Characterization of tritiated JNJ-GluN2B-5 (3-[3H] 1-(azetidin-1-yl)-2-(6-(4-fluoro-3-methyl-phenyl)pyrrolo[3,2-b]pyridin-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-[3H] 1-(azetidin-1-yl)-2-(6-(4-fluoro-3-methyl-phenyl)pyrrolo[3,2-b]pyridin-1-yl)ethanone ([3H]-JNJ- GluN2B-5). In rat cortical membranes, the compound bound to a single site, and the following kinetic parameters were measured; association rate constant Kon = 0.0066 ± 0.0006 min-1 nM-1, dissociation rate constant Koff = 0.0210 ± 0.0001 min-1 indicating calculated KD = Koff/Kon = 3.3 ± 0.4 nM, (mean ± SEM, n = 3). The equilibrium dissociation constant determined from saturation binding experiments in rat cortex was KD of 2.6 ± 0.3 nM (mean ± SEM, n = 3). In contrast to the widely used GluN2B radioligand [3H]-Ro 25-6981, whose affinity Ki for sigma 1 and sigma 2 receptors are 2 and 189 nM, respectively, [3H]-JNJ-GluN2B-5 exhibits no measurable affinity for sigma 1 and sigma 2 receptors (Ki > 10 µM for both) providing distinct selectivity advantages. Anatomical distribution of [3H]-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, [3H]-JNJ-GluN2B-5 is a high-affinity GluN2B radioligand with excellent signal-to-noise ratio and unprecedented selectivity.

Antagonism of the brain P2X7 ion channel attenuates repeated social defeat induced microglia reactivity, monocyte recruitment and anxiety-like behavior in male mice.

Chronic stress is linked to increased anxiety. Repeated social defeat (RSD) in mice causes anxiety that is dependent on activated neurons, reactive microglia, and accumulation of monocytes in the brain. This response requires interactions between the immune system and central nervous system (CNS). Neuronal activation within threat appraisal regions is a key response to RSD, however, it is unclear how microglia become activated. One potential explanation is that microglia express a purinergic non-selective ligand gated adenosine-triphosphate (ATP) receptor 7 (P2X7). Activation of P2X7 promotes the release of chemokines and cytokines, and recruitment of monocytes to the brain. Thus, the purpose of this study was to determine if a novel P2X7 antagonist blocked neuronal and microglia interactions and the corresponding anxiety following RSD. Male mice were administered (i.p.) a P2X7 antagonist, JNJ-54471300, prior to each cycle of RSD. Fourteen hours after RSD, behavioral deficits including social avoidance and anxiety-like were determined. Moreover, several immune parameters were assessed. RSD caused neuronal activation in stress-responsive regions, monocyte production and release, splenomegaly, and social avoidance. These parameters were unaffected by P2X7 antagonism. RSD-associated proportional area of Iba-1+ microglia, monocyte accumulation in the brain, IL-1ß mRNA expression in enriched myeloid cells, plasma IL-6, and anxiety-like behavior were ameliorated by P2X7 antagonism. Gene expression analysis in the hippocampus and amygdala showed regional specific responses to RSD and some were reversed with P2X7 antagonism. Overall, blocking P2X7 activation attenuated RSD-induced microglia reactivity with corresponding reduction in neuroinflammation, monocyte accumulation, and anxiety-like behavior in male mice.

Anti-seizure effects of JNJ-54175446 in the intra-amygdala kainic acid model of drug-resistant temporal lobe epilepsy in mice.

There remains a need for new drug targets for treatment-resistant temporal lobe epilepsy. The ATP-gated P2X7 receptor coordinates neuroinflammatory responses to tissue injury. Previous studies in mice reported that the P2X7 receptor antagonist JNJ-47965567 suppressed spontaneous seizures in the intraamygdala kainic acid model of epilepsy and reduced attendant gliosis in the hippocampus. The drug-resistance profile of this model is not fully characterised, however, and newer P2X7 receptor antagonists with superior pharmacokinetic profiles have recently entered clinical trials. Using telemetry-based continuous EEG recordings in mice, we demonstrate that spontaneous recurrent seizures in the intraamygdala kainic acid model are refractory to the common anti-seizure medicine levetiracetam. In contrast, once-daily dosing of JNJ-54175446 (30 mg/kg, intraperitoneal) resulted in a significant reduction in spontaneous recurrent seizures which lasted several days after the end of drug administration. Using a combination of immunohistochemistry and ex vivo radiotracer assay, we find that JNJ-54175446-treated mice at the end of recordings display a reduction in astrogliosis and altered microglia process morphology within the ipsilateral CA3 subfield of the hippocampus, but no difference in P2X7 receptor surface expression. The present study extends the characterisation of the drug-resistance profile of the intraamygdala kainic acid model in mice and provides further evidence that targeting the P2X7 receptor may have therapeutic applications in the treatment of temporal lobe epilepsy.