DTExtm Gene Expression Analysis Microarray based method - 768 element array

145 genes - 4 elements per gene

12 grids - 64 elements [16 x 4] per grid

All DTExtm genes are involved in drug metabolism, conjugation or transport* interrogate for both reported and unreported drug effects on gene expression

* investigate coordinate regulation of gene expression, protein expression and functional activity

* all genes are RefSeq entries verified by NCBI with published literature history

All DTExtm genes are members of coordinate regulatory pathways * internal control for induction or suppression of gene expression

* control for drug selectivity or specificity in the modulation of gene expression

DTExtm microarray is an ADME gene expression survey and screening method

* analysis of ADME gene expression signature(s) for cell or cell line characterisation and comparison

* analysis of ADME gene expression signature(s) and associated effects in drug treated cells or cell lines

- isogenic cancer cell lines

- hepatocytes

- tissue surrogates [HepaRG, Fa2N-4, etc.]

- tissues [liver, kidney, brain, eye, etc.]

DTExtm microarray analysis is reliable and reproducible* either biological replicates or experimental replicates

ADME and DTExtm Microarray Analysis

The regulation of gene expression of various phase I enzymes, phase

II enzymes and phase III transporters has significant potential impact

on the metabolism, elimination, pharmacokinetics / dynamics,

toxicokinetics / dynamics and drug-drug interactions of many

therapeutic agents, as well as their ability to protect the human body

against exposure to environmental xenobiotics.

(Kong 2002, Guengerich 2003, LeCluyse 2003, Kong 2005)

Regulation of Cytochrome P450 gene expression

CYPs Ligands NXRs Modulators

CYP1 Polycyclic AHs,

Halogenated AHs

AHR [Arnt] 3MC, BNF, OMP

CYP2B Steroids CAR [RXR] PBB, CITCO

CYP2C Prostaglandins,

Steroids

CAR [RXR]

PXR [RXR]

PBB, RIF

CYP3A Steroids PXR [RXR] DEX, RIF [hu],

PCN [rt]

CYP4A Prostaglandins,

FAs

PPAR [RXR] DEX, CLF,

Wy14,643

CYP7A Cholesterol,

BAs

LXR [RXR]

FXR [RXR]

CDCA, APD,

22(R)HC,

T0901317

DTExtm Microarray Analysis

What is DTExtm?

A method that allows the simultaneous analysis of changes in the levels of gene expression of 145 ADME associated genes.

How is DTExtm done?

Total RNA converted to cDNA > cDNA coverted to aRNA > aRNA converted to labelled cDNA > hybridise to DTExtm microarray > scan > quantitation > first pass data analysis > second pass data analysis > differential gene expression analysis [induction, suppression].

Why use DTExtm?

Survey basal level gene expression in various primary cells, cell lines or tissues as a prelude to induction, inhibition or toxicity testing.

Survey coordinated changes in the levels of gene expression in various primary cells, cell lines or tissues as a consequence of drug treatment.

DTExtm Microarray Gene Set

Gene Group Phase Group Members Number

Cytochrome P450s

[CYPs]

I 1A, 1B, 2A, 2B, 2C, 2D, 2E, 3A, 4A,

4B, 7A, 8B, 19A, 27A, 27B

18

Nuclear Xenobiotic

Receptors [NXRs]

I AHR, AR, CAR, FXR, HNF, LXR,

NFE, NRF, PPAR, PXR, RXR, VDR

19

Sulfotransferases

[SULTs]

II 1A, 1B, 1C, 1E, 2A, 2B 6

UDP glucuronosyl

transferases [UGTs]

II 1A, 2A, 2B, 8 6

SLC (uptake)

transporters [SLCs]

III CNT, ENT, LST, NTCP, OAT, OATP,

OCT, OCTN, OST, PEPT, PGT,

URAT

36

ABC (efflux)

transporters [ABCs]

III A, B, C, D, E, F, G 49

Controls 18S, 28S, ACT, B2M, GDH, S28,

SH1, SH2, RPLP0, TUB, VIL1

11

DTExtm Microarray Analysis

What is DTExtm?

A method that allows the simultaneous analysis of changes in the levels of gene expression of 145 ADME associated genes.

How is DTExtm done?

Total RNA converted to cDNA > cDNA converted to aRNA > aRNA converted to labelled cDNA > hybridise to DTExtm microarray > scan > quantitation > first pass data analysis > second pass data analysis > differential gene expression analysis [induction, suppression].

Why use DTExtm?

Survey basal level gene expression in various primary cells, cell lines or tissues as a prelude to induction, inhibition or toxicity testing.

Survey coordinated changes in the levels of gene expression in various primary cells, cell lines or tissues as a consequence of drug treatment.

DTExtm Microarray Analysis

What is DTExtm?

A method that allows the simultaneous analysis of changes in the levels of gene

expression of 145 ADME associated genes.

How is DTExtm done?

Total RNA converted to cDNA > cDNA coverted to aRNA > aRNA

converted to labelled cDNA > hybridise to DTExtm microarray > scan >

quantitation > first pass data analysis > second pass data analysis >

differential gene expression analysis [induction, suppression].

Why use DTExtm?

Survey basal level gene expression of all 145 ADME associated genes in various

primary cells, cell lines or tissues as a prelude to induction, inhibition or toxicity

testing.

Survey coordinated changes in the levels of gene expression in various primary

cells, cell lines or tissues as a consequence of drug treatment.

