Increased clopidogrel response is associated with ...

Clinica Chimica Acta 413 (2012) 417–421

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Increased clopidogrel response is associated with ABCC3 expression: A pilot study André Ducati Luchessi a,⁎, Vivian Nogueira Silbiger b, Alvaro Cerda a, Rosario Dominguez Crespo Hirata a, Angel Carracedo c, Maria Brion c, d, Andres Iñiguez e, Marisol Bravo e, Guillermo Bastos e, Amanda Guerra Morais Rego Sousa f, Mario Hiroyuki Hirata a a

Faculty of Pharmaceutical Science, University of Sao Paulo, Sao Paulo, Brazil Faculty of Pharmacy, University Federal of Rio Grande do Norte, Brazil Grupo de Medicina Xenómica, Fundación Medicina Xenómica, CIBERER-Universidade de Santiago de Compostela, Santiago de Compostela, Spain d Genetics of Cardiovascular and Ophthalmologic Diseases, Hospital-University Complex of Santiago (CHUS), Santiago de Compostela, Spain e Hospital Meixoeiro, Vigo, Spain f Instituto Dante Pazzanese do Estado de Sao Paulo, Sao Paulo, Brazil b c

a r t i c l e

i n f o

Article history: Received 29 August 2011 Received in revised form 11 October 2011 Accepted 11 October 2011 Available online 20 October 2011 Keywords: ABCC3 ABCB1 Pharmacogenomics Clopidogrel Antiplatelet agents

a b s t r a c t Background: The aim of this study was investigate the relationship between ABCB1 and ABCC3 gene expressions in peripheral blood cells (PBC) and the response to clopidogrel in patients with coronary arterial disease (CAD). Methods: Twenty-six male CAD patients (50–70 years) under treatment with clopidogrel (75 mg/day) for at least 5 days were selected. Blood samples were obtained to evaluate platelet reactivity and ABCB1 and ABCC3 mRNA expression. Platelet reactivity was measured in P2Y12 Reaction Units (PRU) using VerifyNow. RNA was extracted from PBC and mRNA levels were measured by qPCR, using GAPD as a reference gene. Results: Platelet response to clopidogrel was categorized in to PRU quartiles. Individuals with PRU values within the first quartile (Q1, b 151 units) were considered good responders, while those who had PRU within the fourth quartile (Q4, PRU > 260) were considered non-responders. ABCC3 was 1.7 times more expressed in Q4 than in Q1 PRU group (p= 0.048). Moreover, CAD patients with low ABCC3 expression (Qe1, b 2.5 × 10− 3) had higher probability to have a good response to clopidogrel (OR: 18.00, 95%CI: 1.90–169.99, p = 0.001). Univariate linear regression analysis demonstrated that low ABCC3 mRNA expression contributed with a reduction of 73 PRU in relation to the patients with expression value higher than 2.5 × 10− 3 (p= 0.027). Neither ABCB1 mRNA levels nor clinical variables studied influenced PRU values. Conclusions: Low ABCC3 mRNA expression in peripheral blood cells is associated with increased clopidogrel response, but further studies are needed to describe the functional relationship of clopidogrel with the ABCC3. Crown Copyright © 2011 Published by Elsevier B.V. All rights reserved.

1. Introduction Clopidogrel is a prodrug that requires hepatic biotransformation by cytochrome P450 to an active metabolite [1]. It is an antagonist of the platelet adenosine diphosphate receptor that prevents platelet agregation [2]. Other important antiagregation drug is a aspirin (acetylsalicylic acid), that action is reduces the activation of platelets by irreversibly acetylating cyclooxygenase-1 (COX-1), and thereby reduces thromboxane A2 by platelets [3]. Treatment with clopidogrel in combination with aspirin can prevent recurrent ischaemic events after an acute coronary syndrome or a percutaneous coronary intervention [4]. However, the response

⁎ Corresponding author at: University of Sao Paulo, Faculty of Pharmaceutical Sciences, Department of Clinical and Toxicological Analyses, Av Prof. Lineu Prestes. 580, B-17, 05508-900, Sao Paulo, SP, Brazil. Tel.: + 55 11 3091 3660; fax: + 55 11 3813 2197. E-mail address: [email protected] (A.D. Luchessi).

