Folia Pharmaceutica Universitatis Carolinae: LII

COVER UNIVERSITAS CAROLINA PRAGENSIS FOLIA PHARMACEUTICA UNIVERSITATIS CAROLINAE ISSN 1210-9495 FOLIA PHARMACEUTICA UNIVERSITATIS CAROLINAE LII LII Folia_LII_obalka.indd 1Folia_LII_obalka.indd 1 05.03.2026 15:1805.03.2026 15:18 FOLIA PHARMACEUTICA UNIVERSITATIS CAROLINAE LII Arranged by Assoc. Prof. RNDr. Veronika Opletalová, Ph.D. Lectured: Assoc. Prof. RNDr. Věra Klimešová, CSc., Assoc. Prof. PharmDr. Jakub Chlebek, Ph D. 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. Pavla Žáčková, CSc., Assoc. Prof. RNDr. Veronika Opletalová, Ph.D. (editor) Published by Charles University Karolinum Press Prague 2026 Typeset by Karolinum Press © Charles University, 2026 ISSN 1210-9495 ISBN 978-80-246-6152-0 ISBN 978-80-246-6164-3 (pdf) https://doi.org/10.14712/9788024661643 Charles University Karolinum Press www.karolinum.cz [email protected] CONTENTS Abstracts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 11th Postgraduate and Postdoc Conference of the Faculty of Pharmacy in Hradec Králové, Charles University, Hradec Králové, 27–28 January 2021 . . . . . . . . . . . . . 7 Bioorganic and Pharmaceutical Chemistry Section . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 Pharmacognosy and Toxicology of Natural Products Section . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 Pharmaceutical Technology Section . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41 Pharmaceutical Analysis and Bioanalytical Chemistry Section . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51 Clinical and Social Pharmacy Section . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95 28th National Students’ Scientific Conference of the Faculty of Pharmacy in Hradec Králové, Charles University, Hradec Králové, 13 April 2021 [online] . . . . . . . . . . 107 Section of Biological Sciences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107 Section of Chemical Sciences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122 Section of Pharmaceutical Technology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137 Section of Social and Clinical Pharmacy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145 Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151 Publications of the Faculty of Pharmacy in Hradec Králové, Charles University in the Year 2021 . . . . . . . . 151 Original Papers and Reviews . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151 Multicenter Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 162 Articles in Journals without Impact Factor and Proceedings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163 Monographies and Textbooks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 164 Patents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 165 Degrees . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 165 Social Happenings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175 In Memory of Assoc . Prof . RNDr . Petr Klemera, CSc . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175 Instructions for Authors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 176 CONTENTS 7 2026 Folia Pharm . Univ . Carol . LII Pag . 7–106 ABSTRACTS 11th POSTGRADUATE AND POSTDOC CONFERENCE OF THE FACULTY OF PHARMACY IN HRADEC KRÁLOVÉ, CHARLES UNIVERSITY, HRADEC KRÁLOVÉ, 27–28 JANUARY 2021 BIOORGANIC AND PHARMACEUTICAL CHEMISTRY SECTION SUPRAMOLECULAR INTERACTION OF TETRAPYRAZINO-PORPHYRAZINES DEMUTH, J., NOVÁKOVÁ, V., ZIMČÍK, P. Department of Pharmaceutical Chemistry and Pharmaceutical Analysis, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic e-mail: demuthj1@faf .cuni .cz Tetrapyrazinoporphyrazines (TPyzPzs), members of the phthalocyanine family, are synthetic planar macromolecules . TPyzPzs have remarkable spectral properties due to their system of aromatic double bonds . These macromolecules can absorb light between 300–700 nm, and afterward, they could release the absorbed energy by emitting fluorescence . Alkylamino substituted TPyzPzs are an interesting subtype of TPyzPzs, changing their spectral properties due to supramolecular interaction . The supramolecular interaction lays in stacking two molecules to each other – J-dimer formation (Fig . 1) . The monoFig. 1 Fig. 1. J-type of aggregate 8 meric alkylamino TPyzPzs have only weak fluorescence because the fast relaxing process quenches it .1 On the other hand, dimeric alkylamino TPyzPzs have good fluorescence with a significant bathmotropic shift. Aggregates could be dissolved by the addition of coordination solvent (e.g ., pyridine, N-methylimidazole), which caused changes in absorption spectra (Fig . 2) .2 This project studied the background for possible further application of alkylamino TPyzPzs . The study was supported by the Czech Science Foundation (Project No. 17-19094S), by the Grant Agency of Charles University (Project No. 1168217) and from the project of Specific Academic Research (SVV 260 547). References 1. NOVÁKOVÁ, V., ZIMČÍK, P., MILETÍN, M. et al .: Phys . Chem . Chem . Phys ., 12, 2010, 2555–2563 . 2. DEMUTH, J., MILETÍN, M., MACHAN, M. et al .: ChemPlusChem, 85, 2020, 527–537 . ANIONIC VERSUS CATIONIC PHTHALOCYANINES FOR PHOTODYNAMIC THERAPY: WHAT A DIFFERENCE THE CHARGE MAKES KOLLÁR, J .,1 MACHÁČEK, M.,2 HALAŠKOVÁ, M .,2 DEMUTH, J .,1 ROHLÍČKOVÁ, M.,2 NOVÁKOVÁ, V .,1 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: kollarj@faf .cuni .cz Phthalocyanines (Pcs) and their aza-analogues represent a promising group of organic dyes with interesting photophysical properties (strong absorption in area over 600 nm and strong singlet oxygen production) highly suitable for the use in photodynamic therapy of cancer . The literature reports on cationic and anionic Pcs for photodynamic therapy suggest systematically significant differences in their photodynamic activity. Fig. 2 Fig. 2. Changes in the absorption spectra 9 This project was focused on the series of anionic and cationic zinc(II) Pcs and investigated their photophysical, physicochemical, binding and biological properties with the aim of finding the parameters and/or factors that may contribute to the substantial difference in photodynamic activity between Pcs bearing opposite charges on peripheral substituents . Four different sets of compounds were introduced into the study, namely anionic hydrophilic, cationic hydrophilic, anionic amphiphilic and cationic amphiphilic to compare both the influence of the charge type and its distribution on the macrocycle core. All Pcs were tested on photodynamic activity in vitro on HeLa cells with different activity for anionic Pcs (EC50 ~ 0.3–10 μM) and cationic Pcs (EC50 ~ 3–50 nM) . The effect of pH, binding to serum proteins, interaction with biomembranes, subcellular localization and relocalization after irradiation were disclosed to be the main factors responsible for lower photoactivity of anionic Pcs .1 The work was supported by the Czech Science Foundation (Project No. 19-14758Y), by the Charles University Project PRIMUS/20/SCI/013 and from the project of Specific Academic Research (SVV 260 547). References 1. KOLLÁR, J., MACHÁČEK, M., HALAŠKOVÁ, M . et al.: J. Med. Chem., 63, 2020, 7616−7632. PREPARATION OF NEW MELTING TEMPERATURE MODIFIERS 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: kostelaf@faf .cuni .cz Melting temperature difference (ΔTm) between complementary and mismatched duplex has a crucial role for discrimination of point mutations or single nucleotide polymorphisms. The ΔTm is decreasing with the length of oligodeoxynucleotide. From this point of view, shorter oligodeoxynucleotide probes are advantageous due to higher ΔTm compared with longer probes . On the other hand, their low melting temperature is the main disadvantage. Melting temperature modifiers are used for elimination of the disadvantage. There are three types of modifiers that can be used for thermal stabilisation of oligodeoxynucleotide duplexes: polyamines1, minor groove binders2 and intercalators .3 In our work we focused on preparation of new acridine derivatives . They were prepared by modified published procedures. Our acridine derivatives were tested in solution and compared to the polyamine (spermine) and well known MGB Hoechst 33258 . Majority of acridine derivatives showed comparable or better activity in solution than spermine or Hoechst 33258 . Two most promising acridine derivatives were selected and covalently attached to the oligodeoxynucleotide probe by click chemistry . Probes were tested at PCR conditions . 16 The study was supported by the Czech Science Foundation (Project No. 18-17868S), by the Grant Agency of Charles University (Project No. 205007) and from the project of Specific Academic Research (SVV 260 547). References 1. BRŮŽA, Z., KRATOCHVÍL, J., POUR, M. et al .: Chem .– Eur . J ., 25, 2019, 8053–8060 . 2. CHEN, S ., LIU, Y ., LIU, Z . et al .: RSC Adv ., 6, 2016, 26412–26420 . 3. FURUTA, T ., FUKUYAMA, Y ., ASAKAWA, Y .: Phytochemistry, 25, 1986, 517–520 . MICHAEL AND ANTI-MICHAEL ADDITIONS TO [3]DENDRALENES PERDOMO, S . M ., ANTAL, R ., POUR, M . Department of Organic and Bioorganic Chemistry, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic e-mail: perdomos@faf .cuni .cz Dendralenes are acyclic cross-conjugated polyenes with an interesting, as yet unexamined reactivity and high potential for further synthetic applications .1 Our research group developed a novel synthesis of variously substituted electron-poor [3]dendralenes with a distinct dissonant character, and discovered an unexpected behaviour of these compounds upon nucleophilic attack . In view of the preliminary results and assuming that elimination of the dissonant nature was the driving force for these transformations, we further focused on various versions of Michael additions to dendralenic structures . In some cases, anti-Michael additions also occurred . Using mild conditions and different combinations of nucleophiles and dendralenes, cyclizations and/or simple additions have been observed. This study was supported by the Czech Science Foundation (Project No. 18-17868S), by the Grant Agency of Charles University (Project No. 1348119) and from the project of Specific Academic Research (SVV 260 547). References 1. HOPF, H ., SHERBURN, M . S .: Angew . Chem . Int . Ed ., 51, 2012, 2298–2338 . Scheme 1. Synthesis of the title compounds 17 STUDY OF 2,5-DISUBSTITUTED 1,3,4-OXADIAZOLES AS POTENTIAL ANTITUBERCULOTICS PFLÉGR, V ., KRÁTKÝ, M ., VINŠOVÁ, J . Department of Organic and Bioorganic Chemistry, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic e-mail: [email protected] Nowadays, small heterocyclic molecules (e.g., oxadiazoles, tetrazole, triazoles) have been studied as potential antituberculotics . Compounds with this structural motif target M. tuberculosis (Mtb .), including resistant strains .1 We have prepared a series of 2,5-disubstituted 1,3,4-oxadiazoles with very promising activity against several mycobacterial strains . Asymmetric 1,2-diacylhydrazides were prepared by the reaction of monosubstituted commercially available or in-house prepared (acyl) hydrazine with a appropriate acyl chloride in the presence of a base using anhydrous tetrahydrofuran (THF) as the solvent . 2,5-Disubstituted oxadiazoles were obtained directly by dehydrative cyclization of 1,2-diacylhydrazides (Scheme 1) . In general, the syntheses gave satisfactory yields . The compounds were evaluated for their in vitro antimycobacterial activity against drugsusceptible Mtb H37Rv and nontuberculous mycobacteria (NTM, M. avium, M. kansasii) . Determining the activity against multidrug-resistant Mtb . as well as selectivity index are under investigation . No activity against Gram-positive and Gram-negative bacteria as well as fungal pathogens was identified. The study was supported by the Czech Science Foundation (Project. No. 20-19638Y) and from the project of Specific Academic Research (SVV 260 547). References VOSÁTKA, R ., KRÁTKÝ, M ., ŠVARCOVÁ, M . et al .: Eur . J . Med . Chem ., 151, 2018, 824–835 . Fig. 11 Scheme 1. Additions of nucleophiles to [3]dendralenes 18 SYNTHESIS OF OMEGA-HYDROXYLATED CERAMIDES USING OLEFINATION REACTIONS ONDREJČEKOVÁ, V., OPÁLKA, L., VÁVROVÁ, K. Department of Organic and Bioorganic Chemistry, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic e-mail: sommerov@faf .cuni .cz Omega-hydroxylated ceramides (O-Cer) belong to a subclass of Cer with ultralong N-acyl chains . Together with other groups of Cer, free fatty acids and cholesterol, these lipids create multilamellar structures in stratum corneum, which are responsible for skin barrier function . O-Cer occur in two forms: free form or bound to the surface of corneocytes where they form a corneocyte lipid envelope (Fig . 1) . The detailed role of covalently bound O-Cer remains unclear . Fig. 12 Scheme 1. Synthesis of the title compounds HO R OH HN O O R = H Free ceramide R = Corneocyte Covalently bound ceramide Ceramide OS O Fig. 1. Structure of free O-Cer and O-Cer covalently bound to the corneocyte The main objective of this project was to optimize the synthetic procedure towards three subclasses of O-Cer, specifically Cer OS, OP and OdS. Complete synthesis of O-Cer has not been reported yet. In this project, we modified a previously published procedure of ultralong Cer synthesis where we focused on an improvement of the most complicated steps of this synthesis . Protected 16-hydroxyhexadecanal as a crucial component for olefination reactions was prepared in four steps from hexadecanolide with high yield . This aldehyde was then connected with various heterocyclic sulfones using olefination reactions, such as Julia and Julia-Kocienski reactions to obtain a protected 32-hydroxydotriacontenoic acid, which is an O-Cer precursor . This change in reaction procedure led to a significant improvement in the reaction yield and to a decrease in costs of starting materials . 19 The study was supported by the Czech Science Foundation (Project No. 19-09135J), by the Grant Agency of Charles University (Project No. 1194119) and from the project of Specific Academic Research (SVV 260 547). LIPID-DECORATED DENDRIMERS FOR STUDYING THE BEHAVIOR OF CORNEOCYTE LIPID ENVELOPE VELISSARI, P .,1 PARASKEVOPOULOS, G .,2 VÁVROVÁ, K .1 1 Department of Organic and Bioorganic Chemistry, 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 e-mail: velissap@faf .cuni .cz Skin is the biggest organ in the human body and protects it from excessive water loss while hampers the entrance of undesired substances, allergens and microbes . Skin’s outermost layer, stratum corneum (SC), holds the principal skin barrier and consists of flattened dead cells, known as corneocytes, embedded in a hydrophobic lipidic matrix . Covalently attached to corneocyte’s surface are ceramides forming the so-called “corneocyte lipid envelope” (CLE) .1 Most biophysical SC models are taking into consideration only the lipidic matrix . In our approach, we are focusing on the development of corneocyte mimicking compounds to create a more complex SC model that would incorporate hydrophilic corneocyte mimicking entities with/without CLE in the lipidic matrix to study the putative scaffolding role of CLE . For this purpose, we plan to decorate fourth generation PAMAM dendrimers with ceramides . For the development of an effective synthetic protocol, PAMAM dendrimers and ceramides were not used directly but were replaced by in-house synthesized dendrimers and long-chain aliphatic alcohols with a terminal triple bond, respectively . A copper-catalyzed azide-alkyne cycloaddition protocol was developed to couple the modified dendrimers with the ceramide mimicking compound. The synthetic protocol has already been established and will be applied to conjugate the PAMAM dendrimers with the ceramides . The study was supported by the Czech Science Foundation (Project No. 19-09135J) and from the project of Specific Academic Research (SVV 260 547). References 1. VÁVROVÁ, K., KOVÁČIK, A., OPÁLKA, L.: Eur. Pharm. J., 64, 2017, 28–35. 20 DESIGN, SYNTHESIS AND BIOLOGICAL EVALUATION OF 3-AMINOPYRAZINE-2-CARBOXAMIDE DERIVATIVES AS POTENTIAL ANTIMICROBIALS PALLABOTHULA, V . S . K ., ZITKO, J . Department of Pharmaceutical Chemistry and Pharmaceutical Analysis, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic e-mail: pallabov@faf .cuni .cz Tuberculosis (TB) remains among the WHO top 10 causes of death despite available treatments1 and BCG vaccine. As part of our ongoing research on pyrazinamide (a first-line antitubercular) derivatives, we report the design and synthesis of novel 3-aminopyrazine2-carboxamide derivatives along with their biological evaluation . Almost 50 compounds were prepared according to Scheme 1 and evaluated for their in vitro activity against various strains of mycobacteria and other strains of pathogenic bacteria and fungi . The active compounds were only from series-1 (R is a substituted phenyl) . The most active compounds we selective towards inhibition of Mtb H37Ra and Mtb H37Rv (over other mycobacterial strains) and exerted MIC (Minimum Inhibitory Concentration) ranging from 1 .98 to 7 .81 µg mL‒1. The final compounds were also studied for cytotoxicity on HepG2 cell line followed by SAR . Title compounds will also be studied as potential inhibitors of (human) prolyl-tRNA synthetase based on their structural similarities to confirmed inhibitors reported in the literature .2 Fig. 14 ! ! !" # # # $ %"C ! ! #%"C # !"' ! ! !" # # !"' $ (F$* (H,-.(/,0N% O 3% 4N R6N(H,070H,0 $ * N8N9.R6/N(H:7HXN #%" C XN<XN!# ' XN%= C (67H !" #$%&$#'( #$%&$#') Scheme 1. Synthesis of final compounds a: acyl chlorides/pyridine/Ar medium, b: 2M ammonia in EtOH The study was supported by the Czech Science Foundation (Project No. 20-19638Y) and from the project of Specific Academic Research (SVV 260 547). References 1. World Health Organization, Global Tuberculosis Report 2020 https://www.who.int/publications/i /item/9789240013131 2. ADACHI, R ., OKADA, K ., SKENE, R . et al .: Biochem . Biophys . Res . Commun ., 488, 2017, 393–399 . 21 INSECT MODEL, GALLERIA MELLONELLA, AS NEW POWERFUL TOOL FOR THE DRUG DISCOVERY RESEARCH KONEČNÁ, K., DIEPOLTOVÁ, A., JANĎOUREK, O., ZÁVESKÁ, V. Department of Biological and Medical Sciences, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic e-mail: konecna@faf .cuni .cz The final and often crucial phase of drug discovery research involves the using of appropriate animal models . Mice and rats are the most frequently chosen models for this purpose. However, in taking into account 3Rs (Replacement, Reduction, and Refinement) criteria, which are considered to be a framework for conducting high-quality science in the academic sector, it is desirable to look for a suitable alternative approach .1 Galleria mellonella represents an insect animal model enjoying increasing popularity in research communities . This animal model is employed in studies focused on mechanisms of microbial pathogenesis and virulence, in discrimination between in vivo high and non/ low-toxicity, or in the study of in vivo efficacy of candidate anti-infective drugs. This model’s undeniable pros are that ethical approval is not needed, financial costs are significantly lower than the mouse or rat model, and no special lab equipment is required.2,3 We are currently experienced in this animal model rearing and its use for in vivo toxicity testing . Optimization of the methodical approaches for in vivo candidate anti-infective drug efficacy testing and in vivo microbial biofilm formation is now in the process. The study was supported by the Czech Science Foundation (Project No. 20-19638Y) and from the project of Specific Academic Research (SVV 260 549). References 1. IGNASIAK, K ., MAXWELL, A .: BMC Res . Notes, 10, 2017, 428 . 2. TSAI, C . J ., LOH, J . M ., PROFT, T.: Virulence, 7, 2016, 1705‒1711. 3. ALLEGRA, E ., TITBALL, R . W ., CARTER, J . et al .: Chemosphere, 198, 2018, 469–472 . STUDY OF BOOSTER EFFECT OF COMPOUNDS IN COMBINATION WITH STANDARD DRUGS USED IN THERAPY OF TUBERCULOSIS JANĎOUREK, O., KONEČNÁ, K., DIEPOLTOVÁ, A. Department of Biological and Medical Sciences, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic e-mail: jando6aa@faf .cuni .cz Tuberculosis is still one of the most threatening health problems all over the world . Although the absolute numbers of new cases are decreasing, problem has arisen with resistant strains of Mycobacterium tuberculosis, which is the causative agent of tuberculosis . These cases are hard to cure and it is necessary to use second line drugs . This treatment is expensive, time consuming and the risk of side effects is high . 22 Treatment of tuberculosis is always based on combination of at least 2 to 4 drugs according to therapy regimen . Using combinations can be very useful as well as tricky . Combinations can improve the effect and lower the risk of resistance development . On the other hand, these combinations mean higher risk of interactions that can result in antagonistic effects of these molecules . Outcome of those factors is failure of therapy that can lead to significant spreading of infection and higher mortality rate. Synthesis of novel compounds is one of the most important steps for tuberculosis eradication. But the chance to find molecule with novel mechanism of action, for which mycobacteria will not be able to evolve resistance, is quite low . So, one opportunity to slow down the spreading is to study interactions between drugs used for treatment and new molecules . One of the methods to study these interactions is called checkerboard assay . It is based on studying combinations of two compounds in various concentrations affecting the result . There are 4 possible interactions expressed as FIC (Fractional Inhibitory Concentration), namely Antagonism, Indifference, Additivity, Synergism . Last three named categories are optimal for practice, but synergism is the most desired . Methodology for antimycobacterial checkerboard assay was optimized in this work . The study was supported by the Czech Science Foundation (Project No. 20-19638Y), by Research programme Development and Study of Drugs (Progress Q42) and from the project of Specific Academic Research (SVV 260 547). TOWARDS TO IN VITRO ANTIBIOFILM ACTIVITY SCREENING – INTRODUCTION OF APPROPRIATE METHODICAL APPROACH FOR STAPHYLOCOCCAL BIOFILM FORMATION DIEPOLTOVÁ, A., KONEČNÁ, K., JANĎOUREK, O., NACHTIGAL, P. Department of Biological and Medical Sciences, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic e-mail: diepolta@faf .cuni .cz Staphylococcus aureus (SA) and Staphylococcus epidermidis (SE) are the most common pathogens from the genus Staphylococcus, causing biofilm-associated infections. Bacteria in biofilms are difficult to eradicate due to their resistance and serve as a reservoir for recurring persistent infections .1 A variety of protocols for in vitro drug activity testing against staphylococcal biofilms has been introduced. However, there are often fundamental differences . In our preliminary study, we developed optimal conditions for staphylococcal biofilm formation on plastic pegs in order to set a methodology for the evaluation of the antibiofilm activity of candidate molecules. The convenience of the plastic pegs lies in their removability from the lid for easy access to multiple equivalent biofilms, and in possibility of in situ detection and quantification by confocal laser microscopy. For the purpose of enhancement in staphylococcal biofilm formation, the impact of peg surface modification with 3 different coating materials was studied as well. An increase of biofilm biomass was evaluated by crystal violet staining method.2 The basic 23 precondition for obtaining relevant and reproducible data regarding antibiofilm activity is the formation of robust biofilms with typical attributes such as the presence of a biofilm matrix . In our study, in vitro conditions revealed that we fully met the preconditions for the SA and methicillin-resistant SA strains . In conclusion, we demonstrated statistically significant enhancement of biofilm formation in all studied staphylococcal strains, including either strong biofilm producer phenotype (SA, methicillin-resistant SA) and weak biofilm producer phenotype (SE). The study was supported by the Czech Science Foundation (Project No. 20-19638Y), and from the project of Specific Academic Research (SVV 260 549). References 1. DONLAN, R . M .: Emerg . Infect . Dis ., 8, 2002, 881–890 . 2. CHRISTENSEN, G . D ., BALDASSARRI, L ., SIMPSON, W . A .: Methods Enzymol . 253, 1995, 477–500 . DERIVATIVES OF QUINOXALINE-2-CARBOXAMIDES AS POTENTIAL ANTIMYCOBACTERIALS BOUZ, G., BOUZ, S., ZITKO, J., DOLEŽAL, M. Department of Pharmaceutical Chemistry and Pharmaceutical Analysis, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic e-mail: bouzg@faf .cuni .cz Despite the established treatments, tuberculosis remains an alarming threat to public health according to WHO .1 Novel agents are needed to overcome the increasing rates of resistance and perhaps achieve eradication . As part of our long-term research on pyrazine derivatives, we prepared a series of N-substituted quinoxaline-2-carboxamides (Fig . 1) and evaluated their in vitro antitubercular activity . Several quinoxaline derivatives were found in the literature to possess antitubercular activity .2 Quinoxaline-2-carboxylic acid was activated by oxalyl chloride and reacted with different anilines or benzylamines in the presence of pyridine at room temperature, overnight with stirring, and obtained crudes were then purified with flash chromatography. In addition to activity assessment, final compounds were screened for their in vitro cytotoxicity on HepG2 liver cancer cell lines . In vitro activity against Mtb H37Ra (represented by MIC) ranged between 3 .91–500 µg mL−1, with most compounds having moderate to good activities (MIC < 15 .625 µg mL−1) . Fig. 15 Fig. 15 Fig. 1. Structures of the studied compounds 24 This work was supported by EFSA-CDN (Reg. No. CZ.02.1.01/0.0/0.0/16_019/00008 41) co-funded by ERDF, by the Czech Science Foundation (Project No. 20-19638Y) and from the project of Specific Academic Research (SVV 260 547). References 1. World Health Organization, Global Tuberculosis Report 2020. https://www.who.int/publications/i/item /9789240013131 2. PERAMAN, R ., KUPPUSAMY, R ., KILLI, S . K . et al .: Int . J . Med . Chem ., 2016, art . 6471352 . PYRAZINE-2-CARBOHYDRAZIDE DERIVATIVES AS POTENTIAL ANTIITUBERCULARS NAWROT, D., ZITKO, J., DOLEŽAL, M. Department of Pharmaceutical Chemistry and Pharmaceutical Analysis, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic e-mail: nawrotd@faf .cuni .cz Tuberculosis, an infectious disease, is a major problem when it comes to number of casualties and growing antimicrobial resistance .1 It is treated by first-line drugs (e.g ., pyrazinamide), but new agents are needed . In 2014, Rodrigues et al .2 tested a series of pyrazine-2-carbohydrazide derivatives as potential anticancer class . Compounds were tested on three different tumor cell lines and their growth inhibition was not satisfactory to develop such compounds as new anticancer agents . This work inspired us to the development our title series and evaluate their antimicrobial activity . Current series is based on pyrazine-2-carbohydrazide that is bound to different heterocycles by imine bond (example of compound: N-benzylidenepyrazine2-carbohydrazide) that will be further derivatized into disubstituted compounds (example N-benzoyl-N′-benzylidenepyrazine-2-carbohydrazide). Compounds were tested for biological activity against selected strains of Mycobacterium, eight fungi strains and eight bacterial strains . The minimum inhibitory concentration (MIC) for tested mycobacterial strains was determined for all tested compounds beside isoniazid, ciprofloxacin and rifampicin as reference standard drugs . Results of the biological testing and structure activity relationships are discussed in the presentation . The study was supported from the project of Specific Academic Research (SVV 260 547). References 1. World Health Organization: Global Tuberculosis Report 2020. https://www.who.int/publications/i/item /9789240013131 2. RODRIGUES, F . A . R ., Da S . BOMFIM, I ., CAVALCANTI, B . C . et. al .: Eur . Chem . Bull ., 3, 2014, 358–361 . 