Genotype- versus phenotype-directed personalization of antiplatelet treatment in Koreans with coronary artery disease undergoing coronary stenting

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Genotype- versus phenotype-directed

personalization of antiplatelet treatment in

Koreans with coronary artery disease

undergoing coronary stenting

by

Sung Gyun Ahn

Major in Medicine

Department of Medical Sciences

The Graduate School, Ajou University

Genotype- versus phenotype-directed

personalization of antiplatelet treatment in

Koreans with coronary artery disease

undergoing coronary stenting

by

Sung Gyun Ahn

A Dissertation Submitted to The Graduate School of

Ajou University in Partial Fulfillment of the Requirements

for the Degree of

Ph. D. of Medicine

Supervised by

Seung-JeaTahk, M.D., Ph.D.

Major in Medicine

Department of Medical Sciences

The Graduate School, Ajou University

This certifies that the dissertation

of Sung Gyun Ahn is approved.

SUPERVISORY COMMITTEE

Joon-Han Shin

Seung-Jae Tahk

Myong-Ho Yoon

So-Yeon Choi

Junghan Yoon

The Graduate School, Ajou University

December, 13

th

, 2013

감사의 글

“소심심고 (素心深考),소박한 마음으로 돌아가서 다시 깊이 생각하라” 수학자 히로나가헤이스케가‘학문의 즐거움’에서 소개한 내용이다. 본인은 1996 년 원주의과대학 졸업 후 수원, 페루우아누꼬, 포항 등에서 내과 전문의, 국제협력의사, 심장내과 분과전문의로서 경험을 쌓고, 졸업 후 14 년이 지난 2000 년에 모교에서 부름을 받아 교수로서 삶을 영위할 수 있는 특권을 누리게 되었다. 토지의 저자 박경리 선생께서 아끼셨던 대지의 근원, 원주 (原州)의 흙을 매일 밟으면서 살고 있음에 감사한다. “Standing on the shoulders of giants”아이작 뉴턴의 고백처럼지금껏인생과 학문분야에서많은 거장과 스승들을 만났고 그들의 어깨 위에서 더 깊고 넓은 학문의 세계를 맛볼 수 있었다.먼저 페루까지 국제전화를해서심장내과 연구강사로받아주시고,심혈관중재학의 A 부터 Z 까지 지도해 주신진정한마에스트로탁승제 교수님, 본인의 첫 SCI 논문의 책임저자로 도움을 주셨던 늘 한결같은 신준한 교수님, 심장내과 chief 전공의 시절부터형님리더십을 보여주셨던 윤명호 교수님, 내과의사로서 첫 걸음마를 잘띌 수 있게 도와주셨던 최소연 교수님께 진심으로 감사를 드린다. 이젠 삶의 터전이 된 원주에서 가족처럼 따뜻하게 맞아주셨고, 지금도많은 도움을 주고 계신 윤정한, 이승환, 유병수, 김장영, 안민수, 윤영진, 이준원 교수님과 이지현 연구강사께도 감사를 드린다.귀찮은 서류작업에 많은 도움을 주신 배현경선생께도 감사의 마음을 전한다. 삶의 이유 중 하나인 가족의 헌신과 배려가 없었다면 지금의 나는 불가능했을 것이다. 보석 같은 아내 장이선과 자랑스런 세 아이들 의진, 의민, 희원이에게 나의 사랑을 듬뿍 전한다. 아들을 위해 매일 눈물로 기도하시고, 아낌없이 주는 나무가 되어주신어머님과, 항상저의 편이 되셔서 격려해주실 뿐만 아니라 때때로 용돈까지 챙겨 주시는 마음 넓은 장인어르신 내외께도 진심으로 감사를 드린다. 지면부족으로 일일이 열거하지 못한 분들께는 앞으로 살면서 차근차근 보답하겠노라고 다짐한다. 마지막으로 나의 주, 나의 하나님께 이 모든 영광을 돌린다.

i

- ABSTRACT -

Genotype- versus phenotype-directed personalization of antiplatelet

treatment in Koreans with coronary artery disease undergoing

coronary stenting

Sung Gyun Ahn

Department of Medical Sciences The Graduate School, Ajou University

(Supervised by Professor Seung-Jea Tahk)

