doktorska disertacija
Tijana Markovič (Avtor), Irena Mlinarič-Raščan (Mentor)

Povzetek

Ovrednotenje imunomodulatornih učinkov spojin je pomemben del predkliničnega razvoja zdravil. Razvoj zanesljivih in reprezentativnih metod za ovrednotenje imunomodulatornih učinkov spojin je zato izrednega pomena. V luči novih smernic in zakonodaje Evropske unije »Direktiva 2010/63/EU« za zaščito živali, ki se uporabljajo v raziskovalne namene, se trendi v napovedovanju učinkovitosti in toksičnosti pomikajo od in vivo testiranja na živalih k in silico in in vitro testnim sistemom. Glavni namen doktorske disertacije je doprinesti k razvoju novih imunomodulatornih učinkovin. V ta namen smo raziskave zasnovali v dveh sklopih. V prvem sklopu smo vzpostavili nov in vitro testni sistem za vrednotenje imunomodulatornega delovanja spojin, v drugem sklopu pa smo preučevali delovanje agonistov receptorja EP4 in monoklonskih protiteles v malignih limfocitih B. Predhodne študije naše raziskovalne skupine so identificirale zgodnje spremembe v transkripcijskih mehanizmih, ki razlikujejo med negativno selekcijo in klonalno ekspanzijo limfocitov B. V kompleksnem transkripcijskem programu, ki ga sproži B-celični receptor (BCR), je bil identificiran gen Ptger4, ki kodira receptor EP4 in pomembno vpliva na odločitev o nadaljnjem poteku življenjskega cikla limfocitov B. Prostaglandin E2 (PGE2) je močan regulator funkcije limfocitov B, ki posreduje pro-vnetne in imunoregulatorne učinke. PGE2 je lipidna molekula, ki ima pomembno vlogo v fizioloških in patofizioloških procesih in deluje preko štirih z G-proteinom sklopljenih podtipov receptorjev EP1, EP2, EP3 in EP4. Vsak od teh receptorjev je pomembna terapevtska tarča z lokalizacijami v različnih tkivih in inducira specifične signalne poti, ki smo jih podrobneje povzeli v prvem poglavju. Receptor EP4 je sklopljen s proteinom G-αS, kar vodi v aktivacijo adenilat ciklaze in posledično povišanje znotrajceličnega cikličnega adenozin monofosfata (cAMP). Poleg klasične od cAMP odvisne poti, vodi aktivacija receptorja EP4 tudi v alternativne poti kot je inhibicija signalizacije preko jedrnega transkripcijskega dejavnika NF-κB. Ekspresija receptorja EP4 ni vezana na posamezen organ ali tkiva. Visoko je izražen v maternici, koži, gastrointestinalnem traktu ter v krvotvornih organih. Modulatorji receptorja EP4 tako predstavljajo potencialna zdravila za zdravljenje ulcerativnega kolitisa, trdnih tumorjev, kardiovaskularnih bolezni in B celičnega limfoma (Markovič et. al, 2017). V prvem delu doktorske naloge smo vzpostavili nov in vitro testni sistem za vrednotenje imunomodulatornih lastnosti spojin na osnovi limfoblasotidnih celičnih linij (LCL). Za vrednotenje celic LCL smo izbrali štiri znane imunomodulatorne spojine: tributilkositrov klorid, ciklosporin A, benzo(a)piren in verapamil hidroklorid ter tri imuno-inertne spojine: uretan, furosemid in manitol. In vitro testni sistem temelji na sposobnosti celic, da se odzivajo na imunomodulatorne spojine. Ugotovili smo, da predstavljajo človeške celice LCL zanesljiv in vitro testni sistem za oceno imunomodulatornih lastnosti spojin, ki lahko razlikuje med imunomodulatornimi in imunsko inertnimi spojinami. Hkrati lahko uporaba panela z desetimi limfoblastoidnimi celičnimi linijami, ki izvirajo iz nesorodnih zdravih posameznikov, omogoča vpogled v inter-individualno variabilnost v odzivu na določeno spojino. Celice LCL tako predstavljajo novo alternativno metodo za vrednotenje imunomodulatornih učinkov spojin (Markovič et. al, 2015). V drugem sklopu raziskav smo vrednotili receptor EP4 kot potencialno terapevtsko tarčo za zdravljenje B celičnih levkemij in limfomov, katerih preživetje je odvisno od konstitutivne aktivacije NF-κB. Znano je, da receptor EP4 