25 DESIGN, SYNTHESIS AND BIOLOGICAL EVALUATION OF PYRAZINE-BASED INHIBITORS OF MYCOBACTERIAL METHIONINE AMINOPEPTIDASE JUHÁS, M ., ZITKO, J . Department of Pharmaceutical Chemistry and Pharmaceutical Analysis, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic e-mail: juhasm@faf .cuni .cz Tuberculosis, caused by Mycobacterium tuberculosis (Mtb), is the number one cause of deaths due to a single infectious agent worldwide . In this project, we present the synthesis and biological evaluation of new potential pyrazine-based inhibitors (Fig . 1) of the prominent drug target mycobacterial methionine aminopeptidase 1 (MtMetAP1) . The activity of all compounds was evaluated against the isoform MtMetAP1a . Overall, high inhibition of the isolated enzyme was observed . However, as described previously1, the activity was strongly dependent on the used metal cofactor . The highest activity was seen in the presence of Ni(II) (IC50 = 0 .6 µM, 2Br substitution), the lowest with Co(II) . Several compounds also showed mediocre in vitro potency against Mtb (MIC = 15 .625 µg mL−1) . Unfortunately, no direct correlation with the activity against the isolated enzyme was seen . Despite the high structural similarities of bacterial and fungal MetAP to mycobacterial MtMetAP1, final compounds did not exert any antibacterial nor antifungal activity . The study was supported by the Czech Science Foundation (Project No. 20-19638Y), and from the project of Specific Academic Research (SVV 260 547). References 1. LU, J . P ., YE, Q . Z .: Bioorg . Med . Chem . Lett ., 20, 2010, 2776–2779 . Fig. 16 Fig. 1. General structure of the synthesized compounds 32 Alkaloids are important group of biologically active secondary metabolites . Many of them inhibit human cholinesterases and therefore have potential to be used in the treatment of Alzheimer’s disease (AD) . For example, Amaryllidaceae alkaloid galanthamine is used in therapy of AD .1 AD is one of the most frequent causes of dementia in the world . AD consist of many cognitive and neuropsychiatric manifestations . Enzymes acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) play important role in the progression of this disease .1 The genus Ficus includes arboreous and evergreen plants belonging to the family Moraceae, that is distributed widely throughout the tropical and subtropical regions . It is represented by about 725 species . Plants from the genus Ficus contain among other alkaloids . These alkaloids display various structures and some of them have interesting biological activities such as anti-inflammatory, antitumor, antifungal, antibacterial and antimalarial .2 Within the phytochemical study, 19 extracts from leaves and stems of 14 species of the genus Ficus were prepared . Dry material was ground and then extracted by boiling in ethanol. Ethanolic extracts were purified by liquid-liquid extraction (ether, ethyl acetate, chloroform) . All extracts were tested on the ability to inhibit human cholinesterases . In testing hBuChE inhibition potency has been demonstrated by the extract AL-700C (74 .82 ± 2 .78% at concentration 50 µg mL−1) . This work was supported by EFSA-CDN (Reg. 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 260 548). References 1. HULCOVÁ, D ., BREITEROVÁ, K ., SIATKA T . et al.: Molecules, 23, 2018, art . 719 . 2. KUBO, M ., YATSUZUKA, W ., MATSUSHIMA, S . et al.: Chem . Pharm . Bull ., 64, 2016, 957–960 . NEW AMARYLLIDACEAE ALKALOIDS FROM NARCISSUS PSEUDONARCISSUS cv . CARLTON AS INSPIRATION FOR THE DEVELOPMENT OF NEW DRUGS FOR ALZHEIMER’S DISEASE AL MAMUN, A .,1 CAHLÍKOVÁ, L.,1 HULCOVÁ, D .,1 MAŘÍKOVÁ, J.2 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: almamuna@faf .cuni .cz Plant alkaloids are one of the most interesting groups of secondary metabolites and are present also in the plants of the family Amaryllidaceae . To date, nearly 600 Amaryllidaceae alkaloids (AA) have been isolated .1 Narcissus pseudonarcissus cv . Carlton (NPC) is an Amaryllidaceae species, exclusively used for the commercial extraction of galanthamine as a drug for the treatment of mild to moderate stages of Alzheimer’s disease (AD) . The 33 treatment of AD is only symptomatic including therapies with acetylcholinesterase (AChE) inhibitors . Butyrylcholinesterase (BuChE) is another cholinesterase (ChE), whose activity increases to 40–90% in the later stage of AD brain .1 So far, thirteen known and four novel AA have been isolated from the alkaloidal extract of NPC and have been screened for AChE, BuChE, and prolyloligopeptidase (POP) inhibition activity . Three new compounds named carltonine A, B, and C demonstrate significant inhibition potential towards hBuChE (IC50 = 910 nM, 31 nM, and 14 .84 µM, respectively) .1 Carltonine A also showed moderate POP inhibition activity (IC50 = 143 µM) .1 The novel compound narciabduliine inhibits both hAChE (IC50 = 3 .29 µM) and hBuChE (IC50 = 3 .44 µM) . The next part of the current study was the preparation of synthetic derivatives structurally inspired by carltonines and screening of their biological activities connected with AD . Narcissus pseudonarcissus cv. Carlton Carltonine A IC50,hBuChE = 0.91 ± 0.02 μM Carltonine B IC50,hBuChE = 0.031 ± 0.001μM FC001 IC50,hBuChE = 0.240 ± 0.03μM Structure detail Aldehyde Tyramine + Fig. 1. Synthesis of structural analogues of carltonins The project was supported from the project of Specific Academic Research (SVV 260 548). References 1. AL MAMUN, A., MAŘÍKOVÁ, J., HULCOVÁ, D. et. al.: Biomolecules, 2020, 10, art . 800 . AMARYLLIDACEAE ALKALOIDS OF NORBELLADINE-TYPE AS INSPIRATION FOR DEVELOPMENT OF SELECTIVE BUTYRYLCHOLINESTERASE INHIBITORS PIDANÝ, F .,1 CAHLÍKOVÁ, L.,1 AL MAMUN, A .,1 HULCOVÁ, D .,2 MAŘÍKOVÁ, J.3 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 3 Department of Organic and Bioorganic Chemistry, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic e-mail: pidanyf@faf .cuni .cz Alzheimer’s disease is a serious and irreversible progressive neurodegenerative disorder, that will reach a prevalence of more than 100 million by 2050 due to the aging of 34 population .1 Pathological changes in the affected brain include: intracellular neurofibrillary tangles, extracellular amyloid plaques, increased oxidative stress, cholinergic disfunction, and others . Cholinergic neurotransmission is terminated by acetylcholine hydrolysis regulated by two enzymes: acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) . With the progression of disease the activity of AChE decreases, while that of BuChE increases .2 The alkaloids carltonin A and B demonstrated exceptional selective inhibition potential towards the enzyme BuChE in tens of nanomoles (IC50 = 0 .031 ± 0 .001µM) . Unfortunately, these alkaloids are present in plant material only in trace amounts . The aim of this work is a preparation of synthetic compounds inspired by alkaloids of the belladinetype with subsequent structure-biological activity relationship study . The work was supported from the project of Specific Academic Research (SVV 260 548). References 1. KOŠAK, U ., BRUS, B ., KNEZ, D . et al.: J. Med. Chem., 61, 2018, 119‒139. 2. MEDEN, A., KNEZ, D., JUKIČ, M. et al.: Chem. Commun., 55, 2019, 3765‒3768. PHYTOCHEMICAL INVESTIGATION OF THE DICRANOSTIGMA FRANCHETIANUM (PRAIN) FEDDE (PAPAVERACEAE) HERB: PRELIMINARY STUDY WIJAYA, V .,1 KOHELOVÁ, E .,1 OPLETAL, L .,1 JENČO, J.1 ŠAFRATOVÁ, M .2 1 ADINACO Research Group, Department of Pharmaceutical Botany, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic 2 ADINACO Research Group, Department of Pharmacognosy, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic e-mail: wijayav@faf .cuni .cz Dicranostigma franchetianum (Prain) Fedde, syn . Chelidonium franchetianum Prain (Papaveraceae) is one of the representatives of the scanty genus Dicranostigma Hook .f . & Thomson . This annual plant grows endemically in the Himalayas and western China and is used as an ornamental plant in Europe . Although it is the source of isoquinoline alkaloids (especially isocorydine) and the hitherto little-studied alkaloids (chelilutin, chelirubin and cheilanthifolin), the plant has not been systematically studied until this time . In primary screening of the summary alkaloid extract for cholinesterases inhibition, the inhibitory potency was high (hAChE/hBChE, IC50 = 1.67 ± 0.11/3.85 ± 0.31 µg mL−1) and together at least 25 alkaloids were found in the extract . The primary ethanol extract was prepared from 11 .8 kg of dry herb (Garden of Medicinal Plants, Faculty of Pharmacy in Hradec Králové), from which alkaloid extracts with different solvents with increased polarity (n-hexane, diethyl ether, ethyl acetate, chloroform, chloroform-ethanol) were prepared. The purified diethyl ether extract of the alkaloidal bases (93 g) was separated by flash chromatography on silica to give 12 combined fractions. From these fractions after purification (+)-isocorydine 1, protopine 2, allocryptopine 3, berberine 4, chelerythrine 5, sanguinarine 6) according to the TLC comparison have been obtained so far . Their inhibi- 35 tory activity on hAChE and hBChE was determined by using recombinant human brain cholinesterases with the following results: hAChE/hBChE (IC50, mM) (1: > 1000/657.1 ± 15 .5, 2: 230.0 ± 21.0/208.9 ± 17.7, 3: 250 .0 ± 25 .2, 4: 0.71 ± 0.10/30.7 ± 3.5, 5,6: not determined so far) . The study was supported by the Research program Development and Study of Drugs (Progres Q42) and from the project of Specific Academic Research (SVV 260 548). SEMISYNTHETIC DERIVATIVES OF AMARYLLIDACEAE ALKALOID HAEMANTHAMINE AS POTENTIAL DRUGS IN THE TREATMENT OF ALZHEIMER’S DISEASE PEŘINOVÁ, R.,1 KOHELOVÁ . E .,1 ŠPULÁK, M .,2 MAŘÍKOVÁ, J.,2 HULCOVÁ, D .,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 Organic and Bioorganic Chemistry, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic e-mail: perinovr@faf .cuni .cz Alzheimer’s disease (AD) is the most prevalent neurodegenerative disease worldwide with complex etiology and multifaceted pathophysiology and data indicate an exponential rise in the number of cases of this disease . The well-known Amaryllidaceae alkaloid (AA) galanthamine is a marketed drug for AD therapy under the commercial name Reminyl© (galanthamine hydrobromide) . Studies also pointed out various pharmacological properties of semisynthetic derivatives of some AA, such as alkaloid haemanthamine (HMT), which is widely distributed through Amaryllidaceae plants . Based on our previous results, where several HMT derivatives demonstrated promising hAChe/hBuChe inhibition potency, we continued the preparation of further HMT semisynthetic derivatives .1 Several new esters showed interesting inhibition of both studied cholinesterases, thus structure-activity relationship (SAR) was also studied .2 Newly prepared compounds were identified by 1D-, 2D-NMR and ESI-MS methods. The most potent compounds were studied in more detail (e.g., type of inhibition, docking studies, logBB etc .) . This work was supported from the project of Specific Academic Research (SVV 260 548). References 1. KOHELOVÁ, E., PEŘINOVÁ, R., MAAFI, N. et al .: Molecules, 24, 2019, art . 1307 . 2. PEŘINOVÁ, R., MAAFI, N., KORÁBEČNÝ, J. et al .: Bioorg . Chem ., 100, 2020, art . 103928 . 36 IN VITRO ANTIAGGREGATION POTENTIAL OF SELECTED ISOQUINOLINE ALKALOIDS PARVIN, M . S .,1 KARLÍČKOVÁ, J.,2 HRUBŠA, M .,3 FADRAERSADA, J .,3 MLADĚNKA, P.,3 MACÁKOVÁ, K .1 1 Department of Pharmacognosy, 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 3 Department of Pharmacology and Toxicology, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic e-mail: parvins@faf .cuni .cz The platelet is an anucleate cell circulating in the bloodstream . The normal platelet function is to regulate haemostasis but in a pathological state, such as endothelial dysfunction, enhanced platelet aggregation can lead to certain cardiovascular diseases such as stroke, acute myocardial infarction etc .1 Some plant secondary metabolites, alkaloids in particular, are promising candidates in the development of antiplatelet drugs .2 So far, fourteen isoquinoline alkaloids have been screened for inhibition of platelet aggregation induced by arachidonic acid and collagen and compared with the standard drug acetylsalicylic acid . The screening was performed by impedance aggregometry using whole human blood . Papaverine and bulbocapnine demonstrated inhibition at the concentration of 80 µM. Their mechanisms of action could be an influence on arachidonic acid cascade including cyclooxygenase-1, thromboxane A2 synthase, and antagonism at thromboxane A2 receptors . The antiaggregation activity of papaverine and bulbocapnine seems to be related to antagonism at thromboxane receptors . In addition to antiplatelet activity, the prothrombin time and activated partial thromboplastin time of all alkaloids have been determined and none of the alkaloids showed anticoagulant effects in human plasma . The study was supported from the project of Specific Academic Research (SVV 260 548). References 1. KARLÍČKOVÁ, J., ŘÍHA, M., FILIPSKÝ, T. et al.: Planta . Med ., 82, 2016, 76–83 . 2. QURRAT-UL-AIN, KHAN, H ., MUBARAK, M . S . et al.: Front . Pharmacol ., 7, 2016, art . 292 . SEMI-SYNTHETIC DERIVATIVES OF AMARYLLIDACEAE ALKALOID AMBELLINE AND THEIR CYTOTOXIC POTENTIAL RITOMSKÁ, A .,1 PEŘINOVÁ, R.,1 MAŘÍKOVÁ, 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 Biochemistry, Faculty of Medicine in Hradec Králové, Charles University, Czech Republic e-mail: ritomska@faf .cuni .cz 37 Amaryllidaceae alkaloid ambelline, belonging to the crinane-type group, lacks any significant biological activity. However, its analogues prepared by the derivatization of C11-hydroxyl group possess various interesting properties (e.g ., inhibitory activity of cholinesterases, antimalarial activity) . As a continuation of our earlier work and extension of the prepared derivatives scale, eleven novel aromatic esters were developed (LC-108, LC-180, LC-181, LC-177, LC-131, LC-189, LC-179, LC-119, LC-110, LC-106, LC178) . To characterize their biological activity spectrum, MTT assays were performed to determine their cytotoxic potential . To predict the structure-activity relationship for further research, substances described in our previous work were also included in this cytotoxicity study .1 Molecules with the most pronounced cytotoxic activity contain a methyl group (LC-108), methoxy group (LC-180, LC-176, LC-104, LC-181), ethoxy group (LC-182), or disubstitution with different functional groups (LC-106) on C11 . The study was supported from the project of Specific Academic Research (SVV 260 548). References 1. MAŘÍKOVÁ, J., RITOMSKÁ, A., KORÁBEČNÝ, J . et al .: J . Nat . Prod ., 83, 2020, 1359–1367 . INDOLE ALKALOIDS FROM VINCA MINOR L . AND THEIR BIOLOGICAL ACTIVITY VRABEC, R .,1 HULCOVÁ, D .,1 MAŘÍKOVÁ, J.,2 KUNEŠ, J .,2 HAVELEK, R .,3 LOČÁREK, M.,4 JENČO, J.,4 ŠAFRATOVÁ, M .,1 OPLETAL, L .4 1 ADINACO Research Group, Department of Pharmacognosy, 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 4 ADINACO Research Group, Department of Pharmaceutical Botany, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic e-mail: vrabecr@faf .cuni .cz Vinca minor L . (Apocynaceae), an evergreen trailing subshrub common in Western and Southern Europe, is a rich monoterpene indole alkaloid source . So far, we have isolated 23 alkaloids from aerial parts of V. minor, 12 of them were reported in this species for the first time. Two alkaloid structures are entirely new. The names cosimonine and vincaminorudeine are proposed, and relative configuration determined. In an ongoing study into the discovery of new anti-neurodegenerative drugs from natural sources, we have tested our isolated compounds to inhibit human acetylcholinesterase (hAChE) and butyrylcholinesterase (hBuChE) . None of the alkaloids were active against hAChE, however, most structures showed interesting inhibition of hBuChE with IC50 < 100 µM . In the later and severe stage of Alzheimer’s disease (AD), the expression of AChE is reduced, but the expression BuChE is increased and takes over its role . BuChE is also involved in other pathological AD processes, such as the production of neurofibrillary tangles 38 and the formation of β-amyloid plaques. The active substances were also evaluated for the probability of a blood-brain barrier penetration (logBB) by in silico computational method . The cytotoxicity of isolated compounds was assessed on the panel of 10 tumorous cell lines. Except for eburnamonine, none of the tested alkaloids showed significant cytotoxic activity . The study was supported by the Research program Development and Study of Drugs (Progres Q42) and from the project of Specific Academic Research (SVV 260 548). AROMATIC DERIVATIVES OF HAEMANTHAMINE-TYPE ALKALOID VITTATINE AS POTENTIAL LIGANDS FOR ALZHEIMER’S DISEASE AL SHAMMARI, L .,1 HULCOVÁ, D .,1,2 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 e-mail: alshamml@faf .cuni .cz Recent research on Amaryllidaceae plant family reports the isolation of more than 600 different Amaryllidaceae alkaloids (AA) with different structure types possessing wide range of biological activities . Among the most important biological activities of AA belong activities associated with Alzheimer’s disease (AD) . Alzheimer’s disease (AD) is the most prevalent neurodegenerative disease worldwide with complex etiology and multifaceted pathophysiology and data indicate an exponential rise in the number of cases of this disease . Galanthamine is the one of the most important AA used for treatment of AD and commercially available under the name Reminyl® (galanthamine hydrobromide) . Based on our previous published work of Hippeastrum x hybridum cv . Ferrari 18 different alkaloids have been isolated so far and they were identified by MS, HRMS and 1Dand 2D-NMR techniques . All alkaloids were tested for their activities associated with AD (hAChE, hBuChE and POP) and their ability to inhibit the growth of several cancer cell lines . Moreover, the isolated amount and the structure of vittatine allowed us to develop and synthesize thirteen new aromatic esters of the haemanthamine-type alkaloid vittatine . All the semisynthetic derivatives were studied for their inhibitory potential against both hAChE and hBuChE . The potential candidates were selected to measure the ability to penetrate the blood-brain barrier (BB) and availability to the CNS . The study was supported from the project of Specific Academic Research (SVV 260 548). References 1. AL SHAMMARI, L., HULCOVÁ, D., MAŘÍKOVÁ, J. et al .: S . Afr . J . Bot ., 136, 2021, 137–146 . 39 SEPARATION OF THE CAROTENOID MYXOXANTHOPHYLL FROM SYNECHOCYSTIS SALINA BY HIGH PERFORMANCE COUNTERCURRENT CHROMATOGRAPHY AND EVALUATION OF ITS IMMUNE-STIMULATING PROPERTIES FÁBRYOVÁ, T .,1,2 NOVÁKOVÁ, M .,1,2 VOKURKOVÁ, D .,3 KOPECKÝ, J .,2 HROUZEK P .,2 CHEEL, J .,2 TŮMOVÁ, L.1 1 Department of Pharmacognosy, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic 2 Laboratory of Algal Biotechnology, The Centre ALGATECH, Institute of Microbiology, The Czech Academy of Sciences, Czech Republic 3 Institute of Clinical Immunology and Allergology, Faculty of Medicine in Hradec Králové, Charles Universtity and University Hospital Hradec Králové, Czech Republic e-mail: fabryovt@faf .cuni .cz Cyanobacterium Synechocystis salina is well-known for biosynthesizing different types of pigments including chlorophyll, carotenoids and phycobiliproteins, many of which have attracted the attention of the industries and researchers due to their varied bio-functionality and applications . One of its synthesized carotenoid pigments is myxoxanthophyll (Fig . 1), a glycosylated monocyclic carotenoid that is rarely found in nature . This yellow pigment has been shown to exhibit antioxidant and anti-hyperglycemic activities .1,2 Moreover, its potential in the prophylactic and/or therapeutic treatment of undesirable conditions due to oxidative processes has also been disclosed .3 To the best of our knowledge, this pigment has not yet been offered commercially, and its bio-functional properties of interest in the food, nutraceutical and cosmetic sectors have not been extensively studied . In this study, myxoxanthophyll was isolated from Synechocystis salina by high performance countercurrent chromatography (HPCCC) and its identity was confirmed using high-performance liquid chromatography connected to a high-resolution tandem mass spectrometry detector with electrospray ionization source (HPLC-ESI-HRMS/MS). In addition, the potential activation effect on immune cells in response to myxoxanthophyll treatment was investigated, measuring cell surface CD69 expression using flow cytometry. An increase in the number of granulocytes was observed after myxoxanthophyll treatment . The presented data may support the potential use of myxoxanthophyll for strengthening the immune system against bacterial and fungal infections . Fig. 19 Fig. 1. Chemical structure of myxoxanthophyll 40 The study was supported by National Sustainability Programme I of the Ministry of Education, Youth and Sports of the Czech Republic (Project No. LO1416) and from the project of Specific Academic Research (SVV 260 548). References 1. GHOSH, T ., BHAYANI, K ., PALIWAL, C . et al.: Front . Mar . Sci ., 3, 2016, art . 146 . 2. STEIGER, S ., SCHÄFER, L ., SANDMANN, G .: J . Photochem . Photobiol . B: Biol ., 52, 1999, 14–18 . 3. JAEGER, C ., SAETTLER, A ., SCHROEDER, K . R . et al.: WO2002024183A1, 28 . 03 . 2002 . DERIVATIVES OF MONTANINE-TYPE ALKALOIDS AND THEIR IMPLICATION TO ALZHEIMER’S DISEASE: SYNTHESIS, BIOLOGICAL ACTIVITY, DOCKING STUDY MAAFI, N .,1 CAHLÍKOVÁ, L.,1 MAŘÍKOVÁ, J.,2 HULCOVÁ, D .3 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 ADINACO Research Group, Department of Pharmacognosy, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic e-mail: negarm@faf .cuni .cz The Amaryllidaceae plant family is one of the most important alkaloid containing plant families with biological properties such as antitumor, antimicrobial, antimalarial, and significant antineurodegenerative activities. Among all Amaryllidaceae alkaloids, montanine-type alkaloids are characterized by 5,11-methanomorphanthridine ring system and known for their potential antiproliferative, antibacterial, antimalarial, antirheumatic and anticholinesterase activities .1 The effect of montanine as an inhibitor of acetylcholinesterase has been reported in a dose-dependent pattern with more than 50% inhibition of enzyme at 1 mM concentration, introducing montanine as a moderate inhibitor of acetylcholinesterase .2 In this study, twenty-eight new derivatives of montanine-type alkaloids were synthesized and evaluated for their ability to inhibit human recombinant acetylcholinesterase (hAChE) and butyrylcholinesterase (hBuChE) . Among all, 3 derivatives of 3-O-methylpancracine showed significant selective inhibitory potency of hAChE (IC50 values of 1 .6 ± 0 .1 µM, 3 .1 ± 0 .2 µM, and 4 .3 ± 0 .5 µM) . Derivatization of 3-O-methylpancracine using 1-piperidinecarbonyl chloride resulted to the most potent hBuChE inhibitor with IC50 of 1 .73 ± 0 .05 µM . The same acyl changes on C4 of montanine led to reduction of inhibitory activity on both enzymes . The study was supported from the project of Specific Academic Research (SVV 260 548). References 1. KOUTOVÁ, D ., MAAFI, N ., HAVELEK, R . et al .: Molecules, 25, 2020, art . 2337 . 2. PAGLIOSA, L . B ., MONTEIRO, S . C ., SILVA, K . B . et al .: Phytomedicine, 17, 2010, 698–701 . 41 PHARMACEUTICAL TECHNOLOGY SECTION THE ROLE OF pH IN THE FORMATION OF SKIN LIPID BARRIER SAGRAFENA, I .,1 NOVÁČKOVÁ, A.,1 PARASKEVOPOULOS, G .,1 PULLMANNOVÁ, P .,2 DWIVEDI, A .,2 MAZUMDER, A .,2 VÁVROVÁ, K .2 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 sagrafei@faf .cuni .cz The skin’s pH has the peculiar characteristic of being near-neutral (pH ~ 7 .4) in deeper layers of the epidermis, while it becomes acidic (pH ~ 5 .5) on the surface of the stratum corneum (SC) .1 As it has been previously suggested, the skin’s “acid mantle” contributes to the protection against pathogens, the activation of enzymes for skin function, the inhibition of enzymes perturbing SC integrity, and finally, the impedance of the proinflammatory cytokines release.2 This study suggests a new role for the skin’s pH, closely related to the formation of the unique multilamellar structure of the lipidic matrix in the SC which is essential for the excellent skin barrier function . Isolated human SC lipids model membranes were treated with buffer pH 5 .5 or pH 7 .4 . X-ray diffraction showed that acidic pH treated lipids formed long periodicity phase (d = 13 .53–13 .72 nm), short periodicity phase (d = 5 .56–5 .60 nm), traces of cholesterol phase and for lateral packing orthorhombic conformation (two reflections at 4.1 and 3.7 nm). On the other hand, pH 7 .4 treated lipids showed poorly resolved diffractograms with traces of long periodicity phase and a prevalent unknown peak at q = 1 .35 nm−1 . The functional consequence of the pH-driven change in lipid microstructure was an increased transepidermal water loss at pH 7 .4 (27 ± 5 .0 g m−2 h) compared to pH 5 .5 (21 ± 5 .2 g m−2 h) in either model lipid membranes or isolated SC . These results are consistent with the diminished barrier function in newborn babies, elderly or patients with atopic dermatitis, where SC acidification is incomplete . The study was supported by the Czech Science Foundation (Project No. 19-09135J), by the Grant Agency of Charles University (Project No. 1156120) and from the project of Specific Academic Research (SVV 260 547). References 1. ELIAS, P . M .: Exp . Dermatol . 26, 2017, 999–1003 . 2. PROKSCH, E .: J . Dermatol ., 45, 2018, 1044–1052 . 48 skin barrier lipids . Our preliminary data have shown that there is a direct dependence of monolayer formation on the number of terminal amino groups of each dendrimer . More specifically, different generations (G2, G3 and G4) of PAMAM dendrimers were studied in different concentrations . Interestingly, when G4 PAMAM dendrimer was used in different concentrations (2.5, 5 or 10 μM), the organization of lipids to monolayer was affected most by the lowest dendrimer concentration . Additional experiments are in progress to evaluate the level of interaction between the different PAMAM generations and the skin barrier lipids . The study was supported by the Czech Science Foundation (Project No. 19-09600S) and from the project of Specific Academic Research (SVV 260 547). References 1. SUN, M ., FAN, A ., WANG, Z . et al.: Soft Matter, 8, 2012, 4301–4305 . 2. DAVE, K ., VENUGANTI, V .: Ther . Deliv ., 8, 2017, 1077–1096 . EFFECT OF POLYMER COMBINATIONS ON THE DISSOLUTION PROFILES OF A MODEL DRUG IN BIORELEVANT MEDIA OGADAH, C., VRANÍKOVÁ, B., MARUSHKA, J., ŠKLUBALOVÁ, Z. Department of Pharmaceutical Technology, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic e-mail: ogadahc@faf .cuni .cz Controlled release drug delivery systems are used for optimizing therapeutic efficacy, with the view of maximizing bioavailability and reducing side effects . The most widely used strategy for controlling the release of drugs involves the use of hydrophilic swellable polymers as the release-retardant material, in the form of matrix systems . In this work, the effect of combining two polymers for the purpose of tailoring the drug release characteristics of matrix systems was studied . Theophylline-loaded matrix tablets using guar gum, hydroxypropyl methylcellulose (HPMC) and their combinations in different ratios without any additional excipients (e.g., fillers, lubricants) were prepared by direct compression . Dissolution studies were performed for 24 hours in three biorelevant media – Fasted state Simulated Gastric, Intestinal and Colon Fluids (FaSSGF, FaSSIF and FaSSCoF) of pH 1 .6, 6 .5 and 7 .8, respectively . The results indicated desired sustained release profiles of all formulations with variation in drug release with respect to the proportion of each polymer . Moreover, it was observed that the pH-independent release may be achieved by combining the polymers . These formulations which are essentially pH-independent could lead to a more predictable dissolution profile, which is desirable in the treatment of ailments where there is a constant fluctuation in the GIT pH (e.g., inflammatory bowel diseases). It is expected that varying the ratios of used polymers would optimize the mucoadhesion potential as well . This aspect will be explored in future studies . 