A phosphodiesterase 4 inhibitor, roflumilast N-oxide, inhibits human lung fibroblast functions in vitro (original) (raw)
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2016
The phosphodiesterase 4 inhibitor roflumilast prevents bleomycin-(BLM-) induced lung fibrosis in animal models. However, its mechanism of action remains unknown. We investigated whether roflumilast N-oxide (RNO), the active metabolite of roflumilast, can modulate in vitro the oxidative effects of BLM on human lung fibroblasts (HLF). In addition, since BLM increases the production of F 2-isoprostanes that have per se fibrogenic activity, the effect of RNO on oxidative stress and fibrogenesis induced by the F 2isoprostane 8-epi-PGF 2 was investigated. HLF were preincubated either with the vehicle or with RNO and exposed to either BLM or 8-epi-PGF 2. Proliferation and collagen synthesis were assessed as [ 3 H]-thymidine and [ 3 H]-proline incorporation. Reactive oxygen species (ROS) and F 2-isoprostanes were measured. NADPH oxidase 4 (NOX4) protein and mRNA were also evaluated. BLM increased both cell proliferation and collagen synthesis and enhanced ROS and F 2-isoprostane production. These effects were significantly prevented by RNO. Also, RNO significantly reduced the increase in both NOX4 mRNA and protein, induced by BLM. Finally, 8-epi-PGF 2 per se stimulated HLF proliferation, collagen synthesis, and NOX4 expression and ROS generation, and RNO prevented these effects. Thus, the antifibrotic effect of RNO observed in vivo may be related to its ability to mitigate ROS generation via downregulation of NOX4.
Mediators of Inflammation, 2013
The phosphodiesterase 4 inhibitor roflumilast prevents bleomycin-(BLM-) induced lung fibrosis in animal models. However, its mechanism of action remains unknown. We investigated whether roflumilast N-oxide (RNO), the active metabolite of roflumilast, can modulate in vitro the oxidative effects of BLM on human lung fibroblasts (HLF). In addition, since BLM increases the production of F 2-isoprostanes that have per se fibrogenic activity, the effect of RNO on oxidative stress and fibrogenesis induced by the F 2isoprostane 8-epi-PGF 2 was investigated. HLF were preincubated either with the vehicle or with RNO and exposed to either BLM or 8-epi-PGF 2. Proliferation and collagen synthesis were assessed as [ 3 H]-thymidine and [ 3 H]-proline incorporation. Reactive oxygen species (ROS) and F 2-isoprostanes were measured. NADPH oxidase 4 (NOX4) protein and mRNA were also evaluated. BLM increased both cell proliferation and collagen synthesis and enhanced ROS and F 2-isoprostane production. These effects were significantly prevented by RNO. Also, RNO significantly reduced the increase in both NOX4 mRNA and protein, induced by BLM. Finally, 8-epi-PGF 2 per se stimulated HLF proliferation, collagen synthesis, and NOX4 expression and ROS generation, and RNO prevented these effects. Thus, the antifibrotic effect of RNO observed in vivo may be related to its ability to mitigate ROS generation via downregulation of NOX4.
PLoS ONE, 2013
Roflumilast is the first phosphodiesterase-4 (PDE4) inhibitor to have been approved for the treatment of COPD. The anti-inflammatory profile of PDE4 inhibitors has not yet been explored in human lung tissues. We investigated the effects of roflumilast and its active metabolite roflumilast-N-oxide on the lipopolysaccharide (LPS)induced release of tumor necrosis factor-alpha (TNF-α) and chemokines by human lung parenchymal explants. We also investigated roflumilast's interaction with the long-acting β 2 -agonist formoterol.
Roflumilast, a phosphodiesterase 4 inhibitor, alleviates bleomycin-induced lung injury
British Journal of Pharmacology, 2009
Background and purpose: The effects of a phosphodiesterase 4 (PDE4) inhibitor, roflumilast, on bleomycin-induced lung injury were explored in 'preventive' and 'therapeutic' protocols and compared with glucocorticoids. Experimental approach: Roflumilast (1 and 5 mg•kg-1 •d-1 , p.o.) or dexamethasone (2.5 mg•kg-1 •d-1 , p.o.) was given to C57Bl/6J mice from day 1 to 14 (preventive) or day 7 to 21 (therapeutic) after intratracheal bleomycin (3.75 U•kg-1). In Wistar rats, roflumilast (1 mg•kg-1 •d-1 , p.o.) was compared with methylprednisolone (10 mg•kg-1 •d-1 , p.o.) from day 1 to 21 (preventive) or from day 10 to 21 (therapeutic), following intratracheal instillation of bleomycin (7.5 U•kg-1). Analyses were performed at the end of the treatment periods. Key results: Preventive. Roflumilast reduced bleomycin-induced lung hydroxyproline, lung fibrosis and right ventricular hypertrophy; muscularization of intraacinar pulmonary vessels was also attenuated. The PDE4 inhibitor diminished bleomycininduced transcripts for tumour necrosis factor (TNFa), transforming growth factor (TGFb), connective tissue growth factor, aI(I)collagen, endothelin-1 and the mucin, Muc5ac, in lung, and reduced bronchoalveolar lavage fluid levels of TNFa, interleukin-13, TGFb, Muc5ac, lipid hydroperoxides and inflammatory cell counts. Therapeutic. In mice, roflumilast but not dexamethasone reduced bleomycin-induced lung aI(I)collagen transcripts, fibrosis and right ventricular hypertrophy. Similar results were found in the rat. Conclusions and implications: Roflumilast prevented the development of bleomycin-induced lung injury, and alleviated the lung fibrotic and vascular remodeling response to bleomycin in a therapeutic protocol, the latter being resistant to glucocorticoids.
