Identification of the significant involvement and mechanistic role of CYP3A4/5 in clopidogrel bioactivation

Article abstract of DOI:10.1021/ml3002067

The clinical response to the antiplatelet prodrug clopidogrel is associated with high intersubject variability and a certain level of therapeutic resistance. Previous studies have suggested that genetic polymorphism of CYP2C19 might be one determinant of clopidogrel efficacy and led to the CYP2C19 genotype-tailored antithrombotic therapy. However, evidence against the role of CYP2C19 from multiple studies implied the involvement of other factors. Here, we report that prodrug activation of the thiophene motif in clopidogrel is attenuated by heavy metabolic attrition of the piperidine motif. CYP3A4/5 was identified to be the enzyme metabolizing the piperidine motif. Inhibiting CYP3A4/5-mediated attrition was shown to potentiate active metabolite formation, which was found to be catalyzed by multiple CYP enzymes. Identifying the significant involvement of CYP3A4/5 and characterizing its mechanistic role in clopidogrel bioactivation might assist future pharmacogenomic studies in exploring the full mechanism underlying clopidogrel efficacy.

Full text of DOI:10.1021/ml3002067

Letter
pubs.acs.org/acsmedchemlett
Identification of the Significant Involvement and Mechanistic Role of
CYP3A4/5 in Clopidogrel Bioactivation
Yaoqiu Zhu*,† and Jiang Zhou‡
†MetabQuest Research Laboratory, 202 Chengfu Road, Beijing 100871, People's Republic of China
‡Analytical Instrumentation Center, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, People's
Republic of China
S
* Supporting Information
ABSTRACT: The clinical response to the antiplatelet prodrug clopidogrel is associated with high intersubject variability and a
certain level of therapeutic resistance. Previous studies have suggested that genetic polymorphism of CYP2C19 might be one
determinant of clopidogrel efficacy and led to the CYP2C19 genotype-tailored antithrombotic therapy. However, evidence
against the role of CYP2C19 from multiple studies implied the involvement of other factors. Here, we report that prodrug
activation of the thiophene motif in clopidogrel is attenuated by heavy metabolic attrition of the piperidine motif. CYP3A4/5 was
identified to be the enzyme metabolizing the piperidine motif. Inhibiting CYP3A4/5-mediated attrition was shown to potentiate
active metabolite formation, which was found to be catalyzed by multiple CYP enzymes. Identifying the significant involvement
of CYP3A4/5 and characterizing its mechanistic role in clopidogrel bioactivation might assist future pharmacogenomic studies in
exploring the full mechanism underlying clopidogrel efficacy.
KEYWORDS: Clopidogrel resistance, prodrug attrition, CYP3A4/5, active metabolite potentiation, piperidine metabolism
he antiplatelet prodrug clopidogrel is bioactivated by
hepatic cytochrome P450 (CYP) enzymes (Scheme 1).
underlying the observed clinical uncertainties of clopidogrel
therapy has been increasing. Previous in vitro studies showed
that CYP2C19 is one of only three CYP enzymes capable of
catalyzing active metabolite formation.6 Additionally, clinical
research has reported correlation between the CYP2C19 low
metabolizer genotype and the decreased antiplatelet re-
sponse.7,8 On the basis of these findings, CYP2C19 genetic
polymorphism is considered to be a key determinant of
clopidogrel efficacy.9
T
Scheme 1. Reported Metabolic Activation and Deactivation
Pathways of Clopidogrel
At the same time, there is also a large body of evidence that
does not support the role of CYP2C19 and the CYP2C19-
tailored clopidogrel therapy.7,10−13 Ample research results have
been reported to support the existence of unidentified
determinants, including genetic factors, for clopidogrel
efficacy.7,14−16 Recently proposed involvement of paraoxo-
nase-1 in clopidogrel bioactivation17 was debated and ruled
out.18−21 To reconcile the current controversy of clinical
variability and guide personalized antithrombotic prodrug
The active metabolite binds with a subtype of adenosine
diphosphate (ADP) receptor P2Y12 and inhibits ADP-induced
platelet aggregation. Clinical antithrombotic responses to
clopidogrel are associated with high intersubject variabilities,
with 20−40% of patients being classified as nonresponders,
poor responders, or resistant to clopidogrel.1−5 Recently,
research aiming to rationalize the biological mechanisms
Received: July 25, 2012
Accepted: September 9, 2012
Published: September 9, 2012
© 2012 American Chemical Society
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ACS Medicinal Chemistry Letters
Letter
therapy, characterization of the genetic factors deciding
clopidogrel efficacy is highly desired.
