The human pituitary adenylate cyclase-activating polypeptide receptor (hPAC1-R), a class B G-protein-coupled receptor (GPCR) identified almost 30 years ago, represents an important pharmacological target in the areas of neuroscience, oncology, and immunology. Despite interest in this target, only a very limited number of small molecule modulators have been reported for this receptor. We herein describe the results of a drug discovery program aiming for the identification of a potent and selective hPAC1-R antagonist. An initial high-throughput screening (HTS) screen of 3.05 million compounds originating from the Bayer screening library failed to identify any tractable hits. A second, completely revised screen using native human embryonic kidney (HEK)293 cells yielded a small number of hits exhibiting antagonistic properties (4.2 million compounds screened). BAY 2686013 (1) emerged as a promising compound showing selective antagonistic activity in the submicromolar potency range. In-depth characterization supported the hypothesis that BAY 2686013 blocks receptor activity in a noncompetitive manner. Preclinical, pharmacokinetic profiling indicates that BAY 2686013 is a valuable tool compound for better understanding the signaling and function of hPAC1-R. SIGNIFICANCE STATEMENT: Although the human pituitary adenylate cyclase-activating polypeptide receptor (hPAC1-R) is of major significance as a therapeutic target with a well documented role in pain signaling, only a very limited number of small-molecule (SMOL) compounds are known to modulate its activity. We identified and thoroughly characterized a novel, potent, and selective SMOL antagonist of hPAC1-R (acting in an allosteric manner). These characteristics make BAY 2686013 an ideal tool for further studies.

Endometriosis in an estrogen-dependent disease that is characterized by the presence of endometrial tissue outside the uterine cavity leading to pain and infertility in many affected women. Highly efficient treatment options which create a hypoestrogenic environment can cause side effects such as hot flushes and bone mass loss that are not favorable for premenopausal women. Previous work has demonstrated that increased local or systemic prolactin seems to be involved in the pathogenesis of endometriosis. Here we examined two prolactin receptor (PRLR) blocking antibodies in a murine endometriosis interna model which relies on the induction of systemic hyperprolactinemia in female SHN mice. The severity of the disease is determined by the degree of endometrial invasion into the myometrium. In this model, endometriosis was inhibited by clinical gold standards such as progestins and anti-estrogenic approaches. PRLR blockade completely inhibited endometriosis in this mouse model to the same extent as the anti-estrogen faslodex or the GnRH antagonist cetrorelix. In contrast to cetrorelix and faslodex, the PRLR antibodies did not decrease relative uterine weights and were thus devoid of anti-estrogenic effects. We therefore hypothesize that PRLR antibodies may present a novel and highly efficient treatment option for endometriosis with a good safety and tolerability profile. Clinical studies are on the way to test this hypothesis.

The alpha(2C)-adrenoreceptor antagonist BAY 1193397 is in development for the oral treatment of diabetic foot ulcers. Safety, tolerability, and pharmacokinetics of BAY 1193397 were investigated in 3 randomized, single-center phase 1 studies in healthy male subjects: a first-in-human study (single oral doses of 0.5-50 mg), a relative bioavailability and food effect study (single doses of 1 and 10 mg), and a multiple-dose escalation study (using 2 and 5 mg twice daily and 10 and 20 mg once daily for 9 consecutive days). BAY 1193397 was rapidly absorbed in the fasted state, peak concentrations were reached between 0.6 and 2 hours. The mean terminal half-life was in the range of 17 to 20 hours. Area under the plasma concentration-time curve and maximum concentration appeared to be dose proportional, with a negligible food effect. There were no high-accumulation effects of BAY 1193397 after repeated dosing. BAY 1193397 was safe and well tolerated. At supratherapeutic plasma concentrations, there were slight transient increases in norepineph rine levels, heart rate, and blood pressure that were more pronounced after a single dose compared to steady state and appeared to be maximum concentration dependent. The results of the presented studies support the conduct of subsequent clinical trials with BAY 1193397 in patients with diabetes and compromised microcirculation.

