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Current
Drug Metabolism
ISSN: 1389-2002
Current Drug Metabolism
Volume 8, Number 4, May 2007
Contents
Expression and Function of Cytochrome P450 in Brain
Drug Metabolism Pp. 297-306
R.P. Meyer, M. Gehlhaus, R. Knoth and B. Volk
[Abstract]
CYP-Mediated Clozapine Interactions: How Predictable
Are They? Pp. 307-313
M. Chetty and M. Murray
[Abstract]
The Non-ABC Drug Transporter RLIP76 (RALBP-1) Plays
a Major Role in the Mechanisms of Drug Resistance
Pp. 315-323
Y.C. Awasthi, R. Sharma, S. Yadav, S. Dwivedi, A. Sharma
and S. Awasthi
[Abstract]
Role of P-Glycoprotein in the Intestinal Absorption
of Tanshinone IIA, a Major Active Ingredient in the Root of
Salvia miltiorrhiza Bunge Pp. 325-340
X.-Y. Yu, S.-G. Lin, Z.-W. Zhou, X. Chen, J. Liang, P.-Q.
Liu, W. Duan, B. Chowbay, J.-Y. Wen, C.-G. Li and S.-F. Zhou
[Abstract]
Evaluation of Drug-Transporter Interactions Using
In Vitro and In Vivo Models Pp.
341-363
C.Q. Xia, M.N. Milton and L.-S. Gan
[Abstract]
Transport of Cryptotanshinone, a Major Active Triterpenoid
in Salvia Miltiorrhiza Bunge Widely Used in the Treatment
of Stroke and Alzheimer’s Disease, Across the Blood-Brain
Barrier Pp. 365-377
X.-Y. Yu, S.-G. Lin, X. Chen, Z.-W. Zhou, J. Liang, W.
Duan, B. Chowbay, J.-Y. Wen, E. Chan, J. Cao, C.-G. Li and
S.-F. Zhou
[Abstract]
A Pharmacokinetic Interaction Between Clarithromycin
and Sirolimus in Kidney Transplant Recipient Pp.
379-381
D. Capone, G. Palmiero, A. Gentile, V. Basile, S. Federico,
M. Sabbatini, M. Potenza, A. Perfetti, M. Pieri and G. Tarantino
[Abstract]
Histone Deacetylase Inhibitors: Molecular and Biological
Activity as a Premise to Clinical Application Pp.
383-394
V. Santini, A. Gozzini and G. Ferrari
[Abstract]
Synergistic Cytotoxic Activity of Recombinant TRAIL
Plus the Non-Genotoxic Activator of the p53 Pathway Nutlin-3
in Acute Myeloid Leukemia Cells Pp. 395-403
P. Secchiero, C. Zerbinati, M.G. di Iasio, E. Melloni,
M. Tiribelli, V. Grill and G. Zauli
[Abstract]
Abstracts
[Back to top]
Expression and Function of Cytochrome P450
in Brain Drug Metabolism
R.P. Meyer, M. Gehlhaus, R. Knoth and B. Volk
Cytochrome P450 (CYP, P450) is the collective term for a superfamily
of heme-containing membrane proteins responsible for the metabolism
of ~ 70 – 80 % of clinically used drugs. Besides the
liver and other peripheral organs, P450 isoforms are expressed
in glial cells and neurons of the brain. To enlighten their
function and significance is a topic of high interest, as
most of the neuroactive drugs used in therapy today are not
only substrates, but also inducers of brain P450s with far
reaching consequences. First of all, brain P450s are regulated
differentially from those in liver. The availability of the
prosthetic heme group appears to be essential for correct
membrane insertion and enzymatic functionality of brain P450s.
Furthermore, although not contributing to body’s overall
drug metabolism, brain P450s fulfil particular functions within
specific cell types of the brain. In astrocytes of brain’s
border lines P450 isoforms are expressed at very high level.
