Multi-targeted Countermeasures against Acute and Delayed Effects of OP Exposure

描述（由申请人提供）：有机磷酸盐（OP）神经毒剂已被部署为战争领域和最近恐怖袭击中的大规模杀伤性武器;对军事人员，平民，急救人员和医疗保健提供者构成重大风险。典型的OP神经毒剂，如沙林、梭曼、塔崩和VX，沿着相关的OP杀虫剂代表了人类已知的一些毒性最大的物质。它们的毒性主要是由于它们能够不可逆地与中枢神经系统中的乙酰胆碱酯酶（AChE）结合， 血这一过程使乙酰胆碱酯酶失活，使其无法在突触中处理乙酰胆碱;导致乙酰胆碱的兴奋毒性水平和毒蕈碱和烟碱受体的超敏反应。这种兴奋性毒性化学物质的过载通过胆碱能毒蛋白使受害者丧失能力，导致癫痫发作、瘫痪、粘液分泌、眼睛刺激、心肺抑制、胃肠道不适，最终死亡。此外，OP暴露引发导致CNS中延迟神经变性的机制的后遗症;包括其他受体和转运蛋白的兴奋性毒性增强以及神经轴突的进行性脱髓鞘和降解。接触OP制剂的案例，无论是故意（战争或恐怖主义）还是意外（农药处理不当），每年超过200万; 全世界每年有数十万人死亡。目前可用的OP中毒对策包括同时给予（通过自动注射器）三种不同的药物：（1）抗胆碱能药物，（2）抗惊厥药物，和（3）含吡啶肟的AChE再激活剂。虽然抗胆碱能和抗惊厥药物的工作，以阻止过量的乙酰胆碱的影响，肟基的再活化剂发挥关键作用的切割磷酸化丝氨酸酯位点释放活性AChE分子。然而，目前使用的试剂由于其带电性质而具有差的ADME性质。因此，当前OP对策中的显著缺陷包括：（1）需要使用复杂的给药方案同时进行多治疗模式治疗，（2）当前使用的吡啶肟的CNS渗透性差，以及（3）当前治疗中的任何一种完全缺乏对抗长期暴露于神经毒剂后表现出的长期神经变性后遗症的功效。已经确定聚（ADP-核糖）聚合酶-1（PARP-1）的抑制剂是有用的神经保护剂，特别是当神经变性由兴奋性毒性损伤引起时。PARP-1的抑制剂已经在临床前和临床上用于心脏缺血、出血性休克综合征、中风、创伤性CNS损伤、糖尿病、炎症和癌症的研究。原型PARP-1抑制剂苯甲酰胺已被证明对梭曼诱导的大鼠脑损伤具有神经保护作用。最近有人提出，OP诱导的长期神经病变疾病的固有多途径复杂性应该用类似的多方面治疗方法来应对。因此，设计具有双药效团的小分子以同时调节两个离散的分子靶标是药物发现中的重要策略。与用多组分药剂配制的鸡尾酒或单个丸剂相比，具有可以同时调节多个靶点的单个药剂具有明显且经证实的优点。通过重叠药效团设计的双重作用单一药剂将具有更好的配体效率（例如，降低的MW、LogP），并且将提供一维代谢、药代动力学和安全性特征，这与共施用两种药物相反，其中由于可能的药物-药物相互作用和差异动力学特征，开发指标复杂。这是该项目的目的是发现新的和机械独特的双重作用剂，这将代表一个突破性的创新对策对OP化学威胁。因此，本申请的第一个创新方面是发现了一种新的方法。 新型PARP-1抑制剂，其还具有作为乙酰胆碱酯酶再活化剂的双重活性;通过在同一分子中合并两种已知的药效团，以提供多种药理学作用和治疗OP中毒的上级总体药理学特征。具有双重PARP-1抑制和AChE再活化活性的单一药物将代表OP中毒的创新和新型治疗，因为：（1）PARP-1抑制活性将通过保护线粒体功能和减少氧化应激和能量危机来减弱自由基形成的兴奋性毒性级联，和（2）带有肟的片段将挽救OP结合的失活AChE，从而降低兴奋毒性乙酰胆碱水平。我们方法的第二个创新方面是设计和合成具有合适药物特征的双重药物，使它们能够穿过血脑屏障，这是目前AChE再激活剂缺乏的理想特征。在该I期活动中成功鉴定了两种体外优化系列的新型双重活性PARP-1抑制剂/AChE再活化剂，这将为OP中毒和神经变性啮齿动物模型的体内PK评估和POC研究提供关键工具化合物。这将为最终的电极导线优化活动奠定基础，重点是在SBIR II期继续资助期间确定具有良好平衡的体内PK参数的临床前电极导线。

DESCRIPTION (provided by applicant): Organophosphate (OP) nerve agents have been deployed as weapons of mass destruction in warfare arenas and in recent terrorist attacks; posing significant risks to military personnel, civilians, first responders and healthcare provider. The classic OP nerve agents such as sarin, soman, tabun, and VX, along with related OP pesticides represent some of the most toxic materials known to humankind. Their toxicity is principally due to their ability to irreversibly bind to acetylcholinesterase (AChE) in the CNS and blood. This process deactivates AChE and renders it unable to process acetylcholine in the synapse; resulting in excitotoxic levels of acetylcholine and hypersensitization of muscarinic and nicotinic receptors. This overload of excitotoxic chemicals incapacitates the victim via cholinergic toxidrome, resulting in seizures, paralysis, mucous secretions, eye irritation, cardiorespiratory depression, GI distress, and eventually, death. Additionally, OP exposure initiates a sequelae of mechanisms leading to delayed neurodegeneration in the CNS; including heightened excitotoxicity at other receptors and transporters and progressive demyelination and degradation of nerve axons. Cases of exposure to OP agents, whether intentional (warfare or terrorism) or accidental (pesticide mishandling), number in excess of 2 million per year; resulting in the deaths of several hundred thousand victims per year worldwide. Currently available countermeasures for OP poisoning involve the simultaneous administration (via autoinjectors) of three different agents: (1) an anticholinergic agent, (2) an anticonvulsant agent, and (3) a pyridinium oxime-bearing AChE reactivator. While the anticholinergic and anticonvulsant agents work to block the effects of excess acetylcholine, the oxime-based reactivator plays the key role of cleaving the phosphorylated serine esteratic site to release the active AChE molecule. However, currently