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Comparative Medicine Clinical Research Center, Wake Forest University School of Medicine, Winston-Salem, North Carolina 27157
Address all correspondence and requests for reprints to: Thomas B. Clarkson, D.V.M., Comparative Medicine Clinical Research Center, Wake Forest University School of Medicine, Medical Center Boulevard, Winston-Salem, North Carolina 27157-1040. E-mail: tclarkso{at}wfubmc
Abstract
This study compared the effects of tibolone, a tissue-specific compound for the treatment of climacteric symptoms and the prevention of osteoporosis, with those of conjugated equine estrogens (CEE) with and without medroxyprogesterone (MPA) on bone mineral density and coronary atherosclerosis (CAA) of postmenopausal cynomolgus monkeys. The groups were tibolone [two doses were used, 0.05 mg/kg (LoTib) and 0.2 mg/kg (HiTib)], CEE (0.042 mg/kg), CEE (0.042 mg/kg) plus MPA (0.167 mg/kg given continuously), and a control group given no treatment for 2 yr. Compared with no treatment, bone mineral density was higher by 6.3% (P = 0.0004) in the LoTib group and by 9.5% (P = 0.02) in the HiTib group compared with 4.3% (P = 0.12) for CEE and 4.5% (P = 0.10) for CEE+MPA. Plasma high density lipoprotein cholesterol was reduced by 49% with HiTib and by 34% with LoTib. There were no differences in CAA between control and HiTib (P = 0.60) or LoTib (P = 0.58). CEE and CEE+MPA both reduced CAA by about 62% (CEE vs. control, P = 0.02; CEE+MPA vs. control, P = 0.01). Despite adverse effects of tibolone on plasma lipoprotein concentrations, there was no increase in CAA, suggesting that tibolone is a cardiovascular-safe treatment for climacteric symptoms and the prevention of osteoporosis.
TIBOLONE
[(7
,17)-17-hydroxy-7-methyl-19-norpregn-5
(10)-en-20-yn-3-one)] has been used widely in several countries
for the treatment of menopausal symptoms (1, 2) and for
the prevention of postmenopausal osteoporosis (3, 4).
Among human and nonhuman primates, tibolone is metabolized into three
biologically active metabolites; the 3ß-hydroxy metabolite and
the 3-hydroxy metabolite have estrogen agonist properties, and the
4-ketoisomer has progestogenic and androgenic
effects. The 4 isomer, produced primarily
within the endometrium, protects the endometrium from the agonist
effects of the two estrogenic metabolites (5, 6).
Although tibolone treatment of postmenopausal women has some beneficial effects on plasma lipid/lipoprotein concentrations [reductions in plasma triglyceride (7, 8) and lipoprotein(a) [Lp(a)] concentrations (9, 10)], concern has arisen about its cardiovascular safety because of reductions in plasma concentrations of high density lipoprotein cholesterol concentrations (HDLC) after both short- and long-term treatment (11). Crona et al. (7) reported that tibolone treatment reduced HDLC by about 30%. The same magnitude of HDLC reduction following tibolone treatment was confirmed in a more recent study by Bjarnason et al. (11).
Using ovariectomized rabbits fed atherogenic diets, Zandberg and colleagues (12) compared the effects of treatment with E2 and tibolone on the extent of atherosclerosis. In that study oral tibolone at all three doses and sc administered E2 decanoate resulted in significantly less atherosclerosis than placebo treatment. However, unlike in women, the HDLC concentrations were not reduced in the tibolone groups, and total cholesterol was reduced by 50–70%. In previous studies performed by our group, we found that despite reductions in HDLC with oral contraceptive treatment, there was no exacerbation of coronary artery atherosclerosis (CAA) (13). Thus, the primary purpose of this study was to determine whether tibolone-induced reductions in HDLC resulted in worsened atherosclerosis.
In a preliminary study we determined that postmenopausal cynomolgus monkeys (Macaca fascicularis) shared with postmenopausal women decreases in HDLC after tibolone treatment (40–50% decreases were observed with monkeys). Reported herein are the results of a long-term study with surgically postmenopausal cynomolgus monkeys designed to evaluate the effects of tibolone on CAA and bone mineral density (BMD) and to compare those effects with the effects of conjugated equine estrogens (CEE) treatment, and treatment with CEE plus medroxyprogesterone (MPA) administered continuously.
