The Decreased in Vivo Clearance of CYP2D6 Substrates by CYP2D6*10 Might Be Caused Not Only by the Low-Expression but Also by Low Affinity of CYP2D6

Archives of Biochemistry and Biophysics Vol. 380, No. 2, August 15, pp. 303–308, 2000 doi:10.1006/abbi.2000.1936, available online at http://www.idealibrary.com on

The Decreased in Vivo Clearance of CYP2D6 Substrates by CYP2D6*10 Might Be Caused Not Only by the Low-Expression but Also by Low Affinity of CYP2D6 Tsuyoshi Fukuda,* Yuko Nishida,* Susumu Imaoka,† Toyoko Hiroi,† Masakazu Naohara,* Yoshihiko Funae,† and Junichi Azuma* ,1 *Clinical Evaluation of Medicines and Therapeutics, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamada-oka Suita, Osaka 565-0871 Japan; and †Department of Chemical Biology, Osaka City University Medical School, Osaka, Japan

Received January 27, 2000, and in revised form May 10, 2000

CYP2D6 exhibits genetic polymorphism with interindividual differences in metabolic activity. We have found a significant influence on the pharmacokinetics of venlafaxine by the CYP2D6*10 allele in a Japanese population. CYP2D6.10, which is translated from CYP2D6*10, has two amino acid substitutions: Pro34 3 Ser and Ser486 3 Thr. In this study, CYP2D6.10 was expressed in Saccharomyces cerevisiae and its catalytic activity for CYP2D6 substrates was investigated. The CYP2D6*10B- and *10C-associated cDNA were isolated from human lymphocyte genotyped as CYP2D6*10. In addition, three forms of CYP2D6, Pro34/Thr486 (PT), Ser34/Ser486 (SS), and Pro34/Ser486 (wild type, CYP2D6.1), were constructed by PCR-site mutagenesis to clarify the effects of the two amino-acid substitutions. The expression of CYP2D6 protein was confirmed by immunoblotting using CYP2D antibody. The absorbance at 450 nm was measured by CO-reduced difference spectra from five all microsome preparations. The CYP2D6 forms with Pro34 3 Ser amino acid substitution were at a lower expression than CYP2D6.1 from the findings of immunoblotting and spectral analysis. The apparent K m values of CYP2D6.1, CYP2D6.10A, and CYP2D6.10C were 1.7, 8.5, and 49.7 ␮M, respectively, for bufuralol 1ⴕhydroxylation, and 9.0, 51.9, and 117.4 ␮M, respectively, for venlafaxine O-demethylation, respectively. The V max values were not significantly different among the three variants. These findings suggest that the decreased in vivo clearance by CYP2D6*10 was caused not only by low expression of but also the increased K m value of CYP2D6. © 2000 Academic Press 1 To whom correspondence should be addressed. Fax: ⫹81-6-68798259. E-mail: [email protected].

0003-9861/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.

Key Words: CYP2D6; polymorphism; CYP2D6*10; expression; venlafaxine.

The CYP2D subfamily has received much attention during the past decade due largely to the involvement of CYP2D6 in humans, particularly as the enzyme metabolizes over 40 clinically important drugs (1). More than 20 genetic polymorphisms exist in human CYP2D6 (2). The frequency of the poor metabolizer (PM) 2 with deficient CYP2D6 activity is reported to be 5 to 10% in Caucasians (3) and less than 1% in Japanese (4). The CYP2D6*10 allele, which includes both the CYP2D6*10A and CYP2D6*10B variants, is widely observed in Japanese (38%, unpublished data) and Chinese (50%) and has two amino-acid substitutions, Proline (Pro)34 3 Serine (Ser) and Ser486 3 Threonine (Thr) (5, 6). In Chinese subjects, the influence of this allele on propranolol disposition (7), formation of morphine from codeine (8), and nortriptiline disposition (9) was reported. In Japanese subjects, we found a significant influence on the pharmacokinetics of venlafaxine by CYP2D6*10 allele (10). The C max and AUC values of venlafaxine in homozygotes for CYP2D6*10 were three- and sixfold higher than those in homozygotes for CYP2D6*1 or *2. Venlafaxine, which is a new antidepressant that selectively inhibits reuptake of norepinephrine and serotonin (11), was O-desmethylated by CYP2D6 in human microsomes (12). 2 Abbreviations used: P450 and CYP, cytochrome P450; RT-PCR, reverse transcriptase-polymerase chain reaction; pBS, pBluescript; SDS, sodium dodecyl sulfate; ODV, O-desmethylvenlafaxine; PM, poor metabolizer.

