Janice E. Chambers - US grants

Organophosphorus (OP) insecticides, widely used in agriculture and public health, display a very wide range of acute toxicity levels and are a potential hazard to many individuals. Metabolism and biochemical protective mechanisms were studied during the original grant period. One of the most significant observations was the presence in the brain of phosphorothionate desulfuration (activation) activity which, while very low compared to liver, correlated directly with acute toxicity level, whereas hepatic activity did not. A second important observation was that the Ca++- dependent A-esterases hydrolyze only a few oxons at low, toxicologically-relevant substrate concentrations, and this hydrolysis contributes to the low toxicity displayed by their corresponding phosphorothionates. The following hypothesis will be investigated in the renewal project: The overall toxicity level of a phosphorothionate insecticide is determined mainly by the level of brain desulfuration activity, with lesser contributions by the detoxication pathways. This hypothesis will be investigated primarily by intensive study in the rat of the monooxygenases in the brain, and the A-esterases in several tissues, primarily the liver. There will be three major specific aims: 1) To characterize the brain monooxygenases responsible for phosphorothionate desulfuration through immunological recognition, substrate specificity and kinetics of brain cytochrome P450; 2) To characterize the A-esterases responsible for oxon hydrolysis by tissue and subcellular distribution and assessment of whether the low affinity and high affinity esterases are identical; and 3) A structure-activity relationship study to characterize the structural requirements of substrates for low brain phosphorothionate activation and for high affinity oxon hydrolysis. The resultant information will be of value in the rational development of safer OP insecticides.

health science research support;

biomedical equipment resource; biomedical equipment purchase;

The candidate, Dr. Janice Chambers, intends to develop a career in biochemical toxicology, with emphasis on insecticide mechanisms of action and metabolism, and other related aspects of neurotoxicology, such as behavior. The current emphasis is on concepts related to the acute toxicity of organophosphorus insecticides, which act by inhibiting acetylcholinesterase (AChE). Her short term career goal is to establish a more productive program in metabolism of organophosphorus insecticides and toxic interactions of these compounds in the organism. Her long term career goal is to establish a recognized research program in insecticide neurotoxicology, with emphasis on mechanisms of action (including metabolism) in vertebrates. The proposed RCDA would reduce current teaching commitments of two courses per semester to two courses per year and likewise reduce committee assignments so that the candidate will be able to devote a far greater percentage of her time to research. This concentration will allow her the time not previously available to learn new methodologies to expand the scope of her research program into logical related areas, to better keep up with new developments in the field, to interact more effectively with her graduate students, and to write manuscripts on completed research results. Mississippi State University will reduce her annual teaching load to two courses in her field (Toxicology and Principles of Toxicology) and will reduce her committee obligations to service on only the Animal Research Committee. The research project to be conducted during the tenure of the RCDA will be an investigation of the monooxygenase-mediated activation and detoxication of phosphorothionate insecticides and the detoxication of the activated metabolites by brain (the target tissue) and liver, along with the potential mechanisms (esterases and other binding proteins) present in liver and blood which would protect the target AChE. This current project (ES 04394) will be expanded later into the areas of structure-activity relationships, toxicokinetics and enzyme kinetics.

This application is a request for support of a symposium entitled "Organophosphates: Chemistry, Fate and Effects", which will be presented in the program of the Division of Agrochemicals of the American Chemical Society during its national meeting in April, 1990. This will be a 2-day symposium with 19 speakers who were selected for their knowledge and expertise in a variety of aspects of organophosphate chemistry and toxicology. The focus of the symposium is the organophosphate anticholinesterases which have wide current applications primarily in agricultural uses and are therefore subjects of numerous acute and chronic exposures to humans occupationally. The symposium is designed to bring together the many diverse areas of current organophosphate research to stimulate discussion among individuals who may not have had the opportunity to conveniently interact before. The initial topic will be chemistry, with discussion of chemical properties and reactivity, environmental stability, and chemistry of actual and potential therapeutic agents. This will be followed by a discussion of fate, with emphasis on disposition and metabolism in mammalian systems. Last will be a discussion of effects including such topics as tolerance and behavioral effects, as well as non- anticholinesterase effects such as immunotoxicity or delayed neuropathy. The symposium will be a unique opportunity to bring researchers together in a forum with organophosphates as the underlying theme, and will promote new understanding and insights into the ramifications on human health of organophosphate exposures.

