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Study of the effect of ABC drug transporters' overexpression on the antiproliferative capacities of selected conventional cytostatics
Malečová, Lucie ; Hofman, Jakub (advisor) ; Trejtnar, František (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Lucie Malečová Supervisor: doc. RNDr. Jakub Hofman, Ph.D. Title of diploma thesis: Study of the effect of ABC drug transporters' overexpression on the antiproliferative capacities of selected conventional cytostatics Overexpression of ABC drug transporters is one of the mechanisms of multiple drug resistance development, which results in therapy failure in cancer. ABC transporters such as ABCB1, ABCG2 and ABCC1 have a wide range of substrates, including several conventional cytostatics. This study aimed to examine the influence of overexpression of the mentioned transporters on the antiproliferation effects of docetaxel, etoposide, paclitaxel, methotrexate, pemetrexed, topotecan and vinblastin. The results were obtained with the MTT assay in which the MDCKII and A431 cell lines with/without overexpression of mentioned transporters were used. A decrease of cytotoxicity in the presence of at least one of the transporters was observed in five of seven tested cytostatics. Only topotecan has been proven to be the victim of overexpression of all of the tested transporters. To prove the applicability of the obtained results, topotecan was used in the follow-up combination study. Within this study, I have...
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Assessment of drug cytotoxicity using various cell viability tests
Kaňková, Kateřina ; Hofman, Jakub (advisor) ; Kozlíková, Magdaléna (referee)
V ANGLICKÉM JAZYCE Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Kateřina Kaňková Supervisor: doc. RNDr. Jakub Hofman, Ph.D. Supervisor-consultant: doc. PharmDr. Ondřej Soukup, Ph.D. Title of diploma thesis: Assessment of drug cytotoxicity using various cell viability tests Nowadays, cell viability assays are an important part of preclinical studies. There is a wide spectrum of different methods that can be used for such purposes. This thesis aims to compare some of the fundamental methods, their accuracy and selectivity, as well as the advantages and drawbacks of their application. Six different drugs were measured using five different cell viability assays - colorimetric (MTT, crystal violet), fluorimetric (propidium iodide, calcein-AM) and luminometric (ATP) on the CHO-K1 cell line. The outcomes of all methods are IC50 values of used drugs assessing their cytotoxicity, including graphic representations of the measurements and standard deviations. Finally, all the methods are compared based on experience gained during the experiment as well as information gained during the theoretical research. Among the most suitable methods belong the MTT and ATP assay. Due to some difficulties during the measurement (e.g. unsuitable for use in...
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Study on interactions of selected targeted drugs with ABC drug transporters
Svobodová, Markéta ; Hofman, Jakub (advisor) ; Čečková, Martina (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Markéta Svobodová Supervisor: doc. RNDr. Jakub Hofman, Ph.D. Title od diploma thesis: Study on interactions of selected targeted drugs with ABC drug transporters Cancer is one of the most common causes of death and its incidence is constantly increasing. Treatment mainly includes a combination of drug therapy, surgery and radiotherapy. A new approach, with huge potential, is the use of targeted anti-tumor therapy. This approach is based on specific interaction with tumor cells that reduces the negative effects occuring in conventional chemotherapy. Despite intensive research, we still face insufficient response to treatment. The main cause of therapy failure is the emergence of multidrug resistance (MDR) and the formation of metastases. The MDR phenomenon is most often associated with the overexpression of ATP binding cassette (ABC) transporters. These are responsible not only for the excessive efflux of anticancer drugs from cells, but also mainly affect their pharmacokinetics. Inhibition of these transporters increases the intracellular concentration of the cytostatic and provide the ability to modulate MDR. Therefore, one of the aims of this work was to investigate the inhibitory activity of...
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Assessment of drug cytotoxicity using various cell viability tests
Kaňková, Kateřina ; Hofman, Jakub (advisor) ; Kozlíková, Magdaléna (referee)
V ANGLICKÉM JAZYCE Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Kateřina Kaňková Supervisor: doc. RNDr. Jakub Hofman, Ph.D. Supervisor-consultant: doc. PharmDr. Ondřej Soukup, Ph.D. Title of diploma thesis: Assessment of drug cytotoxicity using various cell viability tests Nowadays, cell viability assays are an important part of preclinical studies. There is a wide spectrum of different methods that can be used for such purposes. This thesis aims to compare some of the fundamental methods, their accuracy and selectivity, as well as the advantages and drawbacks of their application. Six different drugs were measured using five different cell viability assays - colorimetric (MTT, crystal violet), fluorimetric (propidium iodide, calcein-AM) and luminometric (ATP) on the CHO-K1 cell line. The outcomes of all methods are IC50 values of used drugs assessing their cytotoxicity, including graphic representations of the measurements and standard deviations. Finally, all the methods are compared based on experience gained during the experiment as well as information gained during the theoretical research. Among the most suitable methods belong the MTT and ATP assay. Due to some difficulties during the measurement (e.g. unsuitable for use in...
