Title: 48th Krka Prizes Markovič, Tijana. Evaluation of immune modulation and efficacy of EP4 receptor agonist and monoclonal antibodies in vitro. Mentor: prof. dr. Irena Mlinarič Raščan. Description: Evaluation of the immunomodulatory effects of compounds is an important part of preclinical and toxicological evaluation of medicines. Hence, we established a novel in vitro test system for evaluation of immunotoxic and immunomodulatory properties of compounds based on lymphoblasotid cell lines, which we utilised to evaluate imunomodulatory properties of prostaglandin EP4 receptor agonist, PgE1-OH. Next, EP4 receptor was evaluated as a potential therapeutic target for the treatment of B-cell leukaemia and lymphoma. PgE1-OH reduced the activity of NF-?B in the Burkitt’s lymphoma cell line Ramos, which led to synergistic effects with bortezomib and doxorubicin. PgE1-OH also acted synergistically with monoclonal antibodies rituximab and ofatumumab, for which the induction of complement dependent cytotoxicity in vitro was first confirmed. PgE1-OH induced apoptosis in malignant lymphocytes B (EC50 = 13.53 µM, N = 151) isolated from whole blood of patients with the diagnosis of chronic lymphocytic leukaemia (CLL) after their informed consent was obtained. Of particular importance is the discovery that PgE1-OH acted selectively cytotoxic in vitro in CLL cells obtained from patients who are resistant to standard therapy and carry the deletion of the TP53 gene. Moreover, PgE1-OH acted synergistically with fludarabine, which might offer novel therapeutic options. In conclusion, EP4 receptor was evaluated as a promising therapeutic target for the treatment of B-cell leukaemia and lymphoma.
E.01 National awards
COBISS.SI-ID: 4629617Title: 49th Krka Prizes Urbančič, Dunja. Pharmacogenomic evaluation of susceptibility to thiopurines. Mentor: prof. dr. Irena Mlinarič Raščan. Description: Due to a narrow therapeutic index of immunosuppressive and cytostatic thiopurine drugs, their accurate administration is of crucial importance. However, complex metabolism and mode of action of these drugs underlies a multifaceted challenge in the individualized therapy. In the course of our study we first identified a substrate of thiopurine S-methyltransferase (TPMT), selenocysteine, which could be importantly involved in the biological role of the enzyme. We next aimed to identify novel biological markers of the response to thiopurines. Studying genetic variations in healthy Slovenian population and patients with acute lymphoblastic leukaemia or inflammatory bowel disease, we found specific tandem repeats in the gene for TPMT that are linked with clinically established genetic polymorphisms. The presence of such genetic variants predisposes life-threatening thiopurine related toxicities. By employing in vitro model of lymphoblastoid cell lines derived from several different donors, we further explored TPMT-independent factors of thiopurine response. We learnt that a higher expression of transporter P-glycoprotein in cells predicted the resistance to thiopurines. Additionally, we examined the mechanisms of interactions between substrates and inhibitors of TPMT using in silico approach. Based on the conclusions obtained by molecular docking analysis, we experimentally determined the mixed inhibition mechanism of TPMT by sulfasalazine and showed that sulfasalazine enhanced thiopurine toxicity on different cell models. The results of our research broadened the selection of potential biomarkers of response to thiopurines and indicated the importance of drug-drug interactions, contributing to future improvements of thiopurine treatment
E.01 National awards
COBISS.SI-ID: 4845681Desription: Evaluation of the immunomodulatory effects of the compounds is an important part of preclinical and toxicological evaluation of medicines. Hence, we established a novel in vitro test system for evaluation of immunotoxic and immunomodulatory properties of compounds based on lymphoblasotid cell lines, which we utilized to evaluate imunomodulatory properties of prostaglandin EP4 receptor agonist, PgE1-OH. Next, EP4 receptor was evaluated as a potential therapeutic target for the treatment of B cell leukemia and lymphoma. PgE1-OH reduced the activity of NF-?B in the Burkitt's lymphoma cell line Ramos, which led to synergistic effects with bortezomib and doxorubicin. PgE1-OH also acted synergistically with monoclonal antibodies rituximab and ofatumumab, for which the induction of complement dependent cytotoxicity in vitro was first confirmed. PgE1-OH induced apoptosis in malignant lymphocytes B isolated from whole blood of patients with diagnosis of chronic lymphocytic leukemia (CLL) after informed consent was obtained. Of particular importance is the discovery that PgE1-OH acted selectively cytotoxic in vitro in CLL cells obtained from patients, who are resistant to standard therapy and carry the deletion of the TP53 gene. Moreover, PgE1-OH acted synergistically with fludarabine, which might provide novel therapeutic options. In conclusion, EP4 receptor was evaluated as a promising therapeutic target for the treatment of B cell leukemia and lymphoma.
