1521-009X/43/8/1250–1253$25.00 DRUG METABOLISM AND DISPOSITION Copyright ª 2015 by The American Society for Pharmacology and Experimental Therapeutics
http://dx.doi.org/10.1124/dmd.115.064345 Drug Metab Dispos 43:1250–1253, August 2015
Short Communication Danazol Inhibits Cytochrome P450 2J2 Activity in a Substrateindependent Manner Received March 18, 2015; accepted June 5, 2015
ABSTRACT metabolism in a substrate-independent manner, with IC50 values of 0.05, 0.07, 0.18, and 0.34 mM, respectively. Danazol noncompetitively inhibited CYP2J2-mediated astemizole O-demethylation activities with a Ki value of 0.06 mM. Terfenadone strongly inhibited CYP2J2mediated albendazole, astemizole, and terfenadine metabolism (IC50 < 0.21 mM), whereas it showed weak inhibition against CYP2J2catalyzed ebastine hydroxylase activity (IC50 = 6.04 mM). Telmisartan had no inhibitory effect on CYP2J2-mediated ebastine and terfenadine hydroxylation (IC50 > 20 mM). Taken together, these data suggest that danazol may be used as a CYP2J2 index inhibitor in reaction phenotyping studies.
Introduction
2006). Some P450 inhibitors inhibit enzyme activity in a substratedependent manner. For example, fluconazole inhibits CYP3Amediated midazolam metabolism, whereas it does not suppress CYP3A-mediated metabolism of tacrolimus and testosterone (Zhang et al., 2012). In general, P450 index inhibitors used in reaction phenotyping studies should inhibit each P450 isoform-specific activity in a substrate-independent manner. One example is ketoconazole, a representative CYP3A inhibitor that inhibits the CYP3A-mediated metabolism of several substrates including nifedipine, midazolam, terfenadine, testosterone, and triazolam (Wang et al., 1999; Perloff et al., 2000; Racha et al., 2003; Greenblatt et al., 2011). In this study, we evaluated the inhibitory potential of danazol, hydroxyebastine, telmisartan, and terfenadone (representative CYP2J2 inhibitors) against CYP2J2-catalyzed albendazole hydroxylation, astemizole O-demethylation, ebastine hydroxylation, and terfenadine hydroxylation to find a representative CYP2J2 inhibitor. The results from this study will indicate which CYP2J2 inhibitors are suitable for reaction phenotyping and drug-drug interaction studies.
Cytochrome P450 2J2 (CYP2J2) is the member of CYP2J subfamily in humans. It is highly expressed in extrahepatic organs such as the heart, kidneys, lungs, small intestines, and gastrointestinal tissues (Zeldin et al., 1997; Enayetallah et al., 2004; Delozier et al., 2007). CYP2J2 is involved in the metabolism of arachidonic acid (Zeldin, 2001; Roman, 2002). It metabolizes arachidonic acid to epoxyeicosatrienoic acids, which play a role in homeostasis of the heart, lungs, and kidneys (Fleming, 2001; Kroetz and Zeldin, 2002; Smith et al., 2008; Feng et al., 2013; Wang et al., 2014). In addition to its endogenous role, CYP2J2 also metabolizes medications such as albendazole, astemizole, ebastine, and terfenadine (Matsumoto and Yamazoe, 2001; Hashizume et al., 2002; Matsumoto et al., 2002; Lafite et al., 2006; Liu et al., 2006; Lee et al., 2012; Ren et al., 2013; Wu et al., 2013b). To date, danazol, hydroxyebastine, telmisartan, and terfenadone have been reported as strong CYP2J2 inhibitors (Lafite et al., 2006; Yoon and Liu, 2011; Lee et al., 2012; Ren et al., 2013). However, their inhibitory potential on CYP2J2 activities has been evaluated for only one or two CYP2J2 substrates. For example, danazol and hydroxyebastine were evaluated for CYP2J2-mediated astemizole Odemethylation and terfenadine hydroxylation (Yoon and Liu, 2011; Lee et al., 2012). The CYP2J2 inhibition activity of telmisartan was evaluated for astemizole O-demethylation (Ren et al., 2013), whereas terfenadone was studied for ebastine hydroxylation (Lafite et al.,
This study was supported by grants from the National Research Foundation of Korea, Ministry of Science, ICT and Future Planning [NRF-2014M3A9D9069714]; and Ministry of Education [NRF-2013R1A1A2008442], Republic of Korea. E.L. and Z.W. contributed equally to this work. dx.doi.org/10.1124/dmd.115.064345.
Materials and Methods Chemicals and Reagents. O-desmethyl astemizole, hydroxyebastine, and terfenadone were obtained from Toronto Research Chemicals (North York, Ontario, Canada). Albendazole, astemizole, danazol, ebastine, telmisartan, terfenadine, terfenadine alcohol, b-NADP, MgCl2, KH2PO4, D-glucose-6phosphate, and glucose-6-phosphate dehydrogenase were purchased from Sigma-Aldrich (St. Louis, MO). Solvents were high-pressure liquid chromatography grade (Fisher Scientific Co., Pittsburgh, PA), and the other chemicals were of the highest quality available. Human recombinant CYP2J2 was purchased from BD Biosciences (San Jose, CA). Inhibition Studies Using Recombinant CYP2J2. All incubations were performed in triplicate. The incubation mixtures containing cDNA-expressed CYP2J2 (5 pmol/ml for astemizole, ebastine, and terfenadine; 10 pmol/ml for
ABBREVIATION: CE, collision energy. 1250
Downloaded from dmd.aspetjournals.org at ASPET Journals on August 13, 2015
Cytochrome P450 2J2 (CYP2J2) is an enzyme responsible for the metabolism of endogenous substrates including arachidonic acid, as well as therapeutic drugs such as albendazole, astemizole, ebastine, and terfenadine. Selective inhibitors of CYP2J2 are essential for P450 reaction phenotyping studies. To find representative CYP2J2 index inhibitors, we evaluated the inhibitory potential of danazol, hydroxyebastine, telmisartan, and terfenadone against CYP2J2 activity for four representative CYP2J2 substrates (albendazole, astemizole, ebastine, and terfenadine) using recombinant CYP2J2. Of these four CYP2J2 inhibitors, danazol strongly inhibited CYP2J2mediated albendazole, astemizole, ebastine, and terfenadine
1251
Substrate-Independent Inhibition of P450 2J2 by Danazol
Fig. 1. Inhibitory effects of danazol, hydroxyebastine, telmisartan, and terfenadone (0–20 mM) on CYP2J2mediated albendazole (1 mM) (A), astemizole (0.3 mM) (B), ebastine (5 mM) (C), and terfenadine (0.2 mM) (D) metabolism. Recombinant CYP2J2 was incubated with each CYP2J2 substrate. The activity was expressed as the percentage of the remaining activity compared with a control sample containing no inhibitor. Data are the mean values of triplicate experiments.
