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Bioanalytical method development for the investigation of the role of pyridoxal isonicotinoyl hydrazone in isoniazid intoxication II.
Charvátová, Jana ; Vrbatová, Ivana (advisor) ; Pilařová, Pavla (referee)
Diploma thesis Abstract 52 Abstract Bioanalytical method development for the investigation of role of pyridoxal isonicotinoyl hydrazone in isoniazid intoxication II. Jana Charvátová Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Pharmaceutical Chemistry and Drug Control Heyrovského 1203, 500 05 Hradec Králové Keywords: isoniazid, vitamin B6, hydrazone, hydrophilic interaction chromatography HPLC/UV method was developed for analysis of isoniazid - INH, its main (acetylisoniazid - AcINH) and putative metabolites (pyridoxal isonicotinoyl hydrazone - PIH, pyridoxal isonikotinoyl hydrazone 5-phosphate - PIH5P), vitamins B6 (pyridoxine - PN, pyridoxal - PL and pyridoxal 5-phosphate - PLP) using ZIC-pHILIC stationary phase. The following parameters were modified to assess their impact on the retention of these compounds: composition of mobile phase, percentage content of organic solvent, buffer concentration, pH, flow rate of mobile phase and column temperature. The best available separation of the compounds was achieved using 10mM ammonium formate (pH 3) and ACN (40:60, v/v), flow rate 0.10 ml/min and column temperature 25 řC. Developed method was transferred to HPLC/ESI/MS. Experiments revealed that all analytes gave the higher signal in positive mode and the most abundant ions...
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Development of LC/MS/MS method for determination of ambrisentan, midazolam and its metabolite in human plasma
Klivický, Michal ; Kučera, Radim (advisor) ; Štěrbová, Petra (referee)
Development of LC/MS/MS method for determination of ambrisentan, midazolam and its metabolite in human plasma Diploma thesis Michal Klivický Charles University in Prague, Faculty of Pharmacy in Hradec Králové, Department of Pharmaceutical Chemistry and Drug Control Heyrovského 1203, Hradec Králové. Ambrisentan (ABT) is a new potent drug for treatment of pulmonary arterial hypertension, which is metabolized by CYP3A4. Pharmacokinetic profile of midazolam (MDL) and its main metabolite 1-hydroxymidazolam (OH-MDL) can be used as a marker of CYP3A4 activity. Simultaneous determination of ambrisentan, midazolam and 1-hydroxymidazolam is advantageous for studying of potential influence of CYP3A4 induction or inhibition on ambrisentan pharmacokinetics. HPLC/MS/MS method for their analysis in human plasma was developed and validated. After solid phase extraction on a Strata-X (Phenomenex) SPE column, the analytes were separated on a Synergi MAX-RP (150×4.6 mm, 4 µm) (Phenomenex) analytical column using isocratic elution with acetonitrile/5mM ammonium acetate 41:59 (v/v) as a mobile phase. Detection was achieved using tandem mass spectrometry on a triple quadrupole mass spectrometer. Ambrisentan-d3, midazolam-d5 and 13 C3-1-hydroxymidazolam were used as internal standards. The calibration curves were linear...
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The employment of separation methods in the field of drug analysis IV.
Ondrišíková, Lucie ; Kučera, Radim (advisor) ; Vrbatová, Ivana (referee)
Employment of separation methods in the field of drug analysis IV. Diploma thesis Lucie Ondrišíková Charles University in Prague, Faculty of Pharmacy in Hradec Králové, Department of Pharmaceutical Chemistry and Drug Control, Heyrovského 1203, Hradec Králové Nowadays, high performance liquid chromatography is one of the most progressive analytical methods, which is widely used in all areas of drug analysis. Currently, the majority of analysis is done using silica columns. Despite the positive properties (e.g. available in the widest selection of particle size, pore diameter and surface area; wide selection of types of reversed phases) these columns have some limitations - e.g. there are stable only at pH = 2.0 to 8.0 and the temperature must be lower than 60řC. For these reasons new materials for stationary phases are being developed and tested. Zirconia seems to be very perspective; it is stable at any pH and temperatures up to 200řC. The theoretical part of this thesis deals with both the physical and chemical properties of zirconia from a chromatographic point of view. This part describes characteristics of HPLC zirconia columns in comparison to silica columns as well. The experimental part focuses on studying and comparing the retention behavior of neutral, acid and alkaline substances on Zr-...
