DESCRIPTION (provided by applicant): The acute toxicity of OPs is primarily due to inhibition of acetylcholinesterase (AChE). Current therapy for OP poisoning requires resuscitation with the use of atropine, followed by administration of oximes to reactivate AChE, and benzodiazepines to mitigate neurological complications. However, these antidotes have limited effectiveness and between 10 and 40% of patients, depending on the responsible OP, still die even with intensive care support. Furthermore, acute failure of neuromuscular transmission leads to respiratory failure and profound weakness. This failure of the neuromuscular junction (NMJ) has recently been shown to occur within hours after severe poisoning. The purpose of this grant is to determine the physiologic and immunohistochemical efficacy of the nicotinic receptor antagonist pancuronium in preserving NMJ function and NMJ architecture in a novel rat model of parathion poisoning. This model will mimic the Intermediate Syndrome and consist of intensive care treatment with comprehensive medical therapy, including mechanical ventilation, atropine, 2-PAM, benzodiazepines, and vasopressors. Our central hypothesis is that pharmacologic targeting of the NMJ with pancuronium will improve muscle strength and NMJ structure in a rat model of acute parathion poisoning. Lastly, while these studies are critically applicable to chemical warfare and mass casualty situations, they are also applicable to isolated cases of severe OP poisoning that occur every day in the U.S. and abroad.

PUBLIC HEALTH RELEVANCE: If successful, the proposed research promises to improve public health by mitigating the acute and subacute toxic effects of OP pesticides after intentional or accidental poisoning. __SpecificAimsTextDelimiter__ Specific Aims: This proposal, "Pharmacotherapy to counterACT parathion-induced NMJ dysfunction" responds to the CounterACT Program Announcement PA-11-155. Organophosphorus pesticide (OP) poisoning leads to three main syndromes: 1) acute cholinergic syndrome, 2) OP-induced delayed neuropathy (OPIDN), and 3) the Intermediate Syndrome (IMS). Virtually all OP-related research has focused on the first two entities. IMS, however, is a common complication that remains a major contributor to the high morbidity and mortality in OP poisoning.(1) IMS was first described as a syndrome of paralysis occurring 1-4 days after resolution of the acute cholinergic syndrome.(2) Recent work suggests that IMS occurs much sooner.(3-5) Weakness predominantly affects the limb muscles, respiratory musculature, and those supplied by the cranial nerves. The pathophysiology of IMS is not clearly understood (6-9) but is thought to be due to a persistent excess of ACh at the NMJ.(8, 10) Therefore, interventions targeting the NMJ could yield tremendous dividends in mitigating the toxicity of OPs. The proposed studies have the potential to fundamentally change our management and understanding of acute OP poisoning. The ultimate goal of this investigation is to counter the threat of poisoning from OPs. Despite decades of research into the muscarinic and CNS effects of OP action, no therapy for IMS - short of endotracheal intubation and artificial ventilation - exists. Because research in this area has been hampered by the lack of an animal model for IMS, this proposal has the following goals. First, we propose to develop a new rat model of the Intermediate Syndrome following parathion exposure, utilizing comprehensive critical care treatment including endotracheal intubation for up to 48 hours, atropine, 2- PAM, benzodiazepines, and vasopressors such as norepinephrine. Second, we will study the effect of nicotinic acetylcholine receptor (nAChR) antagonism on in vivo muscle strength and on ex vivo NMJ structure in this rat model. We will combine procedures of clinically relevant measures of muscle strength (such as grip strength testing, rotarod performance, and treadmill exercise endurance) and reproducible concomitant force and repetitive stimulation electromyography (EMG) studies in rats treated with a nAChR antagonist, with mechanistic and molecular imaging techniques of NMJ function. This proposal leverages experience and exciting preliminary results from our ongoing CounterACT R21 that identified progressive NMJ failure occurs within 24 hours after OP poisoning despite conventional therapy. In this study we have found that post-OP poisoning treatment with reversible nAChR antagonists (rocuronium and pancuronium) can preserve NMJ appearance on immunohistochemical studies. Unlike this proposed research, in the preliminary studies all animals were euthanized at 24 hours post-poisoning and no assessments of clinical effectiveness were performed. Moreover, our highly productive, longstanding interdisciplinary research team is uniquely able to perform these studies through exceptional innovation with tremendous impact on the field of acute OP poisoning. The PI is an expert in treating humans suffering from, and in conducting laboratory assessment of, severe acute OP poisoning; Dr. Gaspari is an expert in the neurophysiology of small animal models of OP poisoning; and Dr. Khurana is a leading expert in muscle patho-physiology and NMJ studies. We have the expertise and have developed the methods required to solve the research question. We are proposing to accomplish these studies with the following specific aims: Aim 1: To develop a novel rat model of the IMS after poisoning with parathion; Aim 2: To characterize the NMJ structural effects in this new rat IMS model during the acute poisoning and the recovery period Aim 3: To determine the effect of pancuronium therapy on in vivo muscle strength in this new rat IMS model during the acute poisoning and the recovery period. Aim 4: To determine the effect of pancuronium therapy on NMJ structure in this new rat IMS model Our approach, built upon evidence-based laboratory methods is innovative; we propose to develop a novel rat model of the Intermediate Syndrome using parathion that utilizes comprehensive critical care treatment in a new, realistic small-animal model. By facilitating the rapid assessment of novel antidotal approaches, this investigation will have considerable impact on both research and clinical applications. Lastly, while these studies are critically applicable to chemical warfare and mass casualty situations, they are also applicable to cases of severe OP poisoning that occur every day in the U.S. and abroad.