Materials and Methods

Chemicals.

Racemic mephobarbital was a gift from Yoshitomi Pharmaceutical Co. (Osaka, Japan). S- Mephobarbital was separated from the racemic mixture of mephobarbital by a Chiralcel CA-1 column (20 × 250 mm; Daisel Chemical Co., Tokyo, Japan). The separatedS-mephobarbital was further refined by a STR PREP-ODS_H column (20 × 250 mm; Shimazu, Kyoto, Japan). Cyclobarbital was purchased from Tokyo Kasei Kogyo Co. (Tokyo, Japan). S-Mephenytoin was purchased from Daiichi Pure Chemicals (Tokyo, Japan). Orphenadrine and phenobarbital were purchased from Sigma Chemical Co. (St. Louis, MO). NADP⁺, glucose 6-phosphate, and glucose 6-phosphate dehydrogenase were purchased from Oriental Yeast (Tokyo, Japan). HPLC-grade acetonitrile, analytical-grade coumarin, and other reagents were purchased from Wako Pure Chemical Industries Ltd. (Osaka, Japan).

Enzymes and Antibody.

Ten samples of human liver microsomes were obtained from Japanese patients undergoing partial hepatectomy for treatment of metastatic liver tumors at the Division of General Surgery, Department of Surgery, International Medical Center of Japan (Tokyo, Japan) and were prepared as reported previously (Chiba et al., 1993b). Microsomes prepared from human B-lymphoblastoid cells expressing human CYP1A1 (lot 26), CYP1A2 (lot 50), CYP2A6 (lot 33), CYP2B6 (lot 44), CYP2C8 (lot 20), CYP2C9 (lot 18), CYP2C19 (lot 8), CYP2D6 (lot 22), CYP2E1 (lot 33), and CYP3A4 (lot 41) were obtained from Gentest Corp. (Woburn, MA). cDNA-expressed CYP2A6, CYP2C8, CYP2C9, CYP2D6, CYP2E1, and CYP3A4 were coexpressed with NADPH-CYP reductase in human B-lymphoblastoid cells. Anti-rat CYP2B1 rabbit sera and human CYP2B6 standard, which is a membrane preparation of a human B-lymphoblastoid cell line expressing CYP2B6, were obtained from Gentest Corp.

Assay with Human Liver Microsomes.

The primary incubation medium contained 0.2 or 0.5 mg/ml of human liver microsomes, 0.1 mM EDTA, 100 mM potassium phosphate buffer (pH 7.4), an NADPH-generating system (0.5 mM NADP⁺, 2.0 mM glucose 6-phosphate, 1 I.U./ml of glucose 6-phosphate dehydrogenase, and 4 mM MgCl₂), and 100 μM to 1 mMS-mephobarbital, in a final volume of 250 μl.S-Mephobarbital was dissolved in methanol and added to the incubation mixture at a final methanol concentration of 1%. The mixture was incubated at 37°C for 120 min. After the reaction was stopped by adding 100 μl of cold acetonitrile, 50 μl of cyclobarbital (2.5 μg/ml in methanol) was added as an internal standard. The mixture was centrifuged at 10,000g for 5 min, and 100 μl of supernatant was analyzed by HPLC.

HPLC Conditions.

Phenobarbital determinations were carried out using an HPLC-UV assay method. The HPLC system consisted of an L-6000 pump (Hitachi, Tokyo, Japan), an L-4000 UV detector (Hitachi), an AS-2000 autosampler (Hitachi), a D-2500 integrator (Hitachi), and a CAPCELL PAK C₁₈ UG120 column (4.6 × 250 mm, 5 μm; Shiseido, Tokyo, Japan). The mobile phase consisted of 50 mM potassium dihydrogen phosphate and acetonitrile at a ratio of 75:25, v/v, %-adjusted by concentrated hydrochloric acid to pH 3.75, delivered at a flow rate of 1 ml/min. The eluate was monitored at a wavelength of 204 nm. The column temperature was maintained at 30°C. Calibration curves were generated from 12.5 to 125 pmol by processing the authentic standard substance through the entire procedure. Under these chromatographic conditions, phenobarbital, cyclobarbital, andS-mephobarbital were eluted at 10.5, 13.1, and 25.1 min, respectively. The detection limit of phenobarbital was 2 pmol in an incubation mixture of 250 μl. Phenobarbital was quantified by comparison with the standard curves, using the peak-height ratio method. The intra-assay (n = 6) coefficients of variation were 2.6%.

