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Eef programm1.cdr

th Biannual Meeting
of the Hellenic (Greek)
Society for Basic
& Clinical Pharmacology
Professor Arthur Christopoulos, B.Pharm., Ph.D., Drug Discovery, Monash University, Australia Today's science, tomorrow's medicines Athens 23-24
Registration
Young Investigators Forum
Chairs: Dr. E. Papadimitriou
Interaction of pleiotropin with vascular endothelial growth factor 2.
M. Lamprou, E. Papadimitriou The effect of the olive constituent, oleuropein,on lipid homeostasis: role of PPARá.
F. Malliou, I. Andreadou, K. Kypreos, M. Marselos, E. Iliodromitis,L. Skaltsounis, M. Konstandi Endogenous cannabinoids and the inhibitors of their metabolic enzymesprotect the retina from AMPA-induced excitotoxicity in vivo.
D. Kokona, A. Makriyiannis, A. Zimmer, K. Thermos Differential induction of ALDH1A7 in RR and rr rats isassociated with the promoter structure, but not with nuclearreceptor functionality. K. Touloupi, P. Honkakoski, M. Marselos, P. Pappas Association of ABCB1 gene polymorphisms withdyslipidemia. A.Panderi, D. Agapakis, C. Savopoulos,D. Kouvelas, AI Hatzitolios, A. Goulas Coffee Break IUPHAR Immunopharmacology Section
(sponsored and hosted by the Greek Pharmacological Society)
Chairs: Dr K.Tyligada, Prof F. Levi-Schaffer
Katerina Tiligada, PhD (University of Athens, Greece)The newly founded Immunopharmacology Section of IUPHAR Francesca Levi-Schaffer, PhD (The Hebrew Universityof Jerusalem, Israel, Chair of IUPHAR Immunopharmacology Section):In vitro & in vivo models in immunopharmacology:Open-minded for surprises! Madeleine Ennis, PhD (The Queens University of Belfast, UK):Mast cells important not only in allergic diseases but also otherdiseases! Anastassios Germenis, MD, PhD (University of Thessaly, Greece):Design of agonistc altered peptides for immunothepary:the dawn of Quantum Immunology George Kolios, MD, PhD (University of Thrace):Immunological basis for biological therapies in inflammatorybowel disease Lunch/ Poster viewing Academia-Industry partnerships and interactions:
challenges and opportunities
Chairs: Prof Z. Daifoti, Prof M. Maragoudakis
Dimitrios Tzalis, PhD (Taros Chemicals GmbH):Funding opportunities for Pharmacologists through theEuropean Lead Factory (an Innovative Medicines Initiative) Dimitrios Demos, MBA (President, Panhellenic Union ofPharmaceutical Industry) Funding opportunities and interactionswith industry Round table discussion organized by the HellenicAssociation of Pharmaceutical Companies (SFEE)Barbara Baroutsou, ÌD,PhD (Head of Medical Directors Committee):Biomedical research and pharmaceutical industryAggelos Tsakanikas, PhD (University of Athens, Greece)Research and Innovation as a strategic plan for GreeceKostas Athanasakis, PhD (National School of Public Health)Clinical Research in GreeceVicky Soulandrou (Åuropean Public Law Organization)Removing the obstacles to attract more clinical trials in Greece Coffee Break Publishing and peer-review
Chair Prof. C. Flordellis
Jaap Harten, Ph.D. (Elsevier) Research misconduct and publishing ethics Hot topics
Chairs: Prof. N. Sitaras, Prof. K.Thermos
Elias Lolis, PhD (Yale University, USA): Macrophage migration inhibitoryfactor, associated diseases, and its inhibition Antonis Tsarbopoulos, PhD (University of Athens, Greece):Therapeutic Proteins:The Hunt for Post Translational Modifications Vangelis Manolopoulos, PhD (University of Thrace, Greece):Genotype-guided dosing of anticoagulants Keynote Lecture
Chair: Prof. A. Papapetropoulos
Arthur Christopoulos, PhD (Monash University, Australia):GPCR Allostery and Bias in Drug Discovery Welcome reception Pharmacology education(Session organized by the British Pharmacological Society)Chairs: Prof Flower, Dr. McFadzean Ian McFadzean, PhD (King's College London, UK):How the BPS works to promote education in pharmacology within the UK.
Jess Strangward (British Pharmacological Society): Prescribing SafetyAssessment: Raising the profile of Clinical Pharmacology in the UK Stephen Alexander, PhD (University of Nottingham, UK):The IUPHAR/BPS guide to pharmacology Rod Flower, PhD (William Harvey Research Institute, UK)"Pharmacology 2.0" Translating basic pharmacology to drug developmentChairs: Prof A. Gravanis, Prof N. Sakelaridis Anastassis Perrakis, PhD (The Netherlands Cancer Institute, Amsterdam)Autotaxin: from function and structure towards drugs, and back again Dimitris Skokos, PhD Targeting the Delta-4 Notch ligand(Regeneron Pharmaceuticals, Inc., NY, USA) Demetrios Vassilatis PhD (BRFAA, Greece):Innovative therapies for parkinsons diease Lunch/ Poster viewing Cardiovascular pharmacologyChairs: Prof. D. Kouvelas, Dr I.Andreadou Eddie Weitzberg, PhD (Karolinska Institute, Sweden): Nitrate and nitrite:the journey from degradation products to promising therapeutics Kyriakos Kypreos, PhD (University of Patras, Greece):Revisiting the role of HDL in cardiovascular disease Fabio Di Lisa, PhD (University of Padova, Italy):The role of mitochondria in heart disease Stavros Konstantinides, MD, PhD (University of Thrace, Greece): New oral anticoagulants: mechanisms of action, pharmacological properties and clinical data Neuropharmacology: Towards translational neuropsychopharmacology: Focus on neural plasticity theory of Nervous System Disorders Chairs: Prof P. Papaioannidou, Dr. K. Antoniou Catherine Belzung, PhD (UFR Sciences et Techniques, INSERM 930 and University Francois-Rabelais, Tours,France): Neurogenesis, Stress and Depression Christina Dalla, PhD (University of Athens, Greece): Neuroplasticity modulation: effects of stress, gender and antidepressants Ioannis Charalampopoulos, PhD (University of Crete, Greece): Novel synthetic microneurotrophins: potential therapeutic applications in nervous system Awards and Prizes-EPHAR Poster Award Congress Venue :
The Biomedical Research Foundationof the Academy of AthensSoranou Efesiou 4, 115 27 Athens Greece Language :
Official language English Abstracts :
Deadline for Abstract submission February 15, 2014 Registration Fees :
Regular 30,00€Msc/PhD students 20,00€Undergraduate students: free Certificate of Attendance : Certificates will be sent by e mail to the participants
after the Congress.
A SINGLE IN VIVO EXPOSURE TO FENTANYL INDUCES LONG-LASTING REDUCTION
OF GABAERGIC INHIBITION IN THE CA1 AREA OF THE VENTRAL HIPPOCAMPUS.
E. Kouvaras, N. Pipidou, A. Kantikou and E.K. Asprodini.
Laboratory of Pharmacology, Medical School, University of Thessaly, Larissa, Greece. BACKGROUND & OBJECTIVES
The hippocampal formation is differentiated along its longitudinal axis with the ventral pole
being more susceptible to epileptiform discharges. We have shown previously that in vivo
administration of fentanyl reduces GABAergic inhibition and increases excitability in the CA1
area of the dorsal hippocampus. It was the purpose of the present study to examine the effect
of in vivo fentanyl treatment on the inhibitory transmission and excitability of CA1 pyramidal
neurons of the ventral hippocampus.
METHODS
Male Wistar rats were treated either with saline or fentanyl (4x80 μg/kg s.c. every 15min).
One day after treatment, standard intracellular recordings were obtained from 500μm-thick
transverse ventral hippocampal slices of saline- and fentanyl-treated animals. Synaptic
responses were evoked by stimulation of Schaffer collaterals. Comparisons between treatment
groups were made with the Student's t test.
RESULTS
Fentanyl treatment did not alter basic passive and active membrane characteristics (resting
membrane potential (RMP), input resistance, postsynaptic fi ring patterns) of pyramidal
neurons. The peak amplitude of postsynaptic fast (f-) and slow (s-) inhibitory potentials
(IPSPs), measured at 43.6±2.9 ms and 163.2±12.7 ms, respectively, from the onset of the
synaptic response, was smaller in neurons from fentanyl- (f-IPSP: 2.1±0.9mV; s-IPSP: 1.1±0.3,
n=4) compared to saline-treated animals (f-IPSP: 7.3±1.8mV; s-IPSP: 5.8 ±0.5mV, n=4;
p<0.05) when measured at RMP (saline-treated: -65.0±1.0mV, fentanyl-treated: -65.7±1.8mV;
p=0.38).
CONCLUSIONS
In accordance to our previous fi ndings from the dorsal pole of the hippocampus, preliminary
data from the ventral pole points to the reduction of GABA-mediated inhibition 24 hours
following in vivo exposure to fentanyl.
Acknowledgements: This work was supported by the Ministry of Education (Herakleitos II).
DEVELOPMENT OF LC-MS/MS AND LC-UV BASED ANALYTICAL METHODS FOR
DRUG QUALITY CONTROL ASSSESMENT
P. Agrafi otou, C. Tamvakopoulos Division of Pharmacology & Pharmacotechnology, Biomedical Research Foundation, Academy of Athens BACKGROUND & OBJECTIVES
The drug quality control process is not only important for getting a new drug approved but
it is also an important process for ensuring that drugs that have reached the market can be
periodically monitored in order to ensure their stability. For that reason the development
of validated analytical methods is critical and includes procedures, which demonstrate that
a particular method used for determination of analytes in a drug formulation (tablet), is
reliable and reproducible for the intended use. The fundamental parameters for this purpose
include determination of accuracy, precision, selectivity, sensitivity, reproducibility, LLOQ and
stability.
METHODS
High performance Iiquid chromatography (HPLC) connected to a UV/Vis detector was used in
order to develop and validate methods for the identifi cation and quantifi cation of three known
drugs: acetaminophen, omeprazole and meloxicam in pharmaceutical tablets. Furthermore,
LC system connected to a hybrid triple-quadruple/linear ion trap mass spectrometer was used
for additional evaluation of the drugs examined in terms of quality and purity.
RESULTS
Pharmaceutical tablets containing 15 mg meloxicam (from 5 different manufacturers), 20 mg
omeprazole (5 manufacturers) and 500 mg acetaminophen (4 manufacturers) were dissolved
in the appropriate solvent, fi ltered and then examined in terms of the quantity of the drastic
substance and the purity of the drug using the developed validated methods.
CONCLUSIONS
HPLC methods were developed and validated for the determination of meloxicam,
acetaminophen and omeprazole and were applied successfully to commercially available
tablets. The described methods can be readily used for routine quality control of tablets.
DUAL TARGETING OF TUMOUR ANGIOGENESIS AND GNRH-R EXPRESSING
PROSTATE CANCER CELLS, USING NOVEL SUNITINIB-GNRH CONJUGATES.
O. Argyros1, T. Karampelas1, X. Asvos2, D. Fokas3 and C. Tamvakopoulos1 1Division of Pharmacology-Pharmacotechnology, Biomedical Research Foundation Academy of Athens (BRFAA), 2Laboratory of Medicinal Chemistry, Department of Chemistry, University of Ioannina, Greece3Laboratory of Medicinal Chemistry, Department of Materials Science and Engineering, University of Ioannina, Greece BACKGROUND-OBJECTIVES
The Gonadotropin Releasing Hormone Receptor (GnRH-R) is being pursued as a target in
prostate cancer (CaP) for the selective delivery of GnRH-drug conjugates. Here we present the
synthesis and evaluation of novel Sunitinib ANalogues (SANs) that can be conjugated to GnRH
in order to specifi cally target both the GnRH-R1 expressing cells as well as angiogenesis in
the tumour microenvironment. Crucially, our analogues can be readily conjugated to multiple
targeting peptides in order to treat other types of cancers.
METHODS
Several novel SANs were synthesized to allow for direct conjugation of GnRH to Sunitinib. In
vitro molecular targeting and cytotoxicity of SANs were examined in HUVEC, NIH/3T3 and
androgen-independent CaP cell lines (DU145, PC3) and in transgenic DU145 and PC3 cell
lines engineered to stably express GnRH-R1. Initial pharmacokinetic studies were performed
in mice and blood samples were analysed by LC-MS/MS.
RESULTS
All SANs proved effi cacious in the in vitro assays performed, with one specifi c SAN (named
SAN2), displaying strong inhibition of its molecular targets VEGFR2 and PDGFRβ along
with high anti-proliferative activity against all cell lines. Pharmacokinetic studies following
intraperitoneal delivery revealed that SAN2 shows favorable properties that justify its further
development.
CONCLUSIONS
The newly synthesized SAN2 displayed signifi cant effi cacy advantages in vitro compared to
equimolar doses of other SANs and proved equipotent to the original Sunitinib. The development
in our group of GnRH-R1 stable CaP cell lines and further conjugation of SAN2 to GnRH will
allow us to proceed with proof-of-concept in vivo effi cacy and targeting experiments.
SOLUBLE GUANYL CYCLASE (sGC) ACTIVATION BY NO AND HEME-INDEPENDENT
ACTIVATORS: EVIDENCE FOR DIFFERENTIAL REQUIREMENTS OF RESIDUES IN THE
β1 REGULATORY DOMAIN
Z. Zhou1, A. Marazioti2, S. Topouzis2, A. Giannis3, G. A. Spyroulias2, and A. Papapetropou-los14 1"G.P.Livanos-M.Simou" Laboratories, 1st Department of Critical Care Medicine and Pulmo-nary Services, Evangelismos Hospital, School of Medicine, University of Athens, Greece2Laboratory of Molecular Pharmacology, Department of Pharmacy, University of Patras, Greece3Institut für Organische Chemie, Universität Leipzig, Germany4Department of Pharmacy, University of Athens, Greece BACKGROUND & OBJECTIVES
sGC is a heterodimeric α/β hemoprotein that regulates signalling in response to nitric oxide
(NO). We compared the contribution of the β1 regulatory domain to NO and two chemically
distinct heme-independent activators, namely HMR-1766 and BAY 58-2667.
METHODS
Using computational approaches, we identifi ed residues in the N-terminus of β1 that might
infl uence the response to HMR-1766 and BAY 58-2667. After mutagenesis, sGC function was
analyzed in transient transfection experiments.
RESULTS
While exposure of wt sGC-overexpressing cells to HMR1766 led to higher cGMP levels, BAY
58-2667 was a more potent sGC activator. Mutating His to Phe at β1 105 abolished sGC
responsiveness to sodium nitroprusside (SNP), due to heme loss. In contrast, BAY 58-2667
and HMR-1766 activated heme-free H105F sGC. Simultaneous mutation of Y135/R139 to
Ala lead to an enzyme that was unresponsive to both NO and sGC activators. D45E, P74H
and Y83G mutants exhibited a reduced ability to synthesize cGMP in response to SNP and
HMR-1766. The responsiveness L142G to SNP was reduced, while the effect of HMR-1766
remained unaffected. The R116A mutant exhibited unaltered cGMP-synthesizing ability for
HMR-116, a mild reduction towards SNP, but increased cGMP generation after BAY 58-2667
exposure. P118A exhibited reduced responses to SNP and HMR-116, but unaltered behaviour
towards BAY 58-2267. Exposure of wt enzyme to the heme oxidant ODQ reduced sGC levels;
this effect was totally reversed by BAY 58-2667, but only partially by HMR-1766.
CONCLUSIONS
We have identifi ed key residues that are important for the differential responsiveness of sGC
to NO/heme-independent sGC activators and NO-releasing drugs.
IDENTIFICATION AND DEVELOPMENT OF STRUCTURALLY NOVEL INHIBITORS
AGAINST THE H1047R MUTANT OF PI3Kα
E. Kouvari1, O. Argyros1, P. Marakos2, N.Pouli2, N. Lougiakis2, S.Christoforidis3, E. Mikros2, L.Skaltsounis4 C. Tamvakopoulos11Division of Pharmacology-Pharmacotechnology, Center of Basic Research, Biomedical Research Foundation, Academy of Athens, Athens, Greece.
