http://informahealthcare.com/xen ISSN: 0049-8254 (print), 1366-5928 (electronic) Xenobiotica, Early Online: 1–7 ! 2015 Informa UK Ltd. DOI: 10.3109/00498254.2014.1003113
RESEARCH ARTICLE
Inhibitory mechanisms of celastrol on human liver cytochrome P450 1A2, 2C19, 2D6, 2E1 and 3A4 Chunhuan Jin1, Xin He1,2, Fangliang Zhang1, Lina He1, Junxiu Chen1, Lili Wang1,2, Lijun An1 and Yaowen Fan1 1
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School of Chinese Materia Medica, Tianjin University of Traditional Chinese Medicine, Nankai District, Tianjin, P.R. China and Laboratory of Modern Chinese Medicine, Tianjin, P.R. China
2
Tianjin State Key
Abstract
Keywords
1. The present study was conducted to examine the possibility of herb–drug interaction by celastrol, which is a main compound isolated from Tripterygium wilfordii Hook F. using human liver microsomes with cocktail methods. Focused on its inhibitory manner on the metabolism of model probe substrates of five cytochrome P450 isoenzymes (CYP1A2, CYP2C19, CYP2D6, CYP2E1 and CYP3A4) in vitro which are important with the metabolism of different xenobiotics. 2. The results showed that celastrol inhibited the five types of human cytochrome P450 isoforms, with the IC50 values of 2.65 mM (CYP3A4), 5.99 mM (CYP2C19), 6.27 mM (CYP2D6), 7.66 mM (CYP1A2) and 9.38 mM (CYP2E1), respectively. The data indicated that celastrol acted in different manners as an inhibitor of human cytochrome P450 isoforms, which showed that celastrol not only un-competitively inhibited the CYP1A2 and 2E1 activities, but also competitively inhibited the CYP2C19 and 2D6 activities with Ki values of 1.41, 2.29, 5.27 and 4.21 mM, respectively. Celastrol was also a mixed-type inhibitor of CYP3A4, with Ki and Kis values of 2.02 and 5.49 mM, respectively. 3. Celastrol has the potential to inhibit cytochrome P450 activities and may cause the herb– drug interactions. Therefore, the use of celastrol and its preparations with conventional medicines should thus be taken in to account.
CYP, herb–drug interaction (HDI), inhibition, in vitro
Introduction Tripterygium wilfordii is a plant which belongs to tripterygium genus of Celastraceae. As a traditional Chinese medicine, it has been used as anti-inflammatory drug because of its immunosuppressive action and it is widely used in the treatment of immune-related diseases like rheumatoid arthritis, chronic nephritis and systemic lupus erythematosus (Qiu et al., 2003; Yang et al., 1998). Celastrol (Figure 1), a quinone methide triterpene isolated from plants belonging to the family Celastraceae (used in oriental medicine), is one of the main effective constituents of the Chinese medicine Tripterygium. According to a large number of reports, celastrol has significant pharmacological activities such as anti-inflammatory, immunosuppressant and anti-neoplastic activity (Kim et al., 2009; Faust et al., 2009), since scientists of China and the United States found the new anti-neoplastic mechanism of celastrol through cooperation (Yang et al., 2006), celastrol is considered as a promising anti-cancer Address for correspondence: Xin He, PhD, Professor & Director, School of Chinese Materia Medica, Tianjin University of Traditional Chinese Medicine, 312 Anshanxi Road, Nankai District, Tianjin 300193, P.R. China. Tel: +86-22-5959-6231. Fax: +86-22-5959-6153. E-mail: [email protected]
History Received 6 November 2014 Revised 21 December 2014 Accepted 24 December 2014 Published online 26 March 2015
candidate for drug development (Min et al., 2014). Usually, celastrol is used as a main component of the Chinese patent medicine GTV tablet, with the content of 356 mg in each tablet. In most cases, the medicine is taken orally. The cytochrome P450 is an enzyme family which is responsible for biological transformation of xenobiotics and endogenous substances. Although the composition of Chinese medicine is complicated, its material basis for efficacy still needs to be metabolized through cytochrome P450, thus it might affect the metabolism of other medicines or generate drug–drug interactions (Slaughter & Edwards, 1995). Enzymology has close relationship with drug metabolism, illumination of the pharmacology and toxicology of medicines can benefit from the intra-corporal effect studies of Chinese medicine from the perspective of cytochrome P450 (Johnson et al., 2014). However, the effect mechanism of celastrol on cytochrome P450 and the information on celastrol cause herb–drug interaction (HDI) by inhibition of cytochrome P450 is limited. This experiment studied the inhibition of celastrol on the metabolism of cytochrome P450 enzyme in human liver microsomes with ‘‘cocktail’’ probe drug method (Grimm et al., 2009). Meanwhile, enzyme kinetic studies were carried out to determine the inhibitory manners of
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Enzyme kinetic studies To test the inhibition types of CYP1A2, 2D6, 2C19, 3A4 and 2E1, celastrol were preincubated at 37 C for 15 min with human liver microsomes and substrate probes, and then a 30 min further incubation was started by adding NADPH. The reactions were stopped by adding in two-fold volume (400 ml) of cold methanol containing 50 ng/ml carbamazepine. For kinetic studies, the concentrations of celastrol used were from 0 to 50 mM. The concentrations of probe substrates (phenacetin/ dextromethorphan/ mephenytoin / chlorzoxazone/ midazolam) was 12.5/ 30/ 15/ 6.25/ 25 mM, 25/ 60/ 30/ 12.5/ 50 mM, 50/ 120/ 30/ 25/ 120 mM, 100/ 240/ 120/ 50/ 240 mM.
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Figure 1. Chemical structure of celastrol isolated from Tripterygium wilfordii Hook F.
celastrol on cytochrome P450 to predict the potential HDI of celastrol.
