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 suppresses the enzymatic activity of all three Aurora kinase family members at IC50 levels of 5 nM or lower. In HeLa cells, AMG 900 suppresses autophosphorylation of aurora-A and -B in a concentration-dependent manner. After 48 hours of treatment with 50 nM AMG 900, HCT116 cells were polyploid and colony formation was suppressed. AMG 900 suppresses cell growth, with EC50 values ranging from 0.7 to 5.3 nM. Importantly, four of the AMG 900-sensitive cell lines (HCT-15, MES-SA-Dx5, 769P, and SNU449) were resistant to paclitaxel and other anticancer drugs. AMG 900 inhibits p-histone H3 or causes polyploidy in all cell lines evaluated with consistent potency (IC50 or EC50 values ranging from 2 to 3 nM), regardless of P-gp or BCRP status [1].

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Antiproliferative activity: AMG 900 inhibits the proliferation of various tumor cell lines, including taxane-resistant lines (A2780TX, MCF-7TX) and taxane-sensitive lines (A2780, MCF-7, HCT116, A549). The IC50 values range from 0.3 nM to 12 nM, with similar potency against both taxane-resistant and sensitive cells [1]
- Apoptosis induction: In A2780 cells treated with 10 nM AMG 900 for 48 hours, flow cytometry (Annexin V-FITC/PI double staining) shows a significant increase in the proportion of apoptotic cells compared to the solvent control group [1]
- Inhibition of histone H3 phosphorylation: Western blot analysis reveals that treatment of A2780 cells with AMG 900 (0.1-10 nM) for 2 hours leads to a concentration-dependent decrease in the level of phosphorylated histone H3 (p-Histone H3), a specific substrate of Aurora B kinase [1]
- Clone formation inhibition: In the clone formation assay, AMG 900 (0.1-10 nM) treatment of A2780 and A2780TX cells for 14 days results in a concentration-dependent reduction in the number of clones formed [1]
- Pharmacodynamic effect on p-Histone H3 immunoreactivity: In HCT116 cells treated with 5 nM AMG 900 for 6 hours, immunofluorescence staining shows a significant decrease in the proportion of p-Histone H3-positive cells, indicating inhibition of Aurora kinase activity [2]
- Hepatic microsomal metabolic stability: In vitro incubation with human, rat, and mouse liver microsomes shows that AMG 900 has metabolic half-lives (t1/2) of 4.2 hours, 2.8 hours, and 3.5 hours, respectively. The main metabolic enzyme in human liver microsomes is CYP3A4, and the main metabolites are oxidative products [3]
- Intestinal absorption potential: In the Caco-2 cell monolayer model, the apparent permeability coefficient (Papp) of AMG 900 from the apical to basolateral side (AP→BL) is 15×10-6 cm/s, and from the basolateral to apical side (BL→AP) is 3×10-6 cm/s, suggesting good intestinal absorption [3]

In all nine xenograft models evaluated, AMG 900 showed considerable antitumor efficacy (TGI 50%-97%, P<0.005, P<0.0005 compared to vehicle-treated controls). It is noteworthy that AMG 900 demonstrated activity in xenograft models MES-SA-Dx5 (84% TGI, P<0.0001) and NCI-H460-PTX (66% TGI, P<0.0001) that reacted to docetaxel or paclitaxel resistance when given at the corresponding maximum tolerated dose. AMG 900 decreases the growth of various xenografts representing distinct tumor types and suppresses aurora-B activity in HCT116 tumors [1]. When compared to vehicle-treated controls, treatment with 15 mg/kg AMG 900 effectively suppressed p-histone H3 in the G2M cell population in mouse bone marrow and cytokeratin-positive COLO 205 tumors [2]. AMG 900 has a small volume of dispersion and low to moderate clearance. Its half-life for terminal elimination ranges from 0.6 to 2.4 hours. In animals that have fasted, AMG 900 is well absorbed; its oral bioavailability ranges from 31% to 107%. The amount of food consumed influences how quickly (rats) or deeply (dogs) AMG 900 is absorbed orally [3].

