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

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

INCB018424 (180 mg/kg, orally, twice daily) significantly reduced spleen enlargement and the circulation of inflammatory cytokines in a JAK2V617F-driven mouse model, preferentially targeting and eliminating tumor cells, notably prolonging survival without causing bone marrow suppression or immunosuppression. The survival rate exceeded 90% on Day 22 for these mice. Additionally, in myelofibrosis patients, a 15 mg dosage of Ruxolitinib administered twice daily for 48 weeks resulted in at least a 35% reduction in spleen volume in 28% of patients. Patients in the Ruxolitinib group experienced an overall improvement in quality of life and a reduction in symptoms associated with myelofibrosis.

INCB018424 significantly induces apoptosis in Ba/F3 cells in a dose-dependent manner and effectively and selectively inhibits JAK2V617F-mediated signaling and proliferation in both Ba/F3 and HEL cells. At a concentration of 64 nM, INCB018424 doubles mitochondrial depolarization in Ba/F3 cells. It inhibits the proliferation of erythroid progenitor cells derived from both healthy donors and patients with polycythemia vera, with IC50 values of 407 nM and 223 nM, respectively. Furthermore, INCB018424 demonstrates potent activity in inhibiting the formation of erythroid colonies, with an IC50 of 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