ETS-2004

Institute for Scientific Exchange, Inc. Presents:

4^th INTERNATIONAL CONFERENCE ON EARLY TOXICITY SCREENING:

In Silico, In Vitro and High Throughput Screening Approaches

February 23-25, 2004

San Diego, CA, USA

Symposium Venue: San Diego Mission Valley Marriott

Featuring experts from the following institutions: AstraZeneca; Bayer Crop Science; Charles River Laboratories; Chiron Corp; GlaxoSmithKline; HemoGenix; Institute for In Vitro Sciences Inc.; Johnson & Johnson PRD; Lexicon Genetics; Millennium Pharmaceuticals; National Cancer Institute; NIEHS; Novartis Pharmaceuticals; Partek Incorporated; Pfizer, Inc.; Advanced Pharmaceutical Sciences, Inc.; Promega Corp.; University of Toronto; XenoTech LLC.

Sponsored in part by:

PARTICIPATING EXHIBITORS:

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Overview: Challenges Towards Accurate Prediction of Human Drug Toxicity (Albert P. Li; Advanced Pharmaceutical Sciences, Inc.; Baltimore, MD) It has been estimated that the cost of drug development is $800 million per new drug. One of the reasons for the high cost is failure in clinical trials due to drug toxicity. Furthermore, there are examples of marketed drugs that were withdrawn due to unexpected drug toxicity, not predicted by preclinical and clinical safety trials. An overview of the past problems, present limitations and promising new technologies for the prediction of human drug toxicity will be presented.

Species Differences in Xenobiotic Toxicity (Joe Tomaszewski, National Cancer Institute; Bethesda, MD) One of the constant struggles that faces the toxicologist when evaluating toxicity data on a potential new drug, is how much data is enough to ensure that the Phase I clinical trial in man will be safe. Another is whether the accumulated data is sufficient to predict toxicities in man. One of the confounding issues that must be dealt with in this regard, is the disparity in response that often times occurs in the different animal species used to generate this data. Differences in metabolic rate, size, clearance, body weight, surface area, etc. are major contributors to the toxicological difficulties we face. If one attempts to deal with this conundrum as a regulatory box that must be checked prior to entering the clinic, the outcome often results in failure. One of the keys to dealing with species differences is to utilize all of what is known to explain those differences and to draw upon past experience to help guide the way. This talk will attempt to draw upon past experiences to help understand some of these differences and shed some light on how they can be used to design appropriate studies that produce interpretable data. However, despite our best efforts, toxicities do occur and occur frequently in man that is never predicted by even the most carefully designed studies. This is perhaps one of the reasons that the toxicologist needs newer tools in his armamentarium other than the whole animal to predict human sensitivity.

Nonclinical Testing Predictive for Idiosyncratic Liver Injury (Carl L. Alden; Millennium Pharmaceuticals; Cambridge, MA) Preclinical toxicology and metabolism functions must play a pivotal role in improving speed and decreasing attrition attributable to toxicity, side effects, or DMPK attributes. These functions must provide a clear view of any drug development limiting barriers before committing the enormous resources to develop the fundamental data to gain market access. The technology now exists that enables the near elimination of development failures because of DMPK attributes as well as reduction of attrition attributable to toxicity or side effects from the current benchmark level of 40% to less than 20%. However, the dominant paradigm states that predicting for idiosyncratic liver injury cannot be achieved in nonclinical testing. Idiosyncratic responses can cause withdrawal from the market (e.g. bromfenac, troglitazone) or prevent business success (e.g. zileuton) potentially creating negative impact beyond the lost cost of development. Idiosyncratic liver injury is also generally considered to be non-dose responsive and of low/sporadic incidence requiring sample size for detection that often exceeds the number of patients tested in development. Review of the summary basis of approval for three drugs causing idiosyncratic injury indicates the dominant belief that preclinical testing does not predict for idiosyncratic responses may not be true, at least in these cases. A preclinical testing strategy to avoid idiosyncratic liver injury will be described.

The Design and Interpretation of In Vitro Safety Assays in Discovery (Jim (Jinghai) Xu; Pfizer - Groton Labs; Groton, CT) It is now well-accepted that the identification of safer drug candidates for further development relies on a panel of in vitro safety assays in Discovery. However, key elements in the design of such an in vitro safety panel, and the proper interpretation of these in vitro results, have not been fully addressed. This presentation will focus on the strategies in designing an "optimal" in vitro safety panel. Emphasis will be placed on the use of "optimal" in vitro models, concordance with GLP tests, in vitro in vivo correlations, and proper interpretation of the in vitro results.

