Introduction

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IMI has been an antidepressant for the treatment of major depression for more than 30 years (Sallee and Pollock, 1990). The major metabolic pathways of IMI involve 2-hydroxylation to form 2-OH-IMI and N-demethylation to form DMI, which is further 2-hydroxylated (Rudorfer and Potter, 1987; Sallee and Pollock, 1990). In vitro and in vivo studies (Brøsen and Gram, 1988; Brøsen et al., 1986a and b; Brøsen et al., 1991) have suggested that IMI 2-hydroxylation cosegregates with the genetically determined activity of debrisoquine 4-hydroxylase, designated as CYP2D6. This isoform has shown a number of variant alleles associated with the PMs (Daly et al., 1996) and has been known to be responsible for the metabolism of more than 30 drugs, including tricyclic antidepressants (Dahl and Bertilsson, 1993).

The polymorphic S-mephenytoin 4-hydroxylation, another pharmacogenetic entity, is catalyzed by a CYP2C subfamily (Wrighton et al., 1993; Goldstein et al., 1994; de Morais et al., 1994a; Goldstein and de Morais, 1994) designated as CYP2C19, and CYP2C19m1 and m2 have been reported as the mutant genes causing the deficient isozyme activity (de Morais et al., 1994a and b; Goldstein and de Morais, 1994). This genetic polymorphism also shows a cosegregation with the oxidative metabolism of several clinically important drugs (Goldstein and de Morais, 1994). Furthermore, this pharmacogenetic entity has demonstrated a marked interethnic difference in the incidence of the PM phenotype: approximately 3 to 6% of Caucasian (Wilkinson et al., 1989, Nakamura et al., 1985) and 13 to 23% of Oriental populations (Nakamura et al., 1985, Horai et al., 1989) are PMs of S-mephenytoin 4-hydroxylation. In vivo human panel studies (Skjelbo et al., 1991; Koyama et al., 1994) have reported that IMI N-demethylation cosegregates with the CYP2C19-related pharmacogenetics. For instance, the mean N-demethylation clearance of IMI is reduced by approximately 50% in PMs of S-mephenytoin 4-hydroxylation as compared with EMs. In addition, an in vitro study with Japanese human liver microsomes (Chiba et al., 1994) has shown that there was a significant correlation between the IMI N-demethylase and the S-mephenytoin 4-hydroxylase activities, a result that supports the in vivo results. In contrast, other in vitro studies (Skjelbo and Brøsen, 1992; Lemoine et al., 1993) have suggested that CYP2C19 is not involved in IMI N-demethylation. Consequently, we carefully reappraised the experimental conditions used for the previous in vitro studies (Skjelbo and Brøsen, 1992; Lemoine et al., 1993; Chiba et al., 1994) from which the differing conclusions have been drawn in terms of the possible involvement of CYP2C19 in the metabolism of IMI. Then we became aware of the two possible causes of the conflicting results observed in these in vitro experiments: first, no in vitro study has been conducted with microsomes obtained from the EMs and PMs of S-mephenytoin 4-hydroxylation separately and compared between them. Another possible cause is differences in the substrate concentrations (e.g., therapeutically relevant in vivo or not) used for determining CYP isoforms involved in the metabolic pathways of IMI. Indeed, Yasumori et al. (1993) have shown that different CYP isoforms are involved in the metabolism of diazepam at the low and high substrate concentrations and that the involvement of human CYP2C in the N-demethylation occurs in a substrate concentration-dependent manner. Similarly, Pearce et al. (1996) have documented that different CYP isoforms are involved in the metabolism of lansoprazole with the use of different substrate concentrations.

Thus, in the present study, we intended to reappraise the principal CYP isoforms involved in the major metabolic pathways (N-demethylation and 2-hydroxylation) of IMI at a substrate concentration near the therapeutic range and at a supratherapeutic or highly toxic concentration as a counterpart using human liver microsomes and 11 cDNA-expressed human CYPs.

