Roles of two allelic variants (Arg144Cys and Ile359Leu) of cytochrome P4502C9 in the oxidation of tolbutamide and warfarin by human liver microsomes

Article abstract of DOI:10.1080/004982598239614

1. Tolbutamide methyl hydroxylation and racemic warfarin 7-hydroxylation activities were determined in liver microsomes of 39 Japanese and 45 Caucasians genotyped for the cytochrome P450 (P450 or CYP) 2C9 gene into three groups, namely the wild-type (Arg144·Ile359), and two hererozygous Cys allele (Cys144·Ile359) and Leu allele (Arg144·Leu359) variants. 2. Good correlations were found between tolbutamide methyl hydroxylation and racemic warfarin 7-hydroxylation activities in liver microsomes of Japanese and Caucasians. Humans with the Cys allele CYP2C9 variant, which was detected in 22% of Caucasians, were found to have similar catalytic rates to those of the wild-type in the oxidations of tolbutamide and racemic warfarin, whereas humans with the Leu allele, which was detected in 8% Japanese and 7% Caucasian samples, had lower catalytic rates than those of other two groups. 3. The rates of 6- and 7-hydroxylation of racemic warfarin were correlated well with these of S-warfarin, but not R-warfarin, in human liver microsomes. 4. Both human liver microsomes and recombinant CYP2C9 catalysed 7-hydroxylation of S-warfarin more extensively than those of R-warfarin. K(m)'s for the 7-hydroxylation of S-warfarin were not very different in liver microsomes of humans with these three genotypes. Anti-CYP2C9 antibodies and sulphaphenazole inhibited the 6- and 7-hydroxylation of S-warfarin, but nor R warfarin, by > 90% and the methyl hydroxylation of tolbutamide by about 50%. 5. These results suggest that humans with Leu allele of CYP2C9 have lower V(max)'s for S-warfarin 7-hydroxylation and tolbutamide methyl hydroxylation than those with wild-type and Cys allele CYP2C9, although the K(m)'s are not very different in liver microsomes of these three groups of humans. R-warfarin hydroxylation may be catalysed by P450 enzymes other than CYP2C9 in man.

Full text of DOI:10.1080/004982598239614

xenobiotica , 1998, vol. 28, no. 2, 103±115
Roles of two allelic variants (Arg144Cys and
Ile359Leu) of cytochrome P4502C9 in the oxidation
of tolbutamide and warfarin by human liver
m icrosomes
H. YAMAZAKI, K. INOUE and T. SHIMADA*
Osaka Prefectural Institute of Public Health, 3-69 Nakamichi 1-chome, Higashinari-ku,
Osaka 537, Japan
Received 19 April 1997
1. Tolbutamide methyl hydroxylation and racemic warfarin 7-hydroxylation activities
were determined in liver microsomes of 39 Japanese and 45 Caucasians genotyped for the
cytochrome P450 (P450 or CYP) 2C9 gene into three groups, namely the wild-type
(Arg144[Ile359), and two heterozygous Cys allele (Cys144[Ile359) and Leu allele
(Arg144[Leu359) variants.
2. Good correlations were found between tolbutamide methyl hydroxylation and
racemic warfarin 7-hydroxylation activities in liver microsomes of Japanese and Cau-
casians. Humans with the Cys allele CYP2C9 variant, which was detected in 22 % of
Caucasians, were found to have similar catalytic rates to those of the wild-type in the
oxidations of tolbutamide and racemic warfarin, whereas humans with the Leu allele,
which was detected in 8 % Japanese and 7 % Caucasian samples, had lower catalytic rates
than those of other two groups.
3. The rates of 6- and 7-hydroxylation of racemic warfarin were correlated well with
those of S-warfarin, but not R-warfarin, in human liver microsomes.
4. Both human liver microsomes and recombinant CYP2C9 catalysed 7-hydroxylation
of S-warfarin more extensively than those of R-warfarin. K ’s for the 7-hydroxylation of
m
S-warfarin were not very diåerent in liver microsomes of humans with these three
genotypes. Anti-CYP2C9 antibodies and sulphaphenazole inhibited the 6- and 7-
"
hydroxylation of S-warfarin, but not R-warfarin, by
of tolbutamide by about 50 %.
90 % and the methyl hydroxylation
5. These results suggest that humans with Leu allele of CYP2C9 have lower V ’s for
S-warfarin 7-hydroxylation and tolbutamide methyl hydroxylation than those with wild-
max
type and Cys allele CYP2C9, although the K ’s are not very diåerent in liver microsomes
of these three groups of humans. R-warfarin hydroxylation may be catalysed by P450
enzymes other than CYP2C9 in man.
m
Introduction
Cytochromes P450 (P450 or CYP) in liver microsomes contribute signi®cantly
to the biotransformation of xenobiotic chemicals such as drugs, toxic chemicals, and
carcinogens and some endobiotic chemicals including steroids, fatty acids, prosta-
glandins and vitamins (Gonzalez 1989, Guengerich 1991). P450 comprises a
superfamily of these enzymes and individual forms of P450 have considerable, but
overlapping, substrate speci®cities (Nelson et al. 1996). CYP2C subfamily members
have been shown to constitute the major forms of P450 in microsomal fractions of
human livers and to play an important role in the oxidation of clinically used drugs
and other xenobiotic chemicals (Guengerich and Shimada 1991, Shimada et al.
1994).
In man, at least four CYP2C gene products (CYP2C8, CYP2C9, CYP2C18 and
* Author for correspondence.
±
’
0049±8254}98 $12 00
1998 Taylor & Francis Ltd

