betabloc, betaspan, ciplar, corbeta, inderal etc.

TACROLIMUS
• Erratic oral absorption. bioavailability- 15-20%. 80% bound to erythrocytes.plasma protein binding- 99%.
Extensively metabolised in liver by CYP3A4. t 1/2 is 12-43 hrs, in transplant patients.
362  Practical Manual of Experimental and Clinical Pharmacology

• Dose- liver transplant- 100-200 µg/kg orally in two divided doses within 6 hrs or 10-50µg/kg i/v within 24
hrs.
• Kidney transplant- 150-300 µg/kg orally in two divided doses within 6 hrs or 50-100 µg /kg i/v within 24
hrs. etc.

TROPICAMIDE
• Mydriatic – within 20-40 min of instillation, lasts 6 hrs
• Cycloplegic- maximum effect within 30 min short lasting complete recovery within 6 hrs
• Dose- mydriatic- 1-2 drops 0.5%- 15-20 min prior
• Cycloplegic-1-2 drops-1%- repeated after 5 min
• 0.5%, 1%,eye drops
• Trade name: Mydriacyl etc.
 Appendices  363

APPENDIX IV: EQUIPMENT REQUIRED IN CLINICAL

PHARMACOLOGY LABORATORY

• Ventilator with internal oxygen compressor

• Artificial lung
• Pulse oximeter
• Cardiac monitor
• Defibrillator
• 12 lead ECG machine with ECG electrode and tracer paper
• Oxygen cylinder big size
• Nebulizer with respules
• Venturimask with tubing with oxygen flow meter
• Ambu bag with different size facemask
• Infusion pump
• Glucometer with strips with needle to prick
• EMERGENCY TRAY-Laryngoscope of different size including pediatric size blade
– Endotracheal tube of different size with stellate
– Ryles tube of different size including including infant feeding tube
– Oropharyngeal airway of different size
– Central line with double and single lumen
– Vascular guard and tegaderm of different size
– Mersilk 2-0 size
– Cut down set
– Torch
– Bandages
– 3 way cannula ,iv set ,BT set ,disposable syringes 100 cc, 50 cc, 20 cc, 10 cc loaded and labelled injection
sodium Bicarbonate, Diazepam, Haloperidol, Atropine, Adrenaline, 2% xylocaine, Dexona,
hydrocortisone , Chloropheniramine, calcium gluconate, adenosine, midazolam, pancuronium,
vancuronium, cardrone, verapamil, midazolam, propofol
• EMERGENCY TROLLY-with different compartment leveled for different drugs-inj. Furosemide (lasix),
phenytoin, sodium bicarbonate, calcium gluconate, lorazepam, diazepam, adrenaline, atropine, deryphylline,
2% xylocaine solution, iv fluids, hemacil , iv set, BTset.
• SURGICAL TRAY-betadine solution, savlon solution, spirit, sterilize cotton pack, bandages, cut downset,
surgicare gloves with different size, Foley’s catheter, 2% xylocaine jelly, dressing pad
364  Practical Manual of Experimental and Clinical Pharmacology

APPENDIX V: ANALYTICAL AND MOLECULAR MASS

Important Definitions
• % (w/v) concentration: mass (g) of solute contained in 100 mL solution
• % (w/w) concentration: mass (g) of solute contained in 100 g of sample
• % (v/v) concentration: volume (mL) of solute in 100 mL solution
• Molarity (M) = number of moles of solute in 1 L of solution
• Molality (m) = number of moles per 1 kg of solvent
• Normality (N) = number of equivalents of a substance dissolved in 1 L of solution

Important Conversions
Molarity (M) into normality (N) : (M) X (valency)
mg/L
mg/L into mM in water :
MW

mg/L into M in water :

N into mEq/L :
ppm into mg/L in water :

ppm into mg/m3 :

% into ppm :

where,
M = Molarity
N = Normality
mg/L = milligram/Litre
mM = MilliMolar
MW = Molecular weight
mEq/L = milliEquivalent/litre
ppm = Parts per million
% = Percentage
 Appendices  365

