- Email: [email protected]
Selective inhibitory effects of mollugin on CYP1A2 in human liver microsomes
Food and Chemical Toxicology 51 (2013) 33–37
Contents lists available at SciVerse ScienceDirect
Food and Chemical Toxicology journal homepage: www.elsevier.com/locate/foodchemtox
Selective inhibitory effects of mollugin on CYP1A2 in human liver microsomes Heeyeon Kim a, Hyun Kyu Choi b, Tae Cheon Jeong b, Yurngdong Jahng b, Dong Hyun Kim c, Seung-Ho Lee b, Sangkyu Lee a,⇑ a
College of Pharmacy, Kyungpook National University, Daegu 702-701, South Korea College of Pharmacy, Yeungnam University, Gyeongsan 702-701, South Korea c Pharmacogenomics Research Center, Inje University College of Medicine, Busan 614-735, South Korea b
a r t i c l e
i n f o
Article history: Received 22 May 2012 Accepted 11 September 2012 Available online 18 September 2012 Keywords: Mollugin Cytochrome P450 Herb–drug interaction Inhibition
a b s t r a c t Mollugin originally isolated from Rubia cordifolia is a pharmacological compound for its anti-inflammation, anti-cancer, and anti-viral activity. In the present study, a cocktail probe assay was performed for determination of the selective inhibitory effect of mollugin on cytochrome P450 (CYP) enzymes in human liver microsomes (HLM). Incubation of isoform-specific substrate probes CYPs with mollugin (0–25 lM) in HLM resulted in strong inhibition of CYP1A2-catalyzed phenacetin O-deethylation, showing IC50 values of 1.03 and 3.55 lM without and with pre-incubation, respectively. Mollugin-caused inhibition of phenacetin O-deethylation was concentration-dependent in HLMs, but not time-dependent. In addition, the Lineweaver–Burk plot indicated a typical competitive inhibition. Inhibitory effects of mollugin on human recombinant cDNA-expressed CYP1A1 and 1A2 were comparable. Taken together, the results suggested that mollugin might cause herb–drug interaction through selective inhibition of CYP1A2 in humans receiving herbal medications, including R. cordifolia. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction Because herbs have been considered to be less-toxic than synthesis-based drugs, attentions to develop new drugs originating from natural products have greatly been paid worldwide (Zhou et al., 2004; Kim et al., 2008). However, following an increase in consumption of natural compounds for use as herbal medicines, potential risk related to pharmacokinetic interaction has been reported. For example, potential herb–drug interaction through modulation of cytochrome P450 monooxygenase (CYP), a primary cause of drug–drug or drug–herb interactions, has been reported for St. John’s wort and ginko biloba (Guengerich, 1997; Markowitz et al., 2000, 2003). Involvement of the CYP family is observed in 95% cases of drug– drug interaction associated with modulation of drug metabolism by inductive or inhibitive effects of active components derived from herbs or drugs. In addition, inhibition of CYP has been reported as the cause of 70% of drug interactions (Iwata et al., 2005). Therefore, the possibility of herb–drug interactions, particularly through the inhibitory effects of pharmacological
Abbreviations: HLMs, human liver microsomes; CYP, cytochrome P450.
⇑ Corresponding author. Tel.: +82 53 950 8571; fax: +82 53 950 8557. E-mail address: [email protected] (S. Lee). 0278-6915/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.fct.2012.09.013
components on CYP enzymes should be considered in the process of drug development. Mollugin, a naturally isolated component of Rubia cordifolia (Fig. 1), has been used as a traditional Chinese medicine for decades (Lu et al., 2007; Sastry et al., 2010; Jeong et al., 2012). The pharmacological effects of mollugin have recently been reported in vitro; mollugin exerted neuroprotective and anti-inflammatory effects via up-regulation of heme oxygenase-1 in mouse hippocampal and microglial cells and/or inhibition of NF-jB activation in HT-29 human colonic cells (Kim et al., 2009; Jeong et al., 2011), anti-carcinogenic effects against cytotoxicity via induction of apoptosis in 3T3-L1 preadipocytes and inhibition of DNA topoisomerase I and II (Jun et al., 2011; Jeong et al., 2012) and anti-viral activities via suppression of secretion of hepatitis B surface antigen on human hepatoma cells (Ho et al., 1996). In addition, mollugin also exerted inhibitory effects on arachidonic acid- and collagen-induced platelet aggregation (Chung et al., 1994). Despite reports on the various pharmacological effects of mollugin, its influences on CYP enzymes have not been examined to date to our best knowledge. In this study, we report for the first time the selective inhibitory effects of mollugin on CYP1A1 and 1A2-catalyzed phenacetin O-deethylase in human liver microsomes (HLMs) and human recombinant cDNAexpressed CYPs.
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H. Kim et al. / Food and Chemical Toxicology 51 (2013) 33–37
OH O
2.6. Kinetic analysis
OCH3 O
Mollugin Fig. 1. Chemical structure of mollugin.
2. Materials and methods 2.1. Materials Mollugin tested in present was isolated from R. cordifolia L. (Rubiaceae) (Jeong et al., 2011). Pooled HLMs and human recombinant cDNA-expressed CYP1A1 and 1A2 were purchased from BD Gentest (Woburn, MA). Glucose 6-phosphate, bNADP+ and glucose 6-phosphate dehydrogenase were obtained from Sigma Chemical Co. (St. Louis, MO). All other chemicals used were of analytical grade and were used as received.
Calculation of IC50 values (the concentration of inhibitor causing 50% inhibition of the original enzyme activity) was based on the curves of mean enzyme activity versus inhibitor concentration.