	Materials and Methods

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Chemicals. IMI HCl, DMI HCl and TAO were purchased from Sigma Chemical Co. (St. Louis, MO). 2-OH-IMI HCl and 2-OH-DMI oxalate were a generous gift from Ciba-Geigy (Basel, Switzerland). Mianserin was kindly provided by Organon (Oss, the Netherlands). Furafylline was obtained from Salford Ultrafine Chemicals and Research (Manchester, U.K.). Rac-mephenytoin was generously donated by Dr. Küpfer (University of Bern, Bern, Switzerland). S- and R-mephenytoin were separated on a Chiralcel OJ column (10 µm, 4.6 × 250 mm, Daisel Chemical Co. Ltd., Tokyo, Japan) according to the method of Yasumori et al. (1990). NADP, glucose-6-phosphate and glucose-6-phosphate dehydrogenase were purchased from Oriental Yeast (Tokyo, Japan). Quinidine, acetonitrile and other reagents of analytical grade were obtained from Wako Pure Chemical Industries (Osaka, Japan).

Human liver microsomes. Human liver microsomes were obtained from six patients (aged 53 to 61 years, two males and four females) who underwent partial hepatectomy for metastatic liver tumor(s) in the Division of General Surgery, International Medical Center of Japan, Tokyo, Japan, as reported previously (Chiba et al., 1993b). The use of human livers for this study was approved by the institutional ethics committee. Human liver microsomes were prepared by differential centrifugation as described by Chiba et al. (1993a). After the protein content was measured according to the method of Lowry et al. (1951), the suspended microsomal samples were aliquoted, frozen and stored at 80°C until used.

Incubation conditions. The formation of DMI and 2-OH-IMI from IMI was assessed using the incubation conditions developed in our laboratory (Chiba et al., 1994). Typically, using a 2-ml polypropylene tube, reaction mixtures containing 0.1 or 0.2 mg/ml of human liver microsomes, 0.5 mM NADP, 2.0 mM glucose-6-phosphate, 1 IU/ml of glucose-6-phosphate dehydrogenase, 4 mM MgCl₂, 0.1 mM EDTA, 100 mM potassium phosphate buffer (pH 7.4) and 0.25 to 400 µM of IMI in a total volume of 250 µl were incubated at 37°C for 30 min. The reaction was stopped by adding 100 µl of cold acetonitrile. After the termination of incubation, 10 µl of 25 µM mianserin in methanol as an internal standard and 50 µl of 100 mM potassium phosphate buffer (pH 3.0) were added to the sample. The mixture was centrifuged at 8385 × g for 10 min, and the supernatant was injected onto an HPLC apparatus as described below.

In vitro assessment of S-mephenytoin 4-hydroxylation phenotype and DMI 2-hydroxylation capacity. The R/S value for assessing the S- and R-mephenytoin 4-hydroxylase activities was determined according to the method of Yasumori et al. (1990). 4-Hydroxymephenytoin formed from S- and R-mephenytoin in the incubation mixtures was determined according to the method of Chiba et al. (1993b). The one-component kinetic parameters (K_m, V_max and V_max/K_m) of S-mephenytoin 4-hydroxylation were estimated as reported from our laboratory (Chiba et al., 1993a and b). Individual data on the R/S values and kinetic parameters observed in microsomes from the six human livers are listed in table 1. Among the six microsomal samples used in the present study, two samples (HL-32 and HL-35) were estimated as having been obtained from the putative S-mephenytoin PMs, because the R/S values were >0.7 according to the criteria (Yasumori et al., 1990) that have been validated by Chiba et al. (1993b). In addition, HL-37 was identified as a putative S-mephenytoin PM liver by its lower V_max and V_max/K_m values, which ranged between those obtained from HL-32 and from HL-35 (table 1), although the R/S value obtained from this liver was <0.7 because of the extremely low activity of R-mephenytoin 4-hydroxylation. The remaining three samples were estimated as having been obtained from the putative EMs (table 1).

