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A Pharmacology Primer, 6e
Terry Kenakin, Author
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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

As always . for Debbie.

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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.
Abscissae: fractional receptor occupancy by human calcitonin. Curves
response curve slope, to transient and phasic activity [5] shown for receptors transfected into three cell types: HEK cells, CHO
(Fig. 2.12). cells, and Xenopus laevis melanophores. It can be seen that the different
cell types lead to differing amplification factors for the conversion from
agonist receptor occupancy to tissue response. CHO, Chinese hamster
2.5 System effects on agonist response: ovary; HEK, human embryonic kidney.
full and partial agonists
For any given receptor type, different cellular hosts should for amplification of receptor stimuli. This is illustrated by
have characteristic efficiencies of coupling, and these the strikingly different magnitudes of the receptor reserves
should characterize all agonists for that same receptor for calcitonin and histamine receptors shown in Figs. 2.2
irrespective of the magnitude of the efficacy of the agonists. and 2.3. Fig. 2.13 shows the response produced by human
Different cellular backgrounds have different capabilities calcitonin activation of the human calcitonin receptor type
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the structural, condition of the optic nerve. That such an influence
may be exerted is shown by cases of transverse myelitis low down in
the cord, which, according to Erb and Seguin, were complicated by
double optic-nerve atrophy. The second theory is that the involved
part of the cord and the optic nerve present a similar vulnerability to
the same morbid influences. This is illustrated in some cases of
chronic alcoholic and nicotine poisoning, in ergotism, and in the
spinal affections due to hereditary influences and developmental
defects.

To discuss the nature of the disturbing influence which is responsible

for the most characteristic evidence of the disease, the ataxia, would
be equivalent to reviewing almost every mooted question in spinal
physiology. It is to be borne in mind that ataxia is a collective term
designating any inco-ordination of movement which is independent
of motor paralysis. It may be due to abolition or impairment of tactile
perception; it may be due to loss of the muscular sense; it may be
due to hampered motor co-ordination; and, finally, it may be due to a
disturbance of the space-sense. In my opinion it is only in
exceptional cases that any one of these factors can be positively
excluded. Occasionally, one has been noted when the ataxia was
grave but the tactile sense was unimpaired, or where the muscular
sense was perfect but ataxia was well developed. The difficulty with
most such records is that no discrimination is made as to the kind of
ataxia present. That loss of skill which the patient shows when he
shuts his eyes and attempts to perform certain movements without
their aid is undoubtedly due to diminished sensation, either tactile or
muscular, and usually both. The inability to stand with the eyes
closed is probably a cerebellar phenomenon, and in this respect we
are on the way to return to Duchenne's opinion. It is true that the
cerebellar organ is healthy in most tabic subjects, but its centripetal
informer, the direct cerebellar tract, is either itself involved or affected
in its origin in the columns of Clarke. But, besides the static ataxia
and that motor ataxia which can be neutralized by the use of the eye,
there is another disturbance, which, as Erb and his followers hold,
cannot be accounted for on the strength of any sensory disturbance.
It consists in an interference with the proper succession and rhythm
of movement. It seems as if that automatic mechanism by which the
individual or grouped muscular contractions engaged in locomotion
follow each other with the smoothness of the action of perfectly-
fitting cog-wheels were disturbed; the correct after-movement is
hesitated over or skipped, or even takes place at the wrong moment,
neutralizing some other step in the co-ordination required. The
tendency of physiologists and pathologists is to attribute this form of
ataxia to the disease of the intrinsic co-ordinating apparatus of the
cord itself. The experiments of Tarchanoff on a headless duck, and
the determination of the existence of cursorial co-ordinating tracts
uniting the brachial and lumbar nuclei in mammals, as well as the
observations made on automatic co-ordinate movement in
decapitated criminals, demonstrate the existence in the cord of such
an apparatus. The combination of the ganglionic centres which
underlies this co-ordination is affected by the so-called short tracts of
the cord,48 and it is precisely a portion of these which are involved in
the lesion of the column of Burdach. A number of arguments have
been advanced against regarding the lesion of this column, or
indeed any of the lesions of the posterior column, as explaining the
ataxia-producing effect of tabes. Westphal has interposed some
potent objections. He holds that lesion of these columns will be
found more frequently when examinations shall no longer be limited
to those cases where disease is suspected because ataxia was
observed during life. He found extensive disease of the posterior
columns in sufferers from paretic dementia who did not exhibit the
characteristic ataxic gait of tabes. I believe this objection can be met
by the very cases cited by Westphal in its support. Where the spinal
disorder preceded the cerebral—that is, where paretic dementia
occurred as a complication of tabes dorsalis—true locomotor and
static ataxia had been present before the insanity exploded. On the
other hand, where the spinal disease followed the cerebral, typical
ataxia did not ensue. This would seem to indicate that the
destruction of cortical control is inimical to the development of typical
tabes. Leyden has made a suggestion in the same direction when he
attributes the lesser manifestness of locomotor ataxia in tabic
females to their inferior cerebral organization.49 A more convincing
proof of the correctness of this conclusion is furnished by the fact
that if the pathological process, after destroying the posterior
columns and producing ataxia, invades the voluntary motor tract, the
ataxic symptom becomes less palpable.50 This antagonism between
lateral-column and posterior-column lesion is frequently exemplified
in the combined forms of sclerosis. It would seem, then, that where
the brain is healthy and the controlling voluntary tracts are
unimpaired, the ataxia is aggravated, supporting the beautiful theory
of Adamkiewicz, which assumes that the locomotor ataxia is due to a
disturbance of the balance normally existing between the psycho-
motor centres and those controlling the muscular tone as well as
those mediating reflex excitability.51
48 Intersegmental tracts.

