Background and Purpose Neuroinflammation is increasingly recognized to contribute to drug-resistant epilepsy. Activation of the ATP-gated P2X7 receptor (P2X7R) has emerged as an important upstream mechanism and increased P2X7R expression is present in the seizure focus in rodent models and patients. Pharmacologic antagonism of the P2X7R can attenuate seizures in rodents but this has not been explored in human neuronal networks. Experimental Approach Human neurons were differentiated from two induced pluripotent stem cell (hiPSC) lines. P2X7R function on neurons was assessed via P2X7R agonist BzATP-evoked calcium transients. Acute or chronic in vitro models of epileptiform-like events were generated by exposure of hiPSC cultures to the GABAA receptor antagonist picrotoxin or a cocktail of picrotoxin and neuroinflammatory agents with or without the presence of P2X7R antagonists. Epileptiform-like activity was measured via single cell patch-clamp recordings. Key Results BzATP application (300 µM) resulted in increased calcium influx in hiPSC-derived neurons which was blocked by the P2X7R antagonists JNJ-47965567 (100 nM) and AFC-5128 (30 nM). Single-cell patch-clamp recordings showed that, while treatment with AFC-5128 did not reduce epileptiform-like activity triggered by picrotoxin alone, AFC-5128 reduced the severity of epileptiform-like activity under inflammatory conditions. Notably, epileptiform-like events in the inflammation-primed picrotoxin model were refractory to the anti-seizure medication carbamazepine alone but were reduced by the co-application of carbamazepine with AFC-5128. Conclusion and Implications Our findings demonstrate anti-seizure effects of targeting the P2X7R in a human neuronal network model of epilepsy and suggest P2X7R-based treatments may be an effective add-on therapy for controlling drug-resistant seizures.

Background and Purpose Refractory status epilepticus is a clinical emergency associated with high mortality and morbidity. Increasing evidence suggests neuroinflammatory pathways contribute to the development of drug-refractoriness during status epilepticus. The ATP-gated P2X7 receptor (P2X7R) has been described as potential link between inflammation and increased hyperexcitability. The aim of the present study was to determine the contribution of the P2X7R to drug-refractory status epilepticus and its therapeutic potential. Experimental Approach Status epilepticus was induced via a unilateral microinjection of kainic acid into the amygdala in adult mice. Severity of status epilepticus was compared in animals overexpressing or knock-out in the P2X7R, after inflammatory priming by the pre-injection of bacterial lipopolysaccharide (LPS) and in mice treated with P2X7R-targeting and anti-inflammatory drugs. Key Results P2X7R overexpressing mice were unresponsive to several anticonvulsants (lorazepam, midazolam, phenytoin and carbamazepine) during status epilepticus. P2X7R expression was increased in microglia during drug-refractory status epilepticus, P2X7R overexpression led to a pro-inflammatory phenotype in microglia during status epilepticus and the anti-inflammatory drug minocycline restored normal responsiveness to anticonvulsants in P2X7R overexpressing mice. Pre-treatment of wildtype mice with LPS increased P2X7R levels in the brain and promoted the development of pharmaco-resistant status epilepticus, which was overcome by either a genetic deletion of the P2X7R or the administration of the P2X7R antagonists AFC-5128 or ITH15004. Conclusion and Implications Our results demonstrate that P2X7R-induced pro-inflammatory effects contribute to resistance to pharmacotherapy during status epilepticus and suggest therapies targeting the P2X7R as novel adjunctive treatments for drug-refractory status epilepticus.