Full text of DOI:10.1359/jbmr.2001.16.9.1651

JOURNAL OF BONE AND MINERAL RESEARCH
Volume 16, Number 9, 2001
© 2001 American Society for Bone and Mineral Research
Tibolone Exerts Its Protective Effect on Trabecular Bone
Loss Through the Estrogen Receptor
A.G.H. EDERVEEN and H.J. KLOOSTERBOER
ABSTRACT
Tibolone (Org OD14) has estrogenic, progestogenic, and/or androgenic activity depending on the tissue. In
postmenopausal women, tibolone prevents bone loss without stimulating the endometrium. Tibolone is
effective in preventing trabecular bone loss from the peripheral and axial skeleton of young and old
ovariectomized (OVX) rats by reducing bone turnover, that is, bone resorption, like estrogens. We evaluated
the contribution of the various hormonal activities to tibolone’s bone-conserving effect. Three-month-old OVX
rats received tibolone (125 ␮g/rat or 500 ␮g/rat, twice daily), alone or combined with an antiestrogen,
antiandrogen, or antiprogestogen, and the effects on trabecular bone mass and bone turnover were evaluated.
Sham-operated and control OVX groups were treated with vehicle. The remaining OVX groups received oral
doses of tibolone twice daily, alone or with twice daily (a) antiestrogen ICI 164.384, (b) antiandrogen flutamide,
or (c) antiprogestogen Org 31710. For comparison, the effects of 17␤-estradiol and testosterone were examined
also. After 4 weeks, trabecular bone mineral density (BMD) in the distal femur, plasma osteocalcin, and
urinary deoxypyridinoline/creatinine ratio (Dpyr/Cr) were measured. Tibolone or 17␤-estradiol significantly
blocked ovariectomy-induced loss of trabecular BMD and inhibited bone resorption and bone turnover as
judged by reduced Dpyr/Cr ratio and osteocalcin, respectively. These effects of both compounds were
counteracted by the antiestrogen. This suggests a major involvement of the estrogen receptor in the action of
tibolone on bone metabolism. However, the antiandrogen and the antiprogestogen did not counteract the
effects of tibolone, excluding a major role of the androgenic and progestogenic activities of tibolone in its
action against trabecular bone loss. The results indicate that tibolone acts on bone almost entirely through
activation of the estrogen receptor. (J Bone Miner Res 2001;16:1651–1657)
Key words: estrogenic, ovariectomized rat, progestogen, tibolone, trabecular bone loss
humans to ovarian hormone deficiency. These include the
loss of trabecular bone from both the peripheral and the
axial skeleton skeleton(3–5) and an increase in the plasma
and urinary markers of bone formation and resorption.(6,7)
As in humans, bone loss in the OVX rat also can be reduced
by administration of estrogens.(4,8)
INTRODUCTION
STROGEN DEFICIENCY in postmenopausal women results
in a rate of bone resorption exceeding that of bone
formation, which in turn leads to an increased risk of
skeletal fractures.(1) This progressive bone loss and the
resultant fracture risk can be reduced by estrogen-
E
The ability of estrogens to reduce bone loss has been well
replacement therapy.(2) However, the use of conventional established, although less is known about the effects of
hormone-replacement therapy often is limited by the occur- androgens and progestogens on bone turnover. Tibolone
rence of withdrawal bleeding.
(Org OD14; Livial, NV Organon, Oss, The Netherlands) is
As a model of early postmenopausal bone loss, the ovari- a tissue-specific steroid with estrogenic effects on bone and
ectomized (OVX) rat shows many of the same responses as the vagina but no stimulating effects on the breast and
Department of Pharmacology, Research and Development Labs, NV Organon, Oss, The Netherlands.