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Primer Sequence in Current Investigation Primer name

Sequences for human CYP2D6 a

C1 C2 188N 670A 620 1190 1446A 4268N HindIII-2D6

5⬘-CATTTGGTACTGAGGCAGGT-3⬘ 5⬘-GCAGGCTGGGGACTAGGTA-3⬘ 5⬘-CAGCCCGGGCAGTGGCAGGGGGCCTGGTGGGTAGCGTGCA-3⬘ 5⬘-ACAGCATTCAGCACCTCGCG-3⬘ 5⬘-GGCTGCTGGACCTAGCTCAG-3⬘ 5⬘-CACTCATCACCAACCTGTCA-3⬘ 5⬘-TCACCAGGAAAGCAAAGACA-3⬘ 5⬘-TTGCTTTCCTGGTGAGCCCATCCCCCTATGA-3⬘ 5⬘-AAAAAAGCTTAAAAAAATGGGGCTAGAAGCACTGGTGCCCC-3⬘

a All primers were designed from nucleotide sequences described by Gonzalez et al. (18, 19).

upstream fragment (pBS2D6*10up) and the CYP2D6 cDNA downstream fragment (pBS2D6*10down). These CYP2D6 related cDNAs were verified using a sequencing method. Construction of CYP2D6*10 cDNA. To introduce a HindIII linker to the 5⬘-end of the start codon, one set of primers was used to carry out the PCR with pBS2D6*10up as the template using the #HindIII2D6 and #670A primers. This PCR product was cut out with HindIII and BsmI and inserted into pBS2D6*10down to give full-length 2D6 cDNA (pBS2D6*10p). Then, the construct was cut with BamHI, blunt-ended by T4 DNA polymerase, and ligated with the HindIII linker (pBS2D6*10) (Fig. 1). PCR-based site-mutagenesis for the derivatives. The CYP2D6*10B allele has two mutations (gene numbers 188 and 4268) that lead to two amino-acid changes. PCR-based site mutagenesis was carried out to clarify the effect of these mutations (20). A mutation in exon 1

