Pharmacological Research

pharmacological research

Editor Prof. Viktor BAUER, MD., DSc.

Consulting Editors Michal DUBOVICKÝ, PhD. Assoc. Prof. Magda KOUŘILOVÁ, PhD. Mojmír MACH, PhD. Ja na NAVA ROVÁ, PhD. Prof. Radomír NOSÁĽ, MD., DSc. Ružena SOTNÍKOVÁ, PhD.

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BRATISLAVA 2008 Trends in Pharmacological Research ISBN 978–80–970003–7–0

Published by Institute of Experimental Pharmacology, SASc. Dúbravská cesta 9, SK-841 04 Bratislava, Slovak Republic fax: +421-2-59477 5928 • e-mail: [email protected]

Printed in Slovak Republic

Cover, Interior Design & Typesetting Mojmir Mach, PhD.

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. pharmacological research

Table of Contents

Editorial V. Bauer 7

Brief history of the Institute of Experimental Pharmacology R. Nosáľ 8

Trends in studies of drug metabolism and of related drug–drug interactions P. Anzenbacher, E. Anzenbacherová 11

Smooth muscle tissues as models for study of drug action V. Bauer, R. Sotníková, V. Nosáľová, J. Navarová, Š. Mátyás, V. Pucovský, V. Rekalov, K. Szőcs, J. Nedelčevová, Z. Kyseľová, V. Dytrichová, J. Fatyková, M. Kollárová, L. Máleková, M. Srnová, Z. Stojkovičová, G. Tóthová 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. Bauerová, S. Poništ, K. Valachová, D. Mihalová, L. Šoltés, D. Komendová, V. Tomeková, M. Štrosová, P. Gemeiner, G. Poli 25

Effect of cytochrome P450 induction on drug disposition in isolated rat liver preparation Š. 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. Dřímal, V. Knezl, A. Babulová, L. Bacharová, F. Borovičová, M. Dravecká, P. Gibala, J. Gvozdjak, A. Gvozdjaková, J. Jakubovský, M. Kittová, D. Magna, A. Rybár, F.V. Selecký, R. Sotníková, S. Štolc, K. Strížová, J. Tokárová, R. Nosáľ, A. Sauberer, E. Nikšová, S. Markovič, J. Toroková, A. Puškárová, T. Kollár 41 pharmacological research

New computational approach to mathematical modeling in pharmacological research M. Ďurišová, L. Dedík, M. Tvrdoňová 48

Breeding and testing facility Dobrá Voda A. Gajdošík, A. Gajdošíková, E. Ujházy, D. Golhová, B. Kopecká, V. Krchnárová 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 Králové, Czech Republic) J. Květina 66

The use of electrochemical measurement for real-time monitoring of nitric oxide generation by macrophages in vitro A. Lojek, M. Pekarová, R. Nosáľ, J. Hrbáč 77

H1-antihistamine Dithiaden® suppressed platelet aggregation and oxidative burst of neutrophils in vitro R. Nosáľ, K. Drábiková, V. Jančinová, T. Mačičková, J. Pečivová, M. Petríková, Z. Straková 82

Research focuses of the pharmacology in Martin G. Nosáľová, S. Fraňová, A. Strapková, J. Mokrý, M. Šutovská, V. Sadloňová 88

Trends in pharmacological research – contribution from studies of the membrane transport and cell signaling K. Ondriaš 96

Vasoactive effects of Provinols™ in experimental hypertension O. Pecháňová, I. Bernátová 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. Štefek, P.O. Djoubissie, A. Gajdošík, A. Gajdošíková, M. Jusková, Ľ. Križanová, Z. Kyseľová, M. Májeková, L. Račková, V. Šnirc 109 pharmacological research

New pyridoindoles with antioxidant and neuroprotective actions S. Štolc, V. Šnirc, A. Gajdošíková, A. Gajdošík, Z. Gáspárová, O. Ondrejičková, R. Sotníková, Á. Viola, P. Rapta, P. Jariabka, I. Syneková, M. Vajdová, S. Zacharová, V. Nemček, V. Krchnárová 118

Trends of research in pharmacology M. Tichý, P. Urban 137

Trends in developmental toxicology: Protection of the developing organism – an ever topical issue E. Ujházy, M. Mach, J. Navarová, A. Gajdošíková, A. Gajdošík, J. Janšák, V. Dytrichová, M. Dubovický 140

Angiogenesis A perspective target in cancer therapy L. Varinská, L. Mirossay, J. Mojžiš 151

Cytokine-stimulatory effects of acyclic nucleotide analogues: extrapolation of immunopharmacological data from animal to human cells Z. Zídek, E. Kmoníčková, A. Holý 158

AUTHORS INDEX 166 We would like to express our appreciation and thanks to our financial supporters

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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 question 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), 323–324, 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 pharmacologist 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 pharmacological research (covering a range of sub-groups, such as neuro-, cardiovascular, gastrointestinal, respiratory, etc. pharmacology), pharmacokinetics, toxicology (drug toxicology, adverse 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 toxicology, 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 pharmacological research

Brief history of the Institute of Experimental Pharmacology

Radomír NOSÁĽ Director of the Institute of Experimental Pharmacology, Slovak Academy of Sciences, Dúbravská 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 Dobrá 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 increasing 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 experiments, the Research Breeding Center was founded at Dobrá 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, hormones, 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 Brief history of the Institute of Experimental Pharmacology 9

Department of Pharmacology of Natural Substances of the Institute of Chemistry of SASc in Bratislava including its Research Breeding Laboratory at Dobrá 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 focused on mechanism of action of different endogenous and synthesized substances on synaptic transmission in the peripheral and central nervous system, peripheral and coronary 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 mechanism of action of papaverine in the therapy of some neurosomatic diseases, on pharmacokinetics 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 properties were demonstrated. The research in pharmacodynamics was focused on several areas, 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 pharmacological 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 pharmacokinetics, 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 scientiﬁ c issues have been investigated:

• Study of receptor and non-receptor interactions between cationic amphiphilic substances 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.

Bauer et al. Trends in Pharmacological Research 10 R. Nosáľ

• 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 pathways 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 (embryotoxicity, 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 pharmacological interventions in oxidative stress and proinflammatory reaction-induced injury of the organism. One of the main interests of the Institute is to develop new pharmacotherapeutic approaches to diseases associated with pro-inflammatory processes and oxidative stress. An integral part of R&D activities are studies in the field of biopharmaceutical (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 toxicological mechanisms of action of biologically active substances are investigated at body, organ, 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 Dobrá 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 drug–drug interactions

Pavel ANZENBACHER 1, Eva ANZENBACHEROVÁ 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 characterized by attempts to find molecular basis of function (as well as of dysfunction) of processes 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 understand the mechanisms which determine the ability of individual (so many?) enzymes and proteins of drug transport and metabolism to pursue their function, (ii) to understand the differences in the ability of these proteins to exhibit their action due to genetically determined structural alterations and (iii) to understand the clinical consequences of the presence of these structurally altered (or even nonfunctional or missing) proteins.

P. Anzenbacher & E. Anzenbacherová (2008) Trends in Pharmacological Research (Eds. V. Bauer et al.): 11–14. 12 P. Anzenbacher & E. Anzenbacherová

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 metabolism 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 biotransformation however needs deeper insight into the differences in the structural properties of the genetically determined variants of the CYP enzymes which are present 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 patients 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 2nd 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 Gilbert’s 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 corresponding 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 receptors 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

Copyright © 2008 Institute of Experimental Pharmacology | ISBN 978–80–970003–7–0 Trends in studies of drug metabolism and of related drug-drug interactions 13 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 receptor 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 membrane-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 treatment 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 diseases is known and accepted [6,7], the effect of these polymorphisms on the pharmacokinetics 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 21st century with tools offered by recent developments 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 information of key importance.

Acknowledgment

The financial support from the Grant Agency of the Academy of Sciences of the Czech Republic KAN200200651 is gratefully acknowledged.

Bauer et al. Trends in Pharmacological Research 14 P. Anzenbacher & E. Anzenbacherová

REFERENCES [1] Anzenbacher P, Anzenbacherová E: Cytochromes P450 and metabolism of xenobiotics. Cell Mol Life Sci 2001; 58, 737–747. [2] Skopalík J, Anzenbacher P, Otyepka M: Flexibility of human cytochromes P450: molecular dynamics reveals diff erences between CYPs 3A4, 2C9, and 2A6, which correlate with their substrate preferences. J Phys Chem B 2008; 112: 8165–8173. [3] Anzenbacher P, Anzenbacherová E, Lange R, Skopalík J, Otyepka M: Active sites of cytochromes P450: What are they like? Acta Chim Slov 2008; 55: 63–66. [4] Parkinson A: Biotransformation of xenobiotics. In: Casarett and Doull´s Toxicology, the Basic Science of Poi- sons, Chapter 6. Editor: Klaassen CD. Mc Graw Hill, 2001, p. 133–224. [5] Zhou J, Zhang J, Xie W: Xenobiotic nuclear receptor-mediated regulation of UDP-glucuronosyltransferases. Curr Drug Metabol 2005; 6:289–298. [6] Kimura Y, Morita SY, Matsuo M, Ueda K: Mechanism of multidrug recognition by MDR1/ABCB1. Cancer Sci 2007; 98: 1303–1310. [7] Turgut S, Yaren A, Kursunluoglu R, Turgut G: MDR1 C3435T polymorphism in patients with breast cancer. Arch Med Res 2007; 38: 539–544. [8] Aarnoudse AJ, Dieleman JP, Visser LE, Arp PP, van der Heiden IP, van Schaik RH, Molokhia M, Hofman A,Uitterlinden AG, Stricker BH: Common ATP-binding cassette B1 variants are associated with increased digoxin serum concentration. Pharmacogenet Genomics 2008; 18: 299–305. [9] Fiegenbaum M, da Silveira FR, Van der Sand CR, Van der Sand LC, Ferreira ME, Pires RC, Hutz MH: Th e role of common variants of ABCB1, CYP3A4, and CYP3A5 genes in lipid-lowering efi cacy and safety of simvastatin treatment. Clin Pharmacol Th er 2005; 78, 551–558.

Smooth muscle tissues as models for study of drug action

Viktor BAUER, Ružena SOTNÍKOVÁ, Viera NOSÁĽOVÁ, Jana NAVAROVÁ, Štefan MÁTYÁS, Vladimír PUCOVSKÝ, Vladimír REKALOV, Katalyn SZŐCS, Jana NEDELČEVOVÁ, Zuzana KYSEĽOVÁ, Viera DYTRICHOVÁ, Jozefína FATYKOVÁ†, Mária KOLLÁROVÁ, Lubica MÁLEKOVÁ, Monika SRNOVÁ, Zuzana STOJKOVIČOVÁ, Gizella TÓTHOVÁ Department of Smooth Muscle Pharmacology, Institute of Experimental Pharmacology, Slovak Academy of Sciences, Dúbravská cesta 9, 841 04 Bratislava, Slovak Republic, E-MAIL: [email protected]

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 fundamental physiological, pathophysiological processes and characterize features of drug action [1]. Although the complexity of the SM preparations calls upon certain reservation 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. cholinergic, adrenergic, non-adrenergic, non-cholinergic – NANC) and sensoric nerves; processes 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 Ca2+. Introduction of sophisticated electrophysiological, pharmacological, biochemical, isotope and morphological methods have provided 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 described in papers [1–19].

V. Bauer et al. (2008) Trends in Pharmacological Research (Eds. V. Bauer et al.): 15–24. 16 V. Bauer et al.

Results and discussion

1. Calcium and smooth muscle activities Ca2+ has an extremely important role in regulation of SM activity, because there is 2+ a greater gradient between concentrations of the free extracellular ([Ca ]o) and in- 2+ 2+ tracellular ([Ca ]i) calcium than for other ions. Elevation of [Ca ]i above 0.1 μmol/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. Ca2+ homeostasis is maintained by its influx via selective voltage (VOC, Figure 1A) and receptor (ROC) operated Ca2+ channels and less selective cationic channels, by chemically operated Ca2+ release from intracellular stores and by its active transport out of the cell and to intracellular stores, by Ca2+ pumps and exchange mechanisms. Some SMs (phasic ones, like intestine or portal vein) generate inward Ca2+ current on their entry to the cell, tetrodotoxine (TTX) insensitive action potentials and SM contraction, which are inhibited by Ca2+ channel blockers, indicating the essential role of Ca2+ in these processes [2,3]. Other SMs (tonic ones, like vessels or trachea) generate action potentials 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 eﬀ ect of nifedipine on inward Ca2+ current (A), hypoxia on spontaneous + + transient outward K currents (STOCs) and whole cell current (B) and H2O2 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.

Copyright © 2008 Institute of Experimental Pharmacology | ISBN 978–80–970003–7–0 Smooth muscle tissues as models for study of drug action 17 longed low amplitude membrane depolarization. There is, however, some evidence of 2+ SM contractile proteins activation without participation of an increase in [Ca ]i [3,4]. The following membrane elements participate in maintaining the SM membrane potential 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 Ca2+ (responsible for excitatory junction potentials) or permeable for Na+ and K+ (responsible for prolonged depolarization); voltage dependent Na+ channels (myocardial and neuronal type); voltage dependent K+ channels (transient type, responsible for A current); TEA sensitive and insensitive outward and inward rectifiers; Ca2+ 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 Ca2+ sensitive; ATP sensitive and Ca2+ insensitive responsible for resting membrane potential, M current and S current); second messenger activated voltage and Ca2+ dependent channel (conductive for anions, mainly Cl–); the Na+ pump; Ca2+ pump; Na+ - Ca2+ exchanger; Na+ - H+ exchanger and K+ - Na+ - Cl– exchanger [1,5]. 2+ The effects of Cai may be modified by: its binding (e.g. by chelators); modulation of CaM and its interaction with Ca2+ (e.g. by phenothiazines); influence of MLCK and myosine phosphatase (e.g. by substance A-3) or myosine and actine (e.g. by H2O2). Mechanisms coupled to intracellular Ca2+ stores participate as well in maintaining free 2+ 2+ [Ca ]i. From one of these stores (S) Ca is released by IP3 sensitive (IICR) and ryano- dine sensitive (CICR) channels, while from the other (Sß) only through IICR channels. 2+ 2+ 2+ 2+ IP3 receptor activation released Ca evokes additional Ca releases (Ca induced Ca 2+ 2+ release) by means of CICR channels. Free Cai activates proteinkinase C and by Cai / CaM interaction proteinkinaseII, which phosphorylates VOC and IICR channels and releases further Ca2+. Transport of Ca2+ back to its intracellular stores is materialized by a tapsigargin sensitive Ca2+ pump. If in Sß the concentration of Ca2+ becomes low, calcium influx factor (CIF) is produced. CIF activates Ca2+ release activated channels (CRAC) in plasma membrane and mediates the influx of Ca2+ 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 adjustment of SM tone than actine. Their structural organization grants transfer of contractile protein generated force along the whole tissue. It is believed that the longitudinal 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

Bauer et al. Trends in Pharmacological Research 18 V. Bauer et al.

(CSt) tetanic stimulation is needed. The responses of CSt have features of rebound contractions (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, H2O2, Crb and KCl did not differ from that of LSt. In contrast, isolated CM, poor in MP, loses reactivity to Crb and KCl. Mild reduction of difference between responses of CM and CSt by hexamethonium suggests that ganglionic transmission does not participate in communication 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 α- and β-adrenoceptor agonists and antagonists, we found that postsynaptic α1-drenergic receptors dominate in terminal, while α2-receptors in intermediate and proximal parts of the GPI. The mainly inhibitory β-adrenoceptors are homogenously distributed. The cholinergic and NANC nerve terminals possess modulatory α2- inhibitory receptors [8,9]. Isolation of SM cells under which α1-adrenoceptors do not lose their function was developed using antioxidants (dithiothreitol or taurine), high concentration of bovine serum albumin and the relatively specific enzyme, collagenase Type XI (Sigma). Phenylephrine (PhE) induced α1-adrenergic receptor mediated membrane hyperpolarization in TC and LM of GPI. It enhanced the amplitude of inward Ca2+ 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 Ca2+ 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 circulating hormones (e.g. steroids, catecholamines) the receptors and enzymes are affected 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),

Copyright © 2008 Institute of Experimental Pharmacology | ISBN 978–80–970003–7–0 Smooth muscle tissues as models for study of drug action 19 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 A2), or through G-proteins and enzymes (e.g. ad- enylate cyclase, guanylate cyclase, phospholipase C). The produced second messengers (e.g. cAMP, cGMP, prostaglandins, IP3, DAG) subsequently affecting intracellular receptors (e.g. IICR) or enzymes (e.g. proteinkinases) activate myosinkinase (phosphorylates 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 Mg2+ 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 depolarization 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 innervation. 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, indomethacine (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 prostaglandins do not contribute to the generation of NANC response. SP is probably the excitatory and VIP and NO are the inhibitory NANC transmitters [10,11].

5. Eﬀ ects of ROS ROS produced in cell membrane lipid bilayers, in the electron transport system of mitochondria, 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. H2O2 and superoxide (O2 ) in GPI • 1 LM were more effective than hydroxyl radical ( OH) and singlet oxygen ( O2). 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

Bauer et al. Trends in Pharmacological Research 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 O2 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 O2 generating systems appear to result from the presence and •– •– simultaneous action of at least two different ROS (O2 and H2O2). While O2 inhibited cholinergic and NANC transmission and participated at least in part in the evoked relaxation, H2O2 seems to be responsible for elevation of muscle tone and augmentation 2+ of cholinergic contractions and e.j.p.s, resulting from increase of [Ca ]i, with subsequent augmentation of stimulation of evoked contractions, as well as of the Ca2+ 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-dependent relaxation (EDR) and decrease in EC50 values of Ach. Inhibition of prostaglandin and NO synthesis by Indo and NG-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 resulted in diminished EDR of RA, increased endothelemia, elevated systolic blood pressure 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

Figure 2. Eﬀ ect of mesenteric ischemia (60 min) followed by reperfusion (30 min) on EDR of the SMA before (A) and after INOS with 100 μmol/l L-NAME (B). Relaxation is expressed as % of contraction induced by 1 μmol/l PhE. Data are means ± SEM of 8-12 measurements. *p<0.05, **p<0.001 against sham-operated. 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 tissue, 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 hydroxytoluene (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 induced 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 environment that exists in the intestinal lumen. This barrier is impaired in colitis – a chronically recurrent, inflammatory bowel disease, with a variety of etiological mechanisms,

Bauer et al. Trends in Pharmacological Research 22 V. Bauer et al. e.g. low tissue level of endogenous antioxidants and increased ROS production in oxidative 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 colonic injury, abolish the increase in myeloperoxidase (MPO) activity, attenuate the enhanced vascular permeability and prevent the depletion of GSH. Antioxidant activity of the compounds tested may be partly responsible for the observed protection. Activation of H3 receptors by α-methylhistamine and its prodrug, BP 294, on the model of trini- trobenzene sulphonic acid induced colitis exerted a beneficial effect. The H1 antagonist bisulepin reduced the extent of AA injury and diminished the MPO activity, vascular permeability and gamma-glutamyl transpeptidase activity. By interfering with the action 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 ratio, attenuation of granulocyte infiltration, as well as by reduction of increased respon- siveness of colonic SM to Ach and BaCl2. Dietary supplementation with oral pleuran, a β-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, pentacaine, 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 prostaglandins 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 Ca2+ from its intracellular stores may influence SM tone and activity due to alteration of Ca2+ 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

Copyright © 2008 Institute of Experimental Pharmacology | ISBN 978–80–970003–7–0 Smooth muscle tissues as models for study of drug action 23 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.

