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17β-amino-1,3,5(10)estratrien-3-ol (17βAE2), is the 17β-aminoestrogens prototype possessing anticoagulant activity, contrasting with the procoagulant effects of 17β-estradiol (17βE2). Its estrogenicity profile has not been reported, and it was evaluated by uterotrophic assay, estrogen receptor binding affinity and its ability to induce gene transcription of the human estrogen receptor (hER)α mediated in a Saccharomyces cerevisiae yeast expression system. Additionally, 17βAE2 and 17αAE2 were compared with 17βE2 in HeLa cells co-transfected with expression vectors for hERα or hERβ subtypes and for an estrogen-responsive reporter gene. Immature female CD1 mice and Wistar rats (21 days old) were treated for three days with 17βAE2 (10-5000 μg/kg), 17βE2 (0.001-1000 μg/kg) or vehicle (propylenglycol 10 ml/kg) and uterine weights were estimated. 17βAE2 increased uterine weight in a dose-dependent manner. The effective dose (ED)50 uterine weight values: 17βAE2=552 and 764 μg/kg (17βE2=4.8 and 16 μg/kg) and their relative uterotrophic potency were 0.86 and 2.1 (17βE2=100) in mice and rats, respectively. 17βAE2 competed with [(3)H]E2 for the estrogen receptor. The 17βAE2 relative binding affinities (RBAs) were: 0.074; Ki=2.2×10(-6)M (17βE2=100; Ki=1.6×10(-9)M); 0.029 and Ki=3.8×10(-6)M (17βE2=100; Ki=1.1×10(-9)M) for mice and rats uteri respectively. 17βAE2 activated hERα-mediated β-galactosidase transcription activity in the yeast system co-transfected with hERα gene. 17βAE2 effective concentration (EC)50=1.82 μM (17βE2=2.14 nM) with a relative potency of 0.12 (17βE2=100). These transactivation effects were abolished by the antagonist fulvestrant (ICI 182,780), similarly to 17βE2. 17βAE2 and 17αAE2 bind with low relative affinity to hERα and hERβ. Both induced hER-mediated reporter gene transactivation in a dose-response manner. The overall results provide evidence that 17βAE2 has a weak agonist estrogenic action greatly mediated through the hERβ and to a lesser extent the hERα at genomic level., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The binding of estradiol (E(2)) to estrogen receptors (ER) is followed by conformational changes resulting in coactivator or corepressor recruitment that influences gene transcription. A series of synthetic A-ring reduced 19-nortestosterone-derived progestins has the capacity to selectively bind and activate transcription through the ERalpha. Herein, the molecular mechanisms involved in ER subtype-selective interactions of these compounds as assessed by their effects upon both ERalpha and ERbeta structural conformation and their ability to induce recruitment of steroid receptor coactivator-1 (SRC-1) to ERalpha were investigated. The results demonstrated that all synthetic A-ring 3beta,5alpha-tetrahydro-reduced derivatives of 19-nortestosterone induced an ERalpha trypsin digestion pattern similar to that seen with E(2), without effects upon ERbeta. In addition, these compounds had the ability to recruit SRC-1 to the ligand-binding domain of ERalpha similar to E(2). Our data indicate that A-ring 3beta,5alpha-tetrahydro-reduced 19-nortestosterone-derived progestins behave as selective ERalpha agonists with ligand-receptor structural and functional responses similar to those induced with natural E(2).

Synthetic 19-nortestosterone-derived progestins show affinity for the androgen receptor (AR) and retain varying degrees of androgenic activity. In this study, AR- and progesterone receptor (PR)-dependent transcriptional activation induced by norethisterone (NET), levonorgestrel (LNG) and gestodene (GSD), and their 5alpha-reduced derivatives, including limited trypsin digestion of AR in the presence of natural and synthetic progestins were investigated. The results confirmed the progestogenic activity of the three 19-nortestosterone derivatives, which decreases after reduction of the 4-ene-double bound. These compounds were able to activate AR-dependent reporter gene expression, LNG and GSD being the stronger activators. 5alpha-Reduction of LNG and GSD did not change their androgenic transcriptional activity; however, the activation of AR by 5alpha-NET was four-fold higher than NET. The highest selectivity transcriptional index, as a measure of progestogenicity versus androgenicity, was obtained for NET. The 5alpha-reduced derivatives had values significantly lower than those of their parent compounds. Non-reduced and 5alpha-reduced 19-nortestosterone progestins induced virtually identical proteolysis fragmentation patterns of the AR to those observed with DHT.

Levonorgestrel (LNG), a 19-nor-testosterone derivative, is widely used in contraceptive formulations. This compound does not bind to the estrogen receptor (ER), but it shows estrogen-like effects under in vivo and in vitro conditions. The estrogenicity of LNG may be attributed to its bio-transformation into non-phenolic metabolites. In this study, the ability of A-ring reduced LNG metabolites to activate transcription via an estrogenic mechanism of action, including differences between ER alpha and ER beta subtypes, were investigated. Transactivation assays were performed in HeLa cells transfected with expression vectors for ER alpha and ER beta and an estrogen-responsive reporter gene. Cells were also transfected with expression vectors for both progesterone receptor (PR) isoforms (A or B). As expected, the tetrahydro derivatives of LNG (3 alpha,5 alpha- and 3 beta,5 alpha-LNG) showed significantly lower PR-mediated transcriptional activities through both isoforms when compared with progesterone (P(4)) and LNG. In contrast, the 3 beta,5 alpha-tetrahydro derivative resulted in a significant activation of estrogen-dependent gene transcription. This effect was selectively confined to the ER alpha, since little if any activity could be observed with the ER beta and no antagonistic activities were demonstrated. This study provides structural and molecular clues for the well documented in vitro and in vivo intrinsic estrogenicity of 19-nor-testosterone-derived progestins and ligand requirements for ER alpha recognition.

Estrogenic activities of the two 17beta-aminoestrogen (AE) derivatives, prolame and butolame, were studied upon coagulation, serum luteinizing hormone (LH) and uterine weight, including endometrial morphology in castrated female rats. We have also investigated the ability of these two compounds, as well as another AE pentolame, to activate transcription through the estrogen receptor alpha (ERalpha) and the estrogen receptor beta (ERbeta). Administration of prolame and butolame to castrated animals increased significantly (P < 0.01) the mean clotting time when compared with that obtained in the group of control animals. Butolame was a more potent anticoagulant than prolame (P < 0.01), as judged by their corresponding IC(50) (5.4 +/- 0.65 and 66.6 +/- 2.57 micro;g/animal, respectively). In contrast, estradiol significantly shortened blood clotting times (P < 0.005). Both prolame and butolame caused a significant inhibition of serum LH levels (EC(50) 8.10 +/- 0.79 and 17 +/- 64 microg/animal, respectively), and restored castration-induced reduction in uterine weight of ovariectomized rats (EC(50) 4.14 +/- 1.57 and 17.0 +/- 1.78 microg/animal, respectively). In terms of the effects of prolame, butolame and pentolame in transient transfection assays, all the three AE activated ER dependent reporter gene expression, however, only at high concentrations. Prolame had the highest activity followed by butolame and pentolame. Induction of transcription by these compounds was preferentially mediated through the ERalpha, especially in the case of pentolame where little, if any, activation occurred through the ERbeta. None of the compounds showed antagonistic activities through either ER subtype. The overall data suggest that modifications in the structure and length of the amino-alcohol side-chain at C-17 might have an impact on the affinity and estrogenic intrinsic properties of AE at the level of diverse target tissues.

