KSP (EC50 = 6 nM)

Filanesib（ARRY520；ARRY-520）在多重耐药细胞系中保留活性。 Filanesib (ARRY-520) 抑制 HCT-15、NCI/ADR-RES 和 K562/ADR 细胞增殖的 EC50 分别为 3.7、14 和 4.2 nM。 Filanesib (ARRY-520) (10 nM) 通过 KSP 抑制的典型单极纺锤体结构阻断大多数细胞的有丝分裂[1]。 Filanesib (ARRY-520) (10 nM) 诱导有丝分裂停滞，根据四个细胞中组蛋白 H3 (pHH3) 磷酸化的增加和细胞周期蛋白 B1 的积累来判断[2]。 Filanesib (ARRY-520) 和紫杉醇对 I 型和 II 型细胞表现出相同的细胞毒性作用。对于 ARRY-520，II 型 EOC 细胞在 48 小时时的 GI50 为 0.0015 μM。对于 I 型 EOC 细胞，ARRY-520 在 48 小时时的 GI50 > 3 μM[3]。 Filanesib (ARRY-520) (1 nM) 在 24 小时时可诱导 OCI-AML3 细胞显着的 G2M 细胞周期阻滞[4]。

Filanesib (ARRY520; ARRY-520) (10, 15, 20, 30 mg/kg, ip) 在 UISO-BCA-1 异种移植物中具有活性，并且在皮下注射 HT-29、HCT-116、MDA 的小鼠中也优于紫杉醇-MB-231和A2780异种移植物。 ARRY-520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，在DU145前列腺异种移植模型中也优于多西他赛[1]。 RPMI 8226 肿瘤异种移植物对低剂量 ARRY-520（12.5 mg/kg，腹腔注射）特别敏感[2]。 ARRY-520 浓度分别为 27 mg/kg 和 20 mg/kg 时，可显着抑制 SCID 小鼠 HL60 和 MV4-11 异种移植物中的肿瘤生长[4]。

使用 Filanesib (ARRY-520) 处理指数生长的细胞 (0.4×106/mL) 长达 48 小时。对于组合，将 HL-60 和 HL-60Bcl-2 细胞 (0.4×106/mL) 与 Filanesib (ARRY-520)、ABT-737 或两者一起孵育长达 96 小时。 DMSO用作控制剂。使用膜联蛋白-V-FLUOS 染色试剂盒通过流式细胞术测量磷脂酰丝氨酸来估计细胞凋亡。膜完整性同时通过 7-氨基放线菌素 D (7-AAD) 进行评估。为了测量线粒体膜电位 (MMP) 的变化，将 CMXRos (300 nM) 和 MitoTracker Green (500 nM) 加载到细胞中，在 37°C 下保持 1 小时。然后通过测量 CMXRos 保留来评估 MMP 的损失，同时调整线粒体质量。

Tumor xenografts placed subcutaneously are allowed to expand to a volume of 250–350 mm3. Depending on the size of their tumors, the mice are randomly assigned to groups of three to four and given a single intraperitoneal dose of filanesib (ARRY-520). The mice are put to sleep by CO2 inhalation at different intervals following the drug's administration, and the tumors are removed and put in 10% neutral buffered formalin. The tumors that have been fixed in formalin are processed and paraffin embedded using standard protocols. Tumor sections stained with α-tubulin are used to analyze spindle morphology, and TUNEL stain is used to analyze apoptosis. Algorithms developed in ImagePro software are used to analyze the count of TUNEL positive (apoptotic) cells and monopolar/abnormal spindles in three ×40 fields from each sample.

[1]. Front Pharmacol . 2018 Oct 9:9:1131.

[2]. PLoS One . 2017 Sep 19;12(9):e0184888.

Filanesib is a potent Kinesin Spindle Protein (KSP) inhibitor that caused marked tumor regression in preclinical models of human solid tumors and human leukemias, often leading to durable responses.
Filanesib is a synthetic, small molecule targeting the kinesin spindle protein (KSP) with potential antineoplastic activity. Filanesib specifically inhibits KSP (kinesin-5 or Eg5), resulting in activation of the spindle assembly checkpoint, induction of cell cycle arrest during the mitotic phase, and consequently cell death in tumor cells that are actively dividing. Because KSP is not involved in postmitotic processes, such as neuronal transport, this agent does not cause the peripheral neuropathy that is often associated with tubulin-targeting agents. KSP is an ATP-dependent microtubule motor protein that is essential for the formation of bipolar spindles and the proper segregation of sister chromatids during mitosis.

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Drug Indication
Investigated for use/treatment in cancer/tumors (unspecified).

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Mechanism of Action
KSP has been identified as an attractive drug target against cancer. Cancer results when normal cellular processes go awry and lead to a massive, uncontrolled cell division, proliferation, and growth. Inhibitors of KSP cause mitotic arrest by preventing the formation of bipolar spindle. The monopolar spindle thus prevents the separation of centrosomes, organizes the microtubules from a single locus in the cell, and aligns the chromosomes around this locus. This compound is a highly potent KSP inhibitor that demonstrates subnanomolar potency in both enzymatic and cellular assays and causes mitotic arrest, leading to cell death or apoptosis of the immensely proliferating cancer cells.

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Pharmacodynamics
This compound is a highly potent KSP inhibitor that demonstrates subnanomolar potency in both enzymatic and cellular assays and causes mitotic arrest, leading to cell death or apoptosis of the immensely proliferating cancer cells.

C20H22N4O2F2S

420.47608

420.143

C, 57.13; H, 5.27; F, 9.04; N, 13.32; O, 7.61; S, 7.6

885060-09-3

(R)-Filanesib;885060-08-2;Filanesib TFA;1781834-99-8

44224257

white solid powder

1.3±0.1 g/cm3

511.3±60.0 °C at 760 mmHg

263.0±32.9 °C

0.0±1.3 mmHg at 25°C

1.604

3.27

96.46

1

7

6

29

605

1

C([C@]1(SC(C2C=C(F)C=CC=2F)=NN1C(=O)N(C)OC)C1C=CC=CC=1)CCN

LLXISKGBWFTGEI-FQEVSTJZSA-N

InChI=1S/C20H22F2N4O2S/c1-25(28-2)19(27)26-20(11-6-12-23,14-7-4-3-5-8-14)29-18(24-26)16-13-15(21)9-10-17(16)22/h3-5,7-10,13H,6,11-12,23H2,1-2H3/t20-/m0/s1

(2S)-2-(3-aminopropyl)-5-(2,5-difluorophenyl)-N-methoxy-N-methyl-2-phenyl-1,3,4-thiadiazole-3-carboxamide

Filanesib; ARRY520; ARRY 520; ARRY-520

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: ~76 mg/mL (~200.8 mM)
Ethanol: ~10 mg/mL (~26.4 mM)

配方 1 中的溶解度: ≥ 2.5 mg/mL (5.95 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 25.0 mg/mL澄清DMSO储备液加入到400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 2 中的溶解度: ≥ 2.5 mg/mL (5.95 mM) (饱和度未知) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 mg/mL澄清DMSO储备液加入900 μL 20% SBE-β-CD生理盐水溶液中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 3 中的溶解度: ≥ 2.5 mg/mL (5.95 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 mg/mL 澄清 DMSO 储备液添加到 900 μL 玉米油中并混合均匀。

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配方 4 中的溶解度: 5%DMSO+40%PEG300+5%Tween80+50%ddH2O: 3.8mg/ml

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。