Identification of the human cytochrome P450 enzymes involved in the two oxidative steps in the bioactivation of clopidogrel to its pharmacologically active metabolite

Article abstract of DOI:10.1124/dmd.109.029132

The aim of the current study is to identify the human cytochrome P450 (P450) isoforms involved in the two oxidative steps in the bioactivation of clopidogrel to its pharmacologically active metabolite. In the in vitro experiments using cDNA-expressed huma

Full text of DOI:10.1124/dmd.109.029132

0090-9556/10/3801-92–99$20.00
DRUG METABOLISM AND DISPOSITION
Copyright © 2010 by The American Society for Pharmacology and Experimental Therapeutics
DMD 38:92–99, 2010
Vol. 38, No. 1
29132/3541102
Printed in U.S.A.
Identification of the Human Cytochrome P450 Enzymes Involved in
the Two Oxidative Steps in the Bioactivation of Clopidogrel to Its
Pharmacologically Active Metabolite
Miho Kazui, Yumi Nishiya, Tomoko Ishizuka, Katsunobu Hagihara, Nagy A. Farid,
Osamu Okazaki, Toshihiko Ikeda, and Atsushi Kurihara
Drug Metabolism and Pharmacokinetics Research Laboratories, Daiichi Sankyo Co., Ltd., Tokyo, Japan (M.K., Y.N., T.I., K.H.,
O.O., A.K.); Department of Drug Disposition, Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana (N.A.F.);
and Yokohama College of Pharmacy, Yokohama, Japan (T.I.)
Received June 22, 2009; accepted October 1, 2009
ABSTRACT:
The aim of the current study is to identify the human cytochrome
2-oxo-clopidogrel was 35.8, 19.4, and 44.9%, respectively. The
P450 (P450) isoforms involved in the two oxidative steps in the contribution of CYP2B6, CYP2C9, CYP2C19, and CYP3A4 to the
bioactivation of clopidogrel to its pharmacologically active metab- formation of the active metabolite was 32.9, 6.76, 20.6, and 39.8%,
olite. In the in vitro experiments using cDNA-expressed human respectively. In the inhibition studies with antibodies and selective
P450 isoforms, clopidogrel was metabolized to 2-oxo-clopidogrel, chemical inhibitors to P450s, the outcomes obtained by inhibition
the immediate precursor of its pharmacologically active metabo- studies were consistent with the results of P450 contributions in
lite. CYP1A2, CYP2B6, and CYP2C19 catalyzed this reaction. In the each oxidative step. These studies showed that CYP2C19 contrib-
same system using 2-oxo-clopidogrel as the substrate, detection uted substantially to both oxidative steps required in the formation
of the active metabolite of clopidogrel required the addition of of clopidogrel active metabolite and that CYP3A4 contributed sub-
glutathione to the system. CYP2B6, CYP2C9, CYP2C19, and stantially to the second oxidative step. These results help explain
CYP3A4 contributed to the production of the active metabolite. the role of genetic polymorphism of CYP2C19 and also the effect of
Secondly, the contribution of each P450 involved in both oxidative potent CYP3A inhibitors on the pharmacokinetics and pharmaco-
steps was estimated by using enzyme kinetic parameters. The
contribution of CYP1A2, CYP2B6, and CYP2C19 to the formation of
dynamics of clopidogrel in humans and on clinical outcomes.
Clopidogrel is a thienopyridine antiplatelet agent that has been 2003), although the contribution of these P450s to produce the active
widely used in the management of cardiovascular diseases, including
atherothrombosis, unstable angina, and myocardial infarction (Savi
and Herbert, 2005). Clopidogrel is an inactive prodrug that needs to be
converted to the pharmacologically active metabolite in vivo through
the hepatic metabolism to exhibit the antiplatelet effect (Savi et al.,
1992). Clopidogrel is first converted by the action of cytochrome
P450 (P450) to 2-oxo-clopidogrel (a thiolactone) then in a second step
converted to the pharmacologically active, thiol-containing metabolite
as shown in Fig. 1 (Savi et al., 2000). The P450 isoforms involved in
the bioactivation of clopidogrel have been suggested to be CYP1A2 in
rats (Savi et al., 1994) and CYP3A in humans (Clarke and Waskell,
metabolite was still unclear. In addition, several recent clinical studies
demonstrated that CYP3A4, CYP3A5, and CYP2C19 have a signif-
icant role in the formation of the active metabolite from clopidogrel
(Hulot et al., 2006; Suh et al., 2006; Brandt et al., 2007; Farid et al.,
2007, 2008). Furthermore, Brandt et al. (2007) reported that loss of
function of CYP2C19 due to polymorphisms resulted in decreased
exposure to clopidogrel active metabolite and hence a diminished
effect of clopidogrel on platelet aggregation. Involvement of P450s
would be obvious in the first step of clopidogrel bioactivation (Fig. 1)
based on the chemical structure of 2-oxo-clopidogrel showing intro-
duction of one oxygen atom into clopidogrel. Whereas initially the
second step of this pathway, where 2-oxo-clopidogrel is converted to
the active metabolite, was thought to be a hydrolysis step (Savi et al.,
2000), liver microsomes and glutathione were shown to be needed for
the formation and detection of clopidogrel active metabolite (Pereillo
et al., 2002). However, the P450s needed to catalyze the formation of
the active metabolite were not identified. Unlike what is currently
known with respect to the enzymes involved in the bioactivation of
clopidogrel, the formation of the active metabolite of a new thienopy-
ridine antiplatelet agent, prasugrel, from its corresponding thiolactone,
A portion of this work was previously presented as follows: Kurihara A, Hagi-
hara K, Kazui M, Ozeki T, Farid NA, and Ikeda T (2005) In vitro metabolism of
antiplatelet agent clopidogrel: cytochrome P450 isoforms responsible for two
oxidation steps involved in the active metabolite formation. 13th North American
ISSX Meeting; 2005 Oct 23–27; Maui, Hawaii. International Society for the Study
of Xenobiotics, Washington, DC. [Drug Metab Rev 37 (Suppl 2):99 (abstract).]
Article, publication date, and citation information can be found at
http://dmd.aspetjournals.org.
doi:10.1124/dmd.109.029132.
ABBREVIATIONS: P450, cytochrome P450; LC/MS/MS, liquid chromatography/tandem mass spectrometry; HPLC, high-performance liquid
chromatography; ␤-NADP, ␤-nicotinamide adenine dinucleotide phosphate sodium salt; CLint, intrinsic clearance.
92

P450 ISOFORMS RESPONSIBLE FOR BIOACTIVATION OF CLOPIDOGREL
93
FIG. 1. Biotransformation pathway of clopidogrel leading to its pharmacologically active metabolite via 2-oxo-clopidogrel.
