Institute for Scientific Exchange, Inc. Presents:

Text Box: June 5-6, 2008
ETS-2008
Early Toxicity Screening: Metabolism-Based Toxicity in Drug Development
Red Lion Hotel-Fifth Avenue, Seattle, Washington, USA
Seattle, WA, USA

Featuring experts from the following institutions: Allergan Inc.; Amgen Inc.; APSciences/IVAL; AstraZeneca; Johnson & Johnson PRD; Merck & Co.; Merck Serono Research; Pfizer Global R&D; University of Kansas; University of Toronto; University of Washington; Wyeth Research

ISE, Inc.

8775 Centre Park Drive

#713

Columbia MD 21045

(410) 869-9166

Organizing Chairs:

Albert P. Li, APSciences, Inc. and IVAL, LLC

John Erve, Wyeth Research

Sandy Pang, University of Toronto

Role of Drug Metabolism in Drug-induced Liver Toxicity (Sid Nelson, University of Washington; Seattle, WA) Drug-induced liver injury is one of the most common reasons for failure of drugs in development, and for either removal of drugs from the market or "black-box" warnings on marketed drugs. This presentation will focus on the role of reactive metabolites as a major cause of drug-induced liver injury, on structure-toxicity relationships, and on methods to assess risk from reactive metabolites.

Allergic Reactions to Drugs (Mark P. Grillo, Amgen Inc.; South San Francisco, CA) There are many pharmaceutical scientists today who say that one of the most difficult obstacles to overcome in drug discovery is the prediction, and therefore knowledgeable prevention, of drug-induced idiosyncratic immune-based reactions. The immune system is a highly complex and developed system and is our natural defense in fighting varied pathogenic organisms. The more we have learned about the immune system over the last two decades, due in large part to molecular advancements in immunology, the more it has revealed for researchers to come closer to understanding the mechanism(s) of drug- induced allergic reactions. Allergic reactions to drugs can lead to potentially life-threatening reactions such as asthma, anaphylaxis, dermatitis, hepatitis, hemolytic anemia, lupus, nephritis, Stevens-Johnson syndrome, toxic epidermal necrolysis, urticaria, and vasculitis. These drug-induced immune-based diseases can create an enormous burden to affected patients and to the respective pharmaceutical companies involved in the manufacturing of these drugs. Such toxicities can deprive patients of organ function that sometimes results in fatalities. A functional understanding of the association between the use of some drugs and the unpredictable formation of drug-induced hypersensitivity reactions is of major importance in the pharmaceutical drug discovery and development industry today. Immune-based drug-induced allergic diseases may also present scientific opportunities. Studying them could answer critical questions about the nature of the immune system and potentially provide new strategies as it relates to the discovery of safer drugs. In this presentation, an examination of a set of drug-induced allergic reactions and proposed mechanisms will be reviewed, each mediated by different mechanisms that are driven by the inherently different chemistry and biochemistry of the drug.

Reactive Metabolites and Marketed Drugs: What is the Evidence and What are the Consequences? (John C. L. Erve, Wyeth Research) Among drugs that cause toxicity and have been withdrawn from the market for safety reasons, many have subsequently been shown to generate reactive metabolites. Such observations have associated reactive metabolites with drug toxicity and lead some firms to screen for their presence during early pre-clinical discovery. However, there remain drugs on the market which also give rise to reactive metabolites. This presentation will examine some currently marketed drugs that generate reactive metabolites, including one with an excellent safety profile, and discuss the implications.

Development and Discovery: Global and Focused Tools (Priv. Doz. Dr. Stefan Otto Mueller, Merck Serono Research - NCD/Toxicology; Darmstadt – Germany) Testing for the safety of a potential drug is a challenging task for the toxicologist in the pharmaceutical industry. The emerging new technologies have increased the possibilities but also the expectations to assess relevant and species-specific toxicities of drug candidates. Examples on global expression profiling but also classic tools to enhance toxicity assessment are presented.

Toxicity Screening in Drug Discovery and Early Development (Robert T. Dunn, II, Amgen Inc; Thousand Oaks, CA) The optimal function of early screening studies during lead optimization is to both identify and eliminate toxic compounds and enhance the likelihood of a compound’s success later in development. An essential element to early screens is broad endpoint coverage in order to identify targets of toxicity. Broad endpoint coverage at the cellular level is analogous to endpoint coverage that happens during a typical in vivo study with measurement of dozens of parameters. Cellular endpoints that involve critical organelles (e.g., mitochondria) or pathways related to the life cycle of the cell (e.g., apoptosis) are indispensable. Once attractive leads have been identified, a limited set of compounds should be advanced to efficient, focused, and rapid in vivo screens.

