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Introduction
Droloxifene, a derivative of the triphenylethylene drug
tamoxifen, is a novel selective estrogen receptor modulator
(SERM)[1]. Its higher affinity to the estrogen receptor, higher
anti-estrogenic to estrogenic ratio, more effective inhibition
of cell growth and division in estrogen receptor-positive cell
lines, and lower toxicity give it theoretical advantages over
tamoxifen in the treatment of human breast
cancer[2]. Droloxifene may also be a potentially useful agent for the
treatment of postmenopausal osteoporosis because it can
prevent estrogen deficiency-induced bone loss without
causing uterine hypertrophy[3]. Droloxifene may
have an effect on bone and breast tissue because it induces
apoptosis[4]. The corpus luteum is an ovarian tissue that synthesizes and
secretes progesterone, which plays a key role in the
establishment and maintenance of pregnancy in mammals.
Abnormal regression of the corpus luteum will disturb or even
terminate both the implantation process and early pregnancy.
Apoptosis is involved in the regression of the corpus
luteum in many species[5]. Therefore, better understanding
the compounds that induce the apoptosis of luteal cells may
contribute to the development of new anti-implantation
agents. Our laboratory was the first to report that droloxifene
induceed the apoptosis of rat luteal cells in
vitro and the pre-implantation luteal cells in pregnant
rats[6-8]. Moreover, droloxifene facilitates the apoptosis of luteal cells and
shortens the period of pseudopregnancy in pseudopregnant
rats[9]. These results suggest that droloxifene induces the
regression of the corpus luteum and has potential
anti-implantation effects. Exact equilibrium of estrogen and progesterone
is essential for implantation, and any disturbance in the
effects of these hormones can cause
infertility[10]. As a novel selective estrogen receptor modulator with greater
anti-estrogenic effects, droloxifene seems to interfere with the
effect of estrogen and cause anti-implantation effects.
However, the anti-implantation effect of droloxifene has not
been evaluated and reported on. Therefore, in the present
study, the anti-implantation effect of droloxifene was
evaluated and the relationship between the anti-estrogenic
activity of droloxifene and its anti-implantation effect was
analyzed in rats.
Materials and methods
Drugs and reagents The droloxifene was synthesized
by Prof Peng XIA (Department of Organic Chemistry,
College of Pharmacy, Fudan University, Shanghai) and was
suspended in 1% sodium carboxymethylcellulose (CMC).
Estradiol (E2) was purchased from the Shanghai 9th Pharma
ceutical Factory (Shanghai, China) and was suspended in
corn oil. Serum estrogen and progesterone
radioimmunoassay (RIA) kits were obtained from DEPU Ltd (Tianjin, China).
Animals and treatment Sprague-Dawley rats (body
weight: female, 220-250 g an male 300-350 g, SIPPR/BK
LtdShanghai) were kept in a temperature-controlled
(24-26 °C) and light-regulated (12 h light, 12 h dark) room, and were
given ad libitum access to standard chow and water. The
female animals were cohabited with male animals at a ratio of
2:1. The day that sperm were found in the vaginal smear was
designated as d 1 of pregnancy.
Evaluation of anti-implantation efficacy
Confirmed pregnant female rats were randomly assigned into different
groups and were treated with droloxifene (ig), estradiol
(E2, sc), or 1% CMC (as a control, ig). The doses and treatment
times of the different experiments are shown in the results
section. At autopsy on d 9, the number of animals with
implantation sites in each group was recorded. The Bliss
method was used to calculate the ED95,
ED90, and ED50 of the anti-implantation effect of droloxifene.
Assay of the serum levels of estrogen and progesterone
The pregnant rats were treated orally with 2.5 mg/kg
droloxifene or 1% CMC at 22:00 PM on d 4. Blood samples of
0.5 mL were obtained from the tail veins of pregnant rats at
10:00 AM on d 1, d 2, d 3, d 4, d 5 and d 6, and at 22:00 PM on
d 4. The serum levels of estrogen and progesterone were
measured by RIA according to the manufacturer¡¯s inst-
ructions.
