Ticlopidine with Ginkgo Biloba extract: A Feasible Combination for Patients with Acute Cerebral Ischemia

Thrombosis Research 131 (2013) e147–e153

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Regular Article

Ticlopidine with Ginkgo Biloba extract: A Feasible Combination for Patients with Acute Cerebral Ischemia☆ Ji Man Hong a,⁎, Dong Hoon Shin b, Young Ae Lim c, Jin Soo Lee a, In Soo Joo a a b c

Department of Neurology, Ajou University School of Medicine, Suwon, South Korea Department of Neurology, Gachon University Gil Hospital, Gachon Univerity of Medicine and Science, Incheon, South Korea Department of Laboratory Medicine, Ajou University School of Medicine, Suwon, South Korea

a r t i c l e

i n f o

Article history: Received 24 September 2012 Received in revised form 5 December 2012 Accepted 2 January 2013 Available online 7 March 2013 Keywords: cerebral ischemia Ginkgo biloba extract combination antiplatelet thiennopyridine cytochrome

a b s t r a c t Background: Even though clopidogrel is the most used drug for cardiovascular prevention, resistance occurs in significant numbers. Therefore, we evaluated platelet aggregation ability of thienopyridines in relation with various genotypes. Method: The study population was randomly assigned with clopidogrel (n = 43), ticlopidine (n = 41), or ticlopidine plus Gingko Biloba extract (EGb) (n = 43). Dosage was maintained as 75 mg clopidogrel daily, 250 mg ticlopidine twice daily, and 250 mg ticlopidine plus 80 mg Gingko Biloba extract twice daily. Using multiple electrodes aggregometry, platelet aggregation was measured by activators of adenosine diphosphate (ADP), arachidonic acid (ASP), and thrombin (TRAP) at baseline (T0), 7 days (T1), and 90 days (T2). Side-effects were analyzed in the 3 groups. Inhibition of platelet aggregation (IPA) was defined as percent decrease at T0 and T1. Non-responsiveness (bIPA 20%) was analyzed according to cytochrome P450 polymorphisms. Results: There was no difference of general demographics and platelet aggregation at baseline in all groups. A significant difference of platelet aggregation showed on ADP test in the groups at T1 (28.9±17.2 vs.22.7±11.1 vs. 14.6±10.3%, pb 0.001) and T2 (27.5±24.5 vs.18.3±16.6 vs. 14.4±9.8%, p=0.007), whereas ASP (p=0.064) and TRAP tests (p=0.143) had no differences at T1. Serious adverse events had no differences among the groups (p = 0.902). CYP2C19 *2 alleles had poor responsiveness of clopidogrel (p = 0.038), and not in ticlopidine (p = 0.780). Conclusions: This finding suggests that ticlopidine plus Gingko Biloba extract has sufficient anti-platelet abilities with an acceptable profile of adverse events and CYP2C19 *2 alleles are associated with clopidogrel responsiveness. Published by Elsevier Ltd.

Introduction Ticlopidine and clopidogrel are some of the thienopyridine antiplatelet agents that are used for the prevention of cardiovascular events [1]. Clopidogrel has been preferred to ticlopidine because of its safety and tolerability [2]. Nevertheless, the anti-platelet responsiveness to clopidogrel had considerable variability and current data showed that about 4% to 30% of patients had inadequate responsiveness [3,4]. A meta-analysis reported that 21% of patients had low responses to clopidogrel therapy and were associated with an 8-fold increased risk of all cardiovascular events [5]. The variability in response to clopidogrel has been linked to its cytochrome (CYP) P450 dependent oxidative steps which include

☆ Funding: None. ⁎ Corresponding author at: Department of Neurology, School of Medicine, Ajou University, 5 San, Woncheon-dong, Yongtong-gu, Suwon-si, Kyunggi-do, 442–749, South Korea. Tel.: + 82 31 219 5175; fax: + 82 31 219 5178. E-mail address: [email protected] (J.M. Hong). 0049-3848/$ – see front matter. Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.thromres.2013.01.026

