Trends

........,... .....
Editor
Prof. Viktor BAUER, MD., DSc.
Consulting Editors
Michal DUBOVICK, PhD.
Assoc. Prof. Magda KOUILOV, PhD.
Mojmr MACH, PhD.
Jana NAVAROV, PhD.
Prof. Radomr NOS, MD., DSc.
Ruena SOTNKOV, PhD.
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BRATISLAVA 2008
Trends in Pharmacological Research
ISBN 9788097000370
Published by Institute of Experimental Pharmacology, SASc.
Dbravsk cesta 9, SK-841 04 Bratislava, Slovak Republic
fax: +421-2-59477 5928 e-mail: exfasekr@savba.sk
Printed in Slovak Republic
Cover, Interior Design & Typesetting
Mojmir Mach, PhD.
Copyright 2008 Institute of Experimental Pharmacology
All rights reserved. No part may be reproduced, stored in a retrieval system, or trans-
mitted in any form or by any means, electronic, mechanical, photocopying, recording,
or ortherwise, without prior written permission from the Copyright owners.
Table of Contents
Editorial
V. Bauer 7
Brief history of the Institute of Experimental Pharmacology
R. Nos 8
Trends in studies of drug metabolism and of related drugdrug interactions
P. Anzenbacher, E. Anzenbacherov 11
Smooth muscle tissues as models for study of drug action
V. Bauer, R. Sotnkov, V. Nosov, J. Navarov, . Mtys, V. Pucovsk, V. Rekalov,
K. Szcs, J. Nedelevov, Z. Kyseov, V. Dytrichov, J. Fatykov, M. Kollrov,
L. Mlekov, M. Srnov, Z. Stojkoviov, G. Tthov 15
New ways of supplementary and combinatory therapy of rheumatoid
arthritis (RA) by synthetic and natural substances with antioxidant
properties: New perspectives for routinely administered drugs in RA
K. Bauerov, S. Ponit, K. Valachov, D. Mihalov, L. olts,
D. Komendov, V. Tomekov, M. trosov, P. Gemeiner, G. Poli 25
Effect of cytochrome P450 induction on drug
disposition in isolated rat liver preparation
. Bezek, M. Kukan, T. Trnovec 33
Reflection on animal modeling of human cardiac diseases in preclinical
pharmacology: From measurements of coronary blood flow and cardiac
function to sophisticated approaches of protease-catalyzed soluble
cardiac protein expression with experimental myocardial infarction
J. Dmal, V. Knezl, A. Babulov, L. Bacharov, F. Boroviov, M. Draveck,
P. Gibala, J. Gvozdjak, A. Gvozdjakov, J. Jakubovsk, M. Kittov, D. Magna, A. Rybr,
F.V. Seleck, R. Sotnkov, S. tolc, K. Strov, J. Tokrov, R. Nos, A. Sauberer,
E. Nikov, S. Markovi, J. Torokov, A. Pukrov, T. Kollr 41
........,... .....
New computational approach to mathematical
modeling in pharmacological research
M. uriov, L. Dedk, M. Tvrdoov 48
Breeding and testing facility Dobr Voda
A. Gajdok, A. Gajdokov, E. Ujhzy, D. Golhov, B. Kopeck, V. Krchnrov 58
From comparative interspecies and ontogenetic pharmacokinetics up
to the usage of microcamera-techniques for drug bioavailability studies
(Historicizing comments on three decades of the existence of an experimental
biopharmaceutical research in Hradec Krlov, Czech Republic)
J. Kvtina 66
The use of electrochemical measurement for real-time monitoring
of nitric oxide generation by macrophages in vitro
A. Lojek, M. Pekarov, R. Nos, J. Hrb 77
H1-antihistamine Dithiaden suppressed platelet aggregation
and oxidative burst of neutrophils in vitro
R. Nos, K. Drbikov, V. Janinov, T. Maikov,
J. Peivov, M. Petrkov, Z. Strakov 82
Research focuses of the pharmacology in Martin
G. Nosov, S. Fraov, A. Strapkov, J. Mokr, M. utovsk, V. Sadloov 88
Trends in pharmacological research contribution from
studies of the membrane transport and cell signaling
K. Ondria 96
Vasoactive effects of Provinols in experimental hypertension
O. Pechov, I. Berntov 102
Substituted pyridoindoles as antioxidants and aldose reductase
inhibitors in prevention of diabetic complications: A preclinical study
in vitro and in an animal model of experimental diabetes in vivo
M. tefek, P.O. Djoubissie, A. Gajdok, A. Gajdokov, M. Juskov,
. Krianov, Z. Kyseov, M. Mjekov, L. Rakov, V. nirc 109
........,... .....
New pyridoindoles with antioxidant and neuroprotective actions
S. tolc, V. nirc, A. Gajdokov, A. Gajdok, Z. Gsprov, O. Ondrejikov,
R. Sotnkov, . Viola, P. Rapta, P. Jariabka, I. Synekov, M. Vajdov,
S. Zacharov, V. Nemek, V. Krchnrov 118
Trends of research in pharmacology
M. Tich, P. Urban 137
Trends in developmental toxicology: Protection of the
developing organism an ever topical issue
E. Ujhzy, M. Mach, J. Navarov, A. Gajdokov, A. Gajdok,
J. Jank, V. Dytrichov, M. Dubovick 140
Angiogenesis A perspective target in cancer therapy
L. Varinsk, L. Mirossay, J. Moji 151
Cytokine-stimulatory effects of acyclic nucleotide analogues: extrapolation
of immunopharmacological data from animal to human cells
Z. Zdek, E. Kmonkov, A. Hol 158
AUTHORS INDEX 166
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Editorial
Pharmacology is different from most other biological sciences because it does not
ask how nature works, but rather how can we change nature? Answering this ques-
tion requires the integrated effort of multiple techniques (molecular, biochemical,
cellular and systems-based) to come to a total understanding of the action of a drug.
K. Brune, Trends in Pharmacological Sciences 22(6), 323324, 2001.
Pharmacological investigation has existed for as long as people have been taking drugs
(natural and synthetic compounds). In its simplest sense, pharmacology means what
drugs do to the body and vice versa. One of the most exciting aspects of pharmacology lies
in the unique way it encourages interaction between different scientific approaches. This
book is published on the occasion of the 60th anniversary of the Institute of Experimental
Pharmacology of the Slovak Academy of Sciences. It embraces articles in experimental
pharmacology and toxicology from the viewpoint of the basic scientists, the pharmacolo-
gist or the toxicologist. Our intention was to direct the attention of the medical community
on pharmacological and toxicological aspects of drugs. Moreover, we are presenting recent
developments in studies of drug action which were, are, and are intended to be involved
in the scope of interests of pharmacological institutions in the Slovak and Czech Republic.
In both countries, diverse goals were achieved during the last decades in basic pharmaco-
logical research (covering a range of sub-groups, such as neuro-, cardiovascular, gastrointes-
tinal, respiratory, etc. pharmacology), pharmacokinetics, toxicology (drug toxicology, ad-
verse and side effects, environmental toxicology, food toxicology), and clinical pharmacology.
To characterize effects of biologically active compounds, besides classical and clinical
pharmacology, drug metabolism, pharmacokinetics and analytical and clinical toxicology,
different experimental approaches of various biomedical disciplines, like electrophysiol-
ogy, biochemistry, molecular biology, pharmacogenetics, immunology, molecular toxicol-
ogy, drug epidemiology, pharmacy and clinical pharmacy, among others, were employed
on living matter such as cells, tissues or organs, both in animals and humans. Since some
of the experimentators may not see the whole animal just sense details, and thus instead
of grasping the whole, they dissect it to end up with just a foot, an ear or a piece of the
tail (in other words, perhaps a few G proteins, kinases, lipases or phosphatases, etc.), their
results call upon certain reservation in interpretation. Nevertheless, they opened up new
perspectives and opportunities in drug design and development with or without direct
relevance to therapeutics.
Biomedical research in experimental and clinical pharmacology, targeting drug therapy
and toxicology by exploiting the present knowledge on drug mechanisms of action, fate and
toxicity is a rapidly progressing area. Our aim was to evidence that in our institutions not
only carefully integrated hypotheses are generated but we also wish to stress the importance
of maintaining a critical balance between the molecular understanding of drug targets,
action and safety with their effects and toxicity in the whole animal in health and sickness.
Viktor Bauer
........,... .....
Brief history of the Institute of
Experimental Pharmacology
Radomr NOS
Director of the Institute of Experimental Pharmacology, Slovak Academy of Sciences, Dbravsk cesta 9,
841 04 Bratilsava, Slovak Republic
The Institute of Experimental Pharmacology, Slovak Academy of Sciences, represents
the most advanced research institution in basic and applied pharmacology in Slovakia.
The Institute has played an important role in the development of the scientific fields
pharmacology and toxicology. An integral part of the Institute is the Department
of Toxicology and Animal Breeding located at Dobr Voda near Trnava. It is the only
center for toxicological research in breeding of laboratory animals in Slovakia.
The basis of the contemporary Institute was founded in 1947 when, due to increas-
ing demands on evaluation of the quality of new products in former Czechoslovakia,
the Department of Biological Control of the Chemical and Pharmaceutical Works
Inc. was established in Bratislava. In 1950, the Chemical and Pharmaceutical Works
established the Research Institute for Pharmacy. Its Department of Experimental
Medicine can be considered the actual germ of the present Institute of Experimental
Pharmacology. The Department performed descriptive pharmacological analyses of a
broad spectrum of new substances (e.g. follicular hormone, bee-venom, intravenous
preparation of iron) as well as routine assessments of the toxicity of some biologically
active substances. In 1951, due to lack of healthy and standardized animals for experi-
ments, the Research Breeding Center was founded at Dobr Voda near Trnava. The
Breeding Centre has been supplying experimental animals to institutes of the SASc,
universities and institutes of the Ministries of Health, Agriculture and Industry. In the
same year, the Institute was incorporated into the Research Institute of Pharmacy and
Biochemistry in Prague. Research activities were focused mainly on alkaloids, hor-
mones, purine derivatives, optically active ephedrine and compounds derived from an-
timony for veterinary purposes. In 1953, the Institute was incorporated into the newly
established Slovak Academy of Sciences within the Institute of Chemical Technology
of Organic Compounds. In this period industrial projects were completed and research
work became gradually oriented to basic science. Hypotensive alkaloids and cardioac-
tive glycosides of wild-growing plants in Slovakia were isolated and studied. In 1963,
the Central Laboratory of Pharmacology of the Institute of Organic Chemistry and
Biochemistry of the Czechoslovak Academy of Sciences (CsASc) in Prague and the
9 Brief history of the Institute of Experimental Pharmacology
Bauer et al. Trends in Pharmacological Research
Department of Pharmacology of Natural Substances of the Institute of Chemistry of
SASc in Bratislava including its Research Breeding Laboratory at Dobr Voda, merged
to establish the Institute of Pharmacology of the CsASc with Departments in Prague
and Bratislava. Main interests of the newly formed Department in Bratislava were fo-
cused on mechanism of action of different endogenous and synthesized substances on
synaptic transmission in the peripheral and central nervous system, peripheral and cor-
onary blood-vessels, on myocardial contractility and side effects of antituberculotics.
The year 1969 is important in the history of the Institute. The Slovak Departments
of the Pharmacological Institute of the CsASc became independent and the Institute
of Experimental Pharmacology of the SASc was created. From this time on the main
interests of the Institute were concentrated on receptor specificity and the mechanism
of action of alpha- and beta- adrenoceptor blocking drugs, on the studies of inhibitory
and excitatory modulation of synaptic transmission by biogenic amines, theophylline,
5-hydroxytryptamine and the polyene antibiotic cyanein, on elucidation of the mecha-
nism of action of papaverine in the therapy of some neurosomatic diseases, on phar-
macokinetics of pyrazolidine and xanthine derivatives and beta-adrenoceptor blockers,
and on the relationship between chemical structure and specific mechanism of action
of adrenergic receptor antagonists and carbanilate local anesthetics. On studying the
mechanism of action of carbidine, a new neuroleptic drug stobadine was synthetized,
its membrane stabilizing, alpha-adrenergic receptor-blocking and antihypoxic proper-
ties were demonstrated. The research in pharmacodynamics was focused on several ar-
eas, particularly systemic pharmacology, like cardiovascular and neuropharmacology,
smooth muscle, cellular and biochemical pharmacology. In applied pharmacology the
Institute has a long tradition and is keeping in progress in teratology and pharmacologi-
cal toxicology as documented also by the international symposia on toxicology bienni-
ally organized by the Institute. The two areas on basic teratology and toxicology yielded
important results and provided many reports concerning studies on new drug registra-
tions and production of drugs to be used therapeutically. A similar development holds
true for the research in pharmacokinetics concerning studies in basic pharmacokinet-
ics, particularly in the metabolism and fate of biologically active substances in the living
organism. With the aim to find optimal therapeutical regimens, theoretical modeling
of drug pharmacokinetics in the human and animal body has been introduced.
Over the last decade the following scientic issues have been investigated:
Study of receptor and non-receptor interactions between cationic amphiphilic sub-
stances and isolated cells at molecular and cellular levels (effect of beta-adrenorecep-
tor antagonists and antihistaminic drugs, stobadine and chloroquine on platelets,
polymorphonuclear leukocytes and their interactions).
Preclinical study of the action of compounds affecting generation and/or action of
reactive oxygen species in nervous tissue.
10 R. Nos
Copyright 2008 Institute of Experimental Pharmacology | ISBN 9788097000370
Study of intracellular signalization in the smooth muscles of vessels and in the heart
tissues.
Effect of reactive oxygen species on smooth muscles of intestines, air passages and
vessels.
Mechanisms of absorption, distribution and elimination of drugs, mathematical
modeling of pharmacokinetic processes.
Creation of structural models of biomedical systems, detection of metabolic path-
ways of drugs and development of controlled systems of drug release.
Metabolic changes of xenobiotics; study of chemical and enzymatic mechanisms of
molecular oxygen activation; toxic effects of reactive oxygen species and glucose in
long-term hyperglycemia and protective effect of natural and synthetic antioxidants.
Study of molecular mechanisms of transport and antioxidative properties of selected
antioxidants by means of QSAR method.
Possible negative effects of new drugs on pre- and postnatal development (embryo-
toxicity, teratogenicity and neurobehavioral development of offspring).
Acute and chronic toxicities.
Preclinical study of the original pyridoindole stobadine developed at the Institute
(The Prize of the Slovak Academy of Scienceswas awarded for the year 2000).
At present, the main interest of the Institute is focused on the study of pharmaco-
logical interventions in oxidative stress and proinflammatory reaction-induced injury
of the organism. One of the main interests of the Institute is to develop new pharma-
cotherapeutic approaches to diseases associated with pro-inflammatory processes and
oxidative stress. An integral part of R&D activities are studies in the field of biopharma-
ceutical (medicinal) chemistry focused on the design and synthesis of new pyridoindole
derivatives with anti-inflammatory and anti-radical properties and research of the use
of hyaluronan biopolymers. Pharmacodynamic, pharmacokinetic as well as toxicologi-
cal mechanisms of action of biologically active substances are investigated at body, or-
gan, cellular, membrane, receptor and molecular levels. Potential side toxic effects of
drugs are studied using a battery of toxicity tests.
The Department of Toxicology and Animal Breeding at Dobr Voda possesses
Accreditation for Breeding of Laboratory Animals and Experimentation on Laboratory
Animals from the State Veterinary Administration SR No 7656/02-220, Statement of
GLP Compliance No 23/2000 from Slovak National Accreditation Service (area of ex-
pertise: toxicity studies, carcinogenicity, care and housing of animals) and Statement of
Entry in the Register of Forages SR No 8922.
The Institute plays an importnat role not only in the field of research and development
but also in education of young scientists. In cooperation with Comenius University and
the Slovak Technical University, the Institute educates future pharmacologists, toxi-
cologists and biochemists who become qualified specialists for biomedical research not
only in Slovakia but also in research institutes abroad.
........,... .....
Trends in studies of drug metabolism
and of related drugdrug interactions
Pavel ANZENBACHER
1
, Eva ANZENBACHEROV
2
1
Department of Pharmacology and
2
Department of Medical Chemistry and Biochemistry, Faculty of Medicine
and Dentistry, Palacky University at Olomouc, Hnevotinska 3, 775 15 Olomouc, Czech Republic
Key words: drug metabolism, drug interactions, cytochrome P450, conjugation enzymes,
drug transport, nuclear complexes
Introduction
Last twenty years in life sciences and hence also in pharmacology could be character-
ized by attempts to find molecular basis of function (as well as of dysfunction) of pro-
cesses in living organisms. In medicine, an exponential increase of new findings and
of experimental data has appeared. The amount of new information on the other hand
has caused at least in significant number of colleagues an increasing feeling that either
everything has been already found or that it is almost impossible to keep the pace with
this progress.
The reason for that may be a simple fact that we cannot see the forest because of the
trees, in other words, that the intimate reason for the research in the particular field is
not clearly stated or that it is not explained in a way acceptable even for rather learned
part of the society.
Let us hope that the need for an individualized medicine, and, hence, also for an
individualized pharmacotherapy is generally accepted. The contribution of research in
pharmacogenetic applications in pharmacokinetics, namely, in drug metabolism incl.
its regulation and drug transport is expected here. The most general approach is to see
the organism as a whole, incl. its ability to cope with metabolism of foreign compounds,
with their transport and on the regulation of these processes.
The trends in the field of studies on drug transport and metabolism are (i) to under-
stand the mechanisms which determine the ability of individual (so many?) enzymes
and proteins of drug transport and metabolism to pursue their function, (ii) to un-
derstand the differences in the ability of these proteins to exhibit their action due to
genetically determined structural alterations and (iii) to understand the clinical conse-
quences of the presence of these structurally altered (or even nonfunctional or missing)
proteins.
P. Anzenbacher & E. Anzenbacherov (2008) Trends in Pharmacological Research (Eds. V. Bauer et al.): 1114.
12 P. Anzenbacher & E. Anzenbacherov
Drug metabolism enzymes
Cytochromes P450 (CYP)
The spatial structure of the most of the human liver microsomal cytochromes P450
(CYP) has been determined in last five years by X-ray crystallography, including the
most important enzymes CYP3A4, CYP2C9, CYP2D6 which are responsible for me-
tabolism of approximately three quarters of all drugs biotransformed in the man [1].
The understanding of the ability of these enzymes to metabolize the drugs and other
xenobiotics is apparently determined not only by the spatial structure of the active site
and the access/egress channels by which the compounds enter and leave the active site,
but also by the flexibility of these parts of the molecule [2,3].
Deeper understanding of the function of cytochromes P450 and of the drug bi-
otransformation however needs deeper insight into the differences in the structural
properties of the genetically determined variants of the CYP enzymes which are pres-
ent in significant part of population. For example, the CYP2C9 variant *3 is known to
possess isoleucine instead of leucine in position 359 which leads to lowering of ability
of this protein to metabolize warfarin down to one tenth of the activity of the protein
coded by the normal, wild type allele (www.imm.ki.se/cypalleles). As the number of pa-
tients with this variant allele represents at least 10% of population, the detection of this
genotype is contributing significantly to improvement of warfarin pharmacotherapy.
Enzymes of the 2
nd
phase of drug biotransformation
In majority of cases, the drug is metabolized by enzymes of conjugation phase of drug
metabolism. Here, the glucuronidation is the major pathway; and it is becoming clear
that also these enzymes contribute to individual differences in drug metabolism and
to the need of individualized treatment. The first known example was the increased
hepatotoxicity of paracetamol in individuals with Gilberts syndrome, which is caused
by a lower activity of uridine diphosphate glucuronosyltransferase form 1 (UGT1A1
or UGT1.6) due to mutations in exons 2 and 5 [4]. However, little is known about the
links with this and other genetic variants of the UGT enzymes with defects in drug
metabolism.
Regulation of levels of drug metabolism enzymes, nuclear receptors
The most important mechanism deciding on the levels of the enzymes of the first two
phases of drug (xenobiotic) metabolism is the regulation of transcription of their cor-
responding genes by nuclear receptors and their complementary responsive elements
in the promoter sequences of the corresponding genes. The immediate step leading to
the activation of a receptor is the binding of a regulatory molecule (e.g. a polycyclic
hydrocarbon molecule) to the corresponding receptor. There is a family of these re-
ceptors in the cells (mostly in the liver) which regulate expression of enzymes of both
phases of drug metabolism as well as of the transporting pumps (as the P-glycoprotein
13 Trends in studies of drug metabolism and of related drug-drug interactions
of the multidrug resistance complex, MDR1 or ABCB1). There is a considerable body
of evidence that these receptors are involved in regulation of the enzymes mentioned
in the preceding paragraph; for example, the genetic variants in the UGT enzymes may
well reflect the variants of the regulatory molecules as of the receptors (pregnane X re-
ceptor PXR, constitutive androstane receptor CAR, peroxisome proliferator activated
receptor PPAR) [5].
Systems of active drug transport (ABC pumps)
As it has been introduced in the preceding paragraph, the presence of protein mem-
brane-bound efflux transporters in human cell membranes is one of the factors which
may decide on the available level of a drug in the target tissue, in other words, on
the efficacy of the drug applied. Hence, genetic polymorphisms connected with an
absence or dysfunction of a particular system may be reflected in an ineffective treat-
ment or with a significant decrease in drug efficacy. Whereas the connection of the
polymorphisms of the genes of these proteins with development of many serious dis-
eases is known and accepted [6,7], the effect of these polymorphisms on the pharma-
cokinetics of drugs is known in several cases only. For example, it has been shown that
the common polymorphic variants of the MDR1 protein were associated with higher
digoxin serum concentrations [8], or, that there is an association of the MDR1 gene
polymorphisms and the efficacy and safety of the simvastatin treatment [9].
Conclusion
In other words, the individualization of pharmacotherapy seems to be one of the most
important trends in medicine of the 21
st
century with tools offered by recent devel-
opments in pharmacology, molecular biology, biochemistry and analytical chemistry.
The need for cooperation of specialists in these fields is only stressed by complexity
of the life sciences, however, as it has been written in the introduction, the main aim
should be kept in mind, namely, a significant improvement of the drug efficacy and
safety. Drug metabolism studies aimed at the pharmacogenetics of drug metabolizing
enzymes, on the regulation of these enzymes as well as on the pharmacogenetics of
drug membrane-bound efflux transporting systems will certainly bring many infor-
mation of key importance.
Acknowledgment
The financial support from the Grant Agency of the Academy of Sciences of the Czech
Republic KAN200200651 is gratefully acknowledged.
14 P. Anzenbacher & E. Anzenbacherov
REFERENCES
Anzenbacher P, Anzenbacherov E: Cytochromes P450 and metabolism of xenobiotics. Cell Mol Life Sci [1]
2001; 58, 737747.
Skopalk J, Anzenbacher P, Otyepka M: Flexibility of human cytochromes P450: molecular dynamics reveals [2]
diferences between CYPs 3A4, 2C9, and 2A6, which correlate with their substrate preferences. J Phys Chem
B 2008; 112: 81658173.
Anzenbacher P, Anzenbacherov E, Lange R, Skopalk J, Otyepka M: Active sites of cytochromes P450: What [3]
are they like? Acta Chim Slov 2008; 55: 6366.
Parkinson A: Biotransformation of xenobiotics. In: Casarett and Doulls Toxicology, the Basic Science of Poi- [4]
sons, Chapter 6. Editor: Klaassen CD. Mc Graw Hill, 2001, p. 133224.
Zhou J, Zhang J, Xie W: Xenobiotic nuclear receptor-mediated regulation of UDP-glucuronosyltransferases. [5]
Curr Drug Metabol 2005; 6:289298.
Kimura Y, Morita SY, Matsuo M, Ueda K: Mechanism of multidrug recognition by MDR1/ABCB1. Cancer Sci [6]
2007; 98: 13031310.
Turgut S, Yaren A, Kursunluoglu R, Turgut G: MDR1 C3435T polymorphism in patients with breast cancer. [7]
Arch Med Res 2007; 38: 539544.
Aarnoudse AJ, Dieleman JP, Visser LE, Arp PP, van der Heiden IP, van Schaik RH, Molokhia M, Hofman [8]
A,Uitterlinden AG, Stricker BH: Common ATP-binding cassette B1 variants are associated with increased
digoxin serum concentration. Pharmacogenet Genomics 2008; 18: 299305.
Fiegenbaum M, da Silveira FR, Van der Sand CR, Van der Sand LC, Ferreira ME, Pires RC, Hutz MH: Te role [9]
of common variants of ABCB1, CYP3A4, and CYP3A5 genes in lipid-lowering efcacy and safety of simvasta-
tin treatment. Clin Pharmacol Ter 2005; 78, 551558.
........,... .....
Smooth muscle tissues as models
for study of drug action
Viktor BAUER, Ruena SOTNKOV, Viera NOSOV, Jana NAVAROV, tefan MTYS,
Vladimr PUCOVSK, Vladimr REKALOV, Katalyn SZCS, Jana NEDELEVOV, Zuzana KYSEOV,
Viera DYTRICHOV, Jozefna FATYKOV, Mria KOLLROV, Lubica MLEKOV, Monika SRNOV,
Zuzana STOJKOVIOV, Gizella TTHOV
Department of Smooth Muscle Pharmacology, Institute of Experimental Pharmacology, Slovak Academy of
Sciences, Dbravsk cesta 9, 841 04 Bratislava, Slovak Republic, E-MAIL: viktor.bauer@savba.sk
Key words: smooth muscle, autonomic nerves, epithelium, endothelium, drug actio
Introduction
Direct action of drugs in smooth muscles (SM) or via their innervation, endothelium
or epithelium affects SM tone and/or contractility. Properties common for all types of
SM and special properties of the particular ones, cellular and subcellular organization,
innervation, role of endothelium and epithelium make them suitable to discover fun-
damental physiological, pathophysiological processes and characterize features of drug
action [1]. Although the complexity of the SM preparations calls upon certain reserva-
tion in interpretation of results obtained in vitro and in vivo, with some precaution these
are applicable also for other systems involving similar mechanisms as SMs.
Methods
Our department deals mainly with effects of drugs on autonomous (ANS, i.e. cholin-
ergic, adrenergic, non-adrenergic, non-cholinergic NANC) and sensoric nerves; pro-
cesses linked to epithelium, endothelium and to their biologically active mediators and
modulators; membrane and subcellular receptors and receptor coupled processes; ion
channels; enzymes and availability of Ca
2+
. Introduction of sophisticated electrophysi-
ological, pharmacological, biochemical, isotope and morphological methods have pro-
vided the possibility to elucidate causality and targets of drug action in diseases and
pathological conditions, such as: asthma, gastric ulcer, colitis, ischemia/reperfusion
(I/R), diabetes, oxidative stress, etc. Details of the methods used in our studies are de-
scribed in papers [119].
V. Bauer et al. (2008) Trends in Pharmacological Research (Eds. V. Bauer et al.): 1524.
16 V. Bauer et al.
Results and discussion
1. Calcium and smooth muscle activities
Ca
2+
has an extremely important role in regulation of SM activity, because there is
a greater gradient between concentrations of the free extracellular ([Ca
2+
]
o
) and in-
tracellular ([Ca
2+
]
i
) calcium than for other ions. Elevation of [Ca
2+
]
i
above 0.1 mol/l
and its binding with calmoduline (CaM) activates not only myosine light chain kinase
(MLCK) resulting in SM contraction but also further enzymes affecting SM activity.
Ca
2+
homeostasis is maintained by its influx via selective voltage (VOC, Figure 1A) and
receptor (ROC) operated Ca
2+
channels and less selective cationic channels, by chemi-
cally operated Ca
2+
release from intracellular stores and by its active transport out of
the cell and to intracellular stores, by Ca
2+
pumps and exchange mechanisms. Some
SMs (phasic ones, like intestine or portal vein) generate inward Ca
2+
current on their
entry to the cell, tetrodotoxine (TTX) insensitive action potentials and SM contraction,
which are inhibited by Ca
2+
channel blockers, indicating the essential role of Ca
2+
in
these processes [2,3]. Other SMs (tonic ones, like vessels or trachea) generate action po-
tentials only under specific conditions (e.g. inhibition of membrane conductance for K
+

by tetraethylammonium TEA) and their contraction develops without or during pro-
Figure 1. Patch clamp recording of the eect of nifedipine on inward Ca
2+
current (A), hypoxia on spontaneous
transient outward K
+
currents (STOCs) and whole cell current (B) and H
2
O
2
on single K
+
channel activity evoked
by current RAMPs from +20 to -40 mV (C) (channel conductivity and reversal potential are indicated) of guinea
pig (GP) taenia coli (TC) SM cells.
17 Smooth muscle tissues as models for study of drug action
longed low amplitude membrane depolarization. There is, however, some evidence of
SM contractile proteins activation without participation of an increase in [Ca
2+
]
i
[3,4].
The following membrane elements participate in maintaining the SM membrane po-
tential and alterations of its conductivity: the VOC (L-type, T-type, CRAC-type); ROC;
chemical agonists activated nonselective cationic channels (fast and slow) permeable for
Na
+
and Ca
2+
(responsible for excitatory junction potentials) or permeable for Na
+
and
K
+
(responsible for prolonged depolarization); voltage dependent Na
+
channels (myo-
cardial and neuronal type); voltage dependent K
+
channels (transient type, responsible
for A current); TEA sensitive and insensitive outward and inward rectifiers; Ca
2+
and
voltage dependent maxi- (responsible for transient outward current) and low- conduc-
tance (responsible for oscillations of the membrane potential and STOCs) K
+
channels
(Figure 1B, C); membrane potential independent K
+
channels (receptor operated N
type; ATP and Ca
2+
sensitive; ATP sensitive and Ca
2+
insensitive responsible for rest-
ing membrane potential, M current and S current); second messenger activated voltage
and Ca
2+
dependent channel (conductive for anions, mainly Cl
); the Na
+
pump; Ca
2+

pump; Na
+
- Ca
2+
exchanger; Na
+
- H
+
exchanger and K
+
- Na
+
- Cl

exchanger [1,5].
The effects of Ca
i
2+
may be modified by: its binding (e.g. by chelators); modulation
of CaM and its interaction with Ca
2+
(e.g. by phenothiazines); influence of MLCK and
myosine phosphatase (e.g. by substance A-3) or myosine and actine (e.g. by H
2
O
2
).
Mechanisms coupled to intracellular Ca
2+
stores participate as well in maintaining free
[Ca
2+
]
i
. From one of these stores (S) Ca
2+
is released by IP
3
sensitive (IICR) and ryano-
dine sensitive (CICR) channels, while from the other (S) only through IICR channels.
IP
3
receptor activation released Ca
2+
evokes additional Ca
2+
releases (Ca
2+
induced Ca
2+

release) by means of CICR channels. Free Ca
i
2+
activates proteinkinase C and by Ca
i
2+
/
CaM interaction proteinkinase
II
, which phosphorylates VOC and IICR channels and
releases further Ca
2+
. Transport of Ca
2+
back to its intracellular stores is materialized
by a tapsigargin sensitive Ca
2+
pump. If in S the concentration of Ca
2+
becomes low,
calcium influx factor (CIF) is produced. CIF activates Ca
2+
release activated channels
(CRAC) in plasma membrane and mediates the influx of Ca
2+
to the SM cell.
2. Interaction among smooth muscle layers
SMs are composed of mechanically and electrically coupled cells in close vicinity with
the surrounding connective tissues. Myosine possesses a more substantial role in ad-
justment of SM tone than actine. Their structural organization grants transfer of con-
tractile protein generated force along the whole tissue. It is believed that the longitu-
dinal muscle (LM) of guinea pig ileum (GPI), used for more than a hundred years as
SM model, is liable for isotonic shortening or elongation and isometric raise or loss of
SM tone [6]. The role of the circular muscle (CM) and of the so-called microcosmos
of ANS is neglected, though there are significant variations between reactivities of LM
and CM layers. To evoke contraction of GPI longitudinal muscle strips (LSt) single pulse
stimulation of intramural nerves (ES) is sufficient; while for activation of circular strips
18 V. Bauer et al.
(CSt) tetanic stimulation is needed. The responses of CSt have features of rebound con-
tractions (RC) rather than of primary ones. Dose response curves (DRC) of isoprena-
line (Iso), acetylcholine (Ach) and histamine (Hi) have higher amplitudes on LSt than
on CSt, while the contrary applies in the case of carbachol (Crb) and KCl. The sensitivity
of LM, holding the intramural myenteric plexus (MP), to ES, Iso, Ach, Hi, H
2
O
2
, Crb
and KCl did not differ from that of LSt. In contrast, isolated CM, poor in MP, loses re-
activity to Crb and KCl. Mild reduction of difference between responses of CM and CSt
by hexamethonium suggests that ganglionic transmission does not participate in com-
munication between the muscle layers. Peristaltic activity and rhythmic longitudinal
shortenings evoked by Ach, Crb and KCl are in the case of Crb and KCl transient and
succeeded by elongation of the GPI. Elongation results most likely from the presence of
transversally oriented muscle fibers in CM and the less regular organization of actine
and myosine which endows greater contraction capability compared with LM (parallel
longitudinal arrangement of cells converts to transversal direction on shortening and
slewing around the longitudinal axis)[7]. Yet the possible participation of Cajal cells in
the different reactions of muscle layers can not be excluded.
3. Adrenergic transmission
Using - and -adrenoceptor agonists and antagonists, we found that postsynaptic
1
-drenergic receptors dominate in terminal, while
2
-receptors in intermediate and
proximal parts of the GPI. The mainly inhibitory -adrenoceptors are homogenously
distributed. The cholinergic and NANC nerve terminals possess modulatory
2
- inhibi-
tory receptors [8,9].
Isolation of SM cells under which
1
-adrenoceptors do not lose their function was
developed using antioxidants (dithiothreitol or taurine), high concentration of bo-
vine serum albumin and the relatively specific enzyme, collagenase Type XI (Sigma).
Phenylephrine (PhE) induced
1
-adrenergic receptor mediated membrane hyperpolar-
ization in TC and LM of GPI. It enhanced the amplitude of inward Ca
2+
current, the
frequency and amplitude of voltage and temperature dependent STOCs, elicited low
amplitude sustained outward current, reduced the inward and enhanced the outward
component of the whole cell current. These results are in favor of the assumption that
SM relaxation and membrane hyperpolarization in GPI and TC are realized at least in
part as a consequence of activation of Ca
2+
dependent K
+
conductance [9].
4. NANC transmission
The SM tone and its spontaneous activity are modulated beyond the ion channels and
transport mechanisms, also as a result of receptor and enzyme activation. Besides cir-
culating hormones (e.g. steroids, catecholamines) the receptors and enzymes are affect-
ed also by neurotransmitters such as Ach, noradrenaline (NA), substance P (SP), nitric
oxide (NO), vasoactive intestinal polypeptide (VIP), etc., released from the ANS and
sensoric nerves and by mediators such as endothelium-derived relaxing (EDRF/NO),
contracting (EDCF), and hyperpolarizing (EDHF) factor, eicosanoids, Hi, bradyki-
nine (BK), angiotensin, serotonin (5-HT), endothelin, reactive oxygen species (ROS),
etc. released from nerves, epithelium, or endothelium. Receptors on SM membrane are
coupled to ion channels directly (e.g. chemically activated cation channels), by enzymes
(e.g. tyrosine kinase, phospholipase A
2
), or through G-proteins and enzymes (e.g. ad-
enylate cyclase, guanylate cyclase, phospholipase C). The produced second messengers
(e.g. cAMP, cGMP, prostaglandins, IP
3
, DAG) subsequently affecting intracellular re-
ceptors (e.g. IICR) or enzymes (e.g. proteinkinases) activate myosinkinase (phosphory-
lates myosine resuling in SM contraction), or phosphatase (dephosphorylates myosine
resulting in SM relaxation).
In the presence of atropine and guanethidine, stimulation of NANC nerves evokes in
GPI LM relaxation-contraction with RC, while in GPI CM, TC, GP and cat (C) airways
(AW) relaxation with RC [10,11]. Using microelectrodes and sucrose-gap methods, TTX
and Mg
2+
sensitive NANC excitatory (e.j.p) and inhibitory (i.j.p.) junction potentials
were recorded from GPI LM and NANC i.j.p. from GPI CM, TC and CAW. Rebound de-
polarization was recorded on both layers. Frequency profiles demonstrated that NANC
responses arose at higher frequencies than cholinergic ones. Thus GPI LM possesses in
addition to cholinergic and adrenergic, both excitatory and inhibitory NANC innerva-
tion. The NANC excitation is denser in the terminal, while the NANC inhibition in the
proximal parts of GPI. In contrast the GPI CM and TC possess homogenous NANC
inhibitory innervation. To unveil the nature of NANC transmission we used ATP,
ADP, apamin, TEA, VIP, SP and its derivatives, capsaicine, BK, calcitonin gene-related
peptide, catecholamines, 6-hydroxydopamine, reserpine, Hi, 5-HT, GABA, indometha-
cine (Indo), carbamate local anesthetics, 3,4-diaminopyridine, opioids, dipyridamole,
ROS, inhibitors of NO synthase (INOS) and the method of cross desensitization. Our
results suggest that in the GPI, TC, GPAW and CAW ATP, adenosine and prostaglan-
dins do not contribute to the generation of NANC response. SP is probably the excit-
atory and VIP and NO are the inhibitory NANC transmitters [10,11].
5. Eects of ROS
ROS produced in cell membrane lipid bilayers, in the electron transport system of mi-
tochondria, in cell organelles, like peroxisomes, lysosomes, endoplasmic reticulum, as
well as in the cytoplasm by enzymatic and non-enzymatic reactions are essential for
physiological function, metabolism and defense of the majority of cells and tissues [12].
In GPI LM, TC, GPAW, CAW and rat aorta (RA) ROS evoked contraction, relaxation
or biphasic response. Their amplitudes depended on basal tone (spontaneous or elevated
by Hi, 5-HT or KCl). INOS ameliorated the ROS induced contractions. The ROS effects
were dependent on intact endothelium and mucosa. H
2
O
2
and superoxide (O
2
) in GPI
LM were more effective than hydroxyl radical (
OH) and singlet oxygen (

1
O
2
). During
relaxation, which follows the initial phasic contraction the responses of GPI LM elicited
by Ach and ES were suppressed. The NANC excitation was more sensitive to ROS action
20 V. Bauer et al.
than is NANC inhibition. While native neutrophils did not affect the muscle tone, the
fMLP activated ones (ANT) induced contraction, relaxation or a biphasic response of
RA precontracted by PhE, NA or KCl, due to production of O
2
and its transformation

to other ROS. The muscle tone of SMs changed as the result of direct (altered membrane
activity, e.g. Figure 1B, C) or indirect effects of ROS (elimination of the protective role
of the endothelium, interaction with NO or its production, or with cyclooxygenase and/
or lipoxygenase pathways) [13,14].
CAW possesses diethyldithiocarbamic acid sensitive superoxide dismutase activity.
The multiple actions of O
2

generating systems appear to result from the presence and
simultaneous action of at least two different ROS (O
2

and H
2
O
2
). While O
2

inhib-
ited cholinergic and NANC transmission and participated at least in part in the evoked
relaxation, H
2
O
2
seems to be responsible for elevation of muscle tone and augmentation
of cholinergic contractions and e.j.p.s, resulting from increase of [Ca
2+
]
i
, with subse-
quent augmentation of stimulation of evoked contractions, as well as of the Ca
2+
and
voltage sensitive K
+
conductance[15].
6. Pathologic conditions
Introduction of the above mentioned electrophysiological, biochemical, isotope and
morphological methods have provided the possibility to elucidate causality and targets
of drug action in numerous pathological conditions.
a) Ischemia/Reperfusion (I/R)
Effects of I/R on endothelial function (tested in PhE precontracted rings using Ach)
[13] and production of ROS (detected by luminol enhanced chemiluminiscence) was
evaluated in Wistar rats. Ischemia was induced by clamping of the superior mesenteric
artery (SMA) for 60 min followed by reperfusion (for 5 or 30 min). Ischemia or ischemia
followed by 5 min-reperfusion did not change SMA reactivity and ROS production.
Prolongation of reperfusion to 30 min increased the spontaneous production of ROS in
SMA, which correlated with functional injury, i.e. impairment of endothelium-depen-
dent relaxation (EDR) and decrease in EC
50
values of Ach. Inhibition of prostaglandin
and NO synthesis by Indo and N
G
-nitro-L-arginine methyl ester (L-NAME) attenuated
EDR from sham-operated and I/R groups, suggesting that I/R damages both systems,
the EDRF and EDHF (Figure 2).
b) Experimental diabetes
Circulatory and gastrointestinal disorders have often been reported in diabetes. In
diabetic macroangiopathies hyperglycemia facilitates deterioration of EDR through
increased production of ROS. Ten weeks of streptozotocine (STZ)-induced diabetes re-
sulted in diminished EDR of RA, increased endothelemia, elevated systolic blood pres-
sure and increased concentration of ROS in RA and blood. Incubation of aortic rings
in solution with high glucose concentration led to impairment of EDR. Reduced EDR
in SMA of diabetic rats was not aggravated by I/R and was restored by a derivative of
stobadine (STB), the antioxidant SMe1EC2. In diabetic rats, the I/R induced intestinal
injury was more pronounced (by 63.6%) than in non-diabetic rats. The production of
ROS was unchanged or increased in the vascular and was inhibited in the intestinal tis-
sue, probably as a result of ROS release from the injured mucosa [16].
The extent of gastrointestinal damage accompanying STZ-induced diabetes, namely
the length and number of spontaneous gastric lesions, was significantly reduced after
four-month oral treatment with vitamin E (VitE) plus STB and after butylated hydroxy-
toluene (BHT), but not after VitE alone. There was a trend to increased GSH content by
the antioxidants tested in both control and diabetic rats, with the highest values after
BHT treatment, which caused also a significant increase of proteins in the gastric mucosa.
The phasic and tonic component of the KCl induced contraction and the consequent
papaverine evoked relaxation of the rat ileum were not influenced by diabetes. The Ach
evoked contraction amplitude was augmented and DRC shifted to the left, while Iso in-
duced relaxation was reduced, with DRC shifted to the right. The effect of diabetes was
reduced on Ach action by VitE and by BHT and that on Iso action by VitE and VitE plus
STB. The results indicate differences in mechanisms of action of antioxidants in their
protective effects in diabetes accompanying gastrointestinal disorders [17].
c) Colitis
The colonic mucosa provides an efficient barrier against a potentially harmful environ-
ment that exists in the intestinal lumen. This barrier is impaired in colitis a chroni-
cally recurrent, inflammatory bowel disease, with a variety of etiological mechanisms,
Figure 2. Eect of mesenteric ischemia (60 min) followed by reperfusion (30 min) on EDR of the SMA before (A)
and after INOS with 100 mol/l L-NAME (B). Relaxation is expressed as % of contraction induced by 1 mol/l PhE.
Data are means SEM of 8-12 measurements. *p<0.05, **p<0.001 against sham-operated.
22 V. Bauer et al.
e.g. low tissue level of endogenous antioxidants and increased ROS production in oxida-
tive stress. In rat colitis induced by 4% acetic acid (AA), the developed microcirculatory
ischemia and increased mucosal permeability triggered release of proinflammatory
mediators. Excessive production of ROS contributes to the development of tissue injury.
Locally administered STB, N-acetylcysteine and melatonin can reduce the extent of co-
lonic injury, abolish the increase in myeloperoxidase (MPO) activity, attenuate the en-
hanced vascular permeability and prevent the depletion of GSH. Antioxidant activity of
the compounds tested may be partly responsible for the observed protection. Activation
of H
3
receptors by -methylhistamine and its prodrug, BP 294, on the model of trini-
trobenzene sulphonic acid induced colitis exerted a beneficial effect. The H
1
antagonist
bisulepin reduced the extent of AA injury and diminished the MPO activity, vascular
permeability and gamma-glutamyl transpeptidase activity. By interfering with the ac-
tion of the major mast cell mediator Hi, bisulepin may protect the colonic mucosa. Also
the local anesthetic trapencaine was found to reduce the extent of mucosal injury, as
evidenced by decrease in the rank of gross mucosal damage and in wet/dry weight ra-
tio, attenuation of granulocyte infiltration, as well as by reduction of increased respon-
siveness of colonic SM to Ach and BaCl
2
. Dietary supplementation with oral pleuran,
a -glucan isolated as the main fiber from the fungus Pleurotus ostreatus, possessing
biological response modifier properties, enhanced the intestinal integrity effectively,
scavenged free radicals and exhibited antioxidant defense of the colon [18].
d. Peptic ulcer disease
The protective effect of one of the newly synthesized carbamate local anaesthetics, pen-
tacaine, was found in various models of gastric lesions, induced by phenylbutazone,
Indo, concentrated ethanol, cysteamine, AA, water immersion stress. Mechanism of
pentacaine action was found to be based on stimulated secretion of endogenous pros-
taglandins and of gastric mucus connected with slight antisecretory and free radical
scavenging activity[19].
Conclusions
All our above mentioned results imply that comparable pharmacodynamic effects could
be reached by intervention in different levels of the SM tissue. Increased or reduced
release, uptake or storage of neurotransmitters, epithelial or endothelial mediators,
stimulation or inhibition of membrane receptors, ion channels, G-proteins, production
or elimination of second messengers and corresponding enzymes, as well as release of
Ca
2+
from its intracellular stores may influence SM tone and activity due to alteration
of Ca
2+
availability.
Despite similarities in drug actions on SM, for efficient pharmacotherapy and clari-
fication of side effects, it is not negligible if the hit of the target is selective and whether
it does influence the pathological mechanisms or not. Drug action on processes close
to contractile proteins could affect the activity of other muscles, and action on distant
regulatory mechanisms could alter the activity of other organs as well. SM tissue injury
under pathological conditions (ischemia, diabetes, inflammation, oxidative stress, etc.)
might result also from damage of the relevant nerves, epithelium and endothelium,
which are frequently more vulnerable than SM itself. Thus the selected drug should be
effective on specific pathological mechanisms, which have to be targeted and affected.
Acknowledgments
Our experiments during the last decade were supported by VEGA grants 288, 1017,
2052, 5305, 6024, 7155 and the grant of APVT-20-020802. This work was supported by
VEGA grants 2/0086/08 and the grant of APVV-51-0179052.

