Meclofenamate is a non-steroidal anti-inflammatory drug used in the treatment of mild to moderate pain yet poses a rare risk of hepatotoxicity through an unknown mechanism. 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 while 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 This article has not been copyedited and formatted. The final version may differ from this version.
In 2020, nearly one-third of new drugs on the global market were synthetic cannabinoids including the drug of abuse N-(1-adamantyl)-1-(5-pentyl)-1H-indazole-3-carboxamide (5F-APINACA, 5F-AKB48). Knowledge of 5F-APINACA metabolism provides a critical mechanistic basis to interpret and predict abuser outcomes. Prior qualitative studies identified which metabolic processes occur but not the order and extent of them and often relied on problematic “semi-quantitative” mass spectroscopic (MS) approaches. We capitalized on 5F-APINACA absorbance for quantitation while leveraging MS to characterize metabolite structures for measuring 5F-APINACA steady-state kinetics. We demonstrated the reliability of absorbance and not MS for inferring metabolite levels. Human liver microsomal reactions yielded eight metabolites by MS but only five by absorbance. Subsequent kinetic studies on primary and secondary metabolites revealed highly efficient mono- and dihydroxylation of the adamantyl group and much less efficient oxidative defluorination at the N-pentyl terminus. Based on regiospecificity and kinetics, we constructed pathways for competing and intersecting steps in 5F-APINACA metabolism. Overall efficiency for adamantyl oxidation was 17-fold higher than that for oxidative defluorination, showing significant bias in metabolic flux and subsequent metabolite profile compositions. Lastly, our analytical approach provides a powerful new strategy to more accurately assess metabolic kinetics for other understudied synthetic cannabinoids possessing the indazole chromophore.
Recently, the rising frequency in the abuse of synthetic cannabinoids (SCBs) has resulted in the occurrence of numerous severe adverse effects. We have previously shown that several early generations of SCBs undergo extensive metabolism by cytochrome P450s (P450s) and that their metabolites retain biological activity at CB1 and CB2 receptors. In this study, we examined the oxidative metabolism of 5F‐AKB‐48, one of a new structural generation of SCBs. Activity towards 5F‐AKB‐48 was investigated using various human microsomes followed by studies utilizing multiple recombinant human P450s. Screening with these enzymes showed that only 4, CYP2D6, −2J2, −3A4, and −3A5, biosynthesized the 2 major products, mono‐ and dihydroxylated 5F‐AKB‐48, with activities in the range of 2–5 pmol/min/pmol CYP. CYP3A5 formed the most dihydroxylated metabolite, whereas CYP2J2, highly expressed in cardiovascular tissues, showed the biosynthesized the most monohydroxylated derivative. CYP2D6 produced only monohydroxylated derivatives whereas CYP3A4 produced mostly dihydroxylated ones. To elucidate the roles of CYP3A4 and CYP3A5, additional enzymatic reactions utilized genotyped HLMs containing normal activity for CYP3A4 but low or high activities of CYP3A5. Those experiments resulted in significantly different patterns of oxidized metabolites. An UPLC equipped with UV/Vis detector and/or MS/MS were used to analyze the reactions. The profiles of the products biosynthesized were significantly different between the fluorinated and unfluorinated analogs. As this structural difference is known to increase potency for CB1 receptors, understanding the involved mechanism is essential. In conclusion, extensive metabolic studies of SCBs, including investigation of the mechanism(s) of action leading to toxicity, could result in the development of life saving, efficacious treatments for SCB abuse.Support or Funding Information(INBRE 117‐1006096 2018 AP and NIH/NIDA DA039143 to ARP and PLP)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
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