Effect of CYP2A6 genetic polymorphism on the metabolic conversion of tegafur to 5-fluorouracil and its enantioselectivity

Article abstract of DOI:10.1124/dmd.114.058008

Tegafur (FT), a prodrug of 5-fluorouracil, is a chiral molecule, a racemate of R- and S-isomers, and CYP2A6 plays an important role in the enantioselective metabolism of FT in human liver microsomes (R-FT ? S-FT). This study examined the enantioselective metabolism of FT by microsomes prepared from Sf9 cells expressing wild-type CYP2A6 and its variants (CYP2A6*7,*8, *10, and*11) that are highly prevalent in the Asian population. We also investigated the metabolism of coumarin and nicotine, both CYP2A6 probe drugs, in these variants. Enzyme kinetic analyses showed that CYP2A6.7 (I471T) and CYP2A6.10 (I471T and R485L) had markedly lower V_max values for both enantiomers than wild-type enzyme (CYP2A6.1) and other variant enzymes, whereas K_m values were higher in most of the variant enzymes for both enantiomers than CYP2A6.1. The ratios of V_max and K_m values for R-FT to corresponding values for S-FT (R/S ratio) were similar among enzymes, indicating little difference in enantioselectivity among the wild-type and variant enzymes. Similarly, both CYP2A6.7 and CYP2A6.10 had markedly lower V_max values for coumarin 7-hydroxylase and nicotine C-oxidase activities than CYP2A6.1 and other variant enzymes, whereas K_m values were higher in most of the variant enzymes for both activities than CYP2A6.1. In conclusion, the amino acid substitutions in CYP2A6 variants generally resulted in lower affinity for substrates, while V_max values were selectively reduced in CYP2A6.7 and CYP2A6.10. Consistent R/S ratios among CYP2A6.1 and variant enzymes indicated that the amino acid substitutions had little effect on enantioselectivity in the metabolism of FT. Copyright

Full text of DOI:10.1124/dmd.114.058008

1
521-009X/42/9/1485–1492$25.00
http://dx.doi.org/10.1124/dmd.114.058008
DRUG
M
ETABOLISM AND
DISPOSITION
Drug Metab Dispos 42:1485–1492, September 2014
Copyright ª 2014 by The American Society for Pharmacology and Experimental Therapeutics
Effect of CYP2A6 Genetic Polymorphism on the Metabolic
Conversion of Tegafur to 5-Fluorouracil and Its
Enantioselectivity
Ikuo Yamamiya, Kunihiro Yoshisue, Yuji Ishii, Hideyuki Yamada, and Masato Chiba
Pharmacokinetics Research Laboratories, Taiho Pharmaceutical Co., Ltd., Tsukuba, Japan (I.Y., K.Y., M.C.); and Graduate
School of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan (I.Y., Y.I., H.Y.)
Received March 8, 2014; accepted July 7, 2014
ABSTRACT
Tegafur (FT), a prodrug of 5-fluorouracil, is a chiral molecule, a
m
Vmax and K values for R-FT to corresponding values for S-FT (R/S
racemate of R- and S-isomers, and CYP2A6 plays an important role ratio) were similar among enzymes, indicating little difference in
in the enantioselective metabolism of FT in human liver microsomes enantioselectivity among the wild-type and variant enzymes. Simi-
(
R-FT >> S-FT). This study examined the enantioselective metabo- larly, both CYP2A6.7 and CYP2A6.10 had markedly lower Vmax values
lism of FT by microsomes prepared from Sf9 cells expressing wild- for coumarin 7-hydroxylase and nicotine C-oxidase activities than
type CYP2A6 and its variants (CYP2A6*7, *8, *10, and *11) that are CYP2A6.1 and other variant enzymes, whereas K values were
m
highly prevalent in the Asian population. We also investigated the higher in most of the variant enzymes for both activities than
metabolism of coumarin and nicotine, both CYP2A6 probe drugs, in CYP2A6.1. In conclusion, the amino acid substitutions in CYP2A6
these variants. Enzyme kinetic analyses showed that CYP2A6.7 variants generally resulted in lower affinity for substrates, while Vmax
(
I471T) and CYP2A6.10 (I471T and R485L) had markedly lower Vmax
values for both enantiomers than wild-type enzyme (CYP2A6.1) and Consistent R/S ratios among CYP2A6.1 and variant enzymes
other variant enzymes, whereas K values were higher in most of the indicated that the amino acid substitutions had little effect on
variant enzymes for both enantiomers than CYP2A6.1. The ratios of enantioselectivity in the metabolism of FT.
values were selectively reduced in CYP2A6.7 and CYP2A6.10.
m
Introduction
et al., 1999). To date, at least 44 allelic variants of CYP2A6, designated
as CYP2A6*2 to *45, have been identified, 35 of which have mutations
in their coding regions, as shown in the Human Cytochrome P450
Allele Nomenclature Database (http://www.cypalleles.ki.se/). Therefore,
most of these variations are likely attributed to the genetic poly-
morphisms in human CYP2A6 genes. Among these allelic variants, it is
known that CYP2A6*7, *8, *10, and *11 are highly prevalent, with
frequencies of 9.8, 1.1, 2.2, and 0.5%, respectively, in the Japanese
population (Nakajima et al., 2006). These alleles cause the following
Tegafur (FT) is a prodrug of 5-fluorouracil (5-FU) and has been
used for cancer chemotherapy. As shown in Fig. 1, FT is a chiral
molecule and contains an asymmetric carbon at the 29-position of its
tetrahydrofuran ring. Our previous study clarified that cytochrome
P450 (P450) and thymidine phosphorylase catalyze 59-hydroxylation
and 29-hydrolysis, respectively, of the tetrahydrofuran ring of FT,
followed by sequential decomposition of FT to 5-FU (Yamamiya
et al., 2010). Recently we found that CYP2A6 was responsible for the
enantioselective metabolism of FT. CYP2A6 catalyzed the conversion amino acid substitutions: I471T (CYP2A6*7), R485L (CYP2A6*8),
of both FT enantiomers to 5-FU most efficiently among P450 R485L and I471T (CYP2A6*10), and S224P (CYP2A6*11).
isoforms, and the intrinsic clearance of R-FT was significantly higher
than that of S-FT in human liver microsomes (HLMs) (Damle et al., an amino acid sequence of a particular protein, resulting in decreased
001; Yamamiya et al., 2013). Therefore, CYP2A6-dependent enzymatic activity and/or stability. Most genetic polymorphisms of CYP
Nonsynonymous single nucleotide polymorphisms (SNPs) can change
2
enantioselective metabolism likely results in the lower plasma con- genes reduce enzymatic activities, and the magnitude of effect depends on
centrations of R-FT than those of S-FT in humans after oral admin- the substrate and enantiomers of chiral substrates. For example, CYP2C9
istration of tegafur-uracil, a combination drug consisting of racemic is a major isoform for the hydroxylation of piroxicam and diclofenac.
FT and uracil at a molar ratio of 4:1 (Damle et al., 2001).
While the CYP2C9*3 (I359L) variant has drastically reduced piroxicam
CYP2A6 has been well known as a principal enzyme in the oxi- 59-hydroxylase activity, diclofenac 4-hydroxylase activity is not affected
dative metabolism of nicotine, followed by sequential conversion to (Takahashi et al., 2000). There have also been reports that a single amino
cotinine; however, it has been reported that there are large inter- acid substitution often affects enantioselectivity in the metabolism of
individual and interethnic variations in the cotinine/nicotine ratio in chiral substrates. For example, the enantioselectivity between (+)- and
humans after nicotine treatment (Nakajima et al., 1996, 2006; Yamazaki
(2)-bunitrolol 4-hydroxylase activities is completely reversed by a sub-
stitution of methionine at position 374 with valine in the recombinant
dx.doi.org/10.1124/dmd.114.058008.
