125 I-IP10-CXCR3 ( IC50 = 8 nM ); 125 I-ITAC-CXCR3 ( IC50 = 8.2 nM )
Human CXCR3 (Ki = 1.2 nM, ligand: [125I]-CXCL10) [1]
- Murine CXCR3 (Ki = 4.8 nM, ligand: [125I]-CXCL10) [1]

AMG-487 (AMG487) 通过三种 CXCR3 趋化因子（IP-10 IC50=8 nM、ITAC IC50=15 nM 和 MIG IC50=36 nM）抑制 CXCR3 介导的细胞迁移。此外，AMG 487 还可抑制 ITAC 响应的钙动员 (IC50=5 nM)[1]。 AMG487 (1 μM) 发展成较少的肺转移，并且肺明显小于载体处理的肺[2]。 AMG487 消除 C26 肿瘤细胞的增殖/存活[3]。

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与[125I]-CXCL10竞争性结合人及鼠CXCR3，选择性高；对其他趋化因子受体（CXCR1、CXCR2、CXCR4、CCR1-5）无明显结合活性（Ki > 1000 nM）[1]
- 抑制CXCL10诱导的CXCR3表达细胞钙流反应，IC50 = 3.7 nM [1]
- 抑制CXCL11诱导的人外周血淋巴细胞趋化，IC50 = 5.2 nM [1]
- 抑制CXCL10诱导的鼠4T1乳腺癌细胞（IC50 ≈ 10 nM）和鼠脾细胞（IC50 ≈ 5 nM）趋化[2]
- 阻断CXCL10（IC50 ≈ 8 nM）和CXCL11（IC50 ≈ 6 nM）诱导的鼠CT26结直肠癌细胞趋化[3]

AMG-487 (AMG487) (0.03-10 mg/kg, sc) 以剂量依赖性方式显着减少肺部细胞浸润[1]。 AMG487（5 mg/kg，皮下注射，每天两次）比载体治疗的小鼠产生更少的转移[2]。在这两个模型中，AMG487（5 mg/kg，皮下）治疗的小鼠比对照小鼠表现出更少的肺结节。 AMG487 缩小肿瘤体积[3]。

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在鼠4T1乳腺癌肺转移模型中，口服给予AMG 487（30 mg/kg，每日两次，持续21天），与溶媒对照组相比，肺转移灶数量显著减少约67%[2]
- 在鼠CT26结直肠癌肝转移模型（脾内接种）中，口服给予AMG 487（30 mg/kg，每日两次，持续14天），肝转移灶数量减少约70%；在肺转移模型（尾静脉接种）中，相同给药方案使肺转移灶数量减少约60%[3]
- AMG 487 10 mg/kg每日两次的剂量也能抑制CT26诱导的肝和肺转移，但效果弱于30 mg/kg剂量[3]

然后将细胞在 50 mM Hepes pH 7.5、150 mM NaCl、20 mM EDTA、1 mM PMSF、10 μg/mL 亮抑酶肽、2 μg/mL 抑肽酶和 0.2% NP-40 中裂解并超声处理。将等量的裂解物与 Ac-DEVD-AMC 底物和 caspase-3/7 底物在底物缓冲液（50 mM Hepes、100 mM NaCl、1 mM EDTA、10% 蔗糖、0.5% CHAPS、5 mM 二硫苏糖醇）中混合。微量滴定板。使用分光光度计 Ascent Fluoroskan 在 37°C 下连续测量荧光底物的产生，半胱天冬酶活性（表示为 U/mg 蛋白质）定义为每分钟裂解 1 nmol 底物的酶量。

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CXCR3放射性配体结合实验：制备表达人或鼠CXCR3的细胞膜制剂，与不同浓度的AMG 487孵育15分钟。加入[125I]-CXCL10后，37°C孵育60分钟。通过玻璃纤维滤膜过滤去除未结合配体，检测滤膜的放射性强度。计算结合抑制率，并通过回归分析确定Ki值[1]
- 钙流实验：将表达CXCR3的细胞加载钙敏感染料，与AMG 487预孵育30分钟。加入CXCL10诱导钙内流，通过荧光酶标仪检测细胞内钙浓度变化。根据荧光强度抑制率计算IC50值[1]

结肠癌细胞以 104 个细胞 cm2 的密度接种，并在补充或不补充不同浓度的 rCXCL9、rCXCL10 和 rCXCL11 的血清富集培养基或基础培养基（含 0.1% 牛血清白蛋白，BSA）中孵育指定时间。胰蛋白酶分离、收集和计数或用新鲜培养基重新饲喂3天、收获和计数之前的时间。通过倒置光学显微镜以×20放大倍数观察CRC细胞的形态，并在第7天拍照。

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4T1乳腺癌细胞Transwell趋化实验：将4T1细胞接种到Transwell小室上室，上室加入不同浓度的AMG 487，下室加入CXCL10作为趋化因子。37°C孵育4小时后，固定迁移到小室下表面的细胞，染色后在显微镜下计数。计算趋化抑制率和IC50值[2]
- CT26结直肠癌细胞Transwell趋化实验：将CT26细胞接种到Transwell小室上室，上室加入不同浓度的AMG 487，下室加入CXCL10或CXCL11。37°C孵育4小时后，固定、染色迁移细胞并计数。测定趋化抑制率和IC50值[3]
- 脾细胞趋化实验：分离鼠脾细胞并接种到Transwell小室上室，上室加入AMG 487，下室加入CXCL10。37°C孵育4小时后，计数迁移的脾细胞，计算趋化抑制率[2]