The Role of Early Toxicity Screening in Drug Safety Evaluation (Jessica E. Sutherland, Charles River Laboratories Discovery and Development Services; Worcester, MA) Early toxicity screens are an extremely important component of pharmaceutical safety assessment. A properly designing toxicity screening program can be used to optimize lead compounds in an attempt to identify which should move from discovery into development. Early toxicity screening is also useful during pilot and dose range-finding studies or when formulation decisions are needed. Early toxicity screening is also helpful in identifying unusually toxic agents, target organs, and estimating lethality. Selection of the proper animal model(s), sample size, route of administration, and toxicological end points is vital to the success of these early nonclinical programs.

Immortalized Hepatocytes: A New In Vitro Approach to Early Induction and Hepatotoxicity Screening (Andrew Parkinson, Xenotech LLC; Lenexa, KS) A new human hepatocyte cell line has the potential to solve the problem of supply and variability that restrict the use of human hepatocytes for enzyme induction and other in vitro studies. Our studies with these cells indicate that their induction response at least equals that of the majority of fresh human hepatocytes preparations for the major inducible drug-metabolizing enzymes. Additionally, they demonstrate responses to toxicants and non-toxicants which also conform to expected results; they allow for differentiation between toxicants and non-toxicants among the compounds tested, and reflect concentration-dependence with toxicants.

Predictive Toxicity Screening – Future Directions for In Silico Applications (Dale E. Johnson, Chiron Corp.; Emeryville, CA) Predicting potential human toxicity during the early phases of pharmaceutical R&D continues to be only partially successful. The application of new technologies such as toxicogenomics, proteomics and metabonomics continue to be in the validation stage; whereas high-content screening using human cell-based systems and transgenic animal models may emerge as reasonable alternatives to current approaches. Since In silico applications require the generation of new data involving large sets of chem-tox correlations, cell-based assays may become a preferred approach if reasonable validation programs can be established.

Toxicity Evaluation Using Statistical Analysis and Visualization (Tom Downey, Partek Incorporated; St. Charles, MO) Statistical analysis is useful for finding correlations and patterns in screening data. After the patterns are identified, they can be presented using informative interactive visualization of the data. The result is that the data may be used to create in-silico models to predict toxicity of new compounds prior to selecting which compounds to test in-vitro. This talk will demonstrate the application of statistical methods such as statistical tests, principal components analysis of structure-activity relationships, and identification of patterns and trends in high throughput screening data.

(casual attire, not mandatory, but encouraged because it’s so much fun!)

Choosing an Appropriate Endpoint for In Vitro Cytotoxicity and Apoptosis Assays (Terry Riss, Promega Corporation; Madison, WI) A variety of methods for measuring in vitro toxicity and apoptosis have been adapted for use as automated high throughput screening assays. Choosing the appropriate endpoint to measure, determining the correct toxin exposure time, and multiplexing more than one measurement will improve the quality of data from cell-based assays. I will present examples of homogeneous in vitro cytotoxicity and apoptosis assays that measure different endpoints and describe the advantages and disadvantages of each method.

Early Toxicity Screening by ACMS (Accelerated Cytotoxic Mechanism Screening): The Importance of a Radical Approach (Peter O’Brien, University of Toronto; Toronto, CANADA) Many of the drugs withdrawn from the market appear to be drugs that can be metabolically oxidized or reduced to radicals that cause oxidative stress and mitochondrial toxicity. Currently early toxicity screening methods do not measure the ability of a drug to form toxic radicals. Early prioritization of novel therapeutic drugs should include screening for cellular reactive oxygen species formation and mitochondrial toxicity. ACMS evidence using isolated hepatocytes will be presented to demonstrate that many drugs are toxic because of metabolism to N, C, O or S radicals. High throughput early toxicity screening techniques for prooxidant drug radical formation will be shown and oxidative stress biomarkers for in vivo toxicity will be proposed.

Applications of the Comet Assay in Drug Development (Raymond R. Tice, Integrated Laboratory Systems, Inc.; Research Triangle Park, NC ) The comet assay is used for in vitro genotoxicity testing of drug candidates in early phases of discovery / development. A high degree of agreement between results from cytogenetic tests and the comet assay demonstrates the validity of the biological endpoint measured in the comet assay. Furthermore, the comet assay is used in vivo to evaluate potential genotoxic activity in target organs, to investigate first-site-of contact genotoxicity or to evaluate the relevance of in vitro positive findings.

Optimization of Cell-based Assays for Medium Throughput Screening of Oxidative Stress (Sophie Lautraite; Bayer Crop Science; Sophia Antipolis,
FRANCE) Optimization of cell-based assays for medium throughput of oxidative stress-Identification and development of cell-based assays adapted to medium throughput screening requirements is important when screening chemicals for their potential toxic properties. Rapid fluorometer- and pectrophotometer-based microplate assays allowing the evaluation of oxidative stress have been adapted to hepatocyte cell cultures. The system should be useful in selecting candidate compounds during the pre-development phase of agrochemicals or pharmaceuticals. It should also be of interest for retrospective and explanatory studies of mechanisms underlying toxicity observed in vivo.