APPENDIX VI: LOG CONVERSION TABLE

General Rule for Logarithm
log (XY) = log (X) + log (Y)
log (X/Y) = log (X) – log (Y)
log (X)a = (a) log (X)
Common logarithms are those for which the base (e) = 10. It is denoted by log10 [ ] or log [ ]
Natural logarithms are those for which the base (e) = 2 = 2.72. It is denoted by log e [ ] or ln [ ]
loge [ x] = 2.303 log10[x]
Logarithm Table (for numbers 1 to 5.49)
No. 0.00 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08 0.09
1.0 0.000 0.004 0.009 0.013 0.017 0.021 0.025 0.029 0.033 0.037
1.1 0.041 0.045 0.049 0.053 0.057 0.061 0.064 0.068 0.072 0.076
1.2 0.079 0.083 0.086 0.090 0.093 0.097 0.100 0.104 0.107 0.111
1.3 0.114 0.117 0.121 0.124 0.127 0.130 0.134 0.137 0.140 0.143
1.4 0.146 0.149 0.152 0.155 0.158 0.161 0.164 0.167 0.170 0.173
1.5 0.176 0.179 0.182 0.185 0.188 0.190 0.193 0.196 0.199 0.201
1.6 0.204 0.207 0.210 0.212 0.215 0.217 0.220 0.223 0.225 0.228
1.7 0.230 0.233 0.236 0.238 0.241 0.243 0.246 0.248 0.250 0.253
1.8 0.255 0.258 0.260 0.262 0.265 0.267 0.270 0.272 0.274 0.276
1.9 0.279 0.281 0.283 0.286 0.288 0.290 0.292 0.294 0.297 0.299
2.0 0.301 0.303 0.305 0.307 0.310 0.312 0.314 0.316 0.318 0.320
2.1 0.322 0.324 0.326 0.328 0.330 0.332 0.334 0.336 0.338 0.340
2.2 0.342 0.344 0.346 0.348 0.350 0.352 0.354 0.356 0.358 0.360
2.3 0.362 0.364 0.365 0.367 0.369 0.371 0.373 0.375 0.377 0.378
2.4 0.380 0.382 0.384 0.386 0.387 0.389 0.391 0.393 0.394 0.396
2.5 0.398 0.400 0.401 0.403 0.405 0.407 0.408 0.410 0.412 0.413
2.6 0.415 0.417 0.418 0.420 0.422 0.423 0.425 0.427 0.428 0.430
2.7 0.431 0.433 0.435 0.436 0.438 0.439 0.441 0.442 0.444 0.446
2.8 0.447 0.449 0.450 0.452 0.453 0.455 0.456 0.458 0.459 0.461
2.9 0.462 0.464 0.465 0.467 0.468 0.470 0.471 0.473 0.474 0.476
3.0 0.477 0.479 0.480 0.481 0.483 0.484 0.486 0.487 0.489 0.490
3.1 0.491 0.493 0.494 0.496 0.497 0.498 0.500 0.501 0.502 0.504
3.2 0.505 0.507 0.508 0.509 0.511 0.512 0.513 0.515 0.516 0.517
3.3 0.519 0.520 0.521 0.522 0.524 0.525 0.526 0.528 0.529 0.530
3.4 0.531 0.533 0.534 0.535 0.537 0.538 0.539 0.540 0.542 0.543
3.5 0.544 0.545 0.547 0.548 0.549 0.550 0.551 0.553 0.554 0.555
3.6 0.556 0.558 0.559 0.560 0.561 0.562 0.563 0.565 0.566 0.567
3.7 0.568 0.569 0.571 0.572 0.573 0.574 0.575 0.576 0.577 0.579
3.8 0.580 0.581 0.582 0.583 0.584 0.585 0.587 0.588 0.589 0.590
3.9 0.591 0.592 0.593 0.594 0.595 0.597 0.598 0.599 0.600 0.601
4.0 0.602 0.603 0.604 0.605 0.606 0.607 0.609 0.610 0.611 0.612
4.1 0.613 0.614 0.615 0.616 0.617 0.618 0.619 0.620 0.621 0.622
4.2 0.623 0.624 0.625 0.626 0.627 0.628 0.629 0.630 0.631 0.632
4.3 0.633 0.634 0.635 0.636 0.637 0.638 0.639 0.640 0.641 0.642
4.4 0.643 0.644 0.645 0.646 0.647 0.648 0.649 0.650 0.651 0.652
4.5 0.653 0.654 0.655 0.656 0.657 0.658 0.659 0.660 0.661 0.662
4.6 0.663 0.664 0.665 0.666 0.667 0.667 0.668 0.669 0.670 0.671
4.7 0.672 0.673 0.674 0.675 0.676 0.677 0.678 0.679 0.679 0.680
4.8 0.681 0.682 0.683 0.684 0.685 0.686 0.687 0.688 0.688 0.689
4.9 0.690 0.691 0.692 0.693 0.694 0.695 0.695 0.696 0.697 0.698
5.0 0.699 0.700 0.701 0.702 0.702 0.703 0.704 0.705 0.706 0.707
5.1 0.708 0.708 0.709 0.710 0.711 0.712 0.713 0.713 0.714 0.715
5.2 0.716 0.717 0.718 0.719 0.719 0.720 0.721 0.722 0.723 0.723
5.3 0.724 0.725 0.726 0.727 0.728 0.728 0.729 0.730 0.731 0.732
5.4 0.732 0.733 0.734 0.735 0.736 0.736 0.737 0.738 0.739 0.740
366  Practical Manual of Experimental and Clinical Pharmacology

