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Current Pharmaceutical
Analysis
ISSN: 1573-4129
Current Pharmaceutical
Analysis
Volume 1, Number 3, November 2005
Contents
Electrochemical Biosensors as a Screening Tool of
In Vitro DNA-Drug Interaction Pp.217
Graziana Bagni, Mauro Ravera, Domenico Osella and Marco
Mascini
[Abstract]
State of the Art of Biochemical Markers in Metastatic Bone
Disease and the Role of Bisphosphonates as Therapeutic Agents
Pp. 225
Ioannis Kanakis, Achilleas D. Theocharis, Argiris
V. Noulas and Nikos K. Karamanos
[Abstract]
Current Methods of Gene Expression Analysis and Quantification
Pp.243
Beny Spira
[Abstract]
Bioanalytical Aspects in Characterization and Quantification
of Glucuronide Conjugates in Various Biological Matrices
Pp.251
Ramesh Mullangi, Ravi Kanth Bhamidipati and Nuggehally
R. Srinivas
[Abstract]
Cytostatic Effect of the Nucleoside Analogue 2-Chloroadenosine
on Human Prostate Cancer Cell Line Pp.265
Ilaria Bellezza, Massimiliano Agostini, Lavinia Liguori
and Alba Minelli
[Abstract]
Profiling Drug Membrane Transport via Immobilized
Artificial Membrane Chromatography Pp.273
Jin Sun, Tian-Hong Zhang and Zhong-Gui He
[Abstract]
Advances in Chromatographic Analyses of Fluoroquinolones
in Pharmaceuticals and Biological Samples - A Review Article
Pp.283
Victoria F. Samanidou, Eleni A. Christodoulou and
Ioannis N. Papadoyannis
[Abstract]
Review on Scale-up of Coil Column Countercurrent Chromatographs
Pp.309
Qizhen Du and Yoichiro Ito
[Abstract]
Qualitative and Quantitative Analysis of Histone Acetylation
by Mass Spectrometry Pp. 319
Kangling Zhang, Jennifer Tang and Patrick R. Jones
[Abstract]
Analytical Methods of Bioactive Metabolites Produced
by Plants and Microorganisms Pp.329
Antonio Evidente, Anna Andolfi and Mohamed A. Abouzeid
[Abstract]
Abstracts
[Back to top]
Electrochemical Biosensors as a Screening Tool of
In Vitro DNA-Drug Interaction
Graziana Bagni, Mauro Ravera, Domenico Osella
and Marco Mascini
The interaction of some antitumoural drugs with double stranded
DNA immobilised on screen-printed electrodes was studied as
a screening tool for in vitro DNA-drug interaction.
In this case DNA biosensors are used for qualitative analysis
as a rapid indication of the behaviour of DNA with different
metal complexes and are proposed as an alternative method
of the most common techniques. In fact, DNA based electrochemical
biosensors represent a new research field with interesting
possibilities for practical application in various areas such
as environmental and pharmaceutical screening. Interactions
of DNA with chemicals can result in various types of DNA damage.
Many small molecules show a high affinity for DNA and they
can interact with the nucleic acids immobilised on the electrode
surface. The interactions between DNA and drugs can cause
chemical and conformational modifications of nucleic acids
and thus variation of the electrochemical properties of DNA.
The presence of compounds with affinity for DNA is measured
by their effect on the guanine or adenine oxidation peak.
A great advantage of this kind of biosensors is that they
are cost effective and they can be used with a small portable
potentiostat with disposable screen-printed electrodes (SPE).
