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Cannabis sativa is now known to contain at least 70 compounds that are unique to it and known collectively as cannabinoids (ElSohly and Slade, 2005). One of these is (–)-Δ9-tetrahydrocannabinol (Δ9-THC), the main psychoactive constituent of cannabis, and another is (–)-cannabidiol, which is not psychoactive and exhibits much lower affinity than Δ9-THC for cannabinoid CB1 and CB2 receptors (Showalter et al., 1996; Thomas et al., 1998; Pertwee, 1999; Bisogno et al., 2001; Thomas et al., 2004). Cannabidiol is of interest because it lacks psychoactivity and yet has therapeutic potential, for example for the management of inflammation, anxiety, emesis and nausea, and as a neuroprotective agent (Pertwee, 2004). Indeed, together with Δ9-THC, cannabidiol is a major constituent of Sativex, a medicine that is now licensed in Canada for neuropathic pain associated with multiple sclerosis.
In previous experiments (Pertwee et al., 2002), cannabidiol was found to share the ability of the CB1-selective inverse agonist/antagonist, rimonabant, to increase the amplitude of electrically evoked contractions of the mouse vas deferens (Pertwee et al., 2002), which, for rimonabant at least, is most likely an indication of inverse agonist activity (Pertwee, 2005). Cannabidiol was also found to resemble rimonabant in its ability to antagonize (R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo-[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone (R-(+)-WIN55212)- and (−)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl)cyclohexanol (CP55940)-induced inhibition of electrically evoked contractions of the mouse vas deferens in a competitive, surmountable manner (Pertwee et al., 1995, 2002). Unlike rimonabant, however, cannabidiol produced this antagonism at concentrations well below those at which it binds to CB1 (or CB2) cannabinoid receptors, suggesting that it was competing with R-(+)-WIN55212 and CP55940 for an as yet uncharacterized non-CB1 pharmacological target on nerve terminals. These properties of cannabidiol prompted this current study.
Thus, the present investigation was directed primarily at investigating whether the unexpectedly high potency exhibited by cannabidiol as an antagonist of cannabinoid receptor agonists in the mouse vas deferens extends to cannabinoid receptors in mouse brain tissue and/or to Chinese hamster ovary cells stably transfected with human CB2 receptors (hCB2-CHO) cell membranes. Cannabidiol was compared with rimonabant in the brain tissue experiments and with N-[(1S)-endo-1,3,3-trimethyl bicyclo[2.2.1]heptan-2-yl]-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)-pyrazole-3-carboxamide (SR144528), an established CB2 receptor inverse agonist/antagonist in the experiments performed with hCB2-CHO cell membranes. We also addressed the question of whether cannabidiol behaves as an inverse agonist or as a neutral antagonist at CB1 and/or CB2 receptors. Accordingly, in some experiments cannabidiol was compared with a putative neutral cannabinoid receptor antagonist, the synthetic cannabidiol analogue, O-2654 (Thomas et al., 2004). This compound differs from cannabidiol and rimonabant by behaving as a neutral antagonist of cannabinoid receptor agonists rather than as an inverse agonist in the mouse isolated vas deferens (Pertwee, 2005).
In this study, we report first that cannabidiol can behave as an inverse agonist at the human CB2 receptor. Second, we demonstrate that cannabidiol behaves as a high-potency antagonist of cannabinoid receptor agonists in mouse brain tissue and in membranes from CHO cells transfected with human CB2 receptors. Furthermore, the high potency of cannabidiol as an antagonist of the cannabinoid receptor agonist CP55940 in the hCB2-CHO cell membranes appears to be a consequence of the ability of cannabidiol to behave as an inverse agonist at the hCB2 receptor. Some of the results described in this paper have been presented to the International Cannabinoid Research Society (Thomas et al., 2006).
