Vol. 303, Issue 3, 1044-1051, December 2002

[³H]Methoxymethyl-3-[(2-methyl-1,3-thiazol-4-yl)ethynyl]pyridine Binding to Metabotropic Glutamate Receptor Subtype 5 in Rodent Brain: In Vitro and in Vivo Characterization

Jeffery J. Anderson, Sara P. Rao, Blake Rowe, Darlene R. Giracello, Greg Holtz, Deborah F. Chapman, Lida Tehrani, Margaret J. Bradbury, Nicholas D. P. Cosford and Mark A. Varney

Departments of Neuropharmacology (J.J.A., S.P.R., B.R., D.R.G., G.H., D.F.C., M.J.B., M.A.V.) and Medicinal Chemistry (L.T., N.D.P.C.), Merck Research Laboratories, San Diego, California

The binding of [³H]methoxymethyl-3-[(2-methyl-1,3-thiazol-4-yl)ethynyl]pyridine (methoxymethyl-MTEP), a potent and selective antagonist for metabotropic glutamate (mGlu)5 receptors, was characterized in rat brain both in vitro and in vivo. Nonspecific binding, as defined with 10 µM 2-methyl-6-(phenylethynyl)-pyridine (MPEP), was less than 10% of total binding in rat brain membranes. The binding of [³H]methoxymethyl-MTEP was of high affinity (K_d = 20 ± 2.7 nM), saturable (B_max = 487 ± 48 fmol/mg protein), and to a single site. The mGlu5 antagonists methoxymethyl-MTEP and MPEP displaced [³H]methoxymethyl-MTEP binding with IC₅₀ values of 30 and 15 nM, respectively. In vivo administration of [³H]methoxymethyl-MTEP (50 µCi/kg i.v.) revealed 12-fold higher binding in hippocampus (an area enriched in mGlu5 receptors) relative to cerebellum (an area with few mGlu5 receptors) in rats. Similarly, administration of [³H]methoxymethyl-MTEP to mGlu5-deficient mice demonstrated binding at background levels in forebrain, whereas wild-type littermates exhibited 17-fold higher binding in forebrain relative to cerebellum. Systemic administration of unlabeled mGlu5 antagonists methoxymethyl-MTEP and MPEP to rats reduced the binding of [³H]methoxymethyl-MTEP with ID₅₀ values of 0.8 and 2 mg/kg i.p., respectively, 1 h post-treatment. The mGlu5 agonist 2-chloro-5-hydroxyphenylglycine (CHPG) (0.3, 1, and 3 µmol) dose-dependently increased phosphoinositide (PI) hydrolysis in the hippocampus after i.c.v. administration in rats. CHPG-evoked increases in PI hydrolysis were blocked with MPEP at a dose (10 mg/kg i.p.) that markedly reduced [³H]methoxymethyl-MTEP binding in vivo. These results indicate that [³H]methoxymethyl-MTEP is a selective radioligand for labeling mGlu5 and is useful for studying the binding of mGlu5 receptors in rat brain in vitro and in vivo.