CYP2D6; Vmax for (2)-bunitrolol is higher than that for the (+)-isomer in
ABBREVIATIONS: CDHP, 5-chloro-2,4-dihydroxypyridine; CLint, intrinsic clearance; CPR, NADPH–cytochrome P450 oxidoreductase; FT, tegafur;
-FU, 5-fluorouracil; HLM, human liver microsome; IS, internal standard; P450, cytochrome P450; PCR, polymerase chain reaction; SNP, single
nucleotide polymorphism; SRS, substrate recognition site.
5
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486
Yamamiya et al.
and for CPR, forward, 59-ATGATCAACATGGGAGACTCCCACG-39, and
reverse, 59-TACTCCCTGGACGTGTGGAGCTAG-39. Taq DNA polymerase
was used for the PCR amplification. Each PCR product was ligated into the
pCRII-TOPO vector. A mutation was introduced into CYP2A6 cDNA using
a QuikChange Site-Directed Mutagenesis Kit according to the manufacturer’s
instructions. Each pair of complementary mutagenic primers for creation of the
mutant variants, CYP2A6*7, *8, *10, and *11, is listed in Table 1. The variants
carrying a single mutation, i.e., CYP2A6*7 (I471T), *8 (R485L), and *11
(
S224P), were prepared using CYP2A6*1 (wild-type) cDNA as the template.
To prepare the double mutant, CYP2A6*10, the SNP region of CYP2A6*8 was
inserted into CYP2A6*7. All cDNA clones were sequenced at Hokkaido
System Science to verify the entire coding region, including the mutated site.
The CYP2A6 and CPR cDNAs were together ligated into a pFastBac Dual
vector. Recombinant baculoviruses were constructed using the Bac-to-Bac
Baculovirus Expression System according to the manufacturer’s instructions.
E. coli DH10Bac Competent Cells that harbor bacmid vector DNA were
transformed with the pFastBac Dual plasmid using the heat shock method.
Then the recombinant bacmid DNA was isolated and transfected into Sf9 cells
using Cellfectin II–mediated gene transfer to obtain the recombinant
baculovirus. Culture supernatants containing the baculovirus were collected,
Fig. 1. Chemical structure of FT enantiomers.
the wild-type CYP2D6 enzyme, while the opposite enatioselectivity is
observed in the CYP2D6 variant (Narimatsu et al., 1999).
Despite the fact that CYP2A6 is one of major polymorphic isoforms, as
described above, the functional impact of its SNPs on the enantioselective further cultured, and titered by means of the BacPAK qPCR Titration Kit
metabolism of chiral substrates has not been examined (Fukami et al., (Takara Bio Inc., Shiga, Japan). The viral preparations (three stocks) that
7
2
005; Kimura et al., 2005; Tiong et al., 2010; Han et al., 2012). FT has showed a high titer, .1.3 ꢀ 10 pfu/ml, were used for protein expression. To
prepare control microsomes, Sf9 cells were infected with either the empty
baculovirus or the virus containing only CPR cDNA.
Expression of CYP2A6 and CPR in Sf9 Cells. Sf9 cells were precultured
been approved in several oral formulations for the treatment of various
cancers and is thereby one of the most frequently prescribed drugs for
cancer chemotherapy in Asia (Watanabe, 2013). Therefore, it is important
to examine whether allelic CYP2A6 variants prevalent in Asia affect
kinetics and/or enantioselectivity in the bioactivation of FT to 5-FU.
The aim of this study was to clarify the effects of four genetic
variants of CYP2A6 (CYP2A6*7, *8, *10, and *11) on the kinetics of
6
to a cell density of 1 ꢀ 10 cells/ml at 27°C in Grace’s Insect Medium
containing 10% fetal calf serum and 0.1% Pluronic F-68. The insect cells were
then infected with the recombinant virus carrying both CYP2A6 and CPR at
a multiplicity of infection of 1.0. A mixture of hemin and albumin was added at
2
4 hours after infection so that the final concentration of hemin was 2 mg per
enantioselective conversion of FT to 5-FU. In addition, we charac- 215 mg albumin/ml (Grogan et al., 1995). At 72 hours postinfection, the cells
terized the enzyme functions of these variant proteins for coumarin were harvested by centrifugation and suspended in 50 mM Tris-HCl (pH 7.4)
7
-hydroxylase and nicotine C-oxidase activities as probes of CYP2A6 containing 1.15% KCl and 1 mM EDTA. Insect microsomes were prepared
from cellular homogenate by subsequent differential centrifugation steps at
function.
9
000g for 20 minutes and at 105,000g for 60 minutes. After that, the
microsomes were reconstituted in 10 mM Tris-HCl (pH 7.4) containing 250
mM sucrose and 0.1 mM EDTA. The protein concentration in the microsomal
Materials and Methods
Chemicals. Human liver cDNA library was purchased from BioChain preparation was measured according to the method of Bradford (1976).
Institute (Newark, CA). pCRII-TOPO vector, Bac-to-Bac Baculovirus Expression
Immunoblotting. Microsomal proteins were heat-inactivated and separated
System, pFastBac Dual vector, Cellfectin II, Pluronic F-68, and Grace’s Insect using 12% SDS-PAGE followed by an electrotransfer to a polyvinyldifluoride
Medium were purchased from Life Technologies (Carlsbad, CA). QuikChange membrane using an iBlot blotting system (Life Technologies). The membrane
Site-Directed Mutagenesis Kit was purchased from Agilent Technologies (Santa was blocked with 5% nonfat skim milk in Tris-buffered saline (pH 7.4) for 1
Clara, CA). Taq DNA polymerase was purchased from Promega (Madison, WI). hour and then soaked in either 5000-fold–diluted rabbit anti-CYP2A6
Rabbit anti-human CYP2A6 polyclonal antibody was purchased from Nosan polyclonal antibody or anti-CPR polyclonal antibody for 1 hour. A goat anti-
(
(
Kanagawa, Japan). Rabbit anti-NADPH–cytochrome P450 oxidoreductase rabbit IgG antibody conjugated with horseradish peroxidase (Sigma-Aldrich)
CPR) polyclonal antibody was purchased from Merck KGaA (Darmstadt, was used as a secondary antibody for detection. The peroxidase reaction was
Germany). Primers were synthesized at Hokkaido System Science Co. carried out using ImmunoStar LD (Wako Pure Chemical Industries), and target
(
Hokkaido, Japan). Enantiomers of FT, racemic FT, 5-chloro-2,4- protein was visualized by an ImageQuant LAS4000 Mini imaging system (GE
15
dihydroxypyridine (CDHP), and
N
2
-5-FU were prepared at Taiho Pharmaceu- Healthcare Japan, Tokyo, Japan).
