- Email: [email protected]
Identification of human cytochrome P450 isoforms involved in the 7-hydroxylation of chlorpromazine by human liver microsomes
Life Sciences 67 (2000) 175Ð184
IdentiÞcation of human cytochrome P450 isoforms involved in the 7-hydroxylation of chlorpromazine by human liver microsomes Kazuyoshi Yoshiia, Kaoru Kobayashia,*, Mihoko Tsumujia, Masayoshi Tanib, Noriaki Shimadac, Kan Chibaa a
Laboratory of Biochemical Pharmacology and Toxicology, Faculty of Pharmaceutical Sciences, Chiba University, Yayoi-cho 1-33, Inage-ku, Chiba 263-8522, Japan b Department of General Surgery, International Medical Center of Japan, Tokyo, Japan c Material Technology Research Laboratories, Daiichi Pure Chemicals Co. Ltd., Ibaraki, Japan
Abstract Studies to identify the cytochrome P450 (CYP) isoform(s) involved in chlorpromazine 7-hydroxylation were performed using human liver microsomes and cDNA-expressed human CYPs. The kinetics of chlorpromazine 7-hydroxylation in human liver microsomes showed a simple Michaelis-Menten behavior. The apparent Km and Vmax values were 3.4 6 1.0 mM and 200.5 6 83.7 pmol/min/mg, respectively. The chlorpromazine 7-hydroxylase activity in human liver microsomes showed good correlations with desipramine 2-hydroxylase activity (r 5 0.763, p , 0.05), a marker activity for CYP2D6, and phenacetin O-deethylase activity (r 5 0.638, p , 0.05), a marker activity for CYP1A2. Quinidine (an inhibitor of CYP2D6) completely inhibited while a-naphthoßavone (an inhibitor of CYP1A2) marginally inhibited the chlorpromazine 7-hydroxylase activity in a human liver microsomal sample showing high CYP2D6 activity. On the other hand, a-naphthoßavone inhibited the chlorpromazine 7-hydroxylase activity to 55Ð65% of control in a human liver microsomal sample showing low CYP2D6 activity. Among eleven cDNA-expressed CYPs studied, CYP2D6 and CYP1A2 exhibited signiÞcant activity for the chlorpromazine 7-hydroxylation. The K m values for the chlorpromazine 7-hydroxylation of both cDNA-expressed CYP2D6 and CYP1A2 were in agreement with the Km values of human liver microsomes. These results suggest that chlorpromazine 7-hydroxylation is catalyzed mainly by CYP2D6 and partially by CYP1A2. © 2000 Elsevier Science Inc. All rights reserved. Keywords: Chlorpromazine 7-hydroxylation; Cytochrome P450; Microsomes; Human; cDNA-expressed system
* Corresponding author. Tel.: (81) 43 290 2920; fax: (81) 43 290 2920. E-mail address: [email protected] (K. Kobayashi) 0024-3205/00/$ Ð see front matter © 2000 Elsevier Science Inc. All rights reserved. PII: S 0 0 2 4 - 3 2 0 5 ( 0 0 )0 0 6 1 3 -5
176
K. Yoshii et al. / Life Sciences 67 (2000) 175Ð184
Introduction Chlorpromazine is generally regarded as a prototype phenothiazine antipsychotic agent. The drug is extensively metabolized in the liver, and the main metabolic pathways include oxidative processes such as 7-hydroxylation, N-dealkylation, N-oxidation, S-oxidation, as well as other routes that include conjugation of 7-hydroxylated products (1,2). Among these pathways, the 7-hydroxylation is known as a major pathway of chlorpromazine metabolism in human (2,3). In vitro studies (4,5) with human liver microsomes have suggested that chlorpromazine is a potent competitive inhibitor of cytochrome P450 2D6 (CYP2D6). In addition, an in vivo study (6) indicated that administration of quinidine, a potent inhibitor of CYP2D6, signiÞcantly decreased the urinary excretion of 7-hydroxychlorpromazine in healthy volunteers who received a single dose of chlorpromazine. These data suggest that chlorpromazine is a substrate of CYP2D6 and that the enzyme is involved in the metabolism of chlorpromazine to 7-hydroxychlorpromazine. On the other hand, it has been reported that cigarette smoking increases the clearance of chlorpromazine (7,8). Cigarette smoking is well known to cause marked induction of CYP1A2 and accelerate the metabolism of drugs catalyzed by CYP1A2, such as theophylline (9,10), caffeine (11) and phenacetin (12,13). Therefore, CYP1A2 is thought to be involved in the chlorpromazine metabolism. However, to our knowledge, no detailed study has been reported with respect to the speciÞc isoform(s) of CYP involved in the chlorpromazine metabolism in humans. Therefore, the aim of the present study was to clarify the roles of CYP2D6 and CYP1A2 in chlorpromazine 7-hydroxylation in the human liver. Materials and methods Materials Human liver samples 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). Microsomes were prepared as reported previously (14) and stored at 2808C until use. Microsomes were previously characterized by desipramine 2-hydroxylase activity catalyzed by CYP2D6. Microsomes prepared from human B-lymphoblastoid cells expressing human CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4 and CYP4A11 (Gentest Corp., Woburn, MA, USA) were similarly stored at 2808C until use. Chlorpromazine was a gift from Yoshitomi Pharmaceutical Co. (Osaka, Japan), and 7-hydroxychlorpromazine glucuronide was from Psychopharmacology Research Branch, National Institute of Mental Health (Rockville, Md., USA). The other chemical reagents were purchased from commercial sources. Chlorpromazine 7-hydroxylase activities Typical incubation mixtures contained 0.2 mg/ml microsomal protein, 0.1 mM EDTA, 100 mM potassium phosphate buffer (pH 7.4), an NADPH-generating system (0.5 mM NADP1,
K. Yoshii et al. / Life Sciences 67 (2000) 175Ð184
177
2.0 mM glucose-6-phosphate, 1 IU/ml of glucose-6-phosphate dehydrogenase and 4 mM MgCl2), and 1 to 24 mM of chlorpromazine, in a Þnal volume of 250 ml. The mixture was incubated at 378C for 30 min. After the reaction was stopped by adding 100 ml of cold acetonitrile, 50 ml of amitriptyline (10 mM in methanol) was added to the samples as an internal standard. The mixture was centrifuged at 14,0003g for 10 min, and the supernatant was transferred to an autosampling vial for HPLC analysis. The mobile phase consisted of 50 mM sodium bicarbonate and methanol at a ratio of 20:80 (v/v) and was delivered at a ßow rate of 0.8 ml/min. The analytical column used was a 4.6 3 250 mm CAPCELL PAK C18 UG120 column (Shiseido Co., Tokyo, Japan). The eluate was monitored at a wavelength of 248 nm. Concentrations of 7-hydroxychlorpromazine in the reaction mixture were determined based on calibration curves constructed from a series of standards containing varying known amounts of 7-hydroxychlorpromazine together with the internal standard. 