Recently, expression systems of CYP enzymes have been used to investigate the catalytic activities of the enzymes and to determine the metabolic specificity of chemical compounds (13–16). However, there has been no report on the catalytic property of CYP2D6.10, although Johansson et al. suggested that this variant in COS cells elucidated the decreased activity using bufuralol as a probe and reported that Pro34 3 Ser amino acid substitution caused expression of a more unstable gene product (6). We expressed CYP2D6.1, CYP2D6.10A, and CYP2D6.10C in yeast to compare their properties and metabolic activity for CYP2D6 substrates in the present study. MATERIALS AND METHODS Chemicals. Bufuralol and 1⬘-hydroxybufuralol were obtained from Daiichi Pure Chemicals Co. (Tokyo, Japan). NADPH was purchased from Oriental Yeast Co. (Tokyo, Japan). Other reagents and organic solvents were obtained from Wako Pure Chemical Industries (Tokyo, Japan). The expression vector pGYR1, which has a GAPDH promoter and includes the yeast NADPH-P450 reductase gene (17), was supplied by Dr. Y. Yabusaki of Sumitomo Chemical Co. (Osaka, Japan). Venlafaxine and O-desmethylvenlafaxine were from Wyeth Co. (Tokyo, Japan). Subject. Approval for the genetic study was obtained from the local Institutional Review Board. Informed consent was obtained from the subject. CYP2D6 genotyping analysis was carried out according to Fukuda et al. (11). In order to confirm the genotype, we sequenced the PCR product from the full-CYP2D6 gene of the subject. Isolation of CYP2D6*10 cDNA. The full-length CYP2D6*10 cDNA was isolated from human lymphocytes previously determined by genotyping as having the CYP2D6*10 allele. Total RNA was prepared using Isogen RNA isolation regent (Nippon Gene, Toyama, Japan). cDNA was synthesized using random primer with an AMV RNA-PCR kit ver2.0 (Takara, Siga, Japan) following the manufacturer’s instructions. KOD DNA polymerase (Toyobo, Japan) was used for the polymerase chain reaction (PCR) with two pairs of primers (C1/670A and 620/C2) following the manufacturer’s instructions to amplify two parts of CYP2D6 cDNA. All primers were designed from nucleotide sequences described by Gonzalez et al. (18, 19) in the present study (Table I). Two PCR products blunt-ended by T4 DNA polymerase were inserted into pBluescript (pBS) digested by EcoRV or SmaI to give two vectors containing the CYP2D6 cDNA

FIG. 1. Construction of plasmids carrying CYP2D6 cDNA. Restriction sites are Hd, HindIII; Bm, BsmI; Sa, SalI; Sp, SphI; Nt, NotI. Arrows indicate the amplifying direction. The numbers with the sharp mark by the arrows indicate the primers described under Materials and Methods. pBS2D6*10, pBS carrying full-length 2D6 cDNA with HindIII linker. The asterisk indicates the artificial mutation base in the primer and PCR product. The CYP2D6 downstream fragment including the CYP2D6*10C-related sequence was inserted inversely into pBS at the SmaI site (pBS-2D6*10down-2). The direction was changed using the pGEM vector.

THE EXPRESSION OF CYP2D6*10 FORM

FIG. 2. The structure of CYP2D6 cDNAs expressed in yeast cells. The cDNA isolated from human lymphocytes had four different nucleotides from the wild-type CYP2D6 cDNA reported by Gonzalez et al. (17) (but not Met-type) and reached a consensus with CYP2D6*10B (Ch1) reported by Johansson et al. (6). Four different nucleotides were the following: gene number 188 (lead to Pro34Ser), 1127, 1749 (silent), 4268 (Ser486Thr). CYP2D6*10-PT, -SS, and CYP2D6*1 were constructed by the PCR-based site mutagenesis method to demonstrate the effect of two amino-acid substitutions. The CYP2D6*10-Wt is expected to translate into CYP2D6.1. The CYP2D6*10C sequence was reported by Johansson et al. (6) as CYP2D6Ch2(*10C) that has 13 base substitutions more than Ch1(*10B).

