Profiling Induction of Cytochrome P450 Enzyme Activity by Statins Using a New Liquid Chromatography-Tandem Mass Spectrometry Cocktail Assay in Human Hepatocytes

Human hepatocytes in primary culture are a very useful model to directly assess induction of gene expression by xenobiotics. We developed a cytochrome P450 (P450) activity cocktail assay using model substrates for the seven important P450s 1A2 (phenacetin), 2B6 (bupropion), 2C8 (amodiaquine), 2C9 (tolbutamide), 2C19 (S-mephenytoin), 2D6 (propafenone), and 3A4 (atorvastatin). Metabolite formation was determined by liquid chromatography-tandem mass spectrometry in hepatocyte culture supernatants. Atorvastatin has not been previously assessed as a CYP3A probe drug. We demonstrate highly selective atorvastatin ortho-hydroxylation by CYP3A4 by recombinant P450s. In human liver microsomes ortho-hydroxyatorvastatin formation was highly correlated with CYP3A4 protein content (r(s) = 0.78, p < 0.0001, n = 150). We profiled induction of these P450 activities in primary human hepatocytes after treatment with 30 mu M atorvastatin, lovastatin, pravastatin, rosuvastatin, and simvastatin for 24 to 72 h. Except for pravastatin, all statins induced P450 activities to various degrees, approximately in the order atorvastatin > simvastatin > lovastatin > rosuvastatin. Inducibility of P450s followed the order CYP2C8 > CYP3A4 > CYP2C9 > CYP2B6 > CYP2C19 similar to CYP2D6 > CYP1A2. The strongest induction was observed for amodiaquine N-desalkylation, which was induced approximately 20-fold. Quantitative reverse transcription-polymerase chain reaction confirmed corresponding changes on the mRNA level with even more dramatic induction up to almost 100-fold. These data suggest a broader inducing effect of statins on cytochrome P450s and possibly other absorption, distribution, metabolism, and excretion genes than previously known, thus further emphasizing their drug-drug interaction potential. Our cocktail assay should be helpful for economical use of human hepatocytes in the assessment of P450 induction by drugs and drug candidates.