CTEP (RO4956371)

Kinase Assay [1]
For all filtration radioligand binding assays, membrane preparations expressing the target receptors or receptor combinations are resuspended in radioligand binding buffer (15 mM Tris-HCl, 120 mM NaCl, 5 mM KCl, 1.25 mM CaCl2, and 1.25 mM MgCl2, pH 7.4), and the membrane suspension is mixed with the appropriate concentrations of radioligand and nonlabeled drugs in 96-well plates in a total volume of 200 μL and incubated for 60 min at the appropriate temperature. At the end of the incubation, membranes are filtered onto Whatman Unifilter preincubated with 0.1% polyethyleneimine in ish buffer (50 mM Tris-HCl, pH 7.4) with a Filtermate 196 harvester and washed three times with ice-cold ish buffer. Radioactivity captured on the filter is quantified on a Topcount microplate scintillation counter with quenching correction after the addition of 45 μL of MicroScint 40 per well and shaking for 20 min. The concentration of membranes and incubation time is determined for each assay in pilot experiments.

Animal Administration [1]
Adult male Sprague-Dawley rats (body weight approximately 180-210 g) and male NMRI mice (body weight approximately 25 g) are supplied by Charles River. Rats are group-housed and mice are-single housed in separate holding rooms at controlled temperature (20-22°C) and 12-h light/dark cycle (lights on 6:00 AM). Animals are allowed ad libitum access to food and water, with the exception of those used in the Vogel conflict drinking test, where access to water is limited during the training sessions as described below. All formulations are prepared immediately before use in vehicle, consisting of 0.9% NaCl (w/v) and 0.3% Tween 80 (v/v) solution for oral administration of CTEP, MPEP, MTEP, and fenobam; 0.9% NaCl solution for MPEP and MTEP intravenously; and 30% N-methylpyrrolidone, 42% hydroxypropyl-γ-cyclodextrin, and 28% water for fenobam intravenously. The volume of administration for oral dosing is 5 mL/kg for rats, 10 mL/kg for mice, and 2.5 mL/kg for intravenous applications and 10 mL/kg for subcutaneous applications in mice.

References:
[1]. Lindemann L, et al. CTEP: a novel, potent, long-acting, and orally bioavailable metabotropic glutamate receptor 5 inhibitor. J Pharmacol Exp Ther. 2011 Nov;339(2):474-86. [2]. Michalon A, et al. Chronic pharmacological mGlu5 inhibition corrects fragile X in adult mice. Neuron. 2012 Apr 12;74(1):49-56.