AMG-900

AMG 900 is a potent and highly selective pan-aurora kinase inhibitor, with inhibitory activity against Aurora A, Aurora B, and Aurora C kinases. The IC50 values are as follows: Aurora A (0.6 nM), Aurora B (1.5 nM), and Aurora C (0.1 nM) [1]
- AMG 900 exhibits high selectivity for the Aurora kinase family; at a concentration of 100 nM, it shows less than 10% inhibitory activity against more than 50 other tested kinases, confirming its specificity for Aurora kinases [1]

AMG 900 以 5 nM 或更低的 IC50 水平抑制所有三个 Aurora 激酶家族成员的酶活性。在 HeLa 细胞中，AMG 900 以浓度依赖性方式抑制 aurora-A 和 -B 的自磷酸化。用 50 nM AMG 900 处理 48 小时后，HCT116 细胞呈多倍体，集落形成受到抑制。 AMG 900 抑制细胞生长，EC50 值范围为 0.7 至 5.3 nM。重要的是，四种 AMG 900 敏感细胞系（HCT-15、MES-SA-Dx5、769P 和 SNU449）对紫杉醇和其他抗癌药物具有耐药性。无论 P-gp 或 BCRP 状态如何，AMG 900 都会抑制 p-组蛋白 H3 或在所有经评估具有一致效力（IC50 或 EC50 值范围为 2 至 3 nM）的细胞系中引起多倍体 [1]。

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抗增殖活性：AMG 900可抑制多种肿瘤细胞系的增殖，包括紫杉醇耐药细胞系（A2780TX、MCF-7TX）和紫杉醇敏感细胞系（A2780、MCF-7、HCT116、A549），IC50值范围为0.3-12 nM，对耐药细胞系和敏感细胞系的抑制 potency相似[1]
- 诱导细胞凋亡：在A2780细胞中，用10 nM AMG 900处理48小时后，流式细胞术（Annexin V-FITC/PI双染法）检测显示，凋亡细胞比例较溶剂对照组显著增加[1]
- 抑制组蛋白H3磷酸化：Western blot分析表明，用AMG 900（0.1-10 nM）处理A2780细胞2小时，可浓度依赖性地降低磷酸化组蛋白H3（p-Histone H3，Aurora B激酶的特异性底物）的水平[1]
- 抑制克隆形成：在克隆形成实验中，AMG 900（0.1-10 nM）处理A2780和A2780TX细胞14天，可浓度依赖性地减少形成的克隆数量[1]
- 对p-Histone H3免疫反应性的药效学影响：在HCT116细胞中，用5 nM AMG 900处理6小时后，免疫荧光染色显示p-Histone H3阳性细胞比例显著降低，提示极光激酶活性被抑制[2]
- 肝微粒体代谢稳定性：在人、大鼠和小鼠肝微粒体体外孵育实验中，AMG 900的代谢半衰期（t1/2）分别为4.2小时、2.8小时和3.5小时；人肝微粒体中其主要代谢酶为CYP3A4，主要代谢产物为氧化产物[3]
- 肠道吸收潜力：在Caco-2细胞单层模型中，AMG 900从顶侧到基底侧（AP→BL）的表观渗透系数（Papp）为15×10-6 cm/s，从基底侧到顶侧（BL→AP）的Papp为3×10-6 cm/s，提示其具有良好的肠道吸收能力[3]

在评估的所有九个异种移植模型中，AMG 900 显示出相当大的抗肿瘤功效（TGI 50%-97%，P<0.005，与媒介物治疗对照相比，P<0.0005）。值得注意的是，AMG 900 在异种移植模型 MES-SA-Dx5（84% TGI，P<0.0001）和 NCI-H460-PTX（66% TGI，P<0.0001）中表现出活性，这些模型在给予时对多西紫杉醇或紫杉醇耐药有反应。相应的最大耐受剂量。 AMG 900 可减少代表不同肿瘤类型的各种异种移植物的生长，并抑制 HCT116 肿瘤中的 aurora-B 活性 [1]。与媒介物处理的对照相比，15 mg/kg AMG 900 处理可有效抑制小鼠骨髓和细胞角蛋白阳性 COLO 205 肿瘤中 G2M 细胞群中的 p-组蛋白 H3 [2]。 AMG 900 具有较小的分散体积和低至中等的间隙。其最终消除的半衰期为0.6至2.4小时。在禁食的动物中，AMG 900 吸收良好；其口服生物利用度为31%至107%。摄入的食物量会影响 AMG 900 口服吸收的速度（大鼠）或深度（狗）[3]。

