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Tibolone: a steroid with a tissue-specific mode of action
Journal of Steroid Biochemistry & Molecular Biology 76 (2001) 231– 238 www.elsevier.com/locate/jsbmb
Tibolone: a steroid with a tissue-specific mode of action H.J Kloosterboer * NV Organon, Research and De6elopment Laboratories, P.O. Box 20, 5340 Oss, The Netherlands
Abstract In postmenopausal women tibolone has proved to prevent bone-loss and relieve climacteric symptoms as effectively as estrogens, but it does not stimulate the endometrium and the breast. This clinical profile strongly suggests that tibolone is a compound with tissue-specific action. Tibolone is quickly metabolized into its main active metabolites, 3a and 3b-OH, which are also present in an inactive, sulphated, form. In addition a D4-metabolite is found in circulation. The 3-OH-metabolites bind only to the estrogen receptor while the D4-isomer shows affinity only to the progesterone and androgen receptors. Tibolone prevents bone loss in a similar way to estrogens. Studies on bone mass using anti-estrogen, antiprogestin and anti-androgen in combination with tibolone, confirmed the sole involvement of the estradiol receptor. Increases in skin temperature as well as vaginal atrophy can be prevented by tibolone in a similar way to estrogens. Breast safety studies showed that tibolone clearly inhibited the growth of tumors in a DMBA model. In breast cell lines, tibolone profoundly inhibited sulphatase activity and an increase in apoptosis and decrease in cell proliferation was found. The stimulation of the endometrium is prevented by the local formation of the D4-isomer from tibolone or the 3b-OH-metabolite. We conclude that tibolone acts as a tissue-specific compound by mediating its effects via steroid receptors and enzymatic pathways. This dual effect of tibolone explains it’s positive clinical effects on bone, vagina and brain, and avoids stimulation of the endometrium and breast tissue. © 2001 Elsevier Science Ltd. All rights reserved. Keywords: Tibolone; Steroid receptors; Steroid metabolism; Inhibition; Bone; Sulphatase; Breast; Endometrium; Climacterium; Cardiovascular system
1. Introduction For many years, tibolone has been used for the treatment of climacteric complains and prevention of osteoporosis in postmenopausal women without stimulating the breast and endometrium. This tissue-specific effect of tibolone is difficult to explain from the results of preclinical studies using receptor studies with tibolone itself or tests in classical animal models [1,2]. Tibolone is a steroid with a 3-keto-D5 – 10 configuration, a 7a-methyl substituent and a 17a-ethinyl group. The compound does not have structural characteristics that would explain its estrogenic-like effects on vagina, brain and bone. An aromatic A-ring and a 3-hydroxyl Proceedings of the 14th International Symposium of the Journal of Steroid Biochemistry and Molecular Biology ‘‘Recent Advances in Steroid Biochemistry and Molecular Biology’’ (Quebec, Canada 24 – 27 June 2000). * Tel.: +31-412-662452; fax: +31-412-662514. E-mail address: [email protected] (H.J. Kloosterboer).
group are lacking, which are typical requirements for binding to the estradiol receptor. The estrogenic effects of tibolone as observed on the vagina in the Allen Doisy-test may, therefore, be due to the formation of estrogenic metabolites [2]. In addition to the estrogenic activity, the compound has been shown to possess also progestagenic and androgenic properties in specific models for these activities. The progestagenic activity is found to be low in the McPhail test, which is most likely due to the simultaneous presence of estrogenic activity. The final activity in a tissue of a compound is not only determined by the exposure to the compound itself, but also by the hormonal activity of the metabolites in circulation after passing through the intestine and liver. In addition, intracellular metabolism is important for determining which steroid receptors in a tissue become activated [3]. Ultimately, it is the cellular context including number of receptors as well as the set of co-activators and co-repressors [4] and possibly other non-genomic pathways [5] which determines whether a tissue becomes responsive to treatment. In this review the mode of action of tibolone is described using kinetic
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data, receptor results for tibolone and its metabolites together with its effects in various relevant in vitro and in vivo models showing tibolone’s effects on specific tissues relevant to hormone replacement therapy. The results of these studies clearly explain the tissue-specific effect as seen in the clinic.
2. Kinetics and metabolism After tibolone is administered to women, the compound is quickly metabolised into 3a- and 3b-OH-tibolone by the enzymes 3a/b hydroxysteroid dehydrogenase in the intestine and the liver (see Fig. 1). The metabolism as depicted in the figure has been confirmed with in vitro [6] and in vivo studies. The two OH-metabolites have a half-life of approximately 7 h (data on file). In addition to these two metabolites, a third metabolite — the D4-isomer — is found in the circulation for a short period of time but no half-life could be estimated. This metabolite is formed from tibolone directly by the enzyme 3b-HSD-isomerase or from the OH-metabolites. Only the 3b-OH-tibolone can be a substrate for the enzyme but it is theoretically possible that the D4-isomer is formed from the 3ametabolite via tibolone as these enzymes can act in either directions (see Fig. 1). Kunhz et al. [7] could also not determine a half-life for estradiol after oral administration of 2 mg of estradiol to fertile women, but were able to determine a half-life for estrone, a weak estrogen. However, other investigators [8,9] found a clear half-life for estradiol in postmenopausal women. Estradiol is quickly metabolized, mostly to estrone; in addition to that a large pool of sulphated estrone was found in circulation [10].
