The study was conducted in accordance with ethical standards and regulatory requirements, including the ICMR Guidelines (2017) [18] and the New Drugs and Clinical Trials Rules (2019), India [19]. It also adhered to the ethical principles outlined in the Declaration of Helsinki [20] and the International Council for Harmonisation (ICH) Good Clinical Practice (GCP) guidelines [21].

Although the study was open to both men and women, based on previous evidence indicating no significant differences in rivaroxaban pharmacokinetics between sexes [4][9][10], only male subjects met all the inclusion criteria.

Eligible participants were healthy males aged 18 to 45 years, with good general health confirmed by a complete clinical history obtained within six months prior to study initiation. Subjects were required to have normal laboratory values based on medical history and physical examination at screening, as well as normal vital signs (blood pressure, pulse rate, and axillary temperature) and physical examination findings. Additional inclusion criteria included prothrombin time (PT) and activated partial thromboplastin time (aPTT) within normal ranges; creatinine clearance (CrCl) greater than 50 mL/min; negative test results for hepatic transaminases, hepatitis B and C, HIV, and VDRL; and a normal 12-lead electrocardiogram (ECG) performed within six months prior to study start. Participants were also required to have normal chest radiographs and negative urine drug screening results.

Subjects with a body mass index (BMI) between 18 and 30 kg/m² were eligible for inclusion, as well as non-smokers or smokers who had abstained from smoking for at least 10 hours prior to the start of the study. All participants were informed about the study procedures, including potential risks, laboratory tests, electrocardiograms, and reimbursement for travel and meals. Written informed onsent was obtained from all subjects prior to participation. Exclusion criteria included a history of hypersensitivity to rivaroxaban or any related medications, as well as the presence or history of cardiovascular, renal, hepatic, metabolic, gastrointestinal, neurological, endocrine, hematopoietic, psychiatric, or other organic abnormalities.

Additional exclusion criteria included the use of medications that could interfere with the quantification or pharmacokinetics of the study drug, or the use of potentially toxic medications within 30 days prior to study initiation. Subjects with exposure to known inducers or inhibitors of hepatic enzyme systems were also excluded, as well as those who had taken any medication within 7 days or within five half-lives, whichever was longer prior to the start of the study. Participants were excluded if they had been hospitalized for any reason or had experienced a serious illness within 90 days prior to enrollment, had received any investigational drug within 30 days before the study, or had donated or lost 450 mL or more of blood within 90 days prior to study initiation. Further exclusion criteria included a recent history of drug or alcohol abuse; consumption of caffeine or methylxanthine containing products (e.g., cola beverages containing caffeine, theobromine, or theophylline) within 48 hours prior to dosing; and ingestion of grapefruit juice within 72 hours prior to the study.

This study comprised two independent clinical trials conducted under fasting and fed conditions. Each trial was designed as a randomized, open-label, single-dose, two-treatment, two-sequence, four-period replicate crossover bioequivalence study. Such designs are widely recommended for the evaluation of highly variable drugs, defined by an intra-subject coefficient of variation (ISCV) of ≥30%. The replicate crossover approach enables direct estimation of within-subject variability for the reference product, thereby supporting the application of scaled bioequivalence methods. In this design, each subject received both the test (T) and reference (R) formulations twice, allowing for a more robust characterization of intra-subject variability and improving the precision of bioequivalence assessment.

Both trials were conducted at the Phase I screening unit, clinical research center, and bioanalytical facility of ICBio Clinical Research Private Limited, India. In each trial, subjects were randomly assigned in a 1:1 ratio to one of two treatment sequences: T-R-T-R or R-T-R-T, where T represents the test formulation and R the reference formulation. The randomization schedule was generated SAS^® Statistical Software version 9.4 from SAS Institute, Inc., Cary, USA. Subjects assigned to the T-R-T-R sequence received the test product in the first period, followed by the reference formulation in the second period, continuing in an alternating manner across all four periods. Conversely, subjects in the R-T-R-T sequence received the treatments in the reverse order. A washout period of five days was implemented between consecutive treatment periods in both the fasting and fed studies.

