Significantly Different Covalent Binding of Oxidative Metabolites, Acyl

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Significantly Different Covalent Binding of Oxidative Metabolites, Acyl Glucuronides, and S‑Acyl CoA Conjugates Formed from Xenobiotic Carboxylic Acids in Human Liver Microsomes Malin Darnell,† Katarina Breitholtz,‡ Emre M. Isin,† Ulrik Jurva,† and Lars Weidolf*,† †

CVMD iMed DMPK, ‡Drug Safety & Metabolism, AstraZeneca R&D Mölndal, 431 83 Mölndal, Sweden S Supporting Information *

ABSTRACT: Xenobiotic carboxylic acids may be metabolized to oxidative metabolites, acyl glucuronides, and/or S-acyl-CoA thioesters (CoA conjugates) in vitro, e.g., in hepatocytes, and in vivo. These metabolites can potentially be reactive species and bind covalently to tissue proteins and are generally considered to mediate adverse drug reactions in humans. Acyl glucuronide metabolites have been the focus of reactive metabolite research for decades, whereas drug-CoA conjugates, which have been shown to be up to 40−70 times more reactive, have been given much less attention. In an attempt to dissect the contribution of different pathways to covalent binding, we utilized human liver microsomes supplemented with NADPH, uridine 5′-diphosphoglucuronic acid (UDPGA), or CoA to evaluate the reactivity of each metabolite separately. Seven carboxylic acid drugs were included in this study. While ibuprofen and tolmetin are still on the market, ibufenac, fenclozic acid, tienilic acid, suprofen, and zomepirac were stopped before their launch or withdrawn. The reactivities of the CoA conjugates of ibuprofen, ibufenac, fenclozic acid, and tolmetin were higher compared to those of their corresponding oxidative metabolites and acyl glucuronides, as measured by the level of covalent binding to human liver microsomal proteins. The highest covalent binding was observed for ibuprofenyl-CoA and ibufenacylCoA, to levels of 1000 and 8600 pmol drug eq/mg protein, respectively. In contrast and in agreement with the proposed P450mediated toxicity for these drug molecules, the reactivities of oxidative metabolites of suprofen and tienilic acid were higher compared to the reactivities of their conjugated metabolites, with NADPH-dependent covalent binding of 250 pmol drug eq/mg protein for both drugs. The seven drugs all formed UDPGA-dependent acyl glucuronides, but none of these resulted in covalent binding. This study shows that, unlike studies with hepatocytes or in vivo, human liver microsomes provide an opportunity to investigate the reactivity of individual metabolites.

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INTRODUCTION Toxicity is a frequent cause of drug withdrawal from the market, and prediction of drug toxicity has become a major research area in the global life science community. Understanding the mechanisms leading to toxicity and how to design safe drug candidates may impact positively on productivity in drug discovery and development and, in the end, benefit patients. Reactive drug metabolites may mediate liver injury via covalent modification of biological macromolecules.1,2 One such group of metabolites constitutes the acyl glucuronides formed from xenobiotic carboxylic acids (XCAs). These metabolites have been singled out by regulatory authorities for their reactivity and potential toxicity. Several groups have proposed links between their reactivity and toxicity observed in humans.3,4 The hypothesis that another group of XCA conjugates, the S-acylcoenzyme A thioesters (CoA conjugates), might contribute to the hepatotoxicity observed in humans has also been investigated during the past decade.5−13 It has been shown that the CoA conjugate, or the ATP activated acyl-adenylate intermediate preceding CoA conjugation, can be even more reactive than the © 2015 American Chemical Society

corresponding acyl glucuronide. Therefore, it cannot be ruled out that CoA conjugates can contribute to the hepatoxicity observed in humans.5,14−17 While it is important to understand reactivity and its relevance to toxicity, the study of CoA conjugates with respect to detection and quantification can be difficult, especially in vivo. Due to their physicochemical properties, CoA conjugates do not pass membranes and are not present in plasma, urine, or bile. Thus, tissue biopsy or organ removal is necessary for the analysis and detection of CoA conjugates. Because the stability in solution varies between different XCA conjugates and depends, e.g., on the pH in the in vitro incubation or in the quenching solution used after in vitro or in vivo studies, precautions have to be taken to avoid compromising the bioanalytical quantification data. Following formation, CoA conjugates may be further metabolized via Nacyl transferases to stable xenobiotic amino acid conjugates with, e.g., taurine or glycine, which, when detected in excreta, provides Received: December 12, 2014 Published: March 24, 2015 886

DOI: 10.1021/tx500514z Chem. Res. Toxicol. 2015, 28, 886−896

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Chemical Research in Toxicology evidence of CoA conjugate formation in vivo. In contrast, the absence of xenobiotic amino acid adducts is not evidence of lack of CoA conjugate formation.18 Moreover, XCA CoA conjugates can mimic fatty acid-CoA species and may interfere with lipid metabolism in the mitochondria,19−23 which may lead to the reduction of mitochondrial ATP production and, in the worst case, organ failure or liver tumors.24

In addition to acyl glucuronides and CoA conjugates formed from the carboxylic acid moiety, XCAs can also form potentially reactive metabolites via enzyme-mediated oxidation reactions on other parts of the molecule, which may also bind covalently to macromolecules.2,25 Different types of reactive metabolites may form in whole cell systems. Determining which and to what extent metabolite(s) in a mixture contribute to covalent binding is not straightforward. The reactivity of one metabolite can be estimated by inhibiting its formation, e.g., fatty acids can be utilized to inhibit acyl-CoA synthetases (ACSs) catalyzing the CoA conjugation of XCAs, whereas 1-aminobenzotriazole and (−)-borneol are commonly used as nonspecific inhibitors of P450 and UDP-glucuronosyltransferase (UGT) isoenzymes, respectively.11,13,16 Another approach to separately evaluate the reactivity of different metabolites is to use human liver microsomes, supplemented with reaction-specific cofactors, to generate one type of metabolite in each incubation. However, the amount of formed metabolite and extent of covalent binding in microsomal incubations may be different compared to that in hepatocytes and the in vivo situation due to several differences between these systems. Reactive metabolites that do not bind to microsomal proteins may potentially bind to other proteins present in hepatocytes and in vivo, and secondary metabolism, e.g., amino acid or glutathione conjugation, that may prevent covalent binding in hepatocytes is absent in microsomes.18 Furthermore, all CoA conjugates formed in hepatocytes may not be formed by microsomes because ACS enzymes are located in the plasma membrane, peroxisomes, cytosol, and mitochondria in addition to the endoplasmic reticulum (i.e., microsomes).26,27 Although the results from the microsomal incubations cannot be directly translated into whole cell systems and the in vivo situation, valuable information on the reactivity of different metabolites formed from the same substrate will be achieved.

