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WEDNESDAY, MAY 14

8:30 Chairperson’s Remarks 
William B. Mattes, Ph.D., DABT, Director of Toxicology, The Critical Path Institute

8:40 Integration of Pre-Clinical Studies into the Hepatic Safety Assessment Consortium
William B. Mattes, Ph.D., DABT, Director of Toxicology, The Critical Path Institute
Drug-induced liver injury and failure remains one of the most critical problems in pharmaceutical development. There are well known accounts of those marketed drugs that produce a low and seemingly unpredictable incidence of liver failure. Many drugs in early clinical development elicit increases in common measures of hepatic injury; however, these increases are often transient and the implication for serious liver injury unclear. Neither of these situations is well modeled in preclinical safety testing. On the other hand, preclinical safety testing can identify microscopic pathological changes induced by drug treatment, and screen out those compounds that are frank hepatotoxins. The link connecting all these types of studies is the measurement of various molecules in serum: transaminases, bilirubin, cholesterol and triglycerides. These measurements have proven to have real limitations, with both false positive and false negative signals. Thus a key advance in hepatic safety assessment would be the development of new biomarkers, measurable in all species and offering greater sensitivity and specificity in monitoring liver function and injury. 

9:10 New Pre-Clinical Screens to Detect Drug-Induced Mitochondrial Dysfunction Yield a New Model of Idiosyncratic Organ Toxicity
James Dykens, Ph.D., Senior Principal Scientist, Drug Safety R&D, Pfizer Inc.
Many adverse drug responses are idiosyncratic, related neither to dose nor known pharmacology. Such idiosyncratic drug reactions (IDRs) have been attributed to bio-transformations that yield potentially toxic metabolites, and in many cases, a host of reactive free radicals. Models of IDRs are increasingly sophisticated, but ultimately each has limited predictability. A more parsimonious explanation of many IDRs is that the drug or its metabolites have previously-undetected, off-target, deleterious activities, such as impairment of mitochondrial function. Such mitochondrial toxicity is not revealed by available pre-clinical screens, but we have developed a series of assays to detect it. These high-throughput in vitro assays reveal that many, but not all, members of important drug classes, directly and acutely undermine mitochondrial function. Moreover, the magnitude of such impairment is in accord with the clinical disposition. Loss of mitochondrial function is tolerated until a threshold is achieved where the cell lacks sufficient bioenergetic capacity to survive, resulting in tissue toxicity. In this light, the proximate determinants of many IDRs are arguably off-target effects on mitochondria, coupled with previous organ history and genetics, both of which define the individual’s bioenergetic threshold.

9:40 Prediction of Drug-Induced Liver Injury with Special Emphasis on Pro-Inflammatory Responses
Harri Jarvelainen Principal Scientist; DVM, Ph.D., Assoc Prof, Global Safety Assessment, AstraZeneca R&D Montréal
The concordance of hepatotoxicity of pharmaceuticals in humans and in animals is one of the lowest in toxicology – humans are known to be more sensitive to hepatotoxic effects of pharmaceuticals and also environmental toxicants. It has become increasingly evident that inflammation and the innate immunity system heavily influence the outcome in Drug-Induced Liver Injury (DILI). Resident liver macrophages, Kupffer cells, participate in the injury process through the release of several proinflammatory cytokines such as TNF-α, IL-1ß, TGF-ß and IL-6. In fact, it seems that inhibition of inflammatory responses can completely prevent the development of injury, regardless of the type of experimental DILI model used. Importantly, human immunity system is innately very sensitive to the activation of proinflammatory responses, releasing large amounts of cytotoxic cytokines upon activation. On the other hand, the target cells – hepatocytes – are rendered highly vulnerable to inflammatory insults by a plethora of very common co-existing conditions in humans, such as fatty liver and diabetes. It seems that species used in preclinical toxicology studies, especially rodents, are particularly poor models of liver inflammation: both the mediator cells (Kupffer cells) and the target cells (hepatocytes) seem to be more resistant to activator or stress signals in animals. Thus, an increased understanding on underlying inflammatory mechanisms opens new possibilities to better predict and monitor DILI preclinically and clinically.

