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Study of expression of biotransformation enzymes and transport proteins in kidneys
Felešová, Martina ; Smutná, Lucie (advisor) ; Kuchařová, Monika (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Student: Martina Felešová Supervisor: PharmDr. Lucie Smutná, PhD. Title of diploma thesis: Study of expression of biotransformation enzymes and transport proteins in kidneys Xenobiotics are a part of the everyday life of living organisms. Their source is, among other things, diet, environment, administered medicines, food colorings and others. The living organisms have developed complex metabolic processes that eliminate these substances from the body in order to protect themselves against their negative influence. Within this work, we specifically focused on the effect of the murine Car agonist - TCPOBOP on influencing the expression of biotransformation and transport genes. The essence was the isolation of mRNA from the murine kidneys using the classic phenol-chloroform method, followed by the transcription of mRNA into cDNA. In the last step, the RT-PCR reaction was performed. The results are graphs showing how the administration of TCPOBOP affected the expression of the monitored genes. It is clear from these graphs that TCPOBOP has some effect on the expression of target genes. In the case of Cyp3a11, Cyp2b10, Slc22a2 and Ugt3a2, there was a several-fold increase in expression after...
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The role of drug transporters in activation of pregnane X receptor
Kejvalová, Kateřina ; Smutná, Lucie (advisor) ; Pourová, Jana (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Student: Kateřina Kejvalová Supervisor: PharmDr. Lucie Smutná, Ph.D. Title of diploma thesis: The role of drug transporters in activation of pregnane X receptor The nuclear pregnane X receptor (PXR) is involved in maintaining the homeostasis of endogenous substances as well as in regulation of excretion of exogenous substances. PXR activation regulates the expression of transport and biotransformation enzymes I. and II. phase. Due to the high flexibility of PXR ligand binding domain, it can be activated by a wide range of xenobiotics. Including e.g., rifampicin, well-known PXR agonist causes clinically significant drug-drug interactions. However, rifampicin is not stable under physiological conditions and degradation occurs (into rifampicin quinone, 25-desacetylrifampicin, 3-formylrifamycin SV and rifampicin N-oxide). The aim of our experiments was to compare the interspecies activation of PXR (human, monkey, mouse and rat) by rifampicin and its derivatives. Hence, gene reporter experiments were performed using the HepG2 cell line. Statistically significant results in PXR activation were observed for human hPXR and monkey oPXR for all rifampicin derivatives except 25- desacetylrifampicin. Similarity...
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Vasodilatory effects of 4-methyl catechol: ex vivo mechanism of action
Kuzdřalová, Kateřina ; Pourová, Jana (advisor) ; Smutná, Lucie (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Student: Kateřina Kuzdřalová Supervisor: doc. PharmDr. Jana Pourová, Ph.D. Title of diploma thesis: The mechanism of vasodilation effect of 4-methylcatechol ex vivo Keywords: rat, aorta, flavonoids, 4-methylcatechol, vascular smooth muscle, vasodilation Flavonoids belong to the group of polyphenolic bioactive substances found abundantly in plants, fruits and vegetables. Thanks to their pharmacological and biochemical effects, they play a crucial role for human health, especially in the prevention of numerous diseases - metabolic syndrome, osteoporosis, atherosclerosis and also diseases of a cardiovascular system. The aim of the diploma thesis was to determine the mechanism of the vasorelaxant effect of one of the most important metabolites of flavonoids - 4-methylcatechol. For the research we used a standardized ex vivo method on isolated Wistar rat aortic rings. We were able to confirm the vasodilatory effects of 4-methylcatechol. We also confirmed the fact that the flavonoid metabolite directly affects a vascular smooth muscle. Furthermore, we found that the tested compound potentiates the vasodilatory activity of sodium nitroprusside and forskolin, and that vasodilation depends on the activity of...
