Identification of novel brain-penetrating oxime antidotes for phorate toxicity

7.项目摘要/摘要 许多有机磷(OP)杀虫剂，如甲拌磷(O，O-二乙基S-乙硫甲基 对大鼠经口LD50‘S在低mg/kg范围内有很强的毒性，它们或其活性 代谢产物是乙酰胆碱酯酶(AChE)的有效抑制剂。甲拌磷对雌性的毒性一直更大 以S为例，其经口LD50雌雄分别为3.7mgkg和1.4mgkg。甲拌磷 需要单加氧酶介导的生物活性来激活抗胆碱酯酶代谢产物，类似于一些 其他有机磷杀虫剂，如对硫磷(O，O-二乙基-邻硝基苯基硫酸盐)。我们的预赛 对甲拌磷代谢物甲氧磷(Pho)的研究表明，甲胺磷的时间延迟更长，暴力程度也更高。 与对硫磷的活性代谢物对氧磷(PXN)相比，有中毒迹象。此外，模式还包括 在pho和pxn中，肟介导的胆碱酯酶重新激活不同，这是意想不到的，因为两者 是二乙基磷酸酯，并有望使胆碱酯酶具有相同的二乙基 并因此显示出类似的重新激活模式。PHO的末端硫经历了额外的生物活化 乙醚转化为亚硫醚，然后转化为砜。结合能的估算和不同寻常的初步结果 到目前为止观察到的结果表明，可能存在乙氧基离开基团，而不是预期的 乙硫甲基和脑组织中Pho的缓慢生物激活作用，使其先转化为pho-亚砜，然后再转化为pho- 这些意想不到的初步观测结果可能是由砜引起的。2-PAM是目前FDA- 已批准的肟酸痛复活剂。然而，需要一种更有效的不同的肟类复活剂 用一种非传统的磷化部分以及一种可以渗透到大脑的肟类物质将是 有效的甲胺磷治疗所需的。我们的实验室已经发明并获得专利的新型替代品 苯氧基烷基吡啶肟类化合物，显示了与邻苯二甲酸二异辛酯以及 令人信服的证据表明，在我们的大鼠模型中，有其他OP进入大脑。因此，以下是具体的 目标：目标1.通过质谱学确定Pho或Pho代谢物的离开基团 乙酰胆碱酯酶磷酸化多肽的分析和通过计算建模确定屏障高度 潜在的离开群体。目的2.通过对脑和脑组织的分析，确定生物活化效率 用LC/MS/MS定量测定甲拌磷对几种代谢物的肝脏生物活化动力学。 从我们新的被三种甲胺磷抑制的乙酰胆碱酯酶(AChE)肟库中鉴定更有效的肟类复活剂 代谢物通过体外复活研究和有限的体内甲胺磷存活研究。这样做的结果 R21项目将确定几个经过精选的新肟类，可以在未来的 随后的U01项目研究甲胺磷中毒的有效疗法。

7. Project Summary/Abstract Many of the organophosphate (OP) insecticides, such as phorate (O,O-diethyl S-ethylthiomethyl phosphorodithioate), are highly toxic with rat oral LD50's in the low mg/kg range, and they or their active metabolites are potent inhibitors of acetylcholinesterase (AChE). Phorate is consistently more toxic to females than males; for example, rat oral LD50's for males and females are 3.7 and 1.4 mg/kg, respectively. Phorate requires monooxygenase-mediated bioactivation to active anticholinesterase metabolites, similar to a number of other OP insecticides, such as parathion (O,O-diethyl O-nitrophenyl phosphorothionate). Our preliminary studies with a phorate metabolite phorate-oxon (PHO) have indicated a longer time delay and more violent signs of poisoning than with paraoxon (PXN), the active metabolite of parathion. Additionally, patterns of oxime-mediated cholinesterase reactivation differ between PHO and PXN, which is unexpected because both are diethyl phosphates, and would be expected to phosphylate cholinesterase with the same diethyl moiety and therefore display similar reactivation patterns. PHO undergoes additional bioactivation of its terminal sulfur ether to a sulfoxide, then to a sulfone. Estimates of binding energies and the unusual preliminary results observed thus far have suggested that there may be an ethoxy leaving group instead of the expected ethylthiomethyl group and that the slow bioactivation of PHO in the brain to PHO-sulfoxide and then to PHO- sulfone might be responsible for the unexpected preliminary observations. 2-PAM is the currently FDA- approved oxime AChE reactivator. However the need for a different oxime reactivator that is more effective with an unconventional phosphylating moiety as well as an oxime that can penetrate into the brain will be needed for effective phorate therapy. Our laboratories have invented and patented novel substituted phenoxyalkyl pyridinium oximes that show preliminary evidence of survival efficacy with PHO as well as convincing evidence of entry into the brain with other OP's in our rat model. Therefore the following Specific Aims are proposed: Aim 1. To confirm the leaving group of PHO or PHO metabolites through a mass spectral analysis of AChE-phosphylated peptides and through computational modeling to determine barrier height for the potential leaving groups. Aim 2. To determine bioactivation efficiency through analysis of brain and hepatic bioactivation kinetics for phorate to its several metabolites as quantified by LC/MS/MS. Aim 3. To identify more effective oxime reactivators from our novel oxime library of AChE inhibited by the three phorate metabolites through in vitro reactivation studies and limited in vivo phorate survival studies. The results of this R21 project will be the identification of a few down-selected novel oximes that can be further developed in a subsequent U01 project into effective therapeutics for phorate poisoning.