Materials and Methods
Animals
One hundred and fifty-one premenopausal cynomolgus monkeys (Macaca fascicularis), 6–8 yr of age, were obtained through our collaborative association with the Institut Pertainian Bogor, Indonesia. During a preexperimental phase, the animals were evaluated for their health status, were ovariectomized to make them surgically menopausal, and were fed a moderately atherogenic diet for 10 wk. The diet contained about 19% of calories from protein (casein and lactalbumin), 42% of calories from fat (predominately saturated), 38% of calories from carbohydrate, and 0.28 mg cholesterol/cal. Baseline data, including plasma total cholesterol (TPC), HDLC, low density lipoprotein plus very low density lipoprotein (LDL+VLDL) cholesterol, triglycerides (TG), Lp(a) concentrations, apolipoprotein (Apo) A1, ApoE, and body weight, were collected 9 wk after ovariectomy (1 wk before the start of treatment). Plasma concentrations of E2 and progesterone were also monitored to document completeness of ovariectomy. All procedures involving animals were conducted in compliance with state and federal laws, standards of the U.S. DHHS, and guidelines established by the Wake Forest University institutional animal care and use committee.
Study design
The design of the study was a five-group, parallel-arm design
with the treatments lasting for 24 months. Social groups of animals
(n = 5/group) were randomly assigned to treatment using a permuted
block randomization scheme, with a block size of five. This was done to
minimize differences in time when treatments were initiated, as groups
were started into treatment on a staggered schedule of two social
groups every week. The composition of the diets fed and the amount of
hormones added to those diets are presented in Table 1
.
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0.75 mg; LoTib; n = 30), CEE
alone (Premarin, Wyeth-Ayerst Laboratories, Inc., Philadelphia, PA; n = 28) at a dose for the monkeys
comparable to a dose of 0.625 mg/d for women, or the same dose of CEE
plus MPA at a dose that would be comparable to 2.5 mg/d MPA given in a
continuous regimen (CEE+MPA; n = 29). The doses of the hormones
fed to the monkeys were based on the assumption that women in the
United States eat an average of 1800 kcal/d. Thus, a dose intended to
approximate a woman’s dose was added to 1800 kcal of diet. Monkeys
were fed 120 kcal of diet/kg BW and, therefore, took in about 0.2 or
0.05 mg/kg BW tibolone, 0.042 mg CEE/kg BW, or 0.167 mg/kg BW MPA. This
type of caloric adjustment of dose accounts for differences in
metabolic rates between the monkeys and human subjects. Measurements of body weight and plasma lipids/lipoproteins
Body weight measurement and plasma lipid determinations were made at quarterly intervals. After food was withheld for 18 h, blood was collected into evacuated tubes containing EDTA (final concentration, 1.0 g/liter) for TPC, HDLC, and TG analyses. TPC was measured by enzymatic techniques based on the methods of Allain et al. (14) Plasma TG concentrations were determined by the methods of Fossati and Principe (15). HDLC concentrations were measured using the heparin-manganese precipitation procedure described in the Manual of Laboratory Operations of the Lipid Research Clinics Program (16). The LDL and VLDL cholesterol was calculated as the difference between TPC and HDLC. All analyses were made using a COBAS FARA II autoanalyzer (Roche, Montclair, NJ). The laboratory subscribes to the Centers for Disease Control and Prevention (Atlanta, GA) Lipid Standardization Program.
Blood samples to determine concentrations of apoA-I, apoB, apoE, and Lp(a) were collected at 12-month intervals and put into evacuated tubes containing EDTA (1.5 g/liter, final concentration) and a protease inhibitor cocktail consisting of sodium azide (1.0 g/liter, final concentration), aprotinin (0.4 mg/liter, final concentration), and benzamidine (0.15 g/liter, final concentration). Food was withheld from the animals for 18 h before blood sample collection. ApoA-I (17), ApoB (18), and apoE (19) were quantified by ELISA methods previously described. Lp(a) concentrations were measured using modifications of the ELISA for determining apoA-I that was developed at our Lipoprotein Core Laboratory (20). All samples were analyzed in duplicate, and plasma pools were included with all assays.
