Folia Pharmaceutica Universitatis Carolinae

UNIVERSITAS CAROLINA PRAGENSIS FOLIA PHARMACEUTICA UNIVERSITATIS CAROLINAE ISSN 1210-9495 FOLIA PHARMACEUTICA UNIVERSITATIS CAROLINAE L L folia_L_obalka.indd 1folia_L_obalka.indd 1 15.03.22 9:2415.03.22 9:24 FOLIA PHARMACEUTICA UNIVERSITATIS CAROLINAE L Arranged by Assoc. Prof. RNDr. Veronika Opletalová, Ph.D. Lectured: Assoc. Prof. RNDr. Věra Klimešová, CSc., Assoc. Prof. RNDr. Pavel Doležal, CSc. Editorial Advisory Board: Assoc. Prof. RNDr. Pavel Doležal, CSc., Prof. PharmDr. Martin Doležal, Ph.D., Prof. MUDr. Jaroslav Dršata, CSc., Prof. MUDr. Radomír Hrdina, CSc., Prof. RNDr. Lubomír Opletal, CSc., Assoc. Prof. RNDr. Veronika Opletalová, Ph.D., Assoc. Prof. RNDr. Miroslav Polášek, CSc., Prof. RNDr. Jiří Vlček, CSc., Assoc. Prof. RNDr. Veronika Opletalová, Ph.D. (editor) Published by Charles University Karolinum Press Prague 2022 Typeset by Karolinum Press © Charles University, 2022 ISSN 1210-9495 ISBN 978-80-246-5041-8 ISBN 978-80-246-5202-3 (pdf) https://doi.org/10.14712/9788024652023 Univerzita Karlova Nakladatelství Karolinum www.karolinum.cz [email protected] CONTENTS Abstracts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 9th Postgradual and 7th Postdoctoral Scientific Conference of the Faculty of Pharmacy in Hradec Králové, Charles University, Hradec Králové, 23–24 January 2019 Dedicated to the 50th Anniversary of the Founding of the Faculty of Pharmacy in Hradec Králové . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 Bioorganic and Pharmaceutical Chemistry Section . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 Pharmacognosy and Toxicology of Natural Products Section . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 Pharmaceutical Technology Section . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29 Pharmaceutical Analysis and Bioanalytical Chemistry Section . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 Pathobiochemistry and Xenobiochemistry Section . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55 Pharmacology and Toxicology Section . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65 Clinical and Social Pharmacy Section . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81 27th National Students’ Scientific Conference of the Faculty of Pharmacy in Hradec Králové, Charles University, Hradec Králové, 16–17 April, 2019 . . . . . . . . . . . 87 Section of Biological Sciences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87 Section of Chemical Sciences: Synthetic Part . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108 Section of Chemical Sciences: Analytical Part . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126 Section of Technological Sciences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134 Section of Social and Clinical Pharmacy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143 Social Happenings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149 Jubilee of Prof. RNDr. Eva Kvasničková, CSc. (J. Dršata) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149 PharmDr. Magda Vytřísalová, Ph.D., passed away . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151 Instructions for Authors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153 7 2021 Folia Pharm . Univ . Carol . L Pag . 7–86 ABSTRACTS 9th POSTGRADUAL AND 7th POSTDOCTORAL SCIENTIFIC CONFERENCE OF THE FACULTY OF PHARMACY IN HRADEC KRÁLOVÉ, CHARLES UNIVERSITY, HRADEC KRÁLOVÉ, 23–24 JANUARY 2019 DEDICATED TO THE 50th ANNIVERSARY OF THE FOUNDING OF THE FACULTY OF PHARMACY IN HRADEC KRÁLOVÉ BIOORGANIC AND PHARMACEUTICAL CHEMISTRY SECTION MICROBIAL RESISTANCE – ONE OF THE HOTTEST TOPICS OR THE PAST?! JANĎOUREK, O., KONEČNÁ, K. Department of Biological and Medical Sciences, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic e-mail: [email protected] Microbial resistance is a collocation that has been frequently used all over the world. New bacterial strains that have developed resistance are noticed every year. Number of new cases is rising daily. There are rumours claiming that 10 million people will suffer and die of infectious disease caused by resistant strain in 2050. WHO has started a few programs that should lead to elimination of resistance, e.g. END TB or Global action plan on antimicrobial resistance .1,2 On the other hand, resistant microbes, so called superbugs, have their own mechanisms how to survive our attempts to kill them. One way how to be succesfull in attempts to kill these microbes is to focus on finding novel antimicrobial compounds (new mechanism of action, advantageous pharmacological properties, broader spectrum of pathogens being influenced). Design and synthesis of these compounds is crucial part of process but it is also very important to focus on biological properties such as minimal inhibition concentration, timekill assays or mechanism of action (MoA) determination. MoA is also very important to understand the resistance mechanisms . We are able to determine MoA of potential antibiotic agent in four biochemical pathways – inhibition of cell wall synthesis, inhibition of DNA synthesis, inhibition of RNA synthesis or inhibition of proteosynthesis . This assay is based on the incorporation of radioactively labelled compounds, which are part of studied 8 biochemical pathways. If detected radioactivity is lower, it means that potential antibiotic inhibits this pathway and radioactively labelled molecule cannot be incorporated into the final products. Standards used for this screening are vancomycin (inhibition of cell wall synthesis indicated by 3H labelled N-acetylglucosamine), rifampicin (inhibition of RNA synthesis indicated by 3H labelled uridine), ciprofloxacin (inhibition of DNA synthesis indicated by 3H labelled thymidine), chloramphenicol (inhibition of proteosynthesis indicated by 3H labelled leucine), and chlorhexidine (positive control, inhibition of all mentioned biosynthetic pathways).3 Currently, we are focusing on determining the possible MoA of potential antimycobacterial agents. We have revealed specific biomolecules that are connected with mycobacterial biosynthetic pathways. We have chosen 3H labelled arabinose which is involved in biosynthetic pathway of mycobacterial cell wall synthesis (ethambutol is used as standard – inhibition of arabinosyl transferase), and 3H labelled acetic acid, which is involved in the same pathway (isoniazid is used as standard – inhibiton of mycolic acids synthesis). We also need to optimize MoA medium for culturing mycobacteria (Mycobacterium smegmatis, M. aurum and M. tuberculosis H37Ra), to set MIC for standards (rifampicin, isoniazid, ciprofloxacin, streptomycin, ethambutol, gentamicin, vancomycin, chlorhexidine) and to set time-kill properties. The study was supported by the research program Development and Study of Drugs (Progress Q42). References 1. World Health Organization: Gear up to end TB: Introducing the end TB Strategy., 2015. Available from: https:// apps.who.int/iris/handle/10665/156394. 2. World Health Organization: Global action plan on antimicrobial resistance. WHO 2015. Available from: https://www.who.int/antimicrobial-resistance/publications/global-action-plan/en/. 3. NOWAKOWSKA, J ., GRIESSER, H . J ., TEXTOR, M . et al.: Antimicrob. Agents Chemother., 57 (1), 2013, 333‒342. NEW UNALLOYED METAL NANOCONJUGATES AS CATALYSTS IN SELECTED REDUCTION OR ACETALIZATION REACTIONS FOR GREEN CHEMISTRY AMBROŻKIEWICZ, W. Department of Pharmaceutical Chemistry and Pharmaceutical Analysis, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic e-mail: [email protected] A series of mono, bi and tri-metal nanocatalytic transition metal systems were obtained and investigated. The catalysts included combinations of metals (Re, Ru, Rh, Ir, Pd) deposited on a silica support (SiO2) or on a metal support (Ni or Mo). These are new, never described before, catalytic nanomaterials . The studied nanocatalytic systems may be reproducibly obtained by the described synthetic methods . These heterogeneous nanocatalysts can be used in industrial processes, especially in the reaction of carbon dioxide methanation, ammonia decomposition and glycerol acetalization. 9 In the low-temperature ammonia decomposition reaction, a high activity was obtained by the PdNPs/Ni catalyst which can be used to generate hydrogen in fuel cells. The RuNPs/ Ni catalyst has a high activity in the reaction of low-temperature methanation of carbon oxides and can be used in methane production. Obtained nanocatalysts: Re/SiO2, ReRu/ SiO2, ReIr/SiO2, ReRuIr/SiO2, ReRhIr//SiO2, ReRuRh/SiO2, RuRhIr/SiO2, Ru/Mo, RuRh/ Mo, RuRhIr/Mo and ReRhIr/Mo show high activity in acetalization reactions. Nano-Re supported on SiO2 is a highly active and selective catalyst for the acetalization of glycerol into five-membered cyclic acetals (e.g. solketal) and can be used for the processing of glycerol waste. The tested catalytic systems may have practical application and serve for the development of the described industrial chemical processes, which may bring ecological and economic benefits. References 1. POLANSKI, J., BARTCZAK, P., AMBROŻKIEWICZ, W. et al.: PLoS One, 10 (8), 2015, art. e0136805. 2. KAPKOWSKI, K., AMBROŻKIEWICZ, W., SIUDYGA, T. et al.: Appl . Catal . B: Environ ., 202, 2017, 335–345 . 3. POLANSKI, J ., SIUDYGA, T ., BARTCZAK, P . et al.: Appl . Catal . B: Environ ., 206, 2017, 16–23 . SUPRAMOLECULAR ASSEMBLY OF TETRAPYRAZINOPORPHYRAZINES INTO J-DIMERS DEMUTH, J., MACHAN, M., KÁNTOR, M., ZIMČÍK, P., NOVÁKOVÁ, V. Department of Pharmaceutical Chemistry and Pharmaceutical Analysis, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic e-mail: [email protected] Alkylamino substituted azaphthalocyanines (AzaPcs) have interesting spectral properties – they absorb in a wide range of UV-vis spectrum from 300 to 600 nm and quench fluorescence of other compounds. In non-coordinating solvents, these AzaPcs form J-dimers (Fig. 1) due to their planar aromatic core1 that may affect their application as quenchers in oligodeoxynucleotide probes .2 The tendency to aggregation can be driven by peripheral substitution. The goal of this project was to study the relation between peripheral substitution and stability of J-dimers (expressed as KD) . Therefore, a series of unsymmetrical AzaPcs (Fig. 2) was synthesized. Synthetic pathway included preparation of appropriate precursors, statistical cyclotetramerization, isolation of desired ABBB congener and introduction of zinc cation to the center of metal-free AzaPc. Stability of J-dimers was investigated in toluene by titration with pyridine. Obviously, increasing bulkiness of substituent caused destabilization of J-dimers. The study was supported by Charles University (Project No. 1168217) and from the project of Specific Academic Research (SVV 260 401). References 1. NOVÁKOVÁ, V., ZIMČÍK, P., KOPECKÝ, K., et al.: Eur . J . Org . Chem ., 19, 2008, 3260–3263 . 2. DEMUTH, J., KUČERA, R., KOPECKÝ, K., et al.: Chem.-Eur. J., 24 (38), 2018, 9658–9666. 16 References 1. KRATOCHVÍL, J., NOVÁK, Z., GHAVRE, M. et al.: Org. Lett., 17 (3), 2015, 520–523. 2. BRŮŽA, Z., KRATOCHVÍL, J., HARVEY, J. N. et al.: Chem.-Eur. J., 25 (34), 2019, 8053–8060. SYNTHESIS OF SYMMETRICAL AND UNSYMMETRICAL ANIONIC PHTHALOCYANINES FOR PHOTODYNAMIC THERAPY KOLLÁR, J .,1 MACHÁČEK, M.,2 ZIMČÍK, P.1 1 Department of Pharmaceutical Chemistry and Pharmaceutical Analysis, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic 2 Department of Biochemical Sciences, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic e-mail: [email protected] Phthalocyanines (Pcs) represent a promising group of organic dyes with interesting photophysical properties (strong absorption in area 650–750 nm and strong singlet oxygen production) highly suitable for the use in photodynamic therapy of cancer . The aim of this work was to synthesize symmetrical and unsymmetrical anionic Pcs with 3,5-dicarboxylatophenyl moiety connected with Pc core directly by C-C bond or by ether bridge. Unsymmetrical compounds with amphiphilic character can be incorporated to liposomes that may protect this phthalocyanine from binding to proteins or from aggregation at low pH. Precursors for synthesis of symmetrical and unsymmetrical Pcs, 4,5-disubstituted phthalonitriles, were obtained by Suzuki coupling or nucleophilic substitution with molecules bearing 3,5-dimethyl isophthalate. Symmetrical Pcs were obtained by cyclotetramerization reaction (initiator magnesium butoxide) of one precursor while unsymmetrical Pcs were prepared by statistical condensation of phthalonitrile with 4,5-disubstituted phthalonitrile. Methylesters were completely transesterified to butyl esters during this reaction. Magnesium complexes was converted to metal-free ligands and then to zinc complexes. Basic hydrolysis of ester bonds was the last step of the synthesis. Final Pcs were tested on photodynamic activity in vitro on HeLa cells . Photophysical properties and binding to serum protein of Pcs were studied. The work was supported by the Grant Agency of Charles University (Project No. 1060216) and from the project of Specific Academic Research (SVV 260 401).   Fig.7   17 SYNTHESIS AND OPTIMIZATION OF THE PREPARATION OF 32-HYDROXYDOTRIACONTANOIC ACID SOMMEROVÁ V ., OPÁLKA L ., VÁVROVÁ, K . Department of Organic and Bioorganic Chemistry, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic e-mail: [email protected] The 32-hydroxydotriacontanoic acid forms the backbone of all the acylceramides which belong among ultralong chain ceramides . They are essential components of the stratum corneum and play a crucial role in proper function of the skin barrier. The carboxyl group of this acid is bound to a primary amino group of the sphingoid bases and the ω-hydroxy group is either esterified with linoleic acid to form free ceramides or it can be linked to the surface of corneocytes in the form of covalently bound ceramides . The recent literature describes the synthesis of 32-hydroxydotriacontanoic acid with relatively small yields. The most problematic part of the synthesis is the connection of two shorter fragments leading to the ultralong chain .1 The main aim of this project is to prepare covalently bound ceramides on solid particles and to optimize the reaction conditions. The previously used Wittig reaction was changed for other olefinations, mainly Julia and Julia-Kocienski reactions and their modifications. The highest yields were so far obtained by the modified Julia-Kocienski reaction with (1-cyclohexyl-1H-tetrazol-5yl)sulfonyl derivative of hexadecanoic acid as a starting material. In this case, we were able to increase the yield of this reaction even over 70%, which greatly improved the described reaction pathway. The study was supported from the project of Specific Academic Research (SVV 260 401) and by the Czech Science Foundation (Project No. 16-25687J). References 1. OPÁLKA, L., KOVÁČIK, A., SOCHOROVÁ, M. et al.: Org. Lett., 17 (21), 2015, 5456–5459.   Fig.8   18 INVESTIGATION OF THE COMPOUNDS INFLUENCING THE MELTING TEMPERATURE OF OLIGONUCLEOTIDE PROBES KOSTELANSKÝ, F., HAVLÍNOVÁ, Z., MILETÍN, M., ZIMČÍK, P. Department of Pharmaceutical Chemistry and Pharmaceutical Analysis, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic e-mail: [email protected] Real-time PCR is a widely used method in various research fields. The use of longer probes is often not optimal for mismatch discrimination because of low melting temperature difference between fully complementary and mismatched probes. The use of shorter oligonucleotide probes can be advantageous in this case. On the other hand, low melting temperature of these shorter probes is major complication for practical use in real-time PCR. Minor groove binders (MGB) or intercalating dyes can stabilize the duplex and increase the melting temperature, e.g. biogenic polyamines have strong interaction with DNA and RNA .1 For testing the capability to increase melting temperature, several compounds were selected. Commonly known MGB Hoechst 33258 (1),2 its modified derivative 2, naturally occurring spermine 3, three artificial polyamines (4–6) and 14 acridine derivatives (7) were tested for their capability to increase melting temperature of shorter probes. Modified Hoechst 33258 (2) and 14 acridine derivatives (7) were prepared in our laboratory . The tests revealed interesting increase of the melting point of the probes for several compounds . The study was supported by the Technology Agency of the Czech Republic (TH03010251) and by the Grant Agency of Charles University (Project No. 994218). References 1. OUAMEUR, A. A., TAJMIR-RIAHI, H. A.: J. Biol. Chem., 279 (40), 2004, 42041–42054. 2. LUKINAVIČIUS, G., BLAUKOPF, C., PERSHAGEN, E. et al .: Nat . Commun ., 6, 2015, Article 8497, doi: 10.1038/ncomms9497.   Fig.9   19 STABILITY EVALUATION OF MAGNESIUM COMPLEXES OF PHTHALOCYANINES AND AZAPHTHALOCYANINES UNDER ACIDIC CONDITIONS BERANOVÁ, M., ZIMČÍK, P. Department of Pharmaceutical Chemistry and Pharmaceutical Analysis, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic e-mail: [email protected] Phthalocyanines (Pc) and their azaanaloges (AzaPc) represent macrocyclic compounds with a large system of conjugated bonds. Due to this system, they possess interesting photophysical and photochemical properties such as strong emission of fluorescence or production of singlet oxygen, which make them widely examined as potential diagnostic and therapeutic agents .1 Importantly, the central cation incorporated in the macrocycle changes the relaxation pathways of the excited states on the basis of heavy-atom effect.2 Magnesium is the lightest stable central cation and for this reason its complexes are characterized by strong fluorescence; advantageously used as fluorescent probes or labels.1 However, the instability of magnesium complexes in acidic environment (demetallation to metal-free derivatives) is the main obstacle of wider utilization of these fluorescent dyes. Once a macrocycle is demetallated, it loses its strong fluorescent properties. In this work, we decided to more closely evaluate a demetallation and other decomposition of these compounds in water solution at five different pH ranging 1–7.4. The stability was monitored by absorption spectroscopy for 24 h where characteristic splitting of the Q-band occurred after demetallation . Experiments proved that the more acidic environment, the faster process of the demetallation occurs and that Pcs are less stable than corresponding AzaPcs. We also tested possible protection provided by various delivery systems (liposomes and microemulsions). In particular liposomes showed high level protection where no changes in absorption spectra of AzaPc was detected after 24 h even at the most acidic pH 1. The study was supported from the project of Specific Academic Research (SVV 260 401). References 1. NOVÁKOVÁ, V ., DONZELLO, M . P ., ERCOLANI, C . et al .: Coor . Chem . Rev ., 361, 2018, 1–73 . 2. TUHL, A., MAKHSEED, S., ZIMČÍK, P . et al.: J. Porphyrins Phthalocyanines, 16 (7–8), 2012, 817–825. 20 PHARMACOGNOSY AND TOXICOLOGY OF NATURAL PRODUCTS SECTION AMARYLLIDACEAE ALKALOIDS FROM NARCISSUS PSEUDONARCISSUS cv . DUTCH MASTER AS POTENTIAL DRUGS IN TREATMENT OF ALZHEIMER’S DISEASE HULCOVÁ, D .,1,2 MAŘÍKOVÁ, J.,3 OPLETAL, L .1 CAHLÍKOVÁ, L.1 1 ADINACO Research Group, Department of Pharmaceutical Botany, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic 2 Department of Pharmacognosy, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic 3 Department of Organic and Bioorganic Chemistry, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic e-mail: [email protected] Alzheimer’s disease (AD) is the most prevalent neurodegenerative disease worldwide with complex etiology and multifaceted pathophysiology. It is characterized by massive deposits of amyloid-β peptide, neurofibrillary tangles of the hyperphosphorylated τ-protein and inflammatory mediators leading to neuronal death. It is manifested by damage of cognitive and noncognitive functions .1 The genus Narcissus from the Amaryllidaceae family is mainly distributed in southwestern Europe and North Africa. This family contains special type of Amaryllidaceae alkaloids (AA), possessing a wide range of pharmacological properties such as antitumor, antiviral and acetylcholinesterase (AChE) inhibitory activity. AA galanthamine is used for AD therapy .2 Twenty-two AA of various structural types have been isolated. The bulbs were processed by extraction, followed by column and thin layer preparative chromatography and recrystallization. The chemical structures were elucidated by combination of MS, HRMS and NMR spectroscopic techniques. All isolated compounds were evaluated for their in vitro AChE, butyrylcholinesterase (BuChE), prolyl oligopeptidase (POP) and glycogen synthase kinase-3β (GSK-3β) inhibitory activities. The most important biological profile was demonstrated by narcimatuline (IC50, BuChE = 5 .9 ± 0 .2 µM, IC50, POP = 29 .2 ± 0 .9 µM; IC50, GSK-3β = 20 .8 ± 2 .4 µM) . This project was supported from the projects of Specific Academic Research (SVV 260 412, SVV 260 401) and by Research programme Development and Study of Drugs (Progres Q42). References 1. KUMAR, K ., KUMAR, A ., KEEGAN, R . M . et al .: Biomed . Pharmacother ., 98, 2018, 297–307 . 2. HULCOVÁ, D ., BREITEROVÁ, K ., SIATKA T . et. al.: Molecules, 23 (4), 2018, art. 719, doi: 10.3390/molecules23040719 . 21 BRUNSVIGINE ISOMER AS A POTENTIAL AGENT IN THE TREATMENT OF ONCOLOGICAL DISEASES BREITEROVÁ, K .,1 HOŠŤÁLKOVÁ, A.,1 OPLETAL, L .,1 KOUTOVÁ, D .,3 MAŘÍKOVÁ, J.,2 CAHLÍKOVÁ, L.1 1 ADINACO Research Group, Department of Pharmaceutical Botany, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic 2 Department of Organic and Bioorganic Chemistry, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic 3 Department of Medical Biochemistry, Faculty of Medicine in Hradec Králové, Charles University, Czech Republic e-mail: [email protected] Plants from Amaryllidaceae family are interesting source of specific bioactive compounds –Amaryllidaceae alkaloids (AA). So far, nearly 600 AA of various structural types have been detected . Among the most important biological activities of AA belong those associated with the treatment of Alzheimer’s disease (AD) and cancer. These and other diseases of affluence are becoming increasingly widespread all over the world. From this reason the development of new potential drugs is needed. Galanthamine has already been used as a reversible selective inhibitor of human erythrocytic acetylcholinesterase (HuAChE; IC50 HuAChE = 1 .5 ± 0 .2 µM)1 in patients with AD. Many AA have been screened for their activity to inhibit the growth of different cancer cell lines and active compounds (mainly lycorine and haemanthamine) can be also used as lead structures for a preparation of their semisynthetic analogues . From Narcissus cv. PROFESSOR EINSTEIN summary alkaloidal extract 25 different alkaloids have been isolated so far and they were identified by MS, HRMS and 1Dand 2D-NMR techniques and X-ray. All alkaloids were tested on their activities associated with AD (AChE, BuChE, POP, GSK-3β) and their ability to inhibit the growth of several cancer cell lines. From isolated alkaloids, the newly isolated isomer of brunsvigine gave the best results in the screening . IC50 determination was done (IC50 A549 = 2 .29 ± 0 .43 µM, IC50 SAOS-2 = 2.20 ± 0.25 µM) and now it is undergoing determining of the site of interference in the cell cycle . The study was supported from the projects of Specific Academic Research (SVV 260 292 and SVV 260 412). References 1. MAOMAO, H ., CHUNRONG, Q ., OUDE, G . et al.: RSC Adv., 5 (21), 2015, 16562–16574. 22 PAPAVER RHOEAS: THE SOURCE OF ALKALOIDS FOR NMR ELUCIDATION MAŘÍKOVÁ, J.,1 CHLEBEK, J .,2 KUNEŠ, J .1 1 Department of Organic and Bioorganic Chemistry, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic 2 Department of Pharmaceutical Botany, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic e-mail: [email protected] The presented alkaloids were isolated from Papaver rhoeas (Papaveraceae) at the Department of Pharmaceutical Botany, Faculty of Pharmacy, Hradec Králové . The Papaveraceae family is very rich in specific alkaloids derived mostly from isoquinoline. The crude alkaloid extract displayed a promising inhibitory effect on butyrylcholinesterase. As such, it was further subjected to isolation and structural analysis of its constituents. The isolated substances were characterized and interpreted by employing standard 1H, 13C, gCOSY, gHSQC, gHMBCAD and NOESY experiments on a Varian VNMR S500 spectrometer, supported by EI-MS spectra . Each of the isolated alkaloids was later screened for biological activities on acetylcholinesterase, butyrylcholinesterase and prolyloligopeptidase. Selected compounds were also tested for cytotoxicity . The study was supported by the Czech Science Foundation (Project No. 18-17868S) and from the project of Specific Academic Research (SVV 260 401). ONE-STEP ISOLATION OF LUTEIN FROM GREEN MICROALGAE (CHLORELLA VULGARIS) BY HIGH PERFORMANCE COUNTERCURRENT CHROMATOGRAPHY FÁBRYOVÁ, T .,1,2 CHEEL, J .,2 TŮMOVÁ, L.1 KUBÁČ, D.,2 HROUZEK, P .,2 KOPECKÝ, J.2 1 Department of Pharmacognosy, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic 2 Laboratory of Algal Biotechnology – Centre ALGATECH, Institute of Microbiology, Czech Academy of Sciences, Czech Republic e-mail: [email protected] Lutein is a yellow carotenoid that naturally occurs in green leafy vegetables, orangeyellow fruits, and flowers. It is an antioxidant compound with eye health promoting properties and its utilization as an active ingredient in dietary supplements is currently driving the growing industrial demand for this compound. The flower petals of yellow Marigold (Tagetes erecta L .) represent the most important commercial source of lutein; however, their utilization is limited by seasons, climate, planting area, and the high labor costs .1,2 Green Chlorella vulgaris, an eukaryotic microalga, has recently become a promising alternative feedstock for lutein. So far, the commercial lutein has mainly been obtained from Marigold flowers by solvent extraction, but this procedure has a limited specificity to the target compound. Therefore, the application of an efficient and scalable 23 isolation technique is pivotal for obtaining high-quality commercial lutein . In the present study, a high-performance countercurrent chromatography (HPCCC) method was developed and applied to obtain lutein from C. vulgaris biomass. Different two-phase solvent systems composed of n-heptane, ethanol and water were evaluated for their capacity to provide a proper distribution coefficient (0.5 ≤ K ≤ 2.5) of lutein and for exhibiting both an adequate density difference between the two phases (≥ 0.080 g mL−1) and a short settling time (< 30 s). The two-phase solvent system composed of n-heptane–ethanol–water (5:4:1.5, v/v/v) was selected for the isolation of lutein. In addition, different flow rates (1–8 mL min−1) and sample loadings (200–400 mg of extract) were examined to optimize the HPCCC separation conditions. Under the optimized operating conditions, the lower phase of the selected solvent system was used as the mobile phase at a flow rate of 8 mL min−1, whereas the rotational speed and temperature of the separation column were 1200 rpm and 28 °C, respectively. The retention of the stationary phase at the end of the HPCCC separation was 52%. Overall, 10 mg of lutein with purity of 97% was obtained from 200 mg of C. vulgaris extract . The chemical identity of the target compound was confirmed by high performance liquid chromatography with UV-visible detection (HPLC-DAD) in comparison with an authentic standard. The method described in this study represents a good strategy to efficiently isolate lutein from microalgae biomass and can serve as a good reference to scale up the production of this bioactive compound at pilot and industrial size.   Fig.10   Figure 1. Chromatogram of the HPCCC isolation of lutein from microalgae Chlorella vulgaris . 24 This study was supported by the Grant Agency of Charles University (Project No. 1134217), from the project of Specific Academic Research (SVV 260 294) and by the Technological Agency of the Czech Republic (Project No. TJ01000013). References 1. BERNSTEIN, S ., LI, B ., VACHALI, P . P . et al .: Prog . Retin . Eye Res ., 50, 2016, 34–66 . 