|
Current
Drug Metabolism
ISSN: 1389-2002
Current Drug Metabolism
Volume 9, Number 1, January 2008
Contents
Cell Lines: A Tool for In Vitro Drug Metabolism
Studies Pp. 1-11
M.T. Donato, A. Lahoz, J.V. Castell and M.J. Gómez-Lechón
[Abstract]
Strategies to In Vitro Assessment of Major
Human CYP Enzyme Activities by Using Liquid Chromatography
Tandem Mass Spectrometry Pp. 12-19
A. Lahoz, M.T. Donato, J.V. Castell and M.J. Gómez-Lechón
[Abstract]
Comparison of Kinetic Parameters for Drug Oxidation
Rates and Substrate Inhibition Potential Mediated by Cytochrome
P450 3A4 and 3A5 Pp. 20-33
T. Niwa, N. Murayama, C. Emoto and H. Yamazaki
[Abstract]
Epigenetic Regulation of Genes Encoding Drug Metabolizing
Enzymes and Transporters; DNA Methylation and Other Mechanisms
Pp. 34-38
T. Hirota, H. Takane, S. Higuchi and I. Ieiri
[Abstract]
The Prediction of the Hepatic Clearance of Tanshinone
IIA in Rat Liver Subcellular Fractions: Accuracy Improvement
Pp. 39-45
P. Li, G.-J. Wang, J. Li, Q. Zhang, X. Liu, A. Khlentzos
and M.S. Roberts
[Abstract]
CSF as a Surrogate for Assessing CNS Exposure: An
Industrial Perspective Pp. 46-59
J.H. Lin
[Abstract]
Recent Advances in the In Silico Modelling
of UDP Glucuronosyltransferase Substrates Pp. 60-69
M.J. Sorich, P.A. Smith, J.O. Miners, P.I. Mackenzie and
R.A. McKinnon
[Abstract]
The UDP-Glucuronosyltransferases as Oligomeric Enzymes
Pp. 70-76
M. Finel and M. Kurkela
[Abstract]
Metabolic Pathways of T-2 Toxin Pp. 77-82
V. Dohnal, A. Jezkova, D. Jun and K. Kuca
[Abstract]
Effect of the NMDA-Receptor Antagonist Dextromethorphan
in Infant Rat Pneumococcal Meningitis Pp. 83-88
J. Sellner, R. Ringer, P. Baumann, M.J. Perey, B. Schmitt
and S.L. Leib
[Abstract]
Metabolomics Pp. 89-98
V.S. Gomase, S.S. Changbhale, S.A. Patil and K.V. Kale
[Abstract]
Abstracts
[Back to top]
Cell Lines: A Tool for In Vitro Drug Metabolism Studies
M.T. Donato, A. Lahoz, J.V. Castell and M.J. Gómez-Lechón
Primary cultured hepatocytes are a valuable in vitro
model for drug metabolism studies. However, their widespread
use is greatly hindered by the scarcity of suitable human
liver samples. Moreover, the well-known in vitro
phenotypic instability of hepatocytes, the irregular availability
of fresh human liver for cell harvesting purposes, and the
high batch-to-batch functional variability of hepatocyte preparations
obtained from different human liver donors, seriously complicate
their use in routine testing. To overcome these limitations,
different cell line models have been proposed for drug metabolism
screening. Human liver-derived cell lines would be ideal models
for this purpose given their availability, unlimited life-span,
stable phenotype, and the fact that they are easy to handle.
However, the human hepatoma cells currently used (i.e. HepG2,
Mz-Hep-1) show negligible levels of drug-metabolizing and
do not constitute a real alternative to primary hepatocytes.
Different strategies have been proposed to generate metabolically
competent immortalized hepatocytes (transformation of human
hepatocytes with plasmids encoding immortalizing genes, hepatocyte-like
cells derived from stem cells, cell lines generated from transgenic
animals, hepatocyte/hepatoma hydrid cells). Moreover, recombinant
models heterologously expressing P450 enzymes in different
host cells have been developed and successfully used in drug
metabolism testing. In addition, new strategies have recently
been explored to upregulate the expression of drug-metabolizing
enzymes in cell lines of a human origin (i.e. transfection
with expression vectors encoding key hepatic transcription
factors). Among metabolic-based drug-drug interactions, P450
inhibition seems to be the most important. A major application
of recombinant models expressing a single P450 is the screening
of potential enzyme inhibitors. Therefore, pharmaceutical
companies increasingly make use of cell lines to speed up
the selection of new drugs with favourable pharmacokinetic
and metabolic properties.
