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European Journal of Pharmaceutics and Biopharmaceutics 130 (2018) 272–280
Contents lists available at ScienceDirect
European Journal of Pharmaceutics and Biopharmaceutics
journal homepage: www.elsevier.com/locate/ejpb
Research paper
Influence of the estrus cycle of the mouse on the disposition of SHetA2 after
vaginal administration
T
Sanjida Mahjabeena, Manolya Kukut Hatipoglua, Doris M. Benbrooka,b, Stanley D. Kosankec,
⁎
David Garcia-Contrerasd, Lucila Garcia-Contrerasa,
a
Department of Pharmaceutical Sciences, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA
Department of Obstetrics and Gynecology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA
c
College of Dentistry, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA
d
Medicina Veterinaria, Facultad de Estudios Superiores Cuautitlán, Mexico
b
A R T I C LE I N FO
A B S T R A C T
Keywords:
Cervical dysplasia
SHetA2
Vaginal drug delivery
Tissue absorption
Estrous cycle
Preclinical PK/PD
SHetA2 is a 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 stages
of their estrous cycle, which is known to induce anatomical changes in their gynecological tissues. We investigated the effects 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 AUCcervix (335 μg mL h−1), higher Cmax (121.8 ± 38.7 µg/g) and longer t1/2−1
, 44.6 ± 29.5 µg/g and 3.6 h respectively).
cervix (30.3 h) compared to mice dosed during estrus (120 μg mL h
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.
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 discomfort and can lead to infertility [5,6].
Presently, there are no drug-based treatments in clinical practice for
this disease.
SHetA2 (Fig. 1) is a novel, non-toxic chemotherapeutic agent [7],
with strong chemopreventive activity in human cell-culture [8] and
⁎
murine 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
Corresponding author at: The University of Oklahoma Health Sciences Center 1110 N. Stonewall Ave., Oklahoma City, OK 73126-0901, USA.
E-mail address: lucila-garcia-contreras@ouhsc.edu (L. Garcia-Contreras).
https://doi.org/10.1016/j.ejpb.2018.07.004
Received 22 May 2018; Received in revised form 29 June 2018; Accepted 3 July 2018
Available online 04 July 2018
0939-6411/ © 2018 Elsevier B.V. All rights reserved.
European Journal of Pharmaceutics and Biopharmaceutics 130 (2018) 272–280
S. Mahjabeen et al.
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) > 7.8%) and softening time (less than 30 min) as
outlined by the USP [15].
Fig. 1. Chemical structure of SHetA2.
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).
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.
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.
2.2.3. 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) pro-estrus 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. 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
HS-15 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 ThermoFisher Scientific (Waltham, MA). Protease inhibitor cocktail tablets
were purchased from Sigma Aldrich (St Louis, MO).
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
273
European Journal of Pharmaceutics and Biopharmaceutics 130 (2018) 272–280
S. Mahjabeen et al.
MRTcervix = AUMCcervix/AUCcervix 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.
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 min 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 h 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 12,500g. 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 × 5 mm 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.
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.
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 (Fig. 2A), whereas
most cells in the smear of mice in proestrus stage were nucleated epithelial cells (Fig. 2B). In contrast, leukocytes and cornified epithelial
cells were abundantly present in the smear of mice in metestrus stage
(Fig. 2C), whereas the smear of mice in diestrus stage consisted mainly
of leukocytes (Fig. 2D).
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 10,000g, 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.
3.4. Histology of uterine horns
Fig. 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 (Fig. 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
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):
2.2.9. Data analysis
Pharmacokinetic parameters to characterize the vaginal disposition
of SHetA2 were determined as follows: The Cmax-cervix = maximum
concentration in cervix and Tmax-cervix = time to achieve maximum
concentration in cervix, were determined from the cervix concentration
versus time plot. The AUCcervix = area under the cervix concentration
versus time curve, t1/2-cervix = half-life in cervix, Vz/F = apparent volume
of
distribution/F,
Clcervix/F = Clearance/F,
and
Mouse ID
Black
Red
Blue
Green
274
Observation Day
1
2
3
4
5
6
7
8
9
D
M
P
M
D
M
D
P
P
P
E
E
P
P
E
P
M
M
M
P
P
E
D
P
P
E
D
E
E
M
D
E
M
M
P
D
European Journal of Pharmaceutics and Biopharmaceutics 130 (2018) 272–280
S. Mahjabeen et al.
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 (Tmaxcervix = 0.5 h) in both treatment groups, the maximum concentration
(Cmax) 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.
