Aurora A (IC50 = 1.2 nM); Aurora B (IC50 = 396.5 nM)

Alisertib (MLN 8237) 诱导 MM 细胞中异常的有丝分裂纺锤体、有丝分裂积累以及衰老和死亡，以防止细胞分裂。 aleritetib 上调肿瘤抑制基因 p21 和 p27 以及 p53[1]。辅因子与 Aurora A 结合带来的 ATP 亲和力增强可能是 Alisertib (MLN 8237) 对 T217D/W277E Aurora A/TPX2 复合物活性较低的原因[2]。在各种肿瘤细胞系中，aleretitib (MLN 8237) 抑制细胞生长，IC50 范围为 15 至 469 nM[4]。

Alisertib/MLN8237体内药效学活性：有丝分裂指数增加，双极有丝分裂纺锤体减少，染色体排列异常增加[4]
在携带HCT-116结肠肿瘤异种移植物的雌性裸鼠中，以3、10和30 mg/kg的剂量口服Alisertib/MLN8237，通过血浆和肿瘤浓度测量，可获得显著的生物利用度（补充图S4）。每日一次30mg/kg的剂量是最大耐受剂量（MTD）。[4]
对用递增剂量的Alisertib/MLN8237治疗的HCT-116异种移植物的肿瘤组织进行分析，发现有丝分裂标记物pHisH3随时间和剂量的增加而增加，表明Alisertib/MLN8237抑制了Aurora a（图2A）。有丝分裂标志物下降时的血浆浓度约为1至2μmol/L，表明需要该浓度来抑制体内AAK（补充图S4）。此外，在约6μmol/L的浓度下，pHisH3没有受到抑制，这表明Aurora a在体内对Aurora B的抑制具有显著的选择性。[4]

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Alisertib/MLN8237在实体瘤异种移植物模型中引起肿瘤生长抑制，在淋巴瘤体内模型中引起消退[4]
为了确定Alisertib/MLN8237的体内抗肿瘤活性，对携带固体和血液学人类肿瘤异种移植物的小鼠施用了越来越多剂量的Alisertib/MLN8237。图3A显示了每天口服3、10或30mg/kg阿利替布3周后，皮下HCT-116肿瘤裸鼠的平均肿瘤体积。Alisertib/MLN8237治疗导致3、10和30mg/kg组的剂量依赖性TGI分别为43.3%、84.2%和94.7%。该模型中最大的抗肿瘤反应是肿瘤停滞。所有剂量均耐受良好，30mg/kg组最大体重减轻7.4%。[4]

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在人类多发性骨髓瘤的异种移植小鼠模型中，Alisertib/MLN8237（30mg/kg，口服）显著降低了肿瘤负担并提高了总体生存率[1]。在实体瘤异种移植物模型中，Alisertib/MLN8237（3-30mg/kg；口服；每天一次，持续3周）抑制肿瘤的生长[4]。

蛋白激酶测定和抑制剂[2]
使用Alisertib/MLN8237、VX-680、ZM447439和MLN8054。这些化合物的化学结构如图1 A所示。为了测量Aurora A的活性，在适当的抑制剂存在下，使用组蛋白H3作为底物，在100μM[γ-32P]ATP存在下，在30°C下对纯化的细菌表达的Aurora A进行了25 ng（12.5 nM终浓度，图1 B和补充图S1）或250 ng（125 nM最终浓度，所有其他测定）的测定。对于Aurora A/TPX2测定，包括50 ng TPX2[1−43]肽，代表Aurora A的2倍摩尔过量。Aurora A/TPX2复合物在随后添加抑制剂和ATP之前在激酶反应中形成。对于Plk4测定，在适当的抑制剂存在下，使用髓磷脂碱性蛋白（MBP）作为底物，在100μM[γ-32P]ATP存在下，在30°C下测定250 ng细菌表达的纯化His标记的人催化结构域（氨基酸1-269）20分钟。为了评估组蛋白H3和MBP的磷酸化，通过在p81磷酸纤维素纸上对磷酸化底物进行切伦科夫计数，或在SDS-PAGE后通过磷酸化试剂定量放射性标记掺入。每个实验至少重复三次，每次都有类似的结果。为了确定Aurora A和突变体的Km[ATP]值，对1至200μM[γ-32P]ATP（比活度500 cpm-pmol-1）范围内的数据进行了非线性回归分析。使用Prism软件进行数据分析。

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基于酶和细胞的激酶抑制测定[4]
如Manfredi及其同事所述，进行了Aurora A和Aurora B放射性Flashplate酶测定和基于细胞的测定，以确定Alisertib/MLN8237介导的体外抑制的性质和程度。在基于细胞的测定中，Aurora A活性是通过测量Aurora A在苏氨酸288上的自磷酸化来确定的，而Aurora B活性是通过使用高含量成像测定和如前所述测量组蛋白H3在丝氨酸10上的磷酸化（pHisH3）来确定的。还测试了1μmol/LAlisertib/MLN8237对205种激酶的抑制活性。

