Current
Drug Metabolism
ISSN: 1389-2002
Current Drug Metabolism
Volume 6, Number 5, October 2005
Contents
Effect of Omeprazole on the Hydroxylation of Warfarin Enantiomers in Human: In-Vitro Studies with Liver
Microsomes and cDNA-Expressed Cytochrome P450
Isozymes Pp.399
Q. Zhou, S. Zhou and E. Chan
[Abstract]
Cytochrome P450 Enzymes Mechanism Based Inhibitors: Common
Sub-Structures and Reactivity Pp.413
E. Fontana, P.M. Dansette and S.M. Poli
[Abstract]
Coupling of Conjugating Enzymes and Efflux Transporters: Impact
on Bioavailability and Drug Interactions Pp.455
E.J. Jeong, X. Liu, X. Jia, J. Chen and M. Hu
[Abstract]
CYP1A1 Is a Major Enzyme Responsible for the Metabolism of
Granisetron in Human Liver Microsomes Pp.469
H. Nakamura, N. Ariyoshi, K. Okada, H. Nakasa, K.
Nakazawa and M. Kitada
[Abstract]
Effect of Some Biologically Interesting Substituted Tetrahydro-1,4-Oxazines
on Drug Metabolising Enzymes and on Inflammation Pp.481
E.A. Rekka, A.P. Kourounakis, N. Avramidis and P.N.
Kourounakis
[Abstract]
Metabolism and Disposition of the Antiviral Nucleoside Analogue
AM365 in the Isolated Perfused Rat Liver Pp.487
J. Wang, R.L. Nation, A.M. Evans, S. Cox and D. Shackleford
[Abstract]
Mechanisms of Male Infertility: Role of Antioxidants Pp.495
S.A. Sheweita, A.M. Tilmisany and H. Al-Sawaf
[Abstract]
Utility of Recombinant Cytochrome P450 Enzymes: A Drug Metabolism
Perspective Pp. 503
W. Tang, R.W. Wang and Anthony Y.H. Lu
[Abstract]
Abstracts
[Back to top]
Effect of Omeprazole on the Hydroxylation of Warfarin Enantiomers in Human: In-Vitro Studies with Liver
Microsomes and cDNA-Expressed Cytochrome P450
Isozymes
Q. Zhou, S. Zhou and E. Chan
Clinically observed warfarin-omeprazole interaction has been
found to be associated with the inhibition of R-warfarin hydroxylation
by omeprazole. The present study was conducted in human liver
microsomes and cDNA-expressed cytochrome P450s to assess the
inhibitory potential of omeprazole on the hydroxylation of
warfarin enantiomers, and to identify the cytochrome P450
isozymes involved in the inhibition of hydroxylation of warfarin
enantiomers by omeprazole, and to evaluate the extent to which
the in vitro data is predictive of the actual pharmacokinetic
interaction between warfarin and omeprazole observed in
vivo.
Omeprazole inhibited the formation of R-6-, R-7- and S-7-hydroxywarfarin
with the Ki values of 40, 22 and 116 μM,
re-spectively. Its inhibitory effect was selective towards
R-warfarin. Further study conducted in cDNA-expressed cyto-chrome
P450s (CYPs) demonstrates that the inhibition of the in-vitro
biotransformation of warfarin enantiomers by omeprazole is
attributed to its inhibitory effect on the activities of CYP1A2,
CYP3A4, CYP2C9 and CYP2C19.
The extent of the in vivo warfarin-omeprazole interaction
was underestimated as based on the Ki values obtained
from the in-vitro inhibition study, suggesting an
underestimation of the effective concentration of the inhibitor
at the site of interaction or some other mechanisms involved
in the drug interaction between warfarin and omeprazole.
[Back to top]
Cytochrome P450 Enzymes Mechanism Based Inhibitors: Common
Sub-Structures and Reactivity
E. Fontana, P.M. Dansette and S.M. Poli
The inhibition of human cytochrome P450s (CYPs) is one of
the most common mechanisms which can lead to drug-drug interactions.
The inhibition of CYPs can be reversible (competitive or non-competitive)
or irreversible. Irreversible inhibition usually derives from
activation of a drug by CYPs into a reactive metabolite, which
tightly binds to the enzyme active site, leading to a long
lasting inactivation. This process is called “mechanism
based inhibition” or “suicide inhibition”.
The irreversible inactivation usually implies the formation
of a covalent bond between the metabolite and the enzyme,
which can lead to hapten formation and can in some cases trigger
an autoimmune-response.
For these reasons it is of utmost importance to study the
mechanism of the CYP inhibition of new potential drugs as
early as possible during the drug discovery process.
The literature on CYPs is vast and covers numerous aspects
of their biology and biochemistry, however to our knowledge
there is no general and systematic review focusing on mechanism-based
inhibitors; we have reviewed the literature and compiled all
the available data on chemical entities, which are known to
be CYP suicide inhibitors. Each compound is reported together
with its chemical structure, the CYP isoform and the parameters
describing the inactivation. Literature references are reported
together with their PMID (PubMed ID number) to allow a fast
retrieval of the papers.
