Obtaining exposures of metabolites in preclinical species through plasma pooling and quantitative NMR: addressing metabolites in safety testing (MIST) guidance without using radiolabeled compounds and chemically synthesized metabolite standards.

The recent guidance on “Safety Testing of Drug Metabolites” issued by the U.S. Food and Drug Administration, Center for Drug Evaluation and Research (CDER) has highlighted the importance of identifying and characterizing drug metabolites as early as possible in drug discovery and development. Furthermore, upon identifying significant circulating metabolites in human plasma, it has become important to demonstrate that these metabolites are present at an equal or greater exposure level (area under the curve, AUC) in any one of the preclinical species used in safety testing. Frequently, synthetic standards of metabolites are not available, and hence, obtaining their AUC values can be a challenge. In this report, we demonstrate how combinations of nuclear magnetic resonance (NMR) spectroscopy, liquid chromatography/ultraviolet/mass spectrometry (LC/UV/MS), and plasma pooling methods were used to obtain reliable AUC values of metabolites present in the plasma of preclinical species from short-term safety studi...