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The study of membrane receptors by radioligands binding
Rejhová, Alexandra ; Hudeček, Jiří (advisor) ; Hejnová, Lucie (referee)
Drug addiction, opiates respectively, is a social problem which seriousness is currently on the rise. One of key elements causing addiction is tolerance to increasing doses of drug causing abstinence syndrome during withdrawal and craving. Opioid receptors are members of a large group of receptors coupled with heterotrimeric G-proteins (GPCR), whose properties can be investigated using agonist- stimulated binding [35 S] GTPγS. Many extracellular signals are transferred into a cell through GPCR. Opioid receptor agonists inhibit the activity of adenylyl cyclase and are coupled with G-protein group Gi/Go. This work is devoted to the study of changes in isolated plasma membranes of rat forebrain containing opioid receptors of healthy subjects with membranes acquired from morphine addicted subjects. The rats were long-term morphine treated in increasing doses, to develop the dependency. The comparison is done firstly by binding of [3 H]ouabain to Na,K-ATPase, which proves to be a negative standard of changes, secondly by binding [35 S]GTPγS to G-proteins, thereby providing the functional activity of G-protein in stimulating the binding by the agonist of δ-opioid receptors DADLE or agonist of µ-opioid receptors DAMGO. Furthermore, it has been studied the influence of prostaglandin E1 on binding [35...
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Analysis of market with anabolic-androgenic steroids
Holčapek, František ; Běláčková, Vendula (advisor) ; Chmelová, Pavla (referee)
This thesis deals with the black market of enhancing drugs with a particular focus on androgenic anabolic steroids (AAS). Medical studies agree that these substances in the form and quantity abused by athletes to improve performance are damaging the body and therefore author is looking for recommendations for economic policy on how to reduce rate of this abuse. The study of economic literature (especially the Becker's "Theory of rational addiction") shows that users of AAS are rational, often even more rational than users of other harmful substances, because they abuse these substances with long-term plan. The reason of this purposeful approach is that the desired "delight" is derived from hard-earned success unlike other drugs and therefore abuse of AAS is associated with discipline, calculation and hence a (limited ) rationality. Economists building on Becker 's theory point out to cases where this limitation is so significant that it justifies regulation. This thesis is based on the assumption (supported by studies) that prohibition or penalizing the users themselves are ineffective instruments and therefore is the author looking for alternative solution. The author believes that the main stimulators of demand for AAS are misleading media; benevolent government's approach towards bodybuilding competitions; and finally the prohibition leading to the formation of the black market which makes it impossible for (potential ) users to become optimally informed about health risks etc. This hypothesis is being tested in questionnaire survey distributed mainly through social networks. Finally, the author sets out recommendations for economic policy: that restrictive hand of the state should focus attention in the opposite direction than before i.e., the demand side and thereby subtly demotivate users themselves.
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Methods for Leishmania parasite detection and quantificatio as a tool for study of the pathogen-vector-host interactions
Kobets, Tetyana ; Lipoldová, Marie (advisor) ; Kodym, Petr (referee) ; Kopecký, Jan (referee)
Methods for Leishmania parasite detection and quantification as a tool for study of the pathogen-vector-host interactions Leishmaniasis in human is caused by total 21 species of the intracellular protozoan parasite Leishmania, which are transmitted by about 30 species of phlebotomine sand flies. Besides human, Leishmania can infect a number of vertebrate hosts. The major host cell is the macrophage, in which parasites multiply, eventually rupturing the cell and spreading to uninfected cells. Infected monocytes and macrophages circulating in the peripheral blood are thought to be carriers of the parasite to distal sites. Depending on the infected sites of the body, there are three forms of leishmaniasis: cutaneous, mucocutaneous and visceral. Leishmaniasis is a disease for which we still lack effective, affordable and easy to use drugs. In addition, surveillance and control are also neglected. This thesis summarizes the results of several projects using different approaches for parasite load measurement in the mouse model of leishmaniasis, including two methods that were developed and optimized in our laboratory. Detection and quantification of pathogens belongs to the major topics of the research of various infectious diseases. This parameter is necessary for confirmation of the diagnosis, characterization...
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Kinetics Studies of Collagen I Self-Assembly
Voldánová, Michaela ; Ondreáš, František (referee) ; Jančář, Josef (advisor)
Collagen, the most abundant protein of connective tissues, in various forms has a wide applications due to their diverse biological and chemical properties. One of the forms are collagen hydrogels, which are considered very suitable material for applications in tissue engineering, because they are able to provide biodegradable scaffolds that its properties correspond with living tissues. These systems are used for example as scaffold for targeted drug delivery with controlled release, in combination with cells can be used for the regeneration and reconstruction of tissues and organs. Heating the aqueous solution of collagen leads to spontaneous self-assembly process to variously distributed fibrillar structures, which are at a later stage of fibrillogenesis prerequisite for creating a three-dimensional supporting network, which is the basic building block of the gel. The resulting properties of the hydrogel depend not only on its structure, but also on the conditions which cause self-assembly process. Hydrogels were performed at 37 ° C and physiological pH. Studied structural variable was the concentration of collagen. So far, for the research of self-assembly were used spectrometric methods, which only provide information about kinetics of morphogenesis. In this work to study the kinetics of collagen I self-assembly were used rheological methods, which additionally give information about viscoelastic properties of the resulting material. The obtained experimental data confirmed two-step process of collagen I fibrillogenesis consisting of nucleation and growth process. Rheological hydrogels collagen behaved as a nonlinear yield-pseudoplastic. An attempt was made to molecular interpretation of the results. Using two-parametric Avrami equation was determined the rate of self-assembly for each concentration of collagen and the value of Avrami exponent determining the shape of produced units. The prepared hydrogels were subjected to increasing shear stresses (strain amplitude, shear rate). Larger amplitudes leads to collapse of the hydrogel structure, which is able to again partially regenerated.
