Abstract
Studies to assess the enzyme kinetic behavior and to identify the cytochrome P450 (CYP) isoform(s) involved in the major metabolic pathway (N-demethylation) for citalopram (CIT), a selective serotonin reuptake inhibitor, were performed using human liver microsomes and cDNA-expressed human cytochrome P450 isoforms. TheN-demethylation activities showed significant correlations with the α- and 4-hydroxylation activities of triazolam (r s = 0.818 and 0.851, respectively; P < .01) in 10 different human liver microsomes. Anti-CYP3A antibodies and ketoconazole strongly inhibited CIT N-demethylation. In addition, there was a significant correlation between CITN-demethylation and (S)-mephenytoin 4′-hydroxylation (r s = 0.773, P < .05), although little inhibition was observed in the presence of anti-CYP2C antibodies or (S)-mephenytoin. cDNA-expressed CYP3A4 and CYP2C19 catalyzed CIT N-demethylation, whereas no appreciable activities were observed for CYP1A2, CYP2A6, CYP2B6, CYP2C9, CYP2D6 and CYP2E1. The percentage contributions of CYP3A4 and CYP2C19 to the overall N-demethylation of CIT in human liver microsomes were estimated using a relative activity factor; respective values of 70% and 7% were calculated for microsomes obtained from livers from putative extensive metabolizers for (S)-mephenytoin 4′-hydroxylation. These results suggest that CYP3A4 is the major isoenzyme and CYP2C19 is the minor form involved in the major metabolic pathway for CIT in human liver microsomes.
CIT is a new antidepressant of the selective serotonin reuptake inhibitor class (Hyttel, 1982). This drug is metabolized by N-demethylation to DCIT and didesmethylcitalopram, by deamination and further oxidation to a propionic acid derivative and by N-oxidation to CITN-oxide (Oyehaug and Ostensen, 1984).N-Demethylation is the major metabolic pathway of CIT in humans (Oyehaug and Ostensen, 1984). The N-demethylated metabolites DCIT and didesmethylcitalopram are considered to be less potent than the parent compound as serotonin reuptake inhibitors (Hyttel, 1982).
(S)-Mephenytoin 4′-hydroxylation shows a genetically determined polymorphism (Brøsen, 1990; Küpfer and Preisig, 1984;Wilkinson et al., 1989), and there are marked interethnic differences in the incidence of the PM phenotype; approximately 3 to 6% of Caucasian populations (Alván et al., 1990;Brøsen, 1990; Jacqz et al., 1988; Küpfer and Preisig, 1984; Wedlund et al., 1985; Wilkinson et al., 1989) and 13 to 23% of Oriental populations (Horai et al., 1989; Sohn et al., 1992) are PMs of (S)-mephenytoin 4′-hydroxylation. The isoenzyme responsible for (S)-mephenytoin 4′-hydroxylation has been shown to be CYP2C19 (Goldstein et al., 1994; Wrighton et al., 1993), and two mutations (m1 and m2) in theCYP2C19 gene have been described in Japanese PM subjects (de Morais et al., 1994a,b). The metabolism of several clinically important drugs used in psychoneuropharmacological treatment has been demonstrated to cosegregate with this genetically determined hydroxylation polymorphism (Dahl and Bertilsson, 1993; Spina and Capriti, 1994).
Sindrup et al. (1993) first reported in their human panel study that the metabolism of racemic CIT is at least partially under the pharmacogenetic control of (S)-mephenytoin 4′-hydroxylase. In addition, we recently reported that racemic CIT competitively inhibits (S)-mephenytoin 4′-hydroxylation in human liver microsomes, and we suggested that several selective serotonin reuptake inhibitors are substrates of CYP2C19 (Kobayashiet al., 1995). However, little information is available regarding the metabolism of CIT itself in human liver microsomes. In the present study, we investigated the N-demethylation of CIT in human liver microsomes and cDNA-expressed human P450s, to identify the principal isoform of P450s involved in this major metabolic pathway of CIT.
