Monoclonal Antibody Against Prolactin Receptor: A Randomized Placebo-Controlled Study Evaluating Safety, Tolerability, and Pharmacokinetics of Repeated Subcutaneous Administrations in Postmenopausal Women.

BAY 1158061 is a potent monoclonal prolactin (PRL) receptor antibody, blocking PRL receptor (PRLR)-mediated signaling in a noncompetitive manner, which was tested in a randomized, placebo-controlled multiple dose study in postmenopausal women. The objective was to investigate safety, tolerability, pharmacokinetic characteristics, and effects of BAY 1158061 on serum PRL level. The study consisted of 4 parallel groups receiving up to 3 subcutaneous (sc) administrations of BAY 1158061 or placebo in 2 different dosing regimens. Twenty-nine healthy postmenopausal women were randomized and treated with BAY 1158061 or placebo: 30 mg at 14-day interval (7 participants), 60 mg at 28-day interval (8 participants), 90 mg at 14-day interval (7 participants), and placebo (7 participants). To keep the blinding, all randomized participants received sc injections biweekly (14-day interval) on 3 occasions in the lower abdomen. The PRLR antibody showed a favorable safety and tolerability profile in postmenopausal women with no distinct differences in occurrence of adverse events in BAY 1158061 or placebo-treated participants. BAY 1158061 displayed low immunogenicity with low titers of antidrug antibodies and absence of neutralizing antidrug antibodies. Pharmacokinetics were characterized by slow absorption after sc administration with median peak plasma concentrations 7 to 11 days after first dose and about 2-fold accumulation after repeated dosing every 2 weeks. The apparent mean elimination half-life was 9 to 16 days. The PRL concentration-time profiles over 24 hours showed no differences between verum- and placebo-treated participants. Based on the data obtained, BAY 1158061 is considered a good candidate for further development in endometriosis or other PRL-mediated disease conditions.

Riociguat (BAY 63-2521) and aspirin: a randomized, pharmacodynamic, and pharmacokinetic interaction study.

In preclinical studies, drugs that increase cyclic guanosine monophosphate levels have been shown to influence platelet function/aggregation; however, the effect of riociguat on human platelets is unclear. Aspirin, a platelet inhibitor, is likely to be given concomitantly in patients receiving riociguat. It is therefore important to establish clinically whether (1) riociguat affects platelet function and (2) aspirin and riociguat interact. This randomized, open-label, crossover study investigated potential pharmacodynamic and pharmacokinetic interactions between these drugs in healthy male volunteers (N = 18). There were 3 treatment regimens: a single morning dose of riociguat 2.5 mg, aspirin 500 mg on 2 consecutive mornings, and both treatments together, with riociguat given on the second morning. Fifteen participants were available for pharmacodynamic/pharmacokinetic analysis. There was no effect of riociguat alone on bleeding time, platelet aggregation, and serum thromboxane B2 levels. The effects of aspirin on these parameters were not influenced by concomitant administration of riociguat. The pharmacokinetic profile of riociguat showed interindividual variability, which was independent of aspirin coadministration. Six of 17 participants available for safety evaluation reported at least 1 treatment-emergent adverse event. All adverse events were of mild severity, apart from 1 report of moderate headache. No serious adverse events occurred. In conclusion, riociguat demonstrated no clinically relevant pharmacodynamic or pharmacokinetic interactions with aspirin at the doses used in this study in healthy men; coadministration of riociguat and aspirin should therefore not require any dose adjustment for either drug.

A phase I dose-escalation study of regorafenib (BAY 73-4506), an inhibitor of oncogenic, angiogenic, and stromal kinases, in patients with advanced solid tumors.