MS/MS-based fragmentation analyses and online H-D
exchange experiments were conducted to aid the structural
elucidation as illustrated in Figure 2. The metabolite profiles
were summarized in Table 1. To further pinpoint the location
of metabolism (thiophene or piperidine), the 2- and 3-positions
of the thiophene ring were labeled with deuterium, and the
metabolite profiles of the deuterated clopidogrels were
examined for deuterium loss or retention. On the basis of the
results of the above analyses, the metabolite structures were
tentatively proposed (structural elucidation is further discussed
in the Supporting Information). As shown in Scheme 2, we
propose that the piperidine ring of clopidogrel undergoes
extensive dehydrogenation or hydroxylation to form M3−M7.
Similar dehydrogenation and hydroxylation metabolites have
been reported for ticlopidine, a close analogue of clopidog-
rel.27,28 The proposed piperidine hydroxylation metabolite has
also been reported as a synthetic intermediate of clopidogrel.29
Metabolism of the thiophene moiety of clopidogrel leads to not
only the bioactivation metabolite M2 but also M8, which
contains a 3- or 2-hydroxy-2,3-dihydrothiophene substructure.
As the result of metabolic attrition, formation of the
bioactivation metabolite M2 accounts for only 13% of the
CYP-catalyzed conversion.
Metabolism studies were then conducted in cDNA-expressed
CYP supersomes to phenotype the enzymes that catalyze the
metabolite formation. CYP3A4/5 was identified to be the major
enzyme catalyzing the piperidine oxidation, while all of the
tested CYP enzymes catalyze the thiophene oxidation forming
both M2 and M8 (Figure 3 and Figure S3 in the Supporting
Information). It has long been believed that the first step of the
metabolic activation of clopidogrel, thiophene 2-oxidation
leading to M2 formation, is only catalyzed by CYP2C19,
CYP1A2, or CYP2B6, as concluded from previous in vitro
phenotyping studies in cDNA-expressed CYP supersomes.6
However, the reported experiment contained multiple potential
sources that might cause false negative results showing only
contributions from the three CYP enzymes. The high CYP
isozyme compatibility with clopidogrel revealed here is
consistent with its relatively small size and high lipophilicity.
Among all of the CYP isozymes, CYP3A4/5 has the most
versatile active site to accommodate different substrate binding
conformations,30 which explains why piperidine oxidation is
only found with CYP3A4/5. It is also important to note that
significant attritional metabolism of the thiophene ring is found
to accompany 2-oxidation, which further attenuates the desired
bioactivation pathway. With more CYP enzymes having been
identified to catalyze the formation of M2, the contribution
from each individual CYP enzyme is likely to be less significant.
CYP3A4/5 has been considered as only a negligible player in
clopidogrel bioactivation since its direct contribution to the
active metabolite formation was found to be insignificant as
compared to other CYP enzymes.12,31,32 However, the more
detailed characterization of clopidogrel biotransformation
uncovers the significant involvement of CYP3A4/5 in affecting
the active metabolite formation. As compared to the piperidine
oxidation, the thiophene oxidation catalyzed by CYP3A4/5 is
minor. Therefore, the major role of CYP3A4/5 is catalyzing
prodrug attrition rather than prodrug activation. With a major
portion of the absorbed clopidogrel undergoing esterase-
catalyzed hydrolysis, the remaining prodrug is partitioned
between two groups of CYP enzymes: CYP3A4/5 catalyzes the
deactivating piperidine oxidation, while the other CYP enzymes
catalyze the thiophene metabolism including the desired 2-
Although the prodrug bioactivation pathway (Scheme 1),
conversion from clopidogrel (M0) to 2-oxo-clopidogrel (M2)
and to the active thiol metabolite (M13), has been extensively
investigated,6,22−24 the complete biotransformation of clopi-
dogrel is devoid of full characterization. The methyl ester of
clopidogrel undergoes esterase-catalyzed hydrolysis in the
intestine and liver to form the inactive carboxylic acid
metabolite (M1), which is the major known route of
clopidogrel deactivation and accounts for about 85% of the
prodrug loss.25 It has been hypothesized that the remaining
clopidogrel goes to hepatic enzymes, mainly CYPs, for the
desired metabolic activation. However, the high variability in
active metabolite plasma exposures and clinical responses
suggests that the active metabolite formation might be
accompanied by heavy attrition involving highly variable
metabolizing enzymes. To test this hypothesis, we first
conducted in vitro metabolic studies of clopidogrel in pooled
human liver microsomes (HLM).
