Bifunctional Redox Catalyst & Organic Nitrate for Limb Reperfusion

描述(申请人提供)：急性肢体缺血再灌注损伤(LIRI)的特征是自由基、一氧化氮和超氧阴离子失衡。因此，补充一氧化氮和清除超氧化物都是部分有效的，但同时纠正这两种异常可以更好地保护肌肉损伤。为了满足这一未得到满足的临床需求，Radikal Treeutics(RTX)正在开发一种新型小分子制剂R-100，它由释放一氧化氮的有机硝基血管扩张剂和吡咯烷氮氧化物共价连接而成，用作超氧化物歧化酶模拟物、过氧化氢酶模拟物和过氧亚硝酸盐分解催化剂。这些功能结合在一起，使R-100能够去除有毒的活性氧物种并提供一氧化氮，而不会产生产生过氧亚硝酸盐的混杂效应。在一期SBIR中，我们在一个严重的急性LIRI小鼠模型中证明了R-100复苏可以显著减少肢体组织学损伤、脂质过氧化、中性粒细胞浸润和受损肌肉的百分比。鉴于这些病理生理机制在临床LIRI中的中心地位，我们的第一阶段SBIR数据证明了进展到第二阶段SBIR工作范围的合理性。目的#1：排名R-100及其类似物在LIRI R-100啮齿动物模型(两种对映体)中的效力将与在小鼠LIRI筛选模型中的10个类似物进行比较。对表现最好的3种药物的全剂量反应(20、40、80 mg/kg/天)进行比较，以便将表现最好的药物注释为候选分子(CM)。目的#2：在猪急性LIRI模型上建立CM的药效学(PD)谱。我们将扩大规模并生产99%纯度的CM in，以支持大型动物药效研究和后续的GLP毒理学和安全药理学研究。用血管内球囊结扎股动脉6h建立急性LIRI动物模型(每组6只)，在再灌注前10min直接动脉注射CM(0，10，30，80 mg/kg/d，TID)，然后静脉注射，建立剂量-反应关系。在再灌流后7、14和21天，我们将肢体血流灌注(通过多普勒血流)、神经传导速度(神经传导速度)、肌肉力量(通过步行表面的足底压力)与同时血浆CM浓度进行关联，以构建PD图谱。目标#3：建立静脉注射CM的急性安全性、毒性和耐受性 FDA IND申请所需的GLP毒理学和安全性药理学研究。我们将执行FDA规定的最低要求的一组启用IND的GLP研究，以支持在健康人类志愿者中进行首次人参与、剂量递增、单剂量静脉注射CM研究的申请。遗传毒理学研究将包括：Ames、染色体畸变和大鼠微核分析。安全性药理学研究将包括：1)大鼠神经行为和呼吸研究，以及2)犬心血管研究。大鼠和狗的IV剂量范围发现和2周重复给药毒理学研究，以及相关的毒代动力学测定。

DESCRIPTION (provided by applicant): Acute limb ischemia reperfusion injury (LIRI) is characterized by an imbalance in the free radicals nitric oxide and superoxide anion. Accordingly, replenishment of nitric oxide and removal of superoxide are each partially effective, but greater protection of muscle injury is expected from the simultaneous correction of both abnormalities. To address this unmet clinical need, Radikal Therapeutics (RTX) is developing R-100, a novel small molecule agent formed from the covalent linkage of an organic nitrovasodilator that releases nitric oxide, and a pyrrolidine nitroxide that acts as a superoxide dismutase mimetic, catalase mimic, and peroxynitrite decomposition catalyst. In combination, these functionalities allow R-100 to remove toxic reactive oxygen species and deliver nitric oxide without the confounding effect of producing peroxynitrite. In a Phase 1 SBIR, we demonstrated in a severe murine model of acute LIRI that R-100 resuscitation profoundly reduces limb histologic damage, lipid peroxidation, neutrophil infiltration, and % of injured muscle. Given the centrality of these pathophysiologic mechanisms to clinical LIRI, our Phase 1 SBIR data justify progression to the Phase 2 SBIR scope of work. Aim #1: Rank order the potency of R-100 and its analogues in a rodent model of LIRI R- 100 (both enantiomers) will be compared to 10 analogues in a murine LIRI screening model. The top 3 performing agents will be compared over their full dose-responses (20, 40, 80 mg/kg/day) in order to annoint the best performer as the candidate molecule (CM). Aim #2: Establish the pharmacodynamic (PD) profile of the CM in a porcine model of acute LIRI. We will scale-up and manufacture > 99% pure CM in to support large animal efficacy studies and follow-on GLP toxicology and safety pharmacology investigations. In a large animal model of acute LIRI (n=6 minipigs per group) induced by an endovascular balloon occlusion of the femoral artery for 6 h, we will establish the dose-response of parenteral CM (0, 10, 30, 80 mg/kg/day TID) initiated 10 min before reperfusion via direct arterial infusion, f0llowed by an IV infusion thereafter. At 7, 14, and 21 days after reperfusion we will correlate limb perfusion (by Doppler flow), nerve conduction velocity ("NCV", per electromyography, "EMG"), and muscle strength (by foot pressure on a walking surface) with simultaneous plasma concentrations of CM in order to construct a PD profile. Aim #3: Establish the acute safety, toxicity, and tolerance of IV CM in the minimum set of GLP toxicology and safety pharmacology studies required for FDA IND application. We will carry out the minimum set of required FDA-mandated IND-enabling GLP studies to support application for a first-in-man, dose-escalation, single-dose investigation of IV CM in healthy human volunteers. Genetic toxicology studies will include: the Ames, chromosomal aberration, and rat micronucleus assays. Safety pharmacology investigations will include: 1) rat neurobehavioral and respiratory studies, and 2) canine cardiovascular studies. IV dose range-finding and 2 week repeat-dose toxicology studies in rats and dogs, with associated toxicokinetic determinations.