The study protocol and informed consent to participate were reviewed and approved by the institutional review board of Beijing Shijitan Hospital, China. The study was conducted in accordance with consensus ethics principles derived from international ethics guidelines, including the Declaration of Helsinki, the International Council for Harmonisation (ICH) guidelines for Good Clinical Practice (GCP), and all applicable laws, rules, and regulations.
Healthy Chinese male or female volunteers aged ≥ 18 years were certified as healthy based on a comprehensive clinical assessment that included detailed medical history, comprehensive physical examination, vital signs, electrocardiogram (ECG), and laboratory parameters. Female subjects of childbearing age were required to have negative results on a pregnancy test, and only those who agreed to use an appropriate method of contraception during the study period were included. The subjects were restricted in use of concomitant medications, tobacco and alcohol, and supplements throughout the study.
A randomized, single-center, open-label, three-treatment, three-period, three-sequence, crossover study (study no. BEQ16000/CTR20190376) with a 10-day washout period between administrations under fed conditions was conducted in 171 healthy Chinese subjects at Beijing Shijitan Hospital, Capital Medical University, China. The 171 subjects were randomized into one of three sequences (Test (T)/Reference (R)/R, R/T/R, R/R/T). The randomization list consisted of a block size of 6, with 171 subjects randomly divided into two treatment groups with three periods and three sequences (Fig. 1).
Randomization and Interventions
The test formulation of an FDC containing 75 mg of clopidogrel and 100 mg of enteric ASA was compared with the reference formulations of clopidogrel (Plavix®, 75 mg) tablet and enteric-coated ASA (Bayaspirin®, 100 mg) tablet. The dose levels for the present study were selected based on the therapeutic doses of Plavix® and Bayaspirin® in China. The subjects were administered the investigational medicinal product (IMP) under fed conditions (standard high-fat breakfast of approximately 1000 kcal, with approximately 150, 250, and 500–600 kcal from protein, carbohydrates, and fat, respectively) on day 1 of each period. The subjects were monitored to ensure that their breakfast was completed within 30 min, and then the assigned IMP was administered. No food was allowed for a minimum of 4 h after administration. On days of treatment administration, standard lunch and dinner were given at least 4 and 10 h after IMP administration, respectively. The subjects were followed up for 5–7 days after the last administration of IMP.
The primary objective was to determine the bioequivalence of ASA and clopidogrel for an FDC containing 75 mg of clopidogrel and 100 mg of enteric ASA (test formulation) versus the simultaneous administration of the separate commercial tablets (reference formulations) in healthy Chinese subjects under fed conditions. The secondary objective was to assess the clinical safety of each treatment.
Blood samples were collected at the following time points for ASA/salicylic acid (SA, a metabolite of ASA): 0 h (pre-dosing) and 2, 3, 3.50, 4, 4.50, 5, 5.50, 6, 6.50, 7, 7.50, 8, 10, 12, and 16 h post-dosing and for clopidogrel/SR26334: 0 h (pre-dosing), 0.25, 0.50, 0.75, 1, 1.50, 2, 2.50, 3, 4, 5, 6, 12, 16, and 24 h post-dosing. The parameters assessed were Cmax, tmax, AUClast, AUC for clopidogrel/SR26334 (metabolite of clopidogrel), and ASA/salicylic acid in plasma using non-compartmental methods with Phoenix WinNonlin (Certara USA Inc) version 8.1 (Supplementary Table S1) software. An analysis data set of PK parameters for subjects exposed to a minimum of one dose of IMP formed the Pharmacokinetic Parameter Sets (PKPS), whereas subjects who had a minimum of one evaluable PK parameter from a minimum of one period formed the bioequivalence set (BES). The BES data set was evaluated to determine if the test formulation and the reference formulation were bioequivalent. AUClast and tlast were excluded from the BES data set as the sample collection was incomplete. Similarly, the AUC value was excluded from the BES data set as the percentage of AUC determined by extrapolation was > 20%. However, these values were included in the PKPS data set. When a PK profile contained only one quantifiable concentration, Cmax, tmax, and tlast were excluded from the statistics for BES and PKPS.
