`
`DOI: 10.1111/jth.12829
`
`O R I G I N A L A R T I C L E
`
`Active metabolite concentration of clopidogrel in patients
`taking different doses of aspirin: results of the interaction trial
`
`Y . L I A N G , * J . H I R S H , † J . I . W E I T Z , † ‡ D . S L O A N E , § P . G A O , § G . P A R E , ‡ § J . Z H U * and
`J . W . E I K E L B O O M † ‡ §
`*Department of Emergency Medicine, Cardiovascular Institute and Fuwai Hospital, Chinese Academy of Medical Sciences and Peking Union
`
`Medical College, National Center for Cardiovascular Disease, Beijing, China; †Department of Medicine, McMaster University; ‡Thrombosis
`
`and Atherosclerosis Research Institute (TaARI), Hamilton Health Sciences and McMaster University; and §Population Health Research Institute
`
`(PHRI), Hamilton Health Sciences and McMaster University, Hamilton, ON, Canada
`
`To cite this article: Liang Y, Hirsh J, Weitz JI, Sloane D, Gao P, Pare G, Zhu J, Eikelboom JW. Active metabolite concentration of clopidogrel in
`patients taking different doses of aspirin: results of the interaction trial. J Thromb Haemost 2015; 13: 347–52.
`
`Summary. Background: The CURRENT-OASIS-7
`and
`PLATO trials suggest that the benefit of clopidogrel is
`influenced by the dose of aspirin. Objective: To explore a
`potential pharmacokinetic interaction between aspirin and
`clopidogrel, and determinants of
`clopidogrel active
`metabolite (AM) levels. Methods: In part 1, using a 2 9 2
`factorial design, we randomized patients to clopidogrel
`600 mg loading dose (LD) followed by 150 mg day1 for
`and 75 mg day1
`6 days
`thereafter, or
`clopidogrel
`300 mg LD followed by 75 mg day1 thereafter, and
`compared aspirin at 325 mg or 81 mg day1. In part 2,
`patients were given a 600-mg clopidogrel LD, and were
`randomly allocated to aspirin 325 mg or 81 mg day1.
`We combine the data from the two parts. Blood samples
`were collected 1 h after administration of the study drug.
`Results: We
`randomized
`302
`patients
`(mean
`age
`60.4 9.9 years). Clopidogrel AM levels were similar in
`patients randomized to aspirin 325 or 81 mg (geometric
`mean, 12.70 ng mL1; 95% CI, 10.96–14.72 ng mL1;
`and geometric mean, 12.55 ng mL1; 95% CI, 10.80–
`14.58 ng mL1; P = 0.91). Blood levels of clopidogrel
`were lower in CYP2C19*2 loss-of-function (LOF) carriers
`compared with non-carriers (10.72 ng mL1; 95% CI,
`8.83–13.01 ng mL1; and 15.21 ng mL1; 95% CI, 13.30–
`17.40 ng mL1, respectively; P = 0.003) whereas levels in
`gain of function carriers and non-carriers were similar
`
`Correspondence: Yan Liang, Department of Emergency Medicine,
`Cardiovascular Institute and Fuwai Hospital, Chinese Academy of
`Medical Sciences and Peking Union Medical College, National Cen-
`ter for Cardiovascular Disease, 167 Beilishi Road, Xicheng District,
`Beijing 100037, China.
`Tel.: +86 10 88398987; fax: +86 10 88364591.
`E-mail: fwliangyan@sina.cn
`
`Received 15 September 2014
`Manuscript handled by: P. de Moerloose
`Final decision: F. R. Rosendaal, 13 December 2014
`
`© 2014 International Society on Thrombosis and Haemostasis
`
`(13.31 ng mL1; 95% CI, 11.53–15.35 ng mL1; and
`14.07 ng mL1; 95% CI, 11.74–16.87 ng mL1, respec-
`tively; P = 0.4). Independent baseline predictors of clopi-
`dogrel AM levels were LOF genotype, body mass index,
`diabetes, proton pump inhibitor use and creatinine clear-
`ance, but accounted for only 20% of the variability in
`levels. Conclusion: Aspirin dose does not predict clopido-
`grel AM levels 1 h post-LD. Most of the variability in
`clopidogrel AM levels is not explained by patient charac-
`teristics or CYP2C19 metabolizer status.