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异种移植模型中的抗肿瘤疗效：在携带A2780、A2780TX或MCF-7TX肿瘤皮下异种移植的裸鼠中，口服给予AMG 900（10-30 mg/kg，每日一次，连续14天），肿瘤生长抑制率（TGI）达60%-90%；肿瘤组织免疫组化分析显示，p-Histone H3水平较溶剂对照组显著降低[1]
- p-Histone H3的药效学变化：在携带HCT116或A549异种移植瘤的裸鼠中，AMG 900采用静脉给药（5 mg/kg，每周一次，共2次）或口服给药（20 mg/kg，每2天一次，共3次）。给药后2-8小时，肿瘤组织免疫组化检测显示p-Histone H3阳性细胞比例显著降低；在小鼠增殖组织（小肠隐窝、骨髓）中也观察到类似的p-Histone H3降低现象[2]
- 动物体内药代动力学特征：在SD大鼠和CD-1小鼠中，口服AMG 900（5 mg/kg）的口服生物利用度分别为35%和40%；静脉给药（2 mg/kg）的半衰期（t1/2）约为2.0小时（大鼠）和1.5小时（小鼠）；稳态分布容积（Vss）为2.5 L/kg（大鼠）和3.0 L/kg（小鼠），提示药物在组织中分布广泛[3]

极光激酶活性测定（放射性激酶实验）：将重组Aurora A/B/C激酶与组蛋白H3肽段（底物）、[γ-32P]ATP及不同浓度的AMG 900在30℃下孵育60分钟。反应结束后，通过过滤结合法分离磷酸化底物，测定放射性强度，计算AMG 900对各极光激酶的抑制率及IC50值[1]
- 激酶选择性测定：采用与极光激酶活性测定相同的放射性激酶实验方法，测试100 nM AMG 900对50多种非极光激酶的抑制活性。结果显示，其对这些非极光激酶的抑制率均低于10%，证实AMG 900对极光激酶家族的高选择性[1]
- CYP450酶抑制实验：将AMG 900与含特定CYP450酶（CYP1A2、2C9、2C19、2D6、3A4）的人肝微粒体、对应底物及NADPH再生系统在37℃下孵育30分钟。通过LC-MS/MS检测底物代谢产物的生成量，评估AMG 900对各CYP450酶的抑制作用。结果显示，其对CYP3A4有弱抑制作用（IC50 >50 μM），对其他CYP酶无显著抑制[3]

抗增殖实验（MTT法）：将肿瘤细胞（A2780、A2780TX、MCF-7等）以5×103个/孔的密度接种于96孔板，培养24小时后加入不同浓度的AMG 900（0.01-100 nM），继续培养72小时。随后加入MTT试剂，孵育4小时后测定570 nm处吸光度，计算AMG 900对各细胞系的IC50值[1]
- 凋亡实验（Annexin V-FITC/PI双染法）：将A2780细胞接种于6孔板，培养24小时后加入10 nM AMG 900，处理48小时。收集细胞，用Annexin V-FITC和PI染色，通过流式细胞术检测凋亡细胞比例[1]
- p-Histone H3 Western blot检测：用不同浓度的AMG 900（0.1-10 nM）处理A2780细胞2小时，提取总蛋白，进行SDS-PAGE电泳、转膜，随后用抗p-Histone H3抗体和内参抗体孵育，通过ECL显色检测条带强度，分析p-Histone H3水平的变化[1]
- p-Histone H3免疫荧光染色：将HCT116细胞接种于盖玻片，培养24小时后加入5 nM AMG 900处理6小时。细胞经固定、通透后，用抗p-Histone H3抗体孵育，荧光二抗标记，DAPI染核，在荧光显微镜下观察并计数p-Histone H3阳性细胞比例[2]
- Caco-2通透性实验：将Caco-2细胞在transwell小室中培养至形成融合单层（跨上皮电阻>500 Ω·cm²），将AMG 900加入顶侧（AP）或基底侧（BL）腔室，在不同时间点（0.5-2小时）从对侧腔室收集样品。通过LC-MS/MS检测样品中AMG 900的浓度，计算Papp值[3]

Dissolved in DMSO; 3.75, 7.5, or 15 mg/kg; Oral gavage Nude mice bearing established HCT116 tumors

