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Departments of Bone and Cartilage Biology, and Safety Assessment (SR), SmithKline Beecham Pharmaceuticals, King of Prussia, Pennsylvania 19406
Address all correspondence and requests for reprints to: Mark E. Nuttall, Department of Bone and Cartilage Biology, SmithKline Beecham Pharmaceuticals, 709 Swedeland Road, P.O. Box 1539, King of Prussia, Pennsylvania 19406. E-mail: Mark-E-Nuttall{at}sbphrd.com
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Abstract |
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In this report, we also provide molecular-based evidence to support the observations in vivo of a novel selective estrogen receptor modulator (SERM) mechanism of action in bone and endometrial cells. Idoxifene is an agonist through the estrogen response element (ERE) and exhibits similar postreceptor effects to estrogen in bone-forming osteoblasts. Idoxifene also stimulates osteoclast apoptosis, and these pleiotropic effects ultimately could contribute to the maintenance of bone homeostasis. However, idoxifene differs from estrogen in a tissue-specific manner. In human endometrial cells, where estrogen is a potent agonist through the ERE, idoxifene has negligible agonist activity. Moreover, idoxifene was able to block estrogen induced gene expression in endometrial cells, which is in agreement with the observation in the intact rat study. In the uterus, idoxifene has a pharmacologically favorable profile, lacking agonist and therefore growth-promoting activity. Together with its cholesterol lowering effect and lack of uterotrophic activity, these data suggest that idoxifene may be effective in the prevention of osteoporosis and other postmenopausal diseases without producing unwanted estrogenic effects on the endometrium.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The concept of selective estrogen receptor modulation has been demonstrated for a number of compounds including tamoxifen (7), raloxifene (8), droloxifene (9), GW 5638 (10), and levormeloxifene (11). However, the clinical utility of these agents will depend on the profile of tissue-specific effects, and the extent to which they are translated into in vivo efficacy. A SERM is defined as a compound that has estrogen agonism on one or more of the desired target tissues such as bone or liver and has antagonism and/or minimal agonism (i.e. clinically insignificant) in reproductive tissue such as the breast or uterus (12, 13, 14). Although tamoxifen does act as a SERM, it is also associated with an increased incidence of endometrial cancer (14). Attempts to improve on the pharmacological profile of tamoxifen have resulted in compounds that differ in their estrogen agonist/antagonist characteristics, including the pure estrogen antagonists (12). This suggests that it may be possible to develop a molecule with a desired profile of tissue-specific agonist/antagonist activities.
Idoxifene (pyrrolidino-4-iodotamoxifen, Fig. 1
) is a novel SERM that has a 2.5- to
5-fold greater affinity for the estrogen receptor 
than tamoxifen,
while being significantly less uterotrophic (15, 16). To determine
whether or not idoxifene acts as an estrogen agonist on the skeleton
following estrogen withdrawal, we have investigated its effect in the
Ovx rat model. Here, we report on the effect of idoxifene on bone loss,
uterine weight, and plasma cholesterol using experimental designs for
both prevention of bone loss and intervention in progressive bone
loss.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The rats were then segregated into groups (n = 10) that did not differ in their mean values for lsBMD and ptBMD. Surgery was done following the collection of 24 h urine samples for the determination of baseline pyridinium cross-link excretion by ELISA (Metra Biosystems, Palo Alto, CA, product no. 8001).
The following measurements were made at one or more timepoints during each experiment: lumbar spine and proximal tibial BMD (expressed as a percentage of the baseline BMD for each animal); serum total cholesterol; plasma osteocalcin (Biomedical Technologies Inc., Stoughton, MA); pyridinium cross-link excretion (measurements normalized to creatinine and expressed as a percentage of the baseline measurement, having established no significant differences between groups at baseline by unpaired t test).
Groups of ovariectomized rats received, by oral gavage, a single daily dose of either dosing vehicle (1% aqueous solution (wt/vol) of carboxymethyl cellulose), or idoxifene suspended in vehicle. A group of sham-operated rats was dosed with vehicle.
