INFLUENCE OF THE ESTRUS CYCLE OF THE MOUSE ON THE DISPOSITION OF SHetA2 AFTER VAGINAL ADMINISTRATION

Abstract

SHetA2 is novel compound with the potential to treat cervical dysplasia, but has poor water solubility. A vaginal suppository formulation was able to achieve therapeutic concentrations in the cervix of mice, but these concentrations were variable. Histological analysis indicated that mice in the same group were in different stage of their estrous cycle, which is known to induce anatomical changes in their gynecological tissues. We investigated the effect of these changes on the pharmacokinetics and pharmacodynamics of SHetA2 when administered vaginally. Mice were synchronized to be either in estrous or diestrus stage for administration of the SHetA2 suppository. Pharmacokinetic parameters were calculated from the SHetA2 concentrations vs. time data. The reduction in the expression of cyclin D1 protein in the cervix was used as pharmacodynamic endpoint. Mice dosed during diestrus had a larger AUC_cervix (335 μg.mL.h⁻¹), higher C_max (121.8 ± 38.7 μg/g) and longer t_1/2-cervix (30.3 h) compared to mice dosed during estrus (120 μg.mL.h⁻¹, 44.6 ± 29.5 μg/g and 3.6 h respectively). Therapeutic concentrations of SHetA2 were maintained for 48 h in the cervix of mice dosed during diestrus and for only 12 h in the estrus group. The treatment reduced the expression of cyclin D1 protein in the cervix of mice in the estrus to a larger extent. These results indicate that the estrous cycle of mice influences significantly the disposition of SHetA2 after vaginal administration and may also influence its efficacy.

Graphical abstract

1. INTRODUCTION

Cervical dysplasia is a precancerous condition of the uterine cervix, mainly initiated by high risk papilloma virus (HPV) infection [1]. The prevalence of HPV infection in developed countries is reported to range between 40 % and 80 % in young adult females [2, 3]. This infection can be asymptomatic and resolved by the immune system of the individual, but if the infection persists it can progress towards invasive cervical cancer, which is second most common form of gynecologic cancer worldwide [4]. Because of this possibility, physicians may over treat the patient. Current treatments for cervical dysplasia involve invasive procedures such as ablation therapy and cold knife-conization therapy, which are expensive, cause utter discomfort and can lead to infertility [5, 6]. Presently, there are no drug-based treatments in clinical practice for this disease.

SHetA2 (figure 1) is a novel, non-toxic chemotherapeutic agent [7], with strong chemopreventive activity in human cell-culture [8] and murine HPV-induced tumors [9]. However, it has low oral bioavailability (~10 %) due to its poor water solubility [7]. To overcome these limitations, we employed Quality by Design (QbD) approaches to develop a vaginal suppository formulation for direct delivery to the cervix, the site of drug action [10]. The formulation was optimized using two consecutive designs of experiments (DoE). The composition of a hydrophilic suppository base (PEG 400: PEG3350) and the percentage of solubilizing agent (Kolliphor) were optimized in DoE1 (16 experiments). Subsequently, the optimized hydrophilic base (35 % PEG 400, 60 % PEG 3350 and 5 % Kolliphor) was entered in a second DoE (DoE2), which compared the effect of the type of base (hydrophilic versus lipophilic base (cocoa butter)) and the % drug content on the integrity and disintegration/melting time of the suppository. DoE2 determined that a cocoa butter base and 40 % drug content produced suppositories that remained solid at room temperature and melted in 6 min. We performed proof-of concept pharmacokinetic and pharmacodynamics studies in mice to evaluate the potential of the SHetA2 vaginal suppositories to treat cervical dysplasia [10]. SHetA2 suppositories achieved cervix concentrations significantly higher than its predicted therapeutic concentration (4 μM) and induced a 9-fold decrease in the levels of cyclin D1 protein, a marker of efficacy associated with the prognosis in cervical cancer patients [11]. However, a large variability was observed in the SHetA2 concentrations among mice receiving the same dose, at the same time point, even though the quality control indicated that drug variation in the batch of suppositories was within United States Pharmacopoeia (USP) limits [10]. Thus, we investigated the influence of the anatomical and physiological features of the reproductive system of the mouse on SHetA2 absorption.