49 The study was supported by the Grant Agency of Charles University (Project No. 70119/2019) and from the project of Specific Academic Research (SVV 260 547). SELF-EMULSIFYING DRUG DELIVERY SYSTEMS ENABLE ORAL OLIGONUCLEOTIDE DELIVERY KUBAČKOVÁ, J.,1 HOLAS, O .,1 ZBYTOVSKÁ, J .,1 PÁVEK, P .,2 MULLERTZ, A .3 1 Department of Pharmaceutical Technology, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic 2 Depatment of Pharmacology and Toxicology, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic 3 Department of Pharmacy, University of Copenhagen, Copenhagen, Denmark e-mail: kubackja@faf .cuni .cz Oligonucleotide-based drugs represent a highly specific therapeutic approach. However, oral delivery of these drugs is hampered by their low stability and poor permeability . Therefore, we formulate an oligonucleotide (OND) into a well-established oral self-emulsifying drug delivery system (SEDDS) .1 Hydrophilic OND was effectively complexed by hydrophobic ion paring with a cationic lipid, either dimethyldioctadecylammonium bromide (DDAB) or 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) .2 Resulting hydrophobic complexes enabled loading into a lipid-based SEDDS containing intestinal permeation enhancers . Negatively charged Citrem SEDDS and neutral Standard SEDDS were tested in terms of OND protection against nucleases and permeability in vitro across intestinal Caco-2 cell monolayer . Negative surface charge of Citrem SEDDS was found to interfere with OND complexes and thus diminished its protective effect against nuclease to 16% . Simultaneously, negative charge hinders interactions with negative cell surface resulting in low permeation . Without negative surface charge, neutral Standard SEDDS protected 58% of OND . Due to enhanced interactions with cells, the permeation of OND in Standard SEDDS more than doubled . An appropriate in vivo model could shed more light on performance of these promising novel formulations . The study was supported by EFSA-CDN (Reg. 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 260 547). References 1 . MULLERTZ, A ., OGBONNA, A ., REN, S . et al.: J . Pharm . Pharmacol ., 62, 2010, 1622–1636 . 2. HAUPTSTEIN, S ., PRUFERT, F ., BERNKOP-SCHNURCH, A .: Int . J . Pharm ., 487, 2015, 25–31 . 50 SURFACE ENERGY ANALYSIS OF BINARY MELOXICAM POWDER MIXTURES USING INVERSE GAS CHROMATOGRAPHY BROKEŠOVÁ, J .,1 KOKTAN, J .,2 KUENTZ, M .,3 ŠKLUBALOVÁ, Z .1 1 Department of Pharmaceutical Technology, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic 2 Department of Research and Development, Zentiva, k . s ., Prague, Czech Republic 3 Institut for Pharma Technology, School of Life Sciences in Muttenz, University of Applied Sciences and Arts Northwestern Switzerland, Switzerland e-mail: brokesoj@faf .cuni .cz Nowadays, improving a dissolution rate of poorly water-soluble drugs is one of the major challenges for pharmaceutical research . The aim of this work was to analyze surface energy based on inverse gas chromatography to determine surface properties as a precondition for interactive powder mixtures formation . The interactive powder mixtures of meloxicam (MX, BCS class II) and chitosan (CH) were prepared in different ratios using physical mixing and co-milling . The dispersive and specific components of the surface energy were determined using non-polar (hexane, heptane, octane, nonane) and polar (dichloromethane, ethyl acetate, chloroform, toluene, ethanol, acetone, 1,4-dioxane) probes, respectively . The fractional sample surface coverage (5%) was constant and carrier gas (helium) with the flow rate of 10 mL min−1 was used . The results showed the higher dispersive γD = 46 .5 mJ m−2 and lower specific γSP = 2 .9 mJ m−2 component of MX having an acidic nature, while a lower dispersive γD = 40 .5 mJ m−2 and higher specific γSP = 4 .5 mJ m−2 component was detected for CH with a basic nature . The cohesion forces of the drug particles were in balance with drug-carrier adhesion forces (expressed as the wadh/wcoh ratio that was close to one) indicating the successful production of an interactive mixture and its stability . The total surface energy was found to increase after milling but a massive surface amorphization of drug was not observed, which is beneficial for drug stability. The study was supported by the Grant Agency of Charles University (Project No. 268120/2020), by the Pharmaceutical Applied Research Center (The PARC), Zentiva, k. s. and from the project of Specific Academic Research (SVV 260 547). MICROEMULSIONS FOR DERMAL DELIVERY OF IMIQUIMOD 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: panoutse@faf .cuni .cz 51 Imiquimod (IMQ) is a topically-applied imidazoquinoline used for the treatment of several skin diseases, like actinic keratosis and basal cell carcinoma .1 Traditional formulations restrict IMQ’s efficiency for dermal delivery because of the drug’s poor solubility and low cutaneous permeability .1 The aim of this work was the development and evaluation of an IMQ containing, skin-friendly microemulsion system intented for topical delivery . A pseudo-ternary phase diagram was constructed using a mixture of phospholipids in ethanol as a surfactant system and oleic acid as the oil phase . The physicochemical properties of selected formulations were determined in terms of particle size, conductivity, and rheology . The IMQ skin uptake was evaluated by ex vivo permeation experiment in fullthickness human skin . Two of the prepared formulations were able to deliver the active substance more efficiently than the commercial formulation. In addition, the barrier function of microemulsion treated skin recovered faster than that of the control, as was shown from transepidermal water loss experiments . Infrared spectroscopy showed the possible penetration of the microemulsion components into the skin at a molecular level . In conclusion, the presented findings demonstrate that phospholipid microemulsions could become a promising alternative in the topical administration of IMQ . The study was supported by the Czech Science Foundation (Project No. 19-09600S) and from the project of Specific Academic Research (SVV 260 547). References 1 . MA, M ., WANG, J ., GUO, F . et al .: J . Mater . Sci .: Mater . Med ., 26, 2015, art . 192 . PHARMACEUTICAL ANALYSIS AND BIOANALYTICAL CHEMISTRY SECTION OPEN DATABASE SEARCH FOR ILLUMINATING THE “DARK MATTER” OF BOTTOM-UP PROTEOMICS LENČO, J. Department of Analytical Chemistry, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic e-mail: lenco@faf .cuni .cz Despite the advancements in proteomic instrumentation, a significant portion of MS/ MS spectra from bottom-up analyses still evade identification, even though the spectra quality is sufficient. This “dark matter” of proteomics usually compromises biologically relevant components such as uncommon and rare posttranslational modifications, protein sequence polymorphisms, etc .1 A large fraction of unassigned MS/MS spectra is also a consequence of unexpected protein cleavage sites or peptide modifications during sample handling . We have recently scrutinized the adverse effects of the combination of elevated column temperature, extended in-column residence time, and low pH of the mobile phase 52 on tryptic peptides integrity during LC-MS proteomic analyses . To describe possible peptide modifications in an unbiased fashion, we took advantage of an open database search of MS/MS spectra followed by multivariate statistical analysis. In this way, we revealed that peptides bound to the stationary phase can undergo various artificial modifications. Moreover, we serendipically discovered that peptides can be artificially formylated before LC-MS analyses due to dissolving them in a solution containing a mere 0 .1% formic acid . We utilized the gained experience in the open-database search to clarify what changes to a model protein induces an irradiated phthalocyanine for photodynamic therapy . This 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 260 548). References 1. SKINNER, O . S ., KELLEHER, N . L .: Nat . Biotechnol ., 33, 2015, 717–718 . PERSPECTIVES OF USE OF PROPIONIC ACID TO INCREASE THE SENSITIVITY OF PROTEOMIC BOTTOM-UP LC-MS METHODS NAPLEKOV, D., JADEJA, S., LENČO, J., SKLENÁŘOVÁ, H. Department of Analytical Chemistry, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic e-mail: naplekod@faf .cuni .cz Proteomic LC-MS analyses can identify and quantify proteins and peptides and characterize their modifications, interactions between proteinous and other molecules, etc .1 Proteomic samples are usually of limited quantity, and they consist of an enormous number of analytes present in small concentrations below the level of appropriate MS detection by typical analytical flow LC-ESI-MS. Although nanocolumns with an inner diameter of 0 .075 mm have been successfully implemented to proteomics to introduce peptides via nano-electrospray ionization to a mass spectrometer with the right intensity, they still fail to provide sufficient sensitivity for the most demanding analyses, such as in single-cell proteomics . Currently, several strategies are considered to increase sensitivity in proteomic LC-MS analyses. The modification of the mobile phase composition represents the most straightforward one . We have recently demonstrated that replacing 0 .1% formic acid in the mobile phase by 1% propionic acid significantly increased peak intensities of five standard peptides. Thus, we trust that replacing 0 .1% formic acid in the mobile phase with an optimized concentration of weaker propionic acid will increase the MS sensitivity in real-life LC-MS proteomic analyses . This 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 260 548). 53 References 1. GUPTA, N ., TANNER, S ., JAITLY, N . et al.: Genome Res., 17, 2007, 1362‒1377. EVALUATING THE EFFECT OF LOWER FORMIC ACID CONCENTRATION IN MOBILE PHASE FOR PROTEMIC LC-MS ANALYSIS JADEJA, S., LENČO, J., SKLENÁŘOVÁ, H. Department of Analytical Chemistry, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic e-mail: jadejas@faf .cuni .cz Ideal peak shapes of peptides in proteomic LC-MS analyses have been traditionally obtained when using mobile phases of high ionic strength, but in turn, suppress electrospray ionization . Recent advancements in the stationary phases have introduced the Charged Surface Hybrid (CSH) technology .1 Reversed stationary phase with CSH technology produces peak shapes with a mobile phase of lower ionic strength equivalent to those when a higher ionic strength mobile phase is used . Under the scope of the study, we will evaluate the effect of reduced formic acid concentration in the mobile phase on peak symmetry, MS sensitivity, the extent of peptide identification, and the rate of artificial modification. The data on peak characteristics obtained from the advanced CSH column at the varied formic acid concentration (0 .1% to 0 .01%) were compared with an equivalent reversed-phase column, which is widely used to analyze proteomic samples . A well-characterized set of peptides with varied properties and charge states were included . Because of the surface charged property of the CSH column, much improved peak shapes were obtained at a lower concentration of formic acid when compared to the equivalent reversed-phase column . This 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 260 548). References 1. LAUBER, M . A ., KOZA, S . M ., McCALL, S . A . et al.: Anal. Chem. 9, 2013, 6936‒6944. HPLC-FLD/DAD DETERMINATION OF SALIVARY IMMUNE SYSTEM ACTIVATION MARKERS, URIC ACID AND CREATININE IN CLINICAL RESEARCH VERNEROVÁ, A .,1,2 KUJOVSKÁ KRČMOVÁ, L.1,2 1 Department of Analytical Chemistry, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic 2 Department of Clinical Biochemistry and Diagnostics, University Hospital Hradec Králové, Czech Republic e-mail: verneran@faf .cuni .cz 54 Neopterin, kynurenine and tryptophan are useful and important markers for monitoring the activation of the immune system that accompanies number of diseases such as infection, autoimmune and various malignant diseases . Uric acid (UA), the major urinary nitrogencontaining compound, is the product of purine metabolism in the human body and important antioxidant with albumin and ascorbate in saliva . The main advantages of saliva as a diagnostic biological material are simple, safe, and non-invasive collection . The new chromatographic method using monolithic stationary phase for determination of neopterin, kynurenine, tryptophan, creatinine, and UA in human saliva has been developed and will be presented . Neopterin and tryptophan were detected by fluorescence detection and creatinine, kynurenine, and UA by diode array detection . Filtration as a simple and suitable sample treatment procedure for human saliva was used. This novel HPLC-FLD/DAD method in combination with simple and fast sample preparation procedure was used for testing 56 real saliva samples from patients with cancer (breast, ovarian, colorectal, and renal cancer) and 14 saliva samples from patients with periodontal diseases to monitor early inflammatory response. The study was supported by Ministry of Health of the Czech Republic – conceptual development of research organization (UHHK, 00179906), by the Czech Health Research Council (Project. No. NV18-03-00130) and from the project of Specific Academic Research (SVV 260 548). References 1. NGAMCHUEA, K ., CHAISIWAMONGKHOL, K ., BATCHELOR-McAULEY, C . et al .: The Analyst, 143, 2018, 81‒99. DETERMINATION OF PLATINUM DRUGS IN HUMAN PLASMA AFTER HYPERTHERMIC INTRAPERITONEAL CHEMOTHERAPY TUROŇOVÁ, D.,1,2 KUJOVSKÁ KRČMOVÁ, L.1,2 1 Department of Analytical Chemistry, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic 2 Department of Clinical Biochemistry and Diagnostics, University Hospital Hradec Králové, Czech Republic e-mail: turonovd@faf .cuni .cz Cytoreductive surgery combined with hyperthermic intraperitoneal chemotherapy (HIPEC) is a therapeutic method that maximally removes the tumour followed by intraoperative administration of a warmed chemotherapeutic agent . The approach aims are to maximize the anticancer effect and lower chemotherapy’s systemic side effects . Intraperitoneal application of chemotherapeutics leads to their decreased levels in the bloodstream .1 Therefore we need to develop more sensitive methods, which allow us to detect the lower concentration to monitor therapeutic levels and toxicity . An HPLC-PDA method for the determination and quantification of cisplatin or oxaliplatin in the human plasma will be presented . The aim of the method development was to achieve a simple, fast, and sensitive method that could be applied in standardly equiped routine laboratories . As the derivatization agent diethyldithiocarbamate (DDTC) was used . 55 The separation was carried out in 4 .5 minutes using the C18 core-shell column (100 × 4 .6 mm, 2 .7 µm) combined with F5 column guard . The mobile phase consisted of acetonitrile and water, and the target analyte was detected at 254 nm . The method is compensated by application of palladium chloride as the internal standard. The lower limit of quantification for platinum complexes was 20 ng mL−1 . The study was supported by Ministry of Health of the Czech Republic – conceptual development of research organization (UHHK, 00179906), by the Czech Health Research Council (Project. No. NV18-03-00130) and from the project of Specific Academic Research (SVV 260 548). References 1. WITKAMP, A . J ., De BREE, E ., Van GOETHEM, A . R . et al.: Cancer Treat. Rev., 27, 2001, 365‒374. DEVELOPMENT OF UHPLC METHOD FOR ANALYSIS OF VITAMIN K IN SERUM MRŠTNÁ, 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 Hradec Králové, Czech Republic e-mail: arnoltok@faf .cuni .cz Determination of vitamin K, which plays an important role in the human organism, is a challenge in many aspects of bioanalysis . Although vitamin K is not currently routinely analysed, information on its concentration in the human body is crucial, mainly in terms of possible manifestations related to its deficiency. In the analysis of this vitamin, it is a common problem to detect its low concentrations . It is present only in nanomolar concentrations in human biofluids. In addition to the low concentration, there are also obstacles such as its high photosensitivity, the matrix interference, and its adhesion to plastic and glass surfaces . The use of appropriate sample preparation in combination with chromatographic analysis is a solution for enabling the determination of particular forms of vitamin K .1 The presented UHPLC method using fluorescence detection was developed for determination of four vitamin K homologues (K1, MK4, MK7, MK9) in serum . The chromatographic separation was achieved on KinetexTM C18 column (1.7 μm, 100 × 3 mm) using gradient elution with methanol/propanol (phase B) and H2O (phase A). The flow rate was 0 .5 mL min−1 within a total run time of 13 min . Current results of the method development will be presented . The study was supported by Ministry of Health of the Czech Republic – conceptual development of research organization (UHHK, 00179906) and from the project of Specific Academic Research (SVV 260 548). References 1. SIMES, D . C ., VIEGAS, C . S . B ., ARAÚJO, N . et al .: Nutrients, 12, 2020, art . 138 . 56 OPTIMIZATION OF CAPILLARY ELECTROPHORESIS AND SUPERCRITICAL FLUID CHROMATOGRAPHY METHODS FOR THE SEPARATION OF SILYMARIN RIASOVÁ, P .,1,2 JÁČ, P.,1 POLÁŠEK, M .,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: riasovpe@faf .cuni .cz Silybum marianum has been used since ancient times for the treatment of a variety of disorders . The main active constituent of the plant is silymarin, a mixture of the structurally similar flavonolignans silybin A and B, isosilybin A and B, silychristin, silydianin, and the flavonoid taxifolin. The method optimization processes for the silymarin separation using capillary electrophoresis (CE) and supercritical fluid chromatography (SFC) are covered in this study . The CE method was optimized in terms of concentration and pH of borate buffer, type and concentration of cyclodextrin, organic modifier content, and capillary length. Base-line separation of all flavonolignans was achieved with a background electrolyte containing 100 mM boric acid of pH 9 .0, 5 mM heptakis(2,3,6-tri-O-methyl)-β-cyclodextrin and 10% (v/v) MeOH in a 80.5/72 cm (50 µm id) fused silica capillary, at an applied voltage of 25 kV with UV detection at 220 and 320 nm . The SFC method was optimized on two coupled-column systems . Experimental conditions optimized during SFC method development were modifier type and content, flow rate, additives type and concentration, backpressure, column temperature and sample solvent . Baseline separation of all compounds was not achieved . The best performing method was on a Lux Amylose-1 + Lux Cellulose-3 system, with the following chromatographic conditions: 40% MeOH with 0.1% (v/v) trifluoroacetic acid, flow rate 2.8 mL min−1 backpressure 125 bar and column temperature 30 °C . The resolution of the critical peak pairs silychristin and silydianin, and isosilybin B and A was 0 .85 and 0 .70, respectively . The study was supported by the Mobility Fund of Charles University and from the project of Specific Academic Research (SVV 260 548). NEW GRAPHENE BASED SORBENT FOR SOLID PHASE EXTRACTION LOCHMAN, L., KUČERA, R. Department of Pharmaceutical Chemistry and Pharmaceutical Analysis, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic e-mail: lochmanl@faf .cuni .cz Graphene (G) is two-dimensional sp2 single-atom-thick carbon sheet with hexagonal structure. High specific area (theoretical value 2630 m2 g−1) and the affinity to carbon 57 ring structures via π-π stacking interactions make G and graphene oxide (GO) promising candidates for application in analytical chemistry .1 Several studies used GO or G for preparation of the new sorbents for SPE or SPME, and materials with improved properties were presented .2‒4 The aim of our project is to try to prepare a new alternative sorbent based on graphene . The work is focused on the modification of the titanium oxide particles (ZirChrom-SAX) by graphene . That will be followed by the study of sorbent retention on the mixture of model analytes (e.g ., ibuprofen, lidocaine, propylparaben, metoprolol) with different acidbase properties. Such kind of material is presented for the first time. Obtained results will be compared with the properties of unmodified particles and GO or G in free form . The quality of particle coating together with retention performance will be discussed . The study was supported by the Research programme Development and Study of Drugs (Progres Q42) and from the project of Specific Academic Research (SVV 260 547). References 1. RAO, C . N . R ., SOOD, A . K . (eds .): Graphene: Synthesis, Properties, and Phenomena . Weinheim, Wiley-VCH, 2013 . 2. LIU, Q ., SHI, J ., SUN, J . et al.: Angew. Chem., 50, 2011, 5913‒5917. 3. HUANG, K .-J ., JING, Q .-S ., WEI, C .-Y . et al .: Spectrochim . Acta, Part A: Molecular and Biomolecular Spectroscopy, 79, 2011, 1860‒1865. 4. LI, N ., CHEN, J ., SHI, Y .-P.: Talanta, 191, 2019, 526‒534. DIRECT ELECTROMEMBRANE EXTRACTION OF ANTHRACYCLINES FROM TISSUE SAMPLES BAVLOVIČ PISKÁČKOVÁ, H.,1 KOLLÁROVÁ-BRÁZDOVÁ, P .,2 ŠTĚRBA, M.,2 KUČERA, R.,1 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, Czech Republic e-mail: piskacha@faf .cuni .cz Tissue analysis presents bioanalytical challenge compared to analysis of liquid biological samples . Firstly, homogenization prior sample treatment is required to ensure both representative sample and effective sample extraction. Further difficulty is determination of analyte extractability, as the spiked tissue sample or even spiked homogenate do not exactly mimic drug binding and distribution in intact tissue . Electromembrane extraction (EME) is a hybrid microextraction technique laying between liquid-liquid extraction (LLE) and electrophoresis . The extraction of charged analytes is performed from the aqueous sample through the water immiscible organic supported liquid membrane (SLM) to the acceptor solution . The driving force of the extraction is an electrical potential, which is applied across the SLM . The aim of this study was to optimize the EME for isolation of anthracyclines (ANT) from tissues (liver, heart and skeletal mus- 64 COMPARISON OF IMPURITY PROFILES IN LEVOTHYROXINE TABLETS USING METABOLOMICS BASED UHPLC-HRMS METHOD CATAPANO, M . C .,1 HRUŠKOVÁ, A .,1 GARRIGUES, J . C .,2, NOVÁKOVÁ, L .1 1 Department of Analytical Chemistry, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic 2 Laboratoire I .M .R .C .P, Université Toulouse III-Paul Sabatier, Toulouse, France e-mail: catapanm@faf .cuni .cz The aim of this study was to identify impurities in several formulations of tablets containing levothyroxine using an UHPLC-HRMS metabolomics-based method and to compare the profiles of known and unknown impurities. These impurities may be related to the reported side-effects of levothyroxine tablets . In particular, the method was focused on (1) the identification of known and unknown impurities (molecular or supramolecular) from the active principle ingredient (API) and from the excipients, such as mannitol, lactose, and stearic acid, (2) to the study of molecular interactions between API and the excipients . An important part of this study was the comparison of different metabolomics workflows including experimental design, data analysis, feature detection (peak picking), statistical evaluation, and compound identification. Analysis of levothyroxine samples underwent randomization process in order to prevent technical/instrumental biases. Three different experimental designs were compared in terms of sample preparation and UHPLC-HRMS analysis . Data processing involved testing of different settings (mass accuracy, thresholds) as well as testing different software platforms . The data were acquired in MS full scan mode, both ESI negative and positive mode, and will be used in both targeted and non-targeted data processing . In targeted analysis we confirmed the presence of mannitol stearate in levothyroxine drug formulations containing mannitol as an auxiliary ingredient . It was also possible to identify some impurities, for example levothyroxine-lactose adducts (MW = 1100 .7923) . On the other hand, the presence of supramolecular levothyroxine-mannitol complex and levothyroxine-mannitol adduct was not confirmed in formulations. In the next step, more unknown impurities will be identified, and non-targeted metabolomics will be applied. The study was supported by EFSA-CDN (Reg. 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 260 548). EXTRACTION OF PHENOLIC COMPOUNDS FROM EUCALYPTUS LEAVES USING SUPERCRITICAL FLUID EXTRACTION COUPLED WITH LIQUID CHROMATOGRAPHY/TANDEM MASS SPECTROMETRY HASHEMI, B., PILAŘOVÁ, V., NOVÁKOVÁ, L. Department of Analytical Chemistry, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic e-mail: hashemib@faf .cuni .cz 65 Supercritical fluid is a viable alternative extraction solvent for bioactive compounds from their natural sources such as plants, fruits, and flowers. Carbon dioxide is commonly used as supercritical solvent to extract a wide range of non-polar to mid polar compounds . Different parts of eucalyptus tree including leaves, bark, and stems contain both volatile and non-volatile bioactive compounds that have been widely utilized in pharmaceutical and cosmetic industries. In the first part of the present work, supercritical fluid extraction (SFE) using ethanol as the co-solvent is used for the extraction of different phenolic compounds including phenolic acids and flavonoids from eucalyptus leaves followed by the analysis of the extracted analytes using ultra-high performance liquid chromatography/ tandem mass spectrometry (UHPLC-MS/MS). In the proposed method, the parameters associated with the UHPLC-MS/MS were investigated and optimized first and then factors affecting SFE process will be assessed as well . The electrospray ionization (ESI) parameters were optimized in both ionization modes. MS/MS was performed on triple quadrupole and selected reaction monitoring (SRM) was developed for quantitative analysis of the target phenolics . The calibration curves were prepared to check the linear concentration range and sensitivity. The obtained SFE extract was analyzed using the developed UHPLC-MS/ MS and different phenolic acids (such as protocatechuic acid, gallic acid, caffeic acid, and chlorogenic acid) and flavonoids (including catechin, taxifolin, phloridzin, and quercitrin) were identified among the extracted compounds from eucalyptus leaves. In the second stage, we plan to develop supercritical fluid chromatography tandem mass spectrometry (SFC-MS/MS) method which would include the analysis of both volatile and phenolic compounds extracted from eucalyptus leaves . The study was supported by EFSA-CDN (Reg. 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 260 548). THE DEVELOPMENT OF EXTRACTION METHOD USING SUPERCRITICAL FLUIDS HOLLÁ, M., KHOLOVÁ, A., SKLENÁŘOVÁ, H., ŠATÍNSKÝ, D. Department of Analytical Chemistry, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic e-mail: hollama@faf .cuni .cz Recently, growing interest in using green technologies for the extraction of valuable compounds with antioxidant properties has been observed . Extractions utilizing carbon dioxide belong to this group . By applying pressure and mild temperature above the critical point of liquid CO2 or a mixture of CO2 and more polar co-solvent (ethanol, methanol, water), a supercritical fluid is formed. Supercritical fluids are characterized by higher diffusivity, lower density, viscosity and surface tension . This approach enables the extraction of plant polyphenols under mild conditions and without the use of toxic solvents . Moreover, extractions are performed in the absence of light and air during extraction . Thus, the risk of degradation reactions is minimized . 