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Keywords:

  • clopidogrel;
  • CYP2C19 genotype;
  • time course of platelet aggregation inhibition

Abstract

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. Funding
  7. References
  8. Supporting Information

We evaluated the effect of CYP2C19 genotype over time on the antiplatelet response of clopidogrel in healthy subjects. Seventy subjects enrolled for a pharmacodynamic study and 22 subjects for a pharmacokinetic and pharmacodynamic study took 300 mg clopidogrel on the first day and 75 mg once daily for six consecutive days. The subjects with CYP2C19 poor metabolizers (PM, N = 22) and intermediate metabolizers (IM, N = 37) had significantly delayed time to inhibition of platelet aggregation (IPA) compared with CYP2C19 extensive metabolizers (EM, N = 33) (12 vs. 9 vs. 2 hours as median Tmax, P < .05) after a 300 mg of clopidogrel. During maintenance doses of clopidogrel, IPA values of only CYP2C19 PM subjects were gradually decreased from 30.0 ± 21.9% on day 2 to 23.7 ± 16.6% on day 8 (P > .05 for time effect; P < .05 for time and genotype interaction effect). CYP2C19 PM had decreased Cmax and AUC of thiol metabolite compared with CYP2C19 EM (0.42- and 0.37-fold on day 1, P < .01; 0.39- and 0.34-fold on day 7, P < .01, respectively). Delayed time to reach maximal IPA as well as decreased IPA may influence the increased risk of the acute cardiac events in CYP2C19 PM and IM.

Clopidogrel is one of the most common antiplatelet drugs used to treat and prevent atherothrombotic events.[1] The effects of clopidogrel are mediated by its active thiol metabolite, and clopidogrel is biotransformed into thiol metabolite by the hepatic cytochrome P450 isoforms including CYP2C19, CYP2B6, CYP3A4, and CYP3A5.[2, 3] Recently, PON1 genotype was issued as another genetic biomarker associated with formation of thiol metabolite with serious controversy.[3-7] Additionally, the efflux transporter P-glycoprotein affects clopidogrel bioavailability and active metabolite formation.[8] Thus, polymorphisms leading to functional alterations in genes modulating clopidogrel pharmacokinetics are expected to reduce active metabolite formation and antiplatelet effect leading to poor clinical outcomes.[9, 10] Indeed, carriers of a CYP2C19 loss-of-function (LOF) alleles (*2 and *3 alleles) have a higher rate of subsequent cardiovascular events than noncarriers.[11]

Despite its robust association with poor cardiovascular outcomes of clopidogrel therapy, several questions regarding clinical use of CYP2C19 genotype as a predictive biomarker for personalized antiplatelet therapy remain to be answered. The rapid onset of clopidogrel is generally required to improve early clinical outcomes, particularly during emergencies requiring percutaneous coronary intervention (PCI) for acute myocardial infarction.[12] The onset time for maximal inhibition of platelet aggregation (IPA) is dependent on the dose of clopidogrel. Therefore, CYP2C19 genotypes that influence the plasma concentration of clopidogrel active thiol metabolite are expected to be associated with differential time to maximal IPA after clopidogrel administration.[13] However, there are few reports describing the interaction between CYP2C19 genotypes and time course of antiplatelet aggregation response of clopidogrel.[14]

To address these questions, we evaluated the effects of CYP2C19 genotypes on the time course of platelet aggregation inhibition after clopidogrel administration. In addition, we explored possible associations between clopidogrel pharmacokinetics or pharmacodynamics and genetic variants of PON1, CYP2B6, CYP3A4/5, ABCB1, and P2Y12, which are involved in the formation or action of active clopidogrel metabolites.

Methods

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. Funding
  7. References
  8. Supporting Information

Subjects

The study protocol was approved by the Institutional Review Board of Inje University Busan Paik Hospital, Busan, Korea (IRB approval number: 09–162). All of the subjects provided written informed consent before participating in the study. A total of 92 Korean healthy male subjects enrolled in this study. Inclusion criteria were healthy subjects aged 20–45 years, with a body mass index of 18–25 kg/m2. All subjects were determined to be healthy by medical history, a physical examination, vital signs, and clinical laboratory tests performed within 2 weeks before the start of this study. None of the subjects smoked tobacco or received continuous medication. The mean age of our subjects was 23.2 ± 2.0 years and the mean weight was 69.9 ± 6.9 kg. The subjects were not allowed to take any drugs, use alcohol or caffeine, or drink any grapefruit products for 2 weeks before and during the study period.

