[Description]:
RS 127445 is a novel high affinity, selective 5-HT2B receptor antagonist with pKi of 9.5.
[Related Catalog]:
[Target]
5-HT1A Receptor:5.5 (pKi)
5-HT1B/D Receptor:<6 (pKi)
5-HT3 Receptor:<6 (pKi)
5-HT5 Receptor:<6 (pKi)
5-HT6 Receptor:<6 (pKi)
5-HT2A Receptor:6.3 (pKi)
5-HT2C Receptor:6.4 (pKi)
5-HT2B Receptor:9.5 (pKi)
[In Vitro]
RS-127445 is found to has nanomolar affinity for the 5-HT2B receptor (pKi=9.5±0.1) and 1,000 fold selectivity for this receptor as compared to numerous other receptor and ion channel binding sites. RS-127445 potently displaces [3H]-5-HT from human recombinant 5-HT2B receptors expressed in CHO-K1 cells. The affinity (pKi value) of RS-127445 for the 5-HT2B receptor is 9.5±0.1 (n=9). RS-127445 is selective for the 5-HT2B receptor, having approximately 1000 fold lower affinity for the human recombinant 5-HT2A, 5-HT2C, 5-HT5, 5-HT6 and 5-HT7 receptors, a 5-HT1A receptor in rat brain membranes, a 5-HT1B/D receptor in bovine caudate, and a monoamine uptake site in rabbit platelets. RS-127445 potently blocks the 5-HT (10 nM) evoked increases in intracellular calcium concentrations in the HEK-293 cells expressing the 5-HT2B receptor (pIC50 of 10.4±0.1). In cells expressing human recombinant 5-HT2B receptors, RS-127445 potently antagonizes 5-HT-evoked formation of inositol phosphates (pKB=9.5±0.1) and 5-HT-evoked increases in intracellular calcium (pIC10=10.4±0.1). RS-127445 also blocks 5-HT-evoked contraction of rat isolated stomach fundus (pA2B=9.5±1.1) and (±)α-methyl-5-HT-mediated relaxation of the rat jugular vein (pA2=9.9±0.3)[1].
[In Vivo]
In rats, the fraction of RS-127445 that is bioavailable via the oral or intraperitoneal routes is 14 and 60% respectively. Intraperitoneal administration of RS-127445 (5 mg/kg) produced plasma concentrations predicted to fully saturate accessible 5-HT2B receptors for at least 4 h.RS-127445 (5 mg/kg) is administered to rats by oral, intraperitoneal and intravenous routes. Peak plasma concentrations are rapidly achieved with the highest concentrations being found at the first time-point measured following intravenous and intraperitonael administration (0.08 h) and by 0.25 h following dosing by the oral route of administration. RS-127445 is cleared from plasma with an estimated terminal elimination half-life of approximately 1.7 h. The bioavailability of RS-127445, when administered by the oral and intraperitoneal routes is approximately 14 and 62% of that obtained by intravenous administration. Approximately 60% of an intraperitoneal dose and 14% of the oral dose of RS-127445 (5 mg/kg) is bioavailable[1]. RS-127445 (1-30 mg/kg), dose-dependently reduces faecal output, reaching significance at 10 and 30 mg/kg (n=6-11). In blood and brain, >98% of RS-127445 is protein-bound[2].
[Kinase Assay]
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 (Brinkman P10 disrupter) and centrifugation (48,000×g for 15 min). As previously described, 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 [3H]-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 [3H]-5-HT at 4°C for 120 min. Nonspecific binding is determined using 10 μM 5-HT. For the 5-HT2C receptor, membranes from 3×105 cells are incubated with 0.5 nM [3H]-mesulergine at 32°C for 60 min. Nonspecific binding is determined using 10 μM methysergide. Assays are terminated by vacuum filtration through glass fibre filters (GF/B) which had 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.The selectivity of RS-127445 for 5-HT2B receptors is further examined by testing the compound for affinity at over 100 additional ion channel or receptor binding sites. These studies are conducted by Cerep using standard protocols[1].
[Cell Assay]
HEK-293 cells expressing the human 5-HT2B receptor 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 buffered saline and resuspended in inositol free Ham's F12 media at density of approximately 3×106 cells/mL. RS-127445 (10 μM) is initially dissolved in 10% (v/v) DMSO with 90% inositol free Ham's F12 medium. Subsequent dilutions are made with inositol free Ham's F12 medium. 5-HT is dissolved in inositol free Ham's F12 medium containing 100 mM LiCl and 1 mM ascorbate. RS-127445, vehicle or other antagonists are pre-incubated with 240 μL of cell suspension at 37°C for 20 min. 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 160 μL 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 had 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[1].
[Animal admin]
Rats[1] Male Sprague-Dawley rats (200 g) are used. To compare the plasma kinetics of RS-127445 following different routes of administration, 90 rats are distributed into three treatment groups of 30 rats each. A single dose of RS-127445 (5 mg/kg) dissolved (2.5 mg/mL) in ethanol:propylene glycol : water (10 : 50 : 40, v:v:v), is administered to each rat. At 0.08, 0.25, 0.5, 1, 2, 4, 8 and 24 h after dosing, the rats are anaesthetized and blood samples are collected by cardiac puncture. Mice[2] Adult male C57BL/6J mice (25-30 g) are used. The effects of RS-127445 (1 nM-10 µM, single concentration per tissue, 15 min contact time) or vehicle (5 or 50 µL DMSO) are expressed as the percentage change in amplitude compared with the mean amplitude of four pre-drug, post-EFS contractile responses. The results are analysed using a two-sample equal variance t-test.
[References]
[Related Small Molecules]