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Metabolism of vandetanib by cytochrome P450 expressed in prokaryotic systems
Rodová, Marie ; Indra, Radek (advisor) ; Takácsová, Paulína (referee)
1 Abstract Recently, biologically targeted treatment by another name targeted molecular therapies have begun to be used in the treatment of cancers bearing specific molecular genetic or morphological traits. Vandetanib is an oral anticancer drug that belongs to a group of tyrosine kinases inhibitors. These inhibitors block signal pathway receptors, thereby inhibit growth, stimulate cell death and reduce the spread of cancer. Vandetanib was approved in April 2011 by the US FDA for a treatment of progressive or symptomatic medullary thyroid cancer. It is used in patients with metastatic or inoperable locally advanced cancer. The metabolism of vandetanib was studied in this thesis. Specifically, the kinetics of vandetanib oxidation to N-desmethylvandetanib by human recombinant cytochromes P450 3A4 expressed in the membrane of E. coli (Bactosomes). The effect of the presence of cytochrome b5 and the effect of the level of NADPH: cytochrome P450 reductase activity on the activity of cytochrome P450 3A4 were studied. The demethylated metabolite of vandetanib, N-desmethylvandetanib, was identified and separated by high performance liquid chromatography (HPLC). Enzyme kinetics studies indicate that vandetanib oxidation is affected by both, the level of NADPH:CYP reductase activity and the presence of cyt b5....
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Biocompatible polymer nanomaterials with dexamethasone tailored for anti-inflammation therapy
Starenko, Daniil ; Etrych, Tomáš (advisor) ; Indra, Radek (referee)
Chronic inflammatory diseases are very common and affect lives of many people around the world. Moreover, in long-term effect chronic inflammation can lead to the development of such complications as diabetes, atherosclerosis and oncological diseases. Temporary therapy does not lead to complete recovery of a patient, but can provide a relief of symptoms by means of immunosuppression, which causes many adverse effects, because of long-term and nonselective drug activity. Application of the polymeric drug delivery systems is one of the actively researched ways for the improvement of inflammatory diseases treatment quality, which enables longer circulation, better distribution and controlled release of the drug. Dexamethasone is one of the commonly used glucocorticoid drugs used for treatment of chronic inflammatory diseases. Properties of this drug can be enhanced via polymeric drug delivery systems. Recently, so-called biological treatment, particularly employing monoclonal antibodies, against inflammation was introduced into the clinical use. One of such is infliximab - chimeric monoclonal antibody, which acts like the inhibitor of tumor necrotizing factor α (TNF-α). Presented bachelor thesis focuses on the synthesis and characterization of a polymer conjugates based on...
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Preparation and metabolism of tyrosine kinase inhibitors in nanotransporters
Urbanová, Tereza ; Indra, Radek (advisor) ; Bělonožníková, Kateřina (referee)
Cancer is currently one of the major diseases of civilization, so it is no wonder that in recent decades its research has been a priority for many laboratories. The problem of conventional treatment of oncological diseases, which dates to the beginning of the 1940s, is that it is non-specific to tumor cells and with that is connected a number of side effects. In recent years a new approach to the treatment of this serious disease has emerged that uses various nanotransporters (liposomes, proteins, but also inorganic carbon nanotubes), which can encapsulate cytostatics and release it in a targeted manner around the tumor, thus minimizing the side effects. In this diploma thesis the encapsulation of three cytostatics (lenvatinib, adavosertib and sunitinib) into apoferritin was studied. Furthermore, the effect of the cavity of apoferritin on the biotransformation of encapsulated sunitinib; free and bound sunitinib biotransformation by liposome - bound biotransformation enzymes and last but not least, the release kinetics of sunitinib from the apoferritin central cavity were analysed. Lenvatinib has not been shown to be a suitable cytostatic for encapsulation into the central cavity of apoferritin due to its low solubility and negative charge at basic pH. Adavosertib and sunitinib appear to be more suitable...
