Roflumilast (APTA 2217) is an orally available, long-acting inhibitor of phosphodiesterase (PDE) type 4 (PDE4), with anti-inflammatory and potential antineoplastic activities. Upon administration, roflumilast and its active metabolite roflumilast N-oxide selectively and competitively bind to and inhibit PDE4, which leads to an increase of both intracellular levels of cyclic-3', 5'-adenosine monophosphate (cAMP) and cAMP-mediated signaling.

PDE4B1:0.7 nM, PDE4B2:0.2 nM, PDE4A4:0.9 nM, PDE4C1:3 nM, PDE4A1:0.7 nM

In vitro experiments demonstrate that Roflumilast exhibits immunomodulatory and anti-inflammatory activities. It inhibits the activation of CD4+ T cells by anti-CD28 and anti-CD3 antibodies (IC30: 7 nM), and suppresses the synthesis of IL-2 (IC20: 1 nM), IL-4 (IC30: 7 nM), IL-5 (IC25: 13 nM), and IFN-γ (IC35: 8 nM). Additionally, Roflumilast inhibits LTB4 synthesis in human neutrophils (IC50: 2 nM) and blocks ROS generation stimulated by fMLP in eosinophils and neutrophils, with an IC35 of 7 nM and 4 nM, respectively. It also impedes the production of TNF-α in monocytes (IC40: 21 nM) induced by lipopolysaccharide, as well as in monocyte-derived dendritic cells (IC20: 5 nM).

In vivo experiments demonstrated that Roflumilast reduces oxidative stress. It also confers a degree of resistance to pneumonia associated with COPD in animal models. In mouse lungs exposed to tobacco for seven months, Roflumilast (5 mg/kg/day) led to significant reductions in macrophages, neutrophils, B cells, dendritic cells, CD8+ T cells, and CD4+ T cells by 82%, 78%, 100%, 48%, 88%, and 98%, respectively. Following a 14-day intratracheal administration of bleomycin, Roflumilast (5 mg/kg/day) moderately decreased the augmentation of hydroperoxides in the mice's bronchoalveolar lavage fluid. Post-bleomycin treatment, dosages of Roflumilast proportionately reduced total pulmonary hydroxyproline content, with 5 mg/kg/day inhibiting by 47%, and additionally diminished pulmonary fibrotic damage and transcription of αI(I) collagen.

PDE activity is determined with some modifications. The assay mixture contain 50 mM Tris (pH 7.4), 5 mM MgCl2, 0.5 μM cAMP or cGMP, and [3H]cAMP or [3H]cGMP (about 30,000 cpm/assay), the indicated concentration of the inhibitor and an aliquot of the enzyme solution at a final assay volume of 200 μL. Stock solutions of the compounds are diluted 1:100 (v/v) in the Tris buffer mentioned above; appropriate dilutions are prepared in 1% (v/v) DMSO/Tris buffer, which are diluted 1:2 (v/v) in the assays to obtain the desired final concentrations of the inhibitors at a DMSO concentration of 0.5% (v/v). DMSO itself affected none of the PDE activities. After preincubation for 5 min at 37°C, the reaction is started by the addition of substrate (cAMP or cGMP) and the assays are incubated for further 15 min at 37°C. Then 50 μL of 0.2 N HCl is added to stop the reaction and the assays are left on ice for about 10 min. Following incubation with 25 μg of 5′-nucleotidase (Crotalus atrox snake venom) for 10 min at 37°C, the assays are loaded on QAE Sephadex A-25 (1 mL of bed volume in Poly-Prep chromatography columns). The columns are eluted with 2 mL of 30 mM ammonium formate (pH 6.0) and the eluate is counted for radioactivity. Results are corrected for blank values (measured in the presence of denatured protein) that are below 5% of total radioactivity. The amount of cyclic nucleotides hydrolyzed did not exceed 30% of the original substrate concentration[3].