104
H. Yamazaki et al.
CYP2C19) have been shown to be expressed in the liver and, in some instances, in
extrahepatic organs (Romkes et al. 1991, Goldstein and de Morais 1994). CYP2C9
and 2C19 proteins are the major CYP2C gene products that catalyze oxidation of
the prototype substrates tolbutamide, warfarin and S-mephenytoin in human liver
microsomes (Romkes et al. 1991, Goldstein and de Morais 1991). The molecular
basis of CYP2C19 genetic polymorphism has been studied extensively (de Morais
et al. 1994, Goldstein and de Morais 1994).
Recent studies have shown that there are at least three genotypes of the CYP2C9
gene, namely Arg144[Ile359 (wild-type), Cys144[Ile359 (Cys allele) and Arg-
144[Leu359 (Leu allele) (Wang et al. 1995, Haining et al. 1996, Sullivan-Klose et al.
1996). By analysing genomic DNA isolated from human blood samples it has been
found that these allelic variants may be expressed as the functional proteins in
human liver microsomes. By analysing genomic DNA isolated from livers of
Japanese and Caucasian populations, we have also found that the Cys allele
C
22 % (all
CYP2C9 is detected only in Caucasians with
a
frequency of
C
heterozygotes), whereas the Leu allele was determined with frequencies of
8 %
(all heterozygotes) in both races (Inoue et al. 1997). To determine if these allelic
variants catalyse oxidation of several model substrates at similar rates to those of
native P450 enzymes, many investigators have used recombinant P450 proteins
expressed in heterologous systems into which cDNAs or the modi®ed cDNA
sequences of human P450 genes had been introduced (Gonzalez and Korzekwa
1995, Guengerich et al. 1996).
$&*
The recombinant CYP2C9-Leu allelic variant has been shown to have lower
V
’s and higher K ’s for the oxidation of tolbutamide and S-warfarin than those
max
catalysed by recombinant CYP2C9 and CYP2C9-Cys
m
"%%
(Veronese et al. 1993,
Haining et al. 1996, Sullivan-Klose et al. 1996). Other studies have suggested that
"%%
the recombinant CYP2C9-Cys
variant catalyses the oxidation of S-warfarin 7-
hydroxylation at lower rates with lower K ’s than those of wild-type CYP2C9,
m
although such diåerences were not so signi®cant when tolbutamide methyl
hydroxylation is measured (Rettie et al. 1994). Our previous studies using liver
microsomes of humans who were genotyped for CYP2C9 have indicated that
human samples with the heterozygous Leu allele have lower activities for
tolbutamide methyl hydroxylation than those with the wild-type and heterozygous
Cys allele of CYP2C9 in liver microsomes, although the K ’s were not very diåerent
m
in these three groups (Inoue et al. 1997).
To demonstrate that the allelic variants of CYP2C9 will aåect the catalytic roles
of CYP2C9 in liver microsomes, we further determined and compared the activities
of oxidation of racemic and R- and S-warfarin as well as tolbutamide in human
samples which were genotyped for the CYP2C9 gene into wild-type and two
heterozygous Leu allele and Cys allele CYP2C9 variants (Inoue et al. 1997). Three
Caucasians with the Leu allele and eight Caucasians with the Cys allele CYP2C9
were selected to examine in more detail by comparing the catalytic activities to those
catalysed by humans with wild-type CYP2C9. Kinetic analysis was determined in
liver microsomes of these human samples and by recombinant human P450 enzymes
including CYP2C9 and CYP2C9(Cys). The eåects of sulphaphenazole and anti-
CYP2C9 antibodies on the oxidation of these model substrates are also reported.