For each parameter, differences between groups were assessed by unpaired Student’s t test using Microsoft Corp. Excel.
Dose response study with idoxifene
Before surgery, baseline measurements were made as described
above. Ovariectomized rats received idoxifene at 0, 0.1, 0.2, 0.5, and
1.0 mg/kg·day. A sham-operated group received dosing vehicle. The
doses of idoxifene were based on information obtained from preliminary
dose-ranging studies. Dosing commenced immediately following surgery.
Bone mineral density was measured at weeks 8 and 17. After 2 weeks of
treatment, blood samples were collected for the determination of serum
cholesterol and plasma osteocalcin and 24 h urine samples were
collected for the measurement of pyridinium cross-link excretion.
Uterine wet weight was determined at necropsy.
Effect of intervention with idoxifene on progressive loss of
BMD
In a separate experiment, rats underwent measurement of baseline
BMD and were grouped as described above. Following Ovx or sham
operation, the rats were maintained untreated for 4 weeks, after which
a significant decrease in lumbar spine and proximal tibial BMD was
measured in the Ovx rats. Groups of Ovx rats were then dosed with
idoxifene at 0.5 and 2.5 mg/kg·day or dosing vehicle. The
sham-operated rats were dosed with vehicle alone. Further measurements
of lumbar spine and proximal tibial BMD were made at week 8 (week 4 of
dosing) and week 12 (week 8 of dosing) post surgery.
Effects of idoxifene and estrogen on uterine histology
A separate study was done in which groups of 9–10 rats (female
Sprague-Dawley, 8–9 months of age) were ovariectomized and treated sc
with idoxifene (0.2, 1.0, or 10.0 mg/kg·day), 17ß-estradiol (0.1
mg/kg·day) or vehicle (olive oil) for three months. A sham-operated
group received injections of vehicle. Uteri were collected at necropsy
and fixed in formalin. A cross-section of each uterine horn was
processed, embedded in paraffin, and sectioned at 5 µm and stained
with hematoxylin and eosin for light microscopic evaluation.
Effect of idoxifene on uterine wet weight in intact animals
Three groups of 10 rats each (female Sprague-Dawley, 9–10
months of age) were dosed with either vehicle or idoxifene (0.5, 1.5
mg/kg·day) for 8 weeks. Uterine wet weight was determined at
necropsy.
In vitro experiments
Cell culture and materials. All chemicals were purchased
from Sigma Chemical Co. (St. Louis, MO) unless otherwise
stated. Cell lines used in this study were rat osteosarcoma cells: Ros
17/2.8 (17) and the same cell-type stably transfected with the estrogen
receptor: Ros.smer (18), human osteosarcoma (MG-63) and Hos TE-85 (Hos)
cells (19), endometrial Ishikawa cells (kindly given by Dr. V. C.
Jordan, Northwestern University, Chicago, IL) (20). Human
osteoclastoma derived osteoclast-enriched populations of cells were
used for the apoptosis studies as described previously (21). Ros 17/2.8
cells were grown routinely in Ham’s F-12 medium (Gibco BRL Technologies, Grand Island, NY) supplemented with 5% heat
inactivated FBS (Gibco BRL Technologies, Grand Island, NY)
supplemented with penicillin (10 U/ml), streptomycin (100 mg/ml)
(Gibco BRL Technologies) plus 1.1 mM calcium
chloride and 25 mM HEPES. All other cell lines were
routinely cultured in DMEM supplemented with 10% heat inactivated FBS
(Hyclone Laboratories, Inc., Logan, UT), penicillin (10
U/ml) and streptomycin (100 mg/ml) (Gibco BRL
Technologies). All experiments were performed in phenol red free
Eagle’s MEM (EMEM) containing 10% heat-inactivated charcoal-dextran
stripped FBS (Hyclone Laboratories, Inc., Logan, UT) and
supplemented as above. All cell lines were cultured at 37 C in a
humidified atmosphere 95% air-5% CO2. At confluence cells
were subcultured after exposure to trypsin-EDTA (Gibco BRL
Technologies).
