Organophosphorus (OP) compounds, including both nerve agents and pesticides, represent a serious potential threat to the health of the civilian population. Because they are relatively easy to synthesize, are extremely toxic, and may be difficult to rapidly diagnose or treat in a mass casualty situation, they are considered to be a likely tool for use by terrorists in civilian settings. The CounterACT solicitation identifies a need for a rapid, post-exposure protection that can be easily administered to civilians in a mass casualty scenario. We intent to fulfill this requirement by developing a drug formulation that is amenable to use in an autoinjector, can be delivered via intramuscular injection, will become rapidly bioavailable, will have a sufficient long in vivo residence to protect against slowly metabolized OP compounds, and will have sufficient catalytic efficiency and broad spectrum reactivity to protect against a variety of OP threats. The overarching goal of this U54 Center renewal effort, the """"""""Center for Catalytic Bioscavenger Medical Defense Research II: Discovery, Formulation and Preclinical Evaluation"""""""", is to define, characterize, and transition to the NIH for advanced development a drug formulation that will afford post-exposure protection to victims of OP poisoning. The path we will utilize to accomplish this goal has three major initiatives that will be performed in parallel;the vision for this approach is that at the end of year three all of the concurrent efforts will combine to identify a single best candidate drug which will then become the Center's focus for the remaining two years. The first initiative that the Center will pursue is the generation through rationale design, directed evolution, and high throughput library screening of enzyme variants based on OPH (from fi. diminuta) and/or on a recombinant, bacterially expressible paraoxonase 1 (P0N1). Variants will be screened for broad spectrum activity against multiple OP pesticides and nerve agents. They will also be designed to possess high catalytic efficiency, which will allow for the rapid detoxification of OPs in the blood of exposed individuals. These efforts will identify a single """"""""best variant"""""""" or cocktail of variants that meet the disparate requirements for high catalytic activity and broad spectrum reactivity. The second major initiative of the Center will be the characterization of different encapsulation and formulation approaches that will stabilize enzyme activity in vitro (to promote economical long term storage), will allow rapid (>5 minutes) bioavailability in circulation after an intramuscular injection (to be consistent with the intended use in a mass casualty situation), and to result in long (>48 hour) circulatory stability, ensuring that protection is afforded against both rapid onset OPs like the G agents, slow onset OPs like VX and VR, and pesticides that require in vivo metabolism to be converted into their more toxic forms;to afford protection against different OP compounds with very disparate distribution profiles, we will require both rapid bioavailability and long-lived circulatory stability. The third major initiative will be a determination of the utility of co- administration of conventional therapeutic drugs (atropine, an oxime, and/or an anti-convulsant) as an adjunct that will enhance the therapeutic efficacy of a catalytic scavenger. One of the only published reports detailing the use of a catalytic bioscavenger to provide protection against an OP also included an experiment where atropine and the oxime 2-PAM were co-administered with an enzyme;the amount of protection afforded was substantially greater than was predicted based on the efficacy of either drug therapy alone, suggesting that the use of conventional therapeutic drugs with a catalytic scavenger may result in a synergistic rather than additive level of protection. Public Health Relevance: This initiative will determine if there is therapeutic value in coadministration of conventional drugs with a catalytic scavenger enzyme;there are sound theoretical reasons to believe that such synergy might exist, given that conventional therapies work via a functionally distinct mechanism from catalytic scavengers. Conventional therapy functions predominantly to ameliorate the effects of OP compounds that have distributed out of the blood, while catalytic scavengers rapidly eliminate OP compounds in the blood. The Center will focus on development of enzymes specific for six different OP compounds: the nerve agents VX and VR, and the pesticides parathion, chlorpyrifos, phorate, and dicrotophos. Some work will be done with the G-type nerve agents GA, GB, GD, and GF, but since these agents have exceedingly rapid onset of signs after initial exposure (depending on the dose, death can occur within <10 minutes) and the CounterACT solicitation specifically states that """"""""drugs . . that must be given within a very short period (1-15 minutes) after the insult will be of low priority"""""""", we have chosen to make efforts on G-agents in this proposal of low priority. In this regard, we will examine the potential of co-administration of conventional therapy with enzymes to protect against GB;synergy may provide unexpectedly high levels of protection against GB.
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