TOXICITY BIOMARKERS

1:30-1:55 Genomic Signature Biomarkers for Bile Duct Hyperplasia
Jeffrey F. Waring, Ph.D., Associate Research Fellow, Group Leader, Abbott Laboratories
Bile duct hyperplasia (BDH) is a hepatic change that is typically identified late in drug discovery after significant amounts of resources have been invested in a particular compound. For this reason, a predictive biomarker of BDH would be of definite value in drug discovery. In this study, we evaluated whether predictive and diagnostic gene expression signatures could be generated to predict or confirm bile duct hyperplasia in rats. Both predictive and diagnostic signatures were generated that had high degrees of accuracy. These data suggest that these signatures can reliably identify compounds inducing BDH at an early stage in drug discovery, and ultimately could serve as a bridge for clinical studies. 

1:55-2:20 Comparison of Different Biomarker Endpoints for Carcinogenesis of Aristolochic Acid in Rats 
Tao Chen, Ph.D., Research Toxicologist, Division of Genetic and Reproductive Toxicology, FDA/NCTR
Aristolochic acid (AA) is a potent human carcinogen and has been associated with the development of urothelial cancer in humans, and kidney and forestomach tumors in rodents. To investigate the molecular mechanisms responsible for the tumorigenicity of AA, and to compare the different biomarker endpoints, we determined the gene expression profiles, DNA adduct formation and mutagenicity of AA in the liver (non-target tissue) and kidney (target tissue) of Big Blue rats using a similar treatment protocol that resulted in tumors. AA treatment that eventually resulted in kidney tumors in rats also resulted in significant dose-response increases in DNA adduct formation and mutation induction in kidney. Although the same treatment did not produce tumors in rat liver, it did induce DNA adducts and mutations in liver, albeit at lower levels than in kidney. However, microarray analysis of the gene expression profiles indicated that significant alteration of genes associated with cancers mainly occurred in kidney, but not in liver. Our results suggested that AA is a genotoxic carcinogen through a genotoxic mode action in kidney. Although DNA adducts and mutations can be used as biomarkers for carcinogens, they are not target-specific while gene expression profiles can be better indicatives for turmeric target tissues of carcinogens and offer better explanations for the mode of action.

2:20-2:45 The Relative Performance of Nine Preclinical Urinary Biomarkers for Renal Toxicity Across Twenty Exploratory Studies 
Nagaraja Muniappa, Ph.D., DVM, Senior Investigator, Pathology, Safety Assessment, Merck Research Laboratories 
Ten exploratory kidney toxicity studies and ten other studies that manifested either liver, heart, or skeletal muscle injuries in rats were evaluated with nine urinary biomarkers including Kim-1, Clusterin, Osteopontin, Albumin, Trefoil Factor-3, Renal Papillary Antigen-1, Osteoactivin, Lipocalin-2, and GSTalpha. Novel immuno-based multi-plexed assays were developed on the MesoScale platform to evaluate the biomarkers for their relative sensitivity and specificity to kidney tubular injuries. Scatter plots and receiver operator characteristic (ROC) curves were employed to compare performance across the panel of biomarkers in individual study animals and to track care-fully against histomorphologic changes. Kim-1, Clusterin, and Albumin showed the highest performance overall for detecting tubular injury, while Kim-1 was the most specific marker of the panel for renal injury. This multiplexed approach to evaluating preclinical renal toxicity enhances ability to monitor for drug-induced renal injuries, provides insight into linkages between individual biomarkers and specific histopathologic processes, and provides an unprecedented parallel analysis of leading renal biomarker candidates across individual study animals.

