Nestorone® as a Novel Progestin for Nonoral Contraception:Structure-Activity Relationships and Brain Metabolism Studies

Nestorone® (NES) is a highly potent non-androgenic progestin being developed for contraception. NES is a synthetic progestin that may possess neuroprotective and myelin regenerative potential as an added health benefits. In receptor transactivation experiments, NES displayed greater potency than progesterone to transactivate the human progesterone receptor (hPR). This was confirmed by docking experiments which revealed that NES adopts the same docking position within the PR ligand-binding domain (LBD) as progesterone and forms additional stabilizing contacts between 17α-acetoxy and 16-methylene groups and PR LBD supporting its higher potency than progesterone. The analogue 13-ethyl NES also establishes similar contacts as NES with Met909, leading to comparable potency as NES. In contrast, NES is not stabilized within the human androgen receptor (hAR)-LBD leading to negligible AR transactivation. Since progesterone acts in the brain by both PR-binding and indirectly via the metabolite allopregnanolone binding to GABA_A receptor (GABA_A_R), we investigated if NES is metabolized to 3α, 5α-tetrahydronestorone (3α, 5α-THNES) in the brain and if this metabolite could interact with GABA_A_R. In female mice, low concentrations of reduced NES metabolites were identified by Gas Chromatography-Mass Spectrometry in both plasma and brain. However, electrophysiological studies showed that 3α, 5α-THNES exhibited only limited activity to enhance GABAAR-evoked responses with WSS-1 cells and did not modulate synaptic GABA_A_Rs of mouse cortical neurons. Thus the inability of reduced metabolite of NES (3α, 5α-THNES) to activate GABA_A_R suggests that the neuroprotective and myelin regenerative effects of NES are mediated via PR binding and not via its interaction with the GABA_A_R

Dose-finding study of a 90-day contraceptive vaginal ring releasing estradiol and segesterone acetate.

ObjectiveTo evaluate serum estradiol (E2) concentrations during use of 90-day contraceptive vaginal rings releasing E2 75, 100, or 200 mcg/day and segesterone acetate (SA) 200 mcg/day to identify a dose that avoids hypoestrogenism.Study designWe conducted a multicenter dose-finding study in healthy, reproductive-aged women with regular cycles with sequential enrollment to increasing E2 dose groups. We evaluated serum E2 concentrations twice weekly for the primary outcome of median E2 concentrations throughout initial 30-day use (target ≥40 pg/mL). In an optional 2-cycle extension substudy, we randomized participants to 2- or 4-day ring-free intervals per 30-day cycle to evaluate bleeding and spotting based on daily diary information.ResultsSixty-five participants enrolled in E2 75 (n = 22), 100 (n = 21), and 200 (n = 22) mcg/day groups; 35 participated in the substudy. Median serum E2 concentrations in 75 and 100 mcg/day groups were <40 pg/mL. In the 200 mcg/day group, median E2 concentrations peaked on days 4-5 of CVR use at 194 pg/mL (range 114-312 pg/mL) and remained >40 pg/mL throughout 30 days; E2 concentrations were 37 pg/mL (range 28-62 pg/mL) on days 88-90 (n = 11). Among the E2 200 mcg/day substudy participants, all had withdrawal bleeding following ring removal. The 2-day ring-free interval group reported zero median unscheduled bleeding and two (range 0-16) and three (range 0-19) unscheduled spotting days in extension cycles 1 and 2, respectively. The 4-day ring-free interval group reported zero median unscheduled bleeding or spotting days.ConclusionsEstradiol concentrations with rings releasing E2 200 mcg/day and SA 200 mcg/day avoid hypoestrogenism over 30-day use.ImplicationsA 90-day contraceptive vaginal ring releasing estradiol 200 mcg/day and segesterone acetate 200 mcg/day achieves estradiol concentrations that should avoid hypoestrogenism and effectively suppresses ovulation