7-Hydroxychlorpromazine used as a standard was prepared by hydrolysis of 7-hydroxychlorpromazine glucuronide using b-glucuronidase. Kinetics in human liver microsomes Kinetic studies were performed with microsomes from Þve human livers (HL38, HL40, HL41, HL42 and HL43). In determining kinetic parameters, the chlorpromazine concentration ranged from 1Ð24 mM. All reactions were performed in a linear range with respect to protein concentration and incubation time: 0.2 mg/ml microsomal protein and 30-min incubation time. Eadie-Hofstee plots were constructed for determination of the presence of a mono- or biphasic model. Kinetic parameters were estimated by non-linear least-squares regression analysis of untransformed data. Correlation study Correlations between chlorpromazine 7-hydroxylase activities and the metabolic activities of substrates toward the respective distinct CYP isoforms were studied by using 10 different human liver microsomes. Assays for phenacetin O-deethylation at the substrate concentration of 10 mM (CYP1A2), diclofenac 49-hydroxylation at 10 mM (CYP2C9), S-mephenytoin 49-hydroxylation at 100 mM (CYP2C19), desipramine 2-hydroxylation at 10 mM (CYP2D6), testosterone 6b-hydroxylation at 30 mM (CYP3A4) and chlorpromazine 7-hydroxylation at 10 mM were performed. Metabolites of the in vitro probe substrates described above that were formed in the incubation mixtures were determined according to the respective HPLC assay methods as reported elsewhere (14Ð17). Inhibition study The effects of selective inhibitors or substrates (i.e., compounds acting as competitive inhibitors) for Þve different CYP isoforms on the chlorpromazine 7-hydroxylation at 10 mM in human liver microsomes (HL38) were studied. The isoform-selective inhibitors and alternative substrates (18,19) used in this part of the study were 10 mM a-naphthoßavone (CYP1A), 100 mM sulfaphenazole (CYP2C9), 500 mM mephenytoin (CYP2C19), 1 mM quinidine (CYP2D6) and 25 mM troleandomycin (CYP3A4). The effects of a-naphthoßavone and quinidine on the chlorpromazine 7-hydroxylation at 10 mM in two different human liver mi-
178
K. Yoshii et al. / Life Sciences 67 (2000) 175Ð184
crosomes (HL38 and HL39) were also studied at inhibitor concentrations ranging from 0.01 to 10 mM. Assay with cDNA-expressed CYPs Microsomes from human B-lymphoblastoid cells expressing human CYP1A1 (44 pmol CYP/mg), CYP1A2 (124 pmol CYP/mg), CYP2A6 (103 pmol CYP/mg), CYP2B6 (130 pmol CYP/mg), CYP2C8 (26 pmol CYP/mg), CYP2C9 (80 pmol CYP/mg), CYP2C19 (29 pmol CYP/mg), CYP2D6 (39 pmol CYP/mg), CYP2E1 (198 pmol CYP/mg), CYP3A4 (59 pmol CYP/mg) and CYP4A11 (18 pmol CYP/mg) were used. The reactions were carried out as described above. To examine the role of individual CYP isoforms involved in chlorpromazine 7-hydroxylation, each of the cDNA-expressed CYPs (0.5 mg/ml protein concentration) described above was Þrstly incubated with 100 mM chlorpromazine for 60 min at 378C according to the procedure recommended by the supplier. Kinetic analyses were performed using cDNA-expressed CYP1A2 and cDNA-expressed CYP2D6. Chlorpromazine 7-hydroxylase activities were determined using chlorpromazine concentrations ranging from 2Ð80 mM. All reactions were performed within a linear range with respect to protein concentration and incubation time. Brießy, 0.4 mg/ml of cDNA-expressed CYP1A2 and cDNA-expressed CYP2D6 were incubated for 60 min and 15 min, respectively. Kinetic parameters were estimated as described above. Data analysis Data are expressed as mean 6 SD throughout the text, if not otherwise stated. Correlations between the metabolite formation rates of respective CYP isoform-speciÞc substrates and chlorpromazine 7-hydroxylase activities were examined by the least-squares linear regression method. Results Kinetics in human liver microsomes Eadie-Hofstee plots for chlorpromazine 7-hydroxylase activity in four different human liver microsomes are shown in Fig. 1. In all of the human liver microsomes studied, the EadieHofstee plots were linear. Thus, we were able to estimate the kinetic parameters by Þtting them to a simple Michaelis-Menten equation. The mean (6 SD) kinetic parameters of four human liver microsomes were: apparent Km 5 3.4 6 1.0 mM, Vmax 5 200.5 6 83.7 pmol/ min/mg protein, and Vmax/Km 5 58.8 6 16.1 mL/min/mg protein. Correlation study Fig. 2A shows a signiÞcant correlation (r 5 0.763, p , 0.05) between chlorpromazine 7-hydroxylase activity and desipramine 2-hydroxylase activity in a panel of 10 human liver microsomes. A signiÞcant correlation (r 5 0.638, p , 0.05) was also observed between chlorpromazine 7-hydroxylase activity and phenacetin O-deethylase activity in the same panel of human liver microsomes (Fig. 2B). No signiÞcant correlations were observed be-
K. Yoshii et al. / Life Sciences 67 (2000) 175Ð184
179
Fig. 1. Eadie-Hofstee plots of chlorpromazine 7-hydroxylase activity in human liver microsomes prepared from Þve different subjects. d, HL38, Km 5 4.8 mM, Vmax 5 340.2 pmol/min/mg; m, HL40, Km 5 2.5 mM, Vmax 5 118.5 pmol/min/mg; j, HL41, Km 5 3.4 mM, Vmax 5 191.6 pmol/min/mg; s, HL42, Km 5 4.0 mM, Vmax 5 160.6 pmol/min/mg; n, HL43, Km 5 2.4 mM, Vmax 5 191.6 pmol/min/mg.
tween chlorpromazine 7-hydroxylase activity and catalytic activities of diclofenac 49-hydroxylase (r 5 0.518), S-mephenytoin 49-hydroxylase (r 5 0.523) or testosterone 6b-hydroxylase (r 5 0.402). Inhibition study The CYP-isoform selective probes were screened for inhibitory effects on the chlorpromazine 7-hydroxylase activity in a human liver microsome (HL38). Quinidine, a potent inhibitor of CYP2D6, completely inhibited the chlorpromazine 7-hydroxylase activity (the control activity in the absence of quinidine was 201.7 pmol/min/mg). In contrast, the selective probes for CYP1A (a-naphthoßavone), CYP2C9 (sulfaphenazole) and CYP2C19 (mephenytoin) and CYP3A4 (troleandomycin) exhibited no inhibitory effect or marginal inhibition on the chlorpromazine 7-hydroxylase activity (87.5Ð118.6% of control activity). Subsequently, the concentration-effect relationship of a-naphthoßavone and quinidine on the chlorpromazine 7-hydroxylase activity in two different human liver microsomes (HL38 and HL39) was studied. In our preliminary study, desipramine 2-hydroxylase activity, a marker activity of CYP2D6, in microsomes from HL38 and HL39 was 0.218 and 0.009 nmol/min/mg, respectively. Therefore, the microsomes from HL38 and HL39 were selected as representative microsomes in which the catalytic activity by CYP2D6 was high and low, respectively. In the microsomes from HL38, the chlorpromazine 7-hydroxylase activity was inhibited to an undetectable level by 1 mM quinidine but the effect of a-naphthoßavone was marginal (Fig. 3A). On the other hand, the chlorpromazine 7-hydroxylase activity in the microsomes from HL39 was inhibited to 48% of the control activity by 1 mM quinidine and to 55Ð65% of the control activity by 0.1Ð10 mM a-naphthoßavone (Fig. 3B).