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Trends In Pharmacological Research
p. 2532 (2008)
New ways of supplementary and
combinatory therapy of rheumatoid
arthritis (RA) by synthetic and natural
substances with antioxidant properties
New perspectives for routinely administered drugs in RA
Katarna BAUEROV
1
, Silvester PONIT
1
, Katarna VALACHOV
1
, Danica MIHALOV
1
,
Ladislav OLTS
1
, Denisa KOMENDOV
1
, Veronika TOMEKOV
1
, Miriam TROSOV
2
,
Peter GEMEINER
3
, Giuseppe POLI
4
1
Department of Pharmacology of Inflammation,
2
Department of Biochemical Pharmacology, Institute of
Experimental Pharmacology, Dbravsk cesta 9, 841 04 Bratislava, Slovak Republic, E-MAIL: katarina.bauerova@savba.sk
3
Department of Glycobiotechnology, Institute of Chemistry, Slovak Academy of Sciences, Bratislava, Slovak Republic,
4
Department of Clinical and Biological Sciences, University of Torino, San Luigi Gonzaga Hospital, Torino, Italy
Key words: rheumatoid arthritis, adjuvant arthritis, oxidative stress, hyaluronan, antioxidants,
D-penicillamine, coenzyme Q, methotrexate, transition metals, synovial fluid
Introduction
Oxidative stress has been implicated in various pathological conditions involving sev-
eral diseases and aging [14]. These diseases fall into two groups: (i) the first group
involves diseases characterized by pro-oxidants shifting the thiol/disulphide redox
state and impairment of glucose tolerance the so called mitochondrial oxidative
stress conditions (cancer and diabetes mellitus); (ii) the second group involves dis-
eases characterized by inflammatory oxidative conditions, with enhanced activity of
either NAD(P)H oxidase (leading to atherosclerosis and chronic inflammation includ-
ing rheumatoid arthritis) or xanthine oxidase-induced formation of ROS (implicated in
ischemia and reperfusion injury) [5].
Rheumatoid arthritis (RA) is a common severe joint disease that involves all age
groups. Incidence rates of RA are highest in postmenopausal women and increase stead-
ily with advancing age. In general, the disease progresses and often leads to disability. It
can shorten the patient`s life span by 10 years. The cause of the disease is multifactorial,
including genetic predisposition. It is characterized by typical chronic inflammation,
initiated and maintained via autoimmune mechanisms. The disease is assumed to be
triggered by a microorganism in genetically predisposed subjects [6].
K. Bauerov et al. (2008) Trends in Pharmacological Research (Eds. V. Bauer et al.): 2532.
26 K. Bauerov et al.
The pathogenesis of RA is associated predominantly with the formation of free radi-
cals at the site of inflammation. The inflammatory process develops in the tissue of
the synovium: primary sources of reactive oxygen species (ROS) in RA are leukocytes,
which are recruited to accumulate within the synovium. Oxidants can be produced by
activated macrophages in the synovial membrane and by activated neutrophils in the
synovial cavity [78].
In our studies new ways of supplementary or combinatory RA therapy by synthetic
and natural substances with antioxidant activity were investigated and routine drugs
for basic RA therapy were re-evaluated. For this purpose we used the in vitro model of
hyaluronan degradation by the oxidative system Cu(II) ions plus ascorbate and the in
vivo animal model of RA adjuvant arthritis induced in Lewis rats.
Methodological tools
Hyaluronan degradation by the oxidative system Cu(II) ions plus ascorbate
a simplied in vitro model of conditions in rheumatic synovial uid and cavity
Hyaluronan (HA) is a polyelectrolyte component of the synovial fluid (SF). In SF of
healthy subjects, the HA molar mass is of the order of several mega-Daltons. In RA
patients, the mean HA molar mass is however significantly reduced by ROS [910].
The oxidative system Cu(II) ions plus ascorbate was shown to be a source of hydroxyl
radicals degrading hyaluronan. In our in vitro experiments rotational viscometry was
applied [1112].
Adjuvant arthritis an animal disease model resembling rheumatoid arthritis
In our in vivo experiments we used adjuvant arthritis (AA) an animal model of RA
which allows to monitor the disease processes in the acute phase (days 1421) and in the
subchronic phase (after day 28). The advantages of this model are the many similarities
it has with RA, such as symmetrical joint involvement, persistent joint inflammation,
synovial hyperplasia and a good response to most therapies effective in RA [13].
Design of the experiments. After approval by the local ethics committee, AA was in-
duced in male Lewis rats, weighing 150170 g each, by a single intradermal injection
of heat-inactivated Mycobacterium butyricum in incomplete Freunds adjuvant (Difco
Laboratories, Detroit, MI, USA). In each experimental group 68 animals were used.
Generally, in our experiments, inflammatory, arthritic, and oxidative stress param-
eters were assessed. The experiments included healthy intact animals as reference con-
trols, arthritic animals not treated, and arthritic animals treated with the substances
tested.
In the experiment in which combination therapy with coenzyme Q
10
(CoQ
10
) and
methotrexate (MTX) was evaluated, we monitored one basic clinical parameter: change
of the hind paw volume (HPV). The HPV increase was calculated as the percentage of
increase of HPV on day 28 in comparison with the beginning of the experiment. To
27 New ways of supplementary and combinatory therapy of rheumatoid arthritis
compare the changes of this parameter with the results obtained for selected biochemi-
cal parameters, the treated groups and the untreated arthritis were compared with the
control group (100%). The measurement of biochemical parameters was performed on
day 28 after induction of arthritis, as described below:
Level of plasma protein carbonyl groups, a marker of oxidative modifications of pro-
teins, was determined by enzyme linked immunosorbent assay (ELISA) [14]. The re-
action of 4-hydroxynonenal (HNE) and malondaldehyde (MDA) with proteins is fre-
quently associated with their covalent crosslinking leading to the formation of fluoro-
phores. Thus for determination of the level of HNE- and MDA-protein adducts in plasma
spectrofluorimetric measurement was applied [1516].
We studied the effect of therapy with CoQ
10
in a daily oral dose of 20mg/kg b.w.
alone and in the combinatory therapy with MTX in the oral dose of 0.3 mg/kg b.w.
twice a week. Monotherapy with MTX was also performed as a reference treatment.
The clinical and biochemical data were expressed as arithmetic mean with SEM. For
statistic calculations Students t-test was used.
The lack of relevant understanding concerning the pathogenesis of RA is a major prob-
lem in the introduction of new therapies. Several clinical studies as well as preclinical
animal models of RA have documented an imbalance in the body`s redox homeostasis
to a more pro-oxidative environment, suggesting that therapies that restore the redox
balance may have beneficial effects on the disease process [17]. Bauerov and Bezek [6]
and Jaswal et al. [18] described oxidative stress to be one of the primary factors involved
in the pathogenetic changes during rheumatoid arthritis.
Our simplified in vitro model of local conditions in rheumatic synovium is focused
on applying two components. Transition metal ions, whose selection is related to the
fact that in RA the concentration of copper ions is three times higher compared to nor-
mal synovial fluid [19] and these ions are most efficient in ROS generation. Ascorbate,
a known antioxidant in the physiological concentration 40140 M [20], acts as a pro-
oxidant in the presence of metal ions. In this two-component reactive system, HA was
exposed to the action of glutathione, stobadine and the stobadine derivative SMe1EC2.
HCl. Glutathione was shown to be an excellent free radical scavenger in our study, as
it completely inhibited HA degradation. The antioxidative effect of stobadine and its
derivative SMeEC2.HCl was found only in a relatively high concentration, i.e. 1000 M
(21). After addition of d-PN, a dual antioxidative and pro-oxidative effect was observed.
Figure 1 (curve marked 0) demonstrates that the addition of Cu(II) ions followed by
the admixture of the reductant ascorbate results in a gradual decline of the dynamic
viscosity value of the HA sample. As shown in Figure 1 (curves marked 50, 100, and
200), addition of d-PN dose-dependently prolonged the period of complete inhibition
of the degradation of HA. However, after a certain time namely 30 min at 50 M
d-PN, 40 min at 100 M d-PN, or approximately 90 min at 200 M d-PN a rapid uni-
(curves marked 50 and 100) or bi-phasic (curve marked 200) reduction of the sample
dynamic viscosity was evidenced. The protective effect of this drug, evident only for a
certain time period, may be due to the fact that the drug completely traps
OH radicals
generated from Cu(II) ions plus ascorbate under aerobic conditions. Its pro-oxidative
effect is performed especially due to thiyl radicals generated from its molecule, which
further react with d-penicillamine anions, resulting in novel radical-reactive species.
They can generate further
OH radicals by reducing dioxygen molecules.

In our previous in vivo experiments, we demonstrated the beneficial effect of many
substances with antioxidant properties, administered as monotherapy, on the course of
progression of adjuvant arthritis. Of synthetic molecules, copper complexes [22] and
pyridoindole derivatives were investigated and proved beneficial [2324]. Substances of
natural origin, isolated from plants as arbutin, curcumin, rutin, as well as standardized
plant extracts from Arctostaphylos uva ursi, Boswellia serrata and Zingiber officinale,
were found to improve the biochemical picture of AA [2527]. Antioxidant and anti-
inflammatory activities were established for carboxylated (13)-beta-D-glucan isolated
from Saccharomyces cerevisiae [28] and for beta-(1,3/1,6)-D-glucan (Immunoglukn)
isolated from Pleurotus ostreatus [29]. Glucomannans (GM) from Candida utilis were
also evaluated. The anti-arthritic activity for cell-wall GM was comparable to the effect
of Cyclosporin A and was associated with antioxidant activity in vivo [24, 30]. For the
group of natural substances, however, dosage and application form seem to be crucial
for the therapeutic effect.
Antirheumatic treatment affecting the level of CoQ
10
was found to slow down the
progression of RA [3132]. In our AA experiments, mitochondrial function in the heart
Figure 1. Eects of D-PN (0, 50, 100, or
200 M) on HA degradation induced
by a system containing 1.0 M CuCl
2

plus 100 M ascorbate.
and skeletal muscle and effectivity of supplementation with CoQ
10
was dependent on
the severity of the induced adjuvant arthritis. The results with solubilized CoQ
10
(hy-
drosoluble form) indicate its cardioprotective effect in the experimental model of ad-
juvant arthritis. They may thus be of potential significance in the treatment of patients
with rheumatoid arthritis [3338].
Amelioration of clinical manifestations of the disease is crucial for the effectivity of
antiarthritic therapy. From our results we can conclude that significant reduction of ox-
idative stress in AA is not invariably coincident with a significant reduction in clinical
markers of the disease. On balance then, sole reduction of oxidative stress in AA is not
a sufficient therapeutic intervention. Therefore we recommend the antioxidant therapy
as an additional approach to be applied along with standard therapy of RA. Benefits of
this combined therapy should involve not only better efficacy of basal antirheumatic
treatment but also reduction of side effects resulting from the possibility to lower the
dose of a classical antirheumatic drug.
Based on our results with mitochondrial energetics modification and the observed
anti-inflammatory and antioxidant effects [33, 3536], we chose CoQ
10
as one of the
suitable candidates for combinatory therapy of RA. The aim was to assess if CoQ
10
po-
tentiates the antirheumatic effect of MTX. We observed a reduction of hind paw volume
for the therapy studied. The most pronounced effect was found for the combination of
MTX and CoQ
10
. These promising clinical results were further completed by measure-
ments of HNE- and MDA-protein adducts and protein carbonyls in plasma. We ob-
tained a good agreement of the clinical and biochemical measurements: the effect was
increasing in the order CoQ
10
or MTX alone and the combination of CoQ
10
and MTX.
Moreover, the combination decreased all parameters to the level of the control group
values, being more effective than the individual substances by themselves (Figure 2).
The observed antioxidative effect of MTX is given by its complex immunosuppressive
actions, exerted mainly on neutrophils [39].
Conclusions
In vitro we demostrated the antioxidant or pro-oxidant behavior of d-penicillamine in
the presence of cupric ions and ascorbate in the function of free radical generator. In
further experiments we will study in detail the generation of different radicals, mainly
thiyl radicals and their function in the pro-oxidative effect of d-penicillamine. This
could be of great importance for clarifying its adverse effects. It would be relevant to test
d-penicillamine in the system studied also in combination with other antirheumatics of-
ten used in therapeutic practice, concerning e.g. non-steroidal antiinflammatory drugs.
In vivo we demonstrated that CoQ
10
could potentiate the antiarthritic (decrease of
hind paw volume) as well as the antioxidant effect of methotrexate on the level of oxida-
tion of proteins (suppression of levels of protein carbonyls in plasma) as well as lipoper-
oxidation (suppression of levels of HNE-adducts and MDA-adducts to plasma proteins).
In further experiments we intend to address the questions: what is the mechanism of
this effect and how great could be its impact in decreasing the adverse effects of MTX
depending on lowering its dosage. Other candidates for a combinatory therapy with
MTX will also be evaluated.
Acknowledgments
We are grateful for financial support to agencies VEGA, APVV and COST (projects:
VEGA 2/5051/05, VEGA 2/0090/08, VEGA 2/0003/08, APVV-51-017905, APVV-21-
055205, COST B35) and for personal support and cooperation to all colleagues and
friends in Slovakia and Italy.
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........,... .....
Effect of cytochrome P450
induction on drug disposition in
isolated rat liver preparation
tefan BEZEK
1
, Marin KUKAN
2
, Tom TRNOVEC
2
1
Laboratory of Cell Cultures, Institute of Experimental Pharmacology, SASc.,
Dbravsk cesta 9, 841 04 Bratislava, Slovak Republic, E-MAIL: stefan.bezek@savba.sk
2
Research Base of the Slovak Medical University, Limbov 14, 833 03 Bratislava, Slovak Republic
Key words: pharmacokinetics, cytochrome P450, drug elimination
Introduction
Variability in the protein levels of cytochrome P450 (CYP) enzymes among individuals
has been recognized as an important determinant in drug disposition and pharmaco-
logical response in humans. Cytochrome P450 induction-mediated interaction is one
of the major concerns in clinical practice and for the pharmaceutical industry because
of potential involvement of multidrug therapy. There are two major issues associated
with CYP induction [1]. First, induction may cause a reduction in therapeutic efficacy of
comedications. For drugs whose effect is produced primarily by the parent drug, induc-
tion would increase elimination of the drug, resulting in lower drug concentrations, and
decrease the drugs pharmacological effect. Second, induction may create an undesir-
able imbalance between detoxification and activation as a result of increased formation
of reactive metabolites, leading to an increase in the risk of metabolite-induced toxicity.
In the present paper we present results of the effects of CYP induction on the he-
patobiliary disposition of a xanthine related nootropic drug ethimizol; the thymidine
analogue azidothymidine (AZT), an effective drug in the therapy of immunodeficiency
syndrome, and on tacrine (TA), a potent acetylcholinesterase inhibitor approved for
treatment of mild to moderate senile dementia of the Alzheimers type.
Materials and methods
Hepatobiliary disposition of ethimizol, azidothymidine and tacrine in isolated perfused
liver (IPRL) was carried out according to the method described by Bezek et al. [2,4,5]
and Kukan et al. [3]. Hepatocytes were isolated according to the two-step collagenase
liver perfusion method of Seglen [6].
. Bezek et al. (2008) Trends in Pharmacological Research (Eds. V. Bauer et al.): 3340.
34 S. Bezek et al.
Results
Eects of Cytochrome P-450 induction on drug elimination
1. Elimination of ethimizol in isolated perfused rat liver
The ethimizol disappearance rate was faster in liver preparations from pretreated rats,
Figure 1, top. In accordance with the results from single-pass experiment the effect of
induction was more marked following 3-methylcholanthrene (3-MC) pretreatment. It
may be seen from Figure 1, middle, that after phenobarbitone (PB) pretreatment the
formation of ethimizol metabolite M
1
, (4-carbamoyl-l-ethyl-5-methylcarbamoyl-imi-
dazole) was potentiated in comparison to control rats.
Apparently, after 3-MC pretreatment, the concentrations of me tabolite M
1
peaked
earlier than 5 min after the start of perfusion. Evidently, metabolite M
1
was further me-
tabolized as its concen tration in the closed perfusion system decreased. The rate of its
degradation was the most rapid in the 3-MC-treated group, compared to the other two
groups. Finally, faster formation and subsequent more rapid degradation of the second
metabolite M
Y
was observed for the PB- and 3-MC-treated groups compared to control
animals (Figure 1, bottom). Elimination of ethimizol was inhibited in suspension of
rat hepatocytes by SKF 525-A at concentrations of 10 and 100 M by 63% and 60%, re-
spectively, and by -naphthoflavone by 71% and 85%, respectively. Their simultaneous
addition almost completely inhibited ethimizol biotransformation.
2. Elimination of tacrine in isolated perfused rat liver
The rate of tacrine (TA) elimination increased in livers from PB, isosafrole (ISO), and
3-MC pretreated rats compared to control rat livers with 3-MC pretreatment producing
the greatest effect. Following 30 min of perfusion, no unchanged TA could be detected
in the perfusate of 3-MC pretreated rat livers. TA perfusate AUC
(0120min)
values calcu-
lated for control, PB, ISO, and 3-MC pretreated rat livers are shown in Table 1.
Table 1. The eect of enzyme induction on perfusate AUC
0-120min
of TA following 2 h of liver perfusion [5].
Treatment
AUC
0-120min
(nmol.min/ml) % of control value
Control
1940 135
PB*
1397 322 72
ISO*
1328 294 68
3-MC*
271 20 14
(Mean of three experiments)
*p<0.05
35 Efect of CYP induction on drug disposition in isolated rat liver preparation
Figure 1. Disposition of ethimizol in isolated perfused rat liver system [3]. Eect of PB and 3-MC pretreatment
on elimination of ethimizol and its metabolites M
1
, (4-carbamoyl-1-ethyl-5-methylcarbamoyl-imidazole) and
M
Y
(4,5-di-ethylcarbamoyl -imidazole) Top, ethimizol; middle, metabolite M
1
, bottom, metabolite M
Y
. Livers from
control (untreated) (), PB-treated () and 3-MC treated rats ().
36 S. Bezek et al.
Table 2 illustrates the percentage of TA-derived radioactivity excreted into bile for
control, PB, ISO, and 3-MC pretreated rat livers. 3-MC had the greatest effect on TA-
derived metabolites excreted in bile. PB pretreatment had a minor effect on the biliary
radioactivity profile compared to control, whereas ISO and 3-MC pretreatments showed
the presence of additional polar radioactive metabolites. The percentage of TA-derived
metabolites excreted in bile as glucuronidase-sensitive conjugates were increased in the
pretreated rat livers compared to control livers.
3. Elimination of azidothymidine in isolated perfused rat liver
Isolated perfusion rat liver system with medium recirculation was used to study 3-az-
ido-3-deoxythymidine (AZT) disposition in control (saline) and PB-pretreated rats.
The time courses of AZT and main metabolite in the perfusates are shown in Figure 2.
Based on TLC analysis of perfusate and in excreted bile samples, AZT was metabolised
to 3-amino-3-deoxythymidine (AMT), 3-azido-3-deoxy-5-O-B-D-glucopyranurono-
syl-thymidine (GAZT), and to metabolite of unknown structure.
AMT was the major matabolite in the perfusate accounting for 26.05.0 and 51.02%
(mean S.D.) of administered radioactivity in livers from control and phenobarbitone-
pretreated rats, respectively.
Eects of Cytochrome P-450 induction on drug detoxication and activation
1. Effect of induction on detoxication and activation in IPRL
Ethimizol is metabolised in IPRL into at least six metabolites. In recirculation experi-
ments both PB and 3-MC increased ethimizol elimination, but the effect of the latter
was considerably greater. No toxic ethimizol metabolites were found.
2. Effect of induction on detoxication and activation of TA in IPRL.
Perfusion experiments using 3-MC pretreated livers showed a 3-, 7-, and 8-fold in-
crease in irreversible protein binding to microsomal, cytosolic and total liver pro-
Table 2. Eect of enzyme induction (PB, ISO and 3-MC) on total amount of [
14
C]-TA-derived radioactivity excreted
in bile, and glucuronidase sensitive metabolites following 2 h of liver perfusion. (Mean of three experiments)
[5].
Treatment
Total [
14
C]
(% of dose)
Glucuronidase sensitive metabolites
(% of total metabolites)
Control
7.6 1.2 48.9
PB
11.7 2.9 55.1
ISO
14.8 2.0 51.6
3-MC
46.1* 9.7 62.9
*p<0.05
teins, respectively, compared to control [5]. Only a slight effect was observed on pro-
tein binding in perfusion experiments using PB and ISO pretreated animals (Table 3).
3. Effect of induction on detoxication and activation of AZT in IPRL
Phenobarbitone was capable of both stimulation and detoxification of AZT to GAZT
and bioactivation of AZT to AMT, since AMT metabolite is known to be highly toxic to
human bone marrow cells. This induction was the result of enhancement of AZT me-
tabolism rather than its transport into the cells, since on incubation of AZT (0250 /m)
with rat isolated hepatocytes, a linear relationship between concentration and amount
Figure 2. Disposition of AZT in isolated perfused rat liver system [4]. Eect of phenobarbitone pretreatment on
concentration of AZT and main metabolites in rat liver perfused with [
3
H]-AZT. Control , PB-pretreated
. Each point
represents the mean S.D. from three determinations.
38 S. Bezek et al.
taken up by the cells was shown. In addition, the rate of AZT uptake was not influenced
by active transport inhibitors, potassium cyanide (KCN), 2,4-dinitrophenol (DNP), or
low temperature (4C), which is consistent with a simple diffusion of AZT through the
hepatocellular membrane.
Discussion
Induction of drug-clearance pathways (Phase 1 and 2 enzymes and transporters) can
have important clinical consequences. Inducers can (1) increase the clearance of other
drugs, resulting in a decreased therapeutic effect, (2) increase the activation of pro-
drugs, causing an alteration in their efficacy and pharmacokinetics, and (3) increase the
bioactivation of drugs that contribute to hepatotoxicity via reactive intermediates [7].
The importance of P450s has been widely recognized only recently as several promising
drugs have had to be withdrawn from the market because of life-threatening interac-
tions with other drugs [8].
For drugs whose elimination is cleared primarily by CYP-mediated metabolism,
CYP induction will decrease the therapeutic efficacy as a result of a decrease of systemic
exposure. In some cases, changes in drug dosage are required to attain and maintain a
therapy during the initiation, maintenance, and discontinuation of the coadministra-
tion of a potent CYP inducer. In addition, CYP induction may create an undesirable
imbalance between detoxification and activation, leading to an increase in metabolite-
induced toxicity [1].
Ethimizol was metabolised in an isolated rat liver preparation into at least six metab-
olites. The first step, including demethylation and deethylation, is followed by further
biotransformation into hydroxylated and demethylated secondary metabolites [2]. As
a cause of nonlinear ethimizol elimination, a competitive inhibition by the product(s)
of its metabolism is suggested. Further study provided direct evidence of inhibition of
ethimizol elimination by its primary metabolites M
1
and M
y
[9]. When both inhibitors
were present in the suspension, the effect was additive and resulted in a nearly complete
inhibition of ethimizol metabolism [3]. SKF 52S-A is known as a liver microsomal in-
Table 3. Eect of enzyme inducers (PB, ISO, and 3-MC) on irreversible binding of [
14
C]-TA-derived radioactivity to
liver fractions after 2-h liver perfusion experiments [5].
Treatment
Microsomes Cytosol Homogenate
pmolbound/mg protein
Control 121.3 68.0 29.9 6.7 39.7 11.2
PB 142.4 55.0 55.8* 1.6 65.8* 23.3
ISO 129.0 48.0 49.2* 4.7 55.0* 10.8
3-MC 363.2* 87.0 213.1* 110.0 316.6* 71.9
* p<0.05
hibitor of xenobiotic metabolism in untreated and PB-treated rats, whereas ANF is pri-
marily recognized as discriminating monooxygenase in uninduccd and 3-MC-induced
rat microsomes. ANF has a more overt inhibitory effect on ethimizol metabolism than
SKF 525-A. Since SKF 525-A and ANF had no effect on the uptake of ethimizol by he-
patocytes , their ability to inhibit ethimizol biotransformation apparently operates at a
metabolic level. Partial inhibition of ethimizol metabolism by SKF 525-A or ANF sug-
gests that at least two forms of cytochrome P-450 are involved in ethimizol metabolism
in untreated rats [3].
Phenobarbitone, a well established microsomal inducer, proved to be powerful in-
ducer of AZT metabolic enzymes since pretreatment of rats resulted in a 5.5-fold in-
crease of AZT clearance. In addition, the area under the perfusate concentration-time
curve for AMT and for a metabolite of unknown structure was increased 3- and 10-fold,
respectively, and the amount of AZT-dose excreted in the bile was nearly doubled [4].
While glucuronidation of AZT is thought to be a detoxification process, the stimulation
of AMT formation is a bioactivation process, as the AMT catabolite has been found to
be more toxic to human bone marrow cells than the parent compound [10].
Conclusions
The isolated perfused rat liver model provided us with the opportunity to assess the
effect of pretreatment with PB, ISO, and 3-MC on TA metabolism, excretion, and irre-
versible protein binding in a model more similar to that of the intact rat. 3-MC pretreat-
ment had the greatest effect on TA overall disposition. Further support for the involve-
ment of 3-MC inducible CYP1A enzymes in TA metabolism (primary and sequential
as assessed by 1-OH-TA incubations) and activation was obtained through studies with
metabolic inhibitors and 3-MC pretreated rat hepatocytes. These results suggest that the
use of animal models containing higher levels of expressed and active CYP1A should
be associated with increased TA metabolism and bioactivation and consequently may
be more suitable as toxicology models to investigate the underlying mechanism(s) for
TA hepatotoxicity [5]. P450s are the major enzymes involved in drug metabolism, ac-
counting for ~75%. However, if an individual has an inherent (e.g. genetic) deficiency
of a particular P450 or when P450 is inhibited by another drug, toxicity may develop,
particularly if drug accumulation occurs upon multiple doses. Drug-drug interactions
are recognized to be a major cause of adverse drug reactions [11].
Acknowledgement
This work was supported by the grants VEGA 2/0086/08 and 2/0083/08.
40 S. Bezek et al.
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inhibition on the disposition of the xanthine-related nootropic drug ethimizol in isolated perfused liver and
hepatocytes of rats. Drug Metabolism and Disposition 1990a, 18, 96102.
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Guengerich F.P. Cytochrome P450 and chemical toxicology. Chem. Res. Toxicol. 2008; 21: 7083. [11]
........,... .....
Reflection on animal modeling of
human cardiac diseases in preclinical
pharmacology
From measurements of coronary blood flow and
cardiac function to sophisticated approaches of
protease-catalyzed soluble cardiac protein expression
with experimental myocardial infarction
Jn DMAL, Vladimr KNEZL, Anna BABULOV, Ljuba BACHAROV, Frantika BOROVIOV,
Mria DRAVECK, Pavel GIBALA, Jn GVOZDJAK, Anna GVOZDJAKOV, Jn JAKUBOVSK,
Mria KITTOV, Darius MAGNA, Alfonz RYBR, Frantiek Viliam SELECK, Ruena SOTNKOV,
Svorad TOLC, Katarna STROV, Jana TOKROV, Radomr NOS, Anton SAUBERER, Eva NIKOV,
Slavo MARKOVI, Jozefna TOROKOV, Andrea PUKROV, Tom KOLLR
Department of Cardiovascular Pharmacology, Institute of Experimental Pharmacology, Slovak Academy of
Sciences, Dbravsk cesta 9, 841 04 Bratislava, Slovak Republic, E-MAIL: exfadrim@savba.sk
Key words: coronary blood flow, inflammatory cytokines, heart disease
Although pharmacology is a discipline with a rich and enduring heritage, present day
pharmacology is quite different from the traditional subject in the early 1960s. After the
end of World War II, Frantisek Selecky, one of Prof. P. Duchenne-Marullazs team [1],
(Dept. of Pharmacodynamics, Faculty of Medicine, Clermont Ferrand, France), when
back in Bratislava, became Head of the Department of Pharmacology at the Slovak
Academy of Sciences. He took over a small group, housed in an old mill at Mlynske
Nivy. Selecky became involved in cardiovascular pharmacology and toxicity of digitalis
glycosides.
In 1967, Pavek, Drimal and Selecky [2] published an experimental study on the hemo-
dynamics and controllable hypocalcemia as a basis for reversion of arrhythmias to sinus
rhythm in cardiac glycoside intoxication (Figure 1). The cardiac output, stroke volume,
pulmonary vascular and peripheral resistance was measured with the modern invasive
thermal dilution method following digitalis intoxication in anesthetized dogs. Seleckys
life and career came to a tragic end in 1973. The legendary Czech pharmacologist Prof.
Helena Raskova, with her attractive story of human endotoxin pharmacology, peptides
and endogenous messengers, was the director of the Institute delineating Czechoslovak
J. Dmal et al. (2008) Trends in Pharmacological Research (Eds. V. Bauer et al.): 4147.
42 J. Dmal et al.
pharmacology at its first steps. After being trained in her Department of Pharmacology
(Czechoslovak Academy of Sciences, Prague), as were many of our colleagues, I was
fortunate enough to complete my studies in clinical cardiology with Prof. Jan Brod in
the Research Institute of Cardiovascular Diseases (Prague), and then to continue work
on this exciting topic in the Institute of Experimental Pharmacology in Bratislava. In
the field of cardiovascular pharmacology, three relevant studies, (Figure 2), have been
devoted to the mechanism of action of the human octapeptide angiotensin-II on coro-
nary vascular smooth muscle and myocardial mechanics [35]. In the early 1950s, Sir
James Black (I.C.I., UK) with his chemist John Stephenson had proposed a better thera-
peutic strategy to treat coronary artery disease. Later on they found that synthesis of
the naphtyl-analog of isoproterenol would give a pure antagonist of -receptors [6]. It
was the beginning of a new era in experimental and also in clinical pharmacology. In
1970 we published a detailed account of coronary and cardiac metabolic effects of the
-adrenergic receptor blocking agent oxprenolol (at that time a substance with code
number Ba-5983, in the European literature under the name Trasicor) (Figure 3). The
study of oxprenolol [7] provided the initial support for the sustained multicentric stud-
ies (FDA) before the introduction of oxprenolol on the market in the U.S. (Study was
accomplished for S. Merrell Co. in Fort Washington, USA). Using in part the knowledge
gained from the study of oxprenolol, the following projects at the Institute in Bratislava
involved determination of the effects of the Czechoslovak patent -adrenergic receptor
antagonist Trimepranolol on canine coronary blood flow. Later it focused also on studies
of extracellular transport of -adrenergic receptors in myocardial ischemia [8]. Studies
on the intensity of myocardial depression of a new carbanilate compound [9] contin-
ued together with the analyses of selectivity of the new parent -antagonist compound
Figure 1. Hemodynamic variables
in 11 intact dogs following digoxin
intoxication and EDTA infusion
(mean S.E.). The deviations from
control values signicant at the
p<0.05 level are identied by dots,
those signicant at the p<0.01 levels
are represented by triangles. [2]
43 Refection on animal modeling of human cardiac diseases in preclinical pharmacology
exaprolol. The myocardial selectivity and intensity of myocardial depression were fur-
ther studied in animal experiments with experimentally induced myocardial infarction
in canine and rat hearts. Animal models of human cardiac diseases in cardiovascular
pharmacology were considered to be of major significance. A few comments are neces-
sary to the methodology of the animal model of canine and rat myocardial infarction
used in our studies (Figure 4). The similarity in the wave-front of necrosis (also in ST-
segment elevation) in experimental animals and man support the notion that in acute
myocardial infarction (AMI) necrosis progresses from the endocardium to epicardium,
and that the preservation of vascular -adrenergic signaling delays the development of
heart failure. However, in clinical practice, -antagonists, revascularization for acute
myocardial infarction with thrombolysis, or percutaneous coronary angioplasty are
not used in all hospitals though the personnel would be capable to perform it, because
its results are not easily interpreted. These are still burning questions. There is a large
number of studies on the co-occurrence of many diseases. Some of the underlying
pathophysiology, namely coronary vasoconstriction, is shared between two diseases.
The tone of the coronary smooth muscle endothelial layer is a key determinant of vas-
cular tone. Endothelial cells release several types of protein complexes that affect the
underlying cell layers. Protease-catalyzed protein splicing in ischemic tissue is a bona
fide posttranslational modification and increasing evidence indicates that the proxim-
ity of protein subsets, either in their structure or possibly by the physical constraints of
the local environment, dictates when and where protein splicing reaction will occur.
Martin Rodbell [10] and also Alfred Gilman [11] with their concept of the role pro-
teins play in signal transduction were among the first experimentators who began this
ground-breaking work. It was the beginning of the concept of liberation of growth fac-
Figure 2. Change in coronary perfusion pressure
and arterial pressure following angiotensin during
constant ow perfusion of left anterior descending
coronary artery. Phasic eect of angiotensin. P
coron = perfusion pressure, P art. = arterial blood
pressure in mm Hg. [35]
44 J. Dmal et al.
tors and inflammatory cytokines in the form of soluble proteins, directly in the tissues.
Nobel Laureate A.G. Gilman, in an interview reported in the ASPET journal Molecular
Interventions in 2001, said: This is a new kind of pharmacology we do around here,
it is really biochemistry with purpose [11]. Human cardiac diseases have harbored a
strong interest in the biological communication and cell signaling. Coronary heart dis-
ease after myocardial infarction is currently reconsidered as a syndrome occurring not
only as a result of mechanical dysfunction of the left ventricle, but also due to complex
molecular, neuroendocrine and what is most important also inflammatory changes.
The proinflammatory molecules activated in synergy and redundancy by the innate
Type of Preparation
(Group)
Nature of Response( Influence on Coronary Blood Flow)
Coronary
Blood Flow
Coronary
Smooth Muscle Myocardial Heart Rate
Aortic
Blood Pressure
Right-heart bypass and
constant HR
(group C)
Constrict
()
Constant
output
Constant
rate
Variable
effect
Right-heart bypass
(group B)

Constrict
()
Constant
output
Slowing
of rate ()
Variable
effect
Heart-lung preparation
(group D)

Constrict
()
Block inotr.
effects ()
Slowing
of rate ()
Constant
pressure ()
Intact cardiovascular
system (group A)