REFERENCES

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[14] Mátyás Š., Pucovský V., Bauer V.: Role of epithelium and metabolites of arachidonic cascade in the action of reactive oxygen species on the guinea pig trachea. Jap J Pharmacol 2002; 88: 270–278. [15] Bauer V., Oike M., Tanaka H., Inoue R., Ito Y.: Hydrogen peroxide induced responses of cat tracheal smooth muscle cells. Brit J Pharmacol 1997; 121: 867–874. [16] Nosáľová V., Drábiková K., Zúrová-Nedelčevová J., Jančinová V., Okruhlicová Ľ., Nosáľ R., Sotníková R.: Ischaemia/reperfusion-induced organ injury in low dose streptozotocin diabetes. Neuro Endocrinol Lett 2006; 27: 152–155. [17] Nosáľová V., Bauer V., Gajdošík A., Gajdošíková A., Navarová J.: Streptozotocin diabetes induced gastroin- testrinal damage in rats. Biologia 2000; 55: 91–94. [18] Nosáľová V., Bauer V.: Protective eff ect of stobadine in experimental colitis. Life Sci 1999; 65: 1919–1921. [19] Nosáľová V., Bauer V.: Protective eff ect of trapencaine in acetic-acid-induced colitis in rats. Infl ammophar- macology 1996; 4: 387–398.

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

Katarína BAUEROVÁ 1, Silvester PONIŠT 1, Katarína VALACHOVÁ 1, Danica MIHALOVÁ 1, Ladislav ŠOLTÉS 1, Denisa KOMENDOVÁ 1, Veronika TOMEKOVÁ 1, Miriam ŠTROSOVÁ 2, Peter GEMEINER 3, Giuseppe POLI 4 1 Department of Pharmacology of Inflammation, 2 Department of Biochemical Pharmacology, Institute of Experimental Pharmacology, Dúbravská cesta 9, 841 04 Bratislava, Slovak Republic, E-MAIL: [email protected] 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 several diseases and aging [1–4]. 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 diseases characterized by “inflammatory oxidative conditions”, with enhanced activity of either NAD(P)H oxidase (leading to atherosclerosis and chronic inflammation including 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. Bauerová et al. (2008) Trends in Pharmacological Research (Eds. V. Bauer et al.): 25–32. 26 K. Bauerová et al.

The pathogenesis of RA is associated predominantly with the formation of free radicals 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 [7–8]. 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 simpliﬁ ed in vitro model of conditions in rheumatic synovial ﬂ 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 [9–10]. 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 [11–12].

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 14–21) 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 induced in male Lewis rats, weighing 150–170 g each, by a single intradermal injection of heat-inactivated Mycobacterium butyricum in incomplete Freund´s adjuvant (Difco Laboratories, Detroit, MI, USA). In each experimental group 6–8 animals were used. Generally, in our experiments, inflammatory, arthritic, and oxidative stress parameters were assessed. The experiments included healthy intact animals as reference controls, arthritic animals not treated, and arthritic animals treated with the substances tested. In the experiment in which combination therapy with coenzyme Q10 (CoQ10) 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

Copyright © 2008 Institute of Experimental Pharmacology | ISBN 978–80–970003–7–0 New ways of supplementary and combinatory therapy of rheumatoid arthritis 27 compare the changes of this parameter with the results obtained for selected biochemical 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 proteins, was determined by enzyme linked immunosorbent assay (ELISA) [14]. The reaction of 4-hydroxynonenal (HNE) and malondaldehyde (MDA) with proteins is frequently 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 [15–16]. We studied the effect of therapy with CoQ10 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 Student’s t-test was used.

Results and discussion

The lack of relevant understanding concerning the pathogenesis of RA is a major problem 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]. Bauerová 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 normal synovial fluid [19] and these ions are most efficient in ROS generation. Ascorbate, a known antioxidant in the physiological concentration 40–140 μM [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 μM (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

Bauer et al. Trends in Pharmacological Research 28 K. Bauerová et al. of the degradation of HA. However, after a certain time – namely 30 min at 50 μM d-PN, 40 min at 100 μM d-PN, or approximately 90 min at 200 μM 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 [23–24]. 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 [25–27]. Antioxidant and anti- inflammatory activities were established for carboxylated (1–3)-beta-D-glucan isolated from Saccharomyces cerevisiae [28] and for beta-(1,3/1,6)-D-glucan (Immunoglukán®) 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 CoQ10 was found to slow down the progression of RA [31–32]. In our AA experiments, mitochondrial function in the heart

Figure 1. Eﬀ ects of D-PN (0, 50, 100, or 200 μM) on HA degradation induced by a system containing 1.0 μM CuCl2 plus 100 μM ascorbate.

Copyright © 2008 Institute of Experimental Pharmacology | ISBN 978–80–970003–7–0 New ways of supplementary and combinatory therapy of rheumatoid arthritis 29 and skeletal muscle and effectivity of supplementation with CoQ10 was dependent on the severity of the induced adjuvant arthritis. The results with solubilized CoQ10 (hy- drosoluble form) indicate its cardioprotective effect in the experimental model of adjuvant arthritis. They may thus be of potential significance in the treatment of patients with rheumatoid arthritis [33–38]. 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 oxidative 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, 35–36], we chose CoQ10 as one of the suitable candidates for combinatory therapy of RA. The aim was to assess if CoQ10 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 CoQ10. These promising clinical results were further completed by measurements of HNE- and MDA-protein adducts and protein carbonyls in plasma. We obtained a good agreement of the clinical and biochemical measurements: the effect was increasing in the order – CoQ10 or MTX alone and the combination of CoQ10 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 often used in therapeutic practice, concerning e.g. non-steroidal antiinflammatory drugs. In vivo we demonstrated that CoQ10 could potentiate the antiarthritic (decrease of hind paw volume) as well as the antioxidant effect of methotrexate on the level of oxidation of proteins (suppression of levels of protein carbonyls in plasma) as well as lipoperoxidation (suppression of levels of HNE-adducts and MDA-adducts to plasma proteins).

Bauer et al. Trends in Pharmacological Research 30 K. Bauerová et al.

Figure 2. Comparison of changes of hind paw volume with changes of oxidative stress parameters determined in plasma on day 28 after induction of adjuvant arthritis. Percentage of changes was calculated as the values of parameters determined for treated and for untreated arthritis compared to values of the control group (100%). a- control vs. AA; CoQ; MTX; CoQ+MTX; b- AA vs. CoQ; MTX; CoQ + MTX; 1-extremely signifi cant; 2-very signifi cant; 3-signifi cant; 4-not quite signifi cant; 5-not signifi cant

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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[28] Kogan G, Staško A, Bauerová K, Polovka M, Šoltés L, Brezová V, Navarová J, Mihalová D: Antioxidant properties of yeast (1→3)-β-D-glucan studied by electron paramagnetic resonance spectroscopy and its activity in the adjuvant arthritis. Carbohydr Polym 2005; 61: 18–28. [29] Stančíková M, Rovenský J, Švík K, Utešený J, Bauerová K, Jurčovičová J: Th e eff ects of immunostimulatory drugs on rat adjuvant arthritis. Rheumatologia 2008; 22: 9–13. [30] Mihalová D, Poništ S, Kucharská J, Komendová D, Bauerová K: Total antioxidant status – systemic marker of oxidative stress in adjuvant athritis. Chem Listy 2007; 101: 225–26. [31] Comstock GW, Burke AE, Hoff man SC, Helzlsouer KJ, Bendich A, Masi AT, Norkus EP, Malamet RL, Ger- shwin ME: Serum concentrations of alpha tocopherol, beta carotene, and retinol preceding the diagnosis of rheumatoid arthritis and systemic lupus erythematosus. Ann Rheum Dis 1997; 56: 323–25. [32] Knekt P, Heliövaara M, Aho K, Alft han G, Marniemi J, Aromaa A: Serum selenium, serum alpha-tocopherol, and the risk of rheumatoid arthritis. Epidemiology 2000 Jul; 11: 402–405.

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Effect of cytochrome P450 induction on drug disposition in isolated rat liver preparation

Štefan BEZEK 1, Marián KUKAN 2, Tomáš TRNOVEC 2 1 Laboratory of Cell Cultures, Institute of Experimental Pharmacology, SASc., Dúbravská cesta 9, 841 04 Bratislava, Slovak Republic, E-MAIL: [email protected] 2 Research Base of the Slovak Medical University, Limbová 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 pharmacological 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, induction would increase elimination of the drug, resulting in lower drug concentrations, and decrease the drug’s pharmacological effect. Second, induction may create an undesirable 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 hepatobiliary 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 Alzheimer’s 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].

Š. Bezek et al. (2008) Trends in Pharmacological Research (Eds. V. Bauer et al.): 33–40. 34 S. Bezek et al.

Results

Eﬀ ects 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 M1, (4-carbamoyl-l-ethyl-5-methylcarbamoyl-imidazole) was potentiated in comparison to control rats. Apparently, after 3-MC pretreatment, the concentrations of metabolite M1 peaked earlier than 5 min after the start of perfusion. Evidently, metabolite M1 was further metabolized as its concentration 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 MY 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 μM by 63% and 60%, respectively, and by α-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(0–120min) values calculated for control, PB, ISO, and 3-MC pretreated rat livers are shown in Table 1.

Table 1. The eﬀ ect of enzyme induction on perfusate AUC0-120min of TA following 2 h of liver perfusion [5].

AUC0-120min Treatment (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

Figure 1. Disposition of ethimizol in isolated perfused rat liver system [3]. Eﬀ ect of PB and 3-MC pretreatment on elimination of ethimizol and its metabolites M1, (4-carbamoyl-1-ethyl-5-methylcarbamoyl-imidazole) and MY (4,5-di-ethylcarbamoyl -imidazole) Top, ethimizol; middle, metabolite M1, bottom, metabolite MY. Livers from control (untreated) (), PB-treated () and 3-MC treated rats ().

Bauer et al. Trends in Pharmacological Research 36 S. Bezek et al.

Table 2. Eﬀ ect of enzyme induction (PB, ISO and 3-MC) on total amount of [14C]-TA-derived radioactivity excreted in bile, and glucuronidase sensitive metabolites following 2 h of liver perfusion. (Mean of three experiments) [5]. Total [14C] Glucuronidase sensitive metabolites Treatment (% of dose) (% 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

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’-azido-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 26.0±5.0 and 51.0±2% (mean ± S.D.) of administered radioactivity in livers from control and phenobarbitone- pretreated rats, respectively.

Eﬀ ects of Cytochrome P-450 induction on drug detoxiﬁ cation and activation 1. Effect of induction on detoxication and activation in IPRL Ethimizol is metabolised in IPRL into at least six metabolites. In recirculation experiments 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 increase in irreversible protein binding to microsomal, cytosolic and total liver pro-

Figure 2. Disposition of AZT in isolated perfused rat liver system [4]. Eﬀ ect of phenobarbitone pretreatment on concentration of AZT and main metabolites in rat liver perfused with [3H]-AZT. Control , PB-pretreated . Each point represents the mean ± S.D. from three determinations.

teins, respectively, compared to control [5]. Only a slight effect was observed on protein 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 metabolism rather than its transport into the cells, since on incubation of AZT (0–250 /m) with rat isolated hepatocytes, a linear relationship between concentration and amount

Bauer et al. Trends in Pharmacological Research 38 S. Bezek et al.

Table 3. Eﬀ ect of enzyme inducers (PB, ISO, and 3-MC) on irreversible binding of [14C]-TA-derived radioactivity to liver fractions after 2-h liver perfusion experiments [5]. Microsomes Cytosol Homogenate Treatment 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 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 (4°C), 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 prodrugs, 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 interactions 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 metabolites. 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 M1 and My [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-

Copyright © 2008 Institute of Experimental Pharmacology | ISBN 978–80–970003–7–0 Eff ect of CYP induction on drug disposition in isolated rat liver preparation 39 hibitor of xenobiotic metabolism in untreated and PB-treated rats, whereas ANF is primarily 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 hepatocytes , their ability to inhibit ethimizol biotransformation apparently operates at a metabolic level. Partial inhibition of ethimizol metabolism by SKF 525-A or ANF suggests 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 inducer of AZT metabolic enzymes since pretreatment of rats resulted in a 5.5-fold increase 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 irreversible protein binding in a model more similar to that of the intact rat. 3-MC pretreatment had the greatest effect on TA overall disposition. Further support for the involvement 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, accounting 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.

Bauer et al. Trends in Pharmacological Research 40 S. Bezek et al.

REFERENCES [1] Lin J.H. CYP induction-mediated drug interactions: in vitro assessment and clinical implications. Pharma- ceutical Research, 2006; 23: 1089–1116. [2] Bezek, S., Kukan, M., Kallay, Z., Trnovec, T., Stefek, M., Piotrovskiy, L. B. Disposition of ethimizol, a xanthine- related drug, in perfused rat liver and isolated hepatocytes. Drug Metab. Dispos. 1990; 18: 88–95. [3] Kukan M., Bezek S., Stefek M., Trnovec T., Durišová M., Piotrovskiy L.B.: Th e eff ect of enzyme induction and 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, 96–102. [4] Bezek, S., Kukan M., Bohov P.: Hepatobiliary disposition of 3’-Azido-3’-deoxythymidine (AZT) in the rat: Ef- fect of phenobarbitone induction. J. Pharm. Pharmacol. 1994; 46: 575–580. [5] Bezek S., Kukan M., Pool W.F., Woolf T.F. Th e eff ect of cytochromes P4501A induction and inhibition on the disposition of the cognition activator tacrine in rat hepatic preparations. Xenobiotica, 1996; 26: 935–946. [6] Seglen P.O.: Preparation of isolated rat liver cells. Methods Cell Biol. 1976; 13: 29–83. [7] Hewitt N.J., Lecluyse E.L., Ferguson S.S. Induction of hepatic cytochrome P450 enzymes: methods, mechanisms, recommendations, and in vitro-in vivo correlations. Xenobiotica. 2007; 37: 1196–224. [8] Zuber R., Anzenbacherová E., Anzenbacher P. Cytochromes P450 and experimental models of drug metabolism J.Cell.Mol.Med. 2002; 6: 189–198. [9] Kukan M., Bezek S., Trnovec T., Piotrovskiy L.B.: Th e eff ect of primary metabolites of the xanthine-related nootropic drug ethimizol on its hepatic extraction ratio. Drug Metabolism and Disposition 1990b, 18, 383–385. [10] Cretton, E. M., Xie, M. Y., Bevan, R. J., Goudgaon, N. M., Schinazi, R. F., Sommadossi, J.-P. Catabolism of 3’-azido-3’-deoxythymidine in hepatocytes and liver microsomes with evidence of formation of 3’-amino-3’-deoxythymidine, a highly toxic catabolite for human bone marrow cells. Mol. Pharmacol. 1991; 39: 258–266. [11] Guengerich F.P. Cytochrome P450 and chemical toxicology. Chem. Res. Toxicol. 2008; 21: 70–83.

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

Ján DŘÍMAL, Vladimír KNEZL, Anna BABULOVÁ, Ljuba BACHAROVÁ, Františka BOROVIČOVÁ, Mária DRAVECKÁ, Pavel GIBALA, Ján GVOZDJAK†, Anna GVOZDJAKOVÁ, Ján JAKUBOVSKÝ, Mária KITTOVÁ, Darius MAGNA, Alfonz RYBÁR, František Viliam SELECKÝ†, Ružena SOTNÍKOVÁ, Svorad ŠTOLC, Katarína STRÍŽOVÁ, Jana TOKÁROVÁ, Radomír NOSÁĽ, Anton SAUBERER, Eva NIKŠOVÁ, Slavo MARKOVIČ, Jozefína TOROKOVÁ, Andrea PUŠKÁROVÁ, Tomáš KOLLÁR Department of Cardiovascular Pharmacology, Institute of Experimental Pharmacology, Slovak Academy of Sciences, Dúbravská cesta 9, 841 04 Bratislava, Slovak Republic, E-MAIL: [email protected]

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-Marullaz´s 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. Selecky´s 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. Dřímal et al. (2008) Trends in Pharmacological Research (Eds. V. Bauer et al.): 41–47. 42 J. Dřímal et al.

Figure 1. Hemodynamic variables in 11 intact dogs following digoxin intoxication and EDTA infusion (mean ± S.E.). The deviations from control values signifi cant at the p<0.05 level are identifi ed by dots, those signifi cant at the p<0.01 levels are represented by triangles. [2] 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 coronary vascular smooth muscle and myocardial mechanics [3–5]. In the early 1950s, Sir James Black (I.C.I., UK) with his chemist John Stephenson had proposed a better therapeutic strategy to treat coronary artery disease. Later on they found that synthesis of the naphtyl-analog of isoproterenol would give a pure antagonist of β-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 β-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 studies (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 β-adrenergic receptor antagonist Trimepranolol on canine coronary blood flow. Later it focused also on studies of extracellular transport of β-adrenergic receptors in myocardial ischemia [8]. Studies on the intensity of myocardial depression of a new carbanilate compound [9] continued together with the analyses of selectivity of the new parent β-antagonist compound

Figure 2. Change in coronary perfusion pressure and arterial pressure following angiotensin during constant ﬂ ow perfusion of left anterior descending coronary artery. Phasic eﬀ ect of angiotensin. P coron = perfusion pressure, P art. = arterial blood pressure in mm Hg. [3–5] exaprolol. The myocardial selectivity and intensity of myocardial depression were further 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 necessary 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 β-adrenergic signaling delays the development of heart failure. However, in clinical practice, β-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 vascular 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 proximity 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 proteins 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-

Bauer et al. Trends in Pharmacological Research 44 J. Dřímal et al.

Type of Preparation Nature of Response( Influence on Coronary Blood Flow) (Group) Coronary Coronary Aortic Blood Flow Smooth Muscle Myocardial Heart Rate Blood Pressure Right-heart bypass and Constrict Constant Constant Variable constant HR () output rate effect (group C)

Right-heart bypass Constrict Constant Slowing Variable (group B) () output of rate () effect

Heart-lung preparation Constrict Block inotr. Slowing Constant (group D) () effects () of rate () pressure ()

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

Figure 3. Summary of the eff ects of oxprenolol on coronary blood fl ow in fi ve groups of experiments. [7]

Figure 4. SHR 7 days after myocardial infarction (section).

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 disease 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

Copyright © 2008 Institute of Experimental Pharmacology | ISBN 978–80–970003–7–0 Refl ection on animal modeling of human cardiac diseases in preclinical pharmacology 45 immune system, expressed mostly by inflammatory cells, drive hyperproliferative processes 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 proinflammatory 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 cytokine 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 suggested to describe the origin of systemic and myocardial immune/inflammatory activation. 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 (fi 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-α (TNF) mRNA concentrations were signifi cantly higher in the infarced zone (AAR) then in the noninfarced posterior wall (PW).* Statistically signifi cant increase. [12–16]

Bauer et al. Trends in Pharmacological Research 46 J. Dřímal et al. tein (mcp-1)), resulting in the initiation and progression of the hyperimmune pathological response directly in the myocardium. In our recent studies with adrenomedullin, we used spontaneously hypertensive rats (SHR) and permanent experimental myocardial infarction. This type of studies in cardiac tissues also involves massive activation and release of cytokines and activation of inflammatory response and within 7–10 days hypertrophic remodelation of the myocardium. The overexpression of inflammatory mediators, production of interleukins and the release of tumor necrosis factor-α, 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 prevents 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 nature´s many well kept secrets.