The metabolite profile for tibolone shows similarities to that of norethynodrel [11,12]. However, there are differences due to the presence of a 7a-methyl group in tibolone, which means that 5a reduction of the D4-isomer does not occur [13]. This is important for tissues like the endometrium where progestagenic activity is required to inhibit proliferation. This means that the progestagenic activity remains in the endometrial tissue after initial exposure, but in addition to this, local formation of the D4-isomer from the 3OH-metabolites may continue (see Fig. 1). A study with labeled tibolone has shown that the majority of the metabolites are in the sulphated (inactive) form [14]. The similarity between tibolone and estradiol is striking; both are quickly metabolized to form weak estrogenic compounds and a large pool of sulphated compounds is present. This sulfated pool serves as a source from which active estrogenic metabolites are continually formed by an enzyme, sulphatase, which resides in virtually all tissues. The extent to which this occurs may depend on local enzyme activity and tissue-specific metabolism is an important determinant for drug effects in a tissue. Notably Tang et al. [15] have found that tibolone is exclusively converted into the D4-isomer in endometrial tissue fragments.
3. Receptor activation of tibolone and its metabolites The binding affinities of tibolone and its metabolites to the human estrogen, progesterone and androgen receptor have been assessed using cell lines in order to predict their ultimate tissue responses. The results of the binding studies are depicted in Table 1. Surprisingly, tibolone itself binds to all three receptors al-
Fig. 1. Scheme of metabolism of tibolone to its (active) metabolites. 3a/b-HSD, 3a/b-hydrosteroid dehydrogenase; 3bHSD-iso, 3b-hydroxysteroid dehydrogenase/isomerase.
H.J. Kloosterboer / Journal of Steroid Biochemistry & Molecular Biology 76 (2001) 231–238 Table 1 Binding affinities of tibolone and its metabolites to human nuclear progesterone, androgen and estrogen receptor in MCF-7 cells relative to reference compounds ( =100%)a
Tibolone 3a-OH-metabolite 3b-OH-metabolite D4-isomer
ER (E2 = 100%)
PR (Org 2058= 100%)
AR (DHT= 100%)
1.3 3.2 1.7 n.c.
4.9 n.c. n.c. 12.9
3.2 n.c. n.c. 39.2
a E2, estradiol; DHT, dihydrotestosterone; Org 2058 is a pure synthetic progestin; n.c., no competition.
Fig. 2. Effect of ethinyl estradiol (EE2) and tibolone on bone mineral density (pQCT) in young ovariectomized (OVX) rats after oral treatment for 4 weeks. Mean values 9S.E.M. are given.
though it does not contain the essential structural requirements for binding to these receptors: a phenolicgroup for the estrogen receptor and the 3-keto-D4 structure for the progesterone and androgen receptor. The 3OH-metabolites bind solely to the estrogen receptor. The observed binding of tibolone may at least partly be due to metabolism under the assay conditions used. The affinity of the 3OH-metabolites is low but in transactivation experiments (data not shown) using cells transfected with human estrogen receptor and a promoter–reporter construct, the activity of the OHmetabolites is in the nmol range but tibolone can only do that at 1000 times higher concentrations. Based on the results of the transactivation assay one may conclude that binding studies with the mother compound are of limited value. The D4-isomer shows a clear binding to the progesterone receptor, which is comparable to that of natural progesterone. Transactivation assays with human steroid receptors have shown that the D4-isomer activates both the progesterone receptor and the androgen receptor but not the estrogen receptor [16].
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4. Effects on bone Bone loss belongs next to climacteric complaints as one of the earliest symptoms of the menopause. In contrast to the climacterium women do not immediately experience demineralisation of bone in the menopause as a negative aspect because osteoporosis only becomes manifest when actual fractures occur. In the menopause, bone loss is due to an increase in bone resorption and this effect can be prevented by the use of tibolone [17–19], estrogens [20], SERMs [21] or bisphosphonates [22]. Several studies have shown tibolone to have beneficial effects on bone. In ovariectomized rats, tibolone prevented bone loss in a dose-dependent manner when treatment was started immediately after removal of the ovaries (see Fig. 2). The same level in bone mass density can be reached with tibolone as with an estrogen. Desoxypyridinolin and osteocalcin, bone markers for resorption and formation respectively, remain at normal levels indicating that bone turnover remains normal in ovariectomized treated animals treated with tibolone [23]. In bone cells, estrogen, progestin and androgen receptors are present and it is, therefore, important to investigate which hormonal activity of tibolone is responsible for its beneficial effects on bone. This was investigated by determining whether specific antihormones could prevent the positive effects of tibolone on bone. The anti-estrogen ICI 164.384 prevents tibolone’s action on bone mass while the anti-androgen flutamide and the anti-progestin Org 31710 do not modulate the effects of tibolone [24]. Biochemical bone markers in animals treated with the combination of tibolone and anti-estrogen respond as expected and levels are similar to those following ovariectomy. These anti-hormone studies show that tibolone’s action on bone is mediated via the estrogen receptor. Bone quality under tibolone is not impaired as indicated by compression or the three-bending test. Bone quality remains similar to that as in intact animals [25]. Typical experiments have shown that tibolone acts as a bone resorption inhibitor in a similar way to estrogens.
