Formulation and Encapsulation of Enzymic Countermeasures Against Organophosphorus

描述（由申请人提供）：有机磷（OP）神经毒剂（例如，VX和沙林）和杀虫剂（例如，毒死蜱/杜尔善）是已知毒性最强的化合物之一。由于这些杀虫剂在世界范围内的持续使用，并且因为许多国家都有神经毒剂的库存，OP毒剂代表了恐怖袭击和工业事故的重大大规模伤亡威胁。有机磷中毒的治疗方案导致丧失行为能力，不治疗低剂量接触的后遗症或影响；在大规模伤亡的情况下，这也很困难。解决这一问题的办法是开发一种酶的对策，它可以在OP药剂充分发挥作用之前迅速降解它们。近年来，催化生物清除剂的开发取得了一些进展，例如对氧氧化酶-1 （PON1）的工程突变体，特别是针对G-agents。通过美国陆军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（Weizmann Institute）合作的一项抵消U54拨款，对PON1的几种高活性变体进行了设计、表征和在豚鼠身上进行了初步的药代动力学和保护功效测试。已经描述了对环沙林和一系列G剂具有活性的变异，最近的工作已经确定了几种变异，预计对几种OP农药有效。我们在这里提出初步表征的配方，将直接导致注射酶最有用的大规模伤亡情况。本提案的总体目标是开发一种健壮的、封装的PON1变体，与肌内给药兼容，在大规模伤亡情况下发挥最大效用。Cmax的降低，t1/2的增加，货架稳定性的增强都是纳米颗粒酶制剂的结果。俄勒冈州立大学的蛋白质生化学家和西南研究所（SwRI）的封装和纳米材料专家团队合作实现了这一目标。本研究将根据酶活性和稳定性确定几种PON1变异包封的最佳方法和聚合物。纳米颗粒的溶解度和降解将通过不同大小的聚乙二醇（peg）衍生化来调节。最后，最好的衍生化，封装变体将配制IM管理和体外测试。USAMRICD的中和U54测试核心将用于确定最佳IM配方的药代动力学、保护功效和暴露后功效。这些实验将为项目的更新提供基础

DESCRIPTION (provided by applicant): 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.