Organophosphorus (OP) insecticides, widely used in agriculture and public health, display a very wide range of acute toxicity levels and are a potential hazard to many individuals. Metabolism and biochemical protective mechanisms were studied during the original grant period. One of the most significant observations was the presence in the brain of phosphorothionate desulfuration (activation) activity which, while very low compared to liver, correlated directly with acute toxicity level, whereas hepatic activity did not. A second important observation was that the Ca++- dependent A-esterases hydrolyze only a few oxons at low, toxicologically-relevant substrate concentrations, and this hydrolysis contributes to the low toxicity displayed by their corresponding phosphorothionates. The following hypothesis will be investigated in the renewal project: The overall toxicity level of a phosphorothionate insecticide is determined mainly by the level of brain desulfuration activity, with lesser contributions by the detoxication pathways. This hypothesis will be investigated primarily by intensive study in the rat of the monooxygenases in the brain, and the A-esterases in several tissues, primarily the liver. There will be three major specific aims: 1) To characterize the brain monooxygenases responsible for phosphorothionate desulfuration through immunological recognition, substrate specificity and kinetics of brain cytochrome P450; 2) To characterize the A-esterases responsible for oxon hydrolysis by tissue and subcellular distribution and assessment of whether the low affinity and high affinity esterases are identical; and 3) A structure-activity relationship study to characterize the structural requirements of substrates for low brain phosphorothionate activation and for high affinity oxon hydrolysis. The resultant information will be of value in the rational development of safer OP insecticides.

biomedical equipment purchase;

Organophosphorus (OP) insecticides are widely used and constitute an appreciable exposure hazard, especially for children in rural areas because of the intense agricultural activity. OP insecticides or their metabolites are anticholinesterases, and lead to hyperexcitability in the nervous system because of the accumulation of acetylcholine. OP insecticides, have been implicated in causing behavioral effects in children, including hyperactivity. Perturbation of the cholinergic system by an OP insecticide during nervous system development may cause permanent deficits in cognition since the cholinergic system is involved in learning and memory. It is therefore possible that exposure to OP insecticides may be responsible for cognitive deficits and hyperactivity which could be manifest as reduced learning ability and attention deficit disorder/hyperactivity disorder. Our laboratories have been involved in the study of the metabolism and neurochemical sensitivity of adults and juveniles to OP insecticides. However, our laboratories have not performed behavioral studies on juveniles. Most behavioral studies existing at the present on the effects of OP compounds on juveniles have used high dosages to demonstrate effects. It is still unknown whether levels of insecticides encountered as dietary contaminants or as environmental contarninants from agricultural or domestic uses can lead to behavioral effects. The persistence of the acetylcholinesterase (AChE) inhibition, resulting in part from "aging" (permanent inactivation) of the inhibited enzyme. may also play an important role in the severity of the behavioral deficits. This project will determine whether deficits in behavior occur in juvenile rats treated neonatally with the OP insecticide chlorpyrifos, and whether these deficits correlate with the persistence (including aging) of the brain AChE inhibition. The behaviors to be studied are open field (locomotor) activity (to test mainly for hyperactivity) and a delayed alternation task (to test cognition). The results will indicate whether OP insecticides could be responsible for some of.the learning deficits and hyperactivity observed in schools today, particularly in disadvantaged populations.

