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A Pharmacology Primer
A Pharmacology Primer
Techniques for More Effective and Strategic
Drug Discovery

Fifth Edition

Terry P. Kenakin, PhD

Professor, Department of Pharmacology, University of North Carolina
School of Medicine, United States
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Notices
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understanding, changes in research methods, professional practices, or medical treatment may become necessary.
Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any
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 Dedication

As always . . . for Debbie


Science is an evolving process, so books on science should
routinely be expected to evolve from being “out of date”
to “needing to be updated.” From that standpoint alone, a
book such as “A Pharmacology Primer” should be brought
up to date every so often. But in this particular case, there
are unique reasons why “A Pharmacology Primer” should
be updated. This book describes the role of pharmacology
in drug discovery and also that pharmacology is a unique
discipline made up of an amalgam of biological and chem-
ical disciplines. Therefore, any advancement in any one of
those component disciplines impacts pharmacology and
emerges as its modification; the result is a constantly
changing landscape of possibilities. In this most recent
edition, the advances in structural protein chemistry and
increased understanding of cellular signaling has changed
the landscape of discovery and the way pharmacologists
approach research challenges. Specifically, the “null
method” approaches used in the past, necessary because
of the lack of knowledge of the cellular processes that
drugs modify, are giving way to detailed mechanistic
Preface
models of what actually is going on in pharmacological
systems on a molecular level. This makes comparison of
the verisimilitude of pharmacological data to models, in
the process of determining predictive parameters of drug
activity, a more facile process with the potential to attack
the presently most important reason for new drug candi-
date failure, namely, lack of therapeutic efficacy. There
are at least two reasons for the high (50%) rate of candi-
date failure; one may be with us for some time, namely,
the lack of understanding of exactly what it is we need to
do to correct faulty physiology in disease. However, we
may be able to impact the second reason by doing a better
job of determining the true pharmacological efficacies of
the molecules we advance to the clinic and to better trans-
late the clinical results with pharmacological profiles.
This text is part of the application of new pharmacological
data toward that endpoint.
Terry Kenakin
Research Triangle Park, 2018
xiii
 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 . . ..
—Claude Bernard (1813
78).

1.1 About This Book 1 1.7 System-Independent Drug 1.11 Dose
Response Curves 16
1.2 What is Pharmacology? 1 Parameters: Affinity and 1.12 Chapter Summary and
1.3 The Receptor Concept 3 Efficacy 10 Conclusions 19
1.4 Pharmacological Test Systems 4 1.8 What is Affinity? 13 1.13 Derivations: Conformational
1.5 The Nature of Drug Receptors 7 1.9 The Langmuir Adsorption Selection as a Mechanism of
1.6 Pharmacological Intervention and Isotherm 13 Efficacy 20
the Therapeutic Landscape 7 1.10 What is Efficacy? 15 References 21

1.1 ABOUT THIS BOOK
Essentially this is a book about the methods and tools
used in pharmacology to quantify drug activity. Receptor
pharmacology 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 understand-
ing of the mathematics 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. Understanding the derivation is not a
prerequisite for understanding the application of the meth-
ods 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 mathe-
matical equations is definitely not required for pharmacol-
ogy; 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., α 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.
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
fourth century to denote the term drug or medicine. There
are subdisciplines within pharmacology representing spe-
cialty areas. Pharmacokinetics 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

A Pharmacology Primer. DOI: https://doi.org/10.1016/B978-0-12-813957-8.00001-1

© 2019 Elsevier Inc. All rights reserved. 1
2 Chapter | 1 What is Pharmacology?
discussed specifically in Chapter 9, Pharmacokinetics, of
this book. Pharmaceutics is the study of the chemical for-
mulation of drugs to optimize absorption and distribution
within the body. Pharmacognosy is the study of plant nat-
ural products and their use in the treatment of disease. A
very important discipline in the drug-discovery process is
medicinal chemistry, the study of the production of mole-
cules for therapeutic use. This couples synthetic organic
chemistry with an understanding of how biological infor-
mation 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 discovery—see Chapter 8, The Optimal Design of
Pharmacological Experiments. As put by the great
Canadian physician Sir William Osler (1849
1919; the
“father of modern medicine”), “. . . the desire to take
medicine is perhaps the greatest feature which distin-
guishes man from animals . . ..” Pharmacology as a sepa-
rate science is approximately 120
140 years old. The
relationship between chemical structure and biological
activity began to be studied systematically in the 1860s
[1]. It began when physiologists, using chemicals to probe
physiological systems, became more interested in the
chemical probes than the systems they were probing. By
the early 1800s, physiologists were performing physiolog-
ical studies with chemicals that became pharmacological
studies more aimed at the definition of the biological
activity of chemicals. The first formalized chair of phar-
macology, 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 differentia-
tion 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 func-
tion, structure, and chemical properties of the body brought
about by chemical substances
—W. 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
pharmacodynamic interaction between the drug and the
biological recognition system for that drug. Therefore, a
prerequisite to all of pharmacology is an understanding of
the basic concepts of dose
response 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 biologi-
cal 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
dose
response data generically and demonstrates meth-
ods by which drug activity can be quantified across all
biological systems irrespective of the nature of the biolog-
ical 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 serves 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 test-
ing 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 con-
tained in pharmacology to convert drug behaviors in partic-
ular 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
Experiments (after the meaning of the particular para-
meters 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 individual needs). Since the arrival of the human
genome, receptor pharmacology as a science is more rele-
vant 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%
10% 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
1.3 THE RECEPTOR CONCEPT 3

medicinal chemistry and pharmacology has been eluci-
dated by Paton [2]:
For pharmacology there results a particularly close relationship
with chemistry, and the work may lead quite naturally, with no
special stress on practicality, to therapeutic application, or (in
the case of adverse reactions) to toxicology.
—W. 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
understanding and also in terms of the best method to prog-
ress forward. In the worst circumstance, it is a gap of credi-
bility 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.
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 sys-
tems. They also knew that very small changes in the
chemical composition of these substances could lead to
huge differences in activity. This led to the notion that
something on or in the cell must specifically read the
chemical information 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 (1854
1915,
Fig. 1.1A) proposed the existence of “chemoreceptors”
(actually he proposed a collection of amboreceptors, tri-
ceptors, and polyceptors) on cells for dyes. He also pos-
tulated 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 (1852
1926, Fig. 1.1B), during his studies with
the drugs jaborandi (which contains the alkaloid pilocar-
pine) and atropine, introduced the concept that receptors
were switches that received and generated signals and
that these switches could be activated or blocked by spe-
cific molecules. The originator of quantitative receptor
theory, the Edinburgh pharmacologist Alfred Joseph
Clark (1885
1941, Fig. 1.1C), was the first to suggest
that the data, compiled from his studies of the interac-
tions 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

(A) (B) (C)

FIGURE 1.1 Pioneers of pharmacology. (A) Paul Ehrlich (1854
1915). Born in Silesia, Ehrlich graduated
from Leipzig University to go on to a distinguished career as head of institutes in Berlin and Frankfurt. His stud-
ies with dyes and bacteria formed the basis of early ideas regarding recognition of biological substances by che-
micals. (B) John Newport Langley (1852
1926). 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 (1885
1941). 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 origi-
nator of modern receptor theory, Clark applied chemical laws to biological phenomena. His books on receptor
theory formed the basis of modern pharmacology.
4 Chapter | 1 What is Pharmacology?
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 explana-
tion will, however, explain a tithe of the facts observed.
—A. 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 scien-
tific atmosphere 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.”
Homeopathic theories like the Arndt
Schulz law and
Weber
Fechner 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 (1874
1944), suggested that a general theory of
chemical binding between drugs and cells utilizing recep-
tors was “. . . going too far . . . and . . . not admissible”
[6]. The impact of Clark’s thinking against these concepts
cannot be overemphasized to modern pharmacology.
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 β-adrenoceptor. When this is
Bronchial
muscle relaxation
Cardiac inotropy Vascular relaxation
Uterine muscle Salivary gland
relaxation
secretion
Melatonin
synthesis β-Adrenoceptors Cardiac lusitropy
Pancreatic Cardiac chronotropy
secretion
Lacrimal gland Skeletal muscle
secretion tremor
Decreased Urinary bladder
stomach motility
muscle relaxation
FIGURE 1.2 A sampling of the heterogeneous physiological and phar-
macological response to the hormone epinephrine. The concept of recep-
tors links these diverse effects to a single control point, namely, the
β-adrenoceptor.
understood, a much better idea can be gained as to how
to manipulate these heterogeneous responses for therapeu-
tic benefit; the receptor concept introduced order into
physiology 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. These receptors are named for their character-
istic structure that consists of a single protein chain that
traverses 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 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 pro-
duce effect. In the study of biological targets such as
7TMRs and other receptors, a “system” must be
employed that accepts chemical input and returns biologi-
cal 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 sur-
rogate species receptors for predicting human drug activity,
1.4 PHARMACOLOGICAL TEST SYSTEMS 5

Receptors

Enzymes Ion channels

Drug targets

Nuclear
receptors

DNA
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.

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 α2-adrenoceptors are 89% homologous
and thus considered very similar from the standpoint of
amino acid sequence. Furthermore, the affinities of the α2-
adrenoceptor antagonists atipamezole and yohimbine are
nearly indistinguishable (atipamezole human α2-
C10Ki 5 2.9 6 0.4 nM, mouse α2-4H Ki 5 1.6 6 0.2 nM;
yohimbine human α2-C10Ki 5 3.4 6 0.1 nM, mouse α2-4H
Ki 5 3.8 6 0.8 nM). However, there is a 20.9-fold differ-
ence for the antagonist prazosin (human α2-C10Ki 5 2034
6 350 nM, mouse α2-4H Ki 5 97.3 6 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 sensitiv-
ity 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 hypothesis is
immediately disproven by the first drug that shows differ-
ential 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 predictive of
activity on the target human receptor. This will always be a
drug-specific phenomenon.
The link between animal and human receptors is the
fact that both proteins recognize the endogenous transmit-
ter (e.g., acetylcholine, norepinephrine), and therefore the
hope 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
human receptors. These engineered (recombinant) sys-
tems 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 prob-
lem of differences in genotype has been overcome).
However, cellular signaling is an extremely complex pro-
cess 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 dif-
ferences in phenotype (i.e., properties of a receptor pro-
duced 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 some-
what lesser, leap of faith taken in predicting therapeutic
effects in human tissues 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 recombi-
nant cell systems engineered with human-receptor mate-
rial. This huge technological step greatly improved the
predictability of drug activity in humans, but it should be
noted that there 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
express human receptors (through transfection with
human cDNA) in convenient surrogate host cells (referred
6 Chapter | 1 What is Pharmacology?