The α 2C -adrenoreceptor antagonist BAY 1193397 is in development for the oral treatment of diabetic foot ulcers. Safety, tolerability, and pharmacokinetics of BAY 1193397 were investigated in 3 randomized, single-center phase 1 studies in healthy male subjects: a first-in-human study (single oral doses of 0.5-50 mg), a relative bioavailability and food effect study (single doses of 1 and 10 mg), and a multiple-dose escalation study (using 2 and 5 mg twice daily and 10 and 20 mg once daily for 9 consecutive days). BAY 1193397 was rapidly absorbed in the fasted state, peak concentrations were reached between 0.6 and 2 hours. The mean terminal half-life was in the range of 17 to 20 hours. Area under the plasma concentration–time curve and maximum concentration appeared to be dose proportional, with a negligible food effect. There were no high-accumulation effects of BAY 1193397 after repeated dosing. BAY 1193397 was safe and well tolerated. At supratherapeutic plasma concentrations, there were slight transient increases in norepinephrine levels, heart rate, and blood pressure that were more pronounced after a single dose compared to steady state and appeared to be maximum concentration dependent. The results of the presented studies support the conduct of subsequent clinical trials with BAY 1193397 in patients with diabetes and compromised microcirculation.

BACKGROUND:The protease chymase generates multiple factors involved in tissue remodelling including angiotensin II (Ang II) and has been implicated in the pathophysiology of diabetic kidney disease (DKD). This study investigated the effects of the chymase inhibitor fulacimstat on albuminuria in patients with Type II diabetes mellitus and a clinical diagnosis of DKD.METHODS:In this double-blind, randomized, placebo-controlled trial, patients were on the maximum tolerated dose of either an Ang II receptor blocker or an Ang-converting enzyme inhibitor since at least 3 months before the screening visit. Eligible patients were randomized in a 2:1 ratio to treatment with either 25 mg fulacimstat (n = 99) or placebo (n = 48) twice daily on top of standard of care.RESULTS:The randomized patients had a mean urine albumin-creatinine ratio (UACR) of 131 mg/g (range: 29-2429 mg) and a mean (standard deviation) estimated glomerular filtration rate of 60.8 ± 16.9 mL/min/1.73 m2 before treatment start. Fulacimstat was safe and well tolerated, and achieved mean total trough concentrations that were ∼9-fold higher than those predicted to be required for minimal therapeutic activity. UACR increased by 27.4% [coefficient of variation (CV) 86%] and 3% (CV 88.9%) after 24 weeks of treatment with placebo or fulacimstat, respectively. Analysis of covariance revealed a least square mean UACR ratio (fulacimstat/placebo) of 0.804 (90% CI 0.627-1.030, P = 0.1477), indicating a statistically non-significant UACR reduction of 19.6% after fulacimstat treatment compared with placebo.CONCLUSIONS:Fulacimstat was safe and well tolerated but did not reduce albuminuria in patients with DKD. These findings do not support a therapeutic role for chymase inhibition in DKD.

Background Adverse cardiac remodeling is a major risk factor for the development of post myocardial infarction (MI) heart failure (HF). This study investigates the effects of the chymase inhibitor fulacimstat on adverse cardiac remodeling after acute ST-segment-elevation myocardial infarction (STEMI). Methods In this double-blind, randomized, placebo-controlled trial patients with first STEMI were eligible. To preferentially enrich patients at high risk of adverse remodeling, main inclusion criteria were a left-ventricular ejection fraction (LVEF) <= 45% and an infarct size >10% on day 5 to 9 post MI as measured by cardiac MRI. Patients were then randomized to 6 months treatment with either 25 mg fulacimstat (n = 54) or placebo (n = 53) twice daily on top of standard of care starting day 6 to 12 post MI. The changes in LVEF, LV end-diastolic volume index (LVEDVI), and LV end-systolic volume index (LVESVI) from baseline to 6 months were analyzed by a central blinded cardiac MRI core laboratory. Results Fulacimstat was safe and well tolerated and achieved mean total trough concentrations that were approximately tenfold higher than those predicted to be required for minimal therapeutic activity. Comparable changes in LVEF (fulacimstat: 3.5% +/- 5.4%, placebo: 4.0% +/- 5.0%, P = .69), LVEDVI (fulacimstat: 7.3 +/- 13.3 ml/m(2), placebo: 5.1 +/- 18.9 ml/m(2), P = .54), and LVESVI (fulacimstat: 2.3 +/- 11.2 mL/m(2), placebo: 0.6 +/- 14.8 ml/m(2), P = .56) were observed in both treatment arms. Conclusion Fulacimstat was safe and well tolerated in patients with left-ventricular dysfunction (LVD) after first STEMI but had no effect on cardiac remodeling.

