Your SlideShare is downloading. ×
DTEx 02042009
Upcoming SlideShare
Loading in...5
×

Thanks for flagging this SlideShare!

Oops! An error has occurred.

×
Saving this for later? Get the SlideShare app to save on your phone or tablet. Read anywhere, anytime – even offline.
Text the download link to your phone
Standard text messaging rates apply

DTEx 02042009

617
views

Published on

ADME-Associated Gene Expression Analysis using DTEx microarrays

ADME-Associated Gene Expression Analysis using DTEx microarrays

Published in: Technology

0 Comments
0 Likes
Statistics
Notes
  • Be the first to comment

  • Be the first to like this

No Downloads
Views
Total Views
617
On Slideshare
0
From Embeds
0
Number of Embeds
1
Actions
Shares
0
Downloads
1
Comments
0
Likes
0
Embeds 0
No embeds

Report content
Flagged as inappropriate Flag as inappropriate
Flag as inappropriate

Select your reason for flagging this presentation as inappropriate.

Cancel
No notes for slide

Transcript

  • 1. 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
  • 2. 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)
  • 3. Regulation of Cytochrome P450 gene expression CYPs Ligands NXRs Modulators CYP1 Polycyclic AHs, AHR [Arnt] 3MC, BNF, OMP Halogenated AHs CYP2B Steroids CAR [RXR] PBB, CITCO CYP2C Prostaglandins, CAR [RXR] PBB, RIF Steroids PXR [RXR] CYP3A Steroids PXR [RXR] DEX, RIF [hu], PCN [rt] CYP4A Prostaglandins, PPAR [RXR] DEX, CLF, FAs Wy14,643 CYP7A Cholesterol, LXR [RXR] CDCA, APD, 22(R)HC, BAs FXR [RXR] T0901317
  • 4. 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.
  • 5. DTExtm Microarray Gene Set Gene Group Phase Group Members Number Cytochrome P450s I 1A, 1B, 2A, 2B, 2C, 2D, 2E, 3A, 4A, 18 [CYPs] 4B, 7A, 8B, 19A, 27A, 27B Nuclear Xenobiotic I AHR, AR, CAR, FXR, HNF, LXR, 19 Receptors [NXRs] NFE, NRF, PPAR, PXR, RXR, VDR Sulfotransferases II 1A, 1B, 1C, 1E, 2A, 2B 6 [SULTs] UDP glucuronosyl II 1A, 2A, 2B, 8 6 transferases [UGTs] SLC (uptake) III CNT, ENT, LST, NTCP, OAT, OATP, 36 transporters [SLCs] OCT, OCTN, OST, PEPT, PGT, URAT ABC (efflux) III A, B, C, D, E, F, G 49 transporters [ABCs] Controls 18S, 28S, ACT, B2M, GDH, S28, 11 SH1, SH2, RPLP0, TUB, VIL1
  • 6. 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.
  • 7. 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.
  • 8. 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
  • 9. 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.
  • 10. 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]
  • 11. 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]
  • 12. 1.00 1.50 2.00 2.50 0.00 0.50 3.00 3.50 4.00 abcA7 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 DTEx gene expression in six rifampicin treated human hepatocyte lots
  • 13. Induction of CYP450 gene expression in six rifampicin treated human hepatocyte lots 4.00 3.50 3.00 2.50 2.00 1.50 1.00 0.50 0.00 cyp27A1 cyp19A1 cyp4A11 cyp4A22 cyp2C9 cyp2C8 cyp1B1 cyp8B1 cyp2B6 cyp4B1 cyp2C19 cyp27B1 cyp1A2 cyp7A1 cyp2A6 cyp3A4 cyp2D6 cyp2E1
  • 14. Induction of DTExtm gene expression in six rifampicin treated human hepatocyte lots cyp1A2 cyp1B1 cyp2C8 cyp2C9 cyp2C19 cyp2E1 cyp3A4 0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 Cluster plot and histogram of CYP gene expression induction ratios [range = 0-4x vehicle treated]
  • 15. Coordinate regulation of DTExtm gene expression in RIF treated human hepatocytes 3.50 3.00 2.50 2.00 1.50 2.86 2.11 1.00 1.62 1.54 1.45 1.44 1.37 1.28 1.26 1.07 0.50 0.95 0.87 0.00 PXR CYP3A4 ABCB1 UGT1A1
  • 16. Coordinate regulation of DTExtm gene expression in BNF treated human hepatocytes 3.50 3.00 2.50 2.00 1.50 2.96 2.56 2.51 2.25 2.13 1.00 1.64 1.44 0.50 1.00 0.98 0.92 0.90 0.78 0.00 AHR CYP1A2 ABCC2 UGT1A1
  • 17. Induction of CYP3A4 gene expression and activity in RIF treated human hepatocytes 9 8 7 6 5 4 7.8 3 2 2.4 2.3 1 0 DTEx QPCR P450-Glo
  • 18. Induction of CYP3A4 gene expression and activity in RIF treated HepaRG cells 5 4.5 4 3.5 3 2.5 4.5 4.1 2 1.5 1 1.8 0.5 0 DTEx QPCR P450-Glo
  • 19. 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

×