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

描述（由申请人提供）：我们建议开发一种先前未探索的治疗神经毒剂和农药OP暴露的方法，该方法基于内源性BChE直接在人体循环和组织中的有效再活化。这种方法主要基于OP直接在组织中加速降解，其中OP在暴露期间首先进入和/或积累。肟通过天然存在于肺、肠和血浆中的BChE辅助OP水解，从而为OP随后分布到骨骼肌和CNS中的靶位点提供屏障。作为原理的证明，我们最近证明了肟辅助的、基于BChE的催化OP生物清除剂在体外、离体和体内的功能（Radic等人，Biochem J 450，2013，231 -242）。我们证明，TAB 2 OH（一种新开发的人BChE的肟重激活剂）具有足够的能力在体外快速重激活无活性的共价OP-BChE缀合物，并在与纯化的BChE联合给药时增强对OP暴露小鼠的保护和治疗。我们现在建议利用内源性人BChE天然存在于肺，肠和血浆中的nM浓度，而不是给予纯化的BChE，并仅给予高效的BChE再活化剂，以帮助内源性BChE在OP降解。我们认为这是可能的，因为我们最近开发了一系列基于叔和季咪唑的hBChE再活化剂，其在体外比TAB 2 OH有效数倍。叔胺具有额外的血脑屏障和肺泡膜渗透和口服生物利用度的潜力。我们建议研究新的BChE再激活剂在OP暴露小鼠中的体内功效，并进一步改进其结构以改善其再激活特性。这项R21提出的研究，通过积累在体外动力学参数的再激活和营业额的hBChE在OP的存在下，并采取最佳的化合物在小鼠体内分析，应该揭示的潜力内源性BChE再激活的解毒治疗。它还应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潜力的限制。简而言之，是否存在OP暴露水平，在该水平下，口服或胃肠外肟可有效地重新激活BChE，而无需在田间和非无菌条件下进行繁琐且风险相关的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?