Figure 1. Structures of compounds discussed in this study. An asterisk denotes the position of 14C, and T, the position of 3H.

Table 1. Covalent Binding in Human Hepatocytes and Adverse Drug Reactions in Human28 drug name

CVBa in human hepatocytes (pmol/mg protein)

IBP

44

IFC

154

Fenclozic Acid Suprofen Tienilic Acid

51

Zomepirac

20

Tolmetin

11

a

393 116

DILI pattern or other significant adverse drug reactions in human Very rare liver injury, primarily mainly hepatocellular; cholestatic/mixed also reported; available over the counter Withdrawn due to cholestatic or hepatocellular DILI Development terminated due to cholestatic jaundice in clinical trials Rare hepatocellular DILI Withdrawn due to rare hepatocellular DILI, acute liver failure Withdrawn due to anaphylaxis; renal toxicity also reported. Liver enzyme elevations; very rare hepatocellular DILI; very rare liver failure; DILI warning on label

CVB, covalent binding.

In the present study, we utilized human liver microsomes, supplemented with cofactors, to evaluate separately the reactivity of P450-generated oxidative metabolites, acyl glucuronides, and CoA conjugates formed from seven carboxylic acid drugs (Figures 1 and 2). The development of several such drugs has been terminated in clinical trials, whereas other XCAs have been withdrawn from the market after launch or had their use restricted via black box warnings due to toxicity. Ibuprofen (IBP)

Figure 2. Three incubations were performed in human liver microsomes to separately evaluate the reactivity of different metabolites formed from XCAs. P450, UGT, and ACS enzymes are present in liver microsomes; however, different cofactors need to be supplemented in the incubation in order to form the metabolites as shown in the figure.

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metabolites (100 μL), respectively, as described below. In addition, trapping experiments were performed for tolmetin with the same conditions as those described for reaction III. Before the preincubation, either methoxylamine (MeA), glutathione (GSH), or water (control without trapping agent) were added, and the reactions were started by addition of CoA to all incubations. The final concentrations of MeA and GSH were 5 and 2 mM, respectively. Covalent Binding in Human Liver Microsomes. The method used in this work was a modified version of the covalent binding assays reported previously.28,29 Briefly, at the end of the incubation (60 min), 200 μL aliquots of the incubation mixtures were quenched by mixing with 400 μL of acetone in a 2 mL 96-deep well Nunc plate. The samples were vortex-mixed, another aliquot of acetone (400 μL) was added to each sample, and the samples were mixed again. The plate was refrigerated (5 °C) for 60 min and then centrifuged for 5 min at 500 rpm followed by a gentle vortex (10−20 s). The samples quenched with acetone were harvested onto a Whatman GF/B filter paper, using a Brandel Cell Harvester ML-48TI (Brandel, Gaithersburg, MD, USA), and washed with methanol (80% (v/v) in water). Each filter paper was punched out individually and transferred to a 20 mL glass scintillation vial followed by addition of 1.0 mL of SDS, 5% (w/v), in water. The scintillation vials were kept in a shaking water bath (55 °C) for 20 h. Aliquots (250 μL) containing the dissolved protein in SDS were added to a 10 mL scintillation vial containing 5 mL of liquid scintillation cocktail, and the samples were counted in a Wallac 1409 liquid scintillation counter (PerkinElmer Life and Analytical Sciences, Waltham, MA, USA). The protein concentration of the samples was determined using a standard BCA protein assay kit (Thermo Scientific Pierce, Rockford, IL, USA) according to the manufacturer’s instructions. The covalent binding levels of the incubated test compounds were determined by relating the total amount of radioactivity (pmol equivalents) to the protein concentration in each sample. The significance of differences in the covalent binding with and without cofactors was calculated using a two-tailed Student’s t test. Analysis of Metabolites. Incubation aliquots of 100 μL were terminated by addition of an equal volume of ice-cold ACN containing 0.8% formic acid (reactions I and II) or ice-cold ACN (reaction III). The samples were centrifuged for 5 min at 4000 g and 4 °C, followed by mixing with an equal volume of water. The LC/MS analyses were run on a Waters QToF MS Xevo (Milford, MA, USA), and the spectra were acquired with MSE operating under positive or negative electrospray ionization (ESI) conditions in extended dynamic range mode. The cone voltages were optimized for each substrate and metabolite and are presented in Tables S1−S7. All other MS conditions were the same for all compounds. The m/z range was 80−1200 Th with an acquisition time of 0.1 s, the low collision energy was 6 V, and the high collision energy was ramped from 15 to 35 V. Data acquisition was done in centroid mode. The voltages were set to 0.5 kV (ESI+)/2 kV (ESI−) for the capillary, 30 V (ESI+)/40 V (ESI−) for the sampling cone, and 4 V for the extraction cone. The source block temperature was 120 °C, the electrospray desolvation heater was 450 °C, and the desolvation gas was set to 800 L/h. Leucine-enkephaline was used as lock mass (m/z 278.1141 and 556.2771 for positive ESI and m/z 236.1035 and 554.2615 for negative ESI) for internal calibration at a concentration of 0.5 μg/mL and a flow rate of 20 μL/min. The Acquity ultraperformance liquid chromatography (UPLC) system (Waters) consisted of a column manager set at 45 °C, an auto sample manager, and a binary solvent manager operating at a flow rate of 0.5 mL/min. Chromatographic separations were performed on the UPLC system using an Acquity UPLC BEH C18 column (2.1 × 100 mm, 1.7 μm). A 9 min LC gradient was used to separate the parent compound and the metabolites formed in the NADPH and UDPGA liver microsomal incubations (reactions I and II), whereas a 29 min LC gradient was used to separate the parent compound from the CoA conjugate in the CoA liver microsomal incubations (reaction III). The mobile phases in the 9 min LC gradient consisted of A (water with 0.1% formic acid) and B (ACN), and the LC gradient profile was as follows: a linear increase from 10 to 70% B during 0 to 6 min, from 70 to 90% B during 6 to 6.01 min, then B was kept at 90% during 6.01 to 6.70 min, and then increased to 95% during 0.01 min. B was kept at 95% for 2 min and then decreased from 95% to the

and tolmetin are marketed drugs, whereas the other five drugs included in this study, i.e., ibufenac (IFC), fenclozic acid, tienilic acid, suprofen, and zomepirac, have been withdrawn or terminated before their launch (Table 1).28 The mechanism causing the adverse drug reactions of nonsteroidal antiinflammatory drugs (NSAIDs) and other XCAs is not well understood, and it has been hypothesized that the covalent binding of reactive metabolites to tissue proteins may be responsible or partly responsible for the hepatoxicity observed in humans. The aim of this study was to investigate the reactivity, as determined by covalent binding, of XCAs’ metabolites in order to better understand their potential to contribute to the toxicity observed for these XCAs.

