Current Drug Metabolism, Volume 3, No. 5, 2002
Whole-Body Autoradiography In Drug Discovery Pp.451-462
E.G. Solon, S.K. Balani and F.W. Lee
Analysis of Anticancer Drugs and their
Metabolites by Mass Spectrometry Pp.463-480
Ian A. Blair and Anita Tilve
Pharmacogenomics, Regulation and Signaling
Pathways of Phase I and II Drug Metabolizing Enzymes Pp.481-490
Thomas H. Rushmore and A.-N. Tony Kong
Biological Activities, Mechanisms of Action
and Biomedical Prospect of the Antitumor Ether Phospholipid ET-18-OCH3
(Edelfosine), A Proapoptotic Agent in Tumor Cells Pp.491-525
Consuelo Gajate and
Faustino Mollinedo
The Influence of DMPK as an Integrated
Partner in Modern Drug Discovery Pp.527-550
Robert J. Riley, Iain
J. Martin and Anne E. Cooper
Permeability Characteristics of
Endocrine-Disrupting Chemicals Using an In Vitro Cell Culture Model, Caco-2
Cells Pp.551-557
Yukako Yoshikawa,
Ayako Hayashi, Maya Inai, Akiko Matsushita, Nobuhito Shibata, and Kanji Takada
[Back
to top] Whole-Body Autoradiography In Drug Discovery
E.G. Solon, S.K.
Balani and F.W. Lee
In the
drug discovery process the pharmacokinetic screening, drug stability studies,
evaluation of metabolites, CYP involvement, enzyme induction and inhibition,
and excretion studies play a major role. The use of more sensitive and novel
detection systems have made the discovery process less cumbersome than in
previous years. In particular, the use of whole-body autoradiography (WBA) for
tissue distribution, which was once considered an impractical tool, owing to
the long turn around time (4-10 weeks), is coming to the forefront for rapidly
resolving issues encountered in discovery. In today's research environment
early lead compounds can be radio-labeled and whole-body sections imaged
quickly (3-5 days) using new techniques, which has made 14C- and 3H-WBA a
viable tool. The technique has been used in vivo in species from mice to
monkeys, and ex vivo and/or in vitro in larger animals and humans. WBA has
considerable merit in identifying "pharmacodefficient" compounds and
providing insight on mechanistic questions. WBA data can provide information
related to tissue pharmacokinetics, routes of elimination, CYP or Pgp mediated
drug-drug interactions, tissue distribution, site specific drug localization
and retention, metabolism, clearance, compound solubility issues, routes of
administration, penetration into specific targets (e.g., tumors), tissue
binding (e.g., melanin), and interspecies kinetics. Thus, WBA is quickly
becoming part of the battery of studies conducted during the lead optimization
process to select optimal drug candidates. Examples of the use of the WBA tool
in early discovery are reviewed.
[Back
to top] Analysis
of Anticancer Drugs and their Metabolites by Mass Spectrometry
Ian A. Blair and Anita
Tilve
There
is an increasing awareness that the metabolites of anticancer drugs can
contribute to the pharmacodynamic effects that are observed, which has
stimulated a much greater emphasis on metabolic and pharmacokinetic issues.
This has coincided with the development of electrospray and related atmospheric
pressure ionization mass spectrometry techniques such as ionspray (nebulizer
assisted electrospray), turboionspray (heated nebulizer assisted electrospray) and
atmospheric pressure chemical ionization (nebulization coupled with corona
discharge). The combination of collision induced dissociation and tandem mass
spectrometry coupled with a soft ionization process that produces abundant
molecular species provides very powerful methodology for the trace analysis of
drugs and their metabolites. The present review has emphasized the more
rigorous quantitative applications that have appeared in the literature over
the last five years. It is evident that modern techniques of liquid
chromatography tandem mass spectrometry coupled with stable isotope dilution
methodology have had a profound effect on our ability to analyze anticancer
drugs and their metabolites. As new drugs emerge into the clinic, this
methodology will clearly be the method of choice, particularly when many
samples have to be analyzed over a short time. This approach was beautifully
demonstrated in the study of the novel signal transduction inhibitor, Gleevec
where thousands of clinical samples were analyzed for drug and metabolites over
a relatively short period of time. The need to analyze anticancer drugs and
their metabolites with such prompt turn around times has stimulated even more
rapid approaches to analysis using roboticbased purification methodology and
short LC chromatographic run times.
[Back
to top] Pharmacogenomics, Regulation and Signaling Pathways of Phase I and II
Drug Metabolizing Enzymes
Thomas H. Rushmore and
A.-N. Tony Kong
Drug
or xenobiotics metabolizing enzymes (DMEs or XMEs) play central roles in the
biotransformation, metabolism and/or detoxification of xenobiotics or foreign
compounds, that are introduced to the human body. In general, DMEs protect or
defened the body against the potential harmful insults from the environment.
