Org 27569

Kinase experiment:

Binding assays are performed with the CB1 receptor agonist [3H]CP 55,940 (0.7 nM) and the CB1 receptor antagonist [3H]SR 141716A (1.2 nM), 1 mg/mL BSA and 50 mM Tris buffer containing 0.1 mM EDTA and 0.5 mM MgCl2, pH 7.4, in a total assay volume of 500 μL. Binding is initiated by the addition of mouse brain membranes (30 μg). Assays are carried out at 37°C for 60 min before termination by addition of ice-cold wash buffer (50 mM Tris buffer and 1 mg/mL BSA) and vacuum filtration using a 24-well sampling manifold and Whatman GF/B glass-fiber filters that have been soaked in wash buffer at 4°C for 24 h. Each reaction tube is washed five times with a 4-mL aliquot of buffer. The filters are oven-dried for 60 min and then placed in 5 mL of scintillation fluid, and radioactivity is quantitated by liquid scintillation spectrometry. Specific binding is defined as the difference between the binding that occurs in the presence and absence of 1 μM concentrations of the corresponding unlabeled ligand and is 70 to 80% of the total binding.

Animal experiment:

Following a 1-week acclimation period, CB1 (+/+) and (−/−) mice are food-deprived, given an intraperitoneal injection of Org27569 (30 mg/kg), rimonabant (10 mg/kg; positive control), or vehicle at 23 h, and placed in a plastic cage with access to water. A premeasured amount (2.3-2.6 g) of sweet cereal or standard chow is placed in the test cage from 24 to 26h. All mice receive each treatment condition in a counterbalanced design, with at least 96 h between test days.

References:

[1]. Jing L, et al. Effects of the cannabinoid CB₁ receptor allosteric modulator ORG 27569 on reinstatement of cocaine- and methamphetamine-seeking behavior in rats. Drug Alcohol Depend. 2014 Oct 1;143:251-6.
[2]. Fay JF, et al. A key agonist-induced conformational change in the cannabinoid receptor CB1 is blocked by the allosteric ligand Org 27569. J Biol Chem. 2012 Jul 30.
[3]. Gamage TF, et al. In-vivo pharmacological evaluation of the CB1-receptor allosteric modulator Org-27569. Behav Pharmacol. 2014 Apr;25(2):182-5.
[4]. Price MR, et al. Allosteric modulation of the cannabinoid CB1 receptor. Mol Pharmacol. 2005 Nov;68(5):1484-95.
[5]. Baillie GL, et al. CB(1) receptor allosteric modulators display both agonist and signaling pathway specificity. Mol Pharmacol. 2013 Feb;83(2):322-38.