Pharmacokinetic and pharmacodynamic studies of JAK1, JAK2, and JAK1/2 inhibition. (A) Plasma drug levels (μg/L) in naïve C57BL/6J mice after single oral dose of each inhibitor (120 mg/kg itacitinib, 120 mg/kg fedratinib, 90 mg/kg ruxolitinib). Mean values ± standard deviation (SD) are shown. (B) Intracellular STAT1 phosphorylation measured in bone marrow–derived macrophages (BMDM) cultured with vehicle (methyl cellulose) or itacitinib (2500, 500, or 50 nM), (C) vehicle (methyl cellulose) or fedratinib (1000, 300, or 30 nM), or (D) vehicle (captisol in citrate buffer) or ruxolitinib (1000, 50, or 1 nM) for 1 hour and then stimulated with IFN-γ (30 ng/mL) for 20 minutes. Representative overlayed histogram plots are shown below each graph. Experiments were performed using duplicates. Data are representative of 2 independent experiments. (E) Intracellular STAT1 phosphorylation measured in thioglycolate–elicited peritoneal macrophages after a single oral dose of JAK inhibitor and intraperitoneal (500 ng) injection of IFN-γ, shown as percentage of pSTAT1⁺ F4/80⁺ macrophages. F4/80^lo macrophages were excluded from the gating. (F) Representative histograms (left) and fold-change mean fluorescence intensity (MFI) (right) compared to isotype control. Each data point in panels E-F represents 1 mouse. Data are representative of 2 independent experiments. ∗P < .05, ∗∗∗P < .001, or ∗∗∗∗P < .0001 by pairwise comparison.