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MATERIAL AND METHODS

Chemicals and Reagents. 14C-IBP-racemate, 14C-IFC, 14Cfenclozic acid, 14C-tienilic acid, 3H-tolmetin, 3H-suprofen-racemate, and 3H-zomepirac were prepared by AstraZeneca Global Isotope Chemistry Group, and tolmetin, suprofen, and zomepirac were provided by Compound Management, AstraZeneca R&D Mölndal (Mölndal, Sweden). Ultrapure water was obtained from an in-house water purification system (Elgastat Maxima, Elga), and acetonitrile (ACN) was of LC/MS grade (Rathburn Chemical Ltd., Walkerburn, Scotland). FlowLogic U scintillation fluid used for the beta-RAM analysis was obtained from LabLogic (Sheffield, UK). Acetone (LAB-SCAN analytical sciences, Gliwice, Poland) and methanol (Fisher Chemical, Leicestershire, UK) were of high-performance liquid chromatography (HPLC) grade. Formic acid was purchased from Riedel-de Haën AG (Seelze, Germany), and potassium chloride was obtained from Merck (Darmstadt, Germany). Methoxylamine hydrochloride was purchased from Alfa Aesa (Karlsruhe, Germany). Brij58, ammonium acetate (>99%), trizma base, magnesium chloride, potassium chloride, dimethyl sulfoxide (DMSO), leucine enkephalin, NADPH tetrasodium salt hydrate, UDPGA trisodium salt, CoA sodium salt hydrate, ATP disodium salt, and reduced L-glutathione were purchased from SigmaAldrich (St. Louis, MO, USA). Human liver microsomes from a mix of 150 donors were purchased from BD Gentest (Woburn, MA, USA). Human Liver Microsomal Incubations. Stock solutions (10 mM, 21−23 kBq/μL) of 14C-labeled compounds were prepared in 5% DMSO and 50% ACN. Unlabeled and 3H-labeled compounds were codissolved in 5% DMSO and 50% ACN to adjust the specific radioactivity in incubations to the same concentration (10 mM, 21−23 kBq/μL). The stock solutions were diluted to working solutions of 1 mM (2.1−2.3 kBq/μL) in water. Three different master mixes were prepared in order to form oxidative metabolites (reaction I), acyl glucuronides (reaction II), and CoA conjugates (reaction III) (Figure 2). All master mixes were prepared to give a final microsomal protein concentration of 1 mg/mL in the incubations. Master mix I was prepared to give final incubation concentrations of 0.1 M potassium phosphate buffer (0.1 M, pH 7.4) and 10 mM MgCl2. Master mix II gave final incubation concentrations of 0.05 M Tris-HCl (pH 7.4), 10 mM MgCl2, and 0.5 mg/mL Brij58. Master mix III was prepared to give final incubation concentrations of 0.1 M Tris-HCl (pH 7.4), 15 mM MgCl2, 0.15 M KCl, and 3 mM ATP. All incubations were carried out in 0.5 mL 96-well round-bottomed plates (NUNC TM Serving Life Science, Denmark) at 37 °C, with shaking at 400 rpm for 60 min with a total volume of 400 μL in a Thermostar microplate incubator. After addition of the master mixes, the test compounds were added to give final concentrations of 10 μM in reaction I, 100 μM in reaction II, and 200 μM in reaction III. The final DMSO and ACN concentrations in the incubations were less than 0.1 and 1%, respectively. The plates were preincubated at 37 °C and 400 rpm for 5 min prior to initiating the reactions by addition of cofactors or water (controls without cofactors). The final concentrations of the cofactors were 1 mM NADPH for reaction I, 3 mM UDPGA for reaction II, and 0.4 mM CoA for reaction III. All incubations were performed in triplicate. After 60 min incubation, the samples were split in two aliquots, terminated separately, and used for covalent binding measurements (200 μL) and quantification of 888


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Consequently, concentrations of 10, 100, and 200 μM were used to generate OX metabolites, acyl glucuronides, and CoA conjugates, respectively. Metabolites formed via NADPHdependent oxidative processes, most likely not involving the carboxylic acid moiety, corresponded to the mass of the parent compound with an oxygen atom added (+15.9949 Da (M + 16)) with a mass error less than 10 ppm (Tables S1−S7). Two (M + 16) peaks were detected for both IBP and IFC in the mass chromatograms, but they were observed as one broad unresolved peak in the respective radiochromatogram for each compound. For tolmetin, suprofen, and tienilic acid, only one (M + 16) peak was detected for each compound, which corresponded to one peak in the radiochromatogram. Structure elucidation of the (M + 16) metabolites was not attempted. Characterization of the conjugated metabolites was facile, however, utilizing highresolution accurate mass analysis on the protonated molecules and giving mass increments compared to the parent molecule of +176.0320 Da for the acyl glucuronides and +749.1046 Da for the CoA conjugates (Tables S1−S7). In addition to metabolite profiling, the covalent binding to liver microsomal proteins was determined in all incubation samples. Data on the metabolite formation and covalent binding for each compound are given in Table 2 and Figures 4−10. No oxidative (OX) metabolites, acyl glucuronides, or CoA conjugates were detected in the incubations in the absence of the corresponding cofactors (−NADPH, −UDPGA, and −CoA, respectively) for any of the compounds. However, non-cofactor-dependent covalent binding was observed in the −NADPH, −UDPGA, and −CoA incubations, which increased with higher substrate concentrations (Figures 4−10). The origin of this binding was not investigated further. The incubated substrate concentrations of 10 μM (±NADPH), 100 μM (±UDPGA), and 200 μM (±CoA) correspond to a total amount of 4, 40, and 80 nmol of parent compound in the different incubations, respectively. The amounts of parent consumed and remaining after 60 min incubation are shown in Figure 3. No substrate depletion was

