Formation, reactivity, and antiplatelet activity of mixed disulfide conjugates of clopidogrel

Article abstract of DOI:10.1124/mol.112.084392

In this work, we investigated the formation, reactivity, and antiplatelet activity of various mixed disulfide conjugates of clopidogrel. Our results showed that the production of the active metabolite (AM) from 2-oxoclopidogrel by human liver microsomes (HLMs) is greatly affected by the thiol reductants used. Among the 10 thiol compounds tested, glutathione (GSH) is most efficient in producing the AM at a rate of 167 pmoles AM/ min/mg HLM. Interestingly, no AM but only the mixed disulfide conjugates were formed in the presence of 6-chloropyridazine- 3-thiol (CPT), 2,5-dimethylfuran-3-thiol, and 3-nitropyridine-2- thiol (NPT). The mass spectrometry (MS) and MS2 spectra of the conjugates of these thiol compounds confirmed the presence of a mixed disulfide bond linkage between the AM and the thiol reductants. Kinetic studies revealed that the mixed disulfide conjugates were capable of exchanging thiols with GSH to release the AM with second order rate constants ranging from 1.2 to 28 M21s21. The mixed disulfide conjugates of CPT and NPT showed potent inhibition of platelet aggregation after pretreatment with 1 mM GSH, confirming that the AM is responsible for the antiplatelet activity of clopidogrel. Collectively, our results provide strong support for a cytochrome P450 (P450)-mediated bioactivation mechanism involving the initial formation of a glutathionyl conjugate, followed by thiol-disulfide exchange with another GSH molecule to release the AM. Furthermore, the stable mixed disulfide conjugates identified in this study provide a platform to quantitatively generate the therapeutic AM without the need for P450- mediated bioactivation. This property can be further explored to overcome the interindividual variability in clopidogrel therapy. Copyright

Full text of DOI:10.1124/mol.112.084392

Supplemental material to this article can be found at:
http://molpharm.aspetjournals.org/content/suppl/2013/01/24/mol.112.084392.DC1.html
1521-0111/83/4/848–856$25.00
MOLECULAR PHARMACOLOGY
http://dx.doi.org/10.1124/mol.112.084392
Mol Pharmacol 83:848–856, April 2013
Copyright ª 2013 by The American Society for Pharmacology and Experimental Therapeutics
Formation, Reactivity, and Antiplatelet Activity of Mixed Disulfide
Conjugates of Clopidogrels
Haoming Zhang, D. Adam Lauver, Benedict R. Lucchesi, and Paul F. Hollenberg
Department of Pharmacology, University of Michigan Medical School, Ann Arbor, Michigan
Received December 11, 2012; accepted January 24, 2013
ABSTRACT
In this work, we investigated the formation, reactivity, and GSH to release the AM with second order rate constants
antiplatelet activity of various mixed disulfide conjugates of ranging from 1.2 to 28 M21s21. The mixed disulfide conjugates
clopidogrel. Our results showed that the production of the ac- of CPT and NPT showed potent inhibition of platelet aggrega-
tive metabolite (AM) from 2-oxoclopidogrel by human liver tion after pretreatment with 1 mM GSH, confirming that the AM
microsomes (HLMs) is greatly affected by the thiol reductants is responsible for the antiplatelet activity of clopidogrel. Col-
used. Among the 10 thiol compounds tested, glutathione (GSH) lectively, our results provide strong support for a cytochrome
is most efficient in producing the AM at a rate of 167 pmoles AM/ P450 (P450)-mediated bioactivation mechanism involving
min/mg HLM. Interestingly, no AM but only the mixed disulfide the initial formation of a glutathionyl conjugate, followed by
conjugates were formed in the presence of 6-chloropyridazine- thiol-disulfide exchange with another GSH molecule to re-
3-thiol (CPT), 2,5-dimethylfuran-3-thiol, and 3-nitropyridine-2- lease the AM. Furthermore, the stable mixed disulfide con-
thiol (NPT). The mass spectrometry (MS) and MS2 spectra of jugates identified in this study provide a platform to quantitatively
the conjugates of these thiol compounds confirmed the pres- generate the therapeutic AM without the need for P450-
ence of a mixed disulfide bond linkage between the AM and the mediated bioactivation. This property can be further ex-
thiol reductants. Kinetic studies revealed that the mixed plored to overcome the interindividual variability in clopidogrel
disulfide conjugates were capable of exchanging thiols with therapy.
CYP2C19*2 mutant gene accounts for only 12% of the
variations in response (Shuldiner et al., 2009). Other factors
are likely involved but have not been identified.
Introduction
Clopidogrel (Bristol-Myers Squibb, New York, NY), a
thienopyridine antiplatelet agent, is widely used to prevent
The variable response to clopidogrel therapy is closely
heart attack and stroke in patients suffering from acute
related to the fact that clopidogrel is a prodrug that requires
cardiovascular syndromes or peripheral vascular diseases,
oxidative bioactivation by cytochromes P450 (P450s) to its
particularly among those who undergo percutaneous coronary
pharmacologically active metabolite (AM) (Savi et al., 2000;
intervention. In spite of widespread use, clopidogrel has
Kazui et al., 2010). It is well documented that P450-mediated
shown significant interindividual variability in its efficacy
(Gurbel and Tantry, 2007; Freedman and Hylek, 2009; Sofi
et al., 2011). Nearly one-third of patients do not respond to
clopidogrel therapy (Mason et al., 2005). With the aim of
overcoming this interindividual variability, several studies
bioactivation involves two consecutive oxidative steps (Dansette
et al., 2010, 2012a); clopidogrel is first monoxygenated to
2-oxoclopidogrel, which is, in turn, oxidized to the AM in the
second step. Although it has been argued that esterase PON1
is responsible for converting 2-oxoclopidogrel to the AM
have been carried out in the past decade attempting to (Bouman et al., 2011), increasing evidence supports the idea
that 2-oxoclopidogrel is converted to the AM via a sulfenic acid
intermediate (Dansette et al., 2009, 2010, 2012a,b), as illus-
trated in Scheme 1. According to Scheme 1, 2-oxoclopidogrel is
first oxidized to a sulfenic acid intermediate by P450s. The
highly unstable sulfenic acid is then rapidly reduced by glu-
tathione (GSH) to form a mixed disulfide conjugate (RS-SG)
that is subsequently reduced by another GSH molecule to
form the AM. This is consistent with the observation that
GSH is required for the formation of the AM in human liver
identify genetic markers that correlate with the lack of
response. It has been shown that clopidogrel is less effective
in patients who carry the mutant CYP2C19*2 gene (Shuldiner
et al., 2009; Dick et al., 2011; Sofi et al., 2011); however, the
This work is in part supported by the National Institutes of Health [Grants
AA020090 and CA016954].
