' Syllabus hy , . 41. Introduction to experimental pharmacology. él ¢) Pi P gy: 3 \ 7m 2. Commonly used instruments in experimental pharmacology. 234 oa a Study of common laboratory animals. Vr 3) ly ry animals. gy 4, Maintenance of laboratory animals as per CPCSEA guidelines. 5, Common laboratory techniques: Blood withdrawal, serum and plasma separation, anesthetics and euthgnaya used for animal studies, —~ &) Study of different routes of drugs eae in mice/rats, ~ Q) Study of effect of hepatic microsomal enzyme inducers ‘on the pentobarbital ‘ induced sleeping time in mice. My @ Effect of drugs on ciliary motility of frog oesophagus. > : (3.) tffect of drugs on rabbit eee 2 wv, “ Ge of skeletal muscle relaxants using rota-rod apparatus, *-° Le (a Effect of drugs on locomotor activity using photoactometer. € "7 y hotoactomet 12. Anticonvulsant effect of drugs by MES and PTZ method. “~@ ! 13. Study of stereotype and anti-catatonic activity of drugs on rats/mice. 14, Study of anxiolytic activity of drugs using rats/mice. 15. Study of local anesthetics by different methods. soe 2kContents experimental animals. Experiment Title Page No. No. Guide to use this book GA-G2 1. Introduction t6 experimental pharmacology ppp Pbiectve: To study the role of ‘experimental pharmacology | 13 e~ | inthe process of drug discovery and development, 2 Commonly used instruments in experimental pharmacology | 2.1 - 2.8 Objective: To study some basic instruments used in 21 pharmacology laboratory for experiments using isolated tissues and whole animals. 3. Study of common laboratory animals 31-36 Objective: Study of common laboratory animals in 31 ess “ experimental pharmacology. 4 Maintenance of laboratory animals as per CPCSEA guidelines | 4.1- 4.6 Objective: To study of maintenance of laboratory animals as| 4.2 per CPCSEA guidelines. 5. Common laboratory techniques: Blood withdrawal, serum| 5.1 - 5.14 and plasma separation, anesthetics and euthanasia used for animal studies Objective 1: To study the common laboratory techniques 51 used for withdrawal of blood from laboratory animals. Objective 2: To study the techniques for separation of| 5.7 serum and plasma from collected blood sample Objective 3: To study the anaesthetics used in experimental, 5.9 animals. Objective 4: To. study the euthanasia proceed used in| 5.126. Study of different routes of drugs administration in mice/rats | 6.1 - 6.6 Objective 1: To study different routes of dug] 62 administration in the mice/rats. Objective 2: To study the effect of pentobarbital sodium in] 6.4 mice after administrated through different routes. 3 Study of effect of hepatic microsomat enzyme inducers on| 7.1- 7.4 the pentobarbital induced sleeping tinie in mice Objective: To study the effect of hepatic microsomal] 7.3, enzyme induction on the pentobarbital induced sleeping time in mice. 8. Effect of drugs on ciliary motility of frog oesophagus 81-82 Objective: To find out the effects of cholinergic and] 8.1 anticholinergic drugs on ciliary motility of frog oesophagus. 9. Effect of drugs on rabbit eye 91-94 Objective: To find out the effects of pilocarpine on the| 9.2 rabbit eye. 10. Effects of skeletal muscle relaxants using rota-rod apparatus | 10.1 - 10.4 Objective: To find out the skeletal muscle relaxant effects of | 10.1 diazepam in mice using rota-rod apparatus. 1. Effect of drugs on locomotor activity using photoactometer_| 11.1 - 11.4 Objective: To study the effects of diazepam onlocomotor) 112 activity in mice using photoactometer apparatus. 22. Anticonvulsant effect of drugs by MES and PTZ method 12.1 - 12.6 Objective 1: To study the anticonvulsant activity of 12.3 phenytoin by using maximal electro shock induced) convulsion in rats. Objective 2: To study the anticonvulsant effect of diazepam) 125 by using Pentylenetetrazole (Metrazol) induced convulsion in mice.| 13.1-13.6 2B. Study of stereotype and anti-catatonic activity of drugs on rats/mice Objective 4: To study the amphetamine induced stereotype| 13.3 activity in mice. Objective 2: To study the anti-catatonic activity of Levodopa 13.5 in rats. 1 14. Study of anxiolytic activity of drugs using rats/mice 141-145 | Objective: To study the anxiolytic activity of diazepam in| 143 rats using elevated plus maze apparatus. 15. Study of local anesthetics by different methods 15.1-15.8 Objective 1: To istudy the local aneesthetic effects of | 153 lignocaine on the rabbit eye, Objective 2: To study the local anaesthetic effects by| 15.5 intradermal injection of Lignocaine in Guinea pig. ‘| Objective 3: To study the local anaesthetic property of 15.6 Procaine using foot withdrawal reflex of frog. aKList of Figures Figure No. Title Page No. 1a Pioneers of Pharmacology 15 12 Drug discovery and development processes 19 21 Instruments for recording the contraction of isolated tissue 24 22 | Commonly used instruments: for experiments. using whole} 27 animals 3a Commonly used laboratory animals in pharmacology laboratory 3a 51 Different techniques used for collection of blood from laboratory 5.6 animals 5.2 Differences between serum and plasma 57 5.3 Induction of inhalational anaesthesia in animals 5.11 61 Oral route of drug administration 63 62 Different parenteral route of drug administration 64 71 Schematic illustration of pharmacokinetic and metabolism 72 processes 91 Effect of pupillary muscles 91 92 The pathway for secretion and drainage of the aqueous humour 92 10.1 _| Rota-rod apparatus 10.2 111 | Photoactometer apparatus 12 12.1 Drugs with multiple mechanism 12.2 122 _ | Electro-convulsiometer apparatus 123 123 _| Different phases of convulsion pa | 13.1 | Structure of Dopamine pathways and Dopamine receptors in| 132 | CNS 13.2 _ | Study of stereotype and catatonic responses in mice and rat 13.4 14.1 | Elevated plus-maze apparatus for rat 143 15.1 Structure of ester and amide local anaesthetics as.. 15.2 | The animal models used to study the local anaesthetic property 153 + teeList of Tables anaesthesia procedure Table No. Title Page No. 11 Pharmacologists and their outstanding achievements 45 12 Different phases of drug development 17 13 Key stages of drug discovery and developments 18 3 Uses of different laboratory animals in experimental] 3.3 pharmacology 32 Bidlogical and physiological data of commonly used laboratory| 3.5 animals 5 Different techniques used for collection of blood from laboratory| 5.3 animals 52 Differences between serum and plasma 58 53 Commonly used anaesthetic drugs for laboratory animals 5.11 54 Different euthanasia procedures used for experimental| 5.13 laboratory animals 61 Different routes of drug administration in human 6.2 62 Effect of pentobarbital sodium administration through different] 6.5 routes 71 Effect of phenobarbitone on pentobarbital induced sleeping time| 7.4 in mice eee al Effects of different drugs on ciliary motility on frog oesophagus 82 94 The effects of pilocarpine on the rabbit eye 93 9.2 _| The effects of different drugs on the rabbit eye 94 10:1 | Skeletal muscle félaxant effect of diazepam using rota-rod| 103 | apparatus in mice [Ti [Effect of diazepam on locomotor activity in mice using) 113 Photoactometer ajsparatus a) 121 | Study of anticonvulsant activity of phenytoin by using MES| 124 induced convulsion in rats 122 | Study of anticonvulsant effect of diazepam by using) 126 pentylenetetrazole induced convulsion in mice. 13.1 _| Study of amphetamine induced stereotype activity in mice 13.4 13.2 Anti-catatonic activity of levodopa in rats 13.6 141 | Anxiolytic effect of diazepam by using elevated plus maze| 144 apparatus 15.1 |'Local anaesthetic effect of Lignocaine using surface anaesthesia| 15.4 Procedure ~. 152 | Local anaesthetic effect of Lignocaine using infiltration| 15.6 anaesthesia Drocedure 153 | Local anaesthetic effect of Lignocaine using nerve block] 15.7 eKThe main purpose of the experimental pharmacology is to understand what drugs do-to the living organisms and how their effects can be applied to therapeutics. Animal experiments are an integral part of the pharmacology teaching. But to know the techniques of animal experiments and study the mechanism of action of different drugs, animals need to be tortured and sacrificed. Animal experiments are too much expensive, time consuming and tedious processes. Due to animal welfare regulations and ethics, experiments in animals are also not feasible. Hence, with the changing scenario alternatives to animal experiments are introduced for teaching pharmacology throughout the world. Problem based exercise and computer assisted learning (CAL) with the use of modern audio visual aids including computer simulation models are widely used. This new learning system provides a highly interactive and friendly environment to the practical class. A number of computer simulation models have been designed for pharmacology undergraduate students of medical, pharmacy, veterinary, dental and nursing profession. This book consists of 15 experiments. Each experiment has described the purpose of the experiment, terminology and description on the topic. Each experiment has number of objectives and each objective has. described as principle, requirements, procedure, observation, inference and precautions. At the end of every chapter, a set of strategically designed questions have been provided to test the student's grasp of the text and their ability to analyze and solve the problem. For the effective use of this book, at first interactive demonstrations should be conducted by the departmental faculty, on a particular experiment. After an initial exposure, train the students about CAL and conduct the simulation experiments. A group discussion and debate can be performed on that topic during the experiment. Teachers should ensure that as many students as possible must take part in these activities. Analyze the results of the experiment to draw the conclusions, by using appropriate statistical methods, which are as important as doing the practical correctly. The questions at the end of the each experiment may be attempted by the students. Writing answers for them in the practical record note book is optional. Some of the computer simulation softwares that may be used for experimental pharmacology are as follows: 1. ExPharm, developed by Dr. R. Raveendram, JIPMER, Pondicherry, India. 2. X-cology, an interactive CD-ROM, developed by Dr. C. R. Patel, R. C. Patel College of Pharmacy, Karvand Naka, Dhule, India. 