Metabolite Profiling and Reaction Phenotyping for the in Vitro Assessment of the Bioactivation of Bromfenac

Abstract

Bromfenac is a nonsteroidal anti-inflammatory drug that was approved and subsequently withdrawn from the market because of reported cases of acute hepatotoxicity. Recently, in vitro studies have revealed that bromfenac requires UDPGA and alamethicin supplemented human liver microsomes (HLM) to form a major metabolite, bromfenac indolinone (BI). Bromfenac and BI form thioether adducts through a bioactivation pathway in HLM and hepatocytes. [J. P. Driscoll et al., Chem. Res. Toxicol. 2018, 31, 223−230.] Here, Cytochrome P450 (CYP) and UDPglucuronosyltransferase (UGT) reaction phenotyping experiments using recombinant enzymes were performed on bromfenac and BI to identify the CYP and UGT enzymes responsible for bromfenac's metabolism and bioactivation. It was determined that UGT2B7 converts bromfenac to BI, and that while CYP2C8, CYP2C9, and CYP2C19 catalyze the hydroxylation of bromfenac, only CYP2C9 forms thioether adducts when incubated with NAC or GSH as trapping agents. Although CYP2C9 was shown to form a reactive intermediate, no inhibition of CYP2C9 was observed when an IC 50 shift assay was performed. Reaction phenotyping experiments with BI and recombinant CYP enzymes indicated that CYPs 1A2, 2B6, 2C8, 2C9, 2C19, 2D6, and 3A4 were responsible for the formation of an aliphatic hydroxylated metabolite. An aromatic hydroxylation on the indolinone moiety was also formed by CYP1A2 and CYP3A4. The aromatic hydroxylated BI is a precursor to the quinone methide and quinone imine intermediates in the proposed bioactivation pathway. Through timedependent inhibition (TDI) experiments, it was revealed that BI can cause an IC 50 shift in CYP1A2 and CYP3A4. However, BI does not inhibit the other isoforms that were also responsible for the formation of the aliphatic hydroxylation, an alternative biotransformation that does not undergo further downstream bioactivation. The results of these metabolism studies with bromfenac and BI add to our understanding of the relationship between biotransformation, reactive intermediate generation, and a potential mechanistic link to the hepatotoxicity of this compound.

Cyanide Trapping of Iminium Ion Reactive Metabolites: Implications for Clinical Hepatotoxicity

“…Despite the lack of a known structural alert, it was of interest to see whether the cyanide trapping assay would flag these compounds. Bromfenac is metabolized by UDP-glucuronosyltransferase (UGT) enzymes to form an acyl glucuronide, which then undergoes internal cyclization to form an indolinone metabolite, which is further hydroxylated by CYP enzymes to form quinone methides and quinone imines trappable by N-acetyl cysteine . Felbamate forms an α,β-unsaturated aldehyde. , Labetalol forms GSH conjugates after sulfation via phase II enzymes .…”

The assessment of the potential hepatotoxicity of new drugs by in vitro metabolomics

“…Several studies analyzed the exo-metabolome in culture media to examine hepatotoxic effects of xenobiotics (Rodrigues et al, 2018;Seeger et al, 2019;Wang et al, 2019;Goracci et al, 2020;Manier et al, 2020;Xu et al, 2020;Yadav et al, 2020;Iturrospe et al, 2022). Goracci et al analyzed the culture media supernatant to identify dronedarone, entacapone and metformin metabolites using liver micro tissues competent in both Phase I and II xenobiotic metabolism (Goracci et al, 2020).…”

Biotransformation: Impact and Application of Metabolism in Drug Discovery

“…75 Again, bioactivation to a reactive metabolite is thought causative in bromfenac-associated DILI. A series of publications on bromfenac bioactivation illustrates the complex and unusual nature of its bioactivation involving initial acyl glucuronide formation, which then forms an indolinone metabolite through intramolecular nucleophilic substitution 75,76 (see Figure 20). It is this indolinone that undergoes further CYP-mediated oxidation resulting in a quinone imine and/or quinone methide reactive intermediate.…”