3. Results 3.1. Selective inhibitory effects of mollugin on CYP enzyme activities in HLMs A cocktail of CYP substrate in pooled HLM was used for determination of the inhibitory effect of mollugin on inactivation of CYP isoform-catalyzed activity. Incubation with 0–25 lM of mollugin for 60 min resulted in selective inhibition of the activity of CYP1A2-catalyzed phenacetin O-deethylation; the remaining activity was found to be at 21.2% (Table 1). No significant effects of mollugin on the other CYPs were observed. Measurement of CYP1A2 inhibitory activity from 0 to 25 lM of mollugin was performed with or without pre-incubation for 15 min. IC50 values for inhibition of CYP1A2 were calculated as 1.03 lM without pre-incubation and 3.55 lM with pre-incubation (Fig. 2). Values for control activities were 1960.4 and 1761.0 pmole/min/mg protein without and with pre-incubation, respectively.
2.2. In vitro CYP inhibition A cocktail probe assay was performed using 0.2 mg of HLM in a final incubation volume of 0.1 mL. The incubation mixture contained 0.1 M potassium phosphate buffer (pH 7.4), cocktail probe substrates, mollugin (0.25, 2.5 and 25 lM), and a NADPH-generating system (NGS) containing 0.1 M glucose 6-phosphate, 10 mg/ mL b-NADP+, and 1.0 U/mL glucose-6-phosphate dehydrogenase. The following probe substrates were used in the assay: 80 lM phenacetin for CYP1A2, 2.0 lM coumarin for CYP2A6, 50 lM bupropion for CYP2B6, 5.0 lM amodiaquine for CYP2C8, 10 lM diclofenac for CYP2C9, 20 lM omeprazole for CYP2C19, 5.0 lM dextromethorphan for CYP2D6, 50 lM chlorzoxazone for CYP2E1, and 2.5 lM midazolam for CYP3A4. Following a 5-min pre-incubation period in the absence of the substrate mixture, the incubation reaction mixture was incubated for 60 min at 37 °C. The reaction was stopped by addition of 100 lL acetonitrile with 0.1% formic acid. After mixing and centrifugation at 16,000g for 5 min, a 10 lL aliquot was injected onto a C18 column for LC/MS/MS analysis. 2.3. Selective inhibitory effect of mollugin on CYPA2 activity Following the cocktail probe assay, inhibitory effects of mollugin on CYP1A2 activity were further characterized using phenacetin as a sole substrate in HLMs. To evaluate the possible mechanism-based inactivation of CYP1A2, pooled HLMs (0.2 mg) were pre-incubated with mollugin at 0, 5, 10, and 20 lM in 0.1 M potassium phosphate buffer (pH 7.4) for 0, 5, 10, 15, and 20 min, respectively, in the presence of NGS, followed by addition of 80 lM phenacetin. The incubation was continued for an additional 60 min. To identify the inhibition pattern of CYP1A2, pooled HLMs (0.2 mg) were incubated with mollugin at 0, 10, and 40 lM and 0, 40, and 80 lM phenacetin in 0.1 M potassium phosphate buffer (pH 7.4) for 60 min, respectively. 2.4. Effects of mollugin on human recombinant CYP1A1 and 1A2 enzymes Each assay was performed using 10 pmole of human recombinant cDNA-expressed CYP1A1 and 1A2 in a final incubation volume of 0.1 mL, respectively. The incubation mixture contained 0.1 M potassium phosphate buffer (pH 7.4), mollugin, and a NGS containing 0.1 M glucose 6-phosphate, 10 mg/mL b-NADP+, and 1.0 U/mL glucose-6-phosphate dehydrogenase. Phenacetinat 80 lM was used as substrates.
3.2. Inhibitory mechanism of mollugin in HLMs To investigate the mode of inhibitory action of mollugin for CYP1A2 catalyzed phenacetin O-deethylation, concentration and time-dependent inhibition effects of mollugin was evaluated in HLMs. Mollugin was found to induce strong, dose-dependent inhibition of CYP1A2-catalyzed phenacetin O-deethylation in HLMs; in contrast, the time-dependency of enzyme inhibition was not observed (Fig. 3). The control value was 2286.3 pmole/min/mg protein. A time-dependent inhibition assay was conducted in order to evaluate the possibility of competitive inactivation of CYP1A2 by mollugin. According to the Lineweaver–Burk plots, mollugin showed a typical pattern of competitive inhibition for CYP1A2-catalyzed phenacetin O-deethylation with a Ki value of 3.74 lM in HLMs (Fig. 4). 3.3. Dual effects of mollugin on CYP1A1 and 1A2-catalyzed phenacetin O-deethylation with human recombinant cDNA-expressed CYP1A1 and 1A2 To evaluate the selectivity of the inhibitory effect on mollugin for CYP1A1 and CYP1A2, mollugin at 0–25 lM was incubated with human recombinant cDNA-expressed CYP1A1 and 1A2, respectively. Mollugin comparably inhibited both CYP1A1- and 1A2-catalyzed phenacetin O-deethylase activities, with IC50 values of 7.14 and 5.25 lM, respectively (Fig. 5). Values for control activities of CYP1A1 and 1A2 were 3445.8 and 3264.1 pmole/min/pmole CYP, respectively. Mollugin was found to exert a strong inhibition effect on CYP1A family members in HLM. 4. Discussion
2.5. LC/MS/MS analysis An Accela™ LC system coupled to a TSQ Vantage triple quadrupole mass spectrometer (Thermo Fisher Scientific Inc., USA) equipped with a HESI-II Spray source was used for analysis of the CYP enzyme activity. An InertsilÒ ODS-3, 5 lm (2.1 150 mm, GL science) column was used for LC analysis. Mobile phases consisted of 0.1% formic acid (A) and acetonitrile (B). The initial composition was increased to 90% solvent (B) for 15 min. A gradient program with a flow rate of 230 lL/min was used for HPLC separation. Electrospray ionization was performed in the positive ion mode at 3500 kV of spray voltage. Nitrogen was used as the sheath and auxiliary gas with optimum values of 60 and 20 (arbitrary units), respectively. Vaporizer capillary temperatures were at 150 and 300 °C, respectively.