Constrict
()
Block inotr.
effects ()
Slowing
of rate ()
Decreased
pressure ()
Figure 3. Summary of the eects of oxprenolol on coronary blood ow in ve groups of experiments. [7]
Figure 4. SHR 7 days after myocardial infarction (section).
immune system, expressed mostly by inflammatory cells, drive hyperproliferative pro-
cesses in the myocardial muscle, i.e. hyperexpression of inflammatory products occurs,
mostly growth factors and cytokines, produced directly in the heart. Resultant tissue
injury leads to the release of endogenous danger signals in cells. Practically every
cell in the heart syncytium may do that cells drive tumorigenic hyperproliferation.
Hopefully, we also contributed to this mission with our recent studies of proinflam-
matory cytokines and their soluble receptors expressed by inflammatory cells in the
infarcted canine and rat heart [12,13,14,15,16]. In our laboratory we have concentrated
on new cytokine inhibitors and their molecular signaling mechanisms in experimental
myocardial infarction and chronic heart failure: on attenuation of monocyte and cy-
tokine inflammatory expression in the myocardium. In our view, myocardial infarction
is defined as a phenomenon (or rather phenomena) involving complex stress response,
able to induce pathophysiological changes in cardiac cells. An important question in
this sense is whether the cardiac stress after myocardial ischemia may predispose to
inflammation-induced immune response. Several hypotheses have been recently sug-
gested to describe the origin of systemic and myocardial immune/inflammatory activa-
tion. In this view, we described how the adaptive immune cascade triggers the release
of small-molecule proteins, inflammatory cytokines and their receptors, mitogens (like
angiotensin, endothelin), and chemoattractants (like macrophage chemoattractant pro-
Figure 5. (A) Myocardial concentration of adrenomedullin(AM) in SHR 24 h (rst column), 48 h (second column)
and 7 days(third column) after induction of experimental myocardial infarction (EMI). Control basal concentration
of AM = 5.23 1.9 fmol/mg of protein. PW- posterior wall of the left ventricle, AAR- area at risk from the drainage
of the anterior branch of the left coronary artery, where occlusion were made), MOV lateral wall of the left
ventricle. (B) Cardiac tumor necrosis factor- (TNF) mRNA concentrations were signicantly higher in the infarced
zone (AAR) then in the noninfarced posterior wall (PW).* Statistically signicant increase. [1216]
46 J. Dmal et al.
tein (mcp-1)), resulting in the initiation and progression of the hyperimmune patholog-
ical response directly in the myocardium. In our recent studies with adrenomedullin,
we used spontaneously hypertensive rats (SHR) and permanent experimental myocar-
dial infarction. This type of studies in cardiac tissues also involves massive activation
and release of cytokines and activation of inflammatory response and within 710 days
hypertrophic remodelation of the myocardium. The overexpression of inflammatory
mediators, production of interleukins and the release of tumor necrosis factor-, i.e.
molecular and cellular mobilization of up-regulated proteins produced in the infarcted
heart, both early in the acute phase and later during the inflammatory phase of chronic
cardiac ischemia, are being highlighted in acute experimental myocardial infarction
and in chronic heart failure. Another aim was the elucidation of the complexity of mu-
tual interactions of inflammatory cytokines, and monocyte-chemoattractants with
regulatory proteins in vitro in human cell lines in culture. The challenge is to develop
a therapeutic strategy which recognizes the wisdom of adaptive mechanisms and pre-
vents the excesses in expression of proteins that promote activation of inflammatory
and monocyte-chemoattractive mediators. To achieve these tasks, we are involved in a
project which is to formulate a rational approach to a potential therapeutic opportunity
for promoting more effective cell remodulation. The essays contained herein hopefully
attest to how far our knowledge of cardiovascular pharmacology has advanced and how
far it has yet to go before we approach a more complete understanding of natures many
well kept secrets.
REFERENCES
Duchenne-Marullaz P., Gourgon, R., Luccioni R.: Myocardial infarction: Current problems. (Problemes ac- [1]
tuels poses par lischemie myocardiaque). Semaine des Hopitaux 1981, 57, (3940), 16181619.
Pavek K., Drimal J., Selecky,F.V.: Circulatory efects of disodium EDEATE in digoxin-induced ventricular [2]
tachycardia. Cardiologia(Basel) 1967, 50, 297304.
Drimal J.: Efects of angiotensin-II on coronary smooth muscle. Eur J Pharmacol 1968, 5, 5668. [3]
Drimal J., Pavek K., Selecky,F.V.: Primary and secondary efects of angiotensin-II on coronary circulation. [4]
Cardiologia (Basel) 1969, 54,115.
Drimal J. and Boska D.: Efects of angiotensin-II on myocardial mechaniscs and contractile state of heart [5]
muscle. Eur J Pharmacol 1973, 21, 120136.
Black J.W. and Stephenson J.J.: Pharmacology of a new adrenergic -receptor blocking compound. Lancet [6]
1962, 2, 311314.
Drimal J., Aviado D.M.: Efects of oxprenolol on coronary circulation and cardiac metabolism. J Pharmacol [7]
Exp Ter 1971, 176, 312319.
Drimal J., Knezl V., Magna D., Strizova K.: External transport of beta-adrenergic binding sites in ischemic [8]
myocardium. J Gen Physiol Biophys 1978, 6, 583591.
Knezl V.,Magna D., Sotnikova R., Drimal J.: Efects of a new beta-adrenolytic compound propyl-3-acetyl- [9]
4-(2-1-hydroxy-3-isopropylamino)propoxy)carbanilate on isolated heart muscle. Arzneimittel-Forsch (Drug
Res) 1994, 44, 712.
Rodbell M.(1980) Te role of hormone receptors and GTP-regulatory proteins in membrane transduction. [10]
Nature 1980, 284, 1722.
Gilman A.G., Cross tals: Interview with A.Gilman Mol. Interv 2001, 1, 1421. [11]
Drimal J., Patoprsty V., KovacikV.: Stobadine is a potent modulator of endogenous endothelin in human car- [12]
diac fbroblasts.Life Sci 1999, 65, 19391941.
Drimal J.,Drimal J.Jr., Drimal D.: Enhanced endothelin ET(B) receptor down-regulation in human tumor [13]
cells Eur J Pharmacol 2000, 396, 299304.
Drimal J., DrimalJ.Jr., Drimal D.: Diferences in endothelin-1 (ET) mRNA expression,ET-receptor down-reg- [14]
ulation and signaling in normal human fbroblasts and cancer cell lines.Biology 2005, 60, 112.
Drimal J., Drimal J.Jr.,Drimal D.: Hypoxic stress-enhanced expression and release of adrenomedullin (AM) [15]
and up-regulated AM receptors while glucose starvation reduced AM expression and release and down-reg-
ulated AM receptors in monkey renal cells. Phys Rev 2006, 55, 535542.
Drimal J., Knezl V., Paulovicova E., Drimal D.: Enhanced early afer-myocardial infarction concentration of [16]
TNF- subsequently increased circulating and myocardial adrenomedullin in spontaneously hypertensive
rats. Gen Physiol Biophys 2008, 27, 1218.
........,... .....
New computational approach
to mathematical modeling in
pharmacological research
Mria URIOV
1
, Ladislav DEDK
2
, Martina TVRDOOV
2

1
Institute of Experimental Pharmacology, Slovak Academy of Sciences, Dbravsk cesta 9, 841 04 Bratislava,
Slovak Republic, E-MAIL: maria.durisova@savba.sk
2
Institute of Automation, Measurement and Applied Informatics, Faculty of Mechanical Engineering,
Slovak University of Technology, Bratislava, Slovak Republic
Key words: in silico pharmacokinetics, mathematical modeling
Introduction
The theory of linear time-invariant dynamic systems [1] provides a coherent framework
for utilizing general strategies to formulate mathematical models of complex systems
and to investigate phenomena formalized (visualized) as dynamic systems (LDS). The
modeling approaches based on the LDS theory represent a new promising alternative to
highly diverse modeling approaches conventionally used in pharmacological research,
as they form a uniform framework for building mathematical models of diverse dy-
namic processes, such as drug dissolution [2,3], whole-body disposition behavior of
drugs (e.g. absorption, distribution, elimination, etc.) [46], drug effect [7,8], drug bio-
availability [9], metabolite formation [10], in vitro/in vivo relationships [2], physiological
processes [11], etc.
Theory
Formalism
Drug disposition behavior and effect are dynamic processes characterized by continuous
change over a course of time. From the perspective of the LDS theory, these processes
can be formalized (visualized) as dynamic systems. The dynamic system can be regard-
ed as a mathematical means of formally describing how one state of the dynamic process
develops into another state over a course of time. The way of understanding the term
dynamic outlined above, is dominant in this study [112]. In contrast, in a pharmaco-
logical context the term dynamic is conventionally used in relations to drug actions.
M. uriov et al. (2008) Trends in Pharmacological Research (Eds. V. Bauer et al.): 4857.
49 New computational approach to mathematical modeling in pharmacological research
Model
Models of dynamic systems are mathematical objects utilized to study processes for-
malized as dynamic systems. They can be mathematically described by differential
equations, or by transfer functions [112].
Modeling
In the time-domain, construction of mathematical models of dynamic systems is a time
consuming and complicated task due to lack of a priori information about appropriate
model structures. Model construction can be facilitated through a combination of mod-
eling tools in the complex and time domain [212]. The combined modeling approach
exhibits the advantages: 1) It does not require a pre-knowledge about the LDS; 2) It en-
ables visualization of properties of the LDS that are not readily evident form measured
input-output data, allowing a rapid identification of an appropriate model structure; 3)
It starts with a rapid non-iterative procedure that does not require initial estimates of
model parameters, markedly speeding up modeling procedures; 4) It allows the use of
equal model structures for diverse processes, using the transfer-function model,

(1)
where G, a
0
a
n
, b
1
b
m
and a
0
~1 are model coefficients, and s is Laplace variable.
G, called the system gain, is a ratio of the system output and input in steady-state. G
possesses a practical meaning, determined by the nature of the dynamic system. In
pharmacological research, a reciprocal value of G may serve as an estimator of drug
clearance [2,5,6], or G may serve as an estimator of i) the extent of drug dissolved [3],
ii) sensitivity parameters of vessel responses [7,8], iii) an estimator of the extent of drug
bioavailability [9], etc.
Experimental studies
Whole-body Disposition Drug Behavior
The philosophy behind the modeling of drug disposition behavior using tools of the
LDS theory can be explained by the scheme in Figure 1, showing a hypothetical exam-
ple. In the example it is assumed: 1) a drug is administered intravenously in equal doses
by three different modes: a single-bolus dose, a short-time infusion, and multiple-bolus
doses (INPUTS); 2) Physiological characteristics are time-invariant; 3) The site of mea-
surements of the blood concentration-time profiles of the drug (OUTPUTS) is the same
for all modes of drug administration. If functions, such as poly-exponentials, are fitted
to the output profiles without taking into account mathematical description of the drug
administration (a conventional approach in pharmacological research), the resultant
50 M. uriov et al.
Figure 1. Hypothetical example. A drug is i.v. administered in equal doses by: a single-bolus dose I
sd
(t),
short-time infusion I
inf
(t), multiple-bolus doses I
md
(t), denoted by INPUTS I(t). O
sd
(t), O
inf
(t), O
md
(t),
denoted by OUTPUTS O(t) are the related blood concentration-time profles of the drug.
model functions are different. On the contrary, methods based on the LDS theory allow
the construction of a model that is the same for all administration modes assumed in
Figure 1 [112]. This unique model property can be used e.g. for adjustments of drug
dosing schedules, aimed at reaching and maintaining required drug concentrations in
the body [4].
The whole-body disposition behavior of piroxicam (PXM) was investigated in study
[12], with the aim to construct a physiologically-motivated model of PXM whole-body
disposition behavior. PXM was orally administered to healthy human subjects in 20 mg
capsules Feldene Pfizer. Plasma was analyzed for PXM by HPLC. The model (see Figure
2) was formulated using subjects plasma PXM concentration-time profiles and tools of
modeling, based on the LDS theory. The model was capable of: quantifying fractions
of the PXM dose sequentially disposable for absorption and of estimating time delays
between time when the PXM dose reaches the stomach and time when fractions of the
PXM dose were disposable for absorption. The model adequately approximated plasma
PXM concentration-time profiles, see demonstration in Figure 3.
Drug eect
Effects of biologically active substances are conventionally investigated by measuring
tissue responses and registering them (on analog recorders). Subsequently, descriptive
variables, such as maximum responses, times to reach maximum responses, are
determined by visual inspection of registered profiles, what is potentially inaccurate
and poorly reproducible. State-of-the-art measurement techniques allow investigators
to design automatic circuits for measurement of vessel responses and recording mea-
surements in digital forms on computers that are especially suitable to study rapid pro-
cesses developing over a few minutes.
Such an automatic circuit was designed in work [7], where it was used in a classic
study of a noradrenalin effect on a rat renal artery. Changes in the perfusion-medium
pressure were measured by a pressure transducer and registered on a PC, using a mea-
Figure 2. Physiologically-motivated model for piroxicam (PXM) in humans, after PXM single oral dose I;
i
and f
i
for i = 1, , N, are the time-delays and fractions of the PXM dose respectively; N is the number
of PXM-dose fractions and of the time-delays; MT
ge
and G
1
respectively are the mean-time parameter
and gain of the subsystem that formalizes disintegration, dissolution, and gastric emptying processes;
MT
a
and MT
e
respectively are the mean-time parameters of the absorption and elimination processes;
MT
r
is the mean-time of the circulation process,
r
is the time-delays of the circulation process, f
r
is the
circulated fraction of the PXM dose; C is the resultant plasma concentration-time profle of PXM.
52 M. uriov et al.
surement card (Figure 4). The representative raw registered profile is shown in Figure
5. A data-number reduction was performed [13], see Figure 6. A model of the nora-
drenalin effect (Eq. 1) was developed, using tools of the LSD theory. Thereafter, the
following parameters were estimated: the vessel sensitivity parameter, mean time of
vasoconstrictor response and rate constant of vessel relaxation. The given parameters
are not dependent on noradrenalin doses, if the processes underlying the effect satisfy
the principle of superposition [18]. The response of the model to noradrenalin injec-
tion was determined, see Figure 6.
Figure 3. Modeling results of a representative subject. Plasma concentrations of piroxicam (circles), the
response of the developed model to administration of 20 mg capsules (Feldene Pfzer) (line).
Figure 4. Automatic measurement circuit. LDP102 is a pressure transducer; TZ4620 is a linear recorder;
Adavantech Visidaq is a data acquisition and control software installed in a computer; PCL-818HG a
programmable card for reading input signals.
Physiological system
An intravenous glucose tolerance test (IVGTT) is used to estimate parameters describ-
ing glucose metabolism, in normal and disease states. To evaluate measurements from a
frequently sampled IVGTT, the program MINMOD has been developed [14]. MINMOD
posses the following inherent shortcomings: 1) The model structure is oversimplified,
not based on the body physiology, and does not account for the dynamic nature of
regulatory interaction glucose-insulin. Model parameters are not physiologically trans-
parent; 2) Models are composed of two separate parts. However, the glucose-insulin dy-
Figure 5. Raw registered profle of a vessel response.
Figure 6. Raw registered profle of a vessel response after data-number reduction (points). Model
response to mathematically described noradrenalin injection (line).
54 M. uriov et al.
Figure 7. Physiologically-motivated model for frequently sampled intravenous glucose tolerance test.
IR
g
(t) is the glucose infusion; CP is the cardiopulmonary subsystem; The subsystems x, for x = 1, , 4,
formalize body organs; L is the subsystem formalizing decrease of the hepatic glucose release below the
basal level; LA is the subsystem formalizing the glucose transport through the arterial and venous blood
in the left arm;
x
for x = 2, , 4, L, LA, are the time delays of the related subsystems; MT
x
, for x = 1, ,
4, L, LA, are the mean times of the related subsystems; M
0
is the instantaneous increase in the plasma
glucose amount at the inlet of the subsystem CP, evoked by IR
g
(t); M
x
, for x = 1, , 4, are the increases
in the glucose amount at the inlet of the subsystem CP, evoked by the outputs of the subsystems x;
M
L
is the decrease in the glucose amount at the inlet of the subsystem CP, evoked by the output of
the subsystem L; G
x
, for x = 1, , 4, CP, L, determine the products of the gains and plasma fows of the
related subsystem; C(t) is the increase in the plasma glucose concentration above the basal level at the
outlet of the subsystem CP; C
g,LA
(t) is the increase in the plasma glucose concentration above the basal
level at the outlet of the subsystem LA. C
i,LA
(t) is the increase in the plasma insulin concentration-time
profle that enters the subsystem L.
namic interaction is actually a unified body system, therefore coupling of the two parts
in MINMOD is not appropriate. Consequently, a unified model of the dynamic interac-
tion glucose-insulin would be preferable; 3) MINMOD incorporates a product of two
dimensionally different profiles, leading to a dimensional inconsistency; 4) MINMOD
outcomes are not capable of adequate simulating plasma concentration-time profiles of
glucose that do not smoothly decline towards pre-test levels after reaching peak levels;
5) MINMOD does not incorporate time delay parameters, despite the fact that one of
the assumptions behind MINMOD is that time delays in an insulin action on a glucose
utilization are due to a sluggish insulin transport across capillary endothelium. Instead
of time delays, an artificial non-observable quantity is incorporated in MINMOD to
take into account of a delay in an insulin action.
A frequently sampled IVGTT was performed in healthy subjects. Using tools of the
LDS theory, a physiologically-motivated model of dynamic regulatory interaction glu-
cose-insulin was constructed, see Figure 7. The model is capable of quantifying the
insulin-excitable tissue glucose uptake activity and cessation of hepatic glucose release.
Consequently, it is capable of identifying early pre-diabetic states. The model yields
adequate simulations of plasma glucose concentration-time profiles, see the example in
Figure 8.
Figure 8. Modeling results of a representative
subject. Plasma glucose concentration-time
profle (circles); Responses of the model to the
glucose infusion (lines).
56 M. uriov et al.
Conclusion
This work exemplifies an innovative approach to mathematical modeling in
pharmacological research. The approach gives opportunity to obtain information un-
obtainable with the conventional modeling approaches in pharmacological research,
e.g. information i) on factors that control dynamic mechanisms of entero-hepatic cir-
culation, ii) on linearity/nonlinearity of pharmacological processes, iii) on presence of
early pre-diabetic states [11]. Finally, it is concluded that the approach sketched in this
work, similarly as any modeling approach, ought to undergo formal analysis to establish
its appropriateness and to exclude conflicts with accepted physiological notions.
Acknowledgement
This work was supported by the European Union through the Network of Excellence
BioSim, Contact No. LSHB-CT-2004-005137, and the COST program.
REFERENCES
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........,... .....
Breeding and testing facility Dobr Voda
Andrej GAJDOK, Alena GAJDOKOV, Eduard UJHZY, Daniela GOLHOV,
Bernardna KOPECK, Viera KRCHNROV
Department of Toxicology and Laboratory Animal Breeding, Institute of Experimental Pharmacology,
Slovak Academy of Sciences, 919 54 Dobr Voda, Slovak Republic, E-MAIL: exfatoxi@savba.sk
Key words: laboratory animal, breeding facility, testing facility
Introduction
The biomedical sciences are based on laboratory research, which also includes experi-
ments on animals. In 1951 a number of known scientists, motivated and convened by
Dr. F. V. Seleck from the Chemistry Institute SASc, Bratislava, Slovakia, submitted
a demand to the Slovak government in which they recommended the foundation of an
institute that would concern itself with research into the breeding, maintenance and
availability of healthy and genetically defined laboratory animals. The first breeding
station in Slovakia was formed as a part of the Research Institute for Pharmacy and
Biochemisty at Dobr Voda [1]. The village Dobr Voda is located 30 km north-west
of Trnava. The rural character of the site, which extended over 4 hectares, fitted well
the main aim. It also satisfied the required quality criteria. Dr. F.V. Seleck, Prof. H.
Rakov, Dr. . Mahr and Dr. K. erey supported the growth of this facility during
the 50s and 60s. The first animals introduced to the facility were obtained from West
Germany, Denmark and Switzerland. The facility was comparable with similar facilities
in Europe. Dipl. Ing. E. Kollr was Head of the facility, which became a subsidiary of the
Institute of Experimental Pharmacology SASc for the next 20 years. In this time special-
ized laboratories were established to control the health status of animals (parasitologi-
cal, microbiological, pathological, histopathological). Pavilion No. II was reconstructed
in compliance with the Principles of Good Laboratory Practice (GLP). There were four
production areas protected by a barrier. Breedings of specific pathogen free rats (SPF)
and gnotobiotic rats were started. With the aid of gnotobiotechnique outbred colonies
were re-established. After 1990 the activities of the breeding facility were considerably
diminished due to economic conditions. It was affiliated to the Laboratory of Toxicology
and then to the Department of Toxicology and Laboratory Animals Breeding. Its activi-
ties were divided into animal breeding and in vivo testing of chemical substances. Dr. A.
Gajdok was appointed Head of the Department with a staff of five.
A. Gajdok et al. (2008) Trends in Pharmacological Research (Eds. V. Bauer et al.): 5865.
59 Breeding and testing facility Dobr Voda
Breeding facility (Reg. No. SK CH 40004)
In Pavilion No. II there are now three production areas. They are autonomous for secu-
rity purposes and they produce, under protected conditions, rats, mice, Guinea pigs and
gerbils. Rabbits are kept in the old Pavilion No. I.
Production standards in protected area
Air-conditioned with 10 air changes per hour and continuously monitored environ-
ment with temperature 202 C, relative humidity 5060%, lighting system 12/12
or natural. Cages are in standard dimensions, in polypropylene with noise limiting
construction, easy to clean. Constant animal density. The animals are never given
therapeutic or growth adjuvants. They receive one daily ration of complete foodstuffs
adapted to the species involved (KKZ-P/M and KKZ-M/K, Reg. No. K 400310). Non-
chemically treated drinking water is distributed ad libitum. Bedding is composed of
softwood shaving.
Laboratory animals produced at the breeding facility Dobr Voda
Species and strains of laboratory animals produced at the breeding facility are sum-
marized in Table 1.
The WISTAR rat Dv : WI (SPF Han/Rosice)
Origin
Selected by H.H.Donaldson early in the 20th century, at the Wistar Institute,
Philadelphia, USA. In 1947 imported to Europe. The strain was introduced into the
facility Dobr Voda in 1987 from a colony maintained in VFB Pardubice Rosice, CZ,
which they had obtained from the Zentralinstitut fr Versuchstierkunde, Hannover,
Germany, in the same year.
Characteristics
Albino rat, of medium size but with a good growth rate, docile, easy to handle.
Table 1. Laboratory animals produced at the breeding facility Dobr Voda.
Species Outbred strain Inbred strain
Rats WISTAR LEWIS
SHR
Mice ICR BALB/c
Gerbils MON
Guinea pigs TRIK
DH
Rabbits HIL
60 A. Gajdok et al.
Breeding method
A primary colony is made up of monogamous pairs which reproduce automatically. The
rotation system of mating is used to preserve the genetic stability of the population.
Fields of use
The WISTAR rat is considered a polyvalent animal from an experimental point of view.
Taking into account that this is the oldest strain used in the laboratory, the whole medi-
cal research field has included it in their protocols. Its life span as well as its tumour
pathology make the WISTAR rat an interesting model in long-term studies. In addition,
its good aptitude for learning makes it a very good subject for behavioural studies.
The SHR rat Dv : SHR (N/CrlBR)
Origin
The strain was produced by Prof. Okamoto in the University of Kyoto, Japan in 1964.
He called this strain the Spontaneous Hypertensive Rat. In the 70s it was imported to
USA and Europe. The strain was introduced to the facility Dobr Voda in 2002 from the
Heart Research Institute, SASc, Bratislava, Slovakia.
Characteristics
Albino rat of average size, spontaneously hypertensive.
Breeding method
In monogamous pairs, brother and sister mating.
Fields of use
The SHR rat is a preferred model to study essential hypertension in man, a model for
atherosclerosis and cerebral vascular attacks, for screening of antihypertensive drugs,
a model for erythrocytosis and for insulin resistance.
The LEWIS rat Dv : LEW (Crl BR)
Origin
The strain was produced by Dr. Lewis in 1952 from the Wistar breeding. In 1987 it was
imported to Charles River Laboratories, Germany. The strain was introduced to the
facility Dobr Voda in 2001 from the farm Sulzfeld.
Characteristics
Albino rat of medium size, docile, susceptible to induction of autoimmune diseases,
with increased levels of serum thyroxine, insulin and growth hormone.
Breeding method
Fields of use
The LEWIS rat is a suitable model to study adjuvant-induced arthritis, experimental
myocarditis, myastenia gravis, experimental allergic encephalomyelitis and transplan-
tation.
The ICR mouse Dv : ICR (Ici/Velaz)
Origin
SWISS type mouse, which was introduced into the Institute for Cancer Research in
Philadelphia, USA in 1948. The strain was introduced into the facility Dobr Voda in
1993 from a colony maintained in Velaz Prague, CZ, which they had obtained from Iffa
Credo Co., Italy.
Characteristics
Robust albino mouse with excellent reproductive and maternal characteristics.
Breeding
Fields of use
Most widely used outbred mouse. A suitable model for: oncology, toxicology, aging,
teratology, surgery.
The BALB/c mouse Dv : BALB/c (Crl BR/Muv AT)
Origin
The strain had been selected by Mac Dowell in 1922 from the outbred Bagg albino
strain. In 1974 to Charles River Laboratories from NIH. The strain was introduced into
the facility Dobr Voda in 2006 from the Medical University of Vienna, Austria.
Characteristics
Albino mouse, docile.
Breeding
Fields of use
The BALB/c mouse is a useful strain for a wide range of applications: experimental al-
lergic encephalomyelitis, toxicology, pharmacology, aging, teratology, cardiovascular.
It is the most frequently used strain for the production of monoclonal antibodies (im-
munization and production of ascites) intended for diagnosis or therapy.
62 A. Gajdok et al.
The TRIK Guinea pig Dv : TRIK (Mad/Velaz/Ros)
Origin
The TRIK strain was introduced into the facility Dobr Voda in 1994 from a colony
maintained in VFB Pardubice Rosice, CZ, which they had obtained from Velaz
Prague, CZ. It was brought to the Czech Republic in 1993 from Kleintierfarm Madorin,
Switzerland.
Characteristics
Larg Guinea pig with tri-coloured fur (combination of red, black, white) with good re-
productive characteristics.
Breeding
In panmictic colonies. Mating is carried out in groups (one male and three females).
Fields of use
A suitable model for virology, cancerology, toxicology, pharmacology.
The DH Guinea pig Dv : DH (MRC/Wiga/AnLab)
Origin
Origin at MRC Milhill, UK. To the Czech Republic (AnLab, Prague) from the farm
Charles River Laboratories Wiga, BRD. To the facility Dobr Voda the strain was intro-
duced in 1994.
Characteristics
Large albino Guinea pig, docile, emotional, with excellent breeding performance.
Breeding
In panmictic colonies. Mating is carried out in groups (one male and three females).
Fields of use
Experimental viral infection for control purposes. As experimental models for both an-
imal and human diseases. Testing of carcinogenic activity of certain products. A model
for the study of allergy (bronchospasms provoked by histamine), isolated organs (ileum,
trachea), gastric or duodenal ulcer resulting from histamine.
The Mongolian gerbil Dv : MON (Crl BR)
Origin
Developed from a nucleus colony obtained from the Charles River Laboratories
Germany, farm Sulzfeld, in 1996. Charles River Laboratories obtained the breeding
pairs from the University of Missouri, USA in 1981.
Characteristics
Animals with agouti fur colour, docile, highly prone to epileptiform convulsions.
Breeding
Fields of use
A suitable model for epilepsy, nutrition, oncology, toxicology, hibernation and mainly
for experimental infarction.
The HIL rabbit Dv : HIL
Origin
The strain was developed at VV Nitra, Slovakia. It is a crossbred strain obtained by
mating New Zealand White rabbits and Californian rabbits.
Characteristics
Albino rabbit of medium size, occasionally with black coloured tips of ears.
Breeding
Mating is arranged to take place in the male`s quarters.
Fields of use
Research uses include cardiac surgery and studies of hypertension, infectious diseases,
virology, embryology. The species is used routinely in serology and antibody produc-
tion and, lately, for screening embryotoxic agents and teratogens.
The animals produced at the breeding facility Dobr Voda are designed for experi-
mental purposes at research institutes, universities, etc. A survey of animals produced
over the last six years are shown in Table 2.
Table 2. Numbers of animals produced at the breeding facility Dobr Voda.
Strain Species 2003 2004 2005 2006 2007 2008*
WISTAR rat 2603 2872 3159 3129 2578 1903
LEWIS rat 152 173 354 306 382 90
SHR rat 58 148 204 107 166 34
ICR mouse 4452 4997 4532 2522 3496 1642
BALB/c mouse 0 0 0 0 445 180
TRIK + DH Guinea pig 713 915 671 887 569 414
HIL rabbit 0 0 0 66 124 80
* the first six months are covered
64 A. Gajdok et al.
Testing facility (Reg. No. SK P 30004)
For over 15 years, the Department of Toxicology and Laboratory Animal Breeding has
offered a testing facility for evaluation of test compounds in whole animals. The depart-
ment has been performing safety studies to the standard of Good Labratory Practice
(GLP) since 1992. The studies have been sponsored by the pharmaceutical, industrial
chemical and consumer product industries on European basis. Tests are offered to meet
the requirements of the following agencies:
Slovak Centre for Chemical Substances and Products
Organisation for Economic Co-operation and Development
European Economic Community
European Pharmacopeia
The studies are performed to evaluate the possible effects of acute or repeated admin-
istration of a test material on organism and on various organs or physiological systems.
A survey of studies performed at the testing facility Dobr Voda is shown in Table 3.
Table 3. Chronological review of testing activities at the facility Dobr Voda.
Period Sponsor Type of test substance Type of test
19911993
IEPha SASc, SK Stobadine DP 1031 Chronic toxicity 26 weeks
1994
LIKO V, SK Carboxymethylglucane Acute oral toxicity limit
1995
Slovasfalt, Inc., SK Asphalt MOAS I-S Acute oral toxicity limit
19971998
Slovnaft, Inc., SK Oil Pekol 80
Grease V00
Gease AK1 EP konti
Grease WR
Oil M2T Global
Oil Madit gas
Asphalt varnish R
Acute oral toxicity limit
Acute dermal toxicity
Acute dermal irritation
Acute ocular irritation
19972002
IEPha SASc, SK Streptozotocin
Stobadine
Experimental model of
diabetes mellitus
20012007
IEPha SASc, SK Stobadine derivatives Acute toxicity No. 425 p.o.
Acute toxicity No. 425 i.v.
Acute toxicity No. 425 i.p.
20042005
IEP SASc, SK Magnetic nanoparticles Acute toxicity No. 425 p.o.
2005
Derma Protect
Innovation, Germany
Tincture LCD-DPI Acute dermal toxicity
Acute dermal tolerance
Dermal tolerance 28 days
2006
Pharmacentrum SK Instillation XUN Acute toxicity No. 425 p.o.
Acute toxicity No. 425 i.p.
Oral toxicity 28 days
Ocular tolerance 28 days
20062008
Biovendor, CZ Biovendor A - Z Antibody production
IEPha SASc Institute of Experimental Pharmacology, Slovak Academy of Sciences, Bratislava
IEP SASc Institute of Experimental Physics, Slovak Academy of Sciences, Koice
The most extensive was the chronic toxicity study of stobadine in 19921993 [2]. A 26-
week oral toxicty and a micronucleus assay of the new cardioprotective drug stobadine
(CAS No. 95751-51-2) in the form of dipalmitate salt were performed in Wistar rats.
In the 90s the short term tests were designed mainly for the chemical and foodstuff
industry (oils, greases, asphalts, varnishes, glucane).
In the years 19972002 the testing facility participated in the study of the experimen-
tal model of diabetes mellitus induced by streptozotocin [3].
During the next time period the testing was aimed at acute toxicities of pyridoindole
derivatives [4], acute toxicities of magnetic nanoparticles [5] and at toxicities and local
tolerancies of pharmaceutical products.
In the last two years the testing facility has cooperated with the Czech company
Biovendor Laboratory Medicine. HIL rabbits are immunised and monoclonal antibod-
ies are produced for diagnosis of some human diseases.
Conclusion
Recent biomedical research focused on new strategies in prevention and intervention
of serious human diseases as well as testing/screening of potentially harmful effects of
new chemical compounds, drugs and physical factors can not be conducted without
experimental studies by using specially bred laboratory animals. Animal experimenta-
tion, both for research and teaching, will still be essential in the future for the benefit
and protection of mankind and its environment, and for the preservation of plants and
animal life, as well as for the testing of medicines and other substances.
Animal experiments will only yield useful results if the animals are healthy and in
optimal condition, if they are bred without stress according to their species specific
requirements, and kept accordingly. Neglect of these requirements can lead to faulty ex-
perimental results and cannot be defended on scientific, ethical or economical grounds.
REFERENCES
Balonov T., erey K.: Chov laboratrnych zvierat v SSR. Naa veda 1961; 233. [1]
Gajdokov A., Ujhzy E., Gajdok A., Chalupa I., Blako M., Tomkov A., Lika J., Dubovick M., Bauer [2]
V.: Chronic toxicity and micronucleus assay of the new cardioprotective agent stobadine in rats. Arzneim.
Forsch. Drug Res. 1995; 45(5): 531536.
tefek M., Tribulov N., Gajdok A., Gajdokov A.: Te pyridoindole antioxidant stobadine attenuates his- [3]
tochemical changes in kidney of streptozotocin induced diabetic rats. Acta Histochem. 2002; 104(4): 413
417.
tolc S., nirc V., Mjekov M., Gsprov Z., Gajdokov A., tvrtina S.: Development of the new group of [4]
indole-derived neuroprotective drugs afecting oxidative stress. Cell Mol Neurobiol. 2006; 26(78): 1495
1504.
Gajdokov A., Gajdok A., Konerack M., Zviov V., tvrtina S., Krchnrov V., Kopansk P., [5]
Tomaoviov N., tolc S., Timko M.: Acute toxicity of magnetic nanoparticles in mice. Neuro Endocrinol
Lett. 2006; 27 (Suppl.2): 9699.
........,... .....
From comparative interspecies and
ontogenetic pharmacokinetics up to the
usage of microcamera-techniques for
drug bioavailability studies
(Historicizing comments on three decades of the
existence of an experimental biopharmaceutical
research in Hradec Krlov, Czech Republic)
Jaroslav KVTINA
Stages of organization:
19781985, Department of Experimental Biopharmaceutics within the Institute of Experimental Medicine of the
Czechoslovak Academy of Sciences (SAV);
19851993, Institute of Experimental Biopharmaceutics, SAV;
since 1993, Institute of Experimental Biopharmaceutics, a joint research center of the Academy of Sciences of the
Czech Republic and PRO.MED.CS Praha a.s.
The establishment and the first stages of the development of the academic Institute of
Experimental Biopharmaceutics (EBF) in a way paralleled, after an interval of twenty
years, the history of the Pharmacological Institute of the Czechoslovak Academy of
Sciences (F SAV) in Prague. Similarly as the F had its predecessor in the phar-
macological department established (on the initiative of Prof. Helena Rakov) in the
1950s at the Institute of Organic Chemistry and Biochemistry, SAV, the anamnesis
of the EBF included the experimental biopharmaceutical department established (on
the initiative of Prof. Jaroslav Kvtina) in the second half of the 1970s at the Institute of
Experimental Medicine, SAV. The very first field of research of both these academic
drug-oriented institutions, of both the pharmacological and the biopharmaceutical de-
partment, was pharmacodynamic and toxicological examination of selected substances
developed at different, mainly chemical, laboratories of the Academy. The subsequent
transformations of both these academic subunits were also similar. The Pharmacological
Institute was established as a separate institution within the SAV at the beginning of
the 1960s, forming a joint research unit with the Department of Pharmacology of the
then Faculty of Paediatrics, Charles University (both institutions were headed by H.
Rakov). Similarly, also the EBF was established as a separate unit within the SAV
J. Kvtina (2008) Trends in Pharmacological Research (Eds. V. Bauer et al.): 6676.
67 Pharmacokinetics and drug bioavailability studies
in 1985. The conception of the Institute still included pharmacological-toxicological
service for therapeutically promising substances of academic provenience, but its main
programme became the research of a more fundamental character, oriented to the prin-
ciples of the mechanisms of the fate of the drugs and dosage forms in the organism.
The service research part the EBF established links primarily with the Institute
of Macromolecular Chemistry of the SAV in the development of macromolecular car-
riers of pharmaceuticals and their application to platinum cytostatics of the second and
third generations and with the laboratories of the South-Bohemian Academic Centre,
which participated in the development of the inland original immunosuppressive agent
cyclosporine [examples: 6,35,36,37,48]. The role of the Institute in this cooperation con-
sisted in mapping of the biodistributional and pharmacokinetic indices of the substanc-
es under study, particularly from the standpoint of their organ toxicities and attempts
to positively influence their unwanted side effects. The concrete applied results include,
e.g., participation in the introduction of cis-platinum to therapeutic practice by the then
pharmaceutical manufacturer Lachema and recommendation of nephropathic pre-
vention in cyclosporine immunosuppression. The aims of more fundamental research
lines of the EBF at the first stages were based on the long-term research programme of
the Department of Pharmacology and Toxicology of the Faculty of Pharmacy, Charles
University, in Hradec Krlov, with which the Institute was closely connected (thanks
to several joint laboratories and the interconnected university and academic posts of
J. Kvtina). It was a system of experimental studies oriented to some pharmacokinetic
mechanisms (transbarrier transports of pharmaceuticals, their transport bonds, and
their bioelimination). Their results aimed at pharmacokinetic predictions based on the
physical-chemical and structural characteristics of drug models. The means for experi-
mental acquisition of the data from these sources were comparative aspects classified
according to the individual pharmacokinetic parameters:
first, from the aspect of inter-substance relationships and interactions
[example: 31],
second, from the aspect of inter-species similarities or differences (inter-species
in the sense between available laboratory animal models and the human
being for optimization of transfers of pre-clinical pharmacokinetic data in the
direction to the first administration of the drug to the human proband)
[examples: 2,23,24,29,43],
third, from ontogenetic aspects (with the intent of administration to
rationalize the modifications of therapeutic regimens in geronts) [26],
fourth, from pathophysiological aspects (with the aim to modify the
dosage of drugs in selected situations, in particular in the syndromes of
nephropathy, hepatopathy and malabsorption) [examples: 20,30].
One of the methodical specific elements employed have been confrontations between
the pharmacokinetic results obtained in the systems of isolated perfused organs (the
liver, kidney, intestinal segments) [9,17,18,19] and the whole-organism level findings
68 J. Kvtina
obtained from in vivo experiments. It has opened the ways to trace biotransformation
interstage metabolites developed in the given organ system, which would be difficult or
impossible to detect at the whole-organism level. The studies included, e.g., the anal-
yses of the gradual biodegradation cascade of diazepam and nitrazepam in the liver
[4,12,13,16,38] and the transport mechanisms both in the isolated perfused kidney [ex-
ample: 52] and in the intestinal segments [examples: 12,13].
Besides this methodological tactics, the Institute gained a certain priority in the ex-
ploitation technique QSAR (Quantitative-Structure-Activity-Relationships). It started
to be commonly employed in pharmacodynamic screening from the 1960s as an aid
for more precisely targeted chemical modifications within the framework of testing
the groups of substances with potentially medicinal properties. But it was not until the
1980s when the studies by the authors from the Institute, aimed to predict the relation-
ships between, e.g., chemical structures of series of drug models and their lipophilicity,
transport bindings ans excretion, contributed to the modifications of this classic use of
QSAR towards pharmacokinetic indices [32,33,34,53].
The mosaic of the results obtained in this way (both in the past and more recently)
has yielded several significant outputs. They include, e.g., demonstrations of differences
(not only quantitative but also qualitative) between animal species in biotransforma-
tion mechanisms of a number of drug models [2,23,24,43,52]; biotransformation of me-
salazine is shown as an example (Figure 1). These studies resulted in the preference of
the experimental minipig as the representative of the omnivores (weight, 3035 kg) for
pharmacokinetic interspecies comparative research, even in contradiction with the of-
ficially recommended non-rodents, the dog (beagle) and the primate (Macacus rhe-
sus). Experimental data obtained from different laboratory species became the founda-
tions for a tabular aid usable generally in pharmacokinetic interspecies comparative
research [8].
Generalizing predictions resulting from the pharmacokinetic studies of the intesti-
nal compartment also utilized, besides perfusion techniques, the results from the re-
search of malabsorption. They lead to the verification of excretory differences between
the substances transported transintestinally by the mechanism of simple diffusion and
the substances transported by active carrier systems. Cooperation with morphologists
demonstrated shifts in intestinal cellularity during malabsorption [10,15,21]. The ap-
plication of this technique proved to be useful also in gerontological research by dem-
onstrating that in older age categories (demonstration on laboratory rats) the cellularity
of the intestinal wall is also decreased [26].
The transformation of the EBF into the present form took place in 1993 when the
SAV was dissolved and the Academy of Sciences of the Czech Republic (AVR) had
to slenderize. The institute was transformed into a joint research centre of the AVR
and the pharmaceutical manufacturer PRO.MED.CS Praha a.s. This meant not only or-
ganizational changes in research teams, but also in research tactics. In service research,
pharmacokinetics remained the principal task, but there was a more selective orienta-
tion to oral dosage forms, in particular verification of pharmacokinetic bioequivalenc-
es between the generic preparations developed in the technological laboratories of the
sponsoring pharmaceutical firm and original preparations. The reason of these shifts
was the necessity to cover not only certain particular fields of pharmacological pre-
clinical research, but also clinical studies on human probands. The preclinical research
was concerned with pharmacokinetic modelling, which could precede clinical studies,
ranging from alternative in vitro techniques (e.g. dissolution tests of solid dosage forms,
drug transports in cell culture preparations and in isolated intestinal segments) to ani-
mal whole-organism level studies aimed both at demonstrations of the course of levels
of drugs and their metabolites in the systemic circulation and their organ biodistribu-
tion (here the above-mentioned experience with an animal species, the minipig, proved
useful). The majorities in clinical studies have been pharmacokinetic bioequivalences
between dosage forms of the generic drugs being developed and referential preparations.
For the sake of practical application of this section of research capacity of the Institute,
one of the conditions was to obtain the international certificate of Good Laboratory
Practice for the selected series of preclinical tests. Though these transformations con-