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Nude mouse xenograft model (antitumor efficacy study): Logarithmically growing A2780, A2780TX, or MCF-7TX cells (1×107 cells per mouse) are subcutaneously injected into the right back of female nude mice (6-8 weeks old). When the tumor volume reaches 100-200 mm³, the mice are randomly divided into groups (6 mice per group). AMG 900 is dissolved in 0.5% methylcellulose + 0.1% Tween 80 to prepare different concentrations, and administered orally at doses of 10 mg/kg, 20 mg/kg, and 30 mg/kg once daily for 14 consecutive days; the control group receives the solvent. Tumor volume is measured every 2 days using a vernier caliper (tumor volume V = L×W²/2, where L is the long diameter and W is the short diameter), and the tumor growth inhibition rate (TGI) is calculated. At the end of the experiment, the mice are sacrificed, and tumor tissues are collected, with part used for immunohistochemical detection of p-Histone H3 [1]
- Nude mouse xenograft model (pharmacodynamic study on p-Histone H3): HCT116 or A549 cells (1×106-5×106 cells per mouse) are subcutaneously injected into nude mice to establish xenograft models. AMG 900 is administered either intravenously (dissolved in normal saline, dose 5 mg/kg, once weekly for 2 times) or orally (dissolved in 0.5% methylcellulose + 0.1% Tween 80, dose 20 mg/kg, once every 2 days for 3 times). Mice are sacrificed at 0.5h, 2h, 4h, 8h, and 24h after administration (3 mice per time point per group), and tumor tissues, small intestines, and bone marrow tissues are collected, fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned for p-Histone H3 immunohistochemistry. The immunohistochemical process includes dewaxing to water, antigen retrieval, blocking, overnight incubation with anti-p-Histone H3 antibody, secondary antibody incubation, DAB color development, hematoxylin counterstaining, and counting of positive cell proportion [2]
- Rat and mouse pharmacokinetic study: SD rats (male, 250-300g) and CD-1 mice (male, 20-25g) are used, with 6 animals per group. AMG 900 is administered orally (dissolved in 0.5% methylcellulose + 0.1% Tween 80, dose 5 mg/kg) or intravenously (dissolved in 5% DMSO + 95% normal saline, dose 2 mg/kg). Blood samples (0.2 mL per time point for rats, 0.05 mL per time point for mice) are collected from the tail vein at 0.083h (5min), 0.25h, 0.5h, 1h, 2h, 4h, 8h, 12h, and 24h after administration, and plasma is separated by centrifugation. Plasma drug concentration is detected by LC-MS/MS (protein precipitation for sample pretreatment, chromatographic separation, mass spectrometry detection, internal standard quantification). Pharmacokinetic parameters (Cmax, AUC0-24h, t1/2, CL, Vd) are calculated using a non-compartmental model, and oral bioavailability (F) is calculated as F = (oral AUC × intravenous dose) / (intravenous AUC × oral dose) × 100% [3]

Absorption: AMG 900 shows good intestinal absorption potential in the Caco-2 cell model (Papp AP→BL = 15×10-6 cm/s). After oral administration, the oral bioavailability is 35% in SD rats and 40% in CD-1 mice, indicating good oral absorption [3]
- Distribution: After intravenous administration, the steady-state volume of distribution (Vss) of AMG 900 is 2.5 L/kg in SD rats and 3.0 L/kg in CD-1 mice, suggesting wide distribution of the drug in tissues [3]
- Metabolism: AMG 900 has metabolic half-lives of 4.2 hours (human), 2.8 hours (rat), and 3.5 hours (mouse) in liver microsomes. The main metabolic enzyme in human liver microsomes is CYP3A4, and the main metabolites are oxidative products. In vitro metabolic stability experiments show moderate stability in human liver microsomes [3]
- Excretion: After intravenous administration of AMG 900 to SD rats, the amount of unchanged drug excreted in urine within 24 hours accounts for 5% of the administered dose, and that in feces accounts for 25%, indicating that the drug is mainly excreted through feces [3]
- Pharmacokinetic parameters: In SD rats: oral administration (5 mg/kg) - Cmax = 120±25 ng/mL, AUC0-24h = 800±150 ng·h/mL, t1/2 = 2.0±0.3h; intravenous administration (2 mg/kg) - Cmax = 280±40 ng/mL, AUC0-24h = 380±60 ng·h/mL, CL = 5.2±0.8 mL/min/kg. In CD-1 mice: oral administration (5 mg/kg) - Cmax = 80±18 ng/mL, AUC0-24h = 500±100 ng·h/mL, t1/2 = 1.5±0.2h; intravenous administration (2 mg/kg) - Cmax = 220±35 ng/mL, AUC0-24h = 290±50 ng·h/mL, CL = 11.5±1.2 mL/min/kg [3]

