Introduction

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Mephobarbital has been used as an anticonvulsant since 1932. As shown in Fig. 1, this drug is metabolized by N-demethylation to phenobarbital and by 4-hydroxylation to 4-hydroxymephobarbital, which undergoes further glucuronidation (Butler et al., 1952; Hooper et al., 1981). Mephobarbital is a chiral compound that is commercially available as a racemate of R- and S-mephobarbital. Earlier studies indicated that the pharmacokinetics and metabolism of mephobarbital were extremely different between the enantiomers. After a single oral dose of racemic mephobarbital, the oral clearance of R-mephobarbital was much greater than that of S-mephobarbital in six male volunteers (Lim and Hooper, 1989). This stereoselective disposition of mephobarbital has also been confirmed in subjects of different ages and genders (Hooper and Qing, 1990). In the former study, approximately 50% of R-mephobarbital was recovered in urine as R-4-hydroxymephobarbital, whereas only 7% of S-mephobarbital was converted to the corresponding hydroxy metabolite (Lim and Hooper, 1989).

View larger version (15K): [in this window] [in a new window]   Fig. 1.   Metabolic pathways of mephobarbital in humans.

Küpfer and Branch (1985) reported that the urinary recovery of 4-hydroxymephobarbital after administration of mephobarbital was negligible in poor metabolizers (PM²) of S-mephenytoin. These authors also measured the urinary recovery of 4-hydroxymephobarbital after administration of R- or S-mephobarbital to an extensive metabolizer (EM) of S-mephenytoin and showed that 4-hydroxymephobarbital was detected in urine when R-mephobarbital, but not S-mephobarbital, was administered. These findings suggest that 4-hydroxymephobarbital had exclusively been derived from R-mephobarbital and that its formation from R-mephobarbital cosegregates with the genetically determined activity of S-mephenytoin 4'-hydroxylase, designated as cytochrome P450 2C19 (CYP2C19).

However, to our knowledge, no in vitro data on the hydroxylation of mephobarbital enantiomers have been published. The purpose of this study was to clarify the stereoselective hydroxylation of mephobarbital in relation to S-mephenytoin 4'-hydroxylation in human liver microsomes and cDNA-expressed CYP isoforms.

	Materials and Methods

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Chemicals. Racemic mephobarbital was supplied by Yoshitomi Pharmaceutical Co. (Osaka, Japan), and phenobarbital was supplied by Dainippon Pharmaceutical Co. (Osaka, Japan). 4-Hydroxyphenobarbital was purchased from Aldrich Japan (Tokyo, Japan), and cyclobarbital was purchased from Tokyo Chemical Industry Co. (Tokyo, Japan). R- and S-mephobarbital were separated from the racemic mixture using an HPLC system equipped with a Chiralcel CA-1 column (20 × 250 mm; Daisel Chemical Co., Tokyo, Japan). Ethanol was eluted at flow rate of 4.5 ml/min. The eluate was monitored at a wavelength of 280 nm. The column temperature was maintained at 40°C. 4-Hydroxymephobarbital was prepared from 4-hydroxyphenobarbital via N-methylation using dimethyl sulfate as described by Hiers and Hager (1961). Racemic mephenytoin and 4'-hydroxymephenytoin were kindly donated by Dr. Küpfer (University of Berne, Berne, Switzerland). S- and R-mephenytoin were separated from the racemic mixture of mephenytoin by a Chiralcel OJ column (10 µm, 4.6 × 250 mm; Daisel Chemical Co.) as reported by Yasumori et al. (1990). NADP⁺ and glucose 6-phosphate were purchased from Oriental Yeast Co. (Tokyo, Japan). Glucose-6-phosphate dehydrogenase was purchased from Boehringer Mannheim GmbH (Mannheim, Germany). HPLC-grade acetonitrile and other reagents of analytical grade were purchased from Wako Pure Chemical Industries (Osaka, Japan).