starting conditions at 10% during 8.7 to 8.71 min. The sample volume injected throughout the study was 15 μL. The mobile phases in the 29 min LC gradient consisted of A (5 mM ammonium acetate, pH 6.5) and B (ACN), and the LC gradient profile was as follows: 2% B during 0 to 1 min, a linear increase from 2 to 40% B during 1 to 25 min, and from 40 to 70% B during 25 to 26 min. Then, B was increased from 70 to 90% B during 0.01 min, kept at 90% for 0.70 min, and then increased to 95% during 26.7 to 26.71. After 2 min, B was decreased from 95 to 2% during 28.7 to 28.71 min. The LC/MS and LC/radioactivity monitoring (RAM) analyses were performed separately but during the same day. Radioactivity Detection and Quantification of Metabolites. To quantify the formed metabolites, 15 μL aliquots of each sample were injected and separated using the same systems and conditions as those described above. All LC eluent was directed to a LabLogic β-RAM model 5 radio flow-through detector (Brandon, FL, USA). Active counting mode was chosen with automatic noise reduction, the active counting mode threshold set to 1, and the active counting mode factor set to 3 with a flush time of 8 min. A 200 μL flow cell for homogeneous counting was used. The eluent flow rate was 0.5 mL/min, and the scintillation liquid flow rate was 1.5 mL/min. The β-RAM was controlled by Laura Software (version 4.1.6.65, LabLogic Systems Ltd.), and data were collected in real time to generate radiochromatograms. The raw data were collected as counts and transformed to counts per minute by the software. The radiochromatograms from each sample were integrated for parent compound and separated metabolites. The base point, background, and peak integration parameters were set manually. The results are given as percent of total detected radioactivity during the run. Identification of metabolites in the radiochromatograms was based on the retention time of the peak in the radiochromatogram and the corresponding retention time for the MS peak of the metabolite (Tables S1−S7). The metabolites were absent both in the radiochromatogram and in the MS chromatogram in samples from incubations without cofactors. The amount (pmol) of remaining parent compound and formed metabolites per milligram of microsomal protein was calculated by multiplying the percent of total radioactivity detected for each compound/metabolite with the total amount of parent added to each incubation (4 nmol for reaction I, 40 nmol for reaction II, and 80 nmol for reaction III) and divided by the total amount of protein present in the incubation (0.4 mg): amount parent or metabolite (t = 60 min) =

% radioactivity in peak (t = 60 min) × amount parent (t = 0 min) amount total protein in incubation

For the CoA conjugates, the percent covalently bound of the total amount of CoA conjugates (%CVB) was calculated by dividing the covalently bound amount (pmol eq/mg protein) with the sum of amount metabolite detected in the radiochromatogram (pmol eq/mg protein) and the amount metabolite covalently bound (pmol eq/mg protein).

%CVB =

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CVB metabolite formed + CVB

RESULTS Quantification of Metabolites and Covalent Binding to Human Liver Microsomal Proteins. Each substrate, i.e., racemic 14C-IBP, 14C-IFC, 14C-fenclozic acid, 14C-tienilic acid, 3 H-tolmetin, racemic 3H-suprofen, and 3H-zomepirac, was incubated separately for 60 min in three different human liver microsomal systems with and without the cofactors NADPH, UDPGA, or CoA (Figure 2). For each incubation system, the parent compounds, oxidative (OX) metabolites, acyl glucuronides, and CoA conjugates (CoA) were quantified (Figures 3−10 and Table 2). The formation of OX metabolites, acyl glucuronides, and CoA conjugates occurs at different Km values for the same substrate, and three different concentrations were selected for the incubations to reflect these differences.30−32

Figure 3. Parent compound remaining (red bars) and consumed (black bar) after 60 min incubation. The substrate concentrations of 10 μM (NADPH), 100 μM (UDPGA), and 200 μM (CoA) correspond to a total amount of 4 (NADPH), 40 (UDPGA), and 80 (CoA) nmol parent compound in the different incubations.

observed for any incubation and between 0.36 and 2.72 nmol, 0.8 and 11.2 nmol, and 0.8 and 23.2 nmol were consumed in the +NADPH, +UDPGA, and +CoA incubations, respectively. IBP was metabolized to OX-IBP (+NADPH), IBP acyl glucuronide (+UDPGA), and IBP-CoA (+CoA) in the incubations with each respective cofactor (Figure 4A). The 889


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+NADPH and −NADPH or the +UDPGA and −UDPGA incubations (p > 0.05), although the amounts of formed OX-IBP and the IBP acyl glucuronide were almost equal to and four times higher than the amount of IBP-CoA, respectively (Table 2). These data indicate that IBP-CoA is reactive toward liver microsomal proteins, whereas OX-IBP and IBP acyl glucuronide do not appear to bind covalently to liver microsomal proteins. IFC differs only in structure from IBP by lacking the methyl substituent α to the carbonyl group of the acid function (Figure 1). Both OX-IFC and IFC acyl glucuronide were detected in the incubations with the corresponding cofactors (Figure 5A), but there were no significant differences in the covalent binding with and without cofactors (p > 0.05; Figure 5B). In contrast, the covalent binding observed in the +CoA incubation was the highest noted for all investigated compounds (8600 pmol drug eq/mg protein; p < 0.001; Figure 5B and Table 2). Thus, apart from the significantly higher covalent binding of IFC-CoA compared to that of IBP-CoA, the metabolite and reactivity profiles of IFC and IBP are similar (Table 2). Although no oxidative metabolite of fenclozic acid was detected in the +NADPH incubation (Figure 6A), the covalent binding was higher with +NADPH compared to that with −NADPH (p < 0.001; Figure 6B). No detectable levels of covalent binding was observed in the +UDPGA experiments, suggesting that the detected fenclozic acyl glucuronide did not bind covalently to liver microsomal proteins, whereas detected fenclozic acyl-CoA did (p < 0.001; Figure 6A,B). The amount of observed covalent binding (pmol eq/mg protein) divided by the sum of observed covalent binding (i.e., bound CoA conjugates) and unbound CoA conjugates (pmol eq/mg protein) showed that a similar fraction (13−15%) of the CoA conjugates formed from IBP, IFC, and fenclozic acid mediated the covalent binding, although the total amount bound differed between 200 and 8600 pmol eq/mg protein for the three compounds (Table 2). Tolmetin was metabolized to OX-tolmetin, tolmetin acyl glucuronide, and tolmetinyl-CoA (Figure 7A). The covalent

Figure 4. Formed metabolites from IBP in liver microsomes (A) and covalent binding to liver microsomal proteins (B) during 60 min incubation in the presence (black bar) or absence (patterned bar) of NADPH, UDPGA, and CoA. Each bar represents mean ± SD; n = 3. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

covalent binding in the +CoA incubation (1000 pmol drug eq/ mg protein) was significantly higher compared to that in the −CoA incubation (73 pmol drug eq/mg protein; p < 0.001; Figure 4B and Table 2). On the other hand, no significant differences in the covalent binding were observed between the

Table 2. Metabolite Formation and Covalent Binding in Human Liver Microsomes at t = 60 mina compound

IBP + cofactor IBP − cofactor IFC + cofactor IFC − cofactor Fenclozic + cofactor Fenclozic − cofactor Tolmetin + cofactor Tolmetin − cofactor Tolmetin + CoA/MeA Tolmetin + CoA/GSH Suprofen + cofactor Suprofen − cofactor Tienilic + cofactor Tienilic − cofactor Zomepirac + cofactor Zomepirac − cofactor a

oxidative metabolites (+NADPH)

acyl glucuronides (+UDPGA)

