Characterization of Rat and Human UDP-Glucuronosyltransferases Responsible for the in Vitro Glucuronidation of Diclofenac

C. King*,1, W. Tang*, J. Ngui*, T. Tephly{dagger} and M. Braun*

* Merck & Company, Department of Drug Metabolism, P.O. Box 2000, RY80-A9, Rahway, New Jersey 07065; and {dagger} University of Iowa, Department of Pharmacology, Iowa City, Iowa 52242

Received October 18, 2000; accepted January 18, 2001


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In the current study, the identification of the rat and human UDP-glucuronosyltransferase (UGT) isoforms responsible for the glucuronidation of diclofenac was determined. Recombinant human UGT1A9 catalyzed the glucuronidation of diclofenac at a moderate rate of 166-pmol/min/mg protein, while UGT1A6 and 2B15 catalyzed the glucuronidation of diclofenac at low rates (<20-pmol/min/mg protein). Conversely, human UGT2B7 displayed a high rate of diclofenac glucuronide formation (>500 pmol/min/mg protein). Recombinant rat UGT2B1 catalyzed the glucuronidation of diclofenac at a rate of 250-pmol/min/mg protein. Rat UGT2B1 and human UGT2B7 displayed a similar, low apparent Km value of <15 µM for both UGT isoforms and high Vmax values 0.3 and 2.8 nmol/min/mg, respectively. Using diclofenac as a substrate, enzyme kinetics in rat and human liver microsomes showed that the enzyme(s) involved in diclofenac glucuronidation had a low apparent Km value of <20 µM and a high Vmax value of 0.9 and 4.3 nmol/min/mg protein, respectively. Morphine is a known substrate for rat UGT2B1 and human UGT2B7 and both total morphine glucuronidation (3-O- and 6-O-glucuronides) and diclofenac glucuronidation reactions showed a strong correlation with one another in human liver microsome samples. In addition, diclofenac inhibited the glucuronidation of morphine in human liver microsomes. These data suggested that rat UGT2B1 and human UGT2B7 were the major UGT isoforms involved in the glucuronidation of diclofenac.

Key Words: acyl-glucuronides; UGTs; diclofenac; glucuronidation; NSAIDs.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Glucuronidation is generally considered to be a major detoxification process, which enhances the excretion of xenobiotics and endobiotics from the body. Glucuronides, in many cases, are inert, polar metabolites, but active and reactive metabolites are known. For instance, the central actions of morphine are believed to be mediated by morphine-6-O-glucuronide, which is 50 times more potent as an analgesic than morphine itself (Gong et al., 1991Go; Shimomura et al., 1971Go). Some carboxylic acid acyl-glucuronides, including tolmetin and ketoprofen glucuronides, are known to be chemically reactive metabolites (Hyneck et al., 1988Go; Munafo et al., 1990Go). Acyl-glucuronides have been shown to form covalent adducts with protein, possibly contributing to the toxicity of some non-steroidal anti-inflammatory drugs (NSAIDs; Bailey and Dickenson, 1996). Zomepirac was withdrawn from the U.S. market because of an unexplained anaphylactic reaction (Committee on Government Operations, 1983Go; Samuel, 1981Go). It was also shown to form a covalent adduct with plasma proteins. However, no direct correlation between acyl-glucuronide covalent binding and toxicity has emerged. Even endogenous carboxylic acid-containing compounds, such as bilirubin and bile acids, have been shown to covalently bind to proteins (Ikegawa et al., 1999Go; McDonagh et al., 1984Go; Gautam et al., 1984Go) without evidence of toxicity. Recently, Terrier et al. (1999) showed that the enzymatic facilitators of glucuronidation, the UDP-glucuronosyltransferases (UGTs), were potential targets for drug-protein adducts.