analysis of O-desmethylastemizole and hydroxyalbendazole, mobile phase B was linearly increased from 10% to 50% over 3 minutes, held at 50% for 0.1 minute, and then immediately stepped back down to 10% for re-equilibration for 6 minutes. For the analysis of hydroxyebastine and terfenadine alcohol, mobile phase B was linearly increased from 10% to 50% over 6 minutes, held at 50% for 0.1 minute, and then immediately stepped back down to 10% for reequilibration. The total run time was 10 minutes. For the analysis of acetaminophen, 4-hydroxytolbutamide, 4-hydroxymephenytoin, dextrorphan, and 19-hydroxymidazolam, mobile phase B was linearly increased from 10% to 90% over 7 minutes, held at 90% for 0.1 minute, and then immediately stepped back down to 10% for re-equilibration. The total run time was 10 minutes. The operating parameters of the mass spectrometer detector were as follows: ion spray, 4000 V in positive mode; capillary temperature, 350C; vaporizer temperature, 300C; sheath gas pressure, 35 arbitrary units; auxiliary gas, 10 arbitrary units; and nitrogen gas flow rate, 15 l/min. Quantitation was carried out by selective reaction monitoring. The following selective reaction monitoring transitions were monitored: acetaminophen (m/z 152 . 110, collision energy [CE] 25 eV), O-desmethylastemizole (m/z 445 . 204, CE 30 eV), dextrorphan (m/z 258 . 157, CE 35 eV), hydroxylalbendazole (m/z 282 . 250, CE 20 eV), hydroxyebastine (m/z 486 . 167, CE 42 eV), 4hydroxymephenytoin (m/z 236 . 150, CE 20 eV), 19-hydroxymidazolam (m/ TABLE 1 IC50 values of four CYP2J2 inhibitors against CYP2J2-mediated albendazole, astemizole, ebastine, and terfenadine metabolism in the recombinant CYP2J2 isoform The values are presented as average 6 S.D. (n = 3). IC50 (mM) Inhibitor Albendazole
Danazol Hydroxyebastine Terfenadone Telmisartan N.D., not determined.
0.05 0.22 6 0.07 0.14 6 0.01 0.14 6 0.05
Astemizole
Ebastine
Terfenadine
6 6 6 6
0.34 6 0.13 N.D. 6.04 6 2.51 .20
0.18 6 0.02 0.6 6 0.06 0.21 6 0.05 .20
0.07 0.31 0.07 0.85
0.02 0.11 0.02 0.16
Downloaded from dmd.aspetjournals.org at ASPET Journals on August 13, 2015
albendazole), 0.1 mM phosphate buffer (pH 7.4), substrate (albendazole, 1 mM; astemizole, 0.3 mM; ebastine, 5 mM; or terfenadine, 0.2 mM), and inhibitor [danazol, hydroxyebastine, telmisartan, or terfenadone (0–20 mM)] were preincubated for 5 minutes at 37C. Each substrate concentration was lower than its reported Km value (Matsumoto et al., 2002; Liu et al., 2006; Evangelista et al., 2013; Wu et al., 2013b). The reactions were initiated by the addition of a NADPH-generating system (including 3.3 mM D-glucose-6-phosphate, 500 units/ml glucose-6-phosphate dehydrogenase, 1.3 mM NADP, and 3.3 mM MgCl2). To determine the inhibition constant (Ki values) of danazol for CYP2J2-mediated astemizole O-demethylation in recombinant CYP2J2, danazol (0, 0.02, 0.05, 0.1, 0.15, and 0.2 mM) was added to reaction mixtures containing different concentrations of astemizole (0.1, 0.2, and 0.5 mM). The reaction mixtures (final volume, 100 ml) were incubated at 37C in a thermoshaker. After 20-minute incubation, 50 ml of cold acetonitrile containing the internal standard (mebendazole, 300 nM) was added immediately to terminate the reaction. After centrifugation at 10,000g for 5 minutes, aliquots of the supernatant were injected into a liquid chromatography–tandem mass spectrometry system. Recombinant P450 Inhibition Study. Danazol selectivity on CYP2J2 was assessed by its inhibitory potential against five major CYPs: CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A. The incubation mixtures containing recombinant P450 (5 pmol/ml for CYP1A2, 2C19, 2D6, and 3A; 10 pmol/ml for CYP2C9), 0.1 mM phosphate buffer (pH 7.4), P450-isoform probe substrate (Table 2), and danazol (0–30 mM) were preincubated for 5 minutes at 37C. The reactions were initiated by the addition of a NADPHgenerating system and further incubated for 15 minutes (CYP1A2, CYP2D6, and CYP3A), 20 minutes (CYP2C9), and 40 minutes (CYP2C19). The reaction was terminated by adding 50 ml of cold acetonitrile, and aliquots of the supernatant were injected into a liquid chromatography–tandem mass spectrometry system. Analytical Method. The samples were analyzed using a LCMS-8040 Triple Quadrupole Mass Spectrometer (Shimadzu, Tokyo) equipped with an electrospray ionization interface. The separation was carried out on a Luna C18 Reversed-Phase Column (Phenomenex, Torrance, CA) (50 2 mm i.d., 3 mm particle size). The mobile phases were 0.1% (v/v) formic acid in water (A) and 0.1% (v/v) formic acid in acetonitrile (B) at a flow rate of 0.2 ml/min. For the
1252
Lee et al.
z 342 . 203, CE 25 eV), 4-hydroxytolbutamide (m/z 287 . 89, CE 50 eV), mebendazole (m/z 296 . 264, CE 17 eV), and terfenadine alcohol (m/z 488 . 452, CE 36 eV). The analytical data were processed by LabSolution software, version 2.0 (Shimadzu). Data Analysis. The IC50 values were calculated using the WinNonlin software (Pharsight, Mountain View, CA).
Results and Discussion
TABLE 2 IC50 values of danazol against six recombinant P450 isoform activities The values are presented as average 6 S.D. (n = 3). CYP isoform
Fig. 2. Representative Dixon plots for inhibition of CYP2J2-mediated astemizole O-demethylation by danazol in the recombinant CYP2J2 isoform. An increasing concentration of astemizole [0.1 mM (d), 0.2 mM (s), and 0.5 mM (.)] was incubated with recombinant CYP2J2 (5 pmol/ml) (BD Biosciences, San Jose, CA) and an NADPH-generating system at 37C for 20 minutes in the presence or absence of danazol (0, 0.02, 0.05, 0.1, 0.15, and 0.2 mM). The inhibition data fit to a noncompetitive inhibition model. Data are the mean values of duplicate experiments.