The concentrations of clopidogrel/SR26334 and ASA/SA in plasma samples were analyzed using the validated liquid chromatography–tandem mass spectrometry (LC–MS/MS) method. Measurements of clopidogrel and SR26334 were taken by pretreating the plasma samples with liquid–liquid extraction and protein precipitation, respectively. Plasma samples for determination of ASA and salicylic acid concentrations were pretreated by liquid–liquid extraction. The lower limits of quantification (LLOQs) were 5 pg/ml (clopidogrel), 5 ng/ml (SR26334), 5 ng/ml (ASA), and 100 ng/ml (salicylic acid), and the assays had adequate accuracy and precision in estimating analytes.
Safety Measurements and Analysis
Subjects were carefully monitored by vital signs, physical examinations, laboratory parameters (hematology, coagulation, biochemistry, and urinalysis), and standard 12-lead ECG. Adverse events were graded according to Common Terminology Criteria for Adverse Events (CTCAE) v4.03 and classified by System Organ Class (SOC) or Preferred Term (PT) according to the latest version of the Medical Dictionary for Regulatory Activities (MedDRA) dictionary.
Sample Size Determination
Up to 171 subjects were enrolled to have a minimum of 135 subjects for completion, which was deemed sufficient to provide a 90% overall power for PK evaluation. For the sample size calculation in this study, the within-subject standard deviation (SDw) of clopidogrel and ASA in fed condition was considered similar to the observed SDw in fasting condition based on the completed bioavailability and bioequivalence studies (two published studies and one unpublished study) [16,17,18].
For clopidogrel, the pooled point estimates of the PK parameter ratios were 0.99, 0.98, and 1.10; the estimates of SDW (on the natural log scale) were 0.344, 0.335, and 0.365 for AUClast, AUC, and Cmax, respectively. For ASA, the pooled point estimates of pharmacokinetic (PK) parameter ratios were 1.10, 1.10, and 1.08, and the estimates of SDW (on the natural log scale) were 0.488, 0.416, and 0.696 for AUClast, AUC, and Cmax, respectively (the true ratios and true SDw for sample size calculation are presented in Supplementary Table S2). The power of concluding bioequivalence with 135 subjects is provided in Supplementary Table S2. For ASA, the reference-scaled average bioequivalence (RSABE) approach was used, and the standard ABE approach was applied for clopidogrel. The overall power was calculated as a function of the lowest power in clopidogrel and the lowest power in ASA because AUC and Cmax were assumed to be highly correlated parameters; therefore, no power loss between AUC and Cmax of the same ingredient was considered in the sample size calculation. Hence, the overall power was 90.24% (94% × 96%). In summary, 135 subjects could achieve an overall power of 90% to conclude bioequivalence between formulations (FDC versus individual formulation) for clopidogrel and ASA. Allowing for a 20% dropout rate, the total number of subjects for enrollment was 171.
Bioequivalence Statistical Analysis Plan
PK parameters of clopidogrel, ASA, and their major metabolites were summarized using descriptive statistics for each formulation. Prior to the analyses, all primary end points (Cmax, AUClast, and AUC) were log-transformed. The differences for clopidogrel between the fixed-dose formulations and its individual tablet formulation were assessed on log-transformed parameters with a linear mixed-effects model. The point estimates and 90% confidence intervals (CIs) for the geometric mean ratios (GMRs) of Cmax, AUClast, and AUC between the two formulations were obtained within the mixed-effects model framework and then converted to the ratio scale by antilog transformation. Bioequivalence was concluded if the 90% CI for the ratio was within 0.80–1.25.
Bioequivalence for ASA between the FDC and its individual tablet formulation was assessed using the mixed scaled average bioequivalence approach for Cmax, AUClast, and AUC. For each log-transformed parameter, a linear mixed-effects model was formulated, allowing for treatment-specific within-subject variance to obtain the SDW for the reference formulation. If SDW was < 0.294, the traditional standard ABE analysis was conducted within the mixed model framework. Bioequivalence was concluded if the 90% CI for the formulation ratio (test/reference GMR) was within 0.80–1.25. If SDW was ≥ 0.294, the RSABE method was conducted as described by Haider et al.  (i.e., upper one-sided 95% CI for the RSABE metric). Bioequivalence was concluded if the point estimate (test/reference GMR) fell within the range of 0.80–1.25 and the upper one-sided 95% CI for the RSABE metric was ≤ 0, (with θ = (lnΔ)2/σ2w0 and Δ = 1.25, the usual average BE upper limit for the untransformed test/reference ratio of geometric means, and σw0 = 0.25) All statistical calculations were performed using Statistical Analysis Software (SAS).