CYP2C19 and CYP3A4 [6–8]. Moreover, it has recently been reported that omeprazole, a proton pump inhibitor, decreases the antiplatelet effect of clopidogrel possibly due to the inhibition of CYP2C19 enzyme metabolism [9]. Aleil et al. asserted that ticlopidine can be an alternative agent for patients who have clinical and biological non-responsiveness to clopidogrel [10]. Another study reported that the clopidogrel group had a significantly higher proportion of thrombotic stent occlusion compared to the ticlopidine group; in which the patients had stent implantations using dual anti-platelet regimens of aspirin plus ticlopidine or aspirin plus clopidogrel given for up to 4 weeks [11]. Meanwhile, a study reported that poor responsiveness to either clopidogrel or ticlopidine at steady state was common, whereas non-responders to both drugs were relatively infrequent (3.5%), which suggests that poor response to thienopyridines can be a frequent drug-specific mechanism [12]. The extract of Ginkgo Biloba has been shown to have neuroprotective and antioxidant properties against various cardiovascular and neurological diseases [13,14]. It inhibits platelet aggregation and induces vasodilation as a platelet activating factor antagonist [15]. An experiment

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Table 1 Demographics of clopidogrel, ticlopidine, and ticlopidine-plus groups. Characteristic

General demographics Age, y r Female, n (%) Risk factors, n (%) Hypertension Diabetes mellitus Current smoker Hyperlipidemia Cardiac problem Initial laboratory findings Hemoglobin, g/dl Platelet, ×103 /ul Total cholesterol, mg/dl D-dimer C-reactive protein Stroke subtype LAD SAD CE TIA Unknown NIHSS on admission

Clopidogrel group (n = 43)

Ticlopidine group (n = 41)

Ticlopidine plus group (n = 43)

p

63.6 ± 12.8 19 (44.2)

58.1 ± 12.2 10 (24.4)

62.8 ± 11.3 15 (34.9)

0.083 0.163

36 (85.7) 14 (33.3) 14 (32.6) 10 (23.3) 1 (2.4)

34 (82.9) 8 (19.5) 18 (43.9) 16 (39.0) 2 (4.9)

31 (72.1) 15 (34.9) 18 (41.9) 15 (34.9) 1 (2.3)

0.250 0.238 0.337 0.274 0.751

14.1 ± 1.6 235 ± 64 186.5 ± 41.4 154.5 ± 98.2 0.49 ± 1.25

14.7 ± 1.7 231 ± 83 193.7 ± 36.4 136.7 ± 132.2 0.28 ± 0.55

14.3 ± 1.5 231 ± 64.0 193.3 ± 34.1 204.2 ± 288.7 0.30 ± 0.57

0.122 0.950 0.610 0.238 0.502 0.471

10 (23.3) 25 (55.6) 0 (0.0) 2 (4.7) 6 (14.0) 2.86 ± 2.58

7 (17.1) 26 (63.4) 0 (0.0) 5 (12.2) 3 (7.3) 3.05 ± 2.65

14 (32.6) 20 (46.5) 1 (2.3) 3 (7.0) 5 (11.6) 2.86 ± 2.28

detail [17,18]. Randomization was performed by a computer generational block card randomization procedure. This study was conducted from March 2009 to June 2011. The institutional review board at Ajou University Medical Center approved the protocol and all participants provided written informed consent. Eligible patients were over 29 years of age were within 7 days after symptom onset and had an acute ischemic stroke or a transient ischemic attack (TIA). Patients were eligible for the study if had (i) ischemic stroke confirmed by diffusion weighted imaging, or (ii) transient ischemic attack. We excluded (i) patients who had an intracranial hemorrhage by imaging study, (ii) patients who took previous anti-platelet agents, vitamin K antagonists, factor Xa antagonists, chronic treatment with systemic steroidal and non steroidal anti-inflammatory drugs, (iii) patients who received fibrinolytics within the previous 48 hours, (iv) cognitive impairment interfering with the possibility of obtaining informed consent, pregnancy, and participation in another pharmacological study, (v) peptic ulcer disease and platelet or other hematological abnormality, (vi) patients with a 2-fold upper range value on liver function tests. Specimen Collection and Platelet Function Testing