测定细胞活力和增殖[1]
将MM细胞系、从MM患者骨髓抽吸物中纯化的CD138+肿瘤细胞和健康供者外周血单个核细胞（PBMCs）接种于100 μL完整培养基中，以20 × 104个细胞/孔的密度接种于3个96孔板中。每孔加入MLN8237浓度范围为0.0001 ~ 4μ m，终体积为200 μL。采用3-（4,5-二甲基噻唑-2-基）-2,5-二苯基溴化四唑（MTT）测定细胞活力，并在孵育24、48和72小时采用3[H]-胸苷掺入法测定细胞增殖。用分光光度计在570/630 nm处测定吸光度。
将MM细胞单独或与BM基质细胞、rhIL-6 （10 ng/mL）或rhIGF-1 （25 ng/mL）共同孵育于96孔板中，然后暴露于MLN8237 (0.0001-4μM)中24、48和72小时。孵育最后8小时，细胞用3[H]-胸苷（0.5 μCi）脉冲，收集到玻璃滤光片上，用LKB betatplate闪烁计数器计数。
MM细胞株与DMSO或MLN8237 (0.125 ~ 0.5μ m)联合常规抗MM药物美法兰（2.5 ~ 5μ m）、阿霉素（50 ~ 100nm）、地塞米松（50 ~ 100nm）孵育；并用新型抗mm药物硼替佐米（2.5 ~ 5nm）或来那度胺（0.5 ~ 1μ m）治疗72小时。MTT法测定细胞活力。联合指数（CI）采用CalcuSyn软件进行等线图分析，2.0版本(CI < 1表示协同效应；CI = 1，加性效应；和CI bbb1，无显著组合效应)。

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检测细胞凋亡和衰老[1]
采用荧光素偶联膜联蛋白V和碘化丙啶（PI）染色法检测MLN8237触发的MM细胞凋亡。细胞与0.5至1μM MLN8237或DMSO孵育24至72小时，并根据制造商的方案用荧光素异硫氰酸酯-膜联蛋白V和PI染色。流式细胞术检测凋亡细胞，采用BDFACS-Canto II和FlowJo Version 7.0软件。
在MM1中检测到细胞衰老的诱导。根据制造商的方案，使用衰老β-半乳糖苷酶染色试剂盒，用0.5μM MLN8237处理S细胞和OPM1细胞48小时。用光镜观察β-半乳糖苷酶阳性细胞(原放大×20；徕卡dil)在室温下。

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细胞循环分析[1]
将MM细胞暴露于DMSO或0.5 ~ 1μM MLN8237中24 ~ 72小时，70%乙醇在- 20℃下渗透，50 μg/mL PI和20单位/mL RNase-A孵育。流式细胞术采用BDFACS-Canto II和FlowJo软件分析DNA含量。

Animal/Disease Models: Nude mice bearing HCT-116 colon tumor xenograft[4]
Doses: 3, 10, or 30 mg/kg
Route of Administration: Po; one time/day for 3 weeks
Experimental Results: Resulted in a dose-dependent TGI (tumor growth inhibition) of 43.3%, 84.2%, and 94.7% for the 3, 10, and 30 mg/kg groups, respectively.

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In vivo efficacy studies[4]
Nine in vivo tumor models of different histologies grown subcutaneously or disseminated were developed in either nude or severe combined immunodeficient (SCID) mice. The methods for all in vivo studies have been described previously (32), with the exception of the lymphoma tumor models described below. All mice had access to food and water ad libitum and were housed and handled in accordance with the Guide for the Care and Use of Laboratory Animals and Millennium Institutional Animal Care and Use Committee Guidelines. Mice for all models were dosed orally with Alisertib/MLN8237 for approximately 3 weeks and tumor growth inhibition (TGI) was calculated on the last day of treatment. For all studies, Alisertib/MLN8237 was formulated in 10% 2-hydroxypropyl-β-cyclodextrin and 1% sodium bicarbonate and was dosed orally by gavage on a once-daily or twice-daily schedule.
The cell lines OCI-LY7-Luc, OCI-LY19-Luc, and WSU-DLCL2-Luc were used for lymphoma models; tumor cells were inoculated intravenously into 5- to 8-week-old female SCID (nonobese diabetic SCID; Taconic, in study of OCI-LY7-Luc) mice. Mice bearing the disseminated, CD20-positive, non-Hodgkin's lymphoma model OCI-LY19 were treated with vehicle control (10% 2-hydroxypropyl-β-cyclodextrin and 1% sodium bicarbonate was used for all in vivo studies), alisertib at 20 mg/kg twice daily or 30 mg/kg once daily, or the anti-CD20 monoclonal antibody rituximab (Rituxan) at 10 mg/kg once per week. The lymphoma cell lines stably expressed firefly luciferase, and tumor growth over time was measured using whole-body bioluminescent imaging using Xenogen IVIS 200 imaging system. Fifteen minutes before imaging, mice received an intraperitoneal injection of 150 mg/kg of the substrate Luciferin, which when oxidized by luciferase emits light photons. Mice were imaged both dorsally and ventrally, and photon flux values were summed from both views. The antitumor effects of each treatment group were determined by calculating the percent TGI [(Δ control mean tumor photon flux − Δ treated mean tumor photon flux) × 100/Δ control mean tumor photon flux] at the end of treatment.