`
`Keywords: aspirin; clopidogrel; drug interactions; genetic
`polymorphisms; pharmacokinetics.
`
`Introduction
`
`Low-dose aspirin (75–100 mg daily) is effective for the
`secondary prevention of major cardiovascular events and
`there is no definitive evidence that higher doses provide
`additional benefit. However,
`the results of
`subgroup
`analyses from two recently completed randomized con-
`trolled trials suggest that patients treated with clopidogrel
`have better outcomes if they receive a higher dose of aspi-
`rin compared with a lower dose. Thus, among invasively-
`managed acute
`coronary
`syndrome
`(ACS) patients
`enrolled in the CURRENT OASIS-7 trial, double-dose
`compared with standard-dose clopidogrel reduced the risk
`of cardiovascular (CV) death, myocardial infarction (MI)
`or stroke in those who received aspirin 300–325 mg daily
`but not in those who received aspirin 81–100 mg daily
`(P value for interaction = 0.043) [1]. In ACS patients
`enrolled in the PLATO trial, clopidogrel and ticagrelor
`were similarly effective for prevention of CV death, MI
`or stroke among those treated in North America, most of
`whom received aspirin 325 mg daily, but clopidogrel was
`inferior to ticagrelor among patients treated outside of
`
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`348 Y. Liang et al
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`received aspirin
`North America where most patients
`81 mg daily (P value for interaction = 0.045) [2].
`It
`is uncertain whether the apparent superiority of
`clopidogrel in the presence of higher doses of aspirin is
`real or caused by a play of chance. If real, it could be
`caused by a pharmacokinetic or pharmacodynamic inter-
`action between aspirin dose and clopidogrel, leading to
`an increased clopidogrel response. A preliminary report
`suggested that aspirin up-regulates cytochrome P (CYP)
`450
`enzymes
`(CYP2C19
`and CYP3A), which are
`involved in the biotransformation of clopidogrel to its
`active metabolite (AM) [3], thereby potentially leading
`to higher blood levels of
`the AM of clopidogrel
`in
`patients receiving a higher dose of aspirin. To further
`explore this issue we measured clopidogrel AM levels in
`patients randomly allocated aspirin 81 or 325 mg. Our
`primary objective was to determine whether a higher
`dose compared with low-dose aspirin increases clopido-
`grel AM levels in the blood. We also explored indepen-
`dent
`baseline
`determinants
`of
`clopidogrel
`active
`metabolite levels.
`
`Methods
`
`The interaction study was approved by the Research Eth-
`ics Board of Hamilton Health Sciences and all subjects
`provided written informed consent and were recruited
`from the cardiac rehabilitation group of the General Hos-
`pital, McMaster University. The two parts of the study
`are
`separately registered at ClinicalTrials.gov (NCT
`01102439, NCT 01341964).
`
`Eligibility
`
`Patients were eligible for inclusion if they had coronary
`artery disease (CAD) and were receiving long-term dual
`antiplatelet
`therapy with aspirin and clopidogrel. We
`excluded patients with severe liver or renal dysfunction,
`those taking drugs that might interfere with laboratory
`measurement of the antiplatelet effect of aspirin or clopi-
`dogrel (e.g. non-steroidal anti-inflammatory drugs) and
`those deemed to be at high risk of bleeding.
`
`Study design
`
`Interaction part 1 was a two-by-two factorial randomized
`controlled trial based on the design of the CURRENT
`OASIS-7 trial
`[1]. In the first randomization, eligible
`patients were randomly allocated to receive clopidogrel
`600 mg loading dose followed by 150 mg daily for 6 days
`and 75 mg daily thereafter or clopidogrel 300 mg loading
`dose followed by 75 mg daily. In the second randomiza-
`tion, patients were allocated to receive aspirin 325 or
`81 mg daily. The primary outcome was clopidogrel AM
`levels 1 h after administration of study drugs on days 1, 7
`and 14.
`
`The design of Interaction part 2 was modified based on
`the results of part 1, which demonstrated numerically
`higher blood concentrations of the clopidogrel AM 1 h
`after a 600-mg loading dose of clopidogrel
`in patients
`who received 325 mg aspirin compared with 81 mg aspi-
`rin. In part 2, eligible patients were given a 600-mg load-
`ing dose of clopidogrel and were randomly allocated to
`receive 325 or 81 mg aspirin. Blood samples were col-
`lected for measurement of clopidogrel AM levels 1 h after
`administration of study drugs on day 1.
