Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical sciences Candidate: Bc. Monika Šmídlová Supervisor: RNDr. Eva Novotná, Ph.D. Title of diploma thesis: Effect of selected cytostatics for the treatment of leukemia on the activity of human carbonyl reducing enzymes Key words: reductases, leukemia, cytostatics, inhibition Anthracycline antibiotics, especially daunorubicin, are widely used for the treatment of acute myeloid leukemia (AML) and acute lymphocytic leukemia (ALL). Although the efficacy of these drugs is high, treatment is still limited due to cardiotoxicity and tumor cell resistance to anthracyclines. Mechanisms that contribute to the formation of anthracycline resistance include metabolic biotransformation (reduction) to less efficient secondary alcohols. The reduction is calatyzed by carbonyl reducing enzymes belonging to aldo-keto reductase (AKR) and short chain dehydrogenase/reductase (SDR) superfamilies. The discovery of AKR and SDR inhibitors could help to overcome anthracycline resistance and also reduce cardiotoxicity caused by these drugs. The aim of the diploma thesis was to find out whether all-trans-retinoic acid, cyclophosphamide, cytarabine, cladribine and prednisolone are able to inhibit anthracycline reductases AKR1A1, AKR1B10, AKR1C3, AKR7A2...

This diploma thesis deals with optimization of separation conditions for the four groups of analytes related to 7-deazaadenosine (each group was composed of four derivatized nucleosides) using hydrophilic interaction liquid chromatography. All the sixteen analytes were synthesized as potentially cytostatically active compounds. The effect of the type of stationary phase in the chromatographic column, the ratio of organic and aqueous parts of the mobile phase, pH of the buffer used as the mobile phase component and the concentration of ammonium acetate in the buffer in the range of 5-50 mM were tested during the optimization process. Three stationary phases were tested - bare silica (Spherisorb® Silica column), silica- bonded amide (TSKgel Amide-80 column) and silica-bonded native cyclofructan 6 (Frulic-N column). The dimensions of all columns were 2504.6 mm i.d.; particle size 5 µm. During the optimization of separation the mechanism of HILIC was studied. It was found that the distribution of analytes between the aqueous layer partially coated on the surface of the stationary phase and the mobile phase and also the adsorption of analytes on the stationary phase participated in the retention and separation mechanism in all tested chromatographic systems. The three groups of analytes (SK1, SK3, SK4)...

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...

Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Bc. Jesika Štorkánová Supervisor: RNDr. Eva Novotná, Ph.D. Title of diploma thesis: Study of the cytotoxicity of selected chemotherapeutics for the treatment of leukemia in human tumor cell lines Leukemia represents a diverse group of malignant diseases with a hematopoietic disorder with different prognoses. As the incidence of patients with leukemia is increasing, is an effort to establish the treatment that will lead to successful therapy. One of the basic approaches to the treatment of leukemias is chemotherapy. Today it is known that the effectiveness of chemotherapy is influenced by a number of factors which can significantly affect the treatment strategy and thus decide on the outcome of the treatment itself. An important approach in chemotherapy is the selection of cytostatics with maximum efficacy for oncological disease and elimination cytostatics to which the cells are resistant based on the findings in in vitro conditions. The aim of this diploma thesis was to determine the inhibitory effects of in vitro selected chemotherapeutics in cell tumor lines. For determine the inhibitory effect, HCT116, HepG2 and HL-60 cell lines were selected using a colorimetric method based on the...

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Anna Giannitsi Supervisor: RNDr. Jakub Hofman, Ph.D Title of diploma thesis: The role of biotransformation enzymes in the resistance of cancer cells against standard cytostatics Drug resistance is currently one of the major problems of chemotherapy. Tumor cells are able to defend themselves against the effect of cytostatic drugs due to various mechanisms which leads to a failure of anticancer therapy. The effort to describe new mechanisms of resistance and to develop new therapeutic methods, which would limit this therapeutic obstacle, is logically the subject of many studies. The activity of drug metabolizing enzymes and the subsequent decrease of intercellular concentration of anticancer drugs belongs to one of the possible mechanisms of pharmacokinetic resistance. Enzymes of I. and II. phase of biotransformation participate in this phenomenon. Cytochromes P450, main enzymes of the I. phase, play a major role in the metabolism of many cytostatic agents producing either pharmacologically active or inactive metabolites. Increased expression in tumors and the involvement of individual isoforms into the overall metabolism of cytostatic, which is deactivated by their activity, seems to be one of the...

The work analyzes trends in medicines used in specialized medical centers from ATC (Anatomical-Therapeutic-Chemical Classification System) L01 in Czech Republic from perspective of the State Institute of Drug Control (SUKL) and of Czech Health Insurance Companies (Vseobecna zdravotni pojistovna Ceske republiky as the biggest one, and rest joined in Svaz zdravotnich pojišťoven Ceske republiky). SUKL perspective considers medicines delivered to healthcare facilities and the other side providing information regarding payments for L01 medicines between years 2012-2015. Conclusions presented show slightly decreasing trend in terms of number of packages delivered (935 ths. in 2012 and 931 ths. in 2015). On the other hand, value of medicines has increased from 5 067 mil. CZK in 2012 to 6 568 mil. CZK in 2015. The increase in value and decrease in volume in the same time (while prices remain stable or decreasing) indicates trend in increasing size of packaging or entry of new strength (with more active ingredient and therefore more expensive) e.g. entry of Herceptin 600 mg in 2014. This paper further identifies the most expensive medicines in ATC L01 group and starts a discussion about possible reasons of discrepancies between different sources of data which are for the most expensive medicines in hundreds of mil. CZK in value and ths. of packages in terms of volume (e.g. Avastin in 2014: difference 104 mil. CZK and 5 598 packages).

