High-throughput in vivo discovery of novel countermeasures against organophosphate-induced seizure and status epilepticus using zebrafish

利用斑马鱼高通量体内发现针对有机磷诱发的癫痫发作和癫痫持续状态的新对策

• 批准号: 10457138
• 负责人: Christopher McGraw
• 金额: $ 23.74万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-04-01 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10457138
• 关键词: Acoustics; Acridine Orange; Address; Animal Model; Antiepileptic Agents; Apoptotic; Atropine; Biological Assay; Calcium; Caring; Cells; Chemical Exposure; Chemical Weapons; Chemicals; Chlorpyrifos; Clinical; Complication; Data; Detection; Disease; Dose; Drug Screening; Electrophysiology (science); Event; Exposure to; FDA approved; Fluorescence; Functional disorder; Generalized Epilepsy; Genetic; Human; Intoxication; Levetiracetam; Literature; Lorazepam; Maximum Tolerated Dose; Measures; Medical; Memantine; Modeling; Molecular; Monitor; Organophosphates; Parathion; Pesticides; Pharmaceutical Preparations; Phorate; Property; Public Health; Research; Sarin; Sedation procedure; Seizures; Soman; Standardization; Startle Reaction; Status Epilepticus; TdT-Mediated dUTP Nick End Labeling Assay; Time; Validation; Visual; Whole Organism; Work; Zebrafish; acute toxicity; base; blind; candidate identification; chemical threat; excitotoxicity; experimental study; exposed human population; high throughput screening; improved; in vivo; mass casualty; medical countermeasure; nerve agent; novel; programs; real world application; research clinical testing; response; screening; sedative; tabun; therapeutic candidate; therapy development

PROJECT SUMMARY Chemical threats are a major public health concern and CounterACT recognizes the need to identify new medical countermeasures (MCMs) that improve the public health response to mass casualty events related to chemical threats. Organophosphate (OP) agents are of particular concern due to their wide availability in common pesticides and their use in chemical nerve agents. OP-induced seizures are a serious consequence of OP-intoxication and best agents for treating this complication are unclear. Major gaps include limited opportunities for systematic clinical evaluations in humans prior to mass casualty, drawbacks to existing medication, which may not control seizures or excitotoxicity, and worsen sedation; and lack of direction in discovering new MCMs. To address these gaps, we propose to characterize a zebrafish model of OP-induced seizures and excitotoxicity using chlorpyrifos-oxon (CPO)1 – an active metabolite of the common OP pesticide, chlorpyrifos -- and to advance its use for high-throughput compound screening to identify novel MCMs against OP-induced seizures and excitotoxicity. In Aim 1, we will characterize a larval zebrafish model of CPO-induced seizure and optimize parameters for the identification of novel countermeasures. In Aim 1A, we will confirm seizure activity using both electrophysiology (EEG) as well as noninvasive measures based on calcium fluorescence. In addition, we will assess survival and excitotoxicity as evidenced by the burden of apoptotic cells following seizures using TUNEL assay and less time-intensive acridine orange (AO) assay. In Aim 1B, we will identify the ideal assay parameters for detecting novel countermeasures to CPO-induced seizures by identifying the maximum tolerated dose in larval zebrafish of existing countermeasures followed by a CPO dose-response experiment to identify the conditions that result in 25-50% reduction in CPO-induced seizures versus control. This target is chosen to allow detection of the effect of existing countermeasures while providing dynamic range for the detection of superior countermeasures. In Aim 1C, we will quantify sedation as a function of anti-seizure activity in conventional AEDs, by establishing ED50 for several AEDs in response to CPO-induced seizures; these data will be compared to the sedative effect of AEDs in the visual startle response (VSR) and acoustic startle response (ASR) assays. Finally, in Aim 2, we will implement a whole organism compound screen for novel MCMs against OP-induced seizure and excitotoxicity, and prioritize hits based on ranked paired robust strictly standardized mean difference (SSMD*) and least sedating effects. Taken together, this exploratory R21 is responsive to multiple facets of the CounterACT program and provides a framework for whole organism in vivo discovery of novel countermeasures against CPO that may be extended to additional OP agents.

项目摘要 化学威胁是一个主要的公共卫生问题，并反对认识到有必要确定新的 医疗对策（MCMS）改善公共卫生对大规模伤亡事件的反应 化学威胁。有机磷酸盐（OP）剂特别关注其广泛的可用性 常见的农药及其在化学神经剂中的使用。操作引起的癫痫发作是严重的结果 尚不清楚处理这种并发症的操作局毒性和最佳药物。主要差距包括有限 在大规模伤亡之前，人类进行系统的临床评估机会，现有的缺点 药物可能无法控制癫痫发作或兴奋性毒性和恶化的镇静；并且缺乏方向 发现新的MCM。为了解决这些差距，我们建议表征斑马鱼模型 使用Chlorpyrifos-Oxon（CPO）1的癫痫发作和兴奋性毒性1 - 一种普通农药的活性代谢物， Chlorpyrifos-并提高其用于高通量化合物筛选的用途，以识别针对的新型MCM 操作引起的癫痫发作和兴奋性。在AIM 1中，我们将表征CPO诱导的幼虫斑马鱼模型 癫痫发作并优化参数，以识别新型对策。在AIM 1A中，我们将确认 使用电生理学（EEG）以及基于钙的无创测量的癫痫发作活性 荧光。此外，我们将评估凋亡的燃烧所证明的生存和兴奋性 使用TUNEL分析和较少耗时的杂氨橙（AO）测定后进行癫痫发作后的细胞。在AIM 1B中 我们将确定用于检测新颖的对策以通过 确定现有对策的幼虫斑马鱼中的最大耐受剂量，然后是CPO 剂量反应实验，以确定导致CPO诱导的癫痫发作降低25-50％的条件 与控制。选择此目标以允许检测现有对策的影响 提供动态范围以检测出色的对策。在AIM 1C中，我们将量化镇静 通过建立多个AED的ED50，是传统AED中的抗Seizure活性的函数 CPO引起的癫痫发作；这些数据将与AED在视觉惊吓中的镇静作用进行比较 响应（VSR）和声学惊吓反应（ASR）测定法。最后，在AIM 2中，我们将实施整个 有机体复合屏幕，用于针对操作诱导的癫痫发作和兴奋性毒性的新型MCM，并优先命中命中 基于排名的配对鲁棒严格标准化的平均差异（SSMD*）和最少的镇静效果。 综上 整个生物体在体内发现对CPO的新颖对策的框架 扩展到其他OP代理。