Pharmacological Therapeutic effect

test systems in humans

Human receptors
Animal receptors Human receptors Human receptors Human target cells
Animal tissues Surrogate cells Human target cells under influence
of pathology

Current state-of-the-art

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 bio-
logical techniques to clone and express human receptors in cells, recombinant systems sup-
planted 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 patho-
logical processes.

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.,
robustness, growth rate, stability) and not with any
knowledge of verisimilitude to the therapeutically tar-
geted 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 physio-
logical responses, and that activity can be monitored in
pharmacological experiments. In this sense, human recep-
tors expressed in animal cells are still a theoretical 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 evidence
that a surrogate human cell is any more predictive of a
natural receptor activity than an animal cell when com-
pared 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 recep-
tor) 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 possi-
ble 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 phar-
macologist Sir James Black [10]:
. . . angiogenesis, apoptosis, inflammation, commitment of mar-
row 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.
—J. 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 reasons, an emerging strategy for drug discovery is
the use of natural cellular systems. This approach is dis-
cussed in some detail in Chapter 11, The Drug Discovery
Process.
Even when an active drug molecule is found and
activity is verified in the therapeutic arena, there are fac-
tors 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 β-adrenoceptor bronchodilators [11]). In
the absence of genetic knowledge, these therapeutic fail-
ures for a drug could not easily be averted since they in
1.6 PHARMACOLOGICAL INTERVENTION AND THE THERAPEUTIC LANDSCAPE
essence occurred because of the presence of new biologi-
cal targets not originally considered in the drug-discovery
process. However, as new epidemiological information
becomes available, 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 effi-
cacy. 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 molec-
ular nature of biological targets.
1.5 THE NATURE OF DRUG RECEPTORS
While some biological targets such as DNA are not pro-
tein 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
important property of receptors is that they have a 3-D
structure. Proteins are usually composed of one or more
peptide chains; the composition of these chains makes up
the primary and secondary structure of the protein.
Proteins also are described in terms of a tertiary structure,
which defines their shape in 3-D space, and a quaternary
structure, which defines the molecular interactions
between the various components of the protein chains
(Fig. 1.5). It is this 3-D structure which allows the protein
to function as a recognition site and effector for drugs
and other components of the cell; in essence, the ability
of the protein to function as a messenger, shuttling infor-
mation from the outside world to the cytosol of the cell.
For 7TMRs, the 3-D nature of the receptor forms binding
domains for other proteins such as 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,
7
hormones, and autacoids that carry physiological mes-
sages. This important class of drug target is named for a
characteristic structure consisting of 7TM domains loop-
ing into the extracellular and intracellular space—see
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 channels 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
3-D binding domains on the receptor protein in order to
modify physiological and pathological processes.
1.6 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 (1849
1919) “. . . the prime distinction
between man and other creatures is man’s yearning to
take medicine.” The notion that drugs can be used to cure
disease is as old as history. One of the first written
records of actual “prescriptions” can be found in the
Ebers Papyrus (circa 1550 BC): “. . . for night blindness
in the eyes . . . liver of ox, roasted and crushed out . . .
really excellent!”—see Fig. 1.7. Now it is known that
liver is an excellent source of vitamin A, a prime treat-
ment for night blindness, but that chemical detail was not
known to the ancient Egyptians. Disease can be consid-
ered under two broad categories: those caused by invaders
such as pathogens and those caused by intrinsic break-
down of normal physiological function. The first gener-
ally 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 antiretrovirals 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 path-
ological process and repair of the damage to return to nor-
mal 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
8 Chapter | 1 What is Pharmacology?

Levels of protein (receptor) structure

Primary structure Secondary structure

Sequence of Repeating 3-D units such as
amino acid residues α-helices and β-sheets
(buried main chain H bonds)

Quaternary structure Tertiary structure

Arrangement of Single folded and arranged poly-
separate chains peptide chain, the structure of which is
determined by the amino acids
FIGURE 1.5 Increasing levels of protein structure. A protein has a given amino acid sequence to
make peptide chains. These adopt a 3-D structure according to the free energy of the system.
Receptor function can change with changes in tertiary or quaternary structure.

cardiovascular system consists of a heart able to pump

Extracellular ligands
4
3 2
5
6
1 inability to maintain rhythm (arrhythmia) or loss in
7 strength with concomitant inability to empty (congestive
Signaling proteins
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 interaction of signaling proteins in the cell cytosol. This latter
process results in the initiation of cellular signaling.
deoxygenated blood through the lungs and to pump oxy-
genated blood throughout a circulatory system that does
not unduly resist blood flow. Since the heart requires a
high degree of oxygen itself to function, myocardial
ischemia can be devastating to its function. Similarly, an
heart failure) can be fatal. The latter disease is exacer-
bated by elevated arterial resistance (hypertension). A
wide range of drugs are used to treat the cardiovascular
system, including coronary vasodilators (nitrates), diure-
tics, renin-angiotensin inhibitors, vasodilators, cardiac
glycosides, calcium antagonists, beta and alpha blockers,
antiarrhythmics, 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
1.6 PHARMACOLOGICAL INTERVENTION AND THE THERAPEUTIC LANDSCAPE
FIGURE 1.7 The Ebers Papyrus is a 110-page scroll (20 m long) thought to have been written in 1550 BC but containing
information dating from 3400 BC. It is a record of Egyptian medicine and contains numerous “prescriptions” some of which,
though empirical, are valid therapeutic approaches to diseases.
(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 control-
ling sedation and pain also may require treatment. A wide
range of drugs is used for CNS disorders, including sero-
tonin partial agonists and uptake inhibitors, dopamine
agonists, benzodiazepines, barbiturates, opioids, tricyc-
lics, 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 syndrome can affect
this system. Histamine antagonists, proton pump blockers,
opioid agonists, antacids, and serotonin 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 arthri-
tis, allergies, pain, COPD, asthma, fever, gout, graft rejec-
tion, and problems with chemotherapy. Nonsteroidal
antiinflammatory drugs, aspirin and salicylates, leukotri-
ene antagonists, and histamine receptor antagonists are
used to treat inflammatory disorders. The endocrine sys-
tem produces and secretes hormones crucial to the body
for growth and function. Diseases of this class of organs
can lead to growth and pituitary defects—diabetes;
9
abnormality in thyroid, pituitary, adrenal cortex, and
androgen function; osteoporosis; and alterations in estro-
gen
progesterone balance. The general approach to treat-
ment 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 physiological 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 precur-
sors 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 proliferation 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 (460
357 BC), who saw health
and disease as a balance of four humors (i.e., black and
yellow bile, phlegm, and blood), by the 16th century phar-
macological concepts were being formulated. These could
be stated concisely as the following [13]:
G Every disease has a cause for which there is a spe-
cific remedy.
10 Chapter | 1 What is Pharmacology?

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.

G Each remedy has a unique essence that can be
obtained from nature by extraction (“doctrine of
signatures”).
G The administration of the remedy is subject to a
dose
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 che-
micals. Table 1.1 shows partial lists of hormones and neuro-
transmitters in the body. Many more endogenous chemicals
are involved in normal physiological function. The fact that
so many physiological processes are controlled by chemi-
cals provides the opportunity for chemical intervention.
Thus, physiological signals mediated 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.
1.7 SYSTEM-INDEPENDENT DRUG
PARAMETERS: AFFINITY AND EFFICACY
The process of drug discovery relies on the testing of
molecules in systems to yield estimates of biological activ-
ity 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 for-
ward in the drug development process is to use only
system-independent information. Ideally, scales of biolog-
ical activity should be used that transcend the actual bio-
logical 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 activity to all systems. The
only means to do this is to obtain system-independent
measures of drug activity, namely, affinity and efficacy.
If a molecule in solution associates closely with a recep-
tor protein, it has affinity for that protein. The area where it
is bound is the binding domain or locus. If the same mole-
cule 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 physio-
logically active hormone or neurotransmitter), the molecule
is referred to as an antagonist. Therefore, a pharmacologi-
cally active molecule that blocks physiological effect 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
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
1.7 SYSTEM-INDEPENDENT DRUG PARAMETERS: AFFINITY AND EFFICACY 11

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. 1.8). 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 stimu-
lus
response mechanism or cascade (see Fig. 1.8).
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: Drug
Receptor Theory
for how this term evolved). Thus, every molecule has a
unique value for its intrinsic efficacy (in cases of antago-
nists this could be zero). The different abilities of mole-
cules to induce response are illustrated in Fig. 1.9. This
figure shows dose
response curves for four 5-HT
(hydroxytryptamine) (serotonin) agonists in rat jugular
vein. It can be seen that if response is plotted as a func-
tion 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 capa-
bility of 5-cyanotryptamine to induce response is half that
of 5-HT [14]). These agonists are then said to possess dif-
ferent magnitudes of intrinsic efficacy.
It is important to consider affinity and efficacy as sep-
arately manipulatable properties. Thus, there are chemical
features of agonists that pertain especially to affinity and
other features that pertain to efficacy. Fig. 1.10 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 discov-
ery program was the knowledge that histamine, a natu-
rally occurring autacoid, activates histamine H2 receptors
in the stomach to cause acid secretion. This constant acid
secretion 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 secre-
tion and prevent 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 histamine (i.e., these were separate properties).
As can be seen in Fig. 1.10, molecules were consecutively
synthesized with reduced values of efficacy and enhanced
affinity until the target histamine H2 antagonist cimetidine
Stimulus
A+R AR*
Cellular
Stimulus–response
cascade
Response
FIGURE 1.8 Schematic diagram of response production by an agonist.
An initial stimulus is produced at the receptor as a result of ago-
nist
receptor interaction. This stimulus is processed by the stimu-
lus
response apparatus of the cell into observable cellular response.

