New Insights as to Why Progesterone Receptor Modulators, such as Mifepristone, Seem to Be More Effective in Treating Cancers that Are Devoid of the Classical Nuclear Progesterone Receptor.

Mifepristone treatment for advanced cancer has demonstrated considerable improvement in both length and quality of life in patients who no longer have any other treatment options. The target is the progesterone induced blocking factor (PIBF), which helps the tumor to invade the normal tissue and proliferate and suppress cellular immunity. Most of the benefit has been observed in cancers not associated with the classical nuclear progesterone receptor (nPR). There are data showing that the presence of a nPR may be associated with a better prognosis. Membrane PRs seem to be responsible for PIBF secretion. Mifepristone, possibly fails to block another P associated protein that enables the tumor to proliferate, e.g., the progesterone receptor membrane component-1 (PGRMC-1) protein. One hypothesis is that the nPR helps to inhibit tumor production of PGRMC-1 protein. Thus, mifepristone may inhibit tumor spread by suppressing PIBF, but this may be negated by blocking the nPR, allowing PGRMC-1 levels to increase.

Progesterone receptor membrane component 1 inhibits tumor necrosis factor alpha induction of gene expression in neural cells.

Progesterone membrane receptor component 1 (Pgrmc1) is a cytochrome b5-related protein with wide-ranging functions studied most extensively in non-neural tissues. We previously demonstrated that Pgrmc1 is widely distributed in the brain with highest expression in the limbic system. To determine Pgrmc1 functions in cells of these regions, we compared transcriptomes of control siRNA-treated and Pgrmc1 siRNA-treated N42 hypothalamic cells using whole genome microarrays. Our bioinformatics analyses suggested that Pgrmc1 plays a role in immune functions and likely regulates proinflammatory cytokine signaling. In follow-up studies, we showed that one of these cytokines, TNFα, increased expression of rtp4, ifit3 and gbp4, genes found on microarrays to be among the most highly upregulated by Pgrmc1 depletion. Moreover, either Pgrmc1 depletion or treatment with the Pgrmc1 antagonist, AG-205, increased both basal and TNFα-induced expression of these genes in N42 cells. TNFα had no effect on levels of Rtp4, Ifit3 or Gbp4 mRNAs in mHippoE-18 hippocampal control cells, but Pgrmc1 knock-down dramatically increased basal and TNFα-stimulated expression of these genes. P4 had no effect on gbp4, ifit3 or rtp4 expression or on the ability of Pgrmc1 to inhibit TNFα induction of these genes. However, a majority of the top upstream regulators of Pgrmc1 target genes were related to synthesis or activity of steroids, including P4, that exert neuroprotective effects. In addition, one of the identified Pgrmc1 targets was Nr4a1, an orphan receptor important for the synthesis of most steroidogenic molecules. Our findings indicate that Pgrmc1 may exert neuroprotective effects by suppressing TNFα-induced neuroinflammation and by regulating neurosteroid synthesis.

Discovery of Vilaprisan (BAY 1002670): A Highly Potent and Selective Progesterone Receptor Modulator Optimized for Gynecologic Therapies.

Progesterone plays an important role in the female reproductive system. However, there is also evidence that gynecologic disorders/diseases such as uterine fibroids and endometriosis are progesterone-dependent. Steroidal and non-steroidal selective progesterone receptor modulators (SPRMs) have shown potential for the treatment of such diseases. Steroidal SPRMs, including mifepristone and ulipristal acetate, have proven effective in clinical trials. However, several steroidal SPRMs containing a dimethylamino substituent have been associated with elevated liver enzymes in patients. An earlier drug discovery program identified lonaprisan as a highly selective SPRM that did not show drug-related change in liver enzyme activity. Building on data obtained from that work, here we describe the research program that culminated in the discovery of a novel steroidal SPRM, vilaprisan, which combines an extremely high potency with very favorable drug metabolism and pharmacokinetic properties. Vilaprisan has entered clinical development and is currently undergoing phase 3 clinical trials.

An Evidence-based Approach to the Medical Management of Fibroids: A Systematic Review.

