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Effect of cytochromes P450 on metabolism of anticancer drugs bound into apoferritin nanoparticle
Wilhelm, Marek ; Indra, Radek (advisor) ; Ptáčková, Renata (referee)
Tumour-related diseases are the second most common cause of death in the Czech Republic, right after cardiovascular diseases. Nanomedicine - a novel scientific discipline - shows captivating potential in anticancer treatment with help of so called nanotranporters - nanoparticles capable of transporting other molecules. Encapsulation of a cytostatic drug into a nanoparticle improves its pharmacokinetical and pharmacodynamical properties which helps to reduce adverse side effects on non-tumour healthy tissue. In the scope of this diploma thesis apoferritin - apo-form of ferritin - was studied, since this nanotransporter shows promise for clinical use in anticancer treatment. Effect of hepatic microsomes from premedicated and control rats on biotransformation of doxorubicin cytostatic (Dox) in free and apoferritin nanoparticle-bound forms was investigated at pH 7,4. Over the course of biotransformation two types of metabolites - M1 and M2 - were observed. Regardless of the employed inductor all studied microsomes have exhibited similar metabolism of free doxorubicin and its apoferritin encapsulated form (ApoDox). Our results also imply that doxorubicin can be metabolically processed by rat hepatic microsomes in both free and ApoDox form with similar efficiency. We have also studied biotransformation...
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Expression and purification of protein containing photo-methionine - the use of photo-induced cross-linking to map protein-protein interaction
Tichá, Andrea ; Ptáčková, Renata (advisor) ; Indra, Radek (referee)
Protein 14-3-3ζ is an important protein that plays a role in the regulation of cell survival and keeps cell from stress and possible apoptosis. A photo-activatable group can be incorporated into the protein to detect protein-protein interaction by photo induced crosslinking. The aim of this work was to create protein with incorporated photo-methionine in auxotrophic bacteria E. coli B834 and to optimize expression conditions. Photo-methionine is structurally very similar to natural methionine, so photo-methionine can be incorporated into the protein by host aminoacyl tRNA synthetase. It has been studied how the rate of incorporation varies depending on production time. After 3 or 6 hours, up to 70 % incorporation of photo-methionine was achieved compared to about 50 % after one hour.
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Metabolism of tyrosine kinase inhibitor cabozantinib by rat liver microsomes
Jurečka, Tomáš ; Indra, Radek (advisor) ; Mrízová, Iveta (referee)
Cabozantinib is an anticancer drug that was approved for treatment of progressive thyroid cancer by FDA and EMA organizations. Cabozantinib is a tyrosine kinase inhibitor. It blocks signal pathway receptors that are important for growth of tumors. This bachelor's thesis describes the findings about the metabolism of cabozantinib. It studies metabolism of cabozantinib in hepatic microsomes isolated from various laboratory animals (rat, mouse and rabbit) and impact of particular isoforms of cytochromes P450 (CYP) on metabolism of cabozantinib in rat hepatic microsomes. The bachelor's thesis also describes the optimization of method for separation metabolites of cabozantinib by high performance liquid chromatography (HPLC) and also identification of metabolites using mass spectrometry. Up to three different metabolites were detected by utilizing hepatic microsomes isolated from various laboratory animals. Those were M1, monohydroxycabozantinib and O-desmethylcabozantinib. Mouse microsomes oxidized cabozantinib mainly to O-desmethylcabozantinib and rabbit microsomes metabolised cabozantinib mainly to monohydroxycabozantinib. Microsomes from controlled rats produced two metabolites with the overall majority of monohydroxycabozantinib. The highest number of metabolites was produced by microsomes from...
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Preparation of anticancer drugs bound in apoferritin
Fürbacherová, Pavlína ; Indra, Radek (advisor) ; Koblihová, Jitka (referee)
Cancer is one of the most serious problems, which modern medicine faces. In recent years, nanotechnologies and their use in medicine, has developed greatly. The aim is to make drug administration more effective and help to improve treatment of cancer illnesses. Incorporation of chemical substance into a nanoparticle can solve the problem with low stability of the drug, and/or it help to eliminate side effects. Nanoparticle apoferritin, which was studied in this thesis, is a form of commonly occurring protein ferritin. Its structure contains cavity, that can be used for incorporation of drug. Its chemical structure (high temperature stability and stability at wide pH range, easy manipulation by changing pH) and its biocompatibility makes apoferritin a potentionally suitable transporter. Presented thesis studied apoferritin's ability to incorporate anticancer drug cabozantinib into its structure. Cabozantinib is tyrosine kinase inhibitor which is used for treatment of thyroid cancer, renal cell carcinoma and hepatocellular carcinoma. The effect of final pH to the formation of the complex of apoferritin with cabozantinib, and stability of this complex was also studied in this thesis. Considering the results we can say that apoferritin is able to encapsulate cabozantinib into its inner structure. As we...