CYP2D6, CYP2E1, CYP3A4, and CYP4A11 and microsomes from human
␤-lymphoblastoid cell lines expressing human CYP1A1, CYP1A2, CYP2A6,
CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4, and
CYP4A11 were purchased from BD Biosciences Company.
Production of 2-Oxo-clopidogrel from Clopidogrel. The assays were
performed by using the microsomes from baculovirus/insect cells expressing
human P450s (Supersomes). The incubation mixture contained 3 mg pro-
tein/ml of Supersomes, an NADPH-generating system (2.5 mM ␤-NADP, 25
mM D-glucose 6-phosphate, 10 mM magnesium chloride, and 2 units/ml of
glucose-6-phosphate dehydrogenase), and 500 ␮M clopidogrel in a final vol-
ume of 200 ␮l of potassium phosphate (50 mM) buffer (pH 7.4) for CYP1A1,
CYP1A2, CYP2B6, CYP2C8, CYP2C19, CYP2D6, CYP2E1, and CYP3A4
reactions or in a final volume of 200 ␮l of Tris-HCl (50 mM) buffer (pH 7.4)
for CYP2A6, CYP2C9, and CYP4A11 reactions. A mixture without clopi-
dogrel was preincubated at 37°C for 5 min, and the reaction was started by the
addition of 4 ␮l of a solution of clopidogrel in N,N-dimethylacetamide. After
incubation at 37°C for 30 min, an aliquot of the incubation mixture was
collected and added to 3-fold volumes of ethanol to terminate the reaction. The
mixture was centrifuged at 20,800g at 4°C for 3 min, and 25 ␮l of the
supernatant was injected into the HPLC system to determine the concentrations
of 2-oxo-clopidogrel.
FIG. 2. Structure of an internal standard in assay of 2-oxo-clopidogrel and clopi-
dogrel active metabolite by LC/MS/MS.
was shown to be dependent on CYP3A, CYP2B6, CYP2C9, and
CYP2C19 (Kazui et al., 2001; Rehmel et al., 2006). The aim of this
study was to determine the contribution and the role of various P450s
in the oxidation of clopidogrel to 2-oxo-clopidogrel and the formation
of the active metabolite from 2-oxo-clopidogrel.
Materials and Methods
Production of the Active Metabolite of Clopidogrel from 2-Oxo-clopi-
dogrel. The assays were performed by using the microsomes from ␤-lympho-
blastoid cells expressing human P450s. The incubation mixture contained 2 mg
protein/ml of the microsomes from human ␤-lymphoblastoid cells, an
NADPH-generating system, 1 mM glutathione, and 200 ␮M 2-oxo-clopidogrel
in a final volume of 200 ␮l of potassium phosphate (50 mM) buffer (pH 7.4)
for CYP1A1, CYP1A2, CYP2B6, CYP2C8, CYP2C19, CYP2D6, CYP2E1,
and CYP3A4 reactions or in a final volume of 200 ␮l of Tris-HCl (50 mM)
buffer (pH 7.4) for CYP2A6, CYP2C9, and CYP4A11 reactions. A mixture
without 2-oxo-clopidogrel was preincubated at 37°C for 5 min, and the
reaction was started by the addition of 5 ␮l of a solution of 2-oxo-clopidogrel
in N,N-dimethylacetamide. After the incubation at 37°C for 90 min, an aliquot
of the incubation mixture was collected and mixed with 3-fold volumes of
acetonitrile and 20 ␮l of a solution of butyl p-hydroxybenzoate as the internal
standard (20 ␮g/ml in acetonitrile) to terminate the reaction. The mixture was
centrifuged at 20,800g at 4°C for 3 min, and 20 ␮l of the supernatant was
injected into the HPLC system to determine the concentrations of the active
metabolite.
Determination of the Enzyme Kinetic Parameters for 2-Oxo-clopi-
dogrel Formation. The assay was performed by using the Supersomes of
CYP1A2, CYP2B6, CYP2C19, and the control Supersomes. The incubation
mixture contained 20 pmol P450/0.2 mg protein/ml of Supersomes, an
NADPH-generating system, and 0.625, 1.25, 2.5, 5, 10, 20, and 40 ␮M
clopidogrel in a final volume of 300 ␮l of potassium phosphate (100 mM)
buffer (pH 7.4). A mixture without clopidogrel was preincubated at 37°C for
5 min, and the reaction was started by the addition of 6 ␮l of a solution of
clopidogrel in methanol. After incubation at 37°C for 0 and 1 min, 50 ␮l of the
incubation mixture was collected and added to 200 ␮l of acetonitrile and 50 ␮l
of a solution of R-135766 as the internal standard (2 ␮M in acetonitrile) to
terminate the reaction. The mixture was centrifuged at 20,800g at 4°C for 3
min, and 5 ␮l of the supernatant was injected into LC/MS/MS system to
Materials. Clopidogrel, 2-oxo-clopidogrel, the pharmacologically active
metabolite of clopidogrel and R-135766 (shown in Figs. 1 and 2) were
obtained from Ube Industries, Ltd. (Ube, Japan). R-135766 was used as the
internal standard for assay of the active metabolite and 2-oxo-clopidogrel by
liquid chromatography/tandem mass spectrometry (LC/MS/MS). Butyl p-hy-
droxybenzoate, used as the internal standard for assay of the active metabolite
by high-performance liquid chromatography (HPLC)-UV, was purchased from
Wako Pure Chemical Industries, Ltd. (Tokyo, Japan). Furafylline, omeprazole,
(S)-N-3-benzylnirvanol, sulfaphenazole, ketoconazole, glutathione, ␤-nicotin-
amide adenine dinucleotide phosphate sodium salt (␤-NADP), and D-glucose
6-phosphate were purchased from Sigma-Aldrich (St. Louis, MO). Glucose-
6-phosphate dehydrogenase was purchased from Oriental Yeast Co., Ltd.
(Tokyo, Japan). The reagent used to derivatize clopidogrel active metabolite,
3Ј-methoxyphenacyl bromide, was purchased from Tokyo Kasei Kogyo Co.,
Ltd. (Tokyo, Japan). All other chemicals, reagents, and solvents used were
commercially available and were of guaranteed grade or HPLC grade. Mono-
clonal antibodies to CYP1A2, CYP3A4, CYP2B6, and CYP2C19 were ob-
tained from BD Biosciences Company (Woburn, MA). Polyclonal antibody to
CYP2C9 and the rabbit control serum were obtained from Nosan Corporation
(Yokohama, Japan). The specificity and the selectivity of the monoclonal
antibody to CYP2C19 and CYP3A4 were reported by Krausz et al. (2001) and
Gelboin et al. (1995), respectively. The monoclonal antibody to CYP3A4 used
inhibits not only the activity of CYP3A4 but that of CYP3A5 slightly. The
specificity and selectivity of the monoclonal antibodies to CYP1A2 and
CYP2B6 were published previously (website of BD Biosciences Company,
http://www.bdbiosciences.com/ptProduct.jsp?prodIdϭ460095 for monoclonal
antibody CYP1A2 and http://www.bdbiosciences.com/ptProduct.jsp?prodIdϭ
460089 for monoclonal antibody CYP2B6). In addition, the specificity and
selectivity of the polyclonal antibody to CYP2C9 were reported by Ng et al.