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Study of the effect of ABC drug transporters' overexpression on the antiproliferative capacities of selected conventional cytostatics
Malečová, Lucie ; Hofman, Jakub (advisor) ; Trejtnar, František (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Lucie Malečová Supervisor: doc. RNDr. Jakub Hofman, Ph.D. Title of diploma thesis: Study of the effect of ABC drug transporters' overexpression on the antiproliferative capacities of selected conventional cytostatics Overexpression of ABC drug transporters is one of the mechanisms of multiple drug resistance development, which results in therapy failure in cancer. ABC transporters such as ABCB1, ABCG2 and ABCC1 have a wide range of substrates, including several conventional cytostatics. This study aimed to examine the influence of overexpression of the mentioned transporters on the antiproliferation effects of docetaxel, etoposide, paclitaxel, methotrexate, pemetrexed, topotecan and vinblastin. The results were obtained with the MTT assay in which the MDCKII and A431 cell lines with/without overexpression of mentioned transporters were used. A decrease of cytotoxicity in the presence of at least one of the transporters was observed in five of seven tested cytostatics. Only topotecan has been proven to be the victim of overexpression of all of the tested transporters. To prove the applicability of the obtained results, topotecan was used in the follow-up combination study. Within this study, I have...
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Study of the role of nucleoside transporters in intestinal absorption with the use of in vitro transport method based on the Caco-2 cell line.
Ďurišová, Markéta ; Červený, Lukáš (advisor) ; Hofman, Jakub (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Mgr. Markéta Ďurišová Supervisor: PharmDr. Lukáš Červený, Ph.D. Title of rigorosum thesis: Study of the role of nucleoside transporters in intestinal absorption with the use of in vitro transport method based on the Caco-2 cell line. Perorally used drugs are primarly absorbed in the small intestine. Enterocytes, forming the intestinal barrier, express a large number of transporters, representing both ATP-binding (ABC) and "solute carrier" (SLC) transporters, which can significantly affect the pharmacokinetics of the drugs transmitted by them. For this reason, FDA and EMA are currently focusing on studying these interactions and their effects on drug permeability. The aim of this thesis was to investigate whether the equilibrative nucleoside transporters (ENTs), belonging to the SLC transporters, are involved in the absorption of nucleosides (adenosine and thymidine) and subsequently abacavir, the nucleoside antiretroviral. The in vitro transport method was performed on polarized, Caco-2 cell monolayer. By the statistical analysis of acquired data was found, that although the expression of ENTs was confirmed on the apical side of the Caco-2 cell membrane, the transport of nucleosides [3H]ADE and...
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Testing of the cytotoxicity of potential drugs in the cell lines together with the measurement of their passage through the blood brain barrier by the PAMPA method
Šinaľová, Lucia ; Hofman, Jakub (advisor) ; Jansová, Hana (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Lucia Šinaľová Supervisor: RNDr. Jakub Hofman, Ph.D. Title of diploma thesis: Testing of the cytotoxicity of potential drugs in the cell lines together with the measurement of their passage through the blood brain barrier by the PAMPA method This master thesis focuses on the prediction of tacrine derivatives permeability through blood- brain barrier and their cytotoxicity. We studied tacrine-benzothiazole and tacrine- thiaquinazoline derivates as potential drugs for treatment of Alzheimer's disease. The reason of testing new tacrine derivates was effort to find substances with same effect and lower toxicity, for which was the tacrine discarded from clinical practice. The probable permeability of the studied substances was determined in vitro by the PAMPA method. Based on the permeation coeficient values we have identified that tacrine-benzothiazole derivates A-D (Pe= 8,31- 16,9×10-6 cm/s) and tacrine-thiaquinazoline derivates 1, 3, 4 (Pe= 8,59-14,9×10-6 cm/s) were permeable through blood-brain barrier, tacrine-thiaquinazoline derivate 2 (Pe= 3,79×10-6 cm/s) had uncertain permeability and tacrine-thiaquinazoline derivate 5 (Pe= 2,0×10-6 cm/s) was not permeable. In the pre-clinical evaluation of...
