Tofacitinib (CP-690550) Citrate

Licensed by Pfizer Catalog No.S5001 Synonyms: Tasocitinib Citrate

For research use only.

Tofacitinib citrate (CP-690550, Tasocitinib) is a novel inhibitor of JAK with IC50 of 1 nM, 20 nM and 112 nM against JAK3, JAK2, and JAK1, respectively. Tofacitinib citrate has anti-infection activity.

Tofacitinib (CP-690550) Citrate Chemical Structure

CAS No. 540737-29-9

Selleck's Tofacitinib (CP-690550) Citrate has been cited by 167 publications

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Biological Activity

Description Tofacitinib citrate (CP-690550, Tasocitinib) is a novel inhibitor of JAK with IC50 of 1 nM, 20 nM and 112 nM against JAK3, JAK2, and JAK1, respectively. Tofacitinib citrate has anti-infection activity.
Targets
JAK3 [1]
(Cell-free assay)
JAK2 [4]
(Cell-free assay)
1 nM 20 nM
In vitro

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). [2] Additionally, Tofacitinib citrate prevents IL-15-induced CD69 expression in human and cynomolgus monkey NK and CD8+ T cells in vitro. [3]

Cell Data
Cell Lines Assay Type Concentration Incubation Time Formulation Activity Description PMID
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T-cells NGrBeZZHfW6ldHnvckBie3OjeR?= NHP1Tpg,OzByIH7N NXjiPWJWOSCqch?= NX;RWlBkUW6qaXLpeIlwdiCxZjDKRWsyN0qDS{KvWHlMOiCrbjDoeY1idiCFRESgdI9{cXSrdnWgWEBk\WyuczDhd5Nme3OnZDDhd{BqdmirYnn0bY9vKG:oIFnMOk1{fGmvdXzheIVlKFOWQWSzJJBpd3OyaH;yfYxifGmxbjDheEA,OzByIH7NJJBz\WmwY4XiZZRm\CCob4KgNUBpeiCob3zsc5dm\CCkeTDJUE03KHO2aX31cIF1cW:wIH3lZZN2emWmIHHmeIVzKDNyIH3pcpMh[nliaX3teY5w[myxdDDhcoFtgXOrcx?= MXy8ZUB1[XKpZYS9K39jdGGwazegbJJm\j1paIT0dJM7Ny:ydXLt[YQvdmOkaT7ucI0vdmmqLnfvek8zQTh3MkC2PEc,Ojl6NUKwOlg9N2F-
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Assay
Methods Test Index PMID
Western blot JAK3 / STAT5 / p-STAT5 ; LMP1 / EBNA1 / BZLF1 ; JAK3 / p-JAK3 / STAT3 27732937 26082451
Growth inhibition assay Cell number 27732937
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.[2] 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.[3]

Protocol (from reference)

Kinase Assay:

[1]

  • Enzyme assays:

    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 Research:

[2]

  • Cell lines: FDCP-EpoR JAK2WT and JAK2V617F cell lines
  • Concentrations: 0-4 μM
  • Incubation Time: 72 hours
  • Method:

    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.

  • (Only for Reference)
Animal Research:

[2]

  • Animal Models: Mauritius-origin adult cynomolgus monkeys
  • Dosages: 10, 30 mg/kg/d
  • Administration: Oral gavage
  • (Only for Reference)

Solubility (25°C)

In vitro

DMSO 100 mg/mL warmed
(198.21 mM)
Water Insoluble
Ethanol Insoluble

In vivo

Add solvents to the product individually and in order
(Data is from Selleck tests instead of citations):
0.5% methylcellulose
For best results, use promptly after mixing.

30 mg/mL

* Please note that Selleck tests the solubility of all compounds in-house, and the actual solubility may differ slightly from published values. This is normal and is due to slight batch-to-batch variations.

Chemical Information

Molecular Weight 504.49
Formula

C16H20N6O.C6H8O7

CAS No. 540737-29-9
Storage 3 years -20°C powder
2 years -80°C in solvent
Smiles CC1CCN(CC1N(C)C2=NC=NC3=C2C=CN3)C(=O)CC#N.C(C(=O)O)C(CC(=O)O)(C(=O)O)O

In vivo Formulation Calculator (Clear solution)

Step 1: Enter information below (Recommended: An additional animal making an allowance for loss during the experiment)

mg/kg g μL

Step 2: Enter the in vivo formulation (This is only the calculator, not formulation. Please contact us first if there is no in vivo formulation at the solubility Section.)

% DMSO % % Tween 80 % ddH2O
%DMSO %

Calculation results:

Working concentration: mg/ml;

Method for preparing DMSO master liquid: mg drug pre-dissolved in μL DMSO ( Master liquid concentration mg/mL, Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug. )

Method for preparing in vivo formulation: Take μL DMSO master liquid, next addμL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O, mix and clarify.

Method for preparing in vivo formulation: Take μL DMSO master liquid, next add μL Corn oil, mix and clarify.

Note: 1. Please make sure the liquid is clear before adding the next solvent.
2. Be sure to add the solvent(s) in order. You must ensure that the solution obtained, in the previous addition, is a clear solution before proceeding to add the next solvent. Physical methods such
as vortex, ultrasound or hot water bath can be used to aid dissolving.

Molarity Calculator

Mass Concentration Volume Molecular Weight

Clinical Trial Information

NCT Number Recruitment Interventions Conditions Sponsor/Collaborators Start Date Phases
NCT05082428 Not yet recruiting -- Ulcerative Colitis Pfizer December 1 2021 --
NCT05087589 Recruiting Drug: Tofacitinib Primary Sjögren''s Syndrome Peking University People''s Hospital November 18 2021 Phase 2
NCT04768504 Recruiting Drug: Tofacitinib 10 mg Immune-Mediated Colitis Khashayar Esfahani|Sir Mortimer B. Davis - Jewish General Hospital November 15 2021 Phase 2
NCT04624230 Recruiting Drug: tofacitinib Ulcerative Colitis Pfizer August 12 2021 Phase 3
NCT04496960 Recruiting Drug: tofacitinib|Other: Placebo Sjogren''s Syndrome National Institute of Dental and Craniofacial Research (NIDCR)|National Institutes of Health Clinical Center (CC) May 18 2021 Phase 1|Phase 2
NCT04424303 Recruiting Drug: Tofacitinib Ulcerative Colitis Pfizer December 7 2020 --

(data from https://clinicaltrials.gov, updated on 2022-01-17)

Tech Support

Answers to questions you may have can be found in the inhibitor handling instructions. Topics include how to prepare stock solutions, how to store inhibitors, and issues that need special attention for cell-based assays and animal experiments.

Handling Instructions

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Frequently Asked Questions

Question 1:
What is the difference between the two products (S5001, S2789)?

Answer:
Tofacitinib (S2789) is the base form of Tofacitinib citrate (S5001). The biological activity of these two compound are same. S5001 is better than S2789 for Oral gavage.

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