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- Materials and methods
- Conflict of interest
The results described in this paper indicate that the unexpectedly high potency reported previously for cannabidiol-induced antagonism of cannabinoid agonists in the mouse vas deferens (Pertwee et al., 2002) extends to the brain. The apparent KB values for the antagonism of CP55940 or R-(+)-WIN55212 are at least 35 times lower than the Ki values of cannabidiol for displacement of [3H]CP55940 from mouse brain membranes (Showalter et al., 1996; Thomas et al., 1998, 2004; Bisogno et al., 2001 see also Table 1). However, they are similar to the corresponding apparent KB values (34.0 and 120.3 nM, respectively) obtained for cannabidiol in the mouse vas deferens (Pertwee et al., 2002), suggesting that this cannabinoid may be acting on the same target in the brain as in the vas deferens. Cannabidiol appears to exhibit at least some selectivity as an antagonist of CP55940 and R-(+)-WIN55212, since 1 μM cannabidiol did not antagonize stimulation of [35S]GTPγS binding to mouse brain membranes induced by the μ-, δ- and κ-opioid receptor agonist, morphine (Mignat et al., 1995). We have also found in a previous investigation (Pertwee et al., 2002) that cannabidiol is markedly less potent as an antagonist of DAMGO, a selective μ-opioid receptor agonist, than as an antagonist of R-(+)-WIN55212 or CP55940 in the mouse vas deferens. Although, cannabidiol has been reported to modulate allosterically μ- and δ-opioid receptors (Kathmann et al., 2006), this occurs only at high micromolar concentrations and it is therefore unlikely that this interaction occurred in our experiments.
Rimonabant also exhibited greater potency as an antagonist of CP55940- and R-(+)-WIN55212-induced stimulation of [35S]GTPγS binding to mouse brain membranes than as a CB1 receptor ligand. Thus, the apparent KB values of rimonabant for antagonism of these two cannabinoid receptor agonists were respectively 24 and 7 times lower than the Ki of rimonabant for its displacement of [3H]CP55940 from mouse brain membranes. Interestingly, such a Ki/KB discrepancy has not been detected in the mouse-isolated vas deferens (Pertwee et al., 1995). This may be because rimonabant exhibits greater potency as an antagonist of CP55940 and R-(+)-WIN55212 in brain tissue than in the vas deferens because first, R-(+)-WIN55212 and CP55940 inhibit electrically evoked contractions of this tissue not only by acting through CB1 receptors but also by activating non-CB1 targets (see Pertwee et al., 2002, 2005; Thomas et al., 2005) and because these putative non-CB1 targets exhibit little or no sensitivity to antagonism by rimonabant.
By themselves, cannabidiol and rimonabant both inhibited [35S]GTPγS binding to mouse brain membranes. Cannabidiol exhibited particularly high inverse agonist efficacy, producing inhibition of [35S]GTPγS binding at 10 μM, which greatly exceeded that produced by 10 μM rimonabant. Interestingly, in experiments using assay conditions almost identical to those used in the present investigation, Breivogel et al. (2001) found that cannabidiol (concentration unspecified) did not produce any significant effects on [35S]GTPγS binding to C57BL/6 mouse brain membranes. On the other hand, 10 μM cannabidiol has been reported to inhibit [35S]GTPγS binding to rat cerebellar membranes (Petitet et al., 1998).
The results from our experiments with membranes prepared from CB1-transfected and -untransfected CHO cells suggest that cannabidiol can inhibit [35S]GTPγS binding by interacting with the CB1 receptor as an inverse agonist at 10 μM. However, since cannabidiol-inhibited [35S]GTPγS binding to membranes obtained from mice whose CB1 receptors had been genetically deleted as well as from WT mice, it is likely that this cannabinoid can also inhibit [35S]GTPγS-binding through one or more CB1 receptor-independent mechanisms. This in turn raises the possibility that the inverse effect of 10 μM cannabidiol on MF1 mouse brain membranes greatly exceeded that of 10 μM rimonabant (Figure 3a) because cannabidiol was interacting with more than one pharmacological target in an additive or synergistic manner. That cannabidiol and rimonabant exhibited lower potency as inhibitors of [35S]GTPγS binding to brain membranes when these were obtained from C57BL/6 mice rather than from MF1 mice may be because one or more of their targets was more highly expressed by the MF1 mice.