(gene number 188) was converted into wild-type sequence by PCR with pBS2D6*10 as a template using the M13/pUC universal reverse primer and the #188N primer. This PCR product was digested by SmaI and SalI and inserted into pBS2D6*10 (pBS2D6*10-PT). The other mutation in exon 9 (gene 4268) was converted using two sets of primers. The first set was the #1190 and #1446A primers, and the second set was the #4268N primer and the M13/PUC universal forward primer. Subsequently, these PCR-amplified fragments served as templates to amplify a full-length fragment using the #1190 primer and the M13/PUC universal forward primer. The final PCR product was cut with SphI and NotI and inserted into pBS2D6*10 (pBS2D6*10-SS) or pBS2D6*10-PT (pBS2D6*10-Wt) (Figs. 1 and 2). Construction of CYP2D6*10C cDNA. The sequence of the CYP2D6*10C, which had the gene conversion in exon 9 derived from CYP2D7, was described by Johansson et al. (6). The PCR product including the gene conversion in exon 9 was obtained from human lymphocytes by PCR using the #620 and #C2 primers. First, the fragment was inserted into pBS at the SmaI site and second transformed into pGEM-3Z(⫹) (Promega, USA) cut out with SphI and SalI. Finally, the fragment was cut out with SphI and BamHI from the pGEM plasmid and then inserted into pBS2D6*10p (pBS2D6*10C) (Figs. 1 and 2). Expression of CYP2D isoforms in Saccharomyces cerevisiae. Five CYP2D6-related cDNAs cut out with HindIII from the above vectors were subcloned into pGYR1 expression plasmids (21). The pGYR1 vectors containing CYP2D cDNAs were used for transformation of the Saccharomyces cerevisiae AH 22 strain (22). Enzyme assays. Bufuralol 1⬘-hydroxylation activity was measured according to the method of Chow et al. (23) with some modification. The mixture (0.50 ml) containing 5 pmol of P450 protein and 0.4 mM NADPH in 0.1 M potassium phosphate buffer (pH 7.4) was

305

incubated with bufuralol (final concentration of 1 to 100 ␮M) at 37°C after 3 min of preincubation without bufuralol. The incubation continued with gentle shaking at 37°C for 2 min and the reaction was stopped with 6 M perchloric acid (20 ␮l). Denatured protein was precipitated by centrifugation and the supernatant (100 ␮l) was analyzed by HPLC with a reverse-phase column (STR ODS-II, 4.6 ⫻ 250 mm, Shinwa Chemical Industries LTD, Kyoto, Japan). The column temperature was set at 50°C. The mobile phase was composed of 1 mM perchloric acid/acetonitrile (8:2) and was delivered at a gradient flow rate of 0.5 to 1.5 ml/min for 15 min and a constant flow rate of 1.5 ml/min for 15 min. 1⬘-Hydroxybufuralol was detected by a fluorescent detector (Nanospace SI-1, SHISEIDO, Tokyo Japan) with excitation/emission wavelengths at 252/302 nm. Venlafaxine O-demethylation activity was assayed in an incubation mixture (0.50 ml) containing 1.4 mg of microsomal protein and 0.4 mM NADPH in 0.1 M potassium phosphate buffer (pH 7.4). After 3 min of preincubation at 37°C, the reaction (for 30 min at 37°C) was initiated by the addition of venlafaxine (final concentration of 1 to 500 ␮M). Other conditions for incubation and deproteinization were as for the assay of bufuralol 1⬘-hydroxylation activity. The mobile phase was composed of 0.05 M sodium phosphate buffer (pH 3.0)/ acetonitrile (75:25) delivered at a flow rate of 0.6 ml/min. O-desmethylvenlafaxine was detected fluorometrically at 280 nm by excitation set at 195 nm (12, 24). Other methods. Antibodies against P450 UT-7 (CYP2D1) were prepared as described by Ohishi et al. (25). Immunoblotting was performed as described by Imaoka et al. (26). Microsomal proteins were separated by 7.5% SDS–polyacrylamide gel. The amounts of microsomal protein applied were 25.0 ␮g for yeast microsomes and CYP2D1 (1.0 pmol) was used as the control. The amounts of P450 were estimated according to the method of Omura and Sato (27). The protein concentration was measured according to the method of Lowry et al. (28). NADPH P450-reductase activity was measured according to the method of Yasukochi and Masters (29).

