Cytochromes P450 catalyze both steps of the major pathway of clopidogrel bioactivation, whereas paraoxonase catalyzes the formation of a minor thiol metabolite isomer

Article abstract of DOI:10.1021/tx2004085

The mechanism generally admitted for the bioactivation of the antithrombotic prodrug, clopidogrel, is its two-step enzymatic conversion into a biologically active thiol metabolite. The first step is a classical cytochrome P450 (P450)-dependent monooxygena

Full text of DOI:10.1021/tx2004085

Article
pubs.acs.org/crt
Cytochromes P450 Catalyze Both Steps of the Major Pathway of
Clopidogrel Bioactivation, whereas Paraoxonase Catalyzes the
Formation of a Minor Thiol Metabolite Isomer
Patrick M. Dansette,* Julien Rosi, Gildas Bertho, and Daniel Mansuy
Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques, UMR 8601 CNRS, Universite
Paris Cite, 45 rue des Saints-Peres, 75270 Paris Cedex 06, France
́
Paris Descartes, Sorbonne
́
̀
S
* Supporting Information
ABSTRACT: The mechanism generally admitted for the bioactivation of the antithrombotic prodrug, clopidogrel, is its two-step
enzymatic conversion into a biologically active thiol metabolite. The first step is a classical cytochrome P450 (P450)-dependent
monooxygenation of its thiophene ring leading to 2-oxo-clopidogrel, a thiolactone metabolite. The second step was described as
a P450-dependent oxidative opening of the thiolactone ring of 2-oxo-clopidogrel, with intermediate formation of a reactive
sulfenic acid metabolite that is eventually reduced to the corresponding thiol 4b. A very recent paper published in Nat. Med.
(Bouman et al., (2011) 17, 110) reported that the second step of clopidogrel bioactivation was not catalyzed by P450 enzymes
but by paraoxonase-1(PON-1) and that PON-1 was a major determinant of clopidogrel efficacy. The results described in the
present article show that there are two metabolic pathways for the opening of the thiolactone ring of 2-oxo-clopidogrel. The
major one, that was previously described, results from a P450-dependent redox bioactivation of 2-oxo-clopidogrel and leads to 4b
cis, two previously reported thiol diastereomers bearing an exocyclic double bond. The second, minor one, results from a
hydrolysis of 2-oxo-clopidogrel, which seems to be dependent on PON-1, and leads to an isomer of 4b cis, 4b "endo", in which
the double bond has migrated from an exocyclic to an endocyclic position in the piperidine ring. These results were obtained
from a detailed study of the metabolism of 2-oxo-clopidogrel by human liver microsomes and human sera and analysis by HPLC-
MS under conditions allowing a complete separation of the thiol metabolite isomers, either as such or after derivatization with 3′-
methoxy phenacyl bromide or N-ethyl maleimide (NEM). These results also show that the major bioactive thiol isomer found in
the plasma of clopidogrel-treated patients derives from 2-oxo-clopidogrel by the P450-dependent pathway. Finally, chemical
experiments on 2-oxo-clopidogrel showed that this thiolactone is in equilibrium with its tautomer having a double bond inside
the piperidine ring and that nucleophiles such as CH3O− preferentially react on the thioester function of this tautomer. This
allowed us to understand why 4b cis has to be formed via an oxidative opening of 2-oxo-clopidogrel thiolactone, whereas a
hydrolytic opening of this thiolactone ring leads to the "endo" thiol isomer 4b "endo".
by dimedone.11 In the presence of glutathione (GSH) in excess,
INTRODUCTION
■
these sulfenic acids are reduced into the corresponding thiols
411,12 (Figure 1). These thiol metabolites are generally
considered as the pharmacologically active species that are
responsible for the inhibition of the P2Y12 platelet receptor
after the formation of a covalent disulfide bond with a cysteine
residue of this protein.1−3 Another possible mechanism for this
inactivation of the P2Y12 receptor could be the formation of
such a covalent bond from a reaction of their sulfenic acid
metabolites 3 or of the glutathionyl adducts deriving from their
reaction with GSH, with a cysteine SH residue of the P2Y12
platelet receptor.11,12 The latter mechanism remains to be
established.
The mechanism generally admitted for the bioactivation of
ticlopidine 1a and clopidogrel 1b, two tetrahydrothienopyridine
antithrombotic prodrugs, is their two-step enzymatic con-
version into a biologically active thiol metabolite 1−4(Figure 1).
