Inhibitory effects of silibinin on cytochrome P-450 enzymes in human livermicrosomes

Silibinin, the main constituent of silymarin, a flavonoid drug from silybum marianum used in liver disease, was tested for inhibition of human cytochr ome P-450 enzymes. Metabolic activities were determined in liver microsomes from two donors using selective substrates. With each substrate, incubatio ns were carried out with and without silibinin (concentrations 3.7-300 mu M ) at 37 degrees in 0.1 M KH2PO4 buffer containing up to 3% DMSO. Metabolite concentrations were determined by HPLC or direct spectroscopy. First, sili binin IC50 values were determined for each substrate at respective K-M conc entrations. Silibinin had little effect (IC50>200 mu M) on the metabolism o f erythromycin (CYP3A4), chloroxazone (CYP2E1), S(+)-mephenytoin (CYP2C19), caffeine (CYP1A2) or coumarin (CYP2A6). A moderate effect was observed for high affinity dextromethorphan metabolism (CYP2D6) in one of the microsome s samples tested only (IC50=173 mu M). Clear inhibition was found for denit ronifedipine oxidation (CYP3A4; IC50=29 mu M and 46 mu M) and S(-)-warfarin 7-hydroxylation (CYP2C9; IC50=43 mu M and 45 mu M). When additional substr ate concentrations were tested to assess enzyme kinetics, silibinin was a p otent competitive inhibitor of dextromethorphan metabolism at the low affin ity site, which is not CYP2D6 (K-i,K-c=2.3 mu M and 2.4 mu M). Inhibition w as competitive for S(-)-warfarin 2-hydroxylation (K-i,K-c=18 mu M and 19 mu M) and mainly non-competitive for denitronifedipine oxidation (K-i,K-n=9 m u M and 12 mu M). With therapeutic silibinin peak plasma concentrations of 0.6 mu M and biliary concentrations up to 200 mu M, metabolic interactions with xenobiotics metabolised by CYP3A4 or CYP2C9 cannot be excluded.