1. Toxicogenomics in the USA Pfizer, Global Research & Development Drug Safety Evaluation Ikuo HORII - Molecular toxicological approach in drug discovery and development - Current use of toxicogenomics in preclinical studies under assessing the current limitations and future promise

2. Gene related Factor Environment Diet Life-style Genes Organs Disease Side Effect Medicine s n Toxicity directly caused by DNA damage Critical !!! (Mutagenicity, Carcinogenicity, etc.) n Toxicity indirectly derived from the changes of related gene expression Manageable ! Disease is the outcome of the interaction between genes and environment Molecular Tox. Approach Toxicopanomics Toxicogenomics Toxicoproteomics Metabolomics Most toxicologically relevant outcomes require differential expression of multiple genes

3. Regulatory Tox. Single Admin. Tox. Repeated Admin. Tox. Reproduction Tox. Carcinogeneicity Gene Tox. Specific Tox. etc.. Investigative Tox. Traditional Toxicology for Safety Assessment Whole body assessment - General observation (Lethality, Clinical sign, …) - Body weight, Food consumption - Clinical Pathology (Blood chemistry, Hematology, Urinalysis, …) - Functional assessment (Hepatic/Renal, CV, …) - Histopathology (Organ wt., Morphology, …) In Vitro Alternative Molecular Toxicology - New Science - New Technology

4. Most toxicologically relevant outcomes require differential expression of multiple genes. If toxicity manifested at the level of organism is preceded by altered expression of related genes, its detection can serve as an early warning for subsequent deleterious outcomes Miniaturization and automation of new tools for analysis of gene expression and metabolic networking allow the molecular life of cells to be studied at a more holistic (and complex) level than was previously possible Gene expression and toxicology

5. Targeted Effect Molecular Toxicological Approach for Safety Assessment Tox. on extension of efficacy Tox. out of extension of efficacy Compound Target site ( Efficacy) Target site (Toxicity) Side Effect Central nervous Peripher.nervous Cardiovascular Respiratory Digestive Liver/Kidney Urinary Endocrine Hematopoietic Muscle/Skeletal Skin Sensory etc. Toxicity Toxicological Endpoint Efficacy Safety Molecular Tox. Approach Pharmacogenomics Toxicopanomics

6. Regulatory Toxicology Safety Assessment HTP-Tox. in vivo Pilot Tox. Investigative Tox…. HTP-Tox./DMPK - Molecular tox. - Cell culture New analytics - LC/MS/MS (cassette dose) Molecular-targeting (Genomics) Combinatorial Chemistry Many compounds Small amount of comp. Molecular-targeted comp. Change of toxicological approach Introduction of new technology Candidate CompoundLeadsScreening Nos of Compound Cost / Resources IND(Entry into human) NDA SRA/SRT Toxicogenomics Toxicoproteomics Metabolomics Molecular Toxicology Toxicopanomics + New Science

7. Study Design for Toxicogenomics/Toxicoproteomics Assessment Detection - Gene expression : Gene chip analysis, etc - Protein synthesis : 2-D Electrophoresis, Protein analysis Study Design (Comparison with known toxic-compounds under the database) In vivo Normal(non-treat) & Treated - Non - change In vitro Cell / Organ / Tissue - Up - regulation - Down - regulation Data Analysis Toxicity-related Gene Archives-database Database (Published information)

8. Significance of Toxicogemonics / Toxicoproteomics Approach --- Mechanistic investigation & prediction of toxicity --- Mechanistic Tox. Study Study result - Tox.related gene / protein - Mechanistic related metabolic pathway / action site Tox. Prediction Study - Expression profile of gene / protein in new compounds Prediction of Toxicity - Comparison of profiles with existing ( Strategy of drug discovery ) gene-related toxicological database

9. Genes on the toxicology gene chip Functional group Type of genes Stress responseOncogenes Acute phase response Signal transduction Transcription factors Cell proliferationCell cycle regulation Growth factors and receptor Tumor suppressors ApoptosisCaspases Apoptic regulators DNA damageDNA repair DNA morphology InflammationCytokines Vasoregulators, etc. Oxidative stressGlutathione metabolism Oxidase Protein thioles Drug metabolismCytochrome P450s Glutathione transferase UGT TransporterOrganic Peptide Ion pumps

10. DNA ： Genome Gene-polymorphism (Genomics) (SNPs etc) RNA ： Transcriptome Gene expression profile (Transcriptomics) (mRNA ) Protein ： Proteome Protein synthesis profile (Proteomics) (Molecular function) Biochemicals ： Metabolome Metabolite-pattern profile （ Metabolites) (Metabolomics) (Urine, etc) Toxicogenomics ・ Toxicoproteomics ・ Metabolomics