PURPOSE: Regorafenib is a novel oral multikinase inhibitor of angiogenic (VEGFR1-3, TIE2), stromal (PDGFR-ß, FGFR), and oncogenic kinases (KIT, RET, and RAF). This first-in-man, phase I dose-escalation study assessed the safety, pharmacokinetic, pharmacodynamic, and efficacy profiles of regorafenib in patients with advanced solid tumors. PATIENTS AND METHODS: Patients aged 18 years or older with advanced solid tumors refractory to standard treatment were recruited. Regorafenib was administered orally for 21 days on/seven days off in repeating cycles, until discontinuation due to toxicity or tumor progression. Adverse events (AE) were assessed using National Cancer Institute Common Terminology Criteria for Adverse Events v3.0. Pharmacokinetic profiles were measured after a single dose and on day 21. Pharmacodynamic and efficacy evaluations included tumor perfusion assessment using dynamic contrast-enhanced MRI, plasma cytokines, and tumor response using RECIST (v1.0). RESULTS: Fifty-three patients were enrolled into eight cohorts at dose levels from 10 to 220 mg daily. The recommended dose for future studies was determined to be 160 mg daily, with a treatment schedule of 21 days on/seven days off in repeating 28-day cycles. The most common drug-related grade 3 or 4 AEs were dermatologic AEs (hand-foot skin reaction, rash), hypertension, and diarrhea. Pharmacokinetic analysis revealed a similar exposure at steady state for the parent compound and two pharmacologically active metabolites. Tumor perfusion and plasma cytokine analysis showed biologic activity of regorafenib. Three of 47 evaluable patients achieved a partial response (renal cell carcinoma, colorectal carcinoma, and osteosarcoma). CONCLUSION: Regorafenib showed an acceptable safety profile and preliminary evidence of antitumor activity in patients with solid tumors.

Riociguat (BAY 63-2521) and warfarin: a pharmacodynamic and pharmacokinetic interaction study.

Riociguat (BAY 63-2521) and warfarin are likely to be used concomitantly to treat pulmonary hypertension. The aim of this double-blind, crossover, clinical pharmacological study in 30 healthy volunteers was to investigate potential pharmacodynamic and pharmacokinetic interactions between the 2 drugs. Healthy volunteers took 2.5 mg of oral riociguat or matching placebo 3 times daily for 10 days. A single oral dose of warfarin sodium (25 mg) was given 21 days before the study and on the seventh day of riociguat/placebo treatment. Twenty-one participants valid for safety analysis reported 89 treatment-emergent adverse events, all of mild or moderate severity. No serious adverse events occurred. The most frequently reported treatment-emergent adverse events considered to be drug-related were dyspepsia, headache, flatulence, nausea, and vomiting. Twenty-two participants were valid for pharmacodynamic/pharmaco-kinetic analysis. Riociguat (2.5 mg 3 times daily) had no pharmacodynamic interaction with warfarin. Steady-state plasma levels of riociguat did not affect prothrombin time, factor VII clotting activity, or the pharmacokinetics of warfarin. The single dose of warfarin led to a slight decrease (16%) in maximum concentration of riociguat in plasma, which is not likely to be clinically relevant. Clinical studies will confirm the finding here that combined use of riociguat with warfarin will not require dose adaptation.

IL-10 protects monocytes and macrophages from complement-mediated lysis.

Phagocytes, such as monocytes and macrophages, are important cells of the innate immunity in the defense against microbes. So far, it is unclear how these cells survive at the site of combat against microbes, where a hostile inflammatory environment prevails with strong complement activity. We hypothesized that IL-10, a key cytokine involved in the resolution of inflammation, induces resistance to complement attack. Here, we demonstrate for the first time such a cell-protective effect of IL-10 on human monocytes and macrophages. IL-10 is indeed able to protect these cell types in an in vitro model of complement lysis triggered by an anti-MHCI antibody or by binding of zymosan. Investigating potential underlying mechanisms, we found that IL-10 up-regulated the expression of complement regulatory membrane protein CD59 and the general cell-protective stress protein HO-1 in human monocytes. However, further functional analysis failed to link these individual IL-10-mediated effects with the increased protection from complement lysis. Blocking the protective effect of CD59 with an antibody increased complement lysis but did not abrogate the IL-10-protective effect. Interestingly, chemical interference with HO-1 activity did abrogate the protective effect of IL-10, but siRNA-mediated knockdown of HO-1 did not confirm this observation. Our results suggest that IL-10 generates pathogen-clearing phagocytes, which are resistant to complement lysis and thereby, enabled to survive longer in a hostile inflammatory environment.

Quantification of vascular endothelial growth factor, interleukin-8, and basic fibroblast growth factor in plasma of cancer patients and healthy volunteers - comparison of ELISA and microsphere-based multiplexed immunoassay.