View this table: [in this window] [in a new window]   TABLE 1 Characteristics of six human livers R/S was determined as the ratio of the formation rate of 4-hydroxymephenytoin with 1 mM of R-mephenytoin to that with 1 mM of S-mephenytoin. Microsomal samples with a ratio >0.7 were classified as those derived from the putative PM patients. However, HL-37 was identified as a putative PM liver by its lower Vmax and Vmax/Km values, although the R/S value obtained from this liver was <0.7.

HPLC assay for IMI metabolites and DMI 2-hydroxylation. DMI and 2-OH-IMI formed from IMI and 2-OH-DMI from DMI were assayed by a modification of the HPLC methods of Chiba et al. (1994) and Koyama et al. (1993). Briefly, the assay was carried out with the HPLC system consisting of a model EP-10 pump (Eicom, Kyoto, Japan), a model L-7200 UV detector (Hitachi Ltd., Tokyo, Japan), a model ECD-100 electrochemical detector (Eicom) equipped with a model WE-GC glassy carbon working electrode (Eicom) the applied potential of which was set at 820 mV against an Ag/AgCl electrode (Eicom), a model AS-2000 autosampler (Hitachi), a model D-2500 integrator (Hitachi) and a 4.6 × 250 mm CAPCELL PAK phenyl SG-120 column (Shiseido Co. Ltd., Tokyo, Japan). The mobile phase consisted of acetonitrile-potassium phosphate buffer (0.05 M, pH 3.0) in the proportions 26:74 (v/v) and was delivered at a flow rate of 1.0 ml/min. The column temperature was maintained at 30°C by Thermo Minder SM-05 (TAITEC, Saitama, Japan). The eluate was monitored at the wavelength of 204 nm by UV detection as mentioned above. A 75-µl or 15-µl sample was separately injected into the HPLC system with UV detection or ECD, respectively. Calibration curves were prepared from the metabolite solutions containing concentrations between 10 and 200 ng/ml. The retention times of 2-OH-IMI and DMI formed from IMI and of 2-OH-DMI formed from DMI and mianserin as the internal standard were 9.6, 22.5, 8.6 and 14.6 min, respectively. The detection limits of DMI, 2-OH-IMI and 2-OH-DMI were 4.8 ng/tube, 1.5 ng/tube and 1.5 ng/tube, respectively.

Inhibition study. The inhibitors/substrates used for the respective human P450s were as follows: furafylline for CYP1A2 (Tassaneeyakul et al., 1993), S-mephenytoin for CYP2C19 (Wrighton and Stevens, 1992), quinidine for CYP2D6 (Kobayashi, et al., 1989) and TAO for CYP3A (Watkins et al., 1985). Each of the low and high concentrations of IMI (2 and 400 µM), respectively, was incubated with one of the inhibitor/substrate probes at concentrations ranging from 0.001 to 100 µM, except for S-mephenytoin (i.e., 50-750 µM), under the incubation conditions described above. Incubation mixtures containing furafylline and TAO were preincubated in the presence of the NADPH-generating system at 37°C for 15 min, and the reactions were initiated by the addition of IMI solution. These inhibitors are mechanism-based and therefore require the NADPH-dependent complexation for inactivation (Kunze and Trager, 1993; Tassaneeyakul et al., 1993; Watkins et al., 1985). All other incubation mixtures contained IMI and S-mephenytoin or quinidine simultaneously, and the reactions were initiated by the addition of the NADPH generating system without previous incubation. Experiments were performed with microsomal preparations obtained from the human livers of two different putative PMs and three different putative EMs as listed in table 1. The microsomal sample of one putative PM liver (HL-35, table 1) was not sufficient for the inhibition study but remained available for the in vitro kinetic study.

Data analysis. The Michaelis-Menten kinetic parameters for the formation of DMI and 2-OH-IMI from IMI in microsomes obtained from the putative S-mephenytoin EM livers were estimated by fitting the data to the following equation: where V is the velocity of the formation of DMI or 2-OH-IMI, S is the concentration of IMI in the incubation mixture, K_m1 and K_m2 are the affinity constants for the high- and low-affinity components, and V_max1 and V_max2 are the maximum enzyme velocities for the high- and low-affinity components, respectively. The kinetic parameters were estimated initially by the graphical analysis of Eadie-Hoffstee plots, and the values obtained were used as the first estimate for the nonlinear least-squares regression analysis MULTI (Yamaoka et al., 1981), in which unweighted raw data were fitted to the model equation.

