Vasodilating Nitroxide for Therapy of Limb Ischemia-Perfusion Injury

血管舒张一氧化氮治疗肢体缺血灌注损伤

• 批准号: 8118720
• 负责人: PRAKASH G JAGTAP
• 金额: $ 22.52万
• 依托单位: RADIKAL THERAPEUTICS, INC.
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-05-01 至 2013-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8118720
• 关键词: Acute; Address; Adhesions; Adverse event; American; Amputation; Animal Model; Anticoagulant therapy; Arginine; Biochemical; Biological; Biological Assay; Blinded; Blood Platelets; Blood flow; Canis familiaris; Cardiovascular system; Catheters; Chemicals; Chromosome abnormality; Chronic; Clinical; Consumption; Control Groups; Data; Diffusion; Dose; Drug Delivery Systems; Drug Kinetics; Emergency Situation; Enzymes; Excision; Experimental Models; Fiber; Free Radical Alteration; Free Radicals; Freezing; Gastrocnemius Muscle; Harvest; Histologic; Hospitalization; Hydrogen Peroxide; Hypertension; Individual; Injury; Investigation; Ischemia; Length of Stay; Limb structure; Lipid Peroxidation; Liquid substance; Lower Extremity; Mediating; Medical; Microcirculation; Micronucleus Tests; Mitochondria; Modeling; Morbidity - disease rate; Mus; Muscle; Muscle Fibers; NADPH Oxidase; Neutrophil Activation; Neutrophil Infiltration; Nitric Oxide; Oxidants; Oxidases; Oxidation-Reduction; Pathway interactions; Patients; Perfusion; Peroxidases; Peroxonitrite; Pharmacologic Substance; Pharmacology; Phase; Placebo Control; Poly Adenosine Diphosphate Ribose; Poly(ADP-ribose) Polymerases; Prevention; Production; Prophylactic treatment; Protective Agents; Proteins; Protocols documentation; Pyrrolidines; Randomized; Rattus; Reaction; Reactive Oxygen Species; Reperfusion Injury; Reperfusion Therapy; Reporting; Resuscitation; Rhabdomyolysis; Rodent; Safety; Shock; Small Business Innovation Research Grant; Stress; Superoxide Dismutase; Superoxides; Survivors; Testing; Therapeutic; Therapeutic Intervention; Tissues; Toxicogenetics; Toxicokinetics; Toxicology; Translating; United States National Institutes of Health; Vasodilator Agents; Xanthine Oxidase; Xanthines; arginase; attenuation; base; catalase; catalyst; chemokine; clinically relevant; healthy volunteer; hemodynamics; human NOS3 protein; inflammatory marker; innovation; keratinocyte; macrophage inflammatory protein 2; man; medical schools; mimetics; mortality; neurobehavioral; neutrophil; novel; older patient; paraform; prevent; professor; prophylactic; pyrrolidine; respiratory; response; restoration; small molecule; tetrahydrobiopterin; thrombolysis; treatment effect; vasoconstriction

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.

描述（由申请人提供）：我们正在开发一种新型的细胞保护小分子，以预防肢体缺血 - 再灌注损伤（LIRI）。急性下肢缺血仍然是一个重大问题，死亡率范围从老年患者的9-42％和幸存者的截肢率为20％。尽管发病率如此明显，但在过去的二十年中，治疗策略尚未提高，因为液体复苏，溶栓和抗凝治疗的给药仍是治疗中的主体。 LIRI是通过自由基合成的急性改变而产生的，其中a）一氧化物（NO）由于精氨酸酶-1介导的l-精氨酸的消耗而深刻耗尽，这是内皮NO合成酶（ecnos）的底物的底物，以及二级质量的脱氧于毒性，并由无氧化物的毒性升高，并添加了次数的超氧剂量，并添加了无氧元素的作用。 NAHDP氧化酶。这两个自由基物种的不平衡直接通过过氧亚硝酸盐的产生及其激活聚（ADP-核糖）聚合酶（PARP）的激活，而间接地通过对微循环（通过血管收缩（以及进一步的缺血），缺血性粘合和集体粘合和中性和中性化的影响而对微循环产生影响。 NO的补充和去除超氧化物都是部分有效的，但是通过同时纠正这两种异常，预计肌肉损伤会得到更大的保护。为了满足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，它是由有机硝基脱位剂的共价连接形成的，该硝基脱位剂释放了NO，以及一种吡咯烷硝基氧化物，可作为超氧化物歧化酶模拟酶模拟酶模仿，催化氧化酶模仿和氧化氧化物的decalosite decompositiite decalosite decalosite decalostiite decalosity decalosits decalosts catlyst catlyst catlyst catlyst catlyst catlyst catalyst catalyst catalyst catalyst。结合起来，这些功能使R-100可以去除有毒的活性氧，而没有产生过氧亚硝酸盐的混杂作用。初步数据表明，R-100是：1）大鼠和卵形高血压模型中有效的血管扩张剂； 2）在循环休克的鼠模型中，一种深刻的组织保护剂。第1阶段的特定目的：在LIRI的实验鼠模型中，建立R-100剂量依赖性的功效。与迈克尔·沃特金斯（Michael Watkins）教授（哈佛医学院）一起，我们将在LIRI的鼠模型中对R-100进行随机，单盲，安慰剂对照的调查。麻醉的C57BL6小鼠将受到单侧肢体缺血的1.5小时，然后进行24小时再灌注。在再灌注发作前10分钟，将将假损伤组与R-100（0、20、40和80 mg/kg/day）的治疗进行比较。再灌注24小时后，将对小鼠安乐死，肢体收获，后线肌肉将其放在10％多聚甲醛中进行组织学检查或液体N2中的Snap-Frozen进行生物化学分析。我们预计R-100将剂量依赖性地减少组织损伤，这反映在脂质过氧化，中性粒细胞浸润，横纹肌溶解，聚（ADP-核糖）形成和胃肠道ATP浓度中。这些治疗效果预计将转化为肢体再灌注损伤中的临床终点：1）肢体生存力增加，2）横纹肌溶解减少，3）较短的住院和4）降低全因30天死亡率。 公共卫生相关性：流向肢体的血液流失是需要立即恢复灌注的医疗紧急情况。重新打开血管的疗法可能会矛盾地诱导进一步的组织损伤，称为“再灌注损伤”。没有批准的疗法来预防或治疗这种情况。我们正在开发一种针对再灌注损伤基本机制的新型药物，并旨在作为接受血运重建的患者的预防剂，以恢复流向肢体的血液流动。我们将在急性肢体缺血的临床上的小动物模型中测试该药物。