The first step is a classical cytochrome P450 (P450)-dependent
monooxygenation of their thiophene ring by NADPH and O2
5−8
that leads to the thiolactone metabolites 2a and 2b. It is
mainly catalyzed by P450s 2C19 and 2B6 in the case of
ticlopidine and by P450s 2C19, 1A2, and 2B6 in the case of
clopidogrel.9,10 The second step was described as a P450-
dependent oxidative opening of the thiolactone ring of 2 with
the eventual formation of an active thiol metabolite.9,10 We
have shown that this step was a P450-catalyzed cleavage of the
thiolactone ring of metabolites 2 leading to the formation of
reactive sulfenic acid intermediates 3, which have been trapped
Received: September 26, 2011
Published: November 21, 2011
© 2011 American Chemical Society
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patients.20 The latter results questioned our current under-
standing of clopidogrel bioactivation and cast doubts on a large
body of previous metabolic and genetic association studies.
In order to understand the origin of these contradictory
conclusions, we have reinvestigated the in vitro metabolism of
2-oxo-clopidogrel 2b by human liver microsomes and human
sera using HPLC techniques leading to a complete separation
of the isomers of the thiol metabolite of 2b because such a
separation was not done in most studies previously reported on
the metabolism of clopidogrel or 2-oxo-clopidogrel, including
that of Bouman et al.20 Actually, opening of the thiolactone ring
of 2b may lead to five thiol isomers, two cis diastereomers, 4b
cis, with a Z configuration of the exocyclic double bond, two
trans diastereomers with an E configuration of the exocyclic
double bond, 4b trans, and an “endo” isomer, 4b "endo", in
which the double bond has migrated from an exocyclic to an
endocyclic position in the piperidine ring21 (Figure 2). A very
recent, optimized method for the specific, quantitative
determination of the clopidogrel-derived thiol metabolite
isomers, after derivatization of their thiol function upon
treatment with 3′-methoxy phenacyl bromide, in human plasma
of clopidogrel-treated subjects showed the presence of the cis
thiol isomers as major metabolites and of the “endo” thiol
isomer as a minor component.21 The mode of formation of this
minor "endo" thiol isomer was presently unknown. Moreover,
the authors also showed that one of these cis thiol
diastereomers was very active toward the platelet P2Y12
receptor and was presumably responsible for the antithrom-
botic activity of clopidogrel.21
Figure 1. Different steps reported for the bioactivation of ticlopidine
1a and clopidogrel 1b.11
Pharmacokinetic, pharmacodynamic, and genetic studies
provided strong evidence that in vivo bioactivation of
clopidogrel is closely linked to the P450 system.10,13−19
Actually, P450 2C19 was found to play a key role in clopidogrel
bioactivation by contributing to both steps of this bioactiva-
tion.9,10 A common genetic variant within the CYP(P450) 2C19
gene, the CYP2C19*2 loss of function polymorphism, was
found to be associated with an attenuated response to
clopidogrel and a worse clinical outcome in patients undergoing
coronary stenting and treatment with clopidogrel.14−19
A very recent paper published in Nat. Med.20 concluded that
the second step of clopidogrel bioactivation was not catalyzed
by P450 enzymes but by paraoxonase-1 (PON-1) and that
PON-1 was a major determinant of 1b efficacy. It suggested
that PON-1 Q192R polymorphism may have a key role for the
rate of clopidogrel bioactivation and influence platelet response
to this drug and the risk of stent thrombosis in 1b-treated
The results on the in vitro metabolism of 2b reported in this
article give a detailed, complete description of the very
preliminary data quite recently mentioned in a Correspondence
letter22 that we sent to the Editor of Nature Medicine as an
answer to Bouman et al.20 They show that both P450 enzymes
and PON-1 do catalyze the opening of 2b thiolactone ring but
lead to different isomers of the thiol metabolite and that the
Figure 2. Different possible isomers of clopidogrel-derived thiol 4b and derivatization from a reaction with either 3′-methoxy-phenacyl bromide or
NEM. Compounds 4b cis and 4b trans can exist as two diastereomers as they contain two chiral carbons. The benzylic carbon has an (S)
configuration, as in clopidogrel, and the carbon bearing the S atom can have an (S) or (R) configuration. The formula of the tautomer of 4b “endo”,
4b tk, that could exist in equilibrium with 4b “endo” is also shown, as well as its product of reaction with semicarbazide.
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Figure 3. HPLC profiles of incubations of 2-oxo-clopidogrel with human liver microsomes with (3A) and without (3B) NADPH with MS detection
after derivatization with 3′-methoxy-phenacyl bromide and MS2 spectra (parent ion at m/z = 504) of the three derivatized thiol metabolites, 4b′cis (2
diastereomers) and 4b′ “endo”.
