Quantification of Drug Action V

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 differing affinity of different ligands
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 . Use of
radiolabeled drugs bound to their receptors
may be of help in purifying and
analyzing further the receptor protein.