49 In one out of three female eases I found the active disturbance of gait as severe as
in males, but Leyden's observation is supported by all who have seen a sufficiently
large number of female cases.

50 Not because of paresis altogether, for it diminishes materially out of proportion to

the paresis.

51 Archiv für Psychiatrie, x. p. 545. There is another observation which bears in this
direction: James of Boston observed that absolute deaf-mutes in a large percentage
of cases are insusceptible to vertigo or to the allied phenomenon of sea-sickness.
Certainly, the auditory nerve is a space-sense nerve; its physiological elimination is,
however, accompanied by an immunity against a symptom which may be an evidence
of disturbed space-sense transmission. In like manner, the destruction of the central
perceptive and voluntary centres in the paretic dement inhibits the legitimate results of
posterior spinal sclerosis.

The degeneration of the crossed-pyramid tracts in typical tabes seem to be strictly an

atrophy from disuse, perhaps facilitated by the general malnutrition of the cord. It is
limited to that part supplying the most or solely affected extremities. Thus, where the
lower extremities are alone grossly involved it is totally degenerated in the lumbar
area, and only in its outer parts in higher levels. As if to fortify this comparison by
analogous observations from every great segment of the nervous axis, a similar
inhibiting influence of pyramid lesion on co-ordinating disturbance (muscular sense) is
noted in secondary degeneration of the interolivary layer; when uncomplicated with
 pyramid lesion (Meyer and my own case,) ataxia is present; when so complicated
(Schrader, Homén) it is not observed, even if determinable.

Lissauer52 has recently determined the existence of a degeneration

of certain fine nerve-fibres, apparently derived from the outermost of
the radicles into which the posterior nerve-roots divide on entry. They
are situated on that border of the apex of the posterior horn which is
in contact with the lateral column, and were found degenerated in all
cases except such as were in the initial period. No symptomatic
relation has been claimed for this lesion.
52 Neurologisches Centralblatt, 1885, No. 11.

One of the most important questions which have grown out of the
pathological studies of tabes is the relationship between the lesions
and the not infrequently observed restoration of functions which had
been more or less seriously impaired in an earlier period of the
disease. Even those symptoms which ordinarily comprise the
continuous and essential clinical background of tabes may exhibit
remarkable changes in this direction. I have two well-established
observations—one of tabes of eight years' standing, the other of
more recent date—in which that symptom which, once established,
is the most constant, the reflex iridoplegia, disappeared, to reappear
in two months in one case where it had been associated with
myosis, and to reappear in eight months in the other, repeating this
oscillation the following year. I have now under observation a tabic
patient in the sixth year of his illness who two years ago had a return
of both knee-phenomena to a nearly normal extent, to lose them in
two months, and to regain the reflex on the left side four months ago,
retaining it up to the present. These three cases were of syphilitic
subjects. In a fourth advanced non-syphilitic tabic patient, whose
ataxia had reached a maximal degree, I found a return of both knee-
phenomena for three days after its absence had been established by
medical examiners for over a year, and had probably been a feature
for a much longer period. Hammond the younger and Eulenburg
have reported similar cases. Nothing is more surprising to those
unfamiliar with the progress of this disease than to find gross ataxia
or the electrical pains and anæsthesia to disappear or nearly so; and
the alleged success of more than one remedial measure is based on
the fallacious attributing to the remedy what was really due to the
natural remittence of the disease-process or of its manifestations.
The financial success of quacks and the temporary but rapidly
evanescent popularity of static electricity, Wilsonia belts, and like
contrivances are owing to the hopefulness inspired in the credulous
patient by the mere coincidence of spontaneous improvement and
the administration of a new remedy, supplemented, it may be, by the
influence of mind on body in his sanguine condition. It is to be
assumed that the influences which are at work in provoking the
trophic and visceral episodes of tabes are of an impalpable
character, and that all theorizing regarding the reason of their
preponderance in one and their absence in another case are as
premature as would be any speculation regarding their rapid
development and subsidence in the history of one and the same
case. But we have better grounds for explaining the remissions of
the ataxia and anæsthesia.

