William A. Craig SymposiumISAP Research Meeting

PK/PD and Genomics

David AndesUniversity 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

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

24-72 hours

Susceptible

Fluconazole

1:10 dilutions

Resistant

Reconstruction Population Biology

Resistance Development

0.1, 1, 10%

90, 99, 99.9%

MPA ResistanceDominant Selectable Marker

Figure1. Candida albicans host cell and transformant selected on the basis of Mpar. DNA was extracted and digested with BamHI and NsiI, electrophoresed and blotted. Blot was hybridized with an ARG4 radiolabeled probe upstream of the IMH3r disruption. This blot demonstrates that the IMH3r allele integrated at the ARG4 locus and displaced the NsiI restriction site.

Andes et al AAC 2006;50:2374

Pharmacodynamic Tracking of Mixed Cell Populations

q24 h Total Dose (mg/kg)

0.1 1 10 100 1000

Log

10 C

FU

/Kid

neys

1

2

3

4

5

6

7

FH1 FH5 Plot 2 Upper Specification

q12 h Total Dose (mg/kg)

0.1 1 10 100 1000 10000

1

2

3

4

5

6

7

q6 h Total Dose (mg/kg)

0.1 1 10 100 1000 10000

1

2

3

4

5

6

7

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

MethodKetoconazoleCandida albicansIn vitroIC50 concentration X 4 h

Major Expression Categories• Lipid metabolism• Fatty acid metabolism• Sterol metabolism

CHEMOGENOMICS+

Pharmacodynamics

• Transcriptional signature related to drug exposure considered pharmacodynamically – Concentration– Time

– PD phenomena mechanism– PD resistance development

Effect of Prolonged Exposure to a Sub-MIC Fluconazole Concentration on the Expression of Ergosterol

Pathway Genome in S. cerevisiae

ER

G 1

0E

RG

13

HM

G 2

ER

G 1

2E

RG

08E

RG

19LP

H 1

0CE

RG

20

ER

G 0

9E

RG

01

ER

G 0

7E

RG

11

ER

G 2

4E

RG

25

ER

G 0

6E

RG

02

ER

G 0

3E

RG

05

ER

G 0

4A

CT

INRat

io o

f Exp

osur

e/C

ontr

ol m

RN

A E

xpre

ssio

n

0

1

2

3

4

5control .025xMIC Reference

Effect of Increasing Fluconazole Concentration on theExpression of Ergosterol Pathway Genome in S. cerevisiae

ER

G 1

0E

RG

13

HM

G 2

ER

G 1

2E

RG

08E

RG

19LP

H 1

0CE

RG

20

ER

G 0

9E

RG

01

ER

G 0

7E

RG

11

ER

G 2

4E

RG

25

ER

G 0

6E

RG

02

ER

G 0

3E

RG

05

ER

G 0

4A

CTI

NRat

io o

f Exp

osur

e/C

ontro

l mR

NA

Exp

ress

ion

0

1

2

3

4

5

6

Control 1xMIC 4xMIC Reference

Fluconazole Pharmacodynamic Exposures and Ergosterol Path Response (SC In vitro)

No DrugAt MIC 1 h4x MIC 1 h

No Drug¼ MIC 8 h

AUC of ExposureAt MIC 1h < 4x MIC 1 h = ¼ MIC for 8h

Andes et al ICAAC 2000

 

0.00

1.00

2.00

3.00

4.00

5.00

6.00

7.00

Lo

g R

ati

o C

han

ge i

n E

xp

ressio

n

SUB-MIC

MIC

SUPRA-MIC

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

  

Fluconazole Pharmacodynamics and Transcription Response

of Resistance Genes (C. albicans - In vitro)

Time (hours)

6 8 10 12 14

Fo

ld-C

ha

ng

e

0

2

4

6

8

10

12

16 X MIC4 X MIC1 X MIC0.25 x MIC0.06 x MIC

CDR2 - RT-PCR

6 8 10 12 140.0

0.5

1.0

1.5

2.0

2.5

3.0

16 X MIC4 X MIC1 X MIC0.25 x MIC0.06 X MIC

ERG11 - NORTHERN BLOT

DRUG DRUG

During and Following ExposureLepak et al AAC 2006;50:1311

In vivo PD andTranscriptional Profiling

Homogenize in Water

Lysed mouse cells

Free mouse nucleic acid

Intact Candida DNase

Differential Centrifugation

Supernatant

RnaseRnase inh

Intact Candida – Mouse RNA and DNA

Break YeastIsolate RNA

Candida RNALepak et al AAC 2006;50:1311

Up regulated Down regulated Plasma membrane synthesis/maintenance DNA synthesisCell wall synthesis/maintenance Protein synthesisCell stress responseCarbohydrate metabolism

