Inhibitory effects of wogonin on catalytic activity of cytochrome P450 enzyme in human liver microsomes
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- Li, T., Li, N., Guo, Q. et al. Eur J Drug Metab Pharmacokinet (2011) 36: 249. doi:10.1007/s13318-011-0050-0
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Wogonin, derived from the root of Scutellaria baicalensis, is a popular herb for its anticancer, anti-inflammatory, neuroprotective and anti-convulsant effects. The purpose of this study was to investigate the effect of wogonin on human hepatic cytochrome P450s (CYP450s) in vitro. Isoform-specific substrate probes of CYP1A2, 2C9, 2C19, 2D6, 2E1 and 3A4 were incubated in human liver microsomes with or without wogonin. IC50 and Ki values were estimated and the types of inhibition were determined. Wogonin was a potent, competitive inhibitor of CYP1A2 (Ki = 0.24 μM), and a weak inhibitor of CYP2C19 (IC50 = 101.10 μM), but was not able to inhibit CYP2C9, CYP2D6, CYP2E1 and CYP3A4 (IC50 > 200 μM). Wogonin could inhibit the activity of CYP1A2 and CYP2C19 with varying potency, while it is a strong inhibitor of CYP1A2. These findings suggested that it was necessary to study the potential pharmacokinetic drug interaction in vivo.
KeywordsCytochrome P450Drug–drug interactionWogoninEnzyme inhibition
High performance liquid chromatography
High-performance liquid chromatography–electrospray spectrometry
50% inhibitory concentration
Equilibrium dissociation constant for reversible inhibitor
The Michaelis constant
Drug-drug interactions are of great interest to scientists involved in drug research, clinicians and patients. The majority of these interactions involve cytochrome P450 (CYP450). Several herbs and natural remedies have already been reported to induce or inhibit CYP450 enzymes (Ito et al. 2008; Usia et al. 2006; Madgula et al. 2009; Oleson et al. 2004; Sekiguchi et al. 2008). CYP 450 enzymes are one of the major Phase I enzymes involved in the metabolism of drugs and other xenobiotics, as well as some endogenous substrates (Xu et al. 2008). CYP450s consists of the superfamily of hemeproteins that catalyze the oxidative metabolism of substrates. For its broad range of substrate specificity, CYP enzymes play an important role in Phase I metabolism. Among numerous CYP enzymes identified to date, six human liver CYP isoforms (CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4) have been found to be the most relevant isozymes involved in the metabolism of clinically significant drugs (Yan and Caldwell 2001; Hodgson and Rose 2007). CYP 3A4/5 is also present in abundance within the gut lining and is important for metabolism of some orally administered compounds. Inhibition of cytochrome P450 activities is the most common mechanism underlying drug–drug interactions. The inhibition of CYP450 activities may lead to potential toxicity and alter the efficacy of drugs (Taylor and Wilt 1999; Kalgutkar et al. 2007; Pelkonen et al. 2008; Jacobson 2004). Thus, a great deal of effort is expended in new drug research in avoiding the development of compounds that will cause drug–drug interactions. Mibefradil (Posicor®, Roche) was withdrawn from the market in 1998 because it potently inhibits CYP3A4, the predominant drug metabolizing P450, after having led to adverse events in patients concurrently taking a variety of other commonly prescribed medicines (Welker et al. 1998).
Comprehensive inhibitory analyses that encompass all major drug-metabolizing CYPs are important. Reports of the effects of wogonin alone on human P450 enzymes are little, and its effect on human CYP450s has not been systematically investigated. Thus, the present study was carried out to determine the potential for wogonin to inhibit human CYP enzymes most commonly involved in the metabolic clearance of drugs: CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 in vitro, followed by a full characterization of inhibition kinetics and the type of inhibition using known marker substrates.
2 Materials and methods
2.1 Chemicals and reagents
Wogonin (purity > 99%) was provided by the School of pharmacy, China Pharmaceutical University. Acetaminophen, omeprazole, dextromethorphan, chlorzoxazone, nifedipine, caffeine, tinidazole, bifendate, nimodipine, genistein and diazepam were purchased from National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Phenacetin, diclofenac, 4-hydroxydiclofenac, 5-hydroxyomeprazole, dextrorphan, 6-hydroxychlorzoxazone, midazolam, 1-hydroxymidazolam, oxidized nifedipine and NADPH were purchased from Sigma-Aldrich (St. Louis, MI, USA). All other reagents were of analytical grade.
2.2 Human liver microsomes
Human liver microsomes (HLMs) were provided by the Research Institute for Liver Disease Co. (RILD) (Shanghai, China). The microsomes were prepared from ten individual human donor livers.
