TAK-901

Multitargeted inhibitor with potent activity against Aurora B kinase (IC₅₀ = 1.8 nM, recombinant kinase assay) and moderate activity against VEGFR2 (IC₅₀ = 45 nM) and PDGFRβ (IC₅₀ = 62 nM). It exhibited minimal inhibitory activity against Aurora A (IC₅₀ > 1000 nM) and CDK1 (IC₅₀ > 500 nM), confirming selectivity for Aurora B over other cell cycle-related kinases [1]
- In cellular assays, inhibition of Aurora B-mediated histone H3 (Ser10) phosphorylation showed an EC₅₀ = 5.2 nM in HCT116 colorectal cancer cells [1]

TAK-901 降低细胞组蛋白 H3 磷酸化，并根据 Aurora B 抑制引起多倍体。它还显示出对 Aurora B 的时间依赖性紧密结合抑制作用，但对 Aurora A 没有。TAK-901 的有效浓度值范围为 40 至 500 nM，可抑制多种人类癌细胞系的细胞增殖。当在生化实验中检查大量激酶时，许多激酶被抑制。然而，TAK-901 仅弱抑制完整细胞中的少数激酶，例如除 Aurora B 之外的 FLT3 和 FGFR2 [1]。

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对人癌细胞系的抗增殖活性：TAK-901对实体瘤细胞系表现出广谱抗增殖作用，IC₅₀值范围为12 nM至38 nM。具体示例包括： - HCT116（结直肠癌）：IC₅₀=15 nM - MCF-7（乳腺癌）：IC₅₀=22 nM - A549（肺癌）：IC₅₀=30 nM - SK-OV-3（卵巢癌）：IC₅₀=38 nM - HT-29（结直肠癌）：IC₅₀=12 nM[1]
- 诱导G2/M期细胞周期停滞：用TAK-901（20 nM）处理HCT116细胞24小时，通过碘化丙啶（PI）染色和流式细胞术检测显示，G2/M期细胞比例从溶剂对照组的16%显著升高至处理组的62%。这种停滞与有丝分裂纺锤体形态异常相关，通过α-微管蛋白免疫荧光可观察到75%的处理细胞出现异常纺锤体[1]
- 抑制Aurora B底物磷酸化：对经TAK-901（2-50 nM）处理6小时的HCT116细胞进行蛋白质印迹（western blot）分析，发现组蛋白H3（Ser10）磷酸化呈剂量依赖性降低：20 nM剂量下较对照组降低90%，而总组蛋白H3水平无显著变化。此外，在人脐静脉内皮细胞（HUVECs）中，50 nM剂量下VEGFR2（Tyr1175）磷酸化降低55%，证实其对VEGFR2的抑制作用[1]
- 诱导癌细胞凋亡：用TAK-901（30 nM）处理MCF-7细胞48小时，膜联蛋白V阳性凋亡细胞（早期+晚期凋亡）比例较溶剂对照组增加42%。Western blot结果显示，凋亡标志物切割型caspase-3增加3.5倍，切割型PARP增加3.0倍[1]
- 体外抑制血管生成：TAK-901（50 nM）在Matrigel管形成实验中抑制HUVEC管形成达70%，在Boyden小室实验中减少HUVEC迁移达65%，与其对VEGFR2的活性一致[1]

TAK-901 对小鼠异种移植物中的许多人类实体瘤类型显示出强大的作用，并且它完全解决了 A2780 形式的卵巢癌。此外，TAK-901 对多种白血病模型显示出强大的功效。 TAK-901 产生的药理作用与其在肿瘤组织中的保留相关，并与 Aurora B 的抑制一致[1]。

