Vasodilating Nitroxide for Therapy of Limb Ischemia-Perfusion Injury

描述（由申请人提供）：我们正在开发一种用于预防肢体缺血再灌注损伤（LIRI）的新型细胞保护性小分子。急性下肢缺血仍然是一个重要的问题，老年患者的死亡率为9-42%，幸存者的截肢率为20%。尽管发病率和死亡率如此之高，但在过去的二十年里，治疗策略并没有取得进展，液体复苏、溶栓和抗凝治疗仍然是治疗的主要手段。LIRI是由自由基合成的急性改变产生的，其中a）一氧化氮（NO）被深度耗尽，这是由于脱乙酰酶-1介导的L-精氨酸（内皮NO合酶（ecNOS）的底物）的消耗，和B）超氧阴离子上升到毒性水平，这是由解偶联的ecNOS和黄嘌呤和NAHDP氧化酶的超活化产生的.这两种自由基物质的不平衡通过过氧亚硝酸盐的产生及其对聚（ADP-核糖）聚合酶（PARP）的活化直接损害肌肉，并且通过诱导血管收缩（并因此进一步缺血）、血小板粘附和聚集以及中性粒细胞粘附和脱粒对微循环的影响间接损害肌肉。补充NO和清除超氧化物是部分有效的，但更大的保护肌肉损伤，预计从同时纠正这两种异常。为了解决这一未满足的临床需求，Radikal Therapeutics正在开发R-100，这是一种新型药物，由释放NO的有机硝基血管扩张剂和作为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的吡咯烷氮氧化物共价键合而成。结合起来，这些功能允许R-100去除有毒的活性氧物质并提供NO，而不会产生过氧亚硝酸盐的混淆效应。初步数据显示，R-100是：1）大鼠和绵羊高血压模型中的强效血管扩张剂; 2）循环休克小鼠模型中的深层组织保护剂。I期特定目的：在LIRI实验性小鼠模型中确定R-100预防组织损伤的剂量依赖性疗效。我们将与Michael Watkins教授（哈佛医学院）合作，在LIRI小鼠模型中对R-100进行随机、单盲、安慰剂对照研究。麻醉的C57 BL 6小鼠将经受1.5小时的单侧肢体缺血，随后是24小时的再灌注。在再灌注开始前10分钟，将假损伤组与R-100（0、20、40和80 mg/kg/天）处理组进行比较。再灌注24小时后，将小鼠安乐死，收获肢体，并将后肢后肌置于10%多聚甲醛中进行组织学检查或在液氮中速冻进行生化分析。我们预计R-100将剂量依赖性地减少组织损伤，如脂质过氧化、中性粒细胞浸润、横纹肌溶解、聚（ADP-核糖）形成和腓肠肌ATP浓度的减少所反映的。预期这些治疗效果将转化为肢体再灌注损伤的临床终点：1）肢体活力增加，2）横纹肌溶解减少，3）住院时间缩短，4）全因30天死亡率降低。 公共卫生相关性：肢体血流丧失是一种医疗紧急情况，需要立即恢复灌注。重新打开阻塞血管的治疗可能会矛盾地引起进一步的组织损伤，称为“再灌注损伤”。没有批准的治疗方法来预防或治疗这种疾病。我们正在开发一种针对再灌注损伤基本机制的新药，旨在作为接受血管重建以恢复肢体血流的患者的预防剂。我们将在临床相关的急性肢体缺血小动物模型中测试这种药物。

DESCRIPTION (provided by applicant): We are developing a novel cytoprotective small molecule for the prevention of limb ischemia-reperfusion injury (LIRI). Acute lower extremity ischemia remains a significant problem with a mortality rate ranging from 9-42% in elderly patients and a 20% amputation rate in survivors. Despite such significant morbidity and mortality, therapeutic strategies have not advanced over the past two decades, with the administration of fluid resuscitation, thrombolysis and anticoagulant therapy remaining as the mainstays of treatment. LIRI is produced by an acute alteration in the synthesis of free radicals, wherein a) nitric oxide (NO) is profoundly depleted, due to the arginase-1 mediated consumption of L- arginine, the substrate for endothelial NO synthase (ecNOS), and b) superoxide anion rises to toxic levels, produced by uncoupled ecNOS and hyperactivation of xanthine and NAHDP oxidases. The imbalance of these two free radical species damages muscle directly, via the production of peroxynitrite and its activation of poly(ADP-ribose) polymerase (PARP), and indirectly, via an effect on the microcirculation by inducing vasoconstriction (and thus further ischemia), platelet adhesion and aggregation, and neutrophil adhesion and degranulation. Replenishment of NO 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 is developing R-100, a novel agent formed from the covalent linkage of an organic nitrovasodilator that releases NO, 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 NO without the confounding effect of producing peroxynitrite. Preliminary data reveal that R-100 is: 1) a potent vasodilator in rat and ovine hypertension models; and 2) a profound tissue-protective agent in a murine model of circulatory shock. Phase 1 Specific Aim: Establish the dose-dependent efficacy of R-100 in preventing tissue injury in an experimental murine model of LIRI. In conjunction with Professor Michael Watkins (Harvard Medical School), we will carry out a randomized, single-blinded, placebo-controlled investigation of R-100 in a murine model of LIRI. Anesthetized C57BL6 mice will be subjected to 1.5 h of unilateral limb ischemia, followed by 24 h of reperfusion. A sham injury group will be compared to treatment with R-100 (0, 20, 40, and 80 mg/kg/day) 10 min before the onset of reperfusion. After 24 h of reperfusion, mice will be euthanized, limbs harvested, and the posterior hind-limb muscle placed in 10% paraformaldehyde for histologic examination or snap-frozen in liquid N2 for biochemical analysis. We expect that R-100 will dose-dependently reduce tissue injury, as reflected in reductions in lipid peroxidation, neutrophil infiltration, rhabdomyolysis, poly(ADP-ribose) formation, and gastrocnemius ATP concentration. These treatment effects are expected to translate into clinical endpoints in limb reperfusion injury of: 1) increased limb viability, 2) diminished rhabdomyolysis, 3) shorter hospitalization, and 4) reduction in all-cause 30 day mortality. PUBLIC HEALTH RELEVANCE: Loss of blood flow to a limb is a medical emergency requiring immediate restoration of perfusion. Therapies that reopen blocked vessels may paradoxically induce further tissue damage, known as "reperfusion injury". There are no approved therapies to prevent or treat this condition. We are developing a novel drug that targets the basic mechanisms of reperfusion injury and is intended as a prophylactic agent in patients undergoing revascularization to restore blood flow to the limb. We will test this agent in a clinically-relevant small animal model of acute limb ischemia.