We evaluated the effectiveness of genotype- versus phenotype-directed individualization for use of P2Y12 inhibitors to decrease high on-treatment of platelet

reactivity (HOPR). Sixty-five patients undergoing percutaneous coronary intervention for non-ST elevation acute coronary syndromes were randomly assigned to genotype- or phenotype-directed treatment. All patients were screened for the CYP2C19 *2, *3, or *17 alleles by using Verigene CLO assay (Nanosphere, Northbrook, IL, USA). On-treatment platelet reactivity was measured using the VerifyNow P2Y12 assay (Accumetrics, San Diego, CA, USA). 21 CYP2C19 *2 or *3 carriers (65.6%) or 11 patients with HOPR (33.3%), defined as ≥230 of P2Y12 reaction unit (PRU), were given 90 mg ticagrelor twice

daily; non-carriers or patients without HOPR were given 75 mg clopidogrel daily. The primary endpoint was the percentage of patients with HOPR after 30 days of treatment. PRU decreased following both genotype- and phenotype-directed therapies (242±83 vs. 109±90,

ii

p<0.001 in the genotype-directed group; 216±74 vs. 109±90, p=0.001 in the phenotype-directed group). Five subjects (16.2%) in genotype-phenotype-directed group and one (3.3%) in the phenotype-directed group had HOPR at day 30 (p=0.086). All patients with HOPR at the baseline who received ticagrelor had a PRU value of <230 after 30 days of treatment. Conversely, clopidogrel did not lower the number of patients with HOPR at the baseline. Tailored antiplatelet therapy according to point-of-care genetic and phenotypic testing is feasible and effective in decreasing HOPR after 30 days.

iii

TABLE OF CONTENTS

ABSTRACT ··· ⅰ TABLE OF CONTENTS ··· ⅲ LIST OF FIGURES ··· ⅳ LIST OF TABLES ··· ⅴ ABBREVIATION ··· ⅵ . Ⅰ INTRODUCTION ··· 1 . Ⅱ SUBJECTS AND METHODS ··· 3

A. SUBJECTS ··· 3

1. Trial design and study population ··· 3

B. METHODS ··· 5

1. VerifyNowP2Y12 test ··· 5

2. Verigene CLO assay ··· 5

3. Phenotype- versus genotype-guided antiplatelet regimen ··· 6

4. Definitions and endpoints ··· 6

5. Statistical analysis ··· 7 . Ⅲ RESULTS ··· 8 . Ⅳ DISCUSSION ··· 17 . Ⅴ CONCLUSION ··· 21 REFERENCES ··· 22 국문요약 ··· 29

iv

List of Figures

Fig. 1. Schematic diagram of the study. CYP: cytochrome, PCI: percutaneous coronary intervention, PRU: P2Y12 reaction unit, ST-ACS: ST-elevation acute coronary

syndrome ··· 4

Fig. 2. Comparison of on-treatment of platelet reactivity between the baseline and at 1 month follow-up according to the personalization strategy for antiplatelet therapy (A), and the type of antiplatelet agents (B) ··· 16

v

List of Tables

Table 1. Baseline characteristics ··· 9

Table 2. Comparison of on-treatment platelet reactivity ··· 12

Table 3. On-treatment platelet reactivity according to CYP2C19 genotype ··· 13

Table 4. Comparison of on-treatment platelet reactivity according to type of P2Y12

vi

ABBREVIATION

ACS = Acute coronary syndromes DAPT = Dual antiplatelet therapy DES = Drug-eluting stent

CV = Cardiovascular

CYP = Cytochrome

HOPR = High on-treatment platelet reactivity

MI = Myocardial infarction

1

-I. INTRODUCTION

The advent of P2Y12 inhibitors with antiplatelet effect has expanded the use of

drug-eluting stent (DES) for occlusive coronary artery disease. Dual antiplatelet therapy (DAPT) with aspirin and clopidogrel has been considered as the standard treatment in patients with acute coronary syndromes (ACS) undergoing percutaneous coronary intervention (PCI), because DAPT has been shown to reduce the incidence of myocardial infarction (MI) or death from cardiovascular (CV) causes (Yusulf et al., 2001; Levine et al., 2011). However, despite of great efficacy of DAPT including clopidogrel, incident ischemic events still occured substantially in patients with ACS (Tang et al., 2007).

Clopidogrel is a prodrug that is converted to its active metabolite principally by cytochrome (CYP) P450 2C19, at which time it inhibits platelet activation by irreversible binding with P2Y12 receptor. Genetic polymorphism in ABCB1, CYP2C19 may affect the

absorption and metabolism, respectively of clopidogrel, and consequently alter its pharmacodynamics. As a result, there exists high inter-individual variability in the responsiveness to clopidogrel and on-treatment platelet reactivity (OPR). High OPR (HOPR) with clopidogrel is associated with an increased risk of ischemic events with patients with ACS undergoing PCI (Lee K et al., 2009; Marcucci et al., 2009; Ahn et al., 2012).

