Characterization of Tritiated JNJ-GluN2B-5 (3-[3H] 1-(Azetidin-1-yl)-2-(6-(4-fluoro-3-methyl-phenyl)pyrrolo[3,2-b]py Ridin-1-yl)ethanone), a High Affinity GluN2B Radioligand with Selectivity over Sigma Receptors

Here, we describe the characterization of a radioligand selective for GluN2B-containing NMDA receptors, 3-[H-3] 1-(azetidin-1-yl)-2-(6-(4-fluoro-3-methyl-phenyl)pyrrolo[3,2-b]pyridin-1-yl)ethanone ([H-3]-JNJ- GluN2B-5). In rat cortical membranes, the compound bound to a single site, and the following kinetic parameters were measured; association rate constant K-on = 0.0066 +/- 0.0006 min(-1) nM(-1), dissociation rate constant K-off = 0.0210 +/- 0.0001 min(-1) indicating calculated K-D = K-off/K-on = 3.3 +/- 0.4 nM, (mean +/- SEM, n = 3). The equilibrium dissociation constant determined from saturation binding experiments in rat cortex was K-D of 2.6 +/- 0.3 nM (mean +/- SEM, n = 3). In contrast to the widely used GluN2B radioligand [H-3]-Ro 25-6981, whose affinity K-i for sigma 1 and sigma 2 receptors are 2 and 189 nM, respectively, [H-3]-JNJ-GluN2B-5 exhibits no measurable affinity for sigma 1 and sigma 2 receptors (K-i > 10 mu M for both) providing distinct selectivity advantages. Anatomical distribution of [H-3]-JNJ-GluN2B-5 binding sites in rat, mouse, dog, monkey, and human brain tissue was studied using in vitro autoradiography, which showed high specific binding in the hippocampus and cortex and negligible binding in the cerebellum. Enhanced selectivity for GluN2B-containing receptors translated to a good signal-to-noise ratio in both in vitro radioligand binding and in vitro autoradiography assays. In conclusion, [H-3]-JNJ-GluN2B-5 is a high-affinity GluN2B radioligand with excellent signal-to-noise ratio and unprecedented selectivity.