`Both trials were open-labeled and the computer-based
`randomization codes were created by the pharmacy of the
`General Hospital using sealed opaque envelopes.
`
`Sample collection
`
`After collecting 4 mL of venous blood into EDTA-con-
`taining vacutainer tubes, 25 lL of a 500-mM solution of
`30-methoxyphenacyl bromide
`(MPBr)
`in acetonitrile
`(Sigma–Aldrich, Oakville, ON, Canada) was immediately
`added to stabilize the clopidogrel AM. Samples were then
`subjected to centrifugation at 1300 9 g for 10 min at
`4 °C within 15 min of blood collection, and plasma was
`collected and stored in aliquots at 80 °C until shipped
`to the central laboratory. All samples were packed in dry
`ice in styrofoam shipping containers to ensure that they
`remained frozen for at least 48 h during shipping.
`A blood sample was also collected from all study
`patients for genotyping. Genomic DNA was extracted
`from peripheral-blood leukocytes using standard proce-
`dures (Puregene DNA isolation kit; Merck Eurolab,
`Lyon, France) and stored at 80 °C until analysis.
`
`Laboratory assays
`
`With a structural analog of clopidogrel [4] serving as an
`internal standard, clopidogrel AM concentrations were
`measured using a validated electrospray liquid chromatog-
`raphy-tandem mass spectrometry technique (LC-MS/MS)
`at Sanofi-Aventis laboratories in Paris, France. We report
`results for the H4 isomer because this is a more abundant
`active diastereoisomer [4]. The lower limit of quantification
`is 0.5 ng mL1. Patients with clopidogrel AM levels below
`0.5 ng mL1 were deemed to have a level of 0 ng mL1.
`Genotyping studies were performed at the Thrombosis
`and Atherosclerosis Research Institute in Hamilton using
`TaqManâ Drug Metabolism Genotyping Assays on the
`ViiATM 7 Real-Time PCR System (Life Technologies, Wal-
`tham, MA, USA) to determine the frequency of the com-
`monest single nucleotide polymorphisms associated with
`loss of function (rs4244285 [CYP2C19*2]) and gain of
`function (rs12248560 [CYP2C19*17]) alleles. Results were
`analyzed with ViiATM 7 Software v1.1 (Life Technologies)
`using fluorescence data from pre- and post-PCR readouts.
`Quality control was performed according to the manufac-
`turer’s recommendations.
`
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`Ex. 1043, p. 2 of 6
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`
`
`including AM level, platelet
`laboratory results,
`All
`function and genotype, were detected by the operators
`or technicians, who were blinded to the random alloca-
`tions.
`
`Sample size
`
`We used the results obtained in part 1 of the study, which
`showed numerically higher concentrations of clopidogrel
`AM in patients receiving 600 mg clopidogrel who were
`exposed to higher vs.
`low-dose aspirin (the geometric
`means on day 1 after the loading dose were 14.87 and
`12.26 ng mL1, respectively), to determine that a sample
`size of 292 patients was needed to demonstrate that the
`difference observed was statistically significant with at
`least 80% power (two-sided P < 0.05).
`
`Statistical analyses
`
`We present the results separately for Interaction part 1 and
`then present the combined results for parts 1 and 2 com-
`paring 325 mg of aspirin with 81 mg of aspirin in patients
`who received a 600-mg loading dose of clopidogrel.
`We present continuous data as means and standard
`deviations (SD) if normally distributed or as medians and
`interquartile range (IQR) if not normally distributed, and
`categorical data as frequencies and percentages.
`Blood AM levels were not normally distributed, there-
`fore values were log-transformed before any analysis. We
`explored a possible interaction between aspirin and clopi-
`dogrel in part 1 of the study using the Wald test.
`We performed multiple linear
`regression modeling
`using a backward elimination method to determine inde-
`pendent baseline predictors of blood AM levels. All 19
`baseline variables (shown in Table S1) were included as
`potential
`regression variables and the variables were
`deleted from the model one by one using a cut-off of
`P ≤ 0.1. Model goodness of fit was assessed by the
`R-squared statistic, which measures the percentage of
`explained variation over total variation.