Figure 1:

Chemical structure of SHetA2

Unlike humans that have a menstrual cycle, the reproductive cycle of the mouse, known as estrous cycle, has four different stages: proestrus (P), estrus (E), metestrus (M) and diestrus (D) [12]. Due to differential hormonal regulation, the composition, thickness and structure of the stratified squamous and mucosal epithelium in the gynecologic tissues of mice are variable across the estrous cycle [13]. Histological examination of the uterine horns of the mice treated with the same SHetA2 dose in preliminary studies revealed that mice within the same time point group were in different stages of their estrus cycle.

Currently, there are no published reports describing the effects of anatomical and physiological changes due to estrus cycle on drug disposition after vaginal administration of compounds to mice. A single report from Hsu et al. [14] using an excised vaginal membrane in a diffusion chamber indicated that the permeability coefficients for vidarabine, an antiviral drug with activity against herpes, were 10-100 fold higher during the diestrus stage compared to those in the estrus stage. Therefore, the objective of the present study was to evaluate the influence of the anatomical and physiological changes due to the estrus cycle of the mouse on SHetA2 disposition after vaginal administration, and SHetA2 pharmacodynamic endpoint, the reduction in the levels of cyclin D1 protein.

2. MATERIALS AND METHODS

2.1. Materials

SHetA2 was synthesized by Cayman Chemical company, Inc. under a contract from the Rapid Access to Preventive Intervention Development (RAPID) National Cancer Institute (NCI) program. Cocoa butter was purchased from Nature’s Oils (Streetsboro, OH). Kolliphor was obtained from BASF (Germany). Sterile saline and isoflurane were obtained from Henry Schein Animal Health Inc. Acetonitrile (HPLC grade ≥ 99.5 %), methanol (HPLC grade ≥ 99.5 %), phosphoric acid, hydrochloric acid, crystal violet stain and sodium acetate trihydrate were purchased from Sigma Aldrich (St Louis, MO). Captiva^® filtration equipment was purchased from Agilent Technologies Inc. for extraction of drug from tissues. Mouse cyclin D1 enzyme linked immunosorbent assay kit (ELISA) was purchased from Cedarlane lab (NC). T-PER (tissue protein extraction reagent) was purchased from Thermo-Fisher Scientific (Waltham, MA). Protease inhibitor cocktail tablets were purchased from Sigma Aldrich (St Louis, MO).

2.2. Methods

2.2.1. Suppository manufacturing and quality control

SHetA2 suppositories containing 15 mg/kg body weight dose were manufactured by the fusion-molding method [10]. Suppositories were evaluated for content uniformity (85 % to 115 % of intended content), weight variation (no more than two units having a relative standard deviation (RDS) greater than 7.8 %) and softening time (less than 30 min) as outlined by the USP [15].

2.2.2. Animals

Friend Leukemia Virus B (FVB) female mice of 7 weeks of age (National Cancer Institute Charles River Frederick Research Facility) were used in this study because they are the wild type species for the K14-HPV16 mouse model of cervical neoplasia [16] that will be used in efficacy studies. Animals were housed in a facility in a constant temperature room at 22 ± 1 °C with a 12 h light/12 h dark cycle and provided access to food and water ad libitum. All animal experiments were approved by University of Oklahoma Health Sciences Center Institutional Animal Care and Use Committee (IACUC).

2.2.3. Monitoring estrus cycle by the visual method

The vaginal openings of mice were monitored every 24 h to determine their stage in the estrous cycle as described by Byers et al [17]. According to this method, during proestrus the vaginal opening remains swollen, moist and pink; during estrus, the opening becomes less moist and less swollen; during metestrus, a distinctive white cellular debris is observed, but the tissue is not swollen; whereas during diestrus, the vaginal opening is very narrow and not swollen.