The pharmacokinetic parameters characterizing the disposition of
SHetA2 after vaginal administration are presented in Table 3. The area
under the curve (AUCcervix) 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/FCervix = 2.6 mL/h) compared to that of mice dosed during diestrus (CL/Fcervix = 0.9 mL/h).
Consequently, the elimination half-life (t1/2-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 (MRTcervix) was 8-fold shorter.
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
Diestrus
77% ± 6%
23% ± 6%
35% ± 15%
64% ± 13%
epithelium (Fig. 3B). In contrast, the lumen of the uterine horns of mice
in metestrus stage becomes smaller and the number of mitotic cells
decreases (Fig. 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 (Fig. 3D).
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 Fig. 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
Fig. 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).
275
European Journal of Pharmaceutics and Biopharmaceutics 130 (2018) 272–280
S. Mahjabeen et al.
Fig. 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.
Diestrus group
SHetA2 concentration
in cervix ( g/g)
1000
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).
Estrus group
100
10
Therapeutic
conc • —0
1
PK parameter
0.1
−1
AUCcervix (μg mL h
Cmax (μg/g)
t1/2-cervix (h)
Cl/Fcervix (mL/h)
Vz/Fcervix (mL)
MRTcervix (h)
0.01
0.001
0.0001
0.00001
0
10
20
30
40
)
Estrus group
Diestrus group
120
44.6 ± 29.5
3.6
2.6
13.5
5.2
335
121.8 ± 38.7
30.3
0.9
41.3
41.0
50
Time (h)
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
(Fig. 5A).
Notably, the differences in cyclin D1 levels between groups dosed
during estrus was more significant, 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 (Fig. 5B).
Fig. 4. SHetA2 cervix tissue concentration versus time profile obtained from
FVB female mice treated with 15 mg/kg vaginal suppositories as a function of
diestrus
the estrus stage (n = 5–7 mice, per time point in each treatment) (
estrus group).
group,
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 Fig. 5. Mice dosed during diestrus with SHetA2 suppositories exhibited significantly lower levels of cyclin D1 (approximately
50% lower) at Tmax = 0.5 h compared to untreated mice (Fig. 5A). The
276
European Journal of Pharmaceutics and Biopharmaceutics 130 (2018) 272–280
S. Mahjabeen et al.
Diestrus group
A)
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
(Fig. 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 Cmax-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 Fig. 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
(Fig. 6A) is gradually lost (Fig. 6B) 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, Fig. 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 (Fig. 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, Fig. 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 AUCcervix, longer t1/2-cervix and
MRTcervix time compared to those observed in mice dosed during estrus.
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
Untreated Control
Placebo
SHetA2 (15mg/kg)
suppository
ns
ns
Fold change in
cyclin D1 expression
1.5
**
**
**
ns
1.0
0.5
0.
24
5
ho
ho
ur
ur
0.0
Treatment time (h)
Estrus group
B)
Control
Placebo
1.5
SHetA2 suppository
Fold change in
cyclin D1 expression
**
**
**
**
1.0
0.5
ho
12
0.
5
ho
ur
ur
0.0
Treatment time (h)
Fig. 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).
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
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European Journal of Pharmaceutics and Biopharmaceutics 130 (2018) 272–280
S. Mahjabeen et al.
Fig. 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).
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 Fig. 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 D1 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
study exhibited a similar pattern [10]. Therefore, it is plausible that
SHetA2 may have precipitated at the site of absorption and then dissolved and re-absorbed 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
(Fig. 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 (Fig. 5B). The cyclin D1 reduction was also larger in mice dosed during estrus compared to that
observed during estrus (Fig. 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
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European Journal of Pharmaceutics and Biopharmaceutics 130 (2018) 272–280
S. Mahjabeen et al.
for future efficacy studies in the K14-HPV16 mouse model of cervical
dysplasia.
6. Acknowledgements
This work was funded by the MD Anderson Cancer Center Moon
Shot grant with the help of Dr. Michael Frumovitz, the Stephenson
Cancer Center (SCC) Gynecologic Cancers Program at the OUHSC and
R01 CA196200126-01A1 (DMB and LGC, Co-PIs). The authors are
grateful to SCC tissue pathology core for preparation of uterine horns
histology slides and H&E staining.
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