`function
`of
`The
`associations between
`the
`loss
`(LOF) genotype, gain of function (GOF) genotype and
`blood AM levels were presented with the geometric mean
`and 95% confidence intervals (CIs). Comparisons of clop-
`idogrel AM levels between genotype groups are presented
`unadjusted and adjusted for independent baseline predic-
`tors from the above multiple regression model of clopido-
`grel AM levels.
`Analyses were performed with SAS version 9.2 (SAS
`Institute, Cary, NC, USA) on a Unix operating system. A
`two-tailed P < 0.05 was considered statistically significant.
`
`Results
`
`Results of the interaction trial 349
`
`Part 1 results
`
`Baseline characteristics for patients randomized into part
`1 of the Interaction trial were well matched between treat-
`ment groups (Table S2). The geometric means (95% CI)
`of clopidogrel AM concentration in double-dose clopido-
`grel and higher dose aspirin, double-dose clopidogrel and
`low-dose aspirin, standard-dose clopidogrel and higher
`dose aspirin and standard-dose clopidogrel and low-dose
`aspirin groups 1 h after administration of study drugs
`were 14.87 (8.41‒26.31), 12.26 (7.03‒21.38), 6.91 (4.37‒
`10.92) and 7.78 (4.92‒12.29) ng mL1, respectively, on
`day 1. Although clopidogrel AM levels were numerically
`higher in patients receiving double-dose clopidogrel who
`were exposed to higher vs. low-dose aspirin, the differ-
`ences were not statistically significant (P-value for interac-
`tion = 0.54)
`(Table S3). Based on these results we
`evaluated the effect of 325 or 81 mg aspirin on clopido-
`grel AM levels 1 h after administration of a 600-mg load-
`ing dose of clopidogrel in part 2 of the study.
`
`Part 1 and 2 combined results
`
`of
`characteristics
`characteristics Baseline
`Baseline
`patients randomized to receive higher dose compared with
`low-dose aspirin in the two parts of the study are pre-
`sented in Table 1. The mean age was 60.4 (SD 9.9) years
`and 216 (71.5%) were male. Patients randomized to
`receive higher dose compared with low-dose aspirin were
`
`Table 1 Patient baseline characteristics of Interaction Parts 1 and 2
`combined
`
`Age (years),
`mean SD
`Male, n (%)
`Caucasian, n (%)
`BMI (kg m2),
`mean SD
`Hypertension, n (%)
`Diabetes, n (%)
`Dyslipidemia, n (%)
`History of stroke, n (%)
`History of TIA, n (%)
`Current smoking, n (%)
`Platelet count (9109 per L),
`mean SD
`eCrCl (mL min1),
`mean SD
`ALT (mean SD; U L1)
`Total bilirubin
`(mean SD; lmol L1)
`Statin, n (%)
`PPI, n (%)
`
`Higher dose
`ASA, N = 152
`
`Low-dose
`ASA, N = 150
`
`59.3 9.3
`
`61.5 10.4
`
`115 (75.7)
`140 (92.1)
`30.1 5.5
`
`111 (73.0)
`37 (24.3)
`125 (82.2)
`7 (4.6)
`3 (2.0)
`20 (13.2)
`223.6 (57.6)
`
`101 (67.3)
`144 (96.0)
`30.0 6.0
`
`102 (68.0)
`40 (26.7)
`110 (73.3)
`1 (0.7)
`2 (1.3)
`13 (8.7)
`219.4 (52.1)
`
`102.7 (34.1)
`
`98.1 (32.5)
`
`29.5 (11.6)
`8.4 (3.8)
`
`140 (92.1)
`34 (22.4)
`
`27.4 (10.5)
`9.5 (5.5)
`
`138 (92.0)
`39 (26.0)
`
`Part 1 of the interaction study included 82 patients and
`part 2 included 220 patients.
`
`ASA, acetylsalicylic acid; BMI, body mass index; PPI, proton pump
`inhibitor; TIA, transient ischemic attack; eCrCl, estimated creatinine
`clearance; ALT, alanine transaminase.
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`younger (59.3 9.3 and 61.5 10.4 years, respectively,
`P = 0.04) and were more likely to have had prior stroke
`(4.6% and 0.7%, respectively, P = 0.03).