to clopidogrel has substantial interpatient variability that has been associated with increased risk for recurrent cardiovascular events, including stent thrombosis [5,6]. This variability has been related, at least in part, to polymorphisms in genes involved in pharmacokinetic pathways that affects clopidogrel bioavailability and eficacy [7–9]. But other authors, suggest that variation in expression of ATP-binding cassette (ABC) membrane proteins, are a family of efflux transporters, that can limit intestinal absorption and intracellular concentrations of drugs [10,11]. Variants on genes encoding ABC transporters have been implicated in low drug efficacy and safety [12,13]. The ABCB1 is the first transporter membrane discovered in ABC family and it is responsible for the efflux of a broad range of different compounds including cytostatics, antiviral and lowering-cholesterol drugs [14]. However, the role of ABCB1 in the pharmacokinetic of clopidogrel is not well known. It has been shown that long exposure (12–48 h) to high concentrations of clopidogrel (>10 μmol/l) down regulates ABCB1 in Caco-2 cells, indicating a possible relationship of this protein transport with clinical effectiveness of clopidogrel [7].

0009-8981/$ – see front matter. Crown Copyright © 2011 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.cca.2011.10.018

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A.D. Luchessi et al. / Clinica Chimica Acta 413 (2012) 417–421

Another important member of ABC family is ABCC3 which plays an important role in the efflux of endogenous glucuronides and conjugated drug metabolites across cell membranes [15]. A recent study reported the detection of ABCC3 mRNA and protein expression in human platelets [16]. The authors suggested that ABCC3 and other ABC transporters may influence the transport of drugs on platelet membrane interfering with the drug availability and efficacy. The −211T>C a variant located at the promoter region of the ABCC3 gene, that can influence their gene expression, was described to be associated with the clinical outcome of chemotherapy in lung cancer patients through alteration in the gene expression and consequently in the pharmacological response [17]. This study investigated the relationship between ABCB1 and ABCC3 mRNA expressions in peripheral blood cells (PBC) with clopidogrel response of patients with coronary arterial disease (CAD).

Veriti™ 96-Well Fast Thermal Cycler (Applied Biosystems, Foster City, USA) under conditions described by the supplier. cDNA samples were kept at −20 °C for further quantitative TaqMan® real-time PCR (qPCR) analysis. ABCB1 and ABCC3 mRNA levels were measured by qPCR using glyceraldehyde-3-phosphate dehydrogenase (GAPD) as a reference gene. Primers and probes sequences used for ABCB1, ABCC3 and GAPD mRNA expressions were provided as “Assay on demand” format (Applied Biosystems, Foster City, USA). qPCR assays was carried out in 96-well plates of a 7500 Real-Time PCR System (Applied Biosystems, Foster City, USA) using the following protocol: 40 cycles at 95 °C for 15 s and at 60 °C for 1 min. The relative quantification for each target gene was measured using a comparative Ct method using the 2− deltaCt formula, where deltaCt is the difference between the target gene Ct and the GAPD Ct [18]. 2.4. Statistical analysis

