Pharmacokinetics and Bioequivalence of Dienogest in Healthy Bangladeshi Female Volunteers: An Open-Label, Single-Dose, Randomized, Two-Way Crossover Study

Background: Dienogest is a potential treatment for pelvic pain associated with endometriosis, a condition of significant concern in gynaecology. The current study was conducted as a crossover -randomized bioequivalence as- sessment of two oral Dienogest 2 mg formulations, aiming to provide valu a- ble insights for healthcare professionals and researchers in this field. Objec- tive: The primary aim of this research was to evaluate and compare the pharmacokinetic characteristics of Dienogest 2 mg tablets. Dinogest (D i- enogest 2 mg) tablets, manufactured by Nuvista Pharma Limited in Bangl a- desh, and Visanne (Dienogest 2 mg) tablets, manufactured by Bayer Pharma in Germany, w ere the test and reference formulations, respectively.

and Method: The study was an open -label, balanced, randomized, two treat- ments, two sequences, two periods, two-way crossover, laboratory blind, single oral dose bioequivalence study conducted in healthy adult females under fast- ing conditions. The study was carried out on 13 healthy, non -pregnant female subjects, and all the subjects completed both study periods with a 15 -day wa- shout in between. Randomization was used to assign the test and reference formulations to the subjects. Following each oral administration, a series of blood samples were ob tained at different time intervals from pre -dose to 72 hours post-dose and analyzed for Dienogest concentrations using a validated bio-analytical method. A standard non- compartmental model was used to analyze the pharmacokinetic parameters. The primary pharmacokinetic p a- rameters were peak plasma drug concentration (C max), the area under the How to cite this paper: Mahmud, N., Ahamed, N., Bhowmik , U.K., Tushi , S.A., Ghosh, N., Sahana, N.C., Akram , A. and Reza, M.A. (2024) Pharmacokinetics and Bioequivalence of Dienogest in Healthy Ban- gladeshi Female Volunteers: An Open-Label, Single-Dose, Randomized, Two -Way Cro s- sover Study. Open Journal of Obstetrics and Gynecology, 14, 779-789. https://doi.org/10.4236/ojog.2024.145064 Received: April 12, 2024 Accepted: May 21, 2024 Published: May 24, 2024 Copyright © 2024 by author(s) and Scientific Research Publishing Inc. This work is licensed under the Creative Commons Attribution International License (CC BY 4.0). http://creativecommons.org/licenses/by/4.0/ Open Access N. Mahmud et al. DOI: 10.4236/ojog.2024.145064 780 Open Journal of Obstetrics and Gynecology plasma concentration-time curve from time zero to time t (AUC0–t), and AUC from t = 0 to infinity (AUC 0–∞ ). The other PK parameters included time to reach Cmax (Tmax), terminal elimination rate constant (K el), and half -life (t1/2).

The ratios and 90% CI for the geometric mean test/reference were 95.53% (86.70% - 105.26%) for Cmax, 101.75% (95.42% - 108.49%) for AUC0−t, and 101.54% (95.59% % - 107.87%) for AUC 0−∞ . The formulations were bio e- quivalent sin ce the 90% CIs for the geometric mean test/reference ratios were 80% to 125%, according to the predetermined range of US Food and Drug Ad- ministration (FDA) requirements. Conclusion: This single-dose investigation shows that the Dienogest test and reference formulations exhibited a rate and degree of absorption that met the regulatory requirements for bioequivalence.

