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Current
Drug Metabolism
ISSN: 1389-2002
Current Drug Metabolism
Volume 7, Number 4, May 2006
Contents
Cytochrome P450 Expression in the Liver of Food Producing
Animals Pp. 335-348
C. Ioannides
[Abstract]
Evolution and Function of the NR1I Nuclear Hormone Receptor
Subfamily (VDR, PXR, and CAR) with Respect to Metabolism of
Xenobiotics and Endogenous Compounds Pp. 349-365
E.J. Reschly and M.D. Krasowski
[Abstract]
Investigation of Exenatide Elimination and Its
In Vivo and In Vitro Degradation Pp.
367-374
K. Copley, K. McCowen, R. Hiles, L.L. Nielsen, A.
Young and D.G. Parkes
[Abstract]
Evaluation of 170 Xenobiotics as Transactivators of Human
Pregnane X Receptor (hPXR) and Correlation to Known CYP3A4
Drug Interactions Pp. 375-388
M. Sinz, S. Kim, Z. Zhu, T. Chen, M. Anthony, K. Dickinson
and A.D. Rodrigues
[Abstract]
In Vitro Monitoring Picogram Roxithromycin in Human
Urine Using Flow Injection Chemiluminescence Procedure Pp.
389-395
Z. Song, Y. Liu and X. Xie
[Abstract]
Induction of the Hepatic Cytochrome P450 2B Subfamily
by Xenobiotics: Research History, Evolutionary Aspect, Relation
to Tumorigenesis, and Mechanism Pp. 397-409
H. Yamada, Y. Ishii, M. Yamamoto and K. Oguri
[Abstract]
Functional Expression of Human Cytochrome P450 Enzymes
in Escherichia coli Pp. 411-429
C.-H. Yun, S.-K. Yim, D.-H. Kim and T. Ahn
[Abstract]
Pharmacokinetic Mechanisms for Reduced Toxicity of
Irinotecan by Coadministered Thalidomide Pp. 431-454
X.-X. Yang, Z.-P. Hu, S.Y. Chan, W. Duan, P.C.-L.
Ho, U.A. Boelsterli, K.-Y. Ng, E. Chan, J.-S. Bian, Y.-Z.
Chen, M. Huang and S.-F. Zhou
[Abstract]
Abstracts
[Back to top]
Cytochrome P450 Expression in the Liver of Food Producing
Animals
C. Ioannides
A number of enzyme systems participate in the metabolism
of chemicals, but undoubtedly the most important are the cytochromes
P450 (CYP). It is a versatile enzyme system, capable of metabolising
structurally diverse chemicals. To achieve this broad substrate
specificity it exists as a superfamily of enzymes, each family
being characterised by different substrate specificity; families
CYP1, CYP2 and CYP3 are responsible for the metabolism of
exogenous chemicals. Although our current knowledge of the
expression and function of cytochromes P450 in humans and
laboratory animals is extensive, little is known about this
enzyme system in food-producing animals, despite its dominant
role in the metabolism of veterinary drugs, and the crucial
role it plays in controlling the levels of drug and other
chemical residues in edible tissues and food products that
humans consume, a matter of major current concern. Most studies
dealing with the expression of cytochromes P450 in food-producing
animals utilised substrate probes defined in rats and humans
and/or antibodies raised to rat or human antigens. Such an
approach can prove misleading as it assumes that orthologue
proteins in other animals share the same substrate specificity
and, moreover, although antibodies raised to human or rat
antigens may recognise epitopes in other species, they do
not constitute unequivocal proof that the detected proteins
are structurally identical. Despite these drawbacks, there
is substantial experimental evidence that CYP1, CYP2 and CYP3
families are expressed in food-producing animals, but their
role in the metabolism of veterinary drugs and other xenobiotics
has not been addressed.
[Back to top]
Evolution and Function of the NR1I Nuclear Hormone Receptor
Subfamily (VDR, PXR, and CAR) with Respect to Metabolism of
Xenobiotics and Endogenous Compounds
E.J. Reschly and M.D. Krasowski
The NR1I subfamily of nuclear hormone receptors includes
the 1,25-(OH)2-vitamin
D3
receptor (VDR; NR1I1), pregnane X receptor (PXR; NR1I2), and
constitutive androstane receptor (CAR; NR1I3). PXR and VDR
are found in diverse vertebrates from fish to mammals while
CAR is restricted to mammals. Current evidence suggests that
the CAR gene arose from a duplication of an ancestral PXR
gene, and that PXR and VDR arose from duplication of an ancestral
gene, represented now by a single gene in the invertebrate
Ciona intestinalis. Aside from the high-affinity
effects of 1,25-(OH)2-vitamin
D3
on VDRs, the NR1I subfamily members are functionally united
by the ability to bind potentially toxic endogenous compounds
with low affinity and initiate changes in gene expression
that lead to enhanced metabolism and elimination (e.g., induction
of cytochrome P450 3A4 expression in humans). The detoxification
role of VDR seems limited to sensing high concentrations of
certain toxic bile salts, such as lithocholic acid, whereas
PXR and CAR have the ability to recognize structurally diverse
compounds. PXR and CAR show the highest degree of cross-species
variation in the ligand-binding domain of the entire vertebrate
nuclear hormone receptor superfamily, suggesting adaptation
to species-specific ligands. This review examines the insights
that phylogenetic and experimental studies provide into the
function of VDR, PXR, and CAR, and how the functions of these
receptors have expanded to evolutionary advantage in humans
and other animals.
[Back to top]
Investigation of Exenatide Elimination and Its In Vivo
and In Vitro Degradation
K. Copley, K. McCowen, R. Hiles, L.L. Nielsen, A.
Young and D.G. Parkes
Exenatide is a 39 amino acid incretin mimetic for the
treatment of type 2 diabetes, with glucoregulatory activity
similar to glucagon-like peptide-1 (GLP 1). Exenatide is a
poor substrate for the major route of GLP-1 degradation by
dipeptidyl peptidase-IV, and displays enhanced pharmacokinetics
and in vivo potency in rats relative to GLP-1. The
kidney appears to be the major route of exenatide elimination
in the rat. We further investigated the putative sites of
exenatide degradation and excretion, and identified primary
degradants. Plasma exenatide concentrations were elevated
and sustained in renalligated rats, when compared to sham-operated
controls. By contrast, exenatide elimination and degradation
was not affected in rat models of hepatic dysfunction. In
vitro, four primary cleavage sites after amino acids
(AA)-15, -21, -22 and -34 were identified when exenatide was
degraded by mouse kidney membranes. The primary cleavage sites
of exenatide degradation by rat kidney membranes were after
AA-14, -15, -21, and -22. In rabbit, monkey, and human, the
primary cleavage sites were after AA-21 and -22. Exenatide
was almost completely degraded into peptide fragments <
3 AA by the kidney membranes of the species tested.
The rates of exenatide degradation by rabbit, monkey and human
kidney membranes in vitro were at least 15-fold slower
than mouse and rat membranes. Exenatide (1-14), (1 15), (1-22),
and (23-39) were not active as either agonists or antagonists
to exenatide in vitro. Exenatide (15-39) and (16-39)
had moderate-to-weak antagonist activity compared with the
known antagonist, exenatide (9-39). In conclusion, the kidney
appears to be the primary route of elimination and degradation
of exenatide.
[Back to top]
Evaluation of 170 Xenobiotics as Transactivators of Human
Pregnane X Receptor (hPXR) and Correlation to Known CYP3A4
Drug Interactions
M. Sinz, S. Kim, Z. Zhu, T. Chen, M. Anthony, K. Dickinson
and A.D. Rodrigues
The human transcription factor pregnane X receptor (hPXR)
is a key regulator of enzyme expression, especially cytochrome
P450 3A4 (CYP3A4). Due to the prominence of CYP3A4 in the
elimination of many drugs, the development of high throughput
in vitro models to predict the effect of
drugs on CYP3A4 expression have increased. To better interpret
and predict potential drug-drug interactions due to CYP3A4
enzyme induction, we evaluated 170 xenobiotics in a hPXR transactivation
assay and compared these results to known clinical drug-drug
interactions. Of the 170 xenobiotics tested, 54% of them demonstrated
some level of hPXR transactivation. By taking into consideration
cell culture conditions (solubility, cytotoxicity, appropriate
drug concentration in media), as well as in vivo
pharmacokinetics (therapeutic plasma Cmax,
distribution, route of administration, dosing regimen, liver
exposure, potential to inhibit CYP3A4), the risk potential
of CYP3A4 enzyme induction for most compounds reduced dramatically.