This review offers a quick reference to help predict liabilities
of new chemical entities without carrying out extensive in
vitro work, and will hopefully help in designing safer
drugs.
[Back to top]
Coupling of Conjugating Enzymes and Efflux Transporters:
Impact on Bioavailability and Drug Interactions
E.J. Jeong, X. Liu, X. Jia, J. Chen and M. Hu
Conjugating enzymes are traditionally recognized as one of
the major biological barriers to the entry of xeno-biotics/drugs
into systemic circulation and represent one of the main pathways
for their elimination. Similar to drugs that undergo extensive
phase I metabolism, drugs that undergo extensive conjugation
have poor bioavailability and are more prone to metabolism-based
drug interactions. Previously, enterohepatic recycling is
used to explain why certain xenobiotics have half-lives that
are much longer than expected from intravenous injection studies.
In addition, changes in expression levels of metabolic enzymes
due to chemical induction or suppression are often recognized
as the source of drug interaction or toxicity of pollutants
and carcinogens. These traditional approaches, whereas yielding
highly valuable information, fail to recognize the fact that
many conjugates (especially hydrophilic ones) cannot permeate
the cell membrane. In the present review, we will focus on
the coupling process that involves both conjugating enzymes
and efflux transporters. We will briefly review conjugating
enzymes capable of producing highly hydrophilic metabolic
products. The other focus of this review is on various transporters
capable of moving negatively charged hydrophilic conjugates
across the cellular membrane. Evidence will support the hypothesis
that efficient coupling of the conjugating enzymes and efflux
transporters enables enterohepatic recycling and enteric recycling
processes. Termed as a “revolving door” theory,
the hypothesis focuses on the role played by efflux transporter
capable of modulating the cellular excretion of hydrophilic
metabolites. Coupling process in intestine, liver and kidney
will be discussed with an emphasis on the intestinal coupling
process, since we have just begun to understand it. Biological
consequence and new insights into how coupling process can
impact bioavailability of xenobiotics, biological functions
of drugs and carcinogens, and drug interactions will be discussed.
[Back to top]
CYP1A1 Is a Major Enzyme Responsible for the Metabolism
of Granisetron in Human Liver Microsomes
H. Nakamura, N. Ariyoshi, K. Okada, H. Nakasa, K.
Nakazawa and M. Kitada
Granisetron, a potent 5-HT3 receptor antagonist,
has been reported to be mainly metabolized to 7-hydroxygranisetron
and a lesser extent to 9’-desmethylgranisetron in humans.
A previous study indicated that cytochrome P450 (CYP)3A4 is
a major catalyst of 9’-demethylation, although the major
CYP isoform(s) responsible for 7-hydroxylation are unknown.
To clarify granisetron 7-hydroxylase, the in vitro
metabolism of granisetron using expressed human CYPs and human
liver microsomes was investigated. 7-Hydroxygranisetron was
produced almost exclusively by CYP1A1, while, apparently,
9’-desmethylgranisetron was preferentially produced
by CYP3A4. Marked inter-individual differences in the ratio
of the formation of 7-hydroxygranisetron and 9'-desmethylgranisetron
in human liver microsomes was observed. Granisetron 7-hydroxylase
activity was strongly correlated with benzo[a]pyrene
3-hydroxylase activity (p<0.0001), but not with
testosterone 6β-hydroxylase activity in human liver microsomes.
Furthermore, an anti-human CYP1A1 antibody completely inhibited
7-hydroxylation in human liver microsomes, however, the reaction
was not inhibited at all by an anti-CYP3A4 antibody. On the
other hand, granisetron 9'-demethylase activity correlated
significantly not only with testosterone 6β-hydroxylase
activity (p<0.0001) but also with benzo[a]pyrene
3-hydroxylase activity (p<0.01). Consistent with
this, both the anti-CYP1A1 and anti-human CYP3A4 antibodies
inhibited the 9'-demethylase activity. These data indicate
that CYP1A1 is a major enzyme responsible for the metabolism
of granisetron via a main 7-hydroxylation pathway
and an alternative 9’-demethylation route. This is the
first report demonstrating the substantial contribution of
CYP1A1 to the metabolism of a drug, although its role in the
metabolism of environmental compounds is well established.
[Back to top]
Effect of Some Biologically Interesting Substituted Tetrahydro-1,4-Oxazines
on Drug Metabolising Enzymes and on Inflammation
E.A. Rekka, A.P. Kourounakis, N. Avramidis and P.N.
Kourounakis
The effect on hepatic drug metabolising enzymes was evaluated
for three representative structures (1, 2 and 3) that were
selected from a series of substituted oxazine derivatives
designed to possess particular pharmacological properties
such as analgesic, antioxidant and hypolipidemic activity.