	Results

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Kinetic profiles of IMI metabolism and DMI 2-hydroxylation by human liver microsomes. The individual Eadie-Hoffstee plots for the formation of DMI and 2-OH-IMI from IMI with microsomes obtained from six different human livers are shown in figures 1 and 2 (A to F), respectively. The N-demethylation and 2-hydroxylation of IMI gave a biphasic relationship in microsomes from the putative EMs, except for the 2-hydroxylation in HL-36 (fig. 2F), whereas the kinetics of the two metabolic reactions showed a monophasic profile in those from the putative PMs (figs. 1 and 2, A to C). These results suggest that at least two enzymes are involved in the N-demethylation and 2-hydroxylation of IMI in three and two microsomes, respectively, of the putative EM livers, but not in those of the three putative PM livers.

View larger version (30K): [in this window] [in a new window]   Fig. 1.   Individual Eadie-Hoffstee plots for the N-demethylation of IMI by human liver microsomes obtained from six different human livers.  = data obtained from the putative PMs (panels A to C) and  = data obtained from the putative EMs of S-mephenytoin 4-hydroxylation (D to F).

View larger version (30K): [in this window] [in a new window]   Fig. 2.   Individual Eadie-Hoffstee plots for the 2-hydroxylation of IMI by human liver microsomes obtained from six different human livers. Symbols are the same as those given in fig. 1.

Kinetic analysis of metabolic profiles of IMI by human liver microsomes. Table 2 summarizes the individual and mean (± S.D.) kinetic parameters derived from the Michaelis-Menten theoretical analysis by using the two-enzyme kinetic approach (K_m1, K_m2, V_max1, V_max2, V_max1/K_m1 and V_max2/K_m2) and those derived from the one-enzyme kinetic approach (K_m, V_max and V_max/K_m) observed in microsomes from the six human livers.

Inhibition study. Shown in figure 3 are the mean inhibition study data on IMI N-demethylation using human liver microsomes obtained from two putative PMs and three putative EMs. Furafylline was a potent inhibitor of the N-demethylation in microsomes obtained from the two groups at the low concentration (fig. 3A). The IC₅₀ values gave a large variability and ranged from 0.81 to >100 µM in the five different microsomes. The mean maximum inhibition produced by furafylline on IMI N-demethylation at the low concentration was 58% and 57% in microsomes obtained from the PMs and EMs, respectively. However, furafylline produced a weaker inhibitory effect at the high (fig. 3B) than at the low concentration (fig. 3A). On the other hand, S-mephenytoin inhibited the N-demethylation by 47% in microsomes obtained from the EMs, whereas no inhibitory effect was discernible in those obtained from the PMs at the low concentration (fig. 3C). In contrast, no inhibitory effect by S-mephenytoin on the N-demethylation was seen at the high concentration in microsomes of the two phenotype groups (fig. 3D). Quinidine exhibited a weak inhibition (by about 20%) at the low concentration in the EM group and (by around 15%) at the high concentration in the two groups (fig. 3E and F), respectively. TAO produced a weak inhibition (by <17%) in microsomes from the PMs at the low concentration (fig. 3G), whereas the mean maximum inhibitory effects were about 30% and 35%, respectively, in microsomes obtained from the PMs and EMs at the high concentration (fig. 3H).

View larger version (30K): [in this window] [in a new window]   Fig. 3.   Effects of selective CYP inhibitor and/or substrate probes on the N-demethylation of IMI by human liver microsomes. Data are the mean values of experiments performed with microsomes obtained from the putative PMs (, n = 2) and EMs (, n = 3).

View larger version (30K): [in this window] [in a new window]   Fig. 4.   Effects of specific CYP inhibitor and/or substrate probes on the 2-hydroxylation of IMI by human liver microsomes. Data are the mean values of experiments performed with microsomes obtained from the putative PMs (, n = 2) and EMs (, n = 3).