Statistical analysis Differences in pregnancy rates
between the groups were tested by using the
c2 test. Serum levels of estrogen and progesterone are expressed as
mean±SD and Student¡¯s t-test was used to calculate signi-
ficance.
Results
Optimal administration time for the anti-implantation
effect of droloxifene Pregnant rats were treated orally with
droloxifene with doses ranging from 1.25 to 20.0 mg/kg at
10:00 AM on d 2. Within the treatment time, droloxifene had
an anti-implantation effect in rats
(ED95=17.62 mg/kg and ED50=5.34 mg/kg; Table 1). To determine the optimal
administration time for the anti-implantation effect of droloxifene,
pregnant rats were treated orally with droloxifene at either a
high dose (14 mg/kg) or a low dose (2.5 mg/kg) at 10:00 AM
on d 2, d 3, d 4, or d 5, or at 22:00 PM on d 4. Although the
differences in the anti-implantation rates in different groups
were not significant for the 14 mg/kg groups, the
anti-implantation rates of rats treated with droloxifene at 22:00 PM
on d 4 were the highest in the two dose groups (Table 2).
These results suggest that droloxifene has anti-implantation
effects in rats, and that the optimal administration time is at
22:00 PM on d 4.
ED95, ED90, and
ED50 for the anti-implantation effect of
droloxifene Pregnant rats were treated orally
with drol- oxifene at various doses (10, 5.0, 2.5, 1.25, 0.62, 0.31, or 0.15
mg/kg) at 22:00 PM on d 4. The anti-implantation rates of the
droloxifene groups (0.62-10 mg/kg) were higher than that
observed in the control group (P<0.01; Table 3). There was
a dose-dependent relationship between the
anti-implantation rates and droloxifene doses from 0.15 mg/kg to 5.0
mg/kg. The values of ED95,
ED90 and ED50 were 3.70 mg/kg, 2.63
mg/kg and 0.79 mg/kg, respectively.
Antagonistic effect of external
E2 on the anti-implantation effect of droloxifene
To investigate the antagonistic effect of external
E2 on the anti-implantation effect of droloxifene, the anti-implantation effect of external
E2 only was first evaluated. When rats were treated at 22:00 PM on
d 4 with external E2 at doses of 2.0 µg/kg or 8.0 µg/kg (sc),
significant anti-implantation effects were observed
(P<0.05), whereas at doses of 0.5 µg/kg or 1.0 µg/kg, there was no
anti-implantation effect. For rats treated at 22:00 PM on d 4 with
2.5 mg/kg droloxifene alone or 2.5 mg/kg droloxifene
combined with various doses of E2, there was no difference in
implantation rates, although E2 at higher doses (2.0 or
8.0 µg/kg) reduced the anti-implantation rates (Table 4).
Effect of droloxifene on the serum level of estrogen
during early pregnancy Pregnant rats were treated orally with
2.5 mg/kg droloxifene or 1% CMC at 22:00 PM on d 4. The
rate of implantation was 100% and 0% in the control and
droloxifene groups, respectively. In the control group, the
serum level of estrogen remained at low levels from d 1 to d
3, began to rise on d 3, reached the maximum at 10:00 AM on
d 4, then declined sharply, such that the levels on d 5 and d
6 were similar to those on d 3. The serum estrogen levels in
the droloxifene group between d 1 and d 6 were not
significantly different from those in the control group (Figure 1).
These results indicate that there was a surge of estrogen in
the pregnant rats at 10:00 AM on d 4, and that treatment with
droloxifene at 22:00 AM on d 4 had no effect on the level of
estrogen; however, a significant anti-implantation effect was
induced. Therefore, the anti-implantation effect of droloxifene
in rats appears not to be due to antagonism of a surge in the
secretion of nidatory estrogen.