2:45-3:10 Peptide Biomarkers of Exocrine Pancreatic Toxicity
David C. Thompson, Ph.D., Research Fellow, Drug Safety Research & Development, Pfizer Inc.
Historically, serum amylase and lipase levels have been used to indicate pancreas injury. However, these enzyme levels are often not predictive of pathology. In an effort to discover novel biomarkers of pancreatic acinar cell in-jury, we analyzed serum and pancreas tissue from rats treated with pancreatic toxicants using proteomics methods. Analyses of serum tryptic digests by surface-enhanced laser desorption-ionization mass spectrometry revealed two novel peptide biomarkers that were predictive of pancreatic damage. The peptides were identified as fragments of serum proteins that are susceptible to cleavage by proteases leaking from the pancreas. The pep-tide markers proved to be more sensitive than amylase or lipase in detecting pancreatic damage, especially at early time points following toxicant exposure. These two peptides successfully detected pancreatic injury with multiple toxicants and were useful across several species, including human. They appear to be sensitive indicators of exocrine pancreatic damage that may be useful as safety markers for general pancreatic toxicity in multiple species.

3:10-3:15 Closing Remarks

BIOMARKER ASSAY DEVELOPMENT (CONT.) 

1:30-1:55 Novel Biomarker Assays: From Preclinical Development to Clinical Proof of Concept 
Miro Venturi, Ph.D., Novartis Biologics
This talk will highlight recent developments in the field of biomarker assay and validation thereof, with a specific focus on their utility and applications to accelerate drug development of Biologics. Notably, biomarkers are needed for the establishment of successful PK/PD relationships, for guiding appropriate dose regimen schedules and reducing early stage attrition of selected candidates and/or showing superior behavior in the field of biosimilars. Novel assays and new validation concepts will be illustrated, linked to case studies, whereby biomarkers have greatly enhanced and ameliorated drug development efforts.

1:55-2:20 Considerations in Selection of Assay Platforms and Sample Preparation Methods for PD Biomarkers in Alzheimer Disease Clinical Development 
Yan J. Zhang, Ph.D., Senior Research Investigator, Clinical Biomarker Development, Bioanalytical Science, Bristol-Myers Squibb Co.
The measurement of β-amyloid (Aβ) peptides in plasma and cerebrospinal fluid (CSF) is a potential PD biomarker for therapeutics designed to lower Ab peptides for Alzheimer disease treatment. As Aβ peptides form oligomers and tightly bind a number of plasma proteins, it is challenging to accurately measure the peptides in clinical sam-ples. The presentation will discuss the considerations in selection of assay platforms and sample preparation methods to fit for the needs of clinical studies. We have used both singleplex and multiplex assays in support of the clinical development. The need for sample preparation and the performance on different platforms will be discussed.

2:20-2:45 Developing Multiplexed Assays on the MesoScale Discovery Platform to Assess Rat Critical Target Organ Toxicities
Katerina Vlasakova, Research Associate, Systems Toxicology, Safety Assessment, Merck Research Laboratories
Drug development groups have a growing need for both fast and efficient methods for incorporating toxicity screening of drug candidates early in their animal models. Our group is currently working to develop multiplexed screening assays to allow for monitoring several of these biomarkers simultaneously in preclinical species. Meso-Scale Discovery (MSD) was chosen as the platform for multiplexing these assays due to the following advantages i) the ability to multiplex assays within 96-well formats, ii) the use of carbon-based plates with enhanced anti-body binding capacity, and iii) the detection by electroluminescence using a sensitive CCD camera for greater sensitivity and dynamic range of assays. Here we present several biomarker assays that have been successfully developed to monitor toxicity in kidney, skeletal muscle, heart, and liver, and evaluated using samples from numerous rat toxicology studies. Assay development and qualification will be presented.

2:45-3:10 Strategies to Tackle Matrix Effects in Biomarker Assays 
Ole Lagatie, Ph.D., Scientific Advisor, Bioanalysis, Eurofins Medinet
Many biomarkers are analyzed using immunoassays. During development of such an assay one of the most com-promising problems usually is the presence of matrix effects. These matrix effects are caused by poorly-defined serum or plasma factor that result in inaccurate determination of the analyte in clinical study samples. An over-view will be presented on how to deal with these matrix interferences using two major approaches: adjusting calibration samples to reflect study samples and adjusting the study samples to reflect calibration samples. Examples will be shown based on literature and our own in-house experience.

3:10-3:15 Closing Remarks