Research Project
8249357
DESCRIPTION (provided by applicant): Multiorgan injury and failure due to septic complications result in significant morbidity and mortality, especially in pediatric and elderly patients with severe bacterial infections. Pathogenesis of endotoxic shock/sepsis has not been understood in great detail, moreover its therapy, including antibiotics, remains largely symptomatic and supportive. The endogenous proteins released in circulation in response to endotoxin (lipopolysaccharide, LPS) have been largely considered as a sign of damage, and rarely as an attempt of the living system to counteract and combat the disease. The proteins of acute phase response such as C-reactive protein and TNF-a, have been frequently used as diagnostics of systemic inflammation and septic shock. Others, including ALT, NGAL, and troponin are indicative of more specific organ damage such as liver, kidney, and heart, respectively. The particular role for liver in acute phase protein synthesis has been known for long time. Yet, specific diagnostic markers of liver injury, including induced by endotoxin, remains elusive. In the course of biomarker study for liver injury in response to hepatotoxins including LPS, we found that an enzyme of urea cycle, argininosuccinate synthase (ASS), is a highly sensitive marker and is released in large amounts in blood within 30 minutes after treatment with LPS/D-galactosamine and, to a lesser extent, LPS alone. Given that combination of LPS and D-gal produces significant damage to the liver, the release of ASS, an outer mitochondrial membrane/cytosolic enzyme, can be considered an early sign of hepatic injury, preceding even the increase of ALT or AST in blood. The development and validation of a highly sensitive diagnostic ASS SW ELISA in plasma/serum has been completed in our laboratory. During ASS biomarker studies, we discovered an intriguing phenomenon, the ability of recombinant ASS (rASS) to nearly abolish the damage to mouse macrophages in culture induced by high doses of endotoxin, even when added 1 hour after LPS challenge, and reduce TNF-a release (Prima et al., 2010). Another study reported that ASS actually physically binds LPS. In pilot experiments, we were able to demonstrate that injection of rASS significantly decreased mice mortality treated with high doses of LPS (50 % vs. 100 % at 32 h post-injection). We hypothesize that ASS is a natural component of the endotoxin neutralization system that acts in concert with other antioxidant systems to protect from endotoxin/septic insults. Ultimate Goal of the project is to develop a novel, specific therapy for sepsis/endotoxemia based on the ability of hepatic argininosuccinate synthase (ASS) to ameliorate injurious effects of bacterial endotoxins and aberrant immune response, and mitigate multiple organ injury in human patients. In phase I, following specific aims will be accomplished: Specific Aim 1: Develop, purify and characterize a therapeutic-grade, endotoxin-free recombinant human ASS protein. (1-4 months of the project). Specific Aim 2. Characterize anti-bacterial and anti-inflammatory activity of rASS formulations in vitro. (2-12 month of the project) Specific Aim 3: Examine rASS treatment efficacy in models of sepsis and endotoxic shock in mice and establish the most efficient protocol (8-24 months of the project) Deliverable from phase I: Treatment protocol for endotoxemia/sepsis in mice by rASS. Phase I Milestone: Validation of rASS formulation for treatment of endotoxemia/sepsis in mouse models. Therapeutic grade, LPS-free rASS should be safe for subsequent human clinical trials since it represents an endogenous liver-specific protein. PUBLIC HEALTH RELEVANCE: Multiorgan injury and failure due to septic complications remains a significant cause of morbidity and mortality. We propose to develop a novel, specific therapy for endotoxemia/sepsis based on the ability of recombinant hepatic argininosuccinate synthase (rASS) to ameliorate injurious effects of bacterial endotoxins and aberrant immune response, and mitigate multiple organ injury. In phase I, a therapeutic grade, tag-and endotoxin-free engineered human recombinant ASS will be produced and its efficiency to treat endotoxemia/sepsis in mouse models will be established. Outcome will be a validated rASS formulation and protocol for treatment of endotoxemia/sepsis in mouse models.
