Drug
Metabolism Letters
ISSN: 1872-3128
Drug Metabolism Letters
Volume 1, Number 3, August 2007
Contents
Antioxidants Suppress Th1-Type Immune Response
In Vitro Pp. 166-171
K. Schroecksnadel, B. Fischer, H. Schennach, G. Weiss
and D. Fuchs
[Abstract]
Effect of Neurotransmitters on NADPH-Cytochrome P450
Reductase In Vitro Activity Pp. 172-175
G. Gervasini, C. Martinez, J. Benitez and J.A.G. Agúndez
[Abstract]
cAMP-Mediated Regulation of CYP Enzymes and Its Application
in Chemotherapy Pp. 176-178
Y. Ishikawa, S. Suzuki, K. Otsu, C. Ulucan, K. Iwatsubo
and H. Eguchi
[Abstract]
Application of In-Line Liquid Chromatography-Accurate
Radioisotope Counting-Mass Spectrometry (LC-ARC-MS) to Evaluate
Metabolic Profile of [3H]-Mefenamic
Acid in Rat Plasma Pp. 179-188
W. Lam, C.-M. Loi, J. Atherton, W. Stolle, J. Easter and
A. Mutlib
[Abstract]
PET-Evaluated Transport of [11C]Hydroxyurea
Across the Rat Blood-Brain Barrier-Lack of Influence of Cyclosporin
and Probenecid Pp. 189-194
S. Syvänen, J. Barletta, G. Blomquist, B.
Långström and M. Bergström
[Abstract]
An Indirect Screen for Brain Uptake of 1,2-Diarylethane
Melanocortin 4 Receptor Antagonists in Rats Pp. 195-198
W. Yin, L.-S. Gan, J.-T. Wu, S.K. Balani, H. Yang and
F.W. Lee
[Abstract]
Effect of Recombinant Human Thymosin-α1,
an Immuno Modulating Peptide with 28 Amino Acids, on the Activity
of Cytochrome P450s Pp. 199-204
B. Wang, F. He, Y. Lin, M. Huang and S.-F. Zhou
[Abstract]
Involvement of P-glycoprotein and Multidrug Resistance
Associated Protein 1 in the Transport of Tanshinone IIB, a
Primary Active Diterpenoid Quinone from the Roots of Salvia
miltiorrhiza, Across the Blood-Brain Barrier Pp.
205-217
Z.-W. Zhou, X. Chen, J. Liang, X.-Y. Yu, J.-Y.
Wen and S.-F. Zhou
[Abstract]
Effects of Various Promoter Derived Sequences
on the Cleavage Kinetic of an Hammerhead Ribozyme Directed
Against Cyclin E1 mRNA Pp. 218-225
J. Platz, M. Grassi, A. Kuhn, R. Kandolf, G.
Racchi, R. Farra, B. Dapas, N.A. Pascotto, C. Giansante, N.
Fiotti G. Guarnieri and G. Grassi
[Abstract]
Application of Liquid Chromatography-Accelerator Mass
Spectrometry (LC-AMS) to Evaluate the Metabolic Profiles of
a Drug Candidate in Human Urine and Plasma Pp. 226-231
C. Prakash, C.L. Shaffer, L.M. Tremaine, R.G. Liberman,
P.L. Skipper, J. Flarakos and S.R. Tannenbaum
[Abstract]
Studies of Bioactivity, Conformation and Pharmacokinetic
Profiles of Site-Specific PEGylated Thymosin Alpha 1 Derivatives
Pp. 232-240
J. Qie, J. Ma, L. Wang, X. Xu, J. Zheng, S. Dong,
J. Xie, H. Sun, W. Zhou, C. Qi, X. Zhao, Y. Zhang and K. Liu
[Abstract]
Abstracts
[Back to top]
Antioxidants Suppress Th1-Type Immune Response
In Vitro
K. Schroecksnadel, B. Fischer, H. Schennach,
G. Weiss and D. Fuchs
Inflammation is a hallmark of various diseases like infections,
autoimmune syndromes, cardiovascular and neurodegenerative
disorders, and cancer. Thereby, Th1-type cytokine interferon-γ
is a central pro-inflammatory mediator. Antioxidant compounds
counteract the activation of T-cells and macrophages. This
anti-inflammatory activity could be important for the development
of immunotolerance and for treatment of above-mentioned disorders.
[Back to top]
Effect of Neurotransmitters on NADPH-Cytochrome P450
Reductase In Vitro Activity
G. Gervasini, C. Martinez, J. Benitez and J.A.G. Agúndez
Three neurotransmitters, namely adrenaline, serotonin and
tryptamine inhibit the in vitro activity of several cytochrome
P450 (CYP) isozymes (CYP1A2, CYP2C9, CYP2D6 and CYP3A). In
order to test whether this effect is related to inhibition
of the CYP-coupled NADPH reductase activity, we assayed the
potential inhibitory effect of these neurotransmitters and
their main metabolites on the NADPH reductase activity. Of
the five compounds analyzed: tryptamine, tryptophol, serotonin,
5-hydroxytryptamine and adrenaline, only adrenaline significantly
decreased NADPH reductase activity at the fixed concentration
of 500 μM. However, the effect became negligible when
adrenaline concentration was decreased to 100 μM: whereas
a high inhibitory effect was observed in CYP2D6, CYP2C9 and
CYP3A4 enzyme activities, the NADPH reductase activity remains
unchanged.
This study indicates that the effect of these endogenous neurotransmitters
on CYP enzymes is not related to changes in the reductase
activity. In the light of these findings further studies on
the inhibitory effect of these neurotransmitters on CYP enzymes
can be designed ruling out the modulation of the coupled NADPH
reductase activity as a confounding factor.
[Back to top]
cAMP-Mediated Regulation of CYP Enzymes and Its Application
in Chemotherapy
Y. Ishikawa, S. Suzuki, K. Otsu, C. Ulucan, K. Iwatsubo
and H. Eguchi
Certain anti-cancer prodrugs are subject to cytochrome P450
(CYP)-mediated metabolism and become more active. Because
CYP activity may be regulated by phosphorylation via
adenylyl cyclase/protein kinase A, selective adenylyl cyclase
subtype activators may be utilized in future chemotherapy
to regulate CYP activity as a switch in a tumor tissue-specific
manner.
[Back to top]
Application of In-Line Liquid Chromatography-Accurate
Radioisotope Counting-Mass Spectrometry (LC-ARC-MS) to Evaluate
Metabolic Profile of [3H]-Mefenamic
Acid in Rat Plasma
W. Lam, C.-M. Loi, J. Atherton, W. Stolle, J. Easter and
A. Mutlib
Profiling of rat plasma using a highly sensitive LC-ARC-MS
technique showed that [3H]
mefenamic acid was metabolized to several products, including
a sulfate conjugate and a hydroxylated analogue as major metabolites.
This technique of detecting low levels of radioactivity in
plasma was superior to previously used methods, such as β-RAM
detectors.
[Back to top]
PET-Evaluated Transport of [11C]Hydroxyurea
Across the Rat Blood-Brain Barrier-Lack of Influence of Cyclosporin
and Probenecid
S. Syvänen, J. Barletta, G. Blomquist, B.
Långström and M. Bergström
FThe transport of hydroxyurea, a ribonucleoside reductase
inhibitor, over biological membranes is slow and it has therefore
been suggested that the substance could interact with an active
efflux transporter. The transport of [11C]hydroxyurea
into the rat brain was therefore studied after administration
of the multidrug resistance protein inhibitor probenecid (50
and 150 mg/kg), the P-glycoprotein inhibitor cyclosporin A
(25 mg/kg), hydroxyurea (50, 150 and 450 mg/kg) and mannitol
(25%). None of the intervention drugs affected the brain uptake
of [11C]hydroxyurea. The
brain-to-plasma concentration ratios (KP),
with or without intervention drug, were in the range 0.12-0.25
after 60 min of [11C]hydroxyurea
infusion. [110C]Verapamil,
a P-glycoprotein substrate with low brain penetration, was
used to study the ability of hydroxyurea to inhibit P-glycoprotein.
Administration of hydroxyurea (150 and 450 mg/kg) did not
increase brain concentrations of [11C]verapamil.
It is therefore unlikely that hydroxyurea is a substrate for
or an inhibitor of P-glycoprotein or a substrate for a probenecid
sensitive transport system. The low brain concentrations may
instead be the result of slow uptake due to the hydrophilic
nature of hydroxyurea.