2.2.4. Synchronization of estrous cycle

The estrous cycle in mice was synchronized by the Whitten effect, where female mice are exposed to male pheromones from their urine, which induce them to enter the estrus stage on the third day of exposure [18, 19]. Since urine from male mice was not readily available, we used the soiled bedding from male mice cage for a modified Whitten effect [18]. To verify the stage of the estrus cycle in these mice, vaginal lavage was performed every 24 h on the third, fourth and fifth day of exposure as described below.

2.2.5. Monitoring estrus cycle by observation of vaginal cytology

The vaginal lavage was performed in each mouse under light sedation with isoflurane as described by McLean et al [20]. A sterile pipette tip was filled with approximately 10 μL of sterile saline and inserted gently into the vaginal cavity of the mouse, followed by gentle aspiration, and the procedure repeated three to five times. The collected fluid was then smeared onto a microscope glass slide and air-dried. The slides were stained with crystal violet after drying.

Slides were examined with a microscope to determine the type of cells that were present in the smear. The stages of the estrous cycle were determined according to the percentage of anucleated cornified cells, nucleated epithelial cells and leukocytes presented in the smear as follows [21]. A mouse was determined to be in the: (1) proestrus stage when nucleated epithelial cells were predominant; (2) estrus stage when mainly anucleated cornified cells were present; (3) metestrus stage when three types of cells, leukocytes, cornified, and nucleated epithelial cells, were present; and (4) diestrus stage when the majority of cells present were leukocytes [21].

2.2.6. Histology of the uterine horns

A piece of a uterine horn from each mouse was collected, fixed in 10 % neutralized buffered formalin and embedded in a paraffin block. Afterwards, sections were cut perpendicularly to the transverse axis from the paraffin blocks, so that the lumen and stromal areas were included in each section. Each tissue section was fixed onto a microscope slide and stained with hematoxylin and eosin (H&E) for histological analysis.

2.2.7. Pharmacokinetic study

SHetA2 suppositories (15 mg/kg) were administered vaginally to female mice when they were either in diestrus or estrus stage, in order to determine the influence of the resulting anatomical and physiological changes on the extent of drug absorption. We selected these two stages based on a preliminary study, in which mice in diestrus stage exhibited the highest drug concentrations in the cervix and those in estrus stage exhibited the lowest. Also, it is at these two stages that the hormonal levels are the most different, with diestrus and estrus stages corresponding to the secretory and proliferative phases of the human uterine cycle, respectively [22].

Mice were lightly sedated with isoflurane to facilitate suppository insertion and maintained under sedation for 3 minutes in horizontal position to improve the retention of the suppository. Mice were then transferred to a clean cage lined with paper and observed for 10 additional minutes to account for any possible leakage of the yellow colored suppository. Mice dosed during diestrus were euthanized at 0.5, 1, 4, 8, 12, 24, 36 and 48 h after dosing (n = 5 mice, per time point), whereas mice dosed during estrus stage were euthanized at 0.5, 1, 4, 8 and 12 hours after dosing (n = 5 mice, per time point). Blood was collected by cardiac puncture into heparinized tubes and plasma was separated after centrifugation for 10 min at 12,500 g. The cervix tissues were collected and thoroughly washed by saline to remove unabsorbed drug. Plasma and gynecologic tissues were kept frozen at −80 °C until analysis, where each cervix was divided into two pieces, to be used in pharmacokinetic and pharmacodynamic determinations.

In addition, mice (n =3 mice) were dosed with placebo suppositories (cocoa butter and Kolliphor only) and euthanized at the times of highest and lowest drug concentrations in cervix to be used as controls for the pharmacodynamic endpoint.Cervix tissues were collected, washed with saline and kept frozen at −80 °C.

