Drugreceptor Interactions

The effect of an agonist drug whether it is measured as the ability to fire a neuron, inhibit an enzyme or reduce motor function, increases with the concentration of the drug and the number of receptors it occupies. In fact the magnitude of the response, like that of a chemical reaction, is proportional to the product of the concentration of the reactants, in this case the drug and its receptors, and as such obeys the law of Mass Action. Thus the rate at which a drug [D] combines with the receptor [R] to give occupied receptors (or drug receptor complexes [DR]), can be represented by its rate constant K1 so that

Since the drug-receptor interaction is reversible the drug also dissociates from the receptor at a rate K2 when

The equilibrium constant (KA) for the reaction is thus given by

Neurotransmitters, Drugs and Brain Function. Edited by R. A. Webster ©2001 John Wiley & Sons Ltd

When 50% of the receptors are occupied [R] and [DR], free and occupied receptors, must be equal and cancel out so that KA = D, the concentration of drug required to bind to 50% of the receptors. Thus the lower the concentration of drug required to achieve this occupancy, the greater its affinity. Unfortunately its affinity does not necessarily reflect its potency in producing an effect.

The relationship between the dose (concentration) of a drug and the response it produces provides the so-called dose (or concentration) response curve (DRC). This is hyperbolic but is transformed to a sigmoid shape, which is linear over a large dose range, when the dose is plotted on a log scale (Fig. 5.1). Comparison of the concentrations of two or more drugs required to produce the same response is a measure of their relative potency. In Fig. 5.1 the dose-response curves for drugs A and B are one log unit apart and so A is ten times more potent than B. Often the potency of a drug is defined as the dose or concentration of drug required to produce 50% of the maximal response, i.e. the ED50 dose (or EC50 concentration). Drug C in Fig. 5.1 presents a different DRC. It obviously has agonist activity but since it cannot produce a maximal response it is known as a partial agonist. While such a property may seem unwanted the drug could still produce an adequate effect and avoid the danger of that becoming too great with increasing dose.

There are some points about the dose-response curve that justify consideration.

DOSE OR CONCENTRATION?

When a drug is administered to either humans or animals we obviously know the dose but not the concentration at its site of action. In this instance the relationship between the amount of drug and its effect really is a dose-response curve.

If a drug is added to an in vitro system in an organ or tissue bath then, provided the volume of the bathing solution is known, the concentration of drug can be calculated. Concentration is also known if a tissue is superfused with a prepared drug solution. In these instances, the response reflects drug concentration. Even then, the actual concentration of drug at the receptor site is not really known, since there can be a steep gradient between the concentration of drug in the medium and that at the actual receptor, especially if the drug is only in contact with the tissue for a short time. A proportion of most NTs is likely to be metabolised in, or taken up by, the tissue before reaching the receptor, although this is less likely with synthetic drugs.

POTENCY, AFFINITY AND EFFICACY

When looking at Fig. 5.1 it is pertinent to ask why drug A is more active than drug B. It could be that they are achieving the same response by acting through different receptors and that those targeted by A are either more numerous or better equipped to initiate the response. If they are both acting on the same receptor then obviously A has a more appropriate chemical structure to fit that receptor than B, but whether this has conferred on it a greater ability to combine with the receptor (affinity) or to activate it (efficacy) is unclear. It certainly should not be assumed that the EC50 is a measure of the affinity of the drug for the receptor. All responses are the result of a series of

Figure 5.1 Dose (concentration) response curves for three drugs. Percentage response is plotted against log dose. The curves show that drug A achieves the same responses as drug B but at lower doses. A is in fact ten times more active than B since the same effect (e.g. 50% maximal response, ED50) is obtained with 10"7M (or 0.1 mg) A compared with 10"6 M (or 1.0 mg) B. Drugs A and B can both produce the maximal response and are full agonists. Drug C cannot produce a maximal response even at large doses and is known as a partial agonist