HPLC-MS Studies. These studies were performed on a Surveyor
HPLC instrument coupled to a LCQ Advantage ion trap mass
spectrometer (Thermo, Les Ulis, France), using a Gemini C18 column
(100 × 2.1 mm, 3 μm; Phenomenex) and a gradient starting at 40% B
for 1 min then increasing linearly to 100% B in 15 min (A = 10 mM
ammonium acetate buffer, pH 4.6, and B = CH3CN/CH3OH/H2O
(7:2:1)) at 200 μL/min. Mass spectra were obtained by electrospray
ionization (ESI) in positive ionization mode detection under the
following conditions: source parameters, sheeth gas, 20; auxiliary gas,
5; spray voltage, 4.5 kV; capillary temperature, 200 °C; and capillary
voltage, 15 V; m/z range for MS recorded generally between 300 and
700 (except for exploratory experiments with a wider range of 300−
800). MS2 energy was tested between 20 and 40 eV and was generally
35 eV. For all products, the indicated molecular ions corresponded to
M + H+.
major bioactive thiol isomer found in the plasma of clopidogrel-
treated subjects is formed via two P450-dependent steps. This
article also provides additional data allowing us to understand
why the hydrolytic, paraoxonase-dependent opening of 2b only
leads to the “endo” thiol isomer, whereas P450-catalyzed
oxidative opening of 2b is required to obtain the active cis thiol
isomers.
EXPERIMENTAL PROCEDURES
■
Chemicals and Biochemicals. [8S]-2-oxo-clopidogrel (the for-
mula numerotation is in Figure 6) (SR121883), 2b, 3′-methoxyphe-
nacyl-cis-thiol (SAR206251), 4b′cis, and 4′-bromophenacyl-“endo”-
thiol (SAR195539) were a gift of Sanofi-Aventis (Chilly-Mazarin,
France). The sample of [8S]-2-oxo-clopidogrel, 2b, used in our studies
was an 80:20 mixture of diastereomers, as shown by 1H NMR
spectroscopy (see below). All other products including enzymes were
from Sigma-Aldrich (St. Quentin Fallavier, France).
Chemical Experiments on 2b. Reaction of 2b in CD3OD in
the Presence of K2CO3. Compound 2b (20 mM in CD3OD) was
1
treated at 20 °C with a few crystals of K2CO3, and H NMR spectra
were recorded at t = 0, 5, 10, 15, and 25 min. An aliquot of the
reaction mixture was diluted in CH3CN/CH3COOH/H2O 20:9:1 and
analyzed by HPLC-MS.
Microsomal Incubations. Human microsomes (pool, 10 mg
protein/mL) were obtained from BD-Gentest (Le Pont de Claix,
France). Typical incubations were performed in potassium phosphate
buffer (0.1M, pH 7.4) containing 2 mM CaCl2, 100 mM KF,
microsomes (1 mg protein/mL), 2b (100 μM), and a reducing agent
(20 mM ascorbic acid or 5 mM GSH) with or without the NADPH
generating system (1 mM NADP, 15 mM glucose-6-phosphate, and 2
U/mL of glucose-6-phosphate dehydrogenase) at 37 °C for 30 min.
Reactions were stopped by adding one-half volume of CH3CN/
CH3COOH (9:1), and proteins were removed by centrifugation at
13000g. The KF concentration used previously2,20 to completely
inhibit the esterase-dependent hydrolysis of the methyl ester function
of 2b was 100 mM; this is why we generally used this KF
concentration to have comparable data. Literature data indicated
that 1 mM KF did not significantly inhibit PON-1.23 Identical
microsomal incubations were also performed with 1 and 10 mM KF to
know if high KF concentrations led to inhibitory effects on PON-1
and/or P450 enzymes. These experiments showed that the P450-
dependent metabolite 4b cis formation was not affected by a change of
the KF concentration from 1 to 100 mM. Formation of the PON-1-
dependent metabolite, 4b "endo", only slightly decreased (by about
30%) upon increasing [KF] from 1 to 100 mM.
Reaction of 2b with CH3ONa in CH3OH. A solution of 2 mM 2b in
CH3OH was treated with 120 mM CH3ONa in CH3OH at 20 °C for
45 min. After dilution with 10 vol of 100 mM phosphate buffer at pH
7.4, the final product was analyzed by HPLC-MS, as such or after
derivatization with 3′-methoxy phenacyl bromide as described above. It
was purified by loading on a Seppak C18 column (Waters), washing
with H2O, eluting by CH3OH, and analyzed by 1H NMR (in CD2Cl2)
after the evaporation of CH3OH.
NMR Experiments. 1H NMR spectra of 2b and of 4b "endo"
methyl ester were done on a Bruker AVANCE II 500 spectrometer
(500.16 MHz) at 27 °C. Chemical shifts are given in ppm relative to
(CH3)4Si.
