Otenabant Hydrochloride

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.

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.

Male, 14 week old C57/Bl6/6J mice which has been maintained on a high fat diet (45% kcal from fat) for 6 weeks are selected for the DIO weight loss study. The animals body weights range at least five standard deviations from age-matched chow-fed control animals mean body weight. Mice are singly housed. The mean starting weight of all animals is 38.9±0.5 g. On day 0, mice are randomLy assigned to treatment groups (n=10 per group). Mice are dosed daily with vehicle or 10 mg/kg (p.o.) CP-945,598 over 10 days, starting approximately at 30 min before the start of the 12 h dark cycle. BW and food intake are recorded daily. Analysis of variance and comparison of means are calculated for daily and cumulative FI and cumulative BW measurements. P < 0.05 is considered statistically significant.

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.