An in vitro approach to predict idiosyncratic drug-induced agranulocytosis using myeloperoxidase-derived reactive metabolites

Idiosyncratic drug-induced agranulocytosis (iDIAG) is a rare but potentially life-threatening adverse reaction characterized by an absolute neutrophil count of fewer than 500 cells/μL. Owing to its complex and poorly understood mechanisms, predicting the risk of iDIAG remains a major challenge. We hypothesized that the amount of reactive metabolite (RM) formation mediated by myeloperoxidase (MPO), an enzyme abundantly expressed in neutrophils, may be correlated with iDIAG risk. To test this hypothesis, we developed a novel trapping assay using [³H]glutathione and [¹⁴C]cyanide as soft and hard nucleophilic trapping reagents, respectively, to quantify MPO-mediated RM formation. This assay was applied to 42 compounds, comprising 17 high-risk and 25 low-risk compounds, classified based on clinical safety data. RM formation rates alone were insufficient to reliably distinguish between high- and low-risk groups. However, when combined with systemic exposure data, particularly the area under the concentration-time curve from 0 to 24 hours at steady state (AUC_0-24,ss), predictive performance improved markedly. Nonetheless, the model failed to accurately predict risk for certain compounds, such as pyronaridine and sulfamethoxazole (due to short dosing periods), and aminopyrine and ticlopidine (due to the lack of consideration of highly RMs). Radio-LCMS analysis enabled structural elucidation of RM-generating moieties, identifying 4-aminophenol and N-C(-S)-N moieties as highly reactive substructures. These findings highlight the importance of integrating MPO-mediated RM formation, systemic exposure, dosing duration, and structural alerts for the comprehensive assessment of iDIAG risk. SIGNIFICANCE STATEMENT: To our knowledge (best of), this study provides the first quantitative evaluation of reactive metabolite formation mediated by myeloperoxidase, along with structural insights into reactive subgroups. By integrating reactive metabolite reactivity with systemic exposure and considering dosing duration, it proposes an integrative framework to support early assessment of idiosyncratic drug-induced agranulocytosis risk. This approach could contribute to the early identification and elimination of higher-risk compounds, thereby supporting the development of safer drug candidates during the early stages of drug discovery.

Agranulocytosis; Reactive metabolite; Trapping assay.