Organophosphorus (OP) insecticides are heavily employed in agricultural and residential uses, and have ample opportunity to expose infants and children from food and houses during critical times of their nervous system development. The mechanism of toxicity, inhibition of acetylcholinesterase (AChE), causes hyperactivity in cholinergic pathways, which can elicit compensatory neurochemical mechanisms to modulate the enhanced activity. AChE inhibition is considerably more persistent for the diethyl OP compounds than for the dimethyl OP compounds, leading to greater fluctuations in AChE activity following exposure to a dimethyl insecticide, such as methyl parathion, than to a diethyl insecticide, such as chlorpyrifos. Our preliminary results in rats have indicated more persistent whole brain muscarinic acetylcholine receptor (mAChR) decreases following early postnatal exposures to chlorpyrifos than to methyl parathion. Developmental exposures to OP insecticides cause behavioral deficits. Our preliminary results have indicated decreases in open field activity occurring only after, not during, chlorpyrifos exposures, suggesting possible permanent behavioral deficits from developmental exposures. The biochemical lesion occurs in both the central and peripheral nervous systems because of the distribution of AChE in both, so both central and peripheral effects could contribute to behavioral deficits. The possibility of permanent behavioral defects from the extended neurochemical alterations following AChE inhibition of diethyl OP insecticides has generated the following hypothesis for this project: Neurochemical aberrations and behavioral deficits will be more severe and long lasting from the more persistent phosphorylation of a diethyl OP insecticide (chlorpyrifos) during development than from a dimethyl OP insecticide (methyl parathion). The cholinergic parameters to be studied include: AChE, mAChR (total and surface), choline acetyltransferase, high affinity choline uptake, and adenylyl cyclase in brain regions, and AChE in peripheral tissues. The behavioral parameters to be studied include: developmental markers, surface righting, negative geotaxis, free-fall righting, locomotor activity, rotarod performance, grip strength, and memory and learning in two types of water mazes. The results will indicate whether certain OP insecticide chemistries elicit greater harm to the developing nervous system and whether children are likely to suffer from permanent harm to behavior, including cognition, from developmental exposures to OP insecticides.

[unreadable] DESCRIPTION (provided by applicant): Organophosphorus (OP) insecticides, extensively used agriculturally and residentially, display a variety of chemistries. OP insecticides demonstrate a wide range of acute toxicity levels, which are largely dependent on the compound-specific efficiencies of detoxication. Juveniles are typically more vulnerable than adults to the toxic effects of OP insecticides. The low immature xenobiotic detoxication capacity of juveniles contributes to their enhanced vulnerability. It is logical to conclude that compounds, which are readily detoxified in adults, will display relatively higher toxicity levels in juveniles than compounds, which are not readily detoxified; in other words, the so-called "safer" insecticides (based on low toxicity levels in adults) are of relatively more danger to infants and children than are the "unsafe" insecticides. The objective of this application is to determine the relative degree of vulnerability to toxic effects following exposures to a representative group of OP compounds in juvenile and adult rats, and to characterize the role of detoxication in these vulnerabilities. The hypothesis is: The lower vulnerability of adults to the toxicity of OP insecticides observed at high exposure levels is primarily the result of (he greater effectiveness of their mature detoxication systems; the age-related differences in vulnerability to individual OP compounds will be greater for the compounds which are less toxic to adults, i.e., those traditionally viewed as "safer" This hypothesis was derived from our preliminary findings that acute toxicity levels in both adults and juveniles are strongly related to the levels and the efficiency of several protective esterases. We plan to test our hypothesis by investigating the following two specific aims: 1. Determine for 12 select OP compounds the in vitro efficiency of critical detoxication mechanisms and of target enzyme sensitivity during development; and 2. Determine for these 12 OP compounds the contribution of detoxication mechanisms to the in vivo OP toxicity levels among ages. The project will utilize novel OP compounds synthesized in our laboratories, which demonstrate unique characteristics 0 inhibitory potency plus detoxication potential. The project will investigate the efficiency of hepatic and blood detoxication mechanisms (the protective esterases: carboxylesterases, non-target acetylcholinesterase, butyrylcholinesterase, A-esterase) and target enzyme (brain and peripheral acetylcholinesterase) sensitivity for each test compound. Experiments will be conducted in rats of 2 juvenile ages (1 and 12 days) and adults. We expect that detoxication efficiencies will be compound-specific, that the determination of the efficiencies of these protective esterases to detoxify the OP's will allow a prediction of how greatly the juvenile toxicity levels will differ from those of adults, and that there will be more dramatic age-related differences in compounds which are more readily detoxified. These results will indicate the contribution of detoxication to toxicity level, and will allow more accurate predictions of age-related differences in toxicity.