1651

1652
EDERVEEN AND KLOOSTERBOER
endometrium and progestogenic and androgenic activity weight as the selection parameter was used to allocate the
depending on the tissue.(9,10) There is considerable evidence animals to treatment groups of six. Animals were OVX or
that tibolone prevents bone loss in postmenopausal wom- sham-operated under ether anesthesia. Testosterone-
en.(11,12) This protective effect also is evident in the OVX propionate or the antiestrogen ICI 164.384 were adminis-
rat model.(13–15) Tibolone blocks ovariectomy-induced bone
tered subcutaneously. Vehicle (gelatin, 0.5% wt/vol, and
loss by suppressing both bone resorption and bone turnover
in a similar manner to 17␣-ethinyl estradiol, suggesting it
acts like an estrogen on bone.(13,14,16)
mannitol, 5% wt/vol) and all other treatments were given
orally by gavage (0.25 ml) directly into the stomach from
the day of ovariectomy or sham operation onward. After 4
weeks of treatment, the animals were starved overnight for
16 h (with free access to demineralized water). During this
period, the animals were kept in metabolic cages to collect
urine. Then, the animals were anesthetized with ether and
blood was collected from the abdominal aorta. Heparin
plasma was prepared by centrifugation (3000g) for 10 min-
utes at 4°C and stored at Ϫ20°C for later analysis in two
batches. Trabecular BMD of the dissected right femur was
measured immediately after autopsy.
Antiestrogen experiment: Seventy-two animals were al-
located to 12 treatment groups, each of which received one
treatment regimen. Animals in 10 groups were OVX bilat-
erally and the remaining two groups were sham-operated.
OVX animals received oral doses of vehicle, tibolone
(125 ␮g/rat or 500 ␮g/rat twice daily), or 17␤-estradiol (64
␮g/rat or 250 ␮g/rat twice daily) either with or without ICI
164.384 (500 ␮g/rat twice daily). Sham-operated animals
received vehicle twice daily, with or without ICI 164.384
(500 ␮g/rat twice daily).
Antiandrogen experiment: Seventy-two animals were al-
located to 12 groups, each of which received one treatment
regimen. Animals in 11 groups were OVX bilaterally and
the other was sham-operated. Eight groups of OVX animals
received oral doses of tibolone (125 ␮g/rat or 500 ␮g/rat
twice daily) or testosterone-propionate (50 ␮g/rat or 200
␮g/rat twice daily) either with or without the antiandrogen
flutamide (2000 ␮g/rat twice daily). Two groups of OVX
animals received testosterone-propionate (50 ␮g/rat or 200
␮g/rat twice daily) coadministered with ICI 164.384 (1000
␮g/rat twice daily). Sham-operated animals and the OVX
control group received vehicle twice daily.
Antiprogestogen experiment: Forty-two animals were al-
located to seven groups. Animals in six groups were OVX
bilaterally and the other group was sham-operated. OVX
animals received oral doses of vehicle or tibolone (125
␮g/rat or 500 ␮g/rat twice daily) either with or without the
antiprogestogen Org 31710 (2000 ␮g/rat twice daily).
Sham-operated animals received vehicle twice daily.
Given the in vivo hormonal profile of tibolone, this study
evaluated whether the androgenic or progestogenic proper-
ties of tibolone contribute to its efficacy in preventing
ovariectomy-induced bone loss or whether the estrogenic
activity of tibolone is solely responsible. To study this, the
effects of tibolone alone and in combination with com-
pounds with antihormonal activity were investigated in the
OVX rat model of estrogen deficiency–induced bone loss.
Submaximal doses of tibolone were chosen so that any
stimulating effects by the antihormones also could be ob-
served; the tibolone doses result in comparable exposures of
tibolone and its metabolites to those in humans. The anti-
hormone ICI 164.384 was used as a pure antiestrogen with
no agonist activity(17); flutamide was used as an antiandro-
gen(18); and Org 31710 was used as an antiprogestogen.(19)
The hormones 17␤-estradiol and testosterone, alone and in
combination with their respective antihormones, were in-
cluded as a reference for the efficacy of the antihormones.
The trabecular bone mineral density (BMD) of the distal
femur and biochemical markers of bone resorption (urinary
deoxypyridinoline/creatinine ratio [Dpyr/Cr]) and bone
turnover (plasma osteocalcin) were all assessed.
MATERIALS AND METHODS
Materials
Suspensions of tibolone, 17␤-estradiol, Org 31710
((6␤,11␤,17␤)-11-[4-(dimethylamino) phenyl]-4Ј,5Ј-dihydro-
6-methylspiro[estra-4,9-diene-17,2Ј(3ЈH)-furan]-3-one), or flu-
tamide were made in 0.5% wt/vol gelatin and 5% wt/vol
mannitol. Testosterone-propionate and ICI 164.384 (7␣,
17␤-N-butyl-3,17-dihydroxy-N-methylestra-1,3,5(10)-triene-
7-undecanamide) were prepared in arachis oil. The steroidal
compounds were all synthesized by the Department of Medic-
inal Chemistry, NV Organon, Oss, The Netherlands.