Measurements of plasma concentrations of E2 and estrone sulfate
Two weeks before necropsy, serum for E2 and estrone sulfate determinations was collected from animals fasted for 18 h. These serum samples were frozen at approximately 20 C for later analysis. Estrone sulfate was determined directly in serum by RIA using a kit (DSL 5400) and protocols from Diagnostics Systems Laboratories, Inc. (Webster, TX). For E2 determinations, serum samples (0.5 ml) were first spiked with approximately 6000 dpm [3H]17ß-E2 (NET317, [2,4,6,7-N-3H]17ß-E2; SA, 70–115 Ci (2.96–4.25 TBq)/mmol; NEN Life Science Products, Boston, MA) and then extracted after the addition of ethyl ether (4 ml) and vortexing for 5 min. The aqueous layer was then frozen in a dry ice/isopropanol bath, and the organic phase was decanted. Extracts were dried and reconstituted with the zero standard serum from the RIA kit (DSL 4800, ultrasensitive E2, Diagnostics Systems Laboratories, Inc.), which was used for quantitation. E data were corrected for recovery of the internal standard. Intraassay variability was 10.1% for the estrone sulfate assay and 7.94% for the E2 assay.
Measurements of plasma concentrations of tibolone and its metabolites
After 17 months of treatment, plasma samples were obtained from
the tibolone-treated animals at 1, 1.5, 2, and 4 h after
administration of their dose. The samples were analyzed by
Organon. Tibolone is rapidly metabolized into the 3-
and 3ß-hydroxy metabolites and the 4 isomer.
For the determination of tibolone, an internal standard
(2H5 form of tibolone) was
added to the plasma samples immediately after the sampling to correct
for tibolone instability. For determination of the
4 isomer, the internal standard
(2H3 form of the
4 isomer) was added on the day of extraction.
Samples were extracted with n-hexane. The
n-hexane phase was transferred and evaporated to dryness.
The residue was redissolved in ethanol, evaporated to dryness, and
redissolved in isooctane, from which an aliquot was analyzed by
capillary gas chromatography-mass spectrometry. For determination of
the 3- and 3ß-hydroxy metabolites of tibolone, an internal
standard (2H5 form of the
3-hydroxy metabolite) was added to the plasma samples. The samples
were processed with C18 solid phase extraction,
and after Tri-Sil derivatization and reconstitution in water, they were
reextracted with n-hexane. The n-hexane phase was
transferred and evaporated to dryness. The residue was redissolved in
ethanol, from which an aliquot was analyzed by capillary gas
chromatography-mass spectrometry. Calibration curves were constructed
using weighed linear regression. From the calibration curves, the
concentrations in the study samples were calculated.
Measurements of bone density and bone mineral content (BMC)
Bone density was determined after 100 wk by dual energy x-ray absorptiometry (XR-26, Norland, Fort Atkinson, WI) as described previously (21). BMC (grams) and BMD (grams per cm2) were recorded from lumbar spine 1–5. All scans and their analyses were performed by the same technician.
Necropsy procedures and coronary artery atherosclerosis evaluations
After 24 months of postmenopausal treatment, the monkeys were
killed using sodium pentobarbital (100 mg/kg, iv), a method consistent
with the recommendations of the panel on euthanasia of the American
Veterinary Medical Association. The heart was removed, and the coronary
arteries were perfused for 1 h at 100 mm/Hg pressure using 10%
neutral buffered formalin (Fig. 1A). We took 15 blocks (each 3 mm
in length) cut perpendicular to the long axis of the arteries. Five of
these were serial blocks from the left circumflex (LCX), five were from
the left anterior descending (LAD), and five were from the right
coronary artery (RCA).