Under fasting conditions, each subject received a single oral dose of either the test or reference tablet, administered with 240 mL of water following an overnight fast of at least 10 hours. Subjects remained seated, except for a supine rest period at 2 hours post-dose.

Under fed conditions, subjects received a standardized high-fat meal (≥900 kcal per meal with 50% from fat) 30 minutes prior to drug administration and subsequently followed the same dosing and post-dose procedures as in the fasting state. Water intake was restricted for 1 hour before and 1 hour after drug administration; outside this interval, water was permitted without restriction, ad libitum. Standardized meals (lunch, snack, dinner, and late meal) were provided at 4, 8, 12, and 24 hours post-dose in each study period.

A total of 22 blood samples (6 mL each) were collected from each subject during each period via an indwelling cannula at pre-dose and at 0.50, 1.00, 1.50, 2.00, 2.33, 2.66, 3.00, 3.33, 3.66, 4.00, 4.33, 4.66, 5.00, 5.50, 6.00, 8.00, 10.00, 12.00, 16.00, 24.00, and 48.00 hours post-dose. The 24- and 48-hour samples were obtained by direct venipuncture. These procedures were identical under both fasting and fed conditions.

The total duration of the clinical phase was 19 days, from check-in for Period I to the post-study safety assessment. For the fasting study, the first dose was administered on May 03, 2024 to Period I, and the last dose on May 18, 2024 to Period IV (check in May 02, and post assessment date May 20). For the fed study the first dose was administered on May 07, 2024 to Period I, and the last dose was administrated on May 22, 2024 (check in May 06, and post assessment date May 24, 2024).

Blood samples were collected in pre-labelled K₂EDTA vacutainers and centrifuged at 4000 rpm for 10 minutes at 2˚C - 8˚C. Plasma was separated, appropriately labelled, and stored at −70˚C ± 5˚C until analysis. Prior to analysis, plasma samples, calibration standards, internal standard (IS; rivaroxaban-D4, Vivian Life Sciences Private Limited, Mumbai, India), and quality control (QC) samples were thawed and vortex-mixed. For sample preparation, 250 µL aliquots of plasma were combined with 250 µL of extraction buffer and vortexed. Solid-phase extraction was performed using cartridge-based methodology. The cartridges were conditioned with 1 mL of methanol and equilibrated with 1 mL of water before sample loading. Following loading, cartridges were washed with 1 mL of water and 1 mL of washing solution and then dried. Elution was performed using 800 µL of methanol, and the eluate was subsequently diluted with 200 µL of methanol.

Calibration curve standards were prepared by spiking know concentration of rivaroxaban in human plasma. Preparation of rivaroxaban and IS stock solutions were made as Method SOP N˚ MV.085-00. CC standards were prepared by bulk spiking of CC spiking solutions of analyte using pooled human plasma and stored in deep freezer ICBio II/ULTF/0050 at −70˚C ±15˚C. The acceptance criteria was stablished as deviations from the nominal concentration should be less than or equal to 20% for LLOQ and 15% for all other calibration curve standards (CCS), the coefficient of linear correlation (r) must be ≥0.99, and at least 75% of non-zero CCS should comply with the above criteria including lowest and highest calibration standards. To inter batch calibration standard precision (%CV) the range was 1.81% to 4.19% and Inter-batch calibration standard accuracy (%nominal) was 94.44% to 106.92%.

Calibration standards were prepared over a concentration range of 1.287 to 1204.829 ng/mL, with QC samples spiked with the internal standard. Processed analyte samples, IS, and QC samples were transferred into pre-labelled vials and placed in the autosampler maintained at 10˚C ± 3˚C. Quantification of rivaroxaban was carried out using an LC-ESI-MS/MS system (Shimadzu LCMS-8040, Mumbai, India). Chromatographic separation was achieved using a BDS Hypersil C18 column (4.6 × 50 mm, 5 µm; Thermo Scientific, Mumbai, India). The mass spectrometer was operated in positive electrospray ionization mode. Detection was performed using multiple reaction monitoring (MRM) transitions of m/z 436.10 → 145.05 for rivaroxaban and m/z 440.20 → 145.00 for the internal standard.