One question raised is whether cannabidiol was inhibiting [35S]GTPγS binding to mouse brain membranes because, similar to rimonabant, it can block adenosine A1 receptors when these are being activated by endogenously released adenosine (Savinainen et al., 2003). Thus, Savinainen et al. (2003) have found that at a concentration of 1 μM, the selective A1 receptor antagonist DPCPX prevents rimonabant from inhibiting [35S]GTPγS binding to rat cerebellar membranes. Moreover, cannabidiol has recently been found to inhibit the cellular uptake of adenosine (Carrier et al., 2006), an effect that would be expected to augment any inverse effect arising from A1 receptor blockade. It is unlikely, however, that cannabidiol inhibited [35S]GTPγS binding to brain membranes in the present investigation by acting through A1 receptors. Thus, we have found that 1 μM DPCPX does not alter the ability of 100 nM, 1 or 10 μM cannabidiol to inhibit [35S]GTPγS binding to mouse brain membranes (n=3; data not shown). Moreover the experiments in which Breivogel et al. (2001) found cannabidiol not to inhibit [35S]GTPγS binding to CB1+/+ mouse brain membranes (see above) were performed in the absence of any A1 receptor antagonist and in the presence of much less exogenously added adenosine deaminase (0.004 U ml−1) than in our experiments (0.5 U ml−1).
As in mouse brain membranes, in experiments with hCB2-CHO cell membranes, cannabidiol was also found to act more potently as an antagonist of CP55940-induced stimulation of [35S]GTPγS binding than would be expected from its ability to displace [3H]CP55940 from hCB2-CHO cell membranes. Similar results were obtained with SR144528. SR144528 has been reported previously to behave as an inverse agonist at the CB2 receptor (Bouaboula et al., 1999; Portier et al., 1999; Ross et al., 1999; Rhee and Kim, 2002), and this was confirmed by the results obtained in the present study with hCB2-CHO cell membranes. Cannabidiol also behaved as a CB2 receptor inverse agonist as it shared the ability of SR144528 to induce an inhibition of [35S]GTPγS binding to hCB2-CHO cell membranes when added by itself.
There is evidence from the results obtained in this investigation that this antagonism of CP55940 by 1 μM cannabidiol in the hCB2-CHO cell membrane experiments may have been non-competitive in nature. Thus, 1 μM cannabidiol produced a marked downward displacement of the CP55940 log concentration–response curve for stimulation of [35S]GTPγS binding to hCB2 receptors (Figure 7a) and re-analysis of these data in a manner expected to exclude the effect of cannabidiol by itself (see above) suggests that this downward displacement accounts entirely for the antagonism of CP55940 induced by 1 μM cannabidiol in the hCB2-CHO cell membrane experiments (Figure 10a). In terms of the two-state model (Leff, 1995), it may be that CP55940 stimulates [35S]GTPγS binding to CB2 receptors by shifting the equilibrium between constitutively active (R*) and inactive (R) receptors more towards R*, whereas cannabidiol shifts this equilibrium towards R, thereby ‘physiologically’ opposing the ability of CP55940 to stimulate CB2 receptors. Hence at 1 μM, a concentration at which it induces little displacement of [3H]CP55940 from hCB2 receptors (Figure 6), cannabidiol may have been antagonizing CP55940 at the CB2 receptors entirely through inverse agonism and not at all by direct competition with CP55940 for receptors in the R* state.
Figure 10. [35S]GTPγS binding to membranes from hCB2-CHO cell membranes. The effect of (a) 1 μM cannabidiol (n=4–5) or (b) 100 nM SR144528 (n=5–6) on the mean log concentration–response curve of CP55940 for stimulation of [35S]GTPγS binding to CB2-transfected CHO cell membranes after subtraction of the inhibitory effect induced by either 1 μM cannabidiol or 100 nM SR144528 at the basal level of [35S]GTPγS binding, determined in the absence of any other compound. Each symbol represents the mean percentage increase in [35S]GTPγS binding±s.e.m. After this re-analysis, it was found that 1 μM cannabidiol did not produce a significant rightward shift of the CP55940 log concentration–response curve, whereas 100 nM SR144528 antagonized CP55940 with an apparent KB value of 2.5 nM, with 95% CI of 1.6 and 4.3 nM.