2. Subjects and methods 2.1. Subjects and clinical protocol Twenty six, male individuals with CAD, aged 50 to 70 years, who underwent percutaneous coronary angioplasty (PCA) at the Hospital do Meixoeiro (Vigo, Spain) were enrolled in this study from July to December 2008. All individuals were under clopidogrel therapy (75 mg/day) at least for 5 days prior to PCA and received other medication such aspirin, betablockers, nitrates, statin and pump protons inhibitor. Exclusion criteria: primary PCA; high bleeding risk; chronic renal failure; under thienopyridine, glycoprotein IIb/IIIa inhibitor or warfarin therapy; allergic reaction and also aspirin or clopidogrel contraindication. Information on age, systolic and diastolic pressure, body mass index (BMI), diabetes, Dyslipidemia, tobacco smoking, family history of CAD, medications in use and time from last dose of clopidogrel (TLDC) were recorded. This study was approved by the local Ethics Committee and all subjects voluntarily provided written and informed consent in agreement with Helsinki declaration. 2.2. Determination of platelet reactivity Platelet reactivity was evaluated before PCA by turbidimetric optical detection of platelet aggregation using the VerifyNow P2RY12 assay (Accumetrics, San Diego, USA) according to manufacturer's instructions. Briefly, 2.0 ml of blood samples were drawn from an antecubital vein with 3.2% citrate tubes. After 30 min at room temperature, citrated-blood was transferred into standard cartridges containing fibrinogen-coated micro beads and 20 mmol/l of adenosine diphosphate an agonist of purinergic receptor P2Y, G-protein coupled, 12 (P2RY12). Platelet aggregation occurs and the system converts luminosity transmittance of total blood in a result of platelet response to clopidogrel inhibition, this result is denominated P2RY12 reaction units (PRU). 2.3. ABCB1 and ABCC3 mRNA expressions Total RNA was isolated from whole blood samples with the PAXGene Blood RNA system (PreAnalytiX, Hilden, Germany) by automated Qiacube system (Qiagen, Valencia, USA) following the manufacturer's protocols including a RNase-free DNase (Qiagen, Valencia, CA, USA) step to remove genomic DNA. The quantity of RNA was measured using a Nanodrop ND-1000 Spectrophotometer to give the yield and a 260/280 ratio. Agilent Bioanalyser Lab-on-a-chip RNA chips (Agilent Technologies, Waldbronn, Germany) were also used for each sample to check the quality by calculating RNA Integrity Number (RIN) scores, samples with RIN b 5 were exclude. The RNA samples were stored at −80 °C until later cDNA conversion. Isolated RNA samples were converted to complementary DNA (cDNA) using High-Capacity cDNA Reverse Transcription Kits (Applied Biosystems, Foster City, USA) in a

Statistical analyses were carried out using SPSS 15.0 for windows (SPSS, Chicago, USA). Categorical variables were compared by Chisquare test or Exact Fisher test and continuous variables were compared by Kruskal–Wallis test and t-test or Mann–Whitney test. Platelet aggregation response to clopidogrel and ABCB1 and ABCC3 mRNA expression values were categorized in quartiles for statistic comparison (Q1, Q2, Q3 and Q4 for PRU; and Qe1, Qe2, Qe3 and Qe4 for mRNA expression). Univariate logistic regression analysis was performed to evaluate the variables associated with low mRNA expression levels of ABCC3 (model 1) and ABCB1 (model 2). Low mRNA expression was considered Qe1 group that was compared with the remaining groups (Qe2 + Qe3 + Qe4). In both models, the highest response to clopidogrel (Q1, PRU b 151), age, BMI, hypertension, tobacco smoking, family history of CAD, and TLDC were introduced as independent variables. Diabetes and dyslipidemia had null value in one of the categorical groups, therefore, they were not included as independent variables in logistic regression analysis. Multivariate stepwise logistic regression was performed to evaluate the independent variables associated with low ABCC3 mRNA expression, including age, BMI, TLDC and good response clopidogrel (Q1, PRU b 151), as covariates. Univariate linear regression was performed the relationship between PRU values and other variables, such as low ABCC3 (Qe1 b 2.50 ×10− 3) and ABCB1 (Qe1 b 5.08 × 10− 3) expression values, age, BMI, hypertension, tobacco smoking, and family history of CADTLDC diabetes and dyslipidemia. For all these analyses the significance value was established at the p-valueb 0.050. 3. Results 3.1. Clinical data and response of clopidogrel Clinical characteristics and response to clopidogrel data of the CAD patients are presented in Table 1. Dyslipidemia (88%), hypertension (62%) and patients with BMI> 30 (23%) were the major risk factors for CAD in this sample. Platelet response to clopidogrel was categorized in to PRU quartiles. Individuals with PRU values within the first quartile (Q1, PRU b 151) were considered good responders, while those who had PRU within the fourth quartile (Q4, PRU> 260) were considered nonresponders. Age, BMI, clinical data and TLDC of the CAD patients did not differ among clopidogrel response groups (p> 0.05). 3.2. ABCB1 and ABCC3 mRNA expressions ABCB1 and ABCC3 mRNA expresion levels in PBC were compared among PRU quartile groups, as shown in Fig. 1. ABCC3 was 1.7 times less expressed in the Q1 PRU group (2.7 ± 1.2 × 10 − 3) than in the Q4 PRU (4.5 ± 1.7 × 10 − 3, p = 0.048). On the other hand, no significant differences were observed in ABCB1 expression among PRU quartile values (p = 0.864).


