Baricitinib (INCB028050) is a JAK1 and JAK2 inhibitor (IC50=5.9/5.7 nM) with selective and oral activity. Baricitinib has potential anti-inflammatory, immunomodulatory and anti-tumor activity.

JAK3:>400 nM, TYK2:53 nM, JAK1:5.9 nM, JAK2:5.7 nM

方法: PBMC 细胞用 Baricitinib (0-10000 nM) 预孵育 10 min，加入 IL-6 (10 ng/mL) 刺激细胞 48 h，使用 ELISA assay 检测相关因子。
结果: 在 PBMC 中，Baricitinib 抑制 IL-6 刺激的经典底物 STAT3 的磷酸化以及随后的趋化因子 MCP-1 的产生，IC50 值分别为 44 nM 和 40 nM。[1]
方法: CD19+ B 细胞用 IgM 抗体 (1 µg/mL)、sCD40L (250 ng/mL)、IL-4 (100 ng/mL) 和 Baricitinib (0.05-5 µM) 处理 2-5 天，使用 real-time PCR 检测基因表达水平。
结果: 刺激两天后，发现 Baricitinib 以剂量依赖性方式显著抑制 Aicda 的表达水平，而 Bcl6 的表达水平增加。刺激五天后，Xbp1 和 Irf4 的表达水平也显著降低。在相同条件下从上清液中测量的 IgG 产生量显著减少，这分别取决于刺激两天和五天后 Baricitinib 剂量的增加。[2]

方法: 为研究对自身免疫性关节炎的潜在治疗效用，将 Baricitinib (1-10 mg/kg) 口服给药给胶原诱导性关节炎 (CIA) 的 DBA/1j 小鼠，每天两次，持续 15 天。
结果: 早在给药后 4 天，疾病的临床症状就得到了改善，在研究终止时，与载体对照组相比，临床评分分别降低了 19%、67% 和 61%，这是剂量依赖性的。[1]

Enzyme assays are performed using a homogeneous time-resolved fluorescence assay with recombinant epitope tagged kinase domains (JAK1, 837-1142; JAK2, 828-1132; JAK3, 718-1124; Tyk2, 873-1187) or full-length enzyme (cMET and Chk2) and peptide substrate. Each enzyme reaction is performed with or without test compound (11-point dilution), JAK, cMET, or Chk2 enzyme, 500 nM (100 nM for Chk2) peptide, ATP (at the Km specific for each kinase or 1 mM), and 2.0% DMSO in assay buffer. The calculated IC50 value is the compound concentration required for inhibition of 50% of the fluorescent signal. Additional kinase assays are performed at Cerep using standard conditions at 200 nM. Enzymes tested included: Abl, Akt1, AurA, AurB, CDC2, CDK2, CDK4, CHK2, c-kit, EGFR, EphB4, ERK1, ERK2, FLT-1, HER2, IGF1R, IKKα, IKKβ, JNK1, Lck, MEK1, p38α, p70S6K, PKA, PKCα, Src, and ZAP70[1].

Baricitinib(INCB 028050) is dissolved in stock solutions, and then diluted with appropriate media before use[1]. Human PBMCs are isolated by leukapheresis followed by Ficoll-Hypaque centrifugation. For the determination of IL-6-induced MCP-1 production, PBMCs are plated at 3.3×105 cells per well in RPMI 1640+10% FCS in the presence or absence of various concentrations of INCB028050 (1 nM, 10 nM, 100 nM, 1 μM, and 10 μM). Following preincubation with compound for 10 min at room temperature, cells are stimulated by adding 10 ng/mL human recombinant IL-6 to each well. Cells are incubated for 48 h at 37°C, 5% CO2. Supernatants are harvested and analyzed by ELISA for levels of human MCP-1. The ability of INCB028050 to inhibit IL-6-induced secretion of MCP-1 is reported as the concentration required for 50% inhibition (IC50). Proliferation of Ba/F3-TEL-JAK3 cells is performed over 3 d using Cell-Titer Glo[1].