A Neurosteroid-based Novel Treatment for OP-Intoxication

描述（由申请人提供）：该CounterACT申请的主要目标是开发一种新型治疗药物，可减轻急性暴露于二异丙基氟磷酸盐（DFP）（一种致命的有机磷杀虫剂，被认为是一种可信的化学威胁）引起的发病率和死亡率。有机磷酸盐（OP）是不可逆的胆碱酯酶抑制剂，暴露于DFP和相关军用神经毒剂后的OP中毒导致持续性癫痫发作、癫痫持续状态（SE）和永久性脑损伤。目前针对急性OP中毒的医学对策不足以保护大脑免受SE，SE是持续30分钟或更长时间的长时间癫痫发作活动，伴有显著的神经元损伤， mortality.我们建议，促进紧张性抑制的神经甾体对持续性SE产生快速有效的保护，防止不可逆的脑损伤，并延长治疗窗口。这种新的治疗策略是基于神经甾体的新兴分子机制以及参与SE（OP样药物的常见毒性）的细胞变化。神经类固醇是在大脑内局部合成的类固醇，控制抑制。神经甾体类药物是对抗胆碱能药物诱发癫痫发作最有效的抗惊厥药物。由于OP神经毒剂通过胆碱能过度激活引起持续性癫痫发作和脑损伤，因此提出神经类固醇是OP中毒的有效解毒剂。神经类固醇主要作用于GABA-A受体，介导阶段性和紧张性抑制，通过“设定”基线兴奋性在控制癫痫发作中发挥关键作用。最近的研究表明，SE引起突触抑制的显着减少，突触外（神经类固醇敏感）紧张性抑制的变化最小。因此，突触外GABA-A受体的敏感性增强和突触受体的最大刺激功效使得神经类固醇成为控制OP中毒后SE和神经元损伤的理想新药。这种新的治疗策略以前没有经过测试。我们在OP中毒的DFP模型中的初步研究证明了这种理论治疗的可行性和有希望的疗效。我们推测，神经甾体和选择性药物，提高阶段性和突触外紧张性抑制有效地控制OP中毒诱导的SE和神经元损伤。为了检验这一假设，我们将解决两个具体的目的：（目的1）：确定在DFP中毒中加奈索酮同时增强突触和强直抑制的功效;和（目的2）：确定在DFP中毒中加波沙朵选择性增强突触外强直抑制的功效。在暴露于DFP后40分钟、1小时或2小时给予试验药物。将记录行为和EEG癫痫发作以评估药物疗效，并通过免疫组织化学技术计数神经元密度来评估神经保护作用。拟议的研究将提供新疗法的“疗效证明”。合成神经类固醇，如加奈索酮正在进行临床试验，并已显示出良好的安全记录。因此，这项研究的结果将为神经甾体作为对抗化学威胁的有效对策的快速发展奠定基础。

DESCRIPTION (provided by applicant): The main goal of this CounterACT application is to develop a novel class of therapeutics that will mitigate the morbidity and mortality caused by acute exposure to diisopropylfluorophosphate (DFP), a lethal organophosphate insecticide that is considered a credible chemical threat. Organophosphates (OP) are irreversible cholinesterase inhibitors and OP intoxication following exposure to DFP and related military nerve agents results in persistent seizures, status epilepticus (SE) and permanent brain injury. Current medical countermeasures for acute OP intoxication do not sufficiently protect the brain from SE, a prolonged seizure activity lasting 30 min or longer with significant neuronal injury and mortality. We propose that neurosteroids that promote tonic inhibition produce rapid and effective protection against persistent SE, prevent irreversible brain injury, and extend the therapeutic window. This novel therapeutic strategy is based on the emerging molecular mechanisms of neurosteroids and also cellular changes involved in SE, a common toxicity by OP-like agents. Neurosteroids are steroids synthesized locally within the brain that control inhibition. Neurosteroids are the most powerful anticonvulsants against seizures induced by cholinergic agents. Since OP nerve agents cause persistent seizures and brain damage by cholinergic hyperactivation, it is proposed that neurosteroids are an effective antidote for OP intoxication. Neurosteroids act principally at GABA-A receptors, which mediate phasic and tonic inhibition, that play a critical role in controlling seizures by "setting" the baseline excitabilit. Recent work has shown that SE cause a significant decrease in synaptic inhibition with minimal changes in extrasynaptic (neurosteroid-sensitive) tonic inhibition. Therefore, enhanced sensitivity at extrasynaptic GABA-A receptors and maximally stimulating efficacy at synaptic receptors makes neurosteroids ideal new drugs for controlling SE and neuronal damage following OP intoxication. This novel treatment strategy has not been tested previously. Our preliminary studies in the DFP model of OP intoxication demonstrate the feasibility and promising efficacy of this rationale therapy. We hypothesize that neurosteroids and selective drugs that enhance phasic and extrasynaptic tonic inhibition effectively control OP intoxication-induced SE and neuronal damage. To test this hypothesis, we will address two specific aims: (Aim 1): Determine the efficacy of simultaneous augmentation of synaptic and tonic inhibition by ganaxolone in DFP intoxication; and (Aim 2): Determine the efficacy of selective augmentation of extrasynaptic tonic inhibition by gaboxadol in DFP intoxication. Test drugs will be given 40 min, 1 hr or 2 hr after exposure to DFP. Behavioral and EEG seizures will be recorded for assessment of drug efficacy, and neuroprotection will be assessed by counting neuron density by immunohistochemical techniques. The proposed studies will provide "proof-of-efficacy" of the new therapy. Synthetic neurosteroids such as ganaxolone are undergoing clinical trials and have shown an excellent safety record. Therefore, the outcome of this research will set the stage for rapid development of neurosteroids as effective countermeasures against chemical threats.