180
K. Yoshii et al. / Life Sciences 67 (2000) 175Ð184
Fig. 2. Correlations between chlorpromazine 7-hydroxylase activity and desipramine 2-hydroxylase activity (A) or phenacetin O-deethylase activity (B) in a panel of ten human liver microsomes. Correlations were examined with the least-squares linear regression method.
Study with cDNA-expressed CYPs Microsomes from human B-lymphoblastoid cell lines expressing each of eleven CYP isoforms were examined in terms of the abilities of individual CYP isoforms to catalyze the chlorpromazine 7-hydroxylation. CYP2D6 and CYP1A2 were found to catalyze the reaction (217 and 26 pmol/60 min/pmol CYP, respectively), whereas the other CYP isoforms had little discernible or no effect with regard to the chlorpromazine 7-hydroxylation (Fig. 4). Accordingly, kinetic parameters for the chlorpromazine 7-hydroxylation were estimated for cDNAexpressed CYP2D6 and cDNA-expressed CYP1A2. The Km values of CYP2D6 and CYP1A2 were 4.3 6 1.0 and 5.1 6 2.0 mM, respectively. These Km values were similar to that obtained from human liver microsomes (Table 1). The Vmax value of CYP2D6 was greater that that of CYP1A2 (11.7 6 0.9 vs. 0.7 6 0.1 pmol/min/pmol CYP). cDNA-expressed CYP2D6 showed a greater Vmax/Km value than that of cDNA-expressed CYP1A2 (2.83 6 0.82 and 0.15 6 0.04 mL/min/pmol CYP, respectively). Discussion The results suggest that the formation of 7-hydroxychlorpromazine is predominantly catalyzed by CYP2D6. This conclusion was inferred from the following observations. First, the chlorpromazine 7-hydroxylase activity showed a signiÞcant correlation with desipramine 2-hydroxylase activity, a maker reaction of CYP2D6 (20), in a panel of 10 human liver microsomes (Fig. 2A). Second, 1 mM quinidine, a speciÞc inhibitor of CYP2D6, completely inhibited the chlorpromazine 7-hydroxylase activity in human liver microsomes (Fig. 3A).
K. Yoshii et al. / Life Sciences 67 (2000) 175Ð184
181
Fig. 3. Effects of quinidine and a-naphthoßavone on chlorpromazine 7-hydroxylase activity in two different human liver microsomes. The chlorpromazine 7-hydroxylase activities at 10 mM of substrate concentration were determined using microsomes from HL38 in which desipramine 2-hydroxylase activity is high (A) and HL39 in which desipramine 2-hydroxylase activity is low (B). The control activities in the absence of inhibitors were 212.9 and 157.9 pmol/min/mg for HL38 and HL39, respectively. The concentrations of quinidine (d) and a-naphthoßavone (s) ranged from 0.01Ð10 mM. Each data point represents the mean of duplicate determinations.
Third, cDNA-expressed CYP2D6 catalyzed chlorpromazine 7-hydroxylation (Fig. 4). These in vitro Þndings were in agreement with the in vivo observation (6) that the urinary excretion of 7-hydroxychlorpromazine was decreased by pretreatment with quinidine, a speciÞc inhibitor of CYP2D6, in healthy volunteers who received a single oral dose of chlorpromazine, suggesting CYP2D6 is involved in chlorpromazine 7-hydroxylation. The chlorpromazine 7-hydroxylation was catalyzed by not only cDNA-expressed CYP2D6 but also by cDNA-expressed CYP1A2 (Fig. 4). In addition, the correlation study using a panel of 10 human liver microsomes showed there was a signiÞcant correlation between the chlorpromazine 7-hydroxylase activity and the phenacetin O-deethylase activity, a marker reaction of CYP1A2 (16,21) (Fig. 2B). Moreover, a-naphthoßavone, a speciÞc inhibitor for CYP1A2, also inhibited the chlorpromazine 7-hydroxylase activity in a human liver microsomal sample showing low CYP2D6 activity (Fig. 3B). These results suggest that not only CYP2D6 but also CYP1A2 is responsible for catalyzing the chlorpromazine 7-hydroxylation in human liver microsomes. Previously, some investigators (7,8,22) reported that the clearance of chlorpromazine was elevated by cigarette smoking. It is well established that cigarette smoking causes marked induction of CYP1A2 (13,23). Therefore, these in vivo Þndings are at least partially explained by the present results showing that chlorpromazine 7-hydroxylation in human liver microsomes is catalyzed by a cigarette smoking-inducible enzyme, CYP1A2. The results suggest that both CYP2D6 and CYP1A2 are involved in the chlorpromazine 7-hydroxylation. This is not contradicted to the Þnding that Eadie-Hofstee plots for chlorpro-
182
K. Yoshii et al. / Life Sciences 67 (2000) 175Ð184
Fig. 4. Formation of 7-hydroxychlorpromazine from chlorpromazine in microsomes from human B-lymphoblastoid cells expressing the human CYP isoform. A substrate (100 mM chlorpromazine) was incubated at 378C for 60 min with microsomes (0.5 mg/ml) of human B-lymphoblastoid cells expressing CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4 and CYP4A11, respectively. ND, not detectable.
mazine 7-hydroxylation were linear in all human liver microsomal samples studied (Fig. 1), since the results of cDNA-expressed enzymes indicated that Km values of CYP2D6 and CYP1A2 for chlorpromazine 7-hydroxylation were essentially identical (Table 1). These observations support the notion that both CYP2D6 and CYP1A2 are responsible for the chlorpromazine 7-hydroxylation in human liver microsomes. However, the Vmax/Km value of cDNA-expressed CYP2D6 was approximately 20-fold greater than that of cDNA-expressed Table 1 Kinetic parameters of chlorpromazine 7-hydroxylase activity in microsomes from human B-lymphoblastoid cells expressing CYP1A2 or CYP2D6 Isoforms
Km (mM)
Vmax (pmol/min/pmol CYP)
Vmax/Km (mL/min/pmol CYP)
CYP1A2 CYP2D6
5.1 6 2.0 4.3 6 1.0
0.70 6 0.13 11.7 6 0.91
0.15 6 0.04 2.83 6 0.82
Values are expressed as mean 6 SD from three different experiments.
