Historical milestones in the development of tibolone (Livial®).

For a new chemical entity, tibolone had a very long development period of 25 years before it was finally approved for the treatment of climacteric complaints. The reasons for this long development were its complex and fast metabolism and the poor standardization and sensitivity of analytical techniques and clinical methods. In the beginning of the new millennium, the results of primate studies and dose-finding studies in early postmenopausal women showed that tibolone had clear tissue-selective effects: it prevented hot flushes and bone loss, which are estrogen-related effects, while the estrogen-sensitive organs like breast and endometrium were not stimulated. In tissue, measurements of tibolone metabolites revealed that estrogenic metabolites were present in brain, but these metabolites were found as inactive conjugates in breast and endometrium. Attempts to find new indications for tibolone in large clinical trials failed because these studies were performed in elderly women who had already past the menopause many years ago and so unexpected side-effects became apparent due to altered metabolism and gene activation.

Tibolone rapidly attenuates the GABAB response in hypothalamic neurones.

Tibolone is primarily used for the treatment of climacteric symptoms. Tibolone is rapidly converted into three major metabolites: 3 alpha- and 3beta-hydroxy (OH)-tibolone, which have oestrogenic effects, and the Delta 4-isomer (Delta 4-tibolone), which has progestogenic and androgenic effects. Because tibolone is effective in treating climacteric symptoms, the effects on the brain may be explained by the oestrogenic activity of tibolone. Using whole-cell patch clamp recording, we found previously that 17beta-oestradiol (E(2)) rapidly altered gamma-aminobutyric acid (GABA) neurotransmission in hypothalamic neurones through a membrane oestrogen receptor (mER). E(2) reduced the potency of the GABA(B) receptor agonist baclofen to activate G-protein-coupled, inwardly rectifying K(+) (GIRK) channels in hypothalamic neurones. Therefore, we hypothesised that tibolone may have some rapid effects through the mER and sought to elucidate the signalling pathway of tibolone's action using selective inhibitors and whole cell recording in ovariectomised female guinea pigs and mice. A sub-population of neurones was identified post hoc as pro-opiomelanocortin (POMC) neurones by immunocytochemical staining. Similar to E(2), we have found that tibolone and its active metabolite 3 beta OH-tibolone rapidly reduced the potency of the GABA(B) receptor agonist baclofen to activate GIRK channels in POMC neurones. The effects were blocked by the ER antagonist ICI 182 780. Other metabolites of tibolone (3 alpha OH-tibolone and Delta 4-tibolone) had no effect. Furthermore, tibolone (and 3 beta OH-tibolone) was fully efficacious in ER alpha knockout (KO) and ER beta KO mice to attenuate GABA(B) responses. The effects of tibolone were blocked by phospholipase C inhibitor U73122. However, in contrast to E(2), the effects of tibolone were not blocked by protein kinase C inhibitors or protein kinase A inhibitors. It appears that tibolone (and 3 beta OH-tibolone) activates phospholipase C leading to phosphatidylinositol bisphosphate metabolism and direct alteration of GIRK channel function. Therefore, tibolone may enhance synaptic efficacy through the G(q) signalling pathways of mER in brain circuits that are critical for maintaining homeostatic functions.

In vitro effects of tibolone and its metabolites on human vascular coronary cells.

OBJECTIVES: Tibolone is a tissue selective compound with estrogenic, androgenic and progestogenic properties in classical bioassays. It is used for alleviation of menopausal symptoms and for osteoporosis prophylaxis in postmenopausal women. Only few data are available regarding the effects of tibolone on the cardiovascular system. We investigated therefore the in vitro effects of tibolone and its metabolites on the vasculature under special controlled conditions, using human female coronary endothelial and smooth muscle cells. METHODS: The effect on the production of the following markers in endothelial cells from human female coronary arteries was evaluated: nitric oxide synthase, prostacyclin, endothelin, plasminogen-activator-inhibitor-1 (PAI-1), E-Selectin, Intercellular adhesion molecule (ICAM-1), monocyte attracting protein-1 (MCP-1) and the precursor of matrix metalloproteinase-1 (pro-MMP-1). Tibolone, its metabolites, estradiol (E2), E2/norethisterone (NET) and E2/medroxyprogesterone acetate (MPA) were tested at 0.1 microM and 1 microM. The markers were determined by enzyme immunoassays in the cell supernatant. Cell proliferation of smooth muscle cells from female coronary artery was measured by an adenosine triphosphate-assay. RESULTS: Tibolone, its 3-hydroxy metabolites, E2/NET, E2/MPA and estradiol alone had significant effects on the synthesis of all markers tested. The magnitude of the tibolone effects, however, was mostly smaller than that of E2/NET and E2/MPA. Concerning smooth muscle cells tibolone and its 3-hydroxy metabolites also elicited an inhibition of the proliferation compared to control values. The strongest effect here was found for E2/NET and E2 alone, whereas E2/MPA had no effect. CONCLUSION: The results of this in vitro study conducted with cells of the most important vascular bed with respect to the problem of cardiovascular risk suggest that tibolone can positively influence the vasculature. However, these tibolone effects may depend on intact vascular cells and may vary due to the different atherosclerotic stages of the vessels. Thus, experimental studies are useful to explore mechanisms, but clearly cannot replace clinical studies.