The statistical analysis was performed using the SAS^® Statistical Software version 9.4 from SAS Institute, Inc., Cary, USA. Descriptive statistics were used to summarize plasma concentrations at each sampling time point and pharmacokinetic parameters for each subject. These statistics included the mean, standard deviation, coefficient of variation, geometric mean, median, and range of each product at each adjusted sampling time point. To evaluate the pharmacokinetic parameters, an analysis of variance (ANOVA) was performed on the natural logarithm (ln)-transformed values of C_max, AUC 0-t and AUC 0-inf for rivaroxaban. These parameters were analyzed using SAS^® Statistical Software version 9.4 from SAS Institute, Inc., Cary, USA. ANOVA models were applied for each pharmacokinetic parameter, and the main effects were tested at 0.05 level of significance using mean square, standard deviation, media, minimum and maximum value.

To evaluate bioequivalence, Shuirmann’s Two One-Sided t-test, were employed at a 5% level of significance. These tests compared the average pharmacokinetic parameters for T and R formulations, focusing on the ln-transformed values of C_max, AUC 0-t and AUC 0-inf for rivaroxaban. The 90% confidence intervals (CI) for differences between treatments were calculated, providing a statistical range within which the true value of the difference lies with high confidence. Geometric Mean Ratio (GMR) for the ln-transformed pharmacokinetic parameters were performed. The difference between the T and R formulations was expressed as “Test -Reference” and the ratio of means (T/R) was obtained by taking the anti-log value of difference of the Geometric Mean Ratios (GMRs) for the PK parameters. Intra subject coefficient of variation (ISCV) for each PK variable and the power was calculated.

Sample size was determined based on the intra-subject coefficient of variation (CV%) for rivaroxaban, as reported in the published literature, including Tao et al. [22]-[24]. The intra-subject variability (CV%) for both C_max and AUC was approximately 30% - 33%. Assuming a CV% not exceeding 33% for both primary pharmacokinetic parameters, and that the true T/R ratio would fall within the accepted bioequivalence range (80% - 125%), with an expected ratio of 0.94 - 1.06, a minimum of 34 evaluable subjects was required to demonstrate bioequivalence with a statistical power greater than 90% at a 5% significance level. To account for potential dropouts or withdrawals, additional subjects were enrolled, resulting in a total of 36 subjects under fasting conditions and 40 subjects under fed conditions. Given the particular interest in the fed condition, the sample size for this arm was further increased to ensure an adequate number of evaluable subjects.

The safety of both formulations was evaluated through the assessment of adverse events (AEs). Vital signs, including body temperature, blood pressure, and heart rate, were measured at screening, baseline, and at the end of the study. Twelve-lead electrocardiograms (ECGs) and clinical laboratory tests, including urinalysis, blood biochemistry, and hematology, were performed at screening and at 48 hours post-dose in both bioequivalence studies.

A total of 35 healthy adult male subjects completed all four periods of the fasting study, while 38 subjects completed the fed study. These subjects were included in the pharmacokinetic (PK) analysis and safety evaluation. The demographic characteristics of the subjects who completed the bioequivalence study under fasting and fed conditions are summarized in Table 1.

Table 1. Demographic profile of all subjects that completed the study.

BMI = Body Mass Index, SD = standard deviation, min = minimum value, max = maximum value. N = evaluable subjects/included.

All subjects were Asian, male, non-smokers, and non-alcohol users.