Fibroids are the most common tumor of the female reproductive tract, but approved medical treatments are limited. Patients demand uterine-sparing treatments which preserve fertility and avoid surgery. We systematically reviewed PubMed and Cochrane databases from January 1985 to November 2015 for evidence-based medical therapies for fibroids in the context of disease prevention, treatment of early disease, treatment of symptomatic disease, and preoperative management. We identified 2182 studies, of which 52 studies met inclusion and exclusion criteria. Published data affirm the efficacy of multiple agents, which are promising avenues for the development of medical alternatives to surgery.

Triterpenes from Alisma orientalis act as androgen receptor agonists, progesterone receptor antagonists, and glucocorticoid receptor antagonists.

Alisma orientalis, a well-known traditional medicine, exerts numerous pharmacological effects including anti-diabetes, anti-hepatitis, and anti-diuretics but its bioactivity is not fully clear. Androgen receptor (AR), progesterone receptor (PR), and glucocorticoid receptor (GR) are three members of nuclear receptor superfamily that has been widely targeted for developing treatments for essential diseases including prostate cancer and breast cancer. In this study, two triterpenes, alisol M 23-acetate and alisol A 23-acetate from Alisma orientalis were determined whether they may act as androgen receptor (AR), progesterone receptor (PR), or glucocorticoid receptor (GR) modulators. Indeed, in the transient transfection reporter assays, alisol M 23-acetate and alisol A 23-acetate transactivated AR in dose-dependent manner, while they transrepressed the transactivation effects exerted by agonist-activated PR and GR. Through molecular modeling docking studies, they were shown to respectively interact with AR, PR, or GR ligand binding pocket fairly well. All these results indicate that alisol M 23-acetate and alisol A 23-acetate from Alisma orientalis might possess therapeutic effects through their modulation of AR, PR, and GR pathways.

Blockage of progesterone receptor effectively protects pancreatic islet beta cell viability.

The progesterone receptor (PR), a member of nuclear receptor superfamily, is closely associated with gestational, type 1 and type 2 diabetes. However, the underlying mechanisms remain obscure. Here we found that PR activation increased the pro-inflammatory cytokines (PIC)-induced injury in Min6 cells, and PR blockage with siRNA interference protected the cells from damage. Moreover, the new discovered PR antagonist SC51089 effectively improved cell survival by reducing the PIC-stimulated cell apoptosis in Min6 cells. Immunoblotting assays indicated that either PR agonist progesterone (P4) or PR-B over-expression promoted the PIC-induced reinforces of extracellular-signal-regulated kinase 1/2 phosphorylation (p-Erk) and protein 53 (p53), and the attenuations of protein kinase B phosphorylation (p-AKT) and tumor necrosis factor receptor-associated factor 2 (TRAF2). SC51089 could reverse all the P4- or PR-B over-expression induced effects. In addition, PR siRNA inference based assay further supported that SC51089 protected pancreatic islet beta cells from the PR activation or PIC-induced injury by targeting PR and this protective action was mediated by AKT signaling pathway. To our knowledge, this current work might be the first report on the regulation of PR in pancreatic islet beta cell survival. It is expected that SC51089, as a non-steroid PR antagonist, might also find its potential in anti-diabetic research.

An antiprogestin, CDB4124, blocks progesterone's attenuation of the negative effects of a mild stress on sexual behavior.

These experiments were designed to test the hypothesis that a progesterone receptor antagonist would block progesterone's ability to reduce the negative effects of a 5 min restraint on female rat sexual behavior. Ovariectomized Fischer rats were injected with 10 µg estradiol benzoate. Two days later, rats were injected subcutaneously (sc) with the progesterone receptor antagonist, CDB4124 (17α-acetoxy-21-methoxy-11ß-[4-N,N-dimethyaminopheny]-19-norpregna-4,9-dione-3,20-dione) (60 mg/kg), or vehicle (20% DMSO+propylene glycol). One hour later, rats were injected sc with 500 µg progesterone or vehicle (sesame seed oil). Rats were assigned to one of three different treatment conditions: (1) (ECV) estradiol benzoate, CDB4124, sesame seed oil vehicle, (2) (ECP) estradiol benzoate, CDB4124, progesterone, and (3) (EVP) estradiol benzoate, DMSO/propylene glycol vehicle, progesterone. That afternoon sexual behavior was examined before and after a 5 min restraint experience. Before restraint, lordosis behavior was comparable across treatment conditions but only progesterone-treated rats exhibited proceptive behavior. CDB4124 did not block progesterone's induction of proceptivity. However, after restraint, CDB4124 attenuated the positive effects of progesterone on all sexual behaviors examined. The restraint experience inhibited sexual behavior in rats treated with estradiol benzoate and CDB4124 and in rats treated with estradiol benzoate, CDB4124, and progesterone but not in rats given estradiol benzoate and progesterone without CDB4124. These findings are consistent with the hypothesis that progesterone receptors mediate progesterone's ability to reduce the negative sexual behavioral effects of a mild stressor.