Determination of CYP2A6 and CPR Contents. The CYP2A6 content in
tical Co. (Saitama, Japan). 5-FU, glucose 6-phosphate, acetaminophen, and
nicotine were purchased from Sigma-Aldrich (St. Louis, MO). Magnesium insect microsomes was determined according to the quantitative immunoblot-
chloride hexahydrate, hemin chloride, cotinine, coumarin, propranolol, and 7- ting method (Venkatakrishnan et al., 2000). The quantification standard was
hydroxycoumarin were purchased from Wako Pure Chemical Industries (Osaka, prepared by serially diluting commercially available recombinant CYP2A6 (BD
+
Japan). b-NADP and glucose-6-phosphate dehydrogenase were purchased from Gentest). The linearity of CYP2A6 content was confirmed over 100–800 pmol/
Oriental Yeast Co. (Tokyo, Japan). Other chemicals used were of the highest ml. CPR content was determined as described previously (Fukami et al., 2005).
grade commercially available.
Assay of 5-FU Formation from FT in Insect Microsomes. As 5-FU
Cells and Tissue Preparations. Sf9 insect cells and Escherichia coli formed from FT was subjected to extensive metabolism by dihydro-
DH10Bac Competent Cells were purchased from Life Technologies. Human pyrimidine dehydrogenase contamination in enzyme preparations, a potent
hepatic cytosol (pooled preparation from 20 donors) was purchased from
XenoTech (Lenexa, KS). HLMs (pooled preparation from 150 donors) were
purchased from BD Gentest (San Jose, CA). These preparations were stored at
dihydropyrimidine dehydrogenase inhibitor, CDHP, was always added to
prevent the unintended loss of 5-FU (Ikeda et al., 2000; Yamamiya et al.,
2010, 2013). The reaction mixture (0.1 ml) in the assay of insect microsomal
2
80°C until use.
metabolism contained FT, 50–100 pmol/ml CYP2A6, 0.1 mM CDHP, and an
Isolation of cDNA and Construction of Recombinant Baculoviruses. The NADPH-generating system consisting of 1.3 mM b-NADP , 3.3 mM glucose
+
open reading frames of human CYP2A6 and CPR were amplified from
a human liver cDNA library by means of polymerase chain reaction (PCR)
6-phosphate, 3.3 mM magnesium chloride, and 0.4 units of glucose-6-
phosphate dehydrogenase in 100 mM Tris-HCl (pH 7.4). The total
using the following primers: for CYP2A6, forward, 59-ATGCTGGCCT- concentration of microsomal protein was adjusted to 2 mg/ml by adding
CAGGGATGCTTCTG-39, and reverse, 59-CTACACCATGAGCTTCCTG-39; control microsomes. We ensured that differences in microsomal protein

The Enantioselective Metabolism of FT in CYP2A6 Variants
1487
TABLE 1
Sequences for complementary mutagenic oligonucleotides used in the construction of the indicated variants to generate
CYP2A6 variants
The mutated bases are underlined. The double mutations in CYP2A6*10 were generated by the sequential addition of changes by using
oligonucleotides for CYP2A6*7 and *8.
Amino Acid Alteration
I471T
Allele
Direction
Sequence
CYP2A6*7
Sense
Antisense
Sense
Antisense
Sense
Antisense
59-GTCACCTAAGGACACTGACGTGTCCCCC-39
59-GGGGGACACGTCAGTGTCCTTAGGTGAC-39
59-GCCACGATCCCACTAAACTACACCATGAG-39
59-CTCATGGTGTAGTTTAGTGGGATCGTGGC-39
59-CTCTATGAGATGTTCCCTTCGGTGATG-39
59-CATCACCGAAGGGAACATCTCATAGAG-39
R485L
S224P
CYP2A6*8
CYP2A6*11
V ¼ Vmax ꢀ S=ðKm þ SÞ
ð1Þ
ð2Þ
concentrations between the wild-type and variants did not affect the
determinations of kinetic parameters by adjusting the protein concentrations
with control microsomes. In some cases, HLMs (1 mg/ml) were used as the
enzyme source instead of Sf9 microsomes expressing CYP2A6. The in-
cubation mixture was prewarmed for 5 minutes at 37°C, and then the reaction
was started by adding the substrate. The reaction was continued at 37°C for
V ¼ Vmax1 ꢀ S=ðKm1 þ SÞ þ Vmax2 ꢀ S=ðKm2 þ SÞ
where V is metabolite formation rate; S is substrate concentration in the
incubation mixture; Km1 and Km2 are Michaelis-Menten constants for high- and
low-affinity phases, respectively; and Vmax1 and Vmax2 are maximum velocities
for high- and low-affinity phases, respectively.
Statistical Analysis. Kinetic parameters 6 S.E. of recombinant CYP2A6 and
HLM were calculated by fitting the data on the formation rates of metabolites and
concentrations of substrates to the Michaelis-Menten equation by nonlinear
regression analysis, using Phoenix WinNonlin Professional version 6.1 (Certara,
St. Louis, MO). The intrinsic clearance (CLint) was calculated by dividing Vmax
1
5–60 minutes and stopped by adding 3 volumes of ice-cold acetonitrile.
After centrifugation, the supernatant was collected and stored at 280°C until
quantification of 5-FU. Because a small portion of FT is spontaneously
converted to 5-FU, the amount of 5-FU formed in the incubation with control
microsomes was subtracted from the total yield obtained in the enzymatic
reaction. The 5-FU in samples was quantified by means of a previously
developed method (Yamamiya et al., 2013).
m
by K . Assuming normal distribution as the sample size got larger, statistical
Assay of Coumarin 7-Hydroxylase and Nicotine C-oxidase Activity. In
the assay of coumarin 7-hydroxylase activity, the reaction mixture contained
coumarin, 10 pmol/ml CYP2A6, and an NADPH-generating system in 100 mM
Tris-HCl (pH 7.4). The assay of nicotine C-oxidase activity was performed as
follows. Because the conversion of nicotine-D19(59)-iminium ion, which is
formed from nicotine by CYP2A6 catalysis, to cotinine requires cytosolic
aldehyde oxidase, we added the human hepatic cytosol at a concentration of 3
mg/ml to the reaction mixture. For the assay of coumarin 7-hydroxylase and
nicotine C-oxidase activities, the total concentration of microsomal protein was
adjusted to 0.25 mg/ml by adding control microsomes. In a separate
experiment, HLMs (0.25 mg/ml) were used as the enzyme source instead of
Sf9 microsomes expressing CYP2A6. After preincubation at 37°C for 5
minutes, the reaction was initiated by adding the substrate, and the mixture was
then incubated at 37°C for 10–30 minutes. The reaction was stopped by adding
comparisons of the Michaelis-Menten parameter estimates between the wild-type
and variant enzymes were carried out using the following two-sided test statistic:
z ¼jPw 2 Pvj=√S:E: 2w þ S:E:
2
v
ð3Þ
where P
of the kinetic parameter estimate of the wild-type, respectively; and P
the K or Vmax value of the variant and the standard error of the kinetic parameter
w
and S.E.
w
are the K
m
or Vmax value of the wild-type and the standard error
v
and S.E. are
v
m
estimate of the variant, respectively. A P value of ,0.05 was considered significant.