By employing this overall interpretation strategy, the final
percentage of compounds predicted to significantly induce
CYP3A4 reduced to 5%, all of which are known to cause drug-drug
interactions. Also, this is the first report that identifies
several potent compounds that have the ability to transactivate
hPXR that previously have not been identified, such as terbinafine,
diclofenac, sildenafil, glimepiride, montelukast, and ticlopidine.
[Back to top]
In Vitro Monitoring Picogram Roxithromycin in
Human Urine Using Flow Injection Chemiluminescence Procedure
Z. Song, Y. Liu and X. Xie
A sensitive chemiluminescence method, based on the enhancive
effect of roxithromycin on the chemiluminescence reaction
between luminol and hydrogen peroxide in a flow injection
system, was proposed for the determination of roxithromycin.
The increment of chemiluminescence intensity was linear with
roxithromycin concentration in the range 1.0-1000 pg ml
-1 with the detection limit of 0.3 pg ml -1
(3σ ).
At a flow rate of 2.0 ml min -1,
a complete analytical process could be performed within 0.5
min, including sampling and washing, with a relative standard
deviation of less than 5%. The proposed procedure was applied
successfully in the monitoring of roxithromycin in human urine
without any pre-treatment procedures and it was found that
roxithromycin in urine reached its maximum after orally administrated
for two hours, presenting an excretive ratio of 4.6% in 12
h. With urinary excretion rate method, the total elimination
rate constant k and half-life time t1/2
of roxithomycin was calculated, which was 0.1831, 3.785 h.
[Back to top]
Induction of the Hepatic Cytochrome P450 2B Subfamily
by Xenobiotics: Research History, Evolutionary Aspect, Relation
to Tumorigenesis, and Mechanism
H. Yamada, Y. Ishii, M. Yamamoto and K. Oguri
The cytochrome P450 belonging to the CYP2B subfamily has
long been of great interest because it can be induced by xenobiotics.
While a well known diagnostic ligand-receptor theory explains
the induction of the CYP1A subfamily, the mechanism by which
xenobiotics induce the CYP2B subfamily is not fully understood.
Although the constitutive androstane receptor (CAR) undoubtedly
plays a crucial role in the induction, many questions regarding
the mechanism of CAR activation by xenobiotics have not yet
been answered. It is a puzzle that many structurally-unrelated
chemicals can increase the expression of the CYP2B subfamily.
This may support a mechanism(s) distinct from the signaling
induced by ligand-receptor binding. Indeed, phenobarbital,
a typical inducer, cannot associate with CAR. Thus, no one
is able to answer a fundamental question: what is the initial
target of xenobiotics to produce induced expression of CYP2B
enzymes? In this review, we survey the research history of
CYP2B induction, list the inducers reported so far, and discuss
the mechanism of induction including the target site of inducers.
[Back to top]
Functional Expression of Human Cytochrome P450 Enzymes
in Escherichia coli
C.-H. Yun, S.-K. Yim, D.-H. Kim and T. Ahn
Knowledge regarding cytochrome P450 (P450) is crucial to
the fields of drug therapy and drug development, as well as
in our understanding of the mechanisms underlying the metabolic
activation of potentially toxic and carcinogenic compounds.
Escherichia coli is the most extensively utilized
host in the production of recombinant human P450 enzymes.
However, the recovery of substantial yields of functionally
active P450 proteins remains problematic. Mammalian P450 protein
was first expressed in 1991, via the modification
of the N-terminal amino acid sequences in E. coli
cells. Since that time, a variety of strategies have been
established for the functional expression of recombinant P450s
in E. coli, including N-terminal modification, the
use of molecular chaperones, and culturing at lower temperatures.
In all cases, human P450 expressed in E. coli cells
has been shown to efficiently catalyze the oxidation of representative
substrates at efficient rates. These recombinant P450s are
applicable to studies which estimate the kinetic parameters
of drug oxidation, and have also been used to determine the
metabolic pathways of drugs and carcinogens exploited by human
P450s. Despite the potential of P450s in various pharmaceutical
and biotechnological fields, however, a host of substantial
challenges must be overcome before these enzymes can be routinely
utilized. Intrinsically, these enzymes are not very active,
and exhibit poor stability. In this review, we have described
current developments in the heterologous expression of human
P450 enzymes.
[Back to top]
Pharmacokinetic Mechanisms for Reduced Toxicity of
Irinotecan by Coadministered Thalidomide
X.-X. Yang, Z.-P. Hu, S.Y. Chan, W. Duan, P.C.-L.