0
10
20
30
40
50
60
70
80
90
100
primate dog minipig man
A
U
C

[
g
.
h
/
m
l
]
5-ASA
metabolite (N-acetyl-5-ASA)
Figure 1. Example of (nearly qualitative) di erences in the biotransformation of drugs between animal species
(model drug = mesalazine, i.e. 5-aminobenzoic acid, which is biotransformed to form N-acetyl-5-ASA). AUC levels
of plasma time pro le after oral administration of mesalazine (doses calculated for the body surface). Whereas
the formation of the metabolite (N-acetyl-5-ASA) is relatively very similar in humans and the minipig, in the
primate it is signi cantly lower (lower bioavailability of 5-ASA suggests also its di erent biodistribution), in the
experimental beagle the metabolite was not produced at all.
70 J. Kvtina
verted the prevailing part of the research capacity of the EBF to the formal category
of applied research, several teams of the Institute have kept the character of academic
fundamental research. Similarly as in applied research, also in the part of the activities
which rank among the category of more fundamental research, the Institute has kept its
original orientation, i.e. pharmacokinetics. Nevertheless, the focus was narrowed to the
mechanisms of absorption and biotransformation of xenobiotics in the intestinal tract.
Thank to the symbiosis between the study of pharmacokinetic mechanisms and the
selection of model pharmaceuticals selected according to the interests of the sponsor, the
Institute manages to perform both lines of research declared in the preamble of the pres-
ent-day EBF. The hitherto fifteen-year period of the joint centre of the Academy and a
pharmaceutical manufacturer has laid preclinical and clinical foundations for registra-
tion of about two dozens of generic medicinal preparations, which the firm PRO.MED.
CS introduced to the pharmacotherapeutic market [examples: 39,41,42,44,45,46,47, 49].
And it is possible to present a certain number of results, which contributed to shifts in
some knowledge of general validity.
One of the examples is the research of organ biodistribution of the relatively selective
dopaminergic antagonist sulpiride, which on experimental minipigs has demonstrated
its markedly small penetrability through the hematoencephalic barrier (HEB) and a
concentration higher by one order (in comparison with plasma levels) in the hypoph-
ysis, located outside the HEB. A certain application output was the interpretation of
some side effects of this psychotropic agent [7].
A comparative clinical study of two oral tablet forms of the antihyperlipidemic agent
fenofibrate, in which the determination of the pharmacokinetic parameters was con-
nected with the pharmacodynamic effect (the adjustment of the lipoprotein score), has
shown that for the optimal effect it is decisive to maintain a certain minimal plasma
level of the drug, and not to achieve the highest levels. Besides, the determination of the
binding capacity of fenofibrate with the individual lipoprotein fractions (demonstra-
tion of the highest bond with HD-lipoprotein) formulated the thesis of a certain thera-
peutic feedback autoregulation, consisting in a desired, fenofibrate-induced, relative
increase in HDL and thus with a subsequent decrease in the free (i.e. effective) fraction
of this hypolipidemic agent connected with it [22,40].
The result of one of the preclinical studies was the use of L-carnitine for an im-
provement in cerebral availability of one of the derivatives of tacrine, the original
methoxytacrine (from the synthetic provenience of the Military Medical Academy in
Hradec Krlov). The reason for the development of methoxytacrine is the influence on
Alzheimer symptomatology and optimization of the therapeutic index (in comparison
with the classic tacrine) [3,50, 51].
The research of an original substance with a potential drug-transporting effect, with
the working name of VoKv (coined from the first two letters of the surnames of the
authors, a pharmaceutical chemist and a pharmacologist) had a curious result. It is a
chained biodegradable polysaccharide polymer, which easily produces binding com-
plexes [25]. Experiments on rats managed to demonstrate that the substance was gradu-
ally degraded during the transport through the intestinal wall and that concurrently
with degradation the bound model drug was released [21]. The in vivo experiments on
minipigs then revealed that whereas after oral administration the model drug without
the carrier was eliminated from the systemic circulation within 24 hours, in the case
of its binding to VoKv its plasma level could be demonstrated as late as the 60th hour.
However, on the release from the bond, significantly lower plasma levels were logically
achieved (in comparison with the administration of the model drug alone, concentra-
tion c
max
was decreased to 20%). There remained an open question to demonstrate
whether the same biodegradation process takes place also in the human intestinal wall.
Both authors therefore played, following the unwritten tradition used in the pilot stage
of clinical research of drugs, the roles of the first human probands and tested VoKv
with the bound model drug, which was the relatively little toxic expectorant ambroxol,
on themselves. They used an increased dose of the pharmacologically active ingredient,
which was calculated on the basis of the minipig experiment and which was planned to
achieve the plasma levels close to the therapeutic ones. Though the experiment was suc-
cessful and a high level of the bound model drug was being released from the complex
for more than three days (according to the detected levels in the systemic circulation)
(Figure 2), the assumed levels were exceeded in both probands. The mechanical cal-
culation of the dose was theoretically correct, but it was found later that the increased
concentration of the model drug caused saturation of the binding sites of VoKv and thus
in the first eight hours after the administration, due to the unbound free fraction, un-
expectedly high plasmatic concentrations were achieved. This pilot clinical experiment
had a positive effect later. During the parliamentary proceedings when discussing the
Czech law on the protection of animals this experiment served as one of the arguments
for the codification of legal limits of animal experiments. It was enough to include an-
other animal experiment with the above-mentioned increased dose of the drug before
the above-mentioned clinical experiment and the proof of the consequences (including
the interpretational ones) would be evident.
The general development of, e.g., diagnostic microtechniques has been accompanied
by the shifts in the methodological design of the Institute and thus a more detailed
tracing of drug biodistributions in the in vivo conditions. In cooperation with other
clinical departments in Hradec Krlov, the Institute succeeded to utilize a capsule gas-
trointestinal endoscopic microcamera (size, 0.7 2.5 cm, frequency scanning capacity,
2 pictures / 1 sec.) to specify the fate of solid dosage forms in the individual levels of the
digestive tract of experimental minipigs. It resulted not only in the individual analyses
of particular dosage forms [5,11,14,27], but also in more generally valid arguments for
the necessity of the biological experiment as one of the interlinks in pharmacothera-
peutic innovations, also in contradiction to some recent promotional efforts, purpose-
lessly enforcing the so-called alternative techniques, excluding animal experiments. In
equivalence studies of generic preparations, the principal topic is, e.g., simplification
72 J. Kvtina
A
B
C
0
1000
2000
3000
4000
5000
6000
0 24 48 72 96
Time [hours]
C
o
n
c
e
n
t
r
a
t
i
o
n

[
n
g
/
m
l
]
0
5
10
15
20
25
30
Time [hours]
0
20
40
60
80
100
120
140
160
0 10 20 30 40 50 60 70 80
C
o
n
c
e
n
t
r
a
t
i
o
n

[
n
g
/
m
l
]
A
B
Time [hours]
a)
b)
Figure 2. A preclinical pharmacokinetic study (experimental minipig) versus a pilot clinical study of the drug
carrier VoKv (plasma levels of the model active principle ambroxol after its oral administration both in the free
form and after binding to the VoKv complex):
a) minipigs (n = 6)
left scale + curve A = ambroxol levels after its administration in the form of a substance
right scale + curve B = ambroxol levels after its administration in the form of a complex with VoKv
b) human probands (n = 2)
A = average plasma level of ambroxol after the administration of the therapeutic dose
B, C = plasma levels of ambroxol after its administration in the VoKv complex
(B = proband TV, C = proband JK)
by means of dissolution analyses in test-tube conditions in vitro, which should be,
according to these recommendations, the sufficient parameter in the comparison of
dosage forms of generic preparations. Thanks to the experiments with the microcamera
in preclinical in vivo experimental conditions, it was possible to demonstrate how the
gradually changing size of released particles and their interactions with intra-intestinal
biological substrates influences their absorption transintestinal kinetics, which in the
case of isolated use of dissolution remains a black box. Another preclinical application
of the camera as a relatively non-invasive technique (in addition in combination with
cells-confocal laser endomicroscopy) is used in recent experiments of the teams of the
Institute, which manage to document numerous morphological changes in the diges-
tive tract, induced by pharmacotherapeutic preparations [28].
Conclusion
For the Institute of Experimental Biopharmaceutics, the anniversary of the Institute of
Experimental Pharmacology of the Slovak Academy of Sciences presents an opportuni-
ty not only for recollections and congratulations, but also for expressing a sincere com-
bined wish. May the EFa SAV in Bratislava maintain its high standard in research, and
similarly, may the EBF in Hradec Krlov have a chance to keep, at least partially, the
character of a centre of more fundamental research.
QUOD BONUM FAUSTUM FELIX FORTUNATUMQUE EVENIAT!
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........,... .....
The use of electrochemical measurement
for real-time monitoring of nitric oxide
generation by macrophages in vitro
Antonn LOJEK
1
, Michaela PEKAROV
1
, Radomr NOS
2
, Jan HRB
3

1
Institute of Biophysics of the AS CR, v.v.i., Krlovopolsk 135, 612 65 Brno, Czech Republic, E-MAIL: alojek@ibp.cz
2
Institute of Experimental Pharmacology, SASc., Dbravsk cesta 9, 841 04 Bratislava, Slovak Republic
3
Department of Physical Chemistry, Palack University, Faculty of Science, t. Svobody 26,
771 46 Olomouc, Czech Republic
Key words: RAW 264.7 cells, lipopolysaccharide, nitric oxide, amperometry
Introduction
The production of reactive oxygen and nitrogen species (RONS) by phagocytic cells
belongs to basic microbicidal mechanisms. Microbial invaders are phagocytosed and
destroyed by reactive species inside cells [1]. RONS play also an important role as a sig-
nalling molecule between phagocytes and other cells, and as an intracellular messen-
ger within the phagocytes themselves [2]. However, the excess of extracellular RONS
induces a destruction of the surrounding cells and tissues. Thus, there is an effort to
modulate extracellular RONS and their effects pharmacologically, without disturbing
the microbicidal functions of phagocytes. Luminometric methods enable continual
analysis of the production of reactive oxygen species by phagocytes. In this case, cells
are incubated with a selected activator and a tested substance directly in the measuring
chamber of a luminometer. Such a continual analysis reflects the total reactive oxygen
species production and could be employed to distinguish between its intra- and extra-
cellular components. Two approaches are used for this purpose: I) using luminophores
with different permeability through the cell membrane, II) using antioxidant enzymes
such as superoxide dismutase and catalase [3].
The situation is more complicated in the case of nitric oxide (NO), which is pro-
duced through the conversion of L-arginine into L-citrulline. The biosynthesis of NO
is catalyzed by an increased expression of inducible nitric oxide synthase (iNOS) which
results from the stimulation of cells with lipopolysaccharide (LPS) and/or pro-inflam-
matory cytokines such as tumor necrosis factor-, interferon- and interleukin-1 [4].
Macrophage iNOS is responsible for large quantities of NO which are synthesized over
the period of several hours after cells stimulated with LPS.
A. Lojek et al. (2008) Trends in Pharmacological Research (Eds. V. Bauer et al.): 7781.
78 A. Lojek et al.
Nowadays, it is still difficult to directly measure nitric oxide synthesized in vivo as
well as in in vitro models. In biological systems, NO has a short half-life (26 s) due to its
high reactivity. Furthermore it is produced in relatively small (picomolar to nanomolar)
amounts. Because NO is a free radical gas, it reacts rapidly with cellular components
and other substances such as O
2
, superoxide anion radical, or hydrogen peroxide yield-
ing NO
2
, peroxynitrite (ONOO
), and NO
2
/NO
3
. Among direct methods which have

been successfully applied in vivo and as well as in vitro biological models the electro-
chemical detection of NO can provide high sensitivity and also a good selectivity com-
bined with rapid response enabling real-time monitoring of NO production [5].
The aim of this study was to monitore NO production from macrophages RAW 264.7
stimulated with LPS within a time period of 20 h and to compare the dynamics of NO
production with changes in nitrite concentration in cell supernatants as well as with
changes in the level of iNOS protein expression.
Methods
Cell culture
Murine leukaemic macrophage cell line RAW 264.7 (ATCC, USA) was grown in
Dullbeccos modified Eagles medium (DMEM) supplemented with 10% fetal bovine
serum (FBS) and 1% penicillin-streptomycin (Sigma, USA). After reaching confluence,
the cells were harvested and washed. Viable cells were then counted (Coulter, Coulter
Electronics LTD, England). For direct measurement using a porphyrinic microsensor,
nitrite assay and also the detection of iNOS expression, 800 l of culture medium con-
taining 1 10
6
cells were placed into glass vials and then cultured in an incubator in 5%
CO
2
at 37

C. After 2 hours, DMEM medium in glass vials with adherent macrophages
was replaced by Minimal Eagles medium (MEM) (Sigma, USA) supplemented with
10% FBS. MEM medium contains less bicarbonate than DMEM medium and it is more
suitable for measuring of NO production in closed systems where a lower CO
2
level is
present during the experiment. Macrophages were then stimulated with 5 ng/mL of LPS
(Escherichia coli serotype 0111:B4, Sigma, USA) and placed into glass vials the dimen-
sions of which accurately fit into four-port measurement chamber (WPI, USA). The
selective inhibitor of iNOS enzyme 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine
(AMT) (Sigma, USA) was also used for the experiments.
Measurement of NO release from RAW 264.7 cell line using microsensor
Glass vials with cells were placed into the measurement chamber preheated to constant
temperature of 37

C. Direct measurement of NO was executed using a porphyrinic mi-
crosensor prepared in previously described manner [6] and connected to ISO-NO Mark
II NO meter (WPI, USA). The tip of the porphyrinic microsensor was placed to the
surface of the cell layer under visual control and NO release was recorded for the period
of 20 hours.
79 Real-time monitoring of nitric oxide generation by macrophages in vitro
Determination of nitrites in the cell culture supernatant by Griess reaction
Concentration of nitrites in cell supernatants was measured as an indicator of NO pro-
duction according to Gilliam et al. [7].
Western blot analysis of iNOS
Cell extracts for Western blot analysis of iNOS were prepared from the harvested
macrophages which were settled at the bottom of the glass vials. Western blot analyses
were performed according to procedure described in details by Peivov et al. [8].
Results
Changes in NO production from RAW 264.7 cells stimulated with LPS (5 ng/mL) were
monitored for a period of 20 h. Amperogram (current vs. time curve) documenting the
changes in NO concentration by macrophages is shown in Figure 1 upper panel.
As measured by the microelectrode placed on the surface of adherent macrophages,
NO production started to rise around 56 h of co-incubation of the macrophages with
LPS. In following 2 h a gradual increase in NO production was detected. After reach-
ing the maximum the stabilization of NO production was observed. This phase was
followed by slow decline in NO production lasting for the rest of the experiment. In
the case of unstimulated cells (control experiment) the microsensor did not detect any
changes in recorded signal for the whole duration of the experiment. The electrochemi-
Figure 1. Amperogram showing time course of NO
production by RAW 264.7 cells after stimulation
with LPS. Each point of the curve was constructed
from three individual experiments.
Upper panel: The NO production by 110
6
cells
stimulated with LPS (5 ng/mL) in comparison with
non-stimulated cells.
Lower panel: The measurement of NO production
by stimulated macrophages in the presence of
10 M AMT (the selective inhibitor of iNOS).
80 A. Lojek et al.
cal signal induced by NO was sensitive to AMT, the selective inhibitor of iNOS enzyme.
The injection of AMT (10 M, final concentration) into cell culture in WPI chamber
induced a decline in recorded signal (Figure 1 lower panel).
Monitoring of nitrite concentration in supernatants removed from RAW 264.7 cells
was performed as isolated measurements at 0, 6, 7, 8, 9, 10, 14, 16 and 20 hours after
the stimulation. Stimulation of adherent macrophages with LPS caused gradual nitrite
elevation which began around 6 h after stimulation. The injection of AMT caused ex-
tensive decrease in nitrite accumulation in supernatants (data not shown).
LPS stimulation induced iNOS expression. The results obtained using Western blot
analyses demonstrate that the expression of iNOS began 3 h after the cell stimulation.
The maximum amount of iNOS was present 6 h after the stimulation of the cells (data
not shown). The iNOS protein expression remained unaffected after AMT addition.
Discussion
In this study we focused on one of the direct methods, the electrochemical detection of
NO. Using our own nitric oxide selective microelectrode we were able to monitor the NO
release from stimulated RAW 264.7 macrophage cell line. Only two studies are known
which were focused on monitoring of the NO production from LPS-stimulated RAW
264.7 cells using electrochemical microsensors. However, these studies were limited ei-
ther to the short term duration of the experiment [9] or to isolated NO determinations
at 0, 3, 6, 12 or 24 h after cell stimulation [10]. Our in vitro application of electrochemi-
cal measurement using modified microsensor permitted the continual, real-time and
long term detection of NO production from stimulated macrophages for the first time.
We found out that the expression of iNOS protein started early (approx. 3 h) after the
stimulation of macrophages by LPS. This fact implicates that the up-regulation of iNOS
expression caused by LPS enhanced the NO production in macrophages during the
time of experiment. The NO production, after its onset occurring 56 h after the cells
stimulation (as detected by both microsensor and by detection of nitrites using Griess
reaction) increases rapidly to reach its maximum 67 h after the cell stimulation. At this
point, the iNOS protein expression is at maximum, after which a gradual decrease in
iNOS occurs until approximately 12 h of the experiment. The level of NO production is
stable between 612 h. After this period of time, when a decrease in NO production is
detected by microelectrode, the rate of iNOS expression is accelerated. The changes in
iNOS expression during the time of experiment might have been affected by regulatory
signals and/or molecules, many of which are known to be induced by LPS signalling in
macrophages. The selective inhibitor of iNOS enzyme (AMT) was used for inhibition of
NO production in macrophages. Although the injection of AMT to the vicinity of LPS
stimulated cells producing NO caused significant decline in signal detected by micro-
electrode and also extensive decrease in nitrite accumulation in supernatants, the iNOS
protein expression in our experiments remained unaffected. As it is known [11], AMT
81 Real-time monitoring of nitric oxide generation by macrophages in vitro
interacts with the arginine binding site of iNOS enzyme and this consequently results
into the reduction of NO production.
Conclusions
In conclusion, we proved that our microsensor developed for continual direct detec-
tion of NO production in different in vitro biological models is sufficiently sensitive
for monitoring of NO production from LPS stimulated macrophages. In relation to our
experiments with iNOS inhibitor (AMT) we suppose that microelectrode can be used
as a detector of changes in kinetics of NO production released from stimulated cells.
It should enable to monitor even low potential influence of different pharmacological
agents, iNOS inhibitors and other chemical substances on NO production from stimu-
lated macrophages and other cells.
Acknowledgements
This study was conducted under the research plans AVOZ50040507 and AVOZ50040702
and supported by grants GA CR 524/08/1753, VEGA 2/7019/27 and VTS SK-CZ-0114-07.
REFERENCE
Allen RC, Stjernholm RL, Steele RH.: Evidence for the generation of an electronic excitation state(s) in human polymor- [1]
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Moncada S, Palmer RM, Higgs EA: Nitric oxide: physiology, pathophysiology, and pharmacology. Pharmacol Rev 1991; [2]
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with lipopolysaccharide (LPS) in the presence or absence of interferon- (IFN-): possible heterogenity of iNOS activity. J
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synthase inhibitor is solely determined by their af nity toward the diferent isoenzymes. Mol Pharmacol 2000; 58: 102634.
........,... .....
H
1
-antihistamine Dithiaden suppressed
platelet aggregation and oxidative
burst of neutrophils in vitro
Radomr NOS, Katarna DRBIKOV, Viera JANINOV, Tatiana MAIKOV,
Jana PEIVOV, Margita PETRKOV, Zuzana STRAKOV
Department of Cellular Pharmacology , Institute of Experimental Pharmacology, Slovak Academy of Sciences,
Dbravsk cesta 9, 841 04 Bratislava, Slovak Republic, E-MAIL: radomir.nosal@savba.sk
Key words: Dithiaden, neutrophils, blood platelets, oxidative burst, platelet aggregation
Introduction
Pharmacological activity of antihistamines strongly depends on subtype receptor inter-
actions. Besides their antihistaminic activities, H
1
-receptor antagonists possess other
pharmacological activities: antiiflammatory action, inhibition of blood platelet func-
tion and antioxidative effects [1,2]. Due to their cationic amphiphilic structure many
antihistamines exert pharmacological activities which might result in beneficial side
effects. It has been suggested that the physico-chemical nature of H
1
-antihistamines,
namely lipophilic molecules which carry a positive charge, allows them to associate
with cell membranes and competitively inhibit the binding of second messengers, e.g.
Ca
2+
[1]. In this study we compared the effect of Dithiaden

(DIT) from the 1
st
genera-
tion of H
1
-antihistamines on human blood platelets and neutrophil activation in vitro.
Methods
Blood sampling
In all experiments blood samples from healthy volunteers were drawn from the ante-
cubital vein at the blood bank. The blood was anticoagulated with 3.8% v/w trisodium
citrate in the ratio 9:1 in polypropylene centrifugation tubes.
Blood platelet isolation and aggregation
Blood was centrifugated for 15 min at 200 g and 22 C. Platelet rich plasma (PRP) was
removed, platelets were washed in Tyrode solution by repeated centrifugation and the
suspension was adjusted to obtain 2 10
5
platelets/L. For aggregation studies 450 L
R. Nos et al. (2008) Trends in Pharmacological Research (Eds. V. Bauer et al.): 8287.
83 Dithiaden suppressed platelet aggregation and oxidative burst of neutrophils
of platelet suspension was used together with 20 L of DIT. Aggregation was induced
with calcium ionophore A23187 (final concentration 1.8 mol/L) or with thrombin
(0.05 NIH U/mL). Platelet aggregation was measured in aggregometer Chrono-log as
described earlier [3].
Thromboxane generation
Isolated platelets (450 L, 10
4
platelets/ L) were treated with 20 L of DIT. Stimulation
with either A23187 or thrombin (see above) was stopped with indomethacine and after
centrifugation (14 000 g) the generation of thromboxane was determined by means
of a [
125
I]thromboxane B
2
(TXB
2
) RIA kit in a RIA Multidetector Counter JNG 402
[Tesla, for details see 3].
Neutrophil isolation and measurement of chemiluminescence (CL)
After initial centrifugation and removal of PRP (see Blood sampling) erythrocytes were
sedimented in 3% w/v dextran solution and neutrophils were separated by gradient
centrifugation on Lymphoprep. After hypotonic lysis of contaminating erythrocytes,
neutrophils were resuspended in calcium-magnesium-free phosphate buffered solu-
tion to a final concentration of 10
7
neutrophils/ mL [for details see 4]. Extracellular
CL was measured by luminometer LM-01T (Immunotech, Czech Republic) in samples
containing DIT, neutrophils, PMA (0.05 mol/L), isoluminol and horseradish peroxi-
dase (HRP), each in 50 L aliquots. In experiments where the intracellular CL was
recorded, luminol was used as a luminophore and superoxide dismutase and catalase
were added instead of HRP [5,6].
Superoxide (SO) determination
Superoxide formation was measured in isolated human neutrophils as superoxide
dismutase inhibitable reduction of cytochrome c. Neutrophils were incubated with
DIT and stimulated with PMA. After centrifugation absorbance was measured at
550 nm in Hewlett-Packard 8452A spectrophotometer and evaluated as described
previously [7].
Myeloperoxidase (MPO) release
Neutrophils (210
6
/sample) were preincubated with cytochalasine B (5 g/mL), sub-
sequently with DIT and stimulated with PMA. MPO activity was assayed in the su-
pernatant by determining the oxidation of o-dianisidine in the presence of hydrogen
peroxide in Hewlett-Packard 8452A spectrophotometer at 463 nm [for details see 8].
Statistical analysis
All data are expressed as the mean standard error of the mean (SEM). The data
were analyzed by one-way analysis of variance (ANOVA) and p<0.05 were considered
significant.
84 R. Nos et al.
Materials
Dithiaden: Liva (Czech Republic); hydrogen peroxide (Fluka); luminol, isolumi-
nol, PMA (phorbol-12-myristate-13-acetate), dextran, o-dianisidine, superoxide
dismutase, cytochalasine B, thrombin : Sigma-Aldrich USA; calcium ionophore A23187:
Calbiochem Switzerland; lymphoprep: Nycomed Pharma AS Norway; catalase, horse-
radish peroxidase, cytochrome c: Merck (Darmstadt, Germany); thromboxane B
2
RIA
kit: Institute of Radioisotopes Hungary; Tyrode solution ( pH 7.4; 136.9 mmol/L NaCl;
11.9 mmol/L NaHCO
3;
0.4 mmol/L NaH
2
PO
4
.2 H
2
O; 1 mmol/L MgCl
2
.6H
2
O a 5.6
mmol/L glucose). All other chemicals of analytical grade were from available com-
mercial sources.
Results
Figure 1 demonstrates the effect of DIT on human platelet aggregation (left panel) and
thromboxane generation (right panel). Platelet aggregation was significantly decreased
with 20 mol/L DIT for both stimuli used: A23187 and thrombin. Increasing concen-
tration of DIT to 50 and 100 mol/L resulted in 80% inhibition of aggregation. DIT in
the concentration of 0.1 mol/L significantly decreased TXB
2
generation in platelets
stimulated with thrombin and increase in DIT concentration to 100 mol/L resulted
in 95% inhibition of TXB
2
formation. In platelets stimulated with A23187, DIT was ef-
fective only in the highest concentration used (100 mol/L).
DIT dose-dependently decreased chemiluminescence (CL) of whole human blood
and of isolated neutrophils. Figure 2 demonstrates that DIT in the concentration of 1
mol/L decreased whole blood CL by 20% and increase of the concentration of DIT to
10 and 100 mol/L resulted in CL decrease by 58 and 93%, respectively. The intracel-
lular CL decreased with 0.1, 1, 10 and 100 mol/L DIT by 14, 17, 22 and 91% of the con-
trol, respectively. The extracellular CL significantly decreased with 10 and 100 mol/L
by 58 and 99%, respectively.
Superoxide generation significantly decreased with 100 mol/L DIT by 49%. On the
other hand, MPO liberation decreased significantly with 1, 10 and 100 mol/L DIT by
48, 68 and 82%, respectively.
Discussion
In a concentration-dependent manner, DIT decreased both platelet aggregation and
CL of professional phagocytes in vitro. DIT suppressed platelet aggregation stimulated
both with receptor (thrombin) and receptor-bypassing (A23187) stimuli. Inhibition of
TXB
2
generation with DIT was parallel with inhibition of thrombin-induced aggre-
gation. Since DIT also inhibited arachidonic acid liberation from platelet membranes
and malondialdehyde formation from arachidonate pathway [9], it has been suggested
that DIT, like other cationic amphiphilic drugs, suppressed platelet cytosolic phospho-
lipase A
2
activity [10]. A similar effect was demonstrated for other H
1
-antihistamines,
indicating rather interaction with platelet membrane structure than with platelet H
1
-
receptors. H
1
-antihistamines may interfere with platelet aggregation first at arachidonic
acid pathway, second due to the interference with platelet Ca
2+
mobilization [see inhibi-
tion of A23187-induced aggregation 11].
In addition to the suppression of oxidative burst of blood phagocytes, DIT dose-de-
pendently inhibited CL of isolated neutrophils both at extra- and intracellular site. This
was evaluated by means of two luminophores, luminol and isoluminol [46]. A similar
effect was demonstrated for cationic amphiphilic drugs from different pharmacologi-
cal groups. Decrease in extracellular CL might result from scavenging extracellular re-
active oxygen metabolites, thus contributing to protection of organs and tissues [12].
Inhibition of intracellular CL supported the protective effect of DIT against oxidative
burst, yet this mechanism requires further detailed investigation.
Suppression of MPO completely prevented luminol-enhanced CL, indicating inter-
ference with neutrophil oxidative burst [7]. Dose-dependent inhibition of MPO release
from neutrophil granules correlated with inhibition of CL due to DIT. This effect seems
to be rather nonspecific since the inhibition of MPO was also measured with drugs
from different pharmacological groups and on different types of cells [13].
Inhibition of SO required much higher concentrations of DIT. In general,
H
1
-antihistamines showed none or very low scavenging properties against superoxide
anion, hydroxyl radical and peroxyl radical and this effect could not contribute to the
inhibition of CL [14]. PMA, as non-physiological stimulus of superoxide generation and
myeloperoxidase release, activates protein kinase C (PKC), which is known to be a fam-
ily of isoenzymes differing in structure, co-factor requirement and substrate specific-
ity [15]. PMA-stimulated responses differed in their sensitivity to DIT inhibition, with
Figure 1. Decreased aggregation and thromboxane B
2
formation in human platelets stimulated with thrombin
or Ca
2+
-ionophore A23187 in the presence of dithiaden. Mean SEM, n = 6, *p<0.05, **p<0.01.
86 R. Nos et al.
superoxide production being less sensitive than MPO release. Neither horse radish per-
oxidase nor myeloperoxidase activity was inhibited by DIT in our experimental condi-
tions (data not shown) [16]. It is suggested that the inhibitory effect of DIT is due to its
interaction with signaling pathway and may be mediated by either different degrees of
activation or different isoenzymes responsible for particular physiological responses.
Other possible mechanisms for the inhibitory effect of antihistamines to suppress oxida-
tive burst in professional phagocytes might result from their interference with calcium
ion movement, enzymatic pathways like NADPH-oxidase or second messengers [17].
Conclusions
It can be concluded that DIT, a representative of H
1
-antihistamines, possesses support-
ive pharmacological activity on blood cells which participate in the pathophysiology of
many diseases. First, suggestive inhibition of activated platelets may positively affect
thromboembolic disorders, second, due to suppression of oxidative burst in profession-
al phagocytes, DIT might beneficially contribute to the treatment of inflammation, im-
mune responses and ischemia-reperfusion. All data obtained from in vitro experiments
should be verified in clinical conditions.
Acknowledgements
This work was supported in part with VEGA 2/7019/27, APVV SK-CZ-0114-07,
APVV-51-017905.
Figure 2. Eect of dithiaden on phorbol myristate acetate (PMA) stimulated: chemiluminescence (CL) of human
whole blood, extra- and intracellular CL, myeloperoxidase (MPO) release and superoxide generation in isolated
neutrophils. Mean SEM, n=67, *p<0.05, **p<0.01.
REFERENCES
Leurs R, Church MK, Togliatela MH. H [1]
1
-antihistamines: Inverse agonists, anti-infammatory actions and
cardiac efects. Clin Exper Allergy 2002; 32: 48998.
Nos R, Drbikov K, Janinov V, Petrkov M, Fabryov V. Antiplatelet and antiphagocyte activity of H [2]
1
-
antihistamines. Infamm Res 2005; 54: S1920.
Nos R, Janinov V, Danihelov E. Te H [3]
1
-antihistamine antagonist dithiaden inhibits human platelet
function in vitro. Platelets 1997; 8: 17580.
Drbikov K, Nos R, Janinov V, M, Lojek A. Reactive oxygen metabolite production is inhibited [4]
by histamine and H
1
-histamine antagonist dithiaden in human PMN leukocytes. Free Rad Res 2002; 6:
97580.
Janinov V, Drbikov K, Nos R, Holomov D. Extra- and intracellular formation of reactive oxygen [5]
species by human neutrophils in the presence of pheniramine, chlorpheniramine and brompheniramine.
Neuro Endocrinol Lett 2006; 27(Suppl.2): 1413.
Janinov V, Drbikov K, Nos R, Rakov L, Mjekov M, Holomov D. Te combined luminol/isolu- [6]
minol chemiluminescence method for diferentiating between extracellular and intracellular oxidant pro-
duction by neutrophils. Redox Report 2006; 11: 1106.
Peivov J, Maikov T, Lojek A, Gallov L, M, Nos R, Holomov D. In vitro efect of carvedilol on [7]
professional phagocytes. Pharmacology 2007; 79: 8692.
Peivov J, Maikov T, Lojek A, Gallov L, M, Nos R, Holomov D. Efect of carvedilol on reactive [8]
oxygen species and enzymes linking innate and adaptive immunity. Neuro Endocrinol Lett 2006; 27: 1603.
Nos R, Janinov V, Danihelov E. Te efect of H [9]
1
-histamine antagonist dithiaden inhibits platelet func-
tion in vitro. Platelets 1999; 8: 17580.
Nos R, Janinov V. Cationic amphiphilic drugs and phospholipase A [10]
2
(PLA
2
). Tromb Res 2002; 105:
33945.
Nos R. Antiplatelet and antileukocyte efects of cardiovascular, immunomodulatory and chemotherapeu- [11]
tic drugs. Cardiovasc Hematol Agent Med Chem 2006; 4: 23761.
Drbikov K, Janinov V, Nos R, Solk P, Murn J, Holomov D. On the antioxidant activity of carvedilol [12]
in human polymorphonuclear leukocytes in vitro and ex vivo. Neuro Endocrinol Lett 2006; 26: 13840.
Edwards SW. Te respiratory burst: Te generation of reactive oxygen metabolites and their role in micro- [13]
bial killing. In: Biochemistry and Physiology of the neutrophil. Cambridge University Press 1994, p. 1291.
Krlov J, M, Nos R, Drbikov K, Lojek A. Efect of H [14]
1
-antihistamines on the oxidative burst of rat
phagocytes. Infamm Res 2006; 55: S156.
Hug H and Sarre TF. Protein kinase C isoenzymes: divergence in signal transduction? Biochem J 1993; 291: [15]
32943.
Peivov J, Maikov T, Nos R, Danihelov E. [16] Efect of stobadine on superoxide generation and degranu-
lation of stimulated human polymorphonuclear leukocytes in vitro. Biologia 2000; 55 Suppl.8: 1036.
Lieberman P. Preclinical evidence of azelastine hydrochloride activity. Curr Ter Res Clin Exp 2002; 63: [17]
55671.
........,... .....
Research focuses of the
pharmacology in Martin
Gabriela NOSOV, Soa FRAOV, Anna STRAPKOV, Juraj MOKR,
Martina UTOVSK, Vladimra SADLOOV
Department of Pharmacology Jessenius Medical Faculty in Martin, Comenius University in Bratislava,
Sklabinsk 26, 037 53 Martin, Slovak Republic, E-MAIL: nosalova@jfmed.uniba.sk
Key words: antitussive activity of agents, cough reflex, bronchoconstriction, airways
Introduction
In spite of an intensive progress in research and continual broadening of the knowledge
in the field of respiratory pharmacology, there is still a significant gap in understanding
all the mechanisms participating in the pathophysiology of defense reflexes, such as the
cough reflex and bronchoconstriction.
Our aim has been to investigate the antitussive activity of agents not only for ex-
perimental conditions but mainly for the use in clinical practice. We search for agents
with higher efficacy to suppress the pathological type of the cough reflex with minimal
side effects. We focus our attention not only on synthetic agents but also on vegetable
sources.
To get insight also into molecular mechanisms of their activity, we concentrate on
receptors (NK
1
, NK
2
, vaniloid and opioid receptors, GABA receptors, etc.) and mainly
on ion channels, potassium ion channels, which play the main pathophysiological as
well as regulatory role in defence reflexes of the airways.
It has been known for some years that antihypertensive therapy by angiotensin con-
verting enzyme inhibitors is complicated by chronic dry cough. We decided to study
not only mechanisms of this relationship but also the possibilities of its pharmacologi-
cal restriction.
The relation between the cough reflex and bronchoconstriction has been extensively
studied, yet we still do not know the role of bronchoconstriction in the mechanism
originating the cough reflex. That is why our department has been concerned for sev-
eral years in pharmacological modulation of smooth muscle activity in the airways and
the relationship between smooth muscle contraction and cough reflex, as well as its
modulation by various agents (plant extracts, xanthine derivatives, phosphodiesterase
inhibitors).
G. Nosov et al. (2008) Trends in Pharmacological Research (Eds. V. Bauer et al.): 8895.
89 Research focuses of the pharmacology in Martin
Moreover, we are also interested in problems connected with mechanisms of hyper-
reactivity of the airways. In conditions of our models of experimental hyperreactiv-
ity, we study the effects of modulation of NO level on the response of airways smooth
muscle on three levels: a) the precursor L-arginine supplementation or treatment with
drugs releasing this molecule NO donors, b) activity of enzymes involved in NO ho-
meostasis, c) mechanisms of NO action.
Some epidemiological studies have suggested that dietary factors, including con-
sumption of food containing polyphenolic compounds, might reduce the occurrence
of hyperreactivity symptoms. In view of this fact the purpose of our experiments was
and will be to evaluate effects of polyphenolic compounds on allergen induced hyper-
reactivity of the airways.
Our attention will be focused on pharmacological modulation of airways hyperre-
sponsiveness associated with experimental aspiration of meconium.
Considering the requirement of the faculty concerned with oncologic problems, we
attempt to establish an experimental method for evaluation of anticancer activity of
some agents.
Methods
1. The cough reex:
a) mechanical stimulation of the airways
In our department we have been involved for over 35 years in pharmacological modu-
lation of the cough reflex. We established an original method to evaluate antitussive
activity of cough suppressing agents. The cough reflex was provoked by mechanical
irritation of the airways in conscious cats [1,2].
b) chemical stimulation of the airways
Besides mechanical stimulation of the cough reflex, we also use chemical irritation of
the airways by citric acid. The cough reflex is induced chemically by exposure to 0.3 M
citric acid aerosol for 3 min, in which interval the total number of cough efforts is
counted. The cough effort is defined as sudden enhancement of expiratory flow accom-
panied by a typical cough movement and sound recognized by the trained observer.
The cough response is expressed as the total number of coughs during citric acid expo-
sure, quantifying the intensity of cough reactions.
2. Reactivity of smooth muscles of the airways:
a) in vivo
For better evaluation of the effects of bronchoactive substances in experimental condi-
tions, we have introduced a new method using a double chamber whole body plethys-
mograph in conscious guinea pigs. This method allows to evaluate several parameters,
including specific airway resistance, tidal volume, frequency of breathing and minute
90 G. Nosov et al.
ventilation. By suitable adaptation, it can be even used for monitoring the number of
cough efforts during inhalation of tussigenic aerosols.
b) in vitro
Changes of airway smooth muscle reactivity in vitro conditions on cumulative doses of
contractive mediators (acetylcholine, carbachol and histamine) after administration of
many agents were tested by the method of tissue bath.
3. Hyperreactivity of the airways
We use two models of experimental airways hyperreactivity induced by two different
triggers biological or chemical. The allergen ovalbumin is used as the biological classi-
cal model. The chemical model of airways hyperreactivity was developed in our depart-
ment. The chemical trigger used is the exogenous irritant toluene vapour [3,4,5].
4. Model of experimental premenopausal the mammary carcinogenesis
The aim of this study was to establish an experimental premenopausal model of mam-
mary carcinogenesis that would allow to assess preventive tumour suppressive effects of
some agents. Chemoprevention began 7 days before chemo carcinogen administration
and lasted till the end of every experiment. N-methyl-N-nitrosourea (NMU) was used
as chemo carcinogen to induce mammary carcinogenesis. NMU was injected intraperi-
toneally during the period of 4060 postnatal days to rats [6].
Results
We showed that the method of mechanical stimulation of the airways was suitable for
following antitussive activity of agents from synthetic and vegetable sources [1,7,8]. We
also found that for common screening evaluation of antitussic activity of agents chemi-
cal stimulation of the airways was appropriate.
Our department participated in the choice and experimental study of the first
Czechoslovak non-narcotic antitussive agent dropropisine [9].
We obtained original priority results on our findings that GABA-ergic substances are
able to suppress cough [10,11] and that the mechanism can participate in the action of oth-
er cough-suppressing agents (gabalid, baclofen, diazepam, hypnotics and other agents).
We analysed the antitussive activity of opioid analgesics (codeine, butorphanol, pen-
tazocine, tilidine and tramadol) in normal and pathological conditions [12,13].
Our results showed [14] that openers of potassium ion channels (K
+
ATP
sensitive
Pinacidil and BK
+
Ca
NS1619) inhibited citric acid induced cough. Their effect was
prevented by pretreatment with selective blockers, (K
+
ATP
sensitive glibenclamide and
BK
+
Ca
tetraethylammonium chloride).
Peripherally active agents we studied the cough suppressive effect of some drugs with
local anaesthetic or anti-inflammatory activity [15]. We are interested also in agents
that are able to relax the smooth muscle of the airways and modulate the pathway of
the cough reflex [16,17]. We also demonstrated the antitussive action of vinpocetin, a
selective PDE1 inhibitor, cilostazol, a selective PDE3 inhibitor, and zaprinast, a selective
PDE5 inhibitor, in healthy, non-sensitised guinea pigs. This effect was accompanied by
decreased in vivo airway reactivity to histamine. In ovalbumin-sensitized animals, sig-
nificant antitussive activity was found after administration of vinpocetin, citalopram,
a selective PDE4 inhibitor, and zaprinast, with simultaneous decrease of in vivo airway
reactivity to ovalbumin.
Our attention has been focused on vegetable sources (plant polysaccharides), mainly
on agents which could change the quantity and quality of mucus in the airways and thus
modulate the cough reflex [7,8,1823]. We suppose that flavonoids present in Malva
sylvestris and Verbascum densiflorum participate in their antitussive efficacy [7,8]. We
investigated the protective effect of flavonoids (Provinol), isolated from red wine, on the
tracheal smooth muscle response to bronchoconstrictor mediators and allergens in the
model of ovalbumin-induced hyperreactivity of the airways [24].
Our results confirmed that long-lasting administration of enalapril resulted in a
significant increase of the cough parameters assessed and increased the reactivity of
tracheal smooth muscle to histamine, acetylcholine and KCl. Simultaneous admin-
istration of enalapril and diltiazem and inhaled furosemide significantly decreased
enalapril induced cough, and decreased enalapril induced hyperreactivity of tracheal
smooth muscles [25]. The co-administration of enalapril with inhaled cromoglycate
showed minimal effect in inhibition of ACE-I respiratory side effects.
The deficiency or the decrease of bioavailability of the basic substrate for NO syn-
thesis L-arginine can be one of the factors contributing to airway hyperreactivity.
We recorded a decrease of airway reactivity in animals with toluene-induced bronchial
hyperreactivity after administration of L-arginine in the dose of 300 mg/kg b.w. i.p.
over 3 days. Pre-treatment of animals with L-arginine over 17 days did not affect airway
smooth muscle reactivity to a larger extent [26].
We showed the difference in the NO donor activity to depend on the way of admin-
istration. The effect of NO donors can be evaluated as protective on the whole. It is of
interest that pre-treatment with N-acetylcysteine simultaneously with NO donor raised
the beneficial effect of isosorbide dinitrate, presumably by increasing the intracelullar
thiol level [27].
To change NO production, we used inhibition of NO synthases activity by non-
specific (L-NAME) or specific inhibitors (aminoguanidine). We recorded a significant
decrease of tracheal smooth muscle reactivity after acute L-NAME pre-treatment in tol-
uene-induced hyperreactivity, and the opposite effect an increase of tracheal smooth
muscle reactivity in allergen-induced hyperreactivity after acute and chronic L-NAME
pre-treatment. Lung tissue reactivity was reduced after acute and chronic L-NAME pre-
treatment in toluene-induced hyperreactivity but changes in allergen-induced hyper-
reactivity were not significant.
92 G. Nosov et al.
We investigated the effect of intervention in arginase activity in ovalbumin-induced
airway hyperreactivity after in vitro administration of arginase or of the non-selective
inhibitor of arginase N
-hydroxy-L-arginine (NOHA) [28]. We did not record signifi-