REFERENCES [1] Duchenne-Marullaz P., Gourgon, R., Luccioni R.: Myocardial infarction: Current problems. (Problemes ac- tuels poses par l´ischemie myocardiaque). Semaine des Hopitaux 1981, 57, (39–40), 1618–1619. [2] Pavek K., Drimal J., Selecky,F.V.: Circulatory eff ects of disodium EDEATE in digoxin-induced ventricular tachycardia. Cardiologia(Basel) 1967, 50, 297–304. [3] Drimal J.: Eff ects of angiotensin-II on coronary smooth muscle. Eur J Pharmacol 1968, 5, 56–68. [4] Drimal J., Pavek K., Selecky,F.V.: Primary and secondary eff ects of angiotensin-II on coronary circulation. Cardiologia (Basel) 1969, 54,1–15. [5] Drimal J. and Boska D.: Eff ects of angiotensin-II on myocardial mechaniscs and contractile state of heart muscle. Eur J Pharmacol 1973, 21, 120–136. [6] Black J.W. and Stephenson J.J.: Pharmacology of a new adrenergic β-receptor blocking compound. Lancet 1962, 2, 311–314. [7] Drimal J., Aviado D.M.: Eff ects of oxprenolol on coronary circulation and cardiac metabolism. J Pharmacol Exp Th er 1971, 176, 312–319. [8] Drimal J., Knezl V., Magna D., Strizova K.: External transport of beta-adrenergic binding sites in ischemic myocardium. J Gen Physiol Biophys 1978, 6, 583–591. [9] Knezl V.,Magna D., Sotnikova R., Drimal J.: Eff ects of a new beta-adrenolytic compound propyl-3-acetyl- 4-(2-1-hydroxy-3-isopropylamino)propoxy)carbanilate on isolated heart muscle. Arzneimittel-Forsch (Drug Res) 1994, 44, 7–12. [10] Rodbell M.(1980) Th e role of hormone receptors and GTP-regulatory proteins in membrane transduction. Nature 1980, 284, 17–22.

[11] Gilman A.G., Cross tals: Interview with A.Gilman Mol. Interv 2001, 1, 14–21. [12] Drimal J., Patoprsty V., KovacikV.: Stobadine is a potent modulator of endogenous endothelin in human cardiac fi broblasts.Life Sci 1999, 65, 1939–1941. [13] Drimal J.,Drimal J.Jr., Drimal D.: Enhanced endothelin ET(B) receptor down-regulation in human tumor cells Eur J Pharmacol 2000, 396, 299–304. [14] Drimal J., DrimalJ.Jr., Drimal D.: Diff erences in endothelin-1 (ET) mRNA expression,ET-receptor down-regulation and signaling in normal human fi broblasts and cancer cell lines.Biology 2005, 60, 1–12. [15] Drimal J., Drimal J.Jr.,Drimal D.: Hypoxic stress-enhanced expression and release of adrenomedullin (AM) and up-regulated AM receptors while glucose starvation reduced AM expression and release and down-regulated AM receptors in monkey renal cells. Phys Rev 2006, 55, 535–542. [16] Drimal J., Knezl V., Paulovicova E., Drimal D.: Enhanced early aft er-myocardial infarction concentration of TNF-α subsequently increased circulating and myocardial adrenomedullin in spontaneously hypertensive rats. Gen Physiol Biophys 2008, 27, 12–18.

Bauer et al. Trends in Pharmacological Research pharmacological research

New computational approach to mathematical modeling in pharmacological research

Mária ĎURIŠOVÁ 1, Ladislav DEDÍK 2, Martina TVRDOŇOVÁ 2 1 Institute of Experimental Pharmacology, Slovak Academy of Sciences, Dúbravská cesta 9, 841 04 Bratislava, Slovak Republic, E-MAIL: [email protected] 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 dynamic processes, such as drug dissolution [2,3], whole-body disposition behavior of drugs (e.g. absorption, distribution, elimination, etc.) [4–6], drug effect [7,8], drug bioavailability [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 [1–12]. In contrast, in a pharmacological context the term “dynamic” is conventionally used in relations to drug actions.

M. Ďurišová et al. (2008) Trends in Pharmacological Research (Eds. V. Bauer et al.): 48–57. New computational approach to mathematical modeling in pharmacological research 49

Model Models of dynamic systems are mathematical objects utilized to study processes formalized as dynamic systems. They can be mathematically described by differential equations, or by transfer functions [1–12].

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 modeling tools in the complex and time domain [2–12]. 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, a0…an, b1…bm and a0~1 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 example. 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 measurements 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

Bauer et al. Trends in Pharmacological Research 50 M. Ďurišová et al. 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 [1–12]. 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

Figure 1. Hypothetical example. A drug is i.v. administered in equal doses by: a single-bolus dose Isd(t), short-time infusion Iinf(t), multiple-bolus doses Imd(t), denoted by INPUTS I(t). Osd(t), Oinf(t), Omd(t), denoted by OUTPUTS O(t) are the related blood concentration-time profi les of the drug.

Copyright © 2008 Institute of Experimental Pharmacology | ISBN 978–80–970003–7–0 New computational approach to mathematical modeling in pharmacological research 51 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 eﬀ ect 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 measurements in digital forms on computers that are especially suitable to study rapid processes 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; τi and fi 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; MTge and G1 respectively are the mean-time parameter and gain of the subsystem that formalizes disintegration, dissolution, and gastric emptying processes; MTa and MTe respectively are the mean-time parameters of the absorption and elimination processes; MTr is the mean-time of the circulation process, τr is the time-delays of the circulation process, fr is the circulated fraction of the PXM dose; C is the resultant plasma concentration-time profi le of PXM.

Bauer et al. Trends in Pharmacological Research 52 M. Ďurišová et al.

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® Pfi zer) (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.

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 noradrenalin 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 [1–8]. The response of the model to noradrenalin injection was determined, see Figure 6.

Figure 5. Raw registered profi le of a vessel response.

Figure 6. Raw registered profi le of a vessel response after data-number reduction (points). Model response to mathematically described noradrenalin injection (line).

Physiological system An intravenous glucose tolerance test (IVGTT) is used to estimate parameters describing 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-

Bauer et al. Trends in Pharmacological Research 54 M. Ďurišová et al.

Figure 7. Physiologically-motivated model for frequently sampled intravenous glucose tolerance test. IRg(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; τx for x = 2, …, 4, L, LA, are the time delays of the related subsystems; MTx, for x = 1, …, 4, L, LA, are the mean times of the related subsystems; M0 is the instantaneous increase in the plasma glucose amount at the inlet of the subsystem CP, evoked by IRg(t); Mx, 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; ML is the decrease in the glucose amount at the inlet of the subsystem CP, evoked by the output of the subsystem L; Gx, for x = 1, …, 4, CP, L, determine the products of the gains and plasma fl ows of the related subsystem; ΔC(t) is the increase in the plasma glucose concentration above the basal level at the outlet of the subsystem CP; ΔCg,LA(t) is the increase in the plasma glucose concentration above the basal level at the outlet of the subsystem LA. ΔCi,LA(t) is the increase in the plasma insulin concentration-time profi le 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 interaction 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 glucose-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 profi le (circles); Responses of the model to the glucose infusion (lines).

Bauer et al. Trends in Pharmacological Research 56 M. Ďurišová 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 circulation, 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.

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[12] Tvrdoňová M, Dedík L, Mircioiu C, Miklovičová D, Ďurišová M: Physiologically-motivated time-delay model to account for mechanisms underlying enterohepatic circulation of piroxicam in humans. Basic Clin Phar- macol Toxicol 2008; 59: in press. [13] Purdie N, Province DW, Johnson EA: A convenient assay method for the quality control of peptides and proteins. J Pharm Sci 1999; 88:1242–8. [14] Pacini G, Bergman R N, MINMOD: a computer program to calculate insulin sensitivity and pancreatic re- sponsivity from the frequently sampled intravenous glucose tolerance test, Comput. Methods Programs Biomed 1986; 23: 113–122.

Bauer et al. Trends in Pharmacological Research pharmacological research

Breeding and testing facility Dobrá Voda

Andrej GAJDOŠÍK, Alena GAJDOŠÍKOVÁ, Eduard UJHÁZY, Daniela GOLHOVÁ, Bernardína KOPECKÁ, Viera KRCHNÁROVÁ Department of Toxicology and Laboratory Animal Breeding, Institute of Experimental Pharmacology, Slovak Academy of Sciences, 919 54 Dobrá Voda, Slovak Republic, E-MAIL: [email protected]

Key words: laboratory animal, breeding facility, testing facility Introduction

The biomedical sciences are based on laboratory research, which also includes experiments on animals. In 1951 a number of known scientists, motivated and convened by Dr. F. V. Selecký 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 Dobrá Voda [1]. The village Dobrá 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. Selecký, Prof. H. Rašková, Dr. Š. Mahr and Dr. K. Čerey 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. Kollár was Head of the facility, which became a subsidiary of the Institute of Experimental Pharmacology SASc for the next 20 years. In this time specialized 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 activities were divided into animal breeding and in vivo testing of chemical substances. Dr. A. Gajdošík was appointed Head of the Department with a staff of five.

A. Gajdošík et al. (2008) Trends in Pharmacological Research (Eds. V. Bauer et al.): 58–65. Breeding and testing facility Dobrá Voda 59

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 environment with temperature 20±2 °C, relative humidity 50–60%, 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 Dobrá 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 Dobrá Voda in 1987 from a colony maintained in VÚFB Pardubice – Rosice, CZ, which they had obtained from the Zentralinstitut für 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 Dobrá Voda. Species Outbred strain Inbred strain Rats WISTAR LEWIS –SHR Mice ICR BALB/c Gerbils MON – Guinea pigs TRIK – DH – Rabbits HIL –

Bauer et al. Trends in Pharmacological Research 60 A. Gajdošík 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 medical 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 Dobrá 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 Dobrá 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 In monogamous pairs, brother and sister mating.

Fields of use The LEWIS rat is a suitable model to study adjuvant-induced arthritis, experimental myocarditis, myastenia gravis, experimental allergic encephalomyelitis and transplantation.

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 Dobrá 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 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 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 Dobrá Voda in 2006 from the Medical University of Vienna, Austria.

Characteristics Albino mouse, docile.

Breeding In monogamous pairs, brother and sister mating.

Fields of use The BALB/c mouse is a useful strain for a wide range of applications: experimental allergic 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.

Bauer et al. Trends in Pharmacological Research 62 A. Gajdošík et al.

The TRIK Guinea pig – Dv : TRIK (Mad/Velaz/Ros) Origin The TRIK strain was introduced into the facility Dobrá Voda in 1994 from a colony maintained in VÚFB 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 reproductive 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 Dobrá Voda the strain was introduced 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 animal 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 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 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 VÚŽV 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 production and, lately, for screening embryotoxic agents and teratogens.

The animals produced at the breeding facility Dobrá Voda are designed for experimental 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 Dobrá 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 mouse0000445180 TRIK + DH Guinea pig 713 915 671 887 569 414 HIL rabbit 0 0 0 66 124 80 * the first six months are covered

Bauer et al. Trends in Pharmacological Research 64 A. Gajdošík 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 department 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 administration of a test material on organism and on various organs or physiological systems. A survey of studies performed at the testing facility Dobrá Voda is shown in Table 3.

Table 3. Chronological review of testing activities at the facility Dobrá Voda. Period Sponsor Type of test substance Type of test 1991–1993 IEPha SASc, SK Stobadine DP 1031 Chronic toxicity – 26 weeks 1994 LIKO VÚ, SK Carboxymethylglucane Acute oral toxicity – limit 1995 Slovasfalt, Inc., SK Asphalt MOAS I-S Acute oral toxicity – limit 1997–1998 Slovnaft, Inc., SK Oil Pekol 80 Acute oral toxicity – limit Grease V00 Acute dermal toxicity Gease AK1 EP konti Acute dermal irritation Grease WR Acute ocular irritation Oil M2T Global Oil Madit gas Asphalt varnish R 1997–2002 IEPha SASc, SK Streptozotocin Experimental model of Stobadine diabetes mellitus 2001–2007 IEPha SASc, SK Stobadine derivatives Acute toxicity No. 425 p.o. Acute toxicity No. 425 i.v. Acute toxicity No. 425 i.p. 2004–2005 IEP SASc, SK Magnetic nanoparticles Acute toxicity No. 425 p.o. Acute toxicity No. 425 i.v. 2005 Derma Protect Tincture LCD-DPI Acute dermal toxicity Innovation, Germany Acute dermal tolerance Dermal tolerance – 28 days 2006 Pharmacentrum SK Instillation XUN Acute toxicity No. 425 p.o. Acute toxicity No. 425 i.v. Acute toxicity No. 425 i.p. Oral toxicity – 28 days Ocular tolerance – 28 days 2006–2008 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, Košice

The most extensive was the chronic toxicity study of stobadine in 1992–1993 [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 1997–2002 the testing facility participated in the study of the experimental 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 antibodies 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 experimentation, 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 experimental results and cannot be defended on scientific, ethical or economical grounds.

REFERENCES [1] Balonová T., Čerey K.: Chov laboratórnych zvierat v ČSSR. Naša veda 1961; 233. [2] Gajdošíková A., Ujházy E., Gajdošík A., Chalupa I., Blaško M., Tomášková A., Liška J., Dubovický M., Bauer V.: Chronic toxicity and micronucleus assay of the new cardioprotective agent stobadine in rats. Arzneim. Forsch. Drug Res. 1995; 45(5): 531–536. [3] Štefek M., Tribulová N., Gajdošík A., Gajdošíková A.: Th e pyridoindole antioxidant stobadine attenuates his- tochemical changes in kidney of streptozotocin induced diabetic rats. Acta Histochem. 2002; 104(4): 413– 417. [4] Štolc S., Šnirc V., Májeková M., Gáspárová Z., Gajdošíková A., Štvrtina S.: Development of the new group of indole-derived neuroprotective drugs aff ecting oxidative stress. Cell Mol Neurobiol. 2006; 26(7–8): 1495– 1504. [5] Gajdošíková A., Gajdošík A., Koneracká M., Závišová V., Štvrtina S., Krchnárová V., Kopčanský P., Tomašovičová N., Štolc S., Timko M.: Acute toxicity of magnetic nanoparticles in mice. Neuro Endocrinol Lett. 2006; 27 (Suppl.2): 96–99.

Bauer et al. Trends in Pharmacological Research pharmacological research

Jaroslav KVĚTINA Stages of organization: 1978–1985, Department of Experimental Biopharmaceutics within the Institute of Experimental Medicine of the Czechoslovak Academy of Sciences (ČSAV); 1985–1993, Institute of Experimental Biopharmaceutics, ČSAV; 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 (ÚEBF) in a way paralleled, after an interval of twenty years, the history of the Pharmacological Institute of the Czechoslovak Academy of Sciences (FÚ ČSAV) in Prague. Similarly as the FÚ had its predecessor in the pharmacological department established (on the initiative of Prof. Helena Rašková) in the 1950s at the Institute of Organic Chemistry and Biochemistry, ČSAV, the anamnesis of the ÚEBF included the experimental biopharmaceutical department established (on the initiative of Prof. Jaroslav Květina) in the second half of the 1970s at the Institute of Experimental Medicine, ČSAV. The very first field of research of both these academic drug-oriented institutions, of both the pharmacological and the biopharmaceutical department, 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 ČSAV 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. Rašková). Similarly, also the ÚEBF was established as a separate unit within the ČSAV

J. Květina (2008) Trends in Pharmacological Research (Eds. V. Bauer et al.): 66–76. Pharmacokinetics and drug bioavailability studies 67 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 principles of the mechanisms of the fate of the drugs and dosage forms in the organism. The “service” research part the ÚEBF established links primarily with the Institute of Macromolecular Chemistry of the ČSAV 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 consisted in mapping of the biodistributional and pharmacokinetic indices of the substances 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 prevention in cyclosporine immunosuppression. The aims of more fundamental research lines of the ÚEBF 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 Králové, with which the Institute was closely connected (thanks to several joint laboratories and the interconnected university and academic posts of J. Květina). 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 experimental 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

Bauer et al. Trends in Pharmacological Research 68 J. Květina 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 analyses 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 [example: 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 relationships 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 biotransformation mechanisms of a number of drug models [2,23,24,43,52]; biotransformation of mesalazine is shown as an example (Figure 1). These studies resulted in the preference of the experimental minipig as the representative of the omnivores (weight, 30–35 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 foundations for a tabular aid usable generally in pharmacokinetic interspecies comparative research [8]. Generalizing predictions resulting from the pharmacokinetic studies of the intestinal compartment also utilized, besides perfusion techniques, the results from the research 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 application 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 ÚEBF into the present form took place in 1993 when the ČSAV was dissolved and the Academy of Sciences of the Czech Republic (AVČR) had to ”slenderize”. The institute was transformed into a joint research centre of the AVČR 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-

100

90 5-ASA 80 metabolite (N-acetyl-5-ASA) 70 60 50 40

AUC [μg.h/ml] 30 20 10 0 primate dog minipig man

Figure 1. Example of (nearly qualitative) diff 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 profi 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 signifi cantly lower (lower bioavailability of 5-ASA suggests also its diff erent biodistribution), in the experimental beagle the metabolite was not produced at all.

tion to oral dosage forms, in particular verification of pharmacokinetic bioequivalences 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 preclinical 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 animal 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 biodistribution (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-

Bauer et al. Trends in Pharmacological Research 70 J. Květina verted the prevailing part of the research capacity of the ÚEBF 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 present-day ÚEBF. The hitherto fifteen-year period of the joint centre of the Academy and a pharmaceutical manufacturer has laid preclinical and clinical foundations for registration 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 connected 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 (demonstration of the highest bond with HD-lipoprotein) formulated the thesis of a certain therapeutic “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 improvement in cerebral availability” of one of the derivatives of tacrine, the original methoxytacrine (from the synthetic provenience of the Military Medical Academy in Hradec Králové). 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-

Copyright © 2008 Institute of Experimental Pharmacology | ISBN 978–80–970003–7–0 Pharmacokinetics and drug bioavailability studies 71 plexes [25]. Experiments on rats managed to demonstrate that the substance was gradually 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, concentration cmax 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 successful 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 another 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 Králové, the Institute succeeded to utilize a capsule gastrointestinal 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 pharmacotherapeutic 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

Bauer et al. Trends in Pharmacological Research 72 J. Květina a) 160 30

140 25 120 B 20 100 A 80 15

60 10 40 Concentration [ng/ml] 5 20

0 0 0 1020304050607080 Time [hours] Time [hours]

6000 b)

5000

4000

3000

C 2000 B Concentration [ng/ml] 1000 A

0 024487296 Time [hours]

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)

Copyright © 2008 Institute of Experimental Pharmacology | ISBN 978–80–970003–7–0 Pharmacokinetics and drug bioavailability studies 73 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 digestive 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 opportunity not only for recollections and congratulations, but also for expressing a sincere combined wish. May the ÚEFa SAV in Bratislava maintain its high standard in research, and similarly, may the ÚEBF in Hradec Králové 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

Antonín LOJEK 1, Michaela PEKAROVÁ 1, Radomír NOSÁĽ 2, Jan HRBÁČ 3

1 Institute of Biophysics of the AS CR, v.v.i., Královopolská 135, 612 65 Brno, Czech Republic, E-MAIL: [email protected] 2 Institute of Experimental Pharmacology, SASc., Dúbravská cesta 9, 841 04 Bratislava, Slovak Republic 3 Department of Physical Chemistry, Palacký University, Faculty of Science, tř. 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 signalling molecule between phagocytes and other cells, and as an intracellular messenger 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 extracellular 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 produced 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-inflammatory cytokines such as tumor necrosis factor-α, interferon-γ and interleukin-1β [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.): 77–81. 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 (2–6 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 O2, superoxide anion radical, or hydrogen peroxide yield- – – – ing NO2, peroxynitrite (ONOO ), and NO2 /NO3 . Among direct methods which have been successfully applied in vivo and as well as in vitro biological models the electrochemical detection of NO can provide high sensitivity and also a good selectivity combined 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 Dullbecco’s modified Eagle’s 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 μl of culture medium containing 1 × 106 cells were placed into glass vials and then cultured in an incubator in 5%

CO2 at 37°C. After 2 hours, DMEM medium in glass vials with adherent macrophages was replaced by Minimal Eagle’s 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 CO2 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 dimensions 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 microsensor 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.

