Roflumilast N-Oxide in Combination with Formoterol Enhances the Antiinflammatory Effect of Dexamethasone in Airway Smooth Muscle Cells.

Roflumilast is an orally active phosphodiesterase 4 inhibitor approved for use in chronic obstructive pulmonary disease. Roflumilast N-oxide (RNO) is the active metabolite of roflumilast and has a demonstrated antiinflammatory impact in vivo and in vitro. To date, the effect of RNO on the synthetic function of airway smooth muscle (ASM) cells is unknown. We address this herein and investigate the effect of RNO on ß2-adrenoceptor-mediated, cAMP-dependent responses in ASM cells in vitro, and whether RNO enhances steroid-induced repression of inflammation. RNO (0.001-1,000 nM) alone had no effect on AMP production from ASM cells, and significant potentiation of the long-acting ß2-agonist formoterol-induced cAMP could only be achieved at the highest concentration of RNO tested (1,000 nM). At this concentration, RNO exerted a small, but not significantly different, potentiation of formoterol-induced expression of antiinflammatory mitogen-activated protein kinase phosphatase 1. Consequently, tumor necrosis factor-induced IL-8 secretion was unaffected by RNO in combination with formoterol. However, because there was the potential for phosphodiesterase 4 inhibitors and long-acting ß2-agonists to interact with corticosteroids to achieve superior antiinflammatory efficacy, we examined whether RNO, alone or in combination with formoterol, enhanced the antiinflammatory effect of dexamethasone by measuring the impact on IL-8 secretion. Although RNO alone did not significantly enhance the cytokine repression achieved with steroids, RNO in combination with formoterol significantly enhanced the antiinflammatory effect of dexamethasone in ASM cells. This was linked to increased mitogen-activated protein kinase phosphatase 1 expression in ASM cells, suggesting that a molecular mechanism is responsible for augmented antiinflammatory actions of combination therapeutic approaches that include RNO.

Doxofylline does not increase formoterol-induced cAMP nor MKP-1 expression in ASM cells resulting in lack of anti-inflammatory effect.

The xanthine doxofylline has been examined in clinical trials and shown to have efficacy and greater tolerability than theophylline in asthma and chronic obstructive pulmonary disease. The 'novofylline' doxofylline has demonstrated bronchodilatory and anti-inflammatory actions in in vivo and ex vivo experimental models of respiratory disease. However, there are limited studies in vitro. We address this herein and examine whether doxofylline has anti-inflammatory impact on primary cultures of airway smooth muscle (ASM) cells. We conduct a series of investigations comparing and contrasting doxofylline with the archetypal xanthine, theophylline, and the specific phosphodiesterase (PDE) 4 inhibitor, cilomilast. We confirm that the xanthine drugs do not have action as PDE inhibitors in ASM cells. Unlike cilomilast, doxofylline (and theophylline) do not increase cAMP production in ASM cells induced by long-acting ß2-agonist formoterol. Similar to theophylline, and consistent with the lack of cAMP potentiation, doxofylline does not augment formoterol-induced upregulation of the anti-inflammatory protein mitogen-activated protein kinase phosphatase 1 (MKP-1). However, when we examine the effect of doxofylline on secretion of the interleukin 8 from ASM cells stimulated by tumour necrosis factor (an in vitro surrogate measure of inflammation), there was no repression of inflammation. This is in contrast to the anti-inflammatory impact exerted by theophylline and cilomilast in confirmatory experiments. In summary, our study is the first to examine the effect of doxofylline on ASM cells in vitro and highlights some distinct differences between two key members of xanthine drug family, doxofylline and theophylline.