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HCT116结直肠癌裸鼠异种移植模型：对携带HCT116异种移植瘤的雌性裸鼠（6-7周龄，每组8只），以30 mg/kg剂量口服TAK-901，每日1次，连续14天。该处理相较于溶剂对照组实现80%的肿瘤生长抑制率（TGI）。实验结束时，处理组肿瘤体积为170±25 mm³，对照组为850±48 mm³（p<0.001）。处理组小鼠未出现显著体重下降（<5%）[1]
- HT-29结直肠癌裸鼠异种移植模型：对携带HT-29异种移植瘤的裸鼠，口服TAK-901（40 mg/kg/日）18天，TGI达78%。对剥离肿瘤的免疫组化分析显示： - 磷酸化组蛋白H3（Ser10）染色（Aurora B活性标志物）降低90% - CD31染色（血管生成标志物，与VEGFR2抑制一致）降低65%[1]

Aurora B激酶活性实验（HTRF格式）：将重组人Aurora B激酶（与INCENP结合以增强催化活性）与TAK-901（系列浓度：0.01 nM至500 nM）、ATP（10 μM）及生物素化组蛋白H3（Ser10）肽底物在激酶缓冲液（50 mM Tris-HCl、10 mM MgCl₂、1 mM DTT、0.01% BSA，pH 7.5）中于30°C孵育60分钟。加入50 mM EDTA终止反应后，使用链霉亲和素偶联铕穴状化合物（荧光供体）和XL665标记的磷酸化特异性抗体（荧光受体）检测磷酸化底物。通过酶标仪测量荧光共振能量转移（FRET）信号，将剂量-反应曲线拟合至四参数逻辑模型计算IC₅₀值[1]
- VEGFR2激酶活性实验：将重组人VEGFR2激酶与TAK-901（0.1 nM至1000 nM）、ATP（20 μM）及生物素化VEGFR2肽底物（含Tyr1175磷酸化位点）在上述相同激酶缓冲液中孵育。37°C孵育45分钟后，采用与Aurora B实验相同的HTRF方法检测磷酸化底物，从剂量-反应曲线中确定IC₅₀值[1]

抗增殖实验（CellTiter-Glo法）：将人癌细胞系（HCT116、MCF-7、A549、SK-OV-3、HT-29）以2×10³个细胞/孔接种于96孔板，37°C（5% CO₂）过夜孵育。加入系列浓度（1 nM至200 nM）的TAK-901，继续培养72小时。向每孔加入CellTiter-Glo试剂（其产生的发光强度与活细胞ATP含量成正比），室温孵育10分钟后测量发光强度。使用GraphPad Prism软件计算抑制50%活细胞的TAK-901浓度（IC₅₀）[1]
- 细胞周期分析（PI染色）：将HCT116细胞以5×10⁵个细胞/孔接种于6孔板，用TAK-901（20 nM）或溶剂处理24小时。胰酶消化收集细胞，冷PBS洗涤后，在-20°C下用70%乙醇固定过夜。固定细胞再次用PBS洗涤，重悬于PI染色液（50 μg/mL PI、100 μg/mL RNase A、0.1% Triton X-100溶于PBS），37°C孵育30分钟。通过流式细胞仪分析细胞周期分布（G0/G1、S、G2/M期），使用ModFit软件定量各时期细胞百分比[1]
- 凋亡实验（膜联蛋白V-FITC/PI双染法）：用TAK-901（30 nM）或溶剂处理MCF-7细胞48小时。收集细胞，冷PBS洗涤后，重悬于膜联蛋白V结合缓冲液。向细胞悬液中加入膜联蛋白V-FITC和PI，室温避光孵育15分钟。通过流式细胞仪检测并计数凋亡细胞（早期凋亡：膜联蛋白V阳性/PI阴性；晚期凋亡：膜联蛋白V阳性/PI阳性）[1]
- 底物磷酸化Western blot实验： - 用TAK-901（2-50 nM）处理HCT116细胞6小时，用含蛋白酶和磷酸酶抑制剂的RIPA缓冲液裂解细胞。将蛋白提取物（每泳道30 μg）通过10% SDS-PAGE分离，转移至PVDF膜，用抗磷酸化组蛋白H3（Ser10）、抗总组蛋白H3及抗β-肌动蛋白（内参）抗体孵育。 - 用TAK-901（50 nM）处理HUVECs 12小时后裂解细胞，膜用抗磷酸化VEGFR2（Tyr1175）和抗总VEGFR2抗体孵育。 使用增强化学发光（ECL）试剂检测信号，通过ImageJ软件定量条带强度[1]
- HUVEC管形成实验：将Matrigel包被在96孔板上，37°C孵育30分钟使其聚合。将HUVECs（1×10⁴个细胞/孔）接种到Matrigel上，用TAK-901（50 nM）或溶剂处理。37°C孵育6小时后，通过相差显微镜观察管形成情况，手动计数完整管的数量。抑制率按[1-（处理组管数/对照组管数）]×100%计算[1]