66 In this ongoing study, we aim to develop an extraction method for the extraction of polyphenols from dried apples . Since most of the polyphenols typically present in apples are polar, polar co-solvent is needed . For the evaluation of selected analytes (gallic acid, chlorogenic acid, epicatechin, rutin, phloridzin, phloretin, quercetin, quercitrin, catechin, caffeic acid), the newly developed UHPLC-DAD method was used . In this contribution, the influence of the main tested parameters on the extraction recovery will be discussed . The optimization of SFE was divided into two main parts . The Modde software was used for the preparation of the experimental design . First, Plackett Burman design was used to identify the most significant factors (pressure, temperature, water content in ethanolic co-solvent, CO2/co-solvent ratio). Second, the central composite design was applied as the second step . At this point, a narrower range of main factors was further optimized. The fixed values of minor factors were chosen regarding the highest recoveries of target analytes. The study will continue with extraction kinetics and the influence of glass beads size on extraction recovery . The study was supported by the Grant Agency of Charles University (Project No. 1466119) and from the project of Specific Academic Research (SVV 260 548). SEPARATION POTENTIAL OF CHROMATOGRAPHIC METHODS IN THE ANALYSIS OF STRUCTURALLY SIMILAR STEROIDS GAZÁRKOVÁ, T., HROMÁDKO, J., KOČOVÁ VLČKOVÁ, H., NOVÁKOVÁ, L. Department of Analytical Chemistry, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic e-mail: gazarkota@faf .cuni .cz The full chromatographic separation of a complex group of isomeric and isobaric steroids remains a complicated analytical challenge, even though GC-MS/MS and LC-MS/ MS techniques are well established in their analysis . The analyzed compounds differ only by minor structural modifications. Moreover, a typical loss of one to three water molecules in the MS spectra leads to the formation of additional isobars . To eliminate the observed interferences and correctly quantify the target analytes, it is necessary to achieve full chromatographic separation . The present study aims at the development and optimization of fast and sensitive UHPLC-MS/MS and UHPSFC-MS/MS methods for the analysis of 37 steroids. The analyzed compounds belong to the C19 androstenes, C21 pregnanes, and synthetic steroid groups and generate 11 critical pairs/groups of analytes due to their structural similarity. Therefore, the separation potential of both chromatographic methods will be examined in detail and compared . Comprehensive screening of stationary phases was carried out using generic gradient elution in both chromatographic methods, UHPSFC and UHPLC . A screening of mobile phases and gradient optimization followed in the next step using the stationary phases with the most successful separation of critical pairs . Biological samples of mouse plasma were obtained from the Czech Academy of Sciences, dealing with the project focused on 67 the investigation of changes in levels of expressed steroids due to chronic stress exposure . Hence, the protein precipitation method was developed for a target matrix . Moreover, the physiological levels of some steroids in the plasma make a quantification method even more challenging. Finally, the PP-UHPLC-MS/MS method was selected for validation and the analysis of biological samples . This 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 260 548). AUTOMATED PROCEDURES BASED ON HOMOGENEOUS LIQUID-LIQUID EXTRACTION AND PROTEIN PRECIPITATION USING LAB-IN-SYRINGE APPROACH FIKAROVÁ, K., HORSTKOTTE, B., MACHIÁN, D., SKLENÁŘOVÁ, H. Department of Analytical Chemistry, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic e-mail: [email protected] We report on the development of two new Lab-In-Syringe (LIS)-automated salting-out liquid-liquid extraction (SALLE) methods, one in combination with online solid-phase extraction (SPE), coupled to liquid chromatography (LC) . In both cases, SALLE was carried out in the void of the syringe including a magnetic stirring bar for on-demand homogenous mixing and using acetonitrile as an extraction solvent miscible with water . A saturated salt solution of magnesium sulfate and sodium chloride was used to induce phase separation . This methodology is suitable for the extraciton of moderately polar compounds, in our work, sulfonamide chemotherapeutics . The advantage of the procedure is the compatibility of the used solvent with LC . On the other hand, only small preconcentration factor can be achieved as phase separation can be achieved for approximately balanced volumes of organic and aqeuous phase . To improve both preconcentration factor and extract clean-up, in the first work, the extract was diluted in-syringe with alkaline buffer and the analytes were trapped at pH 10 on an anion-exchange resin cartridge integrated into the LC injection loop, thus achieving a double-stage and orthogonal sample clean-up . Analytes were eluted from the cartridge by the acidic mobile phase in gradient elution mode . Running the separation of the analytes and the two-step preparation of the following sample in parallel allowed reducing the total time of analysis to 13 .5 min . The method was applied to spiked urine samples yielding an average recovery value of 102 .7 ± 7 .4% and limits of detection at low ppb level . A similar procedure can be used for centrifugation-less deproteination of milk samples . In this case, acetonitrile also acts as a precipitating agent . The addition of salt then separates three phases: an organic extract with preconcentrated analyte, precipitated proteins, and the aqueous sample and salt solution mixture . First optimization results from this study will be presented . 68 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 260 548). MAGNETIZATION OF COMMERCIAL HLB PARTICLES FOR AUTOMATIC IN-SYRINGE DISPERSIVE MICRO-SOLID PHASE EXTRACTION OF SURFACE WATER CONTAMINANT GEMUH, C . V ., HORSTKOTTE, B ., SOLICH, P . Department of Analytical Chemistry, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic e-mail: gemuhc@faf .cuni .cz We present a simple approach to magnetic dispersive solid phase microextraction and its automation for the enrichment of water contaminants . SupelTM-Select HLB (hydrophilic modified styrene polymer) beads were magnetized by introducing magnetite nanoparticles (Fe3O4) into the sorbent . For this, the beads were soaked with FeCl2 and FeCl3 and then washed with NH4OH leading to the precipitation of magnetite nanoparticles inside the sorbent as reported elsewhere .1 The so-functionalized sorbent was used in a dispersive solid phase extraction methodology that was automated using the Lab-In-Syringe technique . The methodology followed a previous study on using magnetic nanoparticles .2 In short, sample and bead suspension are aspirated into the syringe void and dispersed by the aid of a magnetic stirring bar placed inside the syringe . By automatic activation and deactivation of magnetic stirring process, the analytes were captured by the dispersed sorbent beads and then retained on the stirring bar that allowed discarding the sample solution after the phase separation. Thereafter, the eluent of 60 : 40 (v/v) acetonitrile : ammonium phosphate buffer was used to elute the analytes of interest . This extraction system was coupled online to a liquid chromatography instrument for the determination of model analytes mebendazole, bisphenol A, benzyl 4-hydroxybenzoate, diclofenac and irgasan . Essential parameters such as extraction time, elution time, composition and volume of eluent, as well as phase separation time were optimized . The developed method will be implemented for the analysis of the model contaminants in surface water (lakes and rivers) . The study was supported by the Grant Agency of Charles University (Project No. 1070120) and from the project of Specific Academic Research (SVV 260 548). References 1. FRESCO-CALA, B ., CÁRDENAS, S ., VALCÁRCEL, M .: J . Chromatogr . A, 1468, 2016, 55–63 . 2. MAYA, F ., CABELLO, C . P ., ESTELA, J . M . et al .: Anal . Chem ., 87, 2015, 7545–7549 . 69 WHAT WE CAN AND CANNOT EXPECT FROM EXTRACTION ON NANOFIBROUS SORBENTS? RAABOVÁ, H .,1 HÁKOVÁ, M .,1 ERBEN, J .,2 ŠATÍNSKÝ, D.1 1 Department of Analytical Chemistry, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic 2 Department of Nonwovens and Nanofibrous Materials, Faculty of Textile Engineering, Technical Univesity of Liberec, Czech Republic e-mail: raabovah@faf .cuni .cz Great interest in innovative sorption materials for solid-phase extraction has brought nanofibrous polymers to the scientific scope. Our research group’s knowledge about nanofibrous polymers gained over years, resulted in the study where the nanofibrous extraction mechanism is discussed . The eight polymers were tested as extraction sorbents for acetylsalicylic acid, moxonidine, metoprolol, propranolol, propafenone, diltiazem, atorvastatin, and amiodarone present in 3 matrixes, including an organic solvent, human serum, and bovine serum albumin . The extraction was carried out in an extraction cartridge manually filled with nanofibers and online coupled with a high-performance liquid chromatography system via a six-port switching valve . The obtained extraction recoveries were used for the evaluation of analyte retention on individual sorbents . The main aim of this study was to determine how this retention is affected by analyte physicochemical properties and matrix complexity . This understanding should lead to a more conscious application of nanofibrous sorbents in extraction techniques and simplified method development . As we found out, the tested polymers provided high extraction efficiency for the compounds with log P exceeding 2. It was also confirmed that lipophilicity is the major driving force in the retention mechanism . Moreover, by direct injection of spiked bovine serum albumin and human serum matrix on nanofibers, some of them confirmed their potential to extract analyte and remove protein macromolecules in one step . 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, by the Czech Science Foundation (Project No. 20-19297S), by the Grant Agency of Charles University (Project No. 766218) and from the project of Specific Academic Research (SVV 260 548). MODERN SORBENTS IN ONLINE SOLID PHASE EXTRACTION COUPLED TO LIQUID CHROMATOGRAPHY FOR DETERMINATION OF BISPHENOLS IN MILK KHOLOVÁ, A., RAABOVÁ, H., LHOTSKÁ, I., ŠATÍNSKÝ, D. Department of Analytical Chemistry, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic e-mail: kholovaa@faf .cuni .cz 70 Various advanced sorbents for online extraction and determination of bisphenol A, bisphenol AF, bisphenol C, bisphenol A diglycidyl ether, and bisphenol F diglycidyl ether in bovine milk samples have been compared . The milk is a complex matrix containing proteins and lipids . Our approach to sample preparation presents a new online method including fast extraction using precolumn coupled to liquid chromatography with fluorescence detection. Five types of fibrous sorbents, including polyethylene microfibers, polypropylene microfibers, polycaprolactone microfibers/nanofibers composite, polycaprolactone microfibers/polyvinylidene difluoride nanofibers composite, and polyamide 6, were compared in terms of extraction and clean-up efficiency with commercially available molecularly imprinted polymers for bisphenols and restricted access media sorbent RP-18 ADS. The polymer fibers filled in a cartridge and also commercial sorbents were directly connected to the HPLC system and the clean-up step and the subsequent chromatography separation optimized . The separation was carried out using analytical column YMC-Triart C18 ExRS (150 × 4.6 mm, particle size 3 µm) followed by fluorescence detection (Ex 273 nm, Em 300 nm) . Solvents suitable for separation were acetonitrile with water under gradient elution, for extraction 15% methanol, and the total flow rate of 1 .0 mL min−1 was used . The sorbents with the best results were used for control of bisphenol contamination in milk packed in plastic bottles . The measured results were compared with the migration limit established by the European Union for BPA 0 .05 mg kg−1 in food contact plastics . The study was supported by the STARSS project (Reg. No. Z.02.1.01/0.0/0.0/15_003/ 0000465) co-funded by ERDF, by the Czech Science Foundation (Project No. 20-19297S), by the Grant Agency of Charles University (Project No. 1134119) and from the project of Specific Academic Research (SVV 260 548). DETERMINATION OF PHENOLIC PROFILE IN FRUIT TREES DURING VEGETATION PERIOD ADAMCOVÁ, A., ŠÍROVÁ, K., ŠILHAVÁ, K., ŠATÍNSKÝ, D. Department of Analytical Chemistry, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic e-mail: adamcoa1@faf .cuni .cz The aim of the study was to determine a spectrum of phenolic compounds and their content in apple and pear tree material – leaves, bark, buds, blossom, and fruit . The methanol extracts were obtained from raw material of different cultivars . The main extracted phenolic compounds from apple (phloridzin, phloretin, chlorogenic acid, and quercetin) and pear (arbutin, rutin, chlorogenic acid and its derivatives) tree material were analyzed by two different validated high performance liquid chromatography methods . Finally, YMC-Triart C18 ExRS 150 × 4.6 mm, particle size 5 μm, pore size 8 nm (apple tree) and ASCENTIS Express RP-Amide 150 × 4.6 mm, particle size 2.7 μm (pear tree) analytical columns were used for analysis . Column temperature was 30 °C and injection volume was 1 μl. The separation was performed with gradient elution at flow rate 1 mL min−1 . 71 The mobile phase consisted of acetonitrile and 0 .1% phosphoric acid . The detection was carried out with diode array detector. To observe changing phenolic profile, sampling was performed in March, June, August, and November during the years 2019 and 2020 . The highest concentration of bioactive compounds was found in leaves followed by buds in spring season in both types of studied trees . In that season, the concentration range was from 164 .25 mg g−1 to 228 .85 mg g−1 in 10 apple tree cultivars and from 85 .04 mg g−1 to 161 .69 mg g−1 in 10 pear tree cultivars . The main phenolic compounds were phloridzin in apple trees, and arbutin and chlorogenic acid in pear trees. This finding can lead to applying fruit tree material as a renewable resources for food supplements or extracts with beneficial effect to human health. The study was supported by the Technology Agency of the Czech Republic (Project No. TJ02000196), by the Grant Agency of Charles University (Project No. 1152120) and from the project of Specific Academic Research (SVV 260 548). ADVANCED CHROMATOGRAPHIC APPROACH IN PHENOLIC COMPOUNDS PROFILING IN ARCHIVE TOKAJ WINES MORAVCOVÁ, P .,1 ŠPÁNIK, I .,2 MACHYŇÁKOVÁ, A.,2 ŠATÍNSKÝ, D.1 1 Department of Analytical Chemistry, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic 2 The Institute of Analytical Chemistry, Faculty of Chemical and Food Technology, Slovak University of Technology in Bratislava, Slovakia e-mail: moravcopa@faf .cuni .cz The presented experiment is focused on the characteristics of archive Tokaj wines, especially in terms of their benefits to human body in the form of antioxidant activity of phenolic compounds .1 The samples of archive Tokaj wine come from vineyards of the Slovak part of the Tokaj wine region . The Tokaj wine region is one of the few areas with a production of special wine made from grapes affected by noble rot Botrytis cinerea under particular environmental conditions, which leads to the production of natural wines with a unique aroma .2 More than 60 archive samples were evaluated in term of phenolic substances profile, including hydroxybenzoic and hydroxycinnamic acids, stilbenes and flavan-3-ols. Eighteen phenolic compounds were identified. Individual phenolic substances, which differ significantly in their physico-chemical properties, were detected by ultra high performance liquid chromatography method with diode array detection . Separation and quantification were finally conducted on a polar C18 column with core-shell particles (150 mm × 3.0 mm, particle size 2.6 µm) using a gradient elution mode at a flow rate of 1 .0 mL min‒1 with a mobile phase consisting of acetonitrile and 0 .1% phosphoric acid at 50 °C . These separation conditions provided reliable validation result with linearity (R > 0 .9996), precision (CV < 3 .91%), recovery (87 .35–118 .67%), which are considered acceptable for application in the characterization of these types of matrices . The study was supported from the project of Specific Academic Research (SVV 260 548). 72 References 1. STAŠKO, A ., POLOVKA, M ., BREZOVÁ, V . et al .: Food Chem ., 96, 2006, 185–196 . 2. MAGYAR, I .: Adv . Food Nutr . Res ., 63, 2011, 147–206 . A VALIDATED UHPLC METHOD FOR THE DETERMINATION OF CAFFEOYLQUINIC AND DI-CAFFEOYLQUINIC ACIDS IN GREEN COFFEE EXTRACTS USING AN RP-AMIDE FUSED-CORE COLUMN MAJOROVÁ, M., FIBIGR, J., ŠATÍNSKÝ, D. Department of Analytical Chemistry, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic e-mail: majorovmic@faf .cuni .cz The aim of the study was to develop and validate new UHPLC method for determination of chlorogenic acids and their di-substituted derivates in nutraceuticals containing the extract of green coffee beans . The developed and validated UHPLC method was used for determination of chlorogenic acids in nutraceuticals Kilostop (Astina Pharm, a .s .), Zelená káva Extra (Medicura Natural), Maxivitalis Zelená káva (Simply You Pharmaceuticals), Vieste Zelená káva Premium (Volt Retail), Zelená káva bylinný extrakt (Topvet), Zelená káva (Vito Life), and Kyselina chlorogenová (Vito Life) . For the analysis, extracts were obtained from the samples of nutraceuticals, using methanol and 5% aqueous formic acid solution (25 : 75, v/v). Extracts were filtrated through 0.22 µl PTFE filters. The analysis, which provided satisfying separation of all derivatives of chlorogenic acid, was performed on the Ascentis Express® RP-Amide (100 × 2.1 mm, particle size 2.7 μm) chromatography column using gradient elution program with mobile phase consisted of mixture of acetonitrile and 5% aqueous solution of formic acid. The separation was performed at flow rate of 0 .9 mL min‒1 and the detection was carried out at wavelength of 325 nm using PDA detector . The column temperature was 30 °C . According to the study, in green coffee bean extract samples, chlorogenic acid was found as major component . This research revealed that green coffee bean extracts are rich source of chlorogenic acids . However, the quality of the tested preparations in term of chlorogenic acid content widely differed according to the producer . The study was supported from the project of Specific Academic Research (SVV 260 548). 73 BIOCHEMISTRY, PHARMACOLOGY AND TOXICOLOGY SECTION A PREDICTIVE CAPABILITY OF 3D PRIMARY HUMAN HEPATOCYTE SPHEROIDS IN DRUG-DRUG INTERACTIONS SMUTNÝ, T ., PÁVEK, P . Department of Pharmacology and Toxicology, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic e-mail: smutt6aa@faf .cuni .cz Drug-drug interactions (DDIs) represent a serious medical concern being associated with a treatment failure or toxicity . CYP3A4 is among the most important drug-metabolizing enzymes (DMEs) as it is involved in biotransformation of approximately 50% of all marketed drugs . Addressing a capacity of drug candidates to induce CYP3A4 function in drug development is a prerequisite of therapeutic success and patient safety . Primary human hepatocytes (PHHs) cultured in 2D monolayer are a gold standard to perform CYP induction studies recommended by regulatory agencies . PHHs, however, undergo dramatic dedifferentiation after seeding losing significantly their metabolic capacity. In contrast, 3D spheroids of PHHs generated on ultra-low adherent plates have been shown to closely mimic physiological phenotype of human liver tissue . Moreover, 3D model can maintain stable expression of DMEs for a few weeks enabling to study a long-term effect of drugs on DME expression pattern . I my talk, I am going to address a role of 3D spheroids in a screening of CYP3A4 inducers putting emphasize on benefits of 3D PHH system such as a high and stable basal expression of CYP3A4 . The limits of in vitro-to-in vivo translation accuracy of CYP3A4 induction in 2D compared to 3D PHH model will be demonstrated on a case of AZD1208 drug candidate . Our in-house experience with 3D spheroids will be also discussed . The study was supported by EFSA-CDN (Reg. 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 260 549). MURINE CONSTITUTIVE ANDROSTANE RECEPTOR (CAR) LIGAND TCPOBOP INDUCED HEPATOMEGALY IN HUMANIZED CAR MICE IS INDEPENDENT OF CAR ACTIVATION ŠKODA, J ., DUŠEK, J ., PÁVEK, P . Department of Pharmacology and Toxicology, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic e-mail: skodajo@faf .cuni .cz TCPOBOP – 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene – is a widely-used prototype constitutive androstane receptor (CAR, Nr1i3) ligand . In mice, TCPOBOP after CAR ac- 80 Photodynamic therapy is a highly specific and clinically approved therapeutic procedure for cancer treatment based on the production of cytotoxic reactive oxygen species upon the light activation of an otherwise nontoxic photosensitizer . For this project, the novel peripherally crowded cationic phthalocyanines (Pcs) containing 8 or 16 pyridyl moieties (neutral or quaternized) were synthesized and their photodynamic properties were further evaluated in detail . Photodynamic activity was demonstrated on several cell lines – malignant (HeLa and MCF-7) and non-malignant (3T3 and EA .hy926) . The results of individual in vitro experiments have shown very high phototoxicity with EC50 up to 47 nM (MCF-7 cells) after irradiation while maintaining desirably low inherent toxicity (in the dark), with TC50 > 600 000 nM . The Pcs were localized intracellularly, primarily in the lysosomes . This led to their membrane rupture after activation of Pcs and induced apoptosis with subsequent secondary necrosis. This fact was confirmed by real-time monitoring of Annexin V binding and the loss of cell membrane integrity . In conclusion, this work has demonstrated that a bulky and rigid arrangement of peripheral cationic substituents is very efficient for providing good photophysical properties and high photodynamic activity in the development of novel photosensitizes . The study was supported by the Czech Science Foundation (Project No. 19-14758Y), by the Charles University Project PRIMUS 20/SCI/013, by the Grant Agency of Charles University (Project No. 1620219) and from the project of Specific Academic Research (SVV 260 550). TOPOISOMERASE II INHIBITORS AND PREVENTION OF ANTRACYCLINE TOXICITY ON CARDIOMYOCYTES KUBEŠ, J .,1 JANSOVÁ, H .,1 KARABANOVICH, G .,2 MELNIKOVÁ, I .,2 JIRKOVSKÁ, A .,1 SKALICKÁ, V .,1 APPLOVÁ, L .,1 ROH, J .,2 ŠIMŮNEK, T.1 1 Department of Biochemical Sciences, 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: kubesja1@faf .cuni .cz Anthracyclines (ANTs) remain indispensable in many cancer treatments . Use of all drugs of this class, however, is limited due to risk of severe cardiotoxicity . To date, dexrazoxane (ICRF-187) is still the only cardioprotective agent approved for clinical use . This compound, therefore, represents the main lead in the search for effective cardioprotective agents . The focus regarding its cardioprotective mechanism has shifted from metal chelation to topoisomerase II (TOP2) modulation, which is the aim of this work . To gain more complex insight into the underlying mechanism, diverse commercially available compounds described in literature as TOP2 inhibitors were screened on primary cultures of neonatal rat cardiomyocytes for ability to prevent damage induced by daunorubicin . Additionally, since there are no data of the compounds being assessed for TOP2 81 inhibition side by side under the same conditions and toward both TOP2 isoforms, the inhibitors of interest were also assayed for their respective inhibitory potency towards both TOP2 isoforms using decatenation assay . Since other aim of this work is also a search for potential lead cardioprotective agent(s), novel analogues of most promising inhibitors were also studied . The combined results support the notion that TOP2 is involved in the development of ANT-induced cardiotoxicity but the whole situation appears to be more complex as there is not a simple and direct correlation between inhibitory potency and cardioprotection . More detailed investigation of modes of TOP2 inhibition seems necessary to properly answer this question . This study was supported by the Grant Agency of Charles University (Project No. 246219), InoMed (Project No. CZ.02.1.01/0.0/0.0/18_069/0010046) co-funded by the European Union and from the project of Specific Academic Research (SVV 260 550). THE EFFECT OF SELECTIVE TOPOISOMERASE BETA INHIBITOR XK469 ON ANTHRACYCLINE CARDIOTOXICITY SKALICKÁ, V., KUBEŠ, J., APPLOVÁ, L., JIRKOVSKÁ, A., ŠIMŮNEK, T. Department of Biochemical Sciences, Faculty of Pharmacy in Hradec Králové, Charles University, wHradec Králové, Czech Republic e-mail: skalicv1@faf .cuni .cz Anthracycline (ANT) cardiotoxicity represents a considerable limitation of several antineoplastic regimes . As much as the ANT anticancer effect, their cardiotoxicity effect excels in its complexity. During their long history, the scientific opinions shifted from the traditionally discussed iron chelation hypothesis to topoisomerase II (TOP2) inhibition . TOP2 occurs in two isoforms with diverse functions . While TOP2 alpha (TOP2A) is a crucial enzyme for cell division and thereby is expressed in replicating cells, TOP2 beta (TOP2B) is present in all cells including quiescent cells . Currently, TOP2B has been massively discussed as the potential mechanism of ANT cardiotoxicity . Although dexrazoxane (DEX) was approved by the FDA as a safe and efficient cardioprotectant, its clinical use is limited only to specific groups of patients. One of the main reasons for the restriction is the concern of the possible negative effect on the antineoplastic activity of ANT in the clinical setting . This interaction (although only speculative) could be based on the non-specific inhibition of both TOP2 isoforms by DEX. The development of TOP2B-specific inhibitor could provide a possible means to the effective cardioprotection avoiding the effect on the cancer cells . XK469 was described in literature as a TOP2B specific inhibitor initially discovered as an antineoplastic drug that proceeded to the second phase of several clinical trials . Nevertheless, its use for cardioprotection has never been studied . Our pilot data showed that XK469 possesses an ability to prevent cardiomyocytes from ANT-induced cell death, although this effect is complicated with the toxicity towards cardiomyocytes in higher doses and prolonged incubation times . We 82 also described the XK469 inhibitory activity on individual TOP2 isoforms . Moreover, its effects on ANT-induced apoptosis and DNA damage were determined by caspase assay, comet assay and assessment of phosphorylation of H2AX in both leukemic HL-60 cells and cardiomyocytes . The study was supported by the Czech Science Foundation (Project No. 18-08169S) and from the project of Specific Academic Research (SVV 260 550). TEPOTINIB REVERSES MULTIDRUG RESISTANCE BY INHIBITING THE EFFLUX FUNCTION OF ABCB1 AND ABCG2 TRANSPORTERS IN VITRO BUDAGAGA, Y .,1 VAGIANNIS, D .,1 ZHANG, Y .,1 SKARKA, A .,2 HOFMAN, J .1 1 Department of Pharmacology and Toxicology, Faculty of Pharmacy in Hradec Kralové, Charles University, Czech Republic 2 Faculty of Science, University of Hradec Králové, Hradec Králové, Czech Republic e-mail: budagagy@faf .cuni .cz ABC (ATP-binding cassette) drug efflux transporters play a crucial part in drug-drug interactions and multidrug resistance (MDR) of cancer cells . Tepotinib, a novel c-MET tyrosine kinase inhibitor, has been recently approved for the treatment of non-small cell lung cancer (NSCLC) . This study aims at the investigation of the inhibitory effect of tepotinib on human ABC transporters and evaluation of its role in MDR in vitro . First, results of our accumulation experiments showed that tepotinib inhibits ABCB1 and ABCG2 transporters . Second, using MTT assay we found that tepotinib can reverse daunorubicin and mitoxantrone resistance mediated by ABCB1 and ABCG2, respectively . Furthermore, bidirectional transport assays designated tepotinib as ABCB1 substrate with no affinity to either ABCC1 or ABCG2. Interestingly, although ABCB1-mediated tepotinib’s efflux was demonstrated in transport experiments, functional overexpression of ABCB1 in several cellular models had no appreciable impact on its antitumor activity . Finally, tepotinib did not change the mRNA levels of ABCB1, ABCG2, and ABCC1 in selected cell lines by qRTPCR assay . To sum up, tepotinib could participate in drug-drug interactions by inhibiting the function of ABCB1 and ABCG2 transporters and potentially reverse the MDR in cancer cells . Nevertheless, follow-up experiments with more sophisticated preclinical models are necessary to verify possible clinical impact of our results . The study was supported by the Czech Science Foundation (Project No. 20-20414Y), by the Grant Agency of Charles University (project No. 334120) and from the project of Specific Academic Research (SVV 260 549). 