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

描述（由申请人提供）：仅在美国，败血症每年影响约70万患者，导致超过21万人死亡。脓毒症是一种临床综合征，由宿主和感染因子之间复杂的相互作用引起，其特征是炎症和凝血级联反应的全身激活。血液动力学改变、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不耦合，与多器官功能障碍的发展有关，是大多数死亡的原因。Navigen相信它有一种独特的方法来预防与败血症相关的血液动力学改变和微循环紊乱——血管泄漏。我们的科学联合创始人Dean Li博士发现了一种新的受体Robo4，它在成熟血管中表达，并在内皮损伤后上调。当Robo4被其蛋白质配体Slit2激活时，在体外和体内都能减少血管泄漏。我们的工作进一步表明，Robo4受体的激活干扰了多种渗透因子（包括tnf - α、白细胞介素和凝血酶）的下游信号级联。我们克隆了一个Slit2的活性片段Slit2N，它似乎与Slit2具有相同的功效，并且比天然蛋白更容易产生。Navigen研究表明，在盲肠结扎穿刺脓毒症动物模型中，Slit2N可有效降低死亡率。由于与蛋白质治疗相关的挑战，李博士试图确定一种可以以类似于Slit2N的方式影响Robo4途径的小分子。李博士及其合作者确定，Robo4激活的关键下游步骤是抑制小GTPase， ADP核糖基化因子6 （ARF6）。Li博士及其同事进一步确定，使用小分子抑制剂NAV838，可以通过抑制ARF-GEF， cytohesin2/ARNO来阻断ARF6。李博士的实验室与Navigen合作，已经证明通过阻断ARF6的ARF-GEF或通过Slit2N抑制Robo4激活来抑制ARF6，产生类似的表型：抑制vegf诱导的内皮细胞迁移。Navigen正在申请资金，以确认Slit2N治疗败血症的疗效，并确定NAV838作为一种小分子，是否可能是比Slit2N更可行的治疗败血症的方法。Navigen预计将把这种优越的化合物提交给IND，以最终实现商业化。拟议的研究旨在实现三个目标：1)建立肺炎假单胞菌（PAP）脓毒症动物模型，滴定抗生素治疗剂量和时间，使7天死亡率达到约50%；2)在CLP和PAP模型中测试疗效并确定Slit2N联合抗生素治疗的最有效剂量（PAP在Aim 1中建立）；3)在CLP和PAP动物模型中测试NAV838联合抗生素是否比Slit2N加抗生素更有效。

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.