2. SUN, Z ., LI, T ., ZHOU, Z . G . et al.: Adv . Biochem . Eng . Biotechnol ., 153, 2016, 37–58 . IMMUNOMODULATORY ACTIVITY OF SCUTELLARIA BAICALENSIS AND AZORELLA COMPACTA VRABEC, R .,1 TŮMOVÁ, L.,1 CHEEL, J .,2 VOKURKOVÁ, D .3 1 Department of Pharmacognosy, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic 2 Laboratory of Algal Biotechnology – Centre ALGATECH, Institute of Microbiology, Czech Academy of Sciences, Czech Republic 3 Institute of Clinical Immunology and Allergology, University Hospital in Hradec Králové, Czech Republic e-mail: [email protected] Infectious diseases are an ever-present threat that can be potentially deadly . A negative role is also accounted to the gradually increasing resistance of microorganisms to antibacterial medicines . Enhancing the immunity against infectious agents is, therefore, important goal of searching for new resources of active substances. These could be found in Scutellaria baicalensis L. (Baical skullcap, family Lamiaceae), one of the medicinal herbs with a long history of usage in traditional Chinese medicine. Immunomodulatory activity of S. baicalensis has already been proven to some extent1, but it is still unknown which kind of extract (ethanolic or aqueous) and in which concentration is more effective and which content substances have the highest share on this activity. Another possible source of immunomodulatory active substances is Azorella compacta Phil. (syn. A. yareta, Llareta, family Apiaceae), a cushion shrub grown at altitudes of the Andes in South America’s puna. Experiments with aqueous extracts proved the antioxidant and immunomodulatory effect of contained polyphenols2, but the effect of the ethanolic extract on immune cells is still unknown. In the presented study, the immunomodulatory activity of extracts from Scutellaria baicalensis and Azorella compacta was investigated through CD69 antigen activation, together with seven main flavonoids from Scutellaria . In addition, the content of three important flavonoids in Baical skullcap (baicalin, baicalein, wogonoside) was evaluated as well. References 1. LI, C .-Y ., HOU, Y .-C ., LEE CHAO, P .-D . et al.: J. Ethnopharmacol., 127 (2), 2010, 292–298. 2. TŮMOVÁ, L., DUČAIOVÁ, Z., CHEEL, J. et al.: Pharmacogn. Mag., 13 (50), 2017, 260–264. 25 ALKALOID PROFILING OF HIPPEASTRUM CULTIVARS BY GC-MS, ISOLATION OF AMARYLLIDACEAE ALKALOIDS, AND EVALUATION FOR THEIR CYTOTOXIC ACTIVITY ALSHAMMARI, L .,1 KUNEŠ, J .,2 HAVELEK, R .,3 CAHLÍKOVÁ, L.1 1 ADINACO Research Group, Department of Pharmaceutical Botany, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic 2 Department of Organic and Bioorganic Chemistry, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic 3 Department of Medical Biochemistry, Faculty of Medicine in Hradec Králové, Charles University, Czech Republic e-mail: [email protected] Six alkaloidal extracts of ornamental varieties of Hippeastrum have been studied for their alkaloid profile by GC/MS. Twenty-one compounds with typical mass spectra of Amaryllidaceae alkaloids (AA) were detected. Nineteen of them were identified based on their mass spectra, retention times and retention indexes. Identified alkaloids belong to the crinine, haemanthamine, galanthamine, homolycorine, lycorine, montanine, and tazettine structural type of AA . Using preparative TLC, five AA have been isolated in pure form from various Hippeastrum cultivars. The compounds were identified by MS, 1D and 2D NMR spectroscopic analyses and by comparison of the obtained data with the literature as montanine (1), vittatine (2), 11-hydroxyvittanine (3), lycorine (4) and hippeastrine (5) . Three of the isolated compounds (montanine, vittatine and hippeastrine) have been screened on a panel of human cancer cells (Jurkat, MOLT-4, A549, HT-29, PANC-1, A2780, HeLa, MCF-7 and SAOS-2) for their in vitro cytotoxic activity. In this work, montanine has been found to display strong cytotoxicity against all tested cancer cell lines, which is in accordance with previously reported results.1 This compound has been selected for determination of IC50 values . The study was supported from the project of Specific Academic Research (SVV 260 412). References 1. GOVINDARAJU, K ., INGELS, A ., HASAN, M . N . et al.: Bioorg. Med. Chem. Lett., 28 (4), 2018, 589–593. 32 A STUDY OF THE DRUG RELEASE FROM MATRIX TABLETS WITH POLYVINYL ALCOHOL DOUBKOVÁ, B .,1 KOMERSOVÁ, A .,2 MUŽÍKOVÁ, J.1 1 Department of Pharmaceutical Technology, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic 2 Department of Physical Chemistry, Faculty of Chemical Technology, University of Pardubice, Czech Republic e-mail: [email protected] The aim of this study was to determine the effect of different retardant concentration on drug release from hydrophilic matrix tablets. At the same time, the influence of the type of dry binder used was investigated. Polyvinyl alcohol was used as the retarding agent at the concentrations of 30, 40 and 50%. α-Lactose monohydrate and microcrystalline cellulose in the ratio of 3:1 in the physical mixture and in MicroceLac® 100 were used as dry binders . MicroceLac® 100 is a coprocessed dry binder. Salicylic acid was used as the model less soluble drug. The tablets were prepared by direct compression method. Dissolution testing was performed using the rotating basket method. The results of the dissolution test were evaluated by nonlinear regression analysis. Increasing additions of polyvinyl alcohol decreased drug release rate . It has been found that the type of dry binder does not affect neither the mechanism nor the release rate of salicylic acid. The dissolution behavior of tablets, which contained the physical mixture or the coprocessed dry binder and the same amount of polyvinyl alcohol was comparable. References 1. PATEL, H ., PANCHAL, D . R ., PATEL, U . et al.: J. Pharm. Sci. Biosci. Res., 1 (3), 2011, 143–151. 2. ZALTE, H. D., SAUDAGAR, R. B.: Int. J. Pharm. Biol. Sci., 3 (4), 2013, 17–29. 3. MUPPALANENI, S., OMIDIAN, H.: J. Develop. Drugs, 2 (3), 2013, art. 112, doi: 10.4172/2329-6631.1000112. 4. SAHA, S., SHAHIWALA, A. F.: Expert. Opin. Drug. Deliv., 6 (2), 2009, 197–208. 5. BAUER, F.: Parteck® SRP 80 for Sustained-Release Applications. PharmTech.com, 2017 [Accessed August, 3, 2018]; Available from: www.pharmtech.com/parteck-srp-80-sustained-release-applications. ORAL DELIVERY OF OLIGONUCLEOTIDES FOR LOCAL TREATMENT OF INFLAMMATORY BOWEL DISEASE KUBAČKOVÁ, J.,1 HOLAS, O .,1 ZBYTOVSKÁ, J .,1 PÁVEK, P .,2 MÜLLERTZ, A .3 1 Department of Pharmaceutical Technology, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic 2 Department of Pharmacology and Toxicology, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic 3 Department of Pharmacy, Faculty of Health and Medical Sciences, University of Copenhagen, Denmark e-mail: [email protected] Inflammatory bowel disease (IBD) includes pathological conditions characterised by inappropriate and sustained activation of the mucosal immune system of the small intestine 33 and/or colon.1 Local treatment is preferred over systemic delivery often accompanied with undesired side effects . In IBD, cationic peptides are expressed in the area and phagocytic immune cells infiltrate the site of inflammation.2 By delivery of an anti-inflammatory acting miRNA oligonucleotide, production of pro-inflammatory cytokines by these immune cells decreases, and the inflammatory process itself is suppressed. However, delivery of unstable negatively charged macromolecular oligonucleotides represents a challenge .3 Self-nanoemulsifying drug delivery system (SNEDDS) has been utilised to deliver a hydrophobic complex of oligonucleotides orally .4 The complexes were prepared from a model 20-nucleotide long oligomer and a cationic lipid dimethyldioctadecylammonium bromide (DDAB) or 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) with yields over 95% for molar ratio 1:60. The size of the complexes was estimated by atomic force microscopy to be 75–127 nm and 33–62 nm, for DOTAP and DDAB complex, respectively. The size of dispersed loaded SNEDDSs, ~200 nm, and negative surface charge enabling passive targeting make the formulation suitable for intended purpose . The study was supported from the project of Specific Academic Research (SVV 260 401) and by the Czech Science Foundation (Project No. 270/53/75302). References 1. PODOLSKY, D. K.: N. Engl. J. Med., 347 (6), 2002, 417–429. 2. HUA, S ., MARKS, E ., SCHNEIDER, J . J . et al.: Nanomedicine, 11 (5), 2015, 1117–1132. 3. LAM, J. K. W., CHOW M. Y. T., ZHANG, Y.: Mol. Ther. Nucleic Acids, 4 (9), 2015, art. e252, doi: 10.1038/ mtna .2015 .23 . 4. MÜLLERTZ, A ., OGBONNA, A ., REN, S . et al.: J. Pharm. Pharmacol., 62 (11), 2010, 1622–1636. NOVEL FORMULATIONS FOR TOPICAL APPLICATION OF IMIQUIMOD NOVÁČKOVÁ, A.,1 ŠVECOVÁ, B .,1 VÁVROVÁ, K .,2 HLADKÝ, P.,1 TIRALA, P .,1 RUDECKÁ, K .,1 ZBYTOVSKÁ, J .1 1 Department of Pharmacutical Technology, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic 2 Department of Organic and Bioorganic Chemistry, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic e-mail: [email protected] Imiquimod (IMQ) is a heterocyclic imidazoquinoline used in the treatment of various viral or neoplastic skin diseases.1 The aim of this study was to prepare suspensions, emulsions and liposomes for dermal administration of 1% IMQ and evaluate its in vitro permeation on human skin. IMQ is commercially available as a cream under the brand name Aldara® (5% IMQ). This formulation possesses undesired disadvantages like low skin permeation, instability and side effects on skin.2 In order to overcome these obstacles, the formulations with lower concentration of IMQ and different permeation enhancers (L-Pro2, DDAK and oleic acid) were prepared. In vitro permeation experiments were carried out in Franz Diffusion Cells on human skin while Aldara® was used as a control. 34 After 8 and 24 hours the concentration of IMQ in each skin layer, as well as in acceptor phase (phosphate buffer, pH 7.4), was determined. In emulsions and suspensions a higher concentration of IMQ was proved in epidermis and lower concentration in the deeper compartments compared to Aldara®. The liposomal formulations did not show such high effect on delivery of IMQ to the epidermis. In the future, optimized liposomal formulations will be developed for better potential delivery of IMQ . The study was supported by the Czech Science Foundation (Project No. 13-23891S), by the Grant Agency of Charles University (Project. No. 184217) and from the project of Specific Academic Research (SVV 260 401). References 1. VIDAL, D.: Mini Rev. Med. Chem., 6 (5), 2006, 499–503. 2. HANNA, E., ABADI, R., ABBAS, O.: Int. J. Dermatol., 55 (8), 2016, 831–844. LIPOSOMES FOR DERMAL DRUG DELIVERY PANOUTSOPOULOU, E .,1 PARASKEVOPOULOS, G .,1 VÁVROVÁ, K .,2 ZBYTOVSKÁ, J .1 1 Department of Pharmaceutical Technology, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic 2 Department of Organic and Bioorganic Chemistry, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic e-mail: [email protected] Over the past years, (trans)dermal delivery of biologically active molecules has become very popular due to its advantages over other routes of administration .1 Different methods and vehicles have been used to improve dermal delivery, of which, liposomes seem to be the most studied .2 Recently, our research group prepared imiquimod-containing liposomal mixtures which were unable to efficiently deliver imiquimod to epidermis in higher concentrations than the commercially available cream (Aldara). Inspired by the previous results, the aim of the present study is, to prepare imiquimod loaded liposomes following an alternative approach and evaluate their ability to deliver the active substance to the skin layers . The study was supported from the project of Specific Academic Research (SVV 260 401) and the Czech Science Foundation (Project No. 19-09600S). References 1. PRAUSNITZ, M. R., LANGER, R.: Nat. Biotechnol., 26 (11), 2008 1261–1268. 2. NOUNOU, M . I ., El-KHORDAGUI, L . K ., KHALAFALLAH, N . A . et al .: Recent Pat . Drug Deliv . Formul ., 2 (1), 2008, 9–18. 35 PHARMACEUTICAL ANALYSIS AND BIOANALYTICAL CHEMISTRY SECTION APPLICATION OF MONOLITHIC COLUMNS IN CLINICAL PRACTICE KUČEROVÁ, K.,1,2 KUJOVSKÁ KRČMOVÁ, L.,1,2 MATYSOVÁ, L .1 1 Department of Analytical Chemistry, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic 2 Department of Clinical Biochemistry and Diagnostics, University Hospital in Hradec Králové, Czech Republic e-mail: [email protected] Monolithic stationary phases and columns have rapidly become highly popular separation media for liquid chromatography . Today, various types of monoliths have been developed. They have different forms and are synthesized by different preparation processes . Their unique properties distinguish them from all other columns, especially the tolerance to high flow rates achievable using only moderate pressures and high speed. This characteristics achieved excellent separation and make the columns irreplaceable in certain areas .1,2 Monolithic columns are used for example in the analysis of biologically active substances such as neopterin, kynurenine, tryptophan and creatinine. Elevated levels of tryptophan, its metabolite kynurenine and neopterin have been observed in disease associated with activation of immune system. Determination of these substances in biological fluids, serum, urine, wound exudates or amniotic fluid, serves to determine the patient response to therapy and clinical status. Monoliths allows large separation capacity, better separation efficiency with low back pressure and analysis of more samples of biological material compared to columns with particles.3,4 Using of monoliths will be presented in determination of different analytes (immune system activation markers) in various biological fluids (serum, amniotic fluid, wound liquid, exudates) used in many clinical studies . The study was supported from the project of Specific Academic Research (SVV 260 412), by Ministry of Health of the Czech Republic – conceptual development of research organization (UHHK, 00179906) and by Ministry of Health of the Czech Republic (Projects No. NV18-03-00130, NV17-29241A and NV17-28882A). References 1. MIYABE, K., GUIOCHON, G.: J. Sep. Sci., 27 (10–11), 2004, 853–873. 2. ŠVEC, F.: Chem. Listy, 98 (5), 2004, 232–238. 3. KUJOVSKÁ KRČMOVÁ, L., ČERVINKOVÁ, B., SOLICHOVÁ, D. et al.: Bioanalysis, 7 (21), 2015, 2751– 2762 . 4. KRČMOVÁ, L., SOLICHOVÁ, D., MELICHAR, B. et al.:Talanta, 85 (3), 2011, 1466–1471. 36 DETERMINATION OF IMPORTANT BIOMARKERS DURING TREATMENT WITH RHEOHEMAPHERESIS IN AGE-RELATED DRY FORM OF MACULAR DEGENERATION VERNEROVÁ, A .,1,2 KUJOVSKÁ KRČMOVÁ, L.,1,2 BLÁHA, M .3 1 Department of Analytical Chemistry, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic 2 Department of Clinical Biochemistry and Diagnostics, University Hospital in Hradec Králové, Czech Republic 3 4th Department of Internal Medicine – Hematology, Faculty of Medicine in Hradec Králové, Charles University, Czech Republic e-mail: [email protected] Age-related macular degeneration (AMD) is the most frequent cause of severe visual lost in people older than 55 years in industrial countries. The disease has two variants, dry and wet form. The most common form is the slowly progressing dry (atrophic) form. Rare (10–20%) rapidly progressing wet form with typical neovascular choroidal membrane is in 80–90% the cause of blindness . Rheohemapheresis is used in the treatment of dry form of AMD with soft drusen. It is a double plasma filtration in which specific highmolecular substances (LDL cholesterol, IgM, α2 macroglobulin, fibronectin, fibrinogen, von Willebrand factor) are removed . Reduction of levels of these substances leads to an improvement of the choroidal microcirculation and reduction of accumulation of lipoproteins which are the major component of soft drusen.1 The potential risc of this treatment is a decrease not only in cholesterol but also in antioxidants, such as vitamin E and other important biomolecules such as vitamin A and D. For this reason, we measured levels of vitamin E (α-tocopherol), the vitamin E/cholesterol ratio in serum and lipoproteins (VLDL, LDL, HDL). Serum vitamin A (retinol) and vitamin D were also measured. These parameters were determined before and after rheohemapheresis.2 The individual lipoprotein layers were obtained by ultracentrifugation. The vitamins were extracted from matrix using liquid-liquid extraction, protein precipitation and filtration. For determination of vitamin E and A in serum a reversed-phase high performance liquid chromatography method using monolithic column and diode-array detection was developed and validated.3 For determination of vitamin D, ultra high performance liquid chromatography coupled with mass spectrometry detection (MS/MS) was used.4 First results from Ministry of Health project NV17-29241A will be presented. The study was supported from the project of Specific Academic Research (SVV 260 412) and by the Ministry of Health of the Czech Republic (Projects No. NV17-29241A and NV17-28882A). All rights reserved. References 1. STUDNIČKA, J.: Interní Med., 10 (5), 2008, 240–244. 2. AUFARTOVÁ, J ., BLÁHA, M ., KASALOVÁ, E . et al.: Biomed. Pap. Med. Fac. Univ. Palacky Olomouc, Czech Repub., 159 (3), 2015, 400–406. 3. URBÁNEK, L ., SOLICHOVÁ, D ., MELICHAR, B . et al.: Anal . Chim . Acta, 573–574, 2006, 267–272 . 4. PLÍŠEK, J., KUJOVSKÁ KRČMOVÁ, L., AUFARTOVÁ, J. et al.: J. Sep. Sci., 36 (23), 2013, 3702–3708. 37 UHPLC-MS/MS IN AN INVESTIGATION OF NOVEL CARDIOPROTECTIVE AGENT JAS-2 BAVLOVIČ PISKÁČKOVÁ, H.,1 BRÁZDOVÁ, P .,2 ŠTĚRBA, M.,2 ŠTĚRBOVÁ KOVAŘÍKOVÁ, P.1 1 Department of Pharmaceutical Chemistry and Pharmaceutical Analysis, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic 2 Department of Pharmacology, Faculty of Medicine in Hradec Králové, Charles University, Hradec Králové, Czech Republic e-mail: [email protected] JAS-2 [4,4′-(Butane-2,3-diyl)bis(piperazine-2,6-dione)] is a novel analogue of dexrazoxane (DEX) which is the only approved cardioprotective agent protecting myocardium against anthracycline-induced toxicity . Despite pilot studies indicate that JAS-2 is more effective in protection of neonatal rat cardiomyocytes from toxic effect of anthracyclines as compared with DEX, its use is limited by poor solubility. Therefore, a prodrug with a code name GK667 was prepared to improve the solubility of JAS-2. A modern analytical method is required to investigate stability of GK-667, its conversion to the active form – JAS-2 as well as its further metabolism. The aim of this work was UHPLC-MS/MS analysis of samples from in vitro experiments aimed at investigation of the prodrug activation as well as plasma from a pilot in vivo study. The analyses were performed using a UHPLC system (Nexera, Shimadzu,) coupled with a triple quadrupole mass spectrometer with ESI ion source (LCMS-8030, Shimadzu). The separation was achieved on Luna Omega Polar column (100 × 3.0 mm, 2.5 μm, Phenomenex) protected with a guard column. A mixture of ammonium formate and acetonitrile in a gradient mode was used as a mobile phase. Plasma samples were treated with precipitation. Cell culture medium was simply diluted with ultra-pure water. Stability study on GK-667 (100 μM, 37 °C) showed similar profile in plasma and DMEM medium. GK-667 is rapidly converted to JAS-2 which is slowly degraded to JAS-2met. Stability of JAS-2 (100 μM, 37 °C) in both matrixes was compared with DEX. After a pilot i.v. administration of GK-667 to rabbits (5 mg/kg i.v ., n =2), only JAS-2 was detected (cmax ≈ 10 μM). GK-667 and JAS2met were bellow LLOQ. The method will be further modified to enhance sensitivity, mainly for JAS-2, to be capable for full PK study in rabbits . The study was supported by the Grant Agency of Charles University (Project. No. 1550217), from the project of Specific Academic Research (SVV 260 412) and the Czech Science Foundation (Project No. 18-08169S). 38 DETERMINATION OF SUDAN DYES IN CHILLI PRODUCTS BY MEKC-MS/MS USING A MS FRIENDLY SURFACTANT MORENO-GONZÁLEZ, D., JÁČ, P., NOVÁKOVÁ, L. Department of Analytical Chemistry, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic e-mail: [email protected] Sudan dyes are phenyl-azoic derivatives widely used as synthetic organic colorants because of their colour fastness and low price. Azo dyes have been extensively used in many industrial applications including oils, solvents, plastics, etc. Sudan I, II, III, and IV have been employed for many years as food colorants in different products, such as chilli powder and sauces, to mimic, intensify, and prolong the appearance of natural red hues because of their intense red-orange colour . The use of these colorants can constitute a serious health risk, thus they are banned for food usage in the European Union since 2004 .1 The aim of this work is to develop a fast and sensitive method for the simultaneous determination of Sudan dyes (I, II, III, and IV) in chilli products such as powder, sauce and paste by micellar electrokinectic chromatography-mass spectrometry (MEKC-MS) employing ammonium perfluorooctanoate as volatile surfactant. MEKC separation and MS detection conditions have been optimized in order to achieve a fast, efficient, and sensitive separation of the four dyes. Target compounds were extracted from chilli samples with acetonitrile and purified using freezing-lipid filtration, achieving excellent results in terms of sample throughput . Analytical performance of the method is satisfactory, obtaining limits of quantification lower than 5 μg kg−1 in all cases . The precision, expressed as relative standard deviation (%, RSD) was below 15.7%. The extraction efficiency for fortified samples ranged from 86.5 to 99.8%, with RSDs lower than 10.3%. Matrix effect was evaluated for all samples studied, being lower than 17% in all cases. Its applicability has been successfully tested in 20 chilli products. The concentration was calculated from the corresponding matrix-matched calibration curve, detecting Sudan I and IV in several samples at 50 and 450 μg kg−1 respectively . The study was supported by the STARSS project (Reg. No. CZ.02.1.01/0.0/0.0/15_003/ 0000465) co-funded by ERDF. References 1. Commission Decision of 21 January 2004 on emergency measures regarding hot chilli and hot chilli product . Off . J . Eur . Union ., L27, 2004, 52–54 . 39 PROTEOMIC ANALYSIS OF ACETYLOME FABRIK, I .,1 VAJRYCHOVÁ, M .,1 BENKOVÁ, M .,1 PŮLKRÁBKOVÁ, L.,1 STULÍK, J.,2 SOUKUP, O .1 1 Biomedical Research Center, University Hospital in Hradec Králové, Czech Republic 2 Department of Molecular Pathology and Biology, Faculty of Military Health Sciences in Hradec Králové, University of Defence, Czech Republic e-mail: [email protected] In terms of numbers, acetylation of lysine ε-amino group (AcK) represents the second most important protein posttranslational modification involved in cell signaling next to phosphorylation .1 Unfortunately, functional role of the majority of acetylated sites (i.e. acetylome) is unknown. The position of protein acetylation remains enigmatic even for immune cells whose signaling cascades must be tightly regulated to prevent immunopathology . In particular, there are no rigorous data regarding acetylome of dendritic cells (DCs) – the most effective antigen-presenting cells responsible for priming of adaptive immunity. Therefore, we decided to analyze acetylation events in primary murine bone marrow-derived DCs (BMDCs) using proteomics. First, we compared AcK sites from BMDCs to those obtained from murine liver tissue. While we were able to identify ˃3,500 AcK sites in liver, only ~1,900 were found in BMDCs. This was probably an issue of sensitivity as AcK sites from some BMDC compartments (e.g. mitochondria) were underrepresented, suggesting low AcK stoichiometry. To further improve the depth of BMDC acetylome, we performed HPLC pre-fractionation of BMDC peptides prior immunoprecipitation step, which helped to increase the number of identified BMDC AcK sites to ~3,000 . As expected from transcriptionally active immune cells, about 3% of detected acetylated BMDC proteins were transcription factors and ˃10% of all AcK sites were reported substrates of nuclear CBP/p300 acetyltransferase. The study was supported by Ministry of Health of the Czech Republic – conceptual development of research organization (UHHK, 00179906). References 1. CHOUDHARY, C ., WEINERT, B . T ., NISHIDA, Y . et al.: Nat. Rev. Mol. Cell. Biol., 15 (8), 2014, 536‒550. CHAOTROPIC AGENTS EFFECTING SEPARATION OF CHIRAL DRUG CANDIDATE K1277 PRCHAL, L .,1 NOVÁK, M .,1,2 DOLEŽAL, R.1 1 Biomedical Research Centre, University Hospital in Hradec Králové, Czech Republic 2 Department of Pharmaceutical Chemistry and Pharmaceutical Analysis, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic e-mail: [email protected] Chirality of drugs is one of major concerns since the half of the last century, when the toxicity of one enantiomer of thalidomide caused deformations in millions of new-born 40 children in many developed countries. As a model drug for enantiomeric separation we used derivate of tacrine conjugated with tryptophan labelled K1277, which was synthetized by our colleagues as a potential anti-Alzheimer drug. The chromatographic separation was performed with Dionex 3000RS UHPLC system with UV detection (254 nm) in reverse phase isocratic mode with chiral Lux Cellulose1 column as a stationary phase and a mobile phase consisting of acetonitrile and water in 45:55 or 40:60 ratio with an addition of various inorganic chaotropic agents in different concentrations. All used agents were sodium salts to minimize interactions of cationic part to the separation. The results we obtained were partly in conclusion with Hofmeister chaotropic series as reviewed by Phechkrajang1 and used by Kazakevich et al.2 and Pan et al.3 in their research of chaotropic behaviour . However with more potent agents the separation was still inconclusive even in the low concentrations. The research was also focused on pH addition to the effects especially in the potent agents as for example BF4− anion, which seemed interesting. In the future we would like to analyse behaviour of K1277 with different conditions influencing enantiomeric separation as well as using computer prediction for optimal conditions and potentially experiment with common chiral drugs. The study was supported by the Czech Science Foundation (Project No. GA16-08554S) References 1. PHECHKRAJANG, C. M.: Mahidol Univ. J. Pharm. Sci., 37 (1–2), 2010, 1–7. 2. KAZAKEVICH, Y. V., LOBRUTTO, R., VIVILECCHIA, R..: J. Chromatogr. A., 1064 (1), 2005, 9–18. 