`
`Clopidogrel AM levels Blood samples to measure clopi-
`dogrel AM levels were taken 60.5 2.7 min after the
`loading dose of clopidogrel and were not significantly dif-
`ferent in patients who received higher dose compared
`with low-dose aspirin (geometric mean, 12.70 ng mL1;
`95% CI, 10.96–14.72 ng mL1; and 12.55 ng mL1; 95%
`10.80–14.58 ng mL1,
`CI,
`respectively;
`P = 0.91)
`(Table 2). Clopidogrel AM levels were below the lower
`limit of quantification in six samples and six samples were
`missing labels; thus clopidogrel AM levels were measur-
`able in 290 (96.0%) samples. Results were similar when
`comparing median levels without adjustment or after
`adjustment for baseline characteristics (P = 0.73).
`
`Independent baseline predictors of clopidogrel AM lev-
`els Table 3 demonstrates that independent baseline pre-
`dictors of clopidogrel AM blood concentrations were
`diabetes, body mass index (BMI), proton pump inhibitor
`(PPI) use, estimated creatinine clearance (eCrCl) and
`LOF carrier, but together these factors accounted for
`only 20% of the variance.
`
`Clopidogrel AM levels according to genotype The suc-
`cessful detection rate for the *2 allele was 95.4% (288/
`302) and for the *17 allele it was 93.7% (283/302). The
`prevalence of CYP 2C19 LOF and GOF alleles in all
`patients was 31.9% and 37.1%, respectively, with no sig-
`nificant difference between high and low-dose aspirin
`groups (Table S4). The geometric mean (95% CI) of
`clopidogrel AM levels in LOF carriers was lower than
`(10.72 ng mL1; 95% CI, 8.83–
`that
`in non-carriers
`13.01 ng mL1; and 15.21 ng mL1; 95% CI, 13.30–
`17.40 ng mL1,
`respectively; adjusted P = 0.003) but
`
`Table 2 Blood levels of the clopidogrel active metabolite in Parts 1
`and 2 combined
`
`Higher dose
`ASA, N = 147*
`
`Low-dose
`ASA, N = 143* P value
`
`Median
`(IQ range, ng mL1)
`Geometric mean†
`(95% CI, ng mL1)
`
`14.45
`(6.58–24.20)
`12.70
`(10.96–14.72)
`
`13.80
`(7.62–20.20)
`12.55
`(10.80–14.58)
`
`0.66
`
`0.91
`
`Table 3 Independent baseline predictors* of
`metabolite concentrations†
`
`clopidogrel active
`
`Variable
`
`CYP2C19
`Allele*2
`carrier vs.
`non-carrier
`BMI
`eCrCl
`Diabetes
`PPI use
`
`Parameter
`estimate‡
`
`Lower
`95% CI
`
`Upper
`95% CI P-value
`
`Partial R
`square (%)
`
`0.36621 0.58
`
`0.15
`
`0.0008
`
`3.59
`
`0.03265 0.05
`0.00361 0.01
`0.37454 0.61
`0.30138 0.54
`
`0.01
`0.00
`0.13
`0.06
`
`0.003
`0.05
`0.003
`0.01
`
`3.26
`1.41
`3.44
`2.12
`
`*Model R-square = 20.2%. †Multivariate regression analysis by the
`backward-elimination method for the model selection. There were 19
`baseline variables included as potential regressor variables (all 16
`baseline variables shown in Table 1 plus aspirin group, CYP2C19 *2
`and *17). ‡Concentration values were log-transformed before analy-
`sis. BMI, body mass index; PPI, proton pump inhibitor; eCrCl, esti-
`mated creatinine clearance.
`
`Table 4 Association between CYP2C19 genotype and clopidogrel
`active metabolite concentrations 1 h after a 600-mg loading dose of
`clopidogrel†
`
`Clopidogrel active
`metabolite* Geometric mean
`(95% CI, ng mL1)
`
`Carriers
`
`Non-
`carriers
`
`P value
`(unadjusted)
`
`P value
`(adjusted)
`
`10.72
`(8.83–13.01)
`
`15.21
`(13.30–17.40)
`
`0.004
`
`0.003‡
`
`14.07
`(11.74–16.87)
`
`13.31
`(11.53–15.35)
`
`0.63
`
`0.40§
`
`Loss of
`function
`(*2 allele)
`Gain of
`function
`(*17 allele)
`
`LOF, loss of function, refers to carriage of one or two *2 alleles;
`GOF, gain of function, refers to carriage of one or two *17 alleles.
