Safety and efficacy of the selective progesterone receptor modulator asoprisnil for heavy menstrual bleeding with uterine fibroids: pooled analysis of two 12-month, placebo-controlled, randomized trials.

STUDY QUESTION: Can asoprisnil, a selective progesterone receptor modulator, provide clinically meaningful improvements in heavy menstrual bleeding (HMB) associated with uterine fibroids with an acceptable safety profile? SUMMARY ANSWER: Uninterrupted treatment with asoprisnil for 12 months effectively controlled HMB and reduced fibroid and uterine volume with few adverse events. WHAT IS KNOWN ALREADY: In a 3-month study, asoprisnil (5, 10 and 25 mg) suppressed uterine bleeding, reduced fibroid and uterine volume, and improved hematological parameters in a dose-dependent manner. STUDY DESIGN, SIZE, DURATION: In two Phase 3, double-blind, randomized, placebo-controlled, multicentre studies, women received oral asoprisnil 10 mg, asoprisnil 25 mg or placebo (2:2:1) once daily for up to 12 months. PARTICIPANTS/MATERIALS, SETTING, METHODS: Premenopausal women ≥18 years of age in North America with HMB associated with uterine fibroids were included (N = 907). The primary efficacy endpoint was the percentage of women who met all three predefined criteria at 12 months or the final month for patients who prematurely discontinued: (1) ≥50% reduction in monthly blood loss (MBL) by menstrual pictogram, (2) hemoglobin concentration ≥11 g/dL or an increase of ≥1 g/dL, and (3) no interventional therapy for uterine fibroids. Secondary efficacy endpoints included changes in other menstrual bleeding parameters, volume of the largest fibroids, uterine volume and health-related quality of life (HRQL). MAIN RESULTS AND THE ROLE OF CHANCE: In all, 90% and 93% of women in the asoprisnil 10-mg and 25-mg groups, respectively, and 35% of women in the placebo group met the primary endpoint (P < 0.001). Similar results were observed at month 6 (P < 0.001). The percentage of women who achieved amenorrhea in any specified month ranged from 66-78% in the asoprisnil 10-mg group and 83-93% in the asoprisnil 25-mg group, significantly higher than with placebo (3-12%, P < 0.001). Hemoglobin increased rapidly (by month 2) with asoprisnil treatment and was significantly higher versus placebo throughout treatment. The primary fibroid and uterine volumes were significantly reduced from baseline through month 12 with asoprisnil 10 mg (median changes up to -48% and -28%, respectively) and 25 mg (median changes up to -63% and -39%, respectively) versus placebo (median changes up to +16% and +13%, respectively; all P < 0.001). Dose-dependent, significant improvements in HRQL (Uterine Fibroid Symptom and Quality of Life instrument) were observed with asoprisnil treatment. Asoprisnil was generally well tolerated. Endometrial biopsies indicated dose- and time-dependent decreases in proliferative patterns and increases in quiescent or minimally stimulated endometrium at month 12 of treatment. Although not statistically significantly different at month 6, mean endometrial thickness at month 12 increased by ~2 mm in both asoprisnil groups compared with placebo (P < 0.01). This effect was associated with cystic changes in the endometrium on MRI and ultrasonography, which led to invasive diagnostic and therapeutic procedures in some asoprisnil-treated women. LIMITATIONS, REASONS FOR CAUTION: Most study participants were black; few Asian and Hispanic women participated. The study duration may have been insufficient to fully characterize the endometrial effects. WIDER IMPLICATIONS OF THE FINDINGS: Daily uninterrupted treatment with asoprisnil was highly effective in controlling menstrual bleeding, improving anemia, reducing fibroid and uterine volume, and increasing HRQL in women with HMB associated with uterine fibroids. However, this treatment led to an increase in endometrial thickness and invasive diagnostic and therapeutic procedures, with potential unknown consequences. STUDY FUNDING/COMPETING INTEREST(S): This trial was funded by AbbVie Inc. (prior sponsors: TAP Pharmaceutical Products Inc., Abbott Laboratories). E.A. Stewart was a site investigator in the Phase 2 study of asoprisnil and consulted for TAP during the design and conduct of these studies while at Harvard Medical School and Brigham and Women's Hospital. She received support from National Institutes of Health grants HD063312, HS023418 and HD074711 and research funding, paid to Mayo Clinic for patient care costs related to an NIH-funded trial from InSightec Ltd. She consulted for AbbVie, Allergan, Bayer HealthCare AG, Gynesonics, and Welltwigs. She received royalties from UpToDate and the Med Learning Group. M.P. Diamond received research funding for the conduct of the studies paid to the institution and consulted for AbbVie. He is a stockholder and board and director member of Advanced Reproductive Care. He has also received funding for study conduct paid to the institution from Bayer and ObsEva. A.R.W. Williams consulted for TAP and Repros Therapeutics Inc. He has current consultancies with PregLem SA, Gedeon Richter, HRA Pharma and Bayer. B.R. Carr consulted for and received research funding from AbbVie. E.R. Myers consulted for AbbVie, Allergan and Bayer. R.A. Feldman received compensation for serving as a principal investigator and participating in the conduct of the trial. W. Elger was co-inventor of several patents related to asoprisnil. C. Mattia-Goldberg is a former employee of AbbVie and may own AbbVie stock or stock options. B.M. Schwefel and K. Chwalisz are employees of AbbVie and may own AbbVie stock or stock options. TRIAL REGISTRATION NUMBER: NCT00152269, NCT00160381 (clinicaltrials.gov). TRIAL REGISTRATION DATE: 7 September 2005; 8 September 2005. DATE OF FIRST PATIENT'S ENROLMENT: 12 September 2002; 6 September 2002.