Effects of a one year reusable contraceptive vaginal ring on vaginal Microflora and the risk of vaginal infection: An open-label prospective evaluation

Background A contraceptive vaginal ring (CVR) containing Nestorone® (NES) and ethinyl estradiol (EE) that is reusable for 1-year (13 cycles) is under development. This study assessed effects of this investigational CVR on the incidence of vaginal infections and change in vaginal microflora. Methods There were 120 women enrolled into a NES/EE CVR Phase III trial and a microbiology substudy for up to 1-year of cyclic product use. Gynecological examinations were conducted at baseline, the first week of cycle 6 and last week of cycle 13 (or during early discontinuation visits). Vaginal swabs were obtained for wet mount microscopy, Gram stain and culture. The CVR was removed from the vagina at the last study visit and cultured. Semi-quantitative cultures for Lactobacillus, Gardnerella vaginalis, Enterococcus faecalis, Staphylococcus aureus, Escherichia coli, anaerobic gram negative rods (GNRs), Candida albicans and other yeasts were performed on vaginal and CVR samples. Vaginal infections were documented throughout the study. Results Over 1-year of use, 3.3% of subjects were clinically diagnosed with bacterial vaginosis, 15.0% with vulvovaginal candidiasis, and 0.8% with trichomoniasis. The detection rate of these three infections did not change significantly from baseline to either Cycle 6 or 13. Nugent scores remained stable. H2O2-positive Lactobacillus dominated vaginal flora with a non-significant prevalence increase from 76.7%at baseline to 82.7% at cycle 6 and 90.2% at cycle 13, and a median concentration of 107 colony forming units (cfu) per gram. Although anaerobic GNRs prevalence increased significantly, the median concentration decreased slightly (104 to 103cfu per gram). There were no significant changes in frequency or concentrations of other pathogens. High levels of agreement between vaginal and ring surface microbiota were observed. Conclusion Sustained use of the NES/EE CVR did not increase the risk of vaginal infection and was not disruptive to the vaginal ecosystem

Effects of a novel estrogen-free, progesterone receptor modulator contraceptive vaginal ring on inhibition of ovulation, bleeding patterns and endometrium in normal women

BACKGROUND: Progesterone receptor modulators (PRMs) delivered by contraceptive vaginal rings provide an opportunity for development of an estrogen-free contraceptive that does not require daily oral intake of steroids. The objective of this proof-of-concept study was to determine whether continuous delivery of 600–800 mcg of ulipristal acetate (UPA) from a contraceptive vaginal ring could achieve 80% to 90% inhibition of ovulation. STUDY DESIGN: This was a prospective, controlled, open-labeled, multicenter international trial to examine the effectiveness and safety of this prototype vaginal ring. Thirty-nine healthy women, 21–40 years old and not at risk of pregnancy, were enrolled at three clinic sites. Volunteers participated in a control cycle, a 12-week treatment period and a post-treatment cycle. Pharmacodynamic effects on follicular function and inhibition of ovulation, effects on endometrium, bleeding patterns and serum UPA levels were evaluated. RESULTS: Mean UPA levels during treatment were nearly constant, approximately 5.1 ng/mL throughout the study. Ovulation was documented in 32% of 111 “4-week treatment cycles.” A correlation was observed between serum UPA and degree of inhibition of ovarian activity. There was no evidence of hyperplasia of endometrium, but PRM-associated endometrial changes were frequently observed (41%). CONCLUSION: In this study, the minimum effective contraceptive dose was not established. Further studies are required testing higher doses of UPA to attain ovulation suppression in a higher percentage of subjects

Differential Signal Transduction of Progesterone and Medroxyprogesterone Acetate in Human Endothelial Cells