Catalytic roles of allelic variants of human CYP2C9
M aterials and m ethods
105
Chemicals
Tolbutamide and racemic warfarin were purchased from Sigma Chemical Co. (St Louis, MO, USA).
S-warfarin and R-warfarin, and their 6- and 7-hydroxylated metabolites, and S-mephenytoin were
obtained from Ultra Fine Chemicals Co. (Manchester, UK) and used without further puri®cation. Other
drug substrates, their metabolites and reagents used were obtained from sources as described previously
or were of the highest quality commercially available (Shimada et al. 1989, 1994, Mimura et al. 1993,
Yamazaki et al. 1994).
Enzyme preparation
Human liver samples were obtained from organ donors or patients undergoing liver resection as
described previously (Mimura et al. 1993, Shimada et al. 1994). Liver microsomes were prepared as
±
described and suspended in 10 m Tris-Cl buåer (pH 7 4) containing 1 0 m EDTA and 20 % glycerol
±
m
m
(v v) (Guengerich 1994).
}
CYP2C9 was puri®ed to electrophoretic homogeneity from human liver microsomes as described
previously (Shimada et al. 1986). Microsomes from human lymphoblast cells expressing CYP2C9 and
CYP2C9(Cys) were purchased from Gentest Co. (Woburn, MA, USA). Recombinant CYP2C8, 2C9,
2C18 and 2C19 expressed in yeast microsomes were obtained from Sumitomo Chemical Co. (Osaka,
Japan). Rabbit anti-P450 antibodies raised against human CYP2C9 were prepared and the IgG fractions
obtained as described (Kaminsky et al. 1981, Shimada et al. 1986). Anti-CYP2C9 antibodies have already
been characterized and shown to inhibit CYP2C9-dependent tolbutamide methyl hydroxylation and
«
CYP2C19-dependent S-mephenytoin 4 -hydroxylation activities in human liver microsomes (Shimada
et al. 1986, Brian et al. 1989).
Enzyme assays
Liver microsomal incubations included microsomes (1 0 mg protein ml) in 50 m potassium
±
m
}
+
phosphate buåer (pH 7 4) containing an NADPH-generating system consisting of 0 5 m NADP ,
±
±
m
±
glucose 6-phosphate and 0 5 unit of glucose 6-phosphate dehydrogenase ml, and various
m
5 m
concentrations of drug substrates (Shimada et al. 1989, 1994). Methyl hydroxylation of tolbutamide
}
«
±
±
m
m
b
(substrate concentration of 2 5 m ), 4 -hydroxylation of S-mephenytoin (0 4 m ), 6 -hydroxylation of
testosterone were determined using high-performance liquid chromatography as previously described
(Shimada et al. 1986, 1994, Knodell et al. 1987, Yamazaki et al. 1996). 6- and 7-Hydroxylation of racemic
and R- and S-warfarin were determined according to the original method with modi®cation (Lang and
±
Bocker 1995). The standard incubation mixture consisted of microsomal protein (0 5 mg protein ml)
}
±
±
±
with substrates (0 10 m ) in a ®nal volume of 0 20 ml 50 m potassium phosphate buåer (pH 7 4)
containing the NADPH-generating system. Incubations were carried out at 37 C for 30 min and
m
m
°
l
terminated by adding 10 l 60% HClO (w v). The separation of hydroxylated warfarin metabolites by
}
%
±
l
HPLC was carried out with a C 5- m analytical column (4 6¬150 mm, Kanto Chemical, Tokyo, Japan)
")
eluted with a mixture of 36 % CH CN (v v) containing 0 04 % aqueous H PO (Lang and Bocker 1995).
P450 contents were estimated spectrally by the methods of Omura and Sato (1964). The contents of
human P450 proteins in liver microsomes were estimated by coupled sodium dodecyl sulphate-
±
}
$
$
%
polyacrylamide gel electrophoresis immunochemical development (Western-blotting) (Guengerich et
al. 1982). The intensities of the immunoblots were measured with an Epson GT-8000 Scanner (Tokyo,
}
Japan) equipped with NIH Image Gel Analysis Program adapted for Macintosh computers. Protein
}
concentrations were estimated by the method of Lowry et al. (1951).
Analysis of genetic polymorphisms in CYP2C9 genes
Genomic DNA was isolated from human livers as described previously (Inoue et al. 1997, Yamazaki
±
et al. 1997). Brie¯y, about 2 g liver was suspended in 10 m Tris-Cl buåer (pH 7 5) containing 10 m
EDTA and 100 m NaCl, and homogenized with a Te¯on-pestle homogenizer. The nuclear fractions,
m
m
m
°
collected by centrifugation at 3500 g for 5 min at 4 C, were washed twice with the latter buåer by
centrifugation at 9000 g for 15 min. Genomic DNA was isolated from the nuclear fraction by a
commercial Nuclei Acid Puri®cation System (Perkin Elmer ABI 341, Norwalk, CT, USA).
The genotyping procedure for the detection of CYP2C9 genetic polymorphism was performed
according to the methods as previously described (Wang et al. 1995, Inoue et al. 1997). Brie¯y, the
l
incubation mixture (®nal volume 25 l) for the PCR reaction consisted of 10x Ex Taq buåer, 4 dNTPs,
±
primers, genomic DNA as a template (25 ng) and 0 5 U Ex Taq polymerase (Takara, Kyoto, Japan). The
DNA fragments after PCR ampli®cation were digested with appropriate restriction endonuclease
enzymes, separated by electrophoresis on a 10 % polyacrylamide gel (Bio-Rad Laboratories, Inc.,
Hercules, CA, USA), and visualized after treatment with SYBRTM Green I nucleic acid gel stain
(Wako, Osaka, Japan).