Logarithm Table (for numbers from 5.5 to 10)

No. 0.00 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08 0.09
5.5 0.740 0.741 0.742 0.743 0.744 0.744 0.745 0.746 0.747 0.747
5.6 0.748 0.749 0.750 0.751 0.751 0.752 0.753 0.754 0.754 0.755
5.7 0.756 0.757 0.757 0.758 0.759 0.760 0.760 0.761 0.762 0.763
5.8 0.763 0.764 0.765 0.766 0.766 0.767 0.768 0.769 0.769 0.770
5.9 0.771 0.772 0.772 0.773 0.774 0.775 0.775 0.776 0.777 0.777
6.0 0.778 0.779 0.780 0.780 0.781 0.782 0.782 0.783 0.784 0.785
6.1 0.785 0.786 0.787 0.787 0.788 0.789 0.790 0.790 0.791 0.792
6.2 0.792 0.793 0.794 0.794 0.795 0.796 0.797 0.797 0.798 0.799
6.3 0.799 0.800 0.801 0.801 0.802 0.803 0.803 0.804 0.805 0.806
6.4 0.806 0.807 0.808 0.808 0.809 0.810 0.810 0.811 0.812 0.812
6.5 0.813 0.814 0.814 0.815 0.816 0.816 0.817 0.818 0.818 0.819
6.6 0.820 0.820 0.821 0.822 0.822 0.823 0.823 0.824 0.825 0.825
6.7 0.826 0.827 0.827 0.828 0.829 0.829 0.830 0.831 0.831 0.832
6.8 0.833 0.833 0.834 0.834 0.835 0.836 0.836 0.837 0.838 0.838
6.9 0.839 0.839 0.840 0.841 0.841 0.842 0.843 0.843 0.844 0.844
7.0 0.845 0.846 0.846 0.847 0.848 0.848 0.849 0.849 0.850 0.851
7.1 0.851 0.852 0.852 0.853 0.854 0.854 0.855 0.856 0.856 0.857
7.2 0.857 0.858 0.859 0.859 0.860 0.860 0.861 0.862 0.862 0.863
7.3 0.863 0.864 0.865 0.865 0.866 0.866 0.867 0.867 0.868 0.869
7.4 0.869 0.870 0.870 0.871 0.872 0.872 0.873 0.873 0.874 0.874
7.5 0.875 0.876 0.876 0.877 0.877 0.878 0.879 0.879 0.880 0.880
7.6 0.881 0.881 0.882 0.883 0.883 0.884 0.884 0.885 0.885 0.886
7.7 0.886 0.887 0.888 0.888 0.889 0.889 0.890 0.890 0.891 0.892
7.8 0.892 0.893 0.893 0.894 0.894 0.895 0.895 0.896 0.897 0.897
7.9 0.898 0.898 0.899 0.899 0.900 0.900 0.901 0.901 0.902 0.903
8.0 0.903 0.904 0.904 0.905 0.905 0.906 0.906 0.907 0.907 0.908
8.1 0.908 0.909 0.910 0.910 0.911 0.911 0.912 0.912 0.913 0.913
8.2 0.914 0.914 0.915 0.915 0.916 0.916 0.917 0.918 0.918 0.919
8.3 0.919 0.920 0.920 0.921 0.921 0.922 0.922 0.923 0.923 0.924
8.4 0.924 0.925 0.925 0.926 0.926 0.927 0.927 0.928 0.928 0.929
8.5 0.929 0.930 0.930 0.931 0.931 0.932 0.932 0.933 0.933 0.934
8.6 0.934 0.935 0.936 0.936 0.937 0.937 0.938 0.938 0.939 0.939
8.7 0.940 0.940 0.941 0.941 0.942 0.942 0.943 0.943 0.943 0.944
 Appendices  367