They form a metabolic barrier regulating drugs´ influx,
modulate blood-flow regulation, and act as signalling enzymes
in inflammation. In neurons, however, P450s apparently have
different function. In specified brain regions such as hypothalamus,
hippocampus and striatum they provide signalling molecules
like steroids and fatty acids necessary for neuronal outgrowth
and maintenance. Induction of these P450s by neuroactive drugs
can alter steroid hormone signalling directly in drug target
cells, which may cause clinically relevant side effects like
reproductive disorders and sexual or mental dysfunction. The
understanding of brain P450 func-tion appears to be of major
interest in long-term drug mediated therapy of neurological
diseases.
[Back to top]
CYP-Mediated Clozapine Interactions: How Predictable
Are They?
M. Chetty and M. Murray
Despite the introduction of newer drugs, the atypical antipsychotic
clozapine remains the most effective drug in psychotic patients
who are resistant to treatment with conventional agents. Optimal
therapeutic responses to clozapine have been reported with
serum concentrations between 350 μg/L
and 1000 μg/L.
Clozapine is frequently combined with other drugs to enhance
efficacy and reduce adverse reactions but pharmacokinetic
interactions can have a significant impact on drug response.
The majority of the interactions with clozapine are reported
to be mediated by cytochrome P450 (CYP) enzymes. CYP1A2 has
a major role in the oxidative metabolism of clozapine, with
a minor contribution from CYP3A4, and possibly CYP2D6, CYP2C9
and CYP2C19. Interactions mediated by potent CYP1A2 inhibitors
(such as fluvoxamine) or inducers (like cigarette smoke) appear
to be consistent, predictable and usually clinically significant.
There are many case reports of interactions between clozapine
and weak CYP1A2 inhibitors or inducers which are also potent
inhibitors or inducers of CYP3A4 or CYP2D6. Researchers often
explain these observations on the basis of the CYP1A2 involvement.
In addition, there are case reports of clinically significant
interactions between clozapine and drugs that are not substrates,
inhibitors or inducers of CYP1A2. These interactions are difficult
to predict and may not be consistent, as reflected by the
conflicting literature reports. Further research to elucidate
individual differences in clozapine metabolism, with the potential
to detect the dominant roles of CYPs other than CYP1A2, may
assist us in predicting these interactions.
[Back to top]
The Non-ABC Drug Transporter RLIP76 (RALBP-1) Plays
a Major Role in the Mechanisms of Drug Resistance
Y.C. Awasthi, R. Sharma, S. Yadav, S. Dwivedi, A. Sharma
and S. Awasthi
RLIP76 or Ral binding protein (RalBP-1) was initially cloned
as a Ral-effector that was proposed as a link between Ral
and Ras pathways. This protein is encoded in humans on chromosome
18p11.3 by a gene with 11 exons and 9 introns and is found
ubiquitously from drosophila to humans. RLIP76 displays inhibitory
GTPase activity toward Rho/Rac class G-protein cdc42 which
is involved in regulation of cytoskeletal organization, lamellipodia,
cell migration and apoptosis via Ras. We have recently
shown that RLIP76 is also a multispecific transporter of chemotherapeutic
agents and glutathione conjugates (GS-E). In human cells RLIP76
accounts for more than two third of the transport activity
for GS-E and drugs as opposed to the ABC-transporters including
MRP1, which account for less than one third of this activity.
Evidence is mounting that RLIP76 is a stress-responsive multi-specific,
non-ABC transporter which represents an entirely novel link
between stress-inducible G-protein signaling, receptor tyrosine-kinase
signaling, endocytosis, heat-shock and stress defense pathways,
and transport mediated drug-resistance. The expression of
RLIP76 is significantly greater in human cancer cells of diverse
origin as compared to the non-malignant cells. Inhibition
of RLIP76, using antibodies towards a cell surface epitope,
or depletion of RLIP76 using either siRNA or anti-sense phosphorothioate
oligonucleotides preferentially causes apoptosis in malignant
cells. Administration of RLIP76 antibodies, siRNA, or anti-sense
oligonucleotides to mice bearing syngeneic B16 mouse melanoma
tumors causes rapid and complete regression of tumors. Studies
summarized in this review strongly suggest that RLIP76 is
a logical target for clinical intervention of not only multi-drug
resistance but also for diseases resulting from oxidative
stress.