RESULTS
■
Metabolism of 2-Oxo-clopidogrel by Human Liver
Microsomes. In order to reinvestigate the metabolic fate of
2b in the presence of human liver microsomes or human sera,
we used HPLC-MS methods allowing a complete separation of
the thiol metabolite isomers, either as such or after
derivatization using either 3′-methoxy- or 4′-bromo- phenacyl
bromide or N-ethyl maleimide (NEM) that lead to more stable
final metabolites.21,24
Incubations of 2-Oxo-clopidogrel with Human Sera. Typical
incubations were performed in potassium phosphate buffer (0.1M, pH
7.4) containing 2 mM CaCl2, 100 mM KF, human serum (1 mg
protein/mL), 2b (100 μM), and a reducing agent (20 mM ascorbic
acid or 5 mM GSH), at 37 °C for 30 min. Reactions were stopped by
adding one-half volume of CH3CN/CH3COOH (9:1) and 4 nmol of
internal standard (4′-bromophenacyl-“endo”-thiol, SAR195539), and
proteins were removed by centrifugation at 13000g.
Analysis of the Thiol Metabolites after Derivatization
with 3′-Methoxy Phenacyl Bromide. Compound 2b (0.1
mM) was incubated for 10 min at 37 °C with human liver
microsomes in the presence of NADPH, which is a required
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Figure 4. HPLC profiles of the incubations of 2-oxo-clopidogrel with human liver microsomes with (A) and without (B) NADPH with MS2
detection at m/z = 358 (37Cl) and MS2 spectra (parent ion at m/z = 356 (35Cl)) of the thiol metabolites 4b cis and 4b “endo”.
cofactor for P450-dependent activities,25 and 100 mM KF to
inhibit the esterase-dependent hydrolysis of the methyl ester
function of 2b,2 and of a reducing agent (20 mM ascorbic acid
or 5 mM GSH) to reduce the sulfenic acid intermediate 3b into
the corresponding thiol 4b.11,26 An HPLC-MS study of the
incubate, after derivatization of the thiol metabolites by
treatment with 3′-methoxy phenacyl bromide, showed the
formation of three thiol isomers derivatives (Figure 3)
exhibiting a molecular ion (ESI+) corresponding to M + H+
and characterized by two peaks at m/z = 504 and 506 with the
ratio expected for the 35Cl and 37Cl isotopes. The two major
thiol isomers exhibited identical MS2 spectra, with major
fragments of the molecular ion (m/z = 504) at m/z = 354 and
324 (Figure 3). Their MS2 spectra were identical to that of an
authentic sample of the 3′-methoxy acetophenone derivative of
the most active cis thiol diastereomer, 4b′cis.21 Moreover, the
HPLC retention time of this authentic compound was identical
to that of the less polar of the two major derivatized thiol
isomer metabolites. The minor derivatized thiol metabolite
exhibited a different MS2 spectrum characterized by a major
fragment of the molecular ion (m/z = 504) at m/z = 212
(Figure 3). The corresponding thiol metabolite was identified
at the level of its 4′-bromo acetophenone derivative, after
treatment of the same microsomal incubation with 4′-bromo
phenacyl bromide and comparison of its MS2 spectrum and
HPLC retention time with those of an authentic sample of the
4′-bromoacetophenone derivative of the "endo" thiol isomer 4b
"endo".21
Analysis of the Thiol Metabolites after in Situ
Derivatization with NEM. NEM is an interesting thiol-
trapping agent because it can be used in situ during the
metabolism of a xenobiotic by liver microsomes.26 Incubation
of 2b with human liver microsomes was done under the above-
mentioned conditions but in the presence of 1 mM NEM; the
reducing agent used in this case was ascorbate in order to avoid
a reaction between NEM and GSH. HPLC-MS analysis of the
incubate showed the formation of three NEM-derivatized thiol
metabolites with a ratio very similar to that observed after the
postreaction derivatization with 3′-methoxy phenacyl bromide
(see Figure 2S in Supporting Information). These three
derivatized thiol metabolites, 4b″ cis and 4b″ "endo" (see
Figure 2 for their formula), were characterized by molecular
ions with two peaks at m/z = 481 and 483 (for 35Cl and 37Cl,
respectively), as expected for NEM adducts of thiols 4b cis (2
diastereomers) and 4b "endo".
Analysis of the Thiol Metabolites without Derivatiza-
tion. A similar HPLC-MS study was also performed on the
thiols themselves (without any derivatization) and showed the
formation of three thiol isomers characterized by a MS
molecular ion with two peaks at m/z = 356 and 358 (for
35Cl and 37Cl, respectively), in a ratio very similar to that found
for their 3′-methoxyacetophenone derivatives (Figures 4 and 3).
