Bcr-Abl (IC50 = 1 nM); Src (IC50 = 0.5 nM); lck (IC50 = 0.4 nM); Yes (IC50 = 0.5 nM); c-kit (IC50 = 5 nM); PDGFRβ (IC50 = 28 nM); p38 (IC50 = 100 nM); Her1 (IC50 = 180 nM); Her2 (IC50 = 710 nM); FGFR-1 (IC50 = 880 nM); MEK (IC50 = 1700 nM)

Dasatinib in LMP2A/MYC double transgenic mice reverses splenomegaly. Dasatinib specifically inhibits the growth of bone marrow B cells that express LMP2A and reduces the size of the spleen in TgE mice. When comparing the Tg6/λ-MYC mice treated with dasatinib to the control group, there is a significant decrease in spleen mass. Dasatinib prevents LMP2A/MYC double transgenic mice from developing lymphadenopathy. In Rag1KO mice engrafted with tumor cells from LMP2A/MYC double transgenic mice, dasatinib reverses splenomegaly. Treatment with dasatinib inhibits Lyn phosphorylation in tumors of B lymphocytes that express LMP2A.[3]

Kinase assays are conducted with both wild-type and mutant glutathione S-transferase (GST)-Abl fusion proteins (c-Abl amino acids 220-498). Before usage, the GST-Abl fusion proteins are liberated from glutathione-Sepharose beads; 5 μM of ATP is present. The GST-Abl kinase domain fusion proteins are treated with LAR tyrosine phosphatase right before being used in kinase autophosphorylation and in vitro peptide substrate phosphorylation assays. Sodium vanadate (1 mM) is added to inactivate LAR phosphatase following a 1-hour incubation period at 30°C. Using phosphotyrosine-specific antibody 4G10 to confirm complete (>95%) dephosphorylation of tyrosine residues and c-Abl antibody CST 2862 to confirm equal loading of GST-Abl kinase, immunoblot analysis comparing untreated GST-Abl kinase to dephosphorylated GST-Abl kinase is routinely performed. The range of dasatinib concentrations for mutant T315I is increased to 1,000 nM. The in vitro peptide substrate phosphorylation assays employ the same concentrations of inhibitor. The three inhibitors are examined against GST-Src kinase and GST-Lyn kinase within these same concentration ranges.

Triple-seeded Ba/F3 cell lines are cultured for 72 hours at increasing dasatinib concentrations. A viability assay based on methanethiosulfonate is used to quantify proliferation. IC50 and IC90 values are given as the average of three separate, quadruple-experimented runs. The ranges of inhibitor concentrations (Dasatinib) are 0 nM to 32 nM. For mutant T315I, the dasatinib concentration range is expanded to 200 nM.

For in vivo experiments, dasatinib was dissolved in DMSO at 60 mg/ml and stored in aliquots at −20°C. On each treatment day, aliquots were thawed and diluted with 5.1% polyethylene glycol (PEG-400; EMD, Fisher) and 5.1% Tween-λ80 (Fisher) immediately before use, as previously described[3]
Wild-type (6–16 weeks old), TgE (6–10 weeks old), λ-MYC (16–20 weeks old), and Tg6/λ-MYC (5–10 weeks old, in a given experiment, age difference of mice was less than two weeks) mice were treated with dasatinib (30 mg/kg intraperitoneally) or equivalent amount of vehicle alone once daily for 14 days. On day 15, the mice were sacrificed, and lymph node tumors and spleens were harvested, documented, processed, and analyzed with flow cytometry or western blotting.[3]

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Dissolved in DMSO; 30 mg/kg; i.p. administration

EμLMP2A (TgE and Tg6 strains), MYC (λ-MYC), and LMP2A/λ-MYC double transgenic mice (Tg6/λ-MYC)

[1]. Cancer Res . 2005 Jun 1;65(11):4500-5.

[2]. Blood . 2006 Jul 1;108(1):286-91.

[3]. Antiviral Res . 2012 Jul;95(1):49-56.

Imatinib, a Bcr-Abl tyrosine kinase inhibitor, is a highly effective therapy for patients with chronic myelogenous leukemia (CML). Despite durable responses in most chronic phase patients, relapses have been observed and are much more prevalent in patients with advanced disease. The most common mechanism of acquired imatinib resistance has been traced to Bcr-Abl kinase domain mutations with decreased imatinib sensitivity. Thus, alternate Bcr-Abl kinase inhibitors that have activity against imatinib-resistant mutants would be useful for patients who relapse on imatinib therapy. Two such Bcr-Abl inhibitors are currently being evaluated in clinical trials: the improved potency, selective Abl inhibitor AMN107 and the highly potent dual Src/Abl inhibitor BMS-354825. In the current article, we compared imatinib, AMN107, and BMS-354825 in cellular and biochemical assays against a panel of 16 kinase domain mutants representing >90% of clinical isolates. We report that AMN107 and BMS-354825 are 20-fold and 325-fold more potent than imatinib against cells expressing wild-type Bcr-Abl and that similar improvements are maintained for all imatinib-resistant mutants tested, with the exception of T315I. Thus, both inhibitors hold promise for treating imatinib-refractory CML.[1]

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Mastocytosis is associated with an activating mutation in the KIT oncoprotein (KITD816V) that results in autophosphorylation of the KIT receptor in a ligand-independent manner. This mutation is inherently resistant to imatinib and, to date, there remains no effective curative therapy for systemic mastocytosis associated with KITD816V. Dasatinib (BMS-354825) is a novel orally bioavailable SRC/ABL inhibitor that has activity against multiple imatinib-resistant BCR-ABL isoforms in vitro that is presently showing considerable promise in early-phase clinical trials of chronic myeloid leukemia (CML). Pharmacokinetic analysis suggests that high nanomolar concentrations of dasatinib can be achieved safely in humans. In this study, we demonstrate significant inhibitory activity of dasatinib against both wild-type KIT and the KITD816V mutation in the nanomolar range in in vitro and cell-based kinase assays. Additionally, dasatinib leads to growth inhibition of a KITD816V-harboring human masto-cytosis cell line. Significantly, dasatinib selectively kills primary neoplastic bone marrow mast cells from patients with systemic mastocytosis while sparing other hematopoietic cells. Computer modeling suggests that the KITD816V mutation destabilizes the inactive conformation of the KIT activation loop to which imatinib binds, but it is not predicted to impair binding of KIT by dasatinib. Based upon our results, further evaluation of dasatinib for the treatment of systemic masto-cytosis in clinical trials is warranted. Moreover, dasatinib may be of clinical utility in other disease settings driven by activating KIT mutations.[2]

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Epstein-Barr virus (EBV) infection and latency has been associated with malignant diseases including nasopharyngeal carcinoma, Hodgkin lymphoma, Burkitt lymphoma, and immune deficiency associated lymphoproliferative diseases. EBV-encoded latent membrane protein 2A (LMP2A) recruits Lyn and Syk kinases via its SH2-domain binding motifs, and modifies their signaling pathways. LMP2A transgenic mice develop hyperproliferative bone marrow B cells and immature peripheral B cells through modulation of Lyn kinase signaling. LMP2A/λ-MYC double transgenic mice develop splenomegaly and cervical lymphomas starting at 8 weeks of age. We reasoned that targeting Lyn in LMP2A-expressing B cells with dasatinib would provide a therapeutic option for EBV-associated malignancies. Here, we show that dasatinib inhibits B cell colony formation by LMP2A transgenic bone marrow cells, and reverses splenomegaly and tumor growth in both a pre-tumor and a syngeneic tumor transfer model of EBV-associated Burkitt lymphoma. Our data support the idea that dasatinib may prove to be an effective therapeutic molecule for the treatment of EBV-associated malignancies.[3]

C22H26CLN7O2S.HCL

524.47

523.132

854001-07-3

Dasatinib;302962-49-8;Dasatinib monohydrate;863127-77-9;Dasatinib-d8;1132093-70-9

11466607

White to off-white solid powder

4.264

4

9

7

34

642

0

Cl.O=C(C1=CN=C(NC2C=C(N3CCN(CCO)CC3)N=C(C)N=2)S1)NC1C(C)=CC=CC=1Cl

MSCGWICDJYLQOJ-UHFFFAOYSA-N

InChI=1S/C22H26ClN7O2S.ClH/c1-14-4-3-5-16(23)20(14)28-21(32)17-13-24-22(33-17)27-18-12-19(26-15(2)25-18)30-8-6-29(7-9-30)10-11-31;/h3-5,12-13,31H,6-11H2,1-2H3,(H,28,32)(H,24,25,26,27);1H

N-(2-chloro-6-methylphenyl)-2-[[6-[4-(2-hydroxyethyl)piperazin-1-yl]-2-methylpyrimidin-4-yl]amino]-1,3-thiazole-5-carboxamide;hydrochloride

BMS-354825 HCl; BMS354825; 854001-07-3; Dasatinib (hydrochloride); Dasatinib HCl; N-(2-chloro-6-methylphenyl)-2-[[6-[4-(2-hydroxyethyl)piperazin-1-yl]-2-methylpyrimidin-4-yl]amino]-1,3-thiazole-5-carboxamide;hydrochloride; N-(2-Chloro-6-methylphenyl)-2-((6-(4-(2-hydroxyethyl)piperazin-1-yl)-2-methylpyrimidin-4-yl)amino)thiazole-5-carboxamide hydrochloride; BMS 354825 hydrochloride; SCHEMBL1705152; BMS354825. Dasatinib HCl; Trade name: Sprycel

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 3.33 mg/mL (6.35 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 33.3 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 3.33 mg/mL (6.35 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 33.3 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 3.33 mg/mL (6.35 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 33.3 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 4% DMSO+30% PEG 300+5% Tween 80+ddH2O: 5 mg/mL

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 (Please use freshly prepared in vivo formulations for optimal results.)