dx.doi.org/10.1124/mol.112.084392.
s
This article has supplemental material available at molpharm.
aspetjournals.org.
ABBREVIATIONS: AM, active metabolite of clopidogrel; AM-MP, derivatized active metabolite of clopidogrel; AUC, area under the curve; BME,
b-mercaptoethanol; CPT, 6-chloropyridazine-3-thiol; DFT, 2,5-dimethylfuran-3-thiol; DTT, dithiothreitol; EIC, extracted ion chromatogram; G6PD,
glucose-6-phosphate dehydrogenase; GSH, glutathione; HLM, human liver microsome; IS, internal standard; Km, Michaelis constant; LC-MS/MS,
liquid chromatography-tandem mass spectrometry; MPB, 2-bromo-39-methoxyacetophenone; MS, mass spectrometry; m/z, mass-to-charge ratio;
NAC, N-acetyl-L-cysteine; NPT, 3-nitropyridine-2-thiol; P450, cytochrome P450; PPP, platelet-poor plasma; PRP, platelet-rich plasma; RS-OH,
sulfenic acid; RS-SG, mixed glutathione disulfide conjugate; RS-SX, mixed disulfide conjugate.
848

Mixed Disulfide Conjugates of Clopidogrel
849
Materials and Methods
Chemicals. (S)-clopidogrel, racemic 2-oxoclopidogrel, and trans-
and cis-clopidogrel-MP were purchased from Toronto Research
Company (Toronto, ON, Canada). GSH, dithiothreitol (DTT), g-L-
glutamyl-L-cysteine, Cys-Gly, L-cysteine, b-mercaptoethanol (BME),
N-acetyl-L-cysteine (NAC), cysteamine hydrochloride, 2,5-dimethyl-
furan-3-thiol, 6-chloropyridazine-3-thiol, 3-nitropyridine-2-thiol, and
2-bromo-39-methoxyacetophenone (MPB) were purchased from Sigma-
Aldrich (St. Louis, MO). Pooled HLMs and cytosol were purchased from
XenoTech (Lenexa, KS).
Determination of the Rate for the Formation of the AM by
HLMs in the Presence of Various Thiol Reductants. To ex-
amine the effects of various thiol reductants on the formation of the
AM, we determined the rates at which the AM was produced. Pro-
duction of the AM was performed in 0.1 ml of 50 mM potassium
phosphate (KPi) buffer (pH 7.4) containing 0.2 mg/ml HLM, 0.1 mM
2-oxoclopidogrel, the NADPH-regenerating system, and 1 mM of each
of the thiol reductants except that 0.3 mM CPT, DFT, or NPT were
used. The reaction was initiated by the addition of 5 units of glucose-6-
phosphate dehydrogenase (G6PD) and incubated at 37°C for 20
minutes. The AM was then derivatized with 4 mM MPB at room
temperature for 10 minutes, followed by acidification with acetic acid
to 3% (v/v). For quantification, 50 pmoles of (S)-clopidogrel was added
into each reaction mixture as internal standard (IS). The derivatized
AM (AM-MP) was quantitated using liquid chromatography-tandem
mass spectrometry (LC-MS/MS).
The mass spectrometry (MS) analyses of the reaction mixture were
performed on an ion-trap mass spectrometer (LCQ DecaXP; Thermo
Fisher Scientific, Waltham, MA) as reported previously (Zhang et al.,
2012). In brief, the analytes were separated on a reverse phase C18
column (Gemini 2Â100 mm, 3 mm; Phenomenex, Torrance, CA) using
a binary mobile phase at a flow rate of 0.2 ml/min. The temperature
of the C18 column was maintained at 40°C using a column heater
(Restek Corporation, Lancaster, PA). The mass spectrometer was
operated in positive electrospray ionization mode with the following
settings: heated capillary temperature, 200°C; spray voltage, 14.5 kV;
sheath gas flow, 60 (arbitrary units); auxiliary gas, 20 (arbitrary
units). The AM-MP and IS were fragmented in the MS through
collision-induced dissociation at 35% energy level. Transitions from
m/z (mass-to-charge ratio) 504 → m/z 354 for the AM-MP and from
m/z 322 → m/z 212 for the IS were used to quantitate the amount of
the AM-MP based on a calibration curve consisting of various
concentrations of cis-clopidogrel-MP.
Analyses of the Mixed Disulfide Conjugates of Clopidogrel
Using LC-MS/MS. The mixed disulfide conjugates were produced by
HLMs for both structural and semiquantitative analyses. Metabolism
of 2-oxoclopidogrel was performed in 0.2 ml of 50 mM KPi buffer (pH
7.4) as previously described except that the concentration of HLMs
was increased to 1 mg/ml. The reaction was incubated at 37°C for 30
minutes and then quenched by the addition of 0.1 ml of 10% of acetic
acid in acetonitrile. The quenched samples were centrifuged at
13,000g for 10 minutes to remove the HLMs. Aliquots of 50 ml
supernatant were loaded on a mass spectrometer to analyze both
the active and conjugate metabolites.