The two major thiol metabolites exhibited the same MS2
spectrum with the main fragments of the molecular ion (m/z
= 356) at m/z = 322, 296, 212, 184, and 172 (Figure 4), which
was identical to that previously observed for the 4b cis
diastereomers.2,11 The minor thiol metabolite showed a
different MS2 spectrum characterized by a main fragment at
m/z = 212 (Figure 4). Thus, this analysis of the thiol
metabolites was in complete agreement with the above analysis
done on their 3′-methoxyacetophenone or NEM derivatives,
showing that the metabolism of 2b by NADPH-supplemented
human liver microsomes mainly led to the cis thiol isomers 4b
cis and to the "endo" thiol isomer 4b "endo" as a minor
product.
The "endo" thiol isomer 4b “endo” is a thioenol that should
exist in equilibrium with its thioketone tautomer 4b tk (Figure
2). Accordingly, microsomal incubation of 2b under the above-
mentioned conditions but in the presence of 5 mM
semicarbazide, an usual reagent for ketones and thioketones,27
led to the formation of a new product characterized by a MS
molecular ion with two peaks at m/z = 397 and 399 (for 35Cl
and 37Cl, respectively). This MS molecular ion and its MS2
fragments at m/z = 379 (M-18, H2O), 336 (379-43, CONH),
and 324 (M-73, NNHCONH2) (see Figure 1S in Supporting
Information) were in good agreement with a semicarbazone
structure resulting from reaction of semicarbazide with 4b tk
(Figure 2).
Effects of the Incubation Conditions on 2-Oxo-
clopidogrel Microsomal Metabolism. When identical
microsomal incubations were done in the absence of
NADPH, there was no formation of 4b cis, and the only
thiol isomer that could be detected was 4b "endo" (Table 1).
Moreover, the addition of 20 μM N-benzylimidazole, a well-
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possible tautomer 2b′, in which the double bond has migrated
within the piperidine ring and is no longer conjugated with the
keto group (Figure 5). The existence of this equilibrium was
Table 1. Formation of 4b cis and 4b “endo” upon the
Metabolism of 2b by Human Liver Microsomes
b
(nmol/mg protein/30 min)
conditions
4b cis
4b “endo”
a
complete system
19
4
0.5 0.2
<0.1
2.2 0.4
2.8 0.4
2.3 + 0.4
2.2 0.4
1.0 0.2
0.5 0.2
0.5 0.5
+ 20 μM N-benzyl-imidazole
− ascorbate
− NADPH
<0.1
− NADPH − CaCl2
− NADPH + 500 μM paraoxon
− NADPH − CaCl2 + 5 mM EDTA
<0.1
<0.1
<0.1
a
Complete system: incubations of 100 μM 2b with human liver
microsomes (1 mg protein/ml) in 0.1 M phosphate buffer at pH 7.4
containing 100 mM KF, 2 mM CaCl2, and 20 mM ascorbic acid, for 30
min at 37 °C; analysis by HPLC-MS after derivatization with 3′-
methoxy-phenacyl bromide, as described in Experimental Procedures.
b
Figure 5. Equilibrium between 2-oxo-clopidogrel 2b and its tautomer
2b′ and their reaction in CD3OD and K2CO3 or with CH3O− in
CH3OH.
mean values SD from at least 3 independent experiments.
known inhibitor of microsomal P450s,28 to the incubation of 2b
with NADPH-supplemented microsomes almost completely
inhibited the formation of the 4b cis diastereomers, whereas it
did not significantly modify the formation of 4b "endo" (Table
1). These data indicated that the formation of the cis thiol
isomers 4b cis is P450-dependent. Since the article of Bouman
et al.20 proposed that thiol 4b would derive from a reaction of
2b with PON-1, identical microsomal incubations without
NADPH were performed in the presence of 500 μM paraoxon,
an usual substrate of PON-1.29,30 Table 1 shows that this led to
an 80% inhibition of 4b "endo" formation. Moreover,
incubations in the absence of CaCl2 led to a marked decrease
of 4b "endo" formation, and addition of 5 mM ethylenediamine
tetraacetate (EDTA) led to an even greater decrease of this
formation (Table 1). PON-1 activity is dependent on the
presence of Ca2+ ions.29 Thus, the above results show that the
formation of 4b cis from 2b is dependent on NADPH and is
catalyzed by P450s, whereas that of their isomer 4b "endo" is
not dependent on NADPH and is catalyzed by an esterase such
as PON-1. This conclusion was completely confirmed by the
results of incubations of 2b under conditions identical to those
of the complete system of Table 1 except for the absence of a
reducing agent (ascorbate or GSH). Under those conditions,
one only observed the formation of 4b "endo" (or its
derivatized products with either 3′-methoxy phenacyl bromide
or NEM) (line 3 of Table 1). This is in agreement with the fact
that formation of 4b "endo" from a simple hydrolysis of 2b
does not require the presence of a reducing agent contrary to
that of 4b cis, which needs the reduction of sulfenic acid 3b
(Figure 1).