5. Effects on the climacterium Vaginal atrophy is an early symptom of estrogen deficiency and can be counteracted by the administration of estrogens. This positive effect of estrogens on the vagina can be diminished by progestins. Tibolone displays a full range of estrogenic effect on the development of vaginal epithelial (see Fig. 3). The enzyme 3b-HSD-isomerase can convert tibolone and the 3bOH-metabolite to the D4-isomer, but from the full estrogenic response seen, it can be concluded that activity of the enzyme is low or maybe even absent in the
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vagina. Clinical studies [26,27] do show that the vagina and the endometrium differ in their response to tibolone; a clear estrogenic response is found in the vagina but the endometrium remains atrophied in the majority of women. When menstrual cycles become irregular in the menopause, strong fluctuations in estrogen levels are probably the cause of hot flushes and mood swings. These phenomena can be prevented not only by estrogens but also by tibolone [26,27]. In both preclinical models [28,29] and clinical studies [26,27], it has been shown that tibolone behaves similarly to estrogens in preventing hot flushes. Genazzani et al. [30,31] have shown that tibolone increases endorphin levels in the pituitary and in plasma and it has been suggested that this may be related to the improvements in mood seen with tibolone in the clinic. Mood improvement can also be induced by androgens [32] and the effects of tibolone on mood may, therefore, be due to the D4-isomer metabolite, which can be locally formed in the brain by the enzyme 3b-hydroxysteroid dehydrogenase (HSD)isomerase [33,34]. Alternatively, the decrease in SHBG
Fig. 3. Effect of tibolone on vaginal cornification in ovariectomized rats after treatment for 10 days with estradiol (E2), estradiol plus progesterone (P4) or tibolone. (sc, subcutaneous treatment; po, oral treatment).
by tibolone [35] may cause a higher free testosterone level, indirectly creating a positive effect on mood.
6. Effects on the endometrium Relevant preclinical models for testing the effects of compounds on the endometrium in order to predict the risk to humans do not exist. This is especially so for compounds in which action is dependent on metabolism. Monkey models or in vitro test systems using human endometrium are the only available options. Markiewicz and Gurpide [36], therefore evaluated the effect of tibolone and its metabolites in a human endometrial model by assessing the effects on estrogenic and progestagenic sensitive parameters. Estradiol 17bdehydrogenase is specifically induced by progestins. Tibolone’s D4-isomer metabolite induces estradiol 17bdehydrogenase as may be expected from its progestagenic activity but, surprisingly, tibolone itself induced the enzyme to a similar extent (Fig. 4). The 3a-OHmetabolite had no effect while the 3b-OH-metabolite, which binds only to the estrogen receptor, showed an intermediate effect. The authors suggested, therefore, that tibolone and the 3b-OH-metabolite were converted to the D4-isomer. The same group [15] showed indeed by labeling experiments that tibolone was converted to the D4-isomer and that the enzyme 3b-HSD-isomerase facilitated this. The accumulation of glycogen has been shown to be enhanced by progestins and tibolone also increases this parameter [36]. In addition, tibolone also suppresses the enhancement of PGF2a secretion by estradiol in fragments of secretory endometrium [36]. The effects on all three parameters (estradiol 17b-dehydrogenase, glycogen and PGF2a) show that tibolone has a progestagenic effect on the endometrium. These studies show that in the endometrium a potent progestagenic compound is formed and that this may counteract the estrogenic activity of the 3a- and 3bOH-metabolites. Progestins are strong anti-mitotic agents and are, therefore, considered to be responsible for the atrophic endometrium observed with the clinical use of tibolone [26,27].
7. Effects on the breast
Fig. 4. Induction of estradiol 17b-dehydrogenase (E2DH activity) in human endometrium fragments by tibolone and its metabolites. Figure modified from results presented in [36].
Several epidemiological studies have led to the conclusion that estrogens cause an increase in the risk of breast cancer and that this effect depends on the duration of use [37]. The effect of tibolone has been investigated in breast tumor cell lines but the effects seen were found to be dependent on the subclone used. Since studies with cell lines are considered to be non-predictive for the effect of a compound on breast tissue, we
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Fig. 5. Effect of tibolone on the development of DMBA-induced mammary tumors using the prophylaxic model. Adapted figure from [38].
Fig. 6. Effect of tibolone on conversion of estrone sulphate to estradiol in T47D cells. Figure prepared from results of Chetrite et al. [42].
