Volume 157, Supplement 1 of the Faculty of Medicine And

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OF THE FACULTY OF MEDICINE AND DENTISTRY OF PALACKÝ UNIVERSITY, OLOMOUC CZECH REPUBLIC

VOLUME 157, SUPPLEMENT 1

PALACKÝ UNIVERSITY PRESS, OLOMOUC 2013 BIOMEDICAL PAPERS

Volume 157, Supplement 1

Published quarterly MK ČR E 12793

Published and printed by Palacký University Press, Olomouc Křížkovského 8, 771 47 Olomouc, IČO 61989592

Olomouc 2013

ISSN 1213-8118 eISSN 1804-7521 63. Czech and Slovak Pharmacological Days September 11–13, 2013 Olomouc, Czech Republic Honorary Committee Miroslav Mašláň (Rector, Palacky University Olomouc) Ladislav Mirossay (Rector, Pavel Jozef Safarik University, Kosice) Milan Kolář (Dean, Faculty of Medicine and Dentistry, Palacky University Olomouc) Roman Havlík (Director, Faculty Hospital Olomouc) Pavel Anzenbacher (Chairman of the Scientiﬁ c Committee)

Scientiﬁ c Committee Pavel Anzenbacher Michal Dubovický Soňa Franová Vladimír Geršl Peter Ondra Petr Pávek Ondřej Slanař Pavel Suchý Vilím Šimánek Jitka Ulrichová

Organizing Committee Pavel Anzenbacher Eva Anzenbacherová Jitka Hybnerová Jana Novaková Jitka Ulrichová Karel Urbánek Rostislav Večeřa

Guest Editor Biomedical Papers Karel Urbánek Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2013 Sep; 157 (Supplement 1) Dear participants,

63. Czech and Slovak Pharmacological Days are Development of experimental techniques has opened taking place again, after six years, in Olomouc, Czech possibilities to answer questions about which we only Republic. It is a honor for all enthusiasts who work in dreamed of in the past. Also, the borders between life the field of pharmacology and related sciences in this sciences seem to be more fuzzy than before which hope- city and a pleasure to invite colleagues from both our fully will contribute to application of approaches formerly sister countries, and enjoy the hospitality and friendship typical for each discipline. of Olomouc and Haná region. The organizers also hope that this Conference will be Pharmacology has evolved during last years into a sci- a good place for exchanging experience in science as well ence with many new applications, and, simultaneously, as in the education of pharmacology in general. with new challenges and tasks to be solved in the future. Thank you for coming to Olomouc. Pharmacology and pharmacogenetics is taken as an example of personalization of medicine in the 21st century. Pavel Anzenbacher

v Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2013 Sep; 157 (Supplement 1)

CONTENTS

Abstracts, Oral presentations ...... S1 Abstracts, Posters ...... S16 Communications ...... S50

vi Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2013 Sep; 157 (Supplement 1):S1–S88.

O-1 O-2 Ethanol and its principle metabolite acetaldehyde Effect of methycobalamin application in patients

affect inward rectifier potassium current IK1 in rat with autism ventricular myocytes Adela Corejovaa, Drahomira Rauovab, Daniela Janosikovac, Marketa Bebarovaa, Peter Matejovica, Michal Pasekb, Veronika Pospisilovad, Maria Mikovae, Juraj Repiskyc, Milena Simurdovaa, Jiri Simurdaa Silvia Lakatosovaf, Anna Hrabovskaa, Jan Kyselovica aDepartment of Physiology, Faculty of Medicine, Masaryk aDepartment of Pharmacology and Toxicology, Faculty of University, Brno, Czech Republic Pharmacy, Comenius University in Bratislava, Bratislava, Slovak bInstitute of Thermomechanics - branch Brno, Czech Academy of Republic Sciences, Brno, Czech Republic bDepartment of Pharmaceutical Analysis and Nuclear Pharmacy, Faculty of Pharmacy, Comenius University in Bratislava, Bratislava, Slovak Republic Alcohol intoxication may induce arrhythmias, most cFaculty of Philosophy and arts, Trnava University, Slovak frequently the atrial fibrillation (AF). Increase of inward Republic rectifier potassium currents including the voltage-gated dAutism center Andreas in Bratislava, Slovak Republic e current IK1 is known to play an important role in the Autism center FRANCESCO in Prešov, Slovak Republic pathogenesis of AF. Data describing effects of ethanol fInstitute of Physiology, Faculty of Medicine, Comenius University and its principle metabolite acetaldehyde on mamma- in Bratislava, Slovak Republic lian IK1 are rare and controversial. Hence, we aimed to analyse IK1-changes in the presence of ethanol and acet- Oxidative stress has been suggested to be one of the aldehyde in enzymatically isolated rat right ventricular key elements in the pathophysiology of autism. An inter- myocytes by the whole cell patch clamp technique at room vention targeted to the glutathion metabolic precursors temperature. Ethanol (0.2–200 mM) and acetaldehyde could improve plasma biomarkers of impaired methyla- (3–300 μM) were applied by the rapid perfusion system tion capacity and improve behavior in patients with au- (each concentration in 3–19 cells). A dual effect of etha- tistic disorder. The aim of our project was to examine nol on IK1 was observed. At very low concentrations up to whether methylcobalamin application would affect the 0.8 mM (∼0.04‰), ethanol inhibited IK1, however, at con- gluthatione redox status and autistic disorder symptoms. centrations above 20 mM (∼0.92‰), ethanol conversely 37 patients with autistic disorder were enrolled. Exclusion stimulated IK1. The effect was voltage-independent. In ac- criteria for subject selection were: Asperger syndrome, cordance with these results, IK1-stimulation was preceded high-functioning autism, epilepsy, selected pharmacother- by a transient IK1-inhibition at the beginning of ethanol apy affecting CNS. Oral form of methylcobalamine was application. 2 and 8 mM ethanol (∼0.09 and 0.37‰, re- given daily at dose 500 μg. Venous blood was collected at spectively) caused inhibition of IK1 in some cells but its d0 and d100 and redox status of glutathione and levels of stimulation in others. Acetaldehyde inhibited IK1 with the homocystein, cystine and cobalamine were determined. concentration causing 50%-inhibition IC50 = 61.1±5.1 μM The psychological profile was defined at d0 and d100 by (the Hill coefficient nH = 1.51±0.18), i.e. IK1-inhibition psychologist using scale. Oral application of methylcobal- seems to be negligible in the clinically relevant plasma amine at the dose 500 μg per day influenced glutathione concentrations of acetaldehyde (in healthy humans usu- redox status. Social interaction was increased, including ally up to 3.7 μM). We conclude that ethanol exerts a dual social responsiveness and eye contact. Oral application of effect on the cardiac IK1. Inhibition of IK1 in some cells and methylcobalamine in patients with autism seems to poten- its stimulation in others might result in the heterogeneity tiate antioxidative mechanisms and leads to the changes of cardiac repolarization with possible arrhythmogenic in the psychological profile of the patients. consequences. It is unlikely that, in healthy humans, acetaldehyde significantly contributes to the arrhythmogen- esis observed after the alcohol consumption. O-3 Molecular forms of cholinesterases in heart

a,b b a ACKNOWLEDGMENT Dominika Dingova , Eric Krejci , Anna Hrabovska aDepartment of Pharmacology and Toxicology, Faculty of This work was supported by the grant project Pharmacy, Comenius University in Bratislava, Slovak Republic NT14301-3/2013. bCentre d’Etude de la Sensori-Motricite, CNRS UMR 8194, Universite Paris Descartes, 45 rue des Saints Peres, 75006 Paris, France

Cholinesterases are important enzymes that are targeted by various xenobiotics. In pharmacology, cholinesterase inhibitors are used in the diagnosis and treatment of skeletal muscle weakness and the therapy of memory

S1 Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2013 Sep; 157 (Supplement 1):S1–S88. decline. Recently, importance of cholinesterases in heart mood disorders during pregnancy and the postpartum has been highlighted by multiple research groups. period. An abnormal stimulation of serotonin receptors as Moreover, a link between changed enzyme activities and a result of an increased synaptic availability of serotonin some cardiovascular diseases has been suggested. and impairment of the activity of serotonin transporters Aim of the present project was to characterize and during the brain development due to administration of localize molecular forms of cholinesterases in heart. these drugs can lead to functional alterations accom- Genetically modified mice lacking different mo- panied by postpartum neurobehavioral dysfunctions. lecular forms of cholinesterases were used in the proj- However, there are lack of knowledge on possible adverse ect. Cholinesterase activities were determined in heart effects of SSRI/SNRI drugs on the functional brain devel- compartments by Ellman’s method. Different molecular opment and behavior of the offspring. In our experimental forms were distinguished in biochemical method of su- study, we focused on adverse reactions of developmental crose gradient. Precise localization of different molecular exposure to venlafaxine (VENF) on early postnatal and forms of cholinesterases in heart was determined in light neurobehavioral development of rat offspring. Our experi- microscopy by modified Karnovsky and Roots staining. mental study with venlafaxine showed that it may interfere Specific monoclonal and polyclonal antibodies were used with brain development by gender-dependant way and af- to visualize cholinesterases by fluorescence microscopy. fect neurobehavioral adaptations of rat offspring in a new We confirmed the presence of multiple molecular environment. forms of acetyl- and butyrylcholinesterase in mouse heart. The highest acetylcholinesterase activity was determined in atria, with dense localization in sino-atrial nodal region. ACKNOWLEDGEMENT Molecular form ratio was balanced. Butyrylcholinesterase is dispersed in all heart regions, while activity is 2.5-fold Supported by the grants VEGA 2/0081/11, VEGA higher in ventricles than in atria. Predominant molecular 2/0084/11 and VEGA 2/0107/12. forms of butyrylcholinesterase are monomers/dimers in all studied heart regions. O-5 Iron-chelating agents and acute myocardial ACKNOWLEDGEMENT infarction: in vitro and in vivo study This project was supported by APVV grant SK- Tomas Filipsky, Michal Riha, Radomir Hrdina, FR-0048-11, VEGA 1/1139/12, UK/294/2013. Premysl Mladenka

Department of Pharmacology and Toxicology, Faculty of O-4 Pharmacy in Hradec Kralove, Charles University in Prague, Hradec Developmental manipulation of monoaminergic Kralove, Czech Republic

systems affects neurobehavioral and Iron (Fe) is an essential element virtually for all liv- neuroendocrine regulations ing organisms. However, Fe is able to elevate the production of reactive oxygen species via Fenton chemistry. Michal Dubovicky, Estera Csaszarova, Eduard Ujhazy, Moreover, its homeostasis is disrupted in acute myocar- Natalia Sedlackova, Mojmir Mach dial infarction (AMI), which results in the promotion of oxidative stress. Because of the mentioned role of Fe in Institute of Experimental Pharmacology and Toxicology, Slovak Academy of Sciences, Dubravska cesta 9, 841 04 Bratislava, AMI, which has been the main cause of morbidity and Slovak Republic mortality worldwide, the use of Fe-chelating agents for therapy of AMI could have a therapeutic potential. In this study, Fe-chelating properties, effects on inhi- Monoamines have been shown to affect a variety of bition of Fenton chemistry of flavonoids and synthetic behavioral functions, such as aggression, sexuality, anxi- 1-phenyl-3-methyl-4-acylpyrazol-5-ones were evaluated. ety, mood or learning. In brain development, they play Moreover, effects of dexrazoxane were analysed in the an important organizational role, including cell division, isoprenaline model of AMI in Wistar: Han rats. migration, synaptogenesis, maturation of the cortex and In flavonoids, the most effective substitution was development of neuroendocrine systems. In males, for 6,7-dihydroxy group presented in baicalein, which was example, serotonin is markedly reduced during the 2nd similarly effective as a standard iron chelator deferoxam- and 3rd postnatal week what is essential for full mascu- ine, but its effect on Fenton chemistry was lower. The linization and defeminization of the brain and behavior. 3-hydroxy-4-keto conformation together with 2,3-double Manipulation of monoaminergic systems by the SSRI bond and the catecholic B ring (e.g. in quercetin) were and/or SNRI antidepressant drugs represents a risk factor associated with a substantial chelation but its effect on for healthy cognitive, emotional and behavioral develop- Fenton chemistry was rather negative. In acylpyrazolones, ment. Drugs of the SSRI/SNRI class belong to the most of particular interest is 2,6-bis[4(1-phenyl-3-methylpyra- frequently prescribed antidepressants in the treatment of zol-5-one)carbonyl]pyridine whose Fe-chelating properties

S2 Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2013 Sep; 157 (Supplement 1):S1–S88. increased when pH was decreasing. Interestingly, Fe- are probably associated with increased membrane turno- chelating properties of dexrazoxane did not play the major ver due to neurodegeneration. We found also dramatic role in the isoprenaline model. Its protective effects were changes of kfor (42%) in VD-rats. Compared to controls, probably mediated by inhibition of late myocardial impair- kfor decreased in VD-rats and this value returned to con- ment and ventricular fibrillation likely due to inhibition trols after simvastatin therapy. The MRS parameters can of myocardial calcium overload. be used as relatively non-invasive in vivo markers for de- Conclusively, detailed in vitro studies are necessary mentia or neurodegenerative diseases and they can pos- for the assessment of potential use of Fe chelators in the sibly predict metabolic disorders of the brain and also catecholamine model of AMI. reflect the effect of therapy not detectable by conventional MRI methods.

ACKNOWLEDGEMENT ACKNOWLEDGEMENT This congress contribution was supported by grant of the Czech Science Foundation (P303/12/G163) and by This work was supported by the grant VEGA Charles University in Prague (GAUK 605712 and SVV 2/0084/11. 263 003). O-7 O-6 Polyphenolic compounds and experimentally Effects of simvastatin and creatine citrate induced allergic asthma treatment in a rat model of dementia by in vivo Ivana Kazimierovaa, Marta Joskovaa, Olga Pechanovab, proton and phosphorus magentic resonance Martina Sutovskaa, Sona Franovaa spectroscopy aDepartment of Pharmacology JFM CU, Martin, Slovak Republic Svatava Kasparovaa, Ladislav Baciaka, Radka Tuskovaa, bDepartment of Normal and Pathological Physiology, Slovac Anton Kebisb, Michal Dubovickyc Academy of Science, Bratislava, Slovak Republic aDepartment of Analytical Chemistry, NMR&MS Laboratoatory, The aim of asthma pharmacotherapy is to find new Faculty of Chemical and Food Technology, Slovak University of sources of drugs able to modulate smooth muscle hyper- Technology, Bratislava, Slovak Republic reactivity and the degree of airways inflammation. bSlovak Medical University c Our experimental work was aimed at the influence of Institute of Experimental Pharmacology and Toxicology, Slovak ® Academy Science, Bratislava, Slovak Republic red-wine polyphenolic compounds (Provinol ) on defence airways reflexes and on inflammation during experimentally induced allergic asthma. We studied the effects of In vivo localized proton magnetic resonance spec- Provinol, and combinations of Provinol with clinically troscopy (1H MRS) has become a powerful and unique used antiasthmatics (budesonide and theophylline), technique to non-invasively investigate brain metabolism We utilized the model of guinea pig airways hyper- of rodents and humans. In our 1H MRS experiments, we reactivity induced by 21 days allergen (ovalbumin-OVA) focused on measurement and quantification of changes administration. During OVA sensitisation the experimen- in the following brain metabolites: N-acetylaspartate, tal animal were treated with Provinol (20 mg/kg/day p.o.), total choline, creatine+phosphocreatine (Cr/PCr), tau- budesonide (1mM by inhalation), theophylline (10mg/kg/ rine, myo-inositol, glutamine/glutamate, and lactate. day i.p.) or by half dose combinations of these substances. In addition, in vivo phosphosus (31P) MR spectroscopy The tracheal smooth muscle reactivity to histamine (10- can be used to continuous monitoring of net changes in 8-10-3 mol.l-1) was examined by in vitro method; The spe- phosphorus-containing metabolites (ATP, PCr, inorga- cific airways resistance (sRaw) was evaluated by in vivo to nic phosphate) in various pathological disorders. Very nebulized histamine (10-6 mol.l-1); The cough reflex was in- useful in application of 31P MRS is the measurement of duced by citric acid aerosol (10-3 mol.l-1). Bronchoalveolar enzyme-catalysed reactions using magnetization transfer lavage fluid (BALF) levels of IL-4, IL-5 and expression techniques, in particular, the rate constant of creatine ki- NOS from lung homogenate were utilized as parameters nase (CK) forward reaction, kfor rat brains in pathological of anti-inflammatory effect of Provinol. conditions. The aim of our study was to investigate effects Our result showed that 21 days administration of of two drugs – simvastatin and creatine citrate in vascular Provinol caused: a significant decrease of sRaw; the de- dementia (VD) rat model by 1H and 31P MR spectroscopy. cline in tracheal smooth muscle contraction amplitude; We used the pathophysiological rat model by permanent suppression of cough reflex. The bronchodilatory and an- 4-vessel occlusion in aged rats (> 15 months). We found titussive effect of Provinol was comparable to theophylline that brain metabolite concentrations varied depending on and budesonide. Furthermore, the half-dose combinations the type of therapy. We achieved a significant increase in of Provinol+theophylline and Provinol+budesonide ex- total choline and taurine levels in VD-rats. These changes ceeded bronchodilatory and antitussive effect substances

S3 Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2013 Sep; 157 (Supplement 1):S1–S88. used in monotherapy. Our results demonstrated the sig- O-9 nificant antiinflammatory effect of Provinol. In conclusion, we can summarize the most important Flavonoids from Morus alba affect cell cycle findings of our experiments: The polyphenolic compound of human cancer cells and inflammatory response Provinol posseses efficient antiasthmatic activity. Provinol had bronchodilatory, antitussive effect, suppressed asth- in macrophage-like cells matic inflammation of the airways. Furthermore, Provinol Peter Kollara, Tomas Bartab, Jan Hosekc, Karel Soucekd,g, amplified the bronchodilatory and antisussive effect of Veronika Muller Zavalovaa, Shushan Artiniane,f, budesonide and theophylline. Rabih Talhouke,f, Karel Smejkalc, Pavel Suchy Jra, Ales Hamplb,g ACKNOWLEDGEMENT aDepartment of Human Pharmacology and Toxicology, Faculty of Pharmacy, University of Veterinary and Pharmaceutical Sciences This work was supported by Centre of Experimental Brno, Palackeho 1-3, CZ-612 42, Brno, Czech Republic and Clinical Respirology II. – ‘‘Project co-financed from bDepartment of Histology and Embryology, Faculty of Medicine, EU sources’’ and by Grant VEGA 1/0030/11. Masaryk University, Kamenice 3, CZ-625 00, Brno, Czech Republic cDepartment of Natural Drugs, Faculty of Pharmacy, University of Veterinary and Pharmaceutical Sciences Brno, Palackeho 1-3, O-8 CZ-612 42, Brno, Czech Republic dDepartment of Cytokinetics, Institute of Biophysics, Academy of ACTH mediated regulation of the human MC2R Sciences of the Czech Republic, v.v.i., Kralovopolská 135, CZ-612 receptor 65 Brno, Czech Republic eNCC, Nature Conservation Center, American University of Beirut, Zuzana Kilianova, Nuria Basora, Marcel D Payet, Beirut, Lebanon Nicole Gallo-Payet fDepartment of Biology, Faculty of Arts and Sciences, American University of Beirut, Beirut, Lebanon Service of Endocrinology and Department of Physiology gInternational Clinical Research Center, St. Anne’s University and Biophysics, 3001, 12e Avenue Nord, Sherbrooke, J1H 5N4 Hospital, Pekařská 53, CZ-656 91, Brno, Czech Republic University of Sherbrooke, Quebec, Canada The root bark of Morus alba L. (MA) is used for its ACTH action on the adrenal gland is mediated by its diuretic, antitussic, antidiabetic, and antipyretic effects receptor termed MC2R belonging to the superfamily of in world traditional medicine. Therefore, Morus species G-protein-coupled receptors (GPCRs) and to the subfam- plants have been intensively studied from phytochemi- ily of melanocortin receptors (MCRs). MC2R is coupled cal point of view and bioactive compounds of flavonoid to heterotrimeric Gs protein leading to cAMP produc- character have been isolated. The aim of this study was to tion and activation of the PKA pathway. Upon prolonged evaluate cytotoxicity of three flavonoids isolated from MA agonist stimulation, these receptors classically uncouple (kuwanon E, cudraflavone B, and 4’-O-methylkuwanon from their effector enzymes – a process called desensitiza- E), and to determine their effects on proliferation of THP- tion – and undergo internalization using complex cellular 1 cells, and on cell cycle progression of cancer cells. Anti- machinery. inflammatory effects were also determined for all three The aim of this study was to investigate MC2R regula- given flavonoids. From the three compounds tested, cu- tion pattern as well as molecular mechanism(s) involved draflavone B showed the strongest effects on cell cycle in these phenomen. C-myc tagged hMC2R was expressed progression and viability of tumor and/or immortalized in the M3 (mouse melanoma) cell line enabling to evi- cells, and also on inflammatory response of macrophage- dence hMC2R both by immunoblotting and by indirect like cells. Kuwanon E and 4’-O-methylkuwanon E exerted immunofluorescence. Stimulation with ACTH induced more sophisticated rather than direct toxic effect on used production of cAMP with EC(50) values ranging from cell types. Our data indicate that mechanisms different 7.6-11.9 nM in transient and stable transfectants, respec- from stress-related or apoptotic signaling pathways are in- tively. Pretreatment with ACTH induced a dose-depen- volved in the action of these compounds. Although further dent loss of cAMP production, maximal desenzitization studies are required to precisely define the mechanisms of occurred after 15min of 10 nM ACTH stimulation and MA flavonoid actions, here we clearly demonstrate their was PKA-dependent. ACTH-induced loss of cAMP pro- effects combing antiproliferative and anti-inflammatory duction was accompanied by receptor sequestration into activity in human cancer and macrophage-like cells, re- intracellular vesicles reaching its maximum after 30-min spectively. Confirmed dual activity of tested prenylated 10 nM ACTH exposure. Immunofluorescence colocaliza- flavonoids could be an inspiration for chemical modification studies revealed that hMC2R were redistributed in tions of their structures or isolation of similar substances intracellular vesicles through a clathrin-dependent, but in order to get more potent agents usable for clinical prac- caveolae-independent, process involving PKA. In conclu- tice in future. sion, the present results indicate that hMC2R undergoes physiologic agonist desenzitization and internalization, both processes being PKA-dependent.

S4 Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2013 Sep; 157 (Supplement 1):S1–S88.

ACKNOWLEDGEMENTS ACKNOWLEDGMENT

Work was supported by IGA University of Veterinary This project was supported by IGA VFU 112/2013/ and Pharmaceutical Sciences Brno (62/2011/FaF, FaF. 3/2010/FaF, and 12/2010/FaF), and Masaryk University (MSM0021622430, CZ.1.05/1.1.00/02.0123), and by project FNUSA-ICRC (CZ.1.05/1.1.00/02.0123) from O-11 the European Regional Development Fund. Tolerability of gentamicin in septic preterm neonates with a patent ductus arteriosus O-10 Jirina Martinkovaa Pavla Pokornab TNFRSF1A and TNFRSF1B gene polymorphisms , aDepartment of Pharmacology, Faculty of Medicine in Hradec and their impact on effectivness of therapy with Kralove, Charles University in Prague, Simkova 870, 50038 Hradec infliximab Kralove, Czech Republic bDepartment of Pediatrics - PICU/NICU, General Faculty Hospital Michal Kolorza, Katerina Wroblovaa, Marian Batovskyb, Prague, Ke Karlovu 2, 1280 Prague, Czech Republic. Vladimir Zborilc, Jana Mokranovaa, Ladislava Bartosovaa Background. A patent ductus arteriosus (PDA) is aDepartment of Human Pharmacology and Toxicology, UVPS Brno, Palackeho 1-3, 612 42 Brno, Czech Republic defined as a shunt between the arterial and the venous bGastroenterological Clinic, University Hospital in Bratislava- system which leads to a negative effect on prerenal circu- Petrzalka, Antolska 11, 851 07 Bratislava, Slovak Republic lation.This condition has possible consequences to renal cDepartment of Internal Medicine and Hepatogastroenterology, and hepatic blood flow. In septic neonates treated with University Hospital, Jihlavska 20, 625 00 Brno, Czech Republic gentamicin (Ge) a PDA may lead to both an impairment of gentamicin clearance and increased vulnerability of Anti-TNFα presents itself as the most effective method the the target organs resulting in enhanced Ge toxicity. of treating Crohn’s disease (CD). Despite the high ben- Aims. The main goal was to assess tolerability of ki- efits there is about a 30% rate of primary non-response. netically guided therapy with Ge in critical ill preterm The main target of infliximab is the soluble form of TNFα neonates (TDM) and to identify the impact of covariates in order to block its pro-inflammatory activity as well as (hypotension and body fluid retention) on the Ge phar- in induction of apoptosis via the TNFα membrane form. macokinetics (PK). The activity of TNFα is mediated by interaction with its Methods. This open-label, prospective study (January receptors (TNFR). Polymorphisms in genes of such recep- 2006 -July 2009) enrolled preterm neonates (GA≤34 tors are expected to interfere with their functions due to weeks) during the first week of life stratified in Group 1 changes in protein structure. (GA >31 weeks) and Group 2 (GA within 34-38 weeks). The aim of this study was to evaluate whether poly- The first and second doses of gentamicin 4 mg/kg were morphisms in TNFRSF1A and TNFRSF1B genes influ- adjusted according to birth weight at a 48-hour postdose ence the efficacy of infliximab therapy. A total of 116 interval, next dosing was kinetically guided as described Caucasian CD patients treated with infliximab were previously (1). Tolerability as acute renal dysfunction genotyped. Therapy effectiveness was determined and during antibiotic therapy was assessed using labora- patients were separated into responders (n=98) and tory examinations (Scr, Surea, EF-Na, EF-Mg, and U-Ca/ non-responders (n=18). The genotypes of TNFRSF1A Ucr). Chronic renal dysfunction monitored over postna- (T4672G, G3794C) and TNFRSF1B (T11695C, T587G) tal year 1-5 considered nephrocalcinosis and ototoxic- was determined by PCR-RFLP. ity. Signs of nephrocalcinosis were regularly examined The frequency of variant alleles of TNFRSF1A was by renal sonography (Acuson Aspen Electric Medical comparable between responders and non-responders. Service). Hearing abnormalities were monitored using Variant allele TNFRSF1B 11695C was more common transient otoacoustic emission recordings (Danax AS 72 in non-responders (41.7% vs. 30.1%). Similarly the fre- Audiometer Echo-screen TDA, CZ). quency of TNFRSF1B 587G allele in non-responders was Results. 54 preterm neonates were enrolled to this 33.3% vs. 18.9% on responders. Homozygotes for variant study as follows: Group 1 (N=32), 18/32 of them with alleles of TNFRSF1B 11695C were found more often PDA, Group 2 (N=20), 4/20 with PDA. Neonates with (P=0.0132; OR 5.74, CI95% 1.6-22.1) in non-responders PDA were treated with ibuprofen (15 kg/mg in 3 con-

(n=5, 27.8%) than in responders (n=6, 6.1%). We suggest secutive days). Mean (SD) gentamicin clearance (CLGe1) that TNFRSF1B 11695C variant allele is associated with estimated after the first Ge dose = 0.47 (0.09) mL/min. low therapeutical effect of infliximab. kg-1 in Group 1, and 0.66 (0.10) mL/min.kg-1 in Group 2, was significantly lower in the former (P=0.005). This difference resulted from PDA and treatment with ibuprofen

as shown by comparison of CLGe1 in PDA neonates : 0.370 (0.250) mg/kg. min-1, vs those in non-PDA neonates: 0.700 (0.240) min.kg-1 (P<0.001). These results were

S5 Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2013 Sep; 157 (Supplement 1):S1–S88. consistent with the incidence of acute renal dysfunction airway reactivity to cumulative doses of acetylcholine in which appeared in Group 1 concerning RIFFLE crite- tracheal and lung tissue strips. These changes have been ria: 41% in PDA and 15% in non-PDA subgroups.Tubular associated with suppression of haematological markers dysfunction appeared in 43% of PDA and 15% of non- of inflammation and apoptosis in animals treated with PDA subgroups. All acute renal changes disappeared in roflumilast, suggesting potential use in diseases associated 3 weeks after the start of antibiotic therapy at the latest. with allergic inflammation. Transient signs of nephrocalcinosis appeared in 2 neonates (Group 1). Signs of ototoxicity were not found. Conclusions. Kinetically guided dosage of gentamicin ACKNOWLEDGEMENT based on plasma concentrations after the first dose should take into account comorbidities leading to changes in PK Supported by project UK/159/2013 and by grant parameteres such as a patent ductus arteriosus. VEGA 1/0030/11.

O-12 O-13 Influence of roflumilast on in vivo and in vitro Preclinical studies of a novel derivative of airway reactivity and apoptosis in ovalbumin- quercetin- inhibitor of aldo-ketoreductases sensitized guinea pigs AKR1B1 and AKR1B10. Implications for chronic Ivana Medvedovaa, Marek Prsoa, Alexandra Eichlerovaa, diabetic complications, inflammatory disorders Juraj Mokrya, Daniela Mokrab, Pavel Mikolkab and cancer aDepartment of Pharmacology, Jessenius Faculty of Medicine in Ivana Milackovaa, Marta Soltesova-Prnovaa, Martin, Comenius University in Bratislava, Sklabinska 26, 036 01 Magdalena Majekovaa, Ruzena Sotnikovaa, Jana Navarovaa, Martin, Slovak Republic a b b bDepartment of Physiology, Jessenius Faculty of Medicine in Lucia Rackova , Beatriz Diez-Dacal , Dolores Perez-Sala , Martin, Comenius University in Bratislava, Sklabinska 26, 036 01 Shabnam Enayatc, Sreepama Banerjeec, Milan Stefekb Martin, Slovak Republic aInstitute of Experimental Pharmacology, Slovak Academy of Introduction. Chronic inflammatory diseases, asso- Sciences, Bratislava, Slovak Republic bDepartment of Chemical and Physical Biology, Centro de ciated with airway obstruction and cough, are usually Investigaciones Biologicas, CSIC, Madrid, Spain treated with bronchodilating and anti-inflammatory drugs. cDepartment of Biological Sciences, Middle East Technical Inhibition of phosphodiesterases (PDE) leads to both of University, Ankara, Turkey these effects and as the persistence of airway inflammation depends on a decrease in apoptosis of T lympho- cytes and eosinophils, PDE inhibitors influence apoptosis Lipid peroxidation-derived aldehydes were found to of immune cells too. In chronic obstructive pulmonary be efficiently reduced by aldo-ketoreductase AKR1B1 to disease, roflumilast, selective PDE4 inhibitor, has been the corresponding alcohols, which mediate intracellular recently approved for the pharmacotherapy. The aim of inflammatory signals. Aldo-ketoreductases AKR1B1 and this study was to evaluate the effect of long-term admin- AKR1B10 are over-expressed in various types of cancers istration of roflumilast (p.o. and inh. way of application) and AKR1B1 is for a long time known like the first en- in experimentally induced allergic inflammation (model zyme of the polyol pathway which is implicated in the of allergic asthma) in guinea pigs. pathogenesis of chronic diabetic complications. The novel Material and Methods. 32 male adult guinea pigs, synthesized chloronaphthoquinone derivative of flavo- divided into 4 groups, have been used in the study. noid quercetin CHNQ showed high inhibition (with the

Control group has been left without sensitization. The IC50 values in micromolar range) of the aldo-ketoreduc- latter 3 groups have been sensitized with ovalbumin over tases: rat lens aldose reductase, human AKR1B1 and two weeks and thereafter treated perorally for 7 days AKR1B10. CHNQ was studied compared with querce- with roflumilast at the daily dose of 0.5 mg/kg b.w. or tin on the pathophysiological models of chronic diabetic by 3 minutes inhalation (5 mg/10 mL), or with vehicu- complications (inhibition of accumulation of sorbitol in lum, respectively. Specific airway resistance measured in isolated rat eye lenses during hyperglycemic conditions), whole-body double-chamber plethysmograph has been model of inflammation (induced ulcerative colitis of rats) used as a marker of in vivo airway reactivity. The in vitro and of cancer model (effect on the proliferation of the reactivity of tracheal and lung smooth muscle has been colon cancer cells HCT-116). The toxicology studies of tested using organ bath method. CHNQ were performed on fibroblast cell cultures. In all Results and Conclusion. Sensitization with ovalbumin three models, CHNQ was shown as an effective inhibitor, has led to significant increase in in vivo and in vitro air- more active than quercetin. No toxicity was observed in way reactivity. Roflumilast reduced both specific airway fibroblasts treated by pharmacologically expected con- resistance after nebulisation of histamine, and in vitro centrations of CHNQ. The results and physicochemical

S6 Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2013 Sep; 157 (Supplement 1):S1–S88. properties not violating Lipinski’s rules, indicate good of α7-/- mice, the effect of neostigmine was impossible bioavailability of CHNQ with prospective pharmacologi- to antagonize by atropine or hexamethonium, suggesting cal application. change of muscarinic receptor signalling. As no changes in heart physiology were observed in β2-/- mice in vivo or vitro, we assume that β2 subunits do not have an obvious ACKNOWLEDGEMENT function in heart physiology or that an effective adapta- tion to the lack of this subunit occurred. This work was supported by COST CM1001, VEGA 2/0067/11, VEGA 2/0030/11, EU Structural Funds (ITMS 26240220040), Middle East Technical ACKNOWLEDGEMENT University Research Funds (BAP-2008-07-02-04), RETIC RD07/0064/0007 from ISCIII, SAF2009-11642 and Work was supported by APVV SK-FR-0048-11, VEGA SAF2012-36519 from MINECO (Spain) to DPS. 1/1139/12 2012-2014, FaF UK/27/2013.

O-14 O-15 The role of nicotinic receptors in heart physiology Elucidation of the transformation of nabumetone Katarina Mrvova, Matej Kucera, Anna Hrabovska to the active metabolite, 6-methoxy- 2-naphthylacetic acid (6-MNA) Department of Pharmacology and Toxicology, Faculty of Pharmacy, Comenius University in Bratislava, Bratislava, Slovak Milan Nobilisa, Jiri Mikuseka, Barbora Szotakovaa, Republic Jiri Kunesa, Milan Poura, Robert Jiraskob, Michal Holcapekb, Cardiac function is controlled by autonomic nervous Eva Anzenbacherovac, Pavel Anzenbacherc, system. The autonomic imbalance with reduced vagal Jaroslav Kvetinad and increased sympathetic activity is associated with cardiac diseases and increased risk for cardiac mortality. aFaculty of Pharmacy, Charles University in Prague, Heyrovskeho Experimental reports have demonstrated that stimula- 1203, 500 05 Hradec Kralove, Czech Republic b tion of vagal nerve is able to improve outcomes for dis- Faculty of Chemical Technology, University of Pardubice, eases like hypertension or heart failure. Vagal activity of Studentská 573, 532 10 Pardubice, Czech Republic cDepartment of Pharmacology, Faculty of Medicine and Dentistry, heart is probably disrupted in ganglion for these diseases, Palacky University Olomouc, Hnevotinska 3, 775 15 Olomouc, where the transmission is mediated by nicotinic receptors Czech Republic (nAChR). Neuronal nAChR can be formed by various dInstitute of Experimental Biopharmaceutics, Joint Reseach combinations of heterologous subunits that determine Center of Academy of Science and PRO.MED.CS Praha a.s., ionic and ligand binding characteristics which are impor- Heyrovskeho 1207, 500 05 Hradec Kralove, Czech Republic tant for physiological functions. The aim of this project was to study the role of α7, β2 and β4 nAChR subunits Nonsteroidal anti-inflammatory prodrug nabum- in heart physiology. etone is after oral administration converted in liver to In our in vivo and in vitro experiments, mice with ab- 6-methoxy-2-naphthylacetic acid (6-MNA), the principal sence of α7 (α7-/-), β2 (β2-/-) or β4 (β4-/-) subunits and metabolite responsible for the NSAID effect. No interme- wild-type mice were used. In vivo hemodynamic measure- diates between nabumetone and 6-MNA have been identi- ments were performed at physiological conditions and fied so far. In our recent study (Nobilis M. et al., J Pharm during β1-adrenergic stimulation by dobutamine. In vitro Biomed Anal 80, 164-172, 2013), a new, as yet unreported experiments were performed on isolated heart perfused phase I metabolite was identified within a thorough study by Langendorff in physiological conditions and during of nabumetone metabolism by human and rat liver micro- application of a non-selective cholinesterase inhibitor, somes. Extracts from these biomatrices were subjected to neostigmine. Cholinergic stimulation was antagonized chiral LLE–HPLC–PDA and achiral LLE–UHPLC–MS/ by atropine or hexamethonium. MS analyses to elucidate the chemical structure of this Physiological parameters of heart were similar in α7-/-, metabolite. The new metabolite (having the elemental

β2-/- and wild-type mice in in vivo and in vitro experiments composition C15H16O3) was identified as 4-(6-methoxy- and even adrenergic stimulation did not reveal any signifi- 2-naphthyl)-3-hydroxy-butan-2-one (3-hydroxy nabum- cant differences. However, β4-/- mice had higher heart rate etone) and its identity was confirmed by the synthesis of in vivo and when recorded in isolated heart. Moreover, the compound. Following the incubation of nabumetone these mice had an atypical response to β1-adrenergic with the rat and human liver microsomal fraction, 3-hy- stimulation. These results suggest that β4 subunit plays droxy nabumetone was formed, but no 6-MNA was detect- an important role in heart rate regulation. All genotypes ed in this biomatrix. On the other hand, when 3-hydroxy responded to neostigmine by a significant decrease in nabumetone was incubated with isolated rat hepatocytes, the heart rate. With exception for α7-/- mice, heart rate 6-MNA was detected as the principal metabolite. Hence, was restored by atropine but not by hexamethonium, 3-hydroxy nabumetone was found to be the missing link which suggests a role of muscarinic receptors. In heart in nabumetone biotransformation to 6-MNA.

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O O O

CH3 CH3 OH OH H3CO H3CO H3CO nabumetone 3-hydroxy nabumetone 6-MNA

ACKNOWLEDGEMENT 100 μM significantly decreased PMA-induced phosphorylation of PKC α/ßII. Support from projects No. P207/10/2048 (Czech The results suggest that resveratrol represents an ef- Science Foundation), No. SVV-267-001 (Charles fective naturally occurring substance with potent pharma- University) and GAČR P303/12/535 is gratefully ac- cological effect on oxidative burst of human neutrophils. knowledged. It should be further investigated for its pharmacological activity against oxidative stress in ischaemia-reperfusion, inflammation and other pathological conditions, particu- O-16 larly neoplasia. On the molecular pharmacology of oxidative burst

inhibition in human neutrophils ACKNOWLEDGEMENT Rado Nosala, Katarina Drabikovaa, Viera Jancinovaa, Tomas Pereckoa, Juraj Harmathab, Jan Smidrkalc This work was supported by projects APVV 0052-10, APVV-0315-07 and VEGA 2/0010/13. aInstitute of Experimental Pharmacology and Toxicology SAS Bratislava, Slovak Republic bInstitute of Organic Chemistry and Biochemistry, CAS Prague, Czech Republic O-17 cInstitute of Chemical Technology, Faculty of Food and Kinetically guided dosage of gentamicin Biochemical Technology, Prague, Czech Republic in neonates treated for perinatal asphyxia Neutrophils are present in high numbers in areas of Pavla Pokornaa, Jirina Martinkovab inflammation, where they constitute an important source of reactive oxygen species (ROS). The massive production aDepartment of Pediatrics - PICU/NICU, General Faculty Hospital of antimicrobial and tumoricidal ROS in an inflamma- Prague, Ke Karlovu 2, 1280 Prague, Czech Republic b tory environment is called “oxidative burst” and plays an Department of Pharmacology, Faculty of Medicine in Hradec important role as the first line of defense against environ- Kralove, Charles University in Prague, Simkova 870, 50038 Hradec Kralove, Czech Republic mental pathogens. There are at least two signalling pathways responsible for induction of neutrophil activaion: one is the protein kinase C (PKC)-mediated pathway Background. Modest therapeutic whole body hypo- stimulated with phorbol-4ß-12ß-myristate-13α-acetate thermia (HT) is frequently used in neonates with moder- (PMA), and the other is the Src family proteintyrosine ate and severe hypoxic-ischemic encephalopathy (HIE). kinase-mediated pathway. Resveratrol (RES), a polyphe- If the neonate was contemporary treated with gentamicin nolic phytoalexin, is one of the most extensively studied (Ge) for sepsis, this new condition might modify the Ge natural products, with wideranging biological activity and pharmacokinetics (PK) and consequently its bactericidal tremendous clinical potential. RES has been shown to efficacy. have antioxidant, anti-inflammatory, anti-proliferative, and Aims. to analyze the PK of Ge in septic neonates suf- anti-angiogenic effects, while those on oxidative stress pos- fering from perinatal asphyxia (PA) and treated with HT sibly being presumably the most important. during the first week of life. Oxidative burst of whole human blood measured by Methods. This open-label, prospective study (January luminol/isoluminol-enhanced chemiluminescence(CL) 2006 - December 2009) enrolled full-term and nearly term and stimulated with PMA, fMLP (N-formyl-methionyl- neonates admitted to the Neonatal Intensive Care Unit leucyl-phenyl-alanine), OpZ(opsonized zymosan) and (NICU). The neonates were treated with Ge for suspected Ca2+-ionophore A23187 was inhibited in a concentration- or proven sepsis and those with PA underwent HT (the dependent way, indicating suppression of both receptor whole body temperature within 33-34 ○C). The first and and nonreceptor activated CL by resveratrol. Results from second doses of gentamicin (4 mg/kg) were adjusted ac- isolated human neutrophils revealed that resveratrol was cording to birth weight at a 48 - hour postdose interval, active extracellularly as well as intracellularly in inhibiting next dosing was kinetically guided as described previously the generation of reactive oxygen species. Liberation of (1). ATP and analysis of apoptosis showed that in the con- Results. 54 full-term/nearly term neonates (birth centrations of 100 μM RES did not change the viability weight 2.5-4.56 kg; GA within 36-42 weeks) were divided and integrity of isolated neutrophils. Western blot analysis to 4 groups: 3 groups of neonates suffering from sepsis documented that resveratrol in concentrations of 10 and and PA were treated as follows: group 1 (14/54) with Ge

S8 Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2013 Sep; 157 (Supplement 1):S1–S88. and HT (within 24 hours after birth); group 2 (8/54) Conventional cultivation methods with drug suscep- with Ge and HT (within 24-72 hours after birth); group 3 tibility testing to AT are slow and complicated, requiring (11/54) with Ge only. Septic neonates without PA (group several consecutive steps for diagnosis. During this time 4) were given Ge (21/54). PK parameters of Ge based patients may be treated inappropriately, drug resistant on 4 plasma concentrations after the first dose were as strains may continue to spread, and amplification of resis- follows: mean (SD) of Ge clearance of group 1-4 reached tance may occur. Therefore rapid diagnosis and identifica- 0.57 (0.24) mL/min.kg-1, 0.81 (0.48 mL/min.kg-1), 0.63 tion of MDR-TB or XDR-TB strains are prerequisites for (0.27 mL/min.kg-1), and 0.91 (0.41) mL/min.kg-1, respec- the worldwide fight against TB. tively. This value was significantly lower in group 1 vs group 4 (P=0.004). Mean (SD) values of Vd Ge in group 1-4 was 0.32 (0.15) L//kg, 0.41 (0.18) L//kg, 0.36 (0.191) O-19 L//kg, and 0.55 (0.40) L//kg), respectively. This PK pa- Issues of oral anticoagulants prescription rameter was significantly higher in group 4 vs group 1 (P=0.045). in elderly patients with atrial fibrillation Conclusions. Dosage of gentamicin should be indi- Veronika Slezakovaa, Maria Potocarovab, Jan Murinb, vidualized according to maturational/pathophysiological Andrej Dukatc, Michal Bozikd Miriam Petrovaa, covariates which might influence the gentamicin PK. Martin Wawrucha O-18 aDepartment of Pharmacology and Clinical Pharmacology, Faculty of Medicine, Comenius University in Bratislava, Sasinkova Diagnostics of resistant forms of tuberculosis – 4, 811 08 Bratislava, Slovak Republic b1st Department of Internal Medicine, Faculty of Medicine, conventional vs. molecular-genetic methods Comenius University, Mickiewiczova 13, 813 69 Bratislava, Slovak a,b b a Republic Igor Porvaznik , Juraj Mokry , Ivan Solovic c2nd Department of Internal Medicine, Faculty of Medicine, aNational Institute of Tuberculosis, Lung Diseases and Thoracic Comenius University, Mickiewiczova 13, 813 69 Bratislava, Slovak Surgery, Vysne Hagy, Slovak Republic Republic d bDepartment of Pharmacology, Jessenius Faculty of Medicine, Department of Traumatology, University Hospital Bratislava, Comenius University in Bratislava, Martin, Slovak Republic Limbova 5, 833 05 Bratislava, Slovak Republic

Despite tuberculosis (TB) currently belongs to rarer Atrial fibrillation (AF) represents a condition that is respiratory diseases in the territory of Slovak Republic associated with 5-fold increase of the risk of stroke com- ranking among countries with its low incidence, the inci- pared to healthy population. The risk of stroke increases dence in some other countries is growing year by the year. with advancing age, as well. Consequently, elderly people More serious is the fact that there appear mycobacterial with AF are an eligible group for prophylactic anticoagu- strains resistant to various anti-tuberculosis drugs (AT) lant therapy. Benefit of oral anticoagulants in patients more frequently. The emergence and spread of multi-drug with AF has been confirmed in all age groups. However, resistant tuberculosis (MDR - TB) and extensively drug- several observational studies showed a substantial under- resistant tuberculosis (XDR - TB) are major challenges utilization of oral anticoagulants in elderly populations for global tuberculosis control, since the treatment of re- with AF. CHADS2 and CHA2DS2-VASc score systems sistant forms is difficult both medically and financially. were created for evaluation of the risk of stroke in indi- In Slovakia, the diagnosis has been relying on standard vidual patient with AF. procedures based on the cultivation of mycobacteria and We carried out an observational study to determine subsequent drug susceptibility testing to antituberculot- the influence of the risk factors for stroke defined by ics (AT) for many years. Since these methods require CHADS2 and CHA2DS2-VASc score on the underutiliza- a lot of time, many times exceeding the time of the initial tion of oral anticoagulant treatment. In our study, 111 phase of tuberculosis treatment onset, it is necessary to patients aged ≥ 65 years with AF without any contrain- use new, time-saving diagnostic options. One of the pos- dication for such therapy were included. There was no sibilities is an automatic system that speeds up the time difference in the values of CHADS2 and CHA2DS2-VASc of cultivation, and the time needed to determine sensitiv- score in the group of warfarin users and non-users. We ity to AT. Even faster and more modern way is the use found significant difference in prescription of anticoagu- of molecular-genetic methods, allowing direct evidence lant treatment between elderly men and women with AF of M. tuberculosis in a biological, or culture material, re- (P<0.001). spectively. Furthermore, with these methods, the evidence of resistance to the two most important anti-tuberculosis drugs - Isoniazid and Rifampicin with MDR - TB, and ethambutol, aminoglycosides and fluoroquinolones in XDR – TB is enabled.

