Patients took taltirelin (5 mg bid) or placebo for 28 to 52 weeks.

HEK-EM 293 (human embryonic kidney) cells stably expressing TRH-R were seeded in black-walled, clear bottomed 96-well plates (Corning, NY, USA) at a density of 60,000 cells/well in DMEM with 10% fetal bovine serum and incubatedfor24hat37◦C in5%CO2. The following day, the culture media was replaced with 100 μl of Hank’s balanced salt solution with 20 mM HEPES, pH 7.5 and the cells were loaded with100 μl of calcium 4 fluorescent dye (Molecular Devices, Sunnyvale, CA, USA) for 1 h at room temperature before addition of compounds .Transient changes in intracellular [Ca++] induced by TALTIRELIN were measured using the FLIPRTETRA system (Molecular Devices, Sunnyvale, CA, USA). Changes in fluorescence were detected at the emission wavelength ranges from 515 to 575 nm. The agonistic responses of ligands were assessed immediately upon their addition in a concentration range from 0.1 nMto30 μM.