JAK2 (IC50 = 2.8 nM); JAK1 (IC50 = 3.3 nM); Tyk2 (IC50 = 19 nM); JAK3 (IC50 = 428 nM)

Ruxolitinib sulfate (INCB018424 sulfate) significantly and selectively enhances apoptosis in a dose-dependent manner, potently and selectively inhibits JAK2V617F-mediated signaling and proliferation, and causes a doubling of cells with depolarized mitochondria in Ba/F3 cells at 64 nM. With an IC50 of 67 nM, roxolitinib exhibits significant effectiveness against the development of erythroid colonies. It also suppresses the proliferation of erythroid progenitors from both normal donors and patients with polycythemia vera, with IC50 values of 407 nM and 223 nM, respectively[1].

Ruxolitinib (INCB018424 sulfate; 180 mg/kg, orally, twice a day) causes a JAK2V617F-driven mouse model to exhibit a significantly prolonged survival without myelosuppressive or immunosuppressive effects. By day 22, it also significantly reduces inflammatory cytokine levels and splenomegaly and preferentially eliminates neoplastic cells[1]. In the double-blind trial of myelofibrosis, 41.9% of patients in the Ruxolitinib group and 0.7% in the placebo group reach the primary end point. Ruxolitinib causes the overall symptom score to improve by 50% or more while maintaining the reduction in spleen volume[2]. When given roxolitinib (15 mg twice daily), patients with myelofibrosis had a reduction in spleen volume of at least 35% in 28% of cases by week 48, compared to 0% in the group receiving the best available therapy. By week 48, the mean palpable spleen length had increased by 4% with the best available therapy, but had dropped by 56% with Ruxolitinib. Patients receiving ruxolitinib showed improvements in overall quality-of-life metrics as well as a decrease in myelofibrosis-related symptoms[3].

Biochemical assays[1]
The kinase domains of human JAK1 (837-1142), JAK2 (828-1132), JAK3 (781-1124), and Tyk2 (873-1187) were cloned by PCR with N-terminal epitope tags. Recombinant proteins were expressed using Sf21 cells and baculovirus vectors and purified with affinity chromatography. JAK kinase assays used a homogeneous time-resolved fluorescence assay with the peptide substrate (-EQEDEPEGDYFEWLE). Each enzyme reaction was carried out with test compound or control, JAK enzyme, 500nM peptide, adenosine triphosphate (ATP; 1mM), and 2.0% dimethyl sulfoxide (DMSO) for 1 hour. The 50% inhibitory concentration (IC50) was calculated as the compound concentration required for inhibition of 50% of the fluorescent signal. Biochemical assays for CHK2 and c-MET enzymes were performed using standard conditions (Michaelis constant [Km] ATP) with recombinantly expressed catalytic domains from each protein and synthetic peptide substrates.

Apoptosis[1] Annexin V staining. Cells were treated for 20 to 24 hours and stained with annexin V and propidium iodide for analysis of early apoptotic and dead cells, respectively. Analysis was performed using a FACSCaliber flow cytometer. Mitochondrial membrane potential. Cells were treated for 24 hours and then incubated with 2μM of the dye JC-1. Analysis was performed by flow cytometry using 488-nm excitation and 530-nm and 585-nm emission filters. JC-1 exhibits potential-dependent accumulation in the mitochondria where its emission is in the red spectrum (590nM). A fluorescence shift from red (590nM) to green (530nM) indicates redistribution of the dye to the cytoplasm resulting from loss of mitochondrial membrane potential, an early marker for apoptosis.

[1]. Preclinical characterization of the selective JAK1/2 inhibitor INCB018424: therapeutic implications for the treatment of myeloproliferative neoplasms. Blood, 2010, 115(15), 3109-3117.

[2]. A double-blind, placebo-controlled trial of ruxolitinib for myelofibrosis. N Engl J Med, 2012, 366(9), 799-807.

[3]. JAK inhibition with ruxolitinib versus best available therapy for myelofibrosis. N Engl J Med. 2012 Mar 1;366(9):787-98.

Background: Ruxolitinib, a selective inhibitor of Janus kinase (JAK) 1 and 2, has clinically significant activity in myelofibrosis.
Methods: In this double-blind trial, we randomly assigned patients with intermediate-2 or high-risk myelofibrosis to twice-daily oral ruxolitinib (155 patients) or placebo (154 patients). The primary end point was the proportion of patients with a reduction in spleen volume of 35% or more at 24 weeks, assessed by means of magnetic resonance imaging. Secondary end points included the durability of response, changes in symptom burden (assessed by the total symptom score), and overall survival.
Results: The primary end point was reached in 41.9% of patients in the ruxolitinib group as compared with 0.7% in the placebo group (P<0.001). A reduction in spleen volume was maintained in patients who received ruxolitinib; 67.0% of the patients with a response had the response for 48 weeks or more. There was an improvement of 50% or more in the total symptom score at 24 weeks in 45.9% of patients who received ruxolitinib as compared with 5.3% of patients who received placebo (P<0.001). Thirteen deaths occurred in the ruxolitinib group as compared with 24 deaths in the placebo group (hazard ratio, 0.50; 95% confidence interval, 0.25 to 0.98; P=0.04). The rate of discontinuation of the study drug because of adverse events was 11.0% in the ruxolitinib group and 10.6% in the placebo group. Among patients who received ruxolitinib, anemia and thrombocytopenia were the most common adverse events, but they rarely led to discontinuation of the drug (in one patient for each event). Two patients had transformation to acute myeloid leukemia; both were in the ruxolitinib group.
Conclusions: Ruxolitinib, as compared with placebo, provided significant clinical benefits in patients with myelofibrosis by reducing spleen size, ameliorating debilitating myelofibrosis-related symptoms, and improving overall survival. These benefits came at the cost of more frequent anemia and thrombocytopenia in the early part of the treatment period. (Funded by Incyte; COMFORT-I ClinicalTrials.gov number, NCT00952289.).

C17H18N6.H2O4S

404.4435

404.127

1092939-16-6

Ruxolitinib;941678-49-5;Ruxolitinib (S enantiomer);941685-37-6;Ruxolitinib phosphate;1092939-17-7

25127111

Typically exists as off-white to gray solids at room temperature

3.894

3

8

4

28

535

1

[H][C@@](C1CCCC1)(N2N=CC(C3=C4C=CNC4=NC=N3)=C2)CC#N.O=S(O)(O)=O

LGJWVXWQCTZSGC-UHFFFAOYSA-N

InChI=1S/C17H18N6.H2O4S/c18-7-5-15(12-3-1-2-4-12)23-10-13(9-22-23)16-14-6-8-19-17(14)21-11-20-16;1-5(2,3)4/h6,8-12,15H,1-5H2,(H,19,20,21);(H2,1,2,3,4)

3-cyclopentyl-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)pyrazol-1-yl]propanenitrile;sulfuric acid

INCB-018424 sulfate, INCB 018424, INCB018424; INC424, INC424, INC-424; INCB18424, INCB 18424, INCB-18424; Jakafi and Jakavi (trade name)

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)