Combination of GSH Trapping and Time-Dependent Inhibition Assays as a Predictive Method of Drugs Generating Highly Reactive Metabolites

Covalent binding (CB) of reactive metabolites (RMs) is potentially involved in severe adverse drug reactions. Because the CB assay is of low throughput and costly, a qualitative trapping assay using agents such as [35S]glutathione (GSH) is often performed in the early stages of drug discovery. Trapping methods alone, however, cannot replace the CB assay. We hypothesized that the time-dependent inhibition (TDI) assay might be complementary to [35S]GSH trapping assay in detecting RMs. We performed CB assays, [35S]GSH trapping assays, and TDI assays for 42 structurally diverse compounds. First, we showed that the [35S]GSH trapping assay alone does not correlate with the extent of CB. Four compounds that [35S]GSH trapping assay failed to detect but showed high extent of CB were inactivators of the enzyme in the TDI assay. There was a tendency for compounds judged as positive in the TDI assay to show a high degree of the extent of CB irrespective of the result of the [35S]GSH trapping assay. Finally, to combine parameters from the two assays, we introduced intrinsic clearance to dscribe the formation of RMs (CLint, RMs). The Spearman rank correlation coefficient between the extent of CB and CLint, RMs was 0.77 ( p < 0.0001), which was better than that for the formation rates of [35S]GS adducts. We therefore demonstrated that a combination of the [35S]GSH trapping and TDI assays is an effective method for detecting compounds potentially capable of generating highly reactive metabolites in the early stages of drug discovery.