Basal level DTExtm gene expression in human hepatocytes

Matrix plot of relative level of gene expression data

Cluster plot of relative level of gene expression data

DTExtm Microarray Analysis

What is DTExtm?

A method that allows the simultaneous analysis of changes in the levels of gene expression of 145 ADME associated genes.

How is DTExtm done?

Total RNA converted to cDNA > cDNA coverted to aRNA > aRNA converted to labelled cDNA > hybridise to DTExtm microarray > scan > quantitation > first pass data analysis > second pass data analysis > differential gene expression analysis [induction, suppression].

Why use DTExtm?

Survey basal level gene expression of all 145 ADME associated genes in various primary cells, cell lines or tissues as a prelude to induction, inhibition or toxicity testing.

Survey coordinated changes in the levels of gene expression for all 145 ADME associated genes in various primary cells, cell lines or tissues as a consequence

of drug treatment.

DTExtm gene expression in six rifampicin treated human hepatocyte lots

Matrix plot of relative level of gene expression data [range = 0-60x actin]

Cluster plot of relative level of gene expression data [range = 0-60x actin]

Induction of DTExtm gene expression in six rifampicin treated human hepatocyte lots

Matrix plot of relative level of gene expression induction [range = 0-4x vehicle treated]

Cluster plot of relative level of gene expression induction [range = 0-4x vehicle treated]

Induction of DTEx gene expression in six rifampicin treated human hepatocyte lots

0.00

0.50

1.00

1.50

2.00

2.50

3.00

3.50

4.00abcA7

abcC8

cyp2C9

NTCP1

PGT

UGT2B17

SLC22A3

abcA1

abcB7

abcF2

RXRG

OATPD

UGT2A1

OATPRP4

abcA8

abcC9

cyp2C19

OAT1

UGT2B15

ORCTL4

abcB2

abcD2

cyp27B1

OATPE

AHR

SULT2B1

abcC4

cyp1B1

ENT2

cyp4A11

abcA3

abcB9

abcG1

PPARA

OATPF

cyp7A1

abcA10

abcC11

cyp2E1

OAT3

cyp8B1

abcA4

abcB10

abcG2

PPARB

cyp4A22

abcA12

abcC12

cyp3A4

OAT4

cyp4B1

abcA5

abcB11

abcG4

PPARG

OCT_1

NRF1

abcC7

cyp2C8

LST2

CAR

OSTB

URAT1

abcB6

abcF1

RXRB

OCT_2

Induction of CYP450 gene expression in six rifampicin treated human hepatocyte lots

0.00

0.50

1.00

1.50

2.00

2.50

3.00

3.50

4.00

cyp2C9

cyp27A1

cyp1A2

cyp2C19

cyp27B1

cyp1B1

cyp2D6

cyp4A11

cyp7A1

cyp2A6

cyp2E1

cyp8B1

cyp4A22

cyp2B6

cyp3A4

cyp4B1

cyp2C8

cyp19A1

Induction of DTExtm gene expression in six rifampicin treated human hepatocyte lots

Cluster plot and histogram of CYP gene expression induction ratios

[range = 0-4x vehicle treated]

0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00

cyp3A4

cyp2E1

cyp2C19

cyp2C9

cyp2C8

cyp1B1

cyp1A2

Coordinate regulation of DTExtm gene expression in RIF treated human hepatocytes

1.261.37

1.28

1.621.44

2.11

0.87

2.86

0.95

1.54

1.07

1.45

0.00

0.50

1.00

1.50

2.00

2.50

3.00

3.50

PXR CYP3A4 ABCB1 UGT1A1

Coordinate regulation of DTExtm gene expression in BNF treated human hepatocytes

0.90 0.92

1.44

2.51

2.132.25

2.56

2.96

0.98 1.00

0.78

1.64

0.00

0.50

1.00

1.50

2.00

2.50

3.00

3.50

AHR CYP1A2 ABCC2 UGT1A1

Induction of CYP3A4 gene expression and activity in RIF treated human hepatocytes

2.4

7.8

2.3

0

1

2

3

4

5

6

7

8

9

DTEx QPCR P450-Glo

Induction of CYP3A4 gene expression and activity in RIF treated HepaRG cells

1.8

4.5

4.1

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

DTEx QPCR P450-Glo

DTExtm Gene Expression Analysis

All DTExtm genes are involved in drug metabolism, conjugation or transport* interrogate for both known and unknown drug effects on gene expression

** CYP3A4 induction by RIF (PXR mediated effects on ABCB1 & UGT1A1)

** different levels of CYP3A4 gene expression and induction in different donors

** CYP1A2 induction by BNF (AHR mediated effects on ABCC2 & UGT1A1)

** RIF (PXR) effects on SULT1A1, 1E1, 2A1, UGT2B, ABCA9, A13 and C13 gene expression and activity?

All DTExtm genes are members of coordinate regulatory pathways * internal control for induction or suppression of gene expression

** coordinate regulation of CYP3A4, UGT1A1 and ABCB1 via PXR activation by RIF

** coordinate regulation of CYP1A2, UGT1A1 and ABCC2 via AHR activation by BNF

* control for drug selectivity or specificity in the modulation of gene expression

* concordance between DTExtm microarray, Q-PCR and functional activity assays for CYP induction studies

DTExtm microarray analysis is reliable and reproducible* either biological replicates or experimental replicates