Tibolone is not converted by human aromatase to 7α-methyl-17α-ethynylestradiol (7α-MEE):

Abstract

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 7α-methyl-17α-ethynylestradiol (7α-MEE) by human recombinant aromatase. Testosterone (T), 17α-methyltestosterone (MT), 19-nortestosterone (Nan), 7α-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, 3α- and 3β-hydroxytibolone were not converted by human aromatase whereas the estrogenic activity formed with the Δ⁴-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 17α-ethynyl-group prevents aromatization of (19-nor)steroids while 7α-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.

Introduction

In humans tibolone displays tissue-specific effects and is used for the treatment of climacteric complaints and the prevention of osteoporosis: it acts as an estrogen on bone, central nervous system and on vagina while no estrogenic stimulation could be found on breast or endometrium [1], [2], [3], [4], [5], [6]. After oral application tibolone is rapidly converted to 3α- and 3β-hydroxytibolone and the Δ⁴-isomer without detecting phenolic A-ring metabolites [7], [8], [9], [10]. The observed estrogenic activities can be mediated by the 3α- and 3β-hydroxytibolones which are shown to bind to the ERα and ERβ at about 30% of the potency of estradiol (E2) [11]. Despite the low binding compared to E2 the estrogenic effects on bone can be explained by the tissue-specific activation of the large amounts of circulating inactive sulfated estrogenic metabolites [10], [11]. However, we also want to investigate to what extent the estrogenic activities of tibolone are due to aromatization of the A-ring of tibolone or the Δ⁴-isomer by the enzyme aromatase, leading to 7α-methyl-17α-ethynylestradiol (7α-MEE).

The enzyme aromatase belongs to the cytochrome P450 superfamily, located in the microsomal fraction and formed from the CYP19 gene [12]. The enzyme complex is composed of two polypeptides, aromatase cytochrome P450 and an NADPH-cytochrome c reductase [13], [14]. CYP19 converts the androgens, androstenedione and testosterone to the phenolic A-ring derivatives, estrone and estradiol, respectively. The aromatization of C19 steroids takes place in three sequential oxidative steps. The first step is the hydroxylation of the C19-methyl group followed by a second hydroxylation on the C19-methyl group resulting in the formation of a 19-oxo-compound. The last step is the cleavage of the bond between C10 and C19 resulting in the formation of formic acid and the aromatized A-ring analogue with specific loss of the 1β- and 2β-hydrogen atoms [15], [16]. CYP19 is widely expressed in human tissues and is found in placenta, ovary, endometrium, bone, and brain [17] but not in human adult liver [18].

In the literature a controversy exists about the aromatization of 19-norsteroids. Since these synthetic steroids lack the C19-methyl, required for the initial step for aromatization, some authors claim that 19-norsteroids cannot be aromatized by (human) aromatase in in vitro systems [19], [20], [21], [22] while other authors claim that aromatization is possible [23], [24], [25], [26]. It is suggested that another type of aromatase enzyme could be involved [19], [27] or that the 19-norsteroids are irreversible or suicide inhibitors of aromatase [28], [29], [30]. It is also not clear whether the 17α-substituted progestagen norethisterone (17α-ethynyl-19-nortestosterone, NET) can be aromatized. In menopausal women some conversion of NET to ethynyl estradiol (EE) has been reported to a degree varying from 0.35 to 2.3%. These conversion rates were considered to be of little clinical significance [31] or small but significant [32], respectively. In a study with postmenopausal women on HRT (2 mg estradiol, 1 mg estriol, and 1 mg NET), a conversion rate of 0.35% of NET to EE was found [33]. In some in vitro systems NET inhibited the aromatase activity [34], [35], [36], [37] whereas in other in vitro systems cell cultures NET could be aromatized to EE [38], [39], [40]. In addition, a recently published study reported that tibolone could be converted to 7α-MEE in young pre-menopausal women [41]. The common detection techniques in these in vitro and in vivo studies were, thin layer chromatography, HPLC, gas chromatography, and radio immuno assays. Gas chromatography requires derivatization of the steroids which may lead to chemical aromatization of some steroids as demonstrated by the conversion of norethynodrel to EE [42]. Therefore, we wanted to investigate whether assays measuring in vitro biological activity would yield more sensitive detection methods. The detection limit of the recently developed analytical assay, LC-MSMS without derivatization, is in the nanomolar range. The bioassays are expected to detect activities in the (sub)picomolar range [11]. We used CHO cells stably transfected with human steroid receptors and a luciferase reporter gene to detect both the reduction and generation of hormonal activities in the cell cultures. LC-MSMS detection was used as the golden standard analytical assay for detection of phenolic A-ring derivatives.

This study investigates whether tibolone, its metabolites, 19-norsteroids and 17α-substituted steroids can interfere with the aromatization of androstenedione or whether these steroids can be aromatized. To detect the reduction in levels of test compounds and the generation of the phenolic A-ring analogue, we have used two different, sensitive, detection methods, bioassays and the conventional analytical LC-MSMS without derivatization.