10:10 Networking Coffee Break in the Exhibit Hall 

10:55 Improvements in In vitro and In vivo Screening Techniques Using Human Hepatoma (HepG2) Cells
Firouz Darroudi, M.D., Ph.D., Department of Toxicogenetics, Leiden University Medical Centre, The Netherlands
HepG2 cell system has been established and employed as the metabolic activation system as well as target for evaluating DNA damage. Data obtained so far elucidated on the potential of HepG2 cells: (1) to detect genotoxic potential of human dietary component (2) to define the mode of action, repair and the biological consequences of these chemicals, (3) to detect and elucidate the role of different enzymes involved and responsible for mutagenic, co- and anti-mutagenic potential of food constituents, (4) to discriminate between structurally related chemicals, carcinogens and non-carcinogens. Gene expression profile analysis revealed that HepG2 cells retain the activities of a number of enzymes, Phase I (CYP 1A1/1A2, 2B) as well as Phase II (GST, UDGPT, NAT), which are usually lost during cultivation. Consequently, it can be concluded that human hepatoma cell system reflects the activation / detoxification of genotoxic carcinogens better than other indicator cells that are currently being used and therefore have an increase predictive value for the identification of mutagens, co- and anti-mutagens, at biologically relevant doses. This assay is more relevant to humans in comparison to existing models in vitro and in vivo, therefore, it is a better alternative system to the use of animals in mutagenicity / genotoxicity testing.

11:25 A Systems Biology-Based Predictive Hepatotoxicity Platform
Kalyanasundaram Subramanian, Ph.D, CSO, Research, Strand Life Sciences Pvt. Ltd.
Hepatotoxicity is one of the main challenges in drug development with 60% of all liver failures caused by drug injury. It is a complex outcome of drug-patient-physiology combining genetic, metabolic and disease related features. Consequently, current predictive systems are limited in their ability to address the nature and impact of hepatotoxicity in humans. We have built a comprehensive model of liver homeostasis and treated toxicity as a case of drug-induced perturbations. The liver pathways involved in energy balance, redox status and bile production and cellular architecture have been simulated using dynamical systems modeling. Our model of the liver has been validated to quantitatively simulate both the “normal” behavior as well as demonstrate the evolution and outcome of drug-induced injury by several drugs. In this talk, we will demonstrate how this model can be used to assess the toxicity potential of leads in drug development.

12:55 Session Break

1:25 Chairperson’s Remarks 
Richard Beger, Ph.D., Center for Metabolomics, National Center for Toxicological Research

1:30 Detection of Drug-Induced Liver Toxicity in Early Drug Discovery Process Using Toxicogenomics Approach
Yi Yang, Ph.D., DABT, Associate Research Investigator, Cellular, Molecular and Exploratory Toxicology, Abbott Laboratories
Despite improved toxicology testing, hepatotoxicity associated with new drug candidates remains a major hurdle in drug discovery. Advances in microarray technology have provided a relatively high-throughput tool to monitor toxicity responses on a genome scale. At our laboratories, we applied toxicogenomics to predict hepatotoxicity potentials and to understand the mechanism. This presentation will highlight some of the workflows involving genomic marker identification. Specific examples will also be given to demonstrate how the technology can be successfully implemented in the early drug discovery process.

2:00 A Protein Chip for Hepatotoxicity
Zhiyuan Hu, Ph.D., Research Scientist, Laboratory of Dr. Leroy Hood, Institute for Systems Biology
We developed a high-throughput, label-free, reusable chip for analyzing serum proteins using Surface Plasmon Resonance (SPR) imaging of antibody microarrays. While a single biomarker is unlikely to accurately indicate drug induced liver injury (DILI), characteristic signatures obtained with a panel of liver-specific (or relatively specific) proteins may be more indicative of the severity of the liver injury. In collaboration with Aviva Systems Biology, we generated rabbit polyclonal antibodies for 75 liver specific proteins with multiple epitopes. Antibodies for liver-specific proteins were printed on Lumera Nanocapture Gold™ microarray chips. C57BL/6J mice were administered 300 mg/kg acetaminophen by i.p. injection. Mouse blood was drawn from acetaminophen treated mice during early toxic effects, maximal toxic effects, and the recovery stages of liver injury. Relative expression profiles were generated, recorded and compared with the histopathology data. Hierarchical clustering of the serum protein levels revealed a distinct hepatotoxic profile corresponding to different levels of severity of liver damage. This antibody chip will be a new tool for pre-clinical drug safety assessment.