In HLM incubation without the CYP cofactor β-
nicotinamide adenine dinucleotide 2′-phosphate (reduced,
NADPH), a significant portion of clopidogrel (M0) undergoes
methyl ester hydrolysis. Because the aim of this study is to
characterize the potential nonhydrolyzing deactivation path-
ways, the selective esterase inhibitor, potassium fluoride, was
included in the incubation.26 The extracted ion chromatograms
of HLM incubation samples are shown in Figure 1. M2 (+O, M
Figure 1. Extracted ion chromatogram of clopidogrel incubation with
HLM. (A) Sample without NADPH and (B) sample with NADPH.
Nonlabeled peaks are extracted isotopic signals of other metabolite or
background signals.
+ H+ = 338) possesses an identical LC retention time and MS/
MS spectrum to the reference compound 2-oxo-clopidogrel.
The structure of M2 was confirmed to be 2-oxo-clopidogrel by
an online H-D exchange experiment in which it undergoes just
one H-D exchange (Table 1). In addition to M2 formation,
metabolite profiling identified eight other NADPH-dependent
metabolites, all showing the 35Cl/37Cl isotopic pattern and
parent-related fragmentation patterns (Table 1). Two isomeric
metabolites of M2 were detected: M3 and M4. Three
dehydrogenation metabolites were detected: M5, M6, and
M7. M8 showed a mass shift of “+18 Da”, which was confirmed
to be “+2H + O” by high-resolution MS analysis. M9 and M10
were proposed to be demethylated products of M5 and M7,
respectively.
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ACS Medicinal Chemistry Letters
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Table 1. Metabolite Profiles of Fragmentation Analysis, Online H-D Exchange, and Deuterated Analogue Experiment Results
b
fragmentation
B
D loss
a
m/z
Δm/z
biotransformation
no. of H-D
A
C
2-d
3-d
fragment of metabolism
M0
M2
M3
M4
M5
M6
M7
M8
M9
M10
322
338
338
338
320
320
318
340
306
304
\
\
1
1
2
2
0
1
0
2
1
+
+
+
+
\
+
+
+
+
+
+
+
+
+
+
\
+16
+16
+16
−2
−2
−4
+18
−16
−18
+O
\
y
y
thiophene
piperidine
piperidine
piperidine
piperidine
piperidine
thiophene
piperidine
piperidine
+O
+
+
+
+
\
n
n
+O
n
n
−2H
−2H
−4H
+O + 2H
−2H−CH2
−4H−CH2
n
n
\
ND
n
ND
n
\
+
\
\
n
n
+
+
n
ND
ND
1
\
n
a
b
Number of H-D exchange of the charged molecular ion. Metabolite of the deuterated clopidogrel shows loss of deuterium; ND, metabolite was
not detected; fragmentation patterns are illustrated in Figure 2.
Figure 2. Fragmentation analysis and online H-D exchange experi-
ment result of M0.
Scheme 2. Proposed CYP-Catalyzed Metabolic Pathways of
Clopidogrel
Figure 3. CYP phenotyping results of M2 (metabolic activation), M8
(thiophene metabolism), and M5 (piperidine metabolism) formation.
based on drug−drug interaction, food factors, age, etc.35,36 To
confirm that the change in CYP3A4/5 activity can impact the
clopidogrel bioactivation pathway, we assayed 2-oxo-clopidog-
rel (M2) formation in HLM with the selective CYP3A4/5
inhibitor ketoconazole (KET). In HLM with KET, the
formation of M5 (the major piperidine oxidation metabolite)
was found to decrease by 80% as compared to the control
sample, which indicates a suppression of the CYP3A4/5-
mediated piperidine oxidation pathways. On the other hand,
the formation of activation metabolite 2-oxo-clopidogrel (M2)
was found to increase by 70%, and M8 formation also increased
by 70%, which confirms that the thiophene oxidation is
significantly potentiated (Figure 4).
oxidation. The partition ratio between the two enzyme groups
will decide the active metabolite conversion efficiency and
could directly impact the therapeutic effect of clopidogrel.
CYP3A4/5 is the most abundant P450 enzyme in human liver.