It is only in the most advanced stages of tabes that the destruction of

the axis-cylinder becomes absolute or nearly so. Contrary to the
opinion of Leyden,53 who held that the tabic sclerosis differs from
disseminated sclerosis in the fact that the axis-cylinder does not
survive the myelin disappearance, it is now generally admitted that a
certain number of exposed or practically denuded axis-cylinders may
be preserved in the sclerotic fields.54 It is on the theory that these
delicate channels may be oppressed at one time, perhaps by
inflammatory or congestive pressure, and relieved at another by its
subsidence, that we may assume them to be the channels through
which the now limited, now liberated, functions are mediated. It is
also reasonable to suppose that vicarious action may supplement
the impaired function, and to some extent overcome the disturbing
factors. This is illustrated by the controlling influence of the visual
function—yea, even of the unconscious and ineffectual co-operation
of completely amaurotic eyes—in neutralizing both locomotor and
static ataxia. One patient who was well advanced in the initial period
of tabes, and who had been encouraged to consider the medical
opinion to that effect as the result of an exaggerated refinement of
diagnosis, made repeated tests of the Romberg symptom in his own
case, and deluded himself into the belief that the physician was
mistaken because he succeeded in practically overcoming it with an
effort that too plainly told its own story; but still he overcame it.
Certain peripheral influences have the power of stimulating the
dormant activity of potentially vicarious tracts, and perhaps also the
blunted activity of those whose function is impaired. The outside
temperature, certain barometric conditions, all may exert an
influence in this direction for good or evil.
53 Op. cit., p. 328, vol. ii.

54 Babinski (Neurologisches Centralblatt, 1885, p. 324) notes this feature, and,

consistently with the findings of most modern observers, discovers much more
resemblance to disseminated sclerosis than to the systemic sclerosis with which
Strümpell and Westphal (in part) incline to classify tabes. Similar objections to the
system-disease theory are advanced by Zacher (Archiv für Psychiatrie, xv. p. 340). I
may not pass over in silence the fact that Babinski considers his observations to
militate also against regarding any phase of the tabic sclerosis as a secondary
process. But while it may fairly be asked that a sclerosis to be regarded as systemic
must be shown to be total, this is not necessary for a secondary process, unless the
primary involvement be total also; and that is not the case in tabes.

ETIOLOGY.—Authorities are now agreed that no single cause can be

regarded as the sole responsible factor in all cases of tabes, and that
a number of etiological influences are combined in the provocation of
this disease in most instances. When the distinctiveness of the
affection was first recognized it was customary to attribute it to
sexual excesses, and the unfortunate sufferer had frequently to bear
the implied reproach of having brought his misery on himself, in
addition to the hopeless prospect which those who followed
Romberg and other authorities of the day held out to him.55
55 This opinion survives in a large portion of the German laity and in French novels.
About the time that the poet Heine was dying from an organic spinal affection two
other prominent literary characters of Paris were affected with tabes. It so happened
that all three were popularly regarded as libidinous, and one of their leading
 contemporaries, whose name escapes me, took occasion to issue a manifesto
addressed to the jeunesse dorée which closed with the apostrophe, “Gardons à nos
moelles.”

Heredity plays a very slight part in the etiology of tabes. Writers of

ten and fifteen years ago attributed a greater importance to it than is
now done. But this was due to the incorporation with tabes of the so-
called family form of locomotor ataxia—a disease which is now
regarded as a distinct affection.56
56 There is but one record of direct heredity (the father and son being affected nearly
at the same time), to my knowledge. It was observed at the Berlin Hospital by Remak
(Berliner klinische Wochenschrift, 1885, No. 7). Both father and son were syphilitic.

More importance may be attached to individual predisposition, but

thus far no distinct formulation of this factor has been attempted
except by Schmeichler,57 who offers the suggestion that there are
persons with a predisposition to the development of connective-
tissue proliferation in various organs of the body, and that in them
tabes and other sclerotic affections are consequently more frequent
than in others. This suggestion appears plausible, but it is
unconfirmed by positive observations.
57 Op. cit.

Sex appears on a superficial view to be one of the most important

elements. It is generally admitted that at most one female becomes
tabic for every ten males who do so. Of 81 cases in private practice,
I observed but 3 females. Rockwell, Seguin, Birdsall, and Putnam
give similar figures. This comparative immunity is probably due to
the fact that the female is less exposed to over-exertion, to surface
chilling of the feet, to the injurious consequences of sexual excess,
and to syphilis58 than the male. As a rule, the affection in females is
more insidiously developed, progresses more slowly, is less marked
by crises and trophic disturbances, and not accompanied by as
severe pains and profound disturbance of co-ordination as is the
corresponding affection in males.
 58 Whether the shorter vitality of the syphilitic female as compared with that of the
male is a factor in diminishing the accumulation of chronic tertiary sequelæ in that
sex, or whether it be the lesser vulnerability of the inferior nervous system, I am
unable to decide from the facts at my disposal. In private and clinical experience I
have been struck by the fact that women affected with syphilis in the same way and
under similar circumstances with tabic syphilitic males develop symptoms of
functional disorder of the brain and cord, such as spinal and cerebro-spinal irritation.
My cases referred to had in no instance any indication of a syphilitic condition or
history, and a distinct and different cause was found in all three.