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 1×106 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 μM AMT (the selective inhibitor of iNOS).

Determination of nitrites in the cell culture supernatant by Griess reaction Concentration of nitrites in cell supernatants was measured as an indicator of NO production 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 Pečivová 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 5–6 h of co-incubation of the macrophages with LPS. In following 2 h a gradual increase in NO production was detected. After reaching 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-

Bauer et al. Trends in Pharmacological Research 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 μM, 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 extensive 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 either 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 electrochemical 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 5–6 h after the cells stimulation (as detected by both microsensor and by detection of nitrites using Griess reaction) increases rapidly to reach its maximum 6–7 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 6–12 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 microelectrode and also extensive decrease in nitrite accumulation in supernatants, the iNOS protein expression in our experiments remained unaffected. As it is known [11], AMT

Copyright © 2008 Institute of Experimental Pharmacology | ISBN 978–80–970003–7–0 Real-time monitoring of nitric oxide generation by macrophages in vitro 81 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 detection 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 stimulated 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.

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Bauer et al. Trends in Pharmacological Research pharmacological research

H1-antihistamine Dithiaden® suppressed platelet aggregation and oxidative burst of neutrophils in vitro

Radomír NOSÁĽ, Katarína DRÁBIKOVÁ, Viera JANČINOVÁ, Tatiana MAČIČKOVÁ, Jana PEČIVOVÁ, Margita PETRÍKOVÁ, Zuzana STRAKOVÁ Department of Cellular Pharmacology , Institute of Experimental Pharmacology, Slovak Academy of Sciences, Dúbravská cesta 9, 841 04 Bratislava, Slovak Republic, E-MAIL: [email protected]

Key words: Dithiaden®, neutrophils, blood platelets, oxidative burst, platelet aggregation

Introduction

Pharmacological activity of antihistamines strongly depends on subtype receptor interactions. Besides their antihistaminic activities, H1-receptor antagonists possess other pharmacological activities: antiiflammatory action, inhibition of blood platelet function 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 H1-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. Ca2+ [1]. In this study we compared the effect of Dithiaden® (DIT) from the 1st generation of H1-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 × 105 platelets/μL. For aggregation studies 450 μL

R. Nosáľ et al. (2008) Trends in Pharmacological Research (Eds. V. Bauer et al.): 82–87. Dithiaden® suppressed platelet aggregation and oxidative burst of neutrophils 83 of platelet suspension was used together with 20 μL of DIT. Aggregation was induced with calcium ionophore A23187 (final concentration 1.8 μmol/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 μL, 104 platelets/ μL) were treated with 20 μL 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 125 of a [ I]thromboxane B2 (TXB2) 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 solution to a final concentration of 107 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 μmol/L), isoluminol and horseradish peroxidase (HRP), each in 50 μL 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 (2×106/sample) were preincubated with cytochalasine B (5 μg/mL), subsequently with DIT and stimulated with PMA. MPO activity was assayed in the supernatant 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.

Bauer et al. Trends in Pharmacological Research 84 R. Nosáľ et al.

Materials

Dithiaden®: Léčiva (Czech Republic); hydrogen peroxide (Fluka); luminol, isoluminol, 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, horseradish peroxidase, cytochrome c: Merck (Darmstadt, Germany); thromboxane B2 RIA kit: Institute of Radioisotopes Hungary; Tyrode solution ( pH 7.4; 136.9 mmol/L NaCl; 11.9 mmol/L NaHCO3; 0.4 mmol/L NaH2PO4.2 H2O; 1 mmol/L MgCl2.6H2O a 5.6 mmol/L glucose). All other chemicals of analytical grade were from available commercial 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 μmol/L DIT for both stimuli used: A23187 and thrombin. Increasing concentration of DIT to 50 and 100 μmol/L resulted in 80% inhibition of aggregation. DIT in the concentration of 0.1 μmol/L significantly decreased TXB2 generation in platelets stimulated with thrombin and increase in DIT concentration to 100 μmol/L resulted in 95% inhibition of TXB2 formation. In platelets stimulated with A23187, DIT was effective only in the highest concentration used (100 μmol/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 μmol/L decreased whole blood CL by 20% and increase of the concentration of DIT to 10 and 100 μmol/L resulted in CL decrease by 58 and 93%, respectively. The intracellular CL decreased with 0.1, 1, 10 and 100 μmol/L DIT by 14, 17, 22 and 91% of the control, respectively. The extracellular CL significantly decreased with 10 and 100 μmol/L by 58 and 99%, respectively. Superoxide generation significantly decreased with 100 μmol/L DIT by 49%. On the other hand, MPO liberation decreased significantly with 1, 10 and 100 μmol/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 TXB2 generation with DIT was parallel with inhibition of thrombin-induced aggregation. Since DIT also inhibited arachidonic acid liberation from platelet membranes and malondialdehyde formation from arachidonate pathway [9], it has been suggested

Figure 1. Decreased aggregation and thromboxane B2 formation in human platelets stimulated with thrombin or Ca2+-ionophore A23187 in the presence of dithiaden. Mean ± SEM, n = 6, *p<0.05, **p<0.01.

that DIT, like other cationic amphiphilic drugs, suppressed platelet cytosolic phospholipase A2 activity [10]. A similar effect was demonstrated for other H1-antihistamines, indicating rather interaction with platelet membrane structure than with platelet H1- receptors. H1-antihistamines may interfere with platelet aggregation first at arachidonic acid pathway, second due to the interference with platelet Ca2+ mobilization [see inhibition of A23187-induced aggregation – 11]. In addition to the suppression of oxidative burst of blood phagocytes, DIT dose-dependently inhibited CL of isolated neutrophils both at extra- and intracellular site. This was evaluated by means of two luminophores, luminol and isoluminol [4–6]. A similar effect was demonstrated for cationic amphiphilic drugs from different pharmacological groups. Decrease in extracellular CL might result from scavenging extracellular reactive 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 interference 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, H1-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 family of isoenzymes differing in structure, co-factor requirement and substrate specificity [15]. PMA-stimulated responses differed in their sensitivity to DIT inhibition, with

Bauer et al. Trends in Pharmacological Research 86 R. Nosáľ et al.

Figure 2. Eﬀ ect 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=6–7, *p<0.05, **p<0.01.

superoxide production being less sensitive than MPO release. Neither horse radish peroxidase nor myeloperoxidase activity was inhibited by DIT in our experimental conditions (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 oxidative 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 H1-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 professional phagocytes, DIT might beneficially contribute to the treatment of inflammation, immune 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.

REFERENCES

[1] Leurs R, Church MK, Togliatela MH. H1-antihistamines: Inverse agonists, anti-infl ammatory actions and cardiac eff ects. Clin Exper Allergy 2002; 32: 489–98.

[2] Nosáľ R, Drábiková K, Jančinová V, Petríková M, Fabryová V. Antiplatelet and antiphagocyte activity of H1- antihistamines. Infl amm Res 2005; 54: S19–20.

[3] Nosáľ R, Jančinová V, Danihelová E. Th e H1-antihistamine antagonist dithiaden inhibits human platelet function in vitro. Platelets 1997; 8: 175–80. [4] Drábiková K, Nosáľ R, Jančinová V, Číž M, Lojek A. Reactive oxygen metabolite production is inhibited by histamine and H1-histamine antagonist dithiaden in human PMN leukocytes. Free Rad Res 2002; 6: 975–80. [5] Jančinová V, Drábiková K, Nosáľ R, Holomáňová D. Extra- and intracellular formation of reactive oxygen species by human neutrophils in the presence of pheniramine, chlorpheniramine and brompheniramine. Neuro Endocrinol Lett 2006; 27(Suppl.2): 141–3. [6] Jančinová V, Drábiková K, Nosáľ R, Račková L, Májeková M, Holomáňová D. Th e combined luminol/isoluminol chemiluminescence method for diff erentiating between extracellular and intracellular oxidant production by neutrophils. Redox Report 2006; 11: 110–6. [7] Pečivová J, Mačičková T, Lojek A, Gallová L, Číž M, Nosáľ R, Holomáňová D. In vitro eff ect of carvedilol on professional phagocytes. Pharmacology 2007; 79: 86–92. [8] Pečivová J, Mačičková T, Lojek A, Gallová L, Číž M, Nosáľ R, Holomáňová D. Eff ect of carvedilol on reactive oxygen species and enzymes linking innate and adaptive immunity. Neuro Endocrinol Lett 2006; 27: 160–3.

[9] Nosáľ R, Jančinová V, Danihelová E. Th e eff ect of H1-histamine antagonist dithiaden inhibits platelet function in vitro. Platelets 1999; 8: 175–80.

[10] Nosáľ R, Jančinová V. Cationic amphiphilic drugs and phospholipase A2 (PLA2). Th romb Res 2002; 105: 339–45. [11] Nosáľ R. Antiplatelet and antileukocyte eff ects of cardiovascular, immunomodulatory and chemotherapeu- tic drugs. Cardiovasc Hematol Agent Med Chem 2006; 4: 237–61. [12] Drábiková K, Jančinová V, Nosáľ R, Solík P, Murín J, Holomáňová D. On the antioxidant activity of carvedilol in human polymorphonuclear leukocytes in vitro and ex vivo. Neuro Endocrinol Lett 2006; 26: 138–40. [13] Edwards SW. Th e respiratory burst: Th e generation of reactive oxygen metabolites and their role in microbial killing. In: Biochemistry and Physiology of the neutrophil. Cambridge University Press 1994, p. 1–291.

[14] Králová J, Číž M, Nosáľ R, Drábiková K, Lojek A. Eff ect of H1-antihistamines on the oxidative burst of rat phagocytes. Infl amm Res 2006; 55: S15–6. [15] Hug H and Sarre TF. Protein kinase C isoenzymes: divergence in signal transduction? Biochem J 1993; 291: 329–43. [16] Pečivová J, Mačičková T, Nosáľ R, Danihelová E. Eff ect of stobadine on superoxide generation and degranu- lation of stimulated human polymorphonuclear leukocytes in vitro. Biologia 2000; 55 Suppl.8: 103–6. [17] Lieberman P. Preclinical evidence of azelastine hydrochloride activity. Curr Th er Res Clin Exp 2002; 63: 556–71.

Bauer et al. Trends in Pharmacological Research pharmacological research

Research focuses of the pharmacology in Martin

Gabriela NOSÁĽOVÁ, Soňa FRAŇOVÁ, Anna STRAPKOVÁ, Juraj MOKRÝ, Martina ŠUTOVSKÁ, Vladimíra SADLOŇOVÁ Department of Pharmacology Jessenius Medical Faculty in Martin, Comenius University in Bratislava, Sklabinská 26, 037 53 Martin, Slovak Republic, E-MAIL: [email protected]

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 experimental 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 (NK1, NK2, 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 converting enzyme inhibitors is complicated by chronic dry cough. We decided to study not only mechanisms of this relationship but also the possibilities of its pharmacological 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 several 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. Nosáľová et al. (2008) Trends in Pharmacological Research (Eds. V. Bauer et al.): 88–95. Research focuses of the pharmacology in Martin 89

Moreover, we are also interested in problems connected with mechanisms of hyperreactivity of the airways. In conditions of our models of experimental hyperreactivity, 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 homeostasis, c) mechanisms of NO action. Some epidemiological studies have suggested that dietary factors, including consumption 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 hyperreactivity of the airways. Our attention will be focused on pharmacological modulation of airways hyperresponsiveness 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 reﬂ ex: a) mechanical stimulation of the airways In our department we have been involved for over 35 years in pharmacological modulation 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 accompanied 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 exposure, 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 conditions, 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

Bauer et al. Trends in Pharmacological Research 90 G. Nosáľová 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 classical model. The chemical model of airways hyperreactivity was developed in our department. 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 mammary 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 40–60 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 chemical 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 other 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

Copyright © 2008 Institute of Experimental Pharmacology | ISBN 978–80–970003–7–0 Research focuses of the pharmacology in Martin 91 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, significant 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 [7,8,18–23]. 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 administration 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 synthesis – 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 administration. 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 nonspecific (L-NAME) or specific inhibitors (aminoguanidine). We recorded a significant decrease of tracheal smooth muscle reactivity after acute L-NAME pre-treatment in toluene-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 pretreatment in toluene-induced hyperreactivity but changes in allergen-induced hyperreactivity were not significant.

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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 Nω-hydroxy-L-arginine (NOHA) [28]. We did not record significant 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 μmol 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-induced 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 respiratory 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 experimental meconium aspiration syndrome (MAS) with significantly increased in vitro airway reactivity [32]. We can state that our model of experimental premenopausal mammary carcinogenesis 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 according 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 accumulation of bradykinin, substance P, prostanoids and another inflammatory neuropeptides in the airways. Our results showed a partially protective effect of diltiazem and enalapril co-administration on the adverse respiratory effects induced by enalapril therapy. Another combination therapy able to minimise the respiratory side effects of ACE inhibitors is co-administration of these antihypertensive drugs with inhaled furosemide. Our study of the mechanisms of hyperreactivity showed that the effect of NOS inhibitors 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 observed changes in airway reactivity with accompanying beneficial effect on vital respiratory 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 hyperreactivity.

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 molecular 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.

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We will keep our orientation on studying the activity of agents influencing the respiratory 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 experimental 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

REFERENCES [1] Korpas J., Nosalova G.: Pharmacotherapy of cough. Osveta Martin, 1991, p. 335. [2] Nosalova G., Strapkova A., Korpas J., Crisciulo D.: Objective assessment of cough suppressants under normal and pathological experimental conditions. Drugs Under Experimental and Clinical Research 1989; 15: 77–81. [3] Strapkova A., Nosalova G., Banovcin P., Giacova, D.: Toluen and smooth muscle of the airways. Prac Lek 1992; 44: 105–9. [4] Strapkova A., Nosalova G., Hanacek J.: Eff ects of irritants on airways reactivity. Centr Eur J Publ Hlth 1996; 4: 54–5. [5] Mokry J., Nosalova G.: Evaluation of the cough refl ex and airway reactivity in toluene- and ovalbumin- in- duced airway hyperresponsiveness. J Physiol Pharmacol 2007; 58 (Supl 5): 419–26. [6] Sadlonova V., Kubatka P., Svecova I., Kajo K., Nosalova G., Sadlonova J.: Tumour suppressive eff ect of letro- zole in mammary carcinogenesis of female rats. Acta Med Martiniana 2007; 7: 9–12. [7] Nosalova G., Capek P., Sutovska M., Franova S., Matulova M.: Antitussive Active Polysaccharides from Or- namental – Medicinal Plants. Floriculture, Ornamental and Plant Biotechnol. 2006; 4: 471–80. [8] Nosalova G., Fraňová S., Mokrý J., Sutovska M.: Pharmacotherapy of cough. In: Cough from Lab to Cloníc. Editet by::Korpas, J., Paintal S., Anand A., Publisher: Ane Books India, 2007, p. 253–311. [9] Nosalova G., Strapkova A., Korpas J.: A new antitussive drug – Ditustat Spofa. Bratisl. Lek. Listy 1982a; 78: 257–64. [10] Nosalova G., Varonos D., Papadopoulous-Daifotis Z.: Cough and central GABA-ergic mechanism. Bratisl. Lek. Listy 1986a; 85: 526–32. [11] Nosalova G., Varonos D., Papadopoulous-Daifotis Z., Visnovsky P., Strapkova A.: A GABA-ergic mechanism in the central control of cough. Acta Physiologica Hungarica 1978; 70: 189–94. [12] Nosalova G., Strapkova A., Korpas J.: Following antitussic activity of analgesic drug tilidine. Bratisl. Lek. Listy 1985c; 84: 653–8. [13] Strapkova A., Nosalova G., Korpas J.: Relationship of antitussic and analgesic activity of various substances. Bratisl. Lek. Listy 1987; 88:538–45. [14] Sutovska M., Nosalova G., Franova S.: Th e role of potassium ion channels in cough and other refl exes of the airways. J. Physiol. Pharmacol 2007; (suppl.5), 58: 673–83. [15] Korpas J., Nosalova, G., Widdicombe, J.G.: Th e antitussive actions of the drug RU-20201 given as an aerosol to cats. J.Pharm. Pharmacol 1978a; 30:563–5. [16] Mokrý J., Nosáľová G.: Relationship between cough and bronchoconstriction in condition hyperreactviity of the airway. Progress of Pharmacology in Slovak republic II, Bratislava 2007; 60–4.