3. PAN, L ., LOBRUTTO, R ., KAZAKEVICH, Y . V . et al.: J. Chromatogr. A., 1049 (1–2), 2004, 63–73. ANALYSIS OF CANNABINOIDS IN DIETARY SUPPLEMENTS AND COSMETIC PRODUCTS USING SUPERCRITICAL FLUID CHROMATOGRAPHY SVOBODOVÁ, Z .,1 PRAŽÁKOVÁ, K.,2 PLACHKÁ, K .,1 NOVÁKOVÁ L .1 1 Department of Analytical Chemistry, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic 2 First Private Language Grammar School, Hradec Králové, Czech Republic e-mail: [email protected] We describe development of an ultra-high-performance supercritical fluid chromatography (UHPSFC) method with PDA and MS detection for analysis of cannabinoids in dietary supplements and cosmetics products . Considering a high number of cosmetics and dietary products available on the market and process of cannabinoid legalization for medical use importance of methods suitable for quality control is growing. The optimization of UHPSFC method was carried out with mixture of six most abundant cannabinoids as reference standards including cannabidiol, Δ-9-tetrahydrocannabinol, cannabigerol, cannabidiolic acid, Δ-9-tetrahydrocannabinolic acid and cannabigerolic acid. Firstly, diol, 2-picolylamine, diethylamine, 1-aminoanthracene, BEH, BEH 2-ethyl pyridine, CSH pentafluorophenyl and HSS C18 SB stationary phases were tested. Secondly, 41 mobile phase modifiers and additives, including methanol, ethanol, mixture of acetonitrile and methanol, 0.1% ammonium hydroxide and 10 mM ammonium formate were employed. Their effects on peak shape, peak resolution, selectivity, retention time and analysis time were evaluated. Other optimized parameters involved gradient elution, column temperature and pressure of back-pressure regulator. Finally, Viridis BEH 2-EP column was selected as optimal stationary phase. In order to separate the mixture of six compounds a gradient elution from 2 to 30% of solvent B was used. Mobile phase consisted of CO2 as solvent A and methanol with 0.1% ammonium hydroxide and 2% water as solvent B. To show the applicability of the proposed method, the determination of the six cannabinoids in various cosmetics products and dietary supplements based on cannabis sativa were carried out. This work was supported by the project EFSA-CDN (No. CZ.02.1.01/0.0/0.0/16_019 /0000841) co-funded by ERDF. ULTRA-HIGH PERFORMANCE SUPERCRITICAL FLUID CHROMATOGRAPHY IN PHARMACEUTICAL QUALITY CONTROL: TACKLING THE METHOD VALIDATION PLACHKÁ, K., KHALIKOVA, M., BABIČOVÁ, B., NĚMCOVÁ, Z., ROUBÍČKOVÁ, L., JUNG, O., ŠVEC, F., NOVÁKOVÁ, L. Department of Analytical Chemistry, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic e-mail: [email protected] First part of the study was focused on developing general achiral screening approach for ultra-high performance supercritical fluid chromatography (UHPSFC) methods. For that, 10 pharmaceutical quality control (QC) mixtures containing active pharmaceutical ingredients (API) and their particular impurities were used. In the end, not only that the screening approach was suggested based on obtained results but also UHPSFC method for each QC mixture was obtained. Even though most of these methods seemed to be sufficient, several of them had to be further optimized to ensure their successful validation and application on API and tablet samples. Several challenges occurred during the optimizations. The necessity of the resolution of API and following impurity equal at least to 3 was the most common one which was especially difficult to solve in mixtures with structurally close compounds. However, also unrepeatable elution of compound eluting close to the dead volume or in the end of gradient set-up and/or shifts of retention times due to column aging were detected. The most frequent optimization adjustments involved changes in gradient program . Moreover, the substitution of Viridis HSS C18 SB for slightly different Acquity UPLC HSS C18 SB and/or addition of acetonitrile into the modifier solution also led to significant changes in selectivity. In case of β-estradiol mixture, the coupling of columns was necessary to ensure sufficient resolution of structurally close compounds. Finally, validation of optimized methods was carried out. Parameters recommended by ICH guidelines Q2 and Q3 including specificity, linearity, range, lower and upper limit of quantification, 48 STUDY OF THE BIOACCESSIBILITY OF PHTHALATES AND BISPHENOL A FROM MICROPLASTICS IN SEAWATER USING AN ON-LINE SWITCHING VALVE HPLC SYSTEM FIKAROVÁ, K .,1 ROSENDE, M .,2 COCOVI SOLBERG, D . J .,2 HORSTKOTTE, B .,1 SKLENÁŘOVÁ, H.,1 MIRÓ, M .2 1 Department of Analytical Chemistry, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic 2 FI-TRACE Team, Faculty of Science, University of the Balearic Islands, Palma de Mallorca Spain e-mail: [email protected] For the first time, an automatic flow system hyphenated to LC is presented for dynamic extraction of microplastics (polypropylene and polyvinyl chloride) with incurred phthalates and Bisphenol A (CRMs). This flow setup was able to mimic leaching of the additives from plastic debris in marine environment. The microplastic particles were packed into a metal column holder, through which surrogate seawater was pumped using flow technique. The bioaccessible analytes were preconcentrated on-line using a 10 mm long C18 monolithic column, placed onto the injection valve of an HPLC system, by this, also removing the seawater matrix. After loading the Bisphenol A and phthalates containing leachate, the HPLC valve was switched to the inject position and the analytes were eluted via an optimized acetonitrile/water gradient followed by UV detection. The entire method including dynamic flow-through extraction from microplastics, on-line preconcentration and clean-up along with the HPLC separation took 24 min. Out of the 8 phthalates in CRM, only the 4 most polar species, as well as Bisphenol A, leached significantly by the seawater. To study dynamic extraction behaviour, 40 fractions were measured for each batch of particles and the elution profiles will be shown. RSD of measurements of standards was ≤ 5% and RSD of the dynamic leaching study was ≤ 11%. K. Fikarová acknowledges financial support from the project of Specific Academic Research (SVV 260 412). M. Miró acknowledges financial support from the Spanish State Research Agency (AEI) through projects CTM2014-56628-C3-3-R (AEI/MINECO/FEDER, UE), CTM2017-84763-C3-3-R (AEI/MINECO/FEDER, UE). The work was also supported by the STARSS project (Reg. No. CZ.02.1.01/0.0/0.0/15_003/0000465) co-funded by ERDF. DETECTION TECHNIQUES FOR ANALYSIS OF PHENOLIC COMPOUNDS IN APPLES HOLLÁ, M., SKLENÁŘOVÁ, H., ŠATÍNSKÝ, D. Department of Analytical Chemistry, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic e-mail: [email protected] Apples represent one of the most worldwide available natural source of phenolic compounds responsible for overall well-being associated with their antioxidant properties. 49 Selection of cultivars with higher content of phenolic compounds can improve their intake by consumption. Fruit matrices complexity and structural similarity of polyphenols increase demands on detection selectivity and sensitivity . This problem can be solved by combination of different detection principles . For this reason, fast screening HPLC method coupled with tandem detection DAD-CAD and DAD-FLD was developed to evaluate phenolic profiles in twenty apple cultivars. Both methods employed a fully porous particle column Luna Omega Polar C18 with stationary phase modified for separation of polar compounds. The use of linear gradient elution allowed rapid chromatographic separation. Benefits, disadvantages, and possibilities of tandem connection HPLC-DAD-CAD and HPLC-DAD-FLD methods for determination of phenolic compounds in fruit extract were summarized. DAD at 280 nm was used for determination of gallic acid, epicatechin, phloridzin, and phloretin; at 254 nm for detection of rutin and quercetin; at 320 nm detection of chlorogenic acid. CAD was universal for all analytes and FLD was used at λem 363 nm and λex 278 nm for detection of gallic acid and epicatechin . The LOD and LOQ values of all detectors were compared. In contrast to DAD, FLD did not met the expectations for sensitive evaluation of analytes and selectivity of CAD was low due to absence of additional spectral data. The study was supported from the project of Specific Academic Research (SVV 260 412) and by the Technology Agency of the Czech Republic (Project No. TJ01000151). INCORPORATION OF NEUTROPHIL ELASTASE INHIBITOR INTO POLYSULFONE MEMBRANE – NEW APPROACHES TO IMPROVE HEMODIALYSIS PATIENTS OUTCOMES KOHLOVÁ, M .,1,2 AMORIM, C . G .,2 ZELENÁ, L .,1 SANTOS-SILVA, A .,3 SOLICH, P .,1 MONTENEGRO, C .2 1 Department of Analytical Chemistry, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic 2 Department of Chemical Sciences, REQUIMTE LAQV, Faculty of Pharmacy, University of Porto, Portugal 3 Department of Biological Sciences, REQUIMTE UCIBIO, Faculty of Pharmacy, University of Porto, Portugal e-mail: [email protected] Chronic kidney disease patients undergoing haemodialysis (HD) suffer from chronic inflammation caused by the disease per se and by the long-term contact with artificial material of HD membrane. As a consequence of inflammation, the patients have elevated risk of cardiovascular diseases, and therefore markedly higher mortality rate, compared to healthy population . One of the promising approaches to diminish inflammation is to inhibit neutrophil elastase, which is excessively released from neutrophils during the HD treatment.1 The elastase inhibition is mediated by the direct contact with elastase inhibitor incorporated into HD membrane. For this purpose, a neutrophil elastase inhibitor with chemical formula C21H20N2O3S (Ki = 0.34 nM) was newly synthetized and incorporated into flat sheet polysulfone membrane during its fabrication process. The ability of modified membrane to 50 diminish elastase activity was evaluated in vitro, using fluorometric assay. Membrane with identical composition, without inhibitor, was used as a blank sample. The preliminary results show promising inhibition of elastase in physiological concentration of healthy population (10 µg L−1). However, the concentration of incorporated inhibitor has to be further optimized in order to reach diminution of elastase in real values found in HD patients (29.6–64.8 µg L−1) .2 The study was supported by the Grant Agency of the Charles University (Project No. 860216) and from the project of Specific Academic Research (SVV 260 412). References 1. BRONZE-DA-ROCHA, E ., SANTOS-SILVA, A.: Int. J. Biol. Sci., 14 (10), 2018, 1343–1360. 2. COSTA, E ., ROCHA, S ., ROCHA-PEREIRA, P . et al .: Am . J . Nephrol ., 28, 2008, 935–940 . COLUMN COUPLING IN SUPERCRITICAL FLUID CHROMATOGRAPHY RIASOVÁ, P .,1 VANDER HEYDEN, Y .,2 MANGELINGS, D .2 1 Department of Analytical Chemistry, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic 2 Department of Analytical Chemistry, Applied Chemometrics and Molecular Modelling, Faculty of Medicine and Pharmacy, Vrije Universiteit Brussel, Belgium e-mail: [email protected] Isomers and stereoisomers are always challenging to separate in pharmaceutical research because they are compounds with similar chemical properties, necessitating a unique selectivity . Coupling columns seems to be a promising approach to solve these selectivity problems. Supercritical Fluid Chromatography (SFC) has been established as a preferred chromatographic technique for chiral separations, and an increased interest in this technique is seen for achiral analyses in recent years. Supercritical fluids possess a lower viscosity compared to liquid mobile phases, which causes a lower pressure drop across the column and easily allows the coupling of several columns in series. However, retention mechanisms in SFC are more complex than in HPLC . The aim of this project is to propose a methodology to predict the retention behaviour of analytes in a coupled column system in SFC. Atenolol, ephedrine, propranolol, mianserin, labetalol, nadolol were used as chiral model analytes, quinine, quinidine as diastereomers, and aminophenol, aminobenzoic acid, aminocresol as examples of structural isomers. A combination of a chiral and an achiral stationary phase was selected for the column coupling, using Lux Cellulose-1, Lux Cellulose-2, Lux Cellulose-3, Lux Cellulose-4, Lux Amylose-2 as chiral phases and Luna NH2, Luna Silica, Synergi Polar RP and FluoroSep-RP Phenyl as achiral phases . The mobile phase was composed of CO2 mixed with 20% (v/v) MeOH, which contained 0.1% (v/v) trifluoroacetic acid and 0.1% (v/v) isopropylamine. Retention factors of the coupled systems were estimated using a prediction formula.1 That takes into account retention factors from individual stationary phases and the effective length of stationary phases . The calculated values of retention factors were compared with the experimental ones. Using 51 the formula, we were able to predict elution order of the analytes in the coupled column system. Future work will focus on increasing the predicting precision of the formula by incorporating other factors that influence the retention behaviour of analytes. The study was supported by Erasmus+ programme and from the project of Specific Academic Research (SVV 260 412). References 1. NYIREDY, S ., SZUCS, Z ., SZEPESY, L .: J . Chromatogr . A, 1157, 2017, 122–130 . THE DEVELOPMENT OF A LIQUID CHROMATOGRAPHY-QUADRUPOLETIME-OF-FLIGHT MASS SPECTROMETRY METHOD FOR DETERMINATION OF FLAVONOIDS, ISOFLAVONOID AND THEIR METABOLITES KOČOVÁ VLČKOVÁ, H.,1 MLADĚNKA, P.,2 SOLICH, P .,1 NOVÁKOVÁ, L .1 1 Department of Analytical Chemistry, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic 2 Department of Pharmacology and Toxicology, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic e-mail: [email protected] Known metabolites of flavonoids and isoflavonoids can be categorized into two major groups: compounds with preserved (iso)flavonoid core and low molecular weight phenolics. The aim of our project is to detect the metabolites of flavonol rutin and isoflavonoid tectoridin in rat plasma samples . Firstly, a suitable method employing ultra-high performance liquid chromatography with quadrupole-time-of-flight mass spectrometry (Q-TOF) for the analysis of tectoridin, rutin and the row of its known metabolites: quercetin, quercetin-3-O-glucuronide, tamarixetin, isorhamnetin, tectorigenin, phloroglucinol, 4-methylcatechol, 3,4-dihydroxyphenylacetic acid, 3-hydroxyphenylacetic acid, homovanilic acid, 3-(3-hydroxyphenyl)propionic acid was developed. A very complex optimization of MS parameters had to be carried out especially due to the low sensitivity for the low molecular weight phenolics. The measurement was carried out in MS scan. The particular setting of Q-TOF analyzer was essential for sufficient sensitivity of phenolics, which was increased 10 times on standard solution. Secondly, the optimization of suitable sample preparation step will be necessary for the application on plasma samples. Based on the purpose of this method the simple and non-selective protein precipitation will be employed. In following step, the effect of specific Q-TOF analyzer setting on the signal to noise will be verified on matrix samples. The final method will be applied to real biological samples of rat plasma for the determination of these metabolites and for the identification of other, potentially unknown, metabolites. The study was supported by the project EFSA-CDN ( No. CZ.02.1.01/0.0/0.0/16_019/0000841 ) co-funded by ERDF, by the Czech Science Foundation (Project. No. 301/17/054095) and from the project of Specific Academic Research (SVV 260 412). 52 BENEFITS AND RISKS OF SEPARATIONS AT ELEVATED TEMPERATURE IN PROTEOMIC ANALYSES USING A CONVENTIONAL-FLOW LIQUID CHROMATOGRAPHY-MASS SPECTROMETRY SYSTEM ŠEMLEJ, T., LENČO, J. Department of Analytical Chemistry, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic e-mail: [email protected] Since nanoelectrospray was introduced nanoflow chromatography hyphenated via a nanoelectrospray to a mass spectrometer has generally been viewed as a de facto standard platform for proteomic applications. Indeed, leveraging nanocolumns with inner diameter of 0.075 mm instead of using conventional-flow columns with inner diameter of 2 .1 mm conveys a theoretical 784-fold gain in sensitivity . Nevertheless, in our recent work we demonstrated that providing the instrumentation and method are adjusted, the amount of sample for a proteomic analysis can be only roughly 5-fold greater when using conventional-flow system.1 Column temperature is one of the parameters that contributed significantly to increasing performance of the conventional-flow system. We noticed, however, that at some point the benefit of elevated temperature to peak shape was redeemed by lower number of identified peptides. We hypothesized that an on-column peptide degradation might occur when peptides were separated at elevated temperature using acidic mobile phases. To this end, we scrutinized the effect of temperature on the stability of a model protein trapped in a reversed phase column. We confirmed that temperature as high as 45 °C in combination with 0.1% formic acid already may induce on-column peptide bonds cleavage . We subsequently carried out data-dependent LC-MS analyses of tryptic peptides at various column temperatures . We found out that besides on-column peptide bonds cleavage, peptides trapped on a stationary reversed phase may undergo various artificial chemical modifications in the presence of 0.1% formic acid in mobile phases . The study was supported by the STARSS project (Reg. No. CZ.02.1.01/0.0/0.0/15_003 /0000465), co-funded by ERDF. References 1. LENČO, J., VAJRYCHOVÁ, M., PIMKOVÁ, K. et al .: Anal . Chem ., 90, 2018, 5381–5389 . 53 DETERMINATION OF PHLORIDZIN AND OTHER PHENOLIC COMPOUNDS IN APPLE LEAVES, BARK AND BUDS BY HPLC ADAMCOVÁ, A., ŠATÍNSKÝ, D., HORNA, A. 1 Department of Analytical Chemistry, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic 2 Institute of Nutrition and Diagnostics, RADANAL Ltd., Pardubice, Czech Republic e-mail: [email protected] The aim of this study was to develop a new liquid chromatography method to determine the content of phenolic compounds in raw material of apple trees – leaves, bark and buds. Raw materials from different apple cultivars were extracted in methanol acidified by 0.1% formic acid in ultrasound bath. Extracted phenolic compounds – phloridzin, phloretin, chlorogenic acid, rutin and quercitrin were subsequently analyzed by high performance liquid chromatography using YMC-Triart C18 ExRS 150 × 4.6 mm × 5µm, 8 nm analytical column. The separation was performed with gradient elution at flow rate 1 ml/min. The mobile phase consisted of acetonitrile and 0.1% phosphoric acid. The detection was performed by DAD at wavelengths 280 nm, 327 nm and 354 nm. The temperature of column space was 30 °C, injection volume was 1 μl. The identification of analytes was achieved by comparing their retention time and spectra with retention time and spectra of standard solutions. The method was validated before quantification of phenolic compounds in the leaves extract. The followed validation parameters in defined ranges were obtained for the tested analytes: the linearity (R2 = 0.994–0.998), repeatability (RSD = 0.34–1.75%), recovery (86.54–123.21%) and precision (RSD = 2.07–4.56%). Phloridzin was found as a dominating phenolic compound in concentrations ranging from 42.74 to 94.96 mg/g in leaves extracts. It covered more than 90% of total phenolic content, followed by quercitrin, chlorogenic acid, phloretin, rutin. Concentration range of phloridzin in bark extract was from 48.45 to 95.88 mg/g and in buds extract from 81.39 to 212.24 mg/g. In conclusion, the present study shows that plant material from apple trees is promising source of phytochemicals with positive effect on human health and could be potentially used for the development of dietary supplements . Acknowledgement to financial support from the project of Specific Academic Research (SVV 260 412) and by the Ministry of Agriculture of the Czech Republic (Project No. QJ1510354). 54 CITRININ-SELECTIVE MOLECULARLY IMPRINTED POLYMER AND ITS USE FOR ON-LINE SOLID PHASE EXTRACTION COUPLED TO LIQUID CHROMATOGRAPHY KHOLOVÁ, A., LHOTSKÁ, I., ŠATÍNSKÝ, D. Department of Analytical Chemistry, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic e-mail: [email protected] New selective molecularly imprinted polymer has been used for extraction in on-line SPE-HPLC to achieve the selective determination of citrinin. Four different imprinted polymers varying in combinations of components prepared by bulk polymerization were evaluated in terms of binding capacity and selectivity . Imprinted polymer prepared from the mixture comprising 1-hydroxy-2-naphtoic acid as the template molecule, acrylamide as the structural monomer, ethylene dimethacrylate as the crosslinker (in a molar ratio of 1:4:16), and acetonitrile as the porogenic solvent exhibited the best properties . Selectivity of this sorbent was confirmed by comparison with the non-imprinted counterpart prepared using the same polymerization carried out in absence of the template. Imprinted polymer was packed in a 20 × 3 mm i.d. steel cartridge and coupled to the on-line SPE-HPLC system through a six-port switching valve. The method for determination of citrinin including the on-line extraction step was then developed and validated. The sample in the form of methanolic extract was loaded, cleaned, and preconcentrated in the imprinted SPE cartridge. Following separation of citrinin from residual interferences was achieved using analytical column Kinetex Biphenyl 100 × 4.6 mm i.d., 5 µm particle size, and fluorescence detection (Ex 335, Em 500 nm). The total analysis time was only 9.50 min. Fully validated method was also applied to analysis of food supplements based on red yeast rice extracts which control is implemented in European legislation. Only minor yet acceptable contamination was found in tested samples. This work was supported by the Charles University Grant Agency (Project No. 726 316 and from the project of Specific Academic Research (SVV 260 412). COMPARISON OF LIPID EXTRACTION METHODS FROM MILK USING 2D LIQUID CHROMATOGRAPHY–MASS SPECTROMETRY JAKUBEC, P .,1 BYRDWELL, C . W .,2 NOVÁKOVÁ L .1 1 Department of Analytical Chemistry Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic 2 Food Composition and Methods Development Lab, Beltsville Human Nutrition Research Center, U .S .D .A ., Agricultural Research Service, Beltsville (MD), United States e-mail: [email protected] Over the past decades, lipid samples were mainly extracted using labor intensive, non-parallelizable methods such as Bligh & Dyer and Folch extraction using toxic 55 non-polar solvents, e.g. chloroform. However, these methods were developed when high-resolution accurate mass measurements instruments were not widely available. In addition, medical interest in short and medium chained triacylglycerols was disclosed recently. Comparison of these methods with more modern ones such as single extraction solvent mixture and methyl tert-butyl ether extraction was carried out. Major benefit of these methods compared to traditional extraction is wider coverage of extracted lipids . Chromatographic system used was two-dimensional liquid chromatography using non-aqueous reverse phase on C18 stationary phase in the first dimension and silver-ion stationary phase in the second dimension. Mass spectra were acquired using parallel outlet to high-resolution accurate mass spectrometer with electrospray ionization and triple-quadrupole with atmospheric pressure chemical ionization in first dimension. Second dimension contained parallel outlet to the two triple-quadrupole with to atmospheric pressure photoionization and ion trap with electrospray ionization. The study was supported by the STARSS project ( Reg. No.CZ.02.1.01/0.0/0.0/15_003/0000465 ) co-funded by ERDF and from the project of Specific Academic Research (SVV 260 412). PATHOBIOCHEMISTRY AND XENOBIOCHEMISTRY SECTION ANTHELMINTICS IN PLANTS SYSLOVÁ, E .,1,2 PODLIPNÁ, R .,2 LANDA, P .,2 SZOTÁKOVÁ, B .1 1 Department of Biochemical Sciences, Faculty of Pharmacy in Hradec Králové, Charles University in Prague, Czech Republic 2 Laboratory of Plant Biotechnology, Institute of Experimental Botany, Czech Academy of Science, Prague, Czech Republic e-mail: [email protected] Anthelmintics, the drugs against parasitic worms, are widely used in human and veterinary medicine, nowadays. The usefulness of anthelmintic drugs is indisputable, but at the same time they pose a risk to ecosystems. With excrements of treated animals, anthelmintics can get into the environment and there affect non-target organisms – free-living invertebrates and wild plants. In our project, we are focused on antiparasitical drugs and their uptake, biotransformation and transcriptional response in plants. The most frequently used anthelmintics (albendazole, fenbendazole, flubendazole, ivermectin, monepantel) are followed, and different plant species are tested, also the model plant Arabidopsis thaliana (wild type, Brassicaceae) . The presented work is the part of this project. The aim of the study is to get information about the effects of anthelmintics on hydroponic cultures of Arabidopsis thaliana presented as changes in plant transcriptome. The broad-spectrum benzimidazole anthelmintic fenbendazole and macrocyclic lactone ivermectin were used. Hydroponic cultures were stressed by 5 µM anthelmintic. The effect was studied after 24 and 72 hours of stress. 56 The microarray analysis was performed. For general expression at the transcription level Agilent-based microarrays were used. Quantitative analysis of whole proteomes comparing fenbendazole treated and control samples was carried out using a LC/MS Thermo Orbitrap Fusion spectrometer . In our study the presence of fenbendazole or ivermectin and theirs metabolites in Arabidopsis thaliana influenced gene expression. It was more affected by both anthelmintic in rosettes than in roots. More than 4.000 proteins were identified by proteomic analysis. Most proteins were identified in leaves of plants stressed by fenbendazole. The study was supported by the Czech Science Foundation (Project No. 18-08452S) and from the project of Specific Academic Research (SVV 260 416). SELECTION AND VALIDATION OF REFERENCE GENES FOR mRNA AND miRNA GENE EXPRESSION STUDIES IN HUMAN LIVER SLICES USING RT-qPCR ZÁRYBNICKÝ, T.,1 AMBROŽ, M.,1 ŠUBRT, Z .,2,3 MATOUŠKOVÁ, P .,1 SKÁLOVÁ, L .,1 BOUŠOVÁ, I .1 1 Department of Biochemical Sciences, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic 2 Department of Surgery, Faculty of Medicine in Hradec Králové, Charles University, Czech Republic 3 Department of Surgery, University Hospital in Hradec Králové, Czech Republic e-mail: [email protected] Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is a frequently used technique for gene expression profiling. RT-qPCR analysis depends on data normalization by endogenous reference gene, which expression is stable and independent on experimental conditions. Precision-cut liver slices (PCLS) are an interesting model due to its multicellular composition, preserved tissue architecture and intercellular communication. Its applicability to human tissues allows us to avoid interspecies differences and directly apply human tissues into multiple experimental designs . PCLS also represent a promising model for gene expression studies. However, there has been no study validating this model for selection of a suitable reference gene (or their combination). Therefore, we decided to perform a validation study, since selection of inappropriate reference gene can influence the trend and deviation of results. Three human liver samples received from surgery were used to obtain PCLS (8 mm diameter, 150 µm thickness), which were cultivated for 24 hours and samples were collected after 0, 4, 8, 12, 18 and 24 hours. As we are interested in induction studies, two cytochrome P450 (CYP) inducers β-naphthoflavone and rifampicine were used as positive controls. To verify the viability of PCLS, ATP content and lactate dehydrogenase leakage were measured. Based on literature review, six candidates (GAPDH, SDHA, ACTB, B2M, HPRT and YHWAZ) and five candidates (miR-16-5p, miR-23b-3p, miR-93-5p, miR152-3p and U6) were selected for mRNA and miRNA validation, respectively. Stability of those genes was compared using RefFinder, a free web tool that uses several other software, such as geNorm, Normfinder, BestKeeper and the comparative Ct method. Stability of 57 selected reference genes for mRNA was validated on expression analysis of CYP3A4 and CYP1A2 . The study was supported by the Czech Science Foundation (Project No. 18-09946S) and from the project of Specific Academic Research (SVV 260 416). PHENOTYPIC SCREENING OF A CHEMICALLY DIVERSE COMPOUND LIBRARY IDENTIFIED TWO COMPOUNDS WITH ANTHELMINTIC ACTIVITY AGAINST HAEMONCHUS CONTORTUS NGUYEN, L . T .,1 HERATH, H . M . P . D .,2 PRESTON, S .,2 KURZ, T .,3 SKÁLOVÁ, L .,1 GASSER, B . R .2 1 Department of Biochemical Sciences, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic 2 Department of Veterinary Biosciences, Melbourne Veterinary School, Faculty of Veterinary and Agricultural Sciences, The University of Melbourne, Australia 3 Institute of Pharmaceutical and Medicinal Chemistry, Heinrich-Heine University Düsseldorf, Düsseldorf, Germany e-mail: [email protected] Due to the widespread development of anthelmintic resistance in Haemonchus contortus, there is a continuing need to discover and develop new anthelmintic drugs to ensure sustainable control of this and related, economically important and pathogenic nematodes of ruminants. With this focus in mind, we screened a compound library consisting of 236 chemicals representing diverse classes such as heterocyclic compounds (e.g. thiazoles, pyrroles, quinolines, pyrimidines, benzo[1,4]diazepines), hydroxamic acid-based metalloenzyme inhibitors, peptidomimetics (bisand tris-pyrimidoneamides, alkoxyamides) and various intermediates. In the present study, we measured the inhibition of larval motility and development of exsheathed third-stage (xL3) and fourth-stage (L4) larvae of H. contortus using an optimized, whole-organism phenotypic screening assay. Of the 236 compounds, we identified two active compounds (called BLK127 and HBK4) that induced phenotypic changes in the worm ex vivo . Compound BLK127 induced an ‘eviscerated’ phenotype in xL3 larvae and also inhibited L4 larvae development . Compound HBK4 exerted a ‘curved’ phenotype in both xL3 and L4 larvae. The findings from this study provide a sound basis for future work on assessing the activity of the compounds identified here on adult stages of H. contortus both ex vivo and in vivo (within the host animal), and against other parasitic worms of veterinary and medical importance. The study was supported from the project of Specific Academic Research (SVV 260 416), by the project EFSA-CDN (No. CZ.02.1.01/0.0/0.0/16_019/0000841) co-funded by ERDF, and by the Strategic Partnerships Fund of Charles University. 