Evidence that the plant cannabinoid Delta9-tetrahydrocannabivarin is a cannabinoid CB1 and CB2 receptor antagonist.

Delta9-tetrahydrocannabivarin (THCV) displaced [(3)H]CP55940 from specific binding sites on mouse brain and CHO-hCB(2) cell membranes (K(i)=75.4 and 62.8 nM, respectively).THCV (1 microM) also antagonized CP55940-induced stimulation of [(35)S]GTPgammaS binding to these membranes (apparent K(B)=93.1 and 10.1 nM, respectively). In the mouse vas deferens, the ability of Delta9-tetrahydrocannabinol (THC) to inhibit electrically evoked contractions was antagonized by THCV, its apparent K(B)-value (96.7 nM) approximating the apparent K(B)-values for its antagonism of CP55940- and R-(+)-WIN55212-induced stimulation of [(35)S]GTPgammaS binding to mouse brain membranes. THCV also antagonized R-(+)-WIN55212, anandamide, methanandamide and CP55940 in the vas deferens, but with lower apparent K(B)-values (1.5, 1.2, 4.6 and 10.3 nM, respectively).THCV (100 nM) did not oppose clonidine, capsaicin or (-)-7-hydroxy-cannabidiol-dimethylheptyl-induced inhibition of electrically evoked contractions of the vas deferens. Contractile responses of the vas deferens to phenylephrine hydrochloride or beta,gamma-methylene-ATP were not reduced by 1microM THCV or R-(+)-WIN55212, suggesting that THCV interacts with R-(+)-WIN55212 at prejunctional sites. At 32 microM, THCV did reduce contractile responses to phenylephrine hydrochloride and beta,gamma-methylene-ATP, and above 3 microM it inhibited electrically evoked contractions of the vas deferens in an SR141716A-independent manner. In conclusion, THCV behaves as a competitive CB(1) and CB(2) receptor antagonist. In the vas deferens, it antagonized several cannabinoids more potently than THC and was also more potent against CP55940 and R-(+)-WIN55212 in this tissue than in brain membranes. The bases of these agonist- and tissue-dependent effects remain to be established.

Allosteric modulation of the cannabinoid CB1 receptor.

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 CB1 receptor. In equilibrium binding assays, the Org compounds significantly increased the binding of the CB1 receptor agonist [3H]CP 55,940 [(1R,3R,4R)-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-4-(3-hydroxypropyl)cyclohexan-1-ol], indicative of a positively cooperative allosteric effect. The same compounds caused a significant, but incomplete, decrease in the specific binding of the CB1 receptor inverse agonist [3H]SR 141716A [N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboximide hydrochloride], indicative of a limited negative binding cooperativity. Analysis of the data according to an allosteric ternary complex model revealed that the estimated affinity of each Org compound was not significantly different when the radioligand was [3H]CP 55,940 or [3H]SR 141716A. However, the estimated cooperatively factor for the interaction between modulator and radioligand was greater than 1 when determined against [3H]CP 55,940 and less than 1 when determined against [3H]SR 141716A. [3H]CP 55,940 dissociation kinetic 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 CB1 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 5'-O-(3-[35S]thio)triphosphate binding assay and the mouse vas deferens assay they elicited a significant reduction in the Emax value for CB1 receptor agonists. The data presented clearly demonstrate, for the first time, that the cannabinoid CB1 receptor contains an allosteric binding site that can be recognized by synthetic small molecule ligands.