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Effect of CDK and FLT3 inhibitors on activity of ABC efflux transporters in vitro, relation to multidrug resistance
Poráč, Jakub ; Čečková, Martina (advisor) ; Hofman, Jakub (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Jakub Poráč Supervisor: doc. PharmDr. Martina Čečková, Ph.D. Title of diploma thesis: Effect of CDK and FLT3 inhibitors on activity of ABC efflux transporters in vitro, relation to multidrug resistance P-gp and BCRP are transmembrane proteins that form part of a large family of ABC transporters. These are ATP-driven transporters, which main task is to eliminate exogenous and endogenous substances and their metabolites from cells of both, healthy and tumour tissues. This activity is often associated with the expulsion of administered therapeutics and multiple drug resistance (MDR) in tumour cells. A promising therapy of cancer represents a newer class of drugs target the tyrosine kinase (TK), and cyclin-dependent kinases (CDK), which are cell enzymes responsible for the processes of proliferation, apoptosis and differentiation. Cyclin- dependent kinase inhibitors (CDKI) are used in the treatment of breast cancer, but at the same time they form a new group of drugs with the potential for use in hematological malignancies. In the treatment of AML, a new successful approach is TK inhibitors (TKI), which target the mutated FLT3 receptor, specifically the recently approved drugs midostaurin and...
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Study on the role of selected cytochrome P450 isoforms in cytostatic resistance at apoptosis level
Moriová, Magdalena ; Hofman, Jakub (advisor) ; Novotná, Eva (referee)
Charles University Faculty of Pharmacy in Hradci Králové Departement of Pharmacology & Toxicology Student: Magdalena Moriová Supervisor: RNDr. Jakub Hofman, Ph.D. Title of diploma thesis: Study on the role of selected cytochrome P450 isoforms in cytostatic resistance at apoptosis level Cytostatic resistance is one of the most problematic obstacles in oncological treatment. Beside pharmacodynamic mechanisms, pharmacokinetic factors play an important role in drug resistance as well. Enzymatic transformation of active substance to inactive metabolite in tumor cells probably belongs to these mechanisms, however, evidences concerning the relevance of this phenomenon are predominantly either indirect and/or affected by interference elements. Using comparative experiments with HepG2 cell lines with/without CYP3A4 overexpression, we focused on the evaluation of the role of this clinically important enzyme in the resistance against docetaxel. Methodologically, it was the assessment of apoptosis induction (activation of caspases 3/7, 8 and 9) using commercial luminescent kits. Our results suggest significant participation of CYP3A4 enzyme on the reduction of docetaxel anticancer efficacy after 48 h from treatment, whereas this effect was not recorded in earlier intervals. These findings perfectly correlate...
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Study on interactions of PARP inhibitors with ABC drug efflux transporters
Dziaková, Lucia ; Hofman, Jakub (advisor) ; Čečková, Martina (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Lucia Dziaková Supervisor: RNDr. Jakub Hofman, Ph.D. Title of diploma thesis: Study on interactions of PARP inhibitors with ABC drug efflux transporters. ATP-binding cassette (ABC) transporters are integral membrane proteins that use the energy obtained from ATP to carry transport of numerous endogenous substrances out of the cells, but attention is drawn primarily to the fact that they transfer also xenobiotics. Their overexpression in tumor tissue contributes to multidrug resistance (MDR), which in most cases leads to therapy failure. Poly(ADP-ribose)polymerase inhibitors (PARPi) represent a promising therapeutic approach in the treatment of cancers that exhibit defects in homologous recombination (HR). This work focuses on four selected PARPi (olaparib, rucaparib, niraparib, veliparib) and their interaction potential towards ABC drug efflux transporters (ABCB, ABCC1, ABCG2). In our work, we worked with MDCKII cells (parent, transduced by the transporters of interest) and utilized the principle of accumulation studies based on the measurement of fluorescence intensity of specific model substrates (hoechst33342, calcein AM, daunorubicin, mitoxantrone). We used established inhibitors of studied...
