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Laboratory of Molecular Endocrinology, CHUL Research Center, Québec, Canada G1V 4G2
Address all correspondence and requests for reprints to: Prof. Fernand Labrie, Laboratory of Molecular Endocrinology, CHUL Research Center, 2705 Laurier Boulevard, Québec, Canada G1V 4G2.
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recently, 7
-alkyl derivatives of estradiol (3, 4, 5),
11ß-amidoalkoxyphenyl estradiols (6), or estradiol 7-alkyl
derivatives possessing additional changes designed to increase their
affinity for the ER and/or increase their bioavailability (7, 8, 9, 10, 11) have
been synthesized and shown to possess pure and potent antiestrogenic
activity in the most rigorous in vitro and in
vivo systems (3, 11), including human breast cancer cells in
vitro (3, 7, 9, 10) and in vivo in nude mice (4, 8, 12, 13).
These 7-alkyl and 11ß-amidoalkoxyphenyl derivatives of estradiol,
however, have low oral bioavailability, thus limiting their
acceptability for the treatment of breast cancer. We have thus
developed a series of even more potent estrogen antagonists that
possess high oral bioavailability in the mouse, rat, monkey, and human.
The present study compares the characteristics of the new antiestrogen
EM-800 (14, 15, 16, 17) on well characterized estrogen-sensitive parameters in
the mouse with the effects of ICI 182 780 (4, 12) and toremifene, an
analog of tamoxifen (18, 19, 20, 21, 22).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Chemicals
EM-800
[(+)-7-pivaloyloxy-3-(4'-pivaloyloxyphenyl)-4-methyl-2-(4''-(2`''-piperidinoethoxy)phenyl)-2H-benzopyran]
(14) and ICI 182 780 were synthesized in the medicinal chemistry
division of our laboratory. Both compounds analyzed under Good
Laboratory Practice (GLP) conditions were more than 99% pure.
Toremifene citrate was provided by Orion Corp. (Farmos, Finland). The
structures of these antiestrogens are illustrated in Fig. 1
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, EM-800 and ICI 182 780 were
injected sc once daily at different doses (0.1, 0.3, 1.0, 3.0, or 10
µg/injection), whereas in
Figs. 6–9, EM-800, ICI 182 780 and
toremifene were administered orally by gavage once daily at a dose of
0.3, 1.0, 3.0, or 10.0 µg/animal. Treatment with the antiestrogens
was initiated 2 days after ovariectomy, whereas treatment with estrone
(0.06 µg, sc, twice daily) was started 3 days later (5 days
postovariectomy). Thereafter, estrone and antiestrogens were
administered in combination for a 6-day period. Compounds were
dissolved in a 50:50 (vol/vol) mixture of polyethylene glycol 600 and
ethanol and administered in a 1% (wt/vol) gelatin-0.9% NaCl solution
(final concentration of polyethylene glycol 600/ethanol was 8%). Mice
in the intact and OVX control groups received the vehicle alone during
the 9-day period. The animals were killed by cervical dislocation on
the 11th morning after ovariectomy. The uteri and vagina were rapidly
dissected and weighed, and then frozen in liquid nitrogen and stored at
-80 C.
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-monothioglycerol, 10% glycerol,
and 10 mM sodium molybdate, pH 7.4). For each group, three
different homogenates were used, and each homogenate was prepared from
a pool of two or three uteri or vaginas. The homogenates were then
centrifuged at 105,000 x g for 60 min at 4 C. The
steroid binding assay was performed with freshly prepared cytosol. The
protein concentration of cytosol was determined using the method of
Bradford (23) with BSA as standard.
Estrogen binding assay: [2,4,6,7-3H]Estradiol
(SA, 87 Ci/mmol) was purchased from New England Nuclear (Boston, MA),
whereas diethylstilbestrol was obtained from Sigma Chemical Co. (St.