The MS analyses were performed as previously described except
that the MS detector was operated in the dependent scan mode. The
precursor ions were scanned from m/z 300–700, whereas the MS2
spectra were obtained from m/z 100 to 700 for the four most abundant
ions. For semiquantitative analysis, 50 pmoles of the IS was spiked
into each sample that had been quenched. The relative amounts of
the AM and respective conjugates were calculated as the AUC
(area under the curve) ratios of the metabolites to that of the IS.
Determination of the Kinetics for the Conversion of the
Mixed Disulfide Conjugates to the AM. To examine the reactivity
of the mixed disulfide conjugates, we determined the kinetics for the
reduction of the mixed disulfide conjugates by GSH. The mixed
disulfide conjugates were generated in 1 ml of 50 mM KPi buffer (pH
Scheme 1. Bioactivation of 2-oxoclopidogrel to the active metabolite of
clopidogrel as proposed by Dansette et al. (2012a). 2-oxo, 2-oxoclopidogrel.
microsomes (HLMs) (Kazui et al., 2010). It is widely accepted
that the AM is responsible for inhibition of platelet aggrega-
tion through covalent modification of platelet P2Y12 receptor
(Algaier et al., 2008; Ding et al., 2003). The anti-platelet
activity of the mixed disulfide conjugate RS-SG remains
untested.
We have previously reported that metabolism of 2-oxoclopidogrel
in the presence of N-acetyl-L-cysteine (NAC) and L-cysteine
led to the formation of both the AM and mixed disulfide
conjugates (Zhang et al., 2012). In addition, we demonstrated
that the mixed disulfide conjugates of NAC and L-cysteine
exchange thiols with GSH and that the equilibrium between
the AM, the AM conjugate and GSH is governed by their redox
potentials. The redox potential of the sulfenic acid interme-
diate is likely to be high because it is a reactive oxidant. In
principle, if the AM is produced according to Scheme 1, the
metabolism of 2-oxoclopidogrel by P450s should generate only
the mixed disulfide conjugate in the presence of thiol re-
ductants that have redox potentials between that of the
sulfenic acid and the mixed disulfide conjugate. Therefore, if
appropriate thiol reductants are used, we hypothesize that
only the mixed disulfide conjugates should be produced as
terminal products. By investigating several thiol reductants
with varying redox potentials, not only can we test the validity
of the mechanism shown in Scheme 1, but we can also form
stable mixed disulfide conjugates to evaluate their antiplate-
let activities and develop more efficacious therapeutic agents
that do not require bioactivation by polymorphic P450s.
Here we investigated the formation, reactivity, and antiplate-
let activity of several mixed disulfide conjugates of clopidogrel.
Our results show that the production of the AM by HLMs is
greatly affected by the thiol reductants present in metabolic
reactions. Both the AM and the respective mixed disulfide
conjugates were observed in the presence of 7 thiol reductants
tested. Interestingly, only mixed disulfide conjugates were
observed in the presence of 6-chloropyridazine-3-thiol (CPT),
2,5-dimethylfuran-3-thiol (DFT), and 3-nitropyridine-2-thiol
(NPT). These 3 conjugates were readily reduced by GSH to
release the AM. Furthermore, the GSH-treated conjugates
exhibited potent inhibition of platelet aggregation induced by
an ADP agonist.

850
Zhang et al.
7.4) containing 1 mg/ml HLM, 0.1 mM 2-oxoclopidogrel, and 0.3 or
1 mM thiol reductants. The reaction was incubated at 37°C for 50
The procedures used to determine ex vivo antiplatelet activity were
similar to those as reported by Abell and Liu (2011) and are in
minutes after initiated by the addition of G6PD. The reaction mixture accordance with the guidelines of the University of Michigan
was then centrifuged at 13,000g to remove the HLMs, the superna- University Committee on the Use and Care of Animals (National
tant was loaded to a preconditioned SPE cartridge (C18, 100 mg/1 ml;
Agilent Technologies, Santa Clara, CA), and the mixed disulfide Unit for Laboratory Animal Medicine provided all veterinary care.
conjugate was eluted with 2 ml of methanol. The eluent was then Male New Zealand white rabbits (2.2–2.9 kg) were used as blood
dried using a Speedvac concentrator, and the dried sample was stored donors. Whole blood was drawn from a central ear artery into a plastic
Institutes of Health publication No. 86-23). The University of Michigan
at 280°C until use. Prior to the kinetic measurements, the dried
samples were first re-dissolved in 0.5 ml of 50 mM KPi buffer (pH 7.4)
syringe containing 3.7% sodium citrate as the anticoagulant (1:10
volume ratio of citrate to blood). A whole blood cell count was de-
and then equilibrated at 37°C for 5 minutes. Small aliquots (1–5 ml) of termined with a Medonic CA620 hematology analyzer (Clinical Diag-
stock GSH and cytosol (when present) solutions were added to the
conjugate samples at 1 mM and 0.2 mg/ml, respectively, to initiate the
nostic Solutions, Inc., Plantation, FL). Platelet-rich plasma (PRP), the
supernatant present after centrifugation of whole blood at 100g for 10
thiol-disulfide exchange reaction. At designated times, an aliquot of minute, was diluted with PPP to achieve
a platelet count of
50 ml of the reaction mixture was withdrawn and mixed with 25 ml
of 10% acetic acid in acetonitrile to terminate the thiol-disulfide
approximately 300,000/ml. PPP was prepared by centrifuging the
remaining blood at 1,500g for 10 minutes and discarding the bottom
exchange reaction. The t50 sample was prepared immediately prior cellular layer. The diluted PRP was divided into 0.5-ml samples,
to the addition of GSH. The amounts of the mixed disulfide conjugates centrifuged again at 170g for 10 minutes, and the resulting
and the AM were analyzed using LC-MS/MS as previously described. supernatant was discarded. The platelet pellets were resuspended
in PPP containing the various chemical inhibitors (prepared as
described previously) and incubated with gentle shaking at 37°C for
60 minutes to modify the P2Y12 receptor. Ex vivo platelet aggregation
was assessed by established nephelometric methods with the use of
a 4-channel aggregometer (BioData PAP-4; BioData Corp., Horsham,
PA) by recording the increase in light transmission through a stirred
suspension of PRP maintained at 37°C. Platelet aggregation was
induced with ADP (10 mM). A subaggregatory concentration of
epinephrine (550 nM) was used to prime the platelets before addition
of the agonist.