`*Clopidogrel active metabolite concentrations were log-transformed
`before analysis. †Combined Interaction part 1 and part 2. ‡Adjusted
`for history of stroke, CYP2C19 *17 genotype, body mass index, esti-
`mated creatinine clearance, hypertension, DM and proton pump
`inhibitor use. §Adjusted for history of stroke, CYP2C19 *2 geno-
`type, body mass index, estimated creatinine clearance, hypertension,
`diabetes and proton pump inhibitor use.
`
`there was no difference in levels between GOF carriers
`and non-carriers (Table 4).
`
`Discussion
`
`*5/152 in the higher dose acetylsalicylic acid (ASA) group and 7/150
`in the low-dose ASA group could not have active metabolite (AM)
`levels measured due to being under the lower limit of quantification
`(LLOQ, six samples) or having the label missing (six samples). †The
`results were consistent after being adjusted for independent baseline
`predictors of clopidogrel AM levels (such as the only significant dif-
`ferences being age and stroke, and other determinants including
`genotype, body mass index, estimated creatinine clearance, hyperten-
`sion, diabetes and proton pump inhibitor use), with P = 0.73.
`
`We found no evidence that the aspirin dose affected clop-
`idogrel AM levels. Independent baseline predictors of
`clopidogrel AM concentrations were CYP2C19 genotype,
`BMI, eCrCl, diabetes history and PPI use. Genetic analy-
`ses showed that LOF allele *2 carriers had lower AM
`concentrations than non-carriers, but there was no differ-
`ence in levels between carriers and non-carriers for the
`GOF allele (*17).
`
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`
`The hypothesis that aspirin dose is a determinant of
`clopidogrel response appeared to be supported by the
`results of a small Chinese study by Chen et al. [3] involving
`18 healthy participants for whom the administration of
`50 mg aspirin once-daily for 14 days appeared to be associ-
`ated with up-regulation of CYP2C19 and CYP3A activity
`(P < 0.001 and P < 0.05, respectively). CYP2C19 and
`CYP3A are involved in the conversion of clopidogrel to its
`active metabolite, and at the outset we hypothesized that
`higher doses of aspirin might lead to greater enzyme up-
`regulation and higher blood concentrations of the clopido-
`grel AM, which in turn could explain improved outcomes
`in patients treated with higher doses of aspirin. However,
`our data provide no evidence in support of this hypothesis.
`The intestinal p-glycoprotein transporter is a determi-
`nant of clopidogrel absorption [5] and Jung et al. [6] dem-
`onstrated that aspirin induces expression of the enzyme
`and reduces clopidogrel absorption in rats. More recently,
`Oh and colleagues showed in healthy volunteers that co-
`administration of aspirin 100 mg once-daily increases
`expression of the p-glycoprotein transporter and is associ-
`ated with reduced blood levels of clopidogrel [7]. However,
`they did not show an effect of aspirin co-administration on
`blood levels of the clopidogrel active metabolite and did
`not examine whether the effect of aspirin on p-glycoprotein
`expression was dose dependent.
`Our study was designed to examine whether aspirin
`dose has an effect on clopidogrel pharmacokinetics and
`cannot exclude an interaction between aspirin and clopi-
`dogrel because all patients received aspirin and we did
`not include a placebo group. The lack of evidence of a
`pharmacokinetic interaction between aspirin dose and
`clopidogrel in our study is consistent with the results of a
`study by Lotfi et al. [8], who found no evidence of an
`interaction between the aspirin dose on the risk of stent
`thrombosis in 5187 patients (7604 stents) treated with
`dual antiplatelet therapy. However, our results do not
`definitively exclude the possibility that a higher dose of
`aspirin might improve the efficacy of combined antiplat-
`elet treatment. Aspirin has a dose-dependent effect on
`platelet thromboxane A2 production and serum levels of
`thromboxane B2 have been linked with an increased risk
`of adverse cardiovascular events. On balance, however,
`we believe that the most likely explanation of the CURE
`and PLATO results that raised the possibility of an inter-
`action is a play of chance.
