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CP-690550 (Tofacitinib) Citrate JAK inibitore

N. Cat.S5001

Il citrato di tofacitinib (CP-690550, Tasocitinib) è un nuovo inibitore di JAK con IC50 di 1 nM, 20 nM e 112 nM rispettivamente contro JAK3, JAK2 e JAK1. Ha anche attività Anti-infection.
CP-690550 (Tofacitinib) Citrate JAK inibitore Chemical Structure

Struttura chimica

Peso molecolare: 504.49

Vai a

Controllo Qualità

Lotto: Purezza: 99.99%
99.99

Coltura cellulare, trattamento e concentrazione di lavoro

Linee cellulari Tipo di saggio Concentrazione Tempo di incubazione Formulazione Descrizione dellattività PMID
Sf9 Function assay 30 mins Inhibition of human GST-fused JAK3 catalytic domain expressed in baculovirus-infected Sf9 cells using polyglutamic acid-tyrosine as substrate after 30 mins by ELISA, Ki = 0.0004 μM. 23668484
Sf9 Function assay 90 mins Inhibition of human recombinant N-terminal GST-tagged JAK3 expressed in Sf9 cells using Biotin-LC-EQEDEPEGDYFEWLE as substrate after 90 mins by TR-FRET assay, IC50 = 0.0006 μM. 22087750
Sf9 Function assay 30 mins Inhibition of human GST-fused JAK1 catalytic domain expressed in baculovirus-infected Sf9 cells using polyglutamic acid-tyrosine as substrate after 30 mins by ELISA, Ki = 0.0007 μM. 23668484
Sf9 Function assay 30 mins Inhibition of human GST-fused JAK2 catalytic domain expressed in baculovirus-infected Sf9 cells using polyglutamic acid-tyrosine as substrate after 30 mins by ELISA, Ki = 0.0007 μM. 23668484
Sf9 Function assay Inhibition of human recombinant GST-tagged JH1 domain (785 to1125) of JAK3 expressed in insect Sf9 cells by ELISA, IC50 = 0.001 μM. 14593182
Sf9 Function assay Inhibition of GST-tagged human JAK3 catalytic domain expressed in Sf9 cells by ELISA, IC50 = 0.001 μM. 21105711
Sf9 Function assay 90 mins Inhibition of human recombinant N-terminal GST-tagged JAK1 expressed in Sf9 cells using Biotin-LC-EQEDEPEGDYFEWLE as substrate after 90 mins by TR-FRET assay, IC50 = 0.003 μM. 22087750
Sf9 Function assay Inhibition of GST-tagged human JAK3 catalytic domain expressed in Sf9 cells by ELISA, IC50 = 0.0033 μM. 21105711
Sf9 Function assay 90 mins Inhibition of human recombinant N-terminal GST-tagged JAK2 expressed in Sf9 cells using Biotin-LC-EQEDEPEGDYFEWLE as substrate after 90 mins by TR-FRET assay, IC50 = 0.004 μM. 22087750
SF21 Function assay 10 mins Inhibition of JAK2 (unknown origin) expressed in SF21 cells using Biotin-KAIETDKEYYTVKD as substrate and [33Pgamma]ATP incubated for 10 mins prior to substrate addition measured after 30 mins by Topcount analysis, IC50 = 0.004 μM. 23541670
insect cells Function assay Inhibition of GST-tagged Jak1 expressed in insect cells using 70 uM ATP, IC50 = 0.0061 μM. 21155605
insect cells Function assay Inhibition of GST-tagged Jak3 expressed in insect cells using 18 uM ATP, IC50 = 0.008 μM. 21155605
T-cells Function assay 72 hrs Inhibition of IL2-induced proliferation of human T cells assessed as [3H]thymidine incorporation after 72 hrs by scintillation counting, IC50 = 0.011 μM. 14593182
insect Function assay Inhibition of GST-tagged Jak2 expressed in insect cells using 20 uM ATP, IC50 = 0.012 μM. 21155605
MO7 Function assay Inhibition of Jak3-mediated IL15-induced Stat5 phosphorylation in human MO7 cells by cell-based assay, IC50 = 0.024 μM. 21155605
Ba/F3 Function assay 60 mins Inhibition of TEL-fused JAK1 expressed in Ba/F3 cells assessed as inhibition of STAT5 phosphorylation after 60 mins by AlphaScreen assay, IC50 = 0.026 μM. 22087750