Materials and methods Chemicals and reagents Celastrol with a purity of > 98% was purchased from the NanJing Langze Bioengineering Institute (Nanjing, China). Human pooled liver microsomes was bought from Reid Ltd, liver disease research (Shanghai, China). Phenacetin/ acetaminophen, dextromethorphan/ dextrorphan, chlorzoxazone/ 6-hydroxy chlorzoxazone, midazolam/ 10 -hydroxy midazolam, mephenytoin/ 4-hydroxy mephenytoin, tolbutamide/ 4-hydroxytolbutamide, b-nicotinamide adeninedinucleotide phosphate (NADPH), K2HPO4H2O, KH2PO4 and dimethyl sulfoxide (DMSO) were purchased from Sigma Chemical Co. (St. Louis, MO). Methanol and acetonitrile were high performance liquid chromatography-grade from Concord Corporation (Tianjin, China). Ultra-pure water was obtained from a Milli-Q Pluswater purification system (Millipore, Bedford, MA). All other regents were of analytical grade. IC50 shift assay Cocktail assay (Obach et al., 2007) was employed to analyze the inhibition of celastrol on CYP1A2, CYP2D6, CYP2E1, CYP3A4 and CYP2C19. Phenacetin (10 mM), dextromethorphan (2.5 mM), chlorzoxazone (20 mM), midazolam (5 mM), mephenytoindue (20 mM) were chosen as probe substrates to corresponding cytochrome P450. The mixtures of 20 ml celastrol (final concentrations were 0, 0.625, 3.125, 6.25, 12.5, 25, 50, 100 mM, respectively) were pre-incubated with 20 ml HLMs (final concentration was 0.25 mg protein/ml) in the presence or absence of the NADPH (40 ml) for 30 min at 37 C. Then, probe substrates (80 ml) and NADPH (1 mM, 100 ml) were added in 20 ml above pre-incubation mixtures and incubated for 15 min under the same conditions. The reactions were stopped by adding in two-fold volume (400 ml) of cold methanol containing 50 ng/ml carbamazepine. The assay was performed in triplicate for all test specimens. The inhibitory effect of samples was assessed from the difference between sample and corresponding control.
LC-MS-MS analysis All chromatography was performed using an America Bio System Inc. LC-MS system API 4000 Qtrap, SER.N: AR26221101 (SHIMADZU LC-20AD pump, SIL-20AC constant temperature automatic sampler, CTO-20A column temperature box, CBM-20A controller, positive ESI ion source, Analyst Software 1.5.2 chromatographic work station). An Acquity UPLC BEH C18 column (3.5 mm, 2.1 mm 50 mm) was used. The column was maintained at ambient temperature (25 C). The mobile phase consisted of 0.1% aqueous formic acid (A) and 0.1% formic acid in methanol (B) with a linear-gradient elution at a flow rate of 0.45 ml/min. The elution program was optimized and conducted as follows: 98–98% (0–0.5 min), 98–2% (0.5–1 min); 2–2% (1–2.5 min); 2–98% (2.5–4 min). The yield of corresponding metabolites was calculated by referring to a standard curve constructed based on known concentrations of the pure metabolites. Statistical analysis IC50 values (concentration of inhibitor to cause 50% inhibition of original enzyme activity) were determined by GraphPad Prism version 5.0 (GraphPad Software, San Diego, CA) using the following equation: Y ¼ Bottom þ
Top Bottom 100% 1 þ 10ðLogEC50XÞ
where X is the numerical of inhibitor concentration, Y is the relative activity (CN%). The IC50 shift values were analyzed by comparing the IC50 values when celastrol preincubated for 15 min in the absence and in the presence of NADPH. Enzyme kinetics were performed using GraphPad Prism 5.0 (GraphPad software Inc., San Diego, CA). Graphical inspection from different plots was used to determine the inhibition constant (Ki) values and modes of celastrol to different cytochrome P450 enzymes. Various concentrations of substrates were plotted against reaction velocities to obtain linear transformation for Primary Lineweaver–Burk Plot, a double-reciprocal plot. Dixon plot for Ki was plotted by the slopes/y-intercepts of regression lines in the Lineweaver– Burk Plot and multiple inhibitor concentrations in competitive inhibition/un-competitive inhibition. Dixon plot for Kis in
Inhibitory mechanisms of celastrol on human liver cytochrome
DOI: 10.3109/00498254.2014.1003113
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Table 1. The IC50 value and the IC50 shift value on each one of five kinds of CYPs in () NADPH or (+) NADPH-generating system with modes of inhibition and Ki values for the inhibition of five CYP isoform-mediated metabolism by celastrol in human liver microsomes (n ¼ 3). IC50 values (mM)
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CYP1A2 CYP2C19 CYP2D6 CYP2E1 CYP3A4
()NADPH
(+)NADPH
Shift (NADPH/ +NADPH)
7.66 5.99 6.27 9.38 2.65
4.71 5.19 5.54 5.42 2.38
1.62 1.15 1.13 1.73 1.11
Mode of inhibition
Ki value (mM)
Kis value (mM)
Un-competitive Competitive Competitive Un-competitive Mix (competitive and non-competitive)
1.41 5.27 4.21 2.29 2.02
– – – – 5.49
Figure 2. The IC50 shift curves of celastrol on the CYP1A2, CYP2C19, CYP2D6, CYP2E1 and CYP3A4 activities in human liver microsomes. The concentrations of celastrol were 0.625 to 100 mM. Values are expressed as mean ± SD of three samples.
mixed type of inhibition was plotted by the y-intercepts of regression lines in the Lineweaver–Burk Plot and multiple inhibitor concentrations. The x-intercept is known to 1/Ki. Experimental data were presented as means ± SD.
