Kinase experiment:

Saturable binding and competition binding studies utilize incubations of 1 hour at 22℃. For saturation studies, membranes from mGluR5-GHEK cells are incubated with increasing concentrations (0.1 to 30 nM) of [3H]AZD9272, in the presence or absence of 10 μM MPEP. In a variation of these studies, saturable [3H]AZD9272 binding is determined in the presence of low concentrations (10 and 20 nM) of MPEP. Consistency of the Bmax in the presence or absence of MPEP supports the interaction of these ligands with a unitary binding site[1].

Cell experiment:

hmGluR5-GHEK cells are seeded onto 96 well plates at 50,000 cells/well in media containing 10 μCi/mL [3H]myo-inositol. Cells are incubated overnight (16 h), then washed three times and incubated for 1 hour at 37℃ in HEPES buffered saline supplemented with 1 unit/mL glutamate pyruvate transaminase and 2 mM pyruvate. Cells are washed once in HEPES buffered saline and pre-incubated for 10 minutes in HEPES buffered saline containing 10 mM LiCl. Antagonist activity is determined by pre-incubating cells with AZD9272 for 10 minutes, then incubating for 30 minutes at 37℃ in the presence of glutamate (EC80, 80 μM). AZD9272 is tested at 10 concentrations between 1 nM and 30 μM, in duplicate. The reaction is terminated by the addition of 0.1 mL perchloric acid (5%) on ice, with incubation at 4℃ for at least 30 minutes[1].

Animal experiment:

Approximately 48 male Wistar rats weighing 240 to 250 g at the beginning of the experiments are housed in pairs, or group housed up to 8 rats per cage, in a colony room with water accessible at all times and lights on between 6:00 AM and 6:00 PM; by restricting access to food, animals are kept at approximately 80% of free feeding weight. All animals are divided into different groups and trained to discriminate cocaine (3.4 mg/kg i.p., 15 minutes), PCP (1.6 mg/kg i.p., 30 minutes), MTEP (2 mg/kg i.p., 30 minutes), or AZD9272 (1.6 mg/kg p.o., 60 minutes) from no drug[1].

AZD 9272 is a brain penetrant mGluR5 antagonist.

AZD 9272 causes a concentration dependent decrease in the magnitude of the intracellular Ca2+ response to 1.5 μM of the mGluR group I selective agonist DHPG in both the human and the rat mGluR5 expressing cell lines. The maximal inhibition is 100%. The mean IC50 (±SD) value at the human mGluR5 is 7.6±1.1 nM (n=13) for AZD9272. The mean IC50 value at the rat mGluR5 is 2.6±0.3 nM (n=3) for AZD9272. In contrast, 10 μM of AZD9272 does not diminish the response to 10 μM ATP in the background GHEK cells. Increasing concentrations of AZD9272 causes a decrease in the potency and the maximal response of DHPG. AZD9272 completely reverses the glutamate-stimulated (EC80, 80 μM) phosphatidyl inositol hydrolysis in human mGluR5-GHEK cells in a concentration-dependent manner, with IC50 of 26±3 nM (n=21)[1].

The clearance of AZD 9272 is low following a single intravenous dose at 3 μmol/kg and AZD 9272 is eliminated from plasma with terminal half-lives between 2 and 6 h. The terminal half-lives following oral dosing are similar to the half-lives following intravenous dosing. The volume of distribution at steady state is intermediate for AZD9272[1]. AZD9272 causes no cocaine-appropriate responding and causes a non-dose-dependent reduction in response rates at higher doses. AZD9272 at 2.84 mg/kg causes greater than 80% and typically more than 99% MTEP-appropriate responding up to 20 hours after dose, with a decline to approximately 20% at 24 hours after dose, yielding a t1/2 of 21.93 hours, and causes no systematic effects on response rates. The first time point at which AZD9272 causes >90% MTEP-appropriate responding is at 30 minutes after dose[2].

[1]. Swedberg MD, et al. AZD9272 and AZD2066: selective and highly central nervous system penetrant mGluR5 antagonists characterized by their discriminative effects. J Pharmacol Exp Ther. 2014 Aug;350(2):212-22. [2]. Raboisson P, et al. Discovery and characterization of AZD9272 and AZD6538-Two novel mGluR5 negative allosteric modulators selected for clinical development. Bioorg Med Chem Lett. 2012 Nov 15;22(22):6974-9.