[17] Nosáľová G., Mokrý J.: Th e mechanism of action of xanthine derivatives and the suppresion of cough. Acta Med Martiniana 2001; 1: 14–8. [18] Nosalova G., Strapkova A., Kardosova A., Capek P., Zathurecky L., Bukovska E.: Antitussive action of extracts and polysaccharides of marshmallow. Pharmazie 1992; 47: 224–6. [19] Nosalova G., Strapkova A., Kardosova A., Capek P.: Antitussive activity of a rhamnogalacturonan isolated from the roots of Althaea offi cinalis L. var. Robusta. J Carbohydrate Chemisty 1993; 12: 589–96. [20] Franova S., Kardosova A., Kostalova D.: Herbal polysaccharides in the therapy of cough. Bratisl. Lek. Listy 1998; 99: 108–10. [21] Nosalova G., Sutovska M., Mokry J., Kardosova A., Capek P., Khan M.: Effi cacy of the herbal substances according to cough refl ex. Minerva Biotechnol. 2005; 17: 141–52. [22] Nosalova G., Mokry J., Hassan K.: Antitussive activity of the fruit extract of Emblica offi cinalis Gaertn. (Eu- phorbiaceae). Phytomedicine 2003; 10: 583–89. [23] Nosalova G., Mokry J., Ather A., Khan MTH.: Antitussive activity of ethanolic extract of paederia foetida (Rubiaceae family) in non-anaesthetized cats. Acta Vet Brno 2007; 76: 27–33. [24] Franova S., Nosalova G., Pechanova O., Sutovska M.: Red wine polyphenolic compounds inhibit tracheal smooth muscle contraction during allergen-induced hyperreactivity of the airways. J. Pharmacy and Phar- macology 2007; 59: 727–32. [25] Franova S., Nosalova G., Antosova M., Nosal S.: Enalapril and diltiazem co-administration and respiratory side eff ects of enalapril. Physiol Res 2005; 54: 515–20. [26] Strapkova A., Antosova M., Nosalova G.: Relation of L-arginine to the airway hyperreactivity. Gen Physiol Biophys 2008; 27: 85–91. [27] Strapkova A., Nosalova G.: Nitric oxide and airway reactivity. Bratisl Lek Listy 2001; 102: 345–50. [28] Antosova M., Strapkova A., Nosalova G., Mokry J.: Eff ect of nitric oxide synthases inhibitors on exogenous irritant-induced bronchial hyper-reactivity in guinea pigs. Gen Physiol Biophys 2006; 25: 137–47. [29] Strapkova A., Antosova M., Nosalova G.: Interaction between nitric oxide and prostanoids in the respiratory system. Bratisl Lek Listy 2006; 107: 52–7. [30] Mokry J., Mokra D., Nosalova G.: Direct in vitro eff ects of meconium on airway reactivity in adult rabbits. Bratisl Lek Listy 2006; 107: 9–12. [31] Mokry J., Mokra D., Nosalova G.: Eff ects of meconium on airway reactivity to histamine and acetylcholine in vitro. J Physiol Pharmacol 2007; 58: 409–17. [32] Mokry J., Mokra D., Antosova M., Bulikova J., Calkovska A., Nosalova G.: Dexamethasone alleviates meconium-induced airway hyperresponsiveness and lung infl ammation in rabbits. Pediatr Pulmonol 2006; 41: 55–60. [33] Sadlonova V., Kubatka P., Kajo K., Nosalova G.: Th e model of premenopausal breast cancer: eff ects of letro- zole. Biomed Pap 2007; 151: 75–7. [34] Nosalova G., Mokry J., Franova S.: Pharmacological modulation of cough refl ex. In: Khan MTH: Lead molecules from natural products: Discovery and New Trends. Advances in phytomedicine 2 – Amsterdam: El- sevier 2006, p. 87–110.

Bauer et al. Trends in Pharmacological Research pharmacological research

Trends in pharmacological research – contribution from studies of the membrane transport and cell signaling

Karol ONDRIAŠ Institute of Molecular Physiology and Genetics, Centre of Excellence for Cardiovascular Research, SASc, Vlárska 5, 833 34 Bratislava, Slovak Republic, e-mail: [email protected]

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 physiological 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 conditions. 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 I–III, 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 molecular 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. Ondriáš (2008) Trends in Pharmacological Research (Eds. V. Bauer et al.): 96–101. Contribution from studies of the membrane transport and cell signaling 97

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 calcium 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 H2S [8]. Results

Intracellular chloride channels and apoptosis Apoptosis has been implicated in a variety of heart diseases, such as myocardial infarction, 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 μmol/l) inhibited the H2O2-induced apoptosis in cardiomyocytes and decreased open probability of the chloride channels derived from the mitochondrial and lysosomal vesicles [2]. These results may contribute to the understanding a possible involvement of intracellular chloride channels in apoptosis and cardioprotection. Similarly, BKA (1–100 μmol/l), ATR and ATR derivative CAT (5–100 μmol/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 DIDS–NPPB versus BKA–CAT effect [2] may by used as a pharmacological tool to categorize chloride channels. We have observed that the chloride channel blocker NPPB activated potassium conductance of potassium-chloride promiscuous channels in mitochondrial membranes [10].

Inhibition of calcium Cav1.3 current Calcium currents (ICa) in inner hair cells (IHCs) are carried by the Cav1.3 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.

Bauer et al. Trends in Pharmacological Research 98 K. Ondriaš

Previously a low sensitivity of the Cav1.3 subtype towards dihydropyridines has been demonstrated [11]. Therefore, an effect of two phenylalkylamines (verapamil and gallopamil) and the benzothiazepine diltiazem on ICa through Cav1.3 channels in mouse IHCs was evaluated [4]. The phenylalkylamines verapamil and gallopamil and the benzothiazepine diltiazem inhibited ICa in IHCs in a concentration-dependent manner [4]. This block was largely reversible. Inhibition of the peak ICa by phenylalkylamines and benzothiazepines was voltage independent. Concentrations of phenylalkylamines and benzothiazepine necessary to inhibit 50% of ICa in IHCs were one order larger compared to concentrations, which inhibited ICa through Cav1.2 channels in native cells or expression systems. However, inhibitory concentrations were in the same range as those required for block of ICa in turtle hair cells. A neurotoxic effect of inorganic mercury (Hg2+) and methylmercury (MeHg) was studied on neuronal Cav3.1 (T-type) calcium channel stably expressed in the human embryonic kidney (HEK) 293 cell line. Hg2+ and MeHg inhibited current through the Cav3.1 calcium channel in concentrations 10 nmol/l and higher with an IC50 of 0.63±0.11 μmol/l. and 13.0±5.0 μmol/l, respectively [5]. The interaction with the Cav3.1 calcium channel may significantly contribute to neuronal symptoms of mercury poisoning during 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 (ICa–v) by antidepressants 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 ICa–v 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 ICa–v, with IC50 values of 8.3, 11.6, 11.7, 23.2, 31.0, 64.5, and 364 μmol/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 antidepressants 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].

H2S releases nitric oxide from nitrosothiols and L1210 leukaemia cells An endogenously produced H2S is involved in neuromodulation, cell proliferation, apoptosis, regulation of cardiac function and cardioprotection, vasorelaxation, hypertension, and septic, endotoxin and haemorrhagic shocks and inflammation processes [12]. Many of the effects are shared with NO. Therefore, the H2S-NO interaction was

– studied. It was observed that H2S 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 H2S was responsible for the NO-releasing effect. We assumed that the releasing effect is responsible for some of H2S 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 overexpression 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 parental cells with a stepwise increasing concentration of vincristine [6,7,13]. Based on P-gp overexpression, both doxorubicin and vincristine induce a common multidrug resistance 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 substance, 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 methylxanthines, 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 vincristine resistance in L1210/VCR cells significantly and in a concentration dependent manner [16]. MDR reversal effect of LY294,002 was accompanied with this compound’s influence on vincristine-induced apoptosis. The results pointed to the possible involvement of PI3K/Akt kinase pathway in modulation of P-gp mediated multidrug resistance in L1210/VCR mouse leukemic cell line [16,17].

Bauer et al. Trends in Pharmacological Research 100 K. Ondriaš

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 connected not only to cardiovascular diseases, but also with cancer is important. – We observed that H2S and HS donor NaHS released NO not only from NO donors but also from rat brain homogenate [10]. From this observation we assumed that H2S and/or HS– releases NO from S-nitrosothiols and/or metal nitrosyl complexes in vivo. This might be supported by numerous reports that H2S 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 H2S 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 H2S 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 gratefully acknowledged.

REFERENCES [1] Malekova L, Kominkova V, Ferko M, Stefanik P, Krizanova O, Ziegelhoff er A, Szewczyk A, Ondrias K: Bong- krekic acid and atractyloside inhibits chloride channels from mitochondrial membranes of rat heart. Bio- chim Biophys Acta 2007; 1767: 31–4.

[2] Malekova L, Tomaskova J, Novakova M, Stefanik P, Kopacek J, Lakatos B, Pastorekova S, Krizanova O, Breier A, Ondrias K: Inhibitory eff ect of DIDS, NPPB, and phloretin on intracellular chloride channels. Pfl ugers Arch 2007; 455: 349–57. [3] Zahradnik I, Minarovic I, Zahradnikova A: Inhibition of the cardiac L-type calcium channel current by antidepressant drugs. J Pharmacol Exp Th er 2008; 324: 977–84.

[4] Tarabova B, Lacinova L, Engel J: Eff ects of phenylalkylamines and benzothiazepines on Cav1.3-mediated Ca2+ currents in neonatal mouse inner hair cells. Eur J Pharmac 2007; 573: 39–8. [5] Tarabova B, Kurejova M, Sulova Z, Drabova M, Lacinova L: Inorganic mercury and methylmercury inhibit the Cav3.1 channel expressed in human embryonic kidney 293 cells by diff erent mechanisms. J Pharm Exp Th er 2006; 317: 418–27. [6] Sulová Z, Macejová D, Seres M, Sedlák J, Brtko J, Breier A. Combined treatment of P-gp-positive L1210/ VCR cells by verapamil and all-trans retinoic acid induces down-regulation of P-glycoprotein expression and transport activity. Toxicol In Vitro. 2008 Feb;22(1):96–105. [7] Breier A, Barancik M, Sulova Z, Uhrik B: P-glycoprotein – implication of metabolism of neoplastic cells and cancer therapy. Curr Cancer Drug Targets 2005; 5: 457–68.

[8] Ondrias K, Stasko A, Cacanyiova S, Sulova Z, Krizanova O, Kristek F, Malekova L, Knezl V, Breier A: H2S and HS- donor NaHS releases nitric oxide from nitrosothiols, metal nitrosyl complex, brain homogenate and murine L1210 leukaemia cells. Pfl ugers Arch 2008; in press, DOI: 10.1007/s00424-008-0519-0 [9] Fliss H, Gattinger D: Apoptosis in ischemic and reperfused rat myocardium. Circ Res 1996; 79: 949–56. [10] Ondrias K, Malekova L, Krizanova O: Potassium-chloride promiscuous channels in mitochondrial membranes. Gen Physiol Biophys 2008; 27: 38–4. 2+ [11] Michna M, Knirsch M, Hoda JC, Muenkner S, Langer P, Platzer J, Striessnig J, Engel J: CaV1.3 (α1D) Ca currents in neonatal outer hair cells of mice. J Physiol 2003; 553: 747–58.

[12] Lowicka E, Beltowski J: Hydrogen sulfi de (H2S)—the third gas of interest for pharmacologists. Pharmacol Reports 2007; 59: 4–24. [13] Bohacova V, Sulova Z, Dovinova I, Polakova E, Barancik M, Uhrık B, Orlicky J, Breier A: L1210 cells culti- vated under the selection pressure of doxorubicin or vincristine express common mechanisms of multidrug resistance based on the overexpression of P-glycoprotein. Toxicol Vitro 2006; 20: 1560–8. [14] Drobna Z, Stein U, Walther W, Barancik M, Breier A: Pentoxifylline infl uences drug transport activity of P- glycoprotein and decreases mdr1 gene expression in multidrug resistant mouse leukemic L1210/VCR cells. Gen Physiol Biophys 2002; 21: 103–9. [15] Kupsakova I, Rybar A, Docolomansky P, Drobna Z, Stein U, Walther W, Barancik M, Breier A: Reversal of P-glycoprotein mediated vincristine resistance of L1210/VCR cells by analogues of pentoxifylline A QSAR study. Eur J Pharmaceut Scien 2004; 21: 283–93. [16] Barancik M, Bohacova V, Sedlak J, Sulova Z, Breier A: LY294,002, a specifi c inhibitor of PI3K/Akt kinase pathway, antagonizes P-glycoprotein-mediated multidrug resistance. Eur J Pharmaceut Scien 2006; 29: 426– 34. [17] Uhrík B, El-Saggan AH, Seres M, Gibalová L, Breier A, Sulová Z: Structural diff erences between sensitive and resistant L1210 cells. Gen Physiol Biophys 2006; 25: 427–38.

Bauer et al. Trends in Pharmacological Research pharmacological research

Vasoactive effects of Provinols™ in experimental hypertension

Oľga PECHÁŇOVÁ and Iveta BERNÁTOVÁ Institute of Normal and Pathological Physiology and Centre of Excellence for Cardiovascular Research, SASc., Sienkiewiczova 1, 813 71 Bratislava, Slovak Republic, E-MAIL: [email protected]

Key words: polyphenols, vascular functions, nitric oxide, vasorelaxation, hypertension

Introduction

Numerous experimental and epidemiological data have documented that selected natural 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 signaling cascades implicated in endothelial apoptosis. Due to their antioxidant properties, 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 [3–5]. This review is focused on the cardiovascular beneficial effects of red wine polyphenolic 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.

Eﬀ ects of Provinols™ on vasorelaxation

It w a s do c u ment e d t h at P rov i nol s™ e l ic it e d endot he l iu m- d e p end ent re l a x at ion of r at femoral artery by the Ca2+-induced increase of NO synthase activity and by protecting NO

O. Pecháňová & I. Bernátová (2008) Trends in Pharmacological Research (Eds. V. Bauer et al.): 102–108. Vasoactive eff ects of Provinols™ in experimental hypertension 103 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-dependent 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-dependent vasorelaxation induced by Provinols™. Determination of NO synthase activity in the vascular tissue demonstrated that administration of Provinols™ at the concentration 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 correlated 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 nitroprusside 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 responses 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 possible 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 Ca2+ ([Ca2+] 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 vasorelaxation. This increase in [Ca2+]i can be due to either an influx of extracellular Ca2+ or a release of Ca2+ from intracellular stores. The relaxation produced by Provinols™ was completely prevented in the presence of the Ca2+-entry blocker verapamil, suggesting that the Ca2+ influx to endothelial cells might be crucial for the relaxation ability of the red wine polyphenolic compounds [6]. Analogically, Andriambeloson et al. [10] reported that red wine polyphenolic compounds produced NO-dependent vasorelaxation of rat aortic rings through an extracellular Ca2+-dependent mechanism. However, it cannot be excluded that a release of Ca2+ 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 [Ca2+]i from the intracellular stores, that was sensitive to the phospholipase C inhibitor. There are at least two mechanisms by which polyphenolic compounds could influence NO release: described stimulation of NO synthase activity and preservation

Bauer et al. Trends in Pharmacological Research 104 O. Pecháňová & I. Bernátová and/or stabilization of NO release under basal conditions. The later mechanism includes 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].

Eﬀ ects 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 endothelium-dependent relaxation and induction of inducible NO synthase and cyclooxygenase 2 genes expressions within the arterial wall [13]. This effect probably involves NO pathway since Provinols™ is able to produce ex vivo endothelium-dependent relaxation, as a result of enhanced NO synthesis as is documented above. Pecháňová et al. [8] provided evidence that oral administration of Provinols™ markedly prevented the increase in blood pressure as well as structural and functional cardiovascular 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, attenuated 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 reported 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 ventricle, aorta, brain and kidney of L-NAME-treated rats, resulting in an increase of conjugated 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 responsible for observed high levels of NO synthase activity. Recently, we have reported that the endothelial NO production caused by Provinols™ was associated with the increase 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 administration 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 activity 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 synthesis induced early vascular inflammatory changes as well as subsequent medial thickening, 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 properties of Provinols™ in vivo.

Eﬀ ects of Provinols™ on the developed hypertension

Bernátová 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™ treatment enhanced the regression of aortic wall thickness and improved the decreased endothelium-dependent relaxation in response to acetylcholine and the increased reactivity of the aorta to norepinephrine in hypertension induced by L-NAME. In addition, 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

Bauer et al. Trends in Pharmacological Research 106 O. Pecháňová & I. Bernátová 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 produced by chronic L-NAME treatment is due to enhanced in vivo NO production subsequent 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 ventricular 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 ventricular 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-κB 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 protective effect of Provinols™ [17,18]. The reduced endothelium-dependent vasodilatation seen in response to acetylcholine 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 endothelium-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 structural 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 system 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 myocardial fibrosis, although it did not reduce L-NAME-induced left ventricular hypertrophy. 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 vasodilatation and endothelium protective properties are the possible mechanisms, by which the intake of Provinols™ and other red wine polyphenolic compounds may reduce cardiovascular risk. Thus, the beneficial effects of plant polyphenols in prevention 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.

REFERENCES [1] Middleton EJR, Kandaswami C, Th eoharides TC: Th e eff ect of plant fl avonoids on mammalian cells: Impli- cations for infl ammation, heart disease and cancer.Pharmacol Rev 52: 673–751, 2000. [2] Wollny T, Aiello L, Di Tommaso D, Bellavia V, Rotilio D, Donati MB, De Gaetano G, Iacoviello L: Modulation of haemostatic function and prevention of experimental thrombosis by red wine in rats: a role for increased nitric oxide production. Br J Pharmacol 127: 747–755, 1999. [3] Curin Y, Andriantsitohaina R: Polyphenols as potential therapeutical agents against cardiovascular diseases. Pharmacol Rep 57: 97–107, 2005. [4] Andriantsitohaina R, Curin Y, Ritz MF, Gerald R, Alves A, Mendelowitsch A, Elbaz M: Polyphenols: protection of neurovascular unit in stroke and inhibition of in-stent-neointimal growth. Physiol Res 54: 51P, 2005.

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[5] Zenebe W, Pecháňová O: Eff ects of red wine polyphenolic compounds on the cardiovascular system. Bratisl Lek Listy 103: 159–165, 2002. [6] Zenebe W, Pecháňová O, Andriantsitohaina R: Red wine polyphenols induce vasorelaxation by increased nitric oxide bioactivity. Physiol Res 52: 425–432, 2003. [7] Andriambeloson E, Magnier C, Haan-Archipoff G, Lobstein A, Anton R, Beretz A, Stoclet JC, Andriantsito- haina R: Natural dietary polyphenolic compounds caused endothelium-dependent vasorelaxation in rat thoracic aorta. J Nutr 128: 2324–2333, 1998. [8] Pecháňová O, Bernátová I, Babál P, Martinez MC, Kyselá S, Štvrtina S, Andriantsitohaina R: Red wine polyphenols prevent cardiovascular alterations in L-NAME-induced hypertension. J Hypertens 22: 1551–1559, 2004a. [9] Cishek MB, Galloway MT, Karim M, German JB, Kappagoda CT: Eff ects of red wine on endothelium dependent-relaxation in rabbits. Clin Sci 93: 507–511, 1997. [10] Andriambeloson E, Stoclet JC, Andriantsitohaina R: Mechanism of endothelial Nitric oxide-dependent vasorelaxation induced by wine polyphenols in rat thoracic aorta. J Cardiovasc Pharmacol 33: 248–254, 1999. [11] Martin S, Andriambelson E, Takeda K, Andriantsitohaina R: Red wine polyphenols increase calcium in bovine aortic endothelial cells: a basis to elucidate signalling pathways leading to nitric oxide production. Br J Pharmacol 135: 1979–1987, 2002. [12] Frankel EN, Kanner J, German JB, Parks E, Kinsella JE: Inhibition of oxidation of human low-density lipoprotein by phenolic substances in red wine. Lancet 341: 454–457, 1993. [13] Diebolt M, Bucher B, Andriantsitohaina R: Wine polyphenols decrease blood pressure, improve NO vasodilatation, and induce gene expression. Hypertension 38:159–165, 2001. [14] Duarte J, Perez-Palencia R, Vargas F, Ocete MA, Perez-Vizcaino F, Zarzuelo A, Tamargo J: Antihypertensive eff ects of the fl avonoid quercetin in spontaneously hypertensive rats. Br J Pharmacol 133: 117–124, 2001. [15] Pecháňová O, Dobešová Z, Čejka J, Kuneš J, Zicha J: Vasoactive systems in L-NAME hypertension: the role of inducible NO synthase. J Hypertens 22:167–173, 2004b. [16] Wallerath T, Poleo D, Li H, Forstermann U: Red wine increases the expression of human endothelial nitric oxide synthase. A mechanism that may contribute to its benefi cial cardiovascular eff ects. J Am Coll Cardiol 41:471–478, 2003. [17] Babál P, Pecháňová O, Bernátová I, Štvrtina S: Chronic inhibition of NO synthesis produces myocardial fi - brosis and arterial media hyperplasia. Histol Histopathol 12:623–629, 1997. [18] Kitamoto S, Egashira K, Kataoka C, Koyanagi M, Katoh M, Shimokawa H, Morishita R, Kaneda Y, Sueishi K, Takeshita A: Increased activity of nuclear factor-κB participates in cardiovascular remodeling induced by chronic inhibition of nitric oxide synthesis in rats. Circulation 102: 806–812, 2000. [19] Bernátová I, Pecháňová O, Babál P, Kyselá S, Štvrtina S, Andriantsitohaina R: Wine polyphenols improve cardiovascular remodeling and vascular function in NO-defi cient hypertension. Am J Physiol 282: H– 42–H948, 2002. [20] Brilla CG, Pick R, Tan LB, Janicki JS, Weber KT: Remodelling of the rat right and left ventricles in experimental hypertension. Circ Res 67: 1355–1364, 1990.