Diclofenac (2-[(2,6-dichlorophenyl)amino]benzene acetate) is an effective and frequently prescribed NSAID, that is metabolized by several drug metabolizing pathways, including cytochrome P450s (CYPs) and UGTs. It has been shown to increase serum transaminases in approximately 15% of patients taking the drug on a regular basis (Ciccolunghi et al., 1978Go), suggesting that diclofenac may cause hepatotoxicity. In mice and rats administered diclofenac by oral gavage, protein adducts were found in the liver and it was suggested they were formed by way of the acyl-glucuronide of diclofenac (Hargus et al., 1994Go; Pumford et al., 1993Go).

Several rat and human UGTs have been identified that catalyze the glucuronidation of carboxylic acid moieties to form an acyl-glucuronide conjugate. For example, rat and human UGT1A1 catalyzes the glucuronidation of bilirubin (Ebner and Burchell, 1993Go; King et al., 1996Go). Human UGTs 1A3, 1A9, and 2B7 and rat UGT2B1 also catalyze the glucuronidation of many NSAIDs, including ketoprofen, ibuprofen, and naproxen (Ebner and Burchell, 1993Go; Green et al., 1998Go; Jin et al., 1993Go; Pritchard et al., 1994Go). The UGT isoform(s) which catalyze(s) the glucuronidation of diclofenac, has (have) not been identified. In the present study, the UGT responsible for the in vitro formation of the acyl-glucuronide of diclofenac was identified by using recombinant UGTs. A correlation was subsequently established between the formation of diclofenac and morphine glucuronides with microsomal preparations from 13 human livers.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals.
[U-14C]-UDP-glucuronic acid (UDPGA) (255 mCi/mmol) was purchased from ICN Pharmaceuticals (Irvine, CA). Morphine sulfate, M3G, M6G, and UDPGA were purchased from Sigma Chemical Co. (St. Louis, MO). Ibuprofen and diclofenac acyl-glucuronide were synthesized by way of the Mitsunobu coupling route (Juteau et al., 1997Go). Protein assay reagents were purchased from Bio-Rad (Hercules, CA). All other reagents were of analytical grade.

Rat and human liver microsomes.
Male and female human livers were purchased from the International Institute for the Advancement of Medicine (Exton, PA). Rats were purchased from Harlan (Indianapolis, IN). Hepatic microsomes from male Sprague-Dawley rats and 7 male and 6 female humans were prepared by standard differential centrifugation methods. All microsomal samples were stored as suspensions in 0.25 M sucrose at –80°C. HEK293 cell membrane preparations were stored as a pellet at –80°C. Membrane preparation procedures have been described previously by King et al. (1997).

Stably expressed UGTs.
The maintenance of HEK293 cells, stably expressing a UGT isoform, have been described previously by others (Cheng et al., 1998Go; Coffman et al., 1997Go; Green et al., 1994Go; King et al., 1998Go). Human UGT1A9 was purchased from Gentest Corporation (Woburn, MA).

Assays.
Microsomal protein was pre-incubated on ice for 5 min in the presence of 0.5 mg CHAPS detergent/mg protein. Total protein in each microsomal assay was 0.1-mg protein/ml. Incubations (100 µl) contained diclofenac (100 µM final concentration) and 50 mM Tris–HCl/10 mM MgCl2 (pH 6.4) to protect against the decomposition of diclofenac glucuronide. Reactions were started with the addition of UDPGA (2 mM final concentration) and incubated at 37°C for 10 min with continuous shaking. Diclofenac assays were terminated by the addition of an equal volume of acetonitrile/1% formic acid.

The glucuronidation of morphine was determined with 2 mM morphine and 50 mM Tris–HCl/10 mM MgCl2 (pH 8.4). The reactions were started with the addition of 10 µl of 20 mM 14C-UDPGA (0.25 µCi/ 100 µl) and incubated at 37°C for 10 min with continuous shaking. Incubations with morphine were terminated by the addition of 5 ml of cold 1 M aqueous ammonium acetate, the analysis of morphine glucuronides has been described previously by Puig and Tephly, 1986.

