BIOPHARMACEUTICS & DRUG DISPOSITION Biopharm. Drug Dispos. 36: 565–574 (2015) Published online 31 October 2015 in Wiley Online Library

(wileyonlinelibrary.com) DOI: 10.1002/bdd.1965

Metabolism of (-)-cis- and (-)-trans-rose oxide by cytochrome P450 enzymes in human liver microsomes Hiroshi Nakahashia, Yuuki Yamamuraa, Atsushi Usamia, Pramoch Rangsunvigitb, Pomthong Malakulb, and Mitsuo Miyazawaa,* a

Department of Applied Chemistry, Faculty of Science and Engineering, Kinki University (Kindai University) Kowakae, Higashiosaka-shi, Osaka, 577-8502, Japan b The Petroleum and Petrochemical College, Chulalongkorn University, Bangkok 10330, Thailand

ABSTRACT: The in vitro metabolism of (-)-cis- and (-)-trans-rose oxide was investigated using human liver microsomes and recombinant cytochrome P450 (P450 or CYP) enzymes for the first time. Both isomers of rose oxide were incubated with human liver microsomes, and the formation of the respective 9-oxidized metabolite were determined using gas chromatography-mass spectrometry (GC-MS). Of 11 different recombinant human P450 enzymes used, CYP2B6 and CYP2C19 were the primary enzymes catalysing the metabolism of (-)-cis- and (-)-trans-rose oxide. CYP1A2 also efficiently oxidized (-)-cis-rose oxide at the 9-position but not (-)-trans-rose oxide. α-Naphthoflavone (a selective CYP1A2 inhibitor), thioTEPA (a CYP2B6 inhibitor) and anti-CYP2B6 antibody inhibited (-)-cis-rose oxide 9-hydroxylation catalysed by human liver microsomes. On the other hand, the metabolism of (-)-trans-rose oxide was suppressed by thioTEPA and anti-CYP2B6 at a significant level in human liver microsomes. However, omeprazole (a CYP2C19 inhibitor) had no significant effects on the metabolism of both isomers of rose oxide. Using microsomal preparations from nine different human liver samples, (-)-9-hydroxy-cis- and (-)-9-hydroxy-trans-rose oxide formations correlated with (S)-mephenytoin N-demethylase activity (CYP2B6 marker activity). These results suggest that CYP2B6 plays important roles in the metabolism of (-)-cis- and (-)-trans-rose oxide in human liver microsomes. Copyright © 2015 John Wiley & Sons, Ltd. Key words: microsomes; rose oxide; monoterpene; P450; CYP2B6

Introduction Rose oxide, the monoterpene cyclic ether, is an important aroma compound with a low flavour threshold, which has been isolated from rose oil [1]. Then four isomers were detected in many plants (e.g. Pelargonium spp. [2], Eucalyptus citriodora [3], Cymbopogon winterianus Jowitt [4] and even in the liquid secretion of the widespread beetle Aromia moschata L. [5]. (-)-cis- and (-)-transRose oxide are contained as the main constituents *Correspondence to: Department of Applied Chemistry, Faculty of Science and Engineering, Kinki University (Kindai University) Kowakae, Higashiosaka-shi, Osaka 577-8502, Japan. E-mail: [email protected]

Copyright © 2015 John Wiley & Sons, Ltd.

in several essential oils, whereas the other two isomers are also known. Especially, (-)-cis- and (-)-trans-rose oxide has been used as a food flavour, in perfumes and cosmetics because it has the characteristic odour properties for rosy notes. Microorganisms are increasingly being used as biocatalysts to alter the structures of organic compounds. It was reported that Aspergillus niger catalyses the conversion of (-)-cis- and (-)-trans-rose oxide to two metabolites, namely 9-hydroxy-rose oxide and 8-carboxylic acid [6]. However, there is no literature on the absorption and excretion of the (-)-cis- and (-)-trans-rose oxide in human body. A variety of aroma components in numerous species of plants have been reported to be oxidized Received 23 April 2015 Revised 12 June 2015 Accepted 22 June 2015