Abstract Introduction Adverse cardiac remodelling represents the most important risk factor for the development of heart failure (HF) after myocardial infarction (MI). Chymase is a protease that generates locally pro-fibrotic factors such as angiotensin II, TGFβ, and matrixmetallproteases that contribute to tissue remodelling. Purpose This phase IIa study examined the effects of the chymase inhibitor fulacimstat on functional parameters of adverse cardiac remodelling after acute MI. Methods A double-blind, multinational, randomized, placebo-controlled study was performed in patients after first STEMI who were treated with primary percutaneous coronary intervention within 24h of symptom onset. To enrich for patients at risk of adverse remodelling, main inclusion criteria were a left-ventricular ejection fraction (LVEF)≤45% and an infarct size>10% on day 5 to 9 post MI as measured by cardiac MRI. On day 6 to 12 post MI, patients were randomized to treatment with either 25 mg fulacimstat (n=54) or placebo (n=53) twice daily on top of standard of care. The changes in LVEF, LVEDVI, and LVESVI from baseline to 6 months of treatment were analyzed by a central blinded cardiac MRI core laboratory. Results Fulacimstat was safe and well tolerated, 64.8% of patients treated with fulacimstat and 75.5% of patients treated with placebo reported treatment emergent adverse events. Fulacimstat achieved exposures that were approximately 10-fold higher than those predicted to be required for minimal therapeutic activity. After six months of treatment, there were no effects of fulacimstat compared to placebo on the changes in LVEF, LVEDVI, and LVESVI (see Table). Analysis of primary efficacy parameters Parameter Placebo Fulacimstat p-value LVEF (%) baseline 37.2±6.1 39.1±5.5 0.15 6 months 41.2±8.4 42.6±8.4 0.45 delta 4.0±5.0 3.5±5.4 0.69 LVEDVI (mL/m2) baseline 80.0±17.1 77.4±18.2 0.51 6 months 85.1±19.1 84.7±23.4 0.94 delta 5.1±18.9 7.3±13.3 0.54 LVESVI (mL/m2) baseline 50.5±13.0 47.3±12.3 0.26 6 months 51.1±16.9 49.6±18.1 0.71 delta 0.6±14.8 2.3±11.2 0.56 Data are given as mean ± standard deviation. Conclusion Fulacimstat was safe and well tolerated in patients with left-ventricular dysfunction (LVD) after first STEMI but had no effect on adverse cardiac remodelling in the experimental setting of this study. Acknowledgement/Funding The study was funded by its sponsor BAYER AG