In this work, screening of some antitumoural drug is reported
as example of in vitro interaction. Preliminary results
have been obtained analysing different type of DNA-interacting
agents as metal (Pt, Ru and Ti) complexes. The drugs analysed
were cis-diaminedichloroplatinum(II) cis-[Pt(Cl)2(NH3)2] (cisplatin);
diamine(1,1-cyclobutanedicarboxylate)platinum(II) [Pt(NH3)2C4H6C2O4] (carboplatinum);cis-diamineplatinum(II)malonate
cis-[Pt(NH3)2 C3H2O4]
(cis-malonate); platinum(bipyridile(pyridine)2)2+
[Pt(bpy)(py)2]2+; titanocene dichloride
[(η5-C5H5)2TiCl2]
(titanocene); titanocenebisglycine [(η5-C5H5)2Ti(gly)2]
and imidazolium trans-imidazoledimethylsulfoxidetetrachloro-ruthenate
[Ru(III)Cl4(DMSO)(Im)][ImH] (NAMI-A). Groove
binding, electrostatic interactions, hydrogen and/or van der
Waals bonds and intercalation of planar condensed aromatic
ring systems between adjacent base pairs are the interactions
that the DNA electrochemical biosensor can detect. Different
response behaviour has been observed with these drugs according
to different interaction with DNA.
[Back to top]
State of the Art of Biochemical Markers in Metastatic
Bone Disease and the Role of Bisphosphonates as Therapeutic
Agents
Ioannis Kanakis, Achilleas D. Theocharis, Argiris V. Noulas
and Nikos K. Karamanos
Metastatic spread of cancer to bone is a frequent complication
in several types of malignancies. Normal bone metabolic process
is affected and the imbalance between bone formation and resorption
defines bone disease in cancer. The major causes of the secondary
development of the disease is either the cancer cell itself
(metastatic or non-metastatic) or cancer therapies. Early
diagnosis of bone metastatic disease is very important for
the survival of patients. The dominant disadvantage of imaging
techniques used for detection of bone metastases is that they
are based on direct anatomic visualization of the metastatic
sites on the skeleton and, therefore, are not able to detect
metastasis in early stages of the disease. Various biomarkers
have been developed and used as indicators of bone formation
or resorption. Substantially, these biochemical markers are
products, released during the bone metabolic cycle and can
be detected in biological samples. The levels of these markers
in serum or urine can be correlated with the diagnosis of
osseous metastases and the bulk of tumor burden and, in addition,
may provide useful data for the patients’ follow-up,
in order to evaluate the response to various treatments. The
biochemical markers often used for monitoring bone formation
involve total and bone-specific alkaline phosphatase, osteocalcin
and procollagen type I extension propeptides, and those for
bone resorption involve urinary calcium, hydroxyproline, hydroxylysine-glycosides,
pyridinium crosslinks, crosslinked telopeptides of type I
collagen, tartarate-resistant acid phosphatase and bone sialoprotein.
For the treatment of bone disease, many therapies have been
developed. Bisphosphonates, compounds based on a phosphorous-carbon-phosphorous
spine similar to endogenous pyrophosphate, have been successfully
used in the treatment of osteoporosis and hypercalcemia of
malignancy. They have powerful anti-resorptive activity by
suppressing the action of osteoclasts, resulting in the reduction
of skeletal events. The analytical methods used for the determination
of biomarkers in biological samples as well as the effect
of bisphosphonates on the drift of metastatic bone disease
are presented in this review.
[Back to top]
Current Methods of Gene Expression Analysis and Quantification
Beny Spira
Gene expression analysis methods have many applications
in the clinical and pharmaceutical fields. They provide tools
to determine the effect and side effects of drugs and xenobiotics,
and alterations in the physiological state of a cell or tissue
under various conditions, among other usages. Here, some of
the methods used to quantify gene expression are reviewed.
These methods are divided into two groups, those that measure
the expression of particular genes, such as northern hybridisation,
ribonuclease protection assay, RT-PCR and real-time RT-PCR
and those used to measure the expression of a broad-range
of genes (mRNA differential display, SAGE and DNA arrays).
The potential uses of these methods as well as their advantages
and disadvantages are discussed.