3. Pharmacological experiments on isolated Guinea pig illium (Ver. 4), developed by Department of Pharmacy, University of Bath (UK). G1)4, Pharmacological experiments on isolated Rabbit jejunum (Ver. 3), developed by Welsh School of Pharmacy (UK). 5. Cardiolab - in Vivo Cardiovascular Pharmacology Simulator (Ver. 3), developed by Biosoft, USA. 6 The Strathclyde Pharmacology Simulation Packages, Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, UK. 7. Simulation of Animal Experiments by Elsevier is a learning package of animal experiments in pharmacology and physiology. It is a computer assisted learning program of animal experiments comprises ExPharm, X-cology, ExPhysiology and Experimental Physiology Simulation Software. Note: As per Pharmacy Couneil of india all laboratory techniques and animal experiments are demonstrated by simulated experiments by software and videos. abe (G.2)EXPERIMENT No. 1 INTRODUCTION TO EXPERIMENTAL PHARMACOLOGY Purpose: At the end of the practical class, the students shall be able to 1. Know about experimental pharmacology. 2. Know about the past and present of pharmacology. 3. Know about the use of experimental pharmacology in drug discovery and development processes. aF Terminology: et The term pharmacology comes from the two Greek words: pharmacon - rug or medicine and logas - the truth about or a rational discussion. Pharmacology is a science of drug. It can also be defined as, (‘study of the effects of drugs on the function of living systems” sIt deals with interaction of exogenously administered chemical molecules (drugs) with leaving system. Two important and interrelated areas of pharmacology are: pharmacokinetics and pharmacodynamics. harmacokinetics: The term pharmacokinetics comes from the Greek word: kinesis - Tovement. Pharmacokinetic is movement of drug in and alteration of drugs by the body. It is the qualitative study of drug movement in, through and out of the body, which includes absorption, distribution, metabolism and excretion (ADME) of the drug. e.g. Digoxin is 70% absorbed orally, 25% bound to plasma proteins, localized in heart, skeletal muscle, liver and kidney; smail fraction is metabolized in liver and excreted in urine. Hence, pharmacokinetics, is what the body does to the drug. armacodynamics: The term pharmacodynamics comes from the Greek word: mics - Power. Pharmacodynamics is the study of drug effects, which includes physiological and biochemical effects of drugs and their mechanisms of action at organ/ system/subcellular/ macromolecular levels. e.g. Adrenaline ~ interacts with B,-adrenoceptors in heart, that is a G-protein coupled receptor, which stimulates adenylyl cyclase and increases intracellular cAMP which leads to cardiac stimulation, Hence, pharmacodynamics is what the drug dyes to the body. i tis: The term drug comes from the French word: Drogue - a dry herb. Drug is any Substance or product that is used or intended to be used to modify or explore physiological systems or pathological states for the benefit of the recipient. Drug is the single active chemical entity present in a medicine that is used for diagnosis, prevention, treatment/cure of a disease. Amcor An application of pharmacological information together with nowledge of the disease for its prevention, mitigation on cure is known as pharmacotherapeutics. It includes selection of the most appropriate drug, dosage and duration of treatment for the specific feattires of a patient. Kicology: It is the study of poisonous effect of drugs and other chemicals with mphasis on detection, prevention and treatment of poisonings. tt also includes the study of adverse effects of drugs. (1.1)Experimental Pharmacology - | 1.2 __ introduction to Experimental Pharmacology Essential Drugs: As per the WHO, essential drugs or medicines can be defined as, "those ‘drugs that satisfy the priority healthcare needs of the population”. They are selected with due regard to public health relevance, evidence on efficacy and safety and comparative cost effectiveness, These are intended to be: available within the context of functioning health systems at all times, available in adequate amounts, in appropriate dosage forms, with assured quality, contains adequate information about efficacy and safety, and at a price the ‘individual and community can afford, Chemotherapy: It is the treatment of systemic infection or. malignancy with specific ‘drugs that have selective toxicity for the infecting organism or malignant cell with no/minimal effects on the host cells, 4 clinical Pharmacology: It is the scientific study which involves laboratory animals ing wide-ranging doses of the study drug to obtain preliminary efficacy, toxicity and pharmacokinetic information. Such tests assist pharmaceutical companies to decide whether a drug candidate has scientific merit for further development as an investigational new drug. Xx Clinical Pharmacology: It is the scientific study of drugs in human. It includes farmacokinetic and pharmacodynamic investigation in healthy volunteers and in patients, for evaluation of efficacy and safety of drugs. Pharmacogenetics: This branch of science deals with the study of genetic variations that alters drug metabolism and drug response. Originally, pharmacogenetics focused on familial idiosyncratic drug reactions, where affected individuals show an abnormal, usually adverse response to a class of drug. Pharmacogenomics: This term overlaps with pharmacogenetics, describing the use of genetic information to guide the choice of drug therapy on an individual basis, ie. the response to therapeutic drugs between individuals can be predicted from their genetic make-up. Ultimately, linking specific gene variations with variations in therapeutic or unwanted effects of a particular drug should enable the tailoring of therapeutic choices on the basis of an individual's genotype. Pharmacoepidemiology: This is the study of drug effects at the population level. It is concerned with the variablity of drug effects between incvicuals in a population, and ween populations. a Pharmacoeconomics: This branch of health economics aims to quantify in economic terms, the cost and benefit of drugs used therapeutically. It arose from the concern of many governments to provide for healthcare from tax revenues, raising questions of what therapesitic procedures represent the best value for money. Damen: This is a science and activities related to the detection, assessment, lerstanding and prevention of adverse effects or any other drug related problem. So this is a science of keeping a watch on adverse reactions.Experimental Pharmacology =I 1.3 __ Introduction to Experimental Pharmacology Objective: To study the role of experimental pharmacology in the process of drug discovery and development. Description: Pharmacology encompasses all fields of biomedicine, though it is a relatively new discipline in the life sciences. The uniqueness of pharmacology is that, it takes a proactive approach to biological systems. Pharmacology is the study of the therapeutic value and/or potential toxicity of chemical agents on biological systems. It targets every aspect of the mechanisms for the chemical actions of both traditional. and novel therapeutic agents. Although drugs have been a subject of ancient interest since ancient times, over the past 100 years or so, drugs have been purified, chemically characterized and vast variety of highly potent and selective new drugs have been developed. Experimental Pharmacology: Past and Present: Distinctions between the useful actions of drugs and their toxic effects were recognized thousands of years ago. As people tried plant, animal, and mineral materials for possible use as foods, they noted both the toxic and the therapeutic actions of some of these materials, Past civilizations contributed to our present knowledge of drugs and drug preparations. In India Ayurveda, Siddha and Unani systems of medicine provide healthcare for a large part of the population. The word Ayurveda is composed of two parts: Ayu (= life) and Veda (= knowledge). Sage Bhardwaja got this science from Lord Indra and documented as Vedas, * hence he is considered as ‘Father of Ayurveda’ by many authors. Scholars of Ayurveda had placed the origins of this science of life at sometime around 6000 BC.;The principles were recorded in great detail in compendia, which are called Samhitas. Ancient Chinese writings and Egyptian medical papyri represent the earliest compilations of pharmacological knowledge. They included rough classifications of diseases to be treated, and recommended prescriptions for such diseases. While other civilizations made their own discoveries of the medicinal value of some plants. The introduction of many drugs from. the new world in the 17" century stimulated experimentation on crude_preparations. These experiments were ~ conducted chiefly to. get some ideas about the possible toxic dosage for such drugs as tobacco, nux vomica, ipecac, cinchona bark and coca leaves. By the 18” century, many such descriptive studies were being conducted. The birth of experimental pharmacology is generally associated with the work of the French physiologist, Francois Magendie, in the early 19" century’ Magendie’s research on strychnine-containing plants clearly established the site of action of these substances as being the spinal cord and provided evidence for the view that drugs and poisons must be absorbed into the blood stream and carried to the site of action before producing their effects. The work of Magendie and his pupil, Claude Bernard, on curare induced muscle relaxation and carbon monoxide poisoning helped to establish some of the techniques and principles of the pharmacology.Experimental Pharmacology - 1 1.4 Introduction to Experimental Pharmacology It was in the German universities during the second. half of the 19% century that pharmacology really began to emerge as a well-defined discipline. The bold vision of Rudolf Buchheim, who was appointed to teach material medica at the University of Dorpat in Estonia, created the first pharmacology laboratory at his own house. He established the first institute of pharmacology at the University of Dorpat in 1847. In 1872, Oswald Schmiedeberg, who received research training in Buchheim’s laboratory, became professor of pharmacology at Strasbourg, regarded as ‘Father of Pharmacology’. Schmiedeberg with his many students from all over the world worked in his pharmacological institute and propounded some of the