Results IC50 shift assay To investigate the inhibitory of celastrol on the five cytochrome P450 isoforms, the probe reaction assays were conducted with varied concentrations of celastrol in ‘‘cocktail’’ assay. And the IC50 shift assay was carried out to confirm if the IC50s were left shift for some of the specific cytochrome P450 isoforms. The concentrations of celastrol ranged from 0.625 to 100 mM. Results suggested that celastrol inhibited human CYP1A2, 2C19, 2D6, 2E1 and 3A4 activities in a concentration-dependent manner. Drugs with IC50 values less than 100 mg/ml are considered potent inhibitor (Usia et al., 2006). The strong inhibitory effects of celastrol on five cytochrome P450 isoforms were observed with IC50 values less than 10 mM (Table 1). The residual activity of
cytochrome P450 isoforms in the present of NADPH (+NADPH) decreased slightly compared with that of the absence of NADPH (NADPH), and the IC50 shift value were 1.62 (CYP1A2), 1.15(CYP2C19), 1.13(CYP2D6), 1.73(CYP2E1), 1.11(CYP3A4), respectively (Figure 2). Grimm et al. (2009) stated that a clear criterion is not established. Whereas most cited IC50 shift value of 10-fold is being clearly a positive finding for mechanism-based inhibition. These results were inferred that after 15-min preincubation of celastrol with human liver microsomes in the presence of NADPH, no obvious shift in IC50 was observed for inhibition of five CYPs, suggesting that celastrol might not be a time-dependent inhibitor of above five cytochrome P450 isoforms. Enzyme kinetic studies The inhibition manners of CYP1A2, 2C19, 3A4, 2D6 and 2E1 activities by celastrol were characterized by enzyme kinetic analysis. Figures 3 and 4 showed the double-reciprocal plots of the enzyme inhibited by different concentrations of
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Figure 3. Primary Lineweaver–Burk plot and the Dixon plot for Ki in the inhibitory effect of CYP1A2, CYP2C19, CYP2D6 and CYP2E1 in human liver microsomes. Each data point represents the mean of three samples.
celastrol. The plots of 1/v versus 1/[S] give a family of lines with different slope and intercept. Figure 3(A) and (G) showed an uncompetitive inhibition model indicating that celastrol bind CYP1A2 and CYP2E1 at a site created after
enzyme–substrate complex (E–S) formation with Ki values were 1.41 mM and 5.27 mM (Figure 3B and H). As shown in Figure 3(C) and (E), the straight lines intersected on the common point of the ordinate axis, suggesting competitive
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DOI: 10.3109/00498254.2014.1003113
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Figure 4. Primary Lineweaver–Burk plot (A) and the Dixon plot for Ki (B) and Kis (C) in the inhibitory effect of CYP2E1 in human liver microsomes. Each data point represents the mean of three samples.
inhibition of celastrol on CYP2C19 and CYP2D6. The Ki values were 4.21 mM and 2.29 mM (Figure 3D and F) determined from the Dixon plots. In addition, Figure 4(A) showed that the family of straight lines intersected in the second quadrant, which indicated celastrol was a mixed-type inhibitor of CYP3A4. The result revealed that the inhibitor bind with free enzymes as well as enzyme–substrate complexes. The Ki value of celastrol on 3A4 was 2.02 mM (Figure 4B). The Kis value of celastrol on CYP3A4 was 5.49 mM (Figure 4C).
Discussion The concurrent administration of herbal medicines and prescription drugs is accepted worldwide. The effect on cytochrome P450 is an essential part of assessment of drug interactions and clinical adverse effects (Fontana et al., 2005). Previous studies seldom assessed the effect of celastrol on liver metabolism. This study was carried out to determine whether celastrol is a selective inhibitor of cytochrome P450 isoforms. According to normal judgment, IC5051 mM suggests strong inhibitory efficiency, 1 mM5IC50510 mM suggests medium inhibitory efficiency, and IC50 > 10 mM suggests weak inhibitory efficiency and may have no clinical meaning. Results showed that celastrol inhibits five cytochrome P450 isofroms with the IC50 values less than 10 mM, suggesting celastrol is a potential inhibitor of cytochrome
P450, and this inhibition was concentration-dependent. Moreover, no significant left-shift in IC50 assay was observed for inhibition of five CYPs, suggesting that celastrol might not be a time-dependent inhibitor of above five cytochrome P450 isoforms. Different approaches are provided and emphasis is placed on the potential of in-vitro inhibition studies using human liver microsomes for drug metabolism and inhibition studies. It sometimes coupled with a cocktail probe assay, which is widely used in evaluation of the potential for drug–drug interactions in the early stage of drug development (Dinger et al., 2014). In the present study, we evaluated the inhibition of celastrol on the activities of cytochrome P450 in human liver microsomes by using five cocktail probes. This cocktail screening experiment improves the efficiency and reduces the test cost. The four-type classification of reversible cytochrome P450 inhibitors was based on different binding sites. Therefore, the order of drug administration might affect drug–drug interactions. Order of apply substrate and the inhibitors could influence the interactions between competitive inhibitors and substrate, but will not influence DDI of non-competitive inhibitor. Un-competitive inhibitors only exert their inhibition effect on metabolic enzymes in the presence of substrate, so order of drug administration would have a relatively great influence on inhibiting effect of this kind of inhibitors. For mixed-type inhibitors, administration order of
