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

Abstract

ABSTRACT: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 (Smephenytoin), 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 orthohydroxyatorvastatin 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 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 ϳ 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.

A Systematic Comparison of the Impact of Inflammatory Signaling on Absorption, Distribution, Metabolism, and Excretion Gene Expression and Activity in Primary Human Hepatocytes and HepaRG Cells

“…This was mainly due to the enormous variability between PHH donors. We observed a decline in basal P450 activities during the first day of culture period, which was previously reported in primary hepatocytes (Feidt et al, 2010;Lübberstedt et al, 2011). In the HepaRG cells, much better preservation of the six measured P450 activities over the investigated culture period of 72 hours was observed, in agreement with previous studies (Kanebratt and Andersson, 2008;Turpeinen et al, 2009;Lübberstedt et al, 2011).…”

“…Measurement of P450 Enzyme Activities. Cytochrome P450 enzyme activities were determined in PHH and HepaRG cell culture supernatants using a liquid chromatography with tandem mass spectrometry-based substrate cocktail assay, as described previously (Feidt et al, 2010). CYP2D6 enzyme activity (propafenone 5-hydroxylation) was not quantifiable in HepaRG cells by liquid chromatography-tandem mass spectrometry cocktail assay and was therefore excluded from further analysis.…”

Effect of nicotine on cytochrome P450 1A2 activity

“…We are not aware of any clinical studies demonstrating that bupropion inhibits CYP1A2 activity. In human liver microsomes in vitro, bupropion incubated with melatonin or phenacetin (probes for CYP1A2) shows no inhibitory effects on CYP1A2-mediated pathways [14][15][16]. Chlorzoxazone and caffeine have been dosed together in several metabolic cocktail approaches to phenotype CYP enzymes without any detected metabolic interactions [17][18][19].Thus, there is no known interaction between the study medications affecting the phenotyping of CYP1A2.…”

Relationship Between Statin Type and Responsiveness to Clopidogrel in Patients Treated with Percutaneous Coronary Intervention: A Subgroup Analysis of the CILON-T Trial

“…A recent study that profiled the induction of the CYP450 activity in human hepatocytes by statins using a new liquid chromatography technique, a tandem mass spectrometry cocktail assay, showed that all statins, except pravastatin, induce the P450 activity to some degree, approximately in the order of atorvastatin ＞ simvastatin ＞ lovastatin ＞ rosuvastatin. The inducibility of P450s occurred in the order of CYP2C8 ＞ CYP3A4 ＞ CYP2C9 ＞ CYP2B6 ＞ CYP2C19 ≈ CYP2D6 ＞ CYP1A2 27) . The higher potential of atorvastatin to induce the CYP450 system may play a role in potentiating the conversion of clopidogrel to its active metabolite and hence its antiplatelet effects.…”