Recombinant human CYP isoform study. Microsomes from yeast cell lines expressing each of 11 human CYP isoforms were examined in terms of the abilities of individual CYPs to catalyze IMI N-demethylation and 2-hydroxylation at the low (2 µM) and high concentrations (400 µM) (table 3). Among the CYP isoforms studied, a substantial catalytic activity for the N-demethylation at the low and high concentrations was observed mainly for CYP2C19 and 2C18. In addition, certain activities for the N-demethylation were detected for CYP1A2, 2D6, 3A4 and 2B6 (>10 pmol/pmol P450/10 min). On the other hand, CYP2D6 and 2C19 exhibited a potent catalytic activity for the 2-hydroxylation at the low concentration. Furthermore, CYP1A2, 2C18, 2B6 and 3A4 showed an appreciable activity for the 2-hydroxylation at the high concentration of IMI (table 3).

View larger version (25K): [in this window] [in a new window]   Fig. 5.   Representative Eadie-Hoffstee plots for the N-demethylation of IMI by microsomes obtained from yeast cells expressing cDNA of CYP1A2 (panel A), 2B6 (panel B), 2C18 (panel C), 2C19 (panel D), 2D6 (panel E) and 3A4 (panel F). Data for the remaining five CYP isoforms are not shown, because these isoforms did not show the catalytic activities.

View larger version (23K): [in this window] [in a new window]   Fig. 6.   Representative Eadie-Hoffstee plots for the 2-hydroxylation of IMI by microsomes obtained from yeast cells expressing cDNA of CYP1A2 (panel A), 2C18 (panel B), 2C19 (panel C), 2D6 (panel D) and 3A4 (panel E). Data for the remaining six CYP isoforms are not shown, because these isoforms did not show the catalytic activities.

	Discussion

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The results of our study with human liver microsomes and recombinant human CYP isoforms, taken together, revealed that CYP2C19 and 1A2 are primarily responsible for IMI N-demethylation at the therapeutically relevant concentration in vivo (2 µM) in microsomes obtained from the S-mephenytoin EM livers, whereas the extent of the contribution of CYP3A4 to this metabolic pathway appears to be minor, if any, in microsomes obtained from the EM and PM livers. However, because certain inhibition occurred for IMI N-demethylation by TAO in microsomes of the EM and PM livers at the high IMI concentration (400 µM) (fig. 3H), CYP3A4 appears to be responsible for the N-demethylation at the highly toxic concentration in vivo. In addition, IMI 2-hydroxylation is mediated mainly via CYP2D6 and partially via CYP2C19 in microsomes of the EM livers. Thus at least two CYP isoforms are involved in the two pathways of IMI in microsomes of the EM livers (i.e., CYP2C19 and 1A2 in the N-demethylation and CYP2D6 and 2C19 in the 2-hydroxylation). This contention appears to be compatible with an apparently biphasic kinetic profile (except for one case in the 2-hydroxylation) in the two metabolic reactions of IMI observed for the EM livers (figs. 1 and 2). On the other hand, in microsomes of the S-mephenytoin PM livers, IMI has to be metabolized by the non-CYP2C19 component isoform(s). In this respect, our human microsomal study suggests that CYP1A2 and 2D6 are most likely to be the isoforms involved in the N-demethylation and 2-hydroxylation, respectively, in microsomes of the PM livers. This contention is not inconsistent with a monophasic kinetic profile in the two metabolic reactions of IMI observed in the three PM livers (figs. 1 and 2). On the basis of the overall results obtained from our in vitro study, we wish to propose a scheme for the CYP isoforms involved in the two major pathways of IMI by microsomes from the two genetically determined CYP2C19-related livers (fig. 7). This scheme is proposed mainly on the basis of the human liver microsomal study data observed at a near-therapeutic IMI concentration (i.e., 2 µM).

