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TITLE PAGE
Identification of Epoxide-Derived Metabolite(s) of Benzbromarone
Kai Wang, Hui Wang, Ying Peng, and Jiang Zheng
School of Pharmacy (K.W., H.W., Y.P.), Key Laboratory of Structure-Based Drug Design & Discovery of Ministry of Education (J.Z.), Shenyang Pharmaceutical
University, Shenyang, Liaoning, P. R. China; and Center for Developmental Downloaded from Therapeutics, Seattle Children’s Research Institute, Division of Gastroenterology and Hepatology, Department of Pediatrics, University of Washington School of Medicine, Seattle, Washington (J.Z.) dmd.aspetjournals.org
at ASPET Journals on September 24, 2021
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RUNNING TITLE PAGE
Running title: Epoxidation of Benzbromarone
Corresponding Authors:
Jiang Zheng, PhD Center for Developmental Therapeutics, Seattle Children's Research Institute, Division of Gastroenterology and Hepatology, Department of Pediatrics, University of Washington, Seattle, WA 98101 Key Laboratory of Structure-Based Drug Design & Discovery of Ministry of
Education, Shenyang Pharmaceutical University, Shenyang, Liaoning, 110016, P. R. Downloaded from China Email: [email protected] Tel: 206-884-7651; Fax: 206-987-7660 dmd.aspetjournals.org Ying Peng, PhD School of Pharmacy, Shenyang Pharmaceutical University, PO Box 21, 103 Wenhua Rode, Shenyang, 110016, P. R. China Email: [email protected]
Tel: +86-24-23986361; Fax: +86-24-23986510 at ASPET Journals on September 24, 2021
The number of text pages: 22 The number of figures: 9 The number of schemes: 2 The number of references: 33 The number of words in the Abstract: 154 The number of words in the Introduction: 464 The number of words in the Discussion: 927
Abbreviations: BBR, benzbromarone; ACN, acetonitrile; NAC, N-acetylcysteine; DEX, dexamethasone; NADPH, β-nicotinamide adenine dinucleotide 2′-phosphate reduced tetrasodium salt; MLMs, mouse liver microsomes; DP, declustering potential; EP, entrance potential; CE, collision energy; CXP, cell exit potential; EPI, enhanced product ion; SIM, selected ion monitoring; MRM, multiple-reaction monitoring; LC-MS/MS, liquid chromatography coupled to tandem mass spectrometry; and MS/MS, tandem mass spectrometry.
2 DMD Fast Forward. Published on January 20, 2016 as DOI: 10.1124/dmd.115.066803 This article has not been copyedited and formatted. The final version may differ from this version.
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Abstract
Benzbromarone (BBR) is a benzofuran derivative that has been a quite useful drug for the treatment of gout. However, it was withdrawn from European markets in 2003, due to reported serious incidents of drug-induced liver injury. BBR-induced hepatotoxicity has been suggested to be associated with the formation of a quinone intermediate. The present study reported epoxide-derived intermediate(s) of BBR. Downloaded from An N-acetylcysteine (NAC) conjugate derived from epoxide metabolite(s) was detected in both microsomal incubations of BBR and urine samples of mice treated dmd.aspetjournals.org with BBR. The NAC conjugate was identified as 6-NAC BBR. Ketoconazole suppressed the bioactivation of BBR to the epoxide intermediate(s), and CYP3A subfamily was the primary enzyme responsible for the formation of the epoxide(s). at ASPET Journals on September 24, 2021 The present study provided new information on metabolic activation of BBR.
3 DMD Fast Forward. Published on January 20, 2016 as DOI: 10.1124/dmd.115.066803 This article has not been copyedited and formatted. The final version may differ from this version.
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Introduction
Gout is a form of inflammatory arthritis caused by elevation of blood urate levels
(a condition known as hyperuricemia) which crystallize and deposit into joints and/or surrounding tissues (Kenneth and Hyon, 2006; Azevedo et al., 2014).
Benzbromarone (BBR) is a benzofuran derivative (as shown in Figure 1) acting as a uricosuric agent by inhibiting urate reabsorption (Shin et al., 2011). Both BBR and Downloaded from 6-hydroxy BBR (a metabolite of BBR) have been reported to show potent human uric acid transporter 1 (hURAT1) inhibition property (Wempe et al., 2011), which made dmd.aspetjournals.org BBR a quite useful anti-gout agent for approximately 30 years in many countries.
But recently, clinical cases of acute liver damage including some fatalities related to
BBR (Wagayama et al., 2000; Arai et al., 2002; Reinders et al., 2007) draw our at ASPET Journals on September 24, 2021 attention to the metabolism profiles of BBR.
Hepatotoxicity is often associated with metabolic activation mediated by cytochromes P450 (He et al., 2015). Debromination was initially considered to be a main bioactivation of BBR in vivo (Broekhuysen et al., 1972; Ferber et al., 1981). It was clarified in 1988 that hydroxylation rather than debromination was the predominant metabolic pathway of BBR (Walter-Sack et al., 1988). Early metabolic studies revealed two major hydroxylated metabolites identified as 1′-hydroxy BBR and 6-hydroxy BBR (De Vries et al., 1993; Walter-Sack et al., 1998). It was also reported that P450s 2C9 (major) and 2C19 (minor) were involved in the formation of the 6-hydroxy BBR metabolite (De Vries et al., 1993) characterized by comparison with synthetic standard (McDonald and Rettie, 2007). Initially, BBR was found to be
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DMD # 66803 a P450 2C9 inhibitor (Marques-Soare et al., 2003; Locuson et al., 2004).