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Table 1 Clinical characteristics according to platelet clopidogrel response. Variables

Number of individuals PRU, units Age, years BMI, kg/m2 Hypertension, % Diabetes, % Dyslipidemia, % Family history of CAD, % Tobacco smoking, % TLDC, h

Quartiles of clopidogrel response Total

Q1

Q2

Q3

Q4

p-value

26 217 ± 62 60 ± 10 29 ± 3 62 (16) 27 (7) 88 (23) 12 (3) 19 (5) 11 ± 9

6 121 ± 42 55 ± 11 29 ± 4 67 (4) 0 (0) 100 (6) 17 (1) 33 (2) 8 ± 10

7 184 ± 16 63 ± 6 28 ± 3 57 (4) 43 (3) 71 (5) 0 (0) 29 (2) 15 ± 8

7 243 ± 9 61 ± 10 30 ± 2 71 (5) 14 (1) 86 (6) 29 (2) 0 (0) 5±7

6 301 ± 46 60 ± 13 29 ± 2 50 (3) 50 (3) 100 (6) 0 (0) 17 (1) 16 ± 8

b0.001 0.735 0.297 0.861 0.151 0.307 0.279 0.390 0.153

Numbers of patients in parenthesis. Individuals were grouped in quartiles according to clopidogrel response using PRU values. Continuous variables are expressed as mean ± SD. Variables were compared by Chi-square or Kruskal–Wallis tests among first (Q1), second (Q2), third (Q3) and fourth (Q4) quartiles of response to clopidogrel. PRU: P2RY12 reaction units; BMI: Body Mass Index; CAD: coronary artery disease; TLDC: time from last dose of clopidogrel.

Variables from individuals with low ABCC3 mRNA levels (Qe1, mRNAb 2.5 × 10 − 3) were compared with those having mRNA expression within the other quartiles (Qe2+ Qe3 + Qe4). Interestingly, low ABCC3 expression (Qe1) was associated with low PRU mean values (p= 0.039) (Table 2). Other clinical variables did not influence ABCC3 mRNA levels in PBC. Univariate logistic regression revealed that CAD patients with low ABCC3 mRNA expression (Qe1, b2.5× 10− 3) have 18 times more probability to be responsive to clopidogrel (OR: 18.000, 95%CI: 1.900–169.991, p= 0.011), as shown in Table 3. Others variables were not associated with PBC ABCC3 mRNA levels in CAD patients. No relationship was found between low ABCB1 mRNA expression (Qe1, b5.1× 10− 3) and clinical variables and response to clopidogrel in CAD patients (Table 3). Multivariate stepwise logistic regression confirmed that patients with low ABCC3 mRNA expression had high probability to have a good response to clopidogrel (OR: 30.755, 95%CI: 1.659–570.247, p = 0.021), whereas other variables included in this model (age, BMI and TCLD) were not related with ABCC3 mRNA levels in PBC (Table 4). Univariate linear regression analysis demonstrated that low ABCC3 mRNA expression contributed with a reduction of 73 PRU in relation to the patients with expression value higher than 2.5 ×10− 3 (p= 0.027) (Table 5). In this model, the variables low ABCB1 mRNA expression, age, BMI, hypertension, diabetes, dyslipidemia, tobacco smoking, and family history of CAD and TLDC did not influence platelet response to clopidogrel. 4. Discussion Several risk factors for CAD such as hypertension, dyslipidemia, diabetes, tobacco smoking and family history of CAD have been found

