The Oestrogenic Effects of Gestodene, a Potent Contraceptive Progestin, Are Mediated by Its A-Ring Reduced Metabolites
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693 The oestrogenic effects of gestodene, a potent contraceptive progestin, are mediated by its A-ring reduced metabolites A E Lemus1,2, V Zaga2, R Santillán2, G A García3, I Grillasca3, P Damián-Matsumura1, K J Jackson4, A J Cooney4, F Larrea2 and G Pérez-Palacios5 1Department of Reproductive Biology, Universidad Autónoma Metropolitana-Iztapalapa, Mexico City, Mexico 2Department of Reproductive Biology, Instituto Nacional de la Nutrición S. Zubirán, Mexico City, Mexico 3Facultad de Química, Universidad Nacional Autónoma de México, Mexico City, Mexico 4Department of Cell Biology, Baylor College of Medicine, Houston, Texas, USA 5Dirección General de Salud Reproductiva, Secretaría de Salud, Mexico City, Mexico (Requests for offprints should be addressed to A E Lemus, Departamento de Biología de la Reproducción, Instituto Nacional de la Nutrición S. Zubirán, Vasco de Quiroga 15, México, D.F., C.P. 14000, Mexico; Email: [email protected]) Abstract Gestodene (17-ethynyl-13-ethyl-17-hydroxy-4,15- assessed by its capability to induce oestrogen-dependent gonadien-3-one) is the most potent synthetic progestin progestin receptors (PR) in the anterior pituitary of currently available and it is widely used as a fertility castrated female rats. regulating agent in a number of contraceptive formulations The results demonstrated that 3GSD and 3GSD were because of its high effectiveness, safety and acceptability. able to activate, in a dose-dependent manner, the hER- The observation that contraceptive synthetic progestins mediated transcription of both the -galactosidase and the exert hormone-like effects other than their progestational CAT reporter genes in the yeast and HeLa cells expression activities, prompted us to investigate whether gestodene systems respectively. In both assays the 3 derivative of (GSD) administration may induce oestrogenic effects, GSD exhibited a significantly greater oestrogenic effect even though the GSD molecule does not interact with than its 3 isomer, while unchanged GSD and 5GSD intracellular oestrogen receptors (ER). were completely ineffective. Neither 3GSD nor 3GSD To assess whether GSD may exert oestrogenic effects exhibited oestrogen synergistic actions. Interestingly, the through some of its neutral metabolites, a series of exper- pure steroidal anti-oestrogen ICI-182,780 diminished imental studies were undertaken using GSD and three of the transactivation induced by 3GSD and 3GSD in the its A-ring reduced metabolites. Receptor binding studies yeast expression system. Furthermore, administration of by displacement analysis confirmed that indeed GSD does 3GSD resulted in a significant increase of oestrogen- not bind to the ER, whereas its 3,5-tetrahydro reduced dependent PR in the anterior pituitaries of castrated rats in derivative (3GSD) interacts with a relative high affinity comparison with vehicle-treated animals. The character- with the ER. The 3,5 GSD isomer (3GSD) also binds istics of the 3GSD-induced PR were identical to those to the ER, though to a lesser extent. The ability of the induced by oestradiol benzoate. A-ring reduced GSD derivatives to induce oestrogenic The overall results demonstrate that 3GSD and its 3 actions was evaluated by the use of two different molecular isomeric alcohol specifically bind to the ER and possess a bioassays: (a) transactivation of a yeast system co- weak intrinsic oestrogenic activity, whereas unmodified transfected with the human ER (hER) gene and GSD does not. The data contribute to a better understand- oestrogen responsive elements fused to the -galactosidase ing of the GSD mechanism of action and allow the reporter vector and (b) transactivation of the hER- hypothesis to be advanced that the slight oestrogen- mediated transcription of the chloramphenicol acetyl like effects attributable to GSD are mediated by its transferase (CAT) reporter gene in a HeLa cells expression non-phenolic, tetrahydro reduced metabolites. system. The oestrogenic potency of 3GSD was also Journal of Endocrinology (2000) 165, 693–702 Introduction generation contraceptive formulations, representing a breakthrough in the field of fertility regulation (Kuhl The availability of novel, very potent synthetic progestins 1996). The most effective of these progestins is gestodene has allowed the development of the so-called third (GSD), a synthetic 19-nor steroid that has been widely Journal of Endocrinology (2000) 165, 693–702 Online version via http://www.endocrinology.org 0022–0795/00/0165–693 2000 Society for Endocrinology Printed in Great Britain Downloaded from Bioscientifica.com at 10/01/2021 04:38:28PM via free access 694 A E LEMUS and others · Oestrogenic effects of gestodene used in low dose combined oral contraceptives (Fotherby exerts