DESCRIPTION (provided by applicant): The Center for Environmental Health Sciences (CEHS) within the CVM-MSU submits an application to develop a COBRE with the overall scientific theme of environmental health, and a specific emphasis on the toxicological effects of pesticides. Our COBRE would be a multi-disciplinary research effort with two overarching goals: 1) to develop individual junior faculty expertise and credibility so that each is recognized as a successful, fully enfranchised member of the community of environmental health scientists with independent, competitive funding support from peer-reviewed mechanisms (primarily R01 grants); and 2) to develop a team of scientists who can compete for programmatic research support, such as a program project grant, for investigation of environmental health problems elicited by agricultural chemicals. Organizationally, an Administrative Core would coordinate overall activities of the COBRE, and be responsible for mentoring the junior scientists in their career development to increase their competitiveness, and for data management activities (data entry and statistical guidance). Four biomedical mechanistic research projects will be based on hypothesis-driven studies of the potential effects of pesticides on the mammalian nervous or endocrine systems at various stages of development (from early development through aging). The mechanisms of toxicity of pesticides will be investigated in animal models (both traditional laboratory rodents as well as new animal models). A Research Resources Core would provide infrastructure to these biomedical projects through animal resources; (Animal Sub-Core) and shared instrumentation (Imaging Sub-Core and Analytical Chemistry Sub-Core). In addition, an epidemiology and exposure assessment project would bring real world perspectives to the biochemical and physiological mechanisms under study. Renovation of laboratory space would occur. The Principal Investigators (PI) of the science projects are junior faculty who demonstrate distinct but complementary areas of expertise, all of whom can contribute individually and programmatically to answering critical questions in the assessment of the impact of pesticides on selected areas of human health. All are committed to developing new or expanded expertise in scientific questions or methodologies useful in the area of environmental health, and that fit the priority research areas of the National Institute of Environmental Health Sciences (NIEHS), for whose support we hope to ultimately compete.

toxicology; environmental health; computer simulation;

DESCRIPTION (provided by applicant): This application for an R21 grant is designed to solidify an interdisciplinary team of basic and clinical researchers in the Center for Environmental Health Sciences at Mississippi State University for research into the environmental factors contributing to the higher mortality of cardiovascular disease (CVD) in the Deep South and among African Americans, and to position this team for participation in larger-scale on- going multi-institutional epidemiological studies. This health disparity in CVD is logically related to risk factors more prevalent in the South (which has a higher proportion of African Americans than other regions in the country.) The South is rural and highly agricultural, so the Southern populations would be expected to be routinely exposed to higher levels of pesticides than populations in many other regions. We hypothesize the following: The effects of pesticide exposure contribute to the development of CVD and are more pronounced in the African American population. Two enzymes that we have studied for many years because of their involvement in the detoxication of many pesticides are paraoxonase (PON1) and carboxylesterase (CaE). The former is known to be associated with HDL particles and the latter has recently been identified as a cholesteryl ester hydrolase that is involved in reverse cholesterol transport;therefore, both of these enzymes are involved in vascular health, and are potential biomarkers of susceptibility to CVD. We have experience biomonitoring urinary pesticide metabolites as an index of current pesticide exposure. We have developed a novel geospatial analysis of pesticide harvested crop records that predicts pesticide exposure to individuals for the previous 35 years of their lives. We have experience in cardiology and access to patients in a cardiology practice. We propose a pilot study to investigate the above listed parameters (i.e., PON1 and CaE in the blood and pesticide metabolites in the urine) of subjects recruited from the cardiology practice, and to calculate strength of associations among these factors, the degree of cardiovascular disease and the pesticide exposure estimated in the geospatial analysis model. This pilot study will be conducted following a year of planning, solidifying the interdisciplinary team, optimizing assays, and developing interactions with the project's consultants. The pilot study will position us to participate in a far more extensive study through interactions with our consultants at the University of Alabama at Birmingham. In addition, we anticipate that we will develop more simplified assay methods for PON1 and CaE that will be more amenable for use as a clinical biomarker.