0.924

LAD=large artery disease, SAD=small artery disease, CE=cardioembolism, TIA=transient ischemic attack.

demonstrated that the antithrombotic and antiplatelet effects were augmented with Ginkgo biloba and ticlopidine [16]. Therefore, we compared the platelet aggregation ability of thienopyridines (ie. clopidogrel, ticlopidine, and ticlopidine plus Ginkgo biloba) and analyzed the association between genetic polymorphisms of cytochrome P450 genes. Methods Study Design and Participants The study population was prospectively assigned to one of three groups in an open-label fashion, to undergo a three-arm parallel treatment regimen with a single dose scheme: 75 mg clopidogrel daily; 250 mg ticlopidine twice daily; and 250 mg ticlopidine plus 80 mg Ginkgo Biloba extract (ticlopidine plus) twice daily. According to previous studies, the adverse effects of ticlopidine were explained in

Venous blood samples were collected in 3.2% sodium citrate (Becton Dickinson, USA) for platelet aggregation test at the following time points: T0 at baseline, before antiplatelet administration; T1 at 7 days after antiplatelet administration; T2 at 90 days after antiplatelet administration. Platelet function tests were performed using Multiplate device (Dynabyte Medical, Munich, Germany) immediately after venipuncture in a single laboratory blind to the genetic profile of the study patients. Agonists for platelet function tests were adenosine diphosphate (ADP test, 6.4 μM), arachidonic acid (ASP test, 0.5 mM), and thrombin (TRAP test, 32 μM). Parameter for analysis of platelet activation was used by the area under the curve (AUC). The mean values of the 2 independent determinations were expressed as the AUC of the aggregation tracing [19,20]. Utilizing a previously reported method [12,21], inhibition of platelet aggregation (IPA) was defined as the percent decrease of AUC with aggregation values which was obtained at baseline (T0) and 7-day administration (T1): [IPA (%inhibition)= (AUCT0 – AUCT1)*100 /(AUC T0)]. Non-responder was defined as the absolute difference between baseline and post-administration IPA b 20% or IPAb 30%. The primary efficacy end point was IPAb 20% on the ADP test [12,22]. Genotyping and Adverse Effect Analysis

Table 2 Platelet function tests of clopidogrel, ticlopidine, and ticlopidine-plus groups. Platelet function tests

Clopidogrel group (n = 43)

Ticlopidine group (n=41)

Ticlopidine plus group (n=43)

p

(T0) ADP test ASP test TRAP test (T1) ADP test ASP test TRAP test (T2) ADP test ASP test TRAP test Non-responder, b20% inhibition Non-responder, b30% inhibition Absolute value, ≥35 AUC (T1) Absolute value, ≥40 AUC (T1)

50 (8 to 101) 75 (1 to 126) 112 (29 to 175) 28 (2 to 84) 81 (7 to 144) 110 (21 to 163) 21 (2 to 121) 83 (21 to 142) 106 (4 to 187) 13 (30.2)

49 (16 to 86) 72 (3 to 111) 95 (6 to 155) 21 (6 to 48) 77 (9 to 122) 92 (6 to 157) 15 (2 to 84) 61 (7 to 148) 77 (4 to 187) 5 (12.2)

43(12 to 113) 72 (2 to 133) 106 (37 to 169) 13 (2 to 61) 65.5 (13 to 122) 92 (9 to 149) 13 (1 to 49) 53.5 (4 to 109) 80 (35 to 146) 3 (7.1)

0.723⁎ 0.686⁎ 0.093⁎ b0.001⁎ 0.064⁎ 0.143⁎ 0.007⁎ 0.001⁎ 0.013⁎ 0.011

17 (39.5)

7 (17.1)

4 (9.5)

0.002

13 (30.2)

7 (17.1)

2 (4.7)

0.007

11 (25.6)

5 (12.2)

1 (2.3)

0.006

Adenosine diphosphate (ADP) test, arachidonic acid (ASP) test, thrombin (TRAP) test. Numbers represent median (minimum to maximum) or count (percentage). ⁎ : Kruskal-Wallis test.

