This document discusses enhancing the bioactivation of the drug cyclophosphamide through differential regulation of drug-metabolizing enzymes CYP2B6 and CYP3A4 by the nuclear receptor constitutive androstane receptor (CAR). It aims to identify selective CAR activators that preferentially induce CYP2B6 over CYP3A4 to improve cyclophosphamide's therapeutic effects and reduce toxicity by changing its metabolism. The document outlines past studies on CAR and PXR regulation of these cytochrome P450 enzymes and the effects of ritonavir on their activity. Validation of selective CYP2B6 inducers on cyclophosphamide biotransformation in humanized animal models is also proposed.
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Wang
1. Enhancing the bioactivation of Cyclophosphamide by hepatic Drug-metabolizing Enzymes Hongbing Wang Department of Pharmaceutical Sciences
2. Cyclophosphamide CYCLOPHOSPHAMIDE Breast cancer Ovarian cancer Leukemia Lymphomas Prostate cancer Lung cancer Multiple myeloma Systemic lupus erythematosus Rheumatoid arthritis ……
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.