T-cells Function assay Inhibition of JAK3/1 in human T cells expressing CD3 assessed as inhibition of IL2-stimulated STAT5a phosphorylation, IC50 = 0.028 μM. 23540648
PBMC Function assay Inhibition IL-7-indcued STAT5 phosphorylation in human PBMC cells by flow cytometry, IC50 = 0.039 μM. 26927423
TF1 Function assay 20 mins Inhibition of JAK1 in human TF1 cells assessed as inhibition of IL6-induced STAT3 phosphorylation incubated for 20 mins followed by IL6 challenge for 30 mins in presence of whole blood, EC50 = 0.043 μM. 23659214
TF1 Function assay Inhibition of JAK1 in human TF1 cells assessed as suppression of IL6-stimulated STAT3 phosphorylation by AlphaScreen assay, INH = 0.045 μM. 26372653
CTLL Function assay Inhibition of Jak3-mediated IL2-induced Stat5 phosphorylation in mouse CTLL cells by cell-based assay, IC50 = 0.048 μM. 21155605
Sf9 Function assay 90 mins Inhibition of human recombinant N-terminal GST-tagged TYK2 expressed in Sf9 cells using Biotin-LC-EQEDEPEGDYFEWLE as substrate after 90 mins by TR-FRET assay, IC50 = 0.052 μM. 22087750
TF1 Function assay Inhibition of JAK1 in human TF1 cells assessed as inhibition of IL2-induced STAT3 phosphorylation, EC50 = 0.053 μM. 22591402
TF1 Function assay 20 mins Inhibition of JAK1 in human TF1 cells assessed as inhibition of IL6-induced STAT3 phosphorylation incubated for 20 mins prior to IL6-induction measured after 30 to 45 mins, EC50 = 0.053 μM. 22698084
TF1 Function assay 20 mins Inhibition of JAK1 in human TF1 cells assessed as inhibition of IL6-induced STAT3 phosphorylation incubated for 20 mins followed by IL6 challenge for 30 mins, EC50 = 0.053 μM. 23659214
Ba/F3 Function assay 60 mins Inhibition of TEL-fused JAK3 expressed in Ba/F3 cells assessed as inhibition of STAT5 phosphorylation after 60 mins by AlphaScreen assay, IC50 = 0.054 μM. 22087750
T-cells Function assay Inhibition of allogenic cells-stimulated proliferation in monkey T cells by mixed lymphocyte reaction method, IC50 = 0.057 μM. 14593182
CD34+ Function assay Inhibition of JAK2 in human CD34+ cells assessed as inhibition of EPO-mediated cell proliferation, IC50 = 0.071 μM. 26927423
TF1 Function assay Inhibition of IL4-induced proliferation of TF1 cells, IC50 = 0.08 μM. 16934457
T-cells Function assay Inhibition of allogenic cells-stimulated proliferation in human T cells by mixed lymphocyte reaction method, IC50 = 0.087 μM. 14593182
TF1 Function assay 20 mins Inhibition of JAK2 in human TF1 cells assessed as inhibition of EPO-induced STAT5 phosphorylation incubated for 20 mins prior to EPO-induction measured after 30 to 45 mins, EC50 = 0.093 μM. 22698084
TF1 Function assay 20 mins Inhibition of JAK2 in human TF1 cells assessed as inhibition of EPO-induced STAT5 phosphorylation incubated for 20 mins followed by IL6 challenge for 30 mins, EC50 = 0.093 μM. 23659214
PBMC Function assay Inhibition of JAK1 in human PBMC cells assessed as inhibition of IL-6-induced MCP1 secretion, IC50 = 0.095 μM. 26927423
TF1 Function assay 2 hrs Inhibition of JAK2 in GMCSF-stimulated human TF1 cells assessed as suppression of STAT5 phosphorylation preincubated for 2 hrs followed by GMCSF stimulation for 50 mins by FACS reader analysis, IC50 = 0.095 μM. 27130359
TF1 Function assay Inhibition of JAK2 in EPO-stimulated human TF1 cells expressing stably integrated beta-lactamase reporter gene under control of STAT5 response elements in interferon regulatory factor 1 gene promoter by fluorescence assay, IC50 = 0.107 μM. 29156136