Formation of the Active Metabolite H4 from the Mixed
Disulfide Conjugates of Clopidogrel. Since the antiplatelet
activity of the AM is closely related to its stereochemistry, we
investigated the formation of the active metabolite H4 from three
mixed disulfide conjugates of CPT, DFT, and NPT because of their
relatively high redox potentials. Due to lack of genuine standards for
the stereoisomers of the AM, we first treated the mixed disulfide
conjugates with DTT to release the AM and then derivatized the AM
with MPB so that we were able to compare the AM-MP derivatives
with trans- and cis-clopidogrel-MP standards. The mixed disulfide
conjugates of CPT, NPT, and DFT were prepared as described in the
preceding section. To release the AM, the conjugate (0.2 ml) was
treated with 2 mM DTT at 37°C for 20 minutes. The reaction mixture
was then mixed with 0.1 ml acetonitrile and derivatized with 4 mM
MPB at room temperature for 10 minutes. The derivatization reaction
was then terminated by the addition of 10 ml acetic acid. The
diastereomers of the derivatized AM, AM-MP, were separated on a
reverse-phase C18 column (50 Â 2.1 mm, 5 mm Kinetex; Phenomenex)
with a binary mobile phase consisting of 0.1% formic acid in water
(solvent A) and 0.1% formic acid in acetonitrile (solvent B). The
gradient was as follows: 35% B for 2 minutes, linearly increasing to 40%
B by 7 minutes and to 90% B by 17 minutes, and then held at 90% B
for 1 minute. The flow rate was 0.3 ml/min. MS analyses of the AM-MP
were performed as described in the quantitation of the AM-MP.
Antiplatelet Activity of the Mixed Disulfide Conjugates of
Clopidogrel. To generate sufficient quantities of the mixed disulfide
conjugates, the metabolism of 2-oxoclopidogrel was performed in 2-ml
reaction mixtures containing 1 mg/ml HLM, 0.1 mM 2-oxoclopidogrel,
the NADPH-regenerating system, and 0.3 mM CPT or NPT or 1 mM
GSH. The reaction was initiated by the addition of 10 units G6PD and
incubated at 37°C for 50 minutes. Two control samples were prepared
in parallel. One control sample did not contain any G6PD (2G6PD),
which was designed to examine whether 2-oxoclopidogrel and other
components present in the HLMs contributed to antiplatelet ac-
tivities. The other control (2SH; minus thiol) did not contain any thiol
reductant, enabling us to examine whether any metabolites other
than the AM and the conjugate interfered with the antiplatelet
activity assay. After an incubation of 50 minutes, the reaction mix-
tures were centrifuged at 13,000g to remove the HLMs. The super-
natants were loaded onto SPE C18 cartridges to enrich the mixed
disulfide conjugates. After extensive washing with water to remove
salts and other water-soluble metabolites, the conjugate samples
were eluted with 2 ml of methanol. The methanolic fractions were
dried using a Speedvac concentrator (Thermo Fischer Scientific), and
the dried samples were then resuspended in 1 ml of platelet-poor
plasma (PPP). Prior to the antiplatelet activity assays, the resus-
pended conjugates were divided into two equal volumes (0.5 ml each),
one of which was treated with 1 mM GSH at 37°C for 30 minutes to
generate the AM. Both samples were then placed on ice until use.
Results
Effects of Thiol Reductants on the Formation of the
Active Metabolite of Clopidogrel. To examine the effects
of thiol reductants, we determined the steady-state rates for
the formation of the AM in the presence of various thiol
reductants. The concentrations of the thiol reductants present
in the metabolic reactions were 1 mM except for CPT, DPT
and NPT. Instead, the concentrations of these three thiol
Fig. 1. Effects of thiol reductants on the formation of the AM of
clopidogrel. The AM was produced in 0.1 ml of 50 mM KPi buffer (pH
7.4) containing 0.2 mg/ml HLM, 0.1 mM 2-oxoclopidogrel, the NADPH-
regenerating system, and the thiol reductants. The concentrations of the
thiol reductants were 1 mM except for CPT, DFT, and NPT, which were
0.3 mM each. The reaction was initiated by the addition of 5 units of G6PD
and incubated at 37°C for 20 minutes. The active metabolite was then
quantitated as the MP derivative as described in Materials and Methods.
The reported rates were averaged over three separate measurements.
Abbreviations for the thiol compounds are provided in Table 1. CG,
L-cysteine-L-glyceine; CYA, Cysteamine; GC, g-L-glutamyl-L-cysteine.

Mixed Disulfide Conjugates of Clopidogrel
851
TABLE 1
formed in the presence of 1 mM NAC. The rate is only ∼7% of
that observed in the presence of 1 mM GSH. No AM was
observed in the presence of CPT, DFT, or NPT. Overall, the
rates for the formation of the AM decrease in the order of
GSH . L-cysteine . Cys-Gly . g-L-glutamyl-L-cysteine . cyste-
amine . BME . NAC . CPT or DFT or NPT. This wide range
of rates underscores the critical role of thiol reductants in the
formation of the AM.