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Mitotic index, spindle bipolarity, and chromosome alignment assays[4]
Mice bearing HCT-116 xenografts were treated orally with a single dose of 3, 10, and 30 mg/kg Alisertib/MLN8237, and tumor samples were removed at specified time points. Frozen tumor tissue sections were stained for the mitotic marker pHisH3, then visualized using immunofluorescence detection and quantified at the indicated time points. The methods used to stain and quantify pHisH3, which is also an Aurora B substrate, have been described previously.

[1]. A novel Aurora-A kinase inhibitor MLN8237 induces cytotoxicity and cell-cycle arrest in multiple myeloma Blood June 24, 2010 vol. 115 no. 25 5202-5213.

[2]. Drug-Resistant Aurora A Mutants for Cellular Target Validation of the Small Molecule Kinase Inhibitors MLN8054 and MLN8237 ACS Chem. Biol., 2010, 5 (6), pp 563-576.

[3]. Aurora Kinase Inhibitors: Current Status and Outlook. Front Oncol. 2015 Dec 21;5:278.

[4]. Characterization of Alisertib (MLN8237), an investigational small-molecule inhibitor of aurora A kinase using novel in vivo pharmacodynamic assays.Clin Cancer Res. 2011 Dec 15;17(24):7614-7624.

4-[[9-chloro-7-(2-fluoro-6-methoxyphenyl)-5H-pyrimido[5,4-d][2]benzazepin-2-yl]amino]-2-methoxybenzoic acid is a benzazepine.
Alisertib is a novel aurora A kinase inhibitor under investigation for the treatment of various forms of cancer.
Alisertib is a second-generation, orally bioavailable, highly selective small molecule inhibitor of the serine/threonine protein kinase Aurora A kinase with potential antineoplastic activity. Alisertib binds to and inhibits Aurora A kinase, which may result in disruption of the assembly of the mitotic spindle apparatus, disruption of chromosome segregation, and inhibition of cell proliferation. Aurora A kinase localizes to the spindle poles and to spindle microtubules during mitosis, and is thought to regulate spindle assembly. Aberrant expression of Aurora kinases occurs in a wide variety of cancers, including colon and breast cancers.

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Drug Indication
For the treatment of various forms of cancer.

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Aurora-A is a mitotic kinase that regulates mitotic spindle formation and segregation. In multiple myeloma (MM), high Aurora-A gene expression has been correlated with centrosome amplification and proliferation; thus, inhibition of Aurora-A in MM may prove to be therapeutically beneficial. Here we assess the in vitro and in vivo anti-MM activity of MLN8237, a small-molecule Aurora-A kinase inhibitor. Treatment of cultured MM cells with MLN8237 results in mitotic spindle abnormalities, mitotic accumulation, as well as inhibition of cell proliferation through apoptosis and senescence. In addition, MLN8237 up-regulates p53 and tumor suppressor genes p21 and p27. Combining MLN8237 with dexamethasone, doxorubicin, or bortezomib induces synergistic/additive anti-MM activity in vitro. In vivo anti-MM activity of MLN8237 was confirmed using a xenograft-murine model of human-MM. Tumor burden was significantly reduced (P = .007) and overall survival was significantly increased (P < .005) in animals treated with 30 mg/kg MLN8237 for 21 days. Induction of apoptosis and cell death by MLN8237 were confirmed in tumor cells excised from treated animals by TdT-mediated dUTP nick end labeling assay. MLN8237 is currently in phase 1 and phase 2 clinical trials in patients with advanced malignancies, and our preclinical results suggest that MLN8237 may be a promising novel targeted therapy in MM.[1]

