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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
Part 1
General Considerations in
Experimental Pharmacology
Introduction to Experimental
1 Pharmacology
EXPERIMENTAL BACKGROUND traditional drugs used in their own practices in

the treatment of several diseases. Thus, ‘materia
Drugs and their use were well started in pre- medica’ the science of drug preparation and
historic era and even the beneficial or toxic effects therapeutic use of drugs began to develop as the
of many plants and animal sources were recog- part of pharmacology. But, limitation in use was
nized. In ancient time, India and China enlist lack of methods for purifying active agents from
many types of remedies, out of which a few are the crude materials and methods for testing
recognized till today as useful drugs. Before 1693, hypotheses.
“Pharmacology” word was not coined but in the In the late 18th and early 19th centuries, François
year 1693 “Samuel Dale” published the 1st edition Magendie and later his student Claude Bernard
of his book entitled as ‘Pharmacologia sen began to develop the methods of experimental
Manuduction ad Materiam Medicam’. 2500BC, animal physiology and pharmacology and was
there were tremendous attempts to introduce recognized as father of physiology and prince of
rational methods of experimentation into drug vivisectors. Antoine Lavoisier proved that
discovery and research, but the limitation of respiration was a form of combustion by using a
thought; which proposed to explain disease guinea pig and Stephen Hales measured blood
without the need for experimentation and obser- pressure in the horse in the same era. In the 1880s,
vation was dominated. The oldest belief was that, Louis Pasteur demonstrated the germ theory of
India and China dominated in the ancient medicine by giving anthrax to sheep. Furthermore,
therapeutics. Indian contribution is with advances in chemistry and development of
“Rigveda” (2500-3000 BC) which is one of the physiology in the 18th, 19th, and early 20th
ancient preparation compendium, followed by centuries contributed to the foundation needed
“Charaka Samhita” written by physician for basic understanding, how drug works at the
“Charaka” and renowned surgeon “Sushruta” organ and tissue levels?
described various preparations of different Real advances in the basic pharmacology
sources. “Pan Tsao” a Materia Medica of China during this time were mainly promoted by the
has contributed of many preparations from the private manufacturers and marketers. But, the
animal, plant or mineral origins. concept of rational therapeutics was started with
The concept of pharmacology began in the randomized controlled clinical trial (compa-
17thcentury, when observation and experi- rative study of drug action in human) which were
mentation began to replace traditional drug use. reintroduced into drug development about 50
Many physicians from Great Britain and of this years ago that it became possible to accurately
continent understood the values of experimen- evaluate therapeutic drug. At the same time major
tation when they applied them to the effects of expansion of research was taken into all the
4  Practical Manual of Experimental and Clinical Pharmacology
biological fields. As new concepts and new particles, nanotubes, micelles, liposomes,
techniques were introduced, information quantum dots, dendrimers, fullerenes, and
accumulated about drug action and their target hydrogels have been developed for the more
on receptor at more molecular level. So, during targeted disease therapy in CVS, CNS, GIT,
the last half-century, many fundamental new chemotherapy, etc.
drug groups and new members of old groups were So, based on previous studies and experience,
introduced. pharmacological products are broadly governed
The studies of the molecular basis for drug by two principles;
action have been seen in last three decades due to 1. All therapies for health should meet the same
rapid growth of information and understanding standards of evidence of efficacy and safety in
in the concerned area. The molecular mechanisms preclinical and clinical studies and
of action of many drugs have now been identified; 2. Substances used as drugs can be toxic under
numerous receptors have been isolated, struc- certain conditions
turally characterized, and cloned. In fact, the use
Pioneers of Pharmacology
of receptor identification methods has led to the
discovery of many orphan receptors for which no • Father of Modern Experimental Medicine
Claude Bernard (1813-1878)
ligand has been discovered. • Father of American Pharmacology
Genetic studies such as, decoding of the John Jacob Abel (1857-1938)
genomes of many species from bacteria to humans • Father of Indian Pharmacology
have led to an emerging area of unsuspected Ram Nath Chopra (1882-1973)
• Father of Clinical Pharmacology
relationships between receptor families. In the Lou Lasagna (1923-2003)
present scenario, pharmacogenomics reveals the • Father of Modern Medicine
relation of the individual’s genetic makeup to his Hippocrates (460 BC-370 BC)
or her response to specific drugs. It helps to • Father of Modern Pharmacology
Oswald Schmiedeberg (1838-1921)
understand individual’s inherited abnormality in • Father of Modern Chemotherapy
their DNA, so it is now possible in the case of Paul Ehrlich (1854-1915)
some inherited diseases to define exactly which
DNA base pairs are abnormal and in which Claude Bernard
chromosome they appear thus making it easy to (July 12, 1813 – February 10, 1878)
target the disease progression.
A French physiologist recognized for his three
One of the most powerful new genetic tech-
major works:
nique is the ability to produce transgenic animals
(e.g. mice) in which the gene for the receptor or its 1. He worked on the functions of the pancreas
gland, and postulated that the pancreatic juice
endogenous ligand has been “knocked out,” i.e.
has great significance in digestion. For this
mutated so that the gene product is absent or
work, he won a prize for experimental
nonfunctional. Homozygous “knock out” mice
physiology from the French Academy of
have complete suppression of that function, while
Sciences.
heterozygous animals have partial suppression.
2. He investigated glycogenic function of the liver.
On the other hand “knock in” mice have been bred
that can over express certain receptors of interest. 3. He established the existence of vasomotor
system.
In last two decades, many nanomedicines like
polymeric nanoparticles, biochips, nanosensors, Note: Claude Bernard is known as the father of
bioreactors, neural stem cells, immune nano- physiology and prince of vivisectors.
Introduction to Experimental Pharmacology  5
John Jacob Abel same year appointed as assistant to

(May 19, 1857 – May 26, 1938) Professor Frerichs at the Berlin Medical
1883 : Ph.D from the University of Michigan. Clinic.
1891 : Founded and chaired the first depart- 1882 : Ehrlich published his method of staining
ment of pharmacology in the United the tubercle bacillus that Koch had
States at the University of Michigan. discovered and also derived the Gram
1893 : Chaired the pharmacology department method of staining bacteria, so much used
at Johns Hopkins University by modern bacteriologists. He has
1897 : Second to isolate epinephrine (First was become Professor.
Napoleon Cybulski in 1895) 1890 : Appointed as one of assistants of Robert
1914 : Isolated amino acids from the blood Koch, Director of the newly established
1926 : Reported the isolation and crystal- Institute for Infectious Diseases where
lization of insulin Ehrlich began the immunological studies
1896 : Appointed as Director of Institute for the
Note: Formulated the idea of the artificial kidney control of therapeutic sera, Steglitz, Berlin
1897 : Appointed as Public Health Officer at
Ram Nath Chopra Frankfurt-am-Main
(August 17, 1882 - June 13, 1973) 1899 : Became Director of the Royal Institute of
1908 : Obtained MD degree from Cambridge Experimental Therapy, Frankfurt, where
University he devoted himself to chemotherapy
1921 : Appointed as the first Professor of 1904 : Ehrlich became honorary Professor of
Pharmacology in the newly established the University of Göttingen, Germany
Calcutta School of Tropical Medicine 1906 : Received Prize of Honor at the XVth
and parallely headed the Department of International Congress of Medicine at
Pharmacology at the Calcutta Medical Lisbon and discovered the structural
College formula of atoxyl, a chemical compound
able to treat sleeping sickness
1941-1957
1908 : Shared Nobel Prize with Metchnikoff the
: Appointed as Director of the Drug
highest scientific distinction
Research Laboratory at Srinagar 1958
1909 : He and his student Sahachiro Hata
onwards; Honorary Scientific Advisor to
developed Salvarsan, a treatment
the Regional Research Laboratory,
effective against syphilis
Jammu
1911 : Liebig Medal of the German Chemical
He first introduced and done systematic study
Society
of Rauwolfia serpentina and had a major
1914 : The Cameron Prize of Edinburgh
contribution in establishing the first National Drug
Research Institute of India, Lucknow (Presently, Note: He coined the term “chemotherapy” and
known as Central Drug Research Institute (CDRI)). popularized the concept of “magic bullet”. He is
He pioneered research on herbal drugs in India. credited with the first empirical observation of the
blood-brain barrier and the development of the
Note: Indian Posts and Telegraphs Department first antibacterial drug in modern medicine.
has issued a commemorative stamp in his honor.
DRUG DEVELOPMENT AND USE OF
Paul Ehrlich ANIMALS: AN OVERVIEW
(March 14, 1854 – August 20, 1915)
1878 : Obtained his Doctor of Medicine (MD); Drug development mainly deals with three stages:
Dissertation on the theory and practice Stage I: Hit and lead compound development
of staining animal tissues and in the phase (Identification of lead compound amongst
the million compounds and selection for further The objective of the lead optimization phase
study) is to identify one or more drug candidates suitable
Stage II: Preclinical studies (Done in form of for further development. Thereafter, the drug
in vitro and in vivo animal experiments) enters into the stage II (preclinical studies),
which involves pharmacodynamics, pharma-
Stage III: Clinical studies (Experiment in human:
cokinetics and toxicity studies (acute toxicity,
Clinical trial Phase 0, I, II, III, IV and V)
chronic toxicity, genotoxicity, etc.) in animals
The process of finding a new drug for a
particular disease, usually involves several steps as well as receptor identification and
beforehand. The discovery of drugs, mainly characterization studies. The aim of the
involves in vitro as well as in vivo studies in preclinical study is to find out the maximum
different species. In late 18th century, pharma- recommended starting dose (MRSD) for the
ceutical companies played an important role in human. This is done by several in vitro (cell line,
research and development (R & D) and due to enzyme inhibition, etc.) and in vivo (animal
their focused and planned activity in the drug model) studies. In stage III, after getting the
discovery R & D grew rapidly. MRSD, dose can be extrapolated and calculated
In the first stage of drug development, target for the safe starting dose (SSD) with application
identification is the most important step. It saves of safety factor. After the selection of SSD,
lots of time. Targeted chemical moiety identification clinical trial begins with the healthy volunteers
also makes some sense. After the target (Phase I), But, there are few exceptions for the
identification, next step is to find lead compounds. Phase I study, in which patients are preferred
Lead compound is identified by the cloning of the instead of healthy volunteers such as cancer,
target protein. Amongst several species sequences, HIV, cystic fibrosis, etc. The complete steps of
human sequence is preferred due to the species to drug development are summarized in the Table
species variation. There is specific lengthy protocol 1.1 and Figure 1.1.
for the development of lead compound, which is beyond
the limit of this chapter. Briefly, cloning of the target
protein, identification of the functional activity of COMMONLY USED EXPERIMENTAL
target protein, combinatorial chemistry study and ANIMALS
high throughput screening (HTS) or ultra high-
throughput screening (UHTS) are mainly involved Selection of an animal model is one of the most
in the identification of hit compound and then lead important steps in any of the experimental
compound for the further stage. After the lead pharmacological study. Animal model preferred
compound identification, the next step is lead for the study must be producing similar disease
compound optimization, which mainly involves
profile as in the human. Hence, suitable animal
increasing the potency of the compound with
model should be selected which follows three main
respect to selectivity, metabolic stability,
objectives:
pharmacokinetics and toxicological effects on its
selected target. 1. Use of an animal phylogenetically closer to
man or
Note: High-throughput screening (HTS) involves assays
performed in a parallel fashion using multi-well assay plates 2. Use of an animal in which the process under
of 96, 384 and above, whereas ultra high-throughput investigation is as close as possible to that in
screening (UHTS) is performed using single-digit microliter
to nanoliter scale or 1536 well plate and more. UHTS is
man,
considered to be more sensitive and can screen more 3. The Anatomy, Physiology and Biochemistry
compounds as compared to the HTS. are considered to be similar
Table 1.1: Drug discovery timeframe and compounds screened

Duration Events during the period Compounds Screened *
(years)*
0-1 Identify targeted disease, budget, research team, drug design,
devise assays and select ‘hits’
1-2 Establish utility of ‘hits’ in animals
2-5 Identify promising ‘hits’; then identify lead compound and optimize
it with (literature, potency, acute toxicity, ADME, synthesis,
feasibility, etc.)
4-5 Patent protection
4-9 IND application, Phase 0, I to III clinical trials
8-11 Regulatory review
10-15 NDA application, Marketing and phase IV and V
15-20 Patent protection expires
* Years and compounds screened are approximate values which may vary with drug to drug
Roughly about $850 Million are required to develop a drug
Note: In the present scenario due to use of high throughput or ultra high throughput screening drug development
timeframe is significantly reduced with better output.
Fig. 1.1: Summary of developmental stages of a drug. Divided into the three stages of drug development includes lead
compound identification and optimization, preclinical studies and clinical studies. (IND= Investigational New Drug; NDA=
New Drug Application; ANDA= Abbreviated New Drug Application)
Broadly experimental animals are divided into have spreaded to several countries. DBA/2 is the
three categories. most ancient of all in bred strains, established by
CC Little in 1909 whereas C57BL/6 was
established by Miss Lathrop after 10 years. Other
commonly used strains C3H, CBA and A were
discovered by LC Strong.
The ability to add and selectively alter the mouse
genome is the potential tool for understanding the
genetic basis of human health and disease. Due to
the large similarity in mice and human genome
(>99% conserved), it provides good model for
research not only on mammalian biology but also
Mouse as an Experimental Animal on a wide variety of human diseases like, cancer,
(Mus musculus) diabetes, ageing, atherosclerosis, immunological
disease, autoimmune disorders, neurological
dysfunction and other endocrine diseases and
several other diseases. Inbred and mutant mice are
ubiquitously accepted as the preferred models for
identifying and understanding inherited human
diseases. Most importantly, its selection as the first
model animal to has its genome sequenced in the Human
Genome.
Knockout and Knock in mice have been
developed for the selective assessment at the
genetic level. In the Knockout mice, selective gene
According to paleontological data, men and mice
is taken out whereas in Knock in mice gene of
have been in contact since the early “Pleistocene”
interest is introduced into the mice. The first
(From two million to 11 thousand years ago). In
knockout mouse was created by Mario R Capecchi,
the biomedical research mice are preferred which Martin Evans and Oliver Smithies in 1989, for
are the smallest rodents used in the laboratory. which they were awarded the Nobel Prize for
They have several advantages over other species, Medicine in 2007. In 2006, Nobel Prize was
e.g. they are easy to keep, handle and require small awarded to Andrew Z. Fire and Craig C Mello for
place for housing. RNA interference, in which genes are silenced or
Mice proved as an invaluable resource in “knocked down” by short pieces of double-
identifying the several alleles which further stranded RNA.
develop in the research of mutagenesis. Mice are
the only known species in which it is possible to Region and Species Found
grow totipotent embryonic stem (ES) cells in vitro, • Asian species: Mus cervicolar, Mus cookee, Mus
and can form germ line once re-injected into a caroli, Mus famulus (India) and Mus fragilicauda
developing embryo. (Thailand-latest strain)
• Western Mediterranean region: Mus spretus
Mice (subgenus Mus) comprise several species • Eastern Mediterranean and Central Europe: Mus
that are similar in size and shape but never spicilegus, Mus macedonicus
hybridize in wild. Region to region, its species • Western Europe and Africa: Mus domesticus
varies which is described in the box below. Mus • India, Eastern Europe, Japan, Russia, Northern
China: Mus musculus
musculus has Indian subcontinent origin but now
 Important Points to Remember  Important Points to Remember

• Smallest laboratory animal • Albino (subgroup, Wistar and Sprague Dawley (SD)
• Common strain Swiss albino mice rat)
• Nude mice: Hairless genetic mutant which lacks • Wistar rat: wide head and the ear is long whereas tail
thymus gland length is less than the body length
• Biege mice: Lack natural killer cells and are susceptible • SD rats: longer and narrow head. Tail is longer than the
to cancer body length
• Mice use their tail to help in thermoregulation • Do not vomit (due to strong sphincter between the
stomach and the esophagus and lack vomiting center)
Rat as an Experimental Animal • Do not have tonsil and gallbladder
(Rattus norvegicus) • Diffuse pancreas, so not a good model for type I diabetes
• Coprophagy (eating their own stool)
• Tail of rat helps in thermoregulation of body
Note: Some important phenotypic differences

between baby rats and mice
1. Baby rat has blunt and broad large head
relative to body whereas mice have triangular,
small head relative to body
2. Baby rat has small ears relative to the head
whereas mice have large ears
Rat is the most commonly used animal in the 3. In the baby rat hind paw and body ratio is
biomedical research. The randomly bred strains larger as compared to mice
are used almost exclusively and are derived from 4. Tail is thick and shorter than body length in
the Norway rat (Rattus norvegicus) which is baby rat while mice have thin and larger or
thought to have originated in the area between same length tail as compared to body
the Caspian Sea and Tobolsk. Among these,
“Wistar rat” and the “Sprague Dawley rat” are Guinea Pig (GP) as an Experimental Animal
preferred because of easy handling, sensitivity and (Cavia porcellus)
low cost. The Albino rat (officially known as the
Pink-Eyed White or PEW) is most likely the very
first mutation to be discovered and purposely bred.
Albino rats were introduced to Great Britain by a
travelling entertainer around the year 1800.
Nude rats resemble nude mice in their lack of a
normal thymus and functionally mature T cells
and are phenotypically hairless with possible
fine-sparse hair growth and most preferred model
in immunological research.
Rat is preferably used in the research of About 400 years ago, Guinea pig (Cavia porcellus)
behavior, pharmacology, physiology, neuro- was introduced into Europe from South America.
sciences, immunogenetics, transplantation, There are 3 major varieties of strains used in the
cancer risk assessment, cardiovascular diseases experimental studies and is the member of rodents
and aging. Further, development and genetic suborder “Hystricomorpha”. Guinea pig is
characterization of inbred, congenic, and herbivorous and eats green foods, seeds and roots,
recombinant strains took place in the United States, but now in many laboratories feed is provided
Japan and other European countries. with a readymade chow diet which fulfills its daily
dietary requirement. But, it is essential to add

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

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

• Uterus resembles humans and exhibiting regular
menstrual periods
• Metabolism approximately same as human
• Ideal model for pharmacokinetic study
• Best for studying drugs acting on CNS (memory, anxiety,
antidepressants, etc.), CVS (antihypertensive,
antianginal, etc.), GIT and fertility
Mammalians are used widely in the biomedical • Require regular check up for rabies, tuberculosis and
research due to the anatomical and physiological timely immunization
Dog as an Experimental Animal

(Canis familiaris)
or rodent, hence they have been extensively used

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

(Felis catus)
Anatomical and physiological similarities
Cat has similar physiological features which are between man and pig makes this animal a good
common with humans than the laboratory rabbit model in many research areas including
pharmacological and toxicological research. fruit flies and Caenorhabditis elegans: nematodes)
Several isolated organ models, investigation of which do not have backbones. Nowadays,
skin permeation and for digestive systems, etc. studying embryo development and genetics make
are few important areas of research with pig as an it useful animal model as in genetic techniques. It
experimental animal. This is preferred because, it is getting popularity into the biomedical research
is small sized at maturation as well as is selecting due to its clear eggs which can be developed
for less hair contains on the body. In Europe, pigs outside the mother’s body and allow watching a
are used in pharmaceutical R & D studies in place zebra fish egg grow into a newly formed fish under
of dogs and primates. a microscope in 2-4 days. The lifespan of zebra
Dissimilarities are in the vascularization (rich fish is considered to be approximately 5 years and
in man, poor in the pig) and in the sebaceous length of the adult fish is about 6 cm.
glands. Humans have mostly eccrine sweat The advantages of this model is that it can be
glands over the body surface, whereas the pig has kept at fairly high densities in a small tank, so it is
only apocrine glands. Similar findings also cheaper to maintain than other experimental
observed while studying the skin of other domestic animals, a single spawning produces 100-200 eggs
mammals. Pig has small lungs in relation to body which are easily collectible, development of eggs is
size and is susceptible to bronchitis and clear and easily observed and manipulated
pneumonia. whereas drawbacks are that it requires an
aquarium to maintain, not closely related to humans
 Important Points to Remember as a mouse or other animal model (not mammalian)
• Alimentary tract resembles human and genetic modification has not been possible as
• Important animal model in cardiovascular research it is in the mouse (Knock out/Knock in).
such as atherosclerosis, MI, etc.
Frog (Rana tigrina)
Zebra Fish (Danio rerio)
Frog is the only tail less experimental amphibian

which is used very commonly in the biomedical
Dr George Streisinger at the University of Oregon research. They are mainly characterized by long
observed that the Zebra fish is a suitable model hind legs, a short oval body, webbed fingers or
for studying vertebrate development and genetics toes and projected eyes. Generally, frogs and toads
in early 1970’s and is considered to be the “Father are two characterized amphibians and have no
of Zebra fish research”. The zebra fish is native to taxonomic basis to make difference, but can be
the freshwater streams of the southeastern differentiated by their appearance. (Toads are dry
Himalayan region such as Eastern India and whereas frog is wet). Anatomically their heart
Pakistan, Bangladesh, Nepal, and Myanmar. contains three chambers which is different from the
Zebra fish are vertebrates and have a backbone other mammals used in the experiment. Barbouruta
like humans which gives more close relation to kalimantanensis (Flat headed Frog) is the first
humans than commonly used invertebrate species of frog which is found without lungs. Some
models, such as insects and worms (Drosophila- frog species like Xenopus laevis was first widely
used in laboratories in pregnancy assays. The were domesticated in India about 2000 BC and
relationship between electricity and the nervous then introduced to Japan via Korea about 300 BC.
system were well studied in frogs by biologist Chickens can be easily bred and housed. So, they
Luigi Galvani. Frogs are generally used in the field are being increasingly used as experimental
of CVS and CNS research, also used in cloning animals. The digestive tract of the chicken is
research and other branches of embryology simple, relatively short and highly efficient.
because frogs are among the closest living relatives The use of chicken as an experimental animal
of man who lack egg shells. Some hybridized frogs was started since the 1960s. They are being tested
are also used in biomedical research such as in many areas of biomedical research such as
Edible Frog (Rana esculenta), which is a hybrid of breeding and genetics, growth, performance
the Pool Frog (R.lessonae) and the Marsh Frog (R. testing, embryology, incubation, fertility, artificial
ridibunda). insemination, hatchability, anatomy, toxicology
There are several important substances like and pharmacology, behavior and welfare,
epibatidine (an alkaloid), a painkiller 200 times physiology, biochemistry, endocrinology and
more potent than morphine and other toxins like neurobiology. Some of the important models
irritants, hallucinogens, convulsants, nerve developed in the chicken are chick comb method,
poisons, and vasoconstrictors which are obtained chicken blood pressure measurement, heart rate,
from different species of frog. Other chemicals EEG and activity through telemetric system,
isolated from the skin of frogs may offer resistance Scleroderma model in chicken, measurement of
to HIV infection which is considered to be a good contractile force of isolated cardiac myocytes, and
target for research. vasodilating activity of chicken. Other few system
involved in the research of chick are angiogenesis
 Important Points to Remember (chick chorioallantoic membrane assay: to test
• Oxygen can pass through their highly permeable skin angiogenesis and inhibition of angiogenesis),
and hence “breathe” largely through their skin catalepsy antagonism in chicken (white Leghorn),
• Camouflage is a common defensive mechanism in frogs in learning and memory (Aversive discrimination
(hiding or color change) in chickens) and model for spontaneous
• Very commonly used in the CVS related experiments
autoimmune thyroiditis (Obese strain chicken: OS
or bioassays
chicken, e.g. UCD-200 chickens). At the finale, the
Chicken (Gallus domesticus) only model for studying Avian diseases.
Pigeon (Columbia livia)
In the experimental pharmacology, chicken are

widely in use due to the ease of availability and Pigeons constitute the family Columbidae within
its organs. The maintenance is quite economical. the order Columbiformes, which include some 300
The room temperature should be around 23°C species. In general, the terms “dove” and “pigeon”
which is achieved by reducing the temperature are used interchangeably. Pigeon is survived on
from 32-33°C (1°-2°C per week) until a temperature seeds, fruit and plants. Pigeons have strong wing
of 23°C is obtained. It is thought that chickens muscles comprises 31-44% of their total body
weight and are amongst the strongest fliers of all whole body grooming. For example: rat, mice,
birds. guinea pig, gerbil, hamster, dog, etc.
Pigeons are mainly preferred in screening Barber: This is the extent of grooming in which
antiemetic activity, cardiovascular diseases such the fur is nibbled off. Animal may barber
as spontaneous arteriosclerosis in pigeons and themselves or another animal. For example: rat,
standardization of cardiac glycosides, CNS such mice, hamster, dog, etc.
as anxiety pigeon method, apomorphine induces Behavioral estrus: It is one type of reproductive
stereotypic behavior in pigeons and screening of behavior in which female entreats male to grab
intravenous anesthetics in pigeon. Bioassay of her, occurs mainly in the vaginal proestrus (12
prolactin through the pigeon crop method is one hour period before ovulation).
of the important methods in prolactin assessment. Box behavior: One type of play or defense behavior
Note: General information of laboratory animals in which two rats stands on their hind legs/paws
and their vital parameters are explained in Tables facing each other nearly nose to nose, and pushes
1.2 and 1.3. or keep paw at each other with their front legs/
paws.
ANIMAL BEHAVIOR AND TERMINOLOGY Chase or pursuit: Animals pursue behavior in
which an animal runs after another animal
Animal behavior: It is defined as the behavioral Chatter: Repetitive grinding of the incisors against
reaction of an animal to any internal or external each other
stimuli. Cannibalism: The act of killing and eating of its
Abnormal appearance: It involves the postures like own species.
head down, tucked abdomen, hunched back, facial Coprophagy: Reingestion of feces by the animal
distortion, or pallor of an animal. Ear wiggle: Female vibrates her ears rapidly in
Allogroom: Rapid little nibbles like activity of one behavioral estrus (about every 4-5 days) to solicit
animal to another, it is head or neck grooming or and maintain mounting behavior by the male.
Table 1.2: General information of laboratory animals
Animal Weight(g) Lifespan Floor space*/ Food intake Water intake Gestation Animal House
(Year) animal (g/day) (ml/day) period (Days) Temperature Humidity
(cm2 × h (cm) (°C) (%)
Mouse 18-40 1.5-3 77.4 × 12 3-5 6-7 19-21 19-23 40-70

Rat 150-400 2-4 187.0 × 14 5-10/100g 10/100g 21-23 19-23 40-70
Hamster 85-150 1-3 103.2 × 12 5-7 10/100g 15.5-16 19-23 40-60
Gerbil 55-100 2-4 103.2 × 12 5-8 4-7 24-26 19-23 30-50
Guinea pig 600-1200 4-8 651.4 × 18 6/100 g 10 /100 g 59-72 18-26 40-70
Rabbit 1000-3500 6-12 270 × 35.5 5/100g 10 /100 g 31-32 16-20 40-60
Chicken 1000-3500 3-5 30.48 × 60.96 125-250 200-300 20-22 18-26 40-70
* Floor space × height (h) is given as per adult animal requirement for housing in centimeter of the cage.
Table 1.3: Vital parameters of different experimental animals

Animal Rectal Heart Blood pressure (mmHg) Blood volume Respiratory Tidal
temperature (°C) rate (min) Systole Diastole (ml/Kg) rate (min) volume (ml)
Mouse 38-39 310-840 133-160 90-110 58.5 60-220 0.18
Rat 36-40 250-450 84-134 60 54-70 70-115 0.6-2
Hamster 37-38 250-500 150 100 78 35-135 0.6-1.4
Gerbil 37-38.5 360 — — 66-78 90 —
Guinea pig 37.2-40 230-380 80-94 55-58 69-75 42-104 2.3-5.3
Rabbit 38.5-40 130-325 90-130 60-90 57-65 30-60 4-6
Flank mark: It is a scent marking olfactory behavior Pup retrieval: Carrying behavior of mother animal.
which is left on the objects in the surroundings by They keep rambling pups in her mouth with its back
rubbing their flanks. For example: rat, mice, dog, etc. to targeted place. For example: rat, cat, dog, etc.
Gallop: Movement of both limbs (left and right) Recumbency: Unusual length of time for pain as
nearly in synchrony. The gallop is fast and symptom.
asymmetric movement at the time of free-flight Sniff: It is an exploratory behavior which involves
(all four limbs off the ground) whereas trot is
cluster of movement sequences with snout and
the movement in which diagonal pairs of legs
head.
move in synchrony (left front and right hind
paw) Solicitation: One of the sexual behaviors of female
in which female skims towards male and runs a
Infanticide: Killing the young one’s of their own
short distance, then wait for a while. “Full
species. For example: rat, mice, guinea pig, etc
solicitation” in which she repeats the same activity,
Lordosis: Female mating posture which is a whereas “partial solicitation” in which she does it
reflexive behavior that is triggered by a touch once
on the lower back, flanks, or genital region.
Squeak: Vocalization response of rodents in pain
Vulva, which normally faces the floor, rotates
or any head grooming and mild social interactions
almost 90° to the vertical, i.e. to backward facing
position. Vocalization: Crying out sound, when palpated
or given some painful stimuli
Mount: This is the male copulatory position, and
is seen when a male mounts a female prior to
ANIMAL CARE, HANDLING AND SEX
mating.
DETERMINATION
Mutilation: Symptom of pain including, licking,
biting, scratching, shaking, or rubbing. Handling and restraining of experimental animals
is an important aspect in practical pharmacology,
Nibble: Rats may nibble their own skin or that of
which includes transferring the cage, feeding,
other rats with their teeth, appear to be combing
treatment (gavages or parenteral) or any other
the fur with their teeth and nibbling the skin
procedure with the animals. In India CPCSEA has
underneath.
given complete guideline for animal caring,
Nip: Light pinching activity which may elicit a housing and handling requirements. As per GLP,
squeak with the teeth whereas skin remains handling the commonly used rodents, there are
unbroken. several points to be keep in mind which will limit
Peep: Brief high pitched sound heard during head the unnecessary stress or strain to the animals.
and body allogrooming.
Precaution to be taken before
Pica: The nausea response of rat called “Pica” handling the animals
but rat cannot vomit (lacks vomiting center).
• Before restrain, first pet or soothe the animal
Piloerection: This is phenomenon in cold, or stress by slow deliberate movements on their body
in which body hair rises or erect on end. • Overcrowding near the animal cage should
Popcorning: Animal show this behavior during be avoided
play or happy moments, they run-around and • Noise should be kept to a minimum as much
jump into the air and land on all fours paws. For as possible
example: Baby guinea pig, dog, cat, etc. • Don’t hold animal too hard, it may face
difficulty in breathing and may die too
Poofing: see Piloerection • Never agitate the animal, it may become violent
Pup-killing: See infanticide for self protection.
Mouse Way 3: Hold the complete body by grabbing back

of the neck by using all fingers. (Forceful gripping
This is the smallest animal in the laboratory and
should be avoided) (Fig. 1.4).
used very commonly.
Way 1: One can handle it with the help of blunt
forceps by grasping the skin behind the neck/
body. This technique is often used to transfer mice
from one cage to another. (Should not hold too
hard, it may injure the tissue) (Fig. 1.2).
Fig. 1.4: Mouse handling technique (way 3)
Rat
Way 1: Lift rat out of the cage by grasping the base
Fig. 1.2: Mouse handling technique (way 1)
of the tail and place on a soft surface. (Hard smooth
surfaces can make the rat tense) (Fig. 1.5).
Way 2: Grasp the base of the tail with one hand
and with the other grasp the loose skin behind its
neck (Fig. 1.3)
Fig. 1.3: Mouse handling technique (way 2) Fig. 1.5: Rat handling technique (way 1)
Way 2 (A and B): Place your index and middle

fingers (way 2A) alongside the rat’s head and your
thumb and ring fingers under its forelegs (way 2B).
Use your index and middle fingers to secure its
head and the remaining fingers to support the body
(Figs 1.6A and B).
Fig. 1.7: Rat handling technique (way 3)

(For color version see Plate 1)
Figs 1.6A and B: Rat handling technique (way 2)

(For color version of Figure 1.6A, see Plate 1)
Way 3: Hold the complete body by grabbing the

back by using complete palm (Fig. 1.7).
Guinea Pig
These are very humble rodents and can be easily
handled because of their docile nature
Way 1(A and B): By using both hands, calmly
grasp it with one hand under the chest and use B
your other hand to support its hindquarters (Figs Figs 1.8A and B: Guinea pig handling technique
1.8A and B) (way 1A and B)
Way 2: Handle guinea pig with one hand, by

holding its hind quarter (Fig. 1.9).
Fig. 1.9: Guinea pig handling technique (way 2)

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

Way 2: Hold the complete body by grabbing back

by using complete palm (Fig. 1.11). Fig. 1.12: Hamster handling technique (way 3)
Rabbit
Way 1: By using single hand, hold the pelvic
region. This technique is mainly used to transfer
rabbits from one cage to another (Fig. 1.13)
Fig. 1.15: Rabbit handling technique (way 3)
Sex Determination in Rodents

Mouse/ Rat
Direction 1: Restrain the mouse/rat and lift the base
of the tail. Sex is most easily determined by anogenital
Fig. 1.13: Rabbit handling technique (way 1)
distance. Males normally have a greater distance
between the anus and urogenital openings. Male mice
Way 2: By using both the hands, hold the complete
also have a larger genital papilla (Fig. 1.16).
hindquarter (Fig. 1.14).
Direction 2: Scrotum can be easily palpable in male
Note: In winter scrotum shrinks inside the body,
hence direction 2 should be followed carefully in
winter.
Guinea Pig
Both male and female guinea pigs display similar
anogenital distances.
Direction 1: The female has detectable pink nipple
at either side, a separate urethal orifice, a vaginal
membrane, a perineal sac, and an anus whereas
male has a penis, a larger perineal sac, and an
anus (Fig. 1.17).
Direction 2: The penis lies just under the skin and
can be inverted with gentle pressure. The testes
Fig. 1.14: Rabbit handling technique (way 2) and penis are palpable in adults.
DIET AND EXPERIMENTAL ANIMALS

Way 3: By using the both hands, calmly grasp it
with one hand supporting back of neck and the The influence of the diet is directly on the health
other hand supporting its hindquarters (Fig. 1.15). of any animal or human. Diet is important in order
Fig. 1.16: Gender identification in male/female Rat
Fig. 1.17: Gender identification in Guniea pig
to maintain health and energy. Main constituents develop the different models of the animals by
of the diet remain the same as for human, i.e. restricting or enhancing the constituents of diet.
proteins, carbohydrates, lipids, fibers, vitamins For example: atherosclerosis model, hypertension
and some ions and elements. Blood, urine model, diabetic model, pancreatitis model, etc.
concentration, pH and extent of ionization of Some species are very susceptible to the specific
compounds all depend on the diet of the type of diet, hence it can be used in the develop-
individual. In the experimental setting, one can ment of certain disease model such as rabbits are
susceptible to hypercholesterolemia and So, 0.25 mg is to be administered as per 10 gm

arteriosclerosis after excessive cholesterol feeding, of mice
pigeons develop spontaneous arteriosclerosis Suppose this has to be given in the volume of 0.1 ml
whereas hypercholesterolemia can be induced in Then, (1) will be written as
rats by daily administration of 1 ml/100 g body 0.25 mg/10 gm/0.1 ml (2)
weight of a cocktail containing in 1 ml peanut oil, Step 3: Now, weighing and dilution of the drug,
100 g cholesterol, 30 g propylthiouracil, and 100 From (2),
g cholic acid by gavages over a period of 7 days. = 0.25 mg/0.1 ml (multiply by 10)
All animal diets are tested in the government = 2.5 mg/1 ml
laboratory for their constituents (Proteins, = 25 mg/10 ml
carbohydrates, fibers, lipids, etc.) before supply to Hence, the solution should be prepared by
any institute or research center. adding 2.5 mg in 1ml or 25 mg in 10 ml of solvent,
which will give the dose of 0.25 mg/10 gm or
DOSE CALCULATION FOR 25 mg/kg of body weight.
EXPERIMENTAL ANIMALS
For Injectables
Sometimes pure powder of the drug is not
available, so student have to use the injectable
form or ampoule of the drug
Step 1: So, in this case two important things should
be kept in mind
Strength of the injection and its solubility in
the solvent
Suppose, morphine is available in the strength
of 15 mg/ml
Calculation 1 Students have to use this strength in rat at a
dose of 5 mg/kg, to show the analgesic activity of
For Powder Drugs
the drug.
One should follow the following steps given So, drug dose = 5 mg/kg
below, for calculating the doses and volume of = 5 mg/1000 gm
injection of any given drug, to be administered in = 0.5 mg/100 gm (for rat) (1)
the animal. That means, 0.5 mg of drug should be
administered in the 100 gm of body weight rat.
Step 1: Think of “What you have?” Suppose this is given in 0.1ml volume
Drug dose, weight of the animal and volume to be Then, (1) can be written as
injected 0.5 mg/100 gm/0.1 ml (2)
Suppose drug dose is 25 mg/kg for mice (Calculate Step 2:
the drug dose in the multiple of 10 gm in case of Dilution of injection, strength 15 mg/ml
mice or 100 gm in case of rat, guinea pig or rabbit) Put 1ml of drug in test tube and then add 2 ml
of solvent in the test tube (solution A)
Step2: Now, test tube contains 15 mg of drug in 3 ml
Hence, 25 mg/kg = 25 mg/1000g Hence, = 15 mg/3 ml
= 0.25 mg/10 gm (mice) (1) = 5 mg/1 ml (3)
= 2.5 mg/100 gm (rat and = 0.5 mg/0.1 ml
higher weight animal) [equivalent to equation (2)]
So, student can use directly 0.1 ml of the If concentration of solution 1 is given in
“solution A”, to provide 0.5 mg of drug in 100 gm mg/ml and the concentration of solution 2 is
of body weight rat. given in µg/ml, then, make both units same
(Annexure-VII).
Calculation 2
Calculation 3
This formula is applicable in most of the cases
and used very commonly, By applying the conversion factor
C1 × V1 = C2 × V2 Sometime, doses of a drug is not known for the

animals, then it may be converted to the respective
Where, animal drug dose by the help of conversion factors
C1 is the concentration of solution 1, developed according to the body surface area.
C2 is the concentration of solution 2, The steps involved are:
V1 is the volume of solution 1,
V2 is the volume of solution 2.
For example
Suppose, you are provided with the 50 ml solution 1. From human drug dose
of a 5 mg/ml concentration and the instructor Suppose, human dose of drug A = 100 mg/
wants you to make it, 10 ml of 3.5 mg/ml. kg/day [for the calculation, dose will be
So, you have calculated either for 20 gm mice or 200 gm rat
C1 = 5 mg/ml V1 = ? = A or 400 gm guinea pig or 1500 gm rabbit (as per
C2 = 3.5 mg/ml V2 = 10 ml animal selection)]
Step I: Dose given = 100 mg/kg
Apply the formula Step II: Convert into absolute dose*
Human dose given = 100 mg/kg = 70 × 100 =
C1 × V 1 = C2 × V2
7000 mg/70 kg
So, 5 mg/ml × A = 3.5 mg/ml × 10 ml
(Absolute dose is converted for 70 kg adult).
3.5 mg/ml × 10 ml * If, a adult dose is taken, then avoid step II.
Hence, A =
5 mg/ml For example, drug dose is 50 mg/day, then it
is assumed that, it is for 50 mg/70 kg of adult.
3.5 × 10 ml Note: Conversion of absolute dose is
= 7 ml
5 important because it keep the dose as per the
Therefore, take 7 ml of the solution 1 in a test body weight of animal/human to make easy
tube and make it volume to the 10 ml by the solvent. calculation such as convet dose mg/kg into
The resultant solution will be 10 ml of 3.5 mg/ml. the mg/70 kg for human, mg/1.5 kg for rabbit,
Note: The units of the solution concentration must mg/400 gm for guinea pig, mg/200 mg for rat
be same such as if it is in mg/ml then both solution and mg/20 mg for mice.
concentrations should be in mg/ml. Step III: Multiply by conversion factor
Step IV: Then, convert into per kg dose according ROUTES OF DRUG ADMINISTRATION IN
to animal EXPERIMENTAL ANIMALS
Mice: 18.2 mg/20 mg = 910 mg/kg/day Feeding or Oral Gavage (Figs 1.18A to E)
Rat: 126 mg/200 gm = 630 mg/kg/day Step I: Hold the rodent in a hand carefully
Guinea pig: 217 mg/400 gm = 542.5 mg/kg/ Step II: Measure the tube length from the nose to
day the last rib of the rodent and mark it
Rabbit: 490 mg/1500 gm = 326.66 mg/kg/day Step III: Give a gentle tight grip at the back of
2. From animal to animal drug dose neck, so that it opens its mouth widely (if possible,
Mice dose of drug A is 5 mg/kg, then, calculate use any hard wooden or plastic gag)
the drug dose for the other animals
Step IV: Push the rodent head slightly upward
The same as example 1 and back to straighten the esophagus and then
Step I: Dose given -= 5 mg/kg either from right or left side of teeth, insert the tube
Step II: Convert into absolute dose by gentle rotation to avoid the resistance (do not
For mice (20 mg) = 5 mg/kg = 0.1 mg/20 mg force the tube in esophagus, this may injure
Step III: Multiply by conversion factor mucous membrane).
Step IV: Then, convert into per kg dose according

to animal
From Step III
Rat: 0.7 mg/200 gm = 3.5 mg/kg/day
Guinea pig: 1.22 mg/400 gm = 1.83 mg/kg/day
A
Rabbit: 2.78 mg/1500 gm = 1.85 mg/kg/day
In the same pattern, if the rat dose is known then
other animal dose can be calculated by using the
above mentioned steps with conversion factor
given below:
• Rat (200 gm) to mice (20 gm) conversion factor
is 0.14
• Rat (200 gm) to guinea pig (400 gm) conversion
factor is 1.74
• Rat (200 gm) to rabbit (1500 gm) conversion
factor is 3.9
Note: After converting the drug dose to
corresponding animals, do as per Calculation 1 B
or Calculation 2 for the volume to be injected. Figs 1.18A and B
C
Figs 1.18A to C: Feeding or oral gavage to the rodent (A) Mice, (B) Rat, (C) Guinea pig
D E
Figs 1.18D and E: Feeding or oral gavage to the (D) Hamster, (E) Rabbit: with wooden gag
Step V: Slowly pass the tube observing for the • Sterile syringes and needles must be used for
swallowing reflex and when desired length of tube any type of injections
has been inserted, inject solution with the help of • Always select the smallest possible gauge (G)
syringe
needle to limit tissue trauma and injection
Note: Recommended in mice and rats but is not discomfort. For example: 25-27G needle is pre-
preferable in guinea pig because they have a small
ferred in adult mice for the injection in tail vein.
palatal ostium which gets easily damaged.
• Aspiration technique is always an important
Injection Site and Techniques aspect before pushing the injection solution at
Following precautions should be taken before the site.
injection:
• Injection sites should be cleaned with a Note: Aspiration is a technique of creating vacuum
suitable disinfectant/antiseptic (isopropyl at the site of injection by pulling piston back to
alcohol, ethanol, spirit, etc.) check the right placement of the needle.
Intraperitoneal (i.p) Injection (Figs 1.19 to 1.22)
Fig. 1.19: Mouse Fig. 1.21: Hamster
Fig. 1.20: Rat Fig. 1.22: Rabbit (But not preferred)

Intravenous (i.v) injection (Figs 1.23 and 1.24)
Fig. 1.23: Cross-section of tail vein and procedure of injection or blood withdrawal
Fig. 1.24: Rabbit marginal vein (Prefered route)
Intramuscular (i.m) Injection (Figs 1.25 and 1.26)
Fig. 1.25: Thigh (Mouse/Rat) Fig. 1.26: Gluteus (Rabbit)

Subcutaneous (s.c) Injection Nap of the Neck (Figs 1.27 to 1.31)
Fig. 1.27. Mouse (Nap of the neck) Fig. 1.28: Rat (Nap of the neck)
Fig. 1.29: Hamster (Nap of the neck) Fig. 1.30: Hamster (Flank)
Fig. 1.31: Rat (Flank)

Intracardiac Injection (Figs 1.32 and 1.33)
Fig. 1.32: Rat Fig. 1.33: Hamster
Note: Other less preferred routes are Intradermal includes handling and restraint, needle size, site and
or Intrathecal. location of the vein and dilation of the vein. Animal
should be restrained which make procedure easy
BLOOD COLLECTION FROM THE and comfortable for both animal and
EXPERIMENTAL ANIMALS experimenter. Sometimes use of anesthesia is
preferred in small animals compared to the larger
Blood/plasma/serum of animals is required for a animals. But, some reports suggest that use of
variety of analytical purposes such as to measure anesthetics may interfere with the hematological
the drug concentration in the pharmacokinetics
and biochemical parameters of the animal still in
study or for the estimation of different biochemical
some situations handling with anesthetic is
or hematological parameters of a study. So the
preferred at the time of blood withdrawal because
experimenter should know the proper technique
it determines the quality and accuracy of the blood
to withdraw the blood sample from the animals.
sample. For example: In stress catecholamine’s
Guideline says that always use the technique
level are increased in the blood which may be
which is causing less distress, pain and discomfort
reduced by the use of anesthetics. The general
to the animal. The collection technique of blood depends
principle for selection of needle size (i.e. length
on the three major objectives,
and bore) is determined by the diameter of the
vein, i.e. bore size selected should have less
diameter than the vein (needle diameter < vein
diameter). In the experimental studies needles
between 10-50 mm in length and 17-27G bore is
preferred for most of species. Meanwhile, dilation
of the vein can be facilitated by several means such
The principle for blood collection from veins as by anesthetic or warming or use of any irritant.
and arteries is almost the same, whereas arteries In conscious animals, however, blood can be more
are preferred only when a large sample has to be easily obtained if the animal is warmed first. Some
withdrawn. Moreover, this is rapid and relatively laboratories also preferred thermostatically warmed
easy. There are some basic principles for collecting box (e.g. made from perspex). Animals are kept at
blood from an experimental animal which 30° C for 10-15 min. (kept under constant
observation in order to prevent hyperthermia). then this requires little more blood. The amount of
Then, selection of the appropriate site is important blood withdrawn is mainly correlated to their body
because it makes the procedure easy and the weight and the total blood volume. Cannulation is done
amount of the blood required is easily obtained. for the multiple sampling which reduces the stress
and trauma to animals. The general information
Agents Used for Vasodilatation during for animal blood withdrawal is explained briefly
Blood Withdrawal in Table 1.4 and procedure for collection of blood
• Anesthetics sample is briefly given in Flow Chart 1.1.
• Dipping the tail of rodents into warm water (temperature
of around 45°C)
• Xylene (use with caution because it has carcinogenic
potency or may causes skin rashes ) and
• Fentanyl/fluanisone and acetyl promazine (mainly in
rabbits)
Volume of blood withdrawn depends on the Adverse Effects Experienced during Blood
analytical process to be used. For example: if it is Withdrawal
to be used in the latest instruments like, HPLC, Blood withdrawal is a technique which requires
PCR, spectrophotometer, ELISA kit, etc. then it is skill and experienced hand. Inexperienced hand
required only in micro liters (μl) and if it is to be and improper technique may induce thrombosis
used in the biochemical parameters assessment (clotting) and phlebitis (inflammation of the vein),
Flow Chart 1.1: Procedure for collection of blood sample
Caution: Sample taken too quickly or abrupt withdrawal of needle may collapse vein
Note: As a general rule, blood may be withdrawn up to 10% of total blood volume of animal (single
withdrawal) and 1% of total blood volume/24 hr for repeated withdrawal
Table 1.4: General information on blood withdrawal in animals

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

withdrawal sample/ 24hr Volume
Abdominal/thoracic 19-21G 1 Up to 15 ml Bruising, hemorrhage,

infection
Cardiac puncture 20-21G 1 1-25 ml ———————
Decapitation — 1 10-20 ml ———————

Rabbit Marginal ear vein/ 19-23G Up to 8 0.5-10 ml Bruising/hemorrhage,
artery infection
Cardiac puncture 19-21G 1 60-200 ml ———————
Cat Cephalic vein 21 or more G Up to 8 (each 2-5 ml Hemorrhage, infection,

leg 4 sample) swelling
Jugular vein 21G (1" long) Up to 8 2-20 ml Infection, hemorrhage and

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

Dog Cephalic vein 21 or more G Up to 8 (each 2-5 ml Hemorrhage, infection,
leg 4 sample) swelling
Jugular vein 21G (1" long) Up to 8 2-20 ml Bruising/hemorrhage,
infection
Cardiac puncture 16G(1.5"long) 1 Up to 900 ml ——————
Pig Cranial vena cava 19-21G- pig 1 in 7 days 5-30 ml Bruising/hemorrhage,

20-21G-minipig infection
Ear vein 21-23G Up to 8/24 hr 1-3 ml Bruising/hemorrhage/

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

Monkey Abdominal vena 19G 1 Up to 10 ml ——————
(Marmoset) cava (Terminal)
Cephalic vein, 1" 22 G Up to 8 (each 5 - 10 ml Bruising/hemorrhage,
leg 4 sample) Infection
Saphenous vein 22 G Up to 8 10% of BW Bruising/hemorrhage,
Infection
Femoral vein 22 G Up to 8 5 - 10 ml Infection, Bruising/
hemorrhage
Jugular vein 22 G Up to 8 5 - 10 ml Bruising/hemorrhage,
Infection
etc. Other adverse effects are hemorrhage, bruising, VARIABILITY OF DRUG RESPONSES IN
thrombosis and infection at the site of needle entry. EXPERIMENTAL ANIMALS
If any of the side effects occurs, then treatment
should be given from the assigned veterinary Species differences in drug sensitivity can often
surgeon. Hemorrhage is not common and can be be explained by differences in their pharma-
associated with the animal which have clotting cokinetic, including quantitative and qualitative
defect. Bruising is mainly due to subcutaneous differences in the ability to detoxify drugs. Sex and
bleeding at the time of venous puncture. age can also modulate the rates of absorption,
Table 1.5: Variability of drug responses

Drug Variable responses
Adrenaline Cat: Stimulates pregnant uterus but contracts non-pregnant uterus; Rabbit: stimulates uterus; Rat:
relax uterus
Alloxan Produces diabetes in most animals but not in guinea pig
Atropine Mice > cat > human > dog= rabbit (100 times of human dose) (sensitivity in order)
ATP Relaxes rabbit anococcygeus muscle whereas contracts rat anococcygeus muscle
Cardiac Rat heart is resistant
glycosides
Dopamine Shows fall in BP of dog (instead rise seen in man and other animal)
Histamine Cat: Produces Bronchodilation and pulmonary vasoconstriction (increases blood pressure); Guinea
pig and human: contract uterus (500 times more sensitive than mice and rat);Hamster stomach
strip is not sensitive to 5HT and histamine, but suitable for the bioassay of prostaglandins
(PGE2 > PGF2)
Insulin Mice susceptibility to insulin induced epilepsy is highly reduced in summer
Morphine Produces depression in rabbit, rat, dog, monkey and man whereas stimulate mice, cat, pig or horses
Pethidine Dog is resistant to pethidine
ATP: Adenosine triphosphate; 5HT: 5-hydroxytryptamine; PGE2: Prostaglandins E2; PGF2: Prostaglandins F2;
metabolism, distribution, and elimination of DISEASES CAUSED BY ANIMALS

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

Zoonotic disease Animal (organism involved) Mode of transmission Treatment
Rat fever Rat (Streptobacillus Animal bite, direct contact Penicillin or erythromycin or
moniliformis ) with secretions of the mouth, tetracyclines for 7-14 days
nose, eye of an infected animal
Ring worm Mice, Rat, guinea pig, Direct or indirect contact with Fluconazole, Itraconazole,
hamster, gerbil etc. skin lesions or infected hair, Ketoconazole and Terbinafine
(Microsporum spp., or fomites
Trichophyton spp., fungal
organisms)
Rabies Dog, cat, rat, mice Animal bite Prophylactic Rabies vaccine at 0, 3,
(Rhabdovirus, Rabies, 7, 14 and 28 days. Booster at 90 days
Hydrophobia)
Herpes B virus Monkey Herpesvirus Bite or direct or indirect Acyclovir
Infection simiae, a DNA herpesvirus contact with infected saliva
or tissues
Hantavirus Mice (Peromyscus spp.) Inhalation of the virus in the Better take precaution and prevention
Infection dust of mice excreta (urine from rodents
and feces) Ribavirin (some benefit)
Tetanus Nearly every laboratory Animal bite Immune globine im injection and
animal penicillin to eradicate the bacteria
Salmonella Dog, cat, ferrets etc Bite or direct or indirect Ciprofloxacin, ceftriaxone and
contact with infected saliva amoxicillin
or tissues
Campylobacter Dog, non-human Bite or direct or indirect Erythromycin, azithromycin and
primates contact with infected saliva Doxycycline
or tissues
Psittacosis Birds Nasal secretions and in the Doxycycline, Azithromycin,
stool from infected birds Erythromycin, Rifampin and
Tetracycline
Cutaneous Amphibians and Fish Direct or indirect contact Amikacin
mycobacteriosis with urine or feces
diseases is greater for those who work with • Prophylactic vaccination of experimenters,
experimental animals. Hence, proper vaccination post-bite treatment of victim and animal
program should be run in the institute. quarantine should be maintained.
To prevent zoonotic disease following
• Avoid direct contact with animal urine and
precautions should be taken while handling the
feces, and wear protective clothing, e.g: gloves
animals
• Regular health testing of animals should be or apron, etc.
done and use respiratory mask during the • Test serum to assess prior exposure
experiment • Post-exposure treatment and assessment
Fig. 1.34: Mode of transfer of zoonoses
Allergy due to Animal Exposure be done to identify those with pre-existing

allergies or IgE antibodies whereas pulmonary
Allergy onset to the experimenter while handling
function testing (PFT) to identify occupational
animal is mainly depending on the duration of
asthma and to evaluate and monitor symptoms
exposure, and allergen exposure concentration.
is advisable.
The common mode allergy is animal fur, skin urine
or faces or saliva (Table 1.7). Allergies develop Prevention
immediately or within 2 years of exposure through
• Ventilation exhaust should be proper
direct contact or inhalation. Immediate allergic
which minimizes the generation of micro-
reaction (Type 1), mediated through release of IgE
organisms
antibody and less commonly, includes IgG-
• Proper cleanup of hazardous dust, fumes, etc.
mediated allergy, which may occur 1-12 h after
which limit the area of contamination by
exposure. Non-IgE-mediated allergy involving
reducing allergens
other immunoglobulin and giving rise to specific
• Avoid crowding of animals per area
pathology, such as extrinsic allergic alveolitis.
• Regular cleaning and disinfection of contami-
Symptoms: Rhinitis, Conjunctivitis, skin effects nated walls, surfaces, etc.
(Urticaria, Wheals and Eczema) • Animal bed should be change at regular basis
Diagnosis of allergy: Skin tests, RAST or enzyme- to avoid the accumulation of urine and faeces
linked immunosorbent assay (ELISA) may in the animal house (act as reservoir of allergen)
• Storage and disposal of animal waste should ANESTHESIA AND EXPERIMENTAL ANIMALS
be proper
• Eating, drinking, smoking, etc should be strictly Aim of the anesthesia used in an experimental
prohibited in contaminated areas. study is to ensure analgesia, amnesia and
immobilization of animal for the ease of handling
Table 1.7: Medium of allergens spread from or any surgical procedure. While anesthetizing the
experimental animal animal one should maintain the three vitals such
as “blood circulation”, “oxygenation” (to ensure
Animal Allergen present
adequate oxygen concentration in the animal’s
Rat Proteins in urine and saliva arterial blood) and “respiration” (to ensure that
Mice Urinary proteins the animal’s ventilation is adequately maintained).
Guinea pigs Urinary proteins (penetrate low into the Anesthetic is commonly used in the experimental
respiratory tract) study so as to make the procedure more simple,
Rabbits Glycoprotein found in the fur (major).
reliable, and reproducible. Generally, all types of
Proteins in urine and saliva (minor)
anesthetics are used in the study (Table 1.8).
Cats Proteins from the sebaceous glands (hair
shafts and saliva) Ether must be used in a fume hood and stored
Dog Proteins found in saliva, hair and skin appropriately, otherwise use of ether as an
anesthetic agent is prohibited.
INHALATIONAL INJECTION LOCAL
• Difficulty in use, requires special • Depth of anesthesia that cannot • Requires experienced person to
apparatus be easily altered use (identifying location and time
• Thermoregulation required, cold • Safe and effective to use to use)
gas may reduce temperature • High first pass metabolism • Onset within 15 minutes of
• Short lasting • Usually, animals under injectable application and may last from 45
• Required maintenance dose anesthesia are not intubated minutes to several hours
• Special arrangement required • No special arrangement required
such as Drop System, gas • No effect on the physiological
scavenging system, etc. property of animal
• Example: Ether, Halothane, • No first pass metabolism
Chloroform, Nitrous oxide, Isoflu-
rane, etc.
• Bypass first pass metabolism
EUTHANASIA METHOD USED IN THE “Euthanasia means the humane killing

EXPERIMENTAL STUDY (sacrifice) of an animal which produces rapid
unconsciousness and subsequent death without
Animals are used in the experimental
pharmacological studies and to assess the drug or minimal pain or distress to animal.”
activity, it requires analyzing the organ (brain, The choice of a method depends on
kidney, lung, etc.), blood (plasma or serum), and species, age, and availability of restraint and
body fluid (CSF, urine, etc.) to conclude the result. skill of the individuals performing euthanasia.
Hence for the purpose, animals have to be It is very important that, in an experimental
sacrificed to obtain the said part of the body. setting, the method of euthanasia must
Therefore, according to the animal care guidelines be primarily consistent with the experimental
they should be euthanized. goals.
Table 1.8: Anesthetic agents used in experimental animals

Anesthetic agent Dose (mg/kg), Route of administration
Mouse Rat Hamster Guinea pig Rabbit Frog* Zebra fish*
α-Chloralose ————— ————— ————— ————— 80 – 100, iv ————— —————

Halothane, 3-4% induction; 3-4% induction; 3-4% induction; 3-4% induction; ————— 1-5% 1-5%
Isoflurane, 1-2% 1-2% 1-2% 1-2%
Enflurane maintenance maintenance maintenance maintenance
Hexobarbital 60, iv or ip 60, iv or ip ————— ————— ————— ————— —————

Ketamine HCl 22-24, im 22-24, im ————— 22-24, im 22-24, im/iv 50-150 50-150
Pentobarbitone 35-50, iv or ip 25-50, iv or ip 35, iv 30-40, iv or ip 30-40, iv or ip 60, dorsal —————
sodium lymph sac
Thiopentone 25-50, iv or ip 20-40, iv or ip 20-40, iv or ip 20-55, iv or ip 20, iv ————— —————

sodium
Urethane# ————— 0.75-1.5g/kg, ip ————— 1.25 -1.5 g/kg, ip 1g/kg, iv or ip ————— —————
Tricaine and ————— ————— ————— ————— ————— 25-100 mg/L 25-100 mg/L
benzocaine
Ketamine (K) + 100 (K) + (60-90) (K) + (80-100) (K) + 40 (K) + 5 (25-50) (K) + ————— —————
Xylazine (5-10)(X) i.p. (6-9) (X), i.p. (7-10) (X), i.p. (X), i.p. (6-10) (X), i.m.
Ketamine (K) + 75 (K) + 1 75 (K) + 100 (K) + 40 (K) + 25 (K) + ————— —————
Medetomidine (M), i.p. 0.5 (M), i.p., 0.25 (M), i.p. 0.5 (M), s.c. 0.5 (M), i.m.
(M) s.c.
# Carcinogen! Use with caution and have prolonged anesthesia

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

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

substance in relation to the functional response
While performing bioassay, sometime responses
of a standard of the same substance. A bioassay
are varied, i.e. sometimes it may increase and
measures the ‘relative activity’ or ‘potency’ of a
sometimes it may decrease, which is calculated
substance and its specific capacity to achieve as error of the procedure. Broadly error is divided
an intended biological effect. In other words, into two categories, i.e. due to biological variation
potency is the quantitative measure of biological or due to methodological error.
activity. It is usually measured as an observable
Biological variation: It is known that animal to
or objectively measurable parameter at a set
animal or individual biological variation occurs
interval from the bioassay starting point (at time
in response to a drug action. The effect of drug
zero). Normally, as potency increases with dose to the tissue response may vary and will fall
increasing concentration of a substance (in in a range. So, when we plot the response on the
solution), the biological activity is directly linked Y-axis and drug dose on the X-axis, the graph
to subdivisions or dilutions of the original obtained is the symmetric frequency distribution
concentration. The most useful bioassays are those curve (Fig. 2.1). So, the response taken is the mean
in which the range of functional responsiveness is of all responses observed.
the scale of readings for the measurable parameter
between the minimum and maximum response which
is relatively large and falls between an effective,
practical range of concentrations/dilutions of a
substance to generate analyzable ‘dose-response’
data.
Fig. 2.1: Symmetric frequency distribution curve, plot of

response versus drug dose
Reason and its correction for biological

variation:
• Downregulation of receptors (repeated
washing of tissue; avoid over washing)
• Loss of tissue sensitivity (change the tissue)
• Animal use of different species, sex, age, weight
and health status (must use same species)
Principles in Short • Laboratory condition may be variable (must be
• To compare a test drug potency with a standard drug, kept constant)
quantitatively • Housing and handling of animals (must be
• To evaluate both test and standard drugs, as identical handled by a qualified experienced staff)
to each other
• To estimate the biological variation of the drug to Methodological error: Usually this error occurs
species and other groups of animals or living beings due to the faulty method selection or due to the
• Methods for comparing therapeutic potency of unknown incorrect procedure, i.e human error and
and standard drug
experimental error.
Bioassay  47
Human error is one which is done by the 5. Estimation of ED50/TD50 and LD50. (Presently
experimenter during the experiment. It may instead of LD50, NOAEL or LD10 is preferred).
happen from the time of tissue selection or
physiological salt solution (PSS) preparation or METHODOLOGY (FLOW CHART 2.1)
during the response recording. In every step, there
are chances of error, so to maintain the standard In academic set-up, traditional organ bath is
conditions for the isolated tissue, be sure regarding commonly in use, which contains inner organ
the accuracy of the procedure. bath, water bath with coiled inlet for physiological
Experimental error may happen due to the salt solution (PSS), stirrer, heating coil, kymograph
faulty procedure selection or due to the calibration with revolving motor, etc. (Fig. 2.2). But in industry
error of the instrument. This may be corrected by more recent sophisticated equipments are
properly balancing the lever and by maintaining available for drug discovery such as multiple
the temperature or pH of the PSS. organ baths (may be up to 8 inner organ bath).
Organ bath assembly was first used by Rudolph
Reasons for Methodological error and its correction Magnus in early 19th century. He standardized
• Lack of standardization of procedure (standar- the set-up with intestinal strips. More or less set is
dize your procedure beforehand) still same with some modification.
• Set-up of apparatus (Check every instrument
used in the experiment for their proper Essential Parts and Uses of Organ Bath
working) Two types of organ baths are designed which got
• Tissue isolation/extraction and preparation
access in most of the laboratories such as,
for the experiment (minimize handling and
1. Single unit organ bath: This set-up was
excess cleaning of the tissue while mounting
designed and developed by Rudolph Magnus.
in the organ bath)
It has only one inner organ bath (Fig. 2.3).
• Preparation of physiological salt solution
2. Double or multiple unit organ baths: The
(PSS) and maintenance of its pH (Follow the
concept of these organ baths were developed
standard procedure to prepare and maintain
by Gaddum. He was developed a double unit
the pH of PSS)
organ bath. It has two inner tissue organ baths,
• Drug preparation or it dilution (while making
but it is not able to replace the conventional
serial dilution of the drug, mix it slowly; Avoid
organ bath. Nowadays, in many industries
the vigorous mixing).
automated multiple unit organ baths are used
APPLICATIONS OF BIOASSAY for more efficient and fast drug discovery
(Fig. 2.4).
According to several pharmacopeias, assay used
for the drug may be varied. For example: Essential Parts of Organ Bath which is
1. Estimate potency of natural drug, for which Required to Record the Response of a
chemical method is not known or established Tissue/Muscle
2. Standardization of drugs of natural origin
(plant and animal origin) whose structure or Rotating drum and kymograph: Rotating drum is
origin is unpredictable also known as Sherrington rotating drum. Its speed
3. Screening of new compound for biological can be adjusted by the attached gear and lever.
activity For the slow contracting tissue, the drum is kept
4. Estimation of biologically active substance like on low speed and for the fast contracting tissue
histamine, acetylcholine, 5-hydroxytrypta- the drum speed should be kept on comparable
mine, adrenaline, bradykinin, substance P, high speed. The standard speed of the drum is 1
prostaglandins, etc. revolution/96 min.
Flow Chart 2.1: Overview of complete steps for bioassay
Kymogram is the paper used to record the Paper is smoked with the help of burning cotton soaked in
response of the tissue/muscle against the known/ benzene or kerosene. The drum is kept on the height
unknown concentration of the drug. Paper has where it gets dark and densely smoked (At center and
two sides, one side is glossy or smooth and other adequate height). Precaution should be taken while smoking
side is rough. While mounting the paper on the drum and should maintain sufficient distance from fire. For
drum, one should make sure that the smooth better writing on the kymograph paper, smoking should
surface is outside (helps or don’t produce be uniform and dense.
Presently in most of the pharmacology laboratories, ink
resistance in the movement of the lever on
kymograph paper is also in use where there is no need to
kymogram) and the rough surface is inside (help smoke the paper. Stylus works as a writer on the kymograph.
in sticking of paper to the drum).
Bioassay  49
1. Power input 12. Stirrer

2. Switch board 13. Reservoir for PSS
3. Heating rod 14. Clip to control the flow of PSS to inner bath
4. Thermostat 15. Stylus or writing point
5. Coiled PSS inlet to inner bath 16. Motor
6. Outer bath water outlet 17. Gear to adjust the speed
7. Inner bath PSS outlet 18. Clutch lever
8. Inner bath with air supply (aerator) and tissue holder 19. Screw to adjust the level of drum
9. Thermometer 20. Drum rotation indicator
10. Recording lever 21. Sherrington rotating drum
11. Fulcrum 22. Lift screw (to move drum up or down)
Fig. 2.2: Schematic presentation of commonly used organ bath set-up
Fig. 2.3: Single unit organ bath Fig. 2.4: Double unit organ bath (For color version see Plate 1)
Outer bath/Outer water jacket: Mainly used to Fixing of the recording: Tracing on kymogram is
store water outside the inner organ bath to fixed with help of fixing solution at the end of
maintain the temperature for the experiment. It is the experiment. Fixing solution is made up of
made up of perspex glass, glass or steel. Perspex shellac and colophony saturated in the alcohol.
is more widely used due to its durability. Sufficient powdered shellac is mixed in the
Inner organ bath: It is transparent and made up of alcohol (methanol) and dissolved till it gets
glass to observe the tissue during experiment. saturated and precipitated then keep solution for
Inner bath has varying capacity from 5 to 50 ml. 5-7 days to settle down the particles and decant
Proper marking is done on the tube to fill the PSS at the remnants to get non-precipitated solution.
fixed level every time. Store the prepared fixing solution in the cool and
dark place (Fig. 2.5).
Tissue holder and oxygen supply: Tissue is attached
inside the inner organ bath with the help of tissue
holder and it also supports the air or oxygen Drug Response Relationship
supply through its motor. Potency comparison of standard and test
Fulcrum: Writing lever is attached to this and help preparation always depends on the response
in free vertical movement to record the response produced by the particular concentration of
of tissue against the drug. standard/test. The dose response is always directly
Heating iron coil and thermostat: Heating coil is co-related to the concentration of the standard/
attached to the outer bath and is made up of iron. test drugs. Students always prefer to make a dose
It maintains the temperature outside the inner response curve (DRC) or concentration response
bath as well as of PSS which moves in the inner curve (CRC) in an ascending order (Fig. 2.6A) (It is
bath through coiled inlet. Whereas “thermostat” less common practice of making in the descending
as name suggests maintains static or constant order due to the lack of known high concentration).
temperature throughout the experiment and But, sometimes it shows, reduced sensitivity of the
avoids wide variation in temperature. tissue. Hence, to retain the tissue sensitivity for a
Stirrer: It gives circulating water to maintain the longer duration, one should vary the dose order
similar temperature throughout the outer organ intermittently (neither ascending nor descending)
bath. (Fig. 2.6B).
Fig. 2.5: Fixing of kymogram; fixing solution is made by dissolving the shellac in methanol and saturate the solution for
5-7 days to settle down the particles at bottom and then filter it and store at cool and dark place)
Bioassay  51
Fig. 2.6A: Ascending dose response plot
Fig. 2.6B: Intermittent order dose response plot
The dose response curve depends on the linear then there is good co-relation with potency of the
regression relationship on log dose of potency of two preparations.
standard and test preparation. The dose response
curve of standard and test preparation should be PHYSIOLOGICAL SALT SOLUTION (PSS)
parallel. (Figs 2.7A to C) This is not only useful as Preparation of PSS depends on tissue selection
to estimation of potency (two parallel lines) but it for the experiment (Table 2.1). PSS is very
also estimates the errors in the method applied. If important to maintain tissue outside the animal
there is no regression of potency, there is no use of body which fulfills their internal environment of
dose response relationship and it is thought that ions and nutrition. This is often called as Ringer
if the mean square for regression is relatively large solution.
Figs 2.7A to C: (A) Correct linear

regression relationship of standard and
test (log dose versus percentage
response plot); (B) Incorrect linear
regression relationship of standard and
test; (C) Guide to select the dose of
standard and test during the experiment
and showing the several effects of drug
on a tissue (supramaximal effect is
commonly known as ceiling effect of
the drug)
Table 2.1: Composition of different physiological salt solution (PSS) [salts in g/10 liters]
Ingredients Tyrode Krebs De Jalon Frog Ringer Ringer Locke McEwens
NaCl 80.0 69 90.0 65.0 90.0 76.0
NaHCO3 10.0 21 5.0 2.0 2.0 21.0
D Glucose 10.0 20 5.0 20.0 10.0 20.0
KH2PO4 – 1.6 – – – –
NaH2PO4 0.50 – – – – 1.44
KCl 2.0 3.6 4.2 1.4 4.2 4.2
MgSO4.7H2O – 2.90 – – – –
MgCl2 1.0 – – – – –
Sucrose – – – – – 5
CaCl2 2.64 3.70 0.8 1.58 3.2 3.0
Aeration Air 95%O2 + 95%O2 + Air O2 95%O2 +
5% CO2 5% CO2 5% CO2
Bioassay  53
Solution prepared with the help of distilled or avoid addition of calcium and glucose which should
double distilled or deionized water. Main be added at the time of experiment.
components of PSS are sodium (Na+), chloride
(Cl–), potassium (K+), magnesium (Mg+), calcium Role of Each Ingredient
(Ca+) and glucose. • Sodium (Na+): One of the major extracellular cations
It is always important to select PSS in which tissue which maintain the electrolyte level in the tissue. It
last longest. Aeration is important for solution with makes solution isotonic by maintaining the osmolarity.
oxygen (O2) or 95% O2 + 5% CO2 (carbogen). This • Potassium (K +): It is the major intracellular cation
having important role in nerve conduction, muscle
process helps to provide O2 to tissue and mix PSS
contraction, etc. Its role is remarkable in maintaining
thoroughly in organ bath. Additionally, carbogen heart rate and rhythm.
is important because, pure O2 may interact with • Calcium chloride (CaCl 2 ): It controls excitability of
bicarbonate (HCO3–) buffer in PSS which will muscle, nerve and glands. Among contraction-
cause CO2 loss and PSS become alkaline. relaxation coupling it also helps in maintaining the
integrity and permeability of the cell. The major role
Certain precautions must be taken, while
remains excitation.
addition of ingredients into the distilled water • Magnesium chloride (MgCl 2): Being second most
(DW)/double distilled water (DDW) or deionized common intracellular cation, its main action to reduce
water (DIW). Calcium chloride should be added the spontaneous activity of tissue, also play an
at last, to prevent precipitation or chelation of the important role in neurotransmission in muscle
contraction.
bicarbonate which make solution turbid or
• Bicarbonate (HCO3–) and Sodium hydrogen phos-
opaque. This may interfere with internal property phate (NaHPo 4): Acts as a buffer.
of solution and may reduce visibility of tissue in • Glucose: Major nutrients for the tissue in ‘in vitro’ set-up.
inner organ bath. Thumb rule for selection of PSS
is that, Tyrode may be used for experiment with Important Points to Remember
non-innervated muscle while Krebs is used for • McEwen solution contains sucrose in addition to
nerve muscle preparation. The pH is important to glucose.
maintain PSS property and should be at range • DeJalon, Frog-Ringer and Ringer-Locke do not contain
magnesium (Mg 2+) and phosphate (PO4–)
7.3-7.4.
• Krebs, DeJalon and McEwen are aerated with 95% O 2
The preparation of PSS is important and the + 5% CO 2 and used for mammalian isolated organ
error in making should not exceed 1%. The (nerve associates) and avian skeletal muscle
physical properties of ingredients should also be • Tyrode specially used for the mammalian smooth
muscle
taken into consideration, such as calcium chloride
• For amphibian tissue, use Frog-Ringer solution
and magnesium chloride are very hygroscopic, • For heart muscle preparation, use Ringer-Locke
so it is best to add them from stock solution. MgSO4 solution
is not hygroscopic in nature and hence used instead of • Krebs solution may be used for any tissue
MgCl2. The reason being exchange of chloride with
sulphate (SO4–) does not make any markedly LEVER AND MAGNIFICATION
difference, because most part of chloride ion is It is the mechanical instrument based on the
fulfilled by NaCl and sulphate (SO4–) ion have no principle of static equilibrium which is derived
harmful effect. from Newton’s laws of motion and statics. Hence,
Preparation of various PSS is given in Table 2.1. working of the lever is denoted by the
Fresh solution is always prepared while it can be
“Load force × length of load arm = Effort force
stored for 24 hr in fridge, but storage can’t be preferred
× length of effort arm.”
for longer time because of microbial growth (due to
presence of glucose). If preparation of solution is Lever has three basic parts (1) effort arm (point
needed and requires storage longer than 24 hr then where you apply the force) (2) load arm (effect
observed due to application of force) and (3) Magnification (Mx) and its Adjustment
fulcrum. Both load arm and the effort arm are measured The transducer which amplifies contraction are
from the fulcrum to the load and effort, respectively mainly divided into two types, namely, when there
(Figs 2.8 to 2.10). is change in the length of tissue recorded after
The lever used in the in vitro bioassays is of the contraction but no change in tone or tension
“type 1 lever”. In most of the experiments, the change recorded, then it is called as isotonic transducer
in the tissue length is measured and recorded by the such as simple writing, frontal writing, etc. whereas
lever on a kymogram (Fig. 2.11). Stylus is the point of the in isometric transducer there is no change in the
lever which touches the kymograph for recording the response length but change in tone or tension of tissue is
(writing point). It is made of stainless steel, aluminum recorded. Such as in the muscle twitching, cramp,
or wood. Lever is attached to fulcrum and mainly used etc. this type of recording needs special set-up.
to magnify the response of the isolated tissue In isotonic set-up magnification of the
preparation. So, lever records and amplifies the recording lever plays important role for a
response obtained during the drug effect on the isolated magnified response. Usually, magnification is
tissue. Magnification can vary for tissue to tissue from kept low in the case of fast contracting tissues
5 to 15 times. The standard rule is that, if tissue whereas it is kept high in the case of slow
preparation is slow contracting magnification is large contracting tissues, so as to record the proper
either 10-15 times but when tissue is fast contracting response. The magnification of the lever is
the magnification may reduce to 5-10 times. calculated as followed (Fig. 2.12):
Fig. 2.8: Fulcrum (F) is located between the input effort (E)
and the output load (L). For example: Bioassay, seesaw,
triceps brachii muscle, Bicycle hand brakes, scissors
(double lever), etc.
Fig. 2.9: Fulcrum (F) is located at the one end and effort
input (E) at the other. The load input (L) is at the middle of ‘E’
and ‘F’. For exmple: Dental elevator, Bottle opener, puss-up,
etc.
Fig. 2.10: Fulcrum (F) is located at the one end and at load
input (L) the other. The effort input (E) is at the middle of ‘L’
and ‘F’. For example: forceps, etc.
Bioassay  55
Simple lever: Made of stainless steel, aluminum or wood Frontal writing lever (FWL): Made of stainless steel,
with stylus* attached to it. and aluminum with stylus* (two arm) attached to it.
Attachment of simple lever should be tangential to the Attachment of FWL should be perpendicular to the smoked
smoked drum drum. It magnified a small contraction of tissue/muscle on
the kymograph
Gimbal lever (GL): Made of stainless steel, or aluminum Sprung lever: Made of stainless steel, or aluminum with
with stylus* attached to it. A roller is fitted in between for stylus* attached to it with help of return spring. Tension of
free movement by the gravity force. Attachment of GL the lever is adjusted with the screw attached at the opposite
should be tangential to the smoked drum. end of stylus. Attachment of simple lever should be
tangential to the smoked drum
A 20 cm
5
B 4 cm
Auxotonic lever (AL): Made of stainless steel, aluminum Torsion lever (TL): Made of stainless steel, aluminum or
or wood with stylus* attached to it. wood with stylus* attached to it.
Attachment of AL should be perpendicular to the smoked Attachment of TL should be tangential to the smoked
drum. drum
Starling heart lever (SHL): Made of stainless steel or

aluminum with detachable stylus attached to it, Attachment
of SHL is perpendicular to the smoked drum.
* Stylus (Writing point): Ideally it should be made up of aluminum or stainless steel but in laboratory, it can be made
by the thin steady film of X-ray or photography film or any hard paper (for temporary use).
Fig. 2.11: Different types of levers, commonly used in experimental pharmacology
Distance from fulcrum to writing point (A) Answer:

Mx = A = 20 cm
Distance from fulcrum to tied tissue (B)
B = 4 cm
Mx = ?
Example: Calculate the magnification of a lever
attached to a fulcrum, if the distance of writing point Mx =
from the fulcrum is 20 cm and the tissue attached to the
Hence, the magnification of attached lever is 5x.
lever is 4 cm apart from the fulcrum?
Fig. 2.12: Measurement of magnification of lever
DOSE CYCLE (DC) AND RESPONSE when there is any baseline shift found. Variability
(biological) of about ±10% is acceptable in the tissue
Principles of the experiment depend on the dose response recording. Tissue may show the
response relationship of test drug as well as “tachyphylaxis”, in which there are decreased
standard drug. So, for measuring responses, it is responses to drugs after repeated dosing.
necessary to first fulfill the basic requirements of While taking response, dose cycle has three
the experiment, i.e. tissue should properly relax important phases, baseline recording, dose
and proper load should be given for the specified response and washing of tissue. Dose cycle is defined
time. Baseline recording is initial step and as the time gap between drug dose additions whereas
important to record, which helps to take a uniform contact time is defined as time duration at which
response of drug. (it can indicate the shift of tissue come in contact with drug. Dose cycle is
baseline; if any) It is not necessary that all tissue commonly of 3 minutes for fast contracting
show the steady straight line responses but few tissues or 5 minutes for slow contracting tissues.
tissues show the spontaneous tissue variability (This includes at least two tissue wash)
which can be observed in the response recording (Fig. 2.13)
Fig. 2.13: Measurement of dose cycle and contact time in slow contracting and fast contracting tissues
Bioassay  57
TYPE OF TISSUE measured. Thus, the observed data are dose units,
e.g.: assay of digitalis in cat. Unlike, the indirect
Preparation of tissues used in the isolated tissue assay the experimental unit receives one or more
experiments are broadly classified in two ways; specified doses of the preparation and the
1. According to tissue/muscle obtained: observed data may be either quantal or
– Smooth muscle preparation, e.g.: Uterus, quantitative responses. Depending on the
tracheal smooth muscle, vas deferens, experimental design, several dose levels of T and
aorta, ileum, ascending or descending S are given to the same or different experimental
colon, etc. units and the mean dose level is selected.
– Skeletal muscle, e.g.: Rectus abdominus Mainly two experimental designs are followed,
muscle, etc. one is crossover, and another is parallel group or
– Cardiac muscle, e.g.: Frog heart etc. completely randomized design.
2. According to response obtained by tissue/ In the procedure, the test or standard
muscle: preparation is infused at a fixed rate into the
– Slow contracting tissue/muscle: For example: circulation of animal until a direct effect is
Frog rectus abdominus muscle, stomach observed in the animal. For example: stopping of
fundus, biventer cervicis muscle of chick, heart beating after the continuous administration
guinea pig tracheal smooth muscle, etc. of digitalis at the constant rate in the assay of
– Fast contracting tissue/muscle: For example: digitalis in cats.
Ileum, uterus, ascending and descending
The threshold dose of standard = Total period
colon, etc.
of infusion × Rate of drug administration
CLASSIFICATION OF BIOASSAY TDS
Concentration of test = ×CSD
Broadly bioassay is classified into three groups TDT
namely, Where,
1. Direct end point assay (DEPA) TDS = Threshold dose of standard
2. Quantal assay (all or none assay)
TDT = Threshold dose of test
3. Graded assay
CSD = Concentration of standard drug
a. Bracketing assay
b. Matching assay
Advantages
c. Interpolation assay
d. Multiple point assay (3-point, 4-point, • Drug effects appear rapidly and are easily
6-point and 8-point) recognized
• Drug effect is directly proportional to drug
Direct End-point Assay dose
• Rapid end-point detection.
In a direct assay, the threshold dose required for
response is determined for each experimental unit.
Disadvantages
The principle of direct assay is to measure direct
response of dose of standard and test preparation. • Only toxicity study or high dose study is
The ratio between these doses estimates the possible
potency of the test preparation relative to the • Dose ranging study cannot be done.
standard.
The response should be clear and easily Quantal Assay (All or None Assays)
recognized and the dose given to the experimental The unknown is compared with the standard
animal should be in such a way that is easily with respect to potency which produces the
quantal affect, i.e. changes is easily recognized used is once but some responses have no
sign or often death. These responses are recorded; permanent effect and can be used in a design of
reason being effect of some drug or stimulus to several test.
any targeted system is not able to record response
quantitatively or the reaction of subject is so Graded Response Assay (GRA)
minimal which cannot be quantified or recorded.
In this type of biological assays, the extent of the
In a quantal assay there is use of dose response
reaction is a function of the dose of drug. Ideally,
relationship, however assay are more closely
the quantitative relation between dosage and
related to direct assay. As a procedure quantal
response would be expressed in terms of an
response to a drug is obtained and percentage of
equation describing the mode of drug action. Most
positive response at each dose is calculated. Most
graded reactions are consistent with the sigmoid
popular example in the drug discovery is
form, approaching asymptotically to a “floor” and
determination of LD50 (Fig. 2.14) and other is assay
a “ceiling.” However, unlike the all-or-none
of insulin by mice convulsion method. The
response, where the number exposed to treatment
response in the quantal assay is varying, i.e. some
is known or can be estimated readily from control
responses are irreversible and hence the animal
groups, the amount of reaction representing
100 % of a graded response would need to be deter-
mined experimentally to solve either formula.
By GRA, potency of a test agonist is determined
by comparing its mean response to standard mean
response. This process is known as ‘analytical
dilution assay’. (Serial dilution of standard/test
drug) (Fig. 2.15).
This assay simply depends on the several
graded responses by exponential increase in the
test dose and which is compared with the
standard graded dose response. GRA is simplest
way of determining potency of a test drug because it
Fig. 2.14: Determination of LD50 does not require statistical analysis.
Fig. 2.15: Serial dilution (descending order)

Bioassay  59
Bracketing Assay (Fig. 2.16) subjective, experimental error is not excluded out
This assay is preferred when test sample volume and there is no sign of parallelism as it lacks dose
is too small. It is the simplest way of GRA, in response relationship. It requires most sensitive
which single or few response (s) is taken by using tissue, so tissue selection is the most important
any test drug concentration. Consequently, this aspect in this assay.
response is bracketed between two responses (one
higher and one lower) of the standard drug. Then
the potency of the test drug is directly calculated
from concentration of standard drug or by Interpolation Assay (Fig. 2.18)
interpolation through dose response curve. This method depends on the assumption of dose
Limitation of the assay is poor precision and response curve. Concentration of unknown is
reliability and also unable to calculate error. interpolated from the dose response curve graph.
At the first step DRC of the standard drug is plotted
Matching Assay (Fig. 2.17) then single or few responses of the test drug are
plotted. The dose of the test drug which comes at
Comparison of potency between the unknown and
the linear log dose-response relationship is
standard drug is done by trial and error method.
interpolated from the dose response plot.
Important part of this method is that response is
matched at only one dose, so it does not needs
Multiple Point Assay
dose response curve of test compound. It requires
very small sample volume, whereas meanwhile The above mentioned methods are not ideal
having several disadvantages such as it is purely because of lack in sensitivity, accuracy and might
Dose of ‘S’
Concentration of T = Conc. of ‘S’
Dose of ‘T’
Fig. 2.16: Bracketing assay; Test compound dose response (T) is bracketed between the two dose responses of
standard compound (S1 and S2), one response more and another is lower than test compound
Fig. 2.17: Matching assay; the test compound response is matched with the selected response of standard compound.
Initially DRC of standard compound is obtained then take a response of fixed volume of test then either higher response
or lower response of standard is selected. In the above figure, matching assay is performed by selecting a higher response
of standard than the test response, hence the test volume is increased slowly to meet the response of standard response,
then concentration of test is find by formula given in the column. (T/T*/T** = Showing increasing dose of test compound to
match the standard response)
doses must be in the linear portion of the dose-

response plot of standard compound, i.e. between
25 to 75% (Dose discrimination is better at these
portions) (Fig. 2.19).
Three-point assay (Fig. 2.20): Method depends on
the latin square randomization of total three
responses selected from DRC prepared for
standard as well as test (2 response from standard
and 1 response from test: response selection is made
between 25-75% of response). For many bioassay,
dose response data can be transformed to generate log
dose transformed response lines to yield as extended a
Fig. 2.18: Interpolation bioassay
linear range as feasible. Estimates of relative potency
are then obtained as the displacement of parallel
involve many methodological errors such as tissue log dose response lines of standard and test
sensitivity error, variable temperature error, compound.
dilution error, etc. So, to correct the above
mentioned limitations, graded response assays S1 S2 T
are preferred. Repeated response recording in Latin square randomization S2 T S1
graded response assays minimize the tissue T S1 S2
sensitivity error and improve the methodological
errors. These assays are performed by the selection Calculation for 3’point assay:
of 1 or more dose responses of test compound and
these responses are compared with 2 or more T – S1 s
Relative potency (M) = log 2
responses of standards. The selection of the test S2 – S1 s1
Bioassay  61
Fig. 2.19: Dose should be selected in the linear portion of the plot (25-75%)
s1
antilog M
t
Fig. 2.20: Three-point assay
Concentration of unknown compound s1 and s2 = Standard drug dose which came

in contact with tissue and given
= the response ‘S1’ and ‘S2’
respectively
= “x” times more concentrated than standard
compound t = Test drug dose which came in
Where, contact with tissue and given the
S1 and S2 = Length of standard dose response ‘T’
response selected between 25-
Note: There are several factors plays role during
75%
T = Length of test dose response the experiments such as biological environmental
selected in between of two or methodological factors. So, there may chances
standard response of some error present during the bioassay. Hence,
the percentage of error is calculated by the formula T1 and T2 = Length of test dose response
given below: selected
s1 and s2 = Doses which produces mean
ACT – OCT
Percentage error (%) = 100 response of S1 and S2
ACT respectively
where, t1 and t2 = Doses which produces mean
ACT = Actual concentration of test response of T1 and T2 res-
OCT = Observed concentration of test pectively
Note: In bioassay, permiciable limit of percentage Concentration of unknown
error is < 10%.
Four-point assays (Fig. 2.21): Method is same as =
3-point assay, only difference is that in this
experiment responses are selected; 2 responses of = ‘x’
standard and 2 of test from the DRC for the So, the concentration of unknow is ‘x’ times
consecutive 16 response of Latin square more strengther than standard
randomization as shown below in figure. This
procedure is more sensitive than 3-point assay Note: There are several factors plays role during
and reduces the error or variability the experiments such as biological environmental
or methodological factors. So, there may chances
S1 S2 T1 T2 of some error present during the bioassay. Hence,
S2 T1 T2 S1 the percentage of error is calculated by the formula
Latin square randomization T1 T2 S1 S2 given below:
T2 S1 S2 T1 ACT – OCT
Percentage error (%) = 100
ACT
where,
M=
ACT = Actual concentration of test
OCT = Observed concentration of test
Where,
S1 and S2 = Length of standard dose Note: In bioassay, permiciable limit of percentage
response selected error is < 10%.
Fig. 2.21: Four-point assay

Bioassay  63
6-point and 8-point Assay In pharmacology, several different types of

antagonist are described such as
These methods of bioassays are generally not
adopted for the experiment purpose because of Competitive antagonist (Antagonists bind to the
the time consuming lengthy procedure. The receptor of agonist but show no efficacy, e.g.
responses obtained for the 6-point is ‘36’ and ‘64’ propranolol, naloxone, etc.) (Figs 2.22A and C).
for 8-point. But, the advantage being reduced error Non-competitive antagonist (Antagonists bind to
and variability of the procedure over other the receptor at sites which is not related to the agonist
methods due to the large number of responses and binding site for example Ca2+ blockers) (Fig. 2.22B).
hence have greater specificity.
Physiological (functional) antagonist (Antagonist
has the opposite biological action of the agonist,
BIOASSAY OF ANTAGONIST
by action on a different receptor, e.g.: salbutamol
a β2-adrenoceptor agonist given in acute asthmatic
The term antagonist refers to any drug which can
attack antagonizes the bronchoconstrictor action.
completely or partially block a response of an
agonist. So, an antagonist is the opposite of an Pharmacokinetic antagonist (Antagonist which
agonist which stimulates an action and these two reduces the free blood concentration of drug at its
are considered as a prime agent in pharmacology. target site either by reducing drug absorption or
Antagonist activity may be reversible or accelerating renal or hepatic elimination)
irreversible depending on antagonist–receptor Chemical antagonist (Particular drug may
complex which in turn depends on the nature of antagonize the action of a second drug by binding
antagonist receptor binding. When assessing an and inactivating the second drug. For example
antagonist, the following points should kept in protamine binds to heparin and makes it
mind such as, unavailable for interactions with proteins
a. To check whether the antagonism is surmoun- involved in the formation of blood clots)
table by increasing the concentration of agonist But, in the practical pharmacology, the identification
or not and of antagonist is limited to the competitive or non-
b. To check whether the antagonism is reversible competitive or physiological antagonists only. For
or not (does agonist regain response, after analyzing the antagonist, a dose-response curve
washing of antagonist) of agonist is made in the absence and presence of a
This is due to identification of the type of fixed concentration of antagonist, which shifts the
antagonism. Such as, if an antagonist is sur- DRC to the right (parallel shift), with the same
mountable or reversible, it is likely to be compe- maximum response and the same shape; for the
titive. competative antagonist.
Antagonist has no effect of its own, so, it is
Experimentally antagonist is divided into the
determined experimentally by the intensity to
two groups, block the agonist activity. The process of dose
1. Preventive determination is same as it is with the agonist.
• When the antagonist used before addition First, the agonist response is obtained thereafter
of agonist, it is called the preventive percentage inhibition is determined by using the
antagonist different doses of antagonist. Sometimes,
2. Curative percentage inhibition is converted into the probit
• In this process of antagonism, first agonist scale against the log dose to make graph more linear
and then antagonist is added (Fig. 2.23).
Fig. 2.22: (A) Cumulative plot showing competitive antagonism (parallel shift) in the presence of antagonism; (B) Cumulative
plot of non-competitive antagonism in the presence of antagonist, (C) Competitive antagonism response plot, showing a
partial response block of an agonist which is regained by the repeated tissue washing
experiment will be 1 X 10–10 M. The exposure time of

antagonist to tissue should be around 15min. but in
the routine experiment it is also seen that 3-5 min
contact time of antagonist with tissue can also elicited
the result.
Hence, various methods are employed for the
identification of competitive antagonist,
1. Schild plot method and pA2, pA10 values
2. Parallel shift of DRC
3. Double reciprocal plot Lineweaver and Burk
Fig. 2.23: Probit plot
Schild Plot Method and pA2, pA10 Values
As a general rule antagonist takes a long-time Generally, potencies of competitive antagonist are
to block an action of agonist. In the experimental expressed as pA2 values or pAx values. Hence
purpose, the use of antagonist concentration is around the term, pA2 is defined as the negative logarithm
10 times less than the agonist concentration, i.e. if to base 10 of the antagonist concentration in molar
agonist used in the concentration of 1 X 10-9 M then units corresponding to a dose-ratio of 2, i.e. the
the concentration of antagonist used in the same concentration that produces a 2-fold shift in the
Bioassay  65
Figs 2.24A and B: (A) Schild plot; (B) Determination of IC50
response of agonist concentration-response curve. experimental units compared to the Schild method
Kd (the dissociation constant of the antagonist for to plot a graph.
the receptor) is the other form of expressing the Once the experiments are completed a series
potencies of competitive antagonists. Procedures of dose ratios (DR) are calculated for observed
(both design and model) for estimating Kd and responses. For example, the ratio of the dose of
pA2 have also been developed from the results of agonist (A’) to produce a specific effect (e.g. half
an antagonist inhibition curve in the presence of maximal effect) in the presence of the antagonist
a fixed concentration of the agonist (Figs 2.24A (B) to the dose required in the absence of the
and B). antagonist (A) is calculated.
Suppose, the antagonist is of competitive type,
then the dose ratio will be expressed as, Parallel Shift Plot (Fig. 2.25)
Dose ratio (d) = 1+ (antagonist)/(Kd) It is the simplest form of experimental approach
d-1 = (antagonist)/(Kd) of identifying an antagonist in which the shift of
log (d-1)= log [(antagonist)/(Kd)] DRC is parallel towards right after addition of
log (d-1)= log(antagonist)- log(Kd) competitive antagonist is seen.
If the antagonist is competitive, then slope will
be 1.0 and the X-intercept and Y-intercept will
both be equal to the Kd of the antagonist. The other
formula to identify the competitive inhibition is
by the difference of pA2 and pA10 (pA2 – pA10). If
the difference (pA2 – pA10) is 0.95 or in the ranges
of 0.8 - 1.20, the inhibition is competitive. (pA2
and pA10, where 2 and 10 is the dose ratio)
The limitation of the above mentioned methods
is that no one applies to evaluate antagonist
potencies for compounds that are not pure
antagonist, i.e. partial and inverse agonists and
antagonists lacking intrinsic activity. So, in these
cases Waud Model is applied for the estimation of
pA2, Kd, and IC50. Waud Method requires fewer Fig. 2.25: Parallel shift plot
Lineweaver-Burk Plot or Woolf-Lineweaver- HUMAN TISSUE BIOASSAY

Burk Plot/graph (Fig. 2.26)
There is a common practice of using animal tissues
This method was developed by Hans Lineweaver in bioassay to ensure the activity of an unknown
and Dean Burk in 1934 for the assessment of compound in institute/industries which provides
enzyme kinetics. Lineweaver-Burk plot is inverse a more convenient tool than using human tissues.
of response plotted against the inverse of dose But, limitation of animal tissue use in the research
concentration of agonist, which was invented to is that it can’t predict accurate outcome in relation
analyze how fast a drug can produce its response, to the human. So, new insight includes bioassay
against a present antagonist. This method applied of human tissue and cytokines in the cell lines
to distinguish between competitive, non- which have very close precision to human.
competitive and uncompetitive antagonists. Human tissue is used in areas such as in vitro/
in vivo, efficacy and toxicological pharmacology. But,
Findings of Different Antagonist major difficulty is that, human tissue is difficult
to obtain, because of many ethical limitations and
• Competitive inhibition: Have the same y- difficult in standardization. Advantages of
intercept as uninhibited (since V max is human tissue over animal tissue are that, it can
unaffected by competitive inhibitors the provide a reliable and sensitive alternative. The
inverse of Vmax also doesn’t change) but there most commonly studied human tissue is vascular
are different slopes and x-intercepts between tissue such as veins (commonly available from
the two data sets. removal of varicosities), cardiac blood vessels
• Noncompetitive inhibition: Produces plots (available after transplant program), large blood
with the same x-intercept as uninhibited (Km vessels (available following major surgery, e.g.:
is unaffected) but different slopes and y- limb amputations or organ removal and small
intercepts. cardiac vessels (can be obtained from the atrial
• Uncompetitive inhibition: Causes different tip, removed to permit access to cannula during
intercepts on both the y- and x-axes but the cardiac surgery which requires bypass).
same slope. Recently, testing on human tissue validated many of the
pathways and proteins involved in the body functions such
as discovery of vascular endothelium as a major modulator
of vascular function and the discovery of the nitric oxide
pathway, the endothelin pathway, vascular endothelial
growth factor, and endothelium-derived hyperpolarizing
factor
Note: Tissue >2 mm exceeds the limits of diffusion and
which is maintained by continuous gassing, perfusion or
cooling at 4ºC.
Collection and storage of human tissue samples:

There are several problems in the collection of fresh
human tissue, namely cost, time and ethical issues
or even a conflict of interest between the surgeon,
pathologist and the researcher. The use of tissue or
storage varies with the quality of tissue and
experimental time, e.g. cardiac and brain tissues
have the shortest ‘experimental window’ and hence
Fig. 2.26: Woolf-Lineweaver Burk plot it is difficult to assay.
Bioassay  67
Brain tissue: Being most important part of body, it Method of field stimulation: A measure of
is most difficult tissue to obtain. The only possible sympathetic drive is assessed by using field-
way out is the tumor tissue removal, which is stimulation. The strain gauge is preferred because
preferably used fresh due to shortest experimental cannulation of small vessels is technically difficult
window. and therefore limits the number of arteries that
can be studied simultaneously. Blood vessel is
Cardiac tissue: Vital part of body, hence one of the
placed between two platinum or silver plates,
difficult human tissue to obtain and only obtained
through which a small voltage is applied. The
after some surgical procedure like cardiac resection,
resulting electrical field stimulates the
transplant or postmortem, whereas, atrial tissues are
sympathetic nerves on the blood vessel to release
freely available from atrial appendage removal
their catecholamine stores, producing a
during cardiac catheterization. Cardiac tissue
measurable vasoconstriction response.
must be used freshly or may be stored in an
arrested state (4°C), to reduce metabolic activity Method of pressure-flow system: A pressure-flow
and avoid degradation. Preservation up to two system is an alternative methodology, where the
weeks (remain functional) can be achieved by vascular tube is cannulated at each end, enabling
submerging tissue into physiological salt solution fluid to be perfused at physiological levels. The
(PSS) and oxygenating at 37.8°C. Experiments may pressure-flow systems offer up to ten times more
be carried out for maximum of 8-12 hours. sensitivity than the strain gauge. In this assay, the
transducers measure pressure and flow rather
Lung tissues (bronchial rings or thin strips of than force. The technique also uses imaging
parenchymal tissue): Respiratory tissue is soft and microscopy to measure changes in the lumen
delicate hence, it gets easily damaged, so requires diameter that correlate directly with vaso-
skilled handling. Lung tissue is most commonly constriction or relaxation.
obtained from tissue removed for the treatment of Strain gauges also alter the natural orien-
lung cancer. Thin strips of parenchyma are used as tation and environment of the tested tissue,
an isolated tissue preparation in an organ bath whereas pressure-flow systems are much less
for the measurement of contraction using a disruptive.
modified strain gauge technique. Isolated bronchial For example, cardiac tissue is cut into strips
rings dissected within 24 hours of surgery retain that are placed in an organ bath and stimulated
the ability to respond to nerve stimulation and using platinum or silver electrodes. Test drugs
can generate an IgE-mediated sensitization of the are added, and the resulting changes in cardiac
responses to bronchoconstrictors. muscle contractility are recorded.
Vascular tissue (arterial tissue, intestine, stomach): Note: In experiment, students may use isolated arterial
Arterial tissues may be obtained from small strip or ring segment, uterus smooth muscle, lung slices
arteries of skin, gut and skeletal muscle after or tracheal rings, colon (mucosal layer) etc. in the human
biopsy, whereas readily available from surgical tissues bioassay experiment.
Temperature condition: 37°-38°C
removal procedures such as tumor. Effect observed: Contractility (In few tissues like isolated
lung slices or colon mucosal layer release of cytokines
Objective and other signaling factors may study)
Technique should be sensitive and reliable
because it mimic in vivo environment equivalent BIOASSAY OF CYTOKINES
to human being.
Bioassay must generate a quantitatively
Method measurable parameter that reproducibly increases
The choice of method depends on the level of with increasing cytokine concentration. Biological
sensitivity required for the procedure. assay (bioassay) of cytokines is important for
biological characterization and potency molecules with complex structure and molecular
determination due to clinic as biotherapeutic composition, which cannot be completely deter-
agents for malignant, infectious or autoimmune mined by physical and chemical methods alone.
diseases. The main reason of bioassay is that these Principle of bioassay is also applied when two
are biologically active proteins and cannot be or more preparations of an individual protein are
completely characterized by physiochemical being compared, they should be identical in the
methods alone. bioassay. Repeatability, intermediate precision,
and reproducibility are the main advantages in
Objective conducting cytokine bioassay. It helps greatly in
that a bioassay has the appropriate specificity,
The parameters most often measured in cytokine
i.e. the measured parameter is exclusively related
bioassays include increase/decrease in (live) cell to the concentration of the biologically active
number, inhibition of virus replication, increase protein.
in (cell) colony number, and increase in cell surface
Note:
antigen expression.
• Reproducibility is always essential for
Cytokines for bioassay: Interleukins (ILs), analytical procedure. This is best achieved by
interferons (IFNs), hematopoietic (colony) identifying and minimizing possible known
stimulating factors (CSFs), tumor necrosis factors variables.
• Presence of other substances such as active
(TNFs), and certain (polypeptide) growth factors
impurities/contaminants may interfere with
(GFs) and many more.
results.
Principle of Cytokines Bioassay Procedure for Cytokines Bioassay
Principle of assaying cytokines depends on the (Flow Chart 2.2)
quantitative comparison of test sample to a Currently, there is large number of cytokines,
standard sample. Most proteins are relatively large mostly rDNA-derived, which are available
Flow Chart 2.2: An example of estimation of IL 6 cytokine by the ELISA method
Note: Washing is an important procedure during the assay, hence protocol of washing should be followed properly
Bioassay  69
commercially for several therapeutic purposes. Log dose conversion

A major problem with clinical-grade cytokine s 1 = 0.2 × 10–7= log (0.2 × 10–7)
products from an assay point of view is that they = log (0.2) + (–7) log 10 = –0.69897 + (–7) x 1
are very highly potent. Their potencies often = –7.69897
exceed 1.0 million IU (MIU) or greater and s 2 = 0.4 × 10–7 = log (0.4 × 10–7)
therefore they require considerable dilution = log (0.4) + (–7) log 10 = –0.39794 + (–7) × 1
before titration in bioassays. ‘Off-plate’ dilutions = –7.39794
are very important step which often involve t1 = 0.2 × 10-3 = log (0.2 x 10–3)
several 10-fold dilution steps. Undiluted = log (0.2) + (–3) log 10 = –0.69897 + (–3) × 1
constituents of the samples may interfere with = –3.69897
bioassay performance.
t2 = 0.4 × 10–3= log (0.4 × 10–3)
Note: Dilution at each stage carried out with = log (0.4) + (–3) log 10 = –0.39794 + (–3) × 1
micropipettes calibrated for precise delivery of the = –3.39794
dilution volume.
S1 S2 T1 T2
EXAMPLE OF PERFORMING A SET OF BIOASSAY S2 T1 T2 S1
Latin square design
T1 T2 S1 S2
Question: 1 T2 S1 S2 T1
Write the Protocol and undertake the 4-point

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

09 + 08
histamine, hence, as the rule in the experiment the = 0.30103
tissue selection will depend on the sensitivity of 18.50 17.50
the tissue which is most sensitive to histamine. 17
Therefore, your tissue of choice should be Guinea = 0.30103 = 0.142153
36
Pig ileum.
The response taken here is approx value (not Conc. of unknown
to scale; made for understanding purpose) s1
Responses (mm) Mean = antilog M
t1
S1 34 38 33 32 34.25 7
S2 52 57 49 53 52.75 0.2 10
= antilog (0.142153)
T1 26 29 23 27 26.25 0.2 10 3
T2 45 43 44 43 43.75
= “x”
Hence, the given solution is the “x” times more Tissue: Rat ileum (best tissue is leech dorsal
potent than standard. muscle or frog rectus abdominis muscle) (Subject
Representation of plotted DRC is shown in to availability in laboratory).
Figure 2.27 and representation of results is shown
Step 3: Selection of PSS: Tyrode
in Figure 2.31.
Step 4: It is fast contracting hence, magnification
Question: 2 should be low
To determine the unknown concentration of given Magnification: 7x
acetylcholine (ACh) by using suitable tissue.
Step 5: Adjust temperature between 35-37°C.
Answer
Step 6: Prepare the tyrode and collect the tissue.
Step 1: Start with aim then write down the Keep tissue in continuous slow oxygenation.
principle of bioassay, procedure and if possible
Step 7: Balance the lever and then tie the tissue
make the flow diagram for the procedure
and adjust the weight by counterweight at other
(explained briefly, see text in Chapter; Flow Chart
side of fulcrum.
2.1).
Step 8: Leave the tissue in bath for relaxation for
Step 2: Commonly all tissues are sensitive to ACh, at least 30 - 45 min, meanwhile change the PSS 3-
but select the tissue which is reliable, sensitive 4 times during the relaxation (chances of pH
and durable. change after prolonged oxygenation).
Aim of the experiment: To determine the unknown concentration of histamine in the given sample in an in vitro tissue
of your preference.
Tissue selected: Guinea pig ileum (Highly sensitive tissue for histamine)
PSS: Tyrode
Dose cycle: 3 min
Temp: 35-37°C
Level: Isotonic (Frontal writing lever)
Magnification: 7x
Inner organ bath volume: 10 ml
Dose cycle: 3 min
Fig. 2.27: Presentation of kymogram after the experiment is over. Write down every variables used in the experiment
Bioassay  71
Step 9: Prepare the standard drug and select the For example: If the drug concentration is (S1= 0.8
experimental design (3-point, 4-point or others). × 10–7) and volume inner organ bath is 10 ml, then the
Step 10: 3-point is selected due to less time concentration of the drug to which tissue come in contact
consumption than 4-point and reduced variability will be (S1= 0.8 X 10-8), so for the calculation (S1= 0.8
than bracketing and matching assay. X 10-8) should be taken and all the concentration will
be changed accordingly.
Response recording (Arbitrary value;
not to scale)
Responses (mm) Mean

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

Question 3 4 times during the relaxation (chances of pH

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

Bioassay  73
DRC response of agonist Antagonist concentration used is 10–10

Reponse in mm % Response of maximal response Antagonist logdose Response
12 44.44444 concentration
16 59.25926 0.2 × 10–10 log (0.2) + (–10) log 10 17
20 74.07407 = –0.69897 – 10 = –10.69897 20
24 88.88889 2R 23
27 100 0.4 × 10–10 log (0.4) – 10 = –0.39794 – 10 10
* If the 2R is selected as the 24 mm response, then = –10.39794 16
targeted ‘R’ response should be ‘12 mm 20
24
0.3 × 10–10 log (0.3) – 10 = –0.52288 – 8 12
–log (antagonist) Response % Response to 2R = –10.52288 16
10.70 17 41.66667 22
10.40 10 83.33333
10.52 12 70.83333
Fig. 2.30: Calculation and determination of pA2 ; Inference: Write down about the finding of the study [Value of –log (antagonist)]
Figs 2.31A to D: Representation of bioassay result by graphical methods. The student can represent their
findings by any of the graph given above
SUGGESTED READING 15. Halpern W, Mulvany MJ, Warshaw DM. Mechanical

properties of smooth muscle cells in the walls of
Bioassay arterial resistance vessels. J Physiol 1978;275:85-101.
1. Allen DD, Caviedes R, Cárdenas AM, Shimahara T, 16. Hulsmann AR, de Jongste JC. Studies of human
Segura-Aguilar J, Caviedes PA. Cell lines as in vitro airways in vitro: A review of the methodology. J
models for drug screening and toxicity studies. Drug Pharmacol Toxicol Methods 1993;30(3):117-32.
Dev Ind Pharm 2005;31(8):757-68. 17. Jung SP, Siegrist B, Wang YZ, Wade MR, Anthony
2. Arunlakshana O, Schild H O. Some quantitative uses CT, Hornick C, etc. Effect of human Angiostatin
of drug antagonists. Brit J Pharmacol 1959;14:48- protein on human angiogenesis in vitro. Angiogenesis
50. 2003;6(3):233-40.
3. Bliss CI, Cattell McK. Biological assay. Annual review 18. Lareau S, Moore R, Mainwood GW, Labow RS, Keon
of physiology 1943;5:479-539. WJ, Deslauriers R. An NMR probe to study function
and metabolism simultaneously in isolated human
4. Bliss CI. The design of biological assays. Annals of
cardiac tissue. Magn Reson Med 1999;20(2):312-18.
the New York Academy of Sciences 1950;52:877-88.
19. National Bioethics Advisory Commission, (2001)
5. Box GEP, Hay WA. A statistical design for the
Ethical and Policy Issues in Research Involving
efficient removal of trends occurring in a comparative
Human Subjects. NBAC In: http://www.
experiment with an application in biological assay. bioethics.gov
Biometrics 1953;9:304-19. 20. Phung MW, Dass CR. In vitro and in vivo assays for
6. Burn JH. The errors of biological assay. Physiological angiogenesis-modulating drug discovery and
Reviews 1930;10:146-69. development. J Pharm Pharmacol 2006;58(2):153-
7. Butcher E C, Berg E L, Kunkel E J. Systems biology in 60.
drug discovery. Nature Biotechnol 2004;22:1253-59. 21. Schild H O. pAx a new scale for the measurement of
8. Cheng Hsien C. The power issue: Determination of drug antagonisum. Br J Pharmac 1947;2:184-206.
KB or Ki from IC50. A closer look at the Cheng- 22. Wadhwa M, Bird C, Dilger P, Mire-Sluis T, Thorpe
Prusoff equation, the Schild plot and related power R, Quantitative biological assays for individual
equations. Journal of Pharmacological and cytokines, in: F. Balkwill (Ed.), Cytokine Cell Biology:
Toxicological Methods 2001;46:61-71. A Practical Approach, Oxford University Press,
9. DeBeer E J. The calculation of biological assay results Oxford, UK 2000;207-39.
by the graphical methods. The all- or-none type of 23. WHO, Recommendations for the preparation,
response. J Pharmacol Exp Ther 1945;85:1-13. characterization and establishment of International
biological reference materials. WHO/BS/04.1995.
10. Eugene M Laska, Morris J Meisner. Statistical
www.who.int/biologicals.
methods and applications of bioassay. Ann. Rev.
Pharmacol. Toxicol 1987;27:385-97.
11. Finney DJ. The principle of bioassay. Journal of the
Bioassay of Antagonism
Royal Statistical Society 1947a;S9:46-91. 1. Arunlakshana O, Schild H O. Some quantitative uses
12. Finney MJ, Karlsson JA, Persson CG. Effects of of drug antagonists. Br J Pharmacol 1959;14:
bronchoconstrictors and bronchodilators on a novel 48-50.
human small airway preparation. Br J Pharmacol 2. Gaddum JH. Theories of drug antagonism.
1985;85(1):29-36. Pharmacol Rev 1957;9:211-18.
3. Ghosh K, Kowal D, Dawson L A, Tasse R. Design
13. Ghelani AM, Holroyde MC, Sheard P. Response of
and models for estimating antagonist potency (pA2,
human isolated bronchial and lung parenchymal
Kd and IC50) following the detection of antagonism
strips to SRS-A and other mediators of asthmatic
observed in the presence of intrinsic activity.
bronchospasm. Br J Pharmacol 1980;71(1):107-12. Neuropharmacology 1999;38(30):361-73.
14. Ghosh K, Kowal D, Dawson L, A and Tasse R. Design 4. Jones RL, Wise1 H, Clark R, Whiting RL, Bley KR.
and models for estimating antagonist potency (pA2, Investigation of the prostacyclin (IP) receptor
Kd and IC50) following the detection of antagonism antagonist RO1138452 on isolated blood vessel and
observed in the presence of intrinsic activity. platelet preparations. Br J Pharmacol 2006;149:
Neuropharmacology 1999;38(3):361-73. 110-20
Bioassay  75
5. Kunchandy J, Kulkarni SK. Apparent pA2 estimation 10. Schild HO. pA, a new scale for the measurement of
of benzodiazepine receptor antagonists. Methods drug antagonisum. Br J Pharmacol 1947;2:184-206.
Find Exp Clin Pharmacol 1986;8:553-55. 11. Shannon HE, Cone EJ, Gorodetsky CW. Morphine-
6. Lazareno S, Birdsall NJM. Estimation of antagonist like discriminative stimulus effects of buprenorphine
Kb from the inhibition curves in functional and demethoxybuprenorphine in rats: Quantitative
experiments: Alternative to the Cheng-Prusoff antagonism by naloxone. J Pharmacol Exp Ther
equation. Trends Pharmacol Sci 1993b;14:37-239. 1984a;229:768-74.
7. Lazareno S, Birdsall NJM. Estimation of competitive 12. Spealman RD. Discriminative-stimulus effects of
antagonist affinity from functional inhibition curves midazolam in squirrel monkeys: Comparison with
using the Gaddum, Schild and Cheng-Prusoff other drugs and antagonism by Ro 15-1788. J
equations. Br J Pharmacol 1993a;109:1110-19. Pharmacol Exp Ther 1985;235:456-62.
8. Martin JR, Jenck F, Moreau JL. Comparison of 13. Takemori AE, Kupferberg HJ, Miller JW. Quantitative
benzodiazepine receptor ligands with partial studies of the antagonism of morphine by nalorphine
agonistic, antagonistic or partial inverse agonistic and naloxone. J Pharmacol Exp Ther 1969;169(1):
properties in precipitating withdrawal in squirrel 39-45.
monkeys. J Pharmacol Exp Ther 1995;275:405-11. 14. Waud DR. Analysis of dose-response curves. In: Daniel
9. Schild HO. Drug antagonism and pA2. Pharmacol EE, Paton, D.M. (Eds.), Methods in Pharmacology,
Rev 1957;9:242-46. vol. 3. Plenum Press, New York, 1975, 471-506.
Commonly Used Instruments
3 in Pharmacology Laboratory
Kymograph (Sherrington-Starling
Kymograph) (Fig. 3.1)
This is the basic instrument used to obtain a
graphical amplified measurable response of
a muscle or tissue (contraction/relaxation)
against a given concentration of drug or stimuli,
hence the resolution of the tissue response is
increased.
It consists of two important parts, i.e. a motor
box and drum. Motor operates drum in a variable
speed according to the need of the experiment or
the tissue attached to the recording lever.
Additionally, speed setting lever or variable speed
clutch lever, spindle and On/Off switch is
attached to motor box.
On/Off switch: This is the main power supply
board and it should remain ‘ON’ while doing the
practical.
Speed setting lever or Variable speed lever: This
lever is made to control the speed of the drum.
This ranges from 0.12-0.25 mm/sec to 320-640
mm/sec, i.e. slow or fast. The speed of the drum
mainly depends on the type of the tissue and dose Fig. 3.1: Kymograph and its different parts
cycle used.
Clutch lever: It disengages or engages the gear. to the electrically amplified impulse to record the
response. Its sensitivity is as high as 5 micro volts
Spindle and screw: Rod like structure which holds
per mm. The complete system is designed with
the drum in the vertical position, where stylus
couplers, matching transducers and pick-ups, and
touches the drum. Height of the drum can be adjusted
main console which includes the chart drive, pen
with lift screw attached at the top of the spindle.
recording system, the amplifier and coupler housing.
Student’s Physiograph (Fig. 3.2) Standard transducer for student physiograph: A
Physiograph is an electronic stimulator which is transducer is an electronic device which is
sensitive enough to convert the mechanical energy prompted by energy from one system (mechanical)
Commonly Used Instruments in Pharmacology Laboratory  77
Analgesiometer (Figs 3.3A and B)

The instrument works on the principle of
observing the pain threshold in the rodents before
and after the drug administration, so useful for
differentiating between centrally acting morphine-
like analgesics and non-opiate analgesics. This is
performed mainly to identify any pain stimulus
threshold in rodents against a radiant heat and to
screen analgesic drug by increasing pain
threshold. Hence, the radiant heat method is being
used for evaluation of systemic analgesic activity
Fig. 3.2: Student’s physiograph of a drug. The end-point of the experiment is considered
(For color version see Plate 1) as escape reaction which is considered to be controlled
by brain. There are two type of analgesiometer used
and converts energy to another. There are several in the animal experiment namely tail flick
types of transducers such as pressure transducer, analgesiometer and hotplate analgesiometer.
respiration belt transducer, force transducer, pulse Physiologically, tail flick is mainly mediated as a
transducer, isotonic transducer, etc. which are spinal reflex whereas hotplate paw withdrawal
very commonly used in the biomedical research. is mainly mediated by the brain. Therefore, the
Coupler is the main device which is attached observation of the escape reaction can be regarded
to the transducer, which should be compatible as a true assessment of the influence of the drug
with transducer and controls the input impulse. acting on the brain.
There are several types of couplers such as strain
gauge coupler which is mainly involved in the Instrument and its Parts
experiment of arterial and venous pulse, blood
pressure recording, BMR, spirometry A. Animal is placed into restrainer and leaving
experiment, in vitro frog heart preparation, the tail exposed outside the restrainer. Clean
isolated tissues, finger movement, etc. Second the tail with the help of cotton soaked in water
is biopotential coupler used in the recording of or ethanol. Then leave the tail for drying and
ECG, EEG, EMG, etc. and other less commonly also to familiarize with the restrainer. Then,
used are pulse/respiration coupler, temperature keep on the “tail flick analgesiometer”. 1/3rd
coupler, etc. tail proximally left due to the thick and
So, the application of Students physiograph is in keratinized skin and then keep tail on the place
several biomedical research fields such as made for tail above hot wire (made up of
recording in cardiovascular physiology (arterial nichrome wire) of the analgesiometer. The time
and venous pulse, blood pressure in dog/rabbit, of tail flick is measured and recorded. The cut-
ECG clinical, effect of vagal stimulation and drugs off time is set-up 15-20 sec for mice whereas in
on perfused isolated frog heart, etc.), the case of rat, cut off time is 20-30 sec to avoid
neuromuscular physiology (muscle twist, strength any further injury to the tail (Fig. 3.3A).
of stimulus, fatigue, isometric contraction, etc.), B. Instrument consists of an electrically heated
experimental respiratory analysis (respiratory surface (made up of iron, aluminum or copper)
movement, lung volumes, tidal volume, IRV, ERV, whose temperature is maintained by the
VC, IC, BMR, etc.), human experiment on ECG thermostat ‘Knob’ at 55° to 56 °C. After
clinical, EMG, EEG, etc. and to see the effect of maintaining the temperature mice are placed
drugs on other in vitro experiments like, isolated on the hotplate and observed for either paw
intestine, isolated uterus, sciatic nerve, etc. licking or jumping reaction. The reaction time
Figs 3.3A and B: (A) Tail flick analgesiometer: Showing on/off switch for power on/off, water inlet and outlet to maintain the
normal temperature surrounding the hot-wire and current control knob which control the hotness of nichrome wire.
(B) Eddy’s Hotplate analgesiometer: Showing on/off switch, Temperature control knob which can be adjusted on the
desired temperature (55-56°C), Thermometer is placed at the corner place for continuous observation of the temperature,
Hotplate on which the animal is kept for observation and lid cover of hotplate
is recorded by a stop-watch. Repeated reading jerks as well as face and forelimb clonus that may
is taken at 20, 60, and 90 minutes after the progress to rearing and falling down (such ictal
drug administration. The cut-off time is set-up behavior is referred to as limbic seizures) whereas,
15-20 sec for mice whereas in the case of rat convulsions mediated by the brainstem involve
cut-off time is 20-30 sec to avoid any further tonic-clonic convulsions. Brainstem seizures also
injury to the paw (Fig. 3.3B). may occur independently of forebrain neural
substrates once generalized tonic-clonic
Practical Application convulsions are induced by transauricular or
corneal electroshock (ES) in rats. The Seizure
Mainly used in the screening of centrally acting severity score followed for screening the
analgesics. However, screening of local anticonvulsant is evaluated by:
anesthetics or screening of muscle relaxant may 0 = no seizure; 1 = forelimb extension without
also be performed. hind limb extension; 2 = complete forelimb
Note: In screening of the analgesic drugs, result may
extension and partial hind limb extension,
misinterpreted with false positive result due to muscle 3 = complete (parallel to the tail) tonic hind limb
relaxant or local anesthetic drugs extension (THLE), 4 = postictal depression.
Instrument and its Parts

Maximal Electroshock Seizure (MES)
Electroshock is applied through a pair of ear-clip
Convulsiometer (Fig. 3.4)
electrodes or corneal electrode, using a sinusoidal
This instrument is used to induce convulsions maximal current of 150 mA, 50 Hz for 0.2 sec for
experimentally which are hypothesized to rat and 12 mA, 50 Hz for 0.2 sec for mice. Animal
originate from the forebrain or brainstem. is housed in acrylic cage, 1 min before the current
Convulsions mediated by the forebrain involve application, and observed for an additional 2 min
clonic spasms, behavioral immobility, myoclonic period. Recordings may be videotaped and
and closed arms entries reflect the relative safety

of closed arms compared with the relative
fearfulness of open arms. Anxiolytics would be
expected to increase the frequency of entries and
time spent into open arms. It is also a good
screening method for learning and memory in
rodents.
Fig. 3.4: Convulsiometer (For color version see Plate 1)
Morris Water Maze (Fig. 3.6)
watched for all stages of seizure. Its only
application is the screening, if the antiepileptic This instrument is designed to evaluate learning
drug acting on tonic-clonic convulsion and animal and memory in rodents and to evaluate effect of
should be screened one week prior to the drugs. The test apparatus consists of a circular
experimental day. fiberglass tank (130 cm in diameter, 50 cm in depth;
dimension for rat). The pool is filled to a height of
Elevated Plus Maze (Fig. 3.5) 30 cm with water at room temperature, 21-22°C.
The pool is divided into four vertical quadrants
This instrument is made for a novel test for the
(Q1, Q2, Q3 and Q4) of equal surface area. A
selective identification of ‘anxiogenic and
transparent escape platform (10 cm in diameter, 29
anxiolytic’ drug effects in rodents. It is also used
cm in height) is placed in a fixed location in the
to evaluate the learning and memory in rodents
tank in the center of one of a quadrant, 1 cm below
in presence or absence of a drug.
the water surface. The platform is not visible from
The plus maze apparatus consists of two open
just above water level. Many extra maze cues
(16 × 5 cm for mice and 50 × 10 cm for the rats) and
surrounding the maze are available for the rats to
two closed arms (16 × 5 × 12 cm for mice and 50 ×
use in locating the escape platform. The maximum
10 × 40 cm for rats), and an open roof with the
cut-off time for learning and memory is 300s.
entire maze elevated (25 cm for mice and 50 cm for
rats) from the floor. The animals are placed Precaution in instrument use: Animals that do not
individually at the center of the elevated plus maze float on the water and search for the platform
with their head facing the open arm. During the 5 should be excluded from the study.
minutes test the preference of the animal for the
first entry, the numbers of entries into the open
Fig. 3.6: Morris water maze for the rat
Passive Avoidance Test (Fig. 3.7)

Fig. 3.5: Plus Maze apparatus for the rat (50 × 10 × 40 cm).
Apparatus consist of Shuttle-box divided into an
The closed arm painted black inside to evoke the night like
situation. All the movements of rat are videotaped in dim illuminated compartment and a dark compartment
lighted calm room of the same size by a wall with a guillotine door.
(TCP) method. This method is always preferred

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

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

Type of centrifuge Speed (rpm) Refrigeration RCF (g) Centrifuge tube used
Density gradient
Material Size
Low speed 1-6000 Some 6000 Glass/Plastic 10-50 ml
High speed 1000-25,000 All (near 4°C) 50,000 Plastic 0.5-2 ml
Ultracentrifuge 20-80,000 All 6,00,000 Plastic 0.25-2 ml
Sophisticated Instruments and Techniques Used in Pharmacology Laboratory  83
Components of Centrifuge
Figs 4.1A and B: (A) Showing the position of angle of rotation tube with axis of rotation axis. Its RCF value variation
with the increasing r/r’from axis of rotation (B) Showing the position of tube on the either side of axis of rotation
Figs 4.2A and B: (A) Showing the process of fixed angle rotor centrifuge and at time its separation in the two layers at
the rest; (B) Process of vertical rotor centrifuge and separation into two layer at the rest
Salient Feature of PCR

Primary application is in vitro amplification of
specific target DNA sequences and being highly
sensitivity. But, the limitation of the process is, it
requires highly skilled person, extremely liable to
contamination and it may not easy to set up a
quantitative assay.
High Performance Liquid Chromatography

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

Flow Chart 4.1: For PCR Run
Flow Chart 4.2: For nucleotide sequencing
Note: This is just a brief introduction to the PCR run and sequencing procedure for beginners. In each step, there are
several factors which have to be considered during the process such as temperature, concentration of PCR mixture,
agarose gel, primer designing, etc.
Fig. 4.4: Steps of polymerase chain reaction (PCR);

Time of extension and elongation is depend on selection of DNA polymerase
Note: Buffer solution: Makes environment favorable to DNA polymerase (It may contains divalent like Mg2+ and Mn2+ and
monovalent like K+ cations)
Basis of Column Selection suitable for the sample load. Smaller packing
particle sizes can provide higher column
Column selection is an important part in the
efficiency, hence give higher resolution. In
HPLC, which has whole influence on the process.
addition, column length gives better separ-
So, the selection of column depends on the several
ability.
factors which are molecular weight of the sample
(Exclusion limit molecular weight), Ability to Note: Smaller packing particle sizes and longer
dissolve the sample (Applicable to packing column length may increase the column pressure;
materials) and molecular weight distribution hence column should be able to hold the pressure.
(Range of calibration curve).
Column inner diameters and packing Basis of Detector Selection
material particle sizes play an important role in The objective of selecting a detector is to sense the
the efficiency of particle. HPLC columns range presence of a sample and send its corresponding
from 20 to 500 mm in length (L) and 1 to 100 mm electrical signal to a computer data system. The
in internal diameter (id). Sample load is selection is depending upon the characteristics
proportional to the column cross-sectional area. and concentrations of the compounds that need
It is necessary to select a column inner diameter to be separated and analyzed such as if,
Fig. 4.5: Components of HPLC (Mobile phase reservoir: Solvent which moves through the column; High-pressure pump:
Used to generate and meter a specified flow rate of mobile phase, typically milliliters per minute; Injector or Autosampler:
Able to inject /introduce the sample into the continuously flowing mobile phase that carries the sample into the HPLC
column; Column: Contains the chromatographic packing material (stationary phase) needed to effect the separation;
Detector: Needed to see the separated compound bands as they elute from the HPLC column. Data processor: detector
connected to computer which records the electrical signal generated to form the chromatogram for a detected sample
which further identified and quantified; Waste mobile phase collection: Mobile phase exiting through detector is collected as
waste)
compound is fluorescent then selects a with smaller particles [1-1.7 micron] and instrumentation,
fluorescence detector whereas if sample absorbs which is designed to deliver mobile phase at very high
ultraviolet light then use UV-absorbance detector. pressure, 15,000 to 100,000 psi is known as ultra-
performance liquid chromatography(UPLC).
Chromatogram Separation method selection depends on
The curve obtained after the electrical signal is resolution (Selectivity of the packing material for
generated and sent it to the computer data the compound of interest), load (Capacity of the
system. It generates a graph on computer screen packing material) and speed (Isolation time).
commonly known as “chromatogram”. It has Further, HPLC performs on the five important
several parts such as baseline i.e. graph separation mode namely (Table 4.2).
obtain by the mobile phase only, peaks, i.e • Normal phase chromatography (NP),
mainly obtained in the presence of the test • Reversed phase chromatography (RP),
substance or even in presence of some impurities
• Size exclusion chromatography (SEC),
(Fig. 4.6).
• Ion exchange chromatography(IEX) and
Important Point to Remember
Efficiency of HPLC is a measure of mechanical separation • Affinity chromatography (AC).
power whereas selectivity of HPLC is a measure of All these modes are summarized in the table.
chemical separation power.
Ultra-performance Liquid Chromatography (UPLC)
Application
HPLC is well known for its selectivity, efficiency and
high resolution due to the improved column technology. HPLC is widely used in biotechnological,
But, if its resolution is increased by the columns biomedical, and biochemical research, but very
Fig. 4.6: HPLC chromatogram
commonly used to determine concentration of detection of mass technique. There are two
drug in plasma/serum in the therapeutic drug common Atmospheric Pressure Ionization (API)
monitoring (TDM) LC/MS processes; Electrospray Ionization (ESI)
HPLC is also used in a variety of other fields and Atmospheric Pressure Chemical Ionization
and industries including the cosmetics, energy, (APCI). Both are soft ionization technique and
food, and environmental studies are compatible with most chromatographic
separations. APCI is regarded as a soft ionization
Liquid Chromatography and Mass- process which results in a mass spectrum typically
Spectrometry (LC-MS) (Fig. 4.7) dominated by a single ion (either + or –) that
In general, coupling of a mass spectrometer to an corresponds directly to the molecular weight of
HPLC system is called LC/MS. So, it is the compound, which is protonated in the
combination of powerful analytical separation positive ion mode (M+H)+ or deprotonated in
technique with a powerful analysis and the negative ion mode (M-H)-.
Table 4.2: Summary of different types of mode of HPLC, its requirement and features
NPC RPC SEC IEC AC
Stationary phase Silica gel (Polar) Silica-ODS Porous Polymer Ion exchange gel Packing with
(Silica-C18) Aqueous porous ligand
(Non-Polar) Polymer
Mobile phase Organic solvent MeOH/Water Organic solvent Buffer solution Buffer solution
(n-Hexane/IPE) (Polar) (THF)Buffer
(100% organic) solution
(Non-Polar)
Type of Adsorption Hydrophobic Gel permeation Ion exchange Affinity
Interaction
Feature Fat-soluble Most widely used Molecular weight Separation of Purification of
distribution ionic substances enzymes and
Protein Separation proteins
Normal phase chromatography (NPC); Reversed phase chromatography (RPC); Size exclusion chromatography (SEC);
Ion exchange chromatography (IEC); Affinity chromatography (AC)
Fig. 4.7: Component of LC-MS
Salient feature of LCMS is that it can detect the Telemetry

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

telemetry is in use for the determination of natural
• Telemetry is used in the experimental as well
as clinical practices. For monitoring tempe- reading in experimental animal or human in
rature clinics. It is hypothesized that the biochemical or
• Cardiovascular parameters (BP, HR, ECG, etc.) physiological parameters may alter during the
• Respiratory parameters (FEV1 , VO 2 con- anxiety or hormonal changes in the animal during
sumption, etc.) the handling or human in variable conditions.
• CNS (EEG etc.) Telemetry is also used clinically for patients who
• Others like locomotor activity, exploratory are at risk of abnormal heart activity or CNS or
behavior, etc. gastrointestinal activity.
Pyrogen Test
5 (In Vivo and In Vitro Methods)
INTRODUCTION intravenously within a period of not more than

ten minutes.
Pyrogen is defined as an agent that causes rise
in temperature which can be polysaccharide or LABORATORY CONDITIONS
protein in nature. The pyrogenic molecules can
be exogenous or endogenous whose presence The test is performed in a quiet room maintained
in injectables may be detrimental to the patients. at a temperature of 24-26°C. The temperature
Effect of infusion of exogenous pyrogens in the should not differ for more than ± 5° C from the
body may vary from mild pyrogenic effect to animal house.
shock. These manifestations underline the
importance of pyrogen testing in injectable APPARATUS
fluids.
A number of in vivo and in vitro methods are 1. Restrainer: Wooden restrainer is used. The
available for pyrogen testing. In vivo methods restrainer is of the size to allow normal sitting
have the advantage of detecting any pyrogenic position of the rabbit.
molecule, which may be protein or poly- 2. Glassware: The glassware used should be
saccharide, or some other unknown molecule sterilized by autoclave at 125°C for 8 minutes.
whereas disadvantage of being too sensitive 3. Syringes and needles: Sterile disposable syringes
and may sometimes give erroneous results due and needles are used for the test.
to biological variations. “In vitro” methods like 4. Recording of temperature: An accurate
Limulus Amebocyte Lysate (LAL) test have also temperature-sensing device, a mercury
been described but they are not cost effective thermometer attached to a probe, calibrated to
and may show variable results with new assure an accuracy of ± 0.1oC is used. The
molecules. Additionally, some protein temperature-sensing probe is inserted into the
molecules like recombinant products may show rectum of the test rabbit to a depth of not less
pyrogenic response just because of their than 7.5 cm, and, after an interval of 5 minutes,
chemistry. Conventional method of in vivo the rabbit’s body temperature is recorded.
pyrogen testing is the method by using rabbits.
TEST ANIMALS
Principle: The test involves the measurement of
the rise in body temperature of rabbits following Healthy, adult white New Zealand rabbits of
the intravenous injection of a sterile solution of either sex, preferably of the same variety, weighing
the substance being examined. It is designed for not less than 1.5 kg, fed on a complete and
products that can be tolerated by the rabbit in a balanced diet and not showing loss of body weight
dose not to exceed 10 ml per kg injected during the week preceding the test are used.
METHOD Procedure
Preliminary Test (Sham Test) The temperature of each animal is recorded at

intervals of thirty minutes, beginning ninety
In case animals are used for the first time in a minutes before the injection of the solution being
pyrogen test or have not been used during the two
examined and is continued for three hours after
previous weeks, conditioning for one to three days
the injection. Thirty minutes immediately
before testing the substance is done. After an
preceding the injection of the test dose, the initial
overnight fast, the animals are examined, by
temperature of each rabbit is recorded, which is
injecting intravenously into them 10 ml/kg of
the mean of two temperature readings recorded
body weight of a pyrogen free saline solution.
for that rabbit at an interval of fifteen minutes in
Water is withheld during the test. The temperature
the thirty-minute period. In any one group of test
of the animals is recorded beginning 90 minutes
before injection and continued for 3 hours after animals, only those animals whose initial
injection of the solution being examined. Any temperatures do not vary by more than +1oC from
animal showing a temperature variation of 0.6oC each other is used.
or more is excluded from the main test (Fig. 5.1). Rabbits usually show a temperature
variation greater than ± 0.2oC between two
Main Test successive readings in the determination of
“initial temperature (IT)” should be excluded from
Main test is done by using a group of three rabbits. the test. The solution being examined is injected
slowly into the marginal vein of the ear of each
Preparation of the Sample rabbit over a period not exceeding ten minutes,
The test substance is dissolved in a pyrogen free unless otherwise prescribed in the monograph.
saline solution. The liquids being examined is The temperature of each animal is recorded at
warmed to approximately 38oC before injection. half-hourly intervals for three hours after the
The amount of the sample to be injected is varying injection. The highest temperature difference of
according to the preparation being examined. The all the recorded different temperatures for a
volume of injection is not being less than 0.5 ml rabbit is taken as its response. When this
per kg and not more than 10 ml per kg of body difference is negative, the result is counted as a
weight. zero response.
Fig. 5.1: In vivo Pyrogen test. Design and selection of rabbits for sham and main test
Pyrogen Test (In Vivo and In Vitro Methods)  93
Interpretation of Results substance with the cells for a specified period of

If the sum of the responses of the group of three time. Concentration of pro-inflammatory
rabbits does not exceed 1.4oC and if the response cytokines (e.g., IL-1α, IL-6) is quantified via a
of any individual rabbit is less than 0.6oC, the cytokine-specific enzyme-linked immunosorbent
preparation being examined passes the test. If the assay (ELISA) by comparison to a standard curve.
response of any rabbit is 0.6oC or more, or if the Using an endotoxin standard curve, the endotoxin
sum of the responses of the three rabbits exceeds content of the product is calculated.
1.4oC, continue the test using five other rabbits. If A product “passes” if the endotoxin content
not more than three of the eight rabbits show is < 0.5 endotoxin units (EU)/mL.
individual responses of 0.6oC or more, and if the Laboratory condition: Cell culture grow at
sum of the responses of the group of eight rabbits specified conditions hence, the laboratory
does not exceed 3.7oC, the preparation being condition is maintained at temperature (37°C ±
examined passes the test. 1°C), humidity (90% ± 10%), Air (5.0% O2 ± 1%
Animals are not used for testing more CO2 in ambient air).
frequently than once every 48 hrs. After the animal Cell culture required are mammalian cell line,
has shown positive response, at least two weeks primary cells, or heparinized whole blood or
are allowed to recover the animal. cryopreserved cells/whole blood and fresh whole
blood (should be used within 4 hrs of collection)
Pyrogen Testing by “in vitro” Method may be also used.
Due to the expertise hand and long time required Note: Cell cultures should be free of contamination
for the experiment, in vivo method is not adopted with bacteria, mycoplasma, or fungi.
for the pyrogen testing in present scenario. So, in
vitro method was developed, which is equally Precaution during the Experiment
good and have been taking comparative less time. Instrument used in the test should be sterile,
It is also preferred due to its high precision, pyrogen free (e.g., pipette tips, pipettes, culture
sensitivity, reliability and reproducibility over the in ware, etc.)
vivo method where chances for inter individual Test substances should be diluted in sterile,
variation is very high. There are parenteral drugs pyrogen-free 0.9% NaCl.
which may contain endotoxin, augment in vivo Medical devices can be directly incubated with
biological activities. Hence, such drugs require the cells in suspension.
not only quantifying endotoxin but also regulate Test substances should be fully solubilized
overall in vivo action of contaminated parenteral (i.e., no visual observation of test substance in the
drugs. Hence, in vitro method, not only establishes dosing solution).
the presence of pyrogen but also endotoxins in Methods
contaminated parenteral drugs.
During the experiment, it is advisable to add three
The performance standards consist of 1)
groups of control namely
essential test method components, 2) reference “Negative control” = to assure the proper
substances, and 3) the comparable accuracy and functioning of the test
reliability that should be achieved. procedure (Vehicle group)
“Positive control” = to assure the enough
Principles sensitivity to a pyrogenic
The principle of in vitro pyrogenicity test methods substances (international
is based predominantly on the human cell type reference standard endotoxin
used, and its assessment for the pyrogen and such as E.coli) and
endotoxin. Specific human-derived cells used in “Benchmark control” = to ensure the test method
the in vitro test method and incubation of test is functioning properly.
The reliability and relevance of each test SUGGESTED READING

method is evaluated with pyrogen-free parenteral
1. Akihiko Yamamoto, Masaki Ochiai, Kazunari
drugs spiked with different concentrations of an
Kamachi, Michiyo Kataoka, Hiromi Toyoizumi,
endotoxin standard. In vitro pyrogenicity test Yoshichika Arakawa, et al. A clinically relevant in
methods may be used to test parenteral drugs, vitro pyrogen test using a human cell line that
biological products and medical devices. have the similar responsiveness to various pyrogens
to that of human peripheral blood cells (hPBC). In:
Test Substance Application and Sample AATEX 14, Proc. 6th World Congress on Alternatives
Collection and Animal Use in the Life Sciences: Special Issue,
2007Aug 21-25; 2007;647-53.
2. Finney DJ. Statistical Methods in Biological Assay.
3rd edn. Charles Griffin and Co., Ltd., London 1978.
3. Indian pharmacopeia; 1996: A-28.
4. Moesby L, Jensen S, Hanse EW, Christensen JD. A
comparative study of Mono Mac 6 cells, isolated
mononuclar cells and Limulus amoebocyte lysate
assay in pyrogen testing, Int J Pharm 1999;191:
141-49.
5. Pool EJ, Johaar G, James S, Petersen I, Bouic P. The
detection of pyrogens in blood products using as ex
vivo whole blood culture assay. J Immunoassay
Interpretation of Result 1998:19;95-111.
6. Thomas H et al. Statement on the varidity of in vitro
Positive control samples curve is used to calculate
pyrogen tests 2006.
the level of relative release cytokine in the sample.
7. Yamamoto A, Ochiai M, Kataoka M, Toyoizumi H,
When, the level of cytokine release is greater than
Horiuchi Y. Development of a Highly Sensitive in
or equal to that induced by the 0.5 EU/mL vitro Assay Method for Biological Activity of
endotoxin standard, then the sample is considered Endotoxin Contamination in Biological Products.
positive for a pyrogen test. Biologicals 2002;30:85-92.
Practical Aspects of
6 Cell Culture
This chapter includes the basic principles and the use of cell culture is very efficient to produce
methodology of cell culture in the pharmacology. the reliable data.
The important advantages are that it facilitates In general, the cell culture is the cultivation or
analysis of biological properties of the host growth of cells outside of the host organism that
organism and effect of several drugs on the system may include eukaryotic or prokaryotic cells. The
at the laboratory setting, which generally not advantage over other method of assessment is that,
readily accessible at the level of the intact it allows direct access to a population of cells
outside the parent organism and the effect is
organism. Historically, cell culture started with
directly seen. But, drawbacks are the architecture
the maintenance of medullary plate of an
of the original tissue is lost and the cells change
embryonic chicken in warm saline by Wilhelm properties over time.
Roux in 1885, whereas, methodology of tissue
culture was established by Ross Granville Harrison. Two Types of Cell Culture (Fig. 6.1)
In the recent times, there is more ethical constraint • Primary cell culture
on the use of animal in the research or experiment, • Cell line culture or subculture (secondary or
when other alternative methods are available. So, tertiary culture or more)
Organ culture: survival up to 3 weeks
Fig. 6.1: Classification of cell culture; primary culture is usually fibroblasts of the dermis or the basal epithelial layer of the
epidermis that gives rise to high density of the cells. These cells represent the largest compartment of proliferating, or
potentially proliferating, cells. Subculture is prepared from the primary culture by rinsing, dissection, and either mechanical
disaggregation or enzymatic digestion in trypsin and/or collagenase
Primary Culture Note: A cell line is the culture that is produced

from subculture of the primary cell culture and
These are the cell type which is explanted directly
further additional subculture. After this increases
from an organism, animals or human, which are
the possibility of cross-contamination, so
capable cell divisions in culture under the
verification of origin is important.
standard maintained conditions. While main-
taining and developing in culture it retains Principles of Cell Culture
characteristics of normal cells as in the host organ.
Because of these reasons, it is the best experimental There are four basic requirements for cell culture
in vitro models to assess the activity of any drug, namely, sterile condition, its medium and nutrition,
which gives rise to the comparable good result principles of cell growth and basic knowledge and
than the other in vitro study. practice of techniques involved in the growth of a
But, the limitation being these are susceptible particular cell type. The basic principles of cell
to contamination and have limited period to grow culture maintenance of a non-adherent cell line and
and eventually senesce and die. Senescence is adherent cell line is almost similar.
determined by a number of intrinsic factors Cryopreservation is other important principle for
regulating cell cycle, such as Rb and p53 and is the storage of cell lines for further subculture.
accompanied by shortening of the telomeres on Principle of Sterile Condition is the most important
the chromosomes. Once the telomeres reach a principle to get the good result. For this biological
safety cabinet should be turned on and allowed to
critical minimum length, the cell can no longer
run for at least 15 min to remove the contaminated
divide. Telomere length is maintained by
air. All work surfaces within the cabinet should be
telomerase, which is down regulated in most
normal cells except germ cells. decontaminated with an appropriate solution
(either 70% ethanol or isopropanol). Antibiotics
Cell Line Culture or Secondary Cell Culture are used sometimes to avoid any contamination in
the cell culture, e.g: Amphotericin B, Ampicillin,
As the name suggest, it is the second culture which Gentamicin, Kanamycin, Penicillin, Streptomycin,
was originally explanted from a donor organism Tetracycline and many more. Mainly antibiotics
and preserved. These cells are terminally are used during collection, transportation, and
differentiated cells and harder to maintain than dissection of biopsy samples because of the
less specialized cells. For example, animal (mouse intrinsic contamination risk involved in these
or rat) cells are easy to maintain than the human operations. The second principle is principle of
cells. These cells do not divide indefinitely and medium or nutrition used, which are required for
eventually their physical characteristics will keep survival and growth of cell culture. Nutrient
changing to mature and die. Therefore, limited medium should be sterile and cannot be autoclaved.
number of subcultivations from these cells is The compounds in nutrient medium are destroyed
possible. Cells always get to a certain density and by the heat of autoclaving. Medium must therefore
stop dividing even if nutrients are present in the be sterilized by passing it through a sterile filter
culture. These limits in the process is defined as small enough in pore size to hold back bacteria and
Hayflick’s limit (1961). mycoplasmas, e.g: membrane filter. Then, the third
Whereas, other type of cells known as “infinite principle is principle of cell growth, majority of cell
cell line.” These cells are easy to maintain, culture carried out by researchers involves the use
manipulate in culture and easy to obtain large of various nonadherent (i.e. P815, EL-4, etc.) or
quantities. So, the other most important is that adherent (i.e. FM3, STO, NIH 3T3, etc.) continuously
these cell lines subcultured indefinitely, i.e. these growing cell lines. The cell population can be
cells are immortalized. grown by selection of the correct medium (e.g.,
Practical Aspects of Cell Culture  97
keratinocyte growth medium (KGM) or MCDB 153

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

Culture Growth Media
(The amount and concentration varies according
to the cell selected for growth)
Ingredients
• Ions: Na + , K + , Mg 2+, Ca 2+, Cl - , CO 2 or
bicarbonate, P+ etc.
• Sugars: Glucose
• Amino acids: 13 essential
• Trace elements: Iron, selenium, zinc, etc.
• Vitamins Fig. 6.2: Cell growth cycle during the cell growth in the
medium and showing the different procedure timeline and
• Fetal calf serum: Usually 10%: Nutralizes the its different phases of growth
trypsin, used to buffer toxic nutrients by
binding them and contains hormone like
Methodology
growth factors or peptide hormones accelerate
the growth. Tissue Collection and Transportation
• Antibiotics: Amphotericin B, Ampicillin,
Gentamicin, Kanamycin, etc.: To prevent the The important step in beginning of the cell culture
growth of bacterial or fungal contamination is collection of samples in a labeled container of
culture medium containing antibiotics. The
Physiological Condition to Maintain the Culture samples should be labeled and stored in a
Temperature : 37°C refrigerator, but freezing of sample should be
pH : 7.2-7.5 avoided. Transfer of sample should be protected,
Humidity : 80-95% preferably double wrapped in a sealed polythene
Gas phase : CO2 (5-10%) or Bicarbonate bag and transferred. (To avoid contamination).
Visible light : Should be kept in dark The following precautions should be taken during
the experiment on the cell line
Cryopreservation is the process which • Switch on the laminar flow and UV source of
involved in the storage of primary or secondary safety cabinet for at least 15 min before
cell culture for further use. Cryopreservation is
experiment
done with a slow cooling, 1°C/min, down to –70°C
• Clean the area with disinfectant (70% ethanol
and thereafter, rapid transfer to a liquid nitrogen
or isopropanol)
freezer High cell density at freezing (1 × 106–1 ×
• Never uncover sterile articles like flask, bottle,
107 cells/ml). Preservative such as glycerol or
dimethyl sulfoxide (DMSO) at 5-10% may be Petri dish, etc. in open air/place.
added to the cell culture to preserve for long • Cover the cell line as soon as procedure is
duration. finished (Never leave it open to the
After cryopreservation if someone wants to use environment)
the sample then rapid thawing and slow dilution, • Sterile pipettes should be taken from the
<“20-fold, in medium is used to dilute the wrapper at the time of use (Sterile pipettes don’t
preservative. need not to be flamed).
• Cells may die with a hot pipette and always • Techniques should be performed as rapidly
use the different pipettes for different bottles. as possible to minimize contamination.
(Do not draw from a different bottle with the
Protocol for the Experiment (Flow Chart 6.1)
same pipette)
• After removing the cap from a bottle, flask, etc. It is necessary to change the growth medium every
third day. Cells require subculturing according to
do not place the cap with the open end upright
their doubling time. Generally, they should be
on the laboratory bench (chances of contami-
subcultured twice a week.
nation) Adherent cells lines require trypsinization while
• Do not hold the opened flask/bottle straight the non-adherent cell lines do not require trypsini-
up into the air. If possible, tilt the container so zation (Figs. 6.3A and B). Generally cells should
that any falling microorganisms fall onto the be splitted 1: 3 to 1: 5 ratio depending upon the cell
lip (Avoid hand contamination) doubling period.
Flow Chart 6.1: Procedure for secondary cell line development
Note: Cells should be grow into the culture flask (T-75 cm2 or T-25 cm2)
Practical Aspects of Cell Culture  99
3. Das RC. Antibodies in biotech: year of the bear. Am

Biotechnol Lab 2003;20:4-6.
4. Finter NB, Garland AJM, Telling RC. Large-scale
mammalian cell culture: a perspective. Bioprocess
Technology 1990;10:1-14.
5. Freshney RI. Cell line provenance. Cytotechnology
2002;39:3-15.
6. Juliano RL. Signal transduction by cell adhesion
Figs 6.3A and B: (A) Adherent cell line growth receptors and the cytoskeleton: functions of integrins,
(B) Non-adherent cell line growth cadherins, selectins, and immunoglobulin-
superfamily members. Annu Rev Pharmacol. Toxicol
2002;42:283-323.
In T-75 cm2 culture flask initially 10-15 lac cells 7. Knazek RA, Gullin P, Kohler PO, Dedrick R. Cell
should be inoculated and the ideal capacity is 20- culture on artificial capillaries. An approach to tissue
25 ml while in T-25 cm2culture flask 2 lac cells growth in vitro. Science 1972;178:65-67.
can be inoculated and can contain culture medium 8. Marcovic O, Marcovic N. Cell cross-contamination
up to 6-8 ml. in cell cultures: the silent and neglected danger. In
vitro Cell Dev Biol 1998;34:108.
9. Pereira RM, Marques CC, Baptista MC, Vasques MI
SUGGESTED READING
and Horta AEM. Embryos and culture cells: A model
1. Beshay NM, Zordoky, Ayman OS. El-Kadi. H9c2 for studying the effect of progesterone. Animal
cell line is a valuable in vitro model to study the drug Reproduction Science 2009;111:31-40.
metabolizing enzymes in the heart. Journal of 10. Zhengshan Zhao, Dee Montgomery-Brock2, Cheng-
Pharmacological and Toxicological Methods 2007; Sheng Lee and Yuanan Lu. Establishment,
56:317-22. characterization and viral susceptibility of 3 new cell
2. Cerni C. Telomeres, telomerase, and myc. An update. lines from snakehead, Channa striatus (Blooch).
Mutat Res 2000;462:31-47. Methods in Cell Science 2003;25:155-66.
Preclinical to Clinical Drug
7 Dose Calculation
INTRODUCTION start of clinical study in drug discovery which

requires special permission through Investi-
The experiment on animals gives an overall idea gational New Drug Application (IND).
about the tested drug (pharmacodynamics, Derivation from the animal dose to the human
pharmacokinetic and toxicology) through various dose of a tested drug is important step due to
methods involved in the process. But, the ultimate chances of toxicities. So, question arises that “How
goal of any drug research is to use the drug in the much dose should be selected, which is safe for
patients care. So, it is most important to perform a the human?” The first dose used in human is
planned scientific study in the humans before, known as “maximum recommended starting
it’s clinical use. Preclinical study is primarily dose” (MRSD) or first human dose (FHD), entry
designed to find a lead compound with desired into human (EIH) or first time in man (FTIM) whose
efficacy and safety for clinical study through derivation initiates clinical trials of new tested
pharmacokinetics and pharmacodynamics data drug in adult healthy volunteers/patients.
obtained from in vitro and in vivo studies (Fig. 7.1).
Hence, the compound selected for the clinical
Requirements for Determination of the
study is based on the bioavailability, volume of
First Human Dose
distribution and tissue penetration, half-life and
its receptor binding study. Phase 0 or phase 1 • Preclinical data, including
clinical studies is end of preclinical study and – Pharmacodynamic studies,
Fig. 7.1: Schematic diagram of dose conversion from the in vitro studies to in vivo (animal)
and finally to in vivo (human) studies
Preclinical to Clinical Drug Dose Calculation  101
– Full toxicological studies of the drug, and to minimize chances of patient exposure to sub-
– Pharmacokinetics data (absorption, distri- therapeutic doses of drugs. So the approach
bution, metabolism, and excretion). applied is usually a multiple ascending dose
(MAD) instead of a single ascending dose (SAD)
Procedure of Starting-dose Calculations study. The principle in the treatment of cancer is
that more acceptance of toxicity to achieve
There are several approaches of extrapolation or
therapeutic benefit so the dose selected as starting-
transformation of doses from preclinical to clinical
dose calculation for anticancer is generally based
(human) study which mainly depend on the drugs
on a dose and dose schedule that have produced
behavior across different species and its
some toxicity in animals rather than on a dose
physiochemical and pharmacological nature. It is
that has been identified as safe in animals or
very important to find a starting dose in human but
NOAEL.
the method should be economical and less time-
consuming. These approaches can vary from Note: The dose selection should be appropriate
scientist to scientist and their training and because cytotoxic compounds have a very low
experience but broadly extrapolation of the first therapeutic index and a steep concentration–
human dose is divided into two categories (Fig. 7.2). response curve for safety.
1. Extrapolation of dose of cytotoxic drugs in
which patients are the target individuals and Procedure
2. Extrapolation of dose of non-cytotoxic drugs The approach is based on the toxic dose low (TDL)
in which healthy volunteers are the target and the concept of lethal dose (LD10) (Fig. 7.3).
individuals
Toxic dose low (TDL): TDL is defined as the lowest
dose of a drug which does not produce any
Cytotoxic Drugs
lethality when its dose is doubled but able to
The main objective of extrapolation of doses for change pathology in hematological, chemical,
cytotoxic drugs involves a therapeutic benefit and clinical, or morphological parameters.
Fig. 7.2: Different categories for extrapolation of safe starting dose

Approach 1: The Process of Calculating the

MRSD by using NOAEL Approach
Fig. 7.3: Representation of therapeutic, toxic and

lethal dose
Animal preferred: Dog or monkey

Drug dosing: Single dose and dosing for 5 days
(Dose is expressed as mg/m2)
Drug dose selection: 1/3rd of TDL
Lethal Dose (LD10)

It is defined as the lowest dose of a drug which
produces lethality to 10% of normal (non-tumor)
mice (LD10) during a specific period of observation
under standard condition. Step 1: No observed adverse effect level
Animal preferred: Mice (NOAEL) determination
Drug dosing : Single dose and dosing for 5 days The NOAEL is a generally accepted benchmark
(Dose is expressed as mg/m2) for safety which is derived from appropriate
Drug dose selection : LD10 (1/10th of LD10) animal models. This can serve as the starting
Note: If the calculated 1/10th of LD10 in mice and point for determining a reasonably safe starting
1/3rd TDL in large animal are different, then the dose (SSD) of a new therapeutic in healthy human
lower of the two doses be used as the starting dose. volunteers. Hence, the NOAEL is defined as the
highest dose of a drug that does not produce significant
Non-cytotoxic Drugs adverse effects.
There are four approaches which are preferred in Besides NOAEL, other more terms which may
the extrapolation of first human dose for non- confuse with is no observed effect level (NOEL)
cytotoxic drugs which is any pharmacodynamic actions of the
• Approach 1: No observable adverse effect level drug and may not raise a safety concern, lowest
(NOAEL) observed adverse effect level (LOAEL) is defined as
• Approach 2: Similar class of drug the dose of a drug at which it shows the lowest
• Approach 3: Pharmacokinetics adverse effect sometime also called as maximum
• Approach 4: Comparative tolerated dose (MTD).
Preclinical to Clinical Drug Dose Calculation  103
Three requisite findings of preclinical without prodromal indicators, variable bio-

toxicology studies that can be used to determine availability, irreversible toxicity, unexplained
the NOAEL are mortality, large variability in doses or AUC levels
1. Overt toxicity (e.g. clinical signs, macro- and eliciting effect, novel therapeutic target and
microscopic lesions); lowered when there is design and conduct of
2. Surrogate markers of toxicity (e.g. serum liver toxicological study is of good quality, therapeutics
enzyme levels); and should be administered by the same route,
3. Exaggerated pharmacodynamic effects. schedule, and duration of administration or
similar toxicity profile.
Step 2: Human equivalent dose (HED) calculation According to Association of Food and Drug Officials
Human equivalent dose (HED) is the dose which (AFDO) of the United States, the maximum starting dose
for the FHD study is the smallest of the following three
is obtained from converting mg/kg dose for each doses:
animal species to the mg/kg dose in human which is • 1/10 of the highest no-observed effect dose in rodents,
equivalent to the animal’s NOAEL on a mg/m2 • 1/6 of the highest no-effect dose in dogs, or
basis. This conversion process mainly depends • 1/3 of the highest no-effect dose in monkeys
on three major factor based on the body Surface
area (mg/m2), body weight (mg/kg), route of Step 5: Consideration of the pharmacologically
administration, etc. While extrapolating the active dose (PAD)
animal dose to the HED, most appropriate method PAD is an in vivo study to compare the dose
is used. decided by the HED and MRSD by the help of
pharmacodynamic model. HED can be derived
Step 3: Most appropriate species selection from a PAD estimate by using a body surface area
conversion factor (BSA-CF). This HED value
After selection of HED, the MRSD is calculated by should be compared directly to the MRSD. If this
using the most appropriate sensitive species pharmacologic HED is lower than the MRSD, it may
(defined as the species in which the minimum be appropriate to decrease the clinical starting dose
HED can be obtained). While determining, 1st for pragmatic or scientific reasons.
HED, human ADME data is obtained by the in Drugs like vasodilators, anticoagulants,
vitro studies. monoclonal antibodies, or growth factors from
which toxicity may arise from magnified
Step 4: Application of safety factor pharmacologic effects. Hence, PAD in these cases
Receiving 1st dose in the human after preclinical may be a more sensitive indicator of potential
study have many ethical issues, hence it is toxicity than the NOAEL and might therefore
important to give a marginally safe dose to the justify lowering the MRSD.
volunteers/patients. So, safety factor is included
Other Approaches
in the calculation of the human first dose.
Practically, for the calculation of MRSD, HED is Approach 2: Similar Class of Drug
divided by safety factor 10. While a safety factor of These are the investigational drugs which have
10 can generally be considered adequate for same chemical class or related chemical structure
protection of human subjects participating in or having similar toxicological profiles. For these
initial clinical trials, this safety factor may not be similar drug profile, first human dose is calculated
appropriate for all cases. The safety factor should as the ratio of an optimal starting dose of the
be raised when there is favorable reason for similar parent drug to its NOAEL dose.
increase such as steep dose-response curve, severe
toxicities, non-monitorable toxicity, toxicities FHDP/NDP = FHDi/NDi
where, pectives,” Journal of Clinical Pharmacology 1995;

FHDP : Optimal starting dose of parent 35:957-66.
compound 3. Brodie BB, Reid WD. Some pharmacological
consequences of species variation in rates of
NDP : No observed adverse effect level
metabolism. Fed Proc 1967;26:1062-70.
of parent drug 4. Freireich EJ, Gehan EA, Rall DP, Schmidt LH, Skipper
FHDi : Starting dose of investigational HE. “Quantitative Comparison of Toxicity of
drug Anticancer Agents in Mouse, Rat, Hamster, Dog,
NDi : No observed adverse effect level Monkey, and Man,”Cancer Chemotherapy Reports
of investigational drug 1966;50:219-44.
5. Goldsmith MA, Slavik M, Carter SK. Quantitative
prediction of drug toxicity in humans from toxicology
Approach 3: Pharmacokinetic
in small and large animals. Cancer Res 1975;35:
In this approach NOAEL and its corresponding 1354-64.
AUC are used. The lowest dose from more than 6. Gomeni R, Bani M, D’Angeli C, Corsi M, Bye A.
Computer-Assisted Drug Development (CADD): An
one animal species are calculated and selected.
emerging technology for designing first-time-in man
The clearance of the drug in humans (CLh) is and proof-of concept studies from preclinical
predicted using allometric scaling. Then, the experiments. Eur J Pharm Sci 2001;13:261-70.
starting dose is calculated as: 7. Lave T, Coassolo P, Reigner B. Prediction of hepatic
metabolic clearance based on interspecies allometric
FHD = AUCp × CLh scaling techniques and in vitro–in vivo correlations.
where Clin Pharmacokinet 1999;36:211-31.
8. Mahmood I, Balian JD. The pharmacokinetic
FHD : First human dose
principles behind scaling from preclinical results to
AUCp : AUC obtained at the NOAEL in phase I protocols. Clin Pharmacokinet 1999;36:
preclinical toxicology studies, and 1-11.
CLh : Clearance in humans predicted by 9. Mahmood I. Allometric issues in drug development.
allometric scaling J Pharm Sci 1999;88:1101-06.
10. Penta JS, Rosner GL, Trump DL. Choice of starting
dose and escalation for phase I studies of antitumor
Approach 4: Comparative
agents. Cancer Chemother Pharmacol 1992;31:
This approach is comparative study of estimation 247-50.
of the starting dose using at least two or more 11. Penta JS, Rozencweig M, Guarino AM, Muggia FM.
Mouse and large animal toxicology studies of twelve
methods and then compares the results to find an antitumor agents: Relevance to starting dose for phase
optimal starting dose. I clinical trials. Cancer Chemother Pharmacol 1979;
3:97-101.
SUGGESTED READING 12. Posvar EL, Sedman AJ. New drugs: First time in
man. J Clin Pharmacol 1989;29:961-66.
1. Bonate PL, Howard D. Critique of prospective 13. Prieur DJ, Young DM, Davis RD, Cooney DA, Homan
allometric scaling: Does the emperor have clothes? J ER, Dixon RL, Guarino AM. Procedures for
Clin Pharmacol 2000;40:335-40. preclinical toxicologic evaluation of cancer
2. Boxenbaum H, DiLea C. “First-Time-in-Human chemotherapeutic agents: Protocols of the laboratory
Dose Selection: Allometric Thoughts and Pers- of toxicology. Cancer Chemother Rep 1973;4:1-6.
Protocol and Thesis Writing
8 for Postgraduate Students
This chapter will provide a brief conceptual

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

• Abstract/Introduction • Abstract/Introduction
• Review of literature • Review of literature
• Materials and methods • Materials and methods
• Statistical analysis • Statistical analysis
• Ethical justification • References
• References • Ethical consideration
• Animal ethics form • Patients information sheet
• Case record form
• Informed consent form
Flow Chart 8.1: Common pathway to write a degree, name of the department and institution
research protocol where the work will conduct.
Note: Title should be properly designed which
should be self explanatory and should never
contain any abbreviation, chemical formula,
propriety names, etc.
Introduction
Composite question: Why the study is needed? Or
what are the rational of doing the study?
The first part of introduction should summarize
the proposed study with the epidemiological
background or prevalence rate if available. (Which
shows the importance of the study?) Further, study
purpose should be described with the favorable
previous study. Next section should give the
background and rationale for initiating the study.
Findings from previous studies must be outlined
including appropriate detailed references to earlier
studies and brief data. If any pilot study conducted
in the same laboratory, then the results should be
included in the later part of introduction. This
section ends with a statement regarding aim and
objectives of study.
Title Page
Composite question: What is the study title? Or who Review of Literature
will conduct the study and where the study will Composite question: What are available data?
be conducted? or How much background information available?
Title page should contains the title of the study, It is an essential part which involves a
name of the candidate and academic degree for collective review and critique in the candidates
which student is making the thesis protocol, name own words of various viewpoints supported by
of the supervisor with his/her highest academic relevant data. The review should be properly
Protocol and Thesis Writing for Postgraduate Students  107
referenced and the method of citing references in Types of Experimental Groups

the text and listing at the end of the text are depend
on the institution guidelines. • Control or normal group (Negative Control): To assess
the impact of external variables (environmental, genetic,
microorganism, etc.) or to recognize the possible effect
Aims and Objectives of unwanted variables without the treatment.
• Vehicle Control group: These groups are kept in the
This section of the protocol gives the ultimate goal experiment to assess whether vehicle alone causes
of the study and the objectives section should give any effects or not. It is given in the same schedule as,
a concise statement to achieve research question. is in the other treatment groups. This group is compared
Hence, the study design must be in such way that with the control or normal animals.
it allow candidate to answer the research • Sham Control group: To assess any effect of the surgical
procedure or special treatment technique such as
objectives. implant on the animals. This group is compared with
the control or normal or vehicle group.
Materials and Methods • Positive Control group: These are the group in which
effect is expected. These are either a disease model
Composite question: Which materials, animals and group in which there are definite physiological changes
method should be used to answer the research are occurred after the chemical or bacteria treatment
question? or it may be a standard treatment* group which also
Explain about the animal use and describe show the definite reduction in the pathology of disease.
• Comparative Control group: It is the group of a standard
about their species, numbers, breed (if applicable), treatment which is directly compares the effect of a
age, sex, and weight. Housing and laboratory test treatment.
condition should be described during experiment *Standard treatment refers to clinically available prototype
such as temperature, humidity, light/dark cycle, drug for a disease. e.g. Phenytoin in the treatment of epilepsy
etc. Properly explain the experimental groups of or best drug treatment available clinically for the same
the study such as control/normal group, negative disease.
control group, treatment group and positive control
group, etc. Write brief paragraph on the disease vention allocation plan for randomized studies
model development and the methods selected for (if any).
the estimation of various parameters to conclude
the study. Ethical Justification
Composite question: Why study is important in the
Statistical Analysis animal? or is any alternative method is available?
Composite question: how the collected data will be If yes, then give the justification of not using the
analyzed? Or which tests are more appropriate to alternative method.
analyze the data? This is a brief paragraph on the method used
In the first section, summarize the study design and importance of the animal use in the study.
and rationale. Thereafter, describe the statistical Justify the total number of animal use and also
plan for each objective described. While selecting give brief on the care and handling of the animal
the statistical tests one should consider the end during the study.
point of the study
If the software will be used for the analysis of References
data, then describe about “What software will be This section contains a list of references for the
used?” study. Institutions have their own style of reference
Note: In clinical research protocol describe about writing hence always refer to your institution
primary and secondary endpoints, sample size guidelines for the style of references used.
and statistical power or precision associated with “Vancouver and Harvard” referencing is very
the sample size, stratification factors and inter- commonly used worldwide.
Example of Vancouver Style
Note: If the authors are more than six, then use the word “et al” (means ‘and others’) after six author and if it is less give
the name of all authors. Reference citation in the protocol is by no. of reference such as above mentioned reference no. is
‘1’, hence it is cited as Superscript.1
Example of Harvard Style
Note: Give the name of all authors. Reference citation in the text is not by the reference number (no reference number),
it is cited as Rawlin et al. (1977) or Rawlin (1977).
In both the referencing style, example given is for journal reference citation. Style for book, newsletter, conferences,
internet, etc. is different in both the styles. There is several other referencing styles which varies with the journal or
publishers
Animal Ethics Form use, surgical procedure adopted and disposal of

the animal if animal requires euthanasia and other
This is an ethical requirement for the approval of
related information.
the research protocol from the Institutional Animal
Ethics Committee (IAEC). It includes the all the Clinical Research Protocol
information regarding the study such as, title of Clinical research protocol is meant for obser-
the study, investigator name and addresses or vational population based research or any
additional investigators name, briefly procedure randomized trial. The typical format and compo-
of the study, justification of the study and animal nents of a Clinical research protocol are:
i. Title page Review of Literature

ii. Abstract/Introduction
It is one of the brief documentation of a collective
iii. Review of literature review and critique in the candidates own words
a. Rationale of the study of various viewpoints supported by relevant data
b. Previous studies on the subject either either of experimental or clinical study. The review
experimental or clinical study should be properly referenced and cited with the
c. Study question reference as per institute guidelines.
iv. Aim and objectives
a. Aim, hypotheses and objectives Study Description
v. Materials and method
Study description involves the outline of the
a. Design and methods
proposed study and its essential components
(Study design, Study population,
are:
Sample size and statistical power,
Subjects, Data collection methods, Data Rationale and Previous Studies on the
management and Statistical analysis) Subject
b. Project Management
(Personnel required, Duration of study An ideal protocol should provide the rationale
and Follow up procedures) and the relevant background information
c. Strengths and limitations related of the proposed study. Rationale should
vi. References indicate in a progressive logical sequence that
vii. Ethical considerations how the study question has emerged. This is
viii. Patient information sheet done by reviewing the relevant literature and
ix. Case Record Form (CRF) current knowledge showing specifically the
x. Patients informed consent ambiguity in knowledge which make the study
xi. Budget worth doing. Any preliminary work done in the
field of proposed study should be included in
xii. Investigators.
the protocol. Rationale should clearly explain
how the proposed study will benefit the
Title Page
community.
Same as the experimental research protocol, this
page contains the title of the study, name of the Study Question
candidate and academic degree for which student
This addresses the issue which one has to
is making the thesis protocol, name of the
pursue during the proposed study. The research
supervisor with his/her highest academic degree,
topic is generally formulated into a question,
name of the department and institution where the
known as study question. The study question
work will conduct.
must clearly explain the objectives of the
proposed study. The necessary steps required
Abstract
to answer the study question should also be
Abstract is a concise summary which provides included in the protocol.
information about the main purpose of the
study, the way in which it will be conducted Aims and Objectives
and its expected benefits. Although in a typical Study question indicates the aim or the goal, which
protocol format, abstract is written on the top is generally very vast. So, this is broken down into
after completion of the studies. smaller questions known as objectives.
Hypotheses Data management and Statistical analysis: This

section deals with data compilation and analysis.
Hypotheses are distinct and clearly defined
This should indicate the procedures for coding
outcomes for any proposed study. Objectives
and entering the data into computer files. It must
should be stated in the form of hypotheses.
mention how the results will be displayed, how
Hypotheses can be written as statements to be
comparisons will be made and which tests will
refuted i.e. no difference between study groups
be used to carry out the statistical analyses in order
and are referred to as Null hypotheses. Alternate
to test the stated hypotheses.
hypotheses are statements assuming the real
difference between the study groups. Thus the
Overall Project Management
research hypotheses provide information about
the comparisons which will be needed to answer This section describes the required personnel,
the study question and establish the format with duration of study and follow-up procedures for
in which one will apply the statistical tests while study participants where appropriate.
interpreting the results.
Strengths and Limitations
Materials and Methods
This section should address the strong points of the
Design and Methods proposed study making it worthy for financial
Study design: Protocol should clearly indicate how support. Along with the criticisms of the proposed
the proposed study will be performed and which study and the compromises made in study design
methods will be adopted. It should mention the while writing protocol should be included.
selected design of study along with the rationale
of choosing the proposed design. References
Study population: Study population means the This section should include all the references
target group in which the study will be carried according to the order of their presentation in the
out. protocol. For the reference style refer to experi-
mental protocol section.
Sample size and statistical power: Protocol must
indicate how big the target group has to be in order ETHICAL CONSIDERATIONS (ICMR GUIDELINE)
to precisely answer the study question. This
should include the assumptions made for This section must state the ethical considerations
calculation and a table of calculation of sample in accordance with the Helsinki Declaration. It
size and power. must indicate the expected hazards of the study,
expected benefits, safety of participants, responsi-
Subjects selection: This should explain the number
bility for liability for injury to participants and
of subjects , criteria for subject selection i.e.
inclusion and exclusion criteria, various inclu- voluntary consent to participate.
sion and exclusion parameters, mechanism of
recruitment of subjects and feasibility of recruit- PATIENT INFORMATION SHEET (PIL)
ment. As the name indicates, this information sheet
Data collection methods: This section should provides all the basic essential information of the
clearly mention how the data will be collected and study to the patients to build up the confidence to
what are the determinants of the proposed study. participate in the study. Such as what is the
Quality control procedures should be involved purpose of the study?, what is the drug used and
and if the procedure is a standard one, it should its indication? What is the procedure to attend
be referenced. the trial? Are there any alternatives of diagnosis
Flow Chart 8.2: Ideal protocol should answer six basic questions stepwise
Why there is need of proposed study?

