Mass cytometry analysis reveals a distinct immune environment in peritoneal fluid in endometriosis: a characterisation study.

BACKGROUND: Endometriosis is a gynaecological condition characterised by immune cell infiltration and distinct inflammatory signatures found in the peritoneal cavity. In this study, we aim to characterise the immune microenvironment in samples isolated from the peritoneal cavity in patients with endometriosis. METHODS: We applied mass cytometry (CyTOF), a recently developed multiparameter single-cell technique, in order to characterise and quantify the immune cells found in peritoneal fluid and peripheral blood from endometriosis and control patients. RESULTS: Our results demonstrate the presence of more than 40 different distinct immune cell types within the peritoneal cavity. This suggests that there is a complex and highly heterogeneous inflammatory microenvironment underpinning the pathology of endometriosis. Stratification by clinical disease stages reveals a dynamic spectrum of cell signatures suggesting that adaptations in the inflammatory system occur due to the severity of the disease. Notably, among the inflammatory microenvironment in peritoneal fluid (PF), the presence of CD69+ T cell subsets is increased in endometriosis when compared to control patient samples. On these CD69+ cells, the expression of markers associated with T cell function are reduced in PF samples compared to blood. Comparisons between CD69+ and CD69- populations reveal distinct phenotypes across peritoneal T cell lineages. Taken together, our results suggest that both the innate and the adaptive immune system play roles in endometriosis. CONCLUSIONS: This study provides a systematic characterisation of the specific immune environment in the peritoneal cavity and identifies cell immune signatures associated with endometriosis. Overall, our results provide novel insights into the specific cell phenotypes governing inflammation in patients with endometriosis. This prospective study offers a useful resource for understanding disease pathology and opportunities for identifying therapeutic targets.

Discovery of the potent non-steroidal glucocorticoid receptor modulator BAY 1003803 as clinical candidate.

We report on the discovery of the new clinical candidate BAY 1003803 as glucocorticoid receptor agonist for the topical treatment of psoriasis or severe atopic dermatitis. In the course of optimizing the amino alcohol series as a highly potent new non-steroidal lead structure, considerations were made as to how physicochemical properties and safety concerns relate to structural motifs. BAY 1003803 demonstrates strong anti-inflammatory activity in vitro paired with a pharmacokinetic profile suitable for topical application.

Discovery and optimization of pyridyl-cycloalkyl-carboxylic acids as inhibitors of microsomal prostaglandin E synthase-1 for the treatment of endometriosis.

Here we report on novel and potent pyridyl-cycloalkyl-carboxylic acid inhibitors of microsomal prostaglandin E synthase-1 (PTGES). PTGES produces, as part of the prostaglandin pathway, prostaglandin E2 which is a well-known driver for pain and inflammation. This fact together with the observed upregulation of PTGES during inflammation suggests that blockade of the enzyme might provide a beneficial treatment option for inflammation related conditions such as endometriosis. Compound 5a, a close analogue of the screening hit, potently inhibited PTGES in vitro, displayed excellent PK properties in vitro and in vivo and demonstrated efficacy in a CFA-induced pain model in mice and in a rat dyspareunia endometriosis model and was therefore selected for further studies.

Discovery of IRAK4 Inhibitors BAY1834845 (Zabedosertib) and BAY1830839.

Interleukin-1 receptor-associated kinase 4 (IRAK4) plays a critical role in innate inflammatory processes. Here, we describe the discovery of two clinical candidate IRAK4 inhibitors, BAY1834845 (zabedosertib) and BAY1830839, starting from a high-throughput screening hit derived from Bayer's compound library. By exploiting binding site features distinct to IRAK4 using an in-house docking model, liabilities of the original hit could surprisingly be overcome to confer both candidates with a unique combination of good potency and selectivity. Favorable DMPK profiles and activity in animal inflammation models led to the selection of these two compounds for clinical development in patients.

Anxiety-related behaviors without observation of generalized pain in a mouse model of endometriosis.