Ho, U.A. Boelsterli, K.-Y. Ng, E. Chan, J.-S. Bian, Y.-Z.
Chen, M. Huang and S.-F. Zhou
The clinical use of irinotecan (CPT-11) is hindered by dose-limiting
diarrhea and myelosuppression. Recent clinical studies indicate
that thalidomide, a known tumor necrosis factor-α
inhibitor, ameliorated the toxicities induced by CPT-11. However,
the mechanisms for this are unknown. This study aimed to investigate
whether combination of thalidomide modulated the toxicities
of CPT-11 using a rat model and the possible role of the altered
pharmacokinetic component in the toxicity modulation using
in vitro models. The toxicity model was constructed
by treatment of healthy rats with CPT-11 at 60 mg/kg per day
by intravenous (i.v.) injection. Body weight, acute and delayed-onset
diarrhea, blood cell counts, and macroscopic and microscopic
intestinal damages were monitored in rats treated with CPT-11
alone or combined therapy with thalidomide at 100 mg/kg administered
by intraperitoneal (i.p.) injection. Single dose and 5-day
multiple-dose studies were conducted in rats to examine the
effects of concomitant thalidomide on the plasma pharmacokinetics
of CPT-11 and its major metabolites SN-38 and SN-38 glucuronide
(SN-38G). The effect of CPT-11 on thalidomide’s pharmacokinetics
was also checked. Rat liver microsomes and a rat hepatoma
cell line, H4-II-E cells, were used to study the in vitro
metabolic interactions between these two drugs. H4-II-E cells
were also used to investigate the effect of thalidomide and
its hydrolytic products on the transport of CPT-11 and SN-38.
In addition, the effect of thalidomide and its hydrolytic
products on rat plasma protein binding of CPT-11 and SN-38
was examined. Administration of CPT-11 by i.v. for 4 consecutive
days to rats induced significant body weight loss, decrease
in neutrophil and lymphocyte counts, severe acute- and delayed-onset
diarrhea, and intestinal damages. These toxicities were alleviated
when CPT-11 was combined with thalidomide. In both single-dose
and 5-day multiple-dose pharmacokinetic study, coadministered
thalidomide significantly increased the area under the plasma
concentration-time curve (AUC) of CPT-11, but the AUC and
elimination half-life (t1/2)
of SN-38 were significantly decreased. However, CPT-11 did
not significantly alter the pharma-cokinetics of thalidomide.
Thalidomide at 25 and 250 μM
and its hydrolytic products at a total concentration of 10
μM
had no significant effect on the plasma protein binding of
CPT-11 and SN-38, except for that thalidomide at 250 μM
caused a significant increase in the unbound fraction (fu)
of CPT-11 by 6.7% (P < 0.05). The hydrolytic products
of thalidomide (total concentration of 10 µM), but not
thalidomide, significantly decreased CPT-11 hydrolysis by
16% in rat liver microsomes (P < 0.01). The formation
of both SN-38 and SN-38G from CPT-11, SN-38 glucuronidation,
or intracellular accumulation of both CPT-11 and SN-38 in
H4-II-E cells followed Michaelis-Menten kinetics with the
one-binding site model being the best fit for the kinetic
data. Coincubation or 2-hr preincubation of thalidomide at
25 μM
and 250 μM
and its hydrolytic products at 10 μM
did not show any significant effects on CPT-11 hydrolysis
and SN-38 glucuronidation. However, preincubation of H4-II-E
cells with thalidomide (250 µM), its hydrolytic products
(total concentration of 10 µM), or phthaloyl glutamic
acid (one major thalidomide hydrolytic product, 10 µM)
significantly increased the intracellular accumulation of
SN-38, but not CPT-11 (P < 0.01). The dose-limiting
toxicities of CPT-11 were alleviated by combination with thalidomide
in rats and the pharmacokinetic modulation by thalidomide
may partially explain its antagonizing effects on the toxicities
of CPT-11. The hydrolytic products of thalidomide, instead
of the parental drug, modulated the hepatic hydrolysis of
CPT-11 and intracellular accumulation of SN-38, probably contributing
to the altered plasma pharmacokinetics of CPT-11 and SN-38.
Further studies are needed to explore the role of both pharmacokinetics
and pharmacodynamic components in the protective effect of
thalidomide against the toxicities of CPT-11. |
