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Presented by Gordon Holt, Ph.D. at the Nonclinical Studies Subcommittee of the Advisory Committee for Pharmaceutical Sc PowerPoint Presentation
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Presented by Gordon Holt, Ph.D. at the Nonclinical Studies Subcommittee of the Advisory Committee for Pharmaceutical Sc

Presented by Gordon Holt, Ph.D. at the Nonclinical Studies Subcommittee of the Advisory Committee for Pharmaceutical Sc

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Presented by Gordon Holt, Ph.D. at the Nonclinical Studies Subcommittee of the Advisory Committee for Pharmaceutical Sc

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

  2. OGS Proteomics Technology: Overview

  3. 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

  4. Comparison of Microexpression Analyses 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

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

  6. 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 clinicallyrelevant early markers of cardiotoxicity?

  7. 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

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

  9. Enriched Normal

  10. RosettaTM 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

  11. 20 10 2 6 10 21 22 23 24 26 27 28 30 32 33 35 36 38 44 45 46 47 0 Fold Change 3 5 7 9 25 29 31 34 37 39 40 41 42 43 -10 -20 Doxorubicin vs control Doxorubicin + ICRF vs. control -30 Doxorubicin treatment markers

  12. 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

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

  14. 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

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

  16. 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

  17. 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

  18. Serum Proteomics • Immunoaffinity enrichment • 30 images • 2,580 MCIs • 21,001 features

  19. 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

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

  21. 17 normals 17 primary breast tumors 17 metastatic breast tumors >> Serum enrichment protocol Breast Cancer - pilot serum marker study

  22. 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 Breast cancer sera - Differentials

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

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