Assay with cDNA-Expressed CYPs.

Microsomes from human B-lymphoblastoid cells expressing human CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4 were used. The reactions were carried out as described for the human liver microsomal study. To examine the role of individual CYP isoforms involved in S-mephobarbitalN-demethylation, each of the cDNA-expressed CYPs (0.2 mg/ml protein concentration) described above was firstly incubated with 1 mMS-mephobarbital for 120 min at 37°C, according to the procedure recommended by the supplier.

Kinetic Analyses.

Kinetic studies were performed with human liver microsomes (n = 2) and cDNA-expressed CYP2B6. TheN-demethylase activities of S-mephobarbital were determined with S-mephobarbital concentrations ranging from 100 μM to 1 mM. All reactions were performed within a linear range with respect to protein concentration and incubation time. Briefly, 0.2 mg/ml of human liver microsomes and cDNA-expressed CYP2B6 were incubated for 120 and 180 min, respectively. Michaelis-Menten kinetic parameters (K_m,V_max, andV_max/K_m) forS-mephobarbital N-demethylation were estimated by nonlinear least-squares regression analysis.

Immunoblotting.

SDS-polyacrylamide gel electrophoresis and immunoblot peroxidase anti-peroxidase staining of human liver microsomes were carried out essentially as described by Laemmli (1970) and Guengerich et al. (1982). Diaminobenzidine was used as a substrate for peroxidase. The intensities of immunostained bands were measured with an MCID image analyzer (Fuji photo film, Tokyo, Japan). Anti-rat CYP2B1 antibodies gave a strong signal with CYP2B6. Although anti-rat CYP2B1 antibodies showed minor cross-reactivities with CYP2C19, CYP2D6, and CYP2E1, immunoreactive CYP2A6, CYP2D6, and CYP2E1 proteins were well resolved from CYP2B6 based on their differences in SDS-polyacrylamide gel electrophoresis mobility.

Correlation Study.

Correlations between the S-mephobarbitalN-demethylase activity at a 200 μM substrate concentration and the catalytic activities of phenacetin O-deethylase (CYP1A2), coumarin 7-hydroxylase (CYP2A6), diclofenac 4′-hydroxylase (CYP2C9), S-mephenytoin 4′-hydroxylase (CYP2C19), desipramine 2-hydroxylase (CYP2D6), chlorzoxazone 6-hydroxylase (CYP2E1), and testosterone 6β-hydroxylase (CYP3A) were studied using microsomes from 10 human livers. The concentrations of CYP isoform-specific substrates used were 0.5 μM for coumarin, 10 μM for phenacetin, diclofenac, or desipramine, 20 μM for chlorzoxazone, 30 μM for testosterone, and 100 μM forS-mephenytoin, respectively. The specific activities for each CYP isoform were determined by HPLC methods reported elsewhere (Chiba et al., 1993b; Yoshimoto et al., 1995; Kobayashi et al., 1998). Specific activity for CYP2B6 was not determined because of the lack of a selective enzyme assay for CYP2B6 (Ekins et al., 1997).

Inhibition Study.

Chemical inhibition

CYP isoform-specific inhibitors or substrates (i.e., compounds able to act as competitive inhibitors) were used to studyN-demethylase activity of S-mephobarbital with 200 μM substrate in human liver microsomes. The inhibitors used in the present study were 100 and 500 μM coumarin (CYP2A6; Yun et al., 1991), 100 and 300 μM orphenadrine (CYP2B6; Reidy et al., 1989), and 100 and 500 μM S-mephenytoin (CYP2C19; Küpfer and Preisig, 1984). The concentrations of coumarin, orphenadrine, andS-mephenytoin were selected on the basis of data reported previously. Briefly, coumarin at a concentration of 100 μM inhibited nicotine C-oxidation catalyzed by CYP2A6 to about 20% of the control (Nakajima et al., 1996), orphenadrine at 500 μM inhibited RP 73401 [3-cyclopentyloxy-N-(3,5-dichloro-4-pyridyl)-4-methoxybenzamide] catalyzed by CYP2B6 to about 35% of the control (Stevens et al., 1997), and S-mephenytoin at 500 μM inhibited omeprazole 5′-hydroxylation catalyzed by CYP2C19 to about 30% of the control (Chiba et al., 1993a). Incubations, except for that of orphenadrine, which was preincubated in the presence of the NADPH-generating system at 37°C for 15 min, were carried out as described above, and the reaction was initiated by the addition of a substrate.