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

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

according to the order of their presentation in
Thesis should clearly indicate how the proposed the protocol as per the style mentioned by the
study was performed and which methods were institute.
adopted. It should mention the selected design of
study along with the rationale of choosing the APPENDICES
proposed design. Ideally, this is the first step to start the
thesis writing. This section should clearly mention Material relevant to the development of the thesis
how the data was collected and what were the like preparation of solutions, buffers, samples of
determinants of the proposed study. Quality control questionnaires, etc. should be included in this
procedures should be involved and if the procedure section.
is a standard one, it should be referenced.
ACKNOWLEDGMENTS
Data Management and Statistical Analysis
In this section, one should acknowledge the
This section deals with data compilation and people for their contribution in pursuing the
analysis. This should indicate the procedures for research project. This may be also given after the
coding and entering the data into computer files. front page at the starting of thesis.
SUGGESTED READING
Note: Before writing the protocol, student/researcher
should perform a small sample study to check the 1. Barletta JF. Conducting a successful residency
hypothesis made, is actually working. These type of study research project. Am J Pharm Educ 2008;72(4):92.
is knwon as “Pilot study”. It is defined as “study conducted 2. Betkerur J. Guideline for writing a research project
on the small number of animal/human to check the synopsis or protocol. Indian J Dermatol Venereol
hypothesis made, procedure and technique used in the Leprol 2008;74(6):687-90.
study with relevant supported background information. If 3. Van Way CW. Writing a scientific paper. Nutr Clin
the data collected is supportive, then start the protocol Pract 2007;22(6):636-40.
writing and then actual experiment and finally thesis writing
4. Betkerur J. Guideline for writing a research project
synopsis or protocol. Indian J Dermatol Venereol
and submission.
Leprol 2008;74(6):687-90.
Toxicology Study
9
Evaluation and development of a new drug General Principles
undergoes numerous procedures in order to get The principle of the toxicity study is to assess all
the safe and efficacious drug. Apart from the other the relative toxic effects of the drug regarding its
studies like pharmacological and pharma- dose, duration and its effect on the organs. Hence,
cokinetic evaluation, toxicity study (also known these studies not only provide adequate thera-
as hyperpharmacology) is also an important and peutic margin of initial dose levels and duration
ethically necessary study during the drug of dosing, but also give guidance for any special
development process. There is species-to-species measures to be taken in initial clinical trials.
variation in the pharmacological responses hence In a large set-up, conducting toxicity studies
during the toxicity study always two or more should comply the following important require-
species are preferred. Protocols must be reviewed ments,
and approved by an Institutional Animal Ethics 1. Should follow the Good Laboratory Practice
Committee for the experiment. (GLP)
2. Studies should be performed by suitably
Objective of the Toxicity Study trained and qualified staff
3. Instruments should be calibrated and
1. Identify any toxic substance prior to clinical
standardized properly and of adequate
use
capacity.
2. Qualitative* and quantitative# assessment of
4. Standard operating procedures (SOPs) should
drug use
be followed.
3. Characterize the cumulative toxicity of the
drug under study (specially, in subchronic or Documentation part is proper and the most important to
save and anticipate should be the results. All documents
chronic study but not predicted by single dose like approval of protocol, raw data, draft report, final report
toxicity) and histology slides and paraffin tissue blocks should be
4. To allow a careful selection of doses for further preserved for a minimum of 5 years after marketing of the
studies (including, carcinogenicity studies) drug
5. Different types of dose identification can be Toxicological study involves toxicodynamics
done. For example maximum lethal dose (MLD), and toxicokinetics. The toxicity studies are
Lethal dose (LD50), maximum tolerated dose conducted as per the duration of clinical use of
(MTD), minimum effective dose (ED50), etc. the drug. So, the study is mainly divided into the
6. Therapeutic index can be calculated (LD50/ three categories, i.e.
ED50)
Acute toxicity study: Single dose toxicity study
* Nature of effect of drug seen in the target organs
# Subacute/subchronic study: Repeated dose from
Drug level (plasma and tissue levels) at which,
drug effects definitely seen and not seen 2 weeks to 6 months
Chronic toxicity study: Repeated dose study from Animal: At least two mammalian species (1rodent
6 months to 12 months or even up to 2 years + 1 non-rodent)
(Younger and still growing animals are
Acute Toxicity Study preferred at the initiation of a subchronic study)
Single-dose Toxicity Studies Treatment and duration: Given in 14, 28, 90 or 180
days (duration depends on therapeutic indication)
Animal: 2 rodent species (mice and rats); in each
• Group I; Control group—Normal or vehicle
group at least 5 animals of either sex
group
Route of administration*: Same as intended for • Group II; Treatment group—drug treatment
humans group
*If the intended route of administration in humans is only Route of administration*: Same as intended for
intravenous, at least one more route should be used in humans
one of the species to ensure systemic absorption of the
drug Doses: At least three graded doses
Doses: At least three graded doses
Selection of Animals
Treatment: Given in a single bolus or several doses
• Rodents must be about six weeks and not more
or by continuous infusion within 24 hours
than eight weeks.
Animals should be observed for 14 days
• Non-rodent (e.g. dog) treatment should begin
Observation: at 4-6 months but no more than 9 months.
1. Signs of intoxication
2. Effect on body weight Selection of Dose
3. Gross pathological changes
• Highest dose should produce observable
Note: It is desirable to include histopathology of toxicity
grossly affected organs. (if any) • Intermediate dose should cause some symptoms,
Calculate the minimum lethal dose (MLD) and but not gross toxicity or death, and should be
maximum tolerated dose (MTD) and lethality dose placed logarithmically between the other two
(LD50) doses
• Lowest dose should not cause observable
Selection of oral dose: Limit of 2 g/kg or 10 times toxicity.
the normal dose that is intended in humans,
whichever is higher is recommended for oral Methods
dosing.
• One rodent (6-10/sex/group; for 14 days) (15-
Repeated Dose Toxicity Studies 30/sex/group; for 90 and 180days)
• One non-rodent species (2-3/sex/group; for
Subacute/subchronic or chronic toxicity study 14 days) (4-6/sex/group; for 90 and 180 days)
falls under these category and being use in the
drug which are intended for the repeated dose in Observation
the clinical setting.
• Sites of injection should be subjected to gross
and microscopic examination if given
Subacute/Subchronic
parenterally.
Aim of the study is to identify target organ toxicity • Non-rodent species: Electrocardiogram and
and establishment of MTD for subsequent studies. fundus examination
Toxicology Study  117
Table 9.1: Analytical parameters which should be assessed • Non-rodent species: Electrocardiogram and
after the subscute/ subchronic/ chronic, toxicity test, listed fundus examination
as follow: • Other, observation parameters should include
• Liver function tests: Total bilirubin, direct or indirect
body weight changes, food/water intake,
bilirubin, bile acids, ammonia blood biochemistry, hematology, and gross
• Hepatocellular leakage enzymes: AST, ALT, SDH, and microscopic studies of all viscera and
LDH tissues (Table 9.1).
• Muscle parameters: Creatine kinase (CK), AST, ALT, Other specific toxicity studies are depend on
LDH
the nature of drug uses such as teratogenicity
• Pancreatic parameters: Amylase, lipase
• Lipid parameters: Cholesterol, triglycerides Cholestatic study, male fertility or female fertility toxicity tests,
enzymes: AP, GGT etc. and some depend on drugs site of use such as
• Calcium, potassium: Often influenced by kidney local toxicity (dermal and photoallergy or dermal
function, kidney parameters should be evaluated phototoxicity, vaginal, ocular or inhalational
concurrently toxicity test, etc.) is done to evaluate the drug in
• Kidney parameters: Urea nitrogen, creatinine urine
rodents or non-rodents.
specific gravity should be evaluated concurrently
when evaluating renal function
Lethality Testing and Calculation
Lethality testing or determination is an essential
• Other, observation parameters should include
regulatory requirement for a drug or biological
cage-side observations, body weight changes,
products before clinical study. It is basically acute
food/water intake, blood biochemistry,
toxicity testing in which death is considered as a
hematology, and gross and microscopic
single end-point and other data or other
studies of all viscera and tissues (Table 9.1).
behavioral parameters are not taken under
Chronic Toxicity Study consideration. Hence, lethal dose of drugs may be
defined as “dose of a given drug which produces
• Animal: 2 species (a rodent and a non-rodent).
mortality in the treated animal, especially in most
• In rodents chronic studies are usually for 6
sensitive species model.” The most commonly
months to 2 years.
calculated lethal dose is median lethal dose or
• In non-rodents chronic studies are usually for
lethal dose 50 (LD50). LD50 is defined as the “dose
1 year but may be longer.
of a given drug which produces mortality in 50% of
Selection of Dose total treated animal preferably in the most sensitive
species model”. LD50 was discovered by Trevan and
• Highest dose should produce observable
Behrens, who identified and read the midpoint
toxicity
on the mortality dose response curve. Lethality
• Intermediate dose should cause some symptoms,
testing is preferred over subchronic or chronic
but not gross toxicity or death, and should be
placed logarithmically between the other two testing because of ease of end-point selection,
doses small procedure and still real and concerned
• Lowest dose should not cause observable toxicity method. For a broader concern, lethality testing is
The length of drug treatment or exposure is done mainly on the 3 doses of selection such as
mainly dependent on the intended period of use one dose which has 100% lethality, second,
in humans. produces marginal lethality and last dose is taken
in between of first 2 doses. Route of administration
Observation is same in all testing animal and preferably,
• Sites of injection should be subjected to gross human intended route. Toxicity is varying
and microscopic examination if given according to route of administration. There is a rule
parenterally. of thumb, i.e. the i.p. LD50 is roughly 30% of oral
and the i.v. is 10% of that of the oral. Duration of 4. Other factors like species, strain or substrain,
the lethality testing ranges from 7 days to 14 days. age, weight, and sex of the animals or even,
Note: Generally, LD 50 is calculated in the animals husbandry condition may interfere with result
(preferably rodents like mice or rat) whereas only one outcome.
direct human LD50 values was determined in “Nazi human There are two methods of calculating the LD50,
experimentation”; is reported.
one is mathematical method and second is
Precautions should be taken while performing graphical method.
the experiment because animals receive a single
calculated dose,
Mathematical Method
1. Animal must be dosed on the same day and
preferably at the same time. Karber’s method is very commonly used because
2. Great care must be taken while calculation, it is simple and does not need to plot the dose
preparation and delivery of the test drug. response curve. It is simplified in the example
3. So, keep laboratory conditions constant for given below,
each animal during the experiment (animal
model chosen must be most sensitive).
Example:
Exp. Dose Dose No.of No. of Mean mortality* DD x MM
Group (mg/kg) (difference (DD)) animals (n) dead animal (MM)
1 3 — 10 0 (A) - —
2 5 2 10 2 (B) 1 2
3 10 5 10 3 2.5 12.5
4 12 2 10 5 4 8
5 15 3 10 7 6.5 19.5
6 20 5 10 10 8.5 42.5
Σ(DD × MM) = 84.5
*Mean mortality = difference of two adjacent no. of dead animal/2

e.g. = (A +B)/2 = (0 + 2)/2 = 1
Σ(DD × MM) = 84.5; then value is divided by the no. of animal in the group (here, n = 10)
Hence, value obtained is 84.5/10 = 8.45
Finally, this value is sustracted from the minimum dose which produces the 100% mortality, i.e. 20 mg/kg
So, LD50 = 20 – 8.45 = 11.55 mg/kg
Graphical Method
This method is well described by the Miller
and Tainter (1944) as well as Litchfield and
Wilcoxon (1949). This is depending on the toxic
log dose response curve and the interpretation
of result is done by the curve. The preferred
method is, first convert the percentage response
into the probit, then plot the curve log dose
on ‘x’-axis and ‘y’-axis contains probit scale
(Fig. 9.1).
The LD50 is the antilog (ld) value which falls
on the ‘x’-axis. Fig. 9.1: Determination of LD50
Toxicology Study  119
Note: Probit analysis is a type of regression used to 4. Finney DJ, Ed. Probit Analysis. Cambridge, England,
analyze binomial response variables. It is most precise Cambridge University Press 1952.
but requires at least two groups of partial responses (i.e. 5. Leo T M van der Ven, Aart Verhoef, Ton van de Kuil,
mortality greater than 0% but less than 100%). Initially, Wout Slob, Pim E G Leonards, Theo J Visser, et al. A
Bliss (1939) developed the idea of probit through 28-day Oral Dose Toxicity Study Enhanced to Detect
transforming the sigmoid dose-response curve to a straight Endocrine Effects of Hexabromocyclododecane in
line dose-response curve which was further refined by Wistar Rats. Toxicol Sci 2006;94(2):281-92.
Finney (1952) 6. Lorke D. A new approach to practical acute toxicity
testing. Arch Toxicol 1983;54:275-87.
7. Phillips J, Gibson W, Yum J, Alden C, Hurd G. Survey
SUGGESTED READING
of the QSAR and in vitro approaches for developing
1. Armitage P, Allen I. Methods of estimating the LD50 non-animal methods to supersede the in vivo. LD50
in quantal response data. J Hygiene 1950;48:398-422. test. Fd. Chem. Toxicol 1990;28:375-94.
2. Bliss C. Some principles of bioassay. Am Scientist 8. Ranjit Madhukar Bidhe, Sangita Ghosh. Acute and
1957;45:449-66. Subchronic (28-day) Oral Toxicity Study in Rats Fed
3. Finney D. The median lethal dose and its estimation. with Novel Surfactants. AAPS PharmSci 2004; 6 (2)
Arch Toxicol 1985;56:215-18. Article 14.
Biomedical Waste Disposal
10
It is important for the student in the laboratory to 7. Avoid personal work like reading, eating,
note that work is always being associated with drinking and smoking in the laboratory.
the potentially toxic, flammable, irritable, painful 8. Always use the clean and dry glassware*
or the carcinogenic substances. So, the dangerous
situation can be developed unexpectedly. With Respect to Experimental Animals
Therefore, one should be aware about the
procedure, careful use of chemicals in the 1. Handle the animal with care (may get aggre-
laboratory and most importantly the other ssive, if get agitated)
important safety concern, facilities and emergency 2. Avoid forceful hold to the animal
action. Moreover, it all includes “Good Laboratory 3. Laboratory should be dim lighted while
Practice (GLP)” (Flow Chart 10.1). performing the experiment e.g.: CNS experi-
In the pharmacology laboratory, students ment, etc.
mainly deal with the animals, chemicals and 4. For any surgical procedure, use the sterile
different types of instruments. So, the students instruments (syringe/needle, forceps, scissor,
should take care of the all the important points etc.) or nicely wiped with 70% alcohol
while working into the laboratory such as: 5. Waste material should be disposed according
to the labeled colored bags.
With Respect to Drug/Chemicals
1. Never work alone/single in the laboratory. *Cleaning of glassware
2. Read the instructions, so, that student should • Most purposes: 0.5% of detergent in water followed by
6-10 water rinses whereas final rinse is with distilled or
be familiar with the chemical and physical
deionised water.
properties such as reactivity, flammability,
• Glassware contaminated with metal ion: Rinse with
toxicity, carcinogenicity and the disposal of concentrated nitric acid, then extensively rinse with
drugs or chemicals used in the experiment. the water
3. Use the apron, gloves, and cap while doing • When Glassware needs to get dried quickly: Rinse
the experiment. with the acetone which quickly evaporates and dries
4. Wear safety glasses with wide side shield to quickly
protect the eye. • Glass or quartz cuvettes: Carefully clean in 0.5%
detergent water in a cuvette washer or in a sonicator
5. Avoid mouth suction to fill pipette or to start
bath (Never use ethanolic KOH or other strong
siphons base which may cause etching)
6. Always be familiar with the safety equip- The use of chromate solutions to clean glassware is
ment like fire extinguisher, eye washes, PROHIBITED due to extreme toxicity of chromates
chemical spill kit, first aid supply, etc. in the including carcinogenicity.
laboratory.
Biomedical Waste Disposal  121
Flow Chart 10.1: To minimize the risk in the laboratory
Waste Disposal Management environment. The wastes material is collected in the

There are different types of material used in the respective “colored bags” in the laboratory and send
pharmacology laboratory such as from simple to the central disposal center to proper disposal of
chemicals to the animals. So, the minimization and biomedical wastes (Table: 10.1).
management of wastes such as chemicals, radio- The other corrective measure to reduce the
active materials, animal tissues/organs, cotton, biomedical wastes is improvements in laboratory
sharpen blades, etc. from the experimental procedure techniques, reduce use of toxic substance or check
should be maintained to ensure the significant for any alternative less/non-hazards chemicals/
reduction in generation of hazardous, radioactive, materials.
and mixed wastes in the environment. There are The waste materials collected in the color
guidelines to dispose the contaminated bio- coded polythene bag (double bagged if required),
degradable or non-biodegradable wastes to reduce then send for incineration or other method of
the contamination and infections in the general destruction.
Table 10.1: Different type of waste disposal bags and their contents
Bag type Contents Example of contents
Yellow Contains infectious and related waste. Cotton, dressing , plaster of Paris(POP), human or
Biodegradable wastes (decomposed by animal anatomical waste, human tissue, organs, body
bacteria or other living organisms) parts, microbiology and biotechnology waste, etc.
Blue Sharp things which can be responsible Glass syringes, needles, surgical blades, shaving blades ,
for infectious diseases etc.
Black Contains non-infectious and related Outdated medicines, contaminated medicines, discarded
waste medicines, packing cover of medicines, waste paper,
general non-infected waste, leftover food and peels of
fruits, etc.
Red Contains infectious and related waste Ryle’s tube, catheter, urinary catheter, suction catheter,
like surgical waste. chest drainage catheter, IV set, blood set and glucose
bottle and contains low level radioactive waste
Violet Cytotoxic drugs and related waste Cytotoxic drugs and related waste, contaminated sharps,
contaminated animal carcasses and cageLinings
SUGGESTED READING 3. National Research Council, Committee on

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

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

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

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

Quantitative Discrete Countable or BP reading, etc. Parametric test Box plot, Scatter
(Numerical) finite number (t-test,correlation plot (2 variables),
and Regression) Dot diagram
Histogram, Stem-
Leaf and Quantile-
Quantile Plot, etc.
Continuous Value lie in Age, height, etc. Parametric test Box plot, Scatter
(Interval/ continuous range (Mean,SD, plot (2 variables),
ratio) ANOVA, z- and Dot diagram
t-tests and Histogram, Stem-
Regression) Leaf and Quantile-
Quantile Plot, etc.
Different Measures of Data So, (7 + 8)/ 2 = “7.5” is median value for above
mentioned data
Mean: This is nothing but the arithmetic average
of any given data. Mean is denotes by the “x–” in Mode: It is very frequently occurring event in a
sample and “µ” in population. given data.
e.g.: 2, 2, 3, 3, 4, 2, 5, 6, 7, 7, 8, 4, 9, 3, 4, 8, 9, 3,
x
. Mean = In the given data ‘3’ is repeated 4 times and
n it is most frequently occurring number. So,
where, x = Observations; “3” is the mode of given data
n = Total number of observations
e.g.: 2, 2, 4, 7, 7, 8, 10, 11, 11,16; Mean of given Measure of Dispersion
data is calculated as:
(2+2+4+7+7+8+10+11+11+16)/10 = 7.8 Range: “Range” = Highest value – Lowest value
That means, range gives the value between the
Median: This is the middle value of any given data, minimum and maximum values of a variable.
when it is arranged in the ascending or descending Understanding this statistic is important in
order. understanding your data, especially for
e.g.: 1, 2, 2, 4, 7, 7, 8, 10, 11, 11, 16 (Odd value) management and diagnostic purposes.
Hence, in the odd numbers of samples, e.g.: 2, 2, 4, 7, 7, 8, 10, 11, 11, 16
middle value is treated as the median. So Highest value= 16 and Lowest value = 2
“7” is median in above data Hence, Range = 16- 2 = 14
1, 2, 4, 6, 7, 8, 10, 11, 12, 16 (Even value)
In the even number of sample, the mean of two Variance: It is a measure of an event that departs
middle values is considered as median value. from expectations, i.e. the dispersion of a group
data around their mean value. It is mean of all Gauss (1777-1855)). In such distribution, variables
deviation score in the data, i.e. it is calculated as have the tendency to cluster around the central
value with a symmetric distribution on the either
side. (Positive and Negative)This type of
distribution is plotted as a normal distribution
where X = individual score curve. The mean, median and the mode
µ = mean of the total score (popu- considered to be equal in such distribution. The
lation) [in sample it is denoted by normal distribution curve has a central tendency
X] and a degree of dispersion. Various methods of
N = number of total observation score analysis are available to make assumptions about
(sometime (N-1) is used instead of normality, including‘t’-tests, analysis of variance
N to eliminate bias (ANOVA) and correlation and regression. Skew
Standard deviation: It is the measure of dispersion is zero in normal distribution but when tail skew
or variability of a data from its mean value, which is towards right it is known as positive skewed
is calculated by the square root of variance. Hence, and when it skew towards left it is known as
it is calculated by the formula negative skewed and therefore measure of central
tendency differs in this context (Fig. 11.3).
Such distribution can be made to normal
distribution by a suitable transformation.
where X = individual score Logically, transformation which makes data
µ = mean of the total score (popu- follow a normal distribution often makes the
lation) [in sample it is denoted by variance uniform as well, and vice versa. Data
X] can be transformed by taking the logarithm, square
N = number of total observation score root, reciprocal, or some other function of the data.
(sometime (N-1) is used instead of In the normal distribution, 68% of data fall
N to elimnate bias between 1 SD, 95.4% of data fall between 2 SDs
Standard Error of Mean (SEM): This is the measure and 99.7% of data fall between 3 SDs. Hence, 95%
of variability, which is defined by the “S/√n”. of population falls within the 1.96 SD (Fig. 11.4).
Kurtosis of normal distribution found to be
Difference between the standard Deviation (SD) zero. (Kurtosis means how distribution is selected)
and Standard Error of Mean (SEM): Standard Relation between the mean, median and mode is
deviation (SD) describes variation in values of well established in the normal distribution Figure 11.3.
individuals which is denoted by ‘S’ whereas Standard
Error of Mean (SEM) describes variation in values Binomial Distribution
of a statistic from one conduct of study to the next
and it is the measure of S/√n. Binomial distribution is applicable to those data
where there are only two possibilities such as
Types of Data Distribution smokers or non-smokers; diabetics or non-
diabetics; male or female; survived or not survived.
Overall data distribution is divided into the two, This type of data have properties like, only two
first continuous variable distribution (normal outcome possible, have fixed number of obser-
distribution) and the discrete variable distribution vation and each event have constant probability
(Binomial and Poisson distribution). to occur. Hence, the binomial distribution curve
has two peaks.
Normal (Gaussian) Distribution
But, in the case of a large observation time
This distribution is also known as Gaussian and corresponding small event, binomial
distribution. (Named after German mathematician, distribution calculation is very tedious and not
Fig. 11.3: Representation of data
Poisson Distribution
This is commonly used distribution in health
sciences defined the distribution of the number of
occurrences “x” of some random event in an
interval of time or space. For example, recruitment
of patients into the clinical trial Phase III studies:
Investigator recruits patients daily according to
inclusion/exclusion criteria. If investigator is
interested in looking the patients enrolled for the
Fig. 11.4: Data and standard deviation study on particular time say, 1-2 months of study
begin, and then data obtained has the poisson
distribution. Hence, it calculates the probability
of event in a fixed interval. In this case event may
occur randomly and independently. This is
calculated by an exponential formula (Fig. 11.6).
Statistical Tests
Statistical test is a mathematical calculation or
analysis of observed and collected data from the
Fig. 11.5: Binomial distribution curve population or sampling. The result obtained, refers
the statistical power of a test. Therefore, statistical
an appropriate method. Hence, describing and power is defined as a measure of the probability
analyzing such event requires “Poisson distri- that a statistical test rejects a false null hypothesis,
bution”. These two, i.e. Binomial and Poisson or in other words, the probability of finding a
distributions are most important discrete probability significant result, if there is any difference. The
distributions (Fig. 11.5). higher the power of a statistical test, the more likely
i.e. quantitative numerical continuous data and the

second which applies on the ordinal or categorical
data or qualitative data (Fig. 11.7).

Equivalent Parametric and non-parametric tests
Parametric test Non-parametric tests
Unpaired student‘t’ test : Mann- Whitney U test
Paired ‘t’ test : Wilcoxon signed rank test
One way ANOVA : Kruskal- Wallis test
Repeated measures ANOVA : Friedman’s test
Pearson correlation coefficient : Spearman rank order
These non-parametric tests are the alternative to the
Fig. 11.6: Poisson distribution corresponding parametric tests in which data is not normally
distributed.
one is to find statistical significance, if the null
Note:
hypothesis is false.
Assumption of the null hypothesis is tested by the p value
Note: which represents the risk observed in the experiment due
• Type I error (α error): When an experimenter concludes to chance. The p value determines the significance or
that there is a difference between the analyzed groups non-significance of a result. (Measure of probability) If one
when, in fact, there is no difference (False positive) fixed the significance level at p value <0.05 that means
• Type II error (β error): When an experimenter concludes chances of 5 observation which may fall outside the range
that there is not a difference between the analyzed i.e. uncertainty of 5%. Other p values which are commonly
groups being studied when, in fact, there is a difference set as significance levels are 0.01 or 0.001. Limitation of
(False negative) the p value is that, it may influence by the study result or
do not show the magnitude of difference in the comparing
As we have seen that there are different types groups.
of data, and for analyzing such data, statistical The alternative to the p value is 95% confidence interval
tests are mainly divided into two groups, one (CI) which represents that 95% chance of the cases to fall
into the range. Most commonly 95% and 99% of the
which applies on the normally distributed data,
Fig. 11.7: Classification of biostatistical tests

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

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

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

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

proportions. Also, because it compares the
Linear regression is a method of estimating or
categorical responses between two or more
predicting a value on some dependent variable
groups, the chi-square statistic can be conducted
given the values of one or more independent
only on actual numbers and not on pre-calculated
variables. Like correlations, statistical regression
percentages or proportions. Chi-square
tells the association or relationship between
summarizes the discrepancy between observed
variables. Unlike with correlations, however, the
and expected frequencies. The smaller the overall
primary purpose of regression is prediction. For
discrepancy is between the observed and expected
example, health of a person is predicted by age,
scores, the smaller the value of the chi-square and
body weight, medical history, disease status,
so vice versa. The chi-square test calculates
marital status, and current behavioral patterns.
approximate p value, hence to make assumption
There are two basic types of regression analysis:
better Yates’ continuity correlation is applied.
simple regression and multiple regression. In simple
regression, we attempt to predict the dependent
variable with a single independent variable. In
multiple regression, one may use any number of
independent variables such as age, body weight,
medical history, etc. in the health of a person, to
predict the dependent variable.
Logistic regression, unlike its linear
This is the test of “2 × 2” table as that of chi-
counterpart, is unique in its ability to predict
square (χ2) test. It directly calculates the probability
dichotomous variables, such as the presence or
(give exact) p value of a data instead of calculating
absence of a specific outcome, based on a specific
statistics to a sampling distribution. Hence, it
set of independent or predictor variables. Like
preferred logically to use the exact probability
correlation, logistic regression provides
rather than approximate χ2 - test. Generally
information about the strength and direction of
this test performed when the sample size is too
the association between the variables. In addition,
low (n < 6). Other test may be use to evaluate the
logistic regression coefficients can be used to
significance of categorical data is Fischer’s exact
estimate odds ratio for each of the independent
test. This is applied to the less sample size (< 5
variables in the model. These odds ratio can tell us
samples) where, chi-square test shows inaccurate
how likely a dichotomous outcome is to occur
evaluation.
given a particular set of independent variables. A
common application of logistic regression is to Present Absent
determine whether and to what degree a set of Yes a b a+b
hypothesized risk factors might predict the onset No c d c+d
of a certain condition. a+c b+d a+b+c+d
χ2)
Chi-square (χ (a b) (c d) (a c) (b d)
p
(abcd)(a b c d)
The inferential statistics that we have discussed
so far (i.e., t-tests, ANOVA) are appropriate only The limitation of χ2- test and Fisher exact test
when the dependent variables being measured is that it does not indicate the strength of an
are continuous (interval or ratio). Chi-square association whereas the experimental and clinical
analysis is often used to examine between-group practices, people more interested in how much
differences on categorical variables, such as more likely an outcome will be, when a treatment
gender, marital status, or grade level. The main or intervention is given or how much is relative
thing to remember is that the data must be nominal risk (risk ratio) is present.
Some other parameters* which depend on the “2 × 2” 3. Altman DG, Whitehead J, Parmar MK, Stenning SP,
table Fayers PM, Machin D. Randomised consent designs
a / (a b) in cancer clinical trials. Eur J Cancer. 1995;31A(12):
Relative risk or risk ratio : 1934-44.
c / (c d)
4. Altman DG. Statistics and ethics in medical research.
Absolute relative risk (ARR) : Collecting and screening data. Br Med J 1980;
281(6252):1399-401.
Odds ratio : 5. Altman DG. Statistics and ethics in medical research.
Misuse of statistics is unethical. Br Med J 1980;
Number need to treat (NNT) : 281(6249):1182-84.
*These are the measures of the adverse or hazards VII—Interpreting results. Br Med J 1980;281(6255):
effect of a drug 1612-14.
VI—Presentation of results. Br Med J 1980; 281
Mann-Whitney ‘U’ Test
(6254):1542-44.
Test is performed when data of two groups have 8. Altman DG. Statistics and ethics in medical research:
measured on an ordinal scale and this test may III How large a sample? Br Med J 1980;281(6251):
applied to large sample size (n>100). It is very 1336-38.
9. Altman DG. Statistics and ethics in medical research:
useful test because it can have high power
Study design. Br Med J 1980;281(6250):1267-69.
approximately 95% compared to unpaired‘t’-test.
10. Altman DG. Statistics and ethics in medical research:
V—Analysing data. Br Med J 1980;281(6253):1473-
Wilcoxon-signed Ranks Test 75.
It corresponds to the paired‘t’ test and have high 11. Altman DG. Statistics: Necessary and important. Br
J Obstet Gynaecol 1986;93(1):1-3.
power approximately 95% compared to paired‘t’-
12. Bland JM, Altman DG. Measurement error
test. Test is performed by calculating difference
proportional to the mean. Br Med J 1996;313(7049):
between pairs then the absolute difference is 106.
ranked. The sum of positive ranks is compared 13. Bland JM, Altman DG. Measurement error. Br Med J
with the sum of the negative ranks. 1996;313(7059):744.
Hence, the groups have no difference if, 14. Bland JM, Altman DG. Multiple significance tests:
Sum of positive ranks = sum of the The Bonferroni method. Br Med J 1995;310(6973):
negative ranks 170.
15. Bland JM, Altman DG. Transformations, means, and
Note: (Software used in biostatistics) confidence intervals. Br Med J 1996;312(7038):1079.
Nowadays, there is increase in application of computer in 16. Bland JM, Altman DG. Transforming data. Br Med J
the biomedical research, so, as in the statistical tests. 1996;312(7033):770.
Reason being, complicated and tedious job to do and thirdly, 17. Gardner MJ, Altman DG. Confidence intervals rather
very low interest of student to perform the statistical tests than P values: Estimation rather than hypothesis
manually. testing. Br Med J (Clin Res Ed) 1986;292(6522):746-
There are several softwares, used in the biostatistics. But,
50.
most commonly used softwares are SPSS, SigmaPlot, in-
stat, BioEstat, Dataplot, graph and prism Macanova, etc.
18. Glantz SA. Biostatistics: How to detect, correct and
prevent errors in the medical literature. Circulation
1980;61(1):1-7.
SUGGESTED READING
19. Godfrey K. Simple linear regression in medical
1. Altman DG, Bland JM. Presentation of numerical research. N Engl J Med 1985 ;313(26):1629-36.
data. Br Med J 1996;312(7030):572. 20. Godfrey K. Statistics in practice. Comparing the
2. Altman DG, Bland JM. Statistics notes: The normal means of several groups. N Engl J Med 1985;313(23):
distribution. Br Med J 1995;310(6975):298. 1450-56.
21. Ludbrook J, Dudley H. Issues in biomedical statistics: 25. Ludbrook J. Statistics in biomedical laboratory and
Analysing 2 × 2 tables of frequencies. Aust N Z J clinical science: Applications, issues and pitfalls. Med
Surg 1994;64(11):780-87. Princ Pract 2008;17(1):1-13.
22. Ludbrook J, Dudley H. Issues in biomedical statistics: 26. Ludbrook J. Statistics in physiology and pharma-
Statistical inference. Aust NZJ Surg 1994;64(9): cology: A slow and erratic learning curve. Clin Exp
630-36. Pharmacol Physiol 2001;28(5-6):488-92.
23. Ludbrook J. Advantages of permutation (rando- 27. Ludbrook J. The presentation of statistics in
mization) tests in clinical and experimental Clinical and Experimental Pharmacology and
pharmacology and physiology. Clin Exp Pharmacol Physiology. Clin Exp Pharmacol Physiol 2008;
Physiol 1994;21(9):673-86. 35(10):1271-74.
24. Ludbrook J. Analysis of 2 x 2 tables of frequencies: 28. Moses LE, Emerson JD, Hosseini H. Analyzing data
Matching test to experimental design. Int J Epidemiol from ordered categories. N Engl J Med. 1984;311(7):
2008;37(6):1430-35. 442-48.
Part 2
Experimental (In Vitro Studies:

Isolated Tissue Preparation)
General Considerations and
Collection of the
12 Tissue/Muscle
General Considerations in Animal Exsanguinations: One of the methods to sacrifice

Experiments the experimental animal by excessive bleeding.
Exposure to animal study and different experi- Decapitation: Process of sacrificing the experi-
mental methodologies is one of the important part mental animals by cutting off the head with the
of the MD (Pharmacology), M Pharm , MBBS and guillotine.
B Pharm syllabus. Several new terminologies have
emerged in experimental pharmacology and Precision: Degree of an exactness of a calculated
student should be familiar with these terms. So, value falling exactly within the range. It shows
this part of the chapter deals with the termi- the refinement of a process, by which experiment
nologies which are commonly used in the is performed. An experimental data is considered
experimental pharmacology. to be excellent, if it is precise and accurate (Figs
12.2A to D)
General Terminology Used in the Reproducibility: It is the measure of a process or
Experimental Pharmacology result which indicates their closeness in the
Acclimatization: Pre-exposure of an animal to the independent set-up, that means repetitive and
particular procedures, the people and laboratory same results are obtained with the same process,
involved in the experiment, before conduct of the method, materials but in distinct laboratory
actual experiment (Fig. 12.1). under different conditions such as different
person, different apparatus, different environ-
Accuracy: Degree of trueness which is measured
ment, etc.
or calculated to its true value. It predominately
depends on the reproducibility or repeatability of Sensitivity: It is a measure for the stimuli which
an experiment. Accuracy is closely related to produces certain reaction or stimulation at
precision but not same (Figs 12.2A to D). minimal strength.
Fig. 12.1: Acclimatize the animal from animal house to the laboratory, person and procedure of the experiment
Figs 12.2A to D: Relation of accuracy and precision: An experiment, in which the accurate value is 20. In (A) all the four
measured value is 20, (B) value measured is not 20, (C) one value is 20 but other is closely related, and (D) all values are
closely related but not 20
Terminology of Experimental Procedure experiment whereas if experiments continue

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

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

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

Acute study: Short duration (day 0 to 1 week).
Subacute or Subchronic study: Short to medium duration (>1week to 12 weeks).
Chronic study: Long duration (>12 weeks to 24 weeks or even more up to 18 months in higher animals
like dog or monkey),
Identification and Collection
13 of Tissue/Muscle
In vitro bioassay is performed in the excised tissue selected to avoid errors in the experiment. The
or muscle. So, the proper identification and procedure is almost same for the tissue collection
collection of the tissue/muscle is the foremost in all experimental rodents. Tissue/muscle are
important procedure for successful bioassay. first selected according to the drug sensitivity,
Collection of the tissue always depends on the thereafter select the animal and continue with the
animal selection as per requirement of the procedures. Maintain the aseptic condition
experiment. The most sensitive tissue should be throughout the procedure.
Collection of Intestinal Tissue/Muscle

Ileum
Identification and Collection of Tissue/Muscle  141
Note:
1. Minimize the tissue damage by keeping the tissue on the cotton soaked with PSS (do not hold it at
the middle part).
2. The procedure remains same in case of guinea pig, rat and rabbit ileum.
Rat Colon (Ascending/Descending)

Ascending Colon
Descending Colon
Rat Uterus
Stomach Fundus
Frog Rectus Abdominis Muscle
Guinea Pig Trachea

Anococcygeus Muscle (Rat)
Vas Deferens (Rat)

Phrenic Nerve Diaphragm (Rat)
Chick Biventer–Cervicis
Note: The animal sensitivity to the drugs used and the most appropriate tissue identification and collection is given briefly
in the Table 13.1.
Figs 13.1A to D: (A) Surgical posture of rodent (rat), arrow denotes the straight head, body and paws, keep complete
body in stretched and straight leveled, (B) Step I to open up the abdominal cavity by midline incision; lift the skin gently to
make short horizontal cut, (C) Position of scissor and forceps to make short horizontal cut), (D) After short horizontal cut,
give the vertical (longitudinal) cut (For color version of Figures 13.1C and D see Plate 2)
Figs 13.1E to J: (E) Vertical cut opens the abdomen (indicated by arrows), (F) Abdominal viscera and its natural positions
(indicated by arrows), (G) Cecum (indicated by arrows), (H) Ileum (indicated by arrows), (I) Ascending colon (indicated
by arrows), (J) Descending colon (indicated by arrows)
M N
Figs 13.1K to N: (K) Position of; (1) Lung, (2) Diaphragm, (3) Fundus, (4) Pylorus, (5) Kidney and (6) Liver, (L) Anatomical
position; (1) Trachea and (2) Esophagus, (M) Localization of trachea (arrows indicate tracheal cartilage rings), (N) Uterus
(For color version of Figures 13.1K to N see Plate 2)
Table 13.1: Different animal tissues/muscles; their identification points and drug sensitivity with predominant receptors present
Animal tissue Identification point of T/M Muscle involved Circular Drug sensitivity Receptor predominantly
(T)/muscle (M) (C) or Longitudinal (L) present in (T/M)
Tracheal chain Present at the ventral neck region, C NA, A and ACh β2-receptor, M2/M3
above esophagus and between L receptors
sternocleidomastoid muscles
Stomach fundus Upper grey part of the stomach L 5-HT > ACh (10 times) > 5HT-D receptor
which attached to the thick and C Histamine (1000 times)
red pylorus Bradykinin
Ileum Ileum connects cecum at the L Histamine > ACh H1, muscarinic receptor
middle part where as large
intestine begins at the distal part
Ascending colon (4-7 cm from the ileocaecum L NA > A β3-receptor, 5-HT2A,
junction) ACh, 5-HT 5-HT4
Decending colon [5-7 cm above rectum, identify by L ACh Muscarinic receptor
Colon
the diagonal muscle strips on the
upper surface]
Anacoccygeus Thin muscle strip arises from the L NA, ACh, 5-HT, IsoP Adrenergic supply
sacral vertebrae and passes to No Histamine NANC
colon end
Vas deferens Attached to epididymis, should L NA and ACh α1-adrenergic receptors and
be distinguish from seminal vesicle muscarinic receptor
Uterus Clear two horn above rectum L Oxytocin, 5-HT, A, NA β, α (after estrogen
connected to ovary treatment*) receptors
ACh M2/M3 receptor
* 0.1 mg/kg stilbestrol, im 24 hr before the experiment with oxytocin/ACh and for 5-HT 0.25 mg/100 g of stilbestrol, i.p for three days before experiment
C- circular; L- longitudinal; alpha2- beta 2 receptor; 5-HT- 5- hydroxy tryptaminergic receptor; IsoP- Isoprenaline; ACh- Acetyl Chosline; H1- Histamine-
1 receptor; NANC- Non-adrenergic non-cholinergic receptor; NA- Noradrenaline; α-Alpha-adrenergic receptor; M2/M3- Muscarinic receptor M2/M3.
Principle of Muscle
14 Contraction
Action potential generated due to chemical, 3. Detachment of the cross bridge from the thin
electrical or mechanical stimuli is responsible for filament, and
muscle contraction. In many studies, it is 4. Energizing the cross bridge so that it can again
confirmed that ability to generate force and attach to a thin filament and repeat the cycle.
movement depends on the interactions of the two
contractile proteins, namely myosin (M) in the Type of Contraction
thick filaments and actin (A) in the thin filaments,
where energy is provided by ATP. Shortening of The classification mainly depends on the role of
the contractile elements in muscle is brought about shortening of the contractile elements in muscle.
by sliding of the thin filaments over the thick Depending on the property of a muscle either
filaments. reduction or no change in the length of muscle,
Each cycle consists of four steps (Fig. 14.1): contraction is classified into the two groups;
1. Attachment of the cross bridge to a thin (actin) 1. Isotonic contraction: The length of muscle
filament, against a constant load is reduced whereas
2. Movement of the cross bridge, producing tone remains same. For example: Bioassay of
tension in the thin filament, guinea pig ileum or other tissues, etc.
Fig. 14.1: Mechanism of muscle contraction

Principle of Muscle Contraction  151
2. Isometric contraction: The length of muscle In the bioassay, contraction/relaxation of the

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

Fast Contracting Smooth
15 Muscle Preparation
EXPERIMENT NO: 15A reagent bottle may vary with

different salt compositions)
Aim
To determine unknown concentration of hista- Precautions before Experimentation
mine by using guinea pig ileum. • Clean the organ bath before starting the
Background experiment specially inner organ bath (chances
of presence of previous drug used)
Guinea pig ileum is most sensitive to histamine. • Balance the writing lever horizontally with the
Histamine, mainly act through the H1 and H2
help of load
receptor. Contractile response of histamine to the
• Prepare PSS for the experiment, while taking
ileum is caused by the H1 receptor found in the
exact quantity of chemicals (1% variability is
ileum, bronchi and capillaries. The assay of the
acceptable)
preparation is based on the magnus method
(1904). Guinea pig ileum has the spontaneous • Add the calcium chloride at the end of PSS
activity and its specificity is improved by using preparation (to avoid any precipitation: PSS
atropine or mepyramine in tyrode. In the assay of should be clear)
histamine, atropine (2 × 10–6 to 10–7 M) and for • Try to minimize the handling of tissue (espe-
acetylcholine (ACh) assay mepyramine (5 × 10–6 cially at the middle part)
to 2 × 10–7) is used for the enhancement of the • Always use the finger to hold the tissue instead
specificity. Some of the endogenous polypeptides of forceps
like angiotensin, bradykinin and substance P are • Maintain the dose cycle properly (tissue
also sensitive to guinea pig ileum. sensitivity depends on the cycle)
• Avoid contact of very high dose of the drug; tissue
Materials and Method may loss sensitivity due to “tachyphylaxis”
(tissue may remain in contractile stage, in
Animal/tissue : Guinea Pig/ileum presence of high drug concentration)
PSS : Tyrode/Ringer
Lever : Frontal writing Methods
Tension/load : upto 1 gm
Step I: Keep the animal (guinea pig) for fasting at
Magnification : 7-10x
least for 24-48 hr.
Aeration air : O2/Carbogen
Temperature : 32-37°C Step II: Sacrifice the guinea pig by stunning (a
Drug : Histamine (Mwt: 307.14) strong blow) on the head then, keep the animal on
(Always check M wt. on the the dissecting board (DB).
Fast Contracting Smooth Muscle Preparation  153
Step III: Fix guinea pig on the DB by tying its legs 3- point, 4-point or matching, etc. (see the example
with the help of thread. given in bioassay chapter for calculation).
Step IV: Cut open the abdomen of guinea pig by a
Inference
vertical midline incision after a small horizontal
cut, and then expose the abdominal contents. Write the findings of the study.
Step V: Identification of the ileum is done by two
Discussion
ways; (1) identify the cecum then come back at least
of 10 cm, or (2) identify stomach, then go forward to Ileum is preferred for the experiment because of
identify the ileum 10 cm before cecum (also see in less mesentery attached to ileum, and nearly all
identification and collection of tissue section). receptors are present. But, 10 cm of ileum attached
to the cecum contains more of α-excitatory receptor
Step VI: Take out the 2 -3 cm or as desired length of is present which should be excluded. Intestine is
the ileum and remaining should be kept in supplied by both parasympathetic and sympa-
refrigerator for the future use. thetic nerves. Muscarinic receptors are predomi-
Step VII: The ileum is trimmed away from nant in parasympathetic action whereas inhi-
mesentery or attached tissues, then clean any bitory activity is mediated by the sympathetic α
waste contains of the ileum by a gentle push of and β- receptors. It is reported that inhibitory α-
PSS by the help of syringe (preferably PSS warmed receptors is present located presynaptically
at 30-35°C. whereas β-receptor (especially β1) are present on
the smooth muscle fibers. Fasting leads to less
Step VIII: Then, the ileum is attached to the one possibility of fecal matter present at the site. The
end with the hook attached to the tissue holding selection of tissue depends on the drug such as
arm and the other end is tie to the lever. guinea pig ileum is most sensitive for the histamine
(Attachment of the tissue should be in the direction or related compounds. Always select the tissue
of the intestine in vivo as far as possible) which is most sensitive to the drug. For example: If
Step IX: The experimental design is selected 3-point, the drug is ACh, then select the dorsal leech muscle
4-point or any other design mentioned. Then, make (if available) or frog rectus abdominis muscle, etc.
the DRC of standard and test drug then plan for For the intestinal preparation most commonly used
species are guinea pig and rabbit. Spontaneous
the selected experimental design accordingly such
activity of the tissue is reduced by performing the
as for 3-point select 2 response of standard and 1
experiment 5-7°C lower than the body temperature.
response of test whereas for 4-point select 2
responses of standard and test each.
SUGGESTED READING
Step X: Prepare the complete graph and calculate
1. Bian X, Burda JE, Carrasquillo M, Galligan JJ.
for determining the unknown concentration of
Postnatal down regulation of inhibitory
given drug. neuromuscular transmission to the longitudinal
Note: Rat or rabbit ileum bioassay procedure is muscle of the guinea pig ileum. Neurogastroenterol
Motil 2009 Apr 13.
same as described in the Guinea pig ileum. But, 2. Foong JP, Bornstein JC. 5-HT antagonists NAN-190
tissue shows variable spontaneous response and SB 269970 block alpha2-adrenoceptors in the
hence the tissue should be relaxed properly. guinea pig. Neuroreport 2009;20(3):325-30.
3. Guagnini F, Cogliati P, Mukenge S, Ferla G, Croci T.
Calculation and Result Tolerance to cannabinoid response on the myenteric
plexus of guinea-pig ileum and human small
The calculation and graph depend on the intestinal strips. Br J Pharmacol 2006;148(8):
method/design of experimentation adopted either 1165-73.
4. Hons IM, Burda JE, Grider JR, Mawe GM, Sharkey PSS : Krebs (AC) /deJalon (DC)
KA. Alterations to enteric neural signaling underlie Lever : Frontal writing
secretory abnormalities of the ileum in experimental Magnification : 4-7x
colitis in the guinea pig. Am J Physiol Gastrointest
Tension/load : 2g
Liver Physiol 2009;296(4):G717-26.
5. Schulz R, Seidl E, Wüster M, Herz A. Opioid
Air : O2 or Carbogen
dependence and cross-dependence in the isolated Temperature : 25°C (AC)/37°C (DC)
guinea-pig ileum. Eur J Pharmacol 1982;84(1-2): 33- Drug : Acetylcholine chloride (ACh)
40. [M. wt: 181.68]
6. Takaki M, Mizutani M, Jin JG, Nakayama S. Slow
hyperpolarizing action of tryptamine on myenteric Precautions before Experimentation
neurons of the isolated guinea-pig ileum. Acta Med
Okayama 1990;44(2):87-91. • Clean the organ bath before starting the
7. Yagasaki O, Sasaki N, Yanagiya I. Evidence of experiment specially inner organ bath (chances
ascending release of acetylcholine from the locally of presence of previous drug used or to avoid
distended guinea pig ileum. Jpn J Pharmacol. 1982; any contamination)
32(5):938-40.
• Balance the frontal writing lever horizontal
with the help of load
EXPERIMENT NO: 15B • Prepare PSS for the experiment, while taking
Aim exact quantity of chemicals (1% variability is
acceptable)
To determine unknown concentration of acetyl- • Add the calcium chloride at the end of PSS
choline (ACh) using rat ascending/descending preparation (to avoid any precipitation: PSS
colon. should be clear)
• Try to minimize the handling of tissue (espe-
Background cially at the middle part)
• Maintain the dose cycle properly (tissue
Initial, few centimeters (cm) of rat colon are usually
sensitivity depend on this cycle)
used for the bioassay of noradrenaline (NA) and
• Identify the ascending or descending colon
adrenaline (Adr) like preparations. Colon is more
properly, otherwise it may interfere with
sensitive to the NA than Adr. ACh assay can be
response.
also performed on the ascending or descending
colon. Its sensitivity may increase by keeping colon Method
at 4°C for 24 hr. Students should keep in mind (Rat ascending colon)
that after removing any tissue from the fridge, it
Step I: Keep the animal (rat) fasting for at least 24
should be allowed to recover the room temperature,
hours
before tying into the organ bath. It is also sensitive
to the substance P and little to prostaglandins and Step II: Sacrifice the rat by stunning (a strong blow)
angiotensin. on the head. After sacrifice, keep the animal on
It is found that the calcium sensing receptor the dissecting board (DB).
(CaSR) is activated by extracellular calcium (Ca2+) Step III: Rat should be fixed on the DB by tying its
and mediates increases in inositol 1,4,5- legs with the help of thread.
trisphosphate which is involved in the contraction
Step IV: Cut open the abdomen of rat by a vertical
and relaxation of the colon. midline incision after a small horizontal cut, and
then expose the abdominal viscera.
Materials and Method
Step V: Identify ascending colon through the
Animal/tissue : Rat/Ascending (AC) or des- cecum. Identify the cecum and then go forward
cending colon (DC) towards the rectum at least 5-7cm.
Step VI: Cut small piece (1.5-3 cm) as per the inner Step X: Select the experimental design and take
organ bath volume and clean it with a lukewarm the responses of the drug.
water or PSS with the help of a syringe.
Step XI: Calculate and make graph of recorded
Step VII: Then tie both the ends with the help of responses.
nylon or cotton thread and tie it to the balanced
horizontal lever. Calculation and Result
Step VIII: Leave the tissue at least for 30 min for The calculation and graph are depend on the
relaxation. method/design of experimentation adopted either
3- point, 4-point or matching, etc. (see the bioassay
Step IX: Then, take the response with the standard
section for calculation).
and test drug (prepared by serial dilution).
Step X: Select the experimental design and take Inference
the responses of the drug.
Write the findings of the study.
Step XI: Calculate recorded responses, according
to the selected experimental design. Discussion
For the ACh most sensitive tissue is dorsal leech
(Rat descending colon)
muscle and the frog rectus abdominis muscle. The
ascending/descending colon is not the suitable
Step I: Keep the animal (rat) fasting for at least 24
tissue for the assay of ACh, where as it may give
hours
erratic responses. Intestine preparations are very
Step II: Sacrifice the rat by stunning (a strong blow) commonly used in the isolated tissue experiment
on the head. After sacrifice, keep the animal on due to the ease of isolation, more handling
the dissecting board (DB). resistant than other tissues which have variable
spontaneous responses.
Step III: Rat is fixed on the DB by tying it legs with
the help of thread.
SUGGESTED READING
Step IV: Cut open the abdomen of rat by a vertical
1. Cheng SX, Okuda M, Hall AE, Geibel JP, Hebert SC.
midline incision after a small horizontal cut, and
Expression of calcium-sensing receptor in rat colonic
then expose the abdominal viscera. epithelium: Evidence for modulation of fluid
Step V: Identify the rectum and then come back at secretion. Am J Physiol Gastrointest Liver Physiol
2002; 283(1): G240-250.
least 5-7 cm for the collection of, descending colon
Step VI: Cut small piece (1.5-3 cm) of DC as per the EXPERIMENT NO: 15C
inner organ bath volume and clean it with a
lukewarm water or PSS with the help of a syringe. Aim
Step VII: Then, tie both the end with the help of To determine unknown concentration of acetyl-
nylon or cotton threads and tie it to the balanced choline (ACh) using rat uterus.
horizontal lever.
Background
Step VIII: Leave the tissue at least for 30 min for
The response of uterus preparation mainly
relaxation.
depends on the animal age due to variation in the
Step IX: Then, take the response with the standard estrus cycle. It is fast contracting tissue and shows
and test drug (prepared by serial dilution). the spontaneous response. It responds to the
minimal dose of ACh at 5 × 10–5M and carbachol • Maintain the dose cycle properly (tissue
at 10–4M. The other drugs like adrenaline (3 × sensitivity depend on this cycle)
10–6 M), noradrenaline (3 × 10–5M), isoprenaline • May give spontaneous response, hence relax
(10 –6 M), ephedrine (10 –3 M) and tyramine the tissue properly.
(10 –3 M) show the specificity to the uterus
preparation. Method
It is important to induce the estrus cycle into
the normal female animal before the experiment. Step I: Female rat (FR) weighing around 150-300 g
Artificially induced estrus cycle have the variable is taken, and 24 hr prior to the experiment, the rat
sensitivity to tested drugs such as for bioassay of is primed with 0.1 mg/kg stilbestrol, IM.
‘oxytocin’ or ACh; rat is treated with stilbestrol
Step II: Sacrifice the FR by stunning (a strong blow)
0.1 mg/kg (20 µg/ml), subcutaneous 24 hr before
on the head. After sacrifice, keep the FR on the
the experiment whereas for assay of ‘5-HT ’ on
dissecting board (DB).
uterus is primed for 3 days at the dose of 0.25 mg/
100 ml. Other drugs like histamine (acting on H1), Step III: FR is fixed on the DB by tying its leg with
adrenaline, and noradrenaline (acting on the help of thread.
β-receptor) are also sensitive to the uterus.
Step IV: Cut open the abdomen of FR by a vertical
midline incision after a small horizontal cut,
and then exposed the abdominal contains (Also
Animal/tissue : Female rat/uterus see in identification and collection of tissue/ muscle
PSS : Krebs/deJalon/McEwen section).
Lever : Frontal writing
Step V: Identify the vaginal orifice.
Tension : 1-4g Step VI: Trace the vaginal orifice interiorly to find
Air : O2 or Carbogen the two horns of the uterus.
Temperature : 32-37°C (at 32°C spontaneous
Step VII: Cut the two horns and now, you have
responses are low)
two samples of the uterus.
Drug : Acetylcholine chloride (ACh)
[M. wt: 181.68] Oxytocin [M. Step VIII: Cut short the tissue according to the inner
wt: 1007.19] organ bath (handle the tissue minimally at the
middle part which can reduce the sensitivity of
Precautions before Experimentation the tissue).
• Clean the organ bath before starting the Step IX: Select the experimental design (matching,
experiment specially inner organ bath (chances bracketing, 3-point, or 4point assay).
Note: Rabbit uterus responses are approximately
• Balance the writing lever horizontal with the
homologous to human. It gives the spontaneous
help of load
pendulum like movement response at the kymo-
graph.
acceptable)
Calculation and Result
• Add the calcium chloride at the end of PSS
preparation (to avoid any precipitation: PSS The calculation and graph depend on the
should be clear) method/design of experimentation adopted either
• Try to minimize the handling of tissue 3- point, 4-point or matching, etc. (see the bioassay
(especially at the middle part) section for calculation).
Inference Background
Write the findings of the study. The guinea pig or rabbit atria (2-4 cm) are generally
preferred for this assay. There are several advan-
Discussion tages of these tissues such as require very less
trimming or slicing of the tissue, thickness of tissue
Drug response to uterus in presence of different
gives easy way to the oxygen to pass through, tissue
estrous cycle differs due to the different receptors
thickness maintained throughout the preparation,
expression. α-adrenergic receptor is prominent have easy separation of right and left atria for
and it causes relaxation which is blocked by experiments using spontaneous beating or stimu-
α-receptor antagonists. Since, uterus has no lated preparation, and size and shape of the atria
inherent tone but the relaxation is observed by gives good contractile tension and stability over
physiological antagonism of the contractile the several hour of the experiment. Isoprenaline,
response of ACh or other drugs. In the estrous salbutamol and ACh shows the sensitivity
stage (uterus vascularity and the size is increased), (contraction) to the atrial tissue.
spontaneous contractility is increased. It is found
that after priming with estradiol, there is excess Materials and Method
expression of α-excitatory adrenoreceptors. Other Animal/tissue : Guinea pig/atria
drugs like 5-HT, noradrenaline, etc. can be PSS : Krebs /Ringer Locke
assayed with the uterus. Prostaglandins also Lever : Frontal writing/starling heart
show the contractile response via PGF2α in the Magnification : 6-8x
uterus smooth muscle of non-pregnant animals Tension : 1g
whereas PGE2 causes relaxation. Air : O2 or Carbogen
Temperature : 30-32°C
SUGGESTED READING Drug : Adrenaline (M. wt.: 183.21)
Isoprenaline HCL (M. wt:
1. Bossmar T, Osman N, Zilahi E, Haj MA, Nowotny 247.72)
N, Conlon JM. Expression of the oxytocin gene, but
Salbutamol (M. wt.: 239.31)
not the vasopressin gene, in the rat uterus during
pregnancy: influence of oestradiol and progesterone.
Precautions before Experimentation
J Endocrinol 2007;193(1):121-26.
2. Kumcu EK, Büyüknacar HS, Göçmen C, Evrüke IC, • Separate atria, ventricle safely, so that AV node
Onder S. Differential effect of neocuproine, a donot get disturbed
copper(I) chelator, on contractile activity in isolated • Clean the organ bath before starting the
ovariectomized non-pregnant rat, pregnant rat and
experiment specially inner organ bath (chances
pregnant human uterus. Eur J Pharmacol 2009 Jan
20.
of presence of previous drug used or to avoid
3. Orescanin-Dusiæ Z, Milovanoviæ S, Blagojeviæ D, any contamination)
Nikoliæ-Kokiæ A, Radojiciæ R, Spasojeviæ I, Spasiæ • Balance the frontal writing lever horizontal
M. Diethyldithiocarbamate potentiates the effects of with the help of load
protamine sulphate in the isolated rat uterus. Redox • Make PSS for the experiment, while taking
Rep 2009;14(2):48-54. exact quantity of chemicals (1% variability is
acceptable)
EXPERIMENT NO: 15D • Add the calcium chloride at the end of PSS
preparation (to avoid any precipitation: PSS
Aim
should be clear)
To determine unknown concentration of adrena- • Try to minimize the handling of tissue
line using guinea pig atria. (especially at the middle part)
Figs15.1A to D: Isolation and separation of atria from the ventricles; (A) Showing heart holded with forcep and making cut
at the atrioventricular junction (shave the AV pacemaker); (B) Isolated atrium from ventricle, dissected into right (Rt) and
left (Lt) atrium; (C) Separate into right (Rt) and left (Lt) atrium, cut through the dotted line; (D) Two pieces (Rt & Lt) of atrium
for the experiment
• Maintain the dose cycle properly (tissue Calculation and Result

sensitivity depend on this cycle) The calculation and graph are depend on the
• Carefully cut and separate ventricles method/design of experimentation adopted either
Method section for calculation).
Step I: Select the guinea pig weighing around 350- Inference

450 g and euthanized by cervical dislocation. Write the findings of the study.
Step II: Lateral thoracotomy is done and identify Discussion
the heart at the right bottom of thoracic.
Atria are highly supplied with the sympathetic and
Step III: Carefully cut the pericardium and remove slightly with parasympathetic nervous system. The
the heart then cut and clean the fat and other β2- receptor is widely present in the atria.
tissue attached to the heart. SUGGESTED READING
Step IV: Transfer the heart to the ice cold PSS so as 1. Chiao H, Caldwell RW. Local cardiac effects of
to reduce the metabolic activity and hence, it can substance P: Roles of acetylcholine and noradrenaline.
Br J Pharmacol 1995;114(2):283-88.
work longer.
2. Hussain M, Chorvatova A, Singh J. Physiological
effects and biochemical properties of a serum protein
Step V: Cut the ventricles carefully so that atria
that produces positive inotropic and chronotropic
pacemaker could not get disturbed. effects on isolated guinea pig atria. Mol Cell Biochem.
2004;261(1-2):201-07.
Step VI: Then, tie the thread on the both side of the 3. Li L, Zhuang FE, Yang L, Zhang CL, Zhao GS, Zhao
atria and hang into the organ bath and attached DK. Effects of osthole on isolated guinea pig heart
to the lever. atria. Zhongguo Yao Li Xue Bao 1995;16(3):251-54.
4. Pousti A, Bakhtiarian A, Najafi R, Deemyad T,
Step VII: Leave the tissue for relaxation for around Brumand K, Hosseini MJ. Effect of sertraline on
ouabain-induced arrhythmia in isolated guinea-pig
30-45 min.
atria. Depress Anxiety 2009 Feb 25.
Step VIII: Select the experimental design (matching, 5. Pousti A, Deemyad T, Malihi G, Brumand K. A
preliminary study on the interaction of fluvoxamine
bracketing, 3-point, or 4-point assay) and start the and adenosine receptor on isolated Guinea-pig atria.
response recording. Int J Neurosci 2006;116(12):1491-99.
6. Royse CF, Royse AG, Rohrlach R, Wright CE, Angus of presence of previous drug used or to avoid
JA. The cardiovascular effects of adrenaline, any contamination)
dobutamine and milrinone in rabbits using • Balance the frontal writing lever horizontal
pressure-volume loops and guinea pig isolated
atrial tissue. Anaesth Intensive Care 2007;35(2):
with the help of load
180-88. • Make PSS for the experiment, while taking
EXPERIMENT NO: 15E acceptable)
Aim • Add the calcium chloride at the end of PSS
To determine the unknown concentration of should be clear)
acetylcholine (ACh) using rat anococcygeus • Try to minimize the handling of tissue (espe-
muscle preparation. cially at the middle part)
Background
sensitivity depend on this cycle).
It is a thin strip of smooth muscle (made of 2
anococcygeus muscle) which arises from sacral Method
vertebrae and reaches to terminal colon (near anus).
The sensitivity of the anococcygeus muscle is to Step I: Sacrifice the rat by stunning (a strong blow)
ACh, noradrenaline, isoprenaline, adenosine and on the head and then transfer to the dissecting
5-HT but insensitive to histamine. The atropine and board (DB).
phentolamine block the contractile property of ACh
and noradrenaline respectively. This assay method Step II: Rat should be fixed on the DB by tying its
is previously described by the Gillespie (1972). The legs with the help of thread.
receptor responsible for the contraction/relaxation Step III: Cut open the abdomen of rat by a vertical
is considered to be prejunctional receptor which
results in enhancement of norepinephrine (NE)
then exposed the abdominal viscera.
release, which further induces contractile response
by activation of postjunctional α1-adrenoreceptors. Step IV: Identify the root of rectum interiorly and
Other receptor found to be responsible is then identify the 2 thin strips of anococcygeus
5-HT1B/1D. muscle which arise from the vertebrae and meet
at the terminal part of colon (anus).
Step V: Cut the rectal colon 2-3 cm from the anus
Animal/tissue : Rat/anococcygeus muscle and clear from the connective tissues and isolate
PSS : Krebs the 2 strips of anococcygeus muscle.
Lever : Frontal writing
Magnification : 7-10x Step VI: Keep in PSS and supply continuous
Tension : up to 1 gm constant air (O2 or carbogen).
Air : O2 or Carbogen Step VII: Prepare the standard and test drugs and
Temperature : 35-37°C select the experimental design.
Drug : Acetyl choline chloride (ACh)
[M. wt: 181.68] Calculation and Result
The calculation and graph are depend on the
Precautions before Experimentation method/design of experimentation adopted either
• Clean the organ bath before starting the 3- point, 4-point or matching, etc. (see the bioassay
experiment specially inner organ bath (chances section for calculation)
Inference hidroalcoholic extract of Pimpinella anisum

(Apiaceae) on rat anococcygeus smooth muscle. J
Write the findings of the study. Ethnopharmacol 2007;110(1):23-29.
Discussion EXPERIMENT NO: 15F