CYP1A2 CYP2C9 CYP2C19 CYP2D6 CYP3A CYP2J2
Substrate
Phenacetin Tolbutamide S-Mephenytoin Dextromethorphan Midazolam Astemizole
Substrate concentration
IC50
mM
mM
10 200 2 5 5 0.3
12 6 3.27 1.1 6 0.24 2.0 6 0.43 . 30 18 6 5.94 0.07 6 0.02
Downloaded from dmd.aspetjournals.org at ASPET Journals on August 13, 2015
Reaction phenotyping and drug-drug interaction studies require that inhibitors for each P450 isoform be both strong and selective. For example, ketoconazole is more extensively used than itraconazole as a CYP3A index inhibitor, even though both ketoconazole and itraconazole have similar inhibitory potentials against CYP3Amediated midazolam 19-hydroxylation (IC50 @ 0.01 mM) (Racha et al., 2003). The reason for this preference is that studies have shown that ketoconazole has a strong inhibitory effect on various CYP3A substrates including midazolam, nifedipine, testosterone, and triazolam (Wang et al., 1999; Perloff et al., 2000; Racha et al., 2003; Greenblatt et al., 2011), while itraconazole has a weak inhibitory effect on CYP3A-mediated terfenadine metabolism (IC50 . 50 mM) (Racha et al., 2003). Another example is montelukast, which is more widely used as a CYP2C8 index inhibitor than pioglitazone, even though pioglitazone is also a good selective inhibitor for CYP2C8 (Khojasteh et al., 2011). Montelukast inhibits CYP2C8-catalyzed amodiaquine, paclitaxel, and repaglinide metabolism in a substrate-independent manner (IC50 = 0.008–0.026 mM), while pioglitazone inhibits CYP2C8-catalyzed amodiaquine, paclitaxel, and repaglinide metabolism with IC50 values of 6.6, 37.6, and 3.8 mM, respectively (VandenBrink et al., 2011). Similar to the previous examples, furafylline, tranylcypromine, ticlopidine, sulfaphenazole, S-benzylnirvanol, quinidine, and diethyldithiocarbamate have been widely used as CYP1A2, CYP2A6, CYP2B6, CYP2C9, CYP2C19, CYP2D6, and CYP2E1 index inhibitors, respectively (Food and Drug Administration drug interaction guidance, 2006; medicine.iupui.edu/ clinpharm/ddis/main-table). Recent studies report that CYP2J2 also plays a role in the biotransformation of xenobiotics, including drugs such as albendazole (Wu et al., 2013b), astemizole (Matsumoto et al., 2002), apixaban (Wang et al., 2010), ebastine (Liu et al., 2006), fenbendazole (Wu
et al., 2013b), ritonavir (Kaspera et al., 2014), and terfenadine (Lafite et al., 2006). Using strong chemical inhibitors to inhibit the biotransformation activity of recombinant P450 isoforms or human liver microsomes is a valuable method for P450 reaction phenotyping studies to characterize the enzymes responsible for drug metabolism (Rodrigues, 1999; Yoon and Liu, 2011). To date, several chemicals including danazol (Lee et al., 2012), decursin (Lee et al., 2014b,c), hydroxyebastine(Yoon and Liu, 2011), telmisartan (Ren et al., 2013), terfenadone (Lafite et al., 2006), thelephoric acid (Wu et al., 2013a), and 49-(p-toluenesulfonylamido)-4-hydroxychalcone (Lee et al., 2014a) have been reported as CYP2J2 inhibitors. However, limited data are available on CYP2J2 index inhibitors to date. In this study, we evaluated the inhibitory potential of strong CYP2J2 inhibitors (danazol, hydroxyebastine, telmisartan, and terfenadone) against various CYP2J2 substrates (albendazole, astemizole, ebastine, and terfenadine) using a recombinant CYP2J2 isoform to find CYP2J2 index inhibitors that can inhibit CYP2J2 activity in a substrateindependent manner. The four compounds tested inhibited CYP2J2-catalyzed metabolism in a substrate-dependent manner. Telmisartan inhibited CYP2J2mediated albendazole hydroxylation and astemizole O-demethylation with IC50 values of 0.14 and 0.85 mM, respectively, while it had no inhibitory effect on the CYP2J2-mediated ebastine and terfenadine hydroxylation (IC50 . 20 mM) (Fig. 1; Table 1). The inhibition of astemizole O-demethylase activity by telmisartan (IC50 = 0.85 mM) was similar to the results of a previous study (IC50 = 0.42 mM) (Ren et al., 2013). Terfenadone showed strong inhibitory potential on CYP2J2-mediated albendazole, astemizole, and terfenadine metabolism (IC50 , 0.21 mM), whereas it showed weak inhibitory potential on the CYP2J2-mediated ebastine hydroxylation (IC50 = 6.04 mM) (Fig. 1; Table 1). The IC50 value of the inhibition of ebastine hydroxylase activity by terfenadone found in our study (6.04 mM) is 8.6-fold higher than that reported by Lafite et al. (2006) (0.7 mM). These differences could have originated from differences in the incubation conditions, such as CYP2J2 concentrations (5 versus 1 pmol CYP2J2/ml) and substrate concentrations (5 versus 1 mM ebastine). Danazol strongly inhibited CYP2J2-mediated albendazole, astemizole, ebastine, and terfenadine metabolism in a substrate-independent manner, with IC50 values of 0.05, 0.07, 0.18, and 0.34 mM, respectively (Fig. 1; Table 1). Hydroxyebastine also inhibited CYP2J2-mediated albendazole, astemizole, and terfenadine metabolism in a substrate-independent manner, with IC50 values of 0.22, 0.31, and 0.6 mM, respectively (Fig. 1; Table 1). However, the inhibitory potentials of danazol were 3.3- to 4.4-fold stronger than those of hydroxyebastine, indicating that danazol is a stronger CYP2J2 inhibitor than hydroxyebastine. Danazol inhibited CYP2J2-mediated astemizole O-demethylation activity with a Ki value of 0.06 mM in a noncompetitive model (Fig. 2). In addition, danazol is a selective
Substrate-Independent Inhibition of P450 2J2 by Danazol inhibitor for CYP2J2 at low concentrations. Although danazol showed inhibitory potential against CYP2C9 (1.1 mM) and CYP2C19 (2 mM) activities, the IC50 value of danazol on CYP2J2-mediated astemizole O-demethylation activity (0.07 mM) was 15-fold lower than those of the other five P450 enzymes (Table 2). Taken together, these data suggest that danazol may be used as a CYP2J2 index inhibitor in reaction phenotyping studies because it strongly inhibits CYP2J2 metabolism in a substrate-independent manner and also selectively inhibits CYP2J2 activity at low concentrations. College of Pharmacy and Research Institute of Pharmaceutical Sciences, Kyungpook National University, Daegu, South Korea
EUNYOUNG LEE ZHEXUE WU JONG CHEOL SHON KWANG-HYEON LIU
References Delozier TC, Kissling GE, Coulter SJ, Dai D, Foley JF, Bradbury JA, Murphy E, Steenbergen C, Zeldin DC, and Goldstein JA (2007) Detection of human CYP2C8, CYP2C9, and CYP2J2 in cardiovascular tissues. Drug Metab Dispos 35:682–688. Enayetallah AE, French RA, Thibodeau MS, and Grant DF (2004) Distribution of soluble epoxide hydrolase and of cytochrome P450 2C8, 2C9, and 2J2 in human tissues. J Histochem Cytochem 52:447–454. Evangelista EA, Kaspera R, Mokadam NA, Jones JP, 3rd, and Totah RA (2013) Activity, inhibition, and induction of cytochrome P450 2J2 in adult human primary cardiomyocytes. Drug Metab Dispos 41:2087–2094. Feng W, Xu X, Zhao G, Li G, Liu T, Zhao J, Dong R, Wang DW, and Tu L (2013) EETs and CYP2J2 inhibit TNF-a-induced apoptosis in pulmonary artery endothelial cells and TGF-b1induced migration in pulmonary artery smooth muscle cells. Int J Mol Med 32:685–693. Fleming I (2001) Cytochrome p450 and vascular homeostasis. Circ Res 89:753–762. Greenblatt DJ, Zhao Y, Venkatakrishnan K, Duan SX, Harmatz JS, Parent SJ, Court MH, and von Moltke LL (2011) Mechanism of cytochrome P450-3A inhibition by ketoconazole. J Pharm Pharmacol 63:214–221. Hashizume T, Imaoka S, Mise M, Terauchi Y, Fujii T, Miyazaki H, Kamataki T, and Funae Y (2002) Involvement of CYP2J2 and CYP4F12 in the metabolism of ebastine in human intestinal microsomes. J Pharmacol Exp Ther 300:298–304. Kaspera R, Kirby BJ, Sahele T, Collier AC, Kharasch ED, Unadkat JD, and Totah RA (2014) Investigating the contribution of CYP2J2 to ritonavir metabolism in vitro and in vivo. Biochem Pharmacol 91:109–118. Khojasteh SC, Prabhu S, Kenny JR, Halladay JS, and Lu AY (2011) Chemical inhibitors of cytochrome P450 isoforms in human liver microsomes: a re-evaluation of P450 isoform selectivity. Eur J Drug Metab Pharmacokinet 36:1–16. Kroetz DL and Zeldin DC (2002) Cytochrome P450 pathways of arachidonic acid metabolism. Curr Opin Lipidol 13:273–283. Lafite P, Dijols S, Buisson D, Macherey AC, Zeldin DC, Dansette PM, and Mansuy D (2006) Design and synthesis of selective, high-affinity inhibitors of human cytochrome P450 2J2. Bioorg Med Chem Lett 16:2777–2780. Lee B, Kang W, Shon J, Park KH, Song KS, and Liu KH (2014a) Potential of 49-(ptoluenesulfonylamide)-4-hydroxychalcone to inhibit the human cytochrome P450 2J2 isoform. J Korean Soc Appl Biol Chem 57:31–34. Lee B, Wu Z, and Liu KH (2014b) Response to comment: “A note on CYP2J2-mediated terfenadine hydroxylation in human liver microsomes”. Food Chem Toxicol 71:286–287.
