Introduction

Pharmacology as an academic discipline, loosely defined as the study of the effects of chemical substances on living systems, is so broad in its sweep that it encompasses all aspects of drug discovery, ranging from the molecular details of the interaction between the drug molecule and its target to the economic and social consequences of placing a new therapeutic agent on the market. In this chapter we consider the more limited scope of ‘classical’ pharmacology, in relation to drug discovery. Typically, when a molecular target has been selected, and lead compounds have been identified which act on it selectively, and which are judged to have ‘drug-like’ chemical attributes (including suitable pharmacokinetic properties), the next stage is a detailed pharmacological evaluation. This means investigation of the effects, usually of a small number of compounds, on a range of test systems, up to and including whole animals, to determine which, if any, is the most suitable for further development (i.e. for nomination as a drug candidate). Pharmacological evaluation typically involves the following:

• Selectivity screening, consisting of in vitro tests on a broad range of possible drug targets to determine whether the compound is sufficiently selective for the chosen target to merit further investigation

• Pharmacological profiling, aimed at evaluating in isolated tissues or normal animals the range of effects of the test compound that might be relevant in the clinical situation. Some authorities distinguish between primary pharmacodynamic studies, concerning effects related to the selected therapeutic target (i.e. therapeutically relevant effects), and secondary pharmacodynamic studies, on effects not related to the target (i.e. side effects). At the laboratory level the two are often not clearly distinguishable, and the borderline between secondary pharmacodynamic and safety pharmacology studies (see below) is also uncertain. Nevertheless, for the purposes of formal documentation, the distinction may be useful

• Testing in animal models of disease to determine whether the compound is likely to produce therapeutic benefit

• Safety pharmacology, consisting of a series of standardized animal tests aimed at revealing undesirable side effects, which may be unrelated to the primary action of the drug. This topic is discussed in Chapter 15.

The pharmacological evaluation of lead compounds does not in general follow a clearly defined path, and often it has no clearcut endpoint but will vary greatly in its extent, depending on the nature of the compound, the questions that need to be addressed and the inclinations of the project team. Directing this phase of the drug discovery project efficiently, and keeping it focused on the overall objective of putting a compound into development, is one of the trickier management tasks. It often happens that unexpected, scientifically interesting data are obtained which beg for further investigation even though they may be peripheral to the main aims of the project. From the scientists’ perspective, the prospect of opening up a new avenue of research is highly alluring, whether the work contributes directly to the drug discovery aims or not. In this context, project managers need to bear in mind the question: Who needs the data and why? – a question which may seem irritatingly silly to a scientist in academia but totally obvious to the commercial mind. The same principles apply, of course, to all parts of a drug discovery and development project, but it tends to be at the stage of pharmacological evaluation that conflicts first arise between scientific aspiration and commercial need.

An important principle in pharmacological evaluation is the use of a hierarchy of test methods, covering the range from the most reductionist tests on isolated molecular targets to much more elaborate tests of integrated physiological function. Establishing and validating such a series of tests appropriate to the particular target and indication being addressed is one of the most important functions of pharmacologists in the drug discovery team. In general, assays become more complicated, slow and expensive, and more demanding of specialist skills as one moves up this hierarchy.

The strengths and weaknesses of these test systems are summarized in Table 11.1.

Table 11.1 Characteristics of pharmacological test systems

Pharmacological characterization of a candidate compound often has to take into account active metabolites, based on information from drug metabolism and pharmacokinetics (DMPK) studies (see Chapter 10). If a major active metabolite is identified, it will be necessary to synthesize and test it in the same way as the parent compound in order to determine which effects (both wanted and unwanted) relate to each. Particular problems may arise if the metabolic fate of the compound shows marked species differences, making it difficult to predict from animal studies what will happen in humans.

Although most of the work involved in pharmacological characterization of a candidate drug takes place before clinical studies begin, it does not normally end there. Both ongoing toxicological studies and early trials in man may reveal unpredicted effects that need to be investigated pharmacologically, and so the discovery team needs to remain actively involved and be able to perform experiments well into the phase of clinical development. They cannot simply wave the compound goodbye once the discovery phase is completed.