(A) (B)
1.5 1.5

1.0 1.0
Force (g)
Force (g)

0.5 0.5

0.0 0.0
−10 −9 −8 −7 −6 −5 −4 0 20 40 60 80 100
Log [agonist] % Receptor occupancy
FIGURE 1.9 Differences between agonists producing contraction of rat jugular vein through activation of
5-HT receptors. (A) Dose
response 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 agonist
receptor complex. Ordinates: force of con-
traction 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)
493
499.
12 Chapter | 1 What is Pharmacology?

. . . we knew the receptor bound histamine, so it was

a matter of keeping affinity and losing efficacy.
— Sir James Black (1996)
pKA
4 5 6 7 8 9 10
1
Histamine
CH2CH2NH2

HN N
2

N-Guanylhistamine
CH2CH2NHCNH2
II
NH2
HN N
3

SKF91581
CH2CH2CH2NHCNHCH3
II
S
HN N
4
Guanidine isostere
H3C CH2SCH2CH2NHCNHCH3
II
+NH2
HN N
5
Burimamide
CH2SCH2CH2NHCNHCH3
II
S
HN N
6
Metiamide
H3C CH2SCH2CH2NHCNHCH3
II
S
HN N
7
Cimetidine
H3C CH2SCH2CH2NHCNHCH3
II
NÐCN
HN N

8
0.0 0.5 1.0
I.A.
FIGURE 1.10 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) 1
33.
1.9 THE LANGMUIR ADSORPTION ISOTHERM 13

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.
1.8 WHAT IS AFFINITY?
The affinity of a drug for a receptor defines the strength
of interaction between the two species. The forces con-
trolling the affinity of a drug for the receptor are thermo-
dynamic (enthalpy as changes in heat and entropy as
changes in the state of disorder). The chemical forces
between the components of the drug and the receptor
vary in importance in relation to the distance of the drug
from the receptor’s binding surface. Thus, the strength of
electrostatic forces (attraction due to positive and nega-
tive 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 bond-
ing (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
between 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 inter-
actions 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 sur-
face, namely, the Langmuir adsorption isotherm.
1.9 THE LANGMUIR ADSORPTION
ISOTHERM
Defined by the chemist Irving Langmuir (1881
1957,
Fig. 1.11), the model for affinity is referred to as the
Langmuir adsorption isotherm. Langmuir, a chemist at
General Electric, was interested in the adsorption of
molecules onto metal surfaces for the improvement of
lighting filaments. He reasoned that molecules had a char-
acteristic rate of diffusion toward a surface (referred to as
condensation and denoted α in his nomenclature) and
also a characteristic rate of dissociation (referred to as
evaporation and denoted as V1; see Fig. 1.11). 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 θ1,
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 2 θ1. The rate of
adsorption toward the surface therefore is controlled by
the concentration of drug in the medium (denoted μ in
Langmuir’s nomenclature) multiplied by the rate of

αμ
θ1 =
αμ + V1

FIGURE 1.11 The Langmuir adsorption isotherm representing the binding of a mole-
cule to a surface. Photo shows Irving Langmuir (1881
1957), 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 (θ1) by a concentration μ.
14
condensation on the surface and the amount of free area
available for binding:
Rate of diffusion toward surface 5 αμð1 2 θ1 Þ: (1.1)
The rate of evaporation is given by the intrinsic rate
of dissociation of bound molecules from the surface mul-
tiplied by the amount already bound:
Rate of evaporation 5 V1 θ1 : (1.2)
Once equilibrium has been reached, the rate of adsorp-
tion equals the rate of evaporation. Equating (1.1) and
(1.2) and rearranging yields
αμ
θ1 5 : (1.3)
αμ 1 V1
This is the Langmuir adsorption isotherm in its origi-
nal form. In pharmacological nomenclature, it is rewritten
according to the convention
½AR ½A
ρ5 5 ; (1.4)
½Rt  ½A 1 KA
where [AR] is the amount of complex formed between
the ligand and the receptor, and [Rt] is the total number
of receptor sites. The ratio ρ refers to the fraction of max-
imal binding by a molar concentration of drug [A] with
an equilibrium 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 α
denoted k1 in receptor 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
drug
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 concentration
of acetylcholine and its action on muscle cells, an 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.
—A. J. Clark (1937).
The term KA is a concentration, and it quantifies affin-
ity. Specifically, it is the concentration that binds to
50% of the total receptor population [see Eq. (1.4) when
[A] 5 KA]. Therefore, the smaller is the KA, the higher is
the affinity. Affinity is the reciprocal of KA. For example,
if KA 5 1028 M, then 1028 M binds to 50% of the recep-
tors. If KA 5 1024 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).
Chapter | 1 What is Pharmacology?
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 visualized for various drug concentrations. The
kinetics of such binding is shown in Fig. 1.12. 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 con-
centration is attained. Fig. 1.12 also shows that the bind-
ing of higher concentrations 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. 1.13A).
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 known.
When the complete curve is defined, the maximal value
of binding can be used to define fractional binding at var-
ious concentrations and thus define the concentration at
which half-maximal binding (binding to 50% of the
receptor population) occurs. This is the equilibrium disso-
ciation constant of the drug
receptor 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. 1.13A that graphical estimation
of both the maximal asymptote and the midpoint is diffi-
cult to perform with the graph in the form shown. A
0.3 nM 1 nM 3 nM
10 nM 30 nM 50 nM
FIGURE 1.12 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 con-
centrations as does the rate of binding.
1.10 WHAT IS EFFICACY?
(A)
1.0
Frac. max.
0.5
0.0
0 10 20 30 40 50
Agonist (nM)
FIGURE 1.13 Dose
response relationship for ligand binding according to the Langmuir adsorption iso-
therm. (A) Fraction of maximal binding as a function of concentration of agonist. (B) Semilogarithmic
form of curve shown in panel A.
much easier format to present binding, or any concentra-
tion
response data, is a semilogarithmic form of the iso-
therm. This allows better estimation of the maximal
asymptote and places the midpoint in a linear portion of
the graph where intrapolation can be done (see
Fig. 1.13B). Dose
response 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., agonists), a signal propor-
tional to bound drug can be observed. The true definition
of a dose
response 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 pharmacologi-
cal jargon as a general depiction of drug and effect. Thus,
a dose
response curve for binding is actually a binding
concentration curve, and an in vitro effect of an agonist
in a receptor system is a concentration
response curve.
1.10 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,
“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 poten-
tial shapes referred to as conformations. A protein stays in
15
(B)
1.0
Frac. max.
0.5
0.0
−10 −9 −8 −7 −6
Log [agonist]
a particular conformation because it is energetically favor-
able 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 structure
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 . . ..
—Lindstrom and Schellman (1959)
Not only are a number of conformations for a given pro-
tein possible, but the protein samples these various confor-
mations 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 molecules, by interact-
ing 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 effi-
cacy). 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 pos-
sible 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 affin-
ity for protein conformations was proposed for oxygen
binding to hemoglobin [17] and for choline derivatives
and nicotinic receptors [18]. Furthermore, assume that
these conformations 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
16
equilibrium association constants Ka and αKa, respec-
tively, for the inactive and active states:
ð1:5Þ
It can be shown that the ratio of the active species Ra
in the presence of a saturating concentration (ρN) of the
ligand vs in the absence of the ligand (ρ0) is given by the
following (see Section 1.13):
ρN αð1 1 LÞ
5 : (1.6)
ρ0 ð1 1 αLÞ
It can be seen that if the factor α is unity (i.e., the
affinity of the ligand for Ra and Ri is equal [Ka 5 αKa]),
then there will be no change in the amount of Ra when
the ligand is present. However, if α 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 pres-
ent. Therefore, 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 recep-
tors 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%
85%).
This concept is demonstrated schematically in
Fig. 1.14. It can be seen that the initial bias in a system of
proteins containing two conformations (square and spheri-
cal) lies far toward the square conformation. When a
ligand (filled circles) enters the system and selectively
binds to the circular conformations, this binding process
removes the circles driving the backward reaction from
circles back to squares. In the absence of this backward
pressure, more square conformations flow into the circu-
lar state to fill the gap. Overall, there is an enrichment of
the circular conformations when unbound and ligand-
bound circular conformations are totaled.
This also can be described in terms of the Gibbs free
energy of the receptor
ligand 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 com-
posed of two conformations ai and ao is given by the fol-
lowing [19]:
X X
ΔGi 5 ΔG0i 2 RT
X (1.7)
3 lnð1 1 Ka;i ½AÞ= lnð1 1 Ka;0 ½AÞ;
Chapter | 1 What is Pharmacology?
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 5 Ka,0, the logarithmic term will not equal zero and the
free
P energy
P of
 the system will change
ΔGi 6¼ ΔG0i . 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 cir-
cumstances, a different conformational bias will be formed
by the differential affinity of the ligand. From these models
comes the concept that binding is not a passive process,
whereby a ligand simply adheres to a protein without
changing it. The act of binding can itself bias the behavior
of the protein. This is the thermodynamic basis of efficacy.
1.11 DOSE
RESPONSE CURVES
The concept of “dose
response” 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 physi-
cians, Paracelsus (1493
1541), that it is only the dose
that makes something beneficial or harmful: “All things
are poison, and nothing is without poison. The Dosis
alone makes a thing not poison.”
Dose
response curves depict the response to an ago-
nist 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, loca-
tion along the concentration axis, slope, and maximal
asymptote (Fig. 1.15). At first glance, the shapes of
dose
response curves appear to closely mimic the line
predicted by the Langmuir adsorption isotherm, and it is
tempting to assume that dose
response curves reflect the
first-order binding and activation of receptors on the cell
surface. However, in most cases, this resemblance is hap-
penstance, and dose
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 depolarization, the single-cell
response to an agonist may be complete depolarization
(in an all-or-none fashion). Taken as a complete collec-
tion, the depolarization profile of the culture where the
cells all have differing thresholds for depolarization
would have a Gaussian distribution of depolarization
thresholds—some cells being more sensitive than others
(Fig. 1.16A). 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 dose
response curve (Fig. 1.16B) that
1.11 DOSE
RESPONSE CURVES 17