Lee B, Wu Z, Sung SH, Lee T, Song KS, Lee MY, and Liu KH (2014c) Potential of decursin to inhibit the human cytochrome P450 2J2 isoform. Food Chem Toxicol 70:94–99. Lee CA, Jones JP, 3rd, Katayama J, Kaspera R, Jiang Y, Freiwald S, Smith E, Walker GS, and Totah RA (2012) Identifying a selective substrate and inhibitor pair for the evaluation of CYP2J2 activity. Drug Metab Dispos 40:943–951. Liu KH, Kim MG, Lee DJ, Yoon YJ, Kim MJ, Shon JH, Choi CS, Choi YK, Desta Z, and Shin JG (2006) Characterization of ebastine, hydroxyebastine, and carebastine metabolism by human liver microsomes and expressed cytochrome P450 enzymes: major roles for CYP2J2 and CYP3A. Drug Metab Dispos 34:1793–1797. Matsumoto S, Hirama T, Matsubara T, Nagata K, and Yamazoe Y (2002) Involvement of CYP2J2 on the intestinal first-pass metabolism of antihistamine drug, astemizole. Drug Metab Dispos 30:1240–1245. Matsumoto S and Yamazoe Y (2001) Involvement of multiple human cytochromes P450 in the liver microsomal metabolism of astemizole and a comparison with terfenadine. Br J Clin Pharmacol 51:133–142. Perloff MD, von Moltke LL, Court MH, Kotegawa T, Shader RI, and Greenblatt DJ (2000) Midazolam and triazolam biotransformation in mouse and human liver microsomes: relative contribution of CYP3A and CYP2C isoforms. J Pharmacol Exp Ther 292:618–628. Racha JK, Zhao ZS, Olejnik N, Warner N, Chan R, Moore D, and Satoh H (2003) Substrate dependent inhibition profiles of fourteen drugs on CYP3A4 activity measured by a high throughput LCMS/MS method with four probe drugs, midazolam, testosterone, nifedipine and terfenadine. Drug Metab Pharmacokinet 18:128–138. Ren S, Zeng J, Mei Y, Zhang JZ, Yan SF, Fei J, and Chen L (2013) Discovery and characterization of novel, potent, and selective cytochrome P450 2J2 inhibitors. Drug Metab Dispos 41:60–71. Rodrigues AD (1999) Integrated cytochrome P450 reaction phenotyping: attempting to bridge the gap between cDNA-expressed cytochromes P450 and native human liver microsomes. Biochem Pharmacol 57:465–480. Roman RJ (2002) P-450 metabolites of arachidonic acid in the control of cardiovascular function. Physiol Rev 82:131–185. Smith HE, Jones JP, 3rd, Kalhorn TF, Farin FM, Stapleton PL, Davis CL, Perkins JD, Blough DK, Hebert MF, and Thummel KE, et al. (2008) Role of cytochrome P450 2C8 and 2J2 genotypes in calcineurin inhibitor-induced chronic kidney disease. Pharmacogenet Genomics 18:943–953. VandenBrink BM, Foti RS, Rock DA, Wienkers LC, and Wahlstrom JL (2011) Evaluation of CYP2C8 inhibition in vitro: utility of montelukast as a selective CYP2C8 probe substrate. Drug Metab Dispos 39:1546–1554. Wang JS, Wen X, Backman JT, Taavitsainen P, Neuvonen PJ, and Kivistö KT (1999) Midazolam alpha-hydroxylation by human liver microsomes in vitro: inhibition by calcium channel blockers, itraconazole and ketoconazole. Pharmacol Toxicol 85:157–161. Wang L, Zhang D, Raghavan N, Yao M, Ma L, Frost CE, Maxwell BD, Chen SY, He K, and Goosen TC, et al. (2010) In vitro assessment of metabolic drug-drug interaction potential of apixaban through cytochrome P450 phenotyping, inhibition, and induction studies. Drug Metab Dispos 38:448–458. Wang X, Ni L, Yang L, Duan Q, Chen C, Edin ML, Zeldin DC, and Wang DW (2014) CYP2J2derived epoxyeicosatrienoic acids suppress endoplasmic reticulum stress in heart failure. Mol Pharmacol 85:105–115. Wu Z, Lee B, Song KS, and Liu KH (2013a) Inhibitory potential of thelephoric acid on CYP2J2 activities in human liver microsome. J Life Sci 23:1126–1132. Wu Z, Lee D, Joo J, Shin JH, Kang W, Oh S, Lee Y, Lee SJ, Yea SS, and Lee HS, et al. (2013b) CYP2J2 and CYP2C19 are the major enzymes responsible for metabolism of albendazole and fenbendazole in human liver microsomes and recombinant P450 assay systems. Antimicrob Agents Chemother 57:5448–5456. Yoon Y and Liu K (2011) Potential of hydroxyebastine and terfenadine alcohol to inhibit the human cytochrome P450 2J2 isoform. J Korean Soc Appl Biol Chem 54:659–666. Zeldin DC (2001) Epoxygenase pathways of arachidonic acid metabolism. J Biol Chem 276: 36059–36062. Zeldin DC, Foley J, Goldsworthy SM, Cook ME, Boyle JE, Ma J, Moomaw CR, Tomer KB, Steenbergen C, and Wu S (1997) CYP2J subfamily cytochrome P450s in the gastrointestinal tract: expression, localization, and potential functional significance. Mol Pharmacol 51: 931–943. Zhang S, Pillai VC, Mada SR, Strom S, and Venkataramanan R (2012) Effect of voriconazole and other azole antifungal agents on CYP3A activity and metabolism of tacrolimus in human liver microsomes. Xenobiotica 42:409–416.
Address correspondence to: Kwang-Hyeon Liu, College of Pharmacy and Research Institute of Pharmaceutical Sciences, Kyungpook National University, 80 Daehakro, Bukgu, Daegu 702-701, South Korea. E-mail: [email protected]
Downloaded from dmd.aspetjournals.org at ASPET Journals on August 13, 2015
Authorship Contributions Participated in research design: Liu. Conducted experiments: Lee, Wu, Shon. Contributed new reagents or analytic tools: Lee, Wu, Shon. Performed data analysis: Lee, Wu, Liu. Wrote or contributed to the writing of the manuscript: Lee, Wu, Liu.