Animals: Sexually mature 3-month-old, virgin female
Wistar rats (Hsd/Cpd:Wu strain; Harlan, The Central Insti-
tute for Breeding of Laboratory Animals, Zeist, The Neth-
erlands) weighing approximately 225 g were used. They
were housed individually under standard conditions of a
14-h light/10-h near-dark cycle in an air-conditioned room
at 21 Ϯ 2°C. The animals had free access to tap water and
were pair-fed with 16 g pelleted food daily (RMH-B diet;
Hope Farms, Linschoten, The Netherlands). All animal pro-
cedures were approved by the local Animal Ethics Com-
mittee.
BMD
At autopsy, the right femur of each animal was excised.
Trabecular BMD in the metaphyseal region (6 mm from the
distal end of the femur) was measured immediately using an
adapted peripheral quantitative computed tomography ma-
chine (Stratec XCT 960A; Stratec, Birkenfield, Germany).
A 360° X-ray scan (resolution 0.148 mm ϫ 0.148 mm) with
a thickness of 1 mm was taken. Intra- and interassay CVs
for the BMD measurements were 2–3%. The XCT-960A
Experimental design
The study comprised three independent experiments. In was calibrated with a standard of hydroxyapatite embedded
all three experiments, a randomized block design with body in acrylic plastic.

TIBOLONE INHIBITS BONE LOSS VIA ESTROGEN RECEPTORS
1653
FIG. 1. Effects of 4 weeks of treatment with tibolone or 17␤-
estradiol alone or in combination with the antiestrogen ICI 164.384 on
femoral trabecular BMD in 3-month-old OVX or sham-operated
(SHM) rats. Tibolone and estradiol doses refer to the total daily dose.
*p Յ 0.05 compared with OVX vehicle; #p Յ 0.05 compared with
corresponding treatment without antiestrogen (two-way ANOVA on
log-transformed data).
Biochemical markers of bone metabolism
Urinary Dpyr was determined by ELISA (Pyrilinks-D,
intra- and interassay CVs, 7.1% and 6.6%, respectively;
Metra Biosystems, Inc., Palo Alto, CA, USA), and urinary
Cr was determined by a standard autoanalyzer. Plasma
osteocalcin was determined by radioimmunoassay (intra-
and interassay CVs, 5.9% and 5.2%, respectively).(20)
FIG. 2. Effects of 4 weeks of treatment with tibolone or 17␤-
estradiol alone or in combination with the antiestrogen ICI 164.384 on
(A) urinary Dpyr/Cr and (B) plasma osteocalcin in 3-month-old OVX
or sham-operated (SHM) rats. Tibolone and estradiol doses refer to the
total daily dose. *p Յ 0.05 compared with OVX vehicle; #p Յ 0.05
compared with corresponding treatment without antiestrogen (two-way
ANOVA on log-transformed data).
Statistical analysis
Geometric means and SEs (SEMs) were calculated. Log-
transformed data were analyzed by two-way analysis of
variance (ANOVA) using SAS software (SAS Institute,
Inc., Cary, NC, USA). All of the groups were compared
with the OVX vehicle recipients; the anti–hormone-treated
groups were compared with the corresponding group with-
out antihormone. Values of p Յ 0.05 were considered
statistically significant.
the OVX group (Fig. 1). When the antiestrogen treatment
was given together with tibolone, 125 ␮g/rat (twice daily),
the protective effect of tibolone was inhibited completely
(p Յ 0.05); the protective effect of the higher dose also was
inhibited, although to a lesser extent (Fig. 1).
Addition of the antiestrogen to the 17␤-estradiol treat-
ment completely inhibited the protective effect of 17␤-
estradiol on ovariectomy-induced bone loss; the inhibition
was significant in the high-dose 17␤-estradiol group (p Յ
0.05) but not in the low-dose group, in which the protective
effect of 17␤-estradiol was small.