Endometriosis is a chronic, hormone-dependent, inflammatory disease, characterized by the presence and growth of endometrial tissue outside the uterine cavity. It is associated with moderate to severe pelvic and abdominal pain symptoms, subfertility and a marked reduction in health-related quality of life. Furthermore, relevant co-morbidities with affective disorders like depression or anxiety have been described. These conditions have a worsening effect on pain perception in patients and might explain the negative impact on quality of life observed in those suffering from endometriosis-associated pain. Whereas several studies using rodent models of endometriosis focused on biological and histopathological similarities with the human situation, the behavioral characterization of these models was never performed. This study investigated the anxiety-related behaviors in a syngeneic model of endometriosis. Using elevated plus maze and the novel environment induced feeding suppression assays we observed the presence of anxiety-related behaviors in endometriosis-induced mice. In contrast, locomotion or generalized pain did not differ between groups. These results indicate that the presence of endometriosis lesions in the abdominal cavity could, similarly to patients, induce profound psychopathological changes/impairments in mice. These readouts might provide additional tools for preclinical identification of mechanisms relevant for development of endometriosis-related symptoms.

Discovery of BAY-390, a Selective CNS Penetrant Chemical Probe as Transient Receptor Potential Ankyrin 1 (TRPA1) Antagonist.

Transient receptor potential ankyrin 1 (TRPA1) is a voltage-dependent, ligand-gated ion channel, and activation thereof is linked to a variety of painful conditions. Preclinical studies have demonstrated the role of TRPA1 receptors in a broad range of animal models of acute, inflammatory, and neuropathic pain. In addition, a clinical study using the TRPA1 antagonist GRC-17536 (Glenmark Pharmaceuticals) demonstrated efficacy in a subgroup of patients with painful diabetic neuropathy. Consequently, there is an increasing interest in TRPA1 inhibitors as potential analgesics. Herein, we report the identification of a fragment-like hit from a high-throughput screening (HTS) campaign and subsequent optimization to provide a novel and brain-penetrant TRPA1 inhibitor (compound 18, BAY-390), which is now being made available to the research community as an open-source in vivo probe.

Extended DNFB-induced contact hypersensitivity models display characteristics of chronic inflammatory dermatoses.

Despite recent developments, there is a high medical need for new treatment options for chronic inflammatory dermatoses like allergic contact dermatitis (ACD) and psoriasis. Particularly, more predictive skin inflammation models are required to facilitate the process of drug discovery. Murine contact hypersensitivity (CHS) models adequately reflect ACD and are also used to characterize therapeutic approaches for psoriasis. Using the hapten 2,4-dinitrofluorobenzene (DNFB), we established new subacute and subchronic DNFB-induced CHS models in C57BL/6 mice, which more closely reflect the characteristics of chronic T-cell-dependent inflammatory dermatoses as pronounced keratinocyte proliferation, strong hypervascularization, immune cell infiltration and overexpression of T cell and inflammatory cytokines. For the subacute DNFB model, we demonstrated anti-inflammatory activity of the glucocorticoid, prednisolone, as well as of neutralization of TNFα, IL-12/IL-23 or IL-18. In the subchronic DNFB-induced CHS model, deficiency for MyD88 and IL-12/IL-35 p35 chain but not IL-12/IL-23 p40 chain led to decreased skin inflammation. Furthermore, as exemplified by the dose-dependently effective therapeutic prednisolone treatment, the subchronic model allows the continuous therapy of a pre-established stable contact dermatitis. Altogether, prolonged DNFB-induced mouse CHS models closely reflect ACD sensitive to glucocorticoids as standard therapy, reveal a more chronic skin inflammation and are responsive to cytokine antagonization.

Circulating tumour cells escape from EpCAM-based detection due to epithelial-to-mesenchymal transition.

