Bioequivalence and pharmacodynamics of a generic dabigatran etexilate capsule in healthy Chinese subjects under fasting and fed conditions

Abstract To assess bioequivalence of a generic dabigatran etexilate capsule in healthy Chinese subjects under fasting and fed conditions. This was an open‐label, single‐center, randomized four‐period crossover study with a 7‐day washout period. A single oral dose of 150 mg generic dabigatran etexilate capsule (test drug) or a commercial dabigatran etexilate capsule (Pradaxa®, reference drug) was given to healthy volunteers under the fasting and fed conditions. Plasma concentrations of total and free dabigatran were detected using a validated HPLC‐MS/MS method. A noncompartmental method was used for pharmacokinetic analysis and established coagulation assays were applied for pharmacodynamic analysis. The 90% CIs of the test/reference ratios of Cmax, AUC0‐t, and AUC0‐∞ for the total dabigatran concentration were 92.57%‐106.58%, 91.63%‐106.32%, and 92.54%‐106.17%, respectively, under fasting condition, and 99.30%‐110.74%, 98.58%‐105.37%, and 97.75%‐103.99%, respectively, under fed conditions. The 90% CIs of the ratios of the parameters for the free dabigatran were 93.18%‐106.98%, 92.13%‐107.10%, 92.89%‐106.48%, respectively, under fasting condition, and 100.05%‐110.89%, 99.37%‐106.23%, 97.59%‐103.98%, respectively, under the fed condition. Additionally, the upper limit of the 90% CIs for σWT/σWR was below 2.5. There were no significant differences in the coagulation parameters including thrombin clotting time, activated partial thromboplastin time, and anti‐IIa activity between the two preparations. The generic dabigatran etexilate capsule is bioequivalent to the brand‐named product in healthy Chinese volunteers under fasting and fed conditions. The two products have comparable pharmacodynamic parameters, with a good safety profile. In addition, food intake influences absorption of both products in a similar way.


| INTRODUC TI ON
Atrial fibrillation (AF) is one of the most prevalent arrhythmia, and it is an independent hazard index for ischemic stroke. 1 The global prevalence of AF adjusted by age was estimated to be 596 and 373 per 100 000 in males and females. 2 Due to population ageing, the age-adjusted prevalence (≥30 years) of AF in China is rising rapidly, reaching to about 0.65%, and 9 million individuals aged ≥60 years are estimated to have AF by 2050. 3 Risk-based anticoagulant therapy is recommended to reducing thromboembolism risk in AF patients. 1,4 Although oral administration of warfarin was proved to reduce stroke incidence in non-valvular AF (NVAF), it presents several drawbacks including long response time, complex drug interactions, and requirement for frequent monitoring. Thus, novel anticoagulants that overcome the drawbacks are needed.
Over the recent years, the treatment of NVAF has advanced rapidly, and several novel oral anticoagulants (NOACs), including dabigatran etexilate, have already applied in clinical practice. 5 As a new oral reversible thrombin inhibitor, dabigatran etexilate is rapidly absorbed and biotransformed into an active form of dabigatran, which directly inhibits thrombin (factor IIa) activity and production. 5 The with a mechanical heart valve or moderate-to-severe mitral stenosis. 4 However, the clinical application of dabigatran etexilate, mainly Pradaxa ® , in Chinese NVAF patients was not satisfactory; the discontinuation rate of dabigatran therapy was higher compared with warfarin, due to its high price that many patients cannot afford. 8 Thus, research and development of generic products of dabigatran etexilate to remove the barrier that the brand-named product of dabigatran etexilate posed and provide the availability and accessibility to dabigatran therapy to Chinese NVAF patients is urgently needed.
Currently, a generic product of dabigatran etexilate (150-mg capsule) is being investigated and developed by Chengdu Brilliant Pharmaceutical Co., Ltd., in China. In order to meet the requirements by the Chinese regulatory authorities for marketing authorization for generic products in China, the present study was carried out to assess bioequivalence (BE) of this generic dabigatran etexilate capsule, with reference to the brand-named product (Pradaxa ® ), in healthy Chinese subjects under fasting and fed conditions. In addition, the pharmacodynamics and safety of this generic product were also assessed.

