Studies on the Metabolism of Troglitazone to Reactive Intermediates in Vitro and in Vivo. Evidence for Novel Biotransformation Pathways Involving Quinone Methide Formation and Thiazolidinedione Ring Scission

Abstract

Therapy with the oral antidiabetic agent troglitazone (Rezulin) has been associated with cases of severe hepatotoxicity and drug-induced liver failure, which led to the recent withdrawal of the product from the U.S. market. While the mechanism of this toxicity remains unknown, it is possible that chemically reactive metabolites of the drug play a causative role. In an effort to address this possibility, this study was undertaken to determine whether troglitazone undergoes metabolism in human liver microsomal preparations to electrophilic intermediates. Following incubation of troglitazone with human liver microsomes and with cDNA-expressed cytochrome P450 isoforms in the presence of glutathione (GSH), a total of five GSH conjugates (M1-M5) were detected and identified tentatively by LC-MS/MS analysis. In two cases (M1 and M5), the structures of the adducts were confirmed by NMR spectroscopy and/or by comparison with an authentic standard prepared by synthesis. The formation of GSH conjugates M1-M5 revealed the operation of two distinct metabolic activation pathways for troglitazone, one of which involves oxidation of the substituted chromane ring system to a reactive o-quinone methide derivative, while the second involves a novel oxidative cleavage of the thiazolidinedione (TZD) ring, potentially generating highly electrophilic alpha-ketoisocyanate and sulfenic acid intermediates. When troglitazone was administered orally to a rat, samples of bile were found to contain GSH conjugates which reflected the operation of these same metabolic pathways in vivo. The finding that metabolism of the TZD ring of troglitazone was catalyzed selectively by P450 3A enzymes is significant in light of the recent report that troglitazone is an inducer of this isoform in human hepatocytes. The implications of these results are discussed in the context of the potential for troglitazone to covalently modify hepatic proteins and to cause oxidative stress through redox cycling processes, either of which may play a role in drug-induced liver injury.

Arabidopsis Glutathione Transferases U24 and U25 Exhibit a Range of Detoxification Activities with the Environmental Pollutant and Explosive, 2,4,6-Trinitrotoluene

“…For conjugates 1 and 2, conjugation at the methyl is likely to occur through the formation of a quinone methide intermediate. While this has been observed previously in GST conjugation to methyl-substituted aromatic rings (Kassahun et al, 2001;Li et al, 2009), this is, to our knowledge, the first report of such a GST conjugate to TNT. For both GST-U24 and GST-U25, catalytic activity increased with pH, with the optimum appearing to be pH 9.5 or greater.…”

New Pioglitazone Metabolites and Absence of Opened-Ring Metabolites in New N-Substituted Thiazolidinedione

“…The use of thiazolidinediones is limited due to their toxicity, and some authors have related this toxicity to the formation of thiazolidinedione ring opening metabolites in troglitazone (Kassahun et al, 2001), which is a common pathway for other thiazolidinediones (Baughman, 2005;Alvarez-Sánchez et al, 2006;Shen et al, 2008). These opened ring metabolites have been previously reported (Uchiyama et al, 2010), and two of them (M-B and M-C) were observed in our work.…”

“…With the recent publication of the Guidance for Metabolite Safety Testing (FDA, 2008), metabolite investigations have become even more important (Baillie, 2008;Leclercq et al, 2009). Considering thiazolidinediones, it was proposed that the ring-opened may be related to formation of reactive metabolites of troglitazone (Kassahun et al, 2001). This ring opening mechanism is common to other thiazolidinediones (Alvarez-Sánchez et al, 2006).…”

Mechanisms of cytochrome P450 substrate oxidation: MiniReview

“…The peripheral site for the substrate has a possible structural basis in some of the X-ray crystal structures of P450 3A4 [31,32], although the possibility FIGURE 5. An example of a bioactivation of a drug (troglitazone) to electrophilic products tagged by glutathione (GSH) [38,39]. that binding at that site is an artifact cannot be dismissed.…”

“…However, these can all be rationalized in the context of the proposed perferryl oxidation chemistry discussed above, and the reader is referred to more extensive reviews published elsewhere [8,36,37]. In a number of cases the, oxidized products are electrophilic and the binding of these to tissue nucleophiles can be related to toxicity ( Figure 5) [38][39][40][41], although demonstration of a causal relationship has proven difficult [42].…”