64 INVESTIGATION OF DNA DAMAGE IN ANTHRACYCLINE-INDUCED CARDIOTOXICITY USING THE COMET ASSAY AND H2AX PHOSPHORYLATION SKALICKÁ, V., JIRKOVSKÁ, A., JIRKOVSKÝ, E., KUBEŠ, J., ŠIMŮNEK, T. Department of Biochemical Sciences, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic e-mail: [email protected] Despite their long history, anthracycline antibiotics (ANT) are still used in clinics in various antineoplastic protocols . Their usage is hampered by several life-threatening side effects such as myelotoxicity and cardiotoxicity . The mechanism of cardiotoxicity remains obscure, limiting the rational discovery of protective strategies . Anthracyclines belong to topoisomerase II (TOPII) poisons giving rise to DNA double strand breaks. Poisoning the TOPIIα in cancer cells provides the basis of their antineoplastic action, but it can also play a role in cardiotoxicity. As TOPIIα expression is minimal in post-mitotic cells, TOPIIβ is the main isoform present in quiescent cells (e.g. cardiomyocytes) and has distinct functions from TOPIIα that involve gene expression regulation and DNA repair. The DNA damage generated in cardiomyocytes by ANTs could lead to the longterm effect on the heart . Moreover, ANTs could also cause oxidative DNA damage through redox-cycling mechanisms . To assess the possible role of DNA damage, several distinct approaches can be employed. Directly, the DNA double strand breaks (DSB) can be assayed by single-cell gel electrophoresis “the comet assay”, where purified nucleoids of single cells trapped in agarose are subjected to electric field. Each DSB loosens the loops in the nucleoid forming a comet-like image. Another method involves the assessment of histone H2AX which is phosphorylated consequently to the DSB formation. These two methods were used to investigate the level of DNA damage caused by ANTs in isolated rat neonatal ventricular cardiomyocytes and HL-60 cells. Furthermore, dexrazoxane as the only approved cardioprotective agent and TOPII catalytic inhibitor was used in analysis ANT-induced DSB formation . The study was supported by the Czech Science Foundation (Project No. 18-08169S) and from the project of Specific Academic Research (SVV 260 416). 65 PHARMACOLOGY AND TOXICOLOGY SECTION INSIGHT INTO A REGULATORY NETWORK OF PREGNANE X RECEPTOR EXPRESSION SMUTNÝ, T., PÁVEK, P. Department of Pharmacology and Toxicology, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic e-mail: [email protected] The pregnane X receptor (PXR, NR1I2) is a key transcription factor involved in the regulation of both endogenous and exogenous metabolism . As a ligand-activated nuclear receptor, PXR responds to the structurally diverse collection of compounds. Following its activation, PXR triggers transcription of CYP3A4, pivotal phase I metabolic enzyme which is estimated to metabolize approximately 50% of all marketed drugs. Of particular interest is the fact that the drug-mediated activation of PXR-CYP3A4 axis may result in clinically significant pharmacokinetic interactions. Although PXR has been studied intensively regarding its transcription function, less is known about exact mechanisms standing behind its own regulation. As previously shown, PXR expression is under the control of activated glucocorticoid receptor (GR) which directly increases a level of PXR transcript. In present follow-up study, we demonstrate that PXR mRNA is not only induced at transcriptional level but also stabilized at posttranscriptional level after activation of GR . An evidence that GR mediates changes in miRNA expression which may subsequently lead to stabilization of PXR mRNA via its 3′-untranslated region will be provided during the lecture. The given postulate will be supported by data gained from gene reporter studies using various reporter vectors, miRNA expression profiling, and qPCR. Overall, major conclusion drawn from our work is that GR increases expression of PXR mRNA by dual mechanisms such as transcriptional activation of PXR from its promoter and induction of post-transcriptional stabilization. The study was supported by the project EFSA-CDN (No.CZ.02.1.01/0.0/0.0/16_019/ 0000841) co-funded by ERDF and from the project of Specific Academic Research (SVV 260 414). 66 NOVEL OBETICHOLIC ACID KETODERIVATIVES AND ISOMERS AS POTENTIAL LIGANDS OF BILE ACID RECEPTORS HORVÁTOVÁ, A .,1 KUDOVÁ, E .,2 KAŠPAR, P .,2 PÁVEK, P .1 1 Department of Pharmacology and Toxicology, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic 2 Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences, Prague, Czech Republic. Metabolic diseases with altered cholesterol and triglyceride levels are serious healthcare problem emerging in western population, and are tightly linked to inflammation. Recently, obeticholic acid (OCA), a potent farnesoid X receptor (FXR) agonist, has been shown to be a promising treatment against inflammatory hepatic disorders. Therefore, we aimed to synthesize new derivatives and isomers of OCA (named A–I) and assess their capacity to activate nuclear receptors involved in metabolic regulation including FXR, vitamin D receptor (VDR), pregnane X receptor (PXR) and constitutive androstane receptor (CAR).1,2 Gene reporter assays were performed to determine their capacity to activate nuclear receptors of interest and changes on expression of target genes were analysed by real time qPCR. Furthermore, these isomers were subjected to LC/MS analysis to determine their stability and possible conversion to OCA in HepG2 cell line and primary human hepatocytes . Our results showed that all derivatives could significantly activate FXR and PXR in therapeutic doses . Compound G is an equally strong ligand of FXR as OCA itself . We have also found that compound H is an activator of VDR. None of the tested compounds was able to activate CAR . Here, we have presented novel ligands of bile acid nuclear receptors derived from OCA. Moreover, we have found a new dual FXR/VDR agonist, compound H, which may have a promising use in the therapy of inflammatory metabolic disorder including steatohepatitis or atherosclerosis .3 The study was supported by the Grant Agency of Charles University (Project. No. 170/50/85006) and from the project of Specific Academic Research (SVV 260 414). References 1. FIORUCCI, S ., DISTRUTTI, E ., RICCI, P . et al.: Expert Opin. Ther. Targets, 18 (12), 2014, 1449–1459. 2. MOLINARO, A., WAHLSTRÖM, A., MARSCHALL, H. U.: Trends Endocrinol. Metab., 29 (1), 2018, 31–41. 3. BOZIC, M ., GUZMÁN, C ., BENET, M . et al.: J. Hepatol., 65 (4), 2016, 748–757. 67 ACTIVATION OF HUMAN CONSTITUTIVE ANDROSTANE RECEPTOR (CAR) BY BENZODIAZEPINES WITHOUT PROLIFERATIVE EFFECT AND LIVER TUMORIGENIC EFFECTS IN HUMAN CELLS ŠKODA, J .,1 DUŠEK, J .,1 HORVÁTOVÁ, A .,1 DOSEDĚL, M.,2 PÁVEK, P .1 1 Department of Pharmacology and Toxicology, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic 2 Department of Social and Clinical Pharmacy, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic e-mail: [email protected] A large number of nongenotoxic chemicals have been shown to increase the incidence of liver tumours in mice by a mode of action involving activation of the constitutive androstane receptor (CAR). Studies with the model CAR activator phenobarbital (PB) have demonstrated that events for mouse liver tumour formation include CAR activation, increased hepatocyte replicative DNA synthesis (RDS), induction of cytochrome P450 CYP2B subfamily enzymes, liver hypertrophy, increased altered hepatic foci and hepatocellular adenomas/carcinomas.1 However, this phenomenon is not confirmed in humans. In this study, we examined drugs widely used in clinics for their interaction with human CAR. We found that some benzodiazepines significantly activate human CAR in gene reporter assay and stimulated CAR translocation in vitro. However, benzodiazepines did not stimulate expression of genes involved in CAR-mediated hepatocyte proliferation in HepaRG cells. Diazepam did not stimulate cell cycle or anti-apoptotic gene expression. We did not find indication about live tumor promoting effect in databases. We can conclude that some benzodiazepines are CAR activators that unlikely stimulate proliferation or liver tumor promotion in humans . The study was supported by the Grant Agency of Charles University (Project. No. 170/50/75006) and from the project of Specific Academic Research (SVV 260 414). References 1. ELCOMBE, C . R ., PEFFER, R . C ., WOLF, D . C . et al.: Crit. Rev. Toxicol., 44 (1), 2013, 64–82. 68 INTERPLAY OF ABCB1, ABCC1 AND OATPs TRANSPORTERS IN TRANSFER OF MARAVIROC ACROSS THE HUMAN PLACENTA TUPOVÁ, L .,1 HIRSCHMUGL, B .,2 PILAŘOVÁ, V.,3 SZEKELY, V .,4 LACZKA, C .,4 NOVÁKOVÁ, L .,3 WADSACK, C .,2 ŠTAUD, F .,1 ČEČKOVÁ, M.1 1 Department of Pharmacology and Toxicology, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic 2 Department of Obstetrics and Gynecology, Medical University of Graz, Austria 3 Department of Analytical Chemistry, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic 4 Research Centre for Natural Sciences, Hungarian Academy of Sciences, Budapest, Hungary e-mail: [email protected] Maraviroc was developed as an inhibitor of HIV entry co-receptor CCR5. Due to importance of CCR5 in physiological signalling processes in organism and also between tumour cells the drug is currently evaluated as a new drug in treatment of many inflammatory and cancer diseases including states that occur in pregnant women. We have recently shown that maraviroc is transported by human ABC drug efflux transporter ABCB1 that is considered as protective component of placental barrier. In this study we aimed to verify the role of this transporter in limiting maraviroc distribution across the human placenta . The method of closed-circuit dual perfusion of maraviroc across human placental cotyledon was performed showing slight decrease of maraviroc concentration in fetal compartment . Presence of ABCB1 inhibitor elacridar and ritonavir abolished this decline, confirming ABCB1-mediated efflux of maraviroc in the feto-maternal direction. However, accelerated transport of maraviroc in materno-fetal direction was also found, suggesting contribution of another transport mechanism in the opposite direction to ABCB1 . Bi-directional transport study in monolayers of MDCKII-ABCC1 cells and accumulation study in A431-OATP2B1, -OATP1A2 and -OATP1B3 were performed, identifying maraviroc as substrate of human ABCC1, OATP1A2 and OATP1B3 . Gene expression of ABCC1 and OATP1A2 was subsequently confirmed in all the perfused placentas as well as in isolated trophoblast and fetal endothelial cells. Our data thereby indicate interplay between ABCB1 acting in feto-maternal direction and other transporters acting in materno-fetal direction, most probably ABCC1 with possible contribution of OATP1A2. These findings may help to understand maraviroc pharmacokinetics in pregnant women and contribute to safer therapy. Moreover, they indicate that the protective role of ABCB1 in the placenta may be covered up by other transporters promoting the transfer in the opposite, materno-fetal direction . The study was supported by the Grant Agency of Charles University (Project No. 616216/C/2016), the Czech Science Foundation (Project No. 17-16169S) and from the project of Specific Academic Research (SVV 260 414). 69 PLACENTAL TRANSPORT AND METABOLISM OF SEROTONIN AND TRYPTOPHAN KARAHODA, R .,1 KASTNER, P .,2 HORÁČKOVÁ, H.,1 ČERVENÝ, L.,1 KUČERA, R.,2 ABAD, C .,1 ŠTAUD, F .1 1 Department of Pharmacology and Toxicology, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic 2 Department of Pharmaceutical Chemistry and Pharmaceutical Analysis, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic e-mail: [email protected] Serotonin (5-HT) is an important neurotransmitter critical for fetal brain development and programming during pregnancy. To date, however, controversies remain on the source of fetal 5-HT. Until recently, it was believed that maternal 5-HT crosses the placenta easily and plays a key role in maintaining fetal brain levels. Nevertheless, recent studies demonstrate that 5-HT can also be synthesized from tryptophan (TRP) within the trophoblast. In addition, kynurenine pathway of TRP metabolism has also been identified in the placenta, giving rise to neuroprotective and neurotoxic metabolites. Furthermore, expression and activity of the rate-limiting enzymes of TRP metabolism in the placenta may be perturbed by maternal conditions such as inflammation, stress, or depression, recently associated with various disorders in fetal neurodevelopment. In our project we aim to characterize and validate several in vitro/ in situ/ex vivo placental models to investigate possible effects of pathologies (e.g. infections) and pharmacotherapy (e.g. antidepressants) on transport and metabolism of TRP and 5-HT in the feto-placental unit . qRT-PCR was utilized to analyze the expression of 29 transporters/enzymes in first trimester and term human placentas, rat term placentas and placental derived cell lines . Furthermore, HPLC analytical methods have been developed for quantification of TRP and 5-HT in biological samples. Dually perfused rat term placenta was employed to investigate placental transport and catabolism of TRP and 5-HT. Finally, 5-HT uptake was quantified using in vitro (BeWo choriocarcinoma cell line) and ex vivo (fresh villous fragments isolated from human term placenta) models . The study was supported by Research programme Development and Study of Drugs (Progres Q42). 70 INTERACTIONS OF ABC TRANSPORTERS AND CYTOCHROME P450 ISOFORMS WITH THE TYROSINE KINASE INHIBITOR ENSARTINIB VAGIANNIS, D ., ŠTAUD, F ., HOFMAN, J . Department of Pharmacology and Toxicology, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic e-mail: [email protected] Ensartinib is a promising small molecule tyrosine kinase inhibitor which is currently evaluated in phase II/III clinical trials for the treatment of solid tumors. In our research, we focused on the interactions of ensartinib with ABC drug efflux transporters and cytochrome P450 (CYP) biotransformation enzymes, important proteins that control pharmacokinetic behavior of a number of drugs and also play the role in cancer multidrug resistance (MDR). First, we assessed the inhibitory effect of ensartinib on human ABCB1, ABCG2 and ABCC1 transporters using accumulation assays in MDCKII cells overexpressing respective ABC transporters. The results of these experiments showed that ensartinib is a potent inhibitor of ABCB1 and ABCG2 . Consequently, the potential of ensartinib to overcome ABC transporter-mediated cytostatic MDR was evaluated in the follow-up MTT drug combination studies . In these assays, ensartinib effectively reversed daunorubicin and mitoxantrone resistance in MDCKII cells with ABCB1 and ABCG2 overexpression, respectively. Furthermore, qRT-PCR gene expression studies were performed in A549, CaCo2, NCI-H1299 and LS174T cellular models; we recorded no significant induction of ABCB1, ABCG2 or ABCC1 genes in all the cell lines following exposure to ensartinib. Finally, using Vivid CYP450 screening kits, we demonstrated that ensartinib is a strong inhibitor of CYP3A4, CYP3A5, CYP2C9 and CYP2C19, a moderate inhibitor of CYP2C8 and CYP2D6 and does not affect the activity of CYP1A2 and CYP2B6 isoforms . In conclusion, our findings indicate that ensartinib exhibits potential to provoke drug-drug interactions and affect MDR phenotype via changes in the activity but not in gene expression of particular ABC transporters and biotransformation enzymes. Moreover, our in vitro combination experiments revealed possible new effective therapeutic approach targeting ABCB1 and/or ABCG2 overexpressing tumors, which could serve as a valuable foundation for future in vivo studies . This work was supported by the Charles University Grant Agency (Project No. 1568218/C/2019), from the project of Specific Academic Research (SVV 260 414) and by the Czech Science Foundation (Project No. 16-26849S). 71 INTERACTION OF PROTEIN KINASE INHIBITORS WITH ABC TRANSPORTERS IN ACUTE MYELOID LEUKEMIA, POTENTIAL ROLE IN DRUG RESISTANCE ŠORF, A .,1 SUCHÁ, S .,1 ŠTEFÁNIKOVÁ, S .,1 SVOREŇ, M.,1 HOFMAN, J .,1 ŠTAUD, F .,1 VÍŠEK, B.,2 ZAVŘELOVÁ, A.,2 ČEČKOVÁ, M.1 1 Department of Pharmacology and Toxicology, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic 2 4th Department of Internal Medicine – Hematology, University Hospital in Hradec Králové, Charles University, Czech Republic e-mail: sorfal @faf.cuni.cz Acute myeloid leukemia (AML) represents a severe hematological malignity, which is, despite the overall therapeutic advances, still characterized by poor prognosis and short survival. These unfavorable characteristics are associated also with the expression of ATP-binding cassette (ABC) transporters in undifferentiated blast cells that drive disease progression and relapses. Our aim was to investigate the interactions of modern protein kinase inhibitors abemaciclib, palbociclib, ribociclib and midostaurin, the novel drugs that have recently gained a breakthrough approval in treatment of both solid tumors and leukemia, with ABC transporters. In vitro and ex vivo approaches comprising cellular models of resistant HL-60 cells and ex vivo peripheral blood mononuclear cells (PBMCs) isolated from de novo diagnosed AML patients were employed. Accumulation studies in HL-60 cells and their ABCB1and ABCG2-overexpressing variants confirmed that all the tested drugs show ability to inhibit efflux of daunorubicin and mitoxantrone, the anticancer drugs approved by US Food and Drug Administration for AML therapy and at the same time substrates of both, ABCB1 and ABCG2 transporters . Furthermore, the ABCG2 inhibitors effectively enhanced number of apoptotic HL-60-ABCG2 cells, when combined with mitoxantrone, as confirmed by Annexin V/propidium iodide double staining . Abemaciclib, ribociclib and midostaurin applied in human plasma-relevant concentrations further increased mitoxantrone accumulation in PBMCs of AML patients . To conclude, our preliminary results indicate the ability of protein kinase inhibitors abemaciclib, ribociclib and midostaurine to interact with ABC transporters in AML patientderived cells. These data will form a basis for our follow up research on AML resistance mechanisms and approaches for their possible overcoming . The study was supported from the project of Specific Academic Research (SVV 260 414) and by the project EFSA-CDN (No. CZ.02.1.01/0.0/0.0/16_019/0000841) co-funded by ERDF. 72 DISCOVERY OF A NOVEL MOUSE CONSTITUTIVE ANDROSTANE (CAR) RECEPTOR AGONIST THAT DOES NOT POSSESS PROLIFERATIVE ACTIVITY CONNECTED WITH NON-GENOTOXIC HEPATOCARCINOGENESIS IN MICE DUŠEK, J .,1 ŠKODA, J .,1 HOLAS, O .,2 HORVÁTOVÁ, A .,1 SMUTNÝ, T.,1 LINHARTOVÁ, L .,1 HIRŠOVÁ, P .,3 KUČERA, O.,4 MIČUDA, S.,5 BRAUENING, A .,6 PÁVEK, P .1 1 Department of Pharmacology and Toxicology, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic 2 Department of Pharmaceutical Technology, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic 3 Laboratory of Liver Pathobiology, Mayo Clinic, Rochester (MN), USA 4 Department of Physiology, Faculty of Medicine in Hradec Králové, Charles University, Czech Republic 5 Department of Pharmacology, Faculty of Medicine in Hradec Králové, Charles University, Czech Republic 6 Department of Food Safety, German Federal Institute for Risk Assessment, Berlin, Germany and Department of Toxicology, University of Tübingen, Tübingen, Germany e-mail: [email protected] Constitutive androstane receptor (CAR) is the primary regulator of drug metabolism and detoxification. CAR activation is connected with mitogenic effects leading to liver hypertrophy and tumorigenesis in rodents . Recently, stilbenoids resveratrol and trans-3,4,5,4′-tetramethoxystilbene (TMS) have been shown to abrogate or alleviate N-nitrosodiethylamine/PB-induced liver carcinogenesis or oxidative stress signaling. Thus, we examined if TMS may be an inverse agonist of mouse Car. Unexpectedly, we have identified TMS as a novel moderate murine Car agonist in cellular reporter gene experiments, in in silico docking experiments as well as in induction experiments in mouse hepatocytes, in AML-12 hepatic cells, or in mice. TMS significantly up-regulates Cyp2b10, Cyp2c29 and Cyp2c59 mRNAs, but down-regulates expression of genes involved in gluconeogenesis and lipogenesis such as Pck1, G6pc, Scd1, Acaca and Fasn in similar degree as TCPOBOP . Importantly, TMS does not induce genes involved in liver proliferation or apoptosis such as Mki67, Foxm1, Myc, Mcl1, Pcna, Bcl2, Bax or Mdm2 in C57BL/6 mice, and has no statistically significant effects on Ki67 and Pcna labeling indexes in mice, but slightly up-regulates Gadd45β mRNA expression . We can thus conclude that TMS is a novel mouse Car ligand with limited effects on hepatocyte proliferation, but controlling Car-target genes involved both in xenobiotic and endobiotic metabolism . The study was supported by the Charles University Grant Agency (Project No. 170/50/75014). 73 RADIOACTIVELY LABELED RAMUCIRUMAB: IN VITRO BINDING AND INTERNALIZATION STUDIES IN VEGFR-2 POSITIVE CELLS JANOUŠEK, J .,1 BÁRTA, P .,2 TREJTNAR, F .1 1 Department of Pharmacology and Toxicology, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic 2 Department of Biophysics and Physical Chemistry, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic e-mail: [email protected] Ramucirumab (RAM) is clinically used fully humanized monoclonal antibody directed against an extracellular domain of vascular endothelial growth factor receptor 2 (VEGFR2). The VEGFR2 is a key receptor responsible for the angiogenesis and was found to be overexpressed in some types of solid tumors (e.g. gastric, pancreatic, breast or lung cancer). RAM binds to the receptor with much greater affinity than its natural ligand and inhibits its function . With proper radiolabeling RAM could be potentially used either as a radiodiagnostic tool for imaging of VEGFR2-positive tumors or in a targeted radiotherapy. The aim of this work was to evaluate the influence of the radiolabeling process on the RAM immunoreactivity . Several methods of either direct or indirect (via chelating agents) radiolabeling and nuclides (99mTc, 131I, and 67Ga) were employed in experiments. All prepared radiopharmaceuticals were tested for radiochemical purity and stability using HPLC and ITLC methods and for in vitro receptor-ligand binding affinity. Two VEGFR2 expressing human cancer cell lines (PC3, SKOV3) were used in the binding study. All employed radiolabeling methods were suitable for the RAM labeling. However, significant differences were observed in radiochemical purity, stability and also in the deterioration of the immunoreactivity of the monoclonal antibody after the radiolabeling process . The direct 99mTc-labeling provided relatively low radiochemical purity, the iodination exhibited low stability and both of these direct labeling methods negatively influenced the immunoreactivity of RAM . All the indirect radiolabeling methods preserved RAM specific binding to the VEGFR2. However, the RAM binding kinetics was slightly altered depending on the radiolabeling conditions . The study was supported by the Charles University Grant Agency (Project No. 998216/C/2016), from the project of Specific Academic Research (SVV 260 414) and by Research programme Development and Study of Drugs (Progres Q42). 80 arterial smooth muscles. Biochanin A was found to be more potent, achieving a lower EC50 and blocking the effect of all four constrictors. These results suggest a positive impact of the isoflavonoids on some cardiovascular pathologies, that needs to be further confirmed by in vivo studies . The study was supported by the Grant Agency of Charles University (Project No. 1080217) and from the project of Specific Academic Research (SVV 260 414). COPPER CHELATORS AND THEIR ABILITY TO BIND ZINC TVRDÝ, V.,1 KARLÍČKOVÁ, J.,2 MLADĚNKA, P.,1 1 Department of Pharmacology and Toxicology, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic 2 Department of Pharmaceutical Botany, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic e-mail: [email protected] Our knowledge about the role of zinc in the human organism has been constantly expanding. It is well know that zinc has many physiological roles and is essential for many enzymes and transcription factors. Its deficiency manifests by many symptoms ranging from immune dysfunction, growth retardation to skin disorders and is mostly caused by insufficient zinc intake in the diet or derangement in GIT absorption. Less is known, that zinc deficiency can follow long-life treatment by metal chelators, which are mostly nonselective . Hence, the aim of this work was to study possible ability of clinically and experimentally used copper chelators to chelate zinc ions by using a competitive spectrophotometric method based on dithizone. The tested compounds included D-penicillamine, trientine and ammonium tetrathiomolybdate (ATTM) that have been clinically used or tested in the treatment of Wilson’s disease, and experimentally tested series of four 8-quinolinoles (8-quinolinol, 5-chloro-7-iodo-8-quinolinol, 5,7-dichloro-8-quinolinol (chloroxine) and 5-nitro-8-quinolinol (nitroxoline). Various physiologically relevant pH levels ranging from 4.5 to 7.5 were simulated. The ability to bind zinc was observed in all of the tested compounds. The most potent clinically used chelator was trientine with approximately 65% zinc binding activity in the molar ratio 1:1 at pH 7.5. However, experimentally used 8-quinolinols showed proportional or even higher binding capacity at lower pH levels. Especially nitroxoline was a very potent zinc chelator at all pH levels. Surprisingly all of the tested compounds showed higher affinity for Zn ion in comparison with ability of D-penicillamine to bind Cu ions . In conclusion we can assume that clinically used copper-chelators as well as ATTM and 8-quinolinols are also relatively potent zinc chelators and hence their longer administration can possibly result in adverse effects associated with zinc deficiency. The study was supported from the project of Specific Academic Research (SVV 260 414). 