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Nintedanib and pirfenidone have been approved for the treatment of idiopathic pulmonary fibrosis (IPF). Both drugs were shown to reduce but not to stop disease progression. Hence the medical need is still high for new effective anti-fibrotic molecules. Arsenic trioxide (ATO), a safe and effective drug used in cancer therapy, decreased pulmonary fibrosis induced by bleomycin in mice. In order to specify the anti-fibrotic properties of ATO, we investigated its effects on main functions of human lung fibroblasts (HLFs). IPF and control HLFs were derived from lung biopsies isolated from patients with IPF or without intertitial lung disease. After pre-treatment with ATO (0.01-1 μM) and stimulation with PDGF-BB (50 ng/ml) or TGF-β1 (1 ng/ml), proliferation, migration and differentiation of HLFs were assessed as well as the cell stress response triggered by ATO. The metalloid did not affect cell viability but it significantly decreased, in a concentration-dependent manner, the proliferation and migration of control and IPF HLFs induced by PDGF-BB. ATO also prevented the up-regulation of α-SMA, Collagen-1 and p-SMAD3 protein levels in TGF-β1-stimulated HLFs. The metalloid effects were associated with stabilization of the transcription factor NRF2 and induction of the antioxidant proteins NQO1 and HO-1 that emphases the stress response developed by ATO-treated HLFs. In conclusions, our results demonstrate that, in vitro, ATO concentrations, in the range of arsenic plasmatic levels measured in patients treated with standard dosing, counteracts the detrimental functions of IPF HLFs. ATO may thus be taken into consideration as a new therapeutic option for IPF treatment.

Pulmonary fibrosis comprises a multitude of rapidly progressing interstitial lung diseases assumed to develop from failure in the process of wound healing. Although TGFβ was reported to play a central role in the pathogenesis, it seems evident that also pro-inflammatory mediators contribute to the initiation and progression of lung fibrosis. In this study, the effect of TGFβ and TNFα on the induction of profibrotic mediators was investigated in human ex vivo lung tissue slices (Precision-Cut Lung Slices; PCLS). PCLS were prepared from tumor-free lung tissue sections and were cultured in the presence of TGFβ, TNFα or combination (TGFβ/TNFα) for up to 48h. Stimulation with TGFβ, TNFα or TGFβ/TNFα lead to specific up- or downregulation of distinct mediators in PCLS as compared to control samples, highlighting the necessity of combining key cytokines for the efficient induction of a pro-fibrotic profile. Upon others, TNFα as well as TGFβ/TNFα stimulation of PCLS lead to specific upregulation of IL-1β, while single stimulation with TGFβ showed no effect. IP10 and IFNγ levels remain unchanged in the presence of TNFα, while TGFβ/TNFα, or TGFβ alone lead to down-modulation of the expression. Stimulation of PCLS with either TNFα or TGFβ showed 2- to 3-fold up-regulation of PAI1. The combination of TNFα and TGFβ lead to further amplification of PAI1 mRNA levels to up to 7-fold. Importantly, ex vivo treatment with Pirfenidone efficiently reverts the TGFβ and TNFα-mediated modulation. We describe here the induction of a specific pro-fibrotic biomarker pattern in PCLS by TGFβ and TNFα stimulation, enabling the investigation of fibrotic signaling pathways with high translational relevance.

Einleitung: Syk ist eine intrazellulare Nicht-Rezeptor-Tyrosinkinase, die jüngst als zentraler Mediator glattmuskulärer Konstriktion identifiziert wurde. Ob Syk eine funktionelle Rolle in der pulmonalen Vasokonstriktion ausübt und sich hieraus mögliche therapeutische Implikationen für die pulmonalarterielle Hypertonie (PAH) ableiten lassen, ist gegenwärtig unbekannt. Methoden: Das vaskuläre Expressionsprofil von Syk wurde sowohl in humanem (PAH versus Nicht-PAH) als auch in murinem Lungengewebe mittels Konfokal-Mikroskopie charakterisiert. Die funktionelle Rolle von Syk in der pulmonalen Vasokonstriktion wurde mittels Einsatz von Syk- und/oder NO-Inhibitoren in humanen Precision-Cut Lung Slices (PCLS) und in isoliert perfundierten und ventilierten Lungen von Wildtyp-Mäusen oder von eNOS- oder PKCa-defizienten Mäusen untersucht. Die Effekte der Syk-Inhibition auf die PAH-assoziierte pulmonalvaskuläre Hyperreagibilität wurden in isolierten Mauslungen nach Induktion pulmonaler TH2-Inflammation analysiert. Ergebnisse: Syk ist sowohl beim Menschen als auch in der Maus in den glatten Gefäßmuskelzellen der Lunge exprimiert. Die Inhibition von Syk hatte eine deutliche Reduktion der Serotonin-, Endothelin-1- und Angiotensin II-induzierten pulmonalen Vasokonstriktion in humanen PCLS und/oder in isoliert perfundierten Mauslungen zur Folge, unabhängig von eNOS oder PKCa. In präkonstringierten Pulmonalgefäßen führte Syk-Inhibition NO-unabhängig zu einer raschen, reversiblen Vasodilatation. Darüber hinaus reduzierte Syk-Inhibition sowohl die hypoxisch pulmonale Vasokonstriktion als auch die TH2-induzierte pulmonalvaskuläre Hyperreagibilität. Diskussion: Syk reguliert die pulmonale Vasokonstriktion unabhängig von NO. Unsere Daten implizieren, dass Syk-Inhibition eine innovative Therapieform in der Behandlung der PAH darstellen könnte.

