A radioimmunoassay for TA-0910, a new metabolically stable thyrotrophin-releasing hormone analogue

Abstract

TA-0910 [1-methyl-(S)-4,5-dihydroorotyl-L-histidyl-L-prolineamide] is a metabolically stable analogue of thyrotrophin-releasing hormone (TRH) and is under clinical investigation as a central nervous system function modulator. A method for determination of its plasma concentrations by radioimmunoassay (RIA) was established. TA-0910 was conjugated to bovine serum albumin and keyhole limpet haemocyanin (KLH) with bis-diazotized benzidine and 1,5-difluoro-2,4-dinitrobenzene as bridging agents. Anti-TA-0910 antisera were prepared by immunizing rabbits with the TA-0910 conjugates and Freund's complete adjuvant. The radiolabelled TA-0910 for RIA was prepared by introducing 125I into the histidine imidazole ring of TA-0910 by the Na125I/chloramine-T method, and purified by reversed phase high-performance liquid chromatography to give a specific radioactivity of 81.4 TBq mmole-1. As the result of testing the cross-reactivity of the antisera with assumed TA-0910 metabolites and TRH, a TA-0910-selective antiserum was obtained from a rabbit immunized with TA-0910-dinitrophenyl-KLH. RIA using this antiserum and the radiolabelled TA-0910 afforded a determination range of 10 pg approximately 5 ng ml-1 plasma. By using this RIA, the time courses of plasma concentrations of unchanged TA-0910 after oral and intravenous administration of TA-0910 were obtained in rats.