cant differences in the reactivity of the tracheal and lung tissue smooth muscle if we
applied arginase in the dose of 75 UI in vitro. In the dose of 5 a 10 mol in vitro NOHA
induced an overall decrease of tracheal and lung tissue smooth muscle reactivity. The
decrease of the contraction amplitude was deepened with higher doses of the inhibitor.
Supplemention with the NO synthesis precursor L-arginine in the dose of 10
4
mol/l
in vitro together with NOHA intensified the decrease of the airways reactivity induced
by the inhibitor arginase.
We tried to detect the interaction of NOS-COX in conditions of exogenous irritant-in-
duced experimental bronchial hyperreactivity by using the COX inhibitor diclofenac or
the direct NO donor molsidomine. The results indicate a possible participation of both
enzymatic systems studied and their interaction in our experimental conditions [29].
Aspiration of meconium in term and post-term neonates can lead to serious respi-
ratory failure associated with inflammation meconium aspiration syndrome. We
found that the administration of meconium suspension to tracheal and lung tissue
strips from adult rabbits and guinea pigs in different concentrations did not increase
airway reactivity [30,31]. On the contrary, in vivo instillation of meconium suspension
to rabbits and subsequent 5-hour-lasting conventional ventilation led to the experi-
mental meconium aspiration syndrome (MAS) with significantly increased in vitro
airway reactivity [32].
We can state that our model of experimental premenopausal mammary carcinogen-
esis may be useful for screening anticancer mammary activity of agents [33].
Discussion
Our results unambiguously showed that the model of mechanical stimulation of the
airways is irreplaceable for evaluation of antitussive activities of agents. Noteworthy
results were obtained in evaluating the antitussive activity of various substances based
on objective methods for cough reflex evaluation. Studies on conscious animals, thus
without any influence of anaesthetics, were included. A big advantage of this method
is the possibility of assessing the efficacy of agents in suppressing cough from laryngo-
pharyngeal and tracheobronchial mucus areas separately in normal and pathological
conditions. Furthermore, there is a possibility to assess the antitussive activity accord-
ing to the number of cough efforts, frequency, intensity of cough attack, and intensity
of maximal cough effort in expiration or in inspiration, what is very important from
the clinical point of view [1,2,7,8,34]. This method has only one disadvantage, i.e. it is
expensive. Regarding the use of chemical stimulation of the airways, antitussive agents
from synthetic and vegetable sources appear to be suitable for screening and using pigs
is cheaper than using cats.
Our results showed that opioids, gabaergic and some types of 5-HT receptors play a
very important role in the mechanism of action of centrally acting antitussive agents.
Presumably, BK
+
Ca
and K
+
ATP
ion channels are also involved in the mechanisms of
cough, antitussive activity of many agents, as well as in airway defence reflexes based
on smooth muscle reactivity and represent an excellent target for the new drugs in the
treatment of airways diseases. Moreover, we found that anti-inflammatory properties of
xanthine derivatives together with their relaxing effect on smooth muscles are effective
in suppression of the cough reflex.
A persistent chronic dry cough is the most common adverse effect of angiotensin
converting enzyme inhibitors (ACE-I) in the therapy of hypertension. The mechanism
of this respiratory adverse effect is related to the inhibition of ACE and the accumula-
tion of bradykinin, substance P, prostanoids and another inflammatory neuropeptides
in the airways. Our results showed a partially protective effect of diltiazem and enal-
april co-administration on the adverse respiratory effects induced by enalapril therapy.
Another combination therapy able to minimise the respiratory side effects of ACE in-
hibitors is co-administration of these antihypertensive drugs with inhaled furosemide.
Our study of the mechanisms of hyperreactivity showed that the effect of NOS in-
hibitors on airway reactivity changes was dependent on the hyperreactivity provoking
factor and the type of therapeutic regimen. Constitutive isoforms of NO synthases have
probably a more significant position in allergen-induced airways hyperreactivity. The
effect of L-arginine supplementation was different depending on the airway level and
pre-treatment duration. The results refer to the importance of optimal L-arginine level
for the control of bronchomotoric tone. The results suggest that arginase may be the
therapeutic target in modification of the airways smooth muscle response to different
impulses. We also found indications on a possible participation of the enzymatic systems
NOS and COX in exogenous irritant-induced experimental bronchial hyperreactivity.
Trying to modulate pharmacologically the meconium aspiration syndrome, we ob-
served changes in airway reactivity with accompanying beneficial effect on vital respira-
tory and cardio-vascular parameters. The administration of anti-inflammatory agents
from different groups dexamethasone, budesonide and aminophylline pointed to
a significant role of anti-inflammatory activity agents in suppression of airway hyper-
reactivity.
Conclusion
We intend to continue with this type of research on antitussive agents and our aim is to
concentrate not only on selecting excellent cough suppressive agents but also on molec-
ular mechanisms which take part in this activity and on combinations of agents which
would decrease side effects after their administration. Our attention will concentrate
on vegetable sources, mainly on vegetable polysaccharides, which appear to be highly
prospective agents in cough suppression.
94 G. Nosov et al.
We will keep our orientation on studying the activity of agents influencing the respi-
ratory tract in in vivo and in vitro conditions, and thus to contribute to the knowledge
of mechanisms of hyperreactivity of the airways. We will also further use our experi-
mental model for studying anticancer activity of agents.
Acknowledgement
The study was supported by Grants of the Agency for Science (VEGA) No. 1/0072/08,
No.1/0073/08, APPV-0030-07 and the Grant of Ministry of Health No. 2005/13-MFN-05
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zole in mammary carcinogenesis of female rats. Acta Med Martiniana 2007; 7: 912.
Nosalova G., Capek P., Sutovska M., Franova S., Matulova M.: Antitussive Active Polysaccharides from Or- [7]
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Lek. Listy 1986a; 85: 52632.
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........,... .....
Trends in pharmacological research
contribution from studies of the
membrane transport and cell signaling
Karol ONDRIA
Institute of Molecular Physiology and Genetics, Centre of Excellence for Cardiovascular Research, SASc,
Vlrska 5, 833 34 Bratislava, Slovak Republic, e-mail: karol@ondrias.sk
Key words: ion channels, neuronal excitability, apoptosis, nitric oxide
Introduction
Scientific orientation of the Institute of Molecular Physiology and Genetics, Slovak
Academy of Sciences, Bratislava, is focused on the molecular basis of elementary physi-
ological functions, with the main orientation on cardiac muscle physiology, membrane
transport and genetics. Research includes electrophysiological and optical studies of the
properties of calcium release channels and their modulation, intracellular chloride and
potassium channels and a role of calcium channels in neuronal excitability. Biochemical
and molecular biology studies are focused towards understanding of the mechanism,
by which selected calcium transport systems (sodium calcium exchanger, intracellular
calcium channels and calcium ATPases) are regulated in normal and pathological con-
ditions. Also, studies on P-glycoprotein (P-gp) mediated multidrug resistance (MDR)
of cancer tissue is in the center of interest. Genetic studies are focused on monogenic
disorders caused by mutations in human genome. Main attention is devoted to those re-
gions, which are involved in serious pathologies, frequent in the population of Slovakia.
Molecular analysis of the blood samples of Slovak patients affected with monogenic
disorders like cystic fibrosis, Duchenne/Becker muscular dystrophy, phenylketonuria,
alkaptonuria, heamophilia A, spinal muscular atrophy type IIII, Huntington chorea,
congenital glaucoma etc., was performed. Based on these results DNA-diagnostics has
been introduced into every-day health service in Slovakia for these disorders.
In some of these studies, pharmacological approach was used to clarify the mo-
lecular basis of elementary physiological functions. In this contribution I will survey
pharmacological approaches using at our institute, particularly in the recent research of
channel function and involvement of P-gp mediated MDR resistance of cancer cells.
K. Ondri (2008) Trends in Pharmacological Research (Eds. V. Bauer et al.): 96101.
97 Contribution from studies of the membrane transport and cell signaling
Methods
Several methods are used to study drug-channel interactions. Channels incorporated
into the bilayer lipid membrane were utilized in the studies devoted to the influence
of bongkrekic acid (BKA), atractyloside (ATR) and chloride channel blockers on single
properties of chloride channels derived from mitochondrial and lysosomal membranes
[1,2]. The whole-cell patch clamp method was used to investigate the inhibition of cal-
cium current by antidepressants in enzymatically isolated rat ventricular myocytes [3]
and mouse IHCs cells [4,5]. A MDR resistant L1210/VCR cells were used for studies of
P-gp regulation [6,7]. Method of the electron paramagnetic resonance spectroscopy of
the spin trap and measurement of the NO oxidation product (which is nitrite) by the
Griess reaction was used to study release of NO from nitrosothiols and cells by H
2
S [8].
Results
Intracellular chloride channels and apoptosis
Apoptosis has been implicated in a variety of heart diseases, such as myocardial in-
farction, ischemia-reperfusion injury, cardiomyopathy, arrhythmias, and dysplasia [9].
It was modulated by chloride channel blockers. Therefore, an involvement of chloride
channels in apoptosis was studied. It was observed that the chloride channel blockers,
5-nitro-2-(phenylpropylamino)-benzoate (NPPB), dihydro-4,4' diisothiocyanostilbene-
2,2'-disulphonic acid (DIDS), and phloretin (100 mol/l) inhibited the H
2
O
2
-induced
apoptosis in cardiomyocytes and decreased open probability of the chloride channels
derived from the mitochondrial and lysosomal vesicles [2]. These results may contrib-
ute to the understanding a possible involvement of intracellular chloride channels in
apoptosis and cardioprotection. Similarly, BKA (1100 mol/l), ATR and ATR deriva-
tive CAT (5100 mol/l) inhibited the chloride channels in side and dose-dependent
manner [1], what may contribute to explanation of their numerous biological effects.
Inhibitory effects of DIDS and NPPB were side dependent, and their target on chloride
channels was better accessible from the cis-side than from the trans-side. On the other
hand, inhibitory effects of BKA and CAT function from the opposite trans-side [1]. The
side-dependent DIDSNPPB versus BKACAT effect [2] may by used as a pharmaco-
logical tool to categorize chloride channels. We have observed that the chloride chan-
nel blocker NPPB activated potassium conductance of potassium-chloride promiscuous
channels in mitochondrial membranes [10].
Inhibition of calcium C
av
1.3 current
Calcium currents (I
Ca
) in inner hair cells (IHCs) are carried by the Ca
v
1.3 subtype
of L-type calcium channels. They play an important role in synaptic transmission of
sound-evoked mechanical stimuli. L-type calcium channels are targets of classes of
the organic blockers dihydropyridines, phenylalkylamines and benzothiazepines.
98 K. Ondria
Previously a low sensitivity of the Ca
v
1.3 subtype towards dihydropyridines has been
demonstrated [11]. Therefore, an effect of two phenylalkylamines (verapamil and gal-
lopamil) and the benzothiazepine diltiazem on I
Ca
through Ca
v
1.3 channels in mouse
IHCs was evaluated [4]. The phenylalkylamines verapamil and gallopamil and the ben-
zothiazepine diltiazem inhibited I
Ca
in IHCs in a concentration-dependent manner [4].
This block was largely reversible. Inhibition of the peak I
Ca
by phenylalkylamines and
benzothiazepines was voltage independent. Concentrations of phenylalkylamines and
benzothiazepine necessary to inhibit 50% of I
Ca
in IHCs were one order larger com-
pared to concentrations, which inhibited I
Ca
through Ca
v
1.2 channels in native cells or
expression systems. However, inhibitory concentrations were in the same range as those
required for block of I
Ca
in turtle hair cells.
A neurotoxic effect of inorganic mercury (Hg
2+
) and methylmercury (MeHg) was
studied on neuronal Ca
v
3.1 (T-type) calcium channel stably expressed in the human
embryonic kidney (HEK) 293 cell line. Hg
2+
and MeHg inhibited current through the
Ca
v
3.1 calcium channel in concentrations 10 nmol/l and higher with an IC
50
of 0.630.11
mol/l. and 13.05.0 mol/l, respectively [5]. The interaction with the Ca
v
3.1 calcium
channel may significantly contribute to neuronal symptoms of mercury poisoning dur-
ing both acute poisoning and long-term environmental exposure.
Inhibition of L-type calcium channel by antidepressants
Antidepressants inhibit many membrane receptors and ion channels, including the
L-type calcium channel. Therefore, an inhibition of calcium current (I
Cav
) by anti-
depressants in enzymatically isolated rat ventricular myocytes using whole-cell patch
clamp was investigated [3]. The molecular mechanism of inhibition was studied by
comparing the voltage and state dependence of antidepressant inhibition of I
Cav
to the
respective properties of calcium antagonists, and by studying the effect of Bay K8644 or
diltiazem on the inhibitory potency of the antidepressants. All selected antidepressants
inhibited calcium currents reversibly and concentration-dependently. At a stimulation
frequency of 0.33 Hz, the antidepressants imipramine, clomipramine, desipramine,
amitriptyline, maprotiline, citalopram, and dibenzepin blocked I
Cav
, with IC
50
values
of 8.3, 11.6, 11.7, 23.2, 31.0, 64.5, and 364 mol/l, respectively. However, the inhibitory
effect of antidepressants was also augmented by diltiazem, suggesting that these drugs
do not compete with diltiazem for a single binding site. These data suggest that antide-
pressants exert their inhibitory action on cardiac L-type calcium channels by a specific
interaction at a receptor site similar to, but distinct from, the benzothiazepine site [3].
H
2
S releases nitric oxide from nitrosothiols and L1210 leukaemia cells
An endogenously produced H
2
S is involved in neuromodulation, cell proliferation,
apoptosis, regulation of cardiac function and cardioprotection, vasorelaxation, hyper-
tension, and septic, endotoxin and haemorrhagic shocks and inflammation processes
[12]. Many of the effects are shared with NO. Therefore, the H
2
S-NO interaction was
studied. It was observed that H
2
S and HS
donor NaHS released NO from nitrosothiols,

namely from GSNO, S-nitroso-N-acetyl-DL-penicillamine (SNAP), from metal nitrosyl
complex nitroprusside (SNP) and from rat brain homogenate and murine L1210 leukae-
mia cells [8], in which HS
, rather than H
2
S was responsible for the NO-releasing effect.
We assumed that the releasing effect is responsible for some of H
2
S biological activities
and that this mechanism might be involved in S-nitrosothiol-signalling reactions [8].
Modulation and overexpression of P-glycoprotein
The spectrum of drugs/substances that are substrates and are extruded by P-gp involves
anthracyclines (e.g. doxorubicin DOX), vinca alkaloids (e.g. vincristine VCR), actin-
omycines (e.g. actinomycin D, dactinomycines), taxols (e.g. paclitaxel), alkylating agents
(mytomycin C), peptide antibiotics (gramicidin, valinomycin), and many others [7].
Development of the most common MDR phenotype associated with a massive over-
expression of P-gp in neoplastic cells may result in more than a hundred-fold higher
resistance of these cells to several drugs. L1210/VCR is a P-gp-positive drug resistant
cell line, in which P-gp overexpression was achieved by repeated cultivation of paren-
tal cells with a stepwise increasing concentration of vincristine [6,7,13]. Based on P-gp
overexpression, both doxorubicin and vincristine induce a common multidrug resis-
tance phenotype in L1210 cells [13]. Relatively little is known about regulation of P-gp
function and expression. Therefore, an effect of drugs on P-gp function and expression
was studied [6].
It was observed that the combined treatment of L1210/VCR cells with all-trans retinoic
acid (ATRA, ligand of retinoic acid receptors, RARs) and verapamil was able to depress
P-gp expression, and consequently its activity. ATRA was not a P-gp-transportable sub-
stance, and thus this effect could not be attributed to verapamil-induced inhibition of
P-gp that would allow ATRA to reach retinoic acid nuclear receptors and activate them [6].
Methylxanthine pentoxifylline (PTX) depressed the P-gp mediated MDR of the
mouse leukemic cell line L1210/VCR [14]. Other methylxanthines like caffeine and
theophylline were found to be ineffective. Studying the capability of 25 methylxan-
thines, which structurally differed in substituents located in positions N1, N3, N7 and
C8, to depress MDR of L1210/VCR cells revealed that for an effective reversal of P-gp
mediated MDR of our cells the existence of a longer polar substituent in the position N1
played a crucial role [15]. The elongation of the substituent in the positions N3 and N7
(from methyl to propyl) increased and in the position C8 (from H to propyl) decreased
the efficacy of xanthines to reverse the vincristine resistance of L1210/VCR cells [14].
LY 294,002, a specific inhibitor of PI3K/Akt kinase pathway, reduced a degree of vin-
cristine resistance in L1210/VCR cells significantly and in a concentration dependent
manner [16]. MDR reversal effect of LY294,002 was accompanied with this compounds
influence on vincristine-induced apoptosis. The results pointed to the possible involve-
ment of PI3K/Akt kinase pathway in modulation of P-gp mediated multidrug resistance
in L1210/VCR mouse leukemic cell line [16,17].
100 K. Ondria
Discussion
A molecular mechanism of about 20% clinically used drugs is based on their interaction
with membrane channels. Reported results and results obtained from the presented
studies indicate that several channels are affected by a single drug and the same channel
is influenced by different drugs [3,6,7,8,11,12]. This makes a problem of a drug-channel
interaction very complex and it needs more studies. Similarly, an understanding that
membrane channels are involved in induction or inhibition of apoptosis that is con-
nected not only to cardiovascular diseases, but also with cancer is important.
We observed that H
2
S and HS
donor NaHS released NO not only from NO donors

but also from rat brain homogenate [10]. From this observation we assumed that H
2
S
and/or HS
releases NO from S-nitrosothiols and/or metal nitrosyl complexes in vivo.

This might be supported by numerous reports that H
2
S shares many biological effects
with NO, for example, both have vasorelaxant, antiinflammatory, cardioprotective,
anti-proliferative or erectile properties. It was assumed that some of the NO-sharing
effects of H
2
S resulted from its ability to release NO in vivo. S-nitrosothiols and NO
transfer reactions between protein and peptide cysteins have been proposed to represent
regulated signaling. From the reported results, it was assumed that H
2
S and/or released
NO from nitrosothiols and so might interplay with NO actions and with S-nitrosothiol
signalling reactions.
Conclusion
MDR of neoplastic tissue represents a serious obstacle in effective chemotherapy of the
cancer. Overexpression of P-gp membrane transport protein with the function to
efflux effectively drugs from intracellular space represents a dominant mechanism
responsible for MDR. Blockade of the function and expression of PGP represents the
way, how to treat the cancer diseases, where MDR plays a significant role. The obtained
results could improve both diagnostics of MDR in cancer patients and effectiveness of
chemotherapy of cancer patients having developed MDR.
Acknowledgements
Financial support by Slovak APVV-0397-07 and VEGA 2/6012/6 grant agencies is grate-
fully acknowledged.
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........,... .....
Vasoactive effects of Provinols
in experimental hypertension
Oga PECHOV and Iveta BERNTOV
Institute of Normal and Pathological Physiology and Centre of Excellence for Cardiovascular Research, SASc.,
Sienkiewiczova 1, 813 71 Bratislava, Slovak Republic, E-MAIL: olga.pechanova@savba.sk
Key words: polyphenols, vascular functions, nitric oxide, vasorelaxation, hypertension
Introduction
Numerous experimental and epidemiological data have documented that selected nat-
ural polyphenols exert protective action on the cardiovascular system. In the coronary
heart disease, the protective effects of polyphenolic compounds include mainly anti-
thrombotic, antiischemic, antioxidant, and vasorelaxant activities [1,2]. Therapeutically
relevant effect of polyphenols may include their ability to interact with the generation
of NO from vascular endothelium, which leads not only to vasodilatation, but also to
the expression of genes that protect the cardiovascular system. Polyphenols, isolated
from red wine particularly, also contribute to the preservation of the integrity of cells
belonging to the vascular wall, mainly those in the endothelium, by acting on the sig-
naling cascades implicated in endothelial apoptosis. Due to their antioxidant proper-
ties, diets supplemented with polyphenolic compounds, might also protect different
tissues against ischemic damage. Polyphenols reduce oxidative and nitrosative stress
leading to cellular death. All these effects of polyphenols might interfere with ath-
erosclerotic plaque development and stability, vascular thrombosis and occlusion and
they might therefore explain their vascular protective properties [35].
This review is focused on the cardiovascular beneficial effects of red wine polyphe-
nolic compounds, Provinols involving (in mg/g of dry powder) 480 proanthocyani-
dins, 61 total anthocyanins, 19 free anthocyanins, 38 catechin, 18 hydroxycinnamic
acids, 14 flavonols and 370 polymeric tannins.
Eects of Provinols on vasorelaxation
It was documented that Provinols elicited endothelium-dependent relaxation of rat fem-
oral artery by the Ca
2+
-induced increase of NO synthase activity and by protecting NO
O. Pechov & I. Berntov (2008) Trends in Pharmacological Research (Eds. V. Bauer et al.): 102108.
103 Vasoactive efects of Provinols in experimental hypertension
from degradation [6]. Because the action of red wine polyphenolic compounds has been
associated with the improvement of endothelium-dependent relaxation and elevation of
NO synthase activity and/or expression in several in vitro and in vivo experiments [7,8],
it may be assumed about possible therapeutic effect of Provinols in diseases associated
with reduced NO bioavailability such as endothelial dysfunction or atherosclerosis.
Zenebe et al. [6] provided the evidence that Provinols elicited endothelium-depen-
dent relaxation in rat femoral artery. The fact that the relaxation abolished by L-NAME
was restored by L-arginine confirmed the involvement of NO in the endothelium-de-
pendent vasorelaxation induced by Provinols. Determination of NO synthase activity
in the vascular tissue demonstrated that administration of Provinols at the concen-
tration of 10
9
to 10
4
mg/ml increased the activity dose-dependently. The maximal
activation of NO synthase was reached at the concentration of 10
4
mg/ml which cor-
related well with the maximal relaxation of the femoral artery induced by Provinols
at the concentration of 10
5
mg/ml.
Moreover, Provinols at the concentration producing the maximal endothelium-
dependent relaxation, restored the relaxation of the femoral artery to acetylcholine
abolished by superoxide

and enhanced partially the relaxant responses of sodium ni-
troprusside suggesting the ability of Provinols to preserve NO from degradation [6,8].
Similarly, red wine polyphenolic compounds caused a dose-dependent relaxation in
rabbit aorta with intact endothelium [9]. The authors documented that relaxation re-
sponses were abolished by L-NAME and were associated with an increase in cGMP
content. Since guanylate cyclase operates as an intracellular receptor for NO, it is possi-
ble that increased concentration of NO was responsible for the enhancement of cGMP
level, which was in agreement with the finding of increased NO synthase activity after
Provinols administration. The increase in intracellular concentration of Ca
2+
([Ca
2+
]
i) represents the critical step for the activation of NO synthase in the endothelial cells
leading to the production of NO and the subsequent endothelium-dependent vasore-
laxation. This increase in [Ca
2+
]i can be due to either an influx of extracellular Ca
2+
or
a release of Ca
2+
from intracellular stores. The relaxation produced by Provinols was
completely prevented in the presence of the Ca
2+
-entry blocker verapamil, suggesting
that the Ca
2+
influx to endothelial cells might be crucial for the relaxation ability of the
red wine polyphenolic compounds [6]. Analogically, Andriambeloson et al. [10] report-
ed that red wine polyphenolic compounds produced NO-dependent vasorelaxation of
rat aortic rings through an extracellular Ca
2+
-dependent mechanism. However, it can-
not be excluded that a release of Ca
2+
from intracellular stores might play a role in the
endothelial NO-dependent relaxation produced by polyphenolic compounds. Indeed,
after red wine polyphenolic compounds administration to the endothelial cell culture,
Martin et al. [11] documented an increase of [Ca
2+
]i from the intracellular stores, that
was sensitive to the phospholipase C inhibitor.
There are at least two mechanisms by which polyphenolic compounds could in-
fluence NO release: described stimulation of NO synthase activity and preservation
104 O. Pechov & I. Berntov
and/or stabilization of NO release under basal conditions. The later mechanism in-
cludes protection of NO from destruction by superoxide and other free radicals. The
antioxidant activity of polyphenols in red wine, grape, green and black tea had been
documented by their inhibitory effects on human low density lipoprotein oxidation
[12]. Provinols restored relaxation of the femoral artery to acetylcholine, which was
abolished by superoxide.

This finding clearly demonstrated that Provinols had an in
vitro antioxidative effect. Moreover, Provinols partially affected the concentration-
response curve for the NO donor sodium nitroprusside-induced relaxation in rings
without endothelium. Both effects were associated with decreased degradation of NO
resulting in the improvement of vasorelaxant responses [6].
Eects of Provinols on the prevention of hypertension
Oral administration of Provinols was able to produce a decrease in blood pressure in
normotensive rats. This hemodynamic effect was associated with enhanced endotheli-
um-dependent relaxation and induction of inducible NO synthase and cyclooxygenase
2 genes expressions within the arterial wall [13]. This effect probably involves NO path-
way since Provinols is able to produce ex vivo endothelium-dependent relaxation, as a
result of enhanced NO synthesis as is documented above.
Pechov et al. [8] provided evidence that oral administration of Provinols mark-
edly prevented the increase in blood pressure as well as structural and functional car-
diovascular changes in the left ventricle and aorta of rats subjected to chronic inhibition
of NO synthesis. Provinols reduced myocardial fibrosis, although it did not affect left
ventricular hypertrophy. In addition, Provinols prevented aortic thickening, attenu-
ated the increase in aortic reactivity to norepinephrine and prevented the decrease in
acetylcholine-induced endothelium-dependent relaxation during NO deficiency in rats.
These alterations were associated with the increased NO synthase activity, the moderate
increase in eNOS expression, and the reduction of oxidative stress, the factors that may
be responsible for the beneficial effect of the Provinols [8].
In general, hypertension is characterized by an increased oxidative stress in various
experimental models, including that produced by chronic inhibition of NO synthesis.
Reduction of hypertension-induced oxidative stress by Provinols could account for its
beneficial effects. Indeed, polyphenolic compounds contained in red wine have been re-
ported to mediate its antihypertensive effects since the reduction of urinary and plasma
values of malonaldehyde has been observed in spontaneously hypertensive rats treated
with polyphenols [14]. We have documented increased oxidative stress in the left ven-
tricle, aorta, brain and kidney of L-NAME-treated rats, resulting in an increase of con-
jugated dienes concentration [8,15]. This increase was partially or completely prevented
when Provinols was given simultaneously with L-NAME. These data strongly suggest
that reduced oxidative stress may contribute to the antihypertensive effect of Provinols
as well as to protection against cardiovascular remodeling in NO-deficient rats.
Chronic Provinols treatment alone or in a combination with L-NAME enhanced
the activity of NO synthase in the heart, aorta and femoral artery to a level higher than
that in the control rats. These data suggest that Provinols is also a potent activator of
NO synthase activity in the cardiovascular system under in vivo conditions [8]. The
moderate increase of eNOS expression by Provinols needs not be the only factor re-
sponsible for observed high levels of NO synthase activity. Recently, we have reported
that the endothelial NO production caused by Provinols was associated with the in-
crease in calcium signaling and the activation of tyrosine kinase pathway within the
endothelial cells [6,11]. Although the eNOS expression was greater in the left ventricle
and aorta from either L-NAME-treated or Provinols-treated rats, the simultaneous ad-
ministration of Provinols with L-NAME did not produce an additive effect on eNOS
expression. It is well accepted that NO by itself is able to elicit the regulation of the activ-
ity or expression of eNOS. The study of Wallerath et al. [16] provides the evidence that
polyphenols of French red wines increased the activity of the eNOS promoter, with the
essential trans-stimulated sequence being located in the proximal 326 base pairs of the
promoter sequence. The eNOS mRNA stability was also increased by red wine [16].
Previously, we and other workers have reported that chronic inhibition of NO syn-
thesis induced early vascular inflammatory changes as well as subsequent medial thick-
ening, vascular and myocardial fibrosis [17,18]. Interestingly, we found that Provinols
treatment partially prevented the increase in myocardial and aortic protein synthesis,
aortic thickening as well as myocardial fibrosis produced by chronic inhibition of NO
synthesis [8]. It is possible that these beneficial effects of Provinols could refer to the
reduced oxidant status and the increased production of NO as indicated by the increase
in NO synthase activity in both cardiac and aortic tissues. For the latter, the enhanced
NO production could contribute to the anti-inflammatory and anti-remodeling prop-
erties of Provinols in vivo.
Eects of Provinols on the developed hypertension
Berntov et al. [19] demonstrated that Provinols even accelerated the decrease in
blood pressure and the improvement of structural and functional cardiovascular
changes after the withdrawal of chronic inhibition of NO synthesis. Provinols treat-
ment enhanced the regression of aortic wall thickness and improved the decreased
endothelium-dependent relaxation in response to acetylcholine and the increased re-
activity of the aorta to norepinephrine in hypertension induced by L-NAME. In addi-
tion, Provinols markedly accelerated the decrease of myocardial fibrosis, although it
did not affect the left ventricular hypertrophy. All Provinols effects were associated
with a decrease in protein synthesis and with an increase of NO synthase activity in
the cardiovascular system of rats previously treated with L-NAME [19].
We have documented that the accelerated blood pressure decrease in Provinols-
treated rats was associated with an increase of NO synthase activity in the left ventricle
and the aorta compared with that of the spontaneous recovery group. In addition, the
increase of NO synthase activity occurred sooner in tissues taken from Provinols-
treated rats than in those from the spontaneous recovery group. In the light of these
findings, it seems that the effect of Provinols on the recovery from hypertension pro-
duced by chronic L-NAME treatment is due to enhanced in vivo NO production sub-
sequent to increased NO synthase activity in both cardiac and vascular tissues of the
rat [19].
As far as the consequences of hypertension are concerned, pressure overload leads
to cardiovascular remodeling comprising myocardial and vascular hypertrophy linked
to changes of the extracellular matrix compartment. An increased collagen deposition
frequently occurs, resulting in fibrosis that is associated with increased myocardial
and vascular stiffness and subsequent abnormalities of cardiac and vascular functions
[20]. It has been suggested that a major factor determining the progression of left ven-
tricular hypertrophy to heart failure is the presence of myocardial fibrosis. As reported
in our previous studies, chronic inhibition of NO synthesis resulted in hypertrophy of
the left ventricle associated with considerable myocardial fibrosis and increased aortic
wall thickness. Increased protein synthesis in both the left ventricle and the aorta was
also observed after L-NAME treatment as was mentioned above. We have found that
three weeks after the cessation of L-NAME treatment the reduction of blood pressure
toward the control value was associated with normalization of protein synthesis in
both the cardiac and aortic tissue without any improvement of cardiac fibrosis, left ven-
tricular hypertrophy, or aortic wall thickness. Thus three weeks of recovery were not
sufficient time to observe the full regression of cardiovascular remodeling, although
blood pressure returned to normal value. It is noteworthy that Provinols treatment
reversed cardiovascular remodeling including myocardial fibrosis, enhanced protein
synthesis and increased aortic wall thickness produced by chronic inhibition of NO
synthesis. The development of vascular remodeling with medial thickening observed
in this model of hypertension has been reported to be a consequence of the absence
of the anti-inflammatory and anti-arteriosclerotic effects of vascular endothelial NO.
The latter effect occurs via the inhibition of the activity of nuclear factor-B by NO.
It cannot be excluded that the inhibition of vascular smooth muscle cell proliferation
through the reduction of transcription factor expression might participate in the pro-
tective effect of Provinols [17,18].
The reduced endothelium-dependent vasodilatation seen in response to acetylcho-
line in aortas from L-NAME-treated rats was potentiated in the Provinols treated
group but not in the spontaneous recovery group [20]. A faster and greater increase of
NO synthase activity was also found in the aorta from the Provinols-treated group
compared with NO synthase activity in aorta from the spontaneous recovery group.
Improved endothelium-dependent vasodilatation is a potential mechanism by which
the ingestion of Provinols and other red wine polyphenolic compounds may reduce
cardiovascular risk.
Conclusions
The present review provide the evidence that Provinols is able to produce ex vivo en-
dothelium-dependent relaxation as a result of enhanced NO synthesis. Administration
of Provinols partially prevents the development of hypertension during NO deficiency.
The effects of Provinols include prevention of myocardial fibrosis, reduction of aortic
wall thickening and improvement of vascular functions. These functional and struc-
tural alterations were associated with significant augmentation of NO production, seen
as the increase of NO synthase activity and eNOS expression. Moreover, it has been
documented that Provinols modulated the oxidative stress in the cardiovascular sys-
tem during NO deficiency.
Oral administration of Provinols induces a faster and more profound decrease of
blood pressure in the recovery from hypertension induced by chronic inhibition of
NO synthesis. This effect of Provinols was associated with a regression in myocar-
dial fibrosis, although it did not reduce L-NAME-induced left ventricular hypertro-
phy. Most interestingly, Provinols treatment reversed the development of aortic wall
hyperplasia, improved endothelium-dependent relaxation, and reduced the increased
vascular reactivity to vasoconstrictor agonist. Improved endothelium-dependent va-
sodilatation and endothelium protective properties are the possible mechanisms, by
which the intake of Provinols and other red wine polyphenolic compounds may re-
duce cardiovascular risk. Thus, the beneficial effects of plant polyphenols in preven-
tion of hypertension may result from their complex influence on the NO balance in
the cardiovascular system.
Acknowledgement
The study was supported by the research grants VEGA 2/6148/26 and 2/7064/28 and
APVV-0586-06, APVV-0538-07, APVV-51-018-04.
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........,... .....
Substituted pyridoindoles as antioxidants
and aldose reductase inhibitors in
prevention of diabetic complications
A preclinical study in vitro and in an animal
model of experimental diabetes in vivo
Milan TEFEK, Paul O. DJOUBISSIE, Andrej GAJDOK, Alena GAJDOKOV, Mria JUSKOV,
udmila KRIANOV, Zuzana KYSEOV, Magdalna MJEKOV, Lucia RAKOV, Vladimr NIRC
Department of Biochemical Pharmacology, Institute of Experimental Pharmacology, Slovak Academy of Sciences,
Dbravsk cesta 9, 841 04 Bratislava, Slovak Republic, E-MAIL: milan.stefek@savba.sk
Key words: diabetes, aldose reductase, animal model, in vitro studies
Introduction
Chronic elevation of blood glucose in diabetes plays a critical role in the development
and progression of major diabetic complications. Prolonged exposure to elevated glu-
cose causes both acute reversible changes in cellular metabolism and long-term irre-
versible changes in stable macromolecules. The injurious effects of hyperglycemia are
characteristically observed in tissues that are not dependent on insulin for glucose entry
into the cell (e.g. eye lens, kidneys, peripheral and autonomic nerves) and, hence, are not
capable of down-regulating glucose transport along with the increase of extracellular
sugar concentrations.
Although multiple biochemical pathways are likely to be responsible for the patho-
genesis of diabetic complications, substantial evidence suggests a key role for non-enzy-
matic glyco-oxidation, oxidative stress and the polyol pathway [1]. Thus, glycation in-
hibitors, antioxidants and aldose reductase inhibitors represent a potential therapeutic
strategy for preventing the onset or the progression of these complications.
In this review we present the pyridoindole antioxidant stobadine, its structural ana-
logues with increased antioxidant activity and modified biological availability, as well
as structurally related carboxymethylated pyridoindoles with antioxidant and aldose
reductase inhibitory activities as prospective agents having a therapeutic potential in
prevention of long-term diabetic complications.
Review
M. tefek et al. (2008) Trends in Pharmacological Research (Eds. V. Bauer et al.): 109117.
110 M. tefek et al.
Stobadine and its structural analogues
Experimental glycation model in vitro
Under conditions of an experimental glycation model in vitro, stobadine significantly
inhibited glycation-related absorbance and fluorescence changes of bovine serum al-
bumin as well as the yield of 2,4-dinitrophenyl-hydrazine-reactive carbonyls with an
efficacy comparable to that of the reference antioxidants Trolox C and 2-keto-4-meth-
iolbutyric acid, and more efficiently than did the glycation inhibitor aminoguanidine.
Since stobadine did not affect the early steps of glycation measured as Amadori product
formation and the covalent binding of glucose, the observed protective effect may be
explained by the ability of the drug to eliminate free radical intermediates of glyco-
oxidation reactions, operative after the preceding glycation steps [24].
Rat model of experimental diabetes in vivo
Establishment of the model
Streptozotocin-induced diabetes in laboratory animals presents an experimental model
whose value is in the elucidation of causal relationships related to human diabetes mel-
litus. In rats, streptozotocin (STZ) induces diabetes when used at doses ranging from
45 to 70 mg/kg. At lower doses, STZ-induced diabetes is not stable, since spontaneous
recovery occurs. Long-term studies on pathological changes related to hyperglycemia
require a stable model of experimentally induced diabetes. Different strains of the same
animal species may differ in sensitivity to the diabetogenic effect of STZ. To estab-
lish the model of experimental diabetes at the Institute of Experimental Pharmacology,
long-term experiments were performed using several different doses of STZ ranging
from 40 to 70 mg/kg i.v. Data were collected on behavioral, functional, morphological
Figure 1. Stobadine and general chemical formula of its structural analogues.
111 Substituted pyridoindoles as antioxidants and aldose reductase inhibitors
and biochemical changes in relation to STZ dosing, showing that young male Wistar
rats, Breeding Facility Dobr Voda (Dv:WI), Slovakia, treated by a single i.v. STZ dose
of 50 or 60 mg/kg developed a persistent disease state characterized by severe hypergly-
cemia with major clinical signs of diabetes mellitus [57].
Stobadine and diabetic complications in experimental diabetes of rats
Cardiovascular system
Long-term treatment of diabetic animals with stobadine attenuated pathological chang-
es in the diabetic myocardium: reduced oxidative damage of myocardial tissue as mea-
sured by conjugated dienes [8], reversed myocardial levels of -tocopherol and coen-
zyme Q
9
near to control values [8,9], reduced elevated activity of superoxide dismutase
in the diabetic myocardium [8], and attenuated angiopathic and atherogenic processes
in the myocardium as assessed by electron microscopy examination [8]. Administration
of stobadine to diabetic rats normalized heart calcium homeostasis, while Ca
2+
-ATPase
was unaffected [10]. On the other side, function of cardiac Na
+
,K
+
-ATPase was dramati-
cally improved by stobadine treatment with regard to Na
+
-handling, thus enabling to
preserve its normal function in regulation of intracellular homeostasis of Na
+
and K
+

