Douglas M. Cerasoli - US grants

The goal of the Organophosphorus Nerve Agent Testing (ONAT) Core, a component of the Center for Catalytic Bioscavenger Medical Defense Research, is to test the mutant enzymes produced by other Research Projects in the Center for in vitro and in vivo efficacy against organophosphorus (OP) nerve agents. The results from this testing will contribute directly to the development of a pretreatment that offers complete protection from poisoning by OPs. The core is located at the US Army Medical Research Institute of Chemical Defense, a singular national resource with a highly trained staff of scientists supported by safety, surety, and environmental specialists who ensure regulatory compliance of experiments involving highly toxic surety agents such as OP nerve agents. The mission of the USAMRICD is the development of medical countermeasures against chemical weapons, which is precisely aligned with the mission of the CounterACT initiative. The ONAT core is uniquely positioned to assess the function of anti-OP bioscavengers against bona fide nerve agents;the use of these highly toxic agents is restricted to a very limited number of research facilities worldwide. The tests to be conducted by the core will include characterization of the OP nerve agent reactivity, specificity, and stereoselectivity of candidate bioscavenger enzymes using a combination of colorimetric, calorimetric, and gas chromatographic/mass spectroscopy approaches. The capacity of different enzymes to persist in the circulation of a model test animal, and the protection from OP toxicity afforded by these enzymes will also be evaluated. The multidisciplinary approach of the ONAT core to assay development and efficacy testing will support the development of a 'next generation'bioscavenger molecule with the potential to bind and destroy OP nerve agents in the blood, before they distribute to the brain, central nervous system, and other toxic targets. In this regard it is important that the USAMRICD, and in particular the PI of the Center as well as the PI for this core component, have experience in both basic science and in early product development, meaning attention will constantly remain focused on not only the scientific issues, but also on the requirements to develop a novel, protein based drug product that will protect against the otherwise toxic effects of OP nerve agent exposure. The goal of the CounterACT program is to create new ways to protect ourselves from toxic chemicals like OP nerve agents. The goal of this core proposal is to test how well these new approaches defend against highly toxic nerve agents. Without the testing that will be conducted by this core, which is unique in its access to OP nerve agents, there will be no way to measure the effectiveness of these new approaches.

DESCRIPTION (provided by applicant): 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.

Two proteins, human plasma BuChE, and a recombinant human BuChE are both in advanced development as potential prophylacitcs for protecting against chemical warfare agents. These proteins react rapidly and stoiciometrically to effectively scavenge the poisons in the blood stream but they require relatively large amounts of protein to provide protection. The challenge of identifying a protein based drug that would require less material while providing improved protection with the advantage of enhanced user acceptance cannot be ignored. Recent efforts the laboratories alinged with the Center have identified several human enzymes, paraoxonase (PON) and a mutant of butyrylcholinesterase that can catalyze the hydrolysis of all nerve agents. As such these are excellent candidate for prophylactic administration to provide protection for first responders to a terrorist attack or military forces in a civilian peacekeeping setting subject to an asymmetric threat. If it were administered intravenously it could also serve as a rapid-onset therapeutic antidote to a segment of the civilian popuation exposed to nerve agents. Preliminary studies have shown that these objectives are both feasible and obtainable. The cost/benefit ratio of such a drug is such that it is an excellent candidate for success and has the potential to be the first in a series of a novel class of drugs. The center will coordinate the overall effort directed toward the enhancement of the the native catalytic activity of these human enzymes to create a viable candidate for transition to advancement to clinical trials within a five year period. This will address a critical gap in the national goal of protecting the civilian population against a terrorist-initiated chemical weapons attack.

Aftercare; Engineering; Medical; Research;

Engineering; improved; Medical; Research; Substrate Specificity;

bioscavenger; Medical; Research;

The goal of the Organophosphorus Nerve Agent Testing (ONAT) Core, a component of the Center for Catalytic Bioscavenger Medical Defense Research, is to test the mutant enzymes produced by other Research Projects in the Center for in vitro and in vivo efficacy against organophosphorus (OP) nerve agents. The results from this testing will contribute directly to the development of a pretreatment that offers complete protection from poisoning by OPs. The core is located at the US Army Medical Research Institute of Chemical Defense, a singular national resource with a highly trained staff of scientists supported by safety, surety, and environmental specialists who ensure regulatory compliance of experiments involving highly toxic surety agents such as OP nerve agents. The mission of the USAMRICD is the development of medical countermeasures against chemical weapons, which is precisely aligned with the mission of the CounterACT initiative. The ONAT core is uniquely positioned to assess the function of anti-OP bioscavengers against bona fide nerve agents; the use of these highly toxic agents is restricted to a very limited number of research facilities worldwide. The tests to be conducted by the core will include characterization of the OP nerve agent reactivity, specificity, and stereoselectivity of candidate bioscavenger enzymes using a combination of colorimetric, calorimetric, and gas chromatographic/mass spectroscopy approaches. The capacity of different enzymes to persist in the circulation of a model test animal, and the protection from OP toxicity afforded by these enzymes will also be evaluated. The multidisciplinary approach of the ONAT core to assay development and efficacy testing will support the development of a 'next generation' bioscavenger molecule with the potential to bind and destroy OP nerve agents in the blood, before they distribute to the brain, central nervous system, and other toxic targets. In this regard it is important that the USAMRICD, and in particular the PI of the Center as well as the PI for this core component, have experience in both basic science and in early product development, meaning attention will constantly remain focused on not only the scientific issues, but also on the requirements to develop a novel, protein based drug product that will protect against the otherwise toxic effects of OP nerve agent exposure.

Medical; Production; Proteins; Research;

The Center recognizes that the participating institutions and the wide variety of disciplines represented offers a uniquely fertile realm for training and education. The Administration & Management Core (AMC) will oversee the activities of the training core through a Education Training Committee (ETC) comprised of the Center I and the Pis of the individual research projects. In addition to the annual symposium as described in section 1.4, the Center will hold a second annual symposium specifically directed to provide the postdoctoral fellows and senior technicians the opportunity to present their work to their peers. The location for these symposia will rotate between the participating institutions to encourage the widest dissemination of information thereby encouraging cross-fertilization to the maximum extent possible. Two of these symposia will be incorporated by the USAMRICD's biennial Bioscience Review, a week-long international meeting focused exclusively on medical chemical defense research. This meeting is the premier international conference addressing all aspects of medical chemical defense and offers the ideal location for members of the Center to present their research findings, exchange ideas, and interact with the world leaders in this field.