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B). The cross-sectional area of the coronary
artery intima, whether normal, containing fatty streaks, or with
plaques, was calculated for all 15 sections. Atherosclerosis extent was
measured as the cross-sectional area of plaque as described previously
(22). Intimal lesions were considered to be plaques if the
thickness was equal to or greater than half the thickness of the
underlying media. Measurements were made blind to treatment by a single
technician with more than 20 yr experience and were randomly
reevaluated by one of us (T. Clarkson). Statistical methods
Analyses were performed using BMDP statistical software (version 7.0, Los Angeles, CA) or Statistical Analysis software (version 6.08, SAS Institute, Inc., Cary, NC). All variables were first evaluated for their distribution and equality of variances between groups. Log transformations were performed for variables that violated the test of equal variances (intimal area, BMD, and BMC).
For data measured at baseline and multiple times during treatment, treatment period averages were used in the analyses because the data were stable across time. The baseline measure of that variable was used as a covariate. The average intimal area was calculated for each artery (mean across blocks) after verifying that the treatment effects were consistent for the length of the artery. The average coronary artery intimal area was calculated as the mean of the RCA, LAD, and LCX means.
CAA measurements were analyzed by ANCOVA using baseline LDL+VLDL cholesterol as a covariate, because it was significantly associated (r = 0.51; P < 0.0001) with outcome atherosclerosis. BMD and BMC were analyzed by analysis of covariance (ANCOVA), adjusting for baseline body weight, which was significantly associated with these outcome measures (BMC: r = 0.56; P < 0.0001; BMD: r = 0.41; P < 0.0001). Multiple regression analyses were performed to evaluate the association between body weight in response to treatment and the BMD and BMC measures.
The data in this report are the means and calculated SEs
retransformed into original units where necessary. One-way ANOVA and
ANCOVA were used to test for differences among groups. t
tests were used for post-hoc between-group comparisons if
the ANOVA or ANCOVA P value was significant. 
0.05 was used to determine statistical significance.
Results
Body weight and plasma lipid, lipoprotein, and Apo concentrations
The body weights of the monkeys in the trial, covaried for
baseline weight, are depicted in Fig. 2.
CEE treatment did not result in increased body weights (2.82 ±
0.04 kg), whereas the addition of MPA to the CEE treatment resulted in
a small body weight increase (2.98 ± 0.04 vs.
2.84 ± 0.04 kg for controls; P = 0.01). Both of
the tibolone-treated groups had greater body weights. The body weight
of the LoTib group was 3.02 ± 0.04 kg (P = 0.002
vs. control), and that of the HiTib group was 3.19 ±
0.04 kg (P = 0.001 vs. control).
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(means for quarterly determinations). All of the data, except those for
apoB, were covaried for baseline values (there were no baseline values
for apoB). Both CEE and CEE+MPA treatment resulted in significantly
lower total plasma cholesterol concentrations (P =
0.002), whereas the total plasma cholesterol concentrations of the two
tibolone-treated groups were not significantly different from the
control value (LoTib and HiTib, P = 0.68 and 0.12,
respectively). Both CEE- and CEE+MPA-treated monkeys had increased
plasma TG concentrations (P = 0.01 and
P = 0.002, respectively). The LoTib-treated monkeys did
not have increased plasma TG concentrations (P = 0.29),
whereas those of the HiTib group were increased (P <
0.0001).
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Of key interest in this study was the effects of the treatments on
plasma concentrations of HDLC. CEE and CEE+MPA treatments resulted in
slightly lower (10%) HDLC compared with the controls. Both LoTib-
and HiTib-treated monkeys had much lower HDLC compared with the control
monkeys (34% and 49% lower than controls; P < 0.0001
for both groups). The ratio of TPC/HDLC was not different for the CEE-
and the CEE+MPA-treated monkeys compared with controls
(P = 0.48 and P = 0.40, respectively).
In contrast, there was a dose-related increase in the TPC/HDLC ratio in
the tibolone-treated groups, an increase compared with controls of 44%
among the LoTib group (P = 0.009) and an increase of
114% among the HiTib group (P < 0.0001).
There were no significant group differences in plasma concentrations of apoB, although the point estimate for the HiTib group was about 24% higher than that for the control group. (There were no baseline apoB measurements made, so the data could not be covaried for baseline concentrations.) Consistent with the low HDLC, plasma concentrations of apoA1 were markedly lower in the two tibolone-treated groups (P = 0.002 for LoTib and P < 0.0001 for HiTib). There were no significant effects of CEE (P = 0.34 vs. control) or CEE+MPA (P = 0.39 vs. control) on apoA1. Plasma apoE concentrations were reduced in all treatment groups. Neither CEE nor CEE+MPA altered plasma concentrations of Lp(a), whereas there were small increases in the tibolone groups, which were more pronounced in HiTib than LoTib.
