Identification of Novel Metabolic Pathways of Pioglitazone in Hepatocytes: N-Glucuronidation of Thiazolidinedione Ring and Sequential Ring-Opening Pathway

The metabolism of [C-14] pioglitazone was studied in vitro in incubations with freshly isolated human, rat, and monkey hepatocytes. Radioactivity detection high-performance liquid chromatography analysis of incubation extracts showed the detection of 13 metabolites (M1-M13) formed in incubations with human hepatocytes. An identical set of metabolites (M1-M13) was also detected in monkey hepatocytes. However, in rat hepatocytes, M1 through M3, M5 through M7, M9 through M11, and M13 were also detected, but M4, M8, and M12 were not detected. The structures of the metabolites were elucidated by liquid chromatography/tandem mass spectrometry using electrospray ionization. Novel metabolites of pioglitazone detected using these methods included thiazolidinedione ring-opened methyl sulfoxide amide (M1), thiazolidinedione ring-opened N-glucuronide (M2), thiazolidinedione ring-opened methyl sulfone amide (M3), thiazolidinedione ring N-glucuronide (M7), thiazolidinedione ring-opened methylmercapto amide (M8), and thiazolidinedione ring-opened methylmercapto carboxylic acid (M11). In summary, based on the results from these studies, two novel metabolic pathways for pioglitazone in hepatocytes are proposed to be as follows: 1) N-glucuronidation of the thiazolidinedione ring of pioglitazone to form M7 followed by hydrolysis to M2, and methylation of the mercapto group of the thiazolidinedione ring-opened mercapto carboxylic acid to form M11; and 2) methylation of the mercapto group of the thiazolidinedione ring-opened mercapto amide to form M8, oxidation of M8 to form M1, and oxidation of M1 to form M3.