The Institute for Scientific Exchange, Inc. Presents:

 

3rd International Symposium Early Toxicity Screening: Early ADME-Tox Screening Approaches December 11, 2002 Philadelphia, Pennsylvania Symposium Venue:  Renaissance Philadelphia Hotel Airport

Please visit us online at www.isciencex.com

Now Open ISEbooks.com – Your online Science book solution

 

Participating Exhibitors:

 

 

 

 

 

Session I:  Prediction of Human Drug Toxicity

(Chair: Albert P. Li)

9:00 AM – 9:30 AM

Overview:  Why did we fail in the prediction of human drug toxicity? (Albert P. Li, Phase 1 Molecular Toxicology, Inc., Santa Fe, NM) There were over thirty drugs that were associated with severe, often fatal, drug toxicity which was realized only after marketing.  In this overview, the current practice in drug development is critically reviewed.  The roles of new technologies such as human in vitro experimental systems, toxicogenomics, toxicoproteomics, and toxicometabolomics, in the early prediction of human drug toxicity will be discussed.

 

9:30 AM – 10:00 AM

Comparative Cytotoxicity Assay Using CYP-expressing Human Liver Cell Lines (Jim Xu; Pfizer - Groton Labs; Groton, CT)

 

10:00 AM – 10:30 AM

High Throughput Screening for Human Liver Based Toxicity (James H. Kelly; Amphioxus Cell Technologies, Inc.; Houston, TX) The success of high throughput drug development has emphasized the need for suitable toxicity and metabolism tools matched to HT discovery and lead optimization.  We have developed a human liver based system using a cell line and simple robust assays that can be applied at the earliest stages of drug discovery.  We have examined several aspects of drug metabolism and present evidence that the system is capable of a wide variety of liver specific metabolism.  Specific inductions of mRNA for cyp1A2 and 3A4 are presented, along with corresponding activity measurements and mechanism based toxicity.  We have screened a small, diverse library to demonstrate the utility of this system and were able to identify compounds with a variety of toxic effects as well as compounds active in altering signaling pathways.   Moreover, by examining series of structurally related compounds, toxicity-related SAR can be developed, yielding real time information for use in new synthesis.  Coupling such high throughput toxicity tools to current discovery efforts will allow simultaneous optimization of both the therapeutic and drug-like properties of new drug candidates. 

 

10:30 AM – 10:45 AM – BREAK

 

Session II:  Emerging Technologies for Toxicity Testing

(Chairs: Chandra Prakash and John Watson)

 

10:45 AM – 11:15 PM

Ion channel high throughput assays for the prediction of human QT prolongation (Keith Tang, Aurora Biomed Inc., Vancouver, B. C., CANADA) Cardiovascular disease and cardiac arrhythmias are one of the most common causes of death in the developed world. Arrhythmias can be associated with myocardial infraction, cardiac hypertrophy, and a rare congenital inherited condition known as long QT syndrome.  The human ether-a-go-go related gene (hERG) is one of the molecular targets of QT syndrome.  Drug interactions with hERG can lead to life threatening arrhythmias. The Ion Channel Reader (ICR) series provides high throughput screening technology based on Atomic Absorption Spectroscopy (AAS), which detects ion channel activity using Flux Assays. ICR offers sensitive, precise and reproducible measurements generating accurate drug rank order similar to that of patch clamp data. Recently, Aurora Biomed has optimized the Rb⁺ Flux Assay to quantify hERG channel inhibition for several known blockers, thus providing a powerful analytical tool for QT liability assessment.

 

11:15 AM – 11:45 AM.

Apoptosis vs. Cytotoxicity vs. Necrosis: Use of Cell-Based Assays to Distinguish Between Different Mechanisms of In Vitro Toxicity (John Watson, Promega Corp., Madison, WI) The use of automated cell based assays for in vitro toxicity screening of chemical compounds is becoming an increasingly important aspect of the drug discovery process.  Historically, in vitro toxicity testing provided information about the dose of a test compound required to kill half of the cells present, but no information was gathered about the mechanism of cell death.  Designing physiologically relevant cell-based assay protocols and measuring the appropriate endpoints during early screening process can help gather information about the mechanism of toxicity of candidate compounds, such as distinguishing between apoptosis and acute necrosis. Cells undergoing the process of apoptosis in vitro eventually loose membrane integrity and show the characteristics of secondary necrosis. We will present results of using a combination of assays to measure different endpoints including: an ATP assay to measure cellular metabolism, a caspase-3/-7 assay to measure apoptosis marker, and LDH release as an indicator of membrane integrity to measure cell viability.