3.2.1. Fasting Condition Efficacy Analysis

Thirty-five (35) subjects were included in the statistical analysis. The mean pharmacokinetic (PK) parameters of rivaroxaban for the reference formulation (R) and the test formulation (T) are summarized in the tables below. The PK parameters presented in Table 2 were individually derived for each subject from the plasma concentration-time profiles of rivaroxaban. A non-compartmental analysis was applied for the estimation of pharmacokinetic parameters. C_max, AUC_0−t, AUC_0−inf, T_max, T_1/2, λ_z (h⁻¹) and Extrapolated AUC (%) of rivaroxaban in plasma concentration are presented in Table 2.

Table 2. Pharmacokinetic parameters after a single rivaroxaban 20 mg oral dose of T and R formulations. Fasting condition. Non-Compartmental model (n = 35).

C_max: maximum plasma concentration; AUC_0−t: area under the plasma concentration–time curve from time 0 to the last measurable concentration; AUC_0−inf: area under the plasma concentration-time curve from time 0 to infinity; T_max: time to reach C_max; λz: terminal elimination rate constant; t_1/2: time required for the plasma concentration to decrease by 50%. Values are expressed as mean ± SD, except for T_max, which is presented as median (range). Min: minimum; max: maximum; h: hours.

The mean C_max, AUC_0–t, and AUC_0–inf values were 329.89 ng/mL, 2700.78 ng*h/mL, and 2830.76 ng*h/mL for the T formulation, compared with 309.91 ng/mL, 2516.17 ng*h/mL, and 2643.16 ng*h/mL for the R formulation. The median T_max was similar between formulations, occurring at 2.55 hours for the T and 2.70 hours for the R formulation.

Figure 1 and Figure 2, show the mean plasma concentration–time profile of rivaroxaban 20 mg oral tablets depicted on arithmetic and logarithmic scales, respectively.

For rivaroxaban, the T/R (T/R) ratio, expressed as GMRs of the ln-transformed PK parameters C_max, AUC_0–t, and AUC_0–inf, were 107.70 (90% CI: 99.85 - 116.17), 109.35 (90% CI: 102.52 - 116.63), and 108.85 (90% CI: 102.48 - 115.61), respectively (Table 3). These values fall within the accepted 90% confidence interval range of 80% - 125%, in accordance with FDA guidelines [23]. The intra-subject coefficient of variation (ISCV) for C_max, AUC_0–t, and AUC_0–inf was 27.26%, 23.04%, and 21.49%, respectively.

The power of the test for Ln-transformed pharmacokinetic parameters C_max was 99.92% and for AUC_0−t and AUC_0−inf 100% respectively, Table 3.

Based on the above results the T formulation is bioequivalent to the R formulation in healthy adults subjects under fasting conditions.

Figure 1. Mean plasma concentration-time profiles of rivaroxaban on an arithmetic scale following a single oral dose in a randomized, crossover study with two treatments, two sequences, and four periods in fasting condition. Test formulations (T1 and T2): Asarap^® 20 mg (Laboratorios Leti S.A.V.). Reference formulations (R1 and R2): Xarelto^® 20 mg (Bayer AG, Germany).

Figure 2. Mean plasma concentration-time profiles of rivaroxaban on a logarithm scale following a single oral dose in a randomized, crossover study with two treatments, two sequences, and four periods in fasting condition. Test formulations (T1 and T2): Asarap^® 20 mg (Laboratorios Leti S.A.V.). Reference formulations (R1 and R2): Xarelto^®20 mg (Bayer AG, Germany).

Table 3. Pharmacokinetic Parameters (C_max, AUC_0−t and AUC_0−inf) of rivaroxaban 20 mg, Fasting condition: Geometric Mean Ratio, 90% Confidence Intervals, ISCV (%) and Power (%) (n = 35).

C_max: maximum plasma concentration, AUC_0−t: area under the plasma concentration–time curve from time 0 to the last measurable concentration; AUC_0−inf: area under the plasma concentration-time curve from time 0 to infinity, CI: confidence intervals, ISCV Intra subject coefficient of variation Ln: natural logarithm.