Pharmacokinetic evaluation of ulipristal acetate for uterine leiomyoma treatment.

INTRODUCTION: Progesterone (P), and its receptors (PRs), play a key role in uterine leiomyoma growth. Selective progesterone receptor modulators exert mixed antagonist and agonist effects on the PRs. Mifepristone, a PR-antagonist, reduces leiomyoma volume and related symptoms. Ulipristal acetate (UPA) exerts a potent antiprogestin activity, with less antiglucocorticoid activity compared to mifepristone. This property provides potential advantages for long-term use. AREAS COVERED: This paper focuses on the effect of UPA on leiomyoma's growth and related symptoms in women. The authors also evaluate UPA's efficacy in reducing leiomyoma's size and menorrhagia in Phase II/III trials. EXPERT OPINION: In the authors' opinion, UPA (5 mg/day) over 3 months can be used to plan the surgery in women with symptomatic leiomyomas. The tolerability and the safety of treatment over a period longer than 3 months have to be evaluated. The results of the follow-up treatment suggest that further studies could successfully evaluate the efficacy and the tolerability of intermittent 3-month courses of treatment.

Ulipristal acetate versus placebo for fibroid treatment before surgery.

BACKGROUND: The efficacy and safety of oral ulipristal acetate for the treatment of symptomatic uterine fibroids before surgery are uncertain. METHODS: We randomly assigned women with symptomatic fibroids, excessive uterine bleeding (a score of >100 on the pictorial blood-loss assessment chart [PBAC, an objective assessment of blood loss, in which monthly scores range from 0 to >500, with higher numbers indicating more bleeding]) and anemia (hemoglobin level of ≤10.2 g per deciliter) to receive treatment for up to 13 weeks with oral ulipristal acetate at a dose of 5 mg per day (96 women) or 10 mg per day (98 women) or to receive placebo (48 women). All patients received iron supplementation. The coprimary efficacy end points were control of uterine bleeding (PBAC score of <75) and reduction of fibroid volume at week 13, after which patients could undergo surgery. RESULTS: At 13 weeks, uterine bleeding was controlled in 91% of the women receiving 5 mg of ulipristal acetate, 92% of those receiving 10 mg of ulipristal acetate, and 19% of those receiving placebo (P<0.001 for the comparison of each dose of ulipristal acetate with placebo). The rates of amenorrhea were 73%, 82%, and 6%, respectively, with amenorrhea occurring within 10 days in the majority of patients receiving ulipristal acetate. The median changes in total fibroid volume were -21%, -12%, and +3% (P=0.002 for the comparison of 5 mg of ulipristal acetate with placebo, and P=0.006 for the comparison of 10 mg of ulipristal acetate with placebo). Ulipristal acetate induced benign histologic endometrial changes that had resolved by 6 months after the end of therapy. Serious adverse events occurred in one patient during treatment with 10 mg of ulipristal acetate (uterine hemorrhage) and in one patient during receipt of placebo (fibroid protruding through the cervix). Headache and breast tenderness were the most common adverse events associated with ulipristal acetate but did not occur significantly more frequently than with placebo. CONCLUSIONS: Treatment with ulipristal acetate for 13 weeks effectively controlled excessive bleeding due to uterine fibroids and reduced the size of the fibroids. (Funded by PregLem; ClinicalTrials.gov number, NCT00755755.).

"True" antiandrogens-selective non-ligand-binding pocket disruptors of androgen receptor-coactivator interactions: novel tools for prostate cancer.