potent oestrogen-like effects through its neutral & Caldwell 1994, Wilde & Balfour 1995). Recently, a tetrahydro reduced metabolites. series of studies (WHO 1995a,b, Jick et al. 1995, Lewis et al. 1996) have suggested that low dose, combined oral contraceptives pills, containing highly potent progestins, Materials and Methods such as GSD, probably carry a small risk of venous thromboembolic disease (VTD) beyond that attributable Steroids and chemicals to combined oral formulations containing less potent Authentic GSD was kindly provided by Schering AG progestins such as norethisterone or levonorgestrel. The (Berlin, Germany) and 5-dihydro GSD (5GSD) was origin of VTD associated with the use of hormonal synthesised by lithium–ammonia reduction of GSD, contraceptives is multifactorial and a number of mech- crystallised from ethyl acetate–hexane and purified by flash anisms has been involved (Inman & Vessey 1968, chromatography (Still et al. 1978). A 60% yield of the pure Vessey et al. 1986, Helmrich et al. 1987, WHO 1989, compound was obtained. The 3,5GSD tetrahydro Vandenbroucke et al. 1994, WHO 1995b, Spitzer et al. derivative (3GSD) was prepared from reduction of 1996, Winkler 1998). The unexpected association 5GSD with -selectride under anhydrous conditions between the use of GSD containing oral contraceptives (Brown & Krifhnamurthy 1972). Sodium hydroxide and and an increased risk of VTD raised the controversial hydrogen peroxide were added, and following extraction question as to whether GSD may exert potent oestrogen with ethyl acetate, a mixture of 3- (98%) and 3- (2%) agonistic or synergistic effects (WHO Scientific Group GSD derivatives were obtained (yield: 95%). Synthesis of 1998), particularly since the GSD molecule does not 3,5 tetrahydro GSD (3GSD) was performed by interact with intracellular oestrogen receptors (ER) sodium borohydride reduction (Bowers et al. 1958) of (Düstenberg et al. 1987). 5GSD (yield: 70%). The molecular structures of GSD Evidence has been accumulated over the last years and its metabolites are depicted in Fig. 1. Chemical indicating that contraceptive synthetic progestins exert purity of GSD and its derivatives was assessed by their potent hormone-like effects other than their progestational melting points, HPLC behaviour, infrared absorption, and activities (Vilchis et al. 1986, Pérez-Palacios et al. 1992, 13C- and 1H-nuclear magnetic resonance. The physical Lemus et al. 1997). The hormonal agonist, synergistic and and spectroscopic constants of the A-ring reduced GSD even antagonistic effects of synthetic progestins are medi- derivatives were as follows: ated either by their interaction with the wrong receptors (Bardin 1983, Chávez et al. 1985, Lemus et al. 1992) or by 5GSD: m.p. 168–170 C; i.r. (KBr) 3362, 3258, 3047, their metabolic conversion products (Larrea et al. 1987, 2948, 2924, 2857, 2086, 1697 cm1; 1H-NMR (300 Lemus et al. 1992). MHz, CDCl3) 5·92 (dd, J=5·7 and 1·5 Hz, 1H), 5·67 This study was aimed at elucidating the role of the (dd, J=5·7 and 3·6 Hz, 1H), 2·60 (S, 1H), 0·867 (t, J=7·5 ff 13 GSD metabolites in determining oestrogen-like e ects. Hz, 3H); C-NMR (75 MHz, CDCl3) 11·11, 20·36, A series of experiments were undertaken to assess 25·60, 29·51, 30·30, 30·37, 33·70, 38·74, 41·21, 43·67, whether non-phenolic A-ring reduced derivatives of 45·59, 47·91, 48·53, 55·80, 56·50, 74·89, 83·06, 83·82, GSD may be involved in mediating its oestrogenic 132·01, 135·56, 211·51; MS (FAB) 313. activity, taking advantage of the fact that GSD, after its 3GSD: m.p. 178–181 C; i.r. (KBr) 3567, 3392, 3252, 1 1 administration, undergoes extensive metabolism in a 3051, 2929, 2089 cm ; H-NMR (300 MHz, CDCl3) similar manner to other 19-nor synthetic progestins 5·93 (dd, J=5·7 and 1·52 Hz, 1H), 5·65 (dd, J=5·7 and (Düstenberg et al. 1987). The in vivo formation of 3,5 3·6 Hz, 1H), 4·09 (m, 1H), 2·60 (s, 1H), 0·85 (t, J=7·5 13 GSD (3GSD) and 3,5 GSD (3GSD) has been Hz, 3H); C-NMR (75 MHz, CDCl3) 11·10, 20·38, demonstrated after the administration of 14C-labelled 23·56, 25·18, 29·60, 30·74, 32·90, 33·45, 36·15, 39·05, GSD to normal women (Düstenberg et al. 1987). 40·54, 46·96, 48·33, 55·87, 56·81, 66·33, 74·68, 83·31, Assessment of the intrinsic oestrogenic potency of GSD 83·83, 132·30, 135·33; MS (FAB) 315. and its derivatives was done by the use of ER binding 3GSD: m.p. 139–141 C; i. r. (KBr) 3373, 3281, 1 1 studies, oestrogen-induced transactivation systems in 2919, 2859 cm ; H-NMR (300 MHz, CDCl3) 5·84 yeast and HeLa cells previously co-transfected with (dd, J=5·5 and 1·2 Hz, 1H), 5·57 (dd, J=5·5 and 3·6 Hz, human ER (hER) expression vectors and their 1H), 3·47 (m, 1H), 2·53 (s, 1H), 0·768 (t, J=7·2 Hz, 3H); 13 cognate reporter vectors, and induction of oestrogen- C-NMR (75 MHz, CDCl3) 10·83, 20·21, 25·37, dependent progestin receptors (PR) in the rat anterior 28·12, 29·42, 30·60, 33·22, 35·27, 38·86, 41·17, 42·86, pituitary.