RESULTS
Antiestrogen experiment
Effects on BMD: Trabecular BMD of the distal femur
Effects on biochemical markers of bone metabolism: Four
decreased significantly (by 68%) in vehicle-treated OVX weeks after ovariectomy, the bone resorption marker, uri-
rats compared with vehicle-treated sham-operated animals nary Dpyr/Cr ratio, and the bone turnover marker, plasma
(p Յ 0.05; Fig. 1). OVX animals that were treated with osteocalcin, had increased markedly in OVX animals com-
either dose of tibolone had significantly greater trabecular pared with sham-operated controls (p Յ 0.05). Both doses
BMD of the distal femur than OVX vehicle recipients (p Յ of tibolone and of 17␤-estradiol significantly inhibited the
0.05). Tibolone, 125 ␮g/rat and 500 ␮g/rat (twice daily) OVX-induced increase in urinary Dpyr/Cr ratio (p Յ 0.05;
prevented 60% and 67% of the OVX-induced bone loss, Fig. 2A), indicating that bone resorption was reduced.
respectively. The OVX-induced loss of trabecular BMD Treatment of control animals with the antiestrogen alone did
also was prevented partially by 17␤-estradiol (24% at 64 not significantly affect the urinary Dpyr/Cr ratio. When the
␮g/rat twice daily and 45% at 250 ␮g/rat twice daily) but antiestrogen was added to the tibolone or 17␤-estradiol
the effect was only significant at the higher dose (p Յ 0.05 treatments, the urinary Dpyr/Cr ratios were increased. This
compared with the OVX vehicle recipients; Fig. 1).
was significant in both tibolone groups (p Յ 0.05). In the
Antiestrogen treatment reduced trabecular BMD in the low-dose tibolone and 17␤-estradiol groups, there was no
sham-operated animals compared with the corresponding longer a statistically significant difference compared with
vehicle recipients (p Յ 0.05) but did not significantly affect the OVX vehicle-treated group.

1654
EDERVEEN AND KLOOSTERBOER
The ability of tibolone to reduce plasma levels of osteo-
calcin was not significantly inhibited by cotreatment with
flutamide (Table 1). The higher dose of testosterone signif-
icantly suppressed plasma osteocalcin levels (p Յ 0.05); this
suppression was significantly blocked by coadministration
of flutamide (p Յ 0.05).
The coadministration of antiestrogen did not inhibit the
action of testosterone on either urinary Dpyr/Cr or plasma
osteocalcin, indicating that the effect of testosterone on
bone metabolism is not caused by extensive aromatization
of testosterone to estradiol.
FIG. 3. Effects of 4 weeks of treatment with tibolone or testosterone
alone and in combination with the antiandrogen flutamide or testoster-
one with the antiestrogen ICI 164.384 on femoral trabecular BMD in
3-month-old OVX or sham-operated (SHM) rats. *p Յ 0.05 compared
with OVX vehicle. Tibolone and testosterone doses refer to the total
daily dose. **p Յ 0.05 compared with corresponding treatment without
antiandrogen; #p Յ 0.05 compared with corresponding treatment with-
out antiestrogen (two-way ANOVA on log-transformed data).
Antiprogestogen experiment
Effects on BMD: Both doses of tibolone significantly
counteracted the effect of ovariectomy on BMD (p Յ 0.05);
the effect of tibolone was not reduced by coadministration
of the antiprogestogen (Fig. 4). In fact, coadministration of
the antiprogestogen induced a small increase in BMD in the
OVX placebo group as well as in both tibolone groups,
reaching statistical significance with the high-dose tibolone
group.
Both doses of tibolone and the high dose of 17␤-estradiol
significantly reduced plasma osteocalcin levels compared
with the OVX control group (p Յ 0.05; Fig. 2B). Addition
of the antiestrogen to tibolone or 17␤-estradiol treatment
increased osteocalcin levels in both tibolone groups and
both 17␤-estradiol groups, although the increase was only
significant with the lower dose of tibolone (p Յ 0.05). There
was also a significant increase in plasma osteocalcin in the
vehicle-treated OVX animals when they were given the
antiestrogen (p Յ 0.05), indicating an increase in bone
turnover.
Effects on biochemical markers of bone metabolism: Ti-
bolone significantly reduced both urinary Dpyr/Cr and
plasma osteocalcin at both doses. Coadministration of the
antiprogestogen did not appreciably change the effect of
high-dose tibolone, although the effect of low-dose tibolone
was reduced to levels that were no longer statistically sig-
nificant (Table 2).
DISCUSSION
The results of this study confirm that tibolone signifi-
cantly inhibits trabecular bone loss via an estrogenic path-
way in the OVX rat model of postmenopausal osteoporosis
Antiandrogen experiment
Effects on BMD: Both tibolone and testosterone- and exclude a major contributory role of its androgenic and
propionate prevented OVX-induced bone loss at both doses progestogenic activities. This is despite all three steroid
(p Յ 0.05). Cotreatment with the antiandrogen flutamide did receptors (androgen, estrogen, and progestogen) being
not block the protective effect of tibolone on bone loss in present in bone cells.