Materials and Methods
Drugs and chemicals.
Racemic CIT hydrobromide and DCIT hydrochloride were kindly supplied by Lundbeck (Copenhagen-Valby, Denmark). Racemic mephenytoin and 4′-hydroxymephenytoin were kind gifts from Dr. Küpfer (University of Berne, Berne, Switzerland). (S)- and (R)-Mephenytoin were separated from the racemic mixture of mephenytoin with a Chiralcel OJ column (10 μm, 4.6 × 250 mm; Daicel Chemical Co., Tokyo, Japan), as reported byYasumori et al. (1990). Triazolam and its metabolites (α- and 4-hydroxytriazolam) were supplied by Nihon Upjohn Co. (Tokyo, Japan). 17α-Ethinylestradiol and ketoconazole were purchased from Sigma Chemical Co. (St. Louis, MO), and cyclobarbital was obtained from Tokyo Kasei Kogyo Co. (Tokyo, Japan). NADP+ and glucose-6-phosphate were purchased from Oriental Yeast Co. (Tokyo, Japan). Glucose-6-phosphate dehydrogenase was obtained from Boehringer Mannheim GmbH (Mannheim, Germany). Quinidine, acetonitrile and other reagents of analytical grade were purchased from Wako Pure Chemical Industries (Osaka, Japan).
Human liver microsomes.
Human liver samples (n = 14) were obtained, as excess material removed during surgery on the liver, from Japanese patients who underwent partial hepatectomy at the Department of General Surgery, International Medical Center of Japan (Tokyo, Japan), as reported from our laboratory (Chiba et al., 1993; Kobayashi et al., 1995). All surgical procedures were performed for the removal of metastatic tumor(s) from the liver. The use of human liver tissue for this study was approved by the Institutional Ethics Committee of the International Medical Center of Japan. Less than 5 min passed between removal of the liver tissue and collection and freezing of samples in liquid nitrogen. The liver parenchymas of the non-tumor-bearing parts used in this study were shown later to be histopathologically normal in all cases. Liver samples obtained from patients with acute or chronic hepatitis, those with cirrhosis or those taking medications known to induce or inhibit the hepatic monooxygenase activity were not included in this study.
Microsomes were prepared by differential centrifugation, and the 105,000 × g pellet was washed and resuspended in 50 mM sodium phosphate buffer (pH 7.4) containing 0.1 mM EDTA. After determination of protein concentration (Lowry et al., 1951), the suspended microsomes were divided into aliquots, frozen and kept at −80°C until used.
Among the 14 microsomal samples used in the present study, four samples were estimated as having been obtained from putative PMs of (S)-mephenytoin, because the R/Sratios for mephenytoin 4′-hydroxylation were >0.7 (Chiba et al., 1993; Yasumori et al., 1990). The characteristics of the 14 human livers and which livers were used for which experiments are listed in table 1.
Characteristics of the 14 human livers
Assay with human liver microsomes.
The basic incubation medium contained 0.1 or 0.2 mg/ml microsomes, 0.5 mM NADP+, 2.0 mM glucose-6-phosphate, 1 IU/ml glucose-6-phosphate dehydrogenase, 4 mM MgCl2, 0.1 mM EDTA, 100 mM potassium phosphate buffer (pH 7.4) and 1 to 500 μM CIT, 100 μM (S)-mephenytoin or 25 μM triazolam, in a final volume of 250 μl. The mixture was incubated at 37°C for 30 min for CIT, 60 min for (S)-mephenytoin and 15 min for triazolam. All reactions were performed in the linear range with respect to protein concentration and incubation time. After the reaction was stopped by 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 for assaying DCIT and 4′-hydroxymephenytoin. To assay the two metabolites of triazolam (i.e., α-hydroxytriazolam and 4-hydroxytriazolam), 50 μl of lorazepam (2.5 μg/ml in methanol) was added as an internal standard. The mixture was centrifuged at 10,000 × g for 5 min, and 50 or 100 μl of supernatant was injected into a HPLC system as described below.