Parent ions and retention times observed for the mixed disulfide
conjugates of clopidogrel in liquid chromatography tandem mass
spectrometry analysis
MH+
Theoreticalb
(m/z)
MH+
RTa
Thiol Compounds
Abbreviation
(m/z)
(min)
Glutathione
L-cysteine
GSH
CYS
CG
661.05
5.47
661.02
475.06
532.10
604.12
431.09
432.07
517.09
500.03
475.05c 5.60c
L-cysteine-L-glyceine
g-L-glutamyl-L-cysteine
Cysteamine
b-mercaptoethanol
N-acetyl-L-cysteine
6-chloropyridazine-
3-thiol
2,5-dimethylfuran-
3-thiol
3-nitropyridine-2-thiol
532.09
604.09
431.05
432.06
517.11
499.99 11.97
482.08 15.80
510.08 12.82
5.44
5.71
6.33
9.65
6.85
Analyses of the Mixed Disulfide Conjugates of Clo-
pidogrel. As shown, formation of the AM was greatly af-
fected by the thiol reductants present. In this experiment, we
examined the effects of the thiol reductants on the formation
of the mixed disulfide conjugates. This is particularly im-
portant to understand the cause for the lack of any AM formed
in the presence of CPT, DFT, and NPT. In marked contrast to
what was observed for the AM, mixed disulfide conjugates
were formed in the presence of all the thiol reductants
examined. The m/z for the parent ions MH1 and the retention
times of these mixed disulfide conjugates are summarized
in Table 1, and the extracted ion chromatograms (EICs) of
four selected mixed disulfide conjugates are presented in
Fig. 2. The parent ions MH1 observed are in excellent
agreement with the theoretical values for these conjugates.
In the presence of BME, four conjugate peaks were ob-
served at m/z 432, eluting from 8.9 to 9.72 minutes (Fig.
2A). These four AM peaks are likely due to the formation of
multiple stereoisomers of clopidogrel, as reported pre-
viously (Pereillo et al., 2002). Two major conjugate peaks
were observed in the presence of CPT and NPT (Fig. 2, C
and D, respectively). However, in the presence of DFT, one
GC
CYA
BME
NAC
CPT
DFT
NPT
482.09
510.06
MH+, parent ions; RT, retention times.
a
Retention time for the most intense peak.
Exact masses calculated from molecular formula.
Data from Zhang et al. (2012).
b
c
reductants were 0.3 mM because of their low Km values
(explained below). As shown in Fig. 1, the AM is formed in the
presence of all but three thiol reductants. The highest rate
for the formation of the AM was observed in the presence of
GSH, an endogenous reductant in the human body. Specifi-
cally, in the presence of 1 mM GSH, the AM is produced at
a rate of 167 pmol AM/min/mg HLM. Likewise, L-cysteine is
∼84% as active as GSH in producing the AM. As observed
previously (Zhang et al., 2012), only a low level of the AM was
Fig. 2. EIC of representative mixed disulfide conjugates of clopidogrel. The mixed disulfide conjugates were produced in 0.2 ml of 50 mM KPi buffer (pH
7.4) containing 1 mg/ml HLMs, 0.1 mM 2-oxoclopidogrel, various thiol reductants, and the NADPH-regenerating system at 37°C for 30 minutes. MS
analyses were performed as described in Materials and Methods. (A) EIC for the BME conjugate at m/z 432.06; (B) EIC for the DFT conjugate at m/z
482.08; (C) EIC for the CPT conjugate at m/z 499.99; (D) EIC for the NPT conjugate at m/z 510.08.

852
Zhang et al.
of GSH greatly favors the formation of the AM. Although both
the AM and the mixed disulfide conjugate were formed in the
presence of BME, it is clear that formation of the conjugate is
favored over the AM. In spite of lack of formation of the AM,
the mixed disulfide conjugates of CPT, DFT, and NPT were
formed in significant quantities. Due to lack of genuine
standards of these conjugates, we were unable to quantitate
the absolute amounts of these conjugates. Caution should be
exercised in comparing the absolute amounts of the con-
jugates based on the AUC ratios because these conjugates
may respond differently to the MS detector.
To determine the chemical structure of these conjugates,
the MS and MS2 spectra were obtained. The MS spectra of all
10 conjugates showed the major MH1 peaks at expected m/z
ratios as summarized in Table 1, along with a pair of 35Cl/37Cl
isotope peaks that are characteristic of the presence of one Cl
atom in clopidogrel. The only exception to this is the CPT
conjugate that contains two chlorine atoms. Its MS and MS2
spectra are shown in Fig. 4. This conjugate was observed at
m/z 499.99, which is within an experimental error of 80 ppm
of the expected m/z value of 500.03. In addition, a strong
isotope peak was also observed at 501.94 with ∼75% of the
intensity of the base peak, indicative of the presence of two
chlorine atoms in this conjugate. The MS2 of the parent ion at
m/z 499.99 showed the formation of multiple daughter ions.
The predominant daughter ion was observed at m/z 353.99,
with other minor ones at m/z 465.90, 211.93, and 183.34. This
fragmentation pattern, in addition to the presence of the
35Cl/37Cl isotope peaks, is consistent with the chemical
structure of the conjugate possessing a mixed disulfide bond
shown in Fig. 4D. The predominant daughter ion m/z 353.99
is assigned to the larger fragment cleaved at the mixed
disulfide bond, as we reported previously for the conjugates of
GSH, NAC, and L-cysteine with clopidogrel (Zhang et al.,
2012). The daughter ions at m/z 212 and 183 are also
characteristic of clopidogrel (Dansette et al., 2012a; Pereillo
et al., 2002) . The MS2 spectrum of the isotope peak at m/z
501.94 provides further evidence for this assignment. As
Fig. 3. Relative amounts of the AM and thiol conjugates of clopidogrel
produced by HLMs. The AM and conjugates were produced in 0.2 ml of
50 mM KPi (pH 7.4) as described in Fig. 2. For these analyses, 50 pmol of
(S)-clopidogrel was spiked into each sample as the IS. Both the AM and
the thiol conjugates were analyzed using LC-MS/MS in the dependent
scan mode as described in Materials and Methods. Open bar represents
AUC ratio of m/z 356 (AM) to m/z 322 (IS); solid bar represents AUC
ratios of respective conjugate to IS. CG, L-cysteine-L-glyceine; CYA, Cys-
teamine; GC, g-L-glutamyl-L-cysteine.
predominant conjugate peak with m/z 482 was observed at
15.8 minutes (Fig. 2B). The Km values for the formation of
the CPT, NPT, and DFT conjugates were determined to be
23, 51, and 30 mM, respectively (unpublished data), which
is significantly lower than a Km of 300 mM for GSH that we
previously reported (Zhang et al., 2012).
