Formulation and Encapsulation of Enzymic Countermeasures Against Organophosphorus

项目主任/首席调查员(最后、第一、中间)：MAGLIERY，Thomas J. 项目摘要/摘要 有机磷(OP)神经毒剂(如VX和沙林)和杀虫剂(如毒死蜱/敌百虫)是 是已知的毒性最强的化合物之一。由于这些杀虫剂在世界各地的持续使用， 由于众所周知，许多国家都有神经毒剂库存，OP毒剂代表着一种重要的 恐怖袭击和工业事故造成的大规模伤亡威胁。骨质疏松症的治疗方案 中毒会导致丧失工作能力，不能治疗后遗症或低剂量暴露的影响；它也是 在大规模伤亡的情况下很难。解决这个问题的一个办法是开发一种酶制剂。 这可能会在OP剂发挥全部作用之前迅速降解它们。最近取得了一些进展 在催化生物清除剂的开发中，例如对氧合酶-1(PON1)的工程突变体， 尤其是针对G-Agents。通过一项反U54拨款，这是美国军队之间的合作 化学防御医学研究所(USAMRICD)、俄亥俄州立大学(OSU)和魏茨曼 研究所已经在几内亚设计、鉴定和测试了几种PON1的高活性变种。 对猪的药代动力学和保护作用进行初步研究。具有抗环沙林活性的变异体和 针对一系列G试剂进行了描述，最近的工作已经确定了预期的几个变体 对几种有机磷农药有效。我们在此建议对配方进行初步表征 这将直接导致在大规模伤亡情况下最有用的可注射酶。 这项提案的总体目标是开发一种健壮的、封装的PON1变体，其配方为 与肌肉注射给药兼容，在大规模伤亡情况下最大限度地发挥效用。降低了Cmax， T1/2的增加和货架稳定性的增强都预计都是由于纳米颗粒的配方 酵素。来自俄亥俄州立大学的蛋白质生物化学家团队和来自 西南研究院(SwRI)已经合作实现了这一目标。这项提案将决定 包埋几种PON1变异体的最佳方法和聚合物 稳定性。纳米颗粒的溶解性和降解性随后将通过衍生化来调节 不同大小的聚乙二醇组分。最后，最好的派生、封装的变体将是 配方用于IM给药，并在体外进行测试。USAMRICD的中和U54测试核心将是 用于确定最佳IM的药代动力学、保护效果和暴露后效果 配方。这些实验将为U54中心的更新项目提供基础 GRANT，或针对选定的PON1变体的最终配方的独立U01。 PHS 398/2590(06/09版)页面续格式页面

Program Director/Principal Investigator (Last, First, Middle): MAGLIERY, Thomas J. PROJECT SUMMARY/ABSTRACT Organophosphorus (OP) nerve agents (e.g., VX and sarin) and pesticides (e.g., chlorpyrifos/Dursban) are among the most toxic compounds known. Because of the continued use of these pesticides around the world, and because many nations are known to have stockpiles of nerve agents, OP agents represent a significant mass casualty threat from both terrorist attacks and industrial accidents. The treatment regimen for OP poisoning results in incapacitation and does not treat sequelae or effects of lower-dose exposures; it is also difficult in mass casualty scenarios. A solution to this problem is development of an enzymic countermeasure that can rapidly degrade the OP agents before they can exert their full effect. There has been recent progress in the development of catalytic bioscavengers, such as engineered mutants of paraoxoase-1 (PON1), particularly against G-agents. Through a CounterACT U54 grant that is a collaboration among the U.S. Army Medical Institute of Chemical Defense (USAMRICD), The Ohio State University (OSU), and The Weizmann Institute, several highly-active variants of PON1 have been engineered, characterized and tested in guinea pigs for preliminary pharmacokinetics and protective efficacy. Variants with activity against cyclosarin and against a spectrum of G agents have been described, and recent work has identified several variants expected to be effective against several OP pesticides. We propose here preliminary characterization of formulations that would directly lead to injectable enzymes most useful in mass casualty situations. The overall goal of this proposal is to develop a robust, encapsulated PON1 variant formulated to be compatible with intramuscular administration for maximum utility in mass casualty situations. Decreased Cmax, increased t1/2, and enhanced shelf stability are all anticipated to result from nanoparticle formulation of the enzyme. A team of protein biochemists from OSU and encapsulation and nanomaterials experts from Southwest Research Institute (SwRI) have partnered to achieve this goal. This proposal will determine the optimum methods and polymers for encapsulation of several PON1 variants in terms of enzyme activity and stability. The solubility and degradation of the nanoparticles will then be tuned by derivatization with polyethyleneglycols (PEGs) of different sizes. Finally, the best derivatized, encapsulated variants will be formulated for IM administration and tested in vitro. The CounterACT U54 Testing Core at USAMRICD will be used to determine pharmacokinetics, protective efficacy, and post-exposure efficacy for the best IM formulation. These experiments will provide the basis for either a project in the renewal of the U54 Center grant, or an independent U01 directed at final formulation of selected PON1 variants. PHS 398/2590 (Rev. 06/09) Page Continuation Format Page