Dienogest, Bioequivalence Study, Endometriosis, Novus CRSL 1. Introduction Endometriosis is a chronic, neuro -inflammatory condition defined as the deve l- opment of endometrial glands and stroma- like lesions outside of the uterus [1] [2]. Endometriosis frequently exhibits symptoms (dysmenorrhea, deep dysp a- reunia, chronic pelvic pain, etc.) that overlap with other gastrointestinal and g y- necologic conditions, making diagnosis m ore difficult [3] . Consequently, for many of these women, the diagnosis of endometriosis can be difficult and leng- thy, and often, the delay results in a reduced qu ality of life [4] . Treatment co n- sists of the surgical removal of lesions and hormonal medication [5] . Endome- triosis is estimated to affect 6% - 10% of women of reproductive age [6]. Treat- ment of endometriosis consists of either medical or surgical management [7] . The surgical management of endometriosis is effective but has several contr o- versial features [8] . Endometriosis must be regarded as a chronic pain disorder with a high recurrence rate, even after surgical removal [9]. Since endometriosis is essentially a hormonal disease, hormonal drug therapy is currently considered an essential and effective therapy [10] . Endometriosis has been treated with various hormones and medicines [11] . Specific medical ther a- pies that are approved for the treatment of endometriosis include gonadotr o- pin-releasing hormone (GnRH) agonists, danazol, the Combined Oral Contr a- ceptive Pill (COCP), and certain progestins [12]. Dienogest is a new generation of progestin that has become one of the most used drugs in all endometriosis phenotypes for long -term treatment [13] [14]. According to the ESHRE (European Society of Human Reproduction and Embry- ology) guidelines, progestins are primarily used as a first-line, long-term treatment that is highly effective and acts on multiple sites of action [15]. Dienogest is almost completely absorbed and has a high oral bioavailability of more than 90% [16]. As it has a relatively short half- life of 10 hours, there is no risk of accumulation of N. Mahmud et al. DOI: 10.4236/ojog.2024.145064 781 Open Journal of Obstetrics and Gynecology the drug in the body even after multiple dosages. Orally administered Dienogest is excreted through urine within 24 hours [17] . Its molecular formula is C20H25NO2, with a molecular weight of 311.4 g/mol [18] . Chemically, Dienogest is described as (17 α-cyanomethyl-17β-hydroxy-estra-4,9-dien-3-one [19]. Its structural formula is displayed in Figure 1 [20]. Peak serum concentrations of approximately 47 nanograms per mL are reached about 1.5 hours after single ingestion [21]. Bioequivalence studies play a crucial role in evaluating a drug ’s efficacy by providing scientific evidence of therapeutic equivalence between different fo r- mulations. If two drugs are bioequivalent, they are expected to be the same for all intents. This study aims to investigate the bioequivalence of test formulations to reference formulations of Dienogest in healthy Bangladeshi female volunteers under fasting conditions [22] . Demonstrating bioequivalence ensures the safety, efficacy, and affordability of generic medications, ultimately benefiting patients, healthcare providers, and healthcare systems alike. 2. Methods 2.1. Study Centre and Study Period/Duration The bioequivalence trial was conducted in 2023 at Novus Clinical Research Se r- vices 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. 2.2. Ethical Consideration The Bangladesh Medical Research Council (BMRC) of the National Research Ethics Committee (NREC) reviewed and a pproved 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 Admini s- tration Law, and the Declaration of Helsinki (and its amendments) were all fo l- lowed during the experiment. Figure 1. Chemical structure of Dienogest. N. Mahmud et al. DOI: 10.4236/ojog.2024.145064 782 Open Journal of Obstetrics and Gynecology 2.3. Identity of Investigational Products 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]. 2.4. Study Subjects 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/m 2 were included in the study. To confirm the eligibility of volunteers, chest radiography, electrocardiography, and laboratory investigations su ch as CBC, Blood glucose, HbA1C, serum creatinine, SGPT, SGOT, Uric Acid, Urea, Lipid profile, routine urine examination, etc., were ca r- ried 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. 2.5. Study Design This study was performed under fasting conditions using a single -centre, ra n- domized-sequence, single-dose, two-period, two-treatment crossover design. E l- igible subjects were randomized to one of the two dosi ng-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. Table 1. Identification of the experimental product (s). IMP details Test product (T) Reference product (R) Trade Name Dinogest Visanne Generic Name Dienogest Dienogest Specification 2 mg/tablet 2 mg/tablet Batch/Lot No. 104223002 WEU6CF Expiry Date Dec’ 2024 Dec’ 2023 Manufacturer Nuvista Pharma Ltd., Dhaka, Bangladesh Bayer Pharma, Germany N. Mahmud et al. DOI: 10.4236/ojog.2024.145064 783 Open Journal of Obstetrics and Gynecology Subjects were checked in the facility the day before the investigation ’s medica- tion 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 p e- riods of the study, which was conside red appropriate as per requirements by the FDA and the EMA [24]. 