Atrasentan (ABT-627) is an endothelin receptor antagonist that can inhibit the activity of ETA (IC50 value is 0.0551 nM).

ETA:0.055 nM

方法：Atrasentan (ABT-627)（ 10μM,24小时） 处理PPC-1-ET 系列细胞，观察细胞生长情况。
结果：Atrasentan 处理显著增加了凋亡细胞的数量 。[2]

方法：Atrasentan （ABT-627）（20 mg/kg ，腹膜内注射） 处理人肿瘤异种移植模型 HT29小鼠，观察Atrasentan对肿瘤缺氧的影响。
结果：Atrasentan 可以明显减少肿瘤缺氧。[1]
方法：单独Atrasentan（20 mg/kg，口服，每天）和单独多西他赛（5 mg / kg，腹腔注射，每3天一次）；ABT-627（20 mg/kg，口服每天）和多西他赛（5 mg/kg，腹腔注射，每 3 天一次）治疗肿瘤异种移植模型小鼠，观察小鼠体内肿瘤生长。
结果：用 Atrasentan + 多西他赛联合治疗小鼠的肿瘤负荷和生长速率显著低于单独使用Atrasentan或多西他赛治疗的小鼠。[2]

Cells are incubated and treated with Atrasentan. They are then washed twice with PBS and lysed in ice-cold lysis buffer [20 mM Tris (pH 7.4), 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 1 mM EGTA, 2.5 mM sodium PPi, 1 mM β-glycerophosphate, 1 mM sodium orthovanadate, 1 μg/mL leupeptin, and 1 mM PMSF]. The extracts are centrifuged to remove cellular debris, and the protein content of the supernatants is determined using the bicinchoninic acid (BCA) protein assay reagent. Proteins (150 μg) are incubated with gentle rocking at 4°C overnight with immobilized Akt antibody cross-linked to agarose hydrazide beads. After the Akt is selectively immunoprecipitated from the cell lysates, the immunoprecipitated products are washed twice with lysis buffer and twice with kinase assay buffer [25 mM Tris (pH 7.5), 10 mM MgCl2, 5 mM β-glycerol phosphate, 0.1 mM sodium orthovanadate, 2 mM DTT] and then resuspended in 40 μL of kinase assay buffer containing 200 μM ATP and 1 μg GSK-3α/β fusion protein. The kinase assay reaction is allowed to proceed at 30°C for 30 min and stopped by the addition of Lamelli SDS sample buffer. Reaction products are resolved by 10% SDS-PAGE, followed by Western blotting with antiphosphorylated GSK-3α/β antibody. For analysis of the total amount of Akt, 40 μg of protein from the lysate samples are resolved by 10% SDS-PAGE, followed by Western blotting with anti-Akt antibody [2].

All three prostate cancer cell lines (LNCaP, C4-2b, and PC-3 cells) are seeded at a density of 3 × 10^3 cells per well in 96-well microtiter culture plates. After overnight incubation, the medium is removed and replaced with a fresh medium containing different concentrations of ABT-627 (0-50 μM) diluted from a 10-mM stock. After 72 h of incubation with the drug, 20 μL of MTT solution (5 mg/mL in PBS) is added to each well and incubated further for 2 h. Upon termination, the supernatant is aspirated and the MTT formazan formed by metabolically viable cells is dissolved in isopropanol (100 μL). The plates are mixed for 30 min on a gyratory shaker, and the absorbance is measured at 595 nm on a plate reader [2].

YM598 (0.3, 1, and 3 mg/kg), atrasentan (0.3, 1, and 3 mg/kg), or 0.5% methylcellulose as vehicle is orally administered to rats with a dosing cannula. The dosing volume of the test substances and vehicle is set at 5 mL/kg. Approximately 20 min after administration of compounds, the rats are anesthetized with sodium pentobarbital, and then pithed and ventilated 30 min after dosing. Approximately 1 h after oral administration of compounds, big endothelin-1 (1 nmol/kg) is intravenously administered, and blood pressure is measured. In these two experiments, the dose of test compound that causes 50% inhibition (ID50) of the big endothelin-1-induced increase in diastolic blood pressure is determined by linear regression analysis [1].