CYT387 sulfate salt

Kinase experiment:

Glutathione-S-transferase (GST)-tagged JAK kinase domains expressed in insect cells are purified before use in a peptide substrate phosphorylation assay. Assays are carried out in 384-well optiplates using an Alphascreen Protein Tyrosine Kinase P100 detection kit and a PerkinElmer Fusion Alpha instrument[1].

Cell experiment:

Ba/F3 cells expressing JAK2V617F (Ba/F3-JAK2V617F) and MPLW515L (Ba/F3-MPLW515L) mutants, as well as CHRF-288-11 (JAK2T875N) and CMK (JAK3A572V) cells are used. The TEL/JAK2 and TEL/JAK3 fusions are generated and introduced into Ba/F3 murine cells. TheTEL/JAK2- or TEL/JAK3-transfected cells are cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calf serum (FCS). Ba/F3 wild-type cells are cultured in RPMI containing 10% FCS supplemented with 5 ng/mL murine IL-3. Proliferation is measured using the Alamar Blue assay after incubating for 72 h at 37°C with 5% CO2[1].

Animal experiment:

Mice[2] On day 32 after bone marrow transplantation (when all mice exhibit severe leukocytosis and erythrocytosis), mice are assigned to 3 groups such that each group has equivalent average body weight and blood counts. Momelotinib (CYT387) is dissolved in NMP (120 mg/mL final; 1-methyl-2-pyrrolidinone, Chromasolv Plus). Subsequently, the Momelotinib/NMP mix is diluted with 0.14M Captisol to a concentration of 6 mg/mL and further diluted with 0.1M Captisol to a final concentration of 4 mg/mL. All 3 groups of mice (n=12 per group) are administered Momelotinib (CYT387) by oral gavage twice daily at 10- to 12-hour intervals from day 34 after bone marrow transplantation to day 82 (end of experiment). Mice receive NMP/Captisol without Momelotinib (CYT387) (0 mg/kg group), 25 mg/kg Momelotinib, or 50 mg/kg Momelotinib. At day 82 after bone marrow transplantation, all mice are euthanized for analysis except for 2 mice each from the 50 mg/kg and 25 mg/kg groups, which are taken off Momelotinib (CYT387) treatment and followed for 45 additional days. For assessment of the effects of Momelotinib (CYT387) on normal blood counts, naive Balb/c mice are administered vehicle control, 50 mg/kg, or 100 mg/kg Momelotinib (CYT387) in an identical fashion as described for the bone marrow transplant experimental mouse cohort. Peripheral blood is drawn at day 14, 28, 42, and 56 and levels of red cells, white cells, reticulocytes, granulocytes, lymphocytes, and monocytes are analyzed.

References:

[1]. Pardanani A, et al. CYT387, a selective JAK1/JAK2 inhibitor: in vitro assessment of kinase selectivity and preclinical studies using cell lines and primary cells from polycythemia vera patients. Leukemia, 2009, 23(8), 1441-1445.
[2]. Tyner JW, et al. CYT387, a novel JAK2 inhibitor, induces hematologic responses and normalizes inflammatory cytokines in murine myeloproliferative neoplasms. Blood, 2010, 115(25), 5232-5240.
[3]. Burns CJ, et al. Phenylaminopyrimidines as inhibitors of Janus kinases (JAKs). Bioorg Med Chem Lett. 2009 Oct 15;19(20):5887-92.