(A)
I "Inactive" II "Activated"
receptors receptors
I

Frequency
II

Conformation

Add ligand
(B)

Frequency
I II

Conformation

(C)
II
Frequency

Conformation

FIGURE 1.14 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.

resembles the Langmuirian binding curve for
drug
receptor binding. The slope of the latter curve
reflects the molecularity of the drug
receptor interaction
(i.e., one ligand binding to one receptor yields a slope of
unity for the curve). In the case of the sequential depolari-
zation of a collection of cells, it can be seen that a nar-
rower range of depolarization thresholds yields a steeper
dose
response curve, indicating that the actual numerical
value of the slope for a dose
response curve cannot be
equated to the molecularity of the binding between
agonist and receptor. In general, shapes of dose
response
curves are completely controlled by cellular factors and
cannot be used to discern drug
receptor mechanisms.
These must be determined indirectly by null methods.
1.11.1 Potency and Maximal Response
There are certain features of agonist dose
response
curves that are generally true for all agonists. The first is
18 Chapter | 1 What is Pharmacology?

that the magnitude of the maximal asymptote is totally
dependent on the efficacy of the agonist and the effi-
ciency of the biological system to convert receptor stimu-
lus into tissue response (Fig. 1.17A). This can be an
extremely useful observation in the drug-discovery pro-
cess 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 asymp-
tote of the dose
response curve for that agonist.
Therefore, if chemists wish to optimize or minimize effi-
cacy in a molecule, they can track the maximal response
to do so. Second, the location, along the concentration
axis of dose
response curves, quantifies the potency of
120
100
asymptote
% Max. response
the agonist (Fig. 1.17B). The potency is the molar con-
centration 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 con-
centration of an agonist required to produce 50% of the
maximal response to the agonist. Thus, an EC50 value of
1 μM indicates that 50% of the maximal response to the
agonist is produced by a concentration of 1 μM of the
agonist (Fig. 1.18). 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 μM of this agonist (Fig. 1.18). Similarly, an EC25 will
be produced by a lower concentration of this same ago-
nist; in this case, the EC25 is 0.5 μM.
Maximal
1.11.2 P-Scales and the Representation of

80
Potency
60
Slope Agonist potency is an extremely important parameter in
40 drug
receptor pharmacology. Invariably it is determined
Threshold from log-dose
response curves. It should be noted that
20 since these curves are generated from semilogarithmic
plots, the location parameter of these curves is log nor-
0 mally distributed. This means that the logarithms of the
−3 −2 −1 0 1 2 3 sensitivities (EC50) and not the EC50 values themselves
Log ([A]/KA) are normally distributed (Fig. 1.19A). Since all statistical
FIGURE 1.15 Dose
response curves. Any dose
response curve can parametric tests must be done on data that come from
be defined by the threshold (where response begins along the concentra- normal distributions, all statistics (including comparisons
tion axis), the slope (the rise in response with changes in concentration), of potency and estimates of errors of potency) must come
and the maximal asymptote (the maximal response). from logarithmically expressed potency data. When log
normally distributed EC50 data (Fig. 1.19B) are converted

(A) (B)
12 120

10 100
Cumulative %

8 80
Frequency

6 60

4 40

2 20

0 0
0 500 1000 0 500 1000
No. of cells No. of cells
FIGURE 1.16 Factors affecting the slope of dose
response curves. (A) Gaussian distributions of the thresh-
olds 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 distributions 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 corresponds to the dose
response curve of steeper slope.
1.12 CHAPTER SUMMARY AND CONCLUSIONS 19

100 100
% Max. response

% Max. response
50 50

Intrinsic activity Potency

f(efficacy) f(efficacy and affinity)

0 0
−2 −1 0 1 −2 −1 0 1
Log ([agonist]/KA) Log ([agonist]/KA)

FIGURE 1.17 Major attributes of agonist dose
response curves. Maximal responses solely reflect efficacy, while the potency (loca-
tion along the concentration axis) reflects a complex function of both efficacy and affinity.

120 then convert to concentration as the last step. For exam-

System maximal response ple, Table 1.2 shows five pEC50 values, giving a mean
100 pEC50 of 8.46 and a standard error of 0.21. It can be seen
that the calculation of the mean as a converted concentra-
% Max. response

80 Maximal response 80%

to the agonist
60
40
EC25
20
0 the standard error, which leads to the values 8.4 and
−9 −8 −7 −6
Log [agonist] : M
FIGURE 1.18 Dose
response curves. Dose
response curve to an ago-
nist that produces 80% of the system maximal response. The EC50 (con-
centration producing 40% response) is 1 μM, the EC25 (20%) is 0.5 μM,
and the EC80 (64%) is 5 μM.
to EC50 data, the resulting distribution is seriously skewed
(Fig. 1.19C). 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 differ-
ent values on the skewed distribution (Fig. 1.19C)]. This
is not true of the symmetrical normal distribution
(Fig. 1.19B).
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 hydrogen ion concentration
(105 M 5 pH 5 5). It is essential to express
dose
response parameters as P-values (2log 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 estimate is to make the calculation as P-values and
tion (EC50 value) leads to an apparently reasonable mean
value of 3.8 nM, with a standard error of 1.81 nM.
EC80
However, the 95% confidence limits (range of values that
40% will include the true value) of the concentration value is
meaningless, in that one of them (the lower limit) is a
EC50 = 1 µM
negative number. The true value of the EC50 lies within
the 95% confidence limits given by the mean 1 2.57 3
−5 −4 −3 20.85 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% confi-
dence limits on the pEC50 are 7.8
9.0. Conversion of
these limits to EC50 values yields 95% confidence limits
of 1
11.8 nM. Thus, the true potency lies between the
values of 1 and 11.8 nM 95% of the time.
1.12 CHAPTER SUMMARY AND
CONCLUSIONS
G Some ideas on the origins and relevance of phar-
macology and the concept of biological “receptors”
are discussed.
G Currently, there are drugs for only a fraction of the
druggable targets present in the human genome.
G While recombinant systems have greatly improved
the drug-discovery process, pathological pheno-
types still are a step away from these drug-testing
systems.
G Because of the fact that drugs are tested in experi-
mental, not therapeutic, systems, system-
independent measures of drug activity (namely,
affinity and efficacy) must be measured in drug
discovery.
20 Chapter | 1 What is Pharmacology?

(A) (B)
10

Frequency
100
6

2
Mean
% Max. response

Stnd. error
95% Conf. limits 7 7.5 8 8.5 9 9.5
50 pEC50
(C)
10

Frequency
6

0
−3 −2 −1 0 1 2 3 2
([A]/KA)
0 5 10 15 20 25 30 35
EC50 : nM

FIGURE 1.19 Log normal distributions of sensitivity of a pharmacological preparation to an agonist. (A)
Dose
response 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 (2log EC50 values). (C) Skewed distribution of
EC50 values converted from the pEC50 values shown in panel B.

G Measures of potency are log normally distributed.

TABLE 1.2 Expressing Mean Agonist Potencies with Only P-scale values (i.e., pEC50) should be used
Error for statistical tests.
pEC50a EC50 (nM)b
8.5 3.16
8.7 2 1.13 DERIVATIONS: CONFORMATIONAL
8.3 5.01 SELECTION AS A MECHANISM OF
8.2 6.31
8.6 2.51 EFFICACY
Mean 5 8.46 Mean 5 3.8
SE 5 0.21 SE 5 1.81 Consider a system containing two receptor conformations
Ri and Ra that coexist in the system according to an allo-
Replicate values of 21/N log EC50’s.
a
b
Replicate EC50 values in nM.
steric constant denoted L:

G Affinity is the strength of binding of a drug to a
receptor. It is quantified by an equilibrium dissoci-
ation constant.
G Affinity can be depicted and quantified with the
Langmuir adsorption isotherm.
G Efficacy is measured in relative terms (having no
absolute scale) and quantifies the ability of a mole-
cule to produce a change in the receptor (most
often leading to a physiological response).
G Dose
response curves quantify drug activity. The
maximal asymptote is totally dependent on effi-
cacy, while potency is due to an amalgam of affin-
ity and efficacy.
Assume that ligand A binds to Ri with an equilibrium
association constant Ka, and Ra by an equilibrium associ-
ation constant αKa. The factor α denotes the differential
affinity of the agonist for Ra (i.e., α 5 10 denotes a 10-
fold greater affinity of the ligand for the Ra state). The
effect of α on the ability of the ligand to alter the equilib-
rium between 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] 1 [ARa])/
[Rtot], where [Rtot] is the total receptor concentration
given by the conservation equation [Rtot] 5 [Ri] 1
[ARi] 1 [Ra] 1 [ARa], is
REFERENCES
Lð1 1 α½A=KA Þ
ρ5 ; (1.8)
½A=KA ð1 1 αLÞ 1 1 1 L
where L is the allosteric constant, [A] is the concentration
of ligand, KA is the equilibrium dissociation constant of
the agonist
receptor complex (KA 5 1/Ka), and α is the
differential affinity of the ligand for the Ra state. It can be
seen that in the absence of agonist ([A] 5 0), ρ0 5 L/
(1 1 L), and in the presence of a maximal concentration
of ligand (saturating the receptors; [A]-N),
ρN 5 (α(1 1 L))/(1 1 αL). The effect of the ligand on
changing the proportion of the Ra state is given by the
ratio ρ/ρ0. This ratio is given by
ρN αð1 1 LÞ
5 : (1.9)
ρ0 ð1 1 αLÞ
Eq. (1.9) indicates that if the ligand has an equal affin-
ity for both the Ri and Ra states (α 5 1), then ρN/ρ0 will
equal unity, and no change in the proportion of Ra will
result from maximal ligand binding. However, if α . 1,
then the presence of the conformationally selective ligand
will cause the ratio ρN/ρ0 to be .1, and the Ra state will
be enriched by presence of the ligand.
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 Chapter 2

How Different Tissues Process

Drug Response

[Nature] can refuse to speak but she cannot give a wrong answer.
— 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. . .
—Werner Heisenberg (1901
76).

