Allosteric modulators bind to receptor sites that are topographically distinct from the agonist-binding (orthosteric) site, and cause a conformational change in the receptor that alters the activity of orthosteric ligands. Often, this conformational change can be detected as a change in the association and/or dissociation characteristics of the orthosteric ligand at its binding site on the receptor. The aim of this study was to investigate allosteric modulation of the cannabinoid CB₁ receptor by three novel compounds from Organon research (ORG 27569-0: 5-Chloro-3-ethyl-1H-indole-2-carboxylic acid [2-(4-piperidin-1-yl-phenyl)-ethyl]-amide; ORG 27759-0: 3-Ethyl-5-fluoro-1H-indole-2-carboxylic acid [2-94-dimethylamino-phenyl)-ethyl]-amide; ORG 29647-1: 5-Chloro-3-ethyl-1H-indole-2-carboxylic acid (1-benzyl-pyrrolidin-3-yl)-amide, 2-enedioic acid salt). We carried out equilibrium radioligand binding experiments and we also utilised an ‘isotopic dilution’ method to measure the dissociation rate constant for [³H]CP55940 (CP55) from mouse brain membranes.

In equilibrium binding experiments [³H]CP55 (0.7nM) was incubated with mouse brain membranes (100µg) for 30 minutes at 37°C in Tris (50mM) containing 1mgml -1 BSA. For dissociation kinetic experiments, [³H]CP55 (0.7nM) was incubated with mouse brain membranes (100µg) for 60 minutes at 25°C in Tris (50mM) containing 1mgml^-1 BSA. Dissociation was visualised by the addition of 1µM CP55 in the presence and absence of test Organon (ORG) compounds. Dissociation times of 0.5 to 120 minutes were used. The dissociation curve for [³H]CP55 was best-fit to a biphasic exponential (P<0.01, Prism 4); the dissociation rate constants (k_-1) for the two “agonist states” are shown in Table 1. In the presence of the Org compounds, the slow k_-1 dissociation rate constant for [³H]CP55 was significantly lower. In equilibrium experiments, the Org compounds significantly increased the level of specific binding of [³H]CP55.

Table 1 : Dissociation rate constants (k_-1) and specific binding (% equilibrium binding) of [³H]CP55 in the presence of Org compounds. Data represent the mean ± SEM (n = 4-8), ***P<0.001 and **P<0.05, one way ANOVA (dissociation) or a one-sample t-test (equilibrium).

These data represent the first evidence of allosteric modulation of agonist binding at the cannabinoid CB₁ receptor. Positive allosteric modulators of the cannabinoid CB₁ receptor may offer a mechanism whereby the beneficial effects of endocannabinoids can be augmented. Furthermore, such drugs would lack the associated psychoactive side-effects associated with CB₁ receptor agonists.