Evaluation of Metabolism-based Drug Toxicity in Human Hepatocytes (Albert P. Li, APSciences, Inc, and IVAL, LLC.; Columbia, MD) Human hepatocytes represent the gold standard for the evaluation of human drug metabolism, and therefore represent an ideal experimental system for the evaluation of human metabolism-based drug toxicity. In our laboratory, we have developed an assay using cryopreserved human hepatocytes, in combination with P450 inhibitors, as an approach to evaluate the role of drug metabolizing enzymes in the drug toxicity. The experimental approach and results with model toxic drugs will be presented.

Tissue Slices for the Evaluation of Metabolism-based Toxicity (Alison Vickers, Allergan Inc.; Irvine CA) Drug-induced organ injury is a multifaceted process, involving numerous cell types and mediators, and remains a significant safety issue in pharmaceutical development and clinical therapy. Tissue slices, an in vitro model representing the multi-cellular, structural and functional features of in vivo tissue, is a promising model for elucidating mechanisms of drug-induced organ injury and for characterizing species susceptibilities. Key cellular pathways causal to organ injury are characterized via gene expression, cell function and morphology. The application of human tissue enhances our ability to focus on the clinical relevance of potential side-effects and biomarkers.

Hepatocyte Assay for the Screening of Canalicular Transporter Inhibitors (Gerry Kenna, AstraZeneca R&D Alderley Park; Mereside, Alderley Park, Macclesfield, Cheshire, England) We have developed novel assays for investigation of drug induced inhibition of canalicular efflux transporters (bsep and mrp2) in rat hepatocytes cultured in sandwich configuration. These use selective fluorescent probe substrates and provide robust data in a physiologically relevant system, which for bsep can be quantified using a cell imaging algorithm. We have also assessed inhibition of the uptake transporter ntcp in the same culture system. Results which be presented which suggest that, for at least some hepatotoxic drugs, inhibition of multiple transporters could contribute to the mechanism of liver injury.

Human Hepatocyte Assay for Canaliculi Inhibitors (Jinghai (Jim) Xu; Pfizer - Groton Labs; Groton, CT) Drug-induced cholestasis is a frequent finding among drugs that induce liver injury in humans. I will discuss an assay system that uses fresh and cryopreserved primary human hepatocyte cultures and automated cellular imaging technology to study mechanisms of drug-induced cholestasis.

Strategies and Techniques for the Characterization of Reactive Metabolites by LC-MS (Shuguang Ma, Amgen Inc.; Thousand Oaks, CA) Characterization of reactive metabolites is critical to designing new drug candidates with an improved toxicological profile. In this presentation, a brief description of LC-MS instrumentation employed for assessing reactive metabolites is followed by a discussion on strategies and techniques for selective and sensitive detection of glutathione trapped reactive metabolites. Techniques for semi-quantitation of reactive metabolite formation are also discussed.

Simultaneous Capturing and High Throughput Screening of Both “Soft” and “Hard” Reactive Metabolites (Zhengyin Yan, Johnson & Johnson Pharmaceutical Research & Development, LLC ; Spring House, PA) As the most used trapping agent for reactive metabolites, glutathione (GSH) has shown low trapping efficiency for “hard” reactive metabolites such as reactive aldehydes. In order to overcome this deficiency, a bi-functional trapping agent (gGSK, g-glutamyl-cystein-lysine) is evaluated as an alternative of GSH for simultaneous trapping both “hard” and “soft” reactive metabolites. In microsomal incubations, “soft” and “hard” reactive metabolites are captured by conjugation to the free thiol and the alphatic amine group of gGSK, respectively, resulting in formation of corresponding stable peptide adducts. Similar to GSH conjugates, all gGSK adducts derived from either “soft” or “hard” reactive metabolites contain a g-glutamyl moiety, and thus undergo a neutral loss (NL) of 129 Da under collision-induced dissociation. As a result, NL tandem MS scan can be utilized as a generic analytical method for rapidly detecting both “hard” or “soft” reactive metabolites. As demonstrated by a number of model compounds, this approach combining with the isotope trapping technique is reliable, sensitive and efficient, and can be potentially used as a high throughput method for simultaneous screening and identifying both “soft” and “hard” reactive metabolites. In comparison with other methods, this approach is highly efficient and suitable in drug discovery for screening a wide variety of compounds for different reactive metabolites.

Metabolites in Safety Testing (Dennis Smith, Pfizer Global Research & Development; Sandwich, Kent, England) ADME studies should be viewed as a component of PK/PD. Percentage-based data should be replaced by absolute quantities/concentrations. This ensures that metabolites (across varying doses and species) are compared appropriately (consistent with the law of mass-action). Structural comparisons, broad ligand binding, historical records of animal and clinical adverse events can be used to answer which circulating metabolites or excreted metabolites represent an additional risk factor to that of the parent drug without the laborious process of synthesizing and testing everything. Further understanding is needed to predict why a metabolite circulates (and at what concentration).

