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A Pharmacology Primer, 6e
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ACADEMIC
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A Pharmacology Primer
Techniques for More Effective and Strategic
Drug Discovery
Sixth Edition

Terry P. Kenakin
Professor of Pharmacology
The University of North Carolina School of Medicine
Chapel Hill, NC, United States
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 Dedication

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Contents
Preface to sixth edition xiii
2.6.3 Differences in receptor density 35
2.6.4 Target-mediated trafficking of
stimulus 37
1. What is pharmacology? 2.7 Receptor desensitization and
1.1 About this book 1 tachyphylaxis 37
1.2 What is pharmacology? 1 2.8 The measurement of drug activity 40
1.3 The receptor concept 3 2.9 Advantages and disadvantages of
1.4 Pharmacological test systems 4 different assay formats 40
1.5 The nature of drug receptors 7 2.10 Drug concentration as an independent
1.6 From the snapshot to the movie 7 variable 41
1.7 Pharmacological intervention and the 2.10.1 Dissimulation in drug
therapeutic landscape 8 concentration 41
1.8 System-independent drug parameters: 2.10.2 Free concentration of drug 43
affinity and efficacy 11 2.11 Chapter summary and conclusions 43
1.9 What is affinity? 13 2.12 Derivations 43
1.10 The Langmuir adsorption isotherm 14 2.12.1 Series hyperbolae can be
1.11 What is efficacy? 15 modeled by a single
1.12 Doseeresponse curves 17 hyperbolic function 44
1.12.1 Potency and maximal response 18 2.12.2 Successive rectangular hyper-
1.12.2 P-scales and the representation bolic equations necessarily
of potency 18 lead to amplification 44
1.13 Chapter summary and conclusions 20 2.12.3 Saturation of any step in a
1.14 Derivations: conformational selection stimulus cascade by two
as a mechanism of efficacy 20 agonists leads to identical
References 21 maximal final responses for
the two agonists 44
2. How different tissues process drug 2.12.4 Procedure to measure free
response drug concentration in the
receptor compartment 45
2.1 The ‘eyes to see’: pharmacologic assays 23 References 45
2.2 The biochemical nature of stimuluse
response cascades 25 3. Drugereceptor theory
2.3 The mathematical approximation of
stimuluseresponse mechanisms 27 3.1 About this chapter 47
2.4 Influence of stimuluseresponse 3.2 Drugereceptor theory 47
cascades on doseeresponse curve 3.3 The use of mathematical models in
slopes 29 pharmacology 48
2.5 System effects on agonist response: 3.4 Some specific uses of models in
full and partial agonists 30 pharmacology 49
2.6 Differential cellular response to 3.5 Mass action building blocks 55
receptor stimulus 33 3.6 Classical model of receptor
2.6.1 Choice of response pathway 33 function 56
2.6.2 Augmentation or modulation of 3.7 The operational model of receptor
stimulus pathway 34 function 57

vii
viii Contents

3.8 Two-state theory 58 4.7.3 Displacement of a radioligand

3.9 The ternary complex model 59 by an allosteric antagonist 92
3.10 The extended ternary complex model 59 4.7.4 Relationship between IC50 and
3.11 Constitutive receptor activity and KI for competitive antagonists 93
inverse agonism 60 4.7.5 Maximal inhibition of binding
3.12 The cubic ternary complex model 62 by an allosteric antagonist 94
3.13 Multistate receptor models and 4.7.6 Relationship between IC50 and
probabilistic theory 63 KI for allosteric antagonists 94
3.14 Chapter summary and conclusions 65 4.7.7 Two-stage binding reactions 94
3.15 Derivations 65 4.7.8 Effect of G-Protein coupling on
3.15.1 Radioligand binding to observed agonist affinity 94
receptor dimers demonstrating 4.7.9 Effect of excess receptor in
cooperative behavior 65 binding experiments: saturation
3.15.2 Effect of variation in an HIV-1 binding curve 94
binding model 66 4.7.10 Effect of excess receptor in
3.15.3 Derivation of the operational binding experiments: displace-
model 67 ment experiments 95
3.15.4 Operational model forcing 4.7.11 Derivation of an allosteric bind-
function for variable slope 67 ing model 95
3.15.5 Derivation of two-state theory 68 References 96
3.15.6 Derivation of the extended
ternary complex model 68 5. Drug targets and drug-target
3.15.7 Dependence of constitutive molecules
activity on receptor density 69
3.15.8 Derivation of the cubic ternary 5.1 Defining biological targets 97
complex model 69 5.2 Specific types of drug targets 100
References 69 5.2.1 G-protein-coupled receptors 100
5.2.2 Ion channels 102
5.2.3 Enzymes 103
4. Pharmacological assay formats:
5.2.4 Nuclear receptors 111
binding
5.2.5 Nucleotide-based drug targets 112
4.1 The structure of this chapter 71 5.3 Small drug-like molecules 114
4.2 Binding theory and experiment 71 5.3.1 Hybrid molecules 116
4.2.1 Saturation binding 74 5.3.2 Chemical sources for potential
4.2.2 Displacement binding 76 drugs 121
4.2.3 Kinetic binding studies 79 5.4 Biologics 126
4.3 Complex binding phenomena: agonist 5.4.1 Replacement proteins 127
affinity from binding curves 80 5.4.2 Eliminating ‘undruggable’ pro-
4.4 Experimental prerequisites for correct teins through PROTACs 130
application of binding techniques 84 5.4.3 Peptides 131
4.4.1 The effect of protein concentra- 5.4.4 Antibodies 135
tion on binding curves 84 5.4.5 Immunotherapy 141
4.4.2 The importance of equilibration 5.4.6 Vaccines 141
time for equilibrium between 5.4.7 Nucleic acidebased drug species 142
two ligands 85 5.5 Summary and conclusions 147
4.5 Binding in allosteric systems 87 References 147
4.6 Chapter summary and conclusions 91 Further reading 149
4.7 Derivations 92
4.7.1 Displacement binding: 6. Agonists: the measurement of affinity
competitive interaction 92 and efficacy in functional assays
4.7.2 Displacement binding:
noncompetitive interaction 92 6.1 Functional pharmacological
experiments 151
 Contents ix

6.2 The choice of functional assays 152 7.3.5 Analyses for partial agonists 201
6.3 Recombinant functional systems 156 7.3.6 The method of Lew and Angus:
6.4 Functional experiments: dissimulation nonlinear regression analysis 203
in time 159 7.4 Noncompetitive antagonism 204
6.5 Experiments in real time versus 7.5 Agonisteantagonist hemiequilibria 208
stop-time 160 7.6 Resultant analysis 210
6.6 Quantifying agonism: the BlackeLeff 7.7 Antagonism in vivo 210
operational model of agonism 162 7.7.1 Antagonists with efficacy in
6.6.1 Affinity-dependent versus vivo 212
efficacy-dependent agonist 7.7.2 Kinetics of target coverage 214
potency 166 7.7.3 Kinetics of dissociation 216
6.6.2 Secondary and tertiary testing 7.7.4 Estimating antagonist
of agonists 168 dissociation with hemiequilibria 219
6.7 Biased signaling 169 7.8 Blockade of indirectly acting agonists 219
6.7.1 Receptor selectivity 175 7.9 Irreversible antagonism 220
6.8 Null analyses of agonism 175 7.10 Chemical antagonism 222
6.8.1 Partial agonists 175 7.11 Chapter summary and conclusions 226
6.8.2 Full agonists 179 7.12 Derivations 227
6.9 Comparing full and partial agonist 7.12.1 Derivation of the Gaddum
activities: Log(max/EC50) 182 equation for competitive
6.10 Chapter summary and conclusions 183 antagonism 227
6.11 Derivations 183 7.12.2 Derivation of the Gaddum
6.11.1 Relationship between the EC50 equation for noncompetitive
and affinity of agonists 183 antagonism 227
6.11.2 Method of Barlow, Scott, and 7.12.3 Derivation of the schild
Stephenson for affinity of equation 228
partial agonists 184 7.12.4 Functional effects of an
6.11.3 Maximal response of a partial inverse agonist with the
agonist is dependent on operational model 228
efficacy 184 7.12.5 pA2 measurement for inverse
6.11.4 System independence of full agonists 228
agonist potency ratios 184 7.12.6 Functional effects of a partial
6.11.5 Measurement of agonist agonist with the operational
affinity: method of Furchgott 184 model 229
6.11.6 Agonism as a positive 7.12.7 pA2 measurements for partial
allosteric modulation of agonists 229
receptoresignaling protein 7.12.8 Method of Stephenson for
interaction to derive partial agonist affinity
DLog(max/EC50) ratios 185 measurement 229
References 187 7.12.9 Derivation of the Method of
Gaddum for noncompetitive
7. Orthosteric drug antagonism antagonism 230
7.12.10 Relationship of pA2 and pKB
7.1 Introduction 189
for insurmountable
7.2 Kinetics of drugereceptor interaction 189
orthosteric antagonism 230
7.3 Surmountable competitive antagonism 192
7.12.11 Resultant analysis 230
7.3.1 Schild analysis 192
7.12.12 Blockade of indirectly acting
7.3.2 Patterns of DoseeResponse
agonists 231
curves that preclude schild
7.12.13 Chemical antagonism:
analysis 197
abstraction of agonist
7.3.3 Best practice for the use of
concentration 231
schild analysis 198
7.12.14 Chemical antagonism:
7.3.4 Analyses for inverse agonists in
abstraction of antagonist
constitutively active receptor
concentration 231
systems 199
References 232
x Contents

8. Allosteric modulation 9.5.1 Predicting agonism 299

9.5.2 Predicting binding 301
8.1 Introduction 233 9.5.3 Drug combinations in vivo 302
8.2 The nature of receptor allosterism 233 9.6 Summary and conclusions 303
8.3 Unique effects of allosteric modulators 235 9.7 Derivations 304
8.4 Functional study of allosteric modulators 240 9.7.1 IC50 Correction Factors:
8.4.1 Phenotypic allosteric modulation competitive antagonists 304
profiles 242 9.7.2 Relationship of pA2 and pKB for
8.4.2 Allosteric agonism 243 Insurmountable Orthosteric
8.4.3 Affinity of allosteric modulators 243 antagonism 304
8.4.4 Negative allosteric modulators 246 9.7.3 Relationship of pA2 and pKB for
8.4.5 Positive allosteric modulators 250 Insurmountable Allosteric
8.4.6 Quantifying PAM activity in vivo 254 Antagonism 305
8.4.7 NAM/PAM induced agonist bias 255 References 305
8.4.8 Optimal assays for allosteric
function 255
10. Pharmacokinetics
8.5 Functional allosteric model with
constitutive activity 256 10.1 Introduction 307
8.6 Internal checks for adherence to the 10.2 Biopharmaceutics 307
allosteric model 257 10.3 The chemistry of “drug-like”
8.7 Methods for detecting allosterism 260 character 308
8.8 Chapter summary and conclusions 262 10.4 Pharmacokinetics 313
8.9 Derivations 262 10.4.1 Drug absorption 313
8.9.1 Allosteric model of receptor 10.4.2 Route of drug
activity 262 administration 319
8.9.2 Effects of allosteric ligands on 10.4.3 General pharmacokinetics 322
response: changing efficacy 263 10.4.4 Metabolism 325
8.9.3 Schild analysis for allosteric 10.4.5 Clearance 330
antagonists 263 10.4.6 Volume of distribution and
8.9.4 Application of Log(Max/R50) half-life 332
values from R50 curves to 10.4.7 Renal clearance 338
quantify the effects of PAMs 264 10.4.8 Bioavailability 340
8.9.5 Quantifying allosterically 10.5 Nonlinear pharmacokinetics 342
mediated induced bias in 10.6 Multiple dosing 343
agonism 264 10.7 Modifying pharmacokinetics through
8.9.6 Functional allosteric model with medicinal chemistry 345
constitutive receptor activity 265 10.8 Practical pharmacokinetics 347
References 266 10.8.1 Allometric scaling 349
10.9 Placement of pharmacokinetic assays
9. The optimal design of in discovery and development 350
pharmacological experiments 10.10 The pharmacokinetics of biologics 352
10.10.1 Absorption 353
9.1 Introduction 269 10.10.2 Duration of action 354
9.2 The optimal design of pharmacological 10.10.3 Antibody PK 355
experiments 269 10.10.4 mRNA PK 355
9.2.1 Drug efficacy 270 10.11 Summary and conclusions 355
9.2.2 Affinity 283 References 356
9.2.3 Orthosteric versus allosteric
mechanisms 292 11. Safety pharmacology
9.3 Null experiments and fitting data to
models 293 11.1 Safety pharmacology 359
9.4 Interpretation of experimental data 296 11.2 Hepatotoxicity 365
9.5 Predicting therapeutic activity in all 11.2.1 Drugedrug interactions 365
systems 299 11.2.2 Direct hepatotoxicity 370
 Contents xi