SHetA2 was extracted from one-half of each cervix tissue (n= 5 per time point) using a Captiva^® filtration system and drug levels were analyzed by HPLC using a method validated in our laboratory [10]. A Waters Alliance HPLC System equipped with Waters Xbrigde C₁₈ 3.5 μm, 2.1 × 150 mm column and Waters Xbridge BEH C₁₈, 3.5 μm, 2.1 × 5mm guard column were used to determine drug concentrations. The mobile phase consisted of acetonitrile and water (80:20 v/v) at flow rate of 0.3 mL/min.

2.2.8. Pharmacodynamic study

The expression of cyclin D1 protein in the cervix of mice treated with SHetA2 suppositories, placebo suppositories and untreated mice was determined by Enzyme Linked Immunosorbant Assay (ELISA). The one-half of the cervix of each mouse (n= 5, per each time point) was homogenized in T-PER reagent (10 μL per each mg of tissue) containing protease inhibitor cocktail. Homogenization was performed in an ice bath using an OMNI-GLH general laboratory homogenizer. Homogenates were centrifuged at 4 °C for 5 min at 10,000 x g, the supernatants were collected and the assay was performed according to manufacturer’s instruction. Each standard blank and test specimen was evaluated in duplicate. The replicates with > 15 % coefficients of variance was eliminated from the analysis. The average of the test samples was compared to the standard curve to derive the cyclin D1 concentration.

2.2.9. Data analysis

Pharmacokinetic parameters to characterize the vaginal disposition of SHetA2 were determined as follows: The C_max-cervix = maximum concentration in cervix and T_max-cervix = time to achieve maximum concentration in cervix, were determined from the cervix concentration versus time plot. The AUC_cervix = area under the cervix concentration versus time curve, t_1/2-cervix = half-life in cervix, Vz/F = apparent volume of distribution/F, Cl_cervix/F = Clearance/F, and MRT_cervix = AUMC_cervix/AUC_cervix were determined by non-compartmental analysis using Phoenix WinNonlin® software. The average SHetA2 cervix concentration of the 5 mice in each time point was used in the calculation.

Statistical analysis of cyclin D1 expression levels in the cervix of mice was performed by Graphpad Prism software using two way ANOVA and Tukey’s multiple comparison test. A p value of less than 0.01 was considered to be statistically significant.

3. RESULTS

3.1. Quality control for suppositories

All manufactured SHetA2 suppositories met USP specifications [23]: content uniformity of 101.3 ± 9.9 % and average weight of 54.4 ± 3.8 mg (relative standard deviation 7.1 %). The average softening time for the suppositories was 5.6 ± 0.1 min.

3.2. Estrus cycle monitoring by visual method

The stages of the estrus cycle for mice in the same cage, as monitored by the physical appearance of their vaginal opening, are presented in table 1. Despite being housed in same cage, the mice exhibited different stages of the estrus cycle. For example, on day 1, two mice were in metestrus, one was in diestrus and one in proestrus. On day 2, one of the mice that was in metestrus remained in that stage whereas the other had already progressed past diestrus into proestrus. Due to the unpredictable variability in the cycle of the mice in the study, it was determined that synchronization of the estrous cycle was needed to reduce variability in the study.

Table 1:

Visual monitoring of estrous cycle in female FVB mice of the same cage, without synchronization (P=proestrus, E=estrus, M=metestrus, D=diestrus):

Observation Day
Mouse ID	1	2	3	4	5	6	7	8	9
Black	D	D	P	P	M	P	P	E	M
Red	M	M	P	P	M	E	E	M	M
Blue	P	D	E	E	M	D	D	D	P
Green	M	P	E	P	P	P	E	E	D

3.3. Monitoring estrus cycle by observation of vaginal cytology

The Whitten effect method was effective to synchronize the estrus cycle in the mice in the study as shown in Table 2. On the third day of exposure to male urine, 77 ± 6 % of the mice were in estrus stage, and on the fourth day 64 ± 13 % of mice were in diestrus stage. Vaginal cytology was also a more unequivocal way to determine the stage of estrus cycle in these mice as the different types of cells are readily recognizable in the smears of vaginal lavages. The smear of mice in estrus stage consisted mostly of cornified epithelial cells (figure 2A), whereas most cells in the smear of mice in proestrus stage were nucleated epithelial cells (figure 2B). In contrast, leukocytes and cornified epithelial cells were abundantly present in the smear of mice in metestrus stage (figure 2C), whereas the smear of mice in diestrus stage consisted mainly of leukocytes (figure 2D).