A DNA construct that comprised of a mouse mammary tumor virus promoter in which the glucocorticoid response elements have been replaced with five copies of a 33-bp vitellogenin estrogen response element was a kind gift from D. McDonnell, (Duke University). This is upstream of the Luciferase reporter gene (MMTV-ERE-Luc) (22). The renilla-Luciferase vector was used to correct for transfection efficiency using the dual-luciferase detection method (Promega Corp., Madison, WI).
Transient transfections
Cells were seeded in either 6-well plates at 1.5 x
105cells/well or in 24-well plates at 1.5 x
104 cells/well in phenol red-free medium. DNA was
introduced into the cell lines by the lipofectin method (Life Technologies, Gaithersburg, MD). Briefly, cells were
cotransfected with 2 µg per well in 6-well plates and 140 ng per well
in 24-well plates of MMTV ERE-Luc and 25 ng of the control
renilla-Luciferase vector (pRL-CMV). Transfection efficiency was
corrected for by co-transfection with a renilla-Luciferase vector,
which uses a different substrate, coelenterazine, for its
bioluminescent readout (Promega Corp.). Cells were
incubated overnight. Transfection medium was then removed and cells
were incubated for 48 or 72 h with or without hormones as
indicated by the figure legends. Cell lysates were prepared as
described in the manufacturer’s protocol for dual-luciferase reporter
assay to assess transfection efficiency (Promega Corp.).
Briefly, cells were washed in PBS and then lysed with 500 µl/well
1 x passive lysis buffer (PLB) for 15 min while rocking sample on
a rocking platform. Lysates were centrifuged for 30 sec at 14,000
x g and the clear lysate was transferred to a tube before
reporter enzyme analysis. Samples (20 µl) were transferred to a
96-well luminescence detection plate and reacted with 100 µl of each
assay reagent (Promega Corp.). Each assay reagent was
injected by a microlumat LB96P luminometer (Wallac, Gaithersburg, MD),
which measured luciferase activity. Results are expressed as relative
light units (RLU).
Apoptosis
To assess apoptosis, osteoclast-enriched cell populations were
prepared as described previously (21) and grown for 72 h in the
presence of either estrogen or idoxifene. Camptothecin was used as a
known inducer of apoptosis. Cell-lysates were prepared and a cell-death
ELISA was performed (Boehringer Mannheim, GmbH,
Germany). We have shown that the results from this ELISA
correlate with other markers of apoptosis, such as cellular
morphological changes and DNA laddering (not shown).
IL-6 production
Human osteosarcoma (MG-63) cells (19) were grown overnight in
phenol redfree DMEM supplemented with 10% charcoal/dextran stripped
FBS. Before treating the cells, the medium was removed, cells were
washed with PBS and medium supplemented with 2% FBS was added. Cells
were then treated as follows: vehicle (dimethylformamide), estrogen
(100 nM), idoxifene (100 nM) for 20 h.
After 20 h, cells were treated with tumor necrosis factor (TNF)
(500 U/ml) for another 24 h. IL-6 production was determined by
BIOTRAK ELISA (Amersham Life Science, Little Chalfont,
UK).
Alkaline phosphatase activity
Cell lysates were obtained by solubilization of the monolayer
cells with 0.1% (vol/vol) Triton X-100. Alkaline phosphatase activity
was determined by colorimetry using p-nitrophenylphosphate as the
substrate (22A ). Results are expressed as optical density absorption at
450 nm for 10 min.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) significantly inhibited the
Ovx-induced loss of lumbar spine BMD (lsBMD). The 0.5 mg/kg·day dose
of idoxifene maintained lsBMD at the sham control value. There were no
significant differences among the 0.2, 0.5 and 1.0 mg/kg·day doses.
