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Characterization of ligand binding to M1 muscarinic acetylcholine receptor using fluorescence anisotropy method
Danková, Hana ; Vokřál, Ivan (advisor) ; Červený, Lukáš (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Student: Hana Danková Supervisors: Prof. Ago Rinken, PhD. MSc. Tõnis Laasfeld PharmDr. Ivan Vokřál, PhD. Title of diploma thesis: Characetrization of ligand binding to M1 muscarinic receptor using fluorescence anisotropy method Muscarinic acetylcholine receptors (mAChRs), members of the superfamily of G-protein coupled receptors (GPCRs), regulate vital physiological processes and are important targets in drug research. Five different subtypes (M1 - M5) have been identified. M1 mAChR is mainly distributed in the central nervous system and is linked to pathophysiology of neurodegenerative diseases. In recent years, fluorescent methods have been frequently used in studies of ligand binding to receptors. The fluorescence anisotropy (FA) is a homogenous assay to characterize ligand binding to receptors. In this work, we have evaluated the FA method with fluorescent ligand MK342 binding to M1 mAChRs expressed on budded baculovirus (BBV) particles. The fluorescence ligand was binding with the high affinity (4,4 nM) to M1 receptor in constructed BBV preparation. The apparent binding affinities (pKi) of eleven classical and three bitopic muscarinic ligands were screened and compared to previously published...
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Comparison of novel amphiphilic anionic phthalocyanine photosensitizers with their hydrophilic analogues
Hasoňová, Kateřina ; Macháček, Miloslav (advisor) ; Vokřál, Ivan (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Bc. Kateřina Hasoňová Supervisor: RNDr. Miloslav Macháček, Ph.D. Title of diploma thesis: Comparison of new amphiphilic anionic photosensitizers based on phthalocyanines with their hydrophilic analogues. Currently, one of the most common causes of death in our population is cancer. There are a number of treatment modalities how to treat tumours, but none of them is ideal, and a large number of people are still dying of cancer. For this reason, intensive research and studies are ongoing, focusing on cancer treatment options. In recent years, photodynamic therapy has been increasingly used to treat both cancerous and non-malignant diseases. It is a modern method that has minimal side effects, is highly selective and minimally invasive. Its principle is a photochemical reaction, which proceeds when a combination of three basic components occurs - light, oxygen and photosensitizer. Separately, these components are nontoxic, but when the photosensitizer is exposed to oxygen, toxic reactive oxygen species are produced, whose subsequently cause destruction of the tumour tissue. A photosensitizer is a compound that is able to absorb radiation of a certain wavelength and then convert it into energy, which...
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Constitutive expression of UDP-glucosyltransferases from Haemonchus contortus
Nekovová, Lucie ; Matoušková, Petra (advisor) ; Vokřál, Ivan (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Lucie Nekovová Supervisor: Ing. Petra Matoušková, Ph.D. Title of diploma thesis: Constitutive expression of UDP-glucosyltransferases from Haemonchus contortus Haemonchosis is one of the most frequent parasitic disease on small ruminants caused by gastrointestinal nematode Haemonchus contortus. Attack of this blood sucking parasite does not mean only serious health damage but also lower productivity and economic losses. Anthelmintics are used as a medical treatment of haemonchosis however they are often insufficient because of worlwide developing resistence. Knowledge of mechanisms causing resistence is still not sufficient, therefore much effort is spent in order to find the factors which could be their source. It appears that changes of biotransformation enzymes and their expression might be one of the reason for resistence. At H. contortus more than 40 genes which code a different types of UDP- glucosyltransferases (UGTs) have been found. UGTs are the main enzymes in nematode which are connected with a deactivation of drugs (glycosylation). Therefore this thesis is focused especially on UGT family with the main goal to analyse changes in expression of these genes of susceptible (ISE) and...