Table 2:

Monitoring of estrus cycle by microscopic examination of vaginal smears from female FVB mice of the same cage, after synchronization by the Whitten effect.

% of mice in each stage	Third day of exposure	Fourth day of exposure
Estrus/Proestrus	77 % ± 6 %	35 % ± 15 %
Diestrus	23 % ± 6 %	64 % ± 13 %

Figure 2:

Cell composition of the vaginal lavages from mice synchronized by the modified Whitten effect showing the four different estrus stages: A) Proestrus (mostly nucleated epithelial cells, marked with red arrow), B) Estrus (mostly cornified epithelial cells, marked with black arrow), C) Metestrus (mostly nucleated epithelial cells, few leukocyte and cornified epithelial cell); D) Diestrus (mostly leukocytes, marked with blue arrow)

3.4. Histology of uterine horns

Figure 3 shows sections of the uterine horns of mice that confirmed the different stages of the estrous cycle: the lumen of the uterine horn of mice in proestrus stage was large with a few mitotic cells in the epithelium (figure 3A), whereas the lumen of the uterine horns of mice in estrus stage is the largest in the cycle and has many mitotic cells in the epithelium (figure 3B). In contrast, the lumen of the uterine horns of mice in metestrus stage becomes smaller and the number of mitotic cells decreases (figure 3C), whereas the lumen of the uterine horns of mice in diestrus stage is the smallest in the cycle, there are no mitotic cells present and the epithelial layer is thin (figure 3D).

Figure 3:

H&E stained slides from transversal sections of the uterine horns of mice receiving a 15 mg/kg SHetA2 vaginal suppository during: A) Proestrus, B) Estrus, C) Metestrus, and D) Diestrus

3.5. Pharmacokinetic study

SHetA2 suppositories melted inside the vaginal cavity of mice within the 3 min that they were kept sedated after insertion and the formulation absorbed in the majority of cases as evidenced by very little to no leakage of the formulation noted within the 10 min that the mice were observed after recovering from anesthesia.

The SHetA2 cervix tissue concentration versus time profile obtained from mice receiving the vaginal suppositories as a function of the estrus stage is shown in figure 4. Administration of the optimized SHetA2 vaginal suppositories achieved concentrations above the therapeutic level (4.0 μM or 1.6 μg/mL) [24] in the cervix of mice in both estrus and diestrus stages, but this concentration was maintained to a different extent in these groups. While therapeutic concentrations of SHetA2 in the cervix of mice treated with the suppository during diestrus were maintained for 48 h, the SHetA2 cervix concentrations in mice treated during estrus fell below therapeutic level after 12 h. Even though the time of maximum SHetA2 concentration was the same (T_max-cervix = 0.5 h) in both treatment groups, the maximum concentration (C_max) was more than threefold higher when mice were dosed during diestrus (121.8 ± 38.7 μg/ml) compared to when mice were dosed during estrus (44.6 ± 29.5 μg/ml). Interestingly, the cervix concentration versus time profile for both groups exhibited an extra peak that occurred at 4 h for the estrus group and at 12 h for the diestrus group.

Figure 4:

SHetA2 cervix tissue concentration versus time profile obtained from FVB female mice treated with 15 mg/kg vaginal suppositories as a function of the estrus stage (n=5-7 mice, per time point in each treatment) (diestrus group, estrus group).