BACKGROUND: Circulating tumour cells (CTCs) have shown prognostic relevance in metastatic breast, prostate, colon and pancreatic cancer. For further development of CTCs as a biomarker, we compared the performance of different protocols for CTC detection in murine breast cancer xenograft models (MDA-MB-231, MDA-MB-468 and KPL-4). Blood samples were taken from tumour bearing animals (20 to 200 mm2) and analysed for CTCs using 1. an epithelial marker based enrichment method (AdnaTest), 2. an antibody independent technique, targeting human gene transcripts (qualitative PCR), and 3. an antibody-independent approach, targeting human DNA-sequences (quantitative PCR). Further, gene expression changes associated with epithelial-to-mesenchymal transition (EMT) were determined with an EMT-specific PCR assay. METHODS: We used the commercially available Adna Test, RT-PCR on human housekeeping genes and a PCR on AluJ sequences to detect CTCs in xenografts models. Phenotypic changes in CTCs were tested with the commercially available "Human Epithelial to Mesenchymal Transition RT-Profiler PCR Array". RESULTS: Although the AdnaTest detects as few as 1 tumour cell in 1 ml of mouse blood spiking experiments, no CTCs were detectable with this approach in vivo despite visible metastasis formation. The presence of CTCs could, however, be demonstrated by PCR targeting human transcripts or DNA-sequences - without epithelial pre-enrichment. The failure of CTC detection by the AdnaTest resulted from downregulation of EpCAM, whereas mesenchymal markers like Twist and EGFR were upregulated on CTCs. Such a change in the expression profile during metastatic spread of tumour cells has already been reported and was linked to a biological program termed epithelial-mesenchymal transition (EMT). CONCLUSIONS: The use of EpCAM-based enrichment techniques leads to the failure to detect CTC populations that have undergone EMT. Our findings may explain clinical results where low CTC numbers have been reported even in patients with late metastatic cancers. These results are a starting point for the identification of new markers for detection or capture of CTCs, including the mesenchymal-like subpopulations.

Cancer therapy monitoring in xenografts by quantitative analysis of circulating tumor DNA.

CONTEXT: Circulating tumor DNA (ctDNA) is a promising biomarker in cancer. MATERIALS AND METHODS: We generated xenograft models of cancer and detected ctDNA in plasma by qRCR targeting human AluJ sequences. RESULTS: Our assay reached single cell sensitivity in vitro and a correlation between ctDNA amount and tumor size was observed in vivo. Treatment with a mitogen activated protein kinase kinase (MEK)-inhibitor (BAY 869766) reduced ctDNA levels. Using this assay, we also confirmed that high levels of cell-free DNA are found in cancer patients compared to healthy individuals. DISCUSSION AND CONCLUSION: We show that ctDNA may be useful biomarker for monitoring tumor growth and treatment response.

High-resolution ultrasound imaging: a novel technique for the noninvasive in vivo analysis of endometriotic lesion and cyst formation in small animal models.

Endometriosis, the presence of endometrial tissue at ectopic sites, is a highly prevalent gynecological disease severely affecting a patient's quality of life. To analyze the mechanisms involved in the disease and to identify new molecular targets for effective therapies, small animal models are an important approach. Herein, we report the first use of high-resolution ultrasound imaging for the in vivo analysis of intraperitoneal endometriotic lesions in mice. This noninvasive technology allows for the repetitive quantitative analysis of growth, cyst development, and adhesion formation of endometriotic lesions with a low intra- and interobserver variability. Moreover, it enables one to easily differentiate between endometrial cysts and stroma. Accordingly, volume measurements of both endometrial cysts and stroma indicated that the initial establishment of endometriotic lesions is associated with enhanced cellular proliferation, followed by a phase of increased secretory activity of endometrial glands. Results of ultrasound analysis correlated well with measurements of lesion volumes by caliper and histology. Importantly, ultrasound imaging could be performed repetitively and noninvasively and reflected best the in vivo situation. The technique could further be demonstrated to successfully monitor the significant inhibition of growth of endometriotic lesions after specific estrogen receptor destabilizator treatment. Thus, high-resolution ultrasound imaging represents an important tool for future preclinical small animal studies, which address the pathophysiology of endometriosis and the development of new treatment strategies.

