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University of Maryland Baltimore
Experimental Therapeutics Symposium 2009

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  • Fortunately before the onset of this aim CITCO was identified by Glaxo group as a selective activator of CAR. Along with RIF, a selective PXR activator, CITCO represent chemical tools to study similarities and differences in CAR and PXR regulation of CYP2B6 and CYP3A4 target genes. In the first experiment, six human hepatocyte preparations were treated with RIF or CITCO and assessed for CYP2B6 andCYP3A4 mRNA induction using Taqman real-time PCR. The top graph shows CYP2B6 induction levels and the bottom CYP3A4. Both RIF and CITCO efficaciously induced CYP2B6. In many livers the magnitudes of induction were similar. For CYP3A4, the PXR activator RIF efficaciously induced CYP3A4. IN contrast, the CAR activator CITCO was associated with weak induction of CYP3A4 compared with CYP2B6. These results suggested that PXR non-selectively regulates CYP2B6 and CYP3A4, and CAR differentially regulates the 2 genes. It was hypothesized that similarities or differences in PXR and CAR binding to CYP2B6 and CYP3A4 respone elements accounted for these induction findings. Before I talk about this, I will review the response elements in the promoters of CYP2B6/3A4 genes.
  • If these drugs were acting through CAR, you would expect them to exhibit similar induction patterns as CITOC in aim 2, that is greater induction of cYP2B6 compared with CYP3a4. All 3 drugs were associated with efficacious CYP2B6 induction at levels similar to RIF and CITCO. However, they exhibited weak induction of CYP3A4 like CITCO. These results provided further indirect evidence that these compounds acted through hCAR. Still more direct evidence was needed. Unfortunately, transfection assays in Hepg2 cells could not be used because of the constitutive activity of CAR in immortalized cells. Therefore we used an in vivo model in mice.
  • Thus the human model suggests that PXR crossregulation of CYP2B6 is greater than CAR crossregulation of CYP3A4. This reflects that PXR can bind to the DR4 in CYP2B6 better than CAR can bind to the ER6 in CYP3A4.
  • Wang

    1. 1. Enhancing the bioactivation of Cyclophosphamide by hepatic Drug-metabolizing Enzymes Hongbing Wang Department of Pharmaceutical Sciences
    2. 2. Cyclophosphamide CYCLOPHOSPHAMIDE Breast cancer Ovarian cancer Leukemia Lymphomas Prostate cancer Lung cancer Multiple myeloma Systemic lupus erythematosus Rheumatoid arthritis ……
    3. 3. Metabolism of Cyclophosphamide
    4. 4. Cytochrome P450s Wang H , et al. Current Drug Metabolism 2008
    5. 5. Transcriptional regulation of CYP2B and CYP3A Wang H. et. al CDM 2003
    6. 6. hCAR selectively induces CYP2B6 over CYP3A4 RIF = Selective hPXR Activator CITCO = Selective hCAR Activator CYP2B6 CYP3A4 Faucette S. et al. JPET 2006
    7. 7. CYP2B6 CYP3A4 Faucette S. et al., JPET 2007
    8. 8. Asymmetric Cross-talk Between PXR and CAR PXR RXR CAR RXR XREM PXRE ER6 PBREM XREM CYP3A4 CYP2B6 Faucette S. et al., JPET 2006
    9. 9. Ritonavir affects the activity of CYP3A4/2B6 Faucette et al., DMD 2004
    10. 10. Potential Clinical Benefit of Differential Regulation of CYP2B6 and CYP3A4 by hCAR dechloroethylCPA (neurotoxic) CYP3A4 CYP2B6 4-hydroxyCPA CAR activator cyclophosphamide phosphoramide mustard + acrolein (therapeutically active) spontaneous Ritonavir + _
    11. 11. LC-Ms/MS chromatograms of CPA & 4-OHCP
    12. 12. Project Aims <ul><li>Identify selective CAR activators as drug targets for preferential induction of CYP2B over CYP3A in vitro </li></ul><ul><li>Evaluate ritonavir and its analogs as alternative drug targets in modulating CYP2B6 induction and CYP3A4 inhibition </li></ul><ul><li>Examine the effects of selective CYP2B6 inducers on the biotransformation of CPA in vitro </li></ul><ul><li>Validate CAR activator-mediated pharmacokinetic changes of CPA in humanized CAR animal model </li></ul>
    13. 13. Acknowledgements <ul><li>Lab Members </li></ul><ul><ul><li>Duan Wang </li></ul></ul><ul><ul><li>Dr. Haishan Li </li></ul></ul><ul><ul><li>Dr. Linhao Li </li></ul></ul><ul><ul><li>Dr. Leslie M. Tompkins </li></ul></ul><ul><ul><li>Antonia Tolson </li></ul></ul><ul><li>Previous lab members: </li></ul><ul><ul><li>Dr. Stephanie Faucette </li></ul></ul><ul><ul><li>Dr. Cornelia Smith </li></ul></ul><ul><li>Collaborators: </li></ul><ul><li>Dr. Michael Colvin </li></ul><ul><li>Dr. Douglas Ross </li></ul><ul><li>Funding Support </li></ul><ul><li>GCC Seed Grant </li></ul><ul><li>NIH </li></ul>