Plasma concentrations of E2 and estrone sulfate
The plasma concentrations (18 h after the last dose) of E2 and
estrone sulfate are summarized in Table 3. As expected, the two groups that
received CEE had significantly increased concentrations of E2 and
estrone sulfate, whereas the two tibolone-treated groups were not
different from the control group.
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The maximum concentration of the tibolone metabolites in blood
samples collected 1 h after treatment are presented as the
measurement of plasma concentrations for the monkeys in this study
(Fig. 3).
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Significant differences in BMD and BMC were found among treatment
groups when treatment values were adjusted for baseline body weight
(Table 4). Both low and high tibolone
treatment groups had significantly higher BMD and BMC than controls.
The CEE and CEE+MPA groups had higher BMD and BMC than the control
group, but this was only of borderline significance for BMD (CEE,
P = 0.12; CEE+MPA, P = 0.10) and BMC
(CEE+MPA, P = 0.13).
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The mean cross-sectional area of the coronary artery intima is
depicted in Fig. 4A. A coronary artery
intimal area value based on a mean of 15 sections (5 from LCX, 5 from
LAD, and 5 from RCA) was calculated for each of the animals. The data
presented in Fig. 4A are the means of those values (±SE)
for the control and each of the treatment groups. Both CEE and CEE+MPA
treatments resulted in markedly reduced intimal areas [CEE, 62%
smaller (P = 0.02); CEE+MPA, 62% smaller
(P = 0.01)]. The intimal areas of the tibolone-treated
groups were not different from those of the control group (LoTib,
P = 0.58; HiTib, P = 0.60).
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is a subanalysis showing the cross-sectional areas of
atherosclerotic plaques (defined as intimal thicknesses equal to or
greater than half the thickness of the underlying media). Among the
control group, 21 of 31 monkeys (68%) had atherosclerotic plaques
present in any of the coronary arteries. Among the CEE-treated
animals, there were 17 of 27 (63%) affected, among CEE+MPA 16 of 29
(55%) were affected, among LoTib 24, of 30 (80%) were affected, and
among HiTib, 21 of 31 (68%) were affected. These differences in
prevalence were not statistically significant, nor were differences in
plaque size (by ANCOVA, P = 0.06) in this subgroup.
However, among CEE-treated animals with coronary artery
atherosclerosis, plaques were 60% smaller compared with controls,
whereas the addition of MPA to CEE treatment attenuated somewhat the
CEE inhibitory effect on atherosclerosis (plaques only 34% smaller
than controls). There was no difference in plaque size in the 2
tibolone-treated subgroups. (LoTib, plaques 24% smaller than controls;
HiTib, plaques 3% larger than controls).
A major objective of this study was to explore if and to what extent
the lower plasma concentrations of HDLC among the tibolone-treated
groups were associated with exacerbation of CAA. Although LoTib and
HiTib treatment resulted in lower plasma HDLCs by 34% and 49%,
respectively, the extent of CAA was not significantly different from
that in the control group. To better understand these findings, we
plotted each monkey’s CAA cross-sectional area against its average
HDLC during treatment and compared that with the same plot for the
control group (Fig. 5, A and B). These
plots show that at the same HDLC concentration, the tibolone groups
have, on the average, less atherosclerosis than the control group.
