Molecular Weight (MW)

504.49

Formula

C16H20N6O.C6H8O7

CAS No.

540737-29-9 (citrate);

Storage

-20℃ for 3 years in powder form

-80℃ for 2 years in solvent

Solubility (In vitro)

DMSO: 100 mg/mL (198.2 mM)

Water: <1 mg/mL

Ethanol: <1 mg/mL

Solubility (In vivo)

0.5% methylcellulose: 30 mg/mL

Synonyms

CP-690550; CP690550; CP 690550; Tasocitinib; Tofacitinib; Xeljanz (Trade name); CP-690550-10; CP-690,550-10; CP-690550 citrate;

In Vitro

In vitro activity: Tofacitinib citrate inhibits IL-2-mediated human T cell blast proliferation and IL-15-induced CD69 expression with IC50 of 11 nM and 48 nM, respectively. Tofacitinib citrate prevents mixed lymphocyte reaction with IC50 of 87 nM. Tofacitinib citrate treatment of murine factor-dependent cell Patersen–erythropoietin receptor (FDCP-EpoR) cells harboring human wild-type or V617F JAK2 leads to prevention of cell proliferation with IC50 of 2.1 μM and 0.25 μM, respectively. Tofacitinib citrate inhibits interleukin-6-induced phosphorylation of STAT1 and STAT3 with IC50 of 23 nM and 77 nM, respectively. Moreover, Tofacitinib citrate generates a significant pro-apoptotic effect on murine FDCP-EpoR cells carrying JAK2VV617F, whereas a lesser effect is observed for cells carrying wild-type JAK2. This activity is coupled with the inhibition of phosphorylation of the key JAK2V617F-dependent downstream signaling effectors signal transducer and activator of transcription (STAT)3, STAT5, and v-akt murine thymoma viral oncogene homolog (AKT). Additionally, Tofacitinib citrate prevents IL-15-induced CD69 expression in human and cynomolgus monkey NK and CD8+ T cells in vitro.

Kinase Assay: The JAK1, JAK2, and JAK3 kinase assays utilize a protein expressed in baculovirus-infected SF9 cells (a fusion protein of GST and the catalytic domain of human JAK enzyme) purified by affinity chromatography on glutathione–Sepharose. The substrate for the reaction is polyglutamic acid-tyrosine [PGT (4:1)], coated onto Nunc Maxi Sorp plates at 100 μg/mL overnight at 37 °C. The plates are washed three times, and JAK enzyme is added to the wells, which contained 100 μL of kinase buffer (50 mM HEPES, pH 7.3, 125 mM NaCl, 24 mM MgCl2) + ATP + 1 mM sodium orthovanadate). For Tofacitinib citrate, it is also added for kinase assay at different doses. After incubation at room temperature for 30 min, the plates are washed three times. The level of phosphorylated tyrosine in a given well is determined by standard ELISA assay utilizing an anti-phosphotyrosine antibody.

Cell Assay: Determination of growth inhibition by Tofacitinib citrate is performed using identical culture conditions for both FDCP-EpoR JAK2WT and JAK2V617F cell lines. Briefly, 1 × 105 cells/mL are cultured in 96-well flat-bottom plates at 37 °C in a humidified 5% CO2 atmosphere using RPMI 1640 supplemented with 1.25% FCS, and 5% WEHI supernatant. Decreased FCS concentration is necessary to prevent binding between Tofacitinib citrate and serum proteins. Growth inhibition assays are terminated by addition of 20 μL CellTiter96 One Solution Reagent. Flat-bottom plates are incubated for an additional 3 hours for MTT assay. Absorbance is determined at 595 nm on a BioTek Synergy-HT microplate reader. Results are the average standard deviation of three independent determinations.

In Vivo

Tofacitinib citrate decrease a delayed-type hyper-sensitivity response and extended cardiac allograft survival in murine models. Furthermore, Tofacitinib citrate treatment of ex-vivo-expanded erythroid progenitors from JAK2V617F-positive PV patients results in specific, antiproliferative (IC50 = 0.2 μM) and pro-apoptotic activity. In contrast, expanded progenitors from healthy controls are less sensitive to Tofacitinib citrate in proliferation (IC50> 1.0 μM), and apoptosis assays. During 2 weeks of Tofacitinib citrate dosing at 10 and 30 mg/kg/d, a significant, time-dependent decrease in NK cell numbers relative to vehicle treatment is observed. Effector memory CD8+ cell numbers in the Tofacitinib citrate-treated group are 55% less than those observed in animals treated with vehicle

Animal model

Mauritius-origin adult cynomolgus monkeys

Formulation & Dosage

Formulated in 0.5% methylcellulose in distilled water; 10, 30 mg/kg/day; Oral gavage

References

J Med Chem. 2010 Dec 23;53(24):8468-84; Cancer Sci. 2008 Jun;99(6):1265-73; J Leukoc Biol. 2004 Dec;76(6):1248-55.

CP-690,550 inhibits signal transducer and activator of transcription (STAT)3 nuclear localization in both murine cell lines. Cancer Sci. 2008 Jun;99(6):1265-73.

Effect of CP-690,550 on ex vivo expanded erythroid progenitors. Cancer Sci. 2008 Jun;99(6):1265-73.

CP-690,550-induced poly (ADP-ribose) polymerase (PARP) and caspase-3 cleavage. Cancer Sci. 2008 Jun;99(6):1265-73.

CP-690,550 modulates immunolocalization of signal transducer and activator of transcription (STAT)3. Cancer Sci. 2008 Jun;99(6):1265-73.