Introduction: In the airways, spleen tyrosine kinase (Syk) promotes inflammation, smooth muscle cell proliferation and contraction (Tabeling, C. et al. Allergy 2017 Jul;72(7):1061-1072). However, little is known about the expression and role of Syk in the vascular compartment of the lung. Here, we analyzed Syk expression and function in the pulmonary vasculature and its possible involvement in the pathogenesis of pulmonary arterial hypertension (PAH). Methods: In human (PAH vs. donor) and murine lungs, Syk expression was assessed by immunofluorescence and spectral confocal microscopy. Syk function was analyzed in human precision-cut lung slices (PCLS) and in isolated perfused lungs of wild-type mice or mice deficient in eNOS, PKCα or mast cells with or without inhibition of Syk, PKC, rho kinase, p38 MAPK and/or NO synthase. Pulmonary vascular hyperresponsiveness was investigated following induction of pulmonary Th2 inflammation. Results: Syk was expressed in pulmonary arterial smooth muscle cells of both control and PAH lungs. Syk inhibition (either with BAY 61-3606 or with BI 1002494) reduced pulmonary vasoconstriction in human PCLS and in isolated mouse lungs independent of eNOS, PKCα or mast cells. In preconstricted lungs, Syk inhibition rapidly reversed vasoconstriction in a NO-independent manner. Pulmonary vascular hyperresponsiveness was markedly diminished following Syk inhibition. Inhibition of p38 MAPK reduced pulmonary vasoconstriction to the same extent as Syk inhibition, and simultaneous inhibition of p38 MAPK and Syk had no additive inhibitory effect when compared to Syk inhibition only. Conclusions: Syk regulates pulmonary vasoconstriction, presumably via p38 MAPK, and may be a promising target in PAH therapy.

Nintedanib (BIBF1120) is a potent, oral, small-molecule tyrosine kinase inhibitor, also known as a triple angiokinase inhibitor, inhibiting three major signaling pathways involved in angiogenesis. Nintedanib targets proangiogenic and pro-fibrotic pathways mediated by the VEGFR family, the fibroblast growth factor receptor (FGFR) family, the platelet-derived growth factor receptor (PDGFR) family, as well as Src and Flt-3 kinases. The compound was identified during a lead optimization program for small-molecule inhibitors of angiogenesis and has since undergone extensive clinical investigation for the treatment of various solid tumors, and in patients with the debilitating lung disease idiopathic pulmonary fibrosis (IPF). Recent clinical evidence from phase III studies has shown that nintedanib has significant efficacy in the treatment of NSCLC, ovarian cancer, and IPF. This review article provides a comprehensive summary of the preclinical and clinical research and development of nintedanib from the initial drug discovery process to the latest available clinical trial data.

Background Spleen tyrosine kinase (Syk) is an intracellular non-receptor tyrosine kinase, which has been implicated as central immune modulator promoting allergic airway inflammation. Syk inhibition has been proposed as a new therapeutic approach in asthma. However, the direct effects of Syk inhibition on airway constriction independent of allergen sensitization remain elusive. Methods Spectral confocal microscopy of human and murine lung tissue was performed to localize Syk expression. The effects of prophylactic or therapeutic Syk inhibition on allergic airway inflammation, hyperresponsiveness and airway remodeling were analyzed in allergen-sensitized and airway-challenged mice. The effects of Syk inhibitors BAY 61-3606 or BI 1002494 on airway function were investigated in isolated lungs of wild-type, PKCα-deficient, mast cell-deficient or eNOS-deficient mice. Results Syk expression was found in human and murine airway smooth muscle cells. Syk inhibition reduced allergic airway inflammation, airway hyperresponsiveness and pulmonary collagen deposition. In naive mice, Syk inhibition diminished airway responsiveness independently of mast cells, or PKCα or eNOS expression and rapidly reversed established bronchoconstriction independently of NO. Simultaneous inhibition of Syk and PKC revealed additive dilatory effects, whereas combined inhibition of Syk and rho kinase or Syk and p38 MAPK did not cause additive bronchodilation. Conclusions Syk inhibition directly attenuates airway smooth muscle cell contraction independent of its protective immunomodulatory effects on allergic airway inflammation, hyperresponsiveness and airway remodeling. Syk mediates bronchoconstriction in a NO-independent manner, presumably via rho kinase and p38 MAPK, and Syk inhibition might present a promising therapeutic approach in chronic asthma as well as acute asthma attacks. This article is protected by copyright. All rights reserved.