Integration of the AUC for each EIC of the conjugates gave
the relative amount of the mixed disulfide conjugates pro-
duced. As shown in Fig. 3, the relative amounts of the mixed
disulfide conjugates varied substantially from each other.
Only a low level of the glutathionyl conjugate was formed,
indicating that metabolism of 2-oxoclopidogrel in the presence
Fig. 4. MS and MS2 spectra of the mixed
disulfide conjugate of CPT. The conjugate
was produced as described in Fig. 2. The MS
and MS2 spectra were obtained using LC-MS/
MS in the dependent-scan mode as described
in Materials and Methods. (A) MS spectrum
of the CPT conjugate; (B) MS2 spectrum of
the parent ion m/z 499.99 for the CPT
conjugate; (C) MS2 spectrum of the parent
ion m/z 501.94 for the CPT conjugate; (D)
Fragmentation pattern of the CPT conjugate
deduced from the MS2 spectrum shown in (B).

Mixed Disulfide Conjugates of Clopidogrel
853
shown in Fig. 4C, a pair of daughter ions, instead of single the kinetic data to a monoexponential function gave first-
ions, were observed two mass units apart, which supports the order rate constants of 0.07, 0.79, 0.43, 1.65, and 0.13
presence of two chlorine atoms. The MS2 spectra for the rest of minute21 for the losses of the mixed disulfide conjugates of
the conjugates exhibited very similar fragmentation patterns, BME, CPT, NAC, DFT, and NPT, respectively. Since these
with the characteristic daughter ions at m/z 354 (unpub- kinetics were determined under pseudo first-order conditions
lished data).
with excess of GSH (1 mM), these rate constants are equiv-
Kinetics for the Reduction of the Mixed Disulfide alent to the second-order rate constants of 1.2, 13, 7.2, 28, and
Conjugates of Clopidogrel by GSH. The kinetics for the 2.2 M21s21, respectively. They also demonstrated the variable
thiol-disulfide exchange reaction between the various mixed reactivity of these conjugates toward GSH. Clearly, the DFT
disulfide conjugates and GSH were determined, and the and CPT conjugates are 10- to 20-fold more reactive than the
results are shown in Fig. 5. Incubation of the conjugates with BME conjugate, respectively. It appears that ∼50% of the
GSH and cytosol led to time-dependent decreases in the BME conjugate still remained even after an incubation of 40
amounts of the mixed disulfide conjugates (Fig. 5A). Fitting minutes, indicating that the thiol-disulfide exchange for this
conjugate had reached equilibrium.
To examine the effect of cytosol and to monitor the
conjugates and the AM simultaneously, we determined the
kinetics for both the formation of the AM and the reduction of
the CPT conjugate in the presence and absence of cytosol. As
shown in Fig. 5B, the decrease in the amount of the CPT
conjugate occurred with concomitant increase in the amount
of the AM with almost identical rate constants. In the
presence of cytosol, the rate constants for the reduction of
the conjugate and for the formation of the AM are 0.73 and
0.50 minute21, respectively. In the absence of cytosol, the
reduction of the CPT conjugate is approximately one-half as
fast, with a rate constant of 0.39 minute21 for the reduction of
the conjugate and 0.35 minute21 for the formation of the AM.
This indicates that cytosol accelerates the reduction of the
thiol-disulfide exchange reaction, as observed previously
(Hagihara et al., 2011, 2012). We confirmed this observation
by use of boiled cytosol, as shown in Supplemental Fig. 1.
Formation of the Active Metabolite H4 from the
Mixed Disulfide Conjugates of cCopidogrel. As shown
in Scheme 1, the active metabolite contains two chiral centers
(C7 and C4) and one double bond (C3-C16). Therefore,
metabolism of racemic 2-oxoclopidogrel could potentially
produce up to eight stereoisomers. However, only four of the
diastereomers, termed H1, H2, H3, and H4, can be separated
on conventional reverse phase C18 columns, whereas the
other four stereoisomers co-elute as enantiomers. It has been
established that H4 is responsible for the antiplatelet activity
in humans, having an S configuration at carbon 7 and a Z (or
cis) configuration at C3–C16 double bond (Savi et al., 2000;
Pereillo et al., 2002; Tuffal et al., 2011). To evaluate the
therapeutic potential of these conjugates, we examined
whether the H4 is formed in the mixed disulfide conjugates;
the results are presented in Fig. 6.
The EIC of m/z 504 for the mixed disulfide conjugate of CPT
exhibited two peaks eluting at 9.4 and 10.1 minutes (Fig. 6C).
Likewise, the EIC of the mixed disulfide conjugate of NPT was
Fig. 5. Kinetics for the reduction of the mixed disulfide conjugates of
almost identical to that of the CPT conjugate, with two peaks
observed at 9.4 and 10.2 minutes (Fig. 6D). The two peaks
observed at 9.4 and 10.2 minutes in the CPT and NPT
conjugates were also observed for the DFT conjugate (Fig.
6E). In addition, an extra peak was observed at 8.0 minutes, and
it appears that a shoulder was also observed at 9.7 minutes.
To determine whether H4 was formed during the conver-
sion of the mixed disulfide conjugates to the AM, we compared
the EICs of the three mixed disulfide conjugates with those of
clopidogrel-MP standards and the AM-MP obtained in the
presence of ascorbic acid and GSH. As shown in Fig. 6A, the
EIC of the trans-clopidogrel-MP exhibited two peaks, at 8.0
clopidogrel by GSH. The mixed disulfide conjugates were prepared from
the reaction mixtures containing 1 mg/ml HLM, 0.1 mM 2-oxoclopidogrel,
the NADPH-regenerating system, and various thiol reductants. The
mixture was purified using SPE C18 cartridges, as described in Materials
and Methods. The purified conjugates were then mixed with 1 mM GSH
and 0.2 mg/ml cytosol (when present). The conjugate remaining and the
AM formed were analyzed using LC-MS/MS. (A) Reduction of the
conjugates of BME (s), CPT (j), NAC (.), DFT (m), and NPT (⬜) by
1 mM GSH in the presence of 0.2 mg/ml cytosol. (B) Reduction of the CPT
conjugate by 1 mM GSH in the presence and absence of 0.2 mg/ml cytosol.
