The bioequivalence trial was conducted in 2023 at Novus Clinical Research Services Limited, a DGDA-approved Contract Research Organization (CRO) in Dhaka, Bangladesh. The clinical stage of the study was performed from August 29 to September 18, 2023, and the analytical stage from October 17 to October 31, 2023.

The Bangladesh Medical Research Council (BMRC) of the National Research Ethics Committee (NREC) reviewed and approved the study protocol and all study documentation on January 22, 2023 (Registration No.: 50730102022). The study was also approved by the Directorate General of Drug Administration (DGDA) on April 13, 2023 (Reference No.: DGDA/CTP-04/2016/8688).

Good Clinical Practice, Good Laboratory Practice, Pharmaceutical Administration Law, and the Declaration of Helsinki (and its amendments) were all followed during the experiment.

Table 1 summarises the investigational products that were used in the research procedure. The doses of Dienogest used in this study were determined based on the recommended dose for endometriosis [23] .

A total of 27 healthy, adult, registered female volunteers were randomly selected for screening from the registered volunteers of Novus Clinical Research Services Limited. Among them, 13 eligible subjects aged 18 to 45 years with a body mass index between 18.5 and 29.99 kg/m² were included in the study. To confirm the eligibility of volunteers, chest radiography, electrocardiography, and laboratory investigations such as CBC, Blood glucose, HbA1C, serum creatinine, SGPT, SGOT, Uric Acid, Urea, Lipid profile, routine urine examination, etc., were carried out before 12 days of the first dosing.

Subjects were excluded from the study if any abnormalities were found in clinical investigations. It was confirmed that the subjects recruited for the study met inclusion and exclusion criteria. Each participant gave written informed consent before the screening, and study-specific informed consent was obtained from each participating subject before check-in.

This study was performed under fasting conditions using a single-centre, randomized-sequence, single-dose, two-period, two-treatment crossover design. Eligible subjects were randomized to one of the two dosing-order subgroups, T/R and R/T. SAS^® (SAS Institute Inc., USA) was used to randomise. The subjects in one sequence group were administered a single tablet of the test formulation with 240 mL of water in the first period, and after a washout period, individuals received a single tablet of the reference formulation in the second phase. The participants in the alternative sequence group received a reference tablet in the first period and a test tablet in the second period. The randomisation code was under controlled access till the completion of the analysis. The analysts were blinded to the sequence of administration of test and reference formulations throughout the analysis procedures.

Subjects were checked in the facility the day before the investigation’s medication was administered in each period to ensure an overnight fast of at least 10 hours. There was a 15-day washout period between two consecutive dosing periods of the study, which was considered appropriate as per requirements by the FDA and the EMA [24] .

The standard meal plan was identical for both study periods, and all in-house subjects received it at 04.00 hours following dosing. Except for one hour before and one hour after dosage, subjects were allowed to drink any amount of water they desired [25] .

Venous blood samples (5 mL) were collected from each subject approximately 22 times through an indwelling cannula to assay. Dienogest from predose to 72 hours postdose at preset time points (0.00 (pre-dose), 0.25, 0.50, 0.75, 1.00, 1.25, 1.50, 1.75, 2.00, 2.50, 3.00, 4.00, 5.00, 6.00, 8.00, 10.00, 12.00, 16.00, 24.00, 36.00, 48.00, and 72.00 hours) and placed in a K₂EDTA tube. Every plasma sample was centrifuged for 10 minutes at 5˚C ± 3˚C at 3500 rpm. Two duplicate tubes containing evenly divided plasma were frozen at −60˚C, one for testing and the other for backup.

Clinical examination and vital sign measurements were carried out to monitor the subjects’ safety at baseline and 1.00, 3.00, 5.00, 7.00, 9.00, 13.00, 26.00, 35.00, 48.00, and 72.00 hours after the dose, as specified in the protocol. However, investigations such as CBC, Blood glucose, HbA1C, serum creatinine, SGPT, SGOT, Uric Acid, Urea, Lipid profile, routine urine examination, etc and physical examinations, including 12-lead ECG and X-rays, were carried out at the time of screening and after the trial. Any adverse effects (AEs) that happened during the trial were tracked. Throughout the study, adverse events were evaluated for their severity, duration, and correlation with the study medication.

Dienogest plasma concentrations were determined using a previously validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. Protein precipitation was used to pretreat plasma samples. Chromatographic separation was done at 40˚C using a Thermo Scientific Hypersil Gold column (4.6 × 50 mm, 5.0 µm). The plasma linearity ranges from 1.000 ng/mL to 200.000 ng/mL. The intra-assay %CV and accuracy (relative error) for Dienogest were 1.30% to 6.22% and 99.5% to 110.2%, respectively, while the inter-assay %CV and accuracy were 3.34% to 4.87% and 103.6% to 107.4%.

The assay sequence was as follows: calibration standards of 1.000, 2.000, 10.000, 20.000, 40.000, 80.000, 160.000, and 200.000 ng/L, volunteers’ plasma samples, and quality-control samples of 3.000, 25.000, 100.0, and 150.00 ng/L throughout all sequences.

Pharmacokinetic parameters were calculated using WinNonlin software, and statistical comparisons of pharmacokinetic parameters were carried out using SAS^® statistical software (Version 9.4; SAS Institute Inc., USA). Pharmacokinetic primary parameters like C_max, AUC_0−t, and AUC_0−∞ and secondary parameters like T_max, t_1/2, K_el, and AUC_{Extrapolation} were determined for all the subjects who had completed both study periods. The two preparations will be bioequivalent if 90% Confidence Intervals (CI) for test/reference ratios of C_max, AUC_(0−t), and AUC_(0−∞) fall between the range of 80% and 125% [26] [27] .

A total of 13 participants were enrolled from 27 screened volunteers, and all finished the clinical phase of the study. Table 2 displays the demographic information of all enrolled subjects.

All Dienogest calibration curve standards are within the acceptance limit (1 - 200 ng/mL). The correlation coefficient was higher than 0.999. There were no visible interferences, and the chromatograms produced were entirely distinct from each other. The method validation followed international guidelines of the Food and Drug Administration (FDA) [28] and the European Medicines Agency (EMA) [29] .

All Adverse Events (AEs) were closely observed and monitored throughout the study. During the clinical stage, mild forms of AEs were observed (subjects 4, 8, and 9 experienced vomiting, and subject 9 had diarrhoea) and resolved spontaneously under medical supervision.

Figure 2 and Figure 3 show the mean plasma concentration-time curves of the

BMI = Body mass index; SD = Standard deviation.

two formulations. The superimposable figures suggest that the two formulations have equivalent mean plasma concentration-time curves.

Table 3 reports the pharmacokinetic results, and Table 4 shows the geometric means, geometric mean ratios, and 90% CIs for the pharmacokinetic parameters of the Dienogest 2 mg tablet.

The impact of formulations, sequences, and periods on log-transformed pharmacokinetic variables was evaluated using the analysis of variance (ANOVA) [30]

C_max: maximum plasma concentration of the drug, AUC_0−t: area under the plasma concentration-time curve from time zero to the time of the last measurable concentration, AUC_0−∞: area under the plasma concentration-time curve from time zero to infinity, T_max: time to reach maximum plasma Concentration, T_1/2: half-life of the drug, K_el: elimination rate constant.

model. No significant period or sequence effects were detected. The ANOVA results are displayed in Table 5.