Study Design

The trial was an open-label, multiple-dose study. All subjects received oral dose of 300 mg clopidogrel (Plavix®; Sanofi-Aventis Korea, Seoul, Korea) on day 1 and 75 mg clopidogrel on days 2–7. Among total 92 subjects, 70 subjects were available for the pharmacodynamic study and 22 subjects were included in the pharmacokinetic and pharmacodynamic study.

On days 1 and 7, blood samples for pharmacokinetic analyses were collected predosing after fasting overnight and at 0, 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4, 6, 8, 10, 12, 24 hours. The subjects were asked to remain in a seated position for 2 hours after taking clopidogrel on days 1 and 7. They were then allowed to perform their usual daily activities in the clinical trial room; however, strenuous activity and exercise were prohibited. Blood samples were also collected to measure ADP-induced platelet aggregation during treatment on day 1 (before the loading dose and at 2, 4, 6, and 12 hours after the dose) and on days 2, 4, 6, and 7 (before the maintenance dose), and on days 8, 10, 14.

Determination of Clopidogrel and Thiol Metabolite Concentrations

The blood samples were centrifuged and stored at −80°C until assayed. Plasma concentrations of clopidogrel and thiol metabolite were analyzed using a validated liquid chromatography-tandem mass spectrometry method (LC-MS/MS). Briefly, plasma samples (0.15 mL) were precipitated by adding two volumes of acetonitrile containing the internal standard (clopidogrel-d4, 3.3 ng/mL). After centrifugation, the supernatant was taken and 0.1 mL of 2 M dithiothreitol was added. The mixtures were then passed through syringe filter (0.44 μm) and a 5-μL aliquot of this solution was injected into an Agilent 1100 series HPLC system (Agilent, Wilmington, DE) coupled to a Qtrap 5500 triple-quadrupole mass spectrometer (AB Sciex, Foster City, CA) equipped with electrospray ionization. Chromatographic separation of the compounds was achieved using a XTerra MS C18 column (particle size 3.5 µm, 2.1 × 50 mm2, i.d.; Waters, Milford, MA) with a mobile phase consisting of 53% acetonitrile in water containing 0.1% formic acid over 2 min. Flow rate was 0.4 mL/min and the retention times for clopidogrel and thiol metabolite were 1.03 and 0.7 min, respectively.

The mass spectrometer was run in the positive mode and m/z 326.2 [RIGHTWARDS ARROW] 216.0 for clopidogrel-d4, m/z 322.1 [RIGHTWARDS ARROW] 212.0 for clopidogrel and m/z 356.1 [RIGHTWARDS ARROW] 155.3 for thiol metabolite were monitored. Calibration curves in the range of 0.025–20 ng/mL for clopidogrel and 0.5–150 ng/mL for the thiol metabolites were established (r = 0.9985 for clopidogrel, r = 0.9957 for thiol metabolite). The intra-day and inter-day coefficients of variation were less than 15%.

Measurement of Platelet Aggregation

The 5 µM ADP-induced platelet aggregation was measured using a Chrono-log Aggregometer (model 700–4DR; Chrono-log, Havertown, PA) with a turbidimetric method, as reported previously.[15] All processes to measure platelet aggregation in all subjects were conducted by one expert in order to reduce inter-analysts variation. Platelet aggregation was expressed as the maximal percentage change of light transmission from baseline using platelet-poor plasma as a reference. The IPA value was calculated from the observed maximal platelet aggregation (MPA) at each scheduled time-point for each treatment asinline image

Pharmacokinetic and Pharmacodynamic Evaluations

The Phoenix WinNonlin version 6.2 software (Pharsight, Mountain View, CA) was used for the pharmacokinetic and pharmacodynamic analyses. We analyzed the plasma concentrations of clopidogrel and its thiol metabolite by noncompartmental analysis. The maximal concentration (Cmax) was estimated directly from the observed plasma concentration–time data. The area under the plasma concentration–time curve for a dosing interval (AUCτ) was calculated using the linear trapezoidal rule.