[Back to top]
Strategies to In Vitro Assessment of Major Human
CYP Enzyme Activities by Using Liquid Chromatographyv Tandem
Mass Spectrometry
A. Lahoz, M.T. Donato, J.V. Castell and M.J. Gómez-Lechón
At the early stage of drug discovery, thousands of new
chemical entities (NCEs) may be screened before a single candidate
can be identified for development. Determining the role of
CYP enzymes in the metabolism of a compound and evaluating
the effect of NCEs on human CYP activities are key issues
in pharmaceutical development as they may explain inter-subject
variability, drug-drug interactions, non-linear pharmacokinetics
and toxic effects. Reliable methods for determining enzyme
activities are needed to characterize an individual CYP enzyme
and to obtain a tool for the evaluation of its role in drug
metabolism in humans. Different liquid chromatography tandem
mass spectrometry methodologies have been developed for the
fast and routine analysis of major in vivo and in
vitro CYPs enzyme activities. The high sensitivity and
selectivity of mass spectrometry allow traditional assays
to be minimized, thus saving time, efforts and money. Therefore
this technology has become the method of choice for the fast
assessment of CYP enzyme activities in early drug discovery
development. Our intention herein is to review the most recent
approaches that have been developed to quickly assess CYPs
activities using in vitro models and liquid chromatography
coupled with mass spectrometry, as well as their application
in early drug discovery.
[Back to top]
Comparison of Kinetic Parameters for Drug Oxidation Rates
and Substrate Inhibition Potential Mediated by Cytochrome
P450 3A4 and 3A5
T. Niwa, N. Murayama, C. Emoto and H. Yamazaki
Cytochrome P450 (P450 or CYP) 3A is one of the most important
P450 subfamilies in terms of its broad substrate specificity
and relatively high abundance in humans. The substrate specificities
of CYP3A4 and CYP3A5 are generally overlapped, but sometimes
could differ from each other. It is still important to understand
drug interactions more precisely in individual subjects. However,
there are few review articles regarding comparative drug oxidation
rates catalyzed by CYP3A4 and CYP3A5 and/or substrate inhibition
potential towards CYP3A4 and CYP3A5. In this article, we summarize
1) Michaelis-Menten constants (Km),
maximal velocities (Vmax),
and intrinsic clearance (Vmax/Km)
values for 63 substrates (94 reactions) mediated by CYP3A4
and/or CYP3A5, 2) inhibition constants (Ki)
and 50% inhibitory concentrations (IC50)
of 18 substrates, and 3) maximum inactivation rate constants
(kinact) of 14 inhibitors
from the literature. The relative contribution of polymorphic
CYP3A5 compared with inducible CYP3A4 varies with the substrates
and the reaction positions of the substrates. Inhibitory effects
of azole antifungal agents and macrolide antibiotics, with
low Ki and/or IC50
values for CYP3A4, are likely to be determinant factors for
predominant drug interactions in humans, although Asian subjects
with relatively high frequency of genetic CYP3A5 expressers
should be carefully treated with CYP3A substrates. The collective
findings in our present survey provide fundamental and useful
information for drug oxidations catalyzed by CYP3A4 and CYP3A5,
in spite of some contradictive kinetic parameters for the
same reactions reported from many laboratories in different
conditions. To understand causal factor(s) and mechanism(s)
for such different reports summarized here is still one of
the hot research topics to be solved in current drug metabolism.
[Back to top]
Epigenetic Regulation of Genes Encoding Drug Metabolizing
Enzymes and Transporters; DNA Methylation and Other Mechanisms
T. Hirota, H. Takane, S. Higuchi and I. Ieiri
Drug metabolizing enzymes and transporters are increasingly
recognized as key determinants of the inter-individual variability
in pharmacokinetic (PK) and pharmacodynamic (PD) outcomes
of clinically important drugs. To date, most studies investigating
this variability have focused on polymorphisms (e.g. SNPs)
in the genes encoding metabolic enzymes and transporters;
however, it has recently been reported that the expression
of some of these genes is under the control of epigenetic
mechanisms. The most common epigenetic mechanism of mammalian
genome regulation is DNA methylation, which does not change
the genetic code but affects gene expression. Owing to its
maintenance of the genomic sequence, DNA methylation is expected
to offer an explanation for the controversial phenotypes of
certain genetic polymorphisms. It has been recognized that
DNA methylation plays a role in the transcriptional regulation
of some PK/PD genes. In this review, we describe the impact
of various epigenetic mechanisms, especially DNA methylation,
on the expression (or activity) of drug metabolizing enzymes
and transporter genes.
