Identification of novel metoclopramide metabolites in humans: in vitro and in vivo studies.

Metoclopramide (MCP) is frequently used to treat gastroparesis. Previous studies have documented MCP metabolism, but systematic structural identification of metabolites has not been performed. The aim of this study was to better understand MCP metabolism in humans. For examination of in vivo metabolism, a single oral 20-mg MCP dose was administered to eight healthy male volunteers, followed by complete urine collection over 24 h. In vitro incubations were performed in human liver microsomes (HLM) to characterize metabolism via cytochromes P450 and UDP-glucuronosyltransferases and in human liver cytosol for metabolism via sulfotransferases. Urine and subcellular incubations were analyzed for MCP metabolites on a mass spectrometer with accurate mass measurement capability. Five MCP metabolites were detected in vivo, and five additional metabolites were detected in vitro. The five metabolites of MCP identified both in vitro and in vivo were an N-O-glucuronide (M1), an N-sulfate (M2), a des-ethyl metabolite (M3), a hydroxylated metabolite (M4), and an oxidative deaminated metabolite (M5). To our knowledge, metabolites M1 and M4 have not been reported previously. M2 urinary levels varied 22-fold and M3 levels varied 16-fold among eight subjects. In vitro studies in HLM revealed the following additional metabolites: two ether glucuronides (M6 and M8), possibly on the phenyl ring after oxidation, an N-glucuronide (M7), a carbamic acid (M9), and a nitro metabolite (M10). Metabolites M6 to M10 have not been reported previously. In conclusion, this study describes the identification of MCP metabolites in vivo and in vitro in humans.

Characterizing the effects of common UDP glucuronosyltransferase (UGT) 1A6 and UGT1A1 polymorphisms on cis- and trans-resveratrol glucuronidation.

The dietary polyphenol resveratrol (RES) exists as cis- and trans-isomers with known stereospecific and stereoselective glucuronidation at the 3 and 4' positions by distinct UGT1A isoforms. We examined cis-RES glucuronidation in various protein sources. UGT1A6 or UGT1A1 genotype-dependent cis-or trans-RES glucuronidation, respectively, was further determined. cis-RES exhibited partial substrate inhibition in UGT1A6 Supersomes and human embryonic kidney 293 cells overexpressing genetically variant UGT1A6 alleles. Cells expressing UGT1A6*4 had the highest activity with a V(max) of 612 +/- 27.36 nmol/min/mg, followed by UGT1A6*3. The *2 allozyme had a higher V(max) (1.6-fold) and K(m) (1.9-fold) than *1. In 51 human liver samples genotyped for UGT1A6, four alleles (frequencies) were identified as *1 (0.58), *2 (0.36), *3 (0.01), and *4 (0.05), leading to assignment of the following genotypes (frequencies): *1/*1 (0.29), *1/*2 (0.45), *1/*3 (0.02), *1/*4 (0.10), and *2/*2 (0.14). Up to 5-fold variability in trans-RES glucuronidation was observed in individual liver samples. In livers stratified by UGT1A6 genotype, a significant difference in cis-RES glucuronidation activity (p < 0.05) was seen between the *2 variants compared with homozygous *1 livers. The trans-RES glucuronidation was quantitated in a human liver bank genotyped for the UGT1A1 TATA box repeat polymorphism. There was no significant difference for formation of trans-RES 3-O-glucuronide. We were surprised to find that trans-RES 4'-O-glucuronide formation was higher in livers with the 7/7 genotype compared with 6/6 and 6/7 (p < 0.05). In conclusion, cis-RES glucuronidation exhibited atypical partial substrate inhibition kinetics in vitro. Whereas cis-RES glucuronidation varied with UGT1A6 genotypes, the UGT1A1*28 polymorphism did not explain variability in trans-RES glucuronidation.

Variable sulfation of dietary polyphenols by recombinant human sulfotransferase (SULT) 1A1 genetic variants and SULT1E1.

Human cytosolic sulfotransferases (SULTs) catalyze the sulfate conjugation of several important endo- and xenobiotics. Among the superfamily of SULT enzymes, SULT1A1 catalyzes the sulfation of small planar phenolic compounds, whereas SULT1E1 has a major role in estrogen conjugation. The human SULT1A1 gene has common single nucleotide polymorphisms that define three allozymes, SULT1A1*1, *2, and *3. The enzyme kinetics of SULT1A1 allozymes and SULT1E1 were characterized for the polyphenolic substrates apigenin, chrysin, epicatechin, quercetin, and resveratrol. Purified recombinant SULT proteins were generated in a baculoviral-insect cell system, and incubated in vitro with each substrate to determine catalytic activity. The effect of polyphenol sulfation was examined in mammalian cell lines stably expressing SULT1E1. For all polyphenols investigated, "normal-activity" SULT1A1*1 allozyme had significantly greater Vmax estimates than SULT1E1, and allele-specific differences in SULT1A1-mediated sulfation were observed. The polymorphic SULT1A1*2 allozyme exhibited low activity toward apigenin, epicatechin, and resveratrol. SULT1A1*1 and *3 acted as normal-activity allozymes for these substrates. Altered cellular proliferation was observed in MCF-7 cells stably expressing SULT1E1 upon treatment with chrysin, quercetin, or resveratrol, thus suggesting inactivation of these compounds by SULT1E1. These results suggest an important role for SULT isozymes and their pharmacogenetics in polyphenol disposition.