[Back to top]
Role of P-Glycoprotein in the Intestinal Absorption
of Tanshinone IIA, a Major Active Ingredient in the Root of
Salvia miltiorrhiza Bunge
X.-Y. Yu, S.-G. Lin, Z.-W. Zhou, X. Chen, J. Liang, P.-Q.
Liu, W. Duan, B. Chowbay, J.-Y. Wen, C.-G. Li and S.-F. Zhou
The extracts from the roots of Salvia miltiorrhiza
Bunge (Danshen) are widely and traditionally used in the treatment
of angina pectoris, acute myocardial infarct, hyperlipidemia
and stroke in China and other Asian countries. In this study,
we have investigated the role of P-glycoprotein (P-gp) in
the intestinal absorption of tanshinone IIA (TSA), a major
active constituent of Danshen, using several in vitro
and in vivo models. The oral bioavailability of TSA
was about 2.9-3.4% in rats, with non-linear pharmacokinetics
when its dosage increased. In a single pass rat intestinal
perfusion model, the permeability coefficients (Papp)
based on TSA disappearance from the luminal perfusates (Plumen)
were 6.2- to 7.2-fold higher (P < 0.01) than those
based on drug appearance in mesenteric venous blood (Pblood).
The Pblood, but
not Plumen, was
significantly increased when co-perfused with verapamil, or
quinidine (both P-gp inhibitors). The uptake and efflux of
TSA in confluent Caco-2 cells were significantly altered in
the presence of verapamil, quinidine, MK-571, or probenecid.
The transport of TSA across Caco-2 monolayers was pH-, temperature-
and ATP-dependent. Furthermore, the transport from the apical
(AP) to basolateral (BL) side of the Caco-2 monolayers was
3.3- to 8.5-fold lower than that from the BL to AP side, but
such a polarized transport was attenuated by co-incubated
verapamil or quinidine. A polarized transport was also observed
in the control MDCKII cells and more apparent in MDR1-MDCKII
monolayers, with the Papp
values of TSA in the BL-AP direction being 7- to 9-fold higher
in MDR1-MDCKII monolayers than those in the control MDCKII
cells. Moreover, TSA significantly inhibited P-gp-mediated
transport of digoxin in P-gp-overexpressing membrane vesicles
with an IC50 of 2.6 μM,
but stimulated vanadate-sensitive P-gp ATPase activity with
estimated Km and
Vmax values of 10.70 ±
0.69 ±M
and 67.65 ±
1.31 nmol/min/mg protein, respectively. TSA was extensively
metabolized to tanshinone IIB (TSB), and two other oxidative
metabolites in rat liver microsomes, but the formation rate
of TSB in rat intestinal microsomes was only about 1/10 of
that in liver microsomes. These findings indicate that TSA
is a substrate and reversing agent for P-gp; and P-gp-mediated
efflux of TSA into the gut lumen and the first-pass metabolism
contribute to the low oral bioavailability. Further studies
are needed to explore the role of other drug transporters
and first-pass metabolism in the low bioavailability of TSA.
[Back to top]
Evaluation of Drug-Transporter Interactions Using
In Vitro and In Vivo Models
C.Q. Xia, M.N. Milton and L.-S. Gan
Drug transporters, including efflux transporters (the ATP
binding cassette (ABC) proteins) and uptake transporters (the
solute carrier proteins (SLC)), have an important impact on
drug disposition, efficacy, drug-drug interactions and toxicity.
Identification of the interactions of chemical scaffolds with
transporters at the early stages of drug development can assist
in the optimization and selection of new drug candidates.