106
H. Yamazaki et al.
Statistical analysis
Kinetic parameters for the tolbutamide methyl hydroxylation and warfarin 7-hydroxylation by
recombinant human P450 enzymes and by liver microsomes were estimated using a KaleidaGraph
program from Synergy Software (Reading, PA, USA) designed for non-linear regression analysis. The
correlations between activities of tolbutamide methyl hydroxylation and warfarin 7-hydroxylation and
contents of individual forms of P450 in diåerent human liver microsomal preparations were analysed
using a linear regression analysis (InStat) program from GraphPad Software (San Diego, CA, USA).
Statistical analysis was analysed by Student’s t-test.
Results
Tolbutamide methyl hydroxylation and warfarin 7-hydroxylation activities by
human liver microsomes
Genomic DNA was isolated from livers of 39 Japanese and 45 Caucasians and
used to examine the genotypes of the CYP2C9 gene using speci®c oligonucleotide
primers as previously described (Inoue et al. 1997). Three genotypes were found in
the CYP2C9 gene, namely Arg144[Ile359 (wild-type), Arg144Cys[Ile359 (Cys
allele) and Arg144[Ile359Leu (Leu allele). The Cys allele was detected only in
C
Caucasians with a frequency of
22 %. The frequencies of the Leu allele of
C
the human samples examined in this study had homozygous Cys144 Cys144 and
CYP2C9 were determined to be
8 % in both Japanese and Caucasians. None of
}
Leu359 Leu359 variants nor the heterozygous Arg144Cys[Ile359Leu variant of
CYP2C9 (Inoue et al. 1997).
}
Tolbutamide methyl hydroxylation and racemic warfarin 7-hydroxylation (in
this case 20 lm warfarin concentration was used) activities were determined in these
human liver microsomes in which three Japanese and three Caucasians were
genotyped to be a heterozygous Leu allele, and ten Caucasians to be a heterozygous
Cys allele of CYP2C9. In both races, humans with the Leu allele of CYP2C9 were
in the lower range of turnover numbers for tolbutamide methyl hydroxylation and
warfarin 7-hydroxylation activities than those with wild-type and Cys allele (®gure
1). The Cys allele of humans detected only in Caucasians showed almost similar
catalytic rates to those of wild-type of CYP2C9. In both races, there were good
correlations between tolbutamide methyl hydroxylation and racemic warfarin 7-
hydroxylation activities.
Activities of 7-hydroxylation of S-warfarin as well as racemic warfarin were
determined in liver microsomes of 35 selected Caucasians in which three were
genotyped to be the Leu allele, eight to be the Cys allele and 24 to be wild-type
CYP2C9, and compared with the levels of total P450 and CYP2C9 and the activities
«
of tolbutamide methyl hydroxylation (®gure 2). S-mephanytoin 4 -hydroxylation
and testosterone 6b-hydroxylation activities were also measured. There were no
signi®cant diåerences in the contents of total P450 and CYP2C9 in liver microsomes
of humans with three CYP2C9 genotypes. In the Caucasian samples, humans with
the Leu allele of CYP2C9 always exhibited very low tolbutamide methyl hy-
droxylation and racemic and S-warfarin 7-hydroxylations, whereas the Cys allele
«
had almost the same rates as the wild-type of CYP2C9. S-mephenytoin 4 -
hydroxylation and testosterone 6b-hydroxylation activities by human live micro-
somes were not very diåerent in the three groups of CYP2C9 genotype.
Activities of S-warfarin 7-hydroxylation were correlated well with those of
racemic warfarin 7-hydroxylation and S-warfarin 6-hydroxylation in liver micro-
somes of 35 Caucasian samples (®gure 3). The turnover numbers of 7-hydroxylation

