Biological Activity
ACT-132577 is the major and pharmacologically active metabolite of macitentan, which is dual ETA/ETB antagonist designed for tissue targeting.
In Vitro
ACT-132577 achieves full inhibition of intracellular calcium increase induced by ET-1 on nonrecombinant cells (primary human pulmonary smooth muscle cells and rat aortic smooth muscle cell line A10 and mouse fibroblast cell line 3T3)[1].
In Vivo
ACT-132577 has a volume of distribution greater than the plasma volume and a longer half-life than its parent compound in the rat[1]. In rat plasma, the mean recovery of ACT-132577 ranges from 82.6% to 90.6%, matrix effect of ACT-132577 in rat plasma ranges from 101.4% to 115.2%[2].
Kinase Assay
The competition binding assay is performed in 200 μL of 50 mM Tris/HCl buffer, pH 7.4, including 25 mM MnCl2, 1 mM EDTA, 0.5% (w/v) bovine serum albumin (fraction V), and 50 μg/mL 4-(2-aminoethyl)benzenesulfonyl fluoride in polypropylene microtiter plates. Membranes containing 0.5 μg of protein (ETA) or 0.2 μg of protein (ETB) are incubated for 2 h at room temperature with 16 pM 125I-ET-1 (8000 cpm) and increasing concentrations of unlabeled test compounds. Maximal and minimal binding is determined in samples without and with 100 nM unlabeled ET-1, respectively. After 2 h of incubation, the membranes are filtered onto filter plates containing GF/C filters. To each well, 50 μL of scintillation cocktail is added, and the filter plates counted in a microplate counter. Test compounds are dissolved, diluted, and added to the assay in dimethyl sulfoxide. The final concentration of dimethyl sulfoxide in the assay is 2.5%, which is found not to interfere with the binding. The IC50 value is calculated as the concentration of antagonist inhibiting 50% of the specific binding of ET-1.
References & source
[1]. Iglarz M, et al. Pharmacology of macitentan, an orally active tissue-targeting dual endothelin receptor antagonist. J Pharmacol Exp Ther. 2008 Dec;327(3):736-45.
[2]. Zhang J, et al. Pharmacokinetic study of ACT-132577 in rat plasma by ultra performance liquid chromatography-tandem mass spectrometry. Int J Clin Exp Med. 2015 Oct 15;8(10):18420-6
Published experimental findings depend on the model and assay conditions. They do not guarantee equivalent performance in every application. For laboratory research use only.