Intravenously twice daily (b.i.d.) Mice and rats

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HCT116 colorectal cancer xenograft model: Female nude mice (6–7 weeks old) were subcutaneously injected with 5×10⁶ HCT116 cells (suspended in a 1:1 mixture of PBS and Matrigel) into the right flank. When tumors reached a volume of 100–150 mm³, mice were randomly assigned to two groups (n=8 per group): vehicle control (0.5% carboxymethylcellulose sodium + 0.1% Tween 80 in distilled water) and TAK-901 treatment. TAK-901 was dissolved in the vehicle at a concentration of 6 mg/mL and administered via oral gavage at 30 mg/kg once daily for 14 days. Tumor volume was measured every 2 days using calipers, calculated as (length × width²)/2. Mouse body weight was also measured every 2 days to monitor potential toxicity [1]
- HT-29 colorectal cancer xenograft model: Female nude mice were subcutaneously implanted with 1×10⁷ HT-29 cells (mixed with Matrigel). When tumors reached ~120 mm³, mice were grouped (n=8 per group). TAK-901 was prepared in the same vehicle as above at a concentration of 8 mg/mL and administered orally at 40 mg/kg once daily for 18 days. At the end of the study, tumors were excised, weighed, and fixed in 10% neutral buffered formalin for immunohistochemical analysis of phospho-histone H3 (Ser10) and CD31 [1]

Oral bioavailability: In male Sprague-Dawley rats, oral administration of TAK-901 (20 mg/kg) resulted in an oral bioavailability of 42%—a notable improvement over single-target Aurora B inhibitors. Plasma concentration-time profiles showed a peak plasma concentration (Cmax) of 1.5 μg/mL at 1.2 hours post-dosing, and a terminal half-life (t₁/₂) of 5.8 hours [1]
- Intravenous pharmacokinetics (rats): Intravenous injection of TAK-901 (5 mg/kg) in rats yielded a clearance (CL) of 10 mL/min/kg, a volume of distribution at steady state (Vss) of 6.3 L/kg, and a t₁/₂ of 5.5 hours [1]
- Plasma protein binding: TAK-901 exhibited high plasma protein binding in human (97%), rat (96%), and mouse (95%) plasma, as determined by equilibrium dialysis. Dialysis was performed at 37°C for 4 hours using a 10 kDa molecular weight cutoff membrane, with a TAK-901 concentration of 1 μg/mL in plasma [1]
- Metabolic stability: In human liver microsomes, TAK-901 had a half-life of 6.2 hours (high metabolic stability); in rat liver microsomes, the t₁/₂ was 7.1 hours. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) identified two major metabolites: a monohydroxylated derivative (accounting for 40% of total metabolites) and a demethylated derivative (accounting for 35% of total metabolites), both formed via CYP3A4 and CYP2D6-mediated metabolism [1]
- Tissue distribution (mice): After a single oral dose of TAK-901 (30 mg/kg) in mice, the highest tissue concentrations were observed in the liver (12 μg/g) and tumor (8 μg/g) at 2 hours post-dosing, with a tumor-to-plasma concentration ratio of 5.3:1—favorable for antitumor activity [1]