2Division of Pharmaceutical Chemistry, Department of Pharmacy, University of Athens, Athens, Greece.
3Laboratory of Biological Chemistry, School of Medicine & Department of Biomedical Research, Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology (BR-IMBB/FORTH), University of Ioannina, Ioannina, Greece.
4Division of Pharmacognosy and Natural Products Chemistry, Department of Pharmacy, University of Athens, Athens, Greece. BACKGROUND & OBJECTIVES
PIK3CA gene, coding for the catalytic subunit of PI3Kalpha (p110α), is one of the most
frequently mutated oncogenes in human tumors. Although several inhibitors against the wild-
type PI3Kα enzyme are currently in clinical trials, none of them is selective for the mutated
form, increasing the risk for potential side effects. Here, we present our efforts to develop
novel inhibitors, targeted against the most common mutation of p110α (H1047R ), using a
library available in the University of Athens, containing natural products and novel synthetic
compounds.
METHODS
Evaluation of the cytotoxicity of 275 compounds of various chemical scaffolds against human
breast cancer cell lines was performed using an in vitro cell viability assay (MTT). A modifi ed
cell-free assay was employed to assess the selectivity of potential inhibitors against the wild-
type or the H1047R mutated enzyme. We also performed initial Pharmacokinetic evaluation of
lead compounds using Liquid Chromatography-Tandem Mass Spectrometry techniques.
RESULTS
Pyridopyrazine 1043 showed potency in the MTT assay (IC 10μΜ) and the cell-free assay
(IC 15μΜ), but no selectivity for the H1047R mutation. Pyrazolopyridines 1113 and 1119 also proved to be very potent but not selective PI3Kα inhibitors in all the in vitro assays with an IC at the low micromolar level. 1043 is orally and intraperitoneally bioavailable in mice and showed good blood exposure after a single dose of 5mg/kg, Cmax(2h)=1.7μM. CONCLUSIONS
Several potent PI3Kα inhibitors were discovered, with favorable pharmacokinetic properties at
preliminary doses. These compounds represent lead chemical structures, certainly worthy of
further evaluation and optimization for the development of specifi c H1047R inhibitors.
3D VASCULARIZED TUMOUR SPHEROIDS:
AN IN VITRO MODEL OF CANCER ANGIOGENESIS.
O Tsigkou1, PK Politis2 and C Tamvakopoulos11Division of Pharmacology-Pharmacotechnology, Biomedical Research Foundation, Academy of Athens, Athens, Greece, 2Center for Basic Research, Biomedical Research Foundation of the Academy of Athens, Athens, Greece.
BACKGROUND & OBJECTIVES
Angiogenesis, the formation of new vessels, is crucial for tumour progression and metastasis.
Typical monolayer cell culture (2D) assays have offered tremendous insight in fundamental
cancer biology and drug development. However, the microenvironment within which cells
reside and interact with the tumour extracellular matrix is more complex. The objective of this
study is to develop 3D vascularized tumour spheroids (TμSph) that recapitulate the tumour
microenvironment and improve the in vitro assessment of cancer therapeutics.
METHODS
For TμSph formation, HUVECs, MSCs and human breast cancer and hepatoma cell line
(MDA, MCF-7, HepG2 and Huh-7) transduced with lentiviruses, were mixed in a fi bronectin
containing collagen gel. TμSph were treated with various concentrations of sorafenib and
cilengitide and their effect on cell metabolic activity, angiogenesis, migration and metastasis
was assessed in vitro.
RESULTS
Microscopy demonstrated the formation of capillary-like network within the TμSph with lumen
like structures (Figure 1). TμSph treated with 0-50μM sorafenib and 0-200μM cilengitide
demonstrated that higher drug concentrations are necessary for destruction of the vascular
network, cell migration and toxicity compared to 2D cell cultures. Gene (real time Reverse
Transcription-Polymerase Chain Reaction (rt RT-PCR) and protein (Western Blotting)
expression profi les and signalling pathways affected by the drugs, and how these compare to
2D cultured cells will also be presented.
CONCLUSIONS
TμSph pose a more promising in vitro drug-screening model compared to 2D standard cell
culture assays.
TARGETED MASS SPECTROMETRY-BASED METHODOLOGIES FOR THE
DETERMINATION OF LIPID BIOMARKERS ASSOCIATED WITH ALLERGIC AIRWAY
INFLAMMATION & ITS RESOLUTION
K. Pyrillou1,2, P. Agrafi otou1, E. Andreakos*2, C. Tamvakopoulos*11Division of Pharmacology – Pharmacotechnology, Center of Basic Research2Laboratory of Immunogenetics, Center of Immunology and TransplantationBiomedical Research Foundation of the Academy of Athens (BRFAA) BACKGROUND & OBJECTIVES
Ω3 and Ω6 polyunsaturated fatty acid (PUFA)-derived lipid mediators (LM) have a signifi cant
contribution in the homeostasis but also in pathological infl ammatory conditions of the lung.
LM have been implicated in multiple infl ammatory mouse models. Such studies attributed
specifi c functions to different families of LM; pro-infl ammatory LM are responsible for the
mounting of an acute infl ammatory response while specialized pro-resolving lipid mediators
(SPMs) regulate its timely resolution. The objective is to study the biosynthesis of selected LM
in a pre-clinical model of allergic airway infl ammation (AAI) and further evaluate their use as
potential biomarkers.
METHODS
AAI was induced in C57Bl/6 mice following sensitization with ovalbumin (OVA) and challenges
with nebulized OVA. Tissues were collected during the resolution phase. LC-MS/MS methods
were developed an applied towards the determination of 13 selected LM with relevance to
AAI, in serum and lung tissue.
RESULTS
Highly sensitive LC-MS/MS methods developed were validated for application in mouse
serum and lung. Analysis of lung tissue obtained during the resolution phase of the AAI model,
revealed increased concentrations of Protectin D1 (PD1) and Protectin DX (PDX) only in
mice with infl amed airways. Their direct upstream biosynthetic precursor, 17-HDHA, was also
upregulated.
CONCLUSIONS
The present study investigated the kinetics of LM production in a self-resolving mouse model
of AAI. SPMs of the Protectins' family showed good correlation with the resolution phase
suggesting their potential use as biomarkers of this process. These fi ndings may be of value
in clinical conditions were pharmacological intervention will be required.
Acknowledgements : This research project is funded by FP7 – PreDicta.
* C.T and E.A share authorship.
(247
NOVEL GNRH-GEMCITABINE CONJUGATES FOR THE TREATMENT OF ANDROGEN-
INDEPENDENT PROSTATE CANCER: TARGETED DRUG DELIVERY COMBINED WITH
PHARMACOKINETIC ENHANCEMENTS
T. Karampelas1, O. Argyros1, N. Sayyad2, A. G. Tzakos2, D. Fokas3, C. Tamvakopoulos1 1) Biomedical Research Foundation, Academy of Athens, Division of Pharmacology-Pharmacotechnology, Soranou Ephessiou Street 4, Athens GR-11527, Greece.
2) Department of Chemistry, Section of Organic Chemistry and Biochemistry, University of Ioannina, Greece.
3) Laboratory of Medicinal Chemistry, Department of Materials Science and Engineering, University of Ioannina, Greece. BACKGROUND AND OBJECTIVES
Gemcitabine, a drug with established effi cacy against a number of solid tumors, has therapeutic
limitations due to its rapid metabolic inactivation. The aim of this study was the development
of an innovative strategy to produce a metabolically stable analogue of gemcitabine that could
also be selectively delivered to prostate cancer (CaP) cells based on cell surface expression
of the Gonadotropin Releasing Hormone-Receptor (GnRH-R).
METHODS
The synthesis and evaluation of conjugated molecules, consisting of gemcitabine linked
to a GnRH agonist, is presented along with results in androgen-independent prostate
cancer models. NMR and ligand binding assays were employed to verify conservation of
microenvironments responsible for binding of novel GnRH-gemcitabine conjugates to the
GnRH-R. In vitro cytotoxicity, cellular uptake and metabolite formation of the conjugates
were examined in CaP cell lines. Evaluation of effi cacy of selected candidates with the best
pharmacokinetic properties was based on a GnRH-R positive androgen-independent CaP
animal model.
CONCLUSIONS
Selected conjugates were effi cacious in the in vitro assays with one of them, namely GSG,
displaying high antiproliferative activity in CaP cell lines along with signifi cant metabolic and
pharmacokinetic advantages in comparison to gemcitabine. Finally, treatment of GnRH-R
positive xenografted mice with GSG, showed a signifi cant advantage in tumor growth inhibition
when compared to gemcitabine.
PRESCRIPTION MEDICATIONS AND HERBAL DRUGS INTERACTIONS
Τsakiri Anastasia, Kotsiou Antonia, Tesseromatis ChristineDepartment of Pharmacology Medical School. University of Athens,Mikras Assias 75, 11527 Greece Correspondent author : Associated Professor Ch. Tesseromatis, Dept. of Pharmacology, Medical School, Athens University, M. Assias 75, 11527, Athens, GreeceTel.: +30210 746 25 73 Fax.: +30210 746 25 55, e-mail: [email protected] ABSTRACT
BACKGROUND AND OBJECTIVES
Plants have been used for disease treatment since Hippocrates era and in recent years
the consumers preference towards herbal preparations presents a remarkable increase.
The herbal supplements and alternative treatments play a signifi cant role in patients' choice
for health maintenance, better physical status, disease fi ght and cosmetic purposes. Both
prescription medications and medicinal plants exert a pharmacodynamic effect on biological
systems and their concurrent administration may cause severe side effects due to interference
interactions. Herbs are organisms containing various active ingredients that exhibit changes
on the biological substrate. Concurrent use of herbs may mimic, magnify, or oppose the
effect of drugs. Potential herb-drug interactions include: bleeding when warfarin is combined
with plants contained coumarin derivate like ginkgo (Ginkgo biloba) garlic (Allium sativum
and other) or serotonin-reuptake inhibitors with St John's wort (Hypericum perforatum)
Moreover St John's wort decreased bioavailability of digoxin, theophylline, cyclosporin, and
phenprocoumon ; combined antidepressants and Panax ginseng induce mania in depressed
patients; anthranoid-containing plants ( senna and cascara) and soluble fi bres ( psyllium) can
decrease the absorption of drugs. Health-care practitioners should caution patients against
mixing herbs and pharmaceutical drugs.
The aim of this paper was to investigate the interactions between prescription medications
and herbal drugs
METHODS
Extensive literature survey on herbs - drugs interactions
RESULTS
Many reports of herb-drug interactions lack laboratory analysis of suspect preparations. The
mechanisms of interactions are induction of cytochrome P450 and isoenzymes CYP3A4,
CYP2C9, CYP1A2.
CONCLUSIONS
Health care practitioners should be aware of the herbal properties and discourage patients,
who take drugs with narrow therapeutic index (warfarin, digoxin, hypoglemic agents,
theophylline, antidepressants) to use herbal products.
IN VITRO SLICE PREPARATION – RELATING FUNCTION TO LABELLING
E. Kouvaras1, 2, N. Pipidou1, A. Kantikou1, G. Koukoulis2 and E.K. Asprodini1.
Laboratory of 1Pharmacology and 2Pathology, Medical School, University of Thessaly, Larissa, Greece. BACKGROUND & OBJECTIVES
Coupling of neuronal functional characteristics to specifi c markers and morphological
attributes has always been challenging in neuroscience research. Based on our recent
fi ndings that immersion fi xation of hippocampal slices exhibits satisfactory histological and
immunohistochemical staining, we used the in vitro slice preparation to study functional
GABAergic inhibition in relation to GABA immunolabelling.
METHODS
Hippocampal transverse slices (500μm thick) were prepared from male Wistar rats and
maintained in oxygenated ACSF. Sharp-electrode intracellular recordings were obtained from
CA1 pyramidal neurons; synaptic responses were evoked by stimulation of Schaffer collaterals.
At the end of the electrophysiological recordings (3-8 hours post brain excision), slices were
fi xed by immersion (4% PFA/0.1Μ PB) and embedded in paraffi n. Consecutive paraffi n 4μm-
thick slices were stained histologically using Eosin/Hematoxylin or immunohistochemically
for GABA using primary polyclonal rabbit antibody against GABA (1:500) and fl uoroscein-
conjugated secondary goat anti-rabbit IgG (H+L) (1:400).
RESULTS
Neurons exhibited basic electrophysiological passive and active membrane properties (resting
membrane potential, input resistance, postsynaptic fi ring patterns), similar to previously
described. Pronounced GABA-mediated synaptic inhibition was associated with the presence
of numerous GABA-immunofl uorescent neurons in stratum radiatum. Within the same layer,
Eosin/Hematoxylin-stained slices demonstrated neurons with normal ultrastructure and clear
and evident nuclei and nucleoli.
CONCLUSIONS
Although preliminary, our results suggest that immersion fi xation may be used for the histological/
immuhistochemical study of tissue which has been previously studied electrophysiologically in
vitro. The broader signifi cance of this method is that neuronal function can be directly related
to diverse markers and mechanisms associated with them.
Acknowledgements: This work was supported by the Ministry of Education (Herakleitos II).
EFFECT OF PROTON PUMP INHIBITORS ON THE PYLORIC SPHINCTER OF THE
RABBIT
G.A. Delis, T.A. Psarra, G.C. Batzias, M. Koutsoviti-Papadopoulou
Laboratory of Pharmacology, School of Veterinary Medicine, Faculty of Health Sciences,
Aristotle University of Thessaloniki, Greece
BACKGROUND & OBJECTIVES
Failure of proton pump inhibitors (PPIs) in effectively treating gastro-esophageal refl ux
disease (GERD) is due to continuing non-acidic refl ux, attributed either to the relaxing effect
of these agents on the lower esophageal sphincter tone, or to the delayed gastric emptying.
Considering that the pyloric sphincter (PS) contractile activity plays an important role in gastric
emptying, this study aimed to investigate the pharmacodynamic effects of omeprazole and
lansoprazole, two currently prescribed PPIs, on the spontaneous contractile activity of the PS
of the rabbit.
METHODS
Following careful removal of the mucosa, rabbit PS strips were prepared and placed in
organ baths containing warmed (37oC) and gassed (95%O -5%CO ) Krebs solution. Full
concentration-response curves of both PPIs were constructed.
RESULTS
The PS strips developed spontaneous phasic contractions. Both PPIs tested, at concentrations
ranging from 10-5 to 10-3 M, produced only a very small decrease in the basal tone, while they
considerably inhibited both the amplitude and frequency of phasic contractions; the latter were
completely suppressed at PPIs concentrations higher than 10-3 M.
CONCLUSIONS
Gastric emptying is a highly synchronized process involving the integration of the propulsive
forces of the antrum contractions and the inhibitory ones of the pyloric muscle. In view of this,
the here observed inhibitory effect of PPIs on the PS contractile activity may lead to reduced
pyloric resistance and to rather facilitate than delay gastric emptying. Conclusively, the PS
response to PPIs does not seem to contribute to the continuing refl ux observed in a proportion
of GERD patients undergoing PPIs treatment.
NOVEL PYRAZOLOPYRIDINES DERIVATIVES AS POTENTIAL ANTI-ANGIOGENIC AGENTS
Michailidou, M.1, Giannouli, V.2, Kostakis, I.K.2, Marakos, P.2, Pouli N.2, Loutrari, H.1
11st Department of Critical Care Medicine & Pulmonary Services, G.P. Livanos and M. Simou Laboratories, Evangelismos Hospital, Medical School, University of Athens 2 Division of Pharmaceutical Chemistry, Department of Pharmacy, University of Athens BACKGROUND & OBJECTIVES
Receptor and non-receptor tyrosine kinases (TK) are essential regulators of normal cell
homeostasis. Dysregulation of these signalling mediators is often associated with serious
clinical disorders including cancer. Inhibitors of certain TK members, such as vascular
endothelial growth factor receptors (VEGFR) which are known to promote tumour-induced
angiogenesis, have been successfully used in cancer therapy. Since quinazoline and
pyridopyrimidine derivatives have been proved to be potent and highly selective inhibitors of
TK, in this work we aimed to synthesize novel substituted pyrazolopyridines and explore their
capability to modulate crucial stages of the angiogenesis process.
