Mitoapocynin, a novel NOX2 inhibitor, mitigates nerve agents induced longterm neurotoxicity

摘要 化学战神经毒剂(CWNA)越来越多地被用于袭击世界各地的平民。目前，我们缺乏 对CWNA幸存者的有效治疗。直到最近，预防因接触CWNA而导致的急性死亡 被当做头等大事。然而，关于沙林袭击幸存者终身健康后果的报道如下 开始浮出水面。与有机磷酸盐(OPs)一样，CWNAs是胆碱酯酶抑制剂，是有效的 致痫药物。在动物模型中，急性CWNA/OP暴露导致癫痫持续状态(SE)和其他 胆碱能症状。目前的医学对策(MCM)，如阿托品、肟类和 安定/咪达唑仑可控制症状，但不能预防长期的神经毒性和共病，主要是 由于持续的氮氧化应激、神经炎症和神经变性。我们预测， 神经保护剂与MCM联合应用可预防CWNA所致的长期神经毒性。我们建议 新型NADPH抑制剂的长期神经保护和抗癫痫作用 在大鼠二异丙基氟磷酸盐(DFP)和神经毒剂梭曼模型中，有丝分裂素(Mitoapocynin)，氧化物酶(NOX2)。 作为概念验证，我们展示了NOX2抑制剂的神经保护和疾病改善特性。 在大鼠DFP和小鼠帕金森病(PD)和癫痫模型中。治疗小鼠或大鼠 红藻氨酸(KA)或DFP诱导SE前的丝裂原蛋白显著预防惊厥发作 癫痫发作表明其具有抗癫痫作用。我们进一步测试了有丝分裂素和脱落素在遥测中的植入 用KA或DFP诱导SE后给予MCM治疗。两种NOX2抑制剂都是血脑屏障 可渗透的。它们阻止了50%的大鼠癫痫的发生，并显著减少了反应性胶质细胞增生和 神经退行性变。虽然我们已经测试了NOX2在大鼠DFP模型中作用的概念证明， 皂苷元的最佳使用剂量过高(300 mg/kg)，这对人类来说是不能推算的 使用。因此，我们开发、表征、体内测试并获得专利(US8962600B2) 丝裂原辅酶原激动素，它的有效剂量比调情素低10-30倍。我们最重要的假设 是丝裂原蛋白能对抗CWNA诱导的长期神经变性、癫痫发生和恢复 大脑功能。我们将在雄性和雌性大鼠DFP模型(特定目标1)中研究这一点并验证 在梭曼模型中(特定目标2)。我们将大鼠暴露于DFP/Soman，用MCM治疗，2小时后用 前三天每天两次口服丝裂原蛋白(30 mg/kg，口服)或赋形剂，6周后进行电池治疗 行为测试。我们将植入一个遥测设备来监测SRS和疾病进展/修改 实时的。为了确定丝裂原蛋白的保护作用，我们将进行脑组织化学研究 神经退行性变和神经炎症，硝基氧化应激分析，以及细胞因子的多重分析。 我们的建议体现了一种针对神经退行性变和癫痫发生的新的翻译方法 并可能为CWNA诱导的长期神经毒性提供一种新的口服活性疗法。

Abstract Chemical warfare nerve agents (CWNA) are increasingly used to attack civilians worldwide. Currently, we lack effective treatment for CWNA survivors. Until recently, preventing acute death due to CWNA exposure had taken a top priority. However, reports on the life-long health consequences of sarin attack survivors are beginning to emerge. Like organophosphates (OPs), CWNAs are cholinesterase inhibitors and potent seizurogenic agents. In animal models, acute CWNA/OP exposure induces status epilepticus (SE) and other cholinergic symptoms. The current medical countermeasures (MCM) such as atropine, oxime, and diazepam/midazolam control symptoms, but do not prevent long-term neurotoxicity and comorbidity, primarily due to persistent nitrooxidative stress, neuroinflammation, and neurodegeneration. We predict that a combination of neuroprotectant and MCM can prevent CWNA-induced long-term neurotoxicity. We propose to investigate the long-term neuroprotective and antiepileptogenic effects of a novel inhibitor of NADPH oxidase (NOX2), mitoapocynin, in rat diisopropylfluorophosphate (DFP) and a nerve agent soman models. As proof of concept, we demonstrated the neuroprotective and disease-modifying properties of NOX2 inhibitors in the rat DFP and the mouse Parkinson's disease (PD) and seizure models. Treating mice or rats with mitoapocynin before the induction of SE with kainate (KA) or DFP significantly prevented the onset of convulsive seizures suggesting its anti-seizure property. We further tested mitoapocynin and diapocynin in telemetry-implanted rats after inducing SE with KA or DFP and treating them with MCM. Both NOX2 inhibitors are blood-brain barrier permeable. They prevented epileptogenesis in >50% rats and significantly reduced reactive gliosis and neurodegeneration. Although we had tested the proof of concept for the role of NOX2 in the rat DFP model, the optimum dose of diapocynin used was too high (300 mg/kg), which cannot be extrapolated for human use. Therefore, we developed, characterized, tested in vivo, and patented (US8962600 B2) the mitoapocynin, which is effective at 10-30 times lesser dose than diapocynin. Our overarching hypothesis is that mitoapocynin counteracts CWNA-induced long-term neurodegeneration, epileptogenesis, and restores brain function. We will investigate this in both male and female rat DFP models (Specific Aim 1) and validate in soman model (Specific Aim 2). We will expose rats to DFP/soman, treat with MCM, and 2h later with mitoapocynin (30 mg/kg, oral) or vehicle twice daily for the first three days, and 6 weeks later conduct a battery of behavioral tests. We will implant a telemetry device to monitor SRS and disease progression/modification in real-time. To determine the protective effect of mitoapocynin, we will conduct brain histochemistry for neurodegeneration and neuroinflammation, nitro-oxidative stress assays, and multiplex assay for cytokines. Our proposal embodies a novel translational approach targeting both neurodegeneration and epileptogenesis and may yield a novel orally-active therapy for CWNA-induced long-term neurotoxicity.