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 (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 (rs = 0.78, p < 0.0001, n = 150). We profiled induction of these P450 activities in primary human hepatocytes after treatment with 30 μM 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 ∼ 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.
Introduction
Cytochromes P450 (P450s) are a large superfamily of hemoproteins that oxidatively metabolize a vast number of endogenous and exogenous substances. In humans, 57 presumably functional genes have been identified, of which approximately one dozen are important for the metabolism of most drugs in clinical use (Lewis, 2004). The expression and activity of these enzymes are highly variable between and within individuals. Factors determining interindividual variability include genetic polymorphisms, sex, age, diseases and environmental factors (Dickins, 2004; Zanger et al., 2008). Within subject variation can occur as a result of hormonal changes, disease states, or, most commonly, as a result of interactions with drugs or other chemicals. Drug-drug interactions were identified as a leading cause of hospitalization and death (Jaquenoud Sirot et al., 2006). Investigation of the conditions under which drug-drug interactions occur, of their clinical relevance, and of the mechanisms that cause them is therefore a highly active field in pharmacological research with relevance for academic researchers, physicians, drug developers, and regulatory authorities. Principal mechanisms of drug-drug interactions are the direct inhibition of enzymatic activity, which may be reversible or irreversible in nature, and induction or repression of gene expression (Bjornsson et al., 2003; Neuvonen et al., 2006).
Preclinical assessment of drugs or drug candidates with respect to their interaction potential has become an integral part of drug development and approval. Numerous experimental systems have been developed to explore effects of chemicals on drug metabolism in vitro, the general goal of such studies being to identify the target proteins that are most potently inhibited or induced to make estimations of clinical relevance (Pelkonen et al., 2008). Inhibition assays for enzyme activity are usually performed with recombinant protein preparations or in human liver microsomes, whereas induction or repression studies require cellular systems. Human hepatocytes in primary culture have become the gold standard for assessment of induction potential, because most cell lines do not express drug-metabolizing enzymes in a regulable way. However, human hepatocytes are a precious resource, difficult to obtain for most researchers and drug companies and not without experimental limitations (Gómez-Lechón et al., 2004). Thus, it is mandatory to use highly sophisticated analytical procedures to extract the highest possible amount of relevant information from each experiment. So-called cocktail assays, in which several enzyme activities are determined in parallel by liquid chromatography-tandem mass spectrometry (LC-MS/MS) are particularly useful in this regard. Although a number of such assays have been published in recent years (Dierks et al., 2001; Testino and Patonay, 2003; Weaver et al., 2003; Kim et al., 2005; Tolonen et al., 2007; Lahoz et al., 2008), there is still a need for optimizing the selection of probe substrates and their composition to get the most significant information.
In this study, we have developed a LC-MS/MS cocktail assay for the purpose of simultaneously determining induction of drug oxidation activities of the seven most relevant drug-metabolizing P450 enzymes in human hepatocytes. We used previously established and well documented probe drugs for six of the seven P450 isoforms. For CYP3A4, the most relevant human drug-metabolizing enzyme, we performed a series of experiments to establish atorvastatin as a selective probe drug. Although it has been known that atorvastatin is metabolized primarily by CYP3A4 (Prueksaritanont et al., 1999), no systematic study to prove the selectivity of this reaction has been published before. The cocktail assay we developed is applicable for in vitro studies in human liver microsomes and in cell culture, and we deem it especially suited for screening activity changes in human primary hepatocytes.
We used our new assay to profile the P450 activity changes that result from treatment of human primary hepatocytes with HMG-CoA reductase inhibitors (statins), and we compared them with prototypical inducers. Although induction of P450s by statins was studied in rat and human hepatocytes as early as 1993, the individual studies were incomplete with respect to the selected statins and P450s (Kocarek et al., 1993; Schuetz et al., 1993; Kocarek and Reddy, 1996; Kocarek et al. 2002). In addition, most previous studies were limited to the quantification of P450 mRNA levels by PCR, and although some studies also included measurements at the protein level, no data on the induction of enzymatic activities have been published to our knowledge. The high sensitivity and simultaneous measurement of P450 activities allowed us to collect a comprehensive data set with an efficient use of human hepatocytes. The results demonstrate a surprisingly broad and potent induction of P450 enzyme activities by most statins.
Materials and Methods
Chemicals and Reagents.
