Kinase experiment:

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℃, the reaction is started by the addition of substrate (cAMP or cGMP) and the assays are incubated for further 15 min at 37℃. 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℃, 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].

Animal experiment:

Mice[4]WT or pIgR?/? mice are used. For studies using Roflumilast, 200 μL of 0.5 mg/mL suspension of Roflumilast or vehicle (4% methylcellulose, 1.3% PEG400 and 5 μg drug per mg animal weight) is administered by oral gavage once daily, 5 days a week for the duration of treatment. Mice are treated daily by oral gavage with 100 μg of Roflumilast (5 μg/g) or vehicle (4% methylcellulose, 1.3% PEG400) for 3 months and lungs are harvested at 12 months of age.

Roflumilast is an orally active, selective inhibitor of phophodiesterase-4 (PDE-4) with IC50 value of 0.11nM [1].

Roflumilast is developed for the treatment of the inflammatory component of COPD since PDE-4 is found mainly in inflammatory cells. In vitro studies show that roflumilast inhibits the production of inflammatory mediators in a variety of human immune cells, suggesting a role for reducing COPD-related inflammation. Roflumilast has a rapid absorption with a bioavailability of ~ 80% and the half-life is approximately 17h after repeated dosing of 500 mcg OD. Pharmacokinetic studies show no significant interactions with the drugs used by COPD patients. Roflumilast is also found to decrease glucose levels in patients with newly diagnosed type-2 diabetes since diabetes is an important comorbidity associated with COPD [2].

References:
[1] Z Huang, JA Mancini. Phosphodiesterase 4 Inhibitors for the Treatment of Asthma and COPD. Current Medicinal Chemistry, 2006.
[2] Tashkin DP. Roflumilast : the new orally active, selective phophodiesterase-4 inhibitor, for the treatment of COPD. Expert Opin Pharmacother. 2014 Jan;15(1):85-96.