The orally available chymase inhibitor BAY 1142524 is currently being developed as a first-in-class treatment for left-ventricular dysfunction after myocardial infarction. Results from 3 randomized, single-center, phase 1 studies in healthy male volunteers examining the safety, tolerability, and pharmacokinetics of BAY 1142524 are summarized. In this first-in-human study, single oral doses of 1-200 mg were administered in fasted state as liquid service formulation or immediate release (IR) tablets. The relative bioavailability and the effect of a high-fat/high-calorie meal were investigated at the 5-mg dose. In a multiple-dose escalation study, doses of 5-50 mg twice daily and 100 mg once daily were given for 5 consecutive days. BAY 1142524 was safe and well tolerated and had no effects on heart rate or blood pressure compared with placebo. BAY 1142524 was absorbed with peak concentration 1-3 hours after administration for IR tablets; it was eliminated from plasma with a terminal half-life of 6.84-12.0 hours after administration of liquid service formulation or IR tablets. Plasma exposures appeared to be dose-linear, with a negligible food effect. There was only low accumulation of BAY 1142524 after multiple dosing. BAY 1142524 exhibited a pharmacokinetic profile allowing for once-daily dosing. The absence of blood pressure effects after administration of BAY 1142524 supports the combination of this novel anti-remodeling drug with existing standard of care in patients with left-ventricular dysfunction after acute myocardial infarction.

The chymase inhibitor fulacimstat is developed as a first‐in‐class treatment option for the inhibition of adverse cardiac remodeling in patients with left ventricular dysfunction (LVD) after acute myocardial infarction (MI). The aim of the study was to examine the safety and tolerability of fulacimstat in patients with LVD after remote MI. A multicenter, multinational randomized, placebo‐controlled study was performed in clinically stable patients (40–79 years of age, left ventricular ejection fraction ≤ 45% because of MI in medical history) who were on stable evidence‐based standard‐of‐care therapies for LVD post‐MI including an angiotensin converting enzyme inhibitor or angiotensin receptor blocker at doses of at least half the recommended target dose. Patients were treated for 2 weeks with either placebo (n = 12) or 4 different doses of fulacimstat (5 mg twice daily, n = 9; 10 mg twice daily, n = 9; 25 mg twice daily, n = 10; 50 mg once daily, n = 9). Fulacimstat was safe and well tolerated at all examined doses. There were no clinically relevant effects on vital signs or potassium levels compared with placebo treatment. Mean plasma concentrations of fulacimstat increased with the administered dose and reached exposures predicted to be therapeutically active. The safety profile and the absence of effects on blood pressure or heart rate in a chronic patient population having similar comorbidities and receiving similar comedication as patients after acute MI support future clinical trials with fulacimstat in patients after acute MI.

The prolactin receptor (PRLR) has been implicated in a variety of physiological processes (lactation, reproduction) and diseases (breast cancer, autoimmune diseases). Prolactin synthesis in the pituitary and extrapituitary sites is regulated by different promoters. Dopamine receptor agonists such as bromocriptine can only interfere with pituitary prolactin synthesis and thus do not induce a complete blockade of PRLR signaling. Here we describe the identification of a human monoclonal antibody 005-C04 that blocks PRLR-mediated signaling at nanomolar concentrations in vitro. In contrast to a negative control antibody, the neutralizing PRLR antibody 005-C04 inhibits signal transducer and activator of transcription 5 phosphorylation in T47D cells and proliferation of BaF3 cells stably expressing murine or human PRLRs in a dose-dependent manner. In vivo application of this new function-blocking PRLR antibody reflects the phenotype of PRLR-deficient mice. After antibody administration female mice become infertile in a reversible manner. In lactating dams, the antibody induces mammary gland involution and negatively interferes with lactation capacity as evidenced by reduced milk protein expression in mammary glands and impaired litter weight gain. Antibody-mediated blockade of the PRLR in vivo stimulates hair regrowth in female mice. Compared with peptide-derived PRLR antagonists, the PRLR antibody 005-C04 exhibits several advantages such as higher potency, noncompetitive inhibition of PRLR signaling, and a longer half-life, which allows its use as a tool compound also in long-term in vivo studies. Therefore, we suggest that this antibody will help to further our understanding of the role of auto- and paracrine PRLR signaling in health and disease.