Pharmacodynamic parameters were estimated in order to evaluate the IPA. The maximal IPA (Emax) and the time to reach Emax (Tmax) after administration of clopidogrel loading dose were determined from the IPA–time data. IPA at steat state (IPAss) was calculated as average of 4 point IPA value during days 4–8. The area under the effect–time curve from time 0 to observation time (AUECt) was calculated from the IPA value versus time curve using the linear trapezoidal rule.

Genotyping

The genotypes of seven genes in this study were determined by single base extension methods; analytical validation for the Korean population was established previously by the PharmacoGenomics Research Center (PGRC), Inje University College of Medicine, Busan, Korea.[16-18] Genomic DNA was used in genotyping for the presence of the following major Korean alleles: CYP2B6*4 (rs2279343), CYP2B6*6 (rs2279343, rs3745274), and CYP2B6*9 alleles (rs3745274), CY2C9*3 (rs1057910), CYP2C19*2 (rs4244285), CYP2C19*3 (rs4986893), CYP2C19*17 (rs12248560), CYP2C9*3 (rs1057910), P2RY12 -43108T>C (rs6787801), CYP3A5*3 (rs776746), and P2RY12 742T>C (rs2046934), P2RY12 -43108T>C (rs6787801) ABCB1 2677G>A/T (rs2032582), ABCB1 3435C>T (rs1045642), and PON1 Q192R (rs662) using an ABI PRISM® genetic analyzer and its mounted GeneMapper® software. All tested genotypes satisfied Hardy–Weinberg equilibrium and showed no statistical significance for all SNPs tested (P > .05) in this study.

The CYP2C19 alleles were classified into three genotype groups according to the number of LOF alleles: CYP2C19 extensive metabolizer (CYP2C19 EM; CYP2C19*1/*1 and heterozygous for CYP2C19*17 allele), CYP2C19 intermediate metabolizer (CYP2C19 IM; CYP2C19*1/*2 and CYP2C19*1/*3), and CYP2C19 poor metabolizer (CYP2C19 PM; CYP2C19*2/*2, CYP2C19*2/*3, and CYP2C19*3/*3).

Statistical Analysis

Continuous variables were expressed as means ± standard deviation. Comparisons of the pharmacokinetic and pharmacodynamic parameters for the different genotype groups were made using Kruskal–Wallis test after assessing the normal distribution of the data for normality. The genotype effect on the time course of IPA values measured repeatedly from days 2 to 8 was analyzed using repeated-measures analyses of variance (RM ANOVA). SAS® ver. 9.2 (SAS Institute, Cary, NC) software was used for the statistical analysis. P values <.006 after Bonferroni adjustment for testing candidate SNPs and P values <.05 for other statistical analysis were considered to indicate statistical significance.

Results

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. Funding
  7. References
  8. Supporting Information

Effects of Candidate SNPs on Clopidogrel Pharmacokinetics and Pharmacodynamics

The mean AUC for clopidogrel and its active thiol metabolite after administration of a clopidogrel loading dose, baseline aggregation, and IPAss were compared among the different genotype groups (Table 1). Only CYP2C19 genotype influenced the AUC of the thiol metabolite and IPA values. No difference was observed when comparing the AUC of clopidogrel and baseline aggregation according to CYP2C19 genotypes. All other tested genotypes of CYP2B6, CYP2C9, CYP3A4, CYP3A5, ABCB1, and P2Y12 showed no significant effect on the AUC of thiol metabolite and IPA value.

Table 1. The Effects of Candidate SNPs in Gene Involved in Clopidogrel Pharmacokinetic and Pharmacodynamic Pathway on Pharmacokinetic and Pharmacodynamic Parameters of Clopidogrel and Its Thiol Metabolite
GenotypeAUC of Clopidogrel (ng h/mL)P-ValueAUC of Thiol Metabolite (ng h/mL)P-ValueBaseline AggregationP-ValueIPAssa (%)P-Value
NMean ± SDNMean ± SDNMean ± SDNMean ± SD
  • AUC of clopidogrel and thiol metabolite is the values from pharmacokinetic parameters at day 1.

  • a

    IPAss is average of inhibition of platelet aggregation (IPA) value during day 4∼ day 8.