indicated by the easy exchange of the vinylic (H3) and allylic
(H7a) hydrogens of 2b with deuteriums in CD3OD in the
1
presence of K2CO3, which was shown by H NMR spectros-
1
copy and MS. A detailed analysis of the H NMR spectrum of
2b in CD3OD, using 2D NMR methods (correlation
spectroscopy (COSY), heteronuclear multiple bond correlation
(HMBC), and heteronuclear single quantum coherence
spectroscopy (HSQC)) allowed us to assign all the signals to
the protons of the molecule. This NMR analysis clearly showed
that 2b was an 80:20 mixture of diastereomers (see for instance
the signals of H3, H8, or H6 in Figure 6A). After the addition
of K2CO3 to the solution of 2b in CD3OD, one observed a
1
progressive disappearance of the H NMR signals at 6.05 and
4.42 ppm of the H3 and H7a protons of 2b and a decrease of
the diastereomers ratio from 80/20 to 50/50 (Figure 6B). The
final spectrum corresponded to a 50:50 mixture of the
diastereomers of 2b deuterated on carbons 3 and 7a (2b″ in
Figure 5). This was not only shown by the disappearance of the
H3 and H7a signals but also by a simplification of the H7
signals, due to the loss of the coupling between H7 and H7a
(Figure 6). Accordingly, the final product (characterized by two
HPLC peaks corresponding to the 50:50 diastereomers
mixture) exhibited an HPLC retention time identical to that
of 2b and a MS molecular ion at m/z = 340 and 342 for 35Cl
and 37Cl, indicating that two deuterium atoms have replaced
two hydrogen atoms in 2b. This formation of 2b″ should result
from a base-catalyzed migration of the double bond of 2b
leading to 2b′, followed by a fast H/D exchange occurring at
the level of the CH2 group α to the COS function of 2b′,
because of the acidity of the allylic hydrogens of this CH2
group, and a base-catalyzed migration of the 2b′ double bond
with the final formation of 2b″. During this deuteration of 2b,
an equilibration of its two diastereomers occurs, leading to the
finally observed 50:50 ratio (Figure 6B).
Metabolism of 2-Oxo-clopidogrel by Human Sera.
Incubation of 100 μM 2-oxo-clopidogrel with human serum (10
mg protein mL−1), which contains PON-1 but not P450s, in
the presence of 100 mM KF for 30 min at 37 °C and the study
of the reaction mixture by HPLC-MS after derivatization using
either 3′-methoxy phenacyl bromide or NEM under the above-
described conditions showed the formation of only one thiol
isomer, 4b "endo" (Figure 2S in Supporting Information).
Chemical Hydrolysis of 2-Oxo-clopidogrel. Several
chemical experiments were done to understand why a simple
hydrolysis of 2b only led to the "endo" thiol isomer 4b "endo".
Actually, the sample of 2b used in our studies was a 80:20
Reaction of 2b with CH3ONa in CH3OH at 20 °C for 45
min led to the opening of the 2b thiolactone ring with the
formation of only one thiol isomer, the methyl ester of 4b
"endo", with an 80% yield. The structure of this product was
established from its mass spectrum (molecular ion with two
peaks at m/z = 370 and 372 for 35Cl and 37Cl, respectively, and
a MS2 main fragment at m/z = 212) and its 1H NMR spectrum
that exhibited characteristic signals at 3.7 and 3.2 ppm for the
1
mixture of diastereomers (as shown by H NMR spectroscopy
and HPLC-MS) that could exist in equilibrium with their
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Figure 6. 1H NMR spectra of 2-oxo-clopidogrel 2b in CD3OD at t = 0 (bottom spectrum) and 15 min after the addition of K2CO3 (top spectrum).
The top spectrum shows that 15 min after the addition of K2CO3, deuteration at position 7a was complete (loss of the H7a signal), whereas that at
position 3 was not (small H3 signal remaining). After 25 min, deuteration at positions 7a and 3 were complete (data not shown).
Figure 7. Different pathways involved in the formation of thiol metabolites 4b cis and 4b “endo” in the metabolism of clopidogrel 1b.
allylic hydrogens α to the CO and amine groups. A detailed
analysis of the 1H NMR spectrum of 4b "endo" methyl ester in
CD2Cl2, using 2D NMR methods (COSY, HMBC, and
HSQC) allowed us to assign all the signals to the protons of
the molecule (data not shown). These data suggest that the
reaction of nucleophiles such as CH3ONa or OH− on 2b
preferentially occurs on the thioester carbon of 2b′, the
tautomer of 2b, which is more electrophilic than the thioester
carbon of 2b which is conjugated to a double bond.