In vivo Time Course Response to FluconazolePerturbation

Lepak et al AAC 2006;50:1311

PAE Model = damage response model in which the plasma membrane and cell wall are structurally and functionallydamaged, followed by a period of recovery manifested by enhanced nucleic acid and protein synthesis to repair the cell.

Up regulatedDNA synthesisProtein synthesis

In vivo Time Course Response to FluconazoleRecovery

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 ArchiveResistance Development

% T>MIC

0 20 40 60 80 100 1202

3

4

5

6a b c d e f g h i j

24-h AUC/MIC

8 32 90 1302

3

4

5

6a1 b1 c1 d1 e1 f1 g1 h1 i1 j1

Lo

g 10 R

es

ista

nt C

FU

/Kid

ne

ys

Cmax/MIC

0.340.661.342.665.32 16.32

3

4

5

6a2 b2 c2 d2 e2 e2 f2 g2 h2 i2 j2

AzolePharmacodynamics And Emergence of ResistancePhenotype

Andes et al AAC 2006;50:2384

TREATMENT REGIMENS MICs (parent K1 MIC 0.5 g/ml) STUDY 1 2 3 4 5 6 7 8

A 1A 2A 3A 4A 5A 6A 7A 8A 0.5 0.5 0.5 0.5 0.5 0.5 1.0 0.5

B 1B 2B 3B 4B 5B 6B 7B 8B 1.0 0.5 0.5 0.5 0.5 1.0 1.0 1.0

C 1C 2C 3C 4C 5C 6C 7C 8C 2.0 0.5 0.5 1.0 0.5 2.0 1.0 1.0

D 1D 2D 3D 4D 5D 6D 7D 8D 1.0 0.5 0.5 2.0 0.25 2.0 1.0 1.0

E 1E 2E 3E 4E 5E 6E 7E 8E 2.0 0.5 0.5 2.0 0.5 4.0 1.0 0.5

F 1F 2F 3F 4F 5F 6F 7F 8F 2.0 0.5 1.0 2.0 1.0 4.0 1.0 0.5

G 1G 2G 3G 4G 5G 6G 7G 8G 4.0 0.5 0.5 2.0 1.0 4.0 2.0 0.5

H 1H 2H 3H 4H 5H 6H 7H 8H 4.0 0.5 0.5 8.0 0.5 8.0 1.0 1.0 I 1I 2I 3I 4I 5I 6I 7I 8I 4.0 0.5 0.5 8.0 0.5 8.0 2.0 1.0

J 1J 2J 3J 4J 5J 6J 7J 8J 4.0 0.5 0.5 8.0 0.5 8.0 1.0 1.0

Andes et al AAC 2006;50:2384

  

% T>MIC0 20 40 60 80 100

Fo

ld C

ha

ng

e i

n E

xp

res

sio

n

0.0

0.5

1.0

1.5

2.0

2.5

3.0 CDR1CDR2

AUC/MIC8 32 90 129

0.0

0.5

1.0

1.5

2.0

2.5

3.0

Peak/MIC0.34 0.66 1.34 2.66 5.32 16.3

0.0

0.5

1.0

1.5

2.0

2.5

3.0

Comparative Quantitative RT-PCR

Resistant Archive

Susceptible Start

Andes et al AAC 2006;50:2384

4J/K1 Up

Protein Synthesis6%

Cell Membrane6%

Cell Cycle2%

Stress Response6%

Other10%

Amino Acid Synthesis15%

Transport15%

Unknown40%

Whole Genome Expression Later Resistance Development

Fluconazole and Candida

4J/K1 Down

Heme Synthesis12%

DNA Synthesis12%

Cell Membrane12%

Unknown18%

Energy Production & Utilization28%

Transport6%

Stress Response6%

Other6%

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.