In vitro activity: Apremilast inhibits TNF-α release by lipopolysaccharide (LPS) with an IC50 of 104 nM (pIC50=6.98±0.2), which almost exactly replicates previous reported TNF-α inhibition by Apremilast on peripheral blood mononuclear cells (PBMCs) (IC50=110 nM) and which is similar to the potency of Apremilast for PDE4 enzymatic inhibition (IC50=74 nM). These results are clearly consistent with the hypothesis that Apremilast inhibits TNF-α by increasing intracellular cAMP levels. PKA, Epac1 and Epac2 knockdowns prevented TNF-α inhibition and IL-10 stimulation by Apremilas.

Kinase Assay: Quantification of cytokines and chemokines was performed using Luminex x-MAP technology (Luminex Corp, Austen TX, USA). Tissue culture supernatants and mouse exudates were analyzed for expression of IL-1α, IL-6 and IL-10 using a Milliplex multi-analyte magnetic bead panel from EMD Millipore (MCYTOMAG-70K, Billerica, MA, USA). Assays were performed according to the kit protocol using the appropriate matrix solution (culture media or PBS for supernatants and exudates, respectively). Data were collected on a Luminex 200 instrument and analyzed using Analyst 5.1 software (Millipore) with four-parameter logistic curve fitting. Samples were assayed in duplicate. All standard curves generated from the known reference cytokine concentrations supplied by the manufacturer had R2 values calculated at or close to 1 and percent recovery between 80 and 120 %. Quality controls included with each kit performed as expected.

Cell Assay: Raw 264.7 cells (100,000) are grown in 96-well plates. After 24 h, cells are stimulated with vehicle (final concentration of 0.025% DMSO) or with Apremilast at the indicated concentrations. After 30 minutes cells are stimulated with LPS 1 μg/mL for 4 h. When studying CGS21680 , SCH58261, ZM241385, BAY60-6583, or GS6201, the adenosine receptor ligands are added 15 minutes before Apremilast. Methotrexate is added 24 h and 1 h before Apremilast. Supernates are then collected and TNF-α levels are quantified with the Mouse TNF-α Quantikine ELISA Kit. IC50 (EC50) calculations are made using non-linear regression, sigmoidal dose-response, constraining the top to 100 % and bottom to 0 %, allowing variable slope, using GraphPad Prism v6.00.