Novel Therapeutic Approach to Acute Lung Injury

DESCRIPTION (provided by applicant): In our Phase 1 application, we described an endothelial specific receptor, Robo4, that is expressed in mature vessels and is upregulated following endothelial injury. Robo4, following activation by its protein ligand, Slit2, reduces agonist induced vascular leak in vitro and in vivo. We provided evidence of Slit2's efficacy in animal models of acute lung injury and described the limitations of Slit2 as an actual therapy due to challenges in protein purification and its inherent instability. The overall objective stated in our Phase 1 application was to develop a new class of biologic agent to block the noncardiac pulmonary edema of ALI/ARDs. Our specific aims were to identify a more stable, more easily purified fragment of Slit2 and to test this fragment in in vitro and in vivo models of acute lung injury. We accomplished these aims with a fragment we will refer to as SlitD1-D2Fc and now propose to advance its development by completing the steps necessary to transition from laboratory to larger-scale production and advancing animal testing for safety, dosing, and efficacy. Our specific aims are: Aim 1: Establish a stable cell line and develop a master cell bank for larger-scale production of recombinant Slit2D1-D2Fc; Aim 2: Convert pilot purification methods to process appropriate for larger scale production of Slit2D1-D2Fc; and Aim 3: Conduct initial pharmacokinetics and safety testing for Slit2D1-D2Fc in rodents and optimize dosing regimens in murine models of endotoxin-induced ALI and systemic sepsis, a condition that often presents with acute lung injury/acute respiratory distress. The primary milestone for our Phase 1 project was the identification of a stable fragment of Slit2 that would lend itself more easily to purification. This has been accomplished with Slit2D1-D2Fc. Furthermore, we advanced our understanding of the mode of action of Robo4 receptor antagonists and our confidence in their efficacy in multiple models of acute lung injury and sepsis, including mortality reduction in animals infected with the H5N1 virus. These studies suggest that activation of Robo4 may provide a common platform for treating multiple infectious agents ranging from endotoxin exposure and gram-negative sepsis to pandemic influenza. Our objective now is to advance the development of SlitD1-D2Fc to the point that it can be transitioned to an outside manufacturer, prove its safety, confirm our hypotheses regarding dosing, and illustrate efficacy in treating both ALI/ARDS and sepsis, conditions that often present hand in hand. Simply put, our objective for this Phase 2 project is to advance SlitD1-D2Fc to lay the foundation for an IND application. PUBLIC HEALTH RELEVANCE: Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) result from a common pathogenic process: pulmonary injury and infection trigger an inflammatory response that results in the release of growth factors and cytokines that increase endothelial and epithelial permeability, permit efflux of inflammatory cells, protein and water from the vascular system into the alveolar space, and suffocate the patient. Slit2D1-D2Fc prevents the increase in endothelial and epithelial permeability and halts the course of events leading to death.