Immunoinhibition.

The immunoinhibition of N-demethylase activity ofS-mephobarbital was examined by preincubating the human liver microsomal samples (0.2 mg/ml) with preimmune IgG or anti-CYP2C IgG in 0.1 M potassium phosphate buffer (pH 7.4) for 30 min at room temperature. S-Mephobarbital and the other reagents of the incubation medium were added, and the reaction was carried out as described above. The anti-CYP2C antibody (2 mg IgG/mg microsomal protein) used in the present study was previously verified to inhibitS-mephenytoin 4′-hydroxylation (CYP2C19) and tolbutamide hydroxylation (CYP2C9) by more than 90% (Kobayashi et al., 1997).

Statistical Analyses.

Correlations between immunoreactive CYP2B6 levels andN-demethylase activities of S-mephobarbital in 10 human liver microsomes were determined by Pearson's product-moment method.

Results

Activity in cDNA-Expressed CYPs.

Microsomes from human B-lymphoblastoid cells expressing each of 10 human CYP isoforms were examined in terms of the abilities of individual CYP proteins to catalyze the N-demethylation ofS-mephobarbital. As shown in Fig.2, of the cDNA-expressed P-450 isoforms studied (CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4), only CYP2B6 catalyzed theN-demethylation of S-mephobarbital (7.9 pmol/min/nmol CYP). The other cDNA-expressed CYP isoforms showed negligible activity (<1 pmol/min/nmol CYP).

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Figure 2

N-Demethylase activity of S-mephobarbital in microsomes from human B-lymphoblastoid cells expressing individual human CYPs.

A substrate (1 mM S-mephobarbital) was incubated at 37°C for 120 min with microsomes (0.2 mg/ml) from human B-lymphoblastoid cells expressing CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4. Each column represents the mean of duplicate experiments. ND, not detectable.

Kinetic Analyses.

Kinetic analyses were performed with cDNA-expressed CYP2B6 and microsomes prepared from two human liver specimens. TheN-demethylase activities of S-mephobarbital of cDNA-expressed CYP2B6 and human liver microsomes exhibited typical Michaelis-Menten kinetics. The kinetic parameters of cDNA-expressed CYP2B6 and two human liver microsomes (HL34 and HL38) forS-mephobarbital N-demethylation are listed in Table 1. The apparentK_m values obtained for two human liver microsomes were close to the apparent K_mvalues obtained for cDNA-expressed CYP2B6 (236 and 276 μM versus 264 μM).

Correlation Study.

As shown in Fig. 3, theN-demethylase activity of S-mephobarbital in 10 human liver microsomes at 200 μM S-mephobarbital showed a strong correlation with immunodetectable CYP2B6 levels (r = 0.920, p < .001). The relationship between the N-demethylase activity ofS-mephobarbital at 200 μM S-mephobarbital and specific activities for CYP isoforms in 10 human liver microsomes is shown in Table 2. TheN-demethylase activity of S-mephobarbital was significantly correlated with catalytic activities of coumarin 7-hydroxylase (r = 0.799, p < .01) andS-mephenytoin 4′-hydroxylase (r = 0.693,p < .05). No significant correlations were observed between N-demethylase activity of S-mephobarbital and catalytic activities of phenacetin O-deethylase (r = 0.249), diclofenac 4′-hydroxylase (r = 0.152), desipramine 2-hydroxylase (r = 0.028), chlorzoxazone 6-hydroxylase (r = 0.299), or testosterone 6β-hydroxylase (r = 0.547).