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The primary objective of this study was to determine whether
reductions in plasma concentrations of HDLC resulting from tibolone
treatment were associated with any exacerbation of diet-induced CAA in
the cynomolgus monkey model. Despite the finding that tibolone
treatment increased plasma concentrations of VLDL+LDL cholesterol (in
HiTib group) and markedly reduced plasma concentrations of HDLC, there
was no exacerbation of CAA compared with the control group. This
finding may be an underestimate of tibolone’s potential for inhibiting
CAA in human subjects, both because the decreases in HDLC were greater
than those seen in human subjects and because human subjects do not
increase LDLC in response to tibolone treatment. Moreover, the monkeys
show an increase in TG levels after tibolone treatment, whereas a
decrease is found in humans (7, 11). CEE treatment was
associated with a reduced amount of CAA in this study, consistent with
what we observed in other studies (23, 24). Our finding of
a lack of CAA exacerbation associated with the tibolone-induced
reductions in HDLC is consistent with our earlier studies, in which a
decrease in HDLC in monkeys treated with contraceptive steroids also
did not exacerbate CAA (13). In our report on that study
we described the plasma lipid-independent effects of ethinyl
E2 that protected the coronary arteries from the deleterious
effects of HDLC reductions. The data obtained in this study with
tibolone would suggest a similar mechanism, with the possibility that
the 3- and 3ß-hydroxy estrogenic metabolites are protecting the
coronary arteries from the low HDLC.
That speculation is consistent with the finding of what appears to be a plasma lipid-independent atherosclerosis protective effect of tibolone in the rabbit model (12). A hypothesis in that study was based on the current understanding of the mechanisms by which E2 inhibits atherosclerosis progression; only about 25% of its effects are on plasma lipid concentrations, and 75% of its effects are plasma lipid independent, presumably by direct effects on arterial metabolism/function (25, 26). A hypothesis was that if tibolone’s estrogenic metabolites had the same beneficial effects as E2 on arterial metabolism/function, arteries should be protected to some extent from the atherogenic lipid concentrations. Using the plasma cholesterol concentrations, one can calculate the predicted arterial cholesterol concentration with the differences between predicted and observed indicating the extent of the benefits to the artery wall of the treatments. Using this approach, E2-treated animals had values 47% less than those predicted, and tibolone-treated animals had values 54% less than those predicted, suggesting that tibolone’s direct arterial benefits might protect against lowered plasma concentrations of HDLC.
We have no metabolic explanation about why high dose tibolone treatment increased the LDL cholesterol of the monkeys whereas it has not been shown to do so among tibolone-treated women. It probably relates to the fact that the hyperlipoproteinemia in the monkey subjects was induced by dietary cholesterol, whereas the LDL cholesterol concentrations of women are only weakly diet dependent and are largely determined by endogenous factors.
Given that tibolone treatment had no adverse effect on coronary artery atherosclerosis of cynomolgus monkeys, it may be that human subjects who do not experience tibolone increases in LDL cholesterol may obtain beneficial effects on coronary artery atherosclerosis. The coronary artery findings also must be considered in relation to the effects of the two interventions on the mammary gland and the endometrium. As a part of this same trial, Cline et al. (27) found that tibolone did not stimulate either the endometrium or breast of the cynomolgus monkeys treated with low or high tibolone, whereas CEE treatment increased both endometrial proliferation and mammary gland epithelial area. CEE+MPA treatment partially inhibited the endometrial effects of the CEE, but tended to increase the mammary epithelial area.
It has been reported previously by our group that the coadministration of MPA (at a dose for monkeys equivalent to 2.5 mg/d continuous for women) markedly attenuated the CEE-associated inhibition of the development of CAA (28). In the present study there is no adverse effect on CAA inhibition by the addition of MPA. If one considers the plaque area of only those animals having plaques, there is a small attenuating effect of MPA added to CEE. However, if one considers atherogenesis to be a continuous process by which treatment might inhibit the rate of progression to atherosclerotic plaques (defined as intimal thickness greater than half the medial thickness), then exclusion of those cases without plaques removes from the analysis those that are most protected. We have endeavored to understand the differences in the outcome of these two studies. In our previous study, reported by Adams et al. (28), CEE alone and CEE+MPA were given as a part of the diet fed in one meal per d. In this current study the amounts of CEE and MPA, while identical to those in the first study, were given in divided doses, again as a part of a meal, half in the morning and half in the afternoon. The Ki67 labeling in the superficial (functional) zone of endometrial glands in the monkeys in the first study by Adams et al. (28) has recently been evaluated by Cline et al. (29). In that study there was about an 18% increase in Ki67 labeling (indicating cellular proliferation) among the animals with CEE treatment and that was reduced to about 0.6% labeling by the coadministration of MPA. In the study reported here, Cline et al. (27) also evaluated Ki67 labeling in the same area of endometrial glands. In this current study CEE resulted in a 45% increase in Ki67 labeling in the endometrium, and coadministration of MPA only reduced Ki67 labeling to 20%. It is our best interpretation that giving MPA as a single dose was more effective in inhibiting endometrial proliferation than giving CEE+MPA in two divided doses. Therefore, it seems that the estrogenic stimulus was higher and the progestogenic effect was reduced in the present study compared with that by Adams et al. (28). The differences between the two studies might suggest that when MPA has the desired antagonistic effect on the endometrium, it attenuates the cardiovascular benefits of CEE. When the progestogenic potency is reduced, the undesirable effects on atherogenesis are reduced.
