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

描述（由申请人提供）：有机磷（OP）化合物是一种强效神经毒性化学品，广泛用于医学、工业和农业;最主要的是作为杀虫剂。神经毒性主要是乙酰胆碱酯酶抑制的结果，可能表现为急性接触或精神症状导致的胆碱能危象和死亡，以及慢性接触后的迟发性神经病。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，这往往不能防止发病或死亡。目前，重组（r）BChE由于其有效的生物清除能力而成为OP毒性的主要暴露前治疗候选物，但由于其与OP的1：1化学计量，如果通过i.m.或皮下途径。为了减少用于递送BChE的全身方法所需的剂量，我们计划将雾化的四聚体rBChE创建到肺中的保护性“BChE生物屏障”，以原位解毒进入的（吸入的）OP，从而防止OP进入体循环并避免通常与吸入暴露相关的中毒。阶段I代表在小鼠中的可行性研究，以检查（i）分别通过微喷雾器或吹入器递送到肺的液体或粉末状PEG-rMaBChE放射性气雾剂的沉积模式（ii）“生物屏障”的持久性和（iii）其提供的针对液体雾化OP杀虫剂对氧磷的50%LD 50的保护程度，如通过RBC AChE和血清BChE的抑制百分比测量的。在II期，将在同源猕猴模型中测试雾化PEG-rMaBChE保护免受OP毒性的功效。不产生抗BChE抗体的同源系统对于重组治疗性蛋白质的准确功效、药代动力学和安全性测试至关重要，所述重组治疗性蛋白质可能需要作为多次施用。在此期间，将生成CHO-K1生产rHuBChE的主细胞库和工作细胞库，并开始生产、纯化和PEG化方案的工艺开发。将在猕猴中测试气化PEG-rHuBChE“生物盾”防止对氧磷毒性的功效，并与同源rMaBChE酶进行比较。预计气雾剂投放将减少保护所需的剂量，因为气雾剂与吸入杀虫剂集中在同一地区。创新的“微型喷雾器”型装置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。 公共卫生相关性：许多糖蛋白是有效的解毒剂，表现出非常重要的生理功能，但不能用作治疗性治疗，因为它们很快从循环中除去，或者如果肌内给药，它们的保护能力需要大剂量。这一项目的目的是将丁酰胆碱酯酶作为气雾剂放入肺部输送装置，使其在肺部沉积，形成“生物屏障”，消除吸入肺部的杀虫剂毒素，防止其进入血液和神经肌肉接头，在那里迅速造成严重毒性。

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.