Assays with membrane preparations from HEK293 cells stably expressing a recombinant UGT isoform or purchased UGT1A9 were conducted in the same manner as noted above for diclofenac and morphine, only in the absence of detergent and with 0.2-mg protein/ml.

The enzyme kinetic analysis of both morphine and diclofenac glucuronidation was determined under optimal conditions with respect to time and protein concentration. Inhibition assays were carried out in the same manner as the morphine assay (above), only 5 (of the 13) human liver microsomes were pooled and pre-incubated with diclofenac (50 µM, 100 µM, or 500 µM) for 3 min on ice.

Instrumentation.
Liquid chromatography-tandem mass spectrometry (LC/MS/MS) was carried out using a Perkin-Elmer Sciex API 3000 tandem mass spectrometer (Toronto, Canada) interfaced with an HPLC system consisting of a Perkin-Elmer (Norwalk, CT) Series 200 quaternary pump and a series 200 autosampler. LC/MS/MS experiments were performed with a Turbo IonSpray® interface with positive ion detection. The source temperature was set at 150°C, the ionization voltage at 5 kV, the orifice potential at 50 V, collision energy at 35 eV and collision gas nitrogen at the manufacturer's setting number of 4. Chromatography was performed on a Betasil C8 column [(4.6 x 50) mm, 5 µm, Keystone Scientific, Wilmington, DE] and the flow rate of the mobile phase was 1 ml/min with 1:25 split. The mobile phase consisted of 1-mM ammonium acetate and 0.1% trifluoroacetic acid in 90% aqueous acetonitrile.

Detection and quantification of diclofenac glucuronide.
Aliquots of samples from incubations with rat or human liver microsomes or recombinant UGTs were mixed with ibuprofen acyl-glucuronide (internal standard, 50 ng) and 4 M urea (1 ml) and transferred to a 96-well plate solid-phase extraction cartridge (Waters CO, Milford, MA) which had been pre-washed with methanol and, subsequently, with water. After loading the samples, the cartridge was washed consecutively with water (300 µl, not retained) and then diclofenac glucuronide was eluted with 90% aqueous acetonitrile (300 µl) containing 0.1% trifluoroacetic acid. The organic eluate was analyzed by LC/MS/MS by multiple reaction monitoring of mass transitions m/z 472->250 (diclofenac-glucuronide) and m/z 383->161 for the internal standard. These mass transitions are specific for the analyte and the internal standard. Diclofenac acyl-glucuronide standard curves were generated over a concentration range of 10 to 10,000 ng/ml in biological matrices.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Glucuronidation of diclofenac by stably expressed UGTs.
The glucuronidation of diclofenac by several stably expressed UGT isoforms was examined to determine which UGTs were responsible for the conjugation (Table 1Go). UGT2B7 catalyzed the glucuronidation of diclofenac at a high rate, 600-pmol/min/mg protein, but at considerably lower rates by UGT1A6 and UGT2B15. Diclofenac was not conjugated in the presence of UGT1A1, 1A4, or 1A8. Diclofenac was also a good substrate for rat UGT2B1.


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TABLE 1 Diclofenac Glucuronidation by Stably Expressed UGTs
 
Enzyme kinetics.
In order to determine which isoforms were specifically involved in the glucuronidation of diclofenac, enzyme kinetic analysis was performed with rat and human liver microsomes and stably expressed UGT2B1 and UGT2B7 (Table 2Go). Rat and human liver microsomes catalyzed the glucuronidation of diclofenac with a high Vmax and low Km. Similarly, stably expressed human UGT2B7 and rat UGT2B1 displayed a high Vmax and low Km with diclofenac as the substrate. These data suggested that rat UGT2B1 and human UGT2B7 were the major enzymes involved in the glucuronidation of diclofenac in liver microsomes.