RESULTS

Isolation of Human CYP2D6 cDNAs The full-length CYP2D6*10B cDNA was isolated from human lymphocytes genotyped as CYP2D6*10type. Figure 2 shows the positions of nucleotide changes in coding regions and amino acid substitutions of CYP2D6 forms, which compared well with previously reported sequence data of wild-type CYP2D6 (18, 19). The nucleotide sequence of this CYP2D6 cDNA was completely identical with those reported by Gonzalez et al. (CYP2D6-Val type) except four nucleotides, that were associated with the CYP2D6*10 allele (6). The fragment related to CYP2D6*10C was obtained by PCR using the primer set 620 and C2. Fourteen nucleotide changes were detected in this fragment, which were identical with CYP2D6*10C described by Johannson et al. (6). Characterization of CYP2D Variants Expressed in Yeast Cells Immunoblotting was performed with antibody against rat CYP2D1 (25). This antibody recognized all CYP2D6 proteins as shown in Fig. 3. Nothing was detected in microsomal protein from yeast cells with pGYR1 vector only similarly reported by Wan et al.

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FUKUDA ET AL. TABLE II

The Contents of P450 and NADPH-P450 Reductase in CYP2D6 Variants Expressed in Yeast Cells

FIG. 3. Immunoblotting of microsomal protein from five CYP2D6 forms. Immunoblotting was carried out for microsomes of yeast cells transformed with recombinant vectors containing CYP2D6*10B (CYP2D6.10A), *10-PT (PT), *10-Wt (CYP2D6.1), *10-SS (SS), and *10C (CYP2D6.10C) cDNAs with antibody against rat CYP2D1 (25). Microsomal proteins were separated by 7.5% SDS–polyacrylamide gel. The amounts of microsomal protein applied were 25.0 ␮g for yeast microsomes and CYP2D1 (1.0 pmol) was used as the control. Nothing was detected in microsomal protein from yeast cells with pGYR1 vector only as previously reported by Wan et al. (21).

(21). The apparent bands of CYP2D6 in microsomal protein from yeast cells expressing CYP2D6*10B (CYP2D6.10A), CYP2D6*10-SS (SS), and CYP2D6*10C (CYP2D6.10C) were thinner than CYP2D6*10-Wt (CYP2D6.1) and CYP2D6*10-PT (PT) (Fig. 3). Typical CO-reduced difference spectra with an absorption maximum at 448 nm were observed with microsomal preparations from all yeast cell types transformed with pGYR1 carrying CYP2D cDNA inserts (Fig. 4), except microsomal preparations from yeast cells transformed with the pGYR1 vector alone (data not shown). The P450 contents of microsomal protein from yeast cells expressing CYP2D6*10B (CYP2D6.10A),

CYP2D6 variants

P450 a (pmol/mg)

NADPH-P450 reductase (unit/mg)

CYP2D6.1 CYP2D6.10A CYP2D6.10C PT b SS

83.1 4.46 5.35 78.5 4.07

0.179 0.169 0.184 0.162 0.239

a

The P450 contents were calculated by the CO-reduced difference spectra in microsomal protein (pmol/mg microsomal protein). b PT, 34Pro and 486Thr; SS, 34Ser and 486Ser.

CYP2D6*10-SS (SS), and CYP2D6*10C (CYP2D6.10C) were very low compared with CYP2D6*10-Wt (CYP2D6.1) and CYP2D6*10-PT (PT). Table II shows the specific contents of P450 calculated from CO-difference spectra and the activities of NADPH P450-reductase in the yeast microsomes. The absorption ratios of 420/450 in these microsomes were larger than that in CYP2D6.1 and PT. Remarkably, CYP2D6.10A, CYP2D6.10C, and SS had the Pro34 3 Ser amino acid substitution. The CO-reduced difference spectra by yeast microsomes from five forms paralleled those in whole cells, respectively, indicating that P450s in CYP2D6.10A and CYP2D6.10C microsomes were not denatured during preparation. NADPH P450-reductase activities of five microsomal preparations were almost similar, although the activity of SS was slightly higher than that of the others. Catalytic Activity of CYP2D Forms

FIG. 4. CO-reduced difference spectra of microsomes of yeast cells transformed with recombinant vectors containing CYP2D6 cDNAs. The spectra were measured in 0.02 M Tris–acetate buffer (pH 7.2) containing 20% glycerol and 0.4% Emulgen 911. The concentrations of microsomal protein were 1.85 (CYP2D6.1), 2.80 (CYP2D6.10A), 2.99 (CYP2D6.10C), 3.72 (PT), and 2.20 (SS) mg/ml, respectively. No absorption at around 450 nm was observed with control microsomes of yeast cells transformed with vector alone.