View larger version (22K): [in this window] [in a new window]   Fig. 7.   Proposed scheme for the involvement of CYP isoforms in the two metabolic pathways of IMI in human liver microsomes of EMs and PMs of S-mephenytoin 4-hydroxylation. The major CYP isoforms are designated with boldface letters, whereas the minor ones are in parentheses. The isoenzyme activities detected only in the recombinant human CYP isoform study (e.g., CYP2B6 and 2C18) are not taken into account in this scheme. Thus the CYP isoforms proposed to be involved in each of the metabolic pathways are derived mainly from the human liver microsomal study with use of the low or near-therapeutic concentration of IMI (2 µM) and partly from the recombinant human CYP isoform study, in which the corresponding isoform activities are detectable.

As shown in figure 3C, the inhibition of IMI N-demethylation by S-mephenytoin in microsomes obtained from the EM livers was <50% at the low concentration, whereas no inhibition was observed in those from the PM livers. This finding indicates that IMI is N-demethylated partially by CYP2C19 in microsomes of the EM livers. In addition, the consistency of the mean K_m values between the high-affinity component enzyme obtained from the S-mephenytoin EM liver microsomes (table 2) and recombinant human CYP2C19 (table 4) provides evidence for the involvement of this CYP isoform in IMI N-demethylation. Accordingly, these results suggest that CYP2C19 is involved partially in the N-demethylation in the EM liver microsomes and that the non-CYP2C19 component isoform(s) might have played a dominant role in this pathway in the PM liver microsomes. Thus our in vitro results strongly support previous in vitro (Chiba et al., 1994) and in vivo studies (Skjelbo et al., 1991; Koyama et al., 1994, 1996) showing that IMI is N-demethylated partially by CYP2C19 in humans.

Furafylline, a CYP1A2 inhibitor (Kunze and Trager, 1993; Tassaneeyakul et al., 1993), exhibited a potent inhibitory effect on IMI N-demethylation by about 60% in microsomes obtained from both the EM and PM livers at the low concentration (fig. 3A), whereas quinidine showed a weak (about 20%) inhibitory effect on the N-demethylation at the low concentration in microsomes of the EM livers (fig. 3E). TAO also showed a weak (<17%) inhibitory effect on this pathway only in microsomes of the PM livers at the low IMI concentration (fig. 3G). These findings indicate that CYP1A2 is another isoform involved in IMI N-demethylation in microsomes of the EM livers and plays a major role in this pathway in those of the PM livers at a near-therapeutic concentration of IMI (fig. 7). However, the possibility cannot be excluded that CYP2D6 and 3A4 are partially involved in IMI N-demethylation by microsomes of the EM and PM livers, respectively (fig. 7). In addition, the data shown in fig. 3B suggest that CYP1A2 is responsible for the N-demethylation at the high IMI concentration (400 µM), a result that agrees with an in vitro human liver microsomal study by Lemoine et al. (1993), who used a high IMI concentration (500 µM) in their inhibition experiments.

The discrepancy with respect to the possible involvement of CYP2C19 in IMI N-demethylation in human liver microsomes between the present and previous studies (Lemoine et al., 1993; Skjelbo and Brøsen, 1992) appears to arise from the differences in the IMI concentrations used in the respective in vitro experiments. Their findings were obtained by using the high substrate concentrations (e.g., 500 and 128-500 µM IMI, respectively), whereas our study was performed at the low drug concentration (2 µM), which was about twice as close to the therapeutic plasma concentration range of IMI (i.e., 200-300 ng/ml or about 0.7-1.1 µM) (Eilers, 1995; Preskorn et al., 1993). We selected the high concentration (400 µM) as a counterpart in order to confirm the previous in vitro observations cited above. We observed that S-mephenytoin inhibited the N-demethylation by 50% at the low IMI concentration in microsomes of the EM livers (fig. 3C), whereas it elicited no inhibition at the high concentration (fig. 3D). The latter finding is in agreement with that reported by Skjelbo and Brøsen (1992). In contrast, the extent of the maximum inhibition on the N-demethylation by TAO, a CYP3A4 inhibitor (Watkins et al., 1985), was significantly (P < .01) greater at the high (33 ± 11%, fig. 3H) than at the low concentration (11 ± 11%, fig. 3G), which suggests that the contribution of CYP3A4 to the N-demethylation would increase with the increasing substrate concentrations. Thus this finding accounts for that of the previous studies (Lemoine et al., 1993; Ohmori, et al., 1993) indicating that CYP3A4 is partially involved in the N-demethylation at the higher IMI concentration (500 and 142 µM, respectively). In this respect, we wish to assert that using a substrate concentration near the therapeutic range in an in vitro study is important for extrapolating the findings to an in vivo situation regarding the search for the possible candidate CYP isoform(s) involved in the metabolic pathway(s) of a drug. Yasumori et al. (1993) and Pearce et al. (1996) have also observed seemingly paradoxical or discrepant results on drugs known to be metabolized by CYP2C19 (i.e., diazepam and lansoprazole, respectively) depending on the different substrate concentrations used for their in vitro human microsomal experiments.