fundamental concepts in pharmacology. His students later occupied 40 academic chairs in pharmacology departments throughout. the world. One of the most eminent of his many distinguished pupils was John Jacob Abel, who brought the new science of experimental pharmacology from Germany to the USA. In the beginning of the 20" century, Paul Ehrlich conceived the idea of specifically seeking special chemical agents with which to treat infections selectively (propounded the "magic bullet” theory), and is thus considered as the ‘Father of Chemotherapy’. In India, the evolution of pharmacology from material medica is a mirror image of globat scenario. Sir Ram Nath Chopra obtained. training at Cambridge University in 1908. After arriving to India, he was appointed as the first professor of pharmacology in 1921 and set up experimental pharmacology laboratory at the Tropical School of Medicine in Kolkata. He made the beginning in pharmacological research of traditional drugs; helped the growth of pharmacology in different medical, dental, pharmacy and veterinary colleges. Hence, Sir Ram Nath Chopra is known as ‘Father of Indian Pharmacology’. M.N. Ghosh, RB. Arora, UK. Sheth, P.C. Dandiya, K.P. Bhargava, B. Mukharji are the other pharmacologists who have contributed a lot for the growth of pharmacology discipline in India by their stupendous work. The progress and contribution of 20" century pharmacology have been immense, with ‘over twenty pharmacologists having received Nobel prizes. Their contributions include discoveries of many important drugs, neurotransmitters and second messengers, as well as an understanding of a number of physiological and biochemical processes. The field of pharmacology in general and the development of highly effective new drugs in particular have increased during the last half of the 20" century. This unprecedented progress has paralleled similar advancement in related disciplines upon which pharmacology builds: molecular biology, biochemistry, physiology, pathology, anatomy and’ the development of new analytical and experimental techniques and instruments. Within the main subject, fall 2 number of compartments e.g. neuropharmacology, imimunopharmacology, molecular pharmacology, chemotherapy, systems —phatmucology, pharmacokinetics, pharmacogenomics, pharmacoepidemiology, pharmacoeconomics etc.Experimental Pharmacology - | 1.5 Introduction to Ex erimental Pharmacology Francois Magendie Claude Bernard “Rudolf Buchheim (1783 - 1855) (1813 - 1878) (1820 - 1879) ‘Oswald Schmiedeberg Paul Ehrlich Sir Ram Nath Chopra (1838 - 1921) (1854 - 1915) (1882 - 973) Fig. 1.1: Pioneers of pharmacology Table 1.1: Pharmacologists and their outstanding achievements Name of Scientist | Outstanding Achievements Francois Magendie (1783-1855) | Pioneer of experimental physiolog) Claude Bernard (1813-1878) | Father of physiolog Rudolf Buchheim (1820-1879) Pioneer of experimental pharmacology Oswald Schmiedeberg (1838-1921) _| Father of pharmacolog) John Jacob Abel (1857-1938)_ | Father of American pharmacology Paul Ehrlich (1854-1915) | Father of chemotherapy Louis Lasagna (1923-2003) | Father of clinical pharmacology ‘Sir Ram Nath Chopra (1882-1973) __| Father of Indian pharmacologyExperimental Pharmacology - | 1.6 __ Introduction to Experimental Pharmacology ‘Types of Experimental Pharmacology: The classical way of pharmacological screening involves sequential testing of new chemical entities or extracts from biological material in isolated organs followed by tests in whole animals, mostly rats and mice but also higher animals if indicated. Most drugs in use nowadays in therapy have been found and evaluated with these methods. The aims of experimental pharmacology are discovery and development of the new drugs, study of the mechanism and site of action of different drugs and study of the toxicity effect of a drug. Experimental pharmacology consists of pre-clinical study and clinical study. 1. Pre-clinical Study: Pre-clinical studies involves in vivo and in vitro experiments using wide range of doses of the sample drug to obtain preliminary efficacy, toxicity and pharmacokinetic information. Such tests assist pharmaceutical companies to decide whether a drug candidate has scientific merit for further development as an investigational new drug (IND). In vivo (animal testing) study is used to measure: how much of a drug is absorbed into the blood, how it is broken down chemically in, the body, the toxicity of the drug and its breakdown products or metabolites, and how quickly the drug and its metabolites are excreted from the body. Short-term testing in animals ranges in duration from 2 weeks to 3 months, depending on the proposed use of the substance. Long-term testing in animals ranges in duration from a few weeks to several years. Some animal testing continues after human tests begin to learn whether long-term use of a drug may cause cancer or birth defects. In vitro (in the test tube) experiments, cell and tissue cultures can be used instead of using animals to test the product ingredients. The development of in vitro methods based on biological ‘materials (for example, skin or other human body cells) that will be suitable for reliably verifying the safety and compatibility ‘of product ingredients. Development of High- throughput screening (HTS), Ultra HTS, automation, robotics and miniaturization techniques, made it feasible to screen 50,000 compounds a day with complex work stations, The development of in. silico methods (in the computer) is to. determine the compatibility of substances on the basis of their chemical structure. Advantages of in vitro tests are fast and cheap, human cells and tissues can be used, transgenic cells carrying human genes can be used, reduction of testing in animals, small\amount of test material is required, lack of systemic effects, reduction of variability between experiments. 2, Clinical Trials: “It is the scientific study of drugs in human. It includes pharmacokinetic and pharmacodynamic investigation in healthy volunteers and in patients. A clinical trial is a research study that tests a new medical treatment or a new way of using an existing treatment to see if it will be a better way to prevent and screen for diagnose or treatment ofExperimental Pharmacology =. 1.7___Introduction to Experimental Pharmacology a disease. We define a clinical trial as a prospective study comparing the effect and value of intervention(s) against a control in human beings. It is only in the past several decades that the clinical trial has emerged ‘as the preferred method in the evaluation of medical interventions. Clinical trials for new drugs. are commonly classified into four phases. Each phase of the drug approval process is treated as a separate trial. The drug-development process will normally proceed through ali four phases over many years. If the drug successfully passes through Phases J, Il and Ill, it will usually be approved.by the national regulatory authority for use in the general population. Phase IV is post-approval (Post marketing surveillance) studies. Now, Phase 0 or micro dosing studies are performed to know the action of drug candidate in human being at early. 4 Table 1.2: Different phases of drug development Pre-clinical phases In vivo and in vitro studies are carried out for pharmacokinetics, toxicity studies and formulating the drug. Phase-I of clinical trial | Small scale trials in 20-80 numbers of healthy volunteers. 4 Studied at single center for knowing the safety tolerability, pharmacokinetics and side effects. Phase-II of clinical trial | Small scale trials in 100-300: numbers of patients as well as healthy volunteers. Studied at single center to assess the C efficacy and dosage. Long term toxicology studies are also carried out, Phase-Iil of clinical trial | Large scale controlled trials in 800-3000 numbers of patients. Y Studied at multicenter for comparing the new drug with the existing drugs. Phase-IV of clinical trial | Post marketing surveillance in patients for detecting any rare or long term ADRs. Use of Experimental Pharmacology in Drug Discovery and Development: The discovery and development of new drugs to provide medicines for treating diseases is the main role of the pharmaceutical industry. It is a challenging and expensive activity of pharmaceutical industry. Biological organisms and especially human beings are extraordinarily complex, and our understanding of how they function at the molecular level remains rudimentary, although considerablé advances in knowledge. have been made in recent decades. Whilst an advanced industrial society was able to plan and deliver a man to the moon following a 10-year program, we are still only able to treat about 60% of cancerExperimental Pharmacology - 1 1.8 ___ Introduction 1o Experimental Pharmacology patients effectively, and do not understand how to correct most mental diseases. Development of new medicines is complex, time consuming and very expensive. The average cost of developing a new drug is estimated to be about US $ 1-12 billion, including expenditures on failed projects. This amount is about four times the price of an Airbus A380 at US $ 270 million, oF five times that of a Boeing B - 787 Dreamliner at US $ 200 million. Total drug development time grew from an average of 8.1 years in the 1960s to 116 years in the 1970s, to 14.2 years in the 1980s, to 15.3 years for drugs approved from 1990 through 1995. Pharmaceutical companies and regulatory authorities are working together to reduce this time span. With the advent of technologies in biological screening procedures of new chemical entities the time involved in drug discovery has gone down in recent years but the cost of drug discovery has touched a new high. Success rate in getting from an initial compound to an approved and commercially available product is very low. Typically, tens of thousands of compounds are screened and tested, and only a handful makes it onto the market as drug products. The statistics is such that, out of the 10,000 compounds that show initial promise, only 0.3% will reach the testing stage for sub-acute study, five will go into human clinical trials, and only one will become an approved drug. Table 1.3: Key stages of drug discovery and developments Drug Discovery | Drug Developments Preclinical study Clinical trials Release of the drug Follow-up monitoring 1. Program selection (choosing a disease to work on) 2. Identification and