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Flow-cytometric analysis of inhibitory effect of novel targeted drugs on the activity of ABC drug efflux transporters
Burianová, Gabriela ; Hofman, Jakub (advisor) ; Červený, Lukáš (referee)
Charles University Faculty of Pharmacy in Hradec Kralove Department of Pharmacology & Toxicology Student: Gabriela Burianova Supervisor: RNDr. Jakub Hofman, Ph.D. Title of diploma thesis: Flow-cytometric analysis of inhibitory effect of novel targeted drugs on the activity of ABC drug efflux transporters Cancer is the second leading cause of death. Cancer treatment often combines conventional chemotherapy, radiation therapy and surgery. More recent approach to treatment is the use of targeted cancer therapy with a greater specificity towards cancer cells. Development of resistance is a major obstacle in the success of chemotherapy. Multidrug resistance (MDR) can be acquired through various mechanisms e.g. overexpression of efflux transporters. ATP binding cassette (ABC) transporters represents a large family of transmembrane proteins that use ATP to pump molecules across the membrane. The three main ABC proteins related to MDR are: P-glycoprotein (ABCB1), multidrug resistance-associated protein 1 (ABCC1) and breast cancer resistance protein (ABCG2). Use of ABC transporter inhibitors increases the amount of chemotherapeutical substrates accumulated within the cells. In this study we evaluated interactions of six synthetic small molecule inhibitors (alisertib, ensartinib, entrectinib, talazoparib,...
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The effect of alisertib and brigatinib on the activity of selected human carbonyl reducing enzymes.
Lakomá, Petra ; Novotná, Eva (advisor) ; Hofman, Jakub (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Petra Lakomá Supervisor: RNDr. Eva Novotná, Ph.D. Title of diploma thesis: The effect of alisertib and brigatinib on the activity of selected human carbonyl reducing enzymes Key words: brigatinib, alisertib, daunorubicin, inhibition, carbonyl-reducing enzymes Protein kinases are enzymes, whose main function is based on a transfer of phosphate group from ATP to protein substrate. This common posttranslational modification is involved in the regulation of intracellular processes and cell signaling. Altered expression of protein kinases is often coupled with a development of cancer. Inhibition of protein kinases may prevent cancer cell proliferation and induce their cell death. The main aim of the diploma thesis was to measure inhibition potential of protein kinase inhibitors, alisertib and brigatinib, against carbonyl-reducing enzymes. Overexpression of carbonyl-reducing enzymes in cancer cells may cause resistance to drugs followed by failure of chemotherapeutic therapy. In case of antracyclin chemotherapeutic daunorubicin, carbonyl-reducing enzymes reduce the carbonyl in C-13 giving rise a primary metabolite daunorubicinol, which has lower cytotoxic effect but higher cardiotoxicity. The effort to...
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Study on impact of selected tyrosine kinase inhibitors on multidrug resistance mediated by ABC drug efflux transporters
Sýkorová, Martina ; Hofman, Jakub (advisor) ; Červený, Lukáš (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Martina Sýkorová Supervisor: RNDr. Jakub Hofman, Ph.D. Title of diploma thesis: Study on impact of selected tyrosine kinase inhibitors on multidrug resistance mediated by ABC drug efflux transporters Tyrosine kinases are an important class of enzymes controlling cell proliferation, carcinogenesis, apoptosis and cell differentiation. Deregulation of these enzymes can transform normal cell into a cancerous one. Blocking their function by tyrosine kinase inhibitors (TKi) is considered a promising treatment for various types of cancer. ATP-binding cassette (ABC) transporters form a family of transmembrane proteins that can transport a wide variety of substrates across biological membranes via ATP-dependent drug efflux pumps. They modulate drug pharmacokinetics, but on the other hand, lead to therapy failure due to overexpression in cancer cells. In our previous study, we evaluated inhibition properties of two selected TKi (alectinib, brivanib) in MDCKII cell lines (parent one and those transduced with human ABCB1, ABCC1 and ABCG2). Alectinib significantly inhibited ABCB1, ABCG2 but not ABCC1 transporter. Brivanib showed triple inhibition of all studied transporters. In the present work, we...
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Role of drug transporters in placental transfer of entecavir
Křečková, Veronika ; Červený, Lukáš (advisor) ; Hofman, Jakub (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Veronika Křečková Supervisor: PharmDr. Lukáš Červený, Ph.D. Title of diploma thesis: Role of drug transporters in placental transfer of entecavir Entecavir (ETV), an analogue of guanosine, is a highly efficient anti-hepatitis B antiviral drug. It is the first-line therapy for both adults and children. Its use in pregnancy is limited due to a number of factors, including lack of data on placental pharmacokinetics. The placental transition of drugs is frequently controlled by drug transporters. ATP-binding (ABC) transporters, P-glycoprotein (P-gp), breast cancer resistance protein (BCRP) or multidrug resistance-associated protein 2 (MRP2) localized in the apical membrane of syncytiotrophoblast and pumping their substrates in the feto-maternal direction belong to most significant determinants of placental pharmacokinetics. Moreover placental transport of nucleoside-derived drugs can be affected by the activity of nucleoside transporters (NTs); equilibrative nucleoside transporters (ENTs) mediate facilitated diffussion, while the concentrative nucleoside transporters (CNTs) control active influx of their substrates. The aim of the diploma thesis was to describe the role of P-gp, BCRP, MRP2 and NTs (ENTs and...