83 THE ESTABLISHMENT OF EX VIVO PRIMARY LUNG TUMOR MODELS AND THEIR APPLICATION FOR TESTING OF DRUG COMBINATIONS VAGIANNIS, D .,1 MORELL, A .,2 ZHANG, Y .,1 BUDAGAGA, Y .,1 HANKE, I .,3 ROZKOŠ, T .,4 HOFMAN, J .1 1 Department of Pharmacology and Toxicology, 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 3 Department of Cardiac Surgery, Faculty of Medicine in Hradec Králové, Charles University and University Hospital Hradec Králové, Czech Republic 4 Fingerland Department of Pathology, Faculty of Medicine in Hradec Králové, Charles University and University Hospital Hradec Králové, Czech Republic e-mail: vagiannd@faf .cuni .cz In this study, we aimed to establish ex vivo explants derived from non-small cell lung cancer (NSCLC) biopsies in cooperation with clinicians . The NSCLC biopsies from patients were obtained immediately after resection, which was followed by collagenase-assisted liberation of tumor cells from tissues . NSCLC cells were separated from physiological cells and fibroblasts and the epithelial origin of cells was confirmed. Once the primary cultures were established, we detected the protein expression levels of ABC drug efflux transporters (ABCB1, ABCG2, and ABCC1) in them. Moreover, transporters’ functional activity was assessed in the samples with detectable expression by employing the accumulation flow-cytometric studies. In addition, in drug combination studies, we demonstrated that tepotinib overcame transporter-mediated resistance to conventional cytostatics in primary explants exhibiting functional activity . Finally, ABCB1, ABCG2 and ABCC1 gene induction studies showed that tepotinib does not have the potential to affect the multidrug resistance phenotype of NSCLC cells . Our results indicate that tepotinib might be a valuable candidate for the combination therapy of NSCLC tumors expressing drug efflux transporters. The study was supported by the Czech Science Foundation (Project No. 20-20414Y), by the Grant Agency of Charles University (Project No. 334120) and from the project of Specific Academic Research (SVV 260 549). The study was approved by the University Hospital Ethics Committee (Document No. 202002 S04P). SILENCING OF SELECTED UDP-GLYCOSYLTRANSFERASE GENES BY RNAi IN HAEMONCHUS CONTORTUS DIMUNOVÁ, D ., MATOUŠKOVÁ, P . Department of Biochemical Sciences, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic e-mail: dimunovd@faf .cuni .cz 84 Drug resistance is a serious problem in many organisms, including parasites . The identification of specific enzymes responsible for drug resistance can lead to a new approach to the treatment . UDP-glycosyltransferases (UGTs), enzymes metabolizing xenobiotics and eobiotics, protect the helminth from the toxic action of anthelmintics by their conversion to inactive glycosides . In our project, we focused on gastrointestinal nematode, a parasite of small ruminants, Haemonchus contortus, and revelation its mechanism of drug resistance . RNA interference (RNAi) was used for the characterization of gene functions in nematode C. elegans . Therefore we tested the usability of this method in Haemonchus contortus . The silencing of selected UGTs can prove their involvement in the metabolism of anthelmintics . The silencing of selected UGTs can be mediated through specific double-stranded siRNA (small interfering RNA), which cause degradation of the corresponding mRNA in vivo and lead to the selected enzymes loss of function. The efficacy of gene suppression by siRNA was optimized . Worms and larvae were exposed to siRNA with different transfection reagents by soaking and subsequently, the RNA was isolated . The suppression of tested genes was determined by qPCR analysis . Despite all the efforts, our selected enzymes were not possible to silence, therefore another method for functional testing has to be employed . The study was supported by the Czech Science Foundation (Project No. 17-11954Y) and from the project of Specific Academic Research (SVV 260 550). USING MACHINE LEARNING IN EVALUATION OF IN VITRO VIABILITY TESTS IN HAEMONCHUS CONTORTUS NGUYEN, L. T., ŽOFKA, M., MAŠÁTOVÁ, E., SKÁLOVÁ, L. Department of Biochemical Sciences, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic e-mail: nguyenli@faf .cuni .cz Haemonchus contortus is a gastrointestinal parasite of sheep with great ability to develop resistance to anthelmintic drugs . With the worsening situation, there is a need for new therapies, testing of drug sensitivity as well as addressing the increases in nematode resistance . At present, there are several viability tests, each having some limitations . Egg hatch test (EHT) and larval development test (LDT) are commonly used in vitro methods which are relatively simple, nevertheless, they require labor-intensive counting of eggs or larvae at different stages under a microscope . In our study, we applied machine learning in the evaluation part of EHT and LDT, which was the object detection and classification from microscopic images . We adopted Mask Region-based Convolutional Neural Network (Mask R-CNN)1 in Python 3, Keras, and TensorFlow model as it is the state-of-the-art approach for object recognition tasks . We leveraged a pre-existing model and re-trained it using our dataset . By generating bounding boxes and segmentation for each instance of an object, the model recognizes eggs from first-stage larvae and/or third-stage larvae. Using the Mask R-CNN methodology, we were able to automate the process to a high degree of accuracy . 85 The study was supported by the Charles University Project UNCE 18/SCI/012 and from the project of Specific Academic Research (SVV 260 550). References 1. HE, K ., GKIOXARI, G ., DOLLÁR, P . et al.: Mask R-CCN . In Proceedings of 2017 IEEE International Conference on Computer Vision (ICCV), Venice, Italy, 22–29 Oct . 2017, pp . 2980–2988 . SERTRALINE AS A NEW POTENTIAL ANTHELMINTICS AGAINST HAEMONCHUS CONTORTUS? ZAJÍČKOVÁ, M.,1 NAVRÁTILOVÁ, M .,1 PRCHAL, L .,2 NGUYEN, L . T .,1 STUCHLÍKOVÁ, R. L.,1 SKÁLOVÁ L .1 1 Department of Biochemical Sciences, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic 2 Biomedical Research Centre, University Hospital Hradec Králové, Czech Republic e-mail: zajickm@faf .cuni .cz Haemonchus contortus is a parasitic nematode of small ruminants which cause problem to many farmers around the world . Grazing management, biological control of pastures, nutritional suplementation, vaccination and selective breading play important role in prevention of this disease . However, chemoterapy still represents the main strategy to control infections caused by H. contortus . Due to wide spread resistance to main clases of anthelmintics (benzimidazoles, macrocyclic lactones, amino-acetonitrile derivative) it is necessary to look for new structures . Synthesis of new chemical entities is complicated, long, and costly process. For this reason, exploiting old structures and finding new indications for already registered drugs represent a good alternative .1 Sertraline is used in human medicine as antidepresant and belongs to the class of selective serotonin reuptake inhibitors . Sertraline showed to be effective against Trichuris muris, Ancylostoma caninum and Schistosoma mansoni .2 In our project we wanted to know if sertraline is also effective against H. contortus . Moreover, we are interested about its biotransformation and hepatotoxicity in the host sheep . The study was supported by the Charles University Grant Agency (Project No. 1568519) and from the project of Specific Academic Research (SVV 260 550). References 1. ZAJÍČKOVÁ, M., NGUYEN, L. T., SKÁLOVÁ, L. et al .: Drug Discov . Today, 25, 2020, 430–437 . 2. WEEKS, J . C ., ROBERTS, W . M ., LEASURE, C . et al .: Sci . Rep ., 8, 2018, art . 975 . 86 METABOLISM OF HELENALIN IN VITRO AND ITS INTERACTION WITH HUMAN CYTOCHROME P450 2A13 ŠADIBOLOVÁ, M .,1 BOUŠOVÁ, I .,1 JUVONEN, R .,2 AURIOLA, S .2 1 Department of Biochemical Sciences, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic 2 School of Pharmacy, Faculty of Health Sciences, University of Eastern Finland, Kuopio, Finland e-mail: sadibolm@faf .cuni .cz Helenalin (HEL) is a sesquiterpene lactone, especially found in Arnica montana and Arnica chamissonis, that demonstrates potent anti-inflammatory activity mediated by direct alkylation of Cys38 within the DNA binding domain of RelA (p65), a key member of the nuclear factor-κB (NF-κB) family of transcription factors. Besides, HEL has been found to exert remarkable anticancer, antibacterial and antiprotozoal activity . HEL is an active component of herbal arnica preparations that have long been used to reduce muscle and joint pain, post-surgical pain as well as to treat minor sports injuries, bruises and swelling associated with trauma, contusions and sprains . Despite the frequent use of these products, neither the fate of HEL in the human organism, nor its interaction with xenobiotic-metabolizing enzymes has been studied so far. To address this issue, we have first investigated the metabolism of HEL and characterized the kinetics of metabolite formation using human and rat liver subcellular fractions and human recombinant cytochrome P450 (CYP) enzymes. UHPLC-MS/MS technique was employed for this purpose. HEL was oxidized into five metabolites by both human and rat liver microsomes, and the oxidation was generally far more efficient in rat microsomes. Moreover, several human CYP isoforms were found to be involved in the oxidation of HEL, namely CYP2A13, 2B6, 3A4, 3A5 and 3A7 . In the following experiments, we have also tested the inhibitory potential of HEL towards human recombinant CYP enzymes . Out of all tested CYPs, the most effective inhibition (the lowest IC50 value) was observed for CYP2A13 . In addition, the inhibition of CYP2A13 was NADPHand time-dependent suggesting that HEL may act as a mechanism-based inhibitor of CYP2A13 . The study was supported by the Czech Science Foundation (Project No. 18-09946S), by the Charles University Grant Agency (Project No. 302120) and from the project of Specific Academic Research (SVV 260 550). UDP-GLYCOSYLTRANSFERASES AND ALBENDAZOLE METABOLISM IN THE JUVENILE STAGES OF HAEMONCHUS CONTORTUS KELLEROVÁ, P ., NAVRÁTILOVÁ, M ., NGUYEN, L . T ., DIMUNOVÁ, D ., RAISOVÁ STUCHLÍKOVÁ, L., ŠTĚRBOVÁ, K., SKÁLOVÁ, L., MATOUŠKOVÁ, P. Department of Biochemical Sciences, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic e-mail: voborilp@faf .cuni .cz 87 The UDP-glycosyltransferases (UGTs), second phase biotransformation enzymes, inactivate xenobiotic substrates . Such transformation is involved in drug-resistance mechanisms in one of the most pathogenic parasites in small ruminants, Haemonchus contortus . In the previous study, we characterized the UGT family in H. contortus, including its nomenclature, phylogeny, and expression in adults – the parasitic life stage .1 However, the drug-metabolism by UGTs in the free-living juvenile stages may as well represent an important defence system against anthelmintics . Due to the almost permanent contact with anthelmintic residues excreted from treated animals in the environment,2 UGTs in juvenile stages may also contribute to drug-resistance development . Therefore, we decided to study the expression of UGTs and the metabolism of albendazole (ABZ) and the primary active ABZ metabolite ABZ sulfoxide (ABZSO) in the eggs, L1s, and L3s larvae of H. contortus . We have also compared the formation of ABZ metabolites and selected UGT transcripts between a sensitive isolate (ISE) and resistant isolates (IRE and multi-resistant WR) . Furthermore, we explored the inducibility of UGTs by ABZ and ABZSO in H. contortus juvenile stages .3 The study was supported by the Czech Science Foundation (Project No. 17-11954Y), by the Charles University projects (PRIMUS/17/SCI/4 and UNCE/18/SCI/012) and from the project of Specific Academic Research (SVV 260 550). References 1. MATOUŠKOVÁ, P ., LECOVÁ, L ., LAING, R . et al .: Int . J . Parasitol ., 8, 2018, 420–429 . 2. PRCHAL, L ., PODLIPNÁ, R ., LAMKA, J . et al .: Environ . Sci . Pollut . Res ., 23, 2016, 13015–13022 . 3. KELLEROVÁ, P ., NAVRÁTILOVÁ, M ., NGUYEN, L . T . et al .: Front . Physiol ., 11, 2020, art . 594116 . TESTING FERN EXTRACTS FRACTIONS ON ANTI-INFLAMMATORY AND ANTHELMINTIC PROPERTIES PAVIČIĆ, A.,1,2 SZOTÁKOVÁ, B .,1 LANGHANSOVÁ, L .2 1 Department of Biochemical Sciences, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic 2 Institute of Experimental Botany, Czech Academy of Sciences, Prague, Czech Republic e-mail: pavicia@faf .cuni .cz Ferns are used in traditional medicine in Asia and therapeutic effects of several fern extracts have been proven . However, there is still space for searching for new bioactive phytochemicals in ferns. Previous results showed significant anti-inflammatory activity of several selected fern species with higher potential in selective COX-1 inhibition comparing to moderate inhibition of COX-2 and 5-LOX activity . Based on these results we decided to make fractionation of extracts of three fern species (Athyrium distentifolium, Dryopteris aemula and Blechnum spicant) by using sequential liquid-liquid techniques .1 Redissolved fractions (n-hexane, chloroform, ethylacetate, n-butanol) and residual aqueous fraction have been tested for anti-inflammatory and anthelmintic activity. Dryopteris aemula showed the highest potential of all fractions in selective COX-1 inhibition, its 88 n-hexane and chloroform fractions showed only moderate selective COX-2 and 5-LOX inhibition . All fern species and their fractionated extracts have been tested for anthelmintic activity using Egg Hatch Test2 with Barber’s pole worms (Haemonchus contortus) . None of the fern samples reduced egg hatching in EHT compared with the control . The study was supported from the project of Specific Academic Research (SVV 260 550). References 1. LANGHANSOVÁ, L ., LANDA, P ., KUTIL, Z . et al.: Food Agric . Immunol ., 28, 2017, 343–353 . 2. Von SAMSON-HIMMELSTJERNA, G ., COLES, G . C ., JACKSON, F . et al .: Parasitol . Res ., 105, 2009, 825-834 . IN VITRO EVALUATION OF THE PHOTODYNAMIC ACTIVITY OF NOVEL BODIPY DYES ROHLÍČKOVÁ, M.,1 KRZEMIEN, W .,2 ZIMČÍK, P.,2 MACHÁČEK, M.1 1 Department of Biochemical Sciences, 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: steklam@faf .cuni .cz Neoplastic diseases are nowadays one of the most common reasons of death in developed countries . Therefore, a great attention is dedicated to the development of new methods for the treatment of those diseases . One of such methods is the photodynamic therapy (PDT) . This is a selective, minimally invasive method with a minimum side effects . Principle of PDT is the application of inactive drug, called photosensitizer (PS), followed by exposure to activating light with suitable wavelength in the presence of molecular oxygen . Therefore, the photodynamic therapy needs three basic components: the PS, light and oxygen .1,2 As it has been said, one of the most important part of PDT is the PS . The objective of this study is assessing the effectiveness of the novel PSs from the group of BODIPY dyes in vitro . All of the studied compounds were evaluated on malignant human cervical cell line (HeLa) and human melanoma cell line SK-MEL-28 . Cytotoxicity after the exposure to activating light (phototoxicity) and intrinsic toxicity in the absence of light (dark toxicity) were determined . Further, the subcellular localization PSs after accumulation in cells was assessed using confocal microscopy and fluorescent organelle-specific probes. All of studied compounds proved to be efficient PSs (EC50 up to 0.14 ± 0.01 μM) with no dark toxicity up to their solubility limit . The study was supported from the project of Specific Academic Research (SVV 260 550). 89 References 1. AGOSTINIS, P ., BERG, K ., CENGEL, K . A . et al .: CA Cancer J . Clin ., 61, 2011, 250–281 . 2. KAMKAEW, A ., LIM, S . H ., LEE, H . B . et al .: Chem . Soc . Rev ., 42, 2013, 77–88 . CAROTUXIMAB AFFECTS ENDOGLIN EXPRESSION AND ADHESION AND TRANSMIGRATION OF MONOCYTES VIA ENDOTHELIAL CELLS IN 7-KETOCHOLESTEROL INDUCED ENDOTHELIAL DYSFUNCTION TRIPSKÁ, K .,1 VICEN, M .,1 HAVELEK, R .,2 IGREJA E SÁ, I . C .,1 EISSAZADEH, S .,1 VITVEROVÁ, B .,1 NACHTIGAL, P .1 1 Department of Biological and Medical Sciences, 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 e-mail: tripskak@faf .cuni .cz Endoglin (Eng) is a transmembrane glycoprotein affecting TGF-β signaling pathway. In the previous study, we demonstrated that treatment of endothelial cells with 7-ketocholesterol (simulating oxidized LDL) leads to the development of endothelial dysfunction with upregulated levels of Eng, cell adhesion molecules (ICAM-1, P/E-selectin), and also increased adhesion and transmigration of monocytes via endothelial cells . Carotuximab (TRC105) is a novel monoclonal antibody that binds Eng with high affinity and block/ inhibits its effects, currently studied in different clinical trials mostly focused on cancer . We hypothesized that carotuximab mediated inhibition of endoglin will affect 7-ketocholesterol induced endothelial dysfunction . Human aortic endothelial cells (HAEC), passage 5, were cultured with appropriate supplements until reaching 90% confluence. Afterwards, cells were treated with or without 300 μg mL‒1 carotuximab for 1 hour followed by addition of 10 µg mL‒1 7-ketocholesterol for another 12 hours . Gene expression of Eng, cell adhesion molecules and Eng transcription factors (KLF6, NF-kB p65 and LXR) was measured by qRT-PCR . Protein levels of Eng, adhesion molecules, MMP-14, pSmads and adhesion assay were quantified by flow cytometry. Soluble endoglin levels were measured by ELISA . Transmigration assay was performed with cell culture inserts and assessed by flow cytometry. We demonstrated that carotuximab was able to prevent 7-ketocholesterol induced Eng and ICAM-1 expression, as wells as adhesion and transmigration of monocytes via endothelial cells . Therefore, we propose that carotuximab might be important in prevention of endothelial dysfunction induced by hypercholesterolemia, but to which extent must be further investigated . This study was supported by the Grant Agency of Charles University (Project No. 1130120) and from the project of Specific Academic Research (SVV 260 549). 96 conducted in acute and community care (71 .1 and 53 .8% vs 34 .5 and 31 .5%, respectively). This review confirmed that PIP is highly prevalent in the CEE region, furthermore, these results will serve as a starting point for our scientific works on analyses of PIP prevalence in several EU and non-EU countries participating in the ESR7 EuroAgeism H2020 project . The study was supported by the EuroAgeism European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No. 764632, by InoMed (Project No. CZ.02.1.01/0.0/0.0/18_069/0010046) co-funded by the European Union, by the Research programme Development and Study of Drugs (Progres Q42) and from the project of Specific Academic Research (SVV 260 551). References 1. RANKIN, A ., CADOGAN, C ., PATTERSON, S . et al .: Cochrane Database Syst . Rev ., 9, 2018, art . CD008165 . 2. ALLDRED, D ., KENNEDY, M ., HUGHES, C . et al .: Cochrane Database Syst . Rev ., 2, 2016, CD009095 . 3. FIALOVÁ, D ., TOPINKOVÁ, E ., GAMBASSI, G . et al.: JAMA, 293, 2005, 1348‒1358. 4. GALLAGHER, P ., LANG, P ., CHERUBINI, A . et al.: Eur. J. Clin. Pharmacol., 67, 2011, 1175‒1188. PRESCRIBING OF HYPNOSEDATIVES IN SENIORS IN ACUTE AND AMBULATORY CARE IN THE CZECH REPUBLIC – INOMED AND EUROAGEISM H2020 PROJECT FINDINGS ANTONENKO, O .,1 PULDOVÁ, K .,1 ZELINKOVÁ, A .,1 HALAČOVÁ, M.,1,2 GREŠÁKOVÁ, S .,1 BRKIĆ, J.1, FIALOVÁ, D .1,3 1 Department of Social and Clinical Pharmacy, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic 2 Department of Clinical Pharmacy, Hospital Na Homolce, Prague, Czech Republic 3 Department of Geriatrics and Gerontology, 1st Faculty of Medicine and General University Hospital in Prague, Charles University, Czech Republic e-mail: antonenol@faf .cuni .cz Insomnia is a frequent problem in older adults, particularly in those suffering from polymorbidity and treated by polypharmacy . The aim of our study was to describe the prevalence of insomnia and patterns of potentially inappropriate prescribing in hypnosedative drug use in acutely hospitalized and ambulatory care seniors in the Czech Republic . Evaluated data comprised comprehensive geriatric characteristics (CGC) of 438 acutely hospitalized and 563 ambulatory care geriatric patients (≥ 65 years) from regionally different study sites in the Czech Republic (Prague, Brno, Hradec Králové) . All patients were prospectively assessed using the EuroAgeism H2020 study protocols . Descriptive statistics was applied in data analysis . On the whole, 34 .6% (n = 151) of acutely hospitalized seniors and 30 .6% (n = 172) in ambulatory care had recorded diagnosis of insomnia in personal history, respectively. The most frequently used hypnosedatives were: antipsychotics e/n (18 .5% and 17 .8%), Z-drugs (16 .2% and 8 .2%) and benzodiazepines (14 .2% and 7 .6%) . We determined also non-geriatric dosing of Z-drugs (10 .5% and 6,0%, respectively) and BZDs (0 .5% and 2 .7%), as well as non-geriatric lenght of drug therapy of Z-drugs (5 .9% 97 and 2 .7%, respectively) and BZDs (5 .3% and 11%, respectively) . Excessive indication of antipsychotics e/n and inappropriate geriatric dosing of Z-drugs and long-term use of BZDs e/n were documented in seniors in the Czech Republic. The study was supported by InoMed (Project No. CZ.02.1.01/0.0/0.0/18_069/0010046) co-funded by the European Union, by the EuroAgeism European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No. 764632, by the Research programme Development and Study of Drugs (Progres Q42) and from the project of Specific Academic Research (SVV 260 551). AGEING IN DEVELOPING COUNTRIES AND PHYSICIAN’S KNOWLEDGE OF EXPLICIT CRITERIA OF PIMs IN OLDER PATIENTS AND BARRIERS TOWARDS APPROPRIATE GERIATRIC PRESCRIBING BANDARI, D . K .,1 BRKIĆ, J.,1 FIALOVA, D .1,2 1 Department of Social and Clinical Pharmacy, Faculty of Pharmacy in Hradec Kralové, Charles University, Czech Republic . 2 Department of Geriatrics and Gerontology, 1st Faculty of Medicine in Prague, Charles University, Czech Republic . e-mail: laxmideepak .pharma@gmail .com Potentially Inappropriate Medication (PIM) use is associated with increased mortality and morbidity in older adults and prescribing patterns are strongly influenced by physicians’ knowledge of geriatric prescribing . We aimed to assess the barriers among physicians to appropriate geriatric prescribing, their knowledge of specific explicit criteria of PIMs and the frequency of use of different information sources and guidelines . A descriptive pilot cross-sectional study was conducted among physicians (n = 256) in two tertiary care teaching hospitals . Knowledge and preferences in usage of different information sources and barriers to appropriate geriatric prescribing were assessed by validated clinical vignettes and by a piloted questionnaire . Pearson’s chi-squared test, Student’s t-test, Mann–Whitney’s U test and logistic regression models were applied using statistical R software (version 4 .0 .3) . Of 201 (78 .5%) respondents, majority were males (63 .2%), 74 .1% received training in geriatric medicine and 39 .8% were currently providing more than once a week care for older adults in long-term care facilities . Only 31.8% of physicians felt confident in appropriate geriatric prescribing and mean score of knowledge of PIMs by clinical vignettes was 3 .5 ± 0 .9 . Multivariate logistic regression revealed the higher odds of good geriatric knowledge in females, age group of 30–39 years, and in physicians that received geriatric training . Positive trend of better scoring in clinical vignettes correlated with the extend of use of PIM criteria . Limited options in drug formularies (80 .6%), lack of acceptable therapeutic alternatives (69 .1%), potential drug-drug interactions (63 .6%) and lack of time (62 .1%) were the top cited barriers to appropriate prescribing in older adults . Educational interventions and interprofessional clinical pharmacy cooperation may improve appropriateness of geriatric prescribing in daily clinical practice . 98 The study was supported by the EuroAgeism European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No. 764632, by InoMed (Project No. CZ.02.1.01/0.0/0.0/18_069/0010046) co-funded by the European Union, by the Research programme Development and Study of Drugs (Progres Q42) and from the project of Specific Academic Research (SVV 260 551). PREVALENCE OF POLYPHARMACY AND RISKS OF POTENTIALLY INAPPROPRIATE MEDICATION USE IN OLDER POPULATION IN A DEVELOPING COUNTRY: A SYSTEMATIC LITERATURE REVIEW AND META-ANALYSIS BHAGAVATHULA, A . S .,1 FIALOVÁ, D .1,2 1 Department of Social and Clinical Pharmacy, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic . 2 Department of Geriatrics and Gerontology, 1st Faculty of Medicine in Prague, Charles University, Czech Republic e-mail: bhagavaa@faf .cuni .cz Aim: This systematic literature review and meta-analysis aimed to investigate the prevalence of polypharmacy and PIM use and major risk factors associated with PIM prescribing in older adults in Ethiopia . Methods: We searched PubMed/MEDLINE, Scopus, Embase, and Google Scholar databases to identify relevant studies published between January 1990 to October 2020 . Observational studies reporting the prevalence and association of risk factors with polypharmacy and PIM use in older population were meta-analyzed . A multilevel meta-analysis was conducted to pool the prevalence estimates, and the risk of PIM use was reported as a relative risk (RR) with 95% confidence interval (CI). Results: We identified by systematic literature review 404 articles. Of those, eight studies fulfilled inclusion criteria, comprising a total sample of 2608 participants. The overall prevalence of polypharmacy and PIM use pooled by meta-analysis in the Ethiopian older population was 33% and 37%, respectively . The risk factors of PIM use were analyzed in the meta-analysis (particularly polymorbidity, polypharmacy, gender, and older age), the older age of 65+ (RR:1.71, 95% CI: 1.16–2.51) was significantly associated with PIM use . Conclusion: This first meta-analysis from a developing country revealed a high prevalence of polypharmacy and PIM use in the Ethiopian older population . There was no awareness to the risk of PIMs in patients with polypharmacy and polymorbidity, and only older age significantly predicted PIM use. Interventions ensuring rational geriatric pharmacotherapy are essential not only in developed countries, but also in developing countries in order to reduce the expected burden of PIM-related geriatric morbidity, higher costs, and mortality . The study was supported by InoMed (Project No. CZ.02.1.01/0.0/0.0/18_069/0010046) co-funded by the European Union, by the EuroAgeism European Union’s Horizon 2020 99 research and innovation programme under the Marie Skłodowska-Curie grant agreement No. 764632, by the Research programme Development and Study of Drugs (Progres Q42) and from the project of Specific Academic Research (SVV 260 551). RISK STRATIFICATION OF PATIENTS FOR POSTOPERATIVE INFECTION AFTER KNEE OR HIP ARTHROPLASTY – PRELIMINARY RESULTS DOMECKÝ, P .,1 REJMAN PATKOVÁ, A .,1 KUČERA, T.,2 ŠPONER P .,2 MALÝ, J .1 1 Department of Social and Clinical Pharmacy, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic 2 Department of Orthopedics, University Hospital Hradec Králové, Czech Republic e-mail: domeckyp@faf .cuni .cz Surgical site infection is a potential complication of all surgical procedures . There is double mortality in patients with developed infection who underwent a surgical procedure compared to non-infected patients . This study aimed to stratify patients according to the risk of postoperative infection and verify the findings in clinical practice. This prospective study has started in March 2020 in the Department of Orthopedics at University Hospital Hradec Králové. The study included patients aged ≥ 18 years who underwent hip or knee arthroplasty. Inflammatory markers, especially neutrophil-to-lymphocyte ratio (NLR), prognostic inflammatory and nutritional index (PINI) and intensive care infection score (ICIS), were analyzed one day before operation (−1D), two days after operation (2D) and in outpatient examination (OE) . 