CYP2B6
*1/*1148.8 ± 7.0>.0514185.0 ± 105.4>.056572.2 ± 11.3>.056544.3 ± 20.4>.05
*1/*689.1 ± 7.7 8191.9 ± 134.5 2772.3 ± 8.5 2738.7 ± 18.6 
CYP2C9
*1/*1208.8 ± 7.2>.0520165.0 ± 88.0.058473.0 ± 10.1>.058441.2 ± 20.3>.05
*1/*329.7 ± 8.8 2403.3 ± 148.0 866.2 ± 12.1 852.3 ± 13.4 
CYP2C19
*1/*1610.6 ± 6.2>.056329.7 ± 114.4.013273.5>.053256.1 ± 11.8 
*3/*1712.8 1262.9 140.5 145.1 
*1/*276.7 ± 5.0 7126.9 ± 40.2 2970.5 ± 10.3 2946.4 ± 14.5 
*1/*3124.9 1166.0 869.4 ± 14.1 845.7 ± 15.4<.001
*2/*2410.2 ± 10.4 4129.9 ± 44.9 1370.9 ± 11.2 1324.2 ± 14.2 
*2/*335.6 ± 0.5 3103.7 ± 36.4 672.0 ± 16.4 624.5 ± 22.0 
*3/*30 0 367.7 ± 11.0 330.1 ± 29.0 
CYP2C19
EM79.5 ± 6.4>.057320.2 ± 107.4<.013372.5 ± 10.6>.053355.8 ± 10.6<.001
IM89.0 ± 7.9 8131.8 ± 39.7 3770.3 ± 11.0 3746.3 ± 14.5 
PM78.3 ± 7.8 7118.6 ± 40.6 2270.8 ± 12.2 2225.1 ± 17.8 
CYP3A5
*1/*127.7 ± 3.2>.052129.2 ± 83.9>.05380.8 ± 5.3>.05329.3 ± 28.1>.05
*1/*378.6 ± 5.5 7226.2 ± 153.2 2973.5 ± 11.7 2946.5 ± 15.2 
*3/*3139.3 ± 8.4 13175.7 ± 93.3 6071.1 ± 9.7 6040.8 ± 21.8 
ABCB1 3435C>T
CC118.1 ± 6.9>.0511186.6 ± 107.6>.055371.8 ± 9.4>.055340.4 ± 22.9>.05
CT811.7 ± 7.9 8210.2 ± 141.8 3071.4 ± 11.6 3044.4 ± 17.7 
TT34.5 ± 1.7 3130.5 ± 22.4 976.5 ± 10.2 943.3 ± 16.5 
P2Y12 742T>C
TT148.6 ± 6.7>.0514195.7 ± 136.4>.056270.2 ± 11.5>.056242.3 ± 20.8>.05
TC78.1 ± 7.6 7164.1 ± 61.2 2975.5 ± 8.0 2942.4 ± 19.2 
CC119.5 1236.6 165.0 151.54 
P2Y12–43108T>C
TT118.3 ± 7.3>.0511150.6 ± 71.3>.053375.8 ± 8.6>.053343.1 ± 18.3>.05
TC99.7 ± 7.9 9220.3 ± 136.6 4571.3 ± 11.3 4538.9 ± 21.2 
CC28.8 ± 1.1 2243.4 ± 201.2 1468.1 ± 10.4 1447.9 ± 20.0 
PON1 Q192R
QQ124.9>.051166.0>.051471.8 ± 10.6>.051447.0 ± 14.1>.05
QR116.3 ± 4.9 11187.8 ± 99.0 3871.3 ± 10.5 3843.8 ± 18.9 
RR1010.2 ± 7.2 10189.4 ± 138.6 4070.9 ± 12.0 4044.6 ± 19.8 

Effects of CYP2C19 Genotype on the Time Course of Platelet Aggregation

The IPA values for a pharmacodynamic assessment according to the CYP2C19 genotype were further evaluated to explore the change in IPA over time (Figure 1). The pharmacodynamic parameters were summarized in Table 2. The IPA value was lowest in CYP2C19 PM, and highest in CYP2C19 EM throughout the study. CYP2C19 PM and IM genotypes had significantly delayed time to maximal IPA compared with CYP2C19 EM (12 (6–24) vs. 9 (4–24) vs. 2 (2–12) hours for median Tmax (range), P = .04) after a 300-mg loading dose of clopidogrel.

image

Figure 1. Mean inhibition of platelet aggregation (IPA)–time profiles after oral administration of 300 mg clopidogrel loading dose and 75 mg clopidogrel for 6 consecutive days (A) (*P < .01, **P < .001 by Kruscal–Wallis test; P > .05 for time effect and P = .03 for time and genotype interaction effect from days 2 to 8 by repeated measured analysis of variance) and larger scale of same curve from 0 to 24 hours after a loading dose of clopidogrel (B).