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non-competitive and competitive mixed inhibitors will cause drug–drug interactions, while administration order of noncompetitive and un-competitive mixed inhibitors does not affect their interactions with the substrate. Ranking the third place in enzyme content, CYP1A2 is mainly distributed in liver and makes up about 13% of cytochrome P450 enzyme in the liver. It takes a part in the metabolism of numerous pro-toxicants and procarcinogens. (Carrillo et al., 2000; Faber et al., 2005; Heeyeon et al., 2013; Shimada et al., 1997). The current study suggested that celastrol act in an un-competitive manner as an inhibitor of CYP1A2 with the Ki and IC50 values of 1.41 mM and 7.66 mM, respectively. Celastrol had the characteristics of an uncompetitive inhibitor in which the inhibitor interacted with the enzyme–substrate complexes. In this case, the binding of the substrates to the actice site of CYP1A2 created a binding site for celastrol. Therefore, celastrol was a CYP1A2 inhibitor and the potential herb–drug interaction with CYP1A2 would be serious. In addition, as celastrol was an un-competitive inhibitor of CYP1A2, it should be noted that combined use of celastrol and CYP1A2 substrate shall be avoided during clinical practice. CYP2C19 is the minor form of the human cytochrome P450 (0.2%) (Shimada et al., 1994), but it has genetic polymorphisms (Goldstein, 2001). The results showed that celastrol strongly inhibited CYP2C19 with an IC50 value of 6.38 mM and a Ki value of 5.27 mM. The competitive inhibition of celastrol on CYP2C19 was obtained, suggesting that celastrol and CYP2C19 substrate compete against each other for the binding site of the enzyme. And the inhibiting effect could be reversed by increasing substrate concentration. Although CYP2D6 is a relatively minor fraction of the total cytochrome P450 content, it catalyzes approximately 30% of the drugs in the market and inhibition of this isoenzyme may result in potential adverse drug interactions in the clinic because the inhibition of this drug-metabolizing enzyme may lead to increased plasma levels of another drug administered concomitantly and result in drug-induced toxicity (Ye et al., 2014). The results suggested that celastrol inhibit CYP2D6 with an IC50 value of 8.14 mM and a Ki value of 4.21 mM. The competitive inhibition of celastrol on CYP2D6 was obtained, suggesting that the use of celastrol may cause interactions with the substrates of CYP2D6, including anti-depressive drugs, such as fluoxetine and amitriptyline, b-receptor-blocking agents, such as metoprolol and propranolol (Fukumoto et al., 2006), and anti-psychotic drugs, such as chlorpromazine, perphenazine, and tamoxifen (Li et al., 2011). As an ethanol-induced cytochrome P450 enzyme, CYP2E1 metabolizes 2% clinically used drugs, and is able to catalyze pro-carcinogen and pro-toxin (Arinc et al., 2007; Zuber et al., 2002). Metabolic reactions catalyzed by CYP2E1 generate reactive oxygen species (reactive oxygen species, ROS), such as oxygen radicals. And rise in intracellular ROS results in lipid peroxidation, which will bind with nucleophile in the cell, like protein, damage cells and cause cytotoxicity or hepatotoxicity (Gonzalez, 2007). Our results suggested that celastrol may un-competitively inhibit CYP2E1 in human liver microsomes with the Ki value was 2.29 mM and the IC50 value was 9.38 mM. That is, celastrol binded only with
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enzyme–substrate complex, but not with free enzymes. High concentration of substrate will increase the occupancy of the active site and the binding site on CYP2E1 for celastrol, and therefore the effective the inhibition. During clinical practice, combined use of celastrol and CYP2E1 substrate should be avoided to defend herb–drug interactions. As an important drug-metabolizing enzyme, CYP3A is able to metabolize about 50% of clinical drugs, and can be inhibited and induced by a variety of drugs. It was once reported that the induction of CYP3A4 expression would change the clearance rate of a variety of clinical drugs, including midazolam, amitriptyline, cyclosporine and oral contraceptive. Our results showed that celastrol inhibited CYP3A4 in human liver microsome with a Ki value of 2.02 mM, an IC50 value of 2.63 mM, and the Kis value of 5.49 mM, celastrol proved to be the mixed-type inhibitor on CYP3A4. As a non-competitive/competitive mixed inhibitor, celastrol could inhibit both free enzyme (E) and enzyme– substrate complex (E–S), and its inhibition on CYP3A4 was strong. The value of Ki was lower than that of Kis, indicating that the affinity of the inhibitor for free enzyme was stronger than that for the enzyme–substrate complexes. Therefore, drug administration order during clinical practice would affect interactions between celastrol and CYP3A4 substrates. The results of this study showed celastrol might affect in vitro the metabolism of drugs which are substrates of the cytochrome P450 isoforms. The dosage of celastrol for cancers and inflammation treatment has been chosen during 0.1–10 mM in some in vitro experiments (Boridy et al., 2014). Previous in vivo studies have studied the inhibitory effects of celastrol on cytochrome P450 activities in rat liver microsomes, which demonstrated that celastrol inhibited the metabolism of CYP1A2, CYP2C11 and CYP3A2 substrate with different mode of inhibition. And because of the poor absorption of pure celastrol, the IC50 and Ki values in that study were higher than the rat plasma concentrations (Min et al., 2014). Zhang et al. (2012) reported that it displayed higher bioavailability for celastrol-containing ‘‘Thunder of God Vine’’ tablets probably owing to the presence of other constituents in the tablet preparations than that pure celastrol administration in rats treated. But the human plasma concentration was seldom reported when celastrol administered orally. Due to the interspecies differences and CYP impression between human and rat, further system study in vivo and in vitro is needed to identify the interactions of celastrol with cytochrome P450 isoform in humans.
Conclusions Results of this study suggest that celastrol could inhibit different isoforms of cytochrome P450, including CYP1A2, CYP2C19, CYP2D6, CYP2E1 and CYP3A4 that exist in reconstructed human liver microsomes with different mechanism. These in vitro experiments on cytochrome P450 isoforms would facilitate further application and development of celastrol as a new anti-cancer drug. However, the effect of herb–drug interactions is still elusive. Therefore, further systematic studies in humans in vitro and in vivo are also needed to identify the interactions of celastrol with cytochrome P450.
DOI: 10.3109/00498254.2014.1003113
Declaration of interest This study was supported by The Natural Science Foundation of Tianjin [12JCZDJC26100]; National Natural Science Foundation of China [NSFC, No.81373890 and No.81430096]; The Program for Changjiang Scholars and Innovative Research Team in University [PCSIRT, No. IRT_14R41]. The authors report no declaration of interest.