An award has been made to Mississippi State University under the direction of Dr. Nikolay M. Filipov for the acquisition of a laser microdissection system for the manipulation and study of various aspects of cell biology. The instrument will benefit a large number of research programs in botany, forestry, immunology, Parasitology, proteomics, and others fields. The instrument provides state-of-the-art capabilities to capture a small number of cells from culture and isolate their internal structures for research on neural development, diseases of fish, plant viruses, and proteomics of chickens. Cells and their structures can be tagged with fluorescent dyes for further study. The microdissection system will be used in several existing courses and summer programs for MSU students. High school students as well as students from a nearby women's college will be involved in the educational programs.

DESCRIPTION (provided by applicant): Organophosphate (OP) anticholinesterases, e.g., nerve agents or insecticides, are potent acetylcholinesterase (AChE) inhibitors. High dose poisoning causes excitotoxicity that leads to seizures and subsequently brain damage. The current therapy in the US includes an oxime reactivator, 2-PAM, which has little, if any, ability t cross the blood-brain barrier (BBB) and consequently cannot stop seizures and prevent brain damage. Our laboratories have developed a library of phenoxyalkyl pyridinium oximes designed to have greater lipophilicity than 2-PAM. Through a paradigm using high sub-lethal dosages of highly relevant surrogates for sarin or VX in rats and administering the novel oximes at 1 hr after OP compound treatment when seizure behavior was on- going, we have demonstrated the ability of a number of these oximes to penetrate the BBB, reactivate AChE, and to attenuate seizure behavior. The objective of this application is to further characterize a limited number of these novel oximes (initially 6, then down-selected to 3) to ultimately select a single lead oxime for an IND application. Additionally we will expand the OP chemistries tested to include an insecticidal chemistry, paraoxon (the active metabolite of parathion; tested first to avoid any confounders that could occur because of the need for bioactivation) and parathion. Endpoints of characterization include brain and peripheral AChE reactivation, seizure attenuation, neurodegeneration (glial fibrillary acidic protein accumulation), oxidative stress (F2-isoprostanes), neuropathology (apoptosis, neuron survival), and behavior. Concurrently molecular dynamics computations will determine if one or two additional oxime structures are predicted to have greater efficacy than our current selections; if so, it/they will be synthesized and substituted for our current selections. The specific aims of the proposed project are: 1. In vivo efficacy characterization aim: To provide expanded measures of neuroprotection on our currently identified BBB-penetrating oximes using the sarin and VX surrogates and also a common insecticidal OP chemistry (i.e., diethyl phosphate) using parathion and its active metabolite paraoxon. Efficacy endpoints for the 4 OP's include: 1a, efficacy screening endpoints with 6 oximes: reactivating rat brain AChE, and the attenuation of seizure behavior and neurodegeneration; and 1b, detailed efficacy characterization endpoints with 3 down-selected oximes: oxidative stress in the brain, neuropathology and behavioral deficits. 2. Pharmacokinetic aim: To determine for the 3 down-selected oximes hepatic in vitro metabolism, plasma protein binding and levels of oxime in the blood and cerebrospinal fluid (obtained through microdialysis). 3. Computational chemistry aim: To model the docking of oxime with AChE and BBB penetration to suggest improved oxime structures. 4. Chemical synthesis aim: To produce sufficient quantities of the test OP's and the test oximes for the experiments proposed, as well as the synthesis of a few new oximes if suggested by the computational chemistry aim. The ultimate outcome will be the identification of the most efficacious novel oxime for further development as a potential substitute for current therapy.