Quinidine inhibited the 2-hydroxylation of IMI almost completely (fig. 4), but not the N-demethylation, at the low concentration (fig. 3), and therefore CYP2D6 is involved dominantly in IMI 2-hydroxylation at the near-therapeutic concentration (fig. 7). This supports the findings from previous in vitro human liver microsomal (Brøsen et al., 1991) and in vivo panel studies (Brøsen and Gram, 1988; Brøsen et al., 1986a and b; Skjelbo et al., 1991; Koyama et al., 1994) that IMI is 2-hydroxylated by CYP2D6. On the other hand, the mean K_m and V_max/K_m values for IMI N-demethylation obtained from recombinant human CYP2D6 were lower and higher, respectively, compared with other human CYPs except for CYP2C19 (table 3). However, the respective CYP2D6 and 2C19 contents are about 1.5% (Shimada et al., 1994) and 0.5% (Goldstein et al., 1994; personal communication with Dr. Goldstein, J.A., National Institutes of Environmental Health Sciences, Research Triangle Park, NC) relative to the total CYPs in human livers. When the V_max/K_m value derived from recombinant human CYP2D6 is adjusted by the CYP2D6 content in human liver microsomes (Shimada et al., 1994), the value is found to be 0.8 times equivalent to that derived from recombinant CYP2C19. Nevertheless, the mean K_m and V_max/K_m values for IMI 2-hydroxylation obtained from CYP2D6 (table 3) were 22 times smaller and 40 times greater, respectively, than those of the N-demethylation (table 4), which suggests that CYP2D6 may have a marked regio-selectivity for the metabolism of IMI. Thus the contribution of CYP2D6 to the N-demethylation is considered to be much less important than its contribution to the 2-hydroxylation.

No inhibitor/substrate probes except quinidine substantially influenced IMI 2-hydroxylation in microsomes obtained from the EM and PM livers (fig. 4). The consistency of the mean K_m values for the 2-hydroxylation between the high-affinity component enzyme in human liver microsomes (K_m1 from the EM and K_m from the PM livers, table 2) and recombinant human CYP2D6 (table 4) further confirms that CYP2D6 plays a major role in IMI 2-hydroxylation. On the other hand, the mean K_m value for the 2-hydroxylation for the low-affinity component (K_m2 in the EM and K_m in the PM livers, table 2) approximated that of recombinant human CYP2C19 (table 4). In addition, the mean V_max/K_m value obtained from recombinant human CYP2C19 was 60 times or 95 times greater, respectively, than that from the CYP1A2 or 3A4 (table 4), which suggests that the non-CYP2D6 component isoform involved in IMI 2-hydroxylation in microsomes of the EM livers appears to be CYP2C19 (fig. 7). However, the contribution of CYP2D6 to the 2-hydroxylation would be much greater than that of CYP2C19 and should account for at least 90% of this metabolic pathway, because the mean V_max/K_m value obtained from CYP2D6 was 20 times greater than that from CYP2C19 (table 4). Moreover, the one-enzyme kinetic profile of IMI 2-hydroxylation (fig. 2F) obtained from HL-36 (an S-mephenytoin EM liver), which showed the greatest V_max and V_max/K_m values for DMI 2-hydroxylation [mediated via CYP2D6 (Spina et al., 1984)] among the six livers (table 1), may suggest the more dominant contribution of CYP2D6 to the 2-OH-IMI formation compared with other S-mephenytoin EM livers (HL-33 and HL-34) that showed a two enzyme-kinetic profile of IMI 2-hydroxylation (fig. 2D and E). This suggestion appears to be supported, because the V_max/K_m ratio of DMI 2-hydroxylation (CYP2D6) to S-mephenytoin 4-hydroxylation (CYP2C19) obtained from HL-36 (the ratio = 10.4) was much greater than those from the other two livers (the ratios = 2.4 and 3.7 in HL-33 and HL-34, respectively, as calculated from the data in table 1). Therefore, it would be difficult to discern a biphasic behavior in the Eadie-Hoffstee plots in the HL-36 microsomes, even if it might have occurred via a minor contribution of CYP2C19 to IMI 2-hydroxylation.