Review 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 ŠTEFEK, Paul O. DJOUBISSIE, Andrej GAJDOŠÍK, Alena GAJDOŠÍKOVÁ, Mária JUSKOVÁ, Ľudmila KRIŽANOVÁ, Zuzana KYSEĽOVÁ, Magdaléna MÁJEKOVÁ, Lucia RAČKOVÁ, Vladimír ŠNIRC Department of Biochemical Pharmacology, Institute of Experimental Pharmacology, Slovak Academy of Sciences, Dúbravská cesta 9, 841 04 Bratislava, Slovak Republic, E-MAIL: [email protected]

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 glucose causes both acute reversible changes in cellular metabolism and long-term irreversible 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 pathogenesis of diabetic complications, substantial evidence suggests a key role for non-enzymatic glyco-oxidation, oxidative stress and the polyol pathway [1]. Thus, glycation inhibitors, 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 analogues 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.

M. Štefek et al. (2008) Trends in Pharmacological Research (Eds. V. Bauer et al.): 109–117. 110 M. Štefek 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 albumin 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-methiolbutyric 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 glycooxidation reactions, operative after the preceding glycation steps [2–4].

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 mellitus. 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 establish 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.

Copyright © 2008 Institute of Experimental Pharmacology | ISBN 978–80–970003–7–0 Substituted pyridoindoles as antioxidants and aldose reductase inhibitors 111 and biochemical changes in relation to STZ dosing, showing that young male Wistar rats, Breeding Facility Dobrá 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 hyperglycemia with major clinical signs of diabetes mellitus [5–7].

Stobadine and diabetic complications in experimental diabetes of rats Cardiovascular system Long-term treatment of diabetic animals with stobadine attenuated pathological changes in the diabetic myocardium: reduced oxidative damage of myocardial tissue as measured by conjugated dienes [8], reversed myocardial levels of α-tocopherol and coenzyme Q9 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 Ca2+-ATPase was unaffected [10]. On the other side, function of cardiac Na+,K+-ATPase was dramatically 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 protection 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], reduced 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 contain material that reacted rapidly at room temperature with p-dimethylaminobenzaldehyde (Ehrlich‘s reagent) to give an adduct with an absorbance spectrum characteristic of the Ehrlich chromogen of pyrrolic nature determined in aging collagens. A significant correlation of the Ehrlich adduct with tendon mechanical strength and collagen fluorescence characteristic of advanced glycation endproducts was observed. The glycation 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 glycation, additional mechanisms may participate in collagen cross-linking in diabetic connective tissues, namely hyperglycemia-induced oxidative stress [7,13,17].

Bauer et al. Trends in Pharmacological Research 112 M. Štefek 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 oxidative 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 conduction 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 failure, 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 significant decrease in contractility of isolated vas deferens elicited by electrical field stimulation, as well as significant increase in contractile response to exogenous noradrenaline when compared with control rats. Administration of stobadine reverted both parameters 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 pentose 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

Copyright © 2008 Institute of Experimental Pharmacology | ISBN 978–80–970003–7–0 Substituted pyridoindoles as antioxidants and aldose reductase inhibitors 113 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 peroxyl radicals. Moreover, the overall antioxidant activity of the compounds was affected 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 Fe2+/EDTA/ H2O2/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 efficient than that of 2-keto-4-methiolbutyric acid (KMBA) and less effective than Trolox. The inhibitory effect of the antioxiodants, expressed as IC50, correlated well with the reciprocal values of corresponding second order rate constants for scavenging OH• radicals [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 peroxyl-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 vivo−in 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].

Bauer et al. Trends in Pharmacological Research 114 M. Štefek et al.

Carboxymethylated pyridoindoles

As shown above, involvement of oxidative stress and the polyol pathway in the etiology 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, structurally 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 IC50 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 Lipinski’s ‘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 complications, 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 applications 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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Bauer et al. Trends in Pharmacological Research pharmacological research

New pyridoindoles with antioxidant and neuroprotective actions

Svorad ŠTOLC 1, Vladimír ŠNIRC 1, Alena GAJDOŠÍKOVÁ 1, Andrej GAJDOŠÍK 1, Zdenka GÁSPÁROVÁ 1, Oľga ONDREJIČKOVÁ 1, Ružena SOTNÍKOVÁ 1, Árpád VIOLA 1, Peter RAPTA 2, Pavol JARIABKA 1, Inge SYNEKOVA 1, Mária VAJDOVÁ 1, Soňa ZACHAROVA 1, Vendel NEMČEK 1, Viera KRCHNÁROVÁ 1 1 Department of Neuropharmacology, Institute of Experimental Pharmacology, SASc., Dúbravská cesta 9, 841 04 Bratislava, Slovak Republic, E-MAIL: [email protected] 2 Faculty of Chemical and Food Technology, Slovak Technical University, Bratislava, Slovak Republic

Key words: acute head trauma, synaptic transmission, hippocampus, α-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 pathological conditions, their production may be overexpressed or improperly counterbal- anced by insufficient capacity of the protective systems [1–5]. Enhancement of antioxidative /antiradical capacity of cells by supplying them with external compounds revealing suitable properties may contribute to limitation of ROS-induced damage [6–9]. 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 lipophilicity, 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 complicated procedures to prepare their water solutions (e.g. in lazaroids) suitable for i.v. use [10–11]. 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 antioxidant and antiradical properties in the Institute of Experimental Pharmacology, Slovak

S. Štolc et al. (2008) Trends in Pharmacological Research (Eds. V. Bauer et al.): 118–136. Pyridoindoles and neuroprotection 119

Academy of Sciences. The research was based on the pyridoindole stobadine, the molecule 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, synthesized, and studied since that. This paper presents some systematic data obtained in investigating the new compounds on their antioxidant properties, α−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 Fe2+/ascorbate system Rat brain homogenates were prepared at 4oC in phosphate/HEPES buffer (pH 7.4) with protease inhibitors (aprotinine, leupeptine, phenylmethylsufonyl fluoride, and pepsta- tine). To 50 μl of 10% homogenate, 10 μl of FeSO4 (5 mmol/l) and 5 μl of ascorbic acid (50 mmol/l) were added and the volume was replenished to 500 μl by potassium-phosphate buffer (50 mmol/l). The mixture was incubated for 30 min at 37 °C. The oxidation induced 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 (pIC50) 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 Lowry’s method. The homogenate was diluted with HEPES buffer to obtain final protein concentration 1 mg/ml. To 1,940 μl of the homogenate, 40 μl of FeSO4 (5 mmol/l) and 20 μl 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 μl 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 induction of oxidation in the final concentration of 30 μmol/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).

Bauer et al. Trends in Pharmacological Research 120 S. Štolc et al.

α-adrenolytic activity Ma le Wistar rats weighing 260 –280 g were k i l led by bleeding out of neck ar teries. The t ho- 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; KH2PO4 1.2; MgSO4 1.2; CaCl2 2.5; NaHCO3 25; glucose 11 – conc. in mmol/l) equilibrated with the mixture O2 + CO2 (95 and 5%, respectively) at pH=7.4. After stabilization (60 min, 37 °C), they were exposed to the α−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 incubation with the drug tested the procedure was repeated. Increasing drug concentrations –7 –5 were used (10 –10 mol/l) and EC50-s were determined. In case of competitive inhibition, the pA2 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 toxicities of selected compounds were measured in ICR mice (females, 28–34 g b.w., 8 weeks old). Each experimental group comprised 8–10 animals receiving the compound tested in a constant volume (10 ml/kg). The dose of the drug administered was changed according to the survival of the animal treated with the drug previously. If a particular animal survived for 24 hours, the higher dose was administered to the following animal, and vice versa (progression factor typically 1.4–1.7). 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 animals followed and special probability ratio exceeded the critical value), the experiment was finished and LD50 values with fiducial limits were expressed [15–17]. 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 occur 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, 23–26.5 g, breed Dobrá 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 assessed 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 decreased (typically to 10 sec, 1 hr after AHT). The animals with SMS > 60 sec were assessed

Copyright © 2008 Institute of Experimental Pharmacology | ISBN 978–80–970003–7–0 Pyridoindoles and neuroprotection 121 separately. Along with each drug-tested group a separate placebo-treated group was always used (n = 50–60, 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 taken from animals five hours after AHT were assessed biochemically. Chemiluminiscence in brain homogenates induced by FeSO4 (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 [21–22], 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 μm) were prepared by conventional technique with a tissue chopper. They were positioned on a support monofile mesh separating water and gas phases in thermostatized incubation chamber (36 °C). The water phase consisted of artificial cerebrospinal fluid (ACSF – NaCl 124; KCl 3.3; KH2PO4 1.25; MgSO4 2.4; CaCl2 2.5; NaHCO3 26; glucose 10 – conc. in mmol/l; pH 7.4), while the gas phase consisted of 95% O2 + 5% CO2. 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 Schäffer 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 O2 in the gas mixture by N2, 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 present in the superfusing medium in suitable concentration throughout the whole experiment. 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 experiments were performed with HEKA PG 284 (Germany) potentiostat under argon using a standard three-electrode arrangement. Standard ABTS and DPPH assays were used for determination of total antioxidant capacity of compounds using UV/Vis/NIR

Bauer et al. Trends in Pharmacological Research 122 S. Štolc 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 CH3 HHHHH H 4aR,9bS(–)-cis 103 H OH H H H H H H (±)-cis 104 H EtOC=O H HH H H H (±)-cis 105 H H H H H H CH3, (CH3)CH3, (CH3)(±)-cis 105 H H H H H H CH3, (CH3)CH3, (CH3)tetrahydro-1H-[4,3-b]indole 106 CH3 HHHHHHH4aR,9bS(–)-cis 107 CH3 PhCH2CH2 HHHHH H (±)-cis 108 CH3 PhCH2 HHHHH H (±)-cis 109 CH3 EtOC=O H H H H H H (±)-cis 110 CH3 Ph(C=O)O H H H CH3C=O H H (±)-cis 111 CH3 HHHHHCH3, (CH3)CH3, (CH3)(±)-cis 112 CH3 HCH3 HH H H H (±)-cis 113 CH3 PhC=O CH3 HH H H H (±)-cis 114 CH3 2-Pyr-C=O CH3 HH H H H (±)-cis 115 CH3 CH3OC=O CH3 HH H H H (±)-cis 116 (SM1M3EC2) CH3 EtOC=O CH3 HH H H H (±)-cis 117 CH3 PrOC=O CH3 HH H H H (±)-cis 118 CH3 iPrOC=O CH3 HH H H H (±)-cis 119 CH3 BuOC=O CH3 HH H H H (±)-cis 120 CH3 iBuOC=O CH3 HH H H H (±)-cis 121 CH3 PhCH2OC=O CH3 HH H H H (±)-cis 122 CH3 4-iPr-PhNHC=O CH3 HH H H H (±)-cis 123 CH3 tBuNHC=S CH3 HH H H H (±)-cis 124 CH3 CH3 NH2 HH H H H (±)-cis 125 CH3 CH3 N(CH3)2 HH H H H (±)-cis 126 CH3 CH3 NO2 HH H H H (±)-cis 127 CH3 CH3 Br H H H H H (±)-cis 128 CH3 CH3 NH2 HH CH3C=O H H (±)-cis 129 CH3 CH3 N(CH3)2 HH CH3C=O H H (±)-cis 130 CH3 EtOC=O H CH3 HH H H (±)-cis 131 OH H H H H H H H (±)-cis 132 OH CH3 HHHHH H (±)-cis 133 (SMe1) CH3OH H H H H H H 4aR,9bS(–)-cis 134 CH3OCH3 HHHHH H 4aR,9bS(–)-cis 135 CH3OCH3C=O H H H H H H (±)-cis 136 CH3OCH3 OC=O H H H H H H (±)-cis 137 (SMe1EC2) CH3OEtOC=O H H H H H H (±)-cis 138 CH3OPrOC=O H H H H H H (±)-cis 139 (SMe1iProC2) CH3O iPrOC=O H H H H H H (±)-cis 140 (SMe1nBuoC2) CH3OBuOC=O H H H H H H (±)-cis 141 (SMe1iBuoC2) CH3O iBuOC=O H H H H H H (±)-cis 142 (SMe1BzoC2) CH3OPhCH2OC=O H H H H H H (±)-cis 143 CH3OH CH3 HH H H H (±)-cis 144 CH3OEtOC=O CH3 HH H H H (±)-cis 145 CH3OPhCH2OC=O CH3 HH H H H (±)-cis 146 CH3OCH3OC=O Br CH3 CH3 HH H (±)-cis 147 CH3OH H CH3 CH3 HH H (±)-cis 148 CH3OCH3OC=O H CH3 CH3 HH H (±)-cis 149 CH3OEtOC=O H CH3 CH3 HH H (±)-cis 150 (SMe1M4M5nProC2) CH3OPrOC=O H CH3 CH3 HH H (±)-cis 151 CH3OBuOC=O H CH3 CH3 HH H (±)-cis 152 CH3O iBuOC=O H CH3 CH3 HH H (±)-cis 153 CH3OPhCH2OC=O H CH3 CH3 HH H (±)-cis 154 iPr EtOC=O H H H H H H (±)-cis 155 Br EtOC=O CH3 CH3 HH H H (±)-cis 156 Br PrOC=O CH3 CH3 HH H H (±)-cis 157 Br PhCH2OC=O CH3 CH3 HH H H (±)-cis 158 H H CH3 CH3 HH H H (±)-cis 159 H CH3OC=O CH3 CH3 HH H H (±)-cis Table continued on next page

drug code (alias) R8 R2 R6 R7 R9 R5 R3, (R3`) R1, (R1`) H4a,H9b

160 H EtOC=O CH3 CH3 HH H H (±)-cis 161 H PrOC=O CH3 CH3 HH H H (±)-cis 162 H iPrOC=O CH3 CH3 HH H H (±)-cis 163 H BuOC=O CH3 CH3 HH H H (±)-cis 164 H iBuOC=O CH3 CH3 HH H H (±)-cis 165 (SM3M4BzoC2) H PhCH2OC=O CH3 CH3 HH H H (±)-cis 166 H H CH3 HCH3 HH H (±)-cis 167 H CH3OC=O CH3 HCH3 HH H (±)-cis 168 H EtOC=O CH3 HCH3 HH H (±)-cis 169 H PrOC=O CH3 HCH3 HH H (±)-cis 170 H iPrOC=O CH3 HCH3 HH H (±)-cis 171 H BuOC=O CH3 HCH3 HH H (±)-cis 172 H H H CH3 CH3 HH H (±)-cis 173 H CH3OC=O H CH3 CH3 HH H (±)-cis 174 H EtOC=O H CH3 CH3 HH H (±)-cis 175 H PrOC=O H CH3 CH3 HH H (±)-cis 176 H iPrOC=O H CH3 CH3 HH H (±)-cis 177 H PhCH2OC=O H CH3 CH3 HH H (±)-cis 178 CH3 CH3 HHHHH H (±)-trans stobadine (SM1M2) CH3 CH3 HHHHH H 4aR,9bS(–)-cis 179 CH3Oi-PrOC=O H CH3 CH3 HH H (±)-cis 180 H BuOC=O CH3 HCH3 HH H (±)-cis 181 H PhCH2OC=O CH3 HCH3 HH H (±)-cis 182 H n-BuOC=O H CH3 CH3 HH H (±)-cis 183 H i-BuOC=O H CH3 CH3 HH H (±)-cis

Shimadzu 3600 spectrometer (Japan). In EPR experiments with Bruker EMX spectrometer, the thermal decomposition of K2S2O8 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.Š.). 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 regression analysis, and the exact test for 2×2 contingency tables were used in statistical assessment 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.

Bauer et al. Trends in Pharmacological Research 124 S. Štolc et al.

Results

Lipid peroxidation The capability of new pyridoindoles and some other selected compounds was measured in brain homogenate in the presence of Fe2+/ascorbate system. Negative logarithms of middle inhibitory concentration (pIC50) determined in whole series are given in Table 2. The compounds were ordered according to their anti-lipoperoxidation potency. 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 stobadine by as much as 1.5–2 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 impairment of CK was tested in rat brain homogenate exposed to Fe2+/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 1. Negative logarithms of middle inhibitory concentration (pIC50) of compounds tested (mol/l), on lipoperoxidation in rat brain homogenate induced by Fe2+/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.

Copyright © 2008 Institute of Experimental Pharmacology | ISBN 978–80–970003–7–0 Pyridoindoles and neuroprotection 125 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 completely identical. Nevertheless, the data indicate a general coherence in the two actions.

α-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 α-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 possess 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 α-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 LD50 (mg/kg) were expressed along with respective GHS characterization (OECD 1998) (Table 4). All 9 stobadine derivatives tested revealed remarkably lower acute toxicity than stobadine, regardless the way of administration. None of these compounds possessed any α-adrenolytic action. It seems that modification of the stobadine molecule aimed at obtaining new molecules with ameliorated acute toxicity was successful.

Figure 2. Factor f expressing inhibitory action of a compound on oxidative impairment of creatine kinase in rat brain homogenate exposed to Fe2+/ascorbic acid system relative to stobadine (dark column, f=1). Abscissa – order No. identical to Table 2 and Figure 1.

Bauer et al. Trends in Pharmacological Research 126 S. Štolc et al.

Table 2. Inhibitory action of compounds tested; pIC50 - negative logarithm of middle inhibitory concentration in lipoperoxidation in rat brain homogenate induced by Fe2+/ascorbic acid system. order error order error No. drug code pIC50 est. f (STB eq) No. drug code pIC50 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 H2SO4 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 non–eff – –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 Fe2+/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 evident 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 3. α-adrenolytic action of new pyridoindoles assessed in rat thoracic aorta rings.

Compound code Type pA2 MRD (%)* Compound code Type pA2 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 H2SO4 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 pA2 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 μmol/l. **only in the highest concentration of the drug used.

Table 4. Acute toxicity of stobadine and its selected derivatives in mice following single dose administration p.o., i.p., and i.v.