8.8 0.944 0.945 0.945 0.946 0.946 0.947 0.947 0.948 0.948 0.949
8.9 0.949 0.950 0.950 0.951 0.951 0.952 0.952 0.953 0.953 0.954
9.0 0.954 0.955 0.955 0.956 0.956 0.957 0.957 0.958 0.958 0.959
9.1 0.959 0.960 0.960 0.960 0.961 0.961 0.962 0.962 0.963 0.963
9.2 0.964 0.964 0.965 0.965 0.966 0.966 0.967 0.967 0.968 0.968
9.3 0.968 0.969 0.969 0.970 0.970 0.971 0.971 0.972 0.972 0.973
9.4 0.973 0.974 0.974 0.975 0.975 0.975 0.976 0.976 0.977 0.977
9.5 0.978 0.978 0.979 0.979 0.980 0.980 0.980 0.981 0.981 0.982
9.6 0.982 0.983 0.983 0.984 0.984 0.985 0.985 0.985 0.986 0.986
9.7 0.987 0.987 0.988 0.988 0.989 0.989 0.989 0.990 0.990 0.991
9.8 0.991 0.992 0.992 0.993 0.993 0.993 0.994 0.994 0.995 1.00
9.9 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00
10.0 1.00
368  Practical Manual of Experimental and Clinical Pharmacology

APPENDIX VII: SI UNIT CONVERSION

Weights and Measure: SI Units

There are two measurement units followed to measure weights and measures i.e. generally the International
System of Units (SI) or the CGS metric units.
Quantity SI Unit Symbol
Length meter M
Mass kilogram Kg
Time second S
Electric Current ampere A
Thermodynamic temperature kelvin K
Amount of Substance mole Mol
Luminous intensity cadela Cd
Absorbed dose of ionising radiation gray Gy
Energy, work, quantity of heat joule J
Pressure pascal Pa
Radioactivity becquerel Bq

SI units: Prefixes used in the names and symbols of the decimal multiples and sub-multiples

Factor Prefix Symbol

9
10 giga G
10 6 mega M
10 3 kilo K
10 2 hecto H
10 1 deca Da
10–1 deci d
10–2 centi c
10–3 milli m
10–6 micro m
10–9 nano n
10–12 pico p
10–15 femto f
10–18 atto a
 Appendices  369

APPENDIX VIII: PRACTICAL EXAMINATION QUESTION PAPER*

MD (Pharmacology)
Long Experiment
1. Write the Protocol and undertake the 4 point bioassay experiment to determine the concentration of
histamine in the given sample in an in vitro tissue of your preference.

Short Experiment
2. Demonstration of analgesic effect of given drug in healthy human volunteers
3. Demonstration of antiepileptic effect of phenytoin in mice
4. Demonstration of straub tail phenomenon

Viva
1. Experimental practical
2. Main theory

DM (Clinical Pharmacology)
Long Experiment
1. Demonstration of β blocker effect in healthy volunteers

Short Experiments
2. Demonstration of acetylator status by isoniazid estimation in healthy volunteers
3. Analyze the given prescription for the patient of bronchial asthma (e.g. corticosteroid in Bronchial asthma)
4. Interpretation of given ECG
a. A 55 year old man with 4 hours of “crushing” chest pain, ECG of patient showed changes in ST , leads
II,III, aVF and in anterior lead , write the diagnosis from ECG and discuss the drug therapy.
b. 45 year old lady complain of palpitations and history of chronic renal failure, ECG showed a wide QRS
tachycardia, write the diagnosis from ECG and discuss the drug therapy.
5. Comment on given drug advertisement (promotional product literature)