Screening for selectivity

The selectivity of a compound for the chosen molecular target needs to be assessed at an early stage. Compounds selected for their potency, for example on a given amine receptor, protease, kinase, transporter or ion channel, are very likely to bind also to related – or even unrelated – molecular targets, and thereby cause unwanted side effects. Selectivity is, therefore, as important as potency in choosing potential development candidates, and a ‘selectivity screen’ is usually included early in the project. The range of targets included in such a screen depends very much on the type of compound and the intended clinical indication. Ligands for monoamine receptors and transporters form a large and important group of drugs, and several contract research organizations (e.g. CEREP, MDL) offer a battery of assays – mainly binding assays, but also a range of functional assays – designed to detect affinity for a wide range of receptors, transporters and channels. In the field of monoamine receptors, for example, it is usually important to avoid compounds that block or activate peripheral muscarinic receptors, adrenergic receptors or histamine (particularly H₁) receptors, because of the side effects that are associated with these actions, and a standard selectivity test battery allows such problems to be discovered early. Recently, several psychotropic and anti-infective drugs have been withdrawn because of sudden cardiac deaths, probably associated with their ability to block a particular type of potassium channel (known as the hERG channel; see Chapter 16) in myocardial cells. This activity can be detected by electrophysiological measurements on isolated myocardial cells, and such a test is now usually performed at an early stage of development of drugs of the classes implicated in this type of adverse reaction.

Interpretation of binding assays

Binding assays, generally with membrane preparations made from intact tissues or receptor-expressing cell lines, are widely used in drug discovery projects because of their simplicity and ease of automation. Detailed technical manuals describing the methods used for performing and analysing drug binding experiments are available (Keen, 1999; Vogel, 2002). Generally, the aim of the assay is to determine the dissociation constant, K_D, of the test compound, as a measure of its affinity for the receptor. In most cases, the assay (often called a displacement assay) measures the ability of the test compound to inhibit the binding of a high-affinity radioligand which combines selectively with the receptor in question, correction being made for ‘non-specific’ binding of the radioligand.

In the simplest theoretical case, where the radioligand and the test compound bind reversibly and competitively to a homogeneous population of binding sites, the effect of the test ligand on the amount of the radioligand specifically bound is described by the simple mass-action equation:

(1)

where B = the amount of radioligand bound, after correcting for non-specific binding, B_max = the maximal amount of radioligand bound, i.e. when sites are saturated, [A] = radioligand concentration, K_A = dissociation constant for the radioligand, [L] = test ligand concentration, and K_L = dissociation constant for the test ligand.

By testing several concentrations of L at a single concentration of A, the concentration, [L]₅₀, needed for 50% inhibition of binding can be estimated. By rearranging equation 1, K_L is given by:

(2)

This is often known as the Cheng–Prusoff equation, and is widely used to calculate K_L when [L]₅₀, [A] and K_A are known. It is important to realize that the Cheng–Prusoff equation applies only (a) at equilibrium, (b) when the interaction between A and L is strictly competitive, and (c) when neither ligand binds cooperatively. However, an [L]₅₀ value can be measured for any test compound that inhibits the binding of the radioligand by whatever mechanism, irrespective of whether equilibrium has been reached. Applying the Cheng–Prusoff equation if these conditions are not met can yield estimates of K_L that are quite meaningless, and so it should strictly be used only if the conditions have been shown experimentally to be satisfied – a fairly laborious process. Nevertheless, Cheng–Prusoff estimates of ligand affinity constants are often quoted without such checks having been performed. In most cases it would be more satisfactory to use the experimentally determined [L]₅₀ value as an operational measure of potency. A further important caveat that applies to binding studies is that they are often performed under conditions of low ionic strength, in which the sodium and calcium concentrations are much lower than the physiological range. This is done for technical reasons, as low [Na⁺] commonly increases both the affinity and the B_max of the radioligand, and omitting [Ca²⁺] avoids clumping of the membrane fragments. Partly for this reason, ligand affinities estimated from binding studies are often considerably higher than estimates obtained from functional assays (Hall, 1992), although the effect is not consistent, presumably because ionic bonding, which will be favoured by the low ionic strength medium, contributes unequally to the binding of different ligands. Consequently, the correlation between data from binding assays and functional assays is often rather poor (see below). Figure 11.1 shows data obtained independently on 5HT₃ and 5HT₄ receptors; in both cases the estimated K_D values for binding are on average about 10 times lower than estimates from functional assays, and the correlation is very poor.

Fig. 11.1 Correlation of binding and functional data for 5HT receptor ligands. (A) 5HT₃ receptors, (B) 5HT₄ receptors.

Data from Heidempergher et al., 1997. Data from Yang et al., 1997.

