Minoru Oguchi

Identification of novel metabolic pathways of pioglitazone in hepatocytes: N-glucuronidation of thiazolidinedione ring and sequential ring opening pathway

The metabolism of [14C]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.

Pharmacokinetics, metabolism, and disposition of rivoglitazone, a novel peroxisome proliferator-activated receptor γ agonist, in rats and monkeys

The pharmacokinetics, metabolism, and excretion of rivoglitazone [(RS)-5-{4-[(6-methoxy-1-methyl-1H-benzimidazol-2-yl)methoxy]benzyl}-1,3-thiazolidine-2,4-dione monohydrochloride], a novel thiazolidinedione (TZD) peroxisome proliferator-activated receptor γ selective agonist, were evaluated in male F344/DuCrlCrlj rats and cynomolgus monkeys. The total body clearance and volume of distribution of rivoglitazone were low in both animals (0.329–0.333 ml per min/kg and 0.125–0.131 l/kg for rats and 0.310–0.371 ml per min/kg and 0.138–0.166 l/kg for monkeys), and the plasma half-life was 4.55 to 4.84 h for rats and 6.21 to 6.79 h for monkeys. The oral bioavailability was high (>95% in rats and >76.1% in monkeys), and the exposure increased dose proportionally. After administration of [14C]rivoglitazone, radioactivity was mainly excreted in feces in rats, whereas radioactivity was excreted in urine and feces with the same ratio in monkeys. Because excreted rivoglitazone in urine and bile was low, metabolism was predicted to be the main contributor to total body clearance. The structures of 20 metabolites (M1–M20) were identified, and 5 initial metabolic pathways were proposed: O-demethylation, TZD ring opening, N-glucuronidation, N-demethylation, and TZD ring hydroxylation. O-Demethylation was the main metabolic pathway in both animals, but N-demethylation and TZD ring hydroxylation were observed only in monkeys. N-Glucuronide (M13) was nonenzymatically hydrolyzed to TZD ring-opened N-glucuronide (M9), and the amount of these metabolites in monkeys was larger than that in rats. In plasma, rivolitazone was observed as the main component in both animals, and O-demethyl-O-sulfate (M11) was observed as the major metabolite in rats but as many minor metabolites in monkeys.

In Vitro Metabolism of Rivoglitazone, a Novel Peroxisome Proliferator-Activated Receptor γ Agonist, in Rat, Monkey, and Human Liver Microsomes and Freshly Isolated Hepatocytes

The in vitro metabolism of rivoglitazone, (RS)-5-{4-[(6-methoxy-1-methyl-1H-benzimidazol-2-yl)methoxy]benzyl}-1,3-thiazolidine-2,4-dione monohydrochloride, a novel thiazolidinedione (TZD) peroxisome proliferator-activated receptor γ selective agonist, was studied in liver microsomes and freshly isolated hepatocytes of rat, monkey, and human as well as cDNA-expressed human cytochrome P450 (P450) and UDP-glucuronosyltransferase (UGT) enzymes. Fourteen metabolites were detected, and these structures were elucidated by liquid chromatography-tandem mass spectrometry. Five initial metabolic pathways of rivoglitazone consisting of four oxidation pathways and one N-glucuronidation pathway were predicted in correspondence with those proposed for in vivo studies using rats and monkeys. In metabolization using liver microsomes, the TZD ring-opened mercapto amide (M22) and TZD ring-opened mercapto carboxylic acid (M23) were identified as the primary metabolite of the TZD ring-opening pathway and its sequential metabolite, which have not been detected previously from in vivo studies. Combination with S-adenosyl-l-methionine was useful to obtain the sequential S-methylated metabolites from the oxidative metabolites. N-Glucuronide and sequential TZD ring-opened metabolites were also found in liver microsomes in the presence of UDP-glucuronic acid. The O-demethyl-O-sulfate (M11), which is the major in vivo metabolite in rats and monkeys, was detected in all species of hepatocytes. In addition, a TZD ring-opened S-cysteine conjugate (M15) was detected in human hepatocytes. From these results, the in vivo metabolic pathways in humans were predicted to be the four oxidation and one N-glucuronidation pathways. The four oxidative metabolites were formed by multiple human P450 enzymes, and N-glucuronide was formed by UGT1A3 and UGT2B7.