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Determination of renal toxicity of antineoplastics in vitro
Zádrapová, Marie ; Maixnerová, Jana (advisor) ; Smutná, Lucie (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Student: Bc. Marie Zádrapová Supervisor: RNDr. Jana Maixnerová, Ph.D. Title of diploma thesis: Determination of renal toxicity of antineoplastics in vitro BRAF inhibitors are important antineoplastics. They work on the principle of inhibition of certain types of protein kinases and turned out to be very efficient for the treatment of melanoma. One of their disadvantages is relatively early onset of resistance; thus, it is important to look for new combinations of drugs that are already in use or work on the development of new structures with similar inhibition efficacy on melanoma cells. Encorafenib and its combination with binimetinib have been shown to be very promising drugs from the group of BRAF inhibitors, however, potential renal toxicity may be a therapeutic limitation. This thesis was focused on the determination of in vitro cytotoxicity of encorafenib on different types of renal cells in three time intervals and on its comparison with two drug standards - amphotericin B and paracetamol. Three types of morphologically and functionally different kidney cells (PODO / TERT256, HK-2 and HEK293) were used for this purpose. The cytotoxic potential was measured by colorimetric method CellTiter 96®...
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Vasodilatory effects of 4-methyl catechol: ex vivo mechanism of action
Kuzdřalová, Kateřina ; Pourová, Jana (advisor) ; Smutná, Lucie (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Student: Kateřina Kuzdřalová Supervisor: doc. PharmDr. Jana Pourová, Ph.D. Title of diploma thesis: The mechanism of vasodilation effect of 4-methylcatechol ex vivo Keywords: rat, aorta, flavonoids, 4-methylcatechol, vascular smooth muscle, vasodilation Flavonoids belong to the group of polyphenolic bioactive substances found abundantly in plants, fruits and vegetables. Thanks to their pharmacological and biochemical effects, they play a crucial role for human health, especially in the prevention of numerous diseases - metabolic syndrome, osteoporosis, atherosclerosis and also diseases of a cardiovascular system. The aim of the diploma thesis was to determine the mechanism of the vasorelaxant effect of one of the most important metabolites of flavonoids - 4-methylcatechol. For the research we used a standardized ex vivo method on isolated Wistar rat aortic rings. We were able to confirm the vasodilatory effects of 4-methylcatechol. We also confirmed the fact that the flavonoid metabolite directly affects a vascular smooth muscle. Furthermore, we found that the tested compound potentiates the vasodilatory activity of sodium nitroprusside and forskolin, and that vasodilation depends on the activity of...
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Study of expression of biotransformation enzymes and transport proteins in kidneys
Felešová, Martina ; Smutná, Lucie (advisor) ; Kuchařová, Monika (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Student: Martina Felešová Supervisor: PharmDr. Lucie Smutná, PhD. Title of diploma thesis: Study of expression of biotransformation enzymes and transport proteins in kidneys Xenobiotics are a part of the everyday life of living organisms. Their source is, among other things, diet, environment, administered medicines, food colorings and others. The living organisms have developed complex metabolic processes that eliminate these substances from the body in order to protect themselves against their negative influence. Within this work, we specifically focused on the effect of the murine Car agonist - TCPOBOP on influencing the expression of biotransformation and transport genes. The essence was the isolation of mRNA from the murine kidneys using the classic phenol-chloroform method, followed by the transcription of mRNA into cDNA. In the last step, the RT-PCR reaction was performed. The results are graphs showing how the administration of TCPOBOP affected the expression of the monitored genes. It is clear from these graphs that TCPOBOP has some effect on the expression of target genes. In the case of Cyp3a11, Cyp2b10, Slc22a2 and Ugt3a2, there was a several-fold increase in expression after...
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The role of drug transporters in activation of pregnane X receptor
Kejvalová, Kateřina ; Smutná, Lucie (advisor) ; Pourová, Jana (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Student: Kateřina Kejvalová Supervisor: PharmDr. Lucie Smutná, Ph.D. Title of diploma thesis: The role of drug transporters in activation of pregnane X receptor The nuclear pregnane X receptor (PXR) is involved in maintaining the homeostasis of endogenous substances as well as in regulation of excretion of exogenous substances. PXR activation regulates the expression of transport and biotransformation enzymes I. and II. phase. Due to the high flexibility of PXR ligand binding domain, it can be activated by a wide range of xenobiotics. Including e.g., rifampicin, well-known PXR agonist causes clinically significant drug-drug interactions. However, rifampicin is not stable under physiological conditions and degradation occurs (into rifampicin quinone, 25-desacetylrifampicin, 3-formylrifamycin SV and rifampicin N-oxide). The aim of our experiments was to compare the interspecies activation of PXR (human, monkey, mouse and rat) by rifampicin and its derivatives. Hence, gene reporter experiments were performed using the HepG2 cell line. Statistically significant results in PXR activation were observed for human hPXR and monkey oPXR for all rifampicin derivatives except 25- desacetylrifampicin. Similarity...