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Study on impact of selected protein kinase inhibitors on drug resistance mediated by cytochromes P450
Janoušková, Adéla ; Hofman, Jakub (advisor) ; Novotná, Eva (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Adéla Janoušková Supervisor: RNDr. Jakub Hofman, Ph.D. Title of diploma thesis: Study on impact of selected protein kinase inhibitors on drug resistance mediated by cytochromes P450 Pharmacokinetic drug resistance often leads to failure of an anticancer therapy. One of the mechanisms is increased efflux of drugs from tumour cells, whereas some studies suggest that increased drug conversion to an inactive metabolite might be another contributing mechanism. The aim of this work was to define the possible role of CYP3A4 and CYP2C8 enzymes in the phenomenon of pharmacokinetic resistance and to investigate the possibility of its modulation by new targeted drugs. In the first part, we used the MTT proliferation method together with HepG2 cells stably transduced with particular human enzymes and demonstrated significant involvement of CYP3A4 in docetaxel resistance. In the following part, we examined the inhibitory effects of four selected tyrosine kinase inhibitors on the CYP3A4 activity in intact cells using a commercial kit. Cobimetinib and dabrafenib showed significant inhibitory activity, while osimertinib and brivanib did not. In the final part, we demonstrated the ability of the first two...
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Study on impact of selected tyrosine kinase inhibitors on multidrug resistance mediated by ABC drug efflux transporters
Sýkorová, Martina ; Hofman, Jakub (advisor) ; Červený, Lukáš (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Martina Sýkorová Supervisor: RNDr. Jakub Hofman, Ph.D. Title of diploma thesis: Study on impact of selected tyrosine kinase inhibitors on multidrug resistance mediated by ABC drug efflux transporters Tyrosine kinases are an important class of enzymes controlling cell proliferation, carcinogenesis, apoptosis and cell differentiation. Deregulation of these enzymes can transform normal cell into a cancerous one. Blocking their function by tyrosine kinase inhibitors (TKi) is considered a promising treatment for various types of cancer. ATP-binding cassette (ABC) transporters form a family of transmembrane proteins that can transport a wide variety of substrates across biological membranes via ATP-dependent drug efflux pumps. They modulate drug pharmacokinetics, but on the other hand, lead to therapy failure due to overexpression in cancer cells. In our previous study, we evaluated inhibition properties of two selected TKi (alectinib, brivanib) in MDCKII cell lines (parent one and those transduced with human ABCB1, ABCC1 and ABCG2). Alectinib significantly inhibited ABCB1, ABCG2 but not ABCC1 transporter. Brivanib showed triple inhibition of all studied transporters. In the present work, we...
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Interaction of natural substances with human aldo-keto reductase 7A2 and other important carbonyl reducing enzymes
Homerová, Andrea ; Novotná, Eva (advisor) ; Hofman, Jakub (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Andrea Homerová Supervisor: RNDr. Eva Novotná, Ph.D. Title of diploma thesis: Interaction of natural substances with human aldo-keto reductase 7A2 and other important carbonyl reducing enzymes Reduction of carbonyl group is one of the phase I metabolism reactions, which is responsible for production of more polar metabolites, enables conjugation in process of biotransformation, excretion of the molecule and it also causes decrease in reactivity and biological activity of the molecule. Endogenous as well as exogenous compounds undergo this reaction and carbonyl reducing enzymes are the ones which possess this reducing activity. Based on the structure, we can divide the enzymes into several groups: short-chain dehydrogenases/reductases, medium-chain dehydrogenases/reductases, aldo-keto reductases and quinone reductases. Inhibition of carbonyl reducing enzymes appears to be a promising aim of research. It is important to take into consideration that by inhibiting carbonyl reducing enzymes it is possible to reduce production of less active, but more toxic metabolites, for example in anthracycline chemoteraputics daunorubicin and doxorubicin and that can lead to change in therapy of cancer. This study...
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