The most important element in creating an acquired predisposition to

tabes is undoubtedly the existence of constitutional syphilis. Some
difference of opinion still exists regarding the proportion of syphilitic
tabic patients, chiefly due to the neglect of Erb—when he first
announced the prevailing view, and which is generally attributed to
him—to differentiate between cases of demonstrated constitutional
syphilis and the so-called spurious or soft chancre. But although
there occurred a reaction against his view which went to as great an
extreme in the opposite direction, the careful and critically registered
statistics accumulated in the mean time strengthen the view that
there are more syphilitic subjects among the tabic than among any
class of sufferers from other nervous affections.59 Reumont, a
physician at Aix-la-Chapelle, to which place syphilitic patients in
general resort in large numbers, found that of 3400 cases of syphilis,
290 had nervous affections, 40 being afflicted with tabes. Bernhardt60
took occasion to examine a group of hospital patients who were free
from tabes, and found that not fully 16 per cent. were syphilitic, while
of 125 tabic patients, over 46 per cent. were determined to have had
positive syphilitic manifestations. Several of those observers who
have paid attention to the question of the syphilitic origin of tabes
have admitted that the more searching their inquiry the larger the
proportion of detected syphilitic antecedent histories. Thus, Rumpf's
earlier table shows 66, and his later 80, per cent. of such
antecedents. This latter figure exactly corresponds to the percentage
of syphilis in my private cases. At a discussion held by members of
the American Neurological Association in 1884, Webber gave 54,
Putnam 49, Rockwell 40, Birdsall 43,61 and Seguin 22 per cent.62 as
the proportion in their experiences.
59 Excepting always those having the distinctive and undisputed syphilitic character.

60 Archiv für Psychiatrie, xv. p. 862.

61 Derived from over five hundred cases which had presented themselves at the clinic
of the College of Physicians and Surgeons.

62 In the Archives of Medicine he tabulates 54 (private) cases as follows:

Chancre alone 23
Chancre followed by secondary symptoms 16
Total of those with history of chancre 39
No history of chancre in 15
Total 54

Of European writers, aside from those already mentioned, Berger

claims 43 per cent., and Bernhardt, in commenting on the increasing
percentage obtained by accurate investigation, reports an additional
series of 7 new cases in private practice, all of which were syphilitic.
Fournier, Voigt, Œhnhausen, and George Fisher estimate the
syphilitic tabic patients at respectively 93, 81, and 72 per cent. of the
whole number. The almost monotonous recurrence of a clear
syphilitic history in my more recent records is such that in private
practice I have come to regard a non-syphilitic tabic patient as the
exception. Among the poorer classes the percentage of discoverable
syphilitic antecedents is undoubtedly much less. The direct exciting
causes of tabes, exposure and over-exertion, are more common with
them and more severe in their operation.

The proof of a relationship between syphilis and tabes dorsalis does

not rest on statistical evidence alone. A number of observations
show that the syphilitic virus is competent to produce individual
symptoms which demonstrate its profound influence on the very
centres and tracts which are affected in tabes. Thus, Finger63
showed that obliteration of the knee-jerk is a frequent symptom of
the secondary fever of syphilis, and that the relation is so intimate
between cause and effect that after the return of the reflex, if there
be a relapse of the fever, the obliteration of the knee-jerk is repeated.
Both the permanent loss of the knee-jerk (Remak) and the peculiar
pupillary symptoms of tabes are sometimes found in syphilitic
subjects who have no other sign of nervous disorder; and Rieger and
Foster64 regard the syphilitic ocular disturbances, even when they
exist independently, as due, like those of tabes, to the spinal, and not
to a primarily cerebral, disturbance. Another argument in favor of the
syphilitic origin of tabes is derived from the occasional remedial
influence of antisyphilitic treatment. The force of this argument is
somewhat impaired by the fact that the same measures occasionally
appear to be beneficial in tabes where syphilis can be excluded. Still,
the results of the mixed treatment in a few cases of undoubted
syphilitic origin are sometimes unmistakable and brilliant.65 As some
cases, even of long standing, yield to such measures, while others,
apparently of lesser gravity and briefer duration, fail to respond to
them, the question as to whether syphilis is a direct cause or merely
a predisposing factor may be answered in this way: That in the
former class it must have been more or less directly instrumental in
provoking the disease, while in the latter class it is to be regarded as
a remote and predisposing factor, to which other causes, not
reached by antisyphilitic treatment, became added. The claim of Erb,
that “tabes dorsalis is probably a syphilitic disease whose outbreak is
determined by certain accessory provocations,” is not subscribed to
unreservedly by a single writer of eminence.
63 “Ueber eine constante nervöse Störung bei florider Syphilis der Secundärperiode,”
Vierteljahrschrift für Dermatologie und Syphilis, viii., 1882.