Newer potent P2Y12 inhibitors, such as prasugrel or ticagrelor have been shown to

be superior to clopidogrel in terms of their ability to decrease events in patients with ACS. However, their benefits were limited because of increased episodes of bleeding (Wiviott et al., 2007; Wallentin et al., 2009). Personalization of antiplatelet treatment for ACS may be

2

-necessary to attain the maximum inhibition of platelet activation with minimum risk of bleeding. The recent development of point-of-care assay kits, VerifyNow P2Y12 (Patti et al., 2008; Price, 2009; Price et al., 2011) or Verigene CLO assays (Buchan et al., 2011), for the assessment of platelet function and CYP2C19 polymorphism, respectively, has enabled the immediate assignment of individualized antiplatelet treatment. However, as yet, studies using high-dose clopidogrel or prasugrel have not conclusively demonstrated a clinical benefit of the phenotype- (Price et al., 2011; Trenk et al., 2012) or genotype-directed personalization of antiplatelet therapy (Roberts et al., 2012) based on residual platelet reactivity, or the presence of CYP2C19 loss-of-function alleles

Therefore, the aim of this study was to investigate the feasibility of point-of-care genotypic and phenotypic testing to guide individualized antiplatelet therapy using ticagrelor in Korean patients with non-ST elevation ACS undergoing PCI. We also compared the effectiveness of genotype-guided versus phenotype-guided antiplatelet therapy in terms of decreasing the number of patients with HOPR after 30 days.

3

-II. SUBJECTS AND METHODS

A. Subjects

1. Trial design and study population

The present study was a single center, prospective, randomized, proof-of-concept trial. The study flow is shown in Fig. 1. The study protocol was approved by the Ethical Review Board of Wonju Severance Christian Hospital (Wonju, South Korea). Informed consent was obtained from all participants.

Between April 10, 2012, and February 06, 2013, 65 patients aged 18-75 were randomly assigned to genotype- or phenotype-directed treatment according to a random-number table. Patients were eligible if they had undergone PCI for non-ST-elevation ACS. The patients presenting with ST-segment elevation MI and who had severe left ventricular dysfunction (ejection fraction, <30%), or cardiogenic shock were excluded from the study. Other exclusion criteria were bradyarrhythmia, a history of transient ischemic or cerebrovascular attacks, a platelet count of <150,000/mL, hematocrit <30%, and a creatinine clearance rate of <30 ml/min. Patients who received a periprocedural thombolytic agent or glycoprotein IIb-IIIa inhibitor, or who planned to use oral anticoagulants or other antiplatelet agent such as cilostazol, were also excluded.

4

-Fig. 1. Schematic diagram of the study. CYP = cyptochrome; PCI = percutaneous coronary intervention; PRU = P2Y12 reaction unit; ST-ACS = ST-elevation acute coronary syndromes.

5

-B. Methods

1. VerifyNow P2Y12 test

OPR was measured using the VerifyNow P2Y12 assay (Accumetrics, San Diego, CA, USA). Blood samples were obtained 12 to 24 hours after PCI and after a 30-day follow-up period. Each sample was placed in a tub containing 3.2% citrate, and the P2Y12 reaction unit

(PRU) was assessed within 2 hours, as previously described (Price, 2009). The HOPR was defined as PRU value ≥230 based on previous studies involving Caucasian subjects (Patti et al., 2008; Bonello et al., 2010). Although studies conducted among Korean subjects revealed that HOPR of an approximate PRU of 270 is necessary to predict future cardiovascular event (Ko et al., 2011; Ahn et al., 2012; Park et al., 2012), we selected a PRU value of 230 as the cutoff, in order to include more patients who might benefit from phenotype-guided antiplatelet therapy.

2. Verigene CLO assay

All patients were screened for the CYP2C19 *2, *3, or *17 allele by using the Verigene CLO assay (Nanosphere, Northbrook, IL, USA). The Verigene CLO assay is an automated sample-to-result microarray-based assay in which DNA extracted from whole blood samples is hybridized to allele-specific probes immobilized on a glass slide. The detection of captured DNA is achieved using nanoparticle-conjugated probes that have been demonstrated to provide excellent sensitivity and that eliminate the need for a target amplification step prior to array hybridization (Jannetto et al., 2010). The Verigene CLO assay accurately identified homozygous and heterozygous *2 and *3 phenotypes with

6

-specificity of 100% and a final call rate 99.7%. The assay automated and can produce a result in approximately 3.5 hours (Saracini et al., 2012).

