Crenolanib (ARO 002) is an orally bioavailable type III tyrosine kinases inhibitor of PDGFRα/β and FLT3 (IC50s: 11, 3.2, and 4 nM).

PDGFRβ:2.1 nmol/L (Kd), PDGFRα:3.2 nmol/L (Kd), FLT3:0.74 nmol/L (Kd)

Crenolanib是一种特异性和强效的RTK抑制剂。对于野生型受体PDGFRA、PDGFRB和FLT3，Crenolanib的Kd分别为3.2、2.1和0.74 nmol/L。在EOL-1细胞系中，Crenolanib强效抑制融合癌基因的激酶活性（IC50值为21 nmol/L），并且显著抑制EOL-1细胞的增殖（IC50: 0.2 pmol/L）[1]。Crenolanib是ABCB1的底物，这一点由ABCB1过表达细胞对Crenolanib的大约五倍的抗性、ABCB1特异性抑制剂PSC-833对这种抗性的逆转以及Crenolanib对ABCB1 ATP酶活性的刺激所证明。相反，Crenolanib不是ABCG2或ABCC1的底物。此外，用药理学相关浓度的500 nM Crenolanib处理FLT3-ITD AML细胞系MV4-11和MOLM-14，并未诱导ABCB1细胞表面表达的上调[2]。Crenolanib治疗消除了HB119细胞以及源于AML患者的FLT3-ITD+细胞系Molm14中FLT3和ERK的磷酸化。50 nmol/L的Crenolanib抑制了包括一位Quizartinib耐药患者（其疾病出现了FLT3-ITD/D835Y突变）的白血病细胞中FLT3的磷酸化[3]。

Crenolanib 显著抑制了肿瘤质量的增长，且在20 mg/kg的治疗剂量下观察到最强的抑制效果。Crenolanib 引发了肿瘤细胞大量的凋亡。此外，应用的 Crenolanib 剂量被接受鼠良好地耐受，治疗期间没有观察到体重损失[4]。来自正在进行的临床试验的相关数据显示，急性髓细胞性白血病患者能够达到足够的 Crenolanib 水平，以体内抑制 FLT3/ITD 及抵抗性赋予的 FLT3/D835 突变体[5]。

Chinese hamster ovary (CHO) cells were transiently transfected with mutated KIT or PDGFRA cDNA constructs and treated with various concentrations of imatinib or crenolanib as previously described. Experiments involving recombinant DNA were carried out using biosafety level 2 conditions in accordance with published guidelines. Protein lysates from cell lines were prepared and subjected to immunoprecipitation using anti-KIT or anti-PDGFRA antibodies followed by sequential immunoblotting for phospho-KIT and total KIT, or phosphotyrosine or total PDGFRA, respectively, as previously reported. Densitometry was carried out to quantify drug effect using Photoshop 5.1 software, with the level of phospho-KIT or phospho-PDGFRA normalized to total protein. Densitometry and proliferation experimental results were analyzed using Calcusyn 2.1 software to mathematically determine the IC50 values. The Wilcoxon rank sum test was used to compare the IC50 values of imatinib and crenolanib for a given mutation [1].

Cells were added to 96-well plates at densities of 20,000 cells per well and incubated with imatinib or crenolanib for 72 hours before measuring cellular proliferation using a 2,3-bis[2-methoxyl-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide (XTT)–based assay [1].

A549 cells were injected into the axillary regions of mice (2×10^6 cells/mouse). When the tumor volumes reached 70 mm^3, the mice were randomly allocated to the control group, low-dose crenolanib group (10 mg/kg), or high-dose crenolanib group (20 mg/kg) (n=6 per group). The vehicle for crenolanib treatment consists of 10% 1-methyl-2-pyrrolidinone and 90% polyethylene glycol 300. The tumor size and mouse body weight were measured every other day for about 2 weeks. The tumor volume was calculated as follows: (mm^3) = (width × width × length)/2. After treatment, the mice were euthanized using carbon dioxide, and the tumors were harvested and analyzed [4].