64 “Auge und Rückenmark,” Graefe's Archiv für Ophthalmologie, Bd. xxvii. iii.

65 In one case already referred to a return of both knee-phenomena and complete

disappearance of locomotor and static ataxia were effected after a duration of four
years. The treatment was neglected and the knee-jerks disappeared, and one has
now returned under the resumed treatment, but accompanied by lightning-like pains.
 At a meeting of the Société médicale des Hôpitaux, held November 10, 1882,
Desplats reported a case in which even better results were obtained. Reumont
(Syphilis und Tabes nach eigenen Erfahrungen, Aachen, 1881) reports 2 out of 36
carefully observed syphilitic cases cured, and 13 as improved under antisyphilitic
treatment.

The question has been raised whether the influence of syphilis is

sufficiently great to justify a clinical demarcation between syphilitic
and non-syphilitic cases. A number of observers, including Reumont,
Leonard Weber, and Fournier, incline to the belief that there are
more atypical forms of tabes in the syphilitic group. Others, including
Rumpf, Krause, and Berger, are unable to confirm this, but the
former admits, what seems to be a general impression among
neurologists, that an early preponderance of ptosis, diplopia, and
pupillary symptoms is more common with syphilitic than with non-
syphilitic tabes. Fournier66 believes that syphilitic patients show more
mental involvement in the pre-ataxic period; but it is evident that he
has based this belief on a study of impure forms. The advent of
tabes in syphilitic cases does not in this respect differ from the rule.
The most protracted and severe diplopia I have yet encountered in a
tabic patient is one, now under observation, in the initial period of the
disease, syphilis being positively excluded as an etiological factor.
66 L'Éncephale, 1884, No. 6.

It seems to be a prevalent opinion that the cases of syphilis in which

tabes is developed include a large proportion of instances in which
the secondary manifestations were slight and unlike that florid
syphilis with well-marked cutaneous and visceral lesions which is
more apt to be followed by transitory or severe vascular affections of
the cord and brain.

Excesses in alcohol, tobacco, and abuse of the sexual function are

among the factors which frequently aggravate the tendency to tabes,
and one or more of them will usually be found associated with the
constitutional factor in syphilitic tabes. Both alcohol and nicotine
have a deleterious effect on nervous nutrition and on the spinal
functions, as is illustrated in the effect of the former in producing
general neuritis, and of both in provoking optic-nerve atrophy and
general paralysis of the insane, not to speak of the pupillary states
which often follow their abuse, and the undeniable existence of a
true alcoholic ataxia. Sexual excesses were, as stated, at one time
regarded as the chief cause: the reaction that set in against this
belief went to the extreme of questioning its influence altogether. It is
to-day regarded as an important aggravating cause in a large
number of cases, and this irrespective of whether it be the result of a
satyriacal irritation of the initial period or a precedent factor. In a
large number of my patients (18 out of 23 in whom this subject was
inquired into) the habit of withdrawing had been indulged in,67 and,
as the patients admitted, with distinct deleterious effects, such as
fulness and throbbing in the lumbo-sacral region, tremor and rigidity,
with tingling or numbness, in the limbs, blurred vision, and
sometimes severe occipital headache; in one case lightning-like
pains in the region of the anus ensued.68
67 Coitus reservatus, the real crime of the Onan of Scripture.

68 Leyden states that coitus in the upright position has been accused of producing
tabes, without mentioning his authority. I have no observation on this subject touching
tabes, but am prepared to credit its bad effect from the account of a masturbator, who
during the orgasm produced while standing felt a distinct shock, like that from a
battery, shooting from the lumbar region into his lower limbs, and causing him to fall
as if knocked down. He consulted me in great alarm—was scarcely able to walk from
motor weakness, and had no knee-phenomenon; in a few weeks it returned, and no
further morbid sign appeared. Masturbators of the worst type occasionally manifest
ataxia, and in three cases I have been able to establish the return of the knee-jerk,
together with other improvements in the spinal exhaustion of these subjects. The loss
and diminution of the patellar jerk, and the frequently associated urinary incontinence,
as well as certain of the peripheral pains found in masturbators, certainly prove that
undue repetition of the sexual act (be it natural or artificial) is competent to affect the
cord in a way that cannot but be injurious in case of a predisposition to tabes, if not
without the latter.