3. Phenotype- versus genotype-guided antiplatelet regimen

Regardless of their prior exposure to clopidogrel, all patients received 300 mg of aspirin and 300 mg of clopidogrel before arriving at the catheterization room. A bolus of unfractionated heparin (70 U/kg) was administered immediately before coronary angiography though the introducer sheath. A second bolus of unfractionated heparin (70 U/Kg) was administered immediately before the PCI. Additional heparin was administered to achieve an activated clotting time of 250–300 s. PCI was performed using balloon predilatation, followed by DES deployment via transradial or transfemoral arteries. The choice regarding the specific type of DES was left to be determined by the operator’s discretion. All patients were given 100 mg of aspirin daily after PCI. Patients with HOPR or who were CYP2C19 *2 or *3 carriers were also given 90 mg of ticagrelor twice daily. Patients without HOPR or who were non-carriers were given of 75 mg clopidogrel daily (Fig. 1).

4. Definitions and endpoints

The primary endpoint was the percentage of patients with HOPR after 30 days of DAPT. The secondary endpoints were 1) PRU, ∆PRU, %IPA, and ∆%IPA, 2) the correlation between the presence of CYP2C19 *2 or *3 carriers and HOPR at baseline, 3) the incidence of bleeding by Bleeding Academic Research Consortium (BARC) definition (Mehran et al., 2011), 4) major adverse cardiac events (MACE) defined as the composite of death from CV

7

-causes, non-fatal myocardial infarction, ischemia-driven target lesion revascularization or stent thrombosis.

5. Statistical analysis

The proportion of patients with HOPR after 1 month of DAPT in the genotype-directed group is assumed to be 5% (Roberts et al., 2012). On a ratio basis of 80%, we calculated that we needed 44 subjects per group to detect a 20% difference the groups. The test statistic used was the two-sided Z test with pooled variance. The significance level of the test was targeted at 0.05. Assuming a dropout rate of 14%, we would therefore need 50 patients each in the genotype- and phenotype-directed groups, forming a total of 100 patients. Enrollment was ceased at 65 patients because the number of Verigene CLO assay kits was insufficient. Analysis was based on intent-to-treat or per-treatment where necessary. All continuous variables are presented as means±SD and were analyzed using the Student’s t-test. Categorical variables are presented as frequencies (percentage), and were analyzed using the Chi-square test or Fisher’s exact test. We compared the change in platelet reactivity between baseline and after 1 month in the genotype-guided and phenotype-guided groups with Student’s t-test. The significance level was defined as p-value <0.05. All analyses were performed with SPSS 20.0 (SPSS Inc., Chicago, Ill, USA).

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-III. RESULTS

Of the 32 patients of the genotype-directed group, 21 CYP2C19 *2 or *3 carriers (65.6%) were administered ticagrelor and 11 CYP2C19 *2 or *3 non-carriers (34.4%) were administered clopidogrel. Of the 33 patients of the phenotype-directed group, 11 patients with HOPR (33.3%) were administered ticagrelor and 22 patients without HOPR (66.7%) were administered clopidogrel. In the genotype-directed group, there were five crossover cases from ticagrelor to clopidogrel because of dyspnea (2 patients), nasal bleeding (1 patient), petechia on the whole body (1 patient), and generalized rash (1 patient). In the phenotype-directed group, one crossover from ticagrelor to clopidogrel occurred due to dyspnea. Sixty-one patients (95.3%) had follow-up Verifynow P2Y12 assay, 31 and 30 in the genotype- and phenotype-directed groups. respectively (Fig. 1). Baseline clinical and procedural characteristics between the groups are presented in Table 1. The two groups were balanced with respect to age, sex, and histories of diabetes mellitus, hypertension, hyperlipidemia, current smoker, and MI. The distribution of discharge medication use between the two groups was similar, except for use of clopidogrel and ticagrelor. Clopidogrel was taken more frequently and ticagrelor less frequently by patients in the phenotype-guided group. (clopidogrel: 34.4% vs. 66.7%, p=0.009, genotype vs. phenotype, p=0.009; ticagrelor: 65.6% vs. 33.3%, genotype vs. phenotype, p=0.009). The indications for PCI, left ventricular ejection fraction, and distribution of vessels stented did not differ between the groups. Procedural variables including the extent of coronary artery disease, stent number, total stent length, and mean stent diameter were also similar.