[Back to top]
The Prediction of the Hepatic Clearance of Tanshinone IIA
in Rat Liver Subcellular Fractions: Accuracy Improvement
P. Li, G.-J. Wang, J. Li, Q. Zhang, X. Liu, A. Khlentzos
and M.S. Roberts
The in vivo hepatic clearance of tanshinone
IIA in the rat was predicted using microsome, cytosol and
S9 fractions combined with two different cofactor systems,
NADPH-regenerating and UDPGA system. Two different models,
the well stirred model and the parallel-tube model, were used
in predicting the in vivo clearance in the rat. The
in vivo clearance of tanshinone IIA was acquired from
a pharmacokinetic study in rat. The results show that the
prediction accuracy acquired from the microsome combined with
the NADPH is poor. The in vivo clearance in the rat
is almost 32 fold higher than the clearance predicted in microsome.
The predicted clearance of the S9 model combined with both
NADPH and UDPGA system is about 4 fold lower than the
in vivo clearance. The predicted clearance of the cytosol
combined with the two cofactor system is about 7 fold lower
than the in vivo clearance. Although the prediction
accuracy acquired from the S9 and cytosol system is not perfect,
the prediction accuracy is improved in these two incubation
systems. Using S9 combined with both the phase I and phase
II metabolism can improve the prediction accuracy.
[Back to top]
CSF as a Surrogate for Assessing CNS Exposure: An Industrial
Perspective
J.H. Lin
For drugs that directly act on targets in the central
nervous system (CNS), sufficient drug delivery into the brain
is a prerequi-site for drug action. Systemically administered
drugs can reach CNS by passage across the endothelium of capillary
vasculatures, the so-called blood-brain barrier (BBB). Literature
data suggest that most marketed CNS drugs have good membrane
permeability and relatively high plasma unbound fraction,
but are not good P-glycoprotein (P-gp) substrates. Therefore,
it is important to use the in vitro parameters of
P-gp function activity, membrane permeability and plasma unbound
fraction as key criteria for lead optimization during the
early stage of drug discovery. Evidence from preclinical and
clinical studies suggests that drug concentration in cerebrospinal
fluid (CSF) appears to be reasonably accurate in predicting
unbound drug concentration in the brain. Therefore, CSF can
be used as a useful surrogate for in vivo assessment
of CNS exposure and provides an important basis for the selection
of drug candidates for entry into development. However, it
is important to point out that CSF drug concentration is not
always an accurate surrogate for predicting unbound drug concentration
in the brain. Depending on the physicochemical properties
of drugs and the site/timing of CSF sampling, the unbound
drug concentration at the biophase within the brain could
differ significantly from the corresponding CSF drug concentration.
[Back to top]
Recent Advances in the In Silico Modelling of UDP
Glucuronosyltransferase Substrates
M.J. Sorich, P.A. Smith, J.O. Miners, P.I. Mackenzie and
R.A. McKinnon
UDP glucurononosyltransferases (UGT) are a superfamily
of enzymes that catalyse the conjugation of a range of structurally
diverse drugs, environmental and endogenous chemicals with
glucuronic acid. This process plays a significant role in
the clearance and detoxification of many chemicals. Over the
last decade the regulation and substrate profiles of UGT isoforms
have been increasingly characterised. The resulting data has
facilitated the prototyping of ligand based in silico
models capable of predicting, and gaining insights into, binding
affinity and the substrate- and regio- selectivity of glucuronidation
by UGT isoforms. Pharmacophore modelling has produced particularly
insightful models and quantitative structure-activity relationships
based on machine learning algorithms result in accurate predictions.
Simple structural chemical descriptors were found to capture
much of the chemical information relevant to UGT metabolism.
However, quantum chemical properties of molecules and the
nucleophilic atoms in the molecule can enhance both the predictivity
and chemical intuitiveness of structure-activity models. Chemical
diversity analysis of known substrates has shown some bias
towards chemicals with aromatic and aliphatic hydroxyl groups.
Future progress in in silico development will depend
on larger and more diverse high quality metabolic datasets.
Furthermore, improved protein structure data on UGTs will
enable the application of structural modelling techniques
likely leading to greater insight into the binding and reactive
processes of UGT catalysed glucuronidation.
[Back to top]
The UDP-Glucuronosyltransferases as Oligomeric Enzymes
M. Finel and M. Kurkela
The UDP-glucuronosyltransferases (UGTs) are integral
membrane proteins of the endoplasmic reticulum that play important
roles in the defense against potentially hazardous xenobiotics.
The UGTs also participate in the metabolism and homeostasis
of many endogenous compounds, including bilirubin and steroid
hormones. Most human UGTs can glucuronidate several substrates
the chemical structures of which may vary significantly. Understanding
the structural basis for the complex substrate specificity
of the UGTs is a major challenge that is hampered by the lack
of sufficient structural information on these enzymes. Nevertheless,
there is currently a broad interest in the structure and function
of the UGTs and here we have focused on their oligomeric state.
