Significantly Different Covalent
Binding of Oxidative
Metabolites, Acyl Glucuronides, and S‑Acyl CoA Conjugates Formed from Xenobiotic Carboxylic Acids
in Human Liver Microsomes
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Abstract
Xenobiotic carboxylic acids may be
metabolized to oxidative metabolites,
acyl glucuronides, and/or S-acyl-CoA thioesters (CoA conjugates) in
vitro, e.g., in hepatocytes, and in vivo. These metabolites can potentially
be reactive species and bind covalently to tissue proteins and are
generally considered to mediate adverse drug reactions in humans.
Acyl glucuronide metabolites have been the focus of reactive metabolite
research for decades, whereas drug-CoA conjugates, which have been
shown to be up to 40–70 times more reactive, have been given
much less attention. In an attempt to dissect the contribution of
different pathways to covalent binding, we utilized human liver microsomes
supplemented with NADPH, uridine 5′-diphosphoglucuronic acid
(UDPGA), or CoA to evaluate the reactivity of each metabolite separately.
Seven carboxylic acid drugs were included in this study. While ibuprofen
and tolmetin are still on the market, ibufenac, fenclozic acid, tienilic
acid, suprofen, and zomepirac were stopped before their launch or
withdrawn. The reactivities of the CoA conjugates of ibuprofen, ibufenac,
fenclozic acid, and tolmetin were higher compared to those of their
corresponding oxidative metabolites and acyl glucuronides, as measured
by the level of covalent binding to human liver microsomal proteins.
The highest covalent binding was observed for ibuprofenyl-CoA and
ibufenacyl-CoA, to levels of 1000 and 8600 pmol drug eq/mg protein,
respectively. In contrast and in agreement with the proposed P450-mediated
toxicity for these drug molecules, the reactivities of oxidative metabolites
of suprofen and tienilic acid were higher compared to the reactivities
of their conjugated metabolites, with NADPH-dependent covalent binding
of 250 pmol drug eq/mg protein for both drugs. The seven drugs all
formed UDPGA-dependent acyl glucuronides, but none of these resulted
in covalent binding. This study shows that, unlike studies with hepatocytes
or in vivo, human liver microsomes provide an opportunity to investigate
the reactivity of individual metabolites