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SHORT COMMUNICATION

SELECTIVITIES OF HUMAN CYTOCHROME P450 INHIBITORS TOWARD RAT P450 ISOFORMS: STUDY WITH cDNA-EXPRESSED SYSTEMS OF THE RAT

	Abstract

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The aim of this study was to determine the selectivities of chemical inhibitors for human cytochrome P450 (P450) isoforms toward the corresponding rat P450 isoforms by using cDNA-expressed rat P450s (CYP1A2, CYP2A1, CYP2C6, CYP2C11, CYP2D2, CYP2E1, CYP3A1, and CYP3A2). Among the inhibitor probes for human P450s used in this study, only sulfaphenazole showed a selective inhibitory effect on the activity of the corresponding rat P450 isoform (CYP2C6). Furafylline also preferentially inhibited the activity of rat CYP1A2. However, methoxalen and ketoconazole more strongly inhibited the activities of other P450 isoforms than those of the corresponding rat P450 isoforms, CYP2A1 and CYP3A1/2, respectively. On the other hand, quinidine and aniline had little effect on the activities of the corresponding rat P450 isoforms, CYP2D2, and rat CYP2E1, respectively. These results suggest that chemical probes that have been used for human P450 isoforms do not always exhibit the same selectivity for the corresponding rat P450 isoforms. However, it appears that sulfaphenazole can be used as a selective inhibitor for rat CYP2C6. In addition, furafylline may also be a relatively selective inhibitor for rat CYP1A2.

Recent advances in research for chemical inhibitors of human P450s have greatly facilitated the characterization of catalytic specificities of individual P450 isoforms involved in drug metabolism. Chemical inhibitors are useful tools for determining the roles of individual P450s involved in drug metabolism in human liver microsomes. However, it is still difficult to determine the roles of P450 isoforms involved in drug metabolism in rat liver microsomes by using chemical inhibitors. This is because the specificities of chemicals used as inhibitor probes for rat P450 isoforms have not been thoroughly evaluated (Eagling et al., 1998).

In the present study, the effects of chemical inhibitors that have been used as inhibitor probes for human P450 isoforms on the corresponding rat P450 isoforms were studied by using cDNA-expressed rat P450s (CYP1A2, CYP2A1, CYP2C6, CYP2C11, CYP2D2, CYP2E1, CYP3A1, and CYP3A2). These isoforms used in this study were selected based on its abundance in rat liver or its significance in metabolism. The chemical inhibitors used in the present study were furafylline, methoxalen, sulfaphenazole, quinidine, aniline, and ketoconazole, which are potent inhibitors of human CYP1A2 (Tassaneeyakul et al., 1994), CYP2A6 (Yamazaki et al., 1994; Koenigs et al., 1997), CYP2C9 (Newton et al., 1995), CYP2D6 (Newton et al., 1995), CYP2E1 (Nakajima et al., 1999), and CYP3A4 (Baldwin et al., 1995; Bourrié et al., 1996), respectively.

	Experimental Procedures

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Chemicals. Ketoconazole was a gift from Janssen Research Foundation (Beerse, Belgium). Midazolam and 4-hydroxymidazolam were gifts from F. Hoffmann-La Roche (Basel, Switzerland). Zaltoprofen was a gift from Zeria Pharmaceutical (Tokyo, Japan). Bufuralol hydrochloride, 1'-hydroxybufuralol and 4-hydroxydiclofenac were purchased from BD Gentest (Woburn, MA). 7-Hydroxytestosterone, 16-hydroxytestosterone, and furafylline were purchased from Ultrafine Chemicals (Manchester, UK). Methoxalen was purchased from BIOMOL Research Laboratories (Plymouth Meeting, PA). p-Nitrophenol was purchased from Nacalai Tesque, Inc. (Kyoto, Japan). Acetaminophen, caffeine, diclofenac, p-nitrocatechol, phenacetin, testosterone, sulfaphenazole, qunidine, and qunine were purchased from Wako Pure Chemicals (Osaka, Japan). Other chemicals were of the highest grade commercially available.