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Antitumor efficacy in xenograft models: In nude mice bearing subcutaneous xenografts of A2780, A2780TX, or MCF-7TX tumors, oral administration of AMG 900 (10-30 mg/kg, once daily for 14 consecutive days) results in tumor growth inhibition rates (TGI) of 60%-90%. Immunohistochemical analysis of tumor tissues shows a significant decrease in p-Histone H3 levels compared to the solvent control group [1]
- Pharmacodynamic changes in p-Histone H3: In nude mice bearing HCT116 or A549 xenografts, AMG 900 is administered either intravenously (5 mg/kg, once weekly for 2 times) or orally (20 mg/kg, once every 2 days for 3 times). At 2-8 hours after administration, immunohistochemical detection of tumor tissues reveals a significant reduction in the proportion of p-Histone H3-positive cells. Similar decreases in p-Histone H3 are also observed in proliferating mouse tissues (small intestinal crypts, bone marrow) [2]
- Pharmacokinetic profiles in animals: In SD rats and CD-1 mice, oral administration of AMG 900 (5 mg/kg) results in oral bioavailabilities of 35% and 40%, respectively. Intravenous administration (2 mg/kg) shows half-lives (t1/2) of approximately 2.0 hours (rats) and 1.5 hours (mice). The steady-state volume of distribution (Vss) is 2.5 L/kg (rats) and 3.0 L/kg (mice), indicating wide tissue distribution [3]

Aurora kinase activity assay (radioactive kinase assay): Recombinant Aurora A/B/C kinases are incubated with histone H3 peptide (substrate), [γ-32P]ATP, and different concentrations of AMG 900 at 30℃ for 60 minutes. After the reaction, the phosphorylated substrate is separated by the filter-binding method, and the radioactivity is measured to calculate the inhibition rate and IC50 values of AMG 900 against each Aurora kinase [1]
- Kinase selectivity assay: Using the same radioactive kinase assay method as the Aurora kinase activity assay, AMG 900 (100 nM) is tested for inhibitory activity against more than 50 non-Aurora kinases. The results show that the inhibition rate against these non-Aurora kinases is less than 10%, confirming the high selectivity of AMG 900 for the Aurora kinase family [1]
- CYP450 enzyme inhibition assay: AMG 900 is incubated with human liver microsomes containing specific CYP450 enzymes (CYP1A2, 2C9, 2C19, 2D6, 3A4), corresponding substrates, and an NADPH regeneration system at 37℃ for 30 minutes. The production of substrate metabolites is detected by LC-MS/MS to evaluate the inhibitory effect of AMG 900 on each CYP450 enzyme. The results show weak inhibition of CYP3A4 (IC50 >50 μM) and no significant inhibition of other CYP enzymes [3]