Catalytic roles of allelic variants of human CYP2C9
107
Figure 1. Correlation between tolbutamide methyl hydroxylation and racemic warfarin 7-hydrox-
ylation activities in liver microsomes of Japanese (A) and Caucasian (B) populations. The
genotypes of the CYP2C9 gene were shown to be wild-type (D ), Cys allele (_ ) and Leu allele (E ).
Figure 2. Contents of total P450 (A) and CYP2C9 (B) and activities of tolbutamide methyl
hydroxylation (C), racemic warfarin 7-hydroxylation (D), S-warfarin 7-hydroxylation (E), S-
«
b
mephenytoin 4 -hydroxylation (F), and testosterone 6 -hydroxylation (G) in liver microsomes of
Caucasians who were genotyped for CYP2C9 gene into wild-type (D ), Cys allele (^) and Leu
allele (E ).
of racemic warfarin was about one-third of those of S-warfarin in human liver
microsomes and it was found that the ratio between activities of S-warfarin 7- and
±
6-hydroxylation was around 3±3 5 in these liver microsomes.
Kinetic analysis of warfarin 7-hydroxylation activities in 35 Caucasians
Kinetic analysis of 7-hydroxylation of racemic, and R- and S-warfarin was
performed in liver microsomes of Caucasian samples genotyped for CYP2C9 gene
(table 1). Apparent K ’s for racemic warfarin 7-hydroxylation were found to be 27,
m
21 and 46 lm in liver microsomes of HL-C15 (wild-type), HL-C31 (Cys allele) and

108
H. Yamazaki et al.
Figure 3. Correlation between activities of 7-hydroxylations of S-warfarin and racemic warfarin (A)
and 6- and 7-hydroxylations of S-warfarin 7-hydroxylation (B) in liver microsomes of Caucasians
who were genotyped for CYP2C9 gene to be wild-type (D ), Cys allele (_ ) and Leu allele (E ).
Table 1. Kinetic analysis of warfarin 7-hydroxylation by liver microsomes of humans genotyped for
CYP2C9.
R,S-warfarin R-warfarin
S-warfarin
Sample
code no.
CYP2C9
genotype
S R ratio
}
max
a
b
K
V
K
V
K
V
of V
m
max
m
max
m
max
±
1 4
±
±
±
5 8
HL-C15
HL-C19
HL-C1
HL-C31
HL-C6
(wild)
(wild)
(Cys)
(Cys)
(Leu)
(Leu)
(Leu)
27
107
161
91
1 0
32
5 8
±
±
±
2 7
22
35
18
55
37
27
8 0
3 0
13
15
±
±
3 8
±
4 5
0 3
±
±
21
46
2 5
30
0 3
±
0 3
±
±
±
0 3
±
9 0
±
6 0
172
129
189
0 7
0 2
±
±
HL-C29
HL-C38
0 1
0 9
±
0 2
±
1 2
a
lm
.
, pmol min mg protein.
K
,
m
b
V
}
}
max
HL-C6 (Leu allele) respectively, with a lower V
for the Leu allele of CYP2C9. K
m
for the 7-hydroxylation of R-warfarin was lower in human samples of the Cys allele
max
than those for the wild-type and Leu allele of CYP2C9. The K ’s were similar for
m
three groups of the CYP2C9 genotype.
The HPLC pro®le of R- and S-warfarin 6- and 7-hydroxylation showed that
liver microsomes of human sample HL-C15 (wild-type of CYP2C9) catalysed the
7-hydroxylation of S-warfarin at much higher rates than those of R-warfarin 7-
hydroxylation, whereas the rates of hydroxylation of R- and S-warfarin by liver
microsomes of human sample HL-C6 (Leu allele) were found to be similar
(®gure 4).
Analysis for the S-warfarin 7-hydroxylation was also performed and compared
in liver microsomes of HL-C19 (wild-type) and recombinant CYP2C9 (®gure 5A),
and of HL-C31 (Cys allele) and recombinant CYP2C9(Cys) (®gure 5B). The K ’s
m
for the 7-hydroxylation were very similar in liver microsomes of wild-type CYP2C9
(human sample of HL-C19) and recombinant CYP2C9(wild), whereas the V
max
determined with recombinant CYP2C9 was much higher than those catalyzed by
liver microsomes of a human sample of HL-C19. The K ’s and V ’s for S-
m
max