11.2.3 Hepatotoxicity in context in 13.2.3 Method of Furchgott for the

vivo 372 measurement of the affinity
11.3 Cytotoxicity 372 of a full agonist 428
11.4 Mutagenicity 374 13.2.4 Schild analysis for the
11.5 hERG activity and Torsades de measurement of competitive
Pointes 376 antagonist affinity 429
11.6 Autonomic receptor profiling and 13.2.5 Method of Stephenson for
off-target effects 376 measurement of partial
11.7 General pharmacology 377 agonist affinity 431
11.8 Clinical testing and drug toxicity 379 13.2.6 Method of Gaddum for
11.9 Summary and conclusions 381 measurement of noncom-
References 381 petitive antagonist affinity 433
13.2.7 Method for estimating affin-
12. The drug-discovery process ity of insurmountable antag-
onist (dextral displacement
12.1 Some challenges for modern drug
observed) 434
discovery 383
13.2.8 Resultant analysis for
12.2 The drug-discovery process 384
measurement of affinity of
12.3 Target-based drug discovery 384
competitive antagonists
12.3.1 Target validation and the
with multiple properties 436
use of chemical tools 385
13.2.9 Measurement of the affinity
12.3.2 Recombinant systems 388
and maximal allosteric
12.4 Systems-based drug discovery 390
constant for allosteric mod-
12.5 High-throughput screening 393
ulators producing surmount-
12.5.1 Structure-based drug design
able effects 436
and virtual screening 404
13.2.10 Method for estimating
12.5.2 Phenotypic screening 405
affinity of insurmountable
12.6 The lead optimization process 409
antagonist (no dextral
12.7 Drug effectiveness 413
displacement observed):
12.7.1 Clinical testing 414
detection of allosteric effect 438
12.7.2 Determining detailed profiles
13.2.11 Measurement of pKB for
of candidate efficacy 416
competitive antagonists
12.7.3 Assays in context 417
from a pIC50 441
12.7.4 Characterization of candidate
13.2.12 Statistical assessment of
efficacies 418
selectivity 442
12.8 Summary and conclusions 419
13.2.13 Measurement of surmount-
References 420
able allosteric antagonism 447
Further reading 422
13.2.14 Measurement of
insurmountable allosteric
13. Selected pharmacological methods antagonism (second
13.1 Binding experiments 423 method) 448
13.1.1 Saturation binding 423 13.2.15 Measurement of PAM
13.1.2 Displacement binding 423 activity 450
13.2 Functional assays 426 Reference 451
13.2.1 Determination of equiactive
concentrations on Dosee
Response curves 426
13.2.2 Method of Barlow, Scott,
Appendix 1: Statistics 453
and Stephenson for
measurement of the affinity Index 483
of a partial agonist 427
This page intentionally left blank
Preface to sixth edition
Pharmacologists almost always are working in systems
they do not fully understand. This has engendered a unique
“null system” of comparisons (before and after drug) that
has sustained the field. Our view of what is actually
happening in our experiment is obtained through our assay,
and as the Nobel Laureate Sir James Black wrote “.The
prismatic qualities of the assay distort our view in obscure
ways and degrees.” (1993; Nobel Lectures: Physiology
and Medicine). What this means to the discipline is that it is
uniquely dependent upon technology unveiling what we do
not understand about physiology and as technology ad-
vances the frontier of understanding, so too does the
perception of pharmacological mechanisms and the effect
of drugs on physiology. In essence, as the acuity of the
pharmacological prism improves, so too does our under-
standing of drug mechanisms. The practical outcome of this
is that a book on pharmacology must be updated every few
years to keep up with the new understanding gained from
technologies “new eyes to see.” This volume has been
updated and has added major chapters on biologics and the
drug discovery process that reflects the changing landscape
of drug therapy as well as views of historical findings
modified by new knowledge.
Terry P. Kenakin Ph.D.
Professor of Pharmacology,
The University of North Carolina School of Medicine,
Chapel Hill, NC, United States
xiii
This page intentionally left blank
Chapter 1
What is pharmacology?
I would in particular draw the attention to physiologists to
this type of physiological analysis of organic systems which
can be done with the aid of toxic agents ..
dClaude Bernard (1813e78).
1.1 About this book
Essentially this is a book about the methods and tools used in
pharmacology to quantify drug activity. Receptor pharma-
cology is based on the comparison of experimental data and
simple mathematical models, with a resulting inference of
drug behavior to the molecular properties of drugs. From this
standpoint, a certain level of understanding of the mathe-
matics involved in the models is useful but not imperative.
This book is structured such that each chapter begins with the
basic concepts and then moves on to the techniques used to
estimate drug parameters, and, finally, for those so inclined,
the mathematical derivations of the models used. Under-
standing the derivation is not a prerequisite for understanding
the application of the methods or the resulting conclusion;
these are included for completeness and are for readers who
wish to pursue exploration of the models. In general, facility
with mathematical equations is definitely not required for
pharmacology; the derivations can be ignored without any
detriment to the use of this book.
Second, the symbols used in the models and deriva-
tions, on occasion, duplicate each other (i.e., a is an
extremely popular symbol). However, the use of these
multiple symbols has been retained, since this preserves the
context of where these models were first described and
utilized. Also, changing these to make them unique would
cause confusion if these methods were to be used beyond
the framework of this book. Therefore, care should be taken
to consider the actual nomenclature of each chapter.
Third, an effort has been made to minimize the need to
cross-reference different parts of the book (i.e., when a
particular model is described, the basics are reiterated
somewhat to minimize the need to read the relevant but
different part of the book in which the model is initially
described). While this leads to a small amount of repeated
description, it is felt that this will allow for a more unin-
terrupted flow of reading and use of the book.
A Pharmacology Primer. https://doi.org/10.1016/B978-0-323-99289-3.00015-4
Copyright © 2022 Elsevier Inc. All rights reserved.
1.2 What is pharmacology?
Pharmacology (an amalgam of the Greek pharmakos,
medicine or drug, and logos, study) is a broad discipline
describing the use of chemicals to treat and cure diseases.
The Latin term pharmacologia was used in the late 1600s,
but the term pharmacum was used as early as the 4th
century to denote the term drug or medicine. In the Greek
translations “Pharmakeia” refers to Sorcery/Witchcraft
which no doubt was evident when particular herbal treat-
ments were effective. There are subdisciplines within
pharmacology representing specialty areas. Pharmacoki-
netics deals with the disposition of drugs in the human
body. To be useful, drugs must be absorbed and transported
to their site of therapeutic action. Drugs will be ineffective
in therapy if they do not reach the organs(s) to exert their
activity; this will be discussed specifically in Chapter 9,
Pharmacokinetics, of this book. Pharmaceutics is the study
of the chemical formulation of drugs to optimize absorption
and distribution within the body. Pharmacognosy is the
study of plant natural products and their use in the treat-
ment of disease. A very important discipline in the drug-
discovery process is medicinal chemistry, the study of the
production of molecules for therapeutic use. This couples
synthetic organic chemistry with an understanding of how
biological information can be quantified and used to guide
the synthetic chemistry to enhance therapeutic activity.
Pharmacodynamics is the study of the interaction of the
drug molecule with the biological target (referred to
generically as the “receptor,” vide infra). This discipline
lays the foundation of pharmacology since all therapeutic
application of drugs has a common root in pharmacody-
namics (i.e., as a prerequisite to exerting an effect, all drug
molecules must bind to and interact with receptors).
The history of pharmacology is tied to the history of
drug discoverydsee Chapter 9, The Optimal Design of
Pharmacological Experiments. As put by the Canadian
physician Sir William Osler (1849e919; the “father of
modern medicine”), “. the desire to take medicine is
perhaps the greatest feature which distinguishes man from
animals ..” Pharmacology as a separate science is
approximately 120e140 years old. The relationship be-
tween chemical structure and biological activity began to be
studied systematically in the 1860s [1]. It began when
1
2 A Pharmacology Primer
physiologists, using chemicals to probe physiological sys-
tems, became more interested in the chemical probes than
the systems they were probing. By the early 1800s, phys-
iologists were performing physiological studies with
chemicals that became pharmacological studies more aimed
at the definition of the biological activity of chemicals. The
first formalized chair of pharmacology, indicating a formal
university department, was founded in Estonia by Rudolf
Bucchiem in 1847. In North America, the first chair was
founded by John Jacob Abel at Johns Hopkins University
in 1890. A differentiation of physiology and pharmacology
was given by the pharmacologist Sir William Paton [2]:
If physiology is concerned with the function, anatomy with
the structure, and biochemistry with the chemistry of the
living body, then pharmacology is concerned with the
changes in function, structure, and chemical properties of
the body brought about by chemical substances
dW.D.M. Paton (1986).
Many works about pharmacology essentially deal in
therapeutics associated with different organ systems in the
body. Thus, in many pharmacology texts, chapters are
entitled drugs in the cardiovascular system, the effect of
drugs on the gastrointestinal (GI) system, the central ner-
vous system (CNS), and so on. However, the underlying
principles for all of these is the same, namely, the phar-
macodynamic interaction between the drug and the bio-
logical recognition system for that drug. Therefore, a
prerequisite to all of pharmacology is an understanding of
the basic concepts of doseeresponse and how living cells
process pharmacological information. This generally is
given the term pharmacodynamics or receptor pharma-
cology, where receptor is a term referring to any biological
recognition unit for drugs (membrane receptors, enzymes,
DNA, and so on). With such knowledge in hand, readers
will be able to apply these principles to any branch of
therapeutics effectively. This book treats doseeresponse data
generically and demonstrates methods by which drug ac-
tivity can be quantified across all biological systems irre-
spective of the nature of the biological target.
A great strength of pharmacology as a discipline is that
it contains the tools and methods to convert “descriptive
data,” i.e., data that serve to characterize the activity of a
given drug in a particular system, to “predictive data.” This
latter information can be used to predict that drug’s activity
in all organ systems, including the therapeutic one. This
defines the drug-discovery process which is the testing of
new potential drug molecules in surrogate systems (where a
potentially toxic chemical can do no lasting harm) before
progression to the next step, namely, testing in human
therapeutic systems. The models and tools contained in
pharmacology to convert drug behaviors in particular
organs to molecular properties (see Chapter 2: How
Different Tissues Process Drug Response) are the main
subject of this book, and the step-by-step design of phar-
macologic experiments to do this are described in detail in
Chapter 8, The Optimal Design of Pharmacological Ex-
periments (after the meaning of the particular parameters
and terms is described in previous chapters).
The human genome is now widely available for drug-
discovery research. Far from being a simple blueprint of
how drugs should be targeted, it has shown biologists that
receptor genotypes (i.e., properties of proteins resulting
from genetic transcription to their amino acid sequence) are
secondary to receptor phenotypes (how the protein interacts
with the myriad of cellular components and how cells tailor
the makeup and functions of these proteins to their indi-
vidual needs). Since the arrival of the human genome, re-
ceptor pharmacology as a science is more relevant than ever
in drug discovery. Current drug therapy is based on less
than 500 molecular targets, yet estimates utilizing the
number of genes involved in multifactorial diseases suggest
that the number of potential drug targets ranges from 2000
to 5000 [3]. Thus, current therapy is using only 5%e10%
of the potential trove of targets available in the human
genome.
A meaningful dialog between chemists and pharma-
cologists is the single most important element of the drug-
discovery process. The necessary link between medicinal
chemistry and pharmacology has been elucidated by
Paton [2]:
For pharmacology there results a particularly close rela-
tionship with chemistry, and the work may lead quite
naturally, with no special stress on practicality, to thera-
peutic application, or (in the case of adverse reactions) to
toxicology.
dW.D.M. Paton (1986).
Chemists and biologists reside in different worlds from
the standpoint of the type of data they deal with. Chemistry
is an exact science with physical scales that are not subject
to system variance. Thus, the scales of measurement are
transferable. Biology deals with the vagaries of complex
systems that are not completely understood. Within this
scenario, scales of measurement are much less constant and
much more subject to system conditions. Given this, a gap
can exist between chemists and biologists in terms of un-
derstanding and also in terms of the best method to progress
forward. In the worst circumstance, it is a gap of credibility
emanating from a failure of the biologist to make the
chemist understand the limits of the data. Usually, however,
credibility is not the issue, and the gap exists due to a lack
of common experience. This book was written in an
attempt to limit or, hopefully, eliminate this gap.
 What is pharmacology? Chapter | 1 3

1.3 The receptor concept
One of the most important concepts emerging from early
pharmacological studies is the concept of the receptor.
Pharmacologists knew that minute amounts of certain
chemicals had profound effects on physiological systems.
They also knew that very small changes in the chemical
composition of these substances could lead to huge dif-
ferences in activity. This led to the notion that something
on or in the cell must specifically read the chemical infor-
mation contained in these substances and translate it into a
physiological effect. This something was conceptually
referred to as the “receptor” for that substance. Pioneers
such as Paul Ehrlich (1854e915, Fig. 1.1A) proposed the
existence of “chemoreceptors” (actually he proposed a
collection of amboreceptors, triceptors, and polyceptors) on
cells for dyes. He also postulated that the chemoreceptors
on parasites, cancer cells, and microorganisms were
different from healthy host and thus could be exploited
therapeutically. The physiologist turned pharmacologist
John Newport Langley (1852e926, Fig. 1.1B), during his
studies with the drugs jaborandi (which contains the alka-
loid pilocarpine) and atropine, introduced the concept that
receptors were switches that received and generated signals
and that these switches could be activated or blocked by
specific molecules. The originator of quantitative receptor
theory, the Edinburgh pharmacologist Alfred Joseph Clark
(1885e941, Fig. 1.1C), was the first to suggest that the
data, compiled from his studies of the interactions of
acetylcholine and atropine, resulted from the unimolecular
interaction of the drug and a substance on the cell surface.
He articulated these ideas in the classic work The Mode of
Action of Drugs on Cells [4], later revised as the Handbook
of Experimental Pharmacology [5]. As put by Clark
It appears to the writer that the most important fact shown
by a study of drug antagonisms is that it is impossible to
explain the remarkable effects observed except by assuming
that drugs unite with receptors of a highly specific pattern
.. No other explanation will, however, explain a tithe of
the facts observed.
dA.J. Clark (1937).
Clark’s next step formed the basis of receptor theory by
applying chemical laws to systems of “infinitely greater
complexity” [4]. It is interesting to note the scientific at-
mosphere in which Clark published these ideas. The
dominant ideas between 1895 and 1930 were based on
theories such as the law of phasic variation essentially
stating that “certain phenomena occur frequently.” Ho-
meopathic theories like the ArndteSchulz law and
WebereFechner law were based on loose ideas around
surface tension of the cell membrane, but there was little
physicochemical basis for these ideas [6]. In this vein,
prominent pharmacologists of the day, such as Walter
Straub (1874e944), suggested that a general theory of
chemical binding between drugs and cells utilizing re-
ceptors was “. going too far . and . not admissible”
[6]. The impact of Clark’s thinking against these concepts
cannot be overemphasized to modern pharmacology.

FIGURE 1.1 Pioneers of pharmacology. (A) Paul Ehrlich (1854e915). Born in Silesia, Ehrlich graduated from Leipzig University to go on to a
distinguished career as head of institutes in Berlin and Frankfurt. His studies with dyes and bacteria formed the basis of early ideas regarding recognition
of biological substances by chemicals. (B) John Newport Langley (1852e926). Though he began reading mathematics and history in Cambridge in 1871,
Langley soon took to physiology. He succeeded the great physiologist M. Foster to the chair of physiology in Cambridge in 1903 and branched out into
pharmacological studies of the autonomic nervous system. These pursuits led to germinal theories of receptors. (C) Alfred J. Clark (1885e941). Beginning
as a demonstrator in pharmacology in King’s College (London), Clark went on to become Professor of pharmacology at University College London. From
there he took the chair of pharmacology in Edinburgh. Known as the originator of modern receptor theory, Clark applied chemical laws to biological
phenomena. His books on receptor theory formed the basis of modern pharmacology.
4 A Pharmacology Primer
It is possible to underestimate the enormous signifi-
cance of the receptor concept in pharmacology until it is
realized how relatively chaotic the study of drug effect
was before it was introduced. Specifically, consider the
myriad of physiological and pharmacological effects of
the hormone epinephrine in the body. As shown in
Fig. 1.2, a host of responses are obtained from the CNS,
cardiovascular system, smooth muscle, and other organs.
It is impossible to see a thread which relates these very
different responses until it is realized that all of these are
mediated by the activation of a single protein receptor,
namely, in this case, the b-adrenoceptor. When this is
understood, a much better idea can be gained as to how to
manipulate these heterogeneous responses for therapeutic
benefit; the receptor concept introduced order into physi-
ology and pharmacology.
Drug receptors can exist in many forms, including cell
surface proteins, enzymes, ion channels, membrane trans-
porters, DNA, and cytosolic proteins (see Fig. 1.3). There
are examples of important drugs for all of these. This book
deals with general concepts which can be applied to a range
of receptor types, but most of the principles are illustrated
with the most tractable receptor class known in the human
genome, namely, seven transmembrane (7TM) receptors
(7TMRs). These receptors are named for their characteristic
structure that consists of a single protein chain that tra-
verses the cell membrane seven times to produce extra-
cellular and intracellular loops. These receptors activate
G-proteins to elicit response, thus they are also
commonly referred to as G-protein-coupled receptors
(GPCRs); this should now be considered a limiting moniker
as these proteins signal to a wide variety of signaling
molecules in the cell and are not confined to G-protein
FIGURE 1.2 A sampling of the heterogeneous physiological and pharmacological response to the hormone epinephrine. The concept of receptors links
these diverse effects to a single control point, namely, the b-adrenoceptor.
effects. There are between 800 and 1000 [7] of these in
the genome [the genome sequence predicts 650 GPCR
genes, of which approximately 190 (on the order of 1% of
the genome of superior organisms) are categorized as
known 7TMRs [8] activated by some 70 ligands]. In the
United States, in 2000, nearly half of all prescription drugs
were targeted toward 7TM receptors [3]. These receptors,
comprising between 1% and 5% of the total cell protein,
control a myriad of physiological activities. They are
tractable for drug discovery because they are on the cell
surface, and therefore drugs do not need to penetrate the
cell to produce effect. In the study of biological targets such
as 7TMRs and other receptors, a “system” must be
employed that accepts chemical input and returns biological
output. It is worth discussing such receptor systems in
general terms before their specific uses are considered.
1.4 Pharmacological test systems
Molecular biology has transformed pharmacology and the
drug-discovery process. As little as 20 years ago, screening
for new drug entities was carried out in surrogate animal
tissues. This necessitated a rather large extrapolation to
span the differences in genotype and phenotype. The belief
that the gap could be bridged came from the notion that the
chemicals recognized by these receptors in both humans
and animals were the same (vide infra). Receptors are
unique proteins with characteristic amino acid sequences.
While polymorphisms (spontaneous alterations in amino
acid sequence, vide infra) of receptors exist in the same
species, in general the amino acid sequence of a natural
ligand-binding domain for a given receptor type largely
may be conserved. There are obvious pitfalls of using

FIGURE 1.3 Schematic diagram of potential drug targets. Molecules can affect the function of numerous cellular components both in the cytosol and on
the membrane surface. There are many families of receptors that traverse the cellular membrane and allow chemicals to communicate with the interior of
the cell.
surrogate species receptors for predicting human drug ac-
tivity, and it never can be known for certain whether
agreement for estimates of activity for a given set of drugs
ensures accurate prediction for all drugs. The agreement is
very much drug and receptor dependent. For example, the
human and mouse a2-adrenoceptors are 89% homologous
and thus considered very similar from the standpoint of
amino acid sequence. Furthermore, the affinities of the a2-
adrenoceptor antagonists atipamezole and yohimbine are
nearly indistinguishable (atipamezole human a2-C10
Ki ¼ 2.9  0.4 nM, mouse a2-4H Ki ¼ 1.6  0.2 nM;
yohimbine human a2-C10 Ki ¼ 3.4  0.1 nM, mouse a2-
4H Ki ¼ 3.8  0.8 nM). However, there is a 20.9-fold
difference for the antagonist prazosin (human a2-C10
Ki ¼ 2034  350 nM, mouse a2-4H Ki ¼ 97.3  0.7 nM)
[9]. Such data highlight a general theme in pharmacological
research, namely, that a hypothesis, such as one proposing
that two receptors which are identical with respect to their
sensitivity to drugs are the same, cannot be proven, only
disproven. While a considerable number of drugs could be
tested on the two receptors (thus supporting the hypothesis
that their sensitivity to all drugs is the same), this hypoth-
esis is immediately disproven by the first drug that shows
differential potency on the two receptors. The fact that a
series of drugs tested show identical potencies may mean
only that the wrong sample of drugs has been chosen to
unveil the difference. Thus, no general statements can be
made that any one surrogate system is completely predic-
tive of activity on the target human receptor. This will al-
ways be a drug-specific phenomenon.
The link between animal and human receptors is the fact
that both proteins recognize the endogenous transmitter
(e.g., acetylcholine, norepinephrine), and therefore the hope
What is pharmacology? Chapter | 1 5
is that this link will carry over into other drugs that
recognize the animal receptor. This imperfect system
formed the basis of drug discovery until human cDNA for
human receptors could be used to make cells express hu-
man receptors. These engineered (recombinant) systems are
now used as surrogate human-receptor systems, and the
leap of faith from animal receptor sequences to human-
receptor sequences is not required (i.e., the problem of
differences in genotype has been overcome). However,
cellular signaling is an extremely complex process and cells
tailor their receipt of chemical signals in numerous ways.
Therefore, the way a given receptor gene behaves in a
particular cell can differ in response to the surroundings in
which that receptor finds itself. These differences in
phenotype (i.e., properties of a receptor produced by
interaction with its environment) can result in differences in
both the quantity and quality of a signal produced by a
concentration of a given drug in different cells. Therefore,
there is still a certain, although somewhat lesser, leap of
faith taken in predicting therapeutic effects in human tis-
sues under pathological control from surrogate recombinant
or even surrogate natural human-receptor systems. For this
reason, it is a primary requisite of pharmacology to derive
system-independent estimates of drug activity that can be
used to predict therapeutic effect in other systems.
A schematic diagram of the various systems used in
drug discovery, in order of how appropriate they are to
therapeutic drug treatment, is shown in Fig. 1.4. As dis-
cussed previously, early functional experiments in animal
tissue have now largely given way to testing in recombinant
cell systems engineered with human-receptor material. This
huge technological step greatly improved the predictability
of drug activity in humans, but it should be noted that there
6 A Pharmacology Primer
FIGURE 1.4 A history of the drug-discovery process. Originally, the only biological material available for drug research was animal tissue. With the
advent of molecular biological techniques to clone and express human receptors in cells, recombinant systems supplanted animal-isolated tissue work. It
should be noted that these recombinant systems still fall short of yielding drug response in the target human tissue under the influence of pathological
processes.
still are many factors that intervene between the genetically
engineered drug-testing system and the pathology of human
disease.
A frequently used strategy in drug discovery is to ex-
press human receptors (through transfection with human
cDNA) in convenient surrogate host cells (referred to as
“target-based” drug discovery; see Chapter 10: Safety
Pharmacology for further discussion). These host cells are
chosen mainly for their technical properties (i.e., robust-
ness, growth rate, stability) and not with any knowledge of
verisimilitude to the therapeutically targeted human cell
type. There are various factors relevant to the choice of
surrogate host cell, such as a very low-background activity
(i.e., a cell cannot be used that already contains a related
animal receptor for fear of cross-reactivity to molecules
targeted for the human receptor). Human receptors are often
expressed in animal surrogate cells. The main idea here is
that the cell is a receptacle for the receptor, allowing it to
produce physiological responses, and that activity can be
monitored in pharmacological experiments. In this sense,
human receptors expressed in animal cells are still a theo-
retical step distanced from the human receptor in a human
cell type. However, even if a human surrogate is used (and
there are such cells available), there is no definitive evi-
dence that a surrogate human cell is any more predictive of
a natural receptor activity than an animal cell when
compared to the complex receptor behavior in its natural
host cell type expressed under pathological conditions.
Receptor phenotype dominates in the end organ, and the
exact differences between the genotypic behavior of the
receptor (resulting from the genetic makeup of the receptor)
and the phenotypic behavior of the receptor (due to the
interaction of the genetic product with the rest of the cell)
may be cell specific. Therefore, there is still a possible gap
between the surrogate systems used in the drug-discovery
process and the therapeutic application. Moreover, most
drug-discovery systems utilize receptors as switching
mechanisms and quantify whether drugs turn on or turn off
the switch. The pathological processes that we strive to
modify may be more subtle. As put by pharmacologist Sir
James Black [10]:
. angiogenesis, apoptosis, inflammation, commitment of
marrow stem cells, and immune responses. The cellular
reactions subsumed in these processes are switch like in
their behavior . biochemically we are learning that in all
these processes many chemical regulators seem to be
involved. From the literature on synergistic interactions, a
control model can be built in which no single agent is
effective. If a number of chemical messengers each bring
information from a different source and each deliver only a
subthreshold stimulus but together mutually potentiate each
other, then the desired information-rich switching can be
achieved with minimum risk of miscuing.
dJ.W. Black (1986).
Such complex end points are difficult to predict from
any one of the component processes leading to yet another
leap of faith in the drug-discovery process. For these rea-
sons, an emerging strategy for drug discovery is the use of
natural cellular systems. This approach is discussed in some
detail in Chapter 11, The Drug Discovery Process.
Even when an active drug molecule is found and ac-
tivity is verified in the therapeutic arena, there are factors
that can lead to gaps in its therapeutic profile. When drugs
are exposed to huge populations, genetic variations in this
population can lead to discovery of alleles that code for
mutations of the target (isogenes), and these can lead to
variation in drug response. Such polymorphisms can lead to

