Clinical Relevance of the Anti-inflammatory Effects of Roflumilast on Human Bronchus: Potentiation by a Long-Acting Beta-2-Agonist

Abstract

Background: Roflumilast is an option for treating patients with severe COPD and frequent exacerbations despite optimal therapy with inhaled drugs. The present study focused on whether the phosphodiesterase (PDE) 4 inhibitor roflumilast and its active metabolite roflumilast N-oxide affect the release of tumor necrosis factor (TNF)-α and chemokines by lipopolysaccharide (LPS)-stimulated human bronchial explants. We also investigated the interactions between roflumilast, roflumilast N-oxide and the β₂-agonist formoterol with regard to cytokine release by the bronchial preparations. Methods: Bronchial explants from resected lungs were incubated with roflumilast, roflumilast N-oxide and/or formoterol and then stimulated with LPS. An ELISA was used to measure levels of TNF-α and chemokines in the culture supernatants. Results: At a clinically relevant concentration (1 nM), roflumilast N-oxide and roflumilast consistently reduced the release of TNF-α, CCL2, CCL3, CCL4, CCL5 and CXCL9 (but not CXCL1, CXCL5, CXCL8 and IL-6) from human bronchial explants. Formoterol alone decreased the release of TNF-α, CCL2, and CCL3. The combination of formoterol with roflumilast (1 nM) was more potent than roflumilast alone for inhibiting the LPS-induced release of TNF-α, CCL2, CCL3, CCL4, and CXCL9 by the bronchial explants. Conclusions: At a clinically relevant concentration, roflumilast N-oxide and its parent compound, roflumilast, reduced the LPS-induced production of TNF-α and chemokines involved in monocyte and T-cell recruitment but did not alter the release of chemokines involved in neutrophil recruitment. The combination of formoterol with roflumilast enhanced the individual drugs' anti-inflammatory effects.

Keywords: beta-2-adrenoceptor agonist; cytokine; human bronchus; lipopolysaccharide; roflumilast.

FIGURE 1

Effects of roflumilast (solid line) and roflumilast N-oxide (dashed line) on the release of TNF-α from LPS-stimulated human bronchial explants. The explants were incubated with roflumilast, roflumilast N-oxide (0.1–1,000 nM) or vehicle before addition of LPS (1 µg/mL) for 24 h. The data are quoted as the mean ± SEM of 8 different experiments and are expressed as the percentage of maximum release of TNF-α following LPS and vehicle (0.1% DMSO).

FIGURE 2

Effects of formoterol alone and combined with roflumilast on LPS-stimulated release of TNF-α, CCL2, CCL3, CCL4 and CXCL9 by human bronchial explants. The explants were incubated with roflumilast (1 and 100 nM), formoterol (10 nM) or vehicle before stimulation with LPS (1 µg/mL) for 24 h. The data are quoted as the mean ± SEM of 7 to 9 different experiments. *p < 0.05, **p < 0.01, ***p < 0.001 vs. LPS, #p < 0.05 and ##p < 0.01.