Aerosol Delivery of a Recombinant Butyrylcholinesterase "BioShield" to Protect Ag

描述（由申请人提供）：有机磷化合物是一种强效的神经毒性化学物质，广泛应用于医药、工业和农业；最明显的是杀虫剂。神经毒性主要是乙酰胆碱酯抑制的结果，急性暴露或慢性暴露后的精神症状和迟发性神经病变可能导致胆碱能危象和死亡。通常，杀虫剂中毒的治疗包括支持性护理和特异性治疗，如阿托品和肟，但往往不能预防发病或死亡。目前，重组(r) BChE因其强大的生物清除能力而成为OP毒性暴露前治疗的主要候选药物，但由于其与OP的化学计量比为1:1，因此如果通过im或皮下途径给药，则需要大剂量。为了减少全身途径递送BChE所需的剂量，我们计划制造一种保护性的四聚体rBChE生物屏障，将其雾化进入肺部，就地解毒进入（吸入）的OPs，从而防止OP进入体循环，避免通常与吸入暴露相关的呼吸。第一阶段是在小鼠中进行可行性研究，以检查(I)分别通过微喷雾器或注入器将液态或粉状PEG-rMaBChE放射雾化器输送到肺部的沉积模式（ii）“生物屏障”的持久性以及（iii）通过对红细胞AChE和血清BChE的抑制百分比测量，它对液态雾化OP杀虫剂对氧磷的50% LD50的保护程度。在II期，雾化PEG-rMaBChE对OP毒性的保护效果将在同源猕猴模型中进行测试。同源系统不产生抗bche抗体，对于可能需要多次给药的重组治疗性蛋白的准确疗效、药代动力学和安全性测试至关重要。在此期间，将产生产生rHuBChE的CHO-K1的主细胞库和工作细胞库，并开始生产，纯化和PEG-ylation协议的工艺开发。雾化PEG-rHuBChE“生物屏蔽”预防对氧磷毒性的效果将在猕猴中进行测试，并与同源的rabche酶进行比较。预计气溶胶输送将减少防护所需的剂量，因为其浓度与吸入杀虫剂的浓度相同。一种创新的“微喷雾器”类型设备的开发将允许在已知使用/释放杀虫剂之前提供用户友好的处理。

DESCRIPTION (provided by applicant): Organophosphorus (OPs) compound are potent neurotoxic chemicals that are widely used in medicine, industry and agriculture; most notably as insecticides. The neurotoxicity which is primarily a result of AChE inhibition, may take the form of cholinergic crisis and death as a consequence of acute exposure or psychiatric symptoms and delayed neuropathy following chronic exposure. Usually, treatment of insecticide poisoning consists of supportive care and specific therapy e.g. atropine and oximes, which often fail to prevent morbidity or death. Currently, recombinant (r) BChE is a leading pre-exposure treatment candidate for OP toxicity due to its potent bioscavenging ability but because of its 1:1 stoichiometry with OP, large doses will be required if delivered by i.m. or subcutaneously routes. In order to reduce the dose required for systemic approaches for delivering BChE, we plan to create a protective "BChE bioshield" of aerosolized tetrameric rBChE into the lungs to detoxify incoming (inhaled) OPs in situ, thus preventing the OP's entry into the systemic circulation and avoiding the respiratoty usually associated with inhalation exposure. Phase I represents a feasibility study in mice to examine (i) the patterns of deposition of either liquid or powdered PEG-rMaBChE radioaerasols delivered to the lungs by Microsprayer or insufflator respectively (ii) the persistence of the "bioshield" and (iii) the degree of protection it affords against 50% LD50 of the liquid aerosolized OP insecticide paraoxon as measured by percent inhibition of RBC AChE and serum BChE. In Phase II, the efficacy of the aerosolized PEG-rMaBChE to protect against OP toxicity will be tested in a homologous macaque model. A homologous system, which does not make anti-BChE antibodies, is critical for accurate efficacy, pharmacokinetics and safety testing of a recombinant therapeutic protein which may be required as a multiple administrations. During this time, Master and Working Cell Banks of CHO-K1 producing rHuBChE will be generated and process development begun on the production, purification and PEG-ylation protocols. The efficacy of an aerasolized PEG-rHuBChE "bioshield" to prevent paraoxon toxicity will be tested in macaques and compared with the homologous rMaBChE enzyme. It is anticipated that aerosol delivery will reduce the dose required for protection because of its concentration in the same areas as the inhaled insecticide. The development of an innovative "Microsprayer" type device would permit a user friendly treatment to be delivered before the known use/release of insecticides. Public Health Relevance: Many glycoproteins which are potent antidotes, exhibit very important physiological functions but cannot be used as therapeutic treatments because they are quickly removed from the circulation or their ability to protect requires large doses if given i.m. The aim of this project is to butyrylcholinesterase as an aerosol in a pulmonary delivery device to that it deposits in the lung and forms a "bioshield" and detoxifies inhaled indecticides poisons in the lung and prevernts them from reaching the blood and the neuromuscular junctions where they can quickly cause severe toxicity.