Tofacitinib

DBA/2 and C57/BL6 mice

3-((3R, 4R)-4-methyl-3-(methyl(7H-pyrrolo[2, 3-d]pyrimidin-4-yl)amino)piperidin-1-yl)-3-oxopropanenitrile

0-4000 nM

0-136 ng/mL

1 nM [], 1 nM [], 1 nM [], 1 nM [], 1 nM [], 1 nM []

In a murine model of heterotopic heart transplantation (DBA2 donor heart into C57/BL6 host), CP-690550 results in a dose-dependent increase in survival of transplanted hearts.The EC50 (drug concentration in blood at which 50% of mice will maintain their graft for >28 days) to be ca.60 ng/mL.CP-690550 prevents rejection of allogeneic kidneys in nonhuman primate (NHPs, macaca fascicularis) (MST of 62 and 83 days for the 50 to 100 ng/ml groups and 200 to 400 ng/ml groups, respectively). [1] Mice chronically dosed with CP-690550 (1.5-15 mg/kg/day) demonstrates dose and time-dependent alterations in lymphocyte subsets when examined by flow cytometry. The most dramatic change observed is a 96% reduction in splenic NK1.1+TCRb-cell numbers following 21 days of treatment. Delayed-type hypersensitivity (DTH) responses in sensitized mice are reduced in a dose-dependent manner following treatment with CP-690550 (1.87–30 mg/kg, s.c.). Extended survival of neonatal Balb/c hearts implanted into the ear pinna of MHC mismahed C3H/HEN mice is observed with CP-690550 monotherapy (10–30 mg/kg/day), but improved upon combination with cyclosporin (10 mg/kg/day). [2]

JAK3 Kinase Assay, A fragment encoding the catalytic domain of human JAK3 (785aa to 1125aa, JH1 catalytic domain) is amplified by PCR from the full length cDNA and cloned into the EcoRI site of the baculovirus transfer vector pVL1393. Recombinant baculovirus is used to infect Sf9 (Spodoptera frugipedra) cells and recombinant GSTJAK3 fusion protein is isolated on glutathione sepharose. The fusion protein is eluted with reduced glutathione and stored in buffer containing 50 mM Tris, pH 7.5, 10 mM DTT and 10% glycerol. JAK3 kinase activity is measured by ELISA as follows: Plates are coated overnight with a random L-glutamic acid and tyrosine co-polymer (4:1) (100 ug/mL). The plates are washed and recombinant JAK3 JH1:GST (100 ng/well) with or without inhibitors was incubated at room temperature for 30 minutes, after which HRP-conjugated PY20 anti-phosphotyrosine antibody (ICN) is added and developed by TMB (3, 3', 5, 5'-tetramethylbenzidine). Other kinases (Table 1) are produced in E. coli or in insect cells, depending upon what is found to be optimal for the given kinase. The catalytic activity of tyrosine kinases is easured using the aftorementioned ELISA, whereas serine/threonine kinases are assayed using radioactive enzyme assays.

312, 37

DMSO 63 mg/mL, Water <1 mg/mL, Ethanol <1 mg/mL