Study design and participants
This study was a randomized, open label, active-controlled, parallel-group, multi-center study. Participants eligible for the study were patients aged 50 years or older with type 2 diabetes mellitus and one or more cardiovascular risk factors (family history of cardiovascular disease, hypertension, smoking history, dyslipidemia, and albuminuria) and without a high risk of bleeding. We excluded participants who were taking cilostazol or aspirin within one month before randomization. Other exclusion criteria included type 1 diabetes mellitus, secondary diabetes, or gestational diabetes; history of macrovascular complication including cardiovascular disease, cerebrovascular disease, and peripheral vascular disease; contraindicated for aspirin or cilostazol; clinically significant thyroid-stimulating hormone value outside the normal range; alanine aminotransferase (ALT) or aspartate aminotransferase (AST) ≥ 2.5 times the upper limit of the normal range; alcohol intake greater than 30 g/day; presence of liver cirrhosis or tumor; continuous use (more than 2 weeks) of antithrombotic agents (Sarpogrelate, Beraprost, Indobufen, Triflusal, Clopidogrel, and Ticlopidine) or nonsteroidal anti-inflammatory drugs within one month or after randomization; current use of wafarin, dicoumarin derivatives, or digoxin; pregnant, nursing, or suspected of being pregnant; and history of gastrectomy. After screening, eligible and consenting participants underwent baseline evaluation (including anthropometric and lifestyle data, vital signs, medical history and concomitant medication, and venous blood and urine samples). All patients were then randomized into two groups: aspirin 100 mg quaque die (QD) or cilostazol 200 mg QD (1:1 matching). The randomization was based on the randomization table for each facility according to registration order. After a 14-day treatment period, participants visited the investigational site and underwent follow-up evaluation (vital signs and venous blood and urine samples); adverse reactions were reported, and the number of remaining pills was determined for evaluation of compliance.
The study was conducted at 2 university hospitals in Korea between October 2016 and July 2019 (ClinicalTrials.gov Identifier: NCT02933788). The study was conducted in accordance with the principles of Good Clinical Practice and was approved by the appropriate institutional review boards and regulatory agencies. All patients provided written informed consent prior to participation.
The primary outcome measure of this study was change in platelet reactivity from baseline to day 14. Platelet reactivity was tested using the Platelet Function Analyzer 100 (PFA-100; Dade Behring, Miami, FL, USA) and the VerifyNow™ Aspirin instrument (Accumetrics Inc., CA, USA). The secondary outcome measures were changes in lipid profiles such as total cholesterol, high density lipoprotein (HDL), low density lipoprotein (LDL), and triglycerides; C-reactive protein (CRP) level; and cluster of differentiation 40 ligand (CD40L) after 14 days of treatment. Safety was assessed by recording major bleeding events (intracranial, gastrointestinal, or other), adverse events (AEs), complete blood count (CBC), blood urea nitrogen (BUN)-to-creatinine (Cr) ratio, and AST/ALT. Adherence to the trial regimen was assessed by pill count, and 70% and lower adherence was defined as nonadherence.
Anthropometric and laboratory measurements
Questionnaires for diabetes duration; current and past medication history; and past medical history for cardiovascular disease, alcohol history, and smoking status were completed. Anthropometric measures (height, weight, waist circumference), systolic blood pressure (SBP), diastolic blood pressure (DBP), heart rate, and clinical laboratory data were assessed at baseline and after 14 days of treatment. Venous blood and urine samples were obtained in the morning after a 12-h overnight fast and 2 h after intake of trial regimen. HbA1c level was measured using high performance liquid chromatography (HPLC). Serum insulin level was measured using an immunoradiometric assay. Insulin resistance was estimated using HOMA-IR, defined as [fasting plasma insulin (mU/L) × fasting plasma glucose (mmol/L)] / 22.5 (12). Beta-cell function was estimated using HOMA – beta, calculated as fasting plasma insulin (µU/ml) × 20/fasting plasma glucose (mmol/L) – 3.5 (12). Chemistry values were determined using standard assays in each local laboratory. Glomerular filtration rate (GFR) was estimated by the Modification of Diet in Renal Disease (MDRD) Study Group formula (13). The levels of CRP and CD40L were measured using a high sensitivity enzyme-linked immunosorbent assay (ELISA) assay (R&D Systems, Minneapolis, MN, USA).
Blood was aspirated under constant vacuum from the sample reservoir through a capillary and a microscopic hole (147 um) in the membrane by PFA-100. Platelet activation evaluated by PFA-100 is based on co-stimulation of platelets by high shearing stress with a capillary and on the contact of platelets with a membrane coated with collagen and epinephrine to form a platelet plug within the hole. Platelet function was quantitated as the time necessary for thrombotic occlusion of a hole in a membrane coated with collagen and epinephrine (closure time; CT) (14). A CT of 193 sec or less indicated normal platelet function, whereas a CT over 300 sec was considered non-closure according to the manufacturer’s cut-off values.
The VerifyNow system evaluates platelet activity by measuring the light absorbance through the sample. VerifyNow contains a lyophilized preparation containing human fibrinogen-coated beads that cross-links with activated platelets. When platelets become activated by the specific agonist, the fibrinogen-coated beads agglutinate with platelets, and light transmission increases. The VerifyNow aspirin test uses arachidonic acid as the specific agonist, which is converted by the cyclooxygenase-1 enzyme (the molecular target of aspirin therapy) into thromboxane A2. The data are output as aspirin response units (ARUs), and a cut-off value of ARU ≥ 550 was accepted to exclude aspirin-induced platelet aggregation according to the manufacturer’s reference values.
The sample size was calculated based on previous studies. The change of platelet aggregation rate (D) between baseline and after administration of aspirin or cilostazol was defined as follows:
According to a previous study, the change of platelet aggregation rate with arachidonic acid after aspirin and cilostazol administration were 0.4504±0.3651 and 0.69 ± 0.4975, respectively (15). A sample size of 58 patients per trial group was estimated to provide the trial with 80% power (2-sided, 5% significance) while considering 10% dropout of study participants.
Categorical variables are expressed as frequency and percentage. Continuous variables are presented as mean ± SD. Continuous variables were analyzed for a normal distribution with the Shapiro–Wilks goodness-of-fit test (using P-value < 0.1 as threshold). Only patients with drug adherence greater than 70% were considered for comparisons. Paired t-tests were used for comparison of normally distributed continuous variables in the same group. Wilcoxon tests were used for paired comparisons of continuous variables not following a normal distribution. The primary and secondary outcomes between the two groups were compared using repeated measures analysis of variance (ANOVA). Safety comparisons were assessed using Chi-square for proportions. All statistical analyses were performed using IBM SPSS software (IBM SPSS version 24.0, Chicago, IL, USA). Two-tailed values of P < 0.05 were considered statistically significant.