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

摘要 化学战神经毒剂（CWNA）越来越多地用于攻击世界各地的平民。目前，我们缺乏 有效治疗CWNA幸存者。直到最近，预防由于接触CWNA而导致的急性死亡 优先考虑的事然而，关于沙林毒气袭击幸存者终身健康后果的报告， 开始出现。与有机磷（OP）一样，CWNA是胆碱酯酶抑制剂， 致瘤剂。在动物模型中，急性CWNA/OP暴露诱导癫痫持续状态（SE）和其他癫痫症状。 胆碱能症状目前的医学对策（MCM），如阿托品，肟， 地西泮/咪达唑仑控制症状，但不能预防长期神经毒性和合并症，主要是 由于持续的硝基氧化应激、神经炎症和神经变性。我们预测， 神经保护剂和MCM的组合可以防止CWNA诱导的长期神经毒性。我们提出 研究新型NADPH抑制剂的长期神经保护和抗癫痫作用 氧化酶（NOX 2），mitoapocynin，在大鼠二异丙基氟磷酸（DFP）和神经毒剂梭曼模型。 作为概念证明，我们证明了NOX 2抑制剂的神经保护和疾病修饰特性 在大鼠DFP和小鼠帕金森病（PD）和癫痫模型中。用以下药物治疗小鼠或大鼠： 在用红藻氨酸（KA）或DFP诱导SE之前，mitoapocynin显著阻止了惊厥的发作。 表明其具有抗癫痫特性。我们进一步测试了米托夹竹桃素和二夹竹桃素在遥测植入 用KA或DFP诱发SE，用MCM治疗。两种NOX 2抑制剂均为血脑屏障 可渗透的它们在>50%的大鼠中预防癫痫发生，并显著减少反应性神经胶质增生， 神经变性虽然我们已经测试了NOX 2在大鼠DFP模型中作用的概念证明， 所用二夹竹桃麻素最佳剂量太高（300 mg/kg），不能外推用于人体 使用.因此，我们开发、表征、体内测试并获得专利（US 8962600 B2）， mitoapocynin，其在比diapocynin小10-30倍的剂量下有效。我们的首要假设是 是mitoapocynin抵消CWNA诱导的长期神经变性，癫痫发生， 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（具体目标2）。我们将大鼠暴露于DFP/梭曼，用MCM处理，2 h后用 mitoapocynin（30 mg/kg，口服）或赋形剂，前三天每天两次，6周后进行一组 行为测试。我们将植入遥测设备，以监测SRS和疾病进展/改善 实时的。为了确定mitoapocynin的保护作用，我们将进行脑组织化学， 神经变性和神经炎症、硝基氧化应激测定和细胞因子的多重测定。 我们的建议体现了一种新的翻译方法，针对神经变性和癫痫发生 并且可能产生用于CWNA诱导的长期神经毒性的新的口服活性疗法。

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.