Subsequently, BBR and four of its analogues were discovered to exhibit extraordinary inhibitory potency for P450 2C19 (Locuson et al., 2004). Idiosyncratic hepatotoxicity of BBR has primarily been suggested to be associated with metabolite
6-hydroxy BBR proposed by McDonald and Rettie (McDonald and Rettie, 2007).
They suggested that sequential oxidation of 6-hydroxy BBR results in a catechol Downloaded from structure, 5,6-dihydroxy BBR, which can be further oxidized to a reactive quinone intermediate capable of adducting protein. However, we believe that the initial dmd.aspetjournals.org epoxidation of BBR may be more important for metabolic activation of BBR.
Epoxide-derived metabolites of many pro-toxicants, such as bromobenzene (Slaughter et al., 1991; Zheng and Hanzlik, 1992a), naphthalene (Zheng et al., 1997; Morisseau at ASPET Journals on September 24, 2021 et al., 2008), styrene (Carlson et al., 2011), and coumarin (Born et al., 2000) are suggested to play important roles in the development of toxicities.
The objectives of this study included (1) characterization of epoxide-derived metabolite(s) of BBR in vitro and in vivo; and (2) identification of cytochromes P450 enzymes responsible for the formation of the metabolite(s). We anticipate that the study would allow us to better understand the mechanisms of idiosyncratic hepatotoxicity of BBR.
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Materials and methods
Chemicals and Materials. Benzbromarone (BBR, purity ＞ 98%) was obtained from Aladdin Industrial Technology Co., Ltd. (Shanghai, China). N-Acetylcysteine
(NAC), ketoconazole, and reduced nicotinamide adenine dinucleotide phosphate
(NADPH) were purchased from Sigma-Aldrich Co. (St. Louis, MO). Recombinant human P450 enzymes were purchased from BD Gentest (Woburn, MA). Downloaded from Dexamethasone (DEX), sodium nitrite, and tin chloride were purchased from the
National Institute for the Control of Pharmaceutical and Biological Products dmd.aspetjournals.org (Shenyang, China). All organic solvents were from Fisher Scientific (Springfield,
NJ). All reagents and solvents were of either analytical or HPLC grade.
Animal Experiments. Male Kunming mice (20 ± 2 g) were obtained from the at ASPET Journals on September 24, 2021
Animal Center of Shenyang Pharmaceutical University. The animals were maintained on standard mouse chow and tap water ad libitum in a 25 °C room with a
12 h dark/light cycle. Mice were individually placed in metabolism cages. After fasting for 12 h with free access to water prior to the experiment, mice were intraperitoneally treated with BBR dissolved in corn oil (10 mL/kg) at 65 mg/kg.
Urine samples were collected from 0 to 24 h after dosing. The control animals treated with corn oil were included. During the experiment, the animals were allowed to free access to food and water. Individual groups (BBR-treated and control) contained four mice. The collected urine samples were stored at -20 °C until analysis.
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Sample Preparation. The collected urine samples were pooled and mixed with triple volumes of acetonitrile (ACN). All samples were vortexed for 3 min and then centrifuged at 16,000 rpm for 10 min at 4 °C. The supernatants were harvested and the ACN was evaporated under a stream of nitrogen gas at 40 °C. The resulting urine samples were extracted with ethyl acetate (Wu et al., 2012). The organic layer was collected and evaporated to dryness under a stream of nitrogen gas at 40 °C. Downloaded from The residues were reconstituted with 100 μL of 50% ACN in water. After centrifugation, the supernatants (5 μL) were injected onto LC-MS/MS for analysis. dmd.aspetjournals.org
Microsomal Incubations. Mouse liver microsomes (MLMs) were prepared in our laboratory according to previous published method (Lin et al., 2007).
Dexamethasone-induced mouse liver microsomes (DEX-induced MLMs) were at ASPET Journals on September 24, 2021 prepared from mice pretreated with DEX (an inducer of CYP3A4) for five consecutive days, using the same procedure above. A stock solution of BBR was prepared in methanol. The incubation mixtures were prepared in a final volume of
0.5 mL phosphate buffer (pH 7.4) containing MLMs or DEX-induced MLMs (1.0 mg
protein/mL), 3.2 mM MgCl2, 75 μM BBR, and 40 mM NAC. Total content of organic solvent was maintained at < 3%. The incubation reactions were initiated by addition of NADPH (1.0 mM). Control samples containing no NADPH were included. After 60 min incubation at 37 °C, the reactions were quenched by adding equal volume of ice-cold ACN. The reaction mixtures were vortex mixed and centrifuged at 16,000 rpm for 10 min to remove precipitated protein. The resulting supernatants were evaporated to dryness under a stream of nitrogen gas at 40 °C and
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DMD # 66803 then reconstituted with 100 μL of 50% ACN in water before injected onto LC-MS/MS for analysis. Each incubation was performed in duplicate.
Human Recombinant P450 Incubations. To determine the specific P450 enzymes involved in the formation of reactive metabolites of BBR, a total of eight human recombinant P450s, including P450s 1A2, 2A6, 2B6, 2C9, 2C19, 2E1, 3A4,
and 3A5, were tested. Conditions were equivalent to the microsomal incubations Downloaded from except for that microsomes were replaced by the individual human recombinant P450 enzymes (20 pmol enzyme with a total volume of 200 µL in each incubation). The dmd.aspetjournals.org experiments were performed in triplicate.