in this sample population, as it has been described previously [19]. However our results indicated that these risk factors did not influence the platelet response to clopidogrel. The categorization in quartiles of PRU values was a better approach to analyze the data of response to clopidogrel. Good responders (PRU b 151) were clearly differenciated from non-responders (PRU > 260), data that are in accordance with those previously reported [20]. Cuisset et al. have evaluated three diferent methods for determination of clopidogrel response (ADP-induced platelet aggregation, Platelet reactivity index Vasodilator-stimulated Phosphoprotein (VASP) and Point of care Verify Now Assay) [21]. Even though, they have shown a good correlation between the results from these methods, there was a poor agreement to identify clopidogrel non-responders. It is likely that the different cutoff values may had influenced these results. In this study, diabetes was not associated with clopidogrel response in CAD patients. However PRU Q1 group did not contain diabetic patients suggesting association with low platelet response to clopidogrel. It has been reported that the relationship between diabetes and poor response to clopidogrel is problably due to an enhanced platelet reactivity found in hyperglycemic status [22]. We also evaluated the ABCB1 and ABCC3 mRNA expressions in PBC and its possible association with platelet response to clopidogrel in CAD patients. Low ABCC3 mRNA expresssors showed better response to clopidogrel, independently of ABCB1 mRNA expression and clinical variables. The mechanisms involved in modulation of ABCC3 mRNA expression in leukocytes are not clear. Bohan et al. have shown that induction of ABCC3 is hepatoprotective in cholestasis and suggested that ABCC3 may be transcriptionally regulated by liver receptor homolog 1 dependent on an intact TNFalpha signaling pathway [23]. Hepatocyte

Fig. 1. ABCC3 and ABCB1 mRNA levels in peripheral blood cells according to clopidogrel response. Patients were grouped in quartiles of PRU values (Q1, Q2, Q3 and Q4). mRNA expression was measured by qPCR using GAPD as reference gene (2− deltaCt method). First PRU quartile (Q1) was compared with the others (Q2, Q3 and Q4) by Mann–Whitney test.

420


Table 2 Clinical characteristics according to ABCC3 mRNA expression. Variables

Table 4 Relationship between ABCC3 expression and clinical variables. Multivariate logistic regression analysis.

Quartiles of ABCC3 mRNA expression Qe1 (6)

ABCC3 mRNA levels Age, years BMI, kg/m2 Hypertension, % Diabetes, % Dyslipidemia, % Family history of CAD, % Tobacco smoking, % TLDC, hours PRU, units

Qe2 + Qe3 + Qe4 (20)

2.1 ± 2.0 × 10− 3 59 ± 14 28 ± 3 67 (4) 0 (0) 100 (6) 17 (1) 17 (1) 12 ± 11 156 ± 75

4.8 ± 2.2 × 10− 3 60 ± 9 29 ± 3 60 (12) 35 (7) 85 (17) 10 (2) 20 (4) 11 ± 9 229 ± 64

p-value b 0.001 0.976 0.387 0.768 ND ND 0.654 0.856 0.976 0.039

Numbers of patients in parenthesis. Individuals were grouped in quartiles according to ABCC3 mRNA expression. Continuous variables are expressed as mean ± SD. mRNA expression was measured by qPCR using GAPD as reference gene (2− deltaCt method). Variables were compared by Chi-square or Mann–Whitney tests between first (Qe1) and the others (Qe2 + Qe3 + Qe4) quartiles. PRU: P2RY12 reaction units; BMI: Body Mass Index; CAD: coronary artery disease; TLDC: time from last dose of clopidogrel; ND: not determined.

nuclear factors (HNFs) have also shown to regulate ABCC3 mRNA in liver tissues from HNF4a-HNull mice. Lu and co-workers have reported that mice lacking HNF4a expression in liver have selective loss of efflux transporters, including ABCC3 mRNA when compared with wild-type mice [24]. Linear regression analysis demonstrated a positive correlation between ABCC3 mRNA expression and response to clopidogrel suggesting that ABCC3 may be involved in clopidogrel bioavailability although we used whole blood cells as study model to analyze a theoretical relationship between clopidogrel and ABCC3 mRNA expression. Many studies uses this model of whole blood cells with act as a marker of the pharmacologic response, since that the expression of these cells may reflect the expression in other cells such as hepatocytes and enterocytes [14,25–27]. Study of Arazi et al., describe that atorvastatin, a drug metabolized in hepatocytes cells, has differential effects on SREBF1a and SCAP mRNA expressions in PBMC that are

Model

Predictor

1

Low ABCC3 mRNA expression PRU Q1 (b151 units) 30.755 Age 0.971 2 BMI, kg/m 1.105 TLDC 0.999