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O-20 O-21 Aryl hydrocarbon receptor and its crosstalk Analysis of non-steroidal anti-inflammatory drug with glucocorticoid receptor in human placental usage and risk perception in hospitalized patients barrier Zoltan Vargaa, Viera Kristovaa, Milan Kriskaa a a c Lucie Stejskalova , Alice Rulcova , Radim Vrzal , aDepartment of Pharmacology and Clinical Pharmacology, Zdenek Dvorakc, Petr Paveka,b Faculty of Medicine, Comenius University in Bratislava, Sasinkova 4, 811 08 Bratislava, Slovak Republic aInstitute of Molecular and Translation Medicine, Faculty of Medicine and Dentistry, Palacky University Olomouc, Drugs from the group of non-steroidal anti-inflam- Hnevotiska 5, 779 00, Olomouc, Czech Republic matory drugs (NSAIDs) belong to the most widely pre- bDepartment of Pharmacology and Toxicology, Faculty of scribed and used pharmacological agents worldwide. Data Pharmacy, Charles University in Prague, Heyrovskeho 1203, gathered in the last decade shows increased incidence of Hradec Kralove, 500 05, Czech Republic c thrombotic events during their administration. The in- Department of Cell Biology and Genetics, Faculty of Science, creased risk of myocardial infarction seems to be a class Palacky University Olomouc, Slechtitelu 11, Olomouc, 779 00, Czech Republic effect of NSAIDs. The magnitude of the risk shows considerable variability within the group. Continuous analysis of NSAID usage with assessment of risk for development Glucocorticoids (dexamethasone and betamethasone) of adverse effects is needed for increasing the safety of are transplacentally administered before 35 weeks of preg- analgesic treatment. For limiting the impact of adverse nancy in fetuses with the risk to be born prematurely to effects on the health of patients, NSAID users should increase fetal pulmonary surfactant production and pre- be informed about the possible adverse effects and their vent infant respiratory distress syndrome. symptoms to ensure early detection and treatment dis- Aryl hydrocarbon receptor (AHR) is a mediator of continuation. In the presented study, we retrospectively the toxicity of particular xenobiotics such as dioxin, poly- analyzed the prescription of NSAIDs in 428 patients in cyclic aromatic hydrocarbons and halogenated biphenyls need of analgesic treatment hospitalized at a department that are involved in tumor initiation and progression. of internal medicine. Factors increasing the risk for ad- The CYP1A1 is the most important xenobiotic-metabo- verse effects were also investigated. A questionnaire study lizing cytochrome P450 enzyme in the human placenta. was conducted to gather information concerning the Importantly, CYP1A1 generates reactive species and its knowledge of NSAID users about adverse effects of the placental activity is elevated in smoking women. CYP1A1 used medication. A total of 251 hospitalized patients and expression is mainly controlled by aryl hydrocarbon recep- a control group of 234 random respondents from a shop- tor ligands. ping center were enrolled into this study. Data were eval- We studied the effects of the glucocorticoids on induc- uated using descriptive statistics, comparisons between ibility of AHR, ARNT, CYP1A1 and other AHR target groups were performed by using of Student’s t-test and genes (CYP1A2, CYP1B1, UGT1A1, BCRP) by proto- chi-squared test. Our results suggest that most patients type AHR ligand 3-methylcholanthrene (3MC) in isolated treated with NSAIDs have factors indicating increased human placental trophoblast culture. risk of development of adverse effects. The results of our We showed that glucocorticoids alone had no effect questionnaire study show limited knowledge of NSAID on activity and protein/mRNA expression of CYP1A1 users about their adverse effects. We consider as alarming and little effect on mRNA expression of other AHR target that only a limited number of respondents were informed genes. However, glucocorticoids significantly augmented by their physician or pharmacist about the possible risks EROD enzymatic activity and significantly stimulated of treatment. More effective communication is needed only CYP1A1 mRNA induction mediated by 3MC. from the side of healthcare professionals to minimize the In conclusion, our data suggest that glucocorticoids long term consequences of adverse effects of NSAIDs. have no effect on AHR target genes expression per se, but they may potentiate CYP1A1 induction in human term placental trophoblast. ACKNOWLEDGMENT

This study was supported by VEGA 1/0501/11. ACKNOWLEDGMENT

Our laboratories are supported by grants CZ.1.05/2.1.00/01.0030 and CZ.1.07/2.3.00/30.0041.

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O-22 O-23 Application of computer-based modeling in the The specific issues in pharmacotherapy in elderly analysis of cardiovascular regulation mechanisms patients – misprescription and underprescription Robert Vojtko, Miriam Petrova, Radoslav Villaris, Martin Wawrucha, Veronika Slezakovaa, Jan Murinb, Viera Kristova Andrej Dukatc, Michal Bozikd, Maria Potocarovab

Department of Pharmacology and Clinical Pharmacology, aDepartment of Pharmacology and Clinical Pharmacology, Faculty of Medicine, Comenius University in Bratislava, Sasinkova Faculty of Medicine, Comenius University in Bratislava, Sasinkova 4, 811 08 Bratislava, Slovak Republic 4, 811 08 Bratislava, Slovak Republic b1st Department of Internal Medicine, Faculty of Medicine, Comenius University, Mickiewiczova 13, 813 69 Bratislava, Slovak Introduction. Even after the replacement of analog Republic recorders of vascular segment responses to vasoactive c2nd Department of Internal Medicine, Faculty of Medicine, stimuli by digital ones evaluation of vessel reactivity still Comenius University, Mickiewiczova 13, 813 69 Bratislava, Slovak relies mainly on several classical descriptive parameters Republic such as amplitude of contractile responses and area under dDepartment of Trauma Surgery, University Hospital Bratislava, the curve. Advanced current digital measurement circuits Limbova 5, 833 05 Bratislava, Slovak Republic allow incomparably more accurate assessment of a wide set of contractile response parameters, whose determina- The specific features of pharmacotherapy in elderly tion was not possible by routine descriptive methods. people results from changes in pharmacokinetics and Aim. To quantify and assess computer-based model pharmacodynamics of drugs, polymorbidity, polyphar- parameters of digitally recorded contractile responses of macy as well as altered compliance. The use of certain perfused rat aterial segments in comparison with para- drugs is accompanied with increased risk of adverse drug meters available by conventional descriptive evaluation. reactions when administered in elderly patients. For this Material and Methods. Our first study analyzed re- reason lists of potentially inappropriate medications were sponses of rat renal arterial segments from control and created. For drugs listed in these lists safer or more effec- diabetic animals to successively increasing bolus doses of tive alternatives are available. The issue of excessive, often noradrenaline (0.1; 0.5; 1; 3; 6; 10 μg). Finally, after pre- potentially inappropriate and also duplicate drug prescrip- contraction there was induced a relaxation by single bolus tion is widely discussed in the literature. On the other dose of acetylcholine (20 μg). Our second study com- hand, less attention is paid to the issue of the absence pared segment responses of renal arteries from healthy of beneficial medication administration. This is a more rats induced by noradrenaline and adrenaline under the frequently occurring issue in comparison with potentially same pattern of stimulation. For the analyses descriptive inappropriate drug prescription. The consequences of the evaluation were used as well as computational modeling absence of administration of beneficial drugs may mani- using methods Levy and Monte Carlo. fest later than the adverse drug reactions of potentially Results. At descriptive evaluation of contractions inappropriate medications. The consequences of absent- we found no significant difference between groups at ing prescription are very serious, e.g. the increased inci- any used dose of neither noradrenaline nor adrenaline. dence of cardio- and cerebrovascular events. A certain Relaxatory responses in the diabetic group showed dis- fear of doctors regarding possible adverse drug reactions tinct decline compared to controls. Digital modeling represents an important reason for non-administration of procedures revealed significant changes of several charac- beneficial medicines. teristic parameters of contractile responses in the diabetic Cardiovascular protective treatment represents an ex- group vs. controls and also in responses to noradrenaline ample of relatively often underused therapy. In our study, vs. adrenaline. 167 patients aged ≥65 years with acute myocardial infarc- Coclusion. The used software design allows to quantify tion or stroke and patients with history of these events large scale of contractile response parameters unavailable were included. Antiplatelet medication was absent in 52 for descriptive evaluations. Thus it seems to be an useful (31.1%) patients without contraindication for antiplatelet tool in comparison with routine descriptive methods. therapy. Underprescription of antiplatelet therapy was common in patients with stroke (P<0.001).

ACKNOWLEDGEMENT

This work was supported by the grant VEGA SR nr. 1/0501/11.

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O-24 O-25

Association of haplotypes HLA-DQ2, HLA-DQ8 BKCa channels and experimental allergic asthma and polymorphism G-308A IN TNF-alpha gene Michaela Kocmalovaa, Maros Oraveca, Marian Adamkovb, with coeliac disease Martina Sutovskaa, Sona Franovaa

Katerina Wroblovaa, Michal Kolorza, Zuzana Horakovaa, aDepartment of Pharmacology, Jessenius Faculty of Medicine in Igor Pavb, Ladislava Bartosovaa Martin, Comenius University in Bratislava, Sklabinska 26, 036 01 Martin, Slovak Republic aDepartment of Human Pharmacology and Toxicology, Faculty bInstitute of Histology and Embryology, Jessenius Faculty of of Pharmacy, UVPS, Brno, Palackeho 1-3, 612 42 Brno, Czech Medicine in Martin, Comenius University in Bratislava, Mala Hora Republic 4, 036 01 Martin, Slovak Republic bGastroenterological Clinic SMU and University Hospital in Bratislava-Petrzalka, Antonska 11, 851 07 Bratislava, Slovak BKCa are universally expressed in organism and they Republic participate in various physiological processes. The channels are activated by both depolarization as well as by

Coeliac disease (CD) is one of the most common calcium concentration increased. BKCa channels play an autoimmune diseases but it is estimated that only 10% important role in airway smooth muscle (ASM). They of patients are diagnosed. Typical abdominal symptoms can be an important regulator of membrane potential and most commonly prevail in children; in adults, usually ex- integral signal in ASM. Regard to above mentioned, this hibit atypical symptoms (anemia, osteoporosis, dermatitis channel activation becomes possible mechanism for the herpetiformis etc.), which are harder to diagnose. asthma treatment. Genetic predispositions play an important role in the The aim of presented studies was to prove whether pathogenesis of CD. CD is associated with HLA-DQ2 long-term therapy by opener of BKCa ion channels NS haplotype (coded DQA1*05/DQB1*02) and HLA-DQ8 1619 inhibit the cough reflex, modulate the ASM reactiv- (DQA1*03/DQB1*0302). Other genes that could be in- ity and influence levels of exhaled NO and mast cells volved in the development of CD are studied, of which infiltration in conditions of experimental allergic inflam- the TNF-α gene is a candidate gene. mation of the airways in guinea pigs. The aim of this study was to create, validate and stan- Presented studies were carried out after treatment of dardize PCR-multiplex methodology for the detection ovalbumine-sensitized guinea pigs by NS1619 for 14 days. of CD risk haplotypes HLA-DQ2 and HLA-DQ8 and Allergic inflammation of airways was induced by repeti- determination of G-308A polymorphism in TNF-α gene tive exposure of guinea pigs to ovalbumine and the degree by PCR-RFLP. Genotypization was performed in 126 pa- of allergic inflammation was determined by histological tients with confirmed diagnosis of CD, 66 family mem- analysis of tracheal and pulmonary samples. The cough bers and 58 healthy subjects. The association between the reflex was induced by 0.3 M citric acid aerosol for 3 min aforementioned genetic variants and the occurence of CD interval, in which total number of coughs was counted. was also explored. ASM reactivity in vivo was expressed as values of specific Statistical analysis confirmed significant differences airway resistance (sRaw) calculated by Pennock. The in- in the occurrence of: fluence of NS1619 on exhaled NO levels and degree of • HLA-DQ2 haplotype among patients and healthy sub- mast cell infiltration visualized by tryptase posivity was jects (P=0.0001) used to prove its anti-inflammatory activity. • HLA-DQ2 haplotype among patients and family mem- NS 1619 significantly reduced sRaw values after long- bers (P=0.0308) term application regardless to used bronchoprovoking • HLA-DQ2 haplotype among family members and substances but this opener didn`t influence cough reflex. healthy subjects (P=0.0001) Immunohistochemical staining of specimens showed in- • HLA-DQ8 haplotype among HLA-DQ2 negative and creasing signs of allergic inflammation during sensitiza- HLA-DQ2 positive patients (P=0.0156) tion procedure as well as significant anti-inflammatory • variant allele 308A among patients and healthy sub- effect of NS 1619. These results were confirmed by indica- jects (P=0.0001) tive measurement of exhaled NO levels. • variant allele 308A among patients and family members (P=0.0188) • variant allele 308A among family members and ACKNOWLEDGMENT healthy subjects (P=0.0046) This work was supported partly by grant VEGA Our results confirmed the association of haplotypes 1/0020/11, VEGA 1/0062/11 and Center of Experimental HLA-DQ2, HLA-DQ8 and G-308A polymorphism in the and Clinical Respirology II co-financed from EU sources. TNF-α gene with coeliac disease.

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O-26 O-27 Improvement of standard anti-arthritic How to prevent adverse drug interactions therapy with N-feruloylserotonine addition to in clinical practice methotrexate in rat adjuvant arthritis Milan Kriska a a a Viera Kuncirova , Silvester Ponist , Frantisek Drafi , Department of Pharmacology and Clinical Pharmacology, Danica Mihalovaa, Marcela Dobiasovaa, Ingrid Tumovab, Faculty of Medicine, Comenius University in Bratislava, Sasinkova Juraj Harmathac, Radomir Nosala, Katarina Bauerovaa 4, Bratislava, Slovak Republic aInstitute of Experimental Pharmacology and Toxicology, Slovak Academy of Sciences, SK-84104, Bratislava, Slovak Republic Polypharmacy in intensive pharmacotherapy repre- bDepartment of Pharmacology and Toxicology, Faculty of sents a typical face of health care in developed countries. Pharmacy, Comenius University in Bratislava, Bratislava, Slovak It is known, that long term usage of more than 5 medi- Republic cines probably produce one adverse interaction. In risky cInstitute of Organic Chemistry and Biochemistry, Academy of groups as elderly patients, others with chronic cardiovas- Sciences of Czech Republic, Prague, Czech Republic cular diseases and DM-2 type patients with complications exceeds consumption more than 10 preparations. Various, Most anti-rheumatics have side-effects when used mostly pharmacopeidemical studies revealed possibil- in higher doses and/or within long-term dosage. ity of 30 to 50% their prevention. These facts forced to Methotrexate (MTX) has become the main immuno- build an effective system of predictive drug selection in suppressive substance used in the treatment of patients therapy with the lowest interaction potential. Projection with rheumatoid arthritis (RA). N-feruloylserotonine and development of an innovative drug involves a search (N-f-5HT) a plant derived natural substance, was found for identification of possible interactive potential on both to posses many positive biological effect: antiprolifera- levels: experimental and clinical. Preregistration random- tive, antioxidative, antiatherogenic as well as inhibition ized studies of IIIb involve evaluation of risk and efficacy of oxidative burst of isolated neutrophils in whole blood. of new drug in treatment schedule containing perspective Combination therapy is expected to have a higher efficacy combinations. without toxicity. The aim of this study was to evaluate the The most important data with regard to possible inter- effects of N-f-5HT in monotherapy and in combination actions in pharmacotherapy are expected to come from therapy with methotrexate (MTX) on disease progression PAAS (Post authorization safety studies), see details in in rat-induced adjuvant arthritis (AA). AA was induced by new legislative from GVP, Commission implementing a single intradermal injection of Mycobacterium butyricum regulation (EU) No 520/2012. in incomplete Freund’s adjuvant to male Lewis rats. The Monitored signals of adverse interactions from these experiments included healthy animals, arthritic animals sources will be further validated in special conditions and without any drug administration, arthritic animals with widely published in EMA, WHO specialized journals. administration of N-f-5HT in the oral daily dose of 15 mg/ Preferential sources of interaction potential are kg b.w., arthritic animals with administration of metho- from ET databases: In printed form are yearly updated trexate in the oral dose of 0.3 mg/kg b.w. twice a week in monographs as Tatro DS, Drug Interaction Facts. and arthritic animals with administration of the combi- Wolters Kluwer Health, Stockey’s Drug Interactions, nation of N-f-5HT and MTX. The following parameters Pharmaceutical Press London. were monitored: clinical (change of hind paw volume The most effective prevention of adverse interactions and arthritic score), biochemical (the activity of cellular will be achieved by implementation of recent results γ-glutamyltransferase in spleen and joint homogenates, of pharmacovigilance and its effective communication activity of glutation reductase in brain homogenate and among health care professionals, physician, pharmacists plasmatic levels of TBARS) and immunological (plas- on one side and patients on the other side. matic levels of CRP, MCP-1, IL-1 and IL-17). Our results showed the beneficial effect of N-f-5HT in combination therapy with MTX. The combination is more effective than MTX alone and it could improve the treatment of RA.

ACKNOWLEDGMENT

Supported by the grants VEGA 2/0045/11, APVV- 0052-10.

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O-28 O-29 Commonly measured biomarkers of oxidative Polymorphisms in protein C gene promoter region stress do not reliably predict cardiovascular and endothelial protein C receptor gene impairment as predisposing factors for venous thrombosis Premysl Mladenkaa, Tomas Filipskya, Jaroslava Vavrovab, Kristyna Horakova, Ladislava Bartosova, Michal Kolorz, Magdalena Holeckovab, Vladimir Palickab, Radomir Hrdinaa Petra Hanzlikova

aDepartment of Pharmacology and Toxicology, Faculty of Department of Human Pharmacology and Toxicology, Faculty of Pharmacy in Hradec Kralove, Charles University in Prague, Pharmacy, UVPS Brno, Czech Republic Heyrovskeho 1203, 500 05 Hradec Kralove, bFaculty of Medicine in Hradec Kralove, Simkova 870, 500 38 The primary abnormalities that are associated with Hradec Kralove and University Hospital Hradec Kralove, Sokolska an increased risk of venous thrombosis (VT) are the in- 581, 500 05 Hradec Kralove, Czech Republic herited deficiencies of protein C. Protein C (PROC), encoded by the PROC gene, acts through its high affinity for Oxidative stress has been linked with cardiovascular binding to its transmembrane receptor (EPCR) encoded diseases in most studies although the pathophysiologi- by the EPCR gene. The objective of the present study was cal relevance has still remained not fully understood. to analyze the link between haplotypes of three polymor- Therefore the aim of this study was to establish the re- phisms in the promoter region of PROC gene, the poly- lationship among different commonly used biomarkers morphism in the EPCR gene and the occurrence of VT. of oxidative stress and cardiovascular dys/function in We genotyped 289 individuals - 126 cases with docu- experimental rats. mented VT and 163 healthy volunteers without a history The data from 145 male Wistar:Han rats, previously of DVT.Genetic analysis was carried out in Laboratory published in original papers analysing effects of iron che- of pharmacogenomiccs, Dept. of Human Pharmacology lators on a model of acute myocardial infarction (AMI), and Toxikology, Faculty of Pharmacy, UVPS, Brno. The were re-analysed in this study. A pathological state simi- occurrence of three polymorphisms in the promoter of lar in many aspects to AMI in humans was induced in PROC gene (-1654C/T, -1641A/G and -1476A/T) was de- a half of these animals by administration of isoprenaline termined by a single PCR reaction, where -1654C/T and (100 mg.kg-1, s.c.). Animals were pre-treated with different -1476A/T were detected by PCR-REA and -1641A/G by iron chelators or solvents in order to mimic treated and allele-specific probes with Real - time PCR. The 6936A/G non-treated population with and without cardiovascular polymorphism in the EPCR was detected by PCR-REA. disorder. The groups of patients and controls were simultaneously As expected, serum cardiac troponin T (cTnT) cor- genotyped for 5 other polymorphisms, which are signifi- related strongly negatively with the cardiac function and cantly linked to a predisposition for VT, i.e. the factor positively with myocardial calcium levels and wet ven- V Leiden. tricles weight. Concerning parameters of oxidative stress, Our results showed that, based on the available knowl- only weak negative associations were found for cTnT and edge, the combination of TAA haplotypes is associated total blood glutathione or serum vitamin C concentra- with a higher plasmatic level of PROC and therefore tions, while no significant correlations were found with with a lower risk of the development of VT. This com- serum vitamin E and plasma TBARS. Similar findings bination occurred with a higher frequency in the control were documented between other parameters of cardiac (P=0.0206). Furthermore, we confirmed a significantly function and biomarkers of oxidative stress. higher (P=0.0066) occurrence of the combination of Conclusively, it appears that commonly used biomark- TAA haplotypes and standard genotype AA EPCR in the ers of oxidative stress may not reliably correspond to the control than in the group of patients.To the contrary, the cardiovascular dys/function. frequency of PROC, CAA and CGT haplotypes was in- significantly higher in the group of patients than in the control. Compared with the genetic variant associated ACKNOWLEDGEMENT with higher PROC levels (TAA), plus the EPCR AA genotype, the genetic variant associated with lower protein C This congress contribution was supported by the levels (CGT and CAA) plus the EPCR AG genotype is grant P303/12/G163 of the Czech Science Foundation, indeed a risk factor for thrombosis. by Charles University (GAUK 39207C and SVV 263 003) and by the project of the Ministry of Health, Czech Republic for conceptual development of research organization 00179906.

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O-30 target AUC of 5000-7000 μg/L.h. All the three patients required another follow-up monitoring with or without Current challenges of body weight based dose adjustment. intravenous busulfan dosing versus dose Conclusions. Our results demonstrate that even after i.v. busulfan administration, interindividual as well as adjustment based on therapeutic drug monitoring intra-individual PK/PD variability is of great concern. Hundie Tesfayea, Romana Branovaa, Petr Rihab, The overall conclusions drawn from these case series Petr Sedlacekb, Jan Vydrac observation is to recommend inter-dose follow-up therapeutic drug monitoring instead of relying on initial dose aDepartment of Medical Chemistry and Clinical biochemistry, predictions as highly required tool to guarantee aimed tar- Division of Clinical Pharmacology, Faculty Hospital in Motol and get with careful interpretation of drug levels considering 2nd Faculty of Medicine, Charles University in Prague, Prague, all influential factors. Thus, it is strongly suggested that Czech Republic follow-up AUC monitoring between doses is may certainly bDepartment of Paediatric Haematology and Oncology, Faculty help to reduce the risk of poor outcomes both in adult nd Hospital in Motol and 2 Faculty of Medicine, Charles University and paediatric HSCT patients. in Prague, Prague, Czech Republic c Institue of Haematology and Blood Transfusion, Prague, Czech Republic O-31 First clinical pharmacology department Introduction. Busulfan in high doses is often used to substitute total body irradiation for bone marrow or hae- in the world mopoietic stem cell transplantation (HSCT) condition- Jan Strojil, Jaroslav Jezdinsky ing therapy. Its considerable pharmacokinetic variability and worrying adverse effect in case of extreme exposure Department of Pharmacology, Faculty of Medicine and Dentistry, warrants pharmacokinetic monitoring in both oral and Palacky University Olomouc, Hnevotinska 3, 775 15 Olomouc, intravenous forms. Previous works suggested that test Czech Republic pharmacokinetic study enables dose prediction for all of the rest doses given every 6 hourly for four days. However, In the past we have published on the topic of the his- extensive case series observations and further studies indi- tory of clinical pharmacology, specifically on the life and cate that the drug shows intra-individual variability chal- work of prof. MUDr. Emil Starkenstein who was a profes- lenging the first dose based prediction. sor of pharmacology at Charles University in Prague and Objectives. The principal aim of this communication is who is considered to be one of the founders of the field to describe typical cases, where dose prediction based on of Clinical Pharmacology. post initial doses concentrations measurement and con- Recent serendipitous finding in the Austrian medical sequent area under the concentration versus time curve journal Therapeutische Monatshefte from 1916 however (AUC) calculation may not be reliable and that may en- led us to reconsider the authorship of the term “clini- danger the principal goal of the intervention. cal pharmacology”. It appears that Dr. Hans Januschke, Patients and Methods. Three exemplary out layer cases born in Opava in 1883, was tasked by then head of the have been processed from patients after the provision of Pharmacology Department, prof. Hand H. Meyer, to informed consent and approval by an independent ethics found a dedicated department of Clinical Pharmacology committee. An adult male at age of 49 years, and two at the Karolinen-Kinderspital in Vienna as early as 1911. children (1 male and 1 female) both at age of 2 years, The children hospital was then headed by prof. C. Frh. respectively have been treated with i.v. busulfan doses on v Pirquet, an interesting figure in his own right. body weight basis according to clinical protocol before The department published several case reports on HSCT. Sampling was started with trough concentration children whose treatment was based on translation of ba- (immediately before the 5th dose) followed by samples im- sic pharmacology research. The department also had an mediately after the end of 2-4 hour lasting infusion (peak), experimental unit where Januschke continued his animal 4h, and 6h from the starting time of the infusion utiliz- research. Literature search did not reveal the fate of the ing limited sample strategy. Busulfan concentrations were department and none of the Austrian pharmacology de- determined by high performance liquid chromatography partments seem to be claiming to be a successor of what

(HPLC). AUC was calculated according to trapezoidal appears to be the first Clinical Pharmacology department rule. in the world. The literature trail ends in 1924. Results. At initial measurement AUC in adult patient The authors plan on publishing an account of the his- was and in a female infant case revealed unacceptably tory in hope that someone knowledgeable of the ultimate low exposure expressed by low AUC 496.4 μg/L-hr. and fate of the department will help us write the ending to an

1284 μg /L-hr), respectively. In contrast, AUC Ctrough-C6 important chapter from the very beginnings of our field. calculated according to the trapezoidal rule in the male child revealed overexposure expressed by AUC Ctrough-C6 11135 μg.L-hr, which is evidently beyond myeloablative

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ACKNOWLEDGMENT effect and its combinations with other nucleosides did not provide any improvement of protective effect of these Supported by Grant IGA UPOL LF 2013_007. nucleosides. Our findings provide in vitro rationale for using uridine or uridine-thymidine ointment in HF syndrome therapy. O-32 Uridine in the ointment could be probably replaced by cy- The protective effect of pyrimidine nucleosides tidine but the thermal instability of this compound (need to be stored in 5°C) makes it a worse candidate for the and their combinations on HaCaT keratinocytes use in clinical praxis. treated with 5-FU: MTT, NTCA and RTCA tests

a b c Jan Hartinger , Pavel Vesely , Eva Matouskova , ACKNOWLEDGEMENT Lubos Petruzelkad,f, Irena Netikovae,f This work was supported by grant NS 9786-4/2008 aHospital Pharmacy, 1st Faculty of Medicine, Charles University in Prague and General Teaching Hospital in Prague, Czech Republic from the Grant Agency of the Ministry of Health of the bDepartment of Cell Biology, Institute of Molecular Genetics, Czech Republic. Academy of Sciences of the Czech Republic, Prague, Czech Republic cPrague Burn Centre, 3rd Faculty of Medicine, Charles University, P-1 Prague, Czech Republic Interaction of rocuronium with human liver dDepartment of Oncology, Hospital Na Bulovce, Prague, Czech Republic cytochromes P450 eOncology and Clinical Pharmacist of Hospital Na Bulovce, a b c Prague, Czech Republic Eva Anzenbacherova , Alena Spicakova , Milan Adamus , fDepartment of Oncology, 1st Faculty of Medicine, Charles Petr Bachledad, Jitka Ulrichovaa, Pavel Anzenbacherb University in Prague and General University Hospital in Prague, Czech Republic aDepartment of Medical Chemistry and Biochemistry, Faculty of Medicine and Dentistry, Palacky University Olomouc, Hnevotinska 3, 775 15 Olomouc, Czech Republic 5-Fluorouracil (5-FU) is one of the most commonly bDepartment of Pharmacology, Faculty of Medicine and used antineoplastic drugs in the therapy of breast, oesoph- Dentistry, Palacky University Olomouc, Hnevotinska 3, 775 15 agus, head and neck and especially colon cancer. Hand- Olomouc, Czech Republic cDepartment of Anesthesiology and Intensive Care Medicine, foot (HF) syndrome (palmar-plantar erythrodysesthesia) University Hospital Olomouc, I.P.Pavlova 6, 775 20 Olomouc, is an adverse effect frequently related with long term i. v. Czech Republic administration of 5-FU and its orally applicable prodrug dDepartment of Surgery II – Vascular and Transplantation capecitabine. According to histology findings it can be Surgery, University Hospital Olomouc, I.P.Pavlova 6, 775 20 characterized as non-specific toxic reaction of keratino- Olomouc, Czech Republic cytes to the presence of cytotoxic agent and its severity can lead even to the interruption of an effective therapy. Rocuronium is a neuromuscular blocking agent acting Experimental practice in some clinics showed that as a competitive antagonist of acetylcholine on nicotinic topical application of 10% uridine ointment is suitable receptors of neuromuscular plate. It is used during bal- for curing HF syndrome. This study is to find out if there anced anesthesia to induce and maintain the neuromus- is evidence for this protective activity of uridine in human cular blockade. Its advantage is a relatively fast onset (1 keratinocytes cultured in vitro. We also tested cytidine, min) and an intermediate duration of its action (30 to deoxycytidine, thymidine and their combinations for the 50 min). It is an aminosteroid derivative with quaternary ability to protect the cells against 5-FU effect. nitrogen atom, with small amount (several per cent of the We measured cellular viability time progression by dose) metabolized to an N-desallyl and 17-desacetyl de- recording cellular adherence with RTCA (real-time cell rivatives. There are indications that this drug may interact analyzer). We also measured metabolic activity of cells with liver microsomal system of cytochromes P450 (CYP) by MTT test and we recorded the changes in cellular mor- as e.g. an increase of rocuronium effect by macrolide anti- phology by NTCA test (non-destructive test of cellular ac- biotics; however, no systematic study on this subject has tivity). The tests were performed on human keratinocyte been published in the literature. In this report, results of cell line HaCaT. an in-vitro inhibition of enzyme activities specific for eight We proved the ability of uridine and cytidine to pro- individual liver microsomal CYP forms are presented. As tect keratinocytes against 5-FU damage in vitro. The level the patients are often premedicated orally with diazepam, of protection was similar for both of these nucleosides. possible interaction of diazepam with rocuronium has While thymidine alone showed only a little protective ef- been also studied, namely, the formation of two main di- fect, the thymidine-uridine and thymidine-cytidine com- azepam metabolites, has been followed in the absence as binations showed better protective activity than uridine well as in the presence of rocuronium. or cytidine only. Deoxycytidine showed no remarkable

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Results have indicated that rocuronium is able to in- rats by invasive method. For ultrashort-acting effect is teract with human liver microsomal cytochromes P450 responsible ester bond in the lateral chain of tested com- by binding to the substrate site hence increasing the high pounds. This group is rapidly hydrolyzed by esterases in spin form of this enzyme. Further studies have shown red blood cells or by free carboxylesterases in plasma a concentration dependent inhibition of human liver mi- (Mokrý P. et al., 38. konf. Syntéza a analýza léčiv, sborník crosomal CYP3A4 form down to 42% with the highest HK,2009:153). rocuronium concentration, 189 microM. This effect has The experiment was performed in vivo with 21 male been confirmed with two substrates of this enzyme, name- Wistar laboratory rats and tested group was divided into ly, with testosterone (formation of 6beta-hydroxytestoster- 3 subgroups: All subgroups (n=3) were administered the one) as well as with diazepam (temazepam formation). dose of 6.0 mg.kg-1 of 2FC2b, 4FC2b or placebo. The Other microsomal CYP forms (CYP1A2, 2A6, 2B6, 2D6, tested agents were administered into vena jugularis and 2E1) have not been inhibited by this drug; however, the the values of systolic blood pressure and heart rate were CYP2C9 and CYP2C19 activities were inhibited down to monitored for 15 min following the administration. For 75-80% of their original activity by the highest concentra- systolic blood pressure invasive monitoring, the „HSE tion of rocuronium. UNIPER UP-100“ by Hugo Sachs Electronic company, To prove the possibility of rocuronium interaction and for the heart rate the „EKG for veterinary purposes“ with other drugs, namely, with diazepam, the effect of were used. Tested agents were compared to placebo. rocuronium on formation of two main diazepam metab- 2FC2b caused statistically significant decrease olites, temazepam (by CYP3A4) and nordiazepam (by (P<0.01) in systolic blood pressure from 1st up to 2.5th action of CYP2C19) in primary culture of human liver minute and from 3rd to 3.5th minute (P<0.05). Compound hepatocytes has been examined. Rocuronium has caused 4FC2b only in 0.5th minute (P<0.01). Effect on the heart an inhibition of both these reactions by 15 and 12%, re- rate of the substance 2FC2b was without significant respectively. sponse but heart rate was significantly decreased from In conclusion, the results open a possibility that in- 0.5th to 3rd minute (P<0.05) by compound 4FC2b. teractions of rocuronium with drugs metabolized by CYP3A4 (and possibly also CYP2C19) may be observed. On the other hand, the effects described here indicate ACKNOWLEDGMENT that the clinical importance is probably limited and that a modulation rather inhibition of the metabolism of these This study was funded by IGA 104/2013/FAF. drugs is observed. P-3 ACKNOWLEDGMENT Drug related problems identified in a community

The authors wish to thank the Internal Grant Agency pharmacy during point-of-care testing of the Czech Ministry of Health projects NT13591 and Martina Balazova, Magdalena Kuzelova NT 13906-4/2012 for financial support. Department of Pharmacology and Toxicology, Faculty of Pharmacy, Comenius University in Bratislava, Odbojarov 10, P-2 832 32 Bratislava, Slovak Republic Antihypertensive and negatively chronotropic Point-of-care testing (PoCT) is a part of professional effect of new -ethyl substitueted practice in community pharmacies in Slovakia. The aim arylcarbonyloxyaminopropanole derivates was to identify drug related problems (DRPs) associated

a b a with lipid-lowering treatment in patients who had their Ondrej Bado , Jan Tengler , Marek Frydrych biochemical parameters tested in a community pharmacy aDepartment of Human Pharmacology and Toxicology, Faculty of of the Faculty of Pharmacy in Bratislava, and to classify Pharmacy, University of Veterinary and Pharmaceutical Sciences, these DRPs according to the PCNE Classification for Palackeho 1-3, 612 42 Brno, Czech Republic DRPs V6.2. bDepartment of Chemical Drugs, Faculty of Pharmacy, University PoCT had been provided to 88 patients (mean age of Veterinary and Pharmaceutical Sciences, Palackeho 1-3, 612 42 64.6 years) in period 11/2011 to 11/2012 Most (88.6%) Brno, Czech Republic of these patients had been tested repeatedly, which resulted in 169 visits in total. There were 40 DRPs associ- Department of Chemical Drugs of the Faculty of ated with lipid-lowering therapy identified, 24 (60.0%) Pharmacy of Veterinary and Pharmaceutical Sciences during the first visits and 16 (40.0%) during the check-up in Brno developed new potential ultrashort-acting beta visits. The most common DRP was a suboptimal effect blockers. These compounds 2FC2a, 2FC2b were used in of therapy (code P1.2 according to PCNE V6.2) in 15 this experiment to pharmacologically evaluate their effect (37.5%) cases. In 10 (25.0%) cases there was a potential on systolic blood pressure and heart rate in normotensive problem of untreated indication (P1.4). In 9 (22.5%)

S17 Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2013 Sep; 157 (Supplement 1):S1–S88. cases the therapy was uneffective (P1.1), mostly because concentrations or high concentrations of the competitors of not taking prescribed lipid-lowering drug. Nonallergic or was not in one case. adverse drug event (P2.1) of statins such as muscle pain On the basis of obtained results, the novel automatic was detected in 5 (12.5%) cases and allergic adverse drug technique has proved its capability to be skillful method event (P2.2) in one (2.5%) case. Six (15.0%) DRPs did for the labeled ligand versus unlabeled ligand competition not require an intervention (I0.0). In other cases of DRPs analysis. Moreover, this technique exceeds well-introduced the interventions were either patient consultation (I2.1), methods for ligand binding characterization thanks to its referral to physician (I2.3) or both. Three (7.5%) DRPs measurement simplicity and low cost runs. were solved completely (O1.0) and 4 (10.0%) partially (O2.0). In 5 (12.5%) cases the DRP was not solved due to lack of cooperation of patient (O3.1). The outcomes ACKNOWLEDGMENT of the rest cases are not known yet (O0.0) as the project still continues. The study was co-financed by the European Social Fund and the state budget of the Czech Republic, project No. CZ.1.07/2.3.00/30.0061. ACKNOWLEDGMENT

This work was supported by Grant FaF UK/6/2013. P-5 The biological effects of flavonoids pomiferin P-4 and osajin in experiment The real-time radioimmunoassay can enhance the Lenka Bartosikova

better detection of ligand competitive reactions Department of Physiology, Faculty of Medicine and Dentistry, in vitro Palacky University Olomouc, Hnevotinska 3, 775 15 Olomouc, Czech Republic Pavel Bartaa, Frantisek Trejtnara, Karl Anderssonb,c aDepartment of Pharmacology and Toxicology, Faculty of The flavonoids pomiferin and osajin were extracted Pharmacy in Hradec Kralove, Charles University in Prague, from infructences of Maclura pomifera, Moraceae. Heyrovskeho 1203, 50005 Hradec Kralove, Czech Republic The aim of the studies was to analyze effects of both bDepartment of Radiology, Oncology and Radiation Sciences, substances under the conditions of preclinical experiment. Uppsala University, SE-751 85 Uppsala, Sweden The cardioprotective effect was performed on isolated, cRidgeview Instruments AB, Uppsala Science Park, Uppsala, modifies Langendorff-perfused rat hearts. The ischemia Sweden was induced by stopping of coronary flow followed by reperfusion. During a prophylactic administration fla- The competitive assays enable to determine the bind- vonoids were applied orally. Biochemical indicators of ing characteristic of new prepared ligands such as radio- oxidative damage in serum and myocardial tissue have labeled monoclonal antibodies as the first step in their been evaluated. The effects of both flavonoids on cardiac pharmacology testing. The better approach lies in in vitro functions also were examined. testing, which serves as the better testing system closely The results demonstrate that prophylactic adminis- modeling the living organism. However, the traditional tration of osajin and pomiferin attenuate the myocardial methods for ligand binding characterization are time dysfunction provoked by ischemia-reperfusion. and material demanding. Disadvantages coming from The renoprotective effect was tested on model of the well-established methods can be circumvented with unilateral ischemia-reperfusion of kidney tissue. During the employment of the real time radioimmunoassay on a prophylactic or therapeutic administration flavonoids previously introduced machine LigandTracer. were applied orally. Selected biochemical markers were The real time detection competitive study ran in the assessed in blood. Renal functions were assessed too. The arrangement of the pre-incubation of cells with radioiodin- kidney tissue samples were used for histopathological ex- ated natural ligand for some time and then followed the amination. addition of unlabeled non-natural ligand (either mono- The results obtained during prophylactic and thera- clonal antibodies or another natural ligand). The binding peutic administration confirmed supposed effects of both domain of epidermal growth factor receptor (EGFR) was substances. used as targeted protein for ligands. The study was per- The antidiabetic and antioxidative effect was moni- formed in vitro with the employment of cancer cell lines tored under the experimental conditions of allox- expressing EGFR in high density. an-induced diabetes mellitus. During a therapeutic The evaluation of measured results revealed the administration flavonoids were applied orally. Selected binding ability of employed unlabeled ligands in the laboratory parameters in serum and in urine were deter- competition with the radioiodinated natural ligand. The mined. Kidney tissue and pancreas samples were taken pre-bound natural ligand was displaced with either low for histopathological analysis.

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The results of biochemical examination show a pro- last TAA administration) and catalase (in the 4th and the tective antioxidative and antidiabetic effect of both fla- 12th week). vonoids. The results of histopathological examination correlate only partially with them. ACKNOWLEDGMENT

P-6 Supported by the project CZ.1.05/2.1.00/03.0076 The influence of chronic administration of from European Regional Development Fund and by the thioacetamide on plasma levels of ALT, AST, Charles University Research Fund (project number P36). GLDH and markers of oxidative stress in liver and P-7 kidneys of female wistar rats Effect of developmental administration a b a Monika Bludovska , Eliska Mistrova , Dana Kotyzova , of antidepressant venlafaxine on selected Magdalena Chottova-Dvorakovab behavioral variables in rat offspring aDepartment of Pharmacology and Toxicology, Faculty of Medicine in Pilsen, Charles University in Prague, Czech Republic Estera Csaszarova, Mojmir Mach, Eduard Ujhazy, bDepartment of Physiology, Faculty of Medicine in Pilsen, Charles Michal Dubovicky University in Prague, Czech Republic Institute of Experimental Pharmacology and Toxicology, Slovak Academy of Science, Dubravska cesta 9, 841 04, Bratislava, Slovak Thioacetamide (TAA) is a well known inducer of he- Republic patic fibrosis and has been often used in experimental chronic hepatotoxicity models due to its ability to produce liver damage similar to human hepatic fibrosis. The highly The estimated prevalence of depression in pregnancy specific hepatotoxic effect is mediated by thioacetamide- ranges from 9 to 16%. Treatment of depression in this S,S-dioxide, the toxic product of TAA oxidation by hepatic critical period raises the question if to treat or not to microsomal enzyme CYP2E1. It is supposed that the gen- treat depression during pregnancy. Consequences of both, eration of reactive oxygen species plays an important role the untreated depression or the antidepressant therapy in TAA induced liver damage and fibrosis. The aim of represent a possible risk for injury of fetal and/or neona- this preliminary experiment was to verify the time course tal development. Selective serotonin re-uptake inhibitor of levels of ALT, AST and GLDH activities in plasma (SSRI) and serotonin and noradrenaline re-uptake inhibi- and markers of oxidative stress in liver and kidneys after tor (SNRI) drugs are commonly used for treatment of chronic TAA administration. maternal depression. Venlafaxine belongs to the SNRI Wistar albino female rats were divided into two antidepressant drugs. The FDA has classified VENF re- groups: control and TAA-treated. TAA was applied in- garding to pregnancy risk as C category of drug, which traperitoneally (200 mg/kg body weight) three times per means that there are no well-controlled studies examining week for a 12-week period. Animals in the control group safety to the developing child. The aim of this study was to received saline. One third of the animals of each group determine the effect of venlafaxine administration during was sacrificed 4 weeks, one third 12 weeks and the re- sensitive functional brain development on neurobehav- maining third 16 weeks after the end of the treatment ioral development of rat offspring. Pregnant Wistar rats period. Blood and tissue samples were collected. Lipid were treated with venlafaxine (oral administration) from peroxidation (LP), reduced glutathione (GSH) and activi- gestation day 15 to day 20 post partum (PP) at the doses ties of glutathione peroxidase (GPx), glutathione reduc- of 7.5, 37.5 and 75 mg/kg. On day 4 PP all litters were tase (GR) and catalase (CAT) were estimated in liver and subjected to „culling“ procedure (number of pups was kidney homogenates. ALT, AST and GLDH levels were reduced to 4 males and 4 females) and on day 21 PP they determined in serum. TAA administration significantly were weaned from their mothers. Animals were tested increased ALT, AST and GLDH activities. The increase in an open field daily in 5 min sessions 5 consecutive in activities of ALT and GLDH persisted 16 weeks, AST days (22-26 PP), in an elevated plus maze (60 PP), in activity was increased only in the 4th week after the treat- a light-dark box (80 PP) and in a forced swimm test (85 ment period. Contrary to the assumption, no increase PP) in individual 5 min sessions. Maternal VENF treat- in LP was observed in liver (a decrease of LP and an ment resulted in a decreased intensity of locomotion in increase in GR activity were even seen in the 16th week). the offspring in the open field test and led to an increased Significant increase in LP was found in kidneys (in the 4th activity in the forced swimm test. In addition, we found re- and the 12th week). This is consistent with the conjecture duced anxiety-like behavior in females compared to males that the renal damage during the development of cirrhosis exposed to VENF. The results suggest that VENF may is possibly mediated by oxidative stress. The only markers gender-dependently interfere with functional development of oxidative damage in liver were significant decreases in of the brain resulting in altered neurobehavioral regula- the activities of GPx (that persisted 16 weeks after the tions and adaptations in a new environment.

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ACKNOWLEDGEMENT ACKNOWLEDGEMENT

The study was supported by the VEGA grants This work was supported by the Grant Agency of the 2/0081/11, 2/0084/11 and 2/0107/12. Charles University in Prague (GAUK 700912/C/2012 and SVV/2013/267-003). P-8 P-9 Cyclin-dependent kinase inhibitors, AT-7519, DINACICLIB, PD 0332991, flavopiridol, and SNS- Incidence of thrombotic complications in patients 032, inhibit ABCB1 and ABCG2 transporters with atrial fibrilation in anamnesis depending on the anticoagulant/antiaggregant therapy Daniela Cihalova, Martina Ceckova, Frantisek Staud Veronika Muller Zavalovaa,b, Vaclav Zizlavskya, Department of Pharmacology and Toxicology, Faculty of b Pharmacy in Hradec Kralove, Charles University in Prague, Marketa Dlouha Heyrovskeho 1203, 500 05 Hradec Kralove, Czech Republic a 2nd Department of Surgery, St. Anne’s University Hospital in Brno and Faculty of Medicine, Masaryk Universiy, Brno, Czech Republic ABCB1 (P-glycoprotein) and ABCG2 (breast cancer b Department of Human Pharmacoloy and Toxicology, Faculty of resistance protein) are the most extensively studied drug Pharmacy, University of Veterinary and Pharmaceutical Sciences, efflux transporters. They contribute to multiple drug re- Brno, Czech Republic sistance in cancer and may be involved in the decreased anticancer efficiency and modified pharmacokinetic/ The atrial fibrilation represents one of the most com- pharmacodynamic properties of therapeutic entities. mon clinically indicated disorders of the cardiac dysrhyth- The cyclin dependent kinase inhibitors (CDKi), AT-7519, mia. Its increasing incidents correlates with increasing dinaciclib, PD 0332991, flavopiridol and SNS-032 are polymorbidity, age and sex of the patients. Nowadays, the novel anticancer agents in phase I/II clinical trials for the number of patients in Czech Republic counts around 1 % treatment of various cancers and their influence on the of the total Czech Republic population. A prevention of function of membrane efflux transporters has not been arterial complications represents an important aspect in described yet. The aim of the present study was to char- treating the possible future worsening of a patient health acterize the inhibitory effect of the selected CDKi, AT- state. The treatment is normally based on the anticoagu- 7519, dinaciclib, PD 0332991, flavopiridol, and SNS-032, lant or antiaggregant therapy. on ABCB1 and ABCG2 efflux activity. The intracellular The sample size involved 66 patients cured for six daunorubicin accumulation in MDCKII-ABCB1 and mi- months in the Second Department of Surgery, St. Anne’s toxantrone accumulation in MDCKII-ABCG2 cells was University Hospital, Brno. Patients indicated with the in- examined by flow cytometry. Parental MDCKII cells were creased risk of the arterial fibrillation were distributed analyzed as a control using both substrates separately. into five groups (four groups according to the farmaco- LY335979 or Ko143 were used as positive control for therapy of the blood coagulation and one more group ABCB1 or ABCG2 inhibition, respectively. The increase of patients for whom the anticoagulant/antiaggregant in the intracellular accumulation of either daunorubicin therapy has not been indicated). A correlation between or mitoxantrone reflects an inhibitory effect. all the five groups has been investigated with respect to The intracellular accumulation of daunorubicin was the applied therapy and the incidence of the thromboem- increased 1.14, 1.49, 2.07, 3.73 and 5.04-fold in the pres- bolic events. ence of AT-7519, dinaciclib, SNS-032, flavopiridol and In the full investigated ensemble of patients, the PD 0332991 at 50 μM concentration, respectively, in thromboembolic complications were indicated for 23 per- MDCKII-ABCB1 cells. The intracellular accumulation sons. In the group treated with warfarin, which in total of mitoxantrone was increased 1.33, 1.91, 2.51, 4.23 and involved 23 patients, seven persons were diagnosed with 4.79-fold in the presence of AT-7519, SNS-032, dinaciclib, the arterial complications what represents 30.4%. In the flavopiridol and PD 0332991 at 50 μM concentration, second group, which in total included 29 persons treated respectively, in the MDCKII-ABCG2 cells. We observed with antiaggregation therapy by means of acetylsalicylic that all CDKi inhibit ABCB1- or ABCG2-mediated ef- acid were 9 patients diagnosed with the thromboembolic flux in a concentration dependent manner, but show no complication. In this group, the relative number of im- significant effects on the parental MDCKII cell line. We pacts represents 31%. Finally, it has been found that in conclude that the inhibition of ABCB1 and/or ABCG2 the group with no anti-coagulant therapy the risk of the transporters by the CDKi could affect the pharmacoki- thromboembolic events increased up to 70%. netic behavior of ABCB1/ABCG2 substrates if adminis- The obtained results suggest that the risk of the com- tered simultaneously. plications is similar in both groups treated by either warfarin or acetylsalicylic acid, which are both common drugs applied in the situations where blood coagulation is an issue. While in the most cases that, in spite of the warfarin

S20 Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2013 Sep; 157 (Supplement 1):S1–S88. treatment, lead to the complications, the reason of the ACKNOWLEDGEMENT unsuccessful therapy could be associated with the non- optimal level of INR. Moreover, we cannot also exclude This work was supported by grants FaF UK/59/2013, a high rate of the drug therapy interactions as a reason of ITMS 2624022071, VEGA 1/0786/11. the anticoagulant or antiaggregant failure. P-11 P-10 Alkaloid boldine is a ligand Impact of diabetic state in patients with end- of farnesoid x receptor stage heart failure and its association with cardiac Michaela Dubeckaa, Marie Zagorovab, Jolana Cermanovab, expression of micrornas Tomas Lahob, Stanislav Micudab, Petr Paveka a a a Gabriel Doka , Peter Krenek , Jan Klimas , aDepartment of Pharmacology and Toxicology, Faculty of b a Eva Gonsalvesova , Jan Kyselovic Pharmacy in Hradec Kralove, Charles University in Prague, Heyrovskeho 1203, Hradec Kralove 500 05, Czech Republic a Department of Pharmacology and Toxicology, Faculty of bDepartment of Pharmacology, Faculty of Medicine in Hradec Pharmacy, Comenius University in Bratislava, Odbojarov 10, Kralove, Charles University in Prague, Simkova 870, Hradec 832 32 Bratislava, Slovak Republic Kralove 500 38, Czech Republic bThe National Institute of Cardiovascular Diseases, Pod Krasnou horkou 1, 833 48 Bratislava, Slovak Republic Boldine is the major alkaloid from Chilean boldo Introduction. Diabetes mellitus is known cardiovascu- tree, and is used in traditional medicine to support bile lar disease risk factor and co-morbidity in heart failure, production, but the scientific data for this effect are lack- linked with progressive end-organ damage. High or un- ing. Recently we found immediate choleretic activity of controlled blood sugar may affect the expression of small boldine in rats, which depended on actual concentration regulatory RNAs - microRNAs, which are fine tuners of of the agent in the bile. multiple aspects of structure and function of the heart In the current study, we analyzed whether boldine ac- as revealed by bioinformatic predictions. The aim of this tivates Farnesoid X receptor (FXR), which controls key study is to determine possible associations or differences genes involved in bile acids synthesis, transport and their between diabetic and nondiabetic patients with end-stage homeostasis in the body. We used gene reporter assays heart failure on the level of various clinical parameters with pFXRE-luc2P reporter construct and the fusion ex- and myocardial microRNAs. pression construct containing the ligand binding domains Methods. Cohort consists of 41 patients with end-stage of human FXR receptor fused to GAL4 to analyze interac- heart failure (14 diabetic, 27 non-diabetic), who under- tion of boldine with human FXR. Experiments have been went heart transplantation. From every patient, we col- performed in HepG2 cells with the constructs treated for lected a set of clinical parameters: age, sex, underlying 24 h with increasing concentrations of boldine alone or cause of heart failure, BMI, blood pressure, analyzed are in combination with chenodeoxycholic acid (CDCA) as also values of ECG, ECHO, cardiac catheterization and a FXR ligand. We found interaction of boldine with FXR serum biochemistry. We assessed the expression of mi- in both experimental systems. We can therefore conclude croRNAs relevant for heart (miR-1, miR-133a, miR-208a, that boldine may be a weak ligand of FXR receptor, which miR-208b, miR-499, miR-29b) by modified real-time PCR correlates with its choleretic effect in rats. in explanted left ventricular samples. Results. Diabetic patients had clearly higher blood sugar (+36%), but also serum levels of urea (+48%) and uric ACKNOWLEDGMENT acid (+36%), as well as body mass index (BMI; +13%). No other of 26 remaining various parameters significantly We acknowledge grant support from Czech Scientific differed between diabetic and non-diabetic population Agency (Centre Excellence project P303-12-G163). of patients with failing hearts. The same is true also for analyzed cardiac microRNAs (miR-1, miR-1, miR-133a, miR-208a, miR-208b, miR-499, miR-29b). Conclusion. This study was based on hypothesis, that higher or uncontrolled glycemia in patients with failing hearts may result in changed molecular frame of the myocardium and thus in worsened prognosis. Contrary to this hypothesis, diabetic patients didn't exhibit differences in expression of analyzed cardiac microRNAs from non-diabetic patients. Conclusively, cause of worsened prognosis for diabetic patients may be more likely the result of damage to other organ than the heart itself.

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P-12 P-13 Participation of opioid receptors in the antitussive Three years of experience of the attention acitivity of Withania somnifera hyperactivity disorder (ADHD) treatment Dana Fleskovaa, Gabriela Nosalovaa, Bimalendu Rayb, with atomoxetine and methylphenidate Ludovit Jureceka, Igor Ondrejkac, Sona Franovaa Zuzana Elgyuttovaa, Alena Vavrovab, Andrea Majkovac, a a aDepartment of Pharmacology, Jessenius Faculty of Medicine Stanislava Jankyova , Tatiana Foltanova in Martin, Comenius University in Bratislava, Martin, Slovak aDepartment of Pharmacology and Toxicology, Faculty of Republic Pharmacy, Comenius University in Bratislava, Odbojarov 10, bNatural Products Laboratory, Department of Chemistry, The 832 32 Bratislava, Slovak Republic University of Burdwan, India bPsychiatric ambulance, Faculty Hospital Nitra, Spitalska 6, cDepartment of Psychiatry, Jessenius Faculty of Medicine in 94901, Nitra, Slovak Republic Martin, Comenius University in Bratislava, Martin, Slovak cDIALOG, spol. s r. o. Tehelna 26, 831 03, Bratislava, Slovak Republic Republic In last decades treatment by plant substances has become one of the leading forms of treatment many diseases Attention hyperactivity disorder (ADHD) is the most of the respiratory system, including cough. Therefore, the frequent psychiatric behavioural disorder in school chil- subject of our interest became plant polysaccharide iso- dren (8-10%). Retrospectively (2010-2013), we analysed lated from Withania somnifera and its antussive activity. 50 patients (82/18 %, M/F), with ADHD. Average age Except of that, we tried to find out what role opioid recep- was 11.9 ±0.4, (5-18 years). Together 48 % of the children tors play in that activity. were from foster homes. The most frequent comorbidi- The experiments were carried out on the concious ties were: F.90 - disturbance of activity and attention male guinea pigs (Trik). The plant substance (Withania (84%), F.80 - specific developmental disorders of speech somnifera) was apllied to the first group of guinea pigs and language (36%) and F.92 - mixed disorders of con- perorally. The cough reflex was evoked by citric acid in duct and emotions (36 %). For the classification of the concentration 0.3 M. To second group of guinea pigs, we comorbidities we used ICD 10. In the pharmacothera- applied 15 min before own application of arabinogalactan py we were analysing ATC class, indication, dosing in (WS), Naloxon hydrochlorid (i.p.). Nonselecitve antago- paediatric indication). As information source about the nist Naloxon methiodid was apllied (15 min before WS) drugs we used the official web side of State Institute for to the third group of animals. Drug Control. All children were treated with sympatho- Polysaccharide isolated from WS showed the ability mimetics atomoxetine (80%) or methylfenidate (52%). after oral administration reduced the parameters of citric The second most present group were antipsychotics (90%, acid induced cough in awaken guinea pigs healthy in vivo risperidone:thiapridal 3:2), and third one antiasthmat- experimental conditions. We showed that on reduction ics (66%, promethazine:cyproheptadine 2:1). Out of the of cough reflex induced by administration of arabino- most frequently used drugs thiapridal was used off la- galactan from Withania somnifera are participated both bel. Because of high prevalence of several comorbidities, centrally and peripherally acting opioid receptors. combined therapy prevailed; most patients (38%) took Our results supported our previous findings that natu- 4 drugs. Potential adverse event was defined as coinci- rally occurring polysaccharides possess antitussive activi- dence of following factors: presence of subjective dis- ty. This study also represents the participation of centrally comfort, presence of this discomfort in the Summary of and peripherally acting opioid receptors on reduction of product characteristics (SmPC) and intervention in the cough reflex induced by Withania somnifera. therapy. Potential adverse events were present in 48% of the patients. Together 50% of the patients who were treated with atomoxetine had potential adverse avents, in meth- ACKNOWLEDGMENT ylphanidate treated patients it was 15.4% of the patients. Most frequent adverse event was anorexia (27%//11.5% This work was supported by Grant LPP-0317-09 and of the atomoxetine/ methylphenidate patients). This the Center of experimental and clinical respirology. physician solved by adding cyproheptadine into the treatment. Hyperkinetic disorder is complex, multifactorial disease. Although the pharmacological possibilities, the success of the treatment depends on the cooperation of multidisciplinary team of professionals and parents.

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P-14 P-15 Long-term consumption of Coca-Cola results The role of ghrelin and orexin systems in development of manifestations of metabolic participation in fear and anxiety behavior testing, syndrome in wistar rats using aversive ultrasonic vocalization in rats Kristina Galkova, Eva Malikova, Eva Kralova, Tereza Havlickova, Pavel Jerabek, Miloslav Krsiak, Jasna Srankova, Peter Krenek, Jan Klimas Magdalena Sustkova-Fiserova

Department of Pharmacology and Toxicology, Faculty of Department of Pharmacology, 3rd Faculty of Medicine, Charles Pharmacy, Comenius University in Bratislava, Odbojarov 10, University in Prague, Prague, Czech Republic 832 32 Bratislava, Slovak Republic Aims. Orexigenic peptides ghrelin and orexin exhibit Background. In rat, the use of Coca cola to induce an central neurobiological effects which are involved in food experimental metabolic syndrome has not been clarified intake, reward, learning, memory, cognition and mood. yet. We aimed to describe the effect of long-term (three Both peptides elicit mostly pro-anxiety effects. Thus we months) consumption of Coca cola on development of wanted to test the potential anxiolytic effects of the ghre- metabolic syndrome in rat. lin receptor antagonist (JMW1959) and orexin receptor Methods. We used 10 weeks old male Wistar rats antagonist (SB334867) which has yet to be done and for receiving a standard diet. Additionally, a group of rats the first time use the aversive ultrasonic vocalization in received a commercially available Coca-Cola beverage rats for testing these peptides mechanisms participation. (CC, n=13), controls (CON, n=7) drank common drink- Methods. One day before the experiment the aversive ing water. We measured weight gain, and plasma glucose behavior was established (i.e. rats were individually ex- (Glc) and cholesterol (Chol) levels by using standard glu- posed in the automatic chamber for 7 min to 6 electro- cometer in capillary blood drops. In addition, systolic and shocks). The aversion was fixed on the next day (1 shock diastolic blood pressures (sBP and dBP) and heart rate during 1 min exposure). After which ghrelin (30; 300 μg/ (HR) were measured by using tail-cuff method. kg) or antagonist (JMW2959 - 0.6, 6; 12 mg/kg, SB334867 Results. We observed a significantly increased body – 1; 10; 20 mg/kg) or saline was i.p. applied and 30 min weight in CC rats (391±12 g; P<0.01) when compared to later the rat was placed into the chamber again for 10 min controls (342±19 g). This was in accordance with sig- (no electro-shocks) and its ultrasonic aversive vocaliza- nificantly increased sBP (CC: 141±4 mmHg vs. CON: tion was automatically measured. The anxiolytic effect 119±7 mmHg; P<0.01) and a tendency of increased dBP was considered compared to the control/saline rats. The (CC: 98±2 mmHg vs. CON: 87±6 mmHg; NS) and signifi- ghrelin antagonist significantly decreased aversive ultra- cantly increased HR (CC: 361±8 mmHg vs. CON: 310±15 sonic vocalization in doses 6 mg/kg (decrease of 74.2%) mmHg; P<0.01). Additionally, we measured a tendency and 12 mg/kg (decrease of 62.8%) versus control. The of increased Glc (CC: 5.65±0.39 mmol/L vs. 4.77±0.51 observed SB33487-induced anxiolytic-like and ghrelin- mmol/L; NS) but stable cholesterol Chol (CC: 4.10±0.06 induced anxiety-like effects were not significant. mmol/L vs. CON: 4.21±0.07 mmol/L; NS). Results. We have found that ghrelin antagonist signifi- Conclusion. Long-term administration of Coca-Cola cantly decreased the rats aversive ultrasonic vocalization, can lead to development of cardiovascular (increased BP which supports the view, that ghrelin antagonism could and HR) as well as metabolic manifestations (impaired be used for the prevention of distress and anxiety-induced glucose homeostasis) of metabolic syndrome in Wistar overeating and drug consumption. rat. Consequently, we propose this model as a reliable experimental model of rat metabolic syndrome. ACKNOWLEDGEMENT

ACKNOWLEDGMENT This study was supported by IGA NT/13687-3/2012, 266705/SVV/2013 and PRVOUK P34. This work was supported by grants APVV- 0887-11, VEGA 1/0981/12, VEGA 1/0564/13 and UKFAF/45/2013.

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P-16 P-17 Resistance of cancer cells to anthracyclines Comparison of methylphenidate use in patients mediated by reductive metabolism: the role of with ADHD hospitalized in clinic of psychiatry, aldo-keto reductase 1C3 Jessenius Medical Faculty, Comenius University Jakub Hofman, Beata Malcekova, Adam Skarka, Eva between years 2009-2012 Novotna, Vladimir Wsol Igor Hrtaneka,b, Igor Ondrejkab , Gabriela Nosalovaa

Department of Biochemical Sciences, Faculty of Pharmacy in aDepartment of Pharmacology, Jessenius Faculty of Medicine in Hradec Kralove, Charles University in Prague, Heyrovskeho 1203, Martin, Comenius University in Bratislava, Martin, Sklabinska 26, 500 05 Hradec Kralove, Czech Republic 036 01, Slovak Republic bClinic of Psychiatry, Jessenius Faculty of Medicine in Martin, Comenius University in Bratislava and Drug resistance belongs to the main obstacles emerg- University Hospital Martin, Kollarova 2, 036 59 Martin, Slovak ing during the cancer chemotherapy. By the conversion Republic of drugs into corresponding less active metabolites, some biotransformation enzymes significantly contribute to this The current study investigated methylphenidate phenomenon. In this study, we investigate possible role (MPH) use and its efficacy in treating ADHD in chil- of aldo-keto reductase 1C3 (AKR1C3) in the resistance dren and adolescent patients hospitalized in Clinic of of cancer cells to anthracyclines. Reducing activity of Psychiatry JFMED CU between the years 2009-2012. AKR1C3 toward anthracyclines was first tested using in- We aimed in use of other most prevalent therapeutic cubations with purified recombinant enzyme prepared in strategies and most prevalent comorbid disorders ac- E. Coli. AKR1C3 was shown to most efficiently catalyse companying ADHD. Efficiency of therapy was evaluated the formation of daunorubicinol, followed by idarubicinol by Clinical Global Impression Scale (CGI), Severity (S) and doxorubicinol. We further examined the reduction and Improvement (I) subscale. The study aimed also in of daunorubicin and idarubicin employing transfection gender and age differences in groups of patients. This of A549, HeLa, MCF7 and HCT 116 cancer cells with retrospective, observational and comparative study evalu- AKR1C3 encoding vector. Production of daunorubi- ate use of MPH in hospitalized patients during 4 years cinol and idarubicinol was greatly accelerated in trans- (2009-2012) from the time when the stimulants were fected cells; this acceleration was significantly blocked registered in SR. Data were obtained from medical re- by 2‘-hydroxyflavanone, a recognised AKR1C3 inhibitor. cords of hospitalized patients, aged 6-17 years. The study To demonstrate the participation of AKR1C3 on anthra- included 104 hospitalizations, 77 patients treated with cycline resistance, we performed MTT proliferation as- MPH. Statistical methods used in this study were: statisti- says employing transfected HeLa, MCF7 and HCT 116 cal analyses, comparative and correlation methods. There cells. Introduction of AKR1C3 into cells significantly is no significant difference in gender and selected period reduced the antiproliferative effect of both daunorubicin in ADHD patients treated by MPH - the proportion of and idarubicin in all three tested cell lines. In conclusion, girls and boys is stable over time. The most frequent dis- our data suggest substantial impact of AKR1C3 on the orders accompanying ADHD were Conduct disorders pharmacokinetic behaviour of daunorubicin and idaru- (51%), Emotional disorders (16.6%) and other specified bicin. In addition, we demonstrate that the reduction of behavioural and emotional disorders (12.6%). There is daunorubicin and idarubicin catalysed by AKR1C3 con- no difference in severity of ADHD of incoming patients tributes to the resistance of cancer cells to anthracycline the most common CGI severity grade is 5 (markedly ill). chemotherapy. Our results should be considered in the There is no significant correlation between ADHD CGI-S clinical use of these anthracyclines. and CGI-I. The most common CGI-I grade was 2 (much improved), scored in 44.6 % of all inpatients. In the period 2009-2010 mono-therapy by MPH prevailed, in the ACKNOWLEDGEMENT period 2011-2012 we recorded the trend of a combination of MPH with another psycho-pharmacotherapy. We found This work is co-financed by the European Social significant difference in use of combination of MPH with Fund and the state budget of the Czech Republic. Project another drug therapy between years 2009-2012 (P=0.020). No. CZ.1.07/2.3.00/30.0061, CZ.1.07/2.3.00/30.0022 and The most often used psychopharmacology were antipsy- CZ.1.07/2.3.00/20.0235. chotics levopromazine (21.2%), chlorprothixene (12.12%) and antihistamine promethazine (12.12%). We did not find a significant relationship between the CGI-I grade of behavioural disturbances and the use of mono-therapy or combination therapy in patients with ADHD and conduct disorder.

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ACKNOWLEDGMENT by dexamethasone in primary human hepatocytes, but not in hepatocyte-derived cell lines. However, neither C/EBPβ This work was supported by the UK Grant nor PGC1α were up-regulated in human hepatocytes by UK/19/2013. dexamethasone. We can conclude that OCT1 is induced only through GR nuclear receptor via an up-regulation of HNF4α in P-18 primary human hepatocytes. Regulation of organic cation transporter 1 (OCT1,

SLC22A1) expression via major nuclear receptors ACKNOWLEDGMENT in primary human hepatocytes

a a a We acknowledge grant support from the Czech Lucie Hyrsova , Alice Rulcova , Lucie Krausova , Scientific Agency (Centre Excellence project P303- Tomas Smutnya, Radim Vrzalb, Zdenek Dvorakb, 12-G163). Ramiro Joverc, Frantisek Trejtnara, Petr Paveka aDepartment of Pharmacology and Toxicology, Faculty of P-19 Pharmacy in Hradec Kralove, Charles University in Prague, Heyrovskeho 1203, 500 05 Hradec Kralove, Czech Republic Hormone replacement therapy after hysterectomy b Department of Cell Biology and Genetics, Faculty of Science, a a b Palacky University Olomouc, Slechtitelu 11, 783 71 Olomouc, Marta Chalupova , Michaela Urbaskova , Petr Fiala , a a Czech Republic Tomas Parak , Pavel Suchy cUnidad Mixta Hepatologia Experimental, Hospital La Fe & a University of Valencia (Dep. Biochemistry and Molecular Biology), Department of Human Pharmacology and Toxicology, Faculty of Av. Campanar, 21, 46009 Valencia, Spain and CIBERehd, Centro Pharmacy, University of Veterinary and Pharmaceutical Sciences, de Investigacion Biomedica en Red de Enfermedades Hepaticas y Brno, Palackeho 1-3, 612 42 Brno, Czech Republic Digestivas, Barcelona, Spain bDepartment of Gynecology and Obstetrics, Hospital Karvina-Raj, Vydmuchov 399/5, 734 12 Karvina-Raj; GynPro s.r.o., Strelnicni Organic cation transporter 1 (OCT1, SLC22A1) is 19/267 Cesky Tesin, Czech Republic a membrane transporter that is important for uptake of numerous cationic drugs into hepatocytes. Its liver- Deterioration of ovarian function due to surgical specific expression in hepatocytes is strongly controlled or physiologic menopause leads to the symptoms of by hepatocyte nuclear factor-4α (HNF4α). Recently we estrogen-deficiency syndrome and lower quality of life. found that dexamethasone through glucocorticoid recep- Hysterectomy with bilateral or unilateral adnexectomy tor (GR) significantly up-regulates OCT1 mRNA and is the most common cause of surgical menopause. protein in primary human hepatocytes. Currently, hormone replacement therapy (HRT) is the Therefore we examined direct (GR response element- most effective agent in the treatment of vasomotor and mediated) and indirect transactivation of OCT1 gene in urogenital symptoms of menopause, and also in the treat- primary human hepatocytes. We also examined which ment of some estrogen-dependent chronic diseases like other liver-enriched transcription factors are involved in osteoporosis. OCT1 gene expression and whether they are regulated The aim of the work was to summarize the findings by dexamethasone. Notably, expression of major nuclear about the pharmacotherapy of estrogen-deficiency syn- receptors HNF4α, Pregnane X receptor, Retinoid X re- drome with focus on patients after hysterectomy with ceptor and Constitutive androstane receptor (CAR) have bilateral or unilateral adnexectomy. The group of the pa- been demonstrated to be augmented by glucocorticoid tients was characterized and prescription analysis with receptor in human liver. evaluation of adverse effects and possible complications Gene reporter construct with 2 kb promoter sequence in the treatment of induced postmenopausal symptoms of the human OCT1 gene was not responsive to gluco- was performed. corticoids in cells cotransfected with GR expression The data about the entire group of patients were ob- construct, but was sensitive for CCAAT/enhancer bind- tained from the medical records at gynecological clinic in ing proteins β (C/EBPβ) and HNF4α cotransfection in Rychvald and Cesky Tesin. The mean age of the patients HepG2 cells. Viral transduction of MZ-Hep1 cells with was 48 years, the most numerous group were women the expression constructs for HNF4α, CCAAT/enhancer from 40 to 49 years. Almost 70% of women underwent binding proteins β (C/EBPβ) and peroxisome proliferator- laparoscopic hysterectomy with unilateral or bilateral ad- activated receptor-γ coactivator 1α (PGC1α), but not with nexectomy. PXR nor CAR, demonstrated significant roles of the tran- HRT was used only in 30% of women after adnexec- scription factors in OCT1 gene induction. Consistently, tomy; the main reasons were apparently worries about we found that expression of OCT1 mRNA in human breast and ovarian cancer, or tromboembolic complica- livers significantly correlates with C/EBPβ and HNF4α tions. Prescription analysis showed that subcutaneous es- mRNAs expression. We observed that HNF4α is induced tradiol 25 mg implant was the most frequently used, the

S25 Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2013 Sep; 157 (Supplement 1):S1–S88. average duration of the treatment was 6.7 years. Adverse ACKNOWLEDGMENT effects were observed in 18.5% of women, mostly nausea, weight gain, cephalea and vertigo. This work was supported by the grants FaF Although there are concerns about adverse effects, UK/12/2011, FaF UK/13/2012 and FaF UK 12/2012. HRT still plays a significant role in the treatment of meno- pausal symptoms. Risks can be decreased and benefits maximized by choice of optimal and individually-tailored P-21 therapeutic regimen. Pharmacokinetics of dexrazoxane and its putative active metabolite in rabbits in relationship to the P-20 cardioprotective effects of the drug The effectivity of additional antioxidant Eduard Jirkovskya, Olga Lencovaa, Jan Stariatb, Jan Buresb, to antidiabetic therapy in diabetic Jaroslav Chladeka, Petra Kovarikovab, Vladimir Gersla, - hypertensive rats Martin Sterbaa

Peter Panek, Eva Kralova, Tatiana Foltanova, aDepartment of Pharmacology, Faculty of Medicine in Hradec Stanislava Jankyova Kralove, Charles University in Prague, Simkova 870, 500 38 Hradec Kralove, Czech Republic Department of Pharmacology and Toxicology, Faculty of bDepartment of Pharmaceutical Chemistry and Drug Analysis, Pharmacy, Comenius University Faculty of Pharmacy in Hradec Kralove, Charles University in Bratislava, Kalinciakova 8, 83232 Bratislava, Slovak Republic in Prague, Heyrovskeho 1203, 500 05 Hradec Kralove, Czech Republic

Hypertension during diabetes is a risk factor for de- Dexrazoxane (DEX) is the only clinically available velopment of endothelial dysfunction. The aim of this cardioprotectant against anthracycline (ANT) cardio- work was to evaluate the effect of combination therapy toxicity. It has been believed to be a pro-drug yielding of diabetic hypertensive rats with antioxidant Pycnogenol a metal chelating metabolite - ADR-925. However, re- and antidiabetic drug metformin drugs on improvement cently this concept has been challenged by the data on of endothelial dysfunction assessed as the reactivity of importance of parent compound-induced inhibition of the vessels. topoisomerase IIβ and thus the role of the metabolite Diabetes was induced in the spontaneous hy- remains unsure. Unfortunately, pharmacokinetics of DEX pertensive rats (SHR) by intraperitoneal injection and its metabolite (ADR-925) is poorly characterized, of streptozotocin (3 x 25 mg/kg/b.w). The treatment with particularly with respect to the myocardium as the site Pycnogenol, metformin and their combination lasted for of action. This hinders our understanding to the PK/PD 6 weeks. After the treatment, the animals were sacrificed relationship and complicates insights into the mechanism and vascular contraction and relaxation responses of the of action. Hence, in this study single DEX administration isolated aorta were registered by SPEL Advanced ISOSYS was given to rabbits (n=20) in a same way in which it has system. been shown cardioprotective before (60 mg/kg, i.p.). The The relaxation responses in untreated diabetic and dia- blood was collected repeatedly after drug administration betic - hypertensive animals were significantly decreased (up to 12 hours) and the animals were randomized for compared to healthy control animals (D 55.36% ± 7.65% termination after 30, 180, 360 and 720 min (n=5, in each / HD 55.54% ± 9.07% / C: 80.61% ± 3.04%, P<0.05). The group) to determine the tissue levels of both agents. DEX relaxation responses of animals treated with Pycnogenol, and ADR-925 were determined in plasma, myocardium, metformin or their combination showed increased vascu- soleus muscle, liver and urine samples using validated lar relaxation response compared to untreated animals. HPLC-MS/MS method and PK analysis was performed

Pycnogenol monotherapy led to a significantly highest by Kinetica 4.0 software. We found mean plasma cmax (406 improvement of vascular relaxation response (HD-P and 51 μM), tmax (0.22 and 2.2 h) for DEX and ADR- 129.26% ± 8.88%, P<0.001). Metformin itself and its 925, respectively and mean t1/2 (DEX) 2.0 h. AUC0-6h combination with Pycnogenol also resulted in a signifi- and AUC0-12h (DEX/ADR-925) ratios were 2.9 and 3.4. cant increase in relaxation of blood vessels compared to In the myocardium, the selected parameters for DEX untreated diabetic - hypertensive animals (HD-M 111.38% and ADR were as follows: cmax (207 and 14 pmol/mg wet ± 7.28%, P<0.01 / HD-PM 108.33% ± 14.45%, P<0.05). weight), tmax (0.5 and 12 h) and AUC0-12h ratio was 5.3. The therapy with Pycnogenol, metformin and their Furthermore, more complex insight was obtained using combination improved vascular relaxation responses that population-based PK approach and the data are being suggest improvement of endothelial dysfunction as well. compared to pharmacodynamic parameters. However the effectivity of additional antioxidant to antidiabetic therapy in diabetic – hypertensive rats was confirmed to be surprisingly lower than monotherapy.

S26 Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2013 Sep; 157 (Supplement 1):S1–S88.

ACKNOWLEDGEMENT P-23 This study was supported by the grant of Czech The evaluation of the reactivating and therapeutic Science Foundation 13-15008S, the Charles University efficacy of two novel bispyridinium oximes (K361, research program PRVOUK P37/5 and SVV 2013-266901. K378) in comparison with the oxime K203 and P-22 trimedoxime in tabun-poisoned rats and mice

Cilia in the respiratory tract during experimental Jiri Kassa, Vendula Sepsova, Martina Tumova, Anna Horova, Kamil Musilek conditions Department of Toxicology, Faculty of Military Health Sciences, a a b Marta Joskova , Vladimira Sadlonova , Dusan Koniar , University of Defence, Trebesska 1575, 500 01 Hradec Kralove, Libor Hargasb, Miroslav Hriankab, Sona Franovaa Czech Republic aDepartment of Pharmacology, Jessenius Faculty of Medicine in Martin, Comenius University in Bratislava, Sklabinska 26, 036 01 The potency of two newly developed bispyridinium Martin, Slovak Republic compounds (K361, K378) to reactivate tabun-inhibited bDepartment of Mechatronics and Electronics, Faculty of acetylcholinesterase and reduce acute toxicity of tabun Electrical Engineering, University of Zilina, Univerzitna 1, 010 26 was compared with the oxime K203 and trimedoxime us- Zilina, Slovak Republic ing in vivo methods. The study determining percentage of reactivation of tabun-inhibited diaphragm acetylcholines- In addition to the airway defense reflexes, mucociliary terase in poisoned rats showed that the reactivating effica- transport is responsible for the protection of the airways cy of the oxime K378 is slightly lower than the reactivating and lungs against excess of mucus and foreign particles. potency of the oxime K203 and trimedoxime while the Cilium is efficient component of this primary innate de- ability of the oxime K361 to reactivate tabun-inhibited fense mechanism. acetylcholinesterase is markedly lower compared to the The purpose of this study was to make the model for oxime K203 and trimedoxime. In the brain, the potency of analysis of ciliary beat frequency in the airway epithelial both newly developed oximes to reactivate tabun-inhibited cells and evaluate the role of cholinergic receptors in the acetylcholinesterase was negligible. The therapeutic ef- ciliary beating during in vitro conditions. ficacy of both newly developed oximes corresponds to The experiment was performed on the tracheal ciliated their weak reactivating efficacy. Their potency to reduce cells of freshly excised mucosa. The trachea of guinea pigs acute toxicity of tabun was significantly lower compared was used. Next microscopic sample examination using to the oxime K203 as well as trimedoxime. In conclusion, light microscope, and high speed camera connected with the reactivating and therapeutic potency of both newly computer was carried out. Short videos were recorded developed oximes does not prevail the effectiveness of the and assessed by ciliary analysis software and following oxime K203 and trimedoxime and, therefore, they are not postanalysis. The ciliary beat frequency was determined suitable for their replacement of commonly used oximes to describe the ciliary beating. Cholinergic response to for the treatment of acute tabun poisoning. metacholine (10-8 mol.L-1) and metacholine (10-8 mol.L-1) plus atropine (10-8 mol.L-1) was monitored after their addition to ciliated cells. ACKNOWLEDGEMENT The experimental model for studying of ciliary kinetics in which cholinergic system plays a role in the modulation The study was supported by the grant of Ministry of of ciliary beating was created by us. Defence - Long-term organization development plan 1011.

P-24 ACKNOWLEDGEMENT Cardiomarkers in isoproterenol–induced model This work was supported by the projects: „Zvýšenie možností kariérneho rastu vo výskume a vývoji v oblasti of cardiotoxicity in rats lekárskych vied“, „Center of Experimental and Clinical Eliska Kolmanova, Ladislava Bartosova, Tomas Parak, respirology II“ and „Measurement of Respiratory Pavel Suchy Epithelium Cilium Kinematics“ co-financed from EU sources and European Social Fund. Department of Human Pharmacology and Toxicology, Faculty of Pharmacy, University of Veterinary and Pharmaceutical Sciences Brno, Palackeho 1-3, 612 42 Brno, Czech Republic

Troponin is a protein, which plays an important role in the muscle contraction. During myocardial ischemia,

S27 Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2013 Sep; 157 (Supplement 1):S1–S88. its cardiospecific isoforms (cTnI and cTnT) are released P-25 from cardiomyocytes into blood where they are determined. Nowadays, troponin I and T are widely used in Relative bioavailability of two molsidomine clinical medicine as a marker of acute coronary syndrome. formulations in healthy volunteers This pilot study focused on the use of cardiomarkers in preclinical testing, where they can help to improve Jiri Kopeckya, Anna Polaskovaa, Ladislav Vyslouzila, primary screening of adverse effects and detect possible Dagmar Lackovaa, Jana Zoulovaa, Jaroslav Chladeka, cardiotoxicity of newly synthesized drugs. We chose tropo- Karel Macekb, Jaroslav Kvetinaa nin I as a marker due to its high sensitivity and specificity, and added two other markers (CK-MB and NT-proBNP) aInstitute of Experimental Biopharmaceutics, Joint Research for comparison. Centre of PRO.MED.CS Praha a.s. and Academy of Sciences of the We induced a model of acute cardiotoxicity by isopren- Czech Republic, Heyrovskeho 1207, 500 03 Hradec Kralove, Czech Republic aline hydrochloride (ISO) in toxic doses. We investigated b nd a group of 15 male Wistar rats, which was divided into 2 Department of Internal Medicine, University Hospital Hradec Kralove, Sokolska 581, 500 05 Hradec Kralove, Czech Republic four subgroups differing in the way of administration (i.v., i.p. or s.c.) and doses of isoprenaline provided by liter- ary sources. Three subgroups were experimental and they "The aim of the open-label, single dose, randomized, were administered isoprenaline, one subgroup was used as crossover (2-sequence, 2-period), clinical study was to a control. We collected blood samples at predetermined compare bioavailability of two oral tablet molsidomine intervals, centrifuged them and stored them at -80°C until (NO-donor used for the management of angina pectoris) the final determination. Cardiomarkers (cTnI, CK-MB formulations (A and B). and NT-proBNP) were assessed in each plasma sample Twenty four healthy volunteers (12 men and 12 wom- using the Dimension ExL analyzer with LM Integrated en, 18-51 years, body mass index 19.1-31.1 kg/m2) received Chemistry System- Siemens® in the laboratory depart- twice a single 4 mg oral dose of molsidomine with a wash- ment of Trauma Hospital of Brno. out period of 7-14 days. Blood samples (10 mL) were tak- The most important finding was the positive outcome, en 0-10 h after drug administration for the measurement which means the possibility of using human kits for the of plasma concentrations of molsidomine (using HPLC detection of cTnI and CK-MB in animals. It confirms method with UV detection). Pharmacokinetic parameters the fact that the structure of troponin is highly conserved were estimated using non-compartmental methods. For across species. Unfortunately, it was impossible to mea- their statistical evaluation, ANOVA, two one-sided tests sure NT-proBNP by the device due to unknown reason. procedure and estimations of 90% confidence intervals The best way of administration, from the perspective (90% CI) were used. of simplicity of application and low mortality, was the The results of the study are presented in the following intraperitoneal application of ISO at the dose 50 mg/ table: kg b.w. The concentration of cTnI and CK-MB in i.p. Pharmacokinetic parameters are expressed as arith- group had an increasing tendency. Troponin showed an metic mean ±s.d. early onset of the release and the curve line describing its concentration dependent on time dropped already after Bioequivalence of the two compared molsidomine for- 6 h. Creatine kinase showed an increasing concentration mulations was proven for all bioavailability parameters (in till the end of our monitoring (24 h). Concentration of the acceptance range 80-125%). markers in the control group was also detectable but in comparison with experimental groups, values of markers in the control group were always lower.

Parameter Formulation A Formulation B Point estimate 90% CI

AUC0-inf [ng.h/ml] 120 ± 65 111 ± 58 109.4 % 97.8 – 122.4 %

AUC0-t [ng.h/ml] 110 ± 60 101 ± 54 110.5 % 98.2 – 124.4 %

AUC0-10 [ng.h/ml] 111 ± 60 102 ± 54 110.6 % 98.3 – 124.4 %

Cmax [ng/ml] 42.5 ± 15.4 43.2 ± 20.5 101.0 % 92.0 – 110.8 %

tmax [min] 33 ± 18 40 ± 23 -10 min -15 to 0 min -1 ke [h ] 0.419 ± 0.100 0.435 ± 0.132 - -

t1/2 [h] 1.76 ± 0.50 1.75 ± 0.59 - -

S28 Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2013 Sep; 157 (Supplement 1):S1–S88.

P-26 P-27 Inhibition of P450 activities by anthocyanidins Lipolytic effects of newly synthesized and anthocyanins aryloxypropanolamine derivatives in vitro Michaela Kopecna Zapletalovaa, Alena Vanduchovaa, Hana Kotolovaa, Katerina Horskaa, Zuzana Markovicovaa, Alena Spicakovaa, Eva Anzenbacherovab, Jan Tyleceka, Tomas Gonecb a Pavel Anzenbacher aDepartment of Human Pharmacology and Toxicology, aDepartment of Pharmacology, Faculty of Medicine and Pharmaceutical Faculty, University of Veterinary and Dentistry, Palacky University Olomouc, Hnevotinska 3, 775 15 Pharmaceutical Sciences Brno, Palackeho 1/3, 612 42 Brno, Czech Olomouc, Czech Republic Republic b bDepartment of Medical Chemistry and Biochemistry, Faculty Department of Chemical Drugs, Pharmaceutical Faculty, of Medicine and Dentistry, Palacky University Olomouc, University of Veterinary and Pharmaceutical Sciences Brno, Hnevotinska 3, 775 15 Olomouc, Czech Republic Palackeho 1/3, 612 42 Brno, Czech Republic

Anthocyanins are a water soluble natural pigments Adipose tissue is a highly metabolically active organ which belong to a large group of polyphenolic named with endocrine/paracrine function involved in regulation flavonoids. They are widely distributed in fruit and of various biological processes including energy metabo- vegetable such as billberries, cranberries, strawberries, lism, neuroendocrine and immune processes. Excess of grapes, and red cabbage but are also found in flowers adipose tissue is related to obesity, insulin resistance and and other plant materials. Anthocyanins are responsible metabolic syndrome development, which is reaching the for cyanic colors ranging from salmon pink through red epidemic proportions and is the main cause of death in and violet to dark blue. There have been over 600 antho- western countries. The use of β3-adrenergic receptor ago- cyanins identified in nature, featuring six common agly- nists represents one of the potential pharmacological ap- cones – anthocyanidins (cyanidin, delphinidin, malvidin, proach in metabolic syndrome treatment. peonidin, petunidin and pelargonidin) and various types In this study, the lipolytic activity of twelve newly syn- of glycosylations. Anthocyanins possess anti-inflamma- thesized aryloxypropanolamine derivatives as potential tory and anti-carcinogenic activity, cardiovascular dis- β₃-agonists were tested using cultured human subcutane- ease prevention, all of which are associated with their ous adipocytes and was compared with Isoproterenol and potent antioxidant property. Interaction of three forms BRL37344 as positive controls. The tested homologous of human hepatic cytochromes P450 - CYP3A4 (testos- series substances differ in substituents on the basic skel- terone 6β-hydroxylation), CYP1A2 (7-ethoxyresorufin eton and were synthesized at Department of Chemical O-deethylation) and CYP2C9 (diclofenac 4’-hydroxyl- Drugs of Pharmaceutical Faculty VFU Brno. The cultured ation) with six anthocyanidins (cyanidin, delphinidin, human subcutaneous preadipocytes Zen-Bio, Inc. were al- malvidin, peonidin, petunidin and pelargonidin) and nine lowed to differentiate for one week under standard differ- anthocyanins (cyanidin-3-glucoside, cyanidin-3-galacto- entiation conditions at 37 °C in 5% CO₂ atmosphere. The side, cyanidin-3-arabinoside, cyanidin-3,5-di-glucoside, tested substances, isoproterenol and BRL-37344 (Sigma), delphinidin-3-glucoside, malvidin-3-glucoside, peonidin- were added at a concentration of 0.0075μmol/L and incu- 3-glucoside, petunidin-3-glucoside and pelargonidin-3-ru- bated at 37 °C for 3 h. Lipolytic activity was assessed as tinoside) were studied using pooled human microsomes. non-esterified fatty acids (NEFA) released by adipocytes. The most influenced activities were interactions of The concentration of NEFA was determined by spectro- CYP3A4 with delphinidin, petunidin, peonidin and pel- photometry at 540 nm. argonidin, CYP1A2 with delphinidin and CYP2C9 with In four substances the rate of lipolysis was higher than pelargonidin, peonidin, malvidin and peonidin. in positive control - isoproterenol , in four substances there were no differences compared to positive control - BRL-37344 observed and in four substances lipolysis ACKNOWLEDGMENT was not affected.

Financial support from Grant Agency of Czech Republic P303/12/G163 project is gratefully acknowledged.

S29 Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2013 Sep; 157 (Supplement 1):S1–S88.

P-28 ACKNOWLEDGMENT The influence of oxidative stress markers in This work was supported by the grants diabetic and hypertensive-diabetic rats by FaFUK/12/2011, FaFUK12/2012 and FaFUK 13/2012. combination of Pycnogenol® and metformin P-29 Anna Krockovaa, Eva Kralovaa, Jana Navarovab, Stanislava Jankyovaa Paroxetine blocks reinstatement of methamphetamine seeking behaviour in an aDepartment of Pharmacology and Toxicology, Faculty of Pharmacy, Comenius University in Bratislava, Kalinciakova 8, animal model of depression 832 32 Bratislava, Slovak Republic a,b a,b a,b bInstitute of Experimental Pharmacology and Toxicology, Slovak Jana Kucerova , Petra Amchova , Zuzana Miklosova , Academy of Sciences, Dubravska cesta 9, 841 04 Bratislava, Alexandra Sulcovaa Slovak Republic aCEITEC - Central European Institute of Technology, Masaryk University, Brno, Czech Republic The oxidative stress formated in connection with bDepartment of Pharmacology, Faculty of Medicine, Masaryk hyperglycaemia has considerable effect in hypertension University, Brno, Czech Republic development in terms of diabetes. An increase of free radicals concentration supports lipid peroxidation and insulin resistance. The aim of work was to evalu- The incidence of methamphetamine (METH) addic- ate MDA and NAGA level changes in control, diabetic tion among individuals with generalised depressive disor- and hypertensive-diabetic rats after administration of der is very high in accordance with the self-medication Pycnogenol®, metformin and their combination. Wistar hypothesis suggesting that symptoms of depression may male rats and spontaneously hypertensive rats were ran- be relieved by the aminergic drugs of abuse. We devel- domly divided into 9 groups. Diabetes was induced in 8 oped an animal model of this comorbidity (IV METH groups by intraperitoneal application of streptozotocin self-administration in olfactory bulbectomy model of de- (25 mg/kg, i. p.) during three consequent days. In the pression in rats) in which we recorded a higher METH last group there were Wistar male rats without diabetes intake and relapse rate. The anticipation for the present (Control). We started administration with Pycnogenol®, study was that the treatment of bulbectomized rats (OBX) metformin and their combination one week later. Levels with antidepressant paroxetine might normalise increased of MDA in liver and NAGA in kidney were evaluated METH intake and relapse tendency. after 6 weeks of therapy. The levels of MDA in liver were Adult male Wistar rats were randomly divided into 2 increased only in diabetic animals compared to control. groups and bilateral OBX/sham (SH) surgery were per- All substances decreased the levels of MDA in diabetic formed. The model of methamphetamine IVSA was initi- (D) and hypertensive–diabetic (HD) group compared ated under fixed ratio schedule of reinforcement using to animals without treatment. The most effectively operant chambers. After reaching a stable METH intake, treatment was metformin in both pathological groups. forced abstinence (no access to the drug) and chronic In contrast to monotherapy the combination of these paroxetine (PRX, dose: 10 mg/kg IP) or vehicle (VEH, drugs was the less effective in the lowering of MDA lev- dose: 3 ml/kg IP, saline) treatment was initiated. After els in liver in D and HD animals. The specific activity 14 days one reinstatement session was performed. Thus, of NAGA in kidney was significantly increased only in there were 4 groups of animals: SH VEH (n=6), SH PRX HD group compared to control and D group. In contrast (n=5), OBX VEH (n=6), OBX PRX (n=5). Statistics: to D rats without treatment the following substances did parametric one-way analysis of variance and paired t-test. not influence the NAGA levels in kidney in monother- The OBX animals exhibited significantly higher ten- apy and in their combination. On the other hand both dency to relapse in terms of METH intake and active substances decreased the NAGA levels in kidney of HD nosepoke responding. Chronic treatment with paroxetine rats compared to HD rats without treatment; the most abolished higher METH intake in the reinstatement ses- significantly Pycnogenol®. The decrease of MDA level sion. In sham operated animals paroxetine did not show and NAGA activity was less effective in groups where significant effects. the combination of treatment was applied compared to These results indicate that paroxetine is able to nor- the other treated animal groups. Diabetic animal groups malize some of the characteristics of the OBX model. responded to the therapy better than diabetic-hypertensive Serotonin is known to potently modulate dopamine levels animal groups. in the reward pathway and inhibition of serotonin transporters was reported to decrease methamphetamine crav- ing. Therefore, investigation of specific serotonergic drugs might be an important step towards finding a novel treatment of drug addiction and relapse prevention.