1253
http://dx.doi.org/10.1124/dmd.115.064345 Drug Metab Dispos 43:1250–1253, August 2015
Short Communication Danazol Inhibits Cytochrome P450 2J2 Activity in a Substrateindependent Manner Received March 18, 2015; accepted June 5, 2015
ABSTRACT metabolism in a substrate-independent manner, with IC50 values of 0.05, 0.07, 0.18, and 0.34 mM, respectively. Danazol noncompetitively inhibited CYP2J2-mediated astemizole O-demethylation activities with a Ki value of 0.06 mM. Terfenadone strongly inhibited CYP2J2mediated albendazole, astemizole, and terfenadine metabolism (IC50 < 0.21 mM), whereas it showed weak inhibition against CYP2J2catalyzed ebastine hydroxylase activity (IC50 = 6.04 mM). Telmisartan had no inhibitory effect on CYP2J2-mediated ebastine and terfenadine hydroxylation (IC50 > 20 mM). Taken together, these data suggest that danazol may be used as a CYP2J2 index inhibitor in reaction phenotyping studies.
Introduction
2006). Some P450 inhibitors inhibit enzyme activity in a substratedependent manner. For example, fluconazole inhibits CYP3Amediated midazolam metabolism, whereas it does not suppress CYP3A-mediated metabolism of tacrolimus and testosterone (Zhang et al., 2012). In general, P450 index inhibitors used in reaction phenotyping studies should inhibit each P450 isoform-specific activity in a substrate-independent manner. One example is ketoconazole, a representative CYP3A inhibitor that inhibits the CYP3A-mediated metabolism of several substrates including nifedipine, midazolam, terfenadine, testosterone, and triazolam (Wang et al., 1999; Perloff et al., 2000; Racha et al., 2003; Greenblatt et al., 2011). In this study, we evaluated the inhibitory potential of danazol, hydroxyebastine, telmisartan, and terfenadone (representative CYP2J2 inhibitors) against CYP2J2-catalyzed albendazole hydroxylation, astemizole O-demethylation, ebastine hydroxylation, and terfenadine hydroxylation to find a representative CYP2J2 inhibitor. The results from this study will indicate which CYP2J2 inhibitors are suitable for reaction phenotyping and drug-drug interaction studies.
Cytochrome P450 2J2 (CYP2J2) is the member of CYP2J subfamily in humans. It is highly expressed in extrahepatic organs such as the heart, kidneys, lungs, small intestines, and gastrointestinal tissues (Zeldin et al., 1997; Enayetallah et al., 2004; Delozier et al., 2007). CYP2J2 is involved in the metabolism of arachidonic acid (Zeldin, 2001; Roman, 2002). It metabolizes arachidonic acid to epoxyeicosatrienoic acids, which play a role in homeostasis of the heart, lungs, and kidneys (Fleming, 2001; Kroetz and Zeldin, 2002; Smith et al., 2008; Feng et al., 2013; Wang et al., 2014). In addition to its endogenous role, CYP2J2 also metabolizes medications such as albendazole, astemizole, ebastine, and terfenadine (Matsumoto and Yamazoe, 2001; Hashizume et al., 2002; Matsumoto et al., 2002; Lafite et al., 2006; Liu et al., 2006; Lee et al., 2012; Ren et al., 2013; Wu et al., 2013b). To date, danazol, hydroxyebastine, telmisartan, and terfenadone have been reported as strong CYP2J2 inhibitors (Lafite et al., 2006; Yoon and Liu, 2011; Lee et al., 2012; Ren et al., 2013). However, their inhibitory potential on CYP2J2 activities has been evaluated for only one or two CYP2J2 substrates. For example, danazol and hydroxyebastine were evaluated for CYP2J2-mediated astemizole Odemethylation and terfenadine hydroxylation (Yoon and Liu, 2011; Lee et al., 2012). The CYP2J2 inhibition activity of telmisartan was evaluated for astemizole O-demethylation (Ren et al., 2013), whereas terfenadone was studied for ebastine hydroxylation (Lafite et al.,
This study was supported by grants from the National Research Foundation of Korea, Ministry of Science, ICT and Future Planning [NRF-2014M3A9D9069714]; and Ministry of Education [NRF-2013R1A1A2008442], Republic of Korea. E.L. and Z.W. contributed equally to this work. dx.doi.org/10.1124/dmd.115.064345.
Materials and Methods Chemicals and Reagents. O-desmethyl astemizole, hydroxyebastine, and terfenadone were obtained from Toronto Research Chemicals (North York, Ontario, Canada). Albendazole, astemizole, danazol, ebastine, telmisartan, terfenadine, terfenadine alcohol, b-NADP, MgCl2, KH2PO4, D-glucose-6phosphate, and glucose-6-phosphate dehydrogenase were purchased from Sigma-Aldrich (St. Louis, MO). Solvents were high-pressure liquid chromatography grade (Fisher Scientific Co., Pittsburgh, PA), and the other chemicals were of the highest quality available. Human recombinant CYP2J2 was purchased from BD Biosciences (San Jose, CA). Inhibition Studies Using Recombinant CYP2J2. All incubations were performed in triplicate. The incubation mixtures containing cDNA-expressed CYP2J2 (5 pmol/ml for astemizole, ebastine, and terfenadine; 10 pmol/ml for
ABBREVIATION: CE, collision energy. 1250
Downloaded from dmd.aspetjournals.org at ASPET Journals on August 13, 2015
Cytochrome P450 2J2 (CYP2J2) is an enzyme responsible for the metabolism of endogenous substrates including arachidonic acid, as well as therapeutic drugs such as albendazole, astemizole, ebastine, and terfenadine. Selective inhibitors of CYP2J2 are essential for P450 reaction phenotyping studies. To find representative CYP2J2 index inhibitors, we evaluated the inhibitory potential of danazol, hydroxyebastine, telmisartan, and terfenadone against CYP2J2 activity for four representative CYP2J2 substrates (albendazole, astemizole, ebastine, and terfenadine) using recombinant CYP2J2. Of these four CYP2J2 inhibitors, danazol strongly inhibited CYP2J2mediated albendazole, astemizole, ebastine, and terfenadine
1251
Substrate-Independent Inhibition of P450 2J2 by Danazol
Fig. 1. Inhibitory effects of danazol, hydroxyebastine, telmisartan, and terfenadone (0–20 mM) on CYP2J2mediated albendazole (1 mM) (A), astemizole (0.3 mM) (B), ebastine (5 mM) (C), and terfenadine (0.2 mM) (D) metabolism. Recombinant CYP2J2 was incubated with each CYP2J2 substrate. The activity was expressed as the percentage of the remaining activity compared with a control sample containing no inhibitor. Data are the mean values of triplicate experiments.
