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Introduction: Recent data demonstrates that inhibition of TRPM2 channels protect the brain against ischemia, specifically in males. A major hindrance to our research has been the lack of specific TRPM2 inhibitors. The current study characterized the specificity and neuroprotective efficacy of a novel TRPM2 inhibitor developed in our laboratory.Methods: Fluorescent calcium imaging was used to determine inhibitor efficacy of the TRPM2 peptide inhibitor (tat-M2NX) in HEK293 cells stably expressing hTRPM2. tat-peptides were detected by immunohistochemistry using an anti-tat antibody. Adult (2-3 mo) and aged (18-20 mo) were subjected to 60 min middle cerebral artery occlusion (MCAo) and injected iv with tat-M2NX (20 mg/kg), control tat-SCR (20 mg/kg) or clotrimazole (CTZ) (30 mg/kg) either 20min prior or 3hr after reperfusion. Infarct size was assessed using TTC staining.Results: Successful TRPM2 inhibition in HEK293 cells was observed by decreased Ca2+ influx following H2O2 exposure. Male mice pre-treated with tat-M2NX had smaller infarct volume compared to tat-SCR; 29.2±4.6% (n=8) vs. 43.4±3.2% (n=8; p<0.01), respectively. No effect of tat-M2NX was observed in female mice. Immunostaining demonstrates tat-M2NX accumulation in striatal and cortical neurons 4-6 hours of IV injection. Importantly, male TRPM2-/- mice were not further protected by tat-M2NX, demonstrating selectivity of our new inhibitor. Administration of tat-M2NX at a delayed time point (3hr) provided significant protection to males when analyzed at 24 hr (28.33 ± 5.1% (n=7) vs. 46.45 ± 3.0% (n=6; p<0.01) or 4 days after MCAo (23.84 ± 4.416, (n=8) vs. 41.41 ± 2.831; (n=8; p<0.01). In contrast, delayed administration of CTZ failed to reduce infarct volume. Finally, we observed that tat-M2NX reduced ischemic injury in aged male mice.Conclusions: These data demonstrate the development of a new peptide inhibitor of TRPM2 channels that provides protection from ischemic stroke in young adult and aged male animals with a relatively wide therapeutic window. Furthermore, we took advantage of TRPM2-/- mice to confirm specificity of tat-M2NX protection.