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Current
Drug Metabolism
ISSN: 1389-2002
Current Drug Metabolism
Volume 7, Number 3, April 2006
Contents
Reactive Intermediates and the Pathogenesis of Adverse Drug
Reactions: The Toxicology Perspective Pp.
219-229
D.E. Amacher
[Abstract]
A Literature Review of Enzyme Kinetic Parameters for CYP3A4-Mediated
Metabolic Reactions of 113 Drugs in Human Liver Microsomes:
Structure-Kinetics Relationship Assessment Pp. 231-249
H.-Z. Bu
[Abstract]
The Impact of In Vitro Binding on In Vitro
- In Vivo Extrapolations, Projections of Metabolic
Clearance and Clinical Drug-Drug Interactions Pp.
251-264
K. Grime and R.J. Riley
[Abstract]
Evaluation of Microsomal Incubation Conditions on CYP3A4-Mediated
Metabolism of Cyclosporine A by a Statistical Experimental
Design Pp. 265-271
M. Hermann, E.T. Kase, E. Molden and H. Christensen
[Abstract]
Candidate Susceptibility Genes in Alzheimer’s Disease
Are at High Risk for Being Forgotten – They Don’t
Give Peace of Mind… Pp. 273-293
A. Palotás and J. Kálmán
[Abstract]
Prediction of Maximum Exposure in Poor Metabolizers
Following Inhibition of Nonpolymorphic Pathways
Pp. 295-299
C. Collins, R. Levy, I. Ragueneau-Majlessi and H. Hachad
[Abstract]
Involvement of Cytoskeleton in AhR-Dependent CYP1A1
Expression Pp. 301-313
Z. Dvorák, R. Vrzal, J. Ulrichová, J.-M. Pascussi,
P. Maurel and
M. Modriansky
[Abstract]
A Critical Evaluation of the Experimental Design of
Studies of Mechanism Based Enzyme Inhibition, with Implications
for In Vitro-In Vivo Extrapolation Pp. 315-334
F. Ghanbari, K. Rowland-Yeo, J.C. Bloomer, S.E. Clarke,
M.S. Lennard, G.T. Tucker and A. Rostami-Hodjegan
[Abstract]
Abstracts
[Back to top]
Reactive Intermediates and the Pathogenesis of Adverse
Drug Reactions: The Toxicology Perspective
D.E. Amacher
Severe adverse drug responses are infrequent but occasionally
serious events that are not readily predictable at the preclinical
development level using only non-human or in vitro
models. A common characteristic of the more serious toxicities
is generation of short-lived and highly reactive electrophilic
species in some individuals. The objective here is to review
the literature for toxicological mechanisms that underlie
known adverse drug reactions and then categorize the biological
consequences of reactive chemical intermediates and radicals
in terms of human risk factors and known metabolic variables.
Xenobiotics described as being associated with rare but potentially
serious adverse events affecting liver, skin, or causing blood
dyscrasias tend to have three of four essential characteristics,
(1) they are capable of forming short-lived reactive intermediates
(RI) or free radicals in target tissues under ideal conditions
that are distinct from primary metabolic products, (2) these
RI escape/overwhelm the detoxification mechanisms associated
with the site of origin or form toxic conjugates, (3) the
unconjugated RI must either selectively damage critical proteins
or other key macromolecules or (4) the RI acts as a hapten
and stimulates an immunological (hypersensitivity) response
or overcomes tolerance. Some risk factors may increase the
probability of susceptibility, but this remains an active
area of research. Because of the complexity of the pathogenesis
of some injuries and the role of individual factors, no highly
predictive in vitro screening methods are available;
however, several methods are evolving that may be used to
reveal mechanisms of action when a serious adverse effect
is encountered.
[Back to top]
A Literature Review of Enzyme Kinetic Parameters for CYP3A4-Mediated
Metabolic Reactions of 113 Drugs in Human Liver Microsomes:
Structure-Kinetics Relationship Assessment
H.-Z. Bu
Cytochrome P450 (CYP) enzymes represent a superfamily of
hemoproteins that are involved in the metabolism of a wide
variety of endogenous and exogenous compounds. For a given
CYP enzyme, kinetic properties of a substrate are usually
related to substrate lipophilicity (log P or log D7.4).