posreduje zaviralne učinke na rast zrelih in nezrelih limfocitov B preko anti-apoptotičnega transkripcijskega dejavnika NF-κB. Z uporabo farmakološkega pristopa smo dokazali, da receptor EP4 inducira preko kaspaz posredovano apoptozo v maligno transformiranih limfocitih B. Ker je povečana aktivnost NF-κB podlaga za razvoj rezistence na številne spojine, smo modulirali to signalno pot preko aktivacije receptorja EP4. Specifični agonist receptorja EP4, PgE1-OH, je znižal aktivnost NF-κB in posledično raven anti-apoptotičnega gena Bcl-XL v celicah Ramos, kar je vodilo v povišano občutljivost celic na z bortezomibom in doksorubicinom inducirane kemoterapevtske učinke. Specifično znižanje od NF-κB odvisnih poti v B-celičnih malignih boleznih odpira nove možnosti za zdravljenje in optimizacijo sedanje terapije z uporabo specifičnih agonistov receptorja EP4 (Gobec et al, 2014). Nadalje smo raziskali vlogo receptorja EP4 v kronični limfocitni levkemiji (KLL). KLL je najpogostejše hematološko maligno obolenje pri odraslih in velja za trenutno neozdravljivo bolezen. Primarne celice smo izolirali iz polne krvi bolnikov z diagnozo KLL, ki so privolili v sodelovanje v raziskavi (dovoljenje KME št. 93/12/10). S pretočno citometrijo smo dokazali višjo ekspresijo receptorja EP4 pri celicah KLL v primerjavi s celicami LCL, pridobljenimi iz krvi zdravih darovalcev. Ugotovili smo, da so citotoksični učinki PgE1-OH posredovani izključno preko receptorja EP4, saj je imel endogeni ligand PGE2, ki se veže na vse 4 EP receptorje, nizek citotoksičen potencial. V skladu s tem je antagonist receptorja EP4 zavrl s PgE1-OH inducirano apoptozo. Citotoksični učinki agonista receptorja EP4, PgE1-OH, so bili časovno in koncentracijsko odvisni, vrednosti EC50 pa so bile 13.53 μM po 24 h (N=151) in 7.21 μM po 48 h (N= 140). PgE1-OH je izkazal inter-individualno variabilnost, kar je v skladu z dejstvom, da je KLL izredno heterogena bolezen. Agonist receptorja EP4 je deloval selektivno na celice KLL v primerjavi s celicami LCL in perifernimi mononuklearnimi celicami (PBMC), pridobljenimi iz krvi zdravih darovalcev. Poleg tega so bile EC50 vrednosti za PgE1-OH po 24 h nižje v primerjavi z fludarabinom. Posebnega pomena je odkritje, da je agonist receptorja EP4 induciral apoptozo pri celicah bolnikov s KLL, ki so rezistentni na standardno citotoksično terapijo in nosijo delecijo TP53 gena. Poleg tega je PgE1-OH pri celicah KLL deloval sinergistično s fludarabinom, kar bi lahko omogočilo nov terapevtski pristop za zdravljenje KLL. Mikro-okolje v KLL je močna gonilna sila za preživetje malignih limfocitov B in rezistenco na apoptozo, zanj pa je značilna konstitutivna aktivacija BCR in povečana koncentracija citokinov. Agonist receptorja EP4 je posredoval imunonomodulatorne učinke, saj je signifikantno znižal izločanje citokinov IL-2, IL-10, TNFα in IFNγ v primerjavi z netretiranimi celicami LCL. Receptor EP4 smo tako ovrednotili kot obetavno terapevtsko tarčo za zdravljenje KLL. V zadnjem delu doktorske disertacije smo vrednotili delovanje monoklonskih protiteles in agonista receptorja EP4 in vitro. Monoklonski protitelesi proti CD20 rituksimab in ofatumumab sta izkazali sinergistično delovanje s PgE1-OH na celični liniji Ramos. Preliminarni rezultati pri celicah KLL prav tako izkazujejo sinergistično delovanje PgE1-OH z anti-CD20 monoklonskimi protitelesi in nadalje potrjujejo pomen EP4 kot potencialne tarče v terapiji KLL. Pri uporabi monoklonskega protitelesa proti CD52, alemtuzumaba, sinergističnih učinkov z agonistom receptorja EP4 nismo zaznali. Ugotovili smo, da agonist receptorja EP4 deluje kemosenzitivno z anti-CD20 monoklonskimi protitelesi na malignih limfocitih B, kar lahko odpira nove možnosti za zdravljenje B-celičnih levkemij in limfomov.