K. Yoshii et al. / Life Sciences 67 (2000) 175Ð184
183
CYP1A2 (Table 1). In human liver microsomes, it has been reported that the contents of CYP2D6 and CYP1A2 are 1.5 6 1.3% and 12.7 6 6.2% of total CYP, respectively (24), indicating CYP2D6 content is 10-fold lower than CYP1A2 content. Accordingly, it was estimated that the contribution of CYP2D6 to the chlorpromazine 7-hydroxylation in human liver microsomes is about two-fold higher than that of CYP1A2. In the human liver microsomal studies, the inhibition of chlorpromazine 7-hydroxylase activity in human liver microsomes by quinidine, a CYP2D6 inhibitor, was more potent than that by a-naphthoßavone, a CYP1A2 inhibitor (Fig. 3A). In addition, the correlation coefÞcient value of chlorpromazine 7-hydroxylase activity with desipramine 2-hydroxylase activity was higher than that with phenacetin O-deethylase activity (Fig. 2). Therefore, both CYP2D6 and CYP1A2 are involved in the 7-hydroxylation of chlorpromazine in human liver microsomes, but the contribution of CYP1A2 to this metabolism appears to be lower than that of CYP2D6. There are large interindividual differences in expression levels of CYP1A2 and CYP2D6 in human liver microsomes. Indeed, CYP1A2 content in microsomes ranged from 0.3 to 100fold of CYP2D6 content in commercially available sources (Gentest). Therefore, the effects of quinidine and a-naphthoßavone on chlorpromazine 7-hydroxylase activity in human liver microsomes were determined with the microsomes from HL38 and HL39, which have a more than 20-fold difference in desipramine 2-hydroxylase activity. Quinidine at 1 mM completely inhibited the chlorpromazine 7-hydroxylase activity in microsomes from HL38 with high catalytic activity by CYP2D6, but the inhibitory effect in microsomes from HL39 with low catalytic activity by CYP2D6 was only 52% (Fig. 3). In contrast, the inhibitory effect by a-naphthoßavone in microsomes from HL39 was greater than that in microsomes from HL38 (Fig. 3). These results are thought to reßect the differences of CYP1A2 and CYP2D6 contents in two human liver microsomes. Therefore, the contribution of CYP1A2 to the chlorpromazine 7-hydroxylation might be signiÞcant in human liver microsomes with low CYP2D6 contents. In conclusion, the results of the present study using human liver microsomes and cDNAexpressed enzymes strongly suggest that the chlorpromazine 7-hydroxylation is catalyzed mainly by CYP2D6 and partially by CYP1A2. To our knowledge, this is the Þrst in vitro study to demonstrate that CYP2D6 and CYP1A2 are responsible for chlorpromazine 7-hydroxylation. In human liver microsomes with low CYP2D6 contents, CYP1A2 may make a signiÞcant contribution to chlorpromazine 7-hydroxylation. Therefore, both the phenotype of CYP2D6 and smoking habits could reßect the ability of chlorpromazine 7-hydroxylation in humans. References 1. E. USDIN, Crc. Crit. Rev. Clin. Lab. Sci. 2 347Ð391 (1971). 2. F. HARTMANN, L.D. GRUENKE, J.C. CRAIG and D.M. BISSELL, Drug Metab. Dispos. 11 244Ð248 (1983). 3. P.K.F. YEUNG, J.W. HUBBARD, E.D. KORCHINSKI and K.K. MIDHA, Eur. J. Clin. Pharmacol. 45 563Ð 569 (1993). 4. S.V. OTTON, T. INABA, W. KALOW, Life Sci. 32 795Ð800 (1983). 5. C. VON BAHR, E. SPINA, C. BIRGERSSON, O. ERICSSON, M. GORANSSON, T. HENTHORN and F. SJOQVIST, Biochem. Pharmacol. 34 2501Ð2505 (1985). 6. G. MURALIDHARAN, J.K. COOPER, E.M. HAWES, E.D. KORCHINSKI and K.K. MIDHA, Eur. J. Clin. Pharmacol. 50 121Ð128 (1996).
184
K. Yoshii et al. / Life Sciences 67 (2000) 175Ð184
7. E.J. PANTUCK, C.B. PANTUCK, K.E. ANDERSON, A.H. CONNEY and A. KAPPAS, Clin. Pharmacol. Ther. 31 533Ð538 (1982). 8. M. CHETTY, R. MILLER and S.V. MOODLEY, Eur. J. Clin. Pharmacol. 46 523Ð526 (1994). 9. J.J. GRYGIEL and D.J. BIRKETT, Clin. Pharmacol. Ther. 30 491Ð496 (1981). 10. S.N. HUNT, W.J. JUSKO and A.M. YURCHAK, Clin. Pharmacol. Ther. 19 546Ð551 (1976). 11. J.A. CARRILLO and J. BENITEZ, Clin. Pharmacol. Ther. 55 293Ð304 (1994). 12. E.J. PANTUCK, K.C. HSIAO, A. MAGGIO, K. NAKAMURA, R. KUNTZMAN and A.H. CONNEY, Clin. Pharmacol. Ther. 15 9Ð17 (1974). 13. D. SESARDIC, A.R. BOOBIS, R.J. EDWARDS and D.S. DAVIES, Br. J. Clin. Pharmacol. 26 363Ð372 (1988). 14. K. CHIBA, K. KOBAYASHI, M. TANI, K. MANABE and T. ISHIZAKI, Drug Metab. Dispos. 21 747Ð749 (1993). 15. K. CHIBA, K. MANABE, K. KOBAYASHI, Y. TAKAYAMA, M. TANI and T. ISHIZAKI, Eur. J. Clin. Pharmacol. 44 559Ð562 (1993). 16. W. TASSANEEYAKUL, D.J. BIRKETT, M.E. VERONESE, M.E. MCMANUS, R.H. TUKEY, L.C. QUATTROCHI, H.V. GELBOIN and J.O. MINERS, J. Pharmacol. Exp. Ther. 265 401Ð407 (1993). 17. K. YOSHIMOTO, H. ECHIZEN, K. CHIBA, M. TANI and T. ISHIZAKI, Br. J. Clin. Pharmacol. 39 421Ð431 (1995). 18. M. BOURRIE, V. MEUNIER, Y. BERGER and G. FABRE, J. Pharmacol. Exp. Ther. 277 321Ð332 (1996). 19. D.J. NEWTON, R.W. WANG and A.Y. LU, Drug Metab. Dispos. 23 154Ð158 (1995). 20. E. SPINA, C. BIRGERSSON, C. VON BAHR, O. ERICSSON, B. MELLSTROM, E. STEINER and F. SJOQVIST, Clin. Pharmacol. Ther. 36 677Ð682 (1984). 21. L.M. DISTLERATH, P.E. REILLY, M.V. MARTIN, G.G. DAVIS, G.R. WILKINSON and F.P. GUENGERICH, J. Biol. Chem. 260 9057Ð9067 (1985). 22. R.K. SALOKANGAS, S. SAARIJARVI, T. TAIMINEN, H. LEHTO, H. NIEMI, V. AHOLA and E. SYVALAHTI, Schizophr. Res. 23 55Ð60 (1997). 23. M. NAKAJIMA, T. YOKOI, M. MIZUTANI, S. SHIN, F.F. KADLUBAR and T. KAMATAKI, Cancer Epidemiol. Biomarkers Prev. 3 413Ð421 (1994). 24. T. SHIMADA, H. YAMAZAKI, M. MIMURA, Y. INUI AND F.P. GUENGERICH, J. Pharmacol. Exp. Ther. 270 414Ð423 (1994).