resistant populations (i.e., resistance of some asthmatics to
the b-adrenoceptor bronchodilators [11]). In the absence of
genetic knowledge, these therapeutic failures for a drug
could not easily be averted since they in essence occurred
because of the presence of new biological targets not
originally considered in the drug-discovery process. How-
ever, as new epidemiological information becomes avail-
able, these polymorphisms can now be incorporated into
the drug-discovery process.
There are two theoretical and practical scales that can
be used to make system-independent measures of drug
activity on biological systems. The first is a measure of the
attraction of a drug for a biological target, namely, its
affinity for a receptor. Drugs must interact with receptors
to produce an effect, and the affinity is a chemical term
used to quantify the strength of that interaction. The sec-
ond is much less straightforward and is used to quantify
the degree of effect imparted to the biological system after
the drug binds to the receptor. This is termed efficacy. This
property was named by Stephenson [12] within classical
receptor theory as a proportionality factor for the tissue
response produced by a drug. There is no absolute scale
for efficacy, but rather it is dealt with in relative terms
(i.e., the ratio of the efficacy of two different drugs on a
particular biological system can be estimated and, under
ideal circumstances, will transcend the system and be
applicable to other systems as well). It is the foremost task
of pharmacology to use the translations of drug effect
obtained from cells to provide system-independent esti-
mates of affinity and efficacy. Before specific discussion
of affinity and efficacy, it is worth considering the mo-
lecular nature of biological targets.
1.5 The nature of drug receptors
While some biological targets such as DNA are not protein
in nature, most receptors are. It is useful to consider the
properties of receptor proteins to provide a context for the
interaction of small molecule drugs with them. An impor-
tant property of receptors is that they have a 3D structure.
Proteins are usually composed of one or more peptide
chains; the composition of these chains makes up the pri-
mary and secondary structure of the protein. Proteins also
are described in terms of a tertiary structure, which defines
their shape in 3D space, and a quaternary structure, which
defines the molecular interactions between the various
components of the protein chains (Fig. 1.5). It is this 3D
structure which allows the protein to function as a recog-
nition site and effector for drugs and other components of
the cell; in essence, the ability of the protein to function as a
messenger, shuttling information from the outside world to
the cytosol of the cell. For 7TMRs, the 3D nature of the
receptor forms binding domains for other proteins such as
What is pharmacology? Chapter | 1 7
G-proteins (these are activated by the receptor and then go
on to activate enzymes and ion channels within the cell; see
Chapter 2: How Different Tissues Process Drug Response)
and endogenous chemicals such as neurotransmitters, hor-
mones, and autacoids that carry physiological messages.
This important class of drug target is named for a charac-
teristic structure consisting of 7TM domains looping into
the extracellular and intracellular spacedsee Fig. 1.6.
These molecules are the main transfer points of information
from the outside to the inside of the cell, and such transfers
occur through changes in the conformation of the receptor
protein (vide infra). For other receptors, such as ion chan-
nels and single transmembrane enzyme receptors, the
conformational change per se leads to a response, either
through an opening of a channel to allow the flow of ionic
current or the initiation of enzymatic activity. Therapeutic
advantage can be taken by designing small molecules to
utilize these binding domains or other 3D binding domains
on the receptor protein in order to modify physiological and
pathological processes.
1.6 From the snapshot to the movie
Drugs interact with living physiology and the outcome of
the interaction is controlled by a combination of the
intrinsic properties of the drug and the sensitivity of the
system to intervention. This being the case, drugs can have
different profiles of activity in different tissues depending
on the tissue sensitivity and setpoint of physiology.
Through the mechanics of mathematical models of drug
activity and the system-independent scales of drug activity
(i.e., affinity, efficacy), pharmacological procedures are
uniquely able to convert a single observation of drug ac-
tivity in a test system (the ‘snapshot’) to a prediction of the
complete realm of activities for that same drug in a range of
tissues of varying setpoints of physiology (the ‘movie’).
This is an essential property of pharmacology in drug dis-
covery as all initial evaluations of new drug activity are
made in isolated systems and assessments of what the new
molecule will do in other systems must be made. In
essence, the cellular host system completely controls what
the experimenter observes regarding the events taking place
at the drug receptor. Drug activity is thus revealed through
a “cellular veil” that can, in many cases, obscure or sub-
stantially modify drugereceptor activity (Fig. 1.7). Minute
signals, initiated either at the cell surface or within the
cytoplasm of the cell, are interpreted, transformed, ampli-
fied, and otherwise altered by the cell to tailor that signal to
its own particular needs. The application of pharmacolog-
ical principles and modeling enable ‘snapshots’ of drug
activity obtained is experiments to guide the progress of
molecules toward drug candidate statusdsee Chapter 3 for
further details.
8 A Pharmacology Primer
FIGURE 1.5 Increasing levels of protein structure. A protein has a given amino acid sequence to make peptide chains. These adopt a 3D structure
according to the free energy of the system. Receptor function can change with changes in tertiary or quaternary structure.
1.7 Pharmacological intervention and
the therapeutic landscape
It is useful to consider the therapeutic landscape with
respect to the aims of pharmacology. As stated by Sir
William Ossler (1849e919) “. the prime distinction be-
tween man and other creatures is man’s yearning to take
medicine.” The notion that drugs can be used to cure dis-
ease is as old as history. One of the first written records of
actual “prescriptions” can be found in the Ebers Papyrus
(c.1550 BCE): “. for night blindness in the eyes . liver
of ox, roasted and crushed out . really excellent!“dsee
Fig. 1.8. Now it is known that liver is an excellent source of
vitamin A, a prime treatment for night blindness, but that
chemical detail was not known to the ancient Egyptians.
Disease can be considered under two broad categories:
those caused by invaders such as pathogens and those
caused by intrinsic breakdown of normal physiological
function. The first generally is approached through the
invader (i.e., the pathogen is destroyed, neutralized, or
removed from the body). The one exception of where the
host is treated when an invader is present is the treatment of
HIV-1 infection leading to AIDS. In this case, while there
are treatments to neutralize the pathogen, such as anti-
retrovirals to block viral replication, a major new approach
is the blockade of the interaction of the virus with the
protein that mediates viral entry into healthy cells, the
chemokine receptor CCR5. In this case, CCR5 antagonists
are used to prevent HIV fusion and subsequent infection.
The second approach to disease requires an understanding
of the pathological process and repair of the damage to
return to normal function.
The therapeutic landscape onto which drug discovery
and pharmacology in general combat disease can gener-
ally be described in terms of the major organ systems of
the body and how they may go awry. A healthy cardio-
vascular system consists of a heart able to pump deoxy-
genated blood through the lungs and to pump oxygenated
blood throughout a circulatory system that does not
unduly resist blood flow. Since the heart requires a high

FIGURE 1.6 Depiction of the structure of seven transmembrane domain
receptors, one of the most if not the most important therapeutic targets
available in the human genome. Chemicals access the receptor through the
extracellular space by binding to the extracellular domains of the protein.
This causes a conformational change in the protein that alters the inter-
action of signaling proteins in the cell cytosol. This latter process results in
the initiation of cellular signaling.
degree of oxygen itself to function, myocardial ischemia
can be devastating to its function. Similarly, an inability to
maintain rhythm (arrhythmia) or loss in strength with
concomitant inability to empty (congestive heart failure)
can be fatal. The latter disease is exacerbated by elevated
arterial resistance (hypertension). A wide range of drugs
are used to treat the cardiovascular system, including
coronary vasodilators (nitrates), diuretics, renine
angiotensin inhibitors, vasodilators, cardiac glycosides,
calcium antagonists, beta and alpha blockers, antiar-
rhythmics, and drugs for dyslipidemia. The lungs must
extract oxygen from the air, deliver it to the blood, and
release carbon dioxide from the blood into exhaled air.
Asthma, chronic obstructive pulmonary disease (COPD),
and emphysema are serious disorders of the lungs and
airways. Bronchodilators (beta agonists), antiin-
flammatory drugs, inhaled glucocorticoids, anticholiner-
gics, and theophylline analogs are used for treatment of
these diseases. The CNS controls all conscious thought
and many unconscious body functions. Numerous dis-
eases of the brain can occur, including depression, anxi-
ety, epilepsy, mania, degeneration, obsessive disorders,
and schizophrenia. Brain functions such as those
What is pharmacology? Chapter | 1 9
controlling sedation and pain also may require treatment.
A wide range of drugs is used for CNS disorders,
including serotonin partial agonists and uptake inhibitors,
dopamine agonists, benzodiazepines, barbiturates, opi-
oids, tricyclics, neuroleptics, and hydantoins. The GI tract
receives and processes food to extract nutrients and
removes waste from the body. Diseases such as stomach
ulcers, colitis, diarrhea, nausea, and irritable bowel syn-
drome can affect this system. Histamine antagonists,
proton pump blockers, opioid agonists, antacids, and se-
rotonin uptake blockers are used to treat diseases of the GI
tract.
The inflammatory system is designed to recognize self
from nonself, and to destroy nonself to protect the body. In
diseases of the inflammatory system, the self-recognition
can break down, leading to conditions in which the body
destroys healthy tissue in a misguided attempt at protection.
This can lead to rheumatoid arthritis, allergies, pain, COPD,
asthma, fever, gout, graft rejection, and problems with
chemotherapy. Nonsteroidal antiinflammatory drugs,
aspirin and salicylates, leukotriene antagonists, and hista-
mine receptor antagonists are used to treat inflammatory
disorders. The endocrine system produces and secretes
hormones crucial to the body for growth and function.
Diseases of this class of organs can lead to growth and
pituitary defectsddiabetes; abnormality in thyroid, pitui-
tary, adrenal cortex, and androgen function; osteoporosis;
and alterations in estrogeneprogesterone balance. The
general approach to treatment is through replacement or
augmentation of secretion. Drugs used are replacement
hormones, insulin, sulfonylureas, adrenocortical steroids,
and oxytocin. In addition to the major organ and physio-
logical systems, diseases involving neurotransmission and
neuromuscular function, ophthalmology, hemopoiesis and
hematology, dermatology, immunosuppression, and drug
addiction and abuse are amenable to pharmacological
intervention.
Cancer is a serious malfunction of normal cell growth.
In the years from 1950 to 1970, the major approach to
treating this disease was to target DNA and DNA pre-
cursors according to the hypothesis that rapidly dividing
cells (cancer cells) are more susceptible to DNA toxicity
than normal cells. Since that time, a wide range of new
therapies based on manipulation of the immune system,
induction of differentiation, inhibition of angiogenesis, and
increased killer T-lymphocytes to decrease cell prolifera-
tion has greatly augmented the armamentarium against
neoplastic disease. Previously, lethal malignancies such as
testicular cancer, some lymphomas, and leukemia are now
curable.
Three general treatments of disease are surgery, genetic
engineering (still an emerging discipline), and pharmaco-
logical intervention. While early medicine was subject to
the theories of Hippocrates (460e357 BCE), who saw
10 A Pharmacology Primer

FIGURE 1.7 The cellular veil. Drugs act on biological receptors in cells to change cellular activity. The initial receptor stimulus usually alters a
complicated system of interconnected metabolic biochemical reactions, and the outcome of the drug effect is modified by the extent of these in-
terconnections, the basal state of the cell, and the threshold sensitivity of the various processes involved. This can lead to a variety of apparently different
effects for the same drug in different cells. Receptor pharmacology strives to identify the basic mechanism initiating these complex events.

FIGURE 1.8 The Ebers Papyrus is a 110-page scroll (20 m long) thought to have been written in 1550 BCE but containing information dating from
3400 BCE. It is a record of Egyptian medicine and contains numerous “prescriptions” some of which, though empirical, are valid therapeutic approaches
to diseases.

health and disease as a balance of four humors (i.e., black
and yellow bile, phlegm, and blood), by the 16th century
pharmacological concepts were being formulated. These
could be stated concisely as the following [13]:
l Every disease has a cause for which there is a specific
remedy.
l Each remedy has a unique essence that can be obtained
from nature by extraction (“doctrine of signatures”).
l The administration of the remedy is subject to a dosee
response relationship.
The basis for believing that pharmacological interven-
tion can be a major approach to the treatment of disease is
the fact that the body generally functions in response to
chemicals. Table 1.1 shows partial lists of hormones and
neurotransmitters in the body. Many more endogenous
chemicals are involved in normal physiological function.
 What is pharmacology? Chapter | 1 11

TABLE 1.1 Some endogenous chemicals controlling normal physiological function.

Neurotransmitters
Acetylcholine 2-Arachidonylglycerol Anandamide
ATP Corticotropin-releasing hormone Dopamine
Epinephrine Aspartate Gamma-aminobutyric acid
Galanin Glutamate Glycine
Histamine Norepinephrine Serotonin
Hormones
Thyroid-stimulating hormone Follicle-stimulating hormone Luteinizing hormone
Prolactin Adrenocorticotropin Antidiuretic hormone
Thyrotropin-releasing hormone Oxytocin Gonadotropin-releasing hormone
Growth-hormone-releasing hormone Corticotropin-releasing hormone Somatostatin
Melatonin Thyroxin Calcitonin
Parathyroid hormone Glucocorticoid(s) Mineralocorticoid(s)
Estrogen(s) Progesterone Chorionic gonadotropin
Androgens Insulin Glucagon
Amylin Erythropoietin Calcitriol
Calciferol Atrial-natriuretic peptide Gastrin
Secretin Cholecystokinin Neuropeptide Y
Insulin-like growth factor Angiotensinogen Ghrelin

Leptin

ATP, adenosine triphosphate.