The pharmacokinetic parameters characterizing the disposition of SHetA2 after vaginal administration are presented in table 3. The area under the curve (AUC_cervix) was almost three-fold higher when mice were dosed during diestrus (335 μg.mL.h⁻¹) than when dosed treated during estrus (120 μg.mL.h⁻¹). These AUC correlated with a faster drug clearance in mice treated during estrus (CL/F_Cervix = 2.6 mL/h) compared to that of mice dosed during diestrus (CL/F_cervix = 0.9 mL/h). Consequently, the elimination half-life (t_1/2-cervix) of SHetA2 from the cervix of mice dosed during estrus (3.6 h) was almost 10-fold shorter than in mice dosed during diestrus (30.3 h) and the mean residence time (MRT_cervix) was 8-fold shorter.

Table 3:

Pharmacokinetic parameters characterizing the disposition of SHetA2 in the cervix tissue after vaginal administration to FVB mice (n = 5-7 mice per time point in each group)

PK Parameter	Estrus group	Diestrus group
AUCcervix(μg.mL.h−1)	120	335
Cmax (μg/g)	44.6 ± 29.5	121.8 ± 38.7
t1/2-cervix(h)	3.6	30.3
Cl/Fcervix (mL/h)	2.6	0.9
Vz/Fcervix(mL)	13.5	41.3
MRTcervix (h)	5.2	41.0

3.6. Pharmacodynamic study

The levels of cyclin D1 protein employed as a pharmacodynamic endpoint to evaluate the effect of SHetA2 levels in cervix tissue are depicted in figure 5. Mice dosed during diestrus with SHetA2 suppositories exhibited significantly lower levels of cyclin D1 (approximately 50 % lower) at T_max = 0.5 h compared to untreated mice (figure 5A). The cyclin D1 expression in mice receiving placebo suppositories was also 15 % lower compared to untreated controls, but this difference was not statistically significant. However, while the cyclin D1 levels in mice receiving the placebo suppositories returned to the same level as untreated controls after 24 h of treatment, the levels of cyclin D1 remained significantly reduced in mice treated with SHetA2 suppositories (figure 5A).

Figure 5:

Cyclin D1 expression level in the cervix of mice treated with 15 mg/kg SHetA2 vaginal suppository: A) diestrus group (n= 3-5 mice, per time point in each treatment), B)estrus group (n= 3-5 mice, per time point in each treatment). Cyclin D1 expression in placebo and treatment group was normalized with the expression level in untreated control (** indicates p value less than 0.005, ns= not significant p values were derived using two-way ANOVA Tukey’s post test, n=3)

Notably, the differences in cyclin D1 levels between groups dosed during estrus was larger, as the cyclin D1 reduction in mice treated with SHetA2 suppositories was statistically lower than that in untreated mice and those receiving placebo suppositories at both 0.5 h and 24 h (figure 5B).

4. DISCUSSION

Cervical cancer affects about a half million women worldwide, but the number of women affected by cervical dysplasia is almost 30-fold larger [25]. Thus, treating cervical dysplasia would have a larger impact on the patient population and potentially reduce the number of patients affected by cervical cancer. To date, only a handful of animal models for cervical dysplasia and cervical cancer have been established, and the majority of these models are based in mice [26]. In particular, the K14-HPV16 transgenic mouse model develops multiple hyper-proliferative and dysplastic lesions that are similar to those observed in human [26, 27]. FVB female mice were employed in the present study, as it is the wild type strain to generate the K14-HPV16 mouse model [27, 28].

Proof-of-concept studies support the potential of vaginal suppositories to deliver SHetA2 as a promising, non-invasive therapy for cervical dysplasia, based on their capability to achieve therapeutic concentrations at the site of action and the reduction of the cyclin D1 protein expression [10]. However, it was necessary to address the cause of the large variations in drug concentrations observed in the cervix from mice within the same experimental group because they could possibly compromise the effectiveness of the proposed treatment.