Of single causes, none exerts so direct and indisputable an influence

on the production of tabes as the action of cold and wet upon the
lower segment of the body. It is usually the case that such exposure
is frequently repeated and combined with over-exertion before the
disease is produced, but it is occasionally possible to trace the very
first symptom of the disease directly to a single exposure. A soldier
who stands up to his knees in a rifle-pit half full of water finds his
limbs numb or tingling; develops slight motor weakness, then
lightning-like pains, and ultimately a typical tabes. In the case of a
peddler who presented an advanced form of the disease, the first
symptoms had developed after a single wetting of his feet: while
walking along one of our watering-places with his wares the swell of
a steamer inundated the beach. He had been subject to perspiring
feet before that, and the perspiration remained checked from that
time on.69 The influence of surface chilling was remarkably manifest
in all three of my female cases. In one of them it was due to frequent
wetting of the feet; in the second, a midwife, the first symptoms
began immediately after standing on a cold hearthstone while
preparing some article needed in a lying-in case. In the third case, a
lady who contracted and safely passed through a scarlatina in her
twenty-eighth year was taken out driving while desquamation was
going on. She became thoroughly chilled, experienced numbness in
the fingers and toes, and from that day on developed a slowly
progressing tabes involving all extremities alike.70
69 Checking of habitual perspiration by violent measures is mentioned by the German
textbook writers as a frequent cause, but occurs quite rarely in the modern tables.

70 In view of the absence of spinal—or, in fact, any nervous—symptoms prior to the

exposure referred to, it does not seem necessary to insist that this was not an
instance of a true post-scarlatinal tabes; and possibly the case thus designated by
Tuczek (Archiv für Psychiatrie, xiii. p. 147) may have been really due to chilling of the
delicate body-surface after desquamation or during that process. The typical form of
myelitis and sclerosis after exanthematous fevers is rather of the disseminated type.

Spinal concussion has been mentioned by a number of authorities

as a possible cause for tabes, as for other forms of sclerotic spinal
disease. In 1 of 81 cases in my own observation the development of
the disorder could be distinctly traced to a railway injury; in 2 a
sudden aggravation was as distinctly referable to a similar cause.71
To what extent railroad travelling, with its attendant continual jarring
of the body, may predispose to the development of tabes or of other
spinal diseases is as yet a matter of mere conjecture. That railroad
travelling exerts a bad influence in some cases of the established
disease is evident; but in others the patients rather like the motion,
and claim to feel benefited by it.
71 A fall from a chair, striking on the back of the latter, while endeavoring to keep a
row of books from coming down in one case, and the shock of the Ashtabula disaster
in the other. The latter patient, the same one who is referred to as describing the
electric-storm sensation in an earlier part of this article, had his foot amputated in
consequence of that disaster; but, like one of the characters in Jacob Faithful, who felt
his toes when the weather changed, though he left both legs at Aboukir, he felt the
terrific pains of the disease in the absent foot as distinctly as in the other. Dumenil and
Petit (Archives de Névrologie, ix. Nos. 25 and 26) relate cases in which a spinal
concussion was the only ascertainable cause.

A number of toxic agents have been charged with producing tabes:

thus, Bourdon maintains this of absinthe; Oppenheim attributes one
case to poisoning by illuminating gas, the exposure to its influence
being immediately followed by a gastric crisis, and this by a
regulation tabes.72 It is supposed that most of the poisons acting on
the cord in this or a similar way, such as arsenic, cyanogen,73
barium, and chloral,74 do not produce a spinal lesion directly, but
through the medium of a secondary cachexia. Of no agent is the
effect in producing tabes so well studied as ergot of rye. It had long
been known that ergot-poisoning provoked certain co-ordinating,
motor, and sensory disturbances, but it was left for Tuczek75 to show
that this vegetable parasite produces a lesion of the spinal cord
which in its character and distribution apes typical posterior sclerosis
so closely as to justify the designation of a tabes ergotica. Possibly,
pellagra, which is sometimes manifested in a similar way,76 may yet
be shown to have a like influence.
72 Archiv für Psychiatrie, xv. p. 861.

73 Bunge, Archiv für experimentelle Pathologie, xii.

 74 Transactions of the Clinical Society of London, xiii. p. 117, 1880.

75 Archiv für Psychiatrie, xiii. p. 148.

76 Bouchard, “Étude d'Anatomie pathologique sur un Cas de Péllagrie,” Gaz. méd. de

Paris, 1864, No. 39.

Among the occasional and exceptional causes of tabes, Leyden and

Jolly mention the puerperal state; Bouchut, diphtheria; and several
instances are recorded in which psychical shock was responsible for
the outbreak of the disease. In a small number of cases I found that
mental worry and anxiety coincided with the period of presumable
origin of the disease.

Age seems to have no special determining influence. It is true that

most sufferers from this disease are men in the prime of life or in the
period following it. But it is precisely at these periods that the
exposure to the recognized causes of tabes is greatest. It seems as
if there were very little liability to the development of tabes after the
fiftieth and before the twenty-fifth year; still, some cases of infantile
tabes have been recorded.77
77 Excluding the so-called family form of locomotor ataxia: 6 rather imperfectly
described cases are cited by Remak (loc. cit.), and 3 additional ones related by
himself. Of the latter, 2 had hereditary syphilis, and of 1 the father was both syphilitic
and tabic.

