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.

ABSTRACT:The UGT1 complex codes for a subfamily of homologous "1A7-like" UDP-glucuronosyltransferases (UGTs), including UGT1A7 and UGT1A8. Little information is available regarding either the substrate specificities or regulation of the UGT1A7-like forms from rats. We compared the activities and tissue expression of UGT1A7 and UGT1A8, which exhibit 77% identity in their amino terminal sequence. UGT1A7 shows broad specificity, catalyzing the glucuronidation of 31 of 40 randomly selected substrates (100 M) at rates >0.1 nmol/mg/min. UGT1A7 substrates included both planar and nonplanar compounds, mono-and polycyclic aromatics, and compounds with bulky side chain ring substitutions. UGT1A8 exhibited a narrower substrate specificity that completely overlapped with UGT1A7. UGT1A8 was most active toward the 1-OH, 4-OH, 5-OH, 6-OH, 7-OH, 10-OH, 11-OH, and 12-OH derivatives of benzo[a]pyrene. Other effective UGT1A8 substrates (>0.1 nmol/mg/ min) included 9-OH-benzo[a]pyrene, 1-naphthol, 4-methylumbelliferone, 7-hydroxycoumarin, chrysin, quercetin, 4-nitrophenol, and estriol. In general, substrates preferred by UGT1A8 were polyaromatic planar structures with nonbulky substituents and a superimposable 1-naphtho ring structure. Studies of the tissue expression of the UGT1A7 and 1A8 mRNAs using RNase protection analysis suggested that each is expressed in liver and kidney of control rats. A major difference is the higher expression of UGT1A7 mRNA in intestine. These studies suggest complementary functions of the UGT1A7 andUGT1A8 forms in xenobiotic metabolism. Further studies are necessary to determine whether their relative contributions change as a function of development, hormonal status, or exposure to inducing agents.

Life-threatening diarrhea afflicts a considerable percentage of patients treated with irinotecan, an anticancer agent with effects elicited through its active metabolite 7-ethyl-10-hydroxycamptothecin (SN-38). The primary detoxification pathway for SN-38 is glucuronidation. The purpose of this study was to evaluate the role that intestinal UDP-glucuronosyltransferases (UGTs) have from hepatic UGTs in modulating this diarrhea. To investigate this, Gunn rats devoid of UGT1A activity were injected with recombinant adenoviral vectors expressing UGT1A1, 1A6, and 1A7, resulting in reconstituted hepatic UGT expression comparable to a heterozygote. Hepatic microsome studies indicated that 4 to 7 days after adenoviral injection, transfected Gunn rats (j/jAV) had SN-38 glucuronide (SN-38G) formation rates three times higher than control heterozygote rats (jϩAV). The adenovirus did not impart any glucuronidating capacity to the intestine in j/jAV rats, whereas jϩAV rats possessed intestinal UGT function. After the administration of 20 mg/kg/day irinotecan i.p. to j/jAV rats 4 days after adenovirus injection, diarrhea ensued before the fourth irinotecan dose. jϩAV rats were spared the diarrhea, and the toxicity was mild compared with the j/jAV rats, as measured by diarrhea scores, weight loss, and histological assessments of the cecum and colon. The pharmacokinetics of irinotecan, SN-38, and SN-38G indicate that the systemic exposure of SN-38 and SN-38G was higher and lower, respectively, in j/jAV rats. Despite this, the biliary excretion of irinotecan and metabolites was similar. Because intestinal UGTs are the main discriminating factor between j/jAV and jϩAV rats, their presence seems to be critical for the gastrointestinal protection observed in jϩAV rats.The UDP-glucuronosyltransferases (UGTs) are a superfamily of metabolic enzymes that catalyze the transfer of UDP-glucuronic acid to endogenous and xenobiotic substrates. Glucuronidation is a detoxification mechanism from several standpoints. It decreases the apparent volume of distribution, increases the molecular weight, and increases substrate specificity for active transport by imparting a negative charge, which are all processes that facilitate substrate elimination from the body (Guillemette, 2003). Furthermore, in most cases, glucuronidation renders the substrate inactive with respect to its pharmacological or physiological target. A Wistar-derived rat model of UGT1A subfamily deficiency, the Gunn rat, has allowed tremendous insight into the importance of the UGT1A family in the metabolism and toxicity of substrates (Wells et al., 2004). The abolition of UGT1A glucuronidation in these rats stems from a frameshift mutation that yields a truncated, nonfunctional protein unable to bind UDP-glucuronic acid (Iyanagi et al., 1989).Although the primary organ of glucuronidation receiving most attention has been the liver, research on intestinal UGTs has shown their importance. Large differences in protein levels of the UGT1A family are not observed between these ...