Roflumilast Prevents the Metabolic Effects of Bleomycin-Induced Fibrosis in a Murine Model
PLOS ONE, 2015
Fibrotic remodeling is a process common to chronic lung diseases such as chronic obstructive pulmonary disease (COPD), pulmonary fibrosis, acute respiratory distress syndrome and asthma. Based on preclinical studies phosphodiesterase 4 (PDE4) inhibitors may exhibit beneficial anti-inflammatory and anti-remodeling properties for the treatment of these respiratory disorders. Effects of PDE4 inhibitors on changes in the lung metabolome in models of pulmonary fibrotic remodeling have not yet been explored. This work studies the effects of the PDE4 inhibitor roflumilast on changes in the lung metabolome in the common murine model of bleomycin-induced lung fibrosis by nuclear magnetic resonance (NMR) metabolic profiling of intact lung tissue. Metabolic profiling reveals strong differences between fibrotic and non-fibrotic tissue. These differences include increases in proline, glycine, lactate, taurine, phosphocholine and total glutathione and decreases in global fatty acids. In parallel, there was a loss in plasma BH4. This profile suggests that bleomycin produces alterations in the oxidative equilibrium, a strong inflammatory response and activation of the collagen synthesis among others. Roflumilast prevented most of these metabolic effects associated to pulmonary fibrosis suggesting a favorable anti-fibrotic profile.
Pulmonary Pharmacology & Therapeutics, 2014
Background: Epithelial to mesenchymal transition (EMT) is under discussion as a potential mechanism of small airway remodelling in COPD. In bronchial epithelium of COPD and smokers markers of EMT were described. In vitro, EMT may be reproduced by exposing well-differentiated human bronchial epithelial cells (WD-HBEC) to cigarette smoke extract (CSE). EMT may be mitigated by an increase in cellular cAMP. Objective: This study explored the effects of roflumilast N-oxide, a PDE4 inhibitor on CSE-induced EMT in WD-HBEC and in primary bronchial epithelial cells from smokers and COPD in vitro. Methods: WD-HBEC from normal donors were stimulated with CSE (2.5%) for 72 h in presence of roflumilast N-oxide (2 nM or 1 mM) or vehicle. mRNA and protein of EMT markers aSMA, vimentin, collagen-1, E-cadherin, ZO-1, KRT5 as well as NOX4 were quantified by real-time quantitative PCR or protein array, respectively. Phosphorylated and total ERK1/2 and Smad3 were assessed by protein array. cAMP and TGFb1 were measured by ELISA. Reactive oxygen species (ROS) were determined by DCF fluorescence, after 30 min CSE (2.5%). Apoptosis was measured with Annexin V/PI labelling. In some experiments, EMT markers were determined in monolayers of bronchial epithelial cells from smokers, COPD versus controls. Results: Roflumilast N-oxide protected from CSE-induced EMT in WD-HBEC. The PDE4 inhibitor reversed both the increase in mesenchymal and the loss in epithelial EMT markers. Roflumilast N-oxide restored the loss in cellular cAMP following CSE, reduced ROS, NOX4 expression, the increase in TGFb1 release, phospho ERK1/2 and Smad3. The PDE4 inhibitor partly protected from the increment in apoptosis with CSE. Finally the PDE4 inhibitor decreased mesenchymal yet increased epithelial phenotype markers in HBEC of COPD and smokers. Conclusions: Roflumilast N-oxide may mitigate epithelialemesenchymal transition in bronchial epithelial cells in vitro.
Roflumilast-N-oxide Induces Surfactant Protein Expression in Human Alveolar Epithelial Cells Type II
PLoS ONE, 2012
Surfactant proteins (SPs) are important lipoprotein complex components, expressed in alveolar epithelial cells type II (AEC-II), and playing an essential role in maintenance of alveolar integrity and host defence. Because expressions of SPs are regulated by cyclic adenosine monophosphate (cAMP), we hypothesized that phosphodiesterase (PDE) inhibitors, influence SP expression and release. Analysis of PDE activity of our AEC-II preparations revealed that PDE4 is the major cAMP hydrolysing PDE in human adult AEC-II. Thus, freshly isolated human AEC-II were stimulated with two different concentrations of the PDE4 inhibitor roflumilast-N-oxide (3 nM and 1 mM) to investigate the effect on SP expression. SP mRNA levels disclosed a large inter-individual variation. Therefore, the experiments were grouped by the basal SP expression in low and high expressing donors. AEC-II stimulated with Roflumilast-N-oxide showed a minor increase in SP-A1, SP-C and SP-D mRNA mainly in low expressing preparations. To overcome the effects of different basal levels of intracellular cAMP, cyclooxygenase was blocked by indomethacin and cAMP production was reconstituted by prostaglandin E2 (PGE2). Under these conditions SP-A1, SP-A2, SP-B and SP-D are increased by roflumilast-N-oxide in low expressing preparations. Roflumilast-N-oxide fosters the expression of SPs in human AEC-II via increase of intracellular cAMP levels potentially contributing to improved alveolar host defence and enhanced resolution of inflammation.