Practically, it is difficult to identify the anoco- Aim
ccygeus muscle. It gets mixed with the surroun-
ding smooth muscles. The only differentiation is, To determine unknown concentration of acetyl-
it has tendinous origin and do not appear soft. choline (ACh) using rat vas deferens
These muscles have a dense adrenergic excitatory
innervation as well as inhibitory innervations in Background
the rabbit, rat and cat. In the studies, it is found Rat and guinea pig are suitable animals for the
that adenosine triphosphate (ATP) have the preparation due to relatively large vas deferens
differential action on different animal such as, is than other animals. For increasing the sensitivity
a powerful inhibitory agent in the rabbit anoco- of preparation animal should be, fed with oats for
ccygeus, whereas in the rat it causes contraction at least for 3 days. Adrenaline, noradrenaline and
while in the cat, requires high concentrations to phenylephrine (µ-sympathomimetic) contract the
produce relaxation. Other like Acetylcholine- vas deferens whose action is blocked by the
sterase-positive fibers and catecholaminergic phentolamine. The method is previously described
fibers are abundant in the anococcygeus. In an by the Henderson et al (1972) and Hart et al (1979).
interesting study, it was found that androgen and
estrogen play an important role in sexual dimor-
phism of the mouse anococcygeus muscle.
Animal/tissue : Rat/vas deferens
SUGGESTED READING PSS : Krebs/Ringer/Tyrode/
1. Creed K E, Gillespie J S. Some electrical properties McEwen
of the rabbit anococcygeus muscle and a Lever : Frontal writing
comparison of the effects of inhibitory nerve Magnification : 6-8x
stimulation in the rat and rabbit. J Physiol 1977; Tension : 0.3-1g
273:137-53. Air : O2 or Carbogen
2. Emre S, Erdem SR, Tuncer M. Does serotonin relax
the rat anococcygeus muscle via 5-HT7 receptors?
Naunyn Schmiedebergs Arch Pharmacol 2000;
Drug : Acetyl choline chloride (ACh)
362(2):96-100. [M. wt: 181.68]
3. Emre-Aydingöz S, Kocaefe C C, Tuncer M. Calcium- : Histamine [M. wt: 307.14]
antagonistic activity of sumatriptan in the rat
anococcygeus muscle. Pharmacology 2002;64(1): 43-
48.
4. Gillespie JS. The rat anococcygeus muscle and its
• Clean the organ bath before starting the
response to nerve stimulation and to some drugs. Br
J Pharmac 1972; 45: 404-16. experiment specially inner organ bath (chances
5. Kulkarni SK, Sharma A. Rat anococcygeus: A of presence of previous drug used)
dynamic smooth muscle preparation for experimental • Balance the writing lever horizontal with the
pharmacology. Methods Find Exp Clin Pharmacol help of load
1994;16(6):379-85.
6. Tirapelli CR, de Andrade CR, Cassano AO, De
Souza FA, Ambrosio SR, da Costa FB, de Oliveira exact quantity of chemicals (1% variability is
AM. Antispasmodic and relaxant effects of the acceptable)
• Add the calcium chloride at the end of PSS receptor specifically β2-adrenoceptors. This effect
preparation (to avoid any precipitation: PSS is found to be antagonized by yohimbine,
should be clear) piperoxan, phentolamine and tolazoline.
• Try to minimize the handling of tissue Contractile effects of sympathomimetic agents
(especially at the middle part) may either antagonize or potentiated by the
• Maintain the dose cycle properly (tissue reversible µ-adrenoceptor antagonists according
sensitivity depend on this cycle) to their doses and duration of contact of drug to
• Carefully remove the connective tissues and the tissue.
blood vessel attached to the vas deferens.
SUGGESTED READING
Method
1. Berdysheva LV and Manukhin BN. Effect of
Step I: Sacrifice the rat by stunning (a strong blow) Activation of Muscarinic Cholinergic Receptors on
on the head and then transfer the animal on the the Kinetics of α1-Adrenergic Contractile Response
dissecting board (DB). of the Rat Vas Deferens. Doklady Biological Sciences
2001;381(1-6):522-25.
Step II: Rat should be fixed on the DB by tying its 2. Minneman KP, Fox AW, Abel PW. Occupancy of
legs with the help of thread. alpha 1-adrenergic receptors and contraction of rat
vas deferens. Mol Pharmacol 1983;23(2):359-68.
Step III: Cut open the abdomen of rat by a vertical
EXPERIMENT NO: 15G
then expose the abdominal viscera.
Step V: Identify the testes then pull it to one side Aim
and vas deferens a white thin tubular structure is To determine unknown concentration of anta-
identified easily. gonist (atropine) using acetylcholine (ACh) as an
Step VI: It is freed out from the epididymis which agonist employing guinea pig ileum preparation
can be distinguished clearly from where it joins by pA2 method.
the urethra. Cut into desired length of tissue.
Background
Step VI: Put into the Petri dish filled with PSS with
proper continuous distinguishable aeration. pA2 is the principle method for studying the
antagonist action for a selected agonist which is
Step VII: Put the tissue into the organ bath and defined as the negative logarithm to base 10 of
perform the experiment as per selected experi- the antagonist concentration (molar units)
mental design. corresponding to a dose-ratio of 2 (i.e. the
concentration that produces a 2-fold shift in the
agonist concentration-response curve). This
The calculation and graph are depend on the method is developed by Schild in 1957. But, the
method/design of experimentation adopted either limitation being Schild design is that it requires
3- point, 4-point or matching, etc. (see the bioassay a large number of experimental units to obtain
section for calculation). even a minimal number of points for the Schild
plot.
Inference
Write the findings of the study. Materials and Method
Animal/tissue : Guinea pig/ileum
Discussion PSS : Tyrode
Contractile responses of rat vas deferens are due Lever : Frontal writing
to the presence of noradrenergic or adrenergic Magnification : 7-10x
Tenin/load : Up to 1 gm Method
Air : O2/Carbogen
Step I: Keep the animal for fasting at least for 24
hours.
Step II: Standard drug DRC is plotted by the same
[M. wt: 181.68]
method as described in the guinea pig ileum
Atropine sulphate [M. wt:
experiment no. 15A.
694.84]
Step III: Select the dose ‘2R’ from the DRC of
Precautions before Experimentation standard drug (between 25-75% of response).
Step IV: Then, take at least three responses of ‘2R’.
experiment specially inner organ bath Step V: Add the 10 times lesser concentration of
atropine (antagonist) and wait for 5-15 min, then
(chances of presence of previous drug used)
without washing add agonist i.e. 2R.
• Balance the writing lever horizontal with the
Step VI: Wash out the antagonist and take the
help of load
responses till the normal response of ‘2R’ is
• Prepare PSS for the experiment, while taking achieved (maintain the dose cycle to preserve the
exact quantity of chemicals (1% variability is sensitivity of the tissue).
acceptable) Step VII: Repeat the step V until the response of
• Add the calcium chloride at the end of PSS the ‘2R’ is about equal to ‘R’(about the half
preparation (to avoid any precipitation: PSS response of ‘2R’.
should be clear) Step VIII: Plot the graph, of % response to 2R versus
• Try to minimize the handling of tissue (espe- -log (antagonist) dose (Figs 15.2A and B)
cially at the middle part)
• Always use the finger to hold the tissue instead Calculation and Result
of forceps After plotting the %response versus –log (anta-
• Maintain the dose cycle properly (tissue gonist), pA2 value is directly extrapolated through
sensitivity depend on this cycle). the graph at which its % response remains 50%.
Example
Figs 15.2A and B: As an example, DRC plot by an agonist thereafter selection of ‘2R’ is shown. After selection of ‘2R’ plot
it at least for the 3 times, then add the antagonist at 10 times lower concentration of agonist and repeat the experiment
as shown in the plot; R1, R2, and R3 are the responses of ‘2R’ after addition of antagonist, (B) Represents the graph plot
of % response to 2R versus –log (antagonist) and determination of pA2 at the X-‘axis’
Slow Contracting Muscle
16
EXPERIMENT NO: 16A the rat fundus due to the lack of H1 receptors in the
stomach muscle. The other drugs which have
Aim sensitivity against the fundus are ACh, bradykinin
To determine unknown concentration of serotonin and prostaglandin (PGE2).
(5-HT) using rat stomach (fundus). The muscle preparation is the important step
in the bioassay using stomach fundus. Both
Background longitudinal and circular muscle may be used in
the experiment which depends on the transverse
Stomach fundus is useful in the bioassay of 5-HT cut made to prepare the tissue (Figs 16.1A to E).
which is considered to be most sensitive tissue
among the three parts of stomach namely, fundus, Materials and Method
corpus and pylorus. Fundus is identified by its gray
color and situated above the pink thick pyloric Animal/tissue : Rat/Stomach fundus
region. Drugs like histamine are little or insensitive to PSS : Tyrode/deJalon/Krebs
Figs 16.1A to E: (A) Showing three parts of stomach; (B) Stomach cut open through lesser curvature and spread on a
paper sheet wetted with PSS, (C) Divided into two parts, if want to preseve the longitudinal muscle, (D) vertical alternate
transverse cut to preserve the longitudinal tissue, (E) Horizontal alternate transverse cut for preserving circular muscle
(donot cut the stomach into two parts if want to see the effect of circular muscle)
Slow Contracting Muscle  165
Lever : Frontal writing/ simple lever Step V: Identify the stomach (just down the line of
Magnification : 7-15x peritoneal cavity touches the liver and spleen.
Tension : 1-4g
Step VI: Cut and separate the stomach attachment
Air : O2/Carbogen
at the end of esophagus and down at the
duodenum.
Drug : Serotonin (5-HT) [M.wt: 176.
218] Step VII: Identify the fundus (grey part) which
Acetylcholine chloride (ACh) discriminates from the pyloric region that is pink
[M. wt: 181.68] in appearance.
Precautions before Experimentation Step VIII: Wash the stomach contents properly with
the PSS and then cut open the fundus by lesser
• Clean the organ bath before starting the curvature.
of presence of previous drug used or to avoid Step IX: Then, cut the fundus at the midline into
any contamination) two equal parts.
• Balance the simple writing lever, tangential to Step X: For preserving the longitudinal muscle the
the rotating drum alternate opposite vertical cut should be given (as
• Prepare PSS for the experiment, while taking shown in the figure) and then tie the thread on the
exact quantity of chemicals (1% variability is either end of the muscle and stretch out the muscle.
acceptable)
Step XI: Then, attach into the organ bath and leave
for relaxation at least 30-45 min.
should be clear) Step XII: Select the experimental design and get
• Try to minimize the handling of tissue (espe- the response with standard and test drug.
• Maintain the dose cycle properly (tissue Calculation and Result
sensitivity depend on this cycle)
The calculation and graph depend on the method /
• Animal should be fasted properly (any food
design of experimentation adopted either
present in the stomach may interfere with the
assay)
section for calculation).
• Carefully clean the fundus from the adjacent
tissues. Inference
Method
Step I: Keep the animal (rat) fasting for at least Discussion
24hr, water is given ad libitum.
Fundus contains the swallowed air and has
Step II: Sacrifice the rat by stunning (a strong blow) functions mainly concerned with pressure
on the head, and then keep on the dissecting board changes. Its preparation is somewhat tedious than
(DB). the other tissue preparations because it require
alternate longitudinal cutting of tissue in which
Step III: Rat is fixed on the DB by tying it legs with
contraction of a circular muscle is measured in a
the help of thread.
longitudinal fashion. It is a slow contracting
Step IV: Cut open the abdomen of rat by a vertical muscle hence, sometimes it needs a stretching
long cut, and then expose the abdominal viscera. weight to bring it to the baseline. Bradykinin
produces slow contraction of stomach fundus

whereas it is a strong stimulator of isolated
intestinal tissue. This tissue well responds to the
tryptamine and prostacycline (PGE2).
SUGGESTED READING
Frog rectus abdominis
1. Cohen ML, Fludzinski LA. Contractile serotonergic muscle (Typical silver grey
receptor in rat stomach fundus. J Pharmacol Exp color; differentiating from the
Ther 1987;243(1):264-69. surrounding muscle)
2. Komada T, Yano S. Pharmacological characterization
of 5-hydroxytryptamine-receptor subtypes in circular
muscle from the rat stomach. Biol Pharm Bull
2007;30(3):508-13.
3. Scarparo HC, Santos GC, Leal-Cardoso JH, Criddle
DN. Selective inhibitory effects of niflumic acid on 5- Fig. 16.2: Identification of frog rectus abdominis muscle
HT-induced contraction of the rat isolated stomach
fundus. Br J Pharmacol 2000;130(3):678-84.
4. Vane JR. A sensitive method for the assay of 5- Animal/tissue : Frog/rectus abdominis muscle
hydroxytryptamine. 1957. Br J Pharmacol 1997; PSS : Ringer
120(4 Suppl):142-47.
Lever : Simple writing lever
5. Yu PL, Fujimura M, Hayashi N, Nakamura T,
Fujimiya M. Mechanisms in regulating the release of Magnification : 10-15x
serotonin from the perfused rat stomach. Am J Tension : 1-2 gm
Physiol Gastrointest Liver Physiol 2001;280(6): Air : O2/Carbogen
G1099-105. Temperature : Room temperature
EXPERIMENT NO: 16B [M. wt: 181.68]
Aim Precautions before Experimentation
To determine unknown concentration of ACh • Frog rectus abdominis muscle is skeletal
using frog rectus abdominis muscle. muscle and requires less precaution during
handling compared to other smooth isolated
Background tissues
Rectus abdominis muscle is a striated skeletal experiment specially inner organ bath (chances
muscle preparation which is sensitive to the ACh of presence of previous drug used)
and curare like substances. But, the most sensitive • Balance the simple writing lever tangentially
muscle for the ACh is considered to be the dorsal leech with smoked drum
muscle. The activity is considered due to presence • Prepare PSS for the experiment with sufficient
of nicotinic muscarinic (NM)-receptor. Mamma- quantity of chemicals (1% variability is
lians muscle fibers are of two types; single or focal acceptable)
-innervated fiber and multiple-innervated fibers. • Add the calcium chloride at the end of PSS
Among which single or focal –innervated fiber preparation (to avoid any precipitation: PSS
shows fast contractility whereas multiple- should be clear)
innervated fibers shows the slow contraction. Frog • Try to minimize the handling of tissue (espe-
rectus abdominis muscle is composed of both cially at the middle part)
fibers but the multiple-innervated fibers dominate • Maintain the dose cycle properly (tissue
and show the slow contraction (Fig. 16.2). sensitivity depend on this cycle)
Method maintaining the room temperature and proper

oxygenation whereas, in summer it gives good
Step I: Pith the frog and lay it on its back on the
result in standard set-up.
frog dissecting board. Pin the four limbs on the
dissecting board (ventral part facing above and
SUGGESTED READING
distal part facing downward).
1. Edge ND. The effect of antiadrenaline compounds
Step II: Remove the abdominal skin and expose on acetylcholine responses of frog rectus abdominis
the rectus abdominis muscle (silver grey, distinct muscle. Br J Pharmacol 1970;38(2):386-93.
from surrounding tissue, at middle lining). 2. Erenmemisoglu A, Tekol Y, Gogusten B.
Neuro-muscular blocking effect of thiamphenicol on
Step III: Dissect out the muscle on the PSS soaked frog rectus abdominis muscle. Gen Pharmacol 1 994;
piece of paper and spread gently. 25(7):1417-20.
3. Karataº Y, Ergün Y, Göçmen C, Seçilmiº A, Singirik
Step IV: Cut into two pieces (preserve the E, Dikmen A, Baysal F. Possible postsynaptic action
longitudinal muscle). of aminoglycosides in the frog rectus abdominis. Acta
Med Okayama 2000;54(2):49-56.
Step V: Tie the thread on the one end and tie it to
4. Kounenis G, Koutsoviti-Papadopoulou M, Elezoglou
the inner organ bath and the second end tied to V. Effect of nizatidine and ranitidine on the D-
the simple lever in the upright position under the tubocurarine neuromuscular blockade in the toad
tension of 1-2 gm. rectus abdominis muscle. Pharmacol Res 1994;
29(2):155-61.
Step VI: Leave tissue for relaxation for at least 5. Nagata M, Kadota K. Acetylcholine bioassay with
45 min. thin strip of frog rectus abdominis muscle. Nippon
Seirigaku Zasshi 1977;39(3):62-64.
Step VII: Mean while prepare the standard and 6. Parle M, Kulkarni SK. Alpha 2 adrenoceptor-
test drug serial dilution. mediated contraction of frog rectus abdominis
muscle. Arch Int Pharmacodyn Ther 1984;271(1):
Step VIII: Start the recording of response by using 122-26.
the suitable experimental design. 7. Premendran SJ, Khapre MD, Sharma ML. Effects of
calcium, strontium, and barium salts on frog rectus
Calculation and Result abdominis. Indian J Exp Biol 1990;28(6):590-91.
8. Ramaswamy S, Geetha VS, Nazimudeen SK,
The calculation and graph depend on the Kameswaran L. Carbachol-induced sensitivity
method/design of experimentation adopted either changes in skeletal muscle and their mechanism of
3- point, 4-point or matching, etc. (see the bioassay action. Eur J Pharmacol 1978;52(2):197-200.
section for calculation). 9. Rao SS, Bhagwat AW, Parmanand VG. Interaction
of acetylcholine and caffeine on the isolated rectus
abdominis of frog (Rana tigrina). Indian J Physiol
Inference
Pharmacol 1978;22(2):155-57.
EXPERIMENT NO: 16C
Discussion
Aim
Frog rectus abdominis muscle is the easiest
To determine unknown concentration of acetyl-
isolated tissue to handle, even safe with new
choline (ACh) using guinea pig trachea.
people. Being the amphibian, it responds under
room temperature and does not require
Background
temperature maintenance. But, season variability
is noted in the response recording. It shows erratic This method is based on the method of Castillo
response in the winter but it is corrected by the and De Beer (1947). The response is taken by the
extraction of at least 6 cm of trachea (containing B. Give the spiral cut to the trachea. Cut is
minimum of 6 rings). Tracheal ring is in “D” form, made at the mid part of cartilage then, tie
and smooth muscle is present in the straight line the thread on the either end and stretch the
of “D” shape. The ring is prepared for the assay tissue (Fig. 16.4C)
by the two means
This preparation is mainly to demonstrate the
1. Separate the ring and tie one another with the
help of thin thread (Fig. 16.3) respiratory dominant β2-adrenoreceptor which
Tie the ring with one another at cartilage, but causes bronchodilation by the adenylcyclase and
it is important to keep in mind that smooth cAMP. Limitation of the tissue preparation is that
muscle should be at longitudinal fashion. it is tedious to prepare and has the slow contrac-
2. A. Longitudinal cut along the mid dorsal tion and relaxation.
surface (cut at mid of cartilage part), then
separate each cut ring. Then, tie the each Materials and Method
cartilage end with one another to form loop
(Figs 16.4A and B) or Animal/tissue : Guinea pig/trachea
PSS : Krebs
Lever : Simple/frontal writing
Tension : Up to 0.5 gm
Air : O2/Carbogen

Fig. 16.3: Separation of tracheal rings and method to • Balance the writing lever horizontal with the
make a chain help of load
• Make PSS for the experiment, while taking
acceptable)
should be clear)
sensitivity depend on this cycle).
Method
Figs 16.4A to C: Another method to prepare trachea for Step I: Guinea pig (GP) is sacrificed under
bioassay anesthesia.
Step II: Fix the GP on the dissecting board and 2. Gok S, Izanli-Paksoy A, Vural K. Contribution of
then shave the hair near the neck region. RhoA kinase and protein kinase C to weak relaxant
effect of pinacidil on carbachol-induced contractions
Step III: The trachea is dissected out and put into in sensitized guinea-pig trachealis. Arch Pharm Res
the petri dish containing Krebs solution (see the 2009;32(2):243-50.
identification and collection of tissue section for detail). 3. Larsson AK, Fumagalli F, DiGennaro A, Andersson
M, Lundberg J, Edenius C, Govoni M, Monopoli A,
Step IV: Tracheal chain is then dissected out by Sala A, Dahlén SE, Folco GC. A new class of nitric
any of the above mentioned methods. oxide-releasing derivatives of cetirizine; pharma-
cological profile in vascular and airway smooth
Step V: Mount the trachea in the inner organ bath muscle preparations. Br J Pharmacol 2007; 151(1):
and give the 45-60 min of relaxation. 35-44.
4. Nascimento NR, Refosco RM, Vasconcelos EC,
Step VI: Select the experimental design (bracketing, Kerntopf MR, Santos CF, Batista FJ, De Sousa CM,
matching, 3-point and 4-point). Fonteles MC. 1,8-Cineole induces relaxation in rat
and guinea-pig airway smooth muscle. J Pharm
Calculation and Result Pharmacol 2009;61(3):361-66.
5. Schaafsma D, Gosens R, Bos IS, Meurs H, Zaagsma
Result and calculation depends on the experi- J, Nelemans SA. Role of contractile prostaglandins
mental design. After getting the complete response and Rho-kinase in growth factor-induced airway
recording, refer the calculation part in bioassay smooth muscle contraction. Respir Res 2005;6:85.
chapter. 6. Tanaka Y, Yamashita Y, Horinouchi T, Koike K.
Adrenaline produces the relaxation of guinea-pig
Discussion airway smooth muscle primarily through the
mediation of beta(2)-adrenoceptors. J Smooth Muscle
The response of this tissue is slow to develop but Res 2005;41(3):153-61.
last for longer period. This preparation is utilized
to study the bronchodilators like theophylline, EXPERIMENT NO: 16D
adrenaline, etc. β-adrenoceptor subtypes which
induced relaxation is identified in smooth muscle Aim
cells of the isolated guinea-pig trachea. Tracheal To determine unknown concentration of acetyl-
preparation is an ideal model to study the choline (ACh) using rat phrenic nerve dia-
contractile drugs like acetylcholine, 5-HT and phragm.
histamine, additionally their antagonism is
studied by several drugs like adrenaline, Background
isoprenaline (ISO), aminophylline, theophylline,
etc. Noradrenaline lacks activity on β2- adrenergic This is primary motor nerve of the diaphragm
receptor, hence absence of relaxation in the which arises mainly from the fourth cervical
presence of the ACh, 5-HT or histamine. In the nerve. Preparation is preferred in the nerve
recent study, role of nitric oxide (NO) as mediated muscle contraction and its response in
bronchodilator is also well studied. presence of agonist and antagonists. This nerve
preparation performs well in Krebs or tyrode
SUGGESTED READING solution at 32-37°C. However, it is seen that the
cholinesterase activity is absent at room temperature.
1. Dellabianca A, Faniglione M, De Angelis S, Tonini S, Cholinergic agonists, anticholinesterase,
Balestra B, Colucci M, Cervio M, Clavenzani P, sympathomimetics or neuromuscular blockers
Chiocchetti R, De Giorgio R, Candura SM. Adenosine
may be used in the preparation. The response
A(1) and A(3) Receptor Agonists Inhibit
Nona-drenergic, Noncholinergic Relaxations in the observed by physostigmine and neostigmine is
Guinea Pig Isolated Trachea. Respiration. 2008 different in the neuromuscular blockage
Dec 11. produced by the d-tubocurarine.
Materials and Method Step V: Separate and clear the thoracic contents,
then identify the phrenic nerve, running from
Animal/tissue : Rat/ phrenic nerve diaphragm
PSS : Krebs /tyrode diaphragm to the thymus gland.
Lever : Preferably spring loaded lever Step VI: Two cuts are given, one at diaphragm and
or simple lever second at the base of the thymus (avoid excessive
Magnification : 10-18x cleaning of the nerve from the muscle).
Tension : 0.5-1g
Air : O2 or Carbogen Step VII: Immediately transfer the nerve to the krebs
Temperature : 35-37°C or tyrode, then attach to the organ bath.
Step VIII: Give proper relaxation for 45-60 min
[M. wt: 181.68]
and select the experimental design and record
Precautions before Experimentation the response by standard and test drugs
respectively.
experiment specially inner organ bath (chances Calculation and Result
• Balance the writing lever horizontal with the The calculation and graph depend on the
help of load method/design of experimentation adopted either
• Prepare PSS for the experiment, while taking 3- point, 4-point or matching, etc. (see the bioassay
exact quantity of chemicals (1% variability is section for calculation).
acceptable)
• Add the calcium chloride at the end of PSS Inference
should be clear)
cially at the middle part) Discussion
• Maintain the dose cycle properly (tissue Rat phrenic nerve–diaphragm preparation is
sensitivity depend on this cycle) commonly used in the experimental
• Carefully remove the connective tissues and pharmacology for evaluation of neuromuscular
blood vessel attached to the phrenic nerve. function. Studies showed 70% of ACh in the
diaphragm contain motor nerve terminal, 10%
Method
intramuscular nerve fibers and 65% acetyl
Step I: Sacrifice the rat by stunning (a strong blow) transferase is in motor terminal with 35% in nerve
on the head and then transfer the animal on the fiber. Studies showed atropine has variable
dissecting board (DB). response with different concentration. In low
Step II: Rat should be fixed on the DB by tying it concentration, it enhanced neuromuscular trans-
legs with the help of thread. mission, possibly via a presynaptic mechanism,
Step III: Cut open the thorax of rat by a vertical however in higher concentration, atropine causes
midline incision, and then exposed the thoracic reduction or it may block transmission.
cavity.
Step IV: Ribs are dissected from the base of the SUGGESTED READING
sternum, and half thorax is cut and removed by 1. Alves-do-Prado W, Prado WA. Neuromuscular
cutting through animal flanks. facilitation induced by muscarinic antagonists in the
rat isolated diaphragm. Gen Pharmacol 1993;24(6): Materials and Method

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

EXPERIMENT NO: 16E
Aim experiment specially inner organ bath (chances
To determine neuromuscular blocking drugs of presence of previous drug used)
using innervated biventer-cervicis preparation of • Balance the writing lever horizontal with the
the chick. help of load
Background exact quantity of chemicals (1% variability is
acceptable)
This preparation is for the study of neuromuscular
blocking agents such as suxamethonium, tubo-
curarine, etc. Muscles contain two types of slow or
should be clear)
twitch fibers which correspond to different
• Try to minimize the handling of tissue
response at the different stimulus. When their nerve
(especially at the middle part)
supply is stimulated electrically, they give twitch
responses which is similar to the rat diaphragm • Maintain the dose cycle properly (tissue
responses whereas its contraction responses are sensitivity depend on this cycle).
similar to the frog rectus muscle. Decamethonium,
suxamethonium, etc. have good specificity for the Method
tissue and give good contractility. Tubocurarine Step I: Select chick weighing about 400-800 g and
chloride is used in the higher concentration to euthanize with sodium pentobarbitone (6 mg/
produce contraction as compared to other drugs. 100g, IM) or chloroform.
Acetylcholine (ACh) is less contractile in this
muscle but produces marked effects in the presence Step II: Give the midline incision on the back of the
of physostigmine or neostigmine. In the presence neck after removal of feathers.
of suxamethonium and ACh, it shows contractile Step III: Identify the biventer-cervicis on the both
and depolarization block which is increased by side of the midline incision just under the skin.
neostigmine whereas tubocurarine has no activity
on slow muscle fiber and neostigmine reversed Step IV: Tie the upper portion with thread and cut
neuromuscular block of twitch muscle. the upper portion then cut the muscle from the
bottom then attach the second thread at the bottom twitch/contraction to different stimuli.
to hang the tissue in inner organ bath. Tubocurarine, suxamethonium, decamethonium,
Step V: Give the relaxation time about 45 min-1 hr. carbachol, etc. may act on the chick biventer
cervicis muscle by releasing acetylcholine at the
Step VI: Make the standard solution and then make synapse which then acts on the postjunctional
serial dilution.
receptors to produce the response.
Step VII: Select the experimental design (matching,
bracketing, 3-point, or 4-point assay). SUGGESTED READINGS
1. Barlow RB, Zoller A. Activity of analogues of de-
The calculation and graph depends on the camethonium on the chick biventer cervicis preparation.
method/design of experimentation adopted either Br J Pharmacol Chemother 1962;19: 485-91.
3- point, 4-point or matching, etc. (see the bioassay 2. Elliott RC. The role of acetylcholine in tetra-
section for calculation). ethylammonium induced contractures of the chick
biventer cervicis muscle in the presence of lidocaine.
Inference Gen Pharmacol 1987;18(1):7-11.
3. Marshall I G. Actions of acetylcholine and carbachol
Write the findings of the study. on the chick biventer cervicis muscle Br J Pharmac
1971;42:462-72.
Discussion
4. Wali FA. Effect of lignocaine on chick biventer cervicis
Biventer cervicis (BC) is an anatomically complex skeletal muscle. Pharmacol Res Commun 1986;18(1):
tendinous muscle and respond differently 31-48.
Cardiac Muscle Preparation
17
EXPERIMENT NO: 17A Precautions
Aim • Animal should be pretreated with heparin

(55-110 µg/kg, i.p or s.c [for rabbit] or heparin
To observe the effect of various drugs on the (500-1000 IU/kg, i.v in rabbit and rat and i.p
isolated heart (Langendorff’s preparation). in guinea pig), to avoid thrombosis in heart, if
mounting take > 2-3 min after the surgical
Background procedure) and then sacrificed after 20-30 mins
In 1897, Oscar Langendorff established the isolated • Maintain the PSS flow rate adequately (Edema
perfused mammalian heart preparation which may develop in cardiac tissues, if the perfusion
pressure is too high) (The expected flow rate
was considered to be the breakthrough in
through the cannula differs depending on
cardiovascular research. This is based on the
species and it ranges from 7-9 ml/min for rat to
principle of retrograde flow in the aorta either at
20 ml/min for rabbit)
constant flow or constant pressure. The entire
• While attaching the heart to the cannula, there
perfusate enters the coronary arteries via the should be already some flow through the aortic
ostia at the aortic root (Fig. 17.1A). The physio- cannula, which helps to avoid air bubbles
logical salt solution (PSS) passes through the entering the coronary arteries
coronary circulation and then, perfusate drains • Insert cannula firmly so that it should not
into the right atrium via the coronary sinus. penetrate into the aortic valve (Fig. 17.1A to C)
Coronary flow rate is measured on volumetric • Maintain the sufficient hydrostatic pressure
determination. by maintaining the distance between reservoir
and heart position.
Animal Required
• Albino Rats (300 gm and at least of 1 yr of age), Method
or Animal is pretreated with the heparin (55-110 µg/
• New Zealand Rabbits (1.5-3 kg and 3 years of kg, i.p or s.c [for rabbit] or heparin (500–1000 IU/kg,
age) or i.v in rabbit and rat and i.p in guinea pig). Animal
• Guinea pig (300-450 gm and 2-3 years of age) is stunned and the thorax is opened immediately
Animal is housed at ambient temperature and the heart exposed. Heart is dissected out and
(23°C ±2°C) under a 12:12 hr light and dark cycle removed as rapidly as possible and transferred
and with free access to tap water and food ad immediately into cold Krebs solution (rat and guinea
libitum. The animals are acclimatized to the pig heart) or McEwens solution (rabbit heart). The
laboratory conditions for at least 1 week prior to ascending aorta is separated gently using forceps
experimentation. from the pulmonary artery and dissect the heart
Figs 17.1A to C: (A) Showing the coronary supply of the rat or rabbit heart; (B) Bilateral coronary supply in guinea pig,
different from the rat or rabbit. It has 2 right and 2 left coronary arteries and (C) Position of cannula in the ascending aorta
(above aortic valve)
with at least 1 cm aorta intact. (Usually the aorta is this to about 18 inches above the heart or alter-
cut just before it divides.) The heart is gently natively, if a peristaltic pump is used set the rate for
squeezed several times to remove blood from the about 15-20 ml/min, reducing it to 3-5 ml/min
heart and prevent formation of thrombus. Trim maintain perfusion chamber and aerated Krebs
away any excess connective and lung tissue from solution in it at 37-38°C.
the heart taking care not to damage the aorta. The
glass cannula (3 mm outer diameter for rats and Attachment of Heart
guinea pig; 4 mm outer diameter for rabbit) tip is
placed 0.5 cm into the aorta and firmly kept in place Attach a heart clip complete with a length of thread
using a thread. It is important to exclude the air to the tip of the ventricles (another small clip may
entrap into the system to prevent air emboli. Initially be bound to the auricles). The thread is passed
side arm is flushed to remove air bubbles from the around a pulley arm vertically below the heart.
apparatus before the experiment is started. Attach the thread to the transducer after passing
Langendorff’s preparation is a constant head it over a second pulley horizontal to first and
reservoir system, used for providing pressure, raise about 20 cm apart and attach to the transducer
Cardiac Muscle Preparation  175
Fig. 17.2: Langendorff apparatus set-up for the assessment of activity of different drugs
which further attach to the coupler of student Recording

physiograph. After the experiment animals are Reading of the rate of heating and of the coronary
disposed in the yellow polythene bags for the flow can most commonly be taken over a period of
incineration. 30 seconds. If the movements are too fast to count,
the kymograph can be run at a high speed for 15-
Instrument 30 seconds before the drug reaches the heart and
in most instances, the effects passes in a few
The recording is done by attaching the thread to a minutes as the drugs are washed through.
strain gauge transducer, an attachment with the Recording of the rate of heating of flow should be
student physiograph. The paper speed can be taken once every 2 minutes. Further, doses should
adjusted as required. The sensitivity of the not be given until the preparation has recovered
recording apparatus to the heart contractions can completely and the rates are steady or until they
be adjusted based on the sample tracings and the have settled down steadily at new control levels.
baseline is adjusted so that the tracing is in middle Note: Important to follow the step: Initially the
of the paper. Standardized speed is maintained. experiment is started with the stimulant drugs
(epinephrine, norepinephrine, isoprenaline and
Drugs calcium in increasing doses), followed with
depressant (acetylcholine, potassium) drugs and
Drug additions by attaching a syringe to the finally antagonists (propranolol and atropine) are
injection port (or into a rubber tubing) preferably added as the effects of the antagonists take a long
keeping the volumes < 0.2 ml. time to wear off.
Observations: The preparation is observed for the EXPERIMENT NO: 17B

inotropic (by measuring the amplitude of contrac-
tions) effect, chronotropic (by measuring the rate of Aim
heart beat) and effect on coronary vascular To determine the effect of different drugs on the
resistance (by measuring the rate of collection of normal and hypodynamic rabbit heart.
drops from the preparation).
Background
1. Based on principle of retrograde perfusion

Hypodynamic heart is defined as the heart
2. Circulation of PSS in isolated heart: Directly delivered exhibiting subnormal power or force than the
to the coronary artery (left and right), collected at normal one. Experimentally, it is developed by
coronary sinus then drained into right ventricle and the supply of the 1/4th of calcium chloride (CaCl2)
then pumped through the pulmonary artery than the required one which reduces the heart
3. Rabbit heart can beat to 9 hour if not treated to the
toxic drugs
rate.
4. Transfer of isolated heart immediately to the ice cold
PSS leads to reduce the metabolic activity as well as Requirements
reduce beating, which results into low requirement of • Animal: Rabbit (2-3 kg)
glucose and oxygen
• Drugs: Digoxin, calcium chloride (CaCl2)
5. Isolated heart reduces work load, hence the cardiac
output in isolated heart cannot be directly correlated • Physiological salt solution (PSS): McEwens
as in in vivo condition solution (For normal heart), ¼th of CaCl2 in
McEwens solution (For the hypodynamic
SUGGESTED READING heart)
1. Beckett PR. The isolated perfused heart preparation:
• Instrument: Physiograph or Starling Heart lever
Two suggested improvements. J Pharm Pharmac and drum.
1970;22:818.
2. Doring HJ, Dehnert H. The isolated perfused heart
Method
according to Langendorff. BVM-Biomesstechnic The entire primary set-up of instrument and the
Verlag, 1987. attachment of the heart is same, as it is in the
3. Fukunami M, Hearse DJ. The inotropic consequences
of cooling: Studies in the isolated rat heart. Heart
Langendorff’s preparation.
and Vessels 1989;5:1-9.
4. Hearse DJ, Sutherland FJ. Experimental models for Recording of the Responses
the study of cardiovascular function and disease. Step I: First record the effect of the drug on the
Pharmacol Res 2000;41(6):597-603. normal heart preparation with the (0.1, 0.2, 0.4
5. Hofer E, Stark U, Stark G, Tritthart HA. Detection
and continuous monitoring of intracardiac low-level
and 0.8 ml. (Digoxin, CaCl2)
potentials from the surface of the Langendorff- Step II: Then, note the dose on the said response
perfused heart. Basic Res Cardiol 1990;85:198-208.
6. Mattsson C, Hoylaerts M, Holmer E, Uthne T, Collen
accordingly.
D. Antithrombotic properties in rabbits of Step III: McEwens solution is replaced by the
heparin and heparin fragments covalently coupled
to human antithrombin III. J Clin Invest 1985;75(4):
hypodynamic McEwens solution and leave for 5-
1169-73. 10 min for the acclimatization of the heart in the
7. Ravelli F, Allessie M. Effects of atrial dilatation on hypodynamic solution.
refractory period and vulnerability to atrial fibrillation
in the isolated Langendorff-perfused rabbit heart. Step IV: Record the response of the drug on the
Circulation 1997;96:1686-95. same concentration as it is used previously in the
8. Stark G, Stark U, Tritthart HA. Acute effects of normal heart.
amiodarone on the pacemaker- and conduction
systems of the Langendorff perfused guinea pig heart. Step V: Note the response of drug after treating
Z Kardiol 1987;76(S2):67. with the hypodynamic solution and note the
dose on the respective response. Then, fix the Step VI: Compare the response of the drug
tracing if used kymograph. on the normal and hypodynamic heart pre-
paration.
Observation and Result

Drug Dose (ml) Normal heart Hypodynamic heart Remarks
Rate Force Rhythm Rate Force Rhythm
Digoxin 0.1
0.2
0.4
0.8
Calcium chloride 0.1
0.2
0.4
0.8
SUGGESTED READING
1. Chapman RA, Niedergerke R. Effects of calcium on
the contraction of the hypodynamic frog heart. J
Physiol 1970;211(2):389-421.
2. Grupp IL, Subramaniam A, Hewett TE, Robbins J,
Grupp G. Comparison of normal, hypodynamic, and
hyperdynamic mouse hearts using isolated work-
performing heart preparations. Am J Physiol Heart
Circ Physiol 1993;265:H1401-10.
EXPERIMENT NO: 17C

Aim
To demonstrate the effect of the inotropic and Fig. 17.3: Three chambered frog heart
chronotropic effects of various drugs on frog heart
normal/hypodynamic)
i. Isolated preparation and mimetic drugs like adrenaline and noradrenaline.
ii. In situ preparation There are few drugs which decreases the rate and
force of contraction and hence they grouped as
Background “negative chronotropic” and “negative inotropic”
drugs respectively like parasympathetic drugs like
The heart is the most common site for the drug target acetylcholine (ACh).
and there are several drugs which influence the
rate, force or rhythm of the heart either as therapeutic REQUIREMENTS
effect or as side effects. Inotropic drugs are those
which increase the force of contraction and the Animal : Frog (50- 70 gm)
response is called as the “positive inotropic Drugs : Adrenaline
response” whereas drugs which increase the heart Noradrenaline
rate are chronotropic drugs and the response is Acetylcholine
known as “positive chronotropic response”. Calcium chloride
Broad classification of drugs into the positive Potassium chloride
inotropic and chronotropic involves sympatho- Atropine
PSS : Frog ringer solution (FRS) and Step VI: The lever used is sterling-heart lever. Heart
hypodynamic solution is attached to the lever with the help of thin thread
(¼CaCl2 in FRS)
Step VII: Then, take the recording with different
Temperature : Room temperature
drugs
Instrument : Physiograph or kymograph
Method for Experiment No: 17(i) Methods for Experiment No: 17(ii)
Step I: Carefully hold the frog with its hind limb Step I: Carefully hold the frog with its hind limb
and then anesthetize the frog by pithing or keep it and then anesthetize the frog by pithing or keep it
into the freezer (-4°C) for about 3-5 min into the freezer (-4°C) for about 3-5 min
Step II: Give a little horizontal cut at the mid of Step II: Give a little horizontal cut at the mid of
abdomen and then a long vertical incision at the abdomen and then give a long vertical incision at
midline above sternum the midline above sternum
Step III: Open the thoracic cage and locate the heart Step III: Open the thoracic cage and locate the heart
Step IV: Carefully remove the pericardium and Step IV: Cannulate the aorta, with a little incision
remove the heart and tie the cannula with aorta in situ
Step V: Clear it from the surrounding tissue and Step V: Take the responses with different drugs
squeeze it gently to remove the blood from the heart using 0.2 ml adrenaline, noradrenaline, ACh,
in cold FRS and then mount it in the organ bath. CaCl2, KCL and atropine.
Observation and Results

Drug Dose (ml) Normal heart Hypodynamic heart Remarks
Rate Force Rhythm Rate Force Rhythm
Adrenaline 0.1
0.2
0.4
0.8
Noradrenaline 0.1
0.2
0.4
0.8
Acetylcholine 0.1
(ACh)
0.2
0.4
0.8
CaCl2 0.1
0.2
0.4
0.8
KCl 0.1
0.2
0.4
0.8
Atropine 0.1
0.2
0.4
0.8
SUGGESTED READING the ventricle of the frog. J Physiol 1968;197(2):

479-509.
1. Broadley KJ. Negative inotropic responses of the
isolated heart of the rat to isoprenaline. Br J Pharmacol 3. Martin Morad, Chris Sanders, James Weiss. The
1972;45(1):123-25. inotropic actions of adrenaline on frog ventricular
2. Graham JA, Lamb JF. The effect of adrenaline on the muscle: Relaxing versus potentiating effects. J Physiol
tension developed in contractures and twitches of 198;311:585-604.
Part 3
Experimental (In Vivo Studies)

Animal Experiment on
18 Central Nervous System (CNS)
EXPERIMENT NO.: 18A
Aim
To demonstrate the effect of pentobarbital on
righting reflex (Hypnosis) in mouse.
Background
Hypnosis is the process of natural sleep and the
drugs or agents inducing it are known as the
hypnotic agents. This concept is applicable to the
patient or human use, but in the animal
experiments, the term “hypnotic” means deeper
stage of central depression which induces Fig. 18.1: Loss of righting reflex in rat (unable to correct
unconsciousness, associated with loss of righting body posture)
reflexes and muscle tone. The “loss of righting
reflex” is the term mainly used to denote the ‘sleep’ Hexabarbital (60 mg/ kg,
of an animal and it is defined as the loss of postural i.p), Diazepam (5-20 mg/kg,
reaction which can’t be corrected when the animal i.p or s.c)
is kept on its back. In the righting reflex animal
turns body in such a way that, its paws or feet are Precautions before Experimentation
pointed at the ground. Righting reflex reaction is
• Laboratory should be dim lighted and noise
dependent on normal vestibular, visual and
free
proprioceptive functions (Fig. 18.1).
• Animal should be marked properly, to avoid
mixing in two groups
• Handle the animals with care (minimize the
Materials stress and pain to animal)
• Observe the animals in a plexi glass chamber
Animal/species : Mouse/Swiss albino
Sex/Body weight : Either sex/ 20-30 g
Methods
Syringe/needle : 1ml/ preferably 24G on-
wards Step 1
Drug : Pentobarbital (50 mg/kg, i.p) • Weigh the animals and mark them properly to
Other drugs : Barbital (180 mg/kg, i.p), distinguish from one another
• Divide animals into two groups (n = 6 in each Step 3

group) • Observe the animals for 45 min to 1 hour
• Observe for the onset of loss of righting reflex
Step 2
and duration of loss of righting reflex
• Group 1: Control group (n = 6); mice are given
saline at the equivalent dose of drug Note: Observe the animals for any other behavior
• Group 2: Treatment group (n = 6); mice are changes during the observation time (sniffing,
given Pentobarbital 50 mg/kg, i.p. rearing, exploratory behavior, etc.)
Observation Table
Sl. Righting reflex Duration of loss of righting
No. Onset (min) (O) Recovery (min) (R) reflex (min) (D= (R) – (O))
Group 1 Group 2 Group 1 Group 2 Group 1 Group 2
1.
2.
3.
4.
5.
6.
Note: Cat is not a suitable animal for the experiment on “loss of righting reflex”. This is because; they
have flexible backbone and no functional clavicle.
Discussion SUGGESTED READING

Evaluation of the righting reflex is an ideal method 1. Liu X, Lee TL, Wong PT. Cyclooxygenase-1 Inhibition
to evaluate drug acting as CNS depressants, since Shortens the Duration of Diazepam-Induced Loss
the entire CNS depressant drugs cause loss of of Righting Reflex in Mouse. Anesth Analg 2006;
righting reflex in animals at certain dose. e.g.: 102:135-40.
2. Simon P, Chermat R, Doaré L, Bourin M, Farinotti R.
Mouse and rat are the ideal models for studying Interactions imprévues de divers psychotropes avec
righting reflex whereas the cat is not a suitable les effets du barbital et du pentobarbital chez la
animal for the experiment “on righting reflex”. souris. J Pharmacol (Paris) 1982:13;241-52.
This is because; they have flexible backbone and
no functional clavicle. There are several methods EXPERIMENT NO: 18B
of doing this experiment, generally putting animal
on its back (distal portion) to correct the position Aim
on its four legs or dropping the animal from a
To demonstrate muscle relaxant property of
certain height and observe either animal touches
diazepam in mouse using rotarod apparatus
ground on its four paws or not, is very commonly
in use. Drugs used in such experiment have CNS
depressant properties such as anesthetics like Background
pentobarbital, thiopental etc. or long of short acting
hypnotics like diazepam, nitrazepam or zolpidem, Rota rod test is used to evaluate fore and hind
zopiclone, triazolam etc. respectively. Studies limb motor coordination of rodents. The apparatus
suggest that when short or long acting hypnotics consists of a horizontal metal rod (coated with
are added with the anaesthetics, it significantly rubber) of 3 cm diameter attached to a motor with
increases the duration of the loss of righting reflex. the speed adjusted to 2 rotations/minute to 6
Animal Experiment on CNS  185
Fig. 18.2: Rota rod apparatus
rotation/min. The rod is 75 cm in length and is • Animal should be marked properly, to avoid
divided into 6 sections by plastic discs, thereby mixing in two groups
allowing the simultaneous testing of 6 mice. The • Handle the animals with care (minimize the
rod is at a height of about 50 cm above the tabletop stress and pain to animal)
in order to discourage the animals to escape from • Precondition the mouse with the procedure
the instrument (Fig. 18.2). The cut off time for the
Methods
test is 2 min. The retention time (sec) for each
mouse/rat is recorded. Step 1
• Weigh the animals and mark properly to
distinguish from one another
• Divide animals into two groups (n = 6 in each
Materials group)
Animal/species : Mice/ Swiss albino Step 2

saline at the equivalent dose of drug
Syringe/needle : 1ml/ preferably 24G on- • Group 2: Treatment group (n = 6); mice are
wards given diazepam at the dose of 3 mg/kg, ip
Drug : Diazepam (3-5mg/kg, i.p)
Step 3
• Observe the animals for 2 min on the rota rod
apparatus
• Laboratory should be dim lighted and noise • Observe the animals, to fall down and note
free the time of falling down
Note: For mouse: Mouse is placed on a 2.5 cm SUGGESTED READING

diameter rod rotating at 3 rev/min. The animal’s
1. Cartmell SM, Gelgor L, Mitchell D. A revised rotarod
muscle coordination is considered to be impaired,
procedure for measuring the effect of antinociceptive
if it falls from the rota rod within 120 sec drugs on motor function in the rat. J Pharmacol
observation period. Methods 1991;26(2):149-59.
2. Dunham NW, Miya T S. A note on a simple approach
Dimensions of Rota Rod for determining neurological deficits in rats and
mouse. J Am Pharmacol Assoc 1957;46:208.
Mouse: Rod is 75 cm in length, 30 mm diameter 3. Liu X, Lee TL, Wong PT. Cyclooxygenase-1 Inhibition
and is divided into 6 sections by plastic discs and Shortens the Duration of Diazepam-Induced Loss
of about 50 cm above the table top. of Righting Reflex in Mouse. Anesth Analg 2006;
102:135-40.
Rat: Rod is 75 cm in length, 60 mm diameter and 4. Stanistaw J. Czuczwar, Kinga K. Borowicz, Zdzistaw
is divided into 6 sections by plastic discs and of Kleinrok, Piotr Tutka, Tomasz Zarnowski,
about 50 cm above the table top. Waldemar A. Turski. Influence of combined
treatment with NMDA and non-NMDA receptor
Observation Table antagonists on electroconvulsions in mouse. Eur J
Pharmacol 1995;281:327-33.
Sl. No. Retention time on Rota rod (cut off time 120 sec)
Pre-drug Post-drug EXPERIMENT NO: 18C
1.
2. Aim
3.
To demonstrate muscle relaxant property of
4.
5. diazepam in mouse using chimney test.
6.
Background
Discussion: Motor coordination is a physiological
Mouse is made to climb backward up a plastic
process to achieve movement which is an essential
Perspex tube (3 cm inner diameter and 25 cm
interaction between neural processes involved in
moving a limb, and the actual limb in movement. length). The animals usually unable to perform
This is mainly processed through peripheral the test within 60s are considered to be positive
nervous system (PNS). It is responsible for both for motor impairment. The internal diameter
the transmission of the efferent to the CNS and varies with the animal’s weight such as for low
the movement of the limb. CNS also plays a vital weight mouse, it requires less diameter and for
role in integrating the afferent feedback and higher weight mouse, it require larger inner
efferent signals. There are several experimental diameter of tube (22-28 mm).
models which are established to study the motor
co-ordination in the rodents such as rotarod Materials and Methods
method, chimney test, grip strength, treadmill Materials
performance etc. In the either tests, rodents (mouse
Animal/species : Mouse/Swiss albino
or rat) are tested for their ability to retain the muscle
co-ordination such as in rotarod test. Mouse/rat Sex/Body weight : Either sex/ 20-30g
should remain on the revolving rod or in chimney Syringe/needle : 1ml/ preferably 24G on-
test; it should climb backward to show the muscle wards
coordination. The drugs which can be screened Drug : Diazepam (3mg/kg, i.p)
through these tests are anesthetics like Pheno-
barbital, etc. centrally active skeletal muscle
relaxants such as benzodiazepam, chlordia- • Laboratory should be dim lighted and noise
zepoxide or diazepam, zolpidem, zopiclone, etc. free
• Animals should be marked properly, to avoid 2. Stanistaw J Czuczwar, Kinga K. Borowicz, Zdzistaw
mixing in two groups Kleinrok, Piotr Tutka, Tomasz Zarnowski,
• Handle the animals with care (minimize the Waldemar A Turski. Influence of combined treatment
with NMDA and non-NMDA receptor antagonists
stress and pain to animal)
on electroconvulsions in mouse. Eur J Pharmacol
• Precondition the mouse with the procedure 1995;281:327-33.
one day prior to the experiment day.
EXPERIMENT NO.: 18D
Methods
Aim
Step 1
• Weigh the animals and mark properly to To demonstrate anti-anxiety effect of diazepam in
distinguish from one another rat using elevated plus maze apparatus
group) Background
Step 2 It is a novel test for the selective identification of
• Group 1: Control group (n = 6); mice are given ‘anxiogenic and anxiolytic’ drug effects in rodents.
saline at the equivalent dose of drug The plus maze apparatus consists of two open
• Group 2: Treatment group (n = 6); mice are (16 × 5 cm for mouse and 50 × 10cm for the rats)
given diazepam at the dose of 3 mg/kg, ip and two closed arms (16 × 5 × 12 cm for mouse and
50 × 10 × 40 cm for rats), and an open roof of the
Step 3 entire maze elevated (25 cm for mouse and 50 cm
• Force mice backwards in a plastic perspex tube for rats) from the floor. The animals are placed
(3 cm inner dimeter, 25 cm length), and make individually at the centre of the elevated plus maze
tube slightly tilted. with their head facing towards the open arm during
• Observe the animal performance for 60 sec, the 90 sec-5 min test. The preference of the animal
(climbing backward into the perspex tube). for the first entry, the number of entries into the
open and closed arms reflect the relative safety of
Observations and Results closed arms as compared with the relative
fearfulness of open arms. Rat/mouse are rodents
Observation Table
and feel safe in dark, hence normal rodents prefer
Sl. Motor Co-ordination (cut off time 60s) dark arm first. Anxiolytics would be expected to
No. Group 1 Group 2 increase the proportion of entries into and time
Present Absent Present Absent spent in open arms (Figs 18.3 and 18.4).
1.
2.
3. Materials
4.
5. Animal/species : Rat/Wistar
6. Sex/Body weight : Either sex/150-250g
Syringe/needle : 1ml/preferably 23G
Discussion Drug : Diazepam (0.5-1 mg/kg, i.p)
Refer to the exp 18b
SUGGESTED READING • Laboratory should be dim lighted and noise
1. Boissier JR, J Tardy, JC Diverres. Une nouvelle method free
simple pour explorer l’action ‘tranquilisante’: le test • Animal should be marked properly, to avoid
de la chimin6e, Med Exp (Basel) 1960;3:81. mixing in two groups
Fig. 18.3: Rat facing towards the open arm in an elevated plus maze (For color version see Plate 3)
Fig. 18.4: Diagram of rat elevated plus maze with dimensions of the arm and height
• Handle the animals with care (minimize the Step 2

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

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

spent in the closed arm. The judgement parameters
There are several methods available for screening
during the experiment are time spent in open arm,
the anxiolytic compounds such as elevated plus latency to enter in open arm, total number of entries
maze, Z-maze, O-maze, Y-maze or radial maze in open arm, and first preference of arm by the
etc. Elevated plus maze is most popularly used in animal (open/closed). The principle is same for
the screening and evaluation of the drug by all above mentioned mazes. Drugs like
decreasing anxiety. It is evaluated by increase in benzodiazepine, alcohol, barbiturates etc. may be
the time spent and exploration time in open arm screened through this method.
SUGGESTED READING four quadrants. A different starting point is

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

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

Sl. no. No. of entries/time spend (sec) Time to reach to
Group 1 Group 2 the platform
Q1 Q2 Q3 Q4 Q1 Q2 Q3 Q4 Group 1 Group 2
1.
2.
3.
4.
5.
6.
Discussion 2. D’Hooge Rudi, Deyn Peter P. De. Applications of

the Morris water maze in the study of learning and
Morris water maze is mainly designed to evaluate memory. Brain Research Reviews 2001;36:60-90.
the drugs acting on learning and memory and 3. Kant G J, Wylier R M, Vasilakis AA, Ghosh S. Effects
was devised by Prof Richard Morris. Morris water of triazolam and diazepam on learning and memory
maze has two advantages over other mazes, (1) as assessed using a water maze. Pharmacol Biochem
rat searches for the escape, so there is no waiting Behav 1996;53(2):317-22.
4. Kant GJ, Wylie RM, Vasilakis AA, Ghosh S. Effects
time and (2) there are no local cues (e.g. olfactory
of triazolam and diazepam on learning and memory
or auditory). Drugs like NMDA receptor as assessed using a water maze. Pharma-co-logy,
antagonists which improve the learning and biochemistry and behavior 1996;53 (2):317-22.
memory whereas benzodiazepines that impair the 5. McNamara RK, Skelton RW. Diazepam impairs
memory, can also be evaluated in the experiment. acquisition but not performance in the Morris water
Study reported that it impairs acquisition but not maze. Pharmacol Biochem Behav 1991;38(3):
retrieval of spatial information and is not due to 651-58.
the sedative, hypothermic or state-dependent
learning effects of diazepam. EXPERIMENT NO: 18F
SUGGESTED READING Aim
1. Arolfo MP, Brioni JD. Diazepam impairs place To demonstrate the anticonvulsant property of
learning in the Morris water maze. Behav Neural Biol diazepam against pentylenetetrazole (PTZ)
1991;55(1):131-36. induced convulsions in mice.
Background equal distribution in each group (Check for

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

Observation Table
Group 1
Sl. no. Onset of Severity of No. of seizure in % of positive Duration of
seizures (sec) seizure (score) 60 min responder* seizure
1.
2.
3.
4.
5.
6.
*Score ≥3 is considered as positive responder
Group 2
Sl. no. Onset of Severity of No. of seizure in % of positive Duration of
seizures (sec) seizure (score) 60 min responder* seizure
1.
2.
3.
4.
5.
6.
Discussion evaluated are phenytoin, valproate, pheno-

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

One to 20 myoclonic body jerks in 10 min; 3 –
• Laboratory should be dim lighted and noise More than 20 body jerks in 10 min; 4 – Clonic
free forelimb convulsions; 5 – Generalized clonic
• Animal should be marked properly, to avoid convulsions with rearing and falling down
mixing in two groups episodes; 6 – Generalized convulsions with tonic
• Handle the animals with care (minimize the extension episodes (same score may be used to
stress and pain to animal) score the PTZ induced seizure also. (Exp. 18F)
Sl no. Onset of Severity of No. of seizure % of positive Duration of

seizure (sec) seizure (score) in 60 min responder seizure (sec)
A B C A B C A B C A B C A B C
1.
2.
3.
4.
5.
6.
Discussion EXPERIMENT NO: 18H

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

• Observe the animals after MES
Materials
• Record whether THLE “present or absent”
Animal/species : Rat/ Wistar • Calculate percentage protection
Sex/Body weight : Male/ 150-250 g Note:
Syringe/needle : 1ml/ preferably 23G 1. All the procedure and scoring is the same for
Drug : Phenytoin (20-25mg/kg, i.p mouse. The only difference is the selection of
or po) electrical stimuli and duration of electrical
stimuli through ear/corneal electrodes
Instrument 2. One can also videotape the whole seizure
Electro convulsiometer: events in rat/mouse and can use the scale to
• (For rat: Intensity of stimulus: (150mA, 50Hz score the seizure events in the slow motion
for 0.2sec) video run (Events takes place in the millisecond
• (For mouse: Intensity of stimulus: (12mA, 50 of time, hence not possible to observe by the
Hz for 0.2 sec) naked eye; only THLE is seen as end point.
Other drugs: Diazepam (3-4 mg/kg, i.p. for mouse) Seizure score
Precautions before Experimentation 0 = no seizure; 1 = forelimb extension without

hind limb extension; 2 = complete forelimb
• Laboratory should be dim lighted and noise extension and partial hind limb extension, 3 =
free complete tonic hind limb extension (THLE) (hind
• Animals should be marked properly, to avoid limb become parallel to the tail) and 4 = post ictal
mixing in two groups depression; See Figs 18.5A and B.
stress and pain to animal) Observations and Results
• Animals should be screened 1 week prior to the
Percentage protection (%) = No. of animals with
experiment
THLE absent / total no. of animal × 100
• Check the instrument and ear electrode for its
proper working before experiment
Observation Table
• Ear should be lubricated with gel or water to
enhance the conductivity of electricity. Sl. Tonic hind limb extension (THLE) % Pro-
no. Present (Yes) Absent (No) tection
Methods Group 1 Group 2 Group 1 Group 2
Step 1 1.
• Weigh the animals and mark properly 2.
• Divide animals into two groups (n = 6 in each 3.
group) 4.
5.
Step II
6.
• Group 1: Control group (n = 6); rats are given
the saline as per body weight
• Group 2: Treatment group (n = 6); rats are given Discussion
phenytoin 20-25 mg/kg, i.p., thereafter 30 min The end point of the experiment is considered as
rats are given electro shock at the intensity of the absence/presence of THLE following a drug
150 mA, 50 Hz for 0.2 sec. (in case of oral test treatment which is a position during the GTCS in
drug, MES induced after 60 min) rodents when tail and both hind limbs are parallel
to each other. It is a subjective measure hence, and D1 to some extent. In the experiment, extra-
mouse/rat should be screened 1 week prior to the pyramidal side effects are identified by catalepsy.
experiment. The animal present with the positive It is an extreme tonus, muscular rigidity which is
THLE is included in the study. One week time is characterized by a tendency to remain in a fixed
given to animal to recover from the excitatory position for long period, hence unable to correct
neuronal discharge in the brain. an externally imposed, unusual posture over a pro-
(Also refer to discussion of experiment 18f) longed period of time (Figs 18.7A to C).
SUGGESTED READING Materials and Methods

1. Blanco MM, Dos Santos Jr JG, Perez-Mendes P, Kohek Materials
SR, Cavarsan CF, Hummel M, Albuquerque C, Mello
LE. Assessment of seizure susceptibility in Animal/species : Rat/ Wistar
pilocarpine epileptic and nonepileptic Wistar rats Sex/Body weight : Male/ 150-250g
and of seizure reinduction with pentylenetetrazole Syringe/needle : 1ml/ preferably 23G
and electroshock models. Epilepsia 2008 Nov 19. Drug : Haloperidol (0.4-4 mg/kg,
2. Rastogi SA, Ticku MK. Involvement of a GABAergic i.p)
mechanism in the anticonvulsant effect of
Instrument : Two Wooden or cardboard
Phenobarbital against maximal electroshock-induced
seizures in rats. Pharmacol Biochem Behav pillar, 10cm apart with a rod
1985;222:141-46. and rod at the height of
3. Stanistaw J, Czuczwar, Kinga K, Borowicz, Zdzistaw 10-20 cm (for mice, height =
Kleinrok, Piotr Tutka, Tomasz Zarnowski, 7-10 cm) (Fig. 18.6)
Waldemar A. Turski. Influence of combined
treatment with NMDA and non-NMDA receptor Precautions before Experimentation
antagonists on electroconvulsions in mouse. Eur J
Pharmacol 1995;281:327-33. • Laboratory should be dim lighted and noise
4. Stephen H Koslow, Lloyd J Roth. Reserpine and free
acetazolamide in maximum electroshock seizure in
• Animal should be marked properly, to avoid
the rat. J Pharmacol Exp Therap 1971;176(3):711-17.
5. Woodbury LA, Davenport VO. Design and use of a mixing in two groups
new electroshock seizure apparatus and analysis of • Handle the animals with care (minimize the
factors altering seizure threshold and pattern. Arch stress and pain to animal)
Int Pharmacodyn 1952;92:97-107. • Condition the animal to the experimental box
for at least 2min, before the experiment (Fig.
EXPERIMENT NO.: 18I 18.6).
Aim
To demonstrate effect of phenothiazine (halo-
peridol) induced catatonia in rat.
Background
Anti-psychotics drugs are well known for their
Fig. 18.6: Dimensions of instrument used to evaluate
extrapyramidal side effects. Present experiment catalepsy in rat
demonstrates extra-pyramidal side effects like
tardive dyskinesia in animal followed by the Methods
phenothiazine (Haloperidol) treatment. Pheno- Step 1
thiazine and butyrophenone neuroleptics act • Weigh the animals and mark properly for
through the blockade of both β and D2 receptors identification
Figs 18.7A to C: Different postures of catatonia produced in the rat
• Divide animals into two groups (n = 6 in each Observation Table

group)
Sl. Cataleptic (Abnormal) Posture (60s)
Step II % of
no. Present Absent
• Group 1: Control group (n = 6); rats are given cataleptic
Group 1 Group 2 Group 1 Group 2 animal
the saline as per body weight
• Group 2: Treatment group (n = 6); rats are given 1.
haloperidol 4 mg/kg, i.p., thereafter 10-15 min 2.
rats expose to the test 3.
4.
Step 3
5.
• Observe the animals behaviour and correction
6.
of its externally induced abnormal posture
(cutoff time 60s)
• Time of posture correction is noted down at Discussion
30, 60, 120 and 360 min Extrapyramidal adverse effects are commonly
• Calculate percentage of responders reported with the antipsychotic drugs like
Note: If rat remains on the bar for 60s, then chlorpromazine, flufenazine, haloperidol etc.
catalepsy is positive They are mediated through dopamine D 2 -
receptor blocked mainly in the nigrostriatal
Observations and Resulte pathway, which leads to dystonia, bradykinesia,
akathisia, dyskinesias etc. These behavioral
Calculation of percentage of cataleptic animals: changes are well studied in rodents by failure
No. of animals showing cataleptic posture to correct the externally exposed abnormal
100
Total number of animals position.
SUGGESTED READING is the presence of the dorsal sacro coccygeus

muscle on the either side of tail originating at the
1. de Sousa Moreira LF, Pinheiro MC, Masur J.
base of spinal cord (Fig. 18.8D). In the previous
Catatonic behavior induced by haloperidol, increased
by retesting and elicited without drug in rats.
study, it showed that either side cut of the muscle
Pharmacology 1982;25(1):1-5. deviate the tail towards the side of attached
2. Cerbo R, Carchedi F, Casacchia M. Role of serotonin muscle. The mode of action is not clear. But various
in catatonia induced with haloperidol and reserpine. studies suggest that straub tail results because of
Boll Soc Ital Biol Sper 1976;52(4):245-50. direct action on the opioids μ-receptor, most
3. Costall B, Naylor RJ. On catalepsy and catatonia importantly μ2–receptor. There are some studies
and the predictability of the catalepsy test for which suggest that, it also results through 5-HT
neuroleptic activity. Psychopharmacologia 1974;
receptors, α 2-(alpha-2) receptors, dopamine
34(3):233-41.
receptors and glucocorticoid receptors as well.
4. Casey DE. Serotoninergic aspects of acute extra
pyramidal syndromes in nonhuman primates.
Psychopharmacol Bull 1989;25:457-59. Materials and Methods
5. Casey DE. Serotonergic and dopaminergic aspects
Materials
of neuroleptic-induced extrapyramidal syndromes
in nonhuman primates. Psychopharmacology 1993; Animal/species : Mice/ albino Swiss
112:S55-S59. Sex/Body weight : Male/ 20-30 g
EXPERIMENT NO.: 18J wards
Aim Drug : Morphine (10-40mg/kg, s.c
or ip)
To demonstrate the Straub tail reaction/pheno-
menon induced by morphine Precautions before Experimentation
• Animals should be marked properly, to avoid
Background mixing in two groups
Simply straub tail means erected tail. The • Handle the animal with care (minimize the
phenomenon was used to check doping i.e. opioid stress and pain to animal)
level in the horses which took part in the race. A • Observe the animal in a plexiglass chamber
subcutaneous injection of morphine hydro- • Laboratory should be noise free (noise may
chloride (10-40 mg/kg, i.p./s.c) into the mouse delay the response)
flank produces a curvature of the tail in the form
of an ‘S’ , following 2 to 15 minutes after the Methods
injection, depending on the dose . Finally, the tail Step 1
curves back along the body of the animal, the tip
• Weigh the animals and mark properly to
touching the center of the head (Figs 18.8A to C).
distinghish from one another
Straub tail phenomenon occurs as a result of
injection of the centrally acting analgesics such
group)
as morphine. But, there are no such studies which
have supporting data for the peripheral analgesic Step II
induced straub tail. • Group 1: Control group (n = 6); mice are given
As straub tail reaction is thought to be due to the saline at the equvalent dose of drug
mechanical contraction of the dorsal sacro coccygeus • Group 2: Treatment group (n = 6); mice are
muscle and electrical stimulation of spinal cord. There given morphine at the dose of 40 mg/kg, s.c.
Figs 18.8A to D: (A) Positive straub tail phenomenon, (B) “S” shaped positive straub tail phenomenon, (C) Extreme
positive straub tail phenomenon, sometimes tail touches the head of mouse and (D) Showing dorsal sacro coccygeus
muscle on the either side of tail root (pointed in the picture) (For color version of Figures 18.8A to C see Plate 3)
Step 3 Note: Straub tail is said to be positive, when the

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

Observation Table
Sl. No. Straub Tail Reaction Straub tail
Group1 Group 2 reaction(Score)
Present Absent Present Absent Group 1 Group 2
1.
2.
3.
4.
5.
6.
Discussion α2-(alpha-2) receptors, dopamine receptors and

Straub tail reaction or phenomenon is the glucocorticoid receptor are well studied in several
characteristic contraction of the dorsal sacro- studies. The tail rise at an angle >30° is considered
coccygeus muscle and electrical stimulation of to be the positive straub tail reaction. There is no
spinal cord in which tail rises than its normal clinical condition resembling this condition hence
position. The role of the μ2–receptor, 5-HT receptors, have only experimental implication.
SUGGESTED READING rat are placed on the hot plate and observed for
either paw licking or jumping reaction. The
1. Anna Capasso, Carmela Casciano and Alberto
reaction time is recorded by a stop-watch.
Loizzo. Dexamethasone reduces morphine induced
straub reaction in mouse. J Pharmacy Pharmacol Repeated reading is taken at 20, 60, and 90
2002;54:983-87. minutes after the drug administration. Cut off time
2. Bilbey D LJ, Salem H, Grossman MH. The anatomical for rat is 20-30 sec and for mice it is 15-20 sec.
basis of the straub phenomenon. Br J Pharmacol
1960;15:540-43. Tail Flick Method
3. Kameyama T, Ukai M, Nabeshima T. Effects of
catecholaminergic or tryptaminergie agents on the Animal is placed into restrainer and leaving the
morphine-induced Straub tail reaction. Jpn J tail exposed outside the restrainer. Clean the tail
Pharmacol 1978:28;249-57. with the help of cotton soaked in water or ethanol.
4. Schwe Fang Pong, Janet Mary Sweetman, Amy Sue Then, leave the tail for drying and also to settle
Pong, John Franklin Carpenter. Evaluation of oral
down the rat/mouse in the restrainer. When rat/
skeletal muscle relaxants in the morphine-induced
straub tail test in mouse. Drug Development mouse setted, then keep restrainer on the “tail flick
Research 2004;11(1):53-57. analgesiometer”. 1/3rd tail proximally left due to
5. Tsutomu Kameyama, Makoto Ukai and Toshitaka the thick and keratinized skin and then keep tail
Nabeshima. Effect of catecholaminergic or trypta- on the place made for tail above hot wire (measure
minergic agents on the morphine induced straub tail the height of tail from wire) of the analgesiometer.
reaction. Japan J Pharmacol 1978;28:249-57. The time of tail flick is measured and recorded. The
6. Zarrindast MR, Alaei-Nia K, Shafizadeh M. On the
cut off time is set up 15-20 sec in case of mouse
mechanism of tolerance to morphine-induced Straub
tail reaction in mouse. Pharmacol Biochem Behav whereas in the case of rat, cut off time is 20-30 sec
2001;69(3-4):419-24. to avoid any further injury to the tail (Fig. 18.9).
EXPERIMENT NO: 18K Materials and Methods
Aim Materials
To demonstrate the analgesic effect of morphine Animal/species : Mice/ albino Swiss

in mouse using hot plate/tail flick method Sex/Body weight : Either sex/ 20-30 g
Background wards
Drug : Morphine (5-7.5 mg/kg, i.p
The method deals on the principle of the thermal or s.c.)
radiation heat. This principle is used in the animal
experiment for the evaluation of the centrally
acting analgesics, and hence this method found
to be the differentiating between the centrally
acting opiates and non-opiates analgesics.
Hotplate Method
The instrument involved is known as “hotplate
analgesiometer”. Instrument consists of an
electrically heated surface (made up of iron,
aluminum or copper) whose temperature is
maintained by the thermostat ‘Knob’ at 55° to
56 °C. After maintaining the temperature mouse/ Fig. 18.9: Tail-flick test in rat
Other drugs : Codeine hydrochloride (30 • Observe for the licking of the paw or jumping
mg/kg s.c.), Pethidine hydro- in case of hot plate or the tail flick in tail flick
chloride (30 mg/kg s.c.) and test and record the time
Phenazone (400 mg/kg s.c.)
Note:
Precautions before Experimentation 1. Record the response at 20, 60 and 90 min after
the saline/drug treatment (Hot Plate test)
• Animals should be marked properly, to avoid 2. Record the response at 30, 60 and 120 min
mixing in two groups after the saline/drug treatment (Tail flick
• Handle the animals with care (minimize the test)
• Check the instrument carefully for any error Observations and Results
and electricity connection
• Carefully check the temperature within the Centrally acting analgesics can be evaluated by
range (chances of burning of paw or tail) [hot the hot plate method where as peripherally acting
plate] analgesics are not effective. e.g: Aspirin showed
• Clean the paw and hot plate for uniform no effect in hot plate method even at very high
temperature distribution doses
• In tail flick method, clean the tail properly, to False positive results: sedatives and muscle
avoid interference with result relaxants (Woolfe and MacDonald, 1944) or
• The time of wire getting red must be substracted psychotomimetics (Knoll, 1967) may give the
from the total time recorded [tail flick] increase reaction time.
• Screening is must for the both tests before the
experiment, if mouse show reaction time more Discussion
than 6 sec, should be excluded from the study
Hot-plate and tail-flick test mimic acute thermal
(selection reaction time for rat is 10 sec).
pain and persistent pain model by the formalin
Methods test. Hot-plate and tail-flick test are two different
methods for evaluation of nociception. These
Step 1 experimental models of pain commonly used to
• Weigh the animals and mark properly tests for response thresholds to high intensity
• Divide animals into two groups (n = 6 in each stimuli (acute pain tests) or persistent pain
group)
models. Tail-flick test is predominantly a spinal
Step II response and Hot-plate is mostly at supraspinal
• Group 1: Control group (n = 6); mice are given level. Studies have conducted to evaluate
the saline at the equvalent dose of drug participation of nitric oxide in the agmatine-
• Group 2: Treatment group (n = 6); mice are mediated potentiation of morphine-induced
given morphine at the dose of 5 mg/kg, s.c. analgesia in mice indicate that agmatine
Step 3 potentiates morphine-induced spinal but not
• Hot plate/tail flick: Observe the animal for 15- supraspinal analgesia, and this effect is
20 seconds (cut off time for mice) 20-30 seconds not mediated by a nitric oxide-dependent
(cut off time for Rat) mechanism.
Observation Table
For hot plate
Sl. Time of paw licking or jumping
No. 20 min 60 min 90 min
1.
2.
3.
4.
5.
6.
For tail flick

Sl. Time of tail flick
No. 30 min 60 min 120 min
1.
2.
3.
4.
5.
6.
SUGGESTED READING in Drug Evaluation. Yearbook Med Publ. Inc., Chicago

1967;305-21.
1. Aanonsen LM, Wilcox GL. Nociceptive action of 6. Luszczki JJ, Czuczwar SJ. Dose-response relationship
excitatory amino acids in the mouse: effects of analysis of vigabatrin doses and their antinociceptive
spinally administered opioids, phencyclidine and effects in the hot-plate test in mouse. Pharmacol Rep
sigma agonists. J Pharmacol Exp Ther 1987;243(1):9- 2008;60(3):409-14.
19. 7. Ung D, Cowan A, Parkman HP, Nagar S. Lack of
2. Abbott FV, Melzack R, Leber BF. Morphine analgesia interaction between metoclopramide and morphine
and tolerance in the tail-flick and formalin tests: dose- in vitro and in mouse. Xenobiotica 2008;38(11):1365-
response relationships. Pharmacol Biochem Behav. 76.
1982;17(6):1213-19. 8. Woolfe G, MacDonald AD. The evaluation of the
3. Abbott FV, Melzack R, Samuel C. Morphine analgesia analgesic action of pethidine hydrochloride (DE-
in tail-flick and formalin pain tests is mediated by MEROL) J Pharmacol Exper Ther 1944;80:300-07.
different neural systems. Exp Neurol 1982;75(3): 9. Zimer PO, Wynn RL, Ford RD, Rudo FG. Effect of
644-51. hot plate temperature on the antinociceptive activity
4. Kambur O, Männistö PT, Viljakka K, Reenilä I, of mixed opioid agonist antagonist compounds.
Lemberg K, Kontinen VK, Karayiorgou M, Gogos Drug Dev Res 1986;7:277-80.
JA, Kalso E. Stress-induced analgesia and morphine
responses are changed in catechol-O-methyl- EXPERIMENT NO.: 18L
transferase-deficient male mouse. Basic Clin
Pharmacol Toxicol 2008;103(4):367-73. Aim
5. Knoll J. Screening and grouping of psycho-
pharmacological agents. In: Siegler PE,Moyer HJ To demonstrate partial global cerebral ischemia
(Eds) Animal and Clinical Pharmacologic Techniques in mice
Background Step 2
Cerebral ischemia leads to a cascade of patho-
physiological processes which contribute to
• Group 2: Treatment group (n = 6); mice are
ischemic cell damage, reduction in oxygen and
given dizocilpine (MK-801) at the dose of 0.1
glucose availability to brain leading to cellular
mg/kg, i.p. 1st dose is given 30 min before the
energy crisis which interrupts the activity of
surgery thereafter, once every day at 24 hr, 48
cellular ion pumps thus disturbing the ionic
hr and 72 hr
gradients homeostasis resulting ultimately to an
increased release of neurotransmitters (mainly Step 3
glutamate) within 1-2 minutes of ischemia. • Brain is removed after 72 hr of cerebral
Glutamate release causes excitation and results ischemia and then following observations are
in early onset seizure. The contribution of over- carried out
stimulation by excitatory amino acids leading to • The infarct area
neurotoxicity and cell death following cerebral • Brain edema
hypoxemia induced by ischemia is well estab-
lished. Activation of NMDA-glutamate receptors Measurement of Brain Edema
results in an increase in free Ca2+ ion which
Brain edema is measured with the wet-dry
triggers a number of potential cytotoxic cascades
method. After the animal is sacrificed by decapi-
including activation of protein kinase-C (PKC),
tation under euthanasia, their brain is removed,
release of platelet activating factor (PAF),
weighed immediately to yield wet weight. After
generation of free radicals and production of
drying in a desiccating oven for 48 h at 70oC, the
nitric oxide.
tissue is reweighed to yield dry weight. The
percentage of water in the tissues is calculated
according to the formula:
Materials
Wet weight – dry weight
Animal/species : Mice/albino Swiss 100
Wet weight
Syringe/needle : Aneurysm clip/bull dog clip
Measurement of Cerebral Infarct Size
Precautions before Experimentation After 72 hr, the animal is sacrificed by decapi-
tation, their brain is removed and infarct size is
• The area of surgery is thoroughly cleaned with
calculated. The brain is kept overnight at (–4°C).
70% ethanol with the sterile cotton swab
Frozen brain is sliced into uniform sections (7-8
• Use sterilized instruments during the surgery
in number per brain) of 1 mm thickness. The slices
• While performing the surgical procedure, the
are immersed in 1% triphenyltetrazolinium
animals are kept warm with the help of Infra-
chloride (TTC) at 37°C in 0.25 M phosphate buffer
red lamp
(pH 8.5) for 5min; tissue sections are dipped in
10% formaldehyde solution for 5 min. Triphenyl-
Method of Cerebral Ischemia Induction
tetrazolinium chloride (TTC) is converted to red
Method formazone pigment and therefore stained the
viable cells deep red. The infarcted cell have lost
Refer Flow Chart 18.1.
the enzyme and cofactor and thus remained
Step 1 unstained dull yellow. The brain slices are
• After ischemia induction, divide animals into placed in between two glass slides. A transparent
two groups (n = 6 in each group) plastic grid with 100 squares per cm2 is placed
Flow Chart 18.1: Procedure for producing cerebral ischemia
over slides. Number of squares falling over non- Observations and Results
stained dull yellow area and total number Sl. no. Cerebral infarct size Brain edema
of squares covered by each brain slice is counted.
Control Treatment Control Treatment
Infarcted area is expressed as a percentage
of total brain volume. For calculation of
infarcted area of a brain, all the brain sections
are studied with the help of magnifying glass
(40 X).
Result is expressed in mean ± SD and is on the duration of ischemia. Therefore, the severity
compared by unpaired t-test (if treatment groups of ischemia has two components: degree of CBF
are more than one and total mean is >2 then one reduction and duration of the ischemic episode.
way ANOVA should be preferred)
SUGGESTED READING
Discussion
There are several other models are used to study 1. Aggarwal R, Medhi B, Pathak A, Dhawan V,
cerebral ischemia or stroke in the published Chakrabarti A. Neuroprotective effect of progesterone
on acute phase changes induced by partial global
literature such as middle cerebral artery occlusion
cerebral ischaemia in mice. J Pharm Pharmacol 2008;
model (MCAO) or unilateral carotid artery ligation 60(6):731-37.
(UCAL). Four vessel occlusion models are also 2. Bochelen, D., Rudin, M. and Saute, A. Calcineurin
used in the number of studies for the producing inhibitors FK506 and SDZASM 981 alleviate the
global cerebral ischemia. The development of outcome of focal cerebral ischemic/reperfusion
ischemic brain damage depends on the reduction injury. J Pharmacol Exp Ther 1999;288:653-59.
of cerebral blood flow (CBF) below critical 3. Dempsey RJ, Baskaya, MK and Doglan, A. Attenu-
ation of brain edema, blood-brain barrier breakdown,
threshold level. So, the decrease of CBF in ischemic
and injury volume by ifenprodil, a polyamine-site
regions may result in an energy failure and further NMDA receptor antagonist, after experimental
lead to an activation of the toxic intracellular traumatic brain injury in rats. Neurosurgery
pathway, additionally the infarct volume depend 2000;47(2):399-406.
19 Cardiovascular System (CVS)
EXPERIMENT NO.: 19A pressure measurement and overall cardiovascular

studies but major limitation lies with cost of the
Aim telemetry apparatus and radio transmitter
To record blood pressure (BP) in rodents (Rat BP). implantation, whereas several advantages over
other methods are accurately obtain blood
Background pressure measurements at the lowest reco-
mmended temperatures and highly sensitive
BP measurement in the animal is same as the human
photoelectric sensor for detection of blood pressure
measurement allowing readings when pulse/flow
pulses. Its software records real time systolic,
disappears during cuff inflation and reappears
diastolic, mean and heart rate and allows complete
during deflation. Mainly, there are two methods of
control of system.
BP measurement which are employed, one is
invasive or direct measurement and other is non-
invasive or indirect method. Direct or invasive
method follows the BP measurement through the
carotid artery. Major limitation, it is tedious work to Materials
maintain the respiratory and surgical procedure.
Animal/species : Rat/Wistar
(Must maintain the aseptic condition) Non- invasive
method includes tail-cuff method which is a
Syringe/needle : 1ml/ preferably 23 G
sensitive and accurate approach for the noninvasive
Drugs : Sodium pentobarbital (50
measurement of blood pressure in conscious or un-
mg/kg i.p.)
conscious rat/mouse. It is simple and inexpensive
but can be misinterpreted due to stress-induced Laboratory conditions: Temperature: 23°C±2°C
changes in blood pressure during animal restraint under a 12:12 hr light: dark cycle (The animals
and heating. Systolic blood pressure (SBP) is still are acclimatized to the laboratory conditions for
preferably measured in rats by the tail-cuff method at least 1 week prior to experimentation.
(Figs 19.1A to D). Several methods of flow/pulse
detection have been described in addition to the Precautions before Experimentation
parent water plethysmograph or its modifications • Handle the animal with care (minimize the
mercury in-rubber or strain gauge detectors, stress and pain to animal)
microphonic and piezoelectric pulse detectors, • Proper training is given to animals to fami-
doppler effect photoelectric and impedance. liarize with the procedure
Now days, telemetry is preferred method • All procedure should be performed by the
employed for conscious rodents/animals blood same person
Figs 19.1A to D: (A) Restrained rat showing attached BP cuff, (B) Close view tail-cuff attachment and
(C) and (D) Showing complete set of BP measurement with cardiogram (For color version of Figure 19.1C, see Plate 3)
Methods
Non-invasive method (Tail cuff method)
Animal Experiment on CVS  209
Invasive method
Step I: For maintaining the ventilation
Note: Pentobarbitone has long duration of action which makes it, anesthesia of choice in the experiment
Step II: Procedure for BP measurement

1. Carotid artery
2. Femoral artery
Procedure for BP measurement through carotid artery
BP measurement through Femoral artery
Recording recorder. Various cardiovascular drugs are added