Next step in the development of mesoprogestins: the preclinical profile of EC313.

Introduction: The pharmacological target for progesterone, different progestins, and Selective Progesterone Receptor Modulators (SPRMs) is the nuclear progesterone receptor (PR). EC313 is a new member of a subgroup of SPRMs, mesoprogestins, which combine especially PR- agonistic and PR-antagonistic activities in one molecule. Methods: The suitable in vivo-model for the differentiation of SPRMs from the subgroup of mesoprogestins is the estrogen-primed juvenile rabbit endometrium assay (McPhail Assay). Remarkably, in contrast to other well-known SPRMs with no agonistic effects in this test, EC313 shows clear partial PR-agonistic effects that are higher than that of the well-known mesoprogestin Asoprisnil which already demonstrated remarkable clinical effectiveness for the treatment of uterine fibroids and endometriosis. The findings from the guinea pig studies presented here can be the impetus for further preclinical development of EC313. This model shows the same features for the termination of pregnancy by antiprogestins such as Mifepristone and Ulipristal acetate (UPA) in humans. Moreover, it is possible to distinguish between progestational and anti-progestational activities in the same experiment. Results: The EC313 treatment reveals PR dominance in the genital tract and inhibits unopposed estrogenic effects. In very high doses (30.0 mg/animal/day subcutaneously (s.c.)) given twice on pregnancy days 43 and 44, no premature labor was induced (in contrast to UPA, dosed at 10.0 and 30. mg/animal/day s.c.). The anti-ovulatory activity of EC313 exceeds that of Ulipristal acetate or Mifepristone. EC313 binds to the steroid receptors in vitro with a similar affinity as the natural ligand progesterone. At the glucocorticoid receptor (GR) EC313 acts as a weak inhibitor. Minor activities at the human androgen receptor (AR) and mineralocorticoid receptor (MR) are considered negligible. No binding to the estradiol receptor was detected. In contrast to some in vitro-receptor findings, estrogenic, anti-estrogenic, androgenic, anti-androgenic, glucocorticoid, and anti-glucocorticoid actions were absent in vivo. The tissue selectivity of EC313 was demonstrated previously by reducing the growth and proliferation of uterine fibroids in animal models (lowest effective dosage 0.1 mg/kg/day s.c.).. As shown in this article, the anti-fibroid activity of EC313 was confirmed with a 10 times lower dosage (0.01 mg/kg/day s.c.). It was also shown that EC313 reduces the growth of endometriotic lesions in a human xenograft immune-deficient (NOD-SCID) mice model with a comparatively very low dosage range. In the aforementioned EC313 activity model, UPA was tested as the reference compound, the clinical effectiveness of which has already been demonstrated. Discussion: For an explanation of these findings, the possibility is discussed that the mixed agonistic/antagonistic feature of EC313 is tissue target-specific based on its super-additive synergism characteristic for active bifunctional agents. In conclusion, the specific pharmacodynamic profile of this compound opens the possibility for the development of a drug with a distinct pharmaco-endocrinological profile against uterine fibroids, endometriosis, and other PR-dependent gynecological diseases.

Dental health, halitosis and mouth breathing in 10-to-15 year old children: A potential connection.

