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 –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

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

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

MPA Resistance Dominant Selectable Marker Andes et al AAC 2006;50:2374

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

Transcriptional Profile Northern Blot Real time RT-PCR Microarray

CHEMOGENOMICS Transcriptional signature related to drug exposure –Target and MOA insight

Liu et al AAC 49:2226, 2005 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

Candida glabrata Proteomic Analysis in Azole Induced Resistance Rogers PD et al. J Antimicrob Chemother 2006;58: proteins Efflux pumps Sterol pathway Carbohydrate metabolism Oxidative stress Post-transcriptional mechanism for Erg11p expression

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

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

Fluconazole Pharmacodynamic Exposures and the Entire Genome (SC In vitro) Andes et al ICAAC 2000

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

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

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

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

Up regulatedDown regulated Plasma membrane synthesis/maintenanceDNA synthesis Cell wall synthesis/maintenanceProtein synthesis Cell stress response Carbohydrate metabolism In vivo Time Course Response to Fluconazole Perturbation Lepak et al AAC 2006;50:1311

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. Up regulated DNA synthesis Protein synthesis In vivo Time Course Response to Fluconazole Recovery Lepak et al AAC 2006;50:1311

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

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

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

Whole Genome Expression Early Resistance Development Fluconazole and Candida Andes et al AAC 2006;50:2384 N = 167 genes

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

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.

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.