CoA conjugates (+CoA)

pmol metabolite/mg protein

pmol CVBb/mg protein





5300 ± 90 N.D.c 2800 ± 270 N.D.c N.D.c N.D.c 1600 ± 150 N.D.c N.A.d

1.1 ± 1.3 1.1 ± 0.6 1.0 ± 0.6 0.4 ± 0.4 27 ± 1.4 1.4 ± 0.4 2.7 ± 0.8 0.6 ± 0.5 N.A.d

26 000 ± 1200 N.D.c 4300 ± 400 N.D.c 2200 ± 750 N.D.c 3500 ± 780 N.D.c N.A.d

8.6 ± 1.1 7.0 ± 0.9 7.6 ± 1.8 6.7 ± 0.9 42 ± 8.0 35 ± 2.3 12 ± 1.5 12 ± 0.8 N.A.d

6700 ± 310 N.D.c 50 300 ± 1900 N.D.c 1300 ± 120 N.D.c N.D.c N.D.c N.A.d

1000 ± 64 73 ± 65 8600 ± 1500 19 ± 6.3 200 ± 30 36 ± 6.0 58 ± 3.6 44 ± 2.0 46 ± 4.8

N.A.d

N.A.d

N.A.d

N.A.d

N.A.d

35 ± 1.5

760 ± 240 N.D.c 1800 ± 160 N.D.c N.D.c

240 ± 8.9 8.4 ± 0.7 260 ± 17 4.8 ± 0.1 23 ± 2.4

21 000 ± 400 N.D.c 400 ± 100 N.D.c 8500 ± 490

120 ± 16 130 ± 4.5 71 ± 3.7 69 ± 2.2 95 ± 2.7

N.D.c N.D.c N.D.c N.D.c N.D.c

180 ± 15 190 ± 11 90 ± 4.7 100 ± 2.8 210 ± 22

N.D.c

9.0 ± 1.5

N.D.c

130 ± 5.8

N.D.c

220 ± 2.8

Mean values ± SD; n = 3. bCVB, covalent binding. cN.D., not detected. dN.A., not available. 890


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Figure 5. Formed metabolites from IFC in liver microsomes (A) and covalent binding to liver microsomal proteins (B) during 60 min incubation in the presence (black bar) or absence (patterned bar) of NADPH, UDPGA, and CoA. Each bar represents mean ± SD; n = 3. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Figure 7. Formed metabolites from tolmetin in liver microsomes (A) and covalent binding to liver microsomal proteins during 60 min incubation in the presence (black bar) or absence (patterned bar) of NADPH, UDPGA, and CoA (B) and the covalent binding in the presence of CoA (black bar), CoA, and methoxylamine (MeA; striped bar) and CoA and glutathione (GSH; white bar) (C). Each bar represents mean ± SD; n = 3. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

binding was higher in the +NADPH compared to that in the −NADPH incubations (p < 0.05), whereas no difference in the covalent binding was observed between the +UDPGA and −UDPGA incubations (Figure 7B). Tolmetinyl-CoA was detected in the mass chromatogram, but the levels were too low for quantification by radiochromatography. Nevertheless, a significantly higher covalent binding was observed in the +CoA compared to that in the −CoA incubations (p < 0.01; Figure 7B). A separate study was designed to establish whether tolmetinylCoA mediated the observed covalent binding. Trapping experiments with MeA and GSH in the presence of CoA resulted in significantly lower covalent binding compared to that in +CoA incubation without any trapping agent (Figure 7C). Not only did covalent binding decrease in these incubations but also the already low levels of the reactive species tolmetinyl-CoA decreased for the MeA incubation and was almost absent in the GSH incubation compared to the incubation with only CoA

Figure 6. Formed metabolites from fenclozic acid in liver microsomes (A) and covalent binding to liver microsomal proteins (B) during 60 min incubation in the presence (black bar) or absence (patterned bar) of NADPH, UDPGA, and CoA. Each bar represents mean ± SD; n = 3. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

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Figure 8. Formed metabolites from suprofen in liver microsomes (A) and covalent binding to liver microsomal proteins (B) during 60 min incubation in the presence (black bar) or absence (patterned bar) of NADPH, UDPGA, and CoA. Each bar represents mean ± SD; n = 3. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Figure 9. Formed metabolites from tienilic acid in liver microsomes (A) and covalent binding to liver microsomal proteins (B) during 60 min incubation in the presence (black bar) or absence (patterned bar) of NADPH, UDPGA, and CoA. Each bar represents mean ± SD; n = 3. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

(data not shown). As with tolmetin, oxidative metabolites as well as acyl glucuronides of suprofen were detected (Figure 8A), whereas the amounts of suprofenyl-CoA formed were sufficient for MS detection but not for quantification by radiochromatography. Only the +NADPH supplemented incubation showed a higher covalent binding compared to that in the incubation without cofactor (p < 0.001; Figure 8B). Tienilic acid formed both an oxidative metabolite and the acyl glucuronide, whereas no CoA conjugate was detected (Figure 9A). A significantly high level of covalent binding (260 pmol/mg protein) was observed in the +NADPH incubation (p < 0.001), whereas no covalent binding to liver microsomal proteins was detected in the +UDPGA incubation (Figure 9B). Furthermore, non-cofactordependent binding was observed in both suprofen and tienilic acid incubations. The only detected metabolite of zomepirac was the zomepirac acyl glucuronide (Figure 10A). However, higher covalent binding was observed in the +NADPH incubation compared to that in −NADPH incubation (p < 0.01; Figure 10B). Noncofactor-dependent binding to liver microsomal proteins was observed and appeared to increase with increasing concentration of zomepirac. The −UDPGA incubation had a significantly higher binding compared to that +UDPGA (p < 0.001; Figure 10B). If the observed binding is due to zomepirac alone, then the higher binding without UDPGA compared to that with UDPGA may be explained by the fact that 15% of zomepirac was consumed with cofactor while all of the drug was available for binding without UDPGA.

