Novel Therapeutic Approach for Sepsis: Blocking the Vascular Response to Cytokine

描述（由申请人提供）：仅在美国，脓毒症影响约700，000例患者，每年导致超过210，000人死亡。脓毒症是由宿主和感染因子之间的复杂相互作用引起的临床综合征，其特征在于炎症和凝血级联反应的全身激活。血流动力学变化、广泛的微循环障碍和细胞改变导致血流和代谢需求之间的解偶联，与多器官功能障碍的发展有关，导致大多数死亡。Navigen认为，它有一种独特的方法来防止血液动力学变化和微循环障碍-血管泄漏-与败血症相关。我们的科学联合创始人Dean Li博士发现了一种新的受体Robo 4，它在成熟血管中表达，并在内皮损伤后上调。Robo 4在被其蛋白质配体Slit 2激活时，可在体外和体内减少血管渗漏。我们的工作进一步说明了Robo 4受体的激活干扰了来自多种渗透性因子（包括TNF-α、白细胞介素和凝血酶）的下游信号级联。我们已经克隆了Slit 2的活性片段Slit 2N，其似乎与Slit 2具有相同的功效，并且比天然蛋白更容易产生。Navigen已证明Slit 2N可有效降低盲肠结扎和穿刺诱导的脓毒症动物模型的死亡率。由于与蛋白质治疗相关的挑战，Li博士试图确定一种小分子，它可以以类似于Slit 2N的方式影响Robo 4通路。Li博士及其合作者确定，Robo 4激活的关键下游步骤是抑制小的GT3，ADP核糖基化因子6（ARF 6）。Li博士及其同事进一步确定，可以使用小分子抑制剂NAV 838通过抑制其ARF-GEF，cytohesin 2/ARNO来阻断ARF 6。Li博士的实验室与Navigen合作，已经表明通过阻断其ARF-GEF或通过Slit 2N抑制Robo 4的激活来抑制ARF 6，产生了类似的表型：抑制VEGF诱导的内皮细胞迁移。Navigen正在寻求该申请的资金，以确认Slit 2N在治疗脓毒症方面的功效，并确定作为一种小分子，NAV 838是否可能是比Slit 2N更可行的治疗脓毒症的方法。Navigen预期将上级化合物推进至IND，以最终商业化。本研究旨在实现3个目标：1）开发由假性肺炎（PAP）诱导的脓毒症的动物模型，其中滴定抗生素治疗剂量和时间以实现约50%的7天死亡率，2）在CLP和PAP模型中测试Slit 2N与抗生素治疗组合的功效并鉴定最有效剂量（目的1中建立的PAP），3）通过在CLP和PAP的动物模型中测试，确定NAV 838与抗生素的组合是否可以具有比Slit 2N加抗生素更大的功效。 公共卫生相关性：脓毒症是由宿主和感染因子之间复杂的相互作用引起的临床综合征，其特征在于包括细胞因子网络和凝血在内的多种炎症途径的全身性激活。与严重脓毒症相关的死亡率在30%至50%之间，脓毒症是手术患者和创伤受害者发病和死亡的主要原因。目前，没有有效的治疗败血症的疗法。

DESCRIPTION (provided by applicant): In the United States alone, sepsis affects approximately 700,000 patients and leads to death of over 210,000 people per year. Sepsis is a clinical syndrome that results from a complex interaction between host and infectious agents, and is characterized by systemic activation of inflammatory and coagulation cascades. Hemodynamic changes, widespread microcirculatory disturbances and cellular alterations, leading to an uncoupling between blood flow and metabolic requirements, are implicated in the development of multiple organ dysfunction, responsible for most of deaths. Navigen believes that it has a unique means of preventing the hemodynamic changes and microcirculatory disturbances - the vascular leak - associated with sepsis. Our scientific co-founder, Dr. Dean Li, identified a novel receptor, Robo4, that is expressed in mature vessels and is upregulated following endothelial injury. Robo4, when activated by its protein ligand, Slit2, reduces vascular leak in vitro and in vivo. Our work has further illustrated that activation of the Robo4 receptor interferes with the downstream signaling cascades from multiple permeability factors including TNF-alpha, interleukins, and thrombin. We have cloned an active fragment of Slit2, Slit2N, which appears to have identical efficacy to Slit2, and is more easily produced than the native protein. Navigen has demonstrated that Slit2N is effective in reducing mortality in the animal model of sepsis induced by cecal ligation and puncture. Due to the challenges associated with protein therapeutics, Dr. Li sought to identify a small molecule that could affect the Robo4 pathway in a manner similar to Slit2N. Dr. Li and collaborators determined that a critical downstream step in Robo4 activation is the inhibition of a small GTPase, ADP ribosylation factor 6 (ARF6). Dr. Li and colleagues further determined that ARF6 can be blocked through inhibition of its ARF-GEF, cytohesin2/ARNO, using a small molecule inhibitor, NAV838. Dr. Li's laboratory, in collaboration with Navigen, has shown that inhibition of ARF6, either by blocking its ARF-GEF or through the inhibitory activation of Robo4 by Slit2N, produced similar phenotypes: inhibition of VEGF-induced migration in endothelial cells. Navigen is seeking funding under this application to confirm the efficacy of Slit2N in the treatment of sepsis and to determine whether NAV838, as a small molecule, may be an even more viable approach to treating sepsis than Slit2N. Navigen would anticipate advancing the superior compound forward to IND for eventual commercialization. The proposed research is intended to accomplish 3 goals: 1) develop animal models of sepsis induced by pseudomonas pneumonia (PAP) in which antibiotic therapy dose and timing are titrated to achieve seven-day mortality of approximately 50%, 2) test efficacy and indentify most efficacious dose of Slit2N combined with antibiotic therapy in models of CLP and PAP (PAP established in Aim 1), 3) determine whether NAV838 combined with antibiotics may have greater efficacy than Slit2N plus antibiotics by testing in animal models of CLP and PAP. PUBLIC HEALTH RELEVANCE: Sepsis is a clinical syndrome that results from a complex interaction between host and infectious agents, and is characterized by systemic activation of multiple inflammatory pathways, including cytokine network and coagulation. Mortality associated with severe sepsis ranges between 30 to 50 percent and sepsis is the leading cause of morbidity and mortality in surgical patients and trauma victims. Currently, there are no effective therapies for the treatment of Sepsis.