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ACKNOWLEDGMENT commonly prescribed 2nd generation AP was olanzapine. Decrease in 1st generation antipsychotics prescription was Supported by Masaryk University Student Grant for seen in 2012 and only slight increases were observed in Specific Research: MUNI/A/0763/2011 and CEITEC prescription of anxiolytics and mood stabilizers in 2012. CZ.1.05/1.1.00/02.0068 from European Regional Development Fund. ACKNOWLEDGMENT

P-30 This work was supported by the UK Grant Depression treatment comparison of adult UK/129/2013. patients hospitalized in year 2008 and 2012 at Clinic of Psychiatry, Jessenius Faculty of Medicine, P-31 Comenius University, Martin Effect of different doses of atropine on gastric Tomas Kulhana,b, Igor Ondrejkab Gabriela Nosalovaa myoelectric activity in experimental pigs a b b b aDepartment of Pharmacology, Jessenius Faculty of Medicine in Martin Kunes , Jaroslav Kvetina , Jan Bures , Ilja Tacheci , c b a Martin, Comenius University in Bratislava, Sklabinska 26, 03601 Michal Pavlik , Stanislav Rejchrt , Kamil Kuca , Martin, Slovak Republic Marcela Kopacovab bClinic of Psychiatry, Jessenius Faculty of Medicine in Martin, Comenius University in Bratislava, Kollarova 2, 03659 Martin, aBiomedical Research Center, University Hospital Hrdaec Kralove, Slovak Republic Sokolska 581, 50005 Hradec Kralove, Czech Republic b2nd Department of Internal Medicine – Gastroenterology, University Hospital Hradec Kralove, Sokolska 581, 500 05 Hradec Primary depression belongs to the most common Kralove, Czech Republic causes leading to psychiatric hospitalization. Tendency cDepartment of Teaching Support, Faculty of Military Health of prescription of broader receptor - neurotransmission Sciences, Trebesska 1575, 500 01 Hradec Kralove, Czech Republic profile substances rises in recent years, utilizing a combination of antidepressants (AD), or antidepressants and Surface electrogastrography (EGG) is a non-invasive antipsychotics (AP) in many cases. Prevalence of the ther- method for clinical assessment of gastric myoelectric apeutic strategies and comorbid disorders accompanying activity. Our group has demonstrated that EGG is also depression were compared in retrospective, observational reliable and feasible in experimental pigs. Porcine EGG study. Medical records of all patients older than 18 years, is fully comparable with that recorded in healthy humans. hospitalized at Clinic of Psychiatry JFM CU Martin The aim of this project was to evaluate the effect of dif- were studied and statistically analyzed in 5 years period. ferent doses of atropine on the gastric myoelectric activity Significantly different gender distributions were witnessed in experimental pigs. Six mature young female experimen- in 2008 when compared with 2012. Significantly different tal pigs (Sus scrofa f. domestica, mean weight 27±1.5 kg) presentations of solitary and recurrent depression in the entered the study three-times within three weeks. years 2008 and 2012 with predominant single episodes of A baseline EGG recording lasted 15 min Intra- depression in 2012 and a prevalence of recurrent depres- muscular atropine 1.5 mg (part A) or 3.0 mg (part B) sion in 2008 were observed. Significant differences in the or 4.5 mg (part C) was administrated. A total of seven degree of depression (acc. to ICD-10) with more severe 15-min intervals were recorded afterwards. Surface cu- degrees of depression in 2012 were found. However, ini- taneous EGG was recorded under general anaesthesia tial disease severity (CGI-S) showed no significant dif- using an Electrogastrography System (MMS, Enschede, ferences between the two compared years. There were the Netherlands). Running spectral analysis (based on significant differences in the rate of improvement at the Fourier transform) was used for the elemental evaluation end of hospitalization (CGI-I) with a marked improve- of the EGG. The results were expressed as running spec- ment in 2008 (1.73 ± 0.65 vs. 1.91 ± 0.64). A significant trum percent activity and the dominant frequency of slow increase in co-morbidities was witnessed in 2012. There waves was set at all intervals of EGG recordings. In part was no significant correlation between severity of disease A, dominant frequency of slow waves increased from the at the start of hospitalization and rate of improvement at basal values (3.0±0.6 cycles per min) to the maximum at the end of stay. No significant differences were evident in 60 min (3.2±0.4) and at 105 min (3.2±0.3), both P<0.001. the use of venlafaxine and escitalopram. A significant rise In part B, dominant frequency of slow waves increased in agomelatine prescriptions was noted in 2012. The most from the basal values (2.7±0.5) to the maximum at 15 common antidepressants prescribed during the study were min (3.0±0.5) and at 75 to 105 min intervals (3.2±0.7 escitalopram, venlafaxine, and trazodone. The 2nd genera- cycles per min), all P<0.001. In part C, there was a de- tion atypical antipsychotic co-medication was significantly crease not significant from the basal values (3.2±0.6) to increased in 2012 (52 vs. 68%, P=0.012), of which sul- the minimum at 75 min. (3.0±0.5 cycles per min.). Other piride experienced the most prominent growth. The most changes of the dominant frequency were low. Heart rate

S31 Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2013 Sep; 157 (Supplement 1):S1–S88. increased after atropine administration in the part A from whereas important transcriptional factor GATA-4 was sig- basal 74±4 beats per min. to the maximum at 65 min nificantly down-regulated in the LV due to the treatment, (111±13; P<0.001), in the part B from 75±5 to the maxi- CARP was inversely regulated. We also found significant mum at 20 min (138±22; P<0.001), in the part C from and persistent up-regulation of desmin expression in the 75±7 to the maximum at 15 min (123±24 beats per min; LV at mRNA level and even higher change at the protein P<0.001). A dose-dependent change of gastric myoelectric level. Furthermore, molecular and morphological remod- activity was found after atropine administration. Two-peak eling in the LV concerned also extracellular matrix with increase of dominant frequency of slow waves was longer marked expression of collagens I and IV, whereas RV and more prominent in the part B (compared with higher showed rather minor changes only. In conclusion, chronic and lower doses of atropine). All changes identified in ANT cardiotoxicity is associated with profound molecular dominant frequency were only minor and mostly fluctu- remodeling of cardiomyocytes and extracellular matrix ated within the normal range. particularly in the LV, while changes in the RV are apparently less pronounced.

ACKNOWLEDGMENT ACKNOWLEDGMENT This study was supported by research project IGA NT14270 from the Ministry of Health. Supported by the grant IGA MZ No. NT13457-4/2012 and the Charles University research program PRVOUK P-32 P37/5. Remodeling of the myocardium in anthracycline P-33 cardiotoxicity: comparison of left and right Interactions of a set of antiviral drugs ventricle with organic anion transporter 1 and organic Olga Lencovaa, Eduard Jirkovskya, Yvona Mazurovab, cation transporter 2 Michaela Adamcovac, Vladimir Gersla, Martin Sterbaa Jana Mandikovaa, Marie Volkovaa, Petr Paveka, aDepartment of Pharmacology, Faculty of Medicine in Hradec Zlatko Janebab, Frantisek Trejtnara Kralove, Charles University in Prague, Simkova 870, 500 38 Hradec Kralove, Czech Republic aDepartment of Pharmacology and Toxicology, Faculty of bDepartment of Histology and Embryology, Faculty of Medicine in Pharmacy in Hradec Kralove, Charles University in Prague, Hradec Kralove, Charles University in Prague, Simkova 870, 500 38 Heyrovskeho 1203, 500 05 Hradec Kralove, Czech Republic Hradec Kralove, Czech Republic bInstitute of Organic Chemistry and Biochemistry AS CR, cDepartment of Physiology, Faculty of Medicine in Hradec Flemingovo namesti 2, 166 10 Prague, Czech Republic Kralove, Charles University in Prague, Simkova 870, 500 38 Hradec Kralove, Czech Republic Transporters play an important role in the fate of xenobiotics and endogenous chemicals in the body. Chronic anthracycline (ANT) cardiotoxicity rep- Renal tubular secretion via organic anion transporter 1 resents a feared complication of cancer chemotherapy (OAT1) and organic cation transporter 2 (OCT2) leads resulting in dilated cardiomyopathy and heart failure. to remove metabolic waste, toxins and drugs in the urine. The aim was to study molecular changes associated with Interestingly, certain drugs such as antivirals can be elimi- myocardial remodeling of the left and right ventricle in nated via both transporters simultaneously. The goal of response to chronic ANT treatment and post-treatment the study was to compare the interactions of a set of anti- follow-up. Chronic cardiotoxicity was induced in rabbits viral drugs with hOAT1 and hOCT2. The HeLa cell line with daunorubicin (DAU 3 mg/kg, weekly, 10 weeks). transiently transfected with hOAT1 and the MDCKII A week after the last drug dose, the animals were ran- cell line transiently transfected with hOCT2 were used domized to sacrifice or follow up for next 10 weeks. The for the experiments. The rate of inhibition of intracellu- chronic treatment with DAU led to significant LV systolic lar accumulation of tritium labelled typical substrate for dysfunction and increase of plasma concentrations of car- OAT1 (para-aminohippuric acid, 3H-PAH) and for OCT2 diac troponin T. The degenerative changes were located (1-methyl-4-phenylpyridinium, 3H-MPP+) induced by ad- mainly in the LV myocardium, while RV myocardium dition of two different concentrations of each tested an- was significantly less affected. Gene expression of thin tiviral drug was evaluated. Among the tested substances, myofilament components revealed only moderate and the highest potency to inhibit hOAT1 exhibited tenofo- rather late changes in the LV, whereas the RV was unaf- vir. Tenofovir and its ester prodrug tenofovir disoproxil fected. In contrast, gene expression of thick myofilament fumarate (TDF) at concentration 100 μM inhibited ac- components was markedly decreased in the LV. We also cumulation of 3H-PAH by 72% and 25%, respectively. found significant and persistent titin down-regulation in Efavirenz, abacavir and carbovir inhibited the accumula- the LV, while this was not the case of the RV. Interestingly, tion of 3H-PAH approximately by 50% at concentration

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1000 μM. In hOCT2 cell model, efavirenz, abacavir and plasma (P<0.05), but Surf+Bud had superior effect (IL-1ß TDF at concentration 100 μM inhibited 3H-MPP+ accu- P<0.009, IL-8 P<0.003). mulation approximately by 25%. Efavirenz and abacavir at In experimental model of MAS, budesonide combined concentration 1000 μM inhibited 3H-MPP+ accumulation with exogenous surfactant improved respiratory param- approximately by 60%. In conclusion, the results demon- eters, lung edema, oxidative damage and inflammation. strated more frequent interactions of the tested antiviral Thus, addition of anti-inflammatory agent may prevent drugs with hOAT1. In most cases, a considerable inhibi- inactivation of surfactant and provide more effective tory effect was found in a relatively high concentration. therapy of MAS. Efavirenz, abacavir and TDF interacted with both tested transporters. ACKNOWLEDGEMENT

ACKNOWLEDGMENT Supported by grants: APVV-0435-11, VEGA 1/0057/11, VEGA 1/0291/12, CEPV II. This work was supported by Charles University in Prague (Project SVV 267 003), grant GAUK No. 360811/ FaF/C-LEK and grant IGA MZ No. NT12398-4/2011. P-35 Differential gene expression of important factors P-34 in human epicardial adipose tissue and left Changes in ventilation and inflammatory ventricular myocardium in end-stage heart failure parameters due to combination therapy Jana Mlynarovaa, Gabriel Dokaa, Peter Musila, at experimental meconium aspiration syndrome Michal Hulmanb, Eva Gonsalvesovab, Jan Kyselovica Pavol Mikolkaa, Daniela Mokraa, Jana Kopincovaa, aDepartment of Pharmacology and Toxicology, Faculty of a b a Pharmacy, Comenius University in Bratislava, Odbojarov 10, Lucia Tomcikova , Jozef Hatok , Andrea Calkovska 832 32 Bratislava, Slovak Republic b aDepartment of Physiology, Jessenius Faculty of Medicine in The National Institute of Cardiovascular Diseases, Pod Krasnou Martin, Comenius University in Bratislava, Mala Hora 4, 036 01 horkou 1, 833 48 Bratislava 37, Slovak Republic Martin, Slovak Republic bDepartment of Medical Biochemistry, Jessenius Faculty of Aims. Epicardial adipose tissue is proposed to play Medicine in Martin, Comenius University in Bratislava, Mala Hora a crucial role in coronary artery disease and atrial fibrila- 4, 036 01 Martin, Slovak Republic tion. In this work we characterize gene expression of several important factors in epicardial adipose tissue (EAT) Meconium aspiration syndrome (MAS) with hypox- and left ventricular myocardium (LV) during development emic respiratory failure is a serious neonatal disease. In of heart failure (HF). this situation, exogenous surfactant administration is the Methods. Samples of EAT and LV were collected from treatment of the first choice. However, aspirated meco- explanted hearts of 29 patients (average age 48±9, 25 men, nium may inactivate surfactant and reduce effect of the 4 women). All patients had been diagnosed with end-stage therapy due to initiation of local inflammation followed HF and underwent heart transplantation. Causes of heart by lung edema, oxidation damage and increased expres- failure were as follows: dilated cardiomyopathy (15), coro- sion of inflammatory mediators. To enhance efficacy of nary artery disease (10) and other causes (4). Samples exogenous surfactant, an anti-inflammatory agent – glu- were collected immediately after heart explantation and cocorticoid budesonide – was added. flash frozen in liquid nitrogen. qRT-PCR was used for New Zealand white rabbits with meconium-induced gene expression analysis. respiratory failure were divided into four groups (n=6 Results. Expression of calcineurin, nuclear factor of in each): without therapy (Mec), with surfactant (Surf), activated T cells 3 and platelet-derived growth factor B with budesonide (Bud), and with combined therapy was significantly higher (170-196%, P<0.05) in EAT com- (Surf+Bud). Respiratory parameters and indexes of oxy- pared to LV. Expression of transforming growth factor genation were registered for additional 5 hours. After sac- β1 (TGFβ1), insuline-like growth factor 1 (IGF1) and rificing animals, lung edema (expressed as wet/dry weight endothelin 1 (ET-1) were several folds higher in EAT ratio), and oxidative damage (thiobarbituric acid-reactive (230%, 1170% and 950% respectively, P<0.05) compared substances, TBARS) and levels of interleukins (IL)-1ß, -6, to LV. A significant 30% (P<0.05) decrease in expression -8 and TNFα in plasma were determined. of vascular endothelial growth factor A was observed in Combined Surf+Bud therapy rapidly improved oxy- EAT. Several genes (IGF1, TGFβ1, ET-1) displayed the genation compared to other groups (P<0.05) and this same differential gene expression profile in EAT and LV effect persisted till the end of experiment. Combined regardless of HF origin and/or diabetes mellitus diagnosis. therapy also reduced lung edema and TBARS (P<0.05). Conclusion. In our work, we identified a significantly All used therapies decreased levels of IL-1ß and IL-8 in different expression of several important factors in EAT

S33 Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2013 Sep; 157 (Supplement 1):S1–S88. and LV of end-stage HF. Finding that EAT highly express ACKNOWLEDGEMENT a number of genes, it is likely that it plays an important role in heart disease, notably in heart failure. This work was supported by the following grants: APVV-0887-11, VEGA 1/0981/12, VEGA 1/0564/13 and UK/331/2013. ACKNOWLEDGEMENT

This contribution is the result of the project implemen- P-37 tation (ITMS 26240220071) supported by the OPRaD Anti-inflammatory effects of budesonide in funded by the ERDF; FaF UK 26/2013. experimental model of acute lung injury P-36 Daniela Mokraa, Pavol Mikolkaa, Hana Pistekovaa, Lucia Tomcikovaa, Andrea Calkovskaa Abnormal respiratory function is not associated aDepartment of Physiology, Jessenius Faculty of Medicine in with altered gene expression of HGF and c-Met in Martin, Comenius University in Bratislava, Mala Hora 4, 036 01 monocrotaline induced pulmonary hypertension Martin, Slovak Republic in wistar rats Acute lung injury (ALI) and its more severe form - Eva Malikova, Kristina Galkova, Jasna Srankova, acute respiratory distress syndrome (ARDS) - are char- Peter Vavrinec, Diana Vavrincova, Peter Krenek, Jan Klimas acterized by dysfunction of pulmonary surfactant, lung edema and inflammation. In the treatment of ALI/ARDS, Department of Pharmacology and Toxicology, Faculty of lung-protecting ventilatory regimes and various anti-in- Pharmacy, Comenius University in Bratislava, Odbojarov 10, flammatory agents may be used. To evaluate its possible 832 32 Bratislava, Slovak Republic benefits, intratracheal glucocorticoid budesonide was given into the lungs of experimental animals with ALI/ Background. An established animal model of pulmo- ARDS. nary arterial hypertension (PAH) is the monocrotaline- Lungs of New Zealand white rabbits were repetitively induced pulmonary hypertension in rats. Respiratory lavaged with saline (30 mL/kg) to induce ALI/ARDS. function of monocrotaline treated rats has not been well Then, animals were treated with budesonide (Pulmicort, documented. Additionally, hepatocyte growth factor 0.25 mg/kg) or were left without therapy (n=6 in each (HGF) with its receptor c-Met plays a role in tissue re- group). All these animals were oxygen-ventilated for ad- generation. We hypothesized that experimentally induced ditional 5 hours. One group of non-ventilated animals PAH affects respiratory system in rats and this might be served as healthy controls (n=6). After sacrificing ani- associated with alterations in HGF/c-Met signalling. mals, total and differential leukocytes in blood, total and Methods. Group of 13 male Wistar rats was injected differential cells in bronchoalveolar lavage fluid (BAL), with monocrotaline (MCR; 60 mg/kg) and 7 control rats lung edema (expressed as wet/dry weight ratio), and levels (CON) received vehicle. Animals were weighted routinely of interleukins (IL)-1ß, -6, -8 and TNFα in plasma were and vital functions were measured using MouseOx meter. determined. After 4 weeks, rats were sacrificed when showing signs of Repetitive lung lavage increased total number of dyspnoe, lethargy and marked weight reduction. cells (P<0.05), particularly neutrophils and eosinophils Results. MCR-treated rats showed a decrease in body (both P<0.001) in the BAL fluid, lung edema formation weight when compared to controls (281±16 g vs. 325±34 g, (P<0.001) and concentrations of pro-inflammatory cyto- P<0.01). Development of pulmonary hypertension was kines (P<0.05) compared to healthy controls. Budesonide associated with significant increase of right ventricular decreased number of neutrophils in BAL fluid (P<0.001), weight to body weight ratio (MCR: 0.86±0.14 mg/g vs. reduced lung edema (P<0.01) and showed trend to de- CON: 0.50±0.03 mg/g, P<0.05) and a trend of increased crease pro-inflammatory cytokines in plasma (P>0.05). lung to body weight ratio (MCR: 8.14±0.91 vs. CON: Budesonide alleviated lung edema and inflammation 6.29±0.68; NS). Oxygen saturation was significantly de- in experimental model of ALI/ARDS, showing further creased in MCR group (91±2 % vs. 95±0.7 %, P<0.05) and perspectives of administration of this drug in the treat- a trend of increased ventilation rate in MCR group was ment of ALI/ARDS in patients. present (113±6 brpm vs 100±2 brpm; NS). We found unchanged mRNA expression of HGF and a lack of mRNA expression c-Met in lung tissue. ACKNOWLEDGEMENT Conclusion. Monocrotaline disrupts the cardiovascular and respiratory system of Wistar rats, which leads to the Supported by grants: APVV-0435-11, VEGA development of PAH. This is not associated with altera- 1/0305/13, CEKR II tions in gene expressions of HGF and c-Met.

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P-38 ACKNOWLEDGEMENT Inhibition of PDE5 by tadalafil influences in Supported by the Project "Center of Experimental and vivo and in vitro airway reactivity in ovalbumin- Clinical Respirology CEKR1 and CEKR2”, co-financed from EU sources, by grant VEGA 1/0030/11, and by proj- sensitized guinea pigs ect UK/159/2013. Juraj Mokrya, Ivana Medvedovaa, Marek Prsoa, Alexandra Eichlerovaa, Pavel Mikolkab, Daniela Mokrab P-39 aDepartment of Pharmacology, Jessenius Faculty of Medicine in Transplacental transport of tenofovir and Martin, Comenius University in Bratislava, Sklabinska 26, 036 01 Martin, Slovak Republic tenofovir disoproxil fumarate bDepartment of Physiology, Jessenius Faculty of Medicine in Zuzana Neumanova, Lukas Cerveny, Frantisek Staud Martin, Comenius University in Bratislava, Mala Hora 4, 036 01 Martin, Slovak Republic Department of Pharmacology and Toxicology, Faculty of Pharmacy in Hradec Kralove, Charles University in Prague, Hradec Introduction. Non-selective inhibition of phosphodi- Kralove, Czech Republic esterases (PDE) via metylxanthine derivatives (theophylline) has been traditionally used in the therapy of chronic Tenofovir disoproxil fumarate (TDF) is a prodrug inflammatory diseases associated with airway obstruction form of tenofovir (TFV), a nucleotide reverse transcrip- (e.g. bronchial asthma and chronic obstructive pulmonary tase inhibitor. TDF-containing regimens are recom- disease – COPD). PDE5 inhibition is widely used in the mended as the first line therapy in HIV infected patients, therapy of erectile dysfunction, pulmonary hypertension including women of childbearing age, and represent an as well as other cardiovascular diseases. However, the ex- acceptable option for prevention of mother-to-child trans- pression of PDE5 was confirmed in several immune cells, mission of HIV (PMTCT). TFV is classified among cat- suggesting its potential role in allergic inflammation. The egory B drugs; it is, therefore, important to have detailed aim of this study was to evaluate the effect of one-week knowledge on transplacental transport of TDF and TFV administration of selective PDE5 inhibitor tadalafil in to assess safety and effectiveness of TDF treatment in experimentally induced allergic inflammation (model of pregnancy. P-glycoprotein (MDR1), breast cancer resis- allergic asthma) in guinea pigs. tance protein (BCRP) and multidrug resistance protein 2 Material and Methods. 24 male adult guinea pigs, di- (MRP2) are the most important drug efflux ABC trans- vided into 4 groups, have been used in the study. Control porters that may affect transplacental pharmacokinetics group has been left without sensitization. The latter 2 by active transport of their substrates from fetal to mater- groups have been sensitized with ovalbumin over two nal circulation. To avoid potential drug-drug interactions weeks and thereafter treated intraperitoneally for 7 days resulting in toxicity or failure of PMTCT it is necessary to with tadalafil at the daily dose of 1.0 mg/kg b.w., or with describe TDF/TFV interactions with these transporters. vehiculum, respectively. Specific airway resistance mea- The aim of study was to investigate transplacental trans- sured in whole-body double-chamber plethysmograph has port of TFV and TDF employing the method of dually been used as a marker of in vivo airway reactivity. The in perfused rat term placenta. Using open-circuit perfusion vitro reactivity of tracheal and lung smooth muscle has system, transport of TFV or TDF was studied in both been tested using organ bath method. fetal-to-maternal and maternal-to-fetal directions. In case Results. Sensitization with ovalbumin has led to sig- of TFV, we observed weak transplacental passage without nificant increase in in vivo and in vitro airway reactivity. involvement of capacity-limited mechanism. Conversely, Tadalafil reduced both specific airway resistance after the transplacental clearances of TDF were concentration- nebulisation of histamine, and in vitro airway reactivity dependent in both directions. Employing closed-circuit to cumulative doses of acetylcholine in tracheal and lung perfusion system, we confirmed saturable transport of tissue strips. These changes have been associated with TDF. Moreover, application of common MDR1 and suppression of haematological markers of inflammation BCRP inhibitor, GF120908, resulted in significant sup- and apoptosis in animals treated with tadalafil. pression of TDF transport from fetus to mother, whereas Conclusions. Selective PDE5 inhibition seems to application of MRPs’ inhibitor, indomethacin, had no ef- play a significant role in allergic airway inflammation. fect on TDF transport. In conclusions, both drugs seem to However, its anti-allergic and possible anti-asthma poten- have restricted placental permeation from mother to fetus. tial needs further testing. Our findings suggest an important role of MDR1 and BCRP in transplacental transport of TDF. These interactions may contribute to potential drug-drug interactions in transplacental pharmacokinetic of this agent.

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ACKNOWLEDGMENT P-41 The study was supported by Grant Agency of Charles Acute poisonings by tricyclic antidepressants University (GAUK 695912/C/2012 and SVV/2013/267- reported to the toxicological information centre 003). in Bratislava P-40 Elena Ondriasovaa, Zuzana Muchovaa, Blazena Caganovab, Silvia Plackovab The effect of chelator on receptor binding and 177 aDepartment of Pharmacology and Toxicology, Faculty of stability in Lu-labeled anti-EGFR monoclonal Pharmacy, Comenius University in Bratislava, Odbojarov 10, antibodies in vitro 832 32 Bratislava, Slovak Republic bNational Toxicological Information Centre, University Hospital Zbynek Novya,c, Alice Laznickovab, Jana Mandikovac, Bratislava, Limbova 5, 833 05 Bratislava, Slovak Republic Milan Laznicekc, Frantisek Trejtnarc According to the National Toxicological Information aLaboratory of Experimental Medicine, Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Centre (NTIC) in Bratislava intoxications by antidepres- Palacky University Olomouc, Hnevotinska 5, 779 00 Olomouc, sants have an increasing tendency. NTIC has still quite of- Czech Republic ten been consulted for advice on tricyclic antidepressants bDepartment of Biophysics and Physical Chemistry, Faculty (TCA) exposures, in spite of the fact that they are not the of Pharmacy in Hradec Kralove, Charles University in Prague, first line therapy in the management of depression since Heyrovskeho 1203, 500 05 Hradec Kralove, Czech Republic the late 1980s. TCA are still valuable in the treatment of cDepartment of Pharmacology and Toxicology, Faculty of severe depression and are also used in the treatment of Pharmacy in Hradec Kralove, Charles University in Prague, chronic pain syndromes, obsessive compulsive disorder Heyrovskeho 1203, 500 05 Hradec Kralove, Czech Republic and panic disorder. Methods. TCA intoxications were analyzed on the Aim. Nowadays, monoclonal antibodies are used in basis of data gathered from telephone consultations and therapy of various diseases. Due to very specific accumu- medical reports forwarded to the NTIC from the whole lation in the target tissue the antibodies could be utilized area of Slovakia in the years 2008-2012. The following as carriers of radioisotopes to the tumors in case of target- data were analyzed: age, sex, intent of exposure (acciden- ed radioimmunotherapy and/or diagnosis. Nevertheless, tal or suicidal), the clinical severity, symptoms, substances essential properties of antibodies such as binding to the co-ingested and treatment of intoxications. target or stability in the organism may be influenced by Results. During the five-year period 940 cases of anti- various structural parameters. In this study we have fo- depressant intoxication were reported, including 126 TCA cused on potential effect of selected chelators on radio- intoxications, mainly by dosulepin, amitriptyline and clo- chemical quality and in vitro receptor binding capacity of mipramine. Intoxications in women (59%) were more fre- two modified monoclonal antibodies. quent than in men. The highest number of poisonings was Materials and Methods. We have selected two mono- in the age range 30-39 years (22%). Intoxications with sui- clonal antibodies - cetuximab and panitumumab, both cidal intent (89%) predominated. The most common poi- ligands of epidermal growth factor receptor (EGFR). soning severity score was PSS1 i.e. mild symptoms (38%). These antibodies were linked with three macrocyclic bi- One lethal combined intoxication was recorded. From the functional chelators (DOTA, NOTA and PTCA) and ra- total number of TCA poisonings 25 were caused by one diolabeled with lutetium-177. The radiochemical stability drug and 101 by combination with other substances. The of the preparations was checked in HPLC system and the most commonly abused substances in combined poison- binding rate to EGFR expressing cell lines (A431, HaCaT ings were benzodiazepines, other antidepressants, antipsy- and HepG2) was examined. chotics and alcohol (21%). The most frequent symptoms Results. The method employed led to very stable ra- of mono-intoxications by TCA were somnolence (32%), diolabeled preparations for each of selected chelators. unconsciousness (20%) and tachycardia (20%). Treatment Binding rate of labeled antibodies varied from 15% to of TCA mono-intoxications consisted mainly of adminis- 45% of applied dose according to used cell line. The re- tration of activated charcoal (48%), gastric lavage (24%) sults showed that the binding to the target cells was not and laxatives (20%). influenced by the molecule of chelator. Conclusion. We conclude that relatively recently developed chelators (NOTA and PCTA) could be useful for radiolabeling of anti-EGFR antibodies with lutetium-177 as well as well-established chelator DOTA. Thus NOTA and PCTA could be convenient tools in future preclinical studies with these radiopharmaceuticals.

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P-42 ACKNOWLEDGMENT Possibilities and risks of using phosphodiesterase Supported by the grants APVV-0435-11, VEGA inhibitors in treatment of meconium aspiration 1/0057/11, ESF 2. syndrome P-43 Hana Pistekovaa, Daniela Mokraa, Ingrid Tonhajzerovaa, Anna Drgovab, Zuzana Visnovcovaa, Martina Repcakovac, Effect of fenugreek seeds enriched diet on Lucia Mikusiakova-Tomcikovac, Andrea Calkovskaa endothelial dysfunction in mild diabetes and its aDepartment of Physiology, Jessenius Faculty of Medicine in relation to nitric oxide and epoxyeicosatrienoic Martin, Comenius University in Bratislava, Mala Hora 4, 036 01 acids pathway Martin, Slovak Republic bDepartment of Medical Biochemistry, Jessenius Faculty of Lenka Pivackova, Jasna Srankova, Beata Gajdacova, Medicine in Martin, Comenius University in Bratislava, Mala Hora Lucia Mensikova, Kristina Galkova, Peter Krenek, Jan Klimas 4, 036 01 Martin, Slovak Republic cDepartment of Clinical Biochemistry, Jessenius Faculty of Department of Pharmacology and Toxicology, Faculty of Medicine in Martin, Comenius University in Bratislava and Pharmacy, Comenius University in Bratislava, Kalinciakova 8, University Hospital, Kollarova 2, 036 01 Martin, Slovak Republic 832 32 Bratislava, Slovak Republic

Ventilation support, administration of exogenous sur- Fenugreek seeds are reported to have strong antihy- factant and inhalation of nitric oxid are standardly used perglycaemic and antioxidant properties. Our aim was to for treatment meconium-induced acute lung injury in investigate effect of the fenugreek seeds enriched diet on newborns. Additionally, there is a possibility to admin- endothelial function and its 2 potent regulators - NO and ister anti-inflammatory drugs such as phosphodiesterase epoxyeicosatrienoic acids (EETs) pathways in rat model (PDE) inhibitors. Aim of this study was to observe and of mild diabetes. to compare effects of non-selective PDE inhibitor ami- Male Wistar rats (total n=40) were randomized into nophylline and selective PDE3 inhibitor olprinone on 4 groups: control (C), diabetes (D), fenugreek (F), fen- cardiovascular parameters. ugreek + diabetes (FD). Mild diabetes was induced by Oxygen-ventilated and anesthetized animals received streptozotocin administration in dose 25 mg/kg/d i.p. for intratracheally 4 ml/kg of meconium suspension (25 mg/ 3 consecutive days. 8-weeks long treatment in the form of mL) or saline. Meconium-instilled animals were treated by a diet enriched with fenugreek seeds (5%) began 2 weeks intravenous administration of aminophylline (2.0 mg/kg) after. Vascular reactivity of aortic rings was tested by ace- delivered at two doses at 0.5 and 2.5 h after meconium tylcholine and sodium nitroprusside. We analyzed mRNA instillation, or olprinone (0.2 mg/kg) given at single dose expressions of epoxygenases (Cyp2j4, Cyp2c23) gener- at 0.5 h after meconium instillation, or were left without ating vasodilatatory EETs and ω-hydroxylases (Cyp4a2, treatment as controls. Cyp4a3) producing vasoconstrictory EETs in aortas using Changes of cardiovascular parameters (blood pressure, qRT-PCR and protein levels of endothelial NO-synthase heart rate, heart rate variability) were evaluated within 5 (eNOS) by Western blotting. min of administration, 5 min after finishing administra- Fenugreek restored blunted endothelium-dependent tion and within 5 hours after treatment. Markers of cardio- relaxation (acetylcholine pD2 - P<0.001). Endothelium- vascular injury [aldosterone, gamma-glutamyltransferase independent relaxation was not affected. Concomitantly, (GGT), aspartate aminotransferase (AST), alanine amino- aortas from diabetic animals exhibited significantly lower transferase (ALT)] and oxidation markers (TBARS, total levels of eNOS protein (P<0.001) and fenugreek was able antioxidant status) were determined in plasma. to attenuate this decrease (P<0.05). mRNA levels of ep- Meconium instillation induced oxidative stress and oxygenases and ω-hydroxylases were not influenced either elevated aldosterone and slightly increased GGT and AST by diabetes or fenugreek treatment. levels. Aminophylline and olprinone reduced oxidation Fenugreek enriched diet normalized vascular func- stress, however, both PDE inhibitors caused short-term in- tion together with eNOS protein levels in diabetic aortas, crease in blood pressure and heart rate, whereas heart rate but did not alter mRNA expression of EETs producing variability remained increased till the end of experiment. enzymes. Thanks to these effects, fenugreek may be ben- As both treatments exerted comparable short-term eficial as adjuvant therapy of diabetes mellitus. cardiovascular changes, their use in the newborns with MAS should be carefully monitored. ACKNOWLEDGMENT

This work was supported by grants FaF UK/37/2013, VEGA 1/0981/12, APVV 0887-11, VEGA 1/0564/13.

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P-44 P-45 Failure of brain energy metabolism after cerebral Comparative study of newly synthesized copper hypoxia-ischaemia in immature rats: in vitro complexes on their anti-inflammatory and mitochondrial and in vivo NMR study antidiabetic activities in rats Katerina Plachaa, Ladislav Baciakb, Zuzana Sumbalovac, Lukas Dobias, Eva Racanska, Ingrid Tumova a,b IvoJuranek Department of Pharmacology and Toxicology, Faculty of aInstitute of Experimental Pharmacology and Toxicology, Slovak Pharmacy, Comenius University in Bratislava, Odbojarov 10, Academy of Sciences, Bratislava, Slovak Republic 832 32 Bratislava, Slovak Republic bIn-vivo NMR Laboratory, Faculty of Chemical and Food Technology, Slovak Technical University, Bratislava, Slovak Copper(II) chelates type of Schiff base with different Republic ligands were objects of considerable attention in many c Pharmacobiochemical Laboratory, Faculty of Medicine, fields of bioorganic chemistry. Significant antiradical, an- Comenius University in Bratislava, Bratislava, Slovak Republic ti-inflammatory, antipyretic as well as antidiabetic activities have been described previously. This work is focused Introduction. Perinatal asphyxia resulting from de- on the study of both anti-inflammatory and antidiabetic creased placental blood flow and/or gas exchange may activities of five mononuclear diaquabis(carboxylato)- lead to perinatal hypoxic-ischaemic (HI) encephalopathy. copper(II) complexes with different aminoacids which Brain injury after HI insult evolves over time, and differ- were projected in effort to alleviate generally high toxicity ent mechanisms are critical during the individual phases. of the cuprates. The anti-inflammatory activity of the com- Pathogenetic mechanisms of perinatal HI encephalopathy pounds was measured plethysmometrically by reduction are now just beginning to be understood. Mitochondrial of rat hind paw edema which was induced by a subplantar function is a critical factor that determines the mode injection of carrageenan. The cytoprotective activity of the of neuronal death. Many studies have been focused on studied compounds on pancreatic β-cells was evaluated investigating alterations of mitochondrial respiration, using modified in vivo oxidative stress model of alloxan- specifically oxidative phosphorylation (OXPHOS), trig- induced diabetes mellitus in rats. All i.p. administered gered by HI insult. Particularly, in main interest is role of compounds in dose of 50 μmol.kg-1 of body weight in mitochondria in apoptosis, failure of energy metabolism, different extent influenced both examinated pathologi- calcium signalling alteration, and reactive oxygen species cal conditions. The average antiedematous activity of generation during hypoxia and reoxygenation. Nowadays, the compounds ranged from 24.3 to 60.2% of the con- non-invasive, non-destructive and real-time-operating tech- trol values. The ability to decrease glucose level in acute niques, namely magnetic resonance imaging and spec- experimental conditions as result of their cytoprotective troscopy, are increasingly utilised in neurological and properties in range 32.2 to 55.1% have been also found. neonatology departments to confirm or refuse cerebral The highest effects in both evaluated parameters showed HI injury. compound IP-5, i.e. diaquabis(DL-β-aminobutyrate-N,O-) Methods. Seven-day-old Wistar rat pups of either sex cupper complex (Fig.1). The antiradical activity of the were subjected to the HII procedure according to Rice compounds is obviously responsive for their protective et al. (1981) with modifications. Brain samples were col- antiphlogistic and antidiabetic properties. However, fur- lected to assess OXPHOS by means of high-resolution ther studies for understanding mechanism of the cytopro- respirometry and non-invasive magnetic resonance mea- tective action of the original compounds are considered. surements. Conclusion. Better understanding of fundamental mechanisms of functional, biochemical and structural changes after HI insult may help in searching new strategies for effective neuroprotection in prophylactic and/or therapeutic intervention.

ACKNOWLEDGEMENT

This work was in part supported by grant VEGA 2/0149/12. Fig. 1. Relative anti-inflammatory and antidiabetic activities of the evaluated compounds.

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P-46 P-47 Involvement of microRNAs in isoproterenol- Efflux of lamivudine in mate1 induced cardiac damage and potential role in expressing MDCK cells diagnosis Josef Reznicek, Martina Ceckova, Marta Lima, Michal Radik, Gabriel Doka, Peter Krenek, Jan Kyselovic, Frantisek Staud Jan Klimas Department of Pharmacology and Toxicology, Faculty of Pharmacy in Hradec Kralove, Charles University in Prague, Department of Pharmacology and Toxicology, Faculty of Heyrovskeho 1203, 500 05 Hradec Kralove, Czech Republic Pharmacy, Comenius University in Bratislava, Odbojarov 10, 832 32 Bratislava, Slovak Republic Multidrug and toxin extrusion proteins (MATEs) and Introduction. Isoproterenol treated rats, a well known human organic cation transporters (OCTs) represent an model of myocardial damage caused by excessive beta- important determinant for the renal and hepatic excretion adrenergic stimulation is linked with dysfunction of con- of mainly cationic drugs. While OCT1 and 2 mediate drug tractile apparatus of the heart, due to expression shift of uptake from the blood to the hepatocytes and renal proxi- cardiac myosin heavy chain isoforms. Dysregulation may mal tubular cells, MATE1 ensures the efflux into bile and be associated with corrupted biogenesis of small endog- urine. Lamivudin, the nucleoside reverse transcriptase in- enous regulatory RNAs, known as microRNAs. hibitor, is one of the most widely used antiretroviral drugs Methods. 18-20 weeks old male Wistar rats were applied in the treatment of HIV-1 infection. Although it treated for 8 days with isoproterenol (ISO; 5 mg/kg; i.p.). is well known that the major route of lamivudine elimina- Using modified real-time PCR, we analyzed in samples tion is the excretion of unchanged drug into the urine, the from left ventricular free wall, skeletal muscle (m. soleus) precise mechanisms involved have not been described yet. and plasma the expression or level of muscle-specific mi- Here we studied interaction of lamivudine with MATE1 croRNAs: miR-1, miR-133a, miR-499 and heart-specific transporter using MDCK cells stably expressing human miR-208a. OCT1, OCT2 and MATE1 as well as double transfected Results. After 8-day beta-adrenergic stimulation the an- MDCK-OCT1/MATE1 and MDCK-OCT2/MATE1 cells imals developed a decrease of left ventricular expression grown in monolayers on cell culture inserts. of all evaluated microRNAs (miR-1, -133a, -208a, -499) of We observed significantly lower accumulation of la- 60% or more. Conversely, expression of these microRNAs mivudine in all cell lines expressing MATE1, while the in skeletal muscle didn't change in comparison with the basolateral to apical transport of lamivudine was greater control group. Similarly to skeletal muscle, plasma level of in the MATE1 expressing cells when compared to the muscle-specific microRNAs (miR-1, miR-133a, miR-499) parent and OCT monotransfected cells. Addition of didn't changed after isoproterenol. But the plasma amount mitoxantrone in a concentration selective for MATE1 of heart-specific miR-208a underwent a massive increase inhibition (2μM) lead to significantly increased cellular from virtually no presence to the level of more than 140- accumulation and reduced the apically directed transport fold higher after isoproterenol treatment. of lamivudine in the cells expressing MATE1. Conclusion. Highly elevated plasma level of cardiac- Our data show that MATE1 affects transcellular specific miR-208a could be a novel marker of heart dam- transport of lamivudine indicating that this antiretrovi- age caused, at least in case of beta-adrenergic stimulation. ral drug is a MATE1 substrate. Based on our results, we It also highlights the significant decrease of left ventricular hypothesize that MATE1 contributes to the excretion of microRNA levels of all evaluated microRNAs, miR-208a lamivudine in kidney. included. We can imply, that muscle-specific microRNAs (miR-1, -133a, -499) are not suitable as diagnostic markers presumably because their primary source is the 100 to ACKNOWLEDGEMENT 150 times more massive skeletal muscle compared to the weight of myocardium. This work was supported by the Grant Agency of the Charles University in Prague (GAUK 1148213/C/2013) and SVV/2013/267-003. ACKNOWLEDGEMENT

This work was supported by grants: FaF UK/59/2013, APVV-0887-11, VEGA 1/0981/12, VEGA 1/0564/13.

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P-48 P-49 Oral quercetin is not able to revert catecholamine Experimental study of potassium and calcium cardiotoxicity ion channels in human pregnant labouring Michal Rihaa, Marie Voprsalovaa, Vladimir Semeckyb, myometrium Magdalena Holeckovac, Jaroslava Vavrovac, Vladimir Vladimira Sadlonovaa, Martina Sutovskaa, Marta Joskovaa, Palickac, Radomir Hrdinaa , Premysl Mladenkaa Karol Dokusb, Jozef Visnovskyb, Marian Adamkovc, a aDepartment of Pharmacology and Toxicology, Faculty of Sona Franova Pharmacy in Hradec Kralove, Charles University in Prague, aDepartment of Pharmacology, Jessenius Faculty of Medicine in Heyrovskeho 1203, 500 05 Hradec Kralove, Czech Republic Martin, Comenius University in Bratislava, Sklabinska 26, 036 01 bDepartment of Biological and Medical Sciences, Faculty of Martin, Slovak Republic Pharmacy in Hradec Kralove, Charles University in Prague, bClinic of Obstetrics and Gynecology, Jessenius Faculty of Heyrovskeho 1203, 500 05 Hradec Kralove, Czech Republic Medicine in Martin, Comenius University Bratislava and Martin bFaculty of Medicine in Hradec Kralove, Simkova 870, 500 38 University Hospital, Kollarova 2, 036 01 Martin, Slovak Republic Hradec Kralove and University Hospital Hradec Kralove, Sokolska cDepartment of Histology and Embryology, Jessenius Faculty of 581, 500 05 Hradec Kralove, Czech Republic Medicine in Martin, Comenius University in Bratislava, Mala Hora 4, 036 01 Martin, Slovak Republic Catecholamines represent essential endogenous substances involved in homeostasis of various organ systems, Myometrium of uterus is characterized by the pres- with particular importance in the cardiovascular system. ence of various ion channels that activity can be modu- A synthetic catecholamine isoprenaline (ISO), with non- lated pharmacologically using the specific agonists or selective β-agonistic activity, has been used in high doses antagonists. The experimental study was focused on po- for induction of a state similar to acute myocardial infarc- tassium KATP and BKCa2+ and calcium CRAC ion channels tion in humans. Despite the complex mechanism of ISO in human pregnant labouring myometrium. cardiotoxicity, some natural compounds have been shown Samples of myometrium were taken from term preg- to protect myocardium in this model. nant women in which the pregnancy had to be terminated This study was aimed at the detailed elucidation of by Caesarean section. Subsequently the samples were the impact of oral pretreatment by flavonole quercetin processed in myometrial strips and placed in organ bath on acute cardiac ISO toxicity. with Krebs-Henseleit solution. Myometrial contraction 23 Wistar:Han male rats of approximate weight 375 g activity was evoked by the application of oxytocin. The were divided into four groups. Gastric gavage was used amplitude of myometrial contractions was assessed after -1 for oral premedication by quercetin (10 mg.kg ) or sol- administration of agonist and antagonist KATP potassium vent daily for 7 days, followed by s.c. administration of ion channels - pinacidil and glybenclamide; agonist and an- -1 ISO (100 mg.kg ) or solvent. Haemodynamic, ECG and tagonist BKCa2+ potassium ion channels - NS1619 and tet- biochemical parameters were analysed, including exami- raethylammonium; and CRAC ion channels antagonist nation of myocardial histology and blood vessels respon- 3-fluropyridine-4-carboxylic acid. siveness. Pinacidil significantly decreased the contractile activity Quercetin pretreatment was not able to prevent ma- of myometrium and its effect was significantly antago- jority of pathophysiological consequences caused by nized by glybenclamide. In contrast, NS1619 and its an- ISO (e.g., stroke volume decrease, cardiac troponin T tagonist tetraethylammonium did not affect significantly release, QRS-T junction elevation, histological impair- the contractile activity of myometrium. 3-fluropyridine- ment), except for left ventricular end-systolic pressure 4-carboxylic acid did not have significant effect on the which was normalized by quercetin in ISO treated ani- amplitude of myometrial contractions, too. mals. Interestingly, quercetin decreased responsiveness of The function of KATP and BKCa2+ potassium ion chan- isolated aorta on vasoconstrictor stimulus in the control nels in human term pregnant myometrium in labour is animals. probably different. Down-regulation of the BKCa2+ potassium ion channels plays probably more important role

to increase uterine contractility than KATP potassium ion ACKNOWLEDGEMENT channels. Low expression of calcium CRAC ion channels did not affect the contractile activity of myometrium in This study was supported by grant of Czech Science experimental conditions. Foundation (project No. P303/12/G163) and by Charles ESF - Zvýšenie možností kariérneho rastu vo výskume University (grant No. 605712C and project SVV 267 003). a vývoji v oblasti lekárskych vied, VEGA 1/0062/11, VEGA 1/0127/13.

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P-50 P-51 Reported adverse drug reactions and off-label Comparison of atomoxetine use in patients with drug use in paediatric population ADHD hospitalized in Clinic of Psychiatry JMF CU in Jana Slaznevaa, Ruzena Kamenskab, Magdalena Kuzelovaa Martin in 2008 and 2012 a,b b a aDepartment of Pharmacology and Toxicology, Faculty of Eva Snircova , Igor Ondrejka Gabriela Nosalova Pharmacy, Comenius University in Bratislava, Kalinciakova 8, aDepartment of Pharmacology, Jessenius Faculty of Medicine in 83232 Bratislava, Slovak Republic Martin, Comenius University in Bratislava, Sklabinska 26, 037 53 bSection of Drug Safety and Clinical Trials, State Institute for Drug Martin , Slovak Republic Control, Kvetna 11, 82508 Bratislava, Slovak Republic bClinic of Psychiatry, Jessenius Faculty of Medicine in Martin, Comenius University in Bratislava and University Hospital Martin, Kolarova 2, 036 59 Martin, Slovak Republic Off-label use of medicinal products in children is common, as few drugs are licensed for use in paediatric population. Concerns rise mainly due to the lack of The current study investigated atomoxetine use and knowledge about safety and effectiveness of drugs used its efficacy in treating ADHD in children and adolescent off-label. Several studies have documented the association patients hospitalized in Clinic of Psychiatry JMF CU between adverse drug reactions (ADRs) and off-label drug Martin in 2008 and 2012. We aimed also in use of other use in children. Serious ADRs from off-label prescribing most prevalent therapeutic strategies, and most preva- of medicinal products appeared in following therapeutic lent comorbid disorders accompanying ADHD. Efficacy groups: allergens, dermatologicals and sex hormones. No of therapy was evaluated by Clinical Global Impression study investigating the extent and characteristics of off- Scale (CGI), Severity (S) and Improvement (I) subscale. label prescribing in the context of ADRs in the Slovak Study aimed also in gender and age differences in groups Republic among children has been conducted yet. of patients. A retrospective, cross-sectional analysis of spontane- This retrospective, observational, comparative study ous ADRs reported to State Institute for Drug Control in evaluate use of atomoxetine (1,2 mg/kg/day) in hospital- the Slovak Republic during 2006-2011 was performed. We ized patients after 5 years period in 2008 and 2012. Data included reports concerning drugs prescribed for patients were obtained from medical records patients, aged 6-17 younger than 18 years. Each ADR was classified accord- years. The study included 389 patients, 192 patients in ing to seriousness and type of reaction. Off-label drug use 2008 (77 with ADHD) and 197 patients in 2012 (76 with was defined as prescription outside the licensed age group ADHD). Statistical methods used in this study were com- specified in Summary of Product Characteristics. We parative and correlation analysis. excluded ADRs related to vaccines, intravenous replace- Use of atomoxetine was significantly higher in 2008, ment solutions, blood products, dietary supplements. but still was a most prevalent treatment prescribed in pa- Out of 1690 ADRs for children reported in the na- tients with ADHD in both years (59.7% vs 35.3%), second tional database, 175 were analysed according to our inclu- one was levomepromazin (9.1 vs 25.0), third one chlor- sion criteria. We identified 14% serious, 70% non-serious prothixen (11.7 vs 15.8) and fourth one methylphenidate ADRs and one fatal case. Most commonly suspected ther- (0.0 vs 26.3). The prevalence of ADHD was higher in apeutic group as a cause of ADRs were anti-infectives for boys than in girls. In 2012 age of patients was significantly systemic use (67%) which were also associated with the higher. Positive correlation was investigated between ini- largest number of serious adverse drug reactions. 6% of tial Severity and global Improvement measured by CGI-S reported ADRs were associated with off-label use. Serious and CGI-I. The most frequent disorders accompanying ADRs occurred in two cases when off-label drugs were ADHD were conduct disorders, emotional disorders and given. other specified behavioural and emotional disorders with onset usually occurring in childhood and adolescence.

ACKNOWLEDGEMENT ACKNOWLEDGMENT This study was supported by grant FaF UK/8/2013. This work was supported by the UK Grant UK/27/2013.

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P-52 P-53 Effect of social stress on the reactivity of the aorta Expression of SDF-1/CXCR4 axis and stem cells of young female rats with different predisposition markers in anthracycline-induced heart failure in to hypertension the rat Ruzena Sotnikovaa, Angelika Puzserovab, Peter Slezakb, Jasna Srankova, Lenka Pivackova, Gabriel Doka, Kristina Peter Balisb, Miroslava Majzunovab, Ima Dovinovab, Iveta Galkova, Eva Malikova, Lucia Mesarosova, Bernatovab Jan Klimas, Jan Kyselovic, Peter Krenek aInstitute of Experimental Pharmacology and Toxicology, Slovak Department of Pharmacology and Toxicology, Faculty of Academy of Sciences, Dubravksa c. 9, 841 04 Bratislava, Slovak Pharmacy, Comenius University in Bratislava, Odbojarov 32, Republic 832 32 Bratislava, Slovak Republic bInstitute of Normal and Pathological Physiology, Slovak Academy of Sciences, Sienkiewiczova 1, 81371 Bratislava, Slovak Republic Introduction. Antracyclines are effective antitumor drugs, but their clinical use is limited by dose-dependent Genetic hypertension and social stress are supposed to cardiotoxicity. Stromal cell-derived factor-1α (SDF-1α) be associated with changes in bioavailability of NO, which and its receptor CXCR4 have a crucial role in the mo- can be manifested by alterations in vascular reactivity. The bilization and homing of the stem cells to ischaemic aim of the work was to study the influence of crowding myocardium, with beneficial effect to myocardial repair. stress on endothelial function of the rat aorta of young SDF-1 can be cleaved and downregulated by dipeptidyl normotensive Wistar-Kyoto rats (WKY) and spontaneous- peptidase-4 (DPP4). ly hypertensive rats (SHR). Five-week-old females of SHR Aim. To determine the expression of the stem cells and WKY rats were exposed to crowding for 2 weeks. markers and genes related to SDF-1/CXCR4 axis that Crowding was induced by reduction of living space from could participate in the regeneration of damaged heart 200 cm2/100 g of body weight (controls) to 70 cm2/100 g after anthracycline administration. of body weight. Endothelium-dependent (acetylcholine- Materials and Methods. Daunorubicin was adminis- induced) relaxation and noradrenaline-induced contrac- tered to male Wistar rats (15mg/kg, i.v., DAU). Control tion of aortic rings were studied in vitro under isometric animals received vehicle. After 8 weeks, animals were sac- conditions. Blood pressure (BP) was measured using rificed. Expression of stem cell factor (SCF), SDF-1α, tail-cuff plethysmography. In the aorta, the NO synthase CXCR4, DPP4, growth factors and markers of cardiac (NOS) activity was measured by conversion of [3H]-L- progenitor (c-kit and MDR1), endothelial progenitors arginine to [3H]-L-citruline and superoxide production by (CD34 and CD133) and mesenchymal (CD44 and the chemiluminiscence method. In control rats, SHRs had CD105) stem cells at mRNA level was determined by significantly higher BP and increased NO and superoxide qRT-PCR in samples of left ventricles. production in the aorta compared to WKY rats. However, Results. We found decreased expressions of CXCR4 no changes in aortic function were observed. Crowding and DPP4 in hearts of DAU group, SDF-1α mRNA levels stress failed to affect BP and vascular function either in were unchanged. DAU group had also decreased expres- SHR or WKY, it however increased significantly aortic sion of vascular endothelial growth factor (VEGF) and NO production in both phenotypes. Superoxide produc- markers of endothelial progenitors and mesenchymal stem tion was increased by stress only in the aorta of WKY. cells. CD44 expression was not altered. We also observed In summary, crowding stress induced changes in NOS a decreased expression of cardiac progenitor cells marker activity and superoxide production in the aortas of yo- c-kit and its ligand SCF, whereas the expression of MDR1, ung female WKY and SHR rats, which however were not another cardiac progenitor cells marker, was increased. sufficient for manifestation of endothelial dysfunction. Conclusion. Daunorubicin did not change the expression of SDF-1α, but it decreased expression of SCF, another chemoattractive factor. This could reduce migration ACKNOWLEDGMENT of stem cells. The observed decreased expression of stem cell markers could reflect their defective migration and This study was supported by the grants Nos. APVV- impaired and decreased regeneration ability of the injured 0523-10, VEGA 2/0084/10 and 2/0086/08. heart. Decreased expression of VEGF in the failing hearts could indicate a defective angiogenesis.

ACKNOWLEDGEMENTS

This work was supported by grants UK/396/2013, VEGA 1/0981/2012 and APVV 0887-11.

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P-54 P-55 Modified cellulose materials in the acute wound Teaching clinical pharmacology in baccalaureate healing nursing programs in the Czech Republic Pavel Suchy, Marta Chalupova, Gabriela Prazanova, Pavlina Strbova a, Zdenka Miksovab, Lenka Mazalovab, Alzbeta Kruzicova, Peter Kollar, Tomas Parak Karel Urbaneka

Department of Human Pharmacology and Toxicology, Faculty of aDepartment of Pharmacology, Faculty of Medicine and Pharmacy, University of Veterinary and Pharmaceutical Sciences, Dentistry, Palacky University, Olomouc, Hnevotinska 3, 775 15 Brno, Palackeho 1–3, 612 42 Brno, Czech Republic Olomouc, Czech Republic bDepartment of Nursing, Faculty of Health Sciences, Palacky University Olomouc, Hnevotinska 3, 775 15 Olomouc, Czech Wound healing is a dynamic and highly integrated Republic process that represents important ability of organism to repair tissue damage in order to restore the proper function. Wound care procedures, mainly dressing techniques, Introduction. Clinical pharmacology is an important have a great impact on the outcome of wound healing to subject of study in university education of general nurses. achieve fast and antiseptic wound closure and improve Courses for nursing students should be more clinical than regeneration of the impaired tissue. those for medical students and should provide informa- Cellulose is a natural polysaccharide that is used in tion on problems encountered by nurses during drug ad- medicine in many indications for its haemostatic and ministration. The aim of this study was to analyze and potential bacteriostatic properties. The effectiveness of compare the pharmacology courses in baccalaureate nurs- newly synthesized modified cellulose derivatives based ing programs at Czech universities with emphasis on the on carboxymethyl cellulose was examined, using a skin specific needs of nursing practice. wound model in rats, with a focus on the rate of healing Methods. Pharmacology training courses of all Czech (wound diameter), tissue reaction and determination of universities providing graduate education for general the cytokines involved in the wound healing process. nurses were analyzed. Both qualitative and quantitative A total of 60 Wistar laboratory rats were divided methods of document analysis were used. The online into 4 groups of 15 animals. Under total anesthesia, skin curricula and course syllabuses were obtained from open circle excision (1.5 cm in diameter) through all the cuta- sources. Moreover, assessment of didactic effectiveness neous layers in the suprascapular region was performed. of recommended pharmacology textbooks for graduate Carboxymethyl cellulose (HHT), carboxymethyl cellulose nursing courses was performed. with iodine (HTB), and as the reference substance car- Results. Teaching Pharmacology in the graduate pro- boxymethyl cellulose with silver (AAG) were used; con- gram of nursing takes place in a total of 14 universities trol group was left without primary dressing. The tested in the Czech Republic. The most common length of the material was applied on the wound, thereafter fixed by course was between 20 – 29 hours (in 42.86% of curricu- stapler. Third of the animals were destroyed after 2, 7 la). Large differences were also found in the content of the and 14 days from the experiment start date. Before the subject. Nearly hundred-percent representation reached euthanasia, blood was obtained by cardiac puncture for the fundamental issues of pharmacology, such as general biochemical examination. Wound and its surroundings principles of pharmacokinetics (92.86%) and pharmaco- were macroscopically observed, skin samples were taken dynamics (78.57%) and special pharmacology selected and subjected to pathohistological and molecular biologi- chapters: treatment of bacterial infections (85.71%), cal examination. Total destruction score as a degree of drugs affecting cardiovascular system (92.86%), anal- inflammation in epidermis, dermis and hypodermis was gesics (78.57%) and drugs affecting autonomic nervous histologically determined. The cytokines TNF-α, TGF-β1 system (71.43%). In contrast, chapters from the clinical and VEGF were detected by the western blot. pharmacology were represented only in minute quanti- Based on this pre-clinical in vivo experiment, HHT ties. This was mainly pharmacotherapy of pain (28.57%) and HTB derivatives showed better characteristics in and pharmacotherapy in the elderly (21.43%), children comparison to conventionally used AAG. They reduced (14.29%) and pregnant and lactating women (21.43%). inflammatory response in the skin with decreased level of The didactic effectiveness of the most frequently used proinflammatory cytokines, and support angiogenesis as Czech textbook, Pharmacology for Students of Health well as the consequent regeneration of the impaired tissue. Sciences, reached 61.11%. Conclusion. Significant differences between pharmacology courses for nurses were found in the length ACKNOWLEDGMENT of courses, their position in the curriculum, course contents and forms of examination. Didactic effectiveness of The study was supported by the grant TAČR used textbooks is insufficient in comparison with those TA01010244. of English-speaking countries. In particular, insufficient attention is paid to specific nursing problems of drug use.

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ACKNOWLEDGEMENT P-57 Supported by Grant IGA UPOL LF 2013_007. Effect of sulforaphane on enzyme activities of human liver microsomal cytochromes P450 P-56 Alena Vanduchovaa, Michaela Kopecna Zapletalovaa, Antibacterial effect of silver nanoparticles Alena Spicakovaa, Eva Anzenbacherovab, in experimental skin infection Pavel Anzenbachera Karel Urbanek, Petra Langerova, Hana Suchankova, aDepartment of Pharmacology, Faculty of Medicine and Dentistry, Palacky University Olomouc, Hnevotinska 3, 775 15 Pavlina Strbova Olomouc, Czech Republic bDepartment of Medical Chemistry and Biochemistry, Faculty Department of Pharmacology, Faculty of Medicine and Dentistry, of Medicine and Dentistry, Palacky University Olomouc, Palacky University Olomouc, Hnevotinska 3, 775 15 Olomouc, Hnevotinska 3, 775 15 Olomouc, Czech Republic Czech Republic

Introduction. Antibacterial activity of silver nanopar- Sulforaphane (SFR) is a phytochemical, which be- ticles is well known. At present, the investigation of this longs to the family of isothiocyanates and is formed from phenomenon is intensifying because of the increase of glucoraphanin (glucosinolate in broccoli) by the action bacterial resistance to antibiotics, caused by their overuse. of myrosinase. The largest amount of SFR occurs espe- The aim of this study was to evaluate antibacterial activity cially in broccoli, broccoli sprouts, but it has been also of liquid and semisolid formulation of silver nanoparticles found in other cruciferous vegetables such as cabbage, on the model of an experimental rat skin infection. cauliflower and kale. SFR, biologically active substance of Methods. Male specific pathogen free Wistar rats broccoli, may protect against various types of cancer, dia- were used and during experiments housed in individually betes or cardiovascular disease. SFR is a possible inhibitor ventilated boxes. Full-thickness skin wounds were made of some major cytochrome P450 enzymes (CYP), which by round scalpel under general anesthesia. Wounds were are taking part in a variety of important reactions of drug inoculated with S. pyogenes strains, obtained from 24-hour metabolism in humans. For this reason, the influence of culture inoculated to 20 ml of meat-pepton suspension SFR on activity or inhibition of some selected human he- and incubated for 24 h at 37 ºC. Topical treatment was ap- patic microsomal CYPs (1A2, 2A6, 2B6, 2C9, 2C8, 2D6, plied once daily for a further 6 days. Animals were divided 2E1 and 3A4) was studied. Our experiments confirmed into 3 groups (n=5), treated by Ag-nanoparticles solution, recent data on inhibition CYP2E1 enzyme activity (chlo- erythromycin solution or placebo or Ag-nanoparticles rzoxazone 6-hydroxylation) by SFR. Significant decrease ointment, neomycin+bacitracin ointment and placebo, was also observed in activities of CYP3A4 (testosterone respectively. Macroscopic healing assessment and smears 6β-hydroxylation) and of CYP2D6 (bufuralol 1’-hydroxyl- for bacterial counts were performed at days 2, 4 and 7 ation). Mechanisms of inhibition remain to be elucidated of the experiment. S. pyogenes strains were identified by by further studies Pastorex Strep set. Results. At the day 7 of the experiments, silver nanoparticles in both liquid and ointment formulation P-58 significantly decreased bacterial counts in comparison Novel analogues of dexrazoxane and ADR-925 for with placebo. Differences in macroscopic wound healing between treated and placebo groups were not significant. protection against anthracycline cardiotoxicity Conclusion. Silver nanoparticles both in solution and Anna Vavrovaa, Jaroslav Roha, Olga Lencova-Popelovab, in ointment show antibacterial activity against experimen- Eduard Jirkovskyb, Katerina Hruskovaa, Eliska Mackovaa, tal Streptoccocci skin wound infection. On the contrary, no a a a effect on the velocity of wound healing was found. Hana Jansova , Pavlína Haskova , Pavla Martinkova , Tomas Eisnera, Marek Kratochvila, Jan Susa, Lucie Tichotovaa, Vladimir Gerslb, Katerina Vavrovaa, Martin Sterbab, ACKNOWLEDGMENT Tomas Simuneka This work was supported by Czech Science aFaculty of Pharmacy in Hradec Kralove, Charles University Foundation project GAP304/10/1316. in Prague, Heyrovskeho 1203, 500 05 Hradec Kralove, Czech Republic bFaculty of Medicine in Hradec Kralove, Charles University in Prague, Simkova 870, 500 38 Hradec Kralove, Czech Republic

Cardiotoxicity as a serious late side-effect compro- mises the clinical usefulness of anthracyclines (ANT).

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Dexrazoxane (DEX) is the only clinically established Expression of cytochrome P450 2C6 was measured in cardioprotective agent, but its use is very limited. liver using real-time PCR (at mRNA level) and Western Traditionally, iron-mediated oxidative stress is believed blotting (at protein level). Formation of diclofenac me- to be the main cause of ANT cardiotoxicity, and DEX- tabolites (typical marker substrate of CYP2C6 enzyme induced cardioprotection is attributed to its hydrolysis activity) was analyzed using HPLC with UV detection. product ADR-925 that can chelate free iron, and thus Silybin in hereditary hypertriglyceridemic rats on prevent cardiac oxidative injury. However, parent struc- HCD diet significantly increased activity of CYP2C6 and ture of DEX is also a catalytic inhibitor of topoisomerase its expression on mRNA level. Expression of CYP2C6 II (TOP2). In this study, novel analogues of DEX (MK- on protein level was affected by silybin consumption in- 15, ES-5) and ADR-925 (KH-TA4, JR-159) were synthe- significantly. Our results suggested that CYP2C6 is up- sized, and their protective activities against daunorubicin regulated by silybin in hereditary hypertriglyceridemic (DAU) cardiotoxicity were examined both in vitro in vivo. rats on high cholesterol diet. In contrast to DEX, none of the novel agents significantly Because cytochrome P450 2C6 is considered to be protected against the DAU cardiotoxicity, and they also a counterpart of human CYP2C9, the results obtained displayed no significant antiproliferative properties. On open the possibility that in humans silybin may affect the other hand, KH-TA4 protected cardiomycytes from the metabolism of drugs metabolized by this cytochrome model oxidative damage, whereas DEX was ineffective. P450. Further studies are needed to elucidate the effects The binding of iron or its mobiliziation from cardiomyo- of silybin on CYP2C9 in humans suffering from meta- cytes not differed from DEX, but unlike DEX the ana- bolic syndrome. logs lacked TOP2 inhibition properties. In conclusion, our data indicate that rather than by antioxidative properties, the interaction with TOP2 may be involved in both ACKNOWLEDGMENT in anthracycline cardiotoxicity as well as it may be indis- pensable for effective pharmacological cardioprotection. This work was supported by Czech Science Foundation project 13-10813S.

ACKNOWLEDGMENT P-60 This publication is co-financed by the European Study of renal clathrin-independent endocytosis Social Fund and the state budget of the Czech Republic. Project No. CZ.1.07/2.3.00/30.0061 and Czech Science of radiolabeled receptor-specific bombesin Foundation (13-15008S). analogues Marie Volkova, Jana Mandikova, Alice Laznickova, P-59 Milan Laznicek, Frantisek Trejtnar

Silybin affects the liver microsomal CYP2C6 Department of Pharmacology and Toxicology, Faculty of in HHTG rats Pharmacy in Hradec Kralove, Charles University in Prague, Heyrovskeho 1203, 500 05 Hradec Kralove, Czech Republic Rostislav Veceraa, Ludmila Kazdovab, Alice Zacharovaa, Zuzana Matuskovaa, Nina Skottovaa, Olena Oliyarnykb, a Bombesin (BN) is a neuropeptide with high affinity Pavel Anzenbacher for the gastrin-releasing peptide (GRP) receptor, which aDepartment of Pharmacology, Faculty of Medicine and is overexpressed by a variety of cancers and provides an Dentistry, Palacky University Olomouc, Hnevotinska 3, 775 15 attractive target for BN/GRP receptor scintigraphy and Olomouc, Czech Republic radionuclide therapy. However, renal accumulation of bDepartment of Metabolism and Diabetes, Division of radiopeptides resulting in toxicological injury may limit Experimental Medicine, Institute for clinical and Experimental potential clinical use of the tested compounds. Renal en- Medicine, Videnska 1958/9, 140 21 Prague, Czech Republic docytosis of the bombesin analogues has usually been studied only as a receptor mediated process. But we tried This study was aimed at investigate of the effect of to investigate the role of macropinocytosis, one of clath- silybin on expression and activity of rat CYP2C6. This rin-independent endocytosis in the renal uptake. cytochrome P450 is considered to be a counterpart of hu- As a fluid-phase endocytosis probe, FITC-labeled dex- man CYP2C9, which metabolizes commonly prescribed tran (FD) was used. We measured the fluorescence of drugs, such as ibuprofen, diclofenac or warfarin. macropinocytosed FD at various concentrations using Male hereditary hypertriglyceridemic rats (accepted LLC-PK1 cells and then determined the influence of es- model of metabolic syndrome) were fed: 1) standard calating concentrations of bombesin on 0.5 mg/mL FD laboratory diet (STD), 2) high cholesterol diet (HCD = macropinocytosis. 5 μM rottlerin was used as a selective STD + 1% of cholesterol w/w + 10% of lard fat w/w), 3) inhibitor of macropinocytosis. high cholesterol diet with silybin (0.5% w/w) for 21 days.

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Bombesin increased the FD endocytosis in LLC-PK anxiolytic effect of flumazenil was observed. The study cells up to a concentration of 0.5 mg/ml. At a concen- was placebo controlled and three different doses of flu- tration of 1 mg/mL the FD endocytosis was declined. mazenil based on literature data were used. While saline Rottlerin which is selective, rapid-acting and irreversible had no influence on the duration and length of recorded inhibitor of fluid phase endocytosis, caused significant amplitudes, flumazenil significantly and dose-dependently lower uptake of 177Lu labeled bombesin analogues com- decreased the total number of vocalizations. pared to the cells treated with bombesines only. The in- There are plenty of preclinical or clinical data showing hibition rate was more than 80% for 177Lu-PCTA-[Lys3] reversal of sedation by flumazenil induced by benzodiaz- bombesin, 45.8% for 177Lu-DOTA-[Pro1,Tyr4]bombesin epines. On the contrary, there are little data showing the and 38.5% for 177Lu-DOTA-[Lys3]bombesin. effect of flumazenil or other antagonists itself. These stud- Based on our results the vesicular uptake is associated ies seem to be in contrary with the effect of flumazenil in with macropinocytosis of the bombesins, but its contribu- humans, where it is believed to possess mainly anxiogenic tion differs according to the used chelating component effect. It seems that in the individuals, who exhibit anxio- or substitution in bombesin molecule. Bombesins are un- genic behavior or in individuals with anticipation anxiety, der this experimental conditions internalized into large flumazenil acts as an anxiolytic agent, while in individuals macropinosomes. We can conclude, that fluid phase en- without any signs of anxiety, flumazenil can really act as docytosis, which is usually called as clathrin-independent anxiogenic agent. endocytosis may play an important role in the accumulation of radiolabeled bombesin analogues in the renal tubular cells. ACKNOWLEDGEMENT

This work was supported by the grant IGA MZ ACKNOWLEDGMENT NS/10503-3 from the Czech Ministry of Health.

The study was supported by Charles University in Prague (Project SVV 267003) and grant GAUK No. P-62 376411/FaF/C-LEK. Boldine enhances bile production in rats Marie Zagorovaa, Zuzana Kadovad, Milos Hrocha, P-61 Jolana Cermanovaa, Jitka Hajkovaa, Pavel Tomsikb, Sedative and anxiolytic properties of flumazenil Tomas Lahoa, Zdenka Kudlackovae, Peter Nachtigale, d d d Martin Votavaa, Jiri Slivaa,b, Ladislav Hessc, Jiri Malekd Petr Pavek , Martina Ceckova , Frantisek Staud , Jaroslav Mokryc, Stanislav Micudaa aDepartment of Pharmacology, 2nd Faculty of Medicine, Charles University in Prague, V Uvalu 84, Prague 5, 150 06 Czech Republic aDepartment of Pharmacology, Faculty of Medicine in Hradec bDepartment of Pharmacology, 3rd Faculty of Medicine, Charles Kralove, Charles University in Prague, Simkova 870, 500 38 Hradec University in Prague, Ruska 87, Prague 10, 10034 Czech Republic Kralove, Czech Republic cInstitute for Clinical and Experimental Medicine, Laboratory of bDepartment of Medical Biochemistry, Faculty of Medicine in Experimental Anaesthesiology, Prague, Czech Republic Hradec Kralove, Charles University in Prague, Simkova 870, 500 38 dDepartment of Anaesthesiology and Critical Care Medicine, Hradec Kralove, Czech Republic 3rd Faculty of Medicine, Charles University in Prague, Ruska 87, cDepartment of Histology and Embryology, Faculty of Medicine in Prague 10, 10034 Czech Republic Hradec Kralove, Charles University in Prague, Simkova 870, 500 38 Hradec Kralove, Czech Republic dDepartment of Pharmacology and Toxicology, Faculty of Flumazenil acts as a benzodiazepine receptor antago- Pharmacy in Hradec Kralove, Charles University in Prague, nist or partial agonist. It competitively inhibits the activ- Heyrovskeho 1203/8, 500 02 Hradec Kralove, Czech Republic ity at the benzodiazepine recognition site on the GABA/ eDepartment of Biological and Medical Sciences, Faculty of benzodiazepine receptor complex, thereby reversing the Pharmacy in Hradec Kralove, Charles University in Prague, effects of benzodiazepines. Heyrovskeho 1203/8, 500 02 Hradec Kralove, Czech Republic Our experiments in rabbits showed that administration of flumazenil (0.1 mg/kg) is associated with sedative and Boldine is the major alkaloid from Chilean boldo tree, anxiogenic effect, which was, in some parameters similar and is used in traditional medicine to support bile produc- to that of midazolam (0.5 mg/kg). In those rabbit, the tion, but the scientific data for this effect are lacking. We loss of righting reflex was achieved approximately after 3 therefore analyzed choleretic potential of the compound minutes irrespective of the administration of midazolam including its possible molecular background. The immedi- of flumazenil. On the other hand, midazolam caused ate and long-term effect of boldine was evaluated in rats more profound decrease of blood pressure and pulse rate. either during 2h i.v. infusion or after 28-day oral pretreat- These results were confirmed by ultrasonic vocalization ment. Constant infusion of boldine instantly increased the test of anxiety in rats. The rats were administered with bile flow 1.4-fold and maintained it stably higher through- various doses of flumazenil (0.1, 1 and 10 mg/kg) and an out the administration. This effect was not associated with

S46 Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2013 Sep; 157 (Supplement 1):S1–S88. the corresponding increase in bile acid or glutathione bili- in rat peritoneal cells (2 x 106/mL). Nitrite and cytokines ary excretion, which indicates that acute choleretic effect levels were determined at the 24-h interval of culture, us- of boldine is not related to the stimulation of either bile ing Griess reagent and ELISA, respectively. acid dependent or independent mechanism of bile forma- It was found that all MWCF possessed the NO- and tion, and it points to osmotic activity of compound itself. cytokine-stimulatory activity, although less expressed Therefore, we analyzed also the concentration of boldine than was the activity of original lysate. The MWCF of in bile and performed the bile clearance study after bo- Gram-positive L. casei were more effective than MWCF lus dose administration of the agent. Indeed, we found of Gram-negative E. coli. The most of the NO-stimulatory a strong relationship between the elevation of bile produc- potential was found to be attributable to the molecules tion and the actual bile excretion of boldine. Nevertheless, of ≤ 3 kDa. The L. casei MWCF-3 kDa accounted for ap- after long term pretreatment, when the bile collection proximately 80% of the NO-stimulatory effect of MWCF- study was performed 24 h after the last administration of 100 kDa. boldine, the production of bile was accelerated despite undetectable levels of the compound in bile or plasma, and without influence on biliary glutathione and bile acid ACKNOWLEDGMENT excretion. In conclusion, our study demonstrated immediate choleretic activity of boldine, which depended on The research was supported by the grant CZ:GA actual concentration of the agent in the bile. Mechanism ČR:303/12/0535 from the Grant Agency of the Czech of increased bile production during long-term administra- Republic. tion of boldine requires further analysis. P-64 ACKNOWLEDGMENT Immunosuppressive pyrimidines:

We acknowledge grant support form the Ministry structure-activity study of Health and Charles University No. IGA NT/13473- Zdenek Zideka, Eva Kmonickovaa, Zlatko Janebab, 3/2012, SVV-2013-266901, SVV/2013/267-003, and Viktor Kolmanb, Petr Jansab PrvoUK P37/05. aDepartment of Pharmacology, Institute of Experimental Medicine, Academy of Sciences, Videnska 1083, 142 20 Prague, P-63 Czech Republic Activation of NO and cytokines by low-molecular bInstitute of Organic Chemistry and Biochemistry, Academy of Sciences, Flemingovo nam. 2, 166 10 Prague 6, Czech Republic weight fractions of lysates from G+ and G– bacteria We synthesized novel series of pyrimidine derivatives

a a a and analysed their immunobiological properties. Effects Eva Kmonickova , Adela Dusilova , Petra Kostecka , of compounds on immune-activated production of nitric Miloslav Kverkab, Zdenek Zideka oxide (NO), prostaglandin E2 (PGE2) and cytokines (interleukin-1, interleukin-6, tumour necrosis factor-α) were aDepartment of Pharmacology, Institute of Experimental Medicine, Academy of Sciences, Videnska 1083, 142 20 Prague, screened under in vitro conditions using mouse peritoneal Czech Republic cells. NO production was activated by lipopolysaccharide bDepartment of Immunology, Institute of Microbiology, Academy (LPS) plus interferon-γ (100 pg/mL/500 pg/mL, respec- of Sciences, Videnska 1083, 142 20 Prague, Czech Republic tively), PGE2 by LPS (10 ng/mL), and cytokines by LPS (5 μg/mL). Pyrimidine derivatives were applied as 50 Immunostimulatory effects of bacterial lysates and μM aqueous solutions, concomitantly with the triggering their fractions differing in the content of molecules of stimuli. The effects were evaluated 24 h afterwards. specified molecular weight (MW) were investigated. The The compounds exhibited inhibitory mode of action lysates were prepared by passing the probiotic bacteria which was strongly dependent on their structure. While Lactobacillus casei DN-114001 and Escherichia coli strain 2-amino-4,6-dihydroxypyrimidines were ineffective as NO Nissle 1917 through the French press followed by lyophi- inhibitors, 2-amino-4,6-dichloropyrimidines suppressed lisation. The lysate fractions differing in the content of NO production by 50%. The highest inhibitory effects molecules of specified MW range were prepared using the were displayed by 4,6-dichloro-2-[(N,N-dimethylamino) MW cutoff microfiltration (centrifugal devices Amicon®, methyleneamino]pyrimidines and 4,6-dichloro-2-formami- Millipore). Four classes of MW cutoff fractions (MWCF) dopyrimidines which inhibited production of NO by ap- were obtained. They contained molecules ≤ 100 kDa, ≤ proximately 95%. The 5-position substituents remained 50 kDa, ≤ 10 kDa, or ≤ 3 kDa. without any significant impact on NO production. The

The effects of original lysates and MWCF on the NO-inhibitory IC50s of most of them were ≤ 5 μM. in vitro production of nitric oxide (NO) and cytokines The compounds were devoid of cytotoxicity. interleukin-1β and tumour necrosis factor-α were analysed

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Besides NO, the pyrimidine derivatives also sup- Results. A model explaining the variability of ND was pressed, though less effectively, production of PGE2 and constructed as follows: cytokines. The mechanism of the action remains to be Normalised dose of tacrolimus = 7.09e^(-3)-9.98e^ established. (-4)×MPA+2.13e^(-4)×Tx-5.92e^(-4)×ATB-2.56e^ (-3)×INTER+1.52e^(-4)×DOSE-2.53e^(-5)×AGE Where MPA is the dose of mycophenolate mofetil in ACKNOWLEDGMENT mg/kg body weight, Tx is time from transplantation in years, ATB is 1 if the patient is treated with an antimicro- The research was supported by the grant CZ: GACR: bial, INTER is 1 is the patient is treated with an interact- 303/12/0172 from the Grant Agency of the Czech ing antimicrobial, DOSE is the dose of tacrolimus in μg/ Republic. kg of body weight, and AGE is patient’s age in years. Presence of an interacting antimicrobial prescription was a highly significant predictor of CI dose/level ratio P-65 and lead on average to a 36% reduction in the dose re- Quantification of drug-drug interactions between quired to achieve therapeutic levels (P<0.01). In 60% of the cases the dose was reduced by the prescribing physi- calcineurin inhibitors and antimicrobial agents cian. Antimicrobials without a reported interaction (con- in kidney transplant recipients in routine clinical trol group) led to a 8% reduction in the dose/level ratio practice (P<0.01). Conclusions: In real-practice settings we quantified Jan Strojila, Jiri Orsagb, Pavel Anzenbachera, Karel Urbaneka, the effects of antimicrobial prescription on CI levels Karel Krejcib and maintenance of therapeutic levels. We constructed a predictive model that describes some of the variability aDepartment of Pharmacology, Faculty of Medicine and in CI levels as seen in routine monitoring after kidney Dentistry, Palacky University Olomouc, Hnevotinska 3, 775 15 transplant. Olomouc, Czech Republic bDepartment of Internal Medicine III – Nephrology, Rheumatology and Endocrinology, Faculty of Medicine and Dentistry, Palacky University Olomouc and University Hospital ACKNOWLEDGMENT Olomouc, I.P.Pavlova 6, 779 00 Olomouc, Czech Republic Supported by grant CZ.1.07/2.3.00/20.0040.

Introduction. Calcineurin inhibitors (CI), tacrolimus and ciclosporin, remain the mainstay of kidney transplant P-66 maintenance immunosuppression. Both are susceptible Influence of cranberry and polyphenon E on gene to multiple drug-drug interactions, especially at the level of metabolism. expresssion of cytochromes P450 (CYP) in mouse Patients. We performed a retrospective analysis of liver

TDM measurements in the past 5 years at a single trans- a a c plant centre in the University Hospital in Olomouc, Czech Barbora Liskova , Pavel Anzenbacher , Jana Nekvindova , Republic. A total of 4,024 measurements of trough con- Veronika Tomankovab, Lenka Jourovab, Hana Bartikovad, d d a centrations (C0) of CIs and MPA were included in the Iva Bousova , Lenka Skalova , Eva Anzenbacherova analysis made in 111 male and 70 female kidney transplant recipients (181 in total). Patients were aged 13 to 72 years (average 45.7), with aDepartment of Pharmacology, Faculty of Medicine and the exception of two patients, all were recipients of grafts Dentistry, Palacky University Olomouc, Czech Republic from deceased donors. Average time from transplant was bDepartment of Medical Chemistry and Biochemistry, Faculty 5.6 years (0.4 to 18). of Medicine and Dentistry, Palacky University Olomouc, Czech Drug measurements were obtained from the hospital Republic cInstitute for Molecular and Translational Medicine, Faculty of information system, information on prescription was as- Medicine and Dentistry, Palacky University Olomouc, Czech certained from prescription database and patient clinical Republic records (including dosing instructions and specific tempo- dDepartment of Biochemical Sciences, Faculty of Pharmacy in rary instructions concerning co-prescription of interacting Hradec Kralove, Charles University in Prague, Hradec Kralove, medication). Czech Republic CI levels were expressed as normalised dose required to reach a unit concentration and a linear regression mod- Polyphenon E is a green tea catechin extract. Many el was constructed to quantify effects of various interact- of the effects of green tea are thought to be due to cat- ing factors (e.g. age, sex, prednisone dose, MPA dose, echin, an especially epigallocatechin gallate (EGCG) as body weight). the main component accounting for 50% of the material. American cranberries (Vaccinium macrocarpon) are

S48 Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2013 Sep; 157 (Supplement 1):S1–S88. a particularly rich source of phenolic phytochemicals, primers of mice CYPs enzymes were selected with high including phenolic acids and flavonoids. Polyphenon E sequence similarity (orthologous) to human CYPs en- and cranberry have influence on human health and may zyme. interfere with the way the body processes certain drugs Our data showed induction of cytochromes P450 ex- using the liver's cytochrome P450 enzyme system. pression in the group B which were fed by diet enriched by Eight months old male NMRI mice were used to this cranberry extract in concentration 2% (w/w) for 4 weeks. experiment and were divided into three groups: The con- Regulation of cytochromes P450 expression and activity trols (A) and to two an experimental group (B, C). Group is a multi-level process that is currently not completely B was treated with 2% (w/w) cranberry extract mixed with understood. diet for 4 weeks. Group C were fed by diet enriched by Polyphenon E in concentration 0.1 % (w/w) for 4 weeks. Control group (A) was fed by standard diet. Effect of both ACKNOWLEDGEMENTS compounds on cytochromes P450 expression was studied using real-time RT-PCR. Relative expression (RE) of This work has been supported by GACR 303/12/G163 a gene of interest was calculated by ddCT method against and LF_2013_008. a selected reference gene (Hprt and 18S rRNA). Specific

S49 EXPERIMENTAL PHARMACOLOGY AND TOXICOLOGY COMMUNICATIONS Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2013 Sep; 157 (Supplement 1):S1–S88. C-1 Activation of NO and cytokines by low-molecular weight fractions of lysates from G+ and G− bacteria Eva Kmonickovaa, Adela Dusilovaa, Petra Kosteckaa, Miloslav Kverkab, Zdenek Zideka

Background. Probiotic bacteria are recognized for favourable effects in the treatment of various pathologies, mainly of gastrointestinal tract, such as ulcerative colitis, pouchitis and diarrhea. The clinical effects have been suggested to result at least in part from immunobiological activities of both live and dead bacteria. The aim of present experiments was to investigate whether and to what extent the spectrum of immunobiological properties of probiotics might be contributed to by molecules characterized by the low-molecular weight. Immunostimulatory effects of bacterial lysates and their fractions differing in the content of molecules of different size were investigated. Methods. The lysates were prepared by passing the probiotic bacteria Lactobacillus casei DN-114001 and Escherichia. coli strain Nissle 1917 through the French press followed by lyophilisation. The lysate fractions differing in the content of molecules of specified molecular weight (MW) range were prepared using the MW cutoff microfiltration (centrifugal devices Amicon®, Millipore). Four classes of MW cutoff fractions (MWCF), devoid of polymeric components of bacterial cell walls such as lipopolysaccharide, lipoteichoic acid and peptidoglycan were obtained. They contained molecules ≤ 100 kDa, ≤ 50 kDa, ≤ 10 kDa, or ≤ 3 kDa. The effects of original lysates and MWCF on the in vitro production of nitric oxide (NO) and cytokines interleukin-1β (IL-1β) and tumour necrosis factor-α (TNF-α) were analysed in rat peritoneal cells (2 x 106/ml). Nitrite and cytokines levels were determined at the 24-h interval of culture, using Griess reagent and ELISA, respectively. Results. It was found that all MWCF possessed the NO- and cytokine-stimulatory activity, although less expressed than was the activity of original lysate. The MWCF of Gram-positive L. casei were more effective than MWCF of Gram-negative E. coli. The most of the NO-stimulatory potential was found to be attributable to the molecules of ≤ 3 kDa. The L. casei MWCF-3 kDa accounted for approximately 80% of the NO-stimulatory effect of MWCF-100 kDa. Irrespective of the bacterial origin, about 42% of the IL-1β-enhancing activity of the crude lysate was contained in the MWCF-3 kDa. The MWCF-3 kDa was responsible for 22% and 52% TNF-α-stimulatory effects of the crude lysates obtained from L. casei and E.coli crude lysates, respectively. Conclusions. The present results provide an unequivocal evidence showing that bacterial lysates contain chemical entities with MW < 100 kDa possessing immunostimulatory properties. They are present in both G+ and G− bacteria. The molecules with MW ≤ 3 kDa are obviously the major contributors to the overall effect. Their share can be estimated to be approximately 55-85%, in dependence on the response and the type of bacteria. The findings warrant further studies which should identify chemical species and determine quantitative aspects of their occurrence in samples of different bacterial origin.

Key words: probiotics, lysate, microfiltration, nitric oxide, cytokines aDepartment of Pharmacology, Institute of Experimental Medicine, Academy of Sciences, Videnska 1083, 142 20, Prague, Czech Republic, bDepartment of Immunology, Institute of Microbiology, Academy of Sciences, Videnska 1083, 142 20, Prague, Czech Republic Corresponding author: Eva Kmonickova, e-mail: [email protected]

INTRODUCTION munobiologically highly active components of their outer membrane such as lipoteichoic acid (LTA) of G+ bacteria, The major medical indications of probiotic bacteria lipopolysaccharide (LPS) of G− bacteria, and peptidogly- are pathologies of gastrointestinal tract, such as ulcerative can (PGN) occurring mainly in G+ species. The aim of colitis, pouchitis and diarrhea1. The favourable clinical present experiments was to investigate whether and to effects have been suggested to result at least in part from what extent the spectrum of immunobiological properties a plethora of their immunobiological activities. Probiotics of probiotics might be contributed to by molecules charac- have been shown to enhance mainly the secretion of Th1 terized by the low-molecular weight. For this purpose, the cytokines and regulatory cytokine interleukin-10 (IL-10) microfiltration fractions of lysates from Lactobacillus casei (ref.2,3). Both Gram-negative (G−) and Gram-positive (G+) DN-114001 and Escherichia coli strain Nissle 1917 varying probiotics are also prominent activators of nitric oxide in the content of chemical entities of different molecular (NO) biosynthesis4,5. The ability of probiotics to modulate weight were prepared. Their ability to enhance production the innate and acquired immune responses is presumed to of NO and cytokines interleukin-1β (IL-1β) and tumour be due to the complex macromolecules represented by im- necrosis factor-α (TNF-α) was analysed.

S51 Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2013 Sep; 157 (Supplement 1):S1–S88.

Untreated Untreated 80 80

Original lysate (L. casei)

60 MWCF-100 kDa 60 Original lysate (E. coli) M) M) μ μ MWCF-50 kDa MWCF-100 kDa MWCF-10 kDa 40 40 MWCF-50 kDa MWCF-3 kDa Nitrite ( Nitrite Nitrite ( Nitrite MWCF-10 kDa MWCF-3 kDa 20 20

0 0

10 -1 10 0 10 1 10 2 10 -1 10 0 10 1 10 2 Concentration of original lysate (μg/mL) Concentration of original lysate (μg/mL)

Fig. 1. In vitro production of NO by rat peritoneal cells (2 x 106/mL) stimulated with lysates and their molecular weight cutoff fractions (MWCF) obtained from the probiotic strains of Lactobacillus casei (A) and Escherichia coli (B). Concentration of nitrites was evaluated at the interval of 24 h of culture using Griess reagent. The points are means ± SEM. Each figure represents one of two identical experiments.

A B 15000 10000 Original lysate: L. casei E.coli Original lysate: L. casei E.coli 12500 MWCF-100 kDa: L. casei E.coli MWCF-100 kDa: L. casei E.coli 8000 MWCF-3 kDa: L. casei E.coli MWCF-3 kDa: L. casei E.coli

10000 6000 Untreated Untreated

7500 (pg/mL) (pg/mL) α β 4000 IL-1 5000 TNF-

2000 2500

0 0 10 0 10 1 10 2 10 0 10 1 10 2 Control Control Concentration of original lysate ( μg/mL) Concentration of original lysate ( μg/mL)

Fig. 2. Secretion of cytokines interleukin-1β (IL-1β) (A) and tumour necrosis factor-α (TNF-α) (B) by rat peritoneal cells (2 x 106/mL) stimulated in vitro with lysates and their molecular weight cutoff fractions (MWCF) obtained from the probiotic strains of Lactobacillus casei and Escherichia coli. Concentration of cytokines was assayed at the interval of 24 h of culture using ELISA. The points are means ± SEM. Each figure represents one of two identical experiments.