2.1 Drug Response as Seen Through 2.5 System Effects on Agonist 2.9 Advantages and Disadvantages
the “Cellular Veil” 23 Response: Full and Partial of Different Assay Formats 41
2.2 The Biochemical Nature of Agonists 30 2.10 Drug Concentration as an
Stimulus
Response Cascades 25 2.6 Differential Cellular Response to Independent Variable 42
2.3 The Mathematical Approximation Receptor Stimulus 33 2.11 Chapter Summary and
of Stimulus
Response 2.7 Receptor Desensitization and Conclusions 44
Mechanisms 28 Tachyphylaxis 38 2.12 Derivations 44
2.4 Influence of Stimulus
Response 2.8 The Measurement of Drug References 46
Cascades on Dose
Response Activity 39 Further Reading 46
Curve Slopes 29

2.1 DRUG RESPONSE AS SEEN THROUGH
THE “CELLULAR VEIL”
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 machinery necessary to convert the stimulus
into an observable response. In this sense, 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 substantially
modify drug
receptor activity (Fig. 2.1). Minute signals,
initiated either at the cell surface or within the cytoplasm
of the cell, are interpreted, transformed, amplified, and
otherwise altered by the cell to tailor that signal to its
own particular needs. In receptor systems where a drug
does produce a response, the relationship between the
binding reaction (drug 1 receptor protein) and the
observed response can be studied indirectly through
observation of the cellular response as a function of drug
concentration (dose
response curve). A general phenom-
enon observed experimentally is that the cellular response
most often is not linearly related to receptor occupancy
(i.e., it does not require 100% occupation of all of the
receptors to produce the maximal cellular response).
Fig. 2.2A shows a functional dose
response curve for
human calcitonin in human embryonic kidney (HEK)
cells transfected with cDNA for human calcitonin recep-
tor 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 calcitonin binding to the receptors (as measured with

A Pharmacology Primer. DOI: https://doi.org/10.1016/B978-0-12-813957-8.00002-3

© 2019 Elsevier Inc. All rights reserved. 23
24 Chapter | 2 How Different Tissues Process Drug Response

Drug cellular response

Drug stimulus

FIGURE 2.1 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 out-
come of the drug effect is modified by the extent of these interconnections, the basal state of the cell,
and the threshold sensitivity of the various processes involved. This can lead to a variety of appar-
ently different effects for the same drug in different cells. Receptor pharmacology strives to identify
the basic mechanism initiating these complex events.

(A) (B)
1.2 1.2
1.0 1.0
Fraction max.

Fraction max.

0.8 0.8
0.6 0.6
0.4 0.4
0.2 0.2
0.0 0.0
−12 −11 −10 −9 −8 −7 −6 0 20 40 60 80 100
Log (calcitonin) % Receptor occupancy
FIGURE 2.2 Binding and dose
response curves for human calcitonin on human calcitonin receptors type 2.
(A) Dose
response curves for microphysiometry responses to human calcitonin in HEK cells (open circles) and
binding in membranes from HEK cells (displacement of [125I]-human calcitonin). (B) Regression of microphy-
siometry responses to human calcitonin (ordinates) upon human calcitonin fractional receptor occupancy (abscis-
sae). 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 rela-
tionship to amylin receptors, Mol. Pharmacol. 52 (1997) 1164
1175.

radioligand 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% to 4%, 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 receptor 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.
2.2 THE BIOCHEMICAL NATURE OF STIMULUS
RESPONSE CASCADES 25

(A) 1.2 (B)

1.2
1.0 1.0

Fraction max.

Fraction max.
0.8 0.8
0.6 0.6
0.4 0.4
0.2 0.2
0.0 0.0
−10 −9 −8 −7 −6 −5 −4 −3 0 1 2 3 4
Log (histamine) : M % Receptor occupancy
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 μM for 3 min; open circles). (B)
Occupancy
response curve for data shown in (A). Ordinates are percentage of maximal response. Abscissae
are calculated receptor occupancy values from an estimated affinity of 20 μM for histamine. Note that maxi-
mal 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) 386
392.