Pharmacological profiling

Pharmacological profiling aims to determine the pharmacodynamic effects of the new compound – or more often of a small family of compounds – on in vitro model systems, e.g. cell lines or isolated tissues, normal animals, and animal models of disease. The last of these is particularly important, as it is intended to give the first real pointer to therapeutic efficacy as distinct from pharmacodynamic activity. It is valuable to assess the activity of the compounds in a series of assays representing increasingly complex levels of organization. The choice of test systems depends, of course, on the nature of the target. For example, characterization of a novel antagonist of a typical G-protein-coupled receptor might involve the following:

• Ligand-binding assay on membrane fragments from a cell line expressing the cloned receptor

• Inhibition of agonist activity in a cell line, based on a functional readout (e.g. raised intracellular calcium)

• Antagonism of a selective agonist in an isolated tissue (e.g. smooth muscle, cardiac muscle). Such assays will normally be performed with non-human tissue, and so interspecies differences in the receptor need to be taken into account. Sometimes specific questions have to be asked about effects on human tissues for particular compounds and then collecting viable tissues to use becomes a major challenge

• Antagonism of the response (e.g. bronchoconstriction, vasoconstriction, increased heart rate) to a selective receptor agonist in vivo. Prior knowledge about species specificity of the agonist and antagonist is important at this stage.

Pharmacological profiling is designed as a hypothesis-driven programme of work, based on the knowledge previously gained about the activity of the compound on its specific target or targets. In this respect it differs from safety pharmacology (see below), which is an open-minded exercise designed to detect unforeseen effects. The aim of pharmacological profiling is to answer the following questions:

• Do the molecular and cellular effects measured in screening assays actually give rise to the predicted pharmacological effects in intact tissues and whole animals?

• Does the compound produce effects in intact tissues or whole animals not associated with actions on its principal molecular target?

• Is there correspondence between the potency of the compound at the molecular level, the tissue level and the whole animal level?

• Do the in vivo potency and duration of action match up with the pharmacokinetic properties of the compound?

• What happens if the drug is given continuously or repeatedly to an animal over the course of days or weeks? Does it lose its effectiveness, or reveal effects not seen with acute administration? Is there any kind of ‘rebound’ after effect when it is stopped?

In vitro profiling

Measurements on isolated tissues

Studies on isolated tissues have been a mainstay of pharmacological methodology ever since the introduction of the isolated organ bath by Magnus early in the 20th century. The technique is extremely versatile and applicable to studies on smooth muscle (e.g. gastrointestinal tract, airways, blood vessels, urinary tract, uterus, biliary tract, etc.) as well as cardiac and striated muscle, secretory epithelia, endocrine glands, brain slices, liver slices, and many other functional systems. In most cases the tissue is removed from a freshly killed or anaesthetized animal and suspended in a chamber containing warmed oxygenated physiological salt solution. With smooth muscle preparations the readout is usually mechanical (i.e. tension, recorded with a simple strain gauge). For other types of preparation, various electrophysiological or biochemical readouts are often used. Vogel (2002) and Enna et al. (2003) give details of a comprehensive range of standard pharmacological assay methods, including technical instructions.

Studies of this kind have the advantage that they are performed on intact normal tissues, as distinct from isolated enzymes or other proteins. The recognition molecules, signal transduction machinery and the mechanical or biochemical readout are assumed to be a reasonable approximation to the normal functioning of the tissue. There is abundant evidence to show that tissue responses to GPCR activation, for example, depend on many factors, including the level of expression of the receptor, the type and abundance of the G proteins present in the cell, the presence of associated proteins such as receptor activity-modifying proteins (RAMPs; see Morfis et al., 2003), the state of phosphorylation of various constituent proteins in the signal transduction cascade, and so on. For compounds acting on intracellular targets, functional activity depends on permeation through the membrane, as well as affinity for the target. For these reasons – and probably also for others that are not understood – the results of assays on isolated tissues often differ significantly from results found with primary screening assays. The discrepancy may simply be a quantitative one, such that the potency of the ligand does not agree in the two systems, or it may be more basic. For example, the pharmacological efficacy of a receptor ligand, i.e. the property that determines whether it is a full agonist, a partial agonist, or an antagonist, often depends on the type of assay used (Kenakin, 1999), and this may have an important bearing on the selection of possible development compounds. Examples that illustrate the poor correlation that may exist between measurements of target affinity in cell-free assay systems, and functional activity in intact cell systems, are shown in Figures 11.1 and 11.2. Figure 11.1 shows the relationship between binding and functional assay data for 5HT₃ and 5HT₄ receptor antagonists. In both cases, binding assays overestimate the potency in functional assays by a factor of about 10 (see above), but more importantly, the correlation is poor, despite the fact that the receptors are extracellular, and so membrane penetration is not a factor. Figure 11.2 shows data on tyrosine kinase inhibitors, in which activity against the isolated enzyme is plotted against inhibition of tyrosine phosphorylation in intact cells, and inhibition of cell proliferation for a large series of compounds. Differences in membrane penetration can account for part of the discrepancy between enzyme and cell-based data, but the correlation between intracellular kinase inhibition and blocking of cell proliferation is also weak, which must reflect other factors.