42 patients (16 women and 26 men) with an average age of 64 .04 ± 10 .21 were included in the study . Hip arthroplasty underwent 31 (73 .8%) and knee arthroplasty 11 (26 .2%) patients . Cefazolin was administered in 88 .1% and vancomycin in 11 .9% of operations . −1D: NLR > 4, PINI score > 21 and ICIS > 4 were identified in 5, 0, and 1 patient, respectively. 2D: NLR > 4, PINI score > 21 and ICIS > 4 were identified in 14, 24, and 4 patients, respectively. OE: NLR > 4, PINI score > 21 and ICIS > 4 were identified in no patient at all. ACS (American Society of Surgeons) risk score > 5% was identified in 9 patients. The postoperative infection was identified in 2 (4.7%) patients and postoperative anemia in 12 (28 .6%) patients . The average length of hospitalization was 11 ± 6 .7 days . Increased inflammatory markers could be suitable for detecting early postoperative infection . However, a more extensive set of patients is needed for further detailed statistical analysis . The study was supported by the project of Specific Academic Research (SVV 260 551). 100 PREVALENCE AND RISK FACTORS OF CARDIOVASCULAR DRUG – DISEASE INTERACTIONS IN SENIORS IN ACUTE AND AMBULATORY CARE IN THE EUROAGEISM H2020 PROJECT KUMMER, I .,1 BRKIĆ, J.1 LUKAČIŠINOVÁ, A.,1 REISSIGOVÁ, J .,2 FIALOVÁ, D .1 1 Department of Social and Clinical Pharmacy, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic 2 Department of Statistical Modeling, Institute of Computer Sciences of the Czech Academy of Sciences, Prague, Czech Republic e-mail: ingrid .kummer2@gmail .com Older patients are vulnerable to drug-disease interactions (DDIs) because of age-related pharmacological changes, polymorbidity, and polypharmacy . The aim of this study was to investigate the prevalence and risk factors of cardiovascular (CVS) DDIs in older adults in ambulatory and acute care facilities in the Czech Republic . The data presented are preliminary. Data of 1152 patients 65+ were collected using the EuroAgeism H2020 protocols in 4 acute care hospitals and 4 ambulances (Jun 2019 – Jan 2020) . Prevalence and risk factors of CVS DDIs were identified according to STOPP/START criteria, the EU(7) PIM list and Beers 2019 criteria. The majority of participants were females (67.4%) and 38.7% were 85+ years old. Polypharmacy (5+ medicines) was identified in 85.3% patients and 90.0% suffered from polymorbidity (4+ diagnoses). Prevalence of at least 1 CVS DDIs was: by START criteria 75 .4%, STOPP criteria 30 .7%, EU(7) PIM criteria 29 .0% and BEERS criteria 21 .9% . Higher odds of being prescribed at least 1 CVS DDIs were in seniors suffering from 2+ CVS diagnoses (OR = 12 .2, 95% CI 7 .3–21 .5, p < 0 .001), in polypharmacy users (OR = 2 .2, 95% CI 1.3–3.9, p = 0.005), and in 85+ years old seniors (OR = 3.1, 95% CI 1.6–6.1, p = 0.001). Our study documented high prevalence of CVS DDIs and risk factors in older patients in different care settings in the Czech Republic . Therefore, safer geriatric prescribing should be promoted, and regular medication check provided by clinical pharmacists . The study was supported by the EuroAgeism European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No. 764632, by InoMed (Project No. CZ.02.1.01/0.0/0.0/18_069/0010046) co-funded by the European Union, by the Research programme Development and Study of Drugs (Progres Q42) and from the project of Specific Academic Research (SVV 260 551). DRUG-RELATED HOSPITAL ADMISSIONS VIA THE EMERGENCY DEPARTMENT: A CROSS-SECTIONAL STUDY OČOVSKÁ, Z.,1 MAŘÍKOVÁ, M.,1,2 KOČÍ, J.,3 VLČEK, J.1,2 1 Department of Social and Clinical Pharmacy, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic 2 Department of Clinical Pharmacy, Hospital Pharmacy, University Hospital Hradec Králové, Czech Republic 3 Department of Emergency Medicine, University Hospital Hradec Králové, Czech Republic e-mail: ocovskaz@faf .cuni .cz 101 Drug-related hospital admissions (DRA) have attracted much research attention worldwide . The aims of this study were to determine the prevalence and preventability of DRA, to identify the implicated medications in DRA and to examine the preventability aspects of DRA . The study examined hospital admissions via the emergency department in the University Hospital Hradec Králové between August and September 2018 . Planned hospitalisations and visits to the emergency room without inpatient hospitalisation were not included . The data were collected retrospectively using electronic medical records . The process of DRA identification included screening for potential adverse drug events which were the main or contributory reason for hospital admissions, causality assessment and assessment of contribution to hospital admission . DRA due to medication errors were considered preventable and DRA due to adverse drug reactions were considered nonpreventable. Anatomical Therapeutic Chemical classification system was used for the classification of medications implicated in DRA. Out of 1204 hospital admissions 193 have been identified as DRA. 144 DRA were related to treatment safety while 49 DRA were related to treatment effectiveness . The prevalence of DRA was 16 .0% (95% CI 14 .0–18 .1) and the preventability of DRA was 54 .4% (95% CI 47 .4–61 .4) . Medication classes most commonly involved in DRA related to treatment safety included antithrombotic agents, antiinflammatory and antirheumatic products, diuretics and antineoplastic agents. Diuretics, antihypertensives, antithrombotic agents and drugs used in diabetes represented the most common medication classes involved in DRA related to treatment effectiveness . Measuring the scope and nature of DRA is essential for the development of risk minimization measures . The relatively high preventability suggests there is a potential to reduce DRA in the future . The study was supported by the Grant Agency of Charles University (Project No. 14120) and from the project of Specific Academic Research (SVV 260 551). ADHERENCE AND BELIEFS ABOUT IMMUNOSUPPRESSANTS IN PATIENTS AFTER KIDNEY TRANSPLANTATION: RESULTS FROM UNICENTRIC FOLLOW-UP STUDY KOŠŤÁLOVÁ, B.,1 MALÁ-LÁDOVÁ, K .,1 KUBĚNA, A. A.,1 HORNE, R .,2 DUSILOVÁ-SULKOVÁ, S .,3 MALÝ, J .1 1 Department of Social and Clinical Pharmacy, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic 2 Centre for Behavioural Medicine, UCL School of Pharmacy, University College London, United Kingdom 3 Haemodialysis Centre, University Hospital Hradec Králové, Czech Republic e-mail: vankovb@faf .cuni .cz Strict medication adherence to immunosuppressants is essential during the whole time after kidney transplantation (KTx) . Therefore, the aim of our study was to evaluate self-reported adherence and beliefs about immunosuppressants over time and links with clinical outcomes such as graft functioning and de novo malignancy . This observational study is a part of the multiphase project TAKTIS (developing, implementing, and testing 102 of an integrated care model for adults after KTx) . Data were collected at the outpatient post-transplant clinic in the University Hospital Hradec Králové . Adult patients at least 4 weeks after KTx were invited for the structured interview, followed by self-administered questionnaire survey . Primary outcomes such as adherence and beliefs about imunosuppressants were measured by validated international tools . Two data collections conducted between 2016 and 2019 involved 134 patients . Non-adherence to immunosuppressants was reported by 18 (13 .4%) patients at the baseline and by 15 (11 .2%) at the followup . The perceived necessity of immunosuppressants decreased while concerns increased over time (p < 0 .001) . Baseline higher concerns were associated with the less than full score in adherence (p = 0 .0445) or with the new onset of cancer . On the contrary, higher baseline needs corresponded with better kidney functioning . Both results remained significant after adjusting for age (p = 0.0493 and p = 0.0059, respectively). As a conclusion, self-reported non-adherence remained similar over the reporting period and beliefs about immunosuppressants corresponded with clinical outcomes related to both underand overimmunosuppression . The study was supported from the project of Specific Academic Research (SVV 260 551). THEORY-DRIVEN DEVELOPMENT OF A MEDICATION ADHERENCE INTERVENTION DELIVERED BY E-HEALTH AND TRANSPLANT TEAM IN ALLOGENEIC STEM CELL TRANSPLANTATION: THE SMILE IMPLEMENTATION SCIENCE PROJECT RIBAUT, J .,1,2 LEPPLA, L .,1,3 TEYNOR, A .,4 VALENTA, S .,1,2 DOBBELS, F .,1,5 ZULLIG, L . L .,6,7 De GEEST, S .1,5 1 Institute of Nursing Science, Department Public Health, Faculty of Medicine, University of Basel, Switzerland 2 Department of Hematology, University Hospital of Basel, Switzerland 3 Departments of Hematology, Oncology and Stem Cell Transplantation, University Medical Center, Freiburg, Germany 4 Department of Computer Science, University of Applied Sciences, Augsburg, Germany 5 Academic Center for Nursing and Midwifery, Department of Public Health and Primary Care, University of Leuven, Belgium 6 Department of Population Health Science, Duke University, Durham (NC), USA 7 Center of Innovation to Accelerate Discovery and Practice Transformation (ADAPT), Durham Veterans Affairs Health Care System, Durham (NC), USA e-mail: janette .ribaut@unibas .ch Medication adherence to immunosuppressants in allogeneic stem cell transplantation (alloSCT) is essential to achieve favorable clinical outcomes .1 Over 600 apps supporting medication adherence exist, yet they lack successful implementation likely because of lack of end-user involvement and theoretical underpinnings in their development and insufficient attention to implementation methods to support the use in real-life. Medication adherence has 3 phases: initiation, implementation and persistence .2 We report the theory-driven development of a medication adherence intervention (implementation and 103 persistence phase) in alloSCT patients as a first step for future digitization and implementation in clinical setting within the SMILe project (Development, implementation and testing of an integrated care model in allogeneic SteM cell transplantatIon faciLitated by eHealth) . We applied the Behavior Change Wheel (BCW) and Capability-Opportunity-Motivation and Behavior (COM-B) model using 3 suggested stages3 followed by one stage added by our team as preparation for digitization of the intervention: (I) Defining the problem in behavioral terms, (II) Identifying intervention options, (III) Identifying content and implementation options, (IV) SMILe Care Model Development. Scientific evidence, data from a contextual analysis and patients’, caregivers’ and clinical experts’ inputs were compiled to work through these steps . (I) Correct immunosuppressant taking and timing were defined as target behaviors. The focus of the intervention was determined within the COM-B dimensions Capability (e.g. lack of routine), Opportunity (e.g. lack of cues) and Motivation (e.g. lack of problem solving) . (II) Five intervention functions were chosen, such as education, training and enablement . (III) Twenty-four behavior change techniques were selected, e.g. action planning and problem solving . (IV) Finally, 17 user stories were developed to guide the SMILeApp’s software development process . Our example on the theory-driven development of an eHealth powered intervention in alloSCT using a rigorous 3+1-stage approach based on BCW, COM-B and agile software development, can be used as methodological guidance for other eHealth intervention developers . Our approach has the potential to enhance the successful implementation and sustained used of eHealth solutions in real-life . The study was supported by the University of Basel, Switzerland. References 1 . GRESCH, B ., KIRSCH, M ., FIERZ, K . et al .: Bone Marrow Transplant ., 52, 2017, 304–306 . 2 . VRIJENS, B ., De GEEST, S ., HUGHES, D . A . et al .: Br . J . Clin . Pharm ., 73, 2012, 691–705 . 3 . MICHIE, S ., Van STRALEN, M . M ., WEST, R .: Implement . Sci ., 6, 2011, art . 42 . ASSOCIATION OF MATERNAL ANTHROPOMETRIC PARAMETERS AND NUTRITION WITH MILK PRODUCTION DURING LACTATION NAJPAVEROVÁ, S .,1 KOVAŘÍK, M.,1 KAČEROVSKÝ, M.,2 ZADÁK, Z .,3 HRONEK, M .1,2 1 Department of Biological and Medical Sciences, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic 2 Department of Obstetrics and Gynecology, University Hospital Hradec Králové, Czech Republic 3 Biomedical Research Centre, University Hospital Hradec Králové, Czech Republic e-mail: najpaves@faf .cuni .cz Human breast milk is a unique source of newborn nutrition and provides a balanced profile of nutrients. Our study is focused on the evaluation of maternal anthropometric parameters, mainly fat-free mass (FFM), adipose tissue mass, body mass index (BMI), and 104 intake of nutritional energy and macronutrients (NEMI) with respect to breast milk production in Czech women, especially at the end of pregnancy and during the nine months after parturition . Fifty-one healthy Czech pregnant women (volunteers from prenatal courses of University Hospital Hradec Králové) were enrolled to this study . Measurements were done in the last pregnancy period (36th–38th gestational week) and in four lactation phases (3 weeks and 3, 6, 9 months postpartum) . Seven-day nutritional records were assessed by the program NutriDan . The bioimpedance spectroscopic method was applied for body composition analysis . The milk sample was sucked by breast pump after 6 hours of nonbreastfeeding . Data showed a positive correlation between NEMI and milk volume (MV) during the four periods postpartum . At the end of pregnancy, protein intake was positively associated with FFM (p < 0 .05) . Greater representation of maternal FFM is related to higher MV production . Women’s adiposity and BMI negatively correlated with MV . In women with BMI above 28 kg m‒2 at the end of pregnancy milk production was reduced below 1 .5 mL per kg of women body weight 3 weeks after parturition . The study was supported by the Grant Agency of Charles University (Project No. 1306218), by Ministry of Health, Czech Republic – conceptual development of research organization (UHHK, 00179906), by the Research programme Development and Study of Drugs (Progres Q42) and from the project of Specific Academic Research (SVV 260 551). PERSISTENCE WITH LIPID LOWERING MEDICINES IN SLOVENIA DVOŘÁČKOVÁ, S.,1 MALÝ, J .,1 LOCATELLI, I .,2 KOS, M .2 1 Department of Social and Clinical Pharmacy, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic 2 Department of Social Pharmacy, Faculty of Pharmacy, University of Ljubljana, Slovenia e-mail: dvoracsi@faf .cuni .cz Lipid lowering medications, specifically the hydroxymethylglutaryl-coenzyme A reductase inhibitors (statins), are considered to be one the most frequently used medications in Europe as well as in the United States .1 The insuficient medication persistence may lead to decompensated lipidemia or cardiovascular events, related healthcare costs and increased mortality in patients with hyperlipidemia .2 The aim of this study was to perform drug utilization analysis of statins (alone or in combinations) between 2015 and 2019 and to assess the medication persistence in a cohort of patients who iniciated the therapy in 2015 and continued until 2019 . For this purpose the Slovenian health claims data on prescription medicines were applied . The database was obtained from the Health Insurance Institute of Slovenia and contained patient’s and physician’s demography, date of refill, number of drug packages refilled and number of defined daily doses (DDD) per package. Medicine consumption was computed as number of DDD per thousand inhabitans per day (DDD/TID). Discontinuation gap was defined as more than 135 days without medication refill. The analysis was conducted using IBM SPSS, ver . 26 and MS Excel . 105 The preliminary results show that the total annual number of statin prescriptions and patients increased during the study period from 796,514 to 890,940 and from 229,543 to 254,842 (cca 13% of Slovenian population), respectively . The patients during the study period comprised averagely 50 .3% males and the mean age was 68 .3 years . The total consumption of statins was 621.33 DDD/TID, highest consumption was registered for rosuvastatin (361.79 DDD/TID) and atorvastatin (179.83 DDD/TID). The study cohort of new patients on therapy in year 2015 contained 30,111 patients (who received 317,877 prescriptions in the period 2015–2019), 68 .7% of these received at least one prescription in 2016 . The discontinuation gap was captured in 38,067 patients between 2015–2019 . The ongoing results are to be expected . Since the worldwide population is expected to age and the most of the statins are available in generic versions, use of statins therapy could also simultaneously increase,1 which would be a cornerstone for further research and focus to importance of optimal medication persistence . The study was supported from the project of Specific Academic Research (SVV 260 551) and ERASMUS+. References 1. MORTENSEN, M . B ., FALK, E ., SCHMIDT, M .: Circ . Cardiovasc . Qual . Outcomes ., 10 (7), 2017, e003811 . 2. DESHPANDE, S ., QUEK, R . G ., FORBES, C . A . et al.: Curr . Med . Res . Opin ., 33, 2017, 769–778 . PHARMACEUTICAL CARE IN GHETTO THERESIENSTADT 1942–1945 ARNDT, T . Department of Social and Clinical Pharmacy, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic e-mail: arndtt@faf .cuni .cz Today the term “pharmaceutical care” includes an individual approach to the patient, orientation to the patient’s pharmacotherapy, and drug problems. However, this definition is applicable even in the specific conditions of the health care system of the ghetto Theresienstadt .1 This work aims to identify medicaments in the ghetto health care system . The information was available from the archives of the Jewish Museum in Prague . The key documents date from 1944–1945 . The information received will be analyzed from the point of view of dosage form, producer, country of origin, the active ingredient, therapeutic indications etc . I compare the analysis with the health and social situation in the ghetto .2 In this way, I have completely analyzed the document Inventory of the drug warehouse in Theresienstadt and overviews of drug deliveries3 and partly group 13 of the so-called “receipts” (Receipt No. 152–277, concerning medicines in Theresienstadt). From the first of these, I identified 332 drugs (manufactured in pharmaceutical factories), 34 pharmaceutical excipients, and 40 plant drugs. I have not identified 52 drugs yet. Of the dosage forms, there were most tablets and dragees (116 pieces), injections (89), and ointments and creams (22) . The groups of analgesics (28), drugs for women’s diseases (22), and chemotherapeu- 112 Albendazole (ABZ) is an anthelmintic drug used for treatment and control of gastrointestinal nematodes in small ruminants . After the treatment, the excrements contain residuals of the drug and its metabolites and if not handled properly they enter the environment . However, the subsequent fate of this drug in the environment is poorly monitored . Our project reveals the circulation of ABZ in the real farm conditions. The field with fodder plants was fertilized with excrements of treated sheep containing ABZ residuals . The grown plants were fed to infected sheep for two weeks . Afterwards, we have detected traces of ABZ in the plants, sheep rumen content, plasma and the excrements, proving the circulation of this environmentally persistent drug . Furthermore, the effect of detected sub-lethal doses of ABZ and the metabolites on the parasites and the host was monitored . The RNA was isolated from liver of sheep exposed to albendazole doses present in fodder plants from the fertilized field for 14 days and reverse transcribed . Individual mRNAs encoding cytochrome P450 isoforms (CYPs) and UDP-glucuronosyltransferase were quantified by real-time polymerase chain reaction (RT-qPCR) and the data were evaluated by comparative delta-Ct method . Results showed that ABZ in very low dose significantly increased CYP1A2 mRNA and decreased CYP3A4 mRNA in sheep . In conclusion, the chronic effect of even very low doses of ABZ present in the environment may enhance its metabolism when proper dose administered, hence cause the ineffective treatment and facilitate the development of resistance in helminths . The study was supported by the Czech Science Foundation (Project No. 18-07724S) and from the project of Specific Academic Research (SVV 260 550). THE EFFECT OF PYRAZINE DERIVATIVE ON SECONDARY METABOLITES CONTENT IN PLANT CULTURE OF SILYBUM MARIANUM (L .) GAERTN IN VITRO BLAHNOVÁ, K., TŮMOVÁ, L. Department of Pharmacognosy, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic e-mail: blahnovk@faf .cuni .cz The aim of this work was to determine the effect of an abiotic elicitor from the class of pyrazine derivates 1-benzyl-3-(pyrazin-2-yl)urea on the production of secondary metabolites (SM) in the plant culture Silybum marianum (L .) Gaertn .1 Elicitation was performed on both callus and suspension cultures . The elicitor was used in three different concentrations. Particular samples were taken after 6, 24, 48, 72 and 168 hours of elicitor influence. After drying, the callus and suspension tissues were extracted with methanol and the content of the monitored secondary metabolites was determined by HPLC . It was also tested if SM are released into the growth medium . Flavonolignans silybinin A and silybinin B were not detected in any of the analyzed samples . The highest production of SM was achieved after elicitation of suspension cultures . Maximum values of the content were reached by flavonoid taxifolin. The most significant increase of the content was determined at silychristin in callus cultures. Only silydianin was released in significant amounts into 113 the growth media of the cultures . It was shown that the elicitor 1-benzyl-3-(pyrazin-2-yl) urea is able to increase the production of SM in both callus and suspension cultures of milk thistle in certain concentrations and duration of action has an effect on the excretion monitored metabolites into the nutrient medium . The study was supported from the project of Specific Academic Research (SVV 260 550). References 1. DOLEŽAL, M., KRÁLOVÁ, K. Synthesis and Evaluation of Pyrazine Derivatives with Herbicidal Activity. In Herbicides, Theory and Applications. Larramendy, M. L., Soloneski, S. (eds.) IntechOpen, 2011, pp. 581‒610. AMARYLLIDACEAE ALKALOIDS AS MODEL STRUCTURES FOR THE DEVELOPMENT OF NEW POTENTIAL DRUGS KNÁPKOVÁ, S ., HULCOVÁ, D . Department of Pharmacognosy, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic email: knapkovs@faf .cuni .cz The family Amaryllidaceae includes a large number of genera of flowering plants as Hippeastrum, Narcissus or Zephyrantes. All of them contain specific Amaryllidaceae alkaloids, which are characteristic of this family due to their chemical structures . The discovery and study of these alkaloids has attracted attention of many scientists due to the diverse biological activity of these compounds, for example cytotoxic, anticholinesterases, antibacterial, antiviral .1 Plants of the genus Hippeastrum have been used in traditional medicine to treat tumors and inflammatory disorders. This use can be explained by the alkaloids, which it contains . It is mainly lycorine, haemantamine and pancristatine . These compounds have an antitumor effect . The species Hippeastrum cv . Ferrari is further rich in the alkaloid vittatine . In some research, simple semisynthetic derivatives of haemanthamine displayed promising inhibitory activities against cholinesterases . For this reason, vittatine was chosen as next lead-structure for preparation of semisyntetic derivatives .2 Seven new derivatives were prepared by esterification of the alkaloid vittatine and two were created in the form of ethers. All of them were identified by NMR and HRMS analysis . Subsequently, the ability of the derivatives to inhibit human acetylcholinesterase and butyrylcholinesterase (BuChE) was studied . Most of the prepared derivatives showed good inhibitory potential against BuChE . The best of them shown activity with IC values 1 .39 ± 0 .08 µM . The study was supported by EFSA CDN (Reg. 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 260 548). References 1. CAHLÍKOVÁ, L., LOČÁREK, M., CHLEBEK, J. et al.: Nat. Prod. Commun., 8, 2013, 781‒785. 2. AL SHAMMARI, L., HULCOVÁ, D., MAŘÍKOVÁ, J. et al.: S. Afr. J. Bot., 136, 2021, 137‒146. 114 INDOLE ALKALOIDS FROM VINCA MINOR AND THEIR BIOLOGICAL ACTIVITY ČEŘOVSKÁ, E.,1 KOHELOVÁ, E .,1 MAŘÍKOVÁ, J.,2 HULCOVÁ, D .,1 CHLEBEK, J .1 1 ADINACO Research Group, 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: cerovsel@faf .cuni .cz Vinca minor L . is an evergreen perennial plant belonging to the Apocynaceae family . Phytochemical screening of its biologically active constituents revealed a wide range of indole alkaloids (IAs) . To date, more than 50 IAs have been reported in V. minor .1 Several of these indole alkaloids, such as vincamine, are frequently used as a remedy due to their cerebrovasodilatory and neuroprotective activity . In addition, some bisindole alkaloids such as vincarubine, exhibit remarkable activity on leukemic cell lines .1,2 The summary ethanolic extract was prepared from dried aerial plants of V. minor (63 kg) and separated by commonly used chromatographic techniques (column chromatography, flash chromatography, preparative TLC). So far, four alkaloids have been obtained from two fractions. The isolated IAs were identified by comparison of obtained analytical data (MS, NMR, optical rotatory) with the literature data. The alkaloids isolated in a sufficient quantity were assayed for their biological activities connected to Alzheimer’s disease (inhibition of cholinesterases, inhibition of prolyloligopeptidase, ability to cross the blood-brain barrier) and anticancer potential (cytotoxicity against panel of tumor and leukemic cell lines). Significant cytotoxic activity was demonstrated by a novel bisindole alkaloid named vincaferugine . The study was supported from the project of Specific Academic Research (SVV 260 548). References 1. ABOUZEID, S ., HIJAZIN, T ., LEWERENZ, L . et al .: Phytochemistry, 168, 2019, 1‒9. 2. PROKSA, B., GROSSMANN, E.: Phytochem. Anal., 2, 1991, 74‒76. ISOLATION OF ALKALOIDS FROM NARCISSUS POETICUS var . RECURVUS AS POTENTIAL DRUGS IN THE TREATMENT OF ALZHEIMER’S DISEASE HASANOVÁ, S ., HULCOVÁ, D . Department of Pharmacognosy, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic e-mail: hasanovs@faf .cuni .cz Plants of the Amaryllidaceae family are known to contain a specific type of compounds, namely the Amaryllidaceae alkaloids . Among these alkaloids, the most important one is galanthamine that is approved for the pharmacological treatment of Alzheimer’s disease (AD) .1 AD is the most prevalent neurodegenerative disease worldwide with complex eti- 115 ology and multifaceted pathophysiology . Based on the various causative factors of AD, several hypotheses, including cholinergic, amyloid, τ-protein, calcium dyshomeostasis, and isoprenoid change, have been put forward . Focused on the cholinergic hypothesis, a deficit of the neurotransmitter acetylcholine (ACh) in the cortex results in a cognitive deterioration . ACh levels can be maintained via inhibition of acetylcholinesterase .2 Important source of Amaryllidaceae alkaloids mentioned above is the genus Narcissus . Extract from Narcissus poeticus var . recurvus was screened at the Department of Pharmaceutical Botany for the first time in 2013.3 Subsequent phytochemical research began with 29 kg of fresh bulbs from which 42 g of alkaloid extract was obtained . About 6 g of lycorine was isolated from this extract. The rest of the extract was evaluated for its alkaloid profile by GS/MS and HPLC. This was followed by separation using flash chromatography. Finally, 18 fractions were obtained which in turn were evaluated by GS/MS and HPLC. Individual fractions are processed by preparative TLC . The study was supported by EFSA-CDN (Reg. 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 260 548). References 1. CAHLÍKOVÁ, L., LOČÁREK, M., CHLEBEK, J. et al.: Nat. Prod. Commun. 8, 2013, 781‒785. 2. HULCOVÁ, D., MAŘÍKOVÁ, J., KORÁBEČNÝ, J. et al.: Phytochemistry, 165, 2019, art . 112055 . 