Pharmacokinetic parameters of tibolone and metabolites in plasma, urine, feces, and bile from ovariectomized cynomolgus monkeys after a single dose or multiple doses of tibolone.

Levels of nonsulfated and sulfated tibolone metabolites were determined in plasma, urine, and feces from six ovariectomized, mature female cynomolgus monkeys after a single dose and multiple p.o. doses (including bile) of tibolone using validated gas chromatography/mass spectrometry and liquid chromatography/tandem mass spectrometry assays. In plasma, the predominant nonsulfated metabolite after single and multiple dosing was the estrogenic 3alpha-hydroxytibolone; levels of the estrogenic 3beta-hydroxytibolone were 10-fold lower and of progestagenic/androgenic Delta(4)-tibolone, 5-fold lower. Tibolone was undetectable. The predominant sulfated metabolite was 3alphaS,17betaS-tibolone; levels of 3betaS,17betaS-tibolone were about 2-fold lower, and monosulfated 3-hydroxymetabolites were about 10-fold lower. After multiple doses, areas under the curve of nonsulfated metabolites were lower (2-fold), and those of sulfated metabolites were 25% higher. In plasma, >95% metabolites were disulfated. In urine, levels of all the metabolites after single and multiple doses were low. After a single dose, high levels of 3beta-hydroxytibolone and the 3-monosulfated metabolites (3betaS,17betaOH-tibolone and 3alphaS,17betaOH-tibolone) were found in feces. After multiple dosing, 3alpha-hydroxytibolone increased, and the ratio of 3alpha/3beta-hydroxytibolone became about 1. The predominant sulfated metabolite was 3alphaS,17betaS-tibolone. Levels of all the metabolites in feces were higher after multiple doses than after a single dose. Levels of nonsulfated and 3-monosulfated metabolites were higher in feces than in plasma. Bile contained very high metabolite levels, except monosulfates. This may contribute to the metabolite content of the feces after multiple doses. 3beta-Hydroxytibolone and 3alphaS,17betaS-tibolone predominated. In conclusion, tibolone had different metabolite patterns in plasma, urine, feces, and bile in monkeys. The bile contributed to the metabolite pattern in feces after multiple doses. The major excretion route was in feces.

Selective tissue distribution of tibolone metabolites in mature ovariectomized female cynomolgus monkeys after multiple doses of tibolone.

Tibolone is a selective tissue estrogenic activity regulator (STEAR). In postmenopausal women, it acts as an estrogen on brain, vagina, and bone, but not on endometrium and breast. Despite ample supporting in vitro data for tissue-selective actions, confirmative tissue levels of tibolone metabolites are not available. Therefore, we analyzed tibolone and metabolites in plasma and tissues from six ovariectomized cynomolgus monkeys that received tibolone (0.5 mg/kg/day by gavage) for 36 days and were necropsied at 1, 1.25, 2.25, 4, 6, and 24 h after the final dose. The plasma and tissue levels of active, nonsulfated (tibolone, 3alpha-hydroxytibolone, 3beta-hydroxytibolone, and Delta(4)-tibolone), monosulfated (3alpha-sulfate,17beta-hydroxytibolone and 3beta-sulfate,17beta-hydroxytibolone), and disulfated (3alpha,17beta-disulfated-tibolone and 3beta,17betaS-disulfated-tibolone) metabolites were measured by validated gas chromatography with mass spectrometry and liquid chromatography with tandem mass spectrometry. Detection limits were 0.1 to 0.5 ng/ml (plasma) and 0.5 to 2 ng/g (tissues). In brain tissues, estrogenic 3alpha-hydroxytibolone was predominant with 3 to 8 times higher levels than in plasma; levels of sulfated metabolites were low. In vaginal tissues, major nonsulfated metabolites were 3alpha-hydroxytibolone and the androgenic/progestagenic Delta(4)-tibolone; disulfated metabolites were predominant. Remarkably high levels of monosulfated metabolites were found in the proximal vagina. In endometrium, myometrium, and mammary glands, levels of 3-hydroxymetabolites were low and those of sulfated metabolites were high (about 98% disulfated). Delta(4)-Tibolone/3-hydroxytibolone ratios were 2 to 3 in endometrium, about equal in breast and proximal vagina, and 0.1 in plasma and brain. It is concluded that tibolone metabolites show a unique tissue-specific distribution pattern explaining the tissue effects in monkeys and the clinical effects in postmenopausal women.