used agents possess poor ADME properties due to their charged nature. Thus, significant deficiencies in current OP countermeasures include: (1) the requirement for simultaneous multi-therapeutic modality treatments with complicated dosing regiments, (2) poor CNS-permeability of currently used pyridinium oximes, and (3) the complete lack of efficacy in any of the current treatments to counteract the long-term neurodegenerative sequelae that manifests after prolonged exposure to nerve agents. It has been established that inhibitors of poly(ADP-ribose)polymerase-1 (PARP-1) are useful neuroprotective agents especially when the neurodegeneration is caused by excitotoxic insult. Inhibitors of PARP-1 have been investigated pre-clinically and clinically for cardiac ischemia, hemorrhagic shock syndrome, stroke, traumatic CNS injury, diabetes, inflammation, and cancer. The prototype PARP-1 inhibitor, benzamide, has been shown to be neuroprotective against soman-induced seizure-related brain damage in the rat. It has been recently suggested that the inherent multiple-pathway complexity of OP-induced long-term neuropathy disorders should be countered with a similarly multi-faceted treatment approach. Hence, the design of small molecules possessing dual- pharmacophores to simultaneously modulate two discreet molecular targets is an important strategy in drug discovery. There is a clear and proven advantage to having a single agent that can modulate multiple targets simultaneously as compared to either a cocktail or a single pill formulated with multi-component agents. A dual acting single agent designed by overlapping pharmacophores would have better ligand efficiency (e.g. reduced MW, LogP) and would provide one dimensional metabolism, pharmacokinetics, and safety profiles as opposed to co-administering two drugs where development metrics are complicated due to possible drug-drug interactions and differential kinetic profiles. It is the aim of this project to discover novel and mechanisticaly unique dual- acting agents which would represent a breakthrough innovative countermeasure against OP chemical threats. Thus, the first innovative aspect of this application is to discover a novel PARP-1 inhibitor that also possesses dual activity as an AChE-reactivator; via merging two known pharmacophores in the same molecule, to provide multiple pharmacological effects and a superior overall pharmacological profile to treat OP-poisoning. A single agent with dual PARP-1 inhibition and AChE-reactivating activity would represent an innovative and novel treatment for OP poisoning because: (1) the PARP-1 inhibition activity would attenuate the excitotoxic cascade of free-radical formation by preserving mitochondrial function and reducing oxidative stress and energetic crisis, and (2) the oxime-bearing fragment would salvage the OP-bound inactivated AChE and thereby reduce excitotoxic acetylcholine levels. The second innovative aspect of our approach is the design and synthesis of dual agents with suitable pharmaceutical profiles, such that they will be able to cross the blood brain barrier, a desired feature that is lacking from current AChE reactivators. The successful identification of two in vitro optimized series of novel, dual-active PARP-1 inhibitor/AChE reactivators in this Phase 1 campaign would provide critical tool compounds for in vivo PK assessment and POC studies in rodent models of OP-intoxication and neurodegeneration. This would set the stage for a final lead optimization campaign focused on identifying pre- clinical leads with well-balanced in vivo PK parameters during an SBIR Phase 2 continuing grant.