Significance of Multiple Bioactivation Pathways for Meclofenamate as Revealed through Modeling and Reaction Kinetics.

Meclofenamate is a nonsteroidal anti-inflammatory drug used in the treatment of mild-to-moderate pain yet poses a rare risk of hepatotoxicity through an unknown mechanism. Nonsteroidal anti-inflammatory drug (NSAID) bioactivation is a common molecular initiating event for hepatotoxicity. Thus, we hypothesized a similar mechanism for meclofenamate and leveraged computational and experimental approaches to identify and characterize its bioactivation. Analyses employing our XenoNet model indicated possible pathways to meclofenamate bioactivation into 19 reactive metabolites subsequently trapped into glutathione adducts. We describe the first reported bioactivation kinetics for meclofenamate and relative importance of those pathways using human liver microsomes. The findings validated only four of the many bioactivation pathways predicted by modeling. For experimental studies, dansyl glutathione was a critical trap for reactive quinone metabolites and provided a way to characterize adduct structures by mass spectrometry and quantitate yields during reactions. Of the four quinone adducts, we were able to characterize structures for three of them. Based on kinetics, the most efficient bioactivation pathway led to the monohydroxy para-quinone-imine followed by the dechloro-ortho-quinone-imine. Two very inefficient pathways led to the dihydroxy ortho-quinone and a likely multiply adducted quinone. When taken together, bioactivation pathways for meclofenamate accounted for approximately 13% of total metabolism. In sum, XenoNet facilitated prediction of reactive metabolite structures, whereas quantitative experimental studies provided a tractable approach to validate actual bioactivation pathways for meclofenamate. Our results provide a foundation for assessing reactive metabolite load more accurately for future comparative studies with other NSAIDs and drugs in general. SIGNIFICANCE STATEMENT: Meclofenamate bioactivation may initiate hepatotoxicity, yet common risk assessment approaches are often cumbersome and inefficient and yield qualitative insights that do not scale relative bioactivation risks. We developed and applied innovative computational modeling and quantitative kinetics to identify and validate meclofenamate bioactivation pathways and relevance as a function of time and concentration. This strategy yielded novel insights on meclofenamate bioactivation and provides a tractable approach to more accurately and efficiently assess other drug bioactivations and correlate risks to toxicological outcomes.