Drugs are injected through the rubber tubing close to note the effect (inotropic/chronotropic etc.) of
to the cannula and a constant volume of saline is these drugs on the heart. After the experiment,
allowed to run each time after injection. The animals are sacrificed under euthanasia and then
recordings are taken on a smoked drum or electronic animal is disposed in a yellow polythene bags for
incineration. In vivo responses to the primary of Laboratory and Clinical Medicine 1971;78:
action of the drug are the main advantage of this 675-82.
preparation unlike the langendorf’s preparation 2. Buñag RD, McCubbin JW, and Page IH. Lack of
correlation between direct and indirect measurements
and hence, the net responses are measured.
of arterial pressure in unanesthetized rats. Cardio-
vascular Research 1971;5:24-31.
Observations and Results 3. Byrom FB and Wilson C. A plethysmographic method
for measuring systolic blood pressure in the intact
Sl. Weight of animal BP HR rat. Journal of Physiology 1938;93:301-04.
No. 4. Fritz M, Rinaldi G. Blood pressure measurement with
1. the tail-cuff method in Wistar and spon-taneously
2. hypertensive rats: Influence of adrenergic- and nitric
3. oxide-mediated vasomotion. Journal of Pharma-
cological and Toxicological Methods 2008; 58:215-
21.
Discussion 5. Hermansen K. A new method for determination of
the systolic blood pressure in conscious rats. Life
There are several methods described for
Sciences 1970;9:1233-37.
measuring BP in rodents. Among those, tail-cuff 6. Lucas J. A modified indirect method of blood pressure
method is very commonly used for measuring measurement in the conscious and anaesthetized rat.
systolic blood pressure (SBP) in rodents. Direct Journal of Physiology 1971;218:1-3.
methods have used tethers and indwelling 7. Maistrello I and Matscher R. Measurement of systolic
catheters connected to blood pressure measure- blood pressure of rats: Comparison of intraarterial
and cuff values. Journal of Applied Physiology 1969;
ment devices, but limitations being are stressful
26:188-93.
and give variable results. The inflation– 8. Van Vliet VN, Chafe LL, Antic V, Schnyder-Candrian
deflation cycle usually involved in the tail-cuff S and Montani JP. Direct and indirect methods used
methods and inflation is characterized by to study arterial blood pressure. Journal of
disappearance of the pulse/flow signal during Pharmacological and Toxicological Methods
cuff and reappearance during deflation. 2000;44:361-73.
9. Williams JR, Harrison TR and Wollmann AA. simple
Clinically, it is used for the diagnosis of pheo- method for determining the systolic blood pressure
chromocytoma. of the unanesthetised rat. Journal of Clinical
Telemetry is a modern technique which Investigation 1939;18:373.
includes an implantable telemetric probe, receiver
and monitoring system and gives very similar EXPERIMENT NO: 19B
results to the tail-cuff method. Telemetry allows
for a longer period of accurate measurement and Aim
gives greater power to the study. So, telemetry is a To record ECG in rodents (rat and mouse).
reliable method of direct measurement and has
been introduced to measure blood pressure (BP) Background
in the conscious unstressed animals. This system
Recording electrocardiogram (ECG) in rodents (rat
simultaneously measures SBP, diastolic blood
and mouse) is commonly used parameter for
pressure (DBP), mean arterial pressure (MAP) and
various pharmacological and toxicological
heart rate (HR).
studies. It provides valuable information about
function and structure of heart. Recording of ECG
SUGGESTED READING
is similar to human ECG but some differences are
1. Buñag R D. Pressor effects of the tail-cuff method in in smaller animals, such as the heart rate is very
awake normotensive and hypertensive rats. Journal high and ST segment is generally absent. Some of
the important aspects of rat electrogram are measure ECG in mouse. It has several advantages
position of the animals, some of the investigators such as it is without anesthesia, surgery and
prefer supine position and others recommend implant.
prone as suitable posture. Mean electrical axis
(MEA) is varied from –22 to +120 in rat. Rat model Materials and Methods
has become relatively invaluable for studying
mechanism of human disease whereas, mouse is Materials
increasingly used in the recent times particularly Animal Species : Swiss mice/Wistar Rat
in drug development and to determine effect of Sex/Body weight : Either sex/20-30 g mice or
gene defect, disease and therapies. Though 150-200 g rat
conventional methods have several disadvan-
tages like requirement of anesthesia, insertion of
pin electrode in the limb, etc. So, new advance
technology has been used in the research like • Handle the animals with care (minimize the
telemetry. It was introduced in 1988, and latest stress and pain to animal)
noninvasive foot plate technology is available to • Keep laboratory calm while recording the ECG
Methods
Observations and Results beings because of difference in body shape

between rat and human chest and large ST
Attach the findings of the study (Electro-
segment is probably because of early ventricular
cardiogram).
repolarization. Selection of anesthetic agents, and
positioning of the animals are important while
Discussion
recording ECG. It has been reported that ECG has
In rat, ECG usually shows presence of more apparent absence of T wave in some of the smaller
prominent QRS complexes as compared to human animals and there is lack of isopotential segment
S and T wave (except in guinea pig) and comple- 2-isopropyl amino ethanol), but in massive dose
xity of QRS wave. it produces myocardial necrosis. Role of
mineralocorticoids (aldosterone) have also been
SUGGESTED READING observed. Studies also indicate increased urinary
excretion of aldosterone in experimentally
1. Xing S, Tsaih SW, Yuan R, Svenson KL, Jorgenson L,
So M, Paigen B, Korstanje R. Genetic influence on
induced myocardial infarction and spontaneous
electrocardiogram time intervals and heart rate in myocardial infarction in human beings. Experi-
aging mice. Am J Physiol Heart Circ Physiol 2009 Apr mentally isoproterenol (a synthetic catecholamine
24. and β adrenergic agonist) induced myocardial
infarction is well established animal model and
EXPERIMENT NO.: 19C resembles to those taking place in human MI.
Aim Materials and Methods

To demonstrate isoproterenol induced myocardial
infarction in rats Materials
Background Animal/species : Rat/ Wistar

Myocardial infarction (MI) is caused due to non- Syringe/needle : 1 ml/ preferably 23G
availability of oxygen to myocardium. Experi- Drug : Isoproterenol (8.5 mg/100
mental studies suggested that production of gm of rat)
catecholamine in cardiac tissue results in
increased oxygen requirement to myocardium by Precautions before Experimentation
increasing cardiac metabolism causing hypoxia. • Animals should be marked properly, to avoid
Experimentally myocardial infarction can be mixing in two groups
produced by injection of epinephrine and • Handle the animals with care (minimize the
isoproterenol (ISO), DIH (1-3-4 dihydroxyphenol- stress and pain to animal)
Methods
Histopathological Examination of Heart functional alterations of cardiac function. Eur J Heart

Fail 2009;11(2):140-46.
Histopathological examination is done and heart
is washed immediately, after removal so that no
EXPERIMENT NO.: 19D
blood should be remaining in the cavity and it is
preserved in 10% formalin. Aim
Histopathological grading is done according
to following criteria. To demonstrate deoxycorticosterone acetate
Grade 0 No change (DOCA) salt induced hypertension in rats
Grade 1 Focal interstitial response
Background
Grade 2 Focal lesions in many sections, con-
sisting of mottled staining and frag- DOCA-salt model is based on the principle of its
mentation of muscle fibers and sequis- sodium and water retention properties in the body,
ting mucoid edema. so it leads to increase in plasma and extracellular
Grade 3 Confluent retrogressive lesion with volume. This model was first developed by the
hyaline necrosis and fragmentation of Seyle et al, 1957. DOCA ultimately leads to
muscle fibers and sequisting mucoid increase the sympathetic activity through the
edema renin-angiotensin-aldosterone system (RAAS)
Grade 4 Massive infarct with occasionally system. Experimental studies suggest that the
acute aneurysm and mural thrombi female and young animals are more prone to the
DOCA induced hypertension.
Sl. Weight ECG HR Histological
No. score Materials
1. Animal/species : Rat/ Wistar
2. Sex/Body weight : Either sex/ 100-150 g
3. Syringe/needle : 1ml/ preferably 23 G
Drug : DOCA (20mg/kg, s.c)
Discussion
Isoproterenol, synthetic sympathomimetics which Methods
produces experimentally induced acute Step 1
myocardial infarction at the high dose by myocyte • Weigh the animals and mark properly to
necrosis. It has predominant action on β-1 receptor distinguish from one another
which causes prolong systolic action, so diastolic • Divide animals into two groups (n = 6 in each
duration is reduced. Endocoronary filling occur group)
during diastole so, heart prone to develop MI.
Isoproterenol is light sensitive, hence solution Step 2
should be prepare in colored beaker or bottle • Group 1: Control group (n = 6); Rats are given
freshly, before use. saline at the equvalent dose of NaCl 1%
solution or propylene glycol 0.5 ml for four
weeks
SUGGESTED READING
• Group 2: Treatment group (n = 6); Rats are
1. Tiwari R, Mohan M, Kasture S, Maxia A, Ballero M. given DOCA 20 mg/kg, s.c. along with NaCl
Cardioprotective potential of myricetin in isopro- 1% sol. orally ad libitum for four weeks
terenol-induced myocardial infarction in wistar rats.
Phytother Res 2009;23 (10):1361-66.
Step 3
2. Krenek P, Kmecova J, Kucerova D, Bajuszova Z, • Observe the animals behavior at baseline,
Musil P, Gazova A, Ochodnicky P, Klimas J, every week and after completion of 4 weeks
Kyselovic J. Isoproterenol-induced heart failure in • Blood pressure, ECG is measured at the
the rat is associated with nitric oxide-dependent beginning (baseline), 1, 2, 3, and 4 weeks

Group 1
Sl. no. BP ECG
0wk 1wk 2wk 3wk 4wk 0wk 1wk 2wk 3wk 4wk
1.
2.
3.
4.
5.
6.
Group 2
Sl. no. BP ECG
0wk 1wk 2wk 3wk 4wk 0wk 1wk 2wk 3wk 4wk
1.
2.
3.
4.
5.
6.
Discussion endopeptidase inhibitor, retrothiophan, on renal

function and blood pressure in conscious
In the DOCA-salt induced hypertension, normotensive Wistar and hypertensive DOCA-salt
vasopressin plays an important role which is rats. J Cardiovasc Pharmacol 1992;20:847-57.
observed elevated in plasma level and urinary 2. Badyal DK, Lata H, Dadhich AP. Animal models of
excretion in several studies. The others are nor- hypertension and effect of drugs. Ind J Pharmacol
epinephrine and angiotensin II, which induce the 2003;35:349-62.
3. Majima M, Katori M, Hanazuka M, Mizogami S,
DOCA –salt hypertension. These rats develop
Nakano T, Nakao Y,Mikami R, Uryu H, Okamura
hypertrophy, excessive collagen deposition,
R,Mohsin SSJ, Oh-Ishi S. Suppression of rat
perivascular and interstitial fibrosis, endothelial desoxycorticosterone-salt hypertension by the
dysfunction, and prolongation of the cardiac kallikrein-kinin system. Hypertension 1991;17:
action potential with hypertension within 4 806-13.
weeks. 4. Schenk J, McNeill JH. The pathogenesis of DOCA-
salt hypertension. J Pharm Toxicol Meth 1992;27:
SUGGESTED READING 161-70.
5. Seyle H, Bois P. the hormonal production of
1. Pham I, el Amrani AI, Fournie-Zaluski MC, Corvol nephrosclerosis and periarteritis nodosa in the
P, Roques B, Michel JB. Effects of the selective primates. Br Med J 1957;1:183-86.
EXPERIMENT NO: 19E Drug : FeCl3(25%) *

Aim *For mice 10% FeCl3 is used.
To demonstrate Ferric Chloride (FeCl3) -induced Methods

thrombosis in rat model. Step 1
• Weigh the animals and anesthetise with
Background sodium pentobarbital (50 mg/kg, i.p.)
FeCl3 is commonly used as an oxidant and causes • Give the midline incision between mandible
endothelial injury, platelet aggregation, and a of the neck and upper end of manubrium
sterni
rapid onset of thrombus formation when it is
• Sternocleidomastoid muscles are gently
directly applied to the blood vessel. The trans-
retracted laterally and locate trachea
migration pathway of the ferric ion plays the main
Step 2
role which shows that some ferric oxide aggregates • Identify the right or left carotid artery and
formed near the developing thrombus in the separate it from vagus and attached tissue
vascular lumen whereas endothelial and smooth • Apply a weighed filter paper (2 × 5 mm) and
muscle injuries are observed in segments of the saturated with 25% FeCl3 solution to the artery
vessel in many studies which came in direct • Paper is allowed to remain at the vessel for 10
contact with the FeCl3. min and then removed and weighed
Step 3
Materials and Methods • The experiment is continued for 60 min after
the induction of thrombosis. Then, thrombus
Materials is removed and weighed
Animal/species : Rat/ Wistar • Blood pressure, blood flow and ECG is
measured at the beginning (baseline), 1, 2, 3
Sex/Body weight : Either sex/ 150-200 g and 4 week (To observe progression of
Syringe/needle : 1 ml/ preferably 23 G thrombosis).
Observation
Sl. no. BP Blood flow ECG
0wk 1wk 2wk 3wk 4wk 0wk 1wk 2wk 3wk 4wk 0wk 1wk 2wk 3wk 4wk
1.
2.
3.
4.
5.
6.
SUGGESTED READING
2. Michael T. Tseng, Alan Dozier, Bodduluri Haribabu
1. Kurz KD, Main BW, Sandusky GE. Rat model of and Uschi M. Graham. Transendothelial migration
arterial thrombosis induced by ferric chloride. Thromb of ferric ion in FeCl3 injured murine common carotid
Res 1990;60:269-80. arteryThrombosis Research 2006;118 (2):275-80.
20 Gastrointestinal Tract (GIT)
EXPERIMENT NO.: 20A Stomach is opened along the greater curvature

and stretched properly and pinned on a wax
Aim plate. The number of ulcers is noted and the
To demonstrate gastric ulcer induction/formation severity recorded with the following scores:
by different methods 0 = no ulcer; 1 = superficial ulcers; 2 = deep ulcers;
3 = perforation
Background
Pylorus ligation method (SHAY method): Animal Materials and Methods
is anesthetized and a midline abdominal incision Materials
is made. The stomach is identified and the pylorus
is ligated operatively. Then, leave the animal in Animal/species :Rat/ Wistar
the individual cages for 19-24 hr. The test Sex/Body weight :Either sex/ 150-250 g
compounds are given either orally (gavage) or Syringe/needle :1 ml/ preferably 23 G
injected subcutaneously 30 min before ligation. Drug :Indomethacin (20 mg/kg,
At specified time, animal is sacrificed under po)
euthanasia. The abdomen is opened and a first Other agent for ulcer : Aspirin (500 mg/kg, po)
ligature is placed around the esophagus close to
the diaphragm to stop the contents in the stomach. Precautions before Experimentation
Then, the stomach is removed, and the contents • Animals should be marked properly, to avoid
are drained in a centrifuge tube. (Volume of the mixing in two groups
gastric content is measured and then, after, • Handle the animals with care (minimize the
centrifugation, acidity is determined by titration stress and pain to animal)
with 0.1N NaOH). • Animals should be fasted for at least 24 hour
Indomethacin induced ulcer: The test drugs are in metabolic cage and water is given ad libitum
administered orally dissolved in 0.1% Tween 80 • Should be housed separately to avoid canni-
solution 10 min prior to oral administration of balism and coprophagy
indomethacin in a dose of 20 mg/kg, dissolved in • While ligation, care must be taken, so that
0.1% Tween 80 solution. Six hours later, the rats neither damage to the blood supply nor stress
are sacrificed by CO2 anesthesia and stomach is to the pylorus occurs.
removed.
Methods
Ethanol induced ulcer: The animals are adminis-
tered 1 ml absolute ethanol orally. Untreated or Step 1
vehicle treated animals are included as control. • Weigh the animals and mark properly
One hour after administration of ethanol, the • Divide animals into two groups (n = 6 in each
animals are euthanized with CO2. group)
Step 2
• Group 1: Control group (n = 6); rat are given
• Group 2: Treatment group (n = 6); rat pylorus
is ligated for 19-24 hr or indomethacin 20 mg/
kg, po or aspirin 500 mg/kg, po
Step 3
• Sacrifice the animal after 19 hrs in case of
pylorus ligation or 6 hrs after indomethacin
or 1 hr after ethanol administration
• Observe for score
Note: Group: 3, may be added in the experimental Fig. 20.1: Arrows indicate gastric ulcer in rat stomach
group, if one wants to see the protective effect of (For color version see Plate 3)
the PPI’s such as omeprazole, rabeprazole etc. or
H-2 blockers such as Ranitidine, cimetidine, etc.
Test drug is administered 30-60 min prior to analgesics induced gastric ulcer, for example
induction of gastric ulcer. indomethacin, aspirin, alcohol induced, etc. or
Ulcer is observed by the following score: pylorus ligation method (SHAY method). All these
0 = no ulcer; 1 = superficial ulcers; 2 = deep ulcers; methods are based on the mechanism of the
3 = perforation. excessive retention or production of gastric acid
(HCl) prostaglandins, histamine, etc. which leads
Observation and Results to oxygen reactive radicals, and other infla-
An ulcer index (UI) is calculated: mmatory mediators, locally at the stomach; can
UI = AU + ASS+PU X 10–1 induce serve ulcer. Severity of ulcer may worsen
AU = average number of ulcers per animal due to the presence ulcer of H. Pylori infection
ASS = average of severity score which is difficult to treat.
PU = percentage of animals with ulcers
SUGGESTED READING
Observation Table
Sl. no. Gastric ulcer 1. Asad M, Shewade DG, Koumaravelou K, Abraham
Group 1 Group 2 BK, Vasu S, Ramaswamy S. Effect of centrally
administered oxytocin on gastric and duodenal ulcers
Score UI Score UI
in rats. Acta Pharmacol Sin 2001;22(6):488-92.
1. 2. Djahanguiri B. The production of acute gastric
2. ulceration by indomethacin in the rat. Scand J
Gastroenterol 1969;4:265-67.
3.
3. Fornai M, Natale G, Colucci R, Tuccori M, Carazzina
4. G, Antonioli L, Baldi S, Lubrano V, Abramo A,
5. Blandizzi C, Del Tacca M. Mechanisms of protection
6. by pantoprazole against NSAID-induced gastric
mucosal damage. Naunyn Schmiedebergs Arch
Discussion Pharmacol 2005;372(1):79-87.
4. Ishihara Masashi, ITO Mikio. Influence of aging on
There are several ideal models to induce the gastric gastric ulcer healing activities of cimetidine and
ulcer which resemble gastric ulcer in humans like omeprazole. Eur J Pharmacol 2002;444:209-15.
Animal Experiment on GIT  219
EXPERIMENT NO: 20B Methods

Step 1
Aim • Weigh the animals and mark properly
To demonstrate cerulein induced acute pan- • Divide animals into two groups (n = 6 in each
creatitis in rat. group)
Step 2
Background • Group 1: Control group (n = 6); Rats are given
Inflammation of the pancreas is known as saline at the equivalent dose of drug
pancreatitis and it is proposed that the enzymes • Group 2: Treatment group (n = 6); Rats are
attack and damage their own tissues which given cerulein at the dose of 40 µg/kg, ip
produce them. Mainly pancreatitis is of two types Step 3
acute and chronic. Acute pancreatitis is infla- • Sacrifice the animals after 2 hr of cerulein
mmation of the pancreas that occurs suddenly administration and remove the pancreas
and usually resolves in a few days with treatment • Observe the following parameters: (1) pan-
creatic edema, (2) Pancreatic morphology
where as chronic pancreatitis is inflammation of
the pancreas that does not heal or improve and it Assessment of Intra-pancreatic Edema
gets worse over time leading to permanent damage. After sacrificing the animal, the pancreas is
It is easy to develop experimental model of the removed and weighed. Then, keep pancreas for
pancreatitis which is developed by the many the desiccation at 95° C for 24 hours.
means but the important are cerulein and L- The difference between the wet and dry tissue
arginine for the practical purpose. weights is calculated and expressed as percentage
of the tissue wet weight.
Materials and Methods (WT – DT)/WT × 100
Materials Where,
WT = weight of wet tissue
Animal/species : Rat/ Wistar DT = weight of dry tissue
Sex/Body weight : Either sex/ 150-250 g Morphological characters is checked for any
Syringe/needle : 1ml/ preferably 23G swelling or abnormal condition.
Drug : Cerulein (40µg/kg, i.p)
Other agent for Observations and Results
pancreatitis : L-arginine monohydro- Observation Table
chloride (5.0g/kg, in 0.9%
Group 1
NaCl, i.p)
Sl. no. Intra-pancreatic edema
Precautions before Experimentation WT DT WT - DT % of the tissue
wet weight
1.
2.
3.
4.
• While locating pancreas one should be careful
because it is diffused and cover a large area in 5.
rats 6.
Group 2 induced by a single intracolonic administration of

Sl. no. Intra-pancreatic edema 20 mg TNBS (volume to be administered is 0.25 ml)
WT DT WT - DT % of the tissue
which is dissolved in 35% ethanol into the
wet weight descending colon (8-10 cm from rectum). Rat is
placed under light ether anesthesia and a rubber
1.
catheter lubricated with lignocaine jelly (outer
2. diameter = 2 mm) is inserted rectally into the colon
3. through anus such that tip is reached 8 cm inside
4. from anus, approximately at the splenic flexure.
5. TNBS is dissolved in 35% ethanol (v/v) and instilled
6. into the lumen of the colon through rubber catheter.
The total volume is expelled with additional air
Discussion and the catheter is removed. Rats are observed for
two weeks and after two weeks rats are sacrificed
In the normal rat, pancreas is extensively diffuse under anesthesia for demonstration of colitis.
and difficult to identify in the peritoneum cavity.
But, in pancreatitis it appears as swollen trans- Materials and Methods
parent structure beneath the stomach and Materials
identified near the duodenum.
Animal/species : Rat/ Wistar
SUGGESTED READING Sex/Body weight : Either sex/ 150-250 g
Syringe/needle : 1 ml/ preferably 23G
1. Frossard JL, Rubbia-Brandt L, Wallig MA, Benathan Drug : Tri Nitro Benzene Sulphonic
M, Ott T, Morel P, Hadengue A, Suter S, Willecke K,
acid (TNBS; 20 mg in 0.25 ml
Chanson M. Severe acute pancreatitis and reduced
acinar cell apoptosis in the exocrine pancreas of
intracolonic)
mouse deficient for the Cx32 gene. Gastroenterology Other agent for
2003;124(2):481-93. colitis : Acetic acid (for 3 days), Dini-
2. Schmidt J, Rattner DW, Lewandrowski K, Compton trochlorobenzene (DNCB),
CC, Mandavilli U, Knoefel WT, Warshaw AL. A dextran sulfate, carrageenan,
better model of acute pancreatitis for evaluating acetic acid, etc
therapy. Ann Surg 1992;215:44-56.
EXPERIMENT NO.: 20C
Aim mixing in two groups
To demonstrate Tri Nitro Benzene Sulphonic acid stress and pain to animal)
(TNBS) induced colitis in rat. • TNBS is irritant and carcinogenic, so handle
with care and wear the gloves while handling
Background • Animals should be fasted for at least 24-48 hr
Inflammatory bowel disease is an idiopathic, (colon should be cleaned of any fecal material).
chronic inflammatory condition, which affects the
Methods
gastrointestinal tract. The mechanism is through
cytokines like TNFα, IL-1 and IL-8 which are Step 1
secreted from macrophages. Due to the lack of • Weigh the animals and mark properly
curative agent, screening of an effective drug is • Divide animals into two groups (n = 6 in each
always in pipeline. Experimentally colitis is group).
Animal Experiment on GIT  221
Step 2 Discussion
Inflammatory bowel disease is a polygenic
35% ethanol at the equivalent dose of drug
• Group 2: Treatment group (n = 6); rats are given disorder that gives rise to multiple clinical
TNBS 20 mg in 0.25 ml dissolved into the 35% subgroups within ulcerative colitis and Crohn’s
ethanol disease. A series of cytokines, like TNF-α, IL-1 and
Step 3 IL-8 are thought to be involved in the process.
• Sacrifice the animals after 14 days after TNBS Among inflammatory cytokines, TNF-α has a
administration and remove 10 cm of descen- broad spectrum of biological effect which plays a
ding colon major role in inflammatory bowel disease. It can
• Cut it longitudinally and open and spread. activate resident macrophages and promote the
Then, observe the gross morphology of colon.
release of other pro-inflammatory mediators
Assessment of Colitis (Enteritis Gross including nitric oxide, prostacyclin and platelet
Morphology Score) activating factors. Among the several experimental
Gross inflammatory index (GII) will be visually models, none of them produced the particularly
assessed for inflammation according to the ulcerative colitis or Crohn’s disease like condi-
following scores: tions; hence it is generally expressed as experi-
0 No inflammatory sign in the whole of 10 cm of mental colitis model and TNBS is very widely used
intestine
to produce experimental colitis.
1 Slight inflammation, and redness, villi visible
less than 15x magnification
2 Intermediate inflammation, discontinuous SUGGESTED READING
hyperemia intermediate redness of villi
3 Intensive inflammation, hyperemia, intensive 1. Levine A, Kenet G, Bruk R, Avni Y, Avinoach I,Aeed
redness of villi H, et al. Effect of heparin on tissue binding activity
Histological examination (HP score) may be of fibroblast growth factor and heparin binding
done to confirm the finding according to the method epidermal growth factor in experimental colitis in
and score described in Levine A et al, 2002. rats. Pediatric Res 2002;51(5):635-40.
2. Medhi B, Prakash A, Avti PK, Saikia UN, Pandhi P,
Khanduja KL. Effect of Manuka honey and
Observation Table sulfasalazine in combination to promote antioxidant
Sl. Colitis Score defense system in experimentally induced ulcerative
no. Group 1 Group 2 colitis model in rats. Indian J Exp Biol 2008;46(8):
583-90.
GII score HP score GII score HP score
3. Prakash A, Medhi B, Avti PK, Saikia UN, Pandhi P,
1. Khanduja KL. Effect of different doses of Manuka
2. honey in experimentally induced inflammatory
3. bowel disease in rats. Phytother Res 2008;22(11):
4. 1511-19.
5. 4. Vogel H Gerhard, Goethe J Wolfgang. Experimental
6. Colitis. In: Drug Discovery and Evaluation Pharma-
GII score: Gross inflammatory index (Score) and HP cological Assay, 2nd edition, Germany, Springer,
score: Histopathological score 2002;896-99.
21 Respiratory System
EXPERIMENT NO.: 21A body plethysmograph is used for measurement of

lung volume which is attached to monitor for the
Aim assessment of parameters (Fig. 21.1).
To measure respiratory volume in guinea pig
using body plethysmograph. Materials and Methods
Materials
Background
Animal/sex : Guinea pig/Male
Body plethysmograph measures the volume of air Weight : 250-650 gm,
present in the lung (while inhale and exhale of
The following baseline investigations are carried
the air), airway resistance, functional residual
out in all the animals:
capacity (FRC), etc. This test is based on the flow
1. Measurement of specific airways conductance
and pressure measurements. Additionally,
in a body plethysmograph.
specific airways conductance and airway/ 2. Measurement of airway response to inhaled
bronchial reactivity to allergen like histamine histamine to quantify baseline bronchial
(inhaled) is carried out using the body plethys- reactivity.
mographic technique which is well described in
Agrawal et al, 1977. An indigenously fabricated Methods
Step 1: Weigh the animal; generally take the
healthy adult guinea pig
Step 2: Group 1*: Control group without any
intervention. 3 animals included in this group
Step 3: Lung volume is assessed
*This experiment just deals with the common way to
assess the lung volume. Other groups may be added
Group 2 may be included in the study if one wants to see
the effect of an allergen. Animals first sensitized to
ovalbumin followed by an inhalation challenge with histamine
(in vivo generation of reactive oxygen species associated
with airway inflammation)
Group 3 may be included if one want to see the effect of
Fig. 21.1: Body plethysmograph; for measuring lung volume therapeutic agents like anti-asthmatics (salbutamol
(for details of body plethysmograph, please refer to “Commonly inhalation), etc. on the allergens induced hypersensitivity
used instrument in pharmacology laboratory” section) reaction.
Animal Experiment on Respiratory System  223
Observations and Result Methods

Write the findings of measurement.
Discussion
Lung volume is measured by the computer monitor
which is attached to the body plethysmograph
directly. Other assessment like bronchial reactivity
and histopathological score may be done for the
histamine treated or intervention after the allergen
induced hypersensitivity reaction.
SUGGESTED READING
1. Agrawal KP. Assessment of airway reactivity in
guinea pigs using a non-invasive body plethys-
mographic technique. Indian J Chest Dis Allied Sci
1977;19(1):3-7.
EXPERIMENT NO.: 21B

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

• Clean the paw with wet cotton
• Mark the paw (the insertion part must be the
same every time).
Fig. 22.1: Plethysmograph; Rat paw is immersed into the Methods

tube’A’ and the reading is checked through the tube ‘B’ (some
set-up have digital display attached to it) Step 1: Weigh the animals and mark properly.
Anti-inflammatory  225
Step 2 Alternative method for measuring paw edema

• Group 1: Control group (n = 6); choose right or
Glass cylinder with an internal diameter of 2 cm and height
left paw of rat as a control which is given saline of 5 cm is attached to the center of a 10 cm diameter Petri
at the equvalent dose of drug dish. Mercury is filled up to 4 centimeters of the cylinder.
• Group 2: Treatment group (n = 6); other paw is Then, the assembly is put on a digital balance with a suitable
given treatment of indomethacin (8 mg/kg, sensitivity.
p.o.), then after 30 min, carrageenan (0.05 ml Volume of paw edema= (W1 – W2)/ G
of 1% solution, s.c. is injected into the plantar Where, W1 = weight of the assembly with the paw
region. immersed
W2 = weight of the assembly without paw
Step 3
immersed
• Observe the animals at 0, 3, 6 and 24 hrs after G = gravity of fluid in the glass cylinder
the drug administration (mercury gravity=13.6)
• Observe the animals behavior carefully and
Note: (1) Take at least two readings, (2) Mark the paw at
calculate the percentage protection after the specific point to immerse every time at the same level
treatment.
Observation Table
Group 1
Sl. Paw edema (0hr) Paw edema (3hr) Paw edema (6hr) Paw edema (24hr)
No. Right paw Left paw Right paw Left paw Right paw Left paw Right paw Left paw
1.
2.
3.
4.
5.
6.
SUGGESTED READING 4. Winter CA, Risley EA, Nuss GW (1963). Anti-

inflammatory and antipyretic activities of
1. Fereidonia M, Ahmadiania A, Semnanianb S, Javan indomethacin, (1-(pchlorobenzoyl)-5-methoxy-2-
M. An accurate and simple method for measurement methyl-indole-3-acetic acid. J Pharmacol Exp Ther
of paw edema. Journal of Pharmacological and 1963;141:369-76.
Toxicological Methods 2000;43:11-14.
2. Ferreira T, Camargo EA, Ribela MT, Damico DC, EXPERIMENT NO: 22B
Marangoni S, Antunes E, De Nucci G, Landucci
Aim
EC. Inflammatory edema induced by Lachesis muta
muta (Surucucu) venom and LmTX-I in To demonstrate analgesic effect of morphine
the rat paw and dorsal skin. Toxicon 2009;53(1): against acetic acid induced writhing in rat.
69-77.
3. Ne•iæ L, Skrbiæ R, Dobriæ S, Stojiljkoviæ MP, Background
Jaæeviæ V, Satara SS, Milovanoviæ ZA, Stojakoviæ
N. Simvastatin and Indomethacin have similar anti- There are few methods to evaluate the peripheral
inflammatory activity in a rat model of acute local analgesic activity. The Randallselitto-Test in
inflammation. Basic Clin Pharmacol Toxicol 2009 rats and writhing tests in mouse are the
Jan 31. commonest. Many of the peripherally acting
analgesics have the anti-inflammatory and • Divide animals into two groups (n = 6 in each
antipyretic effect. The method depends on the group)
principle that an irritant directly administered
Step 2
into the peritoneum cavity of the animal
produces severe pain and irritation in the
ventral part. The writhing is characterized by
• Group 2: Treatment group (n = 6); rats are given
typical stretching behavior of body and rodent
morphine at the dose of 3.5 mg/kg, ip,
tries to touch its ventral part to the ground.
thereafter 10-15 min, acetic acid 0.1 ml of a
0.6% solution, i.p. is given.
Step 3
Materials
• Observe the animals for 45 min in plexiglass
Animal/species : Rat/Wistar chamber
Sex/body weight : Male/150-280 gm • Observe the animals behavior carefully, for (1)
Syringe/needle : 1 ml/preferably 24G on- onset of writhing, (2) duration of writhing and
wards (3) number of writhing or any additional
Drug : Morphine (3.5-4 mg/kg, s.c.) behaviour changes.
Irritant : Acetic acid (0.1 ml of a 0.6%-
1.0% solution, i.p )
Other irritants : Phenylquinone [(0.02% in
1% suspension of carboxy-
methylcellulose (CMC): 0.25
ml, i.p], PGE1 and brady-
kinin may also be used
Other Drugs
screened : Aspirin (20-21 mg/kg, p.o)
Indomethacin (10-20 mg/kg,
i.p.); Amidopyrine 40 mg/kg
p.o.and Phenacetin 80 mg/
kg p.o.

• Laboratory should be dim lighted and noise
free
• Observe the animals in a plexi glass chamber.
Methods
Step 1 Figs 22.2A and B: (A) Showing the stretching of abdomen
• Weigh the animals and mark properly to and rat touches the ventral part to ground and (B) Showing
distinguish from one another the abdominal cramp or discomfort
Anti-inflammatory  227
Observations and Results 2. Ferreira SH, Lorenzetti BB, Corrêa FMA. Central and
peripheral antialgesic action of aspirin-like drugs.
Observation Table Eur J Pharmacol 1978b;53:39-48.
3. Ferreira SH, Nakamura M, DeAbreu Castro MS. The
Writhing response
hyperalgesic effects of prostacyclin and prosta-
Onset Recovery No. of writhing Duration glandin E2. Prostaglandins 1978a;16:31-37.
4. Finck AD, Samaniego E, Ngai SH. Morphine tolerance
1.
decreases the analgesic effects of ketamine in mouse.
2. Anesthesiology 1988;68(3):397-400.
3. 5. Miranda HF, Puig MM, Dursteler C, Prieto JC, Pinardi
G. Dexketoprofen-induced antinociception in animal
4.
models of acute pain: synergy with morphine and
5. paracetamol. Neuropharmacology 2007;52(2):291-96.
6. 6. Rios L, Jacob JJC. Inhibition of inflammatory pain by
naloxone and its N-methyl quaternary analogue. Life
Sci 1982;31:1209-12.
SUGGESTED READING 7. Romer D. Pharmacological evaluation of mild
analgesics. Br J Clin Pharmacol 1980;10:247S-51S.
1. Chiba S, Nishiyama T, Yoshikawa M, Yamada Y. 8. Winter CW, Risley EA, Nuss GW. Carrageenen-
The antinociceptive effects of midazolam on three induced edema in hind paw of the rat as an assay for
different types of nociception in mouse. J Pharmacol anti-inflammatory drugs. Proc Soc Exp Biol Med
Sci 2009;109(1):71-77. 1962;111:544-47.
Local Anesthetics (LA)
23
EXPERIMENT NO: 23 • Mark the control and treatment side properly
to avoid the confusion.
Aim
To demonstrate the effect of any given local Methods
anesthetic (LA) using guinea pig (GP).
Step 1
Background • Weigh the animals and mark properly to
Local anesthetics are agents which block con- distinguish from one another
duction of Na+ by decreasing or preventing the • Remove the hair on its flanks on the both sides.
large transient increase in the permeability of
excitable membranes and at higher concentration Step 2
they also block K+ channels. This is based on the • Control side: (Mark ‘C’): Saline or control jelly
principle of loss of the sensation, even after given is applied at ‘C’ site
an external stimuli. • Treatment side: (Mark ‘T’): Drug is applied
localy at the ‘T’ site
EXPERIMENT 23‘A’
• Anesthetic activity is checked by giving the
Materials and Methods small pinch with forcep on the control and
Materials treatment side and then check the animal
response (squeak response)
Animal/species : Guinea pig
Sex/Body weight : Either sex/250-350 gm
Step 3
Drug : Lignocaine (2% gel)
Instruments : Scissor, razor • Take the responses at the 0, 5, 10, 15, 20, 30, 45
and 60 min after application of gel
Precautions before Experimentation • The effect of the local anesthetic is expressed
• Carefully remove the hair from either side of as ‘Yes’ for the LA effect and ‘No’ for the
flanks with blade absence of the effect.
Local Anesthetics (LA)  229

Observation Table
Sl. No. Time after application of lignocaine gel (min) Remarks
0 5 10 15 20 30 45 60
‘C’ ‘T’ ‘C’ ‘T’ ‘C’ ‘T’ ‘C’ ‘T’ ‘C’ ‘T’ ‘C’ ‘T’ ‘C’ ‘T’ ‘C’ ‘T’
1.
2.
3.
4.
5.
6.
EXPERIMENT 23‘B’ • Fill the abdominal cavity by 10-12 ml of 1%

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

• Expose the sciatic nerve in the abdomen cavity
• Fix two front limbs of frog on the dissecting 1. Arthur H Campbell, Jeanette A Stasse, Geoffrey H
Lord, John E Willson. In vivo evaluation of local
board, in such a way that both hind limbs of
anesthetics applied topically. Journal of Pharma-
the frog hang vertical by outside the board ceutical Sciences 2006;57(12):2045-48.
• Place the leg into the either solution selectively, 2. Ritchie JM, Greengard P. On the mode of action of
i.e. right on normal saline (control) and left in local anesthetics. Ann Rev Pharmacol 1966;6:
the 0.1 N HCl (test) (To check the response) 405-30.
Experiment on Rabbit Eye
24
EXPERIMENT NO: 24A
Aim
To study the effect of different drugs on the rabbit
Fig. 24.2: Pupilometer
eye.
Background eye size. It either increases in size (mydriasis)

or decreases the pupil size (miosis) according
The drugs like miotics (constrict the pupil) and to the drug used in the experiment.
mydriatics (dilate the pupils) are generally screened 3. Corneal reflex: When cornea is touched rabbit
by this method. The role of circular muscle, radial closes the eyes. To check this reflex, a cotton
muscle and ciliary muscle is well-known (Fig. 24.1). tip is prepared and touched at corneoscleral
Effect should be noted on the various para- junction by bringing the corneal tip from the
meters side of the eye. If the cornea is desensitized
1. Effect on conjunctiva: Conjunctiva is usually thin with the local anesthetic, then there will be no
and clear when eyes are healthy which is used corneal reflex.
in the experiment. Changes before and after the 4. Light reflex: It is elicited by pointing the light
drug administration are noted and compared. source on the pupil with the help of torch.
2. Effect on pupil: The original size is measured There is constriction of pupil when rabbit is
with the help of “pupilometer”( it is the scale kept in a darker area or eye is covered with a
cardboard and the size of pupil is measured,
like structure with varying size of round holes,
then light source is pointed on the pupil.
i.e. 0.5 mm, 1 mm, 1.5 mm, 2 mm, 2.5 mm and
Thereafter the size of the pupil is noted and
3 mm) (Fig. 24.2) and after the drug treatment
then compared with the initial reading.
the change in pupil size is noted and then it is
compared with the initial size or the control
Pathway of Vision (Fig. 24.3)
The visual system is responsible for the vision and
is controlled by the central nervous system. It is
composed of many complex pathways through
optic nerve, optic chiasm, optic tract, lateral
geniculate nucleus (LGN) and visual cortex. Vision
is generated by retina (photoreceptors), a layer of
cells at the back of the eye and travel through optic
Fig. 24.1: Mechanism of miosis and mydriasis nerve to visual cortex. Importance of visual system
Experiment on Rabbit Eye  231
Mydriatics : Phenylephrine, Ephedrine

(3-5%)
Anesthetics : Lignocaine (4%)
Instruments : Corneal reflex meter, scissor,
dropper, torch.

• Maintain the aseptic conditions while
instilling the drug
• Do not use the solution, if it is discolored (check
the expiry date of given drug)
• Experiment should be performed in the dim
lighted room
• Check for any eye disease such as conjunc-
tivitis, uvieitis, etc.
Fig. 24.3: Visual pathway; Visual information falls through • Remove the eye lashes carefully.
the cornea to the object left field to the right side of each eye
and right field to the left side of the eye; Optic nerve: Pass Methods
the visual information to the optic chiasm; Optic chiasm:
Step 1
Nerve or fibers of media half of either retina cross in the
optic chiasm; Optic tract: It carries the visual signal of • Weigh and mark the rabbits. Select 3 rabbits
opposite side such as left visual field information pass (one for pupil reflex, second for corneal reflex
through right optic tract and right visual information pass and third for the light reflex)
from left optic tract; LGN: Situated in thalamus and all nerve
synapse here. Then, neurons of the LGN, pass along the • The alternate eye of a rabbit works as control
visual image to the primary visual cortex; Visual cortex: and test group (to avoid individual varia-
After receiving the image in primary cortex through LGN, it tion).
passes to secondary visual cortex (extrastriate visual
cortex) where the visual perception take place about color, Step 2
height, place, motion, distance, etc.
• Restrain the rabbit in the suitable restrainer
is to do many complex tasks such as reception of and fix it for 5 min (to accommodate and
light, the identification and categorization of visual acclimatize rabbit into the restrainer)
objects by forming an image in the retina, assessing • Select the control and test eye according to your
characteristic colors, place, height, distances of an preference (right or left), and mark it with test
objects and guiding body movements to visual clues. eye (TE) for test and control eye (CE) for control
This type of the psychological manifestation of • Eye lashes of both eyes is trimmed out with
visual information is known as ‘visual perception’. scissor and normal saline is put in both the
Interestingly visual system is the same in the higher eyes to make them clean and clear.
animals.
Step 3
Materials and Methods • The drug is instilled* with the help of dropper
in the test eye (not more than 2-3 drops)
Materials
• Then, the response is noted and both test and
Animal : Rabbit (1.5-2.5 kg) control eyes are compared.
Drugs
miotics : Pilocarpine (1-4%), *Note: Volume to be instilled in rabbit eye sac
Physostigmine (1%) should be 40-60 μl.

Observation Table
Time (min) Light reflex Corneal reflex Pupillary size Condition of
conjunctiva
TE CE TE CE TE CE TE CE
0
5
10
20
30
60
SUGGESTED READING 2. Strughold H. The mechanism threshold of the corneal

1. Green K, Downs SJ. Ocular penetration of pilocarpine reflex of the usual laboratory animals. Am J Physiol
in rabbits. Arch Ophthalmol 1975;93(11):1165-68. 1930;94:235-40.
Experimental
25 Pharmacokinetics
EXPERIMENT NO: 25A animal is allowed to take

water ad libitum and free
Aim access to standard food.
To study the pharmacokinetics of phenytoin Anesthesia : Topical lignocaine (4%) (To
following oral single dose administration for 7 minimize discomfort and
days. pain while withdrawing
blood)
Background
Pharmacokinetics is a process by which a drug is
absorbed, distributed, metabolized, and elimi- • Care and handling should be proper
nated through the body. So, during these processes • Route of blood withdrawal should be
drug substances interact with different com- convenient and easily approachable
pounds residing within an animal or system, such • Selection of needle size should be proper to
as nutrients, metabolites, drugs, endogenous minimize the pain and trauma to the marginal
hormones, toxins, etc. Hence, it is important to vein or at site of blood withdrawal.
assess the drug’s interaction which is preferred
for the chronic use such as antiepileptics, anti- Methods
rheumatics, analgesics, etc. Pharmacokinetics in Step I: Rabbits are acclimatized to laboratory and
the human and animals is not exactly the same. person, 1 week prior to the experimentation.
Hence, animal model in this context is rabbit and
it is widely used in pharmacokinetic studies. Step II: Rabbits (n = 6) are administered phenytoin
Special animal ethics permission is required from in a dose of 30 mg/kg/day per oral, at 08:00 hours
CPCSEA for use of monkey, whereas rabbits are for seven consecutive days using an orogastric
easily available through the Institutional Animal tube.
Ethics Committee (IAEC). Step III: On day 7, blood samples (1 ml) are collected
before administration of drug dose (7th day dose)
Materials and Methods of phenytoin at 0 hr and then drug is administered.
Animals : Male white New Zealand Thereafter, blood sample is taken at 0.5, 1, 2, 3, 4,
rabbits (1.5 kg and 2.5 kg) 5, 6, 9, 12 and 24 hours after drug administration
(n=6) i.e. total of 11 samples.
Drug : Phenytoin (30 mg/kg/day) Step IV: Blood sample is drawn from the marginal
Laboratory ear vein after applying anesthesia with topical
conditions : 12 hours day-night cycle at a lignocaine 2% gel. (See instructions for blood collection
temperature of 25 ± 2°C. The in chapter 1).
Step V: Each sample is centrifuged at 3000 rpm for Observation Table

5 minutes and plasma separated and stored at Parameters Drug (Phenytoin)
(–20°C) until HPLC analysis.
Peak plasma concentration
Step VI: Then, phenytoin concentration is (Cmax)
estimated by HPLC method. Time to reach peak plasma
concentration(Tmax-)
Extraction procedure: To 0.2 ml plasma sample/ Absorption constant (ka)
standard sample, add 0.2 ml of 1.0M of sodium Absorption half-life (t½a)
acetate buffer (pH 5.5) and 3.0 ml of chloroform. Elimination constant (kel)
Shake the tubes for 1 min and then centrifuge at Elimination half-life(t½el )
Area under the plasma-
3000rpm for 10 min. Transfer 2.8 ml of drug concentration time
chloroform layer in another test tube and curve (AUC0-24)
evaporate the chloroform at 50°C on a water (AUC0-∞)
bath. Reconstitute the residue in 0.2 ml of mobile
phase to be used for HPLC assay. Inject 100 µl SUGGESTED READING
of this reconstituted solution to HPLC system
1. Medhi B, Sukhija M, Prakash A, Gaikwad S, Bansal
for assay.
V, Pandhi P. Effects of etoricoxib on the pharma-
HPLC conditions: Mobile phase: acetonitrile: cokinetics of phenytoin. Pharmacol Rep 2008;60(2):
methanol: 4mM potassium phosphate buffer (pH 233-37.
6.0) in ratio of 20:40:40(V/V/V)
EXPERIMENT NO: 25B
Flow rate : 1.0 ml/min
Temperature : ambient Aim
AUFS : 0.02
To study the pharmacokinetic interaction of
Detection : UV detector 215 nm phenytoin with etoricoxib after single oral dose
Chart speed : 4 mm/min for 7days.
Injection volume : 100 µl
Sensitivity assay : 0.1 µg/ml Background
Recovery : 98% or more Pharmacokinetic interactions result due to the
Standard : phenytoin 0.5 to 32 µg/ml multiple use of drugs at a time or even with the
food. But, these interactions are more complicated
Step VII: Record the reading and store chrom-
and difficult to predict because the interacting
atogram.
drugs often have unrelated actions. For example:
Step VIII: The following pharmacokinetic para- Phenytoin is used as antiepileptic whereas
meters should be calculated for phenytoin. etoricoxib is used for the acute and chronic pain.
• Peak plasma concentration(Cmax) The interactions are mainly due to alteration of
• Time to reach peak plasma concentration(Tmax) absorption, distribution, metabolism, or excretion
• Absorption constant (ka) which changes the amount and duration of a
drug’s availability at receptor sites. Some drugs
• Absorption half-life (t½a)
cause induction of hepatic mitochondrial
• Elimination constant (kel) enzymes (P-450) such as phenytoin, barbiturates,
• Elimination half-life(t½el ) rifampin, digoxin, etc. and some drugs cause
• Area under the plasma-drug concentration inhibition of hepatic mitochondrial enzymes (P-
time curve (AUC0- 24) and (AUC0-∞). 450) such as TCA, oral contraceptives, isoniazid,
Experimental Pharmacokinetics  235
cimetidine, allopurinol, disulfiram, etc. which can the both drugs then, collect blood samples at 0.5,
influence the pharmacokinetics of several drugs 1, 2, 3, 4, 5, 6, 9, 12 and 24 hours.
metabolized by the same enzyme system.
Step IV: Blood samples are drawn from the
Materials and Methods marginal ear vein after applying anesthesia with
topical lignocaine 2% gel. (See instructions for
Animals : Male white New Zealand blood collection in chapter 1).
rabbits (1.5 kg and 2.5 kg)
(n=6) Step V: Samples are labelled properly, so that there
Drugs : Phenytoin (30 mg/kg/day) is no mixing of samples. Then, each sample is
Etoricoxib (5.6 mg/kg) centrifuged at 3000 rpm for 5 minutes and plasma
Laboratory separated and stored at -20°C until HPLC
conditions : 12 hours day-night cycle at a analysis.
temperature of 25 ± 2°C. The Step VI: Then, phenytoin is estimated by HPLC
animal is allowed to take method.
water ad libitum and free
access to standard food. Extraction procedure: To 0.2 ml plasma sample/
Anesthesia : Topical lignocaine (4%) (To standard sample add 0.2 ml of 1.0M of sodium
minimize discomfort and acetate buffer (pH 5.5) and 3.0 ml of chloroform.
pain) Shake the tubes for 1 min and then centrifuge at
3000 rpm for 10 min. Transfer 2.8 ml of chloroform
Methods layer in another test tube and evaporate the
chloroform at 50°C on a water bath. Reconstitute
Step I: Total 12 rabbits are acclimatized to the residue in 0.2 ml of mobile phase to be used for
laboratory and person, 1 week prior to the HPLC assay. Inject 100 µl of this reconstituted
experimentation. solution to HPLC system for assay.
Step II: On experimental day, rabbits are divided HPLC conditions: For HPLC conditions refer to
into two groups control (phenytion alone) group experiment no. 25A
and treatment group (Phenytoin + etoricoxib). Step VII: Record the readings and store chro-
In control group, rabbits (n=6) are adminis-
matogram
tered phenytoin in a dose of 30 mg/kg/day per
oral at 0900 hours for ten consecutive days using Step VIII: The following pharmacokinetic para-
an orogastric tube. But, in the treatment group meters will be calculated for phenytoin
(n=6) phenytoin is administered in a dose of 30 • Peak plasma concentration(Cmax)
mg/kg/day per oral at 0900 hours for 7 conse- • Time to reach peak plasma concentration(Tmax)
cutive days using an orogastric tube, then next • Absorption constant (ka)
3days etoricoxib 5.6 mg/kg/day, po with same • Absorption half-life (t½a)
dose of phenytoin is administered. • Elimination constant (kel)
Step III: On day 10, blood samples (1 ml) are • Elimination half-life(t½el )
collected before administration of next dose (10th • Area under the plasma-drug concentration
day dose) of phenytoin at 0 hr and then administer time curve (AUC0- 24) and (AUC0-∞).
Observation Table
Parameters Drug
Phenytoin Phenytoin + Etoricoxib
Peak plasma concentration(Cmax)
Time to reach peak plasma concentration(Tmax-)
Absorption constant (ka)
Absorption half-life (t½a)
Elimination constant (kel)
Elimination half-life(t½el )
Area under the plasma-drug concentration
time curve (AUC0- 24)
(AUC0-∞)
SUGGESTED READING 2. Sukhija M, Medhi B, Pandhi P. Effects of artemisinin,

artemether, and arteether on the pharmacokinetics
of phenytoin. Methods Find Exp Clin Pharmacol 2006;
1. Medhi B, Sukhija M, Prakash A, Gaikwad S, Bansal
28(2):89-94.
V, Pandhi P. Effects of etoricoxib on the pharma- 3. Sukhija M, Medhi B, Pandhi P. Effects of artemisinin,
cokinetics of phenytoin. Pharmacol Rep 2008; artemether, arteether on the pharmacokinetics of
60(2):233-37. carbamazepine. Pharmacology 2006;76(3):110-16.
Part 4
Clinical Experiments
Cardiovascular System
26
BLOOD PRESSURE MEASUREMENT AND Phase III : Rhythmic tapping sound (lasts for 12-14 mm Hg
VALIDATION OF SPHYGMOMANOMETER fall)
Phase IV : Muffling/fading of tapping sound (lasts for 4-5
Introduction mm Hg fall)
Phase V : Point at which all sounds disappear-diastolic
Blood pressure (BP) measurement is one of the pressure
important tool for evaluation of cardiovascular
system, which helps in diagnosis and manage- Factors Affecting Blood Pressure
ment of hypertension and related end organ
damages. Hence, monitoring of BP is an essential Several factors may interfere with blood pressure.
and basic step. So, briefly, blood pressure (BP) is BP varies throughout the day according to the time,
the force exerted against blood vessel walls due to season and even the daily routine of individual. It
the blood flow in the vessels. As a result of can rise rapidly while doing physical exercise,
resistance of the blood vessels and the volume of when having an emotional moment or even stress
blood carried into the blood vessel. It is expressed and defense reaction of body to any stimuli.
in millimeters of mercury (mm Hg). The blood
pressure is measured as two end-point, i.e. first Principle of BP Measurement
upper level is systolic pressure (the first sound Principle of blood pressure measurement
heard) and the second is diastolic pressure. (The depends on the compression length of artery
last sound heard) There are 5 phases of sound (brachial in arm, radial in wrist and femoral in
heard (Korotkoff sound). The difference of systolic thigh) which blocks and reopens the artery blood
and diastolic pressure is commonly denoted as flow. The pressure is applied with the help of
“pulse pressure”. (approximately 40 mm Hg). bladder fixed in the cuff and rubber bulb. The
In clinical practice, BP measurement may get systolic and diastolic pressure is measured by the
erroneous (high or low reading) due to the several palpatory, auscultatory or oscillometric method.
reasons such as cuff is too small or cuff too loose,
slow cuff deflation, arm is not at the level of heart, Instrument to Measure the BP
column or dial not at eye level or it may be taken at
the obsolete time in case of anxiety, exercise, after The gold standard instrument for blood pressure
eating, etc. measurement is considered to be mercury
sphygmomanometer. The term developed from the
Korotkoff sounds Greek origin, “sphygmo” denotes pulse, “ manos”
Phase I : First sound, sharp tapping sound-systolic denotes thin and “metron” denotes measure. It
pressure (lasts for 10-12 mm Hg) fall consists of mercury manometer, graduated tube,
Phase II : Soft swishing sound (lasts for 10-15 mm Hg fall)
armlet (Riva Rocci cuff) and air pump.
Fig. 26.1: Parts of sphygmomanometer and the size of the ideal cuff (Riva Rocci)
Cuff (Riva Rocci) (Table 26.1). These errors caused by the non-
calibrated blood pressure apparatus can not be
Selection of BP cuff should be very logical and
reduced by averaging the measurement. This is
scientific. Wrong selection can give false cuff
an error which is difficult to detect and correct.
hypertension or underestimated BP. The recom-
(Another error is the random error which is due to
mended cuff should have air bladder which
biological variability or any stress or emotional
covers at least 80% of the arm circumference and
condition, but it is reduced by averaging a number
width wise it should cover at least 40% of arm
of measurements.)
circumference (Fig. 26.1).
Arm circumference Class Cuff size Formal Calibration of the Pressure Indicator
22 to 26 cm “small adult” 12-22 cm
27 to 34 cm “adult” 16-30 cm
The validation of devices measuring blood
35 to 44 cm “large adult” 16-36 cm pressure is essential. It should be timely calibrated
45 to 52 cm “adult thigh” 16-42 cm and validated at the recommended time for
accurate measurement of blood pressure. The
Rubber bulb and tube: Inflation mode attached to
instrument should be regularly checked for the
the air bladder of the cuff which is at least of 70 cm
air leakage, the condition of cuff, tubes, bulb and
in length.
fittings, rapid exhaust time, scale visibility,
contamination of the glass tube or mercury, cuff
Mercury Reservoir and Manometer: Validation/
inflation and deflation control and security of
calibration of Sphygmomanometer
mercury containment (vapor of mercury is
The manometer provides a simple indication of poisonous).
difference of pressure, the most preferred one is The sphygmomanometer is calibrated at the
mercury manometer which is used to record or indicator from ‘0’ to the maximum pressure (280-
control difference of pressure or fluid flow. 300 mm Hg) on the sphygmomanometer scale at
Accurate measurement of blood pressure requires pressure increments not greater than 50 mm Hg.
the use of validated/calibrated sphygmo- The pressure indicator of all sphygmomanometers
manometer, which requires regular service and should be calibrated by an authorized laboratory
calibration. The calibration is done to avoid any at 0-300 mm Hg. The standard for instrument is
systemic error during the BP measurement set for least uncertainty of measurement and best
Cardiovascular System  241
accuracy, i.e. a least uncertainty of measurement TO PREPARE STANDARD OPERATING

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

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

– Crossed leg (Fig. 26.4C)
• Standing position
– Hand/arm position below heart level (Fig.
26.5A)
i. at the level of heart (26.5B)
ii. above heart level (Fig. 26.5C)
Fig. 26.3B: Supine position
Fig. 26.4C: Sitting with crossed leg

Fig. 26.4A: Recording of BP in sitting position (normal)
Fig. 26.4B: Sitting; cuff position should be at Fig. 26.5A: Standing with hand position below the level of heart
the level of heart (For color version see Plate 4) (cuff level is below heart) (For color version see Plate 4)
functions and rapid changes in the circadian

rhythm. In this population chronodiagnosis of BP
measurement is considered to be more reliable and
reproducible.

Finger Cuff Method (developed by Penaz). This is based
on the principle of the “unloaded arterial wall”. Arterial
pulsation in a finger is detected by a photoplethysmograph
under a pressure cuff. This method gives an accurate
estimate of the changes of systolic and diastolic pressure,
but less than brachial artery pressures. Mainly used for
the ambulatory BP measurement. Advantages being
Fig. 26.5B: Standing with hand position at the heart level monitoring of short change in BP, but cost, inconvenience,
(For color version see Plate 4) and relative inaccuracy for measuring absolute levels of
blood pressure are major limitations.
REGULATION OF BLOOD PRESSURE

Blood pressure regulation (Fig. 26.6) involves
complex interaction of neural, renal and endocrine
mechanism. Arterial blood pressure is generated
as a result of blood flow and the resistance to blood
flow. Major factors affecting blood pressure are
Fig. 26.5C: Standing with hand position above heart level cardiac output (CO) and total peripheral vascular
(For color version see Plate 4) resistance (TPR) of blood vessels. So, blood
pressure (BP) = CO × TPR. CO is the major deter-
Special Population and BP Measurement
minant of SBP, whereas TPR mainly determines
Relationship between obesity and hypertension DBP. Cardiac output and peripheral resistance
is well established since 1930s. Hence, the BP are regulated by the autonomic nervous system
measurement in these populations is an essential (ANS). Blood pressure is regulated by alteration
requirement to maintain the health. Caution, in cardiac output and peripheral resistance
should be taken while measuring the BP, selecting exerted at different target sites like arterioles,
arm circumference and taking bladder dimen- postcapillar venules, kidneys and heart. Myo-
sions. It may give false overestimation of BP (“cuff cardial contractility and heart rate are regulated
hypertension”) if the cuff size is small and vice by both the sympathetic and parasympathetic
versa. Variability in BP is more in the pediatric divisions of the ANS. In contrast, blood vessels
population than in adults. Mercury sphygmo- are not innervated by parasympathetic fibers,
manometer is used in the BP measurement. For therefore the parasympathetic nervous system has
the obese population, pediatric population minimal influence on vascular tone. The neural
appropriate cuff size should be used. Korotkoff control of blood pressure regulation is by
sounds in child aged 1-5 years are not audible parasympathetic neurons that innervate the heart
and hence, murcury sphygmomanometer is not and by three different action of sympathetic
used, so in these age groups doppler, ultrasound, cardiovascular efferents barosensitive, thermo-
or oscillometry would be more sensitive and sensitive and glucosensitive. The barosensitive
reliable. sympathetic efferents are controlled by arterial
BP measurement in the geriatric population is baroreceptors which play a dominant role in
troublesome due to change in the physiological both short-term and long-term blood pressure
Fig. 26.6: Regulation of blood pressure
regulation. The important regulatory systems in pressure activates baroreceptors there by causing
controlling blood pressure are the sympathetic inhibition of cardiac renal vasomotor sympathetic
nervous system and the renin-angiotensin- effect which help in normalization of blood
aldosterone system. A rise in blood pressure pressure.
increases the impulses to the vasomotor center Baroreflex resetting involves both neural and
resulting in decreased output from the center humoral mechanisms.
resulting in compensatory decrease in the efferent Besides these various local hormones and
response of the sympathetic nervous system. metabolites such as ANP, PGs, kinins, NO,
Similarly, a reduction in stretch due to fall in blood endothelins, etc. are involved in regulation of blood
pressure causes reduction in baroreceptor activity. pressure. Nitric oxide in vascular endothelial
The sympathetic baroreflex mechanism is acti- cells, is primarily responsible for controlling
vated by stimulus of mechanoreceptors causes vascular tone and platelet aggregation. The recent
distension of the arterial wall, an increase in blood evidence suggest that nitric oxide may affect
vasodilation not only by activating guanyl cyclase contain family of peptides with natriuretic diuretic,
but also by activating calcium and potassium vasorelaxant and other properties. These consist
channels in vascular smooth muscle cells, nitric of atrial natriuretic peptide (ANP), brain natriuretic
oxide appears to activate these potassium peptide (BNP), and C-type natriuretic peptide. ANP,
channels directly via guanyl cyclase independent a 28 amino acid peptide is synthesized in cardiac
mechanism leading to hyperpolarization of the atrial cells but insignificant amount are also
cells and subsequently causes vasodilatation. synthesized in ventricular cells. It is also
Other factor like endothelin, which is a potent synthesized in the CNS and peripheral nervous
endothelium derived vasoconstrictor, endothelin system and in lungs. Several factors which increase
has got positive inotrophic and chronotropic the release of ANP from the heart are atrial stretch
action on the heart and also contributes to via mechanosensitive ion channels, blood volume
remodeling in the cardiovascular system. Different expansion, head out water emersion changing from
isoforms ET-1, ET-2, ET-3 have been identified. standing to supine position and exercise. Besides
The ET-1 is secreted from the endothelial cells this ANP participates in physiological regulation
which is mainly involved in cardiovascular of sodium excretion and blood pressure, e.g.
actions as paracrine and autocrine locally. ET-1 suppression of ANP production or blockade of its
concentration is very high in the vascular area action impairs the natriuretic response to volume
because it is secreted on the basal side of the expansion and increase blood pressure.
endothelial cells. Endothelins act through several
G protein couple receptors like ET-A and ET-B. Exercise and Blood Pressure
ET-A receptor on vascular smooth cells mediate
Exercise is a planned, structured and repetitive
vasoconstriction whereas ET-B located pre-
bodily movement done to improve or maintain
dominantly on vascular endothelial cells where
one or more components of physical fitness.
they mediate vasodilatation via release of pro-
stacyclin mediate vasoconstriction. Thus endo- Physical fitness includes several components:
thelins produce vasoconstriction by acting cardiorespiratory, muscular strength, flexibility,
through ET-A and ET-B receptors in the vascular endurance and body composition. Exercise can
smooth muscle cells and vasodilation acting be divided into four types:
through ET-B receptors in the endothelium. Other 1. Acute and chronic
mechanism involved are neurohormonal mecha- Acute exercise relates the physiological
nisms of adrenaline and NA influence vascular responses that occur with a single bout of
tone alpha-1 and beta-2 receptors on vascular exercise. Chronic exercise describes person
smooth muscle cells. familiar with repeated bouts of physical
Besides this, (ADH) secreted in response to training.
angiotensin-II affects the vascular tone by acting 2. Aerobic and anaerobic
on its 3 V types of receptors. This mechanism is Aerobic exercise consists of repetitive low-
involved to maintain blood flow at constant level resistance movements that last over a long
and also to maintain vascular tone, so that blood period of time, usually more than 10 minutes,
flow is alter by different metabolites like H+, CO2, such as walking or cycling or it can be
O2, adenosine, lactate, K+, etc. mostly in local described as exercise of muscle movement that
tissue. So, local mechanism of vascular tone uses oxygen to burn both carbohydrates and
regulation is predominant in the vascular beds of fats to produce energy. Anaerobic exercise, on
essential organs which help in maintenance of the other hand, consists of high-resistance,
blood flow and oxygen supply as per demand of low-repetitive movements that last only 1-3
local metabolism in these organs. minutes, interrupted by frequent periods of
Role of natriuretic peptides in regulation of rest between exercise bouts for example weight
blood pressure: Atria and other tissue of mammals lifting, push-ups and sit-ups, etc. or simply it
is an exercise, where muscle movement does goal of submaximal testing is to produce

not require oxygen and only burns carbo- adequate level of exercise stress without
hydrates to produce energy. This builds up physiological and biological strain. Maximal
muscles and gives physical strength through exercise is useful for VO 2 determination,
short bursts of strenuous activity. So an ideal diagnosis and to test the treatment outcome,
exercise program should include both aerobic whereas submaximal exercise is applicable to
and anaerobic exercises. predict VO2 max, to assess the effect of an
3. Isotonic and isometric intervention in the exercise schedule, and to
Isotonic exercise is explained as when a muscle observe the recovery strategies after the
contracts and temporarily shortens with exercise.
movement of attached joint. And isometric The American College of Sports Medicine
exercise can be described when the muscle characterizes aerobic exercise intensity as low,
contracts against a fixed resistance. So, in moderate or high based on elicited rise in heart
isometric exercise, there is no muscle shor- rate. Predicted Maximum Heart Rate (PMHR) is
tening or joint movement, e.g. pushing against calculated by the following formula, PMHR = 220
a wall, trying to a lift a weight, etc. - Age in years. An exercise is categorized as low-
4. Maximal and submaximal intensity if it elicits 35 to 59% of predicted
Maximal exercise testing is considered gold maximum heart rate and moderate-intensity,
standard but as compared to maximal exercise which elicits 60 to 79% of PMHR. Exercise eliciting
testing submaximal exercise test has greater a response greater than this is considered high-
applicability in clinical practice (Table 26.2). intensity.
These tests are used in order to predict Metabolic equivalents (METs) units are usually
maximal aerobic capacity. Application of these used to estimate the oxygen consumption,
tests is selectively based on specific indica- following a specific physical activity. One MET
tions: like person with limited capacity indicates energy cost of sitting quietly. Depending
because of cardiopulmonary, musculoskeletal on physical activity, it varies for example person
and neuromuscular impairments with diffi- engaged in vigorous activity, his or her oxygen
culty during exertion, dyspnea, fatigue, demands will increase, and so do the METs
weakness and pain during daily activities. So, assigned to that activity. Several examples such as
activities like walking slowly, yoga and housework
Table 26.2: Examples of different Submaximal exercises use about 3 METs, fast walking and doubles tennis
use 3 to 6, and jogging, shoveling snow, and singles
Predictive submaximal test
1. Modified Bruce treadmill test
tennis use more than 6. So, in day to day activity,
2. Single-stage submaximal treadmill walking test typical person uses about 3.5 milliliters of oxygen
(SSTWT) per kg of body weight per minute. Aerobic exercise
3. Astrand and Rhyming (A-R) cycle ergometer test intensity can also be categorized based on energy
4. Canadian aerobic fitness test (CAFT) and modified equivalents, measured in METs. One MET is the
CAFT (MCAFT) energy spent at rest to maintain basal bodily
5. 1- Mile tract walking test (Rockport fitness test, 1-
MTW)
functions. (1 MET = 3.5 ml O2/kg/min). An activity
or exercise requiring less than 3 METs is considered
Performance submaximal test
1. Self paced walking test (SPWT) light-intensity, that requires 3-6 METs is considered
2. Modified shuttle waking test (MSWT) as moderate intensity and those exercises which
3. Bag and carry test (BCT) demand more than 6 METs are called high-
4. Time up and go test (TUGT) intensity exercises.
5. 12 minute walk test (12-MWT) Several studies have consistently reported that
6. 6 minute walk test (6-MWT)
regularly performed aerobic exercise of mild-to-
moderate intensity lowers blood pressure and it there are several guidelines available for manage-
is beneficial for patients with essential hyper- ment of hypertension: WHO-International Society
tension. Regular aerobic exercise lowers blood of Hypertension (WHO-ISH), the Joint National
pressure as it involves various physiological Committee (JNC VII) from USA, British Hyper-
mechanisms like decreased sympathetic system tensive Society (BHS IV), Australian, Japanese and
activity alone with potentiating of the baroreceptor Indian guidelines. All these guidelines provide
reflex and reduced arterial stiffness with enhanced the guidance to practitioner based on statistical
total systemic arterial compliance. It also helps in data of particular country’s population. The
increased release of endothelium-derived nitric Indian guideline was released in 2000 and revised
oxide which is probably related to lower plasma in 2007. The Indian guideline is a joint guideline
cholesterol and increased insulin sensitivity. of Hypertensive Society of India, Cardiological
Studies show that reduction in blood pressure Society of India, Indian Medical Association and
with regular aerobic exercise is approximately 8 Association of Physicians of India (API). Most of
to 10 mm Hg for systolic and 7 to 8 mm Hg for the international guideline updated every 4-5
diastolic blood pressure in hypertensive patients. years for example British Hypertensive Society
Several studies have consistently shown that
Guidelines were revised in 1989, 1993, 1999 and
regularly performed aerobic exercise of mild-to-
2004. Though, there are several guidelines
moderate intensity lowers blood pressure in
available but there is no uniformity on cut off
patients with essential hypertension. The exercise
levels of all the guidelines. Recently, optimal blood
is recommended and it is convenient to remember
pressure is considered less than 120/80 mm Hg.
the anagram FITT: Frequency (F), the intensity (I),
the time (T) (or duration), and the type (T) or So, normal blood pressure have been accepted in
exercise. Of the four components of the FITT most of the guidelines is systolic 120-129 and
anagram, the intensity of exercise is by far the most diastolic is 80-84 mm Hg. There is also category
important. Most of the skill in prescribing exercise included in the guideline like high normal which
is related to managing the intensity component. corresponds to systolic 130-139 and diastolic
85-89 mm Hg e.g. in British Hypertensive Society
BLOOD PRESSURE GUIDELINES and Indian Guideline. Similarly, JNC VII used
(TABLE 26.3) term as pre hypertension, mostly JNC VII catego-
Hypertension is the commonest cardiovascular rizes as stage I and stage II hypertension but
disease all over the world. Studies have reported Indian guideline described as stage I, II, and III
that about 25% of world population aged more hypertension which is similar to JNC VI guideline.
than 30 years suffer from hypertension. Presently, Most of the guidelines recommend use of mercury
Table 26.3: Blood pressure and different guidelines

Classification JNC* 7 BHS# IV Indian HTN Guidelines II
<120/80 Normal Optimal Optimal
120-129/80-84 Pre-hypertensive Normal Normal
130-139/85-89 High normal High normal
140-159/90-99 Stage I Stage I Stage I
160-179/100-109 Stage II Stage II Stage II
> 180/110 Stage III Stage III
ISH$ G-1 SBP > 140 SBP 140-159 SBP 140-159
DBP < 90 DBP < 90 DBP < 90
ISH$ G-II SBP > 160 SBP > 160
DBP < 90 DBP < 90
* JNC 7: Joint National Committee on Prevention, Detection, Evaluation, and Treatment of High Blood Pressure
#
BHS (IV): British Hypertensive Society, $ ISH International Society of Hypertension
sphygmomanometer as a gold standard. Besides, International Protocol for validation of blood pressure
this Indian guideline also stresses on daily intake measuring devices in adults. Blood Press Monit 2002;
7(1):3-17.
of salts, alcohol abstinence and described
12. O’Brien E. A century of confusion: Which bladder
metabolic syndromes as per our population. for accurate blood pressure measurement? J Hum
Hypertension 1996;10:565-72.
SUGGESTED READING 13. O’Brien E. A century of confusion: Which bladder
for accurate blood pressure measurement? J Hum
1. Carney SL, Gillies AH, Green SL, Paterson O, Taylor Hypertension 1996;10:565-72.
MS, Smith AJ. Hospital blood pressure measurement:
14. Penaz J. Photoelectric measurement of blood
Staff and device assessment. J Qual Clin Pract 1999;
pressure, volume and flow in the finger. Digest Tenth
19(2):95-98. International Conference Medical Biological
2. Carney SL, Gillies AH, Smith AJ, Smitham S. Hospital Engi-neering. Dresden; 1973:104.
sphygmomanometer use: an audit. J Qual Clin Pract.
15. Pickering TG, Hall JE, Appel LJ, Falkner BE, Graves
1995;15(1):17-22.
J, Hill MN, Jones DW, Kurtz T, Sheps SG, Roccella
3. Coleman A, Freeman P, Steel S, Shennan A. Validation EJ. Recommendations for blood pressure
of the Omron 705IT (HEM-759-E) oscillometric blood measure-ment in humans and experimental animals:
pressure monitoring device according to the British
part 1: blood pressure measurement in humans: A
Hypertension Society protocol. Blood Press Monit
statement for professionals from the Subcommittee
2006;11(1):27-32. of Professional and Public Education of the American
4. Coleman AJ, Steel SD, Ashworth M, Vowler SL, Heart Association Council on High Blood Pressure
Shennan A. Accuracy of the pressure scale of
Research. Circulation 2005;111(5):697-16.
sphygmomanometers in clinical use within primary
16. Pickering TG. Reflections in hypertension. How
care. Blood Press Monit 2005;10(4):181-88. should blood pressure be measured during
5. Dahloff B, Lindholm LH, Hansson L, Schersten B, preg-nancy? J Clin Hypertens (Greenwich) 2005;7(1):
Ekbom T,Wester PO. Morbidity and mortality in the
46-49.
Swedish Trial in Old Patients with Hypertension
17. Shah NC, Sibbritt DW, Heaney S, Sharples J.
(STOP-Hypertension). Lancet 1991;394:405-12. Sphyg-momanometers—an audit in general practice.
6. De Swiet M, Dillon MJ, Little W, O’Brien E, Padfield Aust Fam Physician 2004;33(11):952-54.
PL, Petrie JC. Measurement of blood pressure in
18. Turner MJ, Baker AB, Kam PC. Effects of systematic
children. Recommendations of a working party of
errors in blood pressure measurements on the
the British Hypertension Society. BMJ 1989;299:497. diagnosis of hypertension. Blood Press Monit 2004;
7. De Swiet M, Dillon MJ, Little W, O’Brien E, Padfield 9(5):249-53.
PL, Petrie JC. Measurement of blood pressure in
19. Turner MJ, Irwig L, Bune AJ, Kam PC, Baker AB.
children. Recommendations of a working party of
Lack of sphygmomanometer calibration causes over-
the British Hypertension Society. BMJ 1989;299:497. and under-detection of hypertension: A computer
8. Joffres MR, Hamet P, Rabkin SW, Gelskey D, Hogan simulation study. J Hypertens 2006;24(10):1931-38.
K, Fodor G. Prevalence, control and awareness of
20. van Popele NM, Bos WJ, de Beer NA, van Der Kuip
high blood pressure among Canadian adults.
DA, Hofman A, Grobbee DE, Witteman JC. Arterial
Canadian Heart Health Surveys Research Group. Can stiffness as underlying mechanism of disagreement
Med Assoc J 1992;146(11):1997-2005. between an oscillometric blood pressure monitor and
9. Marshall T, Rouse A. Blood pressure measurement.
a sphygmomanometer. Hypertension 2000;36(4):
Doctors who cannot calibrate sphygmomanometers
484-88.
should stop taking blood pressures. BMJ 2001;
323(7316):806.
10. O’Brien E, Atkins N. A comparison of the British
Regulation of Blood Pressure
Hypertension Society and Association for the 21. Danpney RAL. Central mechanism underlying short
Advancement of Medical Instrumentation protocols and long-term regulation of the cardiovascular
for validating blood pressure measuring devices: Can system. Clinical Exp Pharmacol Physiol 2002;29:
the two be reconciled? J Hypertens 1994;12:1089-94. 261-68.
11. O’Brien E, Pickering T, Asmar R, Myers M, Parati G, 22. Dempsey JA, Sheel AW, St Croix CM, Morgan BJ.
Staessen J, Mengden T, Imai Y, Waeber B, Palatini P, Respiratory influences on sympathetic vasomotor
Gerin W. Working Group on Blood Pressure outflow in humans. Respire Physiol Neurobiol 2002;
Monitoring of the European Society of Hypertension 130:3-20.
23. Di bona GF, Kopp Uc. Neural control of renal Assoc 2007;297:2081-91.
function. Physiol Rev 1997;77:75-197. 39. Foster C, Cadwell K, Crenshaw B, et al. Physical
24. Fruchgott RF. Endothelium derived relaxing factor activity and exercise training prescriptions for
discovery early studies and identification as NO. patients. Cardiol Clin 2001;19:447-57.
Bioscci Rep 1999;19:235. 40. Fu Qi, Vongpatanasin W, Levine BD. Neural and
25. Galie N, Manes A, Branzi A. The endothelin system nonneural Mechanisms for Sex Differences in Elderly
in pulmonary arterial hypertension. Cardiovascular Hypertension – Can Exercise Training Help?
Reseasch 2004;61:227. Hypertension 2008;52:787-94.
26. Godfraind T, Kaba A. Role of calcium in the action 41. Kokkinos PF, Narayan P, Papademetriou V. Exercise
of drug on vascular smooth muscle. Arch Int as Hypertension Therapy: Cardiol Clin 2001;19:
Pharmacodyn Ther 1972;196;S35-S49. 507-16.
27. Guo ZL, Lai HC Lonhust HC. Medullar pathways 42. Labarthe D, Ayala C. Nondrug interventions in
involved in cardiac symphato excitatory reflexes in hypertension prevention and control. Cardiol Clin
the cat. Brain Res 2002;925:55-66. 2002;20:249-63.
28. Guyenet PG. The sympathetic control of blood 43. Vanessa N, Elizabeth D. Submaximal exercise
pressure. Nat Rev Neurosci 2006;7:335-46. testing: Clinical application and interpreatation.
29. Higashi Y, Yoshizumi M. Exercise and endothelial Physical Therapy 2000;80(8):782-807.
function; role of endothelium derived nitric oxide
and oxidative stress in healthy subject and hyper- Blood Pressure Guideline
tensive patients. Pharmacotherapy 2004; 102:87-96.
30. Liu KL. Regulation of renal medularry circulation by 44. Guide to management of hypertension 2008. http://
rennin angiotensin system in genetically hypertensive www.heartfoundation.org.au/Professional_Information/
rats. Clin Exp Pharmacol Physiol 2009 Feb 10. Clinical_Practice/Hypertension.htm
31. Lohmeier TE. The symphatetic nervous system and 45. Guidelines for management of hypertension: Report
long-term blood pressure regulation. Am J hyper- of the fourth working party of the British Hyper-
tension 2001;14:147-54. tension Society, 2004-BHS IV.B Williams, NR Poulter,
32. Pilowsky PM, Good child AK. Baroreceptor reflex MJ Brown et al. Journal of Human Hypertension
pathways and neurotransmitters 10-year on J on 2004;18:139-85.
hypertens 2006;20;1675-88. 46. Guidelines for the Management of Arterial
33. Schreihofer AM, Guyenet PG. The baroreflex and Hyper-tension. The Task Force for the Management
beyond control of symphatetic vasomotor tone by of Arterial Hypertension of the European Society of
GABAergic neurons in the venterolateral medulla. Hypertension (ESH) and of the European. Society of
Clin Exp Pharmacol Physiol 2002;29:514-21. Cardiology (ESC). Authors/Task Force Members:
34. Vallbo AB, Harbarth KE, Wallin BG. micro- Giuseppe Mancia, Co-Chairperson (Italy), Guy de
neuerography; how the technique developed and its Backer, Co- Chairperson(Belgium), Anna Domi-
role in the investigation of the fore the symphatetic niczak (UK), Renata Cifkova (Czech Republic),
nervous system. J Appl Phsiol 2004: 96;1262-69. Robert Fagard ( Belgium), Giuseppe Germano (Italy),
Anthony m. Heagerty (UK), Sverre E. Kjeldsen
Exercise and Blood Pressure (Norway), Stephane Laurent (France), Krzysztof
Narkiewicz (Poland), Luis Ruilope (Spain), Andrzej
35. American College of Sports Medicine, Guidelines for
Rynkiewicz (Poland), Roland E. Schmieder (Ger-
Exercise Testing and Prescriptions, Ed 6. Baltimore,
Lippincott Williams and Wilkins 2000;150. many), Harry A.J. Struijker Boudier (Nether-lands),
36. American College of Sports Medicine. Exercise and Alberto Zanchetti (Italy). Journal of Hypertension
hypertension. Med Sci Sports Exerc 2004;34:533-53. 2007;25:1105-1187.
37. Bavikati VV, Sperling LS, Salmon RD, et al. Effect of 47. Indian hypertension guidelines II. http://www.
Comprehensive Therapeutic Lifestyle Changes in apiindia.org/hypertensioguidelines/hyperhome.htm
Prehypertension. Am J Cardiol 2008;102:1677-80. 48. The Seventh Report of the Joint National Committee
38. Church T, Earnest C, Skinner J, et al. Effects of on Prevention, Detection, Evaluation and Treatment
different doses of physical activity on cardio- of High Blood Pressure US Department of Health
respiratory fitness among sedentary, overweight or and Human Services. National Institutes of Health.
obese post-menopausal women with elevated blood National Heart, Lung and Blood Institute. http://
pressure: A randomized controlled trial. J Am Med www.nhlbi.nih.gov/guidelines/hypertension/jnc7full.pdf
49. World Health Organization, International Society of Materials and Methods

