Pharmacology in Drug Discovery Understanding Drug

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Table of Contents
Cover image
Front-matter
Copyright
Dedication
Foreword
Acknowledgements
Chapter 1. Pharmacology
Chapter 2. Drug Affinity and Efficacy
Chapter 3. Predicting Agonist Effect
Chapter 4. Drug Antagonism
Chapter 5. Allosteric Drug Effects
Chapter 6. Enzymes as Drug Targets
Chapter 7. Pharmacokinetics I
Chapter 8. Pharmacokinetics II
Chapter 9. In Vivo Pharmacology
Chapter 10. Safety Pharmacology
Appendix A. Answers to Chapter Questions
Appendix B. Derivations and Proofs
Index
Front-matter
Pharmacology in Drug Discovery
UNDERSTANDING DRUG RESPONSE
PHARMACOLOGY IN DRUG DISCOVERY
UNDERSTANDING DRUG RESPONSE
Terry P. Kenakin, PhD, Department of Pharmacology, University of North
Carolina School of Medicine, Chapel Hill, NC, USA

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Dedication
As Always … for Debbie
Foreword
Terry Kenakin, Ph.D.
Research Triangle Park, 2011

In the scheme of science, pharmacology is a relatively new scientific

discipline branching off, in the last century, from the older more established
science of physiology. The term can have a range of meanings from the
study of drug action to the design of new drugs. These nuances in
terminology are associated with the way pharmacology is presented and
taught. To a medical student, pharmacology may mean the properties of
therapeutic drugs and the study of how they are used in therapy (i.e.,
therapeutics). To a researcher it may mean the study of drug mechanism of
action. To a scientist working in drug discovery, it may mean the
application of medicinal chemistry to modify physiology for therapeutic
benefit.
This text is designed to introduce all students who may need to interpret a
change in physiology induced by a chemical substance. Physiological
systems customize chemical signal input to their own needs; thus the same
drug can have different effects in different physiological systems.
Pharmacology is unique in that it furnishes the tools to analyze these
different behaviors and trace them to their root cause, i.e., the molecular
mechanism of action. This enables predictions of drug behavior to be made
in all systems, an invaluable tool for drug discovery since almost all drugs
are developed in test systems far removed from the therapeutic one. This
text should enable the reader to interpret drug dose–response data and make
mechanistic inferences at the molecular level.
Acknowledgements
I wish specifically to thank Kristine Jones and April Graham of Elsevier for
patient support of this project and so very much help. I also wish to thank
Dr. Angela Finch, University of Sydney, for valuable comments and
guidance. The excellent artwork of Candy Webster was indispensable in the
production of this book and I wish to thank her for her wonderful efforts. I
am indebted to GlaxoSmithKline for support during the preparation of this
book and to the University of North Carolina School of Medicine for giving
me the means to explore pharmacology and apply it to drug discovery.
Finally, I am very grateful to my wife and family for boundless patience
during the writing of this book.
Chapter 1. Pharmacology

The Chemical Control of Physiology

Outline
Pharmacology and Cellular Drug Response 1
New Terminology 2
Pharmacological Targets 2
Dose–Response Curves 5
Linking Observed Pharmacology With Molecular Mechanism 12
Descriptive Pharmacology: I 15
Summary 16
This chapter discusses how drug response is quantified with dose–response curve and how this leads to classifications of drugs
based on effect (i.e., drugs that produce observed change in cellular processes are termed agonists and those that block such effects
are antagonists). The dependence of observed potency of an agonist upon two drug-related parameters (affinity, efficacy) and two
cell-dependent parameters (target density and efficiency of target coupling) is also discussed. The way that different tissues
process drug stimulus to provide tissue response is considered, along with the null method which can be used to negate cell-
dependent effects on drug activity to provide system-independent indices of drug activity. This is imperative in pharmacology as
drugs are almost always studied in test systems, and not in therapeutic one(s).
Keywords
affinity, agonists, antagonists, dose–response curves, efficacy, null method, potency.