Effect of roflumilast on inflammatory cells in the lungs of cigarette smoke-exposed mice
BMC Pulmonary Medicine, 2008
We reported that roflumilast, a phosphodiesterase 4 inhibitor, given orally at 5 mg/ kg to mice prevented the development of emphysema in a chronic model of cigarette smoke exposure, while at 1 mg/kg was ineffective. Here we investigated the effects of roflumilast on the volume density (V V ) of the inflammatory cells present in the lungs after chronic cigarette smoke exposure.
Pulmonary Pharmacology & Therapeutics, 2010
After more than two decades of research into phosphodiesterase 4 (PDE4) inhibitors, roflumilast (3-cyclopropylmethoxy-4-difluoromethoxy-N-[3,5-di-chloropyrid-4-yl]-benzamide) may become the first agent in this class to be approved for patient treatment worldwide. Within the PDE family of 11 known isoenzymes, roflumilast is selective for PDE4, showing balanced selectivity for subtypes A-D, and is of high subnanomolar potency. The active principle of roflumilast in man is its dichloropyridyl N-oxide metabolite, which has similar potency as a PDE4 inhibitor as the parent compound. The long half-life and high potency of this metabolite allows for once-daily, oral administration of a single, 500-microg tablet of roflumilast. The molecular mode of action of roflumilast--PDE4 inhibition and subsequent enhancement of cAMP levels--is well established. To further understand its functional mode of action in chronic obstructive pulmonary disease (COPD), for which roflumilast is being developed, a series of in vitro and in vivo preclinical studies has been performed. COPD is a progressive, devastating condition of the lung associated with an abnormal inflammatory response to noxious particles and gases, particularly tobacco smoke. In addition, according to the Global Initiative for Chronic Obstructive Lung Disease (GOLD), significant extrapulmonary effects, including comorbidities, may add to the severity of the disease in individual patients, and which may be addressed preferentially by orally administered remedies. COPD shows an increasing prevalence and mortality, and its treatment remains a high, unmet medical need. In vivo, roflumilast mitigates key COPD-related disease mechanisms such as tobacco smoke-induced lung inflammation, mucociliary malfunction, lung fibrotic and emphysematous remodelling, oxidative stress, pulmonary vascular remodelling and pulmonary hypertension. In vitro, roflumilast N-oxide has been demonstrated to affect the functions of many cell types, including neutrophils, monocytes/macrophages, CD4+ and CD8+ T-cells, endothelial cells, epithelial cells, smooth muscle cells and fibroblasts. These cellular effects are thought to be responsible for the beneficial effects of roflumilast on the disease mechanisms of COPD, which translate into reduced exacerbations and improved lung function. As a multicomponent disease, COPD requires a broad therapeutic approach that might be achieved by PDE4 inhibition. However, as a PDE4 inhibitor, roflumilast is not a direct bronchodilator. In summary, roflumilast may be the first-in-class PDE4 inhibitor for COPD therapy. In addition to being a non-steroid, anti-inflammatory drug designed to target pulmonary inflammation, the preclinical pharmacology described in this review points to a broad functional mode of action of roflumilast that putatively addresses additional COPD mechanisms. This enables roflumilast to offer effective, oral maintenance treatment for COPD, with an acceptable tolerability profile and the potential to favourably affect the extrapulmonary effects of the disease.
Pulmonary pharmacology & therapeutics, 2008
Roflumilast, an investigational, targeted phosphodiesterase 4 inhibitor, reduces the in vitro and in vivo inflammatory activity of cells such as neutrophils, eosinophils, macrophages, and monocytes. The aim of this study was to explore the anti-inflammatory properties of roflumilast in a human model of segmental bronchial endotoxin challenge. In a randomized, placebo-controlled, double-blind, single-center parallel-group study, 37 healthy subjects of either sex were treated for 28 days with either oral roflumilast 500 microg once daily or placebo. At day 29, a baseline bronchoalveolar lavage was performed, followed by segmental endotoxin challenge (4 ng/kg) and saline control challenge. After 24h, bronchoalveolar lavage fluid was sampled from the challenged segments and cells were counted and differentiated. After endotoxin challenge, influx of total cells (difference from baseline) in bronchoalveolar lavage of roflumilast-treated subjects was 36% lower than with placebo (p=0.02). C...