Introduction

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The glucuronidation of xenobiotics and endobiotics represents a major route of disposition for these agents in animals. In this process, UDP-glucuronosyltransferases (UGTs¹), residing in the endoplasmic reticulum and nuclear membrane of cells, catalyze the conjugation of exogenous and endogenous lipophilic substrates creating metabolites that are more water soluble and readily excreted. Although the role of glucuronidation has generally been considered to be a dominant feature of detoxification, recent studies have shown that certain glucuronide metabolites may display more potent pharmacologic or toxicological activity than the parent compound. For example, morphine-6-glucuronide is a more active analgesic agent than morphine (Shimomura et al., 1971; Osborne et al., 1988; Osborne et al., 1992), and certain estrogen glucuronides induce hepatic toxicity (Vore et al., 1997). Glucuronides may also serve as part of a therapeutic regimen (Gunning et al., 1994; Nolen et al., 1995).

Two UGT gene families have been identified in humans which encode for proteins that catalyze the glucuronidation of xenobiotics and endobiotics (Mackenzie et al., 1997). The human UGT1 gene family is a complex of 12 unique first exons coding for 9 isoforms, each of which share common second through fifth exons encoding for an identical carboxyl terminal moiety for each protein (Ritter et al., 1992). These isoforms, with the exception of UGT1A5, are important in the metabolism of endobiotics (bilirubin and estrogens), tertiary amines, phenols, and flavonoids (King et al., 2000). It is interesting to note that UGT1A1 catalyzes bilirubin glucuronidation, a critical physiological metabolic reaction, and the glucuronidation of many other xenobiotics such as buprenorphine, an opioid agonist/antagonist, and endobiotics such as estrone and 2-hydroxyestrogen catechols. The UGT2 gene family codes for proteins which are products of separate genes (Mackenzie and Rodbourn, 1990; Haque et al., 1991). These enzymes are very important in drug metabolism, especially UGT2B7. UGT2B7 has a wide distribution in the human organism and reacts with a wide variety of chemical agents and drugs (King et al., 2000) such as morphine, zidovudine, and nonsteroid anti-inflammatory drugs. It has been shown to exist in many extrahepatic tissues, including brain, and to catalyze the glucuronidation of the neurotransmitter serotonin (King et al., 1999), as well as many opioid drugs. The existence of UGT2B7 in the brain is of great importance in the conversion of morphine to the morphine 6-glucuronide, making the requirement for the transport of this conjugate across the blood-brain-barrier and into the brain unnecessary.

The current study was undertaken to study the effects of certain potential inhibitors and alternate substrates on UGTs, especially those known to react with the opioid substrates. Several inhibitors and alternate substrates for UGT2B7 were then studied with UGTs 1A1 and 1A6. The differential inhibition of UGT1A1 catalyzed glucuronidation by these compounds provides evidence for more than one active site in this isoform.

	Materials and Methods

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Chemicals. Losartan was a gift from Merck Laboratories (Rahway, NJ). Morphine, dextrorphan, naltriben, etonitazenyl isothiocyanate, and UDP-[U-¹⁴C] glucuronic acid were purchased from Merck Laboratories, Hoffman LaRoche (Nutly, NJ), Research Biochemicals International (Natick, MA), TOCRIS Chemicals (Ballwin, MO), and ICN Radiochemicals (Costa Mesa, CA). All other aglycone substrates and inhibitors were purchased from Sigma-Aldrich (St. Louis, MO). The protein assay reagents were purchased from Bio-Rad Labs (Hercules, CA). All other reagents were of the highest purity.

Expression of UGTs in Human Embryonic Kidney 293 Cells. The expression of human UGTs 2B7 (Coffman et al., 1997), 1A1 (King et al., 1996), and 1A6 (King et al., 1999) in HK293 cells has been previously described.

Glucuronidation Assays. The membrane preparation for each of the UGTs expressed in HK293 cells and stored at 80°C was previously described (Battaglia et al., 1994). These preparations were thawed and resuspended in 10 mM Tris-buffered saline (pH 7.4) containing 0.5 mM dithiothreitol. Protein concentrations were determined by the Bio-Rad protein assay.