Venous blood samples were taken in tubes containing trisodium EDTA and were promptly centrifuged. The buffy coat was stored in the deep freezer at − 70 °C. Genomic DNA was extracted from peripheral-blood leukocytes by use of standard procedure. Genotyping for CYP 2C19 *2, *3, *17, CYP3A4*18, CYP3A5*3, CYP2B6*4, *6, and *9 alleles, and P2Y12 polymorphisms was performed by a Tag-Man single-nucleotide-polymorphism assay. The bidirectional sequencing or specific polymerase-chain-reaction amplification was done by an automatic DNA sequencer (Applied Biosystems, 3130XL) followed by the use of standard agarose-gel electrophoresis to resolve restrictionfragment-length polymorphisms. After administration, adverse events were classified into serious and non-serious types. Serious type was patients with cardiovascular event or hospitalization and non-serious type was those with dermatologic or other medical problems. Statistical Analysis With a sample of 42 patients per group, sample-size calculation (PASS 2008, USA) were based on an assumption as the equality of

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Table 3 Associations between CYP polymorphisms and ADP responsiveness (n = 127). Genotypes CYP 2C19 *17, -806C>T CC CT TT *2, 681G>A GG GA AA *3, 636G>A GG GA AA CYP2B6 *4, 785A>G AA AG GG *9, 516G>T GG GT TT CYP3A4 *18,878T>C TT TC CC CYP3A5 *3, 6986A>G AA AG GG P2Y12 H1/H2, 742T>C TT TC CC

Non-responder (b20%, n = 21)

Responder (≥20%, n = 106)

20 (95.2) 1 (4.8) 0 (0.0)

106 (100.0) 0 (0.0) 0 (0.0)

9 (42.9) 10 (47.6) 2 (9.5)

60 (56.6) 42 (39.6) 4 (3.8)

15 (71.4) 6 (28.6) 0 (0.0)

85 (80.2) 19 (17.9) 2 (1.9)

15 (71.4) 6 (28.6) 0 (0.0)

72 (67.9) 30 (28.3) 4 (3.8)

15 (71.4) 6 (28.6) 0 (0.0)

80 (75.5) 24 (22.6) 2 (1.9)

19 (90.5) 2 (9.5) 0 (0.0)

105 (99.0) 1 (1.0) 0 (0.0)

2 (9.5) 10 (47.6) 9 (42.9)

8 (7.5) 39 (36.8) 59 (55.7)

14 (66.7) 7 (33.3) 0 (0.0)

78 (73.6) 24 (22.6) 4 (3.8)

P

Non-responder (b30%, n= 28)

Responder (≥30%, n = 99)

27 (96.4) 1 (3.6) 0 (0.0)

99 (100.0) 0 (0.0) 0 (0.0)

14 (50.0) 12 (42.9) 2 (7.1)

55 (55.6) 40 (40.4) 4 (4.0)

19 (67.9) 8 (28.6) 1 (3.6)

81 (81.8) 17 (17.2) 1 (1.0)

22 (78.6) 6 (21.4) 0 (0.0)

65 (65.7) 30 (30.3) 4 (4.0)

22 (78.6) 6 (21.4) 0 (0.0)

73 (73.7) 24 (24.2) 2 (2.1)

26 (92.9) 2 (7.1) 0 (0.0)

98 (99.0) 1 (1.0) 0 (0.0)

2 (7.1)) 15(53.6) 11 (39.3)

8 (8.1) 34 (34.3) 57 (57.6)

19 (67.9) 9 (32.1) 0 (0.0)

73 (73.7) 22 (22.2) 4 (4.1)