2:30 Ice Cream Refreshment Break in the Exhibit Hall 
(Last Chance for Viewing)

3:10 Metabonomics Evaluations of Urine Samples from Multiple Liver Toxicity Studies in Rats
Richard Beger, Ph.D., Branch Chief, Center for Metabolomics, Division of Systems Toxicology, National Center for Toxicological Research
NMR spectral data of urine samples collected on day 2 post-dosing from both control and dosed Sprague-Dawley rats generated from studies of 11 compounds that have been previously associated with liver toxicity were used to build predictive metabonomic models of toxicity. The models, based upon the training set of data had predictive accuracies of approximately 85% in external test sets. The models were built based upon combinations of a total of three NMR integral bin intensities. These bins were determined to be related to the metabolites, N-methylnicotinate, trans-aconitate, and an unknown molecule. Based upon the model results, the metabolite concentrations for N-methylnicotinate and trans-aconitate were determined for the control and high dose groups on days 1 and 2 post-dosing. N-Methylnicotinate was shown to be significantly decreased in day 1 and/or day 2 urine samples by 9 of the 11 liver toxic compounds while trans-aconitate was shown to be significantly decreased in day 1 and/or day 2 urine samples by 8 of the 11 liver toxic compounds. The altered levels of N-methylnicotinate and trans-aconitate correlated with the histopathology results for the 11 toxicity studies and three external toxicity studies of other compounds better than levels of alanine aminotransferase, aspartate aminotransferase, or bilirubin. These results show that N-methylnicotinate and trans-aconitate are potential new and non-invasive pre-clinical biomarkers of toxicity. 

3:50 Qualification of Four Serum Biomarkers of Hepatotoxicity by the Predictive Safety Testing Consortium (PSTC)
Shelli Schomaker, B.A., Senior Scientist, Drug Safety R&D, Pfizer Inc.
The Predictive Safety Testing Consortium (PSTC), a collaboration among 16 pharmaceutical companies with FDA participation, has the goal of qualifying preclinical and clinical safety biomarkers for regulatory acceptance. This consortium, administered by the C-Path Institute, is formally endorsed by the FDA as a “Critical Path Initiative” activity. Several working groups have been formed to advance safety biomarkers for hepatic, renal, and vascular injury as well as carcinogenicity. Member companies use the consortium mechanism to bring forward putative safety biomarkers that have been derived from the literature or discovered in their internal laboratories that have significant supporting data and biological rationale. Cross-validation studies are planned to collaboratively qualify biomarkers for preclinical and ultimately clinical use. This presentation will focus on the advances the Hepatotoxicity Working Group has made towards the qualification of four serum biomarkers of hepatotoxicity: glutamate dehydrogenase, paraoxonase, purine nucleoside phosphorylase, and malate dehydrogenase. The overall value of this PSTC effort extends beyond sharing of costs and intellectual resources as it will also enable a more rapid safety biomarker qualification and direct engagement of FDA on qualification and acceptance criteria for safety biomarkers.

4:20 Serum Bile Acids for the Detection of Hepatotoxicity and Monitoring of Drug Safety
Norman B. Javitt, M.D., Ph.D., Professor of Pediatrics and Medicine, New York University School of Medicine
The transport of bile acid from the basolateral to the canalicular surface of the hepatocyte is a vectorial function requiring specific transporters embedded in each of the surfaces and also an intracellular transport process that interfaces with many other subcellular functions. Hepatocyte injury interferes with the transport process and results in an increase in serum bile acids. As hepatocellular function deteriorates and portal vein blood becomes redistributed, extraction efficiency becomes progressively compromised and elevations in both fasting and postprandial levels provide unique insights on liver status. 

4:50 Human Drug Hepatotoxicity: A Contemporary Clinical Perspective
Victor Navarro, M.D., Associate Professor of Medicine, Department of Hepatology at Thomas Jefferson University
Drug related liver injury (DILI) is a topic of great concern to clinicians, regulators, and pharmaceutical companies. DILI commonly leads to a compound being withdrawn from development; it also confounds safety experts in the post-marketing arena. Any new symptoms following exposure to a drug must raise the concern for DILI. These symptoms that occur in the setting of elevated hepatic transaminases indicate the probability of DILI. The most ominous clinical presentation of DILI comprises jaundice and elevated hepatic transaminases; this combination has been associated with significant morbidity and mortality. Exciting research advances may mitigate the problem of DILI in the future.

5:20pm Close of Conference

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