The question whether or not the UGTs are oligomeric enzymes,
either dimeric or tetrameric, was frequently addressed in
the past, as well as in recent studies. The current knowledge
of protein-protein interactions among the UGTs is limited,
however, primarily due to considerable difficulties in purifying
individual recombinant UGTs as fully active and mono-dispersed
proteins. Such hurdles in studying the oligomeric state of
the UGTs prompted researchers to develop less direct approaches
for examining the quaternary structure of the UGTs and its
functional significance. In this article we have reviewed,
sometimes critically, most of the available studies about
the oligomeric state of the UGTs. Concluding that the UGTs
are oligomeric enzymes, we discuss hetero-oligomerization
among UGTs and its possible implications for the structure,
function and substrate specificity of the enzymes.
[Back to top]
Metabolic Pathways of T-2 Toxin
V. Dohnal, A. Jezkova, D. Jun and K. Kuca
Among the naturally-occurring trichothecenes found in
food and feed, T-2 toxin is the most potent and toxic mycotoxin.
After ingestion of T-2 toxin into the organism, it is processed
and eliminated. Some metabolites of this trichothecene are
equally toxic or slightly more toxic than T-2 itself, and
therefore, the metabolic fate of T-2 toxin has been of great
concern. The main reactions in trichothecene metabolism are
hydrolysis, hydroxylation and deep oxidation. Typical metabolites
of T-2 toxin in an organism are HT-2 toxin, T-2-triol, T-2-tetraol,
3’-hydroxy-T-2, and 3’-hydroxy-HT-2 toxin. There
are significant differences in the metabolic pathways of T-2
toxin between ruminants and non-ruminants. Ruminants have
been more resistant to the adverse effects of T-2 toxin due
to microbial degradation within rumen microorganisms. Some
plant species are resistant to T-2 toxin, while others are
capable of its intake and metabolisation.
[Back to top]
Effect of the NMDA-Receptor Antagonist Dextromethorphan in
Infant Rat Pneumococcal Meningitis
J. Sellner, R. Ringer, P. Baumann, M.J. Perey, B. Schmitt
and S.L. Leib
Excitatory amino acids (EAA) and particularly glutamate
toxicity have been implicated in the pathogenesis of neuronal
injury occurring in bacterial meningitis by activating the
N-methyl-d aspartate (NMDA) receptor complex. Here, we evaluated
the effect of adjuvant treatment with the antitussive drug
dextromethorphan (DM), a non-competitive NMDA receptor antagonist
with neuroprotective potential, in an infant rat model of
pneumococcal meningitis. The experiments were carried out
in postnatal day 6 (P6) and 11 (P11) animals. Pharmacokinetics
of DM and its major metabolite dextrorphan (DO) were performed
for dose finding.
In our study, DM did not alter clinical parameters (clinical
score, motor activity, incidence of seizures, spontaneous
mortality) and cortical neuronal injury but increased the
occurrence of ataxia (P<0.0001). When DM treatment was
started at the time of infection (DM i.p. 15 mg/kg at 0, 4,
8 and 16 hours (h) post infection) in P11 animals, an aggravation
of apoptotic neuronal death in the hippocampal dentate gyrus
was found (P<0.05). When treatment was initiated during
acute pneumococcal meningitis (DM i.p. 15 mg/kg at 12 and
15 h and 7.5 mg/kg at 18 and 21 h after infection), DM had
no effect on the extent of brain injury but reduced the occurrence
of seizures (P<0.03). We conclude that in this infant rat
model of pneumococcal meningitis interference of the EEA and
NMDA pathway using DM causes ataxia, attenuates epileptic
seizures and increases hippocampal apoptosis, but is not effective
in protecting the brain from injury.
[Back to top]
Metabolomics
V.S. Gomase, S.S. Changbhale, S.A. Patil and K.V. Kale
Metabolomics is based on the simultaneous analysis of
multiple low-molecular-weight metabolites from a given sample.
The goals of metabolomics are to catalog and quantify the
myriad small molecules found in biological fluids under different
conditions. The metabolomics represents the collection of
all metabolites in a biological organism, and metabolic profiling
can give an instantaneous 'snapshot' of the physiology of
that cell. Together with the other more established omics
technologies, metabolomics will strengthen its claim to contribute
to the detailed understanding of the in vivo function
of gene products, biochemical analysis, regulatory networks
and more ambitious, the mathematical description and simulation
of the whole cell in the systems biology approach. This phenomenon
will allow the construction of designer organisms for process
application using biotransformation and fermentative approaches
making effective use of single enzymes, whole microbial and
even higher cells and allows the connection of data from genomics,
proteomics to enables coordinating the timing of the analysis
to physiologically important windows.
|