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Metabolism of cabozantinib by enzymes of first phase of biotransformation
Jurečka, Tomáš ; Indra, Radek (advisor) ; Kubíčková, Božena (referee)
Cabozantinib is an anticancer drug that inhibit tyrosine kinases which allow signal pathways important for growth and development of tumors. It is used for treatment of medullary thyroid cancer, hepatocellular carcinoma and kidney cancer. The major enzymes of the first phase of biotransformation that metabolize cabozantinib are cytochromes P450. In this thesis it was studied metabolism of cabozantinib and cytochromes P450 that participated on this metabolism. Hepatic microsomes of rat, mouse and rabbit were used for studying metabolism of cabozantinib in this thesis. It was also focused on the impact of particular isoforms of cytochromes P450 on metabolism of cabozantinib in rat microsomes. Time dependence of cabozantinib conversion in hepatic rat microsomes was also studied. Enzyme kinetics of metabolism of cabozantinib in hepatic rat microsomes, as well as impact of cytochromes P450 inhibitors on the metabolism were included. Metabolites were analyzed by high performance liquid chromatography (HPLC) and mass spectrometry. Formation of metabolites of cabozantinib increased over time to 30 minutes of incubation and with some others to 40 minutes of incubation. Up to five different metabolites were detected in experiments (M1, desmethyl cabozantinib, M3, monohydroxy cabozantinib and cabozantinib...
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Importance of V-ATPase for cancer cell resistence
Suchánková, Kristýna ; Eckschlager, Tomáš (advisor) ; Indra, Radek (referee)
Chemoresistance is one of the main causes of failure of anticancer chemotherapy. Vacuolar-type ATPase (V-ATPase) is an ATP-dependent proton pump involved in the regulation of the pH in cells, cell organelles and the intracellular space. A significant acidification of the extracellular space and intracellular compartments occurs in connection with the metabolism of tumour cells (glucose metabolism, hypoxia, insufficient blood perfusion of the cancer tissue). Basic drugs are transferred into acidic organelles based on the pH gradient, where they are then protonated and accumulated. This mechanism is called lysosomal sequestration and is one of the mechanisms how tumour cells resist to applied drugs, which then do not reach their target site in cancer cell. An increased expression of V-ATPases has been described in relation to chemoresistance and the progression of tumours. This dissertation is focused on observing the membrane subunit V0d from the complex of V-ATPase and the changes in resistance to ellipticine caused by the silencing of this subunit's gene in human neuroblastoma cell lines UKF-NB-4 (sensitive) and UKF-NB-4ELLI (resistant to ellipticine). The expression of the V0d protein was first examined on mRNA level using real-time polymerase chain reaction (RT-PCR). The silencing of selected...
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Study of effects of tyrosine kinase inhibitors and their metabolites on tumour cell lines
Kolárik, Matúš ; Indra, Radek (advisor) ; Vinklářová, Lucie (referee)
Vandetanib, lenvatinib and cabozantinib are inhibitors of receptor tyrosine kinases approved to treat locally advanced or metastatic thyroid gland, kidney and liver cancers. These multi- kinase inhibitors, inhibit phosphorylation of tyrosine moieties of protein, thus modulate cell signalization in cancer cells. Metabolites of vandetanib, lenvatinib and cabozantinib were detected in vitro as well as in vivo in blood and urine. Cytochromes P450 and flavin monooxygenases were identified as primary enzymes participating in metabolism of these drugs. Literature lacks information regarding pharmacological efficacy of vandetanib, lenvatinib and cabozantinib metabolites. The aim of this diploma thesis was the investigation of pharmacological efficacy of N-oxides of vandetanib, lenvatinib and cabozantinib. The viability measurement under normoxic and hypoxic conditions was employed to determined their efficacy. The expression of enzymes of the first phase of xenobiotics metabolism (CYP 450 1A1, 1B1, 3A4 a CYP 450 oxidoreductase) and receptor tyrosine kinases RET and VEGFR2, as well as mechanism of changes in their expression were investigated using western blotting and flow cytometry. High performance liquid chromatography was utilised to investigate possible metabolism of tyrosine kinase inhibitors and...
