In Vitro and In Vivo Metabolic Activation of Obacunone, A Bioactive and Potentially Hepatotoxic Constituent of Dictamni Cortex.

Obacunone is one of the major bioactive constituents from Dictamni cortex, a traditional Chinese medicine widely used in China. Oral administration of obacunone or Dictamni cortex extract has been shown to cause liver injury in rats. Given that obacunone contains a furan ring, which is a structural alert, metabolic activation might be responsible for obacunone-induced liver injury. In this study, bioactivation pathways of obacunone in rat and human liver microsomes were investigated. Obacunone was first metabolized into cis-butene-1,4-dial, and then cis-butene-1,4-dial was captured by glutathione, N-acetyl-cysteine, and N-acetyl-lysine in the microsomal incubation system. A total of 13 adducts derived from the reaction of cis-butene-1,4-dial with glutathione and/or N-acetyl-lysine were detected and structurally identified by liquid chromatography coupled to high-resolution tandem mass spectrometry. The major metabolite (M7) was identified to be the cyclic mono-glutathione conjugate of cis-butene-1,4-dial, which was detected in bile and urine of obacunone-treated rats. M9 and M10, obacunone-derived glutathione-cis-butene-1,4-dial-NAL conjugates, were detected in the microsomal incubations of obacunone fortified with glutathione and N-acetyl-lysine as trapping agents. M3 and M4, pyrroline-2-one derivatives, were also detected in microsomal incubations. Further phenotyping studies indicated that ketoconazole showed a strong inhibitory effect on the production of cis-butene-1,4-dial in a concentration-dependent manner. CYP3A4 was demonstrated to be the primary enzyme responsible for the bioactivation of obacunone by using individual recombinant human CYP450 enzymes. The current study provides an overview of CYP450-dominated bioactivation of obacunone and contributes to the understanding of the role of bioactivation in obacunone-induced liver injury.

In vitro metabolites characterization of ponatinib in human liver microsomes using ultra-high performance liquid chromatography combined with Q-Exactive-Orbitrap tandem mass spectrometry.

Ponatinib is an oral drug for the treatment of chronic myeloid leukemia and acute lymphoblastic leukemia, which has been reported to increase the risk of hepatotoxicity. The aim of this study was to characterize the metabolites of ponatinib in human liver microsomes as well as its reactive metabolites. Ponatinib was incubated with human liver microsomes in the presence of NADPH and trapping agents (glutathione or potassium cyanide). The metabolites were characterized by liquid chromatography in combination with Q-Exactive-Orbitrap-MS. Under the current conditions, six metabolites were detected and structurally identified on the basis of their accurate masses, fragmentation patterns, and retention times. M3 (N-demethylation) was unambiguously identified by matching its retention time and fragment ions with those of its reference standard. N-demethylation and oxygenation were proved to be the predominant metabolic pathways of ponatinib. In addition, two reactive metabolites (cyano adducts) were detected in human liver microsomes in the presence of potassium cyanide and NADPH, suggesting that ponatinib underwent CYP450-mediated metabolic activation, which could be one of the causative mechanisms for its hepatotoxicity. The current study provides new information regarding the metabolic profiles of ponatinib and would be helpful in understanding the effectiveness and toxicity of ponatinib, especially the mechanism of hepatotoxicity.