The fact that so many physiological processes are
controlled by chemicals provides the opportunity for
chemical intervention. Thus, physiological signals medi-
ated by chemicals can be initiated, negated, augmented, or
modulated. The nature of this modification can take the
form of changes in the type, strength, duration, or location
of signal.
1.8 System-independent drug
parameters: affinity and efficacy
The process of drug discovery relies on the testing of mol-
ecules in systems to yield estimates of biological activity in
an iterative process of changing the structure of the molecule
until optimal activity is achieved. It will be seen in this book
that there are numerous systems available to do this, and that
each system may interpret the activity of molecules in
different ways. Some of these interpretations can appear to
be in conflict with each other, leading to apparent capricious
patterns. For this reason, the way forward in the drug
development process is to use only system-independent in-
formation. Ideally, scales of biological activity should be
used that transcend the actual biological system in which the
drug is tested. This is essential to avoid confusion and also
because it is quite rare to have access to the exact human
system under the control of the appropriate pathology
available for in vitro testing. Therefore, the drug-discovery
process necessarily relies on the testing of molecules in
surrogate systems and the extrapolation of the observed ac-
tivity to all systems. The only means to do this is to obtain
system-independent measures of drug activity, namely, af-
finity and efficacy.
If a molecule in solution associates closely with a re-
ceptor protein, it has affinity for that protein. The area where
it is bound is the binding domain or locus. If the same
molecule interferes with the binding of a physiologically
active molecule such as a hormone or a neurotransmitter
(i.e., if the binding of the molecule precludes activity of the
physiologically active hormone or neurotransmitter), the
molecule is referred to as an antagonist. Therefore, a phar-
macologically active molecule that blocks physiological ef-
fect is an antagonist. Similarly, if a molecule binds to a
receptor and produces its own effect, it is termed an agonist.
It also is assumed to have the property of efficacy. Efficacy is
12 A Pharmacology Primer

detected by observation of pharmacological response.
Therefore, agonists have both affinity and efficacy.
Classically, agonist response is described in two stages,
the first being the initial signal imparted to the immediate
biological target, namely, the receptor. This first stage is
composed of the formation, either through interaction with
an agonist or spontaneously, of an active state receptor
conformation. This initial signal is termed the stimulus
(Fig. 1.9). This stimulus is perceived by the cell and pro-
cessed in various ways through successions of biochemical
reactions to the end point, namely, the response. The sum
total of the subsequent reactions is referred to as the
stimuluseresponse mechanism or cascade (see Fig. 1.10).
Efficacy is a molecule-related property (i.e., different
molecules have different capabilities to induce a physio-
logical response). The actual term for the molecular aspect
of response-inducing capacity of a molecule is intrinsic
efficacy (see Chapter 3: DrugeReceptor Theory for how
this term evolved). Thus, every molecule has a unique
value for its intrinsic efficacy (in cases of antagonists this
could be zero). The different abilities of molecules to
induce response are illustrated in Fig. 1.10. This figure
shows doseeresponse curves for four 5-HT (hydroxytryp-
tamine) (serotonin) agonists in rat jugular vein. It can be
seen that if response is plotted as a function of the percent
receptor occupancy, different receptor occupancies for
the different agonists lead to different levels of response.
For example, while 0.6 g force can be generated by
5-HT by occupying 30% of the receptors, the agonist 5-
cyanotryptamine requires twice the receptor occupancy to
generate the same response (i.e., the capability of 5-
cyanotryptamine to induce response is half that of 5-HT
[14]). These agonists are then said to possess different
magnitudes of intrinsic efficacy.

FIGURE 1.9 Schematic diagram of response production by an agonist. An initial stimulus is produced at the receptor as a result of agonistereceptor
interaction. This stimulus is processed by the stimuluseresponse apparatus of the cell into observable cellular response.

FIGURE 1.10 Differences between agonists producing contraction of rat jugular vein through activation of 5-HT receptors. (A) Doseeresponse curves
to 5-HT receptor agonists, 5-HT (filled circles), 5-cyanotryptamine (filled squares), N,N-dimethyltryptamine (open circles), and N-benzyl-5-
methoxytryptamine (filled triangles). Abscissae: logarithms of molar concentrations of agonist. (B) Occupancy response curves for curves shown in
panel A. Abscissae: percent receptor occupancy by the agonist as calculated by mass action and the equilibrium dissociation constant of the agoniste
receptor complex. Ordinates: force of contraction in g. Data drawn from P. Leff, G.R. Martin, J.M. Morse, Differences in agonist dissociation constant
estimates for 5-HT at 5-HT2-receptors: a problem of acute desensitization? Br. J. Pharmacol. 89 (1986) 493e499.

It is important to consider affinity and efficacy as
separately manipulatable properties. Thus, there are chem-
ical features of agonists that pertain especially to affinity
and other features that pertain to efficacy. Fig. 1.11 shows a
series of key chemical compounds made en route to the
histamine H2 receptor antagonist cimetidine (used for
healing gastric ulcers). The starting point for this discovery
program was the knowledge that histamine, a naturally
occurring autacoid, activates histamine H2 receptors in the
stomach to cause acid secretion. This constant acid secre-
tion is what prevents the healing of lesions and ulcers. The
task was then to design a molecule that would antagonize
the histamine receptors mediating acid secretion and pre-
vent histamine H2 receptor activation to allow the ulcers to
heal. This task was approached with the knowledge that
molecules, theoretically, could be made that retained or
even enhanced affinity but decreased the efficacy of hista-
mine (i.e., these were separate properties). As can be seen
in Fig. 1.11, molecules were consecutively synthesized
with reduced values of efficacy and enhanced affinity until
the target histamine H2 antagonist cimetidine was made.
This was a clear demonstration of the power of medicinal
chemistry to separately manipulate affinity and efficacy for
which, in part, the Nobel Prize in Medicine was awarded in
1988.
1.9 What is affinity?
The affinity of a drug for a receptor defines the strength of
interaction between the two species. The forces control-
ling the affinity of a drug for the receptor are thermody-
namic (enthalpy as changes in heat and entropy as changes
FIGURE 1.11 Key compounds synthesized to eliminate the efficacy (burgundy red) and enhance the affinity (green) of histamine for histamine H2
receptors to make cimetidine, one of the first histamine H2 antagonists of use in the treatment of peptic ulcers. Quotation from J.W. Black, A personal view
of pharmacology, Ann. Rev. Pharmacol. Toxicol. 36 (1996) 1e33.
What is pharmacology? Chapter | 1 13
in the state of disorder). The chemical forces between the
components of the drug and the receptor vary in impor-
tance in relation to the distance of the drug from the re-
ceptor’s binding surface. Thus, the strength of
electrostatic forces (attraction due to positive and negative
charges and/or complex interactions between polar
groups) varies as a function of the reciprocal of the dis-
tance between the drug and the receptor. Hydrogen
bonding (the sharing of a hydrogen atom between an
acidic and basic group) varies in strength as a function of
the fourth power of the reciprocal of the distance. Also
involved are van der Waals’ forces (weak attraction be-
tween polar and nonpolar molecules) and hydrophobic
bonds (interaction of nonpolar surfaces to avoid interac-
tion with water). The combination of all of these forces
causes the drug to reside in a certain position within the
protein-binding pocket. This is a position of minimal free
energy. It is important to note that drugs do not statically
reside in one uniform position. As thermal energy varies
in the system, drugs approach and dissociate from the
protein surface. This is an important concept in pharma-
cology as it sets the stage for competition between two
drugs for a single binding domain on the receptor protein.
The probability that a given molecule will be at the point
of minimal free energy within the protein-binding pocket
thus depends on the concentration of the drug available to
fuel the binding process and also the strength of the in-
teractions for the complementary regions in the binding
pocket (affinity). Affinity can be thought of as a force of
attraction and can be quantified with a very simple tool,
first used to study the adsorption of molecules onto a
surface, namely, the Langmuir adsorption isotherm.
14 A Pharmacology Primer
1.10 The Langmuir adsorption isotherm
Defined by the chemist Irving Langmuir (1881e957,
Fig. 1.12), the model for affinity is referred to as the
Langmuir adsorption isotherm. Langmuir, a chemist at
General Electric, was interested in the adsorption of mol-
ecules onto metal surfaces for the improvement of lighting
filaments. He reasoned that molecules had a characteristic
rate of diffusion toward a surface (referred to as conden-
sation and denoted a in his nomenclature) and also a
characteristic rate of dissociation (referred to as evapora-
tion and denoted as V1; see Fig. 1.12). He assumed that the
amount of surface that already has a molecule bound is not
available to bind another molecule. The surface area bound
by molecule is denoted q1, expressed as a fraction of
the total area. The amount of free area open for the binding
of molecule, expressed as a fraction of the total area, is
denoted as 1  q1. The rate of adsorption toward the sur-
face therefore is controlled by the concentration of drug in
the medium (denoted m in Langmuir’s nomenclature)
multiplied by the rate of condensation on the surface and
the amount of free area available for binding:
Rate of diffusion toward surface ¼ amð1  q1 Þ. (1.1)
The rate of evaporation is given by the intrinsic rate of
dissociation of bound molecules from the surface multi-
plied by the amount already bound:
Rate of evaporation ¼ V1 q1 .
Once equilibrium has been reached, the rate of
adsorption equals the rate of evaporation. Equating (1.1)
and (1.2) and rearranging yields
FIGURE 1.12 The Langmuir adsorption isotherm representing the binding of a molecule to a surface. Photo shows Irving Langmuir (1881e957), a
chemist interested in the adsorption of molecules to metal filaments for the production of light. Langmuir devised the simple equation still in use today for
quantifying the binding of molecules to surfaces. The equilibrium is described by condensation and evaporation to yield the fraction of surface bound (q1)
by a concentration m.
am
q1 ¼ . (1.3)
am þ V1
This is the Langmuir adsorption isotherm in its original
form. In pharmacological nomenclature, it is rewritten ac-
cording to the convention
½AR ½A
r¼ ¼ ; (1.4)
½Rt  ½A þ KA
where [AR] is the amount of complex formed between the
ligand and the receptor, and [Rt] is the total number of re-
ceptor sites. The ratio r refers to the fraction of maximal
binding by a molar concentration of drug [A] with an equi-
librium dissociation constant of KA. This latter term is the
ratio of the rate of offset (in Langmuir’s terms V1 and
referred to as k2 in receptor pharmacology) divided by
the rate of onset (in Langmuir’s terms a denoted k1 in re-
ceptor pharmacology).
It is amazing to note that complex processes such as
drugs binding to protein, activation of cells, and observa-
tion of syncytial cellular response should apparently so
closely follow a model based on these simple concepts.
This was not lost on A.J. Clark in his treatise on druge
receptor theory The Mode of Action of Drugs on Cells [4]:
It is an interesting and significant fact that the author in
1926 found that the quantitative relations between the con-
centration of acetylcholine and its action on muscle cells, an
(1.2) action the nature of which is wholly unknown, could be most
accurately expressed by the formulae devised by Langmuir
to express the adsorption of gases on metal filaments.
dA.J. Clark (1937).

The term KA is a concentration, and it quantifies af-
finity. Specifically, it is the concentration that binds to 50%
of the total receptor population [see Eq. (1.4) when [A] ¼
KA]. Therefore, the smaller is the KA, the higher is the
affinity. Affinity is the reciprocal of KA. For example, if
KA ¼ 108 M, then 108 M binds to 50% of the receptors.
If KA ¼ 104 M, a 10,000-fold higher concentration of the
drug is needed to bind to 50% of the receptors (i.e., it is of
lower affinity).
It is instructive to discuss affinity in terms of the
adsorption isotherm in the context of measuring the amount
of receptor bound for given concentrations of drug. Assume
that values of fractional receptor occupancy can be visu-
alized for various drug concentrations. The kinetics of such
binding is shown in Fig. 1.13. It can be seen that initially
the binding is rapid, in accordance with the fact that there
are many unbound sites for the drug to choose. As the sites
become occupied, there is a temporal reduction in binding
until a maximal value for that concentration is attained.
Fig. 1.13 also shows that the binding of higher concentra-
tions of drug is correspondingly increased. In keeping with
the fact that this is first-order binding kinetics (where the
rate is dependent on a rate constant multiplied by the
concentration of reactant), the time to equilibrium is shorter
for higher concentrations than for lower concentrations.
The various values for receptor occupancy at different
concentrations constitute a concentration binding curve
(shown in Fig. 1.14A). There are two areas in this curve of
particular interest to pharmacologists. The first is the
maximal asymptote for binding. This defines the maximal
number of receptive binding sites in the preparation. The
binding isotherm [Eq. (1.4)] defines the ordinate axis as the
fraction of the maximal binding. Thus, by definition, the
maximal value is unity. However, in experimental studies,
real values of capacity are used since the maximum is not
FIGURE 1.13 Time course for increasing concentrations of a ligand with a KA of 2 nM. Initially, the binding is rapid but slows as the sites become
occupied. The maximal binding increases with increasing concentrations as does the rate of binding.
What is pharmacology? Chapter | 1 15
known. When the complete curve is defined, the maximal
value of binding can be used to define fractional binding at
various concentrations and thus define the concentration at
which half-maximal binding (binding to 50% of the re-
ceptor population) occurs. This is the equilibrium dissoci-
ation constant of the drugereceptor complex (KA), the
important measure of drug affinity. This comes from the
other important region of the curve, namely, the midpoint.
It can be seen from Fig. 1.14A that graphical estimation of
both the maximal asymptote and the midpoint is difficult to
perform with the graph in the form shown. A much easier
format to present binding, or any concentrationeresponse
data, is a semilogarithmic form of the isotherm. This allows
better estimation of the maximal asymptote and places the
midpoint in a linear portion of the graph where intra-
polation can be done (see Fig. 1.14B). Doseeresponse
curves for binding are not often visualized, as they require a
means to detect bound (over unbound) drug. However, for
drugs that produce a pharmacological response (i.e., ago-
nists), a signal proportional to bound drug can be observed.
The true definition of a doseeresponse curve is the
observed in vivo effect of a drug given as a dose to a whole
animal or human. However, it has entered into the common
pharmacological jargon as a general depiction of drug and
effect. Thus, a doseeresponse curve for binding is actually
a binding concentration curve, and an in vitro effect of an
agonist in a receptor system is a concentrationeresponse
curve.
1.11 What is efficacy?
The property that gives a molecule the ability to change a
receptor, such that it produces a cellular response, is termed
efficacy. Early concepts of receptors likened them to locks
and keys. As stated by Paul Ehrlich,
16 A Pharmacology Primer
FIGURE 1.14 Doseeresponse relationship for ligand binding according to the Langmuir adsorption isotherm. (A) Fraction of maximal binding as a
function of concentration of agonist. (B) Semilogarithmic form of curve shown in panel A.
Substances can only be anchored at any particular part of
the organism if they fit into the molecule of the recipient
complex like a piece of mosaic finds its place in a pattern.
This historically useful but inaccurate view of receptor
function has in some ways hindered development models of
efficacy. Specifically, the lock-and-key model implies a
static system with no moving parts. However, one feature
of proteins is their malleability. While they have structure,
they do not have a single structure but rather many potential
shapes referred to as conformations. A protein stays in a
particular conformation because it is energetically favorable
to do so (i.e., there is minimal free energy for that
conformation). If thermal energy enters the system, the
protein may adopt another shape in response. Stated by
Linderstrom-Lang and Schellman [15]:
. a protein cannot be said to have “a” secondary struc-
ture but exists mainly as a group of structures not too
different from one another in free energy .. In fact, the
molecule must be conceived as trying every possible
structure ..
dLindstrom and Schellman (1959).
Not only are a number of conformations for a given
protein possible, but the protein samples these various
conformations constantly. It is a dynamic and not a static
entity. Receptor proteins can spontaneously change
conformation in response to variations in the energy of the
system. An important concept here is that small mole-
cules, by interacting with the receptor protein, can bias the
conformations that are sampled. It is in this way that drugs
can produce active effects on receptor proteins (i.e.,
demonstrate efficacy). A thermodynamic mechanism by
which this can occur is through what is known as
conformational selection [16]. A simple illustration can be
made by reducing the possible conformations of a given
receptor protein to just two. These will be referred to as
the “active” (denoted [Ra]) and “inactive” (denoted [Ri])
conformations.
Thermodynamically it would be expected that a ligand
may not have identical affinity for both receptor confor-
mations. This was an assumption in early formulations of
conformational selection. For example, differential affinity
for protein conformations was proposed for oxygen binding
to hemoglobin [17] and for choline derivatives and nico-
tinic receptors [18]. Furthermore, assume that these con-
formations exist in an equilibrium defined by an allosteric
constant L (defined as [Ra]/[Ri]) and that a ligand [A] has
affinity for both conformations defined by equilibrium as-
sociation constants Ka and aKa, respectively, for the inac-
tive and active states.
It can be shown that the ratio of the active species Ra in
the presence of a saturating concentration (rN) of the
ligand versus in the absence of the ligand (r0) is given by
the following (see Section 1.14):
rN að1 þ LÞ
¼ . (1.5)
r0 ð1 þ aLÞ
It can be seen that if the factor a is unity (i.e., the af-
finity of the ligand for Ra and Ri is equal [Ka ¼ aKa]), then
there will be no change in the amount of Ra when the ligand
is present. However, if a is not unity (i.e., if the affinity of
the ligand differs for the two species), then the ratio
necessarily will change when the ligand is present. There-
fore, its differential affinity for the two protein species will
alter their relative amounts. If the affinity of the ligand is
higher for Ra, then the ratio will be >1 and the ligand will
enrich the Ra species. If the affinity for the ligand for Ra is
less than for Ri, then the ligand (by its presence in the
system) will reduce the amount of Ra. For example, if the
affinity of the ligand is 30-fold greater for the Ra state, then
in a system where 16.7% of the receptors are spontaneously
in the Ra state, the saturation of the receptors with this
agonist will increase the amount of Ra by a factor of 5.14
(16.7%e85%).
This concept is demonstrated schematically in Fig. 1.15.
It can be seen that the initial bias in a system of proteins
containing two conformations (square and spherical) lies
 What is pharmacology? Chapter | 1 17

FIGURE 1.15 Conformational selection as a thermodynamic process to bias mixtures of protein conformations. (A) The two forms of the protein are
depicted as circular and square shapes. The system initially is predominantly square. Gaussian curves to the right show the relative frequency of
occurrence of the two conformations. (B) As a ligand (blue dots) enters the system and prefers the circular conformations, these are selectively removed
from the equilibrium between the two protein states. The distributions show the enrichment of the circular conformation at the expense of the square one.
(C) A new equilibrium is attained in the presence of the ligand favoring the circular conformation because of the selective pressure of affinity between the
ligand and this conformation. The distribution reflects the presence of the ligand and the enrichment of the circular conformation.

