[Description]:
Dasatinib (BMS-354825) is a dual Bcr-Abl and Src family tyrosine kinase inhibitor with IC50s of 0.6, 0.8, 79 and 37 nM for Abl, Src, c-Kit and c-KitD816V, respectively.
[Related Catalog]:
[Target]
IC50: 0.6 nM/0.8 nM (AblWT/Src)[1] IC50: 79 nM/37 nM (c-KitWT/c-KitD816V)[2]
[In Vitro]
Dasatinib potently inhibits wild-type Abl kinase and all mutants except T315I over a narrow range (IC50≤1.7 nM). Dasatinib (IC50: 0.8 nM) displays 325-fold greater potency compared with Imatinib against cells expressing wild-type Bcr-Abl in Ba/F3 cells[1].
[In Vivo]
Daily treatment with Dasatinib (50 mg/kg) is initiated on day 10. Using this approach, a significant inhibition of BCPAP orthotopic tumor growth is observed 6 days after treatment (day 16, P=0.014), which is sustained through days 23 and 29 (P=0.0003), compared with vehicle-treated mice[3]. Metabolism studies of Dasatinib (50 mg/kg) in rat suggested that Dasatinib is the major circulating component, whereas multiple metabolites contributed to the remaining 40-60% of the sample radioactivity at 4 h post dose[4].
[Kinase Assay]
Kinase assays using wild-type and mutant glutathione S-transferase (GST)-Abl fusion proteins (c-Abl amino acids 220-498) are done with minor alterations. GST-Abl fusion proteins are released from glutathione-Sepharose beads before use; the concentration of ATP is 5 μM. Immediately before use in kinase autophosphorylation and in vitro peptide substrate phosphorylation assays, GST-Abl kinase domain fusion proteins are treated with LAR tyrosine phosphatase. After 1-hour incubation at 30°C, LAR phosphatase is inactivated by addition of sodium vanadate (1 mM). Immunoblot analysis comparing untreated GST-Abl kinase to dephosphorylated GST-Abl kinase is routinely done 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. The inhibitor concentration ranges for IC50 determinations are 0 to 5,000 nM (Imatinib and AMN107) or 0 to 32 nM (Dasatinib). The Dasatinib concentration range is extended to 1,000 nM for mutant T315I. These same inhibitor concentrations are used for the in vitro peptide substrate phosphorylation assays. The three inhibitors are tested over these same concentration ranges against GST-Src kinase and GST-Lyn kinase.
[Cell Assay]
Ba/F3 cell lines are plated in triplicate and incubated with escalating concentrations of Imatinib, AMN107, or Dasatinib for 72 hours. Proliferation is measured using a methanethiosulfonate-based viability assay (CellTiter96 Aqueous One Solution Reagent). IC50 and IC90 values are reported as the mean of three independent experiments done in quadruplicate. The inhibitor concentration ranges for IC50 and IC90determinations are 0 to 2,000 nM (Imatinib and AMN107) or 0 to 32 nM (Dasatinib). The imatinib concentration range is extended to 6,400 nM for mutants with IC50>2,000 nM. The Dasatinib concentration range is extended to 200 nM for mutant T315I.
[Animal admin]
Mice[3] Male athymic nude mice (25 grams; 5-week old) are used. Dasatinib (50 mg/kg) is prepared for daily oral gavage (5 d/wk) in 80 mM sodium citrate buffer, pH 3.0. For the orthotopic murine model, mice are randomized on day 10 based on bioluminescence activity to receive drug or vehicle. In the metastatic murine model, mice receives dasatinib or vehicle, as described earlier, starting 2 days before intracardiac injection (pretreatment), or on day 11 following randomization (posttreatment). Rats[4] Dasatinib is dosed to male Wistar-Han (WH) rats at 50 mg/kg orally in 0.5% methylcellulose. The automated rat blood collection device is programmed to collect 200 μL of blood at predetermined intervals. At each time point, the accusampler is programmed to directly spot 20 μL of blood twice (two spots) onto the DBS card. The remaining 160 μL of liquid blood is collected into sodium EDTA-containing tubes. Plasma samples are obtained after immediate centrifugation of blood at 11,000 rpm for 5 min. The plasma samples are stored at −80°C until analyses. The DBS samples are dried under room temperature for a minimum of 2 h and stored in a plastic bag in the dessicator until sample analysis.
[References]
[Related Small Molecules]