Glucuronidation and Covalent Protein Binding of Benoxaprofen and Flunoxaprofen in Sandwich-Cultured Rat and Human Hepatocytes
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0090-9556/09/3712-2314–2322$20.00 DRUG METABOLISM AND DISPOSITION Vol. 37, No. 12 Copyright © 2009 by The American Society for Pharmacology and Experimental Therapeutics 28944/3537714 DMD 37:2314–2322, 2009 Printed in U.S.A. Glucuronidation and Covalent Protein Binding of Benoxaprofen and Flunoxaprofen in Sandwich-Cultured Rat and Human Hepatocytes Jennifer Q. Dong and Philip C. Smith Department of Pharmacokinetics and Drug Metabolism, Amgen Inc., Seattle, Washington (J.Q.D.); and Eschelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina (P.C.S.) Received June 15, 2009; accepted September 17, 2009 ABSTRACT: Benoxaprofen (BNX), a nonsteroidal anti-inflammatory drug the hypothesis that part of the covalent binding of all three NSAIDs (NSAID) that was withdrawn because of hepatotoxicity, is more to hepatic proteins is acyl glucuronide-dependent. Moreover, the- toxic than its structural analog flunoxaprofen (FLX) in humans and ses studies confirmed the feasibility of using sandwich-cultured rats. Acyl glucuronides have been hypothesized to be reactive rat hepatocytes for studying glucuronidation and covalent binding metabolites and may be associated with toxicity. Both time- and to hepatocellular proteins. These studies also showed that these in concentration-dependent glucuronidation and covalent binding of vitro methods can be applied using human tissues for the study of BNX, FLX, and ibuprofen (IBP) were determined by exposing sand- acyl glucuronide reactivity. More BNX-protein adduct was formed wich-cultured rat hepatocytes to each NSAID. The levels of gluc- in sandwich-cultured human hepatocytes than FLX-protein ad- uronide and covalent protein adduct measured in cells followed duct, which not only agreed with its relative toxicity in humans but the order BNX > FLX > IBP. These results indicate that 1) BNX- also was consistent with the in vitro findings using rat hepatocyte glucuronide (G) is more reactive than FLX-G, and 2) IBP-G is the cultures. These data support the use of sandwich-cultured human least reactive metabolite, which support previous in vivo studies in hepatocytes as an in vitro screening model of acyl glucuronide rats. The proportional increases of protein adduct formation for exposure and reactivity. BNX, FLX, and IBP as acyl glucuronidation increased also support Many types of acidic drugs form acyl glucuronides, and other individual glucuronides for each drug candidate, a process that can be xenobiotics are metabolized to carboxylic acids (Phase I metabolites), tedious and costly. In addition, hepatic tissue proteins are not present which subsequently undergo Phase II conjugation to form acyl gluc- in the incubation, which makes it less likely to correlate covalent uronides. Often such a glucuronide conjugate constitutes the major protein binding with hepatotoxicity. In vitro models involving hepatic metabolite. The major site of conjugation for most compounds in materials, including liver homogenates, microsomal subcellular prep- humans is believed to be the liver. Modification of critical hepatic aration, and isolated hepatocyte suspensions and cultures, offer many proteins by covalent binding of acidic drugs through reactive acyl advantages over in vitro incubation with model proteins. Not only are glucuronides may provide a basis for direct hepatocyte toxicity or hepatic tissue proteins present, but UDP glucuronosyltransferase immune-mediated adverse reactions (Gillette, 1974; Faed, 1984; (UGT) activities that catalyze the glucuronidation reactions are also Boelsterli, 2002; Bailey and Dickinson, 2003). maintained. However, membrane transport functions are lost or not Some pharmaceutical companies have been conducting in vitro well maintained in isolated hepatocytes or even primary hepatocyte experiments by incubating acyl glucuronides with model proteins or cultures. Although the intact animal model represents undisturbed peptides (Wang et al., 2004) to determine their reactivity, and hence hepatic physiology, high-throughput screenings using in vivo studies possibly predict the relative extent of covalent protein binding in vivo are not very practical. Moreover, extrapolation of animal data to in humans. This method requires chemical synthesis or biosynthesis of humans is often questionable, especially for drug metabolism. We propose that hepatocyte culture with a “sandwich” configura- tion would be a good model for estimating covalent binding to hepatic This work was supported by the National Institutes of Health National Institute of General Medical Sciences [Grant GM61188]. tissue proteins in vivo. Hepatocyte monolayer is cultured between two Article, publication date, and