Loss of lsBMD following Ovx was dependent on a decline in lsBMC, which
was prevented by treatment with idoxifene; there were no differences
among these groups with respect to bone area (data not shown). In the
proximal tibia, all doses of idoxifene (Fig. 2B) significantly
inhibited Ovx-induced loss of BMD. The 0.5 mg/kg·day dose of
idoxifene maintained ptBMD at sham control levels.
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). A dose of 0.5
mg/kg·day was maximally effective, maintaining lsBMD at sham control
levels. Normalization of lsBMD by treatment tended to be due more to
inhibition of the effect of Ovx on BMC than on bone area. Idoxifene
caused a dose-dependent inhibition of the loss of ptBMD (Fig. 3B), the
values for doses of 
0.2 mg/kg·day not differing from sham
control. The effect on ptBMD was due to an effect on ptBMC, not bone
area.
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) and plasma osteocalcin levels (Fig. 4B) measured 2 weeks post ovariectomy. The maximal effect was at 0.5
mg/kg·day, which appeared to maintain bone turnover at sham control
levels. This was maintained at weeks 8 and 17 (data not shown).
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shows the effect of Ovx on
lumbar spine (A) and proximal tibial (B) BMD, and the effect of
intervention with idoxifene (0.5 and 2.5 mg/kg·day) on further
changes in BMD. One month after Ovx, BMD at both sites was
significantly reduced relative to the sham control group. Commencing
treatment with idoxifene at this time completely prevented any further
loss of BMD. Both doses of idoxifene were equally effective.
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). Idoxifene was
maximally effective at 0.5 and 1.0 mg/kg·day, reducing cholesterol
levels below sham control values. This was maintained at weeks 8 and 17
(data not shown).
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). The
uterine wet weight of Ovx rats treated with idoxifene was slightly
greater (P < 0.01) than in vehicle-treated Ovx
controls, although importantly, this effect was not dose dependent.
Significantly, idoxifene inhibited the estrogenic effect seen in the
rat uterus in intact animals at a dose of 1.5 mg/kg·day (Fig. 7B).
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and 2; Fig. 8).
All idoxifene-treated and vehicle-treated Ovx rats showed myometrial
atrophy whereas the majority (8/10) of estrogen-treated Ovx rats showed
myometrial hypertrophy. Mild stromal fibrosis was seen in 4/9
idoxifene-rats at 10.0 mg/kg and in 3/8 rats at 1.0 mg/kg compared with
the moderate to severe fibrosis seen in all estradiol-treated rats. All
sham operated rats (9/9) exhibited moderate stromal fibrosis. Idoxifene
caused a dose-dependent reduction in the number of uterine glands and
caused atrophy of the lining epithelium of the uterus (endometrial
atrophy). The lining epithelium was completely absent in some rats
treated with idoxifene at 10.0 mg/kg. In contrast, estradiol treatment
caused an increase in the number of endometrial glands, endometrial
hyperplasia and dilation of the uterine lumina. Extensive squamous
metaplasia was also present in 2/10 estradiol-treated rats. Endometrial
hyperplasia and focal squamous metaplasia were present in 2/9 sham
control rats.
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). This suggests that idoxifene is
interacting with the osteoblast estrogen receptor to activate the
estrogen response element and therefore behaving as an estrogen agonist
in osteoblasts. Idoxifene at all concentrations tested was an agonist
through this response element in osteoblasts (Fig. 9B) These
observations were confirmed using the natural ERE-linked to luciferase
(23), which gave similar responses to idoxifene and estrogen (not
shown). Also, a viral vector not containing EREs was not induced above
control values in the presence of idoxifene, indicating that the ERE is
required for transcriptional activation upstream of this viral promoter
(not shown). To further confirm that the ERE was absolutely required
for transcriptional activation by idoxifene we transfected the cells
with the osteocalcin promoter (pOCZCAT: -1097 to +34) (24), which does
not contain an ERE and idoxifene was unable to transcriptionally
activate through any of these promoter/reporter constructs that lacked
the ERE (not shown). This extends the classical estrogen receptor
binding studies because receptor binding does not necessarily correlate
with modulation of gene expression in a given tissue (25).
