Oral progesterone is a proven and effective treatment for several uterine disorders [1]. However, hepatic first-pass metabolism and low oral bioavailability (< 10%) have limited its widespread use [2]. Dydrogesterone, a synthetic stereoisomer of the natural progesterone, is considered as an alternative to oral progesterone [3–5]. Dydrogesterone, owing its unique pharmacological features, offers increased affinity for progesterone receptors, decreased affinity for steroid receptors and improved bioavailability compared to oral progesterone [3].

In recent years, dydrogesterone has been widely used in gynecology and obstetrics for the treatment of varied conditions such as irregular mensuration [6], dysmenorrhea [7], threatened or recurrent miscarriage [8] and as a vital component of menopausal hormone therapy [9]. Besides, multiple-doses of conventional dydrogesterone tablets are also recommended for polycystic ovarian syndrome [10], abortion [8], infertility [3], and endometriosis [11]. Dydrogesterone has been shown to be effective in preventing miscarriages by sustaining healthy womb lining and promoting endometrial receptivity [12]. According to recent survey, dydrogesterone 10 mg twice daily and/ or 10 mg thrice daily for duration ranging from few weeks to few months was the most recommended schedule by the gynecologists [13]. Multiple-dose of conventional dydrogesterone for long duration raise concern of low adherence and poor patient compliance [14]. Adherence to dydrogesterone treatment is vital for achieving positive pregnancy outcomes, especially in cases of luteal phase defects, threatened miscarriage, and recurrent pregnancy loss [15]. Reports indicate that nonadherence to long-term treatment is a stern problem, accounting for more than 50% of cases where therapeutic goals are not achieved [16, 17]. The adherence problems further exaggerate when self-administration is required as in case of dydrogesterone treatment. Several existing reports suggested that reducing dosage frequency from twice daily to once daily dosing may improve adherence to therapies among patients and subsequent decreases in health care costs [18]. Thus, several attempts have been made to develop sustained release (SR) dydrogesterone dosage forms [14, 19, 20]. The SR dydrogesterone is expected to provide better therapeutic compliance and treatment adherence thus providing better clinical results. In a recent clinical study, SR dydrogesterone tablet has shown to be bioequivalent to the conventional dydrogesterone tablet [19]. The present study aimed at evaluation of the pharmacokinetics and pharmacodynamics of once-daily, SR dydrogesterone (20 mg) in comparison to twice daily, conventional dydrogesterone (10 mg), in rabbits. In vivo scintigraphy imaging, following oral administration of technetium-99m (99mTc) radiolabeled formulations, was conducted to evaluate the SR behavior. Plasma dydrogesterone was determined at predesignated time points for kinetics. For dynamics, pregnant rabbits were used and endometrial changes were determined using ultrasonography, biochemical changes, and endometrial histopathology post-treatment. Comparison was made against conventional dydrogesterone and animals treated with saline (control).

Dydrogesterone SR tablet (Dydrohope 20 mg, Corona Remedies) and conventional tablets (Duphaston 10 mg, Abbott) were purchased from local pharmacy shop (Kamla Nagar, New Delhi). The stannous chloride was purchased from Sigma Aldrich (St. Louis, MO) and 99mTc was obtained from Baba Atomic Research Centre (Mumbai, India). All other chemicals used were of analytical grade and were purchased from CDH Laboratory (New Delhi, India). Scintigraphy study was conducted at Batra Research Center (New Delhi, India).

Adult female New Zealand White rabbits, 4 to 6 months old, weighed 2.0 to 4.5 kg were used. The rabbits were housed in a controlled environment with 25 ± 2°C temperature, 57 ± 7% relative humidity, a 12-hour light/dark cycle with standard hygienic conditions, free access to fresh tap water, and ad libitum a pelleted diet. All the animal experiment was performed as per the guidelines of Committee for the Purpose of Control and Supervision of Experiments on Animals and with the approved protocol. The animals were acclimatized for two weeks before the experiment.

Scintigraphy and pharmacokinetic study were conducted in non-pregnant rabbits (n = 6). Rabbits were randomly divided into two groups (n = 6). One group received conventional dydrogesterone (10 mg tablet) formulation (CDF group) and other received SR dydrogesterone (20 mg tablet) formulation (SDF group). For scintigraphy, the tablets were radiolabeled with 99mTc using drill and fill method as described previously [21]. Briefly, a hole in the centre of the tablet was made by the help of needle and filled by stannous chloride reduced per technetate. The remaining space of the hole was filled with lactose and completely sealed with wax. The radioactive counts of the tablets were determined using dose calibrator (PTW Curiementor 2, USA). Labeling efficiency and stability were confirmed by instant thin layer chromatography. Radiolabeling was conducted carefully so that each tablet should have nearly 2 MBq of radioactivity. Similarity in dissolution profile of radiolabeled and non-radiolabeled study product validated that the radiolabeling had no impact on dispersion behavior. Radiolabeled tablets were orally administered to animals with feeding sonde such that the tablet was placed close to esophagus avoiding chewing and crushing. Sequential scintigraphic images and blood samples were collected at predesignated time points. Scintigraphy images of the abdominal area were captured with dual head SPECT gamma camera (Millennium MG, GE Healthcare, US) until complete disintegration of the tablet occurred. The images were analyzed using the Genie 4.5 image analysis. Blood samples were collected from marginal ear vein using syringe. The plasma was separated, extracted with methanol and drug content was determined by developed LC-MS/MS method [22]. All the samples were stored at -20°C until analyzed.

