Short Communication Glycerolysis of Acyl Glucuronides as an Artifact of in Vitro Drug Metabolism Incubations

During an investigation of the in vitro glucuronidation of benoxaprofen by human liver S-9 fraction, an unusual drug-related entity possessing a protonated molecular ion that was 74 mass units greater than the parent drug was observed. It was identified as the glycerol ester of benoxaprofen. Formation of this entity required inclusion of uridine diphosphoglucuronic acid (UDPGA) in the incubation, suggesting the formation of benoxaprofen acyl glucuronide followed by transesterification with the glycerol present in the incubation due to its presence as a stabilizer for liver subcellular fractions. Formation occurred during the sample work-up procedure while the samples were subjected to evaporation in vacuo, which does not remove glycerol. Conversion of purified benoxaprofen acyl glucuronide to the glycerol ester was demonstrated in glycerol at 37°C. Other drugs that are converted to acyl glucuronides in vitro (diclofenac, mefenamic acid, tolmetin, and naproxen) were also shown to form corresponding glycerol esters when incubated with human liver S-9 fraction and UDPGA. The potential formation of glycerol esters of carboxylic acid drugs undergoing acyl glucuronidation in vitro represents an experimental artifact to which drug metabolism scientists should be aware. The metabolism of xenobiotics containing carboxylic acid groups to acyl glucuronides is a well known biotransformation reaction (recently reviewed in Skonberg et al., 2008; Stachulski et al., 2006). Acyl glucuronide metabolites have received considerable attention due to evidence suggesting that they are chemically reactive, form adducts with proteins, and potentially cause various toxicities via this mechanism (Yang et al., 2006; Skonberg et al., 2008). Adducts can form directly, with nucleophiles displacing the glucuronic acid moiety via an SN2 reaction (van Breemen and Fenselau, 1985). A second pathway to form adducts first involves migration of the acyl group from the 1-position of the glucuronic acid to the 2-, 3-, and 4-hydroxyl groups. The 1-OH group of glucuronide conjugate can tautomerize to the aldehyde, react with a nucleophilic amine on a macromolecule (e.g., -amino group of lysine residues) to form a Schiff base, followed by a proton shift from the hydroxyl group to the imine yielding an amino ketone (Smith et al., 1990; Ding et al., 1993). This entity is a stable adduct that could potentially serve as an immunogen and yield a toxic response. Benoxaprofen is a nonsteroidal anti-inflammatory agent that was removed from clinical use due to an unacceptably high prevalence of drug-induced hepatotoxicity (Goldkind and Laine, 2006; Smith and Schmid, 2006). The metabolism of benoxaprofen to its acyl glucuronide was demonstrated to be the quantitatively most important metabolic pathway in the human and animal species (Chatfield and Green, 1978), and benoxaprofen acyl glucuronide has been implicated as the entity responsible for toxicity. Covalent bonding of benoxaprofen acyl glucuronide has been demonstrated in vitro and in vivo, with albumin as a prevalent target (Dahms and Spahn-Langguth, 1996; Dong et al., 2005). During the course of an examination of the in vitro metabolism of benoxaprofen, an unusual drug-related entity was observed on HPLC-MS that possessed 74 additional mass units from the parent drug. Further pursuit showed that it was a glycerol ester of benoxaprofen (Fig. 1) and that it could be an artifact. The term metabonate was coined by Beckett in 1971 (Beckett et al., 1971) to describe drug-related chemical entities observed in work-ups or storage of biological samples that can be mistaken for drug metabolites. These entities can arise by intramolecular rearrangements of metabolites (e.g., cyclization) (Erve, 2008) or by reaction of metabolites with constituents of sample work-up such as solvents (e.g., methanol) (Dickinson and King, 1989, 1991; Chabot and Gouyette, 1991). The objective of these studies was to characterize the formation of the glycerol ester of the carboxylic acid group of benoxaprofen, determine how this process arises, and determine whether other COOH-containing drugs can undergo this reaction and form metabonates. Materials and Methods Materials. [C]Benoxaprofen was prepared by a custom synthesis by Nerviano Medical Sciences (Nerviano, Italy). The position of the radioactive atom was the 2-carbon on the benzoxazole. Benoxaprofen was obtained from an in-house library of chemicals at Pfizer (Groton, CT). Alamethicin, uridine diphosphoglucuronic acid (UDPGA), diclofenac, tolmetin, and naproxen were obtained from Sigma-Aldrich (St. Louis, MO). Pentadeuterated glycerol was obtained from Isotec (Miamisburg, OH). Mefanamic acid was obtained from ICN (Aurora, OH). Pooled human liver microsomes and S-9 fraction were obtained from BD Gentest (Woburn, MA). In Vitro Incubation Procedures. Incubations consisted of benoxaprofen (50 M) and pooled human liver S-9 fraction