cDNA-Expressed P450. Microsomes prepared from baculovirus-infected insect cells expressing CYP1A2 (lot 1), CYP2C6 (lot 1), CYP2C11 (lot 1), CYP2D2 (lot 1), CYP3A1 (lot 1), and CYP3A2 (lot 1) and those from human B-lymphoblastoid cells expressing CYP2A1 (lot 7) and CYP2E1 (lot 6) were obtained from BD Gentest. All recombinant P450s were coexpressed with NADPH-P450 oxidoreductase. Recombinant CYP2C6, CYP2C11, CYP3A1, and CYP3A2 were coexpressed with cytochrome b₅.

Incubation Conditions. On the basis of the results of our previous study (Kobayashi et al., 2002), phenacetin O-deethylation (POD), testosterone 7-hydroxylation (T7H), diclofenac 4-hydroxylation (DFH), testosterone 16-hydroxylation (T16H), bufuralol 1'-hydroxylation (BLH), p-nitrophenol 2-hydroxylation (PNPH) and midazolam 4-hydroxylation (MD4H) were chosen as markers for rat CYP1A2, CYP2A1, CYP2C6, CYP2C11, CYP2D2, CYP2E1, and CYP3A1/2-mediated activities, respectively. A typical incubation mixture (0.25 ml total volume) contained 0.1 mM EDTA, 100 mM potassium phosphate buffer (pH 7.4), an NADPH-generating system (0.5 mM NADP⁺, 2 mM glucose 6-phosphate, 1 IU/ml of glucose-6-phosphate dehydrogenase, and 4 mM MgCl₂), a substrate and cDNA-expressed P450. The reaction was initiated by the addition of the NADPH-generating system following a 1-min preincubation at 37°C. All reactions were performed in the linear range with respect to P450 concentration and incubation time. After the reaction had been stopped by the addition of 100 µl of ice-cold acetonitrile, an internal standard was added. The mixtures were centrifuged at 13,000g for 10 min, and the supernatants (each 100 µl) were analyzed by HPLC as described below. The substrate concentration, incubation time, content of cDNA-expressed P450, and amount of internal standard used for each assay are listed in Table 1. Testosterone was dissolved in methanol and added to the incubation mixture at a final methanol concentration of 1%. The other chemicals were dissolved in methanol and added to each test tube. After evaporation with vacuum evaporator, the incubation mixture except microsomes and NADPH-generating system was added, and the compounds were redissolved. Samples for determination of POD activity were evaporated by a vacuum evaporator for 15 min after the centrifugation, and the remaining samples (each 100 µl) were analyzed. Since furafylline and methoxalen are mechanism-based inhibitors, these chemicals were preincubated at 37°C for 30 min with microsomes in the presence of NADPH-generating system before adding substrate.

HPLC Analysis. Determination of respective metabolites was carried out using a Hitachi HPLC system (Tokyo, Japan) consisting of an L-7100 pump, an L-7400 UV detector, an L-7485 intelligent spectrofluorometer, an L-7200 autosampler and a D-7500 integrator and a CAPCELL PAK C₁₈ UG120 column (4.6 mm x 250 mm, 5 µm; Shiseido, Tokyo, Japan). The activities of POD, DFH, BLH, PNPH, MD4H, T7H and T16H were determined as described elsewhere (Kobayashi et al., 2000, 2002).

	Results and Discussion

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Among the chemical inhibitor probes of human P450 isoforms used in this study, only sulfaphenazole showed a selective inhibitory effect on the corresponding rat P450 isoform. As shown in Fig. 1A, sulfaphenazole, a potent inhibitor of human CYP2C9, inhibited only CYP2C6-mediated activity at a concentration as low as 1 µM. Although CYP2C11-mediated activity was also inhibited by sulfaphenazole, it was inhibited to a lesser degree than that of CYP2C6. CYP3A1- and CYP3A2-mediated activities were inhibited by sulfaphenazole but only to 50% of the control level even at the concentration of 100 µM. Very little or no inhibition was observed for CYP1A2-, CYP2A1-, CYP2D2-, and CYP2E1-mediated activities. These findings suggest that sulfaphenazole at low concentrations (<10 µM) could be used as a selective inhibitor probe of CYP2C6.