far toward the square conformation. When a ligand (filled
circles) enters the system and selectively binds to the cir-
cular conformations, this binding process removes the cir-
cles driving the backward reaction from circles back to
squares. In the absence of this backward pressure, more
square conformations flow into the circular state to fill the
gap. Overall, there is an enrichment of the circular con-
formations when unbound and ligand-bound circular con-
formations are totaled.
This also can be described in terms of the Gibbs free
energy of the receptoreligand system. Receptor confor-
mations are adopted as a result of attainment of minimal
free energy. Therefore, if the free energy of the collection
of receptors changes, so too will the conformational
makeup of the system. The free energy of a system
composed of two conformations ai and ao is given by the
following [19]:
X P
DGi ¼ DG0i  RT
X Doseeresponse curves depict the response to an agonist
 lnð1 þ Ka;i ½AÞ=lnð1 þ Ka;0 ½AÞ;
where Ka,i and Ka,0 are the respective affinities of the ligand
for states i and o. It can be seen that unless Ka,i ¼ Ka,0, the
logarithmic term will not equal zero and the free energy of
P P  along the concentration axis, slope, and maximal asymptote
the system will change DGi s DG0i . Thus, if a
ligand has differential affinity for either state, then the
free energy of the system will change in the presence of
the ligand. Under these circumstances, a different confor-
mational bias will be formed by the differential affinity of
the ligand. From these models comes the concept that bind-
ing is not a passive process, whereby a ligand simply ad-
heres to a protein without changing it. The act of binding
can itself bias the behavior of the protein. This is the ther-
modynamic basis of efficacy.
1.12 Doseeresponse curves
The concept of “doseeresponse” in pharmacology has been
known and discussed for some time. A prescription written
in 1562 for hyoscyamus and opium for sleep clearly states,
“If you want him to sleep less, give him less” [13]. It was
recognized by one of the earliest physicians, Paracelsus
(1493e1541), that it is only the dose that makes something
beneficial or harmful: “All things are poison, and nothing is
without poison. The dose [sic] alone makes a thing not
poison.”
(1.6) in a cellular or subcellular system as a function of the
agonist concentration. Specifically, they plot response as a
function of the logarithm of the concentration. They can be
defined completely by three parameters, namely, location
(Fig. 1.16). At first glance, the shapes of doseeresponse
curves appear to closely mimic the line predicted by the
Langmuir adsorption isotherm, and it is tempting to assume
18 A Pharmacology Primer
FIGURE 1.16 Doseeresponse curves. Any doseeresponse curve can be
defined by the threshold (where response begins along the concentration
axis), the slope (the rise in response with changes in concentration), and
the maximal asymptote (the maximal response).
that doseeresponse curves reflect the first-order binding
and activation of receptors on the cell surface. However, in
most cases, this resemblance is happenstance, and dosee
response curves reflect a far more complex amalgam of
binding, activation, and recruitment of cellular elements of
response. In the end, these may yield a sigmoidal curve, but
in reality they are far removed from the initial binding of
drug and receptor. For example, in a cell culture with a
collection of cells with varying thresholds for depolariza-
tion, the single-cell response to an agonist may be complete
depolarization (in an all-or-none fashion). Taken as a
complete collection, the depolarization profile of the culture
where the cells all have differing thresholds for depolari-
zation would have a Gaussian distribution of depolarization
thresholdsdsome cells being more sensitive than others
(Fig. 1.17A). The relationship of depolarization of the
complete culture to the concentration of a depolarizing
agonist is the area under the Gaussian curve. This yields a
sigmoidal doseeresponse curve (Fig. 1.17B) that resembles
the Langmuirian binding curve for drugereceptor binding.
The slope of the latter curve reflects the molecularity of the
drugereceptor interaction (i.e., one ligand binding to one
receptor yields a slope of unity for the curve). In the case of
the sequential depolarization of a collection of cells, it can
be seen that a narrower range of depolarization thresholds
yields a steeper doseeresponse curve, indicating that the
actual numerical value of the slope for a doseeresponse
curve cannot be equated to the molecularity of the binding
between agonist and receptor. In general, shapes of
doseeresponse curves are completely controlled by cellular
factors and cannot be used to discern drugereceptor
mechanisms. These must be determined indirectly by null
methods.
1.12.1 Potency and maximal response
There are certain features of agonist doseeresponse curves
that are generally true for all agonists. The first is that the
magnitude of the maximal asymptote is totally dependent
on the efficacy of the agonist and the efficiency of the
biological system to convert receptor stimulus into tissue
response (Fig. 1.18A). This can be an extremely useful
observation in the drug-discovery process when attempting
to affect the efficacy of a molecule. Changes in chemical
structure that affect only the affinity of the agonist will have
no effect on the maximal asymptote of the doseeresponse
curve for that agonist. Therefore, if chemists wish to opti-
mize or minimize efficacy in a molecule, they can track the
maximal response to do so. Second, the location, along
the concentration axis of doseeresponse curves, quantifies
the potency of the agonist (Fig. 1.18B). The potency is the
molar concentration required to produce a given response.
Potencies vary with the type of cellular system used to
make the measurement and the level of response at which
the measurement is made. A common measurement used to
quantify potency is the EC50, namely, the molar concen-
tration of an agonist required to produce 50% of the
maximal response to the agonist. Thus, an EC50 value of
1 mM indicates that 50% of the maximal response to the
agonist is produced by a concentration of 1 mM of the
agonist (Fig. 1.19). If the agonist produces a maximal
response of 80% of the system maximal response, then
40% of the system maximal response will be produced by
1 mM of this agonist (Fig. 1.19). Similarly, an EC25 will be
produced by a lower concentration of this same agonist; in
this case, the EC25 is 0.5 mM.
1.12.2 P-scales and the representation of
potency
Agonist potency is an extremely important parameter in
drugereceptor pharmacology. Invariably it is determined
from log-doseeresponse curves. It should be noted that
since these curves are generated from semilogarithmic
plots, the location parameter of these curves is log nor-
mally distributed. This means that the logarithms of the
sensitivities (EC50) and not the EC50 values themselves
are normally distributed (Fig. 1.20A). Since all statistical
parametric tests must be done on data that come from
normal distributions, all statistics (including comparisons
of potency and estimates of errors of potency) must come
from logarithmically expressed potency data. When log
normally distributed EC50 data (Fig. 1.20B) are con-
verted to EC50 data, the resulting distribution is seriously
skewed (Fig. 1.20C). It can be seen that error limits on
the mean of such a distribution are not equal [i.e., one
standard error of the mean unit (see Chapter 12: Statistics
and Experimental Design) either side of the mean gives

FIGURE 1.19 Doseeresponse curves. Doseeresponse curve to an
agonist that produces 80% of the system maximal response. The EC50
(concentration producing 40% response) is 1 mM, the EC25 (20%) is
0.5 mM, and the EC80 (64%) is 5 mM.
different values on the skewed distribution (Fig. 1.20C)].
This is not true of the symmetrical normal distribution
(Fig. 1.20B).
One representation of numbers such as potency estimates
is with the P-scale. The P-scale is the negative logarithm of
number. For example, the pH is the negative logarithm of a
What is pharmacology? Chapter | 1 19
FIGURE 1.17 Factors affecting the
slope of doseeresponse curves. (A)
Gaussian distributions of the thresholds
for depolarization of cells to an agonist
in a cell culture. Solid line shows a
narrow range of threshold, and the
lighter line a wider range. (B) Area
under the curve of the Gaussian distri-
butions shown in panel A. These would
represent the relative depolarization of
the entire cell culture as a function of
the concentration of agonist. The more
narrow range of threshold values cor-
responds to the doseeresponse curve of
steeper slope.
FIGURE 1.18 Major attributes of
agonist doseeresponse curves.
Maximal responses solely reflect
efficacy (left), while the potency
(location along the concentration
axis) reflects a complex function of
both efficacy and affinity (right).
hydrogen ion concentration (105 M ¼ pH ¼ 5). It is essential
to express doseeresponse parameters as P-values (log of
the value, as in the pEC50) since these are log normal.
However, it sometimes is useful on an intuitive level to
express potency as a concentration (i.e., the antilog value).
One way this can be done and still preserve the error esti-
mate is to make the calculation as P-values and then convert
to concentration as the last step. For example, Table 1.2
shows five pEC50 values, giving a mean pEC50 of 8.46 and a
standard error of 0.21. It can be seen that the calculation of
the mean as a converted concentration (EC50 value) leads to
an apparently reasonable mean value of 3.8 nM, with a
standard error of 1.81 nM. However, the 95% confidence
limits (range of values that will include the true value) of the
concentration value is meaningless, in that one of them (the
lower limit) is a negative number. The true value of the EC50
lies within the 95% confidence limits given by the mean -
þ 2.57  the standard error, which leads to the values 8.4
and 0.85 nM. However, when pEC50 values are used for
the calculations, this does not occur. Specifically, the mean
of 8.46 yields a mean EC50 of 3.47 nM. The 95% confidence
limits on the pEC50 are 7.8e9.0. Conversion of these limits
to EC50 values yields 95% confidence limits of 1e11.8 nM.
Thus, the true potency lies between the values of 1 and
11.8 nM 95% of the time.
20 A Pharmacology Primer

FIGURE 1.20 Log normal distributions of sensitivity of a pharmacological preparation to an agonist. (A) Doseeresponse curve showing the distribution
of the EC50 values along the log concentration axis. This distribution is normal only on a log scale. (B) Log normal distribution of pEC50 values (log
EC50 values). (C) Skewed distribution of EC50 values converted from the pEC50 values shown in panel B.

l System-independent measures of drug activity coupled

TABLE 1.2 Expressing mean agonist potencies with with pharmacological models of drug mechanisms can
error. combine to convert a single ‘snapshot’ of activity in
pEC50a EC50 (nM)b one system into the complete ‘film’ of what the drug
will do in vivo.
8.5 3.16
l Affinity is the strength of binding of a drug to a receptor.
8.7 2 It is quantified by an equilibrium dissociation constant.
8.3 5.01 l Affinity can be depicted and quantified with the Lang-
8.2 6.31 muir adsorption isotherm.
l Efficacy is measured in relative terms (having no abso-
8.6 2.51
lute scale) and quantifies the ability of a molecule to
Mean ¼ 8.46 Mean ¼ 3.8 produce a change in the receptor (most often leading
SE ¼ 0.21 SE ¼ 1.81 to a physiological response).
a
l Doseeresponse curves quantify drug activity. The
Replicate values of 1/N log EC50’s.
b
Replicate EC50 values in nM. maximal asymptote is totally dependent on efficacy,
while potency is due to an amalgam of affinity and
efficacy.
l Measures of potency are log normally distributed. Only
1.13 Chapter summary and conclusions P-scale values (i.e., pEC50) should be used for statistical
tests.
l Some ideas on the origins and relevance of pharma-
cology and the concept of biological “receptors” are
discussed. 1.14 Derivations: conformational
l Currently, there are drugs for only a fraction of the selection as a mechanism of
druggable targets present in the human genome.
l While recombinant systems have greatly improved the
efficacy
drug-discovery process, pathological phenotypes still Consider a system containing two receptor conformations
are a step away from these drug-testing systems. Ri and Ra that coexist in the system according to an allo-
l Because of the fact that drugs are tested in experimental, steric constant denoted L.
not therapeutic, systems, system-independent measures Assume that ligand A binds to Ri with an equilibrium
of drug activity (namely, affinity and efficacy) must association constant Ka, and Ra by an equilibrium associ-
be measured in drug discovery. ation constant aKa. The factor a denotes the differential

affinity of the agonist for Ra (i.e., a ¼ 10 denotes a 10-fold
greater affinity of the ligand for the Ra state). The effect of
a on the ability of the ligand to alter the equilibrium be-
tween Ri and Ra can be calculated by examining the amount
of Ra species (both as Ra and ARa) present in the system in
the absence of ligand and in the presence of ligand. The
equilibrium expression for ([Ra]þ[ARa])/[Rtot], where
[Rtot] is the total receptor concentration given by the con-
servation equation [Rtot] ¼ [Ri]þ[ARi]þ[Ra]þ[ARa], is
Lð1 þ a½A=KA Þ
r¼ ; (1.7)
½A=KA ð1 þ aLÞ þ 1 þ L
where L is the allosteric constant, [A] is the concentration
of ligand, KA is the equilibrium dissociation constant of
the agonistereceptor complex (KA ¼ 1/Ka), and a is the
differential affinity of the ligand for the Ra state. It can
be seen that in the absence of agonist ([A] ¼ 0), r0 ¼ L/
(1 þ L), and in the presence of a maximal concentration
of ligand (saturating the receptors; [A]/N),
rN¼(a(1 þ L))/(1 þ aL). The effect of the ligand on
changing the proportion of the Ra state is given by the ratio
r/r0. This ratio is given by
rN að1 þ LÞ
¼ . (1.8)
r0 ð1 þ aLÞ
Eq. (1.8) indicates that if the ligand has an equal affinity
for both the Ri and Ra states (a ¼ 1), then rN/r0 will equal
unity, and no change in the proportion of Ra will result
from maximal ligand binding. However, if a > 1, then the
presence of the conformationally selective ligand will cause
the ratio rN/r0 to be > 1, and the Ra state will be enriched
by presence of the ligand.
References
[1] A.-H. Maehle, C.-R. Prull, R.F. Halliwell, The emergence of the
drug-receptor theory, Nat. Rev. Drug Discov. 1 (2002) 1637e1642.
[2] W.D.M. Paton, On becoming a pharmacologist, Annu. Rev. Phar-
macol. Toxicol. 26 (1986) 1e22.
What is pharmacology? Chapter | 1 21
[3] J. Drews, Drug discovery: a historical perspective, Science 287
(2000) 1960e1964.
[4] A.J. Clark, The Mode of Action of Drugs on Cells, Edward Arnold,
London, 1933.
[5] A.J. Clark, A. Heffter, General Pharmacology Handbuch der Exper-
imentellen Pharmakologie, Springer, Berlin, 1937, pp. 165e176, 4.
[6] B. Holmstedt, G. Liljestrand, Readings in Pharmacology, Raven
Press, New York, NY, 1981.
[7] A. Marchese, S.R. George, L.F. Kolakowski, K.R. Lynch,
B.F. O’Dowd, Novel GPCRs and their endogenous ligands:
expanding the boundaries of physiology and pharmacology, Trends
Pharmacol. Sci. 20 (1999) 370e375.
[8] J.C. Venter, M.D. Adams, E.W. Myers, P.W. Li, R.J. Mural,
G.G. Sutton, The sequence of the human genome, Science 291
(2001) 1304e1351.
[9] R. Link, D. Daunt, G. Barsh, A. Chruscinski, B. Kobilka, Cloning of
two mouse genes encoding a2-adrenergic receptor subtypes and
identification of a single amino acid in the mouse a2-C10 homolog
responsible for an interspecies variation in antagonist binding, Mol.
Pharmacol. 42 (1992) 16e17.
[10] J.W. Black, A personal view of pharmacology, Annu. Rev. Phar-
macol. Toxicol. 36 (1996) 1e33.
[11] R. Buscher, V. Hermann, P.A. Insel, Human adrenoceptor poly-
morphisms: evolving recognition of clinical importance, Trends
Pharmacol. Sci. 20 (1999) 94e99.
[12] R.P. Stephenson, A modification of receptor theory, Br. J. Pharma-
col. 11 (1956) 379e393.
[13] S. Norton, Origins of pharmacology, Mol. Interv. 5 (2005) 144e149.
[14] P. Leff, G.R. Martin, J.M. Morse, Differences in agonist dissociation
constant estimates for 5-HT at 5-HT2-receptors: a problem of acute
desensitization? Br. J. Pharmacol. 89 (1986) 493e499.
[15] A. Linderstrom-Lang, P. Schellman, Protein conformation, Enzymes
1 (1959) 443e471.
[16] A.S.V. Burgen, Conformational changes and drug action, Fed. Proc.
40 (1966) 2723e2728.
[17] J.J. Wyman, D.W. Allen, The problem of the haem interaction in
haemoglobin and the basis for the Bohr effect, J. Polym. Sci. 7
(1951) 499e518.
[18] J. Del Castillo, B. Katz, Interaction at end-plate receptors between
different choline derivatives, Proc. Roy. Soc. Lond. B. 146 (1957)
369e381.
[19] E. Freire, Can allosteric regulation be predicted from structure? Proc.
Natl. Acad. Sci. U.S.A. 97 (2000) 11680e11682.
This page intentionally left blank
Chapter 2
How different tissues process drug
response
[Nature] can refuse to speak but she cannot give a wrong
answer.
d Dr. Charles Brenton Hugins (1966).
We have to remember that what we observe is not nature in
itself, but nature exposed to our method of questioning .
dWerner Heisenberg (1901e76).
2.1 The ‘eyes to see’: pharmacologic
assays
If a drug possesses the molecular property of efficacy, then
it produces a change in the receptor that may be detected by
the cell. However, this can occur only if the stimulus is of
sufficient strength and the cell has the amplification ma-
chinery necessary to convert the stimulus into an observ-
able response. In keeping with the mandatory partnership of
the sensitivity of the cell system and the intrinsic power of
the agonist to produce a response, the cellular assay be-
comes a key component that controls the amount of in-
formation that can be gained from the experiment, in
essence, the assay becomes the ‘eyes to see’ the change
imparted to the cell by the drug. Cellular assays can be
natural (and thus the sensitivity and components are set by
Nature) or recombinant whereby the experimenter can
manipulate the levels of response components and thus the
sensitivity of the system. Fig. 2.1 shows some of the factors
that play into the design of a pharmacologic assay whether
as applied to binding studies (Chapter 4) or functional
studies (Chapter 6). When building recombinant systems,
the first option is the type of cell to be used. Different cells
have different components and some of these may be
critical to the response of a given agonist. A case in point is
the response to the hormone amylin which interacts with a
receptor formed by a dimer of the calcitonin receptor and
the membrane protein RAMP3 (Receptor Activity modi-
fying Protein 3). Thus, if a cell is not used that contains
RAMP3, then transfection of calcitonin receptors will not
constitute receptors for amylin and the assay will yield
erroneous results (for further details see Fig. 5.3). Similarly,
the relative stoichiometry of signaling components in a cell
A Pharmacology Primer. https://doi.org/10.1016/B978-0-323-99289-3.00013-0
Copyright © 2022 Elsevier Inc. All rights reserved.
may affect observed bias of agonist response (for further
details see Chapter 6). For complex binding curves
whereby the binding complex is an amalgam of the receptor
with other components (i.e., G-protein), differences in the
complimentary protein can lead to differences in binding
profiles. For instance, overexpression of receptor to the
point where the G-protein components in a cell are insuf-
ficient to produce adequate levels of ternary complex, then
complex binding curves will be produced (for further de-
tails see Fig. 4.19). If cell response is measured, then
various cells reflect changes in function in different ways
thus ‘business rules’ for the definition of what will be
considered drug response must be determined and adhered
to throughout the experiment. A major determinant of the
sensitivity of cells for agonists acting on receptors is the
level of receptor density expressed in the cell, i.e., a high
receptor density produces a sensitive tissue whereas a low
density an insensitive tissue. The impact of functional assay
composition will be considered repeatedly in this book as it
is of paramount importance for the discernment of drug
activity in in vitro systems.
Fig. 2.2A shows a functional doseeresponse curve for
human calcitonin in human embryonic kidney (HEK) cells
transfected with cDNA for human calcitonin receptor type
2 [1]. The response being measured here is the hydrogen
ion release by the cells, a sensitive measure of cellular
metabolism. Also shown (dotted line) is a curve for calci-
tonin binding to the receptors (as measured with radio-
ligand binding). A striking feature of these curves is that the
curve for function is shifted considerably to the left of the
binding curve. Calculation of the receptor occupancy
required for 50% maximal tissue response indicates that
less than 50% occupancy, namely, more on the order of
3%e4%, is needed. In fact, a regression of tissue response
upon the receptor occupancy is hyperbolic in nature
(Fig. 2.2B), showing a skewed relationship between re-
ceptor occupancy and cellular response. This skewed
relationship indicates that the stimulation of the receptor
initiated by binding is amplified by the cell in the process of
response production.
The ability of a given agonist to produce a maximal
system response can be quantified as a receptor reserve.
23
24 A Pharmacology Primer

