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

描述(申请人提供)：有机磷化合物(OPS)是一种强有力的神经毒性化学物质，广泛用于医药、工业和农业；最主要的是作为杀虫剂。神经毒性主要是AChE抑制的结果，可采取胆碱能危象和死亡的形式，作为急性暴露或慢性暴露后的精神症状和延迟性神经病变的结果。通常，杀虫剂中毒的治疗包括支持性护理和特殊治疗，如阿托品和肟类药物，但这往往无法防止发病率或死亡。目前，重组(R)BChE因其强大的生物清除能力而成为OP毒性暴露前治疗的领先候选药物，但由于其与OP的化学计量比为1：1，如果由I.M.提供，将需要大剂量。或皮下途径。为了减少全身途径输送BChE所需的剂量，我们计划创建一种将雾化四聚体rBChE雾化进入肺部的保护性“BChE生物盾牌”，以在原位为传入(吸入)的OP解毒，从而防止OP进入体循环，并避免通常与吸入暴露相关的呼吸。第一阶段是在小鼠身上进行的可行性研究，以检查(I)微喷雾器或喷雾器分别向肺部输送的液体或粉状聚乙二醇单甲醚-rMaBChE放射性气雾剂的沉积模式(Ii)“生物盾牌”的持久性，以及(Iii)通过对RBC AChE和血清BChE的抑制百分比来衡量其对对氧磷雾化液体杀虫剂的50%LD50的保护程度。在第二阶段，将在同源猕猴模型中测试雾化聚乙二醇-rMaBChE对OP毒性的保护效果。一个不产生抗BChE抗体的同源系统对于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，而重组治疗性蛋白可能需要多次给药。在此期间，将建立生产CHO-K1的rHuBChE的主细胞库和工作细胞库，并开始生产、纯化和聚乙二醇化协议的工艺开发。雾化的聚乙二醇-rHuBChE“生物盾牌”预防对氧磷中毒的有效性将在猕猴身上进行测试，并与同源的rMaBChE酶进行比较。预计提供气雾剂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。开发一种创新的“微喷雾器”型装置将允许在已知使用/释放杀虫剂之前提供对用户友好的治疗。 与公共卫生相关：许多糖蛋白是有效的解毒剂，具有非常重要的生理功能，但不能用于治疗，因为它们很快就从循环中移除，或者如果给它们注射，它们的保护能力需要大剂量。该项目的目的是将丁酰胆碱酯酶作为肺部给药装置中的气雾剂，使其沉积在肺部，形成“生物盾牌”，并对吸入的杀菌剂进行解毒，防止它们到达血液和神经肌肉接头，在那里它们很快就会造成严重的毒性。

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.