Viva
1. Clinical practical
2. Main theory

(* This is to provide an overall idea of practical pharmacology examination; it varies Institution to Institution/University)
 INDEX
A B Chimney test 186
Abnormal appearance 16 Barber 16 Chi-square (χ2) 125, 131
Acclimatization 137 Barbital 183, 348 Cholinesterase enzymes 169
Aceclofenac 320, 321, 322, 323 Bicycle ergo meter 256, 258 Chromatogram 87, 320
Acetic acid Binomial distribution 126 Chronobiology 241, 252
colitis 220 Bioassay 45 Cleaning of glass ware 120
writhing 225 3-point assay 60 Clinical trial 3, 6
Acetylcholine 154, 162, 165, 166, 167, 4-point assay 61 Clinomics 39
170, 171, 177, 178 antagonist 62 Colitis 220
anococcygeus 159, 160 bracketing 57, 58 Cold stress test 323
biventer-cervicis171 cytokines 67 Colon 154
fundus 57, 116, 142, 149, 164, 165 error 46 collection 141
heart 158, 177, 178 examples 69 effect of acetylcholine 154
ileum 70, 71,161 human tissue 66 Comparative control group 107
skeletal muscle 166 matching assay 57, 59 Complete Freund’s adjuvant 224
uterus 149, 155, 156, 157 principle 68 Computational pharmacology 338
vas deferens 160, 161, 170 Biomedical waste 120 Confidence interval 128
Acute study 139 Biostatistics 123 Confidence limit 129
Adrenaline 34,157,160,169,177,178 Blood collection/withdrawal 30 Contact time 56, 64
atria 157, Blood pressure measurement 207 Control group 107
heart 157,173,177 animal 207 Convulsiometer 78
uterus 34 error 251 Coprophagy 9, 16, 217
Allogroom 16 exercise 245 Cryopreservation 96, 97
Analgesiometer 77 guideline (Human) 239, 247 Cuff hypertension 243
Analysis of Variance (ANOVA) 125, human 239 Cumulative plot 63
128, 130 regulation 243 Cutaneous mycobacteriosis 35
Analytical toxicology 346 Box behavior 16 Cytokines bioassay 67
Anesthesia 37 BP apparatus 80, 124
Animal BP cuff inflation test 324 D
allergy 36 Bradykinin 149, 165
Data 123
anesthesia 37 Bronchoalveolar lavage (BAL) 223
Decapitation 39, 137
behavior 16
C Dextran sulfate 220
blood collection 30
Diazepam 183, 184, 185,186, 187, 189,
euthanasia 37 Campylobacter 35
190, 192, 196, 291, 295, 331
handling 17 Carbamazepine 357
blood level 348
sex determination 17 blood level 348
solubility 355
Ankle Brachial Index (ABI) 268 HPLC estimation 333
Dibenzyline, see phenoxybenzamine
Anococcygeus Carrageenan
colitis 220 263
Ach 159
inflammatory 224 Diet 21
collection 144
Cat 8, 13, 33, 34, 184 DOCA salt 214
Anococcygeus 144
Catalepsy 197 Dog 8, 13, 17, 33, 34, 37
effect of Ach 159
Catatonia 197 Dopamine 34, 199, 200
Antagonist (Bioassay) 62
Arm position 242, 254 Ceiling effect 51 Dose cycle 56
Ascorbic acid 10 Cell line 95, 138 Dose ratio 64
Aspirin 202, 325, 357 Centrifuge 82 Dose-response curves 50
gastric ulcer 217 Cerebral ischemia 203 Double pith 39
solubility 355 Cerulein 218 Drug
writhing 226 Chick development 5
Atria 157 bi-venter cervicis 171 experimental background 1
Atropine 34, 162, 177 Chicken 8, 15, 95 Drug response relationship 50
372  Practical Manual of Experimental and Clinical Pharmacology
E foot withdrawal reflex 229 arm at heart level 242
ECG rectus abdominis muscle atria 157
animal 211 (collection) 143 calcium chloride 176
clinical 269 Frusemide 303 digoxin 176
ED50 47, 115, frog heart 177
G
Electro convulsiometer 79, 196 hypodynamic 176, 177
Electrodes 78, 195, 196, 272 Gallop 17 Langendorff’s preparation 173
