Constitutive activity of cannabinoid‐2 (CB₂) receptors plays an essential role in the protean agonism of (+)AM1241 and L768242

Abstract

Background and purpose: Cannabinoid-2 (CB2) receptor-selective agonists have shown anti-nociceptive activity in models of neuropathic and inflammatory pain, and the two agonists most widely used, (, have been suggested to be protean agonists. Here we investigated the role of the constitutive activity of CB2 receptors in (+)AM1241 and L768242 protean agonism. Experimental approach: Pharmacological profiles of CB2 receptor ligands were evaluated in Chinese hamster ovary cells expressing recombinant human (hCB2) or rat (rCB2) receptors, by measuring modulation of cAMP. To assess the influence of constitutive activity on pharmacological profile, constitutive activity was abolished by pretreatment with AM630 [(6-iodo-2-methyl-1-[2-(4-morpholinyl)ethyl]-1H-indol-3-yl](4-methoxyphenyl) methanone)], followed by extensive washing. Key results: In cell lines expressing either hCB2 or rCB2 receptors, (+)AM1241 did not reverse forskolin stimulation of cAMP levels. Conversely, L768242 was an inverse agonist at both hCB2 and rCB2 receptors. Abolition of constitutive activity disclosed (+)AM1241 and L768242 agonist activity, while activity of CP55940 [5-(1,1-dimethylheptyl)-2-[(1R,2R,5R)-5-hydroxy-2-(3-hydroxy-propyl)-cyclohexyl]-phenol] was unaffected and AM630 became a neutral antagonist. In presence of constitutively active CB2 receptors, (+)AM1241 antagonized CP55940, but when constitutive activity was abolished, it acted as a partial agonist with additive or antagonistic behaviour, depending on concentration. Conclusions and implications:These results show that (+)AM1241 and L768242 are protean agonists at both hCB2 and rCB2 receptors. Abolition of constitutive activity reveals the agonist activity of these compounds. Thus, differences between in vivo and in vitro profiles of CB2 receptor agonists could be due to different levels of constitutive activity in recombinant versus native CB2 receptors.

Non-Invasive Imaging of the Type 2 Cannabinoid Receptor, Focus on Positron Emission Tomography

“…This is in accordance with data in literature reports, except for GW405833, which is known as a partial agonist (determined in a cAMP assay) [106]. Probably this is due to GW405833 being a protean agonist, as described above [111].…”

“…In a rat neuropathic pain model, GW405833 showed inhibitory effects on the formation of gliosis and inhibited neuropathic pain [110]. GW405833 was recently described as being a protean agonist [111]. As stated in the introduction, the CB 2 R is constitutively active, meaning a fraction of the receptor population attains spontaneously the active confirmation in the absence of a ligand.…”

A PET Brain Reporter Gene System Based on Type 2 Cannabinoid Receptors

“…In our assay, GW405833 shows a 10-fold lower affinity for hCB 2 and behaved as an inverse agonist in our GTPgS assay. Our results are in accordance with those of Yao et al, who reported that GW405833 is a potent inverse agonist in both hCB 2 (16,29,30).…”

Evaluation of Cannabinoid Receptor 2 and Metabotropic Glutamate Receptor 1 Functional Responses Using a Cell Impedance–Based Technology

“…Culture splitting was performed by detaching the cells with 0.5% trypsin/EDTA, and cells were plated at different concentration (see below) and maintained in the incubator at 37°C with 5% CO 2 . The method employed for the generation of hCB2 and hmGluR1 transfected cell lines and their characterization was reported previously (Mancini et al 7 and submitted US patent, respectively).…”