Background
IC 50 value: 0.7nM(Ki for binding)/0.2 nM (Ki for function) Otenabant (CP-945,598) is a recently discovered selective, high affinity, competitive CB1 receptor antagonist that inhibits both basal and cannabinoid agonist-mediated CB1 receptor signaling. Cannabinoid CB1 receptor antagonists exhibit pharmacologic properties favorable for the treatment of metabolic disease. in vitro, ex vivo, and in vivo data indicate that Otenabant (CP-945,598) is a novel CB(1) receptor competitive antagonist that may further our understanding of the endocannabinoid system[1]. in vitro : 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[1]. in vivo: Otenabant (CP-945,598) reverses four cannabinoid agonist-mediated CNS-driven responses (hypo-locomotion, hypothermia, analgesia, and catalepsy) to a synthetic cannabinoid receptor agonist. Otenabant (CP-945,598) exhibits dose and concentration-dependent anorectic activity in two models of acute food intake in rodents, fast-induced re-feeding and spontaneous, nocturnal feeding. Otenabant (CP-945,598) also acutely stimulates energy expenditure in rats and decreases the respiratory quotient indicating a metabolic switch to increased fat oxidation. CP-945,598 at 10 mg/kg promoted a 9%, vehicle adjusted weight loss in a 10 day weight loss study in diet-induced obese mice[1]. After oral administration of a single dose of [(14)C]CP-945,598. Total mean recoveries of the radioactive dose were 97.7, 97.8, and 99.3% from mice, rats, and dogs respectively[2]. Clinical trail: A phase 1 study of the roles of endocannabinoids in insulin secretion and action is recruiting.
Protocol
Kinase experiment: | 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 (10-10 M 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. |
Animal experiment: | 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? |
参考文献: [1]. John R. Hadcock, et al. In vitro and in vivo pharmacology of CP-945,598, a potent and selective cannabinoid CB1 receptor antagonist for the management of obesity. Biochemical and Biophysical Research Communications, 2010; 394;366-371.
[2]. Griffith DA, et al. Discovery of 1-[9-(4-chlorophenyl) -8-(2-chlorophenyl)- 9H-purin-6-yl] -4-ethylaminopiperidine-4-carboxylic acid amide hydrochloride (CP-945,598), a novel, potent, and selective cannabinoid type 1 receptor antagonist. JMedChem. 2009 ;5 |
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