In vitro activity: S-(+)-Rolipram suppresses LPS-induced TNFα expression from human monocyte through inhibiting PDE4 with IC50 about 2 μM. 1 μM S-(+)-Rolipram significantly antagonizes ovalbumin (OA) induced concentration-related contractions of tracheal rings which are isolated from OA-sensitized guinea pigs. S-(+)-Rolipram inhibits PDE4 activity in a CHO-K1 cell line which stably expresses a recombinant full length human PDE-4a with IC50 at 450 nM. Treatment of the human glioma cell line A-172 with Rolipram (including both R- and S-enantiomers of Rolipram) results in increased expression of the cell cycle inhibitors p21 and p27, and decreased activity of cdk2, a cyclindependent kinase essential for cell cycle progression. As a result, the proliferation of A-172 cells is inhibited, with induction of a G1 block. Eventually, Rolipram-treated A-172 cells undergo differentiation, which is followed by apoptotic cell death.

Kinase Assay: The PDE4 selective inhibitor, Rolipram, inhibits immunopurified PDE4B and PDE4D activities similarly, with IC50s of approx. 130 nM and 240 nM respectively. In contrast, Rolipram inhibits immunopurified PDE4A activity with a dramatically lower IC50 of around 3 nM. Rolipram increases phosphorylation of cAMP-response-element-binding protein (CREB) in U937 cells in a dose-dependent fashion, which implies the presence of both high affinity (IC50 approx. 1 nM) and low affinity (IC50 approx. 120 nM) components. Rolipram dose-dependently inhibits the IFN-gamma-stimulated phosphorylation of p38 MAPK in a simple monotonic fashion with an IC50 of approx. 290 nM.

Cell Assay: J774 murine macrophages (ATCC) are cultured at 37°C in 5% CO2 atmosphere in DMEM supplemented with glutamax-1 containing 10% heat-inactivated FBS, 100 U/mL penicillin, 100 μg/mL streptomycin and 250 ng/mL amphotericin B. For experiments, cells are seeded on 24-well plates at a density of 2×105 cells per well. Cell monolayers are grown for 72 h before the experiments are started. Rolipram, IBMX and BIRB 796 are dissolved in DMSO, and 8-Br-cAMP in HBSS. LPS (10 ng/mL) or the compounds of interest at concentrations indicated or the solvent (DMSO, 0.1% v/v) are added to the cells in fresh culture medium containing 10% FBS and the supplements. Cells are further incubated for the time indicated. The effect of LPS and the tested chemicals on cell viability is evaluated by Cell Proliferation Kit II (XTT).