Simvastatin was obtained from Merck (Darmstadt, Germany). Propafenone and 5-hydroxypropafenone hydrochloride were kindly supplied by Knoll (Ludwigshafen, Germany). [2H7]5-Hydroxypropafenone hydrochloride, bupropion hydrochloride, hydroxybupropion hydrochloride, [2H3]hydroxybupropion hydrochloride, 4′-hydroxymephenytoin, and [2H3]4′-hydroxymephenytoin were obtained by chemical synthesis as described previously (Richter et al., 2004). S-Mephenytoin was a kind gift from Professor Urs Meyer (Biozentrum, Basel, Switzerland). Atorvastatin, lovastatin, rosuvastatin, and all other substrates, metabolite standards, and deuterium-labeled internal standards for P450-dependent activities (Table 1) were purchased from Toronto Research Chemicals (North York, ON, Canada). Oligonucleotides were purchased from Metabion International AG (Martinsried, Germany) or Applied Biosystems GmbH (Darmstadt, Germany). Cell culture supplies were obtained from Invitrogen GmbH (Karlsruhe, Germany). Fetal calf serum, DMSO, and dexamethasone were received from Sigma-Aldrich Chemie GmbH (Steinheim, Germany), and insulin was from Aventis (Frankfurt, Germany). All other chemicals were obtained at the highest available grade from Sigma-Aldrich Chemie GmbH.
Standard Solutions.
Stock standard solutions of statins and prototypical inducers were prepared as 30 mM concentration in DMSO, except pravastatin (in water) and phenobarbital (1000 mM in water), and diluted 1:1000 in Williams' medium E for the experiment. Substrates for the cocktail assay were dissolved as 10 mM (bupropion, amodiaquine, and propafenone) or 100 mM (phenacetin, tolbutamide, and S-mephenytoin) stock in organic solvent or water (bupropion and amodiaquine). Atorvastatin was prepared as a 5 mM solution in acetonitrile-water (1:1, v/v). All P450 substrates were diluted in Williams' medium E for the experiments.
Liver Samples and Microsomal Incubations.
Liver tissue and corresponding blood samples were previously collected from patients undergoing liver surgery at the Campus Virchow, Humboldt University (Berlin, Germany). All tissue samples were examined by a pathologist and only histologically nontumorous tissue was used. Clinical patient documentation included age, sex, medical diagnosis, presurgical medication, liver function tests, and alcohol and smoking habits. Patients with hepatitis, cirrhosis, or chronic alcohol use were excluded. The study was approved by the local ethics committee of the Charité, Humboldt University Berlin, following the ethical guidelines of the Declaration of Helsinki, and written informed consent was obtained from all patients. The preparation of human liver microsomes was described before (Wolbold et al., 2003). Microsomal incubation mixtures contained 50 μg of microsomal protein/assay in 0.1 M potassium phosphate buffer (pH 7.4), an NADPH-generating system (5 mM MgCl2, 4 mM glucose 6-phosphate, 0.5 mM NADP+, and 4.0 U/ml glucose-6-phosphate dehydrogenase), and substrates at the indicated concentrations (Table 3). The reaction mixture, in a final volume of 100 μl, was preincubated for 3 min at 37°C in a water bath before reaction was initiated by addition of the NADPH-generating system. After a 15-min incubation, the reactions were terminated by addition of mixed 20 μl of 250 mM formic acid containing the appropriate isotope-labeled internal standards for each drug metabolite. Samples were then cooled in an ice bath to precipitate the proteins. The mixture was vortexed and centrifuged at 16,000g for 5 min. The supernatant was analyzed by LC-MS/MS as described below.
Recombinant Cytochrome P450 Enzymes.
Human recombinantly expressed cytochrome P450 enzymes (CYP1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, 3A4, 3A5, 4F2, 3A4 + cytochrome b5, and 3A5 + cytochrome b5) coexpressing human P450 reductase (Supersomes) were purchased from BD Biosciences Discovery Labware (Bedford, MA). The standard incubation mixture (100 μl) contained 0.1 M potassium phosphate buffer (pH 7.4), an NADPH-generating system as described above, the indicated concentration of substrate (10 or 100 μM atorvastatin), and recombinant expressed P450 enzymes (5 pmol of P450/assay). Incubations were performed according to the manufacturer's instructions. In brief, reactions were started by adding recombinant enzyme into the preincubated reaction mixture (3 min at 37°C), mixing gently, and incubating for 10 min at 37°C in a water bath without agitating the reaction. Otherwise the incubation protocol was similar to microsomal incubations.
P450 Activity Assays and Cocktail Assay Development.
LC-MS/MS analysis.
Metabolites were determined by LC-MS/MS analysis using the respective deuterium-labeled substances as internal standards (Table 1). An Agilent 6460 triple quadrupole mass spectrometer (Agilent, Waldbronn, Germany) coupled to an Agilent 1200 HPLC system consisting of degasser G1379B, binary pump G1312B, well plate sampler G1367D, and column thermostat G1316B was used. The ionization mode was electrospray, polarity positive. Electrospray jetstream conditions were as follows: capillary voltage, 3500 V; drying gas flow, 10 l/min nitrogen; drying gas temperature, 325°C; nebulizer pressure, 20 psi; sheath gas temperature, 350°C; and sheath gas flow, 11 l/min. The mass spectrometer was operated in the multiple reaction monitoring (MRM) mode. MRM transitions, dwell time, fragmentor voltage, and collision energy are summarized in Table 1.
HPLC separation was achieved on a Strategy 5 Pro column (100 × 2.1 mm; Interchim, France) using (A) 10 mM ammonium acetate in water with 1% (v/v) formic acid and (B) acetonitrile as mobile phases at a flow rate of 0.4 ml/min. Gradient runs were programmed as follows: 12% B for the 1st min, a linear increase to 20% B to 3 min, an increase to 55% B to 8 min, an increase to 95% B to 9 min, remaining at 95% B to 11 min, and then reequilibration to 12% B.
Standardization of the analytical assays was performed with calibration samples prepared in cell culture medium or incubation buffer in the concentration range from 0.005 to 5 μM. Calibration curves based on internal standard calibration were obtained by weighted (1/x2) linear regression for the peak area ratio of the analyte to the respective internal standard against the amount of the analyte. The concentration of the analytes in unknown samples was obtained by linear regression analysis. Assay accuracy and precision were determined by analyzing quality control samples that were prepared like the calibration samples. Quality controls over the whole concentration range showed a bias between −14.9 and 10.3% and a relative S.D. (n = 6) less than 10% for all metabolites determined.
Incubation parameters.
Assay parameters including time- and protein linearity, limits of detection, and optimal substrate concentrations were initially determined by using pooled human liver microsomes consisting of approximately 120 individual liver microsome samples. Activities for each substrate were determined in the microsomal pool individually and in combinations with other substrates to adjust the concentrations to minimize mutual inhibition effects.
Isolation, Culture, and Treatment of Primary Human Hepatocytes.
Tissue samples from human liver resections were obtained from patients undergoing partial hepatectomy. Experimental procedures were performed according to the guidelines of the charitable state-controlled foundation, Human Tissue and Cell Research (Regensburg, Germany), and the institutional guidelines for liver resections of tumor patients with primary or secondary liver tumors (Technical University Munich, MRI, Munich, Germany). The use of human hepatocytes for research purposes was approved by the local ethics committees of the Ludwig-Maximilians-University of Munich (Thasler et al., 2003) and the Charité, Humboldt University Berlin, and written informed consent was obtained from all patients. Human hepatocytes were isolated using a modified two-step EGTA/collagenase perfusion procedure as described previously (Weiss et al., 2003; Nussler et al., 2009). Viability of isolated hepatocytes was determined by trypan blue exclusion. Only cell preparations with a viability >80% were used for experiments. The isolated cells were seeded on collagen type I-coated culture dishes at a density of 1 × 105 cells/cm2 in 12-well plates and cultivated at 37°C in a humidified incubator with 5% CO2 in Williams' medium E supplemented with 10% fetal calf serum, 2 mM l-glutamine, penicillin (100 U/ml)/streptomycin (100 μg/ml), 0.032 IU/ml insulin, 0.1% DMSO, and 0.1 μM dexamethasone. Two days after plating, cells were incubated with HMG-CoA reductase inhibitors (atorvastatin, lovastatin, pravastatin, rosuvastatin, and simvastatin) at 30 μM and with prototypical inducers (1 mM phenobarbital and 30 μM rifampicin). Stock solutions were prepared in DMSO except for pravastatin and phenobarbital, which were diluted in water. Controls were treated accordingly with either vehicle DMSO (final concentration 0.1%) or Williams' medium E only. Cell culture media and inducer compounds were replaced daily. At 24, 48, and 72 h after start of treatment, P450 enzyme activity measurements were performed, and hepatocytes were harvested for mRNA analysis.
Determination of Enzyme Activities.
The P450 enzyme activities were measured by incubating the hepatocytes with substrate cocktail for 3 h at 37°C (5% CO2). The final concentration of organic solvent during incubation was less than 1% (v/v). Metabolism of all compounds was linear with time up to 3 h of incubation. Culture supernatants (50 μl) were then collected and mixed with 10 μl of 250 mM formic acid containing the isotope-labeled internal standards for each drug metabolite. Samples were subsequently cooled in an ice bath and centrifuged at 16,000g for 5 min. Supernatants were stored at −20°C until analysis by LC-MS/MS.
Cell Harvest, Preparation of RNA, Reverse Transcription, and Quantitative PCR.
Cells were harvested by directly lysing the cells in the culture dish using Buffer RLT with β-mercaptoethanol from an RNeasy Mini Kit (QIAGEN, Hilden, Germany). Total RNA was prepared using an RNeasy Mini Kit with on-column DNase I treatment according to the supplier's instructions. RNA quality was analyzed with the Agilent Bioanalyzer using the RNA 6000 Nano Lab Chip Kit (Agilent Technologies, Waldbronn, Germany), and only samples with RNA integrity number higher than 8 were used for analysis. Reverse transcription was performed using 0.5 μg of RNA, random hexamers and the TaqMan Reverse Transcription Kit (Applied Biosystems GmbH) according to the supplier's instructions. Relative quantification of P450s 1A2, 2B6, 2C8, 2C9, 2C19, 2D6, and 3A4 mRNA was performed using the standard protocol for the 7500 Real-Time PCR system (Applied Biosystems GmbH). Quantification of CYP1A2 expression was performed with primers designed using Primer Express Software 2.0 (Applied Biosystems GmbH) and 6-carboxyfluorescein-labeled MGB probes, respectively (Applied Biosystems GmbH). Polymerase chain reaction was performed by using 2× Universal PCR Master Mix (Applied Biosystems GmbH) in a final volume of 25 μl. Final sense and antisense primer concentrations were 400 nM, and the probe concentration was 200 nM. The thermal cycling conditions for the assay were 2 min at 50°C, 10 min at 95°C, followed by 40 cycles of 15 s at 95°C and 1 min at 60°C. Previously published TaqMan assays were used for the mRNA quantification of CYP2B6 (Zukunft et al., 2005), CYP2C19 (Burk et al., 2005), CYP2D6 (Toscano et al., 2006), and CYP3A4 Wolbold et al., 2003). All primers and probes used are presented in Table 2. For CYP2C8 and CYP2C9 expression analysis, as well as for 18S rRNA used for normalization, predeveloped TaqMan assays (Applied Biosystems GmbH; 18S, 4319413E; CYP2C8, Hs_00426397_m1; and CYP2C9, Hs_00258314_m1) were used. All P450 mRNA levels were quantified in 10 ng of cDNA template, whereas 18S RNA levels were determined with 25 pg of cDNA template. The original TaqMan results were normalized to the 18S rRNA content in each sample.
Statistical Analyses.
All measurements were performed at least in duplicate, and the mean values were compared and subsequently examined by analysis of variance to determine the influence of inducer on the fold induction compared with the corresponding control. For correlations, nonparametric methods (Spearman's rank correlation coefficient rs) were used. All statistical analyses were performed using the statistics software packages SPSS (version 16.0.2; SPSS, Munich, Germany) and GraphPad Prism (version 4.03; GraphPad Software Inc., San Diego, CA).
Results
Atorvastatin as a Selective Probe Drug for CYP3A4.
In the course of our previous studies on atorvastatin metabolism (Gomes et al., 2009; Riedmaier et al., 2010), we made observations suggesting that atorvastatin hydroxylation could be a rather specific and suitable in vitro phenotyping reaction for CYP3A4 and/or CYP3A5. In agreement with an earlier study (Jacobsen et al., 2000), we found that ortho- and para-hydroxyatorvastatin formation was almost exclusively catalyzed by the CYP3A subfamily isozymes, with negligible activity (corresponding to <2% of the CYP3A4 activity) shown by CYP2C8, but only for the para-hydroxylation (Fig. 1). We found 16-fold (at 10 μM substrate) and 11-fold (at 100 μM substrate) higher activity of CYP3A4 compared with that of CYP3A5 for ortho-hydroxymetabolite formation, whereas the para-metabolite showed no significant activity difference. Cytochrome b5 coexpression increased CYP3A4 activity approximately 6-fold for both reactions, whereas CYP3A5 activity was only enhanced for the ortho- but not for the para-hydroxymetabolite. In conclusion, the data with recombinant P450 enzymes revealed that atorvastatin ortho-hydroxylation is highly selectively catalyzed by CYP3A4, both in the absence and presence of cytochrome b5.
In a pool of human liver microsomes, apparent KM and Vmax values determined for atorvastatin hydroxylation [ortho: KM = 50 μM; Vmax = 467 pmol/(mg · min); para: KM = 60 μM; Vmax = 425 pmol/(mg · min)] were in the same range as those described previously for individual liver microsome samples (Jacobsen et al., 2000). Correlation analysis of data obtained from 150 individual liver microsome samples revealed that atorvastatin ortho- and para-hydroxylation were best correlated to CYP3A4 protein content (rs = 0.78 and 0.76, respectively; p < 0.0001) (Fig. 2A). The correlations to microsomal CYP3A5 content were much less significant (rs = 0.37 and 0.31, respectively; p < 0.0001), and correlations to other P450s were in the range of rs = 0.1 (CYP2D6) to rs = 0.58 (CYP1A2). Other established CYP3A4 probe drugs including verapamil N-demethylation, testosterone 6β-hydroxylation, and dextromethorphan N-demethylation showed correlations to CYP3A4 protein content of rs = 0.70, 0.61, and 0.66, respectively (data not shown). Microsomal atorvastatin ortho- and para-hydroxylation activities were correlated to each other with rs = 0.99 and to norverapamil formation with rs = 0.88 and rs = 0.87, respectively (significance p < 0.0001 for all tests) (Fig. 2B). Taken together, these data demonstrated that atorvastatin ortho-hydroxylation can be used as a highly selective and sensitive marker reaction for CYP3A4, at least for in vitro studies.
Development of a Cytochrome P450 Activity Cocktail Assay.
For the purpose of measuring enzyme induction in human hepatocytes, our aim was to combine the most selective phenotyping reactions for the most significant drug-metabolizing cytochrome P450 enzymes in a single cocktail assay (Fig. 3). Based on experiments in pooled human liver microsomes to establish the assay, we finally selected the seven substrates phenacetin (CYP1A2), bupropion (CYP2B6), amodiaquine (CYP2C8), tolbutamide (CYP2C9), S-mephenytoin (CYP2C19), propafenone (CYP2D6), and atorvastatin (CYP3A4). Further details on metabolism routes and incubation conditions are summarized in Table 3. We also compared the seven activities measured with the cocktail assay in a microsome pool with the activity of each substrate present alone. This comparison showed that under conditions of the cocktail assay, when all seven substrates are present at their optimized concentration, most P450s retained almost their full activity, except for CYP2B6 and CYP2C8 activities, which were reduced by approximately 55 and 50%, respectively (data not shown).
Cytochrome P450 Activities in Human Primary Hepatocytes.
Application of the cocktail activity assay in cultures of primary human hepatocytes allowed us to reliably measure the activities of all seven P450 enzymes simultaneously in 12-well plates, corresponding to 0.4 × 106 cells/incubation. Upon incubation of hepatocyte cultures with the substrate cocktail, time-dependent measurements showed linear product formation for at least 3 h for all substrates. Activities could be determined over a culture period of at least 5 days. During this time, the activities decreased to various extents, depending on the individual culture. In particular CYP2C8, CYP2C9, and CYP2C19 usually showed a stronger reduction compared with the basal activity level (corresponding to 100% on the 2nd day after surgery). In contrast, P450s 1A2, 2B6, and 2D6 displayed more variable behavior with respect to culture duration and hepatocyte donor. Whereas CYP2D6 activity often remained rather constant over time, CYP1A2 and CYP2B6 activity sometimes increased and sometime declined. CYP3A4 enzyme activity in most cultures was reduced to approximately 60% within 3 or 4 days of culture (data not shown).
Induction of P450 Activities by Statins and by Prototypical Inducers.
We treated primary human hepatocytes from various donors with the same concentration (30 μM) of atorvastatin, lovastatin, pravastatin, rosuvastatin and simvastatin for 24 h and up to 96 h. The results of the P450 cocktail activity assay measurements are summarized in Fig. 4 for up to 72 h after treatment. Table 4 shows an overview of the maximal induction seen for mRNA and activities. The prototypical inducers phenobarbital (1 mM) and rifampicin (30 μM) served as well known control inducers for comparison. At 24 h after the start of the incubation, no substantial activity changes were seen for any P450 activity except for CYP2B6, which was already weakly increased for the control inducers. At 48 h, however, induction of P450 activities by phenobarbital was approximately 9-fold for CYP2B6 and 5-fold for CYP3A4. The statin-treated hepatocytes also had clearly increased activities, with atorvastatin showing the strongest effect on CYP2C8 (∼10-fold), CYP3A4 (∼7-fold), and CYP2B6 and CYP2C9 (∼4-fold). The activity increases for CYP1A2, CYP2C19, and CYP2D6 did not exceed ∼2-fold with any of the inducers. Almost equal effects were seen with simvastatin, whereas lovastatin and rosuvastatin had smaller effects. Pravastatin was almost inert as an inducer of enzyme activity.
In contrast to the response to phenobarbital and rifampicin, which did not change much from 48 h to 72 h, statins led to differential and more dynamic responses. For example, atorvastatin increased substantially up to 72 h for CYP2B6 (∼4–11-fold), CYP2C8 (∼10–20-fold), CYP2C9 (∼4–9-fold), and CYP3A4 (∼7–11-fold). The increase between 48 and 72 h was less significant for simvastatin and the other statins. We also measured P450 activities at 96 h after treatment. Most P450 activities declined between 72 and 96 h to the levels of the 48-h time point or less. However, because it is known that prolonged statin treatment is damaging to cultured cells (Yasuda et al., 2005; Bertrand-Thiebault et al., 2007), the decreased activities may be caused by the relatively higher activities in control (untreated) cells rather than reflecting a change in induction (data not shown).
Induction of P450 mRNA by Statins and by Prototypical Inducers.
We used specific quantitative real-time reverse transcription-PCR assays to quantitate the mRNA levels of all seven P450 genes investigated at all time points. The results, presented in Fig. 5, show more dramatic and more dynamic responses compared with those at the activity level (Table 4). Most P450 genes had clearly increased mRNA levels already at 24 h after induction. Between 48 and 72 h, the time profiles were qualitatively comparable to those of the P450 activities, although some notable differences were observed. The maximal induction was between the 48- and the 72-h time points with CYP3A4 being induced over the longest time period among all P450s. The fold induction rates were much higher at the mRNA level, reaching almost 100-fold for the response of CYP2C8 and CYP3A4 in cultures treated with phenobarbital and for the response of CYP3A4 in cultures treated with atorvastatin and simvastatin. Some unexpected observations should also be noted. Although pravastatin was ineffective as an inducer at the activity level, there was a clear inducing response at the mRNA level, especially for the CYP2C mRNAs. However, as indicated by the high error bars of these measurements, these results were very variable between different hepatocyte cultures. Also of interest is the observation that CYP2D6 was not completely inert but showed an induction response of up to ∼5-fold with simvastatin and rosuvastatin. Only CYP1A2 was essentially unaffected by all statins and prototypical inducers throughout all experiments.
Discussion
In this article, we used a newly developed LC-MS/MS-based model substrate cocktail assay to simultaneously measure the time-dependent induction of seven drug-metabolizing cytochrome P450 activities as a response to treatment of primary human hepatocytes with different statins (atorvastatin, lovastatin, pravastatin, rosuvastatin, and simvastatin). Although it has been known for many years that statins induce cytochrome P450 mRNA and protein (Kocarek et al., 1993; Schuetz et al., 1993), our study adds information on the inducing effects of commonly used statins on the activities of the most important drug metabolizing P450s, i.e., 1A2, 2B6, 2C8, 2C9, 2C19, 2D6, and 3A4, on the comparison of enzyme activity versus mRNA induction, and on time-dependent induction profiles. In addition to some new aspects relating to the use of P450 model drugs and the cocktail approach for in vitro assessment of activity, our systematic investigation revealed several interesting novel aspects about the induction of drug-metabolizing P450s by statins, as discussed below.
The P450 activity cocktail assay presented here is focused on the seven most important P450s as judged by their roles in the metabolism of clinically used drugs (Zanger et al., 2008). For CYP1A2 (phenacetin), CYP2B6 (bupropion), CYP2C8 (amodiaquine), CYP2C9 (tolbutamide), and CYP2C19 (S-mephenytoin), we used single established marker substrates (Richter et al., 2004; Walsky and Obach, 2004; Turpeinen et al., 2009) and combined them in a cocktail assay with substrates not used before for CYP2D6 (propafenone) and CYP3A4 (atorvastatin).
For CYP2D6, all previously published cocktail assays used either bufuralol 1′-hydroxylation or dextromethorphan O-demethylation as the model activity. Because both bufuralol and dextromethorphan are substrates for additional P450s and because several reports indicated that CYP2D6 can be inhibited by other model drugs in the cocktail, namely bupropion (Spina et al., 2008) and amodiaquine (Dixit et al., 2007), we tried to avoid these potential interactions by selecting the high-affinity substrate propafenone (Kroemer et al., 1989; Toscano et al., 2006). In our human liver bank, propafenone 5-hydroxylation was highly correlated to CYP2D6 protein amount (rs = 0.72, p < 0.0001, n = 150; data not shown). The very low KM value of 0.5 μM (determined in this study) (Table 3) permits the use of a propafenone concentration that does not interfere with other P450 activities, as shown by microsomal incubations with the substrate cocktail versus single substrates.
For CYP3A4, we established atorvastatin ortho-hydroxylation as a specific marker activity. Although catalytic activities of recombinant P450s for atorvastatin hydroxylation had been reported before (Jacobsen et al., 2000), some potentially important isoenzymes (CYP1A2, CYP2A6, and CYP4F2) and conditions (coexpressed cytochrome b5) were missing in that study, which furthermore lacked microsomal variability data. Therefore, we reinvestigated this question in greater detail using both recombinant enzymes and human liver microsomes. Whereas the newly tested CYP1A2, CYP2A6, and CYp4F2 were completely inactive, we confirmed the finding of the earlier study that CYP2C8 possesses some, albeit very low, catalytic activity for atorvastatin para-hydroxylation. The highly selective atorvastatin ortho-hydroxylation by CYP3A4 was corroborated by the results from correlation analyses of P450 activities and protein amounts determined by immunoblotting. In fact, atorvastatin ortho-hydroxylation was not only best correlated to CYP3A4 among 11 drug-metabolizing P450s analyzed (rs = 0.78), but it was also the best-correlated marker activity for CYP3A4 protein, compared with norverapamil formation (rs = 0.70), testosterone 6β-hydroxylation (rs = 0.61), and dextromethorphan N-demethylation (rs = 0.66; data not shown). An interesting finding was that atorvastatin ortho-hydroxylation showed a high selectivity for recombinantly expressed CYP3A4 compared with CYP3A5, with up to 16-fold activity difference, depending on the conditions. This finding is in contrast to the study by Jacobsen et al. (2000), who observed similar activities for the two isoforms, whereas Park et al. (2008) also reported higher intrinsic clearance of CYP3A4 compared with that of CYP3A5 for ortho- and para-hydroxylation (5- and 2.4-fold, respectively). The selectivity difference was further supported by our liver microsome data, for which the correlation between atorvastatin ortho-hydroxylation and CYP3A4 was much higher compared with that with CYP3A5 (rs = 0.78, versus 0.37, respectively; p < 0.0001). Taken together, these experiments validate atorvastatin as a selective substrate for CYP3A4, which may also be well suited for in vivo studies. Comparison of the P450 activities of the optimized cocktail assay in pooled liver microsomes versus the individual substrates revealed virtually no reduction for P450 activities except for CYP2B6, most likely caused by the presence of S-mephenytoin (Heyn et al., 1996) and CYP2C8, which is probably due to multiple interactions (data not shown). However, as the intention of this assay was mainly assessment of activity induction in human hepatocytes, in which limited interactions should not be of concern, we did not attempt to reduce these interactions further.
In primary cultures of human hepatocytes, the P450 activity cocktail assay proved to be highly valuable because it allowed us to reliably determine activity induction profiles of the seven P450s 1A2, 2B6, 2C8, 2C9, 2C19, 2D6, and 3A4 simultaneously in a 12-well format to compare various statins at different time points. To our knowledge, the first reports of cytochrome P450 induction by the HMG CoA-reductase inhibitor, lovastatin, were published in 1993, showing that lovastatin treatment of rat hepatocytes increased the mRNA levels of P450s 2B2, 2C6, 2C7, 3A1, and 4A1 (Kocarek et al., 1993) and of CYP3A4 in human hepatocytes (Schuetz et al., 1993). Later work showed that simvastatin and fluvastatin also increased the levels of CYP2B, CYP3A, and CYP4A mRNA and immunoreactive protein in rat hepatocytes, whereas pravastatin was without effect (Kocarek and Reddy, 1996). Only one detailed study was performed in human hepatocytes, to our knowledge, showing induction of CYP2B6 and CYP3A mRNA by lovastatin, simvastatin, fluvastatin, and atorvastatin but not pravastatin. The analyses were done after one 24-h treatment and although protein induction was also shown, no activity measurements were performed (Kocarek et al., 2002).
Our data show that the induction of the activities of P450s 2B6, 2C8, 2C9, and 3A4 by atorvastatin are comparable or even exceed those achieved by the model inducers phenobarbital and rifampicin. Although initial increases in mRNA expression were already seen after 24 h of treatment, no significant response was seen for the activities at this early time point. The fact that other studies have observed increased protein after 24 h of treatment may be explained by interindividual responsiveness or by experimental culture conditions. In fact, the observed slight increase in activity for CYP3A4 after 24 h was due to the significant induction observed in only one culture, whereas in all others induction was only visible after 48 h. It is remarkable that CYP2C8 activity induction was strongest among all P450s with a maximum of ∼20-fold reached at 72 h for atorvastatin, and induction by lovastatin and simvastatin also exceeded the effects of phenobarbital and rifampicin (Fig. 4; Table 4). Although rat CYP2C and human CYP2C9 mRNA induction had been reported before in cultured endothelial cells (Fisslthaler et al., 2003; Bertrand-Thiebault et al., 2007), this is the first observation, to our knowledge, of CYP2C8 induction by various statins in human hepatocytes. The mechanism of induction most likely involves the xenosensors CAR and PXR. Thus, El-Sankary et al. (2001) showed in HepG2 cells cotransfected with a CYP3A4 reporter gene and human PXR expression plasmid distinctly higher CYP3A4 expression (2.5–4-fold) after lovastatin, simvastatin, rifampicin, and phenobarbital treatment than without nuclear receptor expression.
Several nuclear receptors have been identified as mediating the xenobiotic-induced transcriptional activation of P450 genes. Both, CAR and PXR have to been shown to induce CYP2C gene transcription in human liver (Chen and Goldstein, 2009). Bertrand-Thiebault et al. (2007) noted that statin induction of CYP2C9 most likely involved CAR rather than PXR, because PXR was not expressed in the endothelial cells used.
Ferguson et al. (2005) demonstrated that nuclear receptors CAR, PXR, glucocorticoid receptor, and hepatocyte nuclear factor-4α (NR2A1) play functional roles in transcriptional regulation of CYP2C8 expression. They identified a CAR/PXR binding site in the 5′-flanking region of the CYP2C8 gene and reported induction of CYP2C8 in human hepatocytes by prototypical PXR ligands including 6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime and rifampicin.
In conclusion, we presented in vitro evidence that atorvastatin ortho-hydroxylation is selectively catalyzed by CYP3A4 and represents a suitable and highly selective marker reaction for this P450. Our cocktail activity assay was designed to simultaneously measure the activity of seven drug-metabolizing P450s in cultures of human hepatocytes. This process allowed us to profile the inducing effects of a set of five statins over a prolonged time. Our data suggest broader and more dynamic effects of statins on P450 expression than previously known, and they establish CYP2C8 as a novel statin-induced P450. These findings should be of interest for basic research on P450 regulation, and they are of clinical relevance with respect to drug-drug interactions.
Acknowledgments.
We gratefully acknowledge the expert technical assistance of Britta Klumpp. We also thank Miia Turpeinen (Oulu, Finland) for help with setting up analytical procedures, Leszek Wojnowski (Mainz, Germany) for analyzing testosterone 6β-hydroxylation in the human liver microsome samples, and Andreas K. Nüssler for organizational support with human hepatocytes. We acknowledge the support of the Foundation for Human Tissue and Cell Research (Regensburg, Germany) for making human liver tissue available for research.
Footnotes
This work was supported in part by the Federal Ministry of Education and Research [German Federal Ministry of Education and Science HepatoSys Network Grants 0313080I, 0313081B, 0313081D]; and the Robert Bosch Foundation, Stuttgart, Germany.
Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
doi:10.1124/dmd.110.033886.
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ABBREVIATIONS:
- P450
- cytochrome P450
- LC-MS/MS
- liquid chromatographic tandem mass spectrometry
- PCR
- polymerase chain reaction
- DMSO
- dimethyl sulfoxide
- MRM
- multiple reaction monitoring
- CAR
- constitutive androstane receptor
- PXR
- pregnane X receptor (NR1I2).
- Received April 12, 2010.
- Accepted June 15, 2010.
- Copyright © 2010 by The American Society for Pharmacology and Experimental Therapeutics