ABSTRACT:Mycophenolic acid (MPA) is the active immunosuppressive metabolite of the anti-organ rejection drug mycophenolate mofetil (MMF) and is implicated in the gastrointestinal toxicity associated with MMF therapy. Intestinal UDP-glucuronosyltransferases (UGT) have been proposed to provide intrinsic resistance against MMF-induced gastrointestinal toxicity by converting MPA to the inactive MPA 7-O-glucuronide. Using an optimized intestinal microsome preparation method that stabilized the intestinal MPA UGT activity, the MPA UGT activity of male Sprague-Dawley rat intestinal microsomes was characterized. A longitudinal gradient similar to that described for other phenolic compounds was observed, with the activity decreasing from the duodenum to the distal small intestine and colon. The catalytic efficiency of MPA glucuronidation decreased from the proximal to distal intestine as a result of decreasing V max and increasing K m . The finding that homozygous Gunn rats lack detectable intestinal MPA UGT activity indicates exclusive roles of UGT1A1, UGT1A6, and/or UGT1A7. Quantitative immunoblotting revealed a parallel between the MPA UGT activity and the content of UGT1A7-like immunoreactivity (18.7 and 7.3 g/mg for duodenum and colon, respectively). In contrast, the lesser MPA-metabolizing UGT, UGT1A1 and UGT1A6, were lower in abundance (1.6-2.1 and 1.7-2.9 g/mg, respectively), and their patterns of longitudinal distribution were distinct from the MPA UGT activity. These data suggest a dominant role of a UGT1A7-like enzyme, presumably UGT1A7 itself, in the catalysis of rat intestinal MPA glucuronidation. Studies are ongoing to investigate the relationship between intestinal UGT1A enzymes and susceptibility to MMF-induced gastrointestinal toxicity.

ABSTRACT:Mycophenolate mofetil (MMF), the prodrug of mycophenolic acid (MPA), is included in current combination immunosuppressive regimens following organ transplant. Treatment with MMF often results in dose-limiting gastrointestinal (GI) side effects. The underlying mechanisms responsible for these side effects are not fully understood, but exposure of the intestinal epithelia to MPA during enterohepatic recycling may be involved. The present study demonstrated that female rats are more susceptible to MMF-induced GI toxicity than male rats. Female Sprague-Dawley rats treated chronically with an oral dose of 50 mg of MPA equivalents/kg/day experienced greater GI toxicity than male rats, as measured by diarrhea grade and weight loss. Intestinal microsomes harvested from the upper jejunum of female rats had approximately 3-fold lower MPA glucuronidation rates compared with male rats. In the remaining areas of the small and large intestine, there was also a trend toward decreased glucuronidation in the female rats. The area under the plasma concentration-time curve (AUC) for MPA following an oral dose of 50 mg of MPA equivalents/kg was roughly similar between genders, whereas the AUC for mycophenolic acid phenolic glucuronide (MPAG) was significantly lower in female rats. Female rats also excreted half of the biliary MPAG as male rats. The greater susceptibility of female rats to MMF-induced gastrointestinal toxicity, despite diminished intestinal MPA exposure via reduced biliary excretion of MPAG, may result from reduced protection of enterocytes by in situ glucuronidation. Likewise, susceptibility to MMF-induced GI toxicity in humans may also result from variable intestinal glucuronidation due to UDP glucuronosyltransferase polymorphisms or differential expression.