Injection of 3×10 5 viable tumor cells s.c. near the right abdominal mammary gland of syngeneic female mice is used to assess local tumor growth and spontaneous metastasis. Tumor diameters are measured twice a week using a caliper, and mice are put to death individually if their s.c. tumor measures 18 mm in diameter or earlier if they appear moribund. Under a dissecting microscope, surface tumor colonies are measured blindly while the lungs are removed and weighed. To assess experimental metastasis, 9×10 4 viable tumor cells are injected intraperitoneally (i.v.) into the lateral tail vein of syngeneic female mice. On day 21 after transplantation, or earlier if the mice appeared moribund, all of the mice are put to death. Under a dissecting microscope, surface tumor colonies are measured blindly while the lungs are removed and weighed. A 50% hydroxypropyl-β-cyclodextrin solution is made; this solution acts as the vehicle at 20%. Following the addition of AMG487 to the 50% solution, the mixture is incubated for two hours in a sonicating water bath with periodic vortexing. To achieve the proper final concentration of AMG487 in 20% hydroxypropyl-β-cyclodextrin, distilled water is added.

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Murine 4T1 breast cancer lung metastasis model: Female BALB/c mice (6-8 weeks old) were intravenously injected with 1×105 4T1 breast cancer cells via the tail vein. AMG 487 was dissolved in 0.5% carboxymethylcellulose sodium plus 0.1% Tween 80. The drug was administered by oral gavage at a dosage of 30 mg/kg, twice daily (morning and evening), starting from day 1 after cell inoculation and continuing for 21 days. At the end of the experiment, mice were euthanized, lung tissues were isolated, fixed, sectioned, and stained with hematoxylin-eosin (HE). The number of surface metastatic nodules on the lungs was counted [2]
- Murine CT26 colorectal carcinoma metastasis model: Female BALB/c mice (6-8 weeks old) were intra-splenically injected with 5×104 CT26 cells to establish the liver metastasis model, or intravenously injected with 1×105 CT26 cells via the tail vein to establish the lung metastasis model. AMG 487 was dissolved in the same vehicle as described above. The drug was administered by oral gavage at dosages of 10 mg/kg or 30 mg/kg, twice daily. For the liver metastasis model, administration lasted for 14 days; for the lung metastasis model, it lasted for 21 days, starting from day 1 after cell inoculation. At the end of the experiment, mice were euthanized, and liver or lung tissues were collected, fixed, sectioned, and stained with HE. The number and area of metastatic nodules were quantified [3]

During the in vivo experiments, oral administration of AMG 487 (10-30 mg/kg, twice daily for up to 21 days) did not cause significant changes in mouse body weight, food intake, or mortality [2, 3]
- No obvious histological abnormalities were observed in major organs (liver, kidney, spleen) of mice treated with AMG 487 compared to the vehicle control group [2, 3]

[1]. Discovery and optimization of a series of quinazolinone-derived antagonists of CXCR3. Bioorg Med Chem Lett. 2007 Jun 15;17(12):3339-43.

[2]. Antagonism of CXCR3 inhibits lung metastasis in a murine model of metastatic breast cancer. Cancer Res. 2006 Aug 1;66(15):7701-7.

[3]. Organ-specific inhibition of metastatic colon carcinoma by CXCR3 antagonism. Br J Cancer. 2009 Jun 2;100(11):1755-64.

AMG 487 is a quinazolinone-derived selective antagonist of CXCR3, which exerts its biological effects by competitively binding to CXCR3 and blocking the interaction between CXCR3 and its ligands (CXCL9, CXCL10, CXCL11) [1, 2, 3]
- The anti-metastatic effect of AMG 487 is mainly mediated by inhibiting CXCR3-dependent chemotaxis and migration of tumor cells, thereby reducing the dissemination of tumor cells to distant organs [2, 3]
- Due to its high selectivity for CXCR3, AMG 487 is a valuable tool compound for studying the role of CXCR3 in tumor metastasis and other CXCR3-mediated pathological processes [1]

C32H28F3N5O4

603.591037750244

603.209

C, 63.68; H, 4.68; F, 9.44; N, 11.60; O, 10.60

473719-41-4

AMG 487 (S-enantiomer); 473720-30-8; (±)-AMG 487; 947536-03-0

24957182

White to yellow solid powder

5.805

99.44

0

10

10

44

997

1

O=C(N([C@@H](C1=NC2=NC=CC=C2C(N1C3=CC=C(OCC)C=C3)=O)C)CC4=CC=CN=C4)CC5=CC=C(OC(F)(F)F)C=C5

WQTKNBPCJKRYPA-OAQYLSRUSA-N

InChI=1S/C32H28F3N5O4/c1-3-43-25-14-10-24(11-15-25)40-30(38-29-27(31(40)42)7-5-17-37-29)21(2)39(20-23-6-4-16-36-19-23)28(41)18-22-8-12-26(13-9-22)44-32(33,34)35/h4-17,19,21H,3,18,20H2,1-2H3/t21-/m1/s1

N-[(1R)-1-[3-(4-ethoxyphenyl)-4-oxopyrido[2,3-d]pyrimidin-2-yl]ethyl]-N-(pyridin-3-ylmethyl)-2-[4-(trifluoromethoxy)phenyl]acetamide

AMG487; AMG-487; AMG 487

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: ~100 mg/mL (~165.7 mM)
Ethanol: ~100 mg/mL

配方 1 中的溶解度: ≥ 2.5 mg/mL (4.14 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 (4.14 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 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 (4.14 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: 5%DMSO + 40%PEG300 + 5%Tween 80 + 50%ddH2O: 5.0mg/ml (8.28mM)

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配方 5 中的溶解度: 5 mg/mL (8.28 mM) in 20% HP-β-CD in 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。
*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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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。