Background: The long-acting β 2 -agonist (LABA) olodaterol was shown to have anti-fibrotic activity in primary human lung fibroblasts (Herrmann et al, Eur Respir J. 2014;44:Suppl.58) however, its anti-fibrotic in vivo efficacy has not yet been demonstrated. Aim: To test the effect of olodaterol in a mouse model of bleomycin induced lung fibrosis. Methods: Bleomycin at a dose of 0.5 mg/kg (1.5-2 U/mg) was intratracheally instilled in C57BL/6 mice on day 0. In a preventive (day 1-20) or therapeutic (day 7 -20) treatment regimen mice were exposed once daily for 5 min to an aqueous solution of olodaterol (1 mg/mL) aerosolized with a jet nebulizer. At the end of the study (day 21) lung density was assessed by micro CT analysis and lung function was measured with a FlexiVent system. Bronchoalveolar lavage fluid (BALF) was analyzed for total and differential cell counts and pro-fibrotic cytokines. Results: The bleomycin challenge induced an increase in lung tissue density, white blood cell (WBC) count and protein concentration in the BAL fluid and a decrease in forced vital capacity (FVC). In the preventive treatment regimen olodaterol attenuated tissue density by 45%, WBC count by 31%, protein by 63% and FVC by 57%. Pro-fibrotic cytokines like TGF-s or MMP-9 were diminished by 57% and 48%, respectively. Therapeutic treatment with olodaterol attenuated the increase in lung density and the decrease in FVC by 10% and 18%, respectively. WBC accumulation was attenuated by 25% and the increase in total protein, TGF-s and MMP-9 levels in BALF was inhibited by 43%, 58% and 49%, respectively. Conclusion: At doses of maximal bronchodilation, olodaterol showed anti-fibrotic properties in a mouse model of bleomycin induced lung fibrosis.

BI 1002494 [(R)-4-{(R)-1-[7-(3,4,5-trimethoxy-phenyl)-[1,6]napthyridin-5-yloxy]-ethyl}pyrrolidin-2-one] is a novel, potent, and selective spleen tyrosine kinase (SYK) inhibitor with sustained plasma exposure after oral administration in rats, which qualifies this molecule as a good in vitro and in vivo tool compound. BI 1002494 exhibits higher potency in inhibiting high-affinity IgE receptor-mediated mast cell and basophil degranulation (IC50 = 115 nM) compared with B-cell receptor-mediated activation of B cells (IC50 = 810 nM). This may be explained by lower kinase potency when the physiologic ligand B-cell linker was used, suggesting that SYK inhibitors may exhibit differential potency depending on the cell type and the respective signal transduction ligand. A 3-fold decrease in potency was observed in rat basophils (IC50 = 323 nM) compared with human basophils, but a similar species potency shift was not observed in B cells. The lower potency in rat basophils was confirmed in both ex vivo inhibition of bronchoconstriction in precision-cut rat lung slices and in reversal of anaphylaxis-driven airway resistance in rats. The different cellular potencies translated into different in vivo efficacy; full efficacy in a rat ovalbumin model (that contains an element of mast cell dependence) was achieved with a trough plasma concentration of 340 nM, whereas full efficacy in a rat collagen-induced arthritis model (that contains an element of B-cell dependence) was achieved with a trough plasma concentration of 1400 nM. Taken together, these data provide a platform from which different estimates of human efficacious exposures can be made according to the relevant cell type for the indication intended to be treated.

Background: Nintedanib a tyrosine kinase inhibitor for PDGFRα and β, FGFR1-3, VEGFR1-3 and Src-family kinaseshas been shown to slow disease progression in idiopathic pulmonary fibrosis (IPF) and has recently been authorized for the treatment of IPF in the US and EU. Objectives: To analyze the anti-fibrotic activity of nintedanib in models of pulmonary and dermal fibrosis associated with SSc. Methods: The effects of nintedanib on migration, proliferation, myofibroblast differentiation and release of extracellular matrix of primary human SSc fibroblasts were analyzed by MTT- and scratch assays, stress fiber staining, qPCR and SirCol assays. The anti-fibrotic effects of nintedanib were evaluated in bleomycin-induced skin fibrosis (Bleo-SF), in murine sclerodermatous chronic graft-versus host disease (GvhD), in tight-skin-1 mice (Tsk-1) and in fos-related antigen-2 transgenic (Fra2) mice. Results: Nintedanib delayed migration, inhibited proliferation, reduced the mRNA levels of Col 1a1, Col 1a2 and fibronectin-1, collagen protein and the basal levels of αSMA and stress fibres in SSc fibroblasts. Preventive as well as therapeutic treatment with nintedanib ameliorated dermal thickening, myofibroblast differentiation and collagen deposition in inflammation-driven models of SSc such as Bleo-SF and GvhD. In models of later, non-inflammatory stages of SSc such as Tsk-1 and Fra2 nintedanib effectively reduced pulmonary as well as dermal fibrosis. Conclusions: Nintedanib effectively inhibits activation of primary human fibroblasts and exerts potent anti-fibrotic effects in mouse models of SSc providing a scientific rationale for clinical trials in SSc.