Shortened treatment duration of glucocorticoid-induced skin atrophy in rats.

Glucocorticoids (GCs) belong to the most widely used anti-inflammatory drugs at all. However, their topical use is limited by their side effect potential, with skin atrophy being the most prominent one. Thus, determining the atrophogenic potential of novel compounds is of importance for drug development. Currently, the most frequently performed model in the base and pharmaceutical research is the hr/hr rat model of GC-induced skin atrophy that lasts for 19 days. In this study, we analysed statistically skin atrophy experiments retrospectively to ascertain (i) the earliest time-point, at which skin atrophy is significantly induced; and (ii) whether the differences between the GC treatment groups change until the end of the experiment. We show here that the treatment duration of rat skin atrophy models might be reduced to 5 days for economical and ethical reasons.

Efomycine M, a new specific inhibitor of selectin, impairs leukocyte adhesion and alleviates cutaneous inflammation.

Specific interference with molecular mechanisms guiding tissue localization of leukocytes may be of great utility for selective immunosuppressive therapies. We have discovered and characterized efomycines, a new family of selective small-molecule inhibitors of selectin functions. Members of this family significantly inhibited leukocyte adhesion in vitro. Efomycine M, which was nontoxic and showed the most selective inhibitory effects on selectin-mediated leukocyte-endothelial adhesion in vitro, significantly diminished rolling in mouse ear venules in vivo as seen by intravital microscopy. In addition, efomycine M alleviated cutaneous inflammation in two complementary mouse models of psoriasis, one of the most common chronic inflammatory skin disorders. Molecular modeling demonstrated a spatial conformation of efomycines mimicking naturally occurring selectin ligands. Efomycine M might be efficacious in the treatment of human inflammatory disorders through a similar mechanism.

Eliapixant is a selective P2X3 receptor antagonist for the treatment of disorders associated with hypersensitive nerve fibers.

ATP-dependent P2X3 receptors play a crucial role in the sensitization of nerve fibers and pathological pain pathways. They are also involved in pathways triggering cough and may contribute to the pathophysiology of endometriosis and overactive bladder. However, despite the strong therapeutic rationale for targeting P2X3 receptors, preliminary antagonists have been hampered by off-target effects, including severe taste disturbances associated with blocking the P2X2/3 receptor heterotrimer. Here we present a P2X3 receptor antagonist, eliapixant (BAY 1817080), which is both highly potent and selective for P2X3 over other P2X subtypes in vitro, including P2X2/3. We show that eliapixant reduces inflammatory pain in relevant animal models. We also provide the first in vivo experimental evidence that P2X3 antagonism reduces neurogenic inflammation, a phenomenon hypothesised to contribute to several diseases, including endometriosis. To test whether eliapixant could help treat endometriosis, we confirmed P2X3 expression on nerve fibers innervating human endometriotic lesions. We then demonstrate that eliapixant reduces vaginal hyperalgesia in an animal model of endometriosis-associated dyspareunia, even beyond treatment cessation. Our findings indicate that P2X3 antagonism could alleviate pain, including non-menstrual pelvic pain, and modify the underlying disease pathophysiology in women with endometriosis. Eliapixant is currently under clinical development for the treatment of disorders associated with hypersensitive nerve fibers.

Preclinical models of endometriosis and interstitial cystitis/bladder pain syndrome: an Innovative Medicines Initiative-PainCare initiative to improve their value for translational research in pelvic pain.

