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UHPLC-MS/MS absolute quantification of cytochrome P450 enzymes in C3A, CACO2 modified cell lines and in human liver microsomes
Rejšková, Lenka ; Szotáková, Barbora (advisor) ; Vokřál, Ivan (referee)
Charles University in Prague, Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences University of Eastern Finland in Kuopio, School of Pharmacy Department of Pharmaceutical Chemistry Candidate: Lenka Rejšková Supervisor: doc. Ing. Barbora Szotáková, Ph.D. Consultant: Prof. Seppo Auriola Title of Diploma thesis: UHPLC-MS/MS absolute quantification of cytochrome P450 enzymes in C3A, CACO2 modified cell lines and in human liver microsomes Cytochrome P450 (CYP) enzymes play a crucial role in drug metabolism. They can be responsible for the failure of treatment, adverse and toxic effects or drug-drug interactions. Modified C3A and CACO2 cell lines with constitutive androstane receptor (CAR) and pregnane X receptor (PXR) might be used for in vitro biotransformation and absorption studies instead of primary cell lines. CYP enzymes should be expressed continuously in these modified cell lines. This study aimed to establish the quantitative method for the analysis of the most common CYP enzymes in human: CYP1A2, CYP2B6, CYP2C9, CYP2C19, CYP2E1, CYP3A5 and CYP3A4 in the above mentioned cell lines and in human liver microsomes. Absolute quantifications of CYP enzymes were carried out by UHPLC in line coupled with tandem mass spectrometry working in scheduled MRM mode. Data assessment was...
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Identification of metabolites of anthelminthics in helminth parasites using mass spectrometry
Komrska, Jan ; Skálová, Lenka (advisor) ; Vokřál, Ivan (referee)
If we want be successful in the treatment of parasitic disease, it is important to get the utmost information on the fate of given drug. The discovery of the metabolism of anthelmintics and in cosequence of participating enzymes can help us fight the rising resistance to given drugs. The aim of our work was to find and identify phase I and phase II metabolites of the anthelminthic drugs albendazole (ABZ), flubendazole (FLU) and mebendazole (MEB) formed in ex vivo incubations by parasitic helminth Dicrocoelium dendriticum, using liquid chromatography-mass spectrometric techniques. In the ex vivo study, D. dendriticum adults were incubated in cultivating medium in presence of benzimidazole drug for 24 hours. After incubation of parasites, both parasite homogenates and medium from the incubation were separately extracted using solid phase extraction. The extracts were analyzed using liquid chromatography-mass spectrometry (LC-MS). We detected in phase I albendazole sulphoxide, reduced flubendazole and reduced mebendazole. As for phase II metabolites, methylderivatives of flubendazole, reduced flubendazole and reduced mebendazole were observed.
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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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Effects of simple sugar consumption on cognitive functions in female rats
Podhorská, Gabriela ; Macháček, Miloslav (advisor) ; Vokřál, Ivan (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Gabriela Podhorská Supervisor: Mgr. Miloslav Macháček, Ph.D. Title of diploma thesis: Effect of simple sugar consumption on cognitive functions in female rats The consumption of simple sugars, especially fructose, increased in the past few decades. Excessive sugar intake has been associated with the development of insulin resistance, hypertriglyceridemia and oxidative stress which lead to the impairment of cognitive functions. Nevertheless, it is still unclear whether these alterations in cognitive functions are exclusively caused by high caloric intake or if they are related to specific characteristics of fructose. Moreover, the mechanisms involved are not yet fully deciphered at the molecular level. Our research group supplemented Sprague-Dawley female rats with 10% w/v glucose in drinking water or with isocaloric fructose solution in a long-term study (7 months), simulating the chronic consumption of sugars in humans. Plasma parameters and expression of proteins, involved in metabolic pathways, were determined in the frontal cortex. Cognitive functions were evaluated through the novel object recognition (NOR) and Morris Water Maze (MWM) tests. Results, obtained in our study, showed a...
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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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Viral hepatitides and their pharmacotherapy
Klímková, Martina ; Mladěnka, Přemysl (advisor) ; Vokřál, Ivan (referee)
15. Abstract Viral hepatitis belongs to the most common infectious diseases in the world by this time. Typical areas of their appearance are countries of South America, South Asia and Africa. They are transmitted parenterally (hepatitis B, C, D) or via the faecal-oral route (hepatitis A, E). Infection starts the immune reaction with production of antibodies that often serve in confirmation of the definitive diagnose. The clinical manifestation of viral hepatitis involves gastrointestinal discomfort, jaundice and actually liver failure and death if the hepatitis is complicated or untreated. Hepatitis A and hepatitis E don not become chronic and patients do not have any health consequences after recovery. Chronic forms of hepatitis B, C, and D cause long-lasting complications. There is no need to use antiviral drugs in the therapy of hepatitis A and E, the symptomatological therapy is sufficient. The other types of hepatitis demand an antiviral therapy. The basic of this therapy are interferon and synthetic nucleoside analogues. Interferons start an appropriate immune reaction and destroy infected hepatocytes and synthetic antivirotics inhibit synthesis of viral nucleic acid. In the treatment of hepatitis B is used pegylated interferon α, ordinarily in combination with nucleoside analogues (lamivudin,...