FIGURE 2.1 Main options available for the design of recombinant assay systems are the type of cell to be used and the level of receptors available to
response to agonists.

FIGURE 2.2 Binding and doseeresponse curves for human calcitonin on human calcitonin receptors type 2. (A) Doseeresponse curves for micro-
physiometry responses to human calcitonin in HEK cells (open circles) and binding in membranes from HEK cells (displacement of [125I]-human
calcitonin). (B) Regression of microphysiometry responses to human calcitonin (ordinates) upon human calcitonin fractional receptor occupancy
(abscissae). Dotted line shows a direct correlation between receptor occupancy and cellular response. HEK, human embryonic kidney. (A) Data from W.-J.
Chen, S. Armour, J. Way, G.C. Chen, C. Watson, P.E. Irving, Expression cloning and receptor pharmacology of human calcitonin receptors from MCF-7
cells and their relationship to amylin receptors, Mol. Pharmacol. 52 (1997) 1164e1175.

The reserve refers to the percentage of receptors not
required for production of maximal response (sometimes
referred to as spare receptors). For example, a receptor
reserve of 80% for an agonist means that the system
maximal response is produced by activation of 20% of the
receptor population by that agonist. Receptor reserves can
be quite striking. Fig. 2.3 shows guinea pig ileal smooth
muscle contractions to the agonist histamine before and
after irreversible inactivation of a large fraction of the re-
ceptors with the protein alkylating agent phenoxybenz-
amine [2]. The fact that the depressed maximum
doseeresponse curve is observed so far to the right of the
control doseeresponse curve indicates a receptor reserve of
98% [i.e., only 2% of the receptors must be activated by
histamine to produce the tissue’s maximal response
(Fig. 2.3B)]. In teleological terms, this may be useful, since
it allows neurotransmitters to produce rapid activation of
organs with minimal receptor occupancy leading to optimal
and rapid control of function.
Receptor reserve is a property of the tissue (i.e., the
strength of amplification of receptor stimulus inherent to the
cells) and it is a property of the agonist (i.e., how much
stimulus is imparted to the system by a given agonist receptor
occupancy). This latter factor is quantified as the efficacy of
the agonist. A high-efficacy agonist need occupy a smaller
fraction of the receptor population than a lower efficacy
agonist to produce a comparable stimulus. Therefore, it is
incorrect to ascribe a given tissue or cellular response system
with a characteristic receptor reserve. The actual value of the
receptor reserve will be unique to each agonist in that system.
For example, Fig. 2.4 shows the different amplification hy-
perbolae of Chinese hamster ovary (CHO) cells transfected
with b-adrenoceptors in producing cyclic adenosine mono-
phosphate (AMP) responses to three different b-adrenoceptor
 How different tissues process drug response Chapter | 2 25

FIGURE 2.3 Guinea pig ileal responses to histamine. (A) Contraction of guinea pig ileal longitudinal smooth muscle (ordinates as a percentage of
maximum) to histamine (abscissae, logarithmic scale). Responses obtained before ( filled circles) and after treatment with the irreversible histamine
receptor antagonist phenoxybenzamine (50 mM for 3 minutes; open circles). (B) Occupancyeresponse curve for data shown in (A). Ordinates are per-
centage of maximal response. Abscissae are calculated receptor occupancy values from an estimated affinity of 20 mM for histamine. Note that maximal
response is essentially observed after only 2% receptor occupancy by the agonist (i.e., a 98% receptor reserve for this agonist in this system). Data
redrawn from T.P. Kenakin, D.A. Cook, Blockade of histamine-induced contractions of intestinal smooth muscle by irreversibly acting agents, Can. J.
Physiol. Pharmacol. 54 (1976) 386e392.

mechanisms in the cell. Each has its own function and

FIGURE 2.4 Occupancyeresponse curves for b-adrenoceptor agonists in
transfected CHO cells. Occupancy (abscissae) calculated from binding af-
finity measured by displacement of [125I]-iodocyanopindolol. Response
measured as increases in cyclic AMP. Drawn from S. Wilson, J.K. Cham-
bers, J.E. Park, A. Ladurner, D.W. Cronk, C.G. Chapman, Agonist potency
at the cloned human beta-3 adrenoceptor depends on receptor expression
level and nature of assay, J. Pharmacol. Exp. Ther. 279 (1996) 214e221.
agonists [3]. It can be seen that isoproterenol requires many
times less receptors to produce 50% response than do both
the agonists BRL 37344 and CGP 12177. This underscores
the idea that the magnitude of receptor reserves is very much
dependent on the efficacy of the agonist (i.e., one agonist’s
spare receptor is another agonist’s essential one).
2.2 The biochemical nature of
stimuluseresponse cascades
Cellular amplification of receptor signals occurs through a
succession of saturable biochemical reactions. Different
receptors are coupled to different stimuluseresponse
operates on its own timescale. For example, receptor
tyrosine kinases (activated by growth factors) phosphory-
late target proteins on tyrosine residues to activate protein
phosphorylation cascades such as mitogen-activated protein
(MAP) kinase pathways. This process, on a timescale on
the order of seconds to days, leads to protein synthesis from
gene transcription with resulting cell differentiation and/or
cell proliferation. Nuclear receptors, activated by steroids,
operate on a timescale of minutes to days and mediate gene
transcription and protein synthesis. This leads to homeo-
static, metabolic, and immunosuppression effects. Ligand-
gated ion channels, activated by neurotransmitters, operate
on the order of milliseconds to increase the permeability of
plasma membranes to ions. This leads to increases in
cytosolic Ca2þ, depolarization, or hyperpolarization of
cells. This in turn results in muscle contraction, release of
neurotransmitters, or inhibition of these processes.
G-protein-coupled receptors (GPCRs) react with a wide
variety of molecules, from small ones such as acetylcholine
to some as large as the protein SDF-1a. Operating on a
timescale of minutes to hours, these receptors mediate a
plethora of cellular processes. A common reaction in the
activation cascade for GPCRs is the binding of the activated
receptor to a trimeric complex of proteins called G-proteins
(Fig. 2.5). These proteinsdcomposed of three subunits
named a, b, and gdact as molecular switches for a number
of other effectors in the cell. The binding of activated re-
ceptors to the G-protein initiates the dissociation of GDP
from the a-subunit of the G-protein complex, the binding of
guanosine monophosphate (GTP), and the dissociation of the
complex into a- and bg-subunits. The separated subunits of
the G-protein can activate effectors in the cell such as ade-
nylate cyclase and ion channels. Amplification can occur at
these early stages if one receptor activates more than one
26 A Pharmacology Primer

FIGURE 2.5 Activation of trimeric G-proteins by activated receptors. An agonist produces a receptor active state that goes on to interact with the G-
protein. A conformational change in the G-protein causes bound GDP to exchange with GTP. This triggers dissociation of the G-protein complex into a-
and bg-subunits. These go on to interact with effectors such as adenylate cyclase and calcium channels. The intrinsic GTPase activity of the a-subunit
hydrolyzes bound GTP back to GDP, and the inactivated a-subunit reassociates with the bg-subunits to repeat the cycle.

FIGURE 2.6 Production of cyclic AMP from ATP by the enzyme adenylate cyclase. Cyclic AMP is a ubiquitous second messenger in cells activating
numerous cellular pathways. The adenylate cyclase is activated by the a-subunit of Gs-protein and inhibited by the a-subunit of Gi-protein. Cyclic AMP is
degraded by phosphodiesterases in the cell.

G-protein. The a-subunit also is a GTPase, which hydrolyzes
the bound GTP to produce its own deactivation. This ter-
minates the action of the a-subunit on the effector. It can be
seen that the length of time for which the a-subunit is active
can control the amount of stimulus given to the effector, and
that this also can be a means of amplification (i.e., one a-
subunit could activate many effectors). The a- and bg-sub-
units then reassociate to complete the regulatory cycle
(Fig. 2.5). Such receptor-mediated reactions generate cellular
molecules called second messengers. These molecules go on
to activate or inhibit other components of the cellular ma-
chinery to change cellular metabolism and state of activation.
For example, the second messenger (cyclic AMP) is generated
by the enzyme adenylate cyclase from ATP. This second
messenger furnishes fuel, through protein kinases, for the
phosphorylation of serine and threonine residues on a number
of proteins such as other protein kinases, receptors, metabolic
enzymes, ion channels, and transcription factors (see Fig. 2.6).
Activation of other G-proteins leads to the activation of
phospholipase C. These enzymes catalyze the hydrolysis of
 How different tissues process drug response Chapter | 2
FIGURE 2.7 Production of second messengers IP3 and DAG through activation of the enzyme phospholipase C. This enzyme is activated by the a-
subunit of Gq-protein and also by bg-subunits of Gi-protein. IP3 stimulates the release of Ca2 from intracellular stores, while DAG is a potent activator of
protein kinase C. DAG, diacylglycerol; IP3, inositol 1,4,5-triphosphate.
phosphatidylinositol 4,5-bisphosphate to 1,2-diacylglycerol
(DAG) and inositol 1,4,5-triphosphate (see Fig. 2.7). This
latter second messenger interacts with receptors on intracel-
lular calcium stores, resulting in the release of calcium into the
cytosol. This calcium binds to calcium sensor proteins such as
calmodulin or troponin C, which then go on to regulate the
activity of proteins such as protein kinases, phosphatases,
phosphodiesterase, nitric oxide synthase, ion channels, and
adenylate cyclase. The second messenger DAG diffuses in the
plane of the membrane to activate protein kinase C isoforms,
which phosphorylate protein kinases, transcription factors, ion
channels, and receptors. DAG also functions as a source of
arachidonic acid, which goes on to be the source of eicosanoid
mediators such as prostanoids and leukotrienes. In general, all
these processes can lead to a case where a relatively small
amount of receptor stimulation can result in a large
biochemical signal. An example of a complete stimuluse
response cascade for the b-adrenoceptor production of blood
glucose is shown in Fig. 2.8 [4].
There are numerous second messenger systems such as
those utilizing cyclic AMP and cyclic guanosine mono-
phosphate (GMP), calcium and calmodulin, phosphoinositides,
and DAG with accompanying modulatory mechanisms. Each
receptor is coupled to these in a variety of ways in different cell
types. Therefore, it can be seen that it is impractical to attempt
to quantitatively define each stimuluseresponse mechanism
for each receptor system. Fortunately, this is not an important
prerequisite in the pharmacological process of classifying ag-
onists, since these complex mechanisms can be approximated
by simple mathematical functions.
2.3 The mathematical approximation of
stimuluseresponse mechanisms
Each of the processes shown in Fig. 2.8 can be described by
a MichaeliseMenten type of biochemical reaction, a stan-
dard generalized mathematical equation describing the
27
interaction of a substrate with an enzyme. Michaelis and
Menten realized in 1913 that the kinetics of enzyme re-
actions differed from those of conventional chemical re-
actions. They visualized the reaction of substrate and an
enzyme yielding enzyme plus product as a form of this
equation: reaction velocity ¼ (maximal velocity of the
reaction  substrate concentration)/(concentration of sub-
strate þ a fitting constant Km). The constant Km (referred to
as the MichaeliseMenten constant) characterizes the
tightness of the binding of the reaction between substrate
and enzyme, essentially a quantification of the coupling
efficiency of the reaction. Km is the concentration at which
the reaction is half the maximal value or, in terms of ki-
netics, the concentration at which the reaction runs at half
its maximal rate. This model forms the basis of enzymatic
biochemical reactions and can be used as a mathematical
approximation of such functions.
As with the Langmuir adsorption isotherm, which in
shape closely resembles MichaeliseMenten type
biochemical kinetics, the two notable features of such re-
actions are the location parameter of the curve along the
concentration axis (the value of Km or the magnitude of the
coupling efficiency factor) and the maximal rate of the re-
action (Vmax). In generic terms, MichaeliseMenten re-
actions can be written in the form
½substract$Vmax ½input$MAX
Velocity ¼ ¼ (2.1)
½substract þ Km ½input þ b
where b is a generic coupling efficiency factor. It can be
seen that the velocity of the reaction is inversely propor-
tional to the magnitude of b (i.e., the lower the value of
b, the more efficiently is the reaction coupled). If it is
assumed that the stimuluseresponse cascade of any given
cell is a series succession of such reactions, there are two
general features of the resultant that can be predicted math-
ematically. The first is that the resultant of the total series of
reactions will itself be of the form of the same hyperbolic
28 A Pharmacology Primer

FIGURE 2.8 Stimuluseresponse cascade for the production of blood glucose by activation of b-adrenoceptors. Redrawn from N.D. Goldberg, G.
Weissman, R. Claiborne, Cyclic nucleotides and cell function, in: G. Weissman, R. Claiborne (Eds.), Cell Membranes Biochemistry, Cell Biology, and
Pathology, H. P. Publishing, New York, NY, 1975, pp. 185e202.

shape (see Section 2.12.1). The second is that the location

parameter along the input axis (magnitude of the coupling
efficiency parameter) will reflect a general amplification
of any single reaction within the cascade (i.e., the magni-
tude of the coupling parameter for the complete series
will be lower than the coupling parameter of any single re-
action; see Fig. 2.9). The magnitude of btotal for the series
sum of two reactions (characterized by b1 and b2) is given
by (see Section 2.12.2):
b1 b2
btotal ¼ . (2.2)
1 þ b2
It can be seen from Eq. (2.2) that for positive nonzero FIGURE 2.9 Amplification of stimulus through successive rectangular
values of b2, btotal < b1. Therefore, the location parameter hyperbolae. The output from the first function (b ¼ 0.3) becomes the input
of the rectangular hyperbola of the composite set of re- of a second function with the same coupling efficiency (b ¼ 0.3) to yield a
more efficiently coupled overall function (b ¼ 0.069). Arrows indicate the
actions in series is shifted to the left (increased potency) of potency for input to yield 50% maximal output for the first function and
that for the first reaction in the sequence (i.e., there is the series functions.
amplification inherent in the series of reactions).
The fact that the total stimuluseresponse chain can be the relationship between the strength of signal imparted
approximated by a single rectangular hyperbola furnishes the to the receptor between two agonists is accurately reflected
basis of using an end-organ response to quantify an agonist by the end-organ response (Fig. 2.10). This is the primary
effect in a nonsystem-dependent manner. An important reason that pharmacologists can circumvent the effects of the
feature of such a relationship is that it is monotonic (i.e., cellular veil and discern system-independent receptor events
there is only one value of y for each value of x). Therefore, from translated cellular events.
 How different tissues process drug response Chapter | 2 29

FIGURE 2.10 The monotonic nature of stimuluseresponse mechanisms. (A) Receptor stimulus generated by two agonists designated 1 and 2 as a
function of agonist concentration. (B) Rectangular hyperbola characterizing the transformation of receptor stimulus (abscissae) into cellular response
(ordinates) for the tissue. (C) The resulting relationship between tissue responses to the agonists as a function of agonist concentration. The general rank
order of activity (2 > 1) is preserved in the response as a reflection of the monotonic nature of the stimuluseresponse hyperbola.