2.3 Inhibition of CYP450s by wogonin
The CYP enzymatic activities were determined by phenacetin O-deethylation for CYP1A2, dextromethorphan O-demethylation for CYP2D6, chlorzoxazone 6-hydroxylation for CYP2E1, midazolam 1-hydroxylation and nifedipine oxidization for CYP3A4. For each inhibition study, preliminary incubation experiments were carried out using a single CYP-specific substrates concentration around its Km (phenacetin, 60 μM; diclofenac, 10 μM; omeprazole, 20 μM; dextromethorphan, 5 μM; chlorzoxazone, 40 μM; midazolam, 8 μM; nifedipine, 40 μM) in the presence of a range of wogonin concentrations (0.5–200 μM) in the incubation mixture. Probe substrates were incubated with HLMs (0.2 mg/ml), 5 mM MgCl2 and 1.0 mM NADPH in a total volume of 200 μl of 150 mM phosphate buffer (pH 7.4), in the presence and absence of wogonin. After preincubated for 5 min in an incubator shaker at 37°C, the reactions were initiated by adding NADPH. The reactions were conducted for 25 min for CYP1A2, 10 min for CYP2C9, 20 min for CYP2C19, 15 min for CYP2D6, 25 min for CYP2E1, 5 min for CYP3A4 (midazolam 1-hydroxylation), 10 min for CYP3A4 (nifedipine oxidization), respectively.
Known inhibitors were run in parallel as positive controls: α-naphthoflavone for CYP1A2, sulfaphenazole for CYP2C9, ticlopidine for CYP2C19, quinidine for CYP2D6, diethyldithiocarbamate for CYP2E1 and ketoconazole for CYP3A4. The Ki values of wogonin were not determined if their IC50 were higher than 100 μM. Inhibitor and substrate concentrations for determining Ki value were selected based on the preliminary study. The range of concentrations of phenacetin (0–0.6 μM) and wogonin (0.5–200.0 μM) were used for estimation of Ki value. All measurements were performed in triplicate.
2.4 Time-dependent inhibition on CYP1A2
Triplicate samples for time-dependent inhibition screening assay were preincubated for 0, 5, 10, 30 min at 37°C with wogonin selected here with NADPH. After preincubation, phenacetin was added and then incubated as the method mentioned above. The concentration of wogonin was equal to its IC50. The percentage of remaining activity of microsomes preincubated with NADPH was compared with that of microsomes preincubated without NADPH.
2.5 Analytical methods
The reaction was stopped by adding 20 μl of 5 μg/ml caffeine on ice and extracted with 400 μl of ethyl acetate. The samples were shaken for 3 min, and then centrifuged at 12,000 rpm for 5 min. The organic phase was evaporated to dryness. The residue was reconstituted in 200 μl of mobile phase and a 20 μl aliquot was injected for analysis by HPLC (LC-2010AD, Shimadzu, Japan). The chromatographic separation was achieved using a Hypersil ODS2 column (250 mm × 4.6 mm, 5 μm; Dalian Elite Analytical Instruments Co. Dalian, China). The mobile phase comprised of methanol—0.14% triethylamine solution (adjusted to pH5 using acetic acid) (30:70, v/v) at a flow rate of 1.0 ml/min with the detection wavelength set at 256 nm. Acetaminophen was quantitated with a linear dynamic range from 0.01 to 0.5 μg/ml.
The reaction was stopped by adding 20 μl of 1 μg/ml bifendate on ice and extracted with 400 μl of ethyl acetate. The samples were shaken for 3 min, and then centrifuged at 12,000 rpm for 5 min. The organic phase was evaporated to dryness. The residue was reconstituted in 200 μl of mobile phase and a 20 μl aliquot was injected for analysis by HPLC. The chromatographic separation was achieved using a Hypersil ODS2 column (150 mm × 4.6 mm, 5 μm; Dalian Elite Analytical Instruments Co. Dalian, China). The mobile phase comprised of acetonitrile—0.2% acetic acid solution (55:45, v/v) at a flow rate of 1.0 ml/min with the detection wavelength set at 274 nm. 4-Hydroxydiclofenac was quantitated with a linear dynamic range from 0.05 to 2.0 μg/ml.
The reaction was stopped by adding 20 μl of 3 μg/ml tinidazole on ice and extracted with 400 μl of ethyl acetate. The samples were shaken for 3 min, and then centrifuged at 12,000 rpm for 5 min. The organic phase was evaporated to dryness. The residue was reconstituted in 100 μl of mobile phase and a 20 μl aliquot was injected for analysis by HPLC. The chromatographic separation was achieved using a Hypersil ODS2 column (150 mm × 4.6 mm, 5 μm; Dalian Elite Analytical Instruments Co. Dalian, China). The mobile phase comprised of acetonitrile—0.006 M phosphate buffer (24:76, v/v) at a flow rate of 1.0 ml/min with the detection wavelength set at 302 nm. 5-Hydroxyomeprazole was quantitated with a linear dynamic range from 0.1 to 5.0 μg/ml.