81 CLINICAL AND SOCIAL PHARMACY SECTION DEVELOPMENT AND IMPLEMENTATION OF INTERVENTIONS TO PROMOTE MEDICATION ADHERENCE IN KIDNEY TRANSPLANT PATIENTS VAŇKOVÁ, B.,1 MALÁ-LÁDOVÁ, K .,1 DUSILOVÁ-SULKOVÁ, S .,2 MALÝ, J.1 1 Department of Social and Clinical Pharmacy, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic 2 Haemodialysis Centre, University Hospital in Hradec Králové, Czech Republic e-mail: [email protected] Adherence to complex therapeutic regimen is essential during the whole posttransplant period . Nevertheless, it decreases over time and changes according to patient’s beliefs and regular care at the clinic .1 The aim is to introduce the design of our study developing and implementing individual multicomponent interventions for supporting medication adherence in patients after kidney transplantation and evaluating their impact on behavioral and transplant outcomes . This single-centre, prospective, interventional study will be undertaken in the University Hospital Hradec Králové in 2019–2020, where all adults on basal immunosuppression (IS) will be approached to mitigate selection bias. Patients at risk of non-adherence to IS will be identified using laboratory (medication level variability index) as well as self-report (BAASIS® questionnaire) measurement of medication adherence, combined with reports by family member or caregivers. These patients will be invited to structured interview with a pharmacist and will receive tailored set of interventions based on their individual barriers in knowledge, medication taking routine or attitudes. Interventions will be developed based on previous research results as well as the needs of the health care facility reflecting all main causes of non-adherence to IS. Study will include the baseline visit and the follow-up after 6 and 12 months. Effectiveness of interventions will be tested on both behavioral (e.g. adherence to IS and self-management, beliefs and knowledge about the treatment) and transplant outcomes (e.g. progression of graft dysfunction, rejection episodes, graft loss) . If interventions proof to be effective, they can be implemented into standard posttransplant care and serve as the template for other clinics . The study was supported from the project of Specific Academic Research (SVV 260 417). References 1. VAŇKOVÁ, B., MALÁ-LÁDOVÁ, K., KUBĚNA, A. A. et al .: Patient Prefer . Adherence, 12, 2018, 2605–2613 . 82 NUTRITIONAL INTAKE OF ENERGY AND SUBSTRATES, THEIR CHANGES IN PREGNANT WOMEN DURING THE LAST DECADE NAJPAVEROVÁ, S .,1 KOVAŘÍK, M.,1 KAČEROVSKÝ, M.,2 HRONEK M .1 1 Department of Biological and Medical Sciences, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic 2 Department of Obstetrics and Gynaecology, University Hospital in Hradec Králové e-mail: [email protected] Malnutrition of the maternal organism during intrauterine fetal development may result in disease in the next life of an individual. The aim of the study was to evaluate the changes in the intake of nutritional energy and substrates (PNES) in Czech pregnant women over the last 10 years and determine the accuracy of the predictive equations for PNES .1 Thirthy-five Czech pregnant women 29 ± 2.79 years old were attended in the pilot study. PNES in individual trimesters of pregnancy was obtained in weekly nutritional records, then evaluated by the computer program NutriDan and compared with values from the predictive equations for PNES .1 In 10 years, PNES (according to the predictive equations) was increased in protein in the 1st trimester of pregnancy by 9.58% (p = 0 .02) and decreased in carbohydrate intakes in all pregnancy periods by 10.06% (p = 0 .04); by 14.60%. (p = 0.0002); by 12.71% (p = 0.0003). Differences in PNES on weekdays does not change despite the expectation . The predictive equations for PNES can be used during pregnancy even after 10 years, except for protein intake in the 1st trimester and carbohydrates throughout pregnancy . The study was supported by the Grant Agency of Charles University (Project. No. 1306218), from the project of Specific Academic Research (SVV 260 417), by Ministry of Health, Czech Republic – conceptual development of research organization (UHHK, 00179906) and by Research programme Development and Study of Drugs (Progres Q42). References 1. HRONEK, M., ZADÁK, Z., HRNČIARIKOVÁ, D. et al.: Nutrition, 25 (9), 2009, 947–953. NO FAULT VACCINE COMPENSATION PROGRAM OVERDUE IN CZECH REPUBLIC – SERIOUS THREAT FOR THE CZECH MEDICAL SYSTEM IN A PROSPECT OF THE NEW CIVIL CODE HONEGR, J .1,2 1 Biomedical Research Center, University Hospital in Hradec Králové, Czech Republic 2 Faculty of Law in Prague, Charles University, Czech Republic e-mail: [email protected] On the first of January 2014 the New Civil Code (Law 89/2012 Coll., Civil Code) has come into the force in the Czech Republic. It has changed many aspects of everyday 83 life, including the problem of liability following adverse events attributed to the no-fault administration of compulsory vaccines . Until then the proprietor of health service facility was held strictly liable, for any damages that aroused from the application of vaccine even if it was caused by the nature of the applied vaccine (§421a of Law 40/1964 Coll., Civil Code). After the new legislation has come into force this is no longer true. Since 2014, the Health service provider should be held liable only, if there is his negligence proved . Thus question comes into mind – who is responsible for such adverse effect now. The position of the Czech government is unclear but it steers towards the manufacturers’ liability. We think that this is the moral hazard. In our opinion, the manufacturer should not be held liable, because he openly states in the SPC that such effects, even rare, could occur. It is the government that demands mandatory vaccination with penalization to offender for those that not conform, so it follows that government should be responsible for any adverse effects caused by such vaccination . Majority of countries united in the OECD have adopted some form of compensation for individuals harmed by compulsory vaccines administration. In this presentation I would try to evaluate such programs with emphasis on the states of comparable size, similar legal systems and comparable level of public health systems and make proposition for such a program tailored for needs of the Czech Health care system . References 1. LOOKER, C ., KELLY, H .: Bull . World Health Organ ., 89, 2011, 371–378 . SPONTANEOUS ADVERSE DRUG REACTIONS REPORTING TO ORAL ANTICOAGULANTS IN THE CZECH REPUBLIC DVOŘÁČKOVÁ, S.,1 ZIMČÍKOVÁ, E.,1 MALÁ-LÁDOVÁ, K .,1 JIRSOVÁ, E .,2 KOLÁŘ, J.,1 MALÝ, J.1 1 Department of Social and Clinical Pharmacy, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic 2 Department of Pharmacovigilance, State Institute for Drug Control, Prague, Czech Republic e-mail: [email protected] Shorter clinical use of direct oral anticoagulants (DOACs) leads to need for yet unknown risks monitoring. The aim of the study was the analysis of spontaneous adverse drug reactions (ADRs) reports to oral anticoagulants (OACs) in the Czech Republic. Retrospective analysis of spontaneous reports of suspected ADRs to OACs from healthcare professionals and patients was conducted. Reports to warfarin, dabigatran, rivaroxaban and apixaban between 01/2005 and 11/2017 were identified in the State Institute for Drug Control (SÚKL) database and quantified as anonymized data in MS Excel 2019. The reports were evaluated by ADR character, seriousness, consequence, and patient’s characteristics. The incidence of ADRs was calculated per exposure units expressed as million defined daily doses (mDDD). Drug utilization data come from the SÚKL database of quarterly reported drug supplies from distributors to healthcare facilities . Descriptive statistics and disproportionality analysis (DA), using reporting odds ratio (ROR) and 99% confidence interval (CI) was performed. 84 SÚKL received 297 reports to OACs with 672 ADRs. DOACs were related to 65% reports, resp. 64% ADRs. The most frequent ADRs were hemorrhagic and thromboembolic events, gastrointestinal and skin disorders. The DA showed that number of DOACs’ ADRs exceeded warfarin (ROR = 10.77, 99% CI = 8.70–13.32) and number of ADRs of dabigatran exceeded rivaroxaban (ROR = 3.90, 99% CI = 2.88–5.29). In 96% of reports serious ADRs were noticed, of which 21 were fatal. In context with higher warfarin utilization, the ratio of ADR reports and mDDD was low; conversely to DOACs. In summary, the number of reports seems to be low, but most ADRs were serious. Higher reporting to DOAC could be a result of shorter time on the market, compared to warfarin long-term use and reduced susceptibility to the ADRs .1 The study was supported from the project of Specific Academic Research (SVV 260 417). References 1. CALDEIRA, D ., RODRIGUES, R ., ABREU, D . et al.: Expert Opin. Drug Saf., 17(4), 2018, 339‒345. FACTORS INFLUENCING ANTIBIOTIC PROPHYLAXIS FOR SURGICAL SITE INFECTION PREVENTION DOMECKÝ, P.,1 HAUSCHKE, L .,2 PATKOVÁ, A .,1 KOLÁŘ, J.,1 MALÝ, J.1 1 Department of Social and Clinical Pharmacy, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic 2 Department of Clinical Pharmacy, Masaryk Hospital in Ústí nad Labem, Regional Health Corporation, Czech republic e-mail: [email protected] Antibiotic prophylaxis (AP) plays an important role in the reduction of surgical site infection (SSI). The aim of the presented study is to verify the use of the Risk Index as one of the tools for the individualization of the patient’s AP in clinical practice. In February 2018, we conducted a cross-sectional study, in which we identified total length of AP as the main problematic area. Special tool – the Risk Index could be used as the basis for the indication and the total length of AP among the common principles (dirty-infected wound, the presence of foreign material, pharmacokinetics of antibiotic etc .) . Before the surgical procedure, we could calculate the ACS (American College of Surgeons) Risk Index, which comprises risk factors (RFs), physical status and the type of surgery. ACS Risk Index estimates the chance of an unfavorable outcome such as SSI or the postoperative complications. After the surgical procedure, the NHSN (National Healthcare Safety Network) Risk Index could be calculated according to the ASA score (classification system that assesses the physical status of patient before surgery into 5 classes), the duration of operation and the wound classification. In 2019, we plan to perform cross-sectional study with an approximate number of 300 patients. In addition to the systematic literature review, we will collect patient and surgical procedure data before, during and after surgery. An AP individualization will be performed. Usability of ACS Risk Index and NHSN Risk Index 85 will be evaluated with the postoperative complication analysis. These will be monitored 30 days after the surgery if no foreign material was used or 1 year if patient obtains foreign material during the surgical procedure. According to these results, the interventions will be arranged to optimize and individualize use of AP. The study was supported from the project of Specific Academic Research (SVV 260 417). ANALYSIS AND MANAGEMENT OF NURSES’ MEDICATION ERRORS DURING DRUG ADMINISTRATION MĚRKOVÁ, V., HOZOVÁ, M., HLOCH, K., MALÁ-LÁDOVÁ, K., MALÝ, J., DOSEDĚL, M. Department of Social and Clinical Pharmacy, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic e-mail: [email protected] During hospitalization, there is a frequent occurrence of various mistakes caused both by healthcare professionals and by patients . A major part of these comprises medication errors during inappropriate drug handling. The key role is played by the possibility of preventing these errors. The aim of the study is to analyze medication errors by direct observation of the nurses during drug preparation and administration. The observation was conducted prospectively during 12 days in an inpatient rehabilitation facility . Three trained pharmacists monitored morning, noon and evening drug administration processes on seven wards. All errors were documented in real time, according to the predefined forms. The form always contained the name, strength, application form and dosage of all drugs prescribed to one patient. Each form was created for each patient individually, based on his or her regular medication . Within the form, the observed errors were further classified into 34 categories, including e.g . storage and labelling of the drug, administration route or drug strength. The obtained data were digitized and analyzed. Nurses (n = 27) administered in total 4662 individual doses, on average 8.18 (SD ± 5.19) doses per patient day (n = 570). Of these doses, 92.8% were administered orally, 5.1% subcutaneously, 1.6% topically, and 0.5% other. Severe misconducts (drug name, or drug strength confusion, and drug omission) occurred in 21 (0.45%) cases, on average 0.037 error per patient day. Patients hospitalized for more than 20 days are more than 50% likely to experience severe nurses’ medication error. The analysis of the observed errors has revealed that nurses commit severe and less severe mistakes, which ultimately can lead to patient harm. It is therefore necessary to focus on their minimization. The study was supported from the project of Specific Academic Research (SVV 260 417). 86 ANALYSIS OF PHARMACOTHERAPY AS ONE OF THE MAIN FALL-RELATED RISK FACTOR – A 12-MONTH PROSPECTIVE STUDY IN HOSPITALS OF SOUTH BOHEMIA REGION VOSÁTKA, J .,1 MALÝ, J.,1 DOSEDĚL, M.,1 BRABCOVÁ, I .,2 HAJDUCHOVÁ, H .,2 BÁRTLOVÁ, S .,2 KUBĚNA, A.,1 KOLÁŘ, J.,1 VLČEK, J.,1 TÓTHOVÁ, V .2 1 Department of Social and Clinical Pharmacy, Pharmaceutical Faculty in Hradec Králové, Charles University, Czech Republic 2 Institute of Nursing, Midwifery and Emergency Care, Faculty of Health and Social Studies, University of South Bohemia in České Budějovice, Czech Republic e-mail: [email protected] Pharmacotherapy belongs among one of the modifiable and predictive risk factors of falls. The aim of the study was to analyze the effect of pharmacotherapy and drug-related factors on falls at hospitalized patients with fall during 2017 at 16 hospital wards of four hospitals in South Bohemia region . The results of this prospective study were collected through online instrument containing data about patients with falls. The data obtained from patient’s medical records (e.g . drug and personal anamnesis, selected laboratory results) were completed with other information (e.g. circumstances of fall, internal and external risk factors). The data analysis was primarily focused on drugs, diagnoses and associated risk factors increasing risk of falls. Potential and individual risks were determined for each patient who experienced fall. The potential risk represented all drugs that showed an increased risk of falls described in current literature or according to its mechanism of action . If a clinical pharmacist could not exclude the drug influence on fall probability, the drug was reported as with individual risk. For data analysis, the descriptive and analytical methods were used. A p-value < 0.05 was considered statistically significant. 280 falls (51.1% women; mean age 77 ± 12), mean 8.8 drugs, and 4.1 drugs with potential or 1.8 individual risk per fall were identified. Drugs affecting the cardiovascular or the central nervous system demonstrated the highest potential risk (> 60%). Use of potential risk drugs were positively associated with increasing age (p = 0.007). The application of pharmacotherapy-related predictive risk factors together with individual approach to the patient can be an effective preventive strategy in drug-induced falls . The study was supported from the project of Specific Academic Research (SVV 260 417) and by the Ministry of Health of the Czech Republic (Project No. 16-33463A). 87 27th NATIONAL STUDENTS’ SCIENTIFIC CONFERENCE OF THE FACULTY OF PHARMACY IN HRADEC KRÁLOVÉ, CHARLES UNIVERSITY, HRADEC KRÁLOVÉ, 16–17 APRIL, 2019 SECTION OF BIOLOGICAL SCIENCES AROMATIC DERIVATIVES OF AMARYLLIDACEAE ALKALOID HAEMANTHAMINE AND THEIR BIOLOGICAL ACTIVITIES HOMOLKOVÁ, L .,1 HAVELEK, R .,2 HULCOVÁ, D .,3 CAHLÍKOVÁ, L.1 1 Department of Pharmaceutical Botany, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic 2 Department of Medical Biochemistry, Faculty of Medicine in Hradec Králové, Charles University, Czech Republic 3 Department of Pharmacognosy, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic e-mail: [email protected] The plants of the Amaryllidaceae family are known to contain a specific type of compounds, namely the Amaryllidaceae alkaloids (AA). Among the many AA, galanthamine has been given a great amount of attention due to the fact that it possesses potent acetylcholinesterase inhibition activity, and is used worldwide for the treatment of Alzheimer’s disease. Some of AA have shown remarkable cytotoxic and antiproliferative activity against diverse types of cancer cells . One of the most interesting compounds is haemanthamine (HA), β-crinine-type of AA, which displays significant in vitro cytotoxic activity against several different types of cancer cell lines (e.g. MOLT-4, Jurkat, HeLa, MCF-7, SK-BR-3, A549, Caco-2, HT-29).1,2 HA was isolated from the bulbs of Narcissus pseudonarcissus cv . Dutch Master in large amounts as a start material for the preparation of its derivatives . Some of the previous prepared derivatives have shown promising cholinesterases inhibitory potencies and cytotoxic activities against wide spectrum of cancer cell lines. Based on these results, we decided to prepare further aromatic semi-synthetic analogues of HA with emphasis on optimizing the structure vs biological activity. Prepared haemanthamine derivatives were tested for their inhibition potency of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) . The cytotoxic activity against selected panel of cancer and healthy cells has been also studied . Derivative LC-90 is the most promissing haemanthamine analogue from the compounds prepared in this study . 2021 Folia Pharm . Univ . Carol . L Pag . 87–148 88 The study was supported from the project of Specific Academic Research (SVV UK 260 412). References 1. HAVELEK, R ., SEIFRTOVÁ, M ., KRÁLOVEC, K . et al.: Phytomedicine, 21 (4), 2014, 479‒490. 2. CEDRÓN, J . C ., RAVELO, Á . G ., LEÓN, L . G . et al.: Molecules, 20 (8), 2015, 13854‒13863. INTERACTIONS OF TAMARIXETIN AND ISORHAMNETIN WITH COPPER LOMOZOVÁ, Z .,1 KARLÍČKOVÁ, J.,1 MLADĚNKA, P.2 1 Department of Pharmaceutical Botany, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic 2 Department of Pharmacology and Toxicology, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic e-mail: [email protected] Copper is an essential trace element involved in many vital metabolic processes. However, unbound copper ions cause damage to various biomolecules and lead to pathological conditions. Disorder of copper homeostasis can be treated with copper chelators.1 Tamarixetin and isorhamnetin are the main O-methylated metabolites of quercetin from the group of flavonoids. Flavonoids belong to plant secondary metabolites and have a positive impact on human health . Chelation of transient metal ions is one of their mechanisms of action .1 Flavonoids may also have negative prooxidant effects, which have been related with their copper reducing activities and formation of free radicals through Fenton reaction .2 In this in vitro study, tamarixetin and isorhamnetin were tested for their interactions with both copper oxidation states at four (patho)physiologically relevant pH conditions (4.5, 5.5, 6.8 and 7.5) by two spectrophotometric methods. Apparently, the 2,3-double bond and the 3-hydroxy-4-keto group in flavonols were efficient for stable copper chelation. In conclusion, compounds showed very good ability to chelate and reduce copper ions, but in acidic condition the chelation effect was slightly lower. The study was supported by the Grant Agency od Charles University (Project. No. 1080217C) and from the project of Specific Academic Research (SVV 260 412). References 1. ŘÍHA, M., KARLÍČKOVÁ, J., FILIPSKÝ, T. et al.: RSC Advances, 4 (62), 2014, 32628–32638. 2. MIRA, L ., FERNANDEZ, M . T ., SANTOS, M . et al.: Free Radic. Res., 36 (11), 2002, 1199–1208. 89 CENTAUREA CYANUS L . ALKALOIDS AND THEIR NEUROPROTECTIVE ACTIVITY DRABBOVÁ, A .,1 MAŘÍKOVÁ, J.,2 KUNEŠ, J .,2 CHLEBEK, J .1 1 Department of Pharmaceutical Botany, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic 2 Department of Organic and Bioorganic Chemistry, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic e-mail: [email protected] Alzheimer’s disease (AD) is the most common neurodegenerative disorder causing dementia. The current portfolio of drugs in use is very limited: there are two therapeutic approaches according to Evidence Based Medicine – cholinesterases inhibitors and N-methyl-D-aspartate blockers. In our screening of natural inhibitors of human acetylcholinesterase (hAChE) and butyrylcholinesterase (hBChE), a summary alkaloidal extract from Centaurea cyanus L . seeds demonstrated promising selective inhibitory activity on hBChE with an IC50 value of 22.62 ± 3.62 µg/mL, but against hAChE was considered inactive (IC50 value > 200 µg/mL). The extract (4.67 g) was fractionated by column chromatography on neutral alumina and nine fractions (A–I) were obtained. Preparative TLC of fraction A (698 mg) on silica gel and C18-reversed phase silica gel led to the isolation of pure compound AD-1 (36 mg), which showed a positive reaction with Dragendorff’s reagent . Based on LC-MS, HRMS, 1H and 13C NMR spectra, and optical rotation its structure was elucidated. The isolation of AD-1 is reported for the first time. The compound was screened for the ability to inhibit hAChE and hBChE and it was found that it is inactive (IC50 values > 100 µM). Furthermore, AD-1 will be tested on the inhibition of selected enzymes such as glycogen synthase kinase-3β, β-secretase, and prolyl oligopeptidase, which play an important role in pathophysiology of AD and represent promising targets for the treatment of this disorder . The study was supported from the project of Specific Academic Research (SVV 260 412). AROMATIC ESTERS OF AMARYLLIDACEAE ALKALOID GALANTHINE AS POTENTIAL DRUGS FOR TREATING ALZHEIMER’S DISEASE KARUMA, T .,1 PEŘINOVÁ, R.,2 HULCOVÁ, D .,1 CAHLÍKOVÁ, L.1 1 Department of Pharmaceutical Botany, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic 2 Department of Pharmacognosy, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic e-mail: [email protected] Alzheimer’s disease (AD) is an irreversible brain disorder that’s progressive and slowly destroys the memory and cognitive skills. AD is the leading cause of dementia in adults and is ranked the third cause of death after cancer and heart disease. Several medications have been approved for treating the symptoms of AD and those that slow down the 96 THE USE OF RNA INTERFERENCE FOR THE MODIFICATION OF DNA TOPOISOMERASE II LEVELS IN CANCER CELLS AND ITS INFLUENCE ON THE ANTINEOPLASTIC EFFECT OF ANTHRACYCLINES KLIEBER, R ., SKALICKÁ, V ., JIRKOVSKÁ, A . Department of Biochemical Sciences, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic e-mail: [email protected] Topoisomerase II (TOP II) is an enzyme that alters the topological state of the DNA double helix during physiological processes through the formation of transient DNA double strand breaks.1 Two TOP II isoforms are known: TOP IIα is essential for proper separation of chromosomes in mitotic cells, whereas TOP IIβ is primarily associated with gene transcription .2 Anthracycline antibiotics (ANT) belong to the group of topoisomerase poisons that stabilize the complex between TOP II and DNA. This prevents the religation of the DNA double strand breaks and thus causes irreversible DNA damage leading to programmed cell death . Although ANT are frequently administered in various antineoplastic protocols (hemato-oncological malignancies, hormone-dependent tumors),3 the therapy still possess a high risk of irreversible cardiotoxicity. The mechanism of cardiotoxicity remains unraveled. However, it has been previously discussed that TOP IIβ inhibition could play there a key role.4 The practical aim of this work was to optimize methodology of RNA interference using siRNA molecules targeting the TOP IIβ isoform in human tumor suspension cell line HL-60. The transfection was performed by the electroporation. Evaluation of TOP IIβ was accomplished both on mRNA level by RT-qPCR and protein level via immunoblotting . Furthermore, antiproliferative effect of daunorubicin as a model ANT was investigated in TOP IIβ depleted cells. The study was supported from the project of Specific Academic Research (SVV 260 416). References 1. SUNTER, N . J ., COWELL, I . G ., WILLMORE, E . et al.: J . Nucleic Acids, 2010, Article 710589, doi: 10.4061/2010/710589. 2. De CAMPOS-NEBEL, M., LARRIPA, I., GONZÁLES-CID, M.: PloS One, 5(9), 2010, e12541, doi: 10.1371 /journal.pone.0012541. 3. BOLLIMPELLI, V . S ., DHOLANIYA, P . S ., KONDAPI, A . K .: Arch . Biochem . Biophys ., 633, 2017, 78–84 . 4. LYU, Y . L ., KERRIGAN, J . E ., LIN, C .-P . et al.: Cancer Res., 67 (18), 2007, 8839–8846. 97 CAN FENBENDAZOLE ENTER THE FOOD CHAIN? MARTÍNKOVÁ, L.,1 RAISOVÁ STUCHLÍKOVÁ, L.,1 SKÁLOVÁ, L .,1 SZOTÁKOVÁ, B .,1 PODLIPNÁ, R .2 1 Department of Biochemical Sciences, Faculty of Pharmacy in Hradec Kralové, Charles University, Czech Republic 2 Laboratory of Plant Biotechnologies, Institute of Experimental Botany, Czech Academy of Science, Czech Republic e-mail: [email protected] Drugs are potentially dangerous environmental contaminants, because they are designed to have biological effects at low concentrations. Especially anthelmintics, veterinary pharmaceuticals used in large amounts in modern husbandry for treatment and prevention of diseases in animals, enter the environment directly via animals excrements left in pastures, used in field fertilization or following direct application in aquaculture.1 Depending on the increased production of soybean in agriculture and use of manure droppings, knowledge of biotransformation and uptake of anthelmintic drugs (e.g. fenbendazole) in soybean is very important .2,3 Plant samples stressed with 10 µM fenbendazole were homogenized and subjected to liquid–liquid extraction. The samples were analysed using UHPLC/MS (QqQ) in positive-ion mode. The analysis of roots, leaves, pods and seeds confirmed the uptake of fenbendazole. Moreover, presence of metabolites substantiate that plant enzymatic systems were able to transform fenbendazole via several reactions. The study was supported by the Czech Science Foundation (Project No.18-07724S) and from the project of Specific Academic Research ( SVV 260 416). References 1. SKÁLOVÁ, L., BOUŠOVÁ, I.: Metabolismus léčiv a jiných xenobiotik (Metabolism of Drugs and Other Xenobiotics) . Praha, Karolinum, 2011 . 2. O sóji (About soybeans), Soja.cz [online]. Available from: https://www.soja.cz/o-soji.html. 3. DOBŠÍKOVÁ, R., ŠIROKÁ, Z., BLAHOVÁ, J.: Farmakologie v produkci potravin pro posluchače Fakulty veterinární hygieny a ekologie (Pharmacology in Food Production for Students of the Faculty of Veterinary Hygiene and Ecology) [online]. VFU Brno, 2012. Available from: https://cit.vfu.cz/ivbp/wp-content/ uploads/2011/07/Farmakologie-v-produkci-potravin.pdf. CHANGES IN EXPRESSION OF CYTOCHROMES P450 AND P-GLYCOPROTEINS IN HAEMONCHUS CONTORTUS AFTER EXPOSITION TO SUBLETHAL DOSES OF DRUGS PASÁK, M ., KELLEROVÁ, P ., MATOUŠKOVÁ, P ., SKÁLOVÁ, L . Department of Biochemical Sciences, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic e-mail: [email protected] The parasite, which causes significant losses of small ruminants through a disease called heamonchosis, is the abomasal nematode Haemonchus contortus.1 The main problem with 98 this parasite is the presence of drug resistance to almost all administered anthelmintics . Ivermectin (IVM) is one of the anthelmintics used in veterinary medicine to eliminate this endoparasite. It is believed that xenobiotic-metabolizing enzymes such as cytochromes P-450 and membrane efflux transporters P-glycoproteins, play a role in resistance in H. contortus .2,3 As the knowledge of the development and mechanism of drug resistance in nematodes is still insufficient, more research is needed. This study focuses on the effect of low concentrations of IVM, which could occur in environmental circulation, on expression of selected cytochromes P450 and P-glycoproteins. Females and males were separated before our experiment. After 4, 12 and 24-hour incubation of nematodes with three different IVM concentrations the levels of selected mRNAs were analyzed by qPCR. The results obtained in nematodes exposed to IVM were compared to the results in control nematodes (exposed to solvent only). Significant IVM-induced changes were found in expression of several genes. Significant sex-differences were observed in inducibility of tested genes. The induction effect of IVM was most pronounced in P-gp 1 genes, thus P-gp 1 might contribute to development of drug resistance in H. contortus . The study was supported by Czech Science Foundation (Project No. 18-07724S) and from the project of Specific Academic Research (SVV 260 416). References 1. TAK, I . R ., DAR, S . A ., DAR, J . S . et al.: Global Veterinaria, 13 (6), 2014, 960–965. 