OVX rats (Fig. 3) but completely and significantly inhibited
In line with previous studies, tibolone significantly inhib-
the protective effect of testosterone (p Յ 0.05). Because ited the negative effects of ovariectomy on femoral trabec-
testosterone can be aromatized to estradiol in some tissues, ular BMD and on the biochemical markers of bone metab-
the antiestrogen also was coadministered with testosterone olism. Coadministration of the antiestrogen with tibolone
to determine whether there was an estrogenic component in generally counteracted the effects of tibolone. However,
the action of testosterone on bone. The addition of anties- with the high dose of tibolone the effect of the antiestrogen
trogen did not reduce the action of testosterone on BMD; was only significant in the case of the urinary Dpyr/Cr ratio,
instead, at the lower dose of testosterone, coadministration suggesting that the dose of antiestrogen may not have been
of antiestrogen significantly increased BMD.
high enough. The antiestrogen counteracted the effect of the
Effects on biochemical markers of bone metabolism: Flu- high dose of 17␤-estradiol on trabecular BMD, indicating
tamide did not abolish the effect of tibolone on the urinary that it was an effective antiestrogen with respect to bone
Dpyr/Cr. The protective effect of tibolone actually was metabolism. These results confirm previous evidence that
enhanced by the use of flutamide at the higher dose of the protective effect of tibolone against bone loss is com-
tibolone (Table 1), an effect that was consistent with the parable with that with estrogen.(21)
BMD observations in these animals (Fig. 3). Like tibolone,
As would be expected, the antiandrogen flutamide coun-
the higher dose of testosterone alone also significantly re- teracted the effects of testosterone on OVX-induced bone
duced OVX-induced increase in urinary Dpyr/Cr. This ef- loss and on the biochemical markers of bone resorption and
fect of testosterone was significantly blocked by cotreat- turnover. However, it did not counteract the effects of
ment with flutamide (p Յ 0.05), indicating an androgen tibolone on any of these markers. These results indicate that
receptor mediated response.
the androgenic activity of tibolone does not play a major

TIBOLONE INHIBITS BONE LOSS VIA ESTROGEN RECEPTORS
1655
␮
␮
FIG. 4. Effects of 4 weeks of treatment with tibolone alone or in
combination with the antiprogestogen Org 31710 on femoral trabecular
BMD in 3-month-old OVX or sham-operated (SHM) rats. Tibolone
doses refer to the total daily dose. *p Յ 0.05 compared with OVX
vehicle; #p Յ 0.05 compared with corresponding treatment without
antiprogestogen.
role in the protective action of tibolone against bone loss.
An interesting new observation is that testosterone can
prevent OVX-induced bone loss and that this effect is
mediated by the androgen receptor and not by the estrogen
receptor via aromatization. Recently, aromatization has
been reported to occur in osteoblast-like cells, which trans-
forms androgens into estrogens.(22) However, our experi-
ments with selective hormone receptor antagonists indicate
that aromatization does not play a major role in the testos-
terone effect. Tibolone itself does not undergo aromatiza-
tion.
␮
␮
Similarly, coadministration of an antiprogestogen did not
counteract the protective effect of tibolone on OVX-induced
bone loss and the bone markers, indicating that the proges-
togenic activity of tibolone does not have an appreciable
role in protecting against bone loss. A progestogen-only
group was not included in this experiment because we had
shown previously that pure progestogen does not prevent
OVX-induced bone loss or inhibit bone resorption.(23)
The tibolone doses used (125 ␮g and 500 ␮g twice daily)
were selected based on earlier experiments indicating that
these doses achieve a submaximal effect. In this study, these
doses only partly protected against OVX-induced bone loss;
this is consistent with our earlier experiments showing that
a dose of 2000 ␮g twice daily is required for complete
protection.(21)
The doses of the antihormones were selected based on
activities reported from in vivo bioassays (unpublished data
on file, NV Organon) and published literature.(17,18) How-
ever, our results indicate that the antiestrogen dose was
submaximal because, although it counteracted the effects of
both tibolone doses on trabecular BMD and on bone turn-
over markers, the effects were statistically significant only
with the lower dose of tibolone.