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stably overexpressed (18).
Transcriptional activation was greater in the Ros.smer cells than the
wild-type cells in the presence of either idoxifene or estrogen
indicating that estrogen receptor- levels determine the magnitude of
transcriptional activation and therefore support the effects of this
compound are through this receptor (Table 3). The number of estrogen receptors in
the stably transfected cells is approximately 2000 sites/cell compared
with the parent cell line that has approximately 200–500 sites/cell
(18). The enhanced transcriptional activity correlates (4- to 6-fold)
with the increased number of receptors. This is in agreement with our
previously reported gel-shift experiments where we showed that
idoxifene will bind to the estrogen receptor- in rat osteoblasts
(26).
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stimulated IL-6
production up to 10,000 fold in osteoblast cell lines (27, 28).
Addition of 17ß-estradiol to the cultures resulted in an inhibition
of TNF-induced production of IL-6 (Fig. 10). We tested the ability of idoxifene
to inhibit TNF-induced IL-6 production in human osteosarcoma MG-63
cells in culture. IL-6 was undetectable without TNF stimulation in
human osteosarcoma cells. Both idoxifene and estrogen inhibited the
TNF-induced IL-6 production (Fig. 10).
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(Ros.smer). Estrogen has previously been shown to
up-regulate alkaline phosphatase activity to a greater extent in the
stably transfected cells (18). Idoxifene, like estrogen, stimulated
alkaline phosphatase activity in both rat osteoblast cell lines (Fig. 11). Significantly, idoxifene and
estrogen both stimulated alkaline phosphatase activity to a greater
extent in the cells stably transfected with the estrogen receptor (Fig. 11).
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). The observed results
with idoxifene were similar to those with estrogen. Both estrogen and
idoxifene at the highest concentrations tested induced apoptosis to the
same extent as the topoisomerase inhibitor camptothecin, a known
inducer of this process. Morphological changes were seen in the
osteoclasts consistent with apoptosis in idoxifene, estrogen and
camptothecin treated cells (not shown). This indicates an
estrogenic-like induction of apoptosis in human osteoclasts by
idoxifene which may play a significant role in the bone sparing effects
of this compound in vivo.
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) which may, at least in part, be
due to the high levels of estrogen receptors reported in endometrial
cells (34). This agonist activity is in agreement with the clinical
observations that estrogen treatment promotes endometrial hyperplasia
(35). Idoxifene had no agonist activity through the ERE, and indeed
prevented the agonist effects of estrogen through the ERE (Fig. 13, A
and B). This correlates with the in vivo studies in the
intact rats (Fig. 7B
), where idoxifene blocked estrogenic effects on
the uterus. We have also observed similar effects of idoxifene in
breast cancer MCF-7 cells (26), suggesting that it is acting as an
antagonist in breast tissue, an effect which may provide important
therapeutic benefits for this compound.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We provide direct evidence that idoxifene is working as an estrogen agonist in osteoblasts in culture, and that the profile of induction of transcription through the ERE in osteoblasts by idoxifene is similar to that of estrogen. To our knowledge these are the first reports of this type of transient reporter experiments without cotransfecting an expression plasmid for the estrogen receptor and therefore may represent a more physiologically relevant cell system. It is not yet known whether idoxifene binds to the recently discovered estrogen receptor; ERß (36), the implications of which are discussed below. In contrast to idoxifene, it has been reported previously that raloxifene is an antagonist of the ERE in osteoblasts (8, 37), and it has a distinct mechanism of action through a raloxifene response element (RRE) (8). It has been proposed that raloxifene is able to exert its biological effect through this non-ERE containing sequence present on the 5'-untranslated region of the human TGFß3 promoter (8). We have confirmed these data indicating no agonist activity of raloxifene through the ERE (not shown). This distinguishes the mechanism of action of idoxifene from that of raloxifene, and aligns idoxifene to the more classical estrogenic mechanism, exerting its biological agonist effect in osteoblasts through the ERE. This is supported by the observation of the direct effects of idoxifene, on osteoblasts, in stimulating alkaline phosphatase activity, a marker associated with bone formation and responsiveness to estrogen.