566 by multiple forms of cytochrome P450 (P450 or CYP) in laboratory animals and humans [7–9]. P450 enzymes have been shown to detoxify and/or toxify these compounds to more polar, and sometimes more reactive, metabolites [10–12]. For example, pulegone, a hepatotoxin in humans [13], is catalysed to a proximate hepatotoxic metabolite, menthofuran, by P450 enzymes [10,11]. Recently, it was reported that a number of monoterpene cyclic ethers, such as 1,8-cineole [14], 1,4-cineole [15], and (+)-menthofuran [11], are metabolized by multiple P450 enzymes. Moreover, it was reported that (+)-menthofuran is a potent mechanism-based inactivator of human CYP2A6 [16]. In the current study, the oxidation of (-)-cis- and (-)-trans-rose oxide by P450 enzymes was examined in microsomes prepared from different human liver samples, and the metabolites formed were analysed by gas chromatography-mass spectrometry (GC-MS). Also we investigated the metabolism of (-)-cis- and (-)-trans-rose oxide by human liver microsomes and recombinant human P450 enzymes.

Material and Methods Chemicals (-)-cis- and (-)-trans-Rose oxide were obtained from Taiyo Perfumery Co. Ltd. The structure was characterized by 1H-NMR, 13C-NMR, specific rotation and MS spectra. The purity of the compounds was judged to be > 95% on analysis with TLC, GC and NMR. α-Naphthoflavone, N,N ′,N′′-triethylenethiophosphoramide (thioTEPA), omeprazole, NADP+, glucose 6-phosphate and glucose 6-phosphate dehydrogenase were purchased from Sigma-Aldrich (St Louis, MO). (-)-9Hydroxy-cis- and (-)-9-hydroxy-trans-rose oxide were isolated and purified from cultured medium on incubation of (-)-cis- and (-)-trans-rose oxide with A. niger [6]. Other reagents and chemicals used in this study were obtained from sources as described previously or were of the highest quality commercially available [17].

(-)-9-Hydroxy-cis-rose oxide Pale yellow oil; EI-MS, m/z (relative intensity) 168 (1), 153 (5), 139 (100), 137 (39.8), 115 (46.2), Copyright © 2015 John Wiley & Sons, Ltd.

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97 (13.4), 81 (22.1), 69 (84.3), 55 (57.2), 43 (39.8), 41 (40.6); IR (KBr, vmax, cm-1) 3409, 2954, 1172, 1068; 1H NMR (CDCl3, 500 MHz) δ 0.95 (3H, d, J = 6.3 Hz, H-11), 1.05 (1H, ddd, J = 11.5, 11.5,13.2 Hz, H-3ax), 1.24 (1H dddd, J = 5.0, 12.5, 12.5, 12.5 Hz, H-5ax), 1.5 1.68 (3H, m, H-3eq, H-4, H-5eq), 1.71 (3H, d, J = 1.5 Hz, H-10), 3.43 (1H, ddd, J = 2.0, 12.5, 12.5 Hz, H-6ax), 3.74 (1H, ddd, J = 2.0, 5.0, 12.5 Hz, H-6eq), 4.00 (2H, s, H-9), 4.05 (1H, ddd, J = 2.0, 8.0, 11.5 Hz, H-2), 5.43 (1H, dq, J = 1.5, 8.0 Hz, H-7); 13C-NMR (CDCl3, 125 MHz) δ 14.1 (C-10), 22.2 (C-11), 30.1 (C-4), 34.3 (C-5), 40.4 (C-3), 67.9 (C-6), 68.0 (C-9), 74.3 (C-2), 126.3 (C-7), 137.7 (C-8).

(-)-9-Hydroxy-trans-rose oxide Pale yellow oil; EI-MS, m/z (relative intensity) 168 (1), 153 (5), 139 (100), 137 (39.8), 115 (46.2), 97 (13.4), 81 (22.1), 69 (84.3), 55 (57.2), 43 (39.8), 41 (40.6); IR (KBr, vmax, cm-1) 3409, 2954, 1172, 1068; 1H NMR (CDCl3, 500 MHz) δ 1.07 (3H, d, J = 7.0, 7.0 Hz, H-11), 1.26 (1H, ddddd, J = 1.2, 3.0, 3.0, 5.0, 13.0 Hz, H-5eq), 1.38 (1H dddd, J = 1.2, 3.5, 6.0, 13.0 Hz, H-3eq), 1.66 (1H ddd, J = 4.8, 8.0, 13.0 Hz, H-3ax), 1.70 (3H, d, J = 1.5, 1.5 Hz, H-10), 1.76 (1H dddd, J = 4.8, 4.8, 8.5, 13.0 Hz, H-5ax),2.02 (m, H-4), 1.71 (3H, d, J = 1.5 Hz, H-10), 3.72 (1H, ddd, J = 3.0, 8.5, 12.0 Hz, H-6ax), 3.76 (1H, ddd, J = 3.0, 4.8, 12.0 Hz, H-6eq), 4.00 (2H, s, H-9), 4.44 (1H, ddd, J = 3.5, 8.0, 8.0 Hz, H-2), 5.56 (1H, d, J = 8.0 Hz, H-7); 13C-NMR (CDCl3, 125 MHz) δ 13.9 (C-10), 19.5 (C-11), 24.8 (C-4), 32.3 (C-5), 37.9 (C-3), 62.2 (C-6), 67.8 (C-9), 68.8 (C-2), 125.4 (C-7), 138.1 (C-8).