Prostate cancer (PCa) therapy typically involves administration of "classical" antiandrogens, competitive inhibitors of androgen receptor (AR) ligands, dihydrotestosterone (DHT) and testosterone (tes), for the ligand-binding pocket (LBP) in the ligand-binding domain (LBD) of AR. Prolonged LBP-targeting leads to resistance, and alternative therapies are urgently required. We report the identification and characterization of a novel series of diarylhydrazides as selective disruptors of AR interaction with coactivators through application of structure and ligand-based virtual screening. Compounds demonstrate full ("true") antagonism in AR with low micromolar potency, selectivity over estrogen receptors α and ß and glucocorticoid receptor, and partial antagonism of the progesterone receptor. MDG506 (5) demonstrates low cellular toxicity in PCa models and dose responsive reduction of classical antiandrogen-induced prostate specific antigen expression. These data provide compelling evidence for such non-LBP intervention as an alternative approach or in combination with classical PCa therapy.

Structural basis for agonism and antagonism for a set of chemically related progesterone receptor modulators.

The progesterone receptor is able to bind to a large number and variety of ligands that elicit a broad range of transcriptional responses ranging from full agonism to full antagonism and numerous mixed profiles inbetween. We describe here two new progesterone receptor ligand binding domain x-ray structures bound to compounds from a structurally related but functionally divergent series, which show different binding modes corresponding to their agonistic or antagonistic nature. In addition, we present a third progesterone receptor ligand binding domain dimer bound to an agonist in monomer A and an antagonist in monomer B, which display binding modes in agreement with the earlier observation that agonists and antagonists from this series adopt different binding modes.

Aromatase promoter I.f is regulated by progesterone receptor in mouse hypothalamic neuronal cell lines.

Aromatase catalyzes the conversion of C(19) steroids to estrogens. Aromatase and progesterone, both of which function at different steps of steroidogenesis, are crucial for the sexually dimorphic development of the fetal brain and the regulation of gonadotropin secretion and sexual interest in adults. The aromatase gene (Cyp19a1) is selectively expressed in distinct neurons of the mouse hypothalamus through a distal brain-specific promoter, I.f, located ∼40 kb upstream of the coding region. However, the regulation of aromatase expression in the brain is not well understood. In this study, we investigated a short feedback effect of progesterone analogues on aromatase mRNA expression and enzyme activity in estrogen receptor α (Esr1)-positive or -negative mouse embryonic hypothalamic neuronal cell lines that express aromatase via promoter I.f. In a hypothalamic neuronal cell line that highly expresses aromatase, progesterone receptor (Pgr), and Esr1, a progesterone agonist, R5020, inhibited aromatase mRNA level and enzyme activity. The inhibitory effect of R5020 was reversed by its antagonist, RU486. Deletion mutants of promoter I.f suggested that inhibition of aromatase expression by progesterone is conferred by the nt -1000/-500 region, and R5020 enhanced binding of Pgr to the nt -800/-600 region of promoter I.f. Small interfering RNA knockdown of Pgr eliminated progesterone-dependent inhibition of aromatase mRNA and enzyme activity. Taken together, progesterone enhances recruitment of Pgr to specific regions of the promoter I.f of Cyp19a1 and regulates aromatase expression in hypothalamic neurons.

Preclinical and clinical pharmacokinetics of PF-02413873, a nonsteroidal progesterone receptor antagonist.