 

11:45 AM – 12:15 PM

HALO - A New Multi-parameter In Vitro Hemotoxicity Testing Platform for All Stages of Drug Development. (Ivan N. Rich; HemoGenix LLC; Irmo, SC) Successful drug development is dependent on early screening and detection of toxicity and efficacy. The need for assay platforms that can predict toxicity and efficacy, before costly preclinical and, more importantly, clinical trials begin, is an absolute requirement. Alternative, in vitro assays can fulfill this requirement. Assay systems that employ fresh human target tissue provide the best and most reliable means to detect early toxicity and efficacy of new drug candidates. To date, routine in vitro hemotoxicity testing has not been used during early screening because the assay systems have been low throughput, labor intensive and expensive to perform. Despite this, the in vitro assays that detect stem cells and their progeny of the blood-forming system, have been demonstrated to provide a high degree of predictability and correlate with effects in the intact animal or patient. The new HALO (Hemotoxicity Assays via Luminescence Output) platform now provides a high-throughput, predictive, multitasking, multilineage and multitarget source testing system that can be used for early toxicity screening and all other stages of drug development. HALO is designed to detect both cell proliferation and apoptosis in all 8 primary blood-forming lineages, as well as stem cells, side-by side, and can compare the effects of compounds on human and a variety of animal target cells simultaneously.

 

12:15 AM – 1:45 PM - LUNCH BREAK

 

1:45 PM – 2:15 PM

In Vitro Toxicology Screening and Toxicity Characterization Using Automated Microscopy (David Monteith; Lilly Research Laboratories; Greenfield, IN) The presentation will provide an outline of strategies and data using automated microscopy systems in the screening and characterization of pharmaceutical toxicities. Specific examples will include evaluation of neurite outgrowth in a neurotox screen and multi-parameter evaluation of lysosomes and mitochondria to characterize hepatotoxicity. Automated microscopy techniques can be a valuable tool to characterize the potential mechanism of toxicity for a compound. While also a powerful tool for in vitro toxicology screening, limitations in throughput of automated microscopy require prioritization of candidate molecules to effectively assist pharmaceutical discovery.

 

2:15 PM – 2:45 PM 

Screening for Xenobiotic-Mediated P450 Induction in the Rat Using a Dedicated DNA Microarray System (Michael D. Kane; Genomic Solutions, Inc.; Ann Arbor, MI) Known inducers of CYP1A1-1A2, CYP3A, and CYP2E1 in rat (beta-naphthoflavone; 40 mg/kg IP, dexamethasone; 50 mg/kg IP, and isoniazid; 100 mg/kg IP, respectively) were dosed daily in rats for 1 or 4 days, and hepatic microsomes and RNA was isolated. Microsomal samples were assayed for enzyme activity. RNA samples were assayed for drug-induced changes in all known P450 isoforms in rat, and other gene products involved in metabolism and hepatic physiology (171 genes or gene families in total) using a dedicated DNA microarray. Beta-naphthoflavone (BNP) treatment resulted in a 23.9- and 26-fold increase in CYP1A1 mRNA on day 1 and 4, respectively, which was accompanied by an 11.4- and 13.5-fold increase in CYP1A1 enzyme activity. A similar response was seen in CYP1A2 following BNP treatment. Similarly, dexamethasone treatment and isoniazid treatment resulted in increases in both mRNA and enzyme activity levels in day 1 and day 4 samples. The DNA microarray also provided information from all study samples on drug-induced changes in many other metabolically relevant genes (mRNAs) from the same sample, augmenting information regarding the response of hepatic tissue to xenobiotic exposure.

 

2:45 PM – 3:15 PM

Novel Probe Substrates for Drug-Metabolizing Cytochrome p450s (James J. Cali, Ph.D.; Promega Corp.; Madison, WI) The modern approach to drug discovery includes the screening of large chemical libraries against targets of interest. Because of the central role of cytochrome p450s (p450) in drug metabolism it is also desirable to screen library compounds for their interactions with the drug metabolizing p450s. Knowledge of p450/drug interactions is predictive of drug disposition in a patient and useful for determining which compounds should be moved forward in the drug development process. P450 screening assays that can be processed in a high throughput mode are particularly attractive. Novel cytochrome p450 substrates will be described that can be used in scalable assays as probes for the effects of new chemical entities on cytochrome p450 activities. Kinetics of the reactions and cytochrome p450 isoform specificity of the substrates will be discussed.

 

3:15 PM – 3:30 PM - BREAK

 

3:30 PM – 4:00 PM-- CANCELLED

Automation and Validation of High Throughput In Vitro Multi-parameter Toxicity Screens (Thomas M. Fletcher III; Southern Research Institute; Birmingham, AL) The success of high throughput screening (HTS) programs at identifying new lead compounds has accentuated the need for related metabolism and toxicology tools to be used in parallel with activity optimization systems.  Examination of a compounds toxicological profile early in the discovery process can allow medicinal chemists to choose one class of compounds for development in favor of another.  We have successfully automated a series of HTS toxicology assays that can be used to provide real time feedback in conjunction with early activity screens.  Data on the optimization and validation of a number of commercially available multiparameter toxicity assay panels on fully automated robotic systems will be presented.  

 

 

4:30 PM – 5:30 PM

Round Table Discussion

 

END OF DAY/SYMPOSIUM