Sf9 Function assay Inhibition of GST-tagged human JAK1 catalytic domain expressed in Sf9 cells by ELISA, IC50 = 0.11 μM. 21105711
T-cells Function assay Inhibition of allogenic cells-stimulated proliferation in mouse T cells by mixed lymphocyte reaction method, IC50 = 0.115 μM. 14593182
ME180 Function assay Inhibition of JAK1 in IL6-stimulated human ME180 cells expressing stably integrated beta-lactamase reporter gene under control of sis-inducible element by fluorescence assay, IC50 = 0.124 μM. 29156136
CTLL-2 Antiproliferative assay 72 hrs Antiproliferative activity against IL-2-stimulated mouse CTLL-2 cells expressing JAK1/JAK3 after 72 hrs by alamar blue assay, IC50 = 0.132 μM. 19762238
HT2 Function assay Inhibition of JAK3 in mouse HT2 cells assessed as suppression of cell growth, IC50 = 0.156 μM. 27771180
DND/L12 Function assay 30 mins Inhibition of JAK3 in human DND/L12 cells after 30 mins by luciferase assay in presence of human serum albumin, IC90 = 0.16 μM. 14593182
T-cells Function assay Inhibition of JAK2/1 in human T cells expressing CD3 assessed as inhibition of IFNgamma-stimulated STAT1 phosphorylation, IC50 = 0.17 μM. 23540648
insect cells Function assay Inhibition of GST-tagged Tyk2 expressed in insect cells using 35 uM ATP, IC50 = 0.176 μM. 21155605
Ba/F3 Function assay 60 mins Inhibition of TEL-fused JAK2 expressed in Ba/F3 cells assessed as inhibition of STAT5 phosphorylation after 60 mins by AlphaScreen assay, IC50 = 0.265 μM. 22087750
TF1 Function assay Inhibition of JAK2 in human TF1 cells assessed as suppression of cell growth, IC50 = 0.2751 μM. 27771180
CD34+ Function assay 45 mins Inhibition of JAK2 homodimer in human CD34+ cells spiked into human whole blood assessed as inhibition of EPO-induced STAT-5 phosphorylation preincubated for 45 mins followed by EPO addition measured after 15 mins by FACS analysis, IC50 = 0.302 μM. 24417533
A2780 Function assay Inhibition of cdk-mediated NPM phosphorylation at thr199 in human A2780 cells 14593182
A2780 Function assay Inhibition of cdk-mediated NPM phosphorylation at thr199 in human A2780 cells 21105711
A2780 Function assay Inhibition of cdk-mediated NPM phosphorylation at thr199 in human A2780 cells 19762238
A2780 Function assay Inhibition of cdk-mediated NPM phosphorylation at thr199 in human A2780 cells 30460842
A2780 Function assay Inhibition of cdk-mediated NPM phosphorylation at thr199 in human A2780 cells 23659214
A2780 Function assay Inhibition of cdk-mediated NPM phosphorylation at thr199 in human A2780 cells 16934457
A2780 Function assay Inhibition of cdk-mediated NPM phosphorylation at thr199 in human A2780 cells 26372653
A2780 Function assay Inhibition of cdk-mediated NPM phosphorylation at thr199 in human A2780 cells 22087750
A2780 Function assay Inhibition of cdk-mediated NPM phosphorylation at thr199 in human A2780 cells 30139575
A2780 Function assay Inhibition of cdk-mediated NPM phosphorylation at thr199 in human A2780 cells 27771180
YT Function assay 30 ng/ml Inhibition of IL2-induced JAK3 phosphorylation in human YT cells at 30 ng/ml by immunoblotting analysis 14593182
YT Function assay 30 ng/ml Inhibition of IL2-induced STAT5A phosphorylation in human YT cells at 30 ng/ml by immunoblotting analysis 14593182
YT Function assay 30 ng/ml Inhibition of IL2-induced STAT5B phosphorylation in human YT cells at 30 ng/ml by immunoblotting analysis 14593182