HPLC conditions.
The determination of DCIT was carried out by a modification of an HPLC method reported previously (Chiba et al., 1993). Briefly, the HPLC system consisted of a model L-6000 pump (Hitachi, Tokyo, Japan), a model L-4000 UV detector (Hitachi), a model AS-2000 autosampler (Hitachi), a model D-2500 integrator (Hitachi) and a 4.6 × 250 mm CAPCELL PAK C18 UG120 column (Shiseido Co., Tokyo, Japan). The mobile phase consisted of 0.05 M potassium dihydrogen phosphate and acetonitrile at a ratio of 70:30 (v/v) and was delivered at a flow rate of 0.8 ml/min. The eluate was monitored at a wavelength of 205 nm. The column temperature was maintained at 30°C. Didesmethylcitalopram, the N-demethyl derivative of DCIT, and CIT N-oxide were not detected under these HPLC conditions. Determination of α- and 4-hydroxytriazolam was carried out as described above, except that the mobile phase consisted of 10 mM potassium phosphate buffer (pH 7.4), acetonitrile and methanol at a ratio of 6:3:1 (v/v), and the eluate was monitored at 220 nm. Calibration curves were generated from 20 to 200 ng/ml by processing the authentic standard substances through the entire procedure. Analytes were quantified by comparison with the standard curves, using the peak-height ratio method. Determination of 4′-hydroxymephenytoin was carried out as reported previously (Chiba et al., 1993). Intraassay (n = 6) coefficients of variation did not exceed 10% for all analytes.
Correlation study.
The N-demethylation activities of CIT were compared with the 4′-hydroxylation activities of (S)-mephenytoin and with the α- and 4-hydroxylation activities of triazolam, using microsomes obtained from 10 human livers. The substrate concentrations used were 1 μM for CIT, 100 μM for (S)-mephenytoin and 25 μM for triazolam. Assays of (S)-mephenytoin 4′-hydroxylation and triazolam α- and 4-hydroxylation activities were performed in duplicate on the same day, with the same set of microsomal preparations.
Inhibition study.
The effects of selective inhibitors or substrates (i.e., compounds acting as competitive inhibitors) of CYP2C19, CYP2D6 and CYP3A on theN-demethylation of CIT (10 and 100 μM) were studied. The isoform-selective inhibitors and alternative substrates used in this part of the study were 100 μM (S)-mephenytoin (CYP2C19) (Küpfer and Preisig, 1984), 10 μM quinidine (CYP2D6) (Guengerich et al., 1986b), 50 μM 17α-ethinylestradiol (CYP3A) (Guengerich, 1988) and 10 μM ketoconazole (CYP3A) (Back and Tjia, 1991; Wrighton and Ring, 1994).
Immunoinhibition study.
Human P450MP (Shimadaet al., 1986) and P450NF (Guengerich et al., 1986a) were purified as reported previously. Polyclonal antibodies to CYP2C and CYP3A were raised in rabbits against human P450MP and P450NF, respectively, as described by Kaminsky et al. (1981). Anti-CYP2C antibodies used in the present study inhibited (S)-mephenytoin 4′-hydroxylation (CYP2C19) and tolbutamide hydroxylation (CYP2C9) by >90%, whereas they did not inhibit testosterone 6β-hydroxylation (CYP3A4) in human liver microsomes. Anti-CYP3A antibodies inhibited testosterone 6β-hydroxylation (CYP3A4) by >80%, whereas they did not inhibit (S)-mephenytoin 4′-hydroxylation (CYP2C19) in human liver microsomes.
The immunoinhibition of CIT N-demethylation was examined by preincubating human liver microsomal samples (0.1 mg/ml) with various concentrations of anti-CYP3A antibodies (0–5 mg IgG/mg microsomal protein) or anti-CYP2C antibodies (0–2 mg IgG/mg microsomal protein) in 0.1 M potassium phosphate buffer (pH 7.4) for 10 min at 37°C. CIT (10 μM) and other components of the incubation medium were added, and the reaction was carried out as described above, except that the incubation period was 60 min.