IdentiÞcation of human cytochrome P450 isoforms involved in the 7-hydroxylation of chlorpromazine by human liver microsomes Kazuyoshi Yoshiia, Kaoru Kobayashia,*, Mihoko Tsumujia, Masayoshi Tanib, Noriaki Shimadac, Kan Chibaa a
Laboratory of Biochemical Pharmacology and Toxicology, Faculty of Pharmaceutical Sciences, Chiba University, Yayoi-cho 1-33, Inage-ku, Chiba 263-8522, Japan b Department of General Surgery, International Medical Center of Japan, Tokyo, Japan c Material Technology Research Laboratories, Daiichi Pure Chemicals Co. Ltd., Ibaraki, Japan
Abstract Studies to identify the cytochrome P450 (CYP) isoform(s) involved in chlorpromazine 7-hydroxylation were performed using human liver microsomes and cDNA-expressed human CYPs. The kinetics of chlorpromazine 7-hydroxylation in human liver microsomes showed a simple Michaelis-Menten behavior. The apparent Km and Vmax values were 3.4 6 1.0 mM and 200.5 6 83.7 pmol/min/mg, respectively. The chlorpromazine 7-hydroxylase activity in human liver microsomes showed good correlations with desipramine 2-hydroxylase activity (r 5 0.763, p , 0.05), a marker activity for CYP2D6, and phenacetin O-deethylase activity (r 5 0.638, p , 0.05), a marker activity for CYP1A2. Quinidine (an inhibitor of CYP2D6) completely inhibited while a-naphthoßavone (an inhibitor of CYP1A2) marginally inhibited the chlorpromazine 7-hydroxylase activity in a human liver microsomal sample showing high CYP2D6 activity. On the other hand, a-naphthoßavone inhibited the chlorpromazine 7-hydroxylase activity to 55Ð65% of control in a human liver microsomal sample showing low CYP2D6 activity. Among eleven cDNA-expressed CYPs studied, CYP2D6 and CYP1A2 exhibited signiÞcant activity for the chlorpromazine 7-hydroxylation. The K m values for the chlorpromazine 7-hydroxylation of both cDNA-expressed CYP2D6 and CYP1A2 were in agreement with the Km values of human liver microsomes. These results suggest that chlorpromazine 7-hydroxylation is catalyzed mainly by CYP2D6 and partially by CYP1A2. © 2000 Elsevier Science Inc. All rights reserved. Keywords: Chlorpromazine 7-hydroxylation; Cytochrome P450; Microsomes; Human; cDNA-expressed system
* Corresponding author. Tel.: (81) 43 290 2920; fax: (81) 43 290 2920. E-mail address: [email protected] (K. Kobayashi) 0024-3205/00/$ Ð see front matter © 2000 Elsevier Science Inc. All rights reserved. PII: S 0 0 2 4 - 3 2 0 5 ( 0 0 )0 0 6 1 3 -5
176
K. Yoshii et al. / Life Sciences 67 (2000) 175Ð184
Introduction Chlorpromazine is generally regarded as a prototype phenothiazine antipsychotic agent. The drug is extensively metabolized in the liver, and the main metabolic pathways include oxidative processes such as 7-hydroxylation, N-dealkylation, N-oxidation, S-oxidation, as well as other routes that include conjugation of 7-hydroxylated products (1,2). Among these pathways, the 7-hydroxylation is known as a major pathway of chlorpromazine metabolism in human (2,3). In vitro studies (4,5) with human liver microsomes have suggested that chlorpromazine is a potent competitive inhibitor of cytochrome P450 2D6 (CYP2D6). In addition, an in vivo study (6) indicated that administration of quinidine, a potent inhibitor of CYP2D6, signiÞcantly decreased the urinary excretion of 7-hydroxychlorpromazine in healthy volunteers who received a single dose of chlorpromazine. These data suggest that chlorpromazine is a substrate of CYP2D6 and that the enzyme is involved in the metabolism of chlorpromazine to 7-hydroxychlorpromazine. On the other hand, it has been reported that cigarette smoking increases the clearance of chlorpromazine (7,8). Cigarette smoking is well known to cause marked induction of CYP1A2 and accelerate the metabolism of drugs catalyzed by CYP1A2, such as theophylline (9,10), caffeine (11) and phenacetin (12,13). Therefore, CYP1A2 is thought to be involved in the chlorpromazine metabolism. However, to our knowledge, no detailed study has been reported with respect to the speciÞc isoform(s) of CYP involved in the chlorpromazine metabolism in humans. Therefore, the aim of the present study was to clarify the roles of CYP2D6 and CYP1A2 in chlorpromazine 7-hydroxylation in the human liver. Materials and methods Materials Human liver samples 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). Microsomes were prepared as reported previously (14) and stored at 2808C until use. Microsomes were previously characterized by desipramine 2-hydroxylase activity catalyzed by CYP2D6. Microsomes prepared from human B-lymphoblastoid cells expressing human CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4 and CYP4A11 (Gentest Corp., Woburn, MA, USA) were similarly stored at 2808C until use. Chlorpromazine was a gift from Yoshitomi Pharmaceutical Co. (Osaka, Japan), and 7-hydroxychlorpromazine glucuronide was from Psychopharmacology Research Branch, National Institute of Mental Health (Rockville, Md., USA). The other chemical reagents were purchased from commercial sources. Chlorpromazine 7-hydroxylase activities Typical incubation mixtures contained 0.2 mg/ml microsomal protein, 0.1 mM EDTA, 100 mM potassium phosphate buffer (pH 7.4), an NADPH-generating system (0.5 mM NADP1,
K. Yoshii et al. / Life Sciences 67 (2000) 175Ð184
177
2.0 mM glucose-6-phosphate, 1 IU/ml of glucose-6-phosphate dehydrogenase and 4 mM MgCl2), and 1 to 24 mM of chlorpromazine, in a Þnal volume of 250 ml. The mixture was incubated at 378C for 30 min. After the reaction was stopped by adding 100 ml of cold acetonitrile, 50 ml of amitriptyline (10 mM in methanol) was added to the samples as an internal standard. The mixture was centrifuged at 14,0003g for 10 min, and the supernatant was transferred to an autosampling vial for HPLC analysis. The mobile phase consisted of 50 mM sodium bicarbonate and methanol at a ratio of 20:80 (v/v) and was delivered at a ßow rate of 0.8 ml/min. The analytical column used was a 4.6 3 250 mm CAPCELL PAK C18 UG120 column (Shiseido Co., Tokyo, Japan). The eluate was monitored at a wavelength of 248 nm. Concentrations of 7-hydroxychlorpromazine in the reaction mixture were determined based on calibration curves constructed from a series of standards containing varying known amounts of 7-hydroxychlorpromazine together with the internal standard. 