The present study revealed that the disposition of SHetA2 after vaginal administration of the same dose was significantly influenced by the stage of the estrus cycle in mice. The difference between the SHetA2 cervix concentrations observed in mice dosed during diestrus and estrus (figure 4) was attributed to differences in the cell composition of the vaginal epithelium during these stages, which in turn influenced drug absorption. During estrus, the epithelium in the vaginal cavity of mice is thick and the superficial layer is formed by large, anuclear cornified epithelial cells [29, 30] that would limit drug absorption. In contrast, during diestrus, the epithelium is thin and is formed by polygonal, plump epithelial cells due to early mucification, which are more likely to absorb drug as indicated by a higher C_max-cervix in the diestrus group.

Another significant difference was the mean residence time of SHetA2 in the cervix of mice, which was 8-fold shorter in mice dosed during estrus (5.2 h), compared to that of mice dosed during diestrus (41.0 h). As a result, SHetA2 concentration in the cervix of mice dosed during estrus fell below therapeutic levels 12 h after the suppository was administrated, whereas, SHetA2 concentration in the cervix of mice dosed during diestrus was maintained at a therapeutic level until 48 h. The possible explanations for these differences are offered in figure 6. The duration of the estrus stage in mice is reported to be 12 h [30, 31], at the end of estrus, the cornified superficial layer of the vaginal epithelium is gradually lost (figure 6A) and becomes completely delaminated at the beginning of metestrus [30]. Consequently, when SHetA2 is administered during the estrus stages, it is likely that most of the drug is lost together with the anucleated cornified cells at the end of the stage, which correlates with cervix concentrations falling below therapeutic levels (12 h, figure 6C). In contrast, the duration of the diestrus stage is 65 h [30, 31], with the epithelial plump cells remaining intact for this period of time (figure 6B) [30]. Thus, when SHetA2 is administered during the diestrus stages, it is likely that the drug remains in the epithelial cell layers with a larger probability to be absorbed, which correlates with cervix concentrations remaining above therapeutic levels for a longer period of time (> 48 h, figure 6C). These differences observed in the disposition of SHetA2 were also reflected in their respective pharmacokinetic parameters (table 3), with mice dosed during diestrus group exhibiting larger AUC_cervix, longer t_1/2-cervix and MRT_cervix time compared to those observed in mice dosed during estrus.

Figure 6:

Schematic diagram of the vaginal epithelium during, (A) estrus and (B) diestrus: (A) The superficial layer of the vaginal epithelium is cornified and this layer is gradually lost at the end of this stage (12 h) [30, 31]. The cornified layer can affect drug absorption and when is lost at the end of the stage, drug may be lost with it. The superficial layer consists of nucleated epithelial cell and the epithelium is intact for the duration of the stage (65 h), therefore a prolonged residence time is expected. (C) Time course of SHetA2 cervix concentration according to length of stages: diestrus (in black line) and estrus (in red line).

An unexpected secondary peak was observed in the SHetA2 cervix tissue concentration versus time profile for both treatment groups. It is unlikely that these peaks are due to enterohepatic recirculation as the drug was under the levels of detection in plasma at all time points and the concentrations are from the cervix tissue. A more plausible explanation is that SHetA2 was first dissolved and then absorbed in tissues, but as the concentration increased inside the tissue, the drug may have precipitated and then dissolved and reabsorbed again. The appearance of double peaks in the plasma concentration versus time profiles following extravascular (mainly oral) administration of drugs has also been reported for other drugs [32–36]. Among these drugs, danazol is poorly soluble in water (0.017 mg/mL) like SHetA2 (0.4 μg/mL) and has a log P value (3.48) similar to that of SHetA2 (3.84) [4, 10]. It has been hypothesized that a portion of the danazol administered orally is rapidly solubilized in the stomach but precipitates in the intestine and might again solubilize aided by bile salts [32]. Jackson et al [37] performed a dissolution study to investigate the precipitation and solubilization kinetics of danazol formulations using nanoparticle tracking analysis and microscopy. They reported that as the drug concentration increased and the solution became saturated, nucleation increased and droplet like precipitates were formed. The dissolution profile of SHetA2 in simulated vaginal fluid reported in our previous study exhibited a similar pattern [10]. Therefore, it is plausible that SHetA2 may have precipitated at the site of absorption and then dissolved and reabsorbed again, but more studies are needed to confirm this assumption.

