Practical Manual of Experimental

and Clinical Pharmacology

Practical Manual of Experimental
and Clinical Pharmacology

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

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

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Practical Manual of Experimental and Clinical Pharmacology

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

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

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

First Edition: 2010

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

Printed at Ajanta Offset

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

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

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

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

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

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

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

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

PART 3: EXPERIMENTAL (IN VIVO STUDIES)

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

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

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

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

PART 4: CLINICAL EXPERIMENTS

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

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

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

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

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