cDNA-expressed UDP-glucuronosyltransferases Research Articles

In vitro metabolism of eupatilin by multiple cytochrome P450 and UDP-glucuronosyltransferase enzymes

Eupatilin, a pharmacologically active flavone derived from Artemisia plants, is extensively metabolized to eupatilin glucuronide, 4-O-desmethyleupatilin and 4-O-desmethyleupatilin glucuronide in human liver microsomes. This study characterized the human liver cytochrome P450 (CYP) and UDP-glucuronosyltransferase (UGT) enzymes responsible for the metabolism of eupatilin. The specific CYPs responsible for O-demethylation of eupatilin to the major metabolite, 4-O-desmethyleupatilin were identified using a combination of correlation analysis, immuno-inhibition, chemical inhibition in human liver microsomes and metabolism by human cDNA-expressed CYP enzymes. UGT enzymes involved in the eupatilin glucuronidation were identified using pooled human liver microsomes and human cDNA-expressed UGT enzymes. Eupatilin was predominantly metabolized by CYP1A2 and, to a lesser extent, CYP2C8 mediated O-demethylation of eupatilin to 4-O-desmethyleupatilin. Eupatilin glucuronidation was catalysed by UGT1A1, UGT1A3, UGT1A7, UGT1A8, UGT1A9, and UGT1A10.

Metabolism and Disposition in Humans of Raltegravir (MK-0518), an Anti-AIDS Drug Targeting the Human Immunodeficiency Virus 1 Integrase Enzyme

Raltegravir is a potent human immunodeficiency virus 1 (HIV-1) integrase strand transfer inhibitor that is being developed as a novel anti-AIDS drug. The absorption, metabolism, and excretion of raltegravir were studied in healthy volunteers after a single oral dose of 200 mg (200 microCi) of [(14)C]raltegravir. Plasma, urine, and fecal samples were collected at specified intervals up to 240 h postdose, and the samples were analyzed for total radioactivity, parent compound, and metabolites. Radioactivity was eliminated in substantial amounts in both urine (32%) and feces (51%). The elimination of radioactivity was rapid, since the majority of the recovered dose was attributable to samples collected through 24 h. In extracts of urine, two components were detected and were identified as raltegravir and the glucuronide of raltegravir (M2), and each accounted for 9% and 23% of the dose recovered in urine, respectively. Only a single radioactive peak, which was identified as raltegravir, was detected in fecal extracts; raltegravir in feces is believed to be derived, at least in part, from the hydrolysis of M2 secreted in bile, as demonstrated in rats. The major entity in plasma was raltegravir, which represented 70% of the total radioactivity, with the remaining radioactivity accounted for by M2. Studies using cDNA-expressed UDP-glucuronosyltransferases (UGTs), form-selective chemical inhibitors, and correlation analysis indicated that UGT1A1 was the main UGT isoform responsible for the formation of M2. Collectively, the data indicate that the major mechanism of clearance of raltegravir in humans is UGT1A1-mediated glucuronidation.

In Vitro Studies of Intestinal Permeability and Hepatic and Intestinal Metabolism of 8-Prenylnaringenin, a Potent Phytoestrogen from Hops (Humulus lupulus L.)

The absorption potential and metabolism of 8-prenylnaringenin (8-PN) from hops (Humulus lupulus L.) were investigated. 8-PN is a potent estrogen with the potential to be used for the relief of menopausal symptoms in women. Monolayers of the human intestinal epithelial cancer cell line Caco-2 and human hepatocytes were incubated with 8-PN to model its intestinal absorption and hepatic metabolism, respectively. The apparent permeability coefficients for 8-PN in the apical-to-basolateral and basolateral-to-apical directions of a Caco-2 monolayer were 5.2 +/- 0.7 x 10(-5) and 4.9 +/- 0.5 x 10(-5) cm/s, respectively, indicating good intestinal absorption via passive diffusion. Both glucuronide and sulfate conjugates of 8-PN were detected in the Caco-2 cell incubations. The 4'-O-glucuronide was the predominant Caco-2 cell metabolite, followed by 7-O-sulfate and 4'-O-sulfate. Both phase I and phase II metabolites of 8-PN were formed by human hepatocytes. The 7-O-glucuronide was the most abundant hepatocyte metabolite, and no sulfate conjugates were detected. Incubations with various cDNA-expressed UDP-glucuronosyltransferases indicated that the isozymes UGT1A1, UGT1A6, UGT1A8, and UGT1A9 were responsible for glucuronidation of 8-PN. Although orally administered 8-PN should be readily absorbed from the intestine, its bioavailability should be reduced significantly by intestinal and hepatic metabolism.

Bile acid glucuronidation by rat liver microsomes and cDNA-expressed UDP-glucuronosyltransferases

Four rat UDP-glucuronosyltransferases (UGTs), UGT2B1, UGT2B2, UGT2B3 and UGT2B6, synthesized in COS-7 cells from appropriate cDNA clones were screened for activity towards a range of bile acids, neutral steroids and retinoic acid. For comparison, as well as optimization of enzymatic assays and product identification, rat liver microsomal preparations from Sprague-Dawley, Fischer 344 and phenobarbital-induced Fischer 344 male rats were also used as enzyme sources. Only two of the expressed proteins, UGT2B1 and UGT2B2, were active in bile acid glucuronidation. UGT2B1 exhibited a high substrate specificity for the carboxyl function of bile acids, whereas UGT2B2 demonstrated less specificity, accepting both hydroxyl and carboxyl functions of bile acids. The preferred substrates for both cloned enzymes were mono-hydroxylated bile acids, followed by di-hydroxylated 6-OH compounds. The levels of UGT activity were sufficient to allow for the identification of the biosynthesized products. The data presented here demonstrate that bile acid glucuronidation is carried out, at least in part, by members of the UGT2B subfamily. Similar results have been obtained previously for neutral steroid glucuronidation. UGT2B3 and UGT2B6 was not involved in BA glucuronidation; none of the cloned enzymes was active toward retinoic acid.