Results
Expression of CYP2A6 and CPR in Sf9 Cells. The protein
an equal volume of ice-cold acetonitrile containing internal standard (IS) to the expression of CYP2A6 variants and CPR in the microsomes prepared
reaction mixture, followed by centrifugation. The supernatant was collected and from Sf9 cells was determined by immunoblot analyses after
stored at 280°C until analysis.
separation by SDS-PAGE (Fig. 2), which were also used to quantify
Concentrations of 7-hydroxycoumarin and cotinine were measured using
CYP2A6 content in each preparation (Table 2). In this study, the
liquid chromatography–tandem mass spectrometry. The analytical system con-
CYP2A6 proteins prepared were named according to the inclusion
sisted of an HP 1100 liquid chromatograph (Agilent Technologies) coupled with
criteria of the Human Cytochrome P450 Allele Nomenclature Data-
an API 4000 triple-quadrupole mass spectrometer (AB Sciex, Framingham, MA)
base. The CPR content was calculated from NADPH–cytochrome
equipped with a Turbo V source and electrospray ionization interface. Positive
electrospray ionization was used for all metabolites and ISs. Sample separation
was performed using a Unison UK-C18 (2.0-mm i.d. ꢀ 50 mm, 3 mm; Imtakt,
Kyoto, Japan) at a flow rate of 0.2 ml/min of the mobile phase consisting of (A)
0.1% formic acid for the 7-hydroxycoumarin assay or 10 mM formic ammonium
for the cotinine assay and (B) acetonitrile at 40°C. The following gradients were
used: in the 7-hydroxycoumarin assay, 20 to 95% B in 5 minutes, 95 to 20% B in
0.1 minute, and 20% B held for 5 minutes; and in the cotinine assay, 10 to 70% B
in 2 minutes, 70 to 10% B in 0.1 minute, and 10% B held for 4 minutes. The
precursor/product ion mass transitions were monitored for determination of the
metabolites and ISs: m/z 163.0/107.0 for 7-hydroxycoumarin, m/z 177.0/98.0 for
cotinine, m/z 260.0/116.0 for propranolol (IS for 7-hydroxycoumarin), and m/z
Fig. 2. Immunoblot analyses of variant CYP2A6 (A) and CPR (B) in insect
microsomes. Microsomes loaded at 10–20 mg of protein per well were subjected to
SDS-PAGE in 12% acrylamide gels and then transferred electrophoretically to
a polyvinyldifluoride membrane. The immunoblots were probed with anti-CYP2A6
and anti-CPR antibodies followed by horseradish peroxidase–conjugated goat anti-
rabbit antibody. The antigen-antibody complexes were detected using a chemilumi-
nescent assay. The commercially available insect microsomes expressing both
CYP2A6 and CPR were loaded onto lane 1 as a positive control. Control Sf9
microsomes expressing neither CYP2A6 nor CPR prepared in this study were loaded
onto lane 2. Lanes 3–8 contained the Sf9 microsomes expressing the following
enzymes: CPR (3), CYP2A6.1 and CPR (4), CYP2A6.7 and CPR (5); CYP2A6.8
and CPR (6), CYP2A6.10 and CPR (7), and CYP2A6.11 and CPR (8).
152.0/110.0 for acetaminophen (IS for cotinine).
Kinetic Analysis. For determining kinetic parameters, FT, coumarin, and
nicotine concentrations varied in ranges between 0.03 and 10 mM, 0.1 and 30,
and 3 and 500 mM, respectively. All reactions were performed in a linear range
of the metabolite formation with respect to enzyme amount and incubation
time. Kinetic parameters were estimated by fitting the data to eq. 1 (monophasic
kinetics) or eq. 2 (biphasic kinetics):

1
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Yamamiya et al.
TABLE 2
Expressions of CYP2A6 and CPR in insect microsomes expressing wild-type
CYP2A6 or its variants
Enzyme (Amino Acid Alteration)
CYP2A6
CPR
CPR/CYP2A6 Ratio
pmol/mg protein
102 21.4
69.8
71.8
59.7
CYP2A6.1 (wild-type)
CYP2A6.7 (I471T)
CYP2A6.8 (R485L)
CYP2A6.10 (I471T and R485L)
CYP2A6.11 (S224P)
0.22
0.22
0.24
0.29
0.25
15.4
17.1
17.4
32.1
130
c reductase activity in each preparation by using reported specific
activity of microsomal CPR (Table 2). Rabbit anti-CYP2A6 and anti-
CPR polyclonal antibodies detected CYP2A6 and CPR expressed in
Sf9 cells with molecular masses of approximately 50 and 80 kDa,
respectively, which corresponded to known molecular weights of
these enzymes (Vermilion and Coon, 1978; Tiong et al., 2010). The
molecular weight of the positive control for CYP2A6 (Fig. 2, lane 1)
was similar to that of microsomes expressing CYP2A6.1 (Fig. 2, lane
4
) or each variant (Fig. 2, lanes 5–8). In contrast, immunoreactivity
with anti-CYP2A6 antibody was absent in the control microsomes
Fig. 2, lane 2) and in the microsomes prepared from cells only
expressing CPR (Fig. 2, lane 3). The expression content of CYP2A6
and CPR) and molar ratio of CPR to CYP2A6 in each sample are
(
(
summarized in Table 2. While the expression contents of CYP2A6
and CPR varied from 59.7 to 130 and from 15.4 to 32.1 pmol/mg of
protein, respectively, the molar ratios of CPR to CYP2A6 were similar
(
0.22–0.29) among microsomal preparations (Table 2).
Kinetic Analyses for Coumarin 7-Hydroxylation and Nicotine
C-Oxidation. For the functional characterization of CYP2A6 variants, Fig. 3. Michaelis-Menten plots of formation of 7-hydroxycoumrain (A) and
coumarin 7-hydroxylase and nicotine C-oxidase activities at different cotinine (B) mediated by wild-type CYP2A6 (CYP2A6.1) and its variants.
Coumarin (0.1–30 mM) and nicotine (3–500 mM) were incubated with CYP2A6
variants (10 pmol/ml) in the presence of an NADPH-generating system at 37°C for
substrate concentrations were fitted to the Michaelis-Menten equation to
obtain kinetic parameters in each preparation (Fig. 3, A and B). To
1
0–30 minutes. When necessary, the protein concentration was adjusted to 0.25 mg/
assess the validity of the probe assays in the insect microsomes, the ml by adding control microsomes. In the assay of nicotine C-oxidase, the human
hepatic cytosol (final concentration, 3 mg/ml) was also added to the reaction
mixture. Kinetic parameters were calculated by curve fitting with the Michaelis-
Menten equation, and the results for coumarin 7-hydroxylation and nicotine C-
kinetic parameters for both reactions in wild-type CYP2A6 (CYP2A6.1)
were also compared with those obtained in HLMs. As listed in Table 3,
CYP2A6.1 and HLMs had the same K
m
values (1.7 mM) for coumarin oxidation are listed in Table 3. Each data point represents an average of triplicate
incubations (mean 6 S.D.) within one experiment.
7
-hydroxylation. Moreover, the K value for nicotine C-oxidation in
m
CYP2A6.1 (72 mM) was similar to that (61 mM) in the high-affinity
component in HLMs. These results indicated that wild-type CYP2A6
(CYP2A6.1) had a catalytic affinity similar to that of the native enzyme
Kinetic Analyses of Enantioselective Conversion of FT to 5-FU.