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TABLE 2 Kinetic Analysis of Diclofenac Glucuronidation by Stably Expressed UGTs and Rat and Human Liver Microsomes Human liver microsomes
 
Determination of diclofenac glucuronidation in human liver microsomes—correlation with morphine glucuronidation.
To further investigate the relative contribution of UGT2B7 in the glucuronidation of diclofenac in human liver microsomes, the glucuronidation of morphine was investigated. Morphine is known to be one of the best substrates for human UGT2B7, and a poor substrate of other human UGTs (Cheng et al. 1999Go; Coffman et al., 1997Go; King et al., 1996Go). Using 13 different samples of human liver microsomes, the glucuronidation of diclofenac and morphine was investigated to determine the levels of UGT enzyme activity. Human liver microsomes catalyzed the glucuronidation of morphine and diclofenac at very high rates to give a correlation coefficient of 0.84. This value suggests that these 2 substrates were conjugated by the same enzyme (Fig. 1Go; Note: There were no statistical differences seen in the glucuronidation of morphine or diclofenac between male and female human liver microsomes; data not shown).



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FIG. 1. Human liver microsomes catalyzed the glucuronidation of morphine and diclofenac at very high rates and gave a correlation coefficient of 0.84. Values are expressed as the mean of duplicate experiments.

 
Inhibition of morphine glucuronidation by diclofenac in pooled human liver microsomes.
With the strong correlation of morphine and diclofenac glucuronidation by the same isoform, the inhibition of morphine by diclofenac was investigated in human liver microsomes. Diclofenac at 50, 100, and 500 µM inhibited the in vitro glucuronidation of morphine by human liver microsomes (Fig. 2Go). These data would suggest that diclofenac and morphine are being glucuronidated by similar enzymes in human liver microsomes.



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FIG. 2. The glucuronidation of morphine was inhibited by diclofenac in human liver microsomes. Assays and analyses are described in the Materials and Methods section. Values are expressed as the mean of duplicate experiments.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Several stably expressed UGTs have been shown to react with a number of carboxylic acid-containing compounds, including the NSAIDs ibuprofen, ketoprofen, and naproxen. These compounds are known to be eliminated from the body primarily by the enzyme-mediated conjugation of the carboxylic acid entity with R-glucuronic acid to form an acyl-glucuronide (Watt et al., 1991Go). Some acyl-glucuronides are unstable molecules and hence they may undergo rearrangement to positional isomers. They may also undergo hydrolysis back to the aglycone (Faed, 1984Go). The side effects of NSAIDs are well documented and include gastric irritation, nephritis (less commonly), acute renal failure, and rarely, severe liver injury. In addition, one particular NSAID, zomepirac, was withdrawn from use because it was implicated in an anaphylactic reaction of unknown etiology. More recently, ketoprofen was shown to bind to several proteins, including a protein of approximately 56 kD corresponding to the molecular weight of a UGT (Terrier et al., 1999Go).

Diclofenac, an NSAID used in the treatment of osteoarthritis and rheumatoid arthritis, has produced adverse hepatic effects, most notably a fulminant hepatic necrosis (Breen et al., 1986Go; Salama et al., 1991Go). However, other studies have shown that the harmful effects associated with diclofenac were more consistent with a direct effect of the drug or one of its metabolites (Iveson et al., 1990Go; Sallie, 1990Go; Scully et al., 1993Go). In short, the underlying mechanism of the liver toxicity associated with diclofenac is not understood as yet. Nevertheless, diclofenac was found to generate protein adducts from the acyl-glucuronide (Pumford et al., 1993Go), and the adducts were shown to retain the glucuronic acid moiety (Hargus et al., 1994Go; Kretz-Rommel and Boelsterli, 1993Go).

