We investigated the pharmacology of three novel compounds, Org 27569 (5-chloro-3-ethyl-1H-indole-2-carboxylic acid [2-(4-piperidin-1-yl-phenyl)-ethyl]-amide), Org 27759 (3-ethyl-5-fluoro-1H-indole-2-carboxylic acid [2-94-dimethylamino-phenyl)-ethyl]-amide), and Org 29647 (5-chloro-3-ethyl-1H-indole-2-carboxylic acid (1-benzyl-pyrrolidin-3-yl)-amide, 2-enedioic acid salt), at the cannabinoid CB 1 receptor. In equilibrium binding assays, the Org compounds significantly increased the binding of the CB 1 receptor agonist, indicative of a positively cooperative allosteric effect. The same compounds caused a significant, but incomplete, decrease in the specific binding of the CB 1 receptor inverse agonist studies also validated the allosteric nature of the Org compounds, because they all significantly decreased radioligand dissociation. These data suggest that the Org compounds bind allosterically to the CB 1 receptor and elicit a conformational change that increases agonist affinity for the orthosteric binding site. In contrast to the binding assays, however, the Org compounds behaved as insurmountable antagonists of receptor function; in the reporter gene assay, the guanosine 5Ј-O-(3-[35 S]thio)triphosphate binding assay and the mouse vas deferens assay they elicited a significant reduction in the E max value for CB 1 receptor agonists. The data presented clearly demonstrate, for the first time, that the cannabinoid CB 1 receptor contains an allosteric binding site that can be recognized by synthetic small molecule ligands.Mammalian tissues express at least two types of cannabinoid receptor, CB 1 and CB 2 , both G protein-coupled (for review, see Howlett et al., 2002). CB 1 receptors are found predominantly at central and peripheral nerve terminals where they mediate inhibition of transmitter release. Endogenous ligands for these receptors also exist. These "endocannabinoids" are all eicosanoids, prominent examples including arachidonoylethanolamide (anandamide) and 2-arachidonoyl glycerol, both of which are synthesized on demand, removed from their sites of action by tissue uptake processes and metabolized by intracellular enzymes (Pertwee and Ross,

Background and purpose: A nonpsychoactive constituent of the cannabis plant, cannabidiol has been demonstrated to have low affinity for both cannabinoid CB 1 and CB 2 receptors. We have shown previously that cannabidiol can enhance electrically evoked contractions of the mouse vas deferens, suggestive of inverse agonism. We have also shown that cannabidiol can antagonize cannabinoid receptor agonists in this tissue with a greater potency than we would expect from its poor affinity for cannabinoid receptors. This study aimed to investigate whether these properties of cannabidiol extend to CB 1 receptors expressed in mouse brain and to human CB 2 receptors that have been transfected into CHO cells. Experimental approach: The [ 35 S]GTPgS binding assay was used to determine both the efficacy of cannabidiol and the ability of cannabidiol to antagonize cannabinoid receptor agonists (CP55940 and R-( þ )-WIN55212) at the mouse CB 1 and the human CB 2 receptor. Key results: This paper reports firstly that cannabidiol displays inverse agonism at the human CB 2 receptor. Secondly, we demonstrate that cannabidiol is a high potency antagonist of cannabinoid receptor agonists in mouse brain and in membranes from CHO cells transfected with human CB 2 receptors. Conclusions and implications:This study has provided the first evidence that cannabidiol can display CB 2 receptor inverse agonism, an action that appears to be responsible for its antagonism of CP55940 at the human CB 2 receptor. The ability of cannabidiol to behave as a CB 2 receptor inverse agonist may contribute to its documented anti-inflammatory properties.

Pregnenolone is considered the inactive precursor of all steroid hormones and its potential functional effects have been largely neglected. The administration of the main active principle of Cannabis sativa (marijuana) Δ9-tetrahydrocannabinol (THC) substantially increases the synthesis of pregnenolone in the brain via the activation of type-1 cannabinoid (CB1) receptor. Pregnenolone then, acting as a signaling specific inhibitor of the CB1 receptor, reduces several effects of THC. This negative feedback mediated by pregnenolone reveals an unknown paracrine/autocrine loop protecting the brain from CB1 receptor over-activation that could open an unforeseen novel approach for the treatment of cannabis intoxication and addiction.

We have previously identified allosteric modulators of the cannabinoid CB 1 receptor (Org 27569, PSNCBAM-1) that display a contradictory pharmacological profile: increasing the specific binding of the CB 1 receptor agonist [ 3 H]CP55940 but producing a decrease in CB 1 receptor agonist efficacy. Here we investigated the effect one or both compounds in a broad range of signaling endpoints linked to CB 1 receptor activation. We assessed the effect of these compounds on CB 1 receptor agonist-induced [35 S]GTPgS binding, inhibition, and stimulation of forskolin-stimulated cAMP production, phosphorylation of extracellular signal-regulated kinases (ERK), and b-arrestin recruitment. We also investigated the effect of these allosteric modulators on CB 1 agonist binding kinetics. -mediated), and inhibition (Ga i -mediated) of cAMP production and b-arrestin recruitment. In contrast, it acts as an enhancer of agonist-induced ERK phosphorylation. Alone, the compound can act also as an allosteric agonist, increasing cAMP production and ERK phosphorylation. We find that in both saturation and kineticbinding experiments, the Org 27569 and PSNCBAM-1 appeared to influence only orthosteric ligand maximum occupancy rather than affinity. The data indicate that the allosteric modulators share a common mechanism whereby they increase available high-affinity CB 1 agonist binding sites. The receptor conformation stabilized by the allosterics appears to induce signaling and also selectively traffics orthosteric agonist signaling via the ERK phosphorylation pathway.