Estrogen receptor-α (ERα) regulates gene transcription through the 2 activation functions (AFs) AF-1 and AF-2. The crucial role of ERαAF-2 was previously demonstrated for endometrial proliferative action of 17β-estradiol (E2). Here, we investigated the role of ERαAF-1 in the regulation of gene transcription and cell proliferation in the uterus. We show that acute treatment with E2 or tamoxifen, which selectively activates ERαAF-1, similarly regulate the expression of a uterine set of estrogen-dependent genes as well as epithelial cell proliferation in the uterus of wild-type mice. These effects were abrogated in mice lacking ERαAF-1 (ERαAF-10). Four weeks of E2 treatment led to uterine hypertrophy and sustained luminal epithelial and stromal cell proliferation in wild-type mice, but not in ERαAF-10 mice. However, ERαAF-10 mice still presented a moderate uterine hypertrophy essentially due to a stromal edema, potentially due to the persistence of Vegf-a induction. Epithelial apoptosis is largely decreased in these ERαAF-10 uteri, and response to progesterone is also altered. Finally, E2-induced proliferation of an ERα-positive epithelial cancer cell line was also inhibited by overexpression of an inducible ERα isoform lacking AF-1. Altogether, these data highlight the crucial role of ERαAF-1 in the E2-induced proliferative response in vitro and in vivo. Because ERαAF-1 was previously reported to be dispensable for several E2 extrareproductive protective effects, an optimal ERα modulation could be obtained using molecules activating ERα with a minimal ERαAF-1 action.

Estrogen replacement is an effective therapy of postmenopausal symptoms such as hot flushes, bone loss, and vaginal dryness. Undesired estrogen effects are the stimulation of uterine and mammary gland epithelial cell proliferation as well as hepatic estrogenicity. In this study, we examined the influence of different estradiol release kinetics on tissue responsivity in ovariectomized (OVX) rats. Pulsed release kinetics was achieved by ip or sc administration of estradiol dissolved in physiological saline containing 10% ethanol (EtOH/NaCl) whereas continuous release kinetics was achieved by sc injection of estradiol dissolved in benzylbenzoate/ricinus oil (1+4, vol/vol). Initial 3-d experiments in OVX rats showed that pulsed ip estradiol administration had profoundly reduced stimulatory effects on the uterus and the liver compared with continuous release kinetics. On the other hand, both administration forms prevented severe vaginal atrophy. Based on these results, we compared the effects of pulsed (sc in EtOH/NaCl) vs. continuous (sc in benzylbenzoate/ricinus oil) estradiol release kinetics on bone, uterus, mammary gland, and liver in a 4-month study in OVX rats. Ovariectomy-induced bone loss was prevented by both administration regimes. However, pulsed estradiol resulted in lower uterine weight, reduced induction of hepatic gene expression, and reduced mammary epithelial hyperplasia relative to continuous estradiol exposure. We conclude that organ responsivity is influenced by different hormone release kinetics, a fact that might be exploited to reduce undesired estradiol effects in postmenopausal women.

Endometriosis is a chronic, estrogen-dependent disease characterized by the presence of ectopic endometrium either in the pelvic cavity (endometriosis externa) or within the uterus (endometriosis interna, adenomyosis). Key symptoms are pelvic pain, dysmenorrhea and infertility. Established rodent animal models used for drug research in endometriosis have certain limitations. Since rodents do not menstruate, they cannot develop endometriosis externa spontaneously, but they suffer from endometriosis interna. There is growing evidence that human endometriosis externa and interna represent two faces of the same disease. Both are estrogen-dependent and respond to similar treatment paradigms. Here, we addressed the question whether a murine endometriosis interna model may also be suitable for the characterization of drugs employed in human endometriosis. We examined the effects of danazol, Faslodex and cetrorelix in SHN mice that developed endometriosis interna after pituitary grafting. The GnRH antagonist cetrorelix and the estrogen receptor antagonist Faslodex, which negatively interfered with estrogen-mediated signaling, completely inhibited endometriosis interna, whereas danazol, an androgenic progestin, showed significant therapeutic activity in the majority of SHN mice. We conclude that this murine endometriosis interna model may be a valuable complement to established endometriosis externa models to support drug research in human endometriosis.

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.