In vitro activity: AVN-492 is a highly specific, selective and orally bioavailable antagonist of the 5-HT6R with Ki of 91 pM. The binding affinity of AVN-492 to 5-HT6R (Ki=91 pM) is more than three orders of magnitude higher than that to bind to the only other target, 5-HT2BR, (Ki=170 nM). Thus, the compound AVN-492 displayed great 5-HT6R selectivity against all other serotonin receptor subtypes, and is extremely specific against any other receptors such as adrenergic, GABAergic, dopaminergic, histaminergic, etc. AVN-492 demonstrates good in vitro and in vivo ADME profile with high oral bioavailability and good brain permeability in rodents. AVN-492 is a very promising tool for evaluating the role the 5-HT6 receptor might play in cognitive and neurodegenerative impairments. AVN-492 is an excellent drug candidate to be tested for treatment of such diseases, and is currently being tested in Phase I trials. The affinity of AVN-492 to bind to 5-HT6R (Ki=91 pM) is more than three orders of magnitude higher than that to bind to the only other target, 5-HT2BR, (Ki=170 nM). Thus, AVN-492 displays great 5-HT6R selectivity against all other serotonin receptor subtypes, and is extremely specific against any other receptors such as adrenergic, GABAergic, dopaminergic, histaminergic, etc

Kinase Assay: AVN-492 is dissolved into 100% DMSO to a concentration of 10mM. This DMSO solution is then diluted 50-fold with either anMQ water or a buffer with corresponding pH. The Caco-2 permeability assay is performed using the MultiScreen 96-well plates. In short, the Caco-2 cells (ATCC, Cat. No HTB-37) are seeded into each well with a porous membrane bottom. The cells are grown for 20-23 days at 37°C in CO2 thermostat until total confluence. The growth medium is changed every 2-3 days. The physical integrity of the cell monolayer established on the well porous membrane is tested using a “leak test” with Lucifer Yellow CH. Permeability ofAVN-492 (200μM) is determined in both directions, the apical-to-basal and basal-to-apical. Permeabilities of comparison compounds Ranitidine (lowpermeability) and Propranolol (high permeability) are measured in apical-to-basal direction only. The Pgp-dependent permeability of Rhodamine 123 (30 μM) is determined with or without the Pgp inhibitor Verapamil (100 μM). To assess participation of the Pgp pump in a possible efflux of AVN-492, the apical-to-basal and basalto-apical permeabilities are also registered in the presence of Verapamil. Concentrations of AVN-322 in donor and acceptor chambers are determined using LC/MS/MS API2000.

Cell Assay: