Vasodilating Nitroxide for Therapy of Limb Ischemia-Perfusion Injury

描述(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严重的问题，老年患者的死亡率为9%-42%，幸存者的截肢率为20%。尽管有如此高的发病率和死亡率，但治疗策略在过去20年中并未取得进展，液体复苏、溶栓和抗凝治疗仍然是治疗的主要手段。LIRI是由于自由基合成的急剧变化而产生的，其中a)由于精氨酸酶-1介导的内皮型一氧化氮合酶(EcNOS)底物L精氨酸的消耗，一氧化氮(NO)被严重消耗，以及b)超氧阴离子上升到毒性水平，由ecNOS解偶联和黄嘌呤和NAHDP氧化酶的过度激活产生。这两种自由基物种的失衡直接损害肌肉，通过产生过氧亚硝酸盐并激活多(ADP-核糖)聚合酶(PARP)，以及通过诱导血管收缩(从而进一步缺血)、血小板黏附和聚集、中性粒细胞黏附和脱颗粒而间接影响微循环。补充NO和清除超氧化物都是部分有效的，但同时纠正这两种异常有望对肌肉损伤产生更大的保护作用。为了满足这一未得到满足的临床需求，Radikal Treeutics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的吡咯烷氮氧化物共价连接而成。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NO，而不会产生过氧亚硝酸根的混杂影响。初步数据显示，R-100是：1)在大鼠和绵羊高血压模型中有效的血管扩张剂；以及2)在小鼠循环休克模型中的深层组织保护剂。第一阶段的特定目标：在实验性LIRI小鼠模型中，建立R-100预防组织损伤的剂量依赖效应。我们将与哈佛医学院的Michael Watkins教授合作，在LIRI的小鼠模型中进行一项随机、单盲、安慰剂对照的R-100研究。麻醉C57BL6小鼠单侧肢体缺血1.5h，再灌注24h。假损伤组与R-100(0、20、40和80 mg/kg/d)治疗组在再灌流开始前10分钟进行比较。再灌注24 h后，处死小鼠，取肢体，将后肢肌肉置于10%多聚甲醛溶液中进行组织学检查或液氮冷冻后进行生化分析。我们预计R-100将剂量依赖性地减少组织损伤，这反映在减少脂质过氧化、中性粒细胞渗透、横纹肌溶解、多(ADP-核糖)形成和腓肠肌ATP浓度。这些治疗效果有望转化为肢体再灌注损伤的临床终点：1)增加肢体存活率，2)减少横纹肌溶解，3)缩短住院时间，4)降低全原因30天死亡率。 公共卫生相关性：肢体血流丧失是一种医疗紧急情况，需要立即恢复血流灌注。重新开放被阻塞的血管的疗法可能会矛盾地导致进一步的组织损伤，即所谓的“再灌流损伤”。目前还没有得到批准的治疗方法来预防或治疗这种情况。我们正在开发一种新药，针对再灌注损伤的基本机制，并打算在接受血管重建术的患者中作为预防性药物，以恢复肢体的血液流动。我们将在一个临床相关的急性肢体缺血的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.