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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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 Contents

Preface xiii 2.6.3 Differences in Receptor Density 36

2.6.4 Target-Mediated Trafficking
of Stimulus 36
1. What is Pharmacology? 1 2.7 Receptor Desensitization and
Tachyphylaxis 38
1.1About This Book 1
2.8 The Measurement of Drug Activity 39
1.2What is Pharmacology? 1
2.9 Advantages and Disadvantages
1.3The Receptor Concept 3 of Different Assay Formats 41
1.4Pharmacological Test Systems 4 2.10 Drug Concentration as an Independent
1.5The Nature of Drug Receptors 7 Variable 42
1.6Pharmacological Intervention 2.10.1 Dissimulation in Drug
and the Therapeutic Landscape 7 Concentration 42
1.7 System-Independent Drug Parameters: 2.10.2 Free Concentration of Drug 43
Affinity and Efficacy 10
2.11 Chapter Summary and Conclusions 44
1.8 What is Affinity? 13
2.12 Derivations 44
1.9 The Langmuir Adsorption Isotherm 13
2.12.1 Series Hyperbolae Can Be
1.10 What is Efficacy? 15
Modeled by a Single Hyperbolic
1.11 Dose
Response Curves 16
Function 45
1.11.1 Potency and Maximal Response 17 2.12.2 Successive Rectangular
1.11.2 P-Scales and the Representation Hyperbolic Equations Necessarily
of Potency 18 Lead to Amplification 45
1.12 Chapter Summary and Conclusions 19 2.12.3 Saturation of Any Step in a Stimulus
1.13 Derivations: Conformational Selection Cascade by Two Agonists Leads
as a Mechanism of Efficacy 20 to Identical Maximal Final
References 21
Responses for the Two Agonists 45
2.12.4 Procedure to Measure Free
Drug Concentration in the
2. How Different Tissues Process Drug Receptor Compartment 45
Response 23 References 46
Further Reading 46
2.1 Drug Response as Seen Through
the “Cellular Veil” 23
2.2 The Biochemical Nature of
3. Drug
Receptor Theory 47
Stimulus
Response Cascades 25
2.3 The Mathematical Approximation of 3.1 About This Chapter 47
Stimulus
Response Mechanisms 28 3.2 Drug
Receptor Theory 48
2.4 Influence of Stimulus
Response 3.3 The Use of Mathematical Models in
Cascades on Dose
Response Curve Pharmacology 49
Slopes 29 3.4 Some Specific Uses of Models in
2.5 System Effects on Agonist Response: Pharmacology 49
Full and Partial Agonists 30 3.5 Classical Model of Receptor Function 55
2.6 Differential Cellular Response to 3.6 The Operational Model of Receptor
Receptor Stimulus 33 Function 56
2.6.1 Choice of Response Pathway 33 3.7 Two-State Theory 58
2.6.2 Augmentation or Modulation 3.8 The Ternary Complex Model 58
of Stimulus Pathway 35 3.9 The Extended Ternary Complex Model 59

vii
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

80 1.11.2 P-Scales and the Representation of

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.