Saracatinib (AZD0530) is a small molecule inhibitor belonging to the Src family kinase inhibitors (IC50=2.7–11 nM), featuring high selectivity, cell permeability, and oral bioavailability, with anti-fibrotic, anti-inflammatory, and potential anti-tumor activities.

Lck:< 4 nM (cell free), EGFR (L858R):5 nM (cell free), Lyn:5 nM (cell free), EGFR (L861Q):4 nM (cell free), c-Src:2.7 nM (cell free), c-YES:4 nM (cell free)

Inhibition of tyrosine kinase activity was examined using an ELISA with recombinant catalytic domains of a panel of receptor and non‐receptor tyrosine kinases (in some cases only part of the catalytic domain was used). This method has been described previously. AZD0530 dose ranges varied depending on the activity versus the particular kinase tested, but were typically 0.001–10μM. Specificity assays against a panel of serine/threonine kinases were performed using a filter capture assay with 32P. Briefly, multidrop 384 plates containing 0.5μL AZD0530 or controls (DMSO alone or pH 3.0 buffer controls) were incubated with 15μL of enzyme plus peptide/protein substrate for 5min before the reaction was initiated by the addition of 10μL of 20mM Mg.ATP. For all enzymes the final concentration was approximated to the Michaelis constant (Km). Assays were carried out for 30min at room temperature before termination by the addition of 5μL orthophosphoric acid. After mixing, the well contents were harvested onto a P81 Unifilter plate, using orthophosphoric acid as the wash buffer. Microcal Origin software was used to interpolate IC50 values by nonlinear regression [1].

Cell proliferation was assessed using a colorimetric 5‐bromo‐2′‐deoxyuridine (BrdU) Cell Proliferation ELISA kit, as described previously. Briefly, cells were plated onto 96‐well plates (1.5×10^4 cells/well), the following day 0.039–20μM AZD0530 in DMSO (at a final concentration of 0.5%) was added and the cells were incubated for 24h. The cells were pulse-labeled with BrdU for 2h and fixed. Cellular DNA was then denatured with the provided solution and incubated with anti-BrdU peroxidase for 90min. Following three washes with phosphate‐buffered saline, tetramethylbenzidine substrate solution was added and the plates were incubated on a plate shaker for 10–30min until the positive control absorbance at 690nm was approximately 1.5 absorbance units [1].

Female athymic mice (nu/nu: Alpk) and rats (RH‐rnu/rnu) were housed and maintained as previously described. Src3T3 and human tumor lines (as indicated in Table 3) were inoculated subcutaneously in the left flank of animals. Tumor growth was monitored by bi‐dimensional caliper measurements twice weekly. The tumor volume was calculated by the following formula: (length×width)×√(length×width)×(π/6) and supported by excision and weighing of tumors at the end of the studies. Dosing started when the average tumor volume reached 0.2–0.5cm3 (except MDA‐MB‐231 and HT29). Animals were treated once daily by oral gavage with either vehicle alone or AZD0530 6.25–50mg/kg for 10–91 days. Tumor growth inhibition was calculated as described previously. For pharmacokinetic and pharmacodynamic analysis animals were humanely sacrificed and samples (plasma and tumor) were collected. Tumor samples were homogenized with 5 volumes of water and extracted with chloroform. Plasma and tumor samples were analyzed for AZD0530 concentration using high‐performance liquid chromatography with tandem mass spectrometric detection after solid‐phase extraction [1].

塞卡替尼, AZD0530

DMSO: 260 mg/mL (479.68 mM), Sonication is recommended.

Ethanol: 29 mg/mL (53.5 mM), Sonication is recommended.

10% DMSO+40% PEG300+5% Tween 80+45% Saline: 5 mg/mL (9.22 mM), Sonication is recommended.