Identification and Characterization of a Novel Ergothioneine Conjugate of Raloxifene

Raloxifene (RX) in the presence of liver microsomes and glutathione (GSH) has shown oxidative bioactivation to reactive intermediates that are conjugated by GSH. L-Ergothioneine (ET) is a naturally occurring sulfhydryl amino acid, similar to GSH, derived from dietary sources with antioxidant properties and reported to accumulate in high concentrations in animals and humans. We hypothesized that ET may have detoxification/conjugation properties similar to GSH. Using rat and human liver microsomes and mouse, rat, dog, monkey, and human hepatocytes, a novel ergothioneine conjugate of raloxifene (RX-ET) (M1) was identified by mass spectrometry. The RX-ET conjugate was further scaled up in rat liver microsomes, isolated, and characterized by high-resolution mass spectrometry and NMR to confirm the structure. A single RX-ET conjugate was characterized and the site of ET conjugation was identified at the C-17 position of RX. The in vivo relevance of this unique conjugate was also established through metabolism studies in intact and bile duct cannulated rats, both untreated and pretreated with ET. In general, the RX-ET conjugate was found in trace amounts in plasma and urine, but in higher concentrations in bile and feces. The major elimination pathway of RX-ET was through biliary elimination. In rats that were pretreated with ET prior to RX administration, significantly larger quantities of ET and RX-ET conjugate were found in in vivo samples. Lastly, these studies suggest that ET conjugation is an additional pathway for scavenging reactive species arising from xenobiotics and may potentially reduce drug-related toxicities. SIGNIFICANCE STATEMENT: Ergothioneine is well known for its antioxidant and free radical scavenging activity. This study identifies its role in conjugating the reactive species arising from the bioactivation of raloxifene in vitro and in vivo suggesting that ergothioneine may act as an additional conjugation pathway similar to glutathione in the disposition of reactive centers or metabolites of xenobiotics.