[Back to top]
Bioanalytical Aspects in Characterization and Quantification
of Glucuronide Conjugates in Various Biological Matrices
Ramesh Mullangi, Ravi Kanth Bhamidipati and Nuggehally
R. Srinivas
The occurrence of phase II metabolism via conjugation
pathway, has been observed for not only several endogenous
molecules such as bilirubin, bile acids, steroid hormones
(androgens and estrogens), retinoids, thyroid hormones, leukotriene
B4, hydroxy metabolites of arachidonic and linoleic
acids, several phenolic neurotransmitters etc. but also for
a number of xenobiotics belonging to diversified chemical
classes. Although topic of focus in this review is pertaining
to glucuronidation, other conjugation phase II reactions are
known to occur via sulfation, acetylation, methylation
or simply via direct addition of glycine or glutathione
adducts. The review covers the importance of glucuronidation
while accounting for various types of known glucuronidation
reactions. The formation of acyl glucuronides and the liability
of acyl glucuronides to form chemically reactive species via
acyl migration mechanism are also covered in this review.
A concise review of the literature on bioanalytical aspects
of the analysis of glucuronides revealed that application
of HPLC with UV, fluorescence, coulometric or mass spectral
detections have been generally practiced for the determination
of several types of glucuronides in biological fluids. Several
examples are provided in this review for the estimation of
glucuronides under various sub-heading such as direct estimation
of nonchiral glucuronides, direct estimation of diastereomeric
glucuronides, estimation followed by derivatization, estimation
by using ion-pairing agent, estimation of mixed glucuronides
by HPLC and/or LC-MS/MS. Additionally, several aspects pertaining
to the development of sound bioanalytical methodology including
aspects for handling procedures and a validation framework
for establishing glucuronide assay are presented.
[Back to top]
Cytostatic Effect of the Nucleoside Analogue 2-Chloroadenosine
on Human Prostate Cancer Cell Line
Ilaria Bellezza, Massimiliano Agostini, Lavinia Liguori
and Alba Minelli
2-Chloroadenosine is an adenosine analog capable of inducing
apoptosis in several cell lines by acting either via
adenosine receptors or via uptake that is followed
by metabolic transformations leading to nucleotide analogs.
DNA-directed nucleotide analogs are antimetabolites effective
in the treatment of a variety of malignancies. Triphosphate
nucleoside analogs show specificity for cells in S-phase,
inhibit DNA synthesis, and kill the cells by mechanisms still
largely unknown. It is likely that cells perceive sublethal
incorporation of analogues into DNA and react by arresting
further DNA synthesis and cell cycle progression. After removal
of the drug, cells resume progression and proliferation, escaping
nucleoside analog toxicity. The induction of cytostasis and
subsequent recovery may mimic a mechanism of resistance to
nucleoside analogs that represents a major problem in cancer
chemotherapy. 2-Chloroadenosine, acting as metabolic precursor
of an S-phase specific nucleoside analogue in human prostate
cancer PC3 cells, inhibited DNA synthesis so that the cell
population accumulated in the S-phase. After release from
the S-phase, proliferation continued and cells exhibited a
lag before resuming proliferation at a rate comparable with
controls. The combination of 2-chloroadenosine with staurosporine,
a cell cycle checkpoint dysregulator, enhances the killing
potential of the antineoplastic agent and diminishes the effective
antineoplastic dose. Hence, the nucleoside analog might be
proposed as an essential component of an effective drug combination
for advanced prostate cancer. This study, as well as providing
the reader with up-to date literature on 2-chloroadenosine,
presents new data hinting at the possible therapeutic use
of this analog.
[Back to top]
Profiling Drug Membrane Transport via Immobilized
Artificial Membrane Chromatography
Jin Sun, Tian-Hong Zhang and Zhong-Gui He
The preclinical and clinical development attrition rates
of new drugs are high due to the unfavorable pharmacokinetic
properties such as poor intestinal absorption and inadequate
target tissue penetration, etc. Drug disposition
performance in nature consists of in vivo sequential
membrane transporting processes and is based on the entry
into and exit of drugs from cell, even for metabolism process
requiring drug to be delivered to the site of metabolic enzymes.
Efficient and reliable high throughput screening that predicts
these membrane permeability properties as early as possible,
in drug discovery and development program is accordingly desirable.
