Gilteritinib (ASP2215) is a FLT3 inhibitor (IC50=0.29 nM) and an AXL inhibitor (IC50=0.73 nM) with ATP-competitive, selective, and oral activity. Gilteritinib exhibits antitumor activity and can be used to treat FLT3 mutation-positive AML.

TYK1/LTK:0.35 nM, c-Kit:230 nM, FLT3:0.29 nM, Axl:0.73 nM, EML4-ALK:1.2 nM

方法：人急性髓细胞白血病细胞 MV4-11 和 MOLM-13 用 Gilteritinib (0.01-100 nM) 处理 5 天，使用 CellTiter-Glo 检测细胞增殖。
结果：Gilteritinib 抑制 MV4-11 和 MOLM-13 细胞的生长，平均 IC50 值分别为 0.92 nM 和 2.9 nM。[1]
方法：人急性髓细胞白血病细胞 Molm14 用 Gilteritinib (5-20 nM) 处理 1-24 h，使用 Western Blot 检测靶点蛋白表达水平。
结果：当与 Molm14 细胞孵育 1 h 时，Gilteritinib 有效地抑制了 FLT3 及其下游信号通路的活性。但是到 24  h，尽管 FLT3 持续受到抑制，但 ERK 信号传导反弹，如 pERK 上调。[2]

方法：为测试体内抗肿瘤活性，将 Gilteritinib (1-10 mg/kg) 口服给药给携带人急性髓细胞白血病肿瘤 MV4-11 的 Nude 小鼠，每天一次，持续二十八天。
结果：在 1 mg/kg/天 (63%)和 3 mg/kg/天 (80%) 剂量下观察到 MV4-11 肿瘤的显着生长抑制。并且在 6 mg/kg/天 (93%) 和 10 mg/kg/天 (100%) 剂量下观察到肿瘤几乎完全消退。[1]

The kinase inhibitory activity of Gilteritinib is tested against a panel of 78 tested kinases using ATP concentrations that are approximately equal to the Km value for each kinase in a TK-ELISA or off-chip mobility shift assay. Initially, two concentrations of Gilteritinib (1 nM and 5 nM) are tested to assess each compound's inhibitory effect on TK activity. Further studies are then conducted using a dose range of Gilteritinib to determine IC50 values for kinases in which activity is inhibited by >50% with 1 nM Gilteritinib as well as for c-KIT. TK-ELISA and MSA assays are used to conduct IC50 studies for FLT3, LTK, AXL, and c-KIT; the HTRF KinEASE-TK assay is performed to assess the IC50 value of echinoderm microtubule-associated protein-like 4-ALK (EML4-ALK)

Gilteritinib is dissolved in DMSO and stored, and then diluted with appropriate media before use.The effect of Gilteritinib on MV4-11 and MOLM-13 cells is assessed using the CellTiter-Glo Luminescent Cell Viability Assay. Subsequent studies are conducted to examine the effect of Gilteritinib and Quizartinib on Ba/F3 cells expressing either FLT3-ITD, FLT3-D835Y, FLT3-ITD-D835Y, FLT3-ITD-F691 L, or FLT3-ITD-F691I. MV4-11 and MOLM-13 cells are treated with DMSO or increasing concentrations of Gilteritinib (0.01, 0.1, 1, 10, and 100 nM) for 5 days, and cell viability is measured using CellTiter-Glo

MiceAntitumor activity is evaluated in nude mice transplanted with MV4-11 AML cells. The pharmacokinetics in xenografted mice is also investigated. MV4-11 xenografted-mice are treated with oral administration of Gilteritinib at 10 mg/kg for 4 days. Treatment of Gilteritinib for 28 days results in dose-dependent inhibition of MV4-11 tumor growth and induces complete tumor regression at more than 6 mg/kg