In vitro activity: ASP015K suppresses the IL-2-induced proliferation of human T cells with greater potency than EPO-induced proliferation of human erythroleukemia cells. In human whole blood, ASP015K inhibits STAT5 phosphorylation (pSTAT5).

Kinase Assay: Human JAK1, JAK2, JAK3, TYK2-domains assays performed using streptavidin-coated 96-well plates. Reaction mixture contained 15 mM Tris-HCl (pH 7.5), 0.01% Tween 20, 2 mM dithiothreitol, 10 mM MgCl2, 250 nM Biotin-Lyn-Substrate-2 (for JAK1, 2 and 3) or Biotin-IRS1-Substrate (for TYK2), and ATP (at final concentrations of 200 μM [JAK1], 10 μM [JAK2], 8 μM [JAK3], and 4 μM [TYK2]). Peficitinib or tofacitinib is dissolved in DMSO. The reaction is initiated by adding the kinase domain, followed by incubation at room temperature for 1 h. Kinase activity is measured as the rate of phosphorylation of Biotin-Lyn-Substrate-2 or Biotin-IRS-Substrate using HRP-conjugated anti-phosphotyrosine antibody (HRP-PY-20) using a phosphotyrosine-specific ELISA. TYK2 kinase assay of Peficitinib is performed with the ATP concentration of 10 μM

Cell Assay: Erythropoietin regulates haematopoiesis (the production of blood cells). Treatment with ASP015K inhibited the human T-cell proliferation induced by IL-2. And this effect was greater than the effect to inhibit EPO-induced human erythroleukemia cell proliferation. In human whole blood, ASP015K concentration-dependently inhibited STAT5 phosphorylation (pSTAT5).