Hypertension Writing Group. Journal of Hypertension
2003;21:1983-92. • Healthy adult volunteers (Willing to give
written informed consent)
CVS EXPERIMENT: 1 • Stethoscope, Mercury sphygmomanometer,
Facilities for Clinical Pharmacology Labora-
Aim tory (Appendix IV).
To measure blood pressure in healthy volunteers. Initial Screening of Volunteers
Background Volunteer is screened for detailed evaluation of
any disease conditions.
Blood pressure is the force exerted by the blood Detailed history should be taken in respect to
against a unit area of the vessel wall. For smoking, alcohol consumption, drug therapy, or
example, if pressure is 100 mm Hg, this means any associated disease conditions.
that the force, exerted is sufficient to push a
column of mercury up to a level of 100 mm Precautions during Experiment
height. Occasionally, pressure is measured in • Subject should be advised for adequate sleep
cm of water. One mm of Hg equals 1.36 cm of over night and light breakfast in the morning
water because the specific gravity of mercury is on the day of experiment (preferably 2 hr
13.6. The blood pressure in the large arteries before)
(Aorta, brachial and others) in the young adult • Avoid caffeinated drink or smoking at least
2 hr before the experiment
rises to a peak value (systolic pressure) of about
• Take 2 or 3 readings, preferably take 2 readings
120 mm of Hg, during each cycle and falls to a
at the time of measurement
maximum value (diastolic) of about 70 mm of
• Volunteer should have a comfortable room,
Hg. The pulse pressure (PP), the difference
sitting in a chair or lying quietly for at least
between SBP and DBP is normally about 40-50 5-10 min, with the arm unconstricted.
mm Hg. The mean arterial pressure is the average • Avoid (or if unavoidable, make a note of it)
pressure throughout the cardiac cycle and is extraneous factors, which may alter the BP.
calculated by For example: Recent smoking/eating, anxiety,
talking, exercise/exertion, cold medication
1 1
DBP + PP or (SBP + 2DBP) like, estrogen, corticosteroid, adernergics such
3 3 as nasal, drops, pentazocine, pupillary
The pressure in vessel below heart level is dilators, phenylephrine, etc.
increased (2 mm Hg at every inch) and that in • Note the time of the day (preferably blood
pressure should be taken at the same time of
vessel above heart is decreased (2 mm Hg at every
the day)
inch) by the effect of gravity. The magnitude of the
• Width of cuff should cover at least 40% of
gravitational effect, the product of the density of
upper arm
blood, the acceleration due to gravity (980 cm/
• Inflatable balloon should cover 80% of the arm
sec2) and the vertical distance above and below circumference
the heart is 0.77 mm Hg/cm at the density of • Place the cuff 1.5-2 cm above the antecubital
normal blood. The pressure in large artery in foot fossa
(105 cm below the heart is 180 mm of Hg (100 + • Record the exact blood pressure, do not round
{0.77 × 105}). off
Source of potential errors (Korotkoff V). However, in adults after exercise

and in children, the diastolic blood pressure
1. Improper equipment
a. Cuff too small/large for length of the arm correlates best with the pressure at which the
b. Inflatable balloon small for arm circumference sound becomes muffled (Korotkoff IV). The
c. Manometer is inaccurate sound of Korotkoff is produced by turbulent
2. Inaccurate reading flow in the brachial artery.
a. Improper placement of cuff
b. Error in value, missing an auscultatory gap or Note: It is always preferred practice to perform
confusion over muffling/disappearance of sound Palpatory method before Auscultatory method. It
c. Variation due to arrhythmia gives accurate blood pressure and heart rate
d. Position of arm not at level of heart
e. Arm held without support
monitoring and reduce variability.
Observer bias may occur with single reading.
Method of Measuring Blood Pressure
1. Palpatory method: The systolic blood pressure
SBP DBP HR
can be determined by inflating an arm cuff and
letting the pressure fall and determining the 1st 2nd 3rd Mean 1st 2nd 3rd Mean
pressure at which the radial pulse first become
palpable. Pressure obtained by this method is
usually 2.5 mm Hg lower than those measured
by the auscultatory method. The importance
of this method is in avoiding the error in
measurement due to auscultatory gap seen in
some subjects.
2. Auscultatory method: Routinely used method
for blood pressure measurement. An inflatable Discussion
cuff (Riva-Rocci cuff) attached to a mercury The measurement of blood pressure is the
manometer (sphygmomanometer) is wrapped important clinical parameter which provides
around the arm and the stethoscope is placed
important information about cardiovascular
over the brachial artery at the elbow (cubital
system under normal and disease conditions.
fossa). The cuff is rapidly inflated until the
Though, there are several instruments available
pressure in it is well above the systolic pressure
but mercury sphygmomanometer is considered
in the brachial artery. The artery is occluded
as ideal one and palpatory method should be
by the cuff pressure and no sound is heard
practiced always before auscultatory mea-
with the stethoscope. The pressure in the cuff is
surement. Blood pressure should be measured in
then released slowly (2-3 mm Hg/sec). The point
both the arms and arm with highest reading
at which systolic pressure in the artery just
should be considered for recording. The normal
exceeds the cuff pressure, a spurt of blood
ratio of SBP, DBP and PP is 3:2:1. Blood pressure
passes through with each heart beat and
synchronically with each beat a tapping sound is usually lower in females by 8-10 mmHg, over
is heard below the cuff. The cuff pressure at weight person tend to have increased blood
which sounds are just heard is the SBP. As the pressure.
cuff pressure is lowered further, the sound
becomes louder dull and muffled, and finally SUGGESTED READING
in most individuals, it disappears. These are 1. De Swiet M, Dillon MJ, Little W, O’Brien E, Padfield
the sounds of Korotkoff. The diastolic blood PL, Petrie JC. Measurement of blood pressure in
pressure in resting adults correlates best with children. Recommendations of a working party of
the pressure at which the sounds disappear the British Hypertension Society. BMJ 1989;299:497.
2. Marshall T, Rouse A. Blood pressure measurement. Materials and Method

Doctors who cannot calibrate sphygmomanometers
should stop taking blood pressures. BMJ 2001; • Healthy adult volunteers (willing to give
323(7316):806. written informed consent)
3. O’Brien E. A century of confusion: Which bladder • Stethoscope, mercury sphygmomanometer,
for accurate blood pressure measurement? J Hum Facilities for Clinical Pharmacology Labo-
Hypertension 1996;10:565-72. ratory (Appendix IV).
4. O’Brien E. A century of confusion: which bladder for
accurate blood pressure measurement? J Hum
Hypertension 1996;10:565-72. Screening of Volunteers
5. Turner MJ, Baker AB, Kam PC. Effects of systematic Volunteers should be screened for detailed
errors in blood pressure measurements on the
evaluation of any disease conditions, detailed
diagnosis of hypertension. Blood Press Monit 2004;
9(5):249-53. history should be taken in respect to smoking,
alcohol consumption, drug therapy, or any
CVS EXPERIMENT: 2 associated disease condition.
Aim Precautions during Experiment

To evaluate chronobiology of blood pressure in Subject should be advised for adequate sleep
healthy volunteers. over night and light breakfast in the morning
on the day of experiment (preferably 2 hr before)
Background Any caffeinated drink or smoking at least 2 hr
before the experiment should be prohibited.
In any biological system, there is rhythm and
cycle of biological events. The blood pressure also Method
shows a circadian rhythm with release of Mercury sphygmomanometer is considered to be
different hormones in the body. Molecular the standard of all the other available methods.
studies suggest that genetic inheritance was
influenced from the environment during the Procedure
course of evolution. Hence, the blood pressure
The blood pressure is monitored in a healthy
and heart rate may vary with change in volunteer over a period of 24 hours at 2 hours
the internal or external environment of the interval. The volunteer should have a normal sleep
body. The chronodiagnosis of a patient over night and is allowed to do his daily routine
provides the accurate measurement of the activities. At the time of each recording the subject
HR and BP whereas one time measurement is rested in a supine position for 5 min, and then
may give the false blood pressure or heart rate readings are taken. Start the experiment at the
value. (Situations like, elder patients, stress, specified time (preferably at 8.00 am and then
anxiety, eating, exercise, etc.) record every 2 hr till 8.00 am, the next day).
Observations
Time (hr) Systolic blood pressure Diastolic blood pressure Heart rate (HR)
(SBP)(mm Hg) (DBP)(mm Hg)
0 (preferable
at 8:00 am)
2
4
6
8
10
12
14
16
18
20
22
24
Observe the time at which blood pressure long-time have become available, the marked
shows highest and lowest recordings of SBP, DBP individual variability of the blood pressure has
and monitor the heart rate. become apparent. The blood pressure taken with
Represent the result, as it given in Figure 26.7. an intra-arterial device continuously over 24 hours
in 20 untreated ambulant hypertensives showed
Statistical Analysis highest value in the mid morning, a progressive
fall during the day and much lower recording
The mean of the all 12 readings should be recorded
during sleep. The readings varied as much as by
and data should be expressed as mean + SD.
40 mm Hg throughout 24 hour. There is a marked
rise in the blood pressure upon awakening,
Discussion
although plasma noradrenaline levels are lower
The Blood pressure is low under basal conditions during sleep, but plasma cortisol and aldosterone
and it achieves peak in the late afternoon, mostly levels rise during the early morning. The rise in
systolic blood pressure and it is significantly low systolic and diastolic blood pressure immediately
while sleeping. As more frequent readings over upon awakening may be as high as 20 mm Hg. A
further rise occurs upon arising and ambulation.
During period of REM sleep the blood pressure
tends to rise a bit and becomes variable.
SUGGESTED READING
1. Kawasaki T, Cugini P, Di Palma L. Chronobiology
approach to human hypertension. Ann Ist Super
Sanita 1993;29(4):679-92.
2. Cugini P, Kavasaki T, Palma LD, et al. Blood pressure
monitoring and chronobiometry: New reference
standard and definitions concerning norm tension
and hypertension. J Health Sci 1989;11:145-62.
3. Cugini P, Kavasaki T, Palma LD, et al. Innovative
Fig. 26.7: Chronobiology of volunteer blood pressure criteria for diagnosing arterial hypertension via blood
pressure monitoring: The chronodiagnosis. J Health cuff position is at the level of the right atrium in
Sci 1991;13:23-34. both positions, the systolic pressure has been
4. Kudzhini P, Kavasaki T, Palma LD, Vatisti P, reported to be 8 mm Hg higher in the supine than
Antonikoli S, Leone D, Uezono K, Sto-onev AG. the upright position. Other posture like crossing
Circadian rhythm of arterial pressure: Chrono-
of leg may raise systolic pressure. The position of
biological criteria for normotension and hypertension.
the arm in respect to the heart level may also vary
Fiziol Cheloveka 1991;17(4):73-79.
5. Cugini P, Lucia P, Di Palma L, Pozzilli P, Re M,
the BP measurements. There is a progressive
Canova R, Gasbarrone L, Cianetti A. Vasoactive increase in the pressure of about 5 mm Hg, when
intestinal peptide: A chronoimmunomodulator? arm position is higher than the heart position,
Biochem Med Metab Biol 1991;46(2):274-76. whereas about 5 mm Hg may fall when the arm
6. Cugini P, Di Palma L, Battisti P, Coppola A, Leone position is below the heart level.
G, Cresci G, Bruscagli G, Lembo T, Angeloni A,
Pelosio A. 24-hour blood pressure: Noninvasive Body Posture (Also see Figs 26.3 to 26.5)
monitoring and biometric analysis in relation to age.
Recenti Prog Med 1991;82(9):463-74. Supine position
7. Cugini P, Cresci G, Di Palma L, Bruscagli G, Battisti Sitting position
P, Lembo T, Angeloni A, Coppola A, Leone G, Pelosio
Other various positions:
A. Recenti Prog Med 1991;82(9):452-62.
8. Cugini P, Lucia P, Di Palma L, Re M, Leone G, Battisti
• Sitting with crossed leg
P, Canova R, Gasbarrone L, Cianetti A. Vasoactive • Standing with hand position below the heart
intestinal peptide fluctuates in human blood with a level.
circadian rhythm. Regul Pept 1991;34(3):141-48. • Standing with hand position at the heart level.
9. Cugini P, Lucia P, Di Palma L, Pozzilli P, Re M, • Standing with hand position above the heart
Canova R, Gasbarrone L, Cianetti A. Temporal level.
interrelationships between circadian rhythms of
vasoactive intestinal peptide and T lymphocyte Materials and Method
subpopulations. J Clin Lab Immunol 1991;34(2):
49-54. • Healthy adult volunteers (willing to give
10. Kawasaki T, Cugini P, Uezono K, Sasaki H, Itoh K, written informed consent)
Nishiura M, Shinkawa K. Circadian variations of • Stethoscope, Mercury sphygmomanometer,
total renin, active renin, plasma renin activity and Facilities for Clinical Pharmacology Labo-
plasma aldosterone in clinically healthy young ratory (Appendix IV).
subjects. Horm Metab Res 1990;22(12):636-39.
Initial Screening of Volunteers
CVS EXPERIMENT: 3
Subject is screened for detailed evaluation of any
Aim disease conditions, detailed history should be
To evaluate the effect of body posture and arm taken in respect to smoking, alcohol con-
position on arterial blood pressure and heart rate. sumption, drug therapy, or any associated
disease conditions.
Background
Precautions during Experiment
Several studies suggest that the change in the body
posture can alter blood pressure to rise or fall. Subject should be advised for adequate sleep over
Ideally, recording of the blood pressure is done in night and light breakfast in the morning day of
supine or in normal relaxed sitting position. experiments (preferably 2 hr before the experiment).
However, diastolic pressure varies about 5 mm Any caffeinated drink, alcohol, cola drinks or
Hg in sitting than the supine position whereas smoking should be avoided at least 2 hr before the
systolic pressure remains same. When the arm experiment.
Procedure twice or thrice and the average should be taken

for accuracy.
Volunteers with history of alcohol consumption
After 15 min of rest in supine position, BP
on previous day, smoking, drug addiction should
should be recorded. Then, volunteers are asked to
be excluded. Those taking any drug especially
stand up immediately and BP at 1 and 3 min are
affecting blood pressure are excluded. Preferred recorded for assessment of postural hypotension.
age is 18-35 years. About 2 hr after a light breakfast The volunteer is then asked to stand up for 5 min
at morning, volunteer are advised to report to the with hand up and BP is recorded. It is again
clinical pharmacology laboratory. Volunteer is measured with hand by the side and hand
asked to lie comfortably for 15 min, after applying hanging after 5 min each. Finally, volunteer is
the blood pressure cuff, BP should be recorded on asked to sit down and BP is recorded after 5 min.
both upper arms and the arm which shows higher If, there is fall in BP, it gives the evidence of postural
blood pressure is selected for subsequent hypotension, and if present, volunteer should be
measurement. Blood pressure should be recorded excluded.

Body posture SBP DBP HR
1st 2nd 3rd Mean 1st 2nd 3rd Mean
Supine
Normal sitting
Sitting with cross leg
Standing with hand position
below the level of heart
at the level of heart
above the level of heart
Discussion the side, pooling occurs in the upper limb still

further. Hence, these situation show increase in
BP and HR usually increase when the volunteer
blood pressure and heart rate. When volunteer
is standing by keeping hands at the heart level.
stands up with arm raised, there is better venous
With hands upward BP is low but HR high. In the
return, no pooling of blood in the upper limb and
sitting position, both BP and HR give intermediate
thus less (lower) sympathetic activity manifesting
results. When the volunteers stand up, there is
a lower blood pressure and heart rate.
pooling of blood in the dependant position, thus
there is decreased venous return. This leads to
SUGGESTED READING
lesser stretching of baroreceptors, sympathetic
activity increases and there is increase in blood 1. Brar KS, Ramesh. Technique of blood pressure
pressure and heart rate due to withdrawal of measurement. MJAFI 2003;59:51-52.
parasympathetic baroreceptor activity. 2. Pickering TG, Hall JE, Appel LJ, Falkner BE, Graves
J, Hill MN, Jones DW, Kurtz T, Sheps SG, Roccella
Baroreceptor is located at the carotid sinus and
EJ. Recommendations for blood pressure mea-
aortic arch. When the volunteer stands up, from surement in humans and experimental animals: Part
sitting position pooling of blood occurs in the 1: blood pressure measurement in humans: A
lower limb. While holding the arm hanging by statement for professionals from the Subcommittee
of Professional and Public Education of the American healthy volunteers (aged 21-50 years) but in the
Heart Association Council on High Blood Pressure clinical setting selection criteria is important and
Research. Hypertension 2005;45(1):142-61. limiting factor which depends on the several
factors include cognitive status, age, weight,
CVS EXPERIMENT: 4 mobility, nutritional status, or use of walking aids.
Aim
To evaluate the effect of propranolol on blood
pressure, heart rate and cardiac workload Initial Screening of Volunteers
following different submaximal exercises (Tread Volunteers are screened for any cardiovascular,
mill test [TMT], Master’s 2 step test, Bicycle ergo respiratory disorder or any conditions which may
meter and Hand Dynamometer) in healthy contraindicate or interfere with exercise protocol.
volunteers. Vertigo is one of the important criteria to screen
the volunteers. Volunteer should be screened for
Background postural hypotension also.
It depends on the validity, reliability, and • Healthy adult volunteers (Willing to give
sensitivity of the instrument and on the objectives written informed consent)
of submaximal exercise, i.e. to induce a state of the • Stethoscope, mercury sphygmomanometer
stress through exercise at which there is no Experiment: 4 A: (Tread mill exercise),
alteration of the biological and physiological Experiment: 4 B: Master’s 2 steps,
parameters. Clinically submaximal exercise Experiment: 4 C: Bicycle ergometer and
testing appears to have greater applicability to Experiment: 4 D: Hand-dynamometer
identify the various diseases like arrhythmia, Drug: β-blocker ( Propranolol: 40 mg PO)
angina, asthma, etc. There are two main categories
of submaximal tests, one is predictive, which Precautions during Experiment
mainly identifies maximal aerobic capacity and
second is performance test which is done to identify • Subject should be advised for adequate sleep
physical capability of volunteers or patients. Sub- over night and light breakfast in the morning
maximal exercise tests can be used to predict on the day of experiments (preferably 2 hr
VO2max, to make diagnosis and assess functional before)
limitations, to assess the outcome of interventions • Avoid caffeinated drink or smoking, at least
such as exercise programs whereas maximal 2 hr before the experiment or any abuse sub-
exercise tests are mainly used to determine VO2max stance
which is used as diagnostic or treatment outcome • Volunteer should get complete instructions of
tools. At the laboratory basis, there are several the procedure and its rehearsal, so as to avoid
instruments through which experimenter can any conditional anxiety of volunteers
determine the BP, HR, RPP and RR of a healthy
• Monitoring equipment should be calibrated
volunteer. Those are treadmill test, Master’s 2 test,
regularly
Bicycle ergometer and hand dynamometer. These
instruments were developed to meet the needs of • Oxygen source and suction device should be
people with various functional limitations, accessible
disabilities and requirements of older, obese • Basic operating procedure of the selected
population. Wrong selection of protocol may instrument should be well explained to the
under stress or over stress the subject. In the volunteers
practical clinical pharmacology, the selection is not • Direction of movement on the master’s 2 step
so important because of the participation of should be clear to the volunteer
• Testing procedure and its consequences

should be discussed beforehand.
Procedure
These studies are performed after taking baseline
values for HR, SBP, DBP, RPP and RR at supine,
sitting and standing, and then subject is asked to
perform submaximal exercise protocol. After
normalization of all the baseline parameters one
tablet of propranolol (40 mg) is given with plain
water to the volunteers. Thereafter, the same
exercise protocol should be repeated following
2 hr post drug administration. The HR, SBP, DBP,
RPP and RR are recorded as described below and
volunteer asked to report any adverse effect.
Meanwhile, exertion, dyspnea, fatigue, weakness,
and pain during exercise should be observed. Fig. 26.8: Treadmill test (TMT)
EXPERIMENT NO: 4(A) grade (16%)-time (3 min); Stage 5: speed 5.0(mph)-

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

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

Values are as mean of 5 volunteers
HR BP RPP RR
Supine
Sitting
Standing
EXPERIMENT: 4(C)
Bicycle Ergometer (Fig. 26.10)
Bicycle Ergometer test is one of the most frequently
used submaximal ergometer tests. This is are
mainly performed to assess the power or work
done by the muscle. This test is preferred due to
easily recordable HR and BP whereas limitations
of the test include the error margin in the predicted
VO2max values and discomfort at the lower part of
some volunteers.
Method
Precondition the volunteer regarding test
procedure, by instructing him/her to warm-up, Fig. 26.10: Bicycle ergometer
and familiarizing with the equipment. Written functional aerobic impairment in cardiovascular
informed consent should be obtained before the disease. Am Heart J 1973; 85: 546 –562.)
experiment. Thereafter, select appropriate At the end of all stages of submaximal exercise
protocol for the submaximal exercise. Volunteer BP, HR and RR are recorded at every 2 min until
is asked to relax for 10 min and then his/her recovery of baseline recordings. From the above
baseline BP, HR and RR are noted. The volunteer values “Rate Pressure Product” is calculated, for
is then subjected to submaximal exercise as the the entire period of the study.
“Bruce protocol”.
Stage 1= 1kg x 150 meters x 2 min RPP= (HR x SBP)/100
Stage 2= 2kg x 150 meters x 2 min
All the above mentioned parameters are
recorded 2 hr after propranolol (40 mg) intake. If
volunteer complain of discomfort, exercise is
(Reference: Bruce RA, Kusumi F, Hosmer D. Maximal stopped quickly and volunteer is asked to relax in
oxygen intake and nomographic assessment of sitting or supine position.

HR BP RPP RR
Discussion a predicted value. Exercise test can also be used to

Myocardial oxygen consumption is determined by advance patients safely to a higher level of
HR, SBP, left ventricular diastolic volume and left performance; also the improvement in exercise
ventricular wall thickness. The RPP increases with capacity demonstrated by an exercise test can be
increasing walk/exercise and is a reliable indicator effective incentive and can encourage risk factor
of myocardial performance. β-blockers decrease the modification.
RPP to allow optimal utilization of oxygen in states
of ischemia. Benzer, “2001”, reported that exercise EXPERIMENT: 4(D)
test is the primary mean used to evaluate the safety Hand Dynamometer (Fig. 26.11)
of participating in an exercise program and to
formulate the exercise prescription. Because of, the Isometric exercise or static exercise generates force
wide scatter of maximal heart rate when plotted with negligible muscle shortening. It also produces
against age, it is much better to determine person’s alterations in cardiovascular parameters. Pressor
actual maximal heart rate by testing, by assigning response is more with isometric exercise, it may
to a target heart rate for training rather than giving be hand grip exercise or weight lifting. β-blockers
middle of four fingers. The subject presses the

dynamometer with maximum isometric effort.
Duration of effort is noted.
Note: Body part should be straight and should
not move forward.
Method
Volunteer is made to sit for 15 min BP, HR and
RPP are recorded until stable values are obtained.
It is recorded on the non-dominant arm. The
subject is asked to perform maximum exercise
Fig. 26.11: Hand dynamometer and technique to hold it using hand dynamometer. The maximum value
is noted down. The subject is then asked to grip
and performs exercise at 25% of maximum
cause decrease in CV response to changes induced
voluntary effort for 90 sec the cuff is inflated at
by the exercise. Volunteer holds dynamometer in 70 sec and BP and HR at 90 sec is noted down,
the dominating hand with the arm at right angles while volunteer is still performing exercise with
and the elbow by the side of the body. The base of the other hand. After stopping exercise BP, HR
the dynamometer should rest on first metacarpal and RPP are recorded for 10 min or till readings
(heel of palm), while the handle should rest on reached back to baseline.

HR/min BP (SBP/DBP) RPP RR
Time(min) Before drug After drug Before drug After drug Before drug After drug Before drug After drug
0
1
2
3
4
5
6
7
10
12
15
17
Experimenter may look for the other con- rise in HR and SBP are slightly less as compared
sequences like, exertion, dyspnea, fatigue, to without drug post exercise values. The
weakness, and pain or stress while conducting recovery of HR and SBP are quicker after drug
test performance. administration. The rise in SBP after exercise is
attenuated by propranolol. There is little effect
Discussion on DBP. The rate pressure product (RPP) is
calculated as:
The baseline heart rate is lower after adminis-
tration of propranolol 40 mg. The post exercise RPP= (HR × SBP)/100
There is large increase in RPP after exercise in in ulnar and median nerve injury: Comparing manual
both the pre drug and post drug session but the muscle strength testing, grip and pinch strength
increase in RPP is less in post drug session after dynamometers and a new intrinsic muscle strength
dynamometer. J Rehabil Med 2004;36(6):273-78.
exercise, also the recovery is quicker. The
10. Wisen AGM, Wohlfart B. A comparison between two
reduction of RPP signifies that propranolol exercise tests on cycle: A computerized test versus
reduces the cardiac work load and O2 demand for the Astrand test. Clin Physiol 1995;15:91-102.
the similar amount of exercise and thereby 11. Wyndham CH. Submaximal tests for estimating
improves exercise tolerance. Volunteers may maximum oxygen intake. Can Med Assoc J 1967;
report giddiness, sedation and heaviness at 30 96:736-45.
min of drug administration. Reduction in sub- 12. Zeballos RJ, Weisman IM. Behind the scenes of
cardiopulmonary exercise testing. Clin Chest Med
maximal exercise responses such as HR, RR, and
1994;15:193-213.
BP can be consistent with improved aerobic
conditioning and in clinical setting interpretation CVS EXPERIMENT: 5
based primarily on the type of test conducted, e.g.
assessment, diagnostic, exercise prescription for Aim
specified outcomes. To evaluate the effect propranolol on mental stress
induced rise in blood pressure and heart rate in
SUGGESTED READING
healthy volunteer.
1. Bruce RA, Kusumi F, Hosmer D. Maximal oxygen
intake and nomographic assessment of functional Background
aerobic impairment in cardiovascular disease. Am
Heart J 1973;85:546-62. Mental stress leads to marginal rise in systolic
2. Bruce RA, Kusumi F, Hosmer D. Maximal oxygen blood pressure in both normal volunteers and
intake and nomographic assess-ment of functional patients with hypertension. This is a result of
aerobic impairment in cardio-vascular disease. Am activation of central sympathetic pathways
Heart J 1973;85:546-62). leading to an increase in peripheral vascular
3. Bruce RA. Exercise testing of patients with coronary
resistance. Earlier studies reported the efficacy of
heart disease: Principles and normal standards for
evaluation. Ann Clin Res 1971;3:323-32. β-adrenoceptor antagonism on the effects of
4. George JD, Vehrs PR, Allsen PE, Fellingham GW, experimental stress in healthy volunteers; it
Fisher AG. Development of a submaximal treadmill showed single oral dose of propranolol (40 mg)
jogging test for fit college-aged individuals. Med Sci can reduce the stress-induced increase in heart
Sports Exerc 1993;25(5):643-47. rate and systolic blood pressure significantly
5. Hartung GH, Krock LP, Crandall CG, Bisson RU, compared to placebo. However minimal effect is
Myhre LG. Prediction of maximal oxygen uptake
documented in diastolic blood pressure following
from submaximal exercise testing in aerobically fit
and nonfit men. Aviat Space Environ Med 1993; β-adrenoceptor blockade like propranolol.
64(8):735-40.
6. Legge BJ, Banister EW. The Astrand-Ryhming
nomogram revisited. J Appl Physiol 1986;61: • Healthy adult volunteers (willing to give
1203-09. written informed consent)
7. Marciniuk DD, Gallagher CG. Clinical exercise testing • Stethoscope, mercury sphygmomanometer,
in interstitial lung disease. Clin Chest Med 1994;15:
Facilities for Clinical Pharmacology Labo-
287-303.
ratory (Appendix IV).
8. Patterson JA, Naughton J, Pietras RJ, Gunnar RM.
Treadmill exercise in assessment of patients with Initial Screening of Volunteers
cardiac disease. Am J Cardiol 1972;30:757-62.
9. Schreuders TA, Roebroeck ME, Jaquet JB, Hovius Volunteer is screened for detailed evaluation of
SE, Stam HJ. Long-term outcome of muscle strength any disease conditions, detailed history should
be taken in respect to smoking, alcohol con- 8. Inflatable balloon should cover 80% of the
sumption, drug therapy, or any associated disease arm circumference
conditions. 9. Place the cuff 2-3 cm above the antecubital
fossa
Precautions for Experiment 10. Read exact pressure, do not round off.
1. Volunteer should be advised for adequate
sleep over night and light breakfast in the Methods
morning day of experiments (preferably
2 hr before) Healthy volunteers are included in the experiment
2. Avoid any caffeinated drink or smoking at following adequate rest and baseline BP and HR
least 2 hr before the experiment readings should be noted every 2 minutes till an
3. Take 2 or 3 readings in every measurement average of 3 recordings are made. The volunteers
4. Volunteer should be sitting on a chair or are administered a mental task for 3 min. The
lying quietly for at least 5-10 min, with the volunteers are asked to continuously go on
relaxed arm subtracting a digit from a 3 digit number in a
5. Avoid (or if unavoidable, make a note of it) recurring fashion and to speak out the subtracted
extraneous factor, which may alter the BP, number, that is noted by the observer. At the end
e.g.: Recent smoking/eating, anxiety, talking, of 3 min, the BP and HR values are noted and no.
exercise/exertion, medication that can of correct and incorrect responses should be
interfere the result of the experiment. recorded. The rise in BP and HR following mental
6. Note the time of the day (preferably blood stress is observed and mean changes in arterial
pressure should be taken at the same time of BP are determined. Then one of the volunteers
the day) receives propranolol 40 mg and other one placebo
7. Width of cuff should cover at least 40% of in a double blind fashion and the same procedure
upper arm. is repeated at 120 min after drug administration.

BP HR Correct response
Pre drug Post drug Pre drug Post drug Pre drug Post drug
1st person
2nd person
3rd person
Discussion plasma half-life has been reported to be between

Propranolol is a non-selective β-adrenergic 10 and 12 hours. Mental stress results in increased
receptor blocker with no autonomic nervous systolic blood pressure and propanolol have been
system activity. It is a competitive antagonist shown to reduce the stress-induced increase in
which specifically competes with beta-adrenergic heart rate and systolic blood pressure to 49.9
receptor stimulating agents for available beta- percent and 8.3 percent respectively compared to
receptor sites. Peak plasma concentrations of 61.0% and 17.4% with placebo in a double blind
propranolol are attained 60 to 90 minutes randomized study in 12 healthy volunteers.
following oral administration and the apparent Several other studies showed the effects of oral
treatment with atenolol and propranolol on blood CVS EXPERIMENT: 6

pressure, heart rate and plasma cyclic adenosine
3':5'-monophosphate (cAMP) in young borderline Aim
hypertensives following psychological stress To evaluate the postural hypotension in the
testing. Studies showed there is a correlation 60-year old male volunteers.
between heart rate and plasma cAMP at rest and
also following psychological stress test β-blockers Background
significantly reduce all the parameters and plasma Postural hypotension is defined as the sudden
cAMP is mostly affected by propranolol. Another drop of blood pressure because of change in body
study using a standardized form of mental stress posture position. Normally this change is
in 21 patients with idiopathic hypertension was compensated by the body, but if the body’s
assessed at baseline and following treatment with response to a change in vertical position is slow
placebo, propranolol , or methyldopa. Studies have or absent, the result is orthostatic hypotension
shown satisfactory control of blood pressure with which indicates the low response of the body
both the drugs but propranolol treatment showed compensation mechanism. If the change in
modification of the pressor response to stress posture causes systolic BP fall below 20 mm Hg
testing ultimately provide more uniform control or diastolic 10 mm Hg then, this shows that the
of blood pressure in patients with essential person is suffering from the postural hypotension.
hypertension. Besides above mentioned effect, the It is clinically important to identify the postural
rise in temperature of the trunk skin is significantly hypotensive person, so that drugs causing the
reduced by propranolol. The self-rating of anxiety, postural hypotension such as vasodilator
alertness and concentration by the volunteers is (glyceryl trinitrate, phenoxybenzamine or phento-
usually unaffected by propranolol. lamine), diuretics (furosemide, ethacrynic acid,
torsemide), etc. should be avoided in the pres-
SUGGESTED READING cription.
1. Dunn FG, Lorimer AR, Lawrie TD. Objective Materials and Methods
measurement of performance during acute stress in • Healthy volunteer of about 60 years of age and
patients with essential hypertension: Assessment of willing to give written informed consent
the effects of propranolol and metoprolol. Clin Sci
• Stethoscope, mercury sphygmomanometer,
(Lond) 1979;57 Suppl 5:413s-15s.
Facilities for Clinical Pharmacology Labo-
2. Dunn FG, Melville DI, Jones JV, Lorimer AR, Lawrie
TD. Standardized stress and hypertension: Com-
ratory (Appendix IV).
parison of effect of propranolol and methyldopa. Br
J Clin Pharmacol 1978;5(3):223-26.
3. González-Gómez A, Garcia-Barreto D, Cabrera R, Volunteer is screened for detailed evaluation of any
Toruncha A, Hernández-Cañero A. Effects of Oral disease conditions, detailed history should be taken
Atenolol and Propranolol on Blood Pressure, Heart in respect to smoking, alcohol consumption, drug
Rate and Plasma Cyclic Adenosine 3':5'-Mono- therapy, or any associated disease conditioned.
phosphate in Borderline Hypertensive Patients.
Pharmacology 1982;25:33-38. Precaution
4. Pandhi P, Sharma PL. Comparative effects of beta,
• Volunteer should be advised for adequate
alpha- and combined beta- plus alpha-adrenoceptor
blocking agents in stress-induced increase in arterial
sleep over night and light breakfast in the
blood pressure. Int J Clin Pharmacol Ther Toxicol morning day of experiments (preferably 2 hr
1987;25(6):297-300. before)
5. Taylor EA , Harrison J, Turner P. Propranolol in • Avoid any caffeinated drink or smoking at
experimentally induced stress. The British Journal of least 2 hr before the experiment
Psychiatry 1981;139:545-49. • Take 2 or 3 readings in each recording
• Volunteer should be sitting on chair or ideally • Width of cuff should cover at least 40% of
in supine position quietly for at least 10-15 upper arm
min, with the arms relaxed • Inflatable balloon should cover 80% of the arm
• Avoid (or if unavoidable, make a note of it) circumference
extraneous factors, which may alter the BP, • Place the cuff 1.5- 2 cm above the antecubital
e.g.: Recent smoking/eating, anxiety, talking, fossa
exercise/exertion, avoid medications which • Read exact pressure, do not round off
can interfere with blood pressure.
• Note the time of the day (preferably blood Method
pressure should be taken at the same time of the For methodology please refer to CVS experiment
day). no. 2.

SBP DBP
1st 2nd 3rd Mean 1st 2nd 3rd Mean
Supine position
Standing Position
Parameters should be recorded associated with postural hypotension, but with

Detailed history should be taken regarding α-blocker like prazosin showed significant risk
volunteer experience with any dizziness, black- of first-dose postural effects, however with the
outs, syncope, etc. newer α1-blockers, for example doxazosin has
Statistical analysis: Data is expressed as mean ± more gradual onset of action.
SD.
SUGGESTED READING
Discussion
1. Christopher J Mathias. Postural hypotension: Causes,
Postural hypotension is associated with
Clinical Features, Investigation, and Management.
increased morbidity and also mortality in elderly Annual Review of Medicine 1999;50:317-36.
people. The postural hypotension can be diag-
2. Ismo Räihä, Sinikka Luutonen, Juhana Piha, Asko
nosed if the systolic blood pressure in decrease
Seppänen, Tuula Toikka, Leif Sourander. Prevalence,
by 20 mm Hg or diastolic blood pressure decrease Predisposing Factors, and Prognostic Importance of
10 mm Hg or more, with or without an increase Postural Hypotension. Arch Intern Med 1995;
in heart or pulse rate, for 3 minutes after standing 155(9):930-35.
from supine posture. The prevalence of postural 3. Peter A Meredith. Is Postural Hypotension a Real
hypotension in older adults is 5-30%, and it is Problem with Antihypertensive Medication?
reportedly higher among hospitalized patients Cardiology 2001;96:19-24.
(52-69%) and patients receiving long-term
hospital care facilities (50%). This may be CVS EXPERIMENT: 7(A)
because of autonomic insufficiency, in case of
Aims
prolonged illness, diabetic polyneuropathy and
patients treated with above mentioned To evaluate the effect of glyceryl trinitrate (GTN)
sympatholytic drugs. Studies have reported that on blood pressure, heart rate in healthy volun-
administration of a β-blocker is not usually teers.
Background other hemodynamic parameters should be carried

Glyceryl trinitrates (GTN), are drugs that release out.
nitric oxide (NO) and are widely used in the
Exclusion Criteria
treatment and prevention of angina. ‘NO’ activates
guanylyl cyclase by increasing cyclic GMP- 1. Known hypersensitivity to GTN and related
dephosphorylation of the myosin light chain and organic nitrate compounds.
the reduction of cystolic (Ca2+) and leads to the 2. Acute circulatory failure associated with
relaxation of smooth muscle cells and additionally marked hypotension (shock).
causes inhibition of platelet aggregation. GTN
3. Conditions associated with elevated intra-
mainly acts in veins then the arterioles. It also
cranial pressure.
increases in subendocardial blood flow which
4. Myocardial insufficiency due to obstruction
causes decreases oxygen consumption leads
(e.g. in the presence of aortic or mitral stenosis
ultimately favors subendocardial perfusion.
or of constrictive pericarditis).
Decreasing peripheral arteriolar resistance
reduces after load and thus myocardial work and 5. Concomitant use of GTN and phospho-
oxygen consumption. diesterase type 5 (PDE5) inhibitors such as
sildenafil is contraindicated, because PDE5
Materials and Methods inhibitors may amplify the vasodilatory effects
of GTN resulting in severe hypotension.
Volunteers are screened for any cardiovascular, Precautions
respiratory disorder or any conditions which may Patients should adequately and cautiously under
interfere with experimental protocol. Volunteers strict medical surveillance and/or hemodynamic
with smoking habit should be excluded. monitoring till completion of experiments and all
Healthy male adult volunteers (willing to give the supportive medical care should be in the
written informed consent) clinical pharmacology laboratory (Appendix IV).
Stethoscope, mercury sphygmomanometer,
Facilities for Clinical Pharmacology Laboratory Methods
(Appendix IV)
After light breakfast volunteers are asked to report
Drug: GTN tablet
to the clinical pharmacology laboratory in the
Inclusion Criteria morning. Initially volunteers are advised to lie
down for 15 min and baseline HR, BP and RR
Healthy volunteers between 18-45 years selected should be noted, the volunteers are given GTN as
for the study. mentioned above. The vital parameters are
No history of nitrate allergy, cardiovascular recorded at baseline, every 15 min till 2 hrs.
disease, psychiatric illness, intake of any drugs Volunteers should be observed for any adverse
especially that can affect BP and HR should be drug reaction like headache, giddiness, allergic
confirmed. Intake of alcohol, smoking, drug abuse, reaction till the completion of experiment. The
dehydration, postural hypotension should be volunteers should be told that in case of any severe
checked for exclusion. Normal ECG, LFT, RFT and headache; GTN tablet should be taken out.

Time (min) Systolic blood pressure Diastolic blood pressure Heart rate (HR) Respiratory rate
(SBP) (mm Hg) (DBP) (mm Hg)
Baseline (0)
15
30
45
60
75
90
105
120
Discussion active substance penetrates the skin and thus

becomes directly bioavailable to the systemic
Fall in SBP and DBP is observed with increase in
circulation at relatively constant concentrations
heart rate and no alteration of respiratory rate is
during the recommended application period. The
seen. GTN is a vasodilator which mainly dilates
nominal rate of nitroglycerin release in vivo is
the veins. Thus there is a systemic vascular
approximately 20-25 μg/cm2 h. It is commonly
resistance reduction, though very mild vasodila-
indicated for Angina Pectoris as monotherapy
tation lead to decreased end diastolic volume.
or in combination with other anti-anginal drugs
There is not much effect on arteries. Pulmonary
such as beta-blockers and/or calcium anta-
vascular resistance is decreased and cardiac
gonists. It is used in congestive heart failure as
output falls. Heart rate tends to rise, so there may
supplementary medication in patients not
be reflex tachycardia which can lead to para-
responding adequately to conventional therapy
doxical anginal pain in patients with ischemic
with digitalis or other positive-inotropic agents
heart disease.
and diuretics. It is also used in prevention of
phlebitis and extravagation secondary to venous
CVS EXPERIMENT: 7(B) cannulation for intravenous fluid and drug
Aim administration when treatment is expected to last
for two days or longer.
To evaluate the effect of glyceryl trinitrate (GTN)
transdermal patches on blood pressure, heart rate Materials and Methods
arterial vasodilatation in healthy volunteers.
Background Volunteers are screened for any cardiovascular,
Glyceryl trinitrate (GTN) transdermal patches respiratory disorder or any conditions which may
available as 1,2,3-Propanetriol trinitrate trans- interfere with experimental protocol. Volunteers
dermal therapeutic system with dose ranging with smoking habit should be excluded.
from 25 to 75 mg. Nitroglycerin patch is an Healthy male adult volunteers (willing to give
organic nitrate derivative which is designed in written informed consent)
flat multilayer system to deliver nitroglycerin Doppler flow meter (Fig. 26.12), Pulse oxymeter,
continuously through a release membrane Facilities for Clinical Pharmacology Laboratory
following application to the skin. In cases where (Appendix IV).
the permeability of the skin is excessive, drug Stethoscope, mercury sphygmomanometer
release is limited by the release membrane. The Drug: Nitroglycerin skin patches
Dosage and Use care should be in the clinical pharmacology

laboratory (Appendix IV).
Volunteers should be given lowest effective dose
(25 mg). The application site should be cleaned 2. The nitroglycerin skin patch contains an
properly and observed for any local irritation. aluminium layer. Therefore nitroglycerin skin
Nitroglycerin skin patch is sealed in a separate patches must be removed before applying
sachet with a tear-off edge to facilitate the magnetic or electrical fields to the body during
application. After removal of the white protective procedures such as MRI, cardiversion or DC
backing, apply the Nitroglycerin skin patches to defibrillation or diathermy treatment.
a clean, non-hairy, dry area of intact skin on the 3. Precautions should be taken carefully as the
trunk or upper arm. Hold the patch in position for possibility of increased frequency of angina
10-20 seconds with the palm of the hand. during patch-off periods in that case use of
concomitant anti-anginal therapy is desirable.
Inclusion Criteria 4. Tolerance to sublingual nitroglycerin: As
Healthy volunteers between 18-45 years are tolerance to nitroglycerin patches develop, the
selected for the study. effect of sublingual nitroglycerin on exercise
No history of nitrate allergy, cardiovascular tolerance may be partially diminished.
disease, psychiatric illness, intake of any drugs
especially that can effect BP and HR should be Methods
confirmed. Intake of alcohol, smoking, drug abuse,
After light breakfast volunteers are asked to
dehydration, postural hypotension should be
report to the clinical pharmacology laboratory
checked for exclusion. Normal ECG, LFT, RFT and
other haemodynamic parameters should be in the morning. Initially volunteers are advised
carried out. to lie down for 15 min and baseline heart rate,
blood pressure and respiratory rate should be be
Exclusion Criteria noted, the volunteers are given GTN as men-
tioned above. The vital parameters are recorded
1. Known hypersensitivity to nitroglycerin and at baseline, every 15 min till 2 hrs. Volunteers
related organic nitrate compounds.
should be observed for any adverse drug reaction
2. Acute circulatory failure associated with
like headache, giddiness, allergic reaction till the
marked hypotension (shock).
completion of experiment. The volunteers should
3. Conditions associated with elevated intra-
cranial pressure. be told that if in case of severe headache,
4. Myocardial insufficiency due to obstruction. Nitroglycerin skin patches should be removed.
5. Concomitant use of nitroglycerin skin patches
and phosphodiesterase type 5 (PDE5) inhi-
bitors such as sildenafil is contraindicated,
because PDE5 inhibitors may amplify the
vasodilatory effects of nitroglycerin skin
patches resulting in severe hypotension.
Precautions
1. Volunteers should be adequately and cau-
tiously under strict medical surveillance and/
or hemodynamic monitoring till completion
of experiments and all the supportive medical Fig. 26.12: Doppler flow meter
Ankle Brachial Index (ABI) ABI should be measure at baseline following

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

Time (min) (SBP) (DBP) (HR) RR ABI Oxygen saturation
ADR
Baseline After Baseline After Baseline After Baseline After Baseline After Baseline After
drug drug drug drug drug drug
SBP= Systolic blood pressure; DBP= Diastolic blood pressure; HR= Heart rate; RR= Respiratory rate; ABI= Ankle
brachial index; ADR= Adverse drug reaction
inhibitors, beta-blockers, diuretics, antihyper-

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

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

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

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

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

HR(beats/min) = 1500/no. of small square
between R-R wave
PR interval: Between beginning of P wave and Q
wave
Normal PR interval
Childhood = 0.1-0.12 sec
Adults = 0.12-0.25 sec
Elderly = 0.14-0.22 sec
QT interval: between onset of Q wave and end of
T wave. It varies with HR.
QTc (corrected QT interval) = QT/√RR
where RR = 60/HR
Fig. 26.16: Triaxial reference system (Polarity (+) and (–) Or QT + 1.75 X (vent.rate – 60).
Fig. 26.17: Cardiac contraction and ECG development

(Arrows indicate movements of electrical current within cardiac muscle)
Fridericia formula= QTc= QT/ (R-Rint)0.33 electrodes through 5000 resistance (important
to cancel out the potential from 3-points)
FDA formula= QT/ (R-Rint)0.37
The selection of the position for 6 unipolar
chest leads is based on the concept that the
Central Terminal of Wilson (CTW)
proximity of the heart to the anterior chest wall
The concept of electrocardiography is more than resulting in the unipolar chest lead functioning
100 years old. It was originally developed by as semi direct leads being influenced by the tissue
WILLEM EINTHOVEN for which he was immediately beneath the electrode. The 6 standard
honored with “Nobel Prize” chest leads (V1 to V6) are recorded by positioning
CTW is the indifferent electrode, and the the exploring chest electrodes.
exploring electrode is the active electrode. Unipolar limb leads may be recorded by a
system in which CTW constitutes the indifferent
Salient Features of CTW electrode and the exploring is on the three active
limb electrodes. These leads are referred to as VR,
• Homogeneity of the body volume conductor VC, and VL. By disconnecting the input to CTW
• Asymmetry of the leads from the extremity being explored an augmen-
• Single equivalent dipole at the center of the tation of the voltage by 50% occurs. This modi-
volume conductor. fication is used inversely for clinical ECG and the
• Constructed by connecting RA, LA, and LL leads are labeled as aVR, aVL and aVF.
Figs 26.18A and B: (A) Central terminal of Wilson (CTW) and chest (C) electrode,
(B) Horizontal plane orientation of unipolar chest lead
Fig. 26.19: Augmented unipolar limb leads (aVR, aVL, aVF)

Position of Chest Electrodes
Fig. 26.20: Position of chest electrodes

Practical Exercise for Cardiovascular System

Practical Exercise 1: A 63-year-old woman with 10 hours of chest pain and sweating, ECG of patient
showed changes in ST, leads V1 - 6, I and aVL, write the diagnosis from ECG and discuss the drug therapy.
Fig. 26.21
Practical Exercise 2: A 55-year-old man with 4 hours of “crushing” chest pain, ECG of patient showed
changes in ST , leads II,III, aVF and in anterior lead , write the diagnosis from ECG and discuss the drug therapy.
Fig. 26.22
Practical Exercise 3: A 76-year-old man complains of breathlessness, ECG showed irregular ventricular
rhythm, sometimes on first look the rhythm may appear regular but on closer inspection it is clearly irregular ,
write the diagnosis from ECG and discuss the drug therapy.
Fig. 26.23
Practical Exercise 4: A 45-year-old lady complains of palpitations and history of chronic renal failure, ECG
showed a wide QRS tachycardia, write the diagnosis from ECG and discuss the drug therapy.
Fig. 26.24
Practical Exercise 5: A male patient of 60 years, known hypertensive with recurrent episode of palpitation
since childhood, with increasing frequency and duration since last four years, ECG shows a regular rhythm of
150 beats per minute or higher, with a narrow QRS complex, write the diagnosis from ECG and discuss the drug
therapy.
Fig. 26.25
Respiratory System
27
RESP. EXPERIMENT: 9 asthma recommend modification of treatment
when PEFR or FEV, drop by 15% or more over
Aim baseline suggesting that PEFR monitoring is as
To compare the effect of salbutamol with placebo reliable as FEV1. Periodic determination of
on peak expiratory flow rate (PEFR) in healthy PEFR in asthmatics helps to find out the
volunteers. severity. Long term daily monitoring is helpful
in the management of such patients for early
Background detection of change in airway obstruction that
meets changes in treatment, for evaluation of
Presently, there is widespread use of peak flow
responses to change the treatment and for
meters for the screening and follow-up of
recognition of air way obstruction.
patients with reversible airway obstruction
which has been advocated since the early 1970s Materials and Method
as a reliable method for evaluating airway
caliber. Peak Expiratory Flow Rate (PEFR) is a
simple bedside test of lung function, and it is Volunteers are screened for any cardiovascular,
most commonly used by the patients with respiratory disorder or any condition which may
asthma. It is popular because of quick and interfere with experimental protocol. Volunteers
simple bedside measurements. Basically, it is a with smoking habit should be excluded.
small tube with a slider device to measure how Healthy adult volunteer of either sex (willing
fast air is driven through the tube by the to give written informed consent)
volunteer exhaling rapidly. Once subject takes Peak flow meter (PEFM) (Mini-Wright).
a deep breath in, and then breaths out as hard
Stethoscope, mercury sphygmomanometer,
and fast as possible, with a good airtight seal of
Facilities for Clinical Pharmacology Laboratory
the lips around the tube. This is best done
(Appendix IV)
standing up; the best of three attempts is usually
taken, with a short rest between attempts. Drug: Salbutamol, Matching placebo
Several reports have shown the strong corre- Inclusion criteria: Healthy volunteer aged 18-40
lation between FEV1 and PEFR. Studies of cross- years of either sex
sectional analyses reported that subjects with
asthma, have correlation coefficients ranging Exclusion criteria: Volunteer with history of
from 0.82 to 0.89. Besides this, PEFR monitoring smoking, alcohol, and any other disease condi-
is relatively simple and inexpensive. Most of tions and drug therapy which can interfere with
guidelines for assessment and treatment of experimental protocol
Respiratory System  281
Precautions for Experiment

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

Peak flow meter was introduced in 1959 and the
original Mini-Wright peak flow meter was
developed in the 1970s by Dr BM Wright, of the
Medical Research Council, and Clement Clarke Figs 27.1A and B: Peak flow meter
International, Harlow, England. Peak flow meter
is a simple device for quick measurement of lung
breakfast. Smoking and caffeine containing
function at bed sides or at home as mini version
beverages should be prohibited. Baseline BP, HR,
is available both for adult and children. Flow
RR and PEFR should be measured with the
meter has short cylinder which made of plastic
subject in the sitting position.
material with an indicator to move in a scale with
number usually express as liters/min. Instru-
ment has a handle in the mouth piece and Procedure for PEFR
opening for air exit from the instrument (Figs Volunteer is asked to hold the PEFM carefully
27.1A and B). so that fingers should not obstruct the scale, slot
and hole of apparatus. Then, volunteers are
Method
instructed to take a deep breath after putting
Volunteers should report to Clinical Pharma- peak flow meter between teeth and lips, then
cology Laboratory early morning with light blow rapidly; average of 3 flow meter readings
should be taken; same procedure should be Discussion: Salbutamol is a β2-agonist, bron-

repeated at 30 min, 1 hr, 2 hr, and 4 hr after chodilator used in the treatment of bronchial
ingestion of blinded drug or placebo. Detailed asthma. Such short acting agents are used in
history should be taken to record adverse drug acute exacerbation of asthma and exercise
reaction (ADR). Measurement of PEFR is done induced asthma. They increase airway caliber.
using variable orifice flow meter. Calibration of PEFR is used to classify patients into various
the meter is expressed in “L/min”. Volunteers levels such as mild intermittent, mild persistent,
are advised to take a deep inspiration and from moderate persistent and severe persistent using
this stage expire fully and vigorously into the a parameter called PEFR variability and calcu-
orifice. Values are similar in standing and sitting lated as
position and nose clip is not essential for this
measurement. PEFR variability =
There are several sources of error for example:
(Evening PEFR – Morning PEFR)
(1) inadequate subject performance, either failure 100
of full inspiration or inadequate expiratory effort (Evening PEFR + Morning PEFR)
(2) error in the instrument, (3) failure to hold 2
instrument upright. Above mentioned minor Using PEFM, clinical pharmacological status of
errors may alter the readings. Hence, it is agents effective in asthma can be done. A 15%
advisable to take either last three of the total 5 change in airway caliber has been proposed as the
readings or the highest obtained reading, one of criteria for modifying treatment. Peak flow
the above methods should be practiced. monitoring is not recommended for children under
12 years.
Normal Range of PEFR
PEFR (L/min) SUGGESTED READING
Adults 350-600
1. Alessandro Brunelli, Francesco Xiume´, Majed
Observations and Result Refai, Michele Salati, Rita Marasco, Valeria Sciarra,
and Armando Sabbatini. Evaluation of Expiratory
Following parameters should be recorded at Volume, Diffusion Capacity, and Exercise Tolerance
different time intervals following administration Following Major Lung Resection A Prospective
of salbutamol and placebo. Follow-up Analysis. CHEST 2007; 131(1):141-147
5. Denyse Gautrin, Luis Carlos D’Aquino, Gilles
Placebo 0 hr 0.5 hr 1 hr 2 hr 4 hr Gagnon, Jean-Luc Malo and André Cartier. Com-
SBP (mm Hg) parison Between Peak Expiratory Flow Rates
DBP (mm Hg) (PEFR) and FEV1 in the Monitoring of Asthmatic
HR (per min) Subjects at an Outpatient Clinic. Chest
RR (per min) 1994;10615:1419-26.
PEFR (L/min) 1. Hansen JE, X-G Sun, Wasserman K. Should forced
Salbutamol 0 hr 0.5 hr 1 hr 2 hr 4 hr expiratory volume in six seconds replace forced vital
SBP (mm Hg) capacity to detect airway obstruction? Eur Respir J
DBP (mm Hg) 2006;27:1244-50.
HR (per min) 4. Mahhumad Inatullaya et al. Peak expiratory flow
RR (per min) rate (PEFR); normal values for people of Multan.
PEFR (L/min) The Professionals 2000;7(4):1-4.
2. Vandevoorde J, Verbanck S, Schuermans D,
Statistical analysis: All the data is expressed as Broekaert L, Devroey D, Kartounian J and incken
mean ±SD and comparison is done by using W. Forced vital capacity and forced expiratory
paired ‘t’ test. (if same volunteer is used pre and volume in six seconds as predictors of reduced total
post drug treatment) lung capacity. Eur Respir J 2008;31:391-95.
RESP. EXPERIMENT: 10 • Basic operating procedure of the selected

Aim volunteers
To evaluate the effect of salbutamol inhalation • Testing procedure and its consequences
in pulmonary function test in healthy male should be discussed beforehand
volunteers. • Training of volunteers for correct use of
spirometer should be explained properly
Background (Volunteer is asked to take deep breath in, then
breath out with maximum force and as hard
Presently, there are large number of pulmonary and as fast as possible, with a good airtight
tests available, from simple to sophisticated one, seal of the lips around the tube, usually done
which provide useful information regarding lung standing up; the best of three attempts is
functions like ventilation, perfusion and diffu- usually taken, with a short rest between
sion of lungs. The PFT is used for different attempts).
reasons such as for diagnosis, to assess effective-
ness of treatment, to evaluate physical fitness Methods
before cardiovascular surgery and in uncommon
situations like for legal opinion for hazardous Volunteers are asked to report early morning before
occupation. breakfast. Smoking and caffeine containing
beverages are prohibited. Baseline BP, HR, RR and
Materials and Methods PFT should be measured. After baseline measure-
ment volunteer is given salbutamol inhalation and
Volunteers are screened for any cardiovascular, PFT and other evaluation should be repeated after
respiratory disorder or any other conditions 30 minutes.
which may interfere with experimental protocol.
Volunteers with smoking habit should be Measurement of Pulmonary Function Test
excluded. At baseline and after treatment following para-
Healthy adult volunteers of either sex (willing meters are recorded: Forced vital capacity (FVC),
to give written informed consent) Forced expiratory volume in half second (FEV0.5),
Computerized Spirometer Forced expiratory volume in one second (FEV1),
Stethoscope, mercury sphygmomanometer Forced expiratory volume in three second (FEV3),
Drug: Salbutamol inhaler Peak expiratory flow rate (PEFM), Mean force
expiratory flow during middle half of the
Precautions for Experiment FVC(FEF2-12), Mean forced expiratory flow rate
• Subject should be advised for adequate sleep between 0.2 and 1.2 liters of volume change
over night and light breakfast in the morning (FEFR25-75), Forced expiratory flow after 25% of
day of experiments (preferably 2 hr before) FVC has been expired (FEF 25%), Forced
• Avoid caffeinated drink or smoking or at least expiratory flow after 50% of FVC has been
2 hr before the experiment or free of any expired (FEF 50%), Forced expiratory flow after
substance abuse 75% of FVC has been expired (FEF 75%), Forced
• Volunteer should get complete instructions expiratory volume ratio, (FEV0.5/FVC), Forced
of the procedure and its rehearsal, so to avoid vital capacity ratio (FEV1/FVC), FEV3/FVC and
any conditional anxiety in the volunteers maximum voluntary ventilation (MVV). To
• Monitoring equipment should be maintained record above mentioned parameters instrument
and regularly calibrated. is calibrated and volunteer’s data entry is done.
• Oxygen source and suction device should be Before measuring, PFT procedure is explained
accessible. in detail to the volunteer and let him/her practice
once or twice. Nasal clip is applied and clean RR (per min)

mouthpiece is attached to the breathing tube then FVC
volunteer is asked to take maximum inspiration FEV0.5
after placing the mouthpiece appropriately FEV1
followed by maximum expiration for forced FEV3
PEFR
expiratory phase.
FEF 2-12
There are several sources of error e.g.: (1) FEF 25-75
inadequate subject performance, either failure of FEF 25%
full inspiration or inadequate expiratory effort FEF 50%
(2) error in the instrument, (3) failure to hold FEF 75%
mouth piece properly. Above mentioned minor FEV0.5/FVC
errors may alter the readings. Hence, it is FEV1/FVC
advisable to take either last three of the total 5 FEV3/FVC
MVV
readings or the highest obtained reading, one of
the above methods should be practiced. Statistical analysis: All the data should be
expressed as mean ± SD, comparison of baseline
Procedure for Inhalation value and following salbutamol inhalation is
The correct use of inhaler is very important and done by paired ‘t’ test (experiment on same
volunteers should be explained in detail about volunteer).
different steps of using inhaler. Inhaler has several
components like plastic body with mouth piece, Discussion
mouth piece cover and canister. Before using Lung function depends on various factors such
inhaler, it should be shaken well and then test fire as effect of posture on vital capacity, which is
shoule be done; means release one puff into air. higher in erect posture as compared to sitting and
The following steps should be followed: After lying posture. Since, more physical effort is
removing mouth piece first clean the mouth piece, applied in erect posture and in this posture
shake adequately, place the inhaler upside down diaphragm is decent it increase capacity of
in one or two finger on the top of the canister. Take thoracic cage with elimination of gravity. So,
the respiration gently by mouth. Inhaler mouth- blood flow to the lung is decreased because of
piece should be placed between teeth and lips
gravitational effect thus ultimately causing
without biting then ask volunteer to start breathing
increased vital capacity on standing. Studies
slowly from mouth, as the volunteer breath in
reported altered PFT in healthy volunteers with
canister should be pressed down to release the one
complete trismus on pulmonary function using
dose with continuously breath in deeply. After
objective and subjective parameters. In healthy
removing the inhaler, hold the breath for at least
volunteers, it was showed that the significant
10 sec or as long as it is comfortable and breathing
should be slow. changes in test data stimulated mild to moderate
pulmonary impairment, whereas patients with
Observations and Result an already impaired pulmonary function
showed a marked deterioration of their initial
Following parameters should be recorded respiratory condition. Besides this, other sophisti-
Parameters 0 hr 0.5 hr cated methods like completely noninvasive
Placebo Salbutamol Placebo Sal- oxygen-enhanced pulmonary function test have
butamol potential for clinical applications in the serial
SBP (mm Hg) diagnosis of lung diseases. Other study reported
DBP (mm Hg) that there is no difference on use of nasal clip on
HR (per min)
PFT.
SUGGESTED READING Healthy adult volunteers of either sex (willing

to give written informed consent)
1. Agarwal D, Gupta PP, Sood S. Evaluation of
Computerized spirometer
pulmonary function testing with and without nose
clip in healthy volunteers. Ind J Allergy Asthma
Stethoscope, mercury sphygmomanometer
Immunol 2004;18(2):83-86. Pulsoxymeter
2. Guleria R, Singh TR, Sinha S, Padhy AK, Gupta K,
Pande JN. Effect of inhalation of salbutamol, Precautions
beclomethasone dipropionate and ipratropium • Subject should be advised to have adequate
bromide on mucociliary clearance in some patients sleep over night and light breakfast in the
with chronic stable bronchial asthma. Indian J Med
morning day of experiments (preferably 2 hr
Res 117, April 2003;158-63.
3. Jakob PM, Wang T, Schultz G, Hebestreit H,
before)
Hebestreit A, Hahn D. Assessment of human • Caffeinated drink or smoking should be
pulmonary function using oxygen-enhanced T(1) avoided at least 2 hr before the experiment
imaging in patients with cystic fibrosis. Magn Reson or free of any substance abuse
Med 2004;51(5):1009-16. • Volunteer should get complete instruction of
4. Spiekerkoetter E, Fabel H, Hoeper MM. Effects of the procedure and its rehearsal, so to avoid
inhaled salbutamol in primary pulmonary hyper- any conditional anxiety in the volunteers
tension Eur Respir J 2002;20:524-28. • Monitoring equipment should be maintained
and regularly calibrated.
RESP. EXPERIMENT: 11 • Oxygen source and suction device should be
accessible.
Aim
• Basic operating procedure of the selected
To evaluate pulmonary function test following instrument should be well explained to the
Stair climbing exercise tolerance test. volunteers
Background should be discussed before hand
• Training of volunteers for correct use and
Altered pulmonary function and exercise detailed explanation of pulmonary function
capacity has been reported in various lung test for example there should be a good air
diseases like bronchial asthma, chronic obstruc- tight seal of the lips around the tube, usually
tive pulmonary disease or as a result of resection done standing up and stair climbing exercise
of lung. Aim of present experiment is to evaluate test.
the changes of pulmonary function and exercise
tolerance using stair climbing test. Stair climbing Stair Climbing Exercise Test
test is popular because it is simple, economical,
The stair climbing test is a symptom-limited
and can be performed in short period. This test
exercise. In this exercise, volunteers are usually
can be applied for multiple evaluations on a large
advised to climb 16 flights of stairs, each flight
number of volunteers or in patients in clinic, since
having 11 steps. Each step is 0.155 m in height.
it more closely resembles to normal daily activity
The volunteers climb at a pace of their own
as compared to cycle ergometry, and cycle
choice, the maximum number of steps and to stop
ergometry is more stressful than any other form
only on exhaustion, limiting dyspnea, leg fatigue,
of exercise test.
or chest pain. During the exercise, pulse rate and
capillary oxygen saturation should be monitored
using a portable pulse-oxymeter. For every
Volunteers are screened for any cardiovascular, volunteer, the number of steps climbed and the
respiratory disorder or any other conditions time taken to complete the test should be
which may interfere with experimental protocol. recorded to calculate different ergometric
Volunteers with smoking is excluded variables: work and VO2 peak.
Method VO 2 peak, mL/kg/min

Volunteers are asked to report early morning FVC
after light breakfast. Smoking and caffeine FVC 0.5
containing beverages are prohibited. Baseline BP, FVC1
HR, RR, oxygen saturation and PFT are measured FVC3
with the subject in the sitting position. Same PEFR
procedure is repeated following exercise. FEF 2-12
FEF 25-75
Measurement of Pulmonary Function Test FEF 25%
FEF 50%
Following parameters are recorded: Forced vital
capacity (FEV), FEV 1 , FEV 1 /FVC, etc. as FEF 75%
mentioned in experiment number 10. To record FEV 0.5/FVC
above mentioned parameters instrument should FEV 1/FVC
be calibrated and volunteer’s data entry should FEV3/FVC
be done. Before measuring the PFT volunteers MVV
should be informed in detail about the procedure
and volunteers should practice once or twice. Statistical Analysis
Nasal clip is applied and clean mouthpiece is Volunteers are evaluated by using pulmonary
attached to the breathing tube then volunteer is function testing (PFT) and stair climbing tests.
asked to take maximum inspiration after placing Pulmonary function test results are expressed
the mouthpiece appropriately followed by as percentage of predicted value according to
maximum expiration for force expiratory phase. age, sex, and height as per standard guide-
There are several sources of error for example: line and all the data should be expressed as
(1) inadequate subject performance, either failure mean ± SD.
of full inspiration or inadequate expiratory effort
(2) error in the instrument, (3) failure to hold Discussion
mouth piece properly. Above mentioned minor
errors may alter the readings. Hence, it is The objectives of the present experiment is to
advisable to take either last three of the total 5 evaluate the changes of pulmonary function
readings or the highest obtained reading, one of following exercise and to evaluate exercise
the above methods should be practiced. tolerance, the stair climbing test used as a form
of exercise testing. Several studies have already
Observations and Results shown a good correlation between stair clim-
bing and cycle ergometry test. It has been found
Following parameters should be recorded that higher values of VO2 peak measured during
Parameters Before exercise After exercise stair climbing. Although the accuracy of the cal-
(Baseline) culation of VO2 peak during the stair climbing
Age, yr test is not universally accepted, and a compa-
Body mass index, kg/m2 rison between the calculated VO2 peak at stair
SBP (mm Hg) climbing test and the VO2 peak measured at
DBP (mm Hg) cycle ergometry should be done. Stair climbing
HR (per min) is commonly used because it is simple, econo-
RR (per min) mical, brief and easier than cycle ergometry, so
in the clinic it can be widely used.
SUGGESTED READING Moxham, Michael I. Polkey. Effect of Severe Isolated

Unilateral and Bilateral Diaphragm Weakness on
1. Holden DA, Rice TW, Stelmach K, et al. Exercise Exercise Performance. Am J Respir and Cri Car Med
testing, 6-min walk, and stair climb in the evaluation 2002;165:1265-70.
of patients at high risk for pulmonary resection. 4. Pollock M, Roa J, Benditt J, et al. Estimation of
ventilator reserve by stair climbing: a study in
Chest 1992;102:1774-79.
patients with chronic airflow obstruction. Chest
2. Morice RC, Peters EJ, Ryan MB, et al. Exercise testing 1993; 104:1378-83.
in the evaluation of patients at high risk for lung 5. Swinburn CR, Wakefield JM, Jones PW. Performance,
resection. Chest 1990;98:54S. ventilation, and oxygen consumption in three different
3. Nicholas Hart, Annabel H Nickol, Derek Cramer, types of exercise tests in patients with chronic
Simon P. Ward, Frédéric Lofaso, Neil B. Pride, John obstructive lung disease. Thorax 1985;40:581-86.
EXERCISE
Excercise No. 1: 39-year-old male presenting with history of occasional breathlessness since 6 months, has been
referred for pulmonary function test (PFT).
Interpret the findings of PFT and discuss the drug therapy?
Name: SR
ID
Diagnosis:
Age: 39 Height: 176 cm Weight: 92 kg Sex: Male
Spirometry Predicted Observed Observed Percent

Value Pre Pred Post Pred Change
FVC L 4.18 3.34 79 3.9 93 16
FEV0.5 L 2.64 1.66 62 1.92 72 15
FEV1 L 3.45 2.29 66 2.6 75 13
FEV3 L 3.94 3.03 76 3.43 87 13
FEV1/FVC % 82 69 84 67 81 –2
FEV3/FVC % 95 91 95 88 92 –3
FEF25-75 L/S 3.62 1.49 41 1.5 41
PEFR L/S 7.82 6.81 87 7.1 90 4
FEF25 L/D 7.24 3.98 54 4.57 63 14
FEF50 L/S 4.32 1.96 45 2.00 46 2
FEF75 L/S 1.77 0.6 33 0.54 30 –10
FET Sec 6.82 7.27 6
FTVC L 3.55 3.39 95 3.83 107 12
PIFR L/S 5.21 4.84 92 4.76 91 –1
FIF50 L/S 4.21 4.65 10
Exercise No. 2: 50-year-old female presenting with history of respiratory difficulty since 6 months, has been
Name:
ID:
Diagnosis:
Age: 50 Height: 160 cm Weight: 63 kg Sex: F

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

Value Pre % Pred
Dlco corr 2326 12.62 54
Dlco Unc 23.26 12.76 54
Va obtps 5.5 2.33 42
DL/VA 5.14 5.41 105
Excercise No. 3: 49 years old male presenting with history of respiratory difficulty since 6 months, has been
Date:
Name:
Diagnosis:
Age: 49 Height: 170 cm Weight: 73 kg Sex: M
Physician: Consulting:

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

Central Nervous System  293
Methodology top etc.) with their index finger for 15 sec.