Many of the organophosphate (OP) anticholinesterases are highly toxic and have been developed as either nerve agents or insecticides. Prolonged acetylcholinesterase (AChE) inhibition results in glutamate-induced seizures with subsequent permanent brain damage. Some OPs are relatively easy to synthesize and could become threat agents of great concern from potential terrorist action where the specific OP employed might not be immediately known. Terrorist actions or accidents could lead to mass casualties in adults and children of both sexes. The current therapy consists of the muscarinic receptor antagonist atropine and an oxime reactivator of the inhibited AChE (2-PAM in the US). However 2-PAM is not broad-spectrum and cannot effectively penetrate the blood brain barrier, so it would leave victims poorly protected against some OP chemistries and would not attenuate the hypercholinergic activity in the brain and resultant brain damage. Therefore an improved oxime therapeutic is needed which can counteract both nerve agent and insecticidal chemistries and can restore brain cholinergic function to attenuate or prevent long-term central nervous system damage, so that both life and brain function may be preserved. Our laboratories have invented and patented (US Patent 9,277,937) a platform of substituted phenoxyalkyl pyridinium oximes that have shown broader based survival efficacy than 2-PAM and also attenuation of signs of seizure-like behavior and neuropathology in male rats exposed to high levels of both nerve agent and insecticidal chemistries. Limited studies in male guinea pigs against sarin have also showed efficacy. Only limited preliminary information exists at present with respect to the novel oximes' pharmacokinetics and no information exists on their therapeutic efficacy in female or juvenile animals. Preliminary data indicate that combinations of a novel oxime and 2-PAM are more efficacious than the single oximes. A combination of these two or combinations of two novel oximes with different specificities for nerve agent and insecticidal chemistries could provide a broader spectrum of efficacy than single oximes and provide a more effective therapeutic in the event of mass casualties induced by an unidentified OP. Therefore this application proposes the generation of additional efficacy data against a highly relevant sarin surrogate (nitrophenyl isopropyl methylphosphonate, NIMP) and paraoxon (PXN; the active metabolite of the insecticide parathion) in adult female and juvenile (both sexes) rats, on our three most efficacious novel oximes and combinations of two oximes. Initial pharmacokinetic and initial oxime toxicity data will be generated. Lastly efficacy tests will be performed in male and female adult guinea pigs with sarin and VX. The goal of this Lead Identification project is to down-select to a lead and an alternate novel oxime or novel oxime binary combination that will be ready to enter into optimization studies through a subsequent Lead Optimization U01 project, that will move the novel oximes into advanced development toward FDA approval.

7. Project Summary/Abstract Many of the organophosphate (OP) insecticides, such as phorate (O,O-diethyl S-ethylthiomethyl phosphorodithioate), are highly toxic with rat oral LD50's in the low mg/kg range, and they or their active metabolites are potent inhibitors of acetylcholinesterase (AChE). Phorate is consistently more toxic to females than males; for example, rat oral LD50's for males and females are 3.7 and 1.4 mg/kg, respectively. Phorate requires monooxygenase-mediated bioactivation to active anticholinesterase metabolites, similar to a number of other OP insecticides, such as parathion (O,O-diethyl O-nitrophenyl phosphorothionate). Our preliminary studies with a phorate metabolite phorate-oxon (PHO) have indicated a longer time delay and more violent signs of poisoning than with paraoxon (PXN), the active metabolite of parathion. Additionally, patterns of oxime-mediated cholinesterase reactivation differ between PHO and PXN, which is unexpected because both are diethyl phosphates, and would be expected to phosphylate cholinesterase with the same diethyl moiety and therefore display similar reactivation patterns. PHO undergoes additional bioactivation of its terminal sulfur ether to a sulfoxide, then to a sulfone. Estimates of binding energies and the unusual preliminary results observed thus far have suggested that there may be an ethoxy leaving group instead of the expected ethylthiomethyl group and that the slow bioactivation of PHO in the brain to PHO-sulfoxide and then to PHO- sulfone might be responsible for the unexpected preliminary observations. 2-PAM is the currently FDA- approved oxime AChE reactivator. However the need for a different oxime reactivator that is more effective with an unconventional phosphylating moiety as well as an oxime that can penetrate into the brain will be needed for effective phorate therapy. Our laboratories have invented and patented novel substituted phenoxyalkyl pyridinium oximes that show preliminary evidence of survival efficacy with PHO as well as convincing evidence of entry into the brain with other OP's in our rat model. Therefore the following Specific Aims are proposed: Aim 1. To confirm the leaving group of PHO or PHO metabolites through a mass spectral analysis of AChE-phosphylated peptides and through computational modeling to determine barrier height for the potential leaving groups. Aim 2. To determine bioactivation efficiency through analysis of brain and hepatic bioactivation kinetics for phorate to its several metabolites as quantified by LC/MS/MS. Aim 3. To identify more effective oxime reactivators from our novel oxime library of AChE inhibited by the three phorate metabolites through in vitro reactivation studies and limited in vivo phorate survival studies. The results of this R21 project will be the identification of a few down-selected novel oximes that can be further developed in a subsequent U01 project into effective therapeutics for phorate poisoning.

