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.

Impacts of diphenylamine NSAID halogenation on bioactivation risks.

Diphenylamine NSAIDs are highly prescribed therapeutics for chronic pain despite causing symptomatic hepatotoxicity through mitochondrial damage in five percent of patients taking them. Differences in toxicity are attributed to structural modifications to the diphenylamine scaffold rather than its inherent toxicity. We hypothesize that marketed diphenylamine NSAID substituents affect preference and efficiency of bioactivation pathways and clearance. We parsed the FDA DILIrank hepatotoxicity database and modeled marketed drug bioactivation into quinone-species metabolites to identify a family of seven clinically relevant diphenylamine NSAIDs. These drugs fell into two subgroups, i.e., acetic acid and propionic acid diphenylamines, varying in hepatotoxicity risks and modeled bioactivation propensities. We carried out steady-state kinetic studies to assess bioactivation pathways by trapping quinone-species metabolites with dansyl glutathione. Analysis of the glutathione adducts by mass spectrometry characterized structures while dansyl fluorescence provided quantitative yields for their formation. Resulting kinetics identified four possible bioactivation pathways among the drugs, but reaction preference and efficiency depended upon structural modifications to the diphenylamine scaffold. Strikingly, diphenylamine dihalogenation promotes formation of quinone metabolites through four distinct metabolic pathways with high efficiency, whereas those without aromatic halogen atoms were metabolized less efficiently through two or fewer metabolic pathways. Overall metabolism of the drugs was comparable with bioactivation accounting for 4-13% of clearance. Lastly, we calculated daily bioload exposure of quinone-species metabolites based on bioactivation efficiency, bioavailability, and maximal daily dose. The results revealed stratification into the two subgroups; propionic acid diphenylamines had an average four-fold greater daily bioload compared to acetic acid diphenylamines. However, the lack of sufficient study on the hepatotoxicity for all drugs prevents further correlative analyses. These findings provide critical insights on the impact of diphenylamine bioactivation as a precursor to hepatotoxicity and thus, provide a foundation for better risk assessment in drug discovery and development.