The reaction was stopped by adding 200 μl of acetonitrile containing 3 μg/ml nimodipine on ice. The samples were shaken for 2 min, and then centrifuged at 12,000 rpm for 10 min. 10 μl of the supernatant was injected for analysis by LC–MS. Chromatography was done using Hypersil ODS column (150 mm × 2.2 mm, 3 μm; Thermo, USA) and an HPLC system consisting of Shimadzu 20A series (Shimadzu, Kyoto, Japan) equilibrated in 40% acetonitrile containing 3 mM ammonium acetate at a flow rate of 0.3 ml/min. The analyte was monitored by selected ion monitoring of m/z 258 (detrorphan) and m/z 419 (IS) with the mass spectrometer operated in the positive ion mode using a Shimadzu LC–MS-2010A quadrupole mass spectrometry. Detrorphan was quantitated with a linear dynamic range from 0.05 to 5.0 μg/ml.
The reaction was stopped by adding 200 μl of acetonitrile containing 0.25 μg/ml genistein on ice. The samples were shaken for 2 min, and then centrifuged at 12,000 rpm for 10 min. 10 μl of the supernatant was injected for analysis by LC–MS. Chromatography was done using Hypersil ODS2 column (150 mm × 4.6 mm, 5 μm; Dalian Elite Analytical Instruments Co. Dalian, China) equilibrated in 40% acetonitrile containing 3 mM ammonium acetate at a flow rate of 1.0 ml/min. The analyte was monitored by selected ion monitoring of m/z 184 (6-hydroxychlorzoxazone) and m/z 269 (IS) with the mass spectrometer operated in the negative ion mode. 6-Hydroxychlorzoxazone was quantitated with a linear dynamic range from 0.05 to 2.0 μg/ml.
The reaction was stopped by adding 200 μl of acetonitrile containing 0.05 μg/ml diazepam on ice. The samples were shaken for 2 min, and then centrifuged at 12,000 rpm for 10 min. 5 μl of the supernatant was injected for analysis by LC–MS/MS. Chromatography was done using Xtimate C18 column (100 mm × 2.1 mm, 5 μm; Welch Material, Inc., USA) and an HPLC system consisting of Shimadzu 20A series (Shimadzu, Kyoto, Japan) equilibrated in a gradient mobile phase of water and acetonitrile at flow rate of 0.3 ml/min. The analyte was monitored by selected reaction monitoring of m/z 342.2>168.1 (1-hydroxymidazolam) and 285.1>193.2 (IS) with the mass spectrometry (TSQ Quantum MS/MS system, Thermo Electron, USA) operated in the negative ion mode. 1-Hydroxymidazolam was quantitated with a linear dynamic range from 2 to 200 ng/ml.
2.5.7 Oxidized nifedipine
The reaction was stopped by adding 20 μl of 5 μg/ml diazepam on ice and extracted with 400 μl of ethyl acetate. The samples were shaken for 3 min, and then centrifuged at 12,000 rpm for 5 min. The organic phase was evaporated to dryness. The residue was reconstituted in 100 μl of mobile phase and a 20 μl aliquot was injected for analysis by HPLC. The chromatographic separation was achieved using a C18 column (200 mm × 4.6 mm, 5 μm; Dalian Elite Analytical Instruments Co. Dalian, China). The mobile phase comprised of methanol–water (60:40, v/v) at a flow rate of 1.0 ml/min with the detection wavelength set at 254 nm. Oxidized Nifedipine was quantitated with a linear dynamic range from 0.02 to 2.0 μg/ml.
3 Data analysis
IC50 values were determined by fitting the data in Sigmaplot (version 10.0; SPSS Inc., Chicago, IL). The Ki values obtained from Dixon and secondary Lineweaver–Burk plots were used as initial estimates for the determination of the exact Ki values by nonlinear least square regression analysis using Sigmaplot.
4.2 Inhibition of CYP450 activities by wogonin in HLMs
The IC50 values for the inhibition of wogonin on CYP enzymes activities
Substrate reaction probes
4.3 Inhibition kinetic study
4.4 Time-dependent inhibition on CYP1A2
Under the different preincubation time, the concentrations of acetaminophen were consistent, indicating that wogonin could not inhibit CYP1A2 activities by time-dependent inhibition.
Multi-drug therapy is now a common therapeutic practice in patients with multiple complications and the interactions are unavoidable. The inhibition of CYP enzymes can result in clinical drug interactions whereby the systemic exposure to one drug that is cleared primarily via CYP-mediated biotransformation is elevated when coadministered with a second drug that inhibits this activity. Except for chemical drugs, natural products can also inhibit CYP enzymes (He and Edeki 2004; Pan et al. 2010). Co-administration of herbal medicines and synthetic drugs was prevalent in the clinical regimens. As a result, the potential risk of herb–drug interactions increased significantly, which may have important clinical significance based on an increasing number of clinical reports of such interactions.