Rats and humans show generally similar responses of
trabecular bone to estrogen deficiency, including the devel-
opment of osteopenia, increased bone turnover, and preven-

1656
EDERVEEN AND KLOOSTERBOER
TABLE 2. EFFECT OF 4 WEEKS OF TREATMENT WITH TIBOLONE ALONE OR IN COMBINATION WITH THE ANTIPROGESTOGEN
ORG 31710 ON BIOCHEMICAL PARAMETERS OF BONE METABOLISM IN 3-MONTH-OLD RATS
Vehicle
Hormone
(sham operated)
Vehicle
Org 31710
Tibolone (250 ␮g)
Tibolone (1000 ␮g)
Antihormone
Vehicle
Vehicle
Vehicle
Org 31710
Vehicle
Org 31710
Urinary Dpyr/Cr (nM/mM)
Plasma osteocalcin (ng/ml)
82*
57*
153
90
172
91
97*
66*
117
84
56*
51*
66*
51*
All animals except those in the “sham-operated” group were OVX. Values expressed as geometric means.
* p Յ 0.05 compared with vehicle-only treatment of OVX rats (two-way ANOVA on log-transformed data).
study on 557 normal postmenopausal women. Calcif Tissue Int
41(Suppl 1):S1–S59.
2. Ettinger B, Genant HK, Cann CE 1985 Long-term estrogen
replacement therapy prevents bone loss and fractures. Ann
Intern Med 102:319–24.
3. Mosekilde L, Danielsen CC, Knudsen UB 1993 The effect of
aging and ovariectomy on the vertebral bone mass and biome-
chanical properties of mature rats. Bone 14:1–6.
4. Wronski TJ, Dann LM, Skott KS, Crooke LR 1989 Endocrine
and pharmacological suppressors of bone turnover protect
against osteopenia in ovariectomized rats. Endocrinology 125:
810–816.
5. Wronski TJ, Yen CF 1991 The ovariectomized rat as an animal
model for postmenopausal bone loss. Cells Mater (Suppl 1):
69–76.
6. Ismail F, Epstein S, Fallon MD, Thomas SB, Reinhardt TA 1988
Serum bone gla protein and the vitamin D endocrine system in the
oophorectomized rat. Endocrinology 122:624–630.
7. Kalu DN, Liu CC, Salerno E, Hollis B, Echon R, Ray M 1991
Skeletal response of ovariectomised rats to low and high doses
of 17␤-estradiol. Bone Miner 14:175–187.
8. Turner RT, Vandersteenhove JJ, Bell NH 1987 The effect of
ovariectomy and 17␤-estradiol on bone histomorphometry in
growing rats. J Bone Miner Res 2:115–122.
9. van der Vies J 1987 Pharmacological studies with (7␣, 17␣)-
17-hydroxy-7-methyl-19-norpregn-5(10)-en-20-yn-3-one (Org
OD14). Maturitas (Suppl 1):15–24.
10. Schot LPC, Kloosterboer HJ, Deckers GHJ 1990 Pharmaco-
logical profile of Org OD14 in experimental animals. Livial:
The Current Status of Research and Therapy. Proceedings of a
symposium held at the 6th International Congress on the
Menopause, Bangkok, Thailand, October 1990. Carnforth
(UK), Parthenon, pp. 11–23.
11. Bjarnason HN, Bjarnason K, Haarbo J, Rosenquist C, Chris-
tiansen C 1996 Tibolone: Prevention of bone loss in late post-
menopausal women. J Clin Endocrinol Metab 81:2419–2422.
12. Rymer J, Chapman MG, Fogelman I 1994 Effect of tibolone
on postmenopausal bone loss. Osteoporos Int 4:314–319.
13. Ederveen AGH, Spanjers CPM, Quaijtaal JHM, Kloosterboer
HJ 2001 Effect of 16 months of treatment with tibolone on
bone mass, turnover, and biomechanical quality in mature
ovariectomized rats. J Bone Miner Res 16:1674–1681.
14. Ederveen AGH, Kloosterboer HJ 1995 a Effect of long-term
treatment with Org OD14, its metabolites or 17␤-estradiol on
bone mass and bone turnover in aged ovariectomized rats.
