| In Vitro | In vitro activity: RS-127445 is a novel high affinity, selective 5-HT2B receptor antagonist devoid of detectable intrinsic activity. RS-127445 is found to have nM affinity and 1000 fold selectivity for the 5-HT2B receptor. RS-127445 is thus among the highest affinity, most selective 5- HT2B receptor ligands. RS-127445 potently blocks the 5-HT evoked increase in inositol phosphate formation and blocks the 5-HT evoked increases in intracellular calcium concentrations with a potency 1000 times greater than that of yohimbine.
Kinase Assay: The selectivity of RS-127445 for 5-HT2B receptors is examined by testing the compound for affinity at over 100 additional ion channel or receptor binding sites. CHO-K1 cells expressing human 5-HT2A, 5-HT2B or 5-HT2C receptors are harvested using 2 mM EDTA in phosphate buffered saline. Cell membranes are prepared by four cycles of homogenization and centrifugation (48,000×g for 15 min). Each assay is established so as to achieve steady state conditions and to optimize specific binding. For the 5-HT2A receptor, membranes from 1×106 cells are incubated with 0.2 nM [3 H]-ketanserin at 32 °C for 60 min. Nonspecific binding is determined using 10 μM methysergide. For the 5-HT2B receptor, membranes from 1.5×106 cells are incubated with 0.2 nM [3 H]-5-HT at 48 °C for 120 min. Nonspecific binding is determined using 10 μM 5-HT. For the 5-HT2Creceptor, membranes from 3×10 5 cells are incubated with 0.5 nM [3 H]-mesuler -gine at 32 °C for 60 min. Nonspecific binding is determined using 10μM methysergide. Assays are terminated by vacuum filtration through glass fibre filters(GF/B) which has been pretreated with 0.1% polyethyleneimine. Total and bound radioactivity is determined by liquid scintillation counting. Greater than 90% specific binding is achieved in each of these assays.
Cell Assay: RS-127445, vehicle or other antagonists are pre-incubated with 240 μl of HEK-293 cells expressing the human 5-HT2B receptor suspension at 37 °C for 20 min. HEK-293 cells are incubated with[3H]-myoinositol (1.67 μCi/ml) in 162 cm2 flasks overnight at 37 °C in an inositol free Ham's F12 medium containing 10% dialyzed foetal bovine serum. The cells are harvested, washed five times with phosphate bufffered saline and resuspended in inositol free Ham's F12 media at density of approximately 3×103 cells/ml. The reactions are initiated by addition of 5-HT. Sixty minutes later, the reactions are terminated by adding 50 μl of ice-cold 20% perchloric acid, chilled in an ice-water bath for 10 min and then neutralized with 160μl of 1 N KOH. Each sample is diluted with 2 ml of 50 mM Tris-HCl, pH 7.4 at room temperature. The aqueous portion (2.2 ml) is transferred onto Dowex AG1X8 columns (1 ml, 1 : 1, w/v) which has been washed with 5 ml of distilled water. The columns are then washed with 18 ml of distilled water and the inositol phosphates are eluted with 3 ml of 1 N HCl. The eluted radioactivity is determined by liquid scintillation spectroscopy using a Packard 1900CA analyzer. |
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