METHODS
Compounds of the general structure shown below:
were synthesized using 2-amino-5-nitro-4-picoline as starting material, which upon suitable transformations was converted to 5-chloropyrazolo[3,4-c]pyridine. A 4-methoxybenzyl group was inserted at N1 of the heterocyclic ring system followed by the addition of a 3-phenyl group. A second chlorine atom was then introduced at position 7 and then both chlorine atoms were successively displaced by suitable amines.
The biological effects of these compounds on the angiogenic phenotype of primary human umbilical vein endothelial cells (HUVEC) were investigated using established in vitro assays of cell proliferation (MTT), migration (transwell) and differentiation into tube-like structures (Matrigel).
RESULTS
Amongst test compounds, three were able to signifi cantly inhibit HUVEC growth, chemotactic
migration and tube-like formation in a concentration-dependent manner, though with a diverse
effi ciency.
CONCLUSIONS
Overall, three novel pyrazolopyridines derivatives with promising anti-angiogenic activities
were identifi ed. Analysis of structure-function relationships allowed detection of optimal
structural requirements for anti-angiogenic activity.
TITLE: DIFFERENT PRODUCTION OF HYDROGEN SULFIDE (H S) IN ACUTE AND
CHRONIC EXPERIMENTAL COLITIS
AUTHORS (FIRST NAME, LAST NAME): A.A. Apostolou *, A.P. Papapetropoulos#, K.K. Karalis * *Developmental Biology Section, Center for Basic Research, Biomedical Research Foundation of the Academy of Athens, Athens, Greece; #Faculty of Pharmacy, Department of Pharmaceutical Chemistry, University of Athens, Athens, Greece BACKGROUND & OBJECTIVES:
Ulcerative colitis (UC) is a chronic Infl ammatory Bowel Disease (IBD), with increasing
incidence particularly in younger ages, and signifi cant impact in the quality of life of the affected
individuals. From recent studies it becomes evident that miscommunication between gut
microbiome and the host innate immunity represents a major part in the pathogenesis of the
disease. Genetics is implicated by affecting the susceptibility, although it has not been shown
to act in a predominant fashion. Because of the complex nature and progress of the disease,
treatment choices remain limited. Further understanding of its pathophysiology may lead to
the identifi cation of more specifi c and effi cient treatment regimens. Hydrogen sulfi de (H S)
is a water- soluble gas produced in many mammalian tissues, including gastrointestinal tract, through metabolism of L-cysteine by enzymes including cystathionine β- synthase (CBS), cystathionine γ- lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (3-MPST). It has been shown that H S can impact on the development of infl ammation, whereas it has been reported to accelerate healing of injured gastrointestinal tissue. Our aim is to characterize the contribution of H S in the development of experimental colitis in mice and its specifi c impact during the infl ammatory and repair phases of the disease.
METHODS
We used C56Bl6 male mice and applied an experimental model of innate immunity- dependent
ulcerative colitis, the Dextran Sodium Sulfate (DSS) model. DSS was administered in their
drinking water and animals were sacrifi ced in different time points. Clinical signs of the disease,
histology and the expression levels of CBS, CSE and 3-MPST enzymes were determined
using Q-PCR and Western Blot in the affected tissue. The statistical analysis of the results
was done by Two-way and One- way ANOVA.
RESULTS
Expression of all three enzymes, i.e. CBS, CSE and 3-MPST, implicated in H S metabolism
was detected over the course of the disease, both at the gene and protein levels. We found that the mRNA and protein levels of all enzymes were signifi cantly increased during the fi rst three days after the onset of the disease coinciding with the active infl ammatory process in the tissue. interestingly, we detected signifi cantly decreased expression in the phase of tissue repair refl ecting the resolution phase of infl ammation. CONCLUSIONS
Our fi ndings indicate a phase of the disease-dependent regulation of CBS, CSE and 3- MPST
enzymes at the transrcriptional and translational level. The signifi cance of the specifi c regulation
of H2S production in the infl amed gut in the pathogenesis of UC is under investigation.
STRUCTURE AND MOLECULAR MECHANISMS OF ACTIVATION OF CRF RECEPTOR
Spyridaki K.1, Matsoukas M.-T. 2, Cordomi A.2, Karageorgos V.1, Fothiadaki M.1,
Mavromoustakos T.3, Margioris A.4, Pardo L.2, Liapakis G.1

1Department of Pharmacology, School of Medicine, University of Crete, Heraklion, 71003,
Greece
2Laboratori de Medicina Computacional, Facultat de Medicina, Universitat Autonoma de
Barcelona, Bellaterra 08193, Spain
3 Laboratory of Organic Chemistry, Department of Chemistry, University of Athens, Athens
11571, Greece
4Laboratory of Clinical Chemistry, School of Medicine, University of Crete, Heraklion, 71003,
Greece
BACKGROUND & OBJECTIVES
The 41-amino-acid peptide corticotropin releasing factor (CRF) plays a key role in the response
of our body to stressful stimuli and the maintenance of homeostasis. These effects are mostly
mediated by its type 1 receptor (CRF R). Malfunctioning of CRF/CRF -neuronal circuits
is closely associated with the appearance of anxiety and depression, whereas small non-peptide CRF R antagonists have been shown to be effective against these neuropsychiatric disorders. The CRF R is a class B G-protein coupled receptor (GPCR), which contains seven alpha helical transmembrane domains (TMs). Here, we aimed to obtain structural information for the third TM (TM3) of the apo state of full length CRF R and to elucidate the molecular mechanisms underlying receptor activation. TM3 was selected because in class A receptors this helix has been shown to form key interactions with ligands, other TMs, and G proteins. METHODS
To accomplish this we used pharmacological, biochemical and computational approaches.
RESULTS
Mutation of Phe203 to various amino-acids affected differentially the ligand binding and
signaling properties of CRF R.
CONCLUSIONS
Phe203 in TM3 plays an important role in CRF R activation, possibly by participating in
functionally important inter-TM interactions. Our results also suggest that Phe203 interacts with non-peptide antagonists, interfering in the interaction network between TM3 and the remaining TMs that stabilize the active state of CRF R. These results provide insight into the molecular mechanisms underlying CRF R activation and also hold great potential for the design of more effi cient small non-peptide CRF R antagonists with anxiolytic and antidepressant properties.
RESISTIVE BREATHING AGGRAVATES ENDOTOXIN- INDUCED LUNG INFLAMMATION:
THE ROLE OF SOLUBLE GUANYLYL CYCLASE
A Pavlidou1,2, C Glynos1, 2, D Toumpanakis1, C Loverdos1, Z Zhou1, V Karavana1, C Magkou3,
A Papapetropoulos2, and T Vassilakopoulos1
1George P. Livanos and Marianthi Simou Laboratories, Evangelismos Hospital, 1st Department of Pulmonary and Critical Care, University of Athens, Athens, Greece. 2Faculty of Pharmacy, University of Athens, Athens, Greece. 3 Department of Histopathology, Evangelismos Hospital, Athens, Greece. BACKGROUND & OBJECTIVES
During exacerbations of chronic obstructi ve pulmonary disease (COPD), increased lung
infl ammation and the accompanying bronchocostriction lead to signifi cant airway narrowing,
i.e. resistive breathing (RB). Thus, we investigated the effect of RB in mice with pre-existing
lung infl ammation by endotoxin inhalation. Since smooth muscle tone is a crucial determinant
of bronchocostriction and involves nitric oxide (NO) signaling through activation of the soluble
guanylyl cyclase (sGC), we aimed to investigate the expression and downstream signaling of
sGC in the context of RB and pre-existing infl ammation.
METHODS
C57BL/6 mice were subjected to RB by restricting tracheal surface area to 50% (tracheal
banding): 1. Sham operated (controls) and TB mice 2. Mice treated with inhaled LPS
pseudomonas aureginosa for 24 hours. 3. Mice treated with inhaled LPS prior TB. After the
end of the experiments the lungs were lavaged; the recovered BALF was used to count cells,
protein concentration and cytokines. We obtained lung tissue samples for immunoblotting
focused on sGC signaling.
RESULTS
Mice subjected to LPS and TB exhibited an increased BALF cellularity, protein content and
cytokines concentration, tumor necrosis factor (TNF)-α, interleukine(IL)-1β and IL-6, compared
with mice treated with inhaled LPS alone. TB downregulated sGC more in pre-existing lung
infl ammation compared with LPS mice.
CONCLUSIONS
TB aggravates pre-existing lung infl ammation in mice and induces a pronounced downregulation
of sGC. Our model of TB might offer new insights by which resistive breathing can enhance
infl ammation in the obstructive lung disease.
CIGARETTE SMOKING ABROGATES THE INFARCT LIMITING EFFECTS OF ISCHEMIC
POSTCONDITIONING THROUGH INSUFFICIENT ACTIVATION OF AKT AND eNOS
SI Bibli1, C Glynos2,3, I Andreadou1, A Chatzianastasiou3, Stavros Topouzis2, EK Iliodromitis4, A Papapetropoulos1 1 Faculty of Pharmacy, University of Athens, Greece;2 Laboratory of Molecular Pharmacology, Department of Pharmacy, University of Patras, Greece;3 1st Department of Critical Care & Pulmonary Services, Faculty of Medicine, University of Athens, Greece;4 2nd Department of Cardiology, "Attikon" General Hospital, Faculty of Medicine, University of Athens, Greece BACKGROUND & OBJECTIVES:
Cigarette smoking is an established risk factor for cardiovascular disease. Ischemic conditioning
decreases myocardial infarct size under physiological conditions, however, comorbidities
ameliorate its benefi cial effects. We investigated the effects of cigarette smoke (CS) exposure
on myocardial infarction and its interference with conditioning mechanisms.
METHODS:
C57BL/6 mice were divided in 2 groups: Control (RA; exposed to Room Air) or exposed to CS
for 4 weeks . Animals from both groups were further divided into 3 subgroups and subjected
to 30 min myocardial isc followed by 2 hours of rep: 1) Control, 2) PreC, 5 min isc-5 min
rep applied before sustained isc and 3) PostC, 3 cycles of 10 sec isc-10 sec rep applied
immediately after sustained isc. The infarct size and area-at-risk were estimated (%I/R).
RESULTS:
CS and RA had similar infarct size (38.3±3.6% vs 40.9±2.2%, p=NS). PreC was benefi cial
for both CS (12.1±1.6%) and RA (17.9±1.8%), p<0.05 vs Control. PostC failed to limit infarct
size in CS animals, versus RA mice (43.3±2.2% vs 17.0±2.7%, p<0.05). Tissue cGMP
concentration, sGC, eNOS, iNOS and p-VASP were increased in CS vs RA. PreC in both
groups and PostC in RA group activated AKT, whereas PostC failed to enhance p-AKT in the
CS group. Enhanced p-eNOS was evident in preconditioned RA and CS, and PostC RA, but
not in PostC CS.
CONCLUSIONS:
Exposure to CS does not per se increase infarct size. Cardioprotection by PreC is preserved
in CS mice, possibly through activation of Akt/eNOS, while the postconditioning benefi t is
lost.
EFFECTS OF ANESTHETIC KETAMINE ON ANXIETY-LIKE BEHAVIOUR IN RATS
N. Pitsikas1, F. Delis2, K. Antoniou2 1Department of Pharmacology, School of Medicine, University of Thessaly, Larissa, Greece 2Department of Pharmacology, School of Medicine, University of Ioannina, Ioannina, Greece BACKGROUND & OBJECTIVES
There is poor experimental evidence concerning the effects of anesthetic doses of the non- competitive N-methyl-D-aspartate (NMDA) receptor antagonist ketamine on anxiety. The present study investigated the effects of anesthetic ketamine (100 mg/kg, i.p.) on anxiety-like behaviour in rats.
For this purpose, the light/dark (LD) and the open fi eld (OF) tests were used. The effects of anesthetic ketamine on motility were assessed using a motor activity chamber. Anesthetic ketamine tested 24 h after treatment induced decreases in the number of transitions between the light and dark chambers and time spent in the light chamber compared to the vehicle-treated animals in the LD test. In the OF test, rats that received anesthetic ketamine spent less time in the periphery and the center of the apparatus compared to their control cohorts. Motor activity of ketamine-treated animals was lower to that displayed by the vehicle- Interestingly, the anesthetic ketamine induced effects on anxiety indices were differentiated following an interval of 48 h post-treatment. The rats displayed an anxiogenic-like profi le in the LD procedure but not in the OF test, while ketamine did not infl uence rats' motor activity. The present results indicate that an anesthetic dose of ketamine induced a decrease in motor activity 24 h post-treatment, while produced an anxiogenic-like profi le that cannot be attributed to changes in motor activity 48 h post-treatment. THE NOVEL DEHYDROEPIANDROSTERONE (DHEA) ANALOGUE BNN27
COUNTERACTED DELAY-DEPENDENT AND SCOPOLAMINE-INDUCED
PERFORMANCE DEFICITS IN A RECOGNITION MEMORY TASK IN RATS
Department of Pharmacology, School of Medicine, University of Thessaly, Larissa, Greece BACKGROUND & OBJECTIVES:
Consistent experimental evidence suggests the involvement of neurosteroid dehydroepiandrosterone (DHEA) in cognition. BNN27 is a novel DHEA analogue, which devoid of steroidogenic activity and its neurotrophic effect has been observed. Its role, however, in learning and memory has not yet established. Thus, the present study was designed to investigate the effects of BNN27 on rats' recognition memory.
For this purpose, the novel object recognition task was used.
Post-training administration of BNN27 (3-10 mg/kg, i.p.) reversed extinction of recognition memory in the normal rat. In addition, BNN27 (3 and 10 mg/kg, i.p.) counteracted the scopolamine (0.2 mg/kg, s.c.)-induced performance defi cits in this recognition memory task. The present results indicate that this novel DHEA analogue may modulates different aspects of recognition memory and suggest that its interaction with the cholinergic system is relevant INDEPENDENT AND SYNERGISTIC EFFECTS OF THE MAPT AND SNCA GENES IN
PARKINSON'S DISEASE AND SCREENING OF COMPOUNDS FOR THE PREVENTION
OF TAU-INDUCED DEFECTS IN DROSOPHILA VISUAL SYSTEM
N. Refenes1,2,4, R. Kreutz2, J. Bolbrinker2, G.Tagaris3, K. Papanikolopoulou4, E. M. C. Skoulakis4, Nikolaos Drakoulis1 1School of Pharmacy, Department of Pharmaceutical Technology, National and Kapodistrian University of Athens, Athens, Greece; 2Institute of Clinical Pharmacology and Toxicology, Charité – Universitätsmedizin Berlin, Berlin, Germany; 3Department of Neurology, "G. Gennimatas" General Hospital, Athens, Greece; 4Institute of Cellular and Developmental Biology, Biomedical Sciences Research Centre "Alexander Fleming" BACKGROUND & OBJECTIVES
Interaction of α-synuclein and protein tau has been proposed as a possible mechanism in
Parkinson's disease (PD). The utility of the Drosophila system in testing candidate compounds
against tau-dependent neuronal dysfunction has been determined.
METHODS
An inversion polymorphism of the microtubule-associated protein tau (MAPT) gene defi nes
two haplotypes, H1 and H2. We set out to determine whether a) H1 haplotype, b) SNPs
included in H1, c) subhaplotypes within H1 defi ned by these SNPs, and d) the synergistic
interaction between the MAPT H1H1 genotype and the SNP rs356219 from the 3′ region
of SNCA (α-synuclein gene), are associated with PD, in patients and control subjects of Greek
and Italian origin. We utilized transgenic fruitfl ies carrying human tau genes expressed under
the UAS(upstream activator sequence)/Gal4 system to investigate the ability of candidate
compounds to inhibit tau-mediated degeneration of photoreceptor neurons.
RESULTS
Overall the frequency of the H1H1 genotype was 63.35 % in PD cases and 56.6% in controls
(OR: 1.33, 95% CI: 0.98-1.79). There was no signifi cant difference between cases and control
subjects in the overall frequency distribution of H1 SNPs, H1 subhaplotypes, SNCA rs356219
GG marker and its combination with MAPT H1H1 genotype. A 1,2,3-dithiazole compound
attenuated the disruption of the regular array of ommatidia in Drosophila eye, but this result
could not be replicated.
CONCLUSIONS
Our data show strong evidence of an association between the H1H1 genotype and PD. The
fi nding from the present study might constitute a basis for the des ign of new compounds with
improved activity in repressing transgenic tau in a Drosophila model.
TETRACYCLINE ANTIBIOTICS AS INHIBITORS OF H S SYNTHESIS
A. Asimakopoulou1, Z. Zhou2, C. Coletta3, C. Szabo3, A. Papapetropoulos2,4 1Dept. of Pharmacy, Lab of Molecular Pharmacology, University of Patras, Patras, Greece; 2‘G.P.Livanos' Laboratory, Department of Critical Care and Pulmonary Services, Faculty of Medicine, University of Athens, Athens, Greece; 3Dept. of Anesthesiology, University of Texas Medical Branch, Galveston, TX, USA4Faculty of Pharmacy, University of Athens, Athens, Greece BACKGROUND & OBJECTIVES:
Hydrogen sulfi de (H S) has emerged as a gaseous signaling molecule with important roles in
neuromodulation, blood pressure regulation, angiogenesis, cellular energetics and apoptosis. H S is produced endogenously by three enzymes in the sulfur metabolic network, namely cystathionine-β-synthase (CBS), cystathionine-γ-lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (3MST). Although several compounds that inhibit CSE are available, no selective inhibitors for CBS or 3MST currently exist. In the present study we screened the LOPAC library for inhibitors of H S-producing enzymes. METHODS:
The methylene blue assay was used to measure H S production from recombinant enzymes.
HCT116 and HT29 cells were used for MTT-based proliferation assays.
RESULTS:
Screening of the LOPAC library yielded several hit compounds that blocked Η S-synthesizing
enzymes to varying extents. Among those, doxycycline and demeclocycline were found to inhibit 3MST by approximately 50% at 100 uM, with doxycycline being a more potent 3MST inhibitor than demeclocycline in the in vitro activity assay. Tetracycline, meclocycline, oxytetracycline, methacycline, tigecycline and minocycline did not affect 3MST activity when used up to 300 uM. From the above-mentioned tetracyclines, tigecycline at 100 uM caused only a slight inhibition of both CBS and CSE; tigecycline additionally inhibited CSE. In a cell-based assay, demeclocycline, but not doxycycline, inhibited the growth of HCT116 and HT29, two colon cancer cell lines in which CBS is highly expressed and H S supports cell CONCLUSIONS:
We conclude that based on its selectivity towards 3MST, demeclocycline could serve as a lead
compound that after undergoing optimization could yield future selective 3MST inhibitors.
ASSOCIATION OF COL5A1, MMP3, COL1A1 GENES WITH INJURIES DURING SPORTS
ACTIVITY IN A GREEK POPULATION OF 739 SUBJECTS
Zouli K., Drakoulis N. National and Kapodistrian University of Athens, School of Health Sciences, Faculty of Pharmacy. BACKGROUND & OBJECTIVES
Tendon and ligaments within the upper and lower limbs are some of the more common sites
of musculoskeletal injuries during physical activity. Several extrinsic and intrinsic factors have
been shown to be associated with these injuries. More recently, studies have suggested that
there is also, at least in part, a genetic component to the Achilles tendon (AT) and anterior
cruciate ligament (ACL) injuries. Sequence variants of the COL5A1, MMP3 and COL1A1
genes showed association with Achilles tendinopathies and ACL ruptures. Those genes
encode for proteins directly involved in biological processes within tendons and ligaments.
METHODS
In this genetic association study, the 739 recruited Greek participants consisted of 378 females
(51,2%) and 361 males (48,8%) aged from 1 to 94 years. The participants were genotyped for
the COL5A1 rs12722 (C/T), MMP3 rs679620 (A/G) and COL1A1 rs1800012 (G/T) sequence
variants. The cohort was divided into 7 subgroups according to the SNP or/and the SNPs
combination examined.
RESULTS
The vast majority of genotype combinations appeared to have a different impact on the
risk of each injury. The genotype combination CT+AG+GG, (over-represented among
our population within the group VII, 14,9%), was associated with increased risk of ACL
rupture (GS=4), although it tended to have a protective effect against Achilles tendinopathy
(GS=1). Furthermore, within the same group, the protective CC+AG+TT combination
and the hazardous TT +GG+GG combination were underrepresented (0,3% and 5,8%, respectively). Similar fi ndings were identifi ed within the other 6 subgroups. CONCLUSIONS
This study demonstrated that chronic Achilles tendinopathy and ACL ruptures are distinctly
different injuries with genetic predisposition. Only a minority of the Greek population examined
seemed to be protected for both injuries. Finally, individuals at increased risk of both ATP and
ACL ruptures were underrepresented among the cohort.
INCIDENCE OF SINGLE NUCLEOTIDE POLYMORPHISM Rs9923231 IN THE VKORC1
GENE, Rs1799853 AND Rs1057910 IN THE CYP2C9 GENE ASSOCIATED WITH
WARFARIN, ON A 614 GREEK VOLUNTEER SAMPLE
Chatziliadi Constantina and Drakoulis NikolaosNational and Kapodistrian University of Athens, School of Health Sciences, Faculty of Pharmacy. BACKGROUND AND OBJECTIVES
We studied the incidence of SNPs in the VKORC1 and CYP2C9 genes associated with
warfarin, in a Greek population. The VKORC1 gene is responsible for the organism's response
to warfarin and CYP2C9 is involved in its metabolism.
METHODS
614 people from the general population took part. Epithelial cells from the the participants' oral
cavity were taken followed by DNA extraction. Genome analysis of all study subjects for the
presence of VKORC1 and CYP2C9 polymorphisms was performed by Real Time PCR.
RESULTS
For the wild-type allele (CC genotype) of Rs9923231, 26,4% of the volunteers were
homozygous, 46,2% heterozygous (CT) and 27,4% homozygous for the mutant allele (TT). For
Rs1799853, 73% were homozygous for the wild-type allele (CC), 24,8% were heterozygous
(CT), and 2,3% were homozygous for the mutant allele (TT) and for Rs1057910, 85% were
homozygous for the wild-type allele (AA), 14,7% heterozygous (AC) and 0,3% homozygous
for the mutant allele (CC). St atistical analysis showed that in heterozygous and even more in
homozygous individuals for the Rs9923231 polymorphism of the VKORC1 gene, the average
daily requirements for warfarin are less compared to individuals lacking VKORC1 mutations.
Individuals with CYP2C9 Rs1799853 and Rs1057910 polymorphisms, have reduced enzyme
activity, metabolize warfarin slowler and thus may require lower doses of warfarin than those
not having these polymorphisms.
CONCLUSIONS
Studying the patients' genotype, individualization of warfarin administered dose is enabled,
in order to be therapeutically effective and safe, with limited risk of toxicity and adverse
reactions.
GENE POLYMORPHISMS PREDISPOSING TO TYPE II DIABETES MELLITUS AND/OR
OBESITY IN 513 GREEKS: FREQUENCY DISTRIBUTION OF TCF7L2, MTNR1B, CDKAL1,
SLC30A8 AND FTO
S.F. Gourzi, K. Tentolouris, N. DrakoulisNational and Kapodistrian University of Athens, School of Health Sciences, Faculty of Pharmacy BACKGROUND AND OBJECTIVES
Diabetes mellitus type 2 is a chronic metabolic disease with pandemic spread worldwide.
Environmental infl uences and genetic background are included in the pathogenesis of the
disease. More than 40 suspected loci have been associated with the risk of developing type 2
diabetes. Four of these (rs7903146, rs10830963, rs7756992, rs13266634) were studied in a
sample of Greek population. Obesity is also a chronic disease and is the most important risk
factor for type 2 diabetes. Many SNPs have been associated with obesity with predominant
the FTO rs9939609, which has been studied in the same sample.
METHODS
For the present study were studied 513 volunteers. The DNA used was collected from buccal
epithelial cells of the oral cavity of the volunteer. It was isolated by the extraction method and
measured by the method of Real Time PCR.
RESULTS
The results have shown that the frequency of alleged dangerous alleles is ranging from 25%
to 70%. There were no differences in the frequencies between both sexes in genotypes and
allele level. The rs9939609 is quite frequent (44% frequency of A), without any differences in
both sexes and has also been associated with type 2 diabetes, although it is still unknown
whether the association is independent or not by obesity.
CONCLUSIONS
Although the genetic basis of type 2 diabetes and obesity is now proven, it is not yet
unfortunately been transferred in daily clinical practice. It is, however, expected in the following
years to contribute signifi cantly to the prevention, diagnosis and treatment of these frequent
diseases.
ANALYSIS OF POLYMORPHISMS RS2476601, RS3761847, RS660895 AND RS7574865
INVOLVED IN CLINICAL AND IMMUNOLOGICAL MANIFESTATIONS OF RHEUMATOID
ARTHRITIS IN 472 INDIVIDUALS.
Karanika Ioanna and Drakoulis NikolaosNational and Kapodistrian University of Athens, School of Health Sciences, Faculty of Pharmacy. BACKGROUND & OBJECTIVES
Rheumatoid Arthritis is a multifactorial disease, the possible genetic components of which
are continuously and systematically researched. The discovery of SNPs (Single Nucleotide
Polymorphism,) by the addition, deletion or change of a base have led to the discovery of new
genetic correlations with the disease. The objective of this particular study is to demonstrate
how the polymorphisms in specifi c loci RS2476601, RS3761847, RS660895 and RS7574865
affect various clinical and immunological manifestations of RA.
METHODS
The analysis was carried out on a sample of 472 healthy individuals from Greece. Genomic
DNA used was isolated from the epithelial cells of the oral cavity and analyzed by Real-Time
Polymerase Chain Reaction.
RESULTS
The genotype of GA RS660895 is 17.37% of the sample, while the GG is the 2.12%. The
RS3761847 occurring in 11, 44% as GG genotype and 44.91% as GA .While RS2476601 is
8.26% having a GA genotype and 0.21% having an AA. The homozygous RS7574865 as TT
is 6.57%, while the GT is 39.40%.
CONCLUSION
All 4 polymorphisms were found in the Greek population, however, RS3761847 alongside
RS7574865 show higher MAF (Minor Allele Frequency) and may be responsible for increased
susceptibility to RA.
GENETIC SUSCEPTIBILITY BIOMARKERS OF ALZHEIMER'S DISEASE DEVELOPMENT:
THE ROLE OF RS7412 AND RS429358 POLYMORPHISMS
Μalliou Maria1,2 Drakoulis Nikolaos21University of Perugia Faculty of Pharmacy, Department of Pharmacology, 2National and Kapodistrian University of Athens, School of Health Sciences, Faculty of Pharmacy BACKGROUND & OBJECTIVES
Alzheimer's disease is a degenerative disease of the central nervous system characterized
by loss of memory, diffi culty in executing daily tasks, anxiety, aggression and at the fi nal
stages the patient falls in coma and death occurs. In this study the polymorphisms rs7412 and
rs429358of apolipoprotein E (ApoE) gene have been analyzed, to reveal their relationship
with Alzheimer's disease, separately and combined on the same allele.
METHODS
DNA from 95 cases, with confi rmed AD diagnosis, and 100 non affected controls, was
isolated by standard DNA extraction and real time Polymerase Chain Reaction was applied
to detect the polymorphisms in all subjects. Statistical analysis was performed and in order
to determinate possible genetic disorder that predisposes to AD the odds ratios (OR) and
95% confi dence intervals (95%CI) of each polymorphism between cases and controls were
evaluated. Further, comparison between male and female within the AD patients was carried
out, to reveal differences in the susceptibility between genders.
RESULTS
In this study OR of polymorphism rs7412, alone, in the Alzheimer population, in male and in
female, were 0.69, 0.61 and 0.85 respectively, as compared to age matched controls. The OR
rs429358 polymorphism, alone, in the Alzheimer group, in male and in female patients were
3.78, 2.91 and 5.90, respectively, as compared to aged matched controls.
CONCLUSIONS
The contribution of ApoE in the development of AD was confi rmed with the exception of the
polymorphism rs7412 when not combined with other polymorphisms. The polymorphism
rs429358 alone or in combination with rs7412 on the same allele predisposes male to
Alzheimer's disease twice as much as compared to females.
HOW DO GENES INFLUENCE PERSONALITY: A RESEARCH IN 830 GREEK
SUBJECTS.
G. Marinos, N. Naziris and N. DrakoulisNational and Kapodistrian University of Athens, School of Health Sciences, Faculty of Pharmacy. BACKGROUND & OBJECTIVES
Intelligence consists of a series of competencies (e.g. perception, learning, adaptation).
Decision making is defi ned as the conscious choice between given options, relating
to a problem. The genetic background and the environment are the key elements for the
personality characteristics of the human being. The object of this study is to determine the
rs324420, rs1800497, rs363050, rs6265, rs1328674 gene effect on these topics, in 830 Greek
Subjects.
METHODS
The population of the volunteers is described, based on genotype, sex, with the respective
frequencies, including the Minor Allele Frequency (MAF). A potential relationship of the
volunteer genus with the above characteristics is checked and fi nally, volunteers are classifi ed;
a volunteer receives +1/-1, for each genotype and haplotype, which enhances/relegates his
intelligence or his decision-making respectively.
RESULTS
No statistically signifi cant gender-characteristics correlation is observed. At a rate of 92.5%,
the volunteers are characterized by prudence and temperance of thought, with only a small
proportion of them (7.5%) being genetically spontaneous and adventurous. Regarding
intelligence, the population is around an average and a little above it, at a rate of 96.3%, while
the edges of the scale suggest only a 0.5% of the volunteers, who, although the "smartest",
somehow lack prudence.
CONCLUSIONS
Individuals with low cognitive ability may be more prudent than others and vice versa, while
the "smartest" ones tend to be more risky, in decision-making. Therefore, intelligence and
decision-making, after all, may be less linked to each other than expected. These results may
be useful in targeted and personalized therapies of relevant diseases.
LOW-FLOW HIGH-OZONE CONCENTRATION DISINFECTION OF DENTAL
IMPRESSIONS
N. Poulis1, A. Kyriacou2, A. Prombonas3, N. Drakoulis1 1National and Kapodistrian University of Athens, School of Health Sciences, Faculty of Pharmacy 2Harokopio University, Athens, Department of Nutrition and Dietetics3 Technological Educational Institute of Athens, Department of Dental Technology, Faculty of Health and Caring Professions BACKGROUND & OBJECTIVES
The disinfection of dental impression materials to ensure no cross-contamination from potential pathogenic bacteria of oral microfl ora is conducted in Dental practice via chemical disinfectants. Low fl ow-high ozone concentration disinfection was used, by means of a prototype device, aiming in elimination of impression material dimensional change problems that current chemical disinfection faces.
Disc shaped dental addition-cured silicone was inoculated with Klebsiella pneumoniae and Staphylococcus aureus, 10mm discs were removed and ozone disinfected for different time intervals, immersion disinfected or served as controls. Disinfection success was examined by using the viable plate count method, while the statistical analysis was conducted via one way-ANOVA (p < 0.05). 120 dental elastomeric impression material specimens (4 material groups) were fabricated according to the ISO 4823 guidelines. Measurements regarding their linear dimensional stability were taken using a measuring microscope before and after ozone disinfection, sodium hypochlorite solution immersion disinfection (0,525%) and benzalkonium chloride solution immersion disinfection (0,3%).
Signifi cant eradication was observed for selected Gram (+) and Gram (-) bacteria after 3 minutes of ozone exposure, leading to complete disinfection of the samples. The lowest linear dimensional changes were found on the ozone disinfected samples which were statistically non-signifi cant.
While immersion disinfection of dental impressions is currently the most widely accepted method of disinfecting dental impressions, low-fl ow high-ozone concentration disinfection provides a quick, effi cient, fully automated alternative method, limiting liquid waste generation. The dimensional stability provided designates this disinfection method highly innovative in the Prosthetic Dentistry fi eld. A precise automated method for impression disinfection is established, relieving the dental team of possible cross-contamination.
BREAST CANCER AND GENETIC ASSOCIATION: A STUDY AMONG 513 NON-AFFECTED
WOMEN OF GREEK ORIGIN.
V. Xenelli, I. Katsa and N. Drakoulis National and Kapodistrian University of Athens, School of Health Sciences, Faculty of Pharmacy. BACKGROUND & OBJECTIVES
Its is well documented that individuals may have increased risk to develop breast cancer
(BRCA), as compared to the general female population, because of the existence of inherited
gene variants. In this study we examined BRCA1 and BRCA2 tumor suppressor gene
polymorphisms (RS1799950, 185delAG, 5331GA, 5382insC, R1751X, RS144848, G4X and
617delT), that may cause deregulation, leading to uncontrolled cell proliferation and tumor
development. Further, genes encoding TNFa and Il6, as indicators of infl ammation, expressing
angiogenic and oncogenic properties, the CHEK2 polymorphism, RS17879961, that leads in
inability to repair DNA damage, the AURKA, RS2273535, polymorphism that is associated
with abnormal segregation of chromosomes, the FGFR2 polymorphisms, RS2981582, and
RS7965399, and the BRCA associated IGF1 gene polymorphism.
METHODS
DNA extraction from buccal epithelial cells and rtPCR was performed in 513 Greek non
affected female subjects (2-91 years). Frequencies of polymorphisms were then calculated.
All individuals gave written consent to use DNA data after anonymization.
RESULTS
BRCA1-RS1799950 revealed 79.49% A:A (wt), 19,45% A:G, and 1,06% G:G genotypes.
All other BRCA1 polymorphisms examined were wt. BRCA2-RS144848 showed genotypic
alteration (G:G) in 5,36% of the population, 54.85% were wt T:T and 39,79% heterozygous G:T.
The TNFa frequencies were, mutated A:A, 0.84%, wild-type G:G, 84,10% and heterozygous
G:A, 15,06%. Il6, showed 55,04% G:G (wt), 38,45% C:G and 6,51% C:C. CHEK2, was 98,90%
T:T (wt) and 1,10% C:T. (No CHEK2-C:C carriers). AURKA, revealed 62,97% A:A, 33,26% T:A
and 3,77% T:T (wt). Finally, FGFR2, was 29,30% C:C (wt), 52,56% C:T, 18,14% T:T, and IGF1
was 92,03% T:T (wt), 7,54% C:T and 0,43% C:C.
CONCLUSIONS
BRCA1 and BRCA2 mutations lead to unsuccessful DNA repair. Homozygous mutated, and
probably also heterozygous females, may have higher risk in developing BRCA and, possibly,
early therapeutic interventions may be more successful, promoting health and enabling family
planning.
CURRENT THERAPEUTIC APPROACHES IN TRIPLE-NEGATIVE BREAST CANCER
Passos ID., Pliakou E., Mironidou-Tzouveleki M.
A' Department of Pharmacology, School of Health Sciences, Faculty of Medicine, Aristotle University of Thessaloniki, Greece.
BACKROUND AND OBJECTIVES
Triple negative breast cancer (TNBC) is an aggressive clinical phenotype characterized
by lack of expression (or minimal expression) of estrogen receptor (ER) and progesterone
receptor (PR) as well as the a bsence of human epidermal growth factor receptor–2 (HER2)
overexpression. It shows substantial overlap with basal-type and BRCA1-related breast
cancers, both of which also have aggressive clinical courses. Because of its expression profi le,
TNBC is not amenable to treatment with hormone therapy and systemic treatment options
are currently limited to cytotoxic chemotherapy. Overall survival, whether in early-stage or
advanced disease, is poor compared with that in patients who have other phenotypes. A
number of targeted approaches to TNBC are undergoing clinical evaluation, including the use
of agents with poly- (ADP-ribose)-polymerase inhibitory properties such as iniparib (BSI-201),
olaparib (AZD2281), and veliparib (ABT-888), antiangiogenic agents such as bevacizumab and
sunitinib, and epidermal growth factor receptor blockers such as cetuximab and erlotinib.
METHODS
The clinical characteristics of TNBC and clinical experience to date with novel targeted agents
under development for this aggressive phenotype is reviewed. The recent literature from
PubMed and Medline databases was reviewed.
RESULTS
Encouraging results with some of these agents have been reported, thereby offering
the promise for improved outcomes in patients with TNBC. In terms of chemotherapeutic
treatment, anthracyclines and taxanes are useful drugs, and high response rates have been
described. The combination with antiangiogenic drugs has also proven useful. Poly- (ADP-
ribose)-polymerase inhibitors, showed favorable results in TN tumors with BRCA mutation.
There are currently several ongoing studies with new drugs including epidermal growth factor
receptor inhibitors, c-kit inhibitors, Raf/Mek/Map kinase inhibitors and mTOR inhibitors, that
have shown promising results.
CONCLUSIONS
In the absence of approved targeted agents for the treatment of TNBC, most new agents
remain experimental. Increased understanding of molecular profi les of TNBC subtypes is
likely to improve therapeutic strategies with targeted agents.
HYDROGEN SULFIDE DONORS REDUCE MONOCYTE CELL ADHESION TO
ENDOTHELIAL MONOLAYER
E. Giannakopoulou1, A. Papapetropoulos2, V.G. Manolopoulos1, A. Tavridou11 Lab. of Pharmacology, Medical School, Democritus University of Thrace, Alexandroupolis2 Faculty of Pharmacy, University of Athens BACKGROUND & OBJECTIVES
Infl ammation and monocyte cell adhesion to endothelium are two key events in the early
stages of atherogenesis. We sought to examine the possible vascular anti-atherosclerotic
effect of two H S donors, GYY4137 and thioglycine, in an in vitro model.
METHODS
EAhy926 cells (3x106) were grown to confl uence in 24-well plates. Cells were serum-starved
for 12h and then incubated with several concentrations of GYY4137 or thioglycine for various
time points. After H S donor treatment, cells were incubated with TNF-α (10ng/ml) for 6h.
Medium was then removed, THP-1 monocytes were counted and seeded on TNF-α activated EAhy926 monolayers and incubated for 1h at 37oC. Non-adherent THP-1 cells were removed and EAhy926 were fi xed with 4% paraformaldehyde. Adhesion was counted on three randomly selected 40x magnifi cation microscopic fi elds/well in four independent experiments.
RESULTS
Control-confl uent EAhy926 showed minimal binding to THP-1 cells, while the adhesion of
THP-1 was signifi cantly increased to TNF-α-stimulated EAhy926. Pre-treatment of EAhy926
with increasing GYY4137 (300μM & 500μM) or thioglycine (100μM, 300μM & 500μΜ)
concentrations for 1h before TNF-α activation caused a concentration dependant inhibition of
THP-1 adhesion up to 69.3±2.2 % and 48.3±2.8 %, respectively. Pre-treatment of EAhy926
with glycine under similar conditions did not attenuate the adhesion of THP-1 to EAhy926
cells.
CONCLUSIONS
H S exerts a potentially protective role in atherosclerotic pathogenesis by inhibiting monocyte
cell adhesion to endothelial cells. Utilization of H S donors might, thus, be a new therapeutic strategy against this pathological process.
AUTOMODULATION OF VASCULAR HISTAMINE LEVELS BY HISTAMINE RECEPTOR
ANTAGONISTS: NOVEL INSIGHTS INTO SYSTEMIC INFLAMMATION
K. Kyriakidis, E. TiligadaDepartment of Pharmacology, Medical School, University of Athens BACKGROUND & OBJECTIVES
The impact of histamine in a variety of important biological functions has been underscored
by the blockbuster success of the H R and H R antagonists. The identifi cation of H R and
H R revived histamine research and new areas are now being explored. This study aimed to investigate the putative peripheral histamine automodulation in the rat blood vessels.
METHODS
Adjuvant arthritis was induced in male Wistar rats by complete Freund's adjuvant and the
animals were scored daily for arthritis signs in the paws. The H R, H R and H R antagonists,
dimethindene, GSK334429 and JNJ7777120 were administered intraperitoneally on day 0 (prophylactic model dosing), the respective controls receiving normal saline. Following sacrifi ce on day 21, the abdominal aorta (AA) and the inferior vena cava (IVC) were dissected out. Tissue histamine content was quantifi ed fl uorophotometrically. RESULTS
Extra-articular infl ammation was evidenced by the signifi cant increases in vascular histamine
levels in arthritic animals. Contrary to dimethindene, GSK334429 and JNJ7777120 increased
dose-dependently the basal histamine levels in normal vessels without inducing arthritic
signs. In adjuvant arthritis, dimethindene and GSK334429, but not JNJ7777120, restored
basal histamine content in the IVC and AA, respectively.
CONCLUSIONS
The fi ndings provide fi rst evidence for the intrinsic regulation of vascular histamine levels
by H R and H R and for the differential involvement of H R and H R in arthritis. The related
feedback mechanisms underlying peripheral histamine regulation in a range of poorly treatable chronic infl ammatory diseases are currently under investigation.
This work was part of the EU FP7 COST BM0806 K CHANNEL ACTIVATION INDUCES ANGIOGENESIS IN VITRO AND IN VIVO
B Umaru, A Pyriochou, A Papapetropoulos and S Topouzis*
Laboratory of Molecular Pharmacology, Department of Pharmacy, University of Patras, 265 04
Patras, Greece *: corresponding author

BACKGROUND AND OBJECTIVES
The angiogenic effects of hydrogen sulfi de depend on the activation of K channels. In
addition, it has been shown that C-type natriuretic peptide (CNP) induces growth responses in endothelial cells. Our aim was to test a) whether direct pharmacological K channel activation or blockade modulates angiogenic-type responses, and b) whether the C-type natriuretic peptide (CNP) affects angiogenesis and if it does, whether this depends on K channel activation.
METHODS
The chicken chorioallantoic membrane (CAM) assay was used as in vivo angiogenesis model.
Human Umbilical Vein Endothelial cells (HUVECs) and the murine brain endothelial cell line
bEnd.3 were used in vitro in Matrigel, proliferation and migration assays.
RESULTS
Treatment of CAM with either the K channel opener SG-209 or with CNP induced robust
angiogenesis that was abolished by the K channel inhibitors Glibenclamide and 5-HD. In vitro, the K channel openers SG-209 and Nicorandil increased cell proliferation, cell migration and Matrigel tube-like formation in both bEnd.3 and in HUVECs. These effects were blocked by K channel inhibition or by p38 and ERK1/2 inhibition. In HUVECs, Glibenclamide or knock-down of the K channel subunit Kir6.1 by siRNA abolished the migratory response to SG-209. Similarly, the effect of CNP on HUVEC Matrigel tube-like formation was blocked by introduction of Kir6.1 subunit siRNA.
CONCLUSIONS
Pharmacological activtion of K channel activation elicits angiogenic responses in vivo
and in vitro.
The vasoactive peptide CNP displays angiogenic properties that depend on K channel CELLULAR STRESS RESPONSE AND AGEING: THE BUDDING YEAST AS AN
EXPERIMENTAL MODEL
M. Kakolyri, B. Delitheos, K. Papamichael, E. TiligadaDepartment of Pharmacology, Medical School, University of Athens BACKGROUND & OBJECTIVES
The budding yeast Saccharomyces cerevisiae is a versatile and powerful model for the
investigation of mechanisms underlying the multicomponent cellular stress response (SR) and
ageing in eukaryotes. Despite the extensive related literature, data on the identifi cation and
functional annotation of the key components that can be modulated by drugs are limited and
largely inconclusive. This study investigated the effects of histamine on the SR and ageing
in yeast, thereby exploring its potential functions in a histamine receptor-free experimental
model and stimulating research on exploitation prospects.
METHODS
Cell growth and viability was determined in cultures grown through to post-logarithmic phase
at 27oC. Thermal preconditioning and heat shock were performed by shifting cells to 37oC
for 2h and 53oC for 30min, respectively. The effects of pharmacologically active agents were
investigated following short- and long-term administration at a range of doses and time points
during growth. Ageing was evaluated by the capacity and rate of cell proliferation, survival and
morphology in various stages of development.
RESULTS
Histamine failed to induce any alteration in yeast proliferation and survival under normal
conditions at any phase of growth. Contrary to the long-term administration, short-term
histamine treatment resulted in a dose-dependent induction of the thermotolerant phenotype
involving alterations in heat shock protein expression and cytoskeletal components.
CONCLUSIONS
The fi ndings provide evidence for the non-receptor-mediated role of histamine to condition
physiological signals in yeast, thus offering the lead for the elucidation of mechanisms
underlying the evolutionary-conserved adaptive and protective processes in eukaryotic cells.
A STUDY OF THE ASSOCIATION OF COMMON ABCB1 POLYMORPHISMS WITH
RESPONSE TO ANTIPSYCHOTIC THERAPY IN A NATURALISTIC SETTING.
G. Papazisis1, D. Antoniadis2, A. Sarrigiannidis2, D. Mpalaris2, A. Panderi3, S. Bargiota2, V.P. Bozikas4, G. Garyfallos2, A. Goulas312nd Department of Pharmacology, 22nd Department of Psychiatry, 31st Department of Pharmacology, 41st Department of Psychiatry, Faculty of Medicine, Aristotle University of Thessaloniki, Thessaloniki, Greece BACKGROUND & OBJECTIVES
Individual response rates to antipsychotic drugs are subject to wide variation which may be
due to genetic factors, among others. P-glycoprotein (P-gp) is an important effl ux pump
which could affect the intracerebral concentration of many antipsychotic drugs, by virtue of its
localization on the blood-brain barrier. Common polymorphisms of the P-gp – coding gene
(ABCB1) have been associated in the past with response to antipsychotic therapy. Here we
report preliminary results from a study of the association of the rs2032582 (2677G>T/A) and
rs1045642 (3435C>T) ABCB1 gene polymorphisms with response to combination therapy of
Greek schizophrenic patients.
METHODS
The study is undertaken in its natural setting and involves the administration of both fi rst
and second generation antipsychotic drugs to consecutive patients of the 2nd Department of
Psychiatry of our Medical School. Dosages are normalized to chlorpromazine equivalents.
Genotyping is done with previously published PCR-RFLP methods. The χ2 test of independence
and ANCOVA tests are used for statistical analysis.
RESULTS
So far, no statistically signifi cant differences are detected between initial responders and non-
responders (defi ned as 30% change in the total PANSS score, from baseline to the end of the
trial), with respect the genotype distribution of either polymorphism. Patients homozygous for
the G allele of rs2032582 appear to receive higher - on average - doses of chlorpromazine
equivalents, but that tendency is still non-signifi cant.
CONCLUSIONS
rs2032582 GG carriers could be more refractory to antipsychotic drug therapy but it is too
early for defi nite conclusions to be drawn.
L-NNA increase L-cysteine- L-NNA inhibits ACh-induced L-NNA does not change induced relaxations in mice relaxations in mice CC NaHS-induced relaxations ***P<0.001 Control vs L-NNA
*P<0.05 Control vs L-NNA
(n=6 and 4; respectively, P>0.05, ns., Control vs L- (n=6 and 4; respectively, 2way Anova, Bonferroni post 2way Anova, Bonferroni respectively, 2way Anova, Bonferroni post hoc test) Wild type
Genetic ablation of eNOS L-NNA increase L-cystein increase L-cystein induced induced H2S production, H2S production in mice CC p<0.001, L –cyst vs L- (n=3,2) (values are given Cyst+L-NNA, Paired t test, as fold increase of basal H S COMPENSATES NO DEFICIENCY IN MURINE CORPUS CAVERNOSUM
Dikmen A.1, d'Emmanuele di Villa Bianca R2, Mitidieri E. 2, Donnarumma E2, Dereli M. 1, Cirino G., Sorrentino R. 2, Yetik-Anacak G1*.
1Ege University Faculty of Pharmacy, Department of Pharmacology, İzmir-TURKEY2 Department of Pharmacy, University of Naples Federico II, Naples, Italy *Corresponding Author E-mail: [email protected] BACKGROUND & OBJECTIVES
Recent studies show that nitric oxide (NO) is required for the exogenous hydrogen sulfi de (H S)
induced vascular relaxation in rat aorta (Coletta et al., 2012) and human CC (d'Emmanuele di Villa Bianca et al., 2009). However it is unknown, if the endogenous H S production and the related relaxation are compensated in the absence of NO.