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As to the antagonism of CP55940 induced by 100 nM SR144528 at the CB2 receptor, this may have been partly competitive in nature and partly a result of inverse agonism. Thus, when the component of SR144528-induced antagonism of CP55940 that seemingly arises from its ability to inhibit [35S]GTPγS binding to hCB2-CHO cell membranes was excluded, a significant SR144528-induced rightward shift in the log concentration–response curve of CP55940 was still apparent (Figure 10b). Although, there still appears to be a downward displacement of the CP55940 log concentration–response curve, this was not found to be statistically significant. Thus, the 95% CI for the bottom of the CP55940 log concentration–response curves in the absence or presence of 100 nM SR144528 overlapped. The apparent KB value of SR144528 calculated from this shift is much closer to the CB2Ki value of SR144528 than the corresponding apparent KB value calculated from the data shown in Figure 7b, however, this recalculated KB value of SR144528 remains significantly less than its CB2Ki value. It is possible, therefore, that the Emax of SR144528 for inhibiting [35S]GTPγS binding to hCB2-CHO cell membranes underestimates its maximal inverse efficacy. This may be because an insufficient proportion of the hCB2 receptors was constitutively active in the absence of CP55940, thereby making it possible for SR144528 to produce a further degree of inverse agonism in the presence of CP55940, which according to the two-state model would be expected to shift the equilibrium for CB2 receptors from R to R* and so increase the number of CB2 receptors in the putative constitutively active R* state (Leff, 1995). This hypothesis is supported by results obtained with O-2654. This ligand does not appear to significantly inhibit [35S]GTPγS binding to hCB2-CHO cell membranes when administered by itself at 1 μM and it antagonized CP55940-induced stimulation of [35S]GTPγS binding to hCB2 receptors with an apparent KB value that does not deviate significantly from its hCB2 Ki value (Table 1). Further experiments will be required to test this hypothesis more fully and also to address the related question of whether the abilities of cannabidiol and rimonabant to behave as inverse agonists in mouse brain membranes accounts at least in part for our finding that these ligands antagonize CP55940-induced stimulation of [35S]GTPγS binding to mouse brain membranes more potently than they displace [3H]CP55940 from such membranes (Table 1).
In conclusion, this paper provides evidence that cannabidiol exhibits unexpectedly high potency in vitro as an antagonist of both CB1 and CB2 receptor agonists and that this antagonism is non-competitive in nature. The mechanism by which cannabidiol antagonized CB1 receptor agonists in our experiments remains to be elucidated, one possibility being that it can also attenuate any responses induced by CP55940 or R-(+)-WIN55212 in brain membranes from CB1−/− mice. It is noteworthy, however, that Breivogel et al. (2001) have reported that in contrast to R-(+)-WIN55212, CP55940 does not stimulate [35S]GTPγS binding to such membranes. As to the high potency displayed by cannabidiol as an antagonist of CB2 receptor activation, our data suggest that this may stem from its ability to induce CB2 receptor inverse agonism at concentrations well below those at which it displaces [3H]CP55940 from these receptors. This action may also contribute to the well-known anti-inflammatory properties of cannabidiol (reviewed in Pertwee, 2004), as there is evidence that CB2 receptor inverse agonism can inhibit immune cell migration (Lunn et al., 2006). This paper also contains further evidence that O-2654 can behave as a neutral CB1 receptor antagonist, at least at concentrations of up to 1 μM, whereas under the same assay conditions cannabidiol and the established inverse agonist, rimonabant, can behave as inverse agonists at concentrations of 1 and 10 μM. O-2654 may also be a neutral CB2 receptor antagonist. Thus, although it inhibited [35S]GTPγS binding to hCB2-CHO cell membranes, it appeared to do so in a CB2 receptor-independent manner.