Brain-penetrating acetylcholinesterase reactivators for several organophosphates

描述(申请人提供)：有机磷(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。大剂量中毒会引起兴奋性毒性，导致癫痫发作，继而造成脑损伤。目前在美国的治疗方法包括一种肟类复活剂2-PAM，它几乎没有通过血脑屏障(BBB)的能力，因此无法阻止癫痫发作和防止脑损伤。我们的实验室已经开发出一个苯氧基烷基吡啶肟库，旨在比2-PAM具有更强的亲油性。通过在大鼠中使用高亚致死剂量的高度相关的沙林或VX替代品，并在1小时后给药新的肟类药物 OP复合治疗当癫痫发作行为持续时，我们已经证明了一些这些肟类物质能够穿透血脑屏障，重新激活AChE，并减轻癫痫发作行为。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个，然后向下选择为3个)，以最终选择用于IND应用的单个铅肟。此外，我们将扩大OP化学测试范围，以包括杀虫化学物质对氧磷(对硫磷的活性代谢物；首先进行测试，以避免由于生物活化的需要而可能出现的任何混杂)和对硫磷。研究的终点包括大脑和外周AChE重新激活、癫痫发作减弱、神经变性(神经纤维酸性蛋白积聚)、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预测一个或两个额外的肟结构比我们当前的选择具有更大的效果；如果是这样，它/它们将被合成并取代我们当前的选择。建议项目的具体目标是：1.体内药效鉴定目的：使用沙林和VX替代物，以及使用对硫磷及其活性代谢物对氧磷的常见杀虫OP化学物质，为我们目前已确定的血脑屏障穿透性肟提供神经保护的扩展措施。4个OP的疗效终点包括：1a，6个肟的疗效筛选终点：重新激活大鼠脑AChE，以及减轻癫痫行为和神经退行性变；以及1b，详细的疗效表征终点，包括3种下选择的肟：大脑中的氧化应激，神经病理和行为缺陷。2.药代动力学目的：测定经微透析法获得的3种已筛选的肟类药物的体外肝脏代谢、血浆蛋白结合力及血液和脑脊液中的肟类物质的含量。3.计算化学目标：模拟肟类与AChE和BBB穿透的对接，以建议改进的肟类结构。4.化学合成目标：为所提出的实验生产足够数量的测试OP和测试肟，以及根据计算化学目标合成一些新的肟类化合物。最终结果将是确定最有效的新型肟作为当前治疗的潜在替代品进行进一步开发。

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.