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Figure 3

Correlation between N-demethylase activity of S-mephobarbital and CYP2B6 levels in 10 human liver microsomes.

The S-mephobarbital N-demethylase activity in 10 human liver microsomes were compared with the levels of immunodetectable CYP2B6. S-Mephobarbital (200 μM) was incubated at 37°C for 120 min with 0.5 mg/ml of human liver microsomes. The relative arbitrary units were determined by the use of an imaging analyzer. The correlation coefficient (r) was calculated by the least-squares regression method.

Inhibition Study.

Chemical inhibition

The effects of coumarin, orphenadrine, and S-mephenytoin onN-demethylase activity of S-mephobarbital were studied by using human liver microsomes (Fig.4). The N-demethylase activity of S-mephobarbital was inhibited to 47 and 29% of control activity by 100 and 300 μM orphenadrine, respectively.S-Mephenytoin at concentrations of 100 and 500 μM inhibited N-demethylase activity ofS-mephobarbital to 83 and 77% of control activity, respectively. The extent of inhibition by coumarin was less than 15%, even at a concentration of 500 μM. In cDNA-expressed CYP2B6, the extent of inhibition by orphenadrine of the N-demethylase activity of S-mephobarbital was similar to that in human liver microsomes (data not shown).

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Figure 4

Effects of coumarin, orphenadrine, and S-mephenytoin on S-mephobarbital N-demethylase activity in human liver microsomes.

The concentration of S-mephobarbital used was 200 μM. Human liver microsomes (0.5 mg/ml) were incubated at 37°C for 120 min. Substrate and inhibitors were dissolved in methanol and added to the incubation mixture at a final methanol concentration of 1%. The control activity in the presence of 1% methanol was 2.5 pmol/min/mg. Each column represents the mean of duplicate experiments.

Immunoinhibition.

The results of the chemical inhibition study showed thatN-demethylase activity of S-mephobarbital was inhibited by around 20% in the presence of 100 or 500 μMS-mephenytoin. This may be because S-mephenytoin is a substrate of not only CYP2C19 (Küpfer and Preisig, 1984) but also CYP2B6 (Heyn et al., 1996), and S-mephenytoin acted as competitive inhibitor of CYP2B6. Therefore, the catalytic contribution of CYP2C19 to S-mephobarbital N-demethylation in human liver microsomes was examined by immunoinhibition study using anti-CYP2C antibody. The results indicated that anti-CYP2C antibody did not inhibit N-demethylase activity ofS-mephobarbital in human liver microsomes. TheN-demethylase activity of S-mephobarbital in the presence of anti-CYP2C IgG (mg IgG/mg microsomal protein) was slightly higher than that of preimmuno IgG (4.4 versus 4.0 pmol/mg/min, mean of duplicate determinations).

Discussion

The results of the present study suggest that CYP2B6 is a principal enzyme responsible for the N-demethylation ofS-mephobarbital in human liver microsomes. The supporting evidence can be summarized as follows: 1) cDNA-expressed CYP2B6 catalyzed S-mephobarbital N-demethylation (Fig.2); 2) the apparent K_m values of human liver microsomes for S-mephobarbitalN-demethylation were close to that of cDNA-expressed CYP2B6 (Table 1); 3) the N-demethylase activity ofS-mephobarbital in 10 human liver microsomes was strongly correlated with immunodetectable CYP2B6 levels (r = 0.920, p < .001, Fig. 3); and 4) orphenadrine at a concentration of 300 μM inhibited the N-demethylase activity of S-mephobarbital in human liver microsomes to 29% of control activity (Fig. 4).

On the other hand, the correlation study using a panel of 10 human liver microsomes indicated that the N-demethylase activity of S-mephobarbital was correlated with coumarin 7-hydroxylase activity, an activity probe for CYP2A6 (Table 2). However, Forrester et al. (1992) showed a highly significant correlation between CYP2A6 and CYP2B6 protein levels in human liver microsomes. The concordance of levels of CYP2A6 and CYP2B6 and the fact that CYP2A6 and CYP2B6 genes are found rather close to each other on human chromosome 19 (Miles et al., 1989) suggest that they might be coordinately regulated. Moreover, results of the present study showed that cDNA-expressed CYP2A6 did not catalyze theS-mephobarbital N-demethylation (Fig. 2), and the extent of inhibition by coumarin at a concentration of 500 μM was slight (Fig. 4). Therefore, the correlation ofS-mephobarbital N-demethylase activity and coumarin 7-hydroxylase activity may be due to coordinate regulation of CYP2B6 and CYP2A6 genes rather than involvement of CYP2A6 in S-mephobarbitalN-demethylation.

