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.

项目概要 化学威胁是一个主要的公共卫生问题，CounterACT 认识到需要识别新的化学威胁 医疗对策（MCM），改善对与以下事件相关的大规模伤亡事件的公共卫生反应 有机磷（OP）制剂因其广泛存在而受到特别关注。 常见杀虫剂及其在化学神经毒剂中的使用会导致OP引起的癫痫发作。 OP 中毒的发生率和治疗该并发症的最佳药物尚不清楚，主要差距有限。 在大规模伤亡之前对人类进行系统临床评估的机会、现有技术的缺点 药物治疗可能无法控制癫痫发作或兴奋性毒性，并且镇静效果恶化且缺乏指导； 为了解决这些差距，我们建议描述 OP 诱导的斑马鱼模型。 使用毒死蜱 (CPO)1（常见 OP 农药的活性代谢物）引起癫痫发作和兴奋性毒性， 毒死蜱——并推进其在高通量化合物筛选中的应用，以识别针对其的新型 MCM OP 诱导的癫痫发作和兴奋性毒性 在目标 1 中，我们将描述 CPO 诱导的斑马鱼幼虫模型。 在目标 1A 中，我们将确认并优化参数以识别新的对策。 使用电生理学 (EEG) 以及基于钙的无创测量方法来测量癫痫活动 此外，我们将评估细胞凋亡负担所证明的存活率和兴奋毒性。 在 Aim 1B 中，使用 TUNEL 测定和时间强度较低的吖啶橙 (AO) 测定来检测癫痫发作后的细胞。 我们将通过以下方法确定理想的测定参数，用于检测 CPO 引起的癫痫发作的新对策 确定现有对策的斑马鱼幼虫的最大耐受剂量，然后采用 CPO 剂量反应实验，以确定导致 CPO 引起的癫痫发作减少 25-50% 的条件 选择该目标是为了在检测现有对策的效果的同时。 为检测卓越对策提供动态范围 在目标 1C 中，我们将量化镇静作用。 作为传统 AED 抗癫痫活性的函数，通过建立几种 AED 的 ED50 作为响应 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 最后，在目标 2 中，我们将实施一个整体。 有机化合物筛选针对 OP 引起的癫痫发作和兴奋性毒性的新型 MCM，并优先考虑命中 基于排名配对的严格标准化平均差 (SSMD*) 和最小镇静效果。 总而言之，这个探索性的 R21 响应了 CounterACT 计划的多个方面，并提供了 整个有机体在体内发现针对 CPO 的新对策的框架 扩展到其他 OP 代理。