BACKGROUND AND PURPOSECannabidiol has been reported to act as an antagonist at cannabinoid CB 1 receptors. We hypothesized that cannabidiol would inhibit cannabinoid agonist activity through negative allosteric modulation of CB 1 receptors. EXPERIMENTAL APPROACHInternalization of CB 1 receptors, arrestin2 recruitment, and PLCβ3 and ERK1/2 phosphorylation, were quantified in HEK 293A cells heterologously expressing CB 1 receptors and in the STHdh Q7/Q7 cell model of striatal neurons endogenously expressing CB 1 receptors. Cells were treated with 2-arachidonylglycerol or Δ 9 -tetrahydrocannabinol alone and in combination with different concentrations of cannabidiol. KEY RESULTSCannabidiol reduced the efficacy and potency of 2-arachidonylglycerol and Δ 9 -tetrahydrocannabinol on PLCβ3-and ERK1/2-dependent signalling in cells heterologously (HEK 293A) or endogenously (STHdh Q7/Q7 ) expressing CB 1 receptors. By reducing arrestin2 recruitment to CB 1 receptors, cannabidiol treatment prevented internalization of these receptors. The allosteric activity of cannabidiol depended upon polar residues being present at positions 98 and 107 in the extracellular amino terminus of the CB 1 receptor. CONCLUSIONS AND IMPLICATIONSCannabidiol behaved as a non-competitive negative allosteric modulator of CB 1 receptors. Allosteric modulation, in conjunction with effects not mediated by CB 1 receptors, may explain the in vivo effects of cannabidiol. Allosteric modulators of CB 1 receptors have the potential to treat CNS and peripheral disorders while avoiding the adverse effects associated with orthosteric agonism or antagonism of these receptors.

Huntington's disease (HD) is a fatal neurodegenerative disorder caused by the presence of an abnormally expanded polyglutamine domain in the N-terminus of huntingtin. We developed a recombinant adeno-associated viral serotype 5 (rAAV5) gene transfer strategy to posttranscriptionally suppress the levels of striatal mutant huntingtin (mHtt) in the R6/1 HD transgenic mouse via RNA interference. Transient cotransfection of HEK293 cells with plasmids expressing a portion of human mHtt derived from R6/1 transgenic HD mice and a short-hairpin RNA directed against the 5' UTR of the mHtt mRNA (siHUNT-1) resulted in reduction in the levels of mHtt mRNA (-75%) and protein (-60%). Long-term in vivo rAAV5-mediated expression of siHUNT-1 in the striatum of R6/1 mice reduced the levels of mHtt mRNA (-78%) and protein (-28%) as determined by quantitative RT-PCR and Western blot analysis, respectively. The reduction in mHtt was concomitant with a reduction in the size and number of neuronal intranuclear inclusions and a small but significant normalization of the steady-state levels of preproenkephalin and dopamine- and cAMP-responsive phosphoprotein 32 kDa mRNA. Finally, bilateral expression of rAAV5-siHUNT-1 resulted in delayed onset of the rear paw clasping phenotype exhibited by the R6/1 mice. These results suggest that a reduction in the levels of striatal mHtt can ameliorate the HD phenotype of R6/1 mice.

The cannabinoid 1 receptor (CB1R) is one of the most widely expressed metabotropic G protein-coupled receptors in brain, and its participation in various (patho)physiological processes has made CB1R activation a viable therapeutic modality. Adverse psychotropic effects limit the clinical utility of CB1R orthosteric agonists and have promoted the search for CB1R positive allosteric modulators (PAMs) with the promise of improved drug-like pharmacology and enhanced safety over typical CB1R agonists. In this study, we describe the synthesis and in vitro and ex vivo pharmacology of the novel allosteric CB1R modulator GAT211 (racemic) and its resolved enantiomers, GAT228 (R) and GAT229 (S). GAT211 engages CB1R allosteric site(s), enhances the binding of the orthosteric full agonist [H]CP55,490, and reduces the binding of the orthosteric antagonist/inverse agonist [H]SR141716A. GAT211 displayed both PAM and agonist activity in HEK293A and Neuro2a cells expressing human recombinant CB1R (hCB1R) and in mouse-brain membranes rich in native CB1R. GAT211 also exhibited a strong PAM effect in isolated vas deferens endogenously expressing CB1R. Each resolved and crystallized GAT211 enantiomer showed a markedly distinctive pharmacology as a CB1R allosteric modulator. In all biological systems examined, GAT211's allosteric agonist activity resided with the R-(+)-enantiomer (GAT228), whereas its PAM activity resided with the S-(-)-enantiomer (GAT229), which lacked intrinsic activity. These results constitute the first demonstration of enantiomer-selective CB1R positive allosteric modulation and set a precedent whereby enantiomeric resolution can decisively define the molecular pharmacology of a CB1R allosteric ligand.

The pharmacological activity of CBD and CBD-DMH in HEK293A cells and their modelled binding sites at CB and CB receptors may explain their in vivo effects and illuminates the difficulties associated with the development of allosteric modulators for CB and CB receptors.

Background:To understand the differential response to cannabinoids, we examined the functional selectivity of type 1 cannabinoid receptor (CB 1 ) agonists in a cell model of striatal neurons. Results: 2-Arachidonylglycerol, ⌬ 9 -tetrahydrocannabinol, and CP55,940 were arrestin2-selective; endocannabinoids and WIN55,212-2 activated G␣ i/o , G␤␥, and G␣ q ; and cannabidiol activated G␣ s independent of CB 1 . Conclusion: Cannabinoids displayed functional selectivity. Significance: CB 1 functional selectivity may be exploited to maximize therapeutic efficacy.