ABSTRACT: Endometriosis (ENDO) and interstitial cystitis/bladder pain syndrome (IC/BPS) are chronic pain conditions for which better treatments are urgently needed. Development of new therapies with proven clinical benefit has been slow. We have conducted a review of existing preclinical in vivo models for ENDO and IC/BPS in rodents, discussed to what extent they replicate the phenotype and pain experience of patients, as well as their relevance for translational research. In 1009 publications detailing ENDO models, 41% used autologous, 26% syngeneic, 18% xenograft, and 11% allogeneic tissue in transplantation models. Intraperitoneal injection of endometrial tissue was the subcategory with the highest construct validity score for translational research. From 1055 IC/BPS publications, most interventions were bladder centric (85%), followed by complex mechanisms (8%) and stress-induced models (7%). Within these categories, the most frequently used models were instillation of irritants (92%), autoimmune (43%), and water avoidance stress (39%), respectively. Notably, although pelvic pain is a hallmark of both conditions and a key endpoint for development of novel therapies, only a small proportion of the studies (models of ENDO: 0.5%-12% and models of IC/BPS: 20%-44%) examined endpoints associated with pain. Moreover, only 2% and 3% of publications using models of ENDO and IC/BPS investigated nonevoked pain endpoints. This analysis highlights the wide variety of models used, limiting reproducibility and translation of results. We recommend refining models so that they better reflect clinical reality, sharing protocols, and using standardized endpoints to improve reproducibility. We are addressing this in our project Innovative Medicines Initiative-PainCare/Translational Research in Pelvic Pain.

Neuropeptide S receptor 1 is a nonhormonal treatment target in endometriosis.

Endometriosis is a common chronic inflammatory condition causing pelvic pain and infertility in women, with limited treatment options and 50% heritability. We leveraged genetic analyses in two species with spontaneous endometriosis, humans and the rhesus macaque, to uncover treatment targets. We sequenced DNA from 32 human families contributing to a genetic linkage signal on chromosome 7p13-15 and observed significant overrepresentation of predicted deleterious low-frequency coding variants in NPSR1, the gene encoding neuropeptide S receptor 1, in cases (predominantly stage III/IV) versus controls (P = 7.8 × 10-4). Significant linkage to the region orthologous to human 7p13-15 was replicated in a pedigree of 849 rhesus macaques (P = 0.0095). Targeted association analyses in 3194 surgically confirmed, unrelated cases and 7060 controls revealed that a common insertion/deletion variant, rs142885915, was significantly associated with stage III/IV endometriosis (P = 5.2 × 10-5; odds ratio, 1.23; 95% CI, 1.09 to 1.39). Immunohistochemistry, qRT-PCR, and flow cytometry experiments demonstrated that NPSR1 was expressed in glandular epithelium from eutopic and ectopic endometrium, and on monocytes in peritoneal fluid. The NPSR1 inhibitor SHA 68R blocked NPSR1-mediated signaling, proinflammatory TNF-α release, and monocyte chemotaxis in vitro (P < 0.01), and led to a significant reduction of inflammatory cell infiltrate and abdominal pain (P < 0.05) in a mouse model of peritoneal inflammation as well as in a mouse model of endometriosis. We conclude that the NPSR1/NPS system is a genetically validated, nonhormonal target for the treatment of endometriosis with likely increased relevance to stage III/IV disease.

Discovery and Characterization of BAY 1214784, an Orally Available Spiroindoline Derivative Acting as a Potent and Selective Antagonist of the Human Gonadotropin-Releasing Hormone Receptor as Proven in a First-In-Human Study in Postmenopausal Women.

The growth of uterine fibroids is sex hormone-dependent and commonly associated with highly incapacitating symptoms. Most treatment options consist of the control of these hormonal effects, ultimately blocking proliferative estrogen signaling (i.e., oral contraceptives/antagonization of human gonadotropin-releasing hormone receptor [hGnRH-R] activity). Full hGnRH-R blockade, however, results in menopausal symptoms and affects bone mineralization, thus limiting treatment duration or demanding estrogen add-back approaches. To overcome such issues, we aimed to identify novel, small-molecule hGnRH-R antagonists. This led to the discovery of compound BAY 1214784, an orally available, potent, and selective hGnRH-R antagonist. Altering the geminal dimethylindoline core of the initial hit compound to a spiroindoline system significantly improved GnRH-R antagonist potencies across several species, mandatory for a successful compound optimization in vivo. In a first-in-human study in postmenopausal women, once daily treatment with BAY 1214784 effectively lowered plasma luteinizing hormone levels by up to 49%, at the same time being associated with low pharmacokinetic variability and good tolerability.