OR

95% CI

p-value

1.659–570.247 0.838–1.126 0.723–1.688 0.997–1.002

0.021 0.696 0.646 0.603

ABCC3 expression was introduced as dependent variables considering as low mRNA expression the first quartile for ABCC3 (Qe1 b 2.5 × 10− 3). mRNA expression was measured by qPCR using GAPD as reference gene (2− deltaCt method). PRU Q1 (b 151 units) represent good platelet response to clopidogrel. PRU: P2RY12 reaction units; BMI: Body Mass Index; CAD: coronary artery disease; TLDC: time from last dose of clopidogrel.

associated with baseline transcription levels, triglycerides response to atorvastatin. Therefore, mRNA expression of ABCC3 may play an important role in determining clopidogrel intracellular concentration and response. The lack of relationship between ABCB1 mRNA expressions in leukocytes, response to clopidogrel and other clinical variables is suggestive that this membrane transporter is not involved in clopidogrel response. However more studies should be carried out to confirm this hypothesis. However, other author investigated the effect of ABCB1 3435CbT polymorphism on ABCB1 expression in PBMCs of 69 individuals treatment with atorvastatin and not found significant association between this polymorphism and ABCB1 mRNA expression [25]. One potential limitation of the present study is the small sample size of clopidogrel responder group to analyze its association with the change in mRNA expression. Therefore the effect of ABCC3 mRNA variants on clopidogrel response should be tested in larger populations for better evaluation of the pharmacogenomic role of these antiplatelet drug and its relationship with membrane transporter. In conclusion, low ABCC3 mRNA expression can be indicative of better response to clopidogrel, independently of ABCB1 mRNA expression and clinical variables. However, the molecular mechanisms involved in regulation of ABCC3 expression and its effect on clopidogrel response or if clopidogrel can be modify directly this gene expression remain to be elucidated.

Table 3 Relationship between ABCB1 and ABCC3 expressions and clinical variables. Model

Predictor

1

Low ABCC3 mRNA expression PRU Q1 (b151 units) 18.000 Age 0.994 2 BMI, kg/m 0.846 Hypertension 0.750 Tobacco smoking 1.000 Family history of CAD 0.556 TLDC 1.000

2

OR

Low ABCB1 mRNA expression PRU Q1 (b151 units) 0.364 Age 0.91 BMI, kg/m2 1.756 Hypertension 0.38 Tobacco smoking 8.568 Family history of CAD 0.259 TLDC 1.002

95% CI

p-value

1.9–169.991 0.905–1.092 0.577–1.240 0.110–5.109 0.600–14.640 0.042–7.457 0.998–1.002

0.011 0.905 0.390 0.769 1.000 0.657 0.852

0.011–11.633 0.759–10.925 0.845–3.649 0.014–10.171 0.100–72.969 0.008–7.88 0.998–1.005

0.567 0.314 0.130 0.564 0.343 0.438 0.246

Univariate logistic regression analysis. ABCC3 and ABCC1 expressions were introduced as dependent variables considering as low mRNA expression the first quartile for ABCC3 (Qe1 b 2.5 × 10− 3) and ABCB1 (Qe1 b 5.1 × 10− 3). mRNA expression was measured by qPCR using GAPD as reference gene (2− deltaCt method). PRU Q1 (b 151 units) represent good platelet response to clopidogrel. PRU: P2RY12 reaction units; BMI: Body Mass Index; CAD: coronary artery disease; TLDC: time from last dose of clopidogrel. PRU: P2RY12 reaction units; BMI: Body Mass Index; CAD: coronary artery disease; TLDC: time from last dose of clopidogrel.

Table 5 Relationship between response to clopidogrel and other variables. Univariate linear regression analysis. Dependent variable

Independent variable

B (SE)

95% CI

R2

p-value

Low ABCC3 expression Low ABCB1 expression Age BMI, kg/m2 Hypertension Diabetes Dyslipidemia Tobacco smoking Family history of CAD TLDC

− 73.9 (31.0)

− 136.0/−9.1

0.15

0.027

− 31.1 (33.8)

− 38.2/100.1

0.03

0.366

0.4 (1.5) 4.1 (5.3) − 6.8 (29.7) 35.4 (31.8) 18.1 (45.2) − 12.2 (19.1) − 0.4 (45.3)

− 2.7/3.4 − 6.7/15.0 − 68.2/64.5 − 30.2/101.1 − 75.1/111.4 − 51.7/27.3 − 93.9/99.1