For efficacy, 18 female rabbits were randomly divided into 3 groups (n = 6): (a) control group treated with 1 ml normal saline twice-daily, (b) CDF group treated with conventional dydrogesterone (1 mg) twice-daily and, (c) SDF group treated with SR dydrogesterone granules (2 mg) once-daily. The pregnancy was induced by mating with male rabbits. The treatment begins with the mating and lasted till the end of first trimester (11–13 days), determined through palpation. The formulations were dispersed in water and fed through oral sonde, directly into esophagus. All the evaluations were made post-treatment, at end of first trimester.

Each animal was properly restrained physically and placed on dorsal recumbency. Hair from the abdomen was gently made wet, with a soaked cotton in water and shaved. The shaved region was cleaned thoroughly with a dry cotton and swabbed with wool soaked in antiseptic solution. Ultrasound gel was then applied on the shaved area and ultrasonography was conducted using ultrasound machine. A transcutaneous probe was used to scan the abdomino-pelvic region. The probe was placed gently on the skin and tilted until a descriptive echographic image was obtained. Endometrial receptivity was assessed by considering the endometrial thickness (distance from the interface between the endometrium and myometrium), volume, contraction or peristalsis and blood flow of the endometrium [23]. We assessed endometrial receptivity by considering the modified endometrial receptivity (ER) scoring system as described previously [22] and shown in Table 1. All the ultrasound parameters were examined by the same doctor using the same ultrasound instrument.

Blood samples (1 ml) were collected from marginal ear vein of each animal using syringe. The samples were collected in heparinized tubes and centrifuged at 2500 rpm for 20 minutes to get plasma. Samples were stored at -20°C until hormonal analyses. We assayed progesterone-induced blocking factor (PIBF), integrin αVβ3 and vascular endothelial growth factor (VEGF) using ELISA kits (Krishgen, India) as per manufacturer protocol. The PIBF, integrin αVβ3 and VEGF was expressed in nanomoles per liter, nanogram per milliliter and picogram per milliliter, respectively.

The change in morphology of endometrium due to dydrogesterone treatment was evaluated using histopathology. Briefly, post-treatment, the animals were euthanatized by intraperitoneal injection of sodium barbital. The abdominal wall was opened longitudinally along the midline, uterus was excised, and the entire endometrium was peeled off from the uterus. The endometrial tissue was fixed in 10% neutral buffered formalin, embedded in paraffin, and cut to 4–5 µ thick sections. The samples were then stained with hematoxylin-eosin for assessment of morphological changes. Sections were observed under light microscope (Olympus) at 400x of original magnification. Ten visual fields were analyzed per tissue section from each animal.

Figure 1 illustrates scintigraphic images of CDF and SDF group animal administered with conventional and SR dydrogesterone tablet, respectively. The scintigraphy images showed distinct in vivo release pattern for conventional and SR tablets. Conventional dydrogesterone tablets (CDF group) remained intact for upto 1 h and disintegrated in duodenum or upper intestine within 2 h. Complete disintegration of conventional tablet occurred within 4 h in small intestine (Fig. 1). In contrast, SR tablets remained intact for upto 6 h. Images showed small fraction of drug release, possibly due to polymer erosion from the tablet surface. Further, tablet disintegrated in small intestine at 8 h followed by drug release. As expected, scintigraphy revealed prolonged disintegration and slow drug release from SR formulation.

Figure 2. Plot of dydrogesterone concentration in plasma as a function of time following oral administration of conventional and SR dydrogesterone to rabbits (n = 6).

The relevant pharmacokinetic parameters are listed in Table 2. The pharmacokinetic results were in corroboration with scintigraphy findings and SR dydrogesterone showed nearly 2.4-fold higher AUC_0-48 compared to conventional formulation. As expected, the t_max of SR dydrogesterone was twice (6 h) as that of conventional dydrogesterone (3 h). Additionally, the C_max of the SR dydrogesterone was significantly (p < 0.05) lower than conventional dydrogesterone. The mean resident time of conventional dydrogesterone (9.06) was significantly (p < 0.05) lower than SR dydrogesterone (18.8). Also, the fluctuation index for SR dydrogesterone was 2.18, which was markedly lower than fluctuation index of conventional dydrogesterone (3.42). The kinetic data confirmed the prolonged release behavior and consistent drug concentration in plasma of SR dydrogesterone.