Author Correction: Amine oxidase 3 is a novel pro-inflammatory marker of oxidative stress in peritoneal endometriosis lesions.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Amine oxidase 3 is a novel pro-inflammatory marker of oxidative stress in peritoneal endometriosis lesions.

Endometriosis is a common gynaecological disease of women in reproductive age, and is thought to arise from retrograde menstruation and implantation of endometrial tissue, mostly into the peritoneal cavity. The condition is characterized by a chronic, unresolved inflammatory process thereby contributing to pain as cardinal symptom in endometriosis. Elevated reactive oxygen species (ROS) and oxidative stress have been postulated as factors in endometriosis pathogenesis. We here set out for a systematic study to identify novel mechanisms and pathways relating to oxidative stress in ectopic peritoneal lesions. Using combined proteomic and transcriptomic approaches, we identified novel targets including upregulated pro-oxidative enzymes, such as amine oxidase 3/vascular adhesion protein 1 (AOC3/VAP1) as well as downregulated protective factors, in particular alkenal reductase PTGR1 and methionine sulfoxide reductase. Consistent with an altered ROS landscape, we observed hemoglobin / iron overload, ROS production and lipid peroxidation in ectopic lesions. ROS-derived 4-hydroxy-2-nonenal induced interleukin IL-8 release from monocytes. Notably, AOC3 inhibitors provoked analgesic effects in inflammatory pain models in vivo, suggesting potential translational applicability.

Induction of Foxp3+ regulatory T cells with histone deacetylase inhibitors.

Histone deacetylase inhibitors are under investigation in the clinic as a new class of anti-cancer therapeutics. While recent studies have also suggested their potential as inhibitors of a wide spectrum of inflammatory reactions, the anti-inflammatory mechanism of action of these compounds is not fully defined. We show here that the histone deacetylase inhibitors MS-275 and SAHA induce the generation of regulatory T cells (Tregs) from anti-CD3/anti-CD28-stimulated human CD4(+)CD25(-) T cells. These Tregs express the regulatory T cell-associated transcription factor Foxp3 and display suppressive activity against CD4(+)CD25(-) T cell proliferation. Topical treatment with histone deacetylase inhibitors also induces Foxp3 expression in the draining lymph nodes and the skin in the context of a murine contact hypersensitivity model. These findings suggest that Treg generation may serve as a novel mechanism by which histone deacetylase inhibitors regulate the immune response, and provide an additional rationale for the use of histone deacetylase inhibitors in the treatment of inflammation.

Discovery and Characterization of the Potent and Selective P2X4 Inhibitor N-[4-(3-Chlorophenoxy)-3-sulfamoylphenyl]-2-phenylacetamide (BAY-1797) and Structure-Guided Amelioration of Its CYP3A4 Induction Profile.

The P2X4 receptor is a ligand-gated ion channel that is expressed on a variety of cell types, especially those involved in inflammatory and immune processes. High-throughput screening led to a new class of P2X4 inhibitors with substantial CYP 3A4 induction in human hepatocytes. A structure-guided optimization with respect to decreased pregnane X receptor (PXR) binding was started. It was found that the introduction of larger and more polar substituents on the ether linker led to less PXR binding while maintaining the P2X4 inhibitory potency. This translated into significantly reduced CYP 3A4 induction for compounds 71 and 73. Unfortunately, the in vivo pharmacokinetic (PK) profiles of these compounds were insufficient for the desired profile in humans. However, BAY-1797 (10) was identified and characterized as a potent and selective P2X4 antagonist. This compound is suitable for in vivo studies in rodents, and the anti-inflammatory and anti-nociceptive effects of BAY-1797 were demonstrated in a mouse complete Freund's adjuvant (CFA) inflammatory pain model.