MATERIAL AND METHODS The fractions of this lysate were further treated using the Amicon® Ultra 0.5 mL centrifugal microfiltration Bacteria, lysates, and low-molecular weight lysate devices (Millipore Corp., Billerica, MA). This procedure fractions allowed to prepare four classes of molecular weight cut- Lactobacillus casei DN-114001 (L. casei) was obtained off microfiltrates (MWCM) containing chemical entities from the Danone Institute, France. Escherichia coli Nissle characterized by MW ≤ 100 kDa, ≤ 50 kDa, ≤ 10 kDa, and 1917 (E. coli; Mutaflor, DSM 6601, serotype 06:K5:H1) ≤ 3 kDa. It was shown previously that even the 100 kDa was supplied by Dr. J. Schulze (Ardeypharm GmbH, microfiltration eliminated the biologically active bacterial Herdecke, Germany). polymers such as LPS, LTA and PGN (ref.6). The crude lysates were prepared from bacteria resuspended in distilled water. They were disrupted by passing Animals and cells three times through a French press (1500 psi), and then Peritoneal cells were obtained by lavage of female lyophilized and diluted to a working concentration of 30 Wistar rats (8-10 wks old; AnLab, Prague, CZ) with 16 mg/mL. To kill possible remnants of viable bacteria, the mL of PBS. The cells were resuspended in complete cul- lysate was heated to 60 °C for 30 min. The sterility of ture medium, and seeded into 96-well round-bottom mi- preparations was checked by both aerobic and anaerobic croplates in 100-μL volumes, 2 x 105 cells/well. Cultures cultivation. Ultimately, the lysates were centrifuged and were maintained at 37 °C, 5% CO2 in humidified Heraeus passed through the 0.22 μm syringe filters.

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Table 1. Biological activities of the molecular weight cutoff fractions (MWCF-100 kDa and MWCF-3 kDa) prepared from the lysates of probiotic strains of Lactobacillus casei and Escherichia coli. The dose-dependent effects on NO and cytokine production (shown in Figs 1 and 2, respectively) were expressed in terms of area under curve (AUC). The AUC of the original, i.e. non-microfiltered lysates were taken as 100%. Differences between samples were evaluated by two-way analysis of variance.

Bacterial samples Response Bacteria original MWCF-100 kDa MWCF-3 kDa lysate 74.6% 64.2% 100% F = 239.2; <0.0001* F = 49.05; <0.0001 L. casei /1,10/ P /1,10/ P MWCF-100 kDa versus MWCF-3 kDa: F = 58.79; P < 0.0001 NO /1,10/ 29.9% 20.4% 100% F = 4461; <0.0001 F = 3069; <0.0001 E. coli /1,10/ P /1,10/ P

MWCF-100 kDa versus MWCF-3 kDa: F/1,10/ = 23.21; P = 0.0007 63.0% 41.1% 100% F = 35.42; = 0.0003 F = 106.30; <0.0001 L. casei /1,8/ P /1,8/ P MWCF-100 kDa versus MWCF-3 kDa: F = 47.68; P = 0.0001 IL-1β /1,8/ 64.8% 42.7% 100% F = 90.49; P<0.0001 F = 1044; P<0.0001 E. coli /1,8/ /1,8/

MWCF-100 kDa versus MWCF-3 kDa: F/1,8/ = 17.46; P = 0.003 39.5% 21.5% 100% F = 83.42; <0.0001 F = 154.50; <0.0001 L. casei /1,8/ P /1,8/ P MWCF-100 kDa versus MWCF-3 kDa: F = 202.1; P<0.0001 TNF-α /1,8/ 74.6% 52.4% 100% F = 72.18; <0.0001 F = 174.70; <0.0001 E. coli /1,8/ P /1,8/ P

MWCF-100 kDa versus MWCF-3 kDa: F/1,8/ = 26.91; P = 0.0008

* Differences against 100% of original, i.e., non-microfiltered lysate. incubator. Complete RPMI-1640 culture medium (Sigma- were done using the Prism program (GraphPad Software, Aldrich) contained 10% heat-inactivated foetal bovine CA). The dose-response relationship was evaluated by serum, 2 mM L-glutamine, 50 μg/mL gentamicin, and means of area under curve (AUC). 5 x 10-5 M 2-mercaptoethanol (all Sigma-Aldrich). Each experimental variant was run in duplicate. The animal welfare and all experimental procedures RESULTS have been approved by the Institution Animal Ethics Committee. NO production Spontaneous production of NO by rat peritoneal cells NO assay was approximately 10 μM. It was substantially and dose- The cells were cultured 24 h in the presence of bac- dependently enhanced by both original lysates and the terial preparations. The concentration of nitrites in cell lysate-derived molecular weight cutoff microfiltration frac- supernatants was taken as a measure of NO production7. tions (MWCF) prepared from the probiotic bacteria L. It was detected by the Griess reagent. A nitrite calibration casei and E. coli. The original, i.e. non-microfiltered lysates curve was used to convert absorbance to μM nitrite. from E. coli were more effective than those from L. casei (Figs 1A and 1B, respectively). Cytokine assay The most potent activators of NO production proved Concentration of cytokines in supernatants was deter- to be the MWCF of L. casei origin (Fig. 1A). Evaluated mined at the interval of 24 h of culture using the ELISA by means of AUC, the effects of the MWCF-100 kDa kits, and following the manufacturer instructions (R&D and MWCF-3 kDa were less expressed than those of the Systems, MN). original lysate (Table 1). The similar NO-augmenting tendency was exhibited Statistical analysis by MWCF derived from the E. coli lysate (Fig. 1B). Analysis of variance with subsequent Bonferroni mul- Nevertheless, in contrast to the very high efficacy of tiple comparison test, and graphical presentation of data the original E. coli lysate, the NO-stimulatory effects of

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MWCF were weak, much weaker than those of L. ca- teria. The low MW molecules, ≤ 3 kDa, are obviously the sei. The onset of the NO-enhancement after the L. casei major contributors to the overall effect of lysates. Their MWCF became apparent when they were prepared from share can be estimated to be approximately 55-85%, in 5-10 μg/mL of the original lysate and NO production dependence on the response and the type of bacteria. increased substantially towards higher MWCF concen- The findings warrant further studies which should identify trations. At least 10-fold higher amount of the original chemical species and determine quantitative aspects of lysate was required to obtain the E. coli MWCF that were their occurrence in samples of different bacterial origin. capable to augment the NO production (Fig. 1A versus 1B). Only the effect of MWCF-100 kDa was significant (P<0.05) if MWCF were prepared from the 50 μg/mL of ABBREVIATIONS lysate. When prepared from the 100 μg/ml of lysate, all MWCF-100, -50, -10, and -3 kDa did induce significant IL-1β, interleukin-1β; LPS, lipopolysaccharide; LTA, production of NO (P<0.001, <0.01, <0.05, and <0.05, re- lipoteichoic acid; MDP, muramyl dipeptide; MW, molecu- spectively). Yet, the response remained very low (Table 1). lar weight; MWCF, molecular weight cutoff microfiltration fraction(s); NO, nitric oxide; PGN, peptidoglycan(s); Cytokine secretion TNF-α, tumour necrosis factor-α. The original lysates prepared from both L. casei and E. coli probiotics activated the secretion of cytokines (Fig. 2). The original lysates of L. casei were significantly ACKNOWLEDGMENT more effective than those of E. coli in activating both

IL-1β (F/1,8/ = 10.84, P=0.011) and TNF-α (F/1,8/ = 64.53, The research was supported by grant CZ:GA P<0.0001) production. ČR:303/12/0535 from the Grant Agency of the Czech The secretion of cytokines was statistically signifi- Republic. cantly enhanced by MWCF as well (Fig. 2), though less effectively than by the original lysates (Table 1). REFERENCES

DISCUSSION AND CONCLUSIONS 1. Damaskos D, Kolios G. Probiotics and prebiotics in inflammatory bowel disease: microflora "on the scope". Br J Clin Pharmacol 2008;65:453-67. At present, reliable data on the diversity and chemi- 2. Cross ML, Ganner A, Teilab D, Fray LM. Patterns of cytokine induc- cal nature of small molecules produced by different types tion by gram-positive and gram-negative probiotic bacteria. FEMS of bacteria and constituting the lysates are largely miss- Immunol Med Microbiol 2004;42:173-80. ing. Based on the available evidence, one of plausible 3. Shida K, Suzuki T, Kiyoshima-Shibata J, Shimada S, Nanno M. Essential roles of monocytes in stimulating human peripheral candidates for the immunostimulatory effects of lysate blood mononuclear cells with Lactobacillus casei to produce cyto- MWCF might be the 492 Da molecule of MDP that has kines and augment natural killer cell activity. Clin Vaccine Immunol been shown to be the minimum constituent responsible 2006;13:997-1003. for many biological activities of PGN (ref.8-12). However, 4. Zídek Z, Kmoníčková E, Kostecká P, Tlaskalová-Hogenová H. Decisive role of lipopolysaccharide in activating nitric oxide and cytokine the participation of MDP on immunobiological effects production by the probiotic Escherichia coli strain Nissle 1917. Folia of MWCF is doubtful, because it was found absent in a Microbiol 2010;55:181-9. cocktail of various distinct mono-, di-, and trimeric muro- 5. Marcinkie wicz J, Ciszek M, Bobek M, Strus, M, Heczko PB, Kurnyta peptides with molecular mass ≤ 3 kDa that were detected M, Biedroń R, Chmielarczyk A. Differential inflammatory mediator 13 response in vitro from murine macrophages to lactobacilli and after the digestion of PGN from L. casei . Like MDP, pathogenic intestinal bacteria. Int J Exp Pathol 2007;88:155-64. other types of muropeptides have been found to possess 6. Zídek Z, Kmoníčková E, Kostecká P, Jansa P. Microfiltration method immunostimulatory properties14,15. Another partial dipep- of removal of bacterial contaminants and their monitoring by nitric tide structure of PGNs is gamma-D-glutamyl-meso-diami- oxide and Limulus assay. Nitric Oxide 2013;28:1-7. 7. Marletta MA, Yoon PS, Iyengar R, Leaf CD, Wishnok JS. Macrophage nopimelic acid which possesses weak immunobiological oxidation of L-arginine to nitrite and nitrate. Biochemistry potential12,16. Furthermore, lactobacilli produce large and 1988;27:8706-11. functionally diverse classes of bacteriocins. The molecu- 8. Adam A, Ellouz F, Ciorbaru R, Petit JF, Lederer E. Peptidoglycan ad- lar mass of the II, III and IV classes is > 10 kDa. The juvants: minimal structure required for activity. Z Immunitatsforsch Exp Klin Immunol 1975;149:341-8. molecular mass of the typeA lantibiotics, belonging to 9. Weidemann B, Schletter J, Dziarski R, Kusumoto S, Stelter F, Rietschel the class I bacteriocins, is within the range 2.164-3.488 ET, Flad HD, Ulmer AJ. Specific binding of soluble peptidoglycan and kDa and that of the typeB lantibiotics ranges from 1.959 muramyldipeptide to CD14 on human monocytes. Infect Immun to 2.041 kDa (ref.17). Although no reliable information 1997;65:858-64. 10. Kim HJ, Y ang JS, Woo SS, Kim, SK, Yun CH, Kim KK, Han SH. on their immunomodulatory effects is available, some of Lipoteichoic acid and muramyl dipeptide synergistically induce them have been found associated with increased expres- maturation of human dendritic cells and concurrent expression of sion of TNF-α (ref.18). proinflammatory cytokines. J Leukoc Biol 2007;81:983-9. In conclusion, the present results provide unequivocal 11. Hosokawa I, Hosokawa Y, Ozaki K, Yumoto H, Nakae H, Matsuo T. Proinflammatory effects of muramyldipeptide on human gingival evidence showing that bacterial lysates contain chemical fibroblasts. J Periodontal Res 2010;45:193-9. entities with MW < 100 kDa possessing immunostimula- 12. Ekman AK, Cardell LO. The expression and function of Nod-like re- tory properties. They are present in both G+ and G− bac- ceptors in neutrophils. Immunology 2010;130:55-63.

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13. Billot-Klein D, Legrand R, Schoot B, van Heijenoort J, Gutmann L. 16. Uehara A, Fujimoto Y, Kawasaki A, Kusumoto S, Fukase K, Takada Peptidoglycan structure of Lactobacillus casei, a species highly re- H. Meso-diaminopimelic acid and meso-lanthionine, amino acids sistant to glycopeptide antibiotics. J Bacteriol 1997;179:6208-12. specific to bacterial peptidoglycans, activate human epithelial cells 14. Hasegawa M, Kawasaki A, Yang K, Fujimoto Y, Masumoto J, Breukink through NOD1. J Immunol 2006;177:1796-804. E, Nuñez G, Fukase K, Inohara N. A role of lipophilic peptidoglycan- 17. Jack RW, Tagg JR, Ray B. Bacteriocins of gram-positive bacteria. related molecules in induction of Nod1-mediated immune respons- Microbiol Rev 1995;59:171-200. es. J Biol Chem 2007;282:11757-64. 18. Zhu LH, Li C, Wu JA, Liang JG, Shi YF. Bacteriocins from lactic acid 15. Pashenko v MV, Popilyuk SF, Alkhazova BI, L'vov VL, Murugin VV, bacteria increases tumor necrosis factor-alpha expression in a rat Fedenko ES, Khaitov RM, Pinegin BV. Muropeptides trigger dis- kidney model of chronic rejection. Transplant Proc 2008;40:3746-7. tinct activation profiles in macrophages and dendritic cells. Int Immunopharmacol 2010;10:875-82.

S55 Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2013 Sep; 157 (Supplement 1):S1–S88. C-2 Interaction of anthocyanins with human liver microsomal cytochromes P450 Michaela Kopecna Zapletalovaa, Alena Vanduchovaa, Alena Spicakovaa, Eva Anzenbacherovab, Pavel Anzenbachera

Aims. Anthocyanins are pigments present in fruit and vegetables. Antocyanins are glycosides; their aglycones are flavonoids, anthocyanidins (the most common are cyanidin, delphinidin, malvidin, peonidin, petunidin, pelargonidin). Sugar part is mostly glucose, galactose, arabinose, or rutinose. Although often used as nutraceutics, detailed studies on possible interactions with human liver drug metabolizing enzymes as cytochromes P450 (CYP) are lacking. In this preliminary report the inhibition of three forms of human liver microsomal CYPs was studied. Methods. Cyanidin-3-glucoside, cyanidin-3-galactoside, cyanidin-3-arabinoside, malvidin-3-glucoside, peonidin- 3-glucoside, petunidin-3-glucoside, delphinidin-3-glucoside, pelargonidin-3-rutinoside and cyanidin-3,5-di-glucoside were added to human liver microsomes to study the inhibition of prototypical enzyme activities of three CYP forms (CYP3A4, testosterone 6β-hydroxylase; CYP2C9, diclofenac 4´- hydroxylase; CYP1A2, 7-ethoxyresorufin O-deethylase) according to standard methods. Results. The CYP3A4 activity was influenced the most being inhibited to about 60% of the original activity by most of the anthocyanins at the highest concentration tested (100 μM) . Inhibition of CYP2C9 enzyme activity was more prominent with delphinidin-3-glucoside and cyanidin-3-galactoside (down to 60% of the original activity), CYP1A2 enzyme was inhibited in a small extent (not more than 25% at the 100 μM concentration of the respective anthocyanin). The results indicate that the interactions of anthocyanins with liver CYP enzymes most probably does not pose any harm to human organism based on drug interactions; however, in case of drugs metabolized by CYP3A4, and an ingestion of large amounts of anthocyanins, a limited increase of drug bioavailability may be observed however probably not clinically significant.

Key words: anthocyanins, cyanidin, malvidin, peonidin, petunidin, delphinidin, pelargonidin, human liver microsomes, cytochrome P450 aDepartment of Pharmacology, Faculty of Medicine and Dentistry, Palacky University Olomouc, 775 15 Olomouc, Czech Republic bDepartment of Medical Chemistry and Biochemistry, Faculty of Medicine and Dentistry, Palacky University Olomouc, 775 15 Olomouc, Czech Republic Corresponding author: Eva Anzenbacherova, e-mail: [email protected]

INTRODUCTION as mono-, di- or trisaccharides. Glucose, galactose and arabinose are the sugars most commonly present in antho- Anthocyanins (Greek anthos, flower and kyanos, cyanins. The most common naturally occurring anthocya- blue) are water soluble natural pigments which belong to nins are the 3-O-glucosides and the 3,5-O-diglucosides of a large group of polyphenolics named flavonoids1. They malvidin, cyanidin, pelargonidin, delphinidin, petunidin are widely distributed in fruits and vegetables such as blue- and peonidin6. berries, cranberries, strawberries, cherries, plums, grapes, and red cabbage but are also found in flowers and other plant materials. Anthocyanins are responsible for cyanic colors ranging from salmon pink through red and violet to dark blue. There have been over 600 anthocyanins identified in nature, featuring six common aglycones – anthocyanidins (cyanidin, delphinidin, malvidin, peonidin, petunidin and pelargonidin). Their usual distribution in fruits2 is: cyanidin 30%, delphinidin 22%, pelargonidin Fig. 1. Structure of anthocyanins tested. 18%, peonidin 7.5%, malvidin 7.5% and petunidin 5% . Daily dietary intake of anthocyanins has been estimated at 82 mg/day in Finland and 12.5 mg per day per person in the United States and can rise several fold depending Anthocyanins have a variety of physiological func- on seasonal and lifestyle factors3,4. Thus, their intake and tions. Consumption of anthocyanins in fruits, vegetables, also bioavailability are important in estimating beneficial wines, jams, and preserves is associated with probable effects of anthocyanins on human health. reduced risks of chronic diseases such as cancer, car- Chemically, anthocyanins are derivatives of 2-phenyl- diovascular diseases, type II diabetes, virus inhibition, benzopyrylium (flavylium cation) (Fig. 1). The anthocy- Alzheimer’s disease6,7. Anthocyanins and other flavonoids anins consist of an aglycone (anthocyanidin), sugar(s), are regarded as important nutraceuticals mainly due to and, in many cases, acyl group(s). Sugars may be present their antioxidant effects, which give them a potential role

S56 Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2013 Sep; 157 (Supplement 1):S1–S88. in prevention of the various diseases associated with oxi- were obtained under approval of the local ethics com- dative stress8,9. mittee and in accordance with the ethic regulations of The bioavailability of the anthocyanins is a main fac- the country of origin (Spain). They were from five males tor for their physiological functions. Anthocyanins are and five females with a protein content of 38.4 mg/mL. either rapidly absorbed from the gastrointestinal tract by For determination of CYP activities, testosterone different mechanisms that may involve specific enzymes and diclofenac were supplied by Sigma Aldrich (Prague, such as bilitranslocases10-12, or are first metabolized by the Czech Republic), ethoxyresorufin was supplied by Fluka gut microflora, leading to anthocyanidins and phenolic (Buchs, Switzerland) and 6β-hydroxytestosterone, 4-hy- acid metabolites13,14. However several studies have demon- droxydiclofenac and 7-ethoxyresorufin were supplied by strated that anthocyanins are very poorly absorbed and Cerilliant Corporation (Round Rock, Texas, USA). are excreted only to a small extent in urine15,16. In feeding studies with animals and humans, typically ca. 0.1% of Methods the quantities ingested, and sometimes much less, has Activities of individual CYP forms were measured been detected in urine. Anthocyanins also undergo me- according to published protocols23. The following mi- tabolism, since part have been found as glucuronidated, crosomal CYP activities were tested: CYP3A4, testos- methylated or sulphated forms17. This anthocyanin me- terone 6β-hydroxylation; CYP1A2, 7-ethoxyresorufin tabolites are delivered again to the gastrointestinal tract O-deethylation; and CYP2C9 activity, diclofenac 4´-hy- via enterohepatic circulation and after reaching the colon droxylation. Final incubation volumes were as follows: are subjected to bacterial metabolism by colon flora18. CYP1A2, 100 μL; CYP2C9, 200 μL nad CYP3A4, 500 The available data imply that the determinants of μL. The reaction mixtures of all CYP activities tested were absorption and excretion are influenced not only by the buffered by 100 mM K-phosphate buffer (pH 7.4). For nature of the sugar moiety but also by the structure of the each enzyme assay, a preliminary experiment was done to 19 anthocyanidin aglycone . The complex array of informa- determine the KM and Vmax for given enzyme reaction and tion on anthocyanin bioavailability obtained with human to obtain the values of substrate concentrations suitable and animal test systems has been reviewed by Prior and for the inhibition experiments. Inhibition experiments Wu20. were routinely performed with six concentrations of the Cytochrome P450 enzymes (P450s, or CYPs) are tested compound (0, 10, 20, 40, 80 and 100 μM in the primarily responsible for most of the drug biotransfor- reaction mixture). The effects of anthocyanins on CYPs mations in variety of tissues such as liver, kidney, brain, activity were determined using the Prominence system lung, and heart21. Most are found in the endoplasmic re- (Shimadzu, Kyoto, Japan). ticulum, but five are localized primarily in mitochondria. P450s are involved in the detoxification of a wide variety of xenobiotics such as drugs, biogenic amines from food RESULTS AND DISCUSSION sources, environmental toxins, and chemical carcinogens, the oxidation of steroids, fatty acids, prostaglandins, leu- Possible interactions of nine anthocyanins (cyanidin- kotrienes, and fat-soluble vitamins22. 3-glucoside, cyanidin-3-galactoside, cyanidin-3-arabino- The current study was undertaken to investigate an side, cyanidin-3,5-di-glucoside, delphinidin-3-glucoside, influence of anthocyanins on enzymatic activity of three malvidin-3-glucoside, peonidin-3-glucoside, petunidin- human liver microsomal CYP forms important for me- 3-glucoside and pelargonidin-3-rutinoside) with activities tabolism of drugs and other xenobiotics. of three forms of P450 enzymes present in human liver microsomes (CYP3A4, CYP1A2 and CYP2C9) were studied. MATERIALS AND METHODS In general, all the anthocyanins tested inhibited activities of CYP enzymes in a concentration-dependent man- Chemicals ner (Fig. 2). The most prominent inhibition was observed Cyanidin-3-O-glucoside chloride (kuromanin chlo- with CYP3A4 activity (testosterone 6β-hydroxylation) ride), cyanidin-3-O-galactoside chloride (idein chloride), by almost all anthocyanins; namely, with delphinidin- cyanidin-3-O-arabinoside, cyanidin-3,5-O-diglucoside chlo- 3-glucoside and cyanidin-3-arabinoside (down to 50% ride (cyanin chloride), delphinidin-3-O-glucoside (myr- of control values) and malvidin-3-glucosides as well as tillin chloride), malvidin-3-O-glucoside chloride (oenin cyanidin-3-glucosides (down to 60% of original values) chloride), peonidin-3-O-glucoside chloride, pelargonidin- at the highest concentration of tested compounds (100 3-O-rutinoside chloride, petunidin-3-O-glucoside chloride μM). The only anthocyanins which did not inhibited the were purchased from Extrasynthese (Genay, France). All CYP3A4 enzyme activity by more than 20% were pelar- of tested compounds were dissolved in distilled water. All gonidin-3-rutinoside and cyanidin-3,5-diglucoside (Fig. 2). other chemicals were supplied by Sigma Aldrich (Prague). Inhibition of CYP2C9 enzyme activity, diclofenac 4´-hydroxylation, was more prominent with delphinidin- Enzyme and substrates 3-glucoside and cyanidin-3-galactoside (down to about Cryopreserved human liver microsomes (pooled) were 60% of the original activity at 100 μM concentration); purchased of Advancell (Barcelona, Spain). Microsomes other anthocyanins were weaker inhibitors of CYP2C9

S57 Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2013 Sep; 157 (Supplement 1):S1–S88.

Fig. 2. Inhibition of CYP activities in human liver microsomes by different anthocyanins (cyanidin-3-glucoside, CY3GL; cyanidin-3-galactoside, CY3GA; cyanidin-3-arabinoside, CY3AR; malvidin-3-glucoside, MA3GL; peonidin-3-glucoside, PN3GL; petunidin-3-glucoside, PT3GL; delphinidin-3-glucoside, DL3GL; pelargonidin-3-rutinoside, PG3RU; cyanidin-3,5- di-glucoside, CY3,5DIGLU). Anthocyanin concentrations 0, 10, 20, 40, 80 and 100 μmol/L.

S58 Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2013 Sep; 157 (Supplement 1):S1–S88. activity (activity decreased by 30 to 20%) (Fig. 2). 4. Chun OK, Chung SJ, Song WO. Estimated dietary flavonoid intake Pelargonidin-3-rutinoside did not inhibit the CYP2C9 and major food sources of U.S. adults. J Nutr 2007;137:1244-52. 5. Kong JM, Chia LS, Goh NK, Chia TF, Brouillard R. Analysis and biologi- enzyme at all. cal activities of anthocyanins. Phytochemistry 2003;64:923-33. Activity of the CYP1A2 form (7-ethoxyresorufin 6. Shih PH, Chan YC, Liao JW, Wang MF, Yen GC. Antioxidant and cog- O-deethylation) was affected by anthocyanins only slightly nitive promotion effects of anthocyanin-rich mulberry (Morus at- (activity decreased about 10%) with the exception of mal- ropurpurea L.) on senescence-accelerated mice and prevention of Alzheimer’s disease. J Nutr Biochem 2010;21:598-605. vidin-3-glucoside exhibiting inhibition of CYP2C9 activity 7. Wang LS, Stoner GD. Anthocyanins and their role in cancer preven- by 30% (Fig. 2). tion. Cancer Lett 2008;269:281-90. The results indicate that – taking into account the rela- 8. Zafra-Stone S, Yasmin T, Baqchi M, Chatterjee A, Vinson JA, Bagchi tively low bioavailability of anthocyanins in the plasma10 D. Berry anthocyanins as novel antioxidants in human health and disease prevention. Mol Nutr Food Res 2007;51(6):675-83. - the interactions of anthocyanins with liver CYP enzymes 9. Kähkönen M, Heinonen M. Antioxidant activity of anthocyanins and most probably does not pose any harm to human organ- their aglycones. J Agric Food Chem 2003;51(3):628-33. ism based on drug interactions. However, in case of a drug 10. McGhie TK, Walton MC. The bioavailability and absorption of an- metabolized by CYP3A4 with a simultaneous ingestion of thocyanins: towards a better understanding. Mol Nutr Food Res 2007;51(6):702-13. large amounts of anthocyanins, a limited increase of bio- 11. Talavera S, Felgines C, Texier O, Besson C, Lamaison J L, Remesy,C. availability of concomitantly taken drug may be observed Anthocyanins are efficiently absorbed from the stomach in anes- which probably would not be also clinically significant. thetized rats. J Nutr 2003;133(12):4178-82. 12. Passamonti S, Vrhovsek U, Vanzo A, Mattivi F. The stomach as a site for anthocyanins absorption from food. FEBS Lett 2003;544(1-3):210- 3. ACKNOWLEDGMENT 13. Tsuda T, Horio F, Osawa T. Absorption and metabolism of cyanidin 3-O-beta-D-glucoside in rats. FEBS Lett 1999;449(2-3):179-82. The financial support from the Grant Agency of the 14. Felgines C, Texier O, Besson C, Fraisse D, Lamaison JL, Remesy C. Blackberry anthocyanins are slightly bioavailable in rats. J Nutr Czech Republic (project P303/12/G163) and from the 2002;132(6):1249-53. Palacky University (project LF_2013_007) is gratefully 15. Manach C, Williamson G, Morand C, Scalbert A, Rémésy C. acknowledged. Bioavailability and bioefficacy of polyphenols in humans. I. Review of 97 bioavailability studies. Am J Clin Nutr 2005;81(1):230-42. 16. Wu X, Cao G, Prior RL. Absorption and metabolism of anthocyanins in elderly women after consumption of elderberry. J Nutr CONFLICT OF INTEREST STATEMENT 2002;132(7):1865-71. 17. Felgines C, Talavéra S, Gonthier MP, Texier O, Scalbert A, Lamaison Author’s conflict of interest disclosure: The authors JL, Rémésy C. Strawberry anthocyanins are recovered in urine as glucuro- and sulfoconjugates in humans. J Nutr 2003;133(5):1296- stated that there are no conflicts of interest regarding the 301. publication of this article. 18. Scalbert A, Morand C, Manach C, Rémésy C. Absorption and metabolism of polyphenols in the gut and impact on health. Biomed Pharmacother 2002;56(6):276-82. 19. Wu X, Pittman HE, McKay S, Prior RL. Aglycones and sugar moieties REFERENCES alter anthocyanin absorption and metabolism after berry consumption in weanling pigs. J Nutr 2005;135(10):2417-24. 1. Takeoka G, Dao L. Anthocyanins. In: Hurst WJ, editor. Methods of 20. Prior RL, Wu X. Anthocyanins: structural characteristics that result in Analysis for Functional Foods and Nutraceuticals. FL: CRC, Boca unique metabolic patterns and biological activities. Free Radic Res Raton; 2002. p. 219–241. 2006;40(10):1014-28. 2. Andersen QM, Jordheim M. The Anthocyanins. In: Andersen QM, 21. Guengerich FP. Human cytochrome P450 enzymes. In: Ortiz de Markham KR, editors. Flavonoids: Chemistry, Biochemistry and Montellano PR, editor. Cytochrome P450. Structure, mechanism and Applications. FL: CRC Press, Boca Raton; 2006. p.471-552 biochemistry. 3rd ed. New York: Kluwer Academic Plenum; 2006. p. 3. Wu X, Beecher GR, Holden JM, Haytowitz DB, Gebhardt SE, Prior RL. 377–530. Concentrations of anthocyanins in common foods in the United 22. Guengerich FP. Cytochromes P450, drugs, and diseases. Mol Interv States and estimation of normal consumption. J Agric Food Chem 2003;3(4):194-204. 2006;54:4069-75. 23. Philips IR, Shephard EA. Cytochrome P450 Protocols, Meth Mol Biol 320, Humana Press, Totowa, NJ; 2006.

S59 Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2013 Sep; 157 (Supplement 1):S1–S88. C-3 Silybin affects the liver microsomal CYP2C6 in HHTg rats Rostislav Veceraa, Alice Zacharovaa, Ludmila Kazdovab, Zuzana Matuskovaa, Nina Skottovaa, Jan Strojila, Olena Oliyarnykb, Pavel Anzenbachera

Aim. This study aims to investigate the effect of silybin on liver expression and activity of rat CYP2C6 under mild liver steatosis. This cytochrome P450 enzyme is considered to be a counterpart of human CYP2C9, which metabolizes commonly prescribed drugs, such as ibuprofen, diclofenac, or warfarin. Methods. Male hereditary hypertriglyceridemic rats (accepted model of metabolic syndrome) were fed: 1) standard laboratory diet (STD), 2) high cholesterol diet (HCD = STD + 1% of cholesterol w/w + 10% of lard fat w/w), 3) high cholesterol diet with silybin (0.5% w/w) for 21 days. Expression of cytochrome P450 2C6 was measured in liver using real-time PCR (at mRNA level) and Western blotting (at protein level). Formation of diclofenac metabolite (typical marker substrate of CYP2C6 enzyme activity) was analyzed using HPLC with UV detection. Results. Silybin in hereditary hypertriglyceridemic rats on HCD diet significantly increased activity of CYP2C6 and its expression on mRNA level. Expression of CYP2C6 on protein level was non-significantly affected by silybin consumption. Our results suggest that CYP2C6 is up-regulated by silybin in hereditary hypertriglyceridemic rats on high cholesterol diet. Conclusion. Since cytochrome P450 2C6 is considered to be a counterpart of human CYP2C9, the results obtained open the possibility that in human silybin may affect the metabolism of drugs metabolized by this cytochrome P450. Further studies are needed to elucidate the effects of silybin on CYP2C9 in humans suffering from metabolic syndrome.

Key words: silybin, cytochrome P450, CYP2C6, hereditary hypertriglyceridemic rat, liver steatosis aDepartment of Pharmacology, Faculty of Medicine and Dentistry, Palacky University Olomouc, Czech Republic bCenter for Experimental Medicine, Institute for clinical and Experimental Medicine, Prague, Czech Republic Corresponding author: Rostislav Vecera, email: [email protected]

INTRODUCTION human CYP2C9 in metabolism of many drugs (ibuprofen, warfarin or diclofenac) is well documented14. Silymarin, a standardized extract from the seeds of the This study aims at investigating effect of silybin (0.5% plant Silybum marianum (Milk Thistle), has been used w/w), administered in high cholesterol and high fat diet in the supportive therapy of liver diseases for centuries. for 3 weeks, on the expression of liver CYP2C6 in he- Silymarin has been promoted as a nutritional supple- reditary hypertriglyceridemic male Wistar (HHTg) rats. ment for healthy liver function1. Its cytoprotective effect These animals selected from Wistar rats exhibit hyper- is believed to be based on antioxidant properties2. In our triglyceridemia, liver steatosis, insulin resistance, and previous studies we have shown a hypolipidemic effect of hypertension and represent an accepted model of meta- silymarin3. Silybin is the main component of silymarin bolic syndrome15,16. In this strain of rats, we have previ- representing about 50% of this extract4. This flavonolig- ously obtained interesting results regarding the beneficial nan has antioxidant5, anti-inflammatory6, anti-viral7, an- properties of silymarin or maca17,18. Experimental high tifibrotic8, anti-cancer9, metabolic4, and other beneficial cholesterol diet (high content of saturated fatty acids and effects. Given that silybin is taken by a lot of patients with cholesterol), is used to simulate the unhealthy diet often metabolic syndrome and liver lipid accumulation we were seen in patients suffering from metabolic syndrome19. interested in its effects on the cytochrome P450 in terms Possible effect of silybin on cytochrome P450 2C6 ex- of possible interactions with concomitantly administered pression could present another complicating factor con- medications. The risk and severity of drug-drug interac- tributing to difficulties in finding the right dose of drugs tions generally rises with increasing dose of the drug and/ metabolized by its human counterpart CYP2C9. or in concomitant administration with drug that may interact with the same transport or metabolic pathway10. It should be noted that these drug-drug interactions pres- MATERIAL AND METHODS ent a leading cause of patient hospitalization and also death11. CYP2C6 is one of the main enzymes of the cy- Experimental animals tochrome P450 (about 20% of the total CYP content) Adult male hereditary hypertriglyceridemic Wistar in rat liver microsomes. This cytochrome P450 can also (HHTg) rats (260 – 280 g of body weight, seven rats in be regarded as a counterpart of human CYP2C9 (ref.12). each group) were maintained under standard laboratory Rat CYP2C6 and human CYP2C9 are known to share conditions. The animals were fed (ad libitum) on: 1) substrates (both catalyze the 7-hydroxylation of warfarin12 standard laboratory diet (=STD), 2) high cholesterol diet and 4´-hydroxylation of diclofenac13). High significance of (HCD), composed of STD + 1% of cholesterol w/w and

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Table 1. Dietary intakes of standard laboratory diet (STD), high cholesterol diet (HCD) and HCD with 0.5% of silybin (HCD+0.5%SB).

Diet STD HCD HCD+0.5%SB

MDC of diet 86.2±3.67 77.8±3.71 79.1±2.64 (g.kg-1 of body weight) MDD of drug - - 395,5±8.4 (mg.kg-1 of body weight) Body weight (g) 340.1±5.5 348.3±6.2 345.3±4.5

Values are means ± SE, n=7; MDC – mean daily consumption of the diet, MDD – mean daily dose of silybin.

expression of CYP2C6 mRNA 200 ++ **

100 relative expression of mRNA (%) mRNA of expression relative 0 STD HCD HCD+0.5%SB

Fig. 1. Expression of CYP2C6 mRNA in liver of HHTg rats. Standard laboratory diet (STD), high cholesterol diet (HCD) and HCD with silybin (HCD + 0.5% SB). Values are means ± SE, n=7; **P<0.02 vs HCD, ++P<0.02 vs HCD.

activity of CYP2C6 1,5

*** 1 CYP450 0,5 nmol of 4´-OH diclofenac/min/nmol 4´-OH diclofenac/min/nmol of nmol 0 STD HCD HCD+0.05%SB

Fig. 2. Activity of CYP2C6 in liver of HHTg rats. Standard laboratory diet (STD), high cholesterol diet (HCD) and HCD with silybin (HCD + 0.5% SB). Values are means ± SE, n=7; ***P<0.01 vs HCD.

S61 Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2013 Sep; 157 (Supplement 1):S1–S88.

Fig. 3. Expression of CYP2C6 protein in liver of HHTg rats. Standard laboratory diet (STD), high cholesterol diet (HCD) and HCD with silybin (HCD + 0.5% SB).Values are means ± SE, n=7.

10% of lard fat and 3) HCD with 0.5% (w/w) of silybin. Activity of CYP2C6 After 21 days of feeding, the rats were fasted overnight. Formation of diclofenac metabolite (4´-hydroxy- Animals were anesthetized by intramuscular administra- diclofenac, formed mainly by CYP2C613) as well as tion of dexmedetomidin (200 μg.kg-1 of body weight) in the levels of diclofenac (standards from Sigma-Aldrich, combination with fentanyl (40 μg.kg-1 of body weight), Prague, Czech Republic) were determined in samples us- followed by administration of diazepam (5 mg.kg-1 of body ing a method by Crespi20. Analyses were performed us- weight). Blood was sampled into EDTA tubes from the ing HPLC with UV detection set at 280 nm (Shimadzu aortic bifurcation. Plasma was separated by centrifuga- Prominence System, Kyoto, Japan). The metabolite was tion (2500 × g, 20 min, 4 °C). Rat liver was removed, separated using LiChrospher 100 RP-18 column (4 x rinsed in ice-cold sucrose solution, and frozen in dry ice. 250 mm ID) with a 5 μm particle size (Merck Millipore, All procedures with animals were approved by the Ethics Darmstadt, Germany) protected by LiChrospher 100 RP- Committee, Ministry of Education, Czech Republic. 18 precolumn (4 x 4 mm ID) of the same origin. 4’-hy- droxydiclofenac was separated at 50 °C and at a flow 1 Real-time PCR procedures ml/min of mobile phase using gradient elution. The mo- A piece of liver tissue sample stabilized in RNA-later bile phase A consisted of 2 mM perchloric acid in a water: (Quiagen, Germantown, USA) was homogenized and acetonitrile (7:3, v/v) and the mobile phase B consisted of subsequently passed through QIAshredder columns to 100% methanol. The gradient steps were: 0-20 min linear eliminate tissue microparticles. RNA was isolated by gradient from 70% to 0% A and from 30% to 100% B; 20- RNeasy Plus Minikit (Quiagen Germantown, USA) which 22 min isocratic at 0% A and 100% B, 22-23 min gradient enable degradation of contaminating genomic DNA. 1 from 0% to 70% A, and from 100% to 30% B; 23-33 min μg of RNA was reverse-transcribed by Transcriptor High isocratic at 70% A and 30% B. Fidelity cDNA Synthesis Kit (Roche, Basel, Switzerland) and random hexamer primers. Thusly synthesized cDNA Western blot procedures was utilized for RT-PCR with the use of a Light Cycler Liver microsomes were obtained using routine pro- 480 SYBR Green Master I mix in a Light Cycler 480 cedure by Lake21. Protein concentration was measured (Roche, Basel, Switzerland). The thermal cycling condi- (BCA Protein Assay Reagent Kit, Pierce, Rockford, USA). tions were: 10 min at 95 °C, followed by 45 cycles at 95 °C 12.5 μg of protein from each sample was mixed with buff- for 10 s, at 58 °C for 15 s, and at 72 °C for 15 s for denatur- er (62.5 mM TRIS, 10% glycerol, 4% mercaptoethanol, ation, annealing, and elongation, respectively. All samples 2% SDS, pH 6.8) in a volume ratio of 1:1 and electro- for RT-PCR were prepared in triplicates. Fluorescence phoresed (discontinuous SDS-polyacrylamide gel; 4% w/w emission (cycle-to-cycle) was monitored and absolute stacking gel, 8% w/w separating gel). Following electro- quantification method was applied to obtain gene expres- phoresis, proteins were transferred onto polyvinylidene sion data. The respective rat primers were designed in our difluoride membrane, Hybond-P (Amersham Biosciences, laboratory and consecutively synthesized by Invitrogen Piscataway, USA). Immunoreactivity was detected after (Life Technologies, Prague, Czech Republic). The follow- 60 min incubation with a primary monoclonal CYP2C6 ing primer sequences were used: antibody (Abcam, Cambridge, UK) and 60 min incuba- CYP2C6 Fw 5‘-GCCTTGTGGAGGAACTGAGG-3‘ tion with a secondary antibody anti-mouse IgG - HRP CYP2C6 Rev 5‘-GCACAGCCCAGGATAAACGT-3‘

S62 Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2013 Sep; 157 (Supplement 1):S1–S88. conjugate (Sigma-Aldrich , Prague, Czech Republic). The results were obtained in models of healthy rats or in vitro. reaction was detected using enhanced chemiluminescence On the other hand, our results in a rat model of human (the manufacturer’s protocol, WB Luminol Reagent, metabolic syndrome (not healthy but HHTg rats) indi- Santa Cruz, USA). The blots were then exposed to medi- cate that in these animals, i.e. under different conditions, cal X-Ray film and scanned (CanoScan Toolbox software, the regulation pathways result in a significantly increased ver. 5.0.). enzyme activity and mRNA expression of CYP2C6. We observed similar differences between healthy Wistar rats Statistical analysis and HHTg rats after rosuvastatin treatment26,27. In nor- All data are expressed as means ± SE, (n=7). motriglyceridemic Wistar rats, rosuvastatin significantly Differences between groups were analysed (analysis of suppressed the liver microsomal CYP2C11 and CYP2C6 variance, ANOVA) followed by the appropriate post-hoc expression, but in HHTg rats rosuvastatin remarkably in- test. Significance threshold was P<0.05. Western blot were duced the expression and activity of CYP2C6. Besides, analysed by ElfoMan software (ver. 2.6, Semecky Inc., both the pathological condition in experimental organ- Prague, Czech Republic). isms and the effects of high cholesterol diet may modulate the final outcome28,29. In primary cultured human hepatocytes, it has been observed that silybin does not RESULTS interact much with concomitantly administered drugs25. On the other hand, Li24 showed that silybin significantly The quantity of feed consumed (standard diet, high- enhanced the effect of loratadine by affecting of CYP3A4, cholesterol diet, and high-cholesterol diet with silybin) suggesting that concurrent use of silybin and loratadine was checked daily per each cage holding one or two ani- should be monitored closely for potential drug interac- mals. The silybin dose (about 390 mg per kg of b.w. per tions. Interpretation of our results, obtained in HHTg day) was chosen in agreement with information published rats, with regard to possible implications in humans is not in literature22. As shown in Table 1., mean daily dietary straightforward. Rat CYP2C6 is not completely identical intake and body weights of rats were not significantly dif- with human CYP2C9, but exhibits a very high sequence ferent between any of the experimental groups. identity with this human cytochrome P450 (ref.12), which HCD diet alone decreased mRNA expression of he- is involved in metabolism of many drugs14. Moreover, it is patic CYP2C6 (Fig. 1). Silybin supplementation of high unclear the precise way in which the pathological condi- cholesterol and high fat diet affected studied cytochrome tion of the experimental organism (metabolic syndrome P450. Unlike the HCD diet alone, silybin in this type of associated with liver steatosis) may affect the mechanism experimental diet significantly increased expression of by which silybin influences the CYP activity and levels. CYP2C6 at the mRNA level to the level found in control group (Fig. 1). On the other hand, at the level of protein, no significant changes were observed (Fig. 3). Fig. CONCLUSION 2 shows the effects of silybin on hydroxylation activity of CYP2C6. In hereditary hypertriglyceridemic rats fed the Our results demonstrate that silybin significantly high cholesterol diet, silybin significantly increased the increased activity and mRNA expression of CYP2C6 diclofenac hydroxylating (4’-hydroxylation) activity cor- in HHTg rats. It may be suggested that silybin has the responding to the CYP2C6 enzyme activity20. It is in good potential to influence levels of drugs metabolized by rat agreement with significant augmentation of its mRNA CYP2C6, especially under conditions associated with expression. metabolic syndrome and ectopic lipid accumulation in liver. However, the clinical relevance of studies performed on experimental animal models is not straightforward. DISCUSSION Further studies are needed to elucidate the effects of silybin on CYP2C9 enzyme in man and also on other Our study shows that administration of the flavono- pleiotropic pathways, including the effect of diet and lignan silybin to hereditary hypertriglyceridemic rats fed pathological state in humans. a high cholesterol diet results in an increase in the expression of mRNA of liver microsomal CYP2C6 enzyme. Positive effect of silybin on CYP2C6 has been confirmed ABBREVIATIONS by a significant rise of its enzymatic activity (is diclofenac 4´-hydroxylation20). This cytochrome P450 is considered ANOVA, analysis of variance; BCA, bicinchoninic to be a counterpart to human CYP2C9 (ref.12). acid; cDNA, complementary deoxyribonucleic acid; CYP, The enzyme activity and sensitivity of CYP2C6 mRNA cytochrome P450; HHTg, hereditary hypertriglyceride- expression to silybin is reported in hereditary hypertriglyc- mia; HRP, horseradish peroxidase; mRNA, messenger eridemic rats (an accepted model of metabolic syndrome) ribonucleic acid; RT-PCR, real-time polymerase chain for the first time. Publications often reports that silybin reaction; SDS, sodium dodecyl sulfate. has an inhibitory effect23,24 or that silybin does not affect the expression of cytochrome P450 (ref.25). These

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ACKNOWLEDGEMENTS 14. Mo SL, Zhou ZW, Yang LP, Wei MQ, Zhou SF. New insights into the structural features and functional relevance of human cytochrome P450 2C9. Part I. Curr Drug Metab 2009;10:1075-126. This work was supported by Czech Science 15. Vrana A, Kazdova L. The hereditary hypertriglyceridemic non- Foundation project 13-10813S. obese rat: an experimental model of human hypertriglyceridemia. Transplant Proc 1990;22:2579. 16. Klimes I, Vrána A, Kunes J, Seböková E, Dobesová Z, Stolba P. Hereditary hypertriglyceridemic rat: a new animal model of meta- CONFLICT OF INTEREST STATEMENT bolic alterations in hypertension. Blood Press 1995;4:137-42. 17. Škottová N, Kazdová L, Oliyarnyk O, Večeřa R, Sobolová L, Ulrichová J: Author’s conflict of interest disclosure: The authors Phenolics-rich extracts from Silybum marianum and Prunella vulgaris stated that there are no conflicts of interest regarding the reduce a high-sucrose diet induced oxidative stress in hereditary hypertriglyceridemic rats. Pharmacol Res 2004;50:123-30. publication of this article. 18. Večeřa R, Orolin J, Škottová N, Kazdová L, Oliyarnyk O, Ulrichová J, Šimánek V: The influence of maca (Lepidium meyenii) on antioxidant status, lipid and glucose metabolism in rat. Plant foods hum nutr REFERENCES 2007;62:59-63. 19. Charlton M, Krishnan A, Viker K, Sanderson S, Cazanave S, McConico A, Masuoko H, Gores G. Fast food diet mouse: novel small animal 1. Wellington K, Jarvis B. Silymarin: a review of its clinical properties in model of NASH with ballooning, progressive fibrosis, and high physi- the management of hepatic disorders. Bio Drugs 2001;15:465-89. ological fidelity to the human condition. Am J Physiol Gastrointest 2. Valenzuela A, Garrido A. Biochemical bases of the pharmacological Liver Physiol 2011;301:G825-34. action of the flavonoid silymarin and of its structural isomer silibinin. 20. Crespi CL, Chang TK, Waxman DJ. Determination of CYP2C9- Biological Research 1994;27:105-12. catalyzed diclofenac 4´-hydroxylation by high-performance liquid 3. Skottova N, Vecera R, Urbanek K, Vana P, Walterova D, Cvak L. Effects chromatography. Methods Mol Biol 2006;320:109-13. of polyphenolic fraction of silymarin on lipoprotein profile in rats fed 21. Lake BG. Preparation and characterization of microsomal frac- cholesterolrich diets. Pharmacological Research 2003;47:17-26. tions for studies on xenobiotic metabolism. In: Snell EK, Mullock B. 4. Loguercio C, Festi D. Silybin and the liver: from basic research to Biochemical toxicology and practical approach. IRL Press: Oxford- clinical practice. World J Gastroenterol 2011;17:2288-301. Washington DC; 1990. p. 183. 5. Trouillas P, Marsal P, Svobodová A, Vostálová J, Gazák R, Hrbác J, 22. Chang JC, Wu YT, Lee WC, Lin LC, Tsai TH. Herb-drug interaction of Sedmera P, Kren V, Lazzaroni R, Duroux JL, Walterová D. Mechanism silymarin or silibinin on the pharmacokinetics of trazodone in rats. of the antioxidant action of silybin and 2,3-dehydrosilybin flavono- Chem Biol Interact 2009;182:227-32. lignans: a joint experimental and theoretical study. J Phys Chem A 23. Zhou SF, Zhou ZW, Yang LP, Cai JP. Substrates, inducers, inhibi- 2008;112:1054-63. tors and structure-activity relationships of human Cytochrome 6. Federico A, Morgillo F, Tuccillo C, Ciardiello F, Loguercio C. Chronic P450 2C9 and implications in drug development. Curr Med Chem inflammation and oxidative stress in human carcinogenesis. Int J 2009;16:3480-675. Cancer 2007;121:2381-86. 24. Li C, Lee MY, Choi JS. Effects of silybinin, CYP3A4 and P-glycoprotein 7. 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Differences in hepatic expression of genes involved in lipid ho- Therapeutic drug monitoring and pharmacogenetic tests as tools meostasis between hereditary hypertriglyceridemic rats and healthy in pharmacovigilance. Drug Saf 2006;29:735-68. Wistar rats and their response to dietary cholesterol. Fod Chem 12. Daniel WA, Haduch A, Syrek M, Boksa J. Direct and indirect inter- Toxicol 2009;47:2624-30. actions between antidepressant drugs and CYP2C6 in the rat 29. Sugatani J, Sadamitsu S, Kurosawa M, Ikushiro S, Sakaki T, Ikari A. liver during long-term treatment. Eur Neuropsychopharmacol Nutritional status affects fluvastatin-induced hepatotoxicity and 2006;16:580-87. myopathy in rats. Drug Metab Dispos 2010;38:1655-64. 13. Leemann, Transon C, Dayer P. Cytochrome P450TB (CYP2C): a major monooxygenase catalyzing diclofenac 4'-hydroxylation in human liver. Life Sci 1993;52:29-34.

