Tofacitinib Citrate (CP-690550 citrate) is a a potent, cell-permeable inhibitor of JAK1/2/3 (IC50s: 1/20/112 nM).

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

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]。

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]。

The JAK1, JAK2, and JAK3 kinase assays utilize a protein expressed in baculovirus-infected SF9 cells (a fusion protein of GST and the catalytic domain of human JAK enzyme) purified by affinity chromatography on glutathione sepharose. The substrate for the reaction was polyglutamic acid-tyrosine [PGT (4:1)], coated onto Nunc Maxi Sorp plates at 100 μg/mL overnight at 37 °C. The plates were washed three times, and JAK enzyme was added to the wells, which contained 100 μL of kinase buffer (50 mM HEPES, pH 7.3, 125 mM NaCl, 24 mM MgCl2) + ATP + 1 mM sodium orthovanadate). After incubation at room temperature for 30 min, the plates were washed three times. The level of phosphorylated tyrosine in a given well was determined by standard ELISA assay utilizing an anti-phosphotyrosine antibody [5].

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].