9

-Table 1. Baseline characteristics

Genotype-directed (n=32) Phenotype-directed (n=33) p Age (years) 60±10 61±9 0.494 Male sex, n (%) 22 (68.8) 26 (78.8) 0.357 Body mass index (kg/m2_{) 25.1±2.7 24.6±3.0 0.467}

Medical history, n (%)

Hypertension 16 (50) 17 (51.5) 0.903 Diabetes mellitus 9 (28.1) 6 (18.2) 0.341 Hyperlipidemia 7 (21.9) 2 (6.1) 0.082 Current smoking 11 (34.4) 13 (39.4) 0.675 Prior myocardial infarction 2 (6.3) 1 (3) 0.613 Discharge medications, n (%)

Aspirin 32 (100) 33 (100)

Clopidogrel 11 (34.4) 22 (66.7) 0.009 Ticagrelor 21 (65.6) 11 (33.3) 0.009 β-blocker 16 (50) 20 (60.6) 0.390 ACE inhibitor or ARB 25 (80.6) 22 (66.7) 0.206 Calcium channel blocker 6 (18.8) 4 (12.1) 0.511

Statin 29 (90.6) 33 (100) 0.114

Indication for procedure, n (%) 0.835 Unstable angina 24 (75) 24 (72.7)

- 10 -

NSTEMI 8 (25) 9 (27.3)

Left ventricular ejection fraction (%) 61±10 64±9 0.323

Vessels stented 0.900

Left main artery 2 (6.3) 1 (3) Left anterior descending artery 15 (46.9) 16 (48.5) Circumflex artery 6 (18.8) 6 (18.2) Right coronary artery 9 (28.1) 10 (30.3) Procedural variables

Vessels/patent 1.3±0.5 1.3±0.5 0.943 Stents/patient 1.8±1.1 1.5±0.8 0.271 Total stent length (mm) 40±26 35±26 0.420 Stent diameter (mm) 3.2±0.4 3.4±1.0 0.478

Values are expressed as number (%) or mean±SD. ACE = angiotensin-converting enzyme; ARB = angiotensin receptor blocker; NSTEMI = non-ST-segment elevation myocardial infarction.

- 11 -

The comparison of OPR according to the personalization strategy of DAPT is as shown in Table 2. At the baseline, there were no differences in OPR, %IPA and the percentage of patients with HOPR between the groups. OPR decreased following both genotype- and phenotype-directed therapies (242±83 vs. 109±90, p<0.001 in the genotype-directed group; 216±74 vs. 109±90, p=0.001 in the phenotype-directed group). At day 30, the percentage of patient with HOPR tended to be higher in the genotype-directed group (16.2% vs. 3.3%, p=0.086). At the 30-day follow-up, PRU and %IPA were similar between the groups. However, ∆PRU was higher in the genotype- versus the phenotype-directed group (156±115 vs. 83±124, p=0.027). This may be due to the fact that more patients were administered ticagrelor in the genotype-directed group (51.6% vs. 33.3%, p=0.009).

Approximately 35% of patients were heterozygous for CYP2C19 *2 or *3 carriers. 23% were homozygous for CYP2C19 *2 or were carriers of CYP2C19 *2/*3 (Table 3). Baseline OPR was higher in patients expressing CYP2C19 *2/*2 or CYP2C19 *2/*3 compared to that in patients expressing wild-type CYP2C19*1/*1 or CYP2C19*1/*17 and in patients heterozygous for CYP2C19 *2 or *3 (280±50 vs. 214±80, p=0.004). However, no difference was found in the baseline OPR between CYP2C19 *2 or *3 non-carriers and those heterozygous for CYP2C19 *2 or *3 (217±84 vs. 209±78, p=0.880). After 30 days, ∆OPR from the baseline to follow-up was higher among patients expressing CYP2C19 *2/*2 or

- 12 -

Table 2. Comparison of on-treatment platelet reactivity

Genotype-directed (n=32) Phenotype-directed (n=33) p Baseline

Patients with PRU values ≥230, n (%) 17 (53.1) 11 (33.3) 0.107

On-treatment platelet reactivity (PRU) 242±83 216±74 0.175

Inhibition of platelet aggregation (%) 21±24 24±20 0.539

Follow-up (Day 30)a _{30±3 31±2 0.796}

Patients with PRU values ≥230, n (%) 5 (16.2) 1 (3.3) 0.086

On-treatment platelet reactivity (PRU) 109±90 131±79 0.337

Change in PRU from baseline 156±115 83±124 0.027

Inhibition of platelet aggregation (%) 66±28 53±31 0.246

Use of ticagrelor 16 (51.6) 10 (33.3) 0.009

At least one copy of CYP2C19 *2 or *3, n (%) 21 (65.6) 17 (51.5) 0.248

CYP2C19 *2/*2 or CYP2C19 *2/*3, n (%) 9 (28.1) 6 (18.2) 0.341

a_{61 patients (95.3%) had follow-up VerifyNow P2Y12 assay: 31 in the genotype-directed}

group and 30 in the phenotype-directed group. CYP = cytochrome; PRU = P2Y12 reaction

