Herman A. M. Verheul

Receptor profiling and endocrine interactions of tibolone

The receptor profiles and in vivo activity of tibolone, and its primary metabolites, Delta(4)-isomer, and 3alpha- and 3beta-hydroxytibolone, were studied and compared to those of structurally related compounds. The Delta(4)-isomer was the strongest binder and activator of the progesterone receptor (PR); tibolone was 10 times weaker in binding and half as potent in transactivation of PR; 3alpha- and 3beta-hydroxytibolone did not bind or activate PR. In rabbits oral tibolone produced a minor progestagenic effect in the endometrium, whereas co-administration of tibolone and the anti-estrogen ICI 164,384 unmasked tibolones progestagenic effect. 3-Hydroxytibolones were the strongest binders and activators of the estrogen receptors (ERs), with greater affinity for ERalpha than for ERbeta. Tibolone showed weaker binding and activation of both ERs and the Delta(4)-isomer has a binding and activation activity of less than 0.1% of E2 for ERalpha or ERbeta. Tamoxifen and 4-hydroxytamoxifen showed partial ERalpha agonistic effects with a maximal response of 12% and raloxifene of 3-5%. Oral administration of 1mg tibolone to ovariectomized rats induced an estrogenic effect on vaginal epithelium. The Delta(4)-isomer was a stronger binder and activator of the androgen receptor (AR) than tibolone; both 3-hydroxytibolones did not bind or activate AR. Introducing a 7alpha-methyl group decreased progestagenic and increased androgenic activity. We conclude that the progestagenic and androgenic activities of tibolone are mediated by the Delta(4)-isomer, and the estrogenic activity, by the 3-hydroxytibolones. The estrogenic activity of the 3-hydroxytibolones masked the progestagenic activity of tibolone in rabbit endometrium. Full estrogenic response was observed in rat vaginal tissue after oral administration of tibolone.

Tibolone: a compound with tissue specific inhibitory effects on sulfatase.

The aim was to test whether sulfatase activity is differently regulated by tibolone in human bone, endometrium and breast cells since selective inhibition of sulfatases in various tissues may contribute to the tissue-specificity of tibolone. Tibolone, its 3 alpha- and 3 beta-hydroxy metabolites and their 3-sulfated forms, and its Delta(4)-isomer strongly (70-90%) inhibited the sulfatase activity in human breast cell lines (two T-47D clones) and intermediately (8-43%) in human endometrial cells (HEC-1A). In contrast, they did not inhibit sulfatase in two human osteoblast-like cell lines (MG 63, HOS TE-85). The specific sulfatase inhibitor, EMATE, showed inhibition in all cell lines. Just as estrone sulfate, 3 alpha-sulfated tibolone was also converted by sulfatase to the unconjugated 3 alpha-hydroxy-tibolone intracellularly in all cell lines. The tissue specific inhibition pattern of sulfatase activity by tibolone and its metabolites suggest that tibolone could be protective against development of mammary carcinomas, whereas it retains favorable estrogenic effects on bone.

Tibolone is not converted by human aromatase to 7α-methyl-17α-ethynylestradiol (7α-MEE):: Analyses with sensitive bioassays for estrogens and androgens and with LC-MSMS

To exclude that aromatization plays a role in the estrogenic activity of tibolone, we studied the effect tibolone and metabolites on the aromatization of androstenedione and the aromatization of tibolone and its metabolites to 7alpha-methyl-17alpha-ethynylestradiol (7alpha-MEE) by human recombinant aromatase. Testosterone (T), 17alpha-methyltestosterone (MT), 19-nortestosterone (Nan), 7alpha-methyl-19-nortestosterone (MENT) and norethisterone (NET) were used as reference compounds. Sensitive in vitro bioassays with steroid receptors were used to monitor the generation of product and the reduction of substrate. LC-MSMS without derivatization was used for structural confirmation. A 10 times excess of tibolone and its metabolites did not inhibit the conversion of androstenedione to estrone by human recombinant aromatase as determined by estradiol receptor assay whereas T, MT, Nan, and MENT inhibited the conversion for 75, 53, 85 and 67%, respectively. Tibolone, 3alpha- and 3beta-hydroxytibolone were not converted by human aromatase whereas the estrogenic activity formed with the Delta4-isomer suggests a conversion rate of 0.2% after 120 min incubation. In contrast T, MT, Nan, and MENT were completely converted to their A-ring aromates within 15 min while NET could not be aromatized. Aromatization of T, MT, Nan and MENT was confirmed with LC-MSMS. Structure/function analysis indicated that the 17alpha-ethynyl-group prevents aromatization of (19-nor)steroids while 7alpha-methyl substitution had no effect. Our results with the sensitive estradiol receptor assays show that in contrast to reference compounds tibolone and its metabolites are not aromatized.