validation of a drug target | Assay development Identification of a “lead compound” ewer Lead optimization Identification of a drug candidate anew The process involves finding out the target that causes the disease. Next, chemical or biological compounds are screened and tested against these targets or assays, which are representative of these targets, to find leading drug candidates for further development. Many new scientific approaches are now used to determine targets (most targets are receptors or enzymes) and obtain the lead compounds; including the use of genomic technology, synthetic chemistry, recombinant DNA (rDNA) technology, laboratory automation and bioinformatics. Tests are performed on the lead compounds in test tubes (in vitro) and on animals (in vivo) to check how they affect the biological systems. The tests,= or re tt. re tic es ts, Experimental Pharmacology = | 1.9 __ Introduction to Exp erimental Pharmacology. often called preclinical research activities, include toxicology, pharmacodynamics and pharmacokinetics, as well as optimization of drug delivery systems. The leading compounds are modified and synthesized to improve their interactions with the targets, or to reduce the toxicity or improve pharmacokinetics performance. At the end of this process, an optimized compound is found and this becomes a potential drug ready for clinical trial in human. The development work has to follow Good Laboratory Practice (GLP) to ensure that proper quality system and ethical considerations are established. Only compounds that satisfy certain performance and safety criteria will proceed to the next stage of clinical trial. Then release of the drug occurs and follow up monitoring is required for confirming the effectiveness of the drug. Drug Preclinical Clinical development Rogulatory| discovery | development approval _— Phase T Phase IT Phase III Phase IV Target selecion. | pharmcokintes.| Phermacokinetios| smatecato | Large-scale | submission | Postmaretng Temtancings "| Shorter’ "| Taercotye | tele patents | contotod'| eftarcato, | urveltace. Lead optimization) toxicology. Side-effects in to assess efficacy] clinical trials and review by| Pramocotgea! | Foruston, | heaty Rassoce, reason, pom. Syntioets ecale-op| vores. Soarebe” Long dn toncoogy Ses 142.5 yoat 415 years—+le 6-7 years——|-__——ple- 1-2 yearo->} 00 Bo corpo +f} =} 474 1 On Doreen Regu [Dur aproved to card compen Beare (hice Fig. 1.2: Drug discovery and development processes After completion of preclinical studies successfully, the investigators files an ‘Investigational New Drug’ application (IND) to the government bodies [such as Central Drugs Standard Control Organization (CDSCO)-in India] for allowance of initial testing in human beings. After the successful clinical trials and laboratory work, the investigators may file a New Drug Application (NDA). Permission to market a drug product will be given by the drug control authority after confirming the drug's safety and effectiveness. After approval, the post marketing surveillance studies in patients are carried out for detecting any rare or long-term adverse reactions.Experimental Pharmacology 1.10 __ Introduction to Experimental Pharmacology ween Who is known as Father of Pharmacology? Describe the role of IND and NDA in drug discovery and development. Discuss the key stages of drug discovery and developments. What is Clinical Pharmacology? What is Pre-clinical study?EXPERIMENT No. 2 COMMONLY USED INSTRUMENTS IN EXPERIMENTAL PHARMACOLOGY Purpose: + At the end of practical class, the students shail be able to 2. Know about various instruments used in experiments on isolated tissues. 2. know about different instruments used in experiments on whole animals. Objective: ~ To.study some basic instruments used in pharmacology laboratory for experiments using isolated tissues and whole animals. Description: Experimental pharmacology includes both, experiments using isolated tissue or organ and experiments using whole animals. These two kinds of experiments require two different kinds of apparatus, organization and skill. The two kinds of experiment thus form quite distinct features and both need clearance from the Institutional Animal Ethics Committee (IAEC) for conducting in the pharmacology laboratory. Instruments for Experiments using Isolated Tissues: 1. Sherrington Recording Drum and Students Organ Bath: Sherrington recording drum and students organ bath assembly are the two parts used for recording of contraction of isolated tissue. An organ bath assembly for intestinal strips was first designed by Rudolph Magnus in 1904. Even today, the assembly remains basically same, Most of the institutions and the research laboratories use the smoked paper or ink pen on white paper for recording the tissues response. Gaddum first designed the student organ bath having two units of inner organ bath and is called double unit organ bath. Sherrington Recording Drum (Kymograph): Sherrington recording drum contains the drum cylinder ‘on which contraction and relaxation of muscles are recorded. It consists of a vertical shaft, which holds the drum cylinder, present on the heavy base. It consists of: (a) Base hoofs with adjustable levelling screws to keep the drum horizontal; (b) Side hoof to turn the drum on its side to make the shaft horizontal; (0) Electrically or pulley driven gear box with fast, slow and neutral gears, and clutch; (d) Contact screw; (e) Contact foil; (f) Drum cylinder, which is made up of a brass or iron cylinder on which a kymograph paper is rolled and smoked, and that is fitted on vertical shaft. (2a)Experimental Pharmacology - | 2.2 Commonly Used Instruments in ...... Organ Bath Assembly: It is the basic requirement for the isolated tissue so that, the tissue can survive during the experimental period. It consists of: (2) An outer jacket (water bath) made up of glass or perspex fitted with thermostat anda small electrical stirrer to keep the uniform heating of water, (b) The inner organ bath made up of glass with variable capacity (10-50 ml) for suspending the tissue in physiological salt solution. (©) An oxygen delivery glass tube which also serves as tissue holder connected to aerator. (d) A reservoir contains the physiological salt solution. () A coil made up of glass or perspex, one end of which is connected to the lower end of the organ bath and the other end to the reservoir having the physiological salt solution. The glass coil is usually of double the capacity of inner organ bath to keep the physiological solution warm before entering the organ bath. (f) The writing lever is used to record the contraction and relaxation of the isolated tissue preparation on smoked paper fixed on circular drum cylinder run at different speed. The writing levers are light in weight, rigid and are generally made up of aluminium, stainless steel, balsa wood or drinking straw. Most of the writing levers belongs to class I-type of mechanical lever, i.e. fulcrum or pivot (lever holder) lies between the writing point and the point of attachment of the tissue. In class Il type of lever the fulcrum lies at one end beyond the writing point and the point of attachment of the tissue. Hence, the writing levers are of two types: (a) Isotonic type, in which change in length due to contraction is recorded while the tension on the muscle remains the same, e.g, simple lever and frontal writing lever and; (b) Isometric type, in which isometric recording measures increase in tension of the tissue when the length of the tissue is kept constant. The latter is used in special circumstances such as.recording muscle twitches produced by electrical stimulation. The common recording levers (Fig. 2.1) used in the laboratory are: (Sideways writing lever or simple lever is the simplest form of lever made up of 4 wood, stainless steel or aluminium, and a stylus or light celluloid writing point is attached at the end of the longer arm. The contrattions are recorded on side- | way as curved lines. | Gi) Frontal writing lever is designed in such a way thill the writing point is hinged and freely rotates on its axel. This helps if fiutini) the tension between the smoked paper and recording tip. The contiactions are recorded frontally as straight line. || Experimental Pharmacology =I 23 Commonly Used Instruments in (iii) Starling’s heart lever is used to record the contraction of the heart, The difference between this and other isotonic levers is that, the fulcrum lies at one al end beyond the point of attachment. | (iv) Brodie’s universal lever is a general utility lever having axis screwed with two | nuts between which the interchangeable levers are clamped. 2. Physiograph and Polygraph: At the present time, kymograph is being replaced by highly sensitive student physiograph and multichannel polygraph recorders. Student physiograph is made up of | Console and Coupler. Console is the main body part of the physiograph. Coupler is plugged into coupler attached space of physiograph. By changing the type of coupler and matching transducers, number of parameters like isometric contraction, isotonic contraction, blood pressure (BP), electrocardiogram (E.CG), electroencephalogram (E.E.G), .electromyogram (EMG), respiratory moverients etc. can be measured. The precision and reliability of such recording is several fold higher as compared to conventional recording devices. Most of the recorders consist of following components: | (a) Transducer: Transducer is a device which receives the signals from any changes in length, pressure, volume or.temperature; and converts it into an electrical potential. With the help of appropriate transducers physiological events such as blood pressure, heart rate, ECG, muscle contraction, body temperature etc. can be accurately recorded using physiographs or polygraphs. {b) Amplifier: This device can amplify a very small signal and used it to cause a pen deflection which is directly proportional to the size of signal. The signal itself may ‘come from an infinite variety of voltage producing sources. It may be generated as another form of energy and translated into an electrical signal by transducer. A wide range of amplifiers are available to meet most requirements. (©) Recording system: Recorder is a chart driven device which moves the chart paper according to required speed with the stylus of a writing element. Student physiograph is a single channel electronic recorder having high sensitivity, precision and accuracy. Its operation is simple as compared to multichannel ‘polygraphs’. 