METHODS
The relaxation responses of murine corpus cavernosum (CC) to ACh, L-Cysteine or sodium
hydrogen sulphide (NaHS) were evaluated in presence or absence of NOS inhibitor Nω-
Nitro-L-arginine (L-NNA) by using a myograph in organ bath experiments. H S production
was assessed by a colorimetric assay. Basal H S production and the expression level of H S producing enzymes were measured in eNOS-/- mice CC, resembling pathologies in erectile dysfunction (ED) and endothelial dysfunction. RESULTS
L-NNA increased the concentration-dependent responses to L-cysteine (the substrate for
H S production) and did not change the relaxation responses to NaHS (a donor of H S),
in mouse CC strips. Furthermore, L-NNA increased the endogenous H S production by L-cysteine in mouse CC. The producing enzymes of H S, CSE and 3-MST, showed an increased expression levels in the genetic ablation of eNOS mouse CC. CONCLUSIONS
Our fi ndings suggest that H S can play a compensatory role in presence of endothelial
dysfunction in mouse CC. THE EFFECT OF HYALURONIC ACID ON WOUND HEALING DEPENDS
MOLECULAR MASS AND INVOLVES MMP-2
Ioannis Bonovolias, Konstantinos Peidis, Sofi a Lazaridou, Iordanis Mimtsoudis, George
Karakiulakis, Eleni Papakonstantinou
Department of Pharmacology, School of Medicine, Aristotle University of Thessaloniki,
Greece
BACKGROUND & OBJECTIVES
Hyaluronic acid (HA) is an extracellular matrix molecule that regulates important cell functions,
such as proliferation and migration and plays an important role in regenerative processes. In
this study, we investigated the effect of low and of high molecular weight HA (LMWHA and
HMWHA) on wound healing and the involvement of matrix metalloproteinase (MMP) -2 in this
process.
METHODS
We used primary cultures of human dermal fi broblasts, and we assessed the effect of HA
(small fragments, 50, 250 and 2500 kDa) on wound healing using the scratch wound assay.
We also evaluated the effect of 4-methyl-umbeliferone (4-MU), an inhibitor of HA synthases
and aristolochic acid (AA-I), an inhibitor of hyaluronidases, on wound healing. Gelatinolytic
activity of MMP-2 was assessed by gelatin zymography. Cell viability and proliferation were
estimated by cell counting.
RESULTS
LMWHA induced the wound healing process in a dose-depended manner. The same results
were also obtained in the presence of 4-MU. On the contrary, HMWHA inhibited wound healing,
in a dose-dependent manner and the same results were also obtained in the presence of AA-I.
Gelatinolytic activity was signifi cantly increased by LMWHA, but not by HMWHA.
CONCLUSIONS
Our results indicate that the effect of HA on wound healing depends on the size of HA and
correlates with increased gelatinolytic activity. The ability of LMWHA to promote wound healing
suggests that it could be used as a pharmacological target for the treatment of delayed or
complicated wound healing.
INTERACTIONS OF THE KAPPA OPIOID RECEPTOR WITH REGULATORS OF G
PROTEIN SIGNALLING
M.-P. Papakonstantinou and Z. Georgoussi Laboratory of Cellular Signalling and Molecular Pharmacology, Institute of Biosciences and Applications, National Centre for Scientifi c Research "Demokritos", Ag. Paraskevi, 15310, Athens BACKGROUND & OBJECTIVES
Opioid receptors (OR) μ, δ, and κ couple to Gi/Go proteins to modulate a variety of
physiological responses in the nervous system. Apart from G proteins, opioid receptor activity
is also controlled by interactions with other proteins that regulate their fi ne tuning opioid [1].
Regulators of G protein Signalling (RGS) comprise a large multifunctional protein family that
accelerate GTP hydrolysis of Gα subunits and modulate G protein coupled receptor (GPCR)
signalling. Previous studies have shown that RGS4 associates with the C-termini of μ- and
δ-opioid receptors (μ-OR, δ-OR) in living cells and plays a key role in Gi/Go protein coupling
selectivity and signaling of these receptors. [2,3]
METHODS AND RESULTS
To deduce whether similar effect also occurs for the kappa opioid receptor (κ-ΟR) and defi ne
the ability of members of B/R4-RGS family to interact with the kappa opioid receptor (κ-ΟR)
we performed pulldown experiments using GST fusion peptides encompassing the carboxyl
terminus of κ-OR. These experiments indicated that RGS2 and RGS4 interact within this
receptor subdomain. Co-immunoprecipitation studies indicated that RGS2 and RGS4 associate
with κ-ΟR constitutively and upon receptor activation and confer selectivity for coupling with a
specifi c subset of G proteins. Functional assays have shown that both members of B/R4-RGS
family attenuate κ-ΟR mediated inhibition of adenylyl cyclase and ERK1,2 phosphorylation,
but present different amplitude in their effect.
CONCLUSIONS
Our results demonstrate that RGS2 and RGS4 are new interacting partners and negative
modulators of κ-ΟR signaling.
This work was supported by the EU grant «Normolife» (LSHC-CT2006-037733) and the
GSRT.
References
[1] Georgoussi, Z., Georganta, E.-M., Milligan, G. (2012) Current Drug Targets, 13, 80-102
[2] Georgoussi Z., Leontiadis L., Mazarakou G., Merkouris M., Hyde K. and Hamm H. (2006),
Cell. Signal.18, 771–782
[3] Leontiadis, L. J., Papakonstantinou, M. P., Georgoussi Z. (2009) Cell. Signal. 21, 1218–
CYCLIN-DEPENDENT KINASE 5 IS INVOLVED IN PLEIOTROPHIN-INDUCED
ENDOTHELIAL CELL MIGRATION
Evgenia Lampropoulou1, Ioanna Logoviti1, Maria Hatziapostolou2,#, Marina Koutsioumpa1, Spyros Skandalis3,4, Philip N. Tsichlis2, Ulf Hellman3, and Evangelia Papadimitriou11Laboratory of Molecular Pharmacology, Department of Pharmacy, University of Patras, GR26504 Patras, Greece; 2Molecular Oncology Research Institute, Tufts Medical Center, Boston, MA 02111, USA; 3Ludwig Institute for Cancer Research, Uppsala University, Uppsala SE-751-05, Sweden; Laboratory of Biochemistry, Department of Chemistry, University of Patras, Greece.
#Current address: Center for Systems Biomedicine, Division of Digestive Diseases, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA, USA BACKGROUND & OBJECTIVES
Cyclin-dependent kinase 5 (CDK5) is a serine/threonine kinase that requires the regulatory
subunits p35 or p39 for activation. CDK5 plays an important role in neuronal migration
and neurite outgrowth and there are studies showing its implication in tumor growth and
angiogenesis. Pleiotrophin (PTN) is a heparin-binding growth factor that induces cell migration
in neuronal, cancer and endothelial cells through its receptor protein tyrosine phosphatase β/ζ
(RPTPβ/ζ) and α β integrin. In the present study, we investigated the role of CDK5 in PTN-
induced endothelial cell migration.
METHODS
Human umbilical vein endothelial cells were used. CDK5 activity was estimated by either
direct measurement of the kinase activity or through its interaction with p35. Migration assays
were performed in 24-well microchemotaxis chambers. RPTPβ/ζ was suppressed using small
interfering RNA. Protein-protein interactions were studied by immunoprecipitation followed by
Western blot or MALDI-TOF analyses, or by proximity ligation assays. Statistical analysis was
performed by unpaired t-test.
RESULTS
Pharmacological inhibition, as well as down-regulation of CDK5 by siRNA, completely abolished
PTN-induced endothelial cell migration. PTN increased CDK5 kinase activity and its interaction
with p35. PTN-induced CDK5 activation seemed to be independent of α β but dependent of
RPTPβ/ζ expression. Interestingly, both CDK5 and p35 were found to interact with RPTPβ/ζ. Activation of c-Src kinase was involved in CDK5 activation, while pharmacological inhibition of CDK5 did not affect PTN-induced β Tyr773 phosphorylation and ERK1/2 activation.
CONCLUSION
CDK5 is a signifi cant regulator of the PTN/RPTPβ/ζ signaling pathway that contributes to
PTN-induced endothelial cell migration.
Acknowledgement: We thank the Advanced Light Microscopy facility of the School of Health Sciences, University of Patras, for using the Leica SP5 confocal microscope.
RECEPTOR PROTEIN TYROSINE PHOSPHATASE BETA/ZETA IS INVOLVED IN VASCULAR
ENDOTHELIAL GROWTH FACTOR-INDUCED ENDOTHELIAL CELL MIGRATION
Marina Koutsioumpa1, Evangelia Poimenidi1, Evangelia Pantazaka1, Christina Theodoropoulou1, Angeliki Skoura2, Vasileios Megalooikonomou2, Nelly Kieffer3, Jose Courty4, Kazuyuki Sugahara5, Evangelia Papadimitriou11Laboratory of Molecular Pharmacology, Department of Pharmacy, University of Patras, Greece; 2Computer Engineering and Informatics Department, University of Patras, Patras, Greece; 3Sino-French Research Centre for Life Sciences and Genomics, CNRS/LIA124, Rui Jin Hospital, Jiao Tong University Medical School, 197 Rui Jin Er Road, Shanghai, 200025, China; 4Laboratoire CRRET, Universite Paris Est Creteil Val de Marne, Avenue du General de Gaulle, 94010 Creteil Cedex, France; 5 Proteoglycan Signaling and Therapeutics Research Group, Faculty of Advanced Life Science, Hokkaido University, Sapporo, Japan BACKGROUND & OBJECTIVES
Receptor protein tyrosine phosphatase beta/zeta (RPTPβ/ζ) is a transmembrane chondroitin
sulfate protein tyrosine phosphatase that acts as a receptor for the growth factor pleiotrophin
(PTN). RPTPβ/ζ interacts with α β on the cell surface and upon binding of PTN leads to c-Src
Tyr530 dephosphorylation, β integrin Tyr773 phosphorylation, cell surface nucleolin (NCL) localization and stimulation of cell migration. c-Src-mediated β Tyr773 phosphorylation is also observed after vascular endothelial growth factor 165 (VEGF ) stimulation of endothelial cells and is essential for VEGF receptor 2 (VEGFR2)-β integrin association and subsequent signaling. In this work we studied whether RPTPβ/ζ is involved in VEGF actions.
METHODS
Human umbilical vein endothelial, human glioma M059K and U87MG, and chinese hamster
ovary cells were used. Migration assays were performed in 24-well microchemotaxis
chambers. RPTPβ/ζ was suppressed using siRNA. Protein-protein interactions were studied
by immunoprecipitation/Western blot, immunofl uorescence or proximity ligation assays.
Statistical analysis was performed by unpaired t-test.
RESULTS
RPTPβ/ζ mediates VEGF -induced c-Src-dependent β Tyr773 phosphorylation, required
for both VEGF -induced VEGFR2-α β interaction and the downstream PI3K activation and cell surface NCL localization. By using immunoprecipitation and proximity ligation assays, RPTPβ/ζ was found not to interact with VEGFR2 but to interact with VEGF. This interaction was interrupted by chondroitin sulphate E (CS-E) and PTN but not bevacizumab. CS-E and down-regulation of RPTPβ/ζ by siRNA inhibited VEGF -induced endothelial cell migration, while PTN decreased the migratory effect of VEGF to the levels of its own effect.
CONCLUSIONS
RPTPβ/ζ is potentially a good target for development of additive or alternative anti-VEGF
therapies.
Acknowledgements: This work was supported by the European Union (European Social Fund – ESF, Heracleitus II (M. Koutsioumpa - E. Papadimitriou), Thales (V. Megalooikonomou) and ΙΚΥ Fellowships of Excellence for Postgraduate studies in Greece - Siemens Program (E. Pantazaka). The authors thank the Advanced Light Microscopy facility of the Medical School, University of Patras for using the Leica SP5 confocal microscope.
PLEIOTROPHIN-INDUCED CELL MIGRATION INVOLVES GENERATION OF REACTIVE
OXYGEN SPECIES
Sotiria Tsirmoula1, Margarita Lamprou1, Maria Hatziapostolou1,#, Nelly Kieffer2 and Evangelia Papadimitriou1,*1Laboratory of Molecular Pharmacology, Department of Pharmacy, University of Patras, GR26504 Patras, Greece; 2Sino-French Research Centre for Life Sciences and Genomics, CNRS/LIA124, Rui Jin Hospital, Jiao Tong University Medical School, 197 Rui Jin Er Road, Shanghai, 200025, China#Current address: Center for Systems Biomedicine, Division of Digestive Diseases, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA, USA BACKGROUND & OBJECTIVES
Pleiotrophin (PTN) is an 18 kDa heparin-binding secreted growth factor that induces cell migration
through binding to both its receptor protein tyrosine phosphatase beta/zeta (RPTPβ/ζ) and integrin
α β . In the present work, we studied the effect of PTN on the generation of reactive oxygen
species (ROS) and the involvement of ROS in PTN-induced cell migration. METHODS
Human umbilical vein endothelial, human glioma M059K, Chinese hamster ovary and human
prostate cancer LNCaP and PC3 cells were used. Intracellular ROS production was assayed
using 5(6)-carboxy-2,7-dichlorodihydrofl uorescein diacetate. Migration assays were performed in
24-well microchemotaxis chambers. RPTPβ/ζ was suppressed using small interfering RNA. The
signifi cance of variability between the results from each group and the corresponding control was
determined by unpaired t-test.
RESULTS
PTN signifi cantly increased endogenous ROS levels in a concentration and time-dependent manner,
while in cells with down-regulated endogenous PTN expression, ROS levels were signifi cantly
decreased. Suppression of RPTPβ/ζ or α β integrin using genetic and pharmacological inhibitors
or inhibition of c-src kinase activity abolished PTN-induced ROS generation. Phosphorylation of β Tyr773 and phosphoinositide 3-kinase (PI3K) or Erk1/2 activation seem to be required, similarly to what has been previously shown for PTN-induced cell migration. Finally, ROS scavenging and xanthine oxidase inhibition completely abolished both PTN-induced ROS generation and cell migration, while NADPH oxidase inhibition had no effect.
CONCLUSIONS
These data suggest that xanthine oxidase-mediated ROS production is required for PTN-induced
cell migration through the cell membrane functional complex of α β and RPTPβ/ζ and activation
of c-src, PI3-K and ERK1/2 kinases. Acknowledgement: This work was supported by the European Union (European Social Fund – ESF) and Greek national funds through the Operational Program "Education and Lifelong Learning" of the National Strategic Reference Framework (NSRF) - Research Funding Program: "Heracleitus II. Investing in knowledge society through the European Social Fund".
DEPRESSION, ANTIDEPRESSANT TREATMENT AND SURROGATE MARKERS OF
ATHEROSCLEROSIS
N. Kokras1,2, C. Dalla2, A.V. Kouzoupis1, C. Papamichael3, G.N. Papadimitriou1, K.S. Stamatelopoulos3.
1. First Department of Psychiatry, Eginition Hospital, University of Athens, Athens, Greece 2. Department of Pharmacology, University of Athens, Athens, Greece.
3. Department of Clinical Therapeutics Alexandra Hospital, University of Athens, Athens, Greece BACKGROUND AND OBJECTIVES
Major Depression is twice more common in women and sex differences exist in antidepressant
pharmacokinetics and pharmacodynamics. Additionally, depression is recognized as a risk
factor for cardiovascular disease. In this study we aim to investigate whether treatment of
male and female depressed patients would impact on surrogate markers of atherosclerosis.
METHODS
In this study, depressed patients treated by their physician with citalopram 20-60mg and
risperidone 0.5-1mg were invited to be examined at baseline and 21 weeks after treatment
initiation. Surrogate markers of atherosclerosis were assessed using a validated non-invasive
protocol.
