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.
Organophosphorus (OP) nerve agents and pesticides are extremely toxic and in wide use or storage worldwide, making the risk of mass casualty accidents or terrorist attacks serious. Current treatments only relieve the most acute symptoms and are difficult to use for large numbers of victims; recent efforts have produced enzymes that could be much more effective. This project will establish the parameters for encapsulation and formulation of these enzymes to make them practical for mass casualty use.