D.10 Educational activities
COBISS.SI-ID: 295575552The present invention belongs to the field of pharmaceutical industry and relates to novel inhibitors of chymotrypsin- like activity of the immunoproteasome (inhibitors of %5i or LMP7 subunit) with a psoralen-based structure in the form of pure diastereoisomers, mixture of diastereomers, in the form of pure enantiomers, mixture of enantiomers, or pharmaceutically acceptable salts, hydrates or solvates thereof. Such compounds are particularly useful for the treatment of diseases that are related with increased activity of the immunoproteasome. The present invention also relates to the preparation of these compounds, use of these compounds, and to the pharmaceutical products that contain these compounds. The characteristic of these compounds is that they bind into the active site of the 5i subunit through specific noncovalent (polar and hydrophobic interactions) or concurrent noncovalent (polar and hydrophobic interactions) and covalent interactions.
F.33 Slovenian patent
COBISS.SI-ID: 4206705Chronic lymphocytic leukemia (CLL) is the most common leukemia in adults in Western world. Disease may have aggressive or indolent clinical course. Standard treatment is based on chemoimmunotherapy, such as FCR scheme, which consists of fludarabine, cyclophosphamide and rituximab. New treatment options include targeted therapy with ibrutinib and idelalisib, which inhibit B-cell receptor (BCR) signaling. The prostaglandin EP4 receptor represents a new potential target for the treatment of B cell leukemias and lymphomas. In this master thesis we evaluated selective EP4 receptor agonists L-902,688 and PgE1-OH. With use of cell viability assay and flow cytometry we demonstrated that they possess cytotoxic effects on CLL cells and act in a time and concentration dependent manner. Applying selective EP4 receptor antagonist CJ-042794 we confirmed that agonists exert their effects through EP4 receptor. EC50 values for L-902,688 and PgE1-OH were significantly lower on CLL cells as compared to lymphoblastoid cell lines (LCL) and peripheral blood mononuclear cells (PBMC), which confirms their selectivity towards malignant B lymphocytes. We also evaluated the most commonly used drug for CLL fludarabine and novel targeted drugs ibrutinib and idelalsib. After 24 h EC50 values for fludarabine were significantly higher as compared to L-902,688 and PgE1-OH and IC50 values for idelalisib were significantly higher only when compared to L-902,688. After 48 h EC50 (IC50) values for fludarabine, ibrutinib and idelalisib were 4,65 µM, 3,15 µM and 8,84 µM, respectively, and were comparable to EC50 for L-902,688 and PgE1-OH. Clinical outcome of therapy is often improved when combining drugs with different mechanism of action, therefore we have decided to evaluate potential synergism of L-902,688 and PgE1-OH with fludarabine, ibrutinib and idelalisib. Combining EP4 receptor agonists with fludarabine and ibrutinib proved to have synergistic outcome at lower concentrations of drugs, whereas idelalisib, which is the least cytotoxic among tested drugs (IC50 was 20,64 after 24 h and 8,84 µM after 48 h), proved to exert synergy in all tested concentrations of drugs. Using annexin V we demonstrated that L-902,688 and PgE1-OH induce apoptosis in malignant B lymphocytes and with CFSE assay we have shown they are also cytostatic. Both compounds inhibited cell division after 48 h, which implies we could also inhibit proliferation of CLL cells in vivo. We have also shown that expression of EP4 receptor is not affected by this two drugs, which confirms the importance of the EP4 receptor as a potential therapeutic target. Selective EP4 receptor agonists exert both cytotoxic and cytostatic effects and synergistic effects are present when combined with novel therapeutics in vitro, therefore this could lead to new treatment options for CLL.
E.01 National awards
COBISS.SI-ID: 4647793