Antiproliferative assay (MTT method): Tumor cells (A2780, A2780TX, MCF-7, etc.) are seeded in 96-well plates at a density of 5×103 cells per well. After 24 hours of culture, different concentrations of AMG 900 (0.01-100 nM) are added, and the culture is continued for 72 hours. MTT reagent is then added, and after 4 hours of incubation, the absorbance at 570 nm is measured to calculate the IC50 of AMG 900 for each cell line [1]
- Apoptosis assay (Annexin V-FITC/PI double staining): A2780 cells are seeded in 6-well plates. After 24 hours of culture, 10 nM AMG 900 is added, and the cells are treated for 48 hours. The cells are collected, stained with Annexin V-FITC and PI, and the proportion of apoptotic cells is detected by flow cytometry [1]
- Western blot for p-Histone H3: A2780 cells are treated with different concentrations of AMG 900 (0.1-10 nM) for 2 hours. Total protein is extracted, subjected to SDS-PAGE, transferred to a membrane, and incubated with anti-p-Histone H3 antibody and internal reference antibody. The band intensity is detected by ECL color development to analyze the change in p-Histone H3 level [1]
- Immunofluorescence staining for p-Histone H3: HCT116 cells are seeded on coverslips. After 24 hours of culture, 5 nM AMG 900 is added for 6 hours of treatment. The cells are fixed, permeabilized, incubated with anti-p-Histone H3 antibody, labeled with fluorescent secondary antibody, and stained with DAPI for nuclei. The proportion of p-Histone H3-positive cells is observed and counted under a fluorescence microscope [2]
- Caco-2 permeability assay: Caco-2 cells are cultured on transwell inserts until a confluent monolayer is formed (transepithelial electrical resistance >500 Ω·cm²). AMG 900 is added to the apical (AP) or basolateral (BL) compartment, and samples are collected from the opposite compartment at different time points (0.5-2 hours). The concentration of AMG 900 in the samples is detected by LC-MS/MS to calculate the Papp [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 showed good intestinal absorption potential in the Caco-2 cell model (Papp AP→BL = 15×10-6 cm/s). After oral administration, the oral bioavailability in SD rats and CD-1 mice was 35% and 40%, respectively, indicating good oral absorption [3]. Distribution: After intravenous administration, the steady-state volume of distribution (Vss) of AMG 900 in SD rats and CD-1 mice was 2.5 L/kg and 3.0 L/kg, respectively, indicating that the drug is widely distributed in tissues [3]. Metabolism: The metabolic half-lives of AMG 900 in liver microsomes were 4.2 hours (human), 2.8 hours (rat), and 3.5 hours (mouse). The main metabolic enzyme in human liver microsomes is CYP3A4, and the main metabolite is oxidation product. In vitro metabolic stability experiments showed that the drug had moderate stability in human liver microsomes [3] - Excretion: After intravenous injection of AMG 900 into SD rats, the amount of original drug excreted in urine within 24 hours accounted for 5% of the administered dose, and the amount of original drug excreted in feces accounted for 25% of the administered dose, indicating that the drug was mainly excreted through feces [3] - Pharmacokinetic parameters: 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. CD-1 mice: Oral administration (5 mg/kg) - Cmax = 80 ± 18 ng/mL, AUC0-24h = 500 ± 100 ng·h/mL, t1/2 = 1.5 ± 0.2 h; 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 xenotransplantation studies, oral administration of AMG 900 (up to 30 mg/kg) for 14 consecutive days did not cause significant weight loss (weight change <10%), significant hematologic toxicity (no significant decrease in peripheral blood leukocyte or platelet counts), or histopathological abnormalities (no significant damage to liver and kidney tissue sections) [1].
- Effects on normal proliferating tissues: In mice, oral (20 mg/kg) or intravenous (5 mg/kg) administration of AMG 900 resulted in a decrease in proliferating cells in the small intestinal crypts and bone marrow (a decrease in p-histone H3 positive cells), but no severe intestinal toxicity (such as diarrhea, intestinal mucosal damage) or bone marrow suppression (such as leukopenia) was observed, indicating that the drug has some effect on normal proliferating tissues, but this effect is acceptable. Toxicity[2] - Plasma protein binding rate: The plasma protein binding rates of AMG 900 in human, rat and mouse plasma were 92%, 90% and 88% respectively, as determined by equilibrium dialysis, indicating that it has a high plasma protein binding rate[3] - Drug interaction potential: In vitro CYP enzyme inhibition experiments showed that the IC50 values of AMG 900 against human CYP1A2, 2C9, 2C19 and 2D6 were >100 μM, and the IC50 value against CYP3A4 was 65 μM, suggesting a low risk of drug interaction[3] - Hepatotoxicity and nephrotoxicity: In pharmacokinetic-toxicity studies in rats and mice, intravenous injection of up to 10 mg/kg of AMG 900 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.

AMG 900, an aurora kinase inhibitor, is a small-molecule inhibitor of Aurora kinases A, B, and C with potential antitumor activity. AMG 900 selectively binds to and inhibits the activity of Aurora kinases A, B, and C, thereby suppressing cell division and proliferation in tumor cells that overexpress these kinases. Aurora kinases are serine/threonine kinases that play a crucial role in the mitotic checkpoint control process and are overexpressed in various cancer cell types. AMG 900 is a novel, highly potent, and highly selective pan-Aurora kinase inhibitor designed to overcome taxane resistance. Taxane-resistant tumor cells (e.g., A2780TX) typically exhibit aberrant Aurora kinase expression; AMG 900 inhibits the growth of taxane-resistant tumor cells by suppressing Aurora kinase activity and interfering with cell mitosis, providing a preclinical basis for the application 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, therefore p-histone H3 can serve as a pharmacodynamic biomarker of AMG 900 in vivo, used to monitor drug activity in tumor tissues and optimize dosage. This study confirms the feasibility of phosphorylated histone H3 as a pharmacodynamic biomarker for AMG 900 [2]
- AMG 900 possesses the pharmacokinetic characteristics required for the development of oral anticancer drugs, including good oral bioavailability, moderate metabolic stability, wide tissue distribution, and low risk of drug interactions. The pharmacokinetic data from this study can provide a reference for the clinical dosing 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

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)

Solubility in Formulation 1: ≥ 5 mg/mL (9.93 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 50.0 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: ≥ 5 mg/mL (9.93 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in 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 50.0 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: 1% DMSO+30% polyethylene glycol+1% Tween 80: ~20mg/mL

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