Unusual Metabolism of Benzethonium by Cytochrome P450

Abstract ID 56418 Poster Board 175 Introduction: Benzethonium (BZT) is a quaternary ammonium antimicrobial used in food-processing, personal care products, antiseptics, and as a preservative in pharmaceutical formulations. BZT-containing cosmetics, creams, and ointments are commonly applied to the skin. In an ADME study conducted by the National Toxicology Program, radiolabeled BZT exhibited high dermal bioavailability (>50%) in rats, with 45% of radioactivity being recovered in feces. Furthermore, the liver contained the highest levels of radioactivity among all tissues. Radioactivity in the liver peaked at 24 hours and persisted out to 7 days after a single dermal application of [¹⁴C]-BZT. Hepatic uptake of dermally absorbed BZT followed by biliary excretion to feces can likely explain these results. Despite widespread human exposure to BZT and the above evidence for significant dermal absorption and hepatic exposure in animals, the metabolism of BZT has received no attention to date. Our objective was to structurally elucidate the metabolites of BZT produced by cytochrome P450 (CYP)-mediated metabolism. Our approach utilized LC-MS-based investigation of the metabolite profile generated by incubating BZT in human liver microsomes (HLM) in the presence or absence of NADPH. High-resolution (time-of-flight) MS/MS fragmentation of parent BZT and the chromatographically resolved metabolites was performed. Results: Incubation of BZT in HLM produced more than 10 NADPH-dependent metabolite peaks with m/z values corresponding to +1 or +2 oxygen atoms, both with and without loss of 2 hydrogen atoms (-2H). MS/MS fragmentation confirmed that metabolism occurs on the 2,4,4-trimethyl-pentan-2-yl (TMP) moiety of BZT. There were at least 5 chromatographically resolved hydroxy metabolites with MS/MS fragmentation supporting the addition of oxygen on the TMP moiety. However, there are only 3 unique carbon positions at which oxygen can be added to the TMP moiety. Hence, in the absence of a chiral column, it should not be possible to observe 5 unique hydroxy metabolite peaks (specifically on the TMP moiety), unless carbon-carbon (C-C) bond rearrangement occurs during the CYP-mediated metabolism of BZT. Furthermore, we observed 5 metabolite peaks corresponding to -2H metabolic modification on the TMP moiety (desaturation without addition of oxygen). However, the TMP moiety of BZT does not contain a pair of vicinal C-H bonds. This further supported a scenario where C-C bond rearrangement must have occurred within the TMP moiety of BZT prior to desaturation. Conclusion: CYP-mediated metabolism of BZT appears to involve intramolecular rearrangement of C-C bonds within the TMP moiety of BZT. Our proposed mechanism begins with hydrogen atom transfer from TMP to P450 compound I (Fe^IV=O) followed by intramolecular rearrangement of the carbon radical intermediate, and oxygen rebound or dehydration to form hydroxy and alkene products, respectively. This unusual metabolism led us to propose several novel metabolites of BZT. Significance: This work builds upon the diverse range of CYP-catalyzed rearrangement reactions reported in the literature that serve as a cornerstone of CYP enzymology. Funding: NIH NIEHS (R01ES031927, P30ES007033, and T32-ES007032)