AIM: The relationship between mouth breathing and dental caries, gingival inflammation, and halitosis in children is contentious with studies reporting positive and negative associations; this study aimed at investigating the effect of mouth breathing on dental, gingival health status, and halitosis. MATERIALS AND METHODS: An observational cross-sectional study was carried out involving 785 randomly selected children and adolescents between the ages of 10 and 15 in the city of Leipzig, Germany (LIFE Child cohort). Caries levels and gingival health status for the upper-right and the lower-left central incisors were assessed by evaluating ICDAS scores and CPI scores, respectively. A standardised questionnaire was used to assess self-reported mouth-breathing habit and halitosis. RESULTS: This study showed a statistically significant association between halitosis and mouth breathing (OR=3.0; 95% CI: 1.5-6.2), and a significant increase in mouth breathing habit in males compared to females (59.7% vs. 40.3%; p<0.05). There were no statistically significant differences in ICDAS scores, orthodontic treatment, CPI scores, or socioeconomic status between the mouth and nasal-breathing groups. CONCLUSION: Mouth breathing habit has no effect on the prevalence of caries or gingivitis based on examining the upper-right central incisor (11) and the lower-left central incisor. However, mouth breathers showed a significant increase in halitosis compared to nasal-breathing individuals.

A 12-month extension study to evaluate the safety and efficacy of asoprisnil in women with heavy menstrual bleeding and uterine fibroids.

STUDY QUESTION: What is the safety and efficacy profile during long-term (12-24 months) uninterrupted treatment with the selective progesterone receptor modulator asoprisnil, 10 and 25 mg in women with heavy menstrual bleeding (HMB) associated with uterine fibroids? SUMMARY ANSWER: Uninterrupted treatment with asoprisnil should be avoided due to endometrial safety concerns and unknown potential long-term consequences. WHAT IS KNOWN ALREADY: Asoprisnil was well tolerated in shorter-term studies and effectively suppressed HMB and reduced fibroid volume. STUDY DESIGN SIZE DURATION: Women with uterine fibroids who had previously received placebo (n = 87) or asoprisnil 10 mg (n = 221) or 25 mg (n = 215) for 12 months in two double-blind studies entered this randomized uncontrolled extension study and received up to 12 additional months of treatment followed by 6 months of post-treatment follow-up. Women who previously received placebo were re-randomized to either asoprisnil 10 or 25 mg for the extension study. This report focuses on the 436 women who received asoprisnil in the double-blind studies and this extension study. Results for women who previously received placebo in the double-blind studies are not described. PARTICIPANTS/MATERIALS SETTING METHODS: Women ≥18 years of age who completed a 12-month, double-blind, placebo-controlled study, had estradiol levels indicating that they were not menopausal and had no endometrial hyperplasia or other significant endometrial pathology were eligible. The safety endpoints were focused on endometrial assessments. The composite primary efficacy endpoint was the proportion of women who demonstrated a response to treatment by meeting all three of the following criteria at the final month for participants who prematurely discontinued or at month 12 for those who completed the study: a reduction from initial baseline to final visit of ≥50% in the menstrual pictogram score, hemoglobin concentration ≥11 g/dl or an increase of ≥1 g/dl from initial baseline at the final visit, and no surgical or invasive intervention for uterine fibroids. Other efficacy endpoints included rates for amenorrhea and suppression of bleeding, changes in fibroid and uterine volume and changes in hematologic parameters. No statistical tests were planned or performed for this uncontrolled study. MAIN RESULTS AND ROLE OF CHANCE: Imaging studies revealed a progressive increase in endometrial thickness and cystic changes that frequently prompted invasive diagnostic procedures. Endometrial biopsy results were consistent with antiproliferative effects of asoprisnil. Two cases of endometrial cancer were diagnosed. At the final month of this extension study (total duration of uninterrupted treatment up to 24 months), the primary efficacy endpoint was achieved in 86 and 92% of women in the asoprisnil 10- and 25-mg groups, respectively. During each month of treatment, amenorrhea was observed in the majority of women (up to 77 and 94% at 10 and 25 mg, respectively). There was a progressive, dose-dependent decrease in the volume of the primary fibroid with asoprisnil 10 and 25 mg (-55.7 and -75.2% median decrease, respectively, from baseline [i.e. the beginning of the placebo-controlled study] to month 12 [cumulative months 12-24] of this extension study). These effects were associated with improvements in quality of life measures. LIMITATIONS REASONS FOR CAUTION: This study was uncontrolled, which limits the interpretation of safety and efficacy findings. The study also had multiple protocol amendments with the addition of diagnostic procedures and, because no active comparator was included, the potential place of asoprisnil in comparison to therapies such as GnRH agonists and surgery cannot be determined. WIDER IMPLICATIONS OF THE FINDINGS: Long-term, uninterrupted treatment with asoprisnil leads to prominent cystic endometrial changes that are consistent with the 'late progesterone receptor modulator' effects, which prompted invasive diagnostic procedures, although treatment efficacy is maintained. Although endometrial cancers were uncommon during both treatment and follow-up, these findings raise concerns regarding endometrial safety during uninterrupted long-term treatment with asoprisnil. This study shows that uninterrupted treatment with asoprisnil should be avoided due to safety concerns and unknown potential long-term consequences. STUDY FUNDING/COMPETING INTERESTS: AbbVie Inc. (prior sponsor, TAP Pharmaceutical Products Inc.) sponsored the study and contributed to the design and conduct of the study, data management, data analysis, interpretation of the data and the preparation and approval of the manuscript. Financial support for medical writing and editorial assistance was provided by AbbVie Inc. M. P. Diamond received research funding for the conduct of the study paid to the institution and is a consultant to AbbVie. He is a stockholder and board and director member of Advanced Reproductive Care. He has also received funding for study conduct paid to the institution for Bayer and ObsEva. E. A. Stewart participated as a site investigator in the phase 2 study of asoprisnil and served as a consultant to TAP Pharmaceuticals during the time of design and conduct of the studies while on the faculty of Harvard Medical School and Brigham and Women's Hospital, Boston, MA. In the last 3 years, she has received support from National Institutes of Health grants HD063312, HS023418 and HD074711. She has served as a consultant for AbbVie Inc., Allergan, Bayer HealthCare AG and Myovant for consulting related to uterine leiomyoma and to Welltwigs for consulting related to infertility. She has received royalties from UpToDate and the Med Learning Group. A.R.W. Williams has acted as a consultant for TAP Pharmaceutical Products Inc. and Repros Therapeutics Inc. He has current consultancies with PregLem SA, Gedeon Richter, HRA Pharma and Bayer. B.R. Carr has served as consultant and received research funding from AbbVie Inc. and Synteract (Medicines360). E.R. Myers has served as consultant for AbbVie Inc., Allergan and Bayer. R.A. Feldman received compensation for serving as a principal investigator and participating in the conduct of the trial. W. Elger was a co-inventor of several patents related to asoprisnil.C. Mattia-Goldberg is a former employee of AbbVie Inc. and owns AbbVie stock or stock options. B.M. Schwefel and K. Chwalisz are employees of AbbVie Inc. and own AbbVie stock or stock options. TRIAL REGISTRATION NUMBER: NCT00156195 at clinicaltrials.gov.