Ključne besede

zdravilne učinkovine;imunomodulatorno delovanje;vrednotenje;metode;limfoblastoidne celične linije;receptor EP4;agonisti;limfom;monoklonska protitelesa;levkemija celic B;

Podatki

Jezik: Slovenski jezik
Leto izida:
Tipologija: 2.08 - Doktorska disertacija
Organizacija: UL FFA - Fakulteta za farmacijo
Založnik: [T. Markovič]
UDK: 615.375(043.3)
COBISS: 295575552 Povezava se bo odprla v novem oknu
Št. ogledov: 147
Št. prenosov: 0
Ocena: 0 (0 glasov)
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Sekundarni jezik: Angleški jezik
Sekundarni naslov: Evaluation of immune modulation and efficacy of EP4 receptor agonist and monoclonal antibodies in vitro
Sekundarni povzetek: Evaluation of the immunomodulatory effects of compounds is an important part of the preclinical development of medicines. The establishment of reliable and representative methods for evaluation of the immunomodulatory effects of compounds is therefore of high importance. In line with current guidelines and legislation of the European Union, Directive 2010/63/EU for the protection of animals used for research purposes, the trends in predicting efficacy and toxicity are moving from in vivo testing on animals to in silico and in vitro testing systems. The main purpose of the doctoral dissertation is to contribute to the development of novel immunomodulatory agents. We designed the research in two parts. First, we established a new in vitro test system for the evaluation of immunomodulatory effects of compounds. Second, the effects of EP4 receptor agonists and monoclonal antibodies were investigated in malignant lymphocytes B. Previous studies of our research group identified early changes in transcription mechanisms that distinguished between negative selection and clonal expansion of lymphocytes B. In the complex transcriptional program triggered by B-cell receptor (BCR), Ptger4 gene, encoding the EP4 receptor, was identified to significantly influence the decision on the life cycle of lymphocytes B. Prostaglandin E2 (PGE2) is a strong regulator of lymphocyte B function, which mediates pro-inflammatory and immunoregulatory effects. PGE2 is a lipid molecule that plays an important role in physiological and pathophysiological processes via four G-protein coupled receptors: EP1, EP2, EP3 and EP4. Each of these receptors is an important therapeutic target with localization in different tissues and induces specific signalling pathways, summarized in the first chapter. The EP4 receptor is coupled to the G-αS protein, leading to the activation of adenylate cyclase and, consequently, the increase in intracellular cyclic adenosine monophosphate (cAMP). In addition to the classical cAMP-dependent pathway, EP4 receptor activation also leads to alternative pathways such as inhibition of signalling via the nuclear transcription factor NF-κB. EP4 receptor expression is not related to a single organ or tissue. It is abundantly expressed in the uterus, skin, gastrointestinal tract and leukocytes. EP4 receptor modulators thus represent potential drugs for the treatment of ulcerative colitis, solid tumours, cardiovascular diseases, and B cell lymphoma (Markovič et. al, 2017). In the first part of the doctoral thesis, we established a new in vitro test system for the evaluation of the immunomodulatory properties of compounds based on lymphoblastoid cell lines (LCL). Four known immunomodulatory compounds were selected for the evaluation: tributyltin chloride, cyclosporine A, benzo (a) pyrene and verapamil hydrochloride, and three immuno-inert compounds: urethane, furosemide and mannitol. The in vitro test system is based on the ability of the cells to respond to immunomodulatory compounds. Human LCL cells thus represent a reliable in vitro method for assessing the immunomodulatory properties of compounds and can distinguish between immunomodulatory and immune-inert compounds. Utilizing a panel of ten LCLs, derived from unrelated healthy individuals allowed insight into inter-individual variability in response to a particular compound. LCL cells thus represent a new alternative method for the evaluation of immunomodulatory effects of the compounds (Markovič et. al, 2015). In the second part of our studies, the