P450 3A Inhibition study. To examine the role of P450 3A subfamily in at ASPET Journals on September 24, 2021 bioactivation of BBR, similar microsomal incubations as described above were performed except for inclusion of ketoconazole at concentrations of 1.0, 10, and 100
μM. The formation of the NAC-BBR conjugate was monitored by LC-MS/MS.
The experiments were performed in triplicate.
Synthesis of 6-NAC BBR. BBR (350 mg, 0.83 mmol) was slowly dissolved in
10 mL of 97% H2SO4 pre-cooled at -15 °C, followed by dropwise addition of 2 mL fuming nitric acid. The reaction mixture was constantly stirred at -15 °C for 4 h.
The mixture was diluted with 20 mL water, and yellowish solid was observed. The solid product was chromatographed over silica. The purified nitration product
(220.5 mg, 0.47 mmol) was heated with SnCl2·2H2O (318.2 mg, 1.41 mmol) in 15 mL of ethanol at 80 °C and refluxed for 4 h. The mixture was neutralized to pH 7 with
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NaHCO3 and extracted with CH2Cl2. The remaining CH2Cl2 layer was washed with water, dried with anhydrous sodium sulfate, and evaporated. After chromatographing on silica gel, the resulting aniline (95 mg, 0.22 mmol) was dissolved in 5 mL of methanol and cooled to -10 ºC. To the aniline solution were 2 drops of concentrated hydrochloric acid added, and the mixture was diazotized by
dropwise addition of NaNO2 (22.4 mg, 0.32 mmol) in 250 μL of water. This cold Downloaded from diazonium solution was then added over 35 min to a stirred solution of NAC (105.8 mg, 0.65 mmol) in 5mL of water held at 65 ºC. After the solution was stirred for 2 h, dmd.aspetjournals.org the mixture was cooled down to room temperature and extracted with CH2C12. The
CH2C12 extracts were chromatographed on silica gel (Zheng and Hanzlik, 1992b).
Further purification by a semi-preparative HPLC system offered 3.2 mg of BBR-NAC at ASPET Journals on September 24, 2021 conjugate. All NMR measurements were obtained at 600 MHZ on a
BRUKER-ARX-600 spectrometer (Switzerland).
LC-MS/MS Method. LC-MS/MS analyses were performed on an AB SCIEX
Instruments 4000 Q-Trap (Applied Biosystems, Foster City, CA) interfaced online with an ekspert ultraLC100 system (Applied Biosystems). The analytical separation
was achieved on an Ultimate XB-C18 column (2.1 × 100 mm, 3 μm, Welch Scientific,
Inc., Shanghai, China) with a flow rate of 0.4 mL/min, and purification by semi-preparative HPLC was achieved on a YMC-Pack ODS-A column (250 × 10 mm,
S-5, 12 nm, YMC Co., Ltd, Japan) with a flow rate of 3 mL/min. The mobile phase consisted of solvent A (0.1% formic acid in ACN) and solvent B (0.1% formic acid in
H2O). A gradient elution was applied for analytical separation：0-2 min, 10% A; 2-8 9 DMD Fast Forward. Published on January 20, 2016 as DOI: 10.1124/dmd.115.066803 This article has not been copyedited and formatted. The final version may differ from this version.
DMD # 66803 min, 10-70% A; 8-9 min, 70-100% A; 9-10 min, 100% A; 10-11 min, 100-10% A;
11-14 min, 10% A. An isocratic elution was used for metabolite purification by semi-preparative HPLC with 45% solvent A for 45 minutes. LC-MS/MS analyses were performed on a 5-μL aliquot of samples. Turbo Ion Spray interface for electrospray ionization was operated in positive ion mode, using the following conditions: ion spray voltage, 5500 V; source temperature, 650°C; curtain gas, 20 psi; Downloaded from ion source gas 1, 50 psi; ion source gas 2, 50 psi; declustering potential (DP), 50 V; entrance potential (EP), 10 V; and cell exit potential (CXP), 5 V. The dmd.aspetjournals.org information-dependent acquisition (IDA) method was utilized to trigger the enhanced product ion (EPI) scans by analyzing multiple reaction monitoring (MRM) signals.
Mass spectrometric analyses were conducted with ion transitions m/z 584→277, at ASPET Journals on September 24, 2021 586→279, and 588→281 for BBR-NAC conjugate derived from epoxide, m/z
616→487, 618→489, and 620→491 for BBR-NAC conjugate derived from the corresponding quinone, and m/z 260.7→116.3 for propranolol (internal standard), respectively. The collision energy was set at 45 eV with a spread of 15 eV. Data were processed using AB SCIEX Analyst software versions 1.6.0 and 1.6.1 (Applied
Biosystems, Foster City, CA).
MS/MS analyses were also conducted on an Agilent 1200 Series Rapid
Resolution LC system equipped with a hybrid quadrupole time-of-flight (Q-TOF) MS system (microQ-TOF; Bruker Corporation, Billerica, MA). Mobile phase A was
ACN with 0.1% (v/v) formic acid, and mobile phase B was water with 0.1% (v/v) formic acid. The mass spectrometric parameters were optimized as follows: end
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DMD # 66803 plate offset, -500 V; capillary voltage, -4500 V; nebulizer gas pressure, 0.3 bar; dry
gas, high-purity nitrogen (N2); dry gas flow rate, 4.0 liters per minute; and gas temperature, 180 °C. The data were analyzed by Bruker Daltonics Data Analysis 3.4 software.