(2003).
Microsomes from Human Liver or from Cells Expressing Isoforms of determine the concentrations of 2-oxo-clopidogrel.
Human P450s. Pooled human liver microsomes from 10 donors were obtained
from the Human and Animal Bridging Research Organization (Tokyo, Japan).
Determination of the Enzyme Kinetic Parameters for the Active Me-
tabolite Formation from 2-Oxo-clopidogrel. The assay was performed by
Supersomes prepared from baculovirus/insect cells expressing human using the Supersomes of CYP3A4, CYP2B6, CYP2C9, CYP2C19, and the
CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, control. The incubation mixture contained 20 pmol P450/0.3 mg protein/ml of

94
KAZUI ET AL.
Supersomes, an NADPH-generating system, 5 mM glutathione and 0.625,
1.25, 2.5, 5, 10, 20, and 40 ␮M 2-oxo-clopidogrel in a final volume of 300 ␮l monoclonal antibody (CYP3A4, CYP2B6, CYP2C19) or 25 mM Tris-HCl
of potassium phosphate (100 mM) buffer (pH 7.4) for CYP2B6, CYP2C19,
buffer (pH 7.5) used as control were mixed and incubated for 20 min on ice.
(pH 7.4). Five microliters of microsomes (20 mg protein/ml) and 10 ␮l of each
and CYP3A4 reactions or in a final volume of 300 ␮l of Tris-HCl (100 mM) When polyclonal antibody to CYP2C9 was used, 5 ␮l of microsomes (20 mg
buffer (pH7.5) for the CYP2C9 reaction. A mixture without 2-oxo-clopidogrel protein/ml) and 10 ␮l of polyclonal antibody to CYP2C9 or rabbit serum used
as a control were mixed and incubated for 10 min at room temperature. After
incubation on ice or room temperature, 113 ␮l of potassium phosphate (100
mM) buffer (pH 7.4), 20 ␮l of glutathione, and 50 ␮l of the NADPH-
generating system were added to that mixture and preincubated at 37°C for 5
min. The reaction was started by the addition of 2 ␮l of a solution of
2-oxo-clopidogrel in methanol and carried out at 37°C for 15 min.
Studies using the selective chemical inhibitors against P450s. Ketocon-
azole, (S)-N-3-benzylnirvanol, omeprazole, and sulfaphenazole were added to
the incubation medium containing 0.5 mg protein/ml of human liver micro-
somes, 100 mM potassium phosphate buffer (pH 7.4), 5 mM glutathione, and
3 ␮M 2-oxo-clopidogrel at a final concentration of 2 ␮M for ketoconazole or
10 ␮M for (S)-N-3-benzylnirvanol, omeprazole, and sulfaphenazole, and pre-
incubated at 37°C for 5 min. The reaction was initiated by the addition of the
NADPH-generating system and carried out for 15 min.
was preincubated at 37°C for 5 min, and the reaction was started by the
addition of 6 ␮l of a solution of 2-oxo-clopidogrel in methanol. After incu-
bation at 37°C for 0, 15, or 30 min, 50 ␮l of the incubation mixture was
collected and added to 198 ␮l of acetonitrile, 2 ␮l of a solution of 3Ј-
methoxyphenacyl bromide (500 mM in acetonitrile) as the derivatization
reagent, and 50 ␮l of a solution of R-135766 as the internal standard (2 ␮M in
acetonitrile) to terminate the reaction. After the mixture was allowed to stand
for 10 min at room temperature, it was centrifuged at 20,800g at 4°C for 3 min,
and 5 ␮l of the supernatant was injected into the LC/MS/MS system (Taka-
hashi et al., 2008) to determine the concentrations of an active metabolite.
Inhibition of Production of 2-Oxo-clopidogrel in Human Liver Micro-
somes. The Km (Michaelis constant) value in 2-oxo-clopidogrel formation
from clopidogrel was determined by using 0.5 mg protein/ml human liver
microsomes as the enzyme source and clopidogrel (at 1.25, 2.5, 5, 10, 20, 40,
80, and 160 ␮M) as substrate in the presence of an NADPH-generating system
at an incubation time of 5 min. This assay was performed in triplicate by using
human liver microsomes. Eadie-Hofstee plots were used to visually detect
deviation from linearity and to make initial estimates of kinetic constants. The
Km value was calculated by WinNonlin nonlinear estimation program (version
4.0.1; Pharsight, Mountain View, CA) as described below under Data Han-
dling. Clopidogrel concentration in the inhibition studies below was set at 4
␮M, which was close to the Km1 value of the high-affinity component.
Inhibition study using the antibodies against P450s. The incubation mixture
contained 0.5 mg protein/ml of human liver microsomes, an NADPH-gener-
ating system, 4 ␮M clopidogrel, and monoclonal antibody to CYP1A2,
CYP2B6, and CYP2C19 in a final volume of 200 ␮l of potassium phosphate
(67 mM) buffer (pH 7.4). Five microliters of microsomes (20 mg protein/ml)
and 10 ␮l of each monoclonal antibody (CYP1A2, CYP2B6, CYP2C19) or 25
mM Tris-HCl buffer (pH 7.5) as control were mixed and incubated for 20 min
on ice. After incubation on ice, 133 ␮l of potassium phosphate (100 mM)
buffer (pH 7.4) and 50 ␮l of the NADPH-generating system were added to that
mixture and preincubated at 37°C for 5 min. The reaction was started by the
addition of 2 ␮l of clopidogrel in methanol and carried out at 37°C for 5 min.
Studies using the selective chemical inhibitors of P450s. (S)-N-3-benzylnir-
vanol and omeprazole were added at a final concentration of 10 ␮M to the
incubation medium consisting of 0.5 mg protein/ml of human liver micro-
somes, 100 mM potassium phosphate buffer (pH 7.4), and 4 ␮M clopidogrel
and preincubated at 37°C for 5 min. The reaction was initiated by the addition
of the NADPH-generating system and carried out for 5 min. In the case of
furafylline, preincubation was performed without clopidogrel for 30 min, and
the reaction was initiated by the addition of the NADPH-generating system and
clopidogrel. After incubation at 37°C for the appropriate times in the inhibition
studies of 2-oxo-clopidogrel formation from clopidogrel, 50 ␮l of the incuba-
tion mixture was collected and treated as described above under Determination
of the Enzyme Kinetic Parameters for 2-Oxo-clopidogrel Formation.
After incubation at 37°C for 15 min in both inhibition studies of the
active metabolite formation from 2-oxo-clopidogrel, 50 ␮l of the incuba-
tion mixture was collected and treated as described under Determination of
the Enzyme Kinetic Parameters for the Active Metabolite Formation from
2-Oxo-clopidogrel.