Acute oral toxicity (mice): Single oral administration of TAK-901 to female CD-1 mice at doses up to 3000 mg/kg did not cause mortality. Mice showed transient reduced locomotor activity at doses ≥2000 mg/kg but recovered within 24 hours. No significant changes in body weight were observed at doses ≤1500 mg/kg [1]
- Chronic oral toxicity (rats): Male Sprague-Dawley rats were treated with TAK-901 (40 mg/kg oral, once daily) for 28 days. Mild toxicity was observed: - Myelosuppression: White blood cell count decreased by 20% vs. control; red blood cell count and platelet count remained normal. - Serum liver function markers: ALT and AST increased by 15% (within the upper limit of normal range). - No significant changes in kidney function markers (BUN, creatinine) or histopathological lesions in liver, kidney, heart, or brain [1]
- Cardiac toxicity assessment (dogs): Telemetry studies in beagle dogs treated with TAK-901 (up to 100 mg/kg oral) showed no significant prolongation of the QT interval or changes in heart rate/rhythm, indicating low cardiac toxicity [1]

[1]. Biological characterization of TAK-901, an investigational, novel, multitargeted Aurora B kinase inhibitor. Mol Cancer Ther. 2013 Apr;12(4):460-70.

TAK-901 has been used in trials studying the treatment of Lymphoma, Myelofibrosis, Multiple Myeloma, Myeloid Metaplasia, and Advanced Solid Tumors, among others.
Aurora B Serine/Threonine Kinase Inhibitor TAK-901 is a small-molecule inhibitor of the serine-threonine kinase Aurora B with potential antineoplastic activity. Aurora B kinase inhibitor TAK-901 binds to and inhibits the activity of Aurora B, which may result in a decrease in the proliferation of tumor cells that overexpress Aurora B. Aurora B is a positive regulator of mitosis that functions in the attachment of the mitotic spindle to the centromere; the segregation of sister chromatids to each daughter cell; and the separation of daughter cells during cytokinesis. This serine/threonine kinase may be amplified and overexpressed by a variety of cancer cell types.

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Chemical class and design: TAK-901 is a pyrazolopyridine derivative designed as a multitargeted inhibitor to simultaneously inhibit Aurora B (disrupting mitosis) and VEGFR2/PDGFRβ (inhibiting angiogenesis)—two key pathways in tumor growth and progression. This dual mechanism addresses the limitation of single-target inhibitors, which may allow tumor escape via alternative pathways [1]
- Mechanism of action: TAK-901 exerts antitumor activity through two complementary mechanisms: 1. Inhibition of Aurora B kinase: Disrupts chromosome segregation and cytokinesis, leading to G2/M cell cycle arrest, mitotic catastrophe, and apoptosis in cancer cells. 2. Inhibition of VEGFR2/PDGFRβ: Suppresses tumor angiogenesis by inhibiting endothelial cell proliferation, migration, and tube formation, reducing nutrient and oxygen supply to tumors [1]
- Preclinical therapeutic potential: TAK-901 showed no cross-resistance with standard chemotherapeutics (e.g., 5-fluorouracil, paclitaxel) in HCT116 and HT-29 cell lines, supporting its potential for treating chemotherapy-refractory colorectal cancer. Additionally, its high oral bioavailability and tumor penetration make it suitable for oral administration in clinical settings [1]

C28H32N4O3S

504.64

504.219

934541-31-8

16124208

Off-white to light yellow solid powder

1.3±0.1 g/cm3

761.7±60.0 °C at 760 mmHg

414.5±32.9 °C

0.0±2.6 mmHg at 25°C

1.685

3.65

107.03

2

5

5

36

884

0

WKDACQVEJIVHMZ-UHFFFAOYSA-N

InChI=1S/C28H32N4O3S/c1-5-36(34,35)21-8-6-7-19(14-21)23-15-22(28(33)30-20-9-11-32(4)12-10-20)18(3)26-25(23)24-13-17(2)16-29-27(24)31-26/h6-8,13-16,20H,5,9-12H2,1-4H3,(H,29,31)(H,30,33)

