Hybrid Katp Channel Opener and Redox Catalyst for Lung Transplantation

描述（由申请人提供）：Radikal Therapeutics（RTX）正在开发一种新型细胞保护剂（R-801），用于预防肺移植（LTX）后的缺血再灌注损伤（IRI）。在重度氧化还原损伤的啮齿动物模型中，R-801可显著减少器官功能障碍、组织梗死和实质炎症。R- 801由2个不同部分的共价融合形成，每个部分均具有已证实的组织保护特性：1）源自吡那地尔的线粒体-K +-ATP通道激活部分，和2）作为超氧化物歧化酶和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结构域。在致死性Cl 2吸入性肺损伤的小鼠模型中，Cl 2暴露后2小时给予R-801可阻断所有组织学损伤（p<0.01），使核NF-kB升高降低76%（p<0.0001），并使IkB水平恢复至超正常水平（p<10-7）。目的#1：在大鼠热缺血性肺IRI模型中确定R- 801相对于其组分功能域的优效性。将Sprague道利（SD）大鼠左肺原位缺血60分钟，再灌注4小时。在局部缺血之前，用IV R-801（0、3、10、30 mg/kg）、羟甲基炔雌醇（HMP; 30 mg/kg）、吡那地尔（30 mg/kg）或HMP和吡那地尔的组合（各30 mg/kg）处理大鼠。一只假大鼠进行开胸手术，但既不缺血也不药物治疗。预期R-801相对于用吡那地尔，HMP， 以及它们的组合，关于组织损伤和炎症。通过检查组织学评分、PMN浸润、脂质过氧化、蛋白质亚硝化、PARP-1活化、核NF-kB、细胞凋亡和氧合（PaO 2）来评估组织损伤。通过检查BAL中的蛋白质、PMN、TNF-α和MIP-1 β来评估炎症。目的#2：确定R- 801在同系大鼠原位LTX模型中的疗效。SD供体大鼠在肺切除前10分钟用R-801或溶剂对照处理。用掺有R-801或溶剂的冷Perfadex冲洗后，将供体肺冷藏12小时，然后进行LTX。在使用左供体肺进行LTX后，受体将立即接受R-801。将评估Wistar大鼠的a）湿/干重量比（W/D）（肺水肿的量度），B）移植物的氧合，c）移植物肺血管阻力，d）动态顺应性，和e）F2-异前列腺素的肺组织分析（脂质过氧化的量度）、组织学和对3-硝基酪氨酸（3-NT）和聚（ADP-核糖）的免疫组织化学反应性。将在3个时间点进行特异性分析：再灌注后1小时，IkB、核p50和有丝分裂原活化蛋白激酶的磷酸化（MAPKs - ERK、p38、JNK）;再灌注后3小时进行RT-PCR，定量肺组织TNF-â、MIP-1 â和Bcl-2的mRNA浓度;再灌注6 h后测定BALF细胞数、蛋白浓度、TNF-α、MIP-1 β、IL-6和IL-1 β。R-801治疗有望降低肺移植后的原发性移植物功能障碍和死亡率。

DESCRIPTION (provided by applicant): Radikal Therapeutics (RTX) is developing a novel cytoprotective agent (R-801) for the prevention of ischemia-reperfusion injury (IRI) following lung transplantation (LTX). In rodent models of severe redox injury, R-801 profoundly reduces organ dysfunction, tissue infarction, and parenchymal inflammation. R- 801 is formed from the covalent fusion of 2 distinct moieties, each with demonstrated tissue protective properties: 1) a mito-K+-ATP channel activating moiety derived from pinacidil, and 2) a pyrrolidine nitroxide domain that acts as superoxide dismutase and catalase mimetics and a peroxynitrite decomposition catalyst. In a murine model of lethal Cl2 inhalational lung injury, R-801 given 2 h after Cl2 exposure blocked all histologic damage (p<0.01), reduced the elevation in nuclear NF-kB by 76% (p<0.0001), and restored the level of IkB¿ to supranormal (p<10-7). Aim #1: Establish the superiority of R- 801 relative to its component functional domains in a rat model of warm-ischemic lung IRI. Left lungs of Sprague Dawley (SD) rats are rendered ischemic in situ for 60 min and reperfused for 4 h. Prior to ischemia, rats are treated with IV R-801 (0, 3, 10, 30 mg/kg), hydroxymethylproxyl (HMP; 30 mg/kg), pinacidil (30 mg/kg), or a combination of HMP and pinacidil (each 30 mg/kg). A sham rat undergoes thoracotomy but neither ischemia nor drug therapy. R-801 is expected to exhibit superior efficacy, relative to treatment with pinacidil, HMP, and their combination, with respect to tissue damage and inflammation. Tissue damage is assessed by examining histologic score, PMN infiltration, lipid peroxidation, protein nitrosation, PARP-1 activation, nuclear NF-kB, apoptosis, and oxygenation (PaO2). Inflammation is assessed by examining BAL for protein, PMNs, TNF-¿, and MIP-1¿. Aim #2: Establish the efficacy of R- 801 in a syngeneic rat model of orthotopic LTX. SD donor rats are treated with R-801 or vehicle control 10 min before lung removal. After flushing with cold Perfadex" spiked with R-801 or vehicle, donor lungs are stored cold for 12h before left LTX. Immediately following LTX with left donor lungs, recipients will receive R-801. Recipient rats will be evaluated for a) wet/dy weight ratio (W/D) (a measure of pulmonary edema), b) oxygenation by the graft, c) graft pulmonary vascular resistance, d) dynamic compliance, and e) lung tissue analysis for F2¿-isoprostane (a measure of lipid peroxidation), histology, and immunohistochemical reactivity to 3-nitrotyrosine (3-NT) and poly(ADP-ribose). Specific analyses will be carried out at 3 time points: at 1 h post reperfusion for IkB¿, nuclear p50, and phosphorylation of mitogen activated protein kinases (MAPKs - ERK, p38, JNK); at 3h post reperfusion for RT-PCR to quantify lung tissue mRNA concentrations of TNF-¿, MIP-1¿, and Bcl-2; and at 6 h post reperfusion for determination of BALF cellularity, protein concentration, TNF-¿, MIP-1¿, IL-6, and IL1-¿. R-801 therapy is expected to translate into decreased primary graft dysfunction and mortality after lung transplant.