steroids 71 ( 2 0 0 6 ) 639–646 available at www.sciencedirect.com journal homepage: www.elsevier.com/locate/steroids Lack of aromatisation of the 3-keto-4-ene metabolite of tibolone to an estrogenic derivative Bindumalini Raobaikady, Michael F.C. Parsons, Michael J. Reed * , Atul Purohit Endocrinology and Metabolic Medicine, Faculty of Medicine, Imperial College, St. Mary’s Hospital, London W2 1NY, UK article info Article history: Received 21 November 2005 Received in revised form 20 March 2006 Accepted 30 March 2006 Keywords: Tibolone Aromatase 7-Methyl norethisterone 7-Methyl ethinylestradiol Hormone-replacement therapy abstract Tibolone is used for the treatment of climacteric symptoms in postmenopausal women. It is metabolised in a tissue-specific manner so that while some metabolites exert estrogenic effects on bone and the CNS, others are thought to protect the breast and endometrium from estrogenic stimulation. Tibolone is a 7-methyl derivative of 19-norethynodrel. Since the introduction of synthetic progestagens for therapeutic use there has been considerable controversy as to whether they can undergo aromatisation to give rise to the potent estrogen, ethinylestradiol. In this study, we examined whether the delta-4-ene (7-methyl norethis- terone) metabolite of tibolone, which has a similar delta-4-ene A-ring structure to that of the estrone precursor, androstenedione, could undergo aromatisation to the potent estrogen, 7-methyl ethinylestradiol. For these studies, JEG-3 choriocarcinoma cells were employed as they have a very high level of aromatase activity. TLC and HPLC procedures were developed to separate phenolic from non-phenolic compounds and were initially used to confirm that JEG-3 cells readily aromatised androstenedione to estrogens (up to 74%). The aromatisation of androstenedione to estrogens by these cells could be completely blocked with the potent aromatase inhibitor letrozole. When [ 3 H] 7-methyl norethisterone was incubated with JEG- 3 cells no evidence for its conversion to [ 3 H] 7-ethinylestradiol was obtained. Radioactivity detected on the TLC plate or HPLC fractions where standard 7-methyl ethinylestradiol was located, revealed that similar levels were present when 7-methyl norethisterone was incu- bated with culture medium alone or with JEG-3 cells in the absence or presence of letrozole. From these investigations, it is concluded that 7-methyl norethisterone does not undergo aromatisation to an estrogenic derivative. © 2006 Elsevier Inc. All rights reserved. 1. Introduction Tibolone (Fig. 1, 1) is a 7-methyl derivative of the syn- thetic progestagen, norethynodrel and is used as a hormone replacement therapy for postmenopausal women [1,2]. In postmenopausal women after its oral administration 75% of the total dose is present in the circulation as sulfated metabo- lites [3]. Tibolone exerts estrogenic effects on bone and the CNS as a result of being metabolised to the 3- and 3-hydroxy tibolone metabolites which have a low affinity for the estro- ∗ Corresponding author. Tel.: +44 207 886 1738; fax: +44 207 886 1790. E-mail address: m.reed@imperial.ac.uk (M.J. Reed). gen receptors (ER)  and  [4,5]. In contrast, it is not considered to exert any estrogenic effects on the breast or endometrium [6]. These differential effects of tibolone are thought to result from the ability of tibolone, and some of its unconjugated and sulfated metabolites, to inhibit steroid sulfatase (STS) activ- ity in breast tissues and the formation of the 3-keto-4-ene isomer (7-methyl norethisterone, 7MeNET, Fig. 1, 2) in the endometrium [7–9]. Since the introduction of synthetic pro- gestagens, such as norethynodrel and norethisterone (NET Fig. 1, 3) in the 1960s, there has been considerable controversy 0039-128X/$ – see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.steroids.2006.03.006