Assay with cDNA-expressed human P450 isoforms.
Microsomes from human B lymphoblastoid cells expressing human CYP1A2, CYP2A6, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4 (Gentest Corp., Woburn, MA) were used. The basic reactions were carried out as described for the human liver microsomal study. To examine the roles of individual P450 isoforms involved in CIT N-demethylation, each of the eight recombinant P450 isoforms (0.5 mg/ml protein concentration) described above was first incubated with 100 μM CIT for 120 min at 37°C, according to the manufacturer’s recommended procedure. The rates of formation of DCIT from CIT were linear at least up to 60 min with both CYP2C19 and CYP3A4, which showed the catalytic ability of CIT N-demethylation under the same conditions. Accordingly, the following studies using recombinant CYP2C19 and CYP3A4 were carried out using an incubation period of 60 min and a protein concentration of 0.5 mg/ml.
Contribution of CYP3A4 and CYP2C19 to CITN-demethylation in human liver microsomes.
The percentage contributions of CYP2C19 and CYP3A4 to CITN-demethylation in human liver microsomes were estimated by application of the RAF proposed by Crespi (1995). This approach makes the assumption that any effects on the rate of metabolism are independent of substrate, i.e., the rank order of rates of metabolism is the same for a particular P450 isoform and the same P450 isoform present in human liver microsomes, and any factor which affects the rate of metabolism for one substrate also does so equally for other substrates (Crespi, 1995). The validity of this assumption has not been rigorously tested, but for most enzymes an appropriate set of test compounds is available (Crespi, 1995). In the present study, we determined the RAF for CYP2C19 (i.e., RAFCYP2C19) as the ratio of the activity of (S)-mephenytoin (100 μM) 4′-hydroxylation, a specific metabolic reaction mediated via CYP2C19 (Goldstein et al., 1994; Wrighton et al., 1993), in human liver microsomes to that with recombinant CYP2C19. Triazolam (25 μM) α-hydroxylation was used for the calculation of the RAF for CYP3A4 (RAFCYP3A4), as a specific metabolic probe of CYP3A4 (Kronbach et al., 1989).
Using RAF, the N-demethylation clearances of CIT by CYP2C19 and CYP3A4 in human liver microsomes (CLCYP2C19 and CLCYP3A4, respectively) are expressed as follows:
The N-demethylation clearance of CIT by human liver microsomes (CLHLM) is the sum of the metabolic clearances by multiple enzymes contributing to CIT N-demethylation. Therefore, the contribution of CYP2C19 and CYP3A4 to the overall clearance by human liver microsomes is estimated from the following equations.
Estimation of CIT N-demethylation clearance with the first-order rate of metabolism.
N-Demethylation clearances of CIT were estimated from the linear portion of the concentration-activity relationship. This is because our preliminary study showed that not only human liver microsomes but also recombinant CYP3A4 exhibited a curvilinear kinetic profile for theN-demethylation of CIT, although the possibility cannot be excluded that CIT enantiomers may show different kinetic profiles for the N-demethylation of CIT. Because we used a racemic mixture of CIT in the present study, we could not analyze these possibly complicated kinetics. Instead, we estimated theN-demethylation clearance of CIT with the first-order rate of metabolism from the linear portion of the concentration-activity relationship. Because the concentration-activity relationship was linear with up to 5 μM CIT with human liver microsomes and CYP3A4 and up to 25 μM CIT with CYP2C19, the parameters were estimated by linear regression analysis over these concentration ranges. All incubations for this estimation were carried out on the same days for each microsomal sample, to avoid errors due to changes in the activities over different storage periods for the microsomes.
Statistical analysis.
