Toxicological evaluation of acyl glucuronides utilizing half-lives, peptide adducts, and immunostimulation assays

•Acyl glucuronides (AGs) is suggested to be involved in the toxicity.•Half-lives and peptide adducts assay exhibited some false-positive.•We found 5 genes induced by AGs using DNA microarray analysis.•Immunostimulation assay using 5 genes as biomarkers showed high accuracy.•The risk assessment of AGs can contribute to improve drug safety.

Chemical reactivity of acyl glucuronides (AGs) is believed to be involved in the toxicity of carboxylic acid-containing drugs. Both direct and immune-mediated toxicity have been suggested as possible mechanisms of toxicity; however, it remains unclear. In the present study, we performed assays of half-lives, peptide adducts, and immunostimulation to evaluate the potential risk of AGs of 21 drugs and analyzed the relationship to the toxic category. AGs of all withdrawn drugs tested in this study showed short half-lives and peptide adducts formation, but so did those of several safe drugs. In contrast, only AGs of withdrawn and warning drugs induced interleukin-8 (IL-8) in human peripheral blood mononuclear cells (hPBMCs). Using a DNA microarray assay, we found that zomepirac AG induced the mRNAs of 5 genes, including IL-8 in hPBMCs. In addition, withdrawn and warning drugs were distinguished from safe drugs by an integrated score of relative mRNA expression levels of 5 genes. The immunostimulation assay showed higher sensitivity, specificity, and accuracy compared with other methods. In preclinical drug development, the evaluation of the reactivity of AGs using half-lives and peptide adducts assays followed by the evaluation of immunostimulation by highly reactive AGs using hPBMCs can contribute to improved drug safety.