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formed in lower amounts; its MS and MS2 characteristics
showed that it was derived from the addition of the thiol
function of 4b "endo" to the activated double bond of NEM
(NEM adduct 4b″"endo" shown in Figures 2 and 7) (data not
shown). The result of this double trapping experiment
completely confirmed that in the presence of NADPH-
supplemented human liver microsomes and a reducing agent
such as ascorbate or GSH, metabolites 4b cis and 4b "endo"
are formed from 2b by two competing pathways, a P450-
dependent oxidative one, occurring via an intermediate sulfenic
acid 3b and an esterase (PON-1)-dependent hydrolytic one,
respectively. Actually, if 4b cis could have been formed by a
simple hydrolytic route, one should have observed the
formation of a NEM adduct of 4b cis (4b″cis of Figure 2)
that was not detected.
Our results showing that the major thiol isomers formed in
the metabolism of 2-oxo-clopidogrel by human liver micro-
somes are 4b cis and that their formation is catalyzed by P450s
are in agreement with those of several previously published
studies of in vitro metabolism of clopidogrel or 2-oxo-
clopidogrel.2,9,11 Formation of the minor isomer 4b "endo" in
these in vitro systems was not reported so far presumably
because either the used HPLC methods did not permit the
separation of 4b cis and 4b "endo" and/or the used incubation
conditions were not adapted for Ca2+-dependent PON-1
activity (EDTA was present in most previously reported
incubation media, and CaCl2 was not added; see last line of
Table 1). It is noteworthy that some xenobiotics, such as
dietary polyphenols and fenofibrate, act as inducers of PON-
133−36 and could have an effect on the formation of 4b "endo"
in clopidogrel-treated patients.
The conclusion concerning the P450-dependent formation of
the 4b cis metabolite is contradictory to that recently drawn by
Bouman et al.20 which excluded the fact that an oxidative P450-
dependent step was involved in the second step of clopidogrel
bioactivation. The latter conclusion, which was deduced from
incubation experiments of 2b with human liver microsomes,
human serum, and recombinant human P450s and esterases
expressed in a human embryonic kidney cell line and HPLC-
MS study of the thiol metabolites, was mainly based on the
following arguments: (i) from all the tested recombinant
enzymes, only recombinant PON-1 and PON-3 were found to
be able to catalyze the formation of thiol metabolites, whereas
P450s seemed to be inactive, and (ii) inhibition of PON-1
greatly inhibited thiol formation by human liver microsomes,
whereas P450 inhibitors did not.20 Actually, the HPLC system
used by Bouman et al. does not seem to separate the thiol
metabolite isomers such as 4b cis and 4b “endo”, and it is
possible that their MS method very preferentially detects 4b
"endo". If this was the case, they would have mainly followed
4b "endo" formation and found, as we did, that this formation
is dependent on PON-1 and not on P450s.
Interestingly, the three thiol isomers that we have detected
upon the metabolism of 2-oxo-clopidogrel by human liver
microsomes in the presence of NADPH are those that were
found to be present in the sera of patients treated with
clopidogrel. Moreover, their proportions were very similar to
those found in clopidogrel-treated patients.21 This suggests that
the major pathway of opening of thiolactone 2b in humans is
the P450-dependent formation of the 4b cis diastereomers,
whereas the formation of 4b "endo" is a minor one. Previous
literature data indicate that the 1b metabolite mainly
responsible for the antiplatelet activity of 1b is one of the 4b
DISCUSSION
■
The aforementioned studies of the metabolism of 2-oxo-
clopidogrel by human liver microsomes and human serum
showed that two different pathways resulting in the opening of
its thiolactone ring are occurring. The major one results from a
P450- and NADPH-dependent oxidation leading to the cis thiol
diastereomers 4b cis. The second, minor one is hydrolytic and
leads to the "endo" thiol isomer 4b “endo” (Figure 7). It occurs
both in liver microsomes and in human sera, depends on the
presence of Ca2+, and is inhibited by paraoxon (Table 1). It is
presumably this metabolic way that was measured by Bouman
et al.20 These authors have compared the activities of various
esterases toward 2b and concluded that PON-1 was mainly
involved in the transformation of 2b into thiols.20 From the
presently available data, it thus seems that the formation of 4b
"endo" from 2b would be mainly catalyzed by PON-1.
It is noteworthy that, under our incubation conditions, thiol
4b cis was stable for at least 1 h and that thiol 4b "endo" had a
half-life of about 25 min. Moreover, under those conditions, we
never observed any conversion of 4b cis into 4b "endo" or of
4b "endo" into 4b cis (data not shown).
From a chemical point of view, how is it possible to
understand why 4b "endo" derives from an hydrolysis of 2b,
while it is necessary to oxidize 2b to produce the cis thiol
isomers 4b cis? Our studies about the chemical hydrolysis of 2-
oxo-clopidogrel under basic conditions, showing that only
"endo" thiol products are formed, suggest that O-nucleophiles
preferentially react on the thioester carbon of the more reactive
tautomer 2b′ that is in equilibrium with 2b. This should be true
for the enzymatic hydrolysis of the thioester function of 2-oxo-
clopidogrel by PON-1 that would explain the formation of only
4b “endo” in incubations of 2-oxo-clopidogrel with liver
microsomes (in the absence of NADPH) and human sera.