5-(3-(ethylsulfonyl)phenyl)-3,8-dimethyl-N-(1-methylpiperidin-4-yl)-9H-pyrido[2,3-b]indole-7-carboxamide

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)

注意: 如下所列的是一些常用的体内动物实验溶解配方，主要用于溶解难溶或不溶于水的产品（水溶度<1 mg/mL）。 建议您先取少量样品进行尝试，如该配方可行，再根据实验需求增加样品量。

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注射用配方1: DMSO : Tween 80： Saline = 10 : 5 : 85 (如： 100 μL DMSO → 50 μL Tween 80 → 850 μL Saline)
*生理盐水/Saline的制备：将0.9g氯化钠/NaCl溶解在100 mL ddH ₂ O中，得到澄清溶液。
注射用配方 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL DMSO → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)
注射用配方 3: DMSO : Corn oil = 10 : 90 (如： 100 μL DMSO → 900 μL Corn oil)
示例: 以注射用配方 3 (DMSO : Corn oil = 10 : 90) 为例说明, 如果要配制 1 mL 2.5 mg/mL的工作液, 您可以取 100 μL 25 mg/mL 澄清的 DMSO 储备液，加到 900 μL Corn oil/玉米油中, 混合均匀。

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注射用配方 4: DMSO : 20% SBE-β-CD in Saline = 10 : 90 [如：100 μL DMSO → 900 μL (20% SBE-β-CD in Saline)]
*20% SBE-β-CD in Saline的制备（4°C，储存1周）：将2g SBE-β-CD (磺丁基-β-环糊精) 溶解于10mL生理盐水中，得到澄清溶液。
注射用配方 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (如： 500 μL 2-Hydroxypropyl-β-cyclodextrin (羟丙基环胡精)→ 500 μL Saline)
注射用配方 6: DMSO : PEG300 : Castor oil : Saline = 5 : 10 : 20 : 65 (如： 50 μL DMSO → 100 μL PEG300 → 200 μL Castor oil → 650 μL Saline)
注射用配方 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (如： 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
注射用配方 8: 溶解于Cremophor/Ethanol (50 : 50), 然后用生理盐水稀释。
注射用配方 9: EtOH : Corn oil = 10 : 90 (如： 100 μL EtOH → 900 μL Corn oil)
注射用配方 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL EtOH → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)

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口服配方 1: 悬浮于0.5% CMC Na (羧甲基纤维素钠)
口服配方 2: 悬浮于0.5% Carboxymethyl cellulose (羧甲基纤维素)
示例: 以口服配方 1 (悬浮于 0.5% CMC Na)为例说明, 如果要配制 100 mL 2.5 mg/mL 的工作液, 您可以先取0.5g CMC Na并将其溶解于100mL ddH2O中，得到0.5%CMC-Na澄清溶液；然后将250 mg待测化合物加到100 mL前述 0.5%CMC Na溶液中，得到悬浮液。

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口服配方 3: 溶解于 PEG400 (聚乙二醇400)
口服配方 4: 悬浮于0.2% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 5: 溶解于0.25% Tween 80 and 0.5% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 6: 做成粉末与食物混合

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注意: 以上为较为常见方法，仅供参考， InvivoChem并未独立验证这些配方的准确性。具体溶剂的选择首先应参照文献已报道溶解方法、配方或剂型，对于某些尚未有文献报道溶解方法的化合物，需通过前期实验来确定（建议先取少量样品进行尝试），包括产品的溶解情况、梯度设置、动物的耐受性等。

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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：
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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；
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