A novel quantitative assessment of formed reactive metabolites by double trapping with [³H]glutathione and...

Drug-induced liver injury often arises from reactive metabolites (RMs) produced in the liver, making it crucial to assess RM formation rates from drug candidates. Conventional assays using glutathione (GSH) effectively trap soft electrophilic RMs but fail to detect hard electrophiles. To address this, we developed a double trapping assay employing [³H]GSH and [¹⁴C]cyanide as soft and hard nucleophilic reagents, respectively. This assay was applied to 25 drugs chosen based on safety profiles. Eight drugs were exclusively trapped by [³H]GSH, while 11 were trapped by [¹⁴C]cyanide or both reagents, demonstrating that a double trapping assay provides a more comprehensive detection method for both soft and hard RMs. Multiplying RM formation rates by daily doses allowed almost complete differentiation between withdrawn/black boxed warning drugs and safer ones. Radio-LCMS analysis provided detailed insights into the substructures of drug candidates responsible for RM production. Interestingly, it was discovered that GSH-based assays sometimes fail to detect certain RMs due to the presence of dithiothreitol in commercial [³H]GSH. This study highlights the efficacy of the double trapping assay using [³H]GSH and [¹⁴C]cyanide in accurately and comprehensively detecting RMs. Furthermore, it offers valuable structural information to minimize RM formation during early drug discovery.