Open circle, formation of AM in the absence of cytosol; open triangle,
formation of the AM in the presence of cytosol; filled circle, reduction of
the conjugate in the absence of cytosol; filled triangle, reduction of
the conjugate in the presence of cytosol. The solid and dashed lines
are nonlinear curve fittings to a single exponential function.

854
Zhang et al.
and 9.7 minutes (dashed line), whereas the EIC of the of H1–H4 (Tuffal et al., 2011), we can conclude that the peaks
cis-clopidogrel-MP exhibited one major peak at 10.2 minutes observed at 8.0, 9.4, 9.7, and 10.2 minutes correspond to H1,
(solid line). The two peaks observed with the trans-clopidogrel- H3, H2 and H4, respectively. It appears that H2 and H3 are
MP are indicative of two pairs of diastereomers representing not completely separated under the chromatographic con-
H1 and H2 (Pereillo et al., 2002; Tuffal et al., 2011). It should ditions we used. Nonetheless, this does not affect our con-
be pointed out that the cis-clopidogrel-MP standard available clusion that the mixed disulfide conjugates of CPT, NPT, and
from the Toronto Research Company contained only one pair DFT are capable of producing the active metabolite H4 in
of the diastereomers (J. Bradley, personal communications); the presence of thiol reductants.
consequently, only one peak was observed at 10.2 minutes.
Antiplatelet Activity of the Mixed Disulfide Conju-
However, it is unclear whether this peak represents H3 or H4. gates of CPT and NPT. As a proof of concept, we elected
To further assist peak assignment, we obtained the EICs in to examine the antiplatelet activities of two of the mixed
the presence of GSH and ascorbic acid. As shown in Fig. 6B disulfide conjugates, the CPT and NPT conjugates, for several
(solid line), the AM-MP obtained in the presence of ascorbic reasons. First, both conjugates can be generated without the
acid exhibited only two peaks, at 9.5 and 10.2 minutes. The formation of any AM, which eliminates any interference from
AM-MP obtained in the presence of GSH exhibited extra the AM during the antiplatelet activity assays. Second, both
peaks in addition to the two observed at 9.5 and 10.2 minutes conjugates exchange thiols with GSH at relatively fast rates,
(dashed line, Fig. 6B). Specifically, a new peak was observed which prevents potential decay of the AM. Third, reduction of
at 8.0 minutes, and a shoulder was observed at 9.7 minutes. It the two conjugates produces the H4 isomer that is known to be
is known that the metabolism of clopidogrel by HLMs in the responsible for the antiplatelet activity in humans. The
presence of GSH produces all four diastereomers (H1–H4), results for the ex vivo antiplatelet activity assays are shown
whereas only H3 and H4 are produced in the presence of in Fig. 7. The percentage of aggregation was normalized to
ascorbic acid (Pereillo et al., 2002; Dansette et al., 2012b; that of PRP to compensate for any variations due to environ-
Zhang et al., 2012). Therefore, it is clear that the peaks at mental factors, such as blood sources and PRP preparations.
9.4 and 10.2 minutes are representative of H3 and H4, while
As shown, the three control samples showed no inhibition
the peaks at 8.0 and 9.7 minutes are representative of H1 and of the platelet aggregation. The first control showed that
H2. On the basis of the order of elution of the MP derivatives free GSH has no effect on platelet aggregation at 1 mM
concentration (GSH, Fig. 7), and the second control showed
that nonmetabolite components present in the reaction
mixtures, such as 2-oxoclopidogrel and related impurities,
did not inhibit platelet aggregation (2G6PD, Fig. 7). It is
Fig. 7. Inhibition of platelet aggregation by the mixed disulfide conjugates
of clopidogrel. The mixed disulfide conjugates were prepared and purified
from the reaction mixtures in the presence of 0.3 mM CPT and NPT, along
with three control samples containing either 1 mM GSH, no thiol reductant,
Fig. 6. Extracted ion chromatograms observed at m/z 504 showing the
formation of the active metabolite H4 from the mixed disulfide conjugates
of clopidogrel. The mixed disulfide conjugates were produced in the HLMs
and purified with SPE cartridges as described in Fig. 5. Prior to MS
analyses, the mixed disulfide conjugates were treated with DTT to release
the AM that was then subsequently derivatized with MPB. The AM-MP
derivatives were analyzed using LC-MS/MS as described in Materials and
Methods. (A) trans- (dashed line) and cis-clopidogrel-MP (solid line)
standards; (B) the AM-MP obtained in the presence of 1 mM GSH (dashed
line) and 1 mM ascorbic acid (solid line); (C) the AM-MP obtained from the
CPT conjugate; (D) the AM-MP obtained from the NPT conjugate; (E) the
AM-MP obtained from the DFT conjugate. The amplitude was multiplied
by two.
or no G6PD. All samples were resuspended in 0.5 ml PPP, some of which
were treated to 1 mM GSH to release the AM. Platelet aggregation was
initiated by the addition of 10 mM ADP and recorded with an aggregometer.
The percentage of aggregation was normalized to that of PRP and averaged
over four separate measurements. For details, see Materials and Methods.