In addition, since xenobiotic metabolism, reactive oxygen
and nitrogen species, atherosclerosis and inflammation are
interrelated and mutually affected, the effects of (2) and
(3) on acute inflammation in vivo and lipoxygenase
activity in vitro were also investigated. It was
found that treatment of rats with (1) caused induction of
cytochrome P450, enhancement of the metabolism of aminopyrine
in vitro and of zoxazolamine and hexobarbital in
vivo. Compound (2) appeared to induce particularly erythromycin
N-demethylation, while (3), a nitric ester, reduced the catalytically
active cytochrome P450, although it increased the metabolism
of specific cytochrome P450 substrates, i.e. 4-nitrophenol
and erythromycin. Compounds (2) and (3), with strong hypolipidemic
and antioxidant properties, reduced acute inflammatory response
in two inflammation models and inhibited li-poxygenase activity
in vitro. These results are helpful in optimising
the biological profile as well as the potential applications
of substituted oxazines
[Back to top]
Metabolism and Disposition of the Antiviral Nucleoside
Analogue AM365 in the Isolated Perfused Rat Liver
J. Wang, R.L. Nation, A.M. Evans, S. Cox and D. Shackleford
The present study was designed to investigate the hepatic
disposition of the prodrug AM365 and the generated antiviral
guanosine analogue, AM188 in the isolated perfused rat liver
(IPL). The livers of rats (n = 12) were isolated and perfused
with Krebs-Henseleit pH 7.4 buffer to which AM365 was added
as a bolus to achieve an initial perfusate concentration of
22.4 µmol/L. During the 120-min period after administration
of AM365, bile was collected in 10-min intervals and perfusate
was collected at the mid-point of these intervals. Concentrations
of AM365 and AM188 in perfusate and bile were quantified by
HPLC. Following administration of AM365, its concentration
in perfusate declined and the concentration of AM188 increased;
the sum of the molar concentrations remained constant. The
clearance and hepatic extraction ratio of AM365 were 3.3 ±
2.4 mL/min and 0.110 ± 0.079, respectively. The cumulative
amount of AM365 excreted in bile during the 120-min perfusion
period was approximately 0.21% of the bolus dose, and 0.36%
of the total amount of AM365 cleared by the liver during the
period. The cumulative amount of AM188 excreted in bile was
about 0.48% of the total amount of AM188 formed during the
perfusion period. In conclusion, AM365 was metabolised to
AM188, which appeared to be the only metabolite and was not
further biotransformed. The biliary excretion of AM365 and
AM188 was negligible.
[Back to top]
Mechanisms of Male Infertility: Role of Antioxidants
S.A. Sheweita, A.M. Tilmisany and H. Al-Sawaf
Defective sperm function is the most common cause of infertility,
and until recently, was difficult to evaluate and treat. Mammalian
spermatozoa membranes are rich in poly unsaturated fatty acids
and are sensitive to oxygen induced damage mediated by lipid
peroxidation. Hence, free radicals and reactive oxygen species
[ROS] are associated with oxidative stress and are likely
to play a number of significant and diverse roles in reproduction.
The excessive gen-eration of reactive oxygen species by abnormal
spermatozoa and by contaminating leukocytes [leukocytospermia]
has been identified as one of the few defined etiologies for
male infertility. Moreover, environmental factors, such as
pesti-cides, exogenous estrogens, and heavy metals may negatively
impact spermatogenesis since male sperm counts were de-clined.
In addition, aging is also likely to further induce oxidative
stress. Limited endogenous mechanisms exist to re-verse these
damages. In a normal situation, the seminal plasma contains
antioxidant mechanisms which are likely to quench these ROS
and protect against any likely damage to spermatozoa. However,
during genitourinary infec-tion/inflammation these antioxidant
mechanisms may downplay and create a situation called oxidative
stress. Assess-ment of such oxidative stress status [OSS]
may help in the medical treatment of male infertility by suitable
antioxidants. The cellular damage in the semen is a result
of an improper balance between ROS generation and scavenging
activities. Therefore, numerous antioxidants such as vitamin
C, vitamin E, glutathione, and coenzyme Q10, have proven beneficial
effects in treating male infertility. A multi-faceted therapeutic
approach to improve male fertility involves identifying harmful
environmental and occupational risk factors, while correcting
underlying nutritional imbalances to encourage optimal sperm
production and function.
[Back to top]
Utility of Recombinant Cytochrome P450 Enzymes: A Drug
Metabolism Perspective
W. Tang, R.W. Wang and Anthony Y.H. Lu
An important role of human cytochrome P450s (P450s) has been
well recognized in the area of drug metabolism and pharmacokinetics.
It has become possible in recent years to express catalytically
active forms of these enzymes in various host systems. The
resulting recombinant human P450s are either purified for
studies of protein structure and the mechanism of catalysis
or isolated in microsomal forms to serve the purposes of P450
phenotyping, metabolic stability screening and inhibitory
potential evaluation. Intact mammalian cells expressing human
enzymes may also be used to test the mutagenic and toxicity
potential of drug candidates. The issue remains, however,
that the data derived from re-combinant P450s are not always
consistent with those generated from human tissue preparations.
The aim of this com-munication is to discuss applications
of recombinant P450s in the drug discovery and development
setting, with an em-phasis on comparison of recombinant and
human liver microsomal systems.
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