The catalytic activity of the human CYP2D6 expressed in yeast was determined with bufuralol and venlafaxine (Table III). The apparent K m values for bufuralol 1⬘-hydroxylation of CYP2D6.1, CYP2D6.10A, and CYP2D6.10C were 1.7, 8.5, and 49.7 ␮M, respectively. The apparent K m value of PT was similar to CYP2D6.1, and the value of SS was similar to CYP2D6.10A, suggesting that Pro34 3 Ser could be the principal factor that determines the apparent K m values. A higher apparent K m was observed for CYP2D6.10C compared with others. The V max values were not different among CYP2D6.1, CYP2D6.10A, and CYP2D6.10C. With regard to venlafaxine O-demethylation, the apparent K m values of CYP2D6.1, CYP2D6.10A, and CYP2D6.10C were 9.0, 51.9, and 117.4, respectively. The apparent K m value of PT for venlafaxine was slightly higher than that of CYP2D6.1. Also, the K m value of SS (30.5 ␮M) for venlafaxine was lower than that of CYP2D6.10A.

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THE EXPRESSION OF CYP2D6*10 FORM TABLE III

Kinetic Parameters of CYP2D6 Variants for Bufuralol 1⬘-Hydroxylation and Venlafaxine O-Demethylation Bufuralol (␮M)

CYP2D6.1 CYP2D6.10A CYP2D6.10C PT SS

Venlafaxine (␮M)

Km

V max

V max/K m

Km

V max

V max/K m

1.7 8.5 49.7 1.6 7.5

13.4 16.5 11.9 13.9 29.6

7.9 1.9 0.2 8.7 3.9

9.0 51.9 117.4 13.2 30.5

1.35 3.69 1.45 2.03 6.32

0.15 0.07 0.01 0.15 0.21

Note. Bufuralol: The mixture (0.50 ml) containing 5 pmol of P450 protein and 0.4 mM NADPH in 0.1 M potassium phosphate buffer (pH 7.4) was incubated with bufuralol (final concentration of 1 to 100 ␮M) at 37°C after 3 min of preincubation. The incubation continued with gentle shaking at 37°C for 2 min. Venlafaxine: The mixture (0.50 ml) containing 1.4 mg of microsomal protein and 0.4 mM NADPH in 0.1 M potassium phosphate buffer (pH 7.4) was used. After 3 min of preincubation at 37°C, the reaction was started by addition of venlafaxine (final concentration of 1 ␮M to 0.5 mM), and the incubation period was 30 min. The mobile phase was composed of 0.05 M sodium phosphate buffer (pH 3.0)/acetonitrile (75:25). O-Desmethylvenlafaxine was detected with excitation/emission wavelengths at 195/280 nm. Values were the mean from three independent experiments. V max, nmol/min/nmol P450; V max/K m , ml/min/nmol P450.