FIGURE 2.11 Agonist-stimulated phosphorylation process with unsaturable dephosphorylation. Panel A shows dephosphorylation (solid ascending
line) as a linear unsaturable process and phosphorylation (descending dotted lines) for a range of agonist concentrations. Rates decrease as the substrate is
depleted. Where these curves intersect denotes a steady-state response (open circles). Panel B: Steady-state responses (ordinates) as a function of agonist
concentration. A sigmoidal curve of slope ¼ 1 describes steady-state responses. Redrawn from J.J. Tyson, K.C. Chen, B. Novak, Sniffers, buzzers, toggles
and blinkers: dynamics of regulatory and signaling pathways in the cell, Curr. Opin. Cell Biol. 15 (2003) 221e231.

2.4 Influence of stimuluseresponse
cascades on doseeresponse curve
slopes
For standard mass action kinetics whereby a single molecule
binds to a single receptor, the resulting binding curves have
Hill coefficient slopes of unity (providing cooperativity is
not present in the binding reactions). However, for agonists
with such simple Langmuirian binding kinetics (slope ¼ 1),
the cellular response curves for that agonist in functional
systems often will have slopes different from unity; this is
not due to cooperativity of binding but rather through signal
processing by the stimuluseresponse cascades in the cell
cytosol [5]. For example, a simple cytosolic biochemical
reaction such as the phosphorylation and dephosphorylation
of an enzyme can change the slope of concentrationeresponse
curve of agonists affecting the reaction. Fig. 2.11 shows
a concentrationeresponse curve for an agonist promoting the
phosphorylation of an enzyme. Fig. 2.11A shows an
ascending solid line depicting the rate of enzyme dephos-
phorylation and multiple descending dotted lines depicting
rates of phosphorylation for different concentrations of
agonist. The open circles represent steady states where the rate
of phosphorylation equals the rate of dephosphorylation; these
are the observed response points for the agonist and are
shown, as a function of agonist concentration, in Fig. 2.11B.
30 A Pharmacology Primer

FIGURE 2.12 Agonist-stimulated phosphorylation process with saturable dephosphorylation process described by MichaeliseMenten kinetics. Curve
descriptions as for Fig. 2.11. Panel B shows that the relationship between steady-state responses and agonist concentration is described by a sigmoid
function of slope ¼ 3. Redrawn from J.J. Tyson, K.C. Chen, B. Novak, Sniffers, buzzers, toggles and blinkers: dynamics of regulatory and signaling
pathways in the cell, Curr. Opin. Cell Biol. 15 (2003) 221e231.

This figure shows a case where the dephosphorylation is

nonsaturable; the slope of the resulting concentratione
response curve has a slope of unity consistent with mass ac-
tion binding kinetics. However, if the dephosphorylation
process is saturable and described by MichaeliseMenten ki-
netics (as might be expected in a cellular biochemical
reaction), then a different pattern emerges. Specifically, the
resulting concentrationeresponse curve (obtained from
the steady-state intersections of the phosphorylation and
dephosphorylation rates) has a slope of 3, considerably steeper
than that mediating agonist binding to the receptor. Thus, it
can be seen that the processing of receptor stimulus by the cell
can control the slopes of concentrationeresponse curves
making inferences about cooperativity fruitless in functional
systems. Depending on the nature of the feedback loops found FIGURE 2.13 Receptor occupancy curves for activation of human
in cellular stimuluseresponse cascades, a wide variety of calcitonin type 2 receptors by the agonist human calcitonin. Ordinates:
response outcomes can result from varying concentratione response as a fraction of the maximal response to human calcitonin.
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2.5 System effects on agonist response: ovary; HEK, human embryonic kidney.
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Different cellular backgrounds have different capabilities calcitonin activation of the human calcitonin receptor type
Another random document with
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the priests that the people must look for instruction concerning the
duty of the present.
The Assyrian records read, on the other hand, as if they were the
work of royal scribes, writing under the direct supervision of the kings
themselves. The gods are described, and their varied relations to the
world below are duly set forth. But the emphasis of the narrative
appears to be given to the glory and the achievements of such great
monarchs as Sargon and Asshurbanipal, as if a long line of scribes,
writing directly for the king’s approval, had continued the chronicles
from reign to reign.
The early literary and religious ideals of China took a very different
form. We find here no priestly autocracy, controlling all intellectual
activities and giving a revelation as to the nature of the universe, the
requirements of the gods, and the obligations of men, obligations
which have never failed to include the strictest obedience to the
behests of the priests, the representatives of the gods. There are no
court chronicles, dictated under royal supervision, and devoted not to
the needs of the people, but to the glorious achievements of the
monarchs. Nor is there any great epic, commemorating the deeds of
heroes and demi-gods. In place of these we find what may be called
a practical system of applied ethics. Confucius was evidently neither
a visionary dreamer nor a poet, nor did he undertake to establish any
priestly or theological authority for his teaching. He gives the
impression of having been an exceptionally clear-headed and
capable thinker, who devoted himself, somewhat as Socrates did a
century later, to studying out the problems affecting the life of the
state and of the individual. With Socrates, however, the chief thing
appears to have been the intellectual interest of the problem, while
with Confucius, the controlling purpose was evidently the welfare of
his fellow-men. It was his aim, as he himself expressed it, through a
rewriting of the wise teachings left us by our ancestors, so as to
adapt them to the understanding of the present generation, to guide
men to wise and wholesome lives, and to prepare them for a better
future.[6]
The work of Confucius stands as the foundation-stone of the
literature, the morals, and the state-craft of China. It was continued
by such writers as Mencius, 350 b.c., and Tsengtze, 320 b.c.
The works of the earlier authors secured, we are told, an immediate
circulation, but we have no knowledge as to the methods employed
for their distribution. It seems probable that in the earlier as in the
later centuries, the authors whose works found approval with the
authorities received directly from the state compensation for their
literary and philosophic labors.
The material used for the earliest known writings was made from
bamboo fibre, and was prepared in the shape of tablets. Early in the
third century b.c. (curiously enough, during the reign of Hwang-ti, the
destroyer of literature), brushes were invented, with which characters
could be traced upon silk. The bamboo was either scratched upon
with a sharp stylus, or the characters were painted upon it with a
dark varnish. Sometimes also the characters were burned into the
bamboo, with a heated metal stylus. India ink was first used in the
seventh century. The invention of paper took place about 100 b.c.,
the first material utilized for the manufacture being bark, fishing-nets,
and rags. Printing from solid blocks was done as early as the first
century a.d. The invention of the art of printing from movable type is
credited to a blacksmith named Pi-Shing. The blacksmith’s first
books were turned out towards the close of the tenth century a.d., or
early in the eleventh century, more than three centuries before the
presses of Gutenberg began their work in Mayence.
The movable type used by Pi-Shing were made of plastic clay. At
the same time, or shortly thereafter, porcelain type were utilized. The
printing from movable type never seems to have developed to such
extent as to supersede block printing. The Emperor Kang-He had
engraved about two hundred and fifty thousand copper type, which
were used for printing the publications of the government. These
type were afterwards melted for use as cash, but were replaced by
his grandson with type made from lead.[7]
There is record of books being printed in Corea (at that time a
province of the Empire) from movable clay type, as early as 1317
a.d.[8]
 Literature has always been an honored profession in China, and
seems even in the earliest times to have attracted a larger proportion
of workers than, during the same period, were engaged in literary
pursuits in any other countries in the world. The mass of literature
was very much added to after the introduction of Buddhism into the
country, which took place during the first century of the Christian era.
Karpeles states that a selection of the early Chinese classics, with
commentaries, undertaken under the direction of one of the
emperors in the eighteenth century, would, it was calculated,
comprise when completed, 163,000 volumes. By the year 1818,
there had been published of the series, 78,731 volumes.[9] From this
enormous mass of material a few books only stand out as
possessing distinctive importance by reason of their influence on the
thought and the life of many generations.
There are the five King and the four Schu, or “books.” The term
“king” means literally a web, a thing woven, or fabricated. Its use in
this connection recalls the ῥαπτός of the Greek rhapsodists, a term
which, originally meaning a thing spun or a yarn, came also to stand
for a literary production of a certain class, a “yarn” that could be
recited. The five King were the “webs” or productions of wise and
holy writers, but the names of these writers have not been
preserved, even as a tradition. The first in order is the Y-king,
already mentioned, the Book of the Developments, which is much
the oldest in the series. The second is the Schu-king or Book of
Chronicles, which begins its narrative with the time of Noah, and
gives the record of the dynasties from 2400 to 721 b.c. In addition to
the historical chronicles, the Schu-king contains, in the form of
dialogues between the emperors and the councillors, the instruction
in the principles of state-craft, in philosophy, in the science of war, in
music, in astronomy, and in general culture. The headings of some
of the chapters recall the matters treated in The Prince of
Machiavelli. The following “royal maxims” do not, however, sound
Machiavellian: “Virtue,” says the great councillor Yih, speaking to the
emperor, “is the foundation of your realm”; “The ruler must lead his
people in the paths of virtue”; “Guard yourself from false shame, and
if you have committed an error, hasten to make frank
acknowledgment of the same. Otherwise you will mislead your
subjects.”[10]
The third of the canonical books is the Schi-king or Book of Songs,
already referred to. This presents the selection made by Confucius
of the hymns, ballads, and folk-songs collected from the earliest
generations. The fourth is the Tschun-tshien, or Spring and Autumn
Year-Book, which is ascribed to Confucius. It is a brief chronicle of
events covering a space of 240 years. The fifth is the Li-ki, or Book
of Ritual, or of Conduct. This gives detailed instructions concerning
the proper ceremonials for all events of life, from the cradle to the
grave.
With these classics should be grouped certain books prepared by
the followers of Confucius, the most important of which, the Lün-yü,
or Conversations, is a record of the instruction given by Confucius to
his pupils in the form of talks. In these conversations we find
questions shaped in a method quite Socratic. With this should be
grouped the Mengtsze, the record of the work of the philosopher
Mencius. His instruction seems, like that of his great forerunner, to
have been very practical in its character. Associated with the earlier
teachings of Confucius, the instruction of Mencius was accepted as
the basis of the moral and the educational system of the nation.
The enormous respect which the Chinese have given to the works
produced during their classical period is believed by authorities like
Williams and Wade to have exercised an influence on the whole
detrimental to the development and to the originality of their later
literature.
The first active literary period preceded Confucius, 500 b.c. From
this period have been preserved the classics already referred to. The
next important epoch is that of the “interpreters,” the counsellors and
the lawgivers, extending from Confucius to Mencius, 350 b.c. They
were followed by a long line of annalists and commentators, whose
work came to an abrupt close with the reign of the Emperor Che
Hwang-ti, 221-226 b.c. Hwang-ti was evidently a man with opinions
of his own. He objected to what seemed to him an exaggerated and
mischievous reverence for the “good old times,” and he proposed to
discourage the laudator temporis acti. He issued an edict directing all
books to be burned excepting those treating of medicine, divination,
and husbandry. This index expurgatorius (possibly the earliest in
history) included all the writings of Confucius and Mencius,
comprising both their original work and their compilations and
editions of the earlier classics. It was further ordered that any one
who dared to mention the Book of History or the Book of Odes
should be put to death. Any one possessing, thirty days after the
issue of the edict, a copy of the books ordered destroyed, was to be
branded and put to labor for four years upon the great wall. This is
probably the most drastic and comprehensive policy for the
suppression of a literature that the world has ever seen. Fortunately,
like similar attempts in later centuries, it was only partially
successful. While the destruction of books was enormous, and while,
of long lists of works, it is probable that all existing copies actually
did disappear, the texts of the most important, including the specially
obnoxious Book of History and Book of Songs, were preserved.
According to one tradition, a large number of the songs were saved
only by having been retained in the memory of public reciters and
their hearers. After the death of the Emperor Che, the text of these
was taken down and again committed to writing. This instance is,
one recalls, fully in line with the methods by which in Greece, before
the general use of writing, the earlier classics were preserved in the
memories of the rhapsodists and their hearers.
It is the opinion of Dr. Williams that the command of the Emperor
Che for the destruction of all books was so thoroughly executed that
“of many classical works not a single copy escaped destruction. The
books were, however, recovered in great part by rewriting them from
the memories of old scholars.... If the same literary tragedy should
be enacted to-day, thousands of persons might easily be found in
China who could rewrite from memory the text and the commentary
of their nine classical works.”
Williams is also my authority for the statement that not only were
the books destroyed as far as copies could be found, but that nearly
five hundred literati were burned alive, in order that no one might
remain to reproach in his writings the emperor for the commission of
so barbarous an act.[11]
One of the most celebrated female writers in China was Pan Whui-
pan, also known as Pan Chao, the sister of the historian Pan Ku,
who wrote the history of the Han dynasty. She was appointed
historiographer after the death of her brother, and completed, about
a.d. 80, his unfinished annals. A little later she wrote the first work in
any language on female education, which was called Nü Kiai or
Female Precepts, and which has formed the basis of many
succeeding books on female education. In the writings of this and of
other Chinese authoresses, instructions in morals and in the various
branches of domestic economy are insisted upon as the first
essentials in the education of women, and as more important than a
knowledge of the classics or of the annals.[12]
1050 a.d. Wang Pih-ho, of the Sung dynasty, compiled for his
private school a horn-book or manual of education, entitled the San-
tsz’ King. The manual is interesting not merely as giving a general
study of the nature of man and the existence of modes of education,
but because it includes a list of books recommended for the student,
a list which gives an impression of the extent of the education and
literature of that date.[13]
The golden age of Chinese literary production is fixed by Sir
Thomas Wade at the period of the Tang dynasty, 620-907 a.d. In 922
a.d. an edition of the classical writers was printed and published
under the instructions of the Emperor. The tendency of writers since
the tenth century has been to devote their energies to commentaries
on the ancient works, and to analyses and interpretations of these
rather than to original production. The writing of historical annals
has, however, gone on with great regularity, and the series of
Chronicles of the Kingdom is very comprehensive in its
completeness.
The rewards of authors are given in the shape of official
appointments and preferments, and of honors and honorariums
bestowed directly by the state. It seems probable that in modern as
in ancient times the writers of China could look for no direct returns
from the circulation of their productions. It is nevertheless the case
that from the time of Confucius to the present day, that is for a period
of two thousand four hundred years, the direct influence of scholars,
thinkers, and writers has been greater in China than in any other part
of the world. The state as a whole and the individual citizen, from the
Emperor down, have, as a rule, been ready to recognize and accept
the authority and the guidance of literary ideals and of intellectual
standards. The case would be paralleled if the French Academy had
existed from the time of Charlemagne to the present day, if the
counsellors and rulers of the state had always been appointed from
the forty, and if the remaining officials of all grades had been
selected by competitive examinations, instituted and supervised by
the forty. The parallel would not be complete, however, unless the
Academy of to-day were still basing its examinations on a codex of
Charlemagne.
The imperial government of China and the Chinese community as a
whole have for many centuries, apparently ever since the time of the
book-burning Hwang-ti, rendered a larger measure of honor (and
also of direct reward as far as this could be given by official station)
to students and scholars, than has been given by any state in the
history of the world. The literary ideal and the literary productions,
the study of which has thus been honored, have, however, been in
the main those of a thousand years or more back. The fact, says
Legge, that the earlier literary period was so fruitful, and that the
works produced in it have been held by later generations in so great
honor, is one cause why original or creative literary productiveness
has been discouraged, and why the later literary activities continue in
so large proportion to take the shape of commentaries. It has also,
he thinks, been an important influence in keeping the language in an
inflexible and undeveloped condition. It was the language of the
fathers, and it would be sacrilege to modify it.
Japan.—The civilization of Japan is an off-shoot or
development of that of China, and the Japanese literature is based
upon Chinese models and standards. The literary relation strikes one
as in some respects similar to that which existed between Great
Britain and the American Colonies, or later with the American States.
The literature of Japan is described, however, as characterized by
much more elasticity, variety, and creative originality than is
possessed by that of China, and in place of stereotyping itself upon
the models of old-time classics, it has shown from century to century
a wholesome power of development.
At one time, says Karpeles, Japan possessed an alphabet of its
own, but later, the Chinese characters were introduced, and were
used together with the older alphabet. It is only the very earliest
writings in which the Japanese characters alone are employed. The
Japanese scribes have from the beginning worked with brushes
rather than with pens, and in so doing, have been able to utilize such
substances as silk, which would have been unsuitable for the work of
the pen. The invention of paper, however, took place at an early
date, possibly simultaneously with its first use in China. Printing from
blocks, and later from type, was promptly introduced from China
early in our era.
According to the native chroniclers, the earliest literary production
of Japan was the work of the two gods Izanaghi and Izanami. These
gods, having created the country, thought it was incomplete without
some poetry, and the poetry was therefore added. Tsurayuki, a poet
of the tenth century, takes the ground that all true expression of
feeling is poetry. The nightingale sings in the wood, the frog croaks
in the pool; each is giving utterance to a feeling, and each, therefore,
is pouring forth a poem. There is no living being, he continues, who
is not a producer of poetry. (This is as startling to us ordinary mortals
as the discovery of Molière’s Monsieur Jourdain that he had been
talking prose all his life without knowing it.) As poetry, says
Tsurayuki, begins with the expression of feeling, it must have come
into existence with the beginning of creation.[14] In the earliest times,
he says, when the gods were poets, the arrangement of sounds into
syllables had not been made, and rhythm had not been invented.
These early divine poems or utterances of the gods are, therefore,
very difficult to understand. Later, however, Susanoo-no-mikoto fixed
sounds into syllables, and then, according to the tenth-century poet,
Japanese literature had its actual beginning, but he does not give us
the date of this useful piece of work. We are inclined to wonder what
the wise Susanoo, etc., did about the announcing of his own name,
say on really formal occasions, before the little matter of the
invention of syllables had been accomplished.
While it is claimed that from prehistoric times there had been in
Japan an active production and a wide distribution of poetry (folk-
songs), the first collection of the “people’s ballads” appears to have
been made as late as 700 a.d. At this time the Emperor, whose
residence was at Nara, took an interest in literature, and during the
quarter century from 700 to 725 a.d. lived “the noble poet” Yamabe-
no-Akahito, and the “wise man of the poets,” Kakino-mo-to-Hito-
Maro. (The god above referred to, who bestowed upon Japan the
invention of syllables, seems to have done his work thoroughly.) The
compilation which took shape during this period is known as the
Man-yo-sin, or the “collection of ten thousand leaves.” The two later
collections are known as The Old and the New Songs of Japan, and
The Hundred Poets.
A special feature in the literature of Japan is the great number of
poetesses. The fashion of women interesting themselves in the
writing of poetry was initiated by the poetic Empress Soto-oro-ime, in
the third century a.d.
The great epic of Japanese literature is the Fei-ke-mono-gatari, that
is The Annals of the Fei-ke Dynasty, which is said to have been
composed in 1083 a.d., and which was sung among the people by
blind rhapsodists. An epic of later date, in twelve books, is credited
to the poet Ikanage. The literary record shows a long series of tales
and romances, which are described as possessing a graceful fancy
and imagination much in advance of Chinese compositions of the
same class.
The theatre has from early times played a very important part in the
social life of Japan, and dramatic composers are held in high honor.
The first dramas written for performance date from about 807 a.d.
The people of Japan have from the early times of Japanese literature
given cordial appreciation to literary producers, and especially poets
and dramatists. The official recognition of literature and of men of
letters appears, however, to have been much less distinctive and
less important than in China. We do not find record of official
positions and preferments being bestowed on the ground of
proficiency in philosophy or literature, or by reason of a knowledge of
the learning of the past; nor have the smaller government places
been distributed by competitive examinations arranged for students
of literature.
The distribution of literature among the people appears to have
been from an early date very general, and the knowledge of the
great classics has certainly been widespread. Of the methods by
which such distribution was accomplished in the early centuries of
literary production we know nothing. It seems probable from certain
references by later authors, that in Japan, as in Greece, the
rhapsodists and reciters were the principal distributors.
Of rewards or compensations given to the earlier Japanese authors
there is no record. The national treasury does not appear to have
been utilized as in China and Assyria. It is possible that the
dramatists may have secured some share of the stage receipts, but
it is probable that the other authors must have contented themselves
with such prestige or honors as came to them from the readers of, or
the listeners to, their compositions.
India.—In India, the typical early literature is the myth. There is
no national epic in the Greek use of the term, in which are described
the doings of heroic men. The literary productions are the work of
poets whose imagination has been impressed with the immensity
and with the mystery of the universe, and whose poetic fancies take
the form of visions. These fancies or visions are concerned with the
doings of the gods, while man plays but a small part in the narrative.
Sanscrit literature is said to date back to the fifteenth century b.c.
The written characters have an origin common with that of the Greek
letters. The oldest existing monuments of Indian script are the edicts
of the King Açoka, cut into the stone at Girnar and elsewhere “so
that they might endure for ever”. They date back to the third century
b.c.
The first literary period of India presents the poetry of the Vedas,
the sacred scriptures of the Sanscrit peoples. The hymns and
invocations comprising the Vedas are supposed to have been
collected about 1000 b.c. This is the date that has by many
authorities been accepted for the collecting of the Homeric poems,
and corresponds nearly with the time fixed for the writing of the
Chinese Book of the Metamorphoses. It also tallies with the period to
which is ascribed the production of the Persian Zend-Avesta.
The term Veda means knowledge, or sacred knowledge. The
collection of the Vedas comprises four divisions. The Rig-Veda, or
Veda of Praises or Hymns; the Sama-Veda, or Veda of Chants or
Tunes; the Yajur-Veda, or Veda of Prayers; and the Atharva-Veda, or
Brahma-Veda.
The second literary period, beginning about the fifth century b.c., is
that of the Folk-Songs, in which the myth becomes legend, and the
gods, approaching a little closer to the earth, assume more nearly
the character of heroes. The third period is that of the classic poets,
whose productions in lyric and dramatic poetry are ranked with the
great works of literature of the world. This period appears to have
reached its height of productiveness between the sixth and tenth
centuries of our era.
The earliest prose works are the theological writings of the
Brahmanic priests, which take the form of commentaries on the
Vedas, and which elucidate the sacred texts, principally from a
sacrificial point of view. The production of these theological
commentaries is supposed to date back to the seventh or sixth
century b.c.
Buddha, or Gautama, philosopher, poet, reformer, and redeemer of
his people, began his work towards the close of the sixth century
b.c. His teachings gave rise to an enormous production of
theological literature in India, Ceylon, China, and Japan.
The information concerning the materials used by the earlier writers
of India, and as to the methods by which their books were placed
before the public, is very meagre. According to Louisy, the use of
diphtherai, or dressed skins, prevailed to some extent. Prepared
palm-leaves were also utilized, particularly by the Buddhist writers of
Ceylon. There appears to have been no general or popular
circulation of the manuscripts. These were costly, and were beyond
the means of any but the very wealthy, while it was also the case
that the knowledge of reading was confined to but limited circles.
It seems probable that the manuscripts were in the main prepared
in the monasteries or temples, and that they were exchanged
between the temples. The teachings of the writers were brought
before the people by preaching or recitations. Certain of the princes
also attached to their courts poets and philosophers, and practically
the only libraries or collections of manuscripts outside of those in the
temples, must have been those contained in the palaces of the few
princes who possessed literary tastes.
There could have been no other way of securing for an author
compensation for his work excepting through princely favors or from
the treasuries of the temples.