In addition to the similarities between estrogen and idoxifene in osteoblasts on a molecular level, we have shown also that they have similar biological effects in human osteoclasts. The ability to stimulate osteoclast apoptosis may be related directly to the antiresorptive effects of both estrogen and idoxifene, and it may be the combination of effects on both osteoblasts and osteoclasts that ultimately leads to the bone-sparing ability of these compounds. 17ß-estradiol promotes apoptosis of murine osteoclasts in vitro and in vivo by 2- to 3-fold (33). It has been reported that anti-TGF-ß antibody inhibited TGF-ß-, estrogen- and tamoxifen-induced osteoclast apoptosis, indicating that TGF-ß might mediate this effect. These studies were done on mixed populations of stromal cells and osteoclasts and therefore the effects may be direct or indirect. Stromal cells are required for osteoclast formation, but they could also mediate osteoclast death if estrogen induced them to produce an apoptosis promoting agent. Estrogen or idoxifene may also act directly to promote osteoclast apoptosis. Functional estrogen receptor expression has been demonstrated in highly enriched populations of osteoclasts and a number of effects such as inhibition of resorption and induction of the apoptosis associated c-fos gene have been observed at estrogen concentrations that stimulate apoptosis (33). The authors proposed that a major role of estrogen before menopause and of estrogen replacement after the menopause is to limit osteoclast numbers and activity through promotion of apoptosis of mature osteoclasts and their precursors. This may well be one of the pleiotropic estrogenic effects in vivo that allows idoxifene to prevent bone loss after ovariectomy.
17ß-estradiol inhibited both TNF-induced IL-6 production and
osteoclast development in primary bone cell cultures derived from
neonatal murine calvariae (27). In the same system the
TNF-stimulated osteoclast development was also suppressed by a
neutralizing monoclonal anti-IL-6 antibody and the authors postulated
that this provided a mechanistic paradigm by which estrogens exert, at
least, part of their antiresorptive influence on the skeleton. Bone and
bone marrow cells produce cytokines, such as IL-6, which act by a
paracrine mechanism in the bone microenvironment to regulate osteoclast
formation. Therefore, the bone loss associated with the removal of
estrogen might be due, at least in part, to the removal of these
inhibitory effects.
Based on these studies we suggest that idoxifene has estrogen agonist effects in bone, in both the osteoblast and osteoclast and that this is the most likely explanation for the prevention of bone loss and inhibition of bone turnover that is seen in in vivo studies. It is tempting to speculate that, because the effects of estrogen in bone are complex and involve multiple target cells (osteoblast and osteoclast) agonism through the classic-ERE is necessary for the complete pleiotropic effects of an estrogenic-compound in bone.
The uterine histology of Ovx rats treated for 3 months with idoxifene (0.2, 1.0, or 10 mg/kg·day) showed myometrial atrophy, decreased numbers of uterine glands and endometrial atrophy, suggesting a lack of estrogenic activity of idoxifene on this tissue. Furthermore, treatment with idoxifene for more than 4 months caused significant prevention of osteopenia while having only a minor, nondose-dependent effect on uterine weight. This observation has been reported for other SERMs such as GW5638 (10) and raloxifene (37). Previously (16) it was shown that idoxifene, at doses similar to those used in the present studies, inhibited the uterotrophic effect of estradiol in immature rats while having only marginal intrinsic uterotrophic activity, and we have confirmed these studies using idoxifene in the intact rat. Taken together, these data suggest that idoxifene is a selective ER agonist in bone while having no ER agonist activity in the uterus, and in fact, is able to block natural estrogen action in the uterus.