LD50 (mg/kg) Compound No. (alias) p.o. i.p. i.v. GHS 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 (LD50). GSH – toxicity category according to Globally Harmonized Hazard System (OECD, 1998). 4 = "mild acute toxicity", 5 = "comparatively low acute toxicity"

Bauer et al. Trends in Pharmacological Research 128 S. Štolc 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, melatonin, PBN, SPBN). Besides diminished sensomotoric impairment expressed as “time on wire” in the time interval of 0–60 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 animals 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 maximum 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 bleeding, and bleeding into brain chambers was significantly reduced in the drug-treated animals in the time period of 1–168 hrs after head injury (Figure 5). In brain homogenates of the animals sacrificed five hours after AHT, a significant acceleration of FeSO4-induced lipoperoxidation assessed as chemiluminiscence was observed. The latent period between inducing lipoperoxidation and the onset of chemiluminiscence 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 period (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, respectively.) 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 compared 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 population spikes evoked in pyramidal neurons by stimulation of Schäffer collaterals. The

Figure 3. Eﬀ ect 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 identiﬁ ed 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 1–168 hours after AHT. Signiﬁ cant 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 bleeding 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.

Bauer et al. Trends in Pharmacological Research 130 S. Štolc 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%. Q order No. Compound code C % SEM% D % SEM% 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 5165 100.0022.42269.1929.16 6142 100.0035.38262.8542.69 7 118 /24 hrs 100.00 37.17 256.49 72.12 8152 100.0021.61254.4049.67 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.

Copyright © 2008 Institute of Experimental Pharmacology | ISBN 978–80–970003–7–0 Pyridoindoles and neuroprotection 131 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 action, the population spike may recover. Such protective activity was expressed as amplitude 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 μmol/l, reaching maximal protection effect (i.e. approx. 70%) in 0.1 μmol/l. Under the given conditions further increase in the drug concentration (up to 1 μmol/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 0–1 V was observed in aqueous solutions compared to DMSO. One or two consecutive products were observed for methoxy-type structures (comp. No. 137, 140). These redox peaks originated 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 kinetics 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 concerning the exceptional ability of these derivatives to scavenge reactive radical species.

Discussion

In accordance with our expectation, the present study showed that suitable modification 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 system Fe2+/ascorbate. The results of electrochemical analysis (voltammetry) supported these observations. The three stobadine derivatives tested revealed complex redox behavior in both non-aqueous and aqueous media, indicating complexity of oxidation of the derivatives. Both ABTS and DPPH tests demonstrated an increase in antioxidant

Bauer et al. Trends in Pharmacological Research 132 S. Štolc et al.

Figure 6. Concentration dependence of eff ect 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 Schäff er 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 concentration (shaded columns) signifi cantly reduced apparently irreversible impairment of synaptic transmission by the hypoxic/reoxygenation conditions.

action in the new derivatives compared to stobadine, corresponding well with the tests in brain homogenates. Moreover, EPR measurements exhibited a strong capacity especially of methoxy-substituted derivatives to eliminate hydroxyl radicals. The findings are in good agreement with [25]. They demonstrated that some pyridoindoles 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 (Σσ+) 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 hydrochloride) (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 α-adrenolytic potency. The fact that the decrease in toxicity was observed regardless the administration 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.

In previous studies, the neuroprotective effect of stobadine was demonstrated in hypoxia/reoxygenation condition [26–27, 23]. Yet some new stobadine derivatives with enhanced antioxidant properties and devoid of α-adrenolytic effect remarkably surpassed 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, inflammation, as well as oxidative stress [28]. Rat hippocampal slices exposed to hypoxia/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 finding 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 antioxidant 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 barriers, 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 compounds (optimal concentrations – No. 137 from 0.1–1 μmol/l, stobadine 3–30 μmol/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.

Bauer et al. Trends in Pharmacological Research 134 S. Štolc 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 hypothesis that antioxidants may interfere with oxidative stress even in brain parenchyma itself. The coincidence of both the neuroprotective and antioxidant actions in some pyridoindoles 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 inflammation [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 resulted in remarkable enhancement of its capacity to inhibit lipoperoxidation and oxidative injury of creatin phosphokinase in rat brain homogenates exposed to Fe2+/ 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 observed. Compared to the model compound stobadine, decreased acute toxicity was observed in all the 9 selected derivatives tested. This decrease was linked to successful elimination of α-adrenolytic activity revealed by stobadine, which is mostly responsible for its hypotensive effect. Neuroprotective action of numerous new derivatives 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 Fe2+-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

Copyright © 2008 Institute of Experimental Pharmacology | ISBN 978–80–970003–7–0 Pyridoindoles and neuroprotection 135 oxidative stress impairment in the given models of acute CNS. Moreover, appropriate modification of the stobadine molecule resulted in decrease of α-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

Miloň TICHÝ, Pavel URBAN National Institute of Public Health, Centre of Occupational Health, Šrobárova 48, 10042 Praha 10, Czech Republic, E-MAIL: [email protected]; [email protected]

Key words: pharmacology, toxicology, chemical mixtures, predicitive methods, QSAR

Pharmacology and toxicology are relatives, although it can seem to somebody only apparently. 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 experimental 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 nowadays 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 between 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 solution 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. Tichý & P. Urban(2008) Trends in Pharmacological Research (Eds. V. Bauer et al.): 137–139. 138 M. Tichý & 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 occupational diseases. This concept of professor Teisinger bore and bears fruits up to day. Professor Nosál, 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 naturally 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 research, too. The studies on metal intoxications and on mobilizing effect of different new chelating agents continue both on experimental level and in subjects occupationally 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 determination 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 methods 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 successfully 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 assessment 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-

Copyright © 2008 Institute of Experimental Pharmacology | ISBN 978–80–970003–7–0 Trends of research in pharmacology 139 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 exposed 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 pharmacological and toxicological activities of individual chemicals but not if they are in mixtures. 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 chemistry 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 techniques. 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.

Bauer et al. Trends in Pharmacological Research pharmacological research

Trends in developmental toxicology Protection of the developing organism – an ever topical issue

Eduard UJHÁZY, Mojmír MACH, Jana NAVAROVÁ, Alena GAJDOŠÍKOVÁ, Andrej GAJDOŠÍK, Jozef JANŠÁK, Viera DYTRICHOVÁ, Michal DUBOVICKÝ Department of Reproductive Toxicology, Institute of Experimental Pharmacology, Slovak Academy of Sciences, Dúbravská cesta 9, 841 04 Bratislava, Slovak Republic, E-MAIL: [email protected]

We would like to dedicate this paper the memory of Dr. Tatiana Balonová

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 almost 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 according to the type of human exposure. The first are three-segment-design studies for reproductive 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, teratogenesis, parturition, weaning, retardation of postnatal growth and functional development have been used as reproductive and developmental parameters of chemicals/drugs [4].

E. Ujházy et al. (2008) Trends in Pharmacological Research (Eds. V. Bauer et al.): 140–150. Protection of the developing organism – an ever topical issue 141

In the Institute of Experimental Pharmacology SASc., Bratislava, the history of experimental teratology began in the 1970s, when this discipline was incorporated into the Department of Toxicology, headed by Dr. Ladislav Vrbovský, CSc. Dr. Tatiana Balonová 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 [5–7]. In cooperation with the Faculty of Natural Sciences, Comenius University, Bratislava, the synthetic bioanalogues of juvenile hormone of insects, Altosid, was tested for its teratogenicity on rats [8]. The cytostatic drug cyclophosphamide was evaluated 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 [9–10]. Further, in light of the aims of Slovakofarma, n.p. Hlohovec, Slovakia, we conducted 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) [13–14], 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 rabbits [18]. Further substances were tested at the Institute, such as local anesthetics (pentacaine, oxetacaine) [19] or the antihistamines pipethiadene, pizotifen maleate [20] and bromadryl in rats [21]. The pyridoindole derivative stobadine (STO), a neuro- and cardioprotective substance with high antioxidant properties, was found promising for long term administration 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 [25–30] and chick embryos [31–32]. Krištofová 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 [28–30]. At present, the newly formed Department of Reproductive Toxicology is concerned with hypoxia/ischemia associated with oxidative stress during developmental stages of

Bauer et al. Trends in Pharmacological Research 142 E. Ujházy et al. rats. Pharmacologically induced chronic intrauterine hypoxia and the model of neonatal anoxia were introduced to study mechanisms of development of hypoxia/ischemia injuries [36–38]. Further, the potential pharmacological intervention by using natural and synthetic antioxidants in maternal and embryofetal disturbation caused by developmental hypoxia/ischemia has been investigated [39–40].

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 postnatal 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 administration of the anticonvulsant PHT during pregnancy in laboratory animals. PHT

Copyright © 2008 Institute of Experimental Pharmacology | ISBN 978–80–970003–7–0 Protection of the developing organism – an ever topical issue 143 teratogenicity is mainly initiated by adverse pharmacological action on the embryonic heart during a sensitive stage of development, resulting in embryonic hypoxia/ischemia [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 necrosis 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 replaced to their mothers. The surviving pups are investigated for neurobehavioral development [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 37ºC water bath for 10–20 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 [45–46].

These experimental models provide great advantages for studying effects of agents, conditions, and new drugs which could ameliorate detrimental effects of hypoxia/ischemia.

Results and discussion

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 reversible, 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 region 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 toxicity 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

Bauer et al. Trends in Pharmacological Research 144 E. Ujházy et al.

Table 1. List of selected drugs/substances tested in the Laboratory of Reproductive Toxicology (1977–2006). Tested substance/drug Animal Dosage Results/developmental toxicity Ref. p.o., 10 and 100 mg/kg, rats Candida albicans 4–16 GD No embryotoxic and teratogenic effect [5] glycoprotein i.v., 15, 30 and 60 mg/kg, rats 8 and 13 GD Candida albicans i.v., 30 and 60 mg/kg, Embryotoxic effect, skull and rib anomalies glycoprotein with rats [6,7] 8 and 13 GD Embryotoxic effect enriched protein fraction p.o., 500 mg/kg, Altosid rats Growth retardation of the pair 13 of ribs [8] 8 GD Cyclophosphamide Embryotoxic and teratogenic effect (gross malfor- i.m., 6.4–40.8 mg/kg, mations of head, extremities and caudum, skeletal mice [9] 11–15 GD anomalies such as synostosis, retardation of ossification) Embryotoxic effect (dose-dependent decrease of p.o., 6.2 and 18.6 mg/kg, fetal organs as well as placental weight), highest rabbits [10] 6–20 GD dose teratogenic effect (exophthalmia, cleft palate/ lip, syndactylism and brachycaudia) p.o., 86, 150, 265, 465 and The two highest doses had maternal and embryofe- Lithium carbonate mice 665 mg/kg, tal effect (increased mortality of dams and fetuses, [11] 1–15 GD increased resorptions) p.o., 11.7, 117.1 and 585.5 The low and middle doses had no adverse effect, Etofylline clofibrate mice mg/kg, the highest dose had embryotoxic effect (decreased [12] 7–16 GD fetal weight, increased postimplantation loss) p.o., 11.7, 117.1 The low and middle doses had no adverse effect, Fenofibrate mice and 585.5 mg/kg, the highest dose had embryotoxic effect (decreased [12] 7–16 GD fetal weight, increased postimplantation loss) mice, p.o., 5, 10 and 20 mg/kg, Exaprolol Skeletal anomalies, more pronounced effect in rats [13] (VULM 111) rats 4–16 GD injection to egg, 0.4, 0.8 chicken and 1.6 mg/egg, Growth stimulating effect on organs and skeleton [14] embryo 5–7 days of incubation p.o., 0.36, 3.6 No embryotoxic effect, the middle and highest Beta-aescine mice and 36 mg/kg, doses increased incidence of skeletal anomalies [15] 7–16 GD (skull and sternebrae) p.o., 30, 120 ACAT inhibitor rats and 300 mg/kg, No embryotoxic and teratogenic effects [16] (VULM 1457) 6–15 GD Calcium antagonist p.o., 5, 50 and 250 mg/kg, rats No embryotoxic and teratogenic effects [17] (VULM 993) 6–15 GD p.o., 5, 10 and 25 mg/kg, MCPA herbicide rabbits No embryotoxic and teratogenic effects [18] 6–27 GD p.o., 1, 10 and 50 mg/kg, Pentacaine rabbits No embryotoxic and teratogenic effects [19] 6–20 GD p.o., 20 mg/kg, Oxetacaine rabbits No embryotoxic and teratogenic effects [19] 6–20 GD p.o., 1, 5 and 10 mg/kg, Embryotoxic effect (decreased fetal and placental Bromadryl rats [21] 2–19 GD weight), no teratogenic effect p.o., 0.24, 0.6 Pipethiadene Embryotoxic effect (decreased fetal weight), mice and 1.2 mg/kg, [20] no teratogenic effect 4–16 GD p.o., 0.24, 0.6 Pizotifen maleate Embryotoxic effect (decreased fetal weight), mice and 1.2 mg/kg, [20] no teratogenic effect 4–16 GD

Tested substance/drug Animal Dosage Results/developmental toxicity Ref. injection to egg, 0.4, 0.8 Quinidine chicken and 1.6 mg/egg, No embryotoxic and teratogenic effects [31] embryo 5–7 days of incubation injection to egg, 0.4, 0.8 Stobadine chicken and 1.6 mg/egg, No embryotoxic and teratogenic effects [31] embryo 5–7 days of incubation chicken 10–3–10–8 mol/l, chick embryo blastoderms at stages No adverse effects on early chick embryogenesis [32] in culture 4–5 HH medium i.v., 1 and 3 mg/kg, Fetotoxic effect (decreased fetal weight), no terato- mice 1, 3, 6, 9 and 12 GD genic effect p.o., 12.2, 61 The middle dose caused reduction of implantation, [24] mice and 122 mg/kg, live fetuses and litter weight, the highest decreased 4–16 GD fetal weight, no teratogenic effect p.o., 5, 15 and 50 mg/kg, males: 70 days before No adverse effects on fertility, survival rate, and rats mating, females: 14 days weight gain of parental aninals, or on prenatal and before mating and during postnatal development of pups gestation and lactation [25] p.o., 5, 15 and 50 mg/kg, rats No embryotoxic and teratogenic effect 4–16 GD p.o., 5, 15 and 50 mg/kg, No adverse effect on reproductive parametres of rats 15 GD–21 PP dams, on survival and development of offspring p.o., 50 mg/kg, No overt effects on dams, embryofetal develop- rats [28] 6–15, 6–20 GD ment or reproductive parameters p.o., 5, 15 and 50 mg/kg, No adverse effects on course of pregnancy, lacta- rats [30] 6 GD–21 PP tion and neurobehavioral development p.o., 50 mg/kg, rats 14 days before mating Subtle alterations of exploratory behavior [29] to 21 PP p.o., 5, 15 and 50 mg/kg, No adverse effect on pre- and postnatal develop- rats [27] 6–21 PP ment of offspring i.v., 2 and 6 mg/kg, Embryotoxic effect (decreased fetal weight), no rats 3, 6, 9 and 12 GD teratogenic effect Maternal toxicity, embryotoxic effect (increased [26] p.o., 5, 15 and 45 mg/kg, rats preimplantation loss, decreased fetal weight), no 2–15 GD teratogenic effect STO crosses placental barrier; higher concentration p.o., [3H] STO, 5 mg/kg, rats STO in the placental and fetal tissue compared to [33] 20 GD maternal plasma STO crosses placental barrier; its concentrations p.o., [ 3H] STO, 5 mg/kg, were higher in fetal plasma compared to values rabbits [34] 20 and 27 GD found in mothers; the highest concentrations were found in the brain and heart STO crosses into milk; however, only 0.39% of the p.o., [3H] STO, 5 mg/kg, rats total radioactivity administered to the lactating rats [49] 10 PP were recovered in the pups p.o., 150 mg/kg, Phenytoin rats Maternal, embryofetal and neurobehavioral toxicity [36] 2–19 GD GD – gestational day, p.o. – oral administration, i.v. – intravenous administration, HH – Hamburger-Hamilton stage in chicken embryo, PP – post partum

Bauer et al. Trends in Pharmacological Research 146 E. Ujházy 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 developmental 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 neurobehavioral disorders and mental diseases during childhood, maturation, or as late as adulthood or senescence. Such diseases include attention deficit-hyperactivity disorder, 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 reactivity 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 variables of oxidative stress in different maternal and fetal organs, such as lysosomal enzyme N-acetyl-ß-D-glucosaminidase (NAGA), glutathione (GSH), lactate, and catecholamines [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 [55–56], is susceptible 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 [57–58]. 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].

The embryo is more susceptible to oxidative stress at key periods in its development, and thus antioxidant defenses are important in modulating oxidative stress-mediated events. A better understanding of these reactions and their consequences is vital in opti- mizing embryonic and fetal development. In our experimental models of chronic intrauterine hypoxia induced by PHT and neonatal anoxia, we evaluated potential protective effects of vitamin E, melatonin and STO. Vitamin E and melatonin failed to ameliorate developmental toxicity induced by PHT and failed to protect rat fetuses [39,59,60]. STO was found to have partial preventive effect on PHT induced toxicity (significantly increased fetal and placental weight, positive influence on some reproductive variables, such as number of live fetuses, resorptions, pre- and postimplantation loss) [61].

Conclusions

Because of the complexity of developmental processes and their interactions and/or interferences that are found only in living animals and humans, in vitro toxicity tests should be an integral part in the investigation of new drugs and chemicals. In vitro test systems fall into 4 categories: established cell lines, primary cell cultures, non-mammalian embryos, and mammalian embryos or primordia [62]. In cooperation with the newly formed Laboratory of Cell Cultures headed by Dr. Štefan Bezek, we began to introduce the model of whole embryo culture (WEC), which is based on cultivation of 9.5-day-old rat embryos in serum [63]. Our future efforts are directed essentially improving dysmorphogenic screening methods. Furthermore, we would like to diversify our basic research by evaluation of abnormal development at various levels ranging from whole organism through organs, tissues, cells, to subcellular and molecular levels using both in vivo and in vitro systems.

Acknowledgement

This work was supported by the grants VEGA 2/0083/08 and 2/0086/08.

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Angiogenesis A perspective target in cancer therapy

Lenka VARINSKÁ, Ladislav MIROSSAY, Ján MOJŽIŠ Department of Pharmacology, Faculty of Medicine, P.J. Šafarik University, Košice, Slovak Republic, E-MAIL: [email protected]

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 normal physiological processes, such as development and reproduction. However, in many disorders the balance between stimulators and inhibitors of angiogenesis is tilted, resulting 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) degradation 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) organization 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. Varinská et al. (2008) Trends in Pharmacological Research (Eds. V. Bauer et al.): 151–157. 152 L. Varinská 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 stabilization of the nascent capillaries. Angiogenesis is a process requiring the coordinated action of a variety of growth factors 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 initiated by neo-angiogenesis [6]. The idea of blocking tumour growth by the inhibition of angiogenesis was put forward in the early 70’s 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 angiogenesis inhibitors.

For the therapeutic inhibition of angiogenesis, three main categories have been identified: 1. direct antiangiogenic drugs that act by targeting the endothelial cells and their functions involved in angiogenesis (proliferation, migration, formation of new vessels); 2. indirect antiangiogenic drugs that thwart the production of angiogenic factors by tumor and microenvironment cells, and/or interfere with extracellular processes; 3. mixed antiangiogenic drugs that may be able to interfere with both endothelial and tumor cells.