Herb extracts and components in herb extracts have been known to either induce or inhibit drug-metabolizing enzymes elsewhere. St. John’s wort and ginko biloba are just few of well-known examples among the numerous cases (Guengerich, 1997; Markowitz et al., 2000, 2003). Therefore, like drug-drug interaction, herb–drug interaction via either induction or inhibition of drugmetabolizing enzymes would also cause a serious adverse drug reaction or toxicity. Meanwhile, herb–drug interaction would have not been considered extensively in the process of drug develop-
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H. Kim et al. / Food and Chemical Toxicology 51 (2013) 33–37 Table 1 Inhibition of CYP450 isoforms by mollugin at 0–25 lM in HLMs. Mollugin (lM)
Relative inhibition (%) CYP1A2
CYP2A6
CYP2B6
CYP2C8
CYP2C9
CYP2C19
CYP2D6
CYP2E1
CYP3A4
0 0.25 2.5 25
100 95.7 78.3 21.2
100 96.3 102.5 85.7
100 96.7 97.0 68.9
100 96.8 114.5 92.5
100 101.7 108.0 88.0
100 101.4 100.9 73.3
100 98.5 101.0 79.5
100 99.4 99.6 79.7
100 100.7 105.0 88.0
To determine the inhibition effect of mollugin on CYP activities, the following probe reactions were used: phenacetin O-deethylation for CYP1A2, coumarin 7-hydroxylation for CYP2A6, bupropion 10 -hydroxylation for CYP2B6, amodiaquine N-deethylation for CYP2C8, diclofenac 40 -hydroxylation for CYP2C9, omeprazole 5-hydroxylation for CYP2C19, dextromethorphan O-demethylation for CYP2D6, chlorzoxazone 6-hydroxylation for CYP2E1, and midazolam 1-hydroxylation for CYP3A4. Each value represents the mean of two determinations and is expressed as percentage of control activity remaining.
Without preincubation With preincubation
80 60 40 20
% of Control activity
% of Control activity
100
100
50
0 0.001
0 0.01
0.01
0.1
1
alpha-Naphtaflavone (µM)
0.1
1
10
Mollugin (µM) Fig. 2. Concentration- and time-dependent inhibition of CYP1A2-catalyzed phenacetin O-deethylase activity by mollugin in HLMs. Data shown represent the mean of duplicate experiments. IC50 value of alpha-naphtaflavone, a positive inhibitor for CYP1A2-catalyzed phenacetin O-deethylase activity was calculated to 0.07 lM.
ment from the natural origin. For an example, mollugin isolated from R. cordifolia has been known for its anti-inflammatory, anticancer, and anti-viral effects (Lu et al., 2007; Sastry et al., 2010; Jeong et al., 2012). Synthetic strategy for mollugin and its structural derivatives with their action mechanisms on NF-jB was also
reported elsewhere (Kim et al., 2009). Meanwhile, possible interaction of mollugin with drug metabolizing enzymes like CYPs has never been studied. In vitro inhibition studies using HLMs, sometimes coupled with a cocktail probe assay, are widely used in evaluation of the potential for drug–drug interactions in the early stage of drug development. In the present study, using 9 cocktail probes, we evaluated the inhibitory effect of mollugin on the activities of CYPs in HLMs. The results showed a selective inhibition by mollugin on CYP1A2catalyzing phenacetin O-deethylation. Next, the inhibition of phenacetin O-deethylation by mollugin was further characterized in a single substrate assay using HLMs and recombinant CYP1A1 and 1A2. In HLMs, mollugin inhibited phenacetin O-deethylase activity with IC50 values of 1.03 and 5.33 lM, without and with pre-incubation, respectively. The results indicated that mollugin has a typical pattern of competitive inhibition mode for CYP1A2 enzyme. From the Lineweaver–Burk plot, the Ki value was calculated to be 3.74 lM in HLMs. In addition, mollugin exhibited dual inhibitory effects for both CYP1A1 and CYP1A2, with IC50 values of 7.14 and 5.25 lM, respectively, when studied with recombinant individual proteins. Therefore, the results clearly suggested that mollugin might inhibit CYP1A2 competitively. CYP1 family is specifically linked with metabolic activation of carcinogens like polycyclic aromatic hydrocarbons (PAH), such as benzo[a]pyrene and 7,12-dimethylbenz[a]anthracene (Slaga et al., 1979). Therefore, selective inhibition of CYP1A family activity might be related to chemopreventive effects. In fact, the inactivation effects on members of the CYP1A family by some natural components linked to important roles for chemopreventive effects in
0 µM 5 µM 10 µM 20 µM
% of control activity
100 80 60 40 20 0 0
5
10
15
20
Preincubation time (min) Fig. 3. Time-course of CYP inhibition by mollugin in HLMs. HLMs were pre-incubated with mollugin and 1 mM of NADPH for 15 min. Data shown represent the mean of duplicate experiments.
