Presented by

Gordon Holt, Ph.D.at the

Nonclinical Studies Subcommitteeof the

Advisory Committee for Pharmaceutical Science

March 9, 2000

OGS Proteomics Technology: Overview

Challenges for Proteomics Validation

Problem

Solution• Sample variability

Process validation

• Low sensitivity

Immunoaffinity enrichment

Subcellular fractionation

Fluorescent dyes

Imaging

• Gel variability

Process validation

Image warping

• Low throughput

Robotics

• Data analysis overload

LIMS management

Genomics Proteomics

Coverage per run All genes 2000 features

Sensitivity limits 1 mol./cell 100 mol./cell

Protein modifications No Yes

Subcellular localization No Yes

Subunits / Complexes No Yes

Clinical samples No Yes

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 proteinsin 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. 1800 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

-30

-20

-10

0

10

20

Fold

Ch

an

ge

3 5 7 9 25 29 31 34 37 39 40 41 42 43

2 6 10 21 22 23 24 26 27 28 30 32 33 35 36 38 44 45 46 47

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 95654

• 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 < 0.05 for > 50% of PEMs

• 63 potential marker proteins identified

• Normal vs primary ........ 16 diff. proteinsNormal vs metastatic ..... 20 diff. proteinsPrimary vs metastatic .... 27 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