Enzymes. Nine samples of human liver microsomes were obtained from Japanese patients undergoing partial hepatectomy for treatment of metastatic liver tumors at the Division of Surgery, International Medical Center of Japan (Tokyo, Japan) and were prepared as reported previously (Chiba et al., 1993). Among the nine microsomal samples used in the present study, two samples were considered to be derived from the CYP2C19-related PM patients, since the R/S ratio for mephenytoin 4'-hydroxylation (i.e., an in vitro phenotyping test) was greater than 0.7 (Yasumori et al., 1990; Chiba et al., 1993). This was subsequently confirmed by the genotyping assessment for two mutated alleles, CYP2C19*2 and CYP2C19*3, which are the most frequently seen CYP2C19 alleles in the Japanese population (Kubota et al. 1996). Genotyping procedures for detection of the CYP2C19*2 and CYP2C19*3 alleles were performed by a polymerase chain reaction-restriction fragment length polymorphism method as described by de Morais et al. (1994a,b) with minor modifications (Kubota et al., 1996). The characteristics of the nine human livers are shown in Table 1.

Assay with Human Liver Microsomes. The primary incubation medium contained 25 or 50 µg of microsomal protein, 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 R-mephobarbital or S-mephobarbital, in a final volume of 250 µl. The mixture was incubated at 37°C for 60 min. After the reaction was stopped by the addition of 100 µl of cold acetonitrile, 50 µl of cyclobarbital (1.25 µg/ml in methanol) was added to the samples as an internal standard. The mixture was centrifuged at 10,000g for 5 min, and 100 µl of the supernatant was analyzed under HPLC conditions as described below.

HPLC Conditions. The determination of 4-hydroxymephobarbital was carried out using a Hitachi HPLC system (Tokyo, Japan) consisting of an L-6000 pump, an L-4000 UV detector, an AS-2000 autosampler, a D-2500 integrator, and a CAPCELL PAK C₁₈ UG120 column (5 µm, 4.6 × 250 mm; Shiseido, Tokyo, Japan). The mobile phase consisted of 50 mM potassium phosphate buffer (pH 5.0) and acetonitrile at the ratio of 75:25 (v/v) and was 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. Under these chromatographic conditions, 4-hydroxymephobarbital and cyclobarbital were eluted at 9.3 and 14.3 min, respectively. The detection limit of 4-hydroxymephobarbital was 2 pmol in an incubation mixture of 250 µl. 4-Hydroxymephobarbital was quantified by comparison with the standard curves, by using the peak-height ratio method. Intra-assay (n = 6) coefficients of variation were less than 7%.

Correlation Studies. The 4-hydroxylase activities of R- and S-mephobarbital were compared with the S-mephenytoin 4'-hydroxylase activity using microsomes obtained from nine human livers. R- and S-mephobarbital were used at a concentration of 100 µM. Assays were performed in duplicate on the same day, with the same set of microsomal preparations. S-Mephenytoin (100 µM) was incubated with 0.2 mg/ml microsomal protein for 60 min. Determination of 4'-hydroxymephenytoin was carried out as reported previously (Chiba et al., 1993).

Immunoinhibition Studies. The immunoinhibition of 4-hydroxylase activity of R-mephobarbital was examined by preincubating the human liver microsomal samples (25 µg of human liver microsomes) with various amounts (0-2 mg of IgG/mg of microsomal protein) of preimmune IgG or anti-CYP2C IgG in 0.1 M potassium phosphate buffer (pH 7.4) for 30 min at room temperature. R-Mephobarbital (100 µM) and the other reagents of the incubation medium were added, and the reaction was carried out as described above. Anti-CYP2C antibody used in the present study inhibited S-mephenytoin 4'-hydroxylase activity (CYP2C19) and tolbutamide hydroxylase activity (CYP2C9) by more than 90%, whereas it did not inhibit testosterone 6-hydroxylase activity (CYP3A) in human liver microsomes (Kobayashi et al., 1997).

Chemical Inhibition Studies. The inhibition of R-mephobarbital 4-hydroxylase activity by S-mephenytoin was studied at the substrate concentrations of 50, 100, and 200 µM in the presence of five concentrations of S-mephenytoin from 0 to 200 µM. The inhibition of S-mephenytoin 4'-hydroxylase activity by R-mephobarbital was studied at the substrate concentrations of 50, 100, and 200 µM in the presence of four concentrations of R-mephobarbital from 0 to 200 µM. Inhibition patterns were determined by a visual inspection of the double reciprocal plots of S-mephenytoin concentration versus the velocity of the reaction. Apparent K_m of each concentration of the putative inhibitors was estimated by a linear regression analysis using unweighted raw data following the Michaelis-Menten kinetics approach. The K_i (inhibition constant) values were determined by an unweighted linear regression analysis using an equation consistent with the competitive inhibition:
where K_m is the substrate concentration at which the reaction velocity is half of the maximum velocity in the absence of an inhibitor, K_{m app} is apparent K_m in the presence of an inhibitor, I is the inhibitor concentration, and K_i is an inhibition constant.