3. Langendorff's Apparatus: Langendorff's apparatus is used to study the effect of different drugs in rabbit or guinea- pig heart. The coronary artery which supplies the blood into the heart arises from the base of is the aorta, just above the aortic valves. If the perfusion is made through the aorta, the © perfusate will go only to the coronary blood vessels provided that the pressure of the perfusate is just sufficient to keep the aortic valves closed. The rabbit or guinea-pig is d injected with heparin sodium (8 mg/kg, s.c.), 60 min prior to sacrifice. Heart is quickly isolated along with the aorta from the body and the aorta is tied to the cannula at the bottom of the perfusion apparatus. Oxygenated Ringer-Locke solution is warmed to 37°C in this apparatus by water circulating in the outer jacket. The thread tied to the hook is passed over pulleys and is connected to the recording system (heart lever). The tension of the lever is adjusted &Experimental Pharmacology - | 24 Commonly Used instruments in . such that it gives maximum contraction. The normal records are taken and various drugs are added to study the effect. The Langendorff's preparation is used to study the autonomic and cardiotonic drugs. This preperation has also been used for the study of the effect of drugs on coronary flow. S5 oe aoe r Sterting’s heart lever Sherrington recording drum Students organ bath Brodie's universal lever Langendorff’s apparatus Students physiograph Fig. 2.1: Instruments for recording the contraction of isolated tissue Instruments for Experiments Using Whole Animals: Number of apparatus are used to study the drug effect in intact animals. Some common apparatus which are generally used in pharmacology laboratory are as follows. 1. Assembly for Recording of Blood Pressure: Poissuille in 1828 designed the mercury manometer and Carl Ludwig in 1847 modified it to allow graphic records to be obtained from a float on the mercury, so that progressive changes in the blood pressure could be studied. Now, also the assembly remains basically same and records only the mean arterial pressure. The mercury manometer consists of a U-shaped glass tube with two vertical limbs about 30 cm in height and 5 mm bore. Half of the tube is filled with mercury. The mercury is displaced equally up in one limb and down in the other, hence to obtain the actual pressure in terms of a mercury column, the displacement recorded must be multiplied by two. To avoid this, a millimeter scale with doubled values, up to 250 mmHg is fitted with the manometer. On the surface of one. limb of the mercury column has a cylindrical floatExperimental Pharmacology - 1 25 Commonly Used Instruments in supporting a long stiff wire, bearing a stylus on its upper end that writes on the rotating smoked paper surface of the kymograph. The other limb of the manometer has a side-tube that is connected through inextensible tube made up of thick rubber or polythene to an arterial cannula. The upper end of the limb is also connected with a reservoir bottle containing some anticoagulant fluid that can be pumped into this limb and through the interconnecting tube to the arterial cannula. At the junction of the side tube, a three way stopcock connects or disconnects the arterial cannula and manometer from the reservoir bottle. Condon’s bicod-pressure manometer is used for recording the blood pressure in small animals (rats and mice) and slightly differs from the conventional manometer. The main limb of the manometer is 28 cm long and 2.5 mm diameter bore. It produces approximately double the sensitivity of the norma! U-shaped manometer. The calibrated scale is fitted so that accurate pressure reading between 0 and 200 mmHg may be obtained. 2. Rotarod Apparatus: Rotarod apparatus is used to study the muscle relaxant property or the muscle grip strength of drugs in mice and rats. Rotarod apparatus consists of a metal axle with a horizontal iron rod, The rod is divided into 2-5 sections for studying the muscle gripping activity in more number of animals (mouse/rat) at a time. Timer is present in each section at the base of the instrument. The rod rotates at variable speed (20-25 rpm is ideal one). The retention time (sec) for each animal is recorded. The length of time that a given animal stays on this rotating rod is a measure of their balance, co-ordination, physical condition, and motor-planning. 3. Photoactometer: Photoactometer or the activity cage is used to study the CNS stimulant and CNS depressant effect of the drug in mice and rats. CNS stimulants and.CNS depressants affect the motor activities in animals. Photoactometer consists of 30 cm long and 30 cm deep box with iron rod at the bottom. It operates on photoelectric cells in which six numbers of light and six numbers of photo cells are connected in circuit with an automatic counter. A photo cell is activated when the rays of light falling on the photo cells are cut-off by animal during crossing the beam of light and the counter counts the number of cut-offs, One mouse can block only one beam of light at a time. More the locomotor activity more is the number of cut-offs, 4. Elevated Plus-Maze: Elevated plus-maze apparatus is used for screening of anxiolytic agents in mice and rats. It is most popularly used because of more simple apparatus, reliable, less time consuming. The plus-maze apparatus consists of two open arms (16 x 5 cm for mouse and 50 x 10 cm for rats; L x W), and two enclosed arms (16 x 5 x 12 cm far mouse and 50 x 10 = 40 cm for rats; L x W x H), open to the top, arranged so that the two open arms are opposite to each other. The maze is elevated to a height of 25 cm for mice and 50 cm for rats from the floor. Rats and mice have dislike for high and open space and prefer enclosed arm. When animals enter open arm, they became immobile, freeze, defecate, have fear like movements and alsoExperimental Pharmacology - | 26 Commonly Used Instruments in plasma cortisol level is reported to be increased, which is similar to anxiety disorder. Motor activity and open arm exploratory time are registered. Benzodiazepines and other anxiolytics decrease motor activity and increase open arm exploratory time. 5. Electro-Convulsiometer: Electro-convulsiometer is an apparatus used to induce convulsions in rats and mice. It is used for screening of anti-epileptic agents. The prescribed electroshock (150 mA to rats and 30 mA to mice for 0.2 sec) is given to the animals through the electrodes placed on ear pina, The electric stimuli produces cortical excitation and induces characteristic seizures in the animals. The maximal electroshock (MES) induced convulsions are divided into three phases i. tonic phase (tonic fiexion and tonic extension), clonic phase and stupor. The duration of each phase of seizure is recorded and the effect of drug on each convulsion is studied. 6. Eddy’s Hot-Plate Analgesiometer: Analgesiometers are used for screening the central analgesic activity of drugs in rats and mice. Eddy's hot plate consists of an électrically heated surface (made up iron, aluminium, copper or a heated glass surface) whose temperature is maintained at 55°C by the thermostat. The hot plate method deals on the principle of the thermal radiation. The paws of mice and rats are very sensitive to heat at temperatures which are not damaging the skin. The animals are placed on the heated surface and the responses are observed. The responses are jumping, withdrawal of the paws and licking of the paws. A cut-off period of 12-15 sec is observed to prevent damage. 7. Tail Flick Analgesiometer: Tail flick apparatus contains a nicrome wire, which can be heated by switching ON the electric current. The heated nicrome wire acts as noxious stimulus. The reaction time {ie. flicks the tail) to radiant heat is measured by placing the mouse in the restrainer_ and keeping the tip of the tail on the analgesiometer (above the nicrome wire) and switching ON the apparatus for heating. Flicking response of tail is taken as the end point. A cut-off period of 12-15 sec. is taken to avoid damage to the tail. It is used for screening the central analgesics in rats and mice. 8. Cook's Pole Climbing Apparatus: It is used for screening of anti-psychotics on condition avoidance response. Cook's pole climbing apparatus consists of transparent chamber with electrified floor and a lid which is attached to the pole. Whole chamber is surrounded by a wooden box. Rats are trained to climb a pole within 30 sec when shock is given. In classical conditioning method, animals trained to act in a certain way (climbing a pole) in response to a signal (buzzer) to avoid @ noxious stimulus. Response to the signal is conditioned resporise while response to noxious stimulus is unconditioned response. The shock is then preceded by a buzzer for 15 sec. This is done for 2-3 times a day for 8 days till rats are trained to clit the pole at the sound of ‘the buzzer. Trained animals are treated with the afiti:pychotic drug; and conditioned response and unconditioned responses are observed.Experimental Pharmacology -1 e e d N a Cook's pole climbing apparatus ‘The mercury manometer Fig. 2.2: Commonly used instruments for experiments using whole animalsExperimental Pharmacology - | 28 Commonly Used instruments it 1. Discuss different instruments used for experiments on isolated tissues. 2. Discuss different types of lever for recording the contraction and relaxation of tissue. 3, Write the uses of rota rod apparatus and photoactometer. g 4, Discuss in detail the assembly for recording blood pressure.EXPERIMENT No. 3 STUDY OF COMMON LABORATORY ANIMALS Purpose: At the end of practical class, the students shall be able to 1. Know about different laboratory animals. 