7-Hydroxychlorpromazine used as a standard was prepared by hydrolysis of 7-hydroxychlorpromazine glucuronide using b-glucuronidase. Kinetics in human liver microsomes Kinetic studies were performed with microsomes from Þve human livers (HL38, HL40, HL41, HL42 and HL43). In determining kinetic parameters, the chlorpromazine concentration ranged from 1Ð24 mM. All reactions were performed in a linear range with respect to protein concentration and incubation time: 0.2 mg/ml microsomal protein and 30-min incubation time. Eadie-Hofstee plots were constructed for determination of the presence of a mono- or biphasic model. Kinetic parameters were estimated by non-linear least-squares regression analysis of untransformed data. Correlation study Correlations between chlorpromazine 7-hydroxylase activities and the metabolic activities of substrates toward the respective distinct CYP isoforms were studied by using 10 different human liver microsomes. Assays for phenacetin O-deethylation at the substrate concentration of 10 mM (CYP1A2), diclofenac 49-hydroxylation at 10 mM (CYP2C9), S-mephenytoin 49-hydroxylation at 100 mM (CYP2C19), desipramine 2-hydroxylation at 10 mM (CYP2D6), testosterone 6b-hydroxylation at 30 mM (CYP3A4) and chlorpromazine 7-hydroxylation at 10 mM were performed. Metabolites of the in vitro probe substrates described above that were formed in the incubation mixtures were determined according to the respective HPLC assay methods as reported elsewhere (14Ð17). Inhibition study The effects of selective inhibitors or substrates (i.e., compounds acting as competitive inhibitors) for Þve different CYP isoforms on the chlorpromazine 7-hydroxylation at 10 mM in human liver microsomes (HL38) were studied. The isoform-selective inhibitors and alternative substrates (18,19) used in this part of the study were 10 mM a-naphthoßavone (CYP1A), 100 mM sulfaphenazole (CYP2C9), 500 mM mephenytoin (CYP2C19), 1 mM quinidine (CYP2D6) and 25 mM troleandomycin (CYP3A4). The effects of a-naphthoßavone and quinidine on the chlorpromazine 7-hydroxylation at 10 mM in two different human liver mi-
178
K. Yoshii et al. / Life Sciences 67 (2000) 175Ð184
crosomes (HL38 and HL39) were also studied at inhibitor concentrations ranging from 0.01 to 10 mM. Assay with cDNA-expressed CYPs Microsomes from human B-lymphoblastoid cells expressing human CYP1A1 (44 pmol CYP/mg), CYP1A2 (124 pmol CYP/mg), CYP2A6 (103 pmol CYP/mg), CYP2B6 (130 pmol CYP/mg), CYP2C8 (26 pmol CYP/mg), CYP2C9 (80 pmol CYP/mg), CYP2C19 (29 pmol CYP/mg), CYP2D6 (39 pmol CYP/mg), CYP2E1 (198 pmol CYP/mg), CYP3A4 (59 pmol CYP/mg) and CYP4A11 (18 pmol CYP/mg) were used. The reactions were carried out as described above. To examine the role of individual CYP isoforms involved in chlorpromazine 7-hydroxylation, each of the cDNA-expressed CYPs (0.5 mg/ml protein concentration) described above was Þrstly incubated with 100 mM chlorpromazine for 60 min at 378C according to the procedure recommended by the supplier. Kinetic analyses were performed using cDNA-expressed CYP1A2 and cDNA-expressed CYP2D6. Chlorpromazine 7-hydroxylase activities were determined using chlorpromazine concentrations ranging from 2Ð80 mM. All reactions were performed within a linear range with respect to protein concentration and incubation time. Brießy, 0.4 mg/ml of cDNA-expressed CYP1A2 and cDNA-expressed CYP2D6 were incubated for 60 min and 15 min, respectively. Kinetic parameters were estimated as described above. Data analysis Data are expressed as mean 6 SD throughout the text, if not otherwise stated. Correlations between the metabolite formation rates of respective CYP isoform-speciÞc substrates and chlorpromazine 7-hydroxylase activities were examined by the least-squares linear regression method. Results Kinetics in human liver microsomes Eadie-Hofstee plots for chlorpromazine 7-hydroxylase activity in four different human liver microsomes are shown in Fig. 1. In all of the human liver microsomes studied, the EadieHofstee plots were linear. Thus, we were able to estimate the kinetic parameters by Þtting them to a simple Michaelis-Menten equation. The mean (6 SD) kinetic parameters of four human liver microsomes were: apparent Km 5 3.4 6 1.0 mM, Vmax 5 200.5 6 83.7 pmol/ min/mg protein, and Vmax/Km 5 58.8 6 16.1 mL/min/mg protein. Correlation study Fig. 2A shows a signiÞcant correlation (r 5 0.763, p , 0.05) between chlorpromazine 7-hydroxylase activity and desipramine 2-hydroxylase activity in a panel of 10 human liver microsomes. A signiÞcant correlation (r 5 0.638, p , 0.05) was also observed between chlorpromazine 7-hydroxylase activity and phenacetin O-deethylase activity in the same panel of human liver microsomes (Fig. 2B). No signiÞcant correlations were observed be-
K. Yoshii et al. / Life Sciences 67 (2000) 175Ð184
179
Fig. 1. Eadie-Hofstee plots of chlorpromazine 7-hydroxylase activity in human liver microsomes prepared from Þve different subjects. d, HL38, Km 5 4.8 mM, Vmax 5 340.2 pmol/min/mg; m, HL40, Km 5 2.5 mM, Vmax 5 118.5 pmol/min/mg; j, HL41, Km 5 3.4 mM, Vmax 5 191.6 pmol/min/mg; s, HL42, Km 5 4.0 mM, Vmax 5 160.6 pmol/min/mg; n, HL43, Km 5 2.4 mM, Vmax 5 191.6 pmol/min/mg.
tween chlorpromazine 7-hydroxylase activity and catalytic activities of diclofenac 49-hydroxylase (r 5 0.518), S-mephenytoin 49-hydroxylase (r 5 0.523) or testosterone 6b-hydroxylase (r 5 0.402). Inhibition study The CYP-isoform selective probes were screened for inhibitory effects on the chlorpromazine 7-hydroxylase activity in a human liver microsome (HL38). Quinidine, a potent inhibitor of CYP2D6, completely inhibited the chlorpromazine 7-hydroxylase activity (the control activity in the absence of quinidine was 201.7 pmol/min/mg). In contrast, the selective probes for CYP1A (a-naphthoßavone), CYP2C9 (sulfaphenazole) and CYP2C19 (mephenytoin) and CYP3A4 (troleandomycin) exhibited no inhibitory effect or marginal inhibition on the chlorpromazine 7-hydroxylase activity (87.5Ð118.6% of control activity). Subsequently, the concentration-effect relationship of a-naphthoßavone and quinidine on the chlorpromazine 7-hydroxylase activity in two different human liver microsomes (HL38 and HL39) was studied. In our preliminary study, desipramine 2-hydroxylase activity, a marker activity of CYP2D6, in microsomes from HL38 and HL39 was 0.218 and 0.009 nmol/min/mg, respectively. Therefore, the microsomes from HL38 and HL39 were selected as representative microsomes in which the catalytic activity by CYP2D6 was high and low, respectively. In the microsomes from HL38, the chlorpromazine 7-hydroxylase activity was inhibited to an undetectable level by 1 mM quinidine but the effect of a-naphthoßavone was marginal (Fig. 3A). On the other hand, the chlorpromazine 7-hydroxylase activity in the microsomes from HL39 was inhibited to 48% of the control activity by 1 mM quinidine and to 55Ð65% of the control activity by 0.1Ð10 mM a-naphthoßavone (Fig. 3B).