In the majority of cases tabes is due to a combination of a number of

the above-mentioned factors. The majority of tabic patients in the
middle and wealthy classes have had syphilis, and of these, in turn,
the majority have been guilty of sexual excesses or perverted sexual
acts, while excesses in tobacco and of alcohol are often superadded.
Among the poorer patients we find syphilis less frequently a factor,
but still present, according to various estimates, in from 20 to 60 per
cent. of the cases. Excesses in tobacco play a lesser, and excesses
in alcohol a larger, part in the supplemental etiology than in the other
class, while exposure to wet and cold and over-exertion are noted in
the majority; indeed, in a fair proportion they are the only assignable
causes.

DIAGNOSIS.—The recognition of advanced tabes dorsalis is one of the

easiest problems of neurological differentiation. The single symptom
which has given one of its names to the disease—locomotor ataxia
—is so manifest in the gait that even the sufferers from the affection
learn to recognize the disease in their fellow-sufferers by the peculiar
walk.78
78 At present I have six tabic patients under treatment, who are acquainted with each
other, and who have made each other's acquaintance in the singular way of
addressing one another on the strength of mutual suffering at Saratoga, at the Hot
Springs of Arkansas, and in New York City.

Although there are other chronic affections of the cord which

manifest ataxia, such as myelitis predominating in the posterior
columns, disseminated sclerosis in a similar distribution, and some
partially recovered cases of acute myelitis, the gait is not exactly like
that of tabes. The uncertainty may be as great, but the peculiarly
stamping and throwing motions are rarely present in these
affections. The clinical picture presented by the ataxic patient, aside
from his gait, is equally characteristic in advanced cases. Absence of
the knee-jerk and other deep reflexes, the bladder paralysis, sensory
disturbance, delayed pain-conduction, trophic disturbances, and
reflex iridoplegia are found in the same combination in no other
chronic disorder of the cord. It is supposable that an imperfect
transverse myelitis in the lumbar part of the cord might produce the
reflex, ataxic, sensory, sexual, and vesical symptoms of ataxia, but
the brachial symptoms found in typical tabes as well as gastric crises
would be absent. The pupillary symptoms would also fail to be
developed, in all probability. It is to be remembered that only
fascicular cord affections can produce a clinical picture exactly like
that of tabes in more than one important respect. In analyzing the
individual symptoms of the early stage the more important differential
features can be most practically surveyed.
The discovery of no single symptom of tabes dorsalis marks so
important an epoch in its study as Westphal's observation that the
knee-phenomenon is usually destroyed in it. Had this symptom not
been detected, so Tuczek admits, ergotin tabes would have eluded
recognition.79 It was claimed by a majority of neurologists at first that
this jerk is always abolished in tabes, but it is now recognized that
there are exceptions, as is shown by cases of Hirt,80 Westphal, and
others, not to mention some well-established cases of its return
during the progress of the disease.
79 It is not to be wondered that, like most new discoveries, that of the pathological
changes of the patellar reflex should have been made the basis of premature
generalizations. The attempt of Shaw (Archives of Medicine) to establish a relation
between disturbances of the speech-faculty and an increased knee-jerk has not met
with any encouragement or confirmation, and has been rebutted by Bettencourt,
Rodrigues (L'Éncephale, 1885, 2), and others.

80 Berliner klinische Wochenschrift, 1886, 10.

The knee-phenomenon is supposed to be a constant attribute of

physiological man. It is difficult to elicit it in children, and frequently
impossible to obtain it in young infants. It also disappears in old age,
without having any special signification, except that this occurrence
seems to be in direct relation to senile involution. In 2403 boys
between the ages of six and thirteen years, Pelizæus81 found it
absent in one only. It is customarily elicited by having the patient
while sitting in a chair throw one leg over the other; hereupon the
ligamentum patellæ is struck a short, quick blow. Under physiological
circumstances the leg is jerked outward involuntarily after an interval
of about one-fifth of a second—one that is scarcely appreciated by
the eye. But if it be found absent by this mode of examination, the
case is not to be regarded as one of absence of the jerk without
further ado. The patient is made to sit on a table, his legs dangling
down and his body leaning back, while he clenches his fists. By this
means the jerk will often be produced where it appears to be
impossible to evolve it by the ordinary means. It is also well to try
different parts of the ligament, and when comparing both sides to
strike on the corresponding spot and in the same direction. Many
subjects who appear to be irresponsive will respond very well when
a point on the outer edge near the tibial insertion is percussed. The
elbow reflex, which has the same signification for the upper extremity
that the knee-jerk has for the lower, is elicited in the same manner.
81 Archiv für Psychiatrie, xv. p. 206.