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Structural-Functional Correlations of Hydroxymethylbilane synthase
Douděrová, Dana
Acute intermittent porphyria (AIP) is an autosomal dominantly inherited disorder, classified as acute hepatic porphyria. It is characterized by a deficiency of hydroxymethylbilane synthase (HMBS, EC 4.3.1.8), the third enzyme in heme biosynthesis. Clinical features include gastrointestinal, neurologic and cardiovascular symptoms, but the most common clinical presentation is abdominal pain caused by neurovisceral crises. The purpose of this study was first to perform molecular analysis of the AIP patients. Once a mutation is detected in a patient, molecular testing is offered to family members. In each affected family, this becomes an important tool for individualised medicine, allowing for careful drug prescription; in addition, it is very important for the asymptomatic carriers to be warned of precipitating factors, thus avoiding an acute attack. The proper DNA diagnostics can be achieved by a combination of a robust and effective pre-screening method and a confirmatory DNA sequencing step. We decided to establish a new generation pre-screening method, which will be highly sensitive and relatively time- and cost-effective. Our method of choice was high-resolution melting (HRM) analysis using the LightScanner instrument. Another important aspect of this project was to study the molecular...
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Cell cycle and differentiation in Giardia Intestinalis
Jiráková, Klára ; Nohýnková, Eva (advisor) ; Doležal, Pavel (referee) ; Müller, Norbert (referee)
Giardia is a unicellular parasitic organism; it is a worldwide cause of human diarrhea. It has minimalistic genome equipment and simplified molecular and metabolic pathways. In this respect, it is a suitable model organism for studying cell cycle regulation and to define the minimal genetic and protein equipment required for the functional reproduction of the eukaryotic cell. Its life cycle comprises of two stages; a pathogenic trophozoite and an infective cyst, which can survive in outer environment. New knowledge about encystation can be therapeutically important because this process is a target for vaccine and drug development. Since cell cycle analysis requires a synchronized population, we studied the effect of the synchronization drug aphidicolin on individual cell characteristics during the cell cycle of Giardia trophozoites. Our results showed that aphidicolin caused inhibition of DNA synthesis and trophozoites were aligned according to their DNA content in G1/S border. Subsequent inhibition of entry into mitosis and cytokinesis indicates, that Giardia has functioning DNA damage checkpoint. Extensive treatment with aphidicolin causes side effects. We detected positive signals for phosphorylated histone H2A which, in mammalian cells, is involved in a signaling pathway triggered as a reaction...
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Voltammetric Determination of Anticancer Drug Flutamide and Its Metabolite 4-Nitro-3-trifluoromethylaniline
Radová, Jitka ; Barek, Jiří (advisor) ; Dejmková, Hana (referee)
The subject of this Diploma Thesis has been the study of electrochemical behavior of anticancer drug flutamide (FLD) and one of its metabolites 4-nitro-3-trifluoromethylaniline (NTMA). The aim was to find and define the optimum conditions for the determination of both compounds at a mercury meniscus modified silver solid amalgam electrode (m-AgSAE) and at a carbon film electrode (CFE) using DC voltammetry (DCV) and differential pulse voltammetry (DPV). This Diploma Thesis follows closely the Bachelor Thesis, defended at the Department of Analytical Chemistry, Faculty of Science, Charles University in Prague. In this Bachelor Thesis, a voltammetric determination of FLD using DCV and DPV at m-AgSAE has been described, with attained limits of quantification (LQs) 5.0·10-6 mol.dm-3 (for DCV) and 3.0·10-6 mol.dm-3 (for DPV). In presented Diploma Thesis, voltammetric behavior of NTMA has been studied in dependence on the pH of the medium of Britton-Robinson (BR) buffer-methanol (9:1). The optimum conditions have been found for the determination of this substance using DCV and DPV at m-AgSAE and CFE in the cathodic area. As the optimum media, BR buffer-methanol (9:1) of resulting pHf 8.3 (for both DCV and DPV at m-AgSAE) and of pHf 5.1 (for both DCV and DPV at CFE) have been chosen. Further, the LQs have...
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