analysis of O-desmethylastemizole and hydroxyalbendazole, mobile phase B was linearly increased from 10% to 50% over 3 minutes, held at 50% for 0.1 minute, and then immediately stepped back down to 10% for re-equilibration for 6 minutes. For the analysis of hydroxyebastine and terfenadine alcohol, mobile phase B was linearly increased from 10% to 50% over 6 minutes, held at 50% for 0.1 minute, and then immediately stepped back down to 10% for reequilibration. The total run time was 10 minutes. For the analysis of acetaminophen, 4-hydroxytolbutamide, 4-hydroxymephenytoin, dextrorphan, and 19-hydroxymidazolam, mobile phase B was linearly increased from 10% to 90% over 7 minutes, held at 90% for 0.1 minute, and then immediately stepped back down to 10% for re-equilibration. The total run time was 10 minutes. The operating parameters of the mass spectrometer detector were as follows: ion spray, 4000 V in positive mode; capillary temperature, 350C; vaporizer temperature, 300C; sheath gas pressure, 35 arbitrary units; auxiliary gas, 10 arbitrary units; and nitrogen gas flow rate, 15 l/min. Quantitation was carried out by selective reaction monitoring. The following selective reaction monitoring transitions were monitored: acetaminophen (m/z 152 . 110, collision energy [CE] 25 eV), O-desmethylastemizole (m/z 445 . 204, CE 30 eV), dextrorphan (m/z 258 . 157, CE 35 eV), hydroxylalbendazole (m/z 282 . 250, CE 20 eV), hydroxyebastine (m/z 486 . 167, CE 42 eV), 4hydroxymephenytoin (m/z 236 . 150, CE 20 eV), 19-hydroxymidazolam (m/ TABLE 1 IC50 values of four CYP2J2 inhibitors against CYP2J2-mediated albendazole, astemizole, ebastine, and terfenadine metabolism in the recombinant CYP2J2 isoform The values are presented as average 6 S.D. (n = 3). IC50 (mM) Inhibitor Albendazole
Danazol Hydroxyebastine Terfenadone Telmisartan N.D., not determined.
0.05 0.22 6 0.07 0.14 6 0.01 0.14 6 0.05
Astemizole
Ebastine
Terfenadine
6 6 6 6
0.34 6 0.13 N.D. 6.04 6 2.51 .20
0.18 6 0.02 0.6 6 0.06 0.21 6 0.05 .20
0.07 0.31 0.07 0.85
0.02 0.11 0.02 0.16
Downloaded from dmd.aspetjournals.org at ASPET Journals on August 13, 2015
albendazole), 0.1 mM phosphate buffer (pH 7.4), substrate (albendazole, 1 mM; astemizole, 0.3 mM; ebastine, 5 mM; or terfenadine, 0.2 mM), and inhibitor [danazol, hydroxyebastine, telmisartan, or terfenadone (0–20 mM)] were preincubated for 5 minutes at 37C. Each substrate concentration was lower than its reported Km value (Matsumoto et al., 2002; Liu et al., 2006; Evangelista et al., 2013; Wu et al., 2013b). The reactions were initiated by the addition of a NADPH-generating system (including 3.3 mM D-glucose-6-phosphate, 500 units/ml glucose-6-phosphate dehydrogenase, 1.3 mM NADP, and 3.3 mM MgCl2). To determine the inhibition constant (Ki values) of danazol for CYP2J2-mediated astemizole O-demethylation in recombinant CYP2J2, danazol (0, 0.02, 0.05, 0.1, 0.15, and 0.2 mM) was added to reaction mixtures containing different concentrations of astemizole (0.1, 0.2, and 0.5 mM). The reaction mixtures (final volume, 100 ml) were incubated at 37C in a thermoshaker. After 20-minute incubation, 50 ml of cold acetonitrile containing the internal standard (mebendazole, 300 nM) was added immediately to terminate the reaction. After centrifugation at 10,000g for 5 minutes, aliquots of the supernatant were injected into a liquid chromatography–tandem mass spectrometry system. Recombinant P450 Inhibition Study. Danazol selectivity on CYP2J2 was assessed by its inhibitory potential against five major CYPs: CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A. The incubation mixtures containing recombinant P450 (5 pmol/ml for CYP1A2, 2C19, 2D6, and 3A; 10 pmol/ml for CYP2C9), 0.1 mM phosphate buffer (pH 7.4), P450-isoform probe substrate (Table 2), and danazol (0–30 mM) were preincubated for 5 minutes at 37C. The reactions were initiated by the addition of a NADPHgenerating system and further incubated for 15 minutes (CYP1A2, CYP2D6, and CYP3A), 20 minutes (CYP2C9), and 40 minutes (CYP2C19). The reaction was terminated by adding 50 ml of cold acetonitrile, and aliquots of the supernatant were injected into a liquid chromatography–tandem mass spectrometry system. Analytical Method. The samples were analyzed using a LCMS-8040 Triple Quadrupole Mass Spectrometer (Shimadzu, Tokyo) equipped with an electrospray ionization interface. The separation was carried out on a Luna C18 Reversed-Phase Column (Phenomenex, Torrance, CA) (50 2 mm i.d., 3 mm particle size). The mobile phases were 0.1% (v/v) formic acid in water (A) and 0.1% (v/v) formic acid in acetonitrile (B) at a flow rate of 0.2 ml/min. For the
1252
Lee et al.
z 342 . 203, CE 25 eV), 4-hydroxytolbutamide (m/z 287 . 89, CE 50 eV), mebendazole (m/z 296 . 264, CE 17 eV), and terfenadine alcohol (m/z 488 . 452, CE 36 eV). The analytical data were processed by LabSolution software, version 2.0 (Shimadzu). Data Analysis. The IC50 values were calculated using the WinNonlin software (Pharsight, Mountain View, CA).
Results and Discussion
TABLE 2 IC50 values of danazol against six recombinant P450 isoform activities The values are presented as average 6 S.D. (n = 3). CYP isoform
Fig. 2. Representative Dixon plots for inhibition of CYP2J2-mediated astemizole O-demethylation by danazol in the recombinant CYP2J2 isoform. An increasing concentration of astemizole [0.1 mM (d), 0.2 mM (s), and 0.5 mM (.)] was incubated with recombinant CYP2J2 (5 pmol/ml) (BD Biosciences, San Jose, CA) and an NADPH-generating system at 37C for 20 minutes in the presence or absence of danazol (0, 0.02, 0.05, 0.1, 0.15, and 0.2 mM). The inhibition data fit to a noncompetitive inhibition model. Data are the mean values of duplicate experiments.