Pharmacokinetic parameters of sulfated tibolone metabolites in postmenopausal women after single and multiple doses of tibolone.

The objective of this study was to determine pharmacokinetic parameters of sulfated tibolone metabolites after single dose and their accumulation after multiple doses of tibolone. Blood samples from postmenopausal women in a single-dose (2.5 mg tibolone), open-label study (n=8) and multiple-dose (placebo, 0.3, 0.625, 1.25, or 2.5 mg/day tibolone for twenty-six cycles of 28 days), randomized, double-blind study (n=15) were analyzed for non-sulfated and sulfated tibolone metabolites by validated gas chromatography-mass spectrometry (GC-MS) and liquid chromatography with tanolam mass spectrometry (LC-MS/MS), respectively. The predominant non-sulfated and sulfated metabolites after a single dose were 3alpha-hydroxy-tibolone and 3alpha,17beta-di-sulfated (di-S)-tibolone. At 3 h, >90% of metabolites were sulfated. Tibolone and Delta(4)-tibolone were detectable for about 6 h. After multiple treatment cycles with different doses, metabolite levels at 10 h were dose-related and levels of di-S metabolites were three- to fivefold higher than after a single dose. Tibolone metabolite levels did not differ between cycles. Inactive di-S tibolone metabolites predominated in blood. No accumulation occurred between cycles 7 and 26.

Molecular analysis of human endometrium: short-term tibolone signaling differs significantly from estrogen and estrogen + progestagen signaling.

Tibolone, a tissue-selective compound with a combination of estrogenic, progestagenic, and androgenic properties, is used as an alternative for estrogen or estrogen plus progesterone hormone therapy for the treatment of symptoms associated with menopause and osteoporosis. The current study compares the endometrial gene expression profiles after short-term (21 days) treatment with tibolone to the profiles after treatment with estradiol-only (E(2)) and E(2) + medroxyprogesterone acetate (E(2) + MPA) in healthy postmenopausal women undergoing hysterectomy for endometrial prolapse. The impact of E(2) treatment on endometrial gene expression (799 genes) was much higher than the effect of tibolone (173 genes) or E(2) + MPA treatment (174 genes). Furthermore, endometrial gene expression profiles after tibolone treatment show a weak similarity to the profiles after E(2) treatment (overlap 72 genes) and even less profile similarity to E(2) + MPA treatment (overlap 17 genes). Interestingly, 95 tibolone-specific genes were identified. Translation of profile similarity into biological processes and pathways showed that ER-mediated downstream processes, such as cell cycle and cell proliferation, are not affected by E2 + MPA, slightly by tibolone, but are significantly affected by E(2). In conclusion, tibolone treatment results in a tibolone-specific gene expression profile in the human endometrium, which shares only limited resemblance to E(2) and even less resemblance to E2 + MPA induced profiles.

The hormone replacement therapy drug tibolone acts very similar to medroxyprogesterone acetate in an estrogen-and progesterone-responsive endometrial cancer cell line.