In this review, we discuss current in vitro and in
vivo models used to investigate the interactions between
drugs and transporters such as P-gp, MRP, BCRP, BSEP, OAT,
OATP, OCT, NTCP, PEPT1/2 and NT. In vitro models
including cell-based, cell-free, and yeast systems as well
as in vivo models such as genetic knockout, gene
deficient and chemical knockout animals are discussed and
compared. The applications, throughput, advantages and limitations
of each model are also addressed in this review.
[Back to top]
Transport of Cryptotanshinone, a Major Active Triterpenoid
in Salvia Miltiorrhiza Bunge Widely Used in the Treatment
of Stroke and Alzheimer’s Disease, Across the Blood-Brain
Barrier
X.-Y. Yu, S.-G. Lin, X. Chen, Z.-W. Zhou, J. Liang, W.
Duan, B. Chowbay, J.-Y. Wen, E. Chan, J. Cao, C.-G. Li and
S.-F. Zhou
Cryptotanshinone (CTS), a major constituent from the roots
of Salvia miltiorrhiza (Danshen), is widely used
in the treatment of coronary heart disease, stroke and less
commonly Alzheimer’s disease. Our recent study indicates
that CTS is a substrate for P-glycoprotein (PgP/MDR1/ABCB1).
This study has investigated the nature of the brain distribution
of CTS across the brain-blood barrier (BBB) using several
in vitro and in vivo rodent models. A polarized
transport of CTS was found in rat primary microvascular endothelial
cell (RBMVEC) monolayers, with facilitated efflux from the
abluminal side to luminal side. Addition of a PgP (e.g. verapamil
and quinidine) or multi-drug resistance protein 1/2 (MRP1/2)
inhibitor (e.g. probenecid and MK-571) in both luminal and
abluminal sides attenuated the polarized transport. In a bilateral
in situ brain perfusion model, the uptake of CTS
into the cerebrum increased from 0.52 ± 0.1% at 1 min
to 11.13 ± 2.36 ml/100 g tissue at 30 min and was significantly
greater than that of sucrose. Co-perfusion of a PgP/MDR1 (e.g.
verapamil) or MRP1/2 inhibitor (e.g. probenecid) significantly
increased the brain distribution of CTS by 35.1-163.6%. The
brain levels of CTS were only about 21% of those in plasma,
and were significantly increased when coadministered with
verapamil or probenecid in rats. The brain levels of CTS in
rats subjected to middle cerebral artery occlusion and rats
treated with quinolinic acid (a neurotoxin) were about 2-
to 2.5-fold higher than the control rats. Moreover, the brain
levels in mdr1α(-/-)
and mrp1(-/-) mice were 10.9- and 1.5-fold higher
than those in the wild-type mice, respectively. Taken collectively,
these findings indicate that PgP and Mrp1 limit the brain
penetration of CTS in rodents, suggesting a possible role
of PgP and MRP1 in limiting the brain penetration of CTS in
patients and causing drug resistance to Danshen therapy and
interactions with conventional drugs that are substrates of
PgP and MRP1. Further studies are needed to explore the role
of other drug transporters in restricting the brain penetration
of CTS and the clinical relevance.
[Back to top]
A Pharmacokinetic Interaction Between Clarithromycin
and Sirolimus in Kidney Transplant Recipient
D. Capone, G. Palmiero, A. Gentile, V. Basile, S. Federico,
M. Sabbatini, M. Potenza, A. Perfetti, M. Pieri and G. Tarantino
Bacterial infection is a frequent event in renal transplant
recipients and often requires the use of antimicrobial agents.
In this paper it is reported an evidence of pharmacokinetic
interaction between clarithromycin and sirolimus in a kidney
transplanted woman, suffering from pulmonary infection sustained
by a bacterial pathogen, in particular Hemophilus Influenzae.
In the present case report, the concomitant administration
of clarithromycin and sirolimus determined impressive increase
of sirolimus trough blood concentrations from 6.2 up to 54
ng/mL and this increase was associated with an acute impairment
of renal function, almost completely reversed upon both drugs
discontinuation.