In this review, enzyme kinetic parameters (Km,
Vmax, and Vmax/Km) of 215
CYP3A4-mediated metabolic reactions of 113 drugs in human
liver microsomes were obtained from the literature, and lipophilicity
values of the 113 drugs were calculated using the ACD/Labs
8.0 program. A low degree of Km- or (Vmax/Km)-lipophilicity
correlation, but no Vmax-lipophilicity correlation,
is exhibited for the CYP3A4-mediated reactions. Overall, Km
decreases, but Vmax/Km increases, with
increasing substrate lipophilicity, and Vmax appears
to be independent of substrate lipophilicity. In other words,
a low Km generally confers a high Vmax/Km
ratio for a substrate. The degree of lipophilicity-kinetics
correlations is related to both reaction types (or reaction
mechanisms) and regiochemical positions (or physicochemical
properties) of the reaction groups of the substrates. Among
the categorized CYP3A4-mediated reactions, the best lipophilicity-kinetics
correlation is achieved for carbon hydroxylation, followed
by N-dealkylation. No or little lipophilicity-kinetics
correlations are seen for N, S-oxidation
and other reactions. Within the hydroxylation group, aliphatic
hydroxylation shows the best lipophilicity-kinetics correlation
while hydroxylation on a carbon atom adjacent to an aromatic
ring does not show any lipophilicity-kinetics correlation.
The detailed structural and kinetic data sets of the human
liver microsomal CYP3A4-mediated reactions represent a specialized
database useful for researchers working in the area of structure-metabolism
relationship modeling and analysis.
[Back to top]
The Impact of In Vitro Binding on In
Vitro - In Vivo Extrapolations, Projections
of Metabolic Clearance and Clinical Drug-Drug Interactions
K. Grime and R.J. Riley
This review provides a vista of the current opportunities
and remaining challenges in the area of in vitro-in
vivo extrapolation, with particular emphasis on drug
binding terms in predictive models, which has been the source
of much controversy. Although the importance of fuinc
(fraction unbound in in vitro incubations) has been
acknowledged for decades, it is not always applied in practice.
This is somewhat disappointing, since although it may be onerous
to measure this term for large numbers of compounds, algorithms
to estimate the term from logD7.4 or logP have
been detailed in the literature. These are sufficiently robust
to negate routine measurement in early drug discovery. Several
groups have recently established convincing relationships
between unbound in vivo and in vitro metabolic
intrinsic clearance (CLint). In the authors’
laboratory, correlations of this type have been constructed
for rat, dog and Man. The use and interpretation of these
models within a drug discovery setting is discussed. The quantitative
prediction of drug-drug interactions from in vitro
cytochrome P450 (CYP) inhibition data remains a challenge.
Although extensive litera-ture databases are at last emerging,
apparent ad hoc use of terms for in vivo
inhibitor concentrations and only occasional consideration
of fuinc may only have confused matters. The effect
of accounting for drug binding on the accuracy of predictions
is reviewed. Other themes including the impact of fuinc
on relative activity factors (RAFs) and how in vitro
data quality and inter-laboratory differences can confound
quantitative human pharmacokinetic predictions are also developed.
[Back to top]
Evaluation of Microsomal Incubation Conditions on CYP3A4-Mediated
Metabolism of Cyclosporine A by a Statistical Experimental
Design
M. Hermann, E.T. Kase, E. Molden and H. Christensen
Objective: The aim of this study was to evaluate the influence
of microsomal incubation conditions on CYP3A4-mediated metabolism
of cyclosporine (CsA) within the limits of previous in
vitro studies by application of a statistical experimental
design.
The effect of changes in microsomal incubation conditions
(NADPH, Mg2+, Cl-, NADPH-regenerating system and
pH) on the formation of the CYP3A4 metabolites AM1 and AM9
from CsA were studied by application of a fractional facto-rial
design. Metabolism was studied in microsomes of transfected
human liver epithelial (THLE) cells specifically expressing
CYP3A4.
Within the conditions tested, a 3-4-fold difference in formation
of CsA metabolites was observed. Formation of both AM1 and
AM9 was favoured by a low Mg2+ concentration (0.5
mM) and no addition of Cl- to the incubation matrix.
However, while a high NADPH concentration (1.75 mM) was the
single most important factor for the formation of AM1, changes
in NADPH concentration between 0.25 and 1.75 mM had no influence
on AM9 formation. Formation of the two metabolites also differed
in their influence by pH changes, as a change in pH from 7.2
to 7.5 significantly increased the formation of AM9, while
formation of AM1 was unaffected by this change.
The present study showed that relatively small changes in
the incubation matrix had a significant influence on the microsomal
CYP3A4-mediated metabolism of CsA. Systematic studies on microsomal
incubation conditions could be a key to improve metabolic
in vitro-in vivo extrapolations in drug development.
[Back to top]
Candidate Susceptibility Genes in Alzheimer’s Disease
Are at High Risk for Being Forgotten – They Don’t
Give Peace of Mind…
A. Palotás and J. Kálmán
Alzheimer’s disease (AD) is a genetically complex and
heterogenous disorder. In a small proportion of cases, mutations
in three determinative (causal) genes are responsible for
autosomal dominant early-onset forms of AD. The majority of
cases, however, is sporadic, late-onset AD with unknown etiology.
The pathology and clinical manifestations of these forms are
influenced by multiple genetic and environmental risk factors.
Over the past decades, a number of candidate genes have been
identified as disease modifiers with conflicting results.
