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Phosphodiesterase (PDE) inhibitors have potential as alternatives or adjuncts to glucocorticoid therapy in asthma. We compared roflumilast (a selective PDE4 inhibitor) with pentoxifylline (a nonselective inhibitor) and dexamethasone in ameliorating the lesions of chronic asthma in a mouse model. BALB/c mice sensitized to ovalbumin were chronically challenged with aerosolized antigen for 6 weeks. During weeks 5 and 6, groups of animals were treated with roflumilast or dexamethasone by daily gavage or with pentoxifylline by daily intraperitoneal injection. Airway hyper-reactivity (AHR) was evaluated by wholebody plethysmography and airway lesions by histomorphometry and immunohistochemistry. Compared with vehicle alone, treatment with roflumilast or dexamethasone significantly reduced accumulation of eosinophils and chronic inflammatory cells, subepithelial collagenization, and thickening of the airway epithelium. Dexamethasone also reduced goblet cell hyperplasia/metaplasia, subepithelial accumulation of transforming growth factor-␤1, and epithelial cytoplasmic immunoreactivity for nuclear factor-B. Treatment with pentoxifylline inhibited only eosinophil recruitment and epithelial thickening. Roflumilast and dexamethasone slightly decreased AHR, whereas this was significantly reduced by pentoxifylline. Thus, in this model of chronic asthma, both roflumilast and dexamethasone were potent inhibitors of airway inflammation and remodeling. Roflumilast did not diminish accumulation of transforming growth factor-␤1, suggesting that it might affect remodeling by mechanisms distinct from glucocorticoids.

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BACKGROUND AND PURPOSEMucociliary malfunction occurs in chronic obstructive pulmonary disease (COPD) and compromised functions of ciliated bronchial epithelial cells may contribute to this. Cigarette smoke, a major risk factor for COPD, impairs ciliary beat frequency (CBF). cAMP augments CBF. This in vitro study addressed, in differentiated, primary human bronchial epithelial cells, whether roflumilast N-oxide, a PDE4 inhibitor, (i) augments CBF; (ii) prevents the reduction in CBF induced by cigarette smoke extract (CSE); and (iii) protects against the loss of the ciliated phenotype following long-term CSE exposure. EXPERIMENTAL APPROACHAir-liquid interface cultured human bronchial epithelial cells were incubated with roflumilast N-oxide and exposed to CSE. CBF was assessed by digital high speed video microscopy (DHSV). Ciliated cells were characterized by b-tubulin IV staining and analyses of Foxj1 and Dnai2 mRNA and protein (real-time quantitative PCR, Western blotting). KEY RESULTSRoflumilast N-oxide concentration-dependently triggered a rapid and persistent increase in CBF and reversed the decrease in CBF following CSE. Long-term incubation of bronchial epithelial cells with CSE resulted in a loss in ciliated cells associated with reduced expression of the ciliated cell markers Foxj1 and Dnai2. The PDE4 inhibitor prevented this loss in the ciliated cell phenotype and the compromised Foxj1 and Dnai2 expression. The enhanced release of IL-13 following CSE, a cytokine that diminishes the proportion of ciliated cells and in parallel, reduces Foxj1 and Dnai2, was reversed by roflumilast N-oxide. CONCLUSION AND IMPLICATIONSRoflumilast N-oxide protected differentiated human bronchial epithelial cells from reduced CBF and loss of ciliated cells following CSE. AbbreviationsCBF, ciliary beat frequency; COPD, chronic obstructive pulmonary disease; CSE, cigarette smoke extract; DHSV, digital high speed video imaging technique; Dnai2, axonemal dynein intermediate polypeptide 2; DUOX, dual oxidase; Foxj1, forkhead transcription factor 1; NAC, N-acetyl cysteine; NOX, NADPH oxidase; ROS, reactive oxygen species BJP British Journal of Pharmacology

Aims/hypothesis The cAMP-degrading phosphodiesterase 4 (PDE4) enzyme has recently been implicated in the regulation of glucagon-like peptide-1 (GLP-1), an incretin hormone with glucose-lowering properties. We investigated whether the PDE4 inhibitor roflumilast elevates GLP-1 levels in diabetic db/db mice and whether this elevation is accompanied by glucose-lowering effects. Methods Plasma GLP-1 was determined in db/db mice after single oral administration of roflumilast or its active metabolite roflumilast-N-oxide. Diabetes-relevant variables including HbA 1c , blood glucose, serum insulin, body weight, food and water intake, and pancreas morphology were determined in db/db mice treated daily for 28 days with roflumilast or roflumilast-N-oxide. Pharmacokinetic/pharmacodynamic analysis clarified the contribution of roflumilast vs its metabolite. In addition, the effect of roflumilast-N-oxide on insulin release was investigated in primary mouse islets.Results Single treatment of db/db mice with 10 mg/kg roflumilast or roflumilast-N-oxide enhanced plasma GLP-1 2.5-and fourfold, respectively. Chronic treatment of db/db mice with roflumilast or roflumilast-N-oxide at 3 mg/kg showed prevention of disease progression. Roflumilast-N-oxide abolished the increase in blood glucose, reduced the increment in HbA 1c by 50% and doubled fasted serum insulin compared with vehicle, concomitant with preservation of pancreatic islet morphology. Furthermore, roflumilast-N-oxide amplified forskolininduced insulin release in primary islets. Roflumilast-N-oxide showed stronger glucose-lowering effects than its parent compound, consistent with its greater effect on GLP-1 secretion and explainable by pharmacokinetic/pharmacodynamic modelling. Conclusions/interpretation Our results suggest that roflumilast and roflumilast-N-oxide delay the progression of diabetes in db/db mice through protection of pancreatic islet physiology potentially involving GLP-1 and insulin activities.