Once in the body, many xenobiotics may induce signal transduction events either
specifically or non-specifically leading to various cellular, physiological and
pharmacological responses including homeostasis, proliferation,
differentiation, apoptosis, or necrosis. For the body to minimize the insults
caused by these xenobiotics, various tissues / organs are well equipped with
diverse DMEs including various Phase I and Phase II enzymes, which are present
in abundance either at the basal level and/or increased / induced after
exposure. To better understand the pharmacogenomic/gene expression profile of
DMEs and the underlying molecular mechanisms after exposure to xenobiotics or
drugs, we will review our current knowledge on DNA microarray technology in
gene expression profiling and the signal transduction events elicited by
various xenobiotics mediated by either specific receptors or non-specific signal
transduction pathways. Pharmacogenomics is the study of genes and the gene
products (proteins) essential for pharmacological or toxicological responses to
pharmaceutical agents. In order to assess the battery of genes that are induced
or repressed by xenobiotics and pharmaceutical agents, cDNA microarray or
oligonucleotide-based DNA chip technology can be a powerful tool to analyze,
simultaneously, the gene expression profiles that are induced or repressed by
xenobiotics. The regulation of gene expression of the various phase I DMEs such
as the cytochrome P450 (CYP) as well as phase II DMEs generally depends on the
interaction of the xenobiotics with the receptors. For instance, the expression
of CYP1 genes can be induced via the aryl hydrocarbon receptor (AhR) which
dimerizes with the AhR nuclear translocator (ARNT), in response to many
polycyclic aromatic hydrocarbon (PAHs). Similarly, the steroid family of orphan
receptors, the constitutive androstane receptor (CAR) and pregnane X receptors
(PXR), heterodimerize with the retinoid X receptor (RXR), transcriptionally
activate the promoters of CYP2B and CYP3A gene expression by xenobiotics such
as phenobarbital-like compounds (CAR) and dexamethasone and rifampin-type of
agents (PXR). The peroxisome proliferator activated receptor (PPAR) which is
one of the first characterized members of the nuclear hormone receptor, also
dimerizes with RXR and it has been shown to be activated by lipid lowering
agent fibrate-type of compounds leading to transcriptional activation of the
promoters on the CYP4A genes. The transcriptional activation of these promoters
generally leads to the induction of their mRNA. The physiological and the
pharmacological implications of common partner of RXR for CAR, PXR, and PPAR
receptors largely remain unknown and are under intense investigations.
For
the phase II DMEs, phase II gene inducers such as phenolic compounds butylated
hydroxyanisol (BHA), tertbutylhydroquinone (tBHQ), green tea polyphenol (GTP),
(-)-epicatechin-3-gallate (EGCG) and the isothiocyanates (PEITC, sulforaphane)
generally appear to be electrophiles. They can activate the mitogen-activated
protein kinase (MAPK) pathway via electrophilic-mediated stress response,
resulting in the activation of bZIP transcription factors Nrf2 which dimerizes
with Mafs and binds to the antioxidant/electrophile response element (ARE/EpRE)
enhancers which are found in many phase II DMEs as well as many cellular
defensive enzymes such as thioredoxins, gGCS and HO-1, with the subsequent induction
of gene expression of these genes. It appears that in general, exposure to
phase I or phase II gene inducers or xenobiotics may trigger a cellular
“stress” response leading to the increase in the gene expression of these DMEs,
which ultimately enhance the elimination and clearance of the xenobiotics
and/or the “cellular stresses” including harmful reactive intermediates such as
reactive oxygen species (ROS), so that the body will remove the “stress”
expeditiously. Consequently, this homeostatic response of the body plays a
central role in the protection of the organism against environmental insults
such as xenobiotics.
Advances
in DNA microarray technologies and mammalian genome sequencing will soon allow
quantitative assessment of expression profiles of all genes in the selected
tissues. The ability to predict phenotypic outcomes from gene expression
profiles is currently in its infancy, however, and will require additional
bioinformatic tools. Such tools will facilitate information gathering from literature
and gene databases as well as integration of expression data with animal
physiology studies. The study of pharmacogenomic/gene expression profile and
the understanding of the regulation and the signal transduction mechanisms
elicited by pharmaceutical agents can be of potential importance during drug
discovery and the drug development.