This drug-drug interaction is due to a likely inhibition of
activity of both cytochrome P450 3A4 and P-glycoprotein. Although
this inter-action could be predicted, it represents the first
reported clinical evidence.
[Back to top]
Histone Deacetylase Inhibitors: Molecular and Biological
Activity as a Premise to Clinical Application
V. Santini, A. Gozzini and G. Ferrari
Epigenetic modifications are reversible chromatin rearrangements
that in normal cells modulate gene expression, without changing
DNA sequence. Alterations of this equilibrium, mainly affecting
the two interdependent mechanisms of DNA methylation and histone
acetylation, are frequently involved in the genesis of cancer.
The histone code, regulating gene expression, is constituted
by the combination of different acetylated lysine residues
of histones. In neoplastic cells, the abundance of deacetylated
histones is usually associated with DNA hypermethylation and
gene silencing. Several compounds, known to have in vitro
antineoplastic activity, have been eventually shown to act
as histone deacetylase inhibitors. Thus, HDAC inhibitors have
been successfully introduced in clinical trials as antitumour
agents. They are classified according to their chemical structures
and are endowed with different specificity and affinity for
the HDACs of classes 1, 2, 4. Among HDAC inhibitors, the most
potent are the hydroxamic acid derivatives, like SAHA, which
has been recently approved for therapy of cutaneous T-cell
lymphomas. Other classes of HDAC inhibitors are short chain
fatty acids (SCFA), benzamides, epoxyketone and non-epoxyketone
containing cyclic tetrapeptides, and hybrid molecules. SCFA,
although widely used (especially valproic acid) and clinically
efficacious, have weak HDAC inhibition constants. Benzamides,
like MS-275, and cyclic peptides, like depsipeptide, have
been studied in numerous clinical trials and demonstrated
low toxicity and activity in solid and haematological neoplasms.
HDAC inhibitors are also potent radiation sensitizers. Their
future in oncology may thus be based on their activity as
single agents and on their synergy with the hypomethylating
drugs and with chemo- and radiotherapeutics.
[Back to top]
Synergistic Cytotoxic Activity of Recombinant TRAIL
Plus the Non-Genotoxic Activator of the p53 Pathway Nutlin-3
in Acute Myeloid Leukemia Cells
P. Secchiero, C. Zerbinati, M.G. di Iasio, E. Melloni,
M. Tiribelli, V. Grill and G. Zauli
To potentiate the response of acute myeloid leukemia (AML)
to TRAIL cytotoxicity, we have adopted a strategy of combining
nutlin-3, a potent non-genotoxic activator of the p53 pathway,
with recombinant TRAIL. The rationale for using such a combination
was that deletions and/or mutations of the p53 gene occur
in only 5-10% of AML and that TRAIL and nutlin-3 activate
the extrinsic and intrinsic pathways of apoptosis, respectively.
TRAIL induced a rapid increase of apoptosis when added to
OCI M4-type and MOLM M5-type AML cells, carrying a wild-type
p53, as well as to NB4 M3-type AML, carrying a mutated p53.
On the other hand, the small molecule activator of the p53
pathway nutlin-3 induced p53 accumulation, cell cycle arrest
and a slow progressive increase of apoptosis in OCI and MOLM
but not in NB4. Of note, nutlin-3 up-regulated the surface
expression of TRAIL-R2 and synergized with TRAIL in inducing
apoptosis in OCI and MOLM as well as in primary M4-type and
M5-type AML blasts, but not in NB4 cells. Moreover, while
nutlin-3 up-regulated the expression of cyclin dependent kinase
inhibitor p21, a p53-target gene mediating cell cycle block
and showing anti-apoptotic activity, the simultaneous addition
of TRAIL plus nutlin-3 induced the caspase-dependent cleavage
of p21. The relevance of p21 down-regulation for sensitizing
AML cells to apoptosis was underscored in knocking-down experiments
with small interfering RNAs. Our data suggest that the combined
treatment of nutlin-3 plus TRAIL might offer a novel therapeutic
strategy for AML.
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