Transcriptional Regulation Of CYP3a4/CYP2B6/CYP2C9Mediated Via Nuclear Receptor PXR By Helicid And Its Metabolites

Objective: More than50%of the clinical drugs are metabolized by CYP3A4inhuman,CYP2B6and CYP2C9also assume16%and25%of drugs clearanceRespectively. CYP3A4, CYP2C9or CYP2B6metabolize almost more than90%ofclinical drugs. The metabolic enzymes often change in the quantity or stability whileinduced by drugs.In drug development,drug metabolism interactions have become anincreasingly important problem.The inductive effect of clinical drug-metabolizingenzymes CYP450is the main reason of drug interactions.In drug research anddevelopment process,it is important to avoid adverse drug interactions by establishing ascreening CYP450inducers model in vitro, eliminating compounds may induceCYP450expression, developping the CYP450-transparent new drugs. This study is aimat establishing and validating an in vitro system to screen drug inducers ofCYP3A4,CYP2B6,CYP2C9mediated via nuclear receptor pregnane X receptor(human pregnant X receptor, hPXR),studying transcriptional regulation of CYP3A4,CYP2B6, CYP2C9mediated via hPXR by traditional Chinese medicine helicid and itstwo metabolites.Methods: In the molecular mechanisms clonging the promoters ofCYP3A4,CYP2B6,CYP2C9which contained the elements that hPXR, a kind of nuclearreceptor,can recognize and bind, and inserting the trans-element to the upstream of firefly luciferase reporter gene of the pGL4.17vectors. Co-transfecting the reportvector and hPXR expression plasmid to HepG2cell line.After24hours, the transfectedcells were treated with helicid of various concentrations (0.004,0.04,0.4μmol/L) and itsmetabolite I and metabolite II (0.0004,0.004,0.04μmol/L) for48h,while rifampin(10μmol/L) was included as the positive control,0.1%DMSO as a negative controlgroup, Cells were lysized and luciferase activity was determined using a Dual-luciferasereporter assay kit.Results: Helicid and its metabolites Ⅰ, Ⅱ at tested concentrations, did notsignificantly increase promoter activities of CYP3A4,CYP2B6,CYP2C9in HepG2cellstransfected with PXR expression plasmid (P>0.05).Conclusion: Nuclear receptor PXR-expressed CYP3A4,CYP2B6,CYP2C9dualluciferase reporter gene platforms were successfully established, and helicid and itsmetabolites Ⅰ, Ⅱ does not significantly induce the transcription ofCYP3A4,CYP2B6,CYP2C9.The establishment of reporter gene system is an efficientand easy mean of detection for large-scale screening CYPs inducers.