No significant difference in dose duration exists between the groups and the dose duration was 11.83 ± 0.83, 12.17 ± 0.75 and 12.0 ± 0.63 days for SDF, CDF and control group, respectively. When comparing the ultrasound parameters of endometrial receptivity between the control and treatment groups, it was found that mean endometrial thickness for control group (9.77 ± 0.46 mm) was significantly less (p < 0.05) than the treatment groups. The mean endometrial thickness for CDF and SDF group animals was 11.9 ± 0.28 mm and 12.07 ± 0.20 mm, respectively (Table 3) and the difference was not statistically significant. In control group, 2 out of 6 animals showed distinct endometrial layering, 2 showed hazy layering and in remaining 2 the layering was absent. Four out of six animals in both CDF and SDF group showed distinct endometrial layering. Further, both the remaining animals in SDF group has hazy layering unlike CDF group where layering was absent in one animal (Table 3).

The endometrial echogenic line results were in agreement with endometrial layering. Homogenous echogenic line was observed in 4 control, 5 CDF and in all 6 SDF group animals. Similarly, endometrial volume greater than 3 ml was observed in 4 out of 6 control animals and in 5 out of 6 animals in both the treatments. Three out of six CDF and SDF group animals showed multifocal blood flow. Further, 2 CDF group animals showed sparse blood flow and in last one the blood flow was not visible. In contrast, all the remaining 3 SDF group animals showed sparse blood flow. Interestingly none of the control animal showed multifocal blood flow and all showed sparse blood flow (Table 3). The representative ultrasonographic image of one animal from each group is presented in Fig. 3.

Consequently, the ER score for each animal of control and treatment groups is shown in Table 4. ER score of the treatment groups was significantly higher (p < 0.05) than control (4.67). The higher ER score of treated animals correlate with improved clinical pregnancy rates and demonstrated the effectiveness of dydrogesterone in endometrial receptivity. Among treatment groups, SDF showed higher ER score (7.0) than CDF group (6.5) but the difference was not statistically significant (p > 0.05).

The endometrial receptivity can be predicted by biochemical assessment of markers such as VEGF, integrin αVβ3 and PIBF. Results (Fig. 4) showed that the all the three biochemicals are positively correlated with dydrogesterone treatment. Improved level of VEGF, integrin αVβ3 and PIBF were observed in treatment groups compared to control (p < 0.05). The level of PIBF and integrin αVβ3 was not significantly different among treatment groups. However, VEGF level of SDF animals was statistically higher (p < 0.05) than CDF animals.

Histopathology images of endometrium of control, CDF and SDF group animals are shown in Fig. 5. No changes in endometrial glands, lining, shape and other microstructures of treatment group animals were observed and images were similar to control animals. Glands are lined by oval to elongated cells having oval nuclei with coarse chromatin and conspicuous nucleoli. Further, glands to stromal ratio was also maintained in treatment group similar to control. The glandular response was better in treatment groups compared to control and score was 2 for treatment group and 1 for control. We observed that the three groups differ in vasculature (Fig. 5). Control group showed few blood vessels, whereas animals treated with dydrogesterone showed moderate to dense vasculature. In both the treatment group the blood vessels were scattered and majority were seen at junction of endometrium and myometrium (Fig. 5). The vascularity score was 1, 2 and 3 for control, CDF and SDF group animals, respectively. The SDF group showed better vascularity compared to other two groups. The results showed that these effects could be due to dydrogesterone which showed pregestational effect mimicking luteal phase.

The preclinical assessments of the present study demonstrated pharmacokinetic and pharmacodynamic equivalence of once-daily SR dydrogesterone and twice-daily conventional dydrogesterone with respect to bioavailability and endometrial receptivity, foreseeing SR dydrogesterone as a valuable, patient compliant alternative to conventional dydrogesterone particularly for chronic reproductive disorders such as luteal phase defects, threatened miscarriage, and recurrent pregnancy loss requiring long-term treatment [14]. Adherence to dydrogesterone treatment is vital for achieving positive pregnancy outcomes and formulation with reduced dosage frequency is expected to improve adherence and patient compliance [15]. Tc-99m scintigraphy along with kinetics was used to assess the in vivo performance of the radiolabeled formulations. Our kinetic data showed 2-fold higher t_max and significantly (p < 0.05) lower C_max of SR dydrogesterone compared to conventional dydrogesterone. The results were consistent with scintigraphy and SR formulation demonstrated delayed disintegration (⁓ 8 h) in small intestine compared to conventional dydrogesterone (⁓ 4 h), which completely disintegrated in stomach or duodenum. In agreement, mean resident time of SR dydrogesterone was nearly twice higher than conventional dydrogesterone. The result suggested prolonged release behavior of SR dydrogesterone. The t_max of conventional dydrogesterone of our study was similar to previous report [24].

