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

Abstract

39-Hydroxy-49-methoxydiclofenac (VI) is a human-specific metabolite known to accumulate in the plasma of patients after repeated administration of diclofenac sodium. Diclofenac also produces glutathioneconjugated metabolites, some of which are human-specific. In the present study, we investigated whether these metabolites could be generated in humanized chimeric mice produced from TK-NOG mice. After a single oral administration of diclofenac to humanized mice, the unchanged drug in plasma peaked at 0.25 hour and then declined with a half-life (t 1/2 ) of 2.4 hours. 49-Hydroxydiclofenac (II) and 39-hydroxydiclofenac also peaked at 0.25 hour and were undetectable within 24 hours. However, VI peaked at 8 hours and declined with a t 1/2 of 13 hours. When diclofenac was given once per day, peak and trough levels of VI reached plateau within 3 days. Studies with administration of II suggested VI was generated via II as an intermediate. Among six reported glutathione-conjugated metabolites of diclofenac, M1 (5-hydroxy-4-(glutathion-S-yl)diclofenac) to M6 (29-(glutathion-S-yl)monoclofenac), we found three dichlorinated conjugates [M1, M2 (49-hydroxy-39-(glutathion-S-yl)diclofenac), and M3 (5-hydroxy-6-(glutathion-S-yl)diclofenac)], and a single monochlorinated conjugate [M4 (29-hydroxy-39-(glutathion-S-yl)monoclofenac) or M5 (49-hydroxy-29-(glutathion-S-yl)monoclofenac)], in the bile of humanized chimeric mice. M4 and M5 are positional isomers and have been previously reported as human-specific in vitro metabolites likely generated via arene oxide and quinone imine-type intermediates, respectively. The biliary monochlorinated metabolite exhibited the same mass spectrum as those of M4 and M5, and we discuss whether this conjugate corresponded to M4 or M5. Overall, humanized TK-NOG chimeric mice were considered to be a functional tool for the study of drug metabolism of diclofenac in humans.

Simulation of human plasma concentration–time profiles of the partial glucokinase activator PF-04937319 and its disproportionate N-demethylated metabolite using humanized chimeric mice and semi-physiological pharmacokinetic modeling

“…Chimeric mice are obtained by transplanting human hepatocytes into immunodeficient mice with liver failure. Since transplanted hepatocytes are considered to be under physiological conditions that closely mimic human hepatic physiology in vivo (compared to in vitro incubation mixtures), the chimeric mouse model has been proposed as a tool for predicting circulating human-specific or disproportionate metabolites (Foster et al, 2014;Grompe & Strom, 2013;Kamimura & Ito, 2016;Kamimura et al, 2010Kamimura et al, , 2015Kitamura & Sugihara, 2014;Peltz, 2013;Sanoh & Ohta, 2014;Scheer & Wilson, 2016;Yoshizato et al, 2012).…”

Expression and inducibility of cytochrome P450s in human hepatocytes isolated from chimeric mice with humanised livers

“…TK-NOG mice with humanised livers have increasingly attracted attention as a potentially useful model for preclinical drug metabolism and pharmacokinetic studies, as they offer good predictivity of the human pharmacokinetics of P450 substrates (Utoh et al, 2016) and show human-specific metabolism (Kamimura et al, 2015). Hepatocytes prepared from these mice, termed Hu-Liver cells, are expected to be useful tools for P450 induction assays and for predicting drug metabolism in humans; however, the molecular characteristics of this cell line have not been clarified.…”

“…The livers of the Hu-Liver mice were shown to be mature, functional human livers, both in terms of gene expression patterns and human-specific drug-metabolising activities (Hasegawa et al, 2011). This Hu-Liver mouse model has been used for studies in various fields, including pharmacokinetics (Nishiyama et al, 2015;Utoh et al, 2016), metabolism (Kamimura et al, 2015(Kamimura et al, , 2017 and toxicity (Suemizu et al, 2018). In addition, another Hu-Liver chimeric mouse model derived from uPA/SCID mice has since been applied to non-clinical safety and metabolism studies (Foster et al, 2014), including the prediction of P450 induction in vivo (Kakuni et al, 2013;Katoh et al, 2005) and human pharmacokinetics (Sanoh and Ohta, 2014).…”

P450-Humanized and Human Liver Chimeric Mouse Models for Studying Xenobiotic Metabolism and Toxicity

“…2013; Bateman et al, 2014;Kamimura et al, 2015;Nakada et al, 2016;Wilson et al, 2018); drug-drug interaction (Hasegawa et al, 2012;Nishimura et al, 2013;Yamazaki et al, 2013;Suzuki et al, 2017;Uchida et al, 2018); human pharmacogenetic studies (Hu et al, 2013;Nishiyama et al, 2015); or other DME studies Inoue et al, 2009;Sanoh et al, 2012aSanoh et al, , 2015Schulz-Utermoehl et al, 2012;Tsukada et al, 2013;Suemizu et al, 2014;Nishiyama et al, 2015;Utoh et al, 2016;Kamimura et al, 2017;Shimizu et al, 2017;Yamazaki-Nishioka et al, 2018). A detailed analysis of all these studies is beyond the scope of this review; we will confine our review to a few examples illustrating the utility of human liver chimeric mice for drug studies.…”