The effect of the estrus cycle was also observed on the pharmacodynamic endpoint marker cyclin D1. Although the levels of cyclin D1 in the cervix of mice treated with SHetA2 during diestrus were significantly reduced at 0.5 h compared to those in untreated controls (figure 5A), this reduction was not statistically different to the reduction of cyclin D1 levels in the cervix of mice treated with placebo suppositories. In contrast, the levels of cyclin D1 in the cervix of mice treated with SHetA2 during estrus were significantly reduced compared to both, placebo treated and untreated mice (figure 5B). The extent of cyclin D1 reduction was also larger in mice dosed during estrus compared to that observed during estrus (figures 5A and 5B). The differences in the pharmacodynamic effect of SHetA2 during estrus and diestrus stages may be explained by the difference in the mitotic activity of the cells of the epithelium during these stages. Cyclin D1 is a cell cycle regulatory protein, which plays essential role in G1 to S phase entry in cell cycle [38]. In G1 phase, cyclin D1 promotes cell cycle progression to S phase upon binding to cyclin dependent kinase 4 and 6. SHetA2 induces G1 cell cycle arrest by degradation of cyclin D1 [24]. In figure 3, a large number of mitotic bodies are observed around the lumen of the uterine horns from mice in estrus stage indicating active cell division and thus more opportunity for SHetA2 activity. In contrast, the mitotic activity around the lumen of the uterine horns from mice in diestrus stage is minimal and thus the reduction in cyclin D levels is minimal. These observations are in agreement with other publications which indicate that the highest mitotic activity in the inner layer of the vaginal epithelium and uterine horns occurs during estrus, decreased during metestrus and its minimal during diestrus [12, 39].

Given the differences in the menstrual cycle of humans and the estrus cycle of mice, it would be interesting to speculate about the implication of the findings of the present study. In contrast to the four stages of the estrus cycle of mice, a regular menstrual cycle has three phases, according to the events in both ovaries and uterus [40]. The thickness and viscosity of the human cervical mucus is altered during these different phases [41], but unlike the mouse, the thickness of the human vaginal epithelium does not drastically change across the cycle. Although a few studies indicate minor changes [39], the clinical relevance of these changes has not been demonstrated. In order to apply the findings of the present study, the menstrual cycle of humans as influenced by its main hormones has been schematically depicted (figure 7A) next to the estrus cycle of the mouse (figure 7B). According to the hormonal levels, the diestrus stage in the mouse corresponds to the human secretory phase, whereas the proestrus and estrus stages correspond to the human proliferative phase of the menstrual cycle [39, 41]. Even though the epithelium of the human vagina and uterus is not significantly affected by the stages, the changes in mucus volume secretion during the menstrual cycle may influence the extent of drug absorption [41]. Consequently, drug absorption may be affected during secretory phase when mucus production is high. Therefore, the ideal time for vaginal administration of drugs to humans would be at the beginning of the secretory phase or right after menses, as shown by the blue arrows in figure 7A and 7B.

Figure 7:

Comparison between human uterine cycle and mouse estrus cycle according to estradiol and progesterone levels: A) hormonal level in human uterine cycle, B) hormonal level in mouse estrous cycle. Blue arrow indicates the ideal time of vaginal drug administration to maximize the drug concentration in gynecological tissues.

5. CONCLUSION

The extent of drug absorption in the cervix of FVB mice appears to be influenced by the stage of the estrus cycle. SHetA2 absorption and residence time in the cervix is maximized during diestrus stage, whereas the pharmacodynamics effects appears to be favored during estrus stage. These effects need to be balanced and taken into account for the design of the dosing regimen with SHetA2 vaginal suppositories for future efficacy studies in the K14-HPV16 mouse model of cervical dysplasia.