Then, total numbers of taps are noted.
Baseline psychomotor functions are assessed
5. Short term Memory: Volunteers are shown
prior to using the following tests. The drug is 10 unrelated objects or 10 words and left for
administered and after that the following tests 1min to memorize the objects. Then, 4hr later,
are repeated at same sequences, and then mean volunteers are given 1 min to recall and write
values before and after treatment are compared. down the objects as many as possible.
1. Card sorting: Volunteer should be given a pack
of cards and is asked to sort then into the
different groups of diamonds, hearts, spades
and clubs as quickly as possible. (fix the card
type among the four or all) Time, to do the task
is noted pre and post treatment.
2. Subjective Assessment: This assessment
done on the basis of VAS score. Volunteers
are assessed for their mood and well being 6. Six letter cancellation: Volunteer is presented
on a series of visual analogue scale (VAS) with a sheet containing randomized letters
with a range from 0-10 or 0-100. The arranged in columns/row. Target six letters
assessment is done for 6 different features. and asked to cancel targeted letters as much
Visual analogue scale (VAS: 0-100) as possible in 2 min in any selected direction
(horizontal/vertical/random) and number of
letters correctly cancelled, attempted and
errors in selection are recorded.
7. Digit symbol substitution test: Test is applied
1. Sleepy—————————————Awake to evaluate cognitive function, attention and
2. Tense—————————————Relaxed psychomotor speed. Volunteers are given a
code table, displaying the correspondence
3. Lethargic———————————Energetic
between pairs of digits (from 1 to 9) and
4. Mentally dull———————————Alert
symbols. They have to fill in blank squares
5. Inability to concentrate——————Ability
with the symbol that is paired to the digit
to concentrate
displayed above the square. The subjects have
6. Anxious————————————Casual to fill in as many squares as possible in 90
seconds.
3. Critical flicker fusion threshold: The CFF
8. Digit span: It is a measure of short term
apparatus consists of a flickering light source
memory in which volunteers have to
against a dark background. A dial is provided
memorize few digit spans from 1-9 and have
in front to adjust the number of flicker per
to recall in a span of time. The order of recall
second. The volunteer is asked to see the
the digit is important such as if the digit
flickering light for a min at 5 Hz. The dial is sequences is 1-4-2-5-3 or 5-2-6-2-3 or 9-2-5-1-
then slowly moved at the rate of 2 Hz/sec. 4. Then the recall is checked in the order of
The volunteer is then asked when the light digits in any sequence. Volunteers are given
appears fused. Volunteer is asked to look at one digit/sec and volunteer have to repeat
this frequency for a minute. Then the rate is the sequence in the correct order.
decreased at 2 Hz/sec till the light appears 9. Immediate recall memory: This task is also
as flickering. This frequency is then noted. known as word list memory task. In which
The average of 3 such reading is noted. volunteers are presented with a list of 10
4. Finger Tapping: Volunteer is asked to tap a related words at the interval of one word/2
particular key (computer/calculation/table sec (18 sec). Then, volunteers are asked to
recall/write down all 10 words after 45 seconds. 11. Pegboard test: The test is used for the gross
The sequences of the word may be checked. movements of hands, fingers and fingertip
10. Hand Stabilometer: The volunteer is asked to dexterity. Volunteers are demonstrated with
insert a probe into the different (descending a board with the space to fix the pegs and
order of diameter) diameter hole. First, instruct instructed (Fig. 28.4). Then, they are asked to
the volunteers and give a trial run, then start fix the entire peg in their correct places. The
the test from the largest diameter hole to the time taken to fix the pegs and percentage of
lowest one. The moment probe touches the correct performances are recorded.
margin of hole, a light indicate and experiment
is ended. The point and time taken to reach
the particular point is noted (Fig. 28.3).
Fig. 28.4: Pegboard test

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

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

Objective evaluation Data should be express as mean ± SD and for
Percentage of impairment (%) comparison is made by unpaired ‘t’ test or
Conclusion: ANOVA, if mean is more than 2.
Discussion study. So, efficiency was evaluated from five recent

Presently, various psychomotor function tests studies to calculate the coefficients of variation for
have been used to measure the effects of different tests of psychomotor function, both during
psychoactive compounds on human behavior and in study days. Study showed saccadic eye
and every test has its own merits and demerits. movement analysis and critical flicker fusion
The psychopharmacologist should be familiar threshold is found to have low coefficients of
with judgement in behavioural terms and variation ( 2·7 to 7·5 %) and visual analogue rating
accurate assessment but at times the range of scales and the digit symbol substitution test are
behavioral activities presented is critical to reported to have much higher coefficients of
evaluate. variation (7·9 to 17·6%). This indicates that
These tests help in evaluation various drugs measures of test variability should be routinely
whether it is psychologically relevant, and it assessed during psychomotor assessment studies
should be evaluated in model of behavior. It is to validate and also to check test methodology.
very much clear that mode and level of activity of
the brain and central nervous system is dependent Note
upon personality, motivation and memory so it is Visual analogue assessment (VAS) can have variation
depending of volunteers/ patients right or left handed person
necessary to examine different factor interfering
because right handed person have more tendency to mark
with performance. Various studies have already toward right side and same thing is applied for left handed
reported effect of different drug on psychomotor person also. So, it is advisable to place VAS in vertical
functions for example, an early recovery time direction.
propofol sedation is similar in elderly and younger
SUGGESTED READING
patients, but recovery of psychomotor function in
the elderly is delayed compared with younger 1. Adam JK, Rmaih WN. A double-blind placebo-
patients. Patients with epilepsy who require high controlled investigation of the psychomotor
CBZ concentrations for optimal control of seizures profile of clopidogrel in healthy volunteers. J
may be at risk of concurrent impairment of Cardiovasc Pharmacol 2008;52(6):507-09.
2. Aitken RCB. Measurement of feelings using
psychomotor function. Study done to evaluate the visual analogue scales. Proceedings of the Royal
effects of various drugs like haloperidol (1 mg), Society of Medicine 1969;62:989-93 Freyd M.
benzhexol (5 mg), diazepam (10 mg) and caffeine The graphic rating scale. Journal of Educational
Psychology 1923;43: 83-102 Hayes, MHS, DG
(400 mg) on subjective and objective measures of
Patterson. Experimental development of the
cognitive and psychomotor function compared graphic rating method. Psychological Bulletin,
with placebo in 20 healthy volunteers showed 1921;18:98-99.
diazepam and benzhexol are associated with 3. Clubley M, Bye CE, Henson TA, Peck AW,
Riddington CJ. Effects of caffeine and cyclizine
significant impairments in subjective alertness,
alone and in combination on human
critical flicker fusion threshold and choice reaction performance, subjective effects and EEG
time (CRT), however, haloperidol could not be activity. Br J Clin Pharmacol 1979;7(2):U57-
distinguished from placebo in psychomotor tests 63.
4. DJ King, G Henry. The effect of neuroleptics on
but it is associated with an apparent improvement cognitive and psychomotor function. A
in CRT (in males) and simple visual reaction time. preliminary study in healthy volunteers. The
Finally, perceptual maze test detected impairment British Journal of Psychiatry 1992;160:647-53.
by benzhexol on processing speed but was not 5. Effect of sodium valproate on carbamazepine
disposition and psychomotor profile in man.
sensitive to any other drug effects. Another study Macphee GJ, Mitchell JR, Wiseman L, McLellan
reported with efficiency of measurement of AR, Park BK, McInnes GT, Brodie MJ. Br J Clin
variability which are generally not coated in the Pharmacol 1988;25(1):59-66.
6. Freyd M. The graphic rating scale. Journal of 11. MF Mannion1, G Lynch2, DJ King. Precision of
Educational Psychology 1923;43:83-102. measures of saccadic eye movements and
7. Hart J, Hill HM, Bye CE, Wilkinson RT, Peck conventional psychomotor function tests.
AW. The effects of low doses of amylobarbitone Human Psychopharmacology 2004;9(1):37-41.
sodium and diazepam on human performance. 12. Murphy SE, Downham C, Cowen PJ, Harmer
Br J Clin Pharmacol 1976; 3(2):289-98. CJ. Direct effects of diazepam on emotional
processing in healthy volunteers. Psycho-
8. Hayes MHS, DG Patterson. Experimental
pharmacology 2008;199(4):503-13.
development of the graphic rating method.
13. Peck AW, Bye CE, Clubley M, Henson T,
Psychological Bulletin, 1921;18:98-99.
Riddington C. A comparison of bupropion
9. Ian Hindmarch. Psychomotor function and
hydrochloride with dexamphetamine and
psy-choactive drugs. ‘Br J Clin Pharmacol
amitriptyline in healthy subjects. Br J Clin
1980;10: 189-209 Pharmacol 1979;7(5):469-78.
10. Lynch G, King DJ, Green JF, Byth W, Wilson- 14. Toshimitsu Kitajima. Recovery of psychomotor
Davis K The effects of haloperidol on visual function after propofol sedation is prolonged in
search, eye movements and psychomotor the elderly. Can J Anesth 2002;49:9;927-31
performance. Psycho-pharmacology (Berl) 15. Wechsler D. WAIS-R manual. New York, NY:
1997;133(3):233-39. Psychological Corporation, 1981.
EXERCISE
Exercise no. 1: 30-year-old female presenting with bilateral hand paresthesia, on examination patients could
lift, left hand more than right on radial three finger for 1st three years. More at night time with pain impairing
sleep also. Possibility of bilateral carpal tunnel syndrome was kept. Plan was to rule out, diabetes and
hypothyroidism. Nerve conduction study was planned and showing normal nerve conduction although latency
is prolonged at left side than right side. Describe your interpretation.
Fig. 28.5
Fig. 28.6
Exercise no. 2: 20-year old female presented with H/o seizure for 10 yr. GTCS with frequency once in every 3-
4 months. (Usually nocturnal seizure with myoclonic jerks. No h/o cognitive decline. Family history was
negative. Describe the EEG findings and discuss pharmacological therapy.
Fig. 28.7
Fig. 28.8
Exercise no. 3: 30-year-old female presented with seizure for past 15 yrs, GTCS at morning and night with
frequency of 1 in 2-3 months. h/o myoclonic jerk is present . Family h/o positive, no history of cognitive
decline. Describe the EEG findings and discuss pharmacological therapy.
Fig. 28.9
Fig. 28.10
Kidney
29
EXPERIMENT NO: 13 Stethoscope, mercury sphygmomanometer,
Facilities for clinical pharmacology laboratory
Aim (Appendix IV)
To evaluate the effect of frusemide on urine Drug: Tablet Frusemide (40 mg)
volume and Na+ and K+ excretion in healthy
volunteers. Precautions
• Subject is advised for adequate sleep over
Background night and light breakfast in the morning on
Thiazide diuretics cause moderate diuresis and day of experiment (preferably 2 hr before)
natriuresis. Loop diuretics cause marked diuresis • Avoid caffeinated drink or smoking or at least
and natriuresis, whereas K+ sparing diuretics 12 hr before the experiment or free of any
produce diuresis and preserve K+. Furosemide substance abuse
(frusemide) is a loop diuretic commonly prescri- • Volunteer should get complete instructions
bed for congestive cardiac failure and edema, of the procedure before the experiment
initially it was used in horse-race to prevent nose • Monitoring equipment is maintained and
bleeding. Presently, furosemide is included in the regularly calibrated
world anti- doping banned drugs list. It acts by • Basic operating procedure of the selected
inhibiting Na+-K+ -2Cl– sympoter in the thick instrument should be well explained to the
ascending limb of loop of Henle, action mostly volunteers.
on distal tubules and has weak carbonic anhy-
drase-inhibiting activity. So, the experiment is Methods
aimed to determine the diuretic and natriuretic
effects of frusemide. The study requires healthy volunteers. Following
preliminary physical examination, volunteer is
Materials and Methods given breakfast and asked to refrain from
smoking tea, coffee or other caffeinated beve-
Initial Screening of Volunteers rages 12 hr before and during study. Fluid intake
Volunteers are screened for any cardiovascular, is regulated and volunteers are adviced to rest
respiratory disorder or any other medical in a room with ambient temperature (25 ± 2ºC)
unstable conditions which may interfere with in clinical pharmacology laboratory.
experimental protocol. Volunteers with smoking
Inclusion Criteria
are excluded.
Healthy adult volunteers of either sex (willing 1. Healthy volunteer aged 18-40 years
to give written informed consent) 2. Willing to give written informed consent.
Exclusion Criteria Detection limit of Na+= 0.0001ppm

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

Na+ conc. of unknown = ?
K+ conc. of unknown = ?
Volunteer No 1
Time Blood pressure Heart rate Urine volume Na+ K+ ADR

0.5 hr
1 hr
1.5 hr
2 hr
3 hr
4 hr
5 hr
6 hr
Volunteer No 2
Time Blood pressure Heart rate Urine volume Na+ K+ ADR

0.5 hr
1 hr
1.5 hr
2 hr
3 hr
4 hr
5 hr
6 hr
Kidney  305
meta stable state and are prone to return to lower 5. Verma AK, da Silva JH, Kuhl DR. Diuretic effects of
energy orbit including ground state and energy subcutaneous furosemide in human volunteers: A
previously absorbed is released as quanta of light, randomized pilot study. Ann Pharmacother 2004;
38(4):544-49.
the wave length of which depends upon the
energy levels which the electrons can attain and
are characteristics of each substance commonly KIDNEY EXPERIMENT: 14
called “emission spectrum”. In emission flame Aim
photometry, the relatively low energy flame
excites a few elements mainly alkali metals. Part To evaluate saluretic, natriuretic and carbonic
of light which is emitted in all directions is anhydrase inhibitory effect of various diuretics
collected by a reflector and falls on a detector. in healthy volunteers.
The light intensity and hence the detector output
is directly proportional to the concentration of Background
substance.
Frusemide causes marked diuresis in terms Excretion of salt and water is important for
of urinary volume. The onset of diuresis is within treatment of condition like peripheral edema
1 hour. The peak effect occurs within the first following congestive heart failure and also used
and second hour. The duration of action is 6-8 for treatment of hypertension. Aim of this
hours. The duration of diuretic effect is approxi- experiment is to calculate saluretic activity by
mately 2 hours. It also causes an increase in Na+ calculating the sum of Na+ and Cl– excretion,
and K+ excretion. Total urine output usually natriuretic activity by the ratio Na+ /K + and
occur within 6 hrs approximately 3500 ml. K+ carbonic anhydrase inhibition from the ratio
and Na+ excretion increases peaking at 1 hr. BP Cl-/(Na+) + (K+). Most commonly used drugs
and HR is not usually affected. Volunteers may are frusemide, thiazide diuretic in the clinical
complain of bad taste and weakness. practice.
SUGGESTED READING Materials and Methods
1. Arancibia A, Nella Gai M, Paulos C, Chávez J, Pinilla
E, Angel N, Ritschel WA. Effects of high altitude
exposure on the pharmacokinetics of furosemide Volunteers are screened for any cardiovascular,
in healthy volunteers. Int J Clin Pharmacol Ther respiratory disorder or any other medical
2004; 42(6):314-20.
2. Klausner EA, Lavy E, Stepensky D, Cserepes E,
unstable conditions which may interfere with
Barta M, Friedman M, Hoffman A. Furosemide experimental protocol. Volunteers with smoking
pharma-cokinetics and pharmacodynamics habit and alcoholics should be excluded.
following gastroretentive dosage form administra- Healthy adult volunteers of either sex (willing
tion to healthy volunteers. J Clin Pharmacol to give written informed consent)
2003;43(7): 711-20. Stethoscope, mercury sphygmomanometer
3. Musso CG, Reynaldi J, Vilas M, De Miguel R,
Imperiali N, Algranati L. Fractional excretion of K,
Drug: Frusemide, thiazide diuretic
Na and Cl following furosemide infusion in healthy,
young and very old people. Int Urol Nephrol 2009 Precautions
Mar 10.
4. Noskov VB, Goncharov IV, Kovachevich IV, Refer to experiment no.: 13
Repenkova LG, Kodratenko SN, Starodubtsev AK.
The pharmacodynamics and pharmacokinetics of Methods
furosemide during ordinary life activities and
during head-down tilt hypokinesia in a healthy The study requires healthy volunteers. After
subject] Eksp Klin Farmakol 1998;61(4):29-33. preliminary physical examination, volunteer is
given breakfast and asked to refrain from smo- One volunteer advise to take frusemide 40 mg
king tea, coffee or other caffeinated beverages 12 with 200 ml of water and other one 25mg with
hr before and during study. Fluid intake is 200ml of water. Voiding of urine is done at every
regulated and volunteers are kept in a room with ½ hr, for 2 hrs and then hourly for 6 hrs. Amount
ambient temperature (25±2ºC) in clinical phar- of water equal to volume of urine each time is
replenished. Samples are assessed for Na+ and
macology laboratory.
K + on flame photometer and Cl - argento-
metrically by potentiometrical end point titration
Inclusion Criteria
method. All the vital parameters BP and HR are
1. Healthy volunteer aged 18-40 years recorded. Following parameters are assessed
2. Willing to give written informed consent saluretic activity from Na+ and Cl– excretion,
natriuretic activity by calculating the ratio, Na+/
Exclusion Criteria K+ and calculation to estimate carbonic anhy-
drase inhibition from the ratio Cl-/(Na+) + (K+)
1. h/o hypersensitivity, i.e. (ion quotient)
2. Electrolyte imbalance and hypertension. (For procedure refer experiment no.:13)

Volunteer No 1
Time Blood Pressure Heart Rate Urine volume Na+ K+ Cl– ADR
0.5 hr
1 hr
1.5 hr
2 hr
3 hr
4 hr
5 hr
6 hr
Volunteer No 2
Time Blood Pressure Heart Rate Urine volume Na+ K+ Cl- ADR
0.5 hr
1 hr
1.5 hr
2 hr
3 hr
4 hr
5 hr
6 hr
Discussion
The saluretic activity is calculated from excretion
Above mentioned diuretics promote excretion of of Na+, Cl-; natriuretic activity and potassium-
salt in the urine as it has saluretic, natriuretic and sparing effect from ratio of Na+/K+, while ratio
minimal effect on carbonic anhydrase inhibition. Cl-/(Na+) + (K+ ) is used to calculate carbonic
Kidney  307
anhydrase inhibition effect. Recent study showed 3. Musso CG, Reynaldi J, Vilas M, De Miguel R,
thiazide and high ceiling diuretics inhibit all Imperiali N, Algranati L. Fractional excretion of K,
mammalian isoforms of carbonic anhydrase. Na and Cl following furosemide infusion in healthy,
young and very old people. Int Urol Nephrol 2009
SUGGESTED READING
Mar 10.
1. Knauf H, Bailey MA, Hasenfuss G, Mutschler E. The 4. Temperini C, Cecchi A, Scozzafava A, Supuran CT.
influence of cardiovascular and anti-inflammatory Carbonic anhydrase inhibitors. Comparison of
drugs on thiazide-induced hemodynamic and chlorthalidone, indapamide, trichloromethiazide,
saluretic effects. Eur J Clin Pharmacol 2006;
and furosemide X-ray crystal structures in adducts
62(11):885-92.
2. Lam NP, Kuk JM, Franson KL, Lau AH. Effect of with isozyme II, when several water molecules
diuretic drugs on creatinine clearance determina- make the difference. Bioorg Med Chem 2009;
tion. Ther Drug Monit 1995;17(2):142-44. 17(3):1214-21.
EXERCISE
Exercise 1: A 9 years old male child, who was operated at the age of 4 days for lumbar myelomeningocele.
Following operation parent notice urinary incontinence at the age of 2 years. All the investigations Hb, urea,
creatinine, USG,MCU,DMSA scan was within normal limit. Based on urodynamic study, a diagnosis of
neurogenic bladder was made and child was put on CIC with regular follow-up. Subsequently in urodynamic
high risk bladder was detected then oxybutynin 5 mg tds started along with CIC for 21 days. Child improved
symptomatically and urodynamically. Following are pre-drug and post oxybutynin urodynamic study. Pre-
drug maximum bladder volume = 87 ml, detrusor leak point pressure = 75 cm H2O, compliance = 1.5 ml/
cmH2O and post-drug maximum bladder volume = 130 ml, detrusor leak point pressure = 40 cmH2O,
compliance = 3.3 ml/ cmH2O. Describe your interpretation?
Pre-drug graph
Fig. 29.1
Post-drug graph
Fig. 29.2
Ophthalmology
30
OPHTHA. EXPERIMENT: 15 Precautions for Experiment
Aim • Subject is advised for adequate sleep over

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

other drugs likely to affect interpretation of
study parameters
4. Coffee, tea or smoking (past 24 hrs) and
alcohol intake (past 48 hrs)
Study Parameters
1. Pupillary diameter is measured with the help
of a pupillometer. It should be measured close
to the right eye with left eye closed with a
card and volunteers are asked to see a distant,
well illuminated object. The point at which 2
holes in the pupillometer just start overlap-
ping each other is taken as pupillary
diameter. Fig. 30.1: Pupillometer
2. Heart rate: Estimated by palpating the radial
artery. Some of the updated models of pupillometer
3. Salivary secretion: Assessed by using rock salt. require complex optometric components. The
‘1g’ of the salt is given to keep under the current method of practice is to manually
tongue and advised to keep there for 1 min, measure these aspects using a bright light, which
then saliva is collected in a measuring stimulates reactivity of the pupil and make a note
cylinder of the dilation compared to the original size of
the pupil. Actual measurements compared with
Pupillometer (Fig. 30.1) a card having different pupil sizes matched
thereon. This method of assessment is time
Pupillometer was developed for the assessment
consuming, and subjective.
of eye shape and condition, monitoring tiredness,
and for the detection of drugs or alcohol in Procedure for measurement of pupillary
human beings. It is a portable instrument that diameter: Before using the pupillometer, instru-
rapidly and simultaneously assesses records and ment is validated. Make sure batteries are
stores pupil size and reactivity to stimulus. It installed and are in operating order. Switch on
provides a rapid diagnostic aid for neurological the pupillometer and then select the different size
assessment of unconscious patients, since pupil option which can be viewed digitally with setting
response is known to be a vital aspect of the of particular eye site or both. Volunteers should
diagnostic process. Regular assessment of the be given detailed instruction to hold the
size, reactivity to light and equality of pupils is pupillometer up to their eyes with proper use of
essential for early recognition of neurological nose rest. Then, volunteers stare through the
deterioration. The main features are to measure view windows and eyepiece, so that the pinpoint
pupil reaction, speed and size in patients with of light is located inside the pupil to calculate
suspected brain injury. The pupilometer mostly Pupillary Diameter.
detects and measures pupil diameter and pupil
response to a light stimulus. In the recent times,
Method
so many models are available and these are with
software helping in the diagnosis of alcohol or All the volunteers are asked to report in the clinical
drug presence and use of this pupillometer pharmacology laboratory in the morning with
requires the active participation from the suspect. good overnight sleep and light breakfast. A
Ophthalmology  311
through medical examination is done before the or placebo in randomized single blinded fashion
experiment. All the procedure is explained in with a glass of water. All the above mentioned
detail. Above mentioned study parameters should parameters should be recorded at frequent
be recorded at baseline before giving the drug, intervals at 0, 0.5, 1, 1.5, 2, 3, 4, 5 and 6 hours
afterward volunteers are given 10 mg of hyoscine following drug intake.

A. Hyoscine
Time (hr) BP (mmHg) H R (beats/min) RR (per min) PD (mm) SS (ml) ADR
0
0.5
1.0
1.5
2.0
3.0
4.0
5.0
6.0
B. Placebo
Time (hr) BP (mmHg) H R (beats/min) RR (per min) PD (mm) SS (ml) ADR
0
0.5
1.0
1.5
2.0
3.0
4.0
5.0
6.0
Blood Pressure (BP) mm Hg, Heart rate (H R) Rate/min, Respiratory rate (RR) Rate/min, Pupillary diameter (PD)
mm, salivary secretion (SS) ml.
Statistical analysis: All the data should be complain of dry mouth, throat, blurred vision,
expressed as mean± SD. Data is compared by the photosensitivity, urination difficulty, flushing,
unpaired ‘t’ test. hallucinations etc. Special precaution should be
Discussion: Hyoscine causes decrease in salivary taken in patients with glaucoma because
secretion. Maximum effect is achieved approxi- hyoscine may precipitate glaucoma in borderline
mately at 3 hrs which returns to baseline level patients. Studies reported oral scopolamine
by 6 hrs which was found significantly different hydrobromide decreases salivation by 70%,
from placebo group in several previous studies. following transdermal hyoscine the magnesium
Similar results may be present with HR and secretion rate is unaltered, whereas sodium,
pupillary diameter in comparison to baseline and potassium, and calcium secretion rates are
when compared to placebo. Volunteers may significantly lowered and no significant modifi-
cation is observed in the electroretinogram in Materials and Method

healthy volunteers following parenteral admini-
stration of hyoscine.
Volunteers are screened for any cardiovascular,
SUGGESTED READING respiratory disorder or any conditions which
may interfere with experimental protocol.
1. Ebert U, Grossmann M, Oertel R, Gramatté T, Kirch Healthy adult volunteers of either sex (willing
W. Pharmacokinetic-pharmacodynamic modeling to give written informed consent)
of the electroencephalogram effects of scopolamine
Pupillometer, Stethoscope, mercury sphy-
in healthy volunteers. J Clin Pharmacol 2001;41(1):
gmomanometer
51-60.
2. Gordon C, Ben-Aryeh H, Attias J, Szargel R, Gutman
Drug: Tropicamide (1%)
D Effect of transdermal scopolamine on salivation.
J Clin Pharmacol 1985;25(6):407-12 Precautions for Experiment
3. Harding GF, Daniels R, Panchal S, Drasdo N, • Subject is advised for adequate sleep over
Anderson SJ. Visual evoked potentials to flash and
night and light breakfast in the morning on
pattern reversal stimulation after administration of
day of experiments (preferably 2 hr before)
systemic or topical scopolamine. Doc Ophthalmol
• Avoid caffeinated drink or smoking or at least
1994;86(3):311-22.
4. Kochiadakis GE, Rombola AT, Kanoupakis EM,
2 hr before the experiment or any abuse
Zuridakis EG, Skalidis EI, Vardas PE. Effect of substance
transdermal scopolamine on heart rate variability • Volunteers should get complete instruction
in patients with severe coronary heart disease. of the procedure and its rehearsal, so to avoid
Pacing Clin Electrophysiol 1996;19(11 Pt 2):1867-71. any conditional anxiety of volunteers
5. Markkanen YJ, Pihlajamäki K. Oral scopolamine • Basic operating procedure should be well
hydrobromide solution as an antisialagogic agent explained to the volunteers
in dentistry. Oral Surg Oral Med Oral Pathol 1987; • Advice the volunteers not to drive unless they
63(4):417-20 can see clearly
6. Renner UD, Oertel R, Kirch W. Pharmacokinetics • Volunteers should not wear soft contact
and pharmacodynamics in clinical use of scopo- lenses following use of tropicamide because
lamine. Ther Drug Monit 2005;27(5):655-65. it contains a preservative which can be
7. Sunahara FA, Farewell J, Mintz L, Johnson WH. absorbed by soft contact lenses and cause eye
Pharmacological interventions for motion sickness:
irritation
cardiovascular effects. Aviat Space Environ Med
1987;58(9 Pt 2):A270-76. Method
After initial screening, volunteers should partici-
OPHTHA. EXPERIMENT: 16
pate in the study as per following inclusion and
Aim exclusion criteria:
To evaluate the effect of Tropicamide (1%) on Inclusion Criteria

pupillary diameter and accommodation reflex. Healthy volunteers aged between 18-45 years of
either sex
Background
Tropicamide, a muscarinic blocker acts as short Exclusion Criteria
acting mydriatic and cycloplegic. The duration • History of Volunteers with: cardiovascular
of action is 4-8 hours so it is mostly preferred disease, significant hepatic/renal dysfuction,
over atropine for examination of lens, vitreous glaucoma, Benign prostatic hypertrophy
humor and retina. (BPH), psychiatric disorders
• Anticholinergic drug taken within past 7 days Assessment of Accommodation

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

Pupillary diameter (mm) Accommodation
Control (Lt eye) Test (Rt eye) Control(Lt eye) Test (Rt eye) ADR
Time
Baseline
5 min
10 min
15 min
30 min
1 hr
2 hr
3 hr
4 hr
5 hr
6 hr
Statistical analysis: All the data should be Discussion

expressed as mean ± SD. Data is analyzed by the Tropicamide is a short acting antimuscarinic drug.
unpaired‘t’ test. The receptors are present in pupillae and because
continuous cholinergic input maintains pupil size. Background

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

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

Pupillary diameter (mm) Near Vision Distance Vision
Time HR BP Control (Lt eye) Test (Rt eye) Control (Lt eye) Test (Rt eye) Control (Lt eye) Test (Rt eye)
0
5
10
15
20
25
30
35
40
45
50
55
1 hr
1.5 hr
2 hr
4 hr
8 hr
In addition, observe for adverse drug reaction (ADR) or salivary secretion if any.
Statistical analysis: All the data should be Background

expressed as mean ± SD. Data is analyzed by
The normal intraocular pressure is maintained at
unpaired’t’ test.
16-23 mm Hg as it is required for the maintenance
of the optical properties of refracting organs. The
Discussion
pressure in the eyeball is mainly because of
Pilocarpine eyedrops produce pupillary contr- elasticity of outer coats, volume of solid and liquid
action in 10 min and the effect is usually contents which maintain the intraocular pressure.
observed up to 4 hrs. Near vision improves The measurement of intraocular pressure can be
whereas distant vision is blurred during the made by various methods like manometry, tono-
same period. The drug produces burning pain metry (digital tonometry, instrumental tonometry
on instillation. Conjuctival hyperemia is like impression tonometry, applanation tono-
develops after 5 min, vague discomfort, pain metry, etc.). These are used to measure the
and blurring of distant vision is noted from 10 intraocular pressure by flattening an area of
to 15 min onwards. Pilocarpine is well-known cornea. There are different factors responsible for
for excessive sweating, salivation, broncho- variations of intraocular pressure like respiration
spasm, bradycardia, hypotension and diarrhea, and pulse which can change the pressure to 1-2
so it has limited clinical use. mm Hg. The intraocular pressure maintains
diurnal variations pressure which is usually high
SUGGESTED READING in the morning then falls in the evening and at
night pressure rises again. There is different
1. Doughty MJ, Lyle WM. A review of the clinical provocative test used in clinical practice to
pharmacokinetics of pilocarpine, moxisylyte
diagnose chronic simple glaucoma and also to find
(thymoxamine), and dapiprazole in the reversal of
diagnostic pupillary dilation. Optom Vis Sci. 1992; out behavior of intraocular tension under stress.
69(5):358-68. Water drinking test is applied since it lowers the
2. Druzgala P, Winwood D, Drewniak-Deyrup M, osmotic tension of blood ultimately raising the
Smith S, Bodor N, Kaminski JJ. New water-soluble intraocular tension.
pilocarpine derivatives with enhanced and sus-
tained muscarinic activity. Pharm Res 1992; 9(3): Materials and Method
372-77.
3. Smith SE, Smith SA, Friedmann AI, Chaston Initial Screening of Volunteers
JM. Comparison of the pupillary, refractive,
and hypotensive effects of Ocusert-40 and pilo-
Volunteers are screened for any systemic
carpine eyedrops in the treatment of chronic disorder or any conditions which may interfere
simple glaucoma. Br J Ophthalmol 1979;63(4): with experimental protocol.
228-32. Healthy adult volunteers of either sex (willing
4. Yie T, Liang L, Chen X. Clinical observation on the to give written informed consent).
effect of 4% pilocarpine gel used one dose per day Schiotz Tonometry, 4% xylocaine, Stetho-
in the treatment of glaucoma. Yan Ke Xue Bao 2000; scope, mercury sphygmomanometer, Facilities
16(2):77-80. for Clinical Pharmacology Laboratory (Appendix
5. Yuan J, Wei H. A clinical observation of the
IV).
therapeutic effects of pilocarpine gel for treatment
of glaucoma. Zhonghua Yan Ke Za Zhi 1998; Precautions for Experiment
34(3):174-77.
OPHTHA. EXPERIMENT: 18 over night and light breakfast in the morning
on day of experiments (preferably 2 hr before)
Aim • Avoid caffeinated drink or smoking or at least
To demonstrate water induced ocular hyper- 2 hr before the experiment or any substance
tension in healthy volunteers. abuse
• Volunteer should get complete instructions of is advised to lie down in supine position without
the procedure, so to avoid any conditional any pillow under the head. Volunteer is asked
anxiety of volunteers to look vertically upward towards roof to relax
• Basic operating procedure should be well the accommodation. The eyelid is separated with
explained to the volunteers. finger without giving any pressure in the eye,
and then globe tonometer is vertically placed on
Method the cornea. The volunteers are advised not to
After initial screening volunteers should parti- move the eye during this time. The pressure is
cipate in the study as per following inclusion and recorded as the pointer reaches the steady state.
exclusion criteria: After recording the pressure, antibiotic should
be applied.
Inclusion Criteria
either sex Ocular pressure (mm Hg)
Time HR BP RR Control eye Test eye
Exclusion Criteria 0
0.5 hr
• History of volunteers with any systemic 1 hr
disease 2 hr
• Volunteers with glaucoma 3 hr
• Anticholinergic drug taken within past 7 days 4 hr
6 hr
• History of substance abuse (narcotics) or any
study parameter. Graphical Representation
• Coffee, tea or smoking (past 24 hrs) and
alcohol intake (past 48 hrs).
Procedure
pharmacology laboratory in the morning with
good over night sleep and light breakfast. A
thorough medical examination is done before the
experiment. All the procedure should be explai-
ned in detail.
Water drinking test: The volunteers are asked to
drink “one liter” of water in 10 min before
breakfast and ocular pressure is measured at
baseline 0, 0.5, 1, 2, 3, 4 and 6 hr. Along with
ocular tension other parameters like blood
pressure, pulse rate, respiratory rate are also
recorded. Fig. 30.2: Representation of result
Measurement of Ocular Tension Statistical analysis: All the data is expressed

After taking written informed consent, cornea is as mean± SD. Data is analyzed by unpaired ‘t’
anesthetized with 4% xylocaine then volunteer test.
Discussion are available like venous congestion test which

increases ocular pressure greater than 10 mm
Provocative tests are commonly used in the
Hg in case of glaucoma.
practice to diagnose chronic simple glaucoma
and to evaluate behavior of intraocular tension SUGGESTED READING
under stress. There are different tests which are
1. Vetrugno M, Sisto D, Trabucco T, Balducci F, Delle
used like water drinking test causes lowering
Noci N, Sborgia C. Water-drinking test in patients
of osmotic tension of blood causing rise of intra- with primary open-angle glaucoma while treated
ocular tension and if it is rise beyond 6 mm Hg, with different topical medications. J Ocul Pharmacol
this indicates glaucoma. Besides this other tests Ther 2005;21(3):250-57.
Clinical Pharmacokinetics
31
EXPERIMENT NO: 19 c. Have voluntarily given written informed
consent and comply with protocol requirement
Aim
d. Clinically normal profile of RBC, WBC and
To study the pharmacokinetics of Aceclofenac DLC (differential leukocytes count)
tablet following single oral dose. e. Clinical normal profile of serum creatinine,
serum aspartate aminotransferase, serum
Background alanine aminotransferase.
f. No change in ECG
Pharmacokinetics is a composite discipline of
g. No abnormality detected in routine exam-
mathematics, physiology and pharmacology
ination of urine.
involved in determination of absorption, distri-
bution, metabolism and elimination of a drug. Its
systemic analysis may predict overall progress of Exclusion Criteria
a drug through the body and the interaction Volunteers are excluded from the study on the
between two or more drugs. Aceclofenac is taken basis of following criteria:
as an example and it is an orally administered a. History of hypersensitivity to any drug
phenyl acetic acid derivatives and potent inhibitor particularly to NSAIDs
of the enzyme cyclooxygenase, which is involved b. Volunteers who have received NSAIDs within
in the production of prostaglandins. It is commonly one week
prescribed for the acute and chronic conditions, c. Volunteers with cardiovascular, respiratory,
e.g. rheumatoid arthritis (RA) and osteoarthritis liver, psychiatric or any other systemic illness
(OA). d. Volunteers who are a regular smokers or
alcoholic
Materials and Methods e. Volunteers who are HIV or Hepatitis B or C
Adult male healthy volunteers, Facilities for positive.
Clinical Pharmacology Laboratory (Appendix IV)
Drug: Aceclofenac (100 mg), HPLC, Centrifuge Precautions
Following submission of written informed
Inclusion Criteria
consent, volunteers should undergo standardized
a. Volunteer age group of l8-45 years screening procedure before initiation of the study
b. Be neither underweight nor overweight as per (Physical and Laboratory examination) one week
chart provided by LIC, India prior to the period of drug administration.
Methods
HPLC METHOD FOR PLASMA ANALYSIS solvent A (consisting of 20% (v/v) 0.005 mol/L
phosphate buffer, 80% (v/v) acetronitrile) and
Sample Preparation solvent B ( 88% (v/v) 0.01 mol/L phosphate buffer
Samples are prepared by adding 0.5 ml sodium and 12% (v/v) acetonitrile, the flow rate should
floride solution (40 mg/mL), 1 ml of 1 mol/L be 1ml/min and injection volume should be 20
phosphoric acid and 0.1 mL internal standard mL and read frequency at UV 275.
solution (0.05 mg ketoprofen) to 1 mL plasma
followed by the addition of 5ml n-hexane/diethyl CHROMATOGRAM
ether (50:50, v/v). The tubes are capped, shaken for Internal standard is ketoprofen and limit of
30 min and then centrifuged at 4000 rpm for 10 min. detection should be 10 ng/mL and the external
The organic layer is transferred into a glass tube standard is bulk solution of aceclofenac.
and evaporated to dryness under a nitrogen stream
at room temperature. Prior to analysis, the residue is DATA RECORDING
dissolved in 120 mL of a solution consisting of 72%,
All data during the conduct of the study should
0.01 mol/L phosphate buffer, 15% acetonitrile, 10%
be directly entered in the data recording forms
methanol, 3% tetrahydrofuran (final pH 2.5), which
and computer generated chromatograms should
is used for HPLC analysis.
be prepared. Data should be entered in software
program to calculate the pharmacokinetic para-
HPLC VARIABLES USED FOR PLASMA meters.
ESTIMATION Results and calculation of pharmacokinetics
Column used in this method is : 250 × 4.6, 5 mm parameters
spherisorb C18 and mobile phase is mixture of Volunteers Vitals: with different time intervals:
Clinical Pharmacokinetics  321
Name: Age: in both young and elderly volunteers. Aceclofenac

Sex: is mainly metabolized by CYP2C9 pathway. The
Address: metabolism of aceclofenac differs in animals and
the human beings. Aceclofenac, additional side-
Time (hr) Plasma level (µg/ml)
chain (esterifies acetoxy) is compared with
0 structurally related diclofenac. 4’ OH aceclofenac
1 is the main metabolite in the plasma and urine
2 after the oral administration of aceclofenac. Other
3 minor metabolites are diclofenac and 4’ OH
4 diclofenac, 5’-OH diclofenac. Aceclofenac gets
5 converted to diclofenac after long time by
6 diclofenac hydroxylate or of hydrolysis of the
8 present compound.
10
Note: Presently, there are different softwares available
12
for pharmacokinetic parameters calculations for example:
16 WinNonlin, NONMEM, etc.
24
Pharmacokinetic parameters: SUGGESTED READING
1. Bioavailability : Rate and extent
1. Bhinge JR, Kumar RV, Sinha VR. A simple and
Concentration time curve sensitive stability-indicating RP-HPLC assay method
Cmax, Tmax for the determination of aceclofenac. J Chromatogr
2. Volume of distribution (Vd) Sci 2008;46(5):440-44.
3. Elimination half life (t1/2 el) 2. Bort R, Ponsonda X,Carrasco E, et al. Metabolism of
aceclofenac in humans. Drug Metab Dispos 1996;
4. Area under curve (AUC0-24 and AUC0-∝)
24(8):834-41.
5. Rate constants (Ka and Kel) 3. Brogden RN, Wiseman LR. Aceclofenac. A review of
a. Absorption rate constant (Ka) its pharmacodynamic properties and therapeutic
b. Elimination rate constant (Kel) potential in the treatment of rheumatic disorders
6. Clearance and in pain management. Drugs 1996;52(1):
Discussion: A newer NSAIDs Aceclofenac, with 113-24.
4. Dooley M, Spencer CM, Dunn CJ. Aceclofenac: A
anti-inflammatory and analgesic properties is reappraisal of its use in the management of pain and
most commonly prescribed in clinical practice. rheumatic disease. Drugs 2001;61(9):1351-78.
Following oral administration of a single 100 mg 5. Hinz B, Auge D, Rau T, Rietbrock S, Brune K, Werner
dose, mean maximum plasma aceclofenac U. Simultaneous determination of Aceclofenac and
concentration (Cmax) of 6.8 to 8.9 mg/L is reduced three of its metabolites in human plasma by high-
in about 1.4 to 2 hours. Cmax and area under the performance liquid chromatography. Biomed Chrom
2003, 17: 268-275.
plasma concentration time curve (AUC) increase
6. Rex N Brogden, Lynda R Wiseman. Aceclofenac: A
linearly after administration of single dose of review of the pharmacodynamic properties and
aceclofenac 50, 100 and 150 mg and the pharma- therapeutic potential in the treatment of Rheumatic
cokinetic properties of the drug are generally disorders and in pain management. Drugs 1996;
unchanged during multiple-dose administration 52(1):113-24.
Miscellaneous
32 Practicals
EXPERIMENT NO: 20 used for the induction of pain do not produce

inflammation or substantial tissue damage and
Aim hence the mechanism, by which NSAIDs mainly
To evaluate the analgesic activity of NSAIDs on act, i.e. by inhibiting prostaglandin synthesis, thus
different human pain models. might not come into play in such a short-time.
To develop an ideal pain model some of the
Background criteria should be fulfilled for example, it should
not cause tissue damage, or psychological injury,
The evaluation of analgesics in volunteers has
it should be simple and reliable without any after
got various difficulties and it is difficult to develop
effect lastly volunteers should have controlled of
an ideal model for example experimental stimulus
cessation during the experiment.
does not produce any emotional response which
is a part of any painful pathological condition.
Precautions before the Experiment
This is because the subject can withdraw the
stimulus whenever the pain became intolerable. • Procedure should be well explained to the
Response is variable because of placebo response, volunteers
is very common in these type of screening. Pain • Eyes should be covered during the experiment
threshold is the time after the stimulus adminis- to avoid time clues and visual distraction
tration the subject starts feeling the painful • Hypersensitivity to the cold or warm should
sensation. Pain tolerance is the point at which the be checked (Complete allergic history should
subject starts feeling the pain which is unbearable be taken)
and subject withdraws hand away from the • Volunteers should be advised for adequate
painful stimulus. Non-steroidal anti-inflam- sleep over night and light breakfast in the
matory drugs (NSAIDs) are most commonly morning day of experiments (preferably 2 hr
prescribed drug for acute or chronic pain and for before)
long-term anti-inflammatory therapy. NSAIDs are • Avoid caffeinated drink, alcohol, cola drinks
believed to modulate inflammation and sensiti- or smoking at least 2 hr before the experiment.
zation of nociceptors by the inhibition of COX 1
and COX 2 resulting in an inhibition of prosta- Materials and Methods
glandins, particularly PGE 1 and PGE 2, which
mainly act locally by promoting inflammation and • Healthy adult volunteers (willing to give
other inflammatory agents. Detection of analgesic written informed consent)
effects of NSAIDs through the experimental pain • Cold/warm water reservoir, ice, and thermo-
models seems to be controversial and less suited. meter (sensitivity ± 0.1°C)
The hypothetical thought that most of the methods Drug: Aceclofenac (100 mg/tablet)
Miscellaneous Practicals  323
Experiment: A
Cold Water Stress
Step 1: Volunteers are asked to immerse their non-
dominant forearm (marked at the specific position)
into a container of warm water (34.5-35.5°C) for
2 min (Fig. 32.1).
Step 2: Then, immediate transfer the arm to
another container of cold water and ice (0.5-1.5°C)
(Fig. 32.1). Volunteers are instructed to leave the
arm immersed in the cold water/ice as long as the
pain could be tolerated (pain tolerance). Cold Fig. 32.1: Cold water stress test
pressor tolerance should be measured in seconds
from immersion in the cold water.
Time of onset of pain and tolerance is recorded
before and after the drug treatment.
Limitation of the Study
Observations: 0 min (baseline), 60 min, 120 min
and 180 min after the drug administration, tests Without inflammation and hyperalgesia, the
are repeated. result is inconsistent and unreliable.
Results
Time of hand withdrawal before drug (Sec) Time of hand withdrawal after drug (Sec)
Volunteer 1
0 min
60 min
120 min
180 min
Volunteer 2
0 min
60 min
120 min
180 min
Note: In this experiment additional parameters such as cardiac parameters (BP, HR, ECG, etc). can be evaluated
Experiment: B marked spots sequentially against a switch

mounted at an aperture, 6 × 6 mm in size, on the
Radiant Heat
stimulator. Without prior notice, a projection
The instrument for delivering radiant heat is kept filament lamp mounted within the stimulator is
at a fixed distance from non-hairy marked skin then turned on by an experimenter. The volunteers
surface of forearm. The subject is told to indicate are instructed to pull their forearm away from the
the time of pain threshold and of maximum pain apparatus as soon as they perceive the radiant
tolerance. heat stimulus as painful and thereby, to indicate
Thermal stimulation radiant heat of a constant their pain threshold. The time elapsing between
intensity is used to induce pain. On the ventral the turning on of the lamp and the withdrawal of
surface of the volunteer dominant forearm, eight the arm from the apparatus, allowing the closure
spots should be marked and numbered from one of the switch is measured, in milliseconds, by a
to eight. The subjects are instructed to press the digital clock and stored on magnetic disk. The
intervals between the applications of the radiant induced pain, reaction time to pain stimuli, fine
heat stimuli ranged randomly between 15 and 25 motor control, or side effects.
sec.
Precautions
Observations: Total of seven measurements (each
• Specialized hairless forearm area should be
lasting 8 min) are carried out at 0, 30, 60, 90, 120,
selected and marked properly
150, and 180 min after drug administration.
• Distance between the heat source and hand is
Measurements: Threshold and tolerance to kept constant
electrically induced pain, threshold to thermally • Hypersensitivity should be checked initially
Volunteer 1
0 min
30 min
60 min
90 min
120 min
150 min
180 min
Volunteer 2
0 min
30 min
60 min
90 min
120 min
150 min
180 min
Experiment: C its edges directly applied to the skin. The cuff is

inflated at the mean arterial pressure and the time
BP Cuff Inflation
at which the pain starts is noted and cuff pressure
In this method, the BP cuff is tied to the arm with is maintained and the time of maximal tolerance
the cap of a cold drink bottle under the cuff with developed is noted.
Volunteer 1
0 min
60 min
120 min
180 min
Volunteer 2
0 min
60 min
120 min
180 min
Experiment: D of hand at 50% of MVC. Time of onset of pain and

Hand Dynamometer time of maximal pain is noted.
The subject is asked to perform his maximal The same protocol is followed 2-3 hr after the
voluntary contraction (MVC) on Smodley’s hand NSAIDs administration (Depends on the half-life
dynamometer. The volunteer is asked to keep grip of drugs).
Volunteer 1
0 min
60 min
120 min
180 min
Volunteer 2
0 min
60 min
120 min
180 min
Other Methods in arterial blood pressure. Int J Clin Pharmacol Ther

Toxicol 1986;24(5):249-53.
The submaximum effort tourniquet method (Smith 5. SF La Vincente1, White1 JM , Somogyi1 AA, Bochner1
et al, 1966) F, CB Chapleo. Enhanced Buprenorphine Analgesia
with the Addition of Ultra-low-dose Naloxone in
Discussion: Presently various human pain models Healthy Subjects. Clinical pharmacology and
are available for example cold-induced pain therapeutics 2008;83(1):144-52.
model, electrically induced pain model, dental 6. Stacher G, Bauer P, Schneider C, Winklehner S,
pain models inducing pain by removal of impac- Schmierer G. Effects of a combination of oral
naproxen sodium and codeine on experimentally
ted or semi-impacted third molars, laser-induced induced pain. Eur J Clin Pharmac 1982a;21:485-90.
pain model, mechanically induced pain model, 7. Stacher G, Steinringer H, Schmierer G, Schneider C,
chemically induced pain model, freezing injury, Winklehner S, Mittelbach G, de Paolis C, Praga C.
pain model, heat-burn injury pain model, and UV Ceruletide increases dose dependently both jejunal
pain models using UVA or UVB radiation, etc. in motor activity and threshold and tolerance to
experimentally induced pain in healthy man. Gut
most of the pain model with human healthy
1984;25:513-19.
volunteers established the comparative efficacy 8. Stacher G, Steinringer H, Schmierer G, Winklehner S,
but some models like in cold-induced pain model Schneider C. Ceruletide increases threshold and
various NSAIDs efficacy was failed to demons- tolerance to experimentally induced pain in healthy
trated consistent analgesic effects. Several studies man. Peptides 1982b;3:955-62.
9. Stacher G, Steinringer H, Winklehner S, Mittelbach G,
reported inter individual variation, significant
Schneider C. Effects of graded oral doses of
influence of gender, etc. meptazinol and pentazocine in comparison with
placebo on experimentally induced pain in healthy
SUGGESTED READING humans. Br J Clin Pharrnac 1983;16:149-56.
10. Telekes A, Holland RL, Peck AW. Indomethacin:
1. Doverty M, White JM, Somogyi AA, Bochner F, Ali Effects of cold-induced pain and the nervous system
R, Ling W. Hyperalgesic responses in methadone in healthy volunteers. Pain 1987;30:321-28.
maintenance patients. Pain 2001;90:91-96. 11. Wolff BB, Kantor TG, Jarvik ME, Laska E. Response
2. Garcia de Jalon PD, Harrison FJ, Johnson KI, Kozma of experimental pain to analgesic drugs; III: codeine,
C, Schnelle K. A modified cold stimulation technique aspirine, secobarbital, and placebo. Clin Pharmacol
for the evaluation of analgesic activity in human Ther 1969;10:217-28.
volunteers. Pain 1985;22:183-89.
3. Jones SF, McQuay HJ, Moore RA, Hand CW. EXPERIMENT NO: 21
Morphine and ibuprofen compared using the cold-
pressure test. Pain 1988;34:117-22. Aim
4. Pandhi P, Sharma PL, Sharma BK, Wahi PL.
Comparative effect of propranolol and labetalol on To evaluate plasma salicylate level by fluorometric
isometric exercise and cold stress induced increase methods in healthy volunteer.
Background Precautions for Experiment

Aspirin, another name acetylsalicylic acid (ASA) • Subject should be advised for adequate sleep
is commonly used as an analgesic for headache, over night and light breakfast in the morning
fever and as an anti-inflammatory agent. Felix on day of experiments (preferably 2 hr before)
Hoffman of Friedrich Bayer and Co., developed a • Avoid caffeinated drink or smoking or at least
stable and better-tolerated form of the drug 2 hr before the experiment or free of any
(acetylsalicylic acid) in 1897, though industrial substance abuse
production of salicylic acid was started in 1859, • Volunteer should get complete instruction of
however early preparations are associated with the procedure
side effects, such as unpleasant taste and • Monitoring equipment should be maintained
dyspepsia. Aspirin acts by inhibiting the cyclo- with regular calibration
oxygenase (COX) activity of prostaglandin (PG), • Basic operating procedure of the selected
which ultimately blocks the metabolism of instrument should be well explained to the
arachidonic acid to cyclic prostanoids such as volunteers.
thromboxane A2 (TXA2). Aspirin acts as anti-
Method: After initial screening volunteers should
platelet because it acetylates serine 529 in the active
participate in the study as per following inclusion
site of the cyclooxygenase-1 enzyme
and exclusion criteria:
(prostaglandin H2 synthase-1), permanently
deactivating it and preventing thromboxane A2 Inclusion Criteria
platelet activation. So in clinical practice it is most
commonly used for preventing thrombotic events, Healthy volunteers aged between 18-45 years of
such as ischemic strokes and acute myocardial either sex.
infarction. Aspirin is rapidly absorbed from the
Exclusion Criteria
gastrointestinal tract and peak plasma concen-
trations are achieved in 30 to 40 minutes, at 60 1. History of volunteers with: cardiovascular
minutes following ingestion of 100 mg of aspirin disease , significant hepatic/renal dysfuction,
which inhibits TXA2 production. Though plasma 2. History of drug allergy particularly to sali-
half-life is only 20 minutes but since platelet cylate
inhibition is of irreversible nature so once daily 3. History of substance abuse (narcotics) or any
dose maintains the antithrombotic action, so it is other drugs likely to affect interpretation of
used for primary and secondary prevention of study parameter.
vascular events. 4. Coffee, tea or smoking (past 24 hours) and
alcohol intake (past 48 hours).
Methods
• Volunteers are screened for any systemic pharmacology laboratory in the morning with good
disorder or any other disease conditions overnight sleep and light breakfast in the morning.
which may interfere with experimental A thorough medical examination should be done
protocol. before the experiment. All the procedure should be
• Healthy adult volunteers of either sex (willing explained in detail. Above mentioned study
to give written informed consent) parameters should be recorded at baseline before
• Stethoscope, mercury sphygmomanometer giving the drug, afterwards volunteers should be
• Spectrofluorimeter given 325 mg of aspirin with a glass of water of 200
• Drug: Aspirin ml. All the above mentioned parameters should be
recorded at frequent intervals at 0, 0.5, 1, 1.5, 2, 3, 4, metabolic pathways. Aspirin has been in the
5 and 6 hours following drug intake. Blood samples clinical practice since > 100 years but presently
are collected at various time interval as mentioned new problem has been started as aspirin resistance
above. where aspirin fail to protect individuals from
Laboratory procedure: Proteins are precipitated thrombotic complications. Though there are no
from plasma with sulphuric acid. Salicylates formal diagnostic criteria but aspirin resistance
are analyzed by mixing a sample of the super- generally describes the failure of aspirin to produce
natants fluids with NaOH and then measuring an expected biological response or the failure of
the fluorescence. The intensity of fluorescence aspirin to prevent atherothrombotic events. The
is proportional to the amount of salicylate incidence of aspirin resistance is 5 to 45% of the
present in the plasma. (Assay method: Jacobs JC, general population but the exact prevalence of
Pesce M. Arthritis Rheumatism, 1978;21:129-31). aspirin resistance is unknown. Measurement of
platelet aggregation, platelet activation, and
Results
bleeding time have all can confirmed variability in
Samples (μg/ml) Spectrofluorimetric reading
patient’s antithrombotic responses to aspirin
(Fluorescence unit)
therapy. Till now mechanism for aspirin resistance
Standard
remains uncertain but probably it is likely due to a
Blank plasma
2.5 combination of clinical, biological, and genetic
5 properties affecting platelet function. Aspirin
10 resistance can improved by educating the patients
20 regarding importance of compliance or by elimi-
40
Test sample (T1)
nating interfering substances like ibuprofen, and
Test sample (T2) avoiding increasing aspirin dose because increa-
sing the dose of aspirin does not enhance COX-1
Calculation inhibition. One can also use alternative drugs, e.g.
clopidogrel but scientific evidence are minimal that
FUT – FUB switching to alternative treatment strategies
Concentration of test sample = × CSS
FUS – FUB improves outcomes. Another problem is variability
in individual response to antiplatelet agents
Where,
FUT = Fluorescence unit of test emerging as a clinical problem: poor respon-
FUB = Fluorescence unit of blank siveness has been associated with an increased
FUS = Fluorescence unit of standard risk of ischemic events, including stent thrombosis.
CSS = Concentration of standard solution SUGGESTED READING
Discussion: Aspirin is a weak acid, following oral 1. Hankey GJ, Eikelboom JW. Aspirin resistance. BMJ
administration little amount is ionized in the 2004;328(7438):477-79.
stomach, approximately 50-80% aspirin is bound 2. Hankey GJ, Eikelboom JW. Aspirin resistance. Lancet
to plasma protein and volume of distribution is 2006;367(9510):606-17.
0.1-0.21 L/kg, 80% aspirin metabolized in the liver 3. Jacobs JC, Pesce M. Micromeasurement of plasma
by conjugation forms salicyluric acid and different salicylate in arthritic children. Arthritis Rheum 1978;
21(1):129-32.
glucuronic acid form. It is excreted by kidney mainly
4. Krasopoulos G, Brister SJ, Beattie WS, Buchanan
in the form of salicyluric acid, with elimination MR. Aspirin “resistance” and risk of cardiovascular
half-life 2-4.5 hours in case of dose < 250 mg, while morbidity: Systematic review and meta-analysis. BMJ
with higher doses > 4 gm half-life increases up to 2008;336(7637):195-98.
15-30 hours. Increased doses switch on from first 5. Tran HA, Anand SS, Hankey GJ, Eikelboom JW
order to zero order because of saturation of Aspirin resistance. Thromb Res 2007;120(3):337-46.
EXPERIMENT NO: 22 • Volunteer should get complete instruction of

the procedure
Aim
• Monitoring equipment should be maintained
To evaluate acetylator status by isoniazid (INH) with regular calibration
estimation in healthy volunteers. • Basic operating procedure of the selected
Background
volunteers.
Biotransformation of many drugs is genetically
determined. Different individuals have different Method: After initial screening, volunteers should
levels of metabolizing enzymes for a particular participate in the study as per following inclusion
drug. Rates of biotransformation of a drug in a and exclusion criteria:
population follows normal distribution but may
be bimodal or trimodal also. INH undergoes Inclusion Criteria
acetylation by N-acetyl transferase enzyme
present in the liver. Rate of metabolism follows Healthy volunteers aged between 18-45 years of
slow and fast acetylator pattern. In Europe, fast either sex.
acetylators constitute 40%, in Japan 85% and in
Asia 85-100%. In India, 30-40% considered as fast Exclusion Criteria
acetylators (half-life 1 hr) and 60-70% as slow
• History of volunteers with: cardiovascular
acetylators (half-life 3 hr). Other drugs, the
disease, significant hepatic/renal dysfuction,
metabolism of which can be affected by acetylator
status are, hydralazine, procainamide, dapsone • History of drug allergy
and some sulphonamides. • History of substance abuse (narcotics) or any
study parameters.
• Coffee, tea or smoking (past 24 hours) and
• Volunteers are screened for any systemic alcohol intake (past 48 hours)
disorder or any conditions which may
interfere with experimental protocol.
Methods
• Healthy adult volunteers of either sex (willing
to give written informed consent) All the volunteers should report to the clinical
• Stethoscope, mercury sphygmomanometer pharmacology laboratory in the morning with
• Spectrofluorimeter
good over night sleep and light breakfast in the
• Drug: INH
morning. A thorough medical examination
Precautions for Experiment should be done before the experiment. All the
procedure should be explain in detailed.
over night and light breakfast in the morning Afterwards the volunteers are asked to take INH
on day of experiments (preferably 2 hr before) 600 mg orally and 2 ml blood sample should be
• Avoid caffeinated drink or smoking or at least collected at 0 hr, 1 hr, 3 hr and 6 hr. Plasma INH
2 hr before the experiment or free of any estimation should be done spectrofluorometri-
substance abuse cally.
Results
Samples (μgm/ml) unit Spectrofluorimetric reading
Standard
Blank plasma
1.25 μg/ml
2.5
5
10
20
Test sample 0 hr 1 hr 3 hr 6 hr
Spectrofluorimetric reading
(Fluorescence unit) Plasma level
of INH
Discussion 2E1 genotype and the suscepti-bility to antituber-

culosis drug-induced hepatitis. Hepatology 2003;
There are different methods for determination of 37(4):924-30
acetylator status of INH: (1) Urinary estimation of 4. Hutchings AD, Routledge PA. A single sample
metabolites: Urine sample is collected at 6 hr after saliva test to determine acetylator phenotype. Br J
a dose of (600 mg) INH. Slow acetylators have Clin Pharmacol 1996;42(5):635-37.
<70% metabolite excreted while fast acetylator 5. Matar KM, Mayet AY, Ayoola EA, Bawazir SA, Al-
have >97% metabolite excreted. (2) Plasma levels: Faleh FZ, Al-Wazzan A. Isoniazid acetylation
INH is given orally 10 mg/kg (600 mg) and blood phenotyping in Saudi Arabs.J Clin Pharm Ther
sample is collected at 0 hr and 6 hr. If INH 2004;29(5):443-47.
6. Najim RA, Al-waizt M, Al-Razzuqi RA. Acetylator
>2.5 μg/ml at 6 hr = slow acetylator and if < 2.5
phenotype in Iraqi patients with allergic contact
μg/ml at 6 hr = fast acetylator. (3) Acetyl INH/
dermatitis. Ann Saudi Med 2005;25(6):473-76.
INH, metabolite ratio: If ratio is <0.48 then it is 7. Pande JN, Pande A, Singh SP. Acetylator status,
slow acetylator and if ratio is >0.77 then it is called drug metabolism and disease. Natl Med J India 2003;
as fast acetylator.(4) t1/2 in INH: Slow acetyaltor: 16(1):24-26.
140-200 min and Fast acetylator: 45-80 min, but 8. Scott EM, Wright RC. Fluorometric determination of
in this test repeated sampling is required. isonicotinic acid hydrazide in serum. J Lab Clin Med
1967;70(2):355-60.
SUGGESTED READING 9. Scott EM, Wright RC. Fluorometric determination of
isonicotinic acid hydrazide in serum. J Lab Clin Med
1. Bozok Cetintaþ V, Erer OF, Kosova B, Ozdemir I, 1967;70(2):355-60.
Topçuoðlu N, Aktoðu S, Eroðlu Z. Determining the 10. Singh SP, Pande JN, Khilnani GC, Kailash S.
relation between N-acetyltransferase-2 acetylator Comparison between serum and urinary sulphadi-
phenotype and antituberculosis drug induced
midine acetylation as predictors of isoniazid
hepatitis by molecular biologic tests. Tuberk Toraks
acetylator status in patients with pulmonary
2008;56(1):81-86.
2. Furet Y, Bechtel Y, Le Guellec C, Bechtel PR, Autret- tuberculosis. Indian J Chest Dis Allied Sci 1996;
Leca E, Paintaud G. Clinical relevance of N- 38(1):5-11.
acetyltransferase type 2 (NAT 2) genetic poly- 11. Sohrani AS, Ahmad B, Janbaz KH. Acetylation
morphism] Therapie 2002;57(5):427-31. percentage method for determination of acetylator
3. Huang YS, Chern HD, Su WJ, Wu JC, Chang SC, status in human volunteers and tuberculous patients.
Chiang CH, Chang FY, Lee SD. Cytochrome P450 Pak J Pharm Sci 1995;8(1):11-16.
EXPERIMENT NO: 23 or intestines, or gastroesophageal reflux disease

(GERD) or slow digestion.
Aim
To evaluate anticholinergic effect of oxybutynin Methods
(30 mg tablet) on salivary secretion in healthy Method 1: The baseline salivation is measured by
volunteers. placing the three dental rolls under the tongue for
2 min. Thereafter, interventional drug is adminis-
Background
tered and wait for 1-2 hr (depending on half-life
Adverse effects of anticholinergic medications are of drug). Salivary secretion is again measured by
well documented. Drugs such as atropine and using three dental rolls which are inserted into
scopolamine inhibit muscarinic receptors and can the buccal cavity under the tongue for 2 min.
produce both peripheral (constipation, dry mouth, The salivary secretion rate is observed at 0 hr
tachycardia, urinary retention, reduced sweating) (baseline), 1, 2, 3, 6 and 12 hr and the evaluation
and central (cognitive and memory impairment, of the salivary flow is done by observing the
confusion, delirium, headache, blurred vision, difference in weight of three dental rolls after and
dizziness, and drowsiness) effects. Other drugs before the intervention drug administration.
like oxybutynin and tolterodine are clinically used Rate of salivary secretion = (Weight of three
to treat symptoms of overactive bladder, such as dental rolls after the treatment – Weight of
incontinence, frequent or urgent urination and three dental rolls at baseline)
increased night-time urination. Oxybutynin is
preferred in the experiment because it causes Method 2: Volunteer should rinse his/her mouth
severe dry mouth. In a study, it was reported that with approximately 60-100 ml of tap water.
¼th of patients who begain oxybutynin treatment Volunteers are allowed to wait for 5-10 min and
had to stop because of dry mouth. then, an accurately weighed 1 × 1 inch square of
paraffin (pre-weight) is placed on each subject’s
Materials and Methods tongue. The subject is then instructed to chew the
paraffin for 2 minutes, after which the accumu-
• Healthy adult volunteers (Willing to give lated saliva and the chewed paraffin is expecto-
written informed consent form) rated into a previously weighed clear dry container
• Cotton dental rolls and weighing balance (beaker, wide mouth glass tube, etc.).
• Oxybutynin extended release (30 mg). The salivary secretion rate is observed at 0 hr
(baseline), 1, 2, 3, 6 and 12 hr and the evaluation
Initial Screening of Volunteers of the salivary flow is done by observing the
Subject is screened for detailed evaluation of any difference in weight of paraffin square after and
disease condition, detailed history should be taken before the intervention drug administration.
in respect to smoking, alcohol consumption, drug Rate of salivary secretion = (Weight of paraffin
therapy, or any associated disease conditioned. and sputum after the treatment - Weight of
paraffin square at baseline)
Precautions for Experiment
Do not give medication to the volunteers who are
Salivary secretion
allergic to oxybutynin, or they have untreated or
Pre-drug(0 hr; Post-drug
uncontrolled glaucoma or are unable to urinate.
baseline) 1 hr 2 hr 3 hr
Volunteers are excluded, in cases of glaucoma,
any liver disease, kidney disease, an enlarged 1st person
2nd person
prostate, ulcerative colitis, blockage in stomach
Discussion with the appearance of an erythema at the site of

Oxybutynin chloride exerts direct antispasmodic injury/injection, followed by development of a
effect on smooth muscles by competitively flare surrounding the site and finally due to fluid
antagonizing the M1, M2, and M3 subtypes of the leakage from capillaries under skin, a wheal forms
muscarinic acetylcholine receptors and inhibits at the site. Hence, identified as redness or swelling
the muscarinic action of acetylcholine on smooth due to release of histamine. Drugs which can be
muscles. As compared to atropine, it exhibits 1/ evaluated are levocetirizine, desloratadine,
5th of the anticholinergic activity of atropine on fexofenadine, loratadine, etc.
the rabbit detrusor muscle, but have 4 to 10 times
the antispasmodic activity. Transdermal patch is Materials and Methods
also available for oxybutynin, but due to less
• Healthy adult volunteers (willing to give
anticolinergic side effects, salivary secretion is
written informed consent form)
affected moderately.
• Facilities for clinical pharmacology laboratory
SUGGESTED READING (Appendix IV)
• Drugs: levocetirizine (5 mg), desloratadine 5
1. Bye CE, Clubley M, Henson T, Peck AW, Smith SA, mg, fexofenadine HCl 180 mg, montelukast
Smith SE. Changes in the human light reflex as a sodium 10 mg, etc. and histamine.
measure of anticholinergic effect of drugs. A
comparison with other measures. Eur J Clin
Pharmacol 1979;15:21-25. Initial Screening of Volunteers
2. Chancellor MB, Appell RA, Sathyan G, Gupta SK. A
Subject is screen for detailed evaluation of any
comparison of the effects on saliva output of
oxybutynin chloride and tolterodine tartrate. Clin disease condition; detailed history should be taken
Ther 2001;23:753-60. in respect to smoking, alcohol consumption, drug
3. Steiner JE, Birnbaum D, Karmeli F, Cohen S. Effect therapy, or any associated disease conditioned.
of Diazepam on Human Salivary Secretion. Digestion
1970;3(5):262-68.
Inclusion Criteria
4. Tripathi SK, Griyappanavar CR, Lal A, Biswas K,
Biswas NR, Sankaranarayanan A, Sharma PL. Volunteer aged 18-40 years, willing to give written
Evaluation of antimuscarinic activity in human informed consent.
volunteers: A teaching aid in clinical pharmacology.
Indian J Physiol Pharmacol 1995;39(2):163-65.
Exclusion Criteria
EXPERIMENT NO: 24 Volunteers should be excluded, if there is h/o any
concomitant chronic or acute illness; or history of
Aim
or current cardiovascular (including cardiac
To demonstrate histamine induced wheal and arrhythmias), respiratory, hepatic, renal, gas-
flare in healthy volunteers. trointestinal, endocrinological, neurological, or
psychiatric disease; anaphylactic shock as well
Background as disorders capable of altering the absorption,
Quantitatively antihistaminic potency of drugs is metabolism or elimination of drugs, or constituting
evaluated by the histamine-induced wheal and a risk factor when taking the trial medication.
flare response. It is an eruption of skin either by In case of concomitant drugs such as cortico-
injury or direct injection of an allergen in the skin. steroids, antihistamines, cromoglycates, or
It is typically characterized by three stages, begins leukotriene antagonists.
Volunteer with habit of heavy caffeine Methods

drinkers (>five cups of coffee, tea, cola, etc. per The volunteers should be selected after proper
day). screening. Volunteers are injected histamine in
Volunteers with exposure to skin irritants or the increasing doses of 0.1, 0.4 and 1.6μg intra-
UV light in last 48 hours before each visit should dermally at the back (at the middle part of both
be excluded. shoulder) and after 3-4 hr, wheal and flare
formation are observed and assessed. Effect of
Precaution antihistaminic drug is analyzed by the same
method. The observation can be performed 15
Volunteers should be evaluated in detail about minutes before and 90 minutes after antihista-
allergy or intolerance to the study drugs-to-drugs minic drugs such as levocetrizine (5 mg) intake.
related to the study procedures, as well as any Other drugs which may be used in the experiment
medicine chemically related to the study drugs or are desloratadine 5mg, fexofenadine HCl 180 mg,
their excipients. montelukast sodium 10 mg, etc.
Observation and Result

Volunteers Histamine
Pre drug(15 minbefore drug intake; baseline) Post-drug (90 min after drug intake)
Wheal Flare Wheal Flare
0.1 0.4 1.6 0.1 0.4 1.6 0.1 0.4 1.6 0.1 0.4 1.6
1 WFB – WFT
2 100
WFB
Percentage (%) of wheel/flare inhibition is antihistaminic drug one should keep in mind that
calculated by, it may provide false negative result if the test is
not done in specified time.
SUGGESTED READING
where 1. Khosla PP, Saha N, Koul A, Chakrabarti A,
WFB = Wheel/flare after histamine Sankaranarayanan A, Sharma PL. Effects of
WFT = Wheel/flare after drug treatment ranitidine alone and in combination with chlor-
pheniramine on histamine-induced wheal and flare
Discussion and psychomotor performance. Indian J Physiol
Pharmacol 1993;37(2):132-34.
It is the direct method of evaluation of anti- 2. Nelly Frossard, Margherita Strolin-Benedetti, Ashok
histaminic activity of a drug. Wheel and flare Purohit, Gabrielle Pauli. Inhibition of allergen-
induced wheal and flare reactions by levocetirizine
method has been used traditionally as a challenge
and Desloratadin. Br J Clin Pharmacol 1965;2:179.
in skin pharmacodynamic studies of the anti- 3. Peck AW, Fowle ASE, Bye C. A comparison of
histaminic activity of new compounds. The subject triprolidine and clemastine on histamine antagonism
is sensitized with allergen, as normally used for and performance tests in man: Implications for the
mechanism of drug induced drowsiness. Eur J clin
the diagnosis of allergy by skin prick testing and Pharmacol 1975;8:455-63.
so, the clinical efficacy may be strengthened by 4. Saha N, Sachdev A, Bhasin DK, Sankaranahyanan
studies using challenge with the specific aller- A, Khosla PP, Singh K, Sharma PL. Clinical
gens. An advantage is that allergen-induced wheal evaluation of the effect of omeprazole, cimetidine,
famotidine and ranitidine on histamine induced
and flare studies recruit allergic subjects rather than cutaneous wheal and flare response. Int J Clin
healthy volunteers. During the screening of a Pharmacol Ther Toxicol 1993;31(7):322-25.
Laboratory Experiments
33 (Assay)
EXPERIMENT NO: 25 stationary phase in contained within a short,

small bored column through which the liquid
Aim mobile phase is pumped at high pressure.
Therapeutic drug monitoring in pharmacology In this chapter few examples are given, so that
(antiepileptic/lithium/digoxin). it will be convenient for students to know, how
TDM is done in the pharmacology laboratory.
Background
(A) Estimation of phenytoin, carbamazepine
Therapeutic drug monitoring (TDM) is an investi- and phenobarbitone by HPLC
gational procedure from last three decades and
found to be very beneficial in the management of Procedure
narrow index therapeutic agents such as anti- Sample preparation
epileptic drugs, digoxin, lithium, antipsychotics,
etc. during the therapy. It has an important place
in the patient’s management and managing their
effects in chronic therapy where clinical symptoms
and signs of toxicity can also be difficult to detect
and interpret. The main objective of the TDM is to
optimize drug therapy by individualizing dose
according to the serum/plasma concentration
and the clinical effects. It is also frequently used
to assess drug toxicity and patient’s compliance.
The most, laboratory procedure for TDM is
performed by the HPLC because of the high
sensitivity or some drug like digoxin is performed
by the chemical/ELISA methods. Hence, all
principle of HPLC separation is applied to the
same; briefly HPLC separation of solute molecule
depends on the distribution of molecules between Chromatographic Conditions
a stationary and a liquid mobile phase. The Mobile phase: Acetonitrile: Methanol : 4 mmK+
relative affinities of solute for two phases may phosphate buffer(pH 6.0) in ratio of:
involve one or more of adsorption, partition, ion 20:40:40 (v/v/v)
exchange or a salvation mechanism and these Flow rate : 1.0 ml/min
determine. The separation characteristics the Temperature : Ambient
AUFS : 0.2 available. Lithium has narrow therapeutic index

Detector : UV detector at 215 nm and the therapeutic level between toxic, thera-
Injection volume : 100 µl peutic and subtherapeutic serum levels are liable
Sensitivity : 0.1 µg/ml to change because of various factors. Individual
Standards : 4-16 µg/ml variation, poor compliance, renal disease,
seasonal variation, etc. are some reasons which
Results make plasma lithium level estimation essential
as a part of better management of patients. It is a
Normal level
monovalent cation which shows a high degree of
Phenytoin 10-20 mg/L, Carbamazepine 4-10 interindividual variation and narrow therapeutic
mg/L, phenobarbitone 15-40 mg/L index. The variability in lithium levels need to be
carefully reviewed with caution, since it can affect
Calculate Concentration of Unknown? the efficacy and safety in the patients. In case of
variation oral dose modification is needed for the
Discussion
management toxicity as well as loss of efficacy.
Several studies have already demonstrated So, the therapeutic drug monitoring (TDM) of
usefulness of TDM in epilepsy management. lithium is essential.
Several tertiary centers reported that increasing
demand of TDM is because of increased awareness Method of Estimation
of physicians and availability of facility in most
Estimation of plasma lithium is commonly done
of the centers. So, in future physician will more
by lithium autoanalyzer. The principle is potential
depend on TDM because of its usefulness,
developed by the lithium ion selective electrode
however it increases the health care cost because
with respect to the reference electrode. Most
of limitation of methodology and most of the drug
sample salts dissociate to their ions and exchange
required sophisticated instruments. In the recent
reaction occurs between the relevant electrode and
time various development has been taken place
the ion which produces a potential between the
for rationalizing the TDM request by compu-
ion selective electrode and the reference one.
terized program with educational program which
Lithium ion selective electrode consists of a
will definitely improve the quality of TDM. Most
neutral carrier- based lithium sensor immobilized
of the TDM laboratories routinly conduct for older
in polyvinyl chloride. It has electrical connection
antiepileptics like phenytoin, carbamazepine,
by silver or silver chloride wire. The reference
phenobarbitone, since it is still widely prescribed.
electrode usually contain shell filled with
Studies reported frequent inter-individual varia-
saturated potassium chloride which is separated
tion in the plasma level/dose ratio for these drugs.
from the sample by a membrane. (For other
Though, there are lot of variations in Western and
methods refer to Brown and Legg et al, 1970. Ann
Asian populations. However, TDM pattern is
Clin Biochem 1970; 7: 13-18.)
similar in developed and developing countries.
Limitation is very few centers publish their TDM Discussion: Therapeutic drug monitoring (TDM),
pattern in the literature. is considered useful in patients suffering from
mood disorders, though it increases the health
care cost. Several studies indicates a pattern of
(B) Estimation of lithium increasing awareness and caution among
physicians while prescribing lithium. TDM
Background
assisted better management of psychiatric
Lithium remains the drug of choice for bipolar patients for early detection and prevention ADR
disorder, though various other drugs are and to avoid lack of therapeutic response. Some
Laboratory Experiments (Assay)  335
of recent studies suggested therapeutic drug Besides ELISA, other chemical methods are
monitoring using salivary plasma lithium levels available for quantitative estimation.
however it was not found beneficial to find the
plasma correlation. The normal therapeutic range SUGGESTED READING
of lithium is 0.6 – 1.2 meq/L. Studies showed,
1. Manpreet Sukhija, Bikash Medhi, Pandhi P. Effects
patients with an early onset of MDP has greater
of artemisinin, artemether, arteether on the pharma-
variation in the lithium levels. It has been reported cokinetics of carbamazepine. Pharmacology,
that variations observed in Italy 10% , 2006;76:110-16.
Netherlands 5% and in a subtropical country 2. Manpreet Sukhija, Bikash Medhi, Pandhi P. Effects
like India seasonal variations observed in plasma of artemisinin, artemether, arteether on the pharma-
lithium levels of up to 25%, with no significant cokinetics of phenytoin. Meth Find Clin Exp
change in the oral lithium dosage. So, lithium Pharmacol,2006,28(3):89-94.
shows marked inter individual variation, genetic 3. Medhi B, Prakash O, Jose VM, Pradhan B, Chakra-
barty S, Pandhi P. Seasonal variation in plasma levels
variability in pharmacokinetics, drug-drug
of lithium in the Indian population-is there need to
interactions, and high levels can lead to adverse
modify dose? Singapore Medical Journal 2008;
effects require TDM in those patients who are on 49(9):724-27.
prophylaxis. 4. Medhi B, Sukhija M, Prakash A, Gaikowad S, Bansal
V, Pandhi P. Effects of Etoricoxib on the Pharma-
Digoxin estimation: The clinical usefulness of the cokinetics of Phenytoin. Pharmacological Reports
measurement of serum digoxin is due to its narrow (Poland Journal of Pharmacology) 2008;60(2):
therapeutic ratio. In addition, individuals may 233-37.
present variable response to digoxin with an 5. Medhi B, Sukhija M, Sumedh G, Vinu M Jose,
apparent increase in susceptibility to toxicity with Chakrabarty S, Pandhi P. Lithium Therapeutic drug
age. ELISA is a sensitive method for digoxin monitoring pattern at a tertiary care hospital in India.
JK Practotioner 2006,13(1):15-17.
quantitation in serum. The activity of the enzyme
6. Prakash A, Medhi B. TDM pattern of the antiepileptic
present on the surface of the well is quantities by drugs in developed and developing countries: An
reaction with a suitable substrate to produce color. overview. Neurosciences 2009;14(3):447-49.
The employment of several serum references of 7. Vinu J, Medhi B, Pandhi P. Antiepileptic Therapeutic
known digoxin concentration permits cons- Drug Monitoring pattern at a tertiary care hospital
truction of a graph of activity and concentration. in India. Nepal Med Coll J 2006;8(2):107-10.
Impact Factor
34
It is the general belief that there is direct Calculation of the IF of a journal for a selected
correlation between impact factor (IF) and journal year depends on the average number of citations
quality ratings among the physicians and of an article getting published in that journal
researchers because it is directly related to the during the last two years from all the published
journal citation frequency. Most importantly, IF articles in that year, i.e. If the IF of a journal is
helps to guide the researcher to choose the best 3.0 in 2008, it reflects that on an average the
journal according to their frequency of citation articles published in 2006 and 2007 were cited
in their field. The IF and citation varies with the 3 times in the collection of all ISI indexed
types of the journals such as journal cited by both journals which was published in 2008.
physicians and researchers have the high IF Hence, the ratio obtained from dividing
(New England Medical journal, The Lancet etc.), citations received in one year (numerator) by paper
followed by the journals cited by either by published during the two previous years (deno-
physician or researchers. minator).
The main purpose of creation of impact IF= CR/PP
factor in the biomedical research field is to Where,
measure the journal’s value by calculating the CR = Citations received in one year
average number of citations per article over a PP = Paper* published during the two pre-
period of time which was initially designed by vious years
Eugene Garfield in 1950’s. It was introduced in *paper: original/research paper, review article,
scientific community as an assessment tool to peer review, proceedings, etc.
evaluate the value of the scientific journal by The recent trend in scientific world is that, if
calculating the number of citations of an article the researchers or physicians want to be recog-
published in particular journal over a specific
nized, they should have a good number of
time period.
publications with good citations. Thus, it has
The terminology of impact factor was first
become mandatory for a scientist to publish their
used in 1961 after the publication in Science
work in journals with high IF. And this criterion
Citation Index (SCI) in 1963. Presently, it is
popularly called Journal Citation Reports (JCR), has lead to the development of the long-time belief
and burgeoning literature using bibliometric that the number only counts. Therefore, a great
measures. In general number of citations of a number of publications make scientists popular
particular article indicates only the mean interest and distinguished in their field. Major criteria in
of scientists for that article thus the IF highlights present time among scientific community, for
average interest of that article which got publish evaluating the status of the scientific journals
in the journal. and also the status of the scientist on the basis of
Impact Factor  337
their publication output, to assess how they are List of leading scientific journals and their impact factor
actively engaged in the research. For example, In (IF)
some countries like South Korea, China, and Journals name Impact factor (2007)
Pakistan, their science ministries are offering
Nature Medicine 31.921
cash rewards to their scientists if they publish NEJM 52.589
papers in journals with high IFs such as Nature, The Lancet 29.887
Science, or Cell, etc. JAMA 25.547
Journal Citation Index (JCR) are used as the BJCP 2.681
JPET 4.003
only evaluation criteria rather than to quanti- Pharmacological Review 18.823
fication of scientific contribution itself. Because Molecular Pharmacology 4.088
original idea of citation analysis was developed Pharmacology Research 9.643
to protect against the uncritical citation of Trends in Pharmacological Sciences 10.4
fraudulent data or even disputed data. Some of Therapeutic Drug Monitoring 3.032 (2006)
study questioned the meaning of IFs, stating that NEJM: New England journal of medicine; BJCP:
they actually represent popularity rather than British journal of clinical pharmacology; JPET:
prestige. Journal of pharmacology and experimental
Eugene Garfield, the inventor of the IF, therapeutics; JAMA: Journal of the American
emphasized that its potential value is primarily medical association.
in the management of library journal collections
to determine their optimum makeup, providing SUGGESTED READING
solid basis for cost-benefit analysis of subscription 1. Garfield E. How can impact factor can be improved?
budgets. Infact inventor of IF never predicted that BMJ 1966;313:413-15.
in scientific community IF will be used as a criteria 2. Garfield E. Use and misuse of citation frequency.
for judging the scientist, quality and providing Curr Contents 1985;43:3-9.
3. Kumar V, Upadhyay S, Medhi B. Impact of Impact
research grant.
factor in Biomedical Research, its use and misuse:
People misuse the IF as there are no specifically an overview. Singapore Med J 2009;50(8):752-55.
defined principles governing its interpretation. 4. Linardi PM, Coelho PMZ, Coster HMA. The impact
The IF is used to measure the importance of factor as a criterion for the quality of scientific
journals as well as researcher potential, for which production is a relative, not absolute measure. Braz
it was never intended, and it is used to make faulty J Med and Biol Res 1996;29:555-61.
5. Moed HF. The impact factor debate the ISI’s uses
comparisons, including journals themselves. So
and limits. Nature 2002;45:731-32.
misuse of IF is a common problem in research field, 6. Rey-Rocha J, Martín-Sempere MJ, Martinez-Frías JY
as scholars have complained about the misuse of López-Vera F. “Some misuses of journal impact factor
the IF since long-time. in research evaluation” Cortex 2001;37(4): 595-97.
Computational
35 Pharmacology
In the changing trend of education, need of approaches are now being used to facilitate the
computer and its knowledge is now necessary in experimental determination of macromolecular
each and every field of science, meanwhile it is structures by aiding in structural refinement based
found very useful tool for explaining any unknown on either nuclear magnetic resonance (NMR) or
or new topic to the students or any scientific body X-ray data. These can also be applied in situations
in a attractive way such as making animated films where experimentally determined structures are
of drug action on a system or showing the not available. With the rapid advance in gene
molecular action of a drug, side effects, transport technology, including the human genome project,
of drugs, etc. Additionally, computer helps in the ability of computational approaches to
demonstration of drug effects on whole animal or accurately predict 3D structures based on primary
on isolated tissues of practical pharmacology for sequence represents an area that is expected to
undergraduate students or postgraduate have a significant impact.
medical/pharmacy students. So, computational Drug design and development is another area
pharmacology or computational therapeutics is a of research in pharmacology and it is correlated
rapidly ubiquitously growing area in the field of with computational biochemistry and biophysics
development of techniques for using software to is having an ever-increasing impact. Compu-
capture, analyze and integrate biological and tational approaches can be used to aid in the
medical data from many diverse sources. The term refinement of drug candidates, systematically
‘in silico’ is an experiment performed by computer changing a drug’s structure to improve its
and is related to the more commonly known in pharmacological properties, as well as in the
vivo and in vitro methods. Among the “3Rs” of identification of novel lead compounds. The latter
Russell and Burch (1959), developed to reduce can be performed via the identification of
the animal use or use with caution in experiment compounds with a high potential for activity from
and research, the replacement with alternative available databases of chemical compounds or
method like in vitro or in silico is the first choice, via de novo drug design approaches, which build
whereas the other two are refinement of methods totally novel ligands into the binding sites of target
to minimize any adverse effects to individual molecules. At the molecular level, computational
animal and reduction of animal use to achieve genomics helps pharmacological studies, about
consistent scientific objectives. These methods are learning of the genomes of cells and sometime uses
increasingly used in discoveries or advances in DNA microarray to identify the genes expressed
pharmacology and therapeutics. Computational in each cell types.
(in silico) methods have been developed and Other important related allied fields are
applied to pharmacology in hypothesis deve- Computational biomodeling, a field concerned with
lopment and testing. Additionally, computational building computational models of biological
Computational Pharmacology  339
systems, Computational biochemistry and biophysics, 4. Balakin KV, Ivanenkov YA, Savchuk NP, Ivaschenko
which make extensive use of structural modeling AA, Ekins S. Comprehensive computational
assessment of ADME properties using mapping
and simulation methods such as molecular
techniques. Curr Drug Disc Tech 2005;2:99-113.
dynamics to explain the kinetics and thermo- 5. Chen YZ, Zhi DG. Ligand–protein inverse docking
dynamics of protein functions, whereas clinomics and its potential use in the computer search of protein
is a bridge between basic biological data and its targets of a small molecule. Proteins 2001b;43:217-
effect in clinical setting. For example certain genes 26.
such as BRCA1 are associated with a higher 6. Dudek AZ, Arodz T, Galvez J. Computational
methods in developing quantitative structure–
probability of developing breast cancer.
activity relationships (QSAR): a review. Comb Chem
High Throughput Screen 2006;9:213-28.
Application of in silico Methods 7. Ekins S, Swaan PW. Development of computational
models for enzymes, transporters, channels and
1. Maintains databases, quantitative structure-
receptors relevant to ADME/TOX. Rev Comp Chem
activity relationships, pharmacophores, 2004;20:333-415.
receptor modeling and other molecular 8. Grzybowski BA, Ishchenko AV, Kim C-K, Topalov
modeling such as data mining and data G, Chapman R, Christianson DW, et al. Combinatorial
analysis which requires a computer. computational method gives new picomolar ligands
2. In silico methods are primarily used alongside for a known enzyme. Proc Natl Acad Sci USA 2002;
the generation of in vitro data both to create 99:1270-73.
9. Kulkarni SA, Zhu J, Blechinger S. In silico techniques
the model and to test it in the discovery and
for the study and prediction of xenobiotic meta-
optimization of new drug with affinity to bolism: a review. Xenobiotica 2005; 35: 955-73.
receptor target, its pharmacokinetics and 10. Langowski J, Long A. Computer systems for the
toxicity properties in addition to physico- prediction of xenobiotic metabolism. Adv Drug Del
chemical characterization. Rev 2002; 54: 407-15.
3. Makes easy correlation of human genome 11. Lemmen C, Lengauer T. Computational methods
through computational and experimental data for the structural alignment of molecules. J Comput
Aided Mol Des 2000;14:215-32.
which can be correlated with all data types.
12. Lipinski CA, Lombardo F, Dominy BW, Feeney PJ.
4. Computational approaches can be used to Experimental and computational approaches to
investigate the energetics associated with estimate solubility and permeability in drug
changes in both conformation and chemical discovery and development settings. Adv Drug Del
structure of the drug. Rev 1997;23:3-25.
13. Mestres J. Computational chemogenomics approaches
SUGGESTED READING to systematic knowledge-based drug discovery. Curr
Opin Drug Discov Dev 2004;7:304-13.
1. Albert A. Relations between molecular structure and 14. Rockey WM, Elcock AH. Rapid computational
biological activity: Stages in the evolution of current identification of the targets of protein kinase
concepts. Ann Rev Pharmacol 1971;11:13-36. inhibitors. J Med Chem 2005;48:4138-52.
2. Aradi I, Erdi P. Computational neuropharmacology: 15. Sieburg HB. Physiological studies in silico. Studies
Dynamical approaches in drug discovery. Trends in the Sciences of Complexity 1990;12:321-42.
Pharmacol Sci 2006;27:240-43. 16. Sung M-H, Simon R. In silico simulation of inhibitor
3. Arý¨ens EJ. Receptors: From fiction to fact. Trends drug effects on nuclear factor-kB pathway dynamics.
Pharmacol Sci 1979;1:11-15. Mol Pharmacol 2004;66:70-75.
Pharmacokinetics/
36 Pharmacodynamics
PHARMACOKINETICS/
PHARMACODYNAMICS (PK/PD)
Pharmacokinetic (PK) and pharmacodynamics
(PD) are the principles and separate part of
pharmacology which always play basic assess-
ment in the drug development. The new concept
of PK/PD model is one which bridges the
principles of both PK and PD. This is a measure of
response of a drug in a postulated time (Fig. 36.1).
In many condition, it is difficult to identify the
minimum effective dose through dose-response
data in a new drug such as antihypertensive,
anticancer drug, prostaglandins, H2-blockers,
respiratory drugs, etc. There are several clinical
factors which have influence on the dose – Fig. 36.1: Pharmacokinetics pharmacodynamics
response-time relationship such as demographics modeling
(age, sex, weight, body weight, body surface area, PK/PD modeling is varying with the com-
lean body mass, etc.) or any disease status. partmental model designed for the analysis. It is
The objectives of PK/PD model are: mainly of two type, follows the classification of
• Forecasting of response of a drug dose pharmacokinetic compartments, i.e. single com-
• Dose selection and dosing interval partment, time –independent PK/PD model and
• Duration of drug response complex PK/PD, time dependent models.
• Estimate therapeutic window The first approach is the simplest and the
• Identify mechanism of action drug concentration is distributed in the one com-
Hence, the pharmacological effect of drug is partment, i.e. blood and it correlates direct
varying accordingly in relation with the plasma pharmacological action, limiting its direct effect
concentration; either it is antihypertensive, anti- in one compartment only. So, in this context there
coagulant or diuretics. Animal study described is no time dependant event. Whereas, the second
the relationship between the concentration of drug approach complex PK/PD is the commonest in
in blood or plasma and drug receptor occupancy vivo approach, which is involving sequential
or functional response, provide clinically useful analysis of the concentration versus time and
test regarding potency, efficacy and duration of effect versus time. This model tells about the dose-
the effect. response-time relationship which gives a
Pharmacokinetics/Pharmacodynamics  341
biophase responses, i.e. compartment where drug 3. Kristensen NR, Madsen H, Ingwersen SH. Using
shows its effect. (Represents half-life, bioavai- stochas-tic di_erential equations for PK/PD model
lability and potency). So, briefly complex PK/ development. J Pharmacokinet Pharmacodyn 2005;
32:109-41.
PD model determine the drug effect in the 4. Lieberman R, Nelson R. Dose-response and
compartment in time course of drug concen- concentration-response relationships: Clinical and
tration. regulatory perspectives. Ther Drug Monit 1993; 15(6):
PK/PD model approach limits, its use in in 498-502.
vivo constants which can be applied to the other 5. Meibohm B, Derendorf H. Basic concepts of
in vivo data calculation but not link with in vitro pharmacokinetic/pharmacodynamic (PK/PD)
modelling. Int J Clin Pharmacol Ther 1997;35(10):
data conversion. The application of PK/PD 401-13.
modeling is documented in cardiovascular (CVS), 6. Schaefer HG, Heinig R, Ahr G, Adelmann H, Tetzloff
central nervous system (CNS), oncology and W, Kuhlmann J. Pharmacokinetic-pharmacodynamic
gastroenterology. modelling as a tool to evaluate the clinical relevance
of a drug-food interaction for a nisoldipine controlled-
release dosage form. Eur J Clin Pharmacol 1997;
SUGGESTED READING 51(6):473-80.
7. Tornoe CW, Jacobsen J L, Pedersen O, Hansen T,
1. Derendorf H, Möllmann H, Hochhaus G, Meibohm Madsen H. Grey-box modelling of pharmacokinetic/
B, Barth J. Clinical PK/PD modelling as a tool in pharmacodynamic systems. J Pharmacokinet
drug development of corticosteroids. Int J Clin Pharmacodyn 2004b;31(5):401-17.
Pharmacol Ther 1997;35(10):481-88. 8. Wagner JG. Kinetics of pharmacologic response. I.
2. Gibb IA, Anderson BJ. Paracetamol (acetaminophen) Proposed relationships between response and drug
pharma-codynamics: Interpreting the plasma concentration in the intact animal and man. J Theor
concentration. Arch Dis Child 2008;93(3):241-47. Biol 1968;20(2):173-201.
Promotional Product
37 Literature
PROMOTIONAL PRODUCT LITERATURE for the researcher, clinicians and other health
professionals, whereas promotional product
The commonest sources for providing information
literature is a form of advertising tool, provided to
from industry to practicing physician are verbal,
the clinician, chemists and other related health
written and computerized, e.g. professional
professional who directly/indirectly related to the
meeting, advertising in journal, e-mail or from
patients health. But, it is different from conven-
medical representative, etc. Lot of money has been
utilized for effective communication to physician. tional advertising in few modes:
Most important aspect of promotional literature 1. Promotional product literature is more
is to look for sources of references which provide selective. Since, it is distributed through
comprehensive update to the physician from controlled means, rather than through general
leading medical national/international journals media placement, the target audience can be
or international organization data like WHO. So, more sharply defined and the message can
product literature is the needful source of be written with the targeted ‘consumer’ in
information regarding the drugs which provide mind.
its complete knowledge about its nature, class, 2. Initial readership is virtually 100%. When a
pharmacological effects and the known side effect, particular consumer group is targeted,
interaction and contraindication for the medical message can be tailored to group which is pre-
practitioners as well as for patients. Hence, disposed to a particular message.
promotional product literature can be defined as 3. There is the opportunity for direct movement
graphic and/or written material prepared by/for into the objective. Because of the established
one party, which is made available to the public interest on the part of the recipient.
for information and distribution, for the purpose In a clinical set-up, a major marketing
of promoting or marketing the particular product technique used by pharmaceutical companies is
or brand. Generally, the sources of product direct-to-physician marketing (DTP). This form of
literature are categorized into several categories, marketing frequently employs promotional
but broadly they are divided into following marketing literature, based on clinical research,
classes:
which serves as an important source of drug
information and may influence the prescribing
behavior of a physician.
Role of Promotional Product Literature