Tibolone, a steroidogenic compound with both estrogenic and progestagenic properties, is used as an alternative for estrogen or estrogen plus progesterone hormone therapy for the treatment of symptoms associated with menopause and osteoporosis. We have evaluated whether the effect of tibolone on a human endometrial cell line is similar to, or comparable with, the effect of estradiol (E(2)), medroxyprogesterone acetate (MPA) or E(2) + MPA treatment. Using stable transfection techniques, the estrogen receptor (ER) expressing human endometrial cancer cell line, ECC1, was altered to also express both progesterone receptors (PRs). These cells were then used to assess growth regulation and expression profiling (Affymetrix U133plus2) under the influence of E(2) (1 nM), MPA (1 nM), E(2) + MPA or tibolone (100 nM). Growth assessment and comparison of profiles indicate that tibolone behaves predominantly like MPA. Furthermore, regulation of prereplication complex genes, such as the minichromosome maintenance genes, could be involved in the observed strong inhibition of growth by tibolone as well as MPA. In addition, in total, 15 genes were found to be specific for tibolone treatment. These genes were predominantly involved in regulation of the cell cycle and differentiation.

Metabolism of exogenous sex steroids and effect on brain functions with a focus on tibolone.

Around the menopause, changes in ovarian secretion of steroids result in changes in brain function: hot flushes and sweating later followed by changes in mood, libido and cognition. The relationship between sex steroids and brain functions are reviewed, with focus on hormonal treatments, in particular tibolone, on the postmenopausal brain and on associations between tissue levels and brain functions. Data on steroid levels in human brain are limited. Exogenous oestrogens alone or combined with progestagens reduce hot flushes and sweating, and may favourably affect anxiety, depression and mood. Testosterone alone or combined with E(2) improves libido and mood. Tibolone reduces hot flushes and sweating, and improves mood and libido, but does not stimulate endometrium or breast, like oestrogens. Tibolone is an ideal compound for studying steroid levels and metabolism in brain in view of its structural differences from endogenous steroids and its extensive metabolism required to express its endocrine effects. Brain levels of tibolone metabolites were measured in ovariectomized cynomolgus monkeys receiving tibolone for 36 days. Compared to serum, higher levels of the oestrogenic 3alpha/beta-hydroxytibolone and the androgenic/progestagenic Delta(4)-tibolone, and lower levels of sulphated metabolites are found in various brain regions. The high levels of oestrogenic metabolites in the hypothalamus explain hot flush reduction. Combined with the presence of Delta(4)-tibolone, the tibolone-induced increase in free testosterone through SHBG reduction explains androgenic effects of tibolone on mood and libido. The levels of tibolone metabolites in the monkey brain support tibolone's effects on brain functions.

Metabolism of tibolone and its metabolites in uterine and vaginal tissue of rat and human origin.

In postmenopausal women, tibolone shows clear tissue differences in its stimulatory effects on the vagina and uterus. In rats, however, it has stimulatory effects on both tissues, with a different, more estrogenic, effect on the uterus than in humans. This may be due to differences in local metabolism. Therefore, in the present study, the metabolism of tibolone was analyzed in incubations of uterine and vaginal tissue from postmenopausal women and ovariectomized rats using radiolabeled tibolone in order to understand the tissue- and species-specific metabolism. In the rat, tibolone (50 nM) was mainly 3alpha-reduced to the estrogenic 3alpha-OH-tibolone in the uterus and vagina. The 3beta-OH tibolone can be isomerized to 3alpha-OH-tibolone with tibolone as intermediate. In contrast, in the same tissues from postmenopausal women, the progestagenic Delta4-isomer and estrogenic 3beta-OH-tibolone were the major metabolites of tibolone. The formation of the Delta4-isomer was higher in uterine tissue. The 3beta-hydroxysteroid dehydrogenase (HSD) inhibitor epostane had no effect on tibolone metabolism in human uterine and vaginal tissue microsomes and HEK293 cells expressing the human 3beta-HSD types 1 and 2 isoforms did not metabolize tibolone. Moreover, the 3beta-reduction of tibolone to 3beta-OH-tibolone was NADPH dependent, while the isomerization of tibolone to the Delta4-isomer did not require a cofactor. It was therefore concluded that human 3beta-HSD isoforms are not involved in the metabolism of tibolone, and that the 3beta-reduction and the Delta5-10 to Delta4 isomerization may be catalyzed by different enzymes. In conclusion, we showed that, in hormone therapy target tissues of the rat as compared with the human, different metabolic pathways for tibolone exist and therefore result in metabolites with different pharmacological properties. The rat is therefore a poor model to predict the effects of tibolone on the uterus in postmenopausal women.

Tibolone and metabolites induce prolactin production in human endometrial stromal cells in vitro: evidence for cell-specific metabolism.

In this study, we assessed the effects of tibolone and its metabolites on the production of a progesterone sensitive parameter, prolactin, in human endometrium stroma cells in vitro. In addition, the metabolism of the compounds by isolated stromal and epithelial cells was evaluated. The reference compounds, progesterone, Org 2058, and DHT all induced prolactin production. Oestradiol also slightly induced prolactin production and enhanced the response to Org 2058. Tibolone and Delta4-tibolone were similar with regard to potency to induce prolactin levels in the culture supernatant. Their potency was lower than that of Org 2058, similar to that of progesterone and higher than that of DHT. The efficacies of tibolone, Delta4-tibolone and Org 2058 were similar (approximately 200-fold induction). The estrogenic tibolone metabolites 3alpha- and 3beta-OH tibolone also significantly stimulated prolactin production. Their potency, however, was low since significance was reached only at the highest concentrations tested. The PR antagonist Org 31710 inhibited both tibolone- and Delta4-tibolone-induced prolactin production. The responses of tibolone and Delta4-tibolone were not affected by co-incubation with the androgen receptor antagonist OH-flutamide. The effect of tibolone, but not Delta4-tibolone, was antagonized approximately 50% in combination with the highest dose (1 microM) estrogen receptor antagonist, ICI 164384. The induction of prolactin by 3alpha- and 3beta-OH tibolone was antagonized most potently by Org 31710, but also by ICI 164384 and OH-flutamide. Tibolone is metabolized differently in epithelial and stromal cells of the human endometrium. The epithelial cells mostly produce the progestagenic/androgenic Delta4-tibolone. The stromal cells produce predominantly the 3beta-OH tibolone, and some Delta4-tibolone, but the net effect observed with regard to prolactin production is progestagenic. When the metabolites 3alpha-OH, 3beta-OH, and Delta4-tibolone were added to the cultures no conversions were observed. The HPLC analyses showed no evidence for the production of sulfated metabolites. In conclusion, the net effects on endometrial stromal cells are predominantly progestagenic. Tibolone is converted by epithelial cells into Delta4-tibolone which displays progestagenic and androgenic activities, whereas in stromal cells also the estrogenic metabolites 3alpha- and 3beta-OH tibolone are formed.

Effects of stable transfection of human fetal osteoblast cells with estrogen receptor-alpha on regulation of gene expression by tibolone.

Tibolone is a synthetic steroid which undergoes tissue selective metabolism into several metabolites having estrogenic, progestogenic or androgenic activities. The effects of 3 alpha-hydroxy tibolone (Org 4094), 3 beta-hydroxy tibolone (Org 30126) and their sulfated metabolites were investigated on human fetal osteoblasts (hFOB). Tibolone had no effect on selected osteoblast marker proteins in estrogen-receptor negative hFOB cells. In contrast, 3 alpha-hydroxy and 3beta-hydroxy tibolone resulted in dose-dependent increases in alkaline phosphatase activity in estrogen receptor (ER) alpha-positive hFOB cells. The maximum increase for both metabolites was comparable to the effects of an optimal dose of 17beta-estradiol, and occurred at 10 muM. At 20 muM, both metabolites increased mRNA levels for alkaline phosphatase and type 1 collagen and protein levels for osteocalcin. Sulfated metabolites of tibolone also increased alkaline phosphatase activity. The estrogen receptor antagonist ICI 182, 780 inhibited stimulation of alkaline phosphatase activity by sulfated and non-sulfated tibolone metabolites, but was more potent on the former. Taken together, these results suggest that stable transfection of ER alpha into hFOB cells confers regulation by 3 alpha-hydroxy and 3beta-hydroxy tibolone metabolites of osteoblast metabolism.

Tibolone and its metabolites inhibit invasion of human mammary carcinoma cells in vitro.

