Glucuronidation of drugs in humanized UDP-glucuronosyltransferase 1 mice: Similarity with glucuronidation in human liver microsomes

Abstract

Uridine 5'-diphosphate-glucuronosyltransferases (UGTs) are phase II drug-metabolizing enzymes that catalyze glucuronidation of various endogenous and exogenous substrates. Among 19 functional human UGTs, UGT1A family enzymes largely contribute to the metabolism of clinically used drugs. While the UGT1A locus is conserved in mammals such as humans, mice, and rats, species differences in drug glucuronidation have been reported. Recently, humanized UGT1 mice in which the original Ugt1 locus was disrupted and replaced with the human UGT1 locus (hUGT1 mice) have been developed. To evaluate the usefulness of hUGT1 mice to predict human glucuronidation of drugs, UGT activities, and inhibitory effects on UGTs were examined in liver microsomes of hUGT1 mice as well as in those of wild-type mice and humans. Furosemide acyl-glucuronidation was sigmoidal and best fitted to the Hill equation in hUGT1 mice and human liver microsomes, while it was fitted to the substrate inhibition equation in mouse liver microsomes. Kinetic parameters of furosemide glucuronidation were very similar between hUGT1 mice and human liver microsomes. The kinetics of S-naproxen acyl-glucuronidation and inhibitory effects of compounds on furosemide glucuronidation in hUGT1 liver microsomes were also slightly, but similar to those in human liver microsomes, rather than in wild-type mice. While wild-type mice lack imipramine and trifluoperazine N-glucuronidation potential, hUGT1 mice showed comparable N-glucuronidation activity to that of humans. Our data indicate that hUGT1 mice are promising tools to predict not only in vivo human drug glucuronidation but also potential drug-drug interactions.

Keywords: Humanized animal model; UDP-glucuronosyltransferase; UGT; drug metabolism; species difference.

Figure 1

Kinetic analyses of furosemide acyl-glucuronide formation in liver microsomes. The substrate concentration-velocity curves (A) and Eadie–Hofstee plots (B) of the furosemide glucuronide formation are shown. Pooled liver microsomes of hUGT1 mice, humans, and regular mice were incubated with 50 μmol/L to 5 mmol/L furosemide and 5 mmol/L UDP-glucuronic acid at 37°C for 30 min. In the substrate concentration-velocity curves, data are the means ± SD of three independent determinations. In the Eadie–Hofstee plots, each data point represents the mean of three independent experiments.

Figure 2

Kinetic analyses of S-naproxen acyl-glucuronide formation in liver microsomes. The substrate concentration-velocity curves (A) and Eadie–Hofstee plots (B) of the S-naproxen glucuronide formation are shown. Pooled liver microsomes of hUGT1 mice, humans, and regular mice were incubated with 25 μmol/L to 2 mmol/L S-naproxen and 5 mmol/L UDP-glucuronic acid at 37°C for 30 min. In the substrate concentration-velocity curves, data are the means ± SD of three independent determinations. In the Eadie–Hofstee plots, each data point represents the mean of three independent experiments.

Figure 3

Inhibitory effects of estradiol (A) and imipramine (B) on furosemide acyl-glucuronide formation by liver microsomes of hUGT1 mice, humans, and regular mice. Residual activities were shown compared to the activity obtained in the absence of inhibitors. Data are the means ± SD of three independent determinations.

Figure 4

Inhibitory effects of serotonin (A) and propofol (B) on furosemide acyl-glucuronide formation by liver microsomes of hUGT1 mice, humans, and regular mice. Residual activities were shown compared to the activity obtained in the absence of inhibitors. Data are the means ± SD of three independent determinations.

Figure 5

Inhibitory effects of AZT on furosemide acyl-glucuronide formation by liver microsomes of hUGT1 mice, humans, and regular mice. Residual activities were shown compared to the activity obtained in the absence of AZT. Data are the means ± SD of three independent determinations.

Figure 6

Imipramine and trifluoperazine N-glucuronidation in liver microsomes of hUGT1 mice, humans, and regular mice. Fold activities were shown compared to the activity obtained in human liver microsomes. Each column represents the mean of three independent determinations. ND: not detected.