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Johnson EF, Connick JP, Reed JR, et al. (2014). Correlating structure and function of drug-metabolizing enzymes: progress and ongoing challenges. Drug Metab Dispos 42:9–22. Kim DY, Park JW, Jeoung D, et al. (2009). Celastrol suppresses allergeninduced airway inflammation in a mouse allergic asthma model. Eur J Pharmacol 612:98–105. Li J, Go¨decke T, Chen SN, et al. (2011). In vitro metabolic interactions between black cohosh (Cimicifuga racemosa) and tamoxifen via inhibition of cytochromes P450 2D6 and 3A4. Xenobiotica 41: 1021–30. Min S, Yu T, Tonggui D, et al. (2014). Inhibitory effects of celastrol on rat liver cytochrome P450 1A2,2C11, 2D6, 2E1 and 3A2 activity. Fitoterapia 92:1–8. Obach RS, Walsky RL, Venkatakrishnan K. (2007). Mechanism-based inactivation of human cytochrome p450 enzymes and the prediction of drug-drug interactions. Drug Metab Dispos 35:246–55. Qiu D, Kao PN. (2003). Immunosuppressive and ant-i inflammatory mechanism of triptolide, the principal active diterpenoid from the Chinese medicinal herb Tripterygium wilfordii Hook. f Drugs R.D 4:1–18. Shimada T, Yamazaki H, Mimura M, et al. (1994). Interindividual variations in human liver cytochrome P-450 enzymes involved in the oxidation of drugs, carcinogens and toxic chemicals: studies with liver micros omes of 30 Japanese and 30 Caucasians. J Pharmacol Exp Ther 270:414–23. Shimada T, Mimura M, Inoue K, et al. (1997). Cytochrome P450dependent drug oxidation activities in live rmicrosomes of various animal species including rats, guinea pigs, dogs, monkeys, and humans. Arch Toxicol 71:401–8. Slaughter RL, Edwards DJ. (1995). Recent advances: the cytochrome P450 enzymes. Ann Pharmacother 29:619–24. Yang H, Chen D, Cui QC, et al. (2006). Celastrol, a triterpene extracted from the Chinese ‘‘Thunder of God Vine,’’ is a potent proteasome inhibitor and suppresses human prostate cancer growth in nude mice. Cancer Res 66:4758–65. Yang Y, Liu Z, Tolosa E, et al. (1998). Triptolide induces apoptotic death of T lymphocyte. Immunopharmacology 40:139–49. Ye LH, He XX, Kong LT, et al. (2014). Identification and characterization of potent CYP2D6 inhibitors in lotus leaves. J Ethnopharmacology 153:190–6. Usia T, Iwata H, Hirastuka A, et al. (2006). CYP3A4 and CYP2D6 inhibitory activities of Indonesian medicinal plants. Phytomedicine 13:67–73. Zhang J, Li CY, Xu MJ, et al. (2012). Oral bioavailability and genderrelated pharmacokinetics of celastrol following administration of pure celastrol and its related tablets in rats. J Ethnopharmacology 144: 195–200. Zuber R, Anzenbacherova´ E, Anzenbacher P. (2002). Cytochromes P450 and experimental models of drug metabolism. J Cell Mol Med 6:189–98.
RESEARCH ARTICLE
Inhibitory mechanisms of celastrol on human liver cytochrome P450 1A2, 2C19, 2D6, 2E1 and 3A4 Chunhuan Jin1, Xin He1,2, Fangliang Zhang1, Lina He1, Junxiu Chen1, Lili Wang1,2, Lijun An1 and Yaowen Fan1 1
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School of Chinese Materia Medica, Tianjin University of Traditional Chinese Medicine, Nankai District, Tianjin, P.R. China and Laboratory of Modern Chinese Medicine, Tianjin, P.R. China
2
Tianjin State Key
Abstract
Keywords
1. The present study was conducted to examine the possibility of herb–drug interaction by celastrol, which is a main compound isolated from Tripterygium wilfordii Hook F. using human liver microsomes with cocktail methods. Focused on its inhibitory manner on the metabolism of model probe substrates of five cytochrome P450 isoenzymes (CYP1A2, CYP2C19, CYP2D6, CYP2E1 and CYP3A4) in vitro which are important with the metabolism of different xenobiotics. 2. The results showed that celastrol inhibited the five types of human cytochrome P450 isoforms, with the IC50 values of 2.65 mM (CYP3A4), 5.99 mM (CYP2C19), 6.27 mM (CYP2D6), 7.66 mM (CYP1A2) and 9.38 mM (CYP2E1), respectively. The data indicated that celastrol acted in different manners as an inhibitor of human cytochrome P450 isoforms, which showed that celastrol not only un-competitively inhibited the CYP1A2 and 2E1 activities, but also competitively inhibited the CYP2C19 and 2D6 activities with Ki values of 1.41, 2.29, 5.27 and 4.21 mM, respectively. Celastrol was also a mixed-type inhibitor of CYP3A4, with Ki and Kis values of 2.02 and 5.49 mM, respectively. 3. Celastrol has the potential to inhibit cytochrome P450 activities and may cause the herb– drug interactions. Therefore, the use of celastrol and its preparations with conventional medicines should thus be taken in to account.
CYP, herb–drug interaction (HDI), inhibition, in vitro
Introduction Tripterygium wilfordii is a plant which belongs to tripterygium genus of Celastraceae. As a traditional Chinese medicine, it has been used as anti-inflammatory drug because of its immunosuppressive action and it is widely used in the treatment of immune-related diseases like rheumatoid arthritis, chronic nephritis and systemic lupus erythematosus (Qiu et al., 2003; Yang et al., 1998). Celastrol (Figure 1), a quinone methide triterpene isolated from plants belonging to the family Celastraceae (used in oriental medicine), is one of the main effective constituents of the Chinese medicine Tripterygium. According to a large number of reports, celastrol has significant pharmacological activities such as anti-inflammatory, immunosuppressant and anti-neoplastic activity (Kim et al., 2009; Faust et al., 2009), since scientists of China and the United States found the new anti-neoplastic mechanism of celastrol through cooperation (Yang et al., 2006), celastrol is considered as a promising anti-cancer Address for correspondence: Xin He, PhD, Professor & Director, School of Chinese Materia Medica, Tianjin University of Traditional Chinese Medicine, 312 Anshanxi Road, Nankai District, Tianjin 300193, P.R. China. Tel: +86-22-5959-6231. Fax: +86-22-5959-6153. E-mail: [email protected]
History Received 6 November 2014 Revised 21 December 2014 Accepted 24 December 2014 Published online 26 March 2015
candidate for drug development (Min et al., 2014). Usually, celastrol is used as a main component of the Chinese patent medicine GTV tablet, with the content of 356 mg in each tablet. In most cases, the medicine is taken orally. The cytochrome P450 is an enzyme family which is responsible for biological transformation of xenobiotics and endogenous substances. Although the composition of Chinese medicine is complicated, its material basis for efficacy still needs to be metabolized through cytochrome P450, thus it might affect the metabolism of other medicines or generate drug–drug interactions (Slaughter & Edwards, 1995). Enzymology has close relationship with drug metabolism, illumination of the pharmacology and toxicology of medicines can benefit from the intra-corporal effect studies of Chinese medicine from the perspective of cytochrome P450 (Johnson et al., 2014). However, the effect mechanism of celastrol on cytochrome P450 and the information on celastrol cause herb–drug interaction (HDI) by inhibition of cytochrome P450 is limited. This experiment studied the inhibition of celastrol on the metabolism of cytochrome P450 enzyme in human liver microsomes with ‘‘cocktail’’ probe drug method (Grimm et al., 2009). Meanwhile, enzyme kinetic studies were carried out to determine the inhibitory manners of
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Enzyme kinetic studies To test the inhibition types of CYP1A2, 2D6, 2C19, 3A4 and 2E1, celastrol were preincubated at 37 C for 15 min with human liver microsomes and substrate probes, and then a 30 min further incubation was started by adding NADPH. The reactions were stopped by adding in two-fold volume (400 ml) of cold methanol containing 50 ng/ml carbamazepine. For kinetic studies, the concentrations of celastrol used were from 0 to 50 mM. The concentrations of probe substrates (phenacetin/ dextromethorphan/ mephenytoin / chlorzoxazone/ midazolam) was 12.5/ 30/ 15/ 6.25/ 25 mM, 25/ 60/ 30/ 12.5/ 50 mM, 50/ 120/ 30/ 25/ 120 mM, 100/ 240/ 120/ 50/ 240 mM.