Combined Genomics, Proteomics and Metabonomics Analysis of Acetaminophen Toxicity in Mouse Liver (François Pognan, AstraZeneca; Wilmington, DE) Acetaminophen has been administered at pharmacological, sub-toxic and toxic doses to overnight fasted mice. Animals have been sacrificed at different time points from 15 minutes to 4 hours post-injection. Genomics analysis in liver has been performed using nylon filter arrays, Affymetrix chips and QT-PCR. Proteomics on liver mitochondrial subfractions has been run using the quantitative fluorescent 2D-DIGE method and metabonomics ¹H NMR spectra from liver tissue, tissue extracts and plasma from mice dosed with acetaminophen have been analyzed separately to identify biochemical changes arising from hepatotoxicity.

Human Hepatocyte Toxicogenomics Assay for the Evaluation of Human Hepatotoxicity (Albert P. Li; PHASE-1 Molecular Toxicology, Inc.; Santa Fe, NM) Accurate prediction of human drug toxicity, especially hepatotoxicity continues to be a challenge in drug development. Animal testing has failed to identify drugs that caused serious hepatotoxicity in human, leading to market withdrawal or serious use limitations of many marketed drugs. Species differences in drug metabolism and sensitivity to drug toxicity are believed to be the major reasons for the lack of animal-human correlation. Freshly isolated human hepatocytes represent a promising experimental system for the evaluation of human hepatotoxicity because of the relevant species (human) and target cells (liver parenchymal cells). Toxicogenomics as an endpoint allows the evaluation of multiple hepatotoxic mechanisms that may not be detected using routine cytotoxicity endpoints. Data with known toxic and nontoxic drugs show that genomics analysis can be used to predict human drug hepatotoxicity.

Human Tissue Slice Toxicogenomics to Evaluate Target Organ Effects (Alison Vickers; Novartis Pharmaceuticals Corporation; E. Hanover, NJ) The 3-dimensional cellular complexity and cellular interactions that are representative of in vivo tissues is achieved in organ slice cultures. Gene expression evaluations of compound induced effects in the potential target tissue are linked with assessments of cell function and morphology to describe the key cellular pathways affected. Application of human tissue enhances our ability to focus on the clinical relevance of a potential outcome and for identifying clinically relevant biomarkers.

Alpha Glutathione S-Transferase – Does it Add Value Over Traditional Markers of Hepatotoxicity in the Wistar Han Rat (Paul Giffen (Hons), GlaxoSmithKline R&D; Hertfordshire UK) The high hepatic concentration of Alpha Glutathione S-Transferase (aGST) has resulted in much interest in its usefulness as a marker of hepatotoxicity. Furthermore, its cytosolic localization, small size and short half-life may potentially offer more information regarding acute hepatocellular integrity than the traditionally measured transaminases. We evaluated aGST as a marker of acute hepatotoxicity in rats treated with experimental hepatotoxicants, in comparison with a series of markers including alanine and aspartate aminotransferase and glutamate dehydrogenase as well as histopathological findings.

Luminescent Assays for Recombinant and Native Cytochromes P450 (James J. Cali, Promega Corp., Madison, WI) Luminescent CYP450 assays were developed that utilize derivatives of beetle luciferin as probe CYP450 substrates. The derivatives were not substrates for the light generating reaction with firefly luciferase but were metabolized by CYP450s to luciferin, which in turn reacted with luciferase to make light. A homogenous luciferase mixture was added directly to conventional CYP450 reactions that used luciferin derivatives as substrates and light output was used to measure CYP450 activity. Luminescent assays detected CYP450 activity in human liver microsomes and in recombinant CYP450 preparations. 6’-deoxyluciferin was hydroxylated to form luciferin by CYP2C9 and dealkylations of luciferin 6’ alkyl and substituted alkyl ethers to form luciferin were catalyzed by CYP1A1, 1A2, 1B1, 2C8, 2C9, 3A4 and 3A7. The luminescent CYP450 assays detected known CYP450 inhibitors with IC₅₀s similar to those reported against conventional substrates. These assays bring the intrinsic strengths of luminescence to the study of CYP450s: they are exquisitely sensitive and unlike fluorescent assays analysis is not confounded by overlap between fluorescent properties of assay components and analytes. The simple, homogenous and robust luminescent assay formats are well suited for rapid screening of multiple compounds against p450 activities.

