Filanesib (ARRY 520) is a selective inhibitor of kinesin spindle protein (KSP, IC50 = 6 nM) with potent anti-proliferative activity.

KSP:6 nM

Filanesib在多种耐药细胞系中展示了抗增殖活性，包括HCT-15、NCI/ADR-RES和K562/ADR，其EC50值分别为3.7、14和4.2 nM。Filanesib（10 nM）通过增加组蛋白H3的磷酸化和四个细胞中cyclin B1的积累，阻断有丝分裂并形成典型的KSP抑制效果所表现的单极纺锤体结构[2]。Filanesib（1 nM）在24小时内显著诱导OCI-AML3细胞发生G2M期细胞周期阻滞[4]。

Filanesib (10, 15, 20, 30 mg/kg, i.p.) 在UISO-BCA-1异种移植物中表现出活性，且在携带皮下HT-29, HCT-116, MDA-MB-231和A2780异种移植物的小鼠中优于紫杉醇。Filanesib在雄激素受体阴性前列腺癌异种移植模型PC-3中优于多西他赛 [1]。RPMI 8226肿瘤异种移植物对低剂量的Filanesib (12.5 mg/kg, i.p.) 特别敏感 [2]。在SCID小鼠的HL60和MV4-11异种移植物中，Filanesib分别以27 mg/kg和20 mg/kg的浓度显著抑制肿瘤生长 [4]。

Exponentially growing cells (0.4×10^6/mL) are treated with ARRY-520 for up to 48 hours. For combination, HL-60 and HL-60Bcl-2 cells (0.4×10^6/mL) are incubated with ARRY-520, ABT-737, or both for up to 96 hours. DMSO is used as the control agent. Apoptosis is estimated by flow cytometry measurements of phosphatidylserine with the Annexin-V-FLUOS Staining Kit. Membrane integrity is simultaneously assessed by 7-amino-actinomycin D (7-AAD). To measure changes in the mitochondrial membrane potential (MMP), cells are loaded with CMXRos (300 nM) and MitoTracker Green (500 nM) for 1 hour at 37°C. The loss of MMP is then assessed by measuring CMXRos retention while simultaneously adjusting for mitochondrial mass [4].

Subcutaneous tumor xenografts are allowed to grow to a volume of 250-350 mm3. The mice are randomized into groups of 3-4 based on tumor size and are given a single dose of ARRY-520 i.p. At various time-points after administration of the drug, the mice are euthanized by CO2 inhalation and the tumors excised and placed in 10% neutral buffered formalin. The formalin-fixed tumors are processed and paraffin-embedded by standard procedures. Spindle morphology is analyzed by staining tumor sections for α-tubulin, and apoptosis is analyzed by TUNEL stain. Monopolar/abnormal spindles and TUNEL positive (apoptotic) cells are counted in three ×40 fields from each sample, analyzed using algorithms developed in software [1].