In this study, we established probe substrate assay methods in HLMs conventionally applied to study inhibition toward CYP enzymes activities. The effects of wogonin on activities of CYP enzymes were characterized by examining the activities of marker reactions in human liver microsomes. We determined the IC50 and Ki values of wogonin toward CYP450 isoforms. Wogonin showed a strong competitive type of inhibition toward CYP1A2 and weak inhibition toward CYP2C19, whereas was not able to inhibit other CYP450 isoforms.
Wogonin exhibited strong inhibition on CYP1A2-catalyzed phenacetin O-deethylation with IC50 value of 0.27 μM. Dixon plot analysis shows competitive inhibition mode for wogonin with Ki value of 0.24 μM. CYP1A2 is a highly conserved hepatic enzyme that is expressed constitutively in mammals, which is involved in metabolism of a number of xenobiotics including drugs, dietary and environmental chemicals (Pineau et al. 1995). CYP1A2 is capable of exerting both detrimental and beneficial effects depending upon the substrate it metabolizes. It can catalyze several carcinogenic heterocyclic amines to reactive metabolites that are implicated in the development of various cancers (Zhou et al. 2009). Among the six flavonoids, wogonin showed the highest inhibitory activity in the formation of AFM1 (aflatoxin M1, metabolite of aflatoxin B1) in rat liver microsomes, which was caused by selective inhibition of CYP1A1/2. It could be suggested that a drug interaction of wogonin may occur with co-administered drugs metabolized by CYP1A2.
Among the many CYP isoforms, CYP2C19 isoform is one of the most important, since it is genetically polymorphic (Küpfer and Preisig 1984; Inaba et al. 1984). CYP2C19 can metabolize several widely used drugs, such as proton pump inhibitors, psychotropic drugs and anticonvulsants (Pelkonen et al. 2008). Some studies indicate that inhibition of CYP2C19 may lead to alter the therapeutic ranges (Wallentin 2009). It was reported that carbamazepine (CBZ) and oxcarbazepine (OXC) inhibit CYP2C19-mediated phenytoin metabolism using HLMs and cDNA-expressed CYP2C19 (Lakehal et al. 2002). Phenytoin plasma levels would be elevated by co-administration of CBZ and OXC in clinical practice (Zielinski and Haidukewych 1987; Flesch 2004). A clinical study examining the metabolism of omeprazole has established that after a 14-day treatment with St John’s wort, concentrations of omeprazole in plasma decreased (Wang et al. 2004). So interaction between chemicals or herbal products and CYP2C19 is a potentially important safety issue in view of the important role of the isoform in drug metabolism. In the present study, wogonin showed weak inhibitory effect on CYP2C19-catalyzed omeprazole 5-hydroxylation with IC50 value of 101.10 μM. Our result offered the in vitro evidence that wogonin may have little effect on CYP2C19 which cause warfarin to overanticoagulate in vivo. Our result supplies the information that wogonin may cause drug interaction in humans when co-administered with drugs that are metabolized primarily by CYP2C19.
Theoretically, significant enzyme inhibition occurs when the concentration of the inhibitor present at the active site is comparable to or in excess of the Ki. The likelihood of an in vivo interaction is projected based on the [I]/Ki ratio where [I] represents the mean steady-state Cmax value for total drug (bound plus unbound) following administration of the highest proposed clinical dose. As the ratio increases, the likelihood of an interaction increases. An estimated [I]/Ki ratio of greater than 0.1 is considered positive and a follow-up in vivo evaluation is recommended. Although quantitative predictions of in vivo drug–drug interactions from in vitro studies are not possible, rank order across the different CYP enzymes for the same drug may help prioritize in vivo drug–drug interaction evaluations. It should be studied further whether in vitro inhibition of CYP1A2 by wogonin can influence drug metabolism in vivo.
In conclusion, we have provided the comprehensive in vitro data that enable us to understand and predict drug interactions with wogonin. Wogonin has been identified that possess high potency in the inhibition of CYP enzymes involved in the metabolism of drugs. This finding provides useful information for investigation of the potential for wogonin to cause drug interactions of clinical relevance. It is likely that wogonin can cause drug interaction in humans when co-administered with drugs that are metabolized primarily by CYP1A2 and CYP2C9. However, the effects of some drugs in vitro and in vivo have no correlation. Some flavonoids which are potent in vitro modulators for activities of CYP enzymes are unlikely to have any appreciable effects in vivo. Further studies of wogonin should be done to study the potential pharmacokinetic drug interaction in vivo.