Catalytic roles of allelic variants of human CYP2C9
109
Figure 4. HPLC pro®le of hydroxylation of R-warfarin (A and C) and S-warfarin (B and D) by liver
microsomes of HL-C15 (wild-type ; A and B) and HL-C6 (Leu allele; C and D). 6-Hydroxylated
(a) and 7-hydroxylated products (b) of R- and S-warfarin are also shown.
Figure 5. Kinetic analysis of S-warfarin 7-hydroxylation catalysed by liver microsomes of human
samples HL-C19 (D ) and HL-C31 (^), which were genotyped for the CYP2C9 gene to be a wild-
type and Cys allele respectively, and by microsomes of human lymphoblastoid cells expressing
CYP2C9 (E ) and CYP2C9(Cys) (_ ). Data are the mean of duplicate determinations.
warfarin 7-hydroxylation by recombinant CYP2C9(Cys) were lower and higher,
respectively, than those catalysed by liver microsomes with HL-C31 (Cys allele). On
analysis using Eadie±Hofstee plots, we found that a single kinetic parameter could
be determined in the 7-hydroxylation of S-warfarin by this human sample (data not
shown).
Tolbutamide methylhydroxylation and warfarin 6- and 7-hydroxylation by
recombinant human CYP2C enzymes
Activities of methyl hydroxylation of tolbutamide and the 6- and 7- hy-
droxylation of R- and S-warfarin were determined in yeast microsomes expressing
human CYP2C8, 2C9, 2C18 and 2C19, and microsomes of human lymphoblastoid

110
H. Yamazaki et al.
Table 2. Methyl hydroxylation of tolbutamide, and 6- and 7-hydroxylation of R- and S-warfarin by
recombinant CYP2C proteins.
Tolbutamide
methyl
R-warfarin
S-warfarin
hydroxylation
(nmol min nmol 6-hydroxylation 7-hydroxylation 6-hydroxylation 7-hydroxylation
P450
enzyme
}
}
P450)
(pmol min nmol P450)
}
}
Yeast microsomes
CYP2C8
CYP2C9
CYP2C18
CYP2C19
!
!
!
!
10
12³2
53³8
14³2
46³3
10
10
10
2³1
23³2
5³1
1³1
740³84
49³3
184³24
!
!
10
10
256³19
410³35
123³11
Human lymphoblast cells
CYP2C9
CYP2C9(Cys)
!
!
26³3
17³2
10
10
13³2
4³1
43³3
244³15
43³3
!
10
Each value represents the mean and range of duplicate or triplicate determinations. Substrate
lm
for R- and S-warfarin.
±
concentrations used were 2 5 m for tolbutamide and 100
m
«
Figure 6. Eåects of anti-CYP2C9 IgG on the activities of S-mephenytoin 4 -hydroxylation (A),
tolbutamide methyl hydroxylation (B), S-warfarin 7-hydroxylation (C), and R-warfarin 7-
hydroxylation (D) by liver microsomes of human samples HL-C15 (E ), HL-C31 (* ), and
HL-C29 (+ ), which were genotyped for the CYP2C9 gene to be a wild-type, Cys allele and Leu
allele respectively. The eåects of preimmune IgG (D ) were also determined. Control activities
«
(pmol products min mg protein) in the absence of antibodies were 108 (S-mephenytoin 4 -
}
}
hydroxylation by HL-C15), 711, 360 and 130 (tolbutamide methyl hydroxylation by HL-C15,
±
HL-C31 and HL-C29), and 11, 8 7 and 1 1 (S-warfarin 7-hydroxylation by HL-C15, HL-C31
±
and HL-C29). R-warfarin 7-hydroxylation was determined only in liver microsomes of HL-C15 ;
±
the turnover rate in the absence of antibodies was 0 8 pmol min mg protein. Data are the mean
}
}
of duplicate determinations.
cells expressing CYP2C9 and CYP2C9(Cys) (table 2). Among the yeast microsomes
expressing CYP2C enzymes examined, CYP2C9 and CYP2C19 were the most
active in catalysing tolbutamide methyl hydroxylation. S-warfarin 6- and 7-
hydroxylation were extensively catalysed by CYP2C9, whereas CYP2C19 was the
most active for the hydroxylations of R-warfarin. In microsomes of human
lymphoblastoid cells, CYP2C9(Cys) was found to be less active in catalysing R- and
S-warfarin 7-hydroxylation as compared with those catalysed by CYP2C9(wild).