Results are expressed as mean ± S.D. throughout the text. Differences in kinetic data and in estimated contributions of P450 isoform(s) between the putative EM and PM livers (table 1) were statistically evaluated using the Mann-WhitneyU test. Correlation coefficients (r s) were determined by the nonparametric technique (Spearman’s rank correlation). A P value of <.05 was considered statistically significant.
Results
Kinetic profile of CIT N-demethylation in human liver microsomes.
Eadie-Hofstee plots for the N-demethylation of CIT (2.5–500 μM) in microsomes obtained from three putative EM (HL-3, -6 and -26) and two putative PM (HL-8 and -29) livers for (S)-mephenytoin are shown in figure 1. The plots showed biphasic curves, suggesting that each reaction showed multiple-enzyme kinetic behavior and/orKm values of the enzyme(s) involved in the CIT N-demethylation were different for the CIT enantiomers. The curves for EM and PM microsomal samples were similar in biphasicity.
Eadie-Hofstee plots for theN-demethylation of CIT in human liver microsomes obtained from three putative EMs (○, ▵, □) and two putative PMs (•, ▴) of (S)-mephenytoin. V, velocity of metabolite formation; S, substrate concentration. The individualR/S ratios were as follows: ○, 0.075; ▵, 0.198; □, 0.162; •, 0.793; ▴, 0.886.
Correlation study.
The correlations in the individual activities for the N-demethylation of CIT vs the α- and 4-hydroxylation of triazolam or the 4′-hydroxylation of (S)-mephenytoin are shown in figure 2. The CIT N-demethylation activities showed a significant correlation with both α- and 4-hydroxylation of triazolam (r s = 0.818 and 0.851, respectively; P < .01) (fig. 2, A and B). Also, a significant correlation was observed between CIT N-demethylation and (S)-mephenytoin 4′-hydroxylation (r s = 0.773; P < .05) (fig. 2C). The (S)-mephenytoin 4′-hydroxylation activities showed a significant correlation with both α- and 4-hydroxylation of triazolam (r s = 0.730 and 0.758, respectively; P < .05, respectively).
Correlations of triazolam α-hydroxylation (A) and 4-hydroxylation (B) and (S)-mephenytoin 4′-hydroxylation (C) with CIT N-demethylation in 10 human liver microsomal preparations, including microsomes obtained from four putative PMs of (S)-mephenytoin.
Inhibition study.
The effects of four inhibitors or substrates on the formation of DCIT at 10 and 100 μM CIT in microsomes obtained from the three putative EM livers (HL-2, -6 and -27) for (S)-mephenytoin are shown in figure 3. Both ketoconazole and 17α-ethinylestradiol inhibited the formation of DCIT by 68% at 10 μM CIT. At 100 μM CIT, these inhibitors showed 74% and 71% inhibition, respectively, of formation of this metabolite from CIT. Quinidine exhibited a minor inhibitory effect on the formation of DCIT at 10 and 100 μM CIT (11% and 9%, respectively). (S)-Mephenytoin also slightly inhibited the formation of DCIT at 10 and 100 μM CIT (17% and 18%, respectively). Interestingly, with microsomes (HL-8, -22 and -29) obtained from the putative PMs of (S)-mephenytoin, no inhibition by (S)-mephenytoin of the formation of DCIT at 100 μM CIT was observed, whereas ketoconazole, 17α-ethinylestradiol and quinidine showed inhibition of 85%, 76% and 16%, respectively.
Mean ± S.D. of percentage inhibition of CITN-demethylation activity at 10 and 100 μM CIT by 17α-ethinylestradiol, ketoconazole, (S)-mephenytoin and quinidine in human liver microsomes obtained from three putative EMs of (S)-mephenytoin (R/S ratios ranging from 0.115 to 0.162).
Immunoinhibition study.