S64 Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2013 Sep; 157 (Supplement 1):S1–S88. C-4 The influence of chronic administration of thioacetamide on levels of ALT, AST and GLDH in serum and markers of oxidative stress in the liver and kidneys of female Wistar rats Monika Bludovskaa, Eliska Mistrovab, Dana Kotyzovaa, Magdalena Chottova-Dvorakovab

Background. Thioacetamide (TAA), a well known inducer of hepatic fibrosis, has been often used in experimental acute and chronic hepatotoxicity models. Reactive oxygen species play an important role in TAA-induced liver damage. Aims. To study the time course of activities of ALT, AST and GLDH in serum and markers of oxidative stress in the liver and kidneys of rats after chronic TAA administration. Methods. Female Wistar rats were treated with TAA (intraperitoneally, 200 mg/kg body weight) for 12 weeks, the controls received saline. Animals were sacrificed 4, 12 or 16 weeks after the treatment discontinuation. Lipid peroxidation (LP), reduced glutathione (GSH) and activities of glutathione peroxidase (GPx), glutathione reductase (GR) and catalase (CAT) were estimated in the liver and kidneys. ALT, AST and GLDH were measured in serum. Results. Significant increases in serum ALT and GLDH persisted for 16 weeks ater the treatment, while serum AST was increased only in animals sacrificed 4 weeks after the treatment cessation. No increase in LP or decrease in GSH was observed in the liver. Furthermore, a decrease in LP and an increase in GR activity appeared in the 16th week. Significant decreases in the activities of catalase and GPx (which persisted in animals sacrificed 12 and 16 weeks after the treatment, respectively) were the only markers of hepatic oxidative damage. In kidneys, LP was significantly increased 4 and 12 weeks after the TAA treatment which implies the importance of oxidative stress in the renal damage that develops as a consequence of liver cirrhosis.

Key words: thioacetamide, liver, kidney, oxidative stress, aminotrasferases, rat aDepartment of Pharmacology and Toxicology, Faculty of Medicine in Pilsen, Charles University in Prague, Czech Republic bDepartment of Physiology, Faculty of Medicine in Pilsen, Charles University in Prague, Czech Republic Corresponding author: Monika Bludovska, e-mail: [email protected]

INTRODUCTION The aim of this preliminary experiment was to study the time course of activities of alanine aminotransferase TAA is a thiono-sulfur containing compound that (ALT), aspartate aminotransferase (AST) and glutamate has been studied and used as a highly specific hepato- dehydrogenase (GLDH) in serum and markers of oxida- toxic substance both in vitro1-3 and (more frequently) in tive stress in the liver and kidneys of rats during the period vivo. Single dose can induce acute hepatic failure4, while of 16 weeks after chronic (12 weeks) TAA administration. chronic oral or intraperitoneal administrations of TAA It has been shown that administration of low intraperi- are established methods in the generation of fibrosis and toneal or oral doses of TAA for 8-14 weeks can induce cirrhosis models in rats5-7. TAA is used for its highly spe- liver injury and dysfunction and cause hepatic fibrosis in cific hepatotoxic effect and the ability to produce liver mice and rats8. damage with histological appearance similar to human hepatic fibrosis8. TAA produces centrilobular necrosis9 and the cytotox- MATERIALS AND METHODS ic effect is mediated by its metabolites, not the compound itself10-11. TAA is rapidly oxidized by hepatic microsomal Chemicals CYP2E1 to acetamide and thioacetamide sulfoxide which Thioacetamide and other reagents were of analytical is further metabolized to thioacetamide-S,S-dioxide. This grade and obtained from Sigma-Aldrich (USA) or as in- highly reactive metabolite binds covalently to intracellular dicated in the specific methods. macromolecules and can initiate necrosis and cause oxidative stress12-15. Bioactivation of TAA and its active metabo- Animals and treatment lite follows saturable kinetics13,16. It was shown that the Female Wistar rats (7–8 weeks of age) were housed generation of reactive oxygen species plays an important at standard laboratory conditions under controlled tem- role in TAA-induced liver damage and the development of perature (22±2 °C), relative humidity (50–60%), and 12-h fibrosis17,18. The ability of antioxidant substances to inhibit light–dark cycles. The animals had free access to water or attenuate the progression of thioacetamide induced and standard pellet rat chow. After 11 days of acclima- liver damage and to decrease its prooxidant effects has tization, forty-six rats were randomly divided into two been proven in many studies19-23.

S65 Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2013 Sep; 157 (Supplement 1):S1–S88. groups: Animals in the treated group were given TAA dase (GPx), glutathione reductase (GR) and catalase (intraperitoneally 200 mg/kg body weight) three times per (CAT) were estimated in liver and kidney homogenates. week over a period of 12 weeks. The dose was based upon Activities of alanine amino transferase (ALT), aspartate literature24,25. Animals in the control group received saline. amino transferase (AST) and glutamate dehydrogenase Animals were sacrificed 4, 12 or 16 weeks after the dis- (GLDH) were determined in serum. TAA treated animals continuation of the treatment. Blood and tissue samples were compared with age-matched controls. were collected and used immediately or stored frozen at The experimental treatment protocol was approved by -70°C until analyzed. Lipid peroxidation (LP), reduced the local Animal Care and Use Committee. glutathione (GSH) and activities of glutathione peroxi-

Table 1. Lipid peroxidation (LP), reduced glutathione (GSH), and activities of glutathione peroxidase (GPx), glutathione reductase (GR) and catalase (CAT) in the liver of thioacetamide-treated rats sacrificed 4, 12 and 16 weeks after the discontinuation of the treatment.

Liver 4 weeks 12 weeks 16 weeks

LP control 91.0±9.2 106.1±10.8 119.5±11.4 (nmol TBARS /g) TAA 85.4±8.6 97.1±14.4 103.1±13.8*

GSH control 3.85±0.42 4.28±0.44 4.58±0.13 (μmol/g) TAA 3.84±0.41 4.37±0.63 4.86±0.38

GPx control 50.1±3.5 45.8±3.8 57.2±4.4 (μmol/g/min) TAA 38.0±5.2*** 40.6±2.5** 49.4±4.5**

GR control 6.76±0.85 5.23±0.33 5.22±0.09 (U/g) TAA 7.07±0.64 5.23±0.57 5.80±0.24**

CAT control 39.8±3.1 41.5±3.2 46.0±3.3 (k/g) TAA 35.7±2.8* 33.8±3.3*** 43.7±3.8

Values are means ±SD; ***P<0.001, **P<0.01 and *P<0.05 vs control group.

Table 2. Lipid peroxidation (LP), reduced glutathione (GSH), and activities of glutathione peroxidase (GPx), glutathione reductase (GR) and catalase (CAT) in kidneys of thioacetamide-treated rats sacrificed 4, 12 and 16 weeks after the discontinuation of the treatment.

Kidneys 4 weeks 12 weeks 16 weeks

LP control 75.2±6.0 79.1±7.0 97.0±23.0 (nmol TBARS /g) TAA 107.1±12.5*** 99.1±9.5** 96.5±6.8

GSH control 4.62±0.34 4.61±0.36 4.74±0.31 (μmol/g) TAA 4.69±0.43 4.60±0.35 4.68±0.33

GPx control 9.4±0.8 9.0±0.3 10.3±0.8 (μmol/g/min) TAA 10.1±1.0 10.7±1.0*** 11.1±0.9

GR control 7.43±0.27 5.92±0.46 7.82±0.59 (U/g) TAA 7.39±0.39 6.40±0.57 7.42±0.63

CAT control 9.8±0.6 10.8±1.2 9.9±0.9 (k/g) TAA 9.7±0.6 12.1±1.4* 11.5±1.6*

Values are means ±SD; ***P<0.001, **P<0.01 and *P<0.05 vs control group.

S66 Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2013 Sep; 157 (Supplement 1):S1–S88.

Biochemical assays Lipid peroxidation (LP) was estimated in liver and kidney homogenates by measuring the products formed in the thiobarbituric acid (TBA) reaction26. Tissue homogenates (0.25 g/2.5 mL of 1.15% potassium chloride (KCl)

were mixed with 1.5 mL of 1% phosphoric acid (H3PO4) and 0.5 mL of 0.6% TBA aqueous solution. The samples were heated at 95 °C for One hour after cooling, 2 mL of n-butanol were added, mixed vigorously and the butanol phase was separated by centrifugation. The absorbance of butanol layer was measured at 520 and 535 nm; the difference between the determinations was used to calculate concentration of TBA reactive substances (TBARS). The results are ex- Fig. 1. Serum alanine aminotransferase (ALT) in thioacetamide- pressed in nmole TBARS/gram of tissue. treated rats sacrificed 4, 12 and 16 weeks after the discontinu- GSH level was estimated in the deproteinized super- ation of the treatment. Values represent means ±SD; *P<0.05 natant fraction of liver and kidney homogenates (0.2 g/ 8 vs control group. mL of 0.02 M EDTA) using 5,5’-dithiobis(2-nitrobenzoic acid) (DTNB, Ellman’s reagent) and reading absorption at 412 nm (ref.27). The results are expressed in μmol GSH/g of tissue. GPx activity was assayed in liver and kidney homogenates by a coupled test system, in which GR is employed for the regeneration of reduced glutathione and butyl hydroperoxide used as the acceptor substrate28. The decrease in NADPH concentration was registered photometrically at 340 nm. The GPx activity is expressed in μmol NADP+/min/g of tissue. GR assay is based on the reduction of oxidized glutathione (GSSG) by NADPH in the presence of glutathione reductase. The formed GSH reacts with 5,5‘-dithiobis(2- nitrobenzoic acid). The increase in absorbance at 412 nm was measured29. The reaction system contained 0.1M Fig. 2. Serum aspartate aminotransferase (AST) in thioacet- phosphate buffer (pH 7.5), 1mM EDTA, 2mM GSSG amide-treated rats sacrificed 4, 12 and 16 weeks after the dis- and 3mM DTNB solution. Reactions were started by the continuation of the treatment. Values represent means ±SD; addition of 2mM NADPH and the increase in absorbance *P<0.05 vs control group. was measured at 412 nm. Catalase activity was estimated according to the meth- 30 od of Aebi et al. by following the decomposition of H2O2 directly by the decrease in extinction of hydrogen peroxide at 240 nm. The activity of catalase is expressed as a rate constant of a first order reaction k per g of tissue. The serum activity of ALT, AST and GLDH were estimated photometrically using the commercial kits (DiaSys Diagnostic System, Germany), according to the manufac- turers’ protocol.

Statistical analysis The results are expressed as means + SD. Significant differences between experimental groups were estimated using unpaired Student’s t test (GraphPad InStat3). Differences between the groups were considered signifi- Fig. 3. Serum glutamate dehydrogenase (GLDH) in thioacet- cant at P<0.05. amide-treated rats sacrificed 4, 12 and 16 weeks after the discontinuation of the treatment. Values represent means ±SD; *P<0.05 vs control group. RESULTS

TAA administration significantly increased serum ALT, AST and GLDH activities. The increase in activi-

S67 Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2013 Sep; 157 (Supplement 1):S1–S88. ties of ALT and GLDH in TAA-treated rats (Fig. 1, Fig. 3) damage to various distal organs35-37 and the increased LP persisted for 16 weeks following the treatment cessation, that was seen in the kidneys of rats in our study is con- increased AST activity was detected only in TAA-treated sistent with the conjecture that the renal damage (as well animals sacrificed 4 weeks after the treatment discontinu- as the impairment of other organs) developed as a conse- ation and returned to control levels in animals sacrificed quence of cirrhosis is presumably mediated by oxidative later (12 and 16 weeks after the treatment) (Fig. 2). stress24 rather than by a direct toxic effect of a hepatotoxic No increase in hepatic LP was observed. Contrary agent. This can be further confirmed by an increase in LP to the assumption, a decrease in LP and an increase in in heart that we have observed as well with some delay GR activity even appeared in the liver of TAA-treated after chronic TAA administration (our not yet published rats in the 16th week following the treatment cessation. findings). In the kidneys, we have also seen an increase in Significant decreases in the activity of GPx, which per- the activities of GPx and catalase. This discrepancy can sisted for 16 weeks after the TAA treatment and in the be explained as a response of these antioxidant enzymes activity of catalase, which was seen in rats sacrificed 4 to the increase in LP that was detected in the kidneys. and 12 weeks after the treatment, were the only markers Such reaction of antioxidant enzymes has already been of oxidative damage in the liver. We have not detected any described4,38. changes in the content of hepatic GSH (Table 1). Significant increase in LP was found in kidneys of rats which were sacrificed 4 and 12 weeks after the treatment ACKNOWLEDGEMENT cessation. Contrarily, a significant increase in the activity of GPx in kidneys (in animals sacrificed 12 weeks after Supported by the grant Charles University Grant the treatment) and in the activity of catalase in kidneys (in Agency - GAUK 99510, project CZ.1.05/2.1.00/03.0076 animals sacrificed 12 and 16 weeks after the treatment) from European Regional Development Fund and by the was also detected. There were no changes in kidney GSH Charles University Research Fund (project number P36). content (Table 2.).

CONFLICT OF INTEREST STATEMENT DISCUSSION Author’s conflict of interest disclosure: The authors Chronic administration of TAA over the period of 12 stated that there are no conflicts of interest regarding the weeks induced liver damage that was characterized by a publication of this article. significant increase in serum activities of ALT and GLDH that persisted for 16 weeks following the treatment discontinuation. The activity of AST was significantly increased REFERENCES only in the animals sacrificed in the 4th week after the treatment. This might reflect the possible tissue repair or 1. Moronvalle-Halley V, Sacre-Salem B, Sallez V, Labbe G, Gautier JC. cessation of injury progression, since the mitochondrial Evaluation of cultured, precision-cut rat liver slices as a model to study druginduced liver apoptosis. Toxicology 2005;207(2):203-14. AST is released only when the cells are severely disinte- 2. Sarkar MK, Sil PC. Hepatocytes are protected by herb Phyllanthus grated31. niruri protein isolate against thioacetamide toxicity. Pathophysiology Oxidative stress has been proven to be involved in the 2007;14(2):113-20. pathogenesis of TAA induced liver damage and a deple- 3. Staňková P, Kučera O, Lotková H, Roušar T, Endlicher R, Cervinková Z. The toxic effect of thioacetamide on rat liver in vitro. Toxicol In Vitro tion of hepatic GSH and an increase in TBARS concen- 2010;24(8):2097-103. tration are typical markers of oxidative stress seen during 4. Reddy PV, Murthy ChR, Reddanna P. Fulminant hepatic failure in- both acute and chronic thioacetamide intoxication32-34. duced oxidative stress in nonsynaptic mitochondria of cerebral In our study, we found neither an increase in LP nor a cortex in rats. Neurosci Lett 2004;368(1):15-20. 5. Li X, Benjamin IS, Alexander B. Reproducible production of thioacet- decrease in GSH content in the liver during the period amide-induced macronodular cirrhosis in the rat with no mortality. from the 4th to the 16th week after the cessation of TAA J Hepatol 2002;36(4):488-93. treatment. However, we have detected some markers of 6. Kučera O, Lotková H, Staňková P, Podhola M, Roušar T, Mezera V, deterioration of hepatic oxidative state since the activity Cervinková Z. Is rat liver affected by non-alcoholic steatosis more susceptible to the acute toxic effect of thioacetamide? Int J Exp of catalase was significantly decreased in animals sacri- Pathol 2011;92(4):281-9. ficed 4 and 12 weeks after the end of the treatment and 7. Low TY, Leow CK, Salto-Tellez M, Chung MC. A proteomic analysis the activity of GPx remained decreased even in the 16th of thioacetamide-induced hepatotoxicity and cirrhosis in rat livers. week after the treatment discontinuation. Contrary to our Proteomics 2004;4(12):3960-74. 8. Wang ME, Chen YC, Chen IS, Hsieh SC, Chen SS, Chiu CH. Curcumin expectations, we saw a decrease in LP and an increase in protects against thioacetamide-induced hepatic fibrosis by attenu- GR activity in the liver in the 16th week after the treat- ating the inflammatory response and inducing apoptosis of dam- ment cessation for which the explanation is not clear at aged hepatocytes. J Nutr Biochem 2012;23(10):1352-66. the moment. 9. Mangipudy RS, Rao PS, Andrews A, Bucci TJ, Witzmann FA, Mehendale HM. Dose dependent modulation of cell death: apop- In animals sacrificed 4 or 12 weeks after the discon- tosis versus necrosis in thioacetamide hepatotoxicity. Int J Toxicol tinuation of TAA treatment, the increase in LP in kidneys 1998;17:193-211. was found. Development of liver cirrhosis can result in 10. Kang JS, Wanibuchi H, Morimura K, Wongpoomchai R, Chusiri Y,

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An herbal fruit, Complement Altern Med [Published online] 2013 March 5 [cited Amomum xanthoides, ameliorates thioacetamide-induced he- 2013 May 10];13:56. doi:10.1186/1472-6882-13-56 patic fibrosis in rat via antioxidative system. J Ethnopharmacol 20. Nissar AU, Farrukh MR, Kaiser PJ, Rafiq RA, Afnan Q, Bhushan S, Adil 2011;135(2):344-50. HS, Subhash BC, Tasduq SA. Effect of N-acetyl cysteine (NAC), an 35. Ramachandran A, Prabhu R, Thomas S, Reddy JB, Pulimood A, organosulfur compound from Allium plants, on experimentally in- Balasubramanian KA. Intestinal mucosal alterations in experimen- duced hepatic prefibrogenic events in wistar rat. Phytomedicine tal cirrhosis in the rat: role of oxygen free radicals. Hepatology 2013 Apr 8. [Epub ahead of print] doi:10.1016/j.phymed.2013.03.009 2002;35(3):622-9. 21. Sarkar MK, Sil PC. Hepatocytes are protected by herb Phyllanthus ni- 36. Barker EA, Smuckler EA. Nonhepatic thioacetamide injury. II. The ruri protein isolate against thioacetamide toxicity. Pathophysiology morphologic features of proximal renal tubular injury.Am J Pathol 2007 Oct;14(2):113-20. 1974;74(3):575-90. 22. Furtado KS, Prado MG, Aguiar E Silva MA, Dias MC, Rivelli DP, 37. Ortega MA, Torres MI, Fernández MI, Rios A, Sánchez-Pozo A, Gil A. Rodrigues MA, Barbisan LF. Coffee and caffeine protect against Hepatotoxic agent thioacetamide induces biochemical and histo- liver injury induced by thioacetamide in male Wistar rats Basic Clin logical alterations in rat small intestine. Dig Dis Sci 1997;42(8):1715- Pharmacol Toxicol 2012;111(5):339-47. 23. 23. de David C, Rodrigues G, Bona S, Meurer L, González-Gallego J, 38. Travacio M, Llesuy S. Antioxidant enzymes and their modification un- Tuñón MJ, Marroni NP. Role of quercetin in preventing thioaceta- der oxidative stress conditions. Free Radic Res Latin Am 1996;48(1- mide-induced liver injury in rats. Toxicol Pathol 2011;39(6):949-57. 2):9-13.

S69 CLINICAL PHARMACOLOGY COMMUNICATIONS Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2013 Sep; 157 (Supplement 1):S1–S88. C-5 Prevalence of thromboembolic complications in patients with atrial fibrillation in relation to a selected antithrombotic therapy Veronika Muller Zavalovaa,b, Vaclav Zizlavskya, Robert Staffaa

Aim. The aim of this work was to find and characterize the correlation between the development of peripheral arterial thromboembolism and the selected medication for the atrial fibrillation-induced coagulopathy. Methods. The evaluated set included 103 patients admitted to the 2nd Department of Surgery, St. Anne’s University Hospital in Brno, during a period of 9 months. Patients were divided into individual groups on the basis of chronic medication of antithrombotic drugs, and the therapy effectiveness was evaluated on the basis of the thromboembolia prevalence. Results. In total, there were 36 thromboembolic complications; in 14 patients, it was a relapse. In the warfarin-administered group, thromboembolia occurred in 31.6 % patients and in the acetylsalicylic acid (ASA)-administered group, in 24.4 % of cases. The highest prevalence of peripheral arterial thromboembolism was observed in the group without any antithrombotic therapy, where this diagnosis was determined in 78.57 % of cases. A significant correlation (P=0.004; OR=7.94; CI 99% 1.183-53.33) was confirmed between the manifestation of coagulopathic states in patients with anticoagulation therapy and unmedicated patients. The smallest incidence of these complications was observed in the group with antiplatelet medication (P=0.0004; OR=11.33; CI 99% 1.693-75.89) compared to unmedicated patients. In the case of warfarin, the pharmacotherapy failure was caused by an insufficiently effective INR, which reached on average 1.42 ± 0.53. Furthermore, a high impact of drug interactions cannot be definitely ruled out, especially in the case of antiplatelet ASA therapy, individually or in combination with clopidogrel.

Key words: atrial fibrillation, thromboembolism, anticoagulation, antiplatelet therapy a2nd Department of Surgery, Center for Vascular Disease, St. Anne´s University Hospital Brno, Faculty of Medicine, Masaryk University, Brno, Czech Republic bDepartment of Human Pharmacology and Toxicology, Faculty of Pharmacy, University of Veterinary and Pharmaceutical Sciences Brno Corresponding author: Veronika Muller Zavalova, e-mail: [email protected]

INTRODUCTION sents an important aspect in the prognosis of patients with atrial fibrillation from the perspective of altering hemo- Atrial fibrillation is one of the most frequent clinically dynamic conditions. Optimally balanced antithrombotic significant disorders of the heart rhythm. The prevalence pharmacotherapy takes into consideration age and sex of of this disease has been growing, and correlates with an patients, heart failure, hypertension, diabetes, incidence increasing polymorbidity, age and sex of patients. To date, of ictus/transitory ischemic attack/thromboembolic com- the number of patients reaches about 2% of population1-3 plications and existence of vascular disease in an anam- and because this figure does not include asymptomatic nesis. Identifying of these clinically significant prediction arrhythmias, the actual number of patients with atrial fi- factors resulted in the creation of CHA2DS2-VASc scoring brillation is probably much higher4,5. The main cause of system9 (Table 1), which, based on scored evaluation of the increased morbidity and mortality in these patients risks, allows individual therapeutic approach. Individual results from up to 6-fold higher risk of thromboembolic categories of a selected antithrombotic therapy are given complications6,7, especially stroke as well as transient isch- by the sum of obtained score. When reaching the value ≥ emic attacks, and embolism in peripheral lower limbs or 2, there is indicated anticoagulation therapy, whereas val- the visceral artery. In clinical practice, atrial fibrillation ues 0-1 prefer antiplatelet or no therapy, depending on the is most often associated with the presence of a very seri- benefits/risks evaluation for every patient. Score interval ous form of stroke with serious neurological deficit, up 1-2 characterizes an area, where can be indicated both an- to 23% 90 days mortality (after 2 years over 45%) and un- tiplatelet and anticoagulation medication. Optimum ther- certain prognosis of patients8. Etiopathogenesis of throm- apy is chosen with respect to other possible limits, which 10 boembolic complications is derived from hemodynamic are not addressed by CHA2DS2-VASc (ref. ). The risk of changes and structural abnormalities of myocardium. hemorrhagic complications (GI tract, brain, urinary tract) Pharmacotherapy is in most patients with atrial fibrilla- significantly increases in connection with antithrombotic tion based on the control of hearth frequency and ad- therapy11-13, and they can be predicted by stratification of equate treatment of related heart disorders, which results anamnestic data using the HAS-BLED score. This sys- in a retardation of progression of anatomically-pathologic tem allows quantification of a potential hemorrhage risk changes of cardiovascular system. Prophylaxis of cardio- due to the evidence of parameters such as hypertension, vascular complications of thromboembolic nature repre- abnormal renal/liver function, stroke, bleeding history or

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Table 1. Risk factor-based approach expressed as a point based scoring system CHA2DS2-VASc . Clinical characteristics of HAS-BLED bleeding risk score.

Risk factor score Risk factor score

C Congestive heart failure/LV dysfunction 1 H Hypertension 1

H Hypertension 1 A Abnormal renal/liver function 1 or 2

A2 Age >75 2 S Stroke 1 D Diabetes mellitus 1 B Bleeding 1

S2 Stroke/TIA/thromboembolism 2 L Labile INRs 1

V Vascular disease 1 E Elderly (e.g. age >65 years) 1

A Age 65–74 1 D Drugs or alcohol (1 point each) 1 or 2

Sc Sex category (i.e. female sex) 1

Maximum score 9 Maximum score 9

CHA2DS2-VASc; score = 1: no antithrombotic therapy recommended; score = 1 antithrombotic therapy with anticoagulation/antiplatelet therapy recommended; score ≥2 anticoagulation therapy recommended. HAS-BLED score ≥3 indicates the risk of bleeding, and regular clinical review is recommended12.

Table 2. Baseline characteristics of the participants, according to the treatment group. (W-warfarin, ASA-acetylsalicylic acid, CLO-clopidogrel)

W ASA ASA+CLO Characteristics no medication 1,5 - 10 mg/day 100 mg/day 100+75 mg/day

Age - yr 73.1±6.96 73.8±9.02 79.0±7.95 77.6±12.56

M - no./total no. (%) 24/38 (63.2 %) 27/45 (60.0 %) 4/6 (66.7 %) 3/14 (21.4 %)

F - no./total no. (%) 14/38 (36.8 %) 18/45 (40.0 %) 2/6 (33.3 %) 11/14 (78.6 %) predisposition, labile INR, elderly (≥65), drugs/alcohol artery in patients included in this study. The necessary concomitantly11. The score 3 and more indicates a high biochemical and clinical examination was carried out risk of hemorrhage and based on this evaluation, it is within 24 hours after a patient’s acceptance, and surgi- necessary to consider some particular anticoagulation or cal intervention was scheduled with respect to the pa- antiplatelet therapy with an aim to effectively decrease the tient’s overall condition. The evaluated group of patients thromboembolia incidence. included 45 women and 58 men, average age 74 years The aim of this study is to compare the frequency of (45-103) (Table 2), and no excluding criteria were applied. life-threatening thromboembolic complications in con- Uneven representation of patients in individual groups is nection with a selected antithrombotic medication, and caused by the actual number of patients treated in this to find correlation between the treatment failure and pos- department. In the monitored group, 4 patients died due sible causal factors. to massive thromboembolic complications and there were 3 major amputation of the limb. Patients with atrial fibrillation in their anamnesis were divided into 4 groups; 3 MATERIALS AND METHODS groups were medicated various chronic antithrombotic pharmacotherapy (warfarin at 1.5 – 10 mg/dose, acetyl- The evaluated set characterizes 103 patients with atrial salicylic acid at 100 mg/dose, acetylsalicylic acid 100 mg fibrillation admitted to the 2nd Department of Surgery, + clopidogrel 75 mg/dose) and 1 group of patients with- St. Anne’s University Hospital in Brno, during a period out indicated antiplatelet/anticoagulation medication. of 9 months (September 2012 to May 2013), regardless Incidence of thromboembolic complications was quanti- the main diagnosis. Due to their main focus, there was fied for individual groups and graphically processed by monitored the incidence of thromboembolia in peripheral Excel® (Microsoft). Odds ratio was used to compare the

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Fig. 1. The occurrence of thromboembolic complications in connection with coagulopathy medication. (W-warfarin, ASA-acetylsalicylic acid, CLO-clopidogrel, ** P<0.01,*** P<0.001). obtained results for individual groups, expressing the risk DISCUSSION ratio of peripheral arterial thrombosis. Statistical analysis of data used Fisher’s exact probability test at statistical Medication therapy of coagulopathy in atrial fibril- significance levels P<0.01 and P<0.001. GraphPad Prism lation has exactly defined recommendations complying

5.00 software (GraphPad Software, San Diego, CA, USA, with CHA2DS2-VASc score. Patients with the score 1-2 www.graphpad.com) was used to evaluate the data. represent a specific group, where the expertise and experience of the physician and psychosocial habits of a patient have a significant impact on the choice of antithrombotic RESULTS medication. The process of considering the risk of bleeding in comparison with thromboembolia in order to opti- Out of the total number of 103 patients with atrial fi- mize the effective therapy requires maximum information. brillation, thromboembolic complications occurred in 36 At present, warfarin therapy has been preferred despite patients, i.e. 34.95% cases. In 14 of them, it was a relapse. numerous dietetic and drug interactions. It was also pre-

The group with anticoagulation warfarin (W) therapy at ferred due to the setting of predictive CHA2DS2-VASc 1.5-10 mg/day included 38 patients, where 12 patients, evaluation score, where most patients were classified in i.e. 31.58%, experienced arterial thrombosis (Fig. 1). The the category of medium-serious and higher risk of throm- INR value obtained within 24 hours after the acceptance boembolic complications with a consequent indication reached on average 1.42 ± 0.53. The second group with of anticoagulation therapy14. Pharmacovigilance studies only antiplatelet therapy by ASA at 100 mg/day includ- confirm up to 64% decrease in the incidence of stroke15 in ed 45 patients (Fig. 1). In this group, thromboembolic connection with a chronic use of warfarin, accompanied complications were observed in 11 patients, i.e. 24.44%. by a relatively small number of hemorrhagic complica- The group with dual antiplatelet by ASA at 100 mg/day tions16. However, the success of this therapy is influenced combined with clopidogrel (CLO) at 75 mg/day, included by optimum therapeutic range INR 2.0-3.0, which was only 6 patients, and thromboembolia occurred in 2 cases achieved in our study group in only 18% of patients. In (33.33%). The highest prevalence of peripheral arterial other cases, INR value did not exceed on average 59.2% thrombosis was observed in the group of patients without of the target range (71% - 47.3%). Despite this fact, the anticoagulation and antiplatelet therapy, where this diag- warfarin therapy seems advantageous in comparison with nosis was revealed in 78.57% of cases. When compared the group of unmedicated patients, and the prevalence of with the warfarin group, these patients are at almost 8 fold cardiovascular complications of thromboembolic nature is higher risk of thromboembolia and the prevalence of this up to 8-fold lower. Antiplatelet therapy by ASA indicated condition in the monitored patients was characterized at in cases, when patients cannot be medicated by warfarin the statistical significance level (P=0.004; OR=7.94; CI due to the negative benefits/risks ratio or if the present 99% 1.183-53.33). In patients with chronic medication condition does not require anticoagulation therapy in par- of ASA at 100 mg/day, there was observed the lowest ticular. Even though the warfarin therapy represents a frequency of thromboembolic complications (P=0.0004; golden standard in the antithrombotic therapy, our results OR=11.33; CI 99% 1.693-75.89), compared with the group imply a higher efficiency of the ASA therapy (24.44% vs without antithrombotic medication, where the risk of the 31.58% in warfarin), which corresponds to 11 fold lower peripheral arterial thrombosis is up to 11 fold higher. probability of the development of cardiovascular thromboembolism. Even though warfarin is a “gold standard” in

S73 Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2013 Sep; 157 (Supplement 1):S1–S88. the antithrombotic therapy, our results imply a higher ef- 3. Go AS, Hylek EM, Phillips KA, Chang Y, Henault LE, Selby JV, Singer fectiveness of the ASA therapy (24.44% vs 31.58% in war- DE. Prevalence of diagnosed atrial fibrillation in adults: national implications for rhythm management and stroke prevention: the farin) in the cardioembolic prevention. This statements Anticoagulation and Risk Factors in Atrial Fibrillation (ATRIA) Study. is also supported by the odds ratio in these two groups, JAMA 2001;285:2370-5. where the incidence of arterial thromboses ratio reaches 4. Kirchhof P, Auricchio A, Bax J, Crijns H, Camm J, Diener HC, Goette 11:8 (ASA:warfarin). Similar results have been reported A, Hindricks G, Hohnloser S, Kappenberger L, Kuck KH, Lip GY, 17 Olsson B, Meinertz T, Priori S, Ravens U, Steinbeck G, Svernhage E, in various clinical studies . Analogy of this therapy with a Tijssen J, Vincent A, Breithardt G. Outcome parameters for trials in more significant prophylactic value of stroke is character- atrial ﬁbrillation: executive summary. Recommendations from a ized by dual antiplatelet (ASA+clopidogrel). This therapy consensus conference organized by the German Atrial Fibrillation brings a significant increase in the risk of hemorrhagic Competence NETwork (AFNET) and the European Heart Rhythm Association (EHRA). Eur Heart J 2007;28:2803-17. complications, reaching the incidence of up to 4.5% pa- 5. Lip GYH, Golding DJ, Nazir M, Beevers DG, Child DL, Fletcher RI. A 18 tients after a year of therapy . The number of observed survey of atrial ﬁbrillation in general practice: the West Birmingham hemorrhages is quantified at similar frequency as in the Atrial Fibrillation Project. Br J Gen Pract 1997;47:285-9. case of warfarin9. In predisposed patients, i.e. patients 6. Castillo Rodríguez JC, Lozano IF.: The change of paradigm in stroke prevention in atrial fibrillation. Challenges and emerging opportuni- with ulcerative gastroduodenal disease in their anamne- ties for the family physician. Aten Primaria 2013;45 Suppl 1:5-17. sis, etc., the danger of such complication is much higher. 7. Lloyd-Jones DM, Wang TJ, Leip EP, Larson MG, Levy D, Vasan RS, They are therefore preventively administered proton pump D’Agostino RB, Massaro JM, Beiser A, Wolf PA, Benjamin EJ. Lifetime inhibitors (PPI - omeprazole, lansoprazole, pantoprazole risk for development of atrial fibrillation: the Framingham Heart Study. Circulation 2004;110:1042-6. and esomeprazole). More and more clinical studies have 8. Tomek A. Fibrilace síní a cévní mozková příhoda. Postgrad Med implied the association between thromboembolic com- 2011;13(1):18-23. plications and co-medication of PPI and clopidogrel18,19, 9. Poçi D, Hartford M, Karlsson T et al. Role of the CHADS2 score in or of PPI and ASA in mono-therapy20. Due to the com- acute coronary syndromes: risk of subsequent death or stroke in patients with and without atrial fibrillation. Chest 2012;141:1431-40. mon co-medication by these drugs, a great emphasis has 10. Lip GYH, Frison L, Halperin J, Lane D. Identifying patients at risk of been given to the clinical research of their interactions. stroke despite anticoagulation. Stroke 2010;41(12):2731-8. Nevertheless, the controversy in the obtained clinical and 11. Pisters R, Lane DA, Nieuwlaat R, de Vos CB, Crijns HJ, Lip GY. A nov- biochemical results does not implicitly proof the signifi- el userfriendly score (HAS-BLED) to assess one-year risk of major bleeding in atrial fibrillation patients: The Euro Heart Survey. Chest cance of the monitored interactions. Yet it was possible 2010;138(5):1093-100. to correlate this combination with the failure of antiplate- 12. Lip GYH. Stroke in atrial fibrillation: epidemiology and thrombopro- let mono- (22.44%) and dual (33.33%) therapy in several phylaxis. J Thromb Haemost 2011;9(1):344-51. patients included in our study group. At the same time, 13. Connolly SJ, Ezekowitz MD, Yusuf S, Eikelboom J, Oldgren J, Parekh A, Pogue J, Reilly PA, Themeles E, Varrone J, Wang S, Alings M, it is necessary to consider the small set of patients with Xavier D, Zhu J, Diaz R, Lewis BS, Darius H, Diener HC, Joyner CD, the combined therapy of ASA and clopidogrel, which has Wallentin L; RE-LY Steering Committee and Investigators. Dabigatran a substantial impact on the statistical evaluation and in- versus warfarin in patients with atrial fibrillation. N Engl J Med sufficiently relevant data, which would not, with a great 2009;361(12):1139-51. 14. Go AS, Hylek EM, Chang Y, Phillips KA, Henault LE, Capra AM, probability, in case of a larger set of patients, reach the fre- Jensvold NG, Selby JV, Singer DE. Anticoagulation therapy for stroke quency of 33.33% of the development of thromboembolia. prevention in atrial fibrillation: how well do randomized trials trans- late into clinical practice? JAMA 2003;290:2685-92. 15. Hart RG, Pearce LA, Aguilar MI. Meta-analysis: antithrombotic therapy to prevent stroke in patients who have nonvalvular atrial fibril- CONFLICT OF INTEREST STATEMENT lation. Ann Intern Med 2007;146:857-67. 16. Mant J, Hobbs FD, Fletcher K, Roalfe A, Fitzmaurice D, Lip GY, Murray Authors’ conflict of interest disclosure: None declared. E. Warfarin versus aspirin for stroke prevention in an elderly community population with atrial fibrillation (the Birmingham Atrial Fibrillation Treatment of the Aged Study, BAFTA): a randomised controlled trial. Lancet 2007;370:493-503. REFERENCES 17. Rengo G, Pagano G, Squizzato A, Moja L, Femminella GD, de Lucia C, Komici K, Parisi V, Savarese G, Ferrara N, Perrone-Filardi 1. European Heart Rhythm Association; European Association for P, Leosco D. Oral anticoagulation therapy in heart failure patients Cardio-Thoracic Surgery, Camm AJ, Kirchhof P, Lip GY, Schotten U, in sinus rhythm: a systematic review and meta-analysis. PLoS One Savelieva I, Ernst S, Van Gelder IC, Al-Attar N, Hindricks G, Prendergast 2013;8(1):e52952;in proces. B, Heidbuchel H, Alfieri O, Angelini A, Atar D, Colonna P, De Caterina 18. Mehta A, Mehta D, Loganathan J, Paladugu N, Bhalodkar NC: R, De Sutter J, Goette A, Gorenek B, Heldal M, Hohloser SH, Kolh Clopidogrel With Proton Pump Inhibitors: Safe or Not? Clin. Cardiol P, Le Heuzey JY, Ponikowski P, Rutten FH. Guidelines for the man- 2011;34(1):528-31. agement of atrial fibrillation: the Task Force for the Management 19. Chen M, Wei JF, Xu YN, Liu XJ, Huang DJ. A meta-analysis of impact of Atrial Fibrillation of the European Society of Cardiology (ESC). of proton pump inhibitors on antiplatelet effect of clopidogrel. European Heart Journal 2010;31:2369-429. Cardiovasc Ther 2012;30(5):227-33. 2. Feyrer R, Ballazhi F, Seitz T, Weyand M, Harig F. Impact of Medical 20. Charlot M. Protonpumpinhibitor use and risk of adverse cardio- Treatment on Long-Term Results after Surgical Ablation of Atrial vascular events in aspirin treated patients with first time myocar- Fibrillation in Cardiac Surgical Patients. Ann Thorac Cardiovasc Surg dial infarction: nationwide propensity score matched study. BMJ 2013; in process. 2011;342:2690-7.

S74 Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2013 Sep; 157 (Supplement 1):S1–S88. C-6 Pharmacogenomics of infliximab therapy, impact of TNFRSF1A and TNFRSF1B gene polymorphisms Michal Kolorza, Katerina Wroblovaa, Jana Mokranovaa, Ladislava Bartosovaa, Petr Diteb, Vladimir Zborilc, Milan Bartosd

Introduction. Anti-TNFα monoclonal antibodies present an effective way of treating Crohn’s disease (CD). Despite their high benefits, there is about 30% rate of a primary non-response. The main target of infliximab is the soluble form of TNFα, which blocks its pro-inflammatory activity and the induction of apoptosis via the TNFα membrane form. The activity of TNFα and balance between its pro-inflammatory and pro-apoptotic effect is mediated by the interaction with its receptors (TNFR). Mechanisms of signaling via TNFα-TNFR interaction has been recently intensively studied from a perspective of selecting appropriate candidates for the infliximab treatment. Aim. The aim of this study was to evaluate whether polymorphisms in TNFRSF1A and TNFRSF1B genes influence the efficacy of the infliximab therapy. Methods. A total of 116 Caucasian CD patients treated with infliximab were genotyped. After initial 10 weeks of the infliximab therapy, effectiveness was determined and patients were divided into responders (n=98) and non-responders (n=18). Genotypes TNFRSF1A (T4672G, G3794C) and TNFRSF1B (T11695C, T587G) were determined by PCR-RFLP. Results. Frequencies of variant alleles of TNFRSF1A were comparable between responders and non-responders. Variant allele TNFRSF1B 11695C was more common in non-responders (41.7% vs. 30.1%). Similarly, the frequency of TNFRSF1B 587G allele in non-responders was 33.3% vs. 18.9% in responders. Homozygotes for variant alleles of TNFRSF1B 11695C were found more often (P=0.013; OR 5.89, CI 95% 1.6-22.1) in non-responders (n=5, 27.8%) than in responders (n=6, 6.1%). Our results imply that TNFRSF1B 11695C variant allele is associated with a low therapeutic effect of infliximab.

Key words: infliximab, single nucleotide polymorphism, pharmacogenomics, TNFR aDepartment of Human Pharmacology and Toxicology, UVPS Brno, Palackeho 1-3, 612 42 Brno, Czech Republic bUniversity Hospital of Ostrava, 17. listopadu 1790, 708 52 Ostrava, Czech Republic cDepartment of Internal Medicine and Hepatogastroenterology, University Hospital, Jihlavska 20, 625 00 Brno, Czech Republic dDepartment of Natural Drugs, UVPS Brno, Palackeho 1-3, 612 42 Brno, Czech Republic Corresponding author: Michal Kolorz, e-mail: [email protected]

INTRODUCTION A basic signaling mechanism at the molecular level is the interaction with TNFR (ref.11). Both TNF forms Biological therapy is today regarded as the most effec- (soluble (sTNF) and membrane-bound (tmTNF)) have tive therapy in various diseases, which pathophysiology an affinity to both types of receptors. Both TNF forms results from excessive immune activation and pathologi- influence cells with TNFR on the membrane and trig- cal prolongation of inflammatory reaction caused by an ger signaling cascade, which results in apoptosis or excessive activation of TNF-alpha. This is also confirmed activation of nuclear factor NF-κB and expression of by the clinical effect of monoclonal antibodies neutraliz- pro-inflammatory genes. Activation of TNFR1 (coded ing TNF-alpha and preventing manifestation of its func- by TNFRSF1A gene) results in the internalization of the tions. Infliximab became the first drug from the group of ligand-receptor complex and association of the “death anti-TNF antibodies, which was used in the CD therapy. domain” with adaptor proteins present in the cell’s cy- However, despite all benefits of the infliximab therapy, toplasm12. In case that after the binding of TNF-alpha to there remain approximately 30% of refractory patients1. TNFR1 there is no internalization of this complex, the A relatively high number of patients, who do not respond intracellular signaling is led via the activation of NF-κB, to the therapy, medical risks and associated high costs of nuclear factor, which influences the expression of several the biological treatment resulted in an intensive research pro-inflammatory genes. Similar process also occurs of factors, which would allow selection of patients with through interaction of sTNF with TNFR2 (coded by the greatest chance for the therapeutic success2-5. In the TNFRSF1B gene) (ref.13). Thus TNFR1 could exert oppos- last decade, there has been an emphasis on the patient’s ing biological effects – pro-apoptotic or pro-inflammatory, genetic profile, besides clinical parameters and biochemi- depending on the activation of caspase or NF-κB signal- cal markers of the disease1,3. Even though the results have ization, respectively14,15. For TNFR2, there is a typical so far been inconsistent2,6-8, the research intensively fo- interaction with tmTNF constituting an inherent part of cuses on the area of genome containing genes for TNF- the TNF-alpha negative reversal regulation of expression. alpha and proteins, which participate in the signaling This provides for the system of reverse signaling, leading processes9,10.