DISCUSSION

We expressed CYP2D6.10 in yeast cells and obtained enough enzyme protein to examine its property and to evaluate the apparent K m and V max for each substrate drug. We found a significant difference of venlafaxine in vivo pharmacokinetics among the different genotypes of CYP2D6 including CYP2D6*10, although the CYP2D6*10 has been categorized into extensive metabolizers (12). The findings of several clinical trials indicate the influence of the CYP2D6*10 genotype on pharmacokinetic parameters varies depending on CYP2D6 substrate drugs. On the other hand, the plasma concentrations of some drugs, i.e., nortriptyltine and haloperidol, are hardly affected by CYP2D6*10 (9, 30). Therefore, we attempted to evaluate the property of the expressed CYP2D6.10 enzyme in vitro for two substrates. The expression of the CYP2D6 protein was confirmed by immunoblotting using CYP2D1 antibody. Furthermore, the absorbance at 450 nm was measured by CO-reduced difference spectra from all five microsome preparations. The CYP2D6 forms containing Ser34 had evidently lower expression than CYP2D6 wild-type from the findings of immunoblotting and spectral analysis. Johansson et al. suggested that the enzyme with amino acid change, Pro34 3 Ser, was a more unstable form or had an improper conformation (6). Our findings may support their suggestion with regard to the immunoblotting and spectral assay findings. Yamazaki et al. reported that a proline-rich region was present following the signal-anchor sequence in the amino-terminal portion of all known microsomal cytochrome P450s, and proposed that the proline residues in the proline-rich region were crucial for the formation of the correct conformation of microsomal P450 molecules (31). In the present study, SS and PT

were expressed to clarify the effect of one of two amino acid substitutions in CYP2D6.10. The findings of SS and PT indicate that the Pro34 3 Ser substitution is more critical than the Ser486 3 Thr substitution for expression in yeast cell. At least, five prolines are conserved between rat CYP2Ds and human CYP2D6 (31), and the Pro 34 in human CYP2D6 is the first residue in this region. Therefore, we expressed a mutant of CYP2D1 with an artificial mutation, Pro 3 Ser, at the first proline residue in the conserved proline-rich region to demonstrate the effect of one amino acid change. The P450 content of the mutant calculated by the CO-reduced difference spectrum in microsomal protein was reduced to 1% that of the wild type (CYP2D1). A catalytic study of the forms of CYP2D6 expressed in yeast was carried out with bufuralol and venlafaxine. The apparent K m for bufuralol was significantly different among CYP2D6.1, CYP2D6.10A, and CYP2D6.10C, although V max values were similar. The additional findings on the K m values of PT and SS indicated that apparent K m values for both substrates were increased mainly by the Pro34 3 Ser amino acid substitution and only secondly by Ser486 3 Thr. The apparent K m value of CYP2D6.10C for bufuralol was much higher than that of CYP2D6.10A and the others, suggesting that gene conversion was critical for bufuralol 1⬘-hydroxylation. Venlafaxine demethylation was measured to examine the effect of the CYP2D6*10 genotype. The apparent K m value of CYP2D6.10A for venlafaxine was fivefold higher than that of CYP2D6.1, although the value of CYP2D6.10C was twofold higher than that of CYP2D6.10A. The difference in the apparent K m between CYP2D6.1 and CYP2D6.10A may be one of the factors that cause the differences in the pharmacokinetics of venlafaxine between homozygotes for CYP2D6*1 and homozygotes for CYP2D6*10.

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In this study using the expression system, it was impossible to precisely extrapolate the clearance from in vitro to in vivo, because decreased metabolic clearance of CYP2D6.10 appeared to be caused by expression itself. However, this study is the first about the affinity of CYP2D6.10 and supported our previous suggestion that CYP2D6*10 influenced the metabolic in vivo clearance of venlafaxine. In addition, the affinity contributes for not only metabolic clearance but also inhibition. This finding also suggests that CYP2D6.10 is less sensitive than CYP2D6.1 to inhibition by bufuralol and venlafaxine. In these points, it is important to investigate the affinity of CYP2D6.10 for different substrates and inhibitors. In conclusion, the findings of the present study suggest that the decreased in vivo clearance of CYP2D6 substrates on the homozygote for CYP2D6*10 could be caused not only by the decreased expression but also by the increased apparent K m value of CYP2D6. Furthermore, these findings indicate that different influences on clearance by CYP2D6.10 might be observed with different substrates. ACKNOWLEDGMENTS We thank Wyeth-Ayerst Research for providing venlafaxine and metabolites. A part of this study was supported by a grant-in-aid from the Ministry of Education, Science, Sports, and Culture of Japan and Research Fellowships of the Japan Society for the Promotion of Science for Young Scientists.

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