This was a single-site, open-label, randomized, balanced, laboratory-blind, single-dose, two-treatment, two-sequence, two-period, two-way crossover oral bioequivalence study of Vonoprazan Fumarate 20 mg tablet manufactured by Beximco Pharmaceuticals Limited, Bangladesh (T) and Takecab 20 mg (Vonoprazan Fumarate 20 mg) tablet of Takeda Pharmaceutical Company Limited, Japan, in healthy adult male human subjects under fasting conditions. SAS was used for randomization and treatment allocation. All the study personnel, as well as the participants, were unblinded to the treatment assigned in each period. Nevertheless, during the analytical procedures, the analyst remains blinded to the sequence of treatment allocation. The clinical investigation was completed over 15 days. Based on Vonoprazan’s reported plasma half-life, which ranges from 7 to 9 hours [32] [33] , a seven-day washout time was considered appropriate for this clinical study since it exceeds the seven half-lives recommended by the FDA [34] .

The study was conducted at one of Bangladesh’s earliest DGDA-approved Contract Research Organizations (CRO). The clinical part of the study was carried out at Novus Clinical Research Services Limited from January 25 to February 04, 2024, while the analytical stage was carried out from February 15 to March 04, 2024.

The study adhered to the most recent versions of the Declaration of Helsinki and the International Council for Harmonization’s current guidelines for good clinical practice [35] [36] . The study documents, including the protocol and consent form, were reviewed and approved by the Bangladesh Medical Research Council (BMRC) of the National Research Ethics Committee (NREC) on November 2023 (Reference No.: BMRC/NREC/2022-2025/272). The study was also approved by the Directorate General of Drug Administration (DGDA) on January 2024 (Reference No.: DGDA/CTP-04/2016/1303).

Table 1 shows the Identification of both Investigational Products.

The participants had to go through a pre-study screening process. Randomly, 48 Bangladeshi volunteers were chosen from Novus’s registered volunteer database if they could participate, communicate well with the researchers, be available during the study period, and give written informed consent. Following a pre-study medical evaluation, 18 healthy participants were enrolled in the study as subjects. Volunteers aged 18 - 55 with a Body Mass Index (BMI) between 18 and 30 kg/m² were recruited for the study [37] . All participants underwent a thorough health assessment, including measurements of blood pressure (BP), pulse rate, temperature, and respiration rate during a physical examination and interview. Additionally, they underwent a 12-lead ECG, chest radiography, hematology and blood chemistry tests, urinalysis, tests for alcohol and drug abuse, and a COVID antigen test. Furthermore, serological examinations included testing for antibodies to HIV, hepatitis B, and hepatitis C. All tests were conducted at the diagnostic center of Novus Clinical Research Services Limited. Selected participants were required to be free of any significant disease or clinically significant abnormal laboratory values upon evaluation of medical history, physical examination, and laboratory tests.

Individuals with known allergies or hypersensitive intolerances to Vonoprazan, chronic illnesses, bleeding disorders, or abnormal health conditions assessed by baseline medication, medical history, and vital signs were excluded from the study. Subjects with difficult vein accessibility were also excluded. Participants were advised to avoid medications impacting liver drug-metabolizing enzymes for at least one month before and during the trial. They were also instructed not to use any prescribed medications for 14 days or over-the-counter (OTC) products for seven days before and during the trial. Participants were asked to abstain from consuming alcohol, beverages, or food containing xanthines for 48 hours before the study and until the final blood sample was collected. Moreover, individuals who had experienced blood loss or donated more than 350 milliliters of blood in the three months before the experiment were ineligible to participate.

On the day before medication administration, eligible participants checked into the clinical pharmacology unit and fasted for 10 hours (except for water) to establish baseline plasma measurements. Randomization was done using SAS (Version 9.4; SAS Institute Ind., USA) by a statistician to allocate individuals to dosing-order subgroups, T/R and R/T. Each participant received a test or reference formulation tablet with 240 milliliters of water. Participants were prohibited from drinking water for one hour before and after drug administration. Following dosing, participants were closely monitored while seated for four hours.

A pre-dose (0.00 hour) pharmacokinetic blood sample was taken on the dosing day, one hour before the study drug’s administration, to establish the baseline plasma level. Blood samples (5 mL) were collected in K₂EDTA tubes from the forearm vein at specific intervals ranging from 0.25 to 48.00 hours after drug administration. The collected samples were subjected to centrifugation (3500 RPM for 10 minutes at 5˚C ± 3˚C) to separate plasma, which was then transferred to polypropylene tubes and stored at −70˚C until liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis was performed.