UNLABELLED: Tibolone is used in postmenopausal women to alleviate menopausal symptoms and to prevent osteoporosis, but it does not stimulate the endometrium and the breast. Up to date, little data are available on the effect of tibolone on breast cancer initiation and progression. OBJECTIVE: In the present in vitro study, we investigated the effect of tibolone and its metabolites (3alpha-OH tibolone, 3beta-OH tibolone, the Delta4 isomer and the sulphated isoform) on invasion of human breast cancer cells. METHODS: The effect on invasion was evaluated in the chick heart invasion assay using MCF-7/6 cells and in the collagen type I invasion assay using T47-D cells. Furthermore, the compounds were tested in aggregation and migration assays. RESULTS: We observed that, at a concentration of 100 microM, tibolone and its 3beta-OH metabolite possess anti-invasive activities in the two different invasion assays. However, this was neither due to effects on cell-cell adhesion nor on motility. In an attempt to probe the mechanism underlying the anti-invasive effect, we found that pro-MMP-9 release was markedly reduced in the supernatant of MCF-7/6 breast cancer cells treated with tibolone, 3alpha-OH tibolone and the Delta4 isomer but, interestingly, not with the sulphated metabolite. CONCLUSION: We conclude that tibolone and its 3beta-OH metabolite have an anti-invasive effect on the tested breast cancer cell lines in vitro. This effect on invasion is not correlated with an effect on cell-cell adhesion or motility but coincides with a decreased release of pro-MMP-9 in the medium.

Molecular portrait of the progestagenic and estrogenic actions of tibolone: behavior of cellular networks in response to tibolone.

Tibolone is a synthetic steroid with estrogenic effects on brain, vagina, and bone without stimulating the endometrium. During tibolone treatment, it is thought that the progestagenic properties of tibolone stimulate cell differentiation, which effectively counterbalances the growth-stimulating effects of the estrogenic properties of tibolone. The objective of this study was to characterize the expression profile that reflects the endometrial responses to the separated estrogenic (growth-inducing) and progestagenic (growth-inhibiting) actions of tibolone, thus gaining insight into the counteracting effect of these properties of tibolone on the endometrium. The estrogenic action of tibolone was studied in the estrogen-responsive ECC1 cell line (expressing estrogen receptor alpha), and the progestagenic action was studied in the progesterone-responsive cell line Ishikawa PRAB-36 (expressing PRA and PRB). The data showed that the progestagenic and estrogenic effects of tibolone produce different expression profiles with a narrow overlap in genes; however, both properties modulate the same biological processes. The final genetic network analysis indicated that the estrogenic effect of tibolone is potentially counterbalanced by the progestagenic metabolite of tibolone via differential regulation of similar cellular processes. For example, both progestagenic and estrogenic properties stimulate proliferation, but they exert the opposite effect on apoptosis. The apoptosis network was stimulated by the progestagenic properties of tibolone; in contrast, the estrogenic effect of tibolone suppressed the apoptosis network. The current results indicate that this differential regulation is realized through modulation of a different group of genes and rarely via contraregulation of the same set of genes.

Tissue-selective effects of tibolone on the breast.

Hormone treatment with an estrogen plus a progestagen (EPT) increases the risk of breast cancer. Both hormone activities are also induced by tibolone. In order to assess the breast safety of tibolone, it was evaluated in several pre-clinical models. The effects were inconclusive in breast cancer cell lines but, in various in vivo models, it did not stimulate the breast. In the 17,12-dimethylbenz(a)anthracene (DMBA) model, tibolone clearly inhibited the growth of breast tumors and, when given prophylactally, far less tumors developed. Ovariectomized monkeys showed no increase in the expression of the proliferation marker Ki67. The effects of tibolone and its metabolites on the steroid metabolizing enzymes in breast tissues were investigated in order to unravel its mode of action in the breast. Tibolone and its metabolites did not inhibit aromatase, but sulfatase was profoundly inhibited. The sulfated 3alpha-OH tibolone metabolite even showed irreversible inhibition of sulfatase. In addition, 17ss-hydroxysteroid dehydrogenase activities were slightly inhibited and sulfotransferase activity was stimulated at low concentrations. The consequence of these effects is that, for both endogenous estrogens and estrogenic-metabolites of tibolone, the equilibrium is preferential for the sulfated forms. The intracellular hormonal milieu tibolone and its metabolites also influence cellular homeostasis. It inhibits cell proliferation of normal breast epithelial cells and stimulates apoptosis. In this respect, tibolone behaves differently from estrogens. Clinical studies have shown that tibolone users experience less breast tenderness and do not show an increase in mammographic density as found with continuous combined EPT. The data concerning tibolone and breast cancer risk are inconclusive and require further investigation.

Progestogenic effects of tibolone on human endometrial cancer cells.