studied the effects on breast tissue in vivo using the 7,12-dimethylbenz[a]anthracene (DMBA) model in rats. After induction of the tumors, treatment with tibolone starting 7 weeks after tumor induction, reduced tumor growth in a similar way as tamoxifen [38]. In a prophylactic protocol, starting treatment at time of tumor induction, tibolone almost completely prevented the initiation of tumor growth (see Fig. 5). The mechanism of action of tibolone on the breast has been studied extensively. Tibolone does not have anti-estrogenic activity at the receptor level and does not possess aromatase-inhibiting effect. Its effect on cellular homeostasis has been studied in breast tumor cell lines and primary human epithelial cells by Gompel et al. [39]. Cell proliferation is inhibited by tibolone and apoptosis, the process of programmed cell death, is stimulated. Using two different methods for assessing the typical features of apoptosis in cells, like blebbing and DNA fragmentation, it was shown that tibolone stimulates apoptosis in breast cells and this was further
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substantiating by the observed decrease in the antiapoptotic protein, bcl-2. Breast tissue contains key enzymes of steroidogenesis like aromatase and 17b-hydroxysteroid dehydrogenase (17b-HSD) together with sulphatase for the formation of active estrogens. High levels of estrone sulphate are present in breast tissue [40] and are the source of active estrogens after the action of the enzyme sulphatase. Inhibition of sulphatase activity may avoid the activation of sulphated estrogenic compounds [41]. Chetrite et al. [42,43] found that the 3OH-metabolites of tibolone are strong inhibitors of sulphatase activity and weak inhibitors of 17b-HSD. The effects of tibolone on the conversion of estrone sulfate to estradiol on sulphatase activity are shown in Fig. 6 representing both the inhibition of the sulphatase and 17b-HSD. Similarly to the inhibition of the formation of estradiol from estrone sulphate, the formation of 3OH-metabolites of tibolone from sulphate conjugates may be inhibited. Since a large pool of sulphated 3OH-tibolone metabolites are found in the circulation, inhibition of this enzyme prevents conversion to a 3OH-metabolite. This leads to lower estrogenic stimulation in the breast, which may be of clinical relevance. It has been shown that women using tibolone have less breast pain and show a lower mammographic density than that seen with conventional HRT [44]. Recently, it was shown that sulphatase inhibition in bone cells does not occur meaning that estrogenic compounds can be formed and act in this tissue [45]. This differential inhibitory effect between breast and bone cells contributes to the tissue-specific action of tibolone. Studies on the differential effects of tibolone and its metabolites have been extended to investigate the sulphated 3-OH-metabolites and the same differentiation was found as previously [46]. Recently, Purohit et al. [47] confirmed that tibolone has sulphatase-inhibiting activity and they even found that the sulphated compounds show irreversible binding in one breast cell line but further studies are needed to clarify the exact type of inhibition expressed by these compounds. The studies with breast cells show that both apoptotic stimulation and sulphatase inhibition are two important factors, which prevent breast stimulation. Comparisons of the effects of tibolone and tamoxifen on the breast show that the two compounds have completely different mechanism of action; tibolone diminishes ligand levels for the receptor while anti-estrogens modify the three-dimensional structure of the estrogen receptor itself (see for a schematic representation Fig. 7). However, both compounds have the effect of avoiding breast stimulations in recipients.
8. Effect of the cardiovascular system and liver The lipid profile of tibolone users is different from that of users of estrogens [48–50]. Tibolone induces a
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decrease in both triglycerides and Lp(a) whereas estrogens give only a decrease in Lp(a). Triglycerides and Lp(a) are considered to be an independent risk factor for cardiovascular disease (CVD). In contrast to the positive effects of tibolone, a decrease in HDL observed which is, at least in males, a negative risk factor in CVD. An extensive debate about the importance of drug-induced HDL-lowering is going on and the use of some HDL-modifying drugs, such as probucol [51], do not at all show an increased risk in myocard infarction. The decrease in HDL may be due to an androgenic effect of the compound on the enzyme, hepatic lipase, caused by D4-metabolite. The androgenic effects of tibolone on the liver leads to a decrease in SHBG [35] and an increase in factors stimulating fibrinolytic activity [52– 54]. The latter observation is another beneficial aspect of tibolone treat-
ment on the cardiovascular system. The direct effects of tibolone on the cardiovascular system have been assessed in cholesterol-fed ovariectomized rabbits. This investigation has shown that tibolone prevents the deposition of cholesterol in the vessel wall (see Fig. 8), lesion formation and the impairment of endothelium dependent smooth muscle relaxation [55]. The effects of tibolone on these parameters show a striking similarity with the effects of estrogens. It seems that the HDL lowering effect by tibolone does not impair the reverse cholesterol transport from peripheral tissues. Simoncini and Genazzani [56] recently showed that tibolone decreased levels of the adhesion molecule VCAM-1 expression in human endothelial cells in a similar way to estrogens, indicating that tibolone acts as an estrogenic compound in this model, due to the action of its 3OH-metabolites.
Fig. 7. Schematic presentation of the mode of action of tibolone and anti-estrogens. Tibolone inhibits the formation of active ligands for the receptor. Anti-estrogens induce a conformational chance of the receptor. ER, estradiol receptor; E2, estradiol; ERE, estrogen responsive element means a negative effect.
Fig. 8. Effect of tibolone on deposition of cholesterol in vessel wall in cholesterol-fed ovariectomized (OVX) rabbits. Cholesterol levels in animals without a diet are set at 0% and those in untreated cholesterol-fed OVX animals at 100%. Figure modified from results presented in [55]. (EE2, ethinyl estradiol and chol, cholesterol).