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Metabolism of flubendazole in human liver subcellular fractions
Holubová, Jana ; Skálová, Lenka (advisor) ; Vokřál, Ivan (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Jana Holubová Supervisor: prof. RNDr. Lenka Skálová, Ph.D. Title of diploma thesis: Metabolism of flubendazole in human liver subcellular fractions Flubendazole (FLU), a benzimidazole anthelmintic drug with a broad spectrum of activity and low toxicity has been used in veterinary as well as human medicine for a long time. Owing its mechanism of action, based on the specific binding to tubulin, FLU is now considered a promising anti-cancer agent. However, we do not have enough information about its biotransformation in human. In our project, subcellular fractions from the liver of 12 human patients (6 males and 6 females) were used to study the stereospecificity, cellular localization, coenzyme preference and possible inter-individual or sex differences in FLU reduction. In addition, the risk of FLU interaction with other drugs was evaluated. Results showed that FLU is predominantly reduced in cytosol and the NADPH coenzyme is preferred. The strict stereospecificity of FLU carbonyl reduction was proven and carbonyl reductase 1 was identified as the main enzyme of FLU reduction in the human liver. A higher reduction of FLU and a higher level of carbonyl reductase 1 protein was found in males than in...
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Study of drug interactions with OATP family transporters using intestinal tissue slices
Čečková, Patrícia ; Vokřál, Ivan (advisor) ; Červený, Lukáš (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Patrícia Čečková Supervisor: PharmDr. Ivan Vokřál, Ph.D. Title of diploma thesis: Study of drug interactions with OATP family transporters using intestinal tissue slices An essential role in the action of orally administered drugs is their absorption through the intestinal barrier. It expresses a variety of transporters, including the OATP2B1 and OATP1A2 influx transporters, belonging to the SLC family. They are located on the apical membrane of enterocytes and allow the flow of endogenous and exogenous substances from the lumen of the intestines to the enterocyte. They affect not only the pharmacokinetics of drugs, but also their safety and efficacy. They represent sites of drug interactions with other drugs/food components that may altered drug efficacy or toxicity. Since FDA (The Food and Drug Administration) and EMA (European Medicines Agency) do not have intestinal OATP transporters included in their guidelines for preclinical studies, there is no single model of interaction study. The limitations of cell models and genetically modified organisms lead to the development of new methods such as the ex vivo method of precision cut intestinal slices (PCIS), which represents a tissue model...
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Haemonchosis as a model parasitosis in sheep - the results of its control during experimental study
Zdeněk, Pavel ; Lamka, Jiří (advisor) ; Vokřál, Ivan (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Student: Pavel Zdeněk Supervisor: prof. RNDr. Jiří Lamka, CSc. Title of diploma thesis: Haemonchosis as a model parasitosis in sheep - the results of its control during experimental study One of the current major issues with breeding domestic animals worldwide is anthelmintic resistance, which damages animals' health and reduces their production. Research workplaces want to identify the mechanisms of resistance development and find out the ways how to prevent it. The aim of this diploma thesis was to describe experimental infection by the model parasite Haemonchus contortus, which was established on Texel breed sheep. H. contortus is one of the important endoparasites; it lives in abomasum of ruminants and feeds with their blood. Faecal samples of all animals entering to the experimental studies were examined by the qualitative ovoscopic method and the animals were thoroughly dewormed by albendazole and monepantel. Then they were infected with larvae L3 of different H. contortus strains. Three strains had been used - ISE (all anthelmintic sensitive), IRE (benzimidazole resistant) and WR (multi-resistant strain). Data of the egg amounts were collected in faecal samples obtained from infected...
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The influence of spinal anesthesia on the degree of DNA damage
Koščáková, Mária ; Kuchařová, Monika (advisor) ; Vokřál, Ivan (referee)
Background: The human organism is exposed daily to many endogenous and exogenous substances that are the source of oxidative damage. Cell structures, including DNA (deoxyribonucleic acid) in the nucleus are damaged due to high concentrations of these substances and accumulation of oxidative stress in cells. The predominance of these damaging processes may later be responsible for human diseases such as cancer, neurodegenerative diseases or heart failure. In our study, we observed oxidative damage at the DNA level due to spinal anesthesia. Methods: Sample processing was performed by comet analysis. The principle consists in fixation of cells (lymphocytes) in the agarose gel, lysis of cell structures for nucleotide release, incubation with specific enzymes and exposure to electrophoresis. Damaged, negatively charged parts of the DNA in the electric field are directed to the positive charged anode, creating a typical comet shape. For visualization, the gels were stained with ethidium bromide (DNA intercalating dye). Results: We have quantified single-strand breaks, oxidized purines and pyrimidines (use of enzymes to detect specific damages). The results are reported in percentage of DNA in the comet's tail. The principle is to compare the intensity of the comet's tail with the total comet intensity....