Prostate cancer is one of the most common cancer-related causes of death among men. Chemotherapy is mainly used for treatment of its later stages, accompanied by unpleasant side effects. So far, the treatment of advanced stages of prostate cancer has not been sufficient, and new more effective alternatives are needed. The application of the TRAIL cytokine, which induces apoptosis in tumor cell, but is not toxic to nonmalignant cells, seems to be a promissing approach. However, TRAIL-based therapy is often limited by the emerging cancer cell resistance. Overcoming the resistance can be achieved by combination therapy of TRAIL with effective sensitizers. Within this work, a combination of TRAIL with platinum-based chemotherapeutic drugs such as cisplatin or its novel derivative LA-12 was applied in order to facilitate the elimination of prostate cancer cells. In the experimental part of this work, using Western blot and flow cytometry analysis it was shown that TRAIL in combination with CDDP or LA-12 effectively enhanced apoptosis in three human prostate cancer cell lines. This effect was accompanied with increased activation/amount of several proapoptotic Bcl-2 family proteins, while no changes in the level of the receptors for TRAIL were observed. These results demonstrated that especially the combination...

<p>Mantle cell lymphoma (MCL) is an aggressive B-cell malignancy with bad prognosis, which predominates in males with advanced age. However, studies of the endocannabinoid system and how it affects tumour behaviour provides the basis for designing innovative therapeutic strategies that could open new opportunities for treatment of patient with MCL. It has earlier been shown that the cannabinoid receptor ligand (R)-(+)-methanandamide (R-MA) induce cell death in MCL by accumulation of ceramide. Ceramide has a pro-apoptotic effect on the cell but could be metabolized by the enzymes glucosylceramide synthase (GCS) and sphingosine kinase 1 (SphK1) to molecules with pro-proliferative effect. Therefore, treatments with R-MA on Jeko-1 MCL cell line were performed in this study to determine interference in the proliferative behaviour as well as in the gene expression of the enzymes GCS and SphK1. In addition, treatments with chemotherapeutic substances, such as doxorubicin or cytarabine (Ara-C), and combinations of R-MA and chemotherapeutic substance, were performed for the same reason. Results showed that the proliferation behaviour of Jeko cells remained unaffected when treated with R-MA, in contrast to the decreased proliferative effects shown when treated with cytostatics or combinations of R-MA and cytostatics. Furthermore, a tendency for up-regulation of GCS and SphK1 expression was recognized when cells were treated with cytostatics or combination of cytostatics and R-MA, in contrast to cells treated with R-MA alone. Although, R-MA alone had a tendency for a small down-regulation of GCS expression, it contributed to a potential elevation of GCS expression when combined with Ara-C or doxorubicin. It is believed that the effect from upregulated levels of the metabolizing enzymes GCS and SphK1 is balanced by, earlier observed, up-regulations of the ceramide synthesis enzymes.</p>

The main goal of this thesis is an introducing and guiding the reader with all important aspects of a proposal of ventilation system for cytostatics laboratory, from theoretical knowledge about the issue via familiarization with an appropriate legislative statute, a demonstration of relevant calculations needed for the project, a proposal in the form of technical reports as well. In this thesis the process of validation, which is needed for installed technology in the cleanroom, is also described. The thesis is supported by drawing documentation of real ventilation project for cytostatics laboratory, which follows the aforementioned technical report and calculations.

Mantle cell lymphoma (MCL) is an aggressive B-cell malignancy with bad prognosis, which predominates in males with advanced age. However, studies of the endocannabinoid system and how it affects tumour behaviour provides the basis for designing innovative therapeutic strategies that could open new opportunities for treatment of patient with MCL. It has earlier been shown that the cannabinoid receptor ligand (R)-(+)-methanandamide (R-MA) induce cell death in MCL by accumulation of ceramide. Ceramide has a pro-apoptotic effect on the cell but could be metabolized by the enzymes glucosylceramide synthase (GCS) and sphingosine kinase 1 (SphK1) to molecules with pro-proliferative effect. Therefore, treatments with R-MA on Jeko-1 MCL cell line were performed in this study to determine interference in the proliferative behaviour as well as in the gene expression of the enzymes GCS and SphK1. In addition, treatments with chemotherapeutic substances, such as doxorubicin or cytarabine (Ara-C), and combinations of R-MA and chemotherapeutic substance, were performed for the same reason. Results showed that the proliferation behaviour of Jeko cells remained unaffected when treated with R-MA, in contrast to the decreased proliferative effects shown when treated with cytostatics or combinations of R-MA and cytostatics. Furthermore, a tendency for up-regulation of GCS and SphK1 expression was recognized when cells were treated with cytostatics or combination of cytostatics and R-MA, in contrast to cells treated with R-MA alone. Although, R-MA alone had a tendency for a small down-regulation of GCS expression, it contributed to a potential elevation of GCS expression when combined with Ara-C or doxorubicin. It is believed that the effect from upregulated levels of the metabolizing enzymes GCS and SphK1 is balanced by, earlier observed, up-regulations of the ceramide synthesis enzymes.