0.167

0.222

0.348

0.741

0.455

0.236

0.662

0.322

0.706

0.702

0.071

0.122

0.561

0.176

0.421

the AUC values at the administration steady state (T1) by one way ANOVA analysis among the three groups. The statistical power of the study was set at 80%, given a standard deviation of 20, with a type I error of 0.05 for platelet aggregation ability. To allow for a 5% drop-out rate, 45 patients were randomly assigned per each group. All analysis was done with data for all patients at randomization minus those lost to follow-up. For the analysis of the differences among the groups, comparison among treatment groups were done with χ 2 test or Fischer's exact test for binary categorical variables. We also assessed continuous variables between groups, including the primary endpoint, with ANOVA test unless otherwise specified. The values were analyzed via the Kruskal-Wallis test among the groups when the data is not normally distributed in the Shapiro-Wilk analysis. Furthermore, a Bonferroni correction for post-hoc analysis was applied to adjust for the comparison of the groups if there were significant differences among the groups at the administration steady states of T1 and T2. Differences between treatment groups in the incidence of adverse events were assessed with χ2 test. Statistical analyses were performed using SPSS software (version 16, Chicago, IL). Differences were considered statistically significant with p values less than 0.05. Results General Demographics Table 1 shows the details relating to the baseline demographics according to the three thienopyridines. Supplementary Fig. 1 shows a trial profile that a total of 184 were screened and 135 patients were enrolled and randomly allocated to the three groups: clopidogrel,

p

0.347

ticlopidine, and ticlopidine plus. The primary analysis (platelet aggregation at T1and T0) was performed in 127 patients. There were no differences of general demographics (sex, age, stroke risk factors, and initial laboratories, and NIH stroke scale) between the groups. Stroke subtypes among the groups showed no difference. At the 3-month follow-up visit, 112 patients were still receiving a maintenance dose of various thienopyridines. Platelet Aggregation Ability Table 2 and Fig. 1 show the platelet aggregation ability among the groups. There was no difference of various platelet aggregation abilities at baseline (T0) in clopidogrel, ticlopidine, and ticlopidine plus groups (ADP test: 49.7± 22.2 vs. 50.3± 19.6 vs. 48.8± 23.3%, p = 0.723; ASP test: 69.6 ± 38.5 vs. 65.8 ± 31.6 vs. 72.3 ± 32.4%, p =0.686; TRAP test: 107.1 ±34.1 vs. 92.2 ±32.2 vs. 105.1 ±33.7%, p = 0.093). At 7 days (T1), clopidogrel, ticlopidine, and ticlopidine plus groups showed a significant difference of platelet aggregation on the ADP test respectively (28.9±17.2 vs. 22.7±11.1 vs. 14.5±10.2%, pb 0.001), whereas there were no significant differences of platelet aggregation power on ASP and TRAP tests (78.7 ± 28.2 vs. 69.2 ± 28.7 vs. 65.8 ± 25.7%, p = 0.064; 101.3 ± 32.2 vs. 86.3 ± 34.9 vs 90.7 ± 30.2%, p = 0.143). In post-hoc analysis on the ADP test at T1, ticlopidine plus group was significantly different from clopidogrel group (ticlopidine plus vs. clopidogrel; p b 0.001). At 90 days (T2), a significant difference of platelet aggregation showed on the ADP test in the groups (27.5 ± 24.5 vs. 18.3 ± 18.6 vs. 14.4 ± 9.8%, p = 0.007), and there also were significant differences of platelet aggregation abilities on ASP and TRAP tests (81.1 ± 29.6 vs.

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Table 4 Associations between CYP polymorphisms and ADP responsiveness according to the class of medication (clopidogrel vs. ticlopidine). Genotypes

CYP 2C19 *17, -806C>T CC CT TT *2, 681G>A GG GA AA *3, 636G>A GG GA AA CYP2B6 *4, 785A>G AA AG GG *9, 516G>T GG GT TT CYP3A4 *18,878T>C TT TC CC CYP3A5 *3, 6986A>G AA AG GG P2Y12 H1/H2, 742T>C TT TC CC

Clopidogrel-based group (n = 43) Non-responder (b20%, n = 13)

Responder (≥20%, n= 30)

13 (100.0) 0 (0.0) 0 (0.0)

30 (100.0) 0 (0.0) 0 (0.0)

4 (30.8) 7 (53.8) 2 (15.4)

18 (60.0) 12 (40.0) 4 (3.8)

10 (76.9) 3 (23.1) 0 (0.0)

24 (80.0) 6 (20.0) 0 (0.0)

