Human cytochrome P4502C8 metabolizes repaglinide to 4'-hydroxyrepaglinide, not 3'-hydroxyrepaglinide

Previous reports have stated that cytochrome P4502C8 (CYP2C8) catalyzes the hydroxylation of repaglinide at the 3-position of the piperidine ring, and this metabolite has been used as a marker for CYP2C8 catalytic activity in vitro and in vivo. However, data shown in the present report demonstrate unequivocally that the actual site of hydroxylation is on the adjacent 4-position. The metabolite was biosynthesized using CYP2C8, isolated, and evaluated by high resolution mass spectrometry and 1D and 2D NMR spectroscopy. Definitive assignment of the structure required 2D heteronuclear single quantum correlation NMR analysis conducted at low temperature and the data for the isolated metabolite were contrasted with data gathered for the synthetic standard of 3'-hydroxyrepaglinide diastereomers. Repaglinide 4'-hydroxylation was measured in pooled human liver microsomes and recombinant CYP2C8 with Michaelis constants of 10.2 and 5.4 μM, respectively. Because repaglinide hydroxylation is an important drug metabolism reaction that probes CYP2C8 activity, it is critical that the correct structure of the CYP2C8 generated metabolite is known. SIGNIFICANCE STATEMENT: Previous studies have misassigned the structure of the main hydroxy metabolite of repaglinide that is generated by human P4502C8 (CYP2C8). This transformation has been used as a marker for CYP2C8 in clinical and in vitro studies. These data unequivocally demonstrate that the absolute structure of the hydroxyrepaglinide metabolite generated by CYP2C8 is 4'-hydroxymetabolite, not 3'-hydroxyrepaglinide as previously claimed.