* Merck & Company, Department of Drug Metabolism, P.O. Box 2000, RY80-A9, Rahway, New Jersey 07065; and
University of Iowa, Department of Pharmacology, Iowa City, Iowa 52242
Received October 18, 2000; accepted January 18, 2001
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ABSTRACT |
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Key Words: acyl-glucuronides; UGTs; diclofenac; glucuronidation; NSAIDs.
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INTRODUCTION |
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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., 1978), 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., 1994
; Pumford et al., 1993
).
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, 1993; King et al., 1996
). Human UGTs 1A3, 1A9, and 2B7 and rat UGT2B1 also catalyze the glucuronidation of many NSAIDs, including ketoprofen, ibuprofen, and naproxen (Ebner and Burchell, 1993
; Green et al., 1998
; Jin et al., 1993
; Pritchard et al., 1994
). 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.
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MATERIALS AND METHODS |
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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., 1998; Coffman et al., 1997
; Green et al., 1994
; King et al., 1998
). 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 TrisHCl/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 TrisHCl/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 472250 (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.
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RESULTS |
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DISCUSSION |
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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., 1986; Salama et al., 1991
). 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., 1990
; Sallie, 1990
; Scully et al., 1993
). 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., 1993
), and the adducts were shown to retain the glucuronic acid moiety (Hargus et al., 1994
; Kretz-Rommel and Boelsterli, 1993
).
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., 1993; Pritchard et al., 1994
). UGT2B7 has also been shown to catalyze the glucuronidation of morphine with very high efficiency (Coffman et al., 1997
, 1998
). 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, 1993; Green et al., 1998
) 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, 1993
). 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., 1998). However, due to the overall abundance of UGT1A3 transcript in the liver (20-fold less than UGT1A1 and 510-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., 1997), 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., 1999
). 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., 1999; Tang et al., 1999a
,b
), further studies are needed to determine the relative contributions of the CYP450s and the UGTs in the expression of diclofenac hepatotoxicity.
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ACKNOWLEDGMENTS |
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NOTES |
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