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) suppresses the three CXCR3 chemokines' ability to migrate cells through CXCR3 (IP-10 IC50=8 nM, ITAC IC50=15 nM, and MIG IC50=36 nM). AMG 487 also suppresses the release of calcium in response to ITAC (IC50=5 nM)[1]. Reduced lung metastases and significantly smaller lungs compared to vehicle-treated lungs are observed with AMG487 (1 μM) treatment[2]. AMG487 stops C26 tumor cells from proliferating or surviving[3].

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Competed with [125I]-CXCL10 for binding to human and murine CXCR3 with high selectivity; showed no significant binding to other chemokine receptors (CXCR1, CXCR2, CXCR4, CCR1-5) with Ki > 1000 nM [1]
- Inhibited CXCL10-induced calcium mobilization in CXCR3-expressing cells with IC50 = 3.7 nM [1]
- Suppressed CXCL11-induced chemotaxis of human peripheral blood lymphocytes with IC50 = 5.2 nM [1]
- Inhibited CXCL10-induced chemotaxis of murine 4T1 breast cancer cells (IC50 ≈ 10 nM) and murine splenocytes (IC50 ≈ 5 nM) [2]
- Blocked CXCL10-induced (IC50 ≈ 8 nM) and CXCL11-induced (IC50 ≈ 6 nM) chemotaxis of murine CT26 colorectal carcinoma cells [3]

AMG-487 (AMG487) (0.03-10 mg/kg, s.c.) significantly reduces cellular infiltration into the lungs in a dose dependent manner[1]. Compared to mice given a vehicle, AMG487 (5 mg/kg, s.c., twice daily) causes fewer metastases to form[2]. In both models, mice treated with AMG487 (5 mg/kg, s.c.) show fewer pulmonary nodules than the control group. AMG487 reduces the tumour volume[3].

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In a murine 4T1 breast cancer lung metastasis model, oral administration of AMG 487 (30 mg/kg, twice daily for 21 days) significantly reduced the number of lung metastatic nodules by ~67% compared to the vehicle control group [2]
- In a murine CT26 colorectal carcinoma liver metastasis model (splenic inoculation), oral administration of AMG 487 (30 mg/kg, twice daily for 14 days) reduced hepatic metastatic nodules by ~70%; in the lung metastasis model (tail vein inoculation), the same dosage regimen decreased lung metastatic nodules by ~60% [3]
- The 10 mg/kg twice-daily dosage of AMG 487 also inhibited CT26-induced liver and lung metastasis, but the effect was less potent than the 30 mg/kg dose [3]

In order to examine the harmful impact of AMG487 on bone marrow cells and how it affects BMDC development, the bone marrow cells are exposed to AMG487 at concentrations of 3 μM, 15 μM, 30 μM, and 60 μM starting on day 0 and getting appropriate doses of AMG487 every three days. After two rounds of washing, LPS is added to the cells to promote maturation. We only add different concentrations of AMG487 (10 μM, 30 μM, and 60 μM) along with the LPS on day 6 in order to study its impact on BMDC activation. In every instance, the vehicle is added in equal parts as a control.

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Radioligand binding assay for CXCR3: Membrane preparations from cells expressing human or murine CXCR3 were incubated with various concentrations of AMG 487 for 15 minutes. [125I]-CXCL10 was then added, and the mixture was incubated at 37°C for 60 minutes. Unbound ligand was removed by filtration through glass fiber filters, and the radioactivity of the filters was measured. The inhibition rate of binding was calculated, and the Ki value was determined by regression analysis [1]
- Calcium mobilization assay: CXCR3-expressing cells were loaded with a calcium-sensitive dye and pre-incubated with AMG 487 for 30 minutes. CXCL10 was added to induce calcium influx, and the change in intracellular calcium concentration was detected by a fluorescence microplate reader. The IC50 value was calculated based on the inhibition of fluorescence intensity [1]

At a density of 10 4 cells cm 2 , colon cancer cells are seeded and then incubated in serum-enriched medium or base medium (containing 0.1% bovine serum albumin, BSA) supplemented or not with different concentrations of rCXCL9, rCXCL10, and rCXCL11 for the indicated times. After that, they are either trypsin-detached, collected, and counted, or they are re-fed with fresh medium for three days, harvested, and counted. On day seven, photos are taken and the morphology of the CRC cells are examined using an inverted optical microscope at ×20 magnification.

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Transwell chemotaxis assay for 4T1 breast cancer cells: 4T1 cells were seeded in the upper chamber of Transwell inserts, and various concentrations of AMG 487 were added to the upper chamber. CXCL10 was added to the lower chamber as a chemoattractant. After incubation at 37°C for 4 hours, cells that migrated to the lower surface of the insert were fixed, stained, and counted under a microscope. The chemotaxis inhibition rate and IC50 value were calculated [2]
- Transwell chemotaxis assay for CT26 colorectal carcinoma cells: CT26 cells were seeded in the upper chamber of Transwell inserts, and AMG 487 at different concentrations was added to the upper chamber. CXCL10 or CXCL11 was added to the lower chamber. The cells were incubated at 37°C for 4 hours, and migrated cells were fixed, stained, and counted. The inhibition rate of chemotaxis and IC50 values were determined [3]
- Splenocyte chemotaxis assay: Murine splenocytes were isolated and seeded in the upper chamber of Transwell inserts. AMG 487 and CXCL10 were added to the upper and lower chambers, respectively. After 4 hours of incubation at 37°C, migrated splenocytes were counted, and the chemotaxis inhibition rate was calculated [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]

In in vivo experiments, oral administration of AMG 487 (10–30 mg/kg, twice daily for 21 days) did not cause significant changes in body weight, food intake, or mortality in mice [2, 3] - No significant histological abnormalities were observed in the major organs (liver, kidney, spleen) of mice treated with AMG 487 compared to the vector 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 selective CXCR3 antagonist that 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 achieved by inhibiting CXCR3-dependent tumor cell chemotaxis and migration, thereby reducing the spread of tumor cells to distant organs [2, 3]. Due to its high selectivity for CXCR3, AMG 487 is an important 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

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

Solubility in Formulation 1: ≥ 2.5 mg/mL (4.14 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: 2.5 mg/mL (4.14 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (4.14 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly..

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Solubility in Formulation 4: 5%DMSO + 40%PEG300 + 5%Tween 80 + 50%ddH2O: 5.0mg/ml (8.28mM)

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Solubility in Formulation 5: 5 mg/mL (8.28 mM) in 20% HP-β-CD in Saline (add these co-solvents sequentially from left to right, and one by one), Suspened solution; with ultrasonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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 (Please use freshly prepared in vivo formulations for optimal results.)