Gastric ulcer 217 rate 254, 268, 309
Electrolyte analyzer 80
picture 221 starling lever 55
Elevated plus maze 79, 187
Ephedrine 156, 231 Gerbil 8, 10 Herpes B virus infection 35
blood withdrawal 32 Hexabarbital 183
Ethanol
euthanasia 39 High performance liquid
35% ethanol 220
70% ethanol 96, 204, general information 16 chromatography (HPLC) 84
vital parameters 16 High throughput screening 6
blood level 348
zoonotic disease 35 Histamine
ulcer 217
Ethical consideration of animals 39 Glyceryl trinitrate ileum 152, 153
blood pressure 264 Hit and lead compound 5
Euthanasia 37
heart rate 264 Hot plate method 201
methods 38
object 38 arterial vasodilatation 266 Human bioassay 66
Good laboratory practice (GLP) 115, 120 Hyper-pharmacology 115
preferred 39
Guinea pig 8, 9 Hypertension
Ex vivo 138
Exercise tolerance test 285 atria 157 experiment 214
collection 158 ocular 316
Exercise
blood withdrawal 32 spontaneous in dog 13
acute and chronic 245
aerobic and anaerobic 245 euthanasia 39 Hypnosis 183
isotonic and isometric 246 general information 16 Hypodynamic heart 177
maximal and submaximal 246 handling 19
ileum 69, 150 I
Experimental animals 6
cat 13 collection 141, 152 ID50 (IC50) 65
chicken 15 experiment 152 Ileum 57, 149
dog 13 popcorning 17 collection 140
frog 14 pA2 161 guinea pig 69, 152
gerbil 10 sex determination 21 pA2 161
guinea pig 9 trachea rat 70, 71
hamster 10 collection 143 Impact factor 336
monkey 12 vital parameters 16 In vitro 138
mouse 8 zoonotic disease 35 In vivo 138
pig 13 H Indomethacin
pigeon 15 carrageenan 224
rabbit 11 5-hydroxy tryptamine (5-HT) 149,
gastric ulcer 217
rat 9 156, 159
Injection site 26
zebra fish 14 stomach fundus 164
Intradermal injection 30
Experimental window 66 Haloperidol 197
Hamster 8, 10, 34 Isolated heart
Exsanguinations 137 Langendorff’s preparation 173
anesthesia 38
F blood withdrawal 32 Isometric
euthanasia 39 contraction 151
False positive 78, 128, 202
Field stimulation 67 general information 16 exercise 246
First human dose 100, 101, 102, 103 handling 20 Isoniazid (INH) 328
Fischer exact test 125, 131 vital parameters 16 Isoprenaline 149, 156,157, 159, 169,
Foot withdrawal reflex 229 zoonotic disease 35 175
Fresh egg white Hand Dynamometer 256, 259, 324 Isoproterenol 213
irritant 224 Hantavirus infection 35 Isotonic
Frog Hayflick’s limit 96 contraction 150
experiment 166 Heart exercise 246
 Index  373
K Nitroglycerin (NTG) Poisson distribution 126, 127
Kindling model 194 patch 266 Polymerase chain reaction (PCR) 84
Knocked down 8 Non-parametric tests 128, 129 Positive control 107
Knocked in 8 Normal distribution 126, 130 Postural hypotension 255, 263, 267, 268
Knocked out 8 Preclinical study 6, 100
O
Kymograph 47 Predicted maximum heart rate 257
fixing 49 Ocular hypertension 316 Procaine HCl 229
Off-plate dilution 68 Product literature 342
ink 49
Orthostatic hypertension 263 Propylene glycol 213
instrument 76
Oxybutynin 330 Protocol 105
L clinical 108
P
Langendorff’s preparation 173 experimental 105
pA2 64 Protocol writing 105
Latin square randomization
example 71 Psittacosis 35
3-point 60
4-point 62 experiment 161 Psychomotor function 291
Pancreatitis p-value 128
Lead compound optimization 6
acute/chronic 219 Pupillary diameter 309
Lethal dose 102
calculation 117 Papillary diameter 311 Pupillometer 230, 310
Parallel shift 64, 65 Pylorus ligation method 217