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The Aurora kinases regulate multiple aspects of mitotic progression, and their overexpression in diverse tumor types makes them appealing oncology targets. An intensive research effort over the past decade has led to the discovery of chemically distinct families of small molecule Aurora kinase inhibitors, many of which have demonstrated therapeutic potential in model systems. These agents are also important tools to help dissect signaling pathways that are orchestrated by Aurora kinases, and the antiproliferative target of pan-Aurora inhibitors such as VX-680 has been validated using chemical genetic techniques. In many cases the nonspecific nature of Aurora inhibitors toward unrelated kinases is well established, potentially broadening the spectrum of cancers to which these compounds might be applied. However, unambiguously demonstrating the molecular target(s) for clinical kinase inhibitors is an important challenge, one that is absolutely critical for deciphering the molecular basis of compound specificity, resistance, and efficacy. In this paper, we have investigated amino acid requirements for Aurora A sensitivity to the benzazepine-based Aurora inhibitor MLN8054 and the close analogue MLN8237, a second-generation compound that is in phase II clinical trials. A crystallographic analysis facilitated the design and biochemical investigation of a panel of resistant Aurora A mutants, a subset of which were then selected as candidate drug-resistance targets for further evaluation. Using inducible human cell lines, we show that cells expressing near-physiological levels of a functional but partially drug-resistant Aurora A T217D mutant survive in the presence of MLN8054 or MLN8237, authenticating Aurora A as a critical antiproliferative target of these compounds.[2]

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Purpose: Small-molecule inhibitors of Aurora A (AAK) and B (ABK) kinases, which play important roles in mitosis, are currently being pursued in oncology clinical trials. We developed three novel assays to quantitatively measure biomarkers of AAK inhibition in vivo. Here, we describe preclinical characterization of alisertib (MLN8237), a selective AAK inhibitor, incorporating these novel pharmacodynamic assays. Experimental design: We investigated the selectivity of alisertib for AAK and ABK and studied the antitumor and antiproliferative activity of alisertib in vitro and in vivo. Novel assays were used to assess chromosome alignment and mitotic spindle bipolarity in human tumor xenografts using immunofluorescent detection of DNA and alpha-tubulin, respectively. In addition, 18F-3'-fluoro-3'-deoxy-l-thymidine positron emission tomography (FLT-PET) was used to noninvasively measure effects of alisertib on in vivo tumor cell proliferation. Results: Alisertib inhibited AAK over ABK with a selectivity of more than 200-fold in cells and produced a dose-dependent decrease in bipolar and aligned chromosomes in the HCT-116 xenograft model, a phenotype consistent with AAK inhibition. Alisertib inhibited proliferation of human tumor cell lines in vitro and produced tumor growth inhibition in solid tumor xenograft models and regressions in in vivo lymphoma models. In addition, a dose of alisertib that caused tumor stasis, as measured by volume, resulted in a decrease in FLT uptake, suggesting that noninvasive imaging could provide value over traditional measurements of response. Conclusions: Alisertib is a selective and potent inhibitor of AAK. The novel methods of measuring Aurora A pathway inhibition and application of tumor imaging described here may be valuable for clinical evaluation of small-molecule inhibitors.[4]

C27H20CLFN4O4

518.92

518.115

C, 62.49; H, 3.88; Cl, 6.83; F, 3.66; N, 10.80; O, 12.33

1028486-01-2

Alisertib sodium;1028486-06-7; 1208255-63-3 (sodium)

24771867

Light yellow to light pink solid powder

1.4±0.1 g/cm3

729.1±70.0 °C at 760 mmHg

394.8±35.7 °C

0.0±2.5 mmHg at 25°C

1.671

5.56

105.93

2

9

6

37

836

0

ZLHFILGSQDJULK-UHFFFAOYSA-N

InChI=1S/C27H20ClFN4O4/c1-36-21-5-3-4-20(29)23(21)25-19-10-15(28)6-8-17(19)24-14(12-30-25)13-31-27(33-24)32-16-7-9-18(26(34)35)22(11-16)37-2/h3-11,13H,12H2,1-2H3,(H,34,35)(H,31,32,33)

4-((9-chloro-7-(2-fluoro-6-methoxyphenyl)-5H-benzo[c]pyrimido[4,5-e]azepin-2-yl)amino)-2-methoxybenzoic acid

MLN-8237; alisertib; MLN8237; MLN 8237

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)

配方 1 中的溶解度: 2.08 mg/mL (4.01 mM) 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), 悬浮液；超声助溶。
例如，若需制备1 mL的工作液，可将100 μL 20.8 mg/mL澄清DMSO储备液加入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: 2.08 mg/mL (4.01 mM) 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), 悬浊液; 超声助溶。
例如，若需制备1 mL的工作液，可将 100 μL 20.8 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: ≥ 2.08 mg/mL (4.01 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 20.8 mg/mL 澄清 DMSO 储备液加入到 900 μL 玉米油中并混合均匀。

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配方 4 中的溶解度: 15% Captisol:5mg/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。