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Table 3. On-treatment platelet reactivity according to CYP2C19 genotype *1/*1 or *1/*17 (n=27) *2 or *3 heterozygote (n=23) *2/*2 or *2/*3 (n=15) p Baseline

Patients with PRU values ≥230, n (%) 10 (38.5) 8 (34.8) 10 (66.7) 0.124

On-treatment platelet reactivity (PRU) 217±84 209±78 279±50*† _0.015

Inhibition of platelet aggregation (%) 28±22 25±24 7±10‡§ _0.008

Follow-up (Day 30)a _{n=26 n=20 n=15}

Patients with PRU values ≥ 230, n (%) 5 (20) 2 (10) 1 (11.1) 0.566

On-treatment platelet reactivity (PRU) 155±76 113±94 91±92 0.063

Change in PRU from baseline 61±106 106±125 180±120∥ 0.005

Inhibition of platelet aggregation (%) 47±28 60±31 67±33 0.095

Ticagrelor use, n (%) 5 (19.2) 10 (50) 11 (73.3) 0.002

a_{Follow-up VerifyNow P2Y12 assay was not available in one patient with CYP2C19 *1/*1 or} CYP2C19 *1/*17 and 3 patients with CYP2C19 *2 or *3 heterozygote. *_{p=0.039 vs.}

CYP2C19 *1/*1 or CYP2C19 *1/*17. †_{p=0.019 vs. CYP2C19 *2 or *3 heterozygote.} ‡_{p=0.007 vs. CYP2C19 *1/*1 or CYP2C19 *1/*17.} §_{p=0.046 vs. CYP2C19 *2 or *3}

heterozygote. ∥_{p = 0.004 vs. CYP2C19 *1/*1 or CYP2C19 *1/*17. Abbreviations as in}

- 14 -

The number of patients with HOPR at baseline was greater in those allocated ticagrelor; however, none had HOPR after 30 days of treatment. The number of patients with HOPR did not change after 30 days of clopidogrel treatment (Table 4). Baseline OPR was significantly higher in patients slated to receive ticagrelor. After 30 days of treatment, OPR was lower (49±30 vs. 179±77, p <0.001) in patients taking ticagrelor compared to clopidogrel (Fig. 2); accordingly, ∆PRU from baseline to follow-up was higher (84±9 vs. 34±26, p <0.001) in patients given ticagrelor. There were no MACEs in either group during the 1-month follow-up period. There were two nuisance BARC type 2 bleeding events in the genotype-directed group related to ticagrelor use.

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Table 4. Comparison of on-treatment platelet reactivity according to type of P2Y12

inhibitor Clopidogrel (n=33) Ticagrelor (n=32) p Baseline

Patients with PRU values ≥230, n (%) 5 (15.2) 23 (82.1) <0.001

On-treatment platelet reactivity (PRU) 192±63 267±77 <0.001

Inhibition of platelet aggregation (%) 32±19 13±21 <0.001

Follow-up (Day 30)a

Patients with PRU values ≥230, n (%) 8 (22.2) 0 0.017

On-treatment platelet reactivity (PRU) 179±77 49±30 <0.001

Inhibition of platelet aggregation (%) 34±26 84±9 <0.001

Change in PRU from baseline 15±64 239±45 <0.001

At least one copy of CYP2C19 *2 or *3, n (%) 11 (33.3) 27 (84.4) <0.001

CYP2C19 *2/*2 or CYP2C19 *2/*3, n (%) 2 (6.1) 13 (40.6) 0.001

a_{At 30 days, 36 patients were given clopidogrel and 25 patients were given ticagrelor.}

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Fig. 2. Comparison of on-treatment of platelet reactivity (OPR) between baseline versus 1–month follow-up (D 30) according to the personalization strategy for antiplatelet therapy (A) and the type of antiplatelet agent (B). OPR decreased following both and phenotype-directed therapy (242±83 vs. 109±90, p <0.001 in the genotype-directed group; 216±74 vs. 109±90, p=0.001 in the phenotype-genotype-directed group). Five subjects (16.2%) in the genotype-directed group and one (3.3%) in the phenotype-directed group had HOPR after 30 days of treatment (p=0.086). After 30 days of treatment, OPR was lower in patients taking ticagrelor compared to clopidogrel (49±30 vs. 179±77, p <0.001).