0.01 0.02 0.01 0.05 0.01 0.02 0.01

0.812 0.439 0.820 0.277 0.692 0.529 0.992

− 0.1/0.1

0.03

0.380

PRU (units)

0.1 (0.1)

ABCC3 and ABCC1 expressions were introduced as independent variables considering as low mRNA expression the first quartile for ABCC3 (Qe1 b 2.5 × 10− 3) and ABCB1 (Qe1 b 5.1 × 10− 3). PRU: P2RY12 reaction units; BMI: Body Mass Index; CAD: coronary artery disease; TLDC: time from last dose of clopidogrel; B (SE): regression coefficient (standard error); CI: confidence interval; R2: coefficient of determination.


Acknowledgments This study was supported by grants from FAPESP-Brazil (2006/ 03487-4), Spanish Health Institute ISCIII (EMER07/018) and DIANA Program of the Fundación Pedro Barrie de la Maza-Spain, which had no role in its design, conduct or reporting. A.D. Luchessi, M. H. Hirata and R. D.C. Hirata are recipients from CNPq-Brazil. V. N. Silbiger was recipient of a fellowship from FAPESP-Brazil. A. Cerda is recipient of a fellowship from CONICYT-Chile. References [1] Taubert D, Kastrati A, Harlfinger S, Gorchakova O, Lazar A, von Beckerath N, et al. Pharmacokinetics of clopidogrel after administration of a high loading dose. Thromb Haemost 2004;92(2):311–6. [2] Sangkuhl K, Klein TE, Altman RB. Clopidogrel pathway. Pharmacogenet Genomics 2010;20(7):463–5. [3] Hankey GJ, Eikelboom JW. Aspirin resistance. Lancet 2006;367(9510):606–17. [4] Smith SC, Allen J, Blair SN, Bonow RO, Brass LM, Fonarow GC, et al. AHA/ACC guidelines for secondary prevention for patients with coronary and other atherosclerotic vascular disease: 2006 update: endorsed by the National Heart, Lung, and Blood Institute. Circulation 2006;113(19):2363–72. [5] Gladding P, Webster M, Ormiston J, Olsen S, White H. Antiplatelet drug nonresponsiveness. Am Heart J 2008;155(4):591–9. [6] Matetzky S, Shenkman B, Guetta V, Shechter M, Bienart R, Goldenberg I, et al. Clopidogrel resistance is associated with increased risk of recurrent atherothrombotic events in patients with acute myocardial infarction. Circulation 2004;109(25):3171–5. [7] Taubert D, von Beckerath N, Grimberg G, Lazar A, Jung N, Goeser T, et al. Impact of P-glycoprotein on clopidogrel absorption. Clin Pharmacol Ther 2006;80(5): 486–501. [8] Mega JL, Close SL, Wiviott SD, Shen L, Hockett RD, Brandt JT, et al. Cytochrome P450 polymorphisms and response to clopidogrel. N Engl J Med 2009;360(21): 2250–1. [9] Feher G, Feher a, Pusch G, Lupkovics G, Szapary L, Papp E. The genetics of antiplatelet drug resistance. Clin Genet 2009;75(1):1–18. [10] Lee C a, Cook J a, Reyner EL, Smith D a. P-glycoprotein related drug interactions: clinical importance and a consideration of disease states. Expert Opin Drug Metab Toxicol 2010;6(5):603–19. [11] Oostendorp RL, Beijnen JH, Schellens JHM. The biological and clinical role of drug transporters at the intestinal barrier. Cancer Treat Rev 2009;35(2):137–47. [12] Rebecchi IMM, Rodrigues AC, Arazi SS, Genvigir FDV, Willrich MAV, Hirata MH, et al. ABCB1 and ABCC1 expression in peripheral mononuclear cells is influenced by gene polymorphisms and atorvastatin treatment. Biochem Pharmacol 2009;77(1):66–75. [13] Ieiri I, Takane H, Hirota T, Otsubo K, Higuchi S. Genetic polymorphisms of drug transporters: pharmacokinetic and pharmacodynamic consequences in pharmacotherapy. Expert Opin Drug Metab Toxicol 2006;2(5):651–74.

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