3. HRSTKA, V.: Neurotropní a antioxidační aktivita vybraných druhů jednoděložných alkaloidních rostlin IV (Neurotropic and antioxidant activity of selected species of monocotyledonous plants containing alkaloids IV) . Diploma thesis, Faculty of Pharmacy in Hradec Králové, Charles University, 2013 . MONITORING OF ALBENDAZOLE TRANSFER FROM OVINE FAECES TO FODDER PLANTS SOCHOVÁ, A ., NAVRÁTILOVÁ, M ., SKÁLOVÁ, L . Department of Biochemical Sciences, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic e-mail: sochovaan@faf .cuni .cz Albendazole (ABZ) belongs to a benzimidazole group of anthelmintic drugs . These drugs are regularly and frequently used to limit and treat parasitic infections in animals . In this way, the consumption of these drugs increases and their negative effects on non-target organisms and the environment is extended .1 We know that the plants can uptake and even biotransform the ABZ in laboratory-controlled conditions .2 The present study monitors the transfer of ABZ and its transformation products (TPs) from the faeces of treated sheep to common fodder plants Medicago sativa and Trifolium pratense . We wanted to know if there is a possibility of transferring these compounds into soil and plants in real field conditions. Our study successfully revealed the occurrence of ABZ TPs (ABZ-SO and ABZ-SO2) in fodder plants. Even two months after the first contact of fodder plants with faeces, ABZ-SO was still present . The highest concentration of TPs was observed in the 1st and 116 2nd week after the application of faeces . Then, the amount of TPs in plants decreased during the time, except in May, where a slight increase was observed, probably due to higher precipitation . The presence of the TPs in fodder plants represents not only a danger to herbivorous invertebrates, but also may play an additional role in the development of ABZ resistance in helminths . The study was supported by the Grant Agency of Charles University (Project No. 1136120) and from the project of Specific Academic Research (SVV 260 550). References 1. DOBŠÍKOVÁ, R., ŠIROKÁ, Z.: Farmakologie v produkci potravin a rezidua léčiv v potravinách (Pharmacology in food production and drug residues in food) [online] . Brno, Veterinární a farmaceutická univerzita Brno, 2014 . 2. RAISOVÁ STUCHLÍKOVÁ, L., NAVRÁTILOVÁ, M., LANGHANSOVÁ, L. et al .: Int . J . Mol . Sci . 21, 2020, art . 5943 . SEMI-SYNTHETIC DERIVATIVES OF β-CARBOLINE ALKALOID HARMINE AND THEIR BIOLOGICAL ACTIVITY FEJTKOVÁ, M .,1 KOHELOVÁ, E .,1 CHLEBEK, J .,1 MAŘÍKOVÁ, J.,2 HULCOVÁ, D .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: fejtkovmar@faf .cuni .cz Alzheimer’s disease (AD) is a serious and irreversible progressive neurodegenerative disorder, that will reach a prevalence of more than 100 million by 2050 due to the aging of population. The main pathological hallmarks of AD are intracellular neurofibrillary tangles, extracellular amyloid plaques, increased oxidative stress and cholinergic dysfunction . Cholinergic neurotransmission is terminated by acetylcholine hydrolysis regulated by two enzymes: acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) .1,2 Alkaloids play an important role in the treatment of AD . Well-known Amaryllidaceae alkaloid galanthamine is a marketed drug for AD therapy under the commercial name Reminyl© .1 β-Carboline alkaloid harmine, isolated from Peganum harmala (Nitrariaceae), lacks any significant activity against human cholinesterases. However, several new derivatives of H3CO harmine N H N CH3 Br R H3CO CH3 N N R 117 harmine prepared by the N-9 derivatization showed interesting inhibition BuChE . Newly prepared compounds were identified by NMR and ESI-MS methods. The most active compounds will be studied in more detail (e.g . type of inhibition, docking studies, logBB etc .) . The study was supported from the project of Specific Academic Research (SVV 260 548). References 1. AL MAMUN, A., MAŘÍKOVÁ, J., HULCOVÁ, D. et al .: Biomolecules, 10, 2020, art . 800 . 2. KOŠAK, U., BRUS, B., KNEZ, D. et al.: J. Med. Chem., 61, 2018, 119‒139. GILTERITINIB AS OCT1 INHIBITOR AND SUBSTRATE: POTENTIAL FOR DRUG-DRUG INTERACTIONS? NOVOTNÁ, K., ŠORF, A., ČEČKOVÁ, M. Department of Pharmacology and Toxicology, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic e-mail: novotnakate@faf .cuni .cz Gilteritinib is one of the recently approved drugs which is primarily used in the treatment of relapsed/refractory acute myeloid leukemia (AML) with mutated FMS-like tyrosine kinase 3 (FLT3) receptor . In this project, gilteritinib was investigated in terms of its ability to interact with solute carriers (SLC) membrane transporters, namely with OCT1 and OCT2 . These membrane proteins play a role in uptake of endogenous compounds and also drugs into the cells of main elimination organs (liver, kidney), but also to cancer cells . In particular, we wanted to examine potential interaction with daunorubicin, a drug traditionally used in AML therapy . First, we performed accumulation study and evaluated, whether gilteritinib is potential inhibitor of OCT1 studying differential uptake of daunorubicin into MDCKII-OCT1 cells based on OCT1 inhibition by gilteritinib . Secondly, the study evaluating the transfer of gilteritinib across the monolayers of MDCKII-OCT1 and control MDCKII-VK cell lines was conducted to test gilteritinib as a potential substrate of this transporter . The obtained data showed that gilteritinib has ability to inhibit the OCT1-mediated transport of daunorubicin into the MDCKII-OCT1 cells . This effect was not observed neither in control cell line, nor MDCKII-OCT2 cells . We further observed enhanced basolateral-to-apical transport of gilteritinib across monolayers of MDCKII-OCT1 cells compared to MDCKII-VK cells . This difference was abolished in the presence of OCT1 inhibitor, suggesting that gilteritinib is a substrate of OCT1 . Results obtained in our study indicate that gilteritinib might be prone to OCT1-mediated pharmacokinetic drugdrug interactions . The hypothesis that combinatory treatment of AML with gilteritinib and daunorubicin could result in decreasing availability of the drugs to the leukemia cells leading to lower efficacy of the treatment should be verified. The study was supported by the Research program Pharmacokinetic mechanisms of drug resistance in acute myeloid leukemia, their affecting and regulation (PRIMUS/20/ MED/010) and from the project of Specific Academic Research (SVV 260 549). 118 NEW INHIBITORS OF TOPOSIOMERASE II – STUDY OF ANTIPROLIFERATIVE EFFECTS AND THEIR INFLUENCE ON ANTITUMOR ACTIVITY OF ETOPOSIDE OHNEMICHLOVÁ, L., KUBEŠ, J., ŠIMŮNEK, T. Department of Biochemical Sciences, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic e-mail: ohnemicl@faf .cuni .cz Cancer is a serious societal health problem, which can affect each of us, and its incidence increases rapidly with age . Anthracycline antibiotics (ANT) are extremely effective drugs that have been used to treat a number of cancers, namely breast cancer, small cell bronchogenic cancer, recurrent ovarian cancer and many more . They have a multimodal mechanism of action, one of them is the inhibition of topoisomerase II (TOP2), an essential cellular enzyme modulating DNA topology . Currently, use of ANTs is limited due to concerns about the occurrence of cardiotoxicity . Etoposide (ETO) is an antitumor agent that acts as topoisomerase poison . ETO is often used in combination therapy with ANTs, but it is not considered cardiotoxic . Dexrazoxane (DEX) is the only approved cardioprotectant against cardiotoxicity of ANTs . However, it does not act as chelating inhibitor, as previously thought, but through inhibition of TOP2 . Although DEX has been shown to be a very potent cardioprotectant, it also has its disadvantages . It has been associated with the occurrence of secondary malignancies, most often with acute myeloid leukemia, less with myelodysplastic syndrome, acute lymphoblastic leukemia or non-Hodgkin’s lymfoma . Hence, it is necessary to examine other agents .1 The aim of the study was to determine whether other inhibitors of TOP2, namely (BNS-22, XK-469, ICRF-193) can affect antiproliferative effect of ETO on the HL-60 leucemic cell line . For the tested agents, concentrations corresponding with their IC50 were determined and on the basis of this concentration their antiproliferative effects were evaluated both alone and in combination with ETO . For determination of cell viability, MTT aasay was used . The results seem promising and some of the agents even show synergistic effect in combination with ETO . The study was supported by the Czech Science Foundation (Project No. 18-08169S) and by the Grant Agency of Charles University (Project No. 246219) and from the project of Specific Academic Research (SVV 260 550). References 1. VEJPONGSA, P., YEH, E. T. H.: Clin. Pharmacol. Ther., 95 (1), 2013, 45‒52. 119 SCREENING OF INHIBITORY ACTIVITY AGAINST CHOLINESTERASES OF VARIOUS SPECIES OF THE GENUS FICUS II JANOVCOVÁ, H .,1 HANUSOVÁ, A .,1 HULCOVÁ, D .,1 CAHLÍKOVÁ, L.2 1 Department of Pharmacognosy, 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: janovcoh@faf .cuni .cz Alkaloids are some of the most interesting substances of secondary metabolism . These compounds are synthesized in plants from amino acids or their derivatives and contain nitrogen in the structure that is incorporated into heterocycle in most cases .1 Alkaloids exhibit a variety of biological activities as cytotoxic, anti-inflammatory, antifungal, antimalarial or anticholinesterase . The anticholinesterase effects of alkaloids include inhibition of human enzymes acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE), which can be used in therapy of Alzheimer’s disease (AD) . AD is the most common form of dementia, manifested by impairment of cognitive and noncognitive functions that eventually lead to death .2 The genus Ficus belongs to the family Moraceae, which is widespread in tropical and subtropical regions of the Western Pacific area. Plants from this genus contain large amounts of substances, including alkaloids .3 For the phytochemical study, nine extracts from leaves and stems from 6 species of the genus Ficus were prepared . Dry material was ground and extracted by boiling in ethanol . Then ethanolic extracts were purified by liquid-liquid extraction (ether, ethyl acetate,chloroform). All extracts were tested for their ability to inhibit AChE and BuChE . Only extract AL708E showed inhibition activity against human BuChE (53 .29 ± 0 .03% at concentration 50 µg mL−1) . The study was supported by EFSA-CDN (Reg. 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 260 548). References 1. HULCOVÁ, D., MAŘÍKOVÁ, J., KORÁBEČNÝ, J. et al .: Phytochemistry, 165, 2019, art . 112055 . 2. CAHLÍKOVÁ, L., MACÁKOVÁ, K., BENEŠOVÁ, N. et al.: Chapter 6 ‒ Natural Compounds (Small Molecules) as Potential and Real Drugs of Alzheimer’s Disease: A Critical Review . In: Studies in Natural Products Chemistry, Vol . 42, Atta-ur-Rahman (ed .), Amsterdam, Elsevier, 2014, pp . 153–194 . 3. KUBO, M ., YATSUZUKA, W ., MATSUSHIMA, S . et al.: Chem. Pharm. Bull., 64, 2016, 957‒960. SCREENING OF INHIBITORY ACTIVITY AGAINST CHOLINESTERASES OF VARIOUS SPECIES OF THE GENUS FICUS I . HANUSOVÁ, A ., JANOVCOVÁ, H ., HULCOVÁ, D . Department of Pharmacognosy, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic e-mail: hanusovaan@faf .cuni .cz 120 Following scientific research focuses on selected Ficus species and their potential usage in treatment of Alzheimer’s disease (AD) thanks to alkaloids contained in them . AD is one of the most common diseases of the elderly and one of the most frequent cause of dementia . Currently, AD is incurable . The only thing that can be done is to slow down progression of the symptoms . Inhibition of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) plays an important role in this process .1,2 Alkaloids are substances of secondary metabolism that are usually synthesized from amino acids or their derivatives and can be found in various plant organs . Alkaloids have a number of interesting biological effects . For example, galanthamine inhibits AChE and therefore it is used for treatment of AD .1 Plants of genus Ficus belonging to Moraceae family include nearly one thousand species . These are evergreen trees that can be found mainly in tropical and subtropical areas of West Pacific. They contain alkaloids of various structures, which have antimalarial, antibacterial and anticancer activity .3 The aim of this research was to screen several extracts as potential sources of substances for the treatment of AD . Ten extracts of 8 selected species of genus Ficus were subjected to a phytochemical study . Dry grinded leaves and stems were extracted by boiling in ethanol. Obtained ethanol extracts were purified by liquid-liquid extraction using ether, ethyl acetate and chloroform . All extracts were tested on the ability to inhibit AChE and BuChE . Ability to inhibit BuChE showed only the extract AL-700C (74 .82 ± 2 .78% at concentration 50 µg mL−1) . The study was supported by EFSA-CDN (Reg. 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 260 548). References 1. HULCOVÁ, D., MAŘÍKOVÁ, J., KORÁBEČNÝ, J. et al .: Phytochemistry, 165, 2019, art . 112055 . 2 . HULCOVÁ, D ., BREITEROVÁ, K ., SIATKA T . et al .: Molecules, 23, 2018, art . 719 . 3. KUBO, M ., YATSUZUKA, W ., MATSUSHIMA, S . et al .: Chem. Pharm. Bull., 64, 2016, 957‒960. MONITORING THE SPREAD OF ALBENDAZOLE FROM THE SHEEP FAECES IN AGRICULTURAL LAND BY LC-MS VRÁBĽOVÁ, D., NAVRÁTILOVÁ, M., SKÁLOVÁ, L. Department of Biochemical Sciences, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic e-mail: vrabldan@faf .cuni .cz The anthelmintic drug albendazole (ABZ) with a broad-spectrum anthelmintic effect is used for the treatment of helminthiasis caused mainly by gastrointestinal worms in veterinary and human medicine . Frequent dose, overdose or underdose make a risk of developing resistance, which can be a serious global problem in the treatment of helminthiasis . The anthelmintics can enter the environment unchanged as a parent compound or as a metabolite through the faeces of treated animals . These chemicals can be absorbed into plants, soil and groundwater and they can have a negative impact on the life and growth of smaller organisms . 121 This experiment aimed to monitor the distribution of albendazole and its transformation products (TPs) albendazole sulfoxide (ABZ-SO) and albendazole sulfone (ABZ-SO2) from the faeces of treated domestic sheep in agricultural land . This was observed from different distances and depths to which the compounds could spread . In general, the concentration of the compounds in the soil was greater in the topsoil than in the bottom layer and closer to the faeces . We also observed the dependence of changes in substance concentration on rainfall (dry and rainy months) . The parent drug and TPs were extracted from soil using the solid-phase dispersion extraction (QuEChERS) and identification and quantification were done by UHPLC-MS . The study was supported by the Grant Agency of Charles University (Project No. 1136120) and from the project of Specific Academic Research (SVV 260 550). References 1. PRCHAL, L ., PODLIPNÁ, R ., LAMKA, J . et al.: Environ . Sci . Pollut . Res ., 23, 2016, 13015–13022 . THE EFFECTS OF TOPOISOMERASE II BETA ON THE SENSITIVITY OF THE CANCER CELLS TO THE ANTINEOPLASTICS JAŠČEVSKÁ, N., SKALICKÁ, V., JIRKOVSKÁ, A. Department of Biochemical Sciences, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic e-mail: jascevsn@faf .cuni .cz Topoisomerase II is a cellular enzyme responsible for solving topological problems of double-stranded DNA. Topoisomerase IIα and IIb isoforms are various gene products having conserved catalytic activities. The TOP IIα isoform is present in proliferating cell, while TOP IIb isoform is predominantly present in non-proliferating cells, especially neurons . Anthracycline antibiotics targeting TOP IIb are currently among the most effective anticancer drugs . The main factor limiting their clinical use is the development of side effects – especially myelotoxicity and cardiotoxicity . The mechanism of anthracyclineinduced cardiotoxicity is still not fully elucidated . Nowadays, the most accepted theory is the ability of anthracyclines to produce reactive oxygen species damaging the heart muscle by oxidation . However, the mechanism is probably multifactorial . Nevertheless, the key role of inhibition of the TOP IIb enzyme, which is present in cardiomyocytes, has been increasingly discussed . The only clinically used cardioprotective is dexrazoxane . Even its mechanism of action is not fully understood . However, new studies suggest the major mechanism of action through TOP IIb located in cardiomyocytes (depletion after dexrazoxane exposure) .1 The practical aim of this project was to investigate the differences in the antiproliferative effects of daunorubicin and dexrazoxane in human cell suspension line of HL-60 tumor cells with various TOP IIb expression . Further to determine the amount of TOP IIα and TOP IIb mRNA in these cell lines by RT-qPCR and the amount of protein by immunoblotting . Primary screening of the designed primers at the TOP IIb enzyme mutation site was also performed . 128 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: stemberma@faf .cuni .cz 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 DielsAlder reaction, in which complex polycyclic compounds can be obtained in single step (Scheme 1, 2) . Fig. 23 Scheme 1. Structure and reactivity of dendralenes Our goal was to prepare new dendralenes (Scheme 2, 5), substituted predominantly with electron withdrawing groups (EWG) . The procedures previously developed by our research group based on Migita-Stille coupling of compounds 3 and 4 .2 Furthermore, their ability to undergo Diels-Alder reaction was investigated (Scheme 2, 6) . Fig. 24 Scheme 2. Preparation and potential reactivity of EWG-[3]dendralenes The study was supported by the Czech Science Foundation (Project No. 18-17868S) and from the project of Specific Academic Research (SVV 260 547). References 1. HOPF, H ., SHERBURN, M . S .: Angew . Chem . Int . Ed ., 51, 2012, 2298–2338 . 2. KRATOCHVÍL, J., NOVÁK, Z., GHAVRE, M. et al .: Org . Lett ., 17, 2015, 520–523 . 129 TESTING OF (−)-N-DODECYL-N-METHYLEPHEDRINIUM BROMIDE AS A CHIRAL SELECTOR IN CAPILLARY ELECTROPHORESIS ENANTIOSEPARATIONS MŰLLEROVÁ, K., JÁČ, P., POLÁŠEK, M. Department of Analytical Chemistry, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic e-mail: mullerovak@faf .cuni .cz Chiral ionic liquid (−)-N-dodecyl-N-methylephedrinium bromide (DMEB) was tested as a selector for chiral separations of selected drugs such as chiral quinolones, ketoprofen, and flurbiprofen by capillary electrophoresis (CE). The effect of several parameters on enantio-separation was examined: (i) type and pH of the separation buffer, (ii) type and amount of organic modifier, and (iii) the concentration of DMEB. When using this chiral selector, only the separation of ofloxacin enantiomers was observed, while the enantio-recognition of other chiral model analytes was not successful under the conditions tested . A CE method for the assay of levofloxacin was developed to demonstrate the potential of DMEB as a chiral selector in quality control of single-isomer drugs . The best separation was achieved with 20 mmol L−1 tris buffer of pH 8 .5 containing 100 mmol L−1 DMEB and 20% (v/v) of acetonitrile as the background electrolyte. The separation took place in 50 µm id fused silica capillary (80.5 cm / 72 cm) at −30 kV with UV detection at 291 nm. The resolution between the peaks of ofloxacin enantiomers was 4.22 ± 0.02 (n = 3). Linearity of the method was proved for the range 10 to 100 µg mL−1 of levofloxacin (y = 0.0305x − 0.0107, R2 = 0.9975), gatifloxacin (40 µg mL−1) was employed as internal standard . The method was applied to the analysis of tablets containing 500 mg of levofloxacin. The content determined was 100.1 ± 4.6% (n = 3) of the declared amount of levofloxacin. Hence, prospective applicability of DMEB as chiral selector in pharmaceutical analysis of single-isomer drugs was demonstrated . The study was supported from the project of Specific Academic Research (SVV 260 548). SYNTHESIS AND DYNAMICS OF DEUTERIUM-LABELED ACYLCERAMIDES HAVRIŠÁK, T ., OPÁLKA, L . Department of Organic and Bioorganic Chemistry, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic e-mail: havrisat@faf .cuni .cz Acylceramides are important components of skin permeability barrier where they are responsible for a formation of the long periodicity lamellar phase and corneocyte lipid envelope . These structures are indispensable for preventing water loss from human body and penetration of undesired components of the environment . Lower levels of acylceramides 130 usually accompany skin diseases like psoriasis and atopic dermatitis . Although acylceramides are essential for proper skin barrier function, little is known about their dynamics in skin . One of the methods allowing us to study the molecular dynamics is solid-state NMR, which requires molecules with partial or complete deuteration . In this project, we decided to synthesize acylceramides with deuteration in their ultralong (32 carbons) chain . First, the synthesis was optimized using 1H compounds and based on this optimization, deuterated acylceramides will be prepared . The synthesis started from butyrolactone and 1,12-dibromododecane which are commercially available in their deuterated form . These starting compounds were transformed into a phosphonium salt and an aldehyde for Wittig reaction, providing a 16-carbon fragment (protected ω-hydroxylated unsaturated acid), which after a modification underwent a second Wittig reaction to create the 32-carbon long chain. This precursor was then esterified with linoleic acid and connected to a sphingoid base to form a final molecule of acylceramide. The optimized synthesis using 1H compounds was performed in 13 steps with approximately 15% overall yield, opening the possibility to synthesize acylceramides with deuteration in half of the ultralong chain (Fig . 1) . In similar manner, acylceramides with deuteration in other half of the chain will be prepared . The study was supported by the Czech Science Foundation (Project No.19-09135J) and from the project of Specific Academic Research (SVV 260 547). SYNTHESIS AND IN VITRO CARDIOPROTECTIVE ACTIVITY OF DEXRAZOXANE ANALOGUES MACUŠ, M ., ROH, J ., KARABANOVICH, G . Department of Organic and Bioorganic Chemistry, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic e-mail: macusm@faf .cuni .cz Anthracylines (ANTs) as daunorubicin, doxorubicin, etc ., are used as anti-cancer drugs . ANTs have a strong anti-tumor effect . Unfortunately, a limiting factor of ANTs use in clinical practice is their serious cardiotoxicity . This side effect can lead to heart failure via irreversible damage of heart muscle cells . The only clinically approved drug used against ANTs-induced cardiotoxicity is bisdioxopiperazine derivative dexrazoxane Fig. 25 Fig. 1. Structure of acylceramides with deuteration in half of the ultralong chain 131 (DEX) . Mechanism of action of DEX is intensively investigated and there are still two main theories: i) chelation of intracellular iron ions that protects hearth against oxidative damage, ii) inhibition/depletion of topoisomerase IIβ in cardiomyocytes. Synthesis and thorough evaluation of new DEX derivatives should help to clarify DEX structure–activity relationship . This study aimed at the preparation of a DEX analogue with a modified linker (EMD). The synthesis of the target compound started from pentane-2,3-dione and there were 6 more steps that lead to erythro and threo forms of 2,3-diaminopentane-N,N,N′,N′-tetraacetic acid, that were separated by column chromatography in the form of tetraesters. The final steps consisted of the cyclization of bisdioxopiperazine rings. Both final diastereomeric products (EMDa and EMDb) were studied for their ability to chelate iron ions, to inhibit TOPIIβ and to protect cardiomyocytes against ANT toxicity. The results of this work contribute to the study of DEX mechanism of action and to the discovery of new and more effective cardioprotective drugs . The study was supported from the project of Specific Academic Research (SVV 260 547). MONITORING OF LIBERATION TESTS FOR THE RELEASE OF CLOTRIMAZOLE FROM NANOFIBERS ERNEST, R., HÁKOVÁ, M., SKLENÁŘOVÁ, H. Department of Analytical Chemistry, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic e-mail: ernestra@faf .cuni .cz The study was focused on the analysis of release profiles of clotrimazole from different types of nanofibers using non-separation flow technique, sequential injection analysis (SIA). Finding ideal conditions for saturation of the fibers with a solution of clotrimazole was another goal. Nanofibers were produced at the Technical University of Liberec. Two types of nanofiber carriers – polymeric (polydioxanone and polycaprolactone) and inorganic (silica) – were employed. Part of the fiber of polydioxanone and polycaprolactone was saturated directly during production in different ratios of monomer and clotrimazole . The saturated fibers were then analyzed in the laboratory. The second part of polydioxFig. 26 Fig. 1. Structure of DEX and synthesis of DEX analogue EMD 132 anone and polycaprolactone together with inorganic nanofibers was produced in a pure state, i.e., without the active substance. These fibers were tested in the laboratory for saturation with an ethanolic solution of clotrimazole at a certain concentration for a certain time . The saturation conditions were changed during the work to closely monitor the relation between the saturation conditions and the released concentration of clotrimazole . The measurements were performed under well-defined conditions that simulated intact healthy human skin at the temperature of 32 °C and pH of the buffer solution of 4 .5 . Under these conditions, nanofiber membranes with bound clotrimazole were placed in 3 Franz cells connected in parallel to a single SIA system and liberated levels of clotrimazole in the acceptor medium were monitored for 135 min . Samples were aspirated from this medium in regular 15 min intervals into a SIA system and the content of clotrimazole was determined online using UV-VIS detection. The resulting release profiles of clotrimazole from individual nanofibers were compared. The main monitored parameters were the release rate of clotrimazole into the acceptor medium, the concentration and the liberation profile of clotrimazole . More detailed results will be discussed in the presentation . The study was supported from the project of Specific Academic Research (SVV 260 548). SYNTHESIS OF WATER-SOLUBLE ANALOGUES OF N-SUBSTITUTED 1,2,4-TRIAZOLES WITH HIGH ANTIMYCOBACTERIAL ACTIVITY VOLFOVÁ, G .,1 STOLAŘÍKOVÁ, J.,2 ROH, J .,1 KARABANOVICH, G .1 1 Department of Organic and Bioorganic Chemistry, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic 2 Laboratory for Mycobacteria and Tuberculosis, Institute for Public Health in Ostrava, Czech Republic e-mail: volfovag@faf .cuni .cz Recently, it was shown that 3,5-dinitrophenyl-1,2,4-triazoles have high activity against susceptible and drug-resistant strains of Mycobacterium tuberculosis with the minimum inhibitory concentration of 0 .06–1 µM .1 Thus, this work aimed at the synthesis of five water-soluble derivatives of 3,5-dinitrophenyland 3-nitro-5-trifluoromethylphenyl1,2,4-triazoles . The synthesis of 4,5-substituted-1,2,4-triazole-3-thiols started from corresponding hydrazides and alkyl isothiocyanates followed by cyclization of obtained thiosemicarbazides in aqueous potassium hydroxide (Scheme 1) . The alkylation of 1,2,4-triazole-3-thiols with piperidine or piperazine containing alkylation agents resulted in five final compounds in moderate yields (39–67%). Moreover, three by-products were isolated . All prepared compounds were evaluated for their antimycobacterial activities against M. tuberculosis My 331/88, M. avium My 330/88 and M. kansasii My 235/80. The current work contributed to the discovery of water-soluble compounds with high efficiency that could be subjected to in vivo evaluation . 