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H. Kim et al. / Food and Chemical Toxicology 51 (2013) 33–37
(B)
(A)
0.25
0.014
Mollugin 0 µM Mollugin 10 µM Mollugin 40 µM
0.012
0.20
Slope
0.010
1/V
0.008 0.006
0.15
0.10
0.004 0.05
0.002 -0.01
0 -0.002
0.01
0.02
0.04
0.03
0.05
0
10
1/S (phenacetin)
20
30
40
Mollugin (µM)
Fig. 4. The Lineweaver–Burke plot (A) and Secondary plot (B) for inhibition of CYP1A2-catalyzed phenacetin O-deethylation by mollugin in HLMs. Activities were obtained following incubation of mollugin at 0 (d), 10 (s), and 40 (.) lM for 60 min. Data shown represent the mean of duplicate experiments.
CYP1A1 CYP1A2
% of control activity
100
Conflict of Interest None declare.
80
Acknowledgements
60
This study was supported by a grant from the Korea Health technology R&D Project, Ministry of Health & Welfare, Republic of Korea (Grant No.: A111345).
40
References
20 0 0.01
0.1
1
10
Mollugin (µM) Fig. 5. Inhibitory effects of mollugin on phenacetin O-deethylation activity in recombinant human CYP1A1 (d) and CYP1A2 (s). Data shown represent the mean of duplicate experiments.
carcinogenesis and chemical toxicities from PAH exposure (Hwang et al., 2008; Pekthong et al., 2008) have been reported in several previous studies. For instance, cacao extracts have been reported to reduce mutagenicity induced by benzo[a]pyrene in vitro through inhibition of CYP1A activity (Ohno et al., 2009) and polysaccharides of edible algae, chalcones, and xanthohumol prenylated flavonoid from Humulus lupulus L. induced inhibition in the carcinogen activator CYP1A (Forejtníková et al., 2005; GamalEldeen et al., 2009). Based on its selective inhibition of CYP1A activity, chemopreventive effect of mollugin should be conducted in the near future. Nevertheless, possible drug interaction of mollugin with CYP1A substrates needs to be investigated. When mollugin was treated orally to mice, hepatic phenacetin O-deethylase activity was significantly reduced as seen in the present in vitro studies (unpublished data). Because CYP1A enzymes are capable of not only carcinogenic PAHs but also some drugs like caffeine and theophylline, alteration of pharmacokinetic properties of these drugs would be inevitable. In this regard, effects of mollugin on caffeine pharmacokinetics are currently under investigation. This investigation would be crucial because effects of a compound have shown weak correlation between in vitro and in vivo as in case of SKF 525-A (Jeong et al., 2004). A well-known CYP inhibitor SKF 525-A inhibits CYP enzymes only in vitro, whereas it significantly induces CYP enzymes when treated more than 1 day.
Chung, M.I., Jou, S.J., Cheng, T.H., Lin, C.N., Ko, F.N., Teng, C.M., 1994. Antiplatelet constituents of formosan Rubia akane. J. Nat. Prod. 57, 313–316. Forejtníková, H., Lunerová, K., Kubínová, R., Jankovská, D., Marek, R., Kares, R., Suchy´, V., Vondrácek, J., Machala, M., 2005. Chemoprotective and toxic potentials of synthetic and natural chalcones and dihydrochalcones in vitro. Toxicology 208, 81–93. Gamal-Eldeen, A.M., Ahmed, E.F., Abo-Zeid, M.A., 2009. In vitro cancer chemopreventive properties of polysaccharide extract from the brown alga, Sargassum latifolium. Food Chem. Toxicol. 47, 1378–1384. Guengerich, F.P., 1997. Role of cytochrome P450 enzymes in drug–drug interactions. Adv. Pharmacol. 43, 7–35. Ho, L.K., Don, M.J., Chen, H.C., Yeh, S.F., Chen, J.M., 1996. Inhibition of hepatitis B surface antigen secretion on human hepatoma cells. Components from Rubia cordifolia. J. Nat. Prod. 59, 330–333. Hwang, Y.P., Han, E.H., Choi, J.H., Kim, H.G., Lee, K.J., Jeong, T.C., Lee, E.S., Jeong, H.G., 2008. Chemopreventive effects of Furan-2-yl-3-pyridin-2-yl-propenone against 7,12-dimethylbenz[a]anthracene-inducible genotoxicity. Toxicol. Appl. Pharmacol. 228, 343–350. Iwata, H., Tezuka, Y., Kadota, S., Hiratsuka, A., Watabe, T., 2005. Mechanism-based inactivation of human liver microsomal CYP3A4 by rutaecarpine and limonin from Evodia fruit extract. Drug Metab. Pharmacokinet. 20, 34–45. Jeong, G.S., Lee, D.S., Kim, D.C., Jahng, Y., Son, J.K., Lee, S.H., Kim, Y.C., 2011. Neuroprotective and anti-inflammatory effects of mollugin via up-regulation of heme oxygenase-1 in mouse hippocampal and microglial cells. Eur. J. Pharmacol. 654, 226–234. Jeong, S.Y., Zhao, B.T., Lee, C.S., Son, J.K., Min, B.S., Woo, M.H., 2012. Constituents with DNA topoisomerases I and II inhibitory activity and cytotoxicity from the roots of Rubia cordifolia. Planta Med. 78, 177–181. Jeong, T.C., Chang, H.W., Lee, E.S., Jeon, T.W., Jeong, H.G., Holsapple, M.P., 2004. SKF 525-A induces cocaine N-demethylase, ethoxyresorufin O-deethylase, and pentoxyresorufin O-dealkylase activities by induction of cytochrome P-450 2B in female B6C3F1 mice. J. Toxicol. Environ. Health A 67, 1955–1970. Jun do, Y., Han, C.R., Choi, M.S., Bae, M.A., Woo, M.H., Kim, Y.H., 2011. Effect of mollugin on apoptosis and adipogenesis of 3T3-L1 preadipocytes. Phytother. Res. 25, 724–731. Kim, H., Kim, K.B., Ku, H.Y., Park, S.J., Choi, H., Moon, J.K., Park, B.S., Kim, J.H., Yea, S.S., Lee, C.H., Lee, H.S., Shin, J.G., Liu, K.H., 2008. Identification and characterization of potent CYP2B6 inhibitors in Woohwangcheongsimwon suspension, an herbal preparation used in the treatment and prevention of apoplexy in Korea and China. Drug Metab. Dispos. 36, 1010–1015. Kim, K.J., Lee, J.S., Kwak, M.K., Choi, H.G., Yong, C.S., Kim, J.A., Lee, Y.R., Lyoo, W.S., Park, Y.J., 2009. Anti-inflammatory action of mollugin and its synthetic derivatives in HT-29 human colonic epithelial cells is mediated through inhibition of NF-kappaB activation. Eur. J. Pharmacol. 622, 52–57.