in HLMs. The K
m
values for coumarin 7-hydroxylation in all CYP2A6 To examine the effect of CYP2A6 polymorphisms on enantioselective
variants (CYP2A6.7, CYP2A6.8, CYP2A6.10, and CYP2A6.11) were FT metabolism, the kinetics of 5-FU formations from R- and S-FT
.4- to 3.2-fold higher than the value in CYP2A6.1, and the Vmax values were compared among CYP2A6 variants. The enantioselective con-
1
in CYP2A6.7 and CYP2A6.10 were significantly lower than that in versions from R- and S-FT to 5-FU catalyzed by CYP2A6 variants
CYP2A6.1 by 4- and 7-fold, respectively (Table 3). As a result, all were fitted by Michaelis-Menten kinetics (Fig. 4, A and B, re-
mutations in the CYP2A6 gene resulted in markedly reduced catalytic spectively), and the obtained kinetic parameters for R- and S-FT
activity (as expressed by CLint or Vmax/K
the decreases in catalytic activity were especially pronounced in 7-hydroxylase and nicotine C-oxidase activities in CYP2A6 variants,
CYP2A6.7 and CYP2A6.10, which were 17 and 4.7%, respecti- the K values for both conversions of R- and S-FT to 5-FU were larger
m
) for coumarin 7-hydroxylation; are listed in Table 4. As observed in the kinetics of coumarin
m
vely, of the wild-type activity in CYP2A6.1 (Table 3). Similar in most of the CYP2A6 variants than that in wild-type CYP2A6
decreases in catalytic activity were observed for nicotine C-oxidase (CYP2A6.1), while the Vmax values for both enantiomers were markedly
activity in allelic variant enzymes (Fig. 3B; Table 3); the Km values lower in CYP2A6.7 and CYP2A6.10 than those in CYP2A6.1 and other
were more than 2-fold higher in all CYP2A6 variants than in CYP2A6.1, variants. These alterations resulted in almost completely impaired
and the Vmax values were markedly lower in CYP2A6.7 and CYP2A6.10 catalytic activities in CYP2A6.7 and CYP2A6.10: 7.2 and 1.3% of
than in CYP2A6.1, resulting in mostly impaired catalytic activities. control activity for the metabolism of R-FT in CYP2A6.7 and
The nicotine C-oxidase activity in CYP2A6.7 was 6.4% of the wild- CYP2A6.10, respectively; 7.7 and 0.6% of control activity for the
type activity. Similarly, CYP2A6.10 showed the minimal activity, metabolism of S-FT in CYP2A6.7 and CYP2A6.10, respectively
without reaching apparent saturation at the highest concentration of (Table 4). Although the degree of alterations in kinetic parameters
5
7
00 mM; the K
85 6 179 mM.
m
value estimated by nonlinear regression analysis was for the metabolism of FT enantiomers differed among the CYP2A6
variants, the ratios of obtained parameters for R-FT to those for S-FT

The Enantioselective Metabolism of FT in CYP2A6 Variants
1489
TABLE 3
Kinetic parameters for coumarin 7-hydroxylase and nicotine C-oxidase activities mediated by wild-type CYP2A6 and its variants
The kinetic parameters were calculated by nonlinear regression analysis. Each kinetic value represents the estimated value 6 S.E.
Kinetic Parameters
Reaction
Enzyme (Amino Acid Alteration)
e
V
max
K
m
CLint
Fold Change
pmol/min per pmol CYP2A6
mM
ml/min per pmol CYP2A6
Coumarin 7-hydroxylase
CYP2A6.1 (wild-type)
CYP2A6.7 (I471T)
CYP2A6.8 (R485L)
CYP2A6.10 (I471T and R485L)
CYP2A6.11 (S224P)
HLMs
CYP2A6.1 (wild-type)
CYP2A6.7 (I471T)
CYP2A6.8 (R485L)
CYP2A6.10 (I471T and R485L)
CYP2A6.11 (S224P)
HLMs
15 6 1
3.4 6 0.1**
13 6 1
2.3 6 0.1**
14 6 1
611 6 30
1.7 6 0.1
2.3 6 0.2*
3.0 6 0.2**
5.5 6 0.5**
4.2 6 0.3**
1.7 6 0.1
72 6 1
242 6 24**
161 6 7**
.500
8.7
1.5
4.4
0.41
3.2
1.0
0.17
0.51
0.047
0.37
N.A.
1.0
0.064
0.58
N.A.
0.77
N.A.
N.A.
b
d
365
Nicotine C-oxidase
47 6 1
0.65
0.041
0.38
N.A.
0.50
10 6 1**
61 6 2**
N.A.
81 6 1**
502 6 14
a
163 6 4**
61 6 2
b
d
8.2
b
c
d
7
96 6 105
1.0 6 0.2
0.78
N.A., not available.
a
CYP2A6.10-mediated formation of cotinine did not reach apparent saturation within the substrate concentration used.
pmol/min per mg protein.
b
c
mM.
d
ml/min per mg protein.
e
m
The ratio of CLint (Vmax/K ) in each CYP2A6 variant to that in CYP2A6.1.
*P , 0.05; **P , 0.01 versus wild-type enzyme.
(
R/S ratio in Table 6) were found to be similar among variants, sug- these amino acid substitutions in CYP2A6 variants reduced the catalytic
gesting that the amino acid substitutions in CYP2A6 variants had little activity for racemic FT to a similar extent as that for the enantiomers
effect on enantioselectivity in the metabolism of FT to 5-FU. Furthermore, (Fig. 4C; Table 5). Among the reduced catalytic activities for the
Fig. 4. Michaelis-Menten plots of 5-FU for-
mation from R-FT (A), S-FT (B), and racemic
FT (C) mediated by wild-type CYP2A6
(
CYP2A6.1) and its variants. FT (0.03–10
mM) was incubated with CYP2A6 variants
50–100 pmol/ml) and 0.1 mM CDHP in the
presence of an NADPH-generating system at
7°C for 15–60 minutes. When necessary, the
(
3
protein concentration was adjusted to 2 mg/ml
by adding control microsomes. Kinetic param-
eters were calculated by curve fitting with the
Michaelis-Menten equation, and the results for
the metabolism of R- and S-FT and of racemic
FT are listed in Tables 4 and 5, respectively.
Each data point represents an average of
triplicate incubations (mean 6 S.D.) within
one experiment.

1
490
Yamamiya et al.
TABLE 4
Kinetic parameters for 5-FU formation from R- and S-FT mediated by wild-type CYP2A6 and its variants
The kinetic parameters were calculated by nonlinear regression analysis. Each kinetic value represents the estimated value 6 S.E.
Kinetic Parameters
Enantiomer
Enzyme (Amino Acid Alteration)
d
V
max
K
m
CLint
Fold Change
pmol/min per pmol CYP2A6
mM
nl/min per pmol CYP2A6
R-FT
CYP2A6.1 (wild-type)
CYP2A6.7 (I471T)
CYP2A6.8 (R485L)
9.1 6 0.2
0.16 6 0.01
0.23 6 0.01**
0.48 6 0.01**
0.64 6 0.03**
0.76 6 0.05**
0.13 6 0.01
2.0 6 0.5
1.3 6 0.1
1.5 6 0.1
3.6 6 0.3**
6.6 6 1.9*
6.6 6 0.7**
1.6 6 0.1
58
4.2
14
0.73
8.0
5.3
1.0
0.072
0.25
0.013
0.14
N.A.
N.A.
1.0
0.077
0.23
0.0060
0.087
N.A.
1.0 6 0.0**
6.9 6 0.1**
0.47 6 0.02**
6.1 6 0.3**
CYP2A6.10 (I471T and R485L)
CYP2A6.11 (S224P)
a
b
c
HLMs
707 6 30
b
c
4
39 6 63
0.22
7.7
S-FT
CYP2A6.1 (wild-type)
CYP2A6.7 (I471T)
CYP2A6.8 (R485L)
CYP2A6.10 (I471T and R485L)
CYP2A6.11 (S224P)
HLMs
9.8 6 0.4
0.88 6 0.04**
6.2 6 0.4**
0.31 6 0.07**
4.4 6 0.4**
0.60
1.7
0.046
0.67c
0.67
b
1072 6 34
N.A., not available.
a
The conversion of R-FT to 5-FU followed biphasic kinetics in HLMs.
pmol/min per mg protein.
b
c
ml/min per mg protein.
d
m
The ratio of CLint (Vmax/K ) in each CYP2A6 variant to that in CYP2A6.1.