View larger version (50K): [in this window] [in a new window]   FIG. 1. Effects of sulfaphenazole (A), furafylline (B), methoxalene (C), ketoconazole (D), aniline (E), and quinidine (F) on CYP1A2-mediated POD, CYP2A1-mediated T7H, CYP2C6-mdiated DFH, CYP2C11-mediated T16H, CYP2D2-mediated BLH, CYP2E1-mediated PNPH, and CYP3A1/2-mediated MD4H activities in cDNA-expressed rat P450s. Substrates (phenacetin, testosterone, diclofenac, bufuralol, p-nitrophenol, and midazolam) were incubated at 37°C with corresponding cDNA-expressed rat P450s. Substrate concentrations, incubation times, and contents of P450 used are shown in Table 1. Each column represents the mean of duplicate experiments. Control activities for each reaction are 10.0 pmol/min/pmol P450 for CYP1A2-meadiated POD activity, 0.6 pmol/min/pmol P450 for CYP2A1-meadiated T7H activity, 15.4 pmol/min/pmol P450 for CYP2C6-meadiated DFH activity, 14.8 pmol/min/pmol P450 for CYP2C11-meadiated T16H activity, 5.9 pmol/min/pmol P450 for CYP2D2-meadiated BLH activity, 8.7 pmol/min/pmol P450 for CYP2E1-meadiated PNPH activity, 1.9 pmol/min/pmol P450 for CYP3A1-meadiated MD4H activity and 5.7 pmol/min/pmol P450 for CYP3A2-meadiated MD4H activity. ND < 2 pmol/min/pmol P450 for POD activity, ND < 0.3 pmol/min/pmol P450 for DFH activity, ND < 0.2 pmol/min/pmol P450 for T7H, T16H, PNPH, and MD4H activities, and ND < 0.02 pmol/min/pmol P450 for BLH activity.

Furafylline, a potent inhibitor of human CYP1A2, potentially inhibited rat CYP1A2-mediated POD activity (Fig. 1B). At concentrations of more than 1 µM, more than 80% of the activity was inhibited. Although CYP2C6-mediated activity was also inhibited by furafylline, the extent of inhibition was less than that of CYP1A2. Furafylline showed a weak inhibitory effect on CYP2A1- and CYP2C11-mediated activities (<30% of the control level at 100 µM), but it showed no apparent inhibitory effect on CYP2D2-, CYP2E1-, CYP3A1/2-mediated activities. These findings suggest that it is possible to use furafylline as a relatively selective inhibitor of rat CYP1A2.

In contrast to sulfaphenazole and furafylline, methoxalen and ketoconazole, which are potent inhibitors of human CYP2A6 and CYP3A1/2, respectively, did not show a selective inhibitory effect on the activities of the corresponding rat P450 isoforms. As shown in Fig. 1C, methoxalen inhibited CYP2A1-mediated activity in a concentration-dependent manner (Fig. 1C). However, more potent inhibitory effects on CYP1A2-, CYP2C6-, and CYP2C11-mediated activities were observed. Similarly, ketoconazole inhibited CYP3A1- and CYP3A2-mediated activities in a concentration-dependent manner (Fig. 1D). However, ketoconazole inhibited CYP1A2- and CYP2C6-mediated activities by more than 50% at a concentration of 10 µM. These findings suggest that methoxalen and ketoconazole are not selective inhibitors of CYP2A1 and CYP3A1/2, respectively.