Figure 4 shows the inhibition of N-demethylation of CIT by polyclonal antibodies raised against CYP3A or CYP2C. The addition of anti-CYP3A reduced the N-demethylation activity of CIT by approximately 70% at a concentration (2 mg IgG/mg microsomal protein) at which >80% of testosterone 6β-hydroxylation was inhibited, whereas (S)-mephenytoin 4′-hydroxylation was not. On the other hand, anti-CYP2C inhibited CIT N-demethylation by <10% at a concentration (2 mg IgG/mg microsomal protein) at which >90% of (S)-mephenytoin 4′-hydroxylation was inhibited. The magnitude of inhibition by both antibodies was similar between the putative EM (HL-5) and PM (HL-29) microsomal samples.
Immunoinhibition of N-demethylation of CIT by anti-CYP3A (A) and anti-CYP2C (B) in human liver microsomes obtained from a putative EM (○) (R/Sratio of 0.204) and a putative PM (•) (R/S ratio of 0.793) of (S)-mephenytoin.
cDNA-expressed human P450 isoform study.
Microsomes from human B lymphoblastoid cell lines expressing each of eight human P450 isoforms were examined in terms of the abilities of individual P450 proteins to catalyze CIT N-demethylation. CYP2C19 and CYP3A4 were found to catalyze the reaction (1.39 and 0.71 pmol/pmol P450/min, respectively), whereas other isoenzymes showed negligible activity for the N-demethylation of CIT (table 2).
N-Demethylation activities for CIT in microsomes from human B lymphoblastoid cells expressing P450 isoforms
Contributions of CYP2C19 and CYP3A4 to CITN-demethylation in human liver microsomes.
The activities of (S)-mephenytoin 4′-hydroxylation and triazolam α-hydroxylation in six different human liver microsomal preparations (HL-5, -6, -8, -22, -29 and -30) ranged from 2.9 to 48.3 pmol/mg/min and from 0.22 to 0.65 nmol/mg/min, respectively. The activities of (S)-mephenytoin 4′-hydroxylation by CYP2C19 and of triazolam α-hydroxylation by CYP3A4 were 4.59 and 1.73 nmol/nmol P450/min, respectively. RAFCYP2C19 and RAFCYP3A4 were thus estimated to range from 0.63 to 10.52 and from 0.13 to 0.38 pmol P450/mg protein, respectively (table 3). The CLrec-CYP2C19 and CLrec-CYP3A4 calculated from the linear ranges were 24.4 and 8.34 μl/nmol P450/min, respectively. Thus, the clearances by CYP2C19 and CYP3A4 contributing to CITN-demethylation in six different human liver microsomes (CLCYP2C19 and CLCYP3A4) were estimated by using equations 1 and 2, respectively; the individual values are listed in table 3. The CLHLM values estimated from the linear region (1–5 μM CIT) ranged from 1.72 to 3.67 μl/mg/min. The mean ± S.D. contributions of CYP2C19 to human microsomal CITN-demethylation, calculated according to equation 3, were 7.35 ± 0.49 and 0.91 ± 0.16% for the microsomes of the putative EM (HL-5, -6 and -30) and PM (HL-8, -22 and -29) livers, respectively. The value for the putative EM microsomes was significantly (P < .05) greater than that for the PM microsomes. For CYP3A4, the mean ± S.D. contributions estimated by using equation 4 were 70.12 ± 15.07 and 58.99 ± 6.46% for the putative EM and PM microsomes, respectively; the values did not significantly differ between the two groups. The sum of the contributions of CYP2C19 and CYP3A4 was 94.26% in HL-5. However, the values in the other microsomes ranged from 52.63 to 71.75%.
