20.500.12556/RUL-187113 Proučevanje sinergističnih učinkov zaviralcev tirozin kinaz Lyn in Lck z monoklonskimi protitelesi proti CD20 in vitro Evaluation of the synergistic effect of Lyn and Lck tyrosine kinase inhibitors with anti-CD20 monoclonal antibodies in vitro Kronična limfocitna levkemija (KLL) je najpogostejša levkemija odraslih, pri kateri ima signalizacija preko B-celičnega receptorja ključno vlogo pri uravnavanju preživetja levkemičnih celic. Ker sta tirozinski kinazi Lyn in Lck pomembna regulatorja signalizacije B-celičnega receptorja, predstavljata obetavni terapevtski tarči za izboljšanje učinkovitosti zdravljenja z monoklonskimi protitelesi proti CD20. Osrednji namen magistrske naloge je bil ovrednotiti citotoksično delovanje sarakatiniba in HY-12072 v kombinaciji z rituksimabom in obinutuzumabom in vitro ter preveriti, ali kombinacije učinkovin izkazujejo sinergistično citotoksično delovanje. Ker je delovanje monoklonskih protiteles proti CD20 povezano z aktivacijo sistema komplementa, smo najprej proučili vpliv humanega seruma na s komplementom posredovano citotoksičnost rituksimaba in obinutuzumaba na celicah MEC-1. Z višanjem koncentracije dodanega humanega seruma iz 0 % na 20 % se je relativna metabolna aktivnost celic MEC-1 po tretiranju z rituksimabom znižala iz 92,0 % na 29,4 %, po tretiranju z obinutuzumabom pa iz 77,5 % na 27,1 %. Proučevali smo tudi citotoksični učinek sarakatiniba in HY-12072 na primarne celice KLL ter njuno kombinirano delovanje z rituksimabom in obinutuzumabom na celice MEC-1. Sarakatinib in HY-12072 sta povzročila koncentracijsko in časovno odvisno znižanje relativne metabolne aktivnosti primarnih celic KLL. Mediana vrednosti IC₅₀ sarakatiniba je po 24 urah znašala 22,52 μM, po 48 urah pa 8,86 μM, mediana vrednosti IC₅₀ HY-12072 pa 3,78 μM po 24 urah oziroma 1,63 μM po 48 urah. Pri vrednotenju kombiniranega delovanja sarakatiniba in HY-12072 z monoklonskima protitelesoma proti CD20 smo pri koncentracijah sarakatiniba 15 in 60 μM in koncentracijah HY-12072 0,15 in 0,6 μM z merjenjem metabolne aktivnosti v kombinaciji z rituksimabom in obinutuzumabom opazili primerljivo znižanje relativne metabolne aktivnosti celic kot posamezne učinkovine. Rezultati so bili skladni z aditivnim delovanjem kombinacije učinkovin in monoklonskih protiteles. Pridobljene ugotovitve prispevajo k boljšemu razumevanju odziva celic KLL na tretiranje s sarakatinibom in HY-12072 v kombinaciji z rituksimabom in obinutuzumabom ter predstavljajo izhodišče za nadaljnje raziskave novih kombiniranih terapevtskih pristopov. Chronic lymphocytic leukemia (CLL) is the most common leukemia in adults, in which B-cell receptor signaling plays a key role in regulating the survival of leukemic cells. As the tyrosine kinases Lyn and Lck are important regulators of B-cell receptor signaling, they represent promising therapeutic targets for improving the efficacy of treatment with anti-CD20 monoclonal antibodies. The main aim of this master’s thesis was to evaluate the cytotoxic effects of saracatinib and HY-12072 in combination with rituximab and obinutuzumab in vitro and to determine whether these drug combinations exhibit synergistic cytotoxic effects. Since the activity of anti-CD20 monoclonal antibodies is associated with activation of the complement system, we first investigated the effect of human serum on complement-mediated cytotoxicity induced by rituximab and obinutuzumab in MEC-1 cells. As the concentration of added human serum increased from 0% to 20%, the relative metabolic activity of MEC-1 cells decreased from 92.0% to 29.4% following treatment with rituximab and from 77.5% to 27.1% following treatment with obinutuzumab. We subsequently evaluated the cytotoxic effects of saracatinib and HY-12072 in primary CLL cells and their combined effects with rituximab and obinutuzumab in MEC-1 cells. In primary CLL cells, saracatinib and HY-12072 induced concentration- and time-dependent decreases in relative metabolic activity. The median IC₅₀ value of saracatinib was 22.52 μM after 24 h and 8.86 μM after 48 h, whereas the median IC₅₀ value of HY-12072 was 3.78 μM after 24 h and 1.63 μM after 48 h. When evaluating the combined effects of saracatinib and HY-12072 with anti-CD20 monoclonal antibodies, relative metabolic activity of MEC-1 cells at saracatinib concentrations of 15 and 60 μM was 81.1% and 30.9%, respectively, in combination with rituximab, and 55.4% and 33.5%, respectively, in combination with obinutuzumab. At HY-12072 concentrations of 0.15 and 0.6 μM, relative metabolic activity was 79.5% and 59.7%, respectively, in combination with rituximab, and 56.3% and 49.0%, respectively, in combination with obinutuzumab. The combinations resulted in decreases in relative metabolic activity comparable to those observed with the individual agents. These findings contribute to a better understanding of the response of CLL cells to treatment with saracatinib and HY-12072 in combination with rituximab and obinutuzumab and provide a basis for further investigation of novel combination therapeutic approaches. kronična limfocitna levkemija družina kinaz Src monoklonska protitelesa proti CD20 zaviralci kinaz sinergizem chronic lymphocytic leukemia Src-family kinases anti-CD20 monoclonal antibodies kinase inhibitors synergism true false false [N. Simšič] Slovenski jezik Angleški jezik Magistrsko delo/naloga 2026-09-09 08:45:21 2026-09-09 08:45:24 2026-09-21 15:36:26 0000-00-00 00:00:00 2026 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 2027-09-07 616.155.392(043.2) 128497 290654467 33627.pdf 33627.pdf 1 57AFF8E95ABA1414DB592C6F6F2081F4 dd36f5e1f1bf651fcaba72939a1a220eeb76cfaf04b6164ec7426c7e5398d7c7 055692d2-ac1a-11f1-8bc5-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=246557 Fakulteta za farmacijo 0 0 0