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 max-
imal 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
receptors with the protein alkylating agent phenoxybenza-
mine [2]. The fact that the depressed maximum
dose
response curve is observed so far to the right of the
control dose
response 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 activa-
tion 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 ago-
nist 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 tis-
sue 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 exam-
ple, Fig. 2.4 shows the different amplification hyperbolae
of Chinese hamster ovary (CHO) cells transfected with
β-adrenoceptors in producing cyclic adenosine monopho-
sphate (AMP) responses to three different β-adrenoceptor
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 ago-
nist’s essential one).
2.2 THE BIOCHEMICAL NATURE OF
STIMULUS
RESPONSE CASCADES
Cellular amplification of receptor signals occurs through
a succession of saturable biochemical reactions. Different
receptors are coupled to different stimulus
response
mechanisms in the cell. Each has its own function and
operates on its own timescale. For example, receptor tyro-
sine kinases (activated by growth factors) phosphorylate
target proteins on tyrosine residues to activate protein
phosphorylation cascades such as mitogen-activated pro-
tein (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 homeostatic, metabolic, and immunosuppression
effects. Ligand-gated ion channels, activated by neuro-
transmitters, operate on the order of milliseconds to
increase the permeability of plasma membranes to ions.
This leads to increases in cytosolic Ca21, depolarization,
or hyperpolarization of cells. This in turn results in mus-
cle contraction, release of neurotransmitters, or inhibition
of these processes.
Another random document with
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 ‘Faint heart never won fair lady,’ she said. ‘I know she likes you.’
‘I hope you won’t think me conceited if I agree with you, but I really
think she does,’ said Kenrick, remembering that curious fainting fit on
the first evening of Miss Harefield’s visit.
He took heart of grace next day, finding Beatrix alone in the library
an hour or so before the late tea. It was a windy afternoon, late in
March, the sky dull and gray, the wood fire glowing redly, Beatrix
seated in her low chair beside the hearth, with a book on her lap,
deep in thought.
‘I don’t wonder you admire Pascal,’ she said, without looking up,
as Kenrick came towards the hearth. ‘His is a most delicate wit.’
‘I am sorry to say I don’t know anything about the gentleman,’ said
Kenrick. ‘Did he write plays or novels?’
‘I beg your pardon. I thought you were Mr. Dulcimer.’
‘You took me for a better man than I am. All those rows of sober
old books are Greek to me—worse than Greek, for I do know that by
sight. I wonder that you can find so much happiness in this dry-as-
dust collection of the dear Vicar’s.’
‘I don’t know about happiness,’ answered Beatrix, with a faint sigh.
‘I find forgetfulness. I suppose that is almost as good.’
‘There cannot be much in your young life that you can wish to
forget,’ said Kenrick.
‘There is very little in it that I care to remember.’
‘Erase it altogether from your memory then, and begin a new life
from to-day,’ said Kenrick, flinging himself head foremost into a gulf
of uncertain issues, like the diver who plunges into the fatal deep to
win the king’s daughter. ‘Let the beginning of a brighter and happier
life date from to-day. You are one of those flowers of earth which
seem to be born to blush unseen. You, who are so worthy of love
and admiration, have lived hidden from those who could admire and
appreciate. But if a real and unmeasured love in the present can
compensate for your losses in the past, that love is yours, Beatrix. I
love you as I never thought I should love. I did not know that it was in
my nature to feel as strongly as I feel for you. Stop—do not answer
me too quickly,’ he cried, reading rejection in her look as she turned
to him with the firelight shining on her face. ‘You will say, perhaps, “I
am rich and you are poor. How am I to believe in your truth?”’
‘I am not capable of thinking meanly of you,’ answered Beatrix.
‘But you ask me what is impossible. I have made up my mind never
to marry.’
‘Will you tell me the reason?’
‘That is my secret.’
‘I am not to be answered so easily, Beatrix. I love you too well to
lose you without a struggle. I have spoken too soon, perhaps. I have
been too precipitate. But I am to go back to India in a few weeks,
and I should like to return with a new happiness—with at least the
promise of your love.’
‘I have no love to give you. If you could see into the bottom of my
heart, you would be horrified at its emptiness. The warmest feeling I
have is gratitude to my friends the Dulcimers. Yes, I think that is the
only human feeling you would discover in my heart. That is why I like
to live among these books. They are a world in themselves. They
give me delight, and ask no love in return.’
‘But I am not like the books, Beatrix; I ask for your love, and I shall
not be easily denied.’
And then he told her his dream about Culverhouse Castle. How
she was to reign there—not like his mother, in silence and seclusion,
but in all the power of youth, beauty, and wealth, a queen of county
society, the centre and focus of a happy world of her own, loved,
admired, and revered.
‘I,’ exclaimed Beatrix—‘I, who have been suspected of poisoning
my father?’
‘That shameful slander has never penetrated beyond this
contemptible hole,’ said Kenrick, very disrespectful to Little Yafford in
the warmth of his indignation. ‘For God’s sake, Beatrix, do not let
that foul scandal weigh in your mind. Perhaps that is the reason you
reject me,’ he added, slow to believe that he had been mistaken
when he fancied himself beloved.
‘No,’ answered Beatrix, ‘but the only man I ever loved rejected me
for that reason.’
‘Oh,’ said Kenrick, deeply mortified.
After this confession he could no longer doubt that he had
mistaken Beatrix’s feelings towards him. He was silent for some
minutes, and then he exclaimed suddenly,—
‘That man was my cousin Cyril?’
‘He was.’
‘Then my cousin Cyril is a mean hound.’
‘Do you want me to hate you?’ cried Beatrix, angrily. ‘He is not
mean. He is all that is good and noble. Why should his pure life be
sullied by the taint that has fallen upon mine? He, a clergyman, could
not afford to take a wife whom men have suspected of evil. He is like
Cæsar. His wife must be above suspicion. He loved me once. He will
love me always, perhaps, a little better than all other women, as I
shall love him to the end of life above all other men. But he has
chosen something better in this life than a woman’s love. He has
given himself to the service of God. No unholy thing must come
within the veil of the temple. Nothing stained, not even with the
suspicion of sin, must enter there. A priest’s wife must be spotless.’
‘If he could suspect you,’ exclaimed Kenrick, vindictively, ‘he is
unworthy——’
‘Oh, for pity’s sake do not suggest that,’ interrupted Beatrix. ‘I
cannot believe that he could suspect me, having once known and
loved me. It was not his suspicion, but the evil thoughts of others,
that parted us.’
‘Then he is a coward,’ cried Kenrick, honestly angry. ‘A man’s
choice of his wife is a question of life or death for himself. He is both
craven and fool if he allows other people to be the arbiters of his
fate.’
 ‘But you do not understand,’ urged Beatrix, pleading for the man
who had broken her heart. ‘It is his office——’
‘His office be——’
He might have said something very shocking if Mrs. Dulcimer had
not come in at this moment. She found Beatrix in tears, and Kenrick
pacing up and down the room with a distracted air. These two facts
indicated that something decisive had happened, and Mrs. Dulcimer
saw from Kenrick’s face that the something was of an unsatisfactory
nature.
‘How provoking!’ she thought. ‘It really seems as if no plan of mine
is to succeed.’
Beatrix did not appear at the tea-table. She sent an apology by
Rebecca. She had a headache, and would go to bed early. Kenrick
was absent-minded, and out of spirits. The meal, usually so cheerful,
was eaten in silence; Mr. Dulcimer had picked up a queer little
seventeenth century copy of Boileau at Great Yafford that morning,
and looked at the tail-pieces and initials as he took his tea.
‘Stop and smoke your cigar here, Ken,’ said Mrs. Dulcimer when
tea was over, and Sir Kenrick was about to follow his host to the
library.
‘But don’t you dislike smoke in this room?’
‘Not for once in a way. Your cigars are very mild, and Rebecca will
air the room well to-morrow morning. I want to have a chat with you.’
‘Delighted,’ said Kenrick, sitting down opposite Mrs. Dulcimer’s
work-table.
He had a shrewd suspicion of what was coming, but he felt that it
would comfort him to pour his woes into a friendly ear. He knew very
well that Mrs. Dulcimer had set her heart upon his marrying Miss
Harefield.
‘What had you been doing to make Beatrix cry?’ asked the Vicar’s
wife, coming straight to the point.
‘I had asked her to be my wife.’
 ‘What, they were tears of joy then?’ cried Mrs. Dulcimer.
‘Quite the contrary. She had rejected me flatly.’
‘Oh, Kenrick! But why?’
‘She did not condescend to enter very minutely into her reasons,
but I believe the principal one is that she doesn’t care for me.’
‘Oh, Kenrick,’ cried Mrs. Dulcimer, in exactly the same tone as
before. ‘What a pity!’
‘Yes, it’s regrettable. If my first thought were her fortune and the
good it would do to Culverhouse, I should deserve my fate. But that
is only my second thought. I love her very dearly. If she were the
poorest little nursery governess in the county I should love her just
the same, and would take her back to India with me, and work for
her and be happy with her all the days of my life.’
‘But her money would pay off those mortgages; as Lady
Culverhouse she would have a leading position in your part of the
country.’
‘She would be admired and adored,’ said Kenrick.
‘It would be in every way such a suitable match,’ protested Mrs.
Dulcimer, a remark she was in the habit of making about every pair
of young people whose footsteps she wished to direct to the
hymeneal altar. ‘Really human nature is very perverse.’
She remembered how ignominiously she had failed in her desire
to benefit Cyril and Bella; and here was this more important scheme
apparently doomed to failure.
‘It is very difficult to serve one’s fellow-creatures,’ she said
presently. ‘But this is not a business to be given up lightly, Kenrick.
This foolish girl is Mr. Dulcimer’s ward, and it is his duty to see her
advantageously settled in life. Now Clement is the very last man to
think of such a thing. He considers he has done his duty when he
has given Beatrix the run of his library.’
‘Yes,’ said Kenrick. ‘It is dear Dulcimer’s only fault to consider
books the beginning, middle, and end of life.’
 ‘Something must be done,’ declared Mrs. Dulcimer, with a sudden
accession of energy. ‘Beatrix ought to marry, and she ought to marry
a man of position. I cannot imagine a more suitable husband than
yourself. Come, Kenrick, be frank with me. You have not told me
everything. There must be some other reason. Don’t you remember
an admission Beatrix made at that dreadful inquest? There was a
love affair of which her father disapproved. Nothing but a prior
attachment could prevent her accepting you. I feel convinced it must
be that.’
‘It is that,’ answered Kenrick. ‘You can keep a secret, I suppose,
Mrs. Dulcimer?’
‘My dear Kenrick, I have kept hundreds.’
This was true, but Mrs. Dulcimer forgot to add how short a time
she had kept them. The Vicar’s wife’s secret of to-day was the town-
crier’s secret of to-morrow.
‘Then I’ll trust you with the clue to the mystery. There is a prior
attachment—to my cousin Cyril.’
‘Good heavens!’ exclaimed Mrs. Dulcimer. ‘Then that is why he
was so indifferent to poor Bella Scratchell, the very girl for him.’
‘He is a contemptible cur,’ said Kenrick.
He went on to abuse his cousin roundly. It was a good thing for
him, no doubt, that Cyril had behaved so badly, for it gave Kenrick
just the chance that Beatrix would put the false lover out of her mind
and marry the true one. He told Mrs. Dulcimer everything.
‘Something must be done,’ she said finally, and she made up her
mind that she, Selina Dulcimer, was the right person to undertake
the task.
 CHAPTER XII.
something must be done.
Before Sir Kenrick’s leave came to an end Mrs. Piper had gone to
the land where there are no sordid cares, no gnawing doubts as to
the honesty of servants, no heart-corroding regrets at the
wastefulness and expenditure of a large household. Mrs. Piper had
gone to that undiscovered country where we may fairly hope that for
those who have lived harmlessly upon earth all is peace. Mr. Piper
drove his smart little pony cart about the country roads and through
the village street as usual, but he wore an altered countenance and
crape to the top of his tall hat. He no longer had a noisy greeting for