Contributions of CYP2C19 and CYP3A4 to CIT N-demethylation estimated by RAFs in human liver microsomes
Discussion
The results suggest that CYP3A4 is primarily responsible for catalyzing the N-demethylation of CIT in human liver microsomes. This conclusion was inferred from the following observations. First, the CIT N-demethylation activities showed a significant correlation with both α- and 4-hydroxylation activities for triazolam, a substrate of CYP3A4 (Kronbach et al., 1989), in microsomes prepared from 10 human livers (fig. 2). Second, ketoconazole and 17α-ethinylestradiol, known as selective inhibitors or substrates of CYP3A (Back and Tjia, 1991; Guengerich, 1988; Wrighton and Ring, 1994), strongly inhibited theN-demethylation of CIT in microsomes obtained from both the putative EM and PM livers for (S)-mephenytoin 4′-hydroxylation (fig. 3). Third, CIT N-demethylation was almost completely inhibited by the addition of anti-CYP3A antibodies (fig. 4). Fourth, the recombinant human CYP3A4 catalyzed CITN-demethylation (table 2).
However, the N-demethylation of CIT was catalyzed not only by recombinant CYP3A4 but also by CYP2C19 (table 2), suggesting that CIT is a substrate of CYP2C19. This observation is consistent with our previous report that this drug competitively inhibited the 4′-hydroxylation of (S)-mephenytoin in human liver microsomes (Kobayashi et al., 1995). This is also consistent with the observation that the N-demethylation activity of CIT was inhibited by (S)-mephenytoin in microsomes obtained from the putative EM livers (fig. 3), whereas no inhibition was observed in the PM microsomes. In addition, theN-demethylation activity of CIT correlated not only with the triazolam α- and 4-hydroxylation activities but also with the (S)-mephenytoin 4′-hydroxylation activity (fig. 2), although the latter correlation might depend on the result that the 4′-hydroxylation activity for (S)-mephenytoin correlated with the α- and 4-hydroxylation activities for triazolam. Moreover, the inhibition of CIT N-demethylation by (S)-mephenytoin in the EM microsomes was <20% (fig. 3), although anti-CYP2C antibodies scarcely inhibited the CITN-demethylation (fig. 4). Taken together, these results suggest that CIT N-demethylation is catalyzed at least in part by CYP2C19 and thus that CYP2C19 contributes to this metabolic pathway of CIT in human liver microsomes to a much lesser extent than does CYP3A4.
Recently, Crespi (1995) proposed a corrective factor (i.e., RAF) to extrapolate the data obtained from cDNA-expressed P450s to those from human liver microsomes. The RAF was applied to interpret the data for the metabolic activation of tobacco smoke-derived nitrosoamine and aflatoxin b1 obtained from cDNA-expressed P450s. However, little information has been available regarding the applicability of the RAF for assessing the magnitude of each of the P450 isoforms involved in the metabolism of therapeutic agents. Therefore, we applied the RAF to estimate the relative contributions of CYP2C19 and CYP3A4 to the overall CIT N-demethylation in each of the human liver microsomal samples used in the study. The estimated contributions of CYP3A4 and CYP2C19 to CITN-demethylation were about 70% and 7%, respectively, in microsomes obtained from the putative EM livers (table 3). This further indicates that CIT N-demethylation is primarily catalyzedvia CYP3A4. On the other hand, <10% of the overallN-demethylation was estimated to be catalyzed viaCYP2C19 (table 3). These findings are consistent with the percentages of inhibition of CIT N-demethylation by CYP3A inhibitors or substrates (68–74%) (fig. 3) and by CYP2C19 substrate (17%) (fig. 3) in human liver microsomes, suggesting that the RAF may be a useful tool for estimating the contributions of human microsomal P450 isoenzymes to drug metabolism from the corresponding cDNA-expressed P450s. However, the concept of the RAF has been proposed based upon several assumptions (Crespi, 1995) (see “Materials and Methods”), and its validity has not been rigorously tested. Therefore, whether the proposed RAF concept would have wide applicability for other in vitro experiments like ours remains unknown and definitely requires further assessment.
With the limitations discussed above, the present approach using the RAF indicated that the CIT N-demethylation activities were almost completely explainable by metabolism via CYP3A4 and CYP2C19 in HL-5. However, some portions of CITN-demethylation could not be explained by CYP3A4 and CYP2C19 alone in the remaining human liver microsomal samples. The findings suggest that enzymes other than CYP3A4 and CYP2C19 are partially involved in CIT N-demethylation in those microsomal samples, although it remains unclear from the present study which P450 isoform(s) may be involved. However, because CITN-demethylation in human liver microsomes was completely inhibited by anti-CYP3A antibodies or selective inhibitors of CYP3A, CYP3A isoform(s) other than CYP3A4 (e.g., CYP3A5) may be responsible for CIT N-demethylation.
