SBIR-STTR Award

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.

Thesaurus Terms:
Acute-Phase Proteins;Acute-Phase Reaction;Acute-Phase State;Affinity;After Care;After-Treatment;Aftercare;Animals;Anti-Bacterial Agents;Anti-Inflammatories;Anti-Inflammatory Agents;Anti-Inflammatory;Antibacterial Agents;Antibiotic Agents;Antibiotic Drugs;Antibiotics;Antiinflammatories;Antiinflammatory Agents;Antioxidants;Apoptosis;Apoptosis Pathway;Argininosuccinate Synthetase;Assay;B Cell Differentiation Factor;B Cell Stimulating Factor 2;B-Cell Differentiation Factor-2;B-Cell Stimulatory Factor-2;Bcdf;Bsf-2;Bsf2;Bsf2 (B Cell Stimulating Factor 2);Bacillus Subtilis;Bacterial Infections;Binding;Binding (Molecular Function);Bioassay;Biologic Assays;Biological Assay;Blood;Blood Circulation;Blood Plasma;Blood Serum;Bloodstream;C Reactive Protein;C-Reactive Protein;Ccl2;Ccl2 Gene;Ccl5;Csif;Csif-10;Cell Culture Techniques;Cell Death, Programmed;Cell Line;Cell Lines, Strains;Cellline;Chemokine (C-C Motif) Ligand 5;Childhood;Chromatography, High Performance Liquid;Chromatography, High Pressure Liquid;Chromatography, High Speed Liquid;Circulation;Clinical Trials;Clinical Trials, Unspecified;Closure By Ligation;Cytokine Synthesis Inhibitory Factor;Cytokine Formation-Inhibiting Factor (Mouse Clone F115 Protein Moiety Reduced);Cytokines, Chemotactic;D-Galactose, 2-Amino-2-Deoxy-;D-Glucose, 4-O-Alpha-D-Glucopyranosyl-;D17s136e;Dif;Development;Diagnostic;Differentiation Factor, B-Cell;Disease;Disorder;Dose;Drug Formulations;E Coli;Elisa;Endotoxemia;Endotoxic Shock;Endotoxins;Ensure;Enterokinase;Enzyme-Linked Immunosorbent Assay;Enzymes;Escherichia Coli;Flr;Failure (Biologic Function);Formulation;Formulations, Drug;Gdcf-2;Gdcf-2 Hc11;Gm-Csf;Gmcsf;Galactosamine;Gamma Interferon;Generalized Growth;Goals;Granulocyte-Macrophage Colony-Stimulating Factor;Growth;Hc11;Hpgf;Hplc;Heart;Hepatic;Hepatocyte-Stimulating Factor;High Pressure Liquid Chromatography;Histamine-Producing Cell-Stimulating Factor;Homologous Chemotactic Cytokines;Hour;Human;Human Engineering;Human, General;Hybridoma Growth Factor;Ifn-Gamma;Ifn-Beta 2;Ifn-G;Ifnb2;Ifng;Il-1;Il-10;Il-6;Il1;Il10;Il10a;Il6 Protein;Inflm;Immune Response;In Vitro;Inflammation;Injection Of Therapeutic Agent;Injections;Injury;Intercrines;Interferon Gamma;Interferon Type Ii;Interferon Gamma (Human Lymphocyte Protein Moiety Reduced);Interferon, Immune;Interferon-Gamma;Interleukin 10 Precursor;Interleukin 6 (Interferon, Beta 2);Interleukin I;Interleukin-1;Interleukin-10;Interleukin-6;Kidney;L-Citrulline[{..}]l-Aspartate Ligase (Amp-Forming);Lps;Laboratories;Ligation;Lipopolysaccharides;Liver;Lymphocyte-Stimulating Hormone;Lytotoxicity;Mcaf;Mcp-1;Mcp1;Mgc17164;Mgc9434;Mgi-2;Macrophage Cell Factor;Maltose;Mammals, Mice;Man (Taxonomy);Man, Modern;Mass Spectrum;Mass Spectrum Analysis;Mice;Miscellaneous Antibiotic;Modeling;Molecular Interaction;Molgramostin;Monitor;Morbidity;Morbidity - Disease Rate;Mortality;Mortality Vital Statistics;Murine;Mus;Myeloid Differentiation-Inducing Protein;N-Terminal;Nh2-Terminal;Organ;Organism;Outcome;Outer Mitochondrial Membrane;Pathogenesis;Patients;Peptide Biosynthesis, Ribosomal;Phase;Photometry/Spectrum Analysis, Mass;Plasma;Plasmacytoma Growth Factor;Preparation;Production;Protein Biosynthesis;Protein Biosynthesis, Ribosomal;Protein Synthesis, Ribosomal;Proteins;Proteins, Specific Or Class, C-Reactive;Protocol;Protocols Documentation;Protocols, Treatment;Puncture Procedure;Punctures;Rantes;Rantes Protein, T-Cell;Rgm;Reactants, Acute-Phase;Recombinants;Regimen;Reporting;Residual;Residual State;Response, Acute-Phase;Reticuloendothelial System, Blood;Reticuloendothelial System, Serum, Plasma;Role;S. Aureus;S.Aureus;Scya2;Scya5;Sds Page;Sds Polyacrylamide Gel Electrophoresis;Sis Cytokines;Sis Delta;Sis-Delta;Sisd;Smc-Cf;Sepsis;Septic Shock;Serum;Serum, Plasma;Small Inducible Cytokine A5;Sodium Dodecyl Sulfate-Page;Sodium Dodecylsulfate-Polyacrylamide Gel Electrophoresis;Spectrometry, Mass;Spectroscopy, Mass;Spectrum Analyses, Mass;Spectrum Analysis, Mass;Staphylococcus Aureus;System;System, Loinc Axis 4;T Helper Factor;T-Cell Specific Protein P288;Tc-Gm-Csf;Tcp228;Tnf;Tnf A;Tnf Gene;Tnfsf2;Testing;Therapeutic;Time;Tissue Growth;Treatment Efficacy;Treatment Protocols;Treatment Regimen;Treatment Schedule;Troponin;Tumor Necrosis Factor Gene;Tumor-Cell Human Gm Colony-Stimulating Factor;Urinary System, Kidney;Validation;Acute Phase Protein;Anti-Bacterial;Anti-Oxidant;Antibacterial;Argininosuccinate Synthase;Arginosuccinate Synthase;Bacterial Disease;Base;Biomarker;Bloodstream Infection;Body System, Hepatic;Chemoattractant Cytokine;Chemokine;Clinical Investigation;Combat;Cultured Cell Line;Cytokine;Cytotoxicity;Disease /Disorder;Disease/Disorder;Elderly Patient;Enteropeptidase;Enzyme Linked Immunosorbent Assay;Experiment;Experimental Research;Experimental Study;Failure;Gene Product;Granulocyte Macrophage Colony Stimulating Factor;Hepatotoxicant;Hepatotoxin;High Performance Liquid Chromatography;Host Response;Immunoresponse;In Vivo;Interferon Beta 2;Interleukin 1;Interleukin 10;Interleukin 6;Lfn-Gamma;Liver-Specific Protein;Living System;Lymphocyte Activating Factor;Macrophage;Mass Spectrometry;Model;Mouse Model;Multiorgan Injury;Novel;Older Patient;Ontogeny;Organ System, Hepatic;Pediatric;Protein Synthesis;Renal;Research Study;Response;Sephadex;Septic;Social Role;Therapeutic Efficacy;Therapeutically Effective;Urea Cycle;Vector

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.