Louis, MO). [3H]Estradiol binding was measured using the
dextran-coated charcoal absorption technique, essentially as previously
described (24, 25, 26, 27). In brief, 0.2-ml aliquots of the cytosol
preparation were incubated with 0.1 ml [3H]estradiol
(final concentration, 3–5 nM) in the presence or absence
of a 100-fold excess of diethylstilbestrol for 3 h at room
temperature. Unbound steroids were separated by incubation for 15 min
at 4 C with 0.3 ml of 0.5% Norit-A and 0.05% dextran T-70 in buffer B
(1.5 mM EDTA disodium salt, 10 mM
-monothioglycerol, and 10 mM Tris-HCl, pH 7.4) and
centrifugation at 3000 x g for 15 min. Aliquots of the
supernatant (0.3 ml) were then taken for radioactivity measurement
after the addition of 10 ml liquid scintillation cocktail.
Enzyme immunoassay (ER-EIA). Preparation of cytosol:Cytosols were prepared as described for the binding assay, except
that vaginal tissue was homogenized in 10 vol of a different buffer (10
mM Tris-HCl, 1.5 mM EDTA disodium salt, 1
mM -monothioglycerol, and 5 mM sodium
molybdate, pH 7.4). For each group, only one homogenate prepared from a
pool of three vaginas was used.
ER-EIA assay: The ER-EIA monoclonal kit from Abbott Laboratories (Diagnostics Division, North Chicago, IL) used for this assay is an enzyme immunoassay based on the sandwich principle, using monoclonal rat antibodies. A detailed description of the method used is given in the instruction manual supplied with the kit. In brief, cytosols (100 µl) were incubated at 4 C for 18 h with antibody (rat anti-ER; D547)-coated polystyrene beads that bind ER proteins. A second antibody (rat anti-ER; H222) conjugated to horseradish peroxidase was then incubated with the beads at 37 C for 1 h to label the bound receptor. A further incubation (30 min at room temperature) of the beads with the enzyme substrate hydrogen peroxide and the color reagent/electron donor o-phenylenediamine·2HCl produced a phenazine polymer chromophore that was measured at 492 nm. Each incubation step was completed by washing the beads with distilled water, and the enzyme reaction was stopped by the addition of 1 N sulfuric acid. A standard curve was obtained by plotting the ER concentration of ER standards (0 and dilutions of 500 fmol/ml ER stock, supplied in the Abbott kit) vs. their absorbance.
Statistical analysis
The effects of drugs and doses were analyzed using a two-factor
nested ANOVA model. When main effects were significant
(P < 0.05), a posteriori pairwise
comparison between the control and treated groups was performed with
Fisher’s least significant difference test (LSMEANS), requiring
P < 0.01 to declare significance. Original scale
values were used to analyze the variables with normally distributed
residuals and respecting the hypothesis of homoscedasticity. If not, a
log or a rank transformation was applied to the measurements before
analysis. The two routes of administration (sc and oral) were analyzed
separately using a common intact group. All data are presented as the
mean ± SEM.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and 3, EM-800 was
approximately 10-fold more potent than ICI 182 780 in blocking the
stimulatory effect of estrone (0.06 µg, sc, twice daily) on uterine
(Fig. 2) and vaginal (Fig. 3) weight, respectively. The P
values associated with the treatment and dose (treatment) nested
effects are reported in Table 1 for both
oral and sc routes. In fact, at the lowest daily dose used (0.1 µg or
0.005 mg/kg), EM-800 caused a 33% decrease (P <
0.0001) in uterine weight, whereas doses of 0.3, 1.0, 3.0, and 10 µg
caused respective 72%, 84%, 91%, and 87% decreases
(P < 0.0001 for all doses vs. control) of
estrone-stimulated uterine weight. ICI 182 780, on the other hand, had
no significant effect at the two lowest doses used and caused 38%,
75%, and 90% decreases in uterine weight at doses of 1.0, 3.0, and 10
µg, respectively (P < 0.0001 at all doses
vs. control).