CPT, metabolites produced in the presence of 0.3 mM CPT; CPT+GSH,
metabolites produced in the presence of 0.3 mM CPT and then treated with
1 mM GSH; 2G6PD, metabolites produced in the absence of G6PD; AM,
1 mM GSH in PRP; GSH, metabolites produced in the presence of 1 mM
GSH; NPT, metabolites produced in the presence of 0.3 mM NPT; NPT+
GSH, metabolites produced in the presence of 0.3 mM NPT and then treated
with 1 mM GSH; PRP, untreated platelet-rich plasma; 2SH, metabolites
produced in the absence of any thiol reductants.

Mixed Disulfide Conjugates of Clopidogrel
855
known that clopidogrel may decompose to byproducts via
nonenzymatic oxidation (Mohan et al., 2008; Fayed et al.,
2009). These byproducts do not seem to have any inhibitory
effects on platelet aggregation. In the third control, we
demonstrated that the metabolites from the reaction mixture,
in the absence of any thiol reductants, have no effects on
platelet aggregation either. However, we did observe ∼60%
inhibition of platelet aggregation in the sample prepared from
the reaction mixture containing GSH (AM, Fig. 7). This is
expected, since metabolism of 2-oxoclopidogrel in the presence
of 1 mM GSH generates the AM as shown in Fig. 1. Incubation
of PRP with the CPT and NPT conjugates did not inhibit
platelet aggregation, indicating that the conjugates them-
selves have no antiplatelet activity (CPT and NPT, Fig. 7). In
marked contrast, incubation of PRP with the CPT and NPT
conjugates that had been treated with 1 mM GSH signifi-
cantly inhibited platelet aggregation by ∼50 and 70%,
respectively. This inhibitory activity most likely arises from
the AM released from the conjugates by GSH. These results
demonstrate that the conjugates of clopidogrel have no anti-
platelet activity and also confirmed that the AM is solely
responsible for the inhibition of platelet aggregation. Further-
more, they demonstrate that it is possible to deliver the AM
without the need for bioactivation by polymorphic P450s. Of
note, the variations in the percentage of aggregation observed
in the GSH, CPT1GSH, and NPT1GSH samples were most
likely due to variations in the concentrations of the AM. It was
estimated that the concentrations of the AM in these samples
were in the range of 1–4 mM (unpublished data).
Scheme 2. Schematic illustration for the formation of the mixed disulfide
conjugate and the AM based on redox potentials. The question marks
indicate unknown redox potentials. CYA, Cysteamine; CYS, L-cysteine.
the formation of the AM, whereas the thiol reductants with
low redox potentials favor the formation of the AM. Of the
10 thiol compounds we examined, only the redox potentials of
four are known: 20.24 V for GSH, 20.22 V for L-cysteine,
20.20 V for cysteamine, and 20.19 V for BME (Lukesh et al.,
2012). It is likely that RS-OH possesses the highest redox
potential because it is a highly reactive oxidant. Although the
redox potentials of CPT, DFT, and NPT are unknown, it is
reasonable to assume that their redox potentials are rela-
tively high. This is because the sulfhydryl groups of CPT,
DFT, and NPT are bonded to conjugated p systems containing
Discussion
We have demonstrated that thiol reductants greatly affect electrophilic atoms, such as a chlorine atom in CPT, an oxygen
the formation of the AM of clopidogrel during the metabolism atom in DFT, and a nitro group in NPT. The chemical
by HLMs. A wide range of rates were observed for the structures of CPT, DFT, and NPT are shown in Fig. 8. The
formation of the AM, depending on the thiol reductants lone pair of electrons of these sulfhydryl groups is dispropor-
present in the metabolic reactions. At one end of the spectrum, tionally delocalized away from the sulfur atom. This is
the AM was produced at a rate of 167 pmol AM/min/mg HLM expected to reduce electron density on the sulfur atoms,
in the presence of GSH, whereas no AM was produced in the consequently leading to an increase in the redox potentials.
presence of CPT, DFT, or NPT (see Fig. 1). Furthermore, we The low redox potential of GSH contributes to the facile
observed the formation of mixed disulfide conjugates in the reduction of RS-OH to form the glutathionyl conjugate RS-SG
presence of all 10 thiol compounds. Analyses of the MS and and subsequent reduction of RS-SG to form the AM. This is
MS2 spectra of the conjugates revealed that, in all cases, the consistent with the highest rate observed for the formation of
AM is bonded to the thiol reductant via a disulfide bond.
the AM in the presence of GSH. The observation that only RS-
The variable rates of the formation for the AM in the SX is formed in the presence of CPT, DFT, and NPT indicates
presence of various thiol reductants can be interpreted on the that reduction of RS-SX by these reductants is prohibited
basis of redox potentials, as illustrated in Scheme 2. As because the redox potential of RS-SX is less than that of CPT,
shown, formation of the mixed disulfide conjugate (RS-SX) DFT, or NPT. The variable rates observed for the formation of
depends on the difference in the redox potentials between the the AM provide direct evidence for the participation of the
sulfenic acid (RS-OH) and the thiol reductant used, whereas thiol reductants and are consistent with the proposed
formation of the AM depends on the difference in redox mechanism shown in Scheme 1.
potentials between the RS-SX and thiol reductant. In other
The relatively high redox potentials of the mixed disulfide
words, the thiol reductants with high redox potentials disfavor conjugates of CPT, DFT, and NPT are further illustrated in the
Fig. 8. Chemical structures and generic names of three
thiol compounds: 6-chloropyridazine-3-thiol (CPT), 2,5-
dimethylfuran-3-thiol (DFT) and 3-nitropyridine-2-thiol
(NPT).

856
Zhang et al.
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Authorship Contributions
Participated in research design: Zhang, Lauver.
Conducted experiments: Zhang, Lauver.
Contributed new reagents or analytic tools: Zhang, Lauver.
Performed data analysis: Zhang, Lauver.
Wrote or contributed to the writing of the manuscript: Zhang,
Lauver, Lucchesi, Hollenberg.
Address correspondence to: Dr. Haoming Zhang, Department of Pharma-
cology, University of Michigan, 1150 West Medical Center Drive, 2220A MSRB
III, Ann Arbor, MI 48109-5632. E-mail: haom@umich.edu