The P2X7 receptor contributes to seizures and inflammation-driven long-lasting brain hyperexcitability following hypoxia in neonatal mice

Background and purpose: Neonatal seizures represent a clinical emergency. However, current anti-seizure medications fail to resolve seizures in ~50% of infants. The P2X7 receptor (P2X7R) is an important driver of inflammation, and evidence suggests that P2X7R contributes to seizures and epilepsy in adults. However, no genetic proof has yet been provided to determine what contribution P2X7R makes to neonatal seizures, its effects on inflammatory signalling during neonatal seizures, and the therapeutic potential of P2X7R-based treatments on long-lasting brain excitability. Experimental approach: Neonatal seizures were induced by global hypoxia in 7-day-old mouse pups (P7). The role of P2X7Rs during seizures was analysed in P2X7R-overexpressing and knockout mice. Treatment of wild-type mice after hypoxia with the P2X7R antagonist JNJ-47965567 was used to determine the effects of the P2X7R on long-lasting brain hyperexcitability. Cell type-specific P2X7R expression was analysed in P2X7R-EGFP reporter mice. RNA sequencing was used to monitor P2X7R-dependent hippocampal downstream signalling. Key results: P2X7R deletion reduced seizure severity, whereas P2X7R overexpression exacerbated seizure severity and reduced responsiveness to anti-seizure medication. P2X7R deficiency led to an anti-inflammatory phenotype in microglia, and treatment of mice with a P2X7R antagonist reduced long-lasting brain hyperexcitability. RNA sequencing identified several pathways altered in P2X7R knockout mice after neonatal hypoxia, including a down-regulation of genes implicated in inflammation and glutamatergic signalling. Conclusion and implications: Treatments based on targeting the P2X7R may represent a novel therapeutic strategy for neonatal seizures with P2X7Rs contributing to the generation of neonatal seizures, driving inflammatory processes and long-term hyperexcitability states.</p

DataSheet1_Anti-seizure effects of JNJ-54175446 in the intra-amygdala kainic acid model of drug-resistant temporal lobe epilepsy in mice.PDF

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.</p

Discovery of 8‑Trifluoromethyl-3-cyclopropylmethyl-7-[(4-(2,4-difluorophenyl)-1-piperazinyl)­methyl]-1,2,4-triazolo[4,3‑<i>a</i>]pyridine (JNJ-46356479), a Selective and Orally Bioavailable mGlu2 Receptor Positive Allosteric Modulator (PAM)

Positive allosteric modulators of the metabotropic glutamate 2 receptor have generated great interest in the past decade. There is mounting evidence of their potential as therapeutic agents in the treatment of multiple central nervous system disorders. We have previously reported substantial efforts leading to potent and selective mGlu2 PAMs. However, finding compounds with the optimal combination of in vitro potency and good druglike properties has remained elusive, in part because of the hydrophobic nature of the allosteric binding site. Herein, we report on the lead optimization process to overcome the poor solubility inherent to the advanced lead <b>6</b>. Initial prototypes already showed significant improvements in solubility while retaining good functional activity but displayed new liabilities associated with metabolism and hERG inhibition. Subsequent subtle modifications efficiently addressed those issues leading to the identification of compound <b>27</b> (JNJ-46356479). This new lead represents a more balanced profile that offers a significant improvement on the druglike attributes compared to previously reported leads

Dual Alleviation of Acute and Neuropathic Pain by Fused Opioid Agonist-Neurokinin 1 Antagonist Peptidomimetics

Herein, the synthesis and biological evaluation of dual opioid agonists–neurokinin 1 receptor (NK1R) antagonists is described. In these multitarget ligands, the two pharmacophores do not overlap, and this allowed maintaining high NK1R affinity and antagonist potency in compounds <b>12</b> and <b>13</b>. Although the fusion of the two ligands resulted in slightly diminished opioid agonism at the μ- and δ-opioid receptors (MOR and DOR, respectively), as compared to the opioid parent peptide, balanced MOR/DOR activities were obtained. Compared to morphine, compounds <b>12</b> and <b>13</b> produced more potent antinociceptive effects in both acute (tail-flick) and neuropathic pain models (von Frey and cold plate). Similarly to morphine, analgesic tolerance developed after repetitive administration of these compounds. To our delight, compound <b>12</b> did not produce cross-tolerance with morphine and high antihyperalgesic and antiallodynic effects could be reinstated after chronic administration of each of the two compounds

4‑Methyl-6,7-dihydro‑4<i>H</i>‑triazolo[4,5‑<i>c</i>]­pyridine-Based P2X7 Receptor Antagonists: Optimization of Pharmacokinetic Properties Leading to the Identification of a Clinical Candidate

The synthesis and preclinical characterization of novel 4-(<i>R</i>)-methyl-6,7-dihydro-4<i>H</i>-triazolo­[4,5-<i>c</i>]­pyridines that are potent and selective brain penetrant P2X7 antagonists are described. Optimization efforts based on previously disclosed unsubstituted 6,7-dihydro-4<i>H</i>-triazolo­[4,5-<i>c</i>]­pyridines, methyl substituted 5,6,7,8-tetrahydro­[1,2,4]­triazolo­[4,3-<i>a</i>]­pyrazines, and several other series lead to the identification of a series of 4-(<i>R</i>)-methyl-6,7-dihydro-4<i>H</i>-triazolo­[4,5-<i>c</i>]­pyridines that are selective P2X7 antagonists with potency at the rodent and human P2X7 ion channels. These novel P2X7 antagonists have suitable physicochemical properties, and several analogs have an excellent pharmacokinetic profile, good partitioning into the CNS and show robust in vivo target engagement after oral dosing. Improvements in metabolic stability led to the identification of JNJ-54175446 (<b>14</b>) as a candidate for clinical development. The drug discovery efforts and strategies that resulted in the identification of the clinical candidate are described herein

4‑Methyl-6,7-dihydro‑4<i>H</i>‑triazolo[4,5‑<i>c</i>]­pyridine-Based P2X7 Receptor Antagonists: Optimization of Pharmacokinetic Properties Leading to the Identification of a Clinical Candidate

The synthesis and preclinical characterization of novel 4-(<i>R</i>)-methyl-6,7-dihydro-4<i>H</i>-triazolo­[4,5-<i>c</i>]­pyridines that are potent and selective brain penetrant P2X7 antagonists are described. Optimization efforts based on previously disclosed unsubstituted 6,7-dihydro-4<i>H</i>-triazolo­[4,5-<i>c</i>]­pyridines, methyl substituted 5,6,7,8-tetrahydro­[1,2,4]­triazolo­[4,3-<i>a</i>]­pyrazines, and several other series lead to the identification of a series of 4-(<i>R</i>)-methyl-6,7-dihydro-4<i>H</i>-triazolo­[4,5-<i>c</i>]­pyridines that are selective P2X7 antagonists with potency at the rodent and human P2X7 ion channels. These novel P2X7 antagonists have suitable physicochemical properties, and several analogs have an excellent pharmacokinetic profile, good partitioning into the CNS and show robust in vivo target engagement after oral dosing. Improvements in metabolic stability led to the identification of JNJ-54175446 (<b>14</b>) as a candidate for clinical development. The drug discovery efforts and strategies that resulted in the identification of the clinical candidate are described herein