By the end of this chapter the reader should be able to understand how drug
response is quantified by the use of dose–response curves, the way in which
different tissues process drug stimulus to provide tissue response and what
qualifies a drug to be classified either as an agonist or antagonist.
Pharmacology and Cellular Drug Response
Pharmacology (from the Greek φάρμακον, pharmakon, “drug” and -λογία, -
logia, the study of) concerns drug action on physiological systems
(physiology from the Greek φύσις, physis, “nature, origin” and -λογία, -
logia is the study of the mechanical, physical and biochemical functions of
living organisms). With regard to the application of pharmacology to the
discovery of drugs for therapeutic benefit, the main focus of
pharmacological theories, procedures and mechanisms relates to the
chemical control of physiological processes. Insofar as the understanding
of these physiological processes benefits the pharmacologic pursuit of
drugs, pharmacology and physiology are intimately related. However, it
will also be seen that complete understanding of the physiologic processes
involved is not a prerequisite to the effective use of pharmacology in the
drug discovery process. In fact, often an operational approach is utilized
whereby the complexity of the physiology is represented by simple
surrogate mathematical functions.
A unique feature of pharmacology is that the effect of the drug is often
observed indirectly, that is, while the drug affects a select biochemical
process in the cell, the outcome to an observer is an overall change in the
state of the whole organism, and this is often the result of multiple
interacting cellular processes. A major aim of pharmacology is to define the
molecular events in initiating drug effects, since these define the action of
drugs in all systems. If quantified correctly, this information can be used to
predict drug effect at the pharmacological target in all systems including
therapeutic one(s). At this point, it is useful to define what is meant by
pharmacological target.
New Terminology
The following new terms will be introduced in this chapter:
• Affinity: The propensity of a drug molecule to associate closely with a
drug target.
• Agonists: Drugs that produce an observable change in the state of a
physiological system.
• Antagonists: Drugs that may not produce a direct effect, but do interfere
with the production of cellular response to an agonist.
• Dose–response curve: The relationship between doses (if the drug is
used in vivo) or concentrations (if used in vitro) of a drug and
pharmacologic effect.
• Drug target: The protein (or in some cases DNA, mRNA) to which a
drug binds to elicit whatever pharmacologic effect it will produce. These
proteins can be seven transmembrane (or one transmembrane) receptors,
enzymes, nuclear receptors, ion channels or transport proteins.
• EC50: Concentration of agonist producing half the maximal response to
the same agonist; usually expressed for calculation and statistical
 manipulation as the pEC50, negative logarithm of the molar concentration
producing 50% response.
• Efficacy: The change in state of the drug target upon binding of a drug.
• Efficiency of target coupling: The relationship between the net quanta
of activation given to a cell and the number of drug targets available for
activation.
• Full agonists: Agonists that produce the full maximal response that the
system can produce.
• Null method: The comparison of equiactive concentrations (or doses) of
drug to cancel the cell-based processing of drug response. The assumption
is that equal responses to a given agonist are processed in an identical
manner by the cell.
• Partial agonists: Agonists that produce a maximal response that is of
lower magnitude than the maximal response that the system can produce
to maximal stimulation.
• pEC50: The negative logarithm of EC50 values. For arithmetic and/or
statistical manipulation, numbers must be normally distributed. This is
true only of pEC50s, not of EC50s; thus all averages, estimates or error and
statistical procedures must use pEC50.
• Potency: The concentration (usually molar) of drug needed to produce a
defined response or effect.
• Target density: The concentration of drug targets at the site of
activation, i.e., on the cell surface for receptors.
Pharmacological Targets
The term “pharmacological target” refers to the biochemical entity to which
the drug first binds in the body to elicit its effect. There are a number of
such entities targeted by drug molecules. In general, they can be proteins
such as receptors, enzymes, transporters, ion channels, or genetic material
such as DNA. The prerequisite for pharmacologic targets is that they have
the ability to discern differences in electronic structure minute enough to be
present in small drug-like molecules; in this regard the most predominant
targets for drugs are protein in nature. Proteins have the tertiary three-
dimensional structure necessary for detailed definition of the electronic
forces involved in small molecule binding. Signals are initiated through
complementary binding of drug molecules to protein conformations that
have a physiological purpose in the cell. The act of these molecules binding
to the protein will change it, and with that change a pharmacologic effect
will occur.
At this point, it is worth considering the beginning and end processes. The
first process is the drug binding to the target. The result(s) of this process
are totally dependent on the affinity and efficacy of the drug. These are drug
parameters unique to its chemical structure. In pharmacologic terms, this is
the most important effect, since it occurs in each and every tissue and organ
possessing the target. Therefore, characterization of this event enables a
general quantification of drug-target activity to be made in the test system,
which will also be true for all systems including the therapeutic one.
Therefore, the characterization of affinity and efficacy become the
primary aim of pharmacologic analysis. However, it can be seen that the
various (and variable) biochemical reactions linking the target to cellular
response intervene, thereby causing a tissue-dependent abstraction of the
link between affinity and efficacy and observed cellular potency. The
magnitude of this abstraction depends upon the number of responding target
units and the efficiency of target coupling.
The major protein target classes are membrane receptors, enzymes, ion
channels and transporter proteins. Of these, the most prominent drug targets
are receptors. While there are a number of types of receptor, one of the most
important from the standpoint of therapeutic drug targets is seven
transmembrane receptors (7TMRs). These are so-called because they span
the cell membrane seven times to form complex recognition domains both
outside and inside the cell. These proteins are capable of recognizing
chemicals such as hormones and neurotransmitters present in the
extracellular space, and transmit signals from these to the cell interior. Due
to the fact that these are on the cell surface and thus exposed to the
extracellular space, these entities were the subject of experiments that
originally defined the receptor concept (see Box 1.1 for history).