CYP1A2 CYP2C9 CYP2C19 CYP2D6 CYP3A CYP2J2
Substrate
Phenacetin Tolbutamide S-Mephenytoin Dextromethorphan Midazolam Astemizole
Substrate concentration
IC50
mM
mM
10 200 2 5 5 0.3
12 6 3.27 1.1 6 0.24 2.0 6 0.43 . 30 18 6 5.94 0.07 6 0.02
Downloaded from dmd.aspetjournals.org at ASPET Journals on August 13, 2015
Reaction phenotyping and drug-drug interaction studies require that inhibitors for each P450 isoform be both strong and selective. For example, ketoconazole is more extensively used than itraconazole as a CYP3A index inhibitor, even though both ketoconazole and itraconazole have similar inhibitory potentials against CYP3Amediated midazolam 19-hydroxylation (IC50 @ 0.01 mM) (Racha et al., 2003). The reason for this preference is that studies have shown that ketoconazole has a strong inhibitory effect on various CYP3A substrates including midazolam, nifedipine, testosterone, and triazolam (Wang et al., 1999; Perloff et al., 2000; Racha et al., 2003; Greenblatt et al., 2011), while itraconazole has a weak inhibitory effect on CYP3A-mediated terfenadine metabolism (IC50 . 50 mM) (Racha et al., 2003). Another example is montelukast, which is more widely used as a CYP2C8 index inhibitor than pioglitazone, even though pioglitazone is also a good selective inhibitor for CYP2C8 (Khojasteh et al., 2011). Montelukast inhibits CYP2C8-catalyzed amodiaquine, paclitaxel, and repaglinide metabolism in a substrate-independent manner (IC50 = 0.008–0.026 mM), while pioglitazone inhibits CYP2C8-catalyzed amodiaquine, paclitaxel, and repaglinide metabolism with IC50 values of 6.6, 37.6, and 3.8 mM, respectively (VandenBrink et al., 2011). Similar to the previous examples, furafylline, tranylcypromine, ticlopidine, sulfaphenazole, S-benzylnirvanol, quinidine, and diethyldithiocarbamate have been widely used as CYP1A2, CYP2A6, CYP2B6, CYP2C9, CYP2C19, CYP2D6, and CYP2E1 index inhibitors, respectively (Food and Drug Administration drug interaction guidance, 2006; medicine.iupui.edu/ clinpharm/ddis/main-table). Recent studies report that CYP2J2 also plays a role in the biotransformation of xenobiotics, including drugs such as albendazole (Wu et al., 2013b), astemizole (Matsumoto et al., 2002), apixaban (Wang et al., 2010), ebastine (Liu et al., 2006), fenbendazole (Wu
et al., 2013b), ritonavir (Kaspera et al., 2014), and terfenadine (Lafite et al., 2006). Using strong chemical inhibitors to inhibit the biotransformation activity of recombinant P450 isoforms or human liver microsomes is a valuable method for P450 reaction phenotyping studies to characterize the enzymes responsible for drug metabolism (Rodrigues, 1999; Yoon and Liu, 2011). To date, several chemicals including danazol (Lee et al., 2012), decursin (Lee et al., 2014b,c), hydroxyebastine(Yoon and Liu, 2011), telmisartan (Ren et al., 2013), terfenadone (Lafite et al., 2006), thelephoric acid (Wu et al., 2013a), and 49-(p-toluenesulfonylamido)-4-hydroxychalcone (Lee et al., 2014a) have been reported as CYP2J2 inhibitors. However, limited data are available on CYP2J2 index inhibitors to date. In this study, we evaluated the inhibitory potential of strong CYP2J2 inhibitors (danazol, hydroxyebastine, telmisartan, and terfenadone) against various CYP2J2 substrates (albendazole, astemizole, ebastine, and terfenadine) using a recombinant CYP2J2 isoform to find CYP2J2 index inhibitors that can inhibit CYP2J2 activity in a substrateindependent manner. The four compounds tested inhibited CYP2J2-catalyzed metabolism in a substrate-dependent manner. Telmisartan inhibited CYP2J2mediated albendazole hydroxylation and astemizole O-demethylation with IC50 values of 0.14 and 0.85 mM, respectively, while it had no inhibitory effect on the CYP2J2-mediated ebastine and terfenadine hydroxylation (IC50 . 20 mM) (Fig. 1; Table 1). The inhibition of astemizole O-demethylase activity by telmisartan (IC50 = 0.85 mM) was similar to the results of a previous study (IC50 = 0.42 mM) (Ren et al., 2013). Terfenadone showed strong inhibitory potential on CYP2J2-mediated albendazole, astemizole, and terfenadine metabolism (IC50 , 0.21 mM), whereas it showed weak inhibitory potential on the CYP2J2-mediated ebastine hydroxylation (IC50 = 6.04 mM) (Fig. 1; Table 1). The IC50 value of the inhibition of ebastine hydroxylase activity by terfenadone found in our study (6.04 mM) is 8.6-fold higher than that reported by Lafite et al. (2006) (0.7 mM). These differences could have originated from differences in the incubation conditions, such as CYP2J2 concentrations (5 versus 1 pmol CYP2J2/ml) and substrate concentrations (5 versus 1 mM ebastine). Danazol strongly inhibited CYP2J2-mediated albendazole, astemizole, ebastine, and terfenadine metabolism in a substrate-independent manner, with IC50 values of 0.05, 0.07, 0.18, and 0.34 mM, respectively (Fig. 1; Table 1). Hydroxyebastine also inhibited CYP2J2-mediated albendazole, astemizole, and terfenadine metabolism in a substrate-independent manner, with IC50 values of 0.22, 0.31, and 0.6 mM, respectively (Fig. 1; Table 1). However, the inhibitory potentials of danazol were 3.3- to 4.4-fold stronger than those of hydroxyebastine, indicating that danazol is a stronger CYP2J2 inhibitor than hydroxyebastine. Danazol inhibited CYP2J2-mediated astemizole O-demethylation activity with a Ki value of 0.06 mM in a noncompetitive model (Fig. 2). In addition, danazol is a selective
Substrate-Independent Inhibition of P450 2J2 by Danazol inhibitor for CYP2J2 at low concentrations. Although danazol showed inhibitory potential against CYP2C9 (1.1 mM) and CYP2C19 (2 mM) activities, the IC50 value of danazol on CYP2J2-mediated astemizole O-demethylation activity (0.07 mM) was 15-fold lower than those of the other five P450 enzymes (Table 2). Taken together, these data suggest that danazol may be used as a CYP2J2 index inhibitor in reaction phenotyping studies because it strongly inhibits CYP2J2 metabolism in a substrate-independent manner and also selectively inhibits CYP2J2 activity at low concentrations. College of Pharmacy and Research Institute of Pharmaceutical Sciences, Kyungpook National University, Daegu, South Korea
EUNYOUNG LEE ZHEXUE WU JONG CHEOL SHON KWANG-HYEON LIU
References Delozier TC, Kissling GE, Coulter SJ, Dai D, Foley JF, Bradbury JA, Murphy E, Steenbergen C, Zeldin DC, and Goldstein JA (2007) Detection of human CYP2C8, CYP2C9, and CYP2J2 in cardiovascular tissues. Drug Metab Dispos 35:682–688. Enayetallah AE, French RA, Thibodeau MS, and Grant DF (2004) Distribution of soluble epoxide hydrolase and of cytochrome P450 2C8, 2C9, and 2J2 in human tissues. J Histochem Cytochem 52:447–454. Evangelista EA, Kaspera R, Mokadam NA, Jones JP, 3rd, and Totah RA (2013) Activity, inhibition, and induction of cytochrome P450 2J2 in adult human primary cardiomyocytes. Drug Metab Dispos 41:2087–2094. Feng W, Xu X, Zhao G, Li G, Liu T, Zhao J, Dong R, Wang DW, and Tu L (2013) EETs and CYP2J2 inhibit TNF-a-induced apoptosis in pulmonary artery endothelial cells and TGF-b1induced migration in pulmonary artery smooth muscle cells. Int J Mol Med 32:685–693. Fleming I (2001) Cytochrome p450 and vascular homeostasis. Circ Res 89:753–762. Greenblatt DJ, Zhao Y, Venkatakrishnan K, Duan SX, Harmatz JS, Parent SJ, Court MH, and von Moltke LL (2011) Mechanism of cytochrome P450-3A inhibition by ketoconazole. J Pharm Pharmacol 63:214–221. Hashizume T, Imaoka S, Mise M, Terauchi Y, Fujii T, Miyazaki H, Kamataki T, and Funae Y (2002) Involvement of CYP2J2 and CYP4F12 in the metabolism of ebastine in human intestinal microsomes. J Pharmacol Exp Ther 300:298–304. Kaspera R, Kirby BJ, Sahele T, Collier AC, Kharasch ED, Unadkat JD, and Totah RA (2014) Investigating the contribution of CYP2J2 to ritonavir metabolism in vitro and in vivo. Biochem Pharmacol 91:109–118. Khojasteh SC, Prabhu S, Kenny JR, Halladay JS, and Lu AY (2011) Chemical inhibitors of cytochrome P450 isoforms in human liver microsomes: a re-evaluation of P450 isoform selectivity. Eur J Drug Metab Pharmacokinet 36:1–16. Kroetz DL and Zeldin DC (2002) Cytochrome P450 pathways of arachidonic acid metabolism. Curr Opin Lipidol 13:273–283. Lafite P, Dijols S, Buisson D, Macherey AC, Zeldin DC, Dansette PM, and Mansuy D (2006) Design and synthesis of selective, high-affinity inhibitors of human cytochrome P450 2J2. Bioorg Med Chem Lett 16:2777–2780. Lee B, Kang W, Shon J, Park KH, Song KS, and Liu KH (2014a) Potential of 49-(ptoluenesulfonylamide)-4-hydroxychalcone to inhibit the human cytochrome P450 2J2 isoform. J Korean Soc Appl Biol Chem 57:31–34. Lee B, Wu Z, and Liu KH (2014b) Response to comment: “A note on CYP2J2-mediated terfenadine hydroxylation in human liver microsomes”. Food Chem Toxicol 71:286–287.
