Previous experiments have shown that (-)-cannabidiol (CBD) attenuates the ability of the CB₁ receptor agonist, WIN55212-2, to inhibit electrically-evoked contractions of the mouse vas deferens (Pertwee et al., 2002). This investigation was directed at establishing whether WIN55212-2 is also antagonized by a CBD analogue, O-2654, that has a 6"-azido-2"-hexyne instead of a pentyl group as its 4' side chain, and at comparing the abilities of CBD and O-2654 to displace [³H]-CP55940 from CB₁ binding sites on mouse brain membranes.

Vasa deferentia were obtained from adult MF1 mice (32 to 45 g) and experiments with these tissues performed as described by Pertwee et al. (2002). O-2654 was dissolved in DMSO and added 90 min before WIN55212-2 which was dissolved in a 1:1 mixture of DMSO and a 0.9% aqueous solution of NaCl. Supramaximal electrical stimuli were applied using 0.5 s trains of 3 pulses (train frequency 0.1 Hz; pulse duration 0.5 ms). Binding experiments were performed with mouse whole brain membranes (100 µg) and [³H]-CP55940 following the method described by Pertwee et al. (2000) for rat brain and cultured cell membranes. The methods used to calculate E_max, K_B and K_i values are also detailed elsewhere (Pertwee et al., 2000; 2002). Values are expressed as means and variability as s.e.mean or 95% confidence limits.

O-2654 (0.32, 1, 6 and 10 µM) antagonized WIN55212-2 in a manner that was concentration-related and that was not accompanied by any significant change in the maximum effect (E_max) of WIN55212-2 (P>0.05; ANOVA followed by Dunnett's test; n=7 to 12). The dextral shifts it produced in the cumulative log concentration response curve of WIN55212-2 did not deviate significantly from parallelism and yielded a Schild plot with a mean slope (0.99±0.16) not significantly different from unity (P>0.1; one-sample t test; n=4). The mean K_B value of O-2654 was calculated to be 85.7 nM with 95% confidence limits of 58.4 and 160.8 nM. Exposure to DMSO for 90 min was accompanied by an increase in mean twitch amplitude of 30.9±14.4% that was not statistically significant (P>0.05; paired t test; n=12) and this mean value did not differ significantly from mean changes in twitch amplitude observed after 90-min exposure to O-2654 at 0.32, 1, 6 or 10 µM, 14.3±14.9%, 15.0±19.0%, 42.3±24.4% and 27.5±16.7% respectively (P>0.05; ANOVA followed by Dunnett's test; n=8 to 12). O-2654 and CBD both displaced [³H]-CP55940 from mouse brain membranes, the mean K_i values with 95% confidence limits shown in brackets being 0.114 µM (0.1 & 0.14 µM; n=5 to 11) and 4.9 µM (2.1 & 11.3 µM; n=5 to 12) respectively.

In conclusion, unlike CBD (Pertwee et al., 2002), O-2654 has a K_B value against WIN55212-2 that is close to its CB₁ K_i value. By itself, O-2654 did not affect the amplitude electrically-evoked contractions of the vas deferens. Hence, it is possible that O-2654 is a "silent" CB₁ receptor antagonist that lacks significant CB₁ efficacy or inverse efficacy.

Pertwee, R.G. et al. (2000) Br. J. Pharmacol., 129, 1577.
Pertwee, R.G. et al. (2002) Eur. J. Pharmacol., 456, 99.

Supported by NIDA and GW Pharmaceuticals.