Box 1.1 The Evolution of the Receptor Concept in Pharmacology

Numerous physiologists and pharmacologists contributed to the
concept of “receptor” as minimal recognition units for chemicals in
cells. Paul Ehrlich (1854–1915) studied dyes and bacteria and
determined that there are “chemoreceptors” (he proposed a
collection of “amboreceptors,” “triceptors” and “polyceptors”) on
parasites, cancer cells and microorganisms that could be exploited
therapeutically.

John Newport Langley (1852–1926), as Chair of the Physiology

Department in Cambridge, studied the drugs jaborandi (containing
the alkaloid pilocarpine) and atropine. He concluded that receptors
were “switches” that received and generated signals and that these
switches could be activated or blocked by specific molecules.
 A. J. Clark (1885–1941), who could be considered the father of
modern receptor pharmacology, was one of the first to suggest from
studies of acetylcholine and atropine that a unimolecular interaction
occurs between a drug and a “substance on the cell.” As stated by
Clark: “… it is impossible to explain the remarkable effects observed
except by assuming that drugs unite with receptors of a highly
specific pattern…”

Historically, while the actual physical nature of receptors was unknown, it

was realized that a distinct entity on the cell surface allows cells to
recognize drugs and read the chemical information encoded in them. Early
concepts of receptors likened them to locks with drugs as keys (i.e., as
stated by the biologist 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…”).
The main value of receptors is that they put order into the previously
disordered world of physiology. For example, it has been observed that the
hormone epinephrine produces a wealth of dissimilar physiological
responses such as bronchiole muscle relaxation, cardiac muscle positive
inotropy, chronotropy and lusitropy, melatonin synthesis, pancreatic,
lacrimal and salivary gland secretion, decreased stomach motility, urinary
bladder muscle relaxation, skeletal muscle tremor and vascular relaxation.
The understanding of how such a vast array of biological responses could
be mediated by a single hormone is difficult until it is realized that these
processes are all mediated by the interaction of epinephrine with a single
receptor protein, in this case the β-adrenoceptor. Thus, when this receptor is
present on the surface of any given cell it will respond to epinephrine, and
the nature of that response will be determined by the encoding of the
receptor excitation produced by epinephrine to the cytosolic biochemical
cascades controlling cellular function. In a conceptual sense, the term
“receptor” can refer to any single biological entity that responds to drugs
(i.e., enzymes, ion channels, transport proteins, DNA and structures in the
nucleus). This information is transmitted through changes in protein shape
(conformation) i.e., the drug does not enter the cell nor does the receptor
change the nature of the drug (as an enzyme would).
Pharmacologic targets can be used to modify physiological processes.
Specifically, chemicals can be used to cause activation, blockade or
modulation of protein receptors and ion channel targets. For enzymes and
transporter proteins the main drug effect is inhibition of ongoing basal
activity of these targets (Chapter 6 discusses these targets in detail). Another
difference between these target classes is location; while receptors, ion
channels and transporter proteins are usually found on the cell surface
(exposed to the extracellular space), enzymes are most often found in the
cytosol of the cell (drugs must enter the cell to act on enzymes). Exceptions
to this general rule are nuclear receptors which reside in the cell nucleus.
Finally, it should be recognized that there are other drug targets present in
the cell, such as DNA, and that chemicals can have physical effects (i.e.,
membrane stabilization) that can change cellular function.