On the other hand, aniline and quinidine, selective inhibitors of human CYP2E1 and CYP2D6, respectively, did not show apparent inhibitory effects on the activities of the corresponding rat P450 isoforms. As shown in Fig. 1E, aniline showed little effect on rat CYP2E1-mediated activity. Aniline inhibited CYP1A2- and CYP2C6-mediated activities, but its effect was weak even at the concentration of more than 100 µM. Quinidine also showed little effect on CYP2D2-mediated activity at a concentration of 10 µM (Fig. 1F), but CYP2C6-mediated activity was inhibited by quinidine even at a concentration of 0.1 µM. Since quinine, a diastereomer of quinidine, is known to be a more efficient inhibitor of rat CYP2D-mediated activity in rat liver microsomes (Kobayashi et al., 1989), the effect of quinine on CYP2D2-mediated activity was examined. As expected, 10 µM of quinine inhibited CYP2D2-mediated activity by more than 90%, but CYP2C6- and CYP2C11-mediated activities were also inhibited by about 70% (data not shown). These findings suggest that not only quinidine but also quinine are not selective inhibitors of CYP2D2.

The results of the present study suggest that considerable differences exist between the selectivities of chemical inhibitors of human and rat P450 orthologues. Boobis et al. (1990) suggested the following three possible reasons for species differences in the effects of chemical inhibitors on drug metabolism in vitro: 1) the active site differs in species, 2) the isoform-catalyzing metabolism differs in species, 3) the inhibition is not via direct competition at the active site and the inhibitory site differs in species. In the present study, cDNA-expressed systems were used for screening of selectivity and comparative potency of several inhibitors. Under these conditions, our data indicated that chemicals used as inhibitor probes of human P450 isoforms are not always appropriate for use as inhibitor probes of rat P450 isoforms. This finding suggests that the active site and the inhibitory site differ in species depending on the isoforms of P450 studied. As shown in Fig. 1, CYP2D2-mediated activities in some cases were higher than 100% of control. Except for assay of testosterone metabolism, organic solvent was not included in the incubation mixture. Therefore, the higher activities did not result from the effect of solvent. In addition, the calibration curves were linear (r > 0.999), although no internal standard was used in the assay for bufuralol 1'-hydroxylation. It was thought that the control activities were slightly low, although the reason is unclear.

In conclusion, it appears that chemical inhibitors used as inhibitor probes of human P450 isoforms do not exhibit the same selectivities in humans and rats. However, it is possible to use sulfaphenazole as a selective inhibitor for rat CYP2C6. Furafylline also appears to be a relatively selective inhibitor for rat CYP1A2.

Finally, caution must be exercised when comparing the effects of inhibitors between rats and humans. In addition, using cDNA-expressed system to evaluate the selectivity of chemical inhibitors cannot present an overall picture, and the selectivity may differ when the inhibitors were used in liver microsomes. Further investigation using chemical inhibitors is needed to determine the roles of individual P450s in drug metabolism by rat liver microsomes.

Kaoru Kobayashi
Kikuko Urashima
Noriaki Shimada
Kan Chiba

Laboratory of Pharmacology and Toxicology, Graduate School of Pharmaceutical Sciences, Chiba University, Chiba (K.K., K.U, K.C); and Daiichi Pure Chemicals Co. Ltd., Tokyo (N.S), Japan

	Footnotes

 
¹ Abbreviations used are: P450, cytochrome P450; POD, phenacetin O-deethylation; T7H, testosterone 7-hydroxylation; DFH, diclofenac 4-hydroxylation; T16H, testosterone 16-hydroxylation; BLH, bufuralol 1'-hydroxylation; PNPH, p-nitrophenol 2-hydroxylation; MD4H, midazolam 4-hydroxylation; HPLC, high-performance liquid chromotography.

Address correspondence to: Dr. Kaoru Kobayashi, Laboratory of Pharmacology and Toxicology, Graduate School of Pharmaceutical Sciences, Chiba University, Yayoi-cho 1-33, Inage-ku, Chiba 263-8522, Japan. E-mail: kaoruk{at}p.chiba-u.ac.jp

	References

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