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Figure 1. Chemical structure of celastrol isolated from Tripterygium wilfordii Hook F.
celastrol on cytochrome P450 to predict the potential HDI of celastrol.
Materials and methods Chemicals and reagents Celastrol with a purity of > 98% was purchased from the NanJing Langze Bioengineering Institute (Nanjing, China). Human pooled liver microsomes was bought from Reid Ltd, liver disease research (Shanghai, China). Phenacetin/ acetaminophen, dextromethorphan/ dextrorphan, chlorzoxazone/ 6-hydroxy chlorzoxazone, midazolam/ 10 -hydroxy midazolam, mephenytoin/ 4-hydroxy mephenytoin, tolbutamide/ 4-hydroxytolbutamide, b-nicotinamide adeninedinucleotide phosphate (NADPH), K2HPO4H2O, KH2PO4 and dimethyl sulfoxide (DMSO) were purchased from Sigma Chemical Co. (St. Louis, MO). Methanol and acetonitrile were high performance liquid chromatography-grade from Concord Corporation (Tianjin, China). Ultra-pure water was obtained from a Milli-Q Pluswater purification system (Millipore, Bedford, MA). All other regents were of analytical grade. IC50 shift assay Cocktail assay (Obach et al., 2007) was employed to analyze the inhibition of celastrol on CYP1A2, CYP2D6, CYP2E1, CYP3A4 and CYP2C19. Phenacetin (10 mM), dextromethorphan (2.5 mM), chlorzoxazone (20 mM), midazolam (5 mM), mephenytoindue (20 mM) were chosen as probe substrates to corresponding cytochrome P450. The mixtures of 20 ml celastrol (final concentrations were 0, 0.625, 3.125, 6.25, 12.5, 25, 50, 100 mM, respectively) were pre-incubated with 20 ml HLMs (final concentration was 0.25 mg protein/ml) in the presence or absence of the NADPH (40 ml) for 30 min at 37 C. Then, probe substrates (80 ml) and NADPH (1 mM, 100 ml) were added in 20 ml above pre-incubation mixtures and incubated for 15 min under the same conditions. The reactions were stopped by adding in two-fold volume (400 ml) of cold methanol containing 50 ng/ml carbamazepine. The assay was performed in triplicate for all test specimens. The inhibitory effect of samples was assessed from the difference between sample and corresponding control.
LC-MS-MS analysis All chromatography was performed using an America Bio System Inc. LC-MS system API 4000 Qtrap, SER.N: AR26221101 (SHIMADZU LC-20AD pump, SIL-20AC constant temperature automatic sampler, CTO-20A column temperature box, CBM-20A controller, positive ESI ion source, Analyst Software 1.5.2 chromatographic work station). An Acquity UPLC BEH C18 column (3.5 mm, 2.1 mm 50 mm) was used. The column was maintained at ambient temperature (25 C). The mobile phase consisted of 0.1% aqueous formic acid (A) and 0.1% formic acid in methanol (B) with a linear-gradient elution at a flow rate of 0.45 ml/min. The elution program was optimized and conducted as follows: 98–98% (0–0.5 min), 98–2% (0.5–1 min); 2–2% (1–2.5 min); 2–98% (2.5–4 min). The yield of corresponding metabolites was calculated by referring to a standard curve constructed based on known concentrations of the pure metabolites. Statistical analysis IC50 values (concentration of inhibitor to cause 50% inhibition of original enzyme activity) were determined by GraphPad Prism version 5.0 (GraphPad Software, San Diego, CA) using the following equation: Y ¼ Bottom þ
Top Bottom 100% 1 þ 10ðLogEC50XÞ
where X is the numerical of inhibitor concentration, Y is the relative activity (CN%). The IC50 shift values were analyzed by comparing the IC50 values when celastrol preincubated for 15 min in the absence and in the presence of NADPH. Enzyme kinetics were performed using GraphPad Prism 5.0 (GraphPad software Inc., San Diego, CA). Graphical inspection from different plots was used to determine the inhibition constant (Ki) values and modes of celastrol to different cytochrome P450 enzymes. Various concentrations of substrates were plotted against reaction velocities to obtain linear transformation for Primary Lineweaver–Burk Plot, a double-reciprocal plot. Dixon plot for Ki was plotted by the slopes/y-intercepts of regression lines in the Lineweaver– Burk Plot and multiple inhibitor concentrations in competitive inhibition/un-competitive inhibition. Dixon plot for Kis in
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Table 1. The IC50 value and the IC50 shift value on each one of five kinds of CYPs in () NADPH or (+) NADPH-generating system with modes of inhibition and Ki values for the inhibition of five CYP isoform-mediated metabolism by celastrol in human liver microsomes (n ¼ 3). IC50 values (mM)
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CYP1A2 CYP2C19 CYP2D6 CYP2E1 CYP3A4
()NADPH
(+)NADPH
Shift (NADPH/ +NADPH)
7.66 5.99 6.27 9.38 2.65
4.71 5.19 5.54 5.42 2.38
1.62 1.15 1.13 1.73 1.11
Mode of inhibition
Ki value (mM)
Kis value (mM)
Un-competitive Competitive Competitive Un-competitive Mix (competitive and non-competitive)
1.41 5.27 4.21 2.29 2.02
– – – – 5.49
Figure 2. The IC50 shift curves of celastrol on the CYP1A2, CYP2C19, CYP2D6, CYP2E1 and CYP3A4 activities in human liver microsomes. The concentrations of celastrol were 0.625 to 100 mM. Values are expressed as mean ± SD of three samples.
mixed type of inhibition was plotted by the y-intercepts of regression lines in the Lineweaver–Burk Plot and multiple inhibitor concentrations. The x-intercept is known to 1/Ki. Experimental data were presented as means ± SD.
