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开发的治疗神经毒剂和农药暴露的方法，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暴露过程中OPs首先进入和/或积累的组织中OPs的加速降解。肟有助于OP被天然存在于肺、肠和血浆中的BChE水解，从而为OPs随后分布到骨骼肌和中枢神经系统的靶点提供了屏障。我们最近证明了肟辅助的、BChE为基础的催化OP生物清除剂在体内、体外和体外的作用原理（Radic等人，生物化学杂志450,2013,231-242）。我们证明了TAB2OH是一种新开发的人BChE肟再激活剂，它有足够的能力在体外快速激活无活性的共价OP- BChE偶联物，并且当与纯化的BChE联合使用时，可以增强OP暴露小鼠的保护和治疗作用。我们现在建议利用天然存在于肺、肠和血浆中的内源性BChE，以nM浓度代替纯化的BChE，并且只使用高效的BChE再激活剂来帮助内源性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叔咪唑和季咪唑的hBChE再激活剂，其体外效率比TAB2OH高几倍。叔胺具有血脑屏障和肺泡膜渗透和口服生物利用度的额外潜力。我们建议研究新型BChE再激活剂在OP暴露小鼠体内的活性，并进一步完善其结构以提高其再激活性能。本R21提出的研究，通过积累在OP存在下hBChE再激活和转化的体外动力学参数，并将最佳化合物在小鼠体内分析，揭示内源性BChE再激活在解毒剂治疗中的潜力。它还应导致确定最有效的给药方案，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，是否存在一个OP暴露水平，在该暴露水平下，用于重新激活BChE的口服或肠外注射肟是有效的，而不必在野外和非无菌条件下诉诸繁琐且风险相关的BChE活性强化？

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?