Figure 10. Formed metabolites from zomepirac in liver microsomes (A) and covalent binding to liver microsomal proteins (B) during 60 min incubation in the presence (black bar) or absence (patterned bar) of NADPH, UDPGA, and CoA. Each bar represents mean ± SD; n = 3. *, p < 0.05; **, p < 0.01; ***, p < 0.001. 892


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DISCUSSION In this study, the reactivities of OX metabolites, acyl glucuronides, and CoA conjugates formed from seven carboxylic acid drugs, i.e., IBP, IFC, fenclozic acid, tolmetin, suprofen, tienilic acid, and zomepirac, were evaluated in human liver microsomal test systems. An experimental approach was designed to reveal which metabolites are most likely to mediate the covalent binding previously reported in cryopreserved human hepatocytes for the seven drugs.28 Human liver microsomes were used because metabolite formation can be preferentially controlled by cofactor supplement and the reactivity of each metabolite can be evaluated separately, whereas several metabolites can contribute in parallel to the covalent binding in hepatocytes. The formation of OX metabolites, acyl glucuronides, and CoA conjugates occurs at different Km values, and the substrate concentrations in this study were 20 and 2 times lower in the ±NADPH and ±UDPGA incubations, respectively, compared to those in the ±CoA incubations to avoid saturation of the enzymes for the different reactions studied.30−32 Thus, the amount formed OX metabolites, acyl glucuronides, and CoA conjugates cannot be directly compared for the same substrate and will be different from whole cell incubations and the in vivo situation, where all metabolizing enzymes will be exposed to the same substrate concentration at any given point in time (Table 2). Nonetheless, our experimental approach provides for valuable information on the reactivity of the different types of metabolites formed for each substrate. Although findings are discussed in detail below, one salient observation is that the reactivity of the CoA conjugates, as measured by covalent binding to microsomal protein, was significantly higher compared to that of the corresponding oxidative metabolite(s). None of the seven formed acyl glucuronides mediated covalent binding. For IBP, only the CoA conjugate mediated covalent binding (1000 pmol/mg protein), although both OX-IBP and IBPglucuronide were formed (Table 2). Previous findings in human liver microsomes, rat hepatocytes, and in vivo in human support the lack of OX-IBP and IBP-glucuronide reactivity.11,33,34 Further support for the formation of IBP-CoA in vivo is given by the presence of the taurine conjugate of IBP that requires the CoA conjugate intermediate for its formation, in human urine.35 The amounts of metabolites formed from IFC and IBP differed significantly, despite the only structural difference being the lack of the methyl group α to the carboxylic acid moiety for IFC. The detected levels of the formed CoA conjugate of IFC were 8-fold higher compared to that of IBP, which may explain the 9-fold higher covalent binding of IFC-CoA. Calculations of the fraction of drug-related material covalently bound compared to the total amount of formed metabolite showed almost equal fractions, i.e., 15 and 13% of the formed IFC-CoA and IBP-CoA mediated covalent binding, respectively. This observation may indicate that the intrinsic reactivity of the two CoA conjugates is comparable. In the present study, racemic IBP was used, which may confound the interpretation of results because it has been shown that the R-form of IBP forms the CoA conjugate to a much greater extent than does the S-form.11,36 Thus, in our liver microsomal system supplemented with CoA, assuming that mainly R-IBP-CoA is formed, the fraction of covalently bound material may or may not be influenced by the presence of S-IBP. In combination with the data generated with NADPH- and UDPGA-mediated metabolism, we can still conclude that CoAmediated metabolism of racemic IBP leads to significant levels of

covalent binding, whereas OX-IBP and IBP acyl glucuronide do not. While the α-methyl group on IBP does not appear to influence the reactivity of the CoA conjugate, one may speculate that the α-methyl group causes steric hindrance when IBP is binding into the active site of ACS enzyme, resulting in less formation of the CoA conjugate compared to that when the αmethyl group is absent (i.e., as for IFC). In contrast, the amount of IBP-glucuronide was 6 times higher compared to that of IFCglucuronide, whereas the NADPH supplemented incubations resulted in approximately two times higher formation of the OXIBP compared to that of OX-IFC (Table 2). In this case, the steric influence of the α-methyl group resulted in a higher amount of metabolites, possibly due to a change in the substrateenzyme affinity or due to the involvement of other isoforms of UGT and P450 that might better accommodate the α-methyl substrate. It has been previously shown that although the concentration of IBP-glucuronide is higher than that of IFC-glucuronide in vivo in the rhesus monkey37 higher levels of protein adducts of IFC were detected in plasma. In the present study, however, we have established that the higher formation of IFC-CoA mediated a higher extent of covalent binding compared to IBP-CoA to human liver microsomal protein. On the other hand, no covalent binding was detected for either of the glucuronides. Bearing in mind that an attempt to correlate in vivo monkey data with in vitro human liver microsomal data is tentative, the apparent difference in covalent binding outcome might be explained by intracellular formation of protein adducts via acyl-CoA. The drug-protein adducts may then escape the cell into circulation. In this case, the protein adducts of IBP and IFC detected in plasma would not necessarily be formed via acyl glucuronides. The higher amount of IFC plasma protein adducts in monkey may then be explained by a higher amount of intracellular IFC-CoA in vivo, mediating the formation of IFC protein adducts. Albumindrug complexes can be transported, in vesicles, across endothelial cells into tumor interstitium.38 Therefore, we speculate that the transport of drug-protein adducts from hepatocytes into plasma may also be possible. Investigations on IFC-CoA levels in liver biopsies or amino acid conjugates of IFC in plasma might provide valuable information to evaluate the potential involvement of IFC-CoA in the formation of protein adducts in vivo. Another possible explanation that supports the involvement of acyl-CoA conjugates rather than acyl glucuronides is that one can assume that highly reactive and potentially toxic metabolites will bind covalently to intracellular proteins soon after formation and may not enter the circulation as the unbound metabolite. Since both IBP and IFC-glucuronides were detected in plasma in the monkey, metabolites of higher reactivity, e.g., the CoA conjugates, could potentially play a more important role in the observed hepatotoxicity for these NSAIDs. Moreover, the higher formation of IFC-CoA, compared to that of IBP-CoA, may contribute to the diverse toxicity outcomes in patients, which resulted in withdrawal of IFC in 1968 due to hepatotoxicity while IBP still can be purchased over the counter.39,40 In this context, one might speculate on the covalent binding of IFC-CoA to microsomal proteins with respect to stoichiometry. The protein concentration in our incubations is 1 mg/mL. Using a rough estimate of 50 kDa as an average molecular weight for the microsomal proteins leads to an estimated amount of 20 nmol in 1 mg of protein. In our study, the CoA-mediated covalent binding of IFC to 1 mg of microsomal protein was determined to be 8.6 nmol. Thus, the estimated ratio of bound drug to protein is almost 1:2 in the case of CoA-dependent binding. Assuming that 893


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contributed.25,28,44,45 However, our results support the reported P450-mediated toxicity,1,2,25,44,45 as only the oxidative metabolites of tienilic acid and suprofen showed reactivity in the liver microsomes, even though the acyl glucuronides were formed to significant levels (Figures 8 and 9). This is in agreement with previous findings for tienilic acid in human liver microsomes supplemented with NADPH and UDPGA.33 In addition, tienilic acid has been reported to cause time-dependent inhibition of P450 2C9.46 In this present study, racemic suprofen was used, but the potential influence of stereoselective formation of metabolites in the three model systems was not considered. Zomepirac and tolmetin, which are structurally similar (Figure 1), both form oxidative metabolites, acyl glucuronides, and CoA conjugates as well as the corresponding taurine and glycine conjugates in vivo in rat livers.8,47,48 However, in the present study, using human liver microsomes, no zomepiracyl-CoA and only trace amounts of tolmetinyl-CoA were detected, suggesting that zomepiracyl-CoA and tolmetinyl-CoA are formed to a greater extent in rats compared to that in human liver microsomes. Alternatively, the ACS enzymes involved in conjugating zomepirac and tolmetin with CoA may reside on the mitochondrial membrane and/or inside the mitochondrion and would not be present in our microsomal incubations.27 Nonetheless, tolmetin microsomal incubations supplemented with CoA showed a higher level of covalent binding compared to that in incubations without CoA, and this was investigated further via coincubation studies with MeA and GSH in the presence of CoA. These incubations resulted in significantly lower covalent binding compared to that in incubations without any trapping agent, implying that MeA and GSH decreased the level of reactive tolmetinyl-CoA available, although the expected tolmetinyl-SG and tolmetinyl-methoxylamide products were not seen in the respective supernatants. Nonetheless, our data suggest that the higher covalent binding observed in the +CoA compared to that in the −CoA incubation may be caused by the reaction of tolmetinyl-CoA with liver microsomal proteins. Furthermore, a higher covalent binding was observed for tolmetin and zomepirac in NADPH-supplemented microsomal incubations, although oxidative metabolism was detected only for tolmetin. GSH adducts of zomepirac and tolmetin in NADPH-supplemented human liver microsomal incubations have previously been identified.49 These GSH adducts were also identified in rat and human hepatocytes as well as in vivo in rat, and the structures of the GSH adducts suggested formation via oxidative pathways.49 Furthermore, both zomepirac and tolmetin adducts have been detected in vivo in human plasma, and the binding was correlated to the exposure of the corresponding acyl glucuronides.3,4 It is noteworthy that, although tolmetin and zomepirac share several structural features, tolmetin is available on the market, whereas zomepirac has been withdrawn due to a high incidence of immunologic and anaphylactic reactions.3 Overall, it appears possible that both the CoA conjugates, the acyl glucuronides, and the oxidative metabolites of tolmetin and zomepirac are important and can mediate the covalent binding to liver proteins and consequently the rare drug-induced liver injury reported for tolmetin and the adverse drug reactions caused by zomepirac. Further studies will be needed for conclusive understanding of the mechanisms of toxicity. In conclusion, bearing in mind that our present study utilized liver microsomes to assess formation and reactivity of metabolites formed from the seven acids studied, it is likely that the covalent binding reported by Thompson et al. in human hepatocytes for suprofen, tienilic acid, and zomepirac is mediated

only accessible nucleophilic moieties are targets for covalent binding, one might also speculate that the number of adducted drug molecules is higher for some protein species compared to others. These considerations speaks highly in favor of valueadding studies using proteomic tools to actually identify the protein targets of the covalent binding to better assess the toxicological impact. Identification of proteins affected may support a prediction of the type and severity of toxic response caused by different reactive species. In addition to IBP and IFC, fenclozic acid was also shown to form reactive CoA conjugates in human liver microsomes (Table 2 and Figure 6). A fraction of 13% of the total amount formed fenclozic acyl-CoA was bound covalently to microsomal protein after 1 h incubation. Compared to IBP-CoA, the amount of fenclozic acyl-CoA formed and the covalent binding measured were both 5 times lower. In comparison with IFC-CoA, the amount and covalent binding of fenclozic acyl-CoA were 39 and 43 times lower, respectively. Interestingly, there seems to be a clear correlation between the amounts of IBP-CoA, IFC-CoA, and fenclozic acyl-CoA formed and the covalent binding of these CoA conjugates, with 13−15% bound for all three CoA conjugates. This finding is in agreement with previous studies in fresh rat hepatocytes where a time-dependent increase of 2phenylpropionyl-CoA and time- and concentration-dependent increases of phenylacetyl-CoA resulted in time- and concentration-dependent increases in covalent binding.5,12 Although fenclozic acid exhibited an excellent preclinical safety profile, it was withdrawn from clinical trials due to humanspecific hepatotoxicity, with its toxic mechanism still unresolved.41 Pickup and co-workers utilized the hepatic reductase null mouse, lacking P450 activity in the liver, to examine the involvement of Phase II metabolism in the toxicity of fenclozic acid. The main metabolites were glycine and taurine conjugates, confirming that CoA-mediated metabolism occurs in vivo, whereas the acyl glucuronide was identified as a minor metabolite. Nevertheless, the covalent binding to hepatic reductase null mouse liver tissue was low (13 pmol drug eq/ mg protein) and does not support the proposal that the toxicity observed in human is mediated via reactive CoA or acyl glucuronide conjugates.42 However, because the observed hepatotoxicity was human-specific, the hepatic reductase null mouse may not be a relevant model for humans, even though metabolism was shifted toward conjugative reactions. For this human-specific hepatotoxicity, other models will be needed to elucidate the mechanism(s) of toxicity.41 Fenclozic acyl glucuronide was shown in a previous study to lack reactivity in liver microsomes,43 as also supported by our studies. Even though no oxidative metabolite was detected in NADPH-supplemented microsomal incubations, higher covalent binding was observed compared to that with microsomes incubated without NADPH (Figure 6B).43 Rodrigues and coworkers43 showed that the NADPH-dependent covalent binding of fenclozic acid was reduced in the presence of the trapping agents cysteine, GSH, cyanide, and MeA, providing indirect evidence for the formation of oxidative metabolite(s) even though the free metabolite(s) was not detectable in the incubation. Suprofen and tienilic acid have been shown to give covalent binding in hepatocytes (Table 1).28 The two compounds were included in our present study in order to investigate whether only the oxidative metabolites, supposed to be involved in the clinical toxicity, mediated the covalent binding observed in human hepatocytes or if acyl glucuronides and/or CoA conjugates also 894


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(6) Li, C., Benet, L. Z., and Grillo, M. P. (2002) Studies on the chemical reactivity of 2-phenylpropionic acid 1-O-acyl glucuronide and S-acylCoA thioester metabolites. Chem. Res. Toxicol. 15, 1309−1317. (7) Li, C., Olurinde, M. O., Hodges, L. M., Grillo, M. P., and Benet, L. Z. (2003) Covalent binding of 2-phenylpropionyl-S-acyl-CoA thioester to tissue proteins in vitro. Drug Metab. Dispos. 31, 727−730. (8) Olsen, J., Li, C., Skonberg, C., Bjørnsdottir, I., Sidenius, U., Benet, L. Z., and Hansen, S. H. (2007) Studies on the metabolism of tolmetin to the chemically reactive acyl-coenzyme A thioester intermediate in rats. Drug Metab. Dispos. 35, 758−764. (9) Sallustio, B. C., Nunthasomboon, S., Drogemuller, C. J., and Knights, K. M. (2000) In vitro covalent binding of nafenopin-CoA to human liver proteins. Toxicol. Appl. Pharmacol. 163, 176−182. (10) Li, C., Grillo, M. P., Badagnani, I., Fife, K. L., and Benet, L. Z. (2008) Differential effects of fibrates on the metabolic activation of 2phenylpropionic acid in rats. Drug Metab. Dispos. 36, 682−687. (11) Grillo, M. P., and Hua, F. (2008) Enantioselective formation of ibuprofen-S-acyl-glutathione in vitro in incubations of ibuprofen with rat hepatocytes. Chem. Res. Toxicol. 21, 1749−1759. (12) Grillo, M. P., and Lohr, M. T. (2009) Covalent binding of phenylacetic acid to protein in incubations with freshly isolated rat hepatocytes. Drug Metab. Dispos. 37, 1073−1082. (13) Grillo, M. P., Wait, J. C. M., Lohr, M. T., Khera, S., and Benet, L. Z. (2010) Stereoselective flunoxaprofen-S-acyl-glutathione thioester formation mediated by acyl-CoA formation in rat hepatocytes. Drug Metab. Dispos. 38, 133−142. (14) Olsen, J., Bjørnsdottir, I., Tjørnelund, J., and Hansen, S. H. (2002) Chemical reactivity of the naproxen acyl glucuronide and the naproxen coenzyme A thioester towards bionucleophiles. J. Pharm. Biomed. Anal. 29, 7−15. (15) Grillo, M. P., and Benet, L. Z. (2002) Studies on the reactivity of clofibryl-S-acyl-CoA thioester with glutathione in vitro. Drug Metab. Dispos. 30, 55−62. (16) Grillo, M. P., Lohr, M. T., and Wait, J. C. M. (2012) Metabolic activation of mefenamic acid leading to mefenamyl-S-acyl- glutathione adduct formation in vitro and in vivo in rat. Drug Metab. Dispos. 40, 1515−1526. (17) Horng, H., and Benet, L. Z. (2013) Characterization of the acyladenylate linked metabolite of mefenamic Acid. Chem. Res. Toxicol. 26, 465−476. (18) Knights, K. M., Sykes, M. J., and Miners, J. O. (2007) Amino acid conjugation: contribution to the metabolism and toxicity of xenobiotic carboxylic acids. Expert Opin. Drug Metab. Toxicol. 3, 159−168. (19) Fromenty, B., and Pessayre, D. (1995) Inhibition of mitochondrial beta-oxidation as a mechanism of hepatotoxicity. Pharmacol. Ther. 67, 101−154. (20) McMurry, L. M., Oethinger, M., and Levy, S. B. (1998) Triclosan targets lipid synthesis. Nature 394, 531−532. (21) Escalada, M. G., Harwood, J. L., Maillard, J., and Ochs, D. (2005) Triclosan inhibition of fatty acid synthesis and its effect on growth of Escherichia coli and Pseudomonas aeruginosa. J. Antimicrob. Chemother. 55, 879−882. (22) Darnell, M., and Weidolf, L. (2013) Metabolism of xenobiotic carboxylic acids: focus on coenzyme A conjugation, reactivity, and interference with lipid metabolism. Chem. Res. Toxicol. 26, 1139−1155. (23) Begriche, K., Massart, J., Robin, M., Borgne-Sanchez, A., and Fromenty, B. (2011) Drug-induced toxicity on mitochondria and lipid metabolism: Mechanistic diversity and deleterious consequences for the liver. J. Hepatol. 54, 773−794. (24) Somchit, N., Sanat, F., Gan, E. H., Shahrin, I. A. W., and Zuraini, A. (2004) Liver injury induced by the non-steroidal anti-inflammatory drug mefenamic acid. Singapore Med. J. 45, 530−532. (25) O’Donnell, J. P., Dalvie, D. K., Kalgutkar, A. S., and Obach, R. S. (2003) Mechanism-based inactivation of human recombinant P450 2C9 by the nonsteroidal anti-inflammatory drug suprofen. Drug Metab. Dispos. 31, 1369−1377. (26) Soupene, E., and Kuypers, F. A. (2008) Mammalian long-chain acyl-CoA synthetases. Exp. Biol. Med. 233, 507−521.

via oxidative metabolites, whereas CoA conjugates seem to mediate covalent binding for IFC, IBP, and tolmetin.28 For fenclozic acid, on the other hand, both oxidative metabolites and CoA conjugates seem to be involved in the observed covalent binding. Interestingly, our experimental approach suggests that no covalent binding to microsomal proteins can be attributed to the acyl glucuronides formed from the seven XCAs even though some of them have previously been shown to react with plasma proteins and albumin.3,4,37 Thus, it is important to acknowledge that metabolites, other than the acyl glucuronide formed from the studied compounds, may mediate adverse drug reactions in humans. However, those reactive metabolites that do not bind to microsomal proteins may potentially bind to other proteins present in hepatocytes, although such investigations are beyond the scope of the work presented here.

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ASSOCIATED CONTENT

S Supporting Information *

Retention times (tR), accurate mass and mass errors of protonated/deprotonated 14C/3H substrates and metabolites, and LC/MS conditions (Tables S1−S7). This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel.: +46 31 776 15 21. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Professor Neal Castagnoli Jr. is gratefully acknowledged for fruitful discussions and input on this manuscript. ABBREVIATIONS ACN, acetonitrile; ACS, acyl-CoA synthetase; CoA conjugate, Sacyl-CoA conjugate; CVB, covalent binding; DILI, drug-induced liver injury; ESI, electrospray ionization; gluc, glucuronide; GSH, glutathione; IBP, ibuprofen; IFC, ibufenac; MeA, methoxylamine; NSAID, nonsteroidal anti-inflammatory drug; OX, oxidative; P450, cytochrome P450; RAM, radioactivity monitoring; tR, retention times; UDPGA, uridine 5′-diphosphoglucuronic acid; UGT, UDP-glucuronosyltransferase; UPLC, ultra performance liquid chromatography; XCAs, xenobiotic carboxylic acids; xenobiotic-CoA, xenobiotic-S-acyl-CoA thioester

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REFERENCES

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Significantly Different Covalent Binding of Oxidative Metabolites, Acyl

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