	Results

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Inhibition of UGT2B7 Activities. UGT2B7 is an important catalyst for the glucuronidation of many important therapeutic agents of diverse chemical structure. Although the substrate reactivity is quite diverse, a previous study using the NMR spectroscopy of an aglycone active site-fusion protein pointed to only one active site (Coffman et al., 2001) for UGT2B7. Studies were performed to explore this further and to determine whether drug-drug interactions were possible by using inhibitors and alternate substrates for UGT2B7. Morphine, one of the morphinan class of opioid substrates, and androsterone, a steroid, were chosen as representative substrates since their glucuronidation is catalyzed by UGT2B7. A number of potential inhibitors of UGT2B7 were tested. One such inhibitor, etonitazenyl, is known as a selective µ opioid antagonist (Burke et al., 1984), which is not glucuronidated by UGT2B7. Figures 1 and 2 show the competitive inhibition of morphine and androsterone by etonitazenyl. The inhibitor constants (K_i) for etonitazenyl were 71 and 141 µM when studied with morphine and androsterone, respectively.

View larger version (21K): [in this window] [in a new window]   Fig. 1.   The competitive inhibition of UGT2B7-mediated morphine glucuronidation by etonitazenyl. The assays were performed under the following optimal conditions for morphine glucuronidation: 37°C for 10 min at pH 8.4. The concentration of UDP-glucuronic acid was 2.0 mM, and the aglycone substrate concentrations were from 0.05 to 5.0 mM. The competitive inhibition of morphine glucuronidation is denoted by the addition of etonitazenyl to the reaction. The data are shown by , control; , 10 µM etonitazenyl; , 50 µM etonitazenyl; and , 100 µM etonitazenyl.

View larger version (22K): [in this window] [in a new window]   Fig. 2.   The competitive inhibition of UGT2B7-mediated androsterone glucuronidation by etonitazenyl. The assays were performed under the following optimal conditions for androsterone glucuronidation: 37°C for 15 min at pH 7.4. The concentration of UDP-glucuronic acid was 2.0 mM, and the aglycone substrate concentrations are from 5 to 50 µM. The competitive inhibition of androsterone glucuronidation is denoted by the addition of etonitazenyl to the reaction. The data are shown by , control; , 50 µM etonitazenyl; and , 100 µM etonitazenyl.

Inhibition of UGT1A1. UGT 1A1 is considered to be the most important enzyme involved in the disposition of bilirubin. However, UGT1A1 also catalyzes the glucuronidation of a number of other endobiotic and xenobiotic substrates which include the catechol estrogen, 2-hydroxyestrone; the anthraquinone, anthraflavic acid; and the opioid, buprenorphine. Since the opioid substrate, buprenorphine, is an excellent substrate for both UGT1A1 and UGT2B7, and since etonitazenyl was an effective competitive inhibitor of morphine glucuronidation catalyzed by UGT2B7, it was of interest to determine whether etonitazenyl might inhibit the UGT1A1-catalyzed glucuronidation of buprenorphine. Etonitazenyl competitively inhibited buprenorphine glucuronidation catalyzed by UGT1A1 (Fig. 3) with a K_i value of 5.5 µM, a value approximately 20-fold lower than that found when used as an inhibitor of UGT2B7. In spite of its profound inhibitory effect on buprenorphine glucuronidation, etonitazenyl concentrations above 100 µM were necessary to show any inhibition with 2-hydroxyestrone and anthraflavic acid (Table 1) and had little to no effect on bilirubin glucuronidation (Fig. 4).

View larger version (28K): [in this window] [in a new window]   Fig. 3.   The competitive inhibition of UGT1A1-mediated buprenorphine glucuronidation by etonitazenyl. The assays were performed under the following optimal conditions for buprenorphine glucuronidation: 37°C for 20 min at pH 7.0. The concentration of UDP-glucuronic acid was 2.0 mM, and the aglycone substrate concentrations were from 10 to 100 µM. The competitive inhibition of buprenorphine glucuronidation was shown by the addition of etonitazenyl to the reaction. The data are shown by  control; , 3.125 µM etonitazenyl; , 6.25 µM etonitazenyl; , 12.5 µM etonitazenyl; and , 25 µM etonitazenyl.

