20.500.12556/RUL-120129 Vrednotenje citotoksičnosti učinkovin za zdravljenje kronične limfocitne levkemije ex vivo Ex vivo evaluation of drug cytotoxicity in treatment of chronic lymphocytic leukemia V medicini se vedno bolj strmi k personalizirani terapiji z namenom zmanjševanja neželenih učinkov zdravil ter obremenitve pacienta in izboljšanja izidov zdravljenja. Pri bolnikih s kronično limfocitno levkemijo (KLL) je prilagoditev terapije še posebej priporočljiva, saj gre za heterogeno in predvsem s starostjo povezano bolezen. Pri starejši populaciji je namreč prisotna še komorbidnost in oslabljenost nekaterih telesnih funkcij. Prilagojena terapija je zaželjena, vendar z in vitro testi težko napovemo in vivo odziv. V razvoju je veliko visoko zmogljivih metod določanja odziva celic ex vivo in vrednotenja citotoksičnosti učinkovin. V magistrski nalogi smo želeli preveriti nov pristop k zdravljenju bolnika s KLL s prilagojeno terapijo z optimizacijo sistema HexascopeHEAMA. Analizirali smo 48 vzorcev krvi bolnikov s KLL. Najprej smo periferne mononuklearne krvne celice izolirali ter inkubirali tri dni v prisotnosti 30 različnih zdravilnih učinkovin (ZU) z različnimi koncentracijami. Za optimizacijo metode smo preverjali vpliv časa inkubacije ter končne koncentracije celic na mikrotitrski plošči ter ovrednotili ponovljivost testa. Za klinični del raziskave smo od teh 48-ih vzorcev krvi izbrali 35 vzorcev in v interni podatkovni bazi KOH UKC Ljubljana poiskali še podatke o genetskih spremembah, stopnji bolezni in prejetih terapijah. Na podlagi zbranih podatkov smo lahko primerljali in ovrednotili naše analize. S citotoksičnim testom HexascopeHEAMA smo ugotovili, da so bili za bolnike s KLL rezultati ob ustrezni koncentraciji celic (10 × 106 /L) na mikrotitrski plošči in dovoljšni infiltraciji limfocitov v krvi (70 %) skladni z zahtevami kakovosti in ponovljivi. Pri napovedi odziva ex vivo z in vivo učinkovitostjo nismo bili uspešni, saj so nam manjkali podatki o dejanskem rezultatu zdravljenja bolnika. Slabost pri izvedbi testa je bil tudi nabor ZU. Bolniki s KLL se pogosto zdravijo s kombinacijo ZU, česar z našim sistemom nismo mogli testirati. Za vpeljavo sistema v vsakdanjo prakso bi bilo morda smiselno razmisliti o spremembi nabora ZU oz., dodati kombinacije (terapevtkse sheme) ZU s katerimi se bolniki zdravijo. Zaradi dejstva, da se pri zdravljenju KLL praviloma uporabljajo tudi monoklonska protitelesa, ima sistem HexascopeHEAMA omejeno uporabnost pri bolnikih s KLL, ker jih ne vključuje. In medicine there is always an incentive to personalize therapy to lower side effects, improve treatment outcomes and reduce the strain on the patient. In patients with chronic lymphocytic leukemia (CLL) the adjustment of the treatment is preferable, as CLL is a heterogeneous and age-related disease. In the presence of comorbidity and impairment of some of the bodily functions in elderly population, tailored therapy is preferable, but with in vitro tests it is difficult to predict an in vivo response. Many high throughput methods are being developed to determine the ex vivo cell response and evaluate the cytotoxicity of active substances. This master’s thesis aims to examine a new approach to the treatment of a patient with CLL with tailored therapy, with optimization of HexascopeHEAMA system. We analyzed 48 blood samples of patients with CLL. Peripheral mononuclear blood cells were firstly isolated, then they were incubated in presence of 30 different active substances with different concentrations. To optimize the method, the influence of the incubation time and the final concentration of cells on the microtiter plate were checked and the reproducibility of the test was evaluated. For the clinical part of the study, 35 samples were selected from these 48 blood samples. Data on genetic mutations, disease stage and therapies received were found in the internal database of UMC Ljubljana. Based on the collected data, it was possible to compare and evaluate the analysis that were carried out. HexascopeHEAMA cytotoxicity test showed that, for patients with CLL with adequate concentration of cells on the microtiter plate and sufficient infiltration of lymphocytes in the blood, the results were in compliance with quality requirements and reproducibility. The prediction of an ex vivo response with in vivo efficacy was not successful, as data on the actual outcome of the patient's treatment were lacking. A weak point in performing the test were also the active substances. Patients with CLL are often treated with a combination of active substances, which was something impossible to test with the used system. In order to introduce the system into everyday practice, it might be reasonable to consider changing the set of active substances or rather, to introduce combinations (therapeutic schemes) of active substances with which patients are treated. Due to the fact that monoclonal antibodies are generally used in the treatment of CLL, the HexascopeHEAMA system has limited utility in patients with CLL, because it does not include them. kronična limfocitna levkemija citotoksičnost ex vivo vrednotenje odpornost/občutljivost na zdravilne učinkovine kromosomske preureditve chronic lymphocytic leukemia cytotoxicity ex vivo evaluation drug resistance/sensitivity chromosomal aberration true false false Slovenski jezik Angleški jezik Magistrsko delo/naloga 2020-09-16 08:45:20 2020-09-16 08:45:30 2022-09-01 03:57:30 0000-00-00 00:00:00 2020 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 1970-01-01 75737 1252.pdf 1252.pdf 1 974EF212ED22C6DF4E4FB2963B26ABA8 432094153756977d36e470a38c333e01d9ee673c17562b39be31114aa8080771 00ca2351-a1ba-11eb-a523-00155dcfd717 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=134885 Fakulteta za farmacijo 0 0 0