Karl-Heinrich Fritzemeier

The novel progestin drospirenone and its natural counterpart progesterone: Biochemical profile and antiandrogenic potential

Drospirenone is a novel progestin under clinical development that is similar to the natural hormone progesterone, combining potent progestogenic with antimineralocorticoid and antiandrogenic activities. This specific pharmacological profile of drospirenone is defined by its pattern of binding affinities to a variety of steroid hormone receptors. In the present study the affinity of drospirenone to the progesterone receptor (PR), the androgen receptor (AR), the glucocorticoid receptor (GR), the mineralocorticoid receptor (MR), and the estrogen receptor (ER) was re-evaluated by steroid binding assays and compared to those obtained for the natural hormone progesterone. Drospirenone displayed high affinity to PR and MR and low binding to AR, similar to progesterone. Unlike progesterone, which showed considerable binding to GR, drospirenone exhibited only low binding to this receptor. Neither drospirenone nor progesterone did bind to the ER. In addition to receptor binding studies, transactivation assays were carried out to investigate the effects of drospirenone and progesterone on AR-, GR-, and MR-mediated induction of transcription. Both progestins showed no androgenic but antiandrogenic activity by inhibiting AR-mediated transcription in a dose-dependent manner. This observation could be confirmed by in vivo experiments carried out with orchiectomized male rats, where the antiandrogenic potency of drospirenone was found to be about five- to ten-fold higher than that of progesterone. In contrast to progesterone, drospirenone was devoid of glucocorticoid activity. Both progestins did not show any antiglucocorticoid action. Furthermore, drospirenone and progesterone both showed considerable antimineralocorticoid activity and weak mineralocorticoid activity.

8-Prenyl naringenin is a potent ERα selective phytoestrogen present in hops and beer

1. IntroductionPhytoestrogens are compounds found in leafy plants andfungi that exhibit estrogenic activity both in vivo and invitro. Chemically they refer to the classes of isoflavones,flavanones, coumestrans and resorcylic acid lactones. Cir-cumstantial evidence suggests that phytoestrogens ingestedwith food have beneficial effects in the prevention of breastcancer, post-menopausal syndrome and atherosclerosis.However, the potency of most of these compounds is rel-atively low compared to 17 -estradiol. Recently, isolationof a potent phytoestrogen (8-prenyl naringenin (8-PN))from the heart wood of an indigenous tree in Thailand(Anaxagorea luzonensis A. Gray) was reported [1]. 8-PNwas also found in Compositae [2] and identified as the estro-genic component in hops (Humulus lupulus L.) and beer [3].We studied in vitro receptor binding of both natu-rally found 8-PN enantiomers 2S(−)8-PN and 2R(+)8-PNcompared to endogenous mammalian estrogens and otherphytoestrogens, using recombinant human ER /ER .Asastriking result we found that both 8-PN enantiomers showhigh affinity and strong selectivity for ER .2S(−)8-PNexhibits an overall higher affinity for both receptors than2R(+)8-PN.Using a mammalian cell-based transient transactivationassay, we were able to demonstrate that 8-PN is the strongestplant-derived ER agonist identified so far, being about 10times more potent than coumestrol and approximately 100times more potent than genistein. Surprisingly and in clearcontrast to genistein, 8-PN is a much weaker agonist ofER than of ER .

Improvement of Endothelial Dysfunction by Selective Estrogen Receptor-α Stimulation in Ovariectomized SHR

Abstract—Both known estrogen receptors, ER&agr; and ER&bgr;, are expressed in blood vessels. To gain further insight into the role of ER&agr; in a functional setting, we investigated the effect of the novel highly selective ER&agr; agonist Cpd1471 on vascular reactivity in ovariectomized spontaneously hypertensive rats (SHR). After ovariectomy or sham operation, 12-week-old female SHR received either 17&bgr;-estradiol (E2, 2 &mgr;g/kg body wt per day), the selective ER&agr; agonist Cpd1471 (30 &mgr;g/kg body wt per day), or placebo. Acetylcholine-induced endothelium-dependent vasorelaxation was significantly blunted in aortas from ovariectomized rats (Rmax, 53%±3% versus sham, 79%±2%; P <0.001). Treatment with E2 or Cpd1471 significantly augmented acetylcholine-induced relaxation in ovariectomized rats (Rmax, 70%±2%; resp, 73%±2%). Endothelium-independent relaxation induced by sodium nitroprusside was not different among the four groups. The contractile response induced by the nitric oxide (NO) synthase inhibitor N&ohgr;-nitro-l-arginine, an index of basal NO formation, was significantly lower in ovariectomized rats compared with sham-operated animals (53±2% versus 77%±5%; P <0.01) and was normalized by both E2 (70%±2%) and Cpd1471 (70%±3%). Aortic endothelial NO synthase (eNOS) expression and phosphorylation of the vasodilator-stimulated phosphoprotein, an index of NO/cGMP-signaling, was reduced in ovariectomized SHR and normalized by E2 and Cpd1471. In SHR after ovariectomy, endothelium-dependent NO-mediated vasorelaxation and eNOS expression are attenuated. The novel selective ER&agr; agonist Cpd1471 prevented these pathophysiological changes to a similar extent as E2. Thus, the pharmacological principle of selective ER&agr; activation mediates positive vascular effects.

Characterization of the novel progestin gestodene by receptor binding studies and transactivation assays.

Gestodene is a novel progestin used in oral contraceptives with an increased separation of progestogenic versus androgenic activity and a distinct antimineralocorticoid activity. This specific pharmacological profile of gestodene is defined by its pattern of binding affinities to a variety of steroid hormone receptors. In the present study the affinity of gestodene to the progesterone receptor (PR), the androgen receptor (AR), the glucocorticoid receptor (GR), the mineralocorticoid receptor (MR) and the estrogen receptor (ER) was re-evaluated by steroid binding assays and compared to those obtained for 3-keto-desogestrel and progesterone. The two synthetic progestins displayed identical high affinity to rabbit PR and similar marked binding to rat AR and GR, while progesterone showed high affinity to PR but only low binding to AR and GR. Furthermore, 3-keto-desogestrel exhibited almost no binding to MR, whereas gestodene, similar to progesterone, showed marked affinity to this receptor. In addition to receptor binding studies, transactivation assays were carried out to investigate the effects of gestodene on AR-, GR- and MR-mediated induction of transcription. In contrast to progesterone, which showed antiandrogenic activity, gestodene and 3-keto-desogestrel both exhibited androgenic activity. Furthermore, all three progestins exhibited weak GR-mediated antagonistic activity. In contrast to progesterone, which showed almost no glucocorticoid activity, gestodene and 3-keto-desogestrel showed weak glucocorticoid action. In addition, gestodene inhibited the aldosterone-induced reporter gene transcription, similar to progesterone, whereas unlike progesterone, gestodene did not induce reporter gene transcription. 3-Keto-desogestrel showed neither antimineralocorticoid nor mineralocorticoid action.

Identification of Selective Estrogen Receptor Modulators by Their Gene Expression Fingerprints

Clinical studies have shown that estrogen replacement therapy (ERT) reduces the incidence and severity of osteoporosis and cardiovascular disease in postmenopausal women. However, long term estrogen treatment also increases the risk of endometrial and breast cancer. The selective estrogen receptor (ER) modulators (SERMs) tamoxifen and raloxifene, cause antagonistic and agonistic responses when bound to the ER. Their predominantly antagonistic actions in the mammary gland form the rationale for their therapeutic utility in estrogen-responsive breast cancer, while their agonistic estrogen-like effects in bone and the cardiovascular system make them candidates for ERT regimens. Of these two SERMs, raloxifene is preferred because it has markedly less uterine-stimulatory activity than either estrogen or tamoxifen. To identify additional SERMs, a method to classify compounds based on differential gene expression modulation was developed. By analysis of 24 different combinations of genes and cells, a selected set of assays that permitted discrimination between estrogen, tamoxifen, raloxifene, and the pure ER antagonist ICI164384 was generated. This assay panel was employed to measure the activity of 38 compounds, and the gene expression fingerprints (GEFs) obtained for each compound were used to classify all compounds into eight groups. The compounds GEF predicted its uterine-stimulatory activity. One group of compounds was evaluated for activity in attenuating bone loss in ovariectomized rats. Most compounds with similar GEFs had similar in vivo activities, thereby suggesting that GEF-based screens could be useful in predicting a compounds in vivo pharmacological profile.

