Atrasentan hydrochloride (ABT-627 hydrochloride) is an antagonist of the endothelin receptor(IC50 = 55.1 μM for ETA).

ETA:55.1 μM

Atrasentan（0-50 μM）显著抑制LNCaP和C4-2b前列腺癌细胞生长。ABT-627与Taxotere联合使用，相比单独使用任一化合物，可显著降低前列腺癌活细胞的数量，并更大程度地下调NF-κB DNA结合活性[2]。Atrasentan显著诱导多种CYPs和药物转运体的表达。它是一种中等强度的P-gp抑制剂（IC50：15.1 μM，在P388/dx细胞中）和一种弱BCRP抑制剂（IC50：59.8 μM，在MDCKII-BCRP细胞中）[3]。

Atrasentan（3 mg/kg, p.o.）通过抑制大型内皮素-1（1 nmol/kg）诱导的加压反应，在去髓大鼠中显示出效果[1]。此外，Atrasentan（10 mg/kg, i.p.）在SCID-hu模型中对C4-2b肿瘤在骨环境中的生长有一定程度的抑制作用[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].