`Previous studies have concluded that genetic polymor-
`phisms in the CYP P450 genes that are involved in the bio-
`transformation of clopidogrel to its AM are an important
`determinant of variable platelet inhibition by clopidogrel
`[9]. Thus, a study of 162 healthy subjects demonstrated that
`the level of the clopidogrel AM in carriers of at least one of
`CYP2C19 reduced-function alleles is 32.4% lower than in
`non-carriers (P < 0.001) [10]. Observational studies have
`suggested that ACS patients treated with clopidogrel who
`are carriers of any LOF CYP 2C19 allele (*2, *3) have a 1.3
`
`© 2014 International Society on Thrombosis and Haemostasis
`
`Results of the interaction trial 351
`
`to 3-fold higher risk of death, MI or stroke and a 2.5 to 5-
`fold higher risk of stent thrombosis compared with non-
`carriers [10–14]. However, all of these studies lacked a con-
`trol group and thus cannot exclude the possibility that the
`reported association between carriage of CYP 2C19 LOF
`alleles and clinical outcome is, at least in part, mediated
`through pathways unrelated to clopidogrel. In contrast to
`the observational studies, in a subgroup analysis of the
`CURE trial, Pare and colleagues demonstrated a consistent
`benefit of clopidogrel over placebo irrespective of the
`CYP2C19 LOF carrier status [15]. Furthermore, Hochhol-
`zer and colleagues reported that the CYP 2C19 LOF alleles
`explain only 5.2% of the observed variation in the platelet-
`inhibition response to clopidogrel and suggested that as yet
`undiscovered genetic polymorphisms and patient factors
`(e.g. age, diabetes and obesity) might be key determinants
`of the variable response to clopidogrel [16]. In the recent
`Pharmacogenomics of Antiplatelet Intervention (PAPI)
`genome-wide association study, the CYP2C19*2 genotype
`accounted for 12% of the variation in the clopidogrel
`response and the addition of age, BMI and lipid levels
`almost doubled the variation in the clopidogrel response
`that could be explained [11]. Our study results are consis-
`tent with the conclusion that polymorphisms involving
`CYP2C19 and clinical factors account for only a small
`fraction of the variable responses to clopidogrel. However,
`emerging evidence indicates that clopidogrel is primarily a
`CYP3A substrate [17–19] and we did not explore the possi-
`ble impact of CYP 3A polymorphisms on clopidogrel
`active metabolite levels.
`An important strength of our study is that we mea-
`sured the concentration of the H4 active metabolite of
`clopidogrel using the most accurate method available.
`The most important limitation is that the study is explor-
`atory and cannot definitively exclude an effect of aspirin
`dose on outcome.
`In summary, our research has demonstrated no evi-
`dence of an interaction between aspirin dose and clopido-
`grel AM concentrations in patients with coronary artery
`disease treated with dual antiplatelet therapy.
`
`Addendum
`
`Y. Liang principal investigator, study design and conduct,
`analysis and interpretation of the data, drafting of first
`version of the manuscript. J. Hirsh and J. I. Weitz study
`design, critical writing and revising the intellectual con-
`tent of the manuscript. D. Sloane critical writing and
`revising the intellectual content of the manuscript. P. Gao
`analysis and interpretation of the data. G. Pare critical
`writing and revising the intellectual content of the manu-
`script. J. Zhu critical writing and revising the intellectual
`content of the manuscript. J. W. Eikelboom study design,
`interpretation of the data, critical writing and revising
`the intellectual content of the manuscript. All authors
`approved the final version of the manuscript.
`
`
`IPR2015-01492
`Panacea Biotec Ltd.
`
`
`
`Ex. 1043, p. 5 of 6
`
`
`
`352 Y. Liang et al
`
`Disclosure of Conflict of Interests
`
`The study was supported by Sanofi-Aventis. J. W. Eikel-
`boom reports grants and honorarium from Astra Zeneca,
`Bayer, Boehringer Ingelheim, Bristol Myers Squibb/Pfizer,
`Daiichi Sankyo, GlaxoSmithKline, Janssen and Sanofi
`Aventis and honorarium from Eli Lilly, outside the sub-
`mitted work.
`
`Supporting Information
`
`Additional Supporting Information may be found in the
`online version of this article:
`
`Table S1. Full linear regression model of baseline factors
`for logH4 in all patients.
`Table S2. Interaction Part 1 baseline characteristics.
`Table S3. Clopidogrel active metabolite concentrations in
`Interaction Part 1* (n = 82).
`Table S4. Distribution of CYP2C19 genotypes according
`to randomized ASA dose groups.
`
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`© 2014 International Society on Thrombosis and Haemostasis
`
`
`IPR2015-01492
`Panacea Biotec Ltd.
`
`
`
`Ex. 1043, p. 6 of 6
`
`
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