[Back
to top]
Biological Activities, Mechanisms of Action and Biomedical Prospect of the
Antitumor Ether Phospholipid ET-18-OCH3 (Edelfosine), A Proapoptotic Agent in
Tumor Cells
Consuelo Gajate and
Faustino Mollinedo
The
antitumor ether lipid ET-18-OCH3 (edelfosine) is the prototype of a new class
of antineoplastic agents, synthetic analogues of lysophosphatidylcholine, that
shows a high metabolic stability, does not interact with DNA and shows a
selective apoptotic response in tumor cells, sparing normal cells. Unlike
currently used antitumor drugs, ET-18- OCH3 does not act directly on the
formation and function of the replication machinery, and thereby its effects
are independent of the proliferative state of target cells. Because of its
capacity to modulate cellular regulatory and signaling events, including those
failing in cancer cells, like defective apoptosis, ET-18-OCH3, beyond its
putative clinical importance, is an interesting model compound for the
development of more selective drugs for cancer therapy. Although ET-18-OCH3
enhances host defense mechanisms against tumors, its major antitumor action
lies in a direct effect on cancer cells, inhibiting phosphatidylcholine
biosynthesis and inducing apoptosis in tumor cells. Recent progress has allowed
unraveling the molecular mechanism underlying the apoptotic action of ET-18-OCH3, leading to the notion that
ET-18-OCH3 is selectively incorporated
into tumor cells and induces cell death by intracellular activation of the cell
death receptor Fas/CD95. This intracellular Fas/CD95 activation is a novel
mechanism of action for an antitumor drug and represents a new way to target
tumor cells in cancer chemotherapy that can be of interest as a new framework
in designing novel antitumor drugs. ET-18-OCH3 and some analogues are
pleiotropic agents that affect additional biomedical important diseases,
including parasitic and autoimmune diseases, suggesting new therapeutic
indications for these compounds.
[Back
to top] The Influence of
DMPK as an Integrated Partner in Modern Drug Discovery
Robert J. Riley, Iain J. Martin and Anne E. Cooper
In
response to the challenge laid down by advances in other drug discovery
functions, DMPK has now established an array of automated, miniaturised in
vitro screens, rapid bioanalytical methodologies and in silico tools with which
to optimise or predict passive absorption, metabolic clearance and minimise
drug-drug interaction potential. The awareness of the pivotal role that
physicochemical properties play in the control of many of these processes has
been key. This review highlights some of these structure-activity relationships
with emphasis on drug absorption, clearance, protein binding and distribution.
However, some fundamental processes remain to be elucidated fully, including
the in vivo impact of non-specific or futile binding in in vitro screens and
the functional significance of intestinal and hepatobiliary transporter
proteins. Transgenic animals should soon add value to our understanding of the
contribution of transporter proteins to drug bioavailability (intestinal and
hepatic drug uptake/efflux) and drug interactions and in validating projections
for Man. Future studies should also focus on the evaluation of the various in
vitro human CYP induction screens available, with particular emphasis on their
predictive value for the clinical scenario.
[Back to top]
Permeability Characteristics of
Endocrine-Disrupting Chemicals Using an In Vitro Cell Culture Model, Caco-2
Cells
Yukako Yoshikawa,
Ayako Hayashi, Maya Inai, Akiko Matsushita, Nobuhito Shibata, and Kanji Takada
The
purpose of this study was to evaluate the permeability characteristics of
endocrine disrupting chemicals utilizing epithelial monolayers of Caco-2 cells.
The drugs tested in this study were bisphenol A (BPA), tert-octylphenol (tOP),
tert-butylphenol (tBP), di(2-ethylhexyl)phthalate (DOP), dibutylphthalate
(DBP), and butylbenzylphthalate (BBP), all of which are used in plastic
materials. The Caco-2 cell line was grown on cell culture inserts with
polyethylene terephthalate membranes, and Hank’s balanced salt solution (HBSS,
pH 7.4) was used for the transport experiments. The barrier properties were
assessed by measuring transepithelial electrical resistance (TEER) using a volt
ohmmeter, and transport of these endocrine disrupting chemicals was examined in
both directions. The permeated amounts of these chemicals within 180 min in the
apical to basolateral (A-to-B) and the basolateral to apical (B-to-A)
directions without verapamil, a P-glycoprotein (P-gp) inhibitor, were in the
rank order of tBP > tOP > BPA > DOP > DBP > BBP and BPA >>
tBP > tOP > DOP > DBP > BBP, respectively. In the presence of 100 mM verapamil, the permeated amounts of BPA, tOP and tBP within 180 min in the
B-to-A direction decreased by 12-, 2.6- and 3.1-fold, respectively. In the case
of phthalate esters, the permeated amount of DOP within 180 min in the B-to-A
direction decreased by 1.6-fold, while that of DBP and BBP showed no
significant changes. The ratios of apparent permeability coefficient of B-to-A
against A-to-B, Papp ratios, for BPA, tOP and tBP were markedly decreased in
the presence of 100 mM verapamil. These findings indicated that both BPA and
alkyl phenols are substrates of the P-gp located in the apical side of Caco-2
cells, and suggested that the P-gp in the small intestine may act as an organic
barrier against BPA and alkyl phenols.