In the present study, the N-demethylase activity ofS-mephobarbital in a panel of human liver microsomes also showed a significant correlation with the S-mephenytoin 4′-hydroxylase activity, an activity probe for CYP2C19 (Table 2). However, cDNA-expressed CYP2C19 did not catalyzeS-mephobarbital N-demethylation (Fig. 2). In addition, anti-CYP2C did not inhibit N-demethylase activity of S-mephobarbital in human liver microsomes. Taken together, these results suggest that the contribution, if any, of CYP2C19 to S-mephobarbital N-demethylation in human liver microsomes is negligible.

The correlation study using a panel of 10 human liver microsomes indicated that the N-demethylase activity ofS-mephobarbital was not correlated with the specific activities of CYP1A2, CYP2C9, CYP2D6, CYP2E1, or CYP3A (Table 2). In agreement with the correlation data, cDNA-expressed CYP1A2, CYP2C9, CYP2D6, CYP2E1, or CYP3A4 failed to catalyze theS-mephobarbital N-demethylation (Fig. 2). These results suggest that CYP1A2, CYP2C9, CYP2D6, CYP2E1, and CYP3A4 play negligible roles in S-mephobarbitalN-demethylation in human liver microsomes.

Previously, Eadie et al. (1978) reported that patients who were pretreated with phenobarbital showed a greater clearance of mephobarbital and a rapid appearance of phenobarbital after administration of mephobarbital than did untreated patients. Phenobarbital has been shown to induce the expression of CYP2B, CYP2C, and CYP3A in rodents (Waxman and Azaroff, 1992). Although the induction of CYP2B expression is thought to be regulated at the transcription level, the molecular mechanisms regulating the gene expression by phenobarbital are still unclear. Recently, Sueyoshi et al. (1999) reported that a nuclear orphan receptor, constitutively activated receptor, regulated the enhancer activity of the phenobarbital-responsive enhancer module located in the 5′-flanking region of the human CYP2B6 gene, as a retinoid X receptor heterodimer. Their data suggest that administration of phenobarbital could induce CYP2B6 in humans as well as in rodents. Therefore, the findings of Eadie et al. (1978) are in good agreement with the present results showing that CYP2B6 mainly catalyzes N-demethylation of S-mephobarbital in human liver microsomes.

CYP2B6 is generally regarded as a minor component of the CYPs in the liver (Shimada et al., 1994) but metabolizes about 3% of drugs in clinical use (Rendic and Di Carlo, 1997). Previous studies using cDNA-expressed CYP2B6 have led to the identification of several substrates for this isoform, including nicotine (Flammang et al., 1992), 7-ethoxycoumarin (Chang et al., 1993), cyclophosphamide (Chang et al., 1993), ifosphamide (Chang et al., 1993; Granvil et al., 1999), 2-chloro-1,1-difluoroethene (Baker et al., 1995),S-mephenytoin (Heyn et al., 1996), 7-ethoxy-4-trifluoromethylcoumarin (Ekins et al., 1997), and testosterone (Ekins et al., 1998). However, the lack of a selective enzyme assay for CYP2B6 has limited investigation of hepatic microsomal CYP2B6 for metabolism of drugs and foreign compounds (Ekins et al., 1997). For example, Ko et al. (1998) showed that not only CYP2B6 but also CYP2C9 contributes to N-demethylation ofS-mephenytoin when a low concentration of the substrate is used. Therefore, N-demethylation ofS-mephobarbital may be a relatively high specific probe for CYP2B6 activity.

In conclusion, the results of the present study using in vitro techniques suggest that S-mephobarbitalN-demethylase activity appears to be mainly catalyzed by CYP2B6 in human liver microsomes.