Enzymes and antibody Human liver microsomes (HG23, HG03, HH18, HG88, HG74, HG06, HK37, HH13, HG64) were obtained from Gentest Co., Inc. (Woburn, MA, USA) and stored at 80 °C. Microsomes were available with complete catalytic assays of the major CYPs: phenacetin-O-deethylase (CYP1A2), coumarin 7-hydroxylase (CYP2A6), (S)-mephenytoin Ndemethylase (CYP2B6), paclitaxel 6α-hydroxylase (CYP2C8), diclofenac 4′-hydroxylase (CYP2C9), (S)-mephenytoin 4′-hydroxylase (CYP2C19), bufuralol 1′-hydroxylase (CYP2D6), chlorzoxazone 6-hydroxylase (CYP2E1), testosterone 6β-hydroxylase (CYP3A4) and lauric acid 12-hydroxylase (CYP4A). CYP2B6Biopharm. Drug Dispos. 36: 565–574 (2015) DOI: 10.1002/bdd

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specific monoclonal antibodies and recombinant human CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4 expressed in Trichoplusia ni cells infected with a baculovirus containing P450 and NADPH-P450 reductase cDNA inserts were obtained from Gentest Co., Inc.; the P450 contents in these systems were used as described in the data sheets provided by the manufacturer. CYP2C19, CYP2E1 and CYP3A4 were also coexpressed with cytochrome b5. Potassium phosphate buffer (100 mM, pH 7.4) was used for all CYP enzymes except CYP2C19 and CYP2B6 (50 mM potassium phosphate buffer, pH 7.4), CYP2A6 and CYP2C9 (100 mM Tris-HCl buffer (pH 7.5)).

Instrumentation The GC-MS analysis was performed on an Agilent 6890N gas chromatograph (Agilent Technologies, Santa Clara, CA, USA) equipped with an Agilent 5973N quadrupole mass selective detector. The metabolites were separated with a HP-5MS nonpolar capillary column (30 m × 0.25 mm i.d., 0.25 mm film thickness, J & W Scientific, Folsom, CA, USA) using helium (at 1.5 ml/min) as the carrier gas. The injector temperature was maintained at 270 °C. The column temperature was programmed as isothermal at 80 °C for 3 min, then raised to 270 °C at a rate of 4 °C/min and held at 270 °C for 5 min. The effluent from the GC column was introduced directly into the source of the ion source via a transfer line (280 °C). The ion source temperature was set at 230 °C. The electron impact (EI) ionization voltage was set to 70 eV and positive charged ions were analysed in full scan mode, applying a scan range of m/z 40–300 amu. The NMR spectra were obtained on a Jeol ECA-500 (500 MHz, 1H; 125 MHz, 13C) spectrometer. The samples were dissolved in chloroform-d with tetramethylsilane (TMS) as an internal standard for 1H-NMR spectra. The residual CHCl3 was used as an internal reference (7.70 ppm) for 13C-NMR spectra measured in CDCl3. The IR spectra were obtained with a FT/IR-470 plus Fourier transform infrared spectrometer. CHCl3 was used as a solvent. Specific rotation was determined with a JASCO DIP-1000 digital polarimeter (Jasco Co., Ltd Japan). Copyright © 2015 John Wiley & Sons, Ltd.