every one, no longer quoted Jeremy Bentham or William Cobbett.
Never was widower more disconsolate than Ebenezer Piper.
Honestly and truly he mourned the careful partner of his youth and
maturity.
‘There wasn’t a finer girl in Great Yafford when me and she was
married,’ he said dolefully, after a brief eulogium of his faithful
Moggie’s domestic virtues.
Mrs. Piper’s monument was to be the glory of the village
churchyard. Mr. Dulcimer was too indulgent and easy to insist upon a
rigid æstheticism in the memorials which the living erected in honour
of the dead. There was a good deal of bad taste in God’s Acre at
Little Yafford, but Mr. Piper was destined to put the cap on the edifice
by the gaudiest and most expensive mausoleum that ever the chief
stonemason of Great Yafford had devised or executed.
It was to be a sarcophagus of the jewel casket shape, with four
twisted columns, like candlesticks, at the corners, and a tall urn
surmounting the lid. Each of the columns was of a different coloured
marble, the urn was dark red serpentine, with a malachite serpent
coiled round it. The urn was supposed to contain Mrs. Piper’s dust,
the serpent indicated that physicians and doctors’ stuff had not been
wanting in the effort to keep Mrs. Piper longer upon earth. Scattered
over the fluted lid of the sarcophagus were to be flowers sculptured
out of coloured marble, and cemented on to the white groundwork.
The sides of the sarcophagus were to be decorated with shields,
richly emblazoned with the Piper arms. Mr. Piper’s arms were his
own composition; his crest a ladder; his motto, Ex sese.
Altogether the monument was to be a wonderful thing, and Mr.
Piper felt a pride in contemplating the sketches which the mason had
caused to be made, and in picturing to himself the effect of the whole
when this great work of art should be finished.
The Piper children, in black frocks, and in a state of semi-
orphanage, were a little more troublesome than they had been in
coloured frocks, and with an invalid mamma as a court of appeal.
They brought the ghost of their lost parent into every argument.
‘I’m sure ma wouldn’t have wished me to learn three verbs in one
morning,’ said Elizabeth Fry.
‘I think ma would have let me off my lessons if I had a sick
headache,’ remonstrated Mary Wolstencroft.
‘I shall do my duty to you whether you like it or no,’ said Bella,
resolutely.
‘Ah, you’d better take care!’ cried Brougham. ‘Ma’s in heaven,
where she can see everything you’re up to, and won’t she make it
disagreeable for you when you get there! If you ever do,’ added the
boy, in a doubtful tone; ‘but I don’t think you stand much chance if
you go on making our lives a misery with Latin grammar.’
Now that poor Mrs. Piper had laid down her load of earthly care,
Miss Scratchell restricted her visits to the Park to purely professional
limits. She entered the schoolroom punctually at nine, and she left it
as punctually at half-past one. She no longer assisted at the
children’s early dinner, a meal which Mr. Piper, when at home,
shared under the name of luncheon. Bella had a keen sense of the
proprieties, and did not care to sit down to luncheon with a
disconsolate widower, or to give Mr. Piper any opportunity to pour his
griefs into her ear, as he would fain have done very often. Mr. Piper
was of a soft and affectionate nature, and when he told his griefs to a
young woman he could not refrain from taking her hand, and even
occasionally squeezing it. This Bella could not possibly permit. She
therefore carefully avoided all conversations about the late Mrs.
Piper, and, as far as was practicable, she avoided Mr. Piper himself.
‘It seems very ’ard,’ complained the widower, ‘that the time when a
man feels lonesomest is a time for everybody to avide him. You
might as well stop, Miss Scratchell, and eat your bit of dinner with
me and the children. You won’t get lamb and sparrowgrass at home.’
‘I know I shall not,’ replied Bella; ‘but I would rather not stay, thank
you, Mr. Piper.’
‘Why not?’
‘My mother wants me at home.’
‘She can’t want you more now than she did when pore Mrs. P——
was alive. You never refused to stop then.’
‘I did not like to refuse dear Mrs. Piper, when she was an invalid,
and wanted every one’s sympathy.’
‘You’re a good-’earted girl,’ said Piper, approvingly. ‘I know what
your motive is. You think it ain’t proper to eat your bit of dinner with
me, now I’m a widower, though there’s all the children to keep you in
countenance. You think it might set the old tabbies up street talking.’
‘It certainly does not require much to do that,’ replied Bella,
smiling. ‘But I really am wanted at home, Mr. Piper, and I mustn’t
stop talking here. I am going to drink tea at the Vicarage this
evening.’
‘Ah!’ sighed Air. Piper, ‘you’re a rum girl. It seems to me that
everybody wants you. I shall send you round a bundle of my early
sparrowgrass.’
‘Pray don’t take the trouble.’
‘Yes, I shall. It costs me about eighteen-pence a stick, so
somebody may as well have the enjoyment of it. But ’orticulture is
my ’obby.’
 It must be observed that although Mr. Piper was a student of
Cobbett, and had taught himself a little Latin, he had never been
able to conquer the mysteries of his own tongue. He still spoke as
bad English as in the days when he was a factory hand, and had
never read a passage of Cobbett’s strong racy prose, or pondered
over a thesis of Bentham’s.
Bella and Beatrix were good friends still, but not such friends as
they had been a year or even six months ago. There was a restraint
on both sides. Beatrix could not have told why it was, but it seemed
to her that there was a change in herself, and a still greater change
in Bella. Bella knew very well what it was that made her
uncomfortable in Miss Harefield’s society. It was the sealed letter in
its hiding-place in Bella’s shabby old bedroom. That sealed letter
weighed like a load of iron upon Bella’s conscience when she found
herself in Beatrix’s company; and yet she was glad that she had
done this thing, if it had been the means of parting Cyril and Beatrix.
She would like to have seen them parted even more irrevocably,
so that under no circumstances could time or chance bring them
together again. She was in this temper of mind when she went to
spend the evening at the Vicarage, after her little talk with Mr. Piper
in the stone portico at the Park.
It was about a week since Sir Kenrick had made his offer and had
been rejected. He had taken a wonderful fancy to fishing for pike
after that catastrophe, and had brought home some very handsome
specimens of that ravenous tribe, for the Vicarage cook to stuff and
bake, and serve with savoury sauces for the three o’clock dinner.
‘I think I shall have to protest, like the highland gillies when they
got too much salmon, if Kenrick goes on bringing home pike in this
way,’ said Mr. Dulcimer, when the cover was lifted and the hungry-
jawed scaly monster appeared before him.
Kenrick was off in the early gray to his fishing grounds, so he and
Beatrix only met of an evening. He was very polite to her, and
evidently bore no malice. Hope was not altogether extinguished in
his breast. He had much confidence in Mrs. Dulcimer, who had said
that something must be done. Kenrick had not the faintest idea what
this inveterate match-maker meant to do, but he felt that her
friendship would stick at nothing which a clergyman’s wife might do
without peril to her soul.
‘Bella,’ said Mrs. Dulcimer after tea, ‘I want to show you the things
I’ve made for the missionary basket. You might be able to help me a
little, perhaps.’
‘I shall be delighted, dear Mrs. Dulcimer,’ answered Bella, inwardly
lamenting that it had pleased God to call her to that station of life in
which her friends always felt themselves justified in asking her to
work for them.
A young woman of fortune like Miss Harefield might be as idle or
as selfish as she pleased. Nobody ever thought of asking for
payment in kind for any favour they showed her; but everybody who
did any kindness to penniless Bella Scratchell wanted to extort
recompense for his or her civility in needlework or some sort of
drudgery.
‘Come up to my room and look at the things, dear,’ said Mrs.
Dulcimer; and then it occurred to Bella that her hostess had
something particular to say to her. She had heard from Cyril that day,
perhaps, or had got news of him by a side wind. Bella’s heart beat
ever so fast at the idea.
They went up to Mrs. Dulcimer’s bedroom, a large old-fashioned
chamber, with an immense four-post bedstead and flowery chintz
curtains, a muslin-draped dressing-table, adorned with a great many
china pots, and a pin-cushion that was a noteworthy feature. Mrs.
Dulcimer’s devotional books—with a great many markers in them,
looking as if they were read immensely—were arranged on either
side of the looking-glass. She used to read Taylor’s ‘Holy Living’
while Rebecca put her hair in papers of an evening. She did not read
the ‘Holy Dying.’ It seemed a great deal too soon for that.
There was a bright fire, and the chintz-covered sofa was wheeled
in front of it. Between the fire and the sofa was Mrs. Dulcimer’s work-
table, and on the table the missionary basket full of ingenious trifles,
useful or useless. Babies’ socks, muffatees, pincushions of every
shape and design, and a variety of the aggravating family of mats.
‘Bella,’ said Mrs. Dulcimer, when they were seated on the sofa, ‘I
have something particular to say to you.’
And then the Vicar’s wife told Bella her plan for marrying Kenrick
and Beatrix, and how Beatrix had refused Kenrick on account of her
attachment to his cousin.
‘Isn’t it a pity, Bella?’ she asked, after lengthily expounding all this.
‘Yes,’ answered Bella, looking thoughtful, ‘they would have suited
each other very well, I should think.’
‘Think!’ cried Mrs. Dulcimer. ‘There’s no thinking about it. They
were made for each other.’
Mrs. Dulcimer’s couples always were made for each other. It is
odd how many of them turned out misfits.
Bella was reflecting that if Beatrix were happily married to Sir
Kenrick Culverhouse, her sin about the sealed letter would weigh
less heavily on her conscience—or, indeed, need not weigh at all.
What can any one ask more than happiness? And, in the eye of the
world, Kenrick was a much more suitable husband for a young
woman of fortune than Cyril could possibly be.
‘Now I have been thinking,’ continued Mrs. Dulcimer, sinking to a
mysterious undertone, ‘that perhaps if Beatrix could be made to think
that Cyril was fickle and inconstant, and that before he left Little
Yafford he had got to care for someone else—you, for instance,’
whispered Mrs. Dulcimer, making a little stab at Bella with her
forefinger, ‘it might cure her of her foolish attachment to him. It is
ridiculous that she should go on caring for a man who doesn’t love
her, when there is a noble young fellow who does love her
passionately, and can make her Lady Culverhouse. If she could only
be made to think that Cyril was fond of you, Bella, without actual
falsehood,’ concluded Mrs. Dulcimer, with a strong emphasis upon
the qualifying adjective, as much as to say that in so good a cause
she would not mind sailing rather near the wind.
 ‘I’m sure I don’t know how it is to be done,’ said Bella, with a
meditative air. ‘Beatrix is so self-opinionated. It is not as if she were a
weak-minded pliable girl. She is as hard as rock.’
‘But you are so clever, Bella. You could manage anything. If I were
to say now that I always thought Cyril was very fond of you—and I
did think so for a long time, as you know, dear—and if you were to
say something that would sustain that idea. We need neither of us
tell an actual story.’
‘Of course not,’ answered Bella, piously. ‘Do you suppose I would
tell a story, dear Mrs. Dulcimer?’
‘Indeed no, my love. I know how truthful you are.’
Thus it was agreed between the Vicar’s wife and her ductile
protégée that, somehow or other, Beatrix was to be persuaded that
her lover had been doubly false to her; false in abandoning her
because evil tongues maligned her, false in preferring another
woman.
 CHAPTER XIII.
‘a smile of thine shall make my bliss.’
By what serpentine twists and windings Bella Scratchell reached the
end she had in view need not be recorded. She was by nature a
creature of many curves, and all her progress in life was devious and
indirect. Enough that she succeeded in making Beatrix Harefield
believe her lost lover false and fickle, and thus undermined the girl’s
respect for the man who had renounced her. So long as Beatrix
could believe that Cyril had sacrificed his heart’s desire to his duty as
priest and teacher, she would have continued to reverence and love
him. Present or absent, he would have remained the one central
figure in her life. From the moment she was persuaded to think him
the shallow lover of a day—or indeed, worse than this, a lover who
had been drawn to her by the lure of her wealth, and who at the
bottom of his heart had always preferred Bella’s lilies and roses—
from that moment she despised him, and concentrated all the forces
of her mind in the endeavour to forget him.
‘I will never pray for him or his work again,’ she vowed to herself,