The Successful Use of an In Vitro Screening Assay for Bone Marrow Cell Toxicity to Predict Human Sensitivity (Joe Tomaszewski, National Cancer Institute; Bethesda, MD) Drugs that are used to treat cancer patients are probably the most toxic drugs that are purposely administered to man for therapeutic purposes. Many of these drugs invariably produce myelosuppression as one of their principal side effects. This bone marrow toxicity is usually manifest as neutropenia and is easily monitored in the clinic. While this toxicity has been mostly associated with alkylating agents and antimetabolites in the past, it is even associated with newer targeted drugs with mechanisms of action such as cdk and HDAC inhibition. The NCI set out to develop in vitro toxicity assays to evaluate various toxicities through the government=s SBIR Program. A collaboration to evaluate bone marrow toxicity was established with the Hipple Cancer Research Corp. Under this program, colony forming unit (CFU) assays were developed for murine and human granulocyte/macrophages, erythrocytes, macrophages, granulocytes and egakaryocytes. These advances were followed up by the development of a limited number of assays using canine, rat and non-human primate marrow. Since neutropenia is the predominant manifestation of myelosuppression in the clinic, the NCI set out to validate the CFU-GM assay in a retrospective study using a number of known myelosuppressive anti-cancer agents. In doing so, the rank order of sensitivity in vitro was compared to the sensitivity of the same species in preclinical and clinical studies and the data was used to calculate a human maximum tolerated dose (MTD). This study has since been expanded and the latest results will be presented. In addition, this assay has also been validated by ECVAM, the European Committee for the Validation of Alternative Methods. In general, the in vitro CFU-GM assay accurately predicts in vivo sensitivity of animals and man. The IC90, rather than the IC50, more accurately predicts in vivo sensitivity and the MTD in man. When the projected human MTD was calculated from murine data alone, 14/21 drugs were correctly predicted within a factor of four. When canine data was added to the calculation, 19/21 drugs were successfully predicted. Thus, in vitro bone marrow data can be used successfully in predicting human sensitivity to myelosuppression produced by anti-cancer agents. As a result, the assay is currently used by the NCI in late discovery/early development to select optimal drug development candidates.

HALO – A High-Throughput, Multifunctional, Multiparameter Hemotoxicity Prediction Testing Platform (Ivan Rich, HemoGenix; Colorado Springs, CO) HALO (Hemotoxicity Assays via Luminescence Output) was introduced by HemoGenix in 2001. HALO is a proliferation assay measuring 14 different lympho-hematopoietic stem and progenitor cells from human, non-human primate, dog, rat and mouse origin simultaneously. Its high-throughput capability allows its use from drug screening to patient monitoring. HALO is becoming an important predictive tool to measure differential toxicity of a wide range of compounds. Recently launched in kit form, HALO is used in the research and clinical arenas.

Evaluation of Neurotoxic Potential Using In Vitro Systems: Limitations and Use of Various Model Systems (G. Jean Harry, National Institute of Environmental Health Sciences; NIH; Research Triangle Park, NC) In neurotoxicology, in vitro models are used to study biological processes in an isolated context and are most useful when a known mechanism of action is evaluated. In vitro tests have the greatest potential to elucidate mechanisms of toxicity, identify target cells of neurotoxicity, and delineate the development of intricate cellular changes however, the inability to recapitulate cell-cell interactions critical for the formation and functioning of the nervous system is a major limitation. When used for screening, end-points must be carefully selected to allow differentiation between cytotoxic, neurotoxic, and pharmacological effects.

Application of Toxicogenomics in Drug
Development (Heike Hellmold, AstraZeneca R&D; Södertälje, SWEDEN) Expectations are high that "omics" techniques will be applied successfully in drug development to reduce attrition due to toxicity. This presentation will discuss current concepts of predictive toxicology and the use of toxicogenomics in mechanistic and predictive toxicology and in the search for biomarkers of toxicity.

11:45 AM – 12:15 PM

In Vitro Evaluation of Toxicity to the Eye and Skin (John W. Harbell, Institute for In Vitro Sciences, Inc.; Gaithersburg, MD) After lead compounds pass initial efficacy and systemic toxicity assessment, additional testing for organ-specific toxicity is performed. Action on the eyes and skin (penetration, metabolism, phototoxicity, and irritation potential) is evaluated to address both patient and worker safety requirements. Ocular irritation assays may use ex vivo (e.g. bovine corneas) or engineered tissue constructs. Dermal penetration/toxicity assays may use human skin (ex vivo and engineered) as well as cultured cells.

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 January 30^th. Poster size should be no larger than 4 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 Sunday February 22nd or before the first session begins on Monday, February 23rd. Poster presenters will have ample time for discussion during breaks and the Welcome reception.

Submit posters abstracts for approval to Nola Mahaney, VP, Operations; ISE, Inc.; 5707 Calverton Street, Suite 2C; Baltimore, MD 21228 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.

4th INTERNATIONAL CONFERENCE ON EARLY TOXICITY SCREENING:

In Silico, In Vitro and High Throughput Screening Approaches

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4^th INTERNATIONAL CONFERENCE ON EARLY TOXICITY SCREENING: