Article pubs.acs.org/JAFC
In Vitro Oxidative Metabolism of Cajaninstilbene Acid by Human Liver Microsomes and Hepatocytes: Involvement of Cytochrome P450 Reaction Phenotyping, Inhibition, and Induction Studies Xin Hua,†,§,∥ Xiao Peng,†,‡,∥ Shengnan Tan,† Chunying Li,†,‡ Wei Wang,†,‡ Meng Luo,†,‡ Yujie Fu,*,†,‡ Yuangang Zu,*,† and Hugh Smyth⊥ †
State Engineering Laboratory of Bio-Resource Eco-Utilization and ‡Collaborative Innovation Center for Development and Utilization of Forest Resources, Northeast Forestry University, Harbin, People’s Republic of China § State Key Laboratory of Veterinary Biotechnology, Division of Bacterial Diseases, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, People’s Republic of China ⊥ College of Pharmacy, The University of Texas at Austin, 1 University Station, A1920, Austin, Texas 78712, United States ABSTRACT: Cajaninstilbene acid (CSA, 3-hydroxy-4-prenyl-5-methoxystilbene-2-carboxylic acid), an active constituent of pigeonpea leaves, an important tropical crop, is known for its clinical effects in the treatment of diabetes, hepatitis, and measles and its potential antitumor effect. In this study, the effect of the cytochrome P450 isozymes on the activity of CSA was investigated. Two hydroxylation metabolites were identified in the study. The reaction phenotype study showed that CYP3A4, CYP2C9, and CYP1A2 were the major cytochrome P450 isozymes in the metabolism of CSA. The metabolic food−drug interaction potential was also evaluated in vitro. The effect of CSA inhibition/induction of enzymatic activities of seven drugmetabolizing CYP450 isozymes in vitro was estimated by high-performance liquid chromatography and liquid chromatography− tandem mass spectrometry analytical techniques. CSA showed different inhibitory effects on different isozymes. CSA reversibly inhibited CYP3A4 and CYP2C9 activities in human liver microsomes with IC50 values of 28.3 and 31.3 μM, respectively, but exhibited no inhibition activities to CYP1A2, CYP2A6, CYP2C19, CYP2D6, and CYP2E1. CSA showed a weak effect on CYP450 enzymes in a time-dependent manner. CSA did not substantially induce CYP1A2, CYP2A6, CYP2B6, CYP2E1, CYP2C9, CYP2C19, CYP2D6, or CYP3A4 at concentrations up to 30 μM in primary human hepatocytes. The results of our experiments may be helpful to predict clinically significant food−drug interactions when other drugs are administered in combination with CSA. KEYWORDS: cajaninstilbene acid, human liver microsomes, recombinant human P450 enzymes, human hepatocytes, food−drug interaction
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INTRODUCTION Pigeonpea (Cajanus cajan (L.) Millsp.) is a very important grain crop of rain-field agriculture. It has been a very popular food in many developing countries; besides that, it is very helpful in medical usage. It is reported that there are many kinds of important active components in pigeonpea, such as flavonoids and stilbenes.1,2 CSA is a novel natural compound extracted from pigeonpea (C. cajan) leaves.3,4 Clinical data suggest CSA can play a role as an antihypoglycemic and also reduce blood triglyceride levels.3,4 Recently, it has also attracted researchers’ attention for its significant anti-inflammatory and analgesic effects.5 Furthermore, a new study reported that CSA was effective in treating postmenopausal osteoporosis.6 In our previous studies, CSA showed significant antioxidant activity.7 It also has been found that CSA possessed antitumor effects, especially its activity in MCF-7 in our latest study.8 On the basis of these results, as a valuable natural antioxidant compound, CSA may potentially be applicable in the health food and medicine industries. To the best of our knowledge, there is no report on CSA metabolism except our previous study, which developed a LC-MS/MS method to determine CSA levels in rat plasma and tissue samples, and the method was applied in pharmacokinetic and tissue distribution studies.9 © 2014 American Chemical Society
According to our previous research, CSA can be rapidly absorbed after oral administration (Tmax, 10.7 ± 0.31 min; t1/2, 51.40 ± 6.54 min). Then it was rapidly and widely distributed in tissues of rats. Therefore, it is necessary to study the metabolism of CSA in vivo and in vitro for future clinical usages. The U.S. Food and Drug Administration (FDA, 2006) recommends that all new chemical entities (NCEs) in development should be characterized with respect to metabolic properties before administration to humans.10 To identify the a new chemical entity’s in vitro characteristics, it is necessary to estimate the role of metabolism in clearance, identify the enzymes involved, clarify structural characterization of metabolites, and evaluate potential food−drug interaction risks associated with the NCE.11−13 Human CYP450 enzymes, especially CYP1A2, CYP2A6, CYP2E1, CYP2C9, CYP2C19, CYP2D6, and CYP3A4, play a key role in the clearance of many drugs. These enzymes are responsible for the metabolism of the Received: Revised: Accepted: Published: 10604
April 8, 2014 September 16, 2014 October 1, 2014 October 1, 2014 dx.doi.org/10.1021/jf501635a | J. Agric. Food Chem. 2014, 62, 10604−10614
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Figure 1. Chemical structures of (A) cajaninstilbene acid (CSA) and (B) isoliquiritigenin (ISL).
majority of drugs.14,15 The activity alteration of these enzymes is the major cause of metabolic food−drug interactions.16 Hence, the aim of our study was to investigate the in vitro metabolism of CSA by human liver microsomes and recombinant human P450 enzymes to identify the CYP450 enzymes involved and evaluate potential CSA food−drug interactions.
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Formation rates of hydroxylation metabolites (M1, M2) from CSA were evaluated in the absence (control) and presence of known CYP450 isoform-specific inhibitors with pooled human liver microsomes. The inhibitors were 20 μM furafylline (CYP1A2 inhibitor), 50 μM pilocarpine (CYP2A6 inhibitor), 50 μM sulfaphenazole (CYP2C9 inhibitor), 5 μM ticlopidine (CYP2C19 inhibitor), 10 μM quinidine (CYP2D6 inhibitor), 50 μM diethylthiocarbamate (CYP2E1 inhibitor), and 5 μM ketoconazole (CYP3A4 inhibitor). The final concentration of acetonitrile in incubations was 0.1%. CSA (20 μM) was preheated for 5 min at 37 °C in an NADPH-generating system. Human liver microsomes (0.5 mg/mL) were added to initiate the reaction and incubated at 37 °C for 15 min. The inhibitors concentrations were chosen selectively for the respective CYP450 isoforms. Inhibition percent of metabolites’ formation rate was calculated by comparing the inhibited activity with uninhibited controls (without inhibitors). Stock solutions of the chemical inhibitors were prepared in acetonitrile. Control solutions were prepared with the same amount of acetonitrile without inhibitors. The conditions for use of these inhibitors have been described in previous publications.20−22 The recombinant human P450 enzyme experiments were performed as described previously with slight modification,23 The incubation system was the same as those applied to the liver microsomal incubations with the exception that microsomes were replaced by recombinant human P450 enzymes CYP1A2, 2A6, 2C9, 2C19, 2D6, 2E1, and 3A4. The enzyme concentration in the incubation system was 50 pmol/mL. The total volume of incubation was 500 μL. The incubation time was 60 min, and the sample treatment method was same as described under Incubations with Human Liver Microsomes for Metabolite Identification. Quantification and Chromatographic Analysis of Metabolites. An LC-MS/MS analysis method was used to quantify CSA and its major metabolites M1 and M2. LC-MS/MS was carried out with an Agilent 1100 series LC system equipped with a UV detector (Agilent Technologies, Palo Alto, CA, USA). An HPLC system consisting of degasser G1379B, binary pump G1312B, a 7725i manual injector, and column thermostat G1316B was used. An HIQ Sil C 18 column (4.6 mm × 250 mm, KYA TEACH, made in Japan) was used. The mobile phase consisted of water and methanol (15:85, v/v) containing 0.1% formic acid. The flow rate was 1.0 mL/min, and the column eluate was monitored at a wavelength of 254 nm. The Agilent 1100 series LC system was series connected with an API5500Q trap triple-stage quadrupole mass spectrometer (Applied Biosystems, Concord, Canada) equipped with an electrospray ionization (ESI) source. The mass spectrometer was operated in negative ion mode with a capillary temperature of 300 °C and spray voltage of −4500 V. The nitrogen gas flow rate, collision energy, and declustering potential were adjusted to give maximum sensitivity for CSA (12 L/min, −25 V, −60 V, respectively).9 The mass spectrometer was operated in multiple reaction monitoring (MRM) mode. For semiquantification of CSA and hydroxylation metabolites (M1, M2), ISL was added to each sample as internal standard and the following MRM transitions were selected on the basis of the fragmentation of (M − H)− ions (results observed in previous MS/MS analyses): 337.1/293.0 for CSA, 353.1/ 279.1 for M1, 353.4/253.9 for M2, and 255.1/119.3 for ISL. An LC-MS/MS quantification method was used to quantify CYP450 enzyme activities in human liver microsomes and primary
MATERIALS AND METHODS
Chemicals and Reagents. Cajaninstilbene acid (CSA, purity = 98%, HPLC grade) was isolated and purified in our laboratory. By comparing the IR, 1H and13C nuclear magnetic resonance (NMR), and MS data with reported data,17 we confirmed the structure of CSA. Isoliquiritigenin (ISL), paclitaxel, glucose 6-phosphate, glucose-6phosphate dehydrogenase, NADPH- and CYP450-specific substrates, inhibitors, and inducers were purchased from Sigma-Aldrich (St. Louis, MO, USA). Human liver microsomes (pooled from 20 donors) and primary human hepatocytes (the human donor was a 47-year-old nonsmoking Chinese female with no known history of human immunodeficiency or virus exposure to hepatitis B or C) were purchased from iPhase Biosciences Co., Beijing, China. Recombinant human P450 enzymes (CYP1A2, CYP2A6, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4) and human CYP-reductase coexpressed in Escherichia coli, supplemented with purified human cytochrome b5, were purchased from Cypex (Dundee, UK). All microsomal preparations were stored at −80 °C until used. The total P450 content, protein concentrations, and specific activity of each P450 isoform were supplied by the manufacturer. All other reagents and chemicals were of analytical grade. The chemical structures of CSA and ISL are shown in Figure 1. Incubations with Human Liver Microsomes for Metabolite Identification. Incubation experiments were performed as described previously with slight modification.18,19 Microsomal incubation mixture in phosphate buffer, pH 7.4 (0.1 mol/L), consisted of CSA (20 μM), 3 mM MgCl2, 0.5 mg/mL microsomal protein, and the NADPH-regenerating system (0.5 mM NADP+, 5 mM glucose 6phosphate, and 0.5 unit of glucose-6-phosphate dehydrogenase/mL). Final volume was 500 μL. Samples were incubated at 37 °C for 5 min before the reaction was initiated by the addition of microsomal proteins. Thereafter, samples were carried out aerobically at 37 °C in a test tube placed on a temperature-controlled heating block for 60 min. To terminate the reaction, 500 μL of acetonitrile containing ISL (100 μL of 50 μM) was added. ISL was added as an internal standard to the incubation sample and vortexed for 3 min, and then the sample was extracted using 1 mL of ethyl acetate. The sample was centrifuged at 12000 rpm for 20 min at room temperature. The supernatant was then transferred to a separate tube and evaporated to dryness under nitrogen. The residues were dissolved in 0.1 mL of methanol and then vortex-mixed for 30 s and centrifuged at high speed for 5 min in an Eppendorf tube. All of the samples were injected into HPLC and LCMS/MS for the analysis of CSA and its metabolites. Identification of P450s Involved in Metabolite Formation from CSA. To determine the isozymes involved in metabolite formation in CSA, the chemical inhibitor and recombinant human P450 enzymes were used. 10605
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Table 1. Experimental Conditions for Measuring Microsomal P450 Activity for Enzyme Inhibition Studies CYP isoform
substrate
3A4 2D6
testosterone dextromethorphan
2C9 2A6 1A2 2C19 2E1 internal standard
tolbutamide coumarin phenacetin S-mephenytoin chlorozoxazone
substrate probe testosterone 6β-hydroxylation dextromethorphan Odemethylation tolbutamide 4′-hydroxylation coumarin 7′-hydroxylation phenacetin O-dealkylation S-mephenytoin 4 -hydroxylation chlorozoxazone 6′-hydroxylation paclitaxel
parent ion
daughter ion
polarity
declustering potential (V)
collision energy (V)
305.5 258.2
269 157
ESI+ ESI+
65 80
25 55
287.1 160.9 151.9 233.4 184 876
171.2 132.1 110.1 190.2 120.1 308.1
ESI+ ESI+ ESI+ ESI− ESI− ESI+
55 65 60 −55 −40 −45
30 35 25 −20 −15 −25
human hepatocytes. The ionization mode was electrospray, polarity positive. Electrospray jet stream conditions were as follows: capillary voltage, −4500 V; drying gas temperature, 300 °C; nebulizer pressure, 20 psi; sheath gas temperature, 350 °C. The mass spectrometer was operated in the MRM mode. MRM transitions, fragmentor voltage, and collision energy of the substrate probes are summarized in Table 1. For structural identification, metabolites and major molecular ion peaks were determined using HPLC-MS, MS/MS, and MS/MS/MS spectra separately. Metabolites were identified on the basis of their accurate mass determination and fragmentation patterns in positive and negative modes. Enzyme Kinetic Analysis of CSA in Human Liver Microsomes and Recombinant Human P450 Enzymes. The enzyme kinetics of CSA metabolism by pooled human liver microsomes and seven recombinant human P450 enzymes (CYP1A2, CYP2A6, CYP2C9, CYP2C19, CYP2D6, CYP2E1, or CYP3A4) was investigated. Kinetic constants were derived from incubations with the following CSA concentrations: 1, 2, 3.9, 7.8, 15.6, 31.3, 62.5, 125, 250, and 500 μM (n = 3). Human liver microsomes and recombinant human P450 enzyme contents were 0.5 mg/mL and 50 pmol/mL, respectively. Incubation times were 30 and 15 min for human liver microsomes and recombinant human P450 enzymes, respectively. Reactions were stopped by adding 500 μL of protein precipitation/internal standard solution and were extracted as previously described. Incubation mixtures without NADPH incubated were used as controls. CSA Inhibition Study of CYP450 Enzymes. The potential of CSA as an inhibitor of major drug-metabolizing CYP450 enzymes was evaluated with human liver microsomes using selective CYP450 probe substrates (Table1) in a reversible and time-dependent manner. Incubation in phosphate buffer, pH 7.4 (0.1 mol/L), was conducted at 37 °C, consisting of human liver microsomes, 0.5 mg/mL, and CYP450 probe substrate with the NADPH-regenerating system. In the assay of CSA inhibition in reversible manner, human liver microsomes were the last reagent added into the incubation system to initiate the reaction; after 10 min, the reaction was terminated by adding an equal volume of acetonitrile (v/v) containing paclitaxel as an internal standard. To examine CSA potential as a time-dependent inhibitor, CSA (20 μM) was preincubated at 37 °C with human liver microsomes and an NADPH-generating system for 30 min. After the preincubation period, the probe substrate was added, and the incubation was continued for 10 min to measure residual CYP450 activity. Reactions were terminated as described above. Samples were analyzed as described under Analytical Methods. The IC50 value was determined by incubation of pooled human liver microsomes (0.5 mg/ mL) with CYP450 probe substrates in the presence and absence of CSA (0.1−100 μM). The results were processed following a method described previously.24 Analysis of CSA To Induce CYP450 Enzymes. To investigate the potential of CSA to induce CYP450 enzymes, the enzyme activities of CYP1A2, CYP2A6, CYP2E1, CYP2C9, CYP2C19, and CYP3A4 in primary human hepatocytes were assessed. Primary human hepatocytes were treated with CSA (1, 10, and 30 μM) or one of three prototypical CYP450 inducers, omeprazole (100 μM), phenobarbital (750 μM), and rifampin (10 μM), as positive controls for 3 days. CSA
and the positive controls were dissolved in DMSO, and hepatocytes treated with DMSO (final concentration = 0.1%, v/v) served as negative controls. Hepatocytes were harvested after 72 h of treatment, and microsomes were prepared from each culture, on the basis of the methods described previously.25 The enzyme activity in microsomal samples was determined by incubating microsomal samples with probe substrates for 10 min at 37 °C. Microsomes were incubated with phenacetin (80 μM), tolbutamide (100 μM), coumarin (100 μM), chlorozoxazone (200 μM), S-mephenytoin (200 μM), and testosterone (250 μM) for various time periods (10−30 min). Then the activities of CYP1A2, CYP2A6, CYP2E1, CYP2C9, CYP2C19, and CYP3A4 were determined, respectively. Samples were analyzed as described under Quantification and Chromatographic Analysis of Metabolites. Hepatocytes’ culture method and sample treatment method were described previously.26 Data Analysis. Nonlinear regression analysis, V = Vmax (S)/(Km + (S)), was used to describe the kinetics of biotransformation of CSA to M1 and M2 in human liver microsomes (GraphPad Prism 5.0).27 V is reaction rate, (S) is substrate concentration, and Km is the Michaelis− Menten constant. The intrinsic clearance (CLint) for formation of each CSA metabolite was calculated by Vmax/Km; it is a parameter commonly used for quantitative in vitro−in vivo allometric scaling.28,29
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RESULTS Identification of Metabolites Formed by Human Liver Microsomes. When CSA was incubated with human liver microsomes in the presence of NADPH, two metabolites (M1 and M2) were detected by HPLC. The formation of the metabolites was dependent on the presence of NADPH, and the yield of metabolite appeared to be linear with respect to incubation time (up to 30 min). However, no metabolite could be clearly observed in the absence of NADPH. Typical chromatograms of CSA and its two main metabolites are shown in Figure 2. The HPLC retention times of metabolites, CSA, and the internal standard (ISL) were 6.8 (M1), 9.1 (M2), 15.7, and 3.9 min, respectively. To characterize M1 and M2, LC-MS/MS and LC-MS/MS/ MS methods were used. MS/MS and MS/MS/MS spectra of M1 and M2 are shown in Figure 3. First, precursor ion scanning identified two potential metabolites that exhibited 353.1 and 353.4 amu in the CSA microsomal incubates that consisted of human liver microsomes and NADPH system (but not in negative control experiments) in addition to the 337.3 amu of CSA. Second, to gain further structural information, MS/MS spectra of these potential metabolites were obtained by collision-induced dissociation of their molecular ions. The MS/ MS spectra detected two major characteristic fragment ions for each potential metabolite: m/z 309.1, 291.2, and 279.1 were obtained from m/z 353.1; m/z 309.3, 253.9, and 238.7 were obtained from m/z 353.4. These transition ions were selected in the MRM modes for identification and quantification of the metabolites: The metabolite with 353.1 amu was measured with 10606
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Figure 2. Representative chromatograms of CSA metabolites (M1 and M2) extracted from human liver microsomal incubates: (A) CSA incubated by human liver microsomes with NADPH-regenerating system; (B) CSA incubated by human liver microsomes without NADPH-regenerating system; MS/MS spectra of (C) M1 and (D) M2, the metabolite of CSA formed by human liver microsomes. ISL was used as an internal standard (IS). The retention times of M1, M2, CSA, and the internal standard (ISL) were 6.8, 9.1, 15.7, and 3.9 min, respectively.