H. Kim et al. / Food and Chemical Toxicology 51 (2013) 33–37 Lu, Y., Liu, R., Sun, C., Pan, Y., 2007. An effective high-speed countercurrent chromatographic method for preparative isolation and purification of mollugin directly from the ethanol extract of the Chinese medicinal plant Rubia cordifolia. J. Sep. Sci. 30, 1313–1317. Markowitz, J.S., DeVane, C.L., Boulton, D.W., Carson, S.W., Nahas, Z., Risch, S.C., 2000. Effect of St. John’s wort (Hypericum perforatum) on cytochrome P-450 2D6 and 3A4 activity in healthy volunteers. Life Sci. 66, 133–139. Markowitz, J.S., Donovan, J.L., Lindsay DeVane, C., Sipkes, L., Chavin, K.D., 2003. Multiple-dose administration of Ginkgo biloba did not affect cytochrome P-450 2D6 or 3A4 activity in normal volunteers. J. Clin. Psychopharmacol. 23, 576–581. Ohno, M., Sakamoto, K.Q., Ishizuka, M., Fujita, S., 2009. Crude cacao Theobroma cacao extract reduces mutagenicity induced by benzo[a]pyrene through inhibition of CYP1A activity in vitro. Phytother. Res. 23, 1134–1139.
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Pekthong, D., Martin, H., Abadie, C., Bonet, A., Heyd, B., Mantion, G., Richert, L., 2008. Differential inhibition of rat and human hepatic cytochrome P450 by Andrographis paniculata extract and andrographolide. J. Ethnopharmacol. 115, 432–440. Sastry, M.N., Claessens, S., Habonimana, P., De Kimpe, N., 2010. Synthesis of the natural products 3-hydroxymollugin and 3-methoxymollugin. J. Org. Chem. 75, 2274–2280. Slaga, T.J., Gleason, G.L., DiGiovanni, J., Sukumaran, K.B., Harvey, R.G., 1979. Potent tumor-initiating activity of the 3,4-dihydrodiol of 7,12dimethylbenz(a)anthracene in mouse skin. Cancer Res. 39, 1934–1936. Zhou, S., Koh, H.L., Gao, Y., Gong, Z.Y., Lee, E.J., 2004. Herbal bioactivation: the good, the bad and the ugly. Life Sci. 74, 935–968.
Contents lists available at SciVerse ScienceDirect
Food and Chemical Toxicology journal homepage: www.elsevier.com/locate/foodchemtox
Selective inhibitory effects of mollugin on CYP1A2 in human liver microsomes Heeyeon Kim a, Hyun Kyu Choi b, Tae Cheon Jeong b, Yurngdong Jahng b, Dong Hyun Kim c, Seung-Ho Lee b, Sangkyu Lee a,⇑ a
College of Pharmacy, Kyungpook National University, Daegu 702-701, South Korea College of Pharmacy, Yeungnam University, Gyeongsan 702-701, South Korea c Pharmacogenomics Research Center, Inje University College of Medicine, Busan 614-735, South Korea b
a r t i c l e
i n f o
Article history: Received 22 May 2012 Accepted 11 September 2012 Available online 18 September 2012 Keywords: Mollugin Cytochrome P450 Herb–drug interaction Inhibition
a b s t r a c t Mollugin originally isolated from Rubia cordifolia is a pharmacological compound for its anti-inflammation, anti-cancer, and anti-viral activity. In the present study, a cocktail probe assay was performed for determination of the selective inhibitory effect of mollugin on cytochrome P450 (CYP) enzymes in human liver microsomes (HLM). Incubation of isoform-specific substrate probes CYPs with mollugin (0–25 lM) in HLM resulted in strong inhibition of CYP1A2-catalyzed phenacetin O-deethylation, showing IC50 values of 1.03 and 3.55 lM without and with pre-incubation, respectively. Mollugin-caused inhibition of phenacetin O-deethylation was concentration-dependent in HLMs, but not time-dependent. In addition, the Lineweaver–Burk plot indicated a typical competitive inhibition. Inhibitory effects of mollugin on human recombinant cDNA-expressed CYP1A1 and 1A2 were comparable. Taken together, the results suggested that mollugin might cause herb–drug interaction through selective inhibition of CYP1A2 in humans receiving herbal medications, including R. cordifolia. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction Because herbs have been considered to be less-toxic than synthesis-based drugs, attentions to develop new drugs originating from natural products have greatly been paid worldwide (Zhou et al., 2004; Kim et al., 2008). However, following an increase in consumption of natural compounds for use as herbal medicines, potential risk related to pharmacokinetic interaction has been reported. For example, potential herb–drug interaction through modulation of cytochrome P450 monooxygenase (CYP), a primary cause of drug–drug or drug–herb interactions, has been reported for St. John’s wort and ginko biloba (Guengerich, 1997; Markowitz et al., 2000, 2003). Involvement of the CYP family is observed in 95% cases of drug– drug interaction associated with modulation of drug metabolism by inductive or inhibitive effects of active components derived from herbs or drugs. In addition, inhibition of CYP has been reported as the cause of 70% of drug interactions (Iwata et al., 2005). Therefore, the possibility of herb–drug interactions, particularly through the inhibitory effects of pharmacological
Abbreviations: HLMs, human liver microsomes; CYP, cytochrome P450.