This study reviews susceptibility genes that are associated
with increased risk of developing AD.
[Back to top]
Prediction of Maximum Exposure in Poor Metabolizers
Following Inhibition of Nonpolymorphic Pathways
C. Collins, R. Levy, I. Ragueneau-Majlessi and H. Hachad
Marked increases in exposure of some substrates have been
noted in poor metabolizers given inhibitors of nonpolymorphic
enzymes. Among the small number of clinical trials conducted
to investigate this problem, a wide variation in the degree
of maximum exposure ratios (area under the curve in poor metabolizers
in the presence of inhibitor/area under the curve in extensive
metabolizers) among the different substrates has been reported,
with some trials reporting profound increases (> tenfold),
and others demonstrating less remarkable changes (< twofold).
The conduct of such trials raises safety concerns for the
trial participants, in addition to other ethical and logistic
concerns; therefore, the possibility was investigated that
maximum exposure (area under the curve in poor metabolizers
in the presence of an inhibitor) could be predicted, and that
substrates susceptible to large increases in exposure could
be identified.
Existing clinical trials were identified by data mining the
literature. A theoretical approach was developed to predict
maximum exposure in poor metabolizers from studies in extensive
metabolizers treated with an inhibitor of the non-polymorphic
pathway. Maximum exposure was predicted in eleven instances
and the mean percentage difference between predicted and observed
was 11.9%. Substrates with a fraction of substrate dose metabolized
by the polymorphic enzyme (fmPOLY) higher than
75% are at greater risk of exhibiting maximum exposure ratios
of more than tenfold.
[Back to top]
Involvement of Cytoskeleton in AhR-Dependent CYP1A1
Expression
Z. Dvorák, R. Vrzal, J. Ulrichová, J.-M. Pascussi,
P. Maurel and
M. Modriansky
Cytochrome P450 (CYP) 1A1 attracts attention mainly because
of its role in production of carcinogenic reactive metabolites
from polycyclic aromatic hydrocarbons such as benzo[a]pyrene,
but recent developments indicate its apparent role in cell
cycle progression. Expression of the enzyme is subject to
regulation by aryl hydrocarbon receptor (AhR). It has been
shown that induction of CYP 1A1 in HepG2 cells and primary
rat hepatocytes by tetrachloro-p-dibenzodioxin (TCDD) is diminished
by colchicine and nocodazole. Both compounds decrease CYP1A1
mRNA, protein, and activity levels in HepG2 cells and mRNA
level in primary rat hepatocytes. Neither compound significantly
affected [3H]-TCDD binding to AhR, thus their effect
on AhR transcriptional activity proceeds via indirect
means. For colchicine and nocodazole are well-known microtubule
interfering agents, we also assessed their effect on microtubule
integrity in both cell types under investigation. Both compounds
disrupt cytoskeleton integrity with differential potency depending
on cell type. The observed suppression of AhR transcriptional
activity by colchicine and nocodazole can be associated with
G2/M cell cycle arrest in HepG2 cells, as demonstrated by
Myt1 protein hyperphosphorylation and FACS analysis. However,
in primary rat hepatocytes, cytoskeleton disruption is independent
of cell cycle while displaying the same influence on AhR-dependent
gene transcription. In our view, this is evidence in favor
of modulatory role of cytoskeleton in AhR-dependent expression.
[Back to top]
A Critical Evaluation of the Experimental Design of
Studies of Mechanism Based Enzyme Inhibition, with Implications
for In Vitro-In Vivo Extrapolation
F. Ghanbari, K. Rowland-Yeo, J.C. Bloomer, S.E. Clarke,
M.S. Lennard, G.T. Tucker and A. Rostami-Hodjegan
The published literature on mechanism based inhibition (MBI)
of CYPs was evaluated with respect to experimental design,
methodology and data analysis. Significant variation was apparent
in the dilution factor, ratio of preincubation to incubation
times and probe substrate concentrations used, and there were
some anomalies in the estimation of associated kinetic parameters
(kinact, KI, r). The impact of the application of inaccurate
values of kinact and KI when extrapolating to the extent of
inhibition in vivo is likely to be greatest for those
compounds of intermediate inhibitory potency, but this also
depends on the fraction of the net clearance of substrate
subject to MBI and the pre-systemic and systemic exposure
to the inhibitor. For potent inhibitors, the experimental
procedure is unlikely to have a material influence on the
maximum inhibition. Nevertheless, the bias in the values of
the kinetic parameters may influence the time for recovery
of enzyme activity following re-synthesis of the enzyme. Careful
attention to the design of in vitro experiments to
obtain accurate kinetic parameters is necessary for a reliable
prediction of different aspects of the in vivo consequences
of MBI. The review calls for experimental studies to quantify
the impact of study design in studies of MBI, with a view
to better harmonisation of protocols.
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