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). The daily
0.1-µg dose of EM-800 caused a 37% (P < 0.0001)
inhibition of estrone-stimulated vaginal weight, whereas doses of 0.3,
1.0, 3.0, and 10 µg caused 66%, 91%, 98%, and 96%
(P < 0.0001 for all groups vs. control)
inhibitions of vaginal weight, respectively. ICI 182 780, on the other
hand, had no detectable effect at the two lowest doses, whereas the
1.0-, 3.0-, and 10-µg daily sc doses caused respective 32%, 53%,
and 90% inhibitions of estrone-stimulated vaginal weight
(P < 0.0001 for all of these groups vs.
control).
As illustrated in Fig. 4, treatment with
EM-800 caused an almost complete loss of ER measured by binding assay
in the uterus of mice supplemented with estrone. The daily 0.3-µg
dose of EM-800 caused a 30% (P < 0.01) loss of
uterine ER levels. With the daily 1.0-µg dose, a 70% loss of uterine
estradiol receptors from 426 ± 31 to 128 ± 14 fmol/mg
protein (P < 0.0001) was observed, whereas further
inhibition was observed at the 3.0- and 10-µg doses, thus decreasing
uterine ER levels to 46 ± 3 fmol/mg protein (89% reduction;
P < 0.0001) and 9 ± 3 fmol/mg protein (98%
reduction; P < 0.0001), respectively. ICI 182 780, on
the other hand, had no significant effect on the same parameter at the
0.3-, 1.0-, and 3.0-µg daily doses, whereas a 65% stimulation of
uterine ER levels (705 ± 105 fmol/mg protein; P
< 0.001) was observed at the highest dose used (10 µg). It can also
be seen in Fig. 4 that ovariectomy increased uterine ERs from 418
± 6 fmol/mg protein in intact animals to 1235 ± 163 fmol/mg
protein (P < 0.0001 vs. intact) in
ovariectomized rats, an increase that was completely reversed by the
administration of estrone to ovariectomized animals.
Comparable effects were observed on vaginal ER levels measured by
binding assay (Fig. 5). Thus, sc
injection of 0.3, 1.0, 3.0, and 10 µg EM-800 caused 40%
(P < 0.01), 82% (P < 0.001), 97%
(P < 0.0001), and 99% (P < 0.0001)
inhibitions of vaginal ER levels, respectively. ICI 182 780, on the
other hand, had no significant effect on this parameter under the assay
conditions used.
We next compared the activities of EM-800, ICI 182 780, and toremifene
administered by the oral route. Whereas EM-800 caused an 18%
(P = NS) inhibition of estrone-stimulated uterine
weight at the daily oral dose of 0.3 µg (Fig. 6), doses of 1.0, 3.0, and 10 µg of the
antiestrogen caused respective 46%, 71%, and 83% inhibitions of
estrone-stimulated uterine weight (P < 0.0001 for all
the three highest doses vs. control). Toremifene, a close
analog of tamoxifen, caused 9% (P = NS), 25%
(P < 0.001), 48% (P < 0.0001), and
51% (P < 0.0001) inhibitions of estrone-stimulated
uterine weight at the 0.3-, 1.0-, 3.0-, and 10-µg doses,
respectively. The only significant inhibitory effect of ICI 182 780,
namely a 21% inhibition (P < 0.01), was observed at
the highest dose used (10 µg daily), indicating at least a 30-fold
lower activity of this compound compared with that of EM-800.
The daily oral administration of EM-800 led to respective 10%
(P = NS), 38%, 64%, and 88% inhibitions of vaginal
weight (P < 0.0001 for the three highest doses used
vs. control) at the 0.3-, 1.0-, 3.0-, and 10-µg doses
(Fig. 7), whereas ICI 182 780 exerted no
significant inhibitory effect on this parameter. Toremifene, on the
other hand, caused 4% (P = NS), 24%, 52%, and 67%
inhibitions of vaginal weight at the 0.3-, 1.0-, 3.0-, and 10-µg
doses, respectively (P < 0.0001 for the three highest
doses used vs. control).
As illustrated in Fig. 8, only oral
administration of the highest dose of EM-800 led to a significant
decrease in uterine ER levels measured by binding assay (58%
reduction; P < 0.01), whereas ICI 182 780 and
toremifene had no effect on this parameter at any of the doses
used.