10 (76.9) 3 (23.1) 0 (0.0)

19 (63.3) 11 (36.7) 0 (0.0)

10 (76.9) 3 (23.1) 0 (0.0)

21 (70.0) 9 (30.0) 0 (0.0)

12 (92.3) 1 (7.7) 0 (0.0)

29 (96.7) 1 (3.3) 0 (0.0)

1 (7.7) 7 (53.8) 5 (38.5)

1 (3.3) 15 (50.0) 14 (46.7)

9 (30.8) 4 (30.8) 0 (0.0)

20 (66.7) 10 (33.3 0 (0.0)

p

Ticlopidine-based group (n = 84) Non-responder (b20%, n = 8)

Responder (≥20%, n = 76)

7 (87.5) 1 (12.5) 0 (0.0)

76 (100.0) 0 (0.0) 0 (0.0)

5 (62.5) 3 (37.5) 0 (0.0)

42 (55.3) 29 (39.5) 4 (5.3)

5 (62.5) 3 (37.5) 0 (0.0)

61 (80.3) 13 (17.1) 2 (2.6)

5 (62.5) 3 (37.5) 0 (0.0)

53 (69.7) 19 (25.0) 4 (5.3)

5 (62.5) 3 (37.5) 0 (0.0)

59 (77.6) 15 (19.7) 2 (2.6)

7 (87.5) 1 (12.5) 0 (0.0)

76 (100.0) 0 (0.0) 0 (0.0)

1 (12.5) 3 (37.5) 4 (50.0)

7 (9.2) 24 (31.6) 45(59.2)

5 (62.5) 3 (37.5) 0 (0.0)

58 (76.3) 14 (18.4) 4 (5.3)

1.000

0.095

0.038

0.780

0.820

0.353

0.382

0.635

0.642

0.474

0.533

0.095

0.765

0.875

0.869

64.4 ± 29.8 vs. 55.3 ± 28.3%, p = 0.001; 99.1 ± 34.4 vs. 75.5 ± 39.7 vs. 82.2 ± 29.46%, p = 0.013). In post-hoc analysis on the ADP test at T2, ticlopidine plus group was also significantly different from clopidogrel group (p=0.009). Interestingly, there was also significant difference between the respective groups on the ASP test (clopidogrel vs. ticlopidine plus; pb 0.001) and the TRAP test (clopidogrel vs ticlopidine; p=0.003).

Cytochrome P450 Polymorphisms and ADP Responsiveness Table 2 shows associations between p450 polymorphisms and ADP responsiveness. Among 127 patients, the non-responder profile had 21 patients (16.5%) with IPA b 20% and 28 patients (22.0%) with IPA b 30%. In the study population, genetic polymorphisms had no association with ADP responsiveness. According to the medication class (clopidogrel vs. ticlopidine) shown in Table 3, genetic variation of the CYP2C19 *2 alleles had a significant association with non-responsiveness of clopidogrel (p=0.038), but no association with ticlopidine-based non-responsiveness (p=0.780) (Fig. 2).

Adverse Events Profile There was no difference of serious adverse events among the groups (p= 0.902). The number of non-serious adverse events was more common in ticlopidine group during the course of the trial (p= 0.048). Reversible skin problem including rash, itching, and pruritis was the leading cause of non-serious adverse events. See Supplementary Data 2.