Pharmacologic effects on cells can include a wide variety of outcomes,
from changes in the mechanical function of cells (i.e., cardiac contractility,
contraction of bronchiole smooth muscle), biochemical metabolic effects
(levels of second messengers such as calcium ion or cyclic AMP) and
modulation of basal activity (level of catalytic degradation of cyclic AMP
by enzymes such as phosphodiesterase, rate of uptake of neuroamines such
as norepinephrine and serotonin).
It is worth considering the process of target choice in the drug discovery
process. Specifically, effective prosecution of any drug target requires a
minimal effort in resources and time (perhaps 1 to 2 years per target), thus it
can be seen how an incorrect choice of target could lead to a serious
dissimulation in the drug discovery process. While there are considerations
in target choice, such as target tractability (how difficult it is to produce a
molecule to alter the behavior of the target), one of the most important
factors is a strong association with the disease that is being treated. It has
been estimated that there are approximately 600 to 1500 possible drug
targets that may be valid to pursue for therapy. These are made up of genes
that are known to be associated with diseases and that also code for protein
that may be modified through binding to a small molecule. [1] No discovery
program could pursue a number of genes close to the number available,
making target validation a very important step in the process. Table 1.1
shows some of the factors involved in the process of target validation, with
particular reference to the problem of HIV-1 viral entry to cause Acquired
Immune Deficiency Syndrome (AIDS). As a preface to the discussion of
cellular drug effect, it is useful to consider the major pharmacological tool
used to quantify it, namely, the dose–response curve.

Table 1.1 Factors Relevant to Target Validation with Reference to AIDS

Factor CCR5 in AIDS Reference
• Target is linked to sensitivity to • CCR5 receptors must be present on cell surface for HIV-1
disease infection [2] and [3]

• Cell level of target alters sensitivity • Down-regulation of CCR5 leads to resistance to HIV-1
and course of disease infection [4]

• Genetically high levels of CCR5 lead to rapid progression

to AIDS [5]

• Interference with target will not lead • CCR5 knockout mouse1 lacks the receptor but is otherwise [6]
to harm healthy

• Ligands for target interfere with • CCR5 interaction with chemokines interfere with HIV-1 [7], [8], [9], [10] and
disease infection [11]

• Patients with high circulating levels of chemokine have

retarded progression to AIDS [12] and [13]

• Δ32 deletion in CCR5 gene leads to lack of receptor [14], [15], [16], [17]
• Specific genetic association
expression and complete resistance to AIDS and [18]
1Genetically altered mouse that does not naturally express the CCR5 receptor.

Dose–Response Curves
A characteristic feature of drugs acting on a specific target in a
physiological system is that there will be a graded increase in response with
an increase in drug concentration (dose). If drug effect can be observed
directly, then the magnitude of effect can be displayed as a function of drug
concentration in the form of a dose–response curve. For example,
epinephrine is known to cause increased heart rate in humans; Fig. 1.1
shows how increasing doses of epinephrine produce increases in heart rate.
The curve-defining dose and resulting observed response can be used as
shorthand to characterize the effect of the drug in the system. This
relationship can then be used to predict what any dose of the drug will do in
the system, in the form of an empirically derived line joining the observed
data points. Figure 1.2 shows the increased heart rate as a function of
epinephrine concentration. The lines joining the data points infer that there
is a continuous relationship between epinephrine dose and heart rate. Such
an empirical relationship can allow interpolation of values, but the