The present in vitro observation that the contribution of CYP2C19 to CIT N-demethylation was <10% in the putative EM liver microsomes is incompatible with findings from the in vivo human panel study by Sindrup et al. (1993). They reported that the N-demethylation clearance of CIT was about 2-fold greater in EM than in PM subjects for (S)-mephenytoin, indicating that about 50% of the total CITN-demethylation was catalyzed by CYP2C19. We cannot offer any reasonable explanation for the discrepancy between the results of the present in vitro study and those of the in vivo study by Sindrup et al. (1993). However, we are tempted to assume that the hepatic microsomes designated as those from putative EM livers in the present in vitro study might have been obtained from Japanese patients heterozygous forCYP2C19. This is because the PM frequency for the 4′-hydroxylation of (S)-mephenytoin is much greater in Japanese populations (22.5%) (Horai et al., 1989), compared with that in Caucasian populations (3–6%) (Alván et al., 1990; Jacqz et al., 1988; Wedlund et al., 1984). Therefore, the frequency of individuals heterozygous for the EM genotype (i.e., wt/m1 orwt/m2) (de Morais et al., 1994a,b) would be estimated to be much greater in Japanese populations, compared with Caucasian populations, implying that the contribution of CYP2C19 to the N-demethylation of CIT might be lower in Japanese human liver microsomes, compared with those from Caucasian subjects. Obviously, this assumption must be confirmed by interethnic in vitro and/or in vivo studies.
CIT is a chiral compound that is commercially available as a racemic mixture. Although (S)-CIT was reported to be metabolized more extensively than (R)-CIT in vivo (Rochatet al., 1995), little information is available concerning the stereoselective metabolism of CIT in in vitro studies. However, the results of our preliminary study indicated that theN-demethylation clearance of (S)-CITvia CYP2C19 is about 2 times greater than that of theR-enantiomer (K. Kobayashi, K. Chiba, T. Yamamoto, T. Ishizaki and Y. Kuroiwa, unpublished observations). Therefore, the discrepancy between the in vitro and in vivo CITN-demethylation activities in humans might be attributable not only to the interethnic differences in CYP2C19 activity but also to stereoselective CIT N-demethylation by CYP2C19.
In conclusion, the present study using human liver microsomes and recombinant human P450 isoforms has strongly suggested that theN-demethylation of CIT is mediated mainly, but not exclusively, via CYP3A4 and partially viaCYP2C19. In addition, the RAF proposed by Crespi (1995) appears to be a tool for estimating the contributions of the two P450 isoforms involved in CIT N-demethylation, thus bridging the gap between human liver microsomal and recombinant human P450 isoform studies.
Footnotes
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Send reprint requests to: Yukio Kuroiwa, Ph.D., Department of Clinical Pharmacy, School of Pharmaceutical Sciences, Showa University, Hatanodai 1–5-8, Shinagawa-ku, Tokyo 142, Japan.
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↵1 This work was supported by a Grant-in-aid for Encouragement of Young Scientists from the Ministry of Education and Science (06772215), the Japan Health Science Foundation (1–7-1-C) and the Drug Innovation Science Project (1–2-10), Tokyo, Japan.
- Abbreviations:
- CIT
- citalopram
- CYP or P450
- cytochrome P450
- DCIT
- desmethylcitalopram
- EM
- extensive metabolizer
- HPLC
- high-performance liquid chromatography
- PM
- poor metabolizer
- RAF
- relative activity factor
- Received April 15, 1996.
- Accepted October 15, 1996.
- The American Society for Pharmacology and Experimental Therapeutics