citation information can be found at layers of Matrigel, a matrix material resembling the mammalian http://dmd.aspetjournals.org. cellular basement membrane, and this has been shown to prolong cell doi:10.1124/dmd.109.028944. viability and has better preservation of liver-specific protein synthesis ABBREVIATIONS: UGT, UDP glucuronosyltransferase; BNX, benoxaprofen; FLX, flunoxaprofen; IBP, ibuprofen; HPLC, high-performance liquid chromatography; PBS, phosphate-buffered saline; SKF-525A, proadifen hydrochloride; LDH, lactate dehydrogenase; ABT, 1-aminobenzotriazole; DMEM, Dulbecco’s modified Eagle’s medium; MCM, modified Chee’s medium; PMSF, phenylmethylsulfonyl fluoride; PH, 1,7-phenanthroline; BNX-G, benoxaprofen glucuronide; FLX-G, flunoxaprofen glucuronide; IBP-G, ibuprofen glucuronide; P450, cytochrome P450; NSAID, nonste- roidal anti-inflammatory drug. 2314 ACYL GLUCURONIDE AND COVALENT BINDING IN HEPATOCYTE CULTURES 2315 Materials and Methods O O R 1 Materials. Rac-BNX was extracted and purified from tablets of Oraflex N HO previously marketed by Eli Lilly & Co. (Indianapolis, IL). [Analysis calculated CH3 for BNX (C16H12ClNO3): C, 63.69; H, 4.01; N, 4.64; Cl, 11.75; O, 15.91. Found: C, 63.05; H, 4.31; N, 4.71; Cl, 11.68; O, 16.25.] The purity of BNX (Ͼ99%) was confirmed based on elemental analysis and analytical high- performance liquid chromatography (HPLC) using UV detection at wave- FIG. 1. Structures of BNX and FLX. BNX was marketed as the racemate; FLX was length of 210 nm. S-FLX, the marketed form, was contributed by Dr. A. marketed as the S-isomer. Forgione (Ravizza Laboratories, Milan, Italy) and was determined to be pure (Ͼ98%) based on HPLC with UV detection. IBP, Triton X-100, EDTA, than primary hepatocytes maintained under conventional culture con- glycerol, glycine, phosphate-buffered saline (PBS), borneol, SKF-525A, lac- ditions (Dunn et al., 1989, 1991). This model system uses biologically tate dehydrogenase (LDH) kit, Percoll, Hanks’ balanced salt solution, and 3 relevant matrix components and therefore is more close to the native dexamethasone were obtained from Sigma-Aldrich (St. Louis, MO). [ H]IBP architecture of the hepatocyte environment. A unique feature of this (1 Ci/mmol) was purchased from ICN (currently Valeant Pharmaceuticals, Costa Mesa, CA). 1,7-Phenanthroline was purchased from Aldrich Chemical sandwiched culture of hepatocytes is the formation of functional bile Co. (Milwaukee, WI). 1-Aminobenzotriazole (ABT) was a gift from Dr. J. canalicular networks formed with rat hepatocytes. In addition, the Mathews at Triangle Research Institute (Research Triangle Park, NC). Proto- normal polarized distribution of several different classes of function- gel, ammonium sulfate, TEMED, dithiothreitol, bromophenol blue, and Tris ally active canalicular transport systems, such as multidrug-resistant were purchased from Bio-Rad Laboratories (Hercules, CA). Collagenase was protein 2, is also re-established in this configuration (LeCluyse et al., obtained from Worthington Biochemicals (Freehold, NJ). Dulbecco’s modified 1994). It has been reported that the hepatobiliary transport of reactive Eagle’s medium (DMEM), modified Chee’s medium (MCM), fetal calf serum, diclofenac glucuronide via multidrug-resistant protein 2 is critical for and insulin were purchased from Invitrogen (Carlsbad, CA). Rat tail collagen diclofenac covalent binding to proteins in the biliary tree (Seitz et al., (type I) was obtained from Collaborative Research (Bedford, MA). Matrigel was purchased from Thermo Fisher Scientific (Waltham, MA). Methanol and 1998). In addition to being a much more biologically relevant in vitro acetonitrile were HPLC grade from Mallinckrodt Baker, Inc. (Phillipsburg, model in terms of morphology and biochemical properties, this sand- NJ). All of the other chemicals used were of reagent grade. Male Sprague- wiched hepatocyte culture system also maintains a better functional Dawley rats (250–350 g) were supplied by Harlan (Indianapolis, IN). Exper- enzyme/transport system than microsomes, with which the hepato- iments were approved by the university’s animal care committee. Human liver cytes can generate metabolites such as acyl glucuronides. Further- microsomal preparations were obtained from BD Gentest (Woburn, MA). more, the availability of sandwiched human hepatocyte cultures will Isolation and Culture of Rat Hepatocytes. Plastic culture dishes (60 mm) allow in vitro evaluation of covalent binding in hepatocytes using were precoated with rat tail collagen, type I (0.1 ml/dish, 1.5 mg/ml), in a human tissues. This is essential for studies of compounds that are gelled state at least 1 day before hepatocyte harvest. To obtain a gelled potentially toxic to humans because direct detection of acyl gluc- collagen substratum, neutralized collagen type I was prepared