*P , 0.05; **P , 0.01 versus wild-type enzyme.
conversion of racemic FT to 5-FU in CYP2A6 variants (Table 5), prepared from transfected Sf9 cells had CYP2A6 protein and catalytic
CYP2A6.7 and CYP2A6.10 had greatly impaired catalytic activities activity with comparable CYP2A6/CPR expression level ratios among
for the metabolism of racemic FT, which were 6.8 and 1.3%, re- the variants, suggesting that the wild-type and variant CYP2A6
spectively, of control activity in CYP2A6.1. As observed in the enzymes were allowed similar access to electrons supplied by CPR
metabolism of CYP2A6 probe drugs, CYP2A6.1 had very similar (Fig. 2; Table 2).
K
m
values for the metabolism of R- and S-FT and racemic FT (0.16,
The enantioselective conversion of FT to 5-FU is exclusively
1
.3, and 0.29 mM, respectively) to those of a high-affinity com- mediated by CYP2A6, and many genetic variants have been identified
ponent in HLMs (0.13, 1.6, and 0.27 mM, respectively), which in the CYP2A6 gene. Most mutations that have emerged in the
predominantly catalyzes the conversion of FT to 5-FU in HLMs CYP2A6 gene are presumed to cause interindividual and interethnic
(
Tables 4 and 5).
variations in CYP2A6-dependent metabolism; indeed, the allele
frequency of CYP2A6 polymorphisms is highly variable between
ethnic groups. For example, African Americans and white Americans
prevalently possess allelic variants including CYP2A6*2, *14, *16,
Discussion
The present study established a baculovirus-Sf9 cell system and *21 alleles, which are not found in the Japanese population
coexpressing CYP2A6 variants and CPR, which allowed the high- (Nakajima et al., 2006). In contrast to American populations, the
level production of active proteins without modifications from full- CYP2A6*4, *7, *8, *9, *10, *11, *13, and *15 alleles are known to be
length P450 cDNAs, and the system was used to analyze the effect of prevalent in Asian populations. Of these, CYP2A6*9, *13, and *15
amino acid substitutions in CYP2A6 on the kinetics of enantiose- have a SNP in the promoter region that results in decreased CYP2A6
lective metabolism of FT in the formation of 5-FU. Microsomes expression, leading to loss of activity (Nakajima et al., 2006). On the
TABLE 5
Kinetic parameters for 5-FU formation from racemic FT mediated by wild-type CYP2A6 and its variants
The kinetic parameters were calculated by nonlinear regression analysis. Each kinetic value represents the estimated value 6 S.E.
Kinetic Parameters
Recombinant Enzyme (Amino Acid Alteration)
d
V
max
K
m
CLint
Fold Change
pmol/min per pmol CYP2A6
mM
nl/min per pmol CYP2A6
CYP2A6.1 (wild-type)
CYP2A6.7 (I471T)
CYP2A6.8 (R485L)
12 6 0
0.29 6 0.01
0.43 6 0.02**
0.58 6 0.02**
0.86 6 0.10**
0.79 6 0.04**
0.27 6 0.02
3.7 6 0.5
40
2.7
14
0.54
7.5
2.6
1.0
1.2 6 0.0**
7.8 6 0.2**
0.46 6 0.03**
5.9 6 0.2**
0.068
0.34
0.013
0.19
N.A.
N.A.
CYP2A6.10 (I471T and R485L)
CYP2A6.11 (S224P)
a
b
c
HLMs
679 6 49
b
c
7
86 6 56
0.21
N.A., not available.
a
The conversion of racemic FT to 5-FU followed biphasic kinetics in HLMs.
pmol/min per mg protein.
b
c
ml/min per mg protein.
d
m
The ratio of CLint (Vmax/K ) in each CYP2A6 variant to that in CYP2A6.1.
*
*P , 0.01 versus wild-type enzyme.

The Enantioselective Metabolism of FT in CYP2A6 Variants
1491
other hand, the CYP2A6*4 allele is a gene deletion type, and its adjacent to helix F, which contributes to the formation of the substrate
homozygous genotype is associated with a complete absence of access site in CYP2A6 (Yano et al., 2005). Therefore, this amino acid
enzymatic activity (Nakajima et al., 2004). For example, a positive change could possibly disrupt the access channel of substrates and modify
correlation exists between expression level of CYP2A6 mRNA and the spatial structure at the putative active site of CYP2A6, leading to
coumarin 7-hydroxylase activity in liver samples from Japanese the alteration of the kinetic properties. The active-site volume of CYP2A6
individuals carrying CYP2A6*1 (wild-type), *4, or *9 alleles (Yoshida is ,25% of the volumes calculated for CYP2C8, CYP2C9, and CYP3A4
et al., 2003). Because the decreasing effects of CYP2A6*4 and *9 (Yano et al., 2005). The markedly small volume of the active site in
alleles identified as regulatory polymorphisms on CYP2A6 activ- CYP2A6 at least reflects differences in the conformation of the peptide
ity would be independent of substrate recognition and substrates, backbone that lead to a relatively low pitch of helices F and G over
the kinetic analyses for these alleles were excluded in the present the top of the active-site cavity as well as tighter packing interactions
study. For the other allelic variants prevalent in Asian populations in the remainder of the molecule. It is therefore likely that an alteration
(
CYP2A6*7, *8, *10, and *11 alleles), it has been reported that these of CYP2A6 structure has a greater effect on the access or binding of
variants have reduced either in vivo or in vitro metabolic activities of substrates responsible for the enzymatic affinity compared with other
CYP2A6 (Daigo et al., 2002; Fukami et al., 2005; Kimura et al., 2005; P450 isoforms.
Han et al., 2012). The effects of individual SNPs (CYP2A6*7 and *8)
on the coding region of the CYP2A6 gene and their combination study resulted in lower affinity of substrate than wild-type CYP2A6,
CYP2A6*10) on enzymatic function or kinetics have not been simul- while the enantioselectivity in the metabolism of FT was almost
taneously explored in recombinant CYP2A6 enzymes (Fukami et al., identical between the wild-type and variants (Tables 4 and 6), and
005; Kimura et al., 2005; Han et al., 2012). Therefore, it was hy- subsequently, the extent of decrease in (or fold change of) catalytic
CYP2A6 polymorphisms by the allelic variants tested in the present
(
2
pothesized that the amino acid substitutions encoded by these allelic activity (CLint) for racemic FT in each variant (Table 5) was similar to
variants potentially affect the previously observed enantioselective con- that for each stereoisomer (Table 4). These findings suggest that
version of FT to 5-FU by altering kinetic parameters for the metabolism polymorphic amino acid residues in CYP2A6 tested in the present
by CYP2A6.
study are not involved in chiral recognition for the binding of FT
A single amino acid change in CYP2A6.7 (I471L) drastically reduced enantiomers: these amino acid residues are not located in the SRS (Xu
max values for all CYP2A6-mediated metabolisms tested in the present et al., 2002; Zhang et al., 2009), and therefore they are less likely
V
study, and the decrease was more pronounced in the CYP2A6*10 allele, involved in the direct interactions with the substrate.
which has two amino acid changes (I471L and R485L) (Tables 3 and 4). It has been reported that the plasma concentrations of racemic FT
This observation is consistent with the previous report that nicotine C- are higher in patients with a variant than those with the wild-type
oxidase activity was markedly reduced in both Japanese and Korean CYP2A6 allele, suggesting that the oral clearance of FT depends on the
subjects heterozygous for CYP2A6*7/CYP2A6*10 and homozygous for CYP2A6 genotype (Daigo et al., 2002; Fujita et al., 2008; Hirose et al.,
CYP2A6*7/CYP2A6*7 (Yoshida et al., 2002). Both residues of I471 and 2010; Chuah et al., 2011). In addition, the positive correlation between
R485 in CYP2A6 are located in the hinge region close to substrate cotinine/nicotine ratio and FT clearance confirmed the importance of
recognition site (SRS) 6 and responsible for the folding of the protein CYP2A6 in FT bioactivation, and it has been suggested that the
around this site. Based on the results in this study, they likely have correlation potentially facilitates prediction of individual 5-FU
distinct roles in catalysis: comparison of kinetic data among CYP2A6.7 concentrations based on a simple nicotine test (Chuah et al., 2011).
