Sinergistično delovanje ibrutiniba in idelalisiba z agonisti receptorja EP4 na celicah kronične limfocitne levkemije : enoviti magistrski študijski program Farmacija

Avsec, Damjan (avtor), Mlinarič-Raščan, Irena (mentor), Markovič, Tijana (komentor)

Kronična limfocitna levkemija (KLL) je najpogostejša levkemija pri odraslih v zahodnem svetu. Bolezen lahko poteka agresivno ali indolentno. Standardno zdravljenje temelji na kemoimunoterapiji, pogosta je shema FCR, ki zajema fludarabin, ciklofosfamid in rituksimab. Nove možnosti zdravljenja vključujejo tarčno zdravljenje z ibrutinibom in idelalisibom, ki zavirata signalizacijo preko B-celičnega receptorja (BCR). Prostaglandinski receptor EP4 predstavlja novo potencialno tarčo za zdravljenje B celičnih levkemij in limfomov. V magistrski nalogi smo ovrednotili selektivna agonista receptorja EP4 L-902,688 in PgE1-OH. Z uporabo testa metabolne aktivnosti in pretočne citometrije smo dokazali, da je citotoksično delovanje na celicah KLL časovno in koncentracijsko odvisno. Z uporabo selektivnega antagonista CJ-042794 smo potrdili, da so učinki agonistov na celicah KLL posredovani