View larger version (42K): [in this window] [in a new window]   Fig. 4.   The effect of buprenorphine and etonitazenyl on UGT1A1-mediated bilirubin glucuronidation. The assays were performed under the following optimal conditions for bilirubin glucuronidation: 37°C for 60 min at pH 7.4. The concentration of UDP-glucuronic acid was 2.0 mM, and the aglycone substrate concentration was 20 µM. The open bars indicate assays performed with etonitazenyl, and the hatched bars indicate assays performed with buprenorphine. The values ± S.D. are from three experiments.

Studies on UGT1A6. Given results obtained with UGT2B7 and UGT1A1, the etonitazenyl inhibition of glucuronidation was considered to be due to interaction with enzyme sites only related to opioid binding or substrates using sites proximate to opioid binding. Inhibition studies were performed with the recombinant human UGT1A6, which does not accept opioid substrates. Etonitazenyl was used as a possible inhibitor with 1-naphthol and 4-methylumbelliferone, two excellent substrates for UGT1A6. As expected, inhibition was not observed with the glucuronidation of either 1-naphthol or 4-methylumbelliferone (data not shown).

	Discussion

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A previous study from this laboratory suggested that the aglycone binding region of UGT2B7 possessed a single site, which could accept a number of chemically divergent substrates (Coffman et al., 2001). The current study supports this idea. K_m values determined for UGT2B7 with substrates such as naloxone, zidovudine, androsterone, and buprenorphine were essentially identical to K_D values obtained for these compounds when they were used to displace morphine binding to the aglycone binding region of the fusion protein used in that study. Furthermore, etonitazenyl inhibition of morphine, androsterone, losartan, and zidovudine glucuronidation was competitive, and K_i values, obtained for etonitazenyl, were comparable irrespective of the substrate studied. Studies using other inhibitors or alternate substrates of UGT2B7 also support the notion that there is one binding domain in UGT2B7. Alternate substrates such as naltriben and morphine, when used as inhibitors, display K_i values similar to their K_m values. Due to the many and diverse substrates for UGT2B7, a major question remains as to whether there might be potential drug-drug interactions, in vivo, involving this protein. This has yet to be answered, but it would seem unlikely, based on the high capacity of UGT2B7 to serve as a catalyst in the glucuronidation of these substrates. Furthermore, no clinical studies have suggested that drug-drug or drug-endobiotic interaction exists.

On the other hand, inhibition studies carried out with recombinant human UGT1A1 suggest that this protein possesses more than one active site. Buprenorphine and bilirubin are two substrates that react with human UGT1A1 with high efficiency. Etonitazenyl, a µ-specific opioid antagonist and an inhibitor of UGT2B7, was chosen to see if it inhibited opioid glucuronidation catalyzed by UGT1A1. Etonitazenyl inhibited the UGT1A1-catalyzed glucuronidation of buprenorphine competitively and with high potency (K_i, 5.5 µM), a value 20-fold lower than that found for the K_i of etonitazenyl on morphine glucuronidation catalyzed by UGT2B7. Suprisingly, etonitazenyl had no effect on bilirubin glucuronidation at concentrations up to 25 µM and required 100 µM or higher to effect a modest inhibition. To test the idea that UGT1A1 had separate active sites for bilirubin and buprenorphine, buprenorphine was used as an inhibitor of bilirubin glucuronidation. Buprenorphine had no significant inhibitory effect on bilirubin glucuronidation except at extraordinary concentrations. Therefore, it appears that distinct binding regions are involved in the reaction of these substrates with UGT1A1. In related studies, neither etonitazenyl nor buprenorphine significantly inhibited the UGT1A1-mediated glucuronidation of estrogen catechols, 17-ethynylestradiol, or anthraflavic acid. Previously, we postulated that patients treated with buprenorphine, possibly for opioid dependence, might experience jaundice due to the possible inhibition of bilirubin glucuronidation (King et al., 2000). It is important to point out that the current study suggests that this would be unlikely because of different binding domains within UGT1A1 for buprenorphine and bilirubin.

Etonitazenyl has been shown here to inhibit a wide range of glucuronidation reactions mediated by UGT2B7 and opioid glucuronidation by UGT1A1. It had no effect on UGT1A6-catalyzed glucuronidation of phenols (1-naphthol) or coumarins (4-methylumbelliferone). Further studies need to be performed with this compound to test its effect on other UGTs, such as UGT1A3 and UGT1A4, which are known to react with amine substrates such as the tertiary amines which are converted to quaternary amine glucuronides (Green and Tephly, 1998).