the quantifier MRM m/z 353.1/279.1 and confirmed with the qualifier MRM transition m/z 353.1/291.2, and the metabolite with 353.4 amu was measured with the quantifier MRM m/z 353.4/253.9 and confirmed with the qualifier MRM 353.4/ 238.7. To determine the structures of M1and M2, the parent drug fragmentation pattern was used as a guide to identify CSA metabolites; metabolic pathways of CSA are shown in Figure 4. M1 and M2 showed the (M − H) ion at m/z 353, which was
16 amu higher than that of the parent compound, suggesting that an oxygen atom was inserted into the molecule of CSA. To further confirm the structure of metabolites M1 and M2, MS/ MS/MS experiments were performed. Fragment ions m/z 309.1, 291.2, 279.1, and 239.4 from M1 and m/z 309.3, 253.9, 250.5, and 238.7 from M2 were analyzed. The major fragment ions of m/z 309.1, 291.2, 279.1, 239.4, 309.3, 253.9, 250.5, and 238.7 are shown in Figure 3. The fragment pathways of M1 and M2 are shown in Figure 5. 10607
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Figure 3. MS/MS and MS/MS/MS spectra of (A) M1 and (B) M2.
Kinetic Analysis of CSA in Human Liver Microsomes. Kinetic analysis for the formation of M1 and M2 was studied with pooled human liver microsomes. The formation rate of M1 and M2 revealed Michaelis−Menten saturation curves. A representative Michaelis−Menten plot for M1 and M2 formation in human liver microsomes is shown in Figure 6.
The apparent Km values of M1 and M2 catalyzed by human liver microsomes are 28.8 and 23.6 μM, respectively (Table 2). Vmax (apparent) values were 141.6 and 122.7 pmol/mg/min, respectively. On average, the apparent intrinsic clearances (Clint) of M1 and M2 formation were 4.9 and 5.2 μL/mg protein/min, respectively. 10608
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Figure 4. Formation and fragmentation pathways of CSA.
Figure 5. Formation and fragmentation pathways of (A) M1 and (B) M2.
Metabolism of CSA by Recombinant Human P450 Enzymes. Kinetic parameters of M1 and M2 from CSA are shown in Table 3. The formation rates of M1 and M2 revealed Michaelis−Menten saturation curves. The Km and Vmax of M1 formation from CSA were 5.9, 9.0, and 12.9 μM and 6.8, 15.0, and 28.9 pmol/mg/min for CYP1A2, CYP2C9, and CYP3A4,
respectively. The Km and Vmax of M2 formation from CSA were 19.7 μM and 104.6 pmol/mg/min, respectively, for CYP2C9. To better assess the relative contribution of the individual P450 isoforms to the in vitro formation of CSA in human liver microsomes, the intrinsic clearances were calculated. The CLint values (Vmax/Km) for the formation of M1 by CYP1A2, 10609
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Figure 6. CSA concentration-dependent formation of (A) M1 and (B) M2 in the incubations with human liver microsomes. Data were fitted to a Michaelis−Menten substrate binding curve.