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Preparation of drugs in nanoparticles for targeted treatment of cancer
Jáklová, Kateřina ; Indra, Radek (advisor) ; Bělonožníková, Kateřina (referee)
The aim of this thesis was to study the ability of the apoferritin to encapsulate drugs vandetanib and etoposid. Vandetanib is an anticancer drug used for the treatment of tumors of the thyroid gland. It acts as an inhibitor of tyrosine kinase receptor. Etoposid, which is widely used for the treatment of malignant blood diseases or solid tumors inhibits type II topoisomerase that regulates topology of DNA. Targeting treatment with nanoparticles can minimize adverse effects connected with both drugs. An apoferritin is a naturally occurring protein that is composed of 24 ferritin subunits. Its structure creates an internal cavity that can be loaded by any compoundands. The structure is remarkably stable and is able to withstand biologically extreme temperatures (up to 70řC) and a wide pH range (pH 2-10). Furthermore, apoferritin can move undetected through the body without any immune response. It is also possible to modify its surface by ligands specific for targeting tissues. The effect of the concentration of apoferritin and the effect of the concentration of the drug on the final concentration of the encapsulated drug was studied. In addition, even the effectiveness of encapsulation was studied. In the case of vandetanib, at a constant concentration of drug and an increasing concentration of...
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Chemo-enzymatic synthesis of antiviral prodrugs
Tupec, Michal ; Stiborová, Marie (advisor) ; Indra, Radek (referee)
Lipases have been widely applied in the manufacture of food products and in some areas of the industry, nowadays they are used in synthetic organic chemistry catalyzing the hydrolytic/esterification reactions under very mild conditions in the field of protecting groups or enantiomer resolution. In this study, the commercial lipase from bacterium Pseudomonas fluorescens was immobilized using the sol-gel process into organosilicate materials with propyl, octyl or phenyl substituents. The highest hydrolytic activity was found in the enzyme on the octyl-derived carrier. The immobilized enzymes differ in their hydrolytic activities on 4-nitrophenyl esters of various lengths. Subsequent experiments revealed quite good pH stability of the enzymes in a buffer (incubations in pH 3 through pH 11), as well as good temperature stability in isooctane (incubations at up to 100 řC). The majority of organic solvents seem to have no substantial effect on the lipase activity. The biocatalytic properties were studied on a model compound from the group of the acyclic nucleoside analogues - 9-(2',3'-dihydroxypropyl)adenine (DHPA). It was found for example that the best acyl donors are vinyl esters, that the lipase shows a preference towards longer vinyl esters, that the reaction proceeds faster in non-polar solvents or that it...
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Mechanism of enzymatic activation of carcinogens and drugs by the system of cytochrome P450
Indra, Radek ; Stiborová, Marie (advisor) ; Souček, Pavel (referee) ; Koblihová, Jitka (referee)
13 Abstract An environmental pollutant and a human carcinogen benzo[a]pyrene (BaP) is after its activation with cytochrome P450 (CYP) able to covalently bind to DNA. In the thesis, one of the target was to investigate an influence of individual components of mixed function monooxygenase (MFO) system on metabolism of benzo[a]pyrene and generation of adducts of activated BaP with DNA. The study was particularly focused to increase our knowledge on the effect of cyt b5 on metabolism of BaP by cytochrome P450 1A1 (CYP1A1) and its potential to serve as a donor of electrons during the reaction cycle of this cytochrome P450. The effect of cyt b5 on generation of BaP metabolites and adducts of BaP with DNA was investigated. In addition the effect of two different expression systems for cytochrome P450 1A1 (prokaryotic and eukaryotic) was also studied. The influence of cyt b5 on oxidation another xenobiotic compound, a plant alkaloid ellipticine that exhibit antitumor activities, was also investigated. Its pharmacological efficiency, as well as side effects depends on its metabolic activation by cytochrome P450. CYP3A4 is very important for ellipticine activation and therefore this enzyme was used in our experiments. Furthermore, a suitability of rat as a model organism mimicking the metabolic fate of BaP...
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Activity of cytochromes P450 1A1, 1A2 and 3A4 expressed in eukaryotic and prokaryotic systems
Indra, Radek ; Stiborová, Marie (advisor) ; Mizerovská, Jana (referee)
Cytochromes P450 (CYP) are a superfamily of heme proteins distributed widely throughout nature, involved in metabolism of a broad variety of substrates and catalyzing a variety of interesting chemical reactions. They play a central role in metabolism of chemotherapeutic agents. Several prodrug antitumor agents have been found as CYP substrates. Ellipticine, an alkaloid found in Apocynaceae plants, is an example of such type of pro-drug. Here, we investigate the efficiencies of human recombinant CYPs expressed in eukaryotic and prokaryotic expression systems, namely in SupersomesTM , microsomes isolated from insect cells transfected with baculovirus construct containing cDNA of human CYP1A1, 1A2 and 3A4 with NADPH:CYP reductase or in Bactosomes, the membrane fraction of E. coli transfected with cDNA of the same human CYP enzymes and NADPH:CYP reductase to oxidize their marker substrates and ellipticine. Cytochrome b5, an aditional component of the mixed function oxidase system, which metabolize xenobiotics was also expressed in some of the systems. The results found in this work demonstrate that human CYP1A1, 1A2 or 3A4 expressed in both eukaryotic and procaryotic systems oxidize their marker substrates (EROD for CYP1A1/2, MROD for CYP1A2 and testosterone 6β-hydroxylation for CYP3A4). They also oxidize...
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