Results IC50 shift assay To investigate the inhibitory of celastrol on the five cytochrome P450 isoforms, the probe reaction assays were conducted with varied concentrations of celastrol in ‘‘cocktail’’ assay. And the IC50 shift assay was carried out to confirm if the IC50s were left shift for some of the specific cytochrome P450 isoforms. The concentrations of celastrol ranged from 0.625 to 100 mM. Results suggested that celastrol inhibited human CYP1A2, 2C19, 2D6, 2E1 and 3A4 activities in a concentration-dependent manner. Drugs with IC50 values less than 100 mg/ml are considered potent inhibitor (Usia et al., 2006). The strong inhibitory effects of celastrol on five cytochrome P450 isoforms were observed with IC50 values less than 10 mM (Table 1). The residual activity of
cytochrome P450 isoforms in the present of NADPH (+NADPH) decreased slightly compared with that of the absence of NADPH (NADPH), and the IC50 shift value were 1.62 (CYP1A2), 1.15(CYP2C19), 1.13(CYP2D6), 1.73(CYP2E1), 1.11(CYP3A4), respectively (Figure 2). Grimm et al. (2009) stated that a clear criterion is not established. Whereas most cited IC50 shift value of 10-fold is being clearly a positive finding for mechanism-based inhibition. These results were inferred that after 15-min preincubation of celastrol with human liver microsomes in the presence of NADPH, no obvious shift in IC50 was observed for inhibition of five CYPs, suggesting that celastrol might not be a time-dependent inhibitor of above five cytochrome P450 isoforms. Enzyme kinetic studies The inhibition manners of CYP1A2, 2C19, 3A4, 2D6 and 2E1 activities by celastrol were characterized by enzyme kinetic analysis. Figures 3 and 4 showed the double-reciprocal plots of the enzyme inhibited by different concentrations of
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Figure 3. Primary Lineweaver–Burk plot and the Dixon plot for Ki in the inhibitory effect of CYP1A2, CYP2C19, CYP2D6 and CYP2E1 in human liver microsomes. Each data point represents the mean of three samples.
celastrol. The plots of 1/v versus 1/[S] give a family of lines with different slope and intercept. Figure 3(A) and (G) showed an uncompetitive inhibition model indicating that celastrol bind CYP1A2 and CYP2E1 at a site created after
enzyme–substrate complex (E–S) formation with Ki values were 1.41 mM and 5.27 mM (Figure 3B and H). As shown in Figure 3(C) and (E), the straight lines intersected on the common point of the ordinate axis, suggesting competitive
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Figure 4. Primary Lineweaver–Burk plot (A) and the Dixon plot for Ki (B) and Kis (C) in the inhibitory effect of CYP2E1 in human liver microsomes. Each data point represents the mean of three samples.
inhibition of celastrol on CYP2C19 and CYP2D6. The Ki values were 4.21 mM and 2.29 mM (Figure 3D and F) determined from the Dixon plots. In addition, Figure 4(A) showed that the family of straight lines intersected in the second quadrant, which indicated celastrol was a mixed-type inhibitor of CYP3A4. The result revealed that the inhibitor bind with free enzymes as well as enzyme–substrate complexes. The Ki value of celastrol on 3A4 was 2.02 mM (Figure 4B). The Kis value of celastrol on CYP3A4 was 5.49 mM (Figure 4C).
Discussion The concurrent administration of herbal medicines and prescription drugs is accepted worldwide. The effect on cytochrome P450 is an essential part of assessment of drug interactions and clinical adverse effects (Fontana et al., 2005). Previous studies seldom assessed the effect of celastrol on liver metabolism. This study was carried out to determine whether celastrol is a selective inhibitor of cytochrome P450 isoforms. According to normal judgment, IC5051 mM suggests strong inhibitory efficiency, 1 mM5IC50510 mM suggests medium inhibitory efficiency, and IC50 > 10 mM suggests weak inhibitory efficiency and may have no clinical meaning. Results showed that celastrol inhibits five cytochrome P450 isofroms with the IC50 values less than 10 mM, suggesting celastrol is a potential inhibitor of cytochrome
P450, and this inhibition was concentration-dependent. Moreover, no significant left-shift in IC50 assay was observed for inhibition of five CYPs, suggesting that celastrol might not be a time-dependent inhibitor of above five cytochrome P450 isoforms. Different approaches are provided and emphasis is placed on the potential of in-vitro inhibition studies using human liver microsomes for drug metabolism and inhibition studies. It sometimes coupled with a cocktail probe assay, which is widely used in evaluation of the potential for drug–drug interactions in the early stage of drug development (Dinger et al., 2014). In the present study, we evaluated the inhibition of celastrol on the activities of cytochrome P450 in human liver microsomes by using five cocktail probes. This cocktail screening experiment improves the efficiency and reduces the test cost. The four-type classification of reversible cytochrome P450 inhibitors was based on different binding sites. Therefore, the order of drug administration might affect drug–drug interactions. Order of apply substrate and the inhibitors could influence the interactions between competitive inhibitors and substrate, but will not influence DDI of non-competitive inhibitor. Un-competitive inhibitors only exert their inhibition effect on metabolic enzymes in the presence of substrate, so order of drug administration would have a relatively great influence on inhibiting effect of this kind of inhibitors. For mixed-type inhibitors, administration order of