NK Immunosuppressive assay 1 to 5 mg/kg 4 weeks Immunosuppressive activity in naive cynomolgus monkey assessed as effect on CD3-CD16+ NK cells at 1 to 5 mg/kg, po for 4 weeks 14593182
T-cells Function assay 5 to 500 nM 1 hr Inhibition of IL2-induced Stat5 phosphorylation in human CD4+ T cells at 5 to 500 nM after 1 hr by Western blot 19053756
SUM149PT Function assay 3 uM 16 hrs Induction of PTPN6 in human SUM149PT cells assessed as inhibition of STAT3 phosphorylation at 3 uM after 16 hrs by Western blotting analysis in presence of pervanadate 24978112
Hs578T Function assay 3 uM 16 hrs Induction of PTPN6 in human Hs578T cells assessed as inhibition of STAT3 phosphorylation at 3 uM after 16 hrs by Western blotting analysis in presence of pervanadate 24978112
T-cells Function assay 50 to 300 nM 1 hr Inhibition of JAK1/JAK3 in human CD4 positive T cells assessed as inhibition of IL4-stimulated STAT6 phosphorylation at 50 to 300 nM preincubated for 1 hr followed by IL-4 stimulation measured after 30 mins by immunoblot analysis 29852068
T-cells Function assay 50 to 300 nM 1 hr Inhibition of JAK1/JAK3 in human CD4 positive T cells assessed as inhibition of IL2-stimulated STAT5 phosphorylation at Tyr-695 residue at 50 to 300 nM preincubated for 1 hr followed by IL-2 stimulation measured after 30 mins by immunoblot analysis 29852068
T-cells Function assay >300 nM 1 hr Inhibition of JAK1/JAK2/TYK2 in human CD4 positive T cells assessed as inhibition of IL6-stimulated STAT3 phosphorylation at >300 nM preincubated for 1 hr followed by IL-6 stimulation measured after 30 mins by immunoblot analysis 29852068
TALL-1 Function assay 1 uM 3 hrs Inhibition of JAK3 in human TALL-1 cells assessed as inhibition of IL-2 induced STAT5 phosphorylation at 1 uM preincubated for 3 hrs followed by IL-2 induction measured after 30 mins by immunoblotting 26258521
BA/F3 Function assay 300 nM 3 hrs Inhibition of JAK3 (unknown origin) expressed in mouse BA/F3 cells assessed as reduction of STAT5 phosphorylation at 300 nM after 3 hrs by immunoblotting analysis 26258521
OCL-AML5 Function assay 1 uM 3 hrs Inhibition of JAK2 in human OCL-AML5 cells assessed as inhibition of GM-CSF induced STAT5 phosphorylation at 1 uM preincubated for 3 hrs followed by GM-CSF induction measured after 30 mins by immunoblotting 26258521
BA/F3 Antiproliferative assay 72 hrs Antiproliferative activity against mouse BA/F3 cells expressing TEL-JAK3 after 72 hrs by cell titer glo assay 26258521
BA/F3 Function assay 1 uM 3 hrs Inhibition of JAK3 (unknown origin) expressed in mouse BA/F3 cells at 1 uM preincubated for 3 hrs followed by pulldown with streptavidin beads by immunoblotting analysis 26258521
Huh7 Function assay 10 uM 30 mins Inhibition of Tyk2 in human Huh7 cells assessed as reduction of IFNalpha5-induced STAT3 phosphorylation at 10 uM pre-incubated for 30 mins before IFNalpha5 stimulation for 30 mins mins by immunoblotting 26231159
Huh7 Function assay 10 uM 30 mins Inhibition of Tyk2 in human Huh7 cells assessed as reduction of basal level STAT3 phosphorylation at 10 uM after 30 mins by immunoblotting 26231159
Huh7 Function assay 1 uM 30 mins Inhibition of Tyk2 in human Huh7 cells assessed as reduction of IFNalpha5-induced STAT3 phosphorylation at 1 uM pre-incubated for 30 mins before IFNalpha5 stimulation for 30 mins mins by immunoblotting 26231159
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Informazioni chimiche, conservazione e stabilità