Kinetic Studies. The 4-hydroxylase activities of R-mephobarbital were determined at substrate concentrations ranging from 40 to 300 µM. All reactions were performed within a linear range with respect to protein concentration and incubation time (i.e., 0.2 mg/ml microsomal protein and 60-min incubation time). The kinetics parameters (K_m and V_max) were estimated by graphic analysis of Eadie-Hofstee plots. The values were subsequently used as initial estimates for nonlinear least-squares regression analysis. The intrinsic clearance values were calculated as V_max/K_m.

Assay with cDNA-Expressed CYPs. Microsomes from human B-lymphoblastoid cells expressing human CYP1A2, CYP2A6, CYP2B6, 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 4-hydroxylation of R- or S-mephobarbital, each of the cDNA-expressed CYPs (0.5 mg/ml of protein concentration) described above was incubated with R- or S-mephobarbital (each at 100 µM) for 2 h at 37°C, according to the procedure recommended by the supplier.

	Results

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4-Hydroxylase Activities of R- and S-Mephobarbital in Human Liver Microsomes. The 4-hydroxylase activities of R- and S-mephobarbital in nine human liver microsomes, including the two CYP2C19-related PM microsomes, are shown in Fig. 2. The 4-hydroxylase activity of R-mephobarbital in the nine human liver microsomes was 3 to 20 times higher than that of S-enantiomer. To assess the stereoselectivity for the 4-hydroxylase activity of mephobarbital, the R/S ratio was determined as the ratio of the formation rate of 4-hydroxymephobarbital from R-mephobarbital to that from S-mephobarbital. The mean (±S.D.) R/S ratio in the EM microsomes was 13.5 ± 5.2, which was 2 to 4 times higher than in the two PM microsomes (3.6 and 6.1).

View larger version (17K): [in this window] [in a new window]   Fig. 2.   4-Hydroxylase activities of R- and S-mephobarbital in human liver microsomes. Substrates (R- and S-mephobarbital) at 100 µM were incubated at 37°C for 60 min with 0.2 mg/ml of human liver microsomes. Each of the columns represents the mean of duplicate experiments. , R-enantiomer; , S-enantiomer.

Correlation Studies. R-Mephobarbital 4-hydroxylase activity in the nine human liver microsomes showed a strong correlation (r = 0.985, p < 0.001) with S-mephenytoin 4'-hydroxylase activity (Fig. 3). However, S-mephobarbital 4-hydroxylase activity did not reach significant correlation with S-mephenytoin 4'-hydroxylase activity (r = 0.655, p > 0.05).

View larger version (19K): [in this window] [in a new window]   Fig. 3.   Correlation of 4-hydroxylase activities of R- and S-mephobarbital with S-mephenytoin 4'-hydroxylase activity in nine human liver microsomes. Substrates at 100 µM were incubated at 37°C for 60 min with 0.2 mg/ml of human liver microsomes. Each of the data points represents the mean of duplicate experiments. The correlation coefficients (r) were calculated by the least-squares regression method. All microsomal samples listed in Table 1 were used in this study.

Immunoinhibition Studies. Figure 4 shows the inhibition of the 4-hydroxylation of R-mephobarbital by a polyclonal antibody raised against CYP2C. In the EM microsomes (HL-7), the addition of the antibody reduced the 4-hydroxylation activity of R-mephobarbital by 85% of the control at a concentration where more than 90% of the 4'-hydroxylation activity of S-mephenytoin was inhibited.

View larger version (22K): [in this window] [in a new window]   Fig. 4.   Immunoinhibition of 4-hydroxylation of R-mephobarbital in human liver microsomes. Human liver microsomes (25 µg of microsomal protein, HL-7) were preincubated with anti-CYP2C IgG () or preimmune IgG () for 30 min at room temperature before incubation with 100 µM R-mephobarbital. The control activity in the absence of antibodies was 0.75 nmol/mg/h. Each of the data points represents the mean of duplicate experiments.