Statistic analysis. Statistic analyses were performed by unpaired Students’s t
-tests, using Graph Pad Prism software. A p value of less than 0.05, 0.01 or 0.001 Downloaded from was considered significantly different.
dmd.aspetjournals.org at ASPET Journals on September 24, 2021
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Results
Mass Spectrometric Behaviors of BBR. To facilitate metabolite identification, we started with mass spectrometric analysis of parent compound BBR in positive ionization mode. The parent drug showed fragment ions of m/z 145, 170, 173, 223,
251, and 279 with [M+H]+ of m/z 425 (Figure 2), which we proposed were derived
from a loss of C10H9O, C6H3BrO, C11H9O2, C5H3Br2, C6H3Br2O, and C7H3Br2O2, Downloaded from respectively.
In Vitro Metabolic Activation of BBR. BBR was incubated in MLMs dmd.aspetjournals.org supplemented with NAC as a trapping agent. One metabolite (retention time = 7.99 min) was detected with a molecular cluster of m/z 584 (50%), 586 (100%), and 588
(50%) (Figure 3B/4B), suggesting an NAC incorporation in BBR. The tandem mass at ASPET Journals on September 24, 2021
(MS/MS) spectrum of the metabolite obtained by MRM-EPI scanning with ion transition m/z 586→455 showed the indicative characteristic neutral loss of 129 Da associated with the cleavage of the NAC moiety (Figure 3E). The product ions at m/z 568 and 544 were derived from the loss of H2O and acetyl group, respectively.
The fragment ion at m/z 279 (loss of C7H3Br2O2) was found to be the same as that of
BBR, indicating that the dibromohydroxybenzoyl ring retained unchanged. This led us to propose that NAC was attached to the benzofuran ring. Considering that the intensity of product ion at m/z 279 was much stronger than that of m/z 455, the rest of
MS/MS spectra of the metabolite were acquired by MRM-EPI scanning with ion transition m/z 586→279 instead of m/z 586→455. The metabolite was also detected in full scan mode (Q1, Figure 4D). No such conjugate was detected in the
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DMD # 66803 microsomal incubation system in the absence of NADPH (Figure 3A), indicating that metabolism was responsible for the formation of the BBR-NAC conjugate. In addition, another metabolite with [M+H]+ of m/z 616, 618, and 620, along with retention time at 7.68 min, was detected in the microsomal mixture in both MRM mode (Figure 4A) and full scan mode (Q1, Figure 4C). The molecular ion matched the molecular weight of BBR-NAC conjugate derived from the quinone intermediate. Downloaded from The corresponding GSH conjugate was reported by McDonald and Rettie (McDonald and Rettie, 2007). dmd.aspetjournals.org
NMR Analysis of Synthetic BBR-NAC Conjugate. To further characterize the metabolite, we chemically synthesized the BBR-NAC conjugate (Scheme 2). Two
BBR-NAC conjugates, presumably 5-NAC BBR and 6-NAC BBR, were obtained in at ASPET Journals on September 24, 2021 the chemical synthesis (Supplemental Figure 1). One product formed in the reaction showed the same chromatographic and mass spectrometric identities (Figure 3D and
3F) as that for the product generated in microsomal incubations. The product was further analyzed by Q-TOF MS in positive mode. It clearly possessed the bromine isotope pattern with a molecular cluster of m/z 583.9346 (50%), 585.9353 (100%), and 587.9336 (50%) (Figure 3G). We succeeded in obtaining 1H-NMR, 13C-NMR, and HMBC NMR spectra of the synthetic BBR-NAC conjugate. The proton NMR spectrum (Figure 5) demonstrated three aromatic proton resonances at 7.30, 7.34, and
7.59 ppm corresponding to the protons at C4, C5 and C1 positions of 6-NAC BBR or
C1, C6 and C4 positions of 5-NAC BBR. Additionally, the HMBC spectrum (Figure
6) showed that C3 had correlations with the protons at 7.34 and 7.59 ppm, and no
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DMD # 66803 correlation was observed between C3 and the proton at 7.30 ppm. Thus, the identity of the conjugate was concluded to be BBR with NAC attached to C6 carbon. In other word, the conjugate was assigned to be 6-NAC BBR.
Urinary Excretion of BBR-NAC Conjugate. To investigate the bioactivation of
BBR in vivo, urinary excretion of BBR-NAC conjugate was monitored by a designed
MRM-EPI template after an intraperitoneal injection of BBR at 65 mg/kg in mice. Downloaded from
As expected, 6-NAC BBR was found in the urine obtained from the animals given
BBR (Figure 7B), and no such urinary metabolite was observed before the treatment dmd.aspetjournals.org
(Figure 7A). The metabolite showed the same retention time as that for the metabolite produced in microsomal incubations (Figure 3B). at ASPET Journals on September 24, 2021 Identification of P450 Enzymes Responsible for Bioactivation of BBR. To determine which P450 enzymes preferentially catalyze the oxidation of BBR, BBR was incubated with individual human recombinant P450 enzymes. As shown in
Figure 8, 6-NAC BBR was detected in the incubations with P450 3A4. Apparently, minor or no 6-NAC BBR was observed in the incubations with the other P450 enzymes tested. The microsome inhibition studies showed that co-incubation with ketoconazole reduced the production of the conjugate in a concentration-dependent manner (Figure 9). These experiments illustrated that CYP3A subfamily was the principal enzyme responsible for the formation of epoxide metabolite(s) of BBR.