Effect of droloxifene on serum levels of progesterone
during early pregnancy in rats Pregnant rats were treated
orally with 2.5 mg/kg droloxifene or 1% CMC at 22:00 PM on
d 4. The rates of implantation were 100% and 0% in the
control and droloxifene groups, respectively. In the control
groups, the serum levels of progesterone rose from 10:00
AM on d1 to 22:00 PM on d 4 and remained at high levels
until d 6. In groups treated with droloxifene at 22:00 PM on
d 4, the levels of progesterone were similar to that of
controls (Figure 2). These results indicate that treatment with
droloxifene at 22:00 PM on d 4 had no effect on the level of
progesterone in early pregnancy.
Discussion
It is well established that an exact equilibrium of
estrogen and progesterone is indispensable for implantation in
rats[10]. A nidatory c surge that occurs on d 4 is essential for
the sensitization of the uterus to induce decidual cell reaction,
the most specific function of the progestational
endom- etrium[11,12]. As a novel selective estrogen receptor
modulator with considerable anti-estrogenic effects, droloxifene
might disturb the hormonal effects and cause an
anti-implantation effect. The present study found that droloxifene
had anti-implantation effects in rats (Tables 1-3) and that
22:00 PM on d 4 was the optimal oral administration time. At this
time there was a good dose-effect relationship between the
anti-implantation rates and droloxifene doses from 0.31
mg/kg to 5.0 mg/kg. The ED95,
ED90 and ED50 of droloxifene were
3.70 mg/kg, 2.63 mg/kg, and 0.79 mg/kg,
respectively.
We found that the serum levels of estrogen in pregnant
rats reached a peak at 10:00 AM on d 4, which indicates that
the nidatory estrogen surge before implantation occurs at
approximately this time. However, the optimal oral
administration time of droloxifene for anti-implantation effects was
at 22:00 PM on d 4, 12 h later than the nidatory estrogen
surge. Therefore, we propose that the anti-implantation
effect of droloxifene is not caused by its interfering with the
nidatory estrogen surge via its anti-estrogenic effect. The
effects of droloxifene are different from those of tamoxifen, a
triphenylethyl compound, which antagonizes the nidatory
estrogen surge[13,14].
In order to further clarify the relationship between the
anti-implantation effect and the anti-estrogenic activity of
droloxifene, the antagonistic effect of external
E2 on the anti-implantation effect of droloxifene was observed in rats. At
first, the anti-implantation effect of external estrogen
(0.5-8.0 µg/kg, sc) was examined after administration at 22:00 PM on
d 4. We found that E2 at doses of 0.5-1.0 µg/kg produced no
anti-implantation effect (Table 4), and had no
antagonistic effect on the anti-implantation effect of droloxifene
(P>0.05; Table 4). When droloxifene was combined with
E2 at higher doses (4.0 or 8.0 µg/kg), the anti-implantation effect
of droloxifene was reduced, but the difference was not
significant according to the c2 test. Therefore, it seems that the
anti-implantation effect of droloxifene may be not related to
its anti-estrogenic activity, especially at physiological doses.
Because an exact equilibrium of estrogen and
progesterone is essential for implantation, and any disturbance in the
effects of these hormones can cause infertility, we
investigated whether droloxifene inhibited implantation by
affecting the serum levels of estrogen and progesterone. We found
that droloxifene had no effect on the serum estrogen and
progesterone levels in early pregnancy when treated at
22:00 PM on d 4. However, in our previous study, we found that
apoptosis of luteal cells and decreases in serum
progesterone levels were induced by treatment with droloxifene at a
dose of 20 mg/kg on d 2 in pregnant
rats[8]. The differences between the two experiments can be explained by the
different doses and administration times. In addition, the period
of observation was too short in the present study.
In conclusion, droloxifene can inhibit implantation in rats
and the optimal oral administration time is 22:00 PM on d 4.
ED90 was 2.63 mg/kg. The anti-implantation effect of
droloxifene is not related to its antiestrogenic activity, or an
antagonistic effect on the nidatory estrogen surge. The
direct inhibition of endometrial receptivity to blastocyst
signal(s) and the apoptosis of luteal cells might be involved in the
anti-implantation mechanism of droloxifene. This
charac-teristic may make droloxifene useful in developing new
contraceptives.
Acknowledgment
We are grateful to Prof Zhi-ping GU for valuable
discussions throughout this study and helpful comments on the
manuscript.
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