Quizartinib (AC220) is a second-generation type II FLT3 tyrosine kinase inhibitor that is orally active and highly selective, inhibiting the autophosphorylation of FLT3-WT and FLT3-ITD (IC50=4.1/1.1 nM). Quizartinib induces apoptosis in tumor cells.

FLT3:1.6 nM (Kd, cell free)

方法：人白血病细胞系 MV4-11 和人恶性黑色素瘤细胞系 A375 用 Quizartinib 处理 72 h，使用 Cell Titer-Blue Cell Viability Assay 检测细胞活力。
结果：Quizartinib 抑制 MV4-11 细胞生长，IC50 为 0.56  nM。Quizartinib 不影响 A375 细胞的增殖，IC50 大于 10000 nM。[1]
方法：AML 细胞系 MV4-11 用 Quizartinib (0.0625-8 nM) 处理 2 h，使用 Western Blot 检测靶点蛋白表达水平。
结果：Quizartinib 对 MV4-11 细胞中的 FLT3 信号通路具有浓度依赖性抑制活性。Quizartinib 有效抑制 FLT3 磷酸化 (IC50=0.50 nM)。Quizartinib 抑制下游分子 SHP-2、STAT5、MEK1/2、ERK1/2 和 AKT的磷酸化。[2]

方法：为检测体内抗肿瘤活性，将 Quizartinib (10 mg/kg，22% hydroxypropyl-β-cyclodextrin, CEP-701 was formulated in 20% gelucire 44/14 in water (vol/vol)) 口服给药给携带 MV4-11 异种移植物的 NU/NU 小鼠，每天一次，持续 28 天。
结果：用 10 mg/kg 的 Quizartinib 治疗导致所有动物的肿瘤快速完全消退，并且在治疗后 60 天的观察期内没有观察到肿瘤再生。[1]

KinomeScan kinase binding assays were performed as previously described. For the FLT3 assay, we used a kinase construct that spanned the catalytic domain only (amino acids 592 to 969 in NP_004110.2). This construct does not include the juxtamembrane domain and is designed to measure the intrinsic binding affinity of the open FLT3 active site for inhibitors [1].

MV4-11 and RS4;11 cells were cultured in Iscove media with 10% fetal bovine serum (FBS) and RPMI complete with 10% FBS, respectively. For proliferation assays, cells were cultured overnight in low serum media (0.5% FBS), then seeded in a 96-well plate at 40 000 cells per well. Inhibitors were added to the cells and incubated at 37°C for 72 hours. Cell viability was measured using the Cell Titer-Blue Cell Viability Assay. To measure inhibition of FLT3 autophosphorylation, cells were cultured in low serum media (0.5% FBS) overnight and seeded at a density of 400 000 cells per well in a 96-well plate the following day. The cells were incubated with inhibitors for 2 hours at 37°C. To induce FLT3 autophosphorylation in RS4;11 cells, 100 ng/mL FLT3 ligand was added for 15 minutes after the 2-hour compound incubation. Cell lysates were prepared and incubated in 96-well plates pre-coated with a total FLT3 capture antibody. The coated plates were incubated with either a biotinylated antibody against FLT3 to detect total FLT3 or an antibody against phosphotyrosines to detect FLT3 autophosphorylation. In both cases, a SULFO-tagged streptavidin secondary antibody was used for electrochemiluminescence detection on the Meso Scale Discovery platform [1].

The model was performed according to published procedures.20 For intravenous bone marrow engraftment, nonobese diabetic/severe combined immunodeficient mice were acclimated for 2 weeks before pretreatment with 150 mg/kg cyclophosphamide delivered intraperitoneally once a day for 2 days. After a 48-hour rest period, animals were given an intravenous injection of 5 × 10^6 MV4-11 cells into the tail vein. AC220 was formulated and delivered as described for pharmacokinetic studies [1].