Brain-penetrating acetylcholinesterase reactivators for several organophosphates

描述（由申请人提供）：有机磷酸（OP）抗胆碱酯酶，例如，神经毒剂或杀虫剂是有效的乙酰胆碱酯酶（AChE）抑制剂。高剂量中毒引起兴奋性中毒，导致癫痫发作和随后的脑损伤。美国目前的治疗包括肟再活化剂2-PAM，其几乎没有穿过血脑屏障（BBB）的能力，因此不能阻止癫痫发作和预防脑损伤。我们的实验室已经开发了苯氧基烷基吡啶肟的库，其被设计为具有比2-PAM更大的亲脂性。通过在大鼠中使用高亚致死剂量的沙林或VX的高度相关替代物并在给药后1小时施用新型肟的范例， 当癫痫发作行为正在进行时，我们已经证明了许多这些肟穿透BBB，重新激活AChE并减弱癫痫发作行为的能力。本申请的目的是进一步表征有限数量的这些新型肟（最初为6种，然后向下选择至3种），以最终选择单个铅肟用于IND申请。此外，我们将扩大测试的OP化学物质，以包括杀虫化学物质、对氧磷（对氧磷的活性代谢物;首先进行测试，以避免由于需要生物活化而可能发生的任何混淆因素）和对氧磷。表征的终点包括脑和外周AChE再活化、癫痫发作减弱、神经变性（胶质细胞酸性蛋白积累）、氧化应激（F2-异前列腺素）、神经病理学（细胞凋亡、神经元存活）和行为。同时，分子动力学计算将确定是否有一个或两个额外的肟结构被预测比我们目前的选择具有更大的功效;如果是这样，它/它们将被合成并取代我们目前的选择。拟议项目的具体目标是：1。体内功效表征目的：使用沙林和VX替代物以及常见的杀虫OP化学（即，磷酸二乙酯）。4种OP的有效性终点包括：1a，6种肟的有效性筛选终点：重新激活大鼠脑AChE，以及癫痫发作行为和神经变性的减弱;和1b，3种下选肟的详细有效性表征终点：脑中的氧化应激、神经病理学和行为缺陷。2.药代动力学目的：测定3种降选肟的体外肝脏代谢、血浆蛋白结合率以及血液和脑脊液（通过微透析获得）中肟的水平。3.计算化学目的：模拟肟与AChE和BBB渗透的对接，以提出改进的肟结构。4.化学合成目的：为了产生足够数量的测试OP和测试肟的实验提出，以及一些新的肟的合成，如果建议的计算化学的目标。最终的结果将是鉴定出最有效的新型肟，以进一步开发为当前治疗的潜在替代品。

DESCRIPTION (provided by applicant): Organophosphate (OP) anticholinesterases, e.g., nerve agents or insecticides, are potent acetylcholinesterase (AChE) inhibitors. High dose poisoning causes excitotoxicity that leads to seizures and subsequently brain damage. The current therapy in the US includes an oxime reactivator, 2-PAM, which has little, if any, ability t cross the blood-brain barrier (BBB) and consequently cannot stop seizures and prevent brain damage. Our laboratories have developed a library of phenoxyalkyl pyridinium oximes designed to have greater lipophilicity than 2-PAM. Through a paradigm using high sub-lethal dosages of highly relevant surrogates for sarin or VX in rats and administering the novel oximes at 1 hr after OP compound treatment when seizure behavior was on- going, we have demonstrated the ability of a number of these oximes to penetrate the BBB, reactivate AChE, and to attenuate seizure behavior. The objective of this application is to further characterize a limited number of these novel oximes (initially 6, then down-selected to 3) to ultimately select a single lead oxime for an IND application. Additionally we will expand the OP chemistries tested to include an insecticidal chemistry, paraoxon (the active metabolite of parathion; tested first to avoid any confounders that could occur because of the need for bioactivation) and parathion. Endpoints of characterization include brain and peripheral AChE reactivation, seizure attenuation, neurodegeneration (glial fibrillary acidic protein accumulation), oxidative stress (F2-isoprostanes), neuropathology (apoptosis, neuron survival), and behavior. Concurrently molecular dynamics computations will determine if one or two additional oxime structures are predicted to have greater efficacy than our current selections; if so, it/they will be synthesized and substituted for our current selections. The specific aims of the proposed project are: 1. In vivo efficacy characterization aim: To provide expanded measures of neuroprotection on our currently identified BBB-penetrating oximes using the sarin and VX surrogates and also a common insecticidal OP chemistry (i.e., diethyl phosphate) using parathion and its active metabolite paraoxon. Efficacy endpoints for the 4 OP's include: 1a, efficacy screening endpoints with 6 oximes: reactivating rat brain AChE, and the attenuation of seizure behavior and neurodegeneration; and 1b, detailed efficacy characterization endpoints with 3 down-selected oximes: oxidative stress in the brain, neuropathology and behavioral deficits. 2. Pharmacokinetic aim: To determine for the 3 down-selected oximes hepatic in vitro metabolism, plasma protein binding and levels of oxime in the blood and cerebrospinal fluid (obtained through microdialysis). 3. Computational chemistry aim: To model the docking of oxime with AChE and BBB penetration to suggest improved oxime structures. 4. Chemical synthesis aim: To produce sufficient quantities of the test OP's and the test oximes for the experiments proposed, as well as the synthesis of a few new oximes if suggested by the computational chemistry aim. The ultimate outcome will be the identification of the most efficacious novel oxime for further development as a potential substitute for current therapy.