BACKGROUND: Vascular endothelial growth factor (VEGF), interleukin-8 (IL-8) and basic fibroblast growth factor (basic FGF) are angiogenic growth factors which may be useful as biomarkers in drug development, where they could give early information on the antiangiogenic activity of novel anticancer compounds. METHODS: We compared two commercially available assays, enzyme linked immunosorbent assay (ELISA) and a multiplexed bead-based immunoassay (xMAP), for the quantification of these factors in plasma samples from more than 100 cancer patients and healthy individuals. RESULTS: For VEGF and IL-8, but not for basic FGF, xMAP was more sensitive than the respective ELISA. This was true for healthy subjects as well as for cancer patients. Intraassay precision was comparable between both assay formats. Linear regression analysis of VEGF concentrations demonstrated a good correlation between ELISA and xMAP. Bland-Altman analysis showed a systematic difference between both assays, with ELISA giving higher concentration values. VEGF levels were higher in female volunteers, and both assays were able to detect this difference. CONCLUSIONS: Multiplexed microsphere-based immunoassays have the potential to substitute ELISA for the detection of proangiogenic growth factors in clinical studies. Their shorter assay times and their ability to quantify multiple analytes in a small sample volume are advantageous.

Frequency of blood CX3CR1-positive natural killer cells correlates with disease activity in multiple sclerosis patients.

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) characterized by enormous variability in its clinical presentation and course, and for which clear diagnostic parameters are lacking. Here we performed an RNA screen in peripheral mononuclear cells from relapsing-remitting (RR) and primary progressive (PP) MS patients compared with healthy donors (HD) that indicated, among other findings, a role for the chemokine receptor CX3CR1 as a diagnostic marker. Gene expression and flow cytometric analyses demonstrated a significantly lower expression of CX3CR1 in MS patients compared with healthy individuals. The subpopulation of cells responsible for causing this reduced expression of CX3CR1 consisted exclusively of natural killer (NK) cells. Importantly, we found a correlation between disease activity and frequency of CX3CR1-positive NK cells in RRMS patients. These findings emphasize the role of NK cells in the development and course of MS and provide evidence for CX3CR1 expression as a marker for MS patients and disease activity.

The target discovery process.

In order to minimise attrition rates in drug development projects, a target discovery process is implemented to select and characterise the most suitable candidate kinase targets, before lead identification and lead optimisation are embarked upon. The process consists of 1) target selection, 2) target assessment, and 3) target validation. This rational approach to target discovery, as a prerequisite for lead discovery, ensures that new therapeutic targets fulfil a set of general criteria, as well as indication-specific, descriptive and functional ones. The approach should ultimately maximise the likelihood of achieving target-selective inhibition by small-molecule inhibitors with minimal in vivo side effects and a therapeutic effect based on a sound biological hypothesis.

Distinct gene expression patterns in a tamoxifen-sensitive human mammary carcinoma xenograft and its tamoxifen-resistant subline MaCa 3366/TAM.

The reasons why human mammary tumors become resistant to tamoxifen therapy are mainly unknown. Changes in gene expression may occur as cells acquire resistance to antiestrogens. We therefore undertook a comparative gene expression analysis of tamoxifen-sensitive and tamoxifen-resistant human breast cancer in vivo models using Affymetrix oligonucleotide arrays to analyze differential gene expression. Total RNAs from the tamoxifen-sensitive patient-derived mammary carcinoma xenograft MaCa 3366 and the tamoxifen-resistant model MaCa 3366/TAM were hybridized to Affymetrix HuGeneFL and to Hu95Av2 arrays. Pairwise comparisons and clustering algorithms were applied to identify differentially expressed genes and patterns of gene expression. As revealed by cluster analysis, the tamoxifen-sensitive and the tamoxifen-resistant breast carcinomas differed regarding their gene expression pattern. More than 100 transcripts are changed in abundance in MaCa 3366/TAM as compared with MaCa 3366. Among the genes that are differentially expressed in the tamoxifen-resistant tumors, there are several IFN-inducible and estrogen-responsive genes, and genes known to be involved in breast carcinogenesis. The genes neuronatin (NNAT) and bone marrow stem cell antigen 2 (BST2) were sharply up-regulated in MaCa 3366/TAM. The differential expression of four genes (NNAT, BST2, IGFBP5, and BCAS1) was confirmed by Taqman PCR. Our results provide the starting point for deriving markers for tamoxifen resistance by differential gene expression profiling in a human breast cancer model of acquired tamoxifen resistance. Finally, genes whose expression profiles are distinctly changed between the two xenograft lines will be further evaluated as potential targets for diagnostic or therapeutic approaches of tamoxifen-resistant breast cancer.

Gene expression profiling of scrapie-infected brain tissue.