Model for Hormonal Emergency Contraception (HEC) in cycling and mated guinea pigs - Studies with the Progesterone Receptor Modulators (PRM) Ulipristal Acetate (UPA/CDB2914) and EC317.

A guinea pig model for new HEC methods is proposed. Two targets for HEC (Hormonal Emergency Contraception), ovulation and conception (post-mating study), were investigated using adjusted PRM treatments: (a) Ovulation inhibition study: Injections on cycle days 10-17, study of ovarian histology on day 18; (b) post-mating study: Injections on cycle days 1 and 2; rate of pregnant females was recorded at autopsy on day 18. P plasma levels permitted assessment of effects on ovulation in non-conceiving animals. RESULTS: (a) All controls had recently ovulated. Statistically significant anti-ovulatory effects (p < 0.05, Fisher's Exact Test) were seen at 10 mg UPA (ulipristal acetate, CDB2914) and ≥0.3 mg EC317; 100% inhibition was found for EC317 at 10, 3, and 1 mg/day. No dosage of UPA was 100% effective. (b) In post-mating studies, 16 of 30 controls were pregnant. Both PRMs (progesterone receptor modulator) exerted inhibitory effects on conception, none on imminent ovulation; 1 of 10 animals had living conceptuses after 10 mg UPA, none following 10 and 1 mg EC317/day, respectively. At pairwise comparison with controls, 10 mg was the lowest effective dosage for UPA (p < 0.05), and 1 mg for EC317 (p < 0.01). P plasma levels: Significantly lower P (p < 0.05) in subsequently pregnant vs non-pregnant controls was found on cycle day 3 or 4; this difference disappeared on day 8 or 9. This stage thus appears vulnerable to hormonal constellations and possibly PRM effects. HEC model: Effects on ovulation and conception were seen at the same dose levels of both PRM. Superior and more consistent effects of EC317 vs UPA (factor ≥10) suggest higher efficacy using EC317 for HEC.

Estradiol prodrugs (EP) for efficient oral estrogen treatment and abolished effects on estrogen modulated liver functions.