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Table 2. Pharmacodynamic Parameters of Clopidogrel and Thiol Metabolite After oral Administration of 300 mg Clopidogrel Loading Dose and 75 mg Clopidogrel for 6 Consecutive Days in Healthy Subjects With CYP2C19 Extensive Metabolizer (EM), Intermediate Metabolizer (IM), and Poor Metabolizer (PM)
ParameterEM (N = 33)IM (N = 37)PM (N = 22)P-Value
  • IPAt, inhibition of platelet aggregation (IPA) value at time t; Emax,D1, maximal IPA after a loading dose of clopidogrel; Tmax, the time to reach Emax; AUECt, area under the effect–time curve from time 0 to time t; IPAss as average of inhibition of platelet aggregation (IPA) value during days 4–8.

  • All data represent as arithmetic mean ± standard deviation, except data for aTmax are expressed as median (range; mean ± standard deviation).

  • b

    P < .05 for IPAD8 vs. IPAD2 in subjects with CYP2C19 PM genotype by Wilcoxon Signed-Rank Test.

After administration of 300 mg loading dose of clopidogrel (Day 1)
IPA4h54.9 ± 19.032.6 ± 11.621.8 ± 16.2.01
Emax (%)62.6 ± 16.254.3 ± 14.846.2 ± 21.4<.01
Tmax (hour)a2 (2–12; 10 ± 6.7)9 (4–24; 11.7 ± 7.3)12 (6–24; 12.4 ± 7.2).04
AUEC6h (%·day)7.1 ± 2.05.6 ± 2.15.1 ± 2.9<.01
AUEC24h (%·day)47.4 ± 12.640.1 ± 13.529.8 ± 16.0<.01
During administration of 75 mg maintenance dose of clopidogrel
IPAD2 (%)51.2 ± 16.543.9 ± 17.030.0 ± 21.9<.01
IPAD8 (%)58.0 ± 13.847.9 ± 14.523.7 ± 16.6b<.01
IPAss (%)55.8 ± 11.746.3 ± 14.525.1 ± 17.8<.01
AUECD14 (%·day)584.4 ± 156.5471.0 ± 148.1275.2 ± 187.1<.01

During maintenance dose of clopidogrel, IPA values of only CYP2C19 PMs were gradually decreased from 30.0 ± 21.9% on day 2 to 23.7 ± 16.6% on day 8 (P < .05, Table 2) while IPA values of CYP2C19 EMs and IMs remained similar to that seen on days 1 or 2 (P > .05 for time effect; P = .03 for time and genotype interaction effect by repeated measures ANOVA, Figure 1A). Therefore, a larger difference IPA between CYP2C19 EM or IM and CYP2C19 PM after 7 days of clopidogrel administration than that after administration of the clopidogrel loading dose was observed.

Effects of CYP2C19 Genotype on Pharmacokinetics of Clopidogrel and Its Metabolite

In order to support pharmacodynamic data, the plasma concentration–time profiles of clopidogrel and its thiol metabolite were compared among the CYP2C19 genotypes (Figure 2) and pharmacokinetic parameters of clopidogrel and thiol metabolite are summarized in Table 3. No difference was observed when comparing the clopidogrel pharmacokinetic parameters among different CYP2C19 genotypes. However, in terms of the thiol metabolite pharmacokinetic parameters, CYP2C19 PM genotypes had decreased Cmax and AUC of thiol metabolite compared with CYP2C19 EM genotypes by 0.42- and 0.37-fold on day 1 (P < .01), and 0.39- and 0.34-fold on day 7 (P < .01 and <.01), respectively.

image

Figure 2. Mean concentration–time profiles of clopidogrel (A) and thiol metabolite (B) after oral administration of 300 mg clopidogrel loading dose and 75 mg clopidogrel for 6 consecutive days

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Table 3. Pharmacokinetic Parameters of Clopidogrel and Thiol Metabolite After Oral Administration of 300 mg Clopidogrel Loading Dose and 75 mg Clopidogrel for 6 Consecutive Days in Healthy Subjects With CYP2C19 Extensive Metabolizer (EM), Intermediate Metabolizer (IM), and Poor Metabolizer (PM)
ParameterEM (N = 7)IM (N = 8)PM (N = 7)P-Value
  1. All data represent as arithmetic mean ± standard deviation.