Catalytic roles of allelic variants of human CYP2C9
111
«
Figure 7. Eåects of sulphaphenazole on the activities of S-mephenytoin 4 -hydroxylation (A),
tolbutamide methyl hydroxylation (B), S-warfarin 7-hydroxylation (C), and R-warfarin 7-
hydroxylation (D) by liver microsomes of human samples HL-C15 (E ), HL-C31 (* ) and HL-
C29 (+ ), which were genotyped for the CYP2C9 gene to be a wild-type, Cys allele and Leu allele
respectively. The eåect of sulphaphenazole on the R-warfarin 7-hydroxylation was also
determined only in liver microsomes of HL-C15. Data are the mean of duplicate determinations.
Figure 8. Eåects of substrate concentration on the sulphaphenazole inhibition of tolbutamide methyl
hydroxylation activities by liver microsomes of human samples HL-C15. Tolbutamide concen-
±
±
±
m
trations determined were 0 025 (D ), 0 25 (_ ) and 2 5 m (E ); control activities in the absence of
sulphaphenazole were 25³5, 140³21 and 420³40 pmol min mg protein respectively. Data are
}
}
the mean of duplicate determinations.
Eåects of anti-CYP2C9 IgG and sulphaphenazole on the oxidation of S-
mephenytoin, tolbutamide and S-warfarin by human liver microsomes
Eåect of antibodies raised against puri®ed human CYP2C9 on the tolbutamide
methyl hydroxylation and S-warfarin 7-hydroxylation activities were determined in
liver microsomes of human samples genotyped for CYP2C9 gene (®gure 6). Eåects
«
of anti-CYP2C9 on the 4 -hydroxylation of S-mephenytoin and the 7-hydroxylation
of R-warfarin were also measured in liver microsomes of a human sample HL-C15
(genotyped for CYP2C19wt wt and wild-type of CYP2C9) and it was found that the
}
antibodies inhibited activities very extensively (®gure 6A). Anti-CYP2C9 IgG was
also found to be very eåective in inhibiting S-warfarin 7-hydroxylation catalysed by

112
H. Yamazaki et al.
liver microsomes of humans genotyped for the wild-type Cys- and Leu allele of
CYP2C9 (®gure 6C). However, the inhibition by anti-CYP2C9 antibodies of
!
tolbutamide methyl hydroxylation by human liver microsomes were
50 % of the
control activities (®gure 6B). Anti-CYP2C9 was found to be non-inhibitory toward
the R-warfarin 7-hydroxylation catalysed by human liver microsomes (®gure 6D).
The eåects of sulphaphenazole, a known inhibitor of CYP2C9 (Brian et al.
1989), were determined under the same experimental conditions described above
(®gure 7). Sulphaphenazole very signi®cantly inhibited the 7-hydroxylation of S-
warfarin and partially inhibited the methyl hydroxylation of tolbutamide, but not
«
the 4 -hydroxylation of S-mephenytoin and 7-hydroxylation of R-warfarin, by
human liver microsomes.
±
When the substrate (tolbutamide) concentrations were decreased from 2 5 to
±
0 025 mm, the eåects of sulphaphenazole on the inhibition of tolbutamide methyl
hydroxylation activities were much more extensive (®gure 8).
Discussion
Recombinant protein engineering has been applied extensively to human P450
research in order to elucidate the mechanisms of expression and catalytic functions
of individual P450 enzymes (Gonzalez 1989, Sandhu et al. 1993, Goldstein and de
Morais 1994, Gonzalez and Korzekwa 1995, Guengerich et al. 1996). Many human
P450 cDNAs and their modi®ed DNA sequences have been introduced into
heterologous organisms such as viruses, bacteria, yeasts, insects and mammalian
cells and the expressed proteins have been used to examine the roles of these P450
enzymes in the oxidation of a number of xenobiotic and endobiotic chemicals and to
study the genetic basis of P450 polymorphisms in man (Gonzalez et al. 1991,
Guengerich et al. 1991, Goldstein and de Morais 1994, Gonzalez and Korzekwa
1995).
Recently, two allelic variant cDNAs of CYP2C9, Cys and Leu alleles, have been
studied in several heterologous systems and the recombinant proteins thus obtained
were used to examine their abilities to oxidize prototypic substrates such as
tolbutamide, phenytoin and warfarin (Yasumori et al. 1991, Kaminsky et al. 1993,
Lopez-Garcia et al. 1993, Sandhu et al. 1993, Veronese et al. 1993, Rettie et al. 1994,
Tracy et al. 1995, Haining et al. 1996, Sullivan-Klose et al. 1996). Haining et al.
(1996) reported that the Leu allele of CYP2C9 catalyses the 6- and 7-hydroxylation
of S-warfarin at slower rates (with higher K ’s) than those of the CYP2C9 wild-
m
type. Similar results have also been reported by others by measuring tolbutamide
methyl hydroxylation, warfarin 7-hydroxylation and phenytoin p-hydroxylation
(Veronese et al. 1993, Sullivan-Klose et al. 1996). In contrast, the Cys allele of
CYP2C9, another variant form, has been reported to have similar catalytic
functions to the wild-type of CYP2C9 for the oxidation of tolbutamide, phenytoin
and warfarin (Veronese et al. 1993, Sullivan-Klose et al. 1996), although there is
some controversy that this allelic variant has been shown to have lower activities for
S-warfarin 7-hydroxylation than those of the CYP2C9 wild-type (Rettie et al. 1994).
Recently variant CYP2C9 forms with the homozygous Leu Leu allele and
}
heterozygous Arg Cys and Ile Leu alleles have been determined to associate with
}
}
the poor metabolizer phenotypes for the oxidations of tolbutamide and phenytoin in
man (Spielberg et al. 1996, Sullivan-Klose et al. 1996). The 6- and 7-hydroxylation
activities of S-warfarin are lower in the CYP2C9 Leu allele protein than the wild