Finally, as shown in Fig. 9, vaginal ER
levels measured by binding assay decreased from 157 ± 12 fmol/mg
protein in estrone-treated rats to 123 ± 5 fmol/mg protein
(P = NS) in animals treated with the 0.3-µg daily
dose of EM-800, whereas daily treatment with the 1.0-, 3.0-, and
10-µg doses reduced vaginal ER levels to 89 ± 0.1 fmol/mg
protein (43% reduction; P < 0.0001), 87 ± 10
fmol/mg protein (45% reduction; P < 0.0001), and
30 ± 4 fmol/mg protein (81% reduction; P <
0.0001), respectively. Toremifene, on the other hand, caused 36%
(P < 0.001), 21% (NS), 47% (P <
0.0001), and 45% (P < 0.0001) inhibitions of vaginal
ER levels at daily doses of 0.3, 1.0, 3.0, and 10 µg, respectively.
ICI 182 780 had no significant effect on vaginal estradiol receptor
levels under the experimental conditions used.
To ensure that the down-regulation of ER levels observed with EM-800
does not result from a failure of [3H]estradiol to
exchange with EM-800 [or its metabolite(s)], we evaluated the effects
of treatments with EM-800, ICI 182 780, and toremifene (administered
orally) on vaginal ER protein levels measured by ER-EIA. As shown in
Table 2, treatment with increasing doses
of EM-800 inhibited vaginal ER protein levels by 26%, 40%, and 71%
at daily doses of 1.0, 3.0, and 10 µg, respectively. As measurements
of ER protein levels by EIA were performed on a pooled tissue
homogenate, no statistical analysis could be performed. In animals
treated with ICI 182 780, only a small 10–11% decrease in vaginal ER
protein levels was observed at doses ranging from 1–10 µg, whereas
treatment with toremifene caused a 27% inhibition of this parameter
only at the highest dose used (10 µg).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In addition, the present data confirm the low oral bioavailability of the steroidal antiestrogen ICI 182 780 (4, 5). Although much higher doses of ICI 182 780 would need to be used to achieve an accurate assessment of the potency of this antiestrogen by the oral route, the present data indicate that ICI 182 780 has less than 3% the activity of EM-800 after oral administration.
On the other hand, the intrinsic estrogenic activity of toremifene, a close analog of tamoxifen (19, 20), is likely to be responsible for the limited maximal ability of this compound to reverse the stimulatory effect of estrone on mouse uterine and vaginal weight. In fact, the maximal inhibitory effect achieved with the daily 10-µg dose of toremifene on uterine weight is limited to about 51% compared with an inhibitory effect of 83% observed for EM-800 at the same dose. The maximal inhibitory effects achieved by toremifene and EM-800 on vaginal weight at the highest dose used (10 µg) were 67% and 88%, respectively (P < 0.0001 between the two compounds). Thus, in addition to its lower potency to neutralize the stimulatory effect of estrone, toremifene at high doses exerts a lower maximal inhibitory effect than can be achieved with EM-800. This situation is analogous to that observed with tamoxifen.
Tamoxifen has long been known not to completely prevent binding of estrogen to the ER (31, 32). In agreement with the present in vivo data, the pharmacology of toremifene has been found to be similar that of tamoxifen (33, 34). In the mouse uterus, toremifene has relatively potent estrogenic activity, whereas its estrogenic activity is weaker in the rat uterus (18, 33). Tamoxifen and toremifene have also shown cross-resistance in the clinic (35).
It can be seen in the present study that the maximal inhibitory effects of EM-800 and ICI 182 780, administered sc, are of similar magnitude, namely 87% and 90% inhibitions of estrogen-stimulated uterine weight and 96% and 90% inhibitions of estrogen-stimulated vaginal weight, respectively. In the rat, at the highest dose used (1 mg/kg·day), treatment with ICI 182 780 for 14 days led to a 92% inhibition of uterine weight compared with the effect of ovariectomy (5). Such data are in agreement with the present finding of a 90% inhibition at the daily 0.5 mg/kg·dose. As mentioned above, the present data achieved with EM-800 and ICI 182 780 are very different from those observed with tamoxifen and toremifene, where the maximal inhibition obtained on uterine weight is about 50%, whereas it is limited to 67% for vaginal weight. As mentioned above, the much lower maximal blockade of estrogen action on these two parameters by tamoxifen and toremifene can be explained by the intrinsic estrogenic activity of these two compounds.
