Quantitative Evaluation of Reactivity and Toxicity of Acyl Glucuronides by [³⁵S]Cysteine Trapping

Acyl glucuronides (AGs) are reactive metabolites of carboxylic acid-containing drugs, which are associated with idiosyncratic toxicity (IDT) such as anaphylaxis, drug-induced liver injury, and so on. In this study, we developed a new in vitro approach for the quantitative assessment of the reactivity of AGs and their toxicity risk. Thirteen test drugs were incubated with human liver microsomes and uridine 5′-diphospho-glucuronic acid in the presence of cysteine (Cys) as a trapping agent. Both acylation and glycation Cys adducts formed from the AGs of the test drugs and were analyzed by ultraperformance liquid chromatography-quadrupole time-of-flight mass spectrometry. Acylation Cys adduct formation can closely reflect the reactivity of AGs to predict their IDT risk. Subsequently, we performed a quantitative trapping assay using radiometric analysis, with [³⁵S]-labeled Cys ([³⁵S]­Cys) as the trapping agent, and the results showed that the test drugs associated with IDT resulted in a high product formation of [³⁵S]­Cys adducts. In conclusion, this approach can be used for the easy and quantitative evaluation of the reactivity of AGs without the need for authentic AG standards and to screen the potential IDT of new chemical entities during the early drug discovery phase.