Accordingly, literature data reported that PON-1 readily
catalyzed the hydrolysis of lactones, whereas it was inactive
on unsaturated lactones involving a less reactive carbonyl
moiety conjugated to a double bond.30 Thus, it seems that the
only way to open the thiolactone ring of 2-oxo-clopidogrel with
the formation of the cis thiol isomers, 4b cis, is to oxidize its S
atom with the intermediate formation of thioester sulfoxide 5b
(Figure 7).11 The thioester carbon of 5b is much more reactive
than that of 2b and should rapidly react with water to give
sulfenic acid 3b, as previously reported.11 Reduction of 3b with
GSH or ascorbic acid26 eventually leads to 4b cis. Formation of
3b in P450-catalyzed metabolism of 2-oxo-clopidogrel is in
agreement with a recent report showing that mechanism-based
inactivation of P450 2B6 by 2-oxo-clopidogrel occurs through
covalent modification of a cysteine residue CYS475.31
This competition between the hydrolysis of 2-oxo-
clopidogrel 2b (via 2b′) leading to 4b "endo" and redox
activation of 2b eventually leading to 4b cis in NADPH-
supplemented human liver microsomes was further confirmed
by an incubation of 2-oxo-clopidogrel with human liver
microsomes in the presence of NADPH under the above-
mentioned conditions except for the absence of a reducing
agent and for the presence of a trapping agent for thiols,
NEM,26 and a trapping agent for sulfenic acids, dime-
done.11,26,32 Under these conditions, the major products
found by an HPLC-MS analysis were the dimedone adducts
6b (two diastereomers in a 50:50 ratio) of the cis sulfenic acid
intermediate 3b, which were previously characterized by MS
11
1
and H and 13C NMR spectroscopy. Another product was
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cis diastereomers.2,21 Consequently, the bioactivation of
clopidogrel should be mainly dependent on P450s. This is in
agreement with several pharmacokinetic and pharmacodynamic
studies showing that the bioactivation of clopidogrel is closely
linked to P450s2,9−11,13,37 and a very recent study showing that
incubation of human platelets with 2-oxo-clopidogrel and
baculosomes containing some human recombinant P450s led
to an inhibition of platelet aggregation by ADP.38 This is also in
agreement with several data showing that a common genetic
variant within the CYP 2C19 gene, the CYP 2C19*2 loss of
function polymorphism, is associated with an attenuated
response to clopidogrel and to a worse clinical outcome in
patients undergoing coronary stenting.14−19 Moreover, even
more recently, eight independent published articles showed no
association of PON-1 Q192R genotype with platelet response
to clopidogrel and risk of stent thrombosis after coronary
stenting,39−46 whereas some of them confirmed the impact of
the CYP 2C19*2 genotype on both platelet effect of
clopidogrel and risk of coronary stenting.39,40,42,46
ABBREVIATIONS
■
EDTA, ethylene diamine tetraacetic acid; HPLC-MS, high
performance liquid chromatography−mass spectrometry; GSH,
glutathione; NEM, N-ethyl maleimide; PON-1, Paraoxonase 1;
P450, cytochrome P450
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CONCLUSIONS
■
Our results show that there are two metabolic pathways for the
opening of the thiolactone ring of 2-oxo-clopidogrel, the one
that was previously described resulting in a P450-dependent
redox bioactivation leading to 4b cis, and a second, hydrolytic,
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4b "endo". Since the most bioactive metabolite of clopidogrel is
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ASSOCIATED CONTENT
■
S
* Supporting Information
HPLC profiles of incubations of 2b with human serum or
human liver microsomes in the presence of several trapping
agents (semicarbazide, NEM and 3'-methoxy-phenacyl bro-
mide). This material is available free of charge via the Internet
at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*Tel: +33 142862191. Fax: +33 142868387. E-mail: patrick.
dansette@parisdescartes.fr.
Funding
Funding for this research was provided by University Paris
Descartes and Centre National de la Recherche Scientifique.
ACKNOWLEDGMENTS
■
We thank Sebastien Roy and Fabrice Hurbin (Sanofi-Aventis)
for a gift of authentic reference compounds, [8S] 2-oxo-
clopidogrel (SR121883), 3′-methoxyphenacyl-cis-thiol
(SAR206251) and 4′-bromophenacyl-“endo”-thiol
(SAR195539), and their analytical data. We thank Justine
Debernardi for her contribution to some laboratory experi-
ments.
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