| In Vitro | In vitro activity: CP-945598 HCl inhibits CB1 receptor with moderate unbound microsomal clearance, low hERG affinity, and adequate CNS penetration. CP-945598 HCl has low affinity with Ki of 7.6 μM for human CB2 receptors.
Kinase Assay: Membranes are prepared from CHOK1 cells stably transfected with the human CB-1 receptor cDNA. GTPγ [35S] binding assays are performed in a 96-well FlashPlate format in duplicate using 100 pM GTPγ [35S] and 10μg membrane per well in assay buffer composed of 50 mM Tris HCl, pH 7.4, 3 mM MgCl2, pH 7.4, 10 mM MgCl2,
20 mM EGTA, 100 mM NaCl, 30 μM GDP, 0.1% bovine serum albumin, and the
following protease inhibitors: 100 μg/mL bacitracin, 100 μg/mL
benzamidine, 5 μg/mL aprotinin, 5 μg/mL leupeptin. The assay mix is then
incubated with increasing concentrations of antagonist (10-10M to 10-5
M) for 10 min and challenged with the cannabinoid agonist CP-55,940 (10
μM). Assays are performed at 30°C for 1 h. The FlashPlates are then
centrifuged at 2000 g for 10 min. Stimulation of GTPγ [35S] binding is then quantified using a Wallac Microbeta. EC50 calculations are done using Prism by GraphPad. Inverse agonism is measured in the absence of agonist.
Cell Assay: Otenabant (CP-945,598) exhibits sub-nanomolar potency at human CB1 receptors in both binding (Ki = 0.7 nM) and functional assays (Ki = 0.2 nM). The compound has low affinity (Ki = 7600 nM) for human CB2 receptors. |
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