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[34] L. Milewick, C.E. Shaw, J.I. Mason, B.R. Carr, C.H. Blomquist, J.L. Thomas, 3b-Hydroxysteroid dehydrogenase activity in tissues of human fetus determined with 5a-androstane-3b, 17b-diol and dehydroepiandrosterone as substrates, J. Steroid Biochem. Mol. Biol. 45 (1993) 525 –537. [35] H.I. Abdallah, D.M. Hart, R. Lindsay, G.H. Beastall, Organon OD14 (tibolone) and menopausal dynamic hormone profiles, Maturitas 8 (1986) 81 –85. [36] L. Markiewicz, E. Gurpide, In vitro evaluation of estrogenic, estrogen antagonistic and progestagenic effects of a steroidal drug (Org OD14) and its metabolites on human endometrium, J. Steroid Biochem. 35 (1990) 535 – 541. [37] V. Beral, On behalf of the collaborative group on hormonal factors in breast cancer, Lancet 350 (1997) 1047 –1059. [38] H.J. Kloosterboer, W.G.E.J. Schoonen, G.H. Deckers, J.G.M. Klijn, Effect of progestagens and Org OD14 in in vitro and in vivo tumor models, J. Steroid Biochem. Mol. Biol. 49 (1994) 311 – 318. [39] A. Gompel, M. Kandouz, M. Siromachkova, A. Lombet, D. Thevenin, M. Mimoun, P. Poitout, The effect of tibolone on proliferation, differentiation and apoptosis in normal breast cells, Gynecol. Endocrinol. 1 (1997) 77 –79. [40] J.R. Pasqualini, G. Chetrite, C. Blacker, M.C. Feinstein, L. Delalonde, M. Talbi, C. Malocke, Concentration of estrone, estradiol, estrone sulfate and evaluation of sulfatase and aromatase activities in pre- and postmenopausal breast cancer patients, J. Clin. Endocr. Metab. 31 (1996) 1460 –1464. [41] L.J. Duncan, A. Purohit, N.M. Howarth, B.V.L. Potter, M.J. Reed, Inhibition of estrone sulfatase activity by estrone-3-0methylthiophosphonate: a potential therapeutic agent in breast cancer, Cancer Res. 53 (1993) 298 – 303. [42] G. Chetrite, H.J. Kloosterboer, J.R. Pasqualini, Effect of tibolone (Org OD14) and its metabolites on estrone sulphatase activity in MCF-7 and T-47D mammary cancer cells, Anticancer Res. 17 (1997) 135 –140. [43] G.S. Chetrite, H.J. Kloosterboer, J.C. Philippe, J.R. Pasqualini, Effects of Org OD14 (Livial®) and its metabolites on 17b-hydroxysteroid dehydrogenase activity in hormone-dependent MCF-7 and T-47D breast cancer cells, Anticancer Res. 19 (1999) 261 – 268. [44] I. Valdivia, D. Ortega, Mammographic density in postmenopausal women treated with tibolone, estriol or conventional hormone replacement therapy, Clin. Drug Invest. 20 (2000) 101 – 107. [45] H.J. Kloosterboer, M.E. de Gooyer, Tibolone inhibits sulfatase
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Tibolone: a steroid with a tissue-specific mode of action H.J Kloosterboer * NV Organon, Research and De6elopment Laboratories, P.O. Box 20, 5340 Oss, The Netherlands
Abstract In postmenopausal women tibolone has proved to prevent bone-loss and relieve climacteric symptoms as effectively as estrogens, but it does not stimulate the endometrium and the breast. This clinical profile strongly suggests that tibolone is a compound with tissue-specific action. Tibolone is quickly metabolized into its main active metabolites, 3a and 3b-OH, which are also present in an inactive, sulphated, form. In addition a D4-metabolite is found in circulation. The 3-OH-metabolites bind only to the estrogen receptor while the D4-isomer shows affinity only to the progesterone and androgen receptors. Tibolone prevents bone loss in a similar way to estrogens. Studies on bone mass using anti-estrogen, antiprogestin and anti-androgen in combination with tibolone, confirmed the sole involvement of the estradiol receptor. Increases in skin temperature as well as vaginal atrophy can be prevented by tibolone in a similar way to estrogens. Breast safety studies showed that tibolone clearly inhibited the growth of tumors in a DMBA model. In breast cell lines, tibolone profoundly inhibited sulphatase activity and an increase in apoptosis and decrease in cell proliferation was found. The stimulation of the endometrium is prevented by the local formation of the D4-isomer from tibolone or the 3b-OH-metabolite. We conclude that tibolone acts as a tissue-specific compound by mediating its effects via steroid receptors and enzymatic pathways. This dual effect of tibolone explains it’s positive clinical effects on bone, vagina and brain, and avoids stimulation of the endometrium and breast tissue. © 2001 Elsevier Science Ltd. All rights reserved. Keywords: Tibolone; Steroid receptors; Steroid metabolism; Inhibition; Bone; Sulphatase; Breast; Endometrium; Climacterium; Cardiovascular system
1. Introduction For many years, tibolone has been used for the treatment of climacteric complains and prevention of osteoporosis in postmenopausal women without stimulating the breast and endometrium. This tissue-specific effect of tibolone is difficult to explain from the results of preclinical studies using receptor studies with tibolone itself or tests in classical animal models [1,2]. Tibolone is a steroid with a 3-keto-D5 – 10 configuration, a 7a-methyl substituent and a 17a-ethinyl group. The compound does not have structural characteristics that would explain its estrogenic-like effects on vagina, brain and bone. An aromatic A-ring and a 3-hydroxyl Proceedings of the 14th International Symposium of the Journal of Steroid Biochemistry and Molecular Biology ‘‘Recent Advances in Steroid Biochemistry and Molecular Biology’’ (Quebec, Canada 24 – 27 June 2000). * Tel.: +31-412-662452; fax: +31-412-662514. E-mail address: [email protected] (H.J. Kloosterboer).
group are lacking, which are typical requirements for binding to the estradiol receptor. The estrogenic effects of tibolone as observed on the vagina in the Allen Doisy-test may, therefore, be due to the formation of estrogenic metabolites [2]. In addition to the estrogenic activity, the compound has been shown to possess also progestagenic and androgenic properties in specific models for these activities. The progestagenic activity is found to be low in the McPhail test, which is most likely due to the simultaneous presence of estrogenic activity. The final activity in a tissue of a compound is not only determined by the exposure to the compound itself, but also by the hormonal activity of the metabolites in circulation after passing through the intestine and liver. In addition, intracellular metabolism is important for determining which steroid receptors in a tissue become activated [3]. Ultimately, it is the cellular context including number of receptors as well as the set of co-activators and co-repressors [4] and possibly other non-genomic pathways [5] which determines whether a tissue becomes responsive to treatment. In this review the mode of action of tibolone is described using kinetic
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data, receptor results for tibolone and its metabolites together with its effects in various relevant in vitro and in vivo models showing tibolone’s effects on specific tissues relevant to hormone replacement therapy. The results of these studies clearly explain the tissue-specific effect as seen in the clinic.
