Background and purpose. Traumatic brain injury (TBI) is an acute brain lesion considered as one of the leading causes of mortality and disability worldwide. After TBI, innate immunity is rapidly activated in response to damage-associated molecular patterns, such as ATP release, recognized by P2X7 purinergic receptors (P2X7R). The P2X7R-NLRP3 inflammasome axis has been identified as one of the main actors in neuroinflammation. Therefore, this study aimed to validate P2X7R as therapeutic target in TBI. Experimental approach. P2X7R was validated through genetic and pharmacological approaches. Six non-nucleotide purine derivatives were evaluated as P2X7R antagonists. Compounds that prevented LPS+ATP-induced IL-1β release from primary glial cultures were investigated in the closed-head injury TBI model in vivo. Finally, we evaluated sP2X7R plasmatic levels in a cohort of TBI patients. Key results. p2x7 -/- mice showed an exaggerated inflammatory response 24 h post-TBI compared to control mice. However, animals treated with the selective P2X7R antagonist JNJ-47965567 (30 mg/kg i.p.) 30 min post-TBI showed improved neurological and inflammatory parameters. The purine derivative ITH15004 was the most potent compound reducing IL-1β production in vitro. When administered in vivo 30 min post-TBI, ITH15004 (1 mg/kg i.p.) improved both neurobehavioral and inflammatory markers at 24 h. In TBI patients, enhanced levels of circulating sP2X7R correlated with the lesion severity 72 h post-TBI and with unfavourable outcomes 24 h post-TBI. Conclusion and implications. These results highlight the importance of P2X7R in the acute phase of TBI and present ITH15004 as a new pharmacological tool to counteract P2X7R-dependent neuroinflammation in vivo.

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.