3 research outputs found
Metabolism of Xenobiotic Carboxylic Acids: Focus on Coenzyme A Conjugation, Reactivity, and Interference with Lipid Metabolism
While xenobiotic carboxylic acids
(XCAs) have been studied extensively
with respect to their enzymatic conversion to potentially reactive
acyl glucuronides with implications to drug induced hepatotoxicity,
the formation of xenobiotic-<i>S</i>-acyl-CoA thioesters
(xenobiotic-CoAs) have been much less studied in spite of data indicating
that such conjugates may be equally or more reactive than the corresponding
acyl glucuronides. This review addresses enzymes and cell organelles
involved in the formation of xenobiotic-CoAs, the reactivity of such
conjugates toward biological macromolecules, and in vitro and in vivo
methodology to assess consequences of such reactivity. Further, the
propensity of xenobiotic-CoAs to interfere with endogenous lipid metabolism,
e.g., inhibition of β-oxidation or depletion of the CoA or carnitine
pools, adds to the complexity of the potential contribution of XCAs
to hepatotoxicity by a number of mechanisms in addition to those in
common with the corresponding acyl glucuronides. On the basis of our
review of the literature on xenobiotic-CoA conjugates, there appear
to be a number of gaps in our understanding of the bioactivation of
XCA both with respect to the mechanisms involved and the experimental
approaches to distinguish between the role of acyl glucuronides and
xenobiotic-CoA conjugates. These aspects are focused upon and described
in detail in this review
Use of HμREL Human Coculture System for Prediction of Intrinsic Clearance and Metabolite Formation for Slowly Metabolized Compounds
Design of slowly
metabolized compounds is an important goal in
many drug discovery projects. Standard hepatocyte suspension intrinsic
clearance (CL<sub>int</sub>) methods can only provide reliable CL<sub>int</sub> values above 2.5 μL/min/million cells. A method that
permits extended incubation time with maintained performance and metabolic
activity of the in vitro system is warranted to allow in vivo clearance
predictions and metabolite identification of slowly metabolized drugs.
The aim of this study was to evaluate the static HμREL coculture
of human hepatocytes with stromal cells to be set up in-house as a
standard method for in vivo clearance prediction and metabolite identification
of slowly metabolized drugs. Fourteen low CL<sub>int</sub> compounds
were incubated for 3 days, and seven intermediate to high CL<sub>int</sub> compounds and a cocktail of cytochrome P450 (P450) marker substrates
were incubated for 3 h. In vivo clearance was predicted for 20 compounds
applying the regression line approach, and HμREL coculture predicted
the human intrinsic clearance for 45% of the drugs within 2-fold and
70% of the drugs within 3-fold of the clinical values. CL<sub>int</sub> values as low as 0.3 μL/min/million hepatocytes were robustly
produced, giving 8-fold improved sensitivity of robust low CL<sub>int</sub> determination, over the cutoff in hepatocyte suspension
CL<sub>int</sub> methods. The CL<sub>int</sub> values of intermediate
to high CL<sub>int</sub> compounds were at similar levels both in
HμREL coculture and in freshly thawed hepatocytes. In the HμREL
coculture formation rates for five P450-isoform marker reactions,
paracetamol (CYP1A2), 1-OH-bupropion (CYP2B6), 4-OH-diclofenac (CYP2C9),
and 1-OH-midazolam (3A4) were within the range of literature values
for freshly thawed hepatocytes, whereas 1-OH-bufuralol (CYP2D6) formation
rate was lower. Further, both phase I and phase II metabolites were
detected and an increased number of metabolites were observed in the
HμREL coculture compared to hepatocyte suspension. In conclusion,
HμREL coculture can be applied to accurately estimate intrinsic
clearance of slowly metabolized drugs and is now utilized as a standard
method for in vivo clearance prediction of such compounds in-house
Significantly Different Covalent Binding of Oxidative Metabolites, Acyl Glucuronides, and S‑Acyl CoA Conjugates Formed from Xenobiotic Carboxylic Acids in Human Liver Microsomes
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