RESULTS
In this on-going-study 35 patients completed their follow-up vascular assessments after
21-24 weeks of treatment. Those patients received a mean citalopram dose of 37 mg and
a mean risperidone dose of 0.72 mg. A remarkable 70% of female patients responded to
treatment in comparison to only 45% of male patients. No signifi cant differences were found in
blood biochemistry, except prolactin, which was increased on average by 50%. Interestingly,
arterial stiffness, a surrogate marker of atherosclerosis, was signifi cantly decreased following
treatment (p<0.001, repeated measures analysis of variance).
CONCLUSIONS
Our preliminary results show that successful pharmacological treatment of major depression
also results in an improvement of arterial stiffness, a surrogate marker of atherosclerosis.
Additionally, a signifi cant sex-difference was obse
rved in response to the combined antidepressant/antipsychotic treatment of major depression.
SEX DIFFERENCES IN RESPONSE TO ENVIRONMENTAL ENRICHMENT: BEHAVIORAL,
NEUROCHEMICAL AND BRAIN PLASTICITY FINDINGS
E. Tzouveka1 , I. Sotiropoulos1,3, N. Kokras1,2, D. Bessinis1, O.F.X. Almeida4, Z. Papadopoulou-Daifoti1 and C. Dalla11. Department of Pharmacology, University of Athens, Greece.
2. First Department of Psychiatry, Eginition Hospital, University of Athens, Greece 3. Life and Health Sciences Research Institute (ICVS), School of Health Sciences, University of Minho, Braga, Portugal4. Neuroadaptations group, Max Planck Institute of Psychiatry, Munich, Germany BACKGROUND AND OBJECTIVES
Early life experiences exert long-lasting effects on neuronal structure, function and connectivity
and thus they play an important role in brain development, behaviour, as well as in the
appearance of neuropsychiatric disorders. Marked sex differences are present in several
neuropsychiatric disorders including depression, Alzheimer's and neurodevelopmental
diseases. Environmental enrichment (EE) has been shown to have benefi cial effects on
brain plasticity and has been proposed as a therapeutic intervention. In the present study, we
investigated sex differences in the effects of EE on cognitive, neurochemical and neuronal
structure indices related to neuropsychiatric disorders.
METHODS
Male and female Wistar rats were reared in a standard or an enriched environment from P0-
P90. Behavioural response was assessed in the open fi eld and the novel object recognition
test. Subsequently, rats were sacrifi ced and the serotonergic, dopaminergic and glutamatergic
neurotransmission was evaluated in the hippocampus. Finally, protein ma rkers indicative of
neuronal plasticity were assessed in the same brain region with Western blot analysis.
RESULTS
In the novel object recognition test both EE males and females had a higher preference index
for the novel object. Corticosterone serum levels were reduced only in males exposed to
EE. Females exposed to EE had low glutamate levels and high serotonergic activity in their
hippocampus. Western blot analysis revealed alterations in cytoskeletal and synaptic proteins,
such as enhanced hippocampal PSD-95 in EE females.
CONCLUSIONS
Present fi ndings reveal the potential signifi cance of male or female sex in supporting the
benefi cial effects of environmental stimuli, which include enhancement of brain plasticity and
cognition.
PREFRONTAL CORTICAL – HIPPOCAMPAL COMMUNICATION AND ANTIDEPRESSANT
TREATMENT IN RATS: EXPLORING SEX DIFFERENCES
V. Kafetzopoulos1, K. Antoniou3 , N. Kokras1,2, I. Sotiropoulos1,4, H. Leite-Almeida4, N. Sousa4, Z. Papadopoulou-Daifoti1 and C. Dalla1 1. Department of Pharmacology, Medical School, National and Kapodistrian University of Athens, Athens, Greece2. First Department of Psychiatry, Eginition Hospital, Medical School, National and Kapodistrian University of Athens, Athens, Greece3. Department of Pharmacology, Medical School, University of Ioannina, Ioannina, Greece4. Life and Health Sciences Research Institute (ICVS), School of Health Sciences, University of Minho, Braga, Portugal BACKGROUND & OBJECTIVES
Depression is twice as much prevalent in women, with sex differences in the onset,
symptomato logy and response to treatment. However, specifi c neurobiological underpinnings
for this differentiation are still under rigorous research. Robust results from our laboratory
indicate that disruption of the prefrontal cortical (PFC) – hippocampal network circuit via
lesion of their interconnecting node, nucleus reuniens (RE), prevents the appearance of
depressive-like behavior in male rats. In the present study, we sought to investigate whether
the antidepressant-mimicking effect of the PFC – hippocampal circuit disruption would be
replicated in female rats.
METHODS
Adult male and female rats received an excitotoxic RE lesion, or a sham-operation. After
one week of recovery, rats were tested in the forced swim test (FST). Between the two FST
sessions, rats received a standard subacute treatment consisting of 3 injections of sertraline
(an SSRI, 10 mg/kg) or clomipramine (a tricyclic antidepressant, 10 mg/kg) or vehicle.
RESULTS
Male and female RE-lesioned rats had lower immobility and enhanced swimming duration
than sham controls in the FST. Sertraline and clomipramine treatment reduced immobility
duration in sham-operated rats, as expected. SSRI treatment in RE-lesioned rats reduced
immobility only in females.
CONCLUSIONS
Our results uncover that the communication of PFC and hippocampus, depending on RE, is
required for the appearance of depressive-like behavior in both sexes. Additionally, following
disruption of the PFC-hippocampus circuit, antidepressant treatment elicits a sexually
dimorphic behavioral profi le.
SUBACUTE AROMATASE INHIBITION EXHIBITS ANTIDEPRESSANT POTENTIAL
IN FEMALE RATS
M. Koronaiou1, N. Pastromas1, V. Kafetzopoulos1, T. Mavridis1, N. Kokras1,2 and C. Dalla1 1. Department of Pharmacology, University of Athens, Athens, Greece.
2. First Department of Psychiatry, Eginition Hospital, University of Athens, Athens, Greece BACKGROUND AND OBJECTIVES
Aromatase inhibitors, such as letrozole, block the production of estrogens and are used
for the treatment of hormone-responsive breast cancer in women. In the present study we
investigated whether decreased estrogen synthesis following letrozole treatment leads to a
depressive-like behavioral response in female rats.
METHODS
Adult cycling female Sprague-Dawley rats were subjected to the forced swim test (FST),
which is a test for antidepressant potential. Females were treated with either vehicle or the
antidepressant fl uoxetine for 28 days, in combination with subacute letrozole (3 injections in
24 hours) or sustained (7 days, 1 injection/day) letrozole treatment. Behavioral response in
the FST was evaluated with the use of the software Kinoscope. Also, gonadal hormone levels
were assayed following behavioral testing.
RESULTS
Immobility duration in the FST was reduced following acute aromatase inhibition, indicating
letrozole's antidepressant potential. Instead, aromatase inhibition for one week did not show
an antidepressant response. Estrogens levels were undetected following letrozole treatment,
as expected. Moreover, serum testosterone levels were elevated following acute letrozole
treatment and this was associated with the decreased depressive behavior in the FST. On the
other hand, progesterone levels explained the increased swimming behavior in the FST.
CONCLUSIONS
These fi ndings indicate that aromatase inhibitors may have psychotropic attributes that
depend on treatment duration. Further preclinical and clinical studies are needed to verify
the value of these fi ndings and evaluate whether there is a role for aromatase inhibitors in
antidepressant response.
CANNABINOID ANTAGONISM BLOCKS THE EXPRESSION OF COCAINE CONDITIONED
PLACE PREFERENCE
F.Delis1, A. Polissidis1, 2, E.Smaragdi1, M.Marselos1, S.Goldberg3 and K. Antoniou1.
1Department of Pharmacology, Medical School, University of Ioannina, 451 10 Ioannina,
Greece
2Biomedical Research Foundation of the Academy of Athens, 115 27 Athens, Greece
3 Preclinical Pharmaclogy Section, Behavioral Neuroscience Branch, National Institute on
Drug Abuse, Baltimore, MD 21224 USA
BACKGROUND AND OBJECTIVES
The potential of endocannabinoid modulation for the treatment of psychostimulant addiction
has been recently suggested. Here we studied the modulatory role of the endocannabinoid
system on the rewarding effects of cocaine, using the conditioned place preference paradigm
in the rat. We tested the potential of cannabinoid CB1 and CB2 agonists and antagonists (i)
to affect the acquisition of cocaine conditioned place preference (Coc CPP), by administering
cannabinoid compounds during cocaine Conditioning and (ii) to affect the expression of
CocCPP, by administering the cannabinoid compounds on Test Day.
METHODS
Coc (20mg/kg) CPP was performed with an unbiased design in a three-compartment
apparatus, with 1 habituation day (15 min free exploration), 8 days of conditioning with
alternating cocaine and saline injections (30 min in black or white chamber), and 1 test day
(15 min free exploration).
RESULTS
The results showed that the cannabinoid inverse agonist/antagonist SR 141716A decreased
the acquisition (at 3mg/kg) and blocked the expression (at 1 and 3 mg/kg) of cocaine CPP.
These effects were not due to direct effects of SR141716A on the conditioning procedure
since SR141716A alone did not produce place reference.
CONCLUSIONS
Our fi ndings show that the endocannabinoid system contributes to the primary rewarding
properties of cocaine and, more so, to the expression of cocaine x environment associations.
These results suggest that pharmacological intervention of the endocannabinoid system may
be of value in the weakening of context-induced cocaine preference.
and blocks the expression of We show that WIN 55212-2 (0.1, 1 mg/kg) and SR 141716A (0.01-3 mg/kg) do not induce place preference. We also show that WIN 55212-2 does not affect the acquisition or the expre LOW DOSES OF ENDOCANNABNOID AGONISTS DIRSUPT OBJECTRECOGNITION VIA
CANNABINOID TYPE 1 RECEPTORS
A. Polissidis1,2, F. Delis1, A. Galanopoulos1, M. Marselos1, N. Pitsikas3, and K. Antoniou 11Dept Pharmacology, Medical School, University of Ioannina, 451 10 Ioannina, Greece2Biomedical Research Foundation of the Academy of Athens, 115 27 Athens, Greece3Dept Pharmacology, Medical School, University of Thessaly, 411 10 Larissa, Greece BACKGROUND AND OBJECTIVES
The therapeutic potential as well as the widespread recreational use of cannabis prompted
us to study whether cannabinoids affect cognitive functions in the rat. We studied the effects
of cannabinoid agonists on novel object recognition (NOR) because (i) this behavioral test is
based on a spontaneous rodent exploratory behavior and (ii) there were limited and confl icting
fi ndings in the literature. One reason behind the inconclusive NOR studies was the use of high,
motor-suppressant doses, which interfered with the exploratory behavior of the animals.
METHODS
Therefore, we determined the effects of endocannabinoid agonists on NOR, using a range
of relatively low doses that did not affect vertical exploratorybehavior (HU 210, 0.001-0.03;
WIN 55212-2, 0.1 – 1mg/kg) or produced a modest decrease in ambulation (WIN 55212-2, 1
mg/kg).We assessed the effects of cannabinoids on (i) short term memory (intertrial interval
1 h), (ii) long-term memory (intertrial interval 24 hrs), and (iii) long-term information retrieval
(intertrial interval 24 hrs).
RESULTS
Cannabinoid agonists (HU210, 0.003 mg/kg and WIN 55212-2, 0.3mg/kg) impaired short-term
NOR memory, without affecting total object exploration time. In addition, WIN 55212-2 (0.3mg/
kg) also impaired long-term NOR memory and long-term NOR retrieval (without affecting total
object exploration time) and these disruptions were prevented if a low dose of SR141716A
(0.03 mg/kg) was administered before WIN55212-2.
CONCLUSIONS
These results show that activation of CB1 receptors affects all components of recognition
memory including consolidation, storage and retrieval and further support the deleterious role
of CB1 activation in cognitive function.
THE ROLE OF ALPHA-ADRENERGIC BLOCKERS IN THE TREATMENT OF CHRONIC
PROSTATITIS / CHRONIC PELVIC PAIN SYNDROME
K. Gkagkalidis¹, S. Kampantais¹, S. Ioannidis¹, C. Toutziaris¹, M. Koptsis¹, I. Pagkos¹,
I. Anagnostou¹, M. Mironidou-Tzouveleki²
1. 1st Department of Urology, Aristotle University of Thessaloniki General Hospital of
Thessaloniki «G. Gennimatas»
2. A' Department of Pharmacology, School of Health Sciences, Faculty of Medicine, Aristotle
University of Thessaloniki, Greece
BACKROUND & OBJECTIVES
Chronic prostatitis/chronic pelvic pain syndrome is a debilitating disease of the male population,
without a confi rmed cause and without a curative treatment. Lower urinary tract symptoms,
pelvic pain and sexual dysfunction are predominant in this entity. All these symptoms in
combination with the absence of drug treatment results in a negative impact on the patients'
quality of life. The purpose of this review is to present the role of alpha-adrenergic blockers in
the treatment of chronic prostatitis/chronic pelvic pain syndrome, their mechanism of action
and also the results of major clinical trials where these drugs have been used.
METHODS
A review of the literature in electronic databases Medline, Scopus and Cochrane Library
was conducted. Additionally, websites of pharmaceutical companies, international urological
guidelines and the most relevant international conferences were researched.
RESULTS
Out of the 51 retrieved articles, 7 were included in the present review. In most of these trials, the
symptoms of patients were suppressed with the administration of a-blocker. In these patients,
drug prescription is recommended for a specifi c long period of time in order to achieve the
optimal result. On the contrary, two studies did not fi nd statistically signifi cant difference in the
improvement of symptoms by the administration of a-blockers compared to placebo.
CONCLUSIONS
Clinical studies including a larger number of patients need to be conducted in order to fi nd
the most appropriate drug treatment. The positive indications that exist, concerning the good
response of patients towards this treatment in combination with its low cost, further support
this need for additional research in this fi eld.
INFLUENCE OF CHRONIC AND SUB-CHRONIC SOY DIET ON THE REPRODUCTIVE
SYSTEM OF ADULT MALE RATS
P. Papaioannidou1, T. Papamitsou2, I. Kiriakidis1, I. Makaronidis1, F. Tsanakalis1, M. Dermentzopoulou211st Department of Pharmacology and 2Department of Histology and EmbryologyMedical Faculty, School of Health Sciences, Aristotle University of Thessaloniki, Thessaloniki, Greece BACKGROUND AND OBJECTIVES
Soy diet may be benefi cial during menopause and old age but may have adverse effects on
reproduction, when consumed during reproductive age. The purpose of this study was to
investigate the effect of chronic and sub-chronic soy diet on the reproductive system of adult
male rats.
METHODS
Four groups of adult male albino Wistar rats, 6 months old, were used: two study and two
control groups. The study groups received ad libidum food enriched in soy protein for 6 weeks
(group 1) and 12 weeks (group 2). The control groups received ad libidum the standard food
for 6 weeks and 12 weeks (groups 3 an4 respectively). All groups were grown under the
same conditions and according to the rules of GLP. The study was approved by the local
Ethics Committee. After sacrifi ce, specimens of the reproductive organs were prepared and
stained for electron microscopic examination. The preparations were observed by two different
persons, blinded to the source of the specimen. Statistical analysis was performed with the
statistical package SPSS.
RESULTS
Ultrastructural changes of the seminiferous epithelium were observed in the study groups;
many spermatocytes appeared with endocytoplasmic and perinuclear oedema. The number
of spermatozoa in the reproductive tract was much lower in group 2 comparatively to group 4;
in group 1 it was similar to the number of spermatozoa in group 3.
CONCLUSIONS
Chronic and sub-chronic soy diet caused ultrastructural changes in the seminiferous
epithelium of adult rats; only chronic soy diet caused adverse effects on spermatogenesis.
ATTITUDES IN ANTIDEPRESSANTS USE IN THESSALONIKI
P. Papaioannidou, A. Ntaralas1st Department of Pharmacology, Medical Faculty, School of Health Sciences, Aristotle University of Thessaloniki, Thessaloniki, Greece BACKGROUND AND OBJECTIVES:
The use of generics in the Greek market of medicines is generally lower than in other European
countries. The purpose of this work was to study the attitudes in antidepressants use, and to
calculate the use of generics in antidepressant sales in a sample from the medicines market
of Thessaloniki.
