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

Abstract

Roflumilast is an orally active phosphodiesterase 4 (PDE4) inhibitor approved for use in chronic obstructive pulmonary disease. Roflumilast N-oxide (RNO) is the active metabolite of roflumilast and has demonstrated anti-inflammatory 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 β2-adrenoceptor-mediated, cAMPdependent responses in ASM cells in vitro, and whether RNO enhances steroid-induced repression of inflammation. RNO (0.001-1000 nM) alone had no effect on AMP production from ASM cells, and significant potentiation of the long-acting β2-agonist formoterol-induced cAMP could only be achieved at the highest concentration of RNO tested (1000 nM). At this concentration, RNO exerted a small, but not significantly different, potentiation of formoterolinduced expression of anti-inflammatory mitogen-activated protein kinase phosphatase 1 (MKP-1). Consequently, tumor necrosis factor (TNF)-induced IL-8 secretion was unaffected by RNO in combination with formoterol. However, as there was the potential for PDE4 inhibitors and LABAs to interact with corticosteroids to achieve superior anti-inflammatory 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. While RNO alone did not significantly enhance cytokine repression achieved with steroids; RNO in combination with formoterol significantly enhanced the anti-inflammatory effect of dexamethasone in ASM cells. This was linked to increased MKP-1 expression in ASM cells, suggesting a molecular mechanism responsible for augmented anti-inflammatory actions of combination therapeutic approaches that include RNO.

Selectively targeting prostanoid E (EP) receptor-mediated cell signalling pathways: Implications for lung health and disease

“…MKP-1upregulation is a common response to elevated cAMP induced by a diverse range of stimuli. In ASM cells we have shown that inflammatory stimuli [134], cAMP elevating agents [133], as well as respiratory medicines such as β2-agonists [133,135] and phosphodiesterase 4 inhibitors [136,137], can all increase MKP-1 via CREB-mediated pathways and perhaps offset the impact of cAMP on cytokine production. In 2016, we used selective antagonists of EP2 (PF-04418948) and EP4 receptors (GW 627368X) to show that PGE2…”

Prostaglandin E2, but not cAMP nor β2-agonists, induce tristetraprolin (TTP) in human airway smooth muscle cells

“…It has been known for some time that β2-agonists have beneficial effects beyond bronchodilation and that they exert antiinflammatory actions in a cAMP-dependent manner [5]. We have shown that this beneficial impact is due in part to the upregulation of MKP-1; that is, in ASM cells, cAMP-elevating agents (including β2-agonists) can also increase expression of anti-inflammatory molecules (MKP-1) to repress inflammation [6][7][8][9]. But what is the effect of cAMP-elevating agents on TTP in ASM cells?…”

“…Secondly, we investigated receptor-mediated pathways and induced production of cAMP as a secondary messenger by activation of Gs-coupled protein receptors (β2-adrenergic receptors and EP2/EP4 prostanoid receptors) with cognate ligands β2-agonists and prostaglandin E2 (PGE2), respectively. In all cases, MKP-1 was used as a positive control as it well-established as being upregulated by cAMP [6][7][8][9]15]. Finally, we investigated the underlying signaling pathways involved.…”

Roles of roflumilast, a selective phosphodiesterase 4 inhibitor, in airway diseases

“…In vitro study has shown that roflumilast N-oxide in combination with formoterol significantly enhanced the effect of dexamethasone in ASM (19). It potentiated formoterol-induced expression of mitogen-activated protein kinase phosphatase 1 (MKP-1).…”