AbstractThe conjugated equine estrogens-only arm of the Women's Health Initiative trial, showing a trend toward protection from heart disease as opposed to women receiving also medroxyprogesterone acetate (MPA), strengthens the debate on the cardiovascular effects of progestins. We compared the effects of progesterone (P) or MPA on the synthesis of nitric oxide and on the expression of leukocyte adhesion molecules, characterizing the signaling events recruited by these compounds. Although P significantly increases nitric oxide synthesis via transcriptional and nontranscriptional mechanisms, MPA is devoid of such effects. Moreover, when used together with physiological estradiol (E2) concentrations, P potentiates E2 effects, whereas MPA impairs E2 signaling. These findings are observed both in isolated human endothelial cells as well as in vivo, in ovariectomized rat aortas. A marked difference in the recruitment of MAPK and phosphatidylinositol-3 kinase explains the divergent effects of the two gestagens. In addition, both P and MPA decrease the adhesiveness of endothelial cells for leukocytes when given alone or with estrogen. MPA is more potent than P in inhibiting the expression of vascular cell adhesion molecule-1 and intercellular adhesion molecule-1. However, when administered together with physiological amounts of glucocorticoids, MPA (which also binds glucocorticoid receptor) markedly interferes with the hydrocortisone-dependent stabilization of the transcription factor nuclear factor κB and with the expression of adhesion molecules, acting as a partial glucocorticoid receptor antagonist. Our findings show significant differences in the signal transduction pathways recruited by P and MPA in endothelial cells, which may have relevant clinical implications

Progesterone receptors: a key for neuroprotection in experimental stroke

Progesterone receptors (PR) are expressed throughout the brain. However, their functional significance remains understudied. Here we report a novel role of PR as crucial mediators of neuroprotection using a model of transient middle cerebral artery occlusion and PR knockout mice. Six hours after ischemia, we observed a rapid increase in progesterone and 5-dihydroprogesterone, the endogenous PR ligands, a process that may be a part of the natural neuroprotective mechanisms. PR deficiency, and even haploinsufficiency, increases the susceptibility of the brain to stroke damage. Within a time window of 24 h, PR-dependent signaling of endogenous brain progesterone limits the extent of tissue damage and the impairment of motor functions. Longer-term improvement requires additional treatment with exogenous progesterone and is also PR dependent. The potent and selective PR agonist Nestorone is also effective. In contrast to progesterone, levels of the neurosteroid allopregnanolone, which modulates -aminobutyric acid type A receptors, did not increase after stroke, but its administration protected both wild-type and PR-deficient mice against ischemic damage. These results show that 1) PR are linked to signaling pathways that influence susceptibility to stroke, and 2) PR are direct key targets for both endogenous neuroprotection and for therapeutic strategies after stroke, and they suggest a novel indication for synthetic progestins already validated for contraception. Although allopregnanolone may not be an endogenous neuroprotective agent, its administration protects the brain against ischemicdamageby signaling mechanisms not involving PR. Collectively, our data clarify the relative roles of PR and allopregnanolone in neuroprotection after stroke.Fil: Liu, Ailing. Institut National de la Santé et de la Recherche Médicale; Francia. Université Paris Sud; FranciaFil: Margaill, Isabelle. Institut National de la Santé et de la Recherche Médicale; Francia. Universite de Paris V; Francia. University Paris Descartes; FranciaFil: Zhang, Shaodong. Institut National de la Santé et de la Recherche Médicale; Francia. Université Paris Sud; FranciaFil: Labombarda, Maria Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Bioquímica Humana; ArgentinaFil: Coqueran, Bérard. Institut National de la Santé et de la Recherche Médicale; Francia. Universite de Paris V; FranciaFil: Delespierre, Brigitte. Université Paris Sud; Francia. Institut National de la Santé et de la Recherche Médicale; FranciaFil: Liere, Philippe. Université Paris Sud; Francia. Institut National de la Santé et de la Recherche Médicale; FranciaFil: Marchand Leroux, Catherine. Institut National de la Santé et de la Recherche Médicale; Francia. Universite de Paris V; FranciaFil: O’Malley, Bert W.. Baylor College of Medicine;Fil: Lydon, John P.. Baylor College of Medicine;Fil: de Nicola, Alejandro Federico. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Sitruk Ware, Regine. The Rockefeller University; Estados UnidosFil: Mattern, Claudia. MetP Pharma AG; SuizaFil: Plotkine, Michel. Universite de Paris V; FranciaFil: Schumacher, Michael. Institut National de la Santé et de la Recherche Médicale; Francia. Université Paris Sud; FranciaFil: Guennoun, Rachida. Université Paris Sud; Francia. Institut National de la Santé et de la Recherche Médicale; Franci