2. Understand the uses of laboratory animals in experimental pharmacology. Terminology: Laboratory animals: Laboratory animals are those animals which can be bred and maintained in the laboratory under suitable conditions by taking the permission of regulatory authorities. Guinea pig has become synonymous to the experimental animals, but many other species are equally useful in the study of drugs. Objective: ‘Study of common laboratory animals in experimental pharmacology. Description: Pre-clinical studies of experimental pharmacology involve laboratory animals using wide-ranging doses of the study drug to obtain preliminary efficacy, toxicity and pharmacokinetic information. Such tests assist pharmaceutical companies to decide whether a drug candidate has scientific merit for further development as an investigational new drug. Experimental animals can be ciassified as: 1, ‘Rodents: Rat, Mouse, Guinea pig, Hamster etc. 2. Non-rodents: Rabbit, Dog, Cat, Pig, Monkey etc. 3. Miscellaneous: Frog, Pigeon, chicken etc. The commonly used laboratory animals in pharmacology laboratory are Frog, Rat, Mice, Guinea pig, Hamster and Rabbit. Cats and dogs are used to study the’ blood pressure experiments. Monkey, pig and some other animals were also used earlier, but now their use has been restricted, However, Beagle dogs are the only strain approved by USFDA for testing of new drugs. Mouse ‘Guniea pig Rabbit Frog Fig. 3.1: Commonly used laboratory animals in pharmacology laboratory a HisExperimental Pharmacology -1 32 Study of Common Laboratory Animals RAT (Rattus norvegius): Rat is a warm blooded rodent which is the most commonly used animal for biomedical research. Rat does not vomit due to lack of vomiting center and tightly presence of lower esophageal sphincter. It has no tonsils and gall bladder in its body. It has diffuse pancreases, thus difficult to perform pancreatectomy to induce type 1 diabetes mellitus. Fundus and pyloric parts of stomach have clear lining between them and gastric acid secretion is continuous, Rat is a coprophagy animal that eats their own stool. It shows resistance to the effect of cardiac glycosides. Oestrus cycle appears at puberty at the age of two to three months and cycle lasts for about four to five days. The cycle can be divided into four stages as follows: (a) Estrus: It lasts for 9-15 h. The vaginal smear shows cornified epithelial cells. It is characterized by sexual receptivity when the female will allow copulation. It ends with ovulation. It can be induced experimentally by administration of diethylstilbestrol ~ (100 mg/kg). (b) Met-estrus: It is about 20 h and occurs shortly after ovulation. The vaginal smear consists of many leucocytes with few cornified epithelial cells. (©) Di-estrus: It is the longest phase of about 60-70 h. The vaginal smear shows = leucocytes only. (d) Proestrus: It lasts for about 12 h. The vaginal smear shows nucleated epithelial cells either single or in groups. Albino Wistar rats and Albino Sprague-Dawley rats have been widely used strains throughout the world. Lewis and porton strains are also used for experimental purposes. Mouse (Mus musculus): Mice are warm blooded rodent which are the smallest laboratory animals..In biomedical research mice are preferred over other species because they can be bred uniformly, cheap and easy to handle, require small place for housing and very sensitive to small doses of 2 drug substances. Due to the large similarity in mice and human genome, it provides good model for research on wide variety of human diseases. Swiss albino mouse is the most widely used strain for pharmacological experiments. Laca and Balb-C strains are also used for experimental purposes. Guniea Pig (Cavia porcellus): Guinea pigs name itself has become synonymous to an experimental animal. Guinea pig is a warm blooded rodent and very docile laboratory animal. It is an herbivorous animal and daily requires vitamin C in diet like human. Guinea pig is highly sensitive to histamine. They are very susceptible to tuberculosis and anaphylactic shock. Penicillin is more toxic to Guinea pig than to the mouse, Guinea pig serum contains an enzyme asparaginase, which has antileukemic action. Hamster (Mesocricetus auratus): Hamsters are a brown to gold colour animal, have chunky body with short legs, a diminutive fluffy tail and a large amount of loose skin covered with dense short soft fur. There are four toes on the front foot and five on the back, They have prominent cheek pouches extending up to the shoulder region. The ears are quite prominent, rounded, usually pigmented and scarcely covered with hair. The most common strain used in pharmacological studies is the Syriat. or Golden hamster, Chinese hamster, European hamster and American hamster.is th ol var ws alls ins est ase and and an nal. gis vctic san rave ared any juite mon ster, Experimental Pharmacology - | 3.3 ‘Study of Common Laboratory Animals Rabbit (Oryctolagus cuniculus): Rabbit is a warm blooded mammalian and a docile animal used for various studies. Some strains of rabbit are resistant to the effect of atropine because they have higher concentrations of atropinesterase enzymes in their blood. In rabbits coitus itself induces secretion of leutenising hormone in females, which leads to ovulation. Progesterone is known to block such ovulation. New Zealand white rabbits are the most common strain used widely in pharmacological studies. Other strains which are also used in studies are the Dutch, Flemish Giant, Himalayan black and some. of the domestic strains, Frog (Rana tigrina): Frog is a cold blooded amphibian which is used very commonly in the biomedical research: Frogs and toads have no taxonomic differences but can be differentiated by their appearances (frog is wet and toad is dry). It has three chambers in its heart, two auricles and one ventricle. The oxygen and moisture are absorbed through their highly permeable skin. Some hybridized frogs are used in biomedical research are Rana esculenta, Rana pipiens, Rana temporaria, Rana lessonae, Rana ridibunda etc. Table 3.1: Uses of different laboratory animals in experimental pharmacology Animals Uses in Experimental Pharmacology Frog (a) Commonly used for bioassays and in pregnancy assays. | (b) Different. drugs acting on central nervous system, neuromuscular LA junction and heart can be studied using frog and its isolated tissues. (Q_Whole frog is also used in screening of central drugs like local anesthetics. Rat (a) The rat and its isolated tissues are used in the screening of drugs acting ‘on central nervous system, cardiovascular system, estrus cycle, mating behaviour, gastric ulcers and acid secretions in stomach. L (b) Rats can be trained properly for various types of work performances in response to a reward or punishment, hence suitable for testing of psychopharmacological agents. 5 (0 -It is’ also useful in the hormonal assay, immunity, transplantation, | immunosuppression, carcinogenicity study and different toxicity studies. (d) The isolated tissues like ileum, trachea, heart, anacoccygius muscle, stomach strip, vas deferens, uterus and phrenic nerve-diaphragm are used in experimental pharmacology. E Mouse (a) Mice are the widely used animals in different toxicity studies. (b) Mice are also used in cancer and genetics research, immunity, / transplantation related experiments. (0) Screening of analgesics, central nervous system and chemotherapeutics | are carried out in this species. | (d) Isolated tissues of mice are rarely used (except vas deferens and ileum) the experimental pharmacology because they are very small and delicate. (e)_They were earlier used in the bioassay of insulinExperimental Pharmacology -| 34 Study of Common Laboratory Animals Animais Uses in Experimental Pharmacology et " (@) It is mainly used in the bioassay of Histamine. (b) They are used in the study of drugs like bronchodilators, local anesthetics, spasmolytic and digitalis. (©) The drugs affecting the hearing activity are also screened using Guinea pigs. (d) They are widely used in immunology. (€) The drugs inducing anaphylaxis and hypersensitivity are screened using Guinea pigs. ( It is also useful in screening of drugs for the treatment of tuberculosis, cholera and amoebiasis. (g) The isolated tissues like ileum, tracheal chain, vas deference and heart are used in experimental pharmacology. Hamster (a) The presence of cheek pouch in hamster makes it useful in immunological research. (b) Syrian hamsters are used in the research field of virology, cancer, genetic, toxicology and reproduction. (©) Chinese hamsters are commonly used in diabetes due to deficiency of B-cells, or presence of defective B-cells in the pancreas. (d) Chinese hamsters have a low chromosome number (22) compared to other laboratory animals due to which they: are used for cytological investigations, genetics and tissue culture. (©) Hamsters are also used in onco virus, influenza virus, respiratory syncytial virus studies and vaccine production. {f) Cheek pouch does not have lymphatic drainage and hence they can be used as a site for tissue transplant such as tumors and grafts. (g) The stomach pouch strips of Syrian hamster have been used for the essay of prostaglandins. Rabbit Ag) It is an ideal animal for pharmacokinetic studies. (@ Rabbits are mainly used. in the py sex hormones. = (b) They have been used in the screening of drugs for diabetes, diphtheria, tuberculosis, cancer and heart diseases. (c) They are commonly used in genetics, nutrition, toxicology, immunology, physiology and reproduction. (d) They are also useful in the study of teratogenicity of the drugs. (e) Isolated tissues like duodenum, ileum and heart are used in experimental pharmacology. (Bioassay of adrenaline can be performed using rabbit ileum. gen testing, in bioassays of insulin (h) Apart from the drugs, effects of creams, cosmetics, special diets and food additives have also been tested in rabbits. (@ The dog is used as a model for cardio vascular research, diabetes mellitus, ulcerative colitis, open heart surgery, organ transplantation, central nervous system, pharmacology and toxicology studies etc. (b) Anaesthetized dogs are commonly used in the study of drugs acting on blood pressure and vascular system.Experimental Pharmacology - | 35 ‘Study of Common Laboratory Animals Animals Uses in Experimental Pharmacology al Cat (a) Cat has distinct nictiating membrane (third eyelid), which is commonly | used in screening of ganglionic blocking drugs. (b) Cats are mainly used in behavioural studies, cardio vascular studies, nerve impulse transmission etc. (0 It is also used in neuropharmacology, toxicology, oncology and chromosomal abnormalities studies. (d) It is also employed for the study of drugs effecting on blood pressure. Monkey | (a) The monkey is used in the fields of virology, parasitology, immunology and immunosuppression. (b) It is also employed for the study of nutrition, reproduction etc.” 4 (© Adult rhesus monkeys are used for N-methyl-4-phenyl-1,2,3,6- | tetrahydropyridine (MPTP) administered model of parkinson’s disease. { Pig (@) They are used in pharmacological and toxicological research. (b) They are used in important research areas like several isolated organ f models, investigation of skin permeation and for digestive systems. Pigeon (@) They are used in screening of antiemetic activity, cardiovascular diseases, 7 CNS disorders and intravenous anaesthetics. ! (b) Bioassay of prolactin through the pigeon crop method is. one of the ql important methods. Chicken (a) They are used in many areas of biomedical research such as breeding el | and genetics, growth, embryology, incubation, fertility, artificial | insemination, toxicology, physiology, biochemistry, endocrinology and e neurobiology. = (b) Chick comb method, heart rate, EEG, memory, angiogenesis, blood n pressure measurement and vasodilating activities like different models can be developed in chicken. i Table 3.2: Biological and physiological data of commonly used laboratory animals yr Parameter Rat_| Mouse | Guin Hamster | Rabbit Adult weight (g) 150-300 | 20-40 | 600-800 80-150. 