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Table 1. Characteristics of the study population.

n (%) Age M/F

Study population 116 38.2 (22-69) 71/45

Responders 98 (84) 38.5 (22-69) 60/38

Clinical and morphological response 51 (44) 38.5 (22-69) 31/20

Clinical response 47 (41) 38.5 (24-63) 29/18

Non-responders 18 (16) 36.4 (25-65) 11/7

Table 2. Clinical criteria for the evaluation of therapeutic effect.

Clinical criteria CDAI Primary responders CMR Symptomatologic improvement, endoscopically <150 and/or; ∆ - 70 and endoscopy conﬁ rmed morphological healing CR Symptomatologic improvement <150 and/or; ∆ - 70 Non-responders Resistance No symptomatology improvement >150 and ∆ < 70

CDAI- Crohn‘s disease activity index; CMR-Clinical and morphological response; CR-Clinical response.

Table 3. Genotype frequencies in study group, primary responders and non-responders.

Non- Primary responders All MAF responders (n=98) CMR (n=51) CR (n=47) (n=18) (n=116) TNFRSF1A -690G>T GG 38 18 20 6 44 GT 44 22 22 11 55 0.384 TT 16 11 5 1 17 -1488G>C GG 23 12 11 3 26 GC 47 21 26 11 58 0.526 CC 28 18 10 4 32 TNFRSF1B 11695C>T CC 45 22 23 8 53 CT 47 25 22 5 52 0.319 TT 642511 587T>C TT 65 35 30 8 73 TC 29 16 13 8 36 0.211 CC 40426

MAF-minor allele frequency; CMR-Clinical and morphological response; CR-Clinical response. to an inhibition of cytokines synthesis and apoptosis of MATHERIAL AND METHODS cells with tmTNF present at the membrane16. In this work, we retrospectively focused on the pos- Patients sible correlation between the infliximab effect in patients In this retrospective study we genotyped 116 unre- with CD and the occurrence of polymorphisms present lated Caucasian (Czech and Slovak) CD patients that in the genes for TNFRSF1A and 1B. were recommended the biological therapy according to standard criteria. All patients were given informed consent before entering the study. The study was approved by The Ethics Committee of University Hospital Brno

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(Czech Republic). Characteristics of the study population Correspondingly, the frequency of TNFRSF1B 587G are shown in Table 1. allele in non-responders was 33.3% vs. 18.9% in respond- Patients received the initial dose of infliximab (5mg/ ers. The frequency of this variant allele was lower in pa- kg/dose) in 0th, 2nd and 6th week. At the end of the first tients with clinical and morphological response 15.7% phase (after 10 weeks), the therapy effectiveness was de- (P=0.031; OR 2.68, CI 95% 1.12-6.44 compared with non- termined. responders).

Clinical criteria After the initial period of therapy, its efficiency was DISCUSSION evaluated by clinical criteria based on endoscopic examination and the CDAI change. Therapeutic response to In this work, we focused on polymorphisms located infliximab was defined as CDAI below 150 or as a drop in a gene sequence for TNFR1 and TNFR2 (TNFRSF1A by more than 70 points, steroid withdrawal or healing of and TNFRSF1B). Infliximab effectiveness after the first 10 fistulas (Table 2). Patients were divided into two groups: weeks of the therapy was studied in 116 patients. In 18 of group of primary responders (n=98) (within this group them (16%), there was no therapeutic response (primary we distinguished patients with clinical and morphologi- non-responders). Other patients (responders) reacted to cal response (n=51) and primary responders with clinical infliximab by an improvement of clinical parameters of response but without morphological healing (n=47) and the disease (n=47; 41%), and 51 patients (43%) also re- group of non-responders (n=18). vealed endoscopically verified morphological healing. An average age of patients included in the study was 38.2 Genotyping years, and there were no significant differences between Patients’ samples were genotyped for SNPs on the group of responders and non-responders (38.5 and TNFRSF1A (T-610G, rs4149570; G-1488C, rs4149569) 36.4, respectively). and TNFRSF1B (T11695C, rs976881; T587G, rs1061622) In the gene for TNFRSF1A, we monitored 2 SNP in the genes by PCR-RFLP. promoter area with an impact on the binding of transcription factors and expression of the receptor protein17. The Statistics frequencies of individual variant alleles were comparable Results were evaluated by Fisher’s exact probability in both groups of patients (38.8% and 36.1% in respond- test and Odds ratio was used for statistical evaluation. ers and 52.6% and 52.8% in non-responders for -690T P value below 0.05 was considered statistically significant. and -1488C, respectively). The frequencies of variant alleles correspond with results of another study, which was monitoring their occurrence in European population1. RESULTS In agreement with other authors7,10, in our study, there was found no association between the presence of variant Study population comprised 116 Caucasian patients alleles and therapeutic effect of the infliximab therapy. treated with infliximab, overall 98 (84%) achieved re- The frequency of the allele TNFRSF1B 11695A was sponse after the initial period of treatment (determined at in responders 30.1%. However, its frequency was signifi- 10th week after primary infliximab administration accord- cantly higher in non-responders (41.7%). The presence ing to standard diagnostic criteria – CDAI and endoscopy of homozygous genotype for this allele was associated examination), and 18 (16%) of patients had primary non- with response failure (P=0.0132). Individuals homozygous response. Genetic distribution of monitored SNPs and mi- for the variant allele totaled 27.8% in the groups of non- nor allele frequencies are shown in Table 3. Frequency of responders, compared with 6.1% in the group of respond- individual genotypes in all the monitored polymorphisms ers. Since this polymorphism is located in the intron 1 corresponded to Hardy-Weinberg equilibrium. sequence, we can hypothesize about the modification of Frequencies of variant alleles of TNFRSF1A were com- posttranslational processes or binding to another poly- parable between responders and non-responders (38.8% morphism in the exon area. However, more studies are and 36.1% for TNFRSF1A 4672G allele, 52.6% and 52.8% necessary to understand this mechanism. The distribution for 3794C allele, respectively). Variant allele TNFRSF1B of variant allele 587G was 18.9% and 33.3% in responders 11695C was more common in non-responders (41.7% vs. and non-responders, respectively. Even though this differ- 30.1%). Homozygotes for variant allele of TNFRSF1B ence in frequencies is considerable and corresponds with 11695C were found more often (P=0.013; OR 5.89, CI frequencies reported in other studies1,4,7,18 our results were 95% 1.6-22.1) in non-responders (n=5, 27.8%) than in not significant (P=0.073). Nevertheless, the frequency of responders (n=6, 6.1%). Homozygous patients for the this variant allele (15.7%) was lowest in patients with clini- variant allele TNFRSF1B 11695C were also more often cal reaction and at the same time morphological healing resistant to the therapy (n=5; 27.8%), compared with pa- (P=0.031), compared with non-responders). The presence tients with clinical and morphological reactions, where of variant allele leads to Met196Arg substitution in the the frequency of this allele was 7.8 % (n=4; P=0.045; OR fourth extracellular cysteine-rich domain. This substitu- 4.52, CI 95% 1.06-19.29), and similarly when compared tion disrupts receptor functions from the perspective of with a group of patients with clinical reaction (n=2; 4.3%, its shedding activity19. Changes in the protein primary P=0.015; OR 8.65, CI 95% 1.5-49.9). sequence leads to a disruption of the ability to induce

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Tumour necrosis factor-a receptor 1 and 2 polymor- 10.1155/2009/591704. phisms in inflammatory bowel disease and their association with 19. Till A, Rosenstiel P, Krippner-Heidenreich A, Mascheretti-Croucher response to infliximab. Aliment Pharmacol Ther 2004;20:303-10. S, Croucher PJ, Schäfer H, Scheurich P, Seegert D, Schreiber S. The 5. Vermeire S, Louis E, Carbonez A, Van Assche G, Noman M, Belaiche Met-196 -> Arg variation of human tumor necrosis factor recep- J, De Vos M, Van Gossum A, Pescatore P, Fiasse R, Pelekmans P, tor 2 (TNFR2) affects TNF-alpha-induced apoptosis by impaired Reynaert H, D'Haens G, Rutgeerts R. Demographic and clinical pa- NF-kappaB signaling and target gene expression. J Biol Chem rameters influencing the short-term outcome of anti-tumor necrosis 2005;280(7):5994-6004. factor (infliximab) treatment in Crohn's disease. Am J Gastroenterol 20. Fabris M, Tolusso B, Di Poi E, Assaloni R, Sinigaglia L, Ferraccioli G. 2002;97:2357-63. 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S78 Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2013 Sep; 157 (Supplement 1):S1–S88. C-7 Current challenges of body weight based intravenous busulfan dosing versus dose adjustment based on therapeutic drug monitoring Hundie Tesfayea, Romana Branovaa, Petr Rihab, Petr Sedlacekb, Jan Vydrac

Background. Busulfan in high doses is often used to substitute total body irradiation for bone marrow or hematopoietic stem cell transplantation (HSCT) conditioning therapy. Its considerable pharmacokinetic variability and worrying adverse effect in case of extreme exposure warrants pharmacokinetic monitoring in both oral and intravenous forms. Previous works suggested that test pharmacokinetic study enables dose prediction for all of the rest doses given every 6 hourly for four days. However, extensive case series observations and further studies indicate that the drug shows intra-individual variability challenging the first dose based prediction. Objectives. The principal aim of this communication is to describe typical cases, where dose prediction based on post initial doses concentrations measurement and consequent area under the concentration versus time curve (AUC) calculation may not be reliable and that may endanger the principal goal of the intervention. Patients and Methods. Data from three exemplary out layer cases have been processed from patients where prae- analysis provision of informed consent of a patient or the guardians in paediatric cases is standard. An adult male at age of 49 years, and two children (1 male and 1 female) both at age of 2 years, respectively have been treated with i.v. busulfan doses on body weight basis according to clinical protocol before HSCT. Sampling was started with trough concentration (immediately before the 5th dose) followed by samples immediately after the end of 2-4 h lasting infusion (peak), 4 h, and 6 h from the starting time of the infusion utilizing limited sample strategy. Busulfan concentrations were determined by high performance liquid chromatography (HPLC). AUC was calculated using the trapezoidal rule. Results. At initial measurement AUC in the adult patient and in the female infant case revealed unacceptably low exposure expressed by low AUC 496.4 μg/L.h and 1284 μg/L.h), respectively. In contrast, AUC Ctrough-C6 calculated according to the trapezoidal rule in the male child revealed overexposure expressed by AUC Ctrough-C6 11135 μg.L.h, which is evidently beyond myeloablative target AUC of 5000-7000 μg/L.h. All the three patients required another follow-up monitoring with or without dose adjustment. Conclusions. Our results demonstrate that even after i.v. busulfan administration, interindividual as well as intra- individual PK/PD variability is of great concern. The over all conclusions drown from these case series observation is to recommend inter-dose follow-up therapeutic drug monitoring instead of relaying on initial dose predictions as highly required tool to guarantee aimed target with careful interpretation of drug levels considering all influential factors. Thus, it is strongly suggested that follow-up AUC monitoring between doses may certainly help to reduce the risk of poor outcomes both in adult and paediatric HSCT patients.

Key words: TDM, busulfan, HSCT, intraindividual variability, dose adjustment AUC aDepartment of Medical Chemistry and Clinical Biochemistry, Division of Clinical Pharmacology, 2nd Faculty of Medicine, Charles University in Prague and Faculty Hospital in Motol, Prague, Czech Republic bDepartment of Paediatric Haematology and Oncology, 2nd Faculty of Medicine, Charles University in Prague and Faculty Hospital in Motol, Prague, Czech Republic cInstitute of Haematology and Blood Transfusion, Prague, Czech Republic

INTRODUCTION The drug is bifunctional alkylating agent, in which two labile methanesulfonate groups are attached to opposite Busulfan is an anti-cancer drug, in use since early ends of a four-carbon alkyl chain. In aqueous media, bu- 1960s. It is a cell cycle non-specific alkylating antineo- sulfan hydrolyzes to release the methanesulfonate groups. plastic agent, in the class of alkyl sulfonates. Its chemical This produces reactive carbonium ions that can alkylate designation is: deoxyribonucleic acid (DNA), which is thought to be re-

1, 4-butanediol dimethanesulfonate (C6H14O6S2 ) sponsible for much of the cytotoxicity of busulfan. Thus; its mechanism of action through alkylation produces guanine-adenine intrastrand crosslinks1. This occurs through a SN2 reaction in which the relatively nucleophilic guanine N7 attacks the carbon adjacent to the mesylate leaving group. This kind of damage cannot be repaired by cellular machinery and thus the cell undergoes apoptosis2. Since Fig. 1. Busulfan. 1970s busulfan high dose is used to replace total body

S79 Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2013 Sep; 157 (Supplement 1):S1–S88. irradiation as myeloablative preparatory regimen before CASE DESCRIPTION 1 bone marrow/hematopoietic stem cell transplantation3. Among the main challenges in this drug use is its wide A 49 year old Caucasian male with myelodysplastic inter- and intra- patient variability especially documented syndrome had been scheduled for umbilical cord blood in oral busulfan pharmacokinetics. Use of the intrave- transfusion (HCT) using busulfan/fludarabine/thiothepa/ nous (I.V.) formulation might reduce this variability by ATG as conditioning regimen. Busulfan 0.8 mg/m2 I.V. eliminating variability in absorption, whereas variability 2 h lasting infusion at 6 hourly intervals was put in the due to drug metabolism remains. Furthermore, systemic protocol. Plasma samples were obtained immediately be- exposure to intravenous busulfan appears to be relatively fore infusion, immediately post infusion, at 4, and 6 h low in children compared with adults at certain doses; from the start of initial dose infusion. The high perfor- however, whether this should dictate a dose increase re- mance liquid chromatography (HPLC) system consisted mains to be investigated in larger studies4. Assessment of of an isocratic pump with a wavelength detector, using pharmacokinetic profiles may allow the characterisation an automatic sampling system (ECOM, Prague, Czech of relationships between pharmacokinetic parameters and Republic) was applied to determine plasma busulfan efficacy and toxicity. Therefore, further studies of phar- concentrations. The ultraviolet detection was carried out macokinetically guided busulfan administration may be at 256 nm and the injected volume was 20 μL and the needed to validate pharmacokinetic-pharmacodynamic chromatograms were processed by Clarity software (Data relationships and to facilitate optimal dosage of the drug Apex) with the retention time 5.8 min for busulfan and in practice5. In clinical practice the interindividual phar- 11.8 min for internal standard, respectively. The calibra- macokinetics and dynamics variability necessitates AUC tion curve was evaluated as linear relation ship between calculation based dose adjustment to achieve the target. the peak area ratio for busulfan and internal standard Early pharmacodynamic studies suggested a significant using the calibration prepared from pooled human se- relationship between high exposure and the occurrence rum spiked with stock solution of busulfan as explained of veno-occlusive disease (VOD) of the liver. However, elsewhere8. Busulfan plasma concentration as measured pharmacodynamic studies are still required to define the by the validated method revealed undetectable in trough relationship between busulfan exposure and optimal trans- or steady state (Css) sample, 194.3 μg/L, 53.9 μg/L, un- plant outcome. For extensive pharmacokinetic study pur- detectable in samples immediately post two hour lasting pose, many samples were used, while some declare also in infusion (C2), at 4 h (C4), and 6 h (C6) from the start theory, only peak and trough levels should be necessary, of infusion, respectively. The AUC drown from these but for assurance of reliability in clinical decision making, concentrations was unacceptably very low bearing only it must be possible to identify outlier values. Dupuis et al.6 496.4 μg/L.h. Despite continuation with the initial dos- for instance recommended that busulfan AUC be calcu- age scheme, samples immediately before the 5th infusion, lated for children using a four-sampling technique. In later immediately after the end of infusion, 4 h after the start publication it was reported that even fewer samples than of 5th infusion, and at 6 h revealed concentrations 556.4 four using 2 or 3 plasma busulfan concentrations can be μg/L, 1604.9 μg/L, 711.7 μg/L, and 430 μg/L respectively used to reliably estimate busulfan AUC after I.V. admin- (Fig. 2). The resulting AUC of approximately 5620 μg/L.h istration in children undergoing HSCT7. In the present as calculated using the trapezoidal rule has been accepted three case studies including an adult and two paediatric for being within the required myeloablative target. patients, the unreliability of fixed dose and initial AUC estimates where busulfan intravenous busulfan has been prescribed as part of a myeloablative regimen prior to HSCT is the described.

Fig. 2. (Case 1) demonstrating significant busulfan blood concentration versus time profile difference in the same subject with the same I.V. dosing regimen in an adult patient.

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Fig. 3. (Case 2) demonstrating significant busulfan blood concentration at trough, 2- 12 h from the start of infusion (C2-C12) profile difference in the same subject with the same total I.V. dose, but at different dosing interval (12 hourly red line versus 6 hourly blue line) in a paediatric female patient illustrating considerable variability.

CASE DESCRIPTION 2 using umbilical cord blood with 9/10 HLA (human leuko- cyte antigen) matched, i.e., with Cw mismatch 4.4x10/5/ The 2 years old girl has been admitted with diagnosis kg CD34+ (cluster of differentiation) cell count has been of Langerhans cell histiocytosis and secondary haemo- conducted. Thereafter, the patient was in excellent clini- phagocytic lymphohistocytosis. After the failure of con- cal state, mostly nonfebrile, in exception of elevation of ventional chemotherapy, the patient has been included in c-reactive protein (CRP) attributed to the underlying the schedule for HSCT to be proceeded by myeloablative disease activity still persistent shortly after HSCT. On preparative regimen, which contained high dose busulfan further follow-up also the patient was in good condition, and fludarabine including prophylaxis against graft versus except for mild sign of mucositis. Since D+1 (1st day after host disease (GVHD) with alemtuzumab (Campath) ad- HSCT), Defibrotide 25mg/kg as prophylaxis against VOD ministration. The patient started I.V. busulfan on actual has been administered. For unexplained CRP elevation, body weight basis, i.e., 2 mg/kg (17 mg/dose at 12 h inter- corticosteroid therapy (1 mg/kg) has been applied. CsA vals with infusion rate of four hours). Blood samples for blood level was also low after the first three days of ad- therapeutic monitoring were collected starting with trough ministration and the dose has been escalated to reach the (before the 4th infusion), then immediately after the end of recommended target. Since D+8 fever episodes dictated the 4th infusion, 8 h, and 12 h from the start time of the combination antibiotic therapy (Amikacin + Targocid + 4th dose. Surprisingly, very low (near the lower detection Meronem). On D+11(11th day after HSCT), Defibrotide limit) steady state concentration (trough) consequently has been terminated for absence of VOD symptoms. leading to unacceptably low exposure expressed by low Corticosteroid therapy, which was terminated on D+15 AUC (1284 μg/L.h) was revealed. After this evidence the (15th day after HSCT), has been restarted at a dose of dosing interval was shortened to 6 hourly regimen with 1 mg/kg/day due to fever up to 39 °C and elevation of the rate of infusion also switched to 2 h (1.2 mg/kg/dose inflammatory markers. The effect of cortico-therapy was i.e., 10.5 mg at 6 hourly intervals (7 doses). Later, the evident after initial dose leading to improvements of clini- AUC of 6139 μg/L.h, which is within the required tar- cal state and laboratory markers. Engraftment with leuko- get, has been achieved. In addition to change of dosing cytes was evident by D+22 (22nd day after HSCT). Later interval, there was a dose escalation by one extra dose during post transplantation period, hemoculture revealed than previously planed. Due extremely low initial busulfan Candida lusitaniae, which has been successfully treated bioavailability, blood sample has been preserved for phar- with combination of posaconazole and mycamine. Later macogenetic analyses or eventual rapid metabolism based follow-up revealed post transplantation lymphoprolefera- on enzyme polymorphism. Further analysis for genetic tive disorder (Epstein Barr Virus (EBV)-induced diffuse polymorphism in this case did not proved genotype, which large B-cell lymphoma (DLBCL), for which the patient may explain the underlying reason for faster elimination of has been successfully treated by totally 8 doses of ritux- the drug given by standard dose (Fig. 3). Allogenic HSCT imab, and now is on regular follow-up management.

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CASE DESCRIPTION 3 episode of haemorrhagic cystitis, most probably of BK virus (a member of polyomavirus) aetiology, otherwise is A 2 year old male infant with acute myeloid leuke- doing well and still in institutional care at the transplanta- mia was scheduled for busulfan myeloablative therapy tion unit. (1.2 mg/kg based on his actual body weight of 11.1 kg). For planed Allogenic HSCT, from sibling (sister) donor, Busulfan + Cyclophosfamide + Melphalan (Bu+Cy+Me) DISCUSSION pre transplantation conditioning regimen including cyclo- sporine-A as prophylaxis against graft versus host disease High-dose busulfan is widely used instead of total body (GVHD) targeting trough concentration of 100-150 μg/L irradiation (TBI) before bone marrow or haemopoietic has been indicated. Busulfan estimated dose of 13 mg stem cell transplantation (HSCT) conditioning regimen has been administered I.V. over 2 hour infusion at 6 hour in both adults and children. Its considerable pharmaco- intervals. Blood samples for therapeutic drug monitoring kinetic variability and worrying adverse effects in case (TDM) purpose have been obtained starting from trough of extreme exposure or therapy failure in case of under- after 4th dose, at peak (immediately after the completion exposure warrant pharmacokinetic monitoring in both of the 5th infusion), at 4 h, and 6 h counted from starting oral and intravenous forms. Previous works suggested that time of the 5th infusion. These four samples were anal- test pharmacokinetic study enables dose prediction for all ysed by HPLC method as explained above and used to 16 doses given every 6 h. However, extensive case series calculate AUC at steady state based on our already de- observations and further studies indicate that the drug veloped limited sampling strategy for TDM of busulfan. shows intra-individual variability challenging the first dose

AUC Ctrough- 6 calculated according to the trapezoidal rule analysis based prediction. Variation in the area under the revealed potential overexposure (11135 μg/L.h) as also concentration/time curve (AUC) of busulfan could results illustrated on Fig. 3. Based on this observation the ini- in substantial risk of over or under treatment with excess tial dose has been reduced by 2 mg per dose. However, risk of toxicity or relapse. Thus intensive therapeutic drug concentrations after the dose adjustment also showed cu- monitoring primarily by determination of drug plasma lev- mulative overexposure, despite evident dose reduction. els using validated methods such as high performance liq- Finally, we decided to further reduce the dose to only 8 uid chromatography (HPLC) as explained elsewhere8 and mg per dose. In this case, after more (approximately 40 followed by AUC calculation to be within required targets %) dose reduction, the AUC still proved to remain above maybe helpful. In conventional sampling regimen for de- the target required on the protocol. As far as outcomes termining AUC after oral administration, over 10 samples is concerned, since 12th day post transplantation, gradual were used to assure accurate tracking of erratic absorp- manifestation of hepatopathy with increased bilirubine- tion. In limited sampling strategy theoretically, only peak mia – max. 70 μmol/L, and liver aminotransaminases and trough levels may be necessary, but for assurance of (GMT max. 11, ALP max. 10, AST 3, ALT 5 μkat/L), all reliability in clinical decision making with possibilities to of which restored within 10 days without further thera- identify outlier values, at least 4 samples may be required9. peutic interventions has been observed. For suspected Based on AUC observed, available guidelines recommend veno-occlusive disease (VOD), defibrotide has been ad- dose escalation or reduction up to 20 % of the initial dose ministered only for several days, and discontinued for not or according to a conventional formula to obtain target fulfilling the VOD criteria (symptoms). To date (28 days AUC between 5000 and 7000 μg/L.h myeloablative range. post transplantation) the child is without signs of liver However, in the demonstrated case of our adult patient impairment, with complete engraftment (of both leuko- (Case 1) such escalation of doses might lead to potentially cyte and thrombocyte elements). The patient suffered an toxic exposure. Provided the same approach of clinical

Fig. 4. (Case 3) demonstrating significant busulfan blood concentration versus time profile difference in the same subject with the same I.V. dosing regimen in paediatric male patient.

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Table 1. Conventional actual body weight based busulfan dosing scheme in children.

Actual body weight (kg) < 9 kg 9 to < 16 kg 16 to 23 kg > 23 to 34 kg > 34 kg

Busulfan dose (mg) 1.0 mg/kg 1.2 mg/kg 1.1 mg/kg 0.95 mg/kg 0.8 mg/kg

and laboratory procedures applied, significant differences lowed. Nevertheless the extended interval dosing in our in concentrations post initial and later doses may be only female patient also showed significantly different AUC explained by intra-individual variability in this adult pa- on the same bodyweight based dose for yet not have been tient as we previously confirmed in paediatric patients well explained reason, meanhile faster metabolism at ini- both on oral and intravenous forms of the drug10. The case tial dose cannot be excluded in this case. According to published just two years ago by Johnson-Davis et al.11 also our observations the relevance of bodyweight based fixed demonstrated significant intra-patient variability especial- dose busulfan dosing scheme requires review or otherwise ly using concomitant drugs warranting more concern to warrants the use of validated limited sampling strategy, further monitor the therapy. McPherson et al.12 reported which may make possible meaningful and more accurate that hepatic veno-occlusive disease occurred in 32% out use of busulfan for pre HSCT preparative regimen9. of 25 patients, but not evidently associated with busulfan AUC. However, low AUC was associated with partial do- nor chimerism, while full donor chimerism has been ob- CONCLUSIONS served in cases within target AUC. Nevertheless, there are other reports suggesting that high plasma concentrations 1) AUC after initial doses of busulfan may not pre- of busulfan have been linked to the occurrence of hepatic dict exposure throughout therapy, due to significant intra- venoocclusive disease, a severe complication associated patient variability conditioned by several patient factors. with a high mortality13. There is an evidence that busulfan 2) Follow-up therapeutic drug monitoring has been pharmacokinetics showed high inter- and intra-individual shown to be highly required tool to guarantee aimed target variability in HSCT using a targeted busulfan/fludarabine in both adult and children patients with careful interpreta- regimen, which indicates the need for intensive monitor- tion of drug levels considering all influential factors. ing and dose adjustment to improve the outcome of HSCT 3) Body weight based fixed dose busulfan dosing (ref.14). Therapeutic monitoring of intravenous busulfan needs review as shortcomings exist and individual ap- is to increase the efficacy and safety of busulfan-based proach is still of vital importance. As overall conclusion, conditioning protocols in paediatric HSCT recipients15. we recommend therapeutic drug monitoring after day 1 of Significant intra-individual variability exists in the appar- application and follow-up between next doses instead of ent oral clearance of busulfan and follow-up therapeutic predicting the pharmacokinetics for the whole 16 doses drug monitoring is recommended particularly if the de- based only on first test dose to secure that the right dose is sired target AUC is narrow16. Interestingly, the paper pub- provided in this challenging intervention due to consider- lished by Bartelink et al.17 expressed that dose adjustment able intra-individual variability. based on AUC of day 1 led to unexpectedly low AUC with unfavourable consequences as demonstrated clearly at least in two of their patients. That is why any changes ACKNOWLEDGEMENTS made based on early monitoring should be followed by another control of blood levels to know actual AUC for Supported by the project (Ministry of Health, Czech eventual dose readjustment as we have published in our Republic) for conceptual development of research orga- preliminary report of limited sample based therapeutic nization 00064203 (University Hospital Motol, Prague, drug monitoring of busulfan in children10. In contrast to Czech Republic) the adult (Case 1), in a paediatric patient (Case 2), to our surprise the AUC later showed unacceptably high and led us to significant dose reduction (approx. by 40% from the REFERENCES initial one). Actual body weight based busulfan dosing schedule (Table 1.) counts with relatively higher dose re- 1. 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Adamcova M. P-32 Eichlerova A. O-12, P-38 Adamkov M. O-25, P-49 Eisner T. P-58 Adamus M. P-1 Elgyuttova Z. P-13 Amchova P. P-29 Enayat S. O-13 Andersson K. P-4 Fiala P. P-19 Anzenbacher P. O-15, P-1, P-26, P-57, P-59, Filipsky T. O-5, O-28 P-65, P-66, C-2, C-3 Foltanova T. P-13, P-20 Anzenbacherova E. O-15, P-1, P-26, P-57, P-66, C-2 Fleskova D. P-12 Artinian S. O-9 Franova S. O-7, O-25, P-12, P-22, P-49 Baciak L. O-6, P-44 Frydrych M. P-2 Bachleda P. P-1 Gajdacova B. P-43 Bado O. P-2 Gallo-Payet N. O-8 Balazova M. P-3 Galkova K. P-14, P-36, P-43, P-53 Balis P. P-52 Gersl V. P-21, P-32, P-58 Banerjee S. O-13 Gonec T. P-27 Barta P. P-4 Gonsalvesova E. P-10, P-35 Barta T. O-9 Hajkova J. P-62 Bartikova H. P-66 Hampl A. O-9 Bartos M. C-6 Hanzlikova P. O-29 Bartosikova L. P-5 Hargas L. P-22 Bartosova L. O-10, O-24, , O-29, P-24, C-6 Harmatha J. O-16, O-26 Basora N. O-8 Hartinger J. O-32 Batovsky M. O-10 Haskova P. P-58 Bauerova K. O-26 Hatok J. P-34 Bebarova M. O-1 Havlickova T. P-15 Bernatova I. P-52 Hess L. P-61 Bousova I. P-66 Hofman J. P-16 Bludovska M. P-6, C-4 Holcapek M. O-15 Bozik M. O-19, O-23 Holeckova M. O-28, P-48 Branova R. O-30, C-7 Horakova Z. O-24 Bures J. P-21, P-31 Horakova K. O-29 Caganova B. P-41 Horova A. P-23 Calkovska A. P-34, P-37, P-42 Horska K. P-27 Ceckova M. P-8, P-47, P-62 Hosek J. O-9 Cermanova J. P-11, P-62 Hrabovska A. O-2, O-3, O-14 Cerveny L. P-39 Hrdina R. O-5, O-28, P-48 Cihalova D. P-8 Hrianka M. P-22 Corejova A. O-2 Hroch M. P-62 Csaszarova E. O-4, P-7 Hrtanek I. P-17 Diez-Dacal B. O-13 Hruskova K. P-58 Dingova D. O-3 Hulman M. P-35 Dite P. C-6 Hyrsova L. P-18 Dlouha M. P-9 Chalupova M. P-19, P-54 Dobias L. P-45 Chladek J. P-21, P-25 Dobiasova M. O-26 Chottova-Dvorakova M. P-6, C-4 Doka G. P-10, P-35, P-46, P-53 Jancinova V. O-16 Dokus K. P-49 Janeba Z. P-33, P-64 Dovinova I. P-52 Jankyova S. P-13, P-20, P-28 Drabikova K. O-16 Jansa P. P-64 Drafi F. O-26 Janosikova D. O-2 Drgova A. P-42 Jansova H. P-58 Dubecka M. P-11 Jerabek P. P-15 Dubovicky M. O-4, O-6, P-7 Jezdinsky J. O-31 Dukat A. O-19, O-23 Jirasko R. O-15 Dusilova A. P-63, C-1 Jirkovsky E. P-21, P-32, P-58 Dvorak Z. O-20, P-18 Joskova M. O-7, P-22, P-49

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Jourova L. P-66 Lencova O. P-21, P-32, P-58 Jover R. P-18 Lima M. P-47 Juranek I. P-44 Liskova B. P-66 Jurecek L. P-12 Macek K. P-25 Kadova Z. P-62 Mackova E. P-58 Kamenska R. P-50 Mach M. O-4, P-7 Kassa J. P-23 Majkova A. P-13 Kasparova S. O-6 Majekova M. O-13 Kazdova L. P-59, C-3 Majzunova M. P-52 Kazimierova I. O-7 Malcekova B. P-16 Kebis A. O-6 Malek J. P-61 Kilianova Z. O-8 Malikova E. P-14, P-36, P-53 Klimas J. P-10, P-14, P-36, P-43, P-46, Mandikova J. P-33, P-40, P-60 P-53 Markovicova Z. P-27 Kmonickova E. P-63, P-64, C-1 Martinkova J. O-11, O-17 Kocmalova M. O-25 Martinkova P. P-58 Kollar P. O-9, P-54 Matejovic P. O-1 Kolman V. P-64 Matouskova E. O-32 Kolmanova E. P-24 Matuskova Z. P-59, C-3 Kolorz M. O-10, O-24, O-29, C-6 Mazalova L. P-55 Koniar D. P-22 Mazurova Y. P-32 Kopacova M. P-31 Medvedova I. O-12 , P-38 Kopecky J. P-25 Mensikova L. P-43 Kopecna Zapletalova M. P-26, P-57, C-2 Mesarosova L. P-53 Kopincova J. P-34 Micuda S. P-11, P-62 Kostecka P. P-63, C-1 Mihalova D. O-26 Kotolova H. P-27 Milackova I. O-13 Kotyzova D. P-6, C-4 Miklosova Z. P-29 Kovarikova P. P-21 Mikolka P. O-12, P-34, P-37, P-38 Kralova E. P-14, P-20, P-28 Mikova M. O-2 Kratochvil M. P-58 Miksova Z. P-55 Krausova L. P-18 Mikusek J. O-15 Krejci E. O-3 Mikusiakova-Tomcikova L. P-42 Krejci K. P-65 Mistrova E. P-6, C-4 Kristova V. O-21, O-22 Mladenka P. O-5, O-28, P-48 Kriska M. O-21, O-27 Mlynarova J. P-35 Krockova A. P-28 Mokra D. O-12, P-34, P-37, P-38, P-42 Krsiak M. P-15 Mokry J. O-12, O-18, P-38, P-62 Kruzicova A. P-54 Mokranova J. O-10, C-6 Krenek P. P-10, P-14, P-36, P-43, P-46, Mrvova K. O-14 P-53 Muchova Z. P-41 Kuca K. P-31 Murin J. O-19, O-23 Kucera M. O-14 Musil P. P-35 Kucerova J. P-29 Musilek K. P-23 Kudlackova Z. P-62 Muller Zavalova V. O-9, P-9, C-5 Kulhan T. P-30 Nachtigal P. P-62 Kuncirova V. O-26 Navarova J. O-13, P-28 Kunes J. O-15 Nekvindova J. P-66 Kunes M. P-31 Netikova I. O-32 Kuzelova M. P-3, P-50 Neumanova Z. P-39 Kverka M. P-63, C-1 Nobilis M. O-15 Kvetina J. O-15, P-25, P-31 Nosal R. O-16, O-26 Kyselovic J. O-2, P-10, P-35, P-46, P-53 Nosalova G. P-12, P-17, P-30, P-51 Lackova D. P-25 Novotna E. P-16 Laho T. P-11, P-62 Novy Z. P-40 Lakatosova S. O-2 Oliyarnyk O. P-59, C-3 Langerova P. P-56 Ondrejka I. P-12, P-17, P-30, P-51 Laznicek M. P-40, P-60 Ondriasova E. P-41 Laznickova A. P-40, P-60 Oravec M. O-25

S86 Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2013 Sep; 157 (Supplement 1):S1–S88.

Orsag J. P-65 Solovic I. O-18 Palicka V. O-28, P-48 Soltesova-Prnova M. O-13 Panek P. P-20 Sotnikova R. O-13, P-52 Parak T. P-19, P-24, P-54 Soucek K. O-9 Pasek M. O-1 Spicakova A. P-1, P-26, P-57, C-2 Pav I. O-24 Snircova E. P-51 Pavek P. O-20, P-11, P-18, P-33, P-62 Srankova J. P-14, P-36, P-43, P-53 Pavlik M. P-31 Staffa R. C-5 Payet MD. O-8 Stariat J. P-21 Pechanova O. O-7 Staud F. P-8, P-39, P-47, P-62 Perecko T. O-16 Stefek M. O-13 Perez-Sala D. O-13 Stejskalova L. O-20 Petrova M. O-19, O-22 Sterba M. P-21, P-32, P-58 Petruzelka L. O-32 Strbova P. P-55, P-56 Pistekova H. P-37, P-42 Strojil J. O-31, P-65, C-3 Pivackova L. P-43, P-53 Suchankova H. P-56 Plackova S. P-41 Suchy P. P-19, P-24, P-54 Placha K. P-44 Suchy P. Jr. O-9 Pokorna P. O-11, O-17 Sulcova A. P-29 Polaskova A. P-25 Sumbalova Z. P-44 Ponist S. O-26 Sus J. P-58 Porvaznik I. O-18 Sustkova-Fiserova M. P-15 Potocarova M. O-19, O-23 Sutovska M. O-7, O-25, P-49 Pospisilova V. O-2 Szotakova B. O-15 Pour M. O-15 Tacheci I. P-31 Prazanova G. P-54 Talhouk R. O-9 Prso M. O-12, P-38 Tengler J. P-2 Puzserova A. P-52 Tesfaye H. O-30, C-7 Racanska E. P-45 Tichotova L. P-58 Rackova L. O-13 Tomankova V. P-66 Radik M. P-46 Tomcikova L. P-34, P-37 Rauova D. O-2 Tomsik P. P-62 Ray B. P-12 Tonhajzerova I. P-42 Rejchrt S. P-31 Trejtnar F. P-4, P-18, P-33, P-40, P-60 Repcakova M. P-42 Tumova I. O-26, P-45 Repisky J. O-2 Tumova M. P-23 Reznicek J. P-47 Tuskova R. O-6 Riha P. O-30, C-7 Tylecek J. P-27 Riha M. O-5, P-48 Ujhazy E. O-4, P-7 Roh J. P-58 Ulrichova J. P-1 Rulcova A. O-20, P-18 Urbanek K. P-55, P-56, P-65 Sadlonova V. P-22, P-49 Urbaskova M. P-19 Sedlacek P. O-30, C-7 Vanduchova A. P-26, P-57, C-2 Sedlackova N. O-4 Varga Z. O-21 Semecky V. P-48 Vavrinec P. P-36 Sepsova V. P-23 Vavrincova D. P-36 Simunek T. P-58 Vavrova A. P-13, P-58 Simurda J. O-1 Vavrova J. O-28, P-48 Simurdova M. O-1 Vavrova K. P-58 Skarka A. P-16 Vesely P. O-32 Skalova L. P-66 Vecera R. P-59, C-3 Skottova N. P-59, C-3 Villaris R. O-22 Slazneva J. P-50 Visnovcova Z. P-42 Slezak P. P-52 Visnovsky J. P-49 Slezakova V. O-19, O-23 Vojtko R. O-22 Sliva J. P-61 Volkova M. P-33, P-60 Smejkal K. O-9 Voprsalova M. P-48 Smidrkal J. O-16 Votava M. P-61 Smutny T. P-18 Vrzal R. O-20, P-18

S87 Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2013 Sep; 157 (Supplement 1):S1–S88.

Vydra J. O-30, C-7 Zacharova A. P-59, C-3 Vyslouzil L. P-25 Zboril V. O-10, C-6 Wawruch M. O-19, O-23 Zidek Z. P-63, P-64, C-1 Wroblova K. O-10, O-24, C-6 Zizlavsky V. P-9, C-5 Wsol V. P-16 Zoulova J. P-25 Zagorova M. P-11, P-62

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STŘEDA 11.9.

14:00 zahájení 63. Farmakologických dnů

14:15–15:00 Zahajovací přednáška Vybrané aspekty vývoje nových léčiv Král V (VŠCHT a Zentiva, Praha)

Sekce experimentální farmakologie a toxikologie:

15:00 Elucidation of the transformation of nabumetone to the active metabolite, 6-methoxy-2-naphthylacetic acid (6-MNA) Nobilis M, Mikušek J, Szotáková B, Kuneš J, Pour M, Jirásko R, Holčapek M, Anzenbacherová E, Anzenbacher P, Květina J 15:20 Zlepšenie štandardnej anti-artritickej liečby pridaním N-feruloyl serotonínu k metotrexátu hodnotené na modeli adjuvantnej artritídy Kuncírová V, Poništ S, Dráﬁ F, Mihalová D, Dobiašová M, Harmatha J, Nosáľ R, Bauerová K 15:40 Ethanol a jeho hlavní metabolit acetaldehyd ovlivňují inward rectiﬁ er draslíkový proud IK1 u komorových srdečních buněk potkana Bébarová M, Matejovič P, Pásek M, Šimurdová M, Šimurda J 16:00 Polyfenolické zmesy a experimentálne vyvolaná alergická astma Kazimierova I, Jošková M, Pecháňová O, Šutovská M, Fraňová S

Přestávka: 16:20–16:40

16:40 BKCa kanály a experimentálna alergická astma Kocmálová M, Oravec M, Adamkov M, Šutovská M, Fraňová S 17:00 Vplyv roﬂ umilastu na in vivo a in vitro reaktivitu dýchacích ciest a apoptózu u ovalbumínom senzibilizova- ných morčiat Medveďová I, Pršo M, Eichlerová A, Mokrý J, Mokrá D, Mikolka P 17:20 Predklinické štúdie nového derivátu kvercetínu-inhibítora aldoketoreduktáz AKR1B1 a AKR1B10. Význam v modeloch chronických diabetických komplikácií, v modeli zápalu a rakoviny Miláčková I, Šoltésová-Prnová M, Májeková M, Sotníková R, Navarová J, Račková L, Díez-Dacal B, Peréz-Sala D, Enayat S, Banerjee S, Štefek M 17:40 Štúdium molekulových foriem cholínesteráz v srdci Dingová D, Krejči E, Hrabovská A 18:00 On the molecular pharmacology of oxidative burst inhibition in human neutrophils Nosál R, Drábiková K, Jančinová V, Perečko T, Harmatha J, Šmidrkal J

19:00 Společenský večer

ČTVRTEK 12. 9.

Sekce experimentální farmakologie a toxikologie – pokračování

9:00 Iron-chelating agents and acute myocardial infarction: in vitro and in vivo study Filipský T, Říha M, Hrdina R, Mladěnka P 9:20 Study of Simvastatin & Creatine citrate therapy in animal model of dementia by in vivo proton and phosphorus magnetic resonance spectroscopy Kašparová S, Bačiak L, Tušková R, Kebis A, Dubovický M 9:40 Developmental manipulation of monoaminergic systems aﬀ ects neurobehavioral and neuroendocrine regulations Dubovický M, Császárová E, Ujházy E, Sedláčková N, Mach M 10:00 Úloha nikotínových receptorov vo fyziológii srdca Mrvová K, Kučera M, Hrabovská A

10:30–11:30 Shromáždění členů ČSEKFT

12:00–14:00 Oběd, Diskuse u posterů

Klinická sekce:

14:00 Farmakoterapie septických nezralých novorozenců v kritickém stavu. Význam komorbidit. 1. gentamicin při současném perzistujícím ductus arteriosus. Martínková J, Pokorná P 14:20 Farmakoterapie septických nezralých novorozenců v kritickém stavu. Význam komorbidit. 2. gentamicin při současné perinatální asfyxiii. Pokorná P, Martínková J 14:40 Analysis of non-steroidal anti-infl ammatory drug usage and risk perception in hospitalized patients Varga Z, Kristová V, Kriška M 15:00 The specifi c issues in pharmacotherapy in elderly patients – misprescription and underprescription Wawruch M, Slezáková V, Murin J, Dukat A, Bozik M, Potocarová M 15:20 Issues of oral anticoagulants prescription in elderly patients with atrial fi brillation Slezáková V, Potocarová M, Murin J, Dukat A, Bozik M, Petrová M, Wawruch M

15:40–16:00 přestávka

16:00 Vieme predchádzať nežiadúcim interakciám v praxi. Kriška M 16:20 Eff ect of methycobalamin application in patients with autism Čorejová A, Rauová D, Jánošíková D, Pospíšilová V, Miková M, Repiský J, Lakatošová S, Hrabovská A, Kyselovič J 16:40 Current challenges of body weight based intravenous busulfan dosing versus dose adjustment based on therapeutic drug monitoring. Tesfaye H, Branova R, Riha J, Sedlacek P, Vydra J 17:00 Impact of diabetic state in patients with end-stage heart failure and its association with cardiac expression of microRNAs. Dóka G, Křenek P, Klimas J, Goncalvesová E, Kyselovič J 17:20 Diff erential gene expression of important factors in human epicardial adipose tissue and left ventricular myocardium in end-stage heart failure. Mlynárová J, Dóka G, Musil P, Hulman M, Goncalvesová E, Kyselovič J 17:40 Eff ect of fenugreek seeds enriched diet on endothelial dysfunction in mild diabetes and its relation to nitric oxide and epoxyeicosatrienoic acids pathway Piváčková L, Šrankova J, Gajdáčová B, Menšíková L, Galková K, Křenek P, Klimas J 18:00 First clinical pharmacology department in the world Strojil J, Jezdinský J

18:30 Volný večer

PÁTEK 13. 9.

Sekce molekulární a buněčné farmakologie:

9:00 Polymorphisms in protein C gene promoter region and endothelial protein C receptor gene as predisposing factors for venous thrombosis Horáková K, Bartošová L, Kolorz M, Hanzlíková P, Wróblová K 9:20 Association of haplotypes HLA-DQ2, HLA-DQ8 and polymorphism G-308A in TNF-alpha gene with coeliac disease Wroblová K, Kolorz M, Horáková Z, Pav I, Bartošová L 9:40 TNFRSF1A and TNFRSF1B gene polymorphisms and their impact on eﬀ ectivness of therapy with inﬂ iximab Kolorz M, Wroblová K, Baťovský M, Zbořil V, Mokřanová J, Bartošová L 10:00 ACTH-mediated regulation of the human MC2R. Kiliánová Z, Basora N, D Payet M, Gallo-Payet N

10:20–10:40 přestávka

10:40 Diagnostika rezistentných foriem tuberkulózy – konvenčné metódy versus molekulárno-genetické metódy Porvazník I, Mokrý J, Solovič I 11:00 Aryl hydrocarbon receptor and its crosstalk with glucocorticoid receptor in human placental barrier Stejskalová L, Rulcová A, Vrzal R, Dvořák Z, Pávek P 11:20 Flavonoids from Morus alba aﬀ ect cell cycle of human cancer cells and inﬂ ammatory response in macrophage-like cells. Kollar P, Bárta T, Hošek J, Souček K, Müller Závalová V, Artinian S, Talhouk R, Šmejkal K, Suchý P Jr, Hampl A 11:40 Commonly measured biomarkers of oxidative stress do not reliably predict cardiovascular impairment Mladěnka P, Filipský T, Vávrová J, Holečková M, Palička V, Hrdina R

Varia:

12:00 Application of computer-based modeling in the analysis of cardiovascular regulation mechanisms Vojtko R, Petrová M, Villaris R, Kristová V 12:20 The Protective Eﬀ ect of Pyrimidine Nucleosides and their Combinations on HaCaT Keratinocytes Treated with 5-FU: MTT, NTCA and RTCA Tests Hartinger J, Veselý P, Matoušková E, Petruželka L, Netíková I

12:40 Závěrečné slovo a ukončení 63. Farmakologických dnů Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. INSTRUCTIONS TO AUTHORS

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