Assay of 2-Oxo-clopidogrel and the Active Metabolite of Clopidogrel by
HPLC-UV. 2-Oxo-clopidogrel and the active metabolite produced in the
human cytochrome P450-expression systems were measured by using an
HPLC system (LC-VP system; Shimadzu Co., Ltd., Kyoto, Japan). Chromato-
graphic separation of 2-oxo-clopidogrel was carried out on a YMC Pack
ODS-A302 column (4.6 ϫ 150 mm, particle size of 5 ␮m, YMC Co., Ltd.,
Tokyo, Japan) at 40°C by using a mobile phase consisting of acetonitrile,
distilled water, and trifluoroacetic acid (35:65:0.02, v/v/v) at a flow rate of 1.0
ml/min. Chromatographic separation of the active metabolite of clopidogrel
was carried out on the same column by using a mobile phase consisting of
acetonitrile, distilled water, and trifluoroacetic acid (38:62:0.02, v/v/v) at a
flow rate of 1.0 ml/min. Detection was carried out at 220 nm. The active
metabolite in the analytical samples treated immediately by acetonitrile was
stable at 4°C for 24 h without derivatization (data not shown).
Assay of 2-Oxo-clopidogrel and the Active Metabolite of Clopidogrel by
Electrospray Ionization-LC/MS/MS System. In the determination of the
enzymatic kinetics parameters and the inhibition studies, 2-oxo-clopidogrel
and the active metabolite were measured by using the method of Takahashi et
al. (2008) with slight modification. The active metabolite of clopidogrel was
derivatized with 3Ј-methoxyphenacyl bromide to stabilize thiol moiety in its
structure. Quattro-LC/MS/MS system (Micromass UK Ltd., Manchester, UK)
was used in the positive-ion detection mode at the electrospray ionization
interface. The peak areas of the m/z 3383183 for 2-oxo-clopidogrel and the
m/z 5043354 for derivatized active metabolite were measured against the
peak areas of the m/z 5483206 for the internal standard (R-135766). Separa-
tion by HPLC was conducted by using an Alliance 2695 Separations Module
(Waters, Milford, MA) with an ODS column (Inertsil ODS-3, 2.1 mm ϫ 150
mm, 5 ␮m; GL Science Inc., Tokyo, Japan) at a flow rate of 0.2 ml/min with
a mobile phase consisting of methanol, distilled water, and trifluoroacetic acid
[710:290:0.5 (v/v/v)].
Inhibition of Production of the Active Metabolite from 2-Oxo-clopi-
dogrel in Human Liver Microsomes. The Km value in the active metabolite
formation from 2-oxo-clopidogrel was determined by using 0.5 mg protein/ml
human liver microsomes as the enzyme source and 2-oxo-clopidogrel (at 1.25,
2.5, 5, 10, 20, 40, 80, and 160 ␮M) as substrate in the presence of the
NADPH-generating system and glutathione (5 mM) at an incubation time of 15
min. This assay was performed in triplicate using human liver microsomes.
Eadie-Hofstee plots were used to visually detect deviation from linearity and
to make initial estimates of kinetic constants. The Km value was calculated by
the WinNonlin nonlinear estimation program (version 4.0.1) as described
below under Data Handling. 2-Oxo-clopidogrel concentration in the inhibition
studies below was set at 3 ␮M, which was close to Km1 value of the
high-affinity component.
Data Handling. All of the calculated values, with the exception of enzyme
kinetics parameters below, were expressed as mean Ϯ S.D. of three experi-
ments throughout Results and Discussion.
Estimation of the enzyme kinetics parameters for 2-oxo-clopidogrel and the
active metabolite formation in the human P450 expression systems. The
reaction rate (V) was calculated according to eq. 1.
generated concentration (␮M) ϫ 1000
V ͑pmol/pmol P450/min͒ ϭ
20 (pmol P450/ml) ϫ incubation time (min)
Inhibition studies using the antibodies against P450s. The incubation mix-
ture contained 0.5 mg protein/ml of human liver microsomes, an NADPH-
generating system, 5 mM glutathione, 3 ␮M 2-oxo-clopidogrel, and monoclo-
nal antibody to CYP3A4, CYP2B6, and CYP2C19 or polyclonal antibody to
(1)
The Michaelis-Menten constant (Km, ␮M) and maximal reaction rate (Vmax
,
pmol/pmol P450/min) were calculated by using WinNonlin Professional (ver-
CYP2C9 in a final volume of 200 ␮l of potassium phosphate buffer (67 mM) sion 4.0.1; Pharsight) based on a pharmacodynamic compiled model (model

P450 ISOFORMS RESPONSIBLE FOR BIOACTIVATION OF CLOPIDOGREL
95
no.101) after an examination of Eadie-Hofstee plots demonstrated a linear
relationship. The calculated Km and Vmax values were expressed as mean Ϯ
S.E. of parameter estimate. The intrinsic clearance (CLint) was calculated as a
ratio of Vmax to Km. The various P450s mediated clearance (CLint, expressed P450
)
in human liver microsomes was determined by using eq. 2. The enzyme
abundance (pmol P450/mg protein) of various P450s in human liver micro-
somes was obtained from the reported data in Rowland Yeo et al. (2004).
CLint, expressed P450 ͑␮l/mg protein/min͒
Vmax ϫ enzyme abundances (pmol P450/mg protein) of various P450s
ϭ
Km
(2)
The contribution ratio (fmP450, %) of each P450 that was responsible for the
production of 2-oxo-clopidogrel and the active metabolite was determined by
using eq. 3.
CLint, expressed P450 for each P450 reaction ϫ 100
f,mP450 ͑%͒ ϭ
(3)
⌺CLint, expressed P450
FIG. 3. Formation of 2-oxo-clopidogrel from clopidogrel in human cytochrome
P450-expression system in the presence of NADPH. The assays were performed by
using the microsomes from baculovirus/insect cells expressing human cytochrome
P450 (Supersomes). Data were expressed as mean Ϯ S.D. of three experiments.
N.D., not determined.
Inhibition of production of 2-oxo-clopidogrel or the active metabolite. The
estimation of the enzyme kinetic parameters for 2-oxo-clopidogrel or the active
metabolite using human liver microsome was calculated by using WinNonlin
nonlinear estimation program (version 4.0.1) and an Eadie-Hofstee plot [x-
axis, V/substrate concentration (␮M); y-axis, V], where the reaction rate V
(pmol/mg protein/min) was calculated according to eq. 4.
