Sequential Metabolism and Bioactivation of the Hepatotoxin Benzbromarone: Formation of Glutathione Adducts From a Catechol Intermediate

Abstract

Benzbromarone (BBR) is a uricosuric agent that has been used as a treatment for chronic gout. Although never approved in the United States, BBR was recently withdrawn from European markets due to several clinical cases linking the drug to an idiosyncratic hepatotoxicity that is sometimes fatal. We report here a possible mechanism of toxicity that involves the bioactivation of BBR through sequential hydroxylation of the benzofuran ring to a catechol, which can then be further oxidized to a reactive quinone intermediate capable of adducting protein. NADPH-supplemented human liver microsomes generated a single metabolite that was identified as 6-OH BBR by comparison with the synthesized chemical standard. CYP2C9 was the major recombinant enzyme capable of catalyzing the formation of 6-OH BBR, although CYP2C19 also showed a lower degree of activity. Further oxidation of either 6-OH BBR or 5-OH BBR by human liver microsomes resulted in the formation of a dihydroxy metabolite with identical chromatographic and mass spectral properties. This product of sequential metabolism of BBR was identified as the catechol, 5,6-dihydroxybenzbromarone. Incubation of the catechol with liver microsomes, in the presence of glutathione, resulted in the formation of two glutathione adducts that could derive from a single ortho-quinone intermediate. Isoform profiling with recombinant human P450s suggested that CYP2C9 is primarily responsible for the formation of this reactive quinone intermediate.

Identification of Epoxide-Derived Metabolite(s) of Benzbromarone

“…Idiosyncratic hepatotoxicity is often associated with metabolic activation mediated by cytochromes P450. A quinone-derived reactive intermediate of BBR was reported by McDonald and Rettie (2007). It was our speculation that the generation of epoxide-derived intermediate(s) would be required before the formation of the quinone-derived reactive intermediate.…”

“…Subsequently, BBR and four of its analogs were discovered to exhibit extraordinary inhibitory potency for P450 2C19 . Idiosyncratic hepatotoxicity of BBR has primarily been suggested to be associated with metabolite 6-hydroxy BBR proposed by McDonald and Rettie (2007), who suggested that sequential oxidation of 6-hydroxy BBR results in a catechol structure, 5,6-dihydroxy BBR, which can be further oxidized to a reactive quinone intermediate capable of adducting protein; however, we believe the initial epoxidation of BBR may be more important for metabolic activation of BBR. Epoxide-derived metabolites of many protoxicants, such as bromobenzene (Slaughter and Hanzlik, 1991;Zheng and Hanzlik, 1992a), naphthalene (Zheng et al, 1997;Morisseau et al, 2008), styrene (Carlson GP, 2011), and coumarin (Born et al, 2000) are suggested to play important roles in the development of toxicities.…”

“…The molecular ion matched the molecular weight of BBR-NAC conjugate derived from the quinone intermediate. The corresponding GSH conjugate was reported by McDonald and Rettie (2007).…”

“…Early metabolic studies revealed two major hydroxylated metabolites identified as 19-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 a P450 2C9 inhibitor (Marques-Soares et al, 2003;.…”

Covalent Binding and Tissue Distribution/Retention Assessment of Drugs Associated with Idiosyncratic Drug Toxicity

“…Most of the drugs used in the present study are known to form various reactive metabolites (Kalgutkar et al, 2005), as shown in Table 1. This includes several types of reactive metabolites, such as quinone imine derived from amodiaquine (Christie et al, 1989); ortho-quinone from benzbromarone (McDonald and Rettie, 2007); quinone methide from tacrine (Madden et al, 1993); nitrenium ion from clozapine (Pirmohamed et al, 1995); iminium ion from nevirapine (Kalgutkar et al, 2005) and zafirlukast (Kassahun et al, 2005); nitroso species from erythromycin (Larrey et al, 1983), phenacetin (Koymans et al, 1990), procainamide (Uetrecht, 1985), and sulfamethoxazole (Naisbitt et al, 2002); arene oxide from imipramine (Masubuchi et al, 1996); acyl glucuronide from valproic acid (Baillie, 1988) and zomepirac (Wang et al, 2001); and radical species from aminopyrine (Uetrecht et al, 1995), flutamide (Kang et al, 2007), and phenytoin (Cuttle et al, 2000). Quinone imine formation from amodiaquine was reported to be caused by autoxidation (Maggs et al, 1987); thus, the high levels of covalent binding without NADPH observed in this study seem to be reasonable.…”