It catalyzes the metabolism of about 50% of drugs used in
human therapy and is known to have genetic poly-
morphism.33,34 Its overall activity can also be highly variable
Figure 4. Selective inhibition of CYP3A4/5-catalyzed piperidine
metabolism potentiates thiophene bioactivation.
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ACS Medicinal Chemistry Letters
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Figure 5. Proposed pathways of clopidogrel active metabolite formation with multiple factors that could significantly impact prodrug efficacy.
The new mechanistic insight on CYP3A4/5 in clopidogrel
bioactivation suggests that an alternative therapeutic strategy to
circumvent clopidogrel resistance can be formulated. We
propose here that the coadministration of clopidogrel with a
selective CYP3A4/5 inhibitor or substrate should be able to
divert more clopidogrel from the attrition pathways to the
activation pathway, which might lead to the same effect as the
loading dose increases and potentiates the active metabolite
plasma exposure. An analogous regimen is the anti-HIV therapy
of Kaletra (lopinavir/ritonavir),37 which has been used for a
decade. In fact, patients might have already benefited from this
strategy by taking one alternative therapy recommended by
FDA: the concurrent use of clopidogrel with cilostazol, another
antiplatelet drug that inhibits type 3 phosphodiesterase.38
Because cilostazol is mainly metabolized by CYP3A4,39 its
adjunctive use might decrease the CYP3A4-catalyzed clopidog-
rel attrition and divert more prodrug to the activation pathway.
This hypothesis is supported by recent clinical research results
showing that adding cilostazol to clopidogrel therapy improves
platelet inhibition in patients with chronic kidney disease
(CKD).40 CKD is a known factor of poor clopidogrel
responsiveness; the clopidogrel resistance (expressed as P2Y12
inhibition) was significantly alleviated following the concurrent
treatment of clopidogrel (75 mg/day) with cilostazol (200 mg/
day), while the clopidogrel doubling treatment (150 mg/day)
without cilostazol did not show improvement.
could impact the already-thin active metabolite formation and
its plasma exposure. To better understand the biological
mechanism underlying clopidogrel efficacy, the following
mechanistic details remain to be further investigated (Figure
5): (1) Does the partition between the thiophene metabolism
pathways involve reductase? (2) Is the hydrolysis of the
thiolactone S-oxide intermediate (M11) in competition with
other nucleophiles like glutathione leading to further attrition?
(3) Does the active metabolite undergo isomerase-catalyzed
tautomerization to its inactive diastereomers? (4) Is the
disposition of the active metabolite regulated by transporters?
(5) In addition to forming a covalent bond with P2Y12 for the
desired efficacy, does the active metabolite form adducts with
off-target cellular and plasma proteins leading to diminished
half-life or plasma exposure? (6) The active metabolite can be
scavenged by glutathione through disulfide bond formation; is
the equilibrium dependent on individual oxidative stress status?
One or more of the above factors might correlate with certain
genotype or disease status and lead to elusive clinical outcomes.
The low bioactivation efficiency due to high prodrug attrition
might exemplify the impact of the factors on active metabolite
plasma exposure.
In summary, we studied the previously unknown metabolic
pathways of clopidogrel. Identification of the significant
involvement and mechanistic role of CYP3A4/5 in active
metabolite formation might help to formulate new therapeutic
strategies to battle clopidogrel resistance. Our research results
also suggest that factors that impact the partition ratio between
the piperidine and the thiophene metabolic pathways might be
determinants of clopidogrel efficacy. The more detailed
characterization of clopidogrel biotransformation presented
The active metabolite (M13) formation from clopidogrel
(M0) has been traditionally referred to as “a two-step process”
(Scheme 1), which might be an understatement of its
mechanistic complexity. Several aspects of this process remain
unknown, and the potential involvement of regulatory factors
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bioactivation of clopidogrel to its pharmacologically active metabolite.
Drug Metab. Dispos. 2010, 38, 92−99.
here might also facilitate future clinical exploration of using the
piperidine and thiophene metabolites as biomarkers to ascertain
an individual's status and delineate multicomponent effects on
clopidogrel efficacy.
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ASSOCIATED CONTENT
* Supporting Information
■
S
(8) Brandt, J. T.; Close, S. L.; Iturria, S. J.; Payne, C. D.; Farid, N. A.;
Ernest, C. S., 2nd; Lachno, D. R.; Salazar, D.; Winters, K. J. Common
polymorphisms of CYP2C19 and CYP2C9 affect the pharmacokinetic
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Thromb. Haemostasis 2007, 5, 2429−2436.
