Taranabant

Kinase experiment:

The binding assay is performed by incubating various concentrations of Taranabant (MK-0364) with 0.5 nM [3H]CP 55,940, 1.5 μg of recombinant human CB1R-CHO membranes (or 0.1 μg of human CB2R-CHO membranes) in 50 mM Tris-HCl, pH 7.4, 5 mM MgCl2, 2.5 mM EDTA, 0.5 mg/mL fatty acid-free bovine serum albumin (BSA), 1× proteinase inhibitor mix, and 1% DMSO. After 1-h incubation at 37°C, the reaction is stopped by filtration, and bound radioligand is separated from free radioligand by washing the filter plate. Total specifically bound radiolabel is approximately 10% of the total added radiolabel. Inhibitory IC50 values are calculated through nonlinear curve fitting, from which Ki values are then calculated. The CB1R density (Bmax=5 pmol/mg based on [3H]CP 55,940 binding) in the recombinant human CB1R-CHO membranes is close to that from rat brain membranes (3-5 pmol/mg)[1].

Animal experiment:

Mice[1] Male C57BL/6N wild-type mice are used. MK-0364 is dissolved or dispersed (with sonication) as a fine homogeneous suspension in 0.225% methylcellulose/10% Tween 80 in water for subsequent oral dosing of mice. All mice are weighed, and vehicle (0.225% methylcellulose/10% Tween 80 in water) or Taranabant (1 or 3 mg/kg) is administered by oral gavage to male mice approximately 30 min before the onset of the dark phase of the light cycle (n=12 per group, age 23 weeks, mean body weight 34.14±0.53 g). Mice are fed ad libitum in the dark phase after dosing. A preweighed aliquot of a highly palatable medium-high fat diet (25% kcal from sucrose, 32% kcal from fat, 4.41 kcal/g) is provided in the food hopper of the cage 5 min before the onset of the dark phase of the light cycle and weighed 2 and 18 h after the onset of the dark phase of the light cycle. In addition, all mice are weighed 18 h after the onset of the dark phase of the light cycle. The study is of crossover design, i.e., vehicle and 1-mk/kg groups are dosed first. After a 4-day washout, the previous vehicle group is dosed with 3 mg/kg Taranabant, and the previous 1-mg/kg group is dosed with vehicle. Rats[1] For acute experiments, male Sprague-Dawley DIO rats are randomized into groups (n=6 rats/group) for compound and vehicle dosing. Rats are weighed 17 h after dosing to determine effects on overnight body weight gain. Taranabant is administered orally to DIO rats 1 h before the start of the dark cycle (3:00 PM) at 0.3, 1, and 3 mg/kg p.o. Vehicle is 10% Tween 80 in water, and dosing volume is 2 mL/kg. Powdered food is provided in food cups that are weighed continuously at 5-min intervals over 18 h, and the data are recorded using a computerized system.

References:

[1]. Fong TM, et al. Antiobesity Efficacy of a Novel Cannabinoid-1 Receptor Inverse Agonist, N-[(1S,2S)-3-(4-Chlorophenyl)-2-(3-cyanophenyl)-1-methylpropyl]-2-methyl-2-{[5-(trifluoromethyl)pyridin-2-yl]oxy}propanamide (MK-0364), in Rodents. J Pharmacol Exp T
[2]. Lin LS, et al. Discovery of N-[(1S,2S)-3-(4-Chlorophenyl)-2- (3-cyanophenyl)-1-methylpropyl]-2-methyl-2- {[5-(trifluoromethyl)pyridin-2-yl]oxy}propanamide (MK-0364), a novel, acyclic cannabinoid-1 receptor inverse agonist for the treatment of obesity. J M