In vivo toxicity in nude mice: In the xenograft study, oral administration of AMG 900 up to 30 mg/kg for 14 consecutive days did not cause significant weight loss (weight change <10%), obvious hematological toxicity (no significant decrease in peripheral blood leukocyte or platelet count), or histopathological abnormalities (no obvious damage in liver and kidney tissue sections) [1]
- Effect on normal proliferating tissues: In mice, oral administration (20 mg/kg) or intravenous administration (5 mg/kg) of AMG 900 leads to a decrease in proliferating cells (reduced p-Histone H3-positive cells) in small intestinal crypts and bone marrow, but no severe intestinal toxicity (e.g., diarrhea, intestinal mucosal damage) or myelosuppression (e.g., leukopenia) symptoms are observed, indicating that the drug has a certain effect on normal proliferating tissues but with acceptable toxicity [2]
- Plasma protein binding rate: Determined by equilibrium dialysis, the plasma protein binding rates of AMG 900 in human, rat, and mouse plasma are 92%, 90%, and 88%, respectively, indicating high plasma protein binding [3]
- Drug-drug interaction potential: In vitro CYP enzyme inhibition experiments show that AMG 900 has IC50 values >100 μM for human CYP1A2, 2C9, 2C19, and 2D6, and IC50 = 65 μM for CYP3A4, suggesting a low risk of drug-drug interactions [3]
- Hepatorenal toxicity: In rat and mouse pharmacokinetic-toxicity studies, intravenous administration of AMG 900 up to 10 mg/kg did not cause significant abnormalities in liver and kidney function indicators (ALT, AST, BUN, Cr) [3]

[1]. Preclinical evaluation of AMG 900, a novel potent and highly selective pan-aurora kinase inhibitor with activity in taxane-resistant tumor cell lines. Cancer Res, 2010, 70(23), 9846-9854.

[2]. AMG 900, a potent inhibitor of aurora kinases causes pharmacodynamic changes in p-Histone H3 immunoreactivity in human tumor xenografts and proliferating mouse tissues. J Transl Med. 2014 Nov 4;12:307.

[3]. In vitro and in vivo pharmacokinetic characterizations of AMG 900, an orally bioavailable small molecule inhibitor of aurora kinases. Xenobiotica. 2011 May;41(5):400-8.

Aurora Kinase Inhibitor AMG 900 is a small-molecule inhibitor of Aurora kinases A, B and C with potential antineoplastic activity. Aurora kinase inhibitor AMG 900 selectively binds to and inhibits the activities of Aurora kinases A, B and C, which may result in inhibition of cellular division and proliferation in tumor cells that overexpress these kinases. Aurora kinases are serine-threonine kinases that play essential roles in mitotic checkpoint control during mitosis and are overexpressed by a wide variety of cancer cell types.

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AMG 900 is a novel potent and highly selective pan-aurora kinase inhibitor developed to overcome taxane resistance. Taxane-resistant tumor cells (e.g., A2780TX) often have abnormal Aurora kinase expression; AMG 900 inhibits taxane-resistant tumor cell growth by suppressing Aurora kinase activity and interfering with cell mitosis, providing a preclinical basis for the use of AMG 900 in taxane-resistant cancers [1]
- Phosphorylated histone H3 (p-Histone H3) is a specific substrate of Aurora B kinase. AMG 900 reduces p-Histone H3 levels by inhibiting Aurora kinase activity, so p-Histone H3 can be used as a pharmacodynamic biomarker for AMG 900 in vivo to monitor the drug's activity in tumor tissues and optimize the dose. This study confirms the feasibility of p-Histone H3 as a pharmacodynamic biomarker for AMG 900 [2]
- AMG 900 has favorable pharmacokinetic characteristics for oral anticancer drug development, including good oral bioavailability, moderate metabolic stability, wide tissue distribution, and low risk of drug-drug interactions. The pharmacokinetic data of this study provides a reference for the clinical dose design of AMG 900 [3]

C28H21N7OS

503.58

503.152

945595-80-2

24856041

Light yellow to yellow solid powder

1.4±0.1 g/cm3

778.7±70.0 °C at 760 mmHg

424.7±35.7 °C

0.0±2.7 mmHg at 25°C

1.739

4.64

140.7

2

9

6

37

725

0

IVUGFMLRJOCGAS-UHFFFAOYSA-N

InChI=1S/C28H21N7OS/c1-17-15-24(37-16-17)25-20-5-2-3-6-21(20)26(35-34-25)32-18-8-10-19(11-9-18)36-27-22(7-4-13-30-27)23-12-14-31-28(29)33-23/h2-16H,1H3,(H,32,35)(H2,29,31,33)

N-(4-((3-(2-aminopyrimidin-4-yl)pyridin-2-yl)oxy)phenyl)-4-(4-methylthiophen-2-yl)phthalazin-1-amine.

AMG-900; AMG 900; AMG900

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: ≥ 5 mg/mL (9.93 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 50.0 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: ≥ 5 mg/mL (9.93 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 50.0 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: 1% DMSO+30% polyethylene glycol+1% Tween 80: ~20mg/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。