Baricitinib phosphate (INCB028050) is a selective orally bioavailable JAK1/JAK2 inhibitor.

JAK1:5.9 nM, JAK2:5.7 nM

在细胞基础实验中，Baricitinib (INCB028050) 显示出强大的JAK信号传导和功能的抑制作用。在PBMCs中，Baricitinib 抑制了由IL-6刺激的典型底物STAT3 (pSTAT3)的磷酸化和随后的趋化因子MCP-1的产生，其IC50值分别为44 nM和40 nM。在分离的天真T细胞中，INCB028050 同样抑制了由IL-23刺激的pSTAT3（IC50=20 nM）。重要的是，这种抑制阻止了由Th17细胞生产的两种病原性细胞因子（IL-17和IL-22）的产生，Th17细胞是具有明显炎症和病原性特征的辅助T细胞亚型，其IC50值为50 nM。与此形成鲜明对比的是，结构相似但无效的JAK1/2抑制剂INCB027753和INCB029843在浓度高达10 μM的测试中，在任何这些实验系统中均无显著效果[1]。

Baricitinib (INCB028050) 的治疗与对照组相比，在2周的治疗期内，以1 mg/kg的剂量抑制了后爪体积增加50%，而以3或10 mg/kg的剂量则抑制超过95%。由于在治疗第0天，即动物出现明显病征时对爪体积的基线测量，对于那些在肿胀明显好转的动物，抑制率可能超过100%[1]。Baricitinib (0.7 mg/天)处理的小鼠，通过H&E染色评估，表现出明显减少的炎症，CD8浸润减少，以及MHC I类和II类表达降低，与对照组相比有显著差异。在与对照组相比，Baricitinib处理的小鼠中，对于鼠和人类斑秃(AA)疾病中关键的效应细胞CD8+NKG2D+细胞大幅减少[2]。

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].