Oral compared to parenteral estrogen administration is characterized by reduced systemic but prominent hepatic estrogenic effects on lipids, hemostatic factors, GH-/IGF I axis, angiotensinogen. In order to avoid such adverse metabolic effects of oral treatment, estradiol (E2) prodrugs (EP) were designed which bypass the liver tissue as inactive molecules. Carbone17-OH sulfonamide [-O2-NH2] substituted esters of E2 (EC508, others) were synthesized and tested for carbonic anhydrase II (CA-II) binding. CA II in erythrocytes is thought to oppose extraction of EP from portal vein blood during liver passage. Ovariectomized (OVX, day minus 14) rats were orally treated once daily from day 1-3. Sacrifice day 4. Uteri were dissected and weighed. Cholesterol fractions and angiotensinogen were determined in plasma. Oral E2 and ethinyl estradiol (EE) generated dose related uterine growth and important hepatic estrogenic effects. EP induced uterine growth at about hundred-fold lower doses. This was possible with almost absent effects on plasma cholesterol or angiotensinogen. Preliminary pharmacokinetic studies with EC508 used intravenous and oral administration in male rats. Resulting blood levels revealed complete oral bioavailability. Further high blood- but low plasma concentrations indicated erythrocyte binding of EC508 in vivo as potential mechanism of low extraction at liver passage. Very high systemic estrogenicity combined with markedly lower or absent adverse hepatic estrogenic effects is evidence for a systemic release of E2 from sulfonamide EP. In conclusion, tested oral EP bypass the liver in erythrocytes furnishing systemic estradiol at hydrolysis. This mechanism avoids the hepatic estrogenic impact of conventional oral estrogen therapy.

Evidence for specific selenium target tissues and new biologically important selenoproteins.

After in-vivo labeling with [75Se]selenite the Se-containing proteins present in rat tissues were investigated by means of SDS-polyacrylamide gel electrophoresis. Thirteen Se-containing proteins or protein subunits with relative molecular weights of 12,100, 15,600, 18,000, 19,700, 22,200, 23,700, 27,800, 33,300, 55,500, 59,900, 64,900, 70,100 and 75,400 were detected in the tissue homogenates. The protein with the molecular weight of 23,700 was the subunit of glutathione peroxidase, which is the only selenoprotein so far known to have biological functions in animals. Most of these proteins were found in all tissues investigated but one was only detected in the testes and the spermatozoa and one was present mainly in the thyroid. With inadequate selenium intake there was a priority supply of the element to the brain, the reproductive and the endocrine organs, and at a molecular level to Se-containing proteins other than glutathione peroxidase. The results suggest important biological functions of these selenoproteins, especially in the specific target tissues.

Binding characteristics of progesterone and antiprogestin ZK 98.299 in human endometrial and myometrial cytosol.

The binding of ZK 98.299, a synthetic progesterone antagonist, with human endometrium and myometrium cytosol was studied and compared with that of progesterone. Progesterone showed specific saturable binding to its receptors in both endometrium and myometrium. ZK 98.299 and progesterone were mutually competitive for binding to progesterone receptors; however, the relative binding affinity of ZK 98.299 was 16% that of progesterone. ZK 98.299 exchanged the progesterone-labelled receptor sites. [3H]ZK 98.299 showed specific binding which was linearly related to the cytosol protein concentration. The binding was not saturable at 15 nM of ligand. The binding capacity and binding affinity of ZK 98.299 receptor was less than that of progesterone. Progesterone also partially displaced the binding of [3H]ZK 98.299. This study suggest that ZK 98.299 and progesterone both bind to the same protein. However, whether ZK 98.299 binds to progesterone receptors alone or even to other functionally related sites is not known. It appears that ZK 98.299 when present in higher concentration than progesterone would be an effective receptor ligand.

The effect of a new antiandrogenic steroid, 6-chloro-17-HYDROXY-1α, 2α-methylenepregna-4,6-diene-3,20-dione acetate (cyproterone acetate) on the sebaceous glands of mice.

The effect of a new antiandrogenic steroid, 6-chloro-17-hydroxy-la,2a-methylenepregna-4,6-diene-3,20-dione acetate (cyproterone acetate) on sebaceous glands of mature male mice as well as mature castrated mice treated with testosterone propionate has been studied qualitatively and quantitatively. A dosage of 1 mg cyproterone acetate every second day for a period of 4 weeks induces a reduction of the relative total sebaceous gland volume of about 80% in intact mice. The stimulant effect of 0.5 mg testosterone propionate every second day and over a period of 4 weeks on sebaceous glands is inhibited by the simultaneous treatment with 1 mg cyproterone acetate every second day. The effect of cyproterone acetate produces a decrease of the number of sebaceous gland alveoli, elicits a reduction of the number of cells per alveolus as well as a decline in cell size. The thickness of the epidermis was decreased in the animals treated with cyproterone acetate. The mechanism of action of this antiandrogen has been explained by a competitive testosterone antagonism since, in contrast to estrogens, the stimulant effect of exogenous testosterone on sebaceous glands is also inhibited.