METHODS:
A sample of antidepressants registered sales was collected from the new registration system,
which has been applied during the last two years in Greece. The sample corresponded to
a small amount of sales from the market of Thessaloniki during the years 2012 and 2013.
All classes of antidepressants and their relative ratios in the sales were estimated, and the
percentage of generics in the sale of each medicine was calculated out of a variety of brand
names in each class of antidepressants. The amount of medicines was estimated in Defi ned
Daily Doses (DDDs) of the reference drug and its generics.
RESULTS:
Selective Serotonin Reuptake Inhibitors (SSRIs) and Serotonin-Norepinephrine Reuptake
Inhibitors (SNRIs) corresponded to 88% of total antidepressants sales (42,000 out of 47,644
DDDs). Generic use corresponded to 28% of total sales and varied greatly, being 14% of
fl uoxetine sale (1,066 out of 7,834 DDDs), 35% of venlafaxine sale (593 out of 1685 DDDs),
36% of sertraline sale (2,972 out of 8,294 DDDs) and 53% of citalopram sale (5,708 out of
10,696 DDDs).
CONCLUSIONS:
The percentage of generics in antidepressants use was low in the studied sample, being
higher than 50% only for citalopram.
SERUM HISTAMINE LEVELS IN ALLERGIC BEEKEEPERS AS POTENTIAL FACTOR TO
DETERMINE THERAPEUTIC STRATEGIES
C. Chliva1, M. Makris1, E. Tiligada1,21Allergy Unit 'D. Kalogeromitros' 2nd Department of Dermatology and Venereology, Attikon University Hospital & 2Department of Pharmacology, Medical School, University of Athens BACKGROUND & OBJECTIVES
Beekeepers, being heavily exposed to honeybee stings, are at increased risk of becoming
allergic, while many outgrow their allergy during beekeeping. The underlying mechanisms
remain elusive, and biomarkers that would distinguish between tolerant and non-tolerant bee-
keepers are not currently available. This preliminary study sought to investigate the value of
serum histamine (sHI) level determination in tolerant and non-tolerant allergic beekeepers as
a marker of prognostic and therapeutic relevance.
METHODS
Eleven medication-free allergic beekeepers were selected, out of 472 volunteers, for the de-
termination of sHI levels, based on a series of exclusion criteria, including allergy history. Six
individuals had the last bee sting experience >200 days (past BS) and 5 during 30 days (re-
cent BS) before blood sampling. sHI levels were quantifi ed fl uorophotometrically and evalu-
ated by appropriate non parametric statistical analyses.
RESULTS
sHI levels in the beekeepers were not statistically different from those determined in 5 non-
beekeeping matched volunteers. The level of sHI was higher in the blood of tolerant compared
to non-tolerant beekeepers, the distribution being wider in the non-tolerant group. Similarly,
sHI levels were higher in the recent compared to the past BS group, while no signifi cant differ-
ence was observed between the tolerant and the non-tolerant past BS subgroups.
CONCLUSIONS
Despite the small sample size, the fi ndings provide fi rst indication for further examining the
potential exploitation of sHI quantifi cation as a prognostic factor that would facilitate the deter-
mination of optimal treatment strategies in allergic beekeepers.
This work was partly supported by the UoA grant 70/4/5900.
RANITIDINE-INDUCED ANAPHYLAXIS: A CASE REPORT
X.S. Aggelides1, M. Makris1, C. Chliva1, K. Papamichael2, E. Tiligada1,21Allergy Unit 'D. Kalogeromitros' 2nd Department of Dermatology and Venereology, Attikon University Hospital & 2Department of Pharmacology, Medical School, University of Athens BACKGROUND & OBJECTIVES
Drug induced anaphylaxis is a constantly increasing medical emergency. We present a case
of a severe anaphylaxis to ranitidine, a commonly used H receptor inverse agonist (H Ria)
and the relevant diagnostic procedure supplemented by blood histamine measurements. METHODS
A young girl was referred to our unit shortly after a severe anaphylactic reaction during
preoperative anesthesia. All drugs used preoperatively were tested through skin prick and
intradermal application. Among them, only ranitidine produced positive reactions and was thus
considered the culprit drug. In order to determine possible cross-reactivity between agents of
the same class, other H Ria were tested, including cimetidine and famotidine, with no positive
reactions whatsoever. Graded challenges -the gold standard of drug allergy diagnosis- to intravenous ranitidine and cimetidine (as a possible therapeutic alternative) were conducted. Histamine levels in both serum and whole peripheral blood were determined fl uorometrically before, 30' after the initiation of any reaction and at the end of an uneventful challenge. RESULTS
The patient's basal serum histamine levels were comparable to those of normal subjects,
whereas basal whole blood histamine levels were higher. During challenge to ranitidine the
patient developed allergic symptoms, including rash, headache and tachycardia, and the
procedure was interrupted. Serum histamine levels were increased from 4.3ng/mL to 17.1ng/
mL, while they remained low after the negative provocation to cimetidine.
CONCLUSIONS
Serum histamine measurement is a useful tool to document anaphylactic reactions especially
in the context of a controlled challenge. In accordance to previous reports, no cross-reactivity
between H Ria was observed.
EXPLORING THE CHARACTERISTICS OF DRUG ALLERGY IN THE GREEK
POPULATION
S. Kogkas1, M. Potika1, O. Tsilochristou1, X. Aggelides1, E. Tiligada1,2 M. Makris1 1Allergy Unit 'D. Kalogeromitros' 2nd Department of Dermatology and Venereology, Attikon University Hospital & 2Department of Pharmacology, Medical School, University of Athens BACKGROUND & OBJECTIVES
Since there are no epidemiologic data on the incidence of drug hypersensitivity reactions
(DHR) in the Greek population, we conducted a descriptive, observational study in order to
document and classify the cases of drug allergy in the outpatient clinic settings of a tertiary
care hospital.
METHODS
Patients visiting the Drug Allergy Outpatient Clinic (OC) of our Unit were enrolled. Data was
retrieved from OC patients only, Emergency Room patients and referrals from other clinics
being excluded.
RESULTS
1129 patients (304 male, 824 females, mean age 45.9±17,9 years) were evaluated. 401
patients (35.6%) experienced an immediate reaction, while 575 patients (50.9%) reported
a non-immediate reaction. Almost one third of the patients (340 pts, 30.1%) reacted to a
single drug (single-reactors), while 519 patients (46%) reported reactions to more than
one drug (multi-reactors). The principal cause of DHR was β-lactams (45.5%), followed by
NSAIDs (13%) and macrolides (6.8%). Within the β-lactam reactors group, 304 (60.6%)
and 196 (39.1%) patients reported a reaction to a synthetic penicillin and to cephalosporins,
respectively. Amoxicillin was involved in the 24% of all DHRs reviewed and was by far the
most frequently involved drug.
CONCLUSIONS
Most of the obtained results are in accordance with the existing literature: female predominance
in drug allergy is universal, and β-lactams and NSAIDs are typically the "usual suspects"
behind DHRs. However, the high rate of allergy to cephalosporins could be considered
as characteristic for the Greek population and possibly correlate to the local physicians'
prescription habits.
NOVEL MULTI – TARGETED COMPOUNDS WITH ACTIVITY IN A HYPERLIPIDAEMIC/
DIABETES II MOUSE MODEL
Pappa E. Maria and Angeliki P. Kourounakis*Department of Medicinal Chemistry, Faculty of Pharmacy, University of Athens15771 Athens, Greece. *[email protected] BACKGROUND AND OBJECTIVES
A correlation exists between oxidative stress, infl ammation, hyperlipidaemia and the
development of atherosclerosis/diabetes II. Simultaneously addressing these targets is
considered benefi cial in the treatment of such multifactorial diseases. The aim of this study is
to evaluate the in vivo activity of two potent (in vitro) derivatives designed as multi – targeted
agents against atherosclerosis/diabetes II.
METHODS
SKH–2 male mice (7 – 8 weeks old) were divided into several groups, receiving either a
standard chow diet or a high fat diet (40% w/w fat) ± multiple low–dose of streptozotocin,
with or without the administration of compounds 1, 2 or reference compounds (at a dose
of 56 μmol/Kg/twice daily for 50 days). At predetermined time points, glucose levels, lipid
parameters, body weight and food consumption were estimated.
RESULTS
The average body weight and food consumption exhibited no signifi cant differences among
groups over time and throughout the experiment. After 50 days of treatment, glucose levels of
mice treated with compound 1 or 2 were decreased by 38% (***P<0.0001) and 41% (***P<0.0001)
compared to control levels. Total cholesterol levels decreased by 51% (***P<0.0001) and 31%
(***P=0.0007), triglyceride levels by 83% (***P<0.0001) and 78% (***P<0.0001), LDL–cholesterol
levels by 52% (**P = 0.005) and 46% (***P=0.0002), while the antiatherogenic index (HDL/
LDL ratio) improved by 40% (*P = 0.033) and 28% (**P=0.001), respectively. Both compounds
exhibited more potent activity, as shown by almost all parameters measured, compared to
reference compounds that were also hereby evaluated.
CONCLUSIONS
Results of these in vivo experiments confi rm the increased activity of the designed multi–
targeted derivatives 1 and 2 compared to reference compounds.
REFERENCES
Lipoic acid: energy production, antioxidant activity and health effects. Ed. M. S. Patel and
L. Packer, CRC Press, 2008. New Multifunctional Di-tert butyl-phenoloctahydro-(pyrido/
benz)oxazine Derivatives with Antioxidant, Antihyperlipidemic, and Antidiabetic Action.
Ladopoulou E, Matralis A, Kourounakis A. J Med Chem. 2013, 56(8):3330. Multi-targeted
drug design approaches for multifactorial diseases: from neurodegenerative to cardiovascular
applications. M. Katselou, A. Matralis and A. Kourounakis. Curr. Med. Chem. 2014. Design
of Novel Potent Antihyperlipidemic Agents with Antioxidant/Anti-infl ammatory Properties;
Exploiting Phenothiazine's Strong Antioxidant Activity. A. Matralis and A. Kourounakis J Med
Chem, 2014.
TARGETING DNA METHYLATION BY GREEN TEA CATECHINS
E.YiannakopoulouDepartment of Medical Laboratories Faculty of Health and Caring Professions, Technological Educational Institute of Athens, [email protected] BACKGROU ND & OBJECTIVES
Recent data have shown strong chemopreventive and possibly cancer chemotherapeutic
effects of green tea catechins against cancer. Although the molecular mechanisms of the
anti-proliferative action of green tea catechins have not been delineated, green tea catechins
seem to be multi-target agents, modulating multiple signalling pathways. In addition green tea
catechins have been reported to modulate epigenetic processes. Aberrant epigenetic alterations
in the genome such as DNA methylation play a signifi cant role in cancer development. Indeed,
epigenetic processes have been recognized as a new target for anticancer drug design. This
review aims to synthesize evidence on the modulation of DNA methylation by green tea
catechins.
METHODS
Electronic databases were searched with the appropriate search terms up to and including
February 2013.
RESULTS
Green tea catechins have been reported to reverse DNA methylation of tumor suppressor
genes and increase transcription of these genes. Green tea catechins and especially EGCG
modulate DNA methylation by attenuating the effect of DNMT1. However, the exact mechanism
of DNMT1 inhibition is not delineated. Suggested mechanisms include direct enzymatic
inhibition, indirect enzymatic inhibition, reduced DNMT1 expression and translation. The
possible effect of green tea catechins on other pathways of DNA methylation i.e. methyl-CpG
binding domain (MBD) proteins has not been investigated. Furthermore, the link between redox
properties and epigenetic modulation by green tea catechins has also not been defi ned.
Conclusion: Since green tea catechins are natural compounds with a rather acceptable safety
profi le, further research on their action as inhibitors of DNA methylation seems worthwhile.
EFFECT OF OPIOIDS ON INTERNEURONS OF THE DORSAL AND VENTRAL RAT
HIPPOCAMPUS
C. Koutsona, E. Kouvaras and E.K. Asprodini.
Labo ratory of Pharmacology, Medical School, University of Thessaly, Larissa, Greece. BACKGROUND & OBJECTIVES
The hippocampus is differentiated along its longitudinal axis in terms of the electrophysiological
properties of its principal neurons, the anatomical characteristics of neuronal network and the
differential role of the neurotransmitters and neuromodulators, including the opioids. The aim
of the present study was to investigate the role of the Ih current of CA1 interneurons in the
functional differentiation of the dorsal and the ventral hippocampus. The effect of morphine on
Ih was also examined.
METHODS
Standard intracellular current clamp recordings were obtained in vitro from CA1 pyramidal
neurons and interneurons of the oriens/alveus border in 500μm-thick transverse slices from
dorsal and ventral rat hippocampus.
RESULTS
Interneurons of the dorsal and ventral hippocampus exhibited similar passive membrane
properties. Conversely, interneurons of the ventral hippocampus showed a stronger Ih compared
to interneurons of the dorsal hippocampus; moreover, Ih kinetics and the characteristics of
the rebound depolarization following Ih activation were distinct between the two populations.
Morphine (30 μΜ) hyperpolarized dorsal and ventral interneurons, reduced Ih current
activation and changed the characteristics of the rebound depolarization; interestingly, in one
interneuron the drug exhibited opposite results (i.e. caused membrane depolarization and Ih
augmentation. Moreover, morphine decreased the frequency of spontaneous interneuronal
fi ring at resting membrane potential and the fi ring frequency elicited in interneurons following
sustained depolarizing pulses.
CONCLUSIONS
Interneurons of the dorsal and ventral hippocampus exhibit different intrinsic characteristics.
Opioids reduce interneuron excitability in both dorsal and ventral hippocampus. Our fi ndings
confi rm the complexity of the modulatory role of the opioids in the hippocampal network.
Acknowledgements: This work was supported by the Ministry of Education (Herakleitos I,
Pythagoras II).
HPLC DETERMINATION OF THE OXCARBAZEPINE ACTIVE METABOLITE
LICARBAZEPINE IN HUMAN SERUM
E. Begas, E Kouvaras and E.K. Asprodini.
Laboratory of Pharmacology, Medical School, University of Thessaly, Larissa, Greece. BACKGROUND & OBJECTIVES
Oxcarbazepine, a keto-derivative of carbamazepine indicated for the monotherapy or
adjunctive treatment of partial seizures, is metabolized to the pharmacologically active
metabolite 10-hydroxycarbazepine, Licarbazepine. In the present study a simple, sensitive
and reliable HPLC method was developed for the determination of Licarbazepine in the serum
of patients undergoing therapy with Oxcarbazepine.
METHODS
250 μL of the Internal Standard (IS) (acetanilide 20μg/mL in methanol) was added in 100
μL serum or spiked samples. The samples were vortexed (15"-30"), left to stand (5') and
centrifuged (13000rpm, 15', 4°C). The supernatant (20μL) was injected in the chromatographic
column (Kromasil; C18; 250x4.6mm ID; 5μm particle size). The mobile phase consisted of
50mM phosphate buffer (pH 4) and acetonitrile 70/30 (v/v), delivered at 30°C, at a fl ow rate
0.8 mL/min. Licarbazepine and the IS were monitored at 210nm.
RESULTS
Licarbazepine and the IS eluted at 7.3±0.04 and 8.2±0.06 min, respectively. The calibration
curve (peak areas of Licarbazepine versus IS) was linear at a range 0.5-50μg/mL. Analytical
variation was 6%. Specifi city was examined in blank serum samples and BIORAD TDM
Quality Control containing 21 antiepileptic and commonly prescribed drugs. No interferences
at the elution times of Licarbazepine or the IS were observed. Moreover, no interferences
were observed at the elution times of 12 medications commonly administered in psychiatric
patients (benzodiazepines, antidepressants, antipsychotics).
CONCLUSIONS
A simple, sensitiv e, reliable and low cost HPLC method was developed for the determination
of Licarbazepine in human samples. The method can be implemented in the Therapeutic Drug
Monitoring of Licarbazepine in epileptic patients and patients with bipolar disorders.
Acknowledgements: This work was supported by the Ministry of Health (KESY).
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