A radioreceptor assay for the analysis of AT1-receptor antagonists. Correlation with complementary LC data reveals a potential contribution of active metabolites.

A reliable and sensitive radioreceptor assay based on rat lung homogenate as receptor preparation was developed to determine the angiotensin-II antagonistic profile of losartan and its main active metabolite EXP 3174 as well as its congeners exemplified by UP 269-6 and SL 91.0102-90 DL. This method proved to be precise with an intra- and interday variability of less than 10% and a limit of quantification < or = 1 ng ml-1. The analysis of the Ki values in protein-free Hepes-buffer versus blank human or rat plasma revealed the distinct high plasma-protein binding of EXP 3174 which consequently caused a dramatic drop of potency from 10-15-fold in the buffer to only about 2-fold in control plasma, when compared to the parent compound losartan and the two congeners investigated. Upon evaluation of clinical samples by both the reported radioreceptor assay (RRA) and the established high-performance liquid chromatography (HPLC), the correlation of the normalized data pairs (concentration equivalents) suggested the contribution of active metabolites to the angiotensin-II antagonistic effect of SL 91.0102-90 DL, but not to the effect of UP 269-6. In the context of an extended preclinical study in rats, the correlation of RRA with the respective HPLC concentration equivalents of losartan and its main active metabolite EXP 3174 confirmed previous findings that only losartan and EXP 3174 exert the angiotensin-II-AT1 receptor blockade without the contribution of other metabolites (P.C. Wong, W.A. Price, A.T. Chiu et al., J. Pharmacol. Exp. Ther. 255 (1990) 211-217).