Dasatinib hydrochloride

Cell experiment:

Ba/F3 cell lines are plated in triplicate and incubated with escalating concentrations of STI571, 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 (STI571 and AMN107) or 0 to 32 nM (Dasatinib). The STI571 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 experiment:

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:

[1]. O'Hare T, et al. In vitro activity of Bcr-Abl inhibitors AMN107 and BMS-354825 against clinically relevant STI571-resistant Abl kinase domain mutants. Cancer Res. 2005 Jun 1;65(11):4500-5.
[2]. Shah NP, et al. Dasatinib (BMS-354825) inhibits KITD816V, an STI571-resistant activating mutation that triggers neoplastic growth in most patients with systemic mastocytosis. Blood. 2006 Jul 1;108(1):286-91.
[3]. Chan CM, et al. Targeted inhibition of Src kinase with dasatinib blocks thyroid cancer growth and metastasis. Clin Cancer Res. 2012 Jul 1;18(13):3580-91.
[4]. Shen Z, et al. Metabolite profiling of dasatinib dosed to Wistar Han rats using automated dried blood spot collection. J Pharm Biomed Anal. 2012 Aug-Sep;67-68:92-7.