133 The study was supported from the project of Specific Academic Research (SVV 260 547). References 1. KARABANOVICH, G ., DUŠEK, J ., SAVKOVÁ, K . et. al .: J . Med . Chem ., 62, 2019, 8115–8139 . EVALUATION OF POSTANTIBIOTIC EFFECT AND POSTANTIBIOTIC SUB-MINIMUM INHIBITORY CONCENTRATION EFFECT OF CONVENTIONAL ANTI-INFECTIVE DRUGS ACTING AGAINST METHICILLIN-RESISTANT STAPHYLOCOCCUS AUREUS FIALOVÁ, K., KONEČNÁ, K., JANĎOUREK, O. Department of Biological and Medical Sciences, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic e-mail: [email protected] Antimicrobial resistance is a global, multifaceted phenomenon with severe consequences, including complicated treatment, health cost, and higher mortality . Therefore, this situation needs to be properly reflected.1 The research and discovery of new anti-infectives represent one of the options for combating this phenomenon . A wide range of parameters is determined in preclinical studies of new candidate anti-infective compounds . Among them, the postantibiotic effect (PAE) and postaantibiotic sub-minimum inhibitory concentration (PAE-SME) should be evaluated . PAE and PAE-SME are important parameters of antibiotic action, widely used as a predictor of pharmacodynamic activity .2 In our study, we have evaluated PAE and PAE-SME of three conventional antibiotics, namely ciprofloxacin, vancomycin and linezolid, acting against methicillin-resistant Staphylococcus aureus . A computerized incubator Bioscreen C was employed in the study of the two parameters mentioned above . For PAE evaluation, bacteria were exposed to three different final concentrations of drugs (5×, 10× and 20× MIC), and in the study of parameter PAE-SME, the subinhibitory concentration corresponding to 0 .1×, 0 .2×, 0 .4×, 0 .6×, and 0 .8× MIC were chosen for evaluation . The study was supported by the Czech Science Foundation (Project No. 20-19638Y). Fig. 27 Fig. 27 Fig. 27 Scheme 1. Synthesis of target trisubstituted 1,2,4-triazoles 134 References 1. FRIEDMAN, N . D ., TEMKIN, E ., CARMELI, Y .: Clin . Microbiol . Infect ., 22, 2016, 416–422 . 2. CHEN, H ., LI, L ., LIU, Y . et al .: Infect . Drug Resist ., 11, 2018, 2107–2115 . CREATION OF A VIRTUAL LIBRARY OF SYNTHETIC COMPOUNDS AND ITS PRACTICAL USAGE FOR DOCKING WITH ALDOSE REDUCTASE VÁVROVÁ, J., KUČEROVÁ-CHLUPÁČOVÁ, M. Department of Pharmaceutical Chemistry and Pharmaceutical Analysis, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic e-mail: vavrovajit@faf .cuni .cz Drug development is a process requiring the analysis of a large amount of data .1 Creating a virtual database of synthesized compounds provides access to primary data concerning structure, results of biological activity studies, and molecular descriptors necessary for druglike prediction . This work represents a continuation of the previous study .2 Microsoft Excel was used to create the database, which includes different structural types, e.g . pyrazine, rhodanine, thiazolidin-2,4-dione, and 1,2,4-oxadiazole derivatives prepared in the research group Design and Development of New Antimicrobial Agents . Molecules are sorted according to structure-similarity, nevertheless, we also provide a spreadsheet containing all compounds in a line-notation ready-to-dock format . To demonstrate this database’s actual usage, a molecular modelling study with enzyme aldose reductase was performed using the software Molecular Operating Environment (MOE). Aldose reductase is the first enzyme in the polyol-pathway, involved in microvascular complications of diabetes . It has become a drug target for aldose reductase inhibitors (ARI), e.g . epalrestat . The main goal of the project was to stress the correlation between in vitro and in silico studies . The isosteric approach was used to predict the binding mode and affinity towards the enzyme in order to test the suitability to synthesize new compounds in the upcoming research . The study was supported from the project of Specific Academic Research (SVV 260 547). References 1. PATRICK, G . L .: An Introduction to Medicinal Chemistry, 6th ed ., Oxford, Oxford University Press, 2017 . 2. KEBAKUILE, L . G . B .: Creation and analysis of in-house database of pyrazine derivatives with potential antimicrobial activity . Diploma thesis . Faculty of Pharmacy in Hradec Králové, Charles University, 2018 . MODIFICATION OF CAPILLARY WALL BY GRAPHENE FOR SEPARATION APPLICATIONS PTÁČKOVÁ, A., KUČERA, R., LOCHMAN, L. Department of Pharmaceutical Chemistry and Pharmaceutical Analysis, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic e-mail: ptackovan@faf .cuni .cz 135 Capillary electrophoresis (CE) is a highly efficient separation method. Substances are separated due to different mobility in an electric field. The CE modes of operation can be modified in different ways, e.g . capillary electrochromatography, micellar electrokinetic chromatography .1 On the other hand, modification of the inner wall of the capillary is believed to substantially improve both separation efficiency and selectivity. Graphene (G) is carbon with a hexagonal structure in form of two-dimensional sp2 single-atom-thick sheets . G seems to be a suitable material for separation application due to its excellent properties such as large surface area together with affinity to carbon ring structures via π-π interactions .2 Our work is focused on the modification of the capillary wall by graphene. The wall of the capillary was modified by the Layer-by-Layer method via layering of differently charged substances bounded by electrostatic forces .3 Chemical coating employing covalent interactions was performed as well .4 Different combinations of polymer (PDDA, PAH, PEI, APTES) and G were used for surface modification. Separation efficiency and selectivity of modified capillaries were studied on the model mixtures of analytes (parabens, nitrophenols, nitroanilines, purines) . Obtained results were compared with commercial unmodified capillary. Improved separation together with prolonged interaction of analytes with G surface was observed . The study was supported by the Research programme Development and Study of Drugs (Progres Q42) and from the project of Specific Academic Research (SVV 260 547). References 1. LAUER, H . H ., ROZING, G . P .: A Primer, Agilent Technologies, Germany 2014 . 2. SITKO, R ., ZAWISZA, B ., MALICKA, E .: Trends Anal . Chem ., 51, 2013, 33–43 . 3. ZHANG, J ., ZHANG, W ., BAO, T . et al .: J . Chromatogr . A, 1339, 2014, 192–199 . 4. QU, Q ., GU, C ., HU, X .: Anal . Chem ., 84, 2012, 8880–8890 . CHOLINESTERASES INHIBITED BY NOVICHOK AGENTS – IN SILICO STUDY OF REACTIVATION POSSIBILITIES VEČEŘA, Z., KUČERA, T. Department of Toxicology and Military Pharmacy, Faculty of Military Health Sciences in Hradec Králové, University of Defence in Brno, Czech Republic e-mail: vecerazb@faf .cuni .cz “Novichoks” is the name of a series of nerve agents (NA) developed in the former Soviet union during the Cold War . Like other nerve agents, Novichok agents irreversibly bind acetylcholinesterase (AChE) and produce a cholinergic toxidrome, but have higher toxicity than older nerve agents .1 Treatment of poisoning caused by nerve agents is based on oxime reactivators of AChE . Mechanism of reactivation involves nucleophilic attack (by oxime group) of phosphorus atom of inhibited AChE .2 The aim of the work was to determine the binding energy of commercially available oxime reactivators into acetylcholinesterase inhibited by novichok, which could be used in the treatment of poisoning . Computational methods such as molecular docking and molecular dynamics were used for the study . 136 Structure of AChE inhibited by organophosphate (OP) were chosen from RSCB PDB (code 5HF9) and OP was replaced (using Chimera software) by 5 selected novichoks designated A230, A232, A234, A242 and A262 . The protein was prepared for molecular modeling . Four commercially available reactivators (pralidoxime, obidoxime, trimedoxime, HI-6) and one experimental reactivator (designated K203) were selected as ligands . They were prepared into docking-friendly pdbqt format (using ChemSketch, Avogadro and AutoDock Tools) . Semiflexible docking was used to determine the best ligand pose at the receptor. The most suitable pose was chosen as initial position for molecular dynamics . Then complexes protein-ligand were prepared and molecular dynamics were performed using Gromacs software . We determined the binding energy of the ligands in the protein . The study was supported by the Faculty of Military Health Sciences and from the project of Specific Academic Research (SVV 260 547). References 1. NEPOVIMOVÁ, E., KUČA, K.: Food Chem. Toxicol., 121, 2018, 343–350. 2. GÓRECKI, L., KORÁBEČNÝ, J., MUSÍLEK, K. et al .: Arch . Toxicol ., 90, 2016, 2831–2859 . SYNTHESIS OF POTENTIAL MYCOBACTERIAL INH-A AND CHOLINESTERASES INHIBITORS BASED ON TRICLOSAN VU, Q . A ., PFLÉGR, V ., KRÁTKÝ, M . Department of Organic and Bioorganic Chemistry, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic e-mail: vuq@faf .cuni .cz The contribution deals with the synthesis and evaluation of new inhibitors of the mycobacterial InhA enzyme and inhibitors of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) . All of the prepared compounds are analogues derived from triclosan, they could be good candidates as potential drugs to use in the treatment of tuberculosis and neurodegenerative diseases, including Alzheimer’s disease .1,2 Eight compounds were prepared in satisfactory yields . All of them were tested for antimicrobial and cholinesterase inhibitory activity . Among all the compounds that we prepared N-[5-chloro-2-(2,4-dichlorophenoxy)phenyl]acetamide showed the best antimicrobial effect against all three selected mycobacteria. Specifically, it had a minimum inhibitory concentration of 31 .25 mg L−1 against M. smegmatis, a minimum inhibitory concentration of 15 .625 mg L−1 against M. aurum, and a minimum inhibitory concentration of 7 .81 mg L−1 against M. tuberculosis H37Ra . This compound also showed the lowest IC50 value for AChE (48.85 μmol L−1) and has a better inhibitory effect against AChE than rivastigmine (56.10 μmol L−1) . The precursor 5-chloro-2-(2,4-dichlorophenoxy)aniline (“amino-triclosan”) showed the best inhibition of BChE (IC50 = 11.93 μmol L−1), which is a better inhibitory activity compared to rivastigmine again . 137 The study was supported by the Czech Science Foundation (Project No. 20-19638Y) and from the project of Specific Academic Research (SVV 260 547). References 1. VOSÁTKA, R ., KRÁTKÝ, M ., VINŠOVÁ, J .: Eur . J . Pharm . Sci ., 114, 2018, 318–331 . 2. KRÁTKÝ, M., ŠTĚPÁNKOVÁ, Š., VORČÁKOVÁ, K. et al .: Molecules, 21, 2016, art . 291 . SECTION OF PHARMACEUTICAL TECHNOLOGY EFFECT OF COMBINATION OF MUCOADHESIVE POLYMERS ON THE BEHAVIOUR OF MATRIX TABLETS IN THE GASTRIC ENVIRONMENT JOHNOVÁ, K., OGADAH, C. U., VRANÍKOVÁ, B. Department of Pharmaceutical Technology, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic e-mail: johnovakar@faf .cuni .cz Colon-targeted drug delivery plays a significant role in the pharmacotherapy of local diseases situated in the large intestine . In terms of sustained drug release, such formulations must be resistant to the acidic environment of the upper gastrointestinal tract . In addition, an enhanced therapeutic efficacy may be achieved by prolonging the residence time of the formulation at the absorption site by means of mucoadhesive drug delivery systems .1 For these reasons, the presented preformulation study investigates the behaviour of matrix tablets based on mucoadhesive polymers in the gastric environment . Knowledge of the behaviour of a drug in acidic pH is important as the surroundings of the inflamed colon may be lower . The selected polymers, guar gum (GG) and hydroxypropyl methylcellulose K15M (HPMC), were used separately or combined in ratios 85 .4 : 14 .6, 50 : 50 and 14 .6 : 85 .4 . The viscosity of polymers dispersions was evaluated in biorelevant dissolution media simulating the gastric environment (FaSSGF) using a rotational rheometer . Subsequently, compacts containing model drug theophylline were prepared and evaluated for their swelling behaviour and dissolution profiles using a USP II apparatus. The obtained data shows that both polymers were able to sustain the drug release from the hydrophilic matrix tablets for up to 24 hours . The mixture 14 .6 : 85 .4 seems to be the most promising as it forms the strongest gel barrier during the first two hours of dissolution. On the contrary, compacts with a high amount of GG perform poorly as a pronounced burst effect may be observed due to its rapid swelling and dissolving of outer, fully hydrated layers into the gastric medium . The study was supported by the Grant Agency of Charles University (Project No. 70119) from the project of Specific Academic Research (SVV 260 547). References 1. CARVALHO, F . C ., BRUSCHI, M . L ., EVANGELISTA, R . C . et al .: Braz . J . Pharm . Sci ., 46, 2010, 1–17 . 144 The study was supported by the Czech Science Foundation (Project No. 19-09135J) and from the project of Specific Academic Research (SVV 260 547). References 1. VÁVROVÁ, K., KOVÁČIK, A., OPÁLKA, L.: Eur . Pharm . J ., 64 (2), 2017, 28–35 . EFFECT OF COMBINATION OF MUCOADHESIVE POLYMERS ON THE BEHAVIOUR OF MATRIX TABLETS IN THE SMALL INTESTINE JANOUŠEK, M., OGADAH, C. U., VRANÍKOVÁ, B. Department of Pharmaceutical Technology, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic e-mail: janoum@faf .cuni .cz Colon-targeted drug delivery is highly desirable due to the direct treatment of local intestinal diseases, drug dose reduction, and minimization of systemic adverse effects . This effect can be achieved by several specific drug delivery systems based on different mechanisms . Generally, the most preferred are orally administered formulations due to the relatively easy manufacturing and high adherence of patients to treatment . These include matrix systems based on mucoadhesive polymers that allow both targeting to the desired site (e.g. colon) and controlled drug release .1 For the above-mentioned reasons, the aim of the presented study was to evaluate the behavior of two mucoadhesive polymers, namely guar gum, hypromellose K15M and their combinations in biorelevant media simulating small intestine fluids (Fasted State Simulated Intestinal Fluid, FaSSIF). The viscosity of the polymer dispersions was measured using a rotational rheometer . Polymer formulations (compacts) containing the model drug theophylline were tested for adhesion (modified balance), swelling index (basket method) and dissolution profiles (paddle apparatus). The obtained results suggested that superior adhesion may be expected in the formulations containing a higher portion of K15M . Moreover, these compacts showed also one of the highest weight gains after 8 hours of the swelling test . Drug dissolution was characterized by controlled release from all prepared matrix tablets over a 24 hour time period . Formulations containing K15M released the drug more constantly in the initial phase in comparison to the formulation comprising guar gum . The study was supported by the Grant Agency of Charles University (Project No. 70119) and from the project of Specific Academic Research (SVV 260 547). References 1. AMIDON, S ., BROWN, J . E ., DAVE, V . S .: AAPS PharmSciTech, 16, 2015, 731–741 . 145 A STUDY OF COMPRESSIBILITY OF DIRECTLY COMPRESSIBLE TABLETING MATERIALS AND TABLETS WITH CARRAGEENAN . NÁPRAVNÍKOVÁ, M., MUŽÍKOVÁ, J. Department of Pharmaceutical Technology, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic e-mail: napravnma@faf .cuni .cz The presented communication deals with the study of compressibility of directly compressible tableting materials with ι-carrageenan.1,2 In formulations, ι-carrageenan is mixed with chitosan, calcium alginate and hypromellose Methocel K15M in the ratios of 1 : 1, 2 : 1 and 3 : 1 . The formulations with salicylic acid as a model drug at a concentration of 20% and sodium stearyl fumarate as a lubricant at a concentration of 1% are also tested . Tablets without the drug are compressed by compression forces of 3, 4 and 5 kN . Tablets with the drug are compressed by compression force of 5 kN and 8 kN . The compressibility is evaluated by energy profile of compression process, another tested parameter is tensile strength of the tablets . The total energy of compression increases with the compression force . The parameter is increased with the addition of chitosan and hypromellose in all ratios as well . With higher proportion of carrageenan in mixtures, its values decrease . The addition of chitosan, calcium alginate, and hypromellose increases values of the energy of plastic deformation and plasticity . The higher values of these parameters indicate improvement of compressibility . Only hypromellose in ratios of 1 : 1 and 2 : 1 to carrageenan increases the strength of carrageenan tablets, calcium alginate significantly reduces it. The model drug salicylic acid reduces all energy values and tensile strength of tablets . The study was supported from the project of Specific Academic Research (SVV 260 547). References 1 . PICKER-FREYER, K . M .: Carrageenans in solid dosage form design . In Pharmaceutical Dosage Forms – Tablets, vol . 2 . Rational Design and Formulation, 3rd ed ., Augsburger, L . L ., Hoag, S . W . (eds .), Boca Raton, CRC Press, 2008, 469–492 . 2. SINGH, K . K .: Carrageenan . In: Handbook of pharmaceutical excipients, 6th ed ., Rowe, R . C ., Sheskey, P . J ., Quinn, M . E . (eds .), London, Pharmaceutical Press, 2009, 122–126 . SECTION OF SOCIAL AND CLINICAL PHARMACY CONSENSUS OF CZECH TERMINOLOGY IN THE FIELD OF MEDICATION ADHERENCE VOŘÍŠKOVÁ, E., KOŠŤÁLOVÁ, B., MALÁ, K. Department of Social and Clinical Pharmacy, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic e-mail: voriskoel@faf .cuni .cz 146 Due to the constantly changing terminology of medication adherence (MA) over the last 50 years, terms are often misused in the literature .1 The aim was to analyze the Czech terminology of MA and to establish a consensus using a Delphi round survey . Czech literature focused on the term of MA and its synonyms was reviewed using bibliographic (PubMed, Bibliographia Medica Čechoslovaca) and full-text (Solen, ProLékaře.cz) databases . A total of 123 articles published between 1998–2020 involved terms “compliance” (69 articles, 57 definitions), “adherence” (78, 51), “persistence” (25, 23), and “concordance” (13, 11), respectively. Based on the identified literature, a list of panelists who were invited for the Delphi round survey as suggested by ABC Taxonomy2 was created . This taxonomy determines 7 terms and their definitions in the field of MA. Out of 106 contacted panelists, 46 responded to the first round during which consensus for 2 definitions from 7 terms in the terminology of MA was established . Further rounds are an ongoing process and the rest of the terms and definitions will be determined. The disunity of using the terminology of MA and related terms showed up as a very common phenomenon of the Czech language . A consensus of Czech terminology will be established after the last round of Delphi survey . The study was supported from the project of Specific Academic Research (SVV 260 551). References 1 . VRIJENS, B ., De GEEST, S ., HUGHES, D . A ., et al.: Br . J . Clin Pharmacol ., 73, 2012, 691–705 . 2. ESPACOMP, Preferred Methods for Translation of the ABC Taxonomy for Medication Adherence. https:// www.espacomp.eu/wp-content/uploads/2020/08/Preferred-Methods-for-Translation-of-the-ABC-Taxonomy -for-Medication-Adherence-31_7_20-FINAL .pdf TRICYCLIC ANTIDEPRESSANTS CONSUMPTION IN THE CZECH REPUBLIC KVĚTENSKÁ, Z., HORKÝ, P. Department of Social and Clinical Pharmacy, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic e-mail: kvetensz@faf .cuni .cz Tricyclic antidepressants are one of the oldest groups of antidepressants . Except for indication for depression, they are used in the therapy of chronic pain or enuresis nocturna in children. Nowadays, tricyclic antidepressants are drugs of the first choice only in some types of neuropathic pain, and in other indications they have been replaced by newer substances with a lower occurrence of side effects. The aim of this work is to find out how utilization of tricyclic antidepressants has developed and what share in utilization of antidepressants they represent . Data for this work were obtained from the State Institute for Drug Control of the Czech Republic, namely from distributors’ report about supplies of medicinal products to pharmacies and healthcare facilities, sellers of selected medicinal products, other distributors and veterinary doctors . Utilization was evaluated for the period from 1.1.2008 to 31.12.2018 with DUR (drug utilization review). For evaluation, a defined daily dose per 1000 inhabitants per day and methods of descriptive statistics were selected . 147 A slight decrease of utilization of tricyclic antidepressants was observed for the watched period, as well as decrease in their share on antidepressatns utilization . The reason for the decrease could be the side effects of tricyclic antidepressants or their possible interactions with other drugs . The study was supported from the project of Specific Academic Research (SVV 260 551). RATIONALITY OF BZD USE IN PHARMACY PRACTICE IN SPAIN AND IN DIFFERENT SETTINGS OF CARE IN THE CZECH REPUBLIC: RESULTS FROM THE INOMED AND THE EUROAGEISM H2020 PROJECTS MAGÁTOVÁ, A .,1 MODAMIO, P .,2 BRKIĆ, J.,1 BRAUN-VIVES, J .,2 MARIŇO, E. L.,2 LUKAČIŠINOVÁ, A.,1 REISSIGOVÁ, J .,3 FIALOVÁ, D .,1,4 1 Department of Social and Clinical Pharmacy, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic 2 Clinical Pharmacy and Pharmaceutical Care Unit, Department of Pharmacy and Pharmaceutical Technology and Physical Chemistry, Faculty of Pharmacy and Food Sciences, University of Barcelona, Barcelona, Spain 3 Department of Statistical Modeling, Institute of Computer Science of the Czech Academy of Sciences, Prague, Czech Republic 4 Department of Geriatrics and Gerontology, 1st Faculty of Medicine in Prague, Czech Republic e-mail: magatova@faf .cuni .cz The aim of the study was to compare the prescription of BZDs in Spanish (SP) and Czech (CZ) sample of older adults. Data of 260 SP community-residing seniors 65+ and of 1602 CZ seniors 65+ were prospectively collected in the EuroAgeism H2020 project in 2018–2019 (by Comprehensive Geriatric Assessment) and analyzed using descriptive statistics (R-software, version 4 .0 .3) . In SP and CZ (community pharmacy samples), there were 62 .4% and 64 .6% of women and the mean age of participants was 71 .74 +/− 6.25 years and 76.61 +/− 7.15 years, respectively. Polypharmacy and excessive polypharmacy were documented in 24 .9% and 3 .8% (p < 0 .001) (CZ) and in 41 .2 % and 13 .1% (p < 0 .001) (SP) of older adults . 36 .2% of seniors used at least one BZDs in SP compared to 4 .9% in CZ (p < 0 .001) . Top 3 most frequently prescribed BZDs were (in SP): lorazepam (16 .5%), lormetazepam (6 .5%) and alprazolam (4 .6%), (in CZ): alprazolam (1 .1%), bromazepam (0 .7%) and oxazepam (0 .4%) . In Spain, extensively high prevalence of BZDs in older adults (35 .4%, p < 0 .001), with expected potential negative consequences, was found in comparison with community pharmacy practice in CZ (2 .4%, p < 0 .001), acute care (18 .0%, p < 0 .001) and ambulatory care (16 .7%, p < 0 .001) . The study was supported by InoMed (Project No. CZ.02.1.01/0.0/0.0/18_069/00100 46, 2019-2022) co-funded by the European Union, the EUROAGEISM H2020-MCSFITN764632, by the Research programme Development and Study of Drugs (Progres Q42) (KSKF-2 Assoc. Prof. Fialová), from the project of Specific Academic Research (SVV 260 551), START programme (Reg. No CZ.02.2.69/0.0/0.0/19_073/0016935) and I-CARE4OLD Horizon 2020 project (Grant agreement ID: 96534). 148 AN ANALYSIS OF DRUG INTERACTIONS BASED ON DRUG INFORMATION CENTRE ENQUIRIES MICHÁLEK, G., ROZSÍVALOVÁ, P., MALÁ, K. Department of of Social and Clinical Pharmacy, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic e-mail: michaleg@faf .cuni .cz Drug information centre (DIC) of the Faculty of Pharmacy in Hradec Králové, Charles University and University Hospital Hradec Králové provides drug information to healthcare professionals in the form of timely and accurate answers to drug-related enquiries, including drug interactions (DI) . This study aimed to analyze enquiries related to DI processed by DIC from 2015 to 2020 . Data were collected from individual enquiries related to drug-drug, drug-herbal and drug-disease interactions . Various parameters assigned to each DI found were analyzed, such as clinical severity or ATC codes of interacting drugs . An analysis based on descriptive statistical methods was carried out . A total of 67 enquiries were analyzed . The three most frequent ATC codes being involved in at least one DI per enquiry were A02BC01 (omeprazole), B01AC06 (acetylsalicylic acid), and C10AA05 (atorvastatin) . Warfarin, venlafaxine and omeprazole were the most frequently interacting drugs regarding the different mechanism of interactions . In contrast, citalopram, furosemide, levothyroxine and omeprazole prevailed in all interacting pairs of every interaction . The majority (62%) of DI were based on pharmacodynamics, while 31% were based on pharmacokinetics . The clinical severity of DI was graded A (Minor) in 23%, B (Moderate) in 67%, and C (Severe) in 10% of cases . The most common potential clinical outcomes of DI were increased risk of adverse effects (18%), elevated plasmatic concentration of a drug (16%), and higher risk of bleeding (13%) . Additionally, a (still ongoing) qualitative analysis of five enquiries sharing a similar clinical topic was carried out. DI constitute a significant portion of enquiries processed by DIC and seem to differ in clinical severity, pharmacological mechanism and potential clinical outcomes . The study was supported from the project of Specific Academic Research (SVV 260 551). THE PREVALENCE OF DRUG-DRUG INTERACTIONS IN PATIENTS ADMITTED TO THE HOSPITAL VIA THE EMERGENCY DEPARTMENT KUKRÁLOVÁ, K .,1 OČOVSKÁ, Z.,1 MAŘÍKOVÁ, M.,1,2 VLČEK, J.1,2 1 Department of Social and Clinical Pharmacy, Faculty of Pharmacy in Hradec Králové, Charles University, Czech Republic 2 Department of Clinical Pharmacy, Hospital Pharmacy, University Hospital Hradec Králové, Czech Republic e-mail: kukralok@faf .cuni .cz The presence of potential drug-drug interactions (DDIs) is common in daily practice and only a small proportion of potential DDIs result in hospitalization of the patients . Nev- 149 ertheless, DDIs represent a significant cause of hospital admissions.1 This cross-sectional study aims to identify DDIs in the medication history of the patients admitted to University Hospital Hradec Králové via the emergency department in August–November 2018 . The objectives of this study are a) to determine the prevalence of hospital admissions with potential DDIs, b) to categorize identified potential DDIs with respect to their mechanism, severity, risk rating, level of documentation and potential outcomes and c) to determine the prevalence of hospital admissions with manifest DDIs . A sample of 375 hospital admissions has been analyzed so far . 300 hospital admissions have been screened for the presence of potential DDIs using Micromedex, Lexicomp and DrugAgency a . s . database of drug-drug interactions . 75 hospital admissions have been excluded from the screening process due to an insufficient number of medications in medication history (< 2). 2273 potential DDIs were identified in 258 hospital admissions (86% of the eligible admissions). The overall prevalence of hospital admissions with identified potential DDI in medication history was 68 .8% (95% CI: 64 .1–73 .5) . 866 different DDI pairs were involved in these potential DDIs. Manifest DDIs which contributed to hospital admission were identified in 17 (4.5%) hospital admissions. The findings will provide a deeper insight into the pharmacoepidemiologic aspects of DDIs . The study was supported by the Grant Agency of Charles University (Project No. 14120) and from the project of Specific Academic Research (SVV 260 551). References 1. DECHANONT, S ., MAPHANTA, S ., BUTTHUM, B . et al.: Pharmacoepidemiol . Drug Saf ., 23, 2014, 489–497 . 151 2026 Folia Pharm . Univ . Carol . LII Pag . 151–174 BIBLIOGRAPHY PUBLICATIONS OF THE FACULTY OF PHARMACY IN HRADEC KRÁLOVÉ, CHARLES UNIVERSITY IN THE YEAR 2021 ORIGINAL PAPERS AND REVIEWS ADAMCOVÁ, A ., HOLST LAURSEN, K ., ZEDERKOPFF BALLIN, N .: Lectin activity in commonly consumed plant-based foods: Calling for method harmonization and risk assessment . Foods, 2021, 10 (11), art . 2796 . AL MAMUN, A., PIDANÝ, F., HULCOVÁ, D., MAŘÍKOVÁ, J., KUČERA, T., SCHMIDT, M., CATAPANO, M. 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