Production of the Active Metabolite of Clopidogrel from 2-Oxo-
clopidogrel. The production of the active metabolite of clopidogrel in
the microsomes of ␤-lymphoblastoid cells expressing the human
P450s was measured by HPLC as shown in Fig. 4, using 2-oxo-
clopidogrel (200 ␮M) as the substrate. Incubation without addition of
glutathione resulted in no detectable production of the active metab-
olite (data not shown). The isoforms CYP2B6, CYP2C9, CYP2C19,
and CYP3A4 converted 2-oxo-clopidogrel to the active metabolite,
and the activity of these P450s ranked in descending order of
CYP2C19, CYP2C9, CYP3A4, and CYP2B6. CYP1A1, CYP1A2,
CYP2A6, CYP2C8, CYP2D6, CYP2E1, and CYP4A11 failed to
catalyze this metabolic reaction.
generated concentration (␮M) ϫ 1000
V ͑pmol/mg protein/min͒ ϭ
0.5 (mg protein/ml) ϫ incubation time (min)
(4)
Eadie-Hofstee plots were used to visually detect deviation from linearity
and to make initial estimates of kinetic constants. Data were then fitted to eq.
5 by using the WinNonlin nonlinear estimation program. S (␮M) is substrate
concentration, Km1 and Vmax1 are the apparent Km and Vmax values for high-
affinity component, and Km2 and Vmax2 are the apparent Km and Vmax values for
the low-affinity component in eq. 5.
The Contribution of Each P450 Involved in the Formation of
the Active Metabolite from Clopidogrel. As the first oxidative step
of clopidogrel bioactivation, the 2-oxo-clopidogrel formation rates
over a range of clopidogrel concentrations were determined in Super-
Vmax1 ϫ S Vmax2 ϫ S
V ϭ
ϩ
(5)
Km1 ϩ S
Km2 ϩ S
The calculated Km and Vmax values were expressed as mean Ϯ S.D. of three
experiments. The enzymatic activity remaining (%) in the presence of the
antibody and the chemical inhibitor was calculated according to eq. 6.
Enzymatic activity remaining ͑%͒
produced concentration (␮M)
with antibody or chemical inhibitor to P450s
ϭ
ϫ 100 (6)
produced concentration (␮M)
without antibody or chemical inhibitor to P450s
The inhibition ratio was calculated according to eq. 7.
Inhibition ratio ͑%͒ ϭ 100 Ϫ enzymatic activity remaining ͑%͒ (7)
Results
Production of 2-Oxo-clopidogrel from Clopidogrel. The abil-
ity to produce 2-oxo-clopidogrel in Supersomes was measured by
HPLC-UV using clopidogrel (500 ␮M) as the substrate. As shown in
Fig. 3, clopidogrel was metabolized to 2-oxo-clopidogrel by CYP1A2,
CYP2B6, and CYP2C19, and the activity of these P450s ranked in
descending order of CYP1A2, CYP2C19, and CYP2B6. Other P450
isoforms (CYP1A1, CYP2A6, CYP2C8, CYP2C9, CYP2D6,
CYP2E1, CYP3A4, and CYP4A11) showed no detectable activity
toward the formation of 2-oxo-clopidogrel.
FIG. 4. Formation of the pharmacologically active metabolite of clopidogrel from
2-oxo-clopidogrel in human cytochrome P450-expression system in the presence of
NADPH and glutathione. The assays were performed by using the microsomes from
␤-lymphoblastoid cells expressing human cytochrome P450. Data were expressed
as mean Ϯ S.D. of three experiments. N.D., not determined.

96
KAZUI ET AL.
TABLE 1
Enzyme kinetic parameters of 2-oxo-clopidogrel formation from clopidogrel
The assays were performed by using the microsomes baculovirus/insect cells expressing human P450 (Supersomes). Km and Vmax values were expressed as mean Ϯ S.E. of parameter
estimate. CLint, expressed P450 and fmP450 (contribution ratio of each P450) values were scaled to eqs. 2 and 3 under Materials and Methods to determine contribution.
Incubation Time
K
Vmax
Vmax/Km
Enzyme Abundances*
CLint, expressed P450
fmP450 %
min
␮M
pmol/pmol P450/min
␮l/pmol P450/min
pmol P450/mg protein
␮l/mg protein/min
CYP1A2
CYP2B6
CYP2C19
1
1
1
1.58 Ϯ 1.35
2.08 Ϯ 0.73
1.12 Ϯ 0.25
2.27 Ϯ 0.46
7.66 Ϯ 0.69
7.52 Ϯ 0.36
1.44
3.68
6.71
52
11
14
74.9
40.5
93.9
35.8
19.4
44.9
* The abundances of CYP1A2, CYP2B6, and CYP2C19 were obtained from the reported data in Rowland Yeo et al. (2004).
TABLE 2
Enzyme kinetic parameters of the formation of the pharmacologically active metabolite of clopidogrel from 2-oxo-clopidogrel
The assays were performed by using the microsomes baculovirus/insect cells expressing human cytochrome P450 (Supersomes). Km and Vmax values were expressed as mean Ϯ S.E. of
parameter estimate. CLint and fmP450 (P450 contribution ratio) values were scaled to eqs. 2 and 3 under Materials and Methods to determine contribution.
Incubation Time
Km
Vmax
Vmax/Km
Enzyme Abundances*
CLint, expressed P450
fmP450 %
min
␮M
pmol/pmol P450/min
␮l/pmol P450/min
pmol P450/mg protein
␮l/mg protein/min
CYP2B6
CYP2C9
CYP2C19
CYP3A4
15
30
15
15
1.62 Ϯ 0.08
18.1 Ϯ 3.8
12.1 Ϯ 2.2
27.8 Ϯ 4.2
2.48 Ϯ 0.03
0.855 Ϯ 0.084
9.06 Ϯ 0.68
3.63 Ϯ 0.29
1.53
11
73
14
16.8
3.45
10.5
20.3
32.9
6.76
20.6
39.8
0.0472
0.749
0.131
155
* The abundances of CYP3A4, CYP2B6, CYP2C19, and CYP2C9 were obtained from the reported data in Rowland Yeo et al. (2004).