2. Kinetics and metabolism After tibolone is administered to women, the compound is quickly metabolised into 3a- and 3b-OH-tibolone by the enzymes 3a/b hydroxysteroid dehydrogenase in the intestine and the liver (see Fig. 1). The metabolism as depicted in the figure has been confirmed with in vitro [6] and in vivo studies. The two OH-metabolites have a half-life of approximately 7 h (data on file). In addition to these two metabolites, a third metabolite — the D4-isomer — is found in the circulation for a short period of time but no half-life could be estimated. This metabolite is formed from tibolone directly by the enzyme 3b-HSD-isomerase or from the OH-metabolites. Only the 3b-OH-tibolone can be a substrate for the enzyme but it is theoretically possible that the D4-isomer is formed from the 3ametabolite via tibolone as these enzymes can act in either directions (see Fig. 1). Kunhz et al. [7] could also not determine a half-life for estradiol after oral administration of 2 mg of estradiol to fertile women, but were able to determine a half-life for estrone, a weak estrogen. However, other investigators [8,9] found a clear half-life for estradiol in postmenopausal women. Estradiol is quickly metabolized, mostly to estrone; in addition to that a large pool of sulphated estrone was found in circulation [10].
The metabolite profile for tibolone shows similarities to that of norethynodrel [11,12]. However, there are differences due to the presence of a 7a-methyl group in tibolone, which means that 5a reduction of the D4-isomer does not occur [13]. This is important for tissues like the endometrium where progestagenic activity is required to inhibit proliferation. This means that the progestagenic activity remains in the endometrial tissue after initial exposure, but in addition to this, local formation of the D4-isomer from the 3OH-metabolites may continue (see Fig. 1). A study with labeled tibolone has shown that the majority of the metabolites are in the sulphated (inactive) form [14]. The similarity between tibolone and estradiol is striking; both are quickly metabolized to form weak estrogenic compounds and a large pool of sulphated compounds is present. This sulfated pool serves as a source from which active estrogenic metabolites are continually formed by an enzyme, sulphatase, which resides in virtually all tissues. The extent to which this occurs may depend on local enzyme activity and tissue-specific metabolism is an important determinant for drug effects in a tissue. Notably Tang et al. [15] have found that tibolone is exclusively converted into the D4-isomer in endometrial tissue fragments.
3. Receptor activation of tibolone and its metabolites The binding affinities of tibolone and its metabolites to the human estrogen, progesterone and androgen receptor have been assessed using cell lines in order to predict their ultimate tissue responses. The results of the binding studies are depicted in Table 1. Surprisingly, tibolone itself binds to all three receptors al-
Fig. 1. Scheme of metabolism of tibolone to its (active) metabolites. 3a/b-HSD, 3a/b-hydrosteroid dehydrogenase; 3bHSD-iso, 3b-hydroxysteroid dehydrogenase/isomerase.
H.J. Kloosterboer / Journal of Steroid Biochemistry & Molecular Biology 76 (2001) 231–238 Table 1 Binding affinities of tibolone and its metabolites to human nuclear progesterone, androgen and estrogen receptor in MCF-7 cells relative to reference compounds ( =100%)a
Tibolone 3a-OH-metabolite 3b-OH-metabolite D4-isomer
ER (E2 = 100%)
PR (Org 2058= 100%)
AR (DHT= 100%)
1.3 3.2 1.7 n.c.
4.9 n.c. n.c. 12.9
3.2 n.c. n.c. 39.2
a E2, estradiol; DHT, dihydrotestosterone; Org 2058 is a pure synthetic progestin; n.c., no competition.
Fig. 2. Effect of ethinyl estradiol (EE2) and tibolone on bone mineral density (pQCT) in young ovariectomized (OVX) rats after oral treatment for 4 weeks. Mean values 9S.E.M. are given.
though it does not contain the essential structural requirements for binding to these receptors: a phenolicgroup for the estrogen receptor and the 3-keto-D4 structure for the progesterone and androgen receptor. The 3OH-metabolites bind solely to the estrogen receptor. The observed binding of tibolone may at least partly be due to metabolism under the assay conditions used. The affinity of the 3OH-metabolites is low but in transactivation experiments (data not shown) using cells transfected with human estrogen receptor and a promoter–reporter construct, the activity of the OHmetabolites is in the nmol range but tibolone can only do that at 1000 times higher concentrations. Based on the results of the transactivation assay one may conclude that binding studies with the mother compound are of limited value. The D4-isomer shows a clear binding to the progesterone receptor, which is comparable to that of natural progesterone. Transactivation assays with human steroid receptors have shown that the D4-isomer activates both the progesterone receptor and the androgen receptor but not the estrogen receptor [16].