Cloning of guinea pig cyclooxygenase-2 and 15-hydroxyprostaglandin dehydrogenase complementary deoxyribonucleic acids: steroid-modulated gene expression correlates to prostaglandin F2 alpha secretion in cultured endometrial cells.

Prostaglandin F2 alpha (PGF2 alpha) secretion is lowest at midcycle and highest on day 15 at luteolysis in the cycling guinea pig uterus and is inversely related to serum progesterone levels. An increase in 17-beta estradiol (E2) occurs only towards the end of the cycle. To investigate the effect of steroids on the control of uterine PGF2 alpha metabolism at the level of gene expression we established a primary cell culture model of day 15 cycling guinea pig endometrial cells. We cloned guinea pig cDNAs for cyclooxygenase 2 (COX-2), 15-hydroxyprostaglandin dehydrogenase (PGDH) that converts PGF2 alpha to biologically inactive 13,14-dihydro-15-keto PGF2 alpha (PGFM) and a fragment of cyclooxygenase-1 (COX-1). They were found to bear 87% and 90% homology at the amino acid level to their human counterparts for COX-2 and PGDH, respectively, retaining all functional sites. Purified epithelial and stromal cell subcultures were primed with medium containing either E2 or medroxyprogesterone acetate (MPA) for 24 h. They were then treated for a further 4 or 24 h either withdrawing the steroid, maintaining the priming steroid, or supplementing with both steroids, before harvesting conditioned media and RNA. Epithelial cells secreted 30-fold more PGF2 alpha compared with stromal cells (e.g. 7.8 +/- 0.7 vs. 0.26 +/- 0.09 pg/ng DNA.24 h), and PGF2 alpha secretion levels were approximately 15-fold higher than those of PGFM (e.g. 7.8 +/- 0.7 vs. 0.45 +/- 0.16 pg/ng DNA.24 h, for epithelial cells). COX-1 transcripts were low and unaffected by treatment in both cell types. COX-2 transcripts were more abundant in epithelial than stromal cells. Steroid-modulated, COX-2 dependent changes in PGF2 alpha secretion were observed. The addition of MPA to E2 primed cells caused a decrease in PGF2 alpha secretion and COX-2 messenger RNA levels after 4 h. Conversely, the addition of E2 to MPA primed epithelial cells led to an increase in PGF2 alpha secretion and COX-2 messenger RNA levels after 4 and 24 h. The withdrawal of E2 caused a fall in PGF2 alpha secretion and COX-2 transcripts after 24 h. In contrast, PGDH transcripts were more abundant in stromal than epithelial cells and were up-regulated by the addition of MPA to E2 primed cells. These in vitro observations are in keeping with the secretory profile seen in vivo in the cycling guinea pig uterus suggesting that 1) the fall of E2 and the coinciding rise in progesterone seen in the early cycle lead to a reduction in PGF2 alpha levels; and 2) the rise of E2 in the late cycle on a progesterone primed uterus is the stimulus for an increase in uterine PGF2 alpha production. Our findings suggest a differential role for uterine stroma and epithelium in vivo whereby the former acts to remove (via PGDH), and the latter to produce (via COX-2) biologically active prostaglandin.

Inhibition of the estradiol-mediated endometrial gland formation by the antigestagen onapristone in rabbits: relationship to uterine estrogen receptors.

There is evidence from previous studies that progesterone antagonists (antigestagens) modify estrogen responses at endometrial and myometrial levels without having affinity to the estrogen receptor (ER). The purpose of the present study was to investigate the influence of the antigestagen onapristone (ZK 98 299) on the uterus in ovariectomized (OVX) estradiol (E2)-substituted rabbits (3.0 micrograms/animal.day). The animals were treated for 8 days with different doses of onapristone (3.0, 10.0, and 30.0 mg/animal.day, sc). Uterine growth was not influenced by onapristone compared to that in OVX E2-substituted controls. However, morphological (light microscopy, transmission electron microscopy) and morphometric criteria indicated that there was a significant dose-dependent inhibition of the estrogen-induced gland formation within the endometrium and degenerative changes in glandular epithelial cells. By contrast, there were morphological signs of activation of the endometrial stroma (proliferation, increased capillarization, and vascularization, edema) above the level of E2-treated animals. A dose-dependent increase in the concentration of uterine cytosolic ER, nuclear ER, and ER mRNA (ER mRNA) was measured in uterine homogenates after onapristone treatment compared to values in OVX E2-substituted controls. Immunocytochemical analysis of ER in uterine sections suggests that the increase in ER after onapristone treatment took place predominantly in the myometrium and surface epithelium. To examine whether the observed interference was mediated via the progesterone receptor (PR), E2-substituted rabbits were treated, in a separate experiment, with onapristone (10.0 mg/animal.day, sc) and various doses of progesterone (1.0, 3.0, and 10.0 mg/animal.day, sc). Progesterone reversed all onapristone-induced changes, indicating that the observed effects were mediated via the PR. The data indicate that the antigestagen onapristone interacts with estrogen action in the absence of the natural PR ligand. The increase in ER and ER mRNA concentrations after onapristone treatment in OVX E2-treated animals suggests that this antigestagen abolished an inhibitory action of the unoccupied PR on ER biosynthesis.