-4 M. concentration 100 mg dose

Figure 5.1 Dose (concentration) response curves for three drugs. Percentage response is plotted against log dose. The curves show that drug A achieves the same responses as drug B but at lower doses. A is in fact ten times more active than B since the same effect (e.g. 50% maximal response, ED50) is obtained with 10"7M (or 0.1 mg) A compared with 10"6 M (or 1.0 mg) B. Drugs A and B can both produce the maximal response and are full agonists. Drug C cannot produce a maximal response even at large doses and is known as a partial agonist cellular events and, with the possible exception of studies on single-channel opening, not a direct measure of receptor occupancy. In any case, the efficacy of the drug must also be considered and since antagonists are devoid of that property their affinity and activity cannot be measured directly through a response (see below).

These problems can be overcome to some extent by using drugs labelled with a radioisotope (generally 3H, 14C or 125I) and then directly determining the amount of label bound when the drug is incubated with samples of the appropriate tissue or, as with the nervous system, fragments of specially prepared isolated neuronal membranes that contain the receptors. Even this approach is not ideal since drugs will combine non-specifically with cellular elements other than the receptor. In practice this can be largely overcome (see Chapter 3). Experimentally, the test tissue is incubated with varying concentrations of the labelled drug (called ligand) until equilibrium is reached. The tissue is then separated from the incubation medium by filtration or centrifugation and dissolved in scintillation fluid which is measured for its radioactivity. This gives the total amount of drug bound, including specific binding to its receptors and any other non-specific tissue binding. The non-specific binding is estimated by running a parallel set of tissue samples incubated with medium containing both the labelled drug and an excess concentration of another unlabelled drug which binds to the same receptor. This should inhibit all the receptor binding of labelled drug. Any residual binding will be to non-specific sites (Fig. 5.2(a)). Subtraction of this non-specific binding from the total binding gives the specific receptor binding for the drug which is a saturable process. The relationship between the amount of ligand bound (B) and its concentration X can be represented, for a preparation where the total number of binding sites is Bmax, as

B — where K is the dissociation (affinity) constant

Concentration of labelled drug Bound (fmol/mg protein)

Figure 5.2 Measurement of specific saturable drug binding, (a) Plot of quantity of labelled drug bound against its increasing concentration in the bathing medium, Subtraction of non-specific from total binding gives the specific binding for the drug, (b) Scatchard plot of B/F (bound/free drug) against level of bound drug (B) gives a straight line the slope of which is 1/KD, while the intercept is Bmax, the maximum number of that drug's binding sites, expressed as fmol/mg tissue protein, For experimental detail see text

Concentration of labelled drug Bound (fmol/mg protein)

Figure 5.2 Measurement of specific saturable drug binding, (a) Plot of quantity of labelled drug bound against its increasing concentration in the bathing medium, Subtraction of non-specific from total binding gives the specific binding for the drug, (b) Scatchard plot of B/F (bound/free drug) against level of bound drug (B) gives a straight line the slope of which is 1/KD, while the intercept is Bmax, the maximum number of that drug's binding sites, expressed as fmol/mg tissue protein, For experimental detail see text

Thus

If B/Xis plotted against B (the Scatchard plot) it should give a straight line (Fig, 5.2(b)) with the slope (1/Kd) giving K and the intercept on the abscissa providing the maximal binding (Bmax), expressed as fmol per mg tissue protein, The steeper the slope, the higher the affinity,

In many binding studies the relative abilities of a series of unlabelled drugs to displace a labelled ligand from a particular receptor is taken as a guide to their affinity for that receptor, This is normally represented as Ki, the concentration of drug required to displace half of the labelled ligand, Its accuracy depends on the chosen ligand only binding to the receptor it is intended to study and no other receptor, It must be emphasised that binding studies only measure the ability of a drug to combine with a receptor, they do not indicate whether it is an agonist or antagonist, Also compared with an antagonist the binding of an agonist may be affected in an uncertain manner by the change in state caused by the activation of the receptor,

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