Presented by Gordon Holt, Ph.D. at the Nonclinical Studies Subcommittee of the Advisory Committee for Pharmaceutical Science March 9, 2000

OGS Proteomics Technology: Overview

Challenges for Proteomics Validation ProblemSolution Sample variabilityProcess validation Low sensitivityImmunoaffinity enrichment Subcellular fractionation Fluorescent dyes Imaging Gel variabilityProcess validation Image warping Low throughputRobotics Data analysis overloadLIMS management

GenomicsProteomics Coverage per runAll genes2000 features Sensitivity limits 1 mol./cell100 mol./cell Protein modificationsNoYes Subcellular localizationNoYes Subunits / ComplexesNoYes Clinical samplesNoYes Comparison of Microexpression Analyses

Identification of Cardiotoxicity and Vasculitis Surrogate Markers Work done in collaboration with Frank Sistare CDER FDA

Investigation of Doxorubicin-Induced Toxicity Doxorubicin (Dxr) background commonly used anticancer agent –effective against childhood leukemia causes dose-related cardiotoxicity –precise toxicity mechanism unknown –metal ions appear to be important metal chelation by ICRF-187 provides significant chemoprotection >> Can proteomics identify clinically relevant early markers of cardiotoxicity?

Doxorubicin cardiotoxicity study design Three rats per group 1 mg/kg doxorubicin per week Treat for 7 weeks Sacrifice animals 24 hrs after last dose Proteomic analyses of plasma –OGS plasma sample SOP –serum enrichment applied

Detection of disease-specific proteins in serum, CSF, synovial fluid Limitation :High-abundance proteins limit sensitivity - albumin - haptoglobin - IgG - transferrin Solution :Immunoaffinity enrichment protocol

Normal Enriched

Rosetta TM Analyses Preliminary study performed on 18 PEMs –one PEM per plasma sample from each rat –approx features in master group –32,400 features screened High-stringency marker selection criteria –98% marker confidence –100% marker incidence on PEMs from given group of three rats

Doxorubicin + ICRF vs. control Doxorubicin treatment markers Fold Change Doxorubicin vs control

Summary of results 34 Dxr treatment markers identified –magnitude up to 28.1-fold >> may be clinically useful Dxr toxicity markers Most Dxr markers normalized by ICRF co- treatment –29 fully normalized –5 partially normalized >> supports ICRF’s protection against Dxr toxicity

Dxr Cardiotoxicity - Preliminary Annotations Lipid metabolism - liposome formation Immune surveillance - complement fixation Wound healing - scar formation - protease inhibition Anti-oxidant metabolism - metal scavenging

Vasculitis study design & status Three rats per group 100 mg/kg SKF Treat for 1, 2, 4, 24 hours Histology completed Proteomic analyses of plasma –OGS plasma sample SOP –serum enrichment applied –PEMs run –differential analysis underway

Identification of Nephrotoxicity Surrogate Markers Work done in collaboration with Quintiles UK

Gentamicin Background  Parenteral aminoglycoside active against gram- negative bacteria  Clinical important toxicity - potential for irreversible cumulative ototoxicity (manifest as hearing loss - initially of high frequencies) and vestibular damage  Reversible nephrotoxicity may occur and acute renal failure reported  Therapeutic index - individual monitoring of plasma concentrations generally required

7 Days of treatment followed by 14 day recovery period Route of administration:intravenous Dose levels:0, 0.1, 1, 10, 40, 60 mg/kg/day Group size:10 male rats per treated group, 20 male rats in control group Blood and urine samples:2, 3 and 8 days Blood Parameters:BUN, creatinine Urine parameters:NAG, ALP, GGT, volume, specific gravity Renal histopathology:standard Proteome samples:420 Study Protocol

 Immunoaffinity enrichment  30 images  2,580 MCIs  21,001 features Serum Proteomics

 Single protein linked to regulation of alternate pathway of complement  Human proximal tubular epithelial cells specifically bind to components of the alternate complement pathway >> Appears at lower dose than identified by conventional means Summary to date

Identification of Breast Cancer Serum Surrogate Markers Work done in collaboration with Prof. C. Coombes CRC London

Breast Cancer - pilot serum marker study 17 normals 17 primary breast tumors 17 metastatic breast tumors >> Serum enrichment protocol

Breast cancer sera - Differentials feature change has p value 50% of PEMs 63 potential marker proteins identified Normal vs primary diff. proteins Normal vs metastatic diff. proteins Primary vs metastatic diff. proteins >> proteins consistent with breast disease stage >> new proteins previously unassociated with breast

Summary: OGS Proteomics Identify disease-specific proteins Identify treatment-specific proteins >> Quantitative and qualitative >> Synergy with genomics data >> Powerful tool for surrogate marker identification

Key Elements for Partnership to Identify Surrogate Markers Institutional mandates of major players –FDA / NIH –academic –industrial Development stage-specific pressures –discovery, validation, commercialization Intellectual property ownership –discoverers (probably) not developers