Clinical laboratory testing (hematology, clinical biochemistry, serology, and urine analysis), physical examinations, vital signs, ECGs, and subject-reported adverse events (AEs) monitoring were all included in the safety assessments. Following administering the medication, vital signs (blood pressure, pulse rate, respiration rate, and body temperature) were recorded at 1.00, 3.00, 5.00, 7.00, 9.00, 11.00, 13.00, 24.00, and 48.00 hours. Throughout the trials, spontaneous reports of adverse events were collected and assessed for severity and correlation with the study medication. Upon completion of the study, clinical laboratory determinations were performed and evaluated to monitor adverse events.

The concentration of Vonoprazan in plasma was measured using protein precipitation and a validated LC-MS/MS method, with Linagliptin as the internal standard. Chromatographic separation utilized an ACE 3 C18-AR column with a size of 4.6 × 50 mm and a 3.0 µm particle size at a column temperature of 30˚C and a flow rate of 0.600 mL/min. The lower limit of quantitation (LLOQ) for Vonoprazan was 0.500 ng/mL, with a dynamic range of 0.500 to 75 ng/mL. The method was found to be linear across this concentration range, with a correlation coefficient greater than 0.99. Quality control (QC) samples at concentrations of 0.500, 2.00, 15.00, 25.00, and 50.00 ng/mL were analyzed and distributed equally among the volunteers’ plasma samples to evaluate method accuracy and precision. Vonoprazan’s within-run precision and accuracy ranged from 2.23% to 9.27% and 96.4% to 120.7%, respectively, whereas its between-run precision and accuracy ranged from 4.16% to 9.57% and 102.4% to 111.8%, respectively.

After completing both study periods, pharmacokinetic analysis was conducted for each participant. Key pharmacokinetic parameters including the maximum observed plasma concentration (C_max), area under the plasma concentration-time curve from administration to the last observed quantifiable concentration at time t (AUC_0-t), area under the plasma concentration-time curve extrapolated to infinity (AUC_0-∞), time to reach maximum observed plasma concentration (T_max), and plasma half-life (t_½) were calculated based on the plasma concentrations of Vonoprazan.

Statistical analysis was conducted using the SAS® statistical software, Version 9.4 of SAS Institute Ind., USA. ANOVA was used to evaluate the log (Ln) transformed pharmacokinetic parameters (AUC_0-t and AUC_0-∞) at a significance level of 5% (α = 0.05). The terms sequence, period, and formulation were used in the ANOVA model. When the 90% confidence intervals (CIs) for the geometric mean ratios of C_max, AUC_0-t, and AUC_0-∞ for both analytes fall between 80% and 125% for the Ln-transformed data, bioequivalence will be declared [38] [39] .

Each of the eighteen enrolled participants who received the Vonoprazan test or reference therapy completed the trial. Table 2 summarizes the baseline demographics of all subjects who completed the study.

Table 3 summarizes the major pharmacokinetic parameters of Vonoprazan under fasting conditions.

Overall, plasma concentrations ( Figure 1 and Figure 2 ) and exposure to Vonoprazan were similar between the two treatments.

Table 4 shows the geometric means, geometric mean ratios (test/reference), 90% confidence interval, and intra-subject percentage CV of the bioequivalence statistics derived from the log-transformed data for C_max, AUC_0–t, and AUC_0–∞. The 90% confidence intervals for Vonoprazan C_max, AUC_0–t, and AUC_0–∞ were 99.47% - 119.53%, 95.58% - 107.50%, and 95.43% - 107.41%, respectively. The 90% confidence intervals of the geometric mean ratios for the three parameters all fell within the predetermined range of 80% to 125%. Therefore, these findings suggest that the bioequivalence requirements were met.

C_max = Maximum plasma drug concentration, AUC_0-t = AUC from time 0 (baseline) to the last measurable concentration, AUC_0-∞ = AUC from baseline extrapolated to infinity.

The ANOVA results ( Table 5 ) show that using logarithmic-transformed data, no sequence, period, or formulation effects were observed (p ≥ 0.5) for any PK parameters.

During the trial, Vonoprazan demonstrated good tolerability. Only two subjects reported adverse events of diarrhea and vertigo, both of which were moderate and not considered possibly or probably related to the study treatment by the investigator. These events did not lead to any premature discontinuation of the trial by participants, nor were any deaths or serious adverse events reported. The laboratory safety tests, 12-lead ECGs, and vital signs showed no clinically significant abnormalities. Additionally, the general health status of all participants was deemed clinically fit after the study’s clinical phase.