The underlying pathomechanisms in prion infections of the central nervous system are still insufficiently understood. The identification of genes with altered expression patterns in the diseased brain may provide insight into the disease development on the molecular level, which ultimately leads to neuronal loss. To provide a detailed analysis of changes in the molecular level in prion disease pathology we used a large-scale gene array based approach, which covers more than 11,000 functionally characterised sequences and expressed sequence tags, for the analysis of gene expression profile alterations in the cortex, medulla, and pons of scrapie-infected mice. The study identified in total 114 genes with altered mRNA levels, the majority of which were previously not known to be affected by the disease. Overall the gene array data demonstrate the presence of a strong inflammatory reaction and stress response, and show similarities to gene expression patterns found in brains affected by Alzheimer's disease and aging, respectively.

Cyr61, a deregulated gene in endometriosis.

Gene expression profiling was performed to identify genes involved in the development of endometriosis. In the secretory phase of the menstrual cycle, several estrogen-regulated genes were up-regulated in endometria of women with endometriosis. The most consistent regulation with one of the highest factors was observed for the Cyr61 gene, which codes for a secreted, cysteine-rich, heparin-binding protein that promotes cell adhesion, migration, and neovascularization. Estrogen responsiveness of endometrial Cyr61 expression was suggested by the higher expression during the proliferative phase and the reduction observed in human endometrial fragments grafted into nude mice subsequently treated with an anti-estrogen. The expression level of Cyr61 was found to be further increased in ectopic endometriotic lesions, as compared to eutopic endometria. In these lesions, an imbalance in expression of the estrogen-converting enzymes 17beta-hydroxysteroid dehydrogenase type 1 and 2 was found, which might explain the elevated Cyr61 level. However, Cyr61 expression was not altered in endometriotic lesions of women treated with a GnRH agonist. These results suggest that Cyr61 may represent a gene characteristic for endometriosis and also play an important role in the development and persistence of endometriotic lesions.

Expression profiling of IL-10-regulated genes in human monocytes and peripheral blood mononuclear cells from psoriatic patients during IL-10 therapy.

Interleukin-10 (IL-10), originally identified as an inhibitor of pro-inflammatory cytokine production, exerts multiple immunomodulatory functions. Its ability to inhibit a Th1 response has been used in clinical trials for the treatment of inflammatory diseases including psoriasis. However, little is known about the molecular mechanisms of IL-10 functions. We aimed at identifying possible mediators of in vitro IL-10 treatment in monocytes by gene chip technology using Hu95a Affymetrix mRNA arrays with 12,000 genes. To prove relevance of the identified genes for the clinical situation we compared these in vitro results with genes being regulated by IL-10 in peripheral blood mononuclear cells from psoriatic patients undergoing IL-10 therapy. A high proportion of the 1,600 genes up-regulated and 1,300 genes down-regulated in vitro was found to be similarly regulated in vivo. Some genes, which were previously unknown to be regulated by IL-10, can be assigned to known IL-10 functions like e.g. the increase of pathogen clearance. Other new potentially immunomodulating genes have been identified to be regulated by IL-10, but their impact needs to be experimentally evaluated. We could confirm a recently reported up-regulation of heme oxygenase-1 (HO-1). However, we demonstrate that the anti-inflammatory mechanisms of IL-10 remain functional even when HO-1 is irreversibly inhibited.

Comparison of proteomic and genomic analyses of the human breast cancer cell line T47D and the antiestrogen-resistant derivative T47D-r.

In search of novel mechanisms leading to the development of antiestrogen-resistance in human breast tumors, we analyzed differences in the gene and protein expression pattern of the human breast carcinoma cell line T47D and its derivative T47D-r, which is resistant toward the pure antiestrogen ZM 182780 (Faslodex trade mark, fulvestrant). Affymetrix DNA chip hybridizations on the commercially available HuGeneFL and Hu95A arrays were carried out in parallel to the proteomics analysis where the total cellular protein content of T47D or T47D-r was separated on two-dimensional gels. Thirty-eight proteins were found to be reproducibly up- or down-regulated more than 2-fold in T47D-r versus T47D in the proteomics analysis. Comparison with differential mRNA analysis revealed that 19 of these were up- or down-regulated in parallel with the corresponding mRNA molecules, among which are the protease cathepsin D, the GTPases Rab11a and MxA, and the secreted protein hAG-2. For 11 proteins, the corresponding mRNA was not found to be differentially expressed, and for eight proteins an inverse regulation was found at the mRNA level. In summary, mRNA expression data, when combined with proteomic information, provide a more detailed picture of how breast cancer cells are altered in their antiestrogen-resistant compared with the antiestrogen-sensitive state.