SBIR-STTR Award

Liver injury induced by hepatic ischemia followed by reperfusion is a major pathogenic cause of liver failure during multiple trauma, thermal injury, hemorrhagic and septic shock, and liver transplantation, which contributes significantly to multiple organ failure and mortality. Current enzymatic assays of serum ALT or LDH are either non-specific or not sensitive enough to detect early stages of liver damage, assess magnitude of parenchymal cell death, or to predict hepatic recovery. Based on liver proteomic degradomics approach, we are developing a panel of novel biomarkers for liver ischemic injury. The most promising biomarker candidate discovered to date is an enzyme of urea/nitric oxide cycle argininosuccinate synthase (ASS). The goals of this proposal are to design, produce, and validate a sandwich (SW) ELISA Kit for quantitative determination of ASS in biological fluids for diagnostics of ischemic liver injury. Specific Aim 1: Design and develop sandwich ELISA for the quantitative detection of argininosuccinate synthase (ASS). Deliverable from Specific Aim 1: Configuration of a prototype ELISA kit for the quantitative detection of ASS in biological fluids. We will develop a SW ELISA, optimize it for sensitive detection and quantification of ASS, including in serum or plasma, and produce this ELISA Kit for preliminary validation in Specific Aim 2. Specific Aim 2: Preliminary validation of the diagnostic utility of the ASS sandwich ELISA in a rat model of liver I/R injury. The objective of this Specific Aim is to examine the utility of the prototype sandwich ELISA for assessment of the magnitude of hepatic ischemic injury and outcome by measuring ASS levels in blood. Deliverable from Specific Aim 2: Diagnostic ELISA Kit for measuring ASS levels in serum for non-invasive diagnostics and monitoring liver I/R injury. The overall result of these efforts will be generation of a validated sandwich ELISA Kit for detection of ASS, and preliminary validation of its utility in experimental ischemia/reperfusion liver injury rat model. Further validation of this ELISA Kit as a reliable diagnostic tool for human ischemic liver Injury will be performed during Phase II SBIR in patients with abdominal and multiple trauma, hemorrhagic and septic shock, and liver transplantation. Liver failure due to various forms of hepatic injury is a significant source of overall mortality. Etiologies of liver injury includes multiple and abdominal trauma from gunshot and blast injuries, drug and alcohol toxicity, hemorrhagic and septic shock, and graft failure after liver transplantation. Enzymatic assays of liver enzymes have traditionally been used as markers of liver injury. However, these diagnostic tests are either not sensitive or non-specific, do not allow for assessment of the magnitude of liver injury, nor do they predict outcome. Thus, there is an explicit clinical need for better non-invasive biomarkers of ischemic liver injury, which can lead to early diagnosis and better treatment. Immunoassay technologies, such as the enzyme-linked immunosorbent assay (ELISA) can be employed to develop a simple, rapid, noninvasive and reliable diagnostic test for liver I/R injury. Amongst other uses, it will be valuable for military medical or emergency responses to national disasters, sepsis and multiple organ failure. In addition, it may provide more information on biochemical and molecular mechanisms contributing to liver injury, recovery of function and/or potential targets for novel therapeutic strategies. We have developed a specific and sensitive biomarker for I/R-induced liver injury that appears, from initial experiments, to be correlated with the severity of damage and may be sensitive to therapeutic intervention. Moreover, this biomarker provides direct assessment of pathobiological mechanisms involved in regulation of nitric oxide synthesis and the urea cycle in the liver that may represent a potential link between liver I/R injury, liver failure, and metabolic encephalopathy during sepsis. Namely, argininosuccinate synthase (ASS) is a crucial liver enzyme. ASS is rapidly up-regulated (within an hour) during ischemia and is released into the circulation. The overall goal of this proposal is to develop a prototype ELISA kit for the quantitative determination of ASS as novel diagnostic biomarker in biological fluids including serum or plasma.

Thesaurus Terms:
biomarker, carbon nitrogen ligase, diagnosis design /evaluation, diagnostic test, enzyme linked immunosorbent assay, injury, liver disorder, liver disorder diagnosis, liver ischemia /hypoxia, reperfusion blood chemistry, early diagnosis, prognosis, protein degradation, protein quantitation /detection, proteomics, serology /serodiagnosis laboratory rat, male

