Peroxynitrite Decomposition Catalyst and Nitric Oxide Donor for Endotoxemia

描述（由申请人提供）：通常认为，自由基一氧化氮（NO）和超氧阴离子生物合成的改变通过诱导微血管缺血和直接器官毒性导致脓毒症中广泛的组织损伤。为了纠正这种自由基失衡，Radikal Therapeutics（RTX）正在开发一种一流的小分子药物（R-100），这是一种基于双功能氧化还原的技术，由2个化学部分的共价键形成：1）释放NO的有机硝基血管扩张剂，2）作为活性氧降解的三功能催化剂的吡咯烷氮氧化物：超氧化物歧化酶模拟物、使过氧化氢解毒的过氧化氢酶模拟物和过氧亚硝酸盐分解催化剂。在LD 100小鼠内毒素血症模型中，脂多糖（LPS）攻击后1小时开始用R-100复苏，可产生100%的存活率，并伴有对终末器官损伤的近乎完全的保护。我们假设R-100比其两种组分功能的总和更上级，并且这两种性质共价融合成单个分子实体为脓毒症治疗创造了强大的商业前景。RTX将通过进行内毒素血症研究来检验这一假设，以确定腹膜内（IP）给予R-100的小鼠的剂量反应、时间窗、作用机制和安全性。目标一：在脂多糖（LPS）攻击后1小时，将3个剂量水平的R-100与媒介物对照进行比较，以确定提供最佳结果的最低剂量（“LDP 00”）。将测量R-100和心脏、肺、肾脏和肝脏代谢物的组织水平，以关联血浆和器官药物摄取。目标二：将R-100的LDPOO剂量与等摩尔剂量的羟甲基异丙基（“HMP”，R-100的氮氧组分）、单硝酸异山梨酯（“ISMN”，典型的单官能NO供体）以及HMP和ISMN的组合进行比较，以验证双官能化合物（R-100）上级其组分官能度的混合物。在LPS激发后1小时开始处理。目标#3：我们将通过在LPS激发后1、2、4和8 h引入R-100来确定治疗时间窗的持续时间。在上述每个目的中，将在LPS给药后48小时检查血清和组织，以确定形态学和生物化学终点，包括脂质过氧化（F21-异前列烷、GSH：GSSG比率）、中性粒细胞浸润、3-硝基酪氨酸（3-NT）、亚硝酸盐/硝酸盐和聚（ADP-核糖）形成、BUN、肌酐、NGAL、AST、ALT、组织学损伤评分和TNF-1、HMGB-1、IL-6、TRP 14、Trx 1、LC 8、I：B1、IL-12、MIP-11、CXCL 9和CXCL 10的血清浓度。目标4：在4小时内，我们将监测LPS激发后1小时给予溶剂对照或R-100的麻醉内毒素血症小鼠的外周动脉血压和心率，以确认治疗方案的血流动力学中性。我们预期R- 100将上级于HMP和ISMN，单独和组合，并且当在LPS激发后6小时开始时将是有效的，从而验证其作为临床脓毒症的潜在治疗剂的效用。 公共卫生相关性：急性肠穿孔和感染引起的脓毒性休克是死亡的主要原因。目前，没有批准的治疗这种情况和预后都很差。我们正在开发一种针对脓毒性休克基本机制的新药，并已在脓毒性炎症实验模型中证明有效。我们现在将在一系列研究中测试这种药物，以确定最佳剂量，确认作用机制，并在疾病发作后建立可能开始治疗的机会窗口。

DESCRIPTION (provided by applicant): Alterations in the biosynthesis of the free radicals nitric oxide (NO) and superoxide anion are generally accepted to contribute to widespread tissue injury in sepsis via their induction of microvacular ischemia and direct organ toxicity. To correct this free radical imbalance, Radikal Therapeutics (RTX) is developing a first- in-class small molecule drug (R-100), a bifunctional redox-based technology formed from the covalent linkage of 2 chemical moieties: 1) an organic nitrovasodilator that releases NO, and 2) a pyrrolidine nitroxide that acts as a trifunctional catalyst of reactive oxygen species degradation: a superoxide dismutase mimetic, a catalase mimic that detoxifies hydrogen peroxide, and a peroxynitrite decomposition catalyst. In an LD100 murine model of endotoxinemia, resuscitation by R-100 starting 1 h after lipopolysaccharide (LPS) challenge produces 100% survival, accompanied by near complete protection against end-organ injury. We hypothesize that R-100 is superior to the sum of its two component functionalities and that the covalent fusion of these two properties into a single molecular entity creates a strong commercial prospect for therapy of sepsis. RTX will test this hypothesis by carrying out endotoxemic studies to establish the dose-response, time-window, mechanism of action, and safety in mice of intraperitoneal (IP) administered R-100. Aim #1: R-100 at 3 dose levels will be compared to vehicle control 1 h post lipopolysaccharide (LPS) challenge, in order to establish the lowest dose providing optimal outcome ("LDPOO"). Tissue levels of R-100 and metabolites from heart, lung, kidney, and liver will be measured, in order to relate plasma and organ drug uptake. Aim #2: the LDPOO dose of R-100 will be compared to equimolar doses of hydroxymethylproxyl ("HMP", the nitroxide component of R-100), isososorbide mononitrate ("ISMN", a classic monofunctional NO donor), and the combination of HMP and ISMN, in order to verify that a bifunctional compound (R-100) is superior to a mixture of its component functionalities. Treatment will be initiated 1 h after LPS challenge. Aim #3: We will determine the duration of the therapeutic time window by introducing R-100 1, 2, 4, and 8 h after LPS challenge. Serum and tissue will be examined 48 h post LPS dosing in each of the above Aims for determination of morphologic and biochemical endpoints, including lipid peroxidation (F21-isoprostane, GSH:GSSG ratio), neutrophil infiltration, 3- nitrotyrosine (3-NT), nitrite/nitrate, and poly(ADP-ribose) formation, BUN, creatinine, NGAL, AST, ALT, histology injury score, and serum concentrations of TNF-1, HMGB-1, IL-6, TRP14, Trx1, LC8, I:B1, IL-12, MIP-11, CXCL9, and CXCL10. Aim #4: Over a 4 h period, we will monitor peripheral arterial blood pressure and heart rate in anesthetized endotoxinemic mice treated with vehicle control or R-100 administered 1 h after LPS challenge, in order to confirm the hemodynamic neutrality of the treatment regimen. We expect that R- 100 will be superior to HMP and ISMN, alone and in combination, and will be effective when initiated 6 h after LPS challenge, thereby verifying its utility as a potential therapeutic for clinical sepsis. PUBLIC HEALTH RELEVANCE: Septic shock resulting from acute bowel perforation and infection is a major cause of mortality. At present, there is no approved therapy for this condition and prognosis is uniformly poor. We are developing a novel drug that targets the basic mechanisms of septic shock, and has proven effective in experimental models of septic inflammation. We will now test this agent in a series of investigations in order to determine the optimal dose, to confirm the mechanism of action, and to establish the window of opportunity after disease onset within which therapy may be initiated.