Effects of estrogens and hormone replacement therapy on breast cancer risk and on efficacy of breast cancer therapies

This review summarises preclinical and clinical data on effects of endogenous and exogenous estrogens on probability of breast cancer diagnosis, and on the course and efficacy of breast cancer therapies. The data indicate that higher endogenous estrogen exposure (e.g. pregnancy, early menarche and late menopause, estrogen levels in future breast cancer patients, obesity) or exogenous estrogens (oral contraceptives; hormone replacement therapies) may be associated with an increased probability of breast cancer diagnosis. However, there is little evidence that estrogens have deleterious effects on the course of breast cancer. Moreover, increased incidence of breast cancer diagnosis after prolonged hormone replacement therapy (HRT) use seems to be associated with clinically less advanced disease. In studies assessing both diagnosis and mortality, HRT is frequently associated with reduced mortality compared to never users. The interaction of progestagens and estrogens on the probability of breast cancer diagnosis is complex and dependent on type of progestagens and regimens employed. Efficacy of current treatment modalities for breast cancer (surgery, irradiation, adjuvant therapy or chemotherapy) is not negatively influenced by estrogens at concentrations considerably higher than those attained with current HRT preparations. Although it cannot be excluded that estrogens increase the probability of breast cancer diagnosis, available data fail to demonstrate that, once breast cancer has been diagnosed, estrogens worsen prognosis, accelerate the course of the disease, reduce survival or interfere with the management of breast cancer. It may therefore be concluded that the prevalent opinion that estrogens and estrogen treatment are deleterious for breast cancer, needs to be revisited. However, results of ongoing prospective, randomised clinical trials with different HRT regimens in healthy women or breast cancer survivors are needed to provide more definite conclusions about risks and benefits of HRT.

Levels of tibolone and estradiol and their nonsulfated and sulfated metabolites in serum, myometrium, and vagina of postmenopausal women following treatment for 21 days with tibolone, estradiol, or estradiol plus medroxyprogestrone acetate

Tibolone has estrogenic effects on the vagina but not on the uterus. To explain this, levels of tibolone and estradiol and their metabolites were determined in serum, myometrium, and vagina. Thirty-four postmenopausal women with uterine prolapse received either no treatment, tibolone, E2 or E2 + medroxyprogesterone acetate (MPA) for 21 days, or a single dose of tibolone. Twenty ± 6 hours after administration, >98% of the 3-hydroxytibolone metabolites in serum and tissues were disulfated. Of the unconjugated metabolites, the estrogenic 3α-hydroxytibolone predominated in serum, whereas the progestagenic/ androgenic Δ 4-tibolone predominated in myometrium and vagina. Levels of disulfated metabolites in serum and tissues were higher (3- to 5-fold) after multiple dosing than after a single dose. Tissue:serum ratios were <1, except for Δ 4-tibolone. In all groups, E2 tissue levels were higher than serum levels; the percentage of serum E1S was >90%. Tibolone did not affect endogenous E1, E2, or E1S levels in serum, but in myometrium and vagina, E1 levels were significantly higher and E1S levels tended to be lower than in controls. Serum and tissue levels of endogenous and exogenous E1, E2, and E 1S were markedly increased 20 hours after E2 or E2 + MPA; the percentage of E1S and tissue:serum ratios were not affected. MPA had no effect on the degree of sulfation of E 1. Compared with serum, tissue levels of E2 were high in all groups; absolute E2 levels in control and tibolone groups were much lower than in the E2 groups. Tibolone metabolite patterns are different in serum, myometrium, and vagina.

Tibolone and 5α-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 tibolones 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.

Estrogen and Tibolone Metabolite Levels in Blood and Breast Tissue of Postmenopausal Women Recently Diagnosed With Early-Stage Breast Cancer and Treated With Tibolone or Placebo for 14 Days

Unlike estrogens plus progestagens, tibolone, a selective tissue estrogenic activity regulator, does not increase breast tenderness and mammographic density. To elucidate this, serum and breast levels of tibolone and estrogenic metabolites are measured. Postmenopausal women (n = 102) with early-stage, ER + ve, primary breast cancer received tibolone or placebo for 14 days in an exploratory, double-blind, randomized trial (STEM carcinoma tissue). Baseline and presurgery sera were collected; tumor tissues were obtained at surgery. E1 (estrone), E2 (estradiol), E1S (estrone-sulfate), tibolone—its nonsulfated, monosulfated, and disulfated 3-hydroxymetabolites—and Δ 4-tibolone were measured by validated gas chromatography and mass spectrometry and liquid chromatography with tandem mass spectrometry assays. More than 12 hours after the final dose, serum E1, E 2, and E1S levels were unchanged with placebo, whereas tibolone significantly increased E1S and the E1S/(E1 + E2) ratio. In tumors, E1 and E2 levels were higher than in serum, and E1S levels were lower, with placebo and tibolone administration. The percentage of E1S was about 90% in serum and 16% in tissue. Tibolone did not affect tissue levels of endogenous estrogens. Serum levels of estrogenic 3α- and 3β-hydroxytibolone, progestagenic/androgenic Δ 4-tibolone, and monosulfate metabolites were low. Serum 3αS,17βS-tibolone and 3 βS,17βS-tibolone levels were 250 and 52 ng/mL, respectively. Tumor levels of 3α- and 3β-hydroxytibolone and Δ 4-tibolone were higher than in serum, but disulfate levels were lower. The percentage of sulfated tibolone metabolites was 99% in serum and 96% in tumor. Serum metabolite patterns of estradiol and tibolone are different from those in tissues and are compatible with neutral effects of tibolone on breast Ki67 expression.