2.6. Standard Meal and Fluid 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]. 2.7. Blood Sampling Venous blood samples (5 mL) were collected from each subject approximately 22 times through an indwelling cannula to assay. Dienogest from predo se 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 K 2EDTA tube. Every plasma sample was centrifuged for 10 minutes at 5 ˚C ± 3˚C at 3500 rpm. Two duplicate tubes co n- taining evenly divided plasma were frozen at − 60˚C, one for testing and the ot h- er for backup. 2.8. Safety Assessment Clinical examination and vital sign measurements were carri ed 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, i n- vestigations such as CBC, Blood glucose, HbA1C, serum creatinine, SGPT, SGOT, Uric Acid, Urea, Lipid profile, routine urine examination, etc and phys i- cal 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 ev a- luated for their severity, duration, and correlation with the study medication. 2.9. Analytical Method Dienogest plasma concentrations were determined using a previously validated liquid chromatography-tandem mass spectrometry (LC -MS/MS) method. Pr o- tein precipitation was used to pretreat plasma samples. Chromatographic sep a- ration 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 sequenc e 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 N. Mahmud et al. DOI: 10.4236/ojog.2024.145064 784 Open Journal of Obstetrics and Gynecology samples, and quality -control samples of 3.000, 25.000, 100.0, and 150.00 ng/L throughout all sequences. 2.10. Pharmacokinetic and Statistical Analyses 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). Pharmacoki netic primary parameters like C max, AUC 0−t, and AUC 0−∞ and secondary parameters like Tmax, t1/2, Kel, and AUCExtrapolation 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 Cmax, AUC(0−t), and AUC(0−∞) fall between the range of 80% and 125% [26] [27]. 3. Results 3.1. General Characteristics of the Subjects A total of 13 participants were enro lled from 27 screened volunteers, and all fi- nished the clinical phase of the study. Table 2 displays the demographic info r- mation of all enrolled subjects. 3.2. Method Validation 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]. 3.3. Tolerability and Safety Assessment 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 spont a- neously under medical supervision. 3.4. Pharmacokinetic Parameters Figure 2 and Figure 3 show the mean plasma concentration- time curves of the Table 2. Demographic Characteristics of the Subjects (n-13). Characteristic Values Age, mean (SD), range, years 26 (5.44), 18 - 35 Weight, mean (SD), range, kg 57.04 (12.54), 40.70 - 77.80 Height, mean (SD), range, cm 152.80 (8.29), 143 - 168 BMI, mean (SD), range, kg/m2 24.30 (4.27), 18.80 - 29.99 BMI = Body mass index; SD = Standard deviation. N. Mahmud et al. DOI: 10.4236/ojog.2024.145064 785 Open Journal of Obstetrics and Gynecology Figure 2. Linear Plot of Mean Plasma Concentration (ng) versus Time (hr). Figure 3. Semilog Plot of Mean Plasma Concentration (ng) versus Time (hr). 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 phar- macokinetic variables was evaluated using the analysis of variance (ANOVA) [30] N. Mahmud et al. DOI: 10.4236/ojog.2024.145064 786 Open Journal of Obstetrics and Gynecology Table 3. Pharmacokinetic parameters (N = 13). Visanne (Dienogest 2 mg) tablets ( Reference Product ) Variable Arithmetic Mean SD CV% Min Median Max Primary Variable Cmax (ng/mL) 55.0587 12.02436 21.8 39.105 51.127 78.118 AUC0− t (hr*ng/mL) 694.8138 194.88638 28.0 408.241 696.563 1100.844 AUC0− ∞ (hr*ng/mL) 727.4534 203.11283 27.9 434.141 729.589 1162.059 Tmax (hr) 1.6346 0.93883 57.4 0.750 1.250 4.000 AUC_% Extrap_obs (%) 4.5198 1.37358 30.4 1.945 4.527 6.452 T½ (hr) 12.8260 3.04074 23.7 8.436 12.320 17.699 Kel (hr− 1) 0.0570 0.01379 24.2 0.039 0.056 0.082 Dienogest 2 mg Tablets ( Test Product ) Secondary Variable Cmax (ng/mL) 52.9390 13.48170 25.5 35.817 51.465 89.783 AUC0− t (hr*ng/mL) 707.0434 186.81099 26.4 440.166 736.734 1007.867 AUC0− ∞ (hr*ng/mL) 738.9579 194.24527 26.3 459.597 768.867 1041.096 Tmax (hr) 2.0192 0.59039 29.2 1.000 2.000 3.000 AUC_% Extrap_obs (%) 4.3629 1.08353 24.8 2.899 4.179 5.932 T½ (hr) 12.6727 2.63849 20.8 9.594 11.491 17.078 Kel (hr− 1) 0.0568 0.01095 19.3 0.041 0.060 0.072 Cmax: maximum plasma concentration of the drug, AUC 0− t: area under the plasma co n- centration-time curve from time zero to the time of the last measurable concentration, AUC0− ∞ : area under the plasma concentration-time curve from time zero to infinity, Tmax: time to reach maximum plasma Concentration, T 1/2: half-life of the drug, K el: elimination rate constant. Table 4. Summary results. Parameter Geometric Least Squares Means (GEOLSM) T/R Ratio (%) 90% Confidence Interval Intra Subject CV (%) Power (%) Test Product