These studies support a therapeutic advantage of idoxifene over estrogen for long-term use for the prevention of postmenopausal disease in women. Idoxifene has no estrogenic-agonist properties in endometrial cells in vitro. Agonism by estrogen in these cells is considered to be the molecular basis of the enhanced proliferation observed clinically during endometrial hyperplasia (38, 39). The effects of estrogen on proliferation of endometrial cells are consistent with the clinical observations of promotion of endometrial hyperplasia (34, 40). The in vitro data are consistent with uterotrophic studies based on the measurement of uterine weight in immature rats and mice, which showed that idoxifene antagonized the effects of estrogen (16). These studies are consistent with both the intact and the Ovx studies presented in this report. In breast cells, estrogen is a strong agonist whereas idoxifene is a potent antagonist (16). Taken together, these results in endometrial cells suggest a favorable therapeutic profile for idoxifene over estrogen in reproductive tissues.
It has been shown that tamoxifen (41) and raloxifene (37) induce conformational changes within the ER that are distinct from estrogen. These specific ligand-receptor complexes were differentially recognized by the cellular transcriptional machinery in a cell-specific context (25). The ER contains two transactivation domains: AF-1 and AF-2 and the relative contributions of these domains differ from cell-to-cell. Tamoxifen functions as an AF-2 antagonist, inhibiting ER activity in cells where AF-2 is dominant. In contrast, it functions as an agonist where AF-1 alone is required. These observations provide a mechanistic basis for cell-specific effects of ER modulators. Idoxifene is an agonist in osteoblasts and osteoclasts that could be explained by this being an AF-1 required environment. However, Willson and co-workers (10) have suggested that this does not strictly correlate in bone where different degrees of AF-1 and AF-2 agonist activity all protect against bone loss. The antagonism in endometrial cells would be explained by a complex between idoxifene and the ER that results in inhibiting ER activity in these cells. This would be analogous to the AF-2 dominant environment in which tamoxifen is a complete antagonist. However, reports of tamoxifen being a mixed agonist/antagonist in the uterus suggest this is not an AF-2 dominant environment.
This mechanistic explanation is probably more complex with the discovery of a new estrogen receptor isoform (ERß) that may have different ligand specificities and tissue distribution and also the discovery of nuclear hormone coactivators and corepressors that may contribute to tissue selectivity of the ER modulators (42). Studies are currently underway to define exactly how idoxifene binds to the ER isoforms in a cell specific context.
The most important benefit of HRT is its cardioprotective effect. Long-term adjuvant treatment with tamoxifen for breast cancer in postmenopausal women was associated with an estrogen-like cardioprotective effect (43) and tamoxifen has also been shown to lower cholesterol in ovariectomized and intact rats (44). The present study demonstrated a dose-responsive reduction in serum total cholesterol following treatment with idoxifene, suggesting that it has the potential for beneficial cardiovascular effects similar to those of HRT or tamoxifen. Although the mechanism of this action of idoxifene has not been studied, it may be similar to that of tamoxifen, which is thought to act by inhibiting cholesterol synthesis (45).
In conclusion, idoxifene showed beneficial effects on bone loss in the axial and appendicular skeleton in the Ovx rat model and has an estrogenic mechanism of action in bone. The pharmacological profile of idoxifene suggests that it behaves like an ER agonist on bone and lipid metabolism while having no ER agonist activity on the uterus at doses that prevent bone loss. This was confirmed in in vitro studies in endometrial cells. Together with its cholesterol lowering effect and lack of uterotrophic activity, these data strongly suggest that although idoxifene may not confer any advantage over estrogen in prevention of osteoporosis, it may provide a better overall profile for the treatment of postmenopausal diseases.
Received May 26, 1998.
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A. M. Badger, S. M. Blake, R. A. Dodds, D. E. Griswold, B. A. Swift, D. J. Rieman, G. B. Stroup, S. J. Hoffman, and M. Gowen Idoxifene, a Novel Selective Estrogen Receptor Modulator, Is Effective in a Rat Model of Adjuvant-Induced Arthritis J. Pharmacol. Exp. Ther., December 1, 1999; 291(3): 1380 - 1386. [Abstract] [Full Text]
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