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Discussion
Benzbromarone is a uricosuric agent that has been used as a treatment for chronic gout. Though effective, serious incidents of BBR-induced idiosyncratic hepatotoxicity including some fatalities have been reported. Benzbromarone was reported to induce impairment of oxidative phosphorylation in cultured HepG2 cells
(Haegler et al., 2015). Arai et al. speculated that administration of allopurinol Downloaded from
(xanthine oxidase enzyme inhibitor) with benzbromarone may accelerate the occurrence of liver dysfunction (Arai et al., 2002). Idiosyncratic hepatotoxicity is dmd.aspetjournals.org often associated with metabolic activation mediated by cytochromes P450. A quinone-derived reactive intermediate of BBR was reported by McDonald and Rettie
(McDonald and Rettie, 2007). It was our speculation that the generation of at ASPET Journals on September 24, 2021 epoxide-derived intermediate(s) would be required before the formation of the quinone-derived reactive intermediate. To seek the epoxide intermediate(s), BBR was incubated in microsomes supplemented with NAC as a trapping agent. We reasoned that the enzymatic epoxidation of BBR would give epoxides 2 and/or 3
(Scheme 1). The adduction of the epoxides with NAC would produce hydroxyl non-aromatic NAC conjugates 4-7 that may further be dehydrated to NAC conjugates
10-12. No non-aromatic NAC conjugates (4-7) were detected in the microsomal mixtures. Instead, one aromatic NAC conjugate was observed in the microsomal mixtures. This conjugate was also found in the urine samples of mice given BBR.
In addition, we chemically synthesized the conjugate which displayed the same chromatographic and mass spectrometric identities as that of the one observed in
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DMD # 66803 microsomal incubations and the urine samples. The conjugate was characterized as
6-NAC BBR (conjugate 11) by NMR. The identification of the NAC conjugate provided a strong evidence for the formation of the epoxide intermediate(s).
The formation of 6-NAC BBR was found to be NADPH-dependent, indicating that the bioactivation of BBR was mediated by cytochromes P450. The recombinant
P450 enzyme studies showed that CYP3A4 was the major enzyme responsible for the Downloaded from generation of the arene epoxide(s) and that decreases in BBR concentrations (75, 25, and 5 μM) did not cause a big loss of the efficiency of CYP3A4 to catalyze the dmd.aspetjournals.org formation of the epoxide metabolite (Supplemental Figure 2). Co-incubation of ketoconazole suppressed the formation of the reactive metabolite(s) in microsomal reactions. Higher activity to catalyze the bioactivation of BBR was found in at ASPET Journals on September 24, 2021 microsomes obtained from DEX-induced mice than that in regular microsomes
(Figure 3C). The observed potentiating effect of DEX on metabolic activation of
BBR, combined with the suppressive effect of ketoconazole on the formation of the epoxide(s) in microsomal reactions with BBR, suggests the participation of CYP3A in the production of the epoxide metabolite(s) of BBR in vitro. Pretreatment with ketoconazole resulted in 60 % decrease in urinary 6-NAC BBR in mice
(Supplemental Figure 3). This indicates that CYP3A-mediated bioactivation of BBR took place also in vivo.
The present study provided new information on the formation of P450-mediated reactive metabolite(s) of BBR, and the metabolic process had not previously been reported. The pathway for bioactivation of BBR presumably involves epoxidation of
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DMD # 66803 the benzofuran ring to epoxide intermediates 2 and/or 3. Four possible hydroxyl non-aromatic NAC conjugates (4-7) would be formed after reaction with NAC.
Sequential dehydration would offer three different aromatic NAC conjugates (10-12).
Interestingly, only one NAC conjugate, i.e. 6-NAC BBR, was observed in microsomal incubations and urine samples. Two episulfonium ions (8 and 9, Scheme 1) as intermediates are proposed for the explanation of the observation. For some reason, Downloaded from the rearrangement of episulfonium ions 8 and 9 exclusively produces aromatic NAC conjugate 11. dmd.aspetjournals.org McDonald and Rettie suggested a possible mechanism of toxicity that involves the bioactivation of BBR through sequential steps of oxidation of the benzofuran ring to a quinone intermediate, an electrophilic species reactive to nucleophilic sites of at ASPET Journals on September 24, 2021 biomolecules. Though quinones are known electrophiles, epoxides 2 and 3 would be the first electrophilic metabolites generated in the line of the metabolic pathway of
BBR. In the present study, the level of the epoxide-derived NAC conjugate was found to be approximately 10-fold higher than that of the conjugate derived from the quinone in microsomes incubated with BBR (Figure 4C and 4D), and little quinone-derived NAC conjugate was detected in the urine samples of animals treated with BBR. The abundance of conjugate 11 observed in microsomal incubations and urine samples suggests that the initial epoxidation might be logically more important in metabolic activation of BBR, although further mechanistic investigation is in need.
The mechanisms underlying idiosyncratic hepatotoxicity remain largely unknown.