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Using of genetically modified cells lines to study transport processes in in vitro kidney models.
Mužíková, Michaela ; Smutná, Lucie (advisor) ; Trejtnar, František (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Student: Michaela Mužíková Supervisor: PharmDr. Lucie Smutná, PhD. Title of diploma thesis: Using of genetically modified cells lines to study transport processes in in vitro kidney models Transport processes in renal cells are mostly secured by proximal tubule transporters. These transporters mediate excretion and reabsorption of wide variety of endogenous and exogenous substances, including xenobiotics. Within the broad spectrum of many drug-transporting proteins, we have chosen the glycoprotein megalin (LRP2), a receptor involved in the reabsorption of albumin and other endogenous substances, such as insulin or hemoglobin. Peptide and aminoglycoside antibiotics represented by e.g., gentamicin and belongs between LRP2 ligands. The diploma thesis deals with the development and utilization of the LRP2 knockout cell lines in accumulation studies. The experiments were performed using human cell lines naturally expressing LRP2, the JEG-3 and HK-2. Several genetically modified cell lines expressing unfunctional LRP2 and consequently exerting harmed ability of model ligand internalization were prepared using the CRISPR/Cas9 technique. The effect was confirmed by accumulation studies with FITC-albumin....
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Study of substance transport via membrane transporters
Kučerová, Zuzana ; Smutná, Lucie (advisor) ; Maixnerová, Jana (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Student: Zuzana Halamová Supervisor: PharmDr. Lucie Smutná, PhD. Title of diploma thesis: Study of substance transport via membrane transporters Wide range of specialized transport proteins expressed mainly in the kidney proximal tubule ensures the excretion of endogenous and exogenous substances. OCT2 (organic cation transporter 2) is localized on the basolateral membrane of the kidney proximal tubule, the transporter transports broad spectrum of structurally different substances. Drug-drug interactions may occur at the level of this transporter due to its broad substrate specificity. Within this diploma thesis the accumulation of the radiolabeled antiviral agent [3 H]lamivudine and the model hOCT2 substrate [3 H]MPP+ was evaluated using accumulation and transport studies. The canine kidney cells MDCKII or human cell line HeLa were used as model cell lines. Mentioned cell lines expressed the human OCT2 transporter stably or transiently. The antibacterial chemotherapy agent trimethoprim was used as competitive inhibitor of OCT2 transporter. The results indicate the lamivudine accumulation in the cells with OCT2 is low compared to accumulation of MPP+ . Accumulation studies showed that lamivudine had a...
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In vitro saturation study of gallium-67 and zirconium-89 labelled monoclonal antibody ramucirumab on SKOV-3 cell line
Holodňáková, Nikola ; Bárta, Pavel (advisor) ; Smutná, Lucie (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Biophysics and Physical Chemistry Student: Nikola Holodňáková Supervisor: Mgr. Pavel Bárta, Ph.D. Name of the work: In vitro saturation study of gallium-67 and zirconium-89 labelled monoclonal antibody ramucirumab on SKOV-3 cell line. Targeted biological treatment becomes more and more important with the development of a new therapy in oncology. It stimulates immune system to eliminate cancer cells. Significant progress has been made since the introduction of monoclonal antibodies. They represent one of the newest possibility used in diagnosis and treatment of tumours. The ability of the monoclonal antibody ramucirumab is to recognize and bind specifically to tumour cell antigens such as the VEGF type 2 receptor (VEGFR-2) and thus to supress angiogenic process. Anti-angiogenic ramucirumab inhibits this receptor via blocking of VEGF binding sites, which prevents the growth of tumours. It is possible to increase antitumor effect of monoclonal antibodies by their combination with other molecules like radionuclides, toxins and cytostatics when forming the so called conjugates. Prepared immunoconjugates serve as diagnostic and therapeutic tools also in Nuclear Medicine. The aim of the experimental work in was the preparation of...
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