Mutual Inhibition Studies. The 4'-hydroxylation of S-mephenytoin in the EM microsomes (HL-7) was competitively inhibited by R-mephobarbital with a K_i value of 34 µM, while the 4-hydroxylation of R-mephobarbital was competitively inhibited by S-mephenytoin with a K_i value of 103 µM.

Kinetics Analyses of R-Mephobarbital 4-Hydroxylase Activity. Typical Eadie-Hofstee plots for R-mephobarbital 4-hydroxylase activity in the EM and PM microsomes of CYP2C19 are shown in Fig. 5. The plots were linear in both samples, indicating that each of the reactions occurred in a simple Michaelis-Menten kinetics fashion. However, the slopes of the 4-hydroxylation markedly differed between the EM and PM microsomes. The Michaelis-Menten kinetics parameters derived from the one-enzyme kinetics approach in the EM (HL-5, -6, -7, and -23) and PM microsomes (HL-8 and -22) of CYP2C19 are listed in Table 2. The apparent K_m values for the 4-hydroxylation were higher in the PM microsomes than in the EM microsomes (457 and 273 µM versus 65 ± 21 µM), while no difference was found between the EM and PM microsomes in V_max values, resulting in the observation that the intrinsic clearance values were 3 to 9 times lower in the PM microsomes than in the EM microsomes (2.3 and 5.3 versus 20.6 ± 5.4 µl/mg/h).

View larger version (17K): [in this window] [in a new window]   Fig. 5.   Representative Eadie-Hofstee plots for R-mephobarbital 4-hydroxylase activity in human liver microsomes. Microsomes were prepared from the liver of an EM (, HL-5) and a PM (, HL-8) of CYP2C19. The following Michaelis-Menten kinetics parameters were calculated from these data: for 4-hydroxylation, Km = 55 and 457 µM and Vmax = 1.5 and 1.1 nmol/h/mg of protein in the EM and PM microsomes, respectively. V, velocity of the metabolite's formation; S, substrate concentration.

Activity in cDNA-Expressed CYPs. Microsomes from human B-lymphoblastoid cells expressing each of the seven human CYP isoforms were examined in terms of the ability of their CYP proteins to catalyze the 4-hydroxylation of R- or S-mephobarbital. The formation of hydroxymephobarbital from R-mephobarbital was catalyzed by only CYP2C19 (1413 pmol/mg/2 h). For the formation of hydroxymephobarbital from S-mephobarbital, all cDNA-expressed CYPs screened showed a negligible activity (< 16 pmol/mg/2 h).

	Discussion

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The pharmacokinetics and metabolism of mephobarbital are recognized to be stereoselective. Küpfer and Branch (1985) reported that 33% of hydroxymephobarbital was recovered in urine after administration of R-mephobarbital, whereas the urinary excretion was negligible after administration of S-mephobarbital. After a single-dose administration of racemic mephobarbital, the oral clearance of R-mephobarbital was much greater than that of S-mephobarbital in six adult male volunteers (Lim and Hooper, 1989). R-Mephobarbital was extensively hydroxylated, and about 50% of R-mephobarbital was recovered in urine as R-hydroxymephobarbital, whereas the elimination of S-mephobarbital was extremely slow, and only 7% of S-mephobarbital was converted to S-hydroxymephobarbital.

In this study using human liver microsomes, the mean 4-hydroxylase activity of R-enantiomer was 3 to 20 times higher than that of S-enantiomer in human liver microsomes (Fig. 2). In the EM microsomes, the mean R/S ratio was 13.5, suggesting that the 4-hydroxylation of mephobarbital was preferential for R-enantiomer. On the other hand, the 4-hydroxylase activities of R-mephobarbital in the two PM microsomes were much lower than those in the EM microsomes (Fig. 2), and the R/S ratios in the two PM microsomes (i.e., 3.6 and 6.1) were also lower than the mean R/S ratio in the EM microsomes. This result suggests that the 4-hydroxylation of mephobarbital is less stereoselective in the PM microsomes. These findings obtained from the present in vitro study are in good agreement with the in vivo observation reported by Küpfer and Branch (1985).

