Cell lines

Primary mononuclear cells isolated from patients with PV or normal control persons

Preparation method

The solubility of this compound in DMSO is >10 mM. General tips for obtaining a higher concentration: Please warm the tube at 37 ℃ for 10 minutes and/or shake it in the ultrasonic bath for a while.Stock solution can be stored below -20℃ for several months.

Reaction Conditions

IC50: erythroid progenitors: 407 nM for normal donors, 223 nM for PV donors myeloid progenitors: 511 nM for normal donors, 444 nM for PV donors 14 days

Applications

Growth of clonogenic progenitors of erythroid (BFU-E) and myeloid origin (CFU-M) was assessed in colony-forming assays in the presence of increasing concentrations of INCB018424. Dose-dependent inhibition of the growth of erythroid and myeloid progenitors was observed with INCB018424. The mean IC50 for INCB018424 against erythroid progenitors was 407 nM for normal donors and 223 nM for PV donors. A similar effect was observed on myeloid progenitors (CFU-M), with IC50 values of 511 nM and 444 nM for control and PV samples, respectively.

Animal models

C57BL/6N mice

Dosage form

Oral administration, 75 mg/kg

Applications

Mice receiving 75 mg/kg ruxolitinib or vehicle 6 hours prior to and 6 hours after injection of OVA/CpG were analyzed for expression of activation markers on CD11c 1CD81 splenic DCs. Lower expression levels of CD40, CD80, CD86 as well as MHC I and II molecules were detected in ruxolitinib-challenged animals. Next, ruxolitinib or vehicle was fed to mice 6 hours prior to as well as 6 hours and 18 hours after priming with OVA/CpG and adoptive transfer of CFSE-labeled OT-I cells. Analysis of transferred CFSE-labeled OT-I T cells revealed reduced proliferation, CD25 expression, and IFN-production in mice pretreated with ruxolitinib.

Other notes

Please test the solubility of all compounds indoor, and the actual solubility may slightly differ with the theoretical value. This is caused by an experimental system error and it is normal.

Ruxolitinib (INCB18424) is a potent and selective JAK1/2 inhibitor with IC50s of 3.3 nM and 2.8 nM in cell-free assays, and has 130-fold selectivity for JAK1/2 over JAK3.

Ruxolitinib potently and selectively inhibits JAK2V617F-mediated signaling and proliferation, markedly increases apoptosis in a dose dependent manner, and at 64 nM results in a doubling of cells with depolarized mitochondria in Ba/F3 cells. Ruxolitinib demonstrates remarkable potency against erythroid colony formation with IC50 of 67 nM, and inhibits proliferating of erythroid progenitors from normal donors and polycythemia vera patients with IC50 values of 407 nM and 223 nM, respectively[1].

Ruxolitinib (180 mg/kg, orally, twice a day) results in survive rate of greater than 90% by day 22 and markedly reduces splenomegaly and circulating levels of inflammatory cytokines, and preferentially eliminated neoplastic cells, resulting in significantly prolonged survival without myelosuppressive or immunosuppressive effects in a JAK2V617F-driven mouse model[1]. In the Ruxolitinib group, the primary end point is reached in 41.9% of patients, as compared with 0.7% in the placebo group in the double-blind trial of myelofibrosis. Ruxolitinib results in maintaining of reduction in spleen volume and improvement of 50% or more in the total symptom score[2]. Ruxolitinib (15 mg twice daily) treatment leads a total of 28% of the patients to have at least a 35% reduction in spleen volume at week 48 in patients with myelofibrosis, as compared with 0% in the group receiving the best available therapy. The mean palpable spleen length has decreased by 56% with Ruxolitinib but has increased by 4% with the best available therapy at week 48. Patients in the ruxolitinib group has an improvement in overall quality-of-life measures and a reduction in symptoms associated with myelofibrosis[3].

References:
[1]. Quintas-Cardama A, et al. Preclinical characterization of the selective JAK1/2 inhibitor INCB018424: therapeutic implications for the treatment of myeloproliferative neoplasms. Blood, 2010, 115(15), 3109-3117.
[2]. Verstovsek S, et al. A double-blind, placebo-controlled trial of ruxolitinib for myelofibrosis. N Engl J Med, 2012, 366(9), 799-807.
[3]. Harrison C, et al. JAK inhibition with ruxolitinib versus best available therapy for myelofibrosis. N Engl J Med. 2012 Mar 1;366(9):787-98.