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Haemonchosis as a model parasitosis in sheep - the results of its control during experimental study
Zdeněk, Pavel ; Lamka, Jiří (advisor) ; Vokřál, Ivan (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Student: Pavel Zdeněk Supervisor: prof. RNDr. Jiří Lamka, CSc. Title of diploma thesis: Haemonchosis as a model parasitosis in sheep - the results of its control during experimental study One of the current major issues with breeding domestic animals worldwide is anthelmintic resistance, which damages animals' health and reduces their production. Research workplaces want to identify the mechanisms of resistance development and find out the ways how to prevent it. The aim of this diploma thesis was to describe experimental infection by the model parasite Haemonchus contortus, which was established on Texel breed sheep. H. contortus is one of the important endoparasites; it lives in abomasum of ruminants and feeds with their blood. Faecal samples of all animals entering to the experimental studies were examined by the qualitative ovoscopic method and the animals were thoroughly dewormed by albendazole and monepantel. Then they were infected with larvae L3 of different H. contortus strains. Three strains had been used - ISE (all anthelmintic sensitive), IRE (benzimidazole resistant) and WR (multi-resistant strain). Data of the egg amounts were collected in faecal samples obtained from infected...
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Study of drug-drug interactions of antiviral drugs on intestinal transporters
Záboj, Zdeněk ; Červený, Lukáš (advisor) ; Vokřál, Ivan (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Zdeněk Záboj Supervisor: PharmDr. Lukáš Červený, Ph.D. Title of diploma thesis: Study of drugs interactions of antiviral drugs with intestinal transporters Sofosbuvir is an antiviral agent widely used in the treatment of chronic hepatitis C. This orally administered prodrug is a designed substrate of ATP-binding (ABC) efflux transporters, P- glycoprotein (ABCB1) and breast cancer resistance protein (ABCG2). ABCB1 and ABCG2 are important determinants of intestinal absorption and are the site of significant pharmacokinetic drug interactions, leading to changes in drug exposure. Pharmacokinetic drug interactions may be undesirable (increasing the toxicity of the treatment) or desirable (allowing dose reduction). Because sofosbuvir is often administered in combination regimens with other anti(retro)virotics, the aim of this thesis was to study the ability to enhance intestinal absorption of sofosbuvir. To study the pharmacokinetic drug interactions on ABCB1 and ABCG2, a widely established in vitro bi-directional transport method through a polarized monolayer formed by the Caco-2 cell line derived from colorectal cancer has been used. We analyzed the drug interactions of sofosbuvir on these efflux...
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Pharmacokinetics of ivermectin in the sheep feces
Sobotová, Dominika ; Vokřál, Ivan (advisor) ; Lamka, Jiří (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Student: Dominika Sobotová Tutor: PharmDr. Ivan Vokřál, Ph.D. Title of diploma thesis: Pharmacokinetics of ivermectin in the sheep feces Key words: ivermectin, pharmacokinetics, sheep, anthelminthic Infection with internal parasites (endoparasites) is one of the most common diseases in sheep. Infection with these parasites mainly with the barber's pole worm (Haemonchus contortus) causes considerable economic losses and has a significant impact on sheep productivity. Anthelmintics, including ivermectin, are used for treatment. Ivermectin belongs to the class of macrocyclic lactones and is characterised by broad spectrum and low toxicity. On the other hand, it poses a risk to the environment in form of residues that are excreted in feces by treated individuals. The aim of this study was to determine the excretion profile of ivermectin in sheep subcutaneously administered in a standard dose 0,2 mg/kg of body weight. UHPLC/MS/MS method was used for the analysis of ivermectin fecal concentration. Based on the obtained results we determined basic pharmacokinetic parameters which includes time to achieve maximum concentration (tmax), maximum concentration (cmax), area under the curve (AUC) and mean residence...
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