BChE reactivators for nerve agent and pesticide OP detoxification in human tissue

描述(由申请人提供)：我们建议开发一种以前未被探索的治疗神经毒剂和农药OP暴露的方法，该方法基于直接在人体循环和组织中有效地重新激活内源性BChE。这种方法在很大程度上是基于OP在暴露期间首先进入和/或积累的组织中OP直接加速降解。在肺、肠和血浆中天然存在的BChE能促进OPs的水解，从而为OPs随后在骨骼肌和中枢神经系统中的靶点分布提供了障碍。作为原理的证明，我们最近在体外、体外和体内证明了基于BChE的催化型OP生物清除剂的功能(Radic等人，Biochem J 450,2013,231-242)。我们证明了一种新开发的人BChE的肟复活剂TAB2OH在体外有足够的能力快速重新激活失活的共价OP-BChE结合物，并与纯化的BChE联合给药，增强了对OP暴露小鼠的保护和治疗。我们现在建议利用肺、肠和血浆中天然存在的内源性BChE，而不是使用纯化的BChE，并且只使用高效的BChE复活剂来帮助内源性BChE降解OP。我们认为这是可能的，因为我们最近开发了一系列基于三元和四元咪唑的hBChE复活剂，它们在体外的效率是TAB2OH的几倍。叔胺具有血脑屏障、肺泡膜渗透和口服生物利用度的额外潜力。我们建议研究新的BChE复活剂在OP暴露的小鼠体内的有效性，并进一步完善它们的结构以改善其复活性能。这项R21建议的研究，通过积累在OP存在下hBChE重新激活和周转的体外动力学参数，并将最佳化合物应用于小鼠体内分析，将揭示内源性BChE在解毒治疗中重新激活的潜力。这也应该导致确定最有效的剂量方案，并评估通过肟辅助催化提高内源性BChE潜力的限度。简而言之，有没有一个OP暴露水平，在这种水平下，口服或非肠道内用于重新激活BChE的肟类药物是有效的，而不必求助于现场和非无菌条件下繁琐且与风险相关的BChE强化活动？

DESCRIPTION (provided by applicant): We propose to develop a previously unexplored approach in therapy of nerve agent and pesticide OP exposure based on efficient reactivation of endogenous BChE directly in human circulation and tissues. This approach is largely based on accelerated OP degradation of OPs directly in tissues where OPs enter and/or accumulate first during an exposure. Oxime assisted OP hydrolysis by BChE naturally present in lungs, intestine and plasma, thus provides a barrier to subsequent distribution of OPs to target sites in skeletal muscle and CNS. We recently demonstrated as a proof of principle, the functioning of oxime assisted, BChE based, catalytic OP bioscavenger in vitro, ex vivo and in vivo (Radic et al., Biochem J 450, 2013,231-242). We demonstrated that TAB2OH, a newly developed oxime reactivator of human BChE had sufficient capacity to rapidly reactivate inactive, covalent OP- BChE conjugates in vitro, and enhance both protection and therapy of OP exposed mice when administered in combination with purified BChE. We now propose to utilize endogenous human BChE naturally present in lungs, intestine and plasma at nM concentrations instead of administering purified BChE, and to administer only highly efficient BChE reactivator to assist endogenous BChE in OP degradation. We assume that this will be possible since we recently developed a series of tertiary and quaternary imidazole based hBChE reactivators that are several fold more efficient in vitro than TAB2OH. The tertiary amines have the additional potential for blood-brain barrier and alveolar membrane permeation and oral bioavailability. We propose to investigate in vivo efficacy of new BChE reactivators in OP exposed mice and further refine their structure to improve their reactivation properties. This R21 proposed research, through accumulating in vitro kinetic parameters of reactivation and turnover of hBChE in the presence of an OP and taking the optimal compounds to an in vivo analysis in mouse, should uncover the potential of endogenous BChE reactivation in antidotal therapy. It should also lead to ascertaining the most efficacious dosage regimen and to assessing the limits of capitalizing on enhancing the potential of endogenous BChE through oxime-assisted catalysis. In short, is there an OP exposure level where oral or parenteral oximes directed to reactivating BChE are efficacious without having to resort to the cumbersome and risk- associated BChE fortification of activity in field and non-sterile conditions?