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4. Effects on bone Bone loss belongs next to climacteric complaints as one of the earliest symptoms of the menopause. In contrast to the climacterium women do not immediately experience demineralisation of bone in the menopause as a negative aspect because osteoporosis only becomes manifest when actual fractures occur. In the menopause, bone loss is due to an increase in bone resorption and this effect can be prevented by the use of tibolone [17–19], estrogens [20], SERMs [21] or bisphosphonates [22]. Several studies have shown tibolone to have beneficial effects on bone. In ovariectomized rats, tibolone prevented bone loss in a dose-dependent manner when treatment was started immediately after removal of the ovaries (see Fig. 2). The same level in bone mass density can be reached with tibolone as with an estrogen. Desoxypyridinolin and osteocalcin, bone markers for resorption and formation respectively, remain at normal levels indicating that bone turnover remains normal in ovariectomized treated animals treated with tibolone [23]. In bone cells, estrogen, progestin and androgen receptors are present and it is, therefore, important to investigate which hormonal activity of tibolone is responsible for its beneficial effects on bone. This was investigated by determining whether specific antihormones could prevent the positive effects of tibolone on bone. The anti-estrogen ICI 164.384 prevents tibolone’s action on bone mass while the anti-androgen flutamide and the anti-progestin Org 31710 do not modulate the effects of tibolone [24]. Biochemical bone markers in animals treated with the combination of tibolone and anti-estrogen respond as expected and levels are similar to those following ovariectomy. These anti-hormone studies show that tibolone’s action on bone is mediated via the estrogen receptor. Bone quality under tibolone is not impaired as indicated by compression or the three-bending test. Bone quality remains similar to that as in intact animals [25]. Typical experiments have shown that tibolone acts as a bone resorption inhibitor in a similar way to estrogens.
5. Effects on the climacterium Vaginal atrophy is an early symptom of estrogen deficiency and can be counteracted by the administration of estrogens. This positive effect of estrogens on the vagina can be diminished by progestins. Tibolone displays a full range of estrogenic effect on the development of vaginal epithelial (see Fig. 3). The enzyme 3b-HSD-isomerase can convert tibolone and the 3bOH-metabolite to the D4-isomer, but from the full estrogenic response seen, it can be concluded that activity of the enzyme is low or maybe even absent in the
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vagina. Clinical studies [26,27] do show that the vagina and the endometrium differ in their response to tibolone; a clear estrogenic response is found in the vagina but the endometrium remains atrophied in the majority of women. When menstrual cycles become irregular in the menopause, strong fluctuations in estrogen levels are probably the cause of hot flushes and mood swings. These phenomena can be prevented not only by estrogens but also by tibolone [26,27]. In both preclinical models [28,29] and clinical studies [26,27], it has been shown that tibolone behaves similarly to estrogens in preventing hot flushes. Genazzani et al. [30,31] have shown that tibolone increases endorphin levels in the pituitary and in plasma and it has been suggested that this may be related to the improvements in mood seen with tibolone in the clinic. Mood improvement can also be induced by androgens [32] and the effects of tibolone on mood may, therefore, be due to the D4-isomer metabolite, which can be locally formed in the brain by the enzyme 3b-hydroxysteroid dehydrogenase (HSD)isomerase [33,34]. Alternatively, the decrease in SHBG
Fig. 3. Effect of tibolone on vaginal cornification in ovariectomized rats after treatment for 10 days with estradiol (E2), estradiol plus progesterone (P4) or tibolone. (sc, subcutaneous treatment; po, oral treatment).
by tibolone [35] may cause a higher free testosterone level, indirectly creating a positive effect on mood.
6. Effects on the endometrium Relevant preclinical models for testing the effects of compounds on the endometrium in order to predict the risk to humans do not exist. This is especially so for compounds in which action is dependent on metabolism. Monkey models or in vitro test systems using human endometrium are the only available options. Markiewicz and Gurpide [36], therefore evaluated the effect of tibolone and its metabolites in a human endometrial model by assessing the effects on estrogenic and progestagenic sensitive parameters. Estradiol 17bdehydrogenase is specifically induced by progestins. Tibolone’s D4-isomer metabolite induces estradiol 17bdehydrogenase as may be expected from its progestagenic activity but, surprisingly, tibolone itself induced the enzyme to a similar extent (Fig. 4). The 3a-OHmetabolite had no effect while the 3b-OH-metabolite, which binds only to the estrogen receptor, showed an intermediate effect. The authors suggested, therefore, that tibolone and the 3b-OH-metabolite were converted to the D4-isomer. The same group [15] showed indeed by labeling experiments that tibolone was converted to the D4-isomer and that the enzyme 3b-HSD-isomerase facilitated this. The accumulation of glycogen has been shown to be enhanced by progestins and tibolone also increases this parameter [36]. In addition, tibolone also suppresses the enhancement of PGF2a secretion by estradiol in fragments of secretory endometrium [36]. The effects on all three parameters (estradiol 17b-dehydrogenase, glycogen and PGF2a) show that tibolone has a progestagenic effect on the endometrium. These studies show that in the endometrium a potent progestagenic compound is formed and that this may counteract the estrogenic activity of the 3a- and 3bOH-metabolites. Progestins are strong anti-mitotic agents and are, therefore, considered to be responsible for the atrophic endometrium observed with the clinical use of tibolone [26,27].
7. Effects on the breast
Fig. 4. Induction of estradiol 17b-dehydrogenase (E2DH activity) in human endometrium fragments by tibolone and its metabolites. Figure modified from results presented in [36].
Several epidemiological studies have led to the conclusion that estrogens cause an increase in the risk of breast cancer and that this effect depends on the duration of use [37]. The effect of tibolone has been investigated in breast tumor cell lines but the effects seen were found to be dependent on the subclone used. Since studies with cell lines are considered to be non-predictive for the effect of a compound on breast tissue, we
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Fig. 5. Effect of tibolone on the development of DMBA-induced mammary tumors using the prophylaxic model. Adapted figure from [38].
