Inclusion and exclusion criteria

Volunteers aged 20–55 years and within 20% of their ideal body weight [IBW (kg) = height (cm) - 100) × 0.9] were enrolled in the study. Those who were not suitable on the basis of physical examinations and routine laboratory tests (blood hematology, biochemistry, prothrombin time, bleeding time, urinalysis) were excluded.

Study design and ethical considerations

The analysis was performed using data from a randomized, open-label, multiple-dose, two-period, two-treatment, comparative crossover study involving 38 healthy adult males. The clinical trial was conducted at Kyungpook National University Hospital Clinical Trial Center (KNUH CTC) to determine the bioequivalence and non-inferiority of two different triflusal formulations, as reported previously [4].

The study was conducted in accordance with the guidelines of Good Clinical Practice and the Declaration of Helsinki and its amendments at the KNUH CTC, and was approved by the institutional review board at KNUH. Written informed consent was obtained from all volunteers before their participation.

Study drugs and dosage regimen

Triflusal capsule (Disgren capsule, 300 mg), as the reference formulation, and triflusal EC capsule (Disgren enteric-coated capsule, 300 mg), as the test formulation were manufactured and provided by Myung-In Pharm. Co., Ltd (Seoul, Republic of Korea). The subjects received the test or reference formulation in multiple doses, followed by a 13-day washout period and subsequent administration of the alternative formulation. During each period, each subject received a single 900 mg oral loading dose on Day 1, followed by a 600 mg/day maintenance dose (given as two 300 mg capsules once daily) from Day 2 to 9.

Blood sampling procedures

For measurement of the plasma concentration of HTB (PK), whole blood samples were obtained at time 0 (pre-dose), and 24, 48, 96, 144, 168, 192, 192.5, 193, 194, 196, 199, 202, and 216 h after the first dose (from Day 1 to Day 10). For platelet aggregation measurements (PD), sampling was performed at time 0 (pre-dose), and 24, 48, 96, 144, 168, 192, 196, 202, and 216 h post-dose. The overall sampling schema is presented in Figure 1.

Blood (7 mL) for PK analysis was collected into tubes containing sodium heparin, and blood (3 mL) for PD analysis was collected into tubes containing 0.109 mmol/L sodium citrate. An aliquot (1 mL) of each plasma sample was placed in a microcentrifuge tube containing 0.1 mL 0.1 M HCl to prevent degradation of triflusal in the plasma. The remaining samples were chilled promptly on crushed ice to maintain the distribution ratio between triflusal and HTB [9].

HTB plasma concentration measurements

Concentrations of HTB, rather than triflusal, were measured in this study because the level of triflusal is too low to be detected after 4 h when administered orally, as reported by Lee et al. [4, 10]. Blood samples were analyzed by high-performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS) [7]. Quantification was performed by multiple reaction monitoring (MRM) transitions. A C18 column was used to separate the components of the samples by chromatography. Linear calibration curves were analyzed in the range of 1 to 300 μg/mL HTB (coefficient of correlation, r = 0.999). The lower limit of quantification of HTB was 1 μg/mL. The intra-day and inter-day % CVs for HTB were each less than 15%. Figure 2 shows the plasma concentrations of HTB with respect to time.

Platelet aggregation assessment

The tubes containing sodium citrate were centrifuged (1,000 rpm, 10 min) and 500 μL of platelet-rich plasma (PRP) was collected as the supernatant. The PRP was transferred to microcentrifuge tubes, which were capped to prevent changes in pH upon exposure to air, and the samples were kept on ice. To obtain 500 μL of platelet-poor plasma (PPP), the remaining sample in the original tubes was centrifuged again (3,000 rpm, 10 min) [11]. Platelet aggregation in PRP was assessed using a Chronolog optical aggregometer (Model 490-4D; Chrono-log, Havertown, PA, USA). Cuvettes were lined up along the two rows of the aggregometer, filled with 250 μL of PRP or PPP, and warmed for 5 min at 37°C [12]. To measure the degree of platelet aggregation, 0.5 μL AA was added to the cuvettes as an agonist, and light transmission aggregometry was observed for 5 min. The aggregometer was calibrated with a cuvette containing PRP, equaling 0% light transmission, as the baseline and with a second cuvette containing PPP, equaling 100% light transmission, as the reference [13]. Based on analyses of the blank plasma samples, which were validated in three individuals, the intra-day and inter-day precisions were calculated as (SD/mean) × 100 (%) and ranged from 2.1 to 6.8% and from 2.1 to 7.6%, respectively [4].

Dataset

In the current study, PK and PD data from 34 healthy volunteers who received the reference drug were used for population PK-PD analysis. In total, 476 HTB plasma concentration data points and 340 platelet aggregation (%) data points were included. The latent variables used as candidate covariates were age, height, and weight, and levels of albumin, creatinine, serum aspartate transaminase (AST), and alanine transaminase (ALT). Creatinine clearance (CLCR), calculated using the Cockcroft-Gault equation, was also included.