Peso molecolare 504.49 Formula

C16H20N6O.C6H8O7

Conservazione (Dalla data di ricezione)
N. CAS 540737-29-9 Scarica SDF Conservazione delle soluzioni stock

Sinonimi Tasocitinib Citrate,CP-690550 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

Solubilità

In vitro
Lotto:

DMSO : 100 mg/mL (198.21 mM)
(Il DMSO contaminato da umidità può ridurre la solubilità. Utilizzare DMSO fresco e anidro.)

Water : Insoluble

Ethanol : Insoluble

Calcolatore di Molarità

Massa Concentrazione Volume Peso molecolare
Calcolatore di Diluizione Calcolatore del Peso Molecolare

In vivo
Lotto:

Calcolatore di formulazione in vivo (Soluzione chiara)

Passo 1: Inserire le informazioni di seguito (Consigliato: Un animale aggiuntivo per tenere conto della perdita durante lesperimento)

mg/kg g μL

Passo 2: Inserire la formulazione in vivo (Questo è solo il calcolatore, non la formulazione. Contattateci prima se non cè una formulazione in vivo nella sezione Solubilità.)

% DMSO % % Tween 80 % ddH2O
%DMSO %

Risultati del calcolo:

Concentrazione di lavoro: mg/ml;

Metodo per preparare il liquido master di DMSO: mg farmaco predissolto in μL DMSO ( Concentrazione del liquido master mg/mL, Vi preghiamo di contattarci prima se la concentrazione supera la solubilità del DMSO del lotto del farmaco. )

Metodo per preparare la formulazione in vivo: Prendere μL DMSO liquido master, quindi aggiungereμL PEG300, mescolare e chiarire, quindi aggiungereμL Tween 80, mescolare e chiarire, quindi aggiungere μL ddH2O, mescolare e chiarire.

Metodo per preparare la formulazione in vivo: Prendere μL DMSO liquido master, quindi aggiungere μL Olio di mais, mescolare e chiarire.

Nota: 1. Si prega di assicurarsi che il liquido sia limpido prima di aggiungere il solvente successivo.
2. Assicurarsi di aggiungere il/i solvente/i in ordine. È necessario assicurarsi che la soluzione ottenuta, nellaggiunta precedente, sia una soluzione limpida prima di procedere allaggiunta del solvente successivo. Metodi fisici come il vortex, gli ultrasuoni o il bagno dacqua calda possono essere utilizzati per facilitare la dissoluzione.

