MIT-WPU | School of Pharmacy

WORLD’S FIRST UNIVERSITY

FOR LIFE TRANSFORMATION

1. INTRODUCTION TO IN VITRO PHARMACOLOGY AND

PHYSIOLOGICAL SALT SOLUTIONS

Dr. Chinmay Deshmukh

Assistant Professor
Email: chinmay.deshmukh@mitwpu.edu.in
 Reference books

1. A Practical book of Pharmacology II by Dr. Pankaj M. Choudhari, Dr. Dheeraj T. Baviskar and Dr.

Prakash Patil, PV books, S Vikas and company (medical publishers) Jalandhar, 2019.

2. Ghosh M.N., Fundamentals Of Experimental Pharmacology, 3rd Edition.

3. Kulkarni S.K., Practical Pharmacology And Clinical Pharmacy, Vallabh Publication,.

4. A Practical book of Pharmacology-II by Hemant Suryawanshi, Mukesh Patel and Sunil Pawar,

Nirali Prakashan, Second edition, January 2020.

 In Vitro Pharmacology
• In vitro pharmacology studies the biological effects of a drug in an isolated environment, such as cell
lines or tissues. It is outside of living organism. This setup conveniently eliminates whole organism
physiological influences allowing for detailed analysis.

• In contrast, In vivo pharmacology is the study of biological effects of a drug in a complex living
organisms and is used to observe the complex physiological effects of a drug. It is within the organism.

• In vitro studies are conducted using component of an organism that have been isolated from their usual
biological surrounding such as microorganisms, cells, or body.

• Microorganisms, cells, tissue, can be studied in artificial culture media therefore these are also called
‘test-tube experiments’ because they are traditionally done in test tubes, flasks, petri dishes etc.

• All the times, the results obtained from in vitro experiments cannot be considered to predict same
reaction of an entire organism in vivo
 Advantages of In-vitro Pharmacology
1. Safety is extremely a crucial stage of the pre-clinical development process. Its purpose is to assess
any potential undesirable effects of the drug on the body’s major systems. Although in the past,
the majority of safety testing of pharmaceuticals was conducted on animals, today, safety tests are
increasingly made using in vitro models that involve isolated cell lines and tissues.

2. In vitro studies permit a species-specific, simpler, more convenient, and more detailed analysis
than can't be done with the whole organism.

3. Drug discovery and development to optimize biological activity of most promising molecules for
further screening.
 Applications of In-vitro Pharmacology
1. Obtain high-quality data on the safety and toxicity of your drug candidate;
2. Identify potential adverse effects early in the drug development process;
3. Assess the potency and efficacy of drug candidate the targeted disease;
4. Gather data on pharmacokinetics and pharmacodynamics of the drug;
5. Study the drug mechanism of action in more depth
6. Evaluate the activity of biosimilar compounds.

7. To study concentration-effect relationship and thereby study drug efficacy.

8. To study acute, subacute and chronic toxicity.

9. Pharmacokinetics

10. Immunotoxicity
 Isolated tissue experiments.
1. In order to see the effect of drugs on tissues such as rectus abdominis muscles of frog, guinea pig
ileum, goat trachea, rat fundus, chicken ileum etc, the tissues must be isolated from animal and
kept in suitable physiological solution throughout the experiment.

2. This tissue is mounted to suitable instrument (e.g organ bath for chicken ileum) and suitable
physiological conditions such as temperature, salts and ions, electrolytes, oxygen are provided to
continue the contraction relaxation pattern as in the body.

3. The dose of suitable drug is given to the tissue under observation. The tissue starts contracting.
E.g. contraction of guinea pig ileum by addition of Acetylcholine.

4. Then with the help of rotating drum and lever, suitable response is produced on kymograph
paper. This procedure is repeated for maximum possible doses till ceiling effect is observed.
 Preparation Receptors commonly studied Bathing solution (Physiological Salt Solution)