Persia.—The first name that comes down to us connected with

the literature of Persia is that of Zoroaster. The Persian form of his
name is Zarathustra, meaning the gold-star. The date of his birth is
said to be more uncertain than that of Homer, but he is supposed to
have lived about 1000 b.c.
He is credited with the authorship of the Gâthas, hymns partly
religious, partly political. To Zoroaster were also revealed the
teachings which later took shape in the sacred scriptures of the
Persians, the Zend-Avesta (commentary-lore). Of these scriptures,
only one division, the Vendidad, has been preserved complete. Of
the other parts only fragments remain. It is estimated that the
Vendidad (which means the regulations against demons) represents
about one twentieth of the original collection.
The oldest portion of the Avesta is the Yasna, or sacrificial liturgy.
This is a grouping together of the commentaries surrounding the
Gâthas. A third division is the Visparad, or the Seasons, in which are
set forth the lists of the objects sacred to each season. A fourth
division is the Yescht-Sade, or little Avesta, comprising prayers and
hymns.
 The monotheistic or dualistic nature of the faith as originally taught
by Zoroaster has, in the later religious writings and practices, been
overlaid and obscured by the different phases of nature worship. Fire
is accepted as the symbol of holiness, but, according to the views of
the educated Parsees, is not itself the thing worshipped.
The existing canon of the Avesta was compiled and published
under the direction of King Sapor II., who reigned 309-330 a.d.
Among the poems of the Avesta we find the legend of which the hero
is Rustem, who stands as the representative of Iran in its long
contest with Turan.
The literature of Persia prior to the fourth century of the Christian
era was probably controlled in great part by the priests. The
exceptions would have been in the case of the court poets or court
historians, writing under the incentive of royal remuneration. It is
probable that songs and recitations were to some extent given to the
public by minstrels or rhapsodists. There is some evidence also of
the development in later centuries of the story-teller or improvisatore,
who made a business of exchanging, for the pence of the public,
stories partly original, but chiefly borrowed from older sources. The
Oriental capacity for story-telling, and the Oriental readiness to
devote an abundance of leisure time to listening to stories, is clearly
indicated not only by modern practices, but also by the history of
such collections as the Arabian Nights. Of this famous series of
tales, neither the nationality nor the date of origin has been fixed with
any degree of certainty. It is probable, however, that the collection
first took shape in Bagdad about 1450 a.d., the date of the invention
of printing. Von Hammer is of opinion that the Bagdad Tales are
based upon a Persian collection called Hezar Afsaneh, The
Thousand Fanciful Stories. From a passage in the Golden Meadows
of El-Mesoudee (quoted by von Hammer) this Persian collection is
known to have been in existence as early as 987 a.d.
It seems probable, as suggested, that the practice of publicly
reciting poems or of narrating stories prevailed in Persia from a very
early date, and constituted here, as in Greece, the first method for
the distribution or the publication of literary compositions. The
material employed for manuscripts was first diphtherai, or skins, and
later papyrus and parchment.
Judæa.—There is a similar lack of evidence concerning the
existence among the Hebrews of anything that could be called
literary property. The great body of the earlier Hebrew literature
belonged, of course, to the class of sacred writings, best known to us
through the books of the Old Testament and of the Apocrypha. In
addition to these, and partly, of course, included with these, were the
various collections of the law and of the comments on the law, while
later years produced the long series of commentaries known to the
reader of to-day under the general name of the Talmud. The various
transcripts required of these writings of the law and the prophets
gave employment to numbers of scribes, who, in the first place,
apparently were usually connected with the Temple, and must have
derived their support from the ecclesiastical revenues, but who later
formed a separate commercial class, receiving payment for their
work as done.
Professor Peters speaks of the age of Hezekiah as the golden age
of Hebrew literature. He quotes the text, Prov. xxv., I, which says that
“the men of Hezekiah translated” or transcribed, or wrote down the
Proverbs of Solomon, as evidently an effort to collect and preserve
the literary treasures of the past. He says, further:
“It is not unnatural to suppose that the writing down of
Solomon’s Proverbs was for the purpose of a library in
Jerusalem, such as the Assyrian kings had long since
collected at Nineveh. The Book of Amos was edited
(somewhere about 711 b.c.) apparently for this library ...
and I suppose Hosea and Micah also to have been edited
about this time and for the same purpose. It was the
formation of this library at just this time and the desire to
collect and preserve all the literary remains of the past,
which led to the collection and preservation of so much of
the literature of the Northern Kingdom, but lately brought
into Judah by the Israelite emigrés. No tales of the valor of
the heroes of Judah, no Judæan folk-lore ante-dating the
 time of David, have been handed down to us; this
literature belonged to the Northern Kingdom. Literary and
antiquarian zeal led to the collection and reception of
these northern tales and poems into Hezekiah’s library ...
where their use in historical works, owing to the awakened
zeal for a knowledge of the past, was assured. So with the
transfer of intellectual activity from Samaria, a new era
begins in Judah, and soon the charming tales and poems
of the north, preserved in the library of Hezekiah, begin to
be woven into the more solid and ambitious works of the
historians and lawyers of Jerusalem.
This literary awakening could not fail to act upon the
priests. They were the custodians of those ancient
religious and legal traditions, which, coming down from the
age of Moses, had grown with, and been modified by,
changing times and conditions. While some portions of the
‘law’ were written, presumably the larger part of it was
handed down mainly by word of mouth.
Moreover, that which was written probably existed in
various independent codes relating to different subjects.
Some of these—such as a tariff of offerings, or tables of
civil and criminal law, like those contained in the Book of
the Covenant—may have been published, or set up at the
Temple gates, where they could be read by the
worshippers. The greater part of the ‘law,’ however, seems
to have been the exclusive, if not esoteric, possession of
the priesthood of the Jerusalem Temple. The literary
activity of the Renaissance made itself felt within the circle
of the priests, leading them to begin to commit to writing
their unwritten law as well as the ancient traditions,
customs, and ceremonies. Thus was commenced the
work which has given us the middle books of the
Pentateuch, as well as much of Genesis and Joshua.”[15]
It appears, therefore, as if the Hebrew literature of the time (the
reign of Hezekiah, covering the period referred to, lasting from 728 to
699 b.c.) consisted substantially of the “law,” that is of the
authoritative teachings of the “church,” and was almost exclusively in
the hands of the priests. They exercised a control, which amounted
practically to an ownership, over the sacred, that is the official,
records of the “law,” and it appears as if the attested copies or
transcripts could be made only with their permission and under their
supervision. It is probable, therefore, that the copyists were attached
to the Temple, and that such moneys as were received from the sale
of their transcripts belonged to the treasury of the Temple,—but the
manner of such sales can only be guessed at, as the records give us
no information. If, however, this understanding of the practice should
prove to be correct, we should have an example, if not of literary
property, at least of a species of “copyright” control.
The severe Jewish law, directing the penalty of death to be inflicted
upon prophets speaking “false words,” or uttering as inspirations of
their own, words which had originated with others, has been quoted
as an early example of regulation of plagiarism, but it appears
evident, says Rénouard,[16] that the crime here to be punished was
not plagiarism but sacrilege, “Vates mendax qui vaticinatur et quæ
non audivit, et quæ ipsi non sunt dicta, ab hominibus est
occidendus.”[17] The utterance of the prophet Jeremiah (c. xxiii. v.
30) evidently refers to the same regulation.
 CHAPTER II.
Greece.

T HE literature of Greece has become the property of the world,

but of the existence of literary property in Greece—that is, of
any system or practice of compensation to writers from their readers
or hearers, either direct or indirect—the traces are very slight; so
slight, in fact, that the weight of authority is against the probability of
such practice having obtained at all.
It is fortunate for the literature of the world that the Greek poets,
dramatists, historians, and philosophers were content to do their
work for the approval of their own generation, for the chance of fame
with the generations to come, or for the satisfaction of the work itself,
as their rewards in the shape of anything more tangible than fame
appear to have been either nothing or something very
inconsiderable.
Clement says: “After the most painstaking researches through the
records left us by the Greeks, we are compelled to conclude that in
none of the Greek states was any recognition ever given under
provision of law, to the right of authors to any control over their own
productions.”[18] Breulier writes: “Literary property, in any sense in
which the term is understood to-day, did not exist at Athens.”[19]
Wilhelm Schmitz concludes that “no such relation as that which to-
day exists between authors and booksellers (publishers) was known
among the Greeks. In none of the writings of the time, do we find the
slightest reference to any such publishing arrangements as Roman
authors in the time of Martial were accustomed to secure.”[20] This
treatise of Schmitz’s is a painstaking and interesting study of the
conditions of Greek literature in classic times and of the relations of
Greek writers to their public, and for certain portions of this chapter I
am largely indebted to the results of his investigations.
 Géraud remarks that in the first development of written language
and literature among the Hebrews and Egyptians, it is easy to
recognize the “fatal influence of the spirit of priestly caste, an
influence from which the Greek peoples were comparatively free.”[21]
The richest literature of antiquity, he goes on to say, is that of
Greece, and it was also in Greece that the art of writing made the
most rapid advances. The teaching of the priests, whether given
through the oracles or not, was purely oral, so that the Greeks did
not come into possession of any body of sacred scriptures such as
formed the original literature of other peoples. On the other hand, the
ardent nature, inquiring and active intellect, and brilliant imagination
of the Greeks, gave an early and rapid development to the arts, to
poetry, and to speculative philosophy.
The old-time tradition credits the introduction of the alphabet in
Greece to Cadmus, and fixes the date of the first Hellenic spelling-
school at about the fifteenth century before Christ. I believe the
authorities are divided as to whether this mythical Cadmus
represents a Phœnician or an Egyptian influence, but this is a
question which need not be considered here. I understand the
philologists are in accord in the conclusion that the Cadmus story
represents, not a first instituting of a Greek alphabet, but merely
certain important modifications in the form of letters already in use.
Birt asserts, as if it were now a settled fact, that while the Greeks
derived their written characters from the Phœnicians, they were
indebted to Egypt for their first ideas in the making of books. There is
a very distinct family resemblance between the Greek characters as
known in literature and those of the Hebrew, Phœnician, and Syriac
alphabets, while the names of the Greek letters Alpha and Beta are
found in all the Semitic dialects. It seems further to be certain that
the earlier peoples of Greece, after for a time having written
perpendicularly according to the fashion of the Chinese, began later
to write from right to left according to the Oriental manner.
The so-called Boustrophedon, a term meaning “turning like oxen
when they plough,” was a method of writing from left to right, and
from right to left in alternate lines. Among the earlier specimens of
this method were the laws of Solon (about 610 b.c.) and the Sigean
inscription (about 600 b.c.). This system represents a period of
transition between the earliest style and that of which the invention is
credited to Pronapides, and is simply the modern European fashion
of writing from left to right. The inscriptions of the Etruscans are
largely written in Boustrophedon. Neither in Greece, however, nor
elsewhere, did this method remain in use for any writings which are
to be classed as literature.
While Greek literature, as far as known to us, must be considered
as beginning with the Homeric poems, the date of which is estimated
by the majority of the authorities at about 900 b.c., there appears to
be no trustworthy example of Greek writing earlier than about 600
b.c. Curiously enough, this specimen was found not in Greece but in
Egypt. Jevons describes it as follows:
“On the banks of the Upper Nile, in the temple of Abu
Simbel, are huge statues of stone, and on the legs of the
second colossus from the south are chipped the names,
witticisms, and records of travellers of all ages, in
alphabets known and unknown. The earliest of the Greek
travellers who have thus left their names were a body of
mercenaries, who seemed to have formed part of an
expedition which was led up the Nile by King
Psammitichus.”[22]
Jevons goes on to give the grounds for the conclusion (based
mainly on the formation of certain of the letters, and in part, of
course, on the references to King Psammitichus) that the inscription
was written, or rather was cut, upon the statue between 620 b.c. and
600 b.c., according as we take the king mentioned to have been the
first or second of his name.[23] We have, then, a date fixing a time at
which the art of writing certainly existed among the Greeks, while it is
further evident that if in the year 600 the art of writing was so well
established that it was understood by a number of mercenaries, it
must have been quite generally diffused through certain classes of
society, and the date for its introduction into Greece must have been
considerably earlier than 600. Jevons knows, however, of no