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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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William A. Craig SymposiumISAP Research Meeting

PK/PD and Genomics

David Andes

University of Wisconsin


PK/PD and Genomics

  • Available tools

  • PK/PD utility


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


Pharmacodynamics and

Genomic Endpoint as Surrogate

CFU

  • In vivo Aspergillosis

  • Voriconazole exposure

GM

RT-PCR

Vallor AC, et al. AAC 52:2593, 2008


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


MPA ResistanceDominant Selectable Marker

Andes et al AAC 2006;50:2374


Pharmacodynamic Tracking of Mixed Cell Populations

Andes et al AAC 2006;50:2374


Microarray

Transcriptional Profile

Northern Blot

Real time RT-PCR


CHEMOGENOMICS

  • Transcriptional signature related to drug exposure

    • Target and MOA insight


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


CHEMOGENOMICS+Pharmacodynamics

  • Transcriptional signature related to drug exposure considered pharmacodynamically

    • Concentration

    • Time

    • PD phenomena mechanism

    • PD resistance development


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


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


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


In vivo Time Course Response to Fluconazole

Perturbation

Up regulatedDown regulated

Plasma membrane synthesis/maintenanceDNA synthesis

Cell wall synthesis/maintenanceProtein synthesis

Cell stress response

Carbohydrate metabolism

Lepak et al AAC 2006;50:1311


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


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


Azole

Pharmacodynamics

And Emergence of

Resistance

Phenotype

Andes et al AAC 2006;50:2384


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

Later Resistance Development

Fluconazole and Candida

N = 69 genes

Andes et al AAC 2006;50:2384


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.


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