11. Timing of gene expression and protein synthesis --- Toxicological assessment point ? --- Toxicological stimulation(Trigger) DNA Signal ｍ RNA mRNA Level Toxicogenomics Appearance of toxicity Protein Protein Level Toxicoproteomics

12. x y z Outbreak of injury Cell injury Repair of injury Metabolic pattern in organism --- Process from toxicity appearance through damage to restoration --- 10 20 10 200 10 0 Metabolic change in injury site

13. liver (steatosis) heart renal medulla renal cortex control Pattern recognition - Combination of changes - Severity Toxicity type & site Pattern analysis Database

14. Ti  (t 1  p 1, t 2  p 1, t 3  p 1…. t i p i ) Gene expression, protein synthesis and metabolism in living body t 1  g 1, t 2  g 1, t 3  g 1, t 1  g 2, t 2  g 2, t 3  g 2, …... t 1  g i, t 2  g i, t 3  g Fn,m Biochemical changes in bio-fluid / cell / organ Metabolomics Toxicological Endpoint up or down Other organs Gene regulations etc. Blood Compound Phase II Metabolism m metabolites Liver t3t3 txtx t2t2 t1t1 T 1, T 2, T 3,etc Phase I Metabolism n metabolites Gene expression Protein synthesis Urine General metabolism

15. Toxicological Endpoint Assessment in Traditional Tox-biomarkers with New Markers of Toxicogenomics, Toxicoproteomics and Metabolomics Toxicological Endpoint Traditional Toxicological Parameters ： Clinical sign, Clinical pathology, Histopathology, etc. Protein synthesis Gene expression (Toxicogenomics) (Toxicoproteomics) Biochemical changes (bio-fluid, cell, organ) (Metabonomics)

16. Safety Database for Tailor-made Medical Treatment Lead compound NDA Candidate compound EIH(IND) Drug Drug discovery Clinical development Toxicity / Side-effect Prediction Countermeasure Safety Assessment Tool Guidance Expert System New data Safety Assessment Database Know - How Management Market Molecular Toxicology Tailor-made Medical Treatment

17. The 2002 Workshop on Pharmacogenetics/Pharmacogenomics in Drug Development and Regulatory Decision-Making --- Sponsored jointly the FDA, DruSafe PhRMA and PWG --- May 16 - 17, 2002 at the University of Maryland, Shady Grove Conference Center Toxicogenomics in Drug Development : Where are we today & where are we going ? Industry and regulatory agencies viewed this meeting as an opportunity to discuss how such data should be included/evaluated in IND and NDA applications. Where are we now ? Where would we like to be ?

18. (1) Is toxicogenomic science and validation technology sufficiently mature to reply upon genomic data for safety decisions and to justify the routine use of genomic data in GLP toxicology studies ? Current toxicogenomic data is not being collected in GLP studies, and the data is difficult to interpret and do not add to standard toxicology assays. However, genomic data is useful in mechanistic studies, and if done with IND compounds, the data should be submitted. There was some consensus that genomic data may be added to standard toxicology data, but we need to explore the “safe harbor” concept with FDA.

19. (2) Where is the value of toxicogenomic data to Industry and the FDA ? The value of toxicogenomic data now is in mechanistic studies and hypothesis testing and not predictive data in risk assessment. Most would like to develop more confidence in data, share data with FDA.

20. (3) How could data from genomic arrays, in conjunction with standard short-term toxicology studies, be used to assist in study design or in species selection for long-term toxicology studies ? The toxicogenomics is not well understood presently to be predictive, especially outside the rat/mouse species, of the human response. The standard toxicology studies need not include or be replaced by genomics, but genomic data may be used to better design of toxicology.

21. (4) Is there a need for guidance in the toxicogenomics area ? If guidance’s existed what wold be their main purpose and what would be the potential impact ? A regulatory guidance document is not necessary at this time. However, standard practice for reviewing data needs to be made transparent and a consensus of how data should be submitted would be useful. Thus, a white paper on how to review genomic data, within FDA, for internal consistency is recommended.

22. (5) Development of “historic databases” in interpreting toxicogenomic findings may be useful if the data are robust and reliable and if toxicogenomics profiles predict toxicities. If this is correct, how should such databases be developed and utilized ? The development of some form of knowledge base rather than a historical database for interpreting toxicogenomic findings. Since the technology is emerging, and data is limited, the potential of genomic data is a “red flag” awareness to evaluate in other toxicological assays.

23. As a conclusion, The application of toxicogenomics disciplines ranges from hypothesis testing of toxicity to safety evaluation. However, validation of the results for use in registration and marketing is limited and can only be evaluated on a case by case basis at the present time. As we progress, the regulatory implications of toxicogenomic data will be transparent and lead to relevant guidance documents.