7. Project Summary/Abstract Many of the organophosphate (OP) anticholinesterases, such as nerve agents, are highly toxic. Terrorist actions or accidents involving OPs could lead to mass casualties with potentially high levels of lethality. The current therapy consists of the muscarinic receptor antagonist atropine and an oxime reactivator of the inhibited acetylcholinesterase (2-PAM in the US). However, 2-PAM is not always effective at saving lives and cannot effectively penetrate the blood brain barrier, so 2-PAM can leave victims poorly protected. An improved oxime therapeutic is needed to counteract nerve agent lethality and assist with neuroprotection, so that both life and brain function may be preserved. Our laboratories have invented, patented and licensed a platform of substituted phenoxyalkyl pyridinium oximes that have shown better survival efficacy than 2-PAM and, unlike 2- PAM, attenuation of signs of seizure-like behavior and neuropathology in rats exposed to high levels of highly relevant nerve agent surrogates. Limited studies in male guinea pigs against sarin have also shown efficacy. With our current CounterACT Lead Identification U01 the efficacious compounds (the ?actives?) have been down-selected to a lead and an alternate, with Oxime 20 being proposed as the Active Pharmaceutical Ingredient (API). The proposed project will build on the present survival efficacy, pharmacokinetic and API toxicity information in rats. Initially a superior vehicle for the API will be developed as a better solvent for the lipophilic API. A pharmacodynamic aim (Aim 1) will determine in rats (both sexes) whether a lower dosage of the API will be effective in promoting survival of lethal dosages of a sarin surrogate (nitrophenyl isopropyl methylphosphonate, NIMP; a G agent chemistry) and a VX surrogate (nitrophenyl ethyl methylphosphonate, NEMP; a V agent chemistry) alone or in combination with 2-PAM. A pharmacokinetic (PK) aim (Aim 2) will determine the PK of the API in the new vehicle, plasma protein binding and hepatic microsomal metabolism in rats of both sexes and will introduce studies of a larger non-rodent test species, the Gottingen minipig, both sexes. An oxime toxicity aim (Aim 3) will investigate dose responses of the API for gross pathological, histopathological, clinical chemistry and hematology adverse results in rats and minipigs of both sexes to identify a Maximum Tolerated Dosage and a No Observed Adverse Effect Level, as well as in vitro genotoxicity and drug-drug interactions for CYPs and transporters. A chemistry aim (Aim 4) will support the previous 3 aims by providing the synthesis of NIMP, NEMP and the API, produce a new vehicle with improved solvent properties, evaluate API stability, and provide initial plans for manufacturing and Chemical Manufacturing Controls. All studies will be non-GLP and will follow FDA guidance from pre-IND meetings. The overarching goal of this Lead Optimization project is to provide optimized pharmacological and toxicological information on our lead oxime in both sexes of two species that will prepare the API to move into advanced development toward FDA approval.