Product Lower Limit (%) Upper Limit (%) Cmax (ng/mL) 51.368 53.773 95.53 86.70 105.26 13.79 96.01 AUC0− t (hr*ng/mL) 680.392 668.707 101.75 95.42 108.49 9.10 99.90 AUC0− ∞ (hr*ng/mL) 711.497 700.698 101.54 95.59 107.87 8.57 99.95 N. Mahmud et al. DOI: 10.4236/ojog.2024.145064 787 Open Journal of Obstetrics and Gynecology Table 5. P-values for sources of variations obtained from the analysis of variance (ANOVA). ANOVA p Values Parameters LCmax LAUC0− t LAUC0− ∞ Sequence 0.4850 0.6515 0.6743 Period 0.4148 0.7241 0.6127 Formulation 0.7197 0.6374 0.6584 model. No significant period or sequence effects were detected. The ANOVA

are displayed in Table 5 . 4. Discussion For Cmax, the ratios of least -squares mean (with 90% confidence intervals) were 95.53% (86.70% - 105.26%). For AUC 0−t and AUC0−∞ , the ratios of least -squares mean (with 90% confidence intervals) were 101.75% (95.42% - 108.49%) and 101.54% (95.59% - 107.87%), respectively. All of the 90% CI of the pharmacokinetic parameters (C max, AUC 0–t, and AUC0–∞ ) were within the bioequivalence acceptable range of 80% to 125%. Moreover, the C max profile of Dienogest 2  mg was almost identical for the test and reference products. The absence of sequence effects in the ANOVA also i n- dicated the absence of a carry-over effect. 5. Limitations There are some limitations in this current study, as with any other bioequiv a- lence study. The results were obtained from healthy adult individuals of a d e- fined age range who were given a single dose of the formulation in compliance with regulatory criteria. The pharmacokinetics might differ among patients in different age groups. The findings of this study may not be general ized to a sp e- cific target population. A non- compartmental model was used to calculate the pharmacokinetic parameters, which are based on certain assumptions about the pharmacokinetic behaviour of the drug, such as uniform distribution and linear kinetics. Any deviations from these presumptions may impact the precision of the model’s predictions. 6. Conclusion The test product Dinogest (Dienogest 2  mg) tablet was unequivocally bioequiv a- lent with the reference product Visanne ® (Dienogest 2  mg) tablet in healt hy adult participants under fasting conditions, per regulatory requirements. Both formulations were well tolerated. Conflicts of Interest The authors declare no conflicts of interest regarding the publication of this paper. N. Mahmud et al. DOI: 10.4236/ojog.2024.145064 788 Open Journal of Obstetrics and Gynecology

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