Clopidogrel
Day 1 (after administration of clopidogrel 300 mg loading dose)
Cmax (ng/mL)5.5 ± 5.14.7 ± 4.45.5 ± 5.2>.05
AUCτ (ng h/mL)9.5 ± 6.49.0 ± 7.98.6 ± 7.8>.05
Day 7 (after administration of clopidogrel 75 mg maintenance dose)
Cmax (ng/mL)0.8 ± 0.71.1 ± 1.70.9 ± 0.4>.05
AUCτ (ng h/mL)1.6 ± 1.32.0 ± 2.51.6 ± 0.7>.05
Thiol metabolite
Day 1 (after administration of clopidogrel 300 mg loading dose)
Cmax (ng/mL)152.2 ± 44.258.3 ± 22.164.2 ± 28.0<.01
AUCτ (ng h/mL)320.2 ± 107.4131.8 ± 39.7118.6 ± 40.6<.01
Day 7 (after administration of clopidogrel 75 mg maintenance dose)
Cmax (ng/mL)45.0 ± 18.624.5 ± 8.117.9 ± 12.5<.01
AUCτ (ng h/mL)87.8 ± 23.743.3 ± 11.230.3 ± 18.8<.01

We observed a significant correlation between the IPAss and AUCτ of thiol metabolite on day 7 (r = 0.82, P < .01; Supplemental Figure S1). However, we did not observe any significant correlation between AUCiτ of thiol metabolite and clopidogrel and between the IPAss and AUCτ of clopidogrel on day 7.

Discussion

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. Funding
  7. References
  8. Supporting Information

We investigated the effects of CYP2C19 genotype on the time course of platelet aggregation after of clopidogrel administration in 92 Korean healthy subjects. In this study, we confirmed that CYP2C19 LOF alleles reduce the active metabolite formation and antiplatelet effects of clopidogrel.[11, 19] And, we found the new finding that CYP2C19 PM and IM genotypes exhibited delayed time to MPA after a clopidogrel loading dose, and the antiplatelet effects of clopidogrel decreased gradually in those with the CYP2C19 PM genotype. These results add to our mechanistic understanding of the effects of CYP2C19 genotype on pharmacokinetics and pharmacodynamics of clopidogrel and its thiol metabolites. In this study, subjects with CYP2C19 PM and IM genotypes had delayed time to MPA after a clopidogrel loading dose, platelet aggregation decreased over time in subjects the CYP2C19 PM genotype compared to those with CYP2C19 EM.

It is well known that the 600 mg loading dose of clopidogrel achieves maximal platelet inhibition at 2 hours while 300 mg dose achieves at 6 hours.[20] Common clinical practice guideline recommend pretreatment with a loading dose of clopidogrel as early as possible before or at the time of PCI.[21] Thus, delayed time to maximum IPA as well as decreased antiplatelet effects in the carriers of CYP2C19 PM and IM (9 and 12 hours of median Tmax in this study) may influence the increased risk of the acute cardiac events that require rapid onset of clopidogrel. Because Tmax to reach maximal response is dose-dependent,[20] higher loading dose (600 or 900 mg) may overcome this delayed Tmax. It remained to be evaluated.

In this study, the subjects with CYP2C19 IM genotype retained the antiplatelet effects of clopidogrel at steady state unlike CYP2C19 PM genotype. The 300 mg loading dose administered to subjects with CYP2C19 IM was not sufficient to alter the pharmacodynamic effects as much as that in the CYP2C19 EM genotype groups, but 75 mg maintenance dose of clopidogrel preserved the antiplatelet effects seen with loading dose administration. In the case of CYP2C19 PM genotype, 75 mg maintenance dose did not keep even the decreased antiplatelet effects of 300 mg loading dose. Simon et al[14] reported that 600 mg loading dose and 150 mg maintenance dose overcame diminished antiplatelet response. However, they did not report the difference in MPA decrease among different CYP2C19 genotype after administration of 75 mg clopidogrel. This discordance may be resulted from that this study has larger sample size, more frequent measurement of platelet aggregation, and conducted in homogeneous same ethnic population compared with previous study although it needs to be confirmed by replication.

