Background: Acyl glucuronides of xenobiotics have been a subject of wide interest from the
pharmaceutical industry with respect to biochemical reactivity, hepatic disposition, and enterohepatic
circulation. The reactivity and lack of stability of an acyl glucuronide for a clinical candidate could
pose major developability concerns. To date, multiple in vitro assays have been published to assess the
risk associated with acyl glucuronides. Despite this fact, the translation of these findings to predicting
clinical safety remains poor.
Methods: In the present investigation, we aimed to provide simplified in vitro strategy to understand
the bioactivation potential of acyl glucuronides of 10 commercial, carboxylic acid containing drugs that
have been categorized as “safe,” “warning,” or “withdrawn” with respect to their marketed use. Acyl
migration was measured as a function of the number of peaks observed in LC-MSn analysis. In addition,
we carried out reactive intermediate trapping studies with glutathione and methoxylamine to identify the
key intermediates in the transacylation bioactivation and glycation pathways, respectively. We also conducted
reaction phenotyping with recombinant UDP-glucuronosyltransferase (UGT) Supersomes® to investigate
if the formation of acyl glucuronides could be linked to specific UGT isoform(s).
Results: Our results were in line with reported values in the literature. Our assay could be used in discovery
research where half-life calculation completely eliminated the need to chemically synthesize the
acyl glucuronide standard for risk assessment. We captured our results for risk assessment in a flow
chart to simplify the various complex in vitro techniques historically presented.
Conclusion: While the compounds tested from “withdrawn” and “warning category” all formed the glutathione
adduct in buffer, none from “safe” category formed the glutathione adduct. In contrast, none of
the compounds tested from any category formed methoxylamine conjugate, a reaction with putative aldehyde
moiety formed via acyl migration. These results, highly favor the nucleophilic displacement as a
cause of the reactivity rather than the acyl migration via aldehyde formation. The workflow presented
could also be applied in the discovery setting to triage new chemical entities of interest.