[Back to top]
An Indirect Screen for Brain Uptake of 1,2-Diarylethane
Melanocortin 4 Receptor Antagonists in Rats
W. Yin, L.-S. Gan, J.-T. Wu, S.K. Balani, H. Yang and
F.W. Lee
Antagonism of the melanocortin 4 receptor (MC4R) has been
proposed as a therapeutic intervention for the prevention
of lean body mass waste, as in cachexia. Pharmacokinetic profiles
of substituted 1,2-diarylethane MC4R antagonists were determined
in rats after a single intravenous (IV) administration at
1 mg/kg. Brain and plasma concentrations of these compounds
were determined at 1 and 4 hours after an oral dose at 10
mg/kg, since oral administration is the intended clinical
dosing route and the pharmacological target is the central
nervous system. The brain to plasma concentration ratios (0.10
– 50) after oral dosing correlated well with Vdss
(2.21 to 81.4 L/kg; R2=0.810)
determined after IV administration. A good correlation was
also observed between the brain AUC0-4hr (119
– 18400 nM*hr) and Vdss
(R2=0.981). Thus, further
screening and ranking of substituted 1,2-diarylethanes for
their brain uptakes could be carried out more efficiently
via the simple and indirect Vdss
screen after intravenous administration in rats.
[Back to top]
Effect of Recombinant Human Thymosin-α1,
an Immuno-Modulating Peptide with 28 Amino Acids, on the Activity
of Cytochrome P450s
B. Wang, F. He, Y. Lin, M. Huang and S.-F. Zhou
There is an increasing application of protein/peptide drugs
in the treatment of various diseases such as cancer and autoimmune
diseases in clinical settings. However, data is scant on the
potential for modulation of cytochrome P450s (CYPs) by these
protein/peptide drugs. In this study, we examined the effects
of recombinant human thymosin-α1
(rh-Tα1)
on hepatic cytochrome P450 (CYP) enzyme activity in rats in
vitro and in vivo. For the in vitro
experiments, rh-Tα1
was incubated with the probe drugs and the liver microsomes
from rats, while rh-Tα1
was administered to rats subcutaneously at 150, 300, or 600
µg/kg daily for two weeks in in vivo studies.
The activities of six rat hepatic CYP enzymes, namely CYP1A2,
CYP2C6, CYP2C11, CYP2D2, CYP2E1, and CYP3A1/2, were determined
by a cocktail of probe drugs including phenacetin (O-deethylation),
tolbutamide (4-hydrolylation), omeprazole (5-hydroxylation),
dextromethorphan (O-demethylation), chlorzoxazone
(6-hydroxylation), and nifedipine (N-dehydrogenation),
respectively. Co-incubation of rh-Tα1
at the concentration of 20 and 50 µmol/l with the liver
microsomes significantly inhibited CYP2E1 activity, whereas
there was no significant effect on the activities of CYP1A2,
CYP2C6, CYP2C11, CYP2D2, and CYP3A1/2. As to in vivo
studies, treatment of rh-Tα1
at either dosage did not significantly alter the liver weight.
However, an ex vivo study demonstrated that the activity
of rat hepatic CYP2E1 was significantly increased by pretreatment
of rh-Tα1
at the three doses for two weeks, and the activities of CYP1A2,
CYP2D2, and CYP3A1/2 were also significantly increased in
rats pretreated with rh-Tα1
at 600 µg/kg. These data indicate that rh-Tα1
can modulate the activities of major rat CYP isoforms, and
further studies are needed to investigate its effect on human
CYP activities and the potential for causing drug interactions.
[Back to top]
Involvement of P-glycoprotein and Multidrug Resistance
Associated Protein 1 in the Transport of Tanshinone IIB, a
Primary Active Diterpenoid Quinone from the Roots of Salvia
miltiorrhiza, Across the Blood-Brain Barrier
Z.-W. Zhou, X. Chen, J. Liang, X.-Y. Yu, J.-Y.
Wen and S.-F. Zhou
Tanshinone IIB (TSB) is a major constituent of Salvia
miltiorrhiza, which is widely used in treatment of cardiovascular
and central nervous system (CNS) diseases such as coronary
heart disease and stroke. This study aimed to investigate
the role of various drug transporters in the brain penetration
of TSB using several in vitro and in vivo
mouse and rat models. The uptake and efflux of TSB in rat
primary microvascular endothelial cells (RBMVECs) were ATPdependent
and significantly altered in the presence of a P-glycoprotein
(P-gp) or multidrug resistance associated protein (Mrp1/2)
inhibitor. A polarized transport of TSB was found in RBMVEC
monolayers with facilitated efflux from the abluminal to luminal
side. Addition of a P-gp inhibitor (e.g. verapamil) in both
abluminal and luminal sides attenuated the polarized transport.