Rat UGT2B1 and human UGT2B7 have been shown to catalyze the glucuronidation of several carboxylic acid containing xenobiotics, including ibuprofen, naproxen and ketoprofen (Jin et al., 1993Go; Pritchard et al., 1994Go). UGT2B7 has also been shown to catalyze the glucuronidation of morphine with very high efficiency (Coffman et al., 1997Go, 1998Go). Similarly, in this study, diclofenac was extensively conjugated but with a much lower Km than morphine conjugation catalyzed by UGT2B7. When human liver microsomes were used to investigate the glucuronidation of diclofenac and morphine, a notable correlation (r = 0.84) was seen with regard to the generation of total morphine glucuronide (i.e., 3 and 6 glucuronides) and diclofenac acyl-glucuronide.

Human UGT1A3 and UGT1A9 have been shown to catalyze the glucuronidation of NSAIDs (Ebner and Burchell, 1993Go; Green et al., 1998Go) UGT1A9 catalyzes the glucuronidation of ibuprofen and ketoprofen at low rates, and in this study, UGT1A9 catalyzed the glucuronidation of diclofenac at a moderate rate. The apparent Km for diclofenac was similar to that found with human liver microsomes and UGT2B7, but UGT1A9 does not catalyze the glucuronidation of morphine (Ebner and Burchell, 1993Go). Unfortunately, there is no specific antibody to UGT1A9 to investigate the relative protein concentration in liver microsomes or of the recombinant isoform.

UGT1A3 also catalyzes the glucuronidation of several xenobiotics containing carboxylic acid moieties. UGT1A3 catalyzed the glucuronidation of morphine at very low rates, but had a Km value of 3 mM (Green et al., 1998Go). However, due to the overall abundance of UGT1A3 transcript in the liver (20-fold less than UGT1A1 and 5–10-fold less than UGT1A4; Mojarrabi, 1996) and the human liver microsomes correlation of diclofenac and morphine glucuronidation, UGT1A3 probably did not substantially contribute to the glucuronidation of diclofenac in vitro.

Kirkwood et al. (1998) showed that diclofenac inhibited the in vitro glucuronidation of dihydrocodeine, and more recently, Ammon et al., 2000, demonstrated that diclofenac inhibits the in vitro glucuronidation of codeine with a Ki value of 7.9 µM. UGT2B7 is the only known enzyme to catalyze the formation of the 6-O-glucuronides of opiates, including codeine (Coffman et al., 1997Go), and diclofenac inhibited the glucuronidation of morphine in this study. These works, along with the present one, strongly suggest that rat UGT2B1 and human UGT2B7 are the main UGT isoforms contributing to the in vitro glucuronidation of diclofenac and morphine. Theoretically, there is the possibility of a drug-drug interaction in vivo, since NSAIDs are co-administered with opioids in post-operative procedures for the treatment of pain (Tighe et al., 1999Go). This possibility seems not to have been studied in a clinical or pre-clinical setting, at least to our knowledge.

In summary, rat UGT2B1 and human UGT2B7 were the major isozymes involved in catalyzing the glucuronidation of diclofenac in rat and human liver microsomes. The Km values for diclofenac glucuronidation by rat and human liver microsomes were similar to those found using stably expressed rat UGT2B1 and human UGT2B7. The extent of morphine glucuronidation that is known to be catalyzed by UGT2B7 was found to correlate with the extent of diclofenac glucuronidation in human liver microsomes. In review of recent evidence suggesting the involvement of CYP450 in diclofenac bioactivation (Shen et al., 1999Go; Tang et al., 1999aGo,bGo), further studies are needed to determine the relative contributions of the CYP450s and the UGTs in the expression of diclofenac hepatotoxicity.


    ACKNOWLEDGMENTS
 
We would like to thank Dr. Ronald Franklin for his critical review of this manuscript.


    NOTES
 
1 To whom correspondence should be addressed at Drug Metabolism and Pharmacokinetics, Merck Research Laboratories, 505 Coast Blvd. South, La Jolla, CA 92037. Fax: (858) 452-9279. E-mail: christopher_king{at}merck.com. Back


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