Identification of estrogen receptor ligands leading to activation of non-genomic signaling pathways while exhibiting only weak transcriptional activity.

Estrogen receptors (ERs) stimulate genomic effects by acting as nuclear transcription factors as well as non-genomic effects by activating distinct cytoplasmic protein kinase cascades. Non-genomic effects have been implicated in numerous cellular processes, such as proliferation, differentiation, apoptosis and vasorelaxation. To exploit non-genomic effects mediated by ERalpha for novel hormone replacement regimens, we screened a focused library of steroid receptor ligands to identify compounds exhibiting properties different from estradiol, i.e. substances that selectively stimulate non-genomic signal transduction pathways while exhibiting low genomic activities. Treatment of breast cancer cells and osteosarcoma cells with estradiol, estren, substance A and substance B led to non-genomic activation of Akt (protein kinase B) and extracellular signal-regulated kinase 1/2 (ERK1/2) signaling cascades mediated by Src (Rous Sarcoma Virus, non-receptor tyrosine kinase) and phosphatidylinositol-3-kinase (PI3K) stimulation. Such compounds leading to prominent Akt/ERK activation but exhibiting only weak genomic properties were applied in vasorelaxation assays, modeling physiological non-genomic ER responses. As expected from PI3K and Src activation data, substances were as effective as estradiol in mediating vasorelaxation. We assume that these pathway-selective estrogen receptor ligands may serve as potent lead structures for novel hormone replacement strategies exhibiting lesser side effects than the existing treatment paradigms.

Medroxyprogesterone acetate but not drospirenone ablates the protective function of 17 beta-estradiol in aldosterone salt-treated rats.

Controversial results obtained from human and animal studies on the prevention of heart disease by estrogens and progestins warrant a better understanding of nuclear hormone receptor function and interaction. To address this issue and taking into account that effects of synthetic progestins are not only referable to action through the progesterone receptor but may also be mediated by other steroid receptors, we characterized cardiovascular function and inflammatory gene expression in aldosterone salt-treated rats on long-term administration of 17β-estradiol, medroxyprogesterone acetate, and drospirenone, a new progestogen exhibiting antimineralocorticoid activity. The complex pattern of cardiovascular injury in ovariectomized Wistar rats induced by chronic aldosterone infusion plus a high-salt diet was significantly attenuated in sham-ovariectomized rats and by coadministration of 17β-estradiol in ovariectomized animals after 8 weeks of continuous treatment. The beneficial role of 17β-estradiol on blood pressure, cardiac hypertrophy, vascular osteopontin expression, perivascular fibrosis, and impaired NO-dependent relaxation of isolated aortic rings was completely abrogated by coadministration of medroxyprogesterone acetate. In contrast, drospirenone was either neutral or additive to 17β-estradiol in protecting against aldosterone salt-induced cardiovascular injury and inflammation. The current results support the hypothesis of complex interactions among estrogen, progesterone, glucocorticoid, androgen, and mineralocorticoid receptor signaling in cardiovascular injury and inflammation. Novel progestins, such as drospirenone, confer superior effects compared with medroxyprogesterone acetate in a model of aldosterone-induced heart disease because of its antimineralocorticoid properties.

Molecular Basis of the Interaction Specificity between the Human Glucocorticoid Receptor and Its Endogenous Steroid Ligand Cortisol

We analyzed the binding of five steroids to the human glucocorticoid receptor (hGR) experimentally as well as theoretically. In vitro, we measured the binding affinity of aldosterone, cortisol, estradiol, progesterone, and testosterone to hGR in competition with the ligand dexamethasone. The binding affinity relative to the endogenous ligand cortisol (100 %) is reduced for progesterone (22 %) and aldosterone (20 %) and is very weak for testosterone (1.5 %) and estradiol (0.2 %). In parallel, we constructed a homology model of the hGR ligand‐binding domain (LBD) based on the crystal structure of the human progesterone receptor (hPR). After docking the five steroids into the hGR model ligand‐binding pocket, we performed five separate 4‐ns molecular dynamics (MD) simulations with these complexes in order to study the complex structures. We calculated the binding affinities with two different approaches (MM/PBSA, FlexX) and compared them with the values of the experimentally determined relative binding affinities. Both theoretical methods allowed discrimination between strongly and weakly binding ligands and recognition of cortisol as the endogenous ligand of the hGR in silico. Cortisol binds most strongly due to a nearly perfect steric and electrostatic complementarity with the hGR binding pocket. Chemically similar ligands such as estradiol, testosterone, and progesterone also fit into the hGR binding pocket, but they are unable to form all those contacts with the amino acids of the protein that are necessary to yield a stable, transcriptionally active receptor conformation. Our analysis thus explains the selectivity of the human glucocorticoid receptor for its endogenous ligand cortisol at a molecular level.

Effects of a pure antiestrogen and progesterone on estrogen-mediated alterations of blood flow and progesterone receptor expression in the aorta of ovariectomized rabbits

There is ample evidence from epidemiological studies that estrogen-replacement therapy protects postmenopausal women against cardiovascular disease. One explanation for this beneficial effect could be the improvement of blood flow under estrogen therapy. By using ultrasound and Doppler color flow mapping we demonstrated in the aorta of ovariectomized rabbits a significant dose-dependent increase in blood flow after treatment with 17beta-estradiol. An increase in blood flow was already observed within 1 h of estradiol treatment and lasted until the end of a 14-day treatment phase. Progesterone did not attenuate the effects of 17beta-estradiol on aortic blood flow. The pure estrogen receptor antagonist ZM 182780, however, dose-dependently reversed the effect of 17beta-estradiol on blood flow after the 14-day treatment phase, but was not able to antagonize the rapid 17beta-estradiol effect on blood flow after 1 h. After killing the animals mRNA and protein expression of the progesterone receptor (PR), a known estrogen-responsive gene in classic target organs, were examined. Analogous to the blood flow results the PR mRNA level increased dose-dependently after 17beta-estradiol treatment, whereas ZM 182780 was able to reverse this effect. Immunohistochemical localization of PR in the aortic wall revealed an increase in immunoreactivity in fibroblasts of the adventitia after 17beta-estradiol treatment. ZM 182780, and to a lesser degree progesterone, reversed the 17beta-estradiol-induced increase in PR immunoreactivity. PR immunoreactivity was further detected in endothelial and smooth muscle cells, but the various hormonal treatments had no discernible effect on the PR mRNA level in these cellular compartments. Our findings in the aorta of OVX rabbits suggest that (a) 17beta-estradiol exhibits a rapid effect on arterial tone, (b) the pure estrogen receptor antagonist ZM 182780 inhibits the 17beta-estradiol effect on blood flow and PR mRNA and (c) progesterone does not attenuate the beneficial effect of estrogens on arterial tone.

11β-Aryl Steroids in the Androstene Series. The Role of the 11β-Region in Steroid Progesterone Receptor Interaction

Abstract The syntheses of 11β-arylandrost-4-en-3-one 24 and the corresponding 9β,19-cyclo derivative 8 are described. Steric interaction between C-19 and the aryl residue effects conformational changes of the steroid ring system that result in reduced affinity for the progesterone receptor. The conformation of 11β-arylandrostenes is discussed in comparison with known antiprogestational steroids.