Identification of novel brain-penetrating oxime antidotes for phorate toxicity

新型脑穿透性肟解毒剂甲拌磷毒性的鉴定

• 批准号: 9633107
• 负责人: Janice Elaine Chambers
• 金额: $ 21.56万
• 依托单位: MISSISSIPPI STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-30 至 2020-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9633107
• 关键词: Acetylcholinesterase; Acetylcholinesterase Inhibitors; Antidotes; Atropine; Attenuated; Behavior; Binding; Biological; Blood - brain barrier anatomy; Brain; Brain Injuries; Butyrylcholinesterase; Cause of Death; Cessation of life; Chemicals; Chemistry; Cholinesterase Inhibitors; Cholinesterases; Computer Simulation; Cytochrome P450; Data; Development; Distal; Ethers; FDA approved; Female; Future; Height; Hepatic; In Vitro; Insecticides; Kinetics; Laboratories; Legal patent; Lethal Dose 50; Libraries; Life; Mediating; Metabolic Activation; Mixed Function Oxygenases; Modeling; Muscarinic Acetylcholine Receptor; Neuraxis; Neurotoxicity Syndromes; Oral; Organophosphates; Oxides; Oximes; Paraoxon; Parathion; Parents; Pattern; Peptides; Peripheral; Phorate; Poisoning; Property; Rattus; Sarin; Savings; Seizures; Series; Sulfones; Sulfoxide; Sulfur; Survivors; Testing; Therapeutic; Time; Toxic effect; Treatment Efficacy; Violence; attenuation; combat; comparative efficacy; dosage; experimental study; improved; in vivo; inorganic phosphate; lipophilicity; liquid chromatography mass spectrometry; male; nerve agent; novel; phosphorodithioic acid; respiratory; response; toxic organophosphate insecticide exposure

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.

7. 项目总结/摘要 许多有机磷 (OP) 杀虫剂，例如甲拌磷（O,O-二乙基 S-乙硫甲基） 二硫代磷酸酯）具有高毒性，大鼠经口 LD50 在低 mg/kg 范围内，并且它们或其活性物质 代谢物是乙酰胆碱酯酶 (AChE) 的有效抑制剂。甲拌磷对雌性的毒性始终更大 比男性；例如，雄性和雌性大鼠经口 LD50 分别为 3.7 和 1.4 mg/kg。甲拌磷 需要单加氧酶介导的生物激活活性抗胆碱酯酶代谢物，类似于许多 其他 OP 杀虫剂，例如对硫磷（O,O-二乙基 O-硝基苯基硫代磷酸酯）。我们的初步 对甲拌磷代谢物甲拌磷 (PHO) 的研究表明，延迟时间更长且更剧烈 中毒迹象比对硫磷的活性代谢物对氧磷 (PXN) 更严重。此外，模式 肟介导的胆碱酯酶再激活在 PHO 和 PXN 之间有所不同，这是出乎意料的，因为两者 是磷酸二乙酯，预计会用相同的二乙基部分磷酸化胆碱酯酶 因此表现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 乙醚转化为亚砜，然后转化为砜。结合能的估计和不寻常的初步结果 迄今为止观察到的结果表明可能存在乙氧基离去基团而不是预期的 乙硫甲基基团，并且 PHO 在大脑中缓慢生物激活为 PHO-亚砜，然后为 PHO- 砜可能是意想不到的初步观察结果的原因。 2-PAM是目前FDA- 批准的肟 AChE 重激活剂。然而，需要一种更有效的不同肟重激活剂 具有非常规磷酸化部分以及可以渗透到大脑中的肟 有效的甲拌磷治疗所需。我们的实验室已发明新型替代品并获得专利 苯氧基烷基吡啶鎓肟显示出 PHO 以及 PHO 的生存功效的初步证据 在我们的大鼠模型中，有令人信服的证据证明其他 OP 可以进入大脑。因此具体如下 提出的目标： 目标 1. 通过质谱确认 PHO 或 PHO 代谢物的离去基团 分析 AChE 磷酸化肽并通过计算模型确定势垒高度 潜在的离去基团。目标 2. 通过分析大脑和 通过 LC/MS/MS 定量甲拌磷对其几种代谢物的肝脏生物活化动力学。目标 3. 至 从我们受三种甲拌磷抑制的 AChE 新型肟库中鉴定出更有效的肟重激活剂 通过体外再激活研究和有限的体内甲拌磷存活研究来检测代谢物。这样做的结果 R21 项目将鉴定一些经过筛选的新型肟，这些肟可以在 随后的 U01 项目研究了甲拌磷中毒的有效治疗方法。