In vitro assay with human liver microsomes The metabolism of (-)-cis- and (-)-trans-rose oxide by human liver microsomes was determined as follows: the standard reaction mixture contained liver microsomes (0.2 mg protein/ml) with 100 μM (-)-cis- and (-)-trans-rose oxide in methanol (at less than 1% v/v in final solvent concentration) in a final volume of 0.5 ml of 100 mM potassium phosphate buffer (pH 7.4) containing an NADPH-generating system (0.5 mM NADP+, 5 mM glucose 6-phosphate, and 0.5 units of glucose 6-phosphate dehydrogenase/ml). The incubation was carried out at 37 °C for 60 min in a shaking water bath, and then terminated by adding 1.5 ml of dichloromethane. The reaction mixture was mixed vigorously for 1 min using a vortex mixer. After centrifugation of the reaction mixture at 3000 rpm for 5 min, the organic layer was analysed by GC-MS.

In vitro assay with cDNA-expressed P450 enzymes Recombinant P450s from T. ni cells expressing 11 individual human P450 enzymes were used. The reactions were carried out as described for the human liver microsomal study. To examine the role of individual recombinant P450 enzymes involved in the metabolism of (-)-cis- and (-)-transrose oxide, each of 11 recombinant P450 enzymes (20 nM) was incubated with 100 μM substrate for 60 min at 37 °C.

Chemical inhibition experiments The inhibitory effects of known P450 enzymeselective inhibitors on the metabolism of (-)-cisand (-)-trans-rose oxide by human liver microsomes and recombinant P450 enzymes were evaluated to determine the P450 enzyme(s) involved in each metabolic pathway. The inhibitors used in the present study were 1–5 μM α-naphthoflavone (a selective CYP1A2 inhibitor), 10–50 μM thioTEPA (a known inhibitor of CYP2B6) and 1–10 μM omeprazole (a CYP2C19 inhibitor). The inhibition experiment was started with a 10 min preincubation at 37 °C of 0.2 mg/ml microsomal proteins or 20 nM recombinant P450 enzymes, inhibitor, 100 mM phosphate buffer (pH 7.4) and NADPH-generating system (0.5 mM Biopharm. Drug Dispos. 36: 565–574 (2015) DOI: 10.1002/bdd

568 NADP+, 5 mM glucose 6-phosphate and 0.5 units of glucose 6-phosphate dehydrogenase/ml), and then 100 mM substrate was added. The reactions were carried out as described for the human liver microsomal study.

Immunoinhibition experiments To further study the metabolism by CYP2B6, the immunoinhibition of 9-hydroxylation activity of (-)-cis- and (-)-trans-rose oxide was examined. After preincubation of human liver microsomes (0.1 mg) with anti-CYP2B6 monoclonal antibody or control serum in 25 mM Tris buffer for 15 min on ice, 100 μM (-)-cis- or (-)-trans-rose oxide and NADPH-generating system in 50 mM potassium phosphate buffer was added, and the reaction was then carried out in an identical manner to the in vitro assay with human liver microsomes.

Correlation test Nine human samples were used to determine the activities of (-)-cis- or (-)-trans-rose oxide metabolite formation and to compare these activities with the activities of known CYP1A2-, CYP2B6-, CYP2C19-catalysed marker reactions in these microsomal preparations, respectively.

Kinetic analysis The kinetic parameters (Vmax and Km) for (-)-cisand (-)-trans-rose oxide metabolism by human liver microsomes and recombinant human P450 enzymes were estimated using a computer program designed for nonlinear regression analysis. Substrate concentrations used for the analysis of (-)-cis- and (-)-trans-rose oxide 9-hydroxylation activities were 50, 100, 150, 200, 300 and 500 μM.

Results Identification of enzymatic products of (-)-cis- and (-)-trans-rose oxide on incubation with human liver microsomes Initially, the metabolism of (-)-cis- and (-)-transrose oxide was examined using human liver microsomes. Figure 1A shows the total ion chromatogram of extracts from the incubation medium of (-)-cis-rose oxide with human liver microsomes in the presence of the NADPH-generating system. Copyright © 2015 John Wiley & Sons, Ltd.