J Bone Miner Res 10:S1;S452 (abstract).
tion of osteopenia by estrogen or bisphosphonate treat-
ment.(5) The OVX rat seems to provide a satisfactory model
for postmenopausal trabecular bone loss, particularly for the
critical early stages of estrogen deficiency. However, it has
some limitations; for example, modeling and longitudinal
bone growth occurs in the juvenile rat skeleton but not in
postmenopausal women.(5) The 52–69% reductions of fem-
oral trabecular BMD in OVX rats compared with their
sham-operated counterparts in the present experiments are
consistent with the expected reduction in trabecular BMD in
estrogen-deficient rats of 50–70% after 4 weeks of estrogen
deficiency.(21)
Tibolone has been shown to prevent OVX-induced tra-
becular bone loss from the peripheral and axial skeletons of
young as well as old OVX rats by suppressing both bone
resorption and bone turnover.(13–15)
Tibolone also suppresses skeletal metabolism and pre-
vents bone loss in postmenopausal women.(11,12) A dosage
of tibolone 2.5 mg daily for 2 years in recently postmeno-
pausal women prevents bone loss and increases spine and
femoral neck bone density.(12) The biochemical data from
this study indicated that tibolone inhibited both bone resorp-
tion and bone formation, suggesting that the effect of tibo-
lone is caused by a suppression of bone remodeling. Thus,
tibolone appears to have effects similar to those of estrogen
on bone biochemistry and density.(7,12)
Tibolone is known to have androgenic and progestogenic
activities in addition to its estrogenic activity,(9,10) all of
which might influence bone metabolism.(24) The results
presented in this study indicate that the protective effects of
tibolone against OVX-induced trabecular bone loss in rats
are caused by its estrogenic activity and are mediated by the
estrogen receptor. The androgenic and progestogenic activ-
ities of tibolone do not appear to play a major role in
preventing OVX-induced bone loss.
ACKNOWLEDGMENTS
This study was funded by NV Organon, Oss, The Neth-
erlands.
15. de Vries ECP, de Rijk EPCT, Ederveen AGH, Kloosterboer HJ
1994 The effect of Org OD14 on bone loss in young and old
ovariectomized rats. Eur J Endocrinol 89(Suppl 2):130 (ab-
stract).
16. Kasugai Y, Ikegami A, Matsuo K, Ohashi M, Sukamoto T,
Hosoi T, Ouchi Y, Orimo H 1998 Effects of tibolone (Org
OD14) treatment for 3 months on ovariectomy-induced os-
REFERENCES
1. Nordin BE, Polley KJ 1987 Metabolic consequences of the
menopause. A cross-sectional, longitudinal, and intervention

TIBOLONE INHIBITS BONE LOSS VIA ESTROGEN RECEPTORS
1657
teopenia in 8-month-old rats on a low calcium diet: Preventive 23. Ederveen AGH, Kloosterboer HJ 1995 A nonaromatisable
testing for 3 months. Bone 22:119–124.
17. Wakeling AE, Bowler J 1987 Steroidal pure anti-estrogens. J
Endocrinol 112:R7–R10.
androgen potentiates the effect of an estrogen on the preven-
tion of ovariectomy-induced bone loss in the rat. Calcif Tissue
Int 56:484 (abstract).
18. Raynaud JP, Ojasoo T 1986 The design and use of sex-steroid 24. Wakley GK, Turner RT 1991 Sex steroids and the regulation
antagonists. J Steroid Biochem Mol Biol 25:811–833.
19. Kloosterboer HJ, Deckers GH, Schoonen WGEF 1994 Phar-
macology of two new very selective antiprogestagens: Org
31710 and Org 31806. Hum Reprod 9:47–52.
20. Verhaeghe J, van Herck E, van Bree R, van Assche FA,
Bouillon R 1989 Osteocalcin during the reproductive cycle in
normal and diabetic rats. J Endocrinol 120:143–151.
21. Ederveen AGH, Kloosterboer HJ 1999 Tibolone, a steroid
with a tissue-specific hormone profile, completely prevents
ovariectomy-induced bone loss in sexually mature rats. J Bone
Miner Res 14:1963–1970.
of bone volume in the rat. Cells Mater (Suppl 1):85–91.
Address reprint requests to:
A.G.H. Ederveen, Ph.D.
Department of Pharmacology
Research and Development Labs
NV Organon
5340 BH Oss, The Netherlands
22. Ebeling PR 1998 Osteoporosis in men. New insights into
aetiology, pathogenesis, prevention and management. Drugs Received in original form September 6, 2000; in revised form
Aging 13:421–434. March 19, 2001; accepted April 4, 2001.