ions [11]. An 8-month supplementation of stobadine to diabetic rats resulted in the pro-
tection of aortic function as well as its ultrastructure [12].
Kidney
Long-term treatment of diabetic animals with stobadine significantly reduced total
proteinuria, albuminuria and enzymuria (N-acetyl-beta-D-glucosaminidase) [13], re-
duced oxidative damage of kidney tissue as shown by decreased conjugated diene level
[13], dramatically improved the function of renal Na,K-ATPase with regard to sodium
handling [14] and also with respect to utilization of ATP [15]. These beneficial effects of
stobadine on the diabetic kidney were supported also by histological findings [16].
Matrix collagen
The pepsin digests of tail tendons from streptozotocin-diabetic rats were found to con-
tain material that reacted rapidly at room temperature with p-dimethylaminobenzalde-
hyde (Ehrlichs reagent) to give an adduct with an absorbance spectrum characteristic
of the Ehrlich chromogen of pyrrolic nature determined in aging collagens. A signifi-
cant correlation of the Ehrlich adduct with tendon mechanical strength and collagen
fluorescence characteristic of advanced glycation endproducts was observed. The glyca-
tion inhibitor aminoguanidine significantly inhibited changes of all three parameters
evaluated. Treatment of diabetic animals with stobadine partially normalized tendon
mechanical strength, while the glycation-related fluorescence and Ehrlich chromogen
absorbance remained unaffected. The results suggest that apart from advanced glyca-
tion, additional mechanisms may participate in collagen cross-linking in diabetic con-
nective tissues, namely hyperglycemia-induced oxidative stress [7,13,17].
112 M. tefek et al.
Eye lens and retina
Long term treatment of diabetic animals with stobadine led to a marked delay in the
development of advanced stages of cataract and at the end of the experiment, the visual
cataract score was significantly decreased in the diabetic groups treated with stobadine.
Biochemical analyses of eye lens proteins showed significant diminution of sulfhydryl
groups and elevation of carbonyl groups in diabetic animals in comparison to healthy
controls. Dietary supplementation with stobadine did not influence the levels of these
biomarkers significantly. Nevertheless, in diabetic animals, stobadine supplementation
significantly attenuated plasma levels of malondialdehyde, an index of systemic oxida-
tive damage [18,19]. Stobadine treatement of diabetic animals significantly inhibited
development of retinal morphological abnormalities and lipid peroxidation even under
a poor glycemic control [20].
Peripheral nerves and brain
Treatment of diabetic animals with stobadine partially prevented decrease in conduc-
tion velocity of sciatic nerve measured in vitro. The protective effect was enhanced by
co-therapy with vitamin E. On the other hand, resistance to ischemic conduction fail-
ure, elevated in diabetic animals, was not affected by any of the drugs studied [21].
Experimentally diabetic rats in an 8-month chronic diabetes model showed signifi-
cant decrease in contractility of isolated vas deferens elicited by electrical field stimula-
tion, as well as significant increase in contractile response to exogenous noradrenaline
when compared with control rats. Administration of stobadine reverted both param-
eters towards control values. The results point to the ability of the antioxidant stobadine
to prevent degenerative changes seen in diabetic sympathetic nerves of vas deferens and
suggest antioxidants as therapeutic agents in reproductive system disability of male dia-
betics [22].
The effects of stobadine treatment on the activities of enzymes related with pen-
tose phosphate pathway and glutathione-dependent metabolism and some of the other
markers of oxidative stress in brain of diabetic rats were determined [23].
Leukocyte function
Stobadine was found effective in prevention of impairment of leukocyte free radical
release function in diabetic rats. The effect of stobadine was comparable with that of
vitamin E. Improvement of the neutrophil bactericidal function in diabetes may reduce
the incidence of clinical bacterial infections in diabetic patients [24].
Models of free radical pathology in vitro
Lipid peroxidation
Stobadine and its structural analogues [25] were found to act as potent scavengers
of peroxyl radicals both in aqueous and lipid phases, the antioxidant activity being
comparable with that of Trolox. Structural changes in the proximity of the indolic
nitrogen were found crucial for the radical scavenging efficiency: aromatization of
the pyridoindole skeleton in dehydrostobadine lowered the antioxidant activity, while
acetylation of the indolic nitrogen completely abolished the ability to scavenge per-
oxyl radicals. Moreover, the overall antioxidant activity of the compounds was af-
fected by their lipid phase availability and basicity. When stobadine and Trolox were
present simultaneously in liposomal incubations, Trolox spared stobadine in a dose-
dependent manner; a direct interaction of Trolox with stobadinyl radical appears to
be a plausible explanation with possible consequences for the antioxidant capacity of
stobadine under in vivo conditions, where recycling of stobadine by vitamin E might
occur [26,27].
Protein oxidation
On exposure to free radicals generated by the Fenton reaction system of Fe
2+
/EDTA/
H
2
O
2
/ascorbate, bovine serum albumin was losing its water solubility depending on
the concentration of the chelated iron. SDS-PAGE analysis proved the presence of dim-
ers and trimers of BSA, accompanied by enhanced bityrosine fluorescence. Stobadine
inhibited the process of albumin insolubilization, the protective effect being more effi-
cient than that of 2-keto-4-methiolbutyric acid (KMBA) and less effective than Trolox.
The inhibitory effect of the antioxiodants, expressed as IC
50
, correlated well with the
reciprocal values of corresponding second order rate constants for scavenging OH
rad-
icals [28].
In an attempt to model the processes of cataractogenesis, the soluble eye lens proteins
were exposed to peroxyl radicals generated in vitro by thermal decomposition of the
azoinitiator AAPH. The radical insult resulted in insolubilization of the lens proteins,
accompanied by the accumulation of free carbonyls, the diminution of sulfhydryls,
accumulation of high molecular weight cross-links and, to a lesser extent, fragments.
The processes of insolubilization and carbonyl formation were significantly inhibited
by stobadine and Trolox. On the other hand, sulfhydryl consumption was much less
sensitive to the antioxidants studied. The results point to a complex mechanism of per-
oxyl-radical-mediated modification of eye lens proteins with implications for cataract
development and indicate a potentially protective role of antioxidants [29].
SDS-PAGE profiles of eye lens proteins showed that both precipitation of soluble eye
lens proteins stressed by free radicals in vitro and progression of diabetic cataract in rats
in vivo were accompanied by significant protein cross-linking. There was a noticeable
contribution of disulfide bridges to protein cross-linking in diabetic eye lens in vivo. In
contrast, under in vitro conditions, when eye lens proteins were exposed to hydroxyl or
peroxyl radicals, the participation of reducible disulfide linkages in the formation of
high molecular products was markedly lower. These in vivoin vitro differences indicate
that the generally accepted role of reactive oxygen species in diabetic cataractogenesis
may be overestimated in connection with the processes of protein cross-linking [30].
114 M. tefek et al.
Carboxymethylated pyridoindoles
As shown above, involvement of oxidative stress and the polyol pathway in the etiol-
ogy of diabetic complications has been generally accepted. Based on the premise that
bifunctional compounds with joint antioxidant/aldose reductase inhibitory activities
could be multifactorially beneficial, new carboxymethylated pyridoindoles, structur-
ally based on stobadin were synthesized (Figure 2) and tested [31].
The novel carboxymethylated congeners of stobadine were characterized as uncom-
petitive inhibitors of aldose reductase, the first enzyme of the polyol pathway, with the
IC
50
values in a micromolar region for the more efficient tetrahydropyridoindole series.
A reasonable degree of selectivity with respect to the closely related aldehyde reductase
was recorded. The inhibitory mode, efficacy and selectivity were preserved even under
the conditions of prolonged STZ-induced experimental diabetes of rats. Antioxidant
action of the novel compounds was documented in a DPPH test and in a liposomal
membrane model, oxidatively stressed by peroxyl radicals. The presence of a basicity
center at the tertiary nitrogen, in addition to the acidic carboxylic function, predisposes
these compounds to form double charged zwitterionic species, characterized with a
maximal distribution ratio in 1-octanol/phosphate lying near the neutral physiological
pH. Indeed, by applying criteria of Lipinskis rule of five, good oral bioavailability of
the novel potential drugs was predicted [31,32].
A property critical for the efficacy of the novel aldose reductase inhibitors in vivo is
how well they penetrate into target tissues. The issue was addressed by measuring their
antioxidant activity in the cellular systems of intact erythrocytes exposed to peroxyl
radicals generated by thermal degradation of the azoinitaitor AAPH in vitro [33].
Figure 2. General chemical structure of carboxymethylated pyridoindoles related to stobadine.
Conclusion
The novel pyridoindoles, structural analogues of stobadine presented in this review, are
expected to have a therapeutic potential in prevention of long-term diabetic complica-
tions, with a prospect of further optimization that could lead to compounds of even
higher potency, ultimately aiming to improve the quality and length of life of diabetic
patients. We believe that it will also be possible to extend the obtained results and ap-
plications to other diseases or pathologies that share with diabetes the involvement of
oxidative stress and the polyol pathway.
Acknowledgement
This work was supported by VEGA Grants No. 2/6026/99, 2/2050/22, 2/5005/25, APVT
Grant No. 20-020802, APVV No. 51-017905, Centaur Pharmaceuticals and COST-B35.
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........,... .....
New pyridoindoles with antioxidant
and neuroprotective actions
Svorad TOLC
1
, Vladimr NIRC
1
, Alena GAJDOKOV
1
, Andrej GAJDOK
1
, Zdenka GSPROV
1
,
Oga ONDREJIKOV
1
, Ruena SOTNKOV
1
, rpd VIOLA
1
, Peter RAPTA
2
, Pavol JARIABKA
1
,
Inge SYNEKOVA
1
, Mria VAJDOV
1
, Soa ZACHAROVA
1
, Vendel NEMEK
1
, Viera KRCHNROV
1
1
Department of Neuropharmacology, Institute of Experimental Pharmacology, SASc., Dbravsk cesta 9,
841 04 Bratislava, Slovak Republic, E-MAIL: stolc@biont.sk
2
Faculty of Chemical and Food Technology, Slovak Technical University, Bratislava, Slovak Republic
Key words: acute head trauma, synaptic transmission, hippocampus, -adrenolytic activity,
acute toxicity, free radical scavengers, cyclic voltammetry, EPR, mouse, rat
Introduction
Numerous biological processes in living organisms are linked with the production of
reactive intermediates. According to the central reactive atom they are called reactive
nitrogen or reactive oxygen species (ROS). Molecules containing an unpaired electron
are free radicals (FR). Being highly reactive, they may easily impair biological molecules,
substantiating thus damage of tissues. ROS are created under physiological conditions,
their action is however limited by natural protective mechanisms. Under certain patho-
logical conditions, their production may be overexpressed or improperly counterbal-
anced by insufficient capacity of the protective systems [15]. Enhancement of antioxi-
dative /antiradical capacity of cells by supplying them with external compounds reveal-
ing suitable properties may contribute to limitation of ROS-induced damage [69].
A number of natural and synthetic compounds with antioxidant and/or antiradical
properties are known. They differ substantially in their affinity to specific ROS by li-
pophilicity, water solubility, side effects, toxicity, etc., which can remarkably determine
their biological effects. High lipophilicity turned out to be a limiting factor in use of
some synthetic compounds with antioxidant action. It was necessary to use rather com-
plicated procedures to prepare their water solutions (e.g. in lazaroids) suitable for i.v. use
[1011]. In spite of great effort in search of molecules with suitable pharmacodynamic
and pharmacokinetic properties, there is still lack of compounds effective enough for
antioxidative protection of biological tissues, suitable especially for medical emergencies
(stroke, infarction, brain trauma, transplantation procedures, tissue preservation, etc.).
Since the 1990s, an extensive search has been made for new compounds with antioxi-
dant and antiradical properties in the Institute of Experimental Pharmacology, Slovak
S. tolc et al. (2008) Trends in Pharmacological Research (Eds. V. Bauer et al.): 118136.
119 Pyridoindoles and neuroprotection
Academy of Sciences. The research was based on the pyridoindole stobadine, the mol-
ecule developed in the Institute, revealing remarkable antioxidant, radical scavenging,
and tissue protective properties. The substance was widely studied (for review see [12]).
A series of new stobadine derivatives with improved properties was projected, synthe-
sized, and studied since that.
This paper presents some systematic data obtained in investigating the new com-
pounds on their antioxidant properties, adrenolytic, and neuroprotective actions, as
well as selected data on their toxicity and antiradical effects. Some of the results have
been included in the Slovak Patent Pending [13].
Material and methods
Lipoperoxidation in rat brain homogenates
exposed to Fe
2+
/ascorbate system
Rat brain homogenates were prepared at 4
o
C in phosphate/HEPES buffer (pH 7.4) with
protease inhibitors (aprotinine, leupeptine, phenylmethylsufonyl fluoride, and pepsta-
tine). To 50 l of 10% homogenate, 10 l of FeSO
4
(5 mmol/l) and 5 l of ascorbic acid (50
mmol/l) were added and the volume was replenished to 500 l by potassium-phosphate
buffer (50 mmol/l). The mixture was incubated for 30 min at 37 C. The oxidation in-
duced was interrupted by 0.35 ml of trichloric acid (20% w/v) with 0.4% (v/v) of 2% (w/w)
ethanolic solution of butylated hydroxytoluene. After centrifugation (6,000 g/5min), the
supernatant was removed and 0.5 ml thiobarbituric acid solution (1.44 % w/v in 0.08
ml/l of NaOH) was added. The supernatant was incubated for 15 min at 80

C. After
cooling, absorption was measured at 534 nm and was considered the measure of lipid
peroxidation. Each drug was tested in at least three different concentrations. Negative
logarithms of middle inhibitory concentration in mol/l with estimate of its error (pIC
50
)
were calculated for each compound.
In vitro-induced oxidative damage of creatin
phosphokinase (CK) in rat brain homogenate
Rat brain homogenate was prepared as above and protein concentration was measured
by Lowrys method. The homogenate was diluted with HEPES buffer to obtain final pro-
tein concentration 1 mg/ml. To 1,940 l of the homogenate, 40 l of FeSO
4
(5 mmol/l)
and 20 l of ascorbic acid (50 mmol/l) were added. The homogenates were incubated
for 30 min at 37 C. The reaction was interrupted by deferroxamine (20 l of 1 mmol/l)
and by cooling to 0 C. The samples were centrifuged at 10,000 g/5 min at 5 C. Activity
of CK was assessed in the supernatant by Sigma Diagnostics Creatine Phosphokinase
Set No. 661. The drugs tested were added to the homogenate immediately before induc-
tion of oxidation in the final concentration of 30 mol/l. In parallel measurements,
the activity of stobadine was always measured. Protective antioxidant activity of each
compound was expressed relative to that of stobadine (=1).
120 S. tolc et al.
-adrenolytic activity
Male Wistar rats weighing 260280 g were killed by bleeding out of neck arteries. The tho-
racic aorta was dissected free and aortic rings were prepared by conventional technique.
The rings were fixed to an isometric tensometric apparatus in organ bath and incubated
in Krebs-bicarbonate solution (NaCl 118; KCl 4.7; KH
2
PO
4
1.2; MgSO
4
1.2; CaCl
2
2.5;
NaHCO
3
25; glucose 11 conc. in mmol/l) equilibrated with the mixture O
2
+ CO
2
(95
and 5%, respectively) at pH=7.4. After stabilization (60 min, 37 C), they were exposed
to the adrenergic agonist phenylephrine in rising concentrations (10
9
10
6
mol/l).
The maximal response attained was used as reference value (100%). After 30 min incu-
bation with the drug tested the procedure was repeated. Increasing drug concentrations
were used (10
7
10
5
mol/l) and EC
50
-s were determined. In case of competitive inhibi-
tion, the pA
2
value was calculated, in case of non-competitive antagonism, a decrease of
maximal contraction to phenylephrine was assessed in the presence of the antagonist.
Acute toxicity in mice
By the up-and-down method [14], oral, intraperitoneal, and intravenous acute toxici-
ties of selected compounds were measured in ICR mice (females, 2834 g b.w., 8 weeks
old). Each experimental group comprised 810 animals receiving the compound tested
in a constant volume (10 ml/kg). The dose of the drug administered was changed ac-
cording to the survival of the animal treated with the drug previously. If a particu-
lar animal survived for 24 hours, the higher dose was administered to the following
animal, and vice versa (progression factor typically 1.41.7). If the stop criterion was
reached, (i.e. if, /a/ three consecutive animals survived after receiving the upper limit
dose of 2,000 mg/kg, /b/ in six consecutively tested animals six turns occurred e.g.
survival-death-survival-death-survival-death, or /c/ after the first turn at least 4 ani-
mals followed and special probability ratio exceeded the critical value), the experiment
was finished and LD
50
values with fiducial limits were expressed [1517]. The clinical
stage of the animals, their behavior, changes in body weight and time of deaths were
registered. The overall observation period was 14 days. Delayed deaths did not oc-
cur in this study. Toxicity of the compounds was categorized according to Globally
Harmonized System [18].
Acute head trauma in mice
Acute head trauma (AHT) was induced in male mice (SWISS, 2326.5 g, breed Dobr
Voda) by defined mechanical insult [19]. The technique was slightly modified by using a
brief halothane anesthesia during AHT. The sensomotoric stage of the animals was as-
sessed by their capability to continue climbing on a horizontally stretched wire 1, 24, and
48 hours after AHT. It was expressed in sec (= Sensomotoric Score, SMS). Mortality of
the animals was also recorded. Only animals able to keep on the wire in the preliminary
test for at least 1 min were accepted to further tests. After AHT, SMS remarkably de-
creased (typically to 10 sec, 1 hr after AHT). The animals with SMS > 60 sec were assessed
separately. Along with each drug-tested group a separate placebo-treated group was al-
ways used (n = 5060, each). The drugs were administered i.v. within 1 min after AHT.
Stobadine (1 mg/kg i.v.) was used as reference compound. All other compounds were
administered in equimolar doses. SMS values in placebo groups were considered to be
100%. SMS higher than this value was interpreted as a protective effect of the given drug.
In separate experiments, brain wet weight was measured in animals up to 168 hours
after AHT, along with brain histopathologic examination. Moreover, brain samples tak-
en from animals five hours after AHT were assessed biochemically. Chemiluminiscence
in brain homogenates induced by FeSO
4
(2.5 mmol/l) was measured by Chronolog
Heawerton Lumiaggregometer (US) [20]. Basal malondialdehyde and total glutathione
levels were measured in the tissue samples according to [2122], respectively. Lactate
content was also determined (Randox kit, UK).
Synaptic transmission in rat hippocampal slices
The technique used was described in detail earlier [23]. Rat hippocampal slices (400 m)
were prepared by conventional technique with a tissue chopper. They were positioned
on a support monofile mesh separating water and gas phases in thermostatized in-
cubation chamber (36