Population PK-PD model development

where C _ij is the j ^th observed value in the i ^th individual, and ϵ _pro,ij and ϵ _add,ij are the residual intra-individual variability with a mean of zero and variances of ${\sigma }_{\mathit{\text{pro}}}^2$ and ${\sigma }_{\mathit{\text{add}}}^2$, respectively. A similar approach was made to the PD observations. The standard errors, which give information on the precision of the parameter estimates, were explored using NONMEM’s “$COVARIANCE” step.

Models were evaluated using diagnostic scatter plots, goodness-of-fit plots, and the log likelihood ratio test (LRT). The results for LRT were considered statistically significant if the objective function value (OFV) decreased by 3.84 (p = 0.05); this was used as a cut-off criterion for model improvement.

where dR/dt is the rate of change of the measured response (R) over time, k _in is the turnover input rate for production of the response, I _max is the fraction representing the maximal capacity by which the drug can inhibit platelet aggregation (0 ≤ I _max  ≤ 1), C is the HTB concentration, IC ₅₀ is the median inhibition concentration, and k _out is the turnover output rate of the response.

Covariate analysis

where the s₀ is an intercept, and s₁(X₁), · · ·, s_p(X_p) are smooth functions that are estimated in a nonparametric fashion. In the equation, candidate covariates with the greatest decrease in the Akaike information criterion were selected as final covariates in the PK model when there was a reduction of at least 3.84 compared with the previous model, and the relationship between the random variable, η, for each parameter and the candidate covariates was improved. Otherwise, they were dropped from the model.

Graphical analyses using goodness-of-fit plots – including observed-versus-population-predicted, observed-versus-individual-predicted, and residual diagnostics – were also conducted to diagnose the degree of model development.

Model evaluation

Bootstrap was recruited to check the robustness of the PK-PD model and parameter estimates. The median value and 95% confidence interval for each parameter were obtained from 1,000 bootstrap dataset. For the PK model, a visual predictive check (VPC) was implemented to further evaluate the accuracy and predictive performance. A total of 1,000 replicate simulation was performed and the 50th (estimated population median), 5th, and 95th percentiles of the created concentrations plotted against time were compared with the overlaid observed concentrations. In addition, predicted concentration-response relationship is plotted with the predicted probability of each concentration data observed at the time of PD sampling.

Sample size determination

We considered the sample size for having an 80% power at a significance level of 0.05 for bioequivalence trials. It was calculated under the condition of the equivalence limit of 22.3%, the true mean difference of 10%, and the standard deviation of 24.2%. The calculation was performed by a formula suggested from Chow et al. [20]. Volunteers were randomly assigned to one of two sequences with simple randomization using SAS software (ver. 9.2; SAS Institute, Inc., Cary, NC, USA).

Statistical analyses

The demographic data were presented using descriptive statistical analysis performed with the standard SPSS package (version 12.0 for Windows, SPSS, College Station, TX, USA). The mean, standard deviation, and range were obtained for each variable.

Study population

Volunteers were recruited from August 2008 to September 2008. In total, thirty-eight volunteers were enrolled in this study. Four subjects dropped out because of severe dental pain, a medication administration error, a missing follow-up urinalysis, or platelet aggregation 1% of baseline in the second period. Data from the remaining 34 subjects were analyzed to construct the PK and PD models. According to the CONSORT flow diagram, Figure 3 shows the subject disposition. The age, height, and weight (means ± SD) of the subjects were 24.1 ± 1.7 years, 176.1 ± 4.9 cm, and 70.8 ± 9.0 kg, respectively (Table 1).

Population PK modeling

where θ₁ and θ₂ were the estimated typical values of CL and V with a median weight (71.65) and θ₄ was the estimated influential factor for weight. Weight produced 30.782 (equivalent to p < 0.001, 1 degree of freedom) decrease in the minimized OFV as well as the change in the random BSV for CL/F from 18.5% to 14.9% and for V/F from 15.6% to 9.5% (as % CV). Because only one demographic variable (weight) was selected as a meaningful covariate, the process of backward covariate exclusion was omitted.

The goodness-of-fit plots for the final PK model are presented in Figure 4. Final estimates from the PK model, explaining the mean value and the between-subject variability, are listed in Table 2. The minimum concentration at steady state (C _ss,min ) was estimated to be 103.5 μg/mL using the values of final population PK parameters; in addition, the estimated value of the accumulation factor was 2.26.

Population PD modeling

Model evaluation

The bootstrap results showed acceptable robustness of the model and are presented together with the final estimates in Table 2. The VPC result (Figure 5) showed that the prediction of the simulated data well-matched the observed concentration-time profiles. This graph, representing a visual internal validation of the model, showed that most of the observed data points were overlaid between the 5th and 95th percentiles.