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 factor (VEGF) alone can substantially suppress tumor growth and angiogenesis in several 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 tyrosine kinase (RTK) inhibitors targeting the VEGF receptors including sunitinib and sorafenib have been developed. Other anti-VEGF agents including VEGF-Trap, a soluble 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 prevent them from binding to their receptors [13]. Tyrosine kinase inhibitors belong to another group of potential antiangiogenic inhibitors. 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-refractory 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].

Bauer et al. Trends in Pharmacological Research 154 L. Varinská et al.

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 extracellular matrix proteins that are associated with the tumorigenic process. MMPs promote 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 current 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 surrounding extracellular matrix. Integrins are a family of cell adhesion molecules consisting of two non-covalently bound transmembrane subunits (alpha and beta). Much research has demonstrated that integrin signaling plays a key role in tumor angiogenesis and metastasis. Integrin alphavbeta3 (αvβ3) is highly expressed on activated endothelial 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 therapy. 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 αvβ3. Antagonists of αvβ3 have been studied most extensively for their antiangiogenic properties [18]. In addition, αvβ3 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 αvβ3 in the pathology of osteolytic diseases, including breast cancer, prostate cancer, and multiple myeloma. Finally, αvβ3 is overexpressed on a variety of different tumor types. For example, a preponderance of data suggests that αvβ3 is found to be overexpressed 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 findings, etaracizumab is presently being investigated in clinical trials of androgen-independent prostate cancer and metastatic melanoma.

Many of the proteins, polypeptides and peptides with antiangiogenic activities are endogenously 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 purposes [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 μg/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 μg/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

Bauer et al. Trends in Pharmacological Research 156 L. Varinská et al. 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 fibrin matrix induced by exposure of human microvascular endothelial cells to VEGF and tumour necrosis factor-α. 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 alterations 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 polyphenols 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).

REFERENCES [1] Carmeliet P. Angiogenesis in health and disease. Nat Med 2003; 9: 653–660. [2] Klagsbrun M, Moses MA. Molecular angiogenesis. Chem Biol 1999; 6: 217–224. [3] Pages G, Milanini J, Richard DE: Signalling angiogenesis via p42/p44 MAP kinase cascade. Ann NY Acad Sci 2000; 902: 187–200. [4] Basbaum CB, Werb Z: Focalized proteolysis: Spatial and temporal regulation of extracellular matrix degradation at the cell surface. Curr Opin Cell Biol 1996; 8: 731–738. [5] Bouïs D, Kusumanto Y, Meijer C: A review on pro- and anti-angiogenic factors as targets of clinical intervention. Pharmacol Res 2006; 53: 89–103. [6] Bergers G, Benjamin LE: Tumorigenesis and the angiogenic switch. Nat Rev Cancer 2003; 3: 401–410. [7] Folkman J. Tumour angiogenesis: therapeutic implications. N Engl J Med 1971; 285: 1182–1186. [8] Kim KJ, Li B, Winer J: Inhibition of vascular endothelial growth factor-induced angiogenesis suppresses tumor growth in vivo. Nature 1993;362: 841–844.

[9] Ferrara N, Gerber HP, LeCouter J: Th e biology of VEGF and its receptors. Nat Med 2003; 9: 669–676. [10] Hurwitz H, Fehrenbacher L, Novotny W: Bevacizumab plus irinotecan, fl uorouracil, and leucovorin for metastatic colorectal cancer. N Engl J Med 2004; 350: 2335–2342. [11] Sandler A, Gray R, Perry MC: Paclitaxel-carboplatin alone or with bevacizumab for non-small-cell-lung cancer. N Engl J Med 2006; 355: 2542–2550. [12] Jain RK, Duda DG, Clark JW: Lessons from phase III clinical trials on anti-VEGF therapy for cancer. Nat Clin Pract Oncol 2006; 3: 24–40. [13] Manetti F, Corelli F, Botta M: Fibroblast growth factors and their inhibitors. Curr Pharm Des 2000; 6: 1897– 924. [14] Chow LQ, Eckhardt SG: Sunitinib: from rational design to clinical effi cacy. J Clin Oncol 2007; 25: 2858–9. [15] Bramhall SR, Schulz J, Nemunaitis J: A double-blind placebo-controlled, randomised study comparing gemcitabine and marimastat with gemcitabine and placebo as fi rst line therapy in patients with advanced pancreatic cancer. Br J Cancer 2002; 87: 161–7. [16] Bissett D, O’Byrne KJ, von Pawel J: Phase III study of matrix metalloproteinase inhibitor prinomastat in non- small-cell lung cancer. J Clin Oncol 2005; 23: 842–9. [17] Tu G, Xu W, Huang H: Progress in the development of matrix metalloproteinase inhibitors. Curr Med Chem. 2008; 15: 1388–95. [18] Kumar CC. Integrin alpha v beta 3 as a therapeutic target for blocking tumor-induced angiogenesis. Curr Drug Targets. 2003; 4: 123–31. [19] Teti A, Migliaccio S, Baron R: Th e role of the avh3 integrin in the development of osteolytic bone metastases: a pharmacological target for alternative therapy? Calcif Tissue Int 2002; 71: 293–9. [20] Ria R, Vacca A, Ribatti D: αvβ3 integrin engagement enhances cell invasiveness in human multiple myeloma. Haematologica 2002; 87: 836–45. [21] McNeel DG, Eickhoff J, Lee FT: Phase I trial of a monoclonal antibody specifi c for avh3 integrin (MEDI-522) in patients with advanced malignancies, including an assessment of eff ect on tumor perfusion. Clin Cancer Res 2005; 11: 7851–60. [22] Shellenberger TD, Wang M, Gujrati M: BRAK/CXCL14 is a potent inhibitor of angiogenesis and a chemotactic factor for immature dendritic cells, Cancer Res 2004; 64: 8262– 8270. [23] Romagnani P, Lasagni L, Annunziato F: CXC chemokines: the regulatory link between infl ammation and angiogenesis, Trends Immunol 2004; 25: 4201– 209. [24] Lee KW, Lee HJ. Th e roles of polyphenols in cancer chemoprevention. Biofactors 2006; 26: 105–121. [25] Mojžiš J, Šarišský M, Pilátová M: In vitro antiproliferative and antiangiogenic eff ects of fl avin7®. Physiol Res. 2008; 57: 413–20. [26] Tomas-Barberen FA, Cliff ord MN. Flavanones, chalcones and dihydrochalcones: nature, occurrence and dietary burden. J Sci Food Agric 2000; 80: 1073–1080. [27] Hsu YL, Kuo PL, Chiang LC: Isoliquiritigenin inhibits the proliferation and induces the apoptosis of human non-small cell lung cancer A549 cells. Clin Exp Pharmacol Physiol. 2004; 31: 414–418. [28] Modzelewska A, Pettit C, Achanta G: Anticancer activities of novel chalcone and bis-chalcone derivatives. Bioorg Med Chem 2006; 14: 3491–3495. [29] Mojžišová G, Mojžiš J, Pilátová M: Antiproliferative and antiangiogenic eff ects of selected chalcones. Acta Pharmacol Sin 2006; 27: 338. [30] Mojžiš J, Varinská L, Perjesi P: Antiangiogenic eff ect of newly synthesized chalcones Eur J Cancer 2007; 5: 87. [31] Varinská, L., Verloop R., Perjesi P: Chalcones and their potential antiangiogenic eff ect. 9th International Conference, Angiogenesis: Basic Science and Clinical Applications, Greece, p. 73, 2008.

Bauer et al. Trends in Pharmacological Research pharmacological research

Cytokine-stimulatory effects of acyclic nucleotide analogues: extrapolation of immunopharmacological data from animal to human cells

Zdeněk ZÍDEK 1, Eva KMONÍČKOVÁ 1, Antonín HOLÝ 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 immunodeficiency 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 treatment 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 [2–7]. The present analysis is focused on the evaluation of immunostimulatory and immunomodulatory effects of acyclic nucleotide analogues in cells of murine and human origin.

Z. Zídek et al. (2008) Trends in Pharmacological Research (Eds. V. Bauer et al.): 158–165. Cytokine-stimulatory eff ects of acyclic nucleotide analogues 159

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 inbred strain C57BL/6 (Charles River Deutschland, Germany). The cells were seeded into 96-well round-bottom microplates (Costar) and maintained at 37 °C, 5% CO2 in humidified Heraeus incubator. The animal PECs were cultured at final density of 2.0 × 106/ml, the human PBMCs at density of 1.0 × 106/ml in complete RPMI-1640 culture medium. It contained 10% heat-inactivated fetal bovine serum, 2 mM L-glutamine, 50 μg/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 chemokines MIP-1α (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 values (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 being statistically highly significant for both MIP-1α (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 biosynthesis which was primarily triggered by IFN-γ (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 secretion 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

Bauer et al. Trends in Pharmacological Research 160 Z. Zídek et al.

A) Secretion of MIP-1 α 500 2500 Coefficient of correlation r = 0.969, P < 0.0001 Mouse PEC Human PBMC

400 2000 MIP-1 HUMAN:

(pg/mL) 300 1500 α α (pg/mL) 200 1000 MICE: MIP-1

100 500

0 . 0 H-3387 H-3040 H-3015 H-3431 H-3002 H-2913 H-3424 H-2952 H-3432 H-2940 H-2989 H-2939 MIC-445 MIC-458 MIC-425 MIC-453 MIC-456 MIC-422 MIC-460 MIC-423 MIC-429 MIC-434 MIC-459 MIC-449 MIC-454 MIC-435 MIC-428 MIC-444 H-PMEA CONTROL Compounds 50 μM

B) Secretion of RANTES 1000 300

Coefficient of correlation r = 0.982, P < 0.0001 RANTES (% HUMAN: Mouse PEC Human PBMC 250 800

200 600 of baseline pg/mL) baseline of 150

400 100 MICE: RANTES (pg/mL)

200 50

0 . 0 H-3002 H-3387 H-3431 H-3015 H-2940 H-3424 H-2913 H-3432 H-2952 H-2989 H-2939 MIC-423 MIC-460 MIC-422 MIC-458 MIC-456 MIC-434 MIC-459 MIC-435 MIC-428 MIC-444 H-PMEA CONTROL Compounds 50 μM Figure 1. In vitro production of chemokines MIP-1α (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 α 60 2500 NO (μM) MIP-1 α (pg/mL) 2250 50 Coefficient of correlation, r = 0.958; P < 0.0001 MIP-1 Human PBMC: 2000

1750 40 1500

30 1250 α

1000 (pg/mL) 20

Mouse PEC:Mouse (μM) Nitrite 750

500 10 250

0 . 0 H-2952 H-2939 H-3432 H-2989 H-2940 H-2913 H-3431 H-3424 H-3002 H-3387 H-3040 H-3015 H-PMEA MIC-444 MIC-428 MIC-449 MIC-454 MIC-459 MIC-435 MIC-434 MIC-460 MIC-456 MIC-453 MIC-423 MIC-422 MIC-429 MIC-425 MIC-458 MIC-445 CONTROL Compounds (μM)

B) Mouse NO / Human RANTES 60 300 pg/mL) baseline of (% S ANTE R : BMC P Human NO (μM) RANTES (% of control) Coefficient of correlation, r = 0.969; P < 0.0001 50 250

40 200

30 150

20 100 Mouse PEC:Mouse (μM) Nitrite

10 50

0 . 0 H-3015 H-3387 H-3431 H-3424 H-2940 H-2913 H-3432 H-2989 H-2952 H-2939 H-PMEA MIC-460 MIC-456 MIC-423 MIC-422 MIC-435 MIC-434 MIC-459 MIC-428 MIC-444 CONTROL Compounds (μM)

Figure 2. Correlation between ability of compounds to upregulate production of NO by mouse peritoneal cells and production of chemokines MIP-1α (A) and RANTES (B) by human peripheral blood mononuclear cells.

Bauer et al. Trends in Pharmacological Research 162 Z. Zídek 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 N6- cyclopropyl-(R)-9-[2-(phosphonomethoxy)propyl]2,6-diaminopurine H-2940 N6-pyrrolidino-9-[2-(phosphonomethoxy)ethyl]2,6-diaminopurine H-2952 N6-cyclopentyl-(R)- 9-[2-(phosphonomethoxy)propyl]2,6-diaminopurine H-2989 N6-isobutyl-9-[2-(phosphonomethoxy)ethyl]2,6-diaminopurine H-3002 N6-dimethylaminoethyl-9-[2-(phosphonomethoxy)ethyl]2,6-diaminopurine H-3015 9-[2-(phosphonomethoxy)ethyl]2,6-diaminopurine H-3387 N6-cyclopropyl-9-[2-(phosphonomethoxy)ethyl]2,6-diaminopurine H-3424 N6-cyclohexylmethyl-9-[2-(phosphonomethoxy)ethyl]2,6-diaminopurine H-3431 N6-cyclopentyl-9-[2-(phosphonomethoxy)ethyl]2,6-diaminopurine H-3432 N6-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-423 2-guanidino-7-(S)-[2-(phosphonomethoxy)propyl]-7H-purine MIC-425 6-guanidino-7-(S)-[2-(phosphonomethoxy)propyl]-7H-purine MIC-428 6-amino-2-guanidino-3-(S)-[2-(phosphonomethoxy)propyl]-3H-purine MIC-429 6-amino-2-guanidino-9-(S)-[2-(phosphonomethoxy)propyl]-9H-purine MIC-434 6-guanidino-9-(S)-[2-(phosphonomethoxy)propyl]-9H-purine MIC-435 2-amino-6-guanidino-9-(S)-[2-(phosphonomethoxy)propyl]-9H-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 MIC-449 2-amino-6-guanidino-7-(S)-[2-(phosphonomethoxy)propyl]-7H-purine MIC-453 6-guanidino-7-(R)-[2-(phosphonomethoxy)propyl]-7H-purine MIC-454 2-amino-6-guanidino-7-(R)-[2-(phosphonomethoxy)propyl]-7H-purine MIC-456 2-guanidino-9-(R)-[2-(phosphonomethoxy)propyl]-9H-purine MIC-458 6-amino-2-guanidino-9-(R)-[2-(phosphonomethoxy)propyl]-9H-purine MIC-459 6-amino-2-guanidino-3-(R)-[2-(phosphonomethoxy)propyl]-3H-purine MIC-460 2-guanidino-7-(R)-[2-(phosphonomethoxy)propyl]-7H-purine MK-417 (2-hydroxy-3-phosphonomethoxypropyl)adenine MK-447 [3-(2,6-diaminoamino-9H-purin-9-yl)-2-hydroxypropyl]methylphosphonic acid

Copyright © 2008 Institute of Experimental Pharmacology | ISBN 978–80–970003–7–0 Cytokine-stimulatory eff ects of acyclic nucleotide analogues 163 human cells (Figures 2A and 2B). The coefficients of correlation between NO production 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-1α 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 immunostimulatory 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 reported 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-1α and MIP-1β, and receptor CXCR4 which binds SDF-1α/β [14, 15]. Chemokines, the natural ligands of these receptors, such as MIP-1α, MIP-1β, 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-γ 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-γ. It concerns mainly TNF-α [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-1α, MIP-1β [22], eotaxin [23], and MCP-1 [24]. On the con-

Bauer et al. Trends in Pharmacological Research 164 Z. Zídek et al. trary, cytokines such as IL-4 and IL-10 [25], TGF-β [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 plausible 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, human monocytes and macrophages are highly refractory to the induction of NO production 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 immunomodulatory effects of compounds in human cell system.

Ackowledgements

The work was supported by grant no. 1M0508.

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Bauer et al. Trends in Pharmacological Research 166 Authors Index

A J Anzenbacherová, Eva 11 Jakubovský, Ján 41 Anzenbacher, Pavel 11 Jančinová, Viera 82 Janšák, Jozef 140 B Jariabka, Pavol 118 Babulová, Anna 41 Jusková, Mária 109 Bacharová, Ljuba 41 K Bauerová, Katarína 25 Bauer, Viktor 7, 15 Kittová, Mária 41 Bernátová, Iveta 102 Kmoníčková, Eva 158 Bezek, Štefan 33 Knezl, Vladimír 41 Borovičová, Františka 41 Kollárová, Mária 15 Kollár, Tomáš 41 D Komendová, Denisa 25 Dedík, Ladislav 48 Kopecká, Bernardína 58 Ditrychová, Viera 15 Krchnarová, Viera 58 Djoubissie, Paul O. 109 Križanová, Ľudmila 109 Drábiková, Katarína 82 Kukan, Marián 33 Dravecká, Mária 41 Květina, Jaroslav 66 Dřímal, Ján 41 Kyseľová, Zuzana 15, 109 Dubovický, Michal 140 Ďurišová, Mária 48 L Dytrichová, Viera 140 Lojek, Antonín 77 F M Fatyková, Jozefína 15 Mach, Mojmír 140 Fraňová, Soňa 88 Mačičková, Tatiana 82 G Magna, Darius 41 Májeková, Magdaléna 109 Gajdošík, Andrej 58, 109, 118, 140 Máleková, Lubica 15 Gajdošíková, Alena 58, 109, 118, 140 Markovič, Slavo 41 GáSpárová, Zdenka 118 Mátyás, Štefan 15 Gemeiner, Peter 25 Mihalová, Danica 25 Gibala, Pavel 41 Mirossay, Ladislav 151 Golhová, Daniela 58 Mojžiš, Ján 151 Gvozdjak, Ján 41 Mokrý, Juraj 88 Gvozdjaková 41 N H Navarová, Jana 15, 140 Holý, Antonín 158 Nedelčevová, Jana 15 Hrbáč, Jan 77 Nemček, Vendel 118 167

Nikšová, Eva 41 Štefek, Milan 109 Nosáľová, Gabriela 88 Stojkovičová, Zuzana 15 Nosáľová, Viera 15 Štolc, Svorad 41, 118 Nosáľ, Radomír 8, 41, 77, 82 Straková, Zuzana 82 Strapková, Anna 88 O Strížová, Katarína 41 Ondrejičková, Oľga 118 Štrosová, Miriam 25 Ondriaš, Karol 96 Šutovská, Martina 88 Synekova, Inge 118 P Szőcs, Katalyn 15 Pecháňová, Oľga 102 T Pečivová, Jana 82 Pekarová, Michaela 77 Tichý, Miloň 137 Petríková, Margita 82 Tokárová, Jana 41 Poli, Giuseppe 25 Tomeková, Veronika 25 Poništ, Silvester 25 Toroková, Jozefína 41 Pucovský, Vladimír 15 Tóthová, Gizella 15 Puškárová, Andrea 41 Trnovec, Tomáš 33 Tvrdoňová, Martina 48 R U Račková, Lucia 109 Rapta, Peter 118 Ujházy, Eduard 58, 140 Rekalov, Vladimír 15 Urban, Pavel 137 Rybár, Alfonz 41 V S Vajdová, Mária 118 Sadloňová, Vladimíra 88 Valachová, Katarína 25 Sauberer, Anton 41 Varinská, Lenka 151 Selecký, František Viliam 41 Viola, Árpád 118 Šnirc, Vladimír 109, 118 Z Šoltés, Ladislav 25 Sotníková, Ružena 15, 41, 118 Zacharova, Soňa 118 Srnová, Monika 15 Zídek, Zdeněk 158