2. GODOY, P ., CHE, H ., BEECH, R . N . et al .: Mol . Biochem . Parasitol ., 204, 2015, 11–15 . 3. LAING, R ., BARTLEY, D . J ., MORRISON, A . A . et al.: Int. J. Parasitol., 45 (4), 2015, 243–251. DNA METHYLATION CHANGES IN OROPHARYNGEAL CARCINOMA BIRKNEROVÁ, N .,1 MATOUŠKOVÁ, P .,1 BARANOVÁ, I .,2 KOVAŘÍKOVÁ, H.,2 LACO, J .,3 VOŠMIK, M .,4 CHMELAŘOVÁ, M.2 1 Department of Biochemical Sciences, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic 2 Institute of Clinical Biochemistry and Diagnostics, Faculty of Medicine in Hradec Králové, Charles University, and University Hospital Hradec Králové, Czech Republic 3 The Fingerland Department of Pathology, Faculty of Medicine in Hradec Králové, Charles University and University Hospital Hradec Králové 4 Department of Oncology and Radiotherapy, Faculty of Medicine in Hradec Králové, Charles University and University Hospital Hradec Králové e-mail: [email protected] Oropharyngeal carcinoma (OP) is a type of head and neck cancer (HNC) emerging in the tissue of base of the tongue, tonsils, soft palate and pharynx. Traditional risk factors include excessive alcohol and tobacco consumption . Recently, human papillomavirus (HPV) has been identified as an additional independent risk factor for the development of these tumors .1 Epigenetic alterations, such as DNA methylation, refer to heritable changes in gene expression that occur without changes in the underlying DNA sequence and can contribute to carcinogenesis .2 The aim of this study was to investigate methylation levels 99 of selected tumor suppressor genes in oropharyngeal squamous cell carcinoma (OPSCC) and compare methylation levels with HPV status. DNA methylation levels of selected tumor suppressor genes were analysed using Methylation-Specific Multiplex Ligation-dependent Probe Amplification (MS-MLPA) in metastatic tumor samples, metastases samples, non-metastatic tumor samples and control tissue samples (non-cancerous palatine tonsils). We considered sample to be methylated above cut-off limit 15% . CADM1 methylation in OPSCC is increased by the presence of HPV infection, which is corresponding to methylation of the CADM1 gene in cervix carcinoma .3 CADM1 gene methylation levels are probably not influenced by metastatic process, because our results show that corresponding metastases has the same methylation status as their primary tumors. Methylation of CADM1 could be used as potential OPSCC biomarker in the future. The study was supported by Ministry of Health, Czech Republic – conceptual development of research organization (UHHK, 00179906) and from the project of Specific Academic Research (SVV 260 416). References 1. ZARAVINOS, A.: Oncotarget, 5 (12), 2014, 3956–3969. 2. Van KEMPEN, P . M ., NOORLAG, R ., BRAUNIUS, W . W . et al.: Epigenetics, 9 (2), 2014, 194–203. 3. Van KEMPEN, P . M . W ., Van BOCKEL, L ., BRAUNIUS, W . W . et al.: Cancer Med., 3 (5), 2014, 1185–1196. METABOLISM OF FLUBENDAZOL IN HUMAN LIVER SUBCELLULAR FRACTIONS HOLUBOVÁ, J .,1 KUBÍČEK, V.,2 SKÁLOVÁ, L .1 1 Department of Biochemical Sciences, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic 2 Department of Biophysics and Physical Chemistry, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic e-mail: [email protected] Flubendazole (FLU), a benzimidazole anthelmintic drug with a broad spectrum of activity and low toxicity, has been used in veterinary as well as human medicine for a long time .1 Owing its mechanism of action, based on the specific binding to tubulin, FLU is now considered a promising anti-cancer agent .2 However, we do not have enough information about its biotransformation in human .3 In our project, subcellular fractions from the liver of 12 human patients (6 males and 6 females) were used to study the stereospecificity, cellular localization, coenzyme preference and possible inter-individual or sex differences in FLU reduction. In addition, the risk of FLU interaction with other drugs was evaluated. Results showed that FLU is predominantly reduced in cytosol and the NADPH coenzyme is preferred. The strict stereospecificity of FLU carbonyl reduction was proven and carbonyl reductase 1 was identified as the main enzyme of FLU reduction in the human liver. A higher reduction of FLU and a higher level of carbonyl reductase 1 protein was found in males than in females, but overall inter-individual variability was relatively low. Hepatic 100 intrinsic clearance of FLU is very low, and FLU had no effect on doxorubicin carbonyl reduction in the liver and in cancer cells. For these reasons, interactions of FLU with other carbonyl bearing drugs are not expected . The obtained results demonstrate the safety of FLU use in human and support FLU repurposing for cancer treatment . The study was supported from the project of Specific Academic Research (SVV 260 416). References 1. MATÉ, M . L ., GEARY, T ., MACKENZIE, C . et al.: J. Vet. Pharmacol. Therap., 40 (5), 2017, 493–499. 2. ČÁŇOVÁ, K., ROZKYDALOVÁ, L., RUDOLF, E.: Acta Medica (Hradec Králové, Czech Republic), 60 (1), 2017, 5–11 . 3. STUCHLÍKOVÁ, L., KRÁLOVÁ, V., LNĚNIČKOVÁ, K. et al.: Drug Test. Anal., 10 (7), 2018, 1139–1146. NEW MODEL OF MUTATED TGR5 AND ITS TREATMENT BY BILE ACIDS DERIVATIVES IN SILICO MOTAMEDI, N .,1 HORVÁTOVÁ, A .,2 KAŠPAR, M .,3 KUDOVÁ, E .,3 DRASTÍK, M.,1 PÁVEK, P .2 1 Department of Biophysics and Physical Chemistry, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic 2 Department of Pharmacology and Toxicology, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic 3 Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Prague, Czech Republic e-mail: [email protected] TGR5 is a member of the G-protein-coupled receptors family that represents a broad group of transmembrane receptors . It plays a role in regulation of cholesterol homeostasis, lipid, and carbohydrate metabolism. That makes TGR5 an interesting target in the pharmacotherapy of diabetes type 2 and obesity . In this study, a homology model of TGR51 was used since no crystal structure has been hitherto available . All observed orientations of cholic acid derivatives in several studies1,2 are consistent and very similar. The 3-α hydroxyl group on the A-ring is oriented towards a polar part of the cavity, forming a hydrogen bond with Glu169. The carboxylic group is facing another polar subdomain on the extracellular opening of the binding pocket probing for Ser270. These observations are in perfect line with our docking results received by employing Auto-Dock Vina software. Based on this we can conclude that Glu169 together with Ser270 are the crucial amino acids determining the proper orientation of a ligand in the pocket. Hydrophobic interaction between the rather nonpolar central part of the binding pocket and the polycyclic skeleton provides further stabilization. Another small nonpolar subdomain (Leu84, Leu87, Leu160, Phe125) hosts ethyl or ethylidene groups bound to the C6 . Macchiarulo et al .3 who propose orientation of ligands towards Tyr89 and Asn93 suggest a different model. This orientation is perpendicular to what we are observing and does not involve any connection of the ligand to Ser270 . The goal of this study is to shed light on this discrepancy by preparing series of mutants . Utilization of molecular docking or use of mutants should help us in further study of novel ligands. 101 The study was supported from the project of Specific Academic Research (SVV 260 401). References 1. SEPE, V ., RENGA, B ., FESTA, C . et al. J. Med. Chem., 57 (18), 2014, 7687–7701. 2. DI LEVA, F . S ., FESTA, C ., RENGA, B . et al.: Sci. Rep., 5, 2015, Article 16605, doi: 10.1038/srep16605. 3. MACCHIARULO, A ., GIOIELLO, A ., THOMAS, C . et al.: ACS Med. Chem. Lett., 4 (12), 2013, 1158–1162. HPLC ANALYSIS OF THE EFFECT OF SILYBIN ON THE FENTON REACTION KLIMKOVÁ, K .,1,2 MERCOLINI, L .,2 MLADĚNKA, P.1 1 Department of Pharmacology and Toxicology, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic 2 Department of Pharmacy and Biotechnology, Alma Mater Studiorum – University of Bologna, Italy e-mail: [email protected] Silymarin, the standardized extract of the milk thistle (Silybum marianum), is a widely used over-the-counter drug recommended for a number of liver diseases . Silymarin contains silybin as the main flavonolignan component. In general, flavonolignans, due to the presence of the flavonoid core functionalized with hydroxy and oxo groups, can interact with transition metals by forming complexes.1 However, the biological behaviour of these complexes is unknown. Tight copper or iron chelation will hinder the redox activity of both metals, while reducing potency can be connected with pro-oxidative effect. Moreover, flavonolignans are direct scavengers of free radicals. The aim of this study was to test the influence of silybin on the ironand copper-driven Fenton reaction, during which hydroxyl radical is produced and hence to determine if silybin acts as an antior pro-oxidant in the presence of these ions . Since compounds can behave differently depending on their ratio to metal, different ratios silybin : metal were tested.2 Salicylic acid was used as an indicator of the hydroxyl radical production . An original method based on HPLC coupled to electrochemical (coulometric) and diode array detectors was developed and applied. In all tested ratios, silybin was able to decrease the production of the hydroxyl radical triggered by both metals and hence only the antioxidant activity was documented. In the case of iron, the highest inhibition was observed at the tested ratio of 1:1, silybin to iron, while progressive anti-oxidative effects of silybin were observed in the case of copper-driven Fenton reaction . Based on these results, the toxicity of silybin based on pro-oxidation via interaction with transition metals is unlikely. The study was supported from the project of Specific Academic Research (SVV 260 414). References 1. TVRDÝ, V., CATAPANO, M. C., RAWLIK, T. et al .: J . Inorg . Biochem ., 189, 2018, 115–123 . 2. MACÁKOVÁ, K., MLADĚNKA, P., FILIPSKÝ, T. et al.: Food Chem., 135 (4), 2012, 2584–2592. 102 CHARACTERIZATION OF LIGAND BINDING TO M1 MUSCARINIC ACETYLCHOLINE RECEPTOR USING FLUORESCENCE ANISOTROPY METHOD DANKOVÁ, H .,1 RINKEN, A .,2 LAASFELD, T .,2 VOKŘÁL, I.1 1 Department of Pharmacology and Toxicology, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic 2 Institute of Chemistry, Faculty of Science and Technology, University of Tartu, Estonia e-mail: [email protected] Methods with detection of fluorescence anisotropy are used in drug screening and kinetic studies of ligands binding to targets . Compared to gold standard radioactive binding, these present simpler, inexpensive and safer option .1 The aim of this work was to evaluate fluorescent anisotropy on M1 muscarinic receptors expressed on surface of baculovirus particles . Apparent potencies expressed as IC50 of eleven classical muscarinic ligands, both agonists and antagonists and three new M2selective ligands were measured and compared to previously published Ki values based on radioactive binding assay. Furthermore, analysis with IQMTools for Matlab was performed to simulate the dynamic process of ligand binding to receptor . IC50 values of muscarinic antagonists were mostly in good agreement with Ki from literature. However, agonists McN-A-343 and acetylcholine showed lower potency in comparison with published Ki . Apparent potency of carbachol could not be determined and UR-MK259 showed intriguing results. Despite some discrepancy, fluorescence anisotropy assay presents highly valuable, homogenous and high throughput method in study of ligands binding to receptor . This study was made by support of Erasmus+ program, which was funded by European Union and from the project of Specific Academic Research (SVV 260 414). References 1. SWONGER, K. N., ROBINSON, A. S.: Curr. Protoc. Protein Sci., 93, 2018, e63, doi: 10.1002/cpps.63. DETERMINATION OF RENAL TOXICITY IN BRAF INHIBITORS IN VITRO ŠIMKOVÁ, M .,1 HRUŠKA, M .,1 MAIXNEROVÁ, J .,2 TREJTNAR, F .2 1 First Private Language Grammar School in Hradec Králové, Czech Republic 2 Department of Pharmacology and Toxicology, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic e-mail: [email protected] One of the most serious malignant skin disease nowadays is melanoma. Targeted treatment based on inhibition of oncogenic mutations of BRAF enzyme using their specific inhibitors, dabrafenib and vemurafenib, has been proved to be a new useful therapeutic procedure .1 However, a renal damage associated with a dysfunction of podocyte cells in 103 the renal glomerulus has recently been observed .2 The aim of this study was to verify the potential cytotoxic effect of dabrafenib and vemurafenib on human kidney cell lines in vitro and compare their quantitative cytotoxic parameters mutually and with comparative agents . The in vitro experiments were performed using two human cell lines representing different types of kidney cells (HEK-293 and PODO/TERT256). The cells were incubated with the tested compounds at different incubation concentrations for 24 h and 48 h. The cellular toxicity of the BRAF inhibitors was compared with a positive control (kidney toxin amphotericin B) and a negative control (non-toxic paracetamol). A colorimetric method based on measurement of cell metabolic activity was employed. The IC50 (drug concentration inhibiting viability to 50%) values were determined by analysis of inhibition curves. The results showed clear toxic effects on kidney cells in both BRAF inhibitors. However, vemurafenib was significantly more toxic in comparison with dabrafenib (even its toxicity is higher than amphotericin B) . The observed differences in toxicity of vemurafenib and dabrafenib towards both cell renal lines were not substantial, which might suggest that the renal damage by BRAF inhibitors might involve more types of kidney cells besides podocytes . The study was supported by Research programme Development and Study of Drugs (Progres Q42) and from the project of Specific Academic Research (SVV 260 414). References 1. RAHMAN, M . A, SALAJEGHEH, A ., SMITH, R . A . et al.: Crit. Rev. Oncol. Hematol., 90 (3), 2014, 220–232. 2. PERICO, L ., MANDALÀ, M ., SCHIEPPATI, A . et al.: Am. J. Kidney Dis., 70 (1), 2017, 145–150. DEVELOPMENT OF A METHOD FOR SCREENING OF COBALT CHELATORS MORAVCOVÁ, M., MLADĚNKA, P. Department of Pharmacology and Toxicology, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic e-mail: [email protected] Cobalt as the internal part of the vitamin B12 is an essential microelement for living organisms including humans. Its lack or excess is associated with pathological conditions. A deficiency of cobalt, which is not very common, could lead even to pernicious anaemia. Cobalt poisoning can be caused, for example, by exposure to cobalt metal dust during the production of tungsten carbide or follows the corrosion of metal hip prostheses. Patients intoxicated by cobalt can develop neurological damage, hypothyroidism and/or cardiomyopathy . The aim of this work is to develop a standardized, rapid and cheap method for the screening of cobalt chelators . For this purpose, spectrophotometric detection using 1-nitroso-2-nafthol-3,6-disulfonic acid disodium salt as the indicator was used. Firstly, it was found that the addition of cobalt ions leads to a clear bathochromic shift of the maximum absorbance of the indicator. The relationship between the absorbance and cobalt concentra- 104 tion was highly linear from 470 to 560 nm at all four pH conditions tested (4.5 to 7.5). The sensitivity of the method was 500 nM at pH 4.5 and even lower at higher pH conditions. Currently, potential cobalt chelators are being tested . In conclusion, a competitive method for screening of cobalt chelation, which as far as we know, has not been available previously, was developed. The study was supported from the project of Specific Academic Research (SVV 260 414). References 1. PAUSTENBACH, D . J ., TVERMOES, B . E ., UNICE, K . M . et al.: Crit. Rev. Toxicol., 43 (4), 2013, 316–362. 2. CATAPANO, M. C., TVRDÝ, V., KARLÍČKOVÁ, J. et al .: Bioorg . Chem ., 77, 2018, 287–292 . ROLE OF SELECTED ABC AND SLC TRANSPORTERS IN TRANSMEMBRANE PERMEABILITY OF MARAVIROC: EFFECT ON TRANSPORT IN PLACENTA MATIAŠKOVÁ, Z., ČEČKOVÁ, M. Department of Pharmacology and Toxicology, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic e-mail: [email protected] Antiretroviral drug maraviroc is an inhibitor of CCR5-trophic HIV virus and belongs to the group of entry inhibitors. Nowadays, maraviroc is administered as a part of combination antiretroviral therapy (cART) primarily in adults and children over the age of two. In pregnant women, it is used to reduce the risk of transmission of HIV to the fetus. The knowledge of interactions of maraviroc with drug transporters in placenta is crucial for optimizing the therapy during pregnancy, in both terms of efficacy and potential adverse effects. Maraviroc is known substrate of ABCB1 transporter, which plays a protective role to the fetus by its efflux activity in the apical membrane of trophoblast. However, the results of our recent study suggest involvement of other transport mechanisms in the maraviroc transplacental pharmacokinetics, especially those operating in the opposite direction to ABCB1 . The aim of this study was to evaluate in in vitro studies whether, besides ABCB1, maraviroc interacts with other transplacental transporters. First, an accumulation study and bidirectional transport of maraviroc across the monolayer of placental BeWo b30 cells was performed. Significant reduction in maraviroc accumulation in the presence of verapamil (100 µM), ritonavir (10 µM) and elacridar (2 µM) suggests that some influx transporters might be involved . On the other hand, an increase of accumulation in the presence of inhibitor MK-571 (50 µM) suggests also involvement of some ABCCs efflux transporter(s). After the following evaluation of transport study, a significant transfer of maraviroc in B-A direction was observed sensitive to the presence of ritonavir and MK-571. In order to identify the interacting transport mechanisms, in vitro studies were performed using MDCKII cells overexpressing human ABCC1 transporter and A431 cells overexpressing human OATP2B1, 1A2 or 1B3 transporter. Substrate affinity of maraviroc to ABCC1, OATP1A2 and OATP1B3 was revealed, but not to OATP2B1. 105 Based on the obtained data we can suggest, that maraviroc interacts with several drug transporters in placenta that could partly reverse the limiting effect of apically localized ABCB1. Considering the fact that antiretroviral therapy is always administered as combination of drugs in developed countries, it can be assumed that maraviroc will be susceptible to drug–drug interactions and this newly obtained data could contribute to better understanding of the transplacental pharmacokinetics of maraviroc and optimization of the therapy . The study was supported from the project of Specific Academic Research (SVV 260 414). EFFECT OF CHELATORS ON ALCOHOL DEHYDROGENASE, A ZINC CONTAINING ENZYME GUTIERRES, C .,1,2 HRUBŠA, M .,1 KREIBICHOVÁ, M .,1 TVRDÝ, V.,1 MLADĚNKA, P.1 1 Department of Pharmacology and Toxicology, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic 2 Degree Course in Pharmacy, Faculty of Pharmacy, University of Lisbon, Portugal e-mail: [email protected] Zinc is an ubiquitous metal present in food, water and air. It is also the second most abundant trace element in living organisms . Zinc is involved in many physiological processes. Among others, zinc is contained in alcohol dehydrogenase (ADH) and is needed for its catalytic role which lies in reversible oxidation of ethanol to acetaldehyde with the concomitant reduction of NAD+ to NADH . Since many, even clinically used, metal chelators are non-selective, they can chelate zinc as well. The aim of this study was hence to test if different chelators can block enzymatic function of ADH. Shortly: different chelators dissolved in DMSO were mixed with ADH from Saccharomyces cerevisiae (0.04 UI/mL), ethanol (3% V/V) and NAD+ (7.2 mM) in a buffer of pH 8.8 at 25 °C. The reaction was monitored for 6 minutes measuring the absorbance at 340 nm, which corresponds to the formed NADH. Our preliminary results with 12 chelators have shown that most of them, in particular hydrophilic chelators, had very low or almost no effect on ADH activity. The most active chelators were nitro or halogen derivatives of 8-hydroxyquinolinole, but their activity at concentrations below 100 µM was quite low. In conclusion, it seems that metal chelators can interfere with ADH only at higher concentrations. The study was supported from the project of Specific Academic Research (SVV 260 414). References 1. MAGONET, E ., HAYEN, P ., DELFORGE, D . et al.: Biochem. J., 287 (2), 1992, 361–365. 2. MARET, W.: Adv. Nutr., 4 (1), 2013, 82–91. 3. VALLEE, B. L., GIBSON, J. G.: J. Biol. Chem., 176 (1), 1948, 435–443. 112 we prepared a series of N-substituted quinoxaline-2-carboxamides, refer to Fig. 1. below. Quinoxaline-2-carboxylic acid was activated by oxalyl chloride and reacted with different anilines in the presence of pyridine at room temperature, overnight with stirring, and then obtained crudes were purified with flash chromatography. Final products were evaluated for in vitro antimicrobial activities against five mycobacterial strains, eight fungal stems, along with four gram positive and four gram negative bacteria of clinical importance. Antimicrobial activity was not observed for any of the prepared compounds. The study was supported from the project of Specific Academic Research (SVV 260 401), as well as by the Grant Agency of Charles University (Project No. C-C3/1572317) and the Czech Science Foundation (Project No. 17-27514Y). References 1. World Health Organization. Global Tuberculosis Report 2018. Available from: https://apps.who.int/iris/handle /10665/274453. SYNTHESIS AND EVALUATION OF POTENTIAL CHOLINESTERASES INHIBITORS HOUNGBEDJI, N .-H .,1 ŠTĚPÁNKOVÁ, Š.,2 KRÁTKÝ, M.1 1 Department of Organic and Bioorganic Chemistry, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic 2 Department of Biological and Biochemical Sciences, Faculty of Chemical Technology, University of Pardubice, Czech Republic e-mail: [email protected] Based on previous knowledge about salicylanilide derivatives [(thio)phosphates, carbamates] proposed as inhibitors of acetyl- (AChE) and butyrylcholinesterase (BChE),1,2 a series of novel salicylanilide-organophosphorus derivatives was designed with this goal. The synthesis consists of MW preparation of salicylanilides followed by reaction with phosphorus reagents to provide esters, 2-substituted 3-(substituted phenyl)-3-hydrobenzo[e] [1,3,2]oxazaphosphinin-4-one 2-oxides/sulfides. Their ability to inhibit both cholinesterFigure 1. The chemical structures of title compounds  Fig.16   113 ases was evaluated using Ellman’s method. AChE was inhibited with IC50 values within the range of 48–66 μM. 5-Chloro-2-{[4-(trifluoromethyl)phenyl]carbamoyl}phenyl diethyl phosphite (Fig. 1) exhibited superior inhibition of BChE (IC50 = 2.37 μM). Additionally, this compound acts as a mixed inhibitor and now, it is under advanced evaluation. The study was supported by the Czech Science Foundation (Project No. 17-27514Y). References 1. VINŠOVÁ, J., KRÁTKÝ, M., KOMLÓOVÁ, M . et al.: Molecules, 19 (6), 2014, 7152‒7168. 2. KRÁTKÝ, M., ŠTĚPÁNKOVÁ, Š., VORČÁKOVÁ, K . et al.: Bioorg. Chem., 58, 2015, 48‒52. INTERMOLECULAR INTERACTIONS STUDIED BY NMR SPECTROSCOPY ŠTOČEK, J. R.,1,2 ČECHOVÁ, L.,1 ŠÁLA, M .,1 DRAČÍNSKÝ, M.1 1 Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences, Prague, Czech Republic 2 Department of Organic and Bioorganic Chemistry, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic e-mail: [email protected]; [email protected] We studied structural adaptations of modified nucleic acid bases induced by intermolecular interactions. Two rotamers depending on the orientation of methylamino group are present in substituted pyrimidine 1 . Separated signals for each rotamer can be observed in 1H NMR spectra. The rotamer ratio can be influenced by interactions with compounds able Figure 1. The most active BChE inhibitor Figure 1. The two rotamers 1A and 1B, the binding partner 2 and formed complex 1B-2  Fig.17    Fig.18   114 to form hydrogen bonds with one rotamer of 1 only. Compounds with the acceptor-donoracceptor (ADA) structure can form three hydrogen bonds with rotamer 1B, i.e . addition of substituted thymine (2) changes the ratio of rotamers in favour of rotamer B (dimer 1B-2) . NMR experiments with variable temperature and concentration of interacting partners may be used for the determination of free energy of complex formation . Similarly, intermolecular interactions change the tautomer equilibria of modified nucleobases significantly. This work was supported by Czech Science Foundation (Project No. 18-11851S). References 1. POHL, R ., SOCHA, O ., ŠÁLA, M . et al.: Eur. J. Org. Chem., 37, 2018, 5128‒5135. PREPARATION OF DENDRALENES SUBSTITUTED WITH ELECTRON-WITHDRAWING GROUPS ŠTEMBEROVÁ M., BRŮŽA Z., POUR M. Department of Organic and Bioorganic Chemistry, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic e-mail: [email protected]  Fig.19   Scheme 1. Structure and reactivity of dendralenes Scheme 2. Potential reactivity of EWG-[3]dendralenes  Fig.20   115 Specific type of branched, cross-conjugated polyenes is called dendralenes1 (from Greek “Dendros” – tree). Their structure (Scheme 1, 1) which, in theory, is not limited by the number [n] of double bonds, is ideal for cycloaddition reactions such as Diels-Alder reaction, in which complex polycyclic compounds can be obtained in single step (Scheme 1, 2) . My goal was to prepare new dendralenes (Scheme 2, 3), substituted with electron withdrawing groups (EWG), based on the procedures previously developed by our research group .2 Furthermore, their ability to undergo Diels-Alder reaction was investigated (Scheme 2, 4) . The study was supported by the Grant Agency of Charles University (Project No. 1054216), the Czech Science Foundation (Project No. 18 17868S) and from the project of Specific Academic Research (SVV 260 401). References 1. HOPF, H., SHERBURN, M. S.: Angew. Chem. Int. Ed., 51 (10), 2012, 2298‒2338. 