p

0.385

Discussion In comparison to the clopidogrel group, ticlopidine-based treatment had a higher proportion of anti-platelet responsiveness shown on the ADP test after administration. Moreover, the ticlopidine plus group had the strongest inhibition response to ADP testing among the groups and this trend continued after 3 months. Serious side-effect profiles in the study population were acceptable and the tendency of adverse events decreased in the ticlopidine plus Gingko group. The non-responders on the ADP test in the clopidogrel group were associated with the carriers of CYP2C19*2 loss-of-function allele, while the ticlopidine group showed no relevance. In the present study, the ticlopidine-based treatment group had higher numbers for responsiveness on the platelet aggregation tests which includes the ADP and ASP pathways [23], and the prevalence was especially higher in the ticlopidine plus group (addition of Ginko biloba). However, the clopidogrel group had a higher prevalence of non-responsiveness to ADP testing and the non-responsiveness rate was consistent with previous studies [3,4]. In addition, the clopidogrel group had a higher proportion of non-responsiveness to ASP and TRAP testing in the present study. Snoep et al. showed that the clopidogrel resistance population is considerable and they are highly susceptible to all cardiovascular events [5]. Moreover, after comparing the effects of clopidogrel and ticlopidine, a recent study showed that clopidogrel based treatments had a significantly higher number of thrombotic events [11]. Another study supported that the poor responsiveness to either clopidogrel or ticlopidine at steady state was common, in which non-responders to both drugs were rare, and a drug-specific mechanism

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P*=0.001 P*=0.093

P*=0.064

P*=0.723

P*=0.143

P*=0.686

P*<0.001

P*=0.013

P*=0.007

Post-hoc analysis

Post-hoc analysis

Post-hoc analysis

T1

Clopidogrel vs. Ticlopidine : Pc=0.168 Clopidogrel vs. Ticlopidine+ : Pc<0.001

Clopidogrel vs. Ticlopidine : Pc=0.396 Clopidogrel vs. Ticlopidine+ : Pc=0.090

Clopidogrel vs. Ticlopidine : Pc=0.129 Clopidogrel vs. Ticlopidine+ : Pc=0.354

T2

Clopidogrel vs. Ticlopidine : Pc=0.222 Clopidogrel vs. Ticlopidine+ : Pc=0.009

Clopidogrel vs. Ticlopidine : Pc=0.066 Clopidogrel vs. Ticlopidine+ : Pc<0.001

Clopidogrel vs. Ticlopidine : Pc=0.003 Clopidogrel vs. Ticlopidine+ : Pc=0.075

Pc = Corrected P by Bonferroni test P*= Kruskal-Wallis test Fig. 1. Changes in platelet aggregation function according to the time sequence (mean ± SEM). There was no difference of various platelet aggregation abilities at baseline (T0) in clopidogrel, ticlopidine, and ticlopidine plus groups (ADP test, p = 0.723; ASP test, p = 0.686; TRAP test, p = 0.093). At 7 days (T1), clopidogrel, ticlopidine, and ticlopidine plus groups showed a significant difference of platelet aggregation on the ADP test respectively (p b 0.001), whereas there were no significant differences of platelet aggregation power on ASP and TRAP tests (p = 0.064 and p = 0.143). At 90 days (T2), a significant difference of platelet aggregation showed on the ADP test in the groups (p = 0.007), and there also were significant differences of platelet aggregation abilities on ASP and TRAP tests (p = 0.001 and p = 0.013).

could be possible for the poor responsiveness to thienpyridines [12]. Although doubling the maintenance dose of clopidogrel enhances platelet inhibition, response to antiplatelet therapy remained variable and 60% of

patients showed poor response [24]. Moreover, a recent large randomized trial reported that high-dose clopidogrel was not superior to a standard dose regimen for a reduction in stent thromboses and other ischemic

(%) P=0.038 P=0.780

Fig. 2. Association of CYP2C19 *2 alleles loss-of-function variant with ADP aggregation in the proportion of reference, responder, and non-responders. Genetic variation of CYP2C19 *2 alleles had a significant association with non-responsiveness of clopidogrel (p = 0.038), but no association with ticlopidine-based non-responsiveness (p = 0.780).