Characterization of molecular changes in endometrium associated with chronic use of progesterone receptor modulators: ulipristal acetate vs. mifepristone

Ulipristal acetate (UPA) is a selective progesterone receptor modulator (PRM), which is used as an emergency contraceptive in women. Recent studies demonstrated the efficacy of an UPA contraceptive vaginal ring (UPA-CVR) as a blocker of ovulation. However, the endometrium of women exposed to UPA over a 6-month period display glandular changes, termed PRM-associated endometrial changes (PAECs). We, therefore, investigated whether UPA-induced PAECs are associated with altered expression of the transcription factor heart- and neural crest derivatives-expressed protein 2 (HAND2) whose downregulation is observed in endometrial epithelial hyperplasia and cancer. Our results showed that while exposure to mifepristone, a well-known PRM, leads to suppression of endometrial HAND2 expression, long-term exposure to UPA-CVR did not cause downregulation of this marker. Further studies, using human primary endometrial stromal cells, confirmed that whereas mifepristone-mediated suppression of HAND2 elevated the levels of its downstream target fibroblast growth factor 18, UPA did not significantly alter the expression of this growth factor. A rationale for the differential regulation of HAND2 by these PRMs was provided by our observation that mifepristone-bound progesterone receptors turn over at a faster rate than those bound to UPA. Collectively, these results support the selective effects of different PRMs and indicate that chronic exposure to UPA does not alter the HAND2 pathway whose dysregulation is linked to complex atypical endometrial hyperplasia and cancer. The results from this study involving a limited number of clinical samples should pave the way for a larger study to determine the safety of UPA for long-term use

Comparative actions of progesterone, medroxyprogesterone acetate, drospirenone and nestorone on breast cancer cell migration and invasion

<p>Abstract</p> <p>Background</p> <p>Limited information is available on the effects of progestins on breast cancer progression and metastasis. Cell migration and invasion are central for these processes, and require dynamic cytoskeletal and cell membrane rearrangements for cell motility to be enacted.</p> <p>Methods</p> <p>We investigated the effects of progesterone (P), medroxyprogesterone acetate (MPA), drospirenone (DRSP) and nestorone (NES) alone or with 17β-estradiol (E2) on T47-D breast cancer cell migration and invasion and we linked some of these actions to the regulation of the actin-regulatory protein, moesin and to cytoskeletal remodeling.</p> <p>Results</p> <p>Breast cancer cell horizontal migration and invasion of three-dimensional matrices are enhanced by all the progestins, but differences are found in terms of potency, with MPA being the most effective and DRSP being the least. This is related to the differential ability of the progestins to activate the actin-binding protein moesin, leading to distinct effects on actin cytoskeleton remodeling and on the formation of cell membrane structures that mediate cell movement. E2 also induces actin remodeling through moesin activation. However, the addition of some progestins partially offsets the action of estradiol on cell migration and invasion of breast cancer cells.</p> <p>Conclusion</p> <p>These results imply that P, MPA, DRSP and NES alone or in combination with E2 enhance the ability of breast cancer cells to move in the surrounding environment. However, these progestins show different potencies and to some extent use distinct intracellular intermediates to drive moesin activation and actin remodeling. These findings support the concept that each progestin acts differently on breast cancer cells, which may have relevant clinical implications.</p