| Study population
A total of 92 healthy subjects aged 18-65 years, body mass index 19-26 kg/m 2 were recruited in this study. During screening visits, all the volunteers were assessed for physical examinations, 12-lead electrocardiogram, laboratory tests (hematology, urinalysis, blood chemistry, and coagulation function), serologic tests (hepatitis B surface antigen, hepatitis C virus antibody, human immunodeficiency virus antibody, and syphilis antibody), vital signs, and past medical history.
Participants agreed to use effective methods of contraception from 2 weeks before screening to 3 months after the end of the trial.
Subjects were excluded from the study if they had (a) abnormal physical examinations, vital signs, electrocardiogram, or laboratory results of clinical significance based on the physician's judgment; (b) any history and/or presence of cardiovascular, endocrine, neurological, gastrointestinal, respiratory, hematological, psychiatric, and hepatic and renal diseases; (c) allergy to any food or drugs; (d) pregnancy or lactation; (e) surgery performed 1 month before this study or planned during this study; (f) blood loss ≥200 mL, blood donation, and/or involvement in any other clinical studies 3 months before this study; (g) heavy consumption of coffee, tea, caffeinated beverage, or alcohol abusers ≥14 units alcohol per week; (h) heavy smokers ≥5 cigarettes per day; (i) drug abusers and positive drug abuse screening test (morphine, tetrahydrocannabinol, methamphetamine, 3,4-methylenedioxyamphetamine, cocaine, and ketamine); (j) treatment of any drugs within the 14 days, or any drugs that affect blood coagulation (namely acetylsalicylic acid, non-steroidal anti-inflammatory drugs, coumarin, etc) within the 30 days before the first administration of the study drug.

| Study design
This clinical trial strictly adheres to the ethical guidelines for human medical research of The Declaration of Helsinki. The protocol of In the fasting cohort, all the subjects were hospitalized 1 day before the study and underwent an over-night fasting for at least 10 hours. The next morning, each subject orally administered a single dose (150-mg capsule) of either the test or reference dabigatran etexilate with 240 mL warm water, and was restricted from drinking for 1 hour before drug administration and 2 hours after administration. For the periods 2-4, underwent the pre-specified treatment sequence and also followed the above procedures.
In the fed cohort, after hospitalization and fasting over-night for at least 10 hours, the subjects were fed with a standard high-fat meal, which contained 800-1000 kcal (500-600 kcal fat, 150 kcal protein, and 250 kcal carbohydrates), 30 minutes before administration of the study drug. Other study procedures were similar with that in the fasting cohort.

| Pharmacokinetic blood sampling and analytical method
Blood samples were collected to the EDTA-K 2 vacuum tube (contain- Analyst® software 1.6.1 was used for MS parameters optimization, data acquisition, and data processing. The standard calibration curves with good linearity were built for both total and free dabigatran within the concentration range of 1.0-300.0 ng/mL. The precision (% CV) was within 6.5%-9.3% for total dabigatran and 2.5%-9.8% for free dabigatran, respectively. The intra-batch accuracy was between 92.1% and 103.1% for total dabigatran and 101.1% and 104.7% for free dabigatran, respectively. The inter-batch accuracy was between 95.7% and 103.5% for total dabigatran and 95.6% and 104.0% for free dabigatran, respectively. The lower limit of quantitation (LOQ) of both total and free dabigatran in the plasma was 1.000 ng/mL. The utilized methods, as indicated by the validation, were suitable for large amounts of biomedical samples.