The recently discovered selective nonsteroidal progesterone receptor (PR) antagonist 4-[3-cyclopropyl-1-(methylsulfonylmethyl)-5-methyl-1H-pyrazol-4-yl]oxy-2,6-dimethylbenzonitrile (PF-02413873) was characterized in metabolism studies in vitro, in preclinical pharmacokinetics in rat and dog, and in an initial pharmacokinetic study in human volunteers. Clearance (CL) of PF-02413873 was found to be high in rat (84 ml · min(-1) · kg(-1)) and low in dog (3.8 ml · min(-1) · kg(-1)), consistent with metabolic stability determined in liver microsomes and hepatocytes in these species. In human, CL was low in relation to hepatic blood flow, consistent with metabolic stability in human in vitro systems, where identified metabolites suggested predominant cytochrome P450 (P450)-catalyzed oxidative metabolism. Prediction of CL using intrinsic CL determined in human liver microsomes (HLM), recombinant human P450 enzymes, and single species scaling (SSS) from pharmacokinetic studies showed that dog SSS and HLM scaling provided the closest estimates of CL of PF-02413873 in human. These CL estimates were combined with a physiologically based pharmacokinetic (PBPK) model to predict pharmacokinetic profiles after oral suspension administration of PF-02413873 in fasted and fed states in human. Predicted plasma concentration versus time profiles were found to be similar to those observed in human over the PF-02413873 dose range 50 to 500 mg and captured the enhanced exposure in fed subjects. This case study of a novel nonsteroidal PR antagonist underlines the utility of PBPK modeling techniques in guiding prediction confidence and design of early clinical trials of novel chemical agents.

Hormone-biological therapy in breast cancer: preclinical evidences, clinical studies and future directions.

BACKGROUND: The development of resistance to endocrine agents is a major issue in the treatment of ER-positive breast cancers patients. The evidence of a cross-talk among signaling pathways and the recent advances in understanding biology of this neoplasm have led to the new concept of hormone-biological therapy. AIMS: Main aim of this review was to examine comprehensively concluded and ongoing clinical trials about endocrine agents combined with biological drugs. Second aim was to provide an up-to-date knowledge of preclinical studies in this flourishing field, considering several in vitro and in vivo models, by focusing on the main signaling pathways involved in breast tumorigenesis. MATERIALS AND METHODS: We performed a literature research for papers published in MEDLINE database until January 2009. We selected the most significant data presented as abstract or poster during the relevant international meetings for breast cancer (ASCO, ESMO and San Antonio Breast Cancer Symposium). Moreover, we systematically reviewed a public available registry of federally and privately supported clinical trials conducted in the United States and around the world to update information about conclusion of trials and ongoing trials not yet published. RESULTS/CONCLUSION: Hormone-biological therapy is a relatively new option in the treatment of breast cancer and some combinations seem to be effective in certain settings. Numerous trials are ongoing and they will help to better clarify the role of different combined therapies. In parallel, cancer biology has a central role in a deeper understanding of this heterogeneous disease and preclinical studies will be necessary to test new compounds and strategies.

In vitro characterization of ZK 230211--A type III progesterone receptor antagonist with enhanced antiproliferative properties.

The progesterone receptor (PR) is a key regulator of female reproductive functions. Compounds with progesterone inhibiting effects (PR antagonists) have found numerous utilities in female reproductive health, ranging from contraception to potential treatment of progesterone-dependent diseases like uterine leiomyomas. Based on in vitro characteristics such as DNA binding activity and partial agonistic transcriptional behavior in the presence of protein kinase A activators (cyclic-AMP), three types of PR modulators with antagonistic properties have been defined. In this study, we analyzed the in vitro characteristics of the PR antagonist ZK 230211 in comparison to the classical antagonists onapristone and mifepristone. We focused on PR actions in genomic signaling pathways, including DNA binding activity, nuclear localization and association with the nuclear receptor corepressor (NCoR) as well as actions in non-genomic signaling, such as the activation of c-Src kinase signaling and cyclin D1 gene promoter activity. ZK 230211 represents a type of PR antagonist with increased inhibitory properties in comparison to mifepristone and onapristone. When liganded to the progesterone receptor, ZK 230211 induces a strong and persistent binding to its target response element (PRE) and increases NCoR recruitment in CV-1 cells. Furthermore, ZK 230211 displays less agonistic properties with regard to the association of PR isoform B and the cytoplasmic c-Src kinase in HeLa cells. It represses T47D cell cycle progression, in particular estradiol-induced S phase entry. In summary, our studies demonstrate ZK 230211 to be a type III progesterone receptor antagonist which is characterized by very strong DNA binding activity and strong antiproliferative effects in the cancer cell lines HeLa and T47D.

Mifepristone treatment improves length and quality of survival of mice with spontaneous leukemia.