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One substrate-derived metabolite was detected that was identified by the mass spectrum as the (-)-9-hydroxy-cis-rose oxide in this assay condition with a GC retention time of 22.52 min. The mass spectrum of (-)-cis-rose oxide-derived metabolite is presented in Figure 1C. Metabolite identification was made on the basis of mass spectral fragmentation pattern and retention time compared with an authentic sample that was isolated and purified from cultured medium on incubation of (-)-cis-rose oxide with A. niger [6]. On the other hand, a similar incubation with (-)-trans-rose oxide revealed the formation of one metabolite in the chromatogram with a retention time of 23.14 min (Figure 1B). The mass spectrum of the (-)-trans-rose oxide-derived metabolite is presented in Figure 1D. The identity of the metabolite was confirmed by analysis of the authentic reference compound that was isolated and purified from cultured medium on incubation of (-)-trans-rose oxide with A. niger [6]. These results identified the metabolite of (-)-trans-rose oxide as (-)-9-hydroxy-trans-rose oxide.

Oxidation of (-)-cis- and (-)-trans-rose oxide by recombinant P450 enzymes Eleven forms of human recombinant P450 enzymes expressed in insect cells were tested for assessment of the catalytic activities to form the 9-hydroxylated metabolite. CYP2B6 and CYP1A2 had the highest activity in catalysing the metabolism of (-)-cis-rose oxide, followed by CYP1A2 and CYP2C19 (Figure 2). On the other hand, (-)-9-hydroxy-trans-rose oxide was formed by CYP2B6 and CYP2C19 (Figure 2). P450 enzymes including CYP1A1, CYP1B1, CYP2A6, CYP2C8, CYP2C9, CYP2D6, CYP2E1 and CYP3A4 had very low activities or activities below the limit of detection. Moreover, three forms of human recombinant P450 enzymes coexpressed with cytochrome b5 were tested similar to the above experiment. However, all P450 enzymes (CYP2C19, CYP2E1 and CYP3A4) had weakly inhibited substrate oxidation activities.

Inhibition of (-)-cis- and (-)-trans-rose oxide metabolism by chemical inhibitors and anti-CYP2B6 To further assess whether the different metabolic pathways of (-)-cis- and (-)-trans-rose oxide were Biopharm. Drug Dispos. 36: 565–574 (2015) DOI: 10.1002/bdd

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Figure 1. GC-MS analysis of (-)-cis- and (-)-trans-rose oxide metabolism catalysed by human liver microsomes. Panel A and B show total ion chromatogram of the substrates (-)-cis- and (-)-trans-rose oxide with human liver microsomes in the presence of an NADPH-generating system, respectively. Panel C and D show the full scan mass spectrum of (-)-9-hydroxy-cis-rose oxide and (-)-9-hydroxy-trans-rose oxide, respectively

Figure 2. Oxidations of (-)-cis- and (-)-trans-rose oxide to respective 9-hydroxylated metabolite by recombinant human P450 enzymes expressed in T. ni cells

catalysed by CYP1A2, CYP2B6 and CYP2C19, the effects of chemical inhibitors of CYP1A2 (α-naphthoflavone), CYP2B6 (thioTEPA) and CYP2C19 (omeprazole) on (-)-cis- and (-)-transrose oxide metabolism by human liver microsomes and P450 enzymes were examined. Initially, the optimal concentrations of α-naphthoflavone, thioTEPA and omeprazole needed to obtain Copyright © 2015 John Wiley & Sons, Ltd.

effective inhibitions of respective 9-hydroxy-rose oxide formation were tested using recombinant CYP1A2, CYP2B6 and CYP2C19. The results of these experiments are shown in Figure 3A, C and F. In the recombinant CYP1A2, α-naphthoflavone was very inhibitory to the formation of (-)-9-hydroxy-cis-rose oxide, whereas in the human CYP2B6 and CYP2C19, thioTEPA and omeprazole Biopharm. Drug Dispos. 36: 565–574 (2015) DOI: 10.1002/bdd

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Figure 3. Effects of α-naphthoflavone (A and B), thioTEPA(C and D), anti-CYP2B6 (E) and omeprazole (F and G) on the formation of (-)-9-hydroxy-cis- (◯) and (-)-9-hydroxy-trans-rose oxide (□) catalysed by recombinant human CYP1A2 (A), CYP2B6 (C), CYP2C19 (F) and human liver microsomes (B, D, E and G). Substrate concentrations used were 100 μM Copyright © 2015 John Wiley & Sons, Ltd.