Since the current in vitro and previous in vivo studies (Küpfer and Branch, 1985; Lim and Hooper, 1989) suggest that the stereoselective metabolism of mephobarbital is mainly attributable to the preferential hydroxylation of R-mephobarbital, we investigated which CYP isoform(s) is involved in the 4-hydroxylation of R-mephobarbital in human liver microsomes. The following results were obtained. First, a significant correlation existed between the activity of R-mephobarbital 4-hydroxylation and that of S-mephenytoin 4'-hydroxylation in the nine human liver microsomes (Fig. 3). Second, the R-mephobarbital 4-hydroxylation was inhibited almost completely by the addition of anti-CYP2C antibody (Fig. 4). Third, there was mutual competitive inhibition of R-mephobarbital 4-hydroxylation and S-mephenytoin 4'-hydroxylation. Fourth, the EM microsomes had a greater mean intrinsic clearance value for R-mephobarbital 4-hydroxylation compared with the respective intrinsic clearance values of the two PM microsomes (Table 2). Fifth, only cDNA-expressed CYP2C19 catalyzed R-mephobarbital 4-hydroxylation, and the other cDNA-expressed CYP isoforms screened showed a negligible activity. These data clearly indicated that CYP2C19 is mainly responsible for the 4-hydroxylation of R-mephobarbital in human liver microsomes.

On the other hand, the 4-hydroxylase activity of S-mephobarbital was not substantially discernible in any of the cDNA-expressed CYPs examined. Therefore, S-mephobarbital is considered to be barely 4-hydroxylated in human liver microsomes, being consistent with an in vivo observation that the excretion of hydroxymephobarbital into urine was negligible in an EM after the administration of S-mephobarbital (Küpfer and Branch, 1985). Previous in vivo studies suggested that most of the circulating phenobarbital seemed to be derived from S-mephobarbital (Küpfer and Branch, 1985; Lim and Hooper, 1989). In addition, our in vitro data indicated that S-mephobarbital was mainly N-demethylated by CYP2B6 (Kobayashi et al., 1999). Taken together, a main metabolic route of S-mephobarbital in vivo and in vitro appears to be the N-demethylation, but not 4-hydroxylation.

The 4'-hydroxylation of mephenytoin by CYP2C19 is known to be stereospecific for S-enantiomer (Goldstein et al., 1994). Our observation that R-mephobarbital is preferentially 4-hydroxylated by CYP2C19 as similar to the 4'-hydroxylation of S-mephenytoin requires an assumptive discussion. The preferential hydroxylation of R-mephobarbital and S-mephenytoin by CYP2C19 may be explained as follows. When the ureido groups of S-mephenytoin and R-mephobarbital are defined as being located in the horizontal position in a three-dimensional model, the 4-position of the phenyl ring (i.e., oxidized site) is located under the ureido surface. CYP2C19 may recognize the carbonyl moiety and phenyl ring of S-mephenytoin and R-mephobarbital precisely in the substrate binding site of CYP2C19. This assumptive explanation appears to be consistent with the proposition by Yasumori et al. (1999).

The present study suggested that R-mephobarbital was extensively 4-hydroxylated via CYP2C19 in human liver microsomes. This finding is consistent with the in vivo observation that the extensive 4-hydroxylation of R-mephobarbital resulted in rapid elimination of this enantiomer in the EM of CYP2C19 (Lim and Hooper, 1989; Hooper and Qing, 1990). At least in the EM of CYP2C19, 4-hydroxylation of mephobarbital seems to be important in the overall metabolism of this drug. In contrast, the PM of CYP2C19 is deficient in R-mephobarbital 4-hydroxylation. In PM of CYP2C19, R-mephobarbital would be anticipated to exhibit a low clearance similar to that of S-mephobarbital. Mephobarbital possesses anticonvulsant property in its own right (Craig and Shideman, 1971), although it is frequently regarded as a prodrug for phenobarbital. Therefore, an exaggerated reaction to this drug might be caused in the PM of CYP2C19.

In closing, to our knowledge, this is the first in vitro study on the hydroxylation of mephobarbital enantiomers using human liver microsomes and cDNA-expressed CYPs, indicating that 4-hydroxylation of mephobarbital more dominantly occur for R-enantiomer than for S-enantiomer and that the 4-hydroxylation of R-mephobarbital is primarily catalyzed by CYP2C19. Because the 4-hydroxylation of R-mephobarbital is a CYP2C19-specific reaction as observed for S-mephenytoin 4'-hydroxylation, we are tempted to propose that R-mephobarbital is an alternative phenotyping probe for assessing CYP2C19 activity in humans.