3.2.2. Fed Condition Analysis of Efficacy

A total of 40 subjects were enrolled in the fed study, of whom 38 completed all four periods of the clinical phase. The pharmacokinetic variables are summarized in Table 4.

Table 4. Pharmacokinetic of mean formulation for rivaroxaban 20 mg. Fed condition Non-Compartmental model (n = 38).

C_max, maximum plasma concentration, AUC_0−t, area under the plasma concentration–time curve from time 0 to the last measurable concentration; AUC_0−inf, area under the plasma concentration-time curve from time 0 to infinity, T_max time to reach C_max, λ_z fraction eliminated per unit of time, T_1/2 time required for plasma concentration to decrease by 50%. *Median (range) min: minimum, max: maximum, h: hours.

Figure 3 and Figure 4 illustrate the mean plasma concentration-time profiles of rivaroxaban 20 mg oral tablets for both the T and R formulations under fed conditions, displayed on arithmetic and logarithmic scales, respectively.

Figure 3. Mean plasma concentration-time profiles of rivaroxaban on an arithmetic scale following a single oral dose in a randomized, crossover study with two treatments, two sequences, and four periods in fed condition. Test formulations (T1 and T2): Asarap^® 20 mg (Laboratorios Leti S.A.V.). Reference formulations (R1 and R2): Xarelto^® 20 mg (Bayer AG, Germany).

Figure 4. Mean plasma concentration-time profiles of rivaroxaban on a logarithm scale following a single oral dose in a randomized, crossover study with two treatments, two sequences, and four periods in fed condition. Test formulations (T1 and T2): Asarap^® 20 mg (Laboratorios Leti S.A.V.). Reference formulations (R1 and R2): Xarelto^® 20 mg (Bayer AG, Germany).

Table 5. Pharmacokinetic Parameters (C_max, AUC_0−t and AUC_0−inf) of rivaroxaban 20 mg. Fed condition Geometric Mean Ratio, 90% Confidence Intervals, ISCV (%) and Power (%) (N = 38).

C_max: maximum plasma concentration, AUC_0−t: area under the plasma concentration-time curve from time 0 to the last measurable concentration; AUC_0−inf: area under the plasma concentration-time curve from time 0 to infinity, CI: confidence intervals, ISCV Intra subject coefficient of variability. Ln: natural logarithm.

All PK parameters (C_max, AUC_0–t, and AUC_0−inf) exhibited GMRs (T/R) and corresponding 90% confidence intervals within the predefined regulatory acceptance range of 80% - 125% [23], thereby demonstrating bioequivalence between the Test (T) and Reference (R) formulations under fed conditions. The within-subject variation (ISCV) for log-transformed PK parameters was estimated using an analysis of variance (ANOVA) model. Under fed conditions, the ISCV was 15.75% for C_max, 12.79% for AUC 0-t and 12.29% for AUC 0-inf parameters. These findings indicate low intra-subject variation, as reflected by the ISCV values, in comparison with the higher variation typically reported under fasting condition study, Table 3 and Table 5 and as described in the literature [4][10]-[13][16][17][22].

3.3.1. Safety Results

Subjects were monitored for adverse events throughout the study (fasting and fed). Vital signs (blood pressure, pulse rate, respiratory rate and ancillary/body temperature) were checked and evaluated before, during and at the end of study. The investigator did clinical examination of the subjects at the time of screening and during the check-out in each period. At the end of these studies, a safety evaluation was done which included hematology and clinical bio-chemistry laboratory. No adverse events or serious adverse events were reported.

3.3.2. Withdrawal and Drop Outs

In the fasting study, subject No. 09 (S09, sequence T-R-T-R) did not report to the clinical site for period II check-in due to a family issue and was therefore considered a dropout. For the fed study, subject S19 (sequence T-R-T-R) did not report to the clinical site for period II, and subject S23 (sequence T-R-T-R) did not report for period III. Both absences were due to personal reasons, and both subjects were considered dropouts from the study.