Toxic effects of metabolites are thought to be one of the possible factors implicated
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(Greer et al., 2010; Xuan et al., 2015). Our work showed that CYP3A4 was the primary P450 enzyme responsible for the metabolic activation of BBR (Figure 8).
Several drugs reportedly eliciting idiosyncratic hepatotoxicities have been documented to be bioactivated mainly by CYP3A4, such as nefazodone (Kalgutkar et al., 2005), zafirlukast (Kassahun et al., 2005), rimonabant (Foster et al., 2013), and amiodarone (Takai et al., 2015; Xuan et al., 2015). CYP3A4 is the major human Downloaded from hepatic P450 enzyme, and the abundance of the enzyme seems unlikely to explain the rare incidence of idiosyncratic events in humans. We speculate that other factors, dmd.aspetjournals.org such as deficiency of glutathione or/and glutathione S-transferases, imbalance between cellular damage and protective responses, and oversensitivity of immune system, could also be involved in the adverse effect. at ASPET Journals on September 24, 2021 In conclusion, the present study demonstrated that microsomal incubations of
BBR generated epoxide intermediate(s) that can be trapped with NAC to produce
6-NAC BBR exclusively. Ketoconazole suppressed the bioactivation of BBR to the epoxide intermediate(s), and CYP3A subfamily was the primary enzyme responsible for the formation of the epoxide(s). The results provide additional information on bioactivation of BBR .
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Authorship contributions
Participated in research design: Zheng.
Conducted experiments: Wang H, Peng.
Performed data analysis: Wang K, Wang H.
Wrote or contributed to the writing of the manuscript: Zheng, Wang H.
Downloaded from
dmd.aspetjournals.org
at ASPET Journals on September 24, 2021
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References
Arai M, Yokosuka O, Fujiwara K, Kojima H, Kanda T, Hirasawa H, and Saisho H
(2002) Fulminant hepatic failure associated with benzbromarone treatment: a case
report. J Gastroenterol Hepatol 17: 625-626.
Azevedo VF, Buiar PG, Giovanella LH, Severo LH, and Carvalho M (2014)
Allopurinol, benzbromarone, or a combination in treating patients with gout: Downloaded from analysis of a series of outpatients. Int J Rheumatol 2014: 1-5.
Born SL, Hu J, and Lehman-McKeeman LD (2000) o-Hydroxyphenylacetaldehyde is dmd.aspetjournals.org a hepatotoxic metabolite of coumarin. Drug Metab Dispos 28:218-223
Broekhuysen J, Pacco M, Sion R, Demeulenaere L, and Van Hee M (1972)
Metabolism of benzbromarone in man. Eur J Clin Pharmacol 4: 125-130. at ASPET Journals on September 24, 2021 Carlson GP (2011) Hepatotoxicity and pneumotoxicity of styrene and its metabolites
in glutathione S-transferase-deficient mice. Drug Chem Toxicol 34: 440-444.
De Vries JX, Walter-Sack I, Voss A, Forster W, Ilisistegui Pons P, Stoetzer F, Spraul M,
Ackermann M, and Moyna G (1993) Metabolism of benzbromarone in man:
structures of new oxidative metabolites, 6-hydroxy- and 1′-oxo-benzbromarone,
and the enantioselective formation and elimination of 1′-hydroxybenzbromarone.
Xenobiotica 23: 1435-1450.
Ferber H, Vergin H, and Hitzenberger G (1981) Pharmacokinetics and
biotransformation of benzbromarone in man. Eur J Clin Pharmacol 19: 431-435.
Foster AJ, Prime LH, Gustafsson F, Temesi DG, Isin EM, Midlöv J, Castagnoli N Jr,
and Kenna JG (2013) Bioactivation of the cannabinoid receptor antagonist
20 DMD Fast Forward. Published on January 20, 2016 as DOI: 10.1124/dmd.115.066803 This article has not been copyedited and formatted. The final version may differ from this version.
DMD # 66803
rimonabant to a cytotoxic iminium ion metabolite. Chem Res Toxicol 26: 124-135.
Greer ML, Barber J, Eakins J, and Kenna JG (2010) Cell based approaches for
evaluation of drug-induced liver injury. Toxicology 268: 125-131.
Haegler P, Grünig D, Berger B, Krähenbühl S, and Bouitbir J (2015) Impaired
mitochondrial function in HepG2 cells treated with
hydroxy-cobalamin[c-lactam]: A cell model for idiosyncratic toxicity. Downloaded from Toxicology 336: 48-58.
He L, Yang A, Cui T, Zhai Y, Zhang F, Chen J, Jin C, Fan Y, Wu Z, Wang L, and He X dmd.aspetjournals.org (2015) Reactive metabolite activation by CYP2C19-mediated rhein hepatotoxicity.
Xenobiotica 45: 361-372.
Kalgutkar AS, Vaz AD, Lame ME, Henne KR, Soglia J, Zhao SX, Abramov YA, at ASPET Journals on September 24, 2021 Lombardo F, Collin C, Hendsch ZS, and Hop CE (2005) Bioactivation of the
nontricyclic antidepressant nefazodone to a. reactive quinone-imine species in
human liver microsomes and recombinant cytochrome P450 3A4. Drug Metab
Dispos 33: 243-253.
Kassahun K, Skordos K, Mclntosh I, Slaughter D, Doss GA, Baillie TA, Yost GS
(2005) Zafirlukast metabolism by cytochrome P450 3A4 produces an electrophilic
alpha,betar-unsaturated iminium species that results in the selective
mechanism-based inactivation of the enzyme. Chem Res Toxicol 18: 1427-1437.
