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

Gene related FactorEnvironment

Diet

Life-style

Genes OrgansDisease

Side Effect

Medicines

Toxicity directly caused by DNA damage Critical !!! (Mutagenicity, Carcinogenicity, etc.)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 differentialexpression of multiple genes

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

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

Targeted Effect

Molecular Toxicological Approach for Safety Assessment

Tox. on extension of efficacy

Tox. out of extension ofefficacy

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

Regulatory Toxicology

Safety Assessment

HTP-Tox. in vivo Pilot Tox. Investigative Tox….

HTP-Tox./DMPKHTP-Tox./DMPK - Molecular tox. - Molecular tox. - Cell culture - Cell cultureNew analyticsNew analytics - LC/MS/MS - LC/MS/MS (cassette dose) (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

ToxicogenomicsToxicoproteomicsMetabolomics

Molecular Toxicology 　 Toxicopanomics + New Science

Study Design for Toxicogenomics/Toxicoproteomics Assessment

Detection - Gene expression : Gene chip analysis, etc - Protein synthesis : 2-D Electrophoresis, Protein analysisStudy 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)

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

Genes on the toxicology gene chip

Functional group Type of genesStress response Oncogenes

Acute phase responseSignal transductionTranscription factors

Cell proliferation Cell cycle regulationGrowth factors and receptorTumor suppressors

Apoptosis CaspasesApoptic regulators

DNA damage DNA repairDNA morphology

Inflammation CytokinesVasoregulators, etc.

Oxidative stress Glutathione metabolismOxidaseProtein thioles

Drug metabolism Cytochrome P450sGlutathione transferaseUGT

Transporter OrganicPeptideIon pumps

　 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

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

xy

z

Outbreak of injury

Cell injury

Repair of injury

Metabolic pattern in organism--- Process from toxicity appearance through damage to restoration ---

10

20

10

20 0

10

0

Metabolic changein injury site

liver (steatosis)

heart

renal medulla

renal cortex

control

Pattern recognition- Combination of changes- Severity

Toxicity type & site

Pattern analysis

Database

Ti(t1p1,t2p1,t3p1….tipi )

Gene expression, protein synthesis and metabolism in living body

t1g1, t2g1, t3g1, t1g2, t2g2, t3g2, …...t1gi, t2gi,

t3gFn,m

Biochemical changes in bio-fluid / cell / organMetabolomics

Toxicological Endpoint

up or down

Other organs

Gene regulations etc.

Blood

Compound Phase II Metabolismm metabolites

Livert3

tx

t2

t1

T1, T2, T3,etc

Phase I Metabolismn metabolites Gene expression

Protein synthesis

Urine

General metabolism

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)

Safety Database for Tailor-made Medical Treatment

Lead compound

NDA

Candidate compound

EIH(IND)

Drug

Drug discovery

Clinical development

Toxicity / Side-effectPrediction

Countermeasure

Safety Assessment ToolGuidance Expert System

New data

Safety Assessment Database

Know - How Management

Market

Molecular Toxicology Tailor-madeMedical Treatment

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 opportunityto discuss how such data should be included/evaluated in IND and NDAapplications.

Where are we now ?Where would we like to be ?

(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.

(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.

(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.

(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.

(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.

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