Ruxolitinib S enantiomer (INCB18424) is the S-enantiomer of Ruxolitinib. Ruxolitinib is the first potent, selective JAK1/2 inhibitor.

JAK1 (cell-free assay):3.3 nM, TYK2 (Cell-free assay):19 nM, JAK2 (cell-free assay):2.8 nM

INCB018424（180 mg/kg,口服,每天两次）显著减少JAK2V617F驱动的小鼠模型中脾肿大和炎性细胞因子的循环水平,并优先消灭肿瘤细胞,显著延长生存期,且未产生骨髓抑制或免疫抑制.上述小鼠在第22天存活率超过90％.15 mg Ruxolitinib处理骨髓纤维化患者,每天两次,在48周时,28％患者的脾脏体积减少至少35％.Ruxolitinib组的患者整体生活质量改善,与骨髓纤维化有关的症状减轻.

INCB018424在Ba/F3细胞中以剂量依赖性方式显著增加细胞凋亡。INCB018424作用于Ba/F3细胞和HEL细胞,有效且选择性抑制JAK2V617F介导的信号和增殖。INCB018424(64 nM) 导致Ba/F3细胞线粒体去极化增加一倍。INCB018424抑制正常供体和真性红细胞增多症患者提供的红系祖细胞增殖,IC50分别为407 nM 和 223 nM。INCB018424表现出对红系细胞集落形成的有效作用,IC50为67 nM。

Recombinant proteins expressed with Sf21 cells and baculovirus vectors are purified with affinity chromatography. JAK kinase assay is done by a homogeneous time-resolved fluorescence assay with the peptide substrate (-EQEDEPEGDYFEWLE). Each enzyme reaction is carried out with Ruxolitinib or control, JAK enzyme, 500 nM peptide, adenosine triphosphate (ATP; 1 mM), and 2% dimethyl sulfoxide (DMSO) for 1 hour. IC50 is the INCB018424 concentration required for inhibition of 50% of the fluorescent signal.

Cell lines: Ba/F3 and HEL cells. Concentrations: 3 μM. Method: Cells are seeded at 2×103/well of white bottom 96-well plates,treated with INCB018424 from DMSO stocks (0.2% final DMSO concentration),and incubated for 48 hours at 37 ℃ in an atmosphere containing 5% CO2.Viability is measured by cellular ATP determination using the Cell-Titer Glo luciferase reagent or viable cell counting.Values are transformed to percent inhibition relative to vehicle control,and IC50 curves are fitted according to nonlinear regression analysis of the data using PRISM GraphPad.

Animal Models: JAK2V617F-driven mouse modelFormulation & . Dosages: 5% dimethyl acetamide,0.5% methylcellulose.180 mg/kg. Administration: Oral gavage