Progestin receptors in brain and pituitary of 20-day-old fetal mice: an autoradiographic study using [125I]progestin.

The distribution of progestin target sites in the brain and pituitary of estrogen-primed 20-day-old fetal mice was investigated by thaw-mount autoradiography. Three pregnant mice were each implanted sc with a Silastic tube containing estrogen on day 17 and ovariectomized on day 19 of gestation. Twenty-four hours after ovariectomy 10 fetuses (5 males and 5 females) were collected and each injected sc with 0.33 microgram/100 g BW [125I]progestin (SA, 2200 Ci/mM). For competition, two additional fetuses were injected with 20 micrograms R5020 1 h before (Z)-17 beta-hydroxy-17 alpha-(2[125I]iodovinyl)4-estren-3-one [( 125I]Progestin) to demonstrate that nuclear uptake and retention of radioactivity were specific for progestin. Two hours after injection of [125I]Progestin all fetuses were mounted, frozen, and sectioned in a cryostat. After 1-37 days of exposure, sections were developed and scanned for labeled cells. Cells with nuclear concentration were found in the male and female preoptic area, within certain nuclear groups in the basal hypothalamus, in the central gray of the midbrain, and in the pituitary. No labeling was detected in the cortex or amygdala. The results indicate that cells in certain regions of the brain and pituitary express progestin receptors at the end of gestation and suggest that progesterone is important for the normal development of these cells.

Progestin receptor cells in the 8-day-old male and female mouse cerebral cortex: autoradiographic evidence for a sexual dimorphism in target cell number.

The present study examined the number and distribution of progestin receptor cells in the 8-day-old male and female cortex and compared cortical labeling with that in the preoptic area and central hypothalamus. Eight-day postnatal mice (four males and four females), treated with estradiol, were each sc injected with 0.32 micrograms/100 g BW [125I]progestin (SA, 2200 Ci/mM). Brains were frozen 2 h after injection of [125I]progestin, sectioned, and processed for thaw-mount autoradiography. Cells with a nuclear concentration of radioactivity were localized in lamina VI of the lateral cortical regions of the male and female brain, while only a few cortical cells were seen in laminae II, III, and V of the suprarhinal, lateral, and cingulate/paracingulate regions. Comparison of the number of labeled cells revealed that the female cortex contained significantly more labeled cells than the male at three of the four levels investigated. Similarly, the number of target cells was higher in the female medial preoptic nucleus, but not in the arcuate nucleus and ventromedial hypothalamic nucleus, while the distributions of labeled cells in the male and female preoptic/hypothalamic regions were comparable. Injection of unlabeled progesterone or R5020 1 h before [125I]progestin reduced the nuclear concentration of radioactivity in all target regions and verified the specificity of [125I]progestin for the progestin receptor. The results of these studies indicate that mouse 8-day-old cortex and preoptic area in the female animal have more progestin receptor cells than those in the male and demonstrate that progestin receptor cells are localized in a region of the cortex known to contain few estrogen target cells. These results further suggest that a sexual dimorphism in progestin cell number may result in a differential effect of progestin on the cortex and preoptic area of the mouse, perhaps establishing a dimorphism in development and function.

Dihydrospirorenone, a new progestogen with antimineralocorticoid activity: effects on ovulation, electrolyte excretion, and the renin-aldosterone system in normal women.