| Pharmacodynamic blood sampling and analysis
The pharmacodynamic (PD) effects of dabigatran etexilate were assessed by measurement of anti-IIa activity, thrombin clotting time (TT), and activated partial thromboplastin time (aPTT). Blood samples were collected at 0, 2 8, and 12 hours after dosing during any one of the four periods for test or reference product. The blood samples were centrifuged at 2500 g for 10 minutes. Plasma was collected and stored at −80°C until testing.
The analyses of aPTT and TT were performed by validated clotting assays with Sysmex CS2000i automatic coagulation analyzer (Wakinohama-Kaigandori, Chuo-ku, Kobe, Japan). Anti-IIa activity was determined by a chromogenic substrate method as previously described. 9

| Safety
The safety of the test and reference dabigatran etexilate capsules was assessed by vital signs monitoring, physical examination, laboratory tests (hematology, biochemistry, and urinalysis), and electrocardiogram and adverse events (AEs) during the study.
Abnormalities that were considered clinically significant by the investigators after randomization were recorded as adverse events.

| Statistical analysis
The C max , AUC 0-t , and AUC 0-∞ of total and free dabigatran were the main observed pharmacokinetic parameters, and the T max of total and free dabigatran was secondary pharmacokinetic parameter. Following logarithmic conversion, analysis of variance (ANOVA) on the main pharmacokinetic parameters was done to estimate the ratio of the test drug to the reference drug and its 90% confidence interval (CI).

| Demographics and baseline characteristics
A total of eligible 92 subjects were recruited, 46 in the fasting cohort, and 46 in the fed cohort. The baseline characteristics, including demographics, height, weight, and body mass index (BMI), are shown in Table 1. Only one subject in the fasting cohort withdrew from the study in the third period 4 hours after dosing of the test product, and the other subjects completed all the periods and were included in the final BE analysis.

| Pharmacokinetics
The plasma concentration-time profile of total and free dabigatran after a single dose of the test and reference dabigatran etexilate under the fasting and fed conditions are shown in Figures 1 and 2, respectively, and the main pharmacokinetic data of total and free dabigatran and the BE analysis are summarized in Tables 2 and 3, respectively. Table 3, the geometric mean ratios (90% CI) of the test-to-reference products for C max , AUC 0-t , and AUC 0-∞ for both total and free dabigatran were all within 80.00%-125.00%. In addition, the upper limit of the 90% CI for the test-to-reference ratio of the within-subject variability (σ WT /σ WR ) was all less than 2.5 when under the fasting and fed conditions. The differences between the test capsule and reference capsule in the T max and elimination half-life (T 1/2 ) values were not statistically significant in both fasting and fed conditions ( Table 2).

As shown in
Based on the FDA guidance, 10 the test dabigatran etexilate capsule was considered bioequivalent to the reference dabigatran etexilate capsule.
As shown in Table 2, food intake seemed to have an influence on the T max of dabigatran. The T max of total and free dabigatran was reached 2 hours after administration in the fasting condition, but 4.5 hours in the fed condition ( Table 2, P < .05).

| Pharmacodynamics
Anti-IIa activity after a single oral dose of dabigatran etexilate capsules under fasting and fed conditions was shown in Table 4. The geometric mean ratios of area under efficacy curve (AUEC) of aPTT, TT, and anti-IIa activity were shown in Table 5. The aPTT, TT, and anti-IIa activity were not significantly different between the two products (all P > .05). Figures 3 and 4, a linear correlation was found between plasma concentrations of total dabigatran and coagulation parameters (ie, aPTT and anti-IIa activity) for both test and reference capsule products under the fasting and fed conditions (P < .05).