Kenneth GS and Hyon C (2006) Epidemiology, risk factors, and lifestyle
modifications for gout. Arthritis Res Ther 8 (Suppl 1): S2.
Lin G, Tang J, Liu XQ, Jiang Y, and Zheng J (2007) Deacetylclivorine: a
21 DMD Fast Forward. Published on January 20, 2016 as DOI: 10.1124/dmd.115.066803 This article has not been copyedited and formatted. The final version may differ from this version.
DMD # 66803
gender-selective metabolite of clivorine formed in female Sprague-Dawley rat liver
microsomes. Drug Metab Dispos 35:607-613.
Locuson CW 2nd, Rock DA, and Jones JP (2004) Quantitative binding models for
CYP2C9 based on benzbromarone analogues. Biochemistry 43: 6948-6958.
Locuson CW 2nd, Suzuki H, Rettie AE, and Jones TP (2004) Charge and substituent
effects on affinity and metabolism of benzbromarone-based CYP2C19 inhibitors. J Downloaded from Med Chem 47: 6768-6776.
Marques-Soares C, Dijols S, Macherey AC, Wester MR, Johnson EF, Dansette PM, dmd.aspetjournals.org and Mansuy D (2003) Sulfaphenazole derivatives as tools for comparing
cytochrome P450 2C5 and human cytochromes P450 2Cs: identification of a new
high affinity substrate common to those enzymes. Biochemistry 42: 6363-6369. at ASPET Journals on September 24, 2021 McDonald MG and Rettie AE (2007) Sequential metabolism and bioactivation of the
hepatotoxin benzbromarone: formation of glutathione adducts from a catechol
intermediate. Chem Res Toxicol 20: 1833-1842.
Morisseau C, Newman JW, Wheelock CE, Hill lii T, Morin D, Buckpitt AR, and
Hammock BD (2008) Development of metabolically stable inhibitors of
mammalian microsomal epoxide hydrolase. Chem Res Toxicol 21: 951-957.
Reinders MK, van Roon EN, Houtman PM, Brouwers JR, and Jansen TL (2007)
Biochemical effectiveness of allopurinol and allopurinolprobenecid in previously
benzbromarone-treated gout patients. Clin Rheumatol 26: 1459-1465.
Shin HJ, Takeda M, Enomoto A, Fujimura M, Miyazaki H, Anazi N, and Endou H
(2011) Interactions of urate transporter URAT1 in human kidney with uricosuric
22 DMD Fast Forward. Published on January 20, 2016 as DOI: 10.1124/dmd.115.066803 This article has not been copyedited and formatted. The final version may differ from this version.
DMD # 66803
drugs. Nephrology 16: 156-162.
Slaughter DE and Hanzlik RP (1991) Identification of epoxide- and quinone-derived
bromobenzene adducts to protein sulfur nucleophiles. Chem Res Toxicol 4:
349-359.
Takai S, Oda S, Tsuneyama K, Fukami T, Nakajima M, and Yokoi T (2015)
Establishment of a mouse model for amiodarone-induced liver injury and analyses Downloaded from of its hepatotoxic mechanism. J Appl Toxicol doi: 10.1002/jat.3141. [Epub ahead of
print] dmd.aspetjournals.org Wagayama H, Shiraki K, Sugimoto K, Fujikawa K, Shimizu A, Takase K, Nakano T,
and Tameda Y (2000) Fatal fulminant hepatic failure associated with
benzbromarone. J Hepatol 32: 874. at ASPET Journals on September 24, 2021 Walter-Sack I, de Vries JX, Ittensohn A, and Raedsch R (1998) Biliary excretion of
benzbromarone and its hydroxilated main metabolites in humans. Eur J Med Res
3:45-49.
Walter-Sack I, Eichelbaum M, de Vries JX, and Weber E (1988) Benzbromarone
biotransformation is not related to polymorphic oxidation of sparteine. Klin
Wochenschr 66: 1097-1098.
Wempe MF, Jutabha P, Quade B, Iwen TJ, Frick MM, Ross IR, Rice PJ, Anzai N, and
Endou H (2011) Developing potent human uric acid transporter 1 (hURAT1)
inhibitors. J Med Chem 54: 2701-2713.
Wu H, Peng Y, Wang S, Wang K, Zhao X, and Jiang F (2012) Metabolism studies
of benzbromarone in rats by high performance liquid
23 DMD Fast Forward. Published on January 20, 2016 as DOI: 10.1124/dmd.115.066803 This article has not been copyedited and formatted. The final version may differ from this version.
DMD # 66803
chromatography-quadrupole time of flight mass spectrometry. J
Chromatogr B 911: 122-132.
Xuan J, Chen S, Ning B, Tolleson WH, and Guo L (2015) Development of
HepG2-derived cells expressing cytochrome P450s for assessing
metabolism-associated drug-induced liver toxicity. Chem Biol Interact doi:
10.1016/j.cbi.2015.10.009. [Epub ahead of print] Downloaded from Zheng J, Cho M, Jones AD, and Hammock BD (1997) Evidence of quinone
metabolites of naphthalene covalently bound to sulfur nucleophiles of proteins of dmd.aspetjournals.org murine clara cells after exposure to naphthalene. Chem Res Toxicol 10: 1008-1014.