2. KRATOCHVÍL, J., NOVÁK, Z., GHAVRE, M. et al.: Org. Lett., 17 (3), 2015, 520‒523. SYNTHESIS OF NITROFURYL-SUBSTITUTED TETRAZOLES AND OXADIAZOLES AS POTENTIAL ANTITUBERCULAR AGENTS BARTOŠ, L ., KARABANOVICH, G ., HRABÁLEK, A ., ROH J . Department of Organic and Bioorganic Chemistry, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic e-mail: [email protected] Tuberculosis (TB) is widespread infectious disease which is mainly caused by Mycobacterium tuberculosis. Referring to the World Health Organization, it is still among the top 10 causes of death in the world. Around 10 million people worldwide suffered from TB and 1.6 million died of TB only in 2017. TB is a leading-killer in the group of HIVpositive people . This work is based on a previous successful discovery of new chemical structures with significant antitubercular activity, namely 1,5and 2,5-disubstituted tetrazoles and 2,5-disubstituted oxadiazoles bearing 3,5-dinitrobenzylsulfanyl fragment, with minimal inhibitory concentration (MIC) values as low as 0.03 µM (i.e. lower MIC compared to Figure 1. General formula of the compounds studied in this work  Fig.21   116 standard medicine used – isoniazid or rifampicin). In this study, we replaced 3,5-dinitrophenyl fragment with 5-nitrofuryl group, which has been identified as a pharmacophore in different types of anti-TB agents . Hence, we prepared a series of nitrofuryl-substituted tetrazoles and oxadiazoles and studied their antimycobacterial activity against Mycobacterium tuberculosis and non-tuberculous M. avium and M. kansasii . The study was supported from the project of Specific Academic Research (SVV 260 401). TOTAL SYNTHESIS OF 6-HYDROXYCERAMIDES AND THEIR BEHAVIOR IN THE MODEL LIPID MEMBRANES MAJCHER, A., OPÁLKA, L., KOVÁČIK, A., VÁVROVÁ, K. Skin Barrier Research Group, Department of Organic and Bioorganic Chemistry; Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic email: [email protected] Ceramides (Cer), the members of sphingolipid family, occur in all human cells and play an important role in cell signalling . In high concentrations, Cer can also be found in the uppermost layer of epidermis called stratum corneum, along with free fatty acids and cholesterol (in equimolar ratio), where they form the intercellular multi-lamellar lipid matrix. The key function of stratum corneum is to ensure a permeability barrier, thus, to provide water and electrolyte homeostasis, and to prevent entry of harmful substances into the organism .1 Cer are composed of a sphingoid base (e.g. sphingosine; C18) and an acyl part derived from long-chain fatty acid (e.g. lignoceric acid; C24) . Cer based on 6-hydroxysphingosine (signed by Motta’s nomenclature as H)2 are one of the most unusual sphingolipids . In contrast to sphingosine-based Cer, 6-hydroxysphingosine-based Cer (H-Cer) are unique only for the epidermis and, in addition, H-Cer are not typical for all mammals .1 Moreover, the function and biosynthesis of H-Cer in the skin is still not completely understood. Several dermatological studies showed that lower concentrations of H-Cer in skin accompany several skin diseases, such as atopic dermatitis.1 The major limitation of understanding the importance and uniqueness of H-Cer is their commercial unavailability . Therefore, the aim of this work was to explore the synthetic route towards H as a precursor of all known H-Cer subclasses . The total synthesis of H was based on the reaction of commercially available tridecanal with trimethylsilyl acetylene. The strategy for the synthesis of H involved an alkynylation of (S)-Garner’s aldehyde (a protected L-serinal) (1) with protected (R)-pentadec-1-yn-3-ol (2) followed by a selective twostep reduction of triple bond to a trans-double bond . In this step, a mild and selective [Cp*Ru(CH3CN)3]PF6-catalyzed Trost’s hydrosilylation followed by protodesilylation was used.3 In conclusion, physiological 6-hydroxylated sphingoid base has been prepared in seven reaction steps with overall yield 36.3%. This base was then used for the preparation of Cer 117 NH (N-lignoceroyl 6-hydroxysphingosine), Cer AH (N-2-hydroxylignoceroyl 6-hydroxysphingosine) and Cer EOH (with omega ester-linked linolenic acid). Additionally, the phase behaviour and biophysical properties of Cer NH have been studied using model lipid membranes. In these experiments, we discovered the specific chain order, different phase transitions of CH2/CD2 chains, tight orthorhombic lateral packing and lowered miscibility of Cer NH with other skin lipids. Since sphingosine exhibits an antimicrobial effect, in the future, we plan to study the antimicrobial effect of H on different cell lines . This work was supported by the Czech Science Foundation (Project No. 19-09135J) and from the project of Specific Academic Research (SVV 260 401). References 1. KOVÁČIK, A., ROH, J., VÁVROVÁ, K.: ChemBioChem., 15 (11), 2014, 1555–1562. 2. KOVÁČIK, A., OPÁLKA, L., ŠILAROVÁ, M. et al .: RSC Adv. 6 (77), 2016, 73343–73350. 3. MOTTA, S ., MONTI, M ., SESANA, S . et al.: Biochim. Biophys. Acta Mol. Basis Dis., 1182 (2), 1993, 147–151. 4. BREIDEN, B., SANDOFF, K.: Biochim. Biophys. Acta Mol. Cell Biol. Lipids, 1841 (3), 2014, 441–452. Scheme 1. Structure and retrosynthesis of physiological 6-hydroxysphingosine, i.e. (2S,3R,4E,6R)-2-aminooctadec-4-ene-1,3,6-triol Scheme 2. (Pie Chart) Relative proportion of ceramides in healthy human skin4  Fig.22    Fig.23   118 SYNTHESIS OF HETEROCYCLIC DENDRALENES ODVÁRKOVÁ, A., BRŮŽA, Z., POUR M. Department of Organic and Bioorganic Chemistry, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic e-mail: [email protected] Dendralenes are acyclic, branched, cross-conjugated polyenes with a specific arrangement of double bonds (Scheme 1, 1) .1 Unlike other polyconjugated systems, the structure of dendralenes requires at least three double bonds, where two of them are conjugated with the middle one but not with each other. The maximal number of double bonds in the molecule is unlimited (Scheme 1, 2) . These compounds have a great potential for further synthesis and their properties have not been thoroughly investigated yet . The aim of my work was to prepare substituted heterocyclic dendralenes 5 (Scheme 2), through coupling of 3 with 4, previously developed by our research group .2 Another aim was to examine their reactivity, especially in Diels-Alder cycloaddition, in which complex, polycyclic compounds 6 are formed. Synthesis and reactivity will be discussed. The study was supported by the Grant Agency of Charles University (Project No. 1054216), the Czech Science Foundation (Project No. 18 17868S) and from the project of Specific Academic Research (SVV 260 401). References 1. HOPF, H., SHERBURN, M. S.: Angew. Chem. Int. Ed., 51 (10), 2012, 2298‒2338. 2. KRATOCHVÍL, J., NOVÁK, Z., GHAVRE, M. et al.: Org. Lett., 17 (3), 2015, 520‒523.  Fig.24   Scheme 1. Structure of dendralenes Scheme 2. Synthesis of the target compounds  Fig.25   119 BETA-SECRETASE-1 INHIBITORS MONTEIRO, A., KUČERA, T. Department of Toxicology and Military Pharmacy, Faculty of Military Health Science in Hradec Králové, University of Defence, Czech Republic e-mail: [email protected] Alzheimer’s disease is a progressive neurodegenerative disease that remains incurable. One of the targets in the search for novel drugs is BACE-1 (β-secretase 1) whose blockage could help to stop the progression. Some of these inhibitors have already been tested in clinical trials. As BACE is a family of isoenzymes, the aim was to find selective BACE-1-inhibitors to minimalize adverse effects that may be caused by inhibition of BACE-2 . Molecular docking was used as a method to predict different interactions between the enzymes and inhibitors that had been already chosen by virtual screening. Appropriate complexes were chosen from RCSB Protein Data Bank and we prepared proteins and ligands for semi-flexible and flexible molecular docking using software Avogadro, Chimera and AutoDock Tools. The computational part was realized by AutoDock Vina. Suitability of the approach was verified by re-docking of ligands co-crystalized with the targets.Selective BACE-1-inhibitors were chosen based on different binding energy with the two enzymes and their subsequent comparison. This study was supported from the project of Specific Academic Research (SVV 260 401). SYNTHESIS OF LANSOPRAZOLE ANALOGUES AS POTENTIAL ANTITUBERCULAR AGENTS MURÍNOVÁ, M., KARABANOVICH, G., HRABÁLEK, A., ROH, J. 1 Department of Organic and Bioorganic Chemistry, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic e-mail: [email protected] Mycobacterium tuberculosis is the bacterium causing serious infectious disease called tuberculosis (TB). Recent studies show an increase of the number of patients suffering from this disease which is the evidence of this bacterium’s resistance to most antibiotics. The drug lansoprazole is well known as an inhibitor of gastric proton pump. However, recent study describes lansoprazole as a promising anti-TB drug candidate. The mechanism of the action is that lansoprazole kills M. tuberculosis by targeting its cytochrome bc1 after intracellular reduction of sulfoxide to lansoprazole sulfide. This active metabolite does not inhibit human H+K+-ATPase thus providing an excellent lead compound for further structural optimization and structure-activity relationship study.1 In our work, we focused on modifying the lansoprazole structure by varying its benzimidazole fragment. Thus we prepared a series of imidazole, 1,2,4-triazole, 1,3,4-oxadiazole, 120 1,3,4-thiadiazole analogues of lansoprazole sulfide and evaluated their antimycobacterial activity against standard M. tuberculosis H37Rv and against non-tuberculous M. avium and M. kansasii . The study was supported from the project of Specific Academic Research (SVV 260 401). References 1. RYBNIKER, J ., VOCAT, A ., SALA, C . et al.: Nat . Commun ., 6, 2015, Article 7659, doi: 10.1038/ncomms8659. TOTAL SYNTHESIS OF NOSTOTREBIN-6 ‒ PROBLEMS AND CHALLENGES VOŘÍŠKOVÁ, E., MATOUŠ, P., VORÁČOVÁ, M., POUR, M. Department of Organic and Bioorganic Chemistry, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic e-mail: [email protected] Nostotrebin-6 (Figure 1) is a polyphenolic secondary metabolite containing the cyclopentenedione moiety isolated from cyanobacteria Nostoc sp . The compound possesses  Fig.26    Fig.27   Figure 1. Nostotrebin-6 121 antimicrobial as well as acetylcholinesterase and butyrylcholinesterase inhibitory activity .1 To the best of our knowledge, no total synthesis has been reported to date, and nostotrebin-6 can be obtained only using a specific method of isolation from natural sources. The aim of this work is to develop and optimize the synthesis of nostotrebin-6 and its analogues, and subject them to biological activity screening . Problems and challenges encountered during synthetic attempts toward the key intermediates and derivatives will be discussed. The study was supported from the project of Specific Academic Research (SVV 260 401), the Grant Agency of Charles University (Project No. 1590119) and the Czech Science Foundation (Project No. 18-17868S). References 1. ZELÍK, P., LUKEŠOVÁ, A., ČEJKA, J. et al.: J. Enzyme Inhib. Med. Chem., 25 (3), 2010, 414–440. ALKALOIDS OF THE AMARYLLIDACEAE FAMILY AND THEIR BIOLOGICAL ACTIVITY I . PUSKÁSOVÁ, D ., ŠAFRATOVÁ, M . Department of Pharmacognosy, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic e-mail: [email protected] The aim of this work was to isolate alkaloids from herbal extract, which was obtained from Narcissus pseudonarcissus cv . Dutch Master plant . The preparation and column chromatography of the extract were performed by PharmDr. Daniela Hulcová, Ph.D., as a part of her doctoral study. Using the preparative TLC method, 2 alkaloids marked as No. 2.1 and 2.2.2 were isolated from the fraction No.4. Their structure was determined using the NMR, GC-MS analysis and optical rotation. After comparing the data obtained with literature, the compounds were identified as (+)-homolycorine and (+)-masonine. Both homolycorine and masonine were subsequently subjected to testing of inhibitory activity against acetylcholinesterase (AChE), butyrylcholinesterase (BuChE), prolyloligopeptidase (POP) and glycogen synthase kinase 3β (GSK-3β). The activity was expressed as IC50 and was compared to IC50 values of the reference substances . Galanthamine (IC50 AChE = 1.7 ± 0.1 μM, IC50 BuChE = 42.3 ± 1.3 μM) and huperzine A (IC50 AChE = 0 .033 ± 0,001 μM, IC50 BuChE > 500 μM) were used as standards to compare the inhibitory activity against AChE and BuChE. While the inhibitory activity against POP was compared to Z-Pro-prolinal (IC50 POP = 2.75×10-3 μM) and berberine (IC50 POP = 42 ± 21 μM), inhibition of GSK-3β was compared to SB-415286 compound (IC50 GSK-3β = 70 nM) . Results for (+)-homolycorine: IC50 AChE = 64 ± 4 μM, IC50 BuChE = 151 ± 20 μM, IC50 POP = 174 ± 41 μM and % inhibition of GSK-3β = 54 ± 1. Inhibitory activity of (+)-masonine: IC50 AChE = 305 ± 34 μM, IC50 BuChE = 229 ± 24 μM, IC50 POP = 314 ± 34 μM,% inhibition of GSK-3β = 66 ± 4 and IC50 GSK-3β = 27.9 ± 0.8 μM. The results found indicate that homolycorine shows moderate inhibitory activity against AChE, and mild activity against BuChE and POP . On the other hand, the inhibition of 128 along with phthalates and polypropylene glycols. These contaminants can interfere with analytes during ionization in the ion source of mass spectrometer, resulting in ion suppression of analytes . Therefore, elimination of contaminants from biological matrix is needed to accurately evaluate metabolomics data. Solid phase extraction pipette tips with porous titanium dioxide as sorbent were employed for PEG elimination. Vaginal swabs were collected from patients suffering from vulvovaginal discomfort caused by Candida albicans. Samples were first extracted in phosphate buffer saline solution. The next step of sample preparation was solid phase extraction. The composition of binding solution, washing buffer, and elution agent and also number of aspiration/expelling repeats in each of these steps were optimized. The liquid chromatography separation was performed on Acquity BEH C18 column using gradient elution with 0.075% formic acid and 0.075% acetonitrile at flow rate of 0.5 mL/min in 13 minutes. Quadrupole time-offlight high resolution tandem mass spectrometer Synapt G2-Si was employed for the data acquisition . 0.1% trifluoroacetic acid in 80% acetonitrile, 50 mM formic acid in 80% acetonitrile, 0.1% formic acid in water, 0.1% trifluoroacetic acid in water, and acetonitrile were tested as binding solutions. Water, dichloromethane, 0.1% formic acid in water, 0.1% formic acid in methanol and 0.1% trifluoroacetic acid in 5% acetonitrile were tested as wash buffers. Finally, 0.1% trifluoroacetic acid in 80% acetonitrile, 50 mM formic acid in 80% acetonitrile, 0 .1% formic acid in 90% acetonitrile, 50% methanol, and 100 mM ammonium hydroxide were tested in elution step. The best combination was evaluated and used as the most appropriate sample preparation step in metabolomics workflow. This work was supported by the STARSS project (Reg. No.CZ.02.1.01/0.0/0.0/15 _003/0000465) co-funded by ERDF, by Ministry of Health of the Czech Republic (Project No. NV15-29225A) and from the project of Specific Academic Research (SVV 260 412). STUDY OF α-BROMOPHENYLACETIC ACID SUITABILITY AS A MODEL ANALYTE FOR CHIRAL SEPARATIONS BUBÁKOVÁ, Z., MORENO-GONZÁLEZ, D., JÁČ, P. Department of Analytical Chemistry, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic e-mail: [email protected] Rizvi and Shamsi reported the employment of α-bromophenylacetic acid (I) dissolved in water/methanol mixture (1:1, v/v) as a model analyte for chiral separations in capillary electrophoresis .1 Recently, Kováčová proved the instability of I in the same solvent as well as in methanol .2 This work was focused to determine the reaction order of the decomposition of 0 .47 mM I by nucleophilic substitution in 50% aqueous methanol . The reaction kinetic study was performed by capillary electrophoresis (CE) in 50 μm (i.d.) polyvinyl alcohol capillary (30 cm/24.5 cm) with UV detection. The I and products of nucleophilic substitution (mandelic acid, α-methoxyphenylacetic acid, and bromide) were separated in 60 mM formate buffer (pH 3.0) at -30 kV; the detection λ was 200 nm. The first order reac- 129 tion kinetics was confirmed by linear and non-linear regression, yielding the rate constant 1.52 × 10−4 ± 2.76 × 10−5 s−1 and 7.89 × 10−5 ± 5.02 × 10−6 s−1, respectively . Additionally, the identity of the degradation products was confirmed by CE coupled to mass spectrometric (MS) detection with electrospray ionization and triple quadrupole analyser. The CE-MS separations carried out in 60 mM formate buffer (pH 3.0) and in 60 mM acetate buffer (pH 5.0) confirmed the results obtained by CE with UV detection.2 Our results provide strong evidence of the instability and fast degradation of I in 50% aqueous methanol indicating that I is not suitable as a model analyte for chiral separations in aqueous methanol medium . The study was supported from the project of Specific Academic Research (SVV 260 412). References 1. RIZVI, S. A. A., SHAMSI, A. S.: Anal. Chem., 78 (19), 2006, 7061–7069. 2. KOVÁČOVÁ, G.: Studium vhodnosti α-bromfenyloctové kyseliny jako modelového analytu pro chirální separace s využitím kapilární elektroforézy (Study of α-bromophenylacetic acid suitability as a model analyte for chiral separations using capillary electrophoresis as a separation technique) . Diploma thesis . Faculty of Pharmacy in Hradec Králové, Charles University, 2018 . DEVELOPMENT OF UHPSFC-PDA METHOD FOR THE DETERMINATION OF ATORVASTATIN AND ITS IMPURITIES NĚMCOVÁ, Z., PLACHKÁ, K., NOVÁKOVÁ, L. Department of Analytical Chemistry, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic e-mail: [email protected] The aim of this study was to develop ultra-high performance supercritical fluid chromatography method (UHPSFC) with PDA detection for the determination of atorvastatin and its potential impurities. Structure similarity of target analytes (stereoisomers, desfluoro and fluoro derivates) predicted their difficult separation. Simultaneously, it was necessary to achieve high sensitivity for the determination of impurities at low concentration levels (0.05% of API) as required by International Conference on Harmonisation (ICH) quality guideline Q3A . After detailed optimization, the separation was finally performed on stationary phase HSS C18 SB . CO2 modified with methanol/acetonitrile (2:1), 15 mM formic acid, 2.5 mM ammonium hydroxide, and 5% water was used as a mobile phase. Gradient was run from 10–18% of modifier in 3.5 min, followed by isocratic step at 18% of modifier until 9 min. Method was validated for API (active pharmaceutical ingredient) and impurities according to the ICH quality guidelines Q2 including linearity, sensitivity, accuracy, precision, and interday accuracy and precision. Method was linear in the range of 0.1–100 µg/mL for atorvastatin and impurity C, 2–100 µg/mL for impurities A and B, 0.2–100 µg/mL for impurity lactone and 0.1–70 µg/mL for impurity D. The accuracy and precision of the method were determined at four different concentration levels for API and at three different concentration levels for impurities. The results for accuracy and precision were 96.6%–104.9%, RSD ≤ 3.7% for impurities and 101.5%–103.2%, RSD ≤ 1.6% for API. The interday accuracy and 130 precision were 96.0%–104.5%, RSD ≤ 3.1% for impurities and 101.1%–103.5%, RSD ≤ 1 .3% for API . In conclusion, this method was found to be suitable for determination of atorvastatin and its impurities in pharmaceutical quality control . The study was supported from the project of Specific Academic Research (SVV 260 412) and the project STARSS (Reg. No. CZ.02.1.01/0.0/0.0/15_003/0000465) co-funded by ERDF. NOVEL AND FAST UHPLC-MS/MS ANALYSIS OF DEXRAZOXANE AND ITS POLAR METABOLITE MOTYČKA, F., BAVLOVIČ PISKÁČKOVÁ, H., ŠTĚRBOVÁ, P. Department of Pharmaceutical Chemistry and Pharmaceutical Analysis, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic e-mail: [email protected] Dexrazoxane (DEX), a bisdioxopiperazine derivative, is the only clinically used drug effective against anthracycline-induced cardiotoxicity . First studies indicated that DEX is a pro-drug bioactivated in cardiomyocytes by enzymatic hydrolysis of piperazine rings to its active metabolite – ADR-925. However, further research revealed that effective cardioprotection induced by bisdioxopiperazine compounds is more likely related to topoisomerase IIβ depletion induced by DEX itself.1 The only bioanalytical method for simultaneous determination of DEX and its metabolite was developed using HPLC-MS/ MS system .2 Nevertheless, the analysis requires 30 min for each run, which does not accomplish requirements for modern bioanalysis . The aim of this project is to develop and validate a fast UHPLC-MS/MS method for determination of DEX and ADR-925 in plasma. The analyses were performed using an UHPLC system coupled to triple quadrupole mass spectrometer with ESI source in positive ion mode (both Shimadzu). Following stationary phases were tested: ZORBAX Bonus-RP (100 mm × 3.0 mm, 1.8 μm), Luna Omega Polar C18 (100 mm × 2.1 mm, 1.6 μm) and Kinetex F5 column (100 mm × 2.1 mm, 1.7 μm). Mixtures of acetonitrile or methanol with different concentrations of ammonium formate or formic acid were tested as mobile phases with various isocratic and gradient elutions. The best results were achieved on the column Kinetex F5 with 1mM ammonium formate and methanol as a mobile phase in a gradient mode. Method was partially validated within the concentration range from 0 .5 to 100 µM for both compounds in plasma . The work was supported from the project of Specific Academic Research (SVV 260 401). References 1. DENG, S ., YAN, T ., JENDRNY, C . et al.: BMC Cancer, 14, 2014, Article 842, doi: 10.1186/1471-2407-14-842. 2. KOVAŘÍKOVÁ, P., PASÁKOVÁ-VRBATOVÁ, I., VÁVROVÁ, A. et al .: J . Pharm . Biomed . Anal ., 76, 2013, 243‒251. 131 HPLC EVALUATION OF l-TRYPTOPHAN AND ITS METABOLITES IN BIOLOGICAL MATERIAL MÁLKOVÁ, K ., KASTNER, P . Department of Pharmaceutical Chemistry and Pharmaceutical Analysis, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic e-mail: [email protected] The purpose of this study was to develop optimized conditions for determination of l-tryptophan and its metabolites (l-kynurenine, kynurenic acid, serotonin, 5-hydroxyindole-3-acetic acid, melatonin) using high performance liquid chromatography . Separation was achieved by the silica gel column Kinetex EVO C18 (100A, 150 × 3 mm, 5 μm) with guard column OPTI-GUARD 1 mm C18 using spectrophotometric and fluorimetric detection. Initial parameters of detection mentioned in the method were for kynurenine (absorbance at 369 nm, 227 nm and fluorescence detection Ex: 369 Em: 475). Detection and elution parameters of the method were further optimized for subsequently added analysed substances on the basis of their individual UV and fluorescence spectra. Different types of mobile phase, different pH of buffer were examined. The final mobile phase consisted of two components: – MF A: water + acetate buffer 0.1 M; pH 4.5; methanol in a ratio 97:3 – MF B: methanol . The separation was performed by gradient elution. The flow rate was 0.5 ml/min. The column temperature was set at 30 °C. The injection volume was 100 μl. Total runtime was 30 min . HPLC analysis was validated according to FDA guidelines. Vanillin was used as the internal standard . Selectivity, stability, linearity, accuracy, precision-repeatability and robustness were measured validation parameters and all found values were within acceptable ranges . The study was supported from the project of Specific Academic Research (SVV 260 401). HPLC METHOD FOR SEPARATION OF CHIRAL IMPURITIES OF DOLUTEGRAVIR LENGVARSKÁ J .,1 ŠTĚRBOVÁ, P.,1 SCRIBA, G .2 1 Department of Pharmaceutical Chemistry and Pharmaceutical Analysis, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic 2 Pharmaceutical/Medical Chemistry, Institute of Pharmacy, Faculty of Biological Sciences, Friedrich Schiller University Jena, Germany email: [email protected] Following ICH Guidelines Q3A(R2) and Q3B(R2), every new drug substance and every new drug product must be checked for pharmaceutical quality based on Good Manufacturing Practice (GMP). 132 In this study, three batches of a new drug substance and one batch of a new drug product (tablets) are tested by validated analytical method using chiral stationary phase Lux Cellulose-4. This HPLC-UV method is able to separate the main substance which is an antiviral drug – sodium salt of dolutegravir and its stereoisomeric impurities as well as some other related substances . The main aim is to establish this method as a future monograph method in The International Pharmacopoeia for impurities testing of dolutegravir sodium . Impurities in pharmaceutical products have potential carcinogenic, mutagenic, or teratogenic effects. Herein even low chiral contamination of enantiomer and also diastereomer developed through the synthesis of the substance can be detected and separated . Therefore, due to this analytical procedure it is possible to insure chiral purity, safety and quality of the drug dolutegravir . The study was supported by Erasmus+, and from the project of Specific Academic Research (SVV 260 401). IN VITRO TRANSDERMAL PERMEATION OF K1280 ČECHOVÁ, L.,1,2, VÁŇOVÁ, N.,1,2 HERMAN, D .2 ¹ Department of Pharmaceutical Chemistry and Pharmaceutical Analysis, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic ² Department of Toxicology and Military Pharmacy, Faculty of Military Health Sciences in Hradec Králové, University of Defence, Czech Republic e-mail: [email protected] K1280 (2-(hydroxymino)-N-[2-(pyrrolidin-1-yl)ethyl]ethanamide) is a new potential nerve agent antidote synthetized in the Department of Toxicology and Military Pharmacy (Faculty of Military Health Sciences). Compared to conventionally used oxime reactivators of acetylcholinesterase, K1280 is an uncharged non-quaternary compound . The absence of the permanently charged nitrogens is believed to improve oxime’s ability to penetrate through biological barriers .1 Permeation of K1280 through skin barrier in vitro was determinated by the Franz diffusion cells using split thickness porcine skin as a model barrier. Full time of the experiment was 24 hours and receptor fluid was collected hourly. Samples of the receptor fluid were measured by optimized LC-MS/MS method. Atropine was utilized as the internal standard . Permeation measures (steady state flux Jss, lag time Tlag and permeation constant Kp) were calculated by SAMPA software. The permeation of K1280 was then compared with standard compound caffeine . Based on the results obtained in this study, the K1280 oxime showed the potential to penetrate through skin in vitro indicating the possible use as a transdermal prophylactic against organophosphate intoxication . The study was supported from the project of Specific Academic Research (SVV 260 401). 133 References 1. SOUKUP, O., KORÁBEČNÝ, J., MALIŇÁK, D. et al.: Med. Chem., 14 (3), 2018, 281–292. UHPLC-MS/MS ANALYSIS OF JAS-2, THE NOVEL ANALOGUE OF DEXRAZOXANE REGULI, A., BAVLOVIČ-PISKÁČKOVÁ, H., ŠTĚRBOVÁ-KOVAŘÍKOVÁ, P. Department of Pharmaceutical Chemistry and Pharmaceutical Analysis, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic e-mail: [email protected] Dexrazoxane (DEX) is a cardioprotective drug clinically used against anthracycline induced cardiotoxicity. 4,4′-(Butane-2,3-diyl)bis(piperazine-2,6-dione) (JAS-2) was synthetized as a novel, more effective analogue of DEX. It was reported that inhibition of topoisomerase II, which seems to be crucial for cardioprotective effect, is mediated by a meso form while the racemic JAS-2 is almost ineffective.1 Nevertheless our in vitro experiments with racemic JAS-2 showed some cardioprotective effect. The aim of this work is 1) to examine the possible role of contamination of racemic JAS-2 with the meso form in the results of in vitro experiments and 2) to develop solid phase extraction (SPE) for JAS-2 and its metabolite (JAS-2met) from plasma. The UHPLC coupled with a triple quadrupole mass spectrometer with ESI+ ion source, Bonus-RP column (100 × 3.0 mm, 1.8 μm) and formic acid (0.25%) with methanol as a mobile phase were used for separation of different forms of JAS-2 . Analysis of JAS-2 and JAS-2met was achieved on Luna Omega Polar column (100 × 3.0 mm, 2.5 μm) with a guard column. A mobile phase containing ammonium formate and acetonitrile were used. Four types of SPE columns (Discovery DSC-PH 100 mg/1 ml, Discovery DSC 18 100 mg/1 ml, Supel Select HLB 30 mg/1 ml, and Hypersep Verify AX 130 mg/1 ml), different types of washing solvents (H2O, 5% MeOH, HCOOH) and elution solvents (ACN, MeOH, ACN + 10% HCOOH) were tested. In racemic JAS-2 we detected less than 0.1% of JAS-2 meso form. Hence, it seems unlikely that this low concentration can be responsible for the inhibition effect. The highest recovery of JAS-2 and JAS-2met from plasma was achieved on Hypersep Verify AX column. Nevertheles, the presence of formic acid, which was necessary for elution of JAS-2met, increased matrix effects and led to the high signal supression of JAS-2met. Therefore, future extractions will be focused only on JAS-2, which is the active substance . The study was supported from the project of Specific Academic Research (SVV 260 401) and by the Grant Agency of Charles University (Project No. 1550217). References 1. SNAPKA, R . M ., WOO, S . H ., BLOKHIN, A . V . et al.: Biochem. Pharmacol., 52 (4), 1996, 543–549. 