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

描述(申请人提供)：有机磷(OP)神经毒剂已被部署为大规模杀伤性武器在战争领域和最近的恐怖袭击中；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。经典的OP神经毒剂，如沙林、梭曼、塔布恩和VX，以及相关的OP杀虫剂，代表了人类已知的一些最有毒的物质。它们的毒性主要是由于它们能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AChE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，并使毒扁豆碱和尼古丁受体超敏。这种过载的兴奋性有毒化学物质通过胆碱能毒素使受害者丧失行为能力，导致癫痫发作、瘫痪、粘液分泌、眼睛刺激、心肺功能抑制、胃肠道痛苦，最终导致死亡。此外，OP暴露会引发导致中枢神经系统迟发性神经变性的机制的后遗症，包括其他受体和转运体的兴奋毒性增加，以及神经轴突的进行性脱髓鞘和降解。接触OP毒剂的案件，无论是故意的(战争或恐怖主义)还是意外的(杀虫剂处理不当)，每年的数量超过200万； 全世界每年有数十万受害者死亡。目前有效的OP中毒的治疗方法包括同时给予三种不同的药物(通过自动注射器)：(1)抗胆碱能药物，(2)抗惊厥药物，和(3)含吡啶肟的AChE复活剂。虽然抗胆碱能和抗惊厥药物可以阻断过量的乙酰胆碱的作用，但基于肟基的复活剂在裂解磷酸化的丝氨酸酯化位点以释放活性AChE分子方面起着关键作用。然而，目前使用的试剂由于其带电性质而具有较差的ADME性能。因此，当前OP对策中的重大缺陷包括：(1)需要同时进行多种治疗方式的复杂给药方案，(2)目前使用的吡啶肟类药物对中枢神经系统的渗透性较差，以及(3)目前任何一种治疗方法在对抗长期暴露于神经毒剂后表现出的长期神经退行性后遗症方面都完全缺乏疗效。现已证实，聚腺苷二磷酸核糖聚合酶-1(PARP-1)的抑制剂是一种有用的神经保护剂，尤其是在兴奋性毒性损伤引起的神经退行性变时。PARP-1的抑制剂已经在临床前和临床上被研究用于心脏缺血、失血性休克综合征、中风、创伤性中枢神经系统损伤、糖尿病、炎症和癌症。PARP-1抑制剂的原型苯甲酰胺已被证明对梭曼诱导的大鼠癫痫相关脑损伤具有神经保护作用。最近有人建议，OP诱导的长期神经病变固有的多途径复杂性应该用类似的多方面治疗方法来对抗。因此，设计具有双药物载体的小分子同时调节两个不同的分子靶点是药物发现中的一个重要策略。与鸡尾酒或由多组分制剂配制的单一药丸相比，拥有一种可以同时调节多个靶点的单一试剂有一个明显且已被证实的优势。由重叠的药效团设计的双作用单药将具有更好的配基效率(例如，降低的Mw，LogP)，并将提供一维代谢、药代动力学和安全性特征，而不是联合使用两种药物，因为可能的药物-药物相互作用和不同的动力学特征导致开发度量复杂。该项目的目标是发现新的和机械上独特的双重作用剂，这将代表着对抗有机磷化学威胁的突破性创新对策。因此，该应用程序的第一个创新方面是发现一个 新型PARP-1抑制剂，也具有AChE复活剂的双重活性；通过将两个已知的药效团合并到同一分子中，提供多种药理作用和优越的整体药理特征来治疗OP中毒。一种具有PARP-1抑制和AChE再激活活性的单一药物将代表一种治疗OP中毒的创新和新的方法，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反应，以及(2)携带肟的片段将挽救OP结合的失活AChE，从而降低兴奋性乙酰胆碱水平。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性的双重制剂，使它们能够通过血脑屏障，这是目前AChE复活剂所缺乏的理想特征。两个体外优化的新型双活性PARP-1抑制剂/AChE复活剂的成功鉴定将为体内PK评估和啮齿动物OP中毒和神经退行性变模型的POC研究提供关键的工具化合物。这将为最终的线索优化活动奠定基础，重点是在SBIR第二阶段持续拨款期间确定具有良好平衡的体内PK参数的临床前线索。

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.