Zheng J and Hanzlik RP (1992a) Dihydroxylated mercapturic acid metabolites of
bromobenzene. Chem Res Toxicol 5: 561-567. at ASPET Journals on September 24, 2021 Zheng J and Hanzlik RP (1992b) Bromo(monohydroxy)phenyl mercapturic acids A
New Class of Mercapturic Acids from Bromobenzene-Treated Rat. Drug Metab
Dispos 20: 688-694.
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Footnotes. This work was supported in part by the National Natural Science Foundation of China [Grant 81373471 and 81430086], and the Natural Science Foundation of Liaoning Province [Grant 2015020738].
K.W. and H.W. equally contributed to the work. Downloaded from dmd.aspetjournals.org at ASPET Journals on September 24, 2021
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Scheme Legends
Scheme 1. Proposed pathways for the formation of BBR-derived NAC conjugates by
P450-mediated epoxidation of BBR.
Scheme 2. Synthetic route of BBR-NAC conjugate.
Figure legends Downloaded from
Figure 1. The chemical structure of benzbromarone (BBR).
Figure 2. MS/MS spectrum of benzbromarone. dmd.aspetjournals.org
Figure 3. Extracted ion (m/z 586→279) chromatograms obtained from LC-Q-Trap
MS analysis of mouse liver microsomal incubations containing BBR and NAC in the at ASPET Journals on September 24, 2021 absence (A) or presence (B) of NADPH. (C) Extracted ion (m/z 586→279) chromatogram obtained from LC-Q-Trap MS analysis of BBR-NAC conjugate generated in DEX-induced liver microsomal incubations. (D) Extracted ion (m/z
586→279) chromatogram obtained from LC-Q-Trap MS analysis of synthetic
BBR-NAC conjugate. (E) MS/MS spectrum obtained from LC-Q-Trap MS analysis of BBR-NAC conjugate generated in microsomal incubation. (F) MS/MS spectrum obtained from LC-Q-Trap MS analysis of synthetic BBR-NAC conjugate. (G)
MS/MS spectrum obtained from Q-TOF MS analysis of synthetic BBR-NAC conjugate.
Figure 4. Extracted ion chromatograms obtained from LC-Q-Trap MS analysis of
BBR-NAC conjugate derived from quinone (A, m/z 618→489) and 6-NAC BBR (B,
26 DMD Fast Forward. Published on January 20, 2016 as DOI: 10.1124/dmd.115.066803 This article has not been copyedited and formatted. The final version may differ from this version.
DMD # 66803 m/z 586→279) generated in microsomal incubations in MRM mode. Extracted ion chromatograms obtained from LC-Q-Trap MS analysis of BBR-NAC conjugate derived from quinone (C, m/z 618→618) and 6-NAC BBR (D, m/z 586→586) generated in microsomal incubations.
Figure 5. 1H NMR spectrum (600 MHz) of BBR-NAC conjugate. Downloaded from Figure 6. HMBC NMR spectrum (600 MHz) of BBR-NAC conjugate.
Figure 7. Extracted ion (m/z 586→279) chromatograms obtained from LC/Q-Trap MS dmd.aspetjournals.org analysis of urine before (A ) and after (B ) treatment with BBR.
Figure 8. Formation of 6-NAC BBR in individual recombinant P450 enzyme
incubations containing NADPH, BBR, and NAC. Data shown represent the mean ± at ASPET Journals on September 24, 2021
SD (n = 3); *p < 0.05, **p < 0.01, ***p < 0.001 were considered significantly different.
Figure 9. Inhibitory effect of ketoconazole at various concentrations on the formation of 6-NAC BBR in mouse liver microsomal incubations. The concentrations of ketoconazole were 1, 10, and 100 μM, respectively. Data shown represent the mean
± SD (n = 3); *p < 0.05, **p < 0.01, ***p < 0.001 compared with group 3A4.
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Scheme 1 DMD Fast Forward. Published on January 20, 2016 as DOI: 10.1124/dmd.115.066803 This article has not been copyedited and formatted. The final version may differ from this version.
Scheme 2 Downloaded from dmd.aspetjournals.org at ASPET Journals on September 24, 2021 DMD Fast Forward. Published on January 20, 2016 as DOI: 10.1124/dmd.115.066803 This article has not been copyedited and formatted. The final version may differ from this version.
Figure 1 Downloaded from dmd.aspetjournals.org at ASPET Journals on September 24, 2021 DMD Fast Forward. Published on January 20, 2016 as DOI: 10.1124/dmd.115.066803 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from dmd.aspetjournals.org at ASPET Journals on September 24, 2021
Figure 2 DMD Fast Forward. Published on January 20, 2016 as DOI: 10.1124/dmd.115.066803 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from dmd.aspetjournals.org at ASPET Journals on September 24, 2021
Figure 3 DMD Fast Forward. Published on January 20, 2016 as DOI: 10.1124/dmd.115.066803 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from dmd.aspetjournals.org
Figure 4 at ASPET Journals on September 24, 2021 DMD Fast Forward. Published on January 20, 2016 as DOI: 10.1124/dmd.115.066803 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from
Figure 5 dmd.aspetjournals.org at ASPET Journals on September 24, 2021 DMD Fast Forward. Published on January 20, 2016 as DOI: 10.1124/dmd.115.066803 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from
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Figure 9 Downloaded from dmd.aspetjournals.org at ASPET Journals on September 24, 2021