containing 20% glycerol (5 mg/ml) with alamethicin (50 g/ml), MgCl2 (5 mM), and UDPGA (5 mM) in 100 mM potassium phosphate buffer at pH 7.5. Alamethicin was mixed with Article, publication date, and citation information can be found at http://dmd.aspetjournals.org. doi:10.1124/dmd.109.027953. ABBREVIATIONS: UDPGA, uridine diphosphoglucuronic acid; HPLC, high-performance liquid chromatography; DMSO, dimethyl sulfoxide; Rt, retention time. 0090-9556/09/3708-1581–1586$20.00 DRUG METABOLISM AND DISPOSITION Vol. 37, No. 8 Copyright © 2009 by The American Society for Pharmacology and Experimental Therapeutics 27953/3492464 DMD 37:1581–1586, 2009 Printed in U.S.A. 1581 at A PE T Jornals on July 7, 2017 dm d.aspurnals.org D ow nladed from S-9 and allowed to stand on ice for 15 min before addition of other components. Incubations were commenced with the addition of UDPGA and run at 37°C in a shaking water bath for 1 h. (Based on this procedure, the concentration of glycerol in the incubation is 5%.) Incubations were terminated with five volumes of CH3CN, the precipitated materials were removed by centrifugation (1700g; 5 min), and the supernatant was evaporated in vacuo in a Genevac vacuum centrifuge (Genevac, Valley Cottage, NY) operated with the settings at “HPLC Fraction” and a temperature that did not exceed 40°C. The residue was reconstituted in HPLC mobile phase (0.2 ml). This incubation procedure was also done for naproxen, diclofenac, mefanamic acid, and tolmetin. Synthesis of Benoxaprofen Glycerol Ester. Benoxaprofen (50 mg) was added to glycerol (10 ml) and heated to 80°C and stirred. Five drops of sulfuric acid were added, and the reaction mixture was stirred for 2 h. The mixture was added to 300 ml of ice-cold water and extracted with CHCl3 (2 50 ml). The combined extracts were dried under nitrogen, and the residue was reconstituted in 0.3 ml of H2O/CH3CN/HCOOH (50:50:0.05). This mixture was injected onto a Waters Novapak C18 (Waters, Milford, MA) semipreparative column (7.9 300 mm; 7 ) equilibrated in aqueous 0.1% HCOOH containing 40% CH3CN at a flow rate of 5 ml/min. The eluent was divided into 1-min fractions, and the fractions containing benoxaprofen 1-glycerol ester were combined and lyophilized to yield 10 mg product. H NMR (600 MHz; DMSO-d6): 8.20 (d, 2H), 7.75 (s, 1H), 7.74 (d, 1H), 7.69 (d, 2H), 7.39 (d, 1H), 4.86 (s, OH, 1H), 4.59 (s, OH, 1H), 4.09 and 4.02 (m, 2 0.5H), 4.00 (m, 1H) 3.97 and 3.90 (m, 2 0.5H), 3.61 (m, 1H), 3.28 (d, 2H), 1.48 (d, 3H). Connectivity of the atoms was confirmed with correlation spectroscopy (COSY) and heteronuclear multiple bond correlation (HMBC) experiments. Biosynthesis of Benoxaprofen Acyl Glucuronide and Reaction with Glycerol. Benoxaprofen (20 M) was incubated with pooled human liver microsomes (2 mg/ml), alamethicin (0.04 mg/ml), UDPGA (6.9 mM), and MgCl2 (5 mM) in 10 ml of potassium phosphate buffer at pH 7.5. The incubation was carried out at 37°C for 1 h followed by addition of CH3CN (10 ml) and centrifugation at 1700g to remove precipitates. Formic acid in water (0.1%; 100 ml) was added to the supernatant, and the resulting mixture was spun in a centrifuge at 40000g to clarify the mixture. The supernatant was loaded onto a Varian Polaris C18 column (4.6 250 mm; 5 ) at 0.8 ml/min using a Jasco (Tokyo, Japan) PU-980 HPLC pump. After loading the entire sample, the column was moved to a Thermo-Finnigan Surveyor quaternary HPLC pumping system (Thermo Fisher Scientific, Waltham, MA) at 0.8 ml/min, and a gradient was applied starting at 30% CH3CN in 0.1% HCOOH for 5 min followed by a linear increase to 90% CH3CN at 25 min. The effluent was collected into 20-s fractions, and the material eluting between 19.0 and 19.7 min contained benoxaprofen acyl glucuronide and was shown to be pure by HPLC. The solvent was removed in vacuo in a conical glass tube. After drying, glycerol (0.05 ml) was added and the tube was sonicated. A small aliquot was removed for immediate HPLC analysis, a second portion was removed, sealed, and placed at 20°C, and the remaining material was sealed and placed in a 37°C water bath. At various timepoints up to 4 days, aliquots were removed and analyzed by HPLC for the formation of benoxaprofen glycerol ester. HPLC-MS/MS. The HPLC-mass spectrometry system consisted of a Surveyor quaternary pump, degasser, autoinjector, and photodiode array detector coupled to a Thermo-Finnigan LTQ ion trap mass spectrometer (Thermo Fisher Scientific). Separation was effected on a Varian Polaris C18 (Varian, Inc., Palo Alto, CA) column (4.6 250 mm; 5 ) using a mobile phase of 0.1% HCOOH in water (“A”) and CH3CN (“B”) at a flow rate of 0.8 ml/min. The mobile phase composition was held at 30% B for 5 min followed by a linear gradient to 90% B at 25 min. The eluent was split, with 0.06 ml/min introduced into the source of the mass spectrometer operated in the positive ion mode. Instrument settings and potentials were adjusted to optimize the signal for the protonated molecular ion for benoxaprofen. Using these conditions, N O