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Preparation of mammalian vectors encoding selected microsomal SDR enzymes
Slezák, Jan ; Hofman, Jakub (advisor) ; Novotná, Eva (referee)
Charles University in Prague Faculty of Pharmacy in Hradci Kralove Department of Biochemical Sciences Candidate: Jan Slezák Superviser: RNDr. Jakub Hofman, Ph.D. Title of diploma thesis: Preparation of mammalian vectors encoding selected microsomal SDR enzymes Microsomal short-chain dehydrogenases/reductases DHRS1, DHRS7 and HSD11b1 (SDR19C1, SDR34C1, SDR26C1) are membrane-bound NAD(P)H-dependent enzymes metabolizing a broad spectrum of carbonyl-bearing substrates. These enzymes from the superfamily of short-chain dehydrogenases/reductases mediate metabolism of endogenous compounds, such as glucocorticoids, retinoids, sfingolipids as well as various xenobiotics. Apart from their biological roles, they participate in the etiology of severe diseases (e.g cancer, Alzheimer disease, obesity etc.). Knowledge of inhibitory and substrate affinity may lead to better understanding of the functions of these enzymes in organism and to the development of new therapeutic approaches. The goal of the present work was to prepare mammalian vectors encoding DHRS1, DHRS7 and HSD11b1. Primer design and cDNA amplification were among the first steps. Primers beared the sequence recognized by restriction endonucleases which was concomitantly present in the multicloning site of the pCI plasmid. Such primer design allowed...
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Reporter gene studies for nanoparticle mediated DNA and siRNA delivery.
Kovářová, Barbora ; Jirkovská, Anna (advisor) ; Hofman, Jakub (referee)
Charles University, Faculty of Pharmacy in Hradec Králové, Department of Biochemical Sciences University of Vienna, Faculty center for Pharmacy, Department of Pharmaceutical Chemistry, Laboratory of MacroMolecular Cancer Therapeutics Candidate: Barbora Kovářová Supervisor (Charles University): PharmDr. Anna Jirkovská, Ph.D. Supervisor (University of Vienna): Univ. Prof. Dipl. Ing. Dr. Manfred Ogris Co-supervisor (University of Vienna): Dr. Haider Sami, Ph.D. Title of diploma thesis: Reporter gene studies for nanoparticle mediated DNA and siRNA delivery Keywords: transfection, plasmid DNA, siRNA, nanoparticles Gene therapy is a promising field offering potential in several currently incurable diseases. Gene therapy is mediated by modulation of gene expression in specific cells by delivering exogenous nucleic acids. One of current tasks of nucleic acid delivery is exploring several synthetic vectors which would have a potential to overcome the disadvantages of commonly used viral vectors. The present study focused on different types of polyethyleneimine-based nanoparticles for plasmid DNA (pDNA) and small interfering RNA (siRNA) delivery. Integration of imaging contrast agents with gene delivery vehicles is advantageous for tracking the gene delivery process both in vivo and in vitro. Gadolinium...
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Interactions of selected anticancer drugs of the MAPK/ERK signaling pathway inhibitors group with the ABC drug transporters
Slatinský, Lukáš ; Čečková, Martina (advisor) ; Hofman, Jakub (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Lukáš Slatinský Supervisor: Assoc. prof. PharmDr. Martina Čečková, Ph.D. Title of diploma thesis: Interactions of selected anticancer drugs of the MAPK/ERK signaling pathway inhibitors group with the ABC drug transporters ABCB1 (Pgp, P-glycoprotein) and ABCG2 (BCRP, breast cancer resistance protein) are members of a transmembrane efflux ATP dependent transporter family, so called ATP-binding cassettes (ABC). Physiologicaly they are expressed in the cellular membrane and protect body tissues against potentially toxic xenobiotics including drugs. They represent also one of the tumor defense mechanisms when being able to efflux a wide variety of cytotoxic drugs out of the cancer cells leading to treatment failure. BRAF protein plays an important regulatory and signal role in MAPK/ERK pathway affecting cell division, differentiation and secretion. Mutations of BRAF lead to overactivity in MAPK/ERK pathway in many cancer cells and can be therefore targeted by anticancer therapy. Cobimetinib and dabrafenib are relatively new anticancer therapeutics inhibiting the signal pathway mentioned above and they are used in treatment of melanoma carrying the BRAF mutation. The aims of this project were to...
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