BACKGROUND: Mifepristone was found to suppress expression of the progesterone-induced blocking factor (PIBF). Progesterone-induced blocking factor suppresses natural killer cell activity. The objective of the present study was to determine if treatment of mice with spontaneous murine lymphocyte leukemia with the progesterone receptor antagonist mifepristone could improve length and quality of life. MATERIALS AND METHODS: Sixty-one mice were gavaged with mifepristone and 33 controls with olive oil. Quality of life was determined by body conditioning score (BCS). Treatment was initiated when the mice were 6 months old. RESULTS: Within 2 weeks of therapy only 11.4% of the mifepristone treated mice died vs. about 50% of controls. The BCS was 5 (highest quality) in 82% of treated mice vs. 11% of controls after 2 weeks of therapy. CONCLUSION: Mifepristone therapy should be further evaluated for treating leukemia and lymphoma.

Regulation of epithelial Na+ channel (ENaC) in the salivary cell line SMG-C6.

Glucocorticoids and mineralocorticoids modulate Na+ transport via epithelial Na+ channels (ENaC). The rat submandibular epithelial cell line, SMG-C6, expresses alpha-ENaC mRNA and protein and exhibits amiloride-sensitive Na+ transport when grown in low-serum (2.5%) defined medium, therefore, we examined the effects of altering the composition of the SMG-C6 cell growth medium on ENaC expression and function. No differences in basal or amiloride-sensitive short-circuit current (Isc) were measured across SMG-C6 monolayers grown in the absence of thyroid hormone, insulin, transferrin, or EGF. In the absence of hydrocortisone, basal and amiloride-sensitive Isc significantly decreased. Similarly, monolayers grown in 10% serum-supplemented medium had lower basal Isc and no response to amiloride. Adding hydrocortisone (1.1 microM) to either the low or 10% serum medium increased basal and amiloride-sensitive Isc, which was blocked by RU486, the glucocorticoid and progesterone receptor antagonist. Aldosterone also induced an increase in alpha-ENaC expression and Na+ transport, which was also blocked by RU486 but not by the mineralocorticoid receptor antagonist spironolactone. Thus, in the SMG-C6 cell line, hydrocortisone and aldosterone increased ENaC expression and basal epithelial Na+ transport. The absence of endogenous ENaC expression in culture conditions devoid of steroids makes the properties of this cell line an excellent model for investigating pathways regulating ENaC expression and Na+ transport.

Ulipristal, a progesterone receptor antagonist as a contraceptive and for the treatment of uterine fibroids.

HRA Pharma, under license from the Research Triangle Institute, is developing ulipristal, a progesterone receptor modulator, for the potential use as a contraceptive and an emergency contraceptive, and for the potential treatment of uterine fibroids. Phase II clinical trials for uterine fibroids and phase III trials for contraception are underway. The drug was expected to be launched in the US and Europe in 2009.

Inhibiting gene expression with peptide nucleic acid (PNA)--peptide conjugates that target chromosomal DNA.

Peptide nucleic acids (PNAs) are nonionic DNA/RNA mimics that can recognize complementary sequences by Watson-Crick base pairing. The neutral PNA backbone facilitates the recognition of duplex DNA by strand invasion, suggesting that antigene PNAs (agPNAs) can be important tools for exploring the structure and function of chromosomal DNA inside cells. However, before agPNAs can enter wide use, it will be necessary to develop straightforward strategies for introducing them into cells. Here, we demonstrate that agPNA-peptide conjugates can target promoter DNA and block progesterone receptor (PR) gene expression inside cells. Thirty-six agPNA-peptide conjugates were synthesized and tested. We observed inhibition of gene expression using cationic peptides containing either arginine or lysine residues, with eight or more cationic amino acids being preferred. Both 13 and 19 base agPNA-peptide conjugates were inhibitory. Inhibition was observed in human cancer cell lines expressing either high or low levels of progesterone receptor. Modification of agPNA-peptide conjugates with hydrophobic amino acids or small molecule hydrophobic moieties yielded improved potency. Inhibition by agPNAs did not require cationic lipid or any other additive, but adding agents to cell growth media that promote endosomal release caused modest increases in agPNA potency. These data demonstrate that chromosomal DNA is accessible to agPNA-peptide conjugates and that chemical modifications can improve potency.