Frog rectus abdominis Ach (nicotinic, Muscarinic) Frog ringer

Guinea pig colon 5 HT Kreb

Guinea pig ileum Ach (muscarinic M3)histaminic H1,3, Neurokinin Kreb or Tyrode

NK 1, Bradykinin B 2

Guinea pig vas deferens EP3 Krebs

Mouse vas deferens Mu kappa and delta opiod, dopamine D2 Kreb

Rabbit jejunum Alpha2 adrenoceptors Tyrode

Rabbit heart Beta1 adrenoceptors Ringer-Locke

Rat or Rabbit aorta Alpha1, adrenocpetors, 5HT2, Neurokinin NK1, Kreb

Bradykinin, Angiotensin

Rat colon Neurokinin Kreb

Rat phrenic nerve Ach Kreb

Rat uterus Beta adrenoceptors, mu opiod, bradykinin Kreb
 Physiological Salt Solutions (PSS)
• Physiological Salt solution (PSS) is artificially prepare solution to keep isolated tissue alive under
experimental conditions. They are substitute of tissue fluid normally present in live organism.
• They provide isotonicity and act as buffer when drug are added.
• PSS supplies nutrients, ions, electrolytes and energy to isolated tissue same as tissue fluid.
• It contains certain concentration of Sodium, Potassium, Calcium, Glucose and Chloride. Each of
this ingredient has its essential role in the solution. These electrolytes are important for muscle
contraction, heart beat, blood clotting, acid and fluid balance in the body
• The aeration with (appropriate gas mixture, Oxygen and Carbon dio oxide) is also bubbled in this
solution to supply the oxygen to cells of tissue.
• The salt solution must be prepared carefully and as per the standard specification or SOP and
errors in preparing it must not exceed 1%
• It is prepared fresh and utilized in 24 hours. It should not be stored due to microbial growth.
• It can be prepared 24 hours in advance if glucose and calcum salts are omitted and only added
when it is used.
 Physiological Salt Solutions
Compound Frog Ringer Ringer De Jalan Tyrode Kreb
1. Sodium chloride (NaCl): to maintain iso-osmolarity, contractility,
(molecular Locke Henslet
isotonicity and excitability.
weight)
2. Potassium chloride (KCl): to maintain ionic balance.
NaCl (58.45)* 110 (6.0)** 154 (9) 154(9) 137(8) 118(6.9)
3. Calcium chloride (CaCl2): to maintain the contractility of
KCl (74.56) 1.9 (0.14) 5.6 (0.42) 5.6 (0.42) 2.7 (0.2) 4.7(0.35)
preparation.
CaCl2 (110.99) 1.1 (0.12) 2.2 (0.24) 0.55 (0.06) 1.8 (0.2) 2.5 (0.28)
4. Sodium bicarbonate (NaHCO3): to provide alkaline pH.
MgCl2 (95.23) --- --- --- 0.1 ---

5. Glucose: to provide energy MgSo4.7H20 ---- ---- ---- -- 1.2 (1.28)

6. Sodium or potassium dihydrogen phosphate (NaH2PO4 NaHCo3 2.4 (0.2) 6.0 (0.5) 6 (0.5) 11.9 (1) 25 (2.1)

KH2PO4): acts as buffer. NaH2PO4 --- --- --- 0.4 (0.05) ---

(119.97)
7. Magnesium chloride or sulphate (MgCl2, MgSo4): to stabilize the
KH2PO4 ---- ---- --- --- 1.2(0.16)
preparation and hence to reduce spontaneous activity.
(136.08)
8. Distilled water: act as vehicle to dissolve various ingredients.
Glucose 11.1(2) 5.55(1) 2.28(0.5) 5.55 5.55
9. Values in brackets represent the quantity in grams for l litre of sol. (180.16)
 Physiological salt solution Tissue for which it is used

Ringer lock’s solution Isolated rabbit heart

Frogs ringer’s solution Frog’s rectus abdominal muscle and leech dorsalis muscle preparation.

Tyrode solution Rabbit intestine and guinea pig ileum

De-Jalon’s solution Rat uterus, duodenum and colon experiments

Kreb Henseleit solution: Guinea Pig tracheal chain preparation and rabbit aortic strip preparation
 Precautions to produce PSS
• Calcium chloride is added at last in the form of solution in order to prevent the precipitation of bicarbonate and isolated tissue

will not live for extended period in cloudy PSS.

• Cloudy PSS also gives erratic response with drugs.

• PSS should be prepared with pure material.

• Quantity of all ingredient must be carefully weighed before preparing PSS

• Ph of PSS varying from 7.3-7.8 depending on organ.

• While taking response the temperature of PSS must be controlled as per requirement. Temperature must be 37 0 C

• Aeration must be given to PSS for tissue survival and response recording.
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