In an in situ rat brain perfusion model, TSB crossed
the blood-brain barrier (BBB) and bloodcerebrospinal fluid
barrier at a greater rate than that for sucrose, and the brain
penetration was increased in the presence of a P-gp or Mrp1/2
inhibitor. The brain levels of TSB were only about 30% of
that in the plasma and it could be increased to up to 72%
of plasma levels when verapamil, quinidine, or probenecid
was co-administered in rats. The entry of TSB to CNS increased
by 67 97% in rats subjected to middle cerebral artery occlusion
or treatment with the neurotoxin, quinolinic acid, compared
to normal rats. Furthermore, The brain levels of TSB in
mdr1a(-/-) and mrp1(-/-) mice were 28- to 2.6-fold
higher than those in the wild-type mice. TSB has limited brain
penetration through the BBB due to the contribution of P gp
and to a lesser extent of Mrp1 in rodents. Further studies
are needed to confirm whether these corresponding transporters
in humans are involved in limiting the penetration of TSB
across the BBB and the clinical relevance.
[Back to top]
Effects of Various Promoter Derived Sequences on the
Cleavage Kinetic of an Hammerhead Ribozyme Directed Against
Cyclin E1 mRNA
J. Platz, M. Grassi, A. Kuhn, R. Kandolf, G.
Racchi, R. Farra, B. Dapas, N.A. Pascotto, C. Giansante, N.
Fiotti G. Guarnieri and G. Grassi
Hammerhead ribozymes (HRz), catalytic RNA molecules capable
of inducing the site-specific cleavage of a phosphodiester
bond within an RNA molecule, are typically introduced into
target cells by specific constructs (such as viral vectors)
able to drive their expression from defined expression cassettes
(promoter). This strategy implies the presence of promoter-derived
sequences bound to the hammerhead ribozyme structure, a fact
which can unpredictably affect HRz cleavage efficiency and
eventually the biological effect.
We explored the effects of promoter-derived sequences on the
cleavage kinetics of an HRz targeted against a relevant cell
cycle regulator, i.e. cyclin E1, implicated in the pathogenesis
of several human diseases including in-stent restenosis and
hepatocellular carcinoma. Sequences derived form the most
commonly used promoters (CMV, T7, Pol I and Pol III promoters)
were added to the minimal HRz structure and their effects
on the cleavage kinetic constants kcat
and Kmevaluated in vitro
under single turn-over conditions, using a mathematical model
we recently developed. The different promoter derived sequences
variably affected HRz cleavage efficiency (kcat/Km)
with those derived from the pol III and from a truncated form
of T7 promoter (T7-S), impairing maximally and minimally kcat/Km,
respectively. Additionally, the extra sequences tend to increase
km and to reduce kcat.
The extent of this effect depends both on the secondary RNA
structure and on the length of the added sequences.
In conclusion, these data, together with further work in cultured
cells, can lead to the selection of optimal expression cassettes
thus contributing to improve HRz efficacy, bringing these
molecules closer to practical applications.
[Back to top]
Application of Liquid Chromatography-Accelerator Mass
Spectrometry (LC-AMS) to Evaluate the Metabolic Profiles of
a Drug Candidate in Human Urine and Plasma
C. Prakash, C.L. Shaffer, L.M. Tremaine, R.G. Liberman,
P.L. Skipper, J. Flarakos and S.R. Tannenbaum
Metabolite profiling of 100- and 1,000-fold diluted urine
and plasma samples from a conventional radiolabeled human
ADME study is described using a highly sensitive LC-AMS technique.
The concentration of radioactivity and the metabolic profiles
in urine and plasma determined using this technique were similar
to those employing standard off-line (i.e. LSC) or in-line
(i.e. β-RAM
or LC-ARC dynamic-flow) radioactivity monitoring techniques.
The results indicate that at a simulated ca. 100 nCi clinical
dose, plasma and urine concentrations of 14C,
as well as their metabolic profiles, may be determined routinely
by LC-AMS. This approach opens the possibility of using LC-AMS
for both the high-throughput quantitation of biological samples
and the generation of high-resolution chromatographic profiles
of complex mixtures at a lower cost than current AMS analyses
that require the conversion of sample carbon to graphite,
a laborious and time consuming process.
[Back to top]
Studies of Bioactivity, Conformation and Pharmacokinetic
Profiles of Site-Specific PEGylated Thymosin Alpha 1 Derivatives
J. Qie, J. Ma, L. Wang, X. Xu, J. Zheng, S. Dong,
J. Xie, H. Sun, W. Zhou, C. Qi, X. Zhao, Y. Zhang and K. Liu
Site-specific mono-PEGylations were performed in different
conformational regions of Thymosin alpha 1 (Tα1)
by introducing one cysteine residue into the chosen site and
coupling with thiol-specific mPEG-MAL reagent. Results demonstrated
that PEGylated sites and regions influenced the conformations
and pharmacokinetic profiles of the peptide greatly with following
order: α-helix,
β-turn,
random coil and terminals, but little on the immunoactivity.
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