⇑ Corresponding author. Tel.: +82 53 950 8571; fax: +82 53 950 8557. E-mail address: [email protected] (S. Lee). 0278-6915/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.fct.2012.09.013
components on CYP enzymes should be considered in the process of drug development. Mollugin, a naturally isolated component of Rubia cordifolia (Fig. 1), has been used as a traditional Chinese medicine for decades (Lu et al., 2007; Sastry et al., 2010; Jeong et al., 2012). The pharmacological effects of mollugin have recently been reported in vitro; mollugin exerted neuroprotective and anti-inflammatory effects via up-regulation of heme oxygenase-1 in mouse hippocampal and microglial cells and/or inhibition of NF-jB activation in HT-29 human colonic cells (Kim et al., 2009; Jeong et al., 2011), anti-carcinogenic effects against cytotoxicity via induction of apoptosis in 3T3-L1 preadipocytes and inhibition of DNA topoisomerase I and II (Jun et al., 2011; Jeong et al., 2012) and anti-viral activities via suppression of secretion of hepatitis B surface antigen on human hepatoma cells (Ho et al., 1996). In addition, mollugin also exerted inhibitory effects on arachidonic acid- and collagen-induced platelet aggregation (Chung et al., 1994). Despite reports on the various pharmacological effects of mollugin, its influences on CYP enzymes have not been examined to date to our best knowledge. In this study, we report for the first time the selective inhibitory effects of mollugin on CYP1A1 and 1A2-catalyzed phenacetin O-deethylase in human liver microsomes (HLMs) and human recombinant cDNAexpressed CYPs.
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H. Kim et al. / Food and Chemical Toxicology 51 (2013) 33–37
OH O
2.6. Kinetic analysis
OCH3 O
Mollugin Fig. 1. Chemical structure of mollugin.
2. Materials and methods 2.1. Materials Mollugin tested in present was isolated from R. cordifolia L. (Rubiaceae) (Jeong et al., 2011). Pooled HLMs and human recombinant cDNA-expressed CYP1A1 and 1A2 were purchased from BD Gentest (Woburn, MA). Glucose 6-phosphate, bNADP+ and glucose 6-phosphate dehydrogenase were obtained from Sigma Chemical Co. (St. Louis, MO). All other chemicals used were of analytical grade and were used as received.
Calculation of IC50 values (the concentration of inhibitor causing 50% inhibition of the original enzyme activity) was based on the curves of mean enzyme activity versus inhibitor concentration.
3. Results 3.1. Selective inhibitory effects of mollugin on CYP enzyme activities in HLMs A cocktail of CYP substrate in pooled HLM was used for determination of the inhibitory effect of mollugin on inactivation of CYP isoform-catalyzed activity. Incubation with 0–25 lM of mollugin for 60 min resulted in selective inhibition of the activity of CYP1A2-catalyzed phenacetin O-deethylation; the remaining activity was found to be at 21.2% (Table 1). No significant effects of mollugin on the other CYPs were observed. Measurement of CYP1A2 inhibitory activity from 0 to 25 lM of mollugin was performed with or without pre-incubation for 15 min. IC50 values for inhibition of CYP1A2 were calculated as 1.03 lM without pre-incubation and 3.55 lM with pre-incubation (Fig. 2). Values for control activities were 1960.4 and 1761.0 pmole/min/mg protein without and with pre-incubation, respectively.
2.2. In vitro CYP inhibition A cocktail probe assay was performed using 0.2 mg of HLM in a final incubation volume of 0.1 mL. The incubation mixture contained 0.1 M potassium phosphate buffer (pH 7.4), cocktail probe substrates, mollugin (0.25, 2.5 and 25 lM), and a NADPH-generating system (NGS) containing 0.1 M glucose 6-phosphate, 10 mg/ mL b-NADP+, and 1.0 U/mL glucose-6-phosphate dehydrogenase. The following probe substrates were used in the assay: 80 lM phenacetin for CYP1A2, 2.0 lM coumarin for CYP2A6, 50 lM bupropion for CYP2B6, 5.0 lM amodiaquine for CYP2C8, 10 lM diclofenac for CYP2C9, 20 lM omeprazole for CYP2C19, 5.0 lM dextromethorphan for CYP2D6, 50 lM chlorzoxazone for CYP2E1, and 2.5 lM midazolam for CYP3A4. Following a 5-min pre-incubation period in the absence of the substrate mixture, the incubation reaction mixture was incubated for 60 min at 37 °C. The reaction was stopped by addition of 100 lL acetonitrile with 0.1% formic acid. After mixing and centrifugation at 16,000g for 5 min, a 10 lL aliquot was injected onto a C18 column for LC/MS/MS analysis. 2.3. Selective inhibitory effect of mollugin on CYPA2 activity Following the cocktail probe assay, inhibitory effects of mollugin on CYP1A2 activity were further characterized using phenacetin as a sole substrate in HLMs. To evaluate the possible mechanism-based inactivation of CYP1A2, pooled HLMs (0.2 mg) were pre-incubated with mollugin at 0, 5, 10, and 20 lM in 0.1 M potassium phosphate buffer (pH 7.4) for 0, 5, 10, 15, and 20 min, respectively, in the presence of NGS, followed by addition of 80 lM phenacetin. The incubation was continued for an additional 60 min. To identify the inhibition pattern of CYP1A2, pooled HLMs (0.2 mg) were incubated with mollugin at 0, 10, and 40 lM and 0, 40, and 80 lM phenacetin in 0.1 M potassium phosphate buffer (pH 7.4) for 60 min, respectively. 2.4. Effects of mollugin on human recombinant CYP1A1 and 1A2 enzymes Each assay was performed using 10 pmole of human recombinant cDNA-expressed CYP1A1 and 1A2 in a final incubation volume of 0.1 mL, respectively. The incubation mixture contained 0.1 M potassium phosphate buffer (pH 7.4), mollugin, and a NGS containing 0.1 M glucose 6-phosphate, 10 mg/mL b-NADP+, and 1.0 U/mL glucose-6-phosphate dehydrogenase. Phenacetinat 80 lM was used as substrates.
