One-electron oxidation of diclofenac by human cytochrome P450s as a potential bioactivation mechanism for formation of 2′-(glutathion-S-yl)-deschloro-diclofenac

Formation of the Accumulative Human Metabolite and Human-Specific Glutathione Conjugate of Diclofenac in TK-NOG Chimeric Mice with Humanized Livers

“…In the present study, similar extracted ion chromatograms were obtained from the bile of the control TK-NOG mice, indicating the same reactions also occur in mouse species. In addition, M6, reported to be generated in vitro by oneelectron oxidation of diclofenac (Boerma et al, 2014), was also detected in mouse bile. In metabolism studies using 3 H-labeled diclofenac, Sarda et al (2012) reported the major metabolites in mouse feces were taurine conjugates of II and diclofenac, whereas Bateman et al (2014) reported the major metabolite in mouse bile was diclofenac acyl glucuronide.…”

“…The chemical structure of M4 was proposed as a 29-hydroxy-39-glutathionyl analog based on the assumption that this monochlorinated metabolite was generated via diclofenac-29,39-oxide, because M4 had not been detected in the incubation mixture of II and NADPH-fortified human liver microsomes. On the other hand, 49-hydroxy-29-(glutathion-S-yl)monoclofenac was proposed by Yu et al (2005), and designated as M5 later (Boerma et al, 2014). Similar to M4, M5 was generated in an incubation mixture of diclofenac and human liver microsomes fortified with glutathione, and its chemical structural assignment was obtained via comparison of mass and 1 H NMR spectra with those of a chemically synthesized authentic standard.…”

“…29-(Glutathion-S-yl)monoclofenac (we designated this conjugate "M6") was found first in incubation mixtures of diclofenac with human liver microsomes (Wen et al, 2008). In vitro studies using various recombinant human P450s fortified with glutathione and human glutathione-S-transferase P1-1 demonstrated that all of the 14 tested P450 isozymes were capable of forming M6 (Boerma et al, 2014). Furthermore, this reaction was NADPH-independent with M6 produced to some extent by flavincontaining monooxygenase or even horseradish peroxidase, suggesting the involvement of one-electron oxidation.…”

“…Metabolic activities of CYP2C9 in the respective hepatocytes were 1.28 (49-hydroxylation of tolbutamide at 400 mM) or 467 (49-hydroxylation of diclofenac at 100 mM) pmol/min per 10 6 viable cells. Reference incubation mixture containing M6 was prepared at VU University Amsterdam (Amsterdam, The Netherlands) as described by Boerma et al (2014). In brief, a final concentration of 500 mM diclofenac was incubated with 100 nM horseradish peroxidase, 100 mM glutathione, and 8 mM human glutathione-S-transferase P1-1 for 1 hour at 37°C.…”

Influence of Proteome Profiles and Intracellular Drug Exposure on Differences in CYP Activity in Donor-Matched Human Liver Microsomes and Hepatocytes

“…75 Likewise, diclofenac was only compensated for CYP2C9, while it is also metabolized by both CYP3A4 and UGT2B7. 76 By monitoring all formed metabolites (e.g., diclofenac is metabolized to 3-,4-,5-, and acyl glucuronide diclofenac by the three enzymes 77 ), the contribution of each enzyme to the drug's metabolism could be better defined and used to improve the activity−protein concentration correlations. In line with this, the CL int for many of the compounds in this study correlated well with several non-probe CYP enzymes in both HLM and HH (Table S4; Figure S4).…”