The absence of the knee-jerk is usually regarded as a suspicious

circumstance in persons of middle life; and where it can be
demonstrated that it has been present years previously and
subsequently disappeared, it is looked upon as of grave import. I,
however, published three years ago an authentic case of
disappearance of the knee-jerk in a physician now in active practice
in New York City who to this day enjoys excellent health and has
developed no other sign of spinal disease. The knee-jerk is also
abolished in a number of conditions not belonging to the domain of
strictly spinal diseases, such as diphtheria, diabetes, secondary
syphilis, and severe cases of intermittent fever. Of these, diabetes
alone can be possibly confounded with tabes dorsalis. The difficulty
of differentiating early tabes and diabetes is enhanced by the fact
that on the one hand there are often ataxic symptoms with diabetes,
while on the other both glycosuria and diabetes insipidus may
complicate tabes. Senator, Frerichs, Rosenstein, Leval-Piquechef,
Charcot, Raymond, Demange, Féré, Bernard, and T. A. McBride all
recognize the occasional presence of the ataxic gait, paræsthesia,
belt sensation, and even fulgurating pains, besides the abolition of
the jerk, in diabetes mellitus.82 In pure cases of diabetes, however, I
am not aware that spinal myosis or the reflex paralytic pupil has
been found.
82 I have now under observation a case of myelitis with predominating sclerosis of the
posterior columns of five years' standing in a merchant who has been under
antidiabetic treatment for eleven years.

Abolition of the knee-jerk is found in all organic diseases of the

spinal cord which destroy any part of the neural arch at the upper
lumbar level, where the translation of the reflex occurs, whether it be
in the posterior root-zones or in the gray matter of the origin of the
crural nerves. Thus, acute or chronic myelitis, disseminated sclerotic
foci of this level, may cause obliteration of the reflex at any time of
the disease; so may acute or chronic anterior poliomyelitis,
neoplasms, and amyotrophic lateral sclerosis of the anterior cornua
type if the destruction of the anterior cornua be complete enough. It
is also found abolished with all diseases of the peripheral nerves—
traumatic and neuritic—which produce absolute motor paralysis of
such nerves.

Among the sources of error possibly incurred in examining for this

important symptom the presence of rheumatism is one. There is
sometimes a tetanic rigidity of the joints which prevents the reflex
from becoming manifest. It is also sometimes found to be absent
immediately after severe epileptic attacks, according to Moeli.83
83 In three examinations after severe attacks of epilepsy I found it normal.

The condition of the pupil is perhaps a more constant sign of early

tabes than the loss of the knee-jerk; at least it has been found well
marked in cases where the jerk had not yet disappeared. It may be
regarded as a rule in neuro-pathology that wherever reflex
iridoplegia is at any time accompanied by other oculo-motor
disturbance, it is either of spinal origin or in exceptional cases due to
disease of the pons varolii. The peculiar character of the pupillary
disturbance of tabes furnishes us with a criterion for distinguishing it
from one affection which in common with it exhibits loss of the knee-
jerk—diphtheria. In diphtheria there is also a reflex disturbance of the
pupil, but it is the reverse of that of early tabes. In the latter reaction
to light is lost, but the accommodative contraction power is retained;
in diphtheria accommodative contraction power is lost, but reaction
to light is retained.

The bladder disturbance has already been described. It is found as a

marked symptom so prominently in no other systemic affection of the
cord, and in few of the non-systemic forms, of sclerosis. In none of
these is it associated with absence of the patellar jerk, reflex
iridoplegia, and fulminating pains, as in tabes, except there be also
some motor paresis. It is the combination of any two of the important
initial symptoms of tabes without paralysis or atrophy that is
regarded as indicative of the disease by most authorities. Thus the
swaying in closing the eyes, if associated with the Argyll-Robertson
pupil, is considered as sufficient to justify the diagnosis of incipient
tabes, even if the knee-jerk be present and fulminating pains and
bladder trouble absent. Undoubtedly, the tabic symptoms must begin
somewhere. But at what point it is justifiable to give a man the
alarming information that he is tabic is a question. I have a number
of neurasthenic subjects now under treatment who have had reflex
iridoplegia for years; in one the knee-jerk is slowly becoming
extinguished; in two it has been becoming more marked after
becoming less; in all the three mentioned there is slight swaying in
closing the eyes and some difficulty in expelling the last drops of
urine while micturating. I do not believe that such a condition justifies
a positive opinion, although the surmise that they are on the road to
developing tabes may turn out correct for all these and for some of
those who have merely reflex iridoplegia.

Incipient tabes cannot be readily confounded with any other chronic

disease of the spinal cord. Some of the cases produced by sudden
refrigeration resemble a beginning myelitis. But the absence of true
paralysis seems to distinguish it from the latter. In all the cases of so-
called acute locomotor ataxia of myelitic origin that I can find a
record of, paralytic symptoms were marked, if not throughout the
disease, at least in the initial period.

Other forms of sclerosis occasionally limited to the posterior columns

imitate the symptoms of tabes. It is unusual, however, for such
sclerosis to be distributed through so great an extent of the posterior
columns as to produce symptoms consistent with tabes in both the
upper and lower extremities. And even where this condition is
complied with, the typical progress so characteristic of tabes is not
adhered to. As previously stated, the progress is weakened by
variations in certain symptoms. Such variations are found in other
forms of sclerosis, but they are not as great, trophic disturbances not
so common, and visceral crises not so violent, as a rule.