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Anticancer drugs in forms of nanoparticles and mechanisms potentiating their anticancer efficiency
Meskařová, Veronika ; Indra, Radek (advisor) ; Bělonožníková, Kateřina (referee)
Cancer has been one of the most common diseases of civilization for centuries. In the 18th century, some cancers were described and the first treatments were proposed. Currently, oncosurgery, chemotherapy, radiotherapy, immunotherapy and hormonal treatment are used to treat cancer. At the same time, efforts are being made to find new anticancer drugs that target tumor cells more selectively. Recently, nanomedicine has also started to be used. This bachelor thesis deals with minimizing the binding of the amount of cytostatic ellipticine to the surface of the nanotransporter apoferritin and achieving higher encapsulation efficiency. Two types of apoferritins at different weight ratios to ellipticine were studied. It has been found that by finding a suitable weight ratio of the two molecules, binding can be minimized, and encapsulation efficiency can be increased. When working with commercial apoferritin, there was a higher encapsulation and a lower binding of ellipticine to the surface at the weight ratio of 1:10. In contrast, when working with recombinant apoferritin, the encapsulation is higher and ellipticine binding are lower at the lower ratio of 1: 2,5. Key words: oncological diseases, anticancer drugs, nanomedicine, cytostatic, nanotransporter, ellipticine, apoferritin [IN CZECH]
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Metabolism of inhibitors of tyrosine kinases, the drugs of new generation
Čillíková, Olívia ; Indra, Radek (advisor) ; Kubíčková, Božena (referee)
Cancer is the second major cause of death after heart-attack in the world. In recent years, research has focused on tyrosine kinase inhibitors (TKIs) as part of targeted chemotherapeutic treatment. Vandetanib is a TKI affecting epidermal growth factor receptor (EGFR), rearrangement during transfection (RET) and vascular endothelial growth factor receptor 2 (VEGFR2). It is primary used for treatment of medullary thyroid cancer. Vandetanib is biotransformed by cytochromes P450 and flavin monooxygenases in human organism. Cytochromes P450 (CYPs) oxidaze vandetanib to only one metabolite, N-desmethyl vandetanib, which exhibits similar efficiency as parental molecule. NADPH is the major cofactor of reaction cycle of CYPs. This bachelor thesis studies the effect of various types of cofactors and pH on oxidation of vandetanib by selected human recombinant cytochromes P450, namely CYP2C8 coexpressed with cyt b5, CYP2D6, CYP3A4 and CYP3A4 coexpressed with cyt b5. Here, we investigate the effect of cofactors NADPH, NADH and their mixture in a 1:1 ratio on the amount of N- desmethyl vandetanib formed during the biotransformation of vandetanib. The effect of pH on the oxidation of vandetanib by CYP 3A4 and CYP 3A4 + b5 was also analysed. We analysed the amount of the metabolite formed at the pH range 7 to 8.5...
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Metabolism of inhibitors of tyrosine kinases, the drugs of new generation
Husák, Ondřej ; Indra, Radek (advisor) ; Černá, Věra (referee)
Vandetanib is orally administrated anti-cancer drug that belongs to the class of tyrosinekinase inhibitors which are used for thyroid cancer treatment. This drug undergoes biotransformation and oxidizes into two metabolites - N desmethyl vandetanib and vandetanib N-oxide. The purpose of this bachelor thesis is to compare the potency of vandetanib to vandetanib N- oxide on neroblastome cell line (UKF-NB-4); the study of metabolic process of vandetanib N- oxid and its effect on vandetanib oxidation. In this bachelor thesis we have confirmed the lower potency of vandetanib N-oxide in comparison to vandetanib. We have also discovered that vandetanib N-oxide undergoes further oxidation into another metabolite via microsomes of phenobarbital treated rats. Data suggest that in the presence of vandetanib N-oxide an increased oxidation of vandetanib to N-desmethyl vandetanib occurs. Further experiments have not confirmed this hypothesis. In higher concentrations of vandetanib, the presence of vandetanib N-oxide did not have any effect on oxidation of vandetanib to N-desmethyl vandetanib. The lower concentration led to inhibition of oxidation of vandetanib.
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Study of tyrosin kinase inhibitor vandetanibe bound in apoferritin and liposomes
Jáklová, Kateřina ; Indra, Radek (advisor) ; Hýsková, Veronika (referee)
5 Abstract In this thesis the anticancer drug vandetanib was studied. Vandetanib is a tyrosine kinase inhibitor affecting signalling of vascular endothelial growth factor receptor (VEGFR), epidermal growth factor receptor (EGFR) or RET protooncogene (REarranged during Transfection). It is primarily used for the treatment of advanced tumors of the thyroid gland. Unfortunately, the usage of vandetanib in the cancer treatment is significantly limited by its toxicity and cardiotoxicity (one of the adverse effects is connected with long QT interval). One way, how to minimize these side effects, is binding a drug into a suitable transporter. Apoferritin and liposomes were used as a transport nanoparticles in this study. The aim of this thesis was to study the stability of the complex of nanoparticle apoferritin with vandetanib molecules (ApoVan) and to study the effect of pH on the release of inhibitor from the ApoVan form. Experiments have shown that ApoVan complex is relatively stable after its storage at 4 řC and - 20 řC for up to 8 weeks. Unfortunately after monitoring the effect of pH on the release of vandetanib from ApoVan, it was found that vandetanib is gradually released from its ApoVan form into the neutral environment at pH 7,4 as well as into the acidic environment at pH 6,5 and the way ApoVan is...
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