We observed an atypical kinetic profile for the N-demethylation by CYP3A4 and 2C18 and for the 2-hydroxylation by CYP2C18 (figs. 5 and 6), which suggests that these metabolic reactions may be related to a positive cooperativity in the substrate activation as discussed below. By reconciling the Akaike's information criterion (Akaike, 1974), we found that these data were better fitted to the Hill than to the Michaelis-Menten equation. The former equation reflects a setting where binding sites can cooperate with each other, and the coefficient N represents the degree of cooperativity (thus N should predict the number of binding sites per enzyme molecule). There is evidence for the binding of two substrate molecules in the CYP3A4 active sites (Shou et al., 1994). Furthermore, CYP3A4 exhibits a positive cooperativity for diazepam 4-hydroxylation and N-demethylation (Andersson et al., 1994) as well as for amitriptyline N-demethylation (Schmider et al., 1995). Accordingly, our finding (fig. 5F) suggests that the CYP3A4-catalyzed IMI N-demethylation is activated by IMI.

Incubations with cDNA-expressed CYP2C18 revealed its relatively high catalytic activity toward IMI (table 3). As indicated by the atypical Eadie-Hoffstee plots for IMI N-demethylation (fig. 5C) and 2-hydroxylation (fig. 6B), the data were also fitted to the Hill equation. The mean N value for CYP2C18 was equivalent to that for CYP3A4 (table 5), which indicates that CYP2C18 may function as an allosteric enzyme in the metabolism of IMI. The S-mephenytoin 4-hydroxylation mediated by CYP2C19 in human liver microsomes is known to be slightly dependent on CYP2C18 (Goldstein and de Morais, 1994). Thus the inhibitory effect on the N-demethylation by S-mephenytoin in microsomes of the EM livers (fig. 3) might be attributable to a competitive inhibition at CYP2C19 and 2C18, although the present study revealed nothing about the extent of the contribution of CYP2C18 to the N-demethylation relative to CYP2C19. However, CYP2C18 cannot be detected in the majority of human liver microsomes using its antibody, and its content, even if it exists, is much lower than that of CYP2C19 (personal communication with Dr. J.A. Goldstein). Thus CYP2C18 would play a minor role, if any, compared with CYP2C19 in the metabolism of IMI.

In conclusion, our study with human liver microsomes and recombinant human CYP isoforms indicates that IMI N-demethylation is mediated mainly by CYP2C19 and CYP1A2 and that 2-hydroxylation exclusively by CYP2D6 and partially by CYP2C19 at the near-therapeutic IMI concentration in microsomes of the S-mephenytoin EM livers (fig. 7). These two major metabolic reactions of IMI appear to be catalyzed mainly by CYP1A2 (partially by CYP3A4) and CYP2D6, respectively, in microsomes of the S-mephenytoin PM livers (fig. 7). These results suggest the importance of an appropriate selection of the substrate or drug concentration, which may be attained as a near-therapeutic level after the usual clinical dose(s) of a drug, in order to assess what CYP isoform(s) are involved in the metabolism of that drug in humans.