Primary, secondary and tertiary product It can be used in following different ways for the
literatures are importantly informative literatures best possible results:
Promotional Product Literature  343
1. Fulfilling requests for information: Many people indications for use together with the dosage and
want detail information regading a product method of use and a brief statement of the
before purchase. So, information can be contraindications, precautions and side effects.
provided in the form of promotional literature
without visiting them personally. Designing Promotional Literature
2. Informational displays: This literature can serve The need and production of the promotional
as a brochure display in a doctor’s office and literature is a result of feedback from market
providing the information to both current research of established need, its attractive
customers and prospects. promotional designing and commercial need.
3. Leaving with prospects following sales meetings:
It is hard to close a sale with just one visit to a Establishing a need: First step requires the
prospect but making repeat visits is also not establishment of the need of literature which can
cost-effective. Hence, leaving a piece of be done by answering some questions like what
promotional literature behind helps in provi- is the specific purpose of the proposed printed
ding additional information to clinician piece.
without making the repeated sales meetings. • Is it absolutely necessary?
4. Use as direct mail pieces: Promotional literature • What is the target audience? And
can be used proactively as direct mail pieces • How big is the “Universe” i.e. checking the
and mailed to people in the target market. demographic information?
5. Promotional literature as a sales tool: A well Deciding on a format: There are many formats
written literature helps a salesperson in which include leaflets, brochures, master
remembering the benefits and features of a brochures, fliers, scanable letters, sales letters, rack
product more easily and gives more effective cards, counter cards, postcards, pocket cards,
sales presentations by illustrating each and meaning and circulars, etc. The choice of one
every point and helps to take business to the which is best for the purpose is influenced by
higher level of sales and profits. various factors like the proposed use, number of
different printed pieces and the budget for
Components of Promotional Product Literature printing.
The Headline Selecting a printer: This should be done prior to
making any moves keeping in mind the quality
The headline is an important part of a printed
and budget.
piece. It sets the tone for the rest of the piece and is
responsible for sales effectiveness. Ideal Characteristics of Promotional Literature
Good promotional literature should inform,
Body Copy
educate, stimulate and direct the reader in the
It is the main body of the literature. It includes the simplest and most concise manner. It should be
text which conveys the advertiser’s message, name focussed, attention-grabbing and benefits-driven.
and address. In case of a pharmaceutical product, It should have conviction value and memorizing
it should include the name of the product value. It should be suggestive and true. Promo-
(normally the brand name), the active ingredients, tional literature should maintain high ethical
using approved names where they exist, the name standards and comply with applicable legal,
and address of the pharmaceutical company or regulatory and professional requirements.
its agent responsible for marketing the product, To ensure the ethical promotional practices,
date of production of the advertisement, Pharmaceutical companies must comply with
“abbreviated prescribing information” which IFPMA (International Federation of Pharma-
should include an approved indication or ceutical Manufacturers and Associations) Code.
The IFPMA Code of Pharmaceutical Marketing promotions and the influence of marketing on
Practices (the “IFPMA Code”) sets out standards prescribing behavior. More emphasis should be
for the ethical promotion of pharmaceutical laid on the evaluation of promotional and other
products to health care professionals to ensure scientific literature while teaching under-
that member companies’ interactions with health graduate students. Sessions of appraisal of
care professionals are appropriate and perceived promotional literature should be conducted for
as such. Effective from January 1st, 2007, this Code interns and resident doctors as they are the ones
replaces the IFPMA Code of Pharmaceutical who usually interact with pharmaceutical
Marketing Practices (Update 2000). In India, representatives. Initiatives should be taken to
standards of promotion are set by the Organization form a body for the review of promotional and
of Pharmaceutical Producers of India (OPPI), other scientific material before it reaches the
which is a premier organization of pharma- target, i.e. doctors or consumers as in direct-to-
ceutical, manufacturers in India. consumer advertising. Government should
establish a national drug policy that is focused
Advantages of Promotional Product Literature on public health problems and is consistent
with general health policies, forming an
1. Introduces a new product in the market.
organization that will evaluate national medica-
2. Increases prospects.
tions and technology together with this policy,
3. Increases sales.
and developing standard treatment guidelines.
4. Fights competition.
One of the methods recommended is the creation
5. Enhances good will of concern.
of an independent, reliable, easily accessible,
6. Educates the target market.
and current source of information for physicians
7. Eliminates middlemen.
(like Internet web pages and drug bulletins). In
8. Supports the salesmanship.
this process, it is important to create resources
9. Raises the standard of living.
to provide physicians an alternative source of
information that is evidence-based. Physicians
Disadvantages of Promotional Product
should equiped themselves with the skills of
Literature
critically appraising and assessing the lite-
1. May be inadequate, altogether inaccurate, rature. The new drug meant for promotion
invalid, unethical, false, misleading, biased, should be preferred over the existing one, if it
and deceptive or based on studies of poor offers clear advantages in terms of safety,
methodological quality. tolerability, efficacy and price, i.e. STEP criteria.
2. Leads to monopoly in a particular brand of The new drug should be relevant to the
product. clinician’s practice in terms of population
3. Creates artificial demand for the product. studied, the disease and the need for new
4. Increases the price of the product as expenses treatment. Obtaining and assessing the quality
on it form the part of the total cost of the product. of references is important and should be
5. May be harmful for the society, as the emphasized. Clinicians should work with
pharmaceutical promotional activities have medical industry representatives to formulate
powerful influences on prescribing behavior evidence-based literature. The methodology of
of the clinicians.
the study should be carefully judged to deter-
6. Enhances the self medication of patients.
mine the authenticity of the evidence. By
critically appraising and assessing the litera-
Current Recommendations
ture, the ultimate goal of medical practice i.e. to
Physicians should be made aware of the limita- ensure the optimum care of the patients can be
tions of the current methods of medical industry achieved.
Promotional Product Literature  345
Check list for evolution of promotional product literature SUGGESTED READING

Name (Active ingredients/ INN/ Generic Yes/No 1. http://www.p2pays.org/ref/16/15687.pdf.
name/ brand name) 2. Cardarelli R, Licciardone JC, Taylor LG. A cross-
Active ingredient/ contents/ doses form/ Yes/No sectional evidence-based review of pharmaceutical
regimen promotional marketing brochures and their under-
Approved therapeutic use Yes/No lying studies: Is what they tell us important and
Dosages from regimen Yes/No true? BMC Fam Pract 2006;7:13.
Adverse drug reaction: minor/ major Yes/No 3. http://www.hubpages.com/hub/Promotional_
Precautions, contraindication and warnings Yes/No Literature.
4. Lal A. Pharmaceutical drug promotion: How it is
Introduction: Drugs food product Yes/No being practiced in India? J Assoc Physicians India
Name and address of manufacturer Yes/No 2001;49:266-73.
Scientific references Yes/No 5. IFPMA Code of Pharmaceutical Marketing Practices
2006 (Revision).
6. OPPI Code of Pharmaceutical Marketing Practices
2007.
Note: 7. Gupta RS, Sharma BD, Bhalla NS. Fundamentals of
FDA has given regulations which specify that commerce theory and functional management. 1st
advertisments are false, lacking in fair balance, or Ed. Kalyani Publishers 1996;440-41.
otherwise misleading if: 8. Sharma RK, Gupta SK, Oberoi M, Sharma R. Principle
and practice of commerce theory and functional
• Using headlines, sub-headlines, or pictorial management. 1st Ed. Kalyani Publishers 1996;
or other graphic material in way that is 436-39.
9. Shetty VV, Karve AV. Promotional literature: How
misleading or erroneous. do we critically appraise? J Postgrad Med 2008;
• Use literature or references inappropriately to 54:217-21.
10. Civaner M. A Proposal for the Prevention of Ethical
support claims in the advertisement. Problems Related to Drug Promotion: A National
• If they make claims about relative safety and Network for Drug Information. Turk J Psyc 2008;
efficacy or about the populations in which the 19:310.
11. Wilkes MS, Doblin BH, Shapiro MF. Pharmaceutical
drug is useful that are not supported by the advertisements in leading medical journals: Experts’
current literatures. assessments. Ann Int Med 1992;116:912-19.
Analytical Toxicology
38
ANALYTICAL TOXICOLOGY Generally, acutely poisoned patients samples
are assessed for general clinical chemistry (blood
This chapter briefly emphasizes on principles and
glucose, blood gases, etc.) and hematology.
practical information on the analysis of chemicals,
Toxicological analyses can play a useful role if
drugs and poisons in biological specimens
the diagnosis is in doubt, the administration of
(serum, plasma, urine, etc.) particularly clinical
antidotes or protective agents is contemplated, or
and forensic specimens. Toxicology is derived
the use of active elimination therapy is being
from Greek word “Toxican” which was used for
considered.
the poisonous substance in which arrow head There are several advantages of analytical
were dipped. Poisonous substances are those toxicology, which have the influence on the
whose cumulative effect leads to the fatality either clinical toxicology, i.e. (1) it gives a correlation of
in low or high dose. Several medicines like, laboratory finding with the clinical diagnosis,
opioids, aspirin, barbiturates, antidepressants, (2) it provide knowledge about the poison, (3) helps
phenothiazine, benzodiazepines, warfarin, in the selection of specific antidote, and (4) therapy
paracetamol, etc. which is effective at therapeutic can be channelize based on the toxicology report.
dose but may produce the toxic effect in the high
doses (Table 38.1). Analytical techniques: Color tests and spectro-
Broadly, toxicology is divided into the different photometry, chromatography and electrophoresis,
subspecialties such as analytical toxicology, clinical mass spectrometry, and immunoassay.
toxicology, environmental toxicology, industrial Methods in assessment of toxicology: In a
toxicology, etc. whereas for pharmacological poisoning case, collection of tissues and the organ
teaching purpose analytical toxicology and clinical in which the poison is suspected to be present
toxicology is important. The analytical toxicology should be kept for the chemical examination. The
may be required to detect, identify, and in measure isolation and identification of unknown poison
a wide variety of compounds in samples from are the very important step in the analysis of any
almost any part of the body or in related materials poisoning. Even small amount of poison present
such as residues in syringes or in soil. Whereas, may changes the physiology of body which often
according to the WHO, “clinical toxicology” is requires the skilled hand and confirmatory results.
defined as the detection, identification and the Complete analytical procedure is divided into
measurement of drugs and other foreign compounds three phases, i.e pre-analytical, analytical, and post-
(xenobiotics) in biological and related specimens to help analytical phases (Fig. 38.1).
in the diagnosis, treatment, prognosis, and prevention Pre-analytical phase is an important procedure
of poisoning. which involves appropriate sample collection (in
Analytical Toxicology  347
The extraction procedure is the important step

in the analysis of toxic compound because further
steps and treatment is depends on the identified
toxins. Toxicological extraction method provides
the suitable concentration of sample to which the
detection technique may be applied. The design
of extraction method is depend on the analytical
aim, the type of the material analyzed and the
nature of the substance to be extracted.
The extraction method depends on the known
as well as the unknown nature of toxins. Whereas,
toxins are mainly classified according to the
Fig. 38.1: Phases of analytical toxicology evaluation
isolation method involved,
1. Volatile poison, isolated by distillation or
diffusion,
appropriate sample tubes) and safe transport, 2. Metallic poisons, isolated by the oxidation of
receipt, and storage of biological samples. the organic matter,
Transfer of samples to the laboratory and then
3. Toxic anions, isolated by dialysis or ion
arranging it for the analysis is also a part of pre-
exchange methods,
analytical phase. Analytical phase is one which
4. Pesticides, direct solvent extraction method
involves complete analytical procedure and
identification in which analysts used tried and 5. Corrosives, e.g: Acids, alkali, etc.
tested procedures to perform the requested or 6. Non-volatile organic substance, isolated by
appropriate analyses to the required degree of solvent extraction and
accuracy and reliability in an appropriate, 7. Miscellaneous poisons like animal poisons,
clinically relevant time-scale. Post-analytical phase e.g: Snake bite, cantharides, etc. or plants
includes reporting results by writing (proforma of poison like Papaver somniferum, Cannabis,
laboratory, telephone, fax, or other electronic Belladona, etc. requiring special extraction
means. technique.
Note: Typically full records of the analysis should be

Reporting of Results
kept for a minimum of 5 years (10 or more years for
medicolegal implications case). Remaining samples The results may be presented in mmol/L, ng/mL,
must be disposed safely in an agreed time-frame. µg/L, and mg/L. But, to avoid any confusion in
units, it is important to generalize the results
Sample Preparation and Methods of Estimation report which should be clear and to maintain
consistency by the laboratory in a particular
The method should be accurate, reliable and
institute or center.
reproducible. Uniform methodology is not
employed and the analytical methods used is Example: Lithium, Thyroxine, and Methotrexate:
depend on local circumstances. There are two Molar units - mmol/L whereas
types of sample/material referred to the toxi- In Scientific Papers and immunoassay results
cological analysis, sample in survival cases and may expressed as ng/mL, µg/L, mg/L, g/L, and
sample in fatal cases. molar (mmol/L, etc.).
Table 38.1: Drugs and chemicals concentrations in blood

Drugs Therapeutic dose (mg/L) Toxic dose (mg/L) Lethal dose (mg/L)
Acetaminophen 10-20 400 1500
Aminophylline 20-100
Barbiturate; Short acting 1 7 10
Intermediate acting 1-5 10-30 30
~
Phenobarbital 10 40-60 80-150
Barbital ~ 10 60-80 100
Carbamazepine 2 8-10
Chloral hydrate 10 100 250
Codeine 25 µg/L
DDT 13 µg/L
Diazepam 0.5-2.5 5-20 >50
Digitoxin 1.7-2.1 µg/L 320 µg/L
Digoxin 0.6-1.3 µg/L 2-9 µg/L
Diphenylhydantoin 5-22 50 100
Ethanol 1.5 g/L > 3.5 g/L
Halothane 200
Iron 500 6
Lead 0.05-1.3 0.7
LSD 1-4 µg/L
Lidocaine 2 6
Lithium 4.2-8.3 13.9 13.9-34.7
Methanol 200 > 890
Morphine 0.1 0.05-4
Nicotine 10 5-52
Propranolol 0.025-0.2 8-12
Quinidine 3-6 10 30-50
Quinine 12
Salicylate 20-100 150-300 500
Theophylline 20-100
Warfarin 1.0-10
SUGGESTED READING Technical Series No 5. Vienna: UN International Drug

Control Programme, 1997
1. Flanagan RJ. SI units - Common sense not dogma is 6. http://www.undcp.org/odccp/
needed. British Journal of Clinical Pharmacology 1995; technical_series_1997-01-01_1.html.
39:589-94. 7. Maurer HH. Systematic toxicological analysis
2. Flanagan RJ. Role of the laboratory in the diagnosis procedures for acidic drugs and/or metabolites
and management of poisoning. In: Medical Toxicology, relevant to clinical and forensic toxicology and/or
Edition 3. Ed. Dart RC. Baltimore: Lippincott, doping control. Journal of Chromatography B 1999;
Williams and Wilkins, 2003: 337-58. 733:3-25.
3. Flanagan RJ, Braithwaite RA, Brown SS, Widdop B, 8. SOFT/AAFS (Society of Forensic Toxicologists/
de Wolff FA. Basic Analytical Toxicology. Geneva: American Academy of Forensic Sciences). Forensic
WHO, 1995. toxicology laboratory guidelines 2002.
4. French - http://www.intox.org/databank/ 9. http://www.softtox.org/guidelines/default.asp
documents/supplem/supp/sup2f.htm. 10. Stewart MJ, Watson ID. Analytical reviews in clinical
5. Flanagan RJ, Streete PJ, Ramsey JD. Volatile substance chemistry: methods for the estimation of salicylate
abuse - Practical guidelines for analytical investigation of and paracetamol in serum, plasma and urine. Annals
suspected cases and interpretation of results. UNDCP of Clinical Biochemistry 1987;24:552-65.
Analytical Toxicology  349
11. Stewart MJ, Watson ID. Analytical reviews in clinical the use of laboratory tests to support poisoned
chemistry: Methods for the estimation of salicylate patients who present to the emergency department.
and paracetamol in serum, plasma and urine. Annals Clinical Chemistry 2003; 49:357-79.
of Clinical Biochemistry 1987;24:552-65. 14. http://www.clinchem.org/cgi/content/full/49/3/
12. Wilson J. External quality assessment schemes for R50.
toxicology. Forensic Science International 2002;128: 15. Duffus JH. Glossary for chemists of terms used in
98-103. toxicology. Pure and applied chemistry, 1993, 65: 2003-
13. Wu AH, McKay C, Broussard LA, Hoffman RS, 2122.
Kwong TC, Moyer TP, Otten EM, Welch SL, Wax P. 16. Stead AH, et al. Standardised thin-layer chro-
National academy of clinical biochemistry laboratory matographic systems for the identification of drugs
medicine practice guidelines: Recommendations for and poisons. Analyst (London), 1982;107:1106-68.
Recent Advances in
39 Pharmacology
TRANSLATIONAL MEDICINE to bed” such as the conventional targeted drug

discovery or “bed to bench” such as biological
Simply, translational medicine is a concept of samples like DNA, blood, or tissue fragments, etc.
translating the laboratory findings to the clinic from the patients and observed the disease cause
i.e. in the patients care. This concept is broadly at molecular level and further helps in develop
known as “bench to bedside”. So, this is the drugs targeted at particular subgroups of disease.
cumulative support of physicians and scientists
to translate findings from the laboratory into better REVERSE PHARMACOLOGY
treatment for patients. This is often named as
“Molecular Medicine” and “Personalized Medicine”, Ayurvedic drugs are generally regarded as safe
which mainly refer to the process of molecular and are commonly used in many acute and
research of drug in laboratory to clinical care. chronic illness. Reverse pharmacology is actually
Objective of translational medicine is to a rediscovery of drugs activity and its mechanism
discover the origin, pathway and mechanism of of action. Hence, reverse pharmacology can be
diseases including the responsible biomarkers. defined as the integrating science of developing
The main aim of such concept is to systematically candidate drugs from a clinical to experiential hits
discover and develop new diagnostics and and leads by transdisciplinary exploratory
therapeutic methods and drugs in short duration studies and ultimately to understand the mecha-
of time. nisms of action at different pathological stages of
The approach used in the translational biological organism. Then, confirm candidate
medicine is unidirectional either from the “bench drugs experimental to clinical use on the basis of
safety, efficacy and acceptability on relevant
science.
The first stage experiential included clinical
trial 2 and 1 as an experience and data from
Fig. 39.2: Three stages of reverse pharmacology:

Fig. 39.1: Bench to bedside concept experiential, exploratory and experimental
Recent Advances in Pharmacology  351
previous uses. Whereas, exploratory studies, the

2nd stage would cover dose-activity in ambulant
patients and selected in vitro and in vivo experi-
mental models to evaluate the key target. The 3rd
stage experimental involves supporting pharma-
coepidemiological data, standardization of
formulation with HPLC pattern, exploratory
human/animal studies, human dose deter-
mination and other experimental parameters like
levels of biological parameters (biochemical,
hematological, tissue, etc.)
Common examples of reverse pharmacology
are Digitalis purpurea (Na + /K + ATPase),
Papaverum somniferum (Opioid receptors), Fig. 39.4: Procedure for microdosing
Cincohona (Antimalarial), Curcuma longa (Anti-
inflammatory, etc.), etc. done on animals. “Microdosing” or “phase 0” is
defined as a microdose which is administered
100 µg of investigational drug or 1/100th of the
pharmacological active dose extrapolated from in
vitro or in vivo animal studies in most sensitive
species, whichever is the lesser.
Microdose is a very low dose compared to the
pharmacological active dose, so its analysis and
assessment depends on the availability of
ultrasensitive analytical methods to measure drug
and its metabolite. (May measure at the level of
picogram to femtogram range).
Analysis is done by the highly sensitive
analytical methods like “Accelerator Mass
Fig. 39.3: Comparative flow diagram of pharmacological Spectrometry” (AMS) and “Positron Emission
research and the reverse pharmacology research Tomography” (PET). The basic step is to label an
investigational drug using the radioisotope
MICRODOSING (PHASE 0) carbon-14 (14C; t1/2 5740 yrs*) for AMS and carbon-
11 (11C: 20 min*) for PET. Both AMS and PET
In the drug development, one of the reasons for quantify the total number of labeled atoms present
drug failures during late developmental phase is in a sample rather than distinguishing between
suboptimal pharmacokinetics (clearance, volume parent drug and metabolite(s).
of distribution, t1/2 , etc.) or change in metabolic Methodology may include the parallel study
status in human. Hence, the pharmacokinetic groups between two and five molecules using
studies before going for the larger trial have shown human subject. Each molecule might be admi-
to be beneficial and a new experimental approach nistered in a crossover design such as an
has been developed, known as microdosing or intravenous (i.v.) dose followed by an oral dose
Phase 0 in human. after a suitable washout period. AMS is used
“Microdosing” study designed, as a solution for determining PK data by taking body samples
of above mentioned problem. Therefore, the over time, processing the samples in the
objectives of the “Microdosing” are to reduce the laboratory and then analyzing their drug
cost and resources spent on non-practicable drugs content. PET provides primarily PD data
through real-time imaging and some limited PK 5. Kutzleb Christian, Busmann Annette, Wendland
data. Thus, pharmacokinetic parameters like Martin, Maronde Erik. Discovery of Novel Regulatory
absorption, Vd, CL, etc. can be obtained. Relative Peptides by Reverse Pharmacology: Spotlight on
Chemerin and the RF-amide Peptides Metastin and
proportion of drug and its metabolites can be
QRFP. Current Protein and Peptide Science
obtained through chromatographic separation 2005;6(3):265-78.
of an extract of blood or plasma followed by 6. Sakurai T. Reverse pharmacology of orexin: From an
analysis of collected chromatography fractions orphan GPCR to integrative physiology. Regul Pept
such as liquid chromatography- mass spectro- 2005;126(1-2):3-10.
metry (LC-MS). 7. Lappin G, Garner RC. Big physics, small doses: The
use of AMS and PET in human microdosing of
Note: *PK data can be obtained for only 2 hr after development drugs. Nature Rev Drug Discovery
drug administration by PET (i.e. 5-6 decay half- 2003;2:233-40.
life) while PK data can be obtained up to 100 days 8. Wilding I, Bell J. Improved early clinical development
after drug administration by using AMS. through human microdosing studies. Drug Discovery
Today 2005;10(13):890-94.
9. Lappin G, Garner RC. Current perspectives of 14C-
SUGGESTED READING
isotope measurement in biomedical accelerator mass
1. Marincola FM. Translational Medicine: A two-way spectrometry. Anal Bioanal Chem 2004;378:356-64.
road, J Transl Med 2003;1(1):1. 10. Aboagye EO, Price PM, Jones T. In vivo pharma-
2. Stacey P Mankoff, Christian Brander, Soldano cokinetics and pharmacodynamics in drug develop-
Ferrone, Francesco M Marincola. Lost in Translation: ment using positron-emission tomography. Drug
Obstacles to Translational Medicine, J Transl Med Discovery Today 2001;6:293-302.
2004;2:14. 11. Bergström M, Grahnén A, Langström B. Positron
3. Clinical and Translational Medicine —http:// emission tomography microdosing: A new concept
www.ctsjournal.com with application in tracer and early clinical drug
4. Pharmacology at a reverse-http://www. express- development. Eur J Clin Pharmacol 2003;59:
pharmaonline.com/20060930/research03.shtml 357-66.
Appendices
APPENDIX I: ABBREVIATIONS
AC Animal Care
AFDO Association of Food and Drug Officials
AMS Accelerator Mass Spectrometry
ANDA Abbreviated New Drug Application
ANOVA Analysis of Variance
AP Arterial Pressure
AVMA American Veterinary Medical Association
AWA Animal Welfare Act
AWIC Animal Welfare Information Center
CAAT Center for Alternatives to Animal Testing
CaSR Calcium Sensing Receptor
CDC Centers for Disease Control and Prevention
CFA Complete Freund’s Adjuvant
CFR Code of Federal Regulations
CIRA Center for Information on Research with Animals
CPCSEA Committee for the Purpose of Control and Supervision of Experimentation on Animals
CRC Concentration Response Curve
DEPA Direct endpoint assay
DMSO Dimethyl Sulfoxide
DRC Dose Response Curve
DTP Direct-To-Physician marketing
ED Effective Dose
ELISA Enzyme-Linked Immunosorbent Assay
EIH Entry Into Human
ESA Endangered Species Act
EU Endotoxin Units
FASEB Federation of American Societies of Experimental Biology
FBR Foundation for Biomedical Research
FDA Food and Drug Administration
FHD First Human Dose
FTIM First Time In Man
GLP Good Laboratory Practices
GCP Good Clinical Practices
GRA Graded Response Assay
HED Human Equivalent Dose
HEPA High-Efficiency Particulate Air Filter
HTS High-Throughput Screening
IACUC Institutional Animal Care and Use Committee
IAEC Institutional Animal Ethics Committee
IBSC/IBC Institutional Biosafety Committee
IBRO International Brain Research organization
ICLAS International Council for Laboratory Animal Science
IFA Incomplete Freund’s Adjuvant
IF Impact Factor
ILAR Institute for Laboratory Animal Research
IND Investigational New Drug
JCR Journal Citation Reports
LAL Limulus Amebocyte Lysate
LAMA Laboratory Animal Management Association
LC-MS Liquid Chromatography and Mass-Spectrometry
LD Lethal Dose
LOAEL Lowest Observed Adverse Effect Level
mAb Monoclonal Antibody
MAD Multiple Ascending Dose
MANOVA Multiple Analysis of Variance
MES Maximal Electroshock Seizure
MLD Minimum Lethal Dose
MRSD Maximum Recommended Starting Dose
MTD Maximum Tolerated Dose
M.wt Molecular Weight
NABR National Association for Biomedical Research
NARRC National Advisory Research Resources Council
NAS National Academy of Sciences
NDA New Drug Application
NIH National Institutes of Health
NOAEL No Observable Adverse Effect Level
OLAW Office of Laboratory Animal Welfare
OHSP Occupational Health and Safety Program
OPPI Organization of Pharmaceutical Producers of India
OSHA Occupational Safety and Health Administration
PAD Pharmacologically Active Dose
PCR Polymerase Chain Reaction
PEFR Peak Expiratory Flow Rate
PEFM Peak Expiratory Flow Meter
PET Positron Emission Tomography
PK/PD Pharmacokinetics/Pharmacodynamics
PSS Physiological Salt Solution
RCF Relative Centrifugal Force
RSC Radiation Safety Committee
SAD Single Ascending Dose
SCAW Scientists Center for Animal Welfare
SCI Science Citation Index
SD Standard Deviation
SEM Standard Error of Mean
SOPs Standard Operating Procedures
SSD Safe Starting Dose
TCP Tail Cuff Pressure
TDL Toxic Dose Low
TDM Therapeutic Drug Monitoring
THLE Tonic Hind Limb Extension
UHTS Ultra High Throughput Screening
UPLC Ultra-performance Liquid Chromatography
US FDA United States Food and Drug Association
WHO World Health Organization
Appendices  355
APPENDIX II: DRUG AND SOLUBILITY
Sl No. Drugs Molecular weight Solvents

a
1 Acetyl choline chloride 181.7 Hygroscopic, freely soluble in water
2 Adrenalineb 183.21 Slightly soluble in water; insoluble in ethanol (95%)
and in ether ; soluble in solutions of mineral acids,
of sodium hydroxide and of potassium hydroxide
3 Adrenaline bitartrate 333.29 Freely soluble in water; slightly soluble in ethanol
(95%); practically insoluble in chloroform and in
ether.
4 Aspirin 180.16 Slightly soluble in water, freely soluble in ethanol
(95%); soluble in chloroform and in ether.
5 Atropine methonitrate 366.42 Freely soluble in water; soluble in ethanol (95%);
practically insoluble in chloroform and in ether
6 Atropine sulphate 694.84 Very soluble in water; freely soluble in ethanol (95%)
and in glycerin; practically insoluble in chloroform
and in ether
7 Caffeine 194.19 Freely soluble in chloroform and in boiling water;
sparingly soluble in water and in ethanol (95%);
slightly soluble in ether
8 Codeine phosphate 406.37 Freely soluble in water; slightly soluble in ethanol
(95%); sparingly soluble in chloroform; practically
insoluble in ether
9 Diazepam 284.74 Freely soluble in chloroform; soluble in ethanol (95%);
very slightly soluble in water
10 Diclofenac sodium 318.13 Freely soluble in methanol; soluble in ethanol (95%);
sparingly soluble in water and in glacial acetic acid;
practically insoluble in ether, in chloroform and in
toluene
11 Diclofenac sodium 318.14 Water for Injection
injection
12 Digoxin 780.95 Practically insoluble in water, freely soluble in
pyridine and in a mixture of equal volumes of
dichloromethane and methanol; slightly soluble in
ethanol (95%).
13 Digoxin injection 780.95 Water for Injection and Ethanol or other suitable
solvents (base of injection)
14 Frusemide 330.74 Soluble in acetone; sparingly soluble in ethanol (95%);
slightly soluble in ether; practically insoluble in
water
15 Haloperidol 375.87 Practically insoluble in water, soluble in chloroform;
sparingly soluble in ethanol (95%).
16 Haloperidol 375.87 Lactic Acid diluted with Water for Injection.
injection
17 Indomethacin 357.79 Insoluble in water ; Soluble in chloroform; sparingly
soluble in ethanol (95%) and in ether
(Contd...)
(Contd...)
Sl No. Drugs Molecular weight Solvents

18 Lithium carbonate 73.89 Soluble in water; practically insoluble in ethanol
(95%).
19 Morphine sulphate 758.83 Soluble in water; freely soluble in hot water; slightly
soluble in ethanol (95%) but more soluble in hot
ethanol (95%); practically insoluble in chloroform
and in ether.
20 Morphine sulphate Water for Injection
injection
21 Omeprazole 345.42 Freely soluble in chloroform; soluble in ethanol (95%)
and in methanol; very slightly soluble in water
22 Oxytocin 1007.20 Soluble in water
23 Oxytocin injection Water for Injection
24 Paracetamol 151.16 Freely soluble in ethanol (95%) and in acetone;
sparingly soluble in water
25. Pentobarbitone Sodium 248.26 Very soluble in water and ethanol
26 Phenobarbital sodium 252.22 Soluble in ethanol (95%) and in ether; very slightly
soluble in water
27 Phenobarbital Sodium 9:1 (Propylene Glycol : Water for Injection)
Injection
28 Phenytoin Sodium 274.25 Soluble in water and ethanol (95%); insoluble in
dichloromethane and in ether
a Should be stored in the air tight container at cool place and immediately close the cap of the bottle after transferring
the Ach from the bottle at the time of weighing.
b Store in tightly-closed and light-resistant containers (decomposes rapidly in presence of moisture and at higher
temperatures) and it is not stable in a neutral or alkaline solution which rapidly becomes red on exposure to air.
Other Solvents
Dimethyl sulfoxide (DMSO), Dichloromethane (DCM), 0.1N HCl or NaOH may use to dissolve the test drug
during the experiment.
Solubility Definitions
• Very soluble: less than 1 part
• Freely soluble: 1-30 parts
• Soluble: 10 – 30 parts
• Sparingly soluble: 30 – 100 parts;
• Slightly soluble: 100 – 1000 parts
• Very slightly soluble: 1000 – 10,000 parts
• Practically insoluble: more than 10,000 parts
Note: Drugs which are not freely soluble in water or warm water, those can be making soluble in DMSO.
The pharmacological activity of the solvent is also considered during the selection of the solvent such as ethanol or
ether have the CNS depressant property hence not preferred as a solvent in the experiment related to the CNS activity
assessment.
Appendices  357
APPENDIX III: LIST OF DRUGS IN CLINICAL

PHARMACOLOGY PRACTICALS
ACECLOFENAC
• Chemically phenylacetic acid derivative
• Well absorbed from GIT
• Peak plasma time-1 – 3 hrs after oral dose
• 99% plasma protein bound
• Plasma elimination half life- 4 hr
• Two third dose excreted in urine as hydroxymetabolite
• Dose-100 mg twice daily p.o. 100 mg once daily in hepatic impairment
• 100 mg. 200 mg-SR. 100 mg BD
• ADR: Indigestion, heartburn, dyspepsia, diarrhea, nausea, abdominal pain, flatulence.
• Trade name- Dolodkind (mankind), Dolokind-SR (mankind), Zerodol etc.
ASPIRIN
• Acetyl salicylic acid
• Rapidly absorbed from GIT, skin. Non-ionised form absorbed in stomach and intestine
• During 1st 20 min after oral dose-aspirin is the dominant form in plasma
• Once absorbed, rapidly converted to salicylate
• 80-90% plasma protein bound, widely distributed
• Volume of distribution (Vd)-170 ml/kg in adults
• Salicylate crosses placental barrier, secreted in breast milk
• Mainly hepatic metabolism
• At 325 mg dose t 1/2 is 2-3 hr, at high dose 15-30 hr
• Excretion- unchanged in urine-30% in alkaline urine, 2% in acidic urine
• Removable by hemodialysis
• Dose- 300- 900 mg every 4-6 hrs for analgesic, anti-inflammatory, antipyretic
• 75-325 mg –as antiplatelet, 50mg, 75mg, 150mg
• ADR: Hyperventilation, bleeding, tinnitus, fluid retention acidosis
• Trade name: Aspicot, colsprin, ecosprin, ASA 50 (german remedies), Loprin -75, Delisprin-150 etc.
CARBAMAZEPINE
• Carbamazepine is related chemically to the tricyclic antidepressants. It is a derivative of iminostilbene with
a carbamyl group at the 5 position; this moiety is essential for potent antiseizure activity. The structural
formula of carbamazepine is slowly and irregularly absorbed from GIT, metabolized in liver – CYP3A4,
CYP2C8c-10,11 epoxide also active. Excreted mainly in urine, widely distributed, 75% plasma protein
bound
• Plasma t1/2 5-26 hr, induces its own metabolism.
• Plasma therapeutic range- 4-12 µg/ml
• Crosses placental barrier, present in breast milk.
• Therapeutic concentrations should be maintained at 6 to 12 µg/ml, although considerable variation occurs.
Side effects referable to the CNS are frequent at concentrations above 9 µg/ml.
• Dose 100-200 mg twice daily can increase 100-200 mg every week upto 0.8-1.2 g daily divided doses, 100,
200,400 mg TAB
• ADR: Stupor or coma, hyperirritability, convulsions, and respiratory depression. Long-term therapy
drowsiness, vertigo, ataxia, diplopia, and blurred vision. Other adverse effects include nausea, vomiting,
serious hematological toxicity (aplastic anemia, agranulocytosis), and hypersensitivity reactions (dermatitis,
eosinophilia, lymphadenopathy, splenomegaly).transient elevation of hepatic transaminases in plasma in

5 to 10% of patients.
• Trade name: Mazetol, tegretol, carbetrol etc.
CHLORPHENIRAMINE
• Slowly absorbed, peak plasma time 2.5- 6 hr following oral administration. 25-50% bioavailability, undergoes
considerable first pass metabolism.
• Widely distributed, enter CNS, excreted in urine. Duration of action 4-6 hrs
• Dose- 4 mg orally 4-6 hourly up to 24 mg max
• I/V 10-20 mg slow
• Trade name: Avil etc.
DIGOXIN
• Absorption from GIT 70-90%
• Therapeutic plasma range- 0.5-2 ng/ml
• Large Vd, concentration in myocardium > plasma
• 20-30% plasma protein bound
• Crosses placenta, CSF, breast milk
• Elimination half life-1.5- 2 days
• Excreted unchanged in urine, not removed by dialysis
• Dose: effect within 2 hrs, max. Within 6 hrs loading dose needed. Steady state concentration (CSS) in sample
taken at least 6 hrs later- 0.5- 2 ng/ml
• Loading dose- 750-1500 µg as a single dose during the initial 24 hrs or in divided daily doses 6 hrly, 250ug
twice daily
• Trade name- Cardioxin, lanoxin etc.
ETORICOXIB
• COX-2 selective inhibitor (106.0 times more selective for COX-2 inhibition over COX-1)
• Bioavailability is 100%
• Plasma Protein binding is 92%
• Metabolism by CYP3A4
• t1/2 is 22 hours
• Drug is excreted through kidney 70% in stool 20%
• It is contraindicated in pregnancy
• Currently it is used in treatment of rheumatoid arthritis, psoriatic arthritis, osteoarthritis, ankylosing
spondylitis, chronic low back pain, acute pain and gout.
• Doses; 60, 90 mg/day for chronic pain and 120 mg/day for acute pain.
• Trade name: Arcoxia etc.
FEXOFENADINE
• Rapidly absorbed orally, peak plasma conc. 2-3 hrs, 60-70% plasma protein bound, elimination t-½ 14 hrs.
Excretion mainly in faeces.
• Dose- 120 mg daily or 60 mg b.d and 180 mg OD for chronic urticaria
• Trade name: Allegra etc.
FUROSEMIDE
• Rapid GI absorption, 60-70% bioavailability,
Appendices  359
• Plasma t1/2 is 2 hr, increased in hepatic and renal failure.

• 99% albumin bound, excreted unchanged in urine, crosses placenta, excreted in breast milk.
• Hemodialysis does not clear it
• Increased free form in heart failure, renal and hepatic impairment.
• Effect of oral dose within 0.5–1 hr,
• Peak at 1-2 hr and last for 4-6 hrs. I/V within 5 min, lasts for 2 hrs
• Dose-oral 40 mg once daily
• I/V: 20-50 mg slowly at 4 mg/min
• Contraindications : Severe Na+ and volume depletion, hypersensitivity to sulfonamides and anuria
• ADR: Hyponatremia, circulatory collapse, thromboembolic episodes, hypochloremic alkalosis, hypokalemia,
hypomagnesemia, cardiac arrhythmias and hypocalcemia, rarely tetany, Ototoxicity, hyperuricemia ,
hyperglycemia , hypercholesteriamia, skin rashes, photosensitivity, paresthesias, bone marrow depression,
and gastrointestinal disturbances.
• Trade name: Lasix, frusenex, diucontin-K, Lasix,oral /iv etc.
GLYCERYL TRINITRATE
• Organic nitrates are polyol esters of nitric acid
• Rapidly absorbed from oral mucosa, GIT and skin
• <100% bioavailability due to pre-systemic clearance
• Sublingual or buccal tablet effect within 1-3 min, transdermal patch or ointment- within 30-60 min, I/V
within 1-2 min.
• Duration of action- sublingual-30-60 min, transdermal patch 24 hr, I/V 3-5 min
• Hydrolysis in plasma, metabolized in liver
• Dose-acute angina- 300-600 µg S/L tab, max 3 doses within 15 min.
• Buccal tablet 1-2 mg, ointment 2%
• Unstable angina-5-10 µg/min to up to 200 µg/min
• Hypertension control 5-25 µg/min
• Tablet for sublingual use-0.3-0.6 mg as needed
• Sublingual spray-0.4 mg as needed
• Chewable tablet 2.5-9 mg 2-4 times daily
• IV 10-20 µg/minute
• Contraindication in raised intracranial tension,tolerance etc.
• ADR: Orthostatic Hypotension, tachycardia, throbbing headache
• Trade name: Angised, nitrocontin, nitroderm, myovit, illisrol, Nitribid, nitrostat, nitrol, nitro- dur etc.
HYOSCINE
• Esters formed by combination of an aromatic acid, tropic acid, and complex organic bases, either tropine
(tropanol) or scopine. Scopine differs from tropine only in having an oxygen bridge between the carbon
atoms designated as 6 and 7
• Readily absorbed from GIT, entirely metabolized in liver, crosses blood brain barrier, placenta
• Absorbed through skin
• Dose: Tab.10 mg, 20 mg I/M OR I/V max. 100mg daily
• 20 mg orally-4 times daily
• 10-20 mg,3-5 times daily,
• IM/IV.20-40 mg,3-5 times daily
• ADR: Increased pulse rate, visual disturbance due to i.v administration
• Trade name: Buscopan etc.
INH- ISONIAZID
• Readily absorbed from GIT
• Peak concentration of 3-7 µg/ ml appear in blood 1-2 hr after a fasting dose of 300 mg orally
• Plasma t ½ is 1-6 hrs
• Primary metabolic route- acetylation is genetically determined. However clinical effectiveness not influenced
by dosing 2 to 3 times per week
• Over 75% appears in urine within 24 hr, crosses BBB, placenta.
• Dose- 300 mg orally empty stomach
• ADR: Rash (2%), fever (1.2%), jaundice (0.6%), and peripheral neuritis (0.2%). Hypersensitivity reaction
manifest with fever, various skin eruptions, hepatitis, and morbilliform, maculopapular, purpuric, and
urticarial rashes. Other are agranulocytosis, eosinophilia, thrombocytopenia, anemia, arthralgia, peripheral
neuritis to isoniazid and occurs in about 2% of patients receiving 5 mg/kg of the drug daily. Precipitate
convulsions in patients with seizure disorders, Muscle twitching, dizziness, ataxia, paresthesias, stupor, and
toxic encephalopathy, euphoria, transient impairment of memory, dryness of the mouth, epigastric distress,
methemoglobinemia, tinnitus, and urinary retention.
• Trade name: ISOKIN, ISONEX, RifacomE-Z, Isokin.tab.100 mg, Isonex.tab. 100mg, Nydrazid etc.
LITHIUM
• Lithium is the lightest of the alkali metals (group Ia); the salts of this monovalent cation similar characteristics
with Na+ and K+.
• Readily absorbed from GIT.
• Peak concentration at 0.5-3 hr after oral dose in modified formulations peak at 2-12 hr after oral dose.
Completely distributed within 6-10 hr. Levels in bone, brain, thyroid more than in serum. Excreted in urine
mainly.
• Elimination half life-20-24 hr. Steady state conc. At 4-7 days. Blood sample to be taken 12 hrs after the last
dose following consistent dosing schedule of 4-7 days.
• Therapeutic level 0.4-1 mmol/liter
• Dose- 450-675 mg bid. or 0.4-1.2 mg in daily divided doses.
• 150, 300, 600 mg lithium carbonate
• ADR: Vomiting, profuse diarrhea, coarse tremor, ataxia, coma, and convulsions, mental confusion,
hyperreflexia, gross tremor, dysarthria, seizures, and cranial nerve and focal neurological signs, progressing
to coma and death. Other toxic effects are cardiac arrhythmias, hypotension, and albuminuria. Other
adverse effects common even in therapeutic dose ranges include nausea, diarrhea, daytime drowsiness,
polyuria, polydipsia, weight gain, fine hand tremor, and dermatological reactions including acne. The
prolonged use of Li+ causes a benign and reversible depression of the T wave of the ECG, an effect not
related to depletion of Na+ or K+. Seizures have been reported in Allergic reactions such as dermatitis and
vasculitis can occur with Li+ administration. mild alopecia. The use of Li+ in pregnancy has been associated
with neonatal goiter, CNS depression, hypotonia (“floppy baby” syndrome), and cardiac murmur, Ebstein’s
malformation.
• Trade name: Licab, eskalith etc.
PHENYTOIN
• It is 5-phenyl or other aromatic substituent appears essential for activity against generalized tonic-clonic
seizures. Alkyl substituent’s in position 5 contribute to sedation, a property absent in phenytoin
• Slowly but completely absorbed from upper intestine, metabolized in liver by cytochrome 2C 9 and 19.
Genetic polymorphism influences metabolism.
• Undergoes eneterohepatic recycling widely distributed. 90% plasma protein bound. Dose dependent halflife.
t1/2 22 hrs. Therapeutic levels- 10-20- ug /ml.
• Dose-3-4 mg/kg daily/150-300 mg daily up to 600 mg, 100 mg capsule
Appendices  361
• Status epilepticus- 10-15 mg/kg slow iv at < 50 mg/min

• ADR: Cardiac arrhythmias, behavioral changes, gingival hyperplasia, osteomalacia, and megaloblastic
anemia. Hirsutism, neutropenia and leucopenia, red-cell aplasia, agranulocytosis, and mild thrombocytopenia,
lymphadenopathy, hypoprothrombinemia. Rarely Stevens-Johnson syndrome. Systemic lupus
erythematosus and potentially fatal hepatic necrosis.
• Trade names: Dilantin, epsolin, eptoin etc.
PHENOBARBITONE
• 5-phenyl-5-ethylbarbituric acid, it has less antiseizure potency than does of phenobarbital, but it is virtually
devoid of hypnotic activity. Rapidly absorbed from GIT, lipid insoluble, peak at 2 hrs after oral and at 4 hr
after IM
• Plasma protein binding-45-60%
• Metabolized in liver, plasma t-half 75-120 hrs, children 21-75 hrs, neonate- prolonged.
• Therapeutic range- 15-40 µg/ml
• Crosses placental barrier
• Dose 60-180 mg daily at night, children 8 mg/kg daily, status epilepticus-10 mg/kg
• ADR: Sedation, the most frequent undesired effect of phenobarbital, nystagmus and ataxia occur at excessive
dosage, irritability and hyperactivity in children, and agitation and confusion in the elderly. Scarlatiniform
or morbilliform rash, possibly with other manifestations of drug allergy, occurs in 1 to 2% of patients.
Hypoprothrombinemia, rarely exfoliative dermatitis.
• Trade name: Gardinal, luminal, luminettes, luminal, mysoline etc.
PILOCARPINE
• Mean elimination half-life-0.7- 1.3 hrs after 5-10 mg oral dose
• Inactivated in plasma and at neuronal synapse
• Glaucoma-0.5-4% eye drop daily 4 times, 1%, 2%, 3%, 4%
• ADR: Precautions, Toxicity, and Contraindications : It is contraindicated in asthma, hyperthyroidism,
coronary insufficiency, and acid-peptic disease and hyperthyroid patients. Other possible undesirable
effects are flushing, sweating, abdominal cramps, belching, a sensation of tightness in the urinary bladder,
difficulty in visual accommodation, headache, and salivation.
• Dry mouth- 5 mg tid
• Trade name: pilocar, carpomiotic, Pilocar/pilodrops, Salagen 5-7.5 mg oral etc.
PROPRANOLOL
• Completely absorbed from GIT, hepatic tissue binding, first pass metabolism
• Peak plasma concentration 1 -2 hr after oral dose. Plasma concentration vary greatly. High lipid solubility,
crosses BBB, placenta.
• 90% plasma protein bound
• Plasma t1/2 is 3-6 hrs, metabolised in liver
• Dose: Hypertension 40—80 mg twice daily
• Pheochromocytoma- 60 mg daily on 3 days before surgery always with alpha blockade angina- 40 mg 2-
3 times daily
• ADR: Bradycardia, asthma, PVD. diabetes
• Trade name: Betabloc, betaspan, ciplar, corbeta, inderal etc.
TACROLIMUS
• Erratic oral absorption. bioavailability- 15-20%. 80% bound to erythrocytes.plasma protein binding- 99%.
Extensively metabolised in liver by CYP3A4. t 1/2 is 12-43 hrs, in transplant patients.
• Dose- liver transplant- 100-200 µg/kg orally in two divided doses within 6 hrs or 10-50µg/kg i/v within 24
hrs.
• Kidney transplant- 150-300 µg/kg orally in two divided doses within 6 hrs or 50-100 µg /kg i/v within 24
hrs. etc.
TROPICAMIDE
• Mydriatic – within 20-40 min of instillation, lasts 6 hrs
• Cycloplegic- maximum effect within 30 min short lasting complete recovery within 6 hrs
• Dose- mydriatic- 1-2 drops 0.5%- 15-20 min prior
• Cycloplegic-1-2 drops-1%- repeated after 5 min
• 0.5%, 1%,eye drops
• Trade name: Mydriacyl etc.
Appendices  363
APPENDIX IV: EQUIPMENT REQUIRED IN CLINICAL

PHARMACOLOGY LABORATORY
• Ventilator with internal oxygen compressor

• Artificial lung
• Pulse oximeter
• Cardiac monitor
• Defibrillator
• 12 lead ECG machine with ECG electrode and tracer paper
• Oxygen cylinder big size
• Nebulizer with respules
• Venturimask with tubing with oxygen flow meter
• Ambu bag with different size facemask
• Infusion pump
• Glucometer with strips with needle to prick
• EMERGENCY TRAY-Laryngoscope of different size including pediatric size blade
– Endotracheal tube of different size with stellate
– Ryles tube of different size including including infant feeding tube
– Oropharyngeal airway of different size
– Central line with double and single lumen
– Vascular guard and tegaderm of different size
– Mersilk 2-0 size
– Cut down set
– Torch
– Bandages
– 3 way cannula ,iv set ,BT set ,disposable syringes 100 cc, 50 cc, 20 cc, 10 cc loaded and labelled injection
sodium Bicarbonate, Diazepam, Haloperidol, Atropine, Adrenaline, 2% xylocaine, Dexona,
hydrocortisone , Chloropheniramine, calcium gluconate, adenosine, midazolam, pancuronium,
vancuronium, cardrone, verapamil, midazolam, propofol
• EMERGENCY TROLLY-with different compartment leveled for different drugs-inj. Furosemide (lasix),
phenytoin, sodium bicarbonate, calcium gluconate, lorazepam, diazepam, adrenaline, atropine, deryphylline,
2% xylocaine solution, iv fluids, hemacil , iv set, BTset.
• SURGICAL TRAY-betadine solution, savlon solution, spirit, sterilize cotton pack, bandages, cut downset,
surgicare gloves with different size, Foley’s catheter, 2% xylocaine jelly, dressing pad
APPENDIX V: ANALYTICAL AND MOLECULAR MASS
Important Definitions
• % (w/v) concentration: mass (g) of solute contained in 100 mL solution
• % (w/w) concentration: mass (g) of solute contained in 100 g of sample
• % (v/v) concentration: volume (mL) of solute in 100 mL solution
• Molarity (M) = number of moles of solute in 1 L of solution
• Molality (m) = number of moles per 1 kg of solvent
• Normality (N) = number of equivalents of a substance dissolved in 1 L of solution
Important Conversions
Molarity (M) into normality (N) : (M) X (valency)
mg/L
mg/L into mM in water :
MW
mg/L into M in water :
N into mEq/L :
ppm into mg/L in water :
ppm into mg/m3 :
% into ppm :
where,
M = Molarity
N = Normality
mg/L = milligram/Litre
mM = MilliMolar
MW = Molecular weight
mEq/L = milliEquivalent/litre
ppm = Parts per million
% = Percentage
Appendices  365
APPENDIX VI: LOG CONVERSION TABLE

General Rule for Logarithm
log (XY) = log (X) + log (Y)
log (X/Y) = log (X) – log (Y)
log (X)a = (a) log (X)
Common logarithms are those for which the base (e) = 10. It is denoted by log10 [ ] or log [ ]
Natural logarithms are those for which the base (e) = 2 = 2.72. It is denoted by log e [ ] or ln [ ]
loge [ x] = 2.303 log10[x]
Logarithm Table (for numbers 1 to 5.49)
No. 0.00 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08 0.09
1.0 0.000 0.004 0.009 0.013 0.017 0.021 0.025 0.029 0.033 0.037
1.1 0.041 0.045 0.049 0.053 0.057 0.061 0.064 0.068 0.072 0.076
1.2 0.079 0.083 0.086 0.090 0.093 0.097 0.100 0.104 0.107 0.111
1.3 0.114 0.117 0.121 0.124 0.127 0.130 0.134 0.137 0.140 0.143
1.4 0.146 0.149 0.152 0.155 0.158 0.161 0.164 0.167 0.170 0.173
1.5 0.176 0.179 0.182 0.185 0.188 0.190 0.193 0.196 0.199 0.201
1.6 0.204 0.207 0.210 0.212 0.215 0.217 0.220 0.223 0.225 0.228
1.7 0.230 0.233 0.236 0.238 0.241 0.243 0.246 0.248 0.250 0.253
1.8 0.255 0.258 0.260 0.262 0.265 0.267 0.270 0.272 0.274 0.276
1.9 0.279 0.281 0.283 0.286 0.288 0.290 0.292 0.294 0.297 0.299
2.0 0.301 0.303 0.305 0.307 0.310 0.312 0.314 0.316 0.318 0.320
2.1 0.322 0.324 0.326 0.328 0.330 0.332 0.334 0.336 0.338 0.340
2.2 0.342 0.344 0.346 0.348 0.350 0.352 0.354 0.356 0.358 0.360
2.3 0.362 0.364 0.365 0.367 0.369 0.371 0.373 0.375 0.377 0.378
2.4 0.380 0.382 0.384 0.386 0.387 0.389 0.391 0.393 0.394 0.396
2.5 0.398 0.400 0.401 0.403 0.405 0.407 0.408 0.410 0.412 0.413
2.6 0.415 0.417 0.418 0.420 0.422 0.423 0.425 0.427 0.428 0.430
2.7 0.431 0.433 0.435 0.436 0.438 0.439 0.441 0.442 0.444 0.446
2.8 0.447 0.449 0.450 0.452 0.453 0.455 0.456 0.458 0.459 0.461
2.9 0.462 0.464 0.465 0.467 0.468 0.470 0.471 0.473 0.474 0.476
3.0 0.477 0.479 0.480 0.481 0.483 0.484 0.486 0.487 0.489 0.490
3.1 0.491 0.493 0.494 0.496 0.497 0.498 0.500 0.501 0.502 0.504
3.2 0.505 0.507 0.508 0.509 0.511 0.512 0.513 0.515 0.516 0.517
3.3 0.519 0.520 0.521 0.522 0.524 0.525 0.526 0.528 0.529 0.530
3.4 0.531 0.533 0.534 0.535 0.537 0.538 0.539 0.540 0.542 0.543
3.5 0.544 0.545 0.547 0.548 0.549 0.550 0.551 0.553 0.554 0.555
3.6 0.556 0.558 0.559 0.560 0.561 0.562 0.563 0.565 0.566 0.567
3.7 0.568 0.569 0.571 0.572 0.573 0.574 0.575 0.576 0.577 0.579
3.8 0.580 0.581 0.582 0.583 0.584 0.585 0.587 0.588 0.589 0.590
3.9 0.591 0.592 0.593 0.594 0.595 0.597 0.598 0.599 0.600 0.601
4.0 0.602 0.603 0.604 0.605 0.606 0.607 0.609 0.610 0.611 0.612
4.1 0.613 0.614 0.615 0.616 0.617 0.618 0.619 0.620 0.621 0.622
4.2 0.623 0.624 0.625 0.626 0.627 0.628 0.629 0.630 0.631 0.632
4.3 0.633 0.634 0.635 0.636 0.637 0.638 0.639 0.640 0.641 0.642
4.4 0.643 0.644 0.645 0.646 0.647 0.648 0.649 0.650 0.651 0.652
4.5 0.653 0.654 0.655 0.656 0.657 0.658 0.659 0.660 0.661 0.662
4.6 0.663 0.664 0.665 0.666 0.667 0.667 0.668 0.669 0.670 0.671
4.7 0.672 0.673 0.674 0.675 0.676 0.677 0.678 0.679 0.679 0.680
4.8 0.681 0.682 0.683 0.684 0.685 0.686 0.687 0.688 0.688 0.689
4.9 0.690 0.691 0.692 0.693 0.694 0.695 0.695 0.696 0.697 0.698
5.0 0.699 0.700 0.701 0.702 0.702 0.703 0.704 0.705 0.706 0.707
5.1 0.708 0.708 0.709 0.710 0.711 0.712 0.713 0.713 0.714 0.715
5.2 0.716 0.717 0.718 0.719 0.719 0.720 0.721 0.722 0.723 0.723
5.3 0.724 0.725 0.726 0.727 0.728 0.728 0.729 0.730 0.731 0.732
5.4 0.732 0.733 0.734 0.735 0.736 0.736 0.737 0.738 0.739 0.740
Logarithm Table (for numbers from 5.5 to 10)

No. 0.00 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08 0.09
5.5 0.740 0.741 0.742 0.743 0.744 0.744 0.745 0.746 0.747 0.747
5.6 0.748 0.749 0.750 0.751 0.751 0.752 0.753 0.754 0.754 0.755
5.7 0.756 0.757 0.757 0.758 0.759 0.760 0.760 0.761 0.762 0.763
5.8 0.763 0.764 0.765 0.766 0.766 0.767 0.768 0.769 0.769 0.770
5.9 0.771 0.772 0.772 0.773 0.774 0.775 0.775 0.776 0.777 0.777
6.0 0.778 0.779 0.780 0.780 0.781 0.782 0.782 0.783 0.784 0.785
6.1 0.785 0.786 0.787 0.787 0.788 0.789 0.790 0.790 0.791 0.792
6.2 0.792 0.793 0.794 0.794 0.795 0.796 0.797 0.797 0.798 0.799
6.3 0.799 0.800 0.801 0.801 0.802 0.803 0.803 0.804 0.805 0.806
6.4 0.806 0.807 0.808 0.808 0.809 0.810 0.810 0.811 0.812 0.812
6.5 0.813 0.814 0.814 0.815 0.816 0.816 0.817 0.818 0.818 0.819
6.6 0.820 0.820 0.821 0.822 0.822 0.823 0.823 0.824 0.825 0.825
6.7 0.826 0.827 0.827 0.828 0.829 0.829 0.830 0.831 0.831 0.832
6.8 0.833 0.833 0.834 0.834 0.835 0.836 0.836 0.837 0.838 0.838
6.9 0.839 0.839 0.840 0.841 0.841 0.842 0.843 0.843 0.844 0.844
7.0 0.845 0.846 0.846 0.847 0.848 0.848 0.849 0.849 0.850 0.851
7.1 0.851 0.852 0.852 0.853 0.854 0.854 0.855 0.856 0.856 0.857
7.2 0.857 0.858 0.859 0.859 0.860 0.860 0.861 0.862 0.862 0.863
7.3 0.863 0.864 0.865 0.865 0.866 0.866 0.867 0.867 0.868 0.869
7.4 0.869 0.870 0.870 0.871 0.872 0.872 0.873 0.873 0.874 0.874
7.5 0.875 0.876 0.876 0.877 0.877 0.878 0.879 0.879 0.880 0.880
7.6 0.881 0.881 0.882 0.883 0.883 0.884 0.884 0.885 0.885 0.886
7.7 0.886 0.887 0.888 0.888 0.889 0.889 0.890 0.890 0.891 0.892
7.8 0.892 0.893 0.893 0.894 0.894 0.895 0.895 0.896 0.897 0.897
7.9 0.898 0.898 0.899 0.899 0.900 0.900 0.901 0.901 0.902 0.903
8.0 0.903 0.904 0.904 0.905 0.905 0.906 0.906 0.907 0.907 0.908
8.1 0.908 0.909 0.910 0.910 0.911 0.911 0.912 0.912 0.913 0.913
8.2 0.914 0.914 0.915 0.915 0.916 0.916 0.917 0.918 0.918 0.919
8.3 0.919 0.920 0.920 0.921 0.921 0.922 0.922 0.923 0.923 0.924
8.4 0.924 0.925 0.925 0.926 0.926 0.927 0.927 0.928 0.928 0.929
8.5 0.929 0.930 0.930 0.931 0.931 0.932 0.932 0.933 0.933 0.934
8.6 0.934 0.935 0.936 0.936 0.937 0.937 0.938 0.938 0.939 0.939
8.7 0.940 0.940 0.941 0.941 0.942 0.942 0.943 0.943 0.943 0.944
Appendices  367
8.8 0.944 0.945 0.945 0.946 0.946 0.947 0.947 0.948 0.948 0.949
8.9 0.949 0.950 0.950 0.951 0.951 0.952 0.952 0.953 0.953 0.954
9.0 0.954 0.955 0.955 0.956 0.956 0.957 0.957 0.958 0.958 0.959
9.1 0.959 0.960 0.960 0.960 0.961 0.961 0.962 0.962 0.963 0.963
9.2 0.964 0.964 0.965 0.965 0.966 0.966 0.967 0.967 0.968 0.968
9.3 0.968 0.969 0.969 0.970 0.970 0.971 0.971 0.972 0.972 0.973
9.4 0.973 0.974 0.974 0.975 0.975 0.975 0.976 0.976 0.977 0.977
9.5 0.978 0.978 0.979 0.979 0.980 0.980 0.980 0.981 0.981 0.982
9.6 0.982 0.983 0.983 0.984 0.984 0.985 0.985 0.985 0.986 0.986
9.7 0.987 0.987 0.988 0.988 0.989 0.989 0.989 0.990 0.990 0.991
9.8 0.991 0.992 0.992 0.993 0.993 0.993 0.994 0.994 0.995 1.00
9.9 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00
10.0 1.00
APPENDIX VII: SI UNIT CONVERSION
Weights and Measure: SI Units

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

9
10 giga G
10 6 mega M
10 3 kilo K
10 2 hecto H
10 1 deca Da
10–1 deci d
10–2 centi c
10–3 milli m
10–6 micro m
10–9 nano n
10–12 pico p
10–15 femto f
10–18 atto a
Appendices  369
APPENDIX VIII: PRACTICAL EXAMINATION QUESTION PAPER*
MD (Pharmacology)
Long Experiment
1. Write the Protocol and undertake the 4 point bioassay experiment to determine the concentration of
histamine in the given sample in an in vitro tissue of your preference.
Short Experiment
2. Demonstration of analgesic effect of given drug in healthy human volunteers
3. Demonstration of antiepileptic effect of phenytoin in mice
4. Demonstration of straub tail phenomenon
Viva
1. Experimental practical
2. Main theory
DM (Clinical Pharmacology)
Long Experiment
1. Demonstration of β blocker effect in healthy volunteers
Short Experiments
2. Demonstration of acetylator status by isoniazid estimation in healthy volunteers
3. Analyze the given prescription for the patient of bronchial asthma (e.g. corticosteroid in Bronchial asthma)
4. Interpretation of given ECG
a. A 55 year old man with 4 hours of “crushing” chest pain, ECG of patient showed changes in ST , leads
II,III, aVF and in anterior lead , write the diagnosis from ECG and discuss the drug therapy.
b. 45 year old lady complain of palpitations and history of chronic renal failure, ECG showed a wide QRS
tachycardia, write the diagnosis from ECG and discuss the drug therapy.
5. Comment on given drug advertisement (promotional product literature)
Viva
1. Clinical practical
2. Main theory
(* This is to provide an overall idea of practical pharmacology examination; it varies Institution to Institution/University)
INDEX
A B Chimney test 186
Abnormal appearance 16 Barber 16 Chi-square (χ2) 125, 131
Acclimatization 137 Barbital 183, 348 Cholinesterase enzymes 169
Aceclofenac 320, 321, 322, 323 Bicycle ergo meter 256, 258 Chromatogram 87, 320
Acetic acid Binomial distribution 126 Chronobiology 241, 252
colitis 220 Bioassay 45 Cleaning of glass ware 120
writhing 225 3-point assay 60 Clinical trial 3, 6
Acetylcholine 154, 162, 165, 166, 167, 4-point assay 61 Clinomics 39
170, 171, 177, 178 antagonist 62 Colitis 220
anococcygeus 159, 160 bracketing 57, 58 Cold stress test 323
biventer-cervicis171 cytokines 67 Colon 154
fundus 57, 116, 142, 149, 164, 165 error 46 collection 141
heart 158, 177, 178 examples 69 effect of acetylcholine 154
ileum 70, 71,161 human tissue 66 Comparative control group 107
skeletal muscle 166 matching assay 57, 59 Complete Freund’s adjuvant 224
uterus 149, 155, 156, 157 principle 68 Computational pharmacology 338
vas deferens 160, 161, 170 Biomedical waste 120 Confidence interval 128
Acute study 139 Biostatistics 123 Confidence limit 129
Adrenaline 34,157,160,169,177,178 Blood collection/withdrawal 30 Contact time 56, 64
atria 157, Blood pressure measurement 207 Control group 107
heart 157,173,177 animal 207 Convulsiometer 78
uterus 34 error 251 Coprophagy 9, 16, 217
Allogroom 16 exercise 245 Cryopreservation 96, 97
Analgesiometer 77 guideline (Human) 239, 247 Cuff hypertension 243
Analysis of Variance (ANOVA) 125, human 239 Cumulative plot 63
128, 130 regulation 243 Cutaneous mycobacteriosis 35
Analytical toxicology 346 Box behavior 16 Cytokines bioassay 67
Anesthesia 37 BP apparatus 80, 124
Animal BP cuff inflation test 324 D
allergy 36 Bradykinin 149, 165
Data 123
anesthesia 37 Bronchoalveolar lavage (BAL) 223
Decapitation 39, 137
behavior 16
C Dextran sulfate 220
blood collection 30
Diazepam 183, 184, 185,186, 187, 189,
euthanasia 37 Campylobacter 35
190, 192, 196, 291, 295, 331
handling 17 Carbamazepine 357
blood level 348
sex determination 17 blood level 348
solubility 355
Ankle Brachial Index (ABI) 268 HPLC estimation 333
Dibenzyline, see phenoxybenzamine
Anococcygeus Carrageenan
colitis 220 263
Ach 159
inflammatory 224 Diet 21
collection 144
Cat 8, 13, 33, 34, 184 DOCA salt 214
Anococcygeus 144
Catalepsy 197 Dog 8, 13, 17, 33, 34, 37
effect of Ach 159
Catatonia 197 Dopamine 34, 199, 200
Antagonist (Bioassay) 62
Arm position 242, 254 Ceiling effect 51 Dose cycle 56
Ascorbic acid 10 Cell line 95, 138 Dose ratio 64
Aspirin 202, 325, 357 Centrifuge 82 Dose-response curves 50
gastric ulcer 217 Cerebral ischemia 203 Double pith 39
solubility 355 Cerulein 218 Drug
writhing 226 Chick development 5
Atria 157 bi-venter cervicis 171 experimental background 1
Atropine 34, 162, 177 Chicken 8, 15, 95 Drug response relationship 50
E foot withdrawal reflex 229 arm at heart level 242
ECG rectus abdominis muscle atria 157
animal 211 (collection) 143 calcium chloride 176
clinical 269 Frusemide 303 digoxin 176
ED50 47, 115, frog heart 177
G
Electro convulsiometer 79, 196 hypodynamic 176, 177
Electrodes 78, 195, 196, 272 Gallop 17 Langendorff’s preparation 173
Gastric ulcer 217 rate 254, 268, 309
Electrolyte analyzer 80
picture 221 starling lever 55
Elevated plus maze 79, 187
Ephedrine 156, 231 Gerbil 8, 10 Herpes B virus infection 35
blood withdrawal 32 Hexabarbital 183
Ethanol
euthanasia 39 High performance liquid
35% ethanol 220
70% ethanol 96, 204, general information 16 chromatography (HPLC) 84
vital parameters 16 High throughput screening 6
blood level 348
zoonotic disease 35 Histamine
ulcer 217
Ethical consideration of animals 39 Glyceryl trinitrate ileum 152, 153
blood pressure 264 Hit and lead compound 5
Euthanasia 37
heart rate 264 Hot plate method 201
methods 38
object 38 arterial vasodilatation 266 Human bioassay 66
Good laboratory practice (GLP) 115, 120 Hyper-pharmacology 115
preferred 39
Guinea pig 8, 9 Hypertension
Ex vivo 138
Exercise tolerance test 285 atria 157 experiment 214
collection 158 ocular 316
Exercise
blood withdrawal 32 spontaneous in dog 13
acute and chronic 245
aerobic and anaerobic 245 euthanasia 39 Hypnosis 183
isotonic and isometric 246 general information 16 Hypodynamic heart 177
maximal and submaximal 246 handling 19
ileum 69, 150 I
Experimental animals 6
cat 13 collection 141, 152 ID50 (IC50) 65
chicken 15 experiment 152 Ileum 57, 149
dog 13 popcorning 17 collection 140
frog 14 pA2 161 guinea pig 69, 152
gerbil 10 sex determination 21 pA2 161
guinea pig 9 trachea rat 70, 71
hamster 10 collection 143 Impact factor 336
monkey 12 vital parameters 16 In vitro 138
mouse 8 zoonotic disease 35 In vivo 138
pig 13 H Indomethacin
pigeon 15 carrageenan 224
rabbit 11 5-hydroxy tryptamine (5-HT) 149,
gastric ulcer 217
rat 9 156, 159
Injection site 26
zebra fish 14 stomach fundus 164
Intradermal injection 30
Experimental window 66 Haloperidol 197
Hamster 8, 10, 34 Isolated heart
Exsanguinations 137 Langendorff’s preparation 173
anesthesia 38
F blood withdrawal 32 Isometric
euthanasia 39 contraction 151
False positive 78, 128, 202
Field stimulation 67 general information 16 exercise 246
First human dose 100, 101, 102, 103 handling 20 Isoniazid (INH) 328
Fischer exact test 125, 131 vital parameters 16 Isoprenaline 149, 156,157, 159, 169,
Foot withdrawal reflex 229 zoonotic disease 35 175
Fresh egg white Hand Dynamometer 256, 259, 324 Isoproterenol 213
irritant 224 Hantavirus infection 35 Isotonic
Frog Hayflick’s limit 96 contraction 150
experiment 166 Heart exercise 246
Index  373
K Nitroglycerin (NTG) Poisson distribution 126, 127
Kindling model 194 patch 266 Polymerase chain reaction (PCR) 84
Knocked down 8 Non-parametric tests 128, 129 Positive control 107
Knocked in 8 Normal distribution 126, 130 Postural hypotension 255, 263, 267, 268
Knocked out 8 Preclinical study 6, 100
O
Kymograph 47 Predicted maximum heart rate 257
fixing 49 Ocular hypertension 316 Procaine HCl 229
Off-plate dilution 68 Product literature 342
ink 49
Orthostatic hypertension 263 Propylene glycol 213
instrument 76
Oxybutynin 330 Protocol 105
L clinical 108
P
Langendorff’s preparation 173 experimental 105
pA2 64 Protocol writing 105
Latin square randomization
example 71 Psittacosis 35
3-point 60
4-point 62 experiment 161 Psychomotor function 291
Pancreatitis p-value 128
Lead compound optimization 6
acute/chronic 219 Pupillary diameter 309
Lethal dose 102
calculation 117 Papillary diameter 311 Pupillometer 230, 310
Parallel shift 64, 65 Pylorus ligation method 217
Lethality testing 117
Parametric tests 128, 129 Pyrogen testing 12, 91
Lignocaine
gel (2%) 228, 233 Passive avoidance test 79 in vivo 91
Paw edema 224 in vitro 93
topical (4%) 233, 235
Peak expiratory flow rate (PEFR) 280
Liquid chromatography and mass- R
spectrometry (LC-MS) 88 Pentobarbital
anesthesia in BP measurement 207 Rabbit 8, 11
Lithium 333
anesthesia in thrombosis model blood withdrawal 33
estimation 334
Local anesthetics (LA) 228 216 general information 16
anesthetic agent 38 handling 21
Lordosis 17
righting reflex 183 oral gavage 26
M Pentylenetetrazole (PTZ) vital parameters 16
Master’s 2 step test 256 GTCS model 191 Rabbit eye 230
Maximal electroshock (MES) 195 kindling model 194 Rabbit ileum 153
Median lethal dose 117, 119 Personalized medicine 350 Rabbit uterus 156
Mental stress 261 Pharmacokinetics Rabies 12, 35
Mepyramine 152 experimental 233 Radiant heat 323
Metabolic equivalents 246 clinical 319 Rat
Micro dosing 351 Phenobarbitone rat fever 35
Molecular medicine 350 HPLC method 333 rat uterus 155
Monkey 8, 12 normal level 334 Rate pressure product 259
blood withdrawal 33 Phenytoin 195, 233, 235 Rectus abdominis muscle
Morphine Phrenic nerve diaphragm collection 143
blood level 348 collection 145 effect of Ach 166
hot plate/ tail flick 201 effect of Ach 169 Regression 131
response 34 Physiological salt solution (PSS) 47 Reverse pharmacology 350
straub tail 199 composition 51, 53 Righting reflex 13, 192
writhing 225 Physostigmine 169, 171, 231 experiment 183
Morris water maze Pica 17 Ring worm 35
experiment 190 Pig 8, 13 Rotarod
instrument 79 blood withdrawal 33 experiment 184
Muscle contraction Pigeon 8, 15
Pilocarpine S
principle 150
Muscle relaxant 184, 186 mitotic 231 Safety factor 103
Myocardial infarction (MI) 213 topical (2%) 314 Salicylate 325
Pilot study 114 blood level 348
N PK/PD modeling 340 Salivary secretion 309, 310, 330
Nictating membrane 13 Plethysmograph 81, 222 Salmonella 35
Schild plot 64, 65, 161 Tetanus 35 U
Seizure score 78 Therapeutic drug monitoring 88, 333
Ulcer 217
Seizure Thesis writing 105, 111
gastric 217, 221
MES 78 Thrombosis
ferric chloride 216 Uterus 34, 57,
PTZ 191
Serotonin Tissue preparation method 135 collection 142
irritant 224 Tonic hind limb extension (THLE) effect of Ach 149, 155
stomach fundus 164 78, 195, 196 effect of oxitocin 149, 156
Sham control group 107 Toxic dose identification 148
SHAY method 217 acute 115, 116
blood level 348 V
Spontaneous activity 53, 152, 153
Stair climbing exercise tolerance chronic 117 Vas deferens 144, 149
test 285 dose calculation 101 effect of Ach 149, 160
Stomach fundus 57, 149 subacute/ subchronic 116 Vasodilatation
collection 142 Toxicity study 115 agent 31
effect of 5-HT 164 Tracheal chain 149, 167 Visual pathway 231
Straub tail reaction 199 collection 167, 168
Student’s physiograph 76 human 67 W
Students‘t’ test 129 identification 148
Translational medicine 350 Waste disposal 120
Subacute study 116
Tread mill test (TMT) 256, 257 Waud method 65
Subchronic study 116
Submaximal exercises 246 Tri Nitro Benzene Sulphonic acid Wheal and flare 331
(TNBS)
T Z
colitis 220
Tail flick method 201 Tropicamide 362 Zebra fish 8, 14
Telemetry 89 topical (1%) 312 anesthesia 38
BP measurement (animal) 207 Tubocurarine 171 Zoonotic disease 34

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Practical Manual of Experimental

and Clinical Pharmacology

JAYPEE BROTHERS MEDICAL PUBLISHERS (P) LTD

Practical Manual of Experimental and Clinical Pharmacology

First Edition: 2010

Printed at Ajanta Offset

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

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

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

PART 3: EXPERIMENTAL (IN VIVO STUDIES)

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

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

PART 4: CLINICAL EXPERIMENTS

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

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

EXPERIMENTAL BACKGROUND traditional drugs used in their own practices in

John Jacob Abel same year appointed as assistant to

Table 1.1: Drug discovery timeframe and compounds screened

 Important Points to Remember  Important Points to Remember

Note: Some important phenotypic differences

dietary requirement. But, it is essential to add

Gerbil as an Experimental Animal

 Important Points to Remember

Dog as an Experimental Animal

or rodent, hence they have been extensively used

Cat as an Experimental Animal

Frog is the only tail less experimental amphibian

Pigeon (Columbia livia)

In the experimental pharmacology, chicken are

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

Table 1.3: Vital parameters of different experimental animals

Mouse Way 3: Hold the complete body by grabbing back

Fig. 1.4: Mouse handling technique (way 3)

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

Fig. 1.7: Rat handling technique (way 3)

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

Way 3: Hold the complete body by grabbing the

Way 2: Handle guinea pig with one hand, by

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

Fig. 1.10: Hamster handling technique (way 1)

Way 2: Hold the complete body by grabbing back

Fig. 1.15: Rabbit handling technique (way 3)

Sex Determination in Rodents

DIET AND EXPERIMENTAL ANIMALS

Fig. 1.16: Gender identification in male/female Rat

Fig. 1.17: Gender identification in Guniea pig

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

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

Step IV: Then, convert into per kg dose according

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

Fig. 1.19: Mouse Fig. 1.21: Hamster

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

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

Fig. 1.24: Rabbit marginal vein (Prefered route)

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

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

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

Fig. 1.31: Rat (Flank)

Intracardiac Injection (Figs 1.32 and 1.33)