C). The water phase consisted of artificial cerebrospinal fluid
(ACSF NaCl 124; KCl 3.3; KH
2
PO
4
1.25; MgSO
4
2.4; CaCl
2
2.5; NaHCO
3
26; glucose
10 conc. in mmol/l; pH 7.4), while the gas phase consisted of 95% O
2
+ 5% CO
2
. ACSF
was equilibrated with the same gas mixture. Both phases were continuously flowing
through the chamber. Their composition could be quickly changed. Bipolar wire elec-
trodes were used to stimulate Schffer collaterals evoking transsynaptically activity
in CA1 pyramidal neurons. Population spikes (PoS) were registered in the region by
glass microelectrode and stored in PC. Their amplitude induced by supramaximal
stimulation was considered to be the measure of efficiency of synaptic transmission.
After replacing O
2
in the gas mixture by N
2,
along with superfusion of the slices with
ACSF equilibrated with the oxygen-free gas mixture and with glucose diminished (4
mmol/l), the PoS were quickly fading. If the hypoxic conditions did not exceed critical
duration (less than 4 min under the given circumstances), the synaptic transmission
failure was reversible. However, if hypoxic conditions were applied for 6 min, the injury
resulted in irreversible transmission failure in most slices. The drugs tested were pres-
ent in the superfusing medium in suitable concentration throughout the whole experi-
ment. Recovery of PoS after 6 min hypoxia and 20 min reoxygenation was assessed.
Electrochemical, UV/Vis, and EPR/spin trapping investigations
Three stobadine derivatives (No. 137, 116, and 140) were studied and compared with
stobadine and trolox. The following methods were used: Cyclovoltammetric experi-
ments were performed with HEKA PG 284 (Germany) potentiostat under argon us-
ing a standard three-electrode arrangement. Standard ABTS and DPPH assays were
used for determination of total antioxidant capacity of compounds using UV/Vis/NIR
122 S. tolc et al.
Table 1. New hexahydro-1H-pyrido[4,3-b]indole derivatives.
drug code (alias) R8 R2 R6 R7 R9 R5 R3, (R3`) R1, (R1`) H4a,H9b
101 H H H H H H H H 4aR,9bS()-cis
102 H CH
3
H H H H H H 4aR,9bS()-cis
103 H OH H H H H H H ()-cis
104 H EtOC=O H H H H H H ()-cis
105 H H H H H H CH
3
, (CH
3
) CH
3
, (CH
3
) ()-cis
105 H H H H H H CH
3
, (CH
3
) CH
3
, (CH
3
) tetrahydro-1H-[4,3-b]indole
106 CH
3
H H H H H H H 4aR,9bS()-cis
107 CH
3
PhCH
2
CH
2
H H H H H H ()-cis
108 CH
3
PhCH
2
H H H H H H ()-cis
109 CH
3
EtOC=O H H H H H H ()-cis
110 CH
3
Ph(C=O)O H H H CH
3
C=O H H ()-cis
111 CH
3
H H H H H CH
3
, (CH
3
) CH
3
, (CH
3
) ()-cis
112 CH
3
H CH
3
H H H H H ()-cis
113 CH
3
PhC=O CH
3
H H H H H ()-cis
114 CH
3
2-Pyr-C=O CH
3
H H H H H ()-cis
115 CH
3
CH
3
OC=O CH
3
H H H H H ()-cis
116 (SM1M3EC2) CH
3
EtOC=O CH
3
H H H H H ()-cis
117 CH
3
PrOC=O CH
3
H H H H H ()-cis
118 CH
3
iPrOC=O CH
3
H H H H H ()-cis
119 CH
3
BuOC=O CH
3
H H H H H ()-cis
120 CH
3
iBuOC=O CH
3
H H H H H ()-cis
121 CH
3
PhCH
2
OC=O CH
3
H H H H H ()-cis
122 CH
3
4-iPr-PhNHC=O CH
3
H H H H H ()-cis
123 CH
3
tBuNHC=S CH
3
H H H H H ()-cis
124 CH
3
CH
3
NH
2
H H H H H ()-cis
125 CH
3
CH
3
N(CH
3
)
2
H H H H H ()-cis
126 CH
3
CH
3
NO
2
H H H H H ()-cis
127 CH
3
CH
3
Br H H H H H ()-cis
128 CH
3
CH
3
NH
2
H H CH
3
C=O H H ()-cis
129 CH
3
CH
3
N(CH
3
)
2
H H CH
3
C=O H H ()-cis
130 CH
3
EtOC=O H CH
3
H H H H ()-cis
131 OH H H H H H H H ()-cis
132 OH CH
3
H H H H H H ()-cis
133 (SMe1) CH
3
O H H H H H H H 4aR,9bS()-cis
134 CH
3
O CH
3
135 CH
3
O CH
3
C=O H H H H H H ()-cis
136 CH
3
O CH
3
OC=O H H H H H H ()-cis
137 (SMe1EC2) CH
3
O EtOC=O H H H H H H ()-cis
138 CH
3
O PrOC=O H H H H H H ()-cis
139 (SMe1iProC2) CH
3
O iPrOC=O H H H H H H ()-cis
140 (SMe1nBuoC2) CH
3
O BuOC=O H H H H H H ()-cis
141 (SMe1iBuoC2) CH
3
O iBuOC=O H H H H H H ()-cis
142 (SMe1BzoC2) CH
3
O PhCH
2
OC=O H H H H H H ()-cis
143 CH
3
O H CH
3
H H H H H ()-cis
144 CH
3
O EtOC=O CH
3 H H H H H ()-cis
145 CH
3
O PhCH
2
OC=O CH
3
H H H H H ()-cis
146 CH
3
O CH
3
OC=O Br CH
3
CH
3
H H H ()-cis
147 CH
3
O H H CH
3
CH
3
H H H ()-cis
148 CH
3
O CH
3
OC=O H CH
3
CH
3
H H H ()-cis
149 CH
3
O EtOC=O H CH
3
CH
3
H H H ()-cis
150 (SMe1M4M5nProC2) CH
3
O PrOC=O H CH
3
CH
3
H H H ()-cis
151 CH
3
O BuOC=O H CH
3
CH
3
H H H ()-cis
152 CH
3
O iBuOC=O H CH
3
CH
3
H H H ()-cis
153 CH
3
O PhCH
2
OC=O H CH
3
CH
3
H H H ()-cis
154 iPr EtOC=O H H H H H H ()-cis
155 Br EtOC=O CH
3
CH
3
H H H H ()-cis
156 Br PrOC=O CH
3
CH
3
H H H H ()-cis
157 Br PhCH
2
OC=O CH
3
CH
3
H H H H ()-cis
158 H H CH
3
CH
3
H H H H ()-cis
159 H CH
3
OC=O CH
3
CH
3
H H H H ()-cis
Table continued on next page
Shimadzu 3600 spectrometer (Japan). In EPR experiments with Bruker EMX spec-
trometer, the thermal decomposition of K
2
S
2
O
8
at 333 K was used as a source of reactive
radicals in the presence of DMPO spin trap.
Compounds tested
The pyridoindoles tested in this study were prepared by one of the authors of the study
(V..). Their chemical structures are shown in Table 1 with their code numbers and
eventual aliases. They were used mostly as mono- or di-hydrochlorides according to the
number of protonizable nitrogens in the molecule, if not otherwise indicated. All other
compounds were obtained from regular commercial sources.
Statistics
The standard Student t-test, ANOVA, Tukey-Kramer comparison test, linear regres-
sion analysis, and the exact test for 22 contingency tables were used in statistical as-
sessment of the data obtained ([24]; GraphPade Software 1994). Means with SEM are
indicated throughout the paper.
Abbreviations
PBN - phenyl-tert-butylnitrone,
SPBN - sodium salt of ortho-phenyl-tert-butylnitrone sulfonic acid,
DHM - 2,3-dihydromelatonin,
MP - methylprednisolone,
ABTS - 2,2-azino-bis(3-ethylenebenzthiazoline-6-sulphonic acid),
DPPH - 2,2-diphenyl1-picrylhydrazyl.
drug code (alias) R8 R2 R6 R7 R9 R5 R3, (R3`) R1, (R1`) H4a,H9b
160 H EtOC=O CH
3
CH
3
H H H H ()-cis
161 H PrOC=O CH
3
CH
3
H H H H ()-cis
162 H iPrOC=O CH
3
CH
3
H H H H ()-cis
163 H BuOC=O CH
3
CH
3
H H H H ()-cis
164 H iBuOC=O CH
3
CH
3
H H H H ()-cis
165 (SM3M4BzoC2) H PhCH
2
OC=O CH
3
CH
3
H H H H ()-cis
166 H H CH
3
H CH
3
H H H ()-cis
167 H CH
3
OC=O CH
3
H CH
3
H H H ()-cis
168 H EtOC=O CH
3
H CH
3
H H H ()-cis
169 H PrOC=O CH
3
H CH
3
H H H ()-cis
170 H iPrOC=O CH
3
H CH
3
H H H ()-cis
171 H BuOC=O CH
3
H CH
3
H H H ()-cis
172 H H H CH
3
CH
3
H H H ()-cis
173 H CH
3
OC=O H CH
3
CH
3
H H H ()-cis
174 H EtOC=O H CH
3
CH
3
H H H ()-cis
175 H PrOC=O H CH
3
CH
3
H H H ()-cis
176 H iPrOC=O H CH
3
CH
3
H H H ()-cis
177 H PhCH
2
OC=O H CH
3
CH
3
H H H ()-cis
178 CH
3
CH
3
H H H H H H ()-trans
stobadine (SM1M2) CH
3
CH
3
179 CH
3
O i-PrOC=O H CH
3
CH
3
H H H ()-cis
180 H BuOC=O CH
3
H CH
3
H H H ()-cis
181 H PhCH
2
OC=O CH
3
H CH
3
H H H ()-cis
182 H n-BuOC=O H CH
3
CH
3
H H H ()-cis
183 H i-BuOC=O H CH
3
CH
3
H H H ()-cis
124 S. tolc et al.
Figure 1. Negative logarithms of middle inhibitory concentration (pIC
50
) of compounds tested (mol/l), on
lipoperoxidation in rat brain homogenate induced by Fe
2+
/ascorbic acid system. Abscissa order No. identical to
Table 2. Means with error estimates are shown. Activity of stobadine (order No. 73) is marked by dark column.
Results
Lipid peroxidation
The capability of new pyridoindoles and some other selected compounds was mea-
sured in brain homogenate in the presence of Fe
2+
/ascorbate system. Negative loga-
rithms of middle inhibitory concentration (pIC
50
) determined in whole series are given
in Table 2. The compounds were ordered according to their anti-lipoperoxidation po-
tency. As shown in Figure 1, most of the new compounds revealed remarkably higher
inhibitory effect on lipoperoxidation than did stobadine, used in the study as reference
pyridoindole. The efficacy of some of the new compounds was surpassing that of sto-
badine by as much as 1.52 orders. Well-known antioxidants, such as PBN, SPBN, and
melatonin, showed under the given circumstances lower antioxidant efficacy than did
even stobadine.
Creatin phoshokinase (CK) oxidative impairment
The inhibitory effect of new pyridoindoles and some other compounds on oxidative im-
pairment of CK was tested in rat brain homogenate exposed to Fe
2+
/ascorbate system.
The results are shown in Table 2. The activity of stobadine was considered to be equal
1 and the activities of all other compounds tested were expressed relative to stobadine.
The proportion factor f >1 indicates higher potency than that of stobadine and vice
versa. As Figure 2 demonstrates, some of the new compounds were found to be more
Figure 2. Factor f expressing inhibitory action of a compound on oxidative impairment of creatine kinase in rat
brain homogenate exposed to Fe
2+
/ascorbic acid system relative to stobadine (dark column, f=1). Abscissa
order No. identical to Table 2 and Figure 1.
effective than stobadine also in this model of oxidative impairment of proteins. The
compounds are ordered in this Figure in the same way as in Figure 1, hence the data
regarding anti-lipoperoxidation and protein protection may be compared. Though the
oxidative system was the same in both models, activities of compounds were not com-
pletely identical. Nevertheless, the data indicate a general coherence in the two actions.
-adrenolytic activity
As shown previously, stobadine revealed hypotensive action, which was considered to
be an undesired side effect. It might be partially linked to its remarkable competitive
-adrenolytic potency. One of the aims in designing new stobadine derivatives was to
find molecules devoid of this action. Indeed, most of the new pyridoindoles did not pos-
sess this property (Table 3). Only four of all the new stobadine derivatives, namely the
compounds No. 106, 108, 127, and 132, had some competitive activity, though weaker
than that observed in stobadine. Moreover, in 10 other derivatives studied, only a weak
non-competitive -adrenergic activity was observed.
Acute toxicity of selected pyridoindole derivatives
Acute toxicity of 10 selected pyridoindoles including stobadine was tested in mice after
p.o., i.p., and i.v. administration. The LD
50
(mg/kg) were expressed along with respec-
tive GHS characterization (OECD 1998) (Table 4). All 9 stobadine derivatives tested
revealed remarkably lower acute toxicity than stobadine, regardless the way of admin-
istration. None of these compounds possessed any -adrenolytic action. It seems that
modification of the stobadine molecule aimed at obtaining new molecules with amelio-
rated acute toxicity was successful.
126 S. tolc et al.
Table 2. Inhibitory action of compounds tested; pIC
50
- negative logarithm of middle inhibitory concentration in
lipoperoxidation in rat brain homogenate induced by Fe
2+
/ascorbic acid system.
order
No. drug code pIC
50
error
est. f (STB eq)
order
No. drug code pIC
50
error
est. f (STB eq)
1 145 6.563 0.018 3.450 46 148 5.509 0.007 1.446
2 121 6.287 0.009 1.962 47 139 5.503 0.013 0.725
3 151 6.274 0.017 1.754 48 146 5.491 0.014 1.710
4 119 6.263 0.009 0.956 49 137 5.487 0.014 2.284
5 165 6.250 0.022 0.958 50 122 5.428 0.04 1.837
6 142 6.246 0.008 1.557 51 129 5.428 0.013 1.676
7 152 6.208 0.016 1.837 52 111 5.388 0.023 1.398
8 120 6.187 0.021 2.063 53 159 5.348 0.007 1.267
9 141 6.141 0.049 3.790 54 143 tartrate 5.328 0.011 2.740
10 107 6.126 0.033 2.011 55 136 5.282 0.037 2.609
11 171 6.105 0.060 56 144 5.120 0.013 1.095
12 150 6.098 0.026 1.465 57 147 5.059 0.010 0.835
13 117 6.084 0.032 1.129 58 166 4.969 0.023
14 182 6.077 0.016 59 133 4.939 0.008 1.573
15 183 6.057 0.012 60 133 tartrate 4.914 0.003 2.559
16 169 6.039 0.037 61 158 4.91 0.026 0.621
17 163 6.032 0.012 2.015 62 131 4.905 0.023 0.624
18 140 6.024 0.016 0.671 63 105 4.852 0.018 0.483
19 177 6.012 0.023 64 114 4.793 0.007 1.591
20 118 6.010 0.011 1.105 65 109 4.787 0.084 1.190
21 175 6.008 0.018 66 DHM 4.76 0.012 1.059
22 156 6.002 0.017 67 134 4.698 0.031 1.133
23 170 5.979 0.024 68 132 4.673 0.038 1.873
24 164 5.964 0.012 0.753 69 106 4.641 0.025 2.051
25 155 5.955 0.034 1.401 70 132 . HBr 4.578 0.028 1.325
26 154 5.949 0.025 2.280 71 104 4.575 0.022 1.159
27 130 5.948 0.015 72 135 4.543 0.011
28 157 5.944 0.001 73 stobadine 4.469 0.023 1
29 149 5.925 0.036 2.570 74 127 4.457 0.03 1.218
30 161 5.909 0.026 1.369 75 PBN 4.242 0.077 0.333
31 176 5.876 0.026 76 178 4.234 0.067
32 168 5.806 0.022 77 103 . HBr 4.203 0.068 0.793
33 116 5.804 0.029 2.426 78 101 4.172 0.010 0.190
34 123 5.799 0.017 1.347 79 trolox 3.930 0.017
35 113 5.796 0.019 1.449 80 102 3.778 0.004 0.094
36 138 5.760 0.008 1.283 81 103 . 1/2 H
2
SO
4
3.514 0.118 0.498
37 153 5.759 0.006 1.388 82 melatonin 2.510 0.053 0
38 162 5.756 0.005 2.072 83 126 1.684 0.533 0.338
39 174 5.731 0.017 84 32 1.201 0.984 0.206
40 108 5.710 0.019 2.296 85 110 1 0.119
41 125 5.620 0.009 1.416 86 128 1 0.591
42 160 5.609 0.009 0.507 87 5-acetyl-stobadine base 1 0.227 (0.046)
43 115 5.551 0.016 2.105 88 methyl-prednisolone noneff 0.010
44 173 5.521 0.018 89 SPBN prooxid. 0.165
45 124 5.509 0.08 1.783
Order No. corresponds to position of a particular compound in Figures 1 and 2. f (STB eq) is a factor expressing drug
potency relative to stobadine (f=1) in inhibition of creatine kinase oxidation impairment induced by Fe
2+
/ascorbic acid
system in rat brain homogenate.
Acute head trauma (AHT) in mice
Several of the pyridoindole derivatives tested were able to diminish significantly the
neurologic deficit (SMS) in mice subjected to standardized AHT. This action was evi-
dent mostly in the acute phase of the injury (1 hr following AHT). The results are sum-
marized in Table 5. For better comparison, changes in SMS are visualized in Figure 3. In
compounds No. 118, 133, 165, 145, 121, and 154, it was possible to recognize improvement
in SMS in comparison to the placebo-treated group even 24 hrs after AHT. With some
compounds the significant protective effect was observed even later (48 hrs, compounds
Table 4. Acute toxicity of stobadine and its selected derivatives in mice following single dose administration
p.o., i.p., and i.v.
Compound No. (alias)
LD
50
(mg/kg)
GHS p.o. i.p. i.v.
stobadine . 2HCl 323.68 164.44 63.16 4
116 (SM1M3EC2 . HCl) >2500.34 1909.85 102.09 5
133 (SMe1 . HCl) >2400.0 1588.55 122.46 5
137 (SMe1EC2 . HCl) >2400.0 1963.36 181.13 5
139 (SMe1iProC2 . HCl) >2300.0 737.90 131.83 5
140 (SMe1nBuoC2 . HCl) >2500.34 2177.70 57.28 5
141 (SMe1iBuoC2 . HCl) >2000.0 73.79 5
142 (SMe1Bzo2 . HCl) >2300.0 2500.34 85.31 5
150 (Me1M4M5nProc2 . HCl) >2000.0 2094.11 384.59 5
165 (SM3M4BzoC2 . HCl) >2000.0 1776.23 33.65 5
Toxicity was expressed as middle lethal dose (LD
50
). GSH toxicity category according to Globally Harmonized Hazard
System (OECD, 1998). 4 = "mild acute toxicity", 5 = "comparatively low acute toxicity"
Table 3. -adrenolytic action of new pyridoindoles assessed in rat thoracic aorta rings.
Compound code Type pA
2
MRD (%)* Compound code Type pA
2
MRD (%)*
stobadine C 7.26 129 n.e.
106 C 6.63 130 n.e.
108 C 6.63 131 n.e.
127 C 6.07 132 . HBr n.e.
132 C < 5 133 n.e.
124 NC 44.9 133 n.e.
142 NC 40.3 134 n.e.
138 NC 34.6 135 n.e.
177 NC 32.4 136 n.e.
101 NC 30.9 137 n.e.
150 NC 22.6 139 n.e.
165 NC 27.6** 140 n.e.
175 NC 22.1** 141 n.e.
161 NC 19.8** 143 tartrate n.e.
159 NC 14.4** 144 n.e.
102 n.e. 145 n.e.
103 base n.e. 147 . 2HCl n.e.
103 . 1/2 H
2
SO
4
n.e. 148 n.e.
104 n.e. 149 n.e.
105 n.e. 151 n.e.
107 n.e. 152 n.e.
109 n.e. 153 n.e.
110 n.e. 154 n.e.
111 n.e. 155 . HBr n.e.
113 n.e. 156 . HBr n.e.
113 n.e. 157 . HBr n.e.
114 n.e. 158 n.e.
115 n.e. 160 n.e.
116 n.e. 162 n.e.
117 n.e. 163 n.e.
118 n.e. 164 n.e.
119 n.e. 166 . 2HCl n.e.
119 n.e. 167 . 2HCl n.e.
120 n.e. 168 n.e.
121 n.e. 169 n.e.
122 n.e. 170 n.e.
123 n.e. 171 n.e.
125 n.e. 173 n.e.
125 . 3HCl n.e. 174 n.e.
126 n.e. 5-acetyl-stobadine n.e.
128 . 2HCl n.e. PBN n.e.
Competitive antagonism (C) is expressed by pA
2
values while non-competitive (NC) action is expressed as decrease in
maximal contraction induced by supramaximal concentrations of phenylepehrine. n.e. no effect. * maximal response
decrease (MRD) observed in presence of compound tested in conc. 10 mol/l. **only in the highest concentration of
the drug used.
128 S. tolc et al.
No. 108, 149). The neuroprotective activity of some of the new pyridoindoles equaled and
even remarkably surpassed the activity of some classical antioxidants (stobadine, me-
latonin, PBN, SPBN). Besides diminished sensomotoric impairment expressed as time
on wire in the time interval of 060 sec, there was an increase in the number of drug-
treated animals exposed to AHT with SMS >60 sec. An increase in the proportion of an-
imals with SMS >60 sec to those with SMS <60 sec was observed with some compounds
not only 1 hr after the trauma but also 24 and even 48 hrs later. The results of repeated
experiments with the same compound in different animals were also included in Table 5.
The action of compound No. 137 on AHT in mice was analyzed in greater detail in a
separate study. AHT induced a significant increase in brain wet weight, with the maxi-
mum occurring five hours after the injury (Figure 4) which could be ascribed to acute
brain edema confirmed by routine histopathology. A single dose of compound No. 137
(1.137 mg/kg i.v. within 1 min after AHT) eliminated the increase in brain wet weight
(Figure 4). Along with this, the incidence of subdural bleeding, brain parenchyma bleed-
ing, and bleeding into brain chambers was significantly reduced in the drug-treated
animals in the time period of 1168 hrs after head injury (Figure 5).
In brain homogenates of the animals sacrificed five hours after AHT, a significant
acceleration of FeSO
4
-induced lipoperoxidation assessed as chemiluminiscence was ob-
served. The latent period between inducing lipoperoxidation and the onset of chemi-
luminiscence was abbreviated from 301.50 25.97 sec in controls to 144.80 23.30 sec
in ATH. Single dose of comp. No. 137 fully prevented the decrease in the latent pe-
riod (301.20 29.66). Accordingly, a significant increase in the rate of chemiluminisce
(mV/min) after AHT in animals not given the drug was fully eliminated by the drug
treatment (controls; AHT and AHT+drug; 1.150 0.131; 1.573 0.070; 1.224 0.084, re-
spectively.) The concentration of malondialdehyde was significantly increased in the
brain of animals after AHT compared to controls (4.181 0.273; 3.167 0.238 nmol/mg
prot., respectively). In drug-treated animals, the MDA brain concentration was reduced
though decrease was not significant (3.803 0.325). The results correspond well with the
significantly decreased level of brain total glutathione level in traumatized animals com-
pared to controls (from 31.034 1.238 to 22.995 1.317 nmol/mg prot.). Administration
of compound No. 137 virtually fully prevented the injury-induced decrease in brain
total glutathione level GSx (31.137 2.820). Moreover, total lactate in mouse brain which
increased after AHT from the control value of 63.288 1.913 to 81.001 4.338 nmol/
mg prot. was also significantly eliminated by compound No. 137 (67.989 2.633). The
effect of compound No. 137 on AHT-induced changes of tissue lactate might indicate
involvement of its action in brain circulation. However, this requires further analysis.
Synaptic transmission in rat hippocampal slices
exposed to hypoxia/reoxygenation
Synaptic transmission in the hippocampal CA1 region can be monitored by popula-
tion spikes evoked in pyramidal neurons by stimulation of Schffer collaterals. The
Figure 3. Eect of new pyridoindoles and some selected antioxidants and/or neuroprotectants on sensomotoric
score (SMS) in mice 1 hour following acute trauma injury. Value of SMS in control groups parallel to drug-treated
groups was considered to be 100%. On abscissa the order No. of drugs tested identical to those in Tab. 5 are
shown. Action of stobadine (order No. 37) is identied by dark column. Protective action of any compound is
proprotional to value exceeding the response in non-treated groups (SMS=100%). Means with S.E.M. are shown.
Figure 4. Changes in brain wet
weight in mice subjected to acute
head trauma (AHT) in time period
1168 hours after AHT. Signicant
decrease in brain edema occurring
5 hours after AHT by single dose of
compound No. 137 (1.137 mg/kg
i.v.) administered within 1 min after
AHT is shown. Means with S.E.M. are
indicated.
Figure 5. Incidence of subdural
bleeding (SDB), brain intraparen-
chymal bleeding (IPB), and bleed-
ing into brain chambers (IChB) in
mice subjected to acute head trama
(AHT) 1-168 hours after AHT. Com-
parison in animals receiving single
dose of compound No. 137 (1.137
mg/kg i.v.) administered within 1
min after AHT with those treated
with placebo.
130 S. tolc et al.
Table 5. Sensomotoric score (SMS) 1 hr after acute head trauma in mice. C SMS in animals receiving i.v. placebo
(saline) was considered to be 100%.
order
No. Compound code C % SEM% D % SEM%
Q
1 hr 24 hrs 48 hrs
1 150 100.00 20.07 402.47 43.91 X
2 PBN 100.00 21.03 371.26 40.39
3 PBN (repeated test) 100.00 15.13 364.12 40.92
4 133 100.00 17.27 308.24 41.36 X X
5 165 100.00 22.42 269.19 29.16
6 142 100.00 35.38 262.85 42.69
7 118 /24 hrs 100.00 37.17 256.49 72.12
8 152 100.00 21.61 254.40 49.67
9 133 /24 hrs 100.00 32.74 253.42 70.52
10 145 100.00 20.58 250.72 35.80 surv X
11 165 /24 hrs 100.00 37.10 249.34 46.28
12 137 100.00 20.96 244.33 50.20 X
13 137 (repeated test) 100.00 17.55 238.22 33.29 X
14 149 100.00 21.13 235.22 34.21 X X X
15 149 /24 hrs 100.00 14.82 234.96 29.64
16 108 /48 hrs 100.00 31.76 234.86 62.30
17 124 100.00 23.30 229.05 39.18
18 121 /24 hrs 100.00 25.20 226.53 37.67
19 121 100.00 20.05 219.93 35.54
20 SPBN 100.00 18.45 217.93 25.91
21 162 100.00 20.16 216.74 31.21 X X
22 DHM 100.00 25.60 215.46 31.04
23 131 100.00 18.06 214.79 33.30
24 143 .tartrate 100.00 18.76 213.06 37.51 X X
25 153 100.00 16.37 211.19 29.67 X
26 127 100.00 25.28 208.15 38.95
27 134 100.00 18.92 203.73 26.38
28 142 /24 hrs 100.00 35.61 197.91 35.88
29 163 100.00 17.08 192.31 32.13 X
30 133 .tartrate 100.00 20.61 191.04 23.91
31 133 (repeated test) 100.00 20.10 188.10 25.56
32 151 100.00 17.77 182.13 31.22 X
33 155 100.00 12.86 181.81 19.90 X X
34 125 100.00 20.25 176.04 24.07
35 154 /24 hrs 100.00 21.15 166.54 24.44
36 melatonin 100.00 16.39 165.14 27.14
37 stobadine 100.00 21.89 164.32 40.32
38 105 100.00 29.52 160.53 33.00 X
39 149 /48 hrs 100.00 15.70 160.22 28.03
40 SPBN (repeated test) 100.00 19.04 159.00 23.22 X
41 154 100.00 20.14 158.65 28.05
42 5-acetylstobadine 100.00 20.49 150.06 17.94
43 155 /24 hrs 100.00 21.39 149.76 59.57
44 108 100.00 17.44 148.65 22.43
45 stobadine (repeated test) 100.00 27.80 141.84 19.26
46 143 .tartrate /24 hrs 100.00 26.73 141.07 32.78
47 118 100.00 23.84 140.90 28.88
48 107 100.00 25.00 139.89 29.11
49 102 100.00 24.32 132.34 26.78
50 MP 100.00 32.24 130.40 25.28
51 111 100.00 16.46 122.11 18.72
52 119 100.00 30.25 113.64 21.00
53 136 100.00 18.59 113.01 22.06
54 145 /24 hrs 100.00 20.29 105.30 24.04
55 153 /24 hrs 100.00 14.34 86.91 15.99 X
56 141 100.00 22.77 70.46 18.05
57 163 /48 hrs 100.00 26.16 68.21 16.90
58 155 /48 hrs 100.00 22.98 66.19 58.48
59 116 100.00 24.10 47.05 13.24
60 107 /24 hrs 100.00 22.56 24.95 7.10
D - SMS in animals treated with single dose of the compound tested in i.v. equimolar to 1 mg/kg of stobadine . 2HCl.
Placebo and compounds were administered within 1 min following trauma. Q - occurrence of significant enhancement
of number of animals with SMS >60 sec relative to those with SMS <60 sec in drug-treated and placebo-treated group
1, 24 and 48 hrs after head trauma. Symbol surv indicates significant increase in number of surviving against non-
surviving animals. Order No. corresponds to position of a particular compound in Figure 3.
neurotransmission is readily decaying during slice exposure to hypoxia/hypoglycemia
conditions within few minutes. If the exposure is long enough (typically six min under
our experimental conditions) the synaptic failure becomes irreversible. If however the
slices are exposed to a sufficient concentration of a drug revealing neuroprotective ac-
tion, the population spike may recover. Such protective activity was expressed as am-
plitude of population spike occurring after a 20-min reoxygenation period, with the
amplitude in the control period being considered 100%. Figure 6 shows the action of
comp. No. 137 in different concentrations on population spike recovery during the 20-
min reoxygenation period. The threshold concentration of the drug was equal or below
0.03 mol/l, reaching maximal protection effect (i.e. approx. 70%) in 0.1 mol/l. Under
the given conditions further increase in the drug concentration (up to 1 mol/l) did not
enhance the protective effect.
Electrochemical, US/VIS, and EPR/spin trapping studies
Experiments with stobadine derivatives No. 116, 137, 140 indicated formation of different
oxidation products closely depending on pyridoindole substitution and on the solvent
used. Oxidation products strongly contributed to the antioxidant and radical scavenging
capacity of the compounds. Complex redox behavior in the potential region 01 V was ob-
served in aqueous solutions compared to DMSO. One or two consecutive products were
observed for methoxy-type structures (comp. No. 137, 140). These redox peaks originat-
ed from the newly formed oxidation products with low redox potentials indicating the
ease of their oxidation and reduction. Standard ABTS and DPPH assays were used for
determination of total antioxidant capacity of samples. Compound No. 116 exhibited the
best hydrogen/electron donating antioxidant action with remarkably higher antioxidant
activity compared to stobadine. Additionally, all stobadine derivatives tested exhibited
higher radical scavenging activity compared to the most frequently used antioxidant
standard trolox. Methoxy-substituted derivatives tested in the EPR showed unusual ki-
netics and a strong elimination of hydroxyl radicals formed in the reaction mixture. The
findings confirmed a special role of the methoxy-substituent on the benzene ring con-
cerning the exceptional ability of these derivatives to scavenge reactive radical species.
Discussion
In accordance with our expectation, the present study showed that suitable modifica-
tion of stobadine resulted in molecules with remarkably higher antioxidant activity
than that observed in stobadine, the model compound. This was well documented by
protection of lipids and proteins exposed to oxidative conditions generated by the sys-
tem Fe
2+
/ascorbate. The results of electrochemical analysis (voltammetry) supported
these observations. The three stobadine derivatives tested revealed complex redox be-
havior in both non-aqueous and aqueous media, indicating complexity of oxidation of
the derivatives. Both ABTS and DPPH tests demonstrated an increase in antioxidant
132 S. tolc et al.
action in the new derivatives compared to stobadine, corresponding well with the tests
in brain homogenates. Moreover, EPR measurements exhibited a strong capacity espe-
cially of methoxy-substituted derivatives to eliminate hydroxyl radicals.
The findings are in good agreement with [25]. They demonstrated that some pyri-
doindoles from the same series were able to scavenge DPPH radical with higher efficacy
than stobadine. Based on SAR analysis, they have suggested that the sum of aromatic
substitution constants (
+
) and hydratation energy were important in enhancing the
radical scavenging property. Moreover, in some new derivatives, they demonstrated an
enhancement of inhibition of lipoperoxidation in dioleylphosphatidyl-choline (DOPC)
liposomes induced by thermal decomposition of 2,2-azobis(2-amidinopropane hydro-
chloride) (AAPH). They related this activity enhancement to an increased lipid-phase
availability, determined mostly by lipophilicity and basicity of the molecules.
The decrease in acute toxicity of the new compounds tested in this study compared to
stobadine might be interpreted in terms of apparently full elimination of -adrenolytic
potency. The fact that the decrease in toxicity was observed regardless the administra-
tion pathway, including injection into central compartment, seems to indicate that it
was not the decrease in bioavailability of the compounds that was responsible for the
decrease in acute toxicity. Further studies aimed specially at pharmacokinetics should
however be done.
Figure 6. Concentration dependence of eect of compoud No. 137 on recovery of population spike (PoS) in
CA1 pyramidal neurons in rat hippocampal slices exposed to reversible hypoxia/hypoglycemia for 6 min. PoS
was evoked by supramaximal stimulation of Scher collaterals and disappeared quickly during hypoxia. In
20-min reoxygenation period in control slices synaptic transmission recoverd to only about one tenth of the
control amplitude (ordinate). Presence of comp. No. 137 in superfusing medium in dependence on its concentra-
tion (shaded columns) signicantly reduced apparently irreversible impairment of synaptic transmission by the
hypoxic/reoxygenation conditions.
In previous studies, the neuroprotective effect of stobadine was demonstrated in
hypoxia/reoxygenation condition [2627, 23]. Yet some new stobadine derivatives with
enhanced antioxidant properties and devoid of -adrenolytic effect remarkably sur-
passed stobadine in this effect, as demonstrated both in in vivo and in vitro models.
The mouse model of AHT is representing a complex brain injury in vivo, which may
involve, among others, tissue edema, blood supply impairment, tissue bleeding, in-
flammation, as well as oxidative stress [28]. Rat hippocampal slices exposed to hy-
poxia/hypoglycemia may represent an in vitro model of ischemia /reperfusion-induced
impairment of brain parenchyma. In both models oxidative stress may participate.
The present study showed that compared to the pyridoindole stobadine, most of its
new derivatives tested were able to ameliorate more effectively neurologic deficits in
mice. The beneficial effect was noticed mostly 1 hour after AHT, in some cases even
later. All these compounds revealed also enhanced antioxidant action. The feasibility
of the relation of antioxidant and neuroprotective effects was supported by the find-
ing of accelerated chemiluminiscence induced in brain homogenates by chemically
triggered lipoperoxidation as well as by increased malondialdehyde concentration,
an indicator of lipoperoxidation, as observed in mouse brain five hours after AHT.
Correspondingly, the decrease in total GSx, a major factor determining tissue antioxi-
dant capacity, was decreased. These changes occurred along with the development of
brain edema and impairment of sensomotoric function and may indicate the presence
of oxidative stress. Accordingly, the new pyridoindole No. 137 with comparatively high
antioxidant activity ameliorated all the indicators studied, which might be interpreted
in terms of the action of the pyridoindole resulting in reduction of oxidative stress
induced by traumatic injury. Decrease in brain lactate might be linked to amelioration
of pathology on the level of brain circulation, which may, yet need not, be related to
oxidative stress.
As no data are available on tissue distribution, penetration across diffusion barri-
ers, tissue distribution, and bioavailability of the new compounds, a precise structure-
activity relationship could not be established. Nevertheless, it can be inferred from
the present findings in general that modification of the stobadine molecule resulting
in enhancement of its antioxidant potency occurs concurrently with enhancement of
neuroprotective action in the mouse AHT model.
Study of the action of compound No. 137 on rat hippocampal slices exposed to the
model of ischemia/reoxygenation (hypoxia/hypoglycemia) proved also the ability of the
compound to protect nervous tissue, similarly as observed with stobadine previously
[23]. However, comparison of the range of effective concentrations of the two com-
pounds (optimal concentrations No. 137 from 0.11 mol/l, stobadine 330 mol/l)
clearly indicated about 30 times higher efficacy of the new pyridoindole compared to
stobadine. That was in accordance with observations in the AHT test, as well as with
comparison of antioxidant potency. The high efficacy of compound No. 137 did not
lead, however, to higher reversibility of synaptic transmission during reoxygenation.
134 S. tolc et al.
The rat hippocampal slice model of hypoxia/reoxygenation induced injury is
less complex than the model of AHT, as circulation is not functional in the slices.
Nevertheless, the results obtained in hippocampal slices seem to support the hypoth-
esis that antioxidants may interfere with oxidative stress even in brain parenchyma it-
self. The coincidence of both the neuroprotective and antioxidant actions in some pyri-
doindoles might be indicative of a relation between the two properties. Nevertheless, a
number of other mechanisms, such as calcium entry inhibition, anti-glutamate action,
etc. can be neither proved nor excluded and require further extensive research.
Stobadine can exert a powerful potecting effect in rat endothelium exposed to
ischemia /reperfusion injury [29] as well as in experimental diabetes [30]. Moreover,
long-term oral administration of stobadine dipalmitate, SMe1 . 2HCl (No. 133) and
SMe1EC2 . HCl (No. 137) to rats with adjuvant arthritis reduced indicators of inflam-
mation [31]. As participation of oxidative stress is assumed in all these conditions,
protective actions of the compounds tested seem to support a general concept of use-
fulness of such compounds in tissue protection.
Conclusions
Antioxidant and antiradical properties of a series of new derivatives (n=82) of the
pyridoindole stobadine are described. Modification in stobadine structure result-
ed in remarkable enhancement of its capacity to inhibit lipoperoxidation and oxi-
dative injury of creatin phosphokinase in rat brain homogenates exposed to Fe
2+
/
ascorbate system. Antiradical efficacy was confirmed in ABTS and DPPH assays in
some of the derivatives and strong elimination of hydroxyl radicals by ESR was ob-
served. Compared to the model compound stobadine, decreased acute toxicity was
observed in all the 9 selected derivatives tested. This decrease was linked to success-
ful elimination of -adrenolytic activity revealed by stobadine, which is mostly re-
sponsible for its hypotensive effect. Neuroprotective action of numerous new deriva-
tives was observed in the mouse model of acute head trauma (AHT). Improvement
in neurologic deficit occurring mostly one hour after AHT was observed after single
i.v. administration of the compounds immediately after AHT. Besides neurologic
impairment, brain edema, decrease in brain total glutathione level, and increase in
Fe
2+
-induced chemilumiscence were observed after AHT, indicating participation of
oxidative stress. These indicators were diminished by one of the stobadine derivatives
tested (comp. No. 137). Neuroprotective action was confirmed in rat hippocampus slices
exposed to reversible 6-min hypoxia/hypoglycemia. Irreversible synaptic transmission
occurring under these conditions was significantly ameliorated in the presence of the
derivative No. 137, the effective concentration range being about 30 times lower than
that in stobadine. It was concluded that enhancement of antioxidant and antiradical
effect of stobadine by appropriate modification of its molecule resulted in concurrent
increase in neuroprotective activity. That was interpreted in terms of amelioration of
oxidative stress impairment in the given models of acute CNS. Moreover, appropriate
modification of the stobadine molecule resulted in decrease of -adrenolytic activity
along with a decrease in acute toxicity. The study is supporting the assumption that
enhancement of neuroprotective action may be, to some extent, related to enhancement
of antioxidant properties established in the series.
Acknowledgements
The study was supported by national grants APVV-51-020802 and APVV-51-017905
awarded by the Slovak Agency for Research and Development (APVV).
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........,... .....
Trends of research in pharmacology
Milo TICH, Pavel URBAN
National Institute of Public Health, Centre of Occupational Health, robrova 48,
10042 Praha 10, Czech Republic, E-MAIL: mtichy@szu.cz; purb@szu.cz
Key words: pharmacology, toxicology, chemical mixtures, predicitive methods, QSAR
Pharmacology and toxicology are relatives, although it can seem to somebody only ap-
parently. However, they are really relatives. The methodology is similar, sometimes may
be different but not in principle. In both cases the main problem is identical: to find in
which way and how chemicals act in organisms and how to change their structure to
fulfil their function: to cure and not to be toxic. The toxicology has taken over experi-
mental methods of pharmacology to treat both sides of chemicals, the good ones and
the bad ones. Both sciences call for quick, simple and economic procedures which make
it possible to predict adverse and toxic effects in advance before meet people as medical
drugs or house ware or through agriculture. Predictive toxicology has been originated
as a discipline.
The concept of the Centre of Occupational Health, as is called the institution nowa-
days former Institute of Industrial Hygiene and Occupational Medicine, was founded
by professor Teisinger. His idea and concept resided in a union of various professions,
professionals of which would be able to wish to solve complex cases of interactions be-
tween health of people and chemical industry, involving pharmacology and pharmacy
to cure occupational diseases. Clinical doctors, pharmacologists, physiologists, statisti-
cians, analytical chemists far to theoretical physicians or electric engineers were able to
meet in professor Teisinger institute, to find a common language and to look for a solu-
tion let us say of problem of silicosis (dust in mines), of exposure to industrial solvents
(general chemical manufacture) or of intoxication by lead (printing houses), mercury
(procedures using electrolysis) or by carbon disulfide (textile industry) or others. This
soup produced enormous results. Only in chemical section eg. development in a world
level and usage of quantum chemistry for predicting carcinogenicity of chemicals or
study on weak interactions vividly important for biological systems but being outsider
for scientists, hand made chromatograph for analysis of air in work places or analysis of
lead in blood samples by polarography, in that time new and modern method, thanks to
friendship with the right physical chemists. Xenobiochemistry was introduced directly
M. Tich & P. Urban(2008) Trends in Pharmacological Research (Eds. V. Bauer et al.): 137139.
138 M. Tich & P. Urban
from the laboratory where cytochrome P450 was discovered and, thus, accompanied
by important contacts. The discipline made it easier to study mechanisms of action of
chemicals. All these activities were accompanied by clinical cure of patients with oc-
cupational diseases.
This concept of professor Teisinger bore and bears fruits up to day. Professor Nosl,
the director of the Institute here in Bratislava asserted the same conception. Personages
from their institutes expanded to various scientific countries, new contacts on high
scientific levels were established, new ideas came, fruitful cooperations arised and natu-
rally new projects. We believe that regardless various discrepancies and confusions the
joint work of reasonable professionals will continue.
The activities of our Centre of Occupational Health support this believe. Beside the
work for Ministry of Health or other state administration the Centre is devoted to re-
search, too. The studies on metal intoxications and on mobilizing effect of different
new chelating agents continue both on experimental level and in subjects occupation-
ally exposed to mercury, lead and aluminum, protective effect of bis-dithiocarbamates
against subacute lethal radiotoxicity of polonium was proved. Knowledge on heterolo-
gous expression and characterization of human cytochrom P450 2A6, identification of
the importance of genetic polymorphism in biotransformation enzymes and NBS1 gene
in development and progression of lymphomas and breast cancer and others was gained
in laboratories of biotransformation. Integrated alternative methods for the determina-
tion of indices of toxicity, including both low organisms or organ cells and computers
with QSAR techniques are developed and work in predictive toxicology laboratory. The
relative neurotropic potency of common industrial solvents related to their toxicoki-
netic characteristics has been experimentally determined.
The Centre participated in IPCS validation study on a basic set of neurobehavioral
screening methods and in development of advanced neurotoxicological techniques.
Methods of early detection of neurotoxic effects of chemicals have been tested and im-
plemented such as EEG, evoked potentials, nerve conduction velocity studies or the
Lanthony test of colour discrimination. In the field of biological monitoring, new meth-
ods were described based on the determination of adducts of reactive chemicals with
blood proteins. Certified reference materials from human urine for creatinine, stress
indicators and aromatic hydrocarbon metabolites for quality control programs are suc-
cessfully produced.
Following the tradition of studies on silicosis, asbestos-related problems have been
newly studied, namely the specification of the type of ventilation disorder and the as-
sessment of the role of CT scan (computerized tomography) in diagnosis of pleural
hyalinosis caused by asbestos. Biological monitoring and health risk of exposure to
methylene-4,4-diphenyldiisocyanate and a survey of the health status of apprentices
exposed to bronchtropic noxae have been performed.
Just for information on the activities which are not so closed to the methodology but
we see them generally important and usable for pharmacological studies (or pharma-
139 Trends of research in pharmacology
ceutical), eventually, too. The Centre is operating three major information systems re-
lating to occupational health: System of Categorization of Work Operations, National
Registry of Occupational Diseases and REGEX, containing workers occupationally ex-
posed to chemical carcinogens.
Returning to toxicological and pharmacological items we are returning to predictive
methods. The scientific community is worried by the fact that we know pharmaco-
logical and toxicological activities of individual chemicals but not if they are in mix-
tures. Especially pharmacologists are aware decades of different activities if a drug acts
alone or in mixture or what is forbidden to eat or to drink if a drug is prescribed. We
are measuring acute toxicity of binary chemical mixtures of different both qualitative
and quantitative composition. Graphical presentation by Raoult from physical chem-
istry is used to visualize the dependence of an activity on molar fraction of a mixture.
Examples, how the toxicity of chemicals is changed with changing molar fraction of a
mixture, are collected. The data collected serve for further analysis by the QSAR tech-
niques. The question is: what physicochemical properties are critical for the fact that the
biological activity of chemicals in mixtures is changed by this and not by that way.
........,... .....
Trends in developmental toxicology
Protection of the developing organism
an ever topical issue
Eduard UJHZY, Mojmr MACH, Jana NAVAROV, Alena GAJDOKOV,
Andrej GAJDOK, Jozef JANK, Viera DYTRICHOV, Michal DUBOVICK
Department of Reproductive Toxicology, Institute of Experimental Pharmacology, Slovak Academy of Sciences,
Dbravsk cesta 9, 841 04 Bratislava, Slovak Republic, E-MAIL: eduard.ujhazy@savba.sk
Key words: developmental toxicity, teratology, neurobehavioral teratology, animal models
Introduction
The aim of developmental toxicology is to detect any adverse effects of chemicals/drugs
on the pregnant female and on the development of the embryo and fetus [1,2]. The
main manifestations of developmental toxicity include: embryolethality, malformation,
growth retardation and functional impairment.
The thalidomide tragedy in the early 1960s alarmed the medical profession about
the dangers to the unborn child exposed to drugs in utero. This drug was widely used
for treatment of nausea and vomiting in pregnant women. In Germany, more than
7,000 children of mothers treated with thalidomide were born with serious congenital
malformations manifested by amelia and focomelia [3]. Thalidomide provided an al-
most perfect example, very well documented the causal relationships between a specific
teratogen and human congenital malformations. This tragedy stimulated an intense
research in the etiology, prevention and treatment of congenital malformations.
Reproductive and developmental toxicity studies are divided into two categories ac-
cording to the type of human exposure. The first are three-segment-design studies for re-
productive and developmental toxicity of drugs. The other category includes multigener-
ation reproduction studies according to the new chemical substance control act. In these
test methods, gametogenesis, estrus cycle, mating behavior, ovulation (luteinization),
fertilization, implantation, embryogenesis in the early gestation period, fetal growth in
the late gestation period, embryonic/fetal death, developmental retardation, teratogen-
esis, parturition, weaning, retardation of postnatal growth and functional development
have been used as reproductive and developmental parameters of chemicals/drugs [4].
We would like to dedicate this paper the memory of Dr. Tatiana Balonov
E. Ujhzy et al. (2008) Trends in Pharmacological Research (Eds. V. Bauer et al.): 140150.
141 Protection of the developing organism an ever topical issue
In the Institute of Experimental Pharmacology SASc., Bratislava, the history of ex-
perimental teratology began in the 1970s, when this discipline was incorporated into the
Department of Toxicology, headed by Dr. Ladislav Vrbovsk, CSc. Dr. Tatiana Balonov
is considered to be a founder of reproductive and teratologic studies at the Institute. The
first experimental studies dealt with possible teratogenic and embryotoxic effects of the
glycoprotein isolated from Candida albicans and its fractions, conducted on mice and
rats [57]. In cooperation with the Faculty of Natural Sciences, Comenius University,
Bratislava, the synthetic bioanalogues of juvenile hormone of insects, Altosid, was test-
ed for its teratogenicity on rats [8]. The cytostatic drug cyclophosphamide was evalu-
ated for its known teratogenic potential in mice and rabbits as a model drug, a positive
control to verify the correctness of methods and procedures used in the Laboratory
[910]. Further, in light of the aims of Slovakofarma, n.p. Hlohovec, Slovakia, we con-
ducted teratogenicity studies of the psychoactive agent lithium carbonate on mice [11],
hypolipidemic agent etofylline clofibrate (VULM), of fenofibrate [12], beta-adrenolytic
agent VULM 111 (exaprolol) [1314], of saponine beta-aescine [15], and of the ACAT
inhibitor VULM 1457 [16]. In cooperation with the Drug Research Institute (VULM),
Modra, Slovakia, the effect of the calcium channel blocker VULM 993 was investigated
in rats [17] and of the herbicide 4-chloro-2-methylphenoxyacetic acid (MCPA) in rab-
bits [18]. Further substances were tested at the Institute, such as local anesthetics (pen-
tacaine, oxetacaine) [19] or the antihistamines pipethiadene, pizotifen maleate [20] and
bromadryl in rats [21].
The pyridoindole derivative stobadine (STO), a neuro- and cardioprotective sub-
stance with high antioxidant properties, was found promising for long term admin-
istration during diseases accompanied with excessive oxidative stress and free radical
formation [22]. Therefore it was subjected to extensive toxicological and teratological
studies in different animal species. The chronic oral toxicity study of STO carried out
along with micronucleus assay in rats did not reveal any evidence of toxic or genotoxic
effects [23]. No signs of teratogenicity were observed in mice [24], rats [2530] and chick
embryos [3132]. Kritofov et al. [33] also observed transplacental transport of
[3H]
STO
across the rat placenta. The distribution of
[3H]
STO in rabbits on gestational days 20 and
27 was determined in maternal and fetal organs after oral administration in a single
dose of 5 mg/kg. During this late period of gestation, the fetal organs, especially the
brain and heart, were saturated with STO and thus in case of oxidative stress STO could
protect these vital organs [34].
At the beginning of the 1990s, the use of Segment I and II methods (one generation
reproduction toxicity tests) [35] was extended to Segment III design (pre- and postnatal
toxicity) complemented by neurobehavioral development evaluation up to adulthood
(neurobehavioral teratology) [30]. Moreover, behavioral toxicology screening tests were
conducted in adult rats of both genders [2830].
At present, the newly formed Department of Reproductive Toxicology is concerned
with hypoxia/ischemia associated with oxidative stress during developmental stages of
142 E. Ujhzy et al.
rats. Pharmacologically induced chronic intrauterine hypoxia and the model of neona-
tal anoxia were introduced to study mechanisms of development of hypoxia/ischemia
injuries [3638]. Further, the potential pharmacological intervention by using natural
and synthetic antioxidants in maternal and embryofetal disturbation caused by devel-
opmental hypoxia/ischemia has been investigated [3940].
Methods
This section presents experimental approaches and methods used in our Department.
These tests represent general principles of developmental toxicity evaluation along with
experimental approaches of basic research.
Teratology studies (Segment II)
The substance tested is administered to pregnant rats during organogenesis (in rats and
mice from day 6 to day 15 of gestation, in rabbits from day 6 to day 20 of gestation). In
rats on day 20 of gestation, in rabbits on day 29 of gestation, the females are sacrificed
and uterine contents are inspected. All live fetuses are examined for external, skeletal
and visceral malformations [35].
Prenatal toxicity studies (Segment III) Neurobehavioral development
This methodological approach is based on exposure of the developing organism to the
substance tested and/or to a physical factor during the sensitive time window of brain
development (perinatal period day 15 of gestation up to day 21 post partum in rats).
Pregnant females are allowed to give birth spontaneously. Newborn pups are evaluated
for their neurobehavioral development from birth until adulthood (maximally up to 6
months). There are batteries of specialized tests based on ethological analysis of animal
behavior. Recommended evaluation concerns somatic growth and maturation, neu-
romotor and reflex development, sensory functions, activity and emotionality levels,
memory and learning [30,37,41].
Methods of developmental toxicology testing a valuable tool to study
hypoxia/ischemia induced changes in prenatal and early postnatal period
On using this type of methods, we are able to study embryofetal and neurobehavioral
alterations due to hypoxia/ischemia acting during the prenatal and/or early postna-
tal period. Several approaches were proposed by researchers to investigate hypoxia/
ischemia-induced injuries during development. In the following section we present our
most used models of developmental hypoxia/ischemia.
Chronic intrauterine hypoxia induced by phenytoin (PHT)
One of the pharmacological approaches to induce developmental oxidative stress is ad-
ministration of the anticonvulsant PHT during pregnancy in laboratory animals. PHT
teratogenicity is mainly initiated by adverse pharmacological action on the embryonic
heart during a sensitive stage of development, resulting in embryonic hypoxia/isch-
emia [42]. Maternal hemodynamic alterations may contribute to embryonic hypoxia,
but these alterations are not of a magnitude which alone could explain the observed
hypoxia-related malformations. Embryonic hypoxia has been associated with specific
pathological changes, such as vascular disruption, hemorrhage, and finally tissue ne-
crosis of embryonic tissues [43]. Tissue necrosis, manifested as malformations in the
fetus at term, may be a direct consequence of hypoxia and/or of reactive oxygen species
(ROS) generation at reoxygenation.
Neonatal anoxia
A different approach to study hypoxia complications during sensitive developmental
stages is exposure of newborn pups (1- or 2-day-old) to an oxygen-free environment
(100% nitrogen content in a glass chamber). After the anoxic insult, all pups are re-
placed to their mothers. The surviving pups are investigated for neurobehavioral devel-
opment [37,44].