1000-3500 Life span (vears) 23 | 15-25 35 23 47 al Suitable ate for eweriment (Months) 15 075 3 1 6 Gestation period (Days) 21-23 | 19-21 | 68-72 16 3132 Daily food intake (g/ 100 g body| 5-10 | 15-20 68 7-10 57 weight) | ad| | | Daily water intake (mi/100 g body 10 S20 10 10 10 a weight) = Rectal temperature (°C) 36-46 | 38-39 | 372-40 37-38 | 385-40 m Heart rates (beats/min) 300-500 | 330-70. | 230-400 7 | Systolic blood pressure (mm Ha) 116 113 | 7 Diastolic blood pressure (mm Hg) 90 a | 47 pa) Blood volume (ml/kg) 6-7 7-9 | 6-12 Respiratory rate (min) 65-180 | 84-230 | 70-104Experimental Pharmacology - | 3.6 ‘Study of Common Laboratory Animals Transgenic Animats: A major part of biomedical research is aimed at understanding human biology at the cellular and molecular level in health and disease. Whereas certain human characteristics are shared by no other species or only by primates, there are many other characteristics that are shared with many species of animals. The'use of animals has therefore become fundamental to all aspects of modern biomedical research from the study of basic biological mechanisms, to the understanding of disease pathology and the development of new medicines for both human and veterinary use. A transgenic animal is one which has beef genetically altered to have specific characteristics, it otherwise would not have. Transgenesis is the process of introducing foreign or exogenous DNA into an animal's genome and transgene is the foreign gene which is introduced in the genome of the animal, In animals, transgenesis either means transferring DNA into the animal or altering DNA already in the animal. Genetic manipulation: by modification of existing genes is known as knock-out technology. Some newer variations of transgenic animals used by investigators are: Knock-in animals (to replace @ gene in the host genome with @ modified version of itself) and Cre-loxP transgenic animal (to disrupt a gene at a specific time of development). Transgenic animals are extensively used to study cancer, obesity, heart diseases, diabetes, arthritis, anxiety, Parkinson disease and aging, Some examples are Ob/Ob mouse used for study obesity, db/db mouse, Goto-Kakizaki rat which is a genetic lean model of type 2 diabetes, Kreesbond dog, Chinese hamster and Celebes black ape are used for type 1 diabetes models. Knockout mice (e.g. COX-2 gene, Angiotensin converting enzyme, D2 receptor and other gene knock out) are valuable tools for studying different human diseases. Transgenesis is required to provide animal models for study of human diseases, improve genetic features of domesticated animals, using farm animals for production of human pharmaceuticals and nutraceuticals, and study the génes regulation and development of animals. . Discuss the commonly used laboratory animals in pharmacology laboratories. 2. What is pre-clinical study? 3. Discuss different biological and physiological data of commonly used laboratory animals. 4. What are'transgenic animals? 5. Discuss the various uses of transgenic animals. akeEXPERIMENT No. 4 MAINTENANCE OF LABORATORY ANIMALS AS PER €PCSEA GUIDELINES Purpose: At the end of practical class, the students shall be able to 1. Realize importance of ethical requirements for preclinical studies. 2. Understand the principle of different guidelines and their importance in animal experiments. 3, Justify the need for proper standards of maintenance and care in the use of animals for research and teaching. Terminology: Committee for Purpose of Control and Supervision of Experiments on Animals (CPCSEA): The Prevention of Cruelty to Animals Act 1960 as amended in 1982, is to prevent the unnecessary pain or suffering on animals, The Central Government has constituted a Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), which ensures that animals are not subjected to unnecessary pain or suffering before, during or after the performance of experiments on them. For this purpose, the Government has made "Breeding of and Experiments on Animals (Control and Supervision) Rules, 1998" as amended during 2001 and 2006, to regulate thé experimentation on animals. Institutional Animal Ethics Committee (AEC): As defined in “Breeding of and Experiments on Animals (Control and Supervision) Rules, 1998": Institutional Animals Ethics Committee means a body comprising of a group of persons recognized and registered by the CPCSEA performed in an establishment which is constituted and operated in accordance with procedures specified for the purpose by the committee. \ Institutional Biosafety Committee (IBSC): The Institutional Biosafety Committee shail be the point for interaction within institution for implementation of the guidelines. Any research project which is likely to have biohazard potential (as envisaged by the guidelines) during the execution stage or which involve the production of either micro-organisms or biologically active molecules that might cause biohazard should be notified to IBSC. IBSC will allow genetic engineering activity on classified organisms only at places where such work should be performed as per guidelines. Provision of suitable safe storage facility of donor, vectors, recipients and other materials involved in experimental work should be made and may be subjected to inspection on accountability. Good Laboratory Practice (GLP) for animal facilities: GLP for animal facilities is intended to assure quality maintenance and safety of animals used in laboratory studies while conducting biomedical and behavioural research and testing of drugs. (4.4)Experimental Pharmacology -1 42 Maintenance of Laboratory Animals ...... Objective: To study the maintenance of laboratory animals as per CPCSEA guidelines. Description: Committee for Purpose of Control and Supervision of Experiments on Animals (CPCSEA): All establishments engaged in research and education involving animals are required to comply with the various guidelines, norms and stipulations set out by CPCSEA. The aim of these guidelines is to ensure humane and ethical treatment of animals, while facilitating legitimate scientific research involving experiments on animals and to make judicious use of animals for experimental purposes. Mandate of CPCSEA: Registration of establishments conducting experiments on animals. Registration of establishments engaged in breeding of laboratory animals. Constitution of IAECs in the establishments registered with CPCSEA. Approval of animal house facilities for small'and large animals. Permission for conducting experiments on large animals. Recommendation for import of animals for experimentations and breeding. Guidelines Issued by CPCSEA: CPCSEA has issued the following guidelines: 1. Guidelines for the Reuse and Rehabilitation of dogs. 2. Guidelines for Constitution/Reconstitutions of IAECs. 3. Care and Management of Equines used in the product of biologicals. 4, Standard operating procedures for IAEC. Maintenance of Laboratory Animals as per CPCSEA Guidelines: CPCSEA has set guidelines for laboratory animal facility. The goal of these guidelines is to promote the humane caré of animals used in biomedical and behavioural research and testing with the basic objective of providing specifications that will enhance anima! well being, quality in the pursuit of advancement of biological knowledge that is relevant to humans and animals. The CPCSEA provides the below mentioned guidelines to be followed by all research institutions in the country. 1. Adequate veterinary care must be provided by a veterinarian or a person who has training or experience in laboratory animal sciences and medicine. Institutions should employ people trained in laboratory animal science or provide for both formal and on-the-job training for the care of animals. It is essential that the animal care staff maintain a high standard of personal cleanliness. 2, All animals must be acquired lawfully as per the CPCSEA guidelines. The transport of animals from one place to another is very important and must be undertaken with care. The main considerations for transport of animals are the mode of transport, the containers, the animal density in cages, food and water during transit, protection from transit infections, injuries and stress. Quarantine is the separation of newly received animals from those already in the facility. An effective quarantine minimizes the chance for introduction of pathogens into an established colony. Po awoor ow asfasd as ms. oth nal Experimental Pharmacology - | 43, Maintenance of Laboratory Animals 3. 10. 