Experimental procedures, detailed results, supplementary
discussion, supporting schemes and figures, and MS and
NMR spectra. This material is available free of charge via the
Internet at http://pubs.acs.org.
(9) U.S. Food and Drug Administration. FDA Drug Safety
Communication: Reduced effectiveness of Plavix (clopidogrel) in
patients who are poor metabolizers of the drug. http://www.fda.gov/
D r u g s / D r u g S a f e t y /
PostmarketDrugSafetyInformationforPatientsandProviders/
ucm203888.htm, 2010.
AUTHOR INFORMATION
Corresponding Author
*Tel: +86-10-6275-7536 or 1-847-530-7238. Fax: +86-10-6275-
2632. E-mail: yaoqiu.zhu@metabquest.com.
■
Author Contributions
́
(10) Pare, G.; Mehta, S. R.; Yusuf, S; Anand, S. S.; Connolly, S. J.;
Y.Z. made the initial discovery, designed the project, conducted
experiments, analyzed the results, and wrote the manuscript.
J.Z. conducted the LC-HRMS experiment, assisted with online
H-D exchange LC-MS experiments, and contributed to
discussion on writing the manuscript.
Hirsh, J.; Simonsen, K.; Bhatt, D. L.; Fox, K. A. A.; Eikelboom, J. W.
Effects of CYP2C19 genotype on outcomes of clopidogrel treatment.
N. Engl. J. Med. 2010, 363, 1704−1714.
(11) Bauer, T.; Bouman, H. J.; van Werkum, J. W.; Ford, N. F.; ten
Berg, J. M.; Taubert, D. Impact of CYP2C19 variant genotypes on
clinical efficacy of antiplatelet treatment with clopidogrel: Systematic
review and meta-analysis. Br. Med. J. 2011, 343, d4588.
Notes
The authors declare no competing financial interest.
(12) Yasar, U.; Bennet, A. M.; Eliasson, E.; Lundgren, S.; Wiman, B.;
De Faire, U.; Rane, A. Allelic variants of cytochromes P450 2C modify
the risk for acute myocardial infarction. Pharmacogenetics 2003, 13,
715−720.
(13) Barker, C. M.; Murray, S. S.; Teirstein, P. S.; Kandzari, D. E.;
Topol, E. J.; Price, M. J. Pilot study of the antiplatelet effect of
increased clopidogrel maintenance dosing and its relationship to
CYP2C19 genotype in patients with high on-treatment reactivity. J.
Am. Coll. Cardiol. Interv. 2010, 3, 1001−1007.
(14) Hulot, J. S.; Collet, J. P.; Silvain, J.; Pena, A.; Bellemain-Appaix,
A.; Barthelemy, O.; Cayla, G.; Beygui, F.; Montalescot, G.
́ ́
ACKNOWLEDGMENTS
■
We are grateful to Dr. Richard B. Silverman (Northwestern
University) for critical reading of the manuscript and Dr.
Hongyu Zhao (Abbott Laboratories) for careful revision of the
manuscript. We thank Dr. Hua Li (Institute of Pharmacology
and Toxicology, Academy of Military Medical Sciences, China)
for providing help with the supersome experiment and Xiu
Zhang (Analytic Instrumentation Center, Peking University)
for helping with NMR analysis. We also thank Pam Beck
(Northwestern University) for her assistance.
Cardiovascular risk in clopidogrel-treated patients according to
cytochrome P450 2C19*2 loss-of-function allele or proton pump
inhibitor coadministration: a systematic meta-analysis. J. Am. Coll.
Cardiol. 2010, 56, 134−143.
ABBREVIATIONS
■
(15) Holmes, D. R., Jr.; Dehmer, G. J.; Kaul, S.; Leifer, D.; O'Gara, P.
T.; Stein, C. M. ACCF/AHA Clopidogrel clinical alert: Approaches to
the FDA boxed warning”: A report of the American College of
Cardiology Foundation Task Force on Clinical Expert Consensus
Documents and the American Heart Association. Circulation 2010,
122, 537−557.
CYP, cytochrome P450; HLM, human liver microsomes;
NADPH, β-nicotinamide adenine dinucleotide 2′-phosphate
(reduced); KET, ketoconazole; CKD, chronic kidney disease
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