As shown in
TT values were beyond the detectable limit in 54 measurements

| Safety
In the fasting cohort, 10 subjects (10/46, 21.7%) experienced 13 AEs, of which 11 AEs were considered possibly related to the drug TA B L E 1 Baseline characteristics of the subjects in the fasting and fed cohorts administration; five and six were likely related to the test and reference products, respectively. All AEs were mild (Grade I), with nasal hemorrhage (n = 3) and abdominal pain (n = 3) being the most frequent. At the last scheduled visit, 12 AEs were completely recovered and one improved. In the fed cohort, nine subjects (9/46, 19.6%) experienced a total of 12 AEs, of which eight were possibly related to the study drugs; five occurred in the test product period and three in the reference product period. All AEs were mild (Grade I), with diarrhea (n = 2) and hyperbilirubinemia (n = 2) being the most frequent.
At the last scheduled visit, 11 AEs were recovered and one improved.
There were no serious AEs or AEs leading to withdrawal in the fasting and fed cohorts. The difference in the incidence of AEs between the test and reference products was not statistically significant.

| D ISCUSS I ON AND CON CLUS I ON S
In the present study, all PK parameters, including C max , AUC 0-t , and AUC 0-∞ , were similar between the test and reference products in healthy Chinese subjects under both fasting and fed conditions. There was no significant difference in the main PK parameters (ie, C max , AUC 0→t , and AUC 0→∞ ) between two dabigatran etexilate capsules under either fasting or fed condition in the present study.
The test-to-reference ratio of geometric mean ratios (90% CI) for the C max , AUC 0-t , and AUC 0-∞ for both total and free dabigatran were within 80.00%-125.00% and the upper limit of 90% CI for σ WT /σ WR was less than 2.5 under fasting and fed conditions. These findings are within the conventional acceptance for bioequivalence evaluation. These main PK parameters of total dabigatran are compared with those data from previous studies. [11][12][13][14] The T max obtained in the present study is consistent with that reported in the Caucasian 11,13 Abbreviations: AUC 0-∞ , area under the curve extrapolated to infinity; AUC 0-t , area under the curve to the last measurable concentration; C max , maximum plasma concentration; GMR, geometric mean ratio; σ WR , intra-individual variability of reference product; σ WT , intra-individual variability of test product. and Japanese subjects. 14 However, the C max and AUC 0-∞ of total dabigatran for both products under the fasting and fed conditions in the present study were approximately 20% higher than that in Caucasian subjects, but similar to that in Japanese. 12 The differences in C max and AUC values between the Chinese and Caucasian may be attributed to several factors such as body weight and serum creatinine, 15 and more importantly the genetic variants. 16 Indeed, it has been demonstrated that there is a gene-dose effect between ABCB1 and CES1 genes and peak concentrations of dabigatran etexilate. 15 In the present study, we investigated the main PK parameters of test and reference preparations under the fed and fasting conditions. Administration with high-fat meals prolonged the median T max of total and free dabigatran from 2 to 4.5 hours, but did not alter the AUC, indicating that food intake may reduce the rate of absorption without impacting the extent of absorption or drug elimination.
The present study revealed that the main coagulation parameters, aPTT, TT, and anti-IIa activity, were comparable between the two products by ANOVA analysis. However, the 95% CIs of geometric mean ratios of some measured coagulation parameters fell outside 80.00%-125.00%, which may be related to the sample size. In this preliminary exploration pharmacodynamic trial, we measured coagulation parameters only in two periods (one R period and one T period).
A four-period trial is expected to more accurately assess the pharmacodynamic equivalence of dabigatran, a highly variable drug. We also found that the aPTT level was correlated with dabigatran concentration although the R 2 value was relatively low compared with anti-IIa activity. This finding is consistent with previous observation that there was no change in the aPTT level during dabigatran therapy. 17 In the present study, TT values exceeded the detectable limit in many subjects, especially in the fasting cohort, which led to missing data in these subjects. It has previously been shown that the dilute thrombin time (dTT) accurately identifies therapeutic and supratherapeutic dabigatran levels, possible due to the fact that dTT has better correlation with dabigatran PK parameters than TT. 17  Abbreviations: aPTT, activated partial thromboplastin time; AUEC, area under efficacy curve; GMR, geometric mean ratio; TT, thrombin clotting time.

ACK N OWLED G M ENTS
We sincerely thank all the volunteers for participating in this clinical study.

CO N FLI C T O F I NTE R E S T
All the authors declare no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The datasets used and analyzed during the current study are available from the corresponding author on reasonable request.