Liver damage and failure due to various forms of hepatic injury is a significant source of overall morbidity and mortality in the US and worldwide. Persistent hepatic injury occurs during viral hepatitis, fatty liver disease (steatohepatitis), drug or alcohol and autoimmune induced hepatitis. Clinical conditions are numerous where hepatic injury is a vital component of multi-organ failure caused by complex trauma including blast injury, septic shock, and graft failure after liver transplantation often leading to death of the patient. Enzymatic assays of serum ALT and AST, widely used as part of a `hepatic function' panel, are neither sensitive nor particularity specific to the liver and do not assess the magnitude of liver injury or predict outcome. If liver damage is imminent, tissue biopsies are routinely used for diagnosis. This indicates an explicit clinical need for non-invasive biomarkers for early detection of liver injury with enhanced diagnostic information. We propose here the continuation of the development of novel liver specific biomarkers which are easily detected in serum and can be used for early diagnosis of patients with liver injury and aid in monitoring of potential liver toxicity in patients during drug treatment. Our biomarkers can also be used in preclinical studies of hepatotoxicity. We identified several novel liver-specific biomarkers in rat models of hepatic injury with the argininosuccinate synthase (ASS) and hepatic estrogen sulfotransferases (EST-1) being the most sensitive markers which rapidly accumulated in blood and correlated with the severity of damage. In Phase I, a prototype SW ELISA for quantifying ASS in serum was developed, and sufficient data were obtained demonstrating the greater biomarker sensitivity and specificity in experimental models of liver injury compared to ALT/AST. Also, we provided the proof-of-concept data indicating the diagnostic and prognostic value of serum ASS in human patients with several types of liver diseases and hepatic injury. In Phase II, a ready-to-use ELISAs Kits for ASS and EST-1 will be developed and validated in preclinical studies in rat models of liver injury and hepatotoxicity. Sufficient supplies of the assay components (e.g. antibodies and antigens) will be produced under quality assured conditions. We will characterize the diagnostic potential of ASS and EST-1 assays in human patients with various types of liver diseases and hepatic injury. The biomarker levels will be that will be correlated with clinical data including liver biopsy, serological and laboratory data with an emphasis on hepatic injury severity, disease progression and outcome. The accuracy, sensitivity, specificity and diagnostic/prognostic values of ASS/EST-1 will be assessed and compared with ALT/AST. These analyses will be used to support a pre-IDE application to the FDA for validation of these assays as diagnostic tools and further commercialization. The deliverables of this Phase II project will be quality assured ELISA assays for two liver injury biomarkers, and clinical data supporting the use of the biomarkers as tools for aid in the diagnosis of liver injury and monitor the magnitude of liver toxicity far beyond information obtained from ALT/AST measurements.

Public Health Relevance:
Liver health is a major component of human wellbeing. Many environmental chemicals, pathogens, drugs or excessive alcohol can harm the liver and affect its function. An estimated 5.5 million Americans have chronic liver diseases, including viral hepatitis, alcoholic liver disease or hepatic cancer, which frequently lead to liver failure and requires liver transplantation. Moreover, increasing numbers of prescription drugs such as cholesterol-lowering drugs and over-the-counter medications, including acetaminophen, can potentially cause liver toxicity and, thereby, require medical liver function checks. For the last 30 years, two enzymes, serum ALT and AST have been used in hepatic function panels to assess liver health. However, ALT/AST are neither sensitive nor specific to detect mild injure, assess magnitude of damage or predict outcome. Banyan Biomarkers is developing predictive bioassays such as ASS and EST-1 that can help determine liver insults early on in patients that suffer from liver toxicity from therapeutic drugs or environmental toxins. These assays will provide a novel diagnostic/prognostic tools and serve as "safety biomarkers." according to the FDA.

Public Health Relevance:


Project Terms:
1,3-Benzodioxole-5-ethanamine, N,alpha-dimethyl-; 3,4 methylenedioxymethamphetamine; APAP; ATGN; Abdomen; Abdominal; Acetamide, N-(4-hydroxyphenyl)-; Acetamidophenol; Acetaminophen; Acetominophen; Active Follow-up; Acute; Affect; Alcohol toxicity; Alcoholic Liver Diseases; Alcohols; American; Aminotransferases; Analysis, Data; Animal Experiments; Animals; Antibodies; Antigens; Argininosuccinate Synthetase; Armed Forces Personnel; Assay; Autoimmune; Autoimmune Hepatitis; Autoimmune Process; Biliary; Bioassay; Biologic Assays; Biological; Biological Assay; Biopsy; Blast Injuries; Blood; Blood Circulation; Blood Plasma; Blood Serum; Bloodstream; Blotting, Western; Body Tissues; Bywaters' syndrome; Carbon Tetrachloride; Cause of Death; Cessation of life; Chemical Class, Alcohol; Cholest-5-en-3-ol (3beta)-; Cholesterol; Chronic; Circulation; Clinical; Clinical Data; Clinical Research; Clinical Study; Collection; Common Rat Strains; Complex; Crush syndrome; Data; Data Analyses; Death; Detection; Development; Development and Research; Diagnosis; Diagnostic; Disease; Disease Progression; Disorder; Drug Prescribing; Drug Prescriptions; Drug or Chemical; Drugs; Drugs, Non-Prescription; Drugs, Nonprescription; EC 2.6.1; ELISA; ESTs; Early Diagnosis; Ecstasy; Ecstasy - drug; Emergencies; Emergency Situation; Ensure; Environmental Toxin; Enzyme-Linked Immunosorbent Assay; Enzymes; Estrone sulfotransferase; Ethanol toxicity; Evaluation; Experimental Models; Experimental Models, Other; Expressed Sequence Tags; FLR; Failure (biologic function); Fatty Liver; Goals; Graft Rejection; Grafting, Liver; Guidelines; HAMA; HCV infection; Half-Life; Half-Lifes; Health; Hepatic; Hepatic Cancer; Hepatic Disorder; Hepatic Failure; Hepatitis; Hepatitis C; Hepatitis C virus infection; Hepatitis, Viral, Non-A, Non-B, Parenterally-Transmitted; Hepatobiliary; Hepatocellular Damage; Hepatotoxic effect; Hepatotoxicity; Human; Human Anti-Mouse Antibody; Human Resources; Human, General; Hydroxyacetanilide; Injury; Ischemia-Reperfusion Injury; L-Citrulline[{..}]L-aspartate ligase (AMP-forming); Laboratories; Lead; Link; Liquid substance; Liver; Liver Failure; Liver Steatosis; Liver Toxicity; Liver Transplant; Liver diseases; MDMA; MOF syndrome; Malignant neoplasm of liver; Mammals, Rabbits; Mammals, Rats; Man (Taxonomy); Man, Modern; Manpower; Measurement; Medical; Medication; Methane, tetrachloro-; Methylenedioxymethamphetamine; Military; Military Personnel; Modeling; Models, Experimental; Molecular; Monitor; Morbidity; Morbidity - disease rate; Mortality; Mortality Vital Statistics; Multiple Organ Failure; N-(4-Hydroxyphenyl)acetanilide; N-Acetyl-p-aminophenol; N-Methyl-3,4-methylenedioxyamphetamine; NAFLD; NANBH; NASH; Non-Prescription Drugs; Non-alcoholic steatohepatitis; OTC Drugs; Organ Dysfunction Syndrome, Multiple; Organ failure; Oryctolagus cuniculus; Outcome; Over-the-Counter Drugs; Oxidative Stress; PT-NANBH; Paracetamol; Parenterally-Transmitted Non-A, Non-B Hepatitis; Patent Medicines; Pathogenesis; Patient Monitoring; Patients; Pattern; Pb element; Peptides; Performance; Pharmaceutic Preparations; Pharmaceutical Preparations; Phase; Plasma; Play; Prescriptions, Drug; Production; Proteins; Proteomics; Quality Control; R & D; R&D; Rabbit, Domestic; Rabbits; Rat; Rattus; Reagent; Recombinant Antibody; Recombinants; Recovery; Recurrence; Recurrent; Reperfusion Damage; Reperfusion Injury; Reperfusion Therapy; Reproducibility; Resolution; Reticuloendothelial System, Blood; Reticuloendothelial System, Serum, Plasma; Role; SBIR; SBIRS (R43/44); Safety; Sampling; Sensitivity and Specificity; Sepsis; Septic Shock; Serologic; Serological; Serum; Serum, Plasma; Severities; Small Business Innovation Research; Small Business Innovation Research Grant; Source; Steatohepatitis; Surrogate Markers; Testing; Tetrachloromethane; Therapeutic; Time; Tissues; Toxic Environmental Agents; Toxic Environmental Substances; Toxic effect on liver cells; Transaminases; Transplant Rejection; Transplantation; Transplantation Rejection; Transplantation of liver; Transplantation, Hepatic; Trauma; Validation; Viral; Viral hepatitis; Western Blotting; Western Blottings; Western Immunoblotting; alcohol induced hepatic injury; alcohol induced liver disorder; alcohol induced liver injury; alcohol-induced hepatic dysfunction; alcohol-induced liver disease; alcohol-induced liver dysfunction; alcohol-mediated liver dysfunction; alcohol-mediated liver injury; argininosuccinate synthase; arginosuccinate synthase; assay development; biomarker; biopsy of liver; bloodstream infection; body system, hepatic; commercialization; design; designing; disease/disorder; drug/agent; early detection; ecstasy; environmental chemical; environmental toxicant; estrogen sulfotransferase; ethanol induced hepatic injury; ethanol induced liver disorder; ethanol induced liver injury; ethanol-induced hepatic dysfunction; ethanol-induced liver disease; ethanol-induced liver dysfunction; ethanol-mediated liver dysfunction; ethanol-mediated liver injury; failure; fluid; follow-up; gene product; graft failure; heavy metal Pb; heavy metal lead; hepatic steatosis; hepatitis non A non B; hepatitis nonA nonB; hepatopathy; hepatotoxicant; hepatotoxin; hepatoxicity; human subject; immunogen; immunogenic; injured; innovate; innovation; innovative; liquid; liver biopsy; liver cancer; liver disorder; liver function; liver ischemia; liver transplantation; malignant liver tumor; multiple organ system failure; new diagnostics; next generation diagnostics; non A non B hepatitis; non A, non B hepatitis; non-A non-B hepatitis; non-A, non-B hepatitis; non-alcohol fatty liver; non-alcohol induced steatohepatitis; non-alcoholic fatty liver; non-alcoholic steato-hepatitis; nonalcohol fatty liver; nonalcoholic fatty liver; nonalcoholic steato-hepatitis; nonalcoholic steatohepatitis; novel; novel diagnostics; organ system, hepatic; p-Acetamidophenol; p-Hydroxyacetanilide; pathogen; personnel; polyclonal antibody; pre-clinical; preclinical; preclinical study; prognostic; protein blotting; prototype; public health relevance; reperfusion; research and development; response; sample collection; social role; specimen collection; tool; transplant