The present study shows that a half-maximal inhibitory effect of estrogen action on mouse uterine weight by ICI 182 780 administered sc is obtained at a dose of 2–3 µg/day or 0.1–0.2 mg/kg·day. These data can be compared with a half-maximal inhibitory dose of 0.5 mg/kg obtained in a recent study, also performed in ovariectomized mice (5). The somewhat higher activity of ICI 182 780 observed in the present study can possibly be explained at least in part by the different vehicles used and the different durations of treatment.
The lower inhibition of vaginal ER protein levels measured by enzyme
immunoassay compared with the protein binding assay indicate that
EM-652, the active metabolite of the prodrug EM-800, occupies part of
the ER binding sites. For ICI 182 780-treated animals, there is no
apparent loss of ER-binding sites or ER protein. Previous studies have
clearly shown a loss of uterine nuclear and cytosolic ER levels
measured by binding assay after administration of the steroidal
antiestrogen ICI 164 384 (36), although ER messenger RNA levels were
unaffected. In fact, previous data have shown that ICI 182 780
increases ER degradation and a loss of ER in the mouse uterus (36, 37).
Moreover, incubation of MCF-7 cells with ICI 182 780 caused a dramatic
fall in ER immunoreactivity on day 2 after addition of the drug
in vitro (38). The loss of ER has been associated with an
increased turnover of the receptor induced by the pure antiestrogen
(37).
The lack of significant inhibition of ER by ICI 182 780 in the present study could be related to the timing of ER assays performed in tissue obtained approximately 24 h after last administration of the antiestrogen and/or some unknown mechanism related, or not, to simultaneous treatment with estrone. It could also be related to the different duration of treatment, namely 9 days, in the present study.
The present data obtained in ovariectomized mice are also in
agreement with our recent findings obtained in intact mice, in which we
found a 57% inhibition of uterine weight with the daily 10-µg oral
dose of EM-800 (0.5 mg/kg·day) (15) and the 42% reduction in
uterine weight observed in rats after 28 days of sc treatment with 0.3
mg/kg·day of ICI 182 780 (12). Comparison of the data from these two
studies suggested that EM-800 given orally was at least as potent as
ICI 182 780 given by sc injection, although different sensitivities
between the rat and mouse could play a role. In fact, the present data
indicate that EM-800 administered by the oral route is 2- to 3-fold
more potent than ICI 182 780 administered sc.
It can be mentioned that although the elimination of estrogens by ovariectomy is well known to lead to an increase in body weight in the rat (39, 40), treatment with ICI 182 780 had no significant effect on body weight (5). Such findings suggest a lack of blockade by ICI 182 780 on the estrogenic mechanisms controlling body weight in analogy with the apparent lack of antiestrogenic activity of ICI 182 780 or ICI 164384 on the inhibitory feedback activity of estrogens on the hypothalamo-pituitary-gonadotropin axis (3, 5, 13, 28).
In summary, the present data show that the orally active antiestrogen EM-800 is the most potent antiestrogen known to date. EM-800 offers the opportunity to test the proposition that a pure antiestrogen should be a valuable improvement in the therapy of breast cancer while eliminating the secondary effects related to the mixed agonist-antagonist estrogenic action of tamoxifen and its analogs, such as toremifene and droloxifene.
Received June 27, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-dihydrotestosterone and antiestrogen N,
n-butyl-N-methyl-11-(16'-chloro-3',17ß-dihydroxy-estra-1',3',5'-(10')triene-7'-yl)undecanamide
in human breast cancer ZR-75–1 cells. Cancer Res 51:2797–2802 protein
expression in human breast cancer. Eur J Cancer 32a:413–416
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