FIG. 5. Kinetic analysis of 2-oxo-clopidogrel formation from clopidogrel in human liver microsomes by an Eadie-Hofstee plot and WinNonlin nonlinear estimation
program. The human liver microsomes were incubated with 1.25 to 160 ␮M clopidogrel at 37°C for 5 min. Data are plotted using an Eadie-Hofstee plots (A) and direct
plots (B). Line in B represents the curve fit to eq. 5 using WinNonlin nonlinear estimation program. Typical results from one of three experiments are shown.
somes containing each P450. The Km values for CYP1A2, CYP2B6, were 1.62 Ϯ 0.08, 18.1 Ϯ 3.8, 12.1 Ϯ 2.2, and 27.8 Ϯ 4.2 ␮M,
and CYP2C19 were 1.58 Ϯ 1.35, 2.08 Ϯ 0.73, and 1.12 Ϯ 0.25 ␮M, respectively (mean Ϯ S.E. of parameter estimate) (Table 2), and
respectively (mean Ϯ S.E. of parameter estimate) (Table 1).
the Vmax values were 2.48 Ϯ 0.03, 0.855 Ϯ 0.084, 9.06 Ϯ 0.68, and
Estimates using eq. 3 of the contribution ratio (fm P450) of 3.63 Ϯ 0.29 pmol/pmol P450/min, respectively (mean Ϯ S.E. of
CYP1A2, CYP2B6, and CYP2C19 to 2-oxo-clopidogrel formation parameter estimate).
were 35.8, 19.4, and 44.9%, respectively, suggesting that the contri-
bution of CYP2C19 to this oxidation process was higher compared CYP2C19, and CYP3A4 reactions are shown in Table 2. Estimates of
with the other two P450s. the fm P450 of CYP2B6, CYP2C9, CYP2C19, and CYP3A4 for the
The CLint,
and fmP450 for CYP2B6, CYP2C9,
expressed P450
As the second oxidative step of clopidogrel, the active metabo- active metabolite formation were 32.9, 6.76, 20.6, and 39.8%, respec-
lite formation rates over a range of 2-oxo-clopidogrel concentra- tively, suggesting that the contribution of CYP3A4 to the active metab-
tions were determined in Supersomes containing each P450. The olite formation was greater compared with the other three P450s.
production of the active metabolite with CYP2C9 was not detected
Inhibition of Production of 2-Oxo-clopidogrel or the Active
adequately at 15-min incubation. Therefore, we used the reaction Metabolite in Human Liver Microsomes. The reaction rate of
rate of the active metabolite at 30 min to calculate the enzyme 2-oxo-clopidogrel formation in human liver microsomes had a bipha-
kinetic parameters for the CYP2C9 reaction. The enzyme kinetics sic pattern in Eadie-Hofstee plots (Fig. 5), indicating the involvement
parameters for the other three P450s reactions were calculated by of multiple enzymes. Accordingly, the kinetic parameters for 2-oxo-
using the reaction rate of the active metabolite formation at 15 min. clopidogrel formation from clopidogrel in human liver microsomes
The Km values for CYP2B6, CYP2C9, CYP2C19, and CYP 3A4 were estimated by using the WinNonlin nonlinear estimation program.

P450 ISOFORMS RESPONSIBLE FOR BIOACTIVATION OF CLOPIDOGREL
97
TABLE 3
and CYP3A4 showed 54.3 Ϯ 3.4, 13.5 Ϯ 4.4, 32.9 Ϯ 4.2, and 31.3 Ϯ
5.3%, respectively (mean Ϯ S.D., n ϭ 3). Sulfaphenazole, the inhib-
itor of CYP2C9 at 10 ␮M, inhibited the active metabolite formation
by 36.2 Ϯ 7.7%. (S)-N-3-Benzylnirvanol and omeprazole, the inhib-
itors of CYP2C19 at 10 ␮M, inhibited this reaction by 35.4 Ϯ 15.2
and 31.3 Ϯ 17.3%, respectively. Ketoconazole, an inhibitor of
CYP3A4 at 2 ␮M, inhibited the formation of the active metabolite by
38.4 Ϯ 8.4% (mean Ϯ S.D., n ϭ 3) (Fig. 6).
Effect of anti-P450 antibodies on the 2-oxo-clopidogrel formation from
clopidogrel and active metabolite formation from 2-oxo-clopidogrel
The assays were performed by using human liver microsomes. Anti-CYP1A2, anti-
CYP2B6, anti-CYP2C19, and anti-CYP3A4 were monoclonal antibodies, and anti-CYP2C9
was polyclonal antibody. The inhibition ratio values were calculated using eqs. 4 and 5 under
Materials and Methods. Data were expressed as mean Ϯ S.D. of three experiments.
Inhibition Ratio
2-Oxo-clopidogrel Formation
Active Metabolite Formation
Discussion
%
Anti-CYP1A2
Anti-CYP2B6
Anti-CYP2C9
Anti-CYP2C19
Anti-CYP3A4
30.9 Ϯ 9.0
26.0 Ϯ 2.5
This study is the first report in which P450s involved in the two
oxidative steps required for the metabolism of clopidogrel to its active
metabolite are identified and their contributions determined.
CYP1A2, CYP2B6, and CYP2C19 are capable of producing 2-oxo-
clopidogrel, whereas the remaining isoforms examined were practi-
cally inactive in this first oxidative step of clopidogrel. Savi et al.
(1994) suggested that rat CYP1A was involved in the active metab-
olite formation from clopidogrel. The present study confirmed that
CYP1A2 does indeed contribute to the first oxidative step of this
metabolic process. Regarding the second metabolic step, formation of
clopidogrel active metabolite was catalyzed by CYP2B6, CYP2C9,
CYP2C19, and CYP3A4. The reaction mixture for the active metab-
olite formation contained glutathione, because it was previously
shown that formation of a thienopyridine’s active metabolite re-
quired the presence of both P450s and a reducing agent such as
glutathione in the incubation mixture (Pereillo et al., 2002; Rehmel
et al., 2006).
When the enzyme kinetic parameters of 2-oxo-clopidogrel forma-
tion by CYP1A2, CYP2B6, and CYP2C19 were calculated, we set up
the incubation time at 1 min, because the reaction rate for the 2-oxo-
clopidogrel formation by CYP2B6 and CYP2C19 appeared to de-
crease after 2-min incubation (data not shown). These decreases in the
enzymatic activity are most likely attributed to mechanism-based
CYP2B6 and CYP2C19 inhibition observed in vitro by clopidogrel
(Richter et al., 2004; Nishiya et al., 2007, 2009; Hagihara et al., 2008).
The enzyme kinetic parameters used in eq. 3 showed that the contri-
bution of each P450 involved in the 2-oxo-clopidogrel formation
decreased in the order CYP2C19 (44.9%) Ͼ CYP1A2 (35.8%) Ͼ
CYP2B6 (19.4%) (Table 1). To further verify the obtained contribu-
tion data from the enzyme kinetic parameters, the inhibition studies
were performed with human liver microsomes, anti-P450 antibodies,
and chemical inhibitors. The inhibition effects of the monoclonal
antibodies on the first oxidation step toward 2-oxo-clopidogrel for-
mation were consistent with the results of the contribution of each
P450 involved in this oxidative step. Anti-CYP1A2, anti-CYP2B6,
and anti-CYP2C19 antibodies inhibited the formation of 2-oxo-clo-
pidogrel by 30.9 Ϯ 9.0, 26.0 Ϯ 2.5, and 42.0 Ϯ 8.1%, respectively. In
addition, the inhibition levels with the chemical inhibitors on the
production of 2-oxo-clopidogrel were consistent with the outcome of
the antibody experiments.
