Induction of cytochrome P450 (CYP) enzymes by xenobiotics is a major concern in clinical practice because of the potential for drug-associated attenuated efficacy, or increased metabolite-associated toxicity, in the presence of concomitantly administered drugs. CYP2B6 is a highly-inducible enzyme in human liver. Recent studies have shown that the relative contribution of CYP2B6 to hepatic CYP content and substrate metabolism are much higher than estimated previously, and suggest that induction of this enzyme may be of therapeutic importance. The long-term objective of this ongoing project is to elucidate the molecular mechanisms governing drug-induced regulation of human hepatic CYP2B6. Results from the current funding period established human (h) PXR as a major transcriptional factor capable of regulating CYP2B6 induction by many compounds. Although it is widely accepted that CAR mediates induction of rodent CYP2B genes, the role of hCAR in human CYP2B6 regulation is far from clear, and direct extrapolation of rodent data to humans is risky due to obvious species differences. The central hypothesis of the proposed studies is that hCAR plays a distinct role compared with hPXR in human CYP2B6 induction, and differs from hPXR in its preferred induction of CYP2B6 over CYP3A4.
The specific aims of the proposed studies are to: 1) define the role of hCAR in the regulation of hepatic CYP2B6 expression, 2) elucidate the molecular mechanisms of differential regulation of CYP2B6 and CYP3A4 by hCAR, 3) characterize the differences between direct and indirect hCAR activators in the mechanisms of hCAR nuclear activation and CYP2B6 induction, and 4) assess the utility of selective hCAR activators to facilitate cyclophosphamide therapeutic activation. A multifaceted experimental approach incorporating protein-protein and protein-DNA binding assays, transfection and hCAR translocation assays in immortalized cell lines and/or human primary hepatocytes, siRNA knockdown of hCAR and hPXR in human hepatocytes, and transient in vivo expression of hCAR in CAR-/- mice will be employed to delineate the overlapping and distinct roles of hCAR versus hPXR in CYP2B6 induction, to compare indirect and direct mechanisms of hCAR nuclear activation and their impact on CYP2B6 induction, and to evaluate the roles of selective hCAR activators in facilitating cyclophosphamide (CPA) bioactivation. Data generated from the proposed in vitro and in vivo studies will provide important insight into the molecular mechanisms of CYP2B6 induction in humans, allowing for improved prediction of drug-drug interactions. In addition, these studies will explore selective modulation of drug metabolism via hCAR-mediated CYP2B6 induction as a means to increase therapeutic efficacy and decrease toxicity of a model CYP2B6 substrate, CPA. ? ? ? ? ? ?
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