Table 2. Kinetic Parameters for the Metabolism of CSA by Human Liver Microsomes metabolite
pathway
Km (μM)
Vmax (pmol/mg/min)
Vmax/Km (μL/mg protein/min)
kinetic model
M1 M2
phenyl hydroxylation phenyl hydroxylation
28.8 23.6
141.6 122.7
4.9 5.2
Michaelis−Menten Michaelis−Menten
Table 3. Kinetic Parameters for the Metabolism of CSA by Recombinant Human P450 Enzymes metabolite
P450
Km (μM)
Vmax (pmol/mg/min)
Vmax/Km (μL/mg protein/min)
% of total CLint
kinetic model
M1
CYP1A2 CYP2C9 CYP3A4 CYP2C9
5.9 9.0 12.9 19.7
6.8 15.0 28.9 104.6
1.2 1.7 2.2 5.3
22.1 31.7 42.6 95.6
Michaelis−Menten Michaelis−Menten Michaelis−Menten
M2
CYP2C9, and CYP3A4 were 1.2, 1.7, and 2.2 μL/mg protein/ min, respectively. The CLint value for the formation of M2 by CYP2C9 was 5.3 μL/mg protein/min. Chemical Inhibition Experiment. Studies were conducted to evaluate the effect of CYP450 enzyme chemical inhibitors on the formation of M1 and M2 in pooled human liver microsomes (the concentration of CSA was 20 μM). According to the result above, M1 and M2 were identified as the metabolites derived from hydroxylation of CSA. Because of the very low yield of metabolites, they have not been separated or synthesized for technical reasons; metabolite formation was quantified by the loss of CSA relative to the internal standard (ISL) in each incubation sample on the basis of the MRM responses. Figure 7 presents the results of CSA biotransformation in the human liver microsome incubations after treatment
with various inhibitors. Among seven kinds of inhibitors, ketoconazole (CYP3A4), sulfaphenazole (CYP2C9), and furafylline (CYP1A2) notably inhibited the formation of M1 from CSA. The percentages of inhibition of M1 formation by CYP1A2, CYP2C9, and CYP3A4 were 26.0, 35.5, and 38.5%, respectively. Sulfaphenazole showed significant inhibition to M2 formation, the percentage of inhibition reaching 97.8%. Percent inhibition of metabolite formation rate by isoformspecific inhibitors was calculated by comparing the inhibited activity with uninhibited controls (without inhibitors). CSA Inhibition Study of CYP450 Enzymes. CSA (0.1− 100 μM) was evaluated for its ability to inhibit CYP450 activity in pooled human liver microsomes using probe substrate. The results are presented in Table 4 and Figure 8. It is indicated that in a reversible manner CSA was an inhibitor of CYP3A4 and CYP2C9 (IC50 values were 28.3 and 31.3 μM, respectively). CSA showed little or no inhibition to CYP1A2, 2A6, 2D6, 2C19, or 2E1 (IC50 ≥ 40 μM). Table 4. CSA Inhibitory Activity against CYP1A2, CYP2A6, CYP2E1, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 in Pooled Human Liver Microsomes IC50 (μM)
Figure 7. Inhibition percent of CSA with chemical inhibitor addition in the incubation system of human liver microsomes. 10610
CYP isoform
0 min of preincubation
30 min of preincubation
3A4 2D6 2C9 2A6 1A2 2C19 2E1
28.3 >100 31.3 >100 >100 >100 43.9
27.2 >100 33.5 >100 >100 >100 44.1
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Figure 8. Effect of CSA on microsomal enzyme activity of CYP3A4, CYP2C9, and CYP2E1 with and without a 30 min preincubation with NADPHfortified human liver microsomes.
Figure 9. Effect of CSA or prototypical inducers on microsomal CYP450 activity in cultured human hepatocytes.
In the assay of time-dependent manner, the IC50 values of CYP3A4 and CYP2C9 were 27.2 and 33.5 μM, respectively. IC50 values of other enzymes were >40 μM. Analysis of CSA Potential To Induce CYP450 Enzymes. To investigate the potential of CSA to induce the expression of CYP450 enzymes, freshly isolated and pretreated human hepatocytes were used. Three preparations of human hepatocytes were cultured and treated with CSA (1, 10, or 30 μM) or one of three prototypical enzyme inducers, omeprazole (100 μM), phenobarbital (750 μM), and rifampin (10 μM), once daily for 3 consecutive days. For the concentrations tested, CSA was not cytotoxic. After treatment in all three preparations of human hepatocytes, prototypical CYP450 enzymes showed obviously sensitive to the inducers, as the result in Figure 9. Treatment with omeprazole produced a marked increase in CYP1A2 (27fold), whereas it showed increases in CYP2C9 (3−4-fold), CYP2C19 (3−10-fold), and CYP3A4 (15−21-fold) after treatment with phenobarbital and rifampin. In the experiment of treatment with human hepatocytes for 3 consecutive days, with the concentration of CSA up to 30 μM, the activities of CYP1A2, CYP2A6, CYP2E1, CYP2C9, CYP2C19 , and
CYP3A4 were almost not affected by CSA. The extent of enzyme activity induction did not reach significant levels,