Meccanismo dazione

Targets/IC50/Ki
JAK3
(Cell-free assay)
1 nM
JAK2
(Cell-free assay)
20 nM
In vitro
Il citrato di Tofacitinib inibisce la proliferazione dei blasti delle cellule T umane mediata da IL-2 e l'espressione di CD69 indotta da IL-15 con IC50 di 11 nM e 48 nM, rispettivamente. Il citrato di Tofacitinib previene la reazione linfocitaria mista con IC50 di 87 nM. Il trattamento di cellule murine Patersen-recettore dell'eritropoietina (FDCP-EpoR) dipendenti da fattori che ospitano JAK2 umano wild-type o V617F con citrato di Tofacitinib porta alla prevenzione della proliferazione cellulare con IC50 di 2,1 µM e 0,25 µM, rispettivamente. Il citrato di Tofacitinib inibisce la fosforilazione di STAT1 e STAT3 indotta dall'interleuchina-6 con IC50 di 23 nM e 77 nM, rispettivamente. Inoltre, il citrato di Tofacitinib genera un significativo effetto pro-apoptotico sulle cellule murine FDCP-EpoR che trasportano JAK2VV617F, mentre un effetto minore è osservato per le cellule che trasportano JAK2 wild-type. Questa attività è accoppiata con l'inibizione della fosforilazione degli effettori di segnalazione a valle chiave dipendenti da JAK2V617F trasduttore di segnale e attivatore di trascrizione (STAT)3, STAT5 e omologo dell'oncogene virale del timoma murino v-akt (AKT). Inoltre, il citrato di Tofacitinib previene l'espressione di CD69 indotta da IL-15 nelle cellule NK e CD8+ T umane e di scimmia cinomolga in vitro.
Saggio chinasico
Saggi enzimatici
I saggi chinasici JAK1, JAK2 e JAK3 utilizzano una proteina espressa in cellule SF9 infettate da baculovirus (una proteina di fusione di GST e il dominio catalitico dell'enzima JAK umano) purificata mediante cromatografia di affinità su glutatione-Sepharose. Il substrato per la reazione è acido poliglicina-tirosina [PGT (4:1)], rivestito su piastre Nunc Maxi Sorp a 100 μg/mL durante la notte a 37 °C. Le piastre vengono lavate tre volte e l'enzima JAK viene aggiunto ai pozzetti, che contenevano 100 μL di tampone chinasico (50 mM HEPES, pH 7,3, 125 mM NaCl, 24 mM MgCl2) + ATP + 1 mM di ortovanadato di sodio. Per CP-690550 (Tofacitinib) Citrate, viene anche aggiunto per il saggio chinasico a diverse dosi. Dopo incubazione a temperatura ambiente per 30 minuti, le piastre vengono lavate tre volte. Il livello di tirosina fosforilata in un dato pozzetto viene determinato mediante saggio ELISA standard utilizzando un anticorpo anti-fosfotirosina.
In vivo
Il citrato di Tofacitinib diminuisce una risposta di ipersensibilità di tipo ritardato e prolunga la sopravvivenza dell'allotrapianto cardiaco in modelli murini. Inoltre, il trattamento con citrato di Tofacitinib di progenitori eritroidi espansi ex vivo da pazienti PV positivi per JAK2V617F si traduce in un'attività antiproliferativa specifica (IC50 = 0,2 μM) e pro-apoptotica. Al contrario, i progenitori espansi da controlli sani sono meno sensibili al citrato di Tofacitinib nei saggi di proliferazione (IC50 > 1,0 μM) e apoptosi. Durante 2 settimane di somministrazione di citrato di Tofacitinib a 10 e 30 mg/kg/die, si osserva una diminuzione significativa e tempo-dipendente del numero di cellule NK rispetto al trattamento con veicolo. Il numero di cellule CD8+ della memoria effettore nel gruppo trattato con citrato di Tofacitinib è inferiore del 55% rispetto a quello osservato negli animali trattati con veicolo.
Riferimenti
  • [4] https://pubmed.ncbi.nlm.nih.gov/14593182/

Applicazioni

Metodi Biomarcatori Immagini PMID
Western blot JAK3 / p-JAK3 / STAT3 LMP1 / EBNA1 / BZLF1 JAK3 / STAT5 / p-STAT5
S5001-WB3
26082451
Growth inhibition assay Cell number
S5001-viability1
27732937

Informazioni sullo studio clinico

(dati da https://clinicaltrials.gov, aggiornato il 2024-05-22)

Numero NCT Reclutamento Condizioni Sponsor/Collaboratori Data di inizio Fasi
NCT06202560 Enrolling by invitation
Frontal Fibrosing Alopecia|Lichen Planopilaris
Institute of Dermatology Thailand
November 29 2023 Not Applicable
NCT05487703 Completed
Arthritis Rheumatoid
Pfizer
November 14 2022 --
NCT05082428 Completed
Ulcerative Colitis
Pfizer
May 30 2022 --
NCT05728008 Completed
Ulcerative Colitis
IRCCS San Raffaele
April 5 2022 --
NCT04768504 Recruiting
Immune-Mediated Colitis
Khashayar Esfahani|Sir Mortimer B. Davis - Jewish General Hospital
March 22 2022 Phase 2

Supporto tecnico

Istruzioni per la manipolazione

Tel: +1-832-582-8158 Ext:3

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Domande Frequenti

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

Risposta:
Tofacitinib (S2789) is the base form of this compound. The biological activity of these two compounds is the same. However, S5001 is better than S2789 for oral gavage.