Immobilized artificial membrane (IAM) chromatography, one
of the biopartitioning modeling systems, covalently bonds
monolayer of phospholipid analogs onto the solid surface of
silica particles and emulates the process of drug–biomembrane
interactions in terms of rapid chromatographic method. It
is a valuably predictive model of drug membrane permeability
and biological activity. The implementation of IAM chromatography
in early drug discovery and development would effectively
lower the preclinical and clinical development attrition rates,
and would be able to increase the fraction of more drug-like
drug candidates. The interaction mechanism of drug–IAM
and its profiling in membrane transport were described in
this review.
[Back to top]
Advances in Chromatographic Analyses of Fluoroquinolones
in Pharmaceuticals and Biological Samples - A Review Article
Victoria F. Samanidou, Eleni A. Christodoulou and Ioannis
N. Papadoyannis
Fluoroquinolones constitute a member of quinolone antibiotics,
an expanding group of synthetic antibiotics, which are effective
towards pathogens resistant to other antibacterials. They
are broadly used for the treatment of several infections,
both in human and veterinary medicine, due to their favourable
kinetics and broad antibacterial activity.
Herein recent advances in chromatographic methods for determination
of fluoroquinolones and their metabolites are reviewed, focusing
on sample pretreatment, chromatographic conditions, detection
techniques, method validation and application to real samples.
Results of published methods are comparatively presented and
criticized.
[Back to top]
Review on Scale-up of Coil Column Countercurrent Chromatographs
Qizhen Du and Yoichiro Ito
Coil column countercurrent chromatography (CC-CCC) has been
intensively used for the separation of natural products. Most
of these separations have been successfully performed on a
laboratory scale. However, scaling-up of this technique for
industrial separations is still a challenge. In the present
paper scale-up of coil column countercurrent chromatographs
with three types of column rotation including high-speed planetary
motion around its horizontal axis, cross-axis medium-speed
planetary motion and slow rotation around its horizontal axis
are reviewed. Up to now, the column capacities of high-speed
countercurrent chromatographs, cross-axis countercurrent chromatographs
and slow rotary countercurrent chromatographs reach 4.6, 2.5
and 40 liters, respectively. An evaluation scaling-up each
type of CC-CCC is made.
[Back to top]
Qualitative and Quantitative Analysis of Histone Acetylation
by Mass Spectrometry
Kangling Zhang, Jennifer Tang and Patrick R. Jones
Acetylation is known to be one of the major post-translational
modifications of histones. The acetylation level of histones
is regulated by the enzymatic activities of HAT and HDAC.
Dozens of structurally divergent classes of HDAC inhibitors
have been identified. They have been shown to induce cell-cycle
arrest, cell differentiation and apoptosis in various cancer
cell lines and to inhibit tumor growth in animals or humans.
Histone deacetylase inhibitors elevate the acetylation level
by blocking or reducing the deacetylation activities of HDACs
reacting with specific acetylated lysine residues. An analytical
method which can ascertain the acetylation or lack of acetylation
at a given lysine residue in a histone is needed to provide
detailed information on the effect of treatment with a HDAC
inhibitor drug. Recently, specific post-translational modification
sites on histones have been identified and studied in detail
by mass spectrometry. In this review, we will summarize the
mass spectrometric methods to identify histone acetylation
sites and quantify the acetylation level. These methods are
applied to the evaluation of the efficacy of histone deacetylase
inhibitor drugs through modulation of histone acetylation.
[Back to top]
Analytical Methods of Bioactive Metabolites Produced
by Plants and Microorganisms
Antonio Evidente, Anna Andolfi and Mohamed A. Abouzeid
HPLC and/or HPTLC, mass spectrometric and immunochemical
methods for the analysis of natural bioactive metabolites
are described. In particular, methods developed for the analysis
of metabolites from plants and microorganisms in complex mixtures
represent an important tool to investigate the biosynthesis
and the mode of action of these metabolites for their large
scale production and for their practical applications.
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