Norethisterone (NET), a 19-nor synthetic progestin, undergoes enzyme-mediated 5alpha-reduction and exerts potent androgenic effects in target organs. To investigate its mode of androgenic action we examined, in a comparative manner, the in vitro metabolism of NET and testosterone (T), as well as the binding affinities to androgen receptors (AR) and the androgenic potency of NET, T, and their 5alpha-reduced derivatives. Bioconversion of [3H]-NET and [3H]-T was studied in rat prostate homogenates, AR binding affinity was assessed in rat ventral prostates using [3H]-mibolerone as the radioligand, and the androgenic potency was evaluated by the increase of beta-glucuronidase activity in the mouse kidney, and by the growth of accessory sex organs in castrated male rats. The results demonstrated that 5alpha-NET displayed a higher AR binding affinity but a significantly lower androgenic potency than unchanged NET. The bioconversion studies indicated that the metabolism of NET was similar to that of T, although to a lesser extent, thus ruling out the possibility that the synthetic progestin metabolizes rapidly into less active derivatives. To investigate the nature of the paradoxical effect of 5alpha-reduction upon the NET molecule, the interaction with AR and the androgenic potency of T, 19-nortestosterone (19norT), 17alpha-ethynyl testosterone (ET) and their 5alpha-reduced derivatives were examined. The results of AR binding studies revealed that 5alpha-reduction of T and ET significantly enhanced their affinities, and that the 5alpha-derivative of 19norT displayed a similar binding affinity to that exhibited by 19norT. In terms of biological activity, the results showed that 5alpha-reduction of T and 19norT significantly increased their androgenic potency, whereas 5alpha-reduction of ET resulted in a significant diminution of its androgenicity in a manner similar to that observed with the 5alpha-reduction of NET. When NET and 19norT were simultaneously administered with 5alpha-dihydrotestosterone they exhibited a potent synandrogenic activity, an effect that was cancelled by their 5alpha-reduction. Interestingly, ET displayed an antiandrogenic activity, an effect that was also suppressed by its 5alpha-reduction. The overall results demonstrated a distinctive, paradoxical effect of 5alpha-reduction upon the NET molecule, which was different from that seen in naturally occurring androgens, and which suggests that the presence of the 17alpha-ethynyl group plays a key role in this phenomenon. The data provided further evidence that the metabolism of synthetic contraceptive progestins modulates the expression of their hormone-like actions.

19-Nor synthetic progestins undergo extensive metabolism at the target cells. The resulting metabolic conversion products interact with putative steroid receptors within the cells, and through those interactions, they may exert either agonistic, synergistic and antagonistic hormonal effects. Studies conducted in our laboratories have disclosed that norethisterone (NET) and D-(1) norgestrel (LNG), two widely used contraceptive progestins, are biotransformed to several A-ring reduced (dihydro and tetrahydro) derivatives. The resulting metabolites 5 alpha-dihydro NET (5 alpha-NET) and 5 alpha-dihydro LNG bind with relative high affinity to the progesterone and androgen receptors. To gain insight into the underlying molecular events mediating the mode of action of NET and its neutral metabolites, we have examined the expression of their biological effects at target organs by using the rabbit uteroglobin gene model and the beta-glucuronidase activity of the mouse kidney. The results of a series of experiments seem to indicate that the enzyme-mediated formation of the 5 alpha (trans A/B ring junction) NET derivative results in a significant diminution of its progestational and androgenic potencies. Furthermore, 5 alpha-NET acquire a potent anti-progestational/contragestational effect as assessed in the female rabbit. These results demonstrated that 5 alpha-reduction of 19-nor progestins exerts a paradoxical effect, at least in terms of their hormone-like effects. The overall data are in line with the concept that metabolism of synthetic progestins at hormone-sensitive organs modulates their mechanisms of action.

Levonorgestrel (LNG) is a synthetic steroid that displays potent progestational and androgenic effects but it lacks estrogen-like activity. To examine the mode of action of this progestin, we studied its metabolism in vitro in target organs and the specific interactions of LNG and its metabolites with putative steroid receptors. The results demonstrated that [3H]LNG was efficiently converted to A-ring reduced derivatives when incubated with rat hypothalamus and pituitary. Under optimal incubation conditions, [3H]5 alpha-dihydro LNG (5 alpha-LNG) and [3H]3 alpha,5 alpha-tetrahydro LNG (3 alpha,5 alpha-LNG) were identified as the major metabolic conversion products, while [3H]3 beta,5 alpha-LNG formation occurred to a lesser extent. A-ring reduction of LNG was NADPH-dependent. Assessment of the relative binding affinities of LNG and its derivatives to progesterone (PR), androgen (AR) and estrogen (ER) receptors by displacement analysis revealed that unchanged LNG binds with high affinity to PR and AR but not to ER. 5 alpha-LNG exhibited a diminished though significant interaction with PR and an enhanced binding affinity for AR as compared with LNG, indicating that 5 alpha-reduction of LNG increases its affinity for AR. The most striking finding was that further reduction of the 5 alpha-LNG molecule at C-3 abolished its binding activity to PR, AR, and even to ER. The overall data provides a plausible explanation for the lack of estrogen agonistic action of LNG and for its potent progestational and androgenic effects.