| In Vitro | In vitro activity: Vactosertib (formerly EW-7197) is a highly potent, selective, and orally available inhibitor of the TGF-β receptor ALK4/ALK5. Vactosertib inhibited ALK5 with IC50 value of 0.013 μM in a kinase assay and with IC50 values of 0.0165 and 0.0121 μM in HaCaT (3TP-luc) stable cells and 4T1 (3TP-luc) stable cells, respectively, in a luciferase assay. Selectivity profiling of Vactosertib using a panel of 320 protein kinases revealed that it is a highly selective ALK5/ALK4 inhibitor with IC50 values of 13 nM and 11 nM respectively. Pharmacokinetic study with vactosertib in rats showed an oral bioavailability of 51%. Vactosertib has potential anti-cancer activity. A phase I clinical study is under way for multiple myeloma and advanced stage solid tumors
Kinase Assay: The 12b (EW-7197) inhibited ALK5 with IC50 value of 0.013 μM in a kinase assay and with IC50 values of 0.0165 and 0.0121 μM in HaCaT (3TP-luc) stable cells and 4T1 (3TP-luc) stable cells, respectively, in a luciferase assay. Selectivity profiling of 12b using a panel of 320 protein kinases revealed that it is a highly selective ALK5/ALK4 inhibitor. Pharmacokinetic study with 12b·HCl in rats showed an oral bioavailability of 51% with high systemic exposure (AUC) of 1426 ng × h/mL and maximum plasma concentration (Cmax) of 1620 ng/mL. Rational optimization of 6 has led to the identification of a highly potent, selective, and orally bioavailable ALK5 inhibitor 12b.
Cell Assay: To establish HaCaT (3TP-luc) and 4T1 (3TP-luc) stable cells, cells were seeded on six-well plates. Cells were allowed to adhere overnight and then transfected with the p3TP-luc (neo) expression plasmid using PEI reagent (Sigma-Aldrich). Transfected cells were cultured for 4 weeks in the presence of G418 (500 μg/mL). Several single clones were isolated and measured luciferase activity. The clone showing response to TGF-β1 treatment was used for reporter assay. HaCaT (3TP-luc) stable cells or 4T1 (3TP-luc) stable cells were seeded at 2.5 × 104 cells/well or 5 × 105cells/well in 96-well plate, respectively, and were allowed to adhere overnight. Cells were concomitantly treated with TGF-β1 (2 ng/mL) and indicated concentrations of ALK5 inhibitors in 0.2% FBS medium and incubated for 24 h at 37 °C in 5% CO2. Cell lysates were prepared using Luciferase Assay System (Promega) according to the manufacturer’s instruction, and luminescence was measured by a luminometer, Micro Lumat Plus (Berthold, Germany). |
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| In Vivo | Pharmacokinetic Study in Rats: Four groups of healthy male Sprague–Dawley rats (7 weeks and 190–210 g) were received 12b·HCl, 12z·HCl, 14f·HCl, and 14m·HCl intravenously at a dose of 2 mg/kg, respectively. Another four groups of rats were orally administered with these four compounds at a dose of 10 mg/kg, respectively. About 0.2 mL of blood samples were collected from the common carotid artery at 0, 0.08, 0.16, 0.25, 0.5, 0.75, 1, 2, and 4 h after intravenous or oral administration. Blood samples were centrifuged at 12000 rpm for 15 min immediately after sampling, and about 100 μL of plasma samples were stored at –70 °C until LC/MS/MS analysis. All animal procedures were approved by the Institutional Animal Ethics Committee of Ewha Womans University, Korea. Protein precipitation was conducted on 100 μL of plasma samples with 3 volumes of CH3CN containing 12a as analytical internal standard. After centrifugation, supernatant was analyzed by API2000 LC-ESI/MS/MS system in a positive MRM mode. Analytical data were processed using Analyst TM1.4.2. (Applied Biosystems, Concord, Canada.) Pharmacokinetic parameters were obtained by noncompartmental analysis of the plasma concentration–time profiles using Kinetica 4.4.1. |
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