54.3 Ϯ 3.4
13.5 Ϯ 4.4
32.9 Ϯ 4.2
31.3 Ϯ 5.3
42.0 Ϯ 8.1
The apparent Km1 and Vmax1 values for the high-affinity component
were 4.70 Ϯ 2.62 ␮M and 144 Ϯ 61.0 pmol/mg protein/min, respec-
tively (mean Ϯ S.D., n ϭ 3). For the low-affinity component, the Km2
was 71.9 Ϯ 30.78 ␮M and the Vmax2 was 1230 Ϯ 220 pmol/mg
protein/min (mean Ϯ S.D., n ϭ 3). Therefore, clopidogrel concentra-
tion in the inhibition studies was set at 4 ␮M, which was close to the
Km1 value of the high-affinity component.
The inhibitory effects of monoclonal antibodies to CYP1A2,
CYP2B6, and CYP2C19 on the production of 2-oxo-clopidogrel were
examined by using human liver microsomes as the enzyme source and
clopidogrel (at Km; 4 ␮M) as the substrate in the presence of an
NADPH-generating system as shown in Table 3. The inhibition ratio
of 2-oxo-clopidogrel formation with monoclonal antibodies to
CYP1A2, CYP2B6, and CYP2C19 were 30.9 Ϯ 9.0, 26.0 Ϯ 2.5, and
42.0 Ϯ 8.1%, respectively (mean Ϯ S.D., n ϭ 3). The inhibitory effect
of monoclonal antibodies to CYP2C19 on this oxidation process was
higher than that of the other two monoclonal antibodies. Moreover,
the effects of chemical inhibitors on the production of 2-oxo-
clopidogrel were also investigated in human liver microsomes
(Fig. 6). Furafylline (10 ␮M), an inhibitor of CYP1A2, inhibited
2-oxo-clopidogrel formation by 34.9 Ϯ 0.5%. (S)-N-3-Benzylnirvanol
and omeprazole, the inhibitors of CYP2C19 at 10 ␮M, also inhibited
the formation of 2-oxo-clopidogrel by 26.9 Ϯ 10.5 and 27.1 Ϯ 9.2%,
respectively (mean Ϯ S.D., n ϭ 3).
The kinetic parameters of clopidogrel active metabolite formation
from 2-oxo-clopidogrel in human liver microsomes were also esti-
mated by using WinNonlin nonlinear estimation program because the
reaction rate of the active metabolite formation in human liver mi-
crosome also demonstrated a biphasic pattern in Eadie-Hofstee plots
(Fig. 7), indicating the involvement of multiple enzymes. The appar-
ent Km1 and Vmax1 values for the high-affinity component were
2.69 Ϯ 0.828 ␮M and 32.4 Ϯ 11.6 pmol/mg protein/min, respectively
(mean Ϯ S.D., n ϭ 3). For the low-affinity component, the Km2 was
94.9 Ϯ 6.05 ␮M and the Vmax2 was 249 Ϯ 41.0 pmol/mg protein/min
(mean Ϯ S.D., n ϭ 3). Based on these results, the 2-oxo-clopidogrel
concentration in the inhibition studies was set at 3 ␮M, which was
close to the Km1 value of the high-affinity component.
With respect to the second oxidation step leading to clopidogrel
active metabolite formation, the inhibitory effects of monoclonal
antibodies to CYP2B6, CYP2C19, CYP3A4, and polyclonal antibody
to CYP2C9 on the production of the active metabolite were examined
by using human liver microsomes as the enzyme source and 2-oxo-
clopidogrel (at Km; 3 ␮M) as the substrate in the presence of an
Likewise, the studies of the formation of clopidogrel active metab-
olite by CYP2B6, CYP2C9, CYP2C19, and CYP3A4 were per-
formed. The possibility of mechanism-based inhibition in the second
oxidation step was not allowed because previous studies demonstrated
that such an inhibition was not involved in this reaction process
(Nishiya et al., 2009).
The enzyme kinetic parameters combined with eq. 3 showed that
the contribution of each P450 involved in the pharmacologically
active metabolite formation decreased in the order CYP3A4
NADPH-generating system and glutathione (5 mM) as shown in (39.8%) Ͼ CYP2B6 (32.9%) Ͼ CYP2C19 (20.6%) Ͼ CYP2C9
Table 3. The inhibition ratio of the active metabolite formation from (6.76%) (Table 2). Anti-CYP2B6, anti-CYP2C9, anti-CYP2C19, and
2-oxo-clopidogrel with antibodies to CYP2B6, CYP2C9, CYP2C19, anti-CYP3A4 antibodies inhibited the formation of the active metab-

98
KAZUI ET AL.
FIG. 6. Effects of the selective chemical inhibi-
tors for CYP1A2 (furafylline), CYP3A4 (keto-
conazole), CYP2C19 [(S)-N-3-benzylnirvanol,
omeprazole], and CYP2C9 (sulfaphenazole) on
the 2-oxo-clopidogrel formation and the active
metabolite formation by pooled human liver mi-
crosome. Data were expressed as mean Ϯ S.D. of
three experiments.
FIG. 7. Kinetic analysis of the active metabolite formation from 2-oxo-clopidogrel in human liver microsome by Eadie-Hofstee plots and WinNonlin nonlinear estimation
program. The human liver microsomes were incubated with 1.25 to 160 ␮M 2-oxo-clopidogrel at 37°C for 15 min. Data are plotted using Eadie-Hofstee plots (A) and direct
plots (B). Line in B represents the curve fit to eq. 5 using WinNonlin nonlinear estimation program. Typical results from one of three experiments are shown.
olite by 54.3 Ϯ 3.4, 13.5 Ϯ 4.4, 32.9 Ϯ 4.2, and 31.3 Ϯ 5.3%, clopidogrel to the active metabolite step. The data specifically point
respectively. Moreover, the inhibition levels of the chemical inhibitors out that CYP2C19 would have a major effect on the formation of
such as sulfaphenazole, (S)-N-3-benzylnirvanol, omeprazole, and ke- active metabolite from clopidogrel, and this observation would ex-
toconazole on the production of the active metabolite from 2-oxo- plain the effect of loss-of-function variants of CYP2C19 on the
clopidogrel exhibited 36.2 Ϯ 7.7, 35.4 Ϯ 15.2, 31.3 Ϯ 17.3, and pharmacokinetics and/or pharmacodynamic response to clopidogrel
38.4 Ϯ 8.4%, respectively. The inhibition levels observed in the (Hulot et al., 2006; Brandt et al., 2007; Mega et al., 2009; Simon et al.,
presence of antibodies or chemical inhibitors corresponded well to the 2009). In these studies, common loss-of-functional polymorphisms of
contribution ratio of each P450 isoform in the second oxidation step. CYP2C19 were associated with decreased exposure in the active
These studies clearly showed that CYP2C19 contributes substan- metabolite of clopidogrel, resulting in decreased pharmacodynamic
tially to both oxidative steps required to the formation of clopidogrel response of clopidogrel, and patients carrying those alleles demon-
active metabolite and that CYP3A4 contributes only to the 2-oxo- strated a higher rate of cardiovascular events while on clopidogrel

P450 ISOFORMS RESPONSIBLE FOR BIOACTIVATION OF CLOPIDOGREL
99
pharmacodynamics of prasugrel and clopidogrel in healthy subjects. Pharmacotherapy 28:
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Gelboin HV, Krausz KW, Goldfarb I, Buters JT, Yang SK, Gonzalez FJ, Korzekwa KR, and
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oxidation step would explain the clinical evidence by CYP3A inhib-
itor on the reduction in clopidogrel active metabolite formation and
the corresponding decrease in the effect of clopidogrel on platelets’
aggregation (Suh et al., 2006; Farid et al., 2007). It is also likely that
CYP3A5 also contributes to clopidogrel’s active metabolite formation
from 2-oxo-clopidogrel as was previously indicated in clinical and in
vitro studies (Suh et al., 2006; Farid et al., 2007; Baker et al., 2008).