134 SECTION OF TECHNOLOGICAL SCIENCES HOMOGENIZATION OF POWDER BLENDS USING A TURBULA MIXER LITOŠOVÁ, M .,1 PALÁT, K .,2 TRPĚLKOVÁ, Ž.,1 ŠKLUBALOVÁ, Z .1 1 Department of Pharmaceutical Technology, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic 2 Department of Organic and Bioorganic Chemistry, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic e-mail: [email protected] Homogeneity is an important quality factor in the pharmaceutical industry because it directly influences the content uniformity and plays an essential role in the efficacy and safety of solid dosage forms .1 In this experimental work, influencing the homogeneity of the powder mixture of acetylsalicylic acid (ASA) and microcrystalline cellulose (Avicel PH102) due to the rotational speed of the mixing vessel in a range of 23–101 rpm and the mixing time was studied using the Turbula shaker mixer. Within time interval 2–62 minutes, the content of ASA was measured by near-infrared spectrometry. The expression of standard deviation was used to evaluate the homogeneity of samples. The best results were detected at a rotational speed of 34 rpm . The study was supported by the Grant Agency of Charles University (Project No. 1286218/2018). References 1. MA, L ., ZHOU, L ., XU, M . et al.: Spectrochim. Acta, Part A, 204, 2018, 783−790. PREPARATION OF BIODEGRADABLE NANOPARTICLES FOR HYDROPHILIC MACROMOLECULAR DRUGS DELIVERY SZANYIOVÁ, A ., HOLAS, O . Department of Pharmaceutical Technology, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic e-mail: [email protected] This study investigates the formulation of nanoparticles containing hydrophilic components (e.g. proteins). Selected material was PLGA based compounds synthesized at Department of Pharmaceutical Technology. Encapsulated compounds were Rhodamine B, FITC labelled dextran and FITC labelled albumin . Selected methods of nanoparticles formulation were double-emulsion technique and nanoprecipitation. Prepared nanoparticles were purified by three cycles of centrifugation and encapsulation efficacy and recovery yield were measured. Effect of different polymers and stabilizers was followed. More specifically, the principal objective was to explore the differences between parameters of the 135 created nanoparticles, such as size, zeta potential and efficacy of encapsulation. Changes in these characteristics were brought about by the chosen polymers, stabilizers, encapsulated compound, length of centrifugation period . Prepared nanoparticles had size ranging between 150–474 nm and zeta potential approximately 30 mV. Even though the main goal of the study was to efficiently encapsulate protein, the amounts of encapsulated albumin were lower compared to rhodamine B or dextran. Main obstacles were presented by separation of nanoparticles from medium. The centrifugation time had a significant impact on the amount of collected nanoparticles. Nanoparticles tended to aggregate during the centrifugation . In case of smaller particles, centrifugation proved to be ineffective way of purification, therefore it was problematic to gain them. From the observation of the two methods working with the same substance (FITC labelled dextran) it is clear, that nanoprecipitation is more suitable for encapsulation of branched polymers, while double-emulsion is more appropriate for the linear PLGA polymer-based nanoparticles . This work supported from the project of Specific Academic Research (SVV 260 401). A STUDY OF TABLETING MATERIALS AND TABLETS WITH THE COMBINATION OF MICROCRYSTALLINE CELLULOSE AND MANNITOL FOR ORALLY DISINTEGRATING TABLETS NOVOTNÁ, A., MUŽÍKOVÁ, J. Department of Pharmaceutical Technology, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic e-mail: [email protected] This work compares the co-processed dry binder Avicel® HFE-102 containing 90% microcrystalline cellulose and 10% mannitol with a physical mixture of related dry binders, microcrystalline cellulose (Avicel® PH-102) and mannitol (Pearlitol® 100SD) in the ratio of 9:1 . Flow properties, compressibility, lubricant sensitivity, tensile strength and disintegration time of tablets were evaluated. Compressibility was evaluated by means of the energy profile of compression process, and lubricant sensitivity by means of the lubricant sensitivity ratio. The results were also compared with the microcrystalline cellulose for direct compression Avicel® PH-102 alone . The flow properties of the co-processed dry binder Avicel® HFE-102 alone and the physical mixture are comparable . Avicel® HFE-102 showes higher values of the energy of plastic deformation, tensile strength of tablets, and a markedly lower lubricant sensitivity than the physical mixture of dry binders. Tablets with the co-processed dry binder Avicel® HFE-102 show short disintegration. Avicel® HFE-102 is suitable for the use in orally dispersible tablets . The study was supported from the project of Specific Academic Research (SVV 260 401). 136 STUDY OF MICROSTRUCTURE OF SKIN BARRIER MODEL USING DEUTERATED CERAMIDES JUHAŠČIK, M., KOVÁČIK, A., OPÁLKA, L., VÁVROVÁ, K. Skin Barrier Research Group, Department of Pharmaceutical Technology, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic e-mail: [email protected] Ceramides (Cer) are sphingolipids, which participate in various biological processes. In mammalian skin, Cer are localized in the uppermost layer of epidermis, the stratum corneum (SC). In this layer, Cer along with cholesterol (Chol) and free fatty acids form multilayer lamellae of intercellular lipid matrix . The skin lipid arrangement in SC is still unclear. To evaluate the skin lipid arrangement, skin membrane models with labelled (deuterated) lipids have been used. Therefore, the aim of this work was to synthesize sphingosine with deuterated chain and Cer based on deuterated sphingosine, i.e., N-lignoceroyl sphingosine-d28 (with lignoceric acid acyl (C24) (d-CerNS) and N-lignoceroyl-d47 sphingosine-d28 (dd-CerNS) and to study their phase behaviour and arrangement in model membranes . Synthesis of deuterated Cer started from elimination of 1-pentadecanol-d31 to obtain a deuterated terminal alkene. Next, vinylation of (S)-Garner’s aldehyde led to an intermediate, which was treated in Grubbs’ metathesis with terminal alkene. The product of Grubbs’ metathesis (protected deuterated sphingosine) was then deprotected under acid conditions; free sphingoid base was acylated by protonated or deuterated lignoceric acid using water soluble carbodiimide . Afterwards, synthesized d-CerNS and dd-CerNS were incorporated into SC model membranes . Model mixtures contained d-CerNS or dd-CerNS, (deuterated) lignoceric acid and Chol in 1:1:1 molar ratio with an addition of cholesteryl sulfate (5wt%). Overall, four types of model membranes with different representation of deuterated methylene (CD2) chains, were studied by temperature depended infrared spectroscopy at temperature from 28 °C to 100 °C. A phase behaviour (conformation, lateral arrangement, and miscibility) of model lipid membranes was investigated. The results of this study could be helpful in explaining the (patho)physiological arrangement of SC lipids. The study was supported by Czech Science Foundation (Project No. 19-09135J) and from the project of Specific Academic Research (SVV 260 401). We thank Mrs. Vencovská for her technical assistance. 137 THE STUDY OF INFLUENCE OF THE MEASUREMENT METHOD ON STATIC ANGLE OF REPOSE OF FREE-FLOWABLE EXCIPIENTS MRÁZKOVÁ, A .,1 TRPĚLKOVÁ, Ž.,2 ŠKLUBALOVÁ, Z .1 Department of Pharmaceutical Technology, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic e-mail: [email protected] Powder flowability plays an essential role in their manufacturing into solid dosage forms .1 Measuring of the static angle of repose (AOR) represents one out of the simple methods of flowability testing. This work is focused on AOR measurement for selected free flowable excipients for direct compression: sorbitol (Merisorb 200), lactose (Excipress GR 150), microcrystalline cellusose (Avicel 200) and dicalcium phosphate anhydrous (DI-CAFOS A150) or for dry powder inhalers: lactose (InhaLac 120). The results obtained using two different equipments for measuring the AOR: automatic tester Erweka and prototype tester for the orifice size 6 mm of a conical hopper were evaluated by analysis of variance (ANOVA, α = 0.05, P < 0.01). Significant difference between methods was detected. According to generally accepted scale,2 tested materials were classified as passable flowing using tester Erweka. On the contrary, good flow behaviour was detected using the protype. The study was supported by the Grant Agency of Charles University (Project No. 1286218/2018) and from the project of Specific Academic Research (SVV 260 401). References 1. LI, Q ., RUDOLPH, V ., WEIGL, B . et al.: Int. J. Pharm., 280 (1–2), 2004, 77–93. 2. Český lékopis 2017 (Czech Pharmacopoeia 2017). Praha, Grada Publishing, 2017. OPTIMIZATION OF DRUG-LOADED NANOPARTICLES PREPARATION KOLÁŘOVÁ, A., HOLAS, O. Department of Pharmaceutical Technology, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic e-mail: [email protected] Two methods of nanoparticles preparation from materials based on of poly(lactic-coglycolic acid) were used. First method was emulsion-sonication, where the particles were formed using high frequency ultra-sound probe. The second method was spontaneous emulsification, where two solvents with different hydrophilicity were used. Nanoparticles were prepared from linear PLGA and PLGA branched by polyacrylic acid. Nanoparticles prepared by emulsion-sonication method were stabilized by three different surfactants – poloxamer Pluronic F127, polysorbate Tween 20 and poly(vinyl) alcohol. Each surfactant was used in three different concentrations. Spontaneous emulsification nanoparticles prepared by spontaneous emulsification were stabilized by poly(vinyl) alcohol. Combination of solvents ethanol:aceton in various ratios was used throughout 144 FUNGEMIA IN THE UNIVERSITY HOSPITAL HRADEC KRÁLOVÉ IN THE PERIOD 2007–2018 HEJKRLÍKOVÁ, E.,1 BUCHTA, V .,2 JÍLEK, P.1 1 Department of Biological and Medical Sciences, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic 2 Department of Clinical Microbiology, University Hospital and Faculty of Medicine in Hradec Králové, Charles University, Czech Republic e-mail: [email protected] Fungemia, presence of fungi in the blood, is a life-threatening condition occurring especially in critically ill and immunocompromised patients . Candida albicans remains the most common cause, although contribution of non-albicans species has increased over past decade .1 Present study aims to describe current epidemiological situation in the University Hospital in Hradec Králové . Data on bloodstream isolates of the yeasts and patient characteristics were obtained from the laboratory information system and further analyzed. From November 2007 to December 2018, fungemia was identified in 235 patients with overall incidence of 0.51 cases per 1000 admissions. Most of fungemic patients (64.3%) were hospitalized in intensive care units. Candida albicans accounted for half of the blood cultures (52.8%) followed by C. glabrata (14.5%), C. tropicalis (8.5%) and C. parapsilosis (6.8%). In general, Candida strains showed high antifungal susceptibility to echinocandins according to CLSI standard breakpoints. On the other hand, increased minimum inhibitory concentrations of azole drugs were observed in some of C. glabrata and C. parapsilosis isolates . Incidence of fungemia, fungal spectrum and susceptibility to antimycotics in the fungi isolated in the University Hospital in Hradec Králové during the follow-up period corresponded to those of other hospitals in the Czech Republic.2 The study was supported from the project of Specific Academic Research (SVV 260 414). References 1. KHATIB, R ., JOHNSON, L . B ., FAKIH, M . G . et al.: Mycoses, 59 (12), 2016, 781‒786. 2. KOCMANOVÁ, I ., LYSKOVÁ, P ., CHRENKOVÁ V . et al.: Epidemiol. Mikrobiol. Imunol., 67 (1), 2018, 3‒10. COMPLEMENTARY AND ALTERNATIVE MEDICINE IN ENQUIRIES OF DRUG INFORMATION CENTER HARAZIMOVÁ, T., ROZSÍVALOVÁ, P., MALÁ, K. Department of Social and Clinical Pharmacy, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic e-mail: [email protected] Drug Information Center (DIC) of the Faculty of Pharmacy in Hradec Králové, and University Hospital in Hradec Králové was established in 1994. Major activity of the 145 center is providing expert information about drugs – primarily in the form of answers on various medicines-related enquiries sent to the center by healthcare professionals . Aims of this study were to analyse enquiries related to complementary and alternative medicine (CAM) in the period 1994–2017 and to evaluate DIC’s activity through feedback from questioners . CAM enquiries were searched in the complete DIC database of enquiries. The analysis was focused on questioner’s profession, region, urgency of enquiry, type of enquiry, professional CAM information resources used or time needed for resolving CAM enquiry . Moreover, the feedback of questioners, who sent the enquiry to DIC from 2015 to 2017, was obtained using an online questionnaire containing 18 items – sociodemographic data and awareness of DIC, quality of answers to enquiries and satisfaction with provided services, and lastly CAM related issues . The total number of accepted CAM enquiries in the study period was 205 (of all 2204 enquiries), with the highest number in 2003 (26; 12.7%). Typical issues concerning CAM enquiries were related to interactions and indications or contraindications of CAM (58; 28.3%). Average time for resolving a CAM enquiry took 141 minutes. The feedback questionnaire was delivered to 94 health professionals with response rate of 40 (42.6%). Majority of respondents (35; 87.5%) were completely satisfied with services, especially because of detailed, comprehensive, clear and understandable answers. Answers were fully or partially used for a specific patient or for the needs of healthcare professionals. CAM related issues were mostly solved by pharmacists (24; 92.5%), especially community pharmacists. Database analysis showed the same pattern – 126 (53.7%) enquiries were sent to DIC by community pharmacists . DIC’s services received good feedback and all of respondents would use its services again including CAM related enquiries sent especially by community pharmacists . The study was supported from the project of Specific Academic Research (SVV 260 417). ANALYSIS OF NON-CARDIAC ADVERSE EVENTS IN RHEUMATIC PATIENTS WITH GLUCOCORTICOID PULSE THERAPY POLLÁKOVÁ, L .,1 JURÍKOVÁ, N.,1 VLČEK, J.,1 SOUKUP, T .,2 PUDIL, R .4 1 Department of Social and Clinical Pharmacy, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic 2 2nd Department of Internal Medicine – Gastroenterology, University Hospital in Hradec Králové, Czech Republic 3 1st Department of Internal Medicine – Cardioangiology, University Hospital in Hradec Králové, Czech Republic e-mail: [email protected] Intravenous glucocorticoid (GC) pulse therapy is effective in life threatening flares of rheumatic diseases and is considered to have low risk of adverse events (AE). However, it is not free of complications and frequency of AE in rheumatic patients is not completely clear, partly owing to additive non-genomic to genomic mechanism, which pulsed GC possess.1 146 The aim of this work is to analyze occurrence of non-cardiac AE in real-life setting, analyze its risk factors and find selected, more susceptible populations with underlying risk factors. Patients were administered 1000 mg methylprednisolone in 3 to 5 doses on alternating days. Analysis includes 278 rheumatic patients with 326 pulse administrations. Majority of patients were women (67%) and median age was 55 years. Patients mostly suffered from connective tissue diseases (n = 191, 59%) and systemic vasculitis (n = 120, 37%). Data were collected retrospectively from their medical records . Fifty-seven non-cardiac AE appeared during 42 (13%) pulse courses and 13 (4%) cases had to be terminated due to serious nature of occurred AE . Most common adverse event was hypertension in 4.6% (n = 15), metabolic disturbances in 4.3% (n = 14) and infections in 2.1% (n = 7) subjects. Common non-cardiac AE were also amylase increase and diarrhea, each appearing in 1.2% (n = 4) subjects. Occurrence of other AE was less common (< 1%). Limitations of this study are based on its retrospective character, as we got to observe only what is being monitored in GC pulse regimen by default. Occurrence of serious AE was common (4%), but when assessing right risk management having all risk factors in mind, our study presents GC pulse therapy as relatively safe regarding non-cardiac AE . The study was supported from the project of Specific Academic Research (SVV 260 417). References 1. SINHA, A., BAGGA, A.: Indian J.Pediatr., 75 (10), 2008, 1057–1066. ANALYSIS OF INDIVIDUAL COUNSELLING WITH PATIENTS IN COMMUNITY PHARMACY BRANDEJSKÁ, T .,1 ŠOLÍNOVÁ, J.,2,3 MALÝ, J.1,3 1 Department of Social and Clinical Pharmacy, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic 2 Pharmacy U Zlatého hada, Cvikov, Czech Republic 3 Section of Clinical Pharmacy, Czech Pharmaceutical Society of the Czech Medical Association of Jan Evangelista Purkyně, Czech Republic e-mail: [email protected] Pharmaceutical care in a community pharmacy can be provided as part of individual consultations. These are focused on drug-related problems (DRPs), monitoring risk factors and evaluating individual risk levels for various diseases and self-medication. The aim of this work was to analyze individual consultations realized in community pharmacy in period of 2009–2019 . Professional individual consultations were provided in the form of a discrete interview between patient and pharmacist in a consulting room of a basic type pharmacy with two dispensing places. Pharmacy is located in a municipality of up to 5,000 inhabitants, where no other pharmacy is available. A written record was derived from 147 each consultation. During the consultation, information from the patient’s history was obtained: demographic data, personal history data, lifestyle, use of medicines and food supplements including dosage information. The identified DRPs were evaluated using the Pharmaceutical Care Network Europe V5.1 classification. The results were described by descriptive statistics . A total of 346 consultations were performed in 148 patients over a defined period. Most patients (81; 55%) attended more than one consultation. In 113 (76%) cases they were women. The mean age of the patients was 68 ± 14.5 years. On average, patient used 7 drugs. Majority of DRPs involved problems with choice of drug (148; 32%) or administration of drugs (143; 31%). The analysis indicated that providing individual consultations in the pharmacy can, among other things, strengthen the culture of safety in use of pharmacotherapy . Conclusions need to be verified on a larger sample of pharmacies. The study was supported from the project of Specific Academic Research (SVV 260 417). ATTITUDES AND BELIEFS ON HUMAN PAPILLOMAVIRUS INFECTION AND VACCINATION ŘÍHOVÁ, A., ZIMČÍKOVÁ, E. Department of Social and Clinical Pharmacy, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic e-mail: [email protected] Human papillomavirus (HPV) infection is the most common sexually transmitted disease. Nearly 80% of world’s population will get HPV infection at some point of their lives. This infection is associated with more than 99% cases of cervical cancer, which is the second most common malignancy of women. The most reliable protection against HPV is vaccination .1,2 The aim of the work was to find out knowledge of high school students about HPV infection and related diseases. In addition, to find out the vaccine coverage rate against HPV and attitudes of students to this vaccination. Data were obtained by survey, which took place at two high schools in the Ústí Region in 2018. Overall, 291 questionnaires were used. Average age of the respondents was 17 years (ranging 15–19 years) and 73.5% of respondents were women. Only one of the students did not know the concept of cervical cancer or penile cancer. More than half of the students (56.7%) have already heard about the human papillomavirus vaccine. The vaccine coverage rate at these schools was 62.5% (60.5% women, 2% men). The most frequently reported reasons for not being vaccinated were related to the doubt about the vaccine efficacy, the fear of undesirable effects and distrust of the vaccination . Compared to men, women had better knowledge about HPV infection, cervical cancer and vaccination. Equally, the knowledge was better with higher age of the respondents. The vaccine coverage rate could be enhanced by increasing awareness at target age groups. 148 Also, better knowledge about vaccination could positively change students’ attitude to the HPV vaccination . The study was supported from the project of Specific Academic Research (SVV 260 417). References 1. CHOVANEC, J., NÁLEŽINSKÁ M.: Onkologie, 8 (6), 2014, 269–274. 2. HAMŠÍKOVÁ, E.: Remedia, 5, 2007, 470–475 149 SOCIAL HAPPENINGS JUBILEE OF Prof. RNDr. EVA KVASNIČKOVÁ, CSc. In May 2019, Professor Emeritus of Charles University, former Vice-Rector of Charles University, former Dean of the Faculty of Pharmacy of Charles University in Hradec Králové Prof. RNDr. Eva Kvasničková, CSc., celebrated 85 years. 150 The native of Pardubice, she graduated from the Faculty of Pharmacy of Masaryk University in Brno in 1958. Then she worked for several years in the biochemical laboratories of the 1st Internal Clinic of the University Hospital in Hradec Králové. This work led her to specialize in biochemistry. She then spent most of her working life with the faculties of Charles University in Hradec Králové. From 1967 to 1971 she was an assistant professor at the Department of Medical Chemistry and Biochemistry of the Faculty of Medicine and in 1971 she moved to the then established Faculty of Pharmacy of Charles University . At the beginning of normalization, this faculty provided a relatively looser political atmosphere for the less politically reliable young teachers like Dr. Kvasničková. Moreover, within the Faculty of Pharmacy, the then small core of the Department of Biochemistry formed a rare friendly intellectual and research microclimate, which shaped its members for years, ideologically, intellectually and culturally, and to which Dr. Kvasničková made a significant contribution . The Faculty of Pharmacy then remained her workplace for another more than forty years. Throughout these years, Professor Kvasničková’s pedagogical work was a basic pillar of teaching biochemical disciplines at the Faculty of Pharmacy . For four decades, she was the main lecturer in General Biochemistry and moreover she established a separate special subject – Xenobiochemistry. At the same time she grew up, especially in the first years under the influence of Prof. Ivo Hais, to be a prominent scientific personality. In line with the goals of pharmaceutical research, she focused on studying the biotransformation of potential drugs and also published most of her scientific work in the field of xenobiochemistry . She received her habilitation in 1988 at the Faculty of Pharmacy in Bratislava, and was appointed professor of biochemistry in 1997 at Charles University. The 1990s were a great impetus for her and finally enabled her to fully develop her organizational and managerial skills. In 1997–1999 she was the Dean of the Faculty of Pharmacy and then from 2000 she was the vice-rector of Charles University for two terms. She applied her important position in Czech drug research mainly as the leading researcher of the Research Centre of Structure and Mechanism of Action of Potential Drugs LN00B125, which has been successfully working under her leadership. She admirably managed the combination of the vice-rector’s position in Prague with pedagogical and research work in Hradec Králové, including the management of graduates and doctoral students. She retired in 2013, but did not lose contact with either the Department of Biochemical Sciences or with the Faculty of Pharmacy. Professor Kvasničková, together with her husband who is a professor of internal medicine, raised two children. Their daughter is a successful medical doctor and their son is a successful pharmaceutical manager . In addition to her pedagogical and scientific achievements, Eva Kvasničková is also a personality with broad cultural interests. Since her youth, she has also been involved in sport activities. She loved skiing, playing tennis, and so far, in proportion to her current physical abilities, she enjoys golf . On behalf of all my colleagues, I wish Professor Kvasničková many more years of health, optimism and much joy of life . Jaroslav Dršata 151 PharmDr. MAGDA VYTŘÍSALOVÁ, Ph.D., PASSED AWAY PharmDr. Magda Vytřísalová, Ph.D., an assistant professor of the Department of Social and Clinical Pharmacy passed away on 31 December 2019 at the age of 40 years. She came from the Moravian town Uničov. Having completed the high school she studied pharmacy at the Faculty of Pharmacy in Hradec Králové and graduated in 2003 . Then she continued with the doctoral study of clinical pharmacy under the supervision of 152 Prof. RNDr. Jiří Vlček, CSc. and successfully defended her doctoral thesis in 2010. Her main research interests included compliance and persistence to treatment of various types of diseases, public health, osteoporosis in the primary care system and drug consumption . With her team, she has published about thirty professional original and summary works in domestic and foreign periodicals indexed not only in the Web of Science and SCOPUS databases . Her publishing activity also included the very successful monograph Clinical Pharmacy II, which together with prof. Jiří Vlček edited. With the start of her doctoral studies, Magda began to engage in pedagogical activities and later as an assistant professor of the department she significantly participated in teaching Social Pharmacy, Professional Information on Medicines and guaranteed the subjects General Principles in Health Care, Pharmacoeconomics and Evaluation of Health Interventions, and Regulatory Affairs in Pharmacy. Since 2003, she has also worked as a pharmacist and especially as a professional editor of the journal Remedia (2006–2012). Magda was a fighter in both her personal and professional life and was able to pursue the goal she had set out with immense tenacity and invention. She showed an innovative approach to teaching and was very close to habilitation. Classical music, playing the piano and dancing were her main personal interests. Her early death hit us hard . Let us dedicate a silent memory to her in our minds . Colleagues from the Department of Social and Clinical Pharmacy 153 INSTRUCTIONS FOR AUTHORS FOR FOLIA PHARMACEUTICA UNIVERSITATIS CAROLINAE Manuscripts should be prepared on the A4 paper, in English, typed in Microsoft Word using Times New Roman font and spacing 1.5. It should be divided into the following sections: Title – Times New Roman 14, left alignment, (Spectrofotometric determination of …). Write the title in lowercase letters and then format it using Font – All Caps (Písmo – Všechna velká). Names of Authors – Times New Roman 12, center alignment, (GASPARIČ, J.,1 MILENA ČERMÁKOVÁ, M.2). Write the names in lowercase letters and then format them using Font – All Caps (Písmo – Všechna velká). Names of Institutions – Times New Roman 10, center alignment), (1 Department of……., Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic). Email address – Times New Roman 10, center alignment, (e-mail: [email protected].) Text – Times New Roman 12, left aligment without indents, starting a new paragraph only by Enter . Bold and Italics may be used . Manuscripts of Original Papers should be divided into sections . Headings of the sections: Abstract – 12, left alignment Keywords – maximum 5 keywords, 12, left alignment (KEYWORDS: extraction – spectrophotometry) Introduction – 12, left alignment Experimental – 12, left alignment . This part may be further subdivided, e.g. Chemistry Materials and Methods General procedure for the preparation of the studied compounds (E)-1-(5-tert-butylpyrazin-2-yl)-3-(3-hydroxyphenyl)prop-2-en-1-one Bioassays Evaluation of antimycobacterial activity Evaluation of photosynthesis-inhibiting activity If it is not absolutely necessary, do not use more than three levels of headlines . Figures must be submitted in black and white and in original size (not more than 12.5 × 18 cm), separately as a supplement. Indicate the placement of the figure in the text. Captions and notes are placed below (10, center alignment) <KoukalFig2.jpg> Fig . 1 . Structures of the studied compounds