An Investigation of Human and Rat Liver Microsomal Mycophenolic Acid Glucuronidation: Evidence for a Principal Role of Ugt1a Enzymes and Species Differences in Ugt1a Specificity

Abstract

ABSTRACT:Mycophenolic acid (MPA; 1,3-dihydro-4-hydroxy-6-methoxy-7-methyl-3-oxo-5-isobenzylfuranyl)-4-methyl-4-hexenoate), the active metabolite of the immunosuppressant prodrug, mycophenolate mofetil, undergoes glucuronidation to its 7-O-glucuronide as a primary route of metabolism. Because differences in glucuronidation may influence the efficacy and/or toxicity of MPA, we investigated the MPA UDP-glucuronosyltransferase (UGT) activities of human liver microsomes (HLMs) and rat liver microsomes with the goal of identifying UGTs responsible for MPA catalysis. HLMs (n ‫؍‬ 23) exhibited higher average MPA glucuronidation rates (14.7 versus 6.0 nmol/mg/min, respectively, p < 0.001) and higher apparent affinity for MPA (K m ‫؍‬ 0.082 mM versus 0.20 mM, p < 0.001) compared with rat liver microsomes. MPA UGT activities were reduced >80% in liver microsomes from Gunn rats. To identify the active enzymes, human and rat UGT1A enzymes were screened for MPA-glucuronidating activity. UGT1A9 was the only human liverexpressed UGT1A enzyme with significant activity and exhibited both high affinity (K m ‫؍‬ 0.077 mM) and high activity (V max ‫؍‬ 28 nmol ⅐ min ؊1 ⅐ mg for rat with three active liver-expressed UGT1A enzymes: 1A1 (medium affinity/capacity), 1A6 (low affinity/medium capacity), and 1A7 (high affinity/capacity). Our data suggest that UGT1A enzymes are the major contributors to hepatic MPA metabolism in both species, but 1A9 is dominant in human, whereas 1A1 and 1A7 are likely the principal mediators in control rat liver. This information should be useful for interpretation of MPA pharmacokinetic and toxicity data in clinical and animal studies.

Regioselective Glucuronidation of Tanshinone IIa after Quinone Reduction: Identification of Human UDP-Glucuronosyltransferases, Species Differences, and Interaction Potential

“…Thus, it is questionable which UGT isoform in rats is mainly involved in the TSA glucuronidation. UGT1A1 and UGT1A7 may be involved in TSA glucuronidation in rats, similar to that found for MPA (Miles et al, 2005). In view of the large species differences in producing M1 and M2, we consider that the specific UGT isoforms responsible for TSA glucuronidation may be different across various species.…”

“…The incubation conditions were the same as those described previously. Propofol, MPA, and their glucuronides were determined based on previous reports (Vree et al, 1999;Miles et al, 2005).…”

Lack of Association between Common Polymorphisms in UGT1A9 and Gene Expression and Activity

“…Even though we could not quantify the rate of formation of FLAVO-7-G using standards, we believe we can detect accurately differences in glucuronidation activities, as the activity measurements were done under linear assay conditions and using a substrate concentration equal to the K m determined under the experimental conditions reported in this study. In addition, a significant and modest correlation was observed between the glucuronidation of flavopiridol and mycophenolic acid, a commonly used UGT1A9 probe Girard et al, 2004;Miles et al, 2005). However, the lack of high correlation suggests that both drugs may be glucuronidated to a certain extent by other UGTs (Hagenauer et al, 2001;Ramirez et al, 2002;Bernard and Guillemette, 2004;Miles et al, 2005;Picard et al, 2005).…”

“…UGT1A9 Glucuronidation Activity. UGT1A9 activities in 46 human liver microsomes were determined using two specific UGT1A9 substrates, mycophenolic acid Miles et al, 2005) and flavopiridol (Hagenauer et al, 2001;Ramirez et al, 2002). Optimal conditions for formation of mycophenolic acid phenyl glucuronide (MPAG) were determined by varying incubation times (0 -60 min), microsomal protein (0.1-1 mg/ml), and substrate concentration (1-1000 M).…”

“…UGT1A9, a member of the UGT1A family, plays an important role in the glucuronidation of flavopiridol (Hagenauer et al, 2001;Ramirez et al, 2002), mycophenolic acid (Mackenzie 2000;Basu et al, 2004;Bernard and Guillemette, 2004;Miles et al, 2005;Picard et al, 2005), propofol (Court, 2005), acetaminophen (Court et al, 2001) and propranolol (Sten et al, 2006). UGT1A9 is expressed in multiple tissues, including liver, kidney, colon, and esophagus (Albert et al, 1999;Tukey and Strassburg, 2000).…”