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events in percutaneous coronary intervention patients [25]. In view of doubling the dose of clopidogrel in patients with poor hereditable metabolism, this method is not a recommended solution to overcome genetic risk [26]. Therefore, our data supports that a ticlopidine-strategy can be an alternative solution for patients with clinical and biological non-responsiveness to clopidogrel. Interestingly, the ticlopidine plus group had the strongest inhibition response to ADP and ASP testing among the groups and this trend continued after 3 months in the present study. Ginkgo biloba extract is one of the most popular herbal products, available in various countries [15]. Because Ginkgo biloba has neuroprotective and antioxidant properties, it has been used in various neurological diseases [13,27,28]. In addition, Ginkgo biloba is a direct inhibitor of platelet aggregation to inhibit APD and collagen-induced platelet aggregation in vitro and shows inhibition of thromboxane A2 synthesis and c-AMP increase in vivo [29–31]. Moreover, some experimental studies demonstrated that the antithrombotic and antiplatelet effects could be enhanced by the combination of antiplatelets and Ginkgo biloba extract [15,16]. Therefore, our data indicate that Ginkgo biloba can potentiate an inhibition property of platelet aggregation as an additional ADP inhibitor and like aspirin; it can directly inhibit thromboxane A2 synthesis. In this study, non-responders of clopidogrel on the ADP test were associated with the CYP2C19 genotypic polymorphism, whereas this genetic connection was not shown in the ticlopidine group. Numerous studies reported that non-responders were associated with genetic polymorphisms in patients with clopidogrel treatment, due to cytochrome P450 dependent oxidation which includes CYP2C19 and CYP3A4 metabolism [6–8]. A recent report has shown omeprazole inhibits the CYP2C19 enzyme, which decreases the antiplatelet effect of clopidogrel [9]. Despite the small population of the present data, our results were consistent with previous studies which had an association with the loss of function in 2* allele variation of the CYP2C19 in clopidogrel unresponsiveness [6]. Moreover, a recent Japanese study provided that ticlopidine is an effective therapeutic option for poor metabolizers of CYP2C19 [32]. Despite the short duration of follow-up, there was no significant difference of the serious or non-serious side-effect profiles among the groups, notably no occurrence of neuropenia in patients with ticlopidine-based regimen. A higher number of adverse effects in ticlopidine therapy can result with the replacement to clopidogrel therapy, although several randomized control trials showed that ticlopidine was more effective than aspirin in preventing strokes [33,34]. In general, drug-induced neutropenia occurs within 3 months [35]. Although there was no neuropenia and no abnormal change of white cell count in the enrolled cases, occurrence rate of hematologic dyscrasia should be investigated in larger clinical trials between ticlopidine plus and clopidogrel regimens. This trial has several limitations. First, it was performed with a relatively small sample size and the follow-up period was short because the laboratory surrogate marker (i.e. platelet aggregation value) was the primary outcome in this study. Therefore, a further larger trial that focuses on other clinical primary outcomes such as cardiovascular events and occurrence rate of hematologic dyscrasia would be required because of the fear of adverse effects like neutropenia, agranulocytosis, thrombotic thrombocytopenic purpura (TTP), and aplastic anemia [17]. Second, this study should be interpreted with caution. Applying the findings of this study to other vascular occlusive diseases (i.e. coronary or peripheral artery disease) is limited due to the fact that the enrolled patients of the present study were restricted to acute ischemic stroke or transient ischemic attack. Nonetheless, ticlopidine and Ginkgo biloba can be a feasible combination in ischemic stroke patients because a new generation of thienopyridines (i.e. prasugrel and ticagleror) has a bleeding tendency and compared to the clopidogrel, has no better outcome in stroke patients [36,37]. Given its potent anti-platelet ability, this combination can be helpful in patients with a greater risk of future vascular events, particularly in polyvascular disease and stent insertion state. Finally, further studies are needed to shed light on the connection

between thienopyridine unresponsiveness and genetic background, since Asians have a higher prevalence of CYP2C19 mutation [38]. This is a pilot study given the small number of participants in each group and it was performed only in a single center. Nevertheless, this study can be the basis for a larger multicenter randomized control trial (Table 4). In conclusion, this study shows that ticlopidine plus Gingko Biloba extract has sufficient anti-platelet abilities and an acceptable profile of adverse events. In addition, the results of this study shed new light on patients with high atherosclerotic risk and their high vascular event rates [39]. Supplementary data to this article can be found online at http:// dx.doi.org/10.1016/j.thromres.2013.01.026. Conflict of Interest Statement All authors declare that they have nothing to declare. References [1] Quinn MJ, Fitzgerald DJ. Ticlopidine and clopidogrel. Circulation 1999;100:1667–72. 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