11. All animals should be observed for signs of illness, injury, or abnormal behaviour. Health and nutritional status of the animals should be properly maintained. Animals should be fed palatable, non-contaminated and nutritionally adequate food daily unless the experimental protocol requires otherwise. Ordinarily animals should have continuous access to fresh, potable, uncontaminated drinking water, according to their particular requirements. Laboratory animals are very sensitive to their living conditions. It is important that, they shall be housed in an isolated building located as far away from human habitations as possible and not exposed, to dust, smoke, noise, wild rodents, insects and birds, The location, building, cages, material and environment of animal rooms are the major factors, which affect the quality of animals. Housing, care, breeding and maintenance of experimental animals is necessary to keep them in physical comfort, good health and behave normally. Bedding should be removed and replaced with fresh materials as often as necessary to keep the animals clean and dry. Sanitation is essential in'an animal facility. Animal rooms, corridors, storage spaces, and other areas should be cleaned with appropriate detergents and disinfectants as often as necessary. The Institute shall maintain SOPs describing procedures/methods adapted with regard to animal husbandry, maintenance, breeding, animal house microbial analysis * and experimentation records. The. animal house should maintain the different records of animals as instructed by CPCSEA. Acceptable experimental techniques and procedures for anaesthesia and euthanasia are applied during the study. : Transgenic animals are used to study the biological functions of specific genes, to develop animal. models for diseases of human or animals, to produce therapeutic products, vaccines and for biological screening. Housing, feeding, ventilation, lighting, sanitation and routine management practices for such animals are similar to those for the other animals of the species as given in guidelines. However, special care’has to be taken with transgenic/gene knockout animals where the animals can become susceptible to diseases. sa All scientists working with laboratory animals must have a deep ethical consideration for the animals they are dealing with. From the ethical point of view it is important that such considerations are taken care at the individual level, at institutional level and finally at the national level. Alternatives to Animal Screening Procedures: 3 R's concept British researchers William Russell and Rex Burch in 1959 formulated this concept of the 3 R's in their book ‘The Principles of Humane Experimental Technique’, which argues that humane science is the best science. Alternative methods fall into three broad categories. These are called the 3 R's: Replacement, Reduction, and Refinement. Since thegr Experimental Pharmacology - 1 44 Maintenance of Laboratory Animals ...... introduction of these principles, they have become widely accepted internationally as the basic principles guiding animal use in research, teaching and testing. Ron Banks added the 4" Rive, Rehabilitation and Reuse of animals. Note: Above mentioned mandate and guidelines are documented in the CPCSEA websites i.e. http://cpesea.nic.in. Institutional Animal Ethics Committee (IAEC): As defined in “Breeding of and Experiments on Animals (Control and supervision) Rules, 1998": Institutional Animals Ethics Committee means a body comprising’ of a group of persons recognized and registered by the CPCSEA. The primary responsibility of a person who has been nominated to represent CPCSEA on IAEC is the well being and welfare of the animals housed or kept for experiments/breeding. ‘Composition and Constitution of IAEC: IAEC includes 8 members as described below: ‘A. Institutional Members (5 members from the institute) 1. AScientist in Charge of Animals Facility 2. AVeterinarian Involved in the Care of Animals 3. A Biological Scientist 4. Two Scientists from Different Biological Disciplines B. CPCSEA Nominated members (3+1 members from outside institute) 1. ACPCSEA Main Nominee 2. ACPCSEA Link Nominee 3, A Scientist from Outside the Institute 4, ASocially Aware Nominee The Chairperson and member secretary of the committee are nominated by the institution and should take care of the functioning of IAEC in accordance with procedures specified for the purpose by the committee. - ‘Objectives of IAEC: (@) Experiments to be avoided wherever it is possible to do so. (b) Experiments on larger animals are avoided when same results can be-achieved in smaller animals. (©) Rationalize usage of animals- 3 R's. (d) Animals to be properly looked after before and after experiments. } Functions of IAEC: (a) IAEC will review research proposals and prevent infliction of unnecessary pain and h sufferings before, during and after experiments on animals, to follow the CPCSEA } guidelines. (b) IAEC reviews and approves alll research proposals involving small laboratory animal experiments with a view to assure quality maintenance and welfare of animals used in pre-clinical researc.Experimental Pharmacology - | 48 Maintenance of Laboratory Animals ...... (©) For experiments on large animals, the IAEC-will forward its recommendation to the CPCSEA, for its approval process. (d) IAEC ensures that experiments shall be performed in every case by or under the supervision of a qualified person and under the responsibility of the Principle Investigator. {e) IAEC reviews the proposals before start of the study as well as monitor the research throughout the study and after completion of the study through annual reports, final report. () IAC team will visit the laboratory in the animal house/respective department where the experiments are conducted. The committee also ensures compliance with all regulatory requirements, applicable guidelines and laws. (g) Monitor and inspect the housing of animals and ensure that it is as per specified standards. Institutional Biosafety Committee (IBSC): The Institutional Biosafety Committee is engaged in hazardous chemical use, genetic engineering research and production activities. This committee shall also examine the proposal on animal experiments involving hazardous agents in addition to its existing functions, IBSC whose members are knowledgeable about hazardous agents, are in place in most of the higher level education, research institutes and in many pharmaceutical industries for safety issues. Institutional Biosafety Committee (BSC) is to be constituted in all centres engaged in genetic engineering research and production activities. The committee will constitute the following. 1. Head of the institute or his nominee. 2. Three or more scientist engaged in DNA work or molecular biology with an outside expert in the relevant discipline. 3. A member with medical qualification - Biosafety officer (in case of work with pathogenic agents/large scale used). 4, One member nominated by Department of Biotechnology (DBT), Government of India. The Main Functions of IBSC: The functions and activities of IBSE include the following: 1. Registration of Biosafety Committee membership composition with Review ‘Committee on Genetic Manipulation (RCGM) and submission of report. ISBC will provide half yearly reports on the ongoing projects to RCGM regarding the observance of the safety guidelines on accidents, risks and on deviations if any. A computerized Central Registry for collation of periodic reports on approved projects will be setup with RCGM to monitor compliance on safeguards as stipulated in the guidelines. Tl. Review and clearance of project proposals falling under restricted category that meets the requirements under the guidelines. IBSC would make efforts to issue clearance certificates quickly on receiving the research proposals from investigators. Il. Tailoring biosafety program to the level of risk assessment. IV. Training of personnel on biosafety. V. Instituting health monitoring program for laboratory personnel, complete medical check-up of personnel working in projects involving work with potentially dangerousExperimental Pharmacology - | 46 Maintenance of Laboratory Animals ...... microorganism should be done prior to starting such projects. Follow up medical check-up including pathological test should be done periodically, annually for scientific workers involved in such projects. Their medical record should be accessible to the RCGM. It will provide half yearly reports on the ongoing projects to RCGM regarding the observance of the safety guidelines on accidents, risks and on deviations if any. VL_Adopting emergency plans. Other Guidelines for Maintenance of Laboratory Animals: The Organization for Economic Cooperation and Development (OECD) and the “International Conference on Harmonization (ICH) are the different organizations that have issued certain guidelines regarding toxicity and safety studies in animals, The OECD has issued a guideline for toxicity testing of various chemicals. OECD is an intergovernmental organization in which representatives of 29 industrialized countries of North America, Europe and the Pacific are involved. The OECD Secretariat located in Paris, France and its work is conducted by various committees and subsidiary groups. The work of the OECD related to chemical safety is carried out in the Environment, Health and Safety Program. The OECD has issued the OECD test guidelines for chemical testing i. collection of methods used to assess the hazards of chemicals and of chemical preparations such as pesticides, These methods cover tests for physical and chemical properties, effects on human health and wild life, accumulation and degradation in the environment. The OECD provides the guidelines on selection of animal species; housing and feeding conditions and preparation of animals for toxicity study. OECD test Guideline 420, 423 and 425 are used for acute toxicity study and 452 for chronic toxicity study. The OECD test guidelines are recognized worldwide as the standard reference tool for chemical testing. The ICH is a sole project that unites the regulatory authorities of Europe, Japan and the United States. The experts from pharmaceutical industries from the above three regions should discuss scientific and technical aspects of product registration. The object of such harmonization is a more economical use of human, animal and material resources and the elimination of unnecessary delay in the global development and availability of new medicines by maintaining the quality, safety, efficacy and regulatory obligations to protect public health. The ICH guidelines are divided into four major categories i.e. a) Quality: relating to chemical and pharmaceutical quality assurance, b) Safety- relating to in vitro and in vivo preclinical studies, ¢) Efficacy- relating to clinical studies, and.d) Multidisciplinary- topics that do not fit uniquely into one of the above categories, Certain guidelines regarding toxicity and safety studies in animals for new chemical or pharmaceutical products are placed in both OECD and ICH. These guidelines are documented in the websites of WWW.vecd.org and WWWiich.org. Ee a Se What is the concept of 3 R's? Define IAEC and IBSC. Discuss CPCSEA guidelines for maintenance of laboratory animals. Discuss OECD and ICH guidelines for toxicity studies. ‘What is GLP?