Development and preliminary validation of a plate-based CB1/CB2 receptor functional assay

Cannabinoid (CB) receptors are being targeted therapeutically for the treatment of anxiety, obesity, movement disorders, glaucoma, and pain. More recently, cannabinoid agonists have displayed antiproliferative activity against breast cancer and prostate cancer in animal models. To study cannabinoid receptor ligands, we have developed a novel plate-based assay that measures internalization of CB1/CB2 receptors by determining the change in the intracellular levels of the radiolabeled agonists: [(3)H]Win55-212-2 for CB1 and [(3)H]CP55-940 for CB2. The developed plate-based assay was validated by determining IC50 values for known antagonists: AM251, AM281, AM630, and AM6545. The data obtained were consistent with previously reported values, thereby confirming that the assay can be used to determine the functional binding activities (IC50) of antagonists for the CB1 and CB2 receptors. In addition, we demonstrated that the plate-based assay may be used for screening against complex matrices. Specifically, we demonstrated that the plate-based assay was able to identify which extracts of several species of the genus Zanthoxylum had activity at the CB1/CB2 receptors.