(
I471T), CYP2A6.8 (R485L), and CYP2A6.10 (I471T and R485L), The kinetic data on FT metabolism reported in this study strongly
variants prevalent in the Asian population, revealed that I471 played support this hypothesis: nearly complete losses in enzymatic activities
a more important role in determining CYP2A6 catalytic capacity (Vmax for the conversion of FT to 5-FU observed in CYP2A6.7 and
for all CYP2A6 substrates than R485, whose substitution alone had little CYP2A6.10 suggested that the efficacy of FT could be compromised
or no effect on the capacity. in patients having these polymorphisms (Tables 4 and 5). CYP2A6
)
Carbon monoxide difference spectral analysis has demonstrated that is also the principal enzyme responsible for the metabolism of pilo-
the I471T substitution reduces the thermal stability of holoenzyme carpine, letrozole, and valproic acid in humans (Di et al., 2009).
(Ariyoshi et al., 2001), which is also likely to contribute to the marked Moreover, it has been reported that plasma concentrations of these drugs
reductions in Vmax of CYP2A6.7 and CYP2A6.10. The replacement of show high interindividual variability and are associated significantly
isoleucine with a hydrophobic side chain to a polar amino acid, with CYP2A6 genotypes (Endo et al., 2008; Tan et al., 2010; Desta
threonine, caused by the CYP2A6*7 and *10 alleles might be expected et al., 2011). Thus, CYP2A6 polymorphisms can alter the pharma-
to change the conformation of CYP2A6, resulting in the decrease of cokinetics of CYP2A6 substrates, resulting in interindividual vari-
heme folding capacity followed by the disordering of the holoprotein ations in their plasma exposures, and hence the association between
structure. On the other hand, the immunoblot analyses in the present the genetic variants and clinical responses, including the therapeutic
study revealed that CYP2A6.7 and CYP2A6.10 retained stable proteins and side effects of the drugs metabolized by CYP2A6, needs to be
consisting of both apo- and holo- forms in the insect microsomes (Fig. investigated in further studies.
2; Table 2). Taken together, the results show that it is possible that the
inactive apoproteins are partly presented in CYP2A6.7 and CYP2A6.10.
However, it is currently unclear which of the catalytic activity, the
stability of the holoprotein, or the heme incorporation in the variant
TABLE 6
R/S ratio of kinetic parameters of FT enantiomers
R/S Ratio
CYP2A6 enzymes has a major effect on the Vmax
.
Recombinant Enzyme (Amino Acid Alteration)
There has been little information on the role of S224, which is located
outside the SRS responsible for the maintenance of CYP2A6 confor-
mation. The S224P substitution affected both Vmax and K , suggesting
m
that this residue contributes to both catalytic capacity and substrate
affinity of CYP2A6 (Tables 3 and 4). The three-dimensional structure as
revealed by X-ray crystallography illustrates that this residue is located
Vmax
Km
CLint
CYP2A6.1 (wild-type)
CYP2A6.7 (I471T)
CYP2A6.8 (R485L)
CYP2A6.10 (I471T and R485L)
CYP2A6.11 (S224P)
0.93
1.1
1.1
1.5
1.4
0.12
0.16
0.13
0.10
0.11
7.6
7.0
8.2
16
12

1
492
Yamamiya et al.
Hirose T, Fujita K, Nishimura K, Ishida H, Yamashita K, Sunakawa Y, Mizuno K, Miwa K,
Nagashima F, and Tanigawara Y, et al. (2010) Pharmacokinetics of S-1 and CYP2A6 genotype
in Japanese patients with advanced cancer. Oncol Rep 24:529–536.
Ikeda K, Yoshisue K, Matsushima E, Nagayama S, Kobayashi K, Tyson CA, Chiba K,
and Kawaguchi Y (2000) Bioactivation of tegafur to 5-fluorouracil is catalyzed by cytochrome
P-450 2A6 in human liver microsomes in vitro. Clin Cancer Res 6:4409–4415.
Kimura M, Yamazaki H, Fujieda M, Kiyotani K, Honda G, Saruwatari J, Nakagawa K,
Ishizaki T, and Kamataki T (2005) Cyp2a6 is a principal enzyme involved in hydroxylation
of 1,7-dimethylxanthine, a main caffeine metabolite, in humans. Drug Metab Dispos 33:
1361–1366.
Nakajima M, Fukami T, Yamanaka H, Higashi E, Sakai H, Yoshida R, Kwon JT, McLeod HL,
and Yokoi T (2006) Comprehensive evaluation of variability in nicotine metabolism and
CYP2A6 polymorphic alleles in four ethnic populations. Clin Pharmacol Ther 80:282–297.
Nakajima M, Yamamoto T, Nunoya K, Yokoi T, Nagashima K, Inoue K, Funae Y, Shimada N,
Kamataki T, and Kuroiwa Y (1996) Role of human cytochrome P4502A6 in C-oxidation of
nicotine. Drug Metab Dispos 24:1212–1217.
In conclusion, the polymorphic CYP2A6 alleles prevalently obser-
ved in the Japanese population (CYP2A6*7, *8, *10, and *11) gen-
erally resulted in lower affinity for CYP2A6 substrates, while Vmax
values were selectively reduced in CYP2A6.7 (I471L) and CYP2A6.10
(
I471L and R485L) for all CYP2A6 substrates. However, CYP2A6
polymorphisms had the same degree of effect on 5-FU formation from
two enantiomers of FT, suggesting that plasma concentrations of these
isomers are altered in parallel among patients with variant alleles of
CYP2A6.
Acknowledgments
Nakajima M, Yoshida R, Fukami T, McLeod HL, and Yokoi T (2004) Novel human CYP2A6
alleles confound gene deletion analysis. FEBS Lett 569:75–81.
Narimatsu S, Kato R, Horie T, Ono S, Tsutsui M, Yabusaki Y, Ohmori S, Kitada M, Ichioka T,
and Shimada N, et al. (1999) Enantioselectivity of bunitrolol 4-hydroxylation is reversed by the
change of an amino acid residue from valine to methionine at position 374 of cytochrome
P450-2D6. Chirality 11:1–9.
The authors thank Ken-ichiro Yoshida (Taiho Pharmaceutical Co.) for
helpful comments on the manuscript.
Authorship Contributions
Participated in research design: Yamamiya, Yoshisue, Ishii, Yamada.
Conducted experiments: Yamamiya.
Contributed new reagents or analytic tools: Yamamiya, Yoshisue, Ishii,
Yamada.
Performed data analysis: Yamamiya.