Lethality testing 117
Parametric tests 128, 129 Pyrogen testing 12, 91
Lignocaine
gel (2%) 228, 233 Passive avoidance test 79 in vivo 91
Paw edema 224 in vitro 93
topical (4%) 233, 235
Peak expiratory flow rate (PEFR) 280
Liquid chromatography and mass- R
spectrometry (LC-MS) 88 Pentobarbital
anesthesia in BP measurement 207 Rabbit 8, 11
Lithium 333
anesthesia in thrombosis model blood withdrawal 33
estimation 334
Local anesthetics (LA) 228 216 general information 16
anesthetic agent 38 handling 21
Lordosis 17
righting reflex 183 oral gavage 26
M Pentylenetetrazole (PTZ) vital parameters 16
Master’s 2 step test 256 GTCS model 191 Rabbit eye 230
Maximal electroshock (MES) 195 kindling model 194 Rabbit ileum 153
Median lethal dose 117, 119 Personalized medicine 350 Rabbit uterus 156
Mental stress 261 Pharmacokinetics Rabies 12, 35
Mepyramine 152 experimental 233 Radiant heat 323
Metabolic equivalents 246 clinical 319 Rat
Micro dosing 351 Phenobarbitone rat fever 35
Molecular medicine 350 HPLC method 333 rat uterus 155
Monkey 8, 12 normal level 334 Rate pressure product 259
blood withdrawal 33 Phenytoin 195, 233, 235 Rectus abdominis muscle
Morphine Phrenic nerve diaphragm collection 143
blood level 348 collection 145 effect of Ach 166
hot plate/ tail flick 201 effect of Ach 169 Regression 131
response 34 Physiological salt solution (PSS) 47 Reverse pharmacology 350
straub tail 199 composition 51, 53 Righting reflex 13, 192
writhing 225 Physostigmine 169, 171, 231 experiment 183
Morris water maze Pica 17 Ring worm 35
experiment 190 Pig 8, 13 Rotarod
instrument 79 blood withdrawal 33 experiment 184
Muscle contraction Pigeon 8, 15
Pilocarpine S
principle 150
Muscle relaxant 184, 186 mitotic 231 Safety factor 103
Myocardial infarction (MI) 213 topical (2%) 314 Salicylate 325
Pilot study 114 blood level 348
N PK/PD modeling 340 Salivary secretion 309, 310, 330
Nictating membrane 13 Plethysmograph 81, 222 Salmonella 35
374  Practical Manual of Experimental and Clinical Pharmacology
Schild plot 64, 65, 161 Tetanus 35 U
Seizure score 78 Therapeutic drug monitoring 88, 333
Ulcer 217
Seizure Thesis writing 105, 111
gastric 217, 221
MES 78 Thrombosis
ferric chloride 216 Uterus 34, 57,
PTZ 191
Serotonin Tissue preparation method 135 collection 142
irritant 224 Tonic hind limb extension (THLE) effect of Ach 149, 155
stomach fundus 164 78, 195, 196 effect of oxitocin 149, 156
Sham control group 107 Toxic dose identification 148
SHAY method 217 acute 115, 116
blood level 348 V
Spontaneous activity 53, 152, 153
Stair climbing exercise tolerance chronic 117 Vas deferens 144, 149
test 285 dose calculation 101 effect of Ach 149, 160
Stomach fundus 57, 149 subacute/ subchronic 116 Vasodilatation
collection 142 Toxicity study 115 agent 31
effect of 5-HT 164 Tracheal chain 149, 167 Visual pathway 231
Straub tail reaction 199 collection 167, 168
Student’s physiograph 76 human 67 W
Students‘t’ test 129 identification 148
Translational medicine 350 Waste disposal 120
Subacute study 116
Tread mill test (TMT) 256, 257 Waud method 65
Subchronic study 116
Submaximal exercises 246 Tri Nitro Benzene Sulphonic acid Wheal and flare 331
(TNBS)
T Z
colitis 220
Tail flick method 201 Tropicamide 362 Zebra fish 8, 14
Telemetry 89 topical (1%) 312 anesthesia 38
BP measurement (animal) 207 Tubocurarine 171 Zoonotic disease 34