180
K. Yoshii et al. / Life Sciences 67 (2000) 175Ð184
Fig. 2. Correlations between chlorpromazine 7-hydroxylase activity and desipramine 2-hydroxylase activity (A) or phenacetin O-deethylase activity (B) in a panel of ten human liver microsomes. Correlations were examined with the least-squares linear regression method.
Study with cDNA-expressed CYPs Microsomes from human B-lymphoblastoid cell lines expressing each of eleven CYP isoforms were examined in terms of the abilities of individual CYP isoforms to catalyze the chlorpromazine 7-hydroxylation. CYP2D6 and CYP1A2 were found to catalyze the reaction (217 and 26 pmol/60 min/pmol CYP, respectively), whereas the other CYP isoforms had little discernible or no effect with regard to the chlorpromazine 7-hydroxylation (Fig. 4). Accordingly, kinetic parameters for the chlorpromazine 7-hydroxylation were estimated for cDNAexpressed CYP2D6 and cDNA-expressed CYP1A2. The Km values of CYP2D6 and CYP1A2 were 4.3 6 1.0 and 5.1 6 2.0 mM, respectively. These Km values were similar to that obtained from human liver microsomes (Table 1). The Vmax value of CYP2D6 was greater that that of CYP1A2 (11.7 6 0.9 vs. 0.7 6 0.1 pmol/min/pmol CYP). cDNA-expressed CYP2D6 showed a greater Vmax/Km value than that of cDNA-expressed CYP1A2 (2.83 6 0.82 and 0.15 6 0.04 mL/min/pmol CYP, respectively). Discussion The results suggest that the formation of 7-hydroxychlorpromazine is predominantly catalyzed by CYP2D6. This conclusion was inferred from the following observations. First, the chlorpromazine 7-hydroxylase activity showed a signiÞcant correlation with desipramine 2-hydroxylase activity, a maker reaction of CYP2D6 (20), in a panel of 10 human liver microsomes (Fig. 2A). Second, 1 mM quinidine, a speciÞc inhibitor of CYP2D6, completely inhibited the chlorpromazine 7-hydroxylase activity in human liver microsomes (Fig. 3A).
K. Yoshii et al. / Life Sciences 67 (2000) 175Ð184
181
Fig. 3. Effects of quinidine and a-naphthoßavone on chlorpromazine 7-hydroxylase activity in two different human liver microsomes. The chlorpromazine 7-hydroxylase activities at 10 mM of substrate concentration were determined using microsomes from HL38 in which desipramine 2-hydroxylase activity is high (A) and HL39 in which desipramine 2-hydroxylase activity is low (B). The control activities in the absence of inhibitors were 212.9 and 157.9 pmol/min/mg for HL38 and HL39, respectively. The concentrations of quinidine (d) and a-naphthoßavone (s) ranged from 0.01Ð10 mM. Each data point represents the mean of duplicate determinations.
Third, cDNA-expressed CYP2D6 catalyzed chlorpromazine 7-hydroxylation (Fig. 4). These in vitro Þndings were in agreement with the in vivo observation (6) that the urinary excretion of 7-hydroxychlorpromazine was decreased by pretreatment with quinidine, a speciÞc inhibitor of CYP2D6, in healthy volunteers who received a single oral dose of chlorpromazine, suggesting CYP2D6 is involved in chlorpromazine 7-hydroxylation. The chlorpromazine 7-hydroxylation was catalyzed by not only cDNA-expressed CYP2D6 but also by cDNA-expressed CYP1A2 (Fig. 4). In addition, the correlation study using a panel of 10 human liver microsomes showed there was a signiÞcant correlation between the chlorpromazine 7-hydroxylase activity and the phenacetin O-deethylase activity, a marker reaction of CYP1A2 (16,21) (Fig. 2B). Moreover, a-naphthoßavone, a speciÞc inhibitor for CYP1A2, also inhibited the chlorpromazine 7-hydroxylase activity in a human liver microsomal sample showing low CYP2D6 activity (Fig. 3B). These results suggest that not only CYP2D6 but also CYP1A2 is responsible for catalyzing the chlorpromazine 7-hydroxylation in human liver microsomes. Previously, some investigators (7,8,22) reported that the clearance of chlorpromazine was elevated by cigarette smoking. It is well established that cigarette smoking causes marked induction of CYP1A2 (13,23). Therefore, these in vivo Þndings are at least partially explained by the present results showing that chlorpromazine 7-hydroxylation in human liver microsomes is catalyzed by a cigarette smoking-inducible enzyme, CYP1A2. The results suggest that both CYP2D6 and CYP1A2 are involved in the chlorpromazine 7-hydroxylation. This is not contradicted to the Þnding that Eadie-Hofstee plots for chlorpro-
182
K. Yoshii et al. / Life Sciences 67 (2000) 175Ð184
Fig. 4. Formation of 7-hydroxychlorpromazine from chlorpromazine in microsomes from human B-lymphoblastoid cells expressing the human CYP isoform. A substrate (100 mM chlorpromazine) was incubated at 378C for 60 min with microsomes (0.5 mg/ml) of human B-lymphoblastoid cells expressing CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4 and CYP4A11, respectively. ND, not detectable.
mazine 7-hydroxylation were linear in all human liver microsomal samples studied (Fig. 1), since the results of cDNA-expressed enzymes indicated that Km values of CYP2D6 and CYP1A2 for chlorpromazine 7-hydroxylation were essentially identical (Table 1). These observations support the notion that both CYP2D6 and CYP1A2 are responsible for the chlorpromazine 7-hydroxylation in human liver microsomes. However, the Vmax/Km value of cDNA-expressed CYP2D6 was approximately 20-fold greater than that of cDNA-expressed Table 1 Kinetic parameters of chlorpromazine 7-hydroxylase activity in microsomes from human B-lymphoblastoid cells expressing CYP1A2 or CYP2D6 Isoforms
Km (mM)
Vmax (pmol/min/pmol CYP)
Vmax/Km (mL/min/pmol CYP)
CYP1A2 CYP2D6
5.1 6 2.0 4.3 6 1.0
0.70 6 0.13 11.7 6 0.91
0.15 6 0.04 2.83 6 0.82
Values are expressed as mean 6 SD from three different experiments.