and the vow had all the savagery of a pagan oath. ‘His name shall
never pass my lips or find a place in my heart. It shall be to me as if
such a man had never lived.’
From this time there was a marked change in her manner. It was
brighter, gayer, harder than it had been before. That mournful
resignation which had distinguished her since her father’s death
gave place to a proud indifference, a careless scorn of all things and
all men, save the few friends she liked and trusted. That disgust of
life which attacks most of us at odd times, and which sometimes
afflicts even the young, had seized upon her. All things in this world
were hateful to her. Solomon, sated with wealth and glory, could not
have felt the emptiness of earthly joys more deeply than this girl of
nineteen, whose lips had scarcely touched the cup of life. She knew
herself rich, and with all good things at her disposal—beautiful
enough to command the love of men; and yet, because that one man
whom she loved had proved false and unworthy, she turned with a
sickened soul from all that earth held of hope or pleasure. Unhappily
she had not yet learned to look higher for comfort. She was not
irreligious. She firmly believed all her Church taught her to believe,
but she had not learned, like Hezekiah, to lay her trouble before the
Lord. She locked up her grief in her own heart, as something apart
from her spiritual life; and she went on conforming outwardly to all
the duties of religion, but deriving no inward solace from her faith.
Beatrix was in this mood when Mrs. Dulcimer, delighted at Bella’s
speedy success, but opining, nevertheless, that something more
must be done, was seized with a happy idea.
‘Kenrick,’ she exclaimed at tea one evening, when Kenrick had
announced his intention of going to have one more peep at
Culverhouse Castle before he embarked on board the P. and O.
steamship that was to carry him on the first stage of his journey to
India—‘Kenrick,’ cried Mrs. Dulcimer, with an excited air, ‘I really
think it is the oddest thing in the world.’
‘What, dear Mrs. Dulcimer?’ asked Kenrick, while everybody else
looked curious.
‘Why, that after knowing you all these years, and hearing you talk
so much about Culverhouse Castle, we should never have seen it.’
‘I don’t know whom you mean by we,’ said Mr. Dulcimer, ‘but I beg
to say that I spent three weeks at Culverhouse in one of my long
vacations, and a capital time I had there. The Avon is one of the
finest salmon rivers I ever fished in.’
‘Ah, that was in Kenrick’s father’s time,’ said Mrs. Dulcimer; ‘but
though you may be perfectly quainted with the place, Clement, I
have never seen it.’
‘That is your own fault,’ exclaimed Kenrick. ‘Nothing would make
me happier than to receive you there. It would be something in the
style of the famous reception at Wolf’s Crag, perhaps, especially if it
were in the close time for salmon; but you should have a hearty
welcome, and I shouldn’t feel my position so keenly as the Master of
Ravenswood felt his.’
‘There would be no Lucy Ashton in the case,’ put in the Vicar,
innocently.
‘And should we really not put you out if we came?’ asked Mrs.
Dulcimer.
‘Not the least in the world. You would have to live as plainly as
Eton boys, that is all. My housekeeper can roast a joint and boil a
potato. I think she might even manage a bit of fish, and a rhubarb
tart. We would not quite starve you, and I know you would be
charmed with the dear old place; but if you are coming you must
make up your minds very quickly. My time is up on the 24th.’
‘We could make up our minds in half an hour, if Clement would
consent,’ answered Mrs. Dulcimer, ‘It would be such a delightful
change for Beatrix. Mr. Namby has been recommending her a
change of air and scene for ever so long; and it is much too cold for
the sea-side. A week in Hampshire would do her a world of good.’
‘Pray do not think of me,’ said Beatrix, ‘I had rather go home while
you are away.’
‘I thought this was your home now, Beatrix,’ remonstrated the
Vicar.
‘It is the only house that has ever seemed like home,’ the heiress
answered, sadly.
‘Of course you will go with us, if we go,’ exclaimed Mrs. Dulcimer.
‘You are our adopted daughter, and we expect you to go everywhere
with us. We don’t even consult you. It is quite a matter of course. I
have set my heart on seeing Culverhouse Castle, and the visit will be
the very thing to do you good. I am sure Mr. Namby would say so if I
asked him about it. So, Clement dear, if you would let Mr. Rodger do
duty for just one Sunday, we might spend ten days at Culverhouse
very easily.’
Mr. Rodger was the new curate, a painstaking youth, with sandy
hair and a large round face like the setting sun.
 Mr. Dulcimer was at first disinclined to listen to his wife’s
suggestions. The journey was long and expensive, and there
seemed to be no justifiable reason for undertaking it; but the Vicar
was an indulgent husband, and he was very fond of salmon fishing,
so the discussion ended by his giving his consent, and it was
arranged that he and the two ladies should join Sir Kenrick at the
castle two days after the young man’s arrival there.
Beatrix consented to go to Culverhouse, just as she would have
consented to go to Buxton, Harrogate, or Scarborough, if Mrs.
Dulcimer had wished her to go there. That disgust of life which had
taken possession of her, since the overthrow of her faith in Cyril, left
her indifferent to all things. She let her maid pack a portmanteau,
and get all things ready for the journey. The girl, Mary, who had
waited upon her at the Water House, had accompanied her to the
Vicarage. She was not an accomplished attendant, but she was
faithful, and Beatrix liked her.
Culverhouse Castle was six miles from a railway station; one of its
chief merits, as Kenrick asserted proudly. He was standing on the
platform when the train arrived, and received his guests with as
much enthusiasm as if he had not seen them for a year or so. He
had a carriage ready to drive them across to Culverhouse.
It was a lovely drive in the spring evening, the sun setting behind
the wooded hills, and all the soft rustic scene steeped in warm yellow
light. Culverhouse was on the edge of the New Forest, and the road
from the station to the castle went through a region of alternate
pasture and woodland. Meadows and banks were yellow with
primroses; the earliest ferns were showing their tender green; the
dog-violets shone like jewels amongst the grass; and the woods
were full of white wind-flowers that shivered at every whisper of the
April breeze. To Beatrix it all seemed very lovely. She breathed more
freely in this unknown world, where nobody had ever spoken evil of
her. There was an infinite relief in having left Little Yafford.
When Culverhouse Castle rose before them on the other side of
the river, Beatrix thought it the loveliest place she had ever seen.
The Avon widened to a smooth lake, and beyond it rose the grave
old Gothic towers, like a castle in a fairy tale. Beatrix turned to
Kenrick, with the kindest smile she had ever bestowed upon him.
‘It is a delicious old place,’ she exclaimed. ‘I cannot wonder that
you are proud of it.’
Kenrick was delighted. His face glowed with pride of race and love
for the house of his birth. They were driving through the little village
street, all the old men and women, young men and maidens, doing
them obeisance as they passed. Then they crossed the bridge and
drove under the gateway, which was a couple of centuries older than
the castle itself, and a minute later Kenrick passed into the banquet-
hall of his ancestors, with Beatrix on his arm. He had offered his arm
to Mrs. Dulcimer, but that match-making matron had bidden him take
care of Miss Harefield, so he had the happiness of leading Beatrix
across the threshold. ‘Jest as if they’d been married and he’d been
a-bringin’ she home,’ old Betsy Mopson said afterwards to her
husband, gardener and man-of-all-work. ‘Her be a rare beauty, her
be.’
Kenrick had done wonders in his two days of preparation. He had
got in a brace of apple-faced young women, from the village, one a
housemaid out of place, the other her younger sister, still on her
promotion, but ready to do anything she was bidden. The old rooms
had been furbished up. Traces of decay were still visible in every
part of the house, but dust and cobwebs had been swept away, and
a general air of freshness and purity pervaded the good old rooms.
Beatrix was enraptured with everything. She seemed to forget her
sorrows amidst these new surroundings. Her life had been spent in a
prison-house, and this first taste of liberty was sweet. After all,
perhaps, even for her, deserted and cast off by the one man she had
ever loved, life held something worth having.
Kenrick led his young guest all round the ruins next morning,
before breakfast. They were both early risers, and had found each
other in the garden before Mr. and Mrs. Dulcimer had left their
rooms. They went into the cloistered quadrangle, where the roses
flourished in summertime, and where now the wallflowers flashed
golden and ruby upon the old gray stones, with colours as vivid as
the stained glass that had once filled the place with rainbow light.
Kenrick showed Beatrix the plan of the vanished abbey—the nave
here, the transept there, the chancel and apse beyond. Everything
was indicated by stones embedded at intervals in the close-cropped
turf, where the sheep browsed happily, unconscious of the sanctified
splendour that had preceded them, the white-robed choir, and
swinging censers, the banners and jewelled crosiers that had passed
beneath the Gothic arches which had once spanned that fair
pasture. Kenrick seemed as sorry for the evanishment of the abbey
as if he had been a papist of the deepest dye.
‘It is dreadful to think that a great part of the house is built out of
the abbey stones,’ he said. ‘I sometimes wonder it doesn’t tumble on
our heads. But tradition says the monks of Culverhouse were lazy
and ignorant, and that there was only one book, an ancient treatise
upon Hunting and Fishing, found in this abode of monastic learning,
when its treasures were confiscated.’
Beatrix had explored every inch of the grounds before the long-
disused gong, which in days past had called poor lonely Lady
Culverhouse to her anchorite repasts, sounded hoarsely from the
hall. Mr. and Mrs. Dulcimer were standing in the porch, scenting the
morning air, when Kenrick and his companion went in.
‘How well the dear girl looks!’ said the Vicar’s wife; ‘the change
has done her good already. You are enjoying Culverhouse, are you
not, Beatrix?’
‘I am very glad to be away from Little Yafford,’ Beatrix answered,
frankly.
‘In that case you ought never to go back,’ said Kenrick.
‘What a selfish remark!’ exclaimed Mrs. Dulcimer, hypocritically.
‘How do you suppose I am to exist without Beatrix, after having had
her as my adopted daughter for the last three months?’
‘What do you think of the weather for salmon-fishing?’ asked the
Vicar, contemplating the bright blue sky with a discontented look that
was hardly becoming in a Christian. ‘We could do with a little more
cloud, couldn’t we, Ken? But, as time is short, we must make the
best of things. I shall expect you to set off with me directly after
breakfast.’
‘I shall be delighted,’ answered Kenrick; but he did not mean to
give up his day to salmon-fishing.
He contrived to set the Vicar going, in a spot where there was
every chance of good sport, and then, under the pretence of having
orders to give about the dinner, ran home across the low-lying water
meadows like a boy let loose from school. He found Mrs. Dulcimer
expounding the chief features of the mansion—which she had never
seen before—to Beatrix, while Betty Mopson stood by in attendance
upon them, and made a running commentary, in a Hampshire
dialect, which was like a foreign language to the strangers from the
north.
‘Hah! Lady Culverhouse wur a good ’ooman,’ said Mrs. Mopson.
‘Thur bean’t many like she. This be the room where hur died. Her
wur a rale lady. And Sir Kenrick, him takes after she.’
Kenrick came in time to hear his praises. He sent Betty back to her
kitchen.
‘We shall not get a decent luncheon if you waste all your morning
chattering here, Betty,’ he said, and Betty departed, grinning and
ducking, and with a fixed idea that the young lady with the dark
‘haiyur,’ was to be the next Lady Culverhouse.
Kenrick spent a happy day in attendance upon the two ladies. He
forgot everything, in the intoxicating delight of the present, forgot that
this holiday in life was to be of the briefest, and that a fortnight hence
he was to be tossing off Gibraltar in a Peninsular and Oriental
steamer. Beatrix seemed happy also, or, at least, she appeared to be
in a condition of placid contentment which was not unpromising.
The Vicar was successful with his rod, and came home radiant.
Betty Mopson had surpassed herself in the preparation of a
substantial English dinner. Everything went smoothly and well with
Sir Kenrick.
Next day he carried off his guests to see some of the lions of the
neighbourhood—a fine old abbey church sorely neglected—a castle