Tofacitinib Citrate

JAK1:112 nM (cell free)|JAK2:20 nM (cell free)|JAK3:1 nM (cell free)

CP-690,550治疗显著延长了与对照组相比的移植物存活时间。12只动物中有4只接受CP-690,550治疗（每个剂量组各两只）存活至研究结束，且肾功能正常，根据组织病理学判断只有轻度排斥反应[1]。用tofacitinib单药治疗小鼠能够抑制对由细菌蛋白质Pseudomonas exotoxin A衍生的免疫毒素以及模型抗原钥孔血蓝蛋白的抗体（Ab）反应。实验显示，在免疫后21天观察到对两种抗原IgG1滴度的千倍降低。Tofacitinib治疗还导致CD127+前B细胞数量减少[4]。

虽然Tofacitinib (CP-690,550) 对JAK3的抑制作用极为强大（酶抑制效力为1 nM），但其对JAK2和JAK1的抑制作用分别低20至100倍。CP-690,550能以比对GM-CSF诱导的增殖30倍更强的效力抑制IL-2诱导的增殖。CP-690,550在使用鼠、猴或人细胞的混合淋巴细胞反应中展现出强大的抑制效果。与其作用机制一致，这些细胞活性与CP-690,550阻断IL-2诱导的JAK3及其关键底物STAT5的磷酸化能力相关[1]。在治疗含有人类野生型或V617F JAK2的鼠类因子依赖细胞Patersen-erythropoietin receptor (FDCP-EpoR)时，CP-690,550能抑制细胞增殖，其IC50分别为2.1 μg/ml和0.25 μg/ml。对JAK2(V617F)-阳性PV患者体外扩增的红细胞祖细胞进行CP-690,550治疗，表现出特异的抗增殖（IC50：0.2 μg/ml）和促凋亡活性[2]。Tofacitinib对JAK3的药理抑制作用与IMA在CML细胞中的抗肿瘤效果协同增强[3]。

DMSO:55 mg/mL (109.02 mM)

Apoptotic cells were detected by flow cytometry using recombinant human Annexin-V conjugated with allophycocyanin. Briefly, after exposure to CP-690,550 for different periods of time, cells were washed in Ca2+-free PBS and resuspended in 100 μL of binding buffer (10 mM HEPES pH 7.4; 0.15 M NaC1; 5 mM KCl; 1 mM MgCl2; 1.8 mM CaCl2) to which Annexin-V-APC had been previously added. Cells were incubated for 20 min in the dark at room temperature, washed and resuspended in 0.3 mL binding buffer. Cells were analyzed on a FACSCalibur flow cytometer equipped with the Cell Quest Pro software [2].

Mice received tofacitinib in PEG300 (100 mg/ml) or vehicle alone (PEG300) by osmotic pump infusion (Alzet Model 2004, 0.25 μl/hour, 28 days). Four days prior to immunization, mice were anesthetized and their dorsal surface was shaved. A one cm incision was made on the back to create a subcutaneous pocket and insert the pump. The incision site was closed with wound clips. Mice were injected weekly (i.p.) with SS1P recombinant immunotoxin (RIT; 5 μg/mouse) beginning on day 0; control mice received injections of saline alone. Every week before SS1P or vehicle immunization, ~50 μl of blood was drawn to obtain serum samples. Sera were stored at ?80°C until analyzed [4].