生物活性

KX2-391 is a Src inhibitor that is directed to the Src substrate pocket. KX2-391 shows steep dose-response curves against various hepatic cell lines. KX2-391 is found to inhibit certain leukemia cells that are resistant to current commercially available chemicals.

KX2-391has been shown to potently inhibit Src-catalyzed trans-phosphorylation of focaladhesion kinase (FAK), Shc, paxillin, and Src kinase autophosphorylation IC50of approximately 20 nM.[1]

Cellviability of KX2-391

体外研究

体内研究

4% DMSO+30% PEG 300+ddH2O

激酶实验

细胞实验

Drugs[4]

KX-01 was initially dissolved in dimethyl sulfoxide(DMSO) and stored at –80 °C.

Celllines and cell culture

Human breast cancer cell lines (BT-474,BT-549, HCC1937, Hs578T, MCF7, MDA-MB-231, MDA-MB-468, SK-BR-3, and T47D) werecultured in RPMI-1640 medium supplemented with 10% fetal bovine serum and10μg/mL gentamicin at 37°C under 5% CO2.

Cellgrowth inhibitory assay

Cells were seeded in 96-well plates andincubated overnight at 37°C under 5% CO2. The cells were exposed to increasingconcentrations of KX-01 or paclitaxel for 3 days. After drug treatment, 50μL of3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolim bromide solution was addedto each well and the plates were incubated for 4 hours at 37°C. Afterdissolving the formazan crystals with 150μL of DMSO, the absorbance of eachwell was measured at 540 nm using a VersaMax microplate reader. The absorbanceand IC50 of KX-01 were analyzed using the SigmaPlot software. Six replicateswere included in each dose and at least three independent experiments wereconducted.

Woundhealing assay

Cells were seeded in 6-well plates andincubated overnight at 37°C under 5% CO2. The cell monolayer wasthen scratched with a pipette tip and the cells were incubated with mediumalone or containing 20 nmol/L KX-01. After 48 hours, the plates were examinedby light microscopy to monitor restoration of the cell monolayer. Thepercentage of the filled gap was analyzed using the SigmaPlot software.

动物实验

Tumor xenograft studies and immunohistochemistry[5]

Breeder pairs of NOD SCID gamma mice were werehoused in sterile cages and maintained in pathogen-free aseptic rooms, whilebeing fed autoclaved food pellets and water ad libitum. Xenograft tumors were establishedby injection of 1 × 106MDA-B6 or MDA-pc3 in 100μL PBS into themammary fat pads of 4–6 week old female animals. Treatments with KX2-391 wereinitiated when tumors became palpable. Mice were fed with either KX2-391 (5mg/kg) in DMSO/water solvent or a matching volume of the solvent. Treatmentswere administered orally twice a day. Digital caliper measurements were takenevery 3 days and tumor volume was calculated by the formula A/2*B2(where A and B were the long and short diameters of the tumor respectively). Atthe end of the experiments animals were sacrificed and tumors were removed.Tumors were fixed in 10% buffered formalin for paraffin embedding.

For the immunohistochemical staining,tumors were dissected and fixed in 10% neutral- buffered formalin for 24–48 h.The tumors were paraffin embedded, sectioned to 4μm thickness, and affixed onthe slide. Simultaneous dewaxing and antigen retrieval was performed on the DakoPT Link using Target Retrieval Solution-High pH. Staining was performed on theDako Autostainer Link using anti-CD34 antibody and the Dako FLEX DAB+DetectionKit. In each stained tumor section, 12, 3, 6 and 9 o’clock fields were imagedat 100 × magnification and the density of stained blood vessels per field was quantifiedusing the Image-Pro Premier software.

不同实验动物依据体表面积的等效剂量转换表（数据来源于FDA指南）

例如，已知某工具药用于小鼠的剂量为88 mg/kg , 则用于大鼠的剂量换算方法：将88 mg/kg 乘以小鼠的Km系数（3），再除以大鼠的Km系数（6），得到该药物用于大鼠的等效剂量44 mg/kg。

参考文献

[1] Naing A, Cohen R, Dy GK, et al. A phase I trial of KX2-391, a novel non-ATP competitive substrate-pocket- directed SRC inhibitor, in patients with advanced malignancies. Invest New Drugs. 2013;31(4):967-973.
[2] Lau GM, Lau GM, Yu GL, et al. Expression of Src and FAK in hepatocellular carcinoma and the effect of Src inhibitors on hepatocellular carcinoma in vitro. Dig Dis Sci. 2009;54(7):1465-1474.
[more]

体内溶解度
5mg/mL

Clinical Trial Information ( data from http://clinicaltrials.gov )

注：以上所有数据均来自公开文献，并不保证对所有实验均有效，数据仅供参考。

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