Lee B, Wu Z, Sung SH, Lee T, Song KS, Lee MY, and Liu KH (2014c) Potential of decursin to inhibit the human cytochrome P450 2J2 isoform. Food Chem Toxicol 70:94–99. Lee CA, Jones JP, 3rd, Katayama J, Kaspera R, Jiang Y, Freiwald S, Smith E, Walker GS, and Totah RA (2012) Identifying a selective substrate and inhibitor pair for the evaluation of CYP2J2 activity. Drug Metab Dispos 40:943–951. Liu KH, Kim MG, Lee DJ, Yoon YJ, Kim MJ, Shon JH, Choi CS, Choi YK, Desta Z, and Shin JG (2006) Characterization of ebastine, hydroxyebastine, and carebastine metabolism by human liver microsomes and expressed cytochrome P450 enzymes: major roles for CYP2J2 and CYP3A. Drug Metab Dispos 34:1793–1797. Matsumoto S, Hirama T, Matsubara T, Nagata K, and Yamazoe Y (2002) Involvement of CYP2J2 on the intestinal first-pass metabolism of antihistamine drug, astemizole. Drug Metab Dispos 30:1240–1245. Matsumoto S and Yamazoe Y (2001) Involvement of multiple human cytochromes P450 in the liver microsomal metabolism of astemizole and a comparison with terfenadine. Br J Clin Pharmacol 51:133–142. Perloff MD, von Moltke LL, Court MH, Kotegawa T, Shader RI, and Greenblatt DJ (2000) Midazolam and triazolam biotransformation in mouse and human liver microsomes: relative contribution of CYP3A and CYP2C isoforms. J Pharmacol Exp Ther 292:618–628. Racha JK, Zhao ZS, Olejnik N, Warner N, Chan R, Moore D, and Satoh H (2003) Substrate dependent inhibition profiles of fourteen drugs on CYP3A4 activity measured by a high throughput LCMS/MS method with four probe drugs, midazolam, testosterone, nifedipine and terfenadine. Drug Metab Pharmacokinet 18:128–138. Ren S, Zeng J, Mei Y, Zhang JZ, Yan SF, Fei J, and Chen L (2013) Discovery and characterization of novel, potent, and selective cytochrome P450 2J2 inhibitors. Drug Metab Dispos 41:60–71. Rodrigues AD (1999) Integrated cytochrome P450 reaction phenotyping: attempting to bridge the gap between cDNA-expressed cytochromes P450 and native human liver microsomes. Biochem Pharmacol 57:465–480. Roman RJ (2002) P-450 metabolites of arachidonic acid in the control of cardiovascular function. Physiol Rev 82:131–185. Smith HE, Jones JP, 3rd, Kalhorn TF, Farin FM, Stapleton PL, Davis CL, Perkins JD, Blough DK, Hebert MF, and Thummel KE, et al. (2008) Role of cytochrome P450 2C8 and 2J2 genotypes in calcineurin inhibitor-induced chronic kidney disease. Pharmacogenet Genomics 18:943–953. VandenBrink BM, Foti RS, Rock DA, Wienkers LC, and Wahlstrom JL (2011) Evaluation of CYP2C8 inhibition in vitro: utility of montelukast as a selective CYP2C8 probe substrate. Drug Metab Dispos 39:1546–1554. Wang JS, Wen X, Backman JT, Taavitsainen P, Neuvonen PJ, and Kivistö KT (1999) Midazolam alpha-hydroxylation by human liver microsomes in vitro: inhibition by calcium channel blockers, itraconazole and ketoconazole. Pharmacol Toxicol 85:157–161. Wang L, Zhang D, Raghavan N, Yao M, Ma L, Frost CE, Maxwell BD, Chen SY, He K, and Goosen TC, et al. (2010) In vitro assessment of metabolic drug-drug interaction potential of apixaban through cytochrome P450 phenotyping, inhibition, and induction studies. Drug Metab Dispos 38:448–458. Wang X, Ni L, Yang L, Duan Q, Chen C, Edin ML, Zeldin DC, and Wang DW (2014) CYP2J2derived epoxyeicosatrienoic acids suppress endoplasmic reticulum stress in heart failure. Mol Pharmacol 85:105–115. Wu Z, Lee B, Song KS, and Liu KH (2013a) Inhibitory potential of thelephoric acid on CYP2J2 activities in human liver microsome. J Life Sci 23:1126–1132. Wu Z, Lee D, Joo J, Shin JH, Kang W, Oh S, Lee Y, Lee SJ, Yea SS, and Lee HS, et al. (2013b) CYP2J2 and CYP2C19 are the major enzymes responsible for metabolism of albendazole and fenbendazole in human liver microsomes and recombinant P450 assay systems. Antimicrob Agents Chemother 57:5448–5456. Yoon Y and Liu K (2011) Potential of hydroxyebastine and terfenadine alcohol to inhibit the human cytochrome P450 2J2 isoform. J Korean Soc Appl Biol Chem 54:659–666. Zeldin DC (2001) Epoxygenase pathways of arachidonic acid metabolism. J Biol Chem 276: 36059–36062. Zeldin DC, Foley J, Goldsworthy SM, Cook ME, Boyle JE, Ma J, Moomaw CR, Tomer KB, Steenbergen C, and Wu S (1997) CYP2J subfamily cytochrome P450s in the gastrointestinal tract: expression, localization, and potential functional significance. Mol Pharmacol 51: 931–943. Zhang S, Pillai VC, Mada SR, Strom S, and Venkataramanan R (2012) Effect of voriconazole and other azole antifungal agents on CYP3A activity and metabolism of tacrolimus in human liver microsomes. Xenobiotica 42:409–416.
Address correspondence to: Kwang-Hyeon Liu, College of Pharmacy and Research Institute of Pharmaceutical Sciences, Kyungpook National University, 80 Daehakro, Bukgu, Daegu 702-701, South Korea. E-mail: [email protected]
Downloaded from dmd.aspetjournals.org at ASPET Journals on August 13, 2015
Authorship Contributions Participated in research design: Liu. Conducted experiments: Lee, Wu, Shon. Contributed new reagents or analytic tools: Lee, Wu, Shon. Performed data analysis: Lee, Wu, Liu. Wrote or contributed to the writing of the manuscript: Lee, Wu, Liu.
1253