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inhibited those by about 50%. Likewise, the results of inhibition experiments on the metabolism of (-)-cis- and (-)-trans-rose oxide in liver microsomes of human sample HH18 with various concentrations of chemical inhibitors are shown in Figure 3B, D and G. In the human sample HH18, α-naphthoflavone was very inhibitory to the formation of (-)-9-hydroxy-cis-rose oxide, whereas thioTEPA inhibited these about 50%, but omeprazole did not show inhibitory effect. In addition, to further study metabolism by CYP2B6, the immunoinhibition of 9-hydroxylation activity of (-)-cis- and (-)-trans-rose oxide was also examined. The results are presented in Figure 3E. In the human sample HH18, anti-CYP2B6 inhibited to a marked extent the formation of (-)-9-hydroxy-trans-rose oxide and inhibited about 50% that of (-)-9-hydroxy-cis-rose oxide.

microsomes of human sample HH18. The kinetic parameters are shown in Table 1. The Vmax values for the oxidation of (-)-cis- and (-)-trans-rose oxide by human liver microsomes were similar, and the turnover numbers for the formation of (-)-9-hydroxy-cis-rose oxide was higher than for the formation of (-)-9-hydroxy-trans-rose oxide. The Km values for the 9-hydroxylation of (-)-cis-rose oxide with CYP1A2 and CYP2B6 were very similar (160.6 μM and 159.2 μM, respectively), and the Km value for the formation of (-)-9-hydroxy-cis-rose oxide with CYP2C19 was high (104.9 μM). For the 9-hydroxylation of (-)-trans-rose oxide, the Km value for 9-hydroxy formation by CYP2B6 was higher than CYP2C19. The Vmax values for both isomers of 9-hydroxlation with CYP2B6 were higher than those with CYP1A2 and CYP2C19.

Correlations of (-)-9-hydroxy-cis- and (-)-9-hydroxy-trans-rose oxide formations with selective P450 activities

Discussion

As shown in Figure 4B, the 9-hydroxylation activity of (-)-cis-rose oxide showed a correlation with CYP2B6-specific (S)-mephenytoin N-demethylase activity (r = 0.691). In contrast, less significant correlations were found with the phenacetin-Odeethylase activity of CYP1A2 (r = 0.233) and (S)-mephenytoin 4′-hydroxylase activity of CYP2C19 (r = 0.363) (Figure 4A and C). On the other hand, the relationship between the 9-hydroxylation activity of (-)-trans-rose oxide and specific activities for CYP2B6 and CYP2C19 in nine individual human liver microsomes is shown in Figure 4D and E. There was a good correlation between (-)-9-hydroxy-trans-rose oxide formation and (S)-mephenytoin N-demethylase activity (r = 0.943), whereas no significant correlation was observed between 9-hydroxylation activity of (-)-trans-rose oxide and (S)-mephenytoin 4′-hydroxylase activity (r = 0.146) (Figure 4D and E).

Kinetic analysis of the (-)-cis- and (-)-trans-rose oxide Kinetic analysis of the hydroxylation of (-)-cis- and (-)-trans-rose oxide was performed with recombinant CYP1A2, CYP2B6, CYP2C19 and liver Copyright © 2015 John Wiley & Sons, Ltd.

In this study, it was found that both isomers of (-)-cis- and (-)-trans-rose oxide were oxidized at the 9-position in human liver microsomes, respectively (Figure 5). No any other metabolic products were detected under this assay condition. The GC-MS analysis suggested the formation of (-)-9-hydroxy-cis- and (-)-9-hydroxy-trans-rose oxide, which is identical to those produced by A. niger as a biocatalyst [6]. In the case of (-)-cis-rose oxide, CYP2B6 is a principal enzyme involved in the metabolism by human liver microsomes, whereas CYP1A2 and CYP2C19 also catalysed (-)-cis-rose oxide 9-hydroxylation. The supporting evidence can be summarized as follows: (1) in 11 recombinant human P450 systems, CYP2B6 had the highest catalytic activity of (-)-cis-rose oxide 9-hydroxylation, followed by CYP1A2 and CYP2C19; (2) αnaphthoflavone, a selective CYP1A2 inhibitor, thioTEPA, a CYP2B6 inhibitor, and anti-CYP2B6 inhibited the (-)-cis-rose oxide 9-hydroxylation, catalysed by human liver microsomes in a concentration-dependent manner, while omeprazole, a CYP2C19 inhibitor, weakly inhibited the metabolism of (-)-cis-rose oxide; (3) there was a high correlation between CYP2B6 marker activity (S)-mephenytoin N-demethylase) and (-)-cis-rose oxide 9-hydroxylation activity in liver microsomes Biopharm. Drug Dispos. 36: 565–574 (2015) DOI: 10.1002/bdd

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Figure 4. Correlation between activities of CYP1A2 (A), CYP2B6 (B and D) and CYP2C19 (C and E) catalytic activities of liver microsomes of nine human samples

of nine human samples examined. These results suggest that CYP2B6 is a major enzyme in (-)-cisrose oxide 9-hydroxylation in human liver microsomes. In our results, (-)-cis-rose oxide 9hydroxylation was suppressed not only by CYP2B6 inhibitor and anti-CYP2B6 but also by CYP1A2 inhibitor. The level of expression of CYP1A2 enzymes in human liver microsomes has been reported to be significantly higher than those of CYP2B6 in 30 Japanese and 30 Caucasians determined [17]. However, the kinetic analysis showed that the Vmax/Km ratio of (-)-cis-rose oxide 9-hydroxylation for CYP2B6 (381 μl/min/ nmol P450) was 1.5-fold higher than that for CYP1A2 (253 μl/min/nmol P450). Collectively, Copyright © 2015 John Wiley & Sons, Ltd.

these suggest that CYP2B6 has probably a more important role than CYP1A2 in catalysing (-)-cisrose oxide 9-hydroxylation by human liver microsomes. On the other hand, CYP2B6 was identified as a major enzyme involved in the metabolism of (-)-trans-rose oxide with the following lines of evidence: (1) with the use of 11 recombinant human P450 enzymes, CYP2B6 had the highest catalytic activity of (-)-trans-rose oxide 9hydroxylation, followed by CYP2C19; (2) thioTEPA and anti-CYP2B6 inhibited the (-)-trans-rose oxide 9-hydroxylation catalysed by human liver microsomes in a concentration-dependent manner, while omeprazole weakly inhibited the Biopharm. Drug Dispos. 36: 565–574 (2015) DOI: 10.1002/bdd

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Table 1. Kinetic analysis of the hydroxylation of (-)-cis- and (-)-trans-rose oxide by human liver microsomes and recombinant human P450 enzymes expressed in T. ni cells Oxidation of (-)-cis- and (-)-trans-rose oxide Formation of (-)-9-hydroxy-cis-rose oxide Enzyme source HH18 Human CYP1A2 Human CYP2B6 Human CYP2C19

Km (μM) 168.7 160.6 159.2 104.9

Vmax a

1.63 40.67b 60.67 9.58

Formation of (-)-9-hydroxy-trans-rose oxide

Vmax/Km c

9.67 253.0d 381.0 91.3

Km (μM)

Vmax

Vmax/Km

101.8 73.80 107.7

1.87 154.0 13.71

18.4 2087.0 127.0

Substrate concentrations used were 50, 100, 150, 200, 300 and 500 μM. a Vmax expressed in nmol/min/mg protein for human liver microsomes. b Vmax expressed in nmol/min/nmol P450 for recombinant human P450 enzymes. c Intrinsic clearance (Vmax/Km) expressed in ml/min/mg protein for human liver microsomes. d Intrinsic clearance (Vmax/Km) expressed in ml/min/nmol P450 for recombinant P450 enzymes.

Figure 5. Oxidation of (-)-cis- and (-)-trans-rose oxide to (-)-9-hydroxy-cis- and (-)-9-hydroxy-trans-rose oxide by human liver microsomal P450 enzymes

metabolism of (-)-trans-rose oxide; (3) there was a good correlation between CYP2B6 marker activity and (-)-trans-rose oxide 9-hydroxylation activity in nine human liver microsomes; (4) the Vmax/Km ratio of (-)-trans-rose oxide 9-hydroxylation for CYP2B6 (2087 μl/min/nmol P450) was much higher than that for CYP2C19 (127 μl/min/nmol P450). In conclusion, the results of the present study using in vitro experiments suggest that both (-)-cis- and (-)-trans-rose oxide isomers are metabolized to respective (-)-cis- and (-)-trans-9-hydroxyrose oxide by human liver microsomal P450 enzymes. CYP2B6 was found to play an important role in catalysing (-)-cis- and (-)-trans-rose oxide 9-hydroxylation in human liver microsomes. Copyright © 2015 John Wiley & Sons, Ltd.

Conflict of Interest The authors have declared that there is no conflict of interest.

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Biopharm. Drug Dispos. 36: 565–574 (2015) DOI: 10.1002/bdd