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non-competitive and competitive mixed inhibitors will cause drug–drug interactions, while administration order of noncompetitive and un-competitive mixed inhibitors does not affect their interactions with the substrate. Ranking the third place in enzyme content, CYP1A2 is mainly distributed in liver and makes up about 13% of cytochrome P450 enzyme in the liver. It takes a part in the metabolism of numerous pro-toxicants and procarcinogens. (Carrillo et al., 2000; Faber et al., 2005; Heeyeon et al., 2013; Shimada et al., 1997). The current study suggested that celastrol act in an un-competitive manner as an inhibitor of CYP1A2 with the Ki and IC50 values of 1.41 mM and 7.66 mM, respectively. Celastrol had the characteristics of an uncompetitive inhibitor in which the inhibitor interacted with the enzyme–substrate complexes. In this case, the binding of the substrates to the actice site of CYP1A2 created a binding site for celastrol. Therefore, celastrol was a CYP1A2 inhibitor and the potential herb–drug interaction with CYP1A2 would be serious. In addition, as celastrol was an un-competitive inhibitor of CYP1A2, it should be noted that combined use of celastrol and CYP1A2 substrate shall be avoided during clinical practice. CYP2C19 is the minor form of the human cytochrome P450 (0.2%) (Shimada et al., 1994), but it has genetic polymorphisms (Goldstein, 2001). The results showed that celastrol strongly inhibited CYP2C19 with an IC50 value of 6.38 mM and a Ki value of 5.27 mM. The competitive inhibition of celastrol on CYP2C19 was obtained, suggesting that celastrol and CYP2C19 substrate compete against each other for the binding site of the enzyme. And the inhibiting effect could be reversed by increasing substrate concentration. Although CYP2D6 is a relatively minor fraction of the total cytochrome P450 content, it catalyzes approximately 30% of the drugs in the market and inhibition of this isoenzyme may result in potential adverse drug interactions in the clinic because the inhibition of this drug-metabolizing enzyme may lead to increased plasma levels of another drug administered concomitantly and result in drug-induced toxicity (Ye et al., 2014). The results suggested that celastrol inhibit CYP2D6 with an IC50 value of 8.14 mM and a Ki value of 4.21 mM. The competitive inhibition of celastrol on CYP2D6 was obtained, suggesting that the use of celastrol may cause interactions with the substrates of CYP2D6, including anti-depressive drugs, such as fluoxetine and amitriptyline, b-receptor-blocking agents, such as metoprolol and propranolol (Fukumoto et al., 2006), and anti-psychotic drugs, such as chlorpromazine, perphenazine, and tamoxifen (Li et al., 2011). As an ethanol-induced cytochrome P450 enzyme, CYP2E1 metabolizes 2% clinically used drugs, and is able to catalyze pro-carcinogen and pro-toxin (Arinc et al., 2007; Zuber et al., 2002). Metabolic reactions catalyzed by CYP2E1 generate reactive oxygen species (reactive oxygen species, ROS), such as oxygen radicals. And rise in intracellular ROS results in lipid peroxidation, which will bind with nucleophile in the cell, like protein, damage cells and cause cytotoxicity or hepatotoxicity (Gonzalez, 2007). Our results suggested that celastrol may un-competitively inhibit CYP2E1 in human liver microsomes with the Ki value was 2.29 mM and the IC50 value was 9.38 mM. That is, celastrol binded only with
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enzyme–substrate complex, but not with free enzymes. High concentration of substrate will increase the occupancy of the active site and the binding site on CYP2E1 for celastrol, and therefore the effective the inhibition. During clinical practice, combined use of celastrol and CYP2E1 substrate should be avoided to defend herb–drug interactions. As an important drug-metabolizing enzyme, CYP3A is able to metabolize about 50% of clinical drugs, and can be inhibited and induced by a variety of drugs. It was once reported that the induction of CYP3A4 expression would change the clearance rate of a variety of clinical drugs, including midazolam, amitriptyline, cyclosporine and oral contraceptive. Our results showed that celastrol inhibited CYP3A4 in human liver microsome with a Ki value of 2.02 mM, an IC50 value of 2.63 mM, and the Kis value of 5.49 mM, celastrol proved to be the mixed-type inhibitor on CYP3A4. As a non-competitive/competitive mixed inhibitor, celastrol could inhibit both free enzyme (E) and enzyme– substrate complex (E–S), and its inhibition on CYP3A4 was strong. The value of Ki was lower than that of Kis, indicating that the affinity of the inhibitor for free enzyme was stronger than that for the enzyme–substrate complexes. Therefore, drug administration order during clinical practice would affect interactions between celastrol and CYP3A4 substrates. The results of this study showed celastrol might affect in vitro the metabolism of drugs which are substrates of the cytochrome P450 isoforms. The dosage of celastrol for cancers and inflammation treatment has been chosen during 0.1–10 mM in some in vitro experiments (Boridy et al., 2014). Previous in vivo studies have studied the inhibitory effects of celastrol on cytochrome P450 activities in rat liver microsomes, which demonstrated that celastrol inhibited the metabolism of CYP1A2, CYP2C11 and CYP3A2 substrate with different mode of inhibition. And because of the poor absorption of pure celastrol, the IC50 and Ki values in that study were higher than the rat plasma concentrations (Min et al., 2014). Zhang et al. (2012) reported that it displayed higher bioavailability for celastrol-containing ‘‘Thunder of God Vine’’ tablets probably owing to the presence of other constituents in the tablet preparations than that pure celastrol administration in rats treated. But the human plasma concentration was seldom reported when celastrol administered orally. Due to the interspecies differences and CYP impression between human and rat, further system study in vivo and in vitro is needed to identify the interactions of celastrol with cytochrome P450 isoform in humans.
Conclusions Results of this study suggest that celastrol could inhibit different isoforms of cytochrome P450, including CYP1A2, CYP2C19, CYP2D6, CYP2E1 and CYP3A4 that exist in reconstructed human liver microsomes with different mechanism. These in vitro experiments on cytochrome P450 isoforms would facilitate further application and development of celastrol as a new anti-cancer drug. However, the effect of herb–drug interactions is still elusive. Therefore, further systematic studies in humans in vitro and in vivo are also needed to identify the interactions of celastrol with cytochrome P450.
DOI: 10.3109/00498254.2014.1003113
Declaration of interest This study was supported by The Natural Science Foundation of Tianjin [12JCZDJC26100]; National Natural Science Foundation of China [NSFC, No.81373890 and No.81430096]; The Program for Changjiang Scholars and Innovative Research Team in University [PCSIRT, No. IRT_14R41]. The authors report no declaration of interest.
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