Dihydrospirorenone (DHSP; 6 beta,7 beta,15 beta,16 beta-dimethylen-3-oxo-17- alpha-pregn-4-en-21,17-carbolacton) is an aldosterone antagonist 8 times as potent as spironolactone in the rat. It is also a progestogen that suppresses ovulation in normal women at a daily dosage of 2 mg. The effects of this dosage on the renin-aldosterone system and sodium and potassium balances were investigated in two experiments. In study I, 12 healthy women received a diet with 100 mmol sodium and 60-70 mmol potassium per day from days 3-13 of their normal menstrual cycles. Six women took 2 mg DHSP; 6 others received placebo from days 8-13 of the cycle. Sodium excretion in the DHSP group rose from a mean of 79 to 98.5 +/- 8.3 mmol/day during medication. Placebo had no effect. The difference between average sodium excretion rates in subjects treated with DHSP or placebo was close to significance (P = 0.053). Potassium excretion did not change. Weight loss was slightly greater after DHSP than placebo treatment. PRA and plasma and urinary aldosterone rose significantly during DHSP medication. In study II, 12 women on a free diet were studied during a control and a treatment cyle. From days 5-25 of the second cycle, they took 2 mg DHSP (n = 6) or 1 mg cyproterone acetate. Both compounds suppressed ovulation and the rise in progesterone. During cycle 1, sodium excretion, PRA, and aldosterone were higher in the luteal than in the follicular phase, probably due to an antialdosterone effect of progesterone. DHSP reversed this pattern of natriuresis by inducing a significant early natriuresis and a rise in PRA and aldosterone. Cyproterone acetate only abolished differences in natriuresis between the follicular and luteal phases and the rise of PRA and plasma aldosterone in the luteal phase. We conclude that DHSP may be a suitable partner of ethinyl estradiol as a constituent of an oral contraceptive, since its progestogenic and antialdosterone profile is similar to that of progesterone. Other synthetic progestogens are devoid of an antialdosterone effect. The antialdosterone effect of DHSP may help prevent sodium retention and a rise in blood pressure in susceptible women.

Lack of binding of gestodene to estrogen receptor in human breast cancer tissue.

Competition studies with progesterone and estradiol receptors of human myometrial tissue as well as of mammary cancer tissue showed that gestodene bound with high affinity to the progesterone receptor, as did other synthetic and natural progestogens. However, gestodene did not bind to the estradiol receptor. The relative binding affinities of all tested synthetic and natural ligands showed no organ-specific differences and no differences between neoplastically transformed and normal tissues.

15,15-Ketals of natural prostaglandins and prostaglandin analogues. Synthesis and biological activities.

The synthesis is described of new 15,15-ethylene ketals of natural prostaglandins and prostaglandin analogues. Especially the crystalline trisamine salt of the 15,15-ethylene ketal of 15-dehydro-16-phenoxy-17,18,19,20-tetranorprostaglandin F2alpha is a very active inducer of luteolysis in laboratory animals and cattle.

Nonsteroidal antiinflammatory agents. 14. Synthesis and pharmacological profile of 6-chloro-5-(cyclopentylmethyl)indan-1-carboxylic acid.

The preparation of 6-chloro-5-(cyclopentylmethyl)indan-1-carboxylic acid is described. This acid has good anti-inflammatory and analgesic activities without producing irritation in the gastrointestinal tract up to the highest tested dose.

Aldosterone antagonists. 4. Synthesis and activities of steroidal 6,6-ethylene-15,16-methylene 17-spirolactones.

Several steroidal 6,6-ethylene-15,16-methylene 17-spirolactones were synthesized to find new progestogens that exhibit both progestational and antimineralocorticoidal activities. The influence of substituents in the 10- and 13-position of the steroidal framework on both properties was investigated. It was found that only compound 12, carrying methyl groups at the 10- and 13-positions, possesses high progestational and antimineralocorticoidal activity.

Structure-activity studies of configurationally rigid arylprostaglandins.

Potent, albeit nonselective, smooth-muscle stimulant activity has been previously reported for 16-phenoxy- and 17-phenylprostaglandins, a finding that led to the design and development of the tissue-selective uterine stimulant sulprostone. As an extension of this work, analogues incorporating the 16-phenoxy and 17-phenyl substituents into the rigid indanyl, tetrahydronaphthyl, dihydrobenzofuryl, and dihydrobenzopyranyl ring systems were prepared and evaluated for uterine stimulant activity in vitro and diarrheal effects in vivo. Since these cyclic groups, with the exception of the indanyl, contain a chiral center, both optical antipodes were prepared. These studies demonstrate that ring size, heteroatom, and absolute configuration at C-16 are important determinants for potency and selectivity.

N-(Methanesulfonyl)-16-phenoxyprostaglandincarboxamides: tissue-selective, uterine stimulants.

In an effort to develop tissue-selective prostaglandin analogues resistant to the metabolic inactivating pathways of the natural materials, hybrid compounds modified both at C-1 with a sulfonimide moiety and in the n-amylcarbinol side chain with substituted phenoxy groups were synthesized and evaluated in a variety of in vitro models. Several of these analogues exhibited potent, tissue-selective, uterine stimulant activity, a finding subsequently confirmed in clinical studies with one member of this series, N-(methanesulfonyl)-16-phenoxy-omega-tetranor-PGE2-carboxamide (CP-34089/ZK-57671, sulprostone).