Catalytic roles of allelic variants of human CYP2C9
113
CYP2C9 protein, with the former protein having higher K ’s for these two reactions
m
(Haining et al. 1996, Sullivan-Klose et al. 1996). Previously, we analysed genomic
DNA of livers from 39 Japanese and 45 Caucasians to determine the genotypes for
the CYP2C9 gene and found that the heterozygous Cys allele mutation is detected
only in Caucasians, with frequencies of about 22 %, whereas the Leu allele is
detected in both Japanese and Caucasian populations, with frequencies of about 8 %
each. It should be mentioned, however, that none of the human samples examined
had homozygous Cys144 Cys144 and Leu359 Leu359 variants nor heterogenous
}
}
Arg144Cys[Ile359Leu variant of CYP2C9 (Inoue et al. 1997). In these studies we
have shown that human samples with the heterozygous Leu allele have lower
activities for tolbutamide methyl hydroxylation than those with the wild-type and
heterozygous Cys allele of CYP2C9 in liver microsomes, although the K ’s were
m
similar in these three groups (Inoue et al. 1997).
In this study we further examined the roles of variant forms of CYP2C9 in
human liver microsomes towards the oxidation of tolbutamide, racemic, R-,
and S-warfarin. The results presented showed that 6- and 7-hydroxylation of S-
warfarin were catalysed principally by CYP2C9 in human liver microsomes with the
latter reaction being catalysed better by human CYP2C9 proteins than the 6-
hydroxylation reaction. The Leu allele of CYP2C9 was suggested to have lower
turnover numbers for S-warfarin 7-hydroxylation than those catalysed by wild-type
and Cys alleles of CYP2C9, although the K ’s in these groups of CYP2C9 genotypes
m
were not very diåerent. It was also determined that the apparent K ’s and V ’s for
m
max
S-warfarin 7-hydroxylation in man with the Cys- and wild-types were relatively
similar. These results are in contrast with the previous ®ndings that recombinant
CYP2C9(Leu) and CYP2C9(Cys) have higher K ’s than those catalysed by
m
recombinant CYP2C9 in the oxidation of S-warfarin (Veronese et al. 1993, Rettie
et al. 1994, Haining et al. 1996).
Although there was a good correlation between the 7-hydroxylation of racemic
warfarin and S-warfarin in liver microsomes from Caucasian samples, the turnover
numbers of S-warfarin 7-hydroxylation were always higher than those of racemic
warfarin 7-hydroxylation by these human liver microsomes. Since the CYP2C9 in
human liver microsomes catalysed about 5±10-fold higher rates for 7-hydroxylation
of S-warfarin than those of R-warfarin, it is possible that the R-enantiomer may be
inhibitory for the hydroxylation of the S-enantiomer when the racemic warfarin was
used as a substrate. Our results also suggested that R-warfarin was catalysed by
other P450 enzymes rather than CYP2C9 in human liver microsomes since R-
warfarin 7-hydroxylation activities were not inhibited by anti-CYP2C9 and
sulphaphenazole. Of the recombinant human CYP2C enzymes examined, CYP2C19
was found to be the most active for the R-warfarin 7-hydroxylation. However, anti-
«
CYP2C9 that inhibited S-mephenytoin 4 -hydroxylation very signi®cantly did not
aåect the R-warfarin 7-hydroxylation by liver microsomes, suggesting that the
reaction was catalysed principally by other P450 enzymes rather than the CYP2C
enzymes (Rettie et al. 1992, Zhang et al. 1995).
In conclusion, the present results have shown that the Leu allele of CYP2C9 has
lower V ’s for S-warfarin 7-hydroxylation and tolbutamide methyl hydroxylation
max
than those catalysed by wild-type and Cys allele, whereas the K ’s are similar in
m
these three genotypes. CYP2C9 catalyses the 7-hydroxylation of S-warfarin better
than that of R-warfarin, and R-warfarin hydroxylation may be catalysed by P450
enzymes other than CYP2C9 in man.

114
H. Yamazaki et al.
Acknow ledgem ents
This work was supported in part by Grants from the Ministry of Education,
Science, and Culture of Japan, the Ministry of Health and Welfare of Japan, and the
Developmental and Creative Studies from Osaka Prefectural Government. We
thank Dr F. P. Guengerich, Vanderbilt University School of Medicine, for
providing the human liver samples.
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