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William A. Craig Symposium ISAP Research Meeting

William A. Craig Symposium ISAP Research Meeting. PK/PD and Genomics David Andes University of Wisconsin. PK/PD and Genomics. Available tools PK/PD utility. PK/PD and Genomics Tool Use. Sequence analysis Detect resistance mutations Genetic reporters e.g. GFP, selectable markers

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William A. Craig Symposium ISAP Research Meeting

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  1. William A. Craig SymposiumISAP Research Meeting PK/PD and Genomics David Andes University of Wisconsin

  2. PK/PD and Genomics • Available tools • PK/PD utility

  3. PK/PD and GenomicsTool Use • Sequence analysis • Detect resistance mutations • Genetic reporters e.g. GFP, selectable markers • Track mixed cell populations • Track expression of gene of interest • Transcriptional profiling • Single gene • Track expression of gene of interest • Surrogate organism burden endpoint • Genome • Investigate expression of entire genome • Proteomics • Investigate translation of genome

  4. Pharmacodynamics and Genomic Endpoint as Surrogate CFU • In vivo Aspergillosis • Voriconazole exposure GM RT-PCR Vallor AC, et al. AAC 52:2593, 2008

  5. Fluconazole Susceptible 90, 99, 99.9% 18 Treatment Regimens X 24-72 hours 1:10 dilutions Resistant 0.1, 1, 10% Reconstruction Population Biology Resistance Development Fluconazole

  6. MPA ResistanceDominant Selectable Marker Andes et al AAC 2006;50:2374

  7. Pharmacodynamic Tracking of Mixed Cell Populations Andes et al AAC 2006;50:2374

  8. Microarray Transcriptional Profile Northern Blot Real time RT-PCR

  9. CHEMOGENOMICS • Transcriptional signature related to drug exposure • Target and MOA insight

  10. Genomic Response of Candida to Triazole • Method • Ketoconazole • Candida albicans • In vitro • IC50 concentration X 4 h • Major Expression Categories • Lipid metabolism • Fatty acid metabolism • Sterol metabolism Liu et al AAC 49:2226, 2005

  11. CHEMOGENOMICS+Pharmacodynamics • Transcriptional signature related to drug exposure considered pharmacodynamically • Concentration • Time • PD phenomena mechanism • PD resistance development

  12. No Drug ¼ MIC 8 h No Drug At MIC 1 h 4x MIC 1 h Fluconazole Pharmacodynamic Exposures and Ergosterol Path Response (SC In vitro) AUC of Exposure At MIC 1h < 4x MIC 1 h = ¼ MIC for 8h Andes et al ICAAC 2000

  13. Fluconazole Pharmacodynamic Exposures and the Entire Genome (SC In vitro) Genes with pharmacodynamic response Andes et al ICAAC 2000

  14. Resistance Genes and Drug Exposure – Pharmacodyanmic Consideration • Examine the relationship between defined fluconazole pharmacodynamic exposures and the expression of ‘resistance’ genes in C. albicans

  15. During and Following Exposure Lepak et al AAC 2006;50:1311

  16. Homogenize in Water In vivo PD andTranscriptional Profiling Differential Centrifugation Rnase Rnase inh Lysed mouse cells Free mouse nucleic acid Intact Candida DNase Supernatant Break Yeast Isolate RNA Intact Candida – Mouse RNA and DNA Candida RNA Lepak et al AAC 2006;50:1311

  17. In vivo Time Course Response to Fluconazole Perturbation Up regulated Down regulated Plasma membrane synthesis/maintenance DNA synthesis Cell wall synthesis/maintenance Protein synthesis Cell stress response Carbohydrate metabolism Lepak et al AAC 2006;50:1311

  18. In vivo Time Course Response to Fluconazole Recovery Up regulated DNA synthesis Protein synthesis PAE Model = damage response model in which the plasma membrane and cell wall are structurally and functionally damaged, followed by a period of recovery manifested by enhanced nucleic acid and protein synthesis to repair the cell. Lepak et al AAC 2006;50:1311

  19. Fluconazole Susceptible C. albicans K1 8 Treatment Regimens X 72 hours 1:10 dilutions Pharmacodynamic Archive Resistance Development Re-infect, Treat, Collect X 10 Archive A1, B1, C1, through J1 Fluconazole

  20. Azole Pharmacodynamics And Emergence of Resistance Phenotype Andes et al AAC 2006;50:2384

  21. Andes et al AAC 2006;50:2384

  22. Comparative Quantitative RT-PCR Resistant Archive Susceptible Start Andes et al AAC 2006;50:2384

  23. Whole Genome Expression Later Resistance Development Fluconazole and Candida N = 69 genes Andes et al AAC 2006;50:2384

  24. Whole Genome Expression During Resistance Development • 4E = 4-fold less susceptible (Day 15) • Up = protein synthesis • Down = energy production and utilization • 4J = 16-fold less susceptible (Day 30) • Up = amino acid and carbohydrate transport and cell membrane maintenance • Down = energy production and utilization Model: The expression of these genes suggest cell membrane changes may contribute to resistance or may could simply represent a response to cell-damaging conditions.

  25. PK/PD and Genomics • Pharmacodynamics consideration impacts genome expression answer • Genomic tools provide resistance tracking tools, surrogate endpoints, and insight into mechanism of PD phenomena.

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