Non-sophisticated model of perinatal asphyxia
This approach is a model of perinatal asphyxia in humans. Pregnant rats are sacrificed
on day 20 of gestation. The uteruses are placed into 37C water bath for 1020 min.
After the anoxic insult, the pups are resuscitated and the surviving pups are adopted by
foster mothers. After that the neurobehavioral development of pups until adulthood is
investigated [4546].
These experimental models provide great advantages for studying effects of agents, con-
ditions, and new drugs which could ameliorate detrimental effects of hypoxia/ischemia.
In no other field of medicine is the therapeutic risk higher than in the treatment of
pregnant women. While in adults most of the unexpected side effects of drugs are re-
versible, they may be irreversible for the embryo and can lead to abnormalities in the
newborn [47]. The current methodology used in developmental toxicology is derived
from basic teratology research. We are able to detect the embryotoxic and teratogenic
potential of the majority of substances, identify the beginning of the embryotoxic re-
gion regarding the dose, and to determine the relationship between dose and effect
[1,48]. Table 1 shows the most important results from reproductive studies conducted at
the Department during almost 30 years.
In the periods of the 1970s and 1980s, standard reproductive and developmental tox-
icity studies of new chemical entities were conducted. It is highly relevant to know the
fate of the drug both in the maternal body and developing embryo and fetus, and also to
144 E. Ujhzy et al.
Table 1. List of selected drugs/substances tested in the Laboratory of Reproductive Toxicology (19772006).
Tested
substance/drug Animal Dosage Results/developmental toxicity Ref.
Candida albicans
glycoprotein
rats
p.o., 10 and 100 mg/kg,
416 GD
No embryotoxic and teratogenic effect [5]
rats
i.v., 15, 30 and 60 mg/kg,
8 and 13 GD
Candida albicans
glycoprotein with
enriched protein fraction
rats
i.v., 30 and 60 mg/kg,
8 and 13 GD
Embryotoxic effect, skull and rib anomalies
Embryotoxic effect
[6,7]
Altosid rats
p.o., 500 mg/kg,
8 GD
Growth retardation of the pair 13 of ribs [8]
Cyclophosphamide
mice
i.m., 6.440.8 mg/kg,
1115 GD
Embryotoxic and teratogenic effect (gross malfor-
mations of head, extremities and caudum, skeletal
anomalies such as synostosis, retardation
of ossification)
[9]
rabbits
p.o., 6.2 and 18.6 mg/kg,
620 GD
Embryotoxic effect (dose-dependent decrease of
fetal organs as well as placental weight), highest
dose teratogenic effect (exophthalmia, cleft palate/
lip, syndactylism and brachycaudia)
[10]
Lithium carbonate mice
p.o., 86, 150, 265, 465 and
665 mg/kg,
115 GD
The two highest doses had maternal and embryofe-
tal effect (increased mortality of dams and fetuses,
increased resorptions)
[11]
Etofylline clofibrate mice
p.o., 11.7, 117.1 and 585.5
mg/kg,
716 GD
The low and middle doses had no adverse effect,
the highest dose had embryotoxic effect (decreased
fetal weight, increased postimplantation loss)
[12]
Fenofibrate mice
p.o., 11.7, 117.1
and 585.5 mg/kg,
716 GD
The low and middle doses had no adverse effect,
the highest dose had embryotoxic effect (decreased
fetal weight, increased postimplantation loss)
[12]
Exaprolol
(VULM 111)
mice,
rats
p.o., 5, 10 and 20 mg/kg,
416 GD
Skeletal anomalies, more pronounced effect in rats [13]
chicken
embryo
injection to egg, 0.4, 0.8
and 1.6 mg/egg,
57 days of incubation
Growth stimulating effect on organs and skeleton [14]
Beta-aescine mice
p.o., 0.36, 3.6
and 36 mg/kg,
716 GD
No embryotoxic effect, the middle and highest
doses increased incidence of skeletal anomalies
(skull and sternebrae)
[15]
ACAT inhibitor
(VULM 1457)
rats
p.o., 30, 120
and 300 mg/kg,
615 GD
No embryotoxic and teratogenic effects [16]
Calcium antagonist
(VULM 993)
rats
p.o., 5, 50 and 250 mg/kg,
615 GD
MCPA herbicide rabbits
p.o., 5, 10 and 25 mg/kg,
627 GD
Pentacaine rabbits
p.o., 1, 10 and 50 mg/kg,
620 GD
Oxetacaine rabbits
p.o., 20 mg/kg,
620 GD
Bromadryl rats
p.o., 1, 5 and 10 mg/kg,
219 GD
Embryotoxic effect (decreased fetal and placental
weight), no teratogenic effect
[21]
Pipethiadene
mice
p.o., 0.24, 0.6
and 1.2 mg/kg,
416 GD
Embryotoxic effect (decreased fetal weight),
no teratogenic effect
[20]
Pizotifen maleate
mice
p.o., 0.24, 0.6
and 1.2 mg/kg,
416 GD
Embryotoxic effect (decreased fetal weight),
no teratogenic effect
[20]
Tested
substance/drug Animal Dosage Results/developmental toxicity Ref.
Quinidine
chicken
embryo
and 1.6 mg/egg,
Stobadine
chicken
embryo
and 1.6 mg/egg,
chicken
embryo
in culture
medium
10
3
10
8
mol/l, chick
blastoderms at stages
45 HH
No adverse effects on early chick embryogenesis [32]
mice
1, 3, 6, 9 and 12 GD
Fetotoxic effect (decreased fetal weight), no terato-
genic effect
[24]
mice
p.o., 12.2, 61
and 122 mg/kg,
416 GD
The middle dose caused reduction of implantation,
live fetuses and litter weight, the highest decreased
fetal weight, no teratogenic effect
rats
p.o., 5, 15 and 50 mg/kg,
males: 70 days before
mating, females: 14 days
before mating and during
gestation and lactation
No adverse effects on fertility, survival rate, and
weight gain of parental aninals, or on prenatal and
postnatal development of pups
[25]
rats
p.o., 5, 15 and 50 mg/kg,
416 GD
No embryotoxic and teratogenic effect
rats
p.o., 5, 15 and 50 mg/kg,
15 GD21 PP
No adverse effect on reproductive parametres of
dams, on survival and development of offspring
rats
p.o., 50 mg/kg,
615, 620 GD
No overt effects on dams, embryofetal develop-
ment or reproductive parameters
[28]
rats
p.o., 5, 15 and 50 mg/kg,
6 GD21 PP
No adverse effects on course of pregnancy, lacta-
tion and neurobehavioral development
[30]
rats
p.o., 50 mg/kg,
14 days before mating
to 21 PP
Subtle alterations of exploratory behavior [29]
rats
p.o., 5, 15 and 50 mg/kg,
621 PP
No adverse effect on pre- and postnatal develop-
ment of offspring
[27]
rats
3, 6, 9 and 12 GD
Embryotoxic effect (decreased fetal weight), no
teratogenic effect
[26]
rats
p.o., 5, 15 and 45 mg/kg,
215 GD
Maternal toxicity, embryotoxic effect (increased
preimplantation loss, decreased fetal weight), no
teratogenic effect
rats
p.o., [
3
H] STO, 5 mg/kg,
20 GD
STO crosses placental barrier; higher concentration
STO in the placental and fetal tissue compared to
maternal plasma
[33]
rabbits
p.o., [
3
H] STO, 5 mg/kg,
20 and 27 GD
STO crosses placental barrier; its concentrations
were higher in fetal plasma compared to values
found in mothers; the highest concentrations were
found in the brain and heart
[34]
rats
p.o., [
3
H] STO, 5 mg/kg,
10 PP
STO crosses into milk; however, only 0.39% of the
total radioactivity administered to the lactating rats
were recovered in the pups
[49]
Phenytoin rats
p.o., 150 mg/kg,
219 GD
Maternal, embryofetal and neurobehavioral toxicity [36]
GD gestational day, p.o. oral administration, i.v. intravenous administration,
HH Hamburger-Hamilton stage in chicken embryo, PP post partum
146 E. Ujhzy et al.
find out whether the drug crosses biological barriers (placenta, blood brain barrier) and
passes into the milk of lactating mothers. We performed series of studies using various
biomodels to detect placental passage of STO and its distribution in individual organs of
the mother and fetus [34], as well as its transition to the milk of lactating rat mothers [49].
In the 1990s, we began to apply a more complex approach in studying the effects of
chemicals. In our laboratory, we introduced neurobehavioral methods to investigate
potential effects of chemicals and other factors such as hypoxia/ischemia and stressful
stimuli on behavioral development of offspring up to adulthood or even senescence
[50]. Effects of chemicals can be manifested at various levels and in different develop-
mental stages (so-called long-term and/or delayed effects). In many cases, theses effects
are not observable immediately or shortly after birth. They start to be apparent as neu-
robehavioral disorders and mental diseases during childhood, maturation, or as late as
adulthood or senescence. Such diseases include attention deficit-hyperactivity disor-
der, mental retardation, autism, schizophrenia, depression or anxiety [51]. Moreover,
functional deficit of the brain can be masked or hidden due to marked plasticity
and action of homeostatic mechanisms in the developing brain. These functional and
neurobehavioral deficits that may be inapparent in everyday life, can be unmasked
/ can appear as a reaction to chemical substances, drugs and/or intensive stressful
events. In experimental conditions these hidden alterations can be detected by using
specific challenge treatments, such as pharmacological challenge (amphetamine, clo-
nidine) or exposure to stressful stimuli [52]. The neuroendocrine system is extremely
sensitive to various factors. Developmental neuroendocrine alterations were found to be
linked with affective disorders (bipolar disease and major depression) and anxiety. In
our study performed in cooperation with the Institute of Experimental Endocrinology
SASc, Bratislava, we found that prenatal PHT administration resulted in increased reac-
tivity of the neuroendocrine system to stressful stimuli in rats aged 18 months [50].
In the last decade, we became interested in experimental modeling of hypoxia/
ischemia during the pre- and neonatal period. We have been studying consequences
of these insults not only at the morphological and neurobehavioral level but also at
the biochemical and neuroendocrine level. We investigated selected biochemical vari-
ables of oxidative stress in different maternal and fetal organs, such as lysosomal en-
zyme N-acetyl--D-glucosaminidase (NAGA), glutathione (GSH), lactate, and cat-
echolamines [29,38,53,54].
Pregnancy exhibits increased susceptibility to hypoxia/ischemia insults associated
with oxidative stress. During particular periods in development, the embryo and fetus,
which is insufficiently equipped with antioxidative enzymatic systems [5556], is suscep-
tible to oxidative stress. Injuries induced by oxidative stress can be manifested in organs
with a high energetic demand and with active metabolism, such as CNS, myocardium,
liver, lungs and retina [5758]. Anomalies of the skeleton, intrauterine growth retardation
and functional abnormalities, such as neurological, behavioral, emotional and cognitive
disorders can occur as a consequence of developmental hypoxia/ischemia [36,37,39].
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........,... .....
Angiogenesis
A perspective target in cancer therapy
Lenka VARINSK, Ladislav MIROSSAY, Jn MOJI
Department of Pharmacology, Faculty of Medicine, P.J. afarik University, Koice, Slovak Republic,
E-MAIL: jan.mojzis@upjs.sk
Key words: blood-vessel growth, tumour angiogenesis, therapy, plant polyphenols, chalcones
Introduction
Angiogenesis, the process of new blood-vessel growth, plays an essential role in nor-
mal physiological processes, such as development and reproduction. However, in many
disorders the balance between stimulators and inhibitors of angiogenesis is tilted, re-
sulting in an angiogenesis switch. The best-known conditions in which angiogenesis is
switched on are malignant, ocular and inflammatory disorders, but many additional
processes are also affected [1]. Understanding of the basic mechanisms of blood vessels
formation is necessary for the establishment of the effective therapeutic strategies for
amelioration of diseases.
Basic steps in angiogenesis
The process of angiogenesis can be divided into the following four main steps (1) degra-
dation of the basement membrane of existing blood vessels; (2) migration of endothelial
cells toward the angiogenic stimulus; (3) proliferation of the endothelial cells leading
to the formation of solid endothelial cell sprouts in the stromal space; and (4) organi-
zation of endothelial cells into capillary tubes and vascular loops with the formation
of tight junctions and the deposition of new basement membrane [2]. Endothelial cell
proliferation occurs early in angiogenesis, and continues as the new capillary sprout
elongates. Activation of PI3K/Akt promotes endothelial cell survival and proliferation
through modulation of numerous cell cycle regulators, including cyclinD1, p27 and
Bcl-X2. MAPK signalling pathways (ERK1/2, p38 and JNK) mediate growth factor and
mechanical force-induced proliferation of endothelial cells [3]. Proteolysis of basement
membrane matricellular components is necessary to promote endothelial cell invasion
into the surrounding interstitial matrix. The degradation of the extracellular matrix
is under control of proteolytic enzymes and their inhibitors. The balance between
proteases and their inhibitors determines if controlled lysis, leading to angiogenesis,
L. Varinsk et al. (2008) Trends in Pharmacological Research (Eds. V. Bauer et al.): 151157.
152 L. Varinsk et al.
can occur [4]. The new sprouts form a lumen by the process of intracellular vascular
fusion or by stabilization of several cells around a central lumen. The final step is stabi-
lization of the nascent capillaries.
Angiogenesis is a process requiring the coordinated action of a variety of growth fac-
tors and cell-adhesion molecules in endothelial and mural cells [5].
Tumour angiogenesis as a therapeutic target
Angiogenesis is considered a key step in tumour growth, invasion, and metastasis.
Tumours remain avascular and latent for

years; however, tumour growth can be initi-
ated by neo-angiogenesis

[6]. The idea of blocking tumour growth by the inhibition
of angiogenesis was put forward in the early 70s by Judah Folkman [7]. Since a close
relationship between tumour growth and angiogenesis has been clarified, and since
the angiogenic mechanism has subsequently been elucidated, various anti-angiogenic
inhibitors for use in cancer treatment have been studied. Angiogenesis does not initi-
ate malignancy but promotes tumour progression and metastasis. Unlike tumour cells,
endothelial cells (ECs) are considered to be genetically stable. This led to the notion that
acquired resistance to such drugs may not develop as readily, if at all. During the last
15 years, substantial effort has been dedicated to identifying compounds that can be
used to either prevent insurgence of primary tumours in subjects at high risk to develop
cancer or prevent tumour relapse after surgical removal.
Antiangiogenic therapy
As it was mentioned above, targeting tumor angiogenesis to treat cancer has been the
focus of intense research in recent decades. The resulting increase in our knowledge
of cancer biology has lead to the development of several new classes of investigational
agents that inhibit the angiogenic process. While many clinical trials on antiangiogenic
compounds have had disappointing results, the recent approval of the first effective
drug targeting tumor vessels has revived interest in further drug development for an-
giogenesis inhibitors.
For the therapeutic inhibition of angiogenesis,
three main categories have been identified:
direct antiangiogenic drugs that act by targeting the 1.
endothelial cells and their functions involved in angiogenesis
(proliferation, migration, formation of new vessels);
indirect antiangiogenic drugs that thwart the production of angiogenic factors by 2.
tumor and microenvironment cells, and/or interfere with extracellular processes;
mixed antiangiogenic drugs that may be able to interfere 3.
with both endothelial and tumor cells.
153 Angiogenesis a perspective target in cancer therapy
As outlined above, angiogenesis is a highly coordinated process that is regulated
by multiple interactions of angiogenic and angiostatic factors. Therefore, blocking a
single angiogenic molecule was expected to have little or no impact on tumor growth.
However, in apparent contrast with this view, experiments with neutralizing antibodies
and other inhibitors demonstrated that blockade of vascular endothelial growth fac-
tor (VEGF) alone can substantially suppress tumor growth and angiogenesis in sev-
eral models [8]. These encouraging findings prompted efforts for the development of
therapies aimed at targeting VEGF and several pharmacologic approaches have been
developed to inhibit the VEGF axis, based on targeting the ligands (mainly VEGF) or
the receptors (VEGFR-1 and VEGFR-2) [9].
So far, bevacizumab (Avastin), a humanized variant of an anti-VEGF neutralizing
monoclonal antibody (mAb), is the first antiangiogenic agent to be approved by the
Food and Drug Administration (FDA) for the treatment of cancer [8]. Bevacizumab was
approved for the treatment of metastatic colorectal cancer [10] and non-small cell lung
cancer [11] in combination with chemotherapy.
Although the bevacizumab is by far the most extensively studied agent, several other
antiangiogenic drugs are also being evaluated as anticancer therapies.
In addition to agents blocking VEGF itself, a variety of small molecule receptor ty-
rosine kinase (RTK) inhibitors targeting the VEGF receptors including sunitinib and
sorafenib have been developed. Other anti-VEGF agents including VEGF-Trap, a solu-
ble receptor targeting VEGF, VEGF-B and PlGF; an antisense oligonucleotide VEGF-AS
targeting VEGF, VEGF-C and VEGF-D are at various stages of clinical development
[12]. Overall, characterization of VEGF signaling pathway led to the identification of
several target molecules with promising therapeutic potentials.
Inhibition of angiogenesis has been shown with mAbs also against other proangio-
genic growth factors, such as bFGF, suramin, suradistas and their derivates, which bind
to and complex aFGF, bFGF as well as platelet derived growth factor (PDGF) and pre-
vent them from binding to their receptors [13].
Tyrosine kinase inhibitors belong to another group of potential antiangiogenic in-
hibitors. Sunitinib is an oral small molecular tyrosine kinase inhibitor that exhibits
potent antiangiogenic and antitumor activity. Other tyrosine kinase inhibitors such as
SU6668 and SU5416 (semaxanib) demonstrated poor pharmacologic properties and
limited efficacy. Therefore, sunitinib was rationally designed and chosen for its high
bioavailability and its nanomolar-range potency against the antiangiogenic receptor
tyrosine kinases (RTKs) vascular endothelial growth factor receptor (VEGFR) and
platelet-derived growth factor receptor (PDGFR). Clinical activity was demonstrated
in neuroendocrine, colon, and breast cancers in phase II studies, whereas definitive
efficacy has been demonstrated in advanced renal cell carcinoma and in imatinib-re-
fractory gastrointestinal stromal tumours, leading to US FDA approval of sunitinib for
treatment of these two diseases. Studies investigating sunitinib alone in various tumor
types and in combination with chemotherapy are ongoing [14].
Another class of agents that initially showed promising pre-clinical data is that of
matrix metalloproteinase inhibitors (MMPI). Matrix metalloproteinases (MMPs) are a
family of zinc-dependent proteinases involved in the degradation and remodeling of ex-
tracellular matrix proteins that are associated with the tumorigenic process. MMPs pro-
mote tumor invasion and metastasis, regulating host defense mechanisms and normal
cell function. Thus, MMPIs are expected to be useful for the treatment of diseases such
as cancer, osteoarthritis, and rheumatoid arthritis. A vast number of MMPIs have been
developed in recent years. Marimastat is the first oral nonselective MMPI tested in the
clinic. However, the results of phase III trials with marimastat in pancreatic and small
cell lung cancer (SCLC) have not been encouraging because of presence of disturbing
toxicity (musculoskeletal disorders), associated with the absence of clinical benefit [15].
In addition, a recently reported randomized trial testing the addition of prinomastat (a
potent inhibitor of MMP-2, MMP-3 and MMP-13) to chemotherapy in NSCLC failed
to show any advantage in patient outcomes [16]. With the failure of these inhibitors in
clinical trials, more efforts have been directed to the design of specific inhibitors with
different Zn-binding groups. The review of Tu and co-workers [17] summarizes the cur-
rent status of MMPIs, the design of small molecular weight MMPIs , a brief description
of available three-dimensional MMP structures, a review of the proposed therapeutic
utility of MMPIs, and a clinical update of compounds that have entered clinical trials
in humans.
Angiogenesis depends on the adhesive interactions of endothelial cells with the sur-
rounding extracellular matrix. Integrins are a family of cell adhesion molecules con-
sisting of two non-covalently bound transmembrane subunits (alpha and beta). Much
research has demonstrated that integrin signaling plays a key role in tumor angiogen-
esis and metastasis. Integrin alphavbeta3 (v3) is highly expressed on activated en-
dothelial cells and tumor cells but is not present in resting endothelial cells and most
normal organ systems, which makes it a suitable target for anti-angiogenic cancer ther-
apy. Etaracizumab is a monoclonal antibody that was chosen for its unique ability to
selectively target multiple and different cell types, all of which are relevant to cancer
pathophysiology. The target for etaracizumab is v3. Antagonists of v3 have been
studied most extensively for their antiangiogenic properties [18]. In addition, v3 is
expressed on tumor cells and osteoclasts and is believed to play an important role in
bone metastasis and subsequent resorption [19]. These findings suggest a potential role
for v3 in the pathology of osteolytic diseases, including breast cancer, prostate cancer,
and multiple myeloma. Finally, v3 is overexpressed on a variety of different tumor
types. For example, a preponderance of data suggests that v3 is found to be overex-
pressed in metastatic melanoma, glioma, multiple myeloma, ovarian, renal, and breast
cancer [20]. The clinical trials indicated that etaracizumab may have effects on tumor
perfusion and may exhibit clinical activity in renal cell cancer [21]. Based on these find-
ings, etaracizumab is presently being investigated in clinical trials of androgen-inde-
pendent prostate cancer and metastatic melanoma.
Many of the proteins, polypeptides and peptides with antiangiogenic activities are en-
dogenously produced during normal or pathological situations and fall into two classes
of molecules: mediators and regulators or inflammation (cytokines and chemokines)
and matrix proteins and derived fragments. Some of these proteins and polypeptides
have been produced as recombinant proteins or synthetic peptides for therapeutic pur-
poses [e.g. 22,23].
Our results
Angiogenesis is a common and key target of most naturally occurring chemopreventive
molecules, where they most likely suppress the angiogenic switch in premalignant tu-
mors, a concept we termed angioprevention. Several hypotheses have been suggested to
explain beneficial effects of increased consumption of vegetables and fruits on human
health. An attractive hypothesis is that vegetables and fruits contain compounds that
have protective effects, independent of those of known nutrients and micronutrients.
Plant polyphenols, a large group of natural antioxidants ubiquitous in a diet high in
vegetables and fruits, certainly are serious candidates [24].
Flavin7 (F7) is a nutritional supplement containing flavonoids and resveratrol as the
main active compounds. We found that exept of its antiproliferative and proapoptotic
effects, F7 possess also antiangiogenic properties. In non-toxic doses (40 to 4 g/ml)
it inhibited endothelial cell migration and capillary tube formation what indicates its
potential antiangiogenic properties. Moreover, F7 also inhibited the activity of matrix
metalloproteinases (MMPs), preferentially MMP-9, at the doses of 100 to 4 g/ml. Our
data suggest that F7 possesses marked antiangiogenic properties in vitro [25].
Research in the field of anticancer effect of polyphenols has focused on flavonoids,
as common components of the human diet. Nevertheless, many fruits and vegetables
are rich dietary sources of chalcones and dihydrochalcones and these compounds could
make even a greater contribution to the total daily intake of natural polyphenolics than
the more extensively studied flavonoids [26].
Chalcones are precursors of the flavonoids in higher plants and they display a wide
variety of pharmacological effects, including antiproliferative and anticancer, activities
[27,28].
In our department we tested several synthetic derivatives of chalcone for their
antiangiogenic effects. From compounds tested, E-2-(4-methoxybenzylidene)-1-
benzosuberone possess significant antiangiogenic effect. The cytotoxic effect of this
compound was concentration-dependent and HUVECs survival significantly decreased
at c=10
4
10
6
mol.l
1
. Furthermore, it completely inhibited

capillary tube formation in
non-toxic concentrations (10
7
10
8
mol.l
1
). Moreover, in concentration 10
7
mol.l
1
it
blocks also endothelial cell migration. Gelatin zymography revealed that this chalcone
reduced MMP-9 activity in HUVECs in a concentration-dependent manner. Inhibitory
effect on MMP-2 activity was observed only at the highest concentration. Vascular
endothelial growth factor (VEGF) secretion was significantly reduced in cancer cells
treated by this chalcone at concetrations 10
6
and 10
7
mol.l
1
[29,30].
Another perspective compound with antiangiogenic activity is 4-hydroxychalcone.
This chalcone, at the concentration 10
4
mol.l
1
(non-toxic concentration), completely
inhibited the formation of capillary-like tubular structures in a three dimensional fi-
brin matrix induced by exposure of human microvascular endothelial cells to VEGF
and tumour necrosis factor-. However the morphology of the endothelial monolayer
covering the fibrin matrix was not affected. It was accompanied by a decrease in uroki-
nase-type plasminogen activator accumulation in the conditioned medium. At the
same concentration we observed the inhibition of VEGF-induced migration of human
endothelial cells as well as their differentiation into tube structures in Matrigel [31].
Conclusions
Angiogenesis inhibitors are likely to change the face of medicine in the next decade.
The chemopreventive agents that selectively

interfere with particular biochemical al-
terations occurring

in tumour cells or those acting on the highly specialized biology

of
endothelial cells during neovascularization deserve special

attention. Understanding
the basic principles by which natural compounds inhibit angiogenesis may lead to the
development of new therapeutic strategies, in addition to supporting the role of poly-
phenols as cancer chemopreventive agent.
Acknowledgement
This work was supported by the Slovak Research and Development Agency under the
contract No. APVV-0325-07 and by the Slovak Grant Agency for Science (grant No.
1/4236/07).
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........,... .....
Cytokine-stimulatory effects of acyclic
nucleotide analogues: extrapolation
of immunopharmacological data
from animal to human cells
Zdenk ZDEK
1
, Eva KMONKOV
1
, Antonn HOL
2
1
Institute of Experimental Medicine and
2
Institute of Organic Chemistry and Biochemistry, Academy of Sciences
of the Czech Republic, v.v.i., Prague, Czech Republic
Key words: acyclic nucleotide analogues; chemokines; nitric oxide
Introduction
Acyclic nucleotide analogues are antivirals effective against replication of both DNA-
viruses and retroviruses [1]. They suppress the multiplication of herpes simplex virus
type-1 and -2, human herpes virus type 6, cytomegalovirus, varicella zoster virus,
Epstein-Barr virus, human papilloma virus, adeno- and pox-viruses, Moloney sar-
coma virus, hepatitis B virus, Friend leukemia virus, human immunodeficiency virus
(HIV) types 1 and 2, simian immunodeficiency virus, and feline immunodeficien-
cy virus. The antiviral activity of acyclic nucleotide analogues is assumed to be due
mainly to the suppression of cellular DNA synthesis mediated by inhibition of repli-
cative DNA-polymerases. The oral prodrugs of the prototype compounds, i.e. 9-(R)-
[2-(phosphonomethoxy)propyl]adenine (tenofovir) and 9-[2-(phosphonomethoxy)
ethyl]adenine (adefovir) were approved by FDA and EMEA for treatment of AIDS
(Viread) and hepatitis B (Hepsera), respectively. Another important representative
of acyclic nucleoside phosphonates is cidofovir (Vistide) which is approved for treat-
ment of cytomegalovirus retinitis in AIDS patients.
We have shown recently that a number of acyclic nucleotide analogues are endowed
with the potential to activate secretion of cytokines including the anti-HIV effective
chemokines and up-regulate biosynthesis of virustatic molecule of nitric oxide (NO)
in a murine model of immunobiological screening [27].
The present analysis is focused on the evaluation of immunostimulatory and im-
munomodulatory effects of acyclic nucleotide analogues in cells of murine and hu-
man origin.
Z. Zdek et al. (2008) Trends in Pharmacological Research (Eds. V. Bauer et al.): 158165.
159 Cytokine-stimulatory efects of acyclic nucleotide analogues
Materials and methods
Acyclic nucleotide analogues (Table 1) were synthesized in-house (Institute of Organic
Chemistry and Biochemistry) by the previously described procedures [8].
The sources of human peripheral blood mononuclear cells (PBMCs) were buffy coats
acquired from healthy donors (provided by the Institute of Hematology and Blood
Transfusion, Prague). The PBMCs were separated by Ficoll-Paque gradient centrifugation.
Pooled mouse peritoneal cells (PECs) were collected from the female mice of the in-
bred strain C57BL/6 (Charles River Deutschland, Germany).
The cells were seeded into 96-well round-bottom microplates (Costar) and main-
tained at 37 C, 5% CO
2
in humidified Heraeus incubator. The animal PECs were cul-
tured at final density of 2.0 10
6
/ml, the human PBMCs at density of 1.0 10
6
/ml in
complete RPMI-1640 culture medium. It contained 10% heat-inactivated fetal bovine
serum, 2 mM L-glutamine, 50 g/ml gentamicin, and 5 10
5
M 2-mercaptoethanol
(all Sigma).
Concentration of chemokines in supernatants of mouse PECs and human PBMCs
was determined by enzyme-linked immunoabsorbent assay (ELISA) kits (R&D Systems,
MN). The length of culture was 16 h.
The concentration of nitrites in supernatants of mouse PECs was taken as a measure
of NO production [9]. It was detected after the 24-h culture, using a Griess reagent.
Results
Several acyclic nucleotide analogues have been found to be potent activators of chemok-
ines MIP-1 (Figure 1A) and RANTES (Figure 1B). Since considerable inter-individual
differences (n = 9) were found in control values of human RANTES, ranging from 186
to 1228 pg/mL, the effects of compounds were expressed in percents of the baseline val-
ues (Figure 1B, right axis). The most prominent stimulators of the chemokines proved
to be the compounds H-2939, H-2940, H-2952, H-2989, H-3432, MIC-428, MIC-444
(for chemical names, see Table 1). The effects of individual acyclic nucleotide analogues
were very similar in mouse PECs and human PBMCs, the coefficients of correlation be-
ing statistically highly significant for both MIP-1 (r = 0.969, p<0.0001) and RANTES
(r = 0.982, p<0.0001).
The constitutive production of NO produced by mouse PECs was barely detectable
(p>0.05), and it remained unchanged in the presence of the acyclic nucleotide analogues
alone (data not shown). However, a number of them were able to up-regulate NO bio-
synthesis which was primarily triggered by IFN- (Figures 2A and 2B). This activity
was typical for the compounds exhibiting the chemokine-enhancing effects. Not sur-
prisingly, the extent of NO production on one side and the range of chemokine secre-
tion on the other one were found to be statistically significantly correlated. This holds
true for not only the production of chemokines by mouse cells (not shown), but also by
160 Z. Zdek et al.
A) Secretion of MIP-1
C
O
N
T
R
O
L
.
H
-
3
3
8
7
H
-
3
0
4
0
H
-
P
M
E
A
M
I
C
-
4
4
5
M
I
C
-
4
5
8
M
I
C
-
4
2
5
M
I
C
-
4
5
3
M
I
C
-
4
5
6
M
I
C
-
4
2
2
M
I
C
-
4
6
0
M
I
C
-
4
2
3
H
-
3
0
1
5
H
-
3
4
3
1
H
-
3
0
0
2
M
I
C
-
4
2
9
M
I
C
-
4
3
4
H
-
2
9
1
3
M
I
C
-
4
5
9
M
I
C
-
4
4
9
M
I
C
-
4
5
4
M
I
C
-
4
3
5
H
-
3
4
2
4
H
-
2
9
5
2
H
-
3
4
3
2
H
-
2
9
4
0
M
I
C
-
4
2
8
H
-
2
9
8
9
H
-
2
9
3
9
M
I
C
-
4
4
4
0
100
200
300
400
500
Mouse PEC Human PBMC
0
500
1000
1500
2000
2500
Coefcient of correlation r = 0.969, P < 0.0001
Compounds 50 M
M
I
C
E
:

M
I
P
-
1

(
p
g
/
m
L
)
H
U
M
A
N
:

M
I
P
-
1

(
p
g
/
m
L
)
B) Secretion of RANTES
C
O
N
T
R
O
L
.
M
I
C
-
4
2
3
M
I
C
-
4
6
0
M
I
C
-
4
2
2
H
-
3
0
0
2
H
-
P
M
E
A
H
-
3
3
8
7
M
I
C
-
4
5
8
M
I
C
-
4
5
6
H
-
3
4
3
1
H
-
3
0
1
5
H
-
2
9
4
0
H
-
3
4
2
4
M
I
C
-
4
3
4
H
-
2
9
1
3
M
I
C
-
4
5
9
M
I
C
-
4
3
5
H
-
3
4
3
2
H
-
2
9
5
2
M
I
C
-
4
2
8
H
-
2
9
8
9
M
I
C
-
4
4
4
H
-
2
9
3
9
0
200
400
600
800
1000
Mouse PEC Human PBMC
0
50
100
150
200
250
300
Coefcient of correlation r = 0.982, P < 0.0001
Compounds 50 M
M
I
C
E
:

R
A
N
T
E
S

(
p
g
/
m
L
)
H
U
M
A
N
:

R
A
N
T
E
S

(
%

o
f

b
a
s
e
l
i
n
e

p
g
/
m
L
)
Figure 1. In vitro production of chemokines MIP-1 (A) and RANTES (B) following 16-h cultivation of human
(n = 8) and mouse (n = 20) cells in presence of test compounds.
A) Mous e NO / Human MIP-1
C
O
N
T
R
O
L
.
H
-
3
0
4
0
H
-
3
0
1
5
H
-
P
M
E
A
M
I
C
-
4
4
5
H
-
3
3
8
7
M
I
C
-
4
2
5
M
I
C
-
4
5
8
H
-
3
0
0
2
M
I
C
-
4
6
0
M
I
C
-
4
5
6
M
I
C
-
4
5
3
M
I
C
-
4
2
3
M
I
C
-
4
2
2
M
I
C
-
4
2
9
H
-
3
4
3
1
H
-
3
4
2
4
M
I
C
-
4
3
5
M
I
C
-
4
3
4
H
-
2
9
4
0
H
-
2
9
1
3
M
I
C
-
4
4
9
M
I
C
-
4
5
4
M
I
C
-
4
5
9
H
-
3
4
3
2
H
-
2
9
8
9
M
I
C
-
4
2
8
H
-
2
9
5
2
H
-
2
9
3
9
M
I
C
-
4
4
4
0
10
20
30
40
50
60
NO (M)
MIP-1 (pg/mL)
0
250
500
750
1000
1250
1500
1750
2000
2250
2500
Coefcient of correlation, r = 0.958; P < 0.0001
Compounds (M)
M
o
u
s
e

P
E
C
:

N
i
t
r
i
t
e

(
M
)
H
u
m
a
n

P
B
M
C
:

M
I
P
-
1

(
p
g
/
m
L
)
B) Mouse NO / Human RANTES
C
O
N
T
R
O
L
.
H
-
3
0
1
5
H
-
P
M
E
A
H
-
3
3
8
7
M
I
C
-
4
6
0
M
I
C
-
4
5
6
M
I
C
-
4
2
3
M
I
C
-
4
2
2
H
-
3
4
3
1
H
-
3
4
2
4
M
I
C
-
4
3
5
M
I
C
-
4
3
4
H
-
2
9
4
0
H
-
2
9
1
3
M
I
C
-
4
5
9
H
-
3
4
3
2
H
-
2
9
8
9
M
I
C
-
4
2
8
H
-
2
9
5
2
H
-
2
9
3
9
M
I
C
-
4
4
4
0
10
20
30
40
50
60
NO (M)
RANTES (% of control)
0
50
100
150
200
250
300
Coefcient of correlation, r = 0.969; P < 0.0001
Compounds (M)
M
o
u
s
e

P
E
C
:

N
i
t
r
i
t
e

(
M
)
H
u
m
a
n

P
B
M
C
:

R
A
N
T
E
S

(
%

o
f

b
a
s
e
l
i
n
e

p
g
/
m
L
)
Figure 2. Correlation between ability of compounds to upregulate production of NO by mouse peritoneal cells
and production of chemokines MIP-1 (A) and RANTES (B) by human peripheral blood mononuclear cells.
162 Z. Zdek et al.
Table 1. Chemical names and codes of test compounds.
Appendix: List of compounds
H-2913 9-[2-(phosphonomethoxy)propyl]adenine (tenofovir)
H-2939 N
6
- cyclopropyl-(R)-9-[2-(phosphonomethoxy)propyl]2,6-diaminopurine
H-2940 N
6
-pyrrolidino-9-[2-(phosphonomethoxy)ethyl]2,6-diaminopurine
H-2952 N
6
-cyclopentyl-(R)- 9-[2-(phosphonomethoxy)propyl]2,6-diaminopurine
H-2989 N
6
-isobutyl-9-[2-(phosphonomethoxy)ethyl]2,6-diaminopurine
H-3002 N
6
-dimethylaminoethyl-9-[2-(phosphonomethoxy)ethyl]2,6-diaminopurine
H-3015 9-[2-(phosphonomethoxy)ethyl]2,6-diaminopurine
H-3387 N
6
-cyclopropyl-9-[2-(phosphonomethoxy)ethyl]2,6-diaminopurine
H-3424 N
6
-cyclohexylmethyl-9-[2-(phosphonomethoxy)ethyl]2,6-diaminopurine
H-3431 N
6
-cyclopentyl-9-[2-(phosphonomethoxy)ethyl]2,6-diaminopurine
H-3432 N
6
-cycloctyl-9-[2-(phosphonomethoxy)ethyl]2,6-diaminopurine
H-PMEA 9-[2-(phosphonomethoxy)ethyl]adenine (adefovir)
MIC-422 2-guanidino-9-(S)-[2-(phosphonomethoxy)propyl]-9H-purine
MIC-428 6-amino-2-guanidino-3-(S)-[2-(phosphonomethoxy)propyl]-3H-purine
MIC-444 2-amino-6-guanidino-9-(R)-[2-(phosphonomethoxy)propyl]-9H-purine
MIC-445 6-guanidino-9-(R)-[2-(phosphonomethoxy)propyl]-9H-purine
MK-417 (2-hydroxy-3-phosphonomethoxypropyl)adenine
MK-447 [3-(2,6-diaminoamino-9H-purin-9-yl)-2-hydroxypropyl]methylphosphonic acid
human cells (Figures 2A and 2B). The coefficients of correlation between NO produc-
tion by mouse PECs and chemokine production by human PBMCs reached the values of
r = 0.958 (p<0.0001), and r = 0.969 (p<0.0001) for MIP-1 and RANTES, respectively.
Discussion
It is believed that effectiveness of chemotherapy, presently a prevailing strategy to treat
virus infections, might be improved by concomitant enhancement of innate immune
defence functions. Hopefully, the active variant of immunopharmacological control
of viral infections, including HIV [10], might be mediated by an agent-stimulated,
-enhanced, or -restored production of natural factors of nonspecific immune defence
system, such as cytokines and chemokines.
Our original data show that acyclic nucleotide analogues are potent immunostimu-
latory agents, and may thus be considered as a novel generation of antivirotics with
combined antimetabolic and immunomodulatory modes of action. Interestingly, none
of the commonly used dideoxynucleotides which have been approved for treatment of
AIDS, i.e. 3-azido-2,3-dideoxythymidine (AZT; zidovudine), 2,3-dideoxyinosine
(ddI; didanosine), and 2,3-dideoxycytidine (ddC; zalcitabine) [11] have so far been re-
ported to exhibit immunomodulatory activity.
Cytokines and chemokines play a pivotal role in control of viral infections. Seeking
drugs that would restore impaired immune effectiveness and/or stimulate factors of
immune defence, including cytokines has therefore become a permanent challenge of
pharmacological research.
As what concerns HIV, a plethora of cytokines have been implicated in control of the
infection, exhibiting both up- and down-regulatory effects [12]. The most promising
targets for therapeutic interventions are chemokines and chemokine receptors [13]. The
main chemokine co-receptors for the entry of T cell line-tropic and macrophage-tropic
HIV-1 isolates are CCR5 that binds chemokines RANTES, MIP-1 and MIP-1, and re-
ceptor CXCR4 which binds SDF-1/ [14, 15]. Chemokines, the natural ligands of these
receptors, such as MIP-1, MIP-1, RANTES, MCP-2, SDF-1, and eotaxin, have been
shown to block the entry of certain HIV strains into its target cells.
Antiviral activity of many cytokines is mediated by enhanced production of NO.
High-output NO production by cells depends largely or entirely on activation of iNOS
and results from all transcriptional, post-transcriptional and post-translational action
of cytokines. A direct NO-stimulatory function is possessed by IFN- that triggers NO
production on its own [16]. Although other cytokines may occasionally stimulate NO
by themselves, they mainly provide an additional signal for activation of NO by IFN-.
It concerns mainly TNF- [17], IL-1, which plays a central role in regulation of hepatic
iNOS activity [18], IL-2, a major iNOS activator in NK cells [19], IL-12 [20], IL-17 [21],
chemokines RANTES, MIP-1, MIP-1 [22], eotaxin [23], and MCP-1 [24]. On the con-
164 Z. Zdek et al.
trary, cytokines such as IL-4 and IL-10 [25], TGF- [26], M-CSF and G-CSF [27, 28] are
generally considered inhibitors of NO production.
The ability of acyclic nucleotide analogues to activate secretion of cytokines is a plau-
sible explanation for their up-regulatory effects on NO production by mouse PECs. It
should be mentioned that while human cells produce large amounts of NO in vivo, hu-
man monocytes and macrophages are highly refractory to the induction of NO produc-
tion under conditions in vitro [29].
Conclusions
Acyclic nucleotide analogues are potent stimulators of cytokine production. Concerning
both qualitative and quantitative measures of their secretion, the effects of compounds
with intrinsic immunostimulatory potential are very similar in mouse and human cells.
The extent of cytokine production is tightly correlated with the enhancement of NO
produced by mouse peritoneal cells. The NO platform can thus be employed as a re-
liable, rapid and economical pivotal screening allowing prediction of immunomodu-
latory effects of compounds in human cell system.
Ackowledgements
The work was supported by grant no. 1M0508.
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166
Authors Index
J
Jakubovsk, Jn 41
Janinov, Viera 82
Jank, Jozef 140
Jariabka, Pavol 118
Juskov, Mria 109
K
Kittov, Mria 41
Kmonkov, Eva 158
Knezl, Vladimr 41
Kollrov, Mria 15
Kollr, Tom 41
Komendov, Denisa 25
Kopeck, Bernardna 58
Krchnarov, Viera 58
Krianov, udmila 109
Kukan, Marin 33
Kvtina, Jaroslav 66
Kyseov, Zuzana 15, 109
L
Lojek, Antonn 77
M
Mach, Mojmr 140
Maikov, Tatiana 82
Magna, Darius 41
Mjekov, Magdalna 109
Mlekov, Lubica 15
Markovi, Slavo 41
Mtys, tefan 15
Mihalov, Danica 25
Mirossay, Ladislav 151
Moji, Jn 151
Mokr, Juraj 88
N
Navarov, Jana 15, 140
Nedelevov, Jana 15
Nemek, Vendel 118
A
Anzenbacherov, Eva 11
Anzenbacher, Pavel 11
B
Babulov, Anna 41
Bacharov, Ljuba 41
Bauerov, Katarna 25
Bauer, Viktor 7, 15
Berntov, Iveta 102
Bezek, tefan 33
Boroviov, Frantika 41
D
Dedk, Ladislav 48
Ditrychov, Viera 15
Djoubissie, Paul O. 109
Drbikov, Katarna 82
Draveck, Mria 41
Dmal, Jn 41
Dubovick, Michal 140
uriov, Mria 48
Dytrichov, Viera 140
F
Fatykov, Jozefna 15
Fraov, Soa 88
G
Gajdok, Andrej 58, 109, 118, 140
Gajdokov, Alena 58, 109, 118, 140
GSprov, Zdenka 118
Gemeiner, Peter 25
Gibala, Pavel 41
Golhov, Daniela 58
Gvozdjak, Jn 41
Gvozdjakov 41
H
Hol, Antonn 158
Hrb, Jan 77
167
Nikov, Eva 41
Nosov, Gabriela 88
Nosov, Viera 15
Nos, Radomr 8, 41, 77, 82
O
Ondrejikov, Oga 118
Ondria, Karol 96
P
Pechov, Oga 102
Peivov, Jana 82
Pekarov, Michaela 77
Petrkov, Margita 82
Poli, Giuseppe 25
Ponit, Silvester 25
Pucovsk, Vladimr 15
Pukrov, Andrea 41
R
Rakov, Lucia 109
Rapta, Peter 118
Rekalov, Vladimr 15
Rybr, Alfonz 41
S
Sadloov, Vladimra 88
Sauberer, Anton 41
Seleck, Frantiek Viliam 41
nirc, Vladimr 109, 118
olts, Ladislav 25
Sotnkov, Ruena 15, 41, 118
Srnov, Monika 15
tefek, Milan 109
Stojkoviov, Zuzana 15
tolc, Svorad 41, 118
Strakov, Zuzana 82
Strapkov, Anna 88
Strov, Katarna 41
trosov, Miriam 25
utovsk, Martina 88
Synekova, Inge 118
Szcs, Katalyn 15
T
Tich, Milo 137
Tokrov, Jana 41
Tomekov, Veronika 25
Torokov, Jozefna 41
Tthov, Gizella 15
Trnovec, Tom 33
Tvrdoov, Martina 48
U
Ujhzy, Eduard 58, 140
Urban, Pavel 137
V
Vajdov, Mria 118
Valachov, Katarna 25
Varinsk, Lenka 151
Viola, rpd 118
Z
Zacharova, Soa 118
Zdek, Zdenk 158

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OH) and singlet oxygen (

and its transformation

OH radicals by reducing dioxygen molecules.

. Among direct methods which have

-hydroxy-L-arginine (NOHA) [28]. We did not record signifi-

donor NaHS released NO from nitrosothiols,

donor NaHS released NO not only from NO donors

releases NO from S-nitrosothiols and/or metal nitrosyl complexes in vivo.

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