The decreasing trend of IPA in subjects with CYP2C19 PM over time may be related to the delayed onset of cardiac events after clopidogrel administration in a clinical situation. This suggests that an additional clopidogrel dose may be guided differently depending on CYP2C19 IM and CYP2C19 PM genotype. Some reports based on clinical presentation not CYP2C19 genotypes have suggested that patients undergoing PCI and taking double dose of clopidogrel have significantly decreased stent thrombosis.[22] However, recent reports based on CYP2C19 genotype such as Clinical Pharmacogenetics Implementation Consortium Guidelines[23] and Dutch Pharmacogenetics Working Group Guideline[24] have recommended for subjects with CYP2C19 IM and PM genotypes to use of an alternative agent (e.g., prasugrel, ticagrelor) if not contraindicated. Mega et al reported that higher dose of clopidogrel in subjects with CYP2C19 IM genotype resulted in comparable platelet inhibition compared to the standard 75 mg dose in those with CYP2C19 EM genotype.[25] However, in CYP2C19 PM genotype, even 300 mg clopidogrel did not result in comparable degrees of platelet inhibition compared to the standard 75 mg dose in CYP2C19 EM genotype. Accordingly, the patients with the CYP2C19 IM genotype may benefit from both higher dose of clopidogrel and more potent ADP receptor blockers such as prausgrel or ticagrelor, but those with the CYP2C19 PM genotype benefit from alternative therapy rather than a higher dose of clopidogrel, particularly those with acute coronary syndromes receiving PCI.

Some studies have reported a negative correlation between clopidogrel and IPA.[26] However, we did not observe any correlation between clopidogrel and IPA, or between clopidogrel and its thiol metabolite. Only less than 15% of Clopidogrel is biotransformed to thiol metabolite, mainly to inactive carboxyl metabolite via carboxyl esterase. Decreased formation of the thiol metabolite may not be associated with a higher plasma level of clopidogrel due to formation of carboxyl metabolite by the high capacity carboxyl esterase.

Bouman et al[3] suggested that PON1 is involved in the formation of the active thiol metabolite of clopidogrel, and thus PON1 Q192R genotype was significantly associated with poor antiplatelet effect and clinical outcome of clopidogrel therapy. However, soon thereafter, several reports showed that PON1 genotype had no effect on the antiplatelet effects of clopidogrel.[5, 27] It has been also reported that PON1 plays a major role in formation of endo-thiol metabolite, while CYP2C19 lead to the formation of active cis-thiol metabolite.[4] Our results confirmed that PON1 genotype was not associated with the antiplatelet effects and pharmacokinetic parameters of clopidogrel. Because the contribution of the endo-thiol metabolite concentration level to total thiol concentration level is minimal (20-fold lower than that of cis-thiol metabolite),[27] there seemed to be no marked difference between total thiol concentration and cis-thiol metabolite concentration. Furthermore, no effects of candidate SNPs in genes involved in clopidogrel pharmacokinetic and pharmacodynamic pathways including CYP2B6, CYP2C9, CYP3A5, ABCB1, and P2Y12 on clopidogrel response were observed, which is consistent with other studies.[9, 11]

In conclusion, CYP2C19 PM and IM genotypes exhibited delayed time to MPA after a clopidogrel loading dose, and the antiplatelet effects of clopidogrel decreased gradually in those with the CYP2C19 PM genotype during maintenance dose of clopidogrel.

Funding

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. Funding
  7. References
  8. Supporting Information

This study was supported by grant A111218-11-PG02 from the National Project for Personalized Genomic Medicine, Korea Health 21 R&D Project, Ministry for Health & Welfare and supported by the Korea Science and Engineering Foundation (KOSEF) grant funded by the Ministry of Education, Science and Engineering (MOEST; No. R13-2007-023-00000-0).

References

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. Funding
  7. References
  8. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. Funding
  7. References
  8. Supporting Information

Additional supporting information may be found in the online version of this article at the publisher's web-site.

FilenameFormatSizeDescription
jcph225-sm-0001-SuppFig-S1.pdf55KFigure S1. Correlation between AUCτ of thiol metabolite and clopidogrel on day 1 (A), between AUCτ of thiol metabolite and clopidogrel on day 7 (B), between mean IPA value during maintenance dose of clopidogrel (IPAss) and AUCτ of clopidogrel on day 7 (C), and between IPAss and AUCτ of thiol metabolite on day 7 (D).

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