The tibolone-treated groups had significantly higher BMC and BMD relative to the control group, whereas the CEE or CEE+MPA groups were not significantly different. Consistent with BMC and BMD measures, the bone strength of the midshaft femur was also greater in tibolone-treated groups relative to the control group, whereas the CEE and CEE+MPA groups were not significantly different (30). These finding are consistent with observations reported by Kasugai et al. (31) and Yoshitake et al. (32), who found that tibolone treatment of ovariectomized rats fed a low calcium diet inhibited bone loss. Yoshitake et al. (32) also found that tibolone treatment improved bone mechanical and histomorphometric indexes of osteopenia. As body weight was increased by tibolone treatment, and higher body weight may increase BMD by the increased load-bearing, multiple regression analysis was performed to determine whether there were treatment effects on BMD and BMC that were independent of effects on body weight (i.e. residual effects of treatment). Treatment body weight accounted for 33% of the variability in BMD and 52% of the variability in BMC. After adjusting for body weight measured at the time of dual energy x-ray absorptiometry, there were no longer significant differences between treatment groups, suggesting that there were not significant effects of treatment on BMD or BMC that were independent of the effect on body weight. As improvements in BMD and BMC occur together with increased body weights in our monkey study, we cannot separate the two phenomena. Lippuner et al. (4) compared the effects of tibolone treatment with those of traditional hormone replacement therapy (2 mg/d E2, orally, plus sequential oral dydrogesterone, 10 mg/d for 14 d of a 28-d cycle) and transdermal hormone replacement therapy (E2 by patch releasing 50 µg/d and with the same sequential dose of oral dydrogesterone as the oral group) for prevention of bone loss in postmenopausal women. It was found that tibolone treatment was generally equivalent to either conventional oral hormone replacement therapy or transdermal hormone replacement therapy for the prevention of bone loss and was without effect on body mass index. That clinical observation would suggest that the prevention of bone loss occurs independently of effects on body mass; thus, in the current study one should not conclude a causal relationship between body weight and bone density resulting from tibolone treatment.
The bone-protective effect of tibolone observed in this study was most
likely related to the estrogenic effects of the 3- and 3ß-hydroxy
metabolites. Ederveen and Kloosterboer (33), in studies
with ovariectomized rats, found that the bone-protective effect of
tibolone could be blocked by the coadministration of an anti-E, but not
by an antiandrogen or antiprogestogen.
The increase in body weights of the tibolone-treated monkeys was of interest to us. Unfortunately, we cannot rule out small differences in calories consumed. Using dual energy x-ray absorptiometry, Shadoan et al. (34) studied the body composition of the monkeys in this study. Among both LoTib and HiTib groups the increase in body weight was due to a significant increase in lean mass (P = 0.001) and a nonsignificant increase in fat mass (P = 0.32). These findings are consistent with a report from Hanggi et al. (35), who found that tibolone treatment of postmenopausal women increased lean body mass, but not body fat mass.
There are no precise plasma measurements that can be used to monitor the dose of CEE used. We added CEE to the diet on a caloric basis, intending to mimic a dose for women of 0.625 mg/d. Previously, we reported plasma concentrations of E2 in cynomolgus monkeys given that dose of CEE to be 600–640 pmol/liter (23). We now know that those concentrations were erroneously high because components of CEE interfere with the direct RIA for E2. In the study reported here the sera from CEE-treated monkeys were extracted before E2 assay to remove interfering CEE components. Using extraction before assay, we found the plasma E2 concentrations of the CEE-treated monkeys to be 58–65 pmol/liter. Although various estimates are given for E2 concentrations in CEE-treated women, Lobo (36) presents a literature consensus of 145 pmol/liter for those given 0.625 mg/d and 70 pmol/liter for those given 0.3 mg/d. These concentrations for women were determined at variable times after the medication was taken. Our observations of the monkeys were made 18 h after administration of the drugs, suggesting that our dose of CEE was probably greater than a comparable dose for women of 0.3 mg and certainly was not greater than a dose of 0.625 mg/d.