Wrote or contributed to the writing of the manuscript: Yamamiya, Yoshisue,
Ishii, Yamada, Chiba.
Takanashi K, Tainaka H, Kobayashi K, Yasumori T, Hosakawa M, and Chiba K (2000) CYP2C9
Ile359 and Leu359 variants: enzyme kinetic study with seven substrates. Pharmacogenetics 10:
9
5–104.
Tan L, Yu JT, Sun YP, Ou JR, Song JH, and Yu Y (2010) The influence of cytochrome oxidase
CYP2A6, CYP2B6, and CYP2C9 polymorphisms on the plasma concentrations of valproic
acid in epileptic patients. Clin Neurol Neurosurg 112:320–323.
Tiong KH, Yiap BC, Tan EL, Ismail R, and Ong CE (2010) Functional characterization of
cytochrome P450 2A6 allelic variants CYP2A6*15, CYP2A6*16, CYP2A6*21, and
CYP2A6*22. Drug Metab Dispos 38:745–751.
Venkatakrishnan K, von Moltke LL, Court MH, Harmatz JS, Crespi CL, and Greenblatt DJ
(
2000) Comparison between cytochrome P450 (CYP) content and relative activity approaches
References
to scaling from cDNA-expressed CYPs to human liver microsomes: ratios of accessory proteins
as sources of discrepancies between the approaches. Drug Metab Dispos 28:1493–1504.
Vermilion JL and Coon MJ (1978) Purified liver microsomal NADPH-cytochrome P-450 re-
ductase. Spectral characterization of oxidation-reduction states. J Biol Chem 253:2694–2704.
Watanabe T (2013) Evidence produced in Japan: tegafur-based preparations for postoperative
chemotherapy in breast cancer. Breast Cancer 20:302–309.
Xu C, Rao YS, Xu B, Hoffmann E, Jones J, Sellers EM, and Tyndale RF (2002) An in vivo pilot
study characterizing the new CYP2A6*7, *8, and *10 alleles. Biochem Biophys Res Commun
2
Yamamiya I, Yoshisue K, Ishii Y, Yamada H, and Yoshida K (2013) Enantioselectivity in the
cytochrome P450-dependent conversion of tegafur to 5-fluorouracil in human liver micro-
somes. Pharmacol Res Perspect 1:e00009.
Yamamiya I, Yoshisue K, Matsushima E, and Nagayama S (2010) Formation pathways of
gamma-butyrolactone from the furan ring of tegafur during its conversion to 5-fluorouracil.
Drug Metab Dispos 38:1267–1276.
Yamazaki H, Inoue K, Hashimoto M, and Shimada T (1999) Roles of CYP2A6 and CYP2B6 in
nicotine C-oxidation by human liver microsomes. Arch Toxicol 73:65–70.
Yano JK, Hsu MH, Griffin KJ, Stout CD, and Johnson EF (2005) Structures of human micro-
somal cytochrome P450 2A6 complexed with coumarin and methoxsalen. Nat Struct Mol Biol
1
Ariyoshi N, Sawamura Y, and Kamataki T (2001) A novel single nucleotide polymorphism
altering stability and activity of CYP2a6. Biochem Biophys Res Commun 281:810–814.
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of
protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254.
Chuah B, Goh BC, Lee SC, Soong R, Lau F, Mulay M, Dinolfo M, Lim SE, Soo R, and Furuie T,
et al. (2011) Comparison of the pharmacokinetics and pharmacodynamics of S-1 between
Caucasian and East Asian patients. Cancer Sci 102:478–483.
90:318–324.
Daigo S, Takahashi Y, Fujieda M, Ariyoshi N, Yamazaki H, Koizumi W, Tanabe S, Saigenji K,
Nagayama S, and Ikeda K, et al. (2002) A novel mutant allele of the CYP2A6 gene
(
CYP2A6*11) found in a cancer patient who showed poor metabolic phenotype towards
tegafur. Pharmacogenetics 12:299–306.
Damle BD, Narasimhan NI, and Kaul S (2001) Stereoselective metabolism and pharmacokinetics
of tegafur. Biopharm Drug Dispos 22:45–52.
Desta Z, Kreutz Y, Nguyen AT, Li L, Skaar T, Kamdem LK, Henry NL, Hayes DF, Storniolo
AM, and Stearns V, et al. (2011) Plasma letrozole concentrations in postmenopausal women
with breast cancer are associated with CYP2A6 genetic variants, body mass index, and age.
Clin Pharmacol Ther 90:693–700.
Di YM, Chow VD, Yang LP, and Zhou SF (2009) Structure, function, regulation and poly-
morphism of human cytochrome P450 2A6. Curr Drug Metab 10:754–780.
Endo T, Nakajima M, Fukami T, Hara Y, Hasunuma T, Yokoi T, and Momose Y (2008) Genetic
polymorphisms of CYP2A6 affect the in-vivo pharmacokinetics of pilocarpine. Pharmaco-
genet Genomics 18:761–772.
Fujita K, Yamamoto W, Endo S, Endo H, Nagashima F, Ichikawa W, Tanaka R, Miya T, Araki K,
and Kodama K, et al. (2008) CYP2A6 and the plasma level of 5-chloro-2, 4-dihydroxypyridine are
determinants of the pharmacokinetic variability of tegafur and 5-fluorouracil, respectively, in
Japanese patients with cancer given S-1. Cancer Sci 99:1049–1054.
Fukami T, Nakajima M, Higashi E, Yamanaka H, Sakai H, McLeod HL, and Yokoi T (2005)
Characterization of novel CYP2A6 polymorphic alleles (CYP2A6*18 and CYP2A6*19) that
affect enzymatic activity. Drug Metab Dispos 33:1202–1210.
2:822–823.
Yoshida R, Nakajima M, Nishimura K, Tokudome S, Kwon JT, and Yokoi T (2003) Effects of
polymorphism in promoter region of human CYP2A6 gene (CYP2A6*9) on expression level
of messenger ribonucleic acid and enzymatic activity in vivo and in vitro. Clin Pharmacol Ther
7
4:69–76.
Yoshida R, Nakajima M, Watanabe Y, Kwon JT, and Yokoi T (2002) Genetic polymorphisms in
human CYP2A6 gene causing impaired nicotine metabolism. Br J Clin Pharmacol 54:
5
Zhang ZG, Liu Y, Guengerich FP, Matse JH, Chen J, and Wu ZL (2009) Identification of amino
acid residues involved in 4-chloroindole 3-hydroxylation by cytochrome P450 2A6 using
screening of random libraries. J Biotechnol 139:12–18.
11–517.
Grogan J, Shou M, Andrusiak EA, Tamura S, Buters JT, Gonzalez FJ, and Korzekwa KR (1995)
Cytochrome P450 2A1, 2E1, and 2C9 cDNA-expression by insect cells and partial purification
using hydrophobic chromatography. Biochem Pharmacol 50:1509–1515.
Han S, Choi S, Chun YJ, Yun CH, Lee CH, Shin HJ, Na HS, Chung MW, and Kim D (2012)
Functional characterization of allelic variants of polymorphic human cytochrome P450 2A6
Address correspondence to: Ikuo Yamamiya, Pharmacokinetics Research Labo-
ratories, Taiho Pharmaceutical Co., Ltd., 3 Okubo, Tsukuba, Ibaraki 300-2611, Japan.
E-mail: i-yamamiya@taiho.co.jp
(
CYP2A6*5, *7, *8, *18, *19, and *35). Biol Pharm Bull 35:394–399.