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IV. DISCUSSION

This was the first study that demonstrated the feasibility and effectiveness of antiplatelet therapy tailored by point-of-care genetic or platelet function testing to decrease the OPR in patients undergoing PCI. The major findings in the present study were that OPR was effectively decreased following both genotype- and phenotype-directed therapy and compared to clopidogrel, ticagrelor caused higher decrease in OPR regardless of the

CYP2C19 genotype.

Heightened platelet reactivity contributes to the occurrence of ischemic events especially in certain clinical subsets such as ACS, chronic kidney disease, or diabetes mellitus. HOPR with clopidogrel treatment has also been proposed to be a predictor of ischemic CV events in patients undergoing DES implantation. To date, there is little consensus on the treatment options to overcome HOPR. Studies examining the effect of high-versus standard-dose clopidogrel have yielded discrepant findings. Mehta et al. noted a reduction in CV events and stent thrombosis in patients given double-dose clopidogrel, whereas Price et al. found no effect on CV outcomes (Mehta et al., 2010; Price et al., 2011). Price et al. attributed this difference to the selection only of patients with HOPR in their study, whereas Mehta et al. enrolled patients regardless of platelet reactivity. Although the addition of cilostazol to DAPT lowered PRU levels more than DAPT, the effect of triple versus double therapy on composite CV outcomes was inconsistent in patients undergoing PCI (Chen et al., 2009; Suh et al., 2011). The more potent antiplatelet agents, prasugrel or ticagrelor, have been shown to be superior to clopidogrel in terms of reducing CV events, but with a higher occurrence of bleeding episodes (Wiviott et al., 2007; Wallentin et al.,

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2009), which is associated with a higher risk of CV events (Eikelboom et al., 2006). In the present study, ticagrelor substantially reduced OPR after 30 days. However, two patients ceased taking ticagrelor use because of nasal bleeding and the appearance of petechiae on the whole body. Because of the concern of the increased risk of bleeding, the use of parasugrel or ticagrelor is not yet widespread in Korea, even among the ACS population after PCI. Further study is therefore warranted on the effects of reduced doses of prasugrel or ticagrelor on the clinical endpoints in Korean ACS patients.

The use of point-of-care testing to tailor antiplatelet therapy according to risk stratification for bleeding and ischemia represents an ideal strategy for ACS patients. Among these tests, the VerifyNow P2Y12 assay to measure OPR is increasingly being used given the evidence suggesting an association between HOPR and CV events (Patti et al., 2008; Bonello et al., 2010). However, there are several shortcomings to measuring OPR: 1) OPR is a surrogate marker only representing the inhibition of P2Y12 receptor mediated platelet

activation, not whole platelet activity; 2) OPR cannot be measured in patients treated with glycoprotein IIb-IIIa inhibitors or thrombolysis; 3) PRU value varies with elapsed time following ACS or PCI (Ahn et al., 2011; Campo et al., 2011); 4) the cutoff for HOPR varies according to patient ethnicity (Ko et al., 2011; Ahn et al., 2012); and 5) the relationship between low OPR and bleeding events has yet to be definitively established. Moreover, trials of personalization of antiplatelet therapy according to the result of VerifyNow P2Y12 assay have failed to demonstrate any improvement in outcomes (Price et al., 2011; Collet et al., 2012; Trenk et al., 2012).

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By comparison, genotyping can be carried out under any clinical circumstances with no limitations with respect to timing. In previous studies, CYP2C19 * or *3 carriers have been shown to have higher OPR and poor clinical outcomes, compared with CYP2C19

*2 or *3 non-carriers (Shuldiner et al., 2009). However, genotyping has also its drawbacks.

Clopidogrel is a pro-drug that is converted to an active metabolite in two hepatic steps involving CYP1A2, 2C9, 2C19, 3A4/5, not only by 2C19. To date, point-of-care clopidogrel genetic testing is limited to CYP2C19 single nucleotide polymorphism (Buchan et al., 2011; Roberts et al., 2012); yet the CYP2C19 *2 genotype only accounts for approximately 12% of the variation in clopidogrel response (Shuldiner et al., 2009). The feasibility of genotype-guided prasugrel use has been proven in a proof-of-concept trial (Roberts et al., 2012), but the clinical benefit of genotype-guided antiplatelet therapy has yet to be demonstrated.

Although genetic testing and platelet function testing may provide additive information in selecting an antiplatelet regimen for patients undergoing PCI, their routine use is not recommended in current practice guidelines (Levine et al., 2011). Genotyping may be an improved guide for chronic antiplatelet therapy, while platelet function testing is more useful in acute settings. Nevertheless, the benefits of genotype- and phenotype-directed individualization of antiplatelet therapy to improve clinical outcomes requires validation though further randomized controlled trials.

A potential limitation of the study relates to the early termination of the trial due to the unavailability of the Verigene CLO assay during the study. Furthermore, crossover rate from ticagrelor to clopidogrel is higher in the genotype- versus the phenotype-directed group, possibly due to the higher number of patients allocated ticagrelor in the genotype-directed

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group. Moreover, there was no standard care control group comparing genotype- versus phenotype-directed antiplatelet therapy. Finally, our analyses were based on a single determination of PRU value, which is subject to random measurement error and may have underestimated the strength of the associations.

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V. CONCLUSION

In summary, tailoring the use of P2Y12 inhibitors based on point-of-care genetic or

phenotypic testing is feasible and seems to be efficacious in terms of decreasing HOPR after 30 days.

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- 29 - - 국문요약 -

관상동맥스텐트 시술을 받은 한국인 관상동맥질환 환자에서

유전자형 혹은 표현형에 따른 개인형 맞춤 항혈소판제 치료

아주대학교 대학원의학과 안 성 균 (지도교수: 탁 승 제) 유전자형 혹은 표현형 현장검사에 의한 개인형 맞춤 항혈소판 치료 후, 잔여 혈소판 활성도가 얼마나 효과적으로 감소되는지를 비교하였다. 2012 년 4 월 2013 년 2 월까지 ST 분절 비상승 급성 관동맥증후군으로 관상동맥 스텐트 시술을 받은 65 명의 환자들을 유전자형 혹은 표현형 맞춤 치료군으로 무작위 할당하였다. 모든 환자에서 Verigene CLO assay (Nanosphere, Northbrook, IL, USA)를 통해 CYP2C19*2, *3, 혹은 *17 단일 염기다형성을 측정하였고, 클로피도그렐에 대한 혈소판 활성도는 VerifyNow P2Y12 assay (Accumetrics, San Diego, CA, USA)를 사용하여 측정하였다. 높은 혈소판 반응성 (High on-treatment platelet reactivity; HOPR)은 클로피도그렐 사용 후 P2Y12 반응단위

(P2Y12 Reaction Unit; PRU)가 230 이상인 경우로 정의하였다. CYP2C19 *2, *3 유전자형을 나타내는 21 명의 환자 (65.6%)들과, HOPR 을 보이는 11 명의 환자들(33.3%)에게 티카그렐러 90mg 을 하루에 두번씩 투여하였고, CYP2C19

- 30 - 75mg 을 하루에 한번씩 투여하였다. 일차 종점은 항혈소판제 치료 30 일 이후에 HOPR 을 가지고 있는 환자들의 비율이었다. 30 일째 유전자형 맞춤 치료군에서 5 명의 환자 (16.2%)에서, 표현형 맞춤 치료군에서는 1 명(3.3%)의 환자에서 HOPR 을 가지고 있었다 (p=0.086). 양군 모두 시작보다 1 달 후 HOPR 의 빈도가 의미 있게 감소하였으며 (유전자형 맞춤 치료군: 53.1% vs. 16.2%, 치료 1 일 vs. 치료 30 일, p=0.022; 표현형 맞춤 치료군: 33.3% vs. 3.3%, 치료 1 일 vs. 치료 30 일, p=0.012), PRU 도 의미 있게 감소하였다 (유전자형 맞춤 치료군: 242±83 vs. 109±90, 치료 1 일 vs. 치료 30 일, p<0.001; 표현형 맞춤 치료군: 216±74 vs. 109±90, 치료 1 일 vs. 치료 30 일, p=0.001). 티카그렐러를 투여 받은 모든 환자에서 치료 후 30 일에 HOPR 은 관찰되지 않았다. 반면, 클로피도그렐 투여 받은 환자에서는 치료 하루와 30 일 사이에 높은 혈소판 활성도에 차이가 없었다. 결론적으로 유전자형 혹은 표현형 현장검사에 의한 개인형 맞춤 항혈소판제 치료는 임상에서 쉽게 적용할 수 있으며, 치료 30 일 후 HOPR 을 감소시키는 데에 효과적이다. 핵심어: 항혈소판 약제, 유전자형 검사, 혈소판 기능검사, 현장검사