Tibolone, a synthetic steroid acting in a tissue-specific manner and used in hormone replacement therapy, is converted into three active metabolites: a Delta(4) isomer (exerting progestogenic and androgenic effects) and two hydroxy metabolites, 3 alpha-hydroxytibolone (3 alpha-OH-tibolone) and 3beta-OH-tibolone (exerting estrogenic effects). In the present study an endometrial carcinoma cell line (Ishikawa PRAB-36) was used to investigate the progestogenic properties of tibolone and its metabolites. This cell line contains progesterone receptors A and B, but lacks estrogen and androgen receptors. When tibolone was added to the cells, complete conversion into the progestogenic/androgenic Delta(4) isomer was observed within 6 d. Furthermore, when cells were cultured with tibolone or when the Delta(4) isomer or the established progestagen medroxyprogesterone acetate was added to the medium, marked inhibition of growth was observed. Interestingly, 3 beta-OH-tibolone also induces some inhibition of growth. These growth inhibitions were not observed in progesterone receptor-negative parental Ishikawa cells, and progestagen-induced growth inhibition of PRAB-36 cells could readily be reversed using the antiprogestagen Org-31489. Upon measuring the expression of two progesterone-regulated genes (fibronectin and IGF-binding protein-3), tibolone, the Delta(4) isomer and medroxyprogesterone acetate showed similar gene expression regulation. These results indicate that tibolone, the Delta(4) metabolite, and to some extent 3 beta-OH-tibolone exert progestogenic effects. Tibolone and most likely 3 beta-OH-tibolone are converted into the Delta(4) metabolite.

Tibolone and 5alpha-dihydrotestosterone alone or in combination with an antiandrogen in a rat breast tumour model.

Tibolone was combined with the antiandrogen flutamide to determine whether the inhibition of tumour growth in the prophylactic 7,12-dimethylbenz(a)anthracene (DMBA) rat model could be attributed to androgenic properties of one of its metabolites. The mean tumour load after tibolone (0.25 or 1.0 mg/kg twice daily orally for 10 weeks) was 125 and 255 versus 718 mm2 for placebo. The mean number of tumours were 1.2 and 2.0 versus 5.8, respectively. Combined with flutamide (10 mg/kg twice daily orally) both doses of tibolone did not result in an increase compared to placebo, but in significantly lower tumour loads (160 and 64 versus 718 mm2, respectively) and smaller numbers of tumours (0.8 and 1.0 versus 5.8, respectively). The differences between tibolone monotherapy and the combination groups with flutamide were not statistically significant indicating that flutamide did not reverse tibolone's inhibition of tumour growth. The positive control, 5alpha-dihydrotestosterone (DHT), entirely suppressed tumour development and flutamide abolished the inhibitory effect of DHT. Thus, unlike DHT, tibolone does not exert its beneficial effect in DMBA-induced tumours via the androgen receptor, but acts via different mechanisms.

Pros and cons of existing treatment modalities in osteoporosis: a comparison between tibolone, SERMs and estrogen (+/-progestogen) treatments.

Tibolone, selective estrogen receptor modulators (SERMs) like tamoxifen and raloxifene, and estrogen (+/-progestogen) treatments prevent bone loss in postmenopausal women. They exert their effects on bone via the estrogen receptor (ER) and the increase in bone mass is due to resorption inhibition. The effect of SERMs on bone mineral density is less than that with the other treatments, but the SERM raloxifene still has a positive effect on vertebral fractures. In contrast to tibolone and estrogens (+/-progestogen), SERMs do not treat climacteric complaints, whilst estrogen plus progestogen treatments cause a high incidence of bleeding. Estrogen plus progestogen combinations have compromising effects on the breast. Tibolone and SERMs do not stimulate the breast or endometrium. Unlike SERMs, tibolone does not possess antagonistic biological effects via the ER in these tissues. Estrogenic stimulation in these tissues is prevented by local metabolism and inhibition of steroid metabolizing enzymes by tibolone and its metabolites. SERMs and estrogen (+/-progestogen) treatments increase the risk of venous thromboembolism (VTE), whilst estrogen (+/-progestogen) combinations have unwanted effects on cardiovascular events. So far, no detrimental effects of tibolone have been observed with respect to VTE or cardiovascular events. The clinical profile of tibolone therefore has advantages over those of other treatment modalities. It is also clear that tibolone is a unique compound with a specific mode of action and that it belongs to a separate class of compounds that can best be described as selective, tissue estrogenic activity regulators (STEARs).