Safety Testing of Metabolites: Is it Necessary? (Sandy Pang, University of Toronto; Toronto, ON Canada) Metabolites are of interest in drug development and post market surveillance due to the untoward reactions and toxicity associated with bioactivation. Hence, there is the need to explore the behaviors of metabolites with regard to risk assessment and safety issues. The document, Safety in Metabolite Testing, MIST, suggests monitor of metabolites when the % in the circulation is demonstrable. Others suggest examination of metabolites, especially at higher doses of drug, when the aggregate amount of metabolite detected is large. This presentation shows that there are differences in kinetics between formed and preformed metabolites in the liver, kidney, and intestine. Reasons are differences in permeability of the precursor drug and the metabolite, formation of different secondary/tertiary metabolites in formation organs due enzyme heterogeneity or enzyme coupling, absence of glomerular filtration for formed metabolite, and unique flow patterns of the intestine. Hence, toxicity may not be duplicated by administration of the metabolite due these named differences. The concentration-time profiles, obtained upon administration of the preformed metabolite, however, strengthen modeling of drug and metabolite data, and allow prediction of metabolite levels or toxicity within target organs via modeling and simulations.

Filling and Mining the Reactive Metabolite Target Protein Database (Robert Hanzlik, University of Kansas; Lawrence, KS) The toxic effects of many drugs and chemicals stem from their biotransformation to chemically reactive metabolites that bind covalently to proteins. Many target proteins have now been identified and their identities collected in the "Reactive Metabolite Target Protein Database," an on-line resource. Bioinformatic mining of this database is beginning to reveal possible connections between the covalent labeling of target proteins and the ultimate production of cytotoxic responses.

About the Institute for Scientific Exchange

The mission of The Institute for Scientific Exchange, Inc. is to advance science via communication – (i. e. symposia, training courses, publications). The events held by the Institute are highly selective, timely, and of the highest professional caliber. One major goal of the Institute, as exemplified by this symposium, is to foster communication among industrial, regulatory, and academic practitioners. Please visit our web site at www.isciencex.com.

Poster Presentations are always encouraged. Please submit your poster abstract for approval by the organizing board by May 15^th. Poster size should be no larger than 3 feet high by 7 feet long. Abstracts of posters will be included in the participant binder and in the ISE website. There is no formal poster presentation scheduled. All posters will remain displayed throughout the conference. Please be prepared to display your poster during registration on Monday, June 19^th or before the first session begins on Tuesday, June 20^th. Poster presenters will have ample time for discussion during breaks and the Panel discussions. Submit posters abstracts for approval to Nola Mahaney, VP, Operations; ISE, Inc.; 8775 Centre Park Drive, #713, MD 21045 or fax at (410) 869-9560 or email file attachment to nola@isciencex.com. Approved poster presenters are responsible for completing a conference attendance registration form and payment of fee (visit www.isciencex.com/register.htm) and for the shipping of the poster itself. Please contact Nola Mahaney for any questions or concerns. Please refer to “Travel Information” for hotel address and shipping information.

General Information: Conveniently located in downtown Seattle, The Red Lion Hotel on Fifth Avenue offers an upscale, boutique hotel experience, personal, award-winning service, first-class meeting space, and - perhaps most importantly - breathtaking views of the Cascade Mountains, Elliott Bay, the Puget Sound and the Emerald City itself. Whether you're planning a family trip, coming to town on business, or just looking to take a little time off from the hustle and bustle of your daily life, we guarantee you'll find The Red Lion Hotel on Fifth Avenue to be an excellent choice of accommodations. http://redlion.rdln.com/HotelLocator/HotelOverview.aspx?metaID=32

Payment

Payment may be made by check or credit card. Checks should be made in US $, payable to Institute for Scientific Exchange, Inc. Mail to: ISE, Inc., 8775 Center Park Drive; #713, Columbia, MD 21045, USA

Cancellation Policy

All cancellations are subjected to a $250.00 cancellation fee. Longer than 30 days, 100% refund (less cancellation fee). Less than 30 days, no refund but registration may be transferred to another person. All refund requests must be in writing. All refunds will be issued after the meeting has occurred. No refunds requests will be accepted after May 2, 2008. Please submit cancellation and refund requests including transferring of registration to:

Email to nola@isciencex.com with remittance to: ISE, Inc. 8775 Centre Park Drive; #713, Columbia, MD 21045, USA; FAX No: (410) 869-9560. Payment may be made by check in US$, payable to Institute for Scientific Exchange, Inc. or by credit card.