EP4 receptor was evaluated as a potential therapeutic target for the treatment of B cell leukaemia and lymphoma, which survival depends on the constitutive activation of NF-κB. EP4 receptor activation mediates the inhibitory effects on the growth of mature and immature lymphocytes B via anti-apoptotic transcription factor NF-κB. Using a pharmacological approach, we demonstrated that EP4 receptor agonist induces caspase-mediated apoptosis in malignantly transformed lymphocytes B. Since the increased activity of NF-κB is the basis for the development of resistance, this signalling pathway was modulated via the activation of the EP4 receptor. The specific EP4 receptor agonist, PgE1-OH, reduced the activity of NF-κB and, consequently, the level of the anti-apoptotic Bcl-XL in Ramos cells, which led to increased susceptibility of cells to bortezomib and doxorubicin-induced chemotherapeutic effects. A specific inhibition of NF-κB dependent pathways in B-cell malignancies thus opens new possibilities for the treatment and optimization of current therapies using specific EP4 receptor agonists (Gobec et al, 2014). Next, the role of EP4 receptor in chronic lymphocytic leukemia (CLL) was investigated. CLL is the most common hematologic malignant disease diagnosed in adults in Western world and is currently considered an incurable disease. Primary cells were isolated from the full blood of patients with diagnosis of CLL after informed consent was obtained (KME approval No. 93/12/10). Higher expression of EP4 receptor on CLL cells compared to LCL cells derived from healthy donors was detected by flow cytometry. The cytotoxic effects of PgE1-OH were mediated solely via EP4 receptor as the endogenous ligand PGE2, which binds to all 4 EP receptors, had low cytotoxic potential. Accordingly, the EP4 receptor antagonist prevented PgE1-OH-induced apoptosis. The cytotoxic effects of the PgE1-OH were time- and concentration-dependent, the EC50 values were 13.53 μM after 24 h (N = 151) and 7.21 μM after 48 h (N = 140). PgE1-OH has shown inter-individual variability, which is consistent with the fact that KLL is very heterogeneous disease. The EP4 receptor agonist was selective to CLL cells compared to LCL cells and peripheral mononuclear cells (PBMC) derived from the blood of healthy donors. In addition, EC50 values after 24 h for PgE1-OH were significantly lower compared to EC50 values for fludarabine. Of particular importance is the discovery that EP4 receptor agonist induced apoptosis in CLL cells obtained from patients which carry a deletion of the TP53 gene, who are known to be resistant to first-line therapy, including fludarabine. In addition, PgE1-OH acted synergistically with fludarabine in primary CLL cells, which might provide a novel therapeutic approach for the treatment of B-cell CLL. CLL microenvironment is a powerful driving force for the survival of malignant lymphocytes B and resistance to apoptosis. It is characterized by constitutive activation of BCR and increased cytokine concentration. The EP4 receptor agonist mediated immunomodulatory effects, as it significantly reduced the secretion of IL-2, IL-10, TNFα, and IFNγ cytokines compared to untreated LCL cells. The EP4 receptor was thus evaluated as a promising therapeutic target for CLL. In the final part of the doctoral dissertation we evaluated the activity of monoclonal antibodies and EP4 receptor agonists in vitro. Monoclonal antibodies against CD20 rituximab and ofatumumab acted synergistically with PgE1-OH in cell line Ramos. Preliminary results on CLL cells also proved synergistic action with anti-CD20 monoclonal antibodies, further confirming the importance of EP4 receptor as a prospective therapeutic target. The synergistic effects with EP4 receptor agonist and monoclonal antibody against CD52, alemtuzumab, were not detected. We have shown that EP4 receptor agonist chemosensitizes malignant B cells to anti-CD20 monoclonal antibodies-induced apoptosis, which might open new possibilities for the treatment of B-cell malignancies.
Sekundarne ključne besede: Imunomodulatorji;Disertacije;
Vrsta dela (COBISS): Doktorska disertacija
Komentar na gradivo: Univ. v Ljubljani, Fak. za farmacijo
Strani: X, 169 str.
ID: 15645134