3.2. Inhibitory mechanism of mollugin in HLMs To investigate the mode of inhibitory action of mollugin for CYP1A2 catalyzed phenacetin O-deethylation, concentration and time-dependent inhibition effects of mollugin was evaluated in HLMs. Mollugin was found to induce strong, dose-dependent inhibition of CYP1A2-catalyzed phenacetin O-deethylation in HLMs; in contrast, the time-dependency of enzyme inhibition was not observed (Fig. 3). The control value was 2286.3 pmole/min/mg protein. A time-dependent inhibition assay was conducted in order to evaluate the possibility of competitive inactivation of CYP1A2 by mollugin. According to the Lineweaver–Burk plots, mollugin showed a typical pattern of competitive inhibition for CYP1A2-catalyzed phenacetin O-deethylation with a Ki value of 3.74 lM in HLMs (Fig. 4). 3.3. Dual effects of mollugin on CYP1A1 and 1A2-catalyzed phenacetin O-deethylation with human recombinant cDNA-expressed CYP1A1 and 1A2 To evaluate the selectivity of the inhibitory effect on mollugin for CYP1A1 and CYP1A2, mollugin at 0–25 lM was incubated with human recombinant cDNA-expressed CYP1A1 and 1A2, respectively. Mollugin comparably inhibited both CYP1A1- and 1A2-catalyzed phenacetin O-deethylase activities, with IC50 values of 7.14 and 5.25 lM, respectively (Fig. 5). Values for control activities of CYP1A1 and 1A2 were 3445.8 and 3264.1 pmole/min/pmole CYP, respectively. Mollugin was found to exert a strong inhibition effect on CYP1A family members in HLM. 4. Discussion
2.5. LC/MS/MS analysis An Accela™ LC system coupled to a TSQ Vantage triple quadrupole mass spectrometer (Thermo Fisher Scientific Inc., USA) equipped with a HESI-II Spray source was used for analysis of the CYP enzyme activity. An InertsilÒ ODS-3, 5 lm (2.1 150 mm, GL science) column was used for LC analysis. Mobile phases consisted of 0.1% formic acid (A) and acetonitrile (B). The initial composition was increased to 90% solvent (B) for 15 min. A gradient program with a flow rate of 230 lL/min was used for HPLC separation. Electrospray ionization was performed in the positive ion mode at 3500 kV of spray voltage. Nitrogen was used as the sheath and auxiliary gas with optimum values of 60 and 20 (arbitrary units), respectively. Vaporizer capillary temperatures were at 150 and 300 °C, respectively.
Herb extracts and components in herb extracts have been known to either induce or inhibit drug-metabolizing enzymes elsewhere. St. John’s wort and ginko biloba are just few of well-known examples among the numerous cases (Guengerich, 1997; Markowitz et al., 2000, 2003). Therefore, like drug-drug interaction, herb–drug interaction via either induction or inhibition of drugmetabolizing enzymes would also cause a serious adverse drug reaction or toxicity. Meanwhile, herb–drug interaction would have not been considered extensively in the process of drug develop-
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H. Kim et al. / Food and Chemical Toxicology 51 (2013) 33–37 Table 1 Inhibition of CYP450 isoforms by mollugin at 0–25 lM in HLMs. Mollugin (lM)
Relative inhibition (%) CYP1A2
CYP2A6
CYP2B6
CYP2C8
CYP2C9
CYP2C19
CYP2D6
CYP2E1
CYP3A4
0 0.25 2.5 25
100 95.7 78.3 21.2
100 96.3 102.5 85.7
100 96.7 97.0 68.9
100 96.8 114.5 92.5
100 101.7 108.0 88.0
100 101.4 100.9 73.3
100 98.5 101.0 79.5
100 99.4 99.6 79.7
100 100.7 105.0 88.0
To determine the inhibition effect of mollugin on CYP activities, the following probe reactions were used: phenacetin O-deethylation for CYP1A2, coumarin 7-hydroxylation for CYP2A6, bupropion 10 -hydroxylation for CYP2B6, amodiaquine N-deethylation for CYP2C8, diclofenac 40 -hydroxylation for CYP2C9, omeprazole 5-hydroxylation for CYP2C19, dextromethorphan O-demethylation for CYP2D6, chlorzoxazone 6-hydroxylation for CYP2E1, and midazolam 1-hydroxylation for CYP3A4. Each value represents the mean of two determinations and is expressed as percentage of control activity remaining.