It is expected that SR formulation allows at least 2-fold reduction in dosage frequency compared to conventional formulation [25]. Concordantly, SR dydrogesterone demonstrated 2.4-fold higher bioavailability (AUC_0-48) compared to conventional dydrogesterone. Notably, bioavailability was obtained after single-dose administration of 20 mg SR and 10 mg conventional dydrogesterone. The dose normalized AUC showed that the SR dydrogesterone was bioequivalent to conventional dydrogesterone, though former performed slightly better. The present findings were in corroboration with a recent phase III trial where extended release dydrogesterone (20 mg) showed efficacy analogous to immediate release dydrogesterone (10 mg) [19]. Further, our preclinical kinetic data was comparable, if not similar, to a recent clinical bioequivalence study by Sharma AD et al. [24].

Importantly, the lower fluctuation index and higher mean resident time for SR dydrogesterone ensures more consistent concentrations of dydrogesterone in the systemic circulation over prolonged period. Our scintigraphy and kinetic data provide clear evidence that SR dydrogesterone (20 mg) is a viable once-daily alternative to twice-daily conventional dydrogesterone. Further, SR dydrogesterone with simpler dosage regimen could improve adherence and patient compliance [16, 17].

We compared efficacy following treatment of rabbits with either SR dydrogesterone (SDF group) or conventional dydrogesterone (CDF group). The comparison was also made with control rabbits treated with normal saline. The treatment duration was decided considering dydrogesterone use in the first trimester of pregnancy [15, 26] and short gestation period (32 ± 1 day) of rabbits [27]. Further, it is difficult to determine pregnancy in rabbits therefore treatment started with the mating instead of pregnancy and the data showed that the mean dosing duration between the groups did not differ significantly. To evaluate the effect of dydrogesterone on endometrial receptivity, ultrasound assessment, biochemical analysis and histopathology was conducted [28–31]. All the evaluations were done post-treatment at the end of first trimester. The endometrial thickness, layering, echo, volume, and blood flow was assessed by ultrasonography and ER scores were obtained for each animal. Significantly higher ER score and PIBF, integrin αVβ3 and VEGF levels in both the treatment groups compared to control, provide substantial evidence for the role of dydrogesterone in improvement of endometrial receptivity [15]. Higher ER scores generally indicate a more receptive endometrium, suggesting a higher probability of implantation [27]. Similarly, PIBF prevent embryo from immune attack and integrin αVβ3, a cell adhesion molecule, helps in implantation [29]. Notably, VEGF over expression in first trimester, regulate the development of new blood vessels required for fetal growth [30]. The similar dydrogesterone effects on endometrium and hormone levels were reported previously [15]. Though the ER scores and biochemical pregnancy markers are significantly lower in control group but all are within the normal range. This is expected, since we have used normal pregnancy model and not the animal models of adverse pregnancy outcomes [32]. Importantly, our results demonstrated that SR dydrogesterone is equally effective as conventional dydrogesterone except VEGF levels, which were higher (p < 0.05) in SR dydrogesterone treated animals. Additionally, the levels of all the pregnancy markers were within range and confirms the safety of dydrogesterone treatment.

In conformity, cross-section image of endometrium of SDF animals also showed dense blood vessels. Further, no cellular or morphological abnormality was observed in endometrium of treated animals and the results were similar to control animals. The findings clearly suggested that dydrogesterone is beneficial for implantation and that once-daily SR dydrogesterone treatment is equally, if not more, effective to twice-daily conventional dydrogesterone in enhancing endometrial receptivity during first trimester.

Firstly, the studies were conducted in rabbits having short gestation period (31–33 days), unlike human with long gestation period (9 months). Secondly, the treatment started with initiation of mating not with pregnancy since it is difficult to determine pregnancy in rabbit. Lastly, the comparison was made with control group, however baseline values of same animals were not considered.

Once-daily, SR dydrogesterone represent a significant advancement in the management of chronic reproductive diseases especially endometriosis, luteal phase defects, threatened miscarriage, and recurrent pregnancy loss. SR dydrogesterone with reduced dose frequency would have better treatment adherence, thus providing better pregnancy outcomes. The more consistent blood levels achieved via SR dydrogesterone could lead to better symptom control and better clinical outcomes. The incorporation of SR technology into dydrogesterone therapy thus enhances its clinical utility, providing a convenient and effective solution for patients suffering from chronic reproductive diseases.

The authors have no relevant financial or non-financial conflict of interest to disclose.

No funding was received for this study.