Moreover, in the present study, the inhibition ratio of the active
metabolite formation from 2-oxo-clopidogrel with monoclonal anti-
body to CYP3A4 was lower than that with CYP3A4/5 inhibitor
ketoconazole, suggesting some contribution by CYP3A5.
Krausz KW, Goldfarb I, Buters JT, Yang TJ, Gonzalez FJ, and Gelboin HV (2001) Monoclonal
antibodies specific and inhibitory to human cytochromes P450 2C8, 2C9, and 2C19. Drug
Metab Dispos 29:1410–1423.
Mega JL, Close SL, Wiviott SD, Shen L, Hockett RD, Brandt JT, Walker JR, Antman EM,
Macias W, Braunwald E, et al. (2009) Cytochrome P-450 polymorphism and response to
clopidogrel. N Engl J Med 360:354–362.
Nishiya Y, Hagihara K, Kurihara A, Okudaira N, Farid NA, Okazaki O, and Ikeda T (2009)
Comparison of mechanism-based inhibition of human cytochrome P450 2C19 by ticlopidine,
clopidogrel and prasugrel. Xenobiotica 39:836–843.
Nishiya Y, Hagihara K, Ito T, Tajima M, Miura S, Kurihara A, Farid NA, and Ikeda T (2009)
Mechanism-based inhibition of human cytochrome P450 2B6 by ticlopidine, clopidogrel, and
the thiolactone metabolite of prasugrel. Drug Metab Dispos 37:589–593.
Ng PS, Imaoka S, Hiroi T, Osada M, Niwa T, Kamataki T, and Funae Y (2003) Production of
inhibitory polyclonal antibodies against cytochrome P450s. Drug Metab Pharmacokinet
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Pereillo JM, Maftouh M, Andrieu A, Uzabiaga MF, Fedeli O, Savi P, Pascal M, Herbert JM,
Maffrand JP, and Picard C (2002) Structure and stereochemistry of the active metabolite of
clopidogrel. Drug Metab Dispos 30:1288–1295.
Richter T, Mu¨rdter TE, Heinkele G, Pleiss J, Tatzel S, Schwab M, Eichelbaum M, and Zanger
UM (2004) Potent mechanism-based inhibition of human CYP2B6 by clopidogrel and ticlo-
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Rowland Yeo K, Rostami-Hodjegan A, and Tucker GT (2004) Abundance of cytochromes P450
in human liver: a meta-analysis. Br J Clin Pharmacol 57:687–688.
Rehmel JL, Eckstein JA, Farid NA, Heim JB, Kasper SC, Kurihara A, Wrighton SA, and Ring
BJ (2006) Interactions of two major metabolites of prasugrel, a thienopyridine antiplatelet
agent, with the cytochromes P450. Drug Metab Dispos 34:600–607.
Savi P, Herbert JM, Pflieger AM, Dol F, Delebassee D, Combalbert J, Defreyn G, and Maffrand
JP (1992) Importance of hepatic metabolism in the antiaggregating activity of the thienopy-
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Savi P, Combalbert J, Gaich C, Rouchon MC, Maffrand JP, Berger Y, and Herbert JM (1994)
The antiaggregating activity of clopidogrel is due to a metabolic activation by the hepatic
cytochrome P450–1A. Thromb Haemost 72:313–317.
Savi P, Pereillo JM, Uzabiaga MF, Combalbert J, Picard C, Maffrand JP, Pascal M, and Herbert
JM (2000) Identification and biological activity of the active metabolite of clopidogrel.
Thromb Haemost 84:891–896.
In conclusion, we demonstrated that the pharmacologically active
metabolite of clopidogrel is produced from clopidogrel by two suc-
cessive oxidation processes, with the first step from clopidogrel to
2-oxo-clopidogrel being catalyzed by CYP1A2, CYP2B6, and/or
CYP2C19 and the second step from 2-oxo-clopidogrel to the active
metabolite being catalyzed by CYP3A4, CYP2B6, CYP2C9, and/or
CYP2C19. In addition, we indicated that the contributions of
CYP2C19 and CYP3A4 were more important relative to the other
P450s in the pharmacologically active metabolite formation by esti-
mating the enzyme kinetic parameters and inhibition effects of the
chemical inhibitors and the antibody to P450s. These findings would
support the results of several clinical drug-drug interaction studies
with clopidogrel, and studies on the effect of genetic polymorphism
on the pharmacodynamic response of clopidogrel, and studies on the
effect of genetic polymorphism of CYP2C19 on the clinical outcomes
in patients treated with clopidogrel (Hulot et al., 2006; Suh et al.,
2006; Brandt et al., 2007; Farid et al., 2007, 2008; Mega et al., 2009;
Simon et al., 2009).
Acknowledgments. We thank Dr. Mary Pat Knadler of Eli Lilly
and Company for helpful comments on this manuscript. The co-author
Nagy A. Farid has retired from Eli Lilly and Company.
Savi P and Herbert JM (2005) Clopidogrel and ticlopidine: P2Y12 adenosine diphosphate-
receptor antagonists for the prevention of atherothrombosis. Semin Thromb Hemost 31:174–
183.
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JA, and Salazar DE (2007) Cytochrome P450 3A inhibition by ketoconazole affects prasugrel
and clopidogrel pharmacokinetics and pharmacodynamics differently. Clin Pharmacol Ther
81:735–741.
Address correspondence to: Miho Kazui, Drug Metabolism and Pharmacoki-
netics Research Laboratories, Daiichi Sankyo Co., Ltd., 1-2-58 Hiromachi, Shina-
gawa-Ku, Tokyo, 140-8710, Japan. E-mail: kazui.miho.yk@daiichisankyo.co.jp
Farid NA, Small DS, Payne CD, Jakubowski JA, Brandt JT, Li YG, Ernest CS, Salazar DE,
Konkoy CS, and Winters KJ (2008) Effect of atorvastatin on the pharmacokinetics and