Fig. 6. Effect of tibolone on conversion of estrone sulphate to estradiol in T47D cells. Figure prepared from results of Chetrite et al. [42].
studied the effects on breast tissue in vivo using the 7,12-dimethylbenz[a]anthracene (DMBA) model in rats. After induction of the tumors, treatment with tibolone starting 7 weeks after tumor induction, reduced tumor growth in a similar way as tamoxifen [38]. In a prophylactic protocol, starting treatment at time of tumor induction, tibolone almost completely prevented the initiation of tumor growth (see Fig. 5). The mechanism of action of tibolone on the breast has been studied extensively. Tibolone does not have anti-estrogenic activity at the receptor level and does not possess aromatase-inhibiting effect. Its effect on cellular homeostasis has been studied in breast tumor cell lines and primary human epithelial cells by Gompel et al. [39]. Cell proliferation is inhibited by tibolone and apoptosis, the process of programmed cell death, is stimulated. Using two different methods for assessing the typical features of apoptosis in cells, like blebbing and DNA fragmentation, it was shown that tibolone stimulates apoptosis in breast cells and this was further
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substantiating by the observed decrease in the antiapoptotic protein, bcl-2. Breast tissue contains key enzymes of steroidogenesis like aromatase and 17b-hydroxysteroid dehydrogenase (17b-HSD) together with sulphatase for the formation of active estrogens. High levels of estrone sulphate are present in breast tissue [40] and are the source of active estrogens after the action of the enzyme sulphatase. Inhibition of sulphatase activity may avoid the activation of sulphated estrogenic compounds [41]. Chetrite et al. [42,43] found that the 3OH-metabolites of tibolone are strong inhibitors of sulphatase activity and weak inhibitors of 17b-HSD. The effects of tibolone on the conversion of estrone sulfate to estradiol on sulphatase activity are shown in Fig. 6 representing both the inhibition of the sulphatase and 17b-HSD. Similarly to the inhibition of the formation of estradiol from estrone sulphate, the formation of 3OH-metabolites of tibolone from sulphate conjugates may be inhibited. Since a large pool of sulphated 3OH-tibolone metabolites are found in the circulation, inhibition of this enzyme prevents conversion to a 3OH-metabolite. This leads to lower estrogenic stimulation in the breast, which may be of clinical relevance. It has been shown that women using tibolone have less breast pain and show a lower mammographic density than that seen with conventional HRT [44]. Recently, it was shown that sulphatase inhibition in bone cells does not occur meaning that estrogenic compounds can be formed and act in this tissue [45]. This differential inhibitory effect between breast and bone cells contributes to the tissue-specific action of tibolone. Studies on the differential effects of tibolone and its metabolites have been extended to investigate the sulphated 3-OH-metabolites and the same differentiation was found as previously [46]. Recently, Purohit et al. [47] confirmed that tibolone has sulphatase-inhibiting activity and they even found that the sulphated compounds show irreversible binding in one breast cell line but further studies are needed to clarify the exact type of inhibition expressed by these compounds. The studies with breast cells show that both apoptotic stimulation and sulphatase inhibition are two important factors, which prevent breast stimulation. Comparisons of the effects of tibolone and tamoxifen on the breast show that the two compounds have completely different mechanism of action; tibolone diminishes ligand levels for the receptor while anti-estrogens modify the three-dimensional structure of the estrogen receptor itself (see for a schematic representation Fig. 7). However, both compounds have the effect of avoiding breast stimulations in recipients.
8. Effect of the cardiovascular system and liver The lipid profile of tibolone users is different from that of users of estrogens [48–50]. Tibolone induces a
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decrease in both triglycerides and Lp(a) whereas estrogens give only a decrease in Lp(a). Triglycerides and Lp(a) are considered to be an independent risk factor for cardiovascular disease (CVD). In contrast to the positive effects of tibolone, a decrease in HDL observed which is, at least in males, a negative risk factor in CVD. An extensive debate about the importance of drug-induced HDL-lowering is going on and the use of some HDL-modifying drugs, such as probucol [51], do not at all show an increased risk in myocard infarction. The decrease in HDL may be due to an androgenic effect of the compound on the enzyme, hepatic lipase, caused by D4-metabolite. The androgenic effects of tibolone on the liver leads to a decrease in SHBG [35] and an increase in factors stimulating fibrinolytic activity [52– 54]. The latter observation is another beneficial aspect of tibolone treat-
ment on the cardiovascular system. The direct effects of tibolone on the cardiovascular system have been assessed in cholesterol-fed ovariectomized rabbits. This investigation has shown that tibolone prevents the deposition of cholesterol in the vessel wall (see Fig. 8), lesion formation and the impairment of endothelium dependent smooth muscle relaxation [55]. The effects of tibolone on these parameters show a striking similarity with the effects of estrogens. It seems that the HDL lowering effect by tibolone does not impair the reverse cholesterol transport from peripheral tissues. Simoncini and Genazzani [56] recently showed that tibolone decreased levels of the adhesion molecule VCAM-1 expression in human endothelial cells in a similar way to estrogens, indicating that tibolone acts as an estrogenic compound in this model, due to the action of its 3OH-metabolites.
Fig. 7. Schematic presentation of the mode of action of tibolone and anti-estrogens. Tibolone inhibits the formation of active ligands for the receptor. Anti-estrogens induce a conformational chance of the receptor. ER, estradiol receptor; E2, estradiol; ERE, estrogen responsive element means a negative effect.
Fig. 8. Effect of tibolone on deposition of cholesterol in vessel wall in cholesterol-fed ovariectomized (OVX) rabbits. Cholesterol levels in animals without a diet are set at 0% and those in untreated cholesterol-fed OVX animals at 100%. Figure modified from results presented in [55]. (EE2, ethinyl estradiol and chol, cholesterol).
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