title: Roflumilast N-oxide reverses corticosteroid resistance in neutrophils from patients with chronic obstructive pulmonary disease.
abstract: BACKGROUND
  Glucocorticoid functions are markedly impaired in patients with chronic obstructive pulmonary disease (COPD). The phosphodiesterase 4 inhibitor roflumilast N-oxide (RNO) is the active metabolite of roflumilast approved as a treatment to reduce the risk of exacerbations in patients with severe COPD.
  
  
  OBJECTIVE
  We sought to characterize the differential effects of RNO versus corticosteroids and their potential additive/synergistic effect in neutrophils from patients with COPD, thus providing scientific rationale for the combination of roflumilast with corticosteroids in the clinic.
  
  
  METHODS
  Peripheral blood neutrophils were isolated from patients with COPD (n = 32), smokers (n = 7), and healthy nonsmokers (n = 25). Levels of IL-8, matrix metallopeptidase 9 (MMP-9), and biomarkers of glucocorticoid resistance were determined by using ELISA and RT-PCR. Neutrophils were incubated with dexamethasone (0.1 nmol/L to 1 μmol/L), RNO (0.1 nmol/L to 1 μmol/L), or the combination of 1 nmol/L RNO plus 10 nmol/L DEX and stimulated with LPS (1 μg/mL) or cigarette smoke extract 5%; levels of IL-8, MMP-9, and other biomarkers were measured at the end of the incubation period.
  
  
  RESULTS
  Peripheral neutrophils from patients with COPD showed a primed phenotype with an increased basal release of IL-8 and MMP-9 and expressed a corticosteroid resistance molecular profile characterized by an increase in phosphoinositide 3-kinase δ, macrophage migration inhibitory factor, and glucocorticoid receptor β expression and a decrease in HDAC activity and mitogen-activated protein kinase phosphatase 1 expression. RNO demonstrated robust anti-inflammatory effects on neutrophils from patients with COPD, reversing their resistance to corticosteroids. The combination of RNO and dexamethasone showed additive/synergistic effects, which were consistent with the reversal of corticosteroid-resistant molecular markers by RNO.
  
  
  CONCLUSIONS
  RNO reverses corticosteroid resistance and shows strong anti-inflammatory effects alone or in combination with corticosteroids on neutrophils from patients with COPD.
authors.affiliation: Clinical Research Unit (UIC), University General Hospital Consortium, Valencia, Spain; Department of Biotechnology, Universidad Politécnica de Valencia, Valencia, Spain; Research Foundation of General Hospital of Valencia, Valencia, Spain. Electronic address: xmilara@hotmail.com.
authors.forename: Javier
authors.initials: J
authors.lastname: Milara
authors.affiliation: Research Foundation of General Hospital of Valencia, Valencia, Spain; CIBERES, Health Institute Carlos III, Valencia, Spain.
authors.forename: Javier
authors.initials: J
authors.lastname: Lluch
authors.affiliation: Research Foundation of General Hospital of Valencia, Valencia, Spain; Department of Pharmacology, Faculty of Medicine, University of Valencia, Valencia, Spain.
authors.forename: Patricia
authors.initials: P
authors.lastname: Almudever
authors.affiliation: Forest Research Institute, Jersey City, NJ.
authors.forename: Jose
authors.initials: J
authors.lastname: Freire
authors.affiliation: Forest Research Institute, Jersey City, NJ.
authors.forename: Qian
authors.initials: Q
authors.lastname: Xiaozhong
authors.affiliation: Clinical Research Unit (UIC), University General Hospital Consortium, Valencia, Spain; Research Foundation of General Hospital of Valencia, Valencia, Spain; Department of Pharmacology, Faculty of Medicine, University of Valencia, Valencia, Spain; CIBERES, Health Institute Carlos III, Valencia, Spain.
authors.forename: Julio
authors.initials: J
authors.lastname: Cortijo
mesh_terms: D000305:Adrenal Cortex Hormones / Q000494:pharmacology*; D000368:Aged; D000631:Aminopyridines / Q000494:pharmacology*; D001549:Benzamides / Q000494:pharmacology*; D015415:Biomarkers / Q000378:metabolism; D045424:Complex Mixtures / Q000302:isolation & purification / Q000494:pharmacology; D003521:Cyclopropanes / Q000494:pharmacology; D003907:Dexamethasone / Q000494:pharmacology*; D004351:Drug Resistance / Q000187:drug effects*; D004357:Drug Synergism; D005260:Female; D015870:Gene Expression / Q000187:drug effects; D006655:Histone Deacetylases / Q000378:metabolism; D006801:Humans; D016209:Interleukin-8 / Q000378:metabolism; D019746:Intramolecular Oxidoreductases / Q000378:metabolism; D008070:Lipopolysaccharides / Q000494:pharmacology; D008263:Macrophage Migration-Inhibitory Factors / Q000378:metabolism; D008297:Male; D020780:Matrix Metalloproteinase 9 / Q000378:metabolism; D008875:Middle Aged; D054639:Mitogen-Activated Protein Kinase Phosphatases / Q000378:metabolism; D009504:Neutrophils / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D058988:Phosphodiesterase 4 Inhibitors / Q000494:pharmacology*; D061251:Primary Cell Culture; D029424:Pulmonary Disease, Chronic Obstructive / Q000378:metabolism / Q000473:pathology; D014026:Nicotiana / Q000737:chemistry
chemical_list: D000305:Adrenal Cortex Hormones; D000631:Aminopyridines; D001549:Benzamides; D015415:Biomarkers; D045424:Complex Mixtures; D003521:Cyclopropanes; D016209:Interleukin-8; D008070:Lipopolysaccharides; D008263:Macrophage Migration-Inhibitory Factors; D058988:Phosphodiesterase 4 Inhibitors; D003907:Dexamethasone; D019869:Phosphatidylinositol 3-Kinases; D054639:Mitogen-Activated Protein Kinase Phosphatases; D020780:Matrix Metalloproteinase 9; D006655:Histone Deacetylases; D019746:Intramolecular Oxidoreductases; C515598:MIF protein, human; C517734:roflumilast N-oxide
keywords: Roflumilast; chronic obstructive pulmonary disease; corticosteroid resistance; dexamethasone; neutrophil