Vasodilating Nitroxide for Therapy of Limb Ischemia-Perfusion Injury

描述（由申请人提供）：我们正在开发一种新的细胞保护性小分子，用于预防肢体缺血再灌注损伤（LIRI）。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，但在过去二十年中，治疗策略并没有进步，液体复苏、溶栓和抗凝治疗仍然是主要的治疗方法。LIRI是由自由基合成的急性改变产生的，其中a)由于精氨酸酶1介导的L-精氨酸的消耗，一氧化氮（NO）被严重耗尽，内皮NO合成酶（ecNOS）的底物，b)超氧阴离子上升到毒性水平，由解偶联的ecNOS和黄嘌呤和NAHDP氧化酶的过度激活产生。这两种自由基的失衡直接损害肌肉，通过产生过氧亚硝酸盐及其对聚（adp -核糖）聚合酶（PARP）的激活，间接损害肌肉，通过诱导血管收缩（从而进一步缺血）、血小板粘附和聚集、中性粒细胞粘附和脱粒而影响微循环。NO的补充和超氧化物的清除都是部分有效的，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，由一种释放NO的有机硝基血管扩张剂和一种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类氮氧化物的共价键形成。综上所述，这些功能允许R-100去除有毒活性氧并输送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在大鼠和绵羊高血压模型中是一种有效的血管扩张剂；2)在小鼠循环休克模型中的深层组织保护剂。目的：建立R-100在实验性小鼠LIRI模型中预防组织损伤的剂量依赖性作用。与Michael Watkins教授（哈佛医学院）合作，我们将在小鼠LIRI模型中进行随机、单盲、安慰剂对照的R-100研究。麻醉后的C57BL6小鼠单侧肢体缺血1.5 h，再灌注24 h。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）治疗组进行比较。再灌注24 h后，将小鼠安乐死，取肢，后肢后肌置于10%多聚甲醛中进行组织学检查或在N2液中快速冷冻进行生化分析。我们预计R-100会剂量依赖性地减少组织损伤，这反映在脂质过氧化、中性粒细胞浸润、横纹肌溶解、聚腺苷核糖（adp -核糖）形成和腓肠肌ATP浓度的减少上。这些治疗效果有望转化为肢体再灌注损伤的临床终点：1)增加肢体活力，2)减少横纹肌溶解，3)缩短住院时间，4)降低全因30天死亡率。

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.