Lobo (36) has also summarized the plasma estrone sulfate concentrations of women given 0.625 mg/d oral estrone sulfate. A plateau of about 57 nmol/liter estrone sulfate was found at 4 h and remained essentially unchanged up to 8 h. In our cynomolgus monkeys we found plasma concentrations of estrone sulfate of about 20–24 nmol/liter after an overnight fast, again suggesting that our CEE dose was on the low side of 0.625 mg/d CEE.
We chose the dose of the LoTib and the HiTib groups to approximate the
range of tibolone doses given to women (1.25–2.5 mg/d). Based on the
monkey pharmacokinetic data, we believe that this objective was
achieved. The maximum concentrations of the 3-hydroxy metabolite of
the monkeys were about 5 and 7 ng/ml after 1 h compared with those
in women of 7 and 14 ng/ml after 1.25 and 2.5 mg tibolone, respectively
(Meuleman, D., personal communication). For the 3ß-hydroxy
metabolite, we found levels of 1.8 ng/ml (LoTib) and 4.2 ng/ml (HiTib)
compared with those in women of 2.0 ng/ml (1.25 mg) and 3.7 ng/ml (2.5
mg; Meuleman, D., personal communication). There was very good
agreement with the 4 isomer. We found levels
of 0.25 ng/ml (LoTib) and 0.48 ng/ml (HiTib) compared with those in
women of 0.26 ng/ml (1.25 mg) and 0.41 ng/ml (2.5 mg; Meuleman, D.,
personal communication).
In summary, the observations made in this long-term trial with surgically postmenopausal cynomolgus monkeys provide evidence that the effects of tibolone on bone are comparable to those of CEE and CEE+MPA and may have advantages over CEE and CEE+MPA for breast and endometrial safety, but do not share CEE’s benefits on CAA. There was, however, no exacerbation of atherosclerosis with tibolone treatment, even though there was a reduction in HDLC concentrations that was similar to or greater than that seen in women.
Acknowledgments
We thank Dick Meuleman, Ph.D. (Organon), for his scholarly and thoughtful input at many stages of this study. We also thank Matthew Dwyer, Timothy Vest, Maryanne Post, Misti Binns, Sam Rankin, and Joel Collins for their excellent technical contributions.
Footnotes
This work was supported in part by a grant from Organon (Oss, The Netherlands). This work was presented in part at the Experimental Biology 2001 Meetings, Orlando, Florida, March 31-April 4, 2001, and in abstract form (Proc Soc Exp Biol Med, 2001, in press).
Abbreviations: ANCOVA, Analysis of covariance; Apo, apolipoprotein; BMC, bone mineral content; BMD, bone mineral density; CAA, coronary atherosclerosis; CEE, conjugated equine E; HDLC, high density lipoprotein cholesterol; HiTib, 0.2 mg/kg tibolone; LAD, left anterior descending; LCX, left circumflex; LDL, low density lipoprotein; LoTib, 0.05 mg/kg tibolone; Lp(a), lipoprotein(a); MPA, medroxyprogesterone; RCA, right coronary artery; TG, triglycerides; TPC, total plasma cholesterol; VLDL, very low density lipoprotein.
Received March 21, 2001.
Accepted August 2, 2001.
References
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) and LoTib
group (
). B, Coronary artery intimal area (square millimeters)
plotted against the average HDL cholesterol level (millimoles per
liter) during the treatment period for the control group
(
). In both panels, linear
regression lines are fitted to the data (control, solid
line; LoTib or HiTib, broken line). The
differences in the regression lines suggest that the tibolone-treated
groups have, on the average, less atherosclerosis than the control
group when they have similar HDL cholesterol concentrations.