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In vitro assessment of cytotoxic action of novel potential nitro group-containing potential antitubercular agents
Turoňová, Dorota ; Macháček, Miloslav (advisor) ; Vokřál, Ivan (referee)
Charles University Faculty of Pharmacy in Hradec Králové Depatrment of Biochemical Sciences Candidate: Bc. Dorota Turoňová Supervisor: RNDr. Miloslav Macháček, Ph.D. Consultant: doc. PharmDr. Jaroslav Roh, Ph.D. Title of diploma thesis: In vitro assessment of cytotoxic action of novel potencial nitro group-containing antitubercular agents More than 10 million new cases of tuberculosis occur every year. Lack of efficient antituberculotic drugs and their unavailability, poor diagnostic and especially inappropriate treatment are drawbacks complicating and even precluding eradication of tuberculosis. An already serious disorder has become even more serious in the last years, especially because of the occurrence of drug-resistant tuberculosis, especially MDR-TB and XDR-TB. Due to chromosomal mutations that make bacteria resistant to drugs it is still necessary to develop new effective drugs to replace those in current use, which are often toxic and associated with the occurrence of side effects. The research group at the Department of Organic and Bioorganic Chemistry, Faculty of Pharmacy in Hradec Králové, Charles University, which has been researching new antitubercular agents for many years, has created new structures of aromatic nitrocompounds with very high antimycobacterial effect. The aim of this...
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Metabolism of monepantel in parazites and their hosts
Valát, Martin ; Skálová, Lenka (advisor) ; Vokřál, Ivan (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Martin Valát Supervisor: prof. RNDr. Lenka Skálová, Ph.D. Title of diploma thesis: Metabolism of monepantel in parazites and their hosts Monepantel (MOP) belongs to a new class of anthelmintic drugs - amino-acetonitrile derivatives. They differ from the commonly used broad-spectrum drugs (makrocyclic lactones, benzimidazoles and imidazothiazoles) in chemical structure and mechanism of action. Because of frequent use of these classes the resistance in many pathogenic parasites was developed. The aim of this study was identification and comparison of phase I and II metabolites of MOP biotransformation in parasites (Haemonchus contortus - sensitive strain ISE and multi-resistant strain WR) and in their hosts - sheep (Ovis aries) through in vivo and ex vivo study. Ultra-high performance liquid chromatography with tandem mass spectrometry technique (UHPLC-MS/MS) was used for identification of MOP metabolites. In sheep, 13 metabolites of phase I and II biotransformation of MOP were detected in in vivo study, 7 of them have not been described previously. In parazites ex vivo, only 4 metabolites of phase I MOP biotransformation were found. Following biotransformation reactions of MOP were...
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Optimalization of Leishmania antigen formulations for the diagnosis of canine leishmaniasis.
Danková, Barbora ; Němečková, Ivana (advisor) ; Vokřál, Ivan (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biological and Medical Sciences Student: Barbora Danková Supervisor of diploma thesis: RNDr. Ivana Němečková, Ph.D. Specialized Supervisor: Prof. Anabela Cordeiro da Silva Title of diploma thesis: Optimalization of Leishmania antigen formulations for the diagnosis of canine leishmaniasis Leishmaniasis is a vector-borne disease caused by a protozoan parasite of the genus Leishmania and is prevalent in 98 countries worldwide. Reliable and accurate tests are necessary for laboratory diagnosis of Leishmania infection because of the wide spectrum of clinical characteristics and symptoms and high rate of asymptomatic infections that may occure. Serological diagnosis of canine leishmaniasis (CanL), especially enzyme-linked immunosorbent assay (ELISA), proved to be usefull tool. Data obtained from ELISA assay for the detection of antibodies against selected recombinant A, B, C, D, E and F proteins were evaluated in infected canine sera from Portugal and Brazil. This work presents the best formulation of recombinant proteins for serological diagnosis. We found that antigen C showed high level of sensitivity and specificity in recognition of positive and asymptomatic sera infected with CanL. Key words: canine leishmaniasis,...
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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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