2 10 20 100 140 mg/kg

C. Dose-frequency relationship

C. Dose-frequency relationship

Concentration-Effect Relationship (A)

The relationship between the concentration of a drug and its effect is determined in order to define the range of active drug concentrations (potency) and the maximum possible effect (efficacy). On the basis of these parameters, differences between drugs can be quantified. As a rule, the therapeutic effect or toxic action depends critically on the response of a single organ or a limited number of organs, e.g., blood flow is affected by a change in vascular luminal width. By isolating critical organs or tissues from a larger functional system, these actions can be studied with more accuracy; for instance, vasoconstrictor agents can be examined in isolated preparations from different regions of the vascular tree, e.g., the portal or saphenous vein, or the mesenteric, coronary, or basilar artery. In many cases, isolated organs or organ parts can be kept viable for hours in an appropriate nutrient medium sufficiently supplied with oxygen and held at a suitable temperature.

Responses of the preparation to a physiological or pharmacological stimulus can be determined by a suitable recording apparatus. Thus, narrowing of a blood vessel is recorded with the help of two clamps by which the vessel is suspended under tension.

Experimentation on isolated organs offers several advantages:

1. The drug concentration in the tissue is usually known.

2. Reduced complexity and ease of relating stimulus and effect.

3. It is possible to circumvent compensatory responses that may partially cancel the primary effect in the intact organism — e.g., the heart rate increasing action of norepinephrine cannot be demonstrated in the intact organism, because a simultaneous rise in blood pressure elicits a counter-regulatory reflex that slows cardiac rate.

4. The ability to examine a drug effect over its full rage of intensities — e.g.,

Lullmann, Color Atlas of Pharmacology © 2000 Thieme it would be impossible in the intact organism to follow negative chrono-tropic effects to the point of cardiac arrest.

Disadvantages are:

1. Unavoidable tissue injury during dissection.

2. Loss of physiological regulation of function in the isolated tissue.

3. The artificial milieu imposed on the tissue.

Concentration-Effect Curves (B)

As the concentration is raised by a constant factor, the increment in effect diminishes steadily and tends asymptotically towards zero the closer one comes to the maximally effective concentra-tion.The concentration at which a maximal effect occurs cannot be measured accurately; however, that eliciting a half-maximal effect (EC50) is readily determined. It typically corresponds to the inflection point of the concentration-response curve in a semilogarith-mic plot (log concentration on abscissa). Full characterization of a concentration-effect relationship requires determination of the EC50, the maximally possible effect (Emax), and the slope at the point of inflection.

B. Concentration-effect relationship

Concentration-Binding Curves

In order to elicit their effect, drug molecules must be bound to the cells of the effector organ. Binding commonly occurs at specific cell structures, namely, the receptors. The analysis of drug binding to receptors aims to determine the affinity of ligands, the kinetics of interaction, and the characteristics of the binding site itself.

In studying the affinity and number of such binding sites, use is made of membrane suspensions of different tissues. This approach is based on the expectation that binding sites will retain their characteristic properties during cell homogenization. Provided that binding sites are freely accessible in the medium in which membrane fragments are suspended, drug concentration at the "site of action" would equal that in the medium. The drug under study is radiolabeled (enabling low concentrations to be measured quantitatively), added to the membrane suspension, and allowed to bind to receptors. Membrane fragments and medium are then separated, e.g., by filtration, and the amount of bound drug is measured. Binding increases in proportion to concentration as long as there is a negligible reduction in the number of free binding sites (c = 1 and B = 10% of maximum binding; c = 2 and B = 20%). As binding approaches saturation, the number of free sites decreases and the increment in binding is no longer proportional to the increase in concentration (in the example illustrated, an increase in concentration by 1 is needed to increase binding from 10 to 20 %; however, an increase by 20 is needed to raise it from 70 to 80%).

The law of mass action describes the hyperbolic relationship between binding (B) and ligand concentration (c). This relationship is characterized by the drug's affinity (1/Kd) and the maximum binding (Bmax), i.e., the total number of binding sites per unit of weight of membrane homogenate.

Kd is the equilibrium dissociation constant and corresponds to that ligand concentration at which 50 % of binding sites are occupied. The values given in (A) and used for plotting the concentration-binding graph (B) result when KD -10.

The differing affinity of different li-gands for a binding site can be demonstrated elegantly by binding assays. Although simple to perform, these binding assays pose the difficulty of correlating unequivocally the binding sites concerned with the pharmacological effect; this is particularly difficult when more than one population of binding sites is present. Therefore, receptor binding must not be implied until it can be shown that

• binding is saturable (saturability);

• the only substances bound are those possessing the same pharmacological mechanism of action (specificity);

• binding affinity of different substances is correlated with their pharmacological potency.

Binding assays provide information about the affinity of ligands, but they do not give any clue as to whether a ligand is an agonist or antagonist (p. 60). Use of radiolabeled drugs bound to their receptors may be of help in purifying and analyzing further the receptor protein.

Types of Binding Forces

Unless a drug comes into contact with intrinsic structures of the body, it cannot affect body function.

Covalent bond. Two atoms enter a covalent bond if each donates an electron to a shared electron pair (cloud). This state is depicted in structural formulas by a dash. The covalent bond is "firm", that is, not reversible or only poorly so. Few drugs are covalently bound to biological structures. The bond, and possibly the effect, persist for a long time after intake of a drug has been discontinued, making therapy difficult to control. Examples include alkylating cytostatics (p. 298) or organo-phosphates (p. 102). Conjugation reactions occurring in biotransformation also represent a covalent linkage (e.g., to glucuronic acid, p. 38).

Noncovalent bond. There is no formation of a shared electron pair. The bond is reversible and typical of most drug-receptor interactions. Since a drug usually attaches to its site of action by multiple contacts, several of the types of bonds described below may participate.

Electrostatic attraction (A). A positive and negative charge attract each other.

Ionic interaction: An ion is a particle charged either positively (cation) or negatively (anion), i.e., the atom lacks or has surplus electrons, respectively. Attraction between ions of opposite charge is inversely proportional to the square of the distance between them; it is the initial force drawing a charged drug to its binding site. Ionic bonds have a relatively high stability.

Dipole-ion interaction: When bond electrons are asymmetrically distributed over both atomic nuclei, one atom will bear a negative (8-), and its partner a positive (8+) partial charge. The molecule thus presents a positive and a negative pole, i.e., has polarity or a dipole. A partial charge can interact electrostatically with an ion of opposite charge.

Dipole-dipole interaction is the electrostatic attraction between opposite partial charges. When a hydrogen atom bearing a partial positive charge bridges two atoms bearing a partial negative charge, a hydrogen bond is created.

A van der Waals' bond (B) is formed between apolar molecular groups that have come into close proximity. Spontaneous transient distortion of electron clouds (momentary faint dipole, 88) may induce an opposite dipole in the neighboring molecule. The van der Waals' bond, therefore, is a form of electrostatic attraction, albeit of very low strength (inversely proportional to the seventh power of the distance).

Hydrophobic interaction (C). The attraction between the dipoles of water is strong enough to hinder intercalation of any apolar (uncharged) molecules. By tending towards each other, H2O molecules squeeze apolar particles from their midst. Accordingly, in the organism, apolar particles have an increased probability of staying in nonaqueous, apolar surroundings, such as fatty acid chains of cell membranes or apolar regions of a receptor.


+ I Binding site

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