Without preincubation With preincubation
80 60 40 20
% of Control activity
% of Control activity
100
100
50
0 0.001
0 0.01
0.01
0.1
1
alpha-Naphtaflavone (µM)
0.1
1
10
Mollugin (µM) Fig. 2. Concentration- and time-dependent inhibition of CYP1A2-catalyzed phenacetin O-deethylase activity by mollugin in HLMs. Data shown represent the mean of duplicate experiments. IC50 value of alpha-naphtaflavone, a positive inhibitor for CYP1A2-catalyzed phenacetin O-deethylase activity was calculated to 0.07 lM.
ment from the natural origin. For an example, mollugin isolated from R. cordifolia has been known for its anti-inflammatory, anticancer, and anti-viral effects (Lu et al., 2007; Sastry et al., 2010; Jeong et al., 2012). Synthetic strategy for mollugin and its structural derivatives with their action mechanisms on NF-jB was also
reported elsewhere (Kim et al., 2009). Meanwhile, possible interaction of mollugin with drug metabolizing enzymes like CYPs has never been studied. In vitro inhibition studies using HLMs, sometimes coupled with a cocktail probe assay, are widely used in evaluation of the potential for drug–drug interactions in the early stage of drug development. In the present study, using 9 cocktail probes, we evaluated the inhibitory effect of mollugin on the activities of CYPs in HLMs. The results showed a selective inhibition by mollugin on CYP1A2catalyzing phenacetin O-deethylation. Next, the inhibition of phenacetin O-deethylation by mollugin was further characterized in a single substrate assay using HLMs and recombinant CYP1A1 and 1A2. In HLMs, mollugin inhibited phenacetin O-deethylase activity with IC50 values of 1.03 and 5.33 lM, without and with pre-incubation, respectively. The results indicated that mollugin has a typical pattern of competitive inhibition mode for CYP1A2 enzyme. From the Lineweaver–Burk plot, the Ki value was calculated to be 3.74 lM in HLMs. In addition, mollugin exhibited dual inhibitory effects for both CYP1A1 and CYP1A2, with IC50 values of 7.14 and 5.25 lM, respectively, when studied with recombinant individual proteins. Therefore, the results clearly suggested that mollugin might inhibit CYP1A2 competitively. CYP1 family is specifically linked with metabolic activation of carcinogens like polycyclic aromatic hydrocarbons (PAH), such as benzo[a]pyrene and 7,12-dimethylbenz[a]anthracene (Slaga et al., 1979). Therefore, selective inhibition of CYP1A family activity might be related to chemopreventive effects. In fact, the inactivation effects on members of the CYP1A family by some natural components linked to important roles for chemopreventive effects in
0 µM 5 µM 10 µM 20 µM
% of control activity
100 80 60 40 20 0 0
5
10
15
20
Preincubation time (min) Fig. 3. Time-course of CYP inhibition by mollugin in HLMs. HLMs were pre-incubated with mollugin and 1 mM of NADPH for 15 min. Data shown represent the mean of duplicate experiments.
36
H. Kim et al. / Food and Chemical Toxicology 51 (2013) 33–37
(B)
(A)
0.25
0.014
Mollugin 0 µM Mollugin 10 µM Mollugin 40 µM
0.012
0.20
Slope
0.010
1/V
0.008 0.006
0.15
0.10
0.004 0.05
0.002 -0.01
0 -0.002
0.01
0.02
0.04
0.03
0.05
0
10
1/S (phenacetin)
20
30
40
Mollugin (µM)
Fig. 4. The Lineweaver–Burke plot (A) and Secondary plot (B) for inhibition of CYP1A2-catalyzed phenacetin O-deethylation by mollugin in HLMs. Activities were obtained following incubation of mollugin at 0 (d), 10 (s), and 40 (.) lM for 60 min. Data shown represent the mean of duplicate experiments.
CYP1A1 CYP1A2
% of control activity
100
Conflict of Interest None declare.
80
Acknowledgements
60
This study was supported by a grant from the Korea Health technology R&D Project, Ministry of Health & Welfare, Republic of Korea (Grant No.: A111345).
40
References
20 0 0.01
0.1
1
10
Mollugin (µM) Fig. 5. Inhibitory effects of mollugin on phenacetin O-deethylation activity in recombinant human CYP1A1 (d) and CYP1A2 (s). Data shown represent the mean of duplicate experiments.
carcinogenesis and chemical toxicities from PAH exposure (Hwang et al., 2008; Pekthong et al., 2008) have been reported in several previous studies. For instance, cacao extracts have been reported to reduce mutagenicity induced by benzo[a]pyrene in vitro through inhibition of CYP1A activity (Ohno et al., 2009) and polysaccharides of edible algae, chalcones, and xanthohumol prenylated flavonoid from Humulus lupulus L. induced inhibition in the carcinogen activator CYP1A (Forejtníková et al., 2005; GamalEldeen et al., 2009). Based on its selective inhibition of CYP1A activity, chemopreventive effect of mollugin should be conducted in the near future. Nevertheless, possible drug interaction of mollugin with CYP1A substrates needs to be investigated. When mollugin was treated orally to mice, hepatic phenacetin O-deethylase activity was significantly reduced as seen in the present in vitro studies (unpublished data). Because CYP1A enzymes are capable of not only carcinogenic PAHs but also some drugs like caffeine and theophylline, alteration of pharmacokinetic properties of these drugs would be inevitable. In this regard, effects of mollugin on caffeine pharmacokinetics are currently under investigation. This investigation would be crucial because effects of a compound have shown weak correlation between in vitro and in vivo as in case of SKF 525-A (Jeong et al., 2004). A well-known CYP inhibitor SKF 525-A inhibits CYP enzymes only in vitro, whereas it significantly induces CYP enzymes when treated more than 1 day.
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