K. Yoshii et al. / Life Sciences 67 (2000) 175Ð184
183
CYP1A2 (Table 1). In human liver microsomes, it has been reported that the contents of CYP2D6 and CYP1A2 are 1.5 6 1.3% and 12.7 6 6.2% of total CYP, respectively (24), indicating CYP2D6 content is 10-fold lower than CYP1A2 content. Accordingly, it was estimated that the contribution of CYP2D6 to the chlorpromazine 7-hydroxylation in human liver microsomes is about two-fold higher than that of CYP1A2. In the human liver microsomal studies, the inhibition of chlorpromazine 7-hydroxylase activity in human liver microsomes by quinidine, a CYP2D6 inhibitor, was more potent than that by a-naphthoßavone, a CYP1A2 inhibitor (Fig. 3A). In addition, the correlation coefÞcient value of chlorpromazine 7-hydroxylase activity with desipramine 2-hydroxylase activity was higher than that with phenacetin O-deethylase activity (Fig. 2). Therefore, both CYP2D6 and CYP1A2 are involved in the 7-hydroxylation of chlorpromazine in human liver microsomes, but the contribution of CYP1A2 to this metabolism appears to be lower than that of CYP2D6. There are large interindividual differences in expression levels of CYP1A2 and CYP2D6 in human liver microsomes. Indeed, CYP1A2 content in microsomes ranged from 0.3 to 100fold of CYP2D6 content in commercially available sources (Gentest). Therefore, the effects of quinidine and a-naphthoßavone on chlorpromazine 7-hydroxylase activity in human liver microsomes were determined with the microsomes from HL38 and HL39, which have a more than 20-fold difference in desipramine 2-hydroxylase activity. Quinidine at 1 mM completely inhibited the chlorpromazine 7-hydroxylase activity in microsomes from HL38 with high catalytic activity by CYP2D6, but the inhibitory effect in microsomes from HL39 with low catalytic activity by CYP2D6 was only 52% (Fig. 3). In contrast, the inhibitory effect by a-naphthoßavone in microsomes from HL39 was greater than that in microsomes from HL38 (Fig. 3). These results are thought to reßect the differences of CYP1A2 and CYP2D6 contents in two human liver microsomes. Therefore, the contribution of CYP1A2 to the chlorpromazine 7-hydroxylation might be signiÞcant in human liver microsomes with low CYP2D6 contents. In conclusion, the results of the present study using human liver microsomes and cDNAexpressed enzymes strongly suggest that the chlorpromazine 7-hydroxylation is catalyzed mainly by CYP2D6 and partially by CYP1A2. To our knowledge, this is the Þrst in vitro study to demonstrate that CYP2D6 and CYP1A2 are responsible for chlorpromazine 7-hydroxylation. In human liver microsomes with low CYP2D6 contents, CYP1A2 may make a signiÞcant contribution to chlorpromazine 7-hydroxylation. Therefore, both the phenotype of CYP2D6 and smoking habits could reßect the ability of chlorpromazine 7-hydroxylation in humans. References 1. E. USDIN, Crc. Crit. Rev. Clin. Lab. Sci. 2 347Ð391 (1971). 2. F. HARTMANN, L.D. GRUENKE, J.C. CRAIG and D.M. BISSELL, Drug Metab. Dispos. 11 244Ð248 (1983). 3. P.K.F. YEUNG, J.W. HUBBARD, E.D. KORCHINSKI and K.K. MIDHA, Eur. J. Clin. Pharmacol. 45 563Ð 569 (1993). 4. S.V. OTTON, T. INABA, W. KALOW, Life Sci. 32 795Ð800 (1983). 5. C. VON BAHR, E. SPINA, C. BIRGERSSON, O. ERICSSON, M. GORANSSON, T. HENTHORN and F. SJOQVIST, Biochem. Pharmacol. 34 2501Ð2505 (1985). 6. G. MURALIDHARAN, J.K. COOPER, E.M. HAWES, E.D. KORCHINSKI and K.K. MIDHA, Eur. J. Clin. Pharmacol. 50 121Ð128 (1996).
184
K. Yoshii et al. / Life Sciences 67 (2000) 175Ð184
7. E.J. PANTUCK, C.B. PANTUCK, K.E. ANDERSON, A.H. CONNEY and A. KAPPAS, Clin. Pharmacol. Ther. 31 533Ð538 (1982). 8. M. CHETTY, R. MILLER and S.V. MOODLEY, Eur. J. Clin. Pharmacol. 46 523Ð526 (1994). 9. J.J. GRYGIEL and D.J. BIRKETT, Clin. Pharmacol. Ther. 30 491Ð496 (1981). 10. S.N. HUNT, W.J. JUSKO and A.M. YURCHAK, Clin. Pharmacol. Ther. 19 546Ð551 (1976). 11. J.A. CARRILLO and J. BENITEZ, Clin. Pharmacol. Ther. 55 293Ð304 (1994). 12. E.J. PANTUCK, K.C. HSIAO, A. MAGGIO, K. NAKAMURA, R. KUNTZMAN and A.H. CONNEY, Clin. Pharmacol. Ther. 15 9Ð17 (1974). 13. D. SESARDIC, A.R. BOOBIS, R.J. EDWARDS and D.S. DAVIES, Br. J. Clin. Pharmacol. 26 363Ð372 (1988). 14. K. CHIBA, K. KOBAYASHI, M. TANI, K. MANABE and T. ISHIZAKI, Drug Metab. Dispos. 21 747Ð749 (1993). 15. K. CHIBA, K. MANABE, K. KOBAYASHI, Y. TAKAYAMA, M. TANI and T. ISHIZAKI, Eur. J. Clin. Pharmacol. 44 559Ð562 (1993). 16. W. TASSANEEYAKUL, D.J. BIRKETT, M.E. VERONESE, M.E. MCMANUS, R.H. TUKEY, L.C. QUATTROCHI, H.V. GELBOIN and J.O. MINERS, J. Pharmacol. Exp. Ther. 265 401Ð407 (1993). 17. K. YOSHIMOTO, H. ECHIZEN, K. CHIBA, M. TANI and T. ISHIZAKI, Br. J. Clin. Pharmacol. 39 421Ð431 (1995). 18. M. BOURRIE, V. MEUNIER, Y. BERGER and G. FABRE, J. Pharmacol. Exp. Ther. 277 321Ð332 (1996). 19. D.J. NEWTON, R.W. WANG and A.Y. LU, Drug Metab. Dispos. 23 154Ð158 (1995). 20. E. SPINA, C. BIRGERSSON, C. VON BAHR, O. ERICSSON, B. MELLSTROM, E. STEINER and F. SJOQVIST, Clin. Pharmacol. Ther. 36 677Ð682 (1984). 21. L.M. DISTLERATH, P.E. REILLY, M.V. MARTIN, G.G. DAVIS, G.R. WILKINSON and F.P. GUENGERICH, J. Biol. Chem. 260 9057Ð9067 (1985). 22. R.K. SALOKANGAS, S. SAARIJARVI, T. TAIMINEN, H. LEHTO, H. NIEMI, V. AHOLA and E. SYVALAHTI, Schizophr. Res. 23 55Ð60 (1997). 23. M. NAKAJIMA, T. YOKOI, M. MIZUTANI, S. SHIN, F.F. KADLUBAR and T. KAMATAKI, Cancer Epidemiol. Biomarkers Prev. 3 413Ð421 (1994). 24. T. SHIMADA, H. YAMAZAKI, M. MIMURA, Y. INUI AND F.P. GUENGERICH, J. Pharmacol. Exp. Ther. 270 414Ð423 (1994).