Identification of Epoxide-Derived Metabolite(s) of Benzbromarone

Wang, Kai; Wang, Hui; Peng, Ying; Zheng, Jiang

doi:10.1124/dmd.115.066803

Cited by 19 publications

(47 citation statements)

References 29 publications

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“…McDonald and Rettie (2007) first suggested that BBR was sequentially oxidized by CYP2C9 to a catechol which can be further oxidized to a reactive o-quinone intermediate identified as glutathione (GSH) conjugates in liver microsomal systems. Previous work from our laboratory revealed that BBR was metabolized to an epoxide intermediate(s) through the initial epoxidation of the benzofuran ring by CYP3A (Wang et al, 2016b). In our following studies, mercapturic acid excreted from urine and hepatic protein adduction derived from the epoxide intermediate (s) were observed in BBR-treated mice, which confirmed the epoxidation metabolism of BBR in vivo (Wang et al, 2016a,b).…”

Section: Introductionsupporting

confidence: 85%

Glutathione Conjugation and Protein Adduction Derived from Oxidative Debromination of Benzbromarone in Mice

Wang

Gong

et al. 2019

Drug Metab Dispos

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Benzbromarone (BBR), a uricosuric agent, has been known to induce hepatotoxicity, and its toxicity has a close relation to cytochrome P450-mediated metabolic activation. An oxidative debromination metabolite of BBR has been reported in microsomal incubations. The present study attempted to define the oxidative debromination pathway of BBR in vivo. One urinary mercapturic acid (M1) and one glutathione (GSH) conjugate (M2) derived from the oxidative debromination metabolite were detected in BBR-treated mice after solid phase extraction. M1 and M2 shared the same chromatographic behavior and mass spectral identities as those detected in N-acetylcysteine/GSHand BBR-fortified microsomal incubations. The structure of M1 was characterized by chemical synthesis, along with mass spectrometry analysis. In addition, hepatic protein modification that occurs at cysteine residues (M93) was observed in mice given BBR. The observed protein adduction reached its peak 4 hours after administration and occurred in a dose-dependent manner. A GSH conjugate derived from oxidative debromination of BBR was detected in livers of mice treated with BBR, and the formation of the GSH conjugate apparently took place earlier than the protein adduction. In summary, our in vivo work provided strong evidence for the proposed oxidative debromination pathway of BBR, which facilitates the understanding of the mechanisms of BBR-induced hepatotoxicity. SIGNIFICANCE STATEMENT This study investigated the oxidative debromination pathway of benzbromarone (BBR) in vivo. One urinary mercapturic acid (M1) and one glutathione (GSH) conjugate (M2) derived from the oxidative debromination metabolite were detected in BBR-treated mice. M1 and M2 were also observed in microsomal incubations. The structure of M1 was characterized by chemical synthesis followed by mass spectrometry analyses. More importantly, protein adduction derived from oxidative debromination of BBR (M93) was observed in mice given BBR, and occurred in dose-and time-dependent manners. The success in detection of GSH conjugate, urinary N-acetylcysteine conjugate, and hepatic protein adduction in mice given BBR provided solid evidence for in vivo oxidative debromination of BBR. The studies allowed a better understanding of the metabolic activation of BBR.

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Section: Introductionsupporting

confidence: 85%

Glutathione Conjugation and Protein Adduction Derived from Oxidative Debromination of Benzbromarone in Mice

Wang

Gong

et al. 2019

Drug Metab Dispos

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“…The observation of a high volume of protein adduction derived from BBR, combined with the finding of more potent hepatotoxicity induced by BBR, led us to propose that epoxidation at C6 of BBR is a key step for hepatotoxicity of BBR. Our previous study demonstrated that the reaction of the epoxide metabolite(s) of BBR with GSH contributes to the depletion of GSH (Wang et al, 2016b). The present study found that treatment of BBR caused significant elevation in serum AST activity in opposition to a decrease in hepatic and plasma GSH content.…”

Section: Discussionsupporting

confidence: 56%

“…Liver tissues (0.2 g) harvested 30 minutes postadministration were homogenized in 2.0 ml of phosphate buffer (pH 7.4). The resulting homogenates were denatured by heating in a water bath at 60°C for 30 minutes and then digested overnight with a mixture of chymotrypsin (1.0 mg/ml) and Pronase E (2.0 mg/ml), according to our previous study (Wang et al, 2016b). The supernatants (5 ml) were subjected to the LC-MS/MS system for analysis.…”

Section: Determination Of In Vivo Protein Adductions Derived From Bbrmentioning

confidence: 99%

Metabolic Epoxidation Is a Critical Step for the Development of Benzbromarone-Induced Hepatotoxicity

et al. 2017

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Benzbromarone (BBR) is effective in the treatment of gout; however, clinical findings have shown it can also cause fatal hepatic failure. Our early studies demonstrated that CYP3A catalyzed the biotransformation of BBR to epoxide intermediate(s) that reacted with sulfur nucleophiles of protein to form protein covalent binding both in vitro and in vivo. The present study attempted to define the correlation between metabolic epoxidation and hepatotoxicity of BBR by manipulating the structure of BBR. We rationally designed and synthesized three halogenated BBR derivatives, fluorinated BBR (6-F-BBR), chlorinated BBR (6-Cl-BBR), and brominated BBR (6-Br-BBR), to decrease the potential for cytochrome P450-mediated metabolic activation. Both in vitro and in vivo uricosuric activity assays showed that 6-F-BBR achieved favorable uricosuric effect, while 6-Cl-BBR and 6-Br-BBR showed weak uricosuric efficacy. Additionally, 6-F-BBR elicited much lower hepatotoxicity in mice. Fluorination of BBR offered advantage to metabolic stability in liver microsomes, almost completely blocked the formation of epoxide metabolite(s) and protein covalent binding, and attenuated hepatic and plasma glutathione depletion. Moreover, the structural manipulation did not alter the efficacy of BBR. This work provided solid evidence that the formation of the epoxide(s) is a key step in the development of BBR-induced hepatotoxicity.

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“…The final metabolite of this molecule is ortho-quinone, which may be related to hepatotoxicity [43]. Other studies have reported the presence of an intermediate epoxide metabolite, which has been demonstrated to be hepatotoxic in murine models [44,45]. Another study reported that the addition of a fluorine molecule in the 6 position of benzbromarone reduced the production of these metabolites, reducing its toxicity in mice [46].…”

Section: A Mechanism For Hepatotoxicity: Animal Models and In Vitro Smentioning

confidence: 99%

Benzbromarone in the treatment of gout

Azevedo

Kos²,

Vargas-Santos

et al. 2019

Adv Rheumatol

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Background: Benzbromarone is a uricosuric drug that has been used in the treatment of gout over the last 30 years. Due to its potent inhibition of the dominant apical (luminal) urate exchanger in the human proximal tubule URAT1, it reduces the urate reabsorption, diminishing serum urate levels and therefore preventing gout flares. Main body of the abstract: Through several clinical trials, Benzbromarone has been proved effective and safe, inclusive in patients with chronic kidney disease and as combination therapy with allopurinol. Due to hepatotoxicity reports, it was withdrawn from the European market by the manufacturer, however many authors have questioned the product's withdrawal due to a lack of clinical evidence in order to support its hepatotoxicity. Benzbromarone is still available in several European countries, New Zealand, Brazil and several other countries. Despite the product's marketing over more than 20 years after the first hepatotoxicity reports, we have found only five reports in our literature search, and no prospective or retrospective study correlating hepatotoxicity with benzbromarone use. Short conclusion: Benzbromarone is a safe and effective molecule for the treatment of gout. However, due to in vitro and in vivo data related to hepatotoxicity, it is prudent to prescribe it with some caution, especially for patients with an already known liver condition.

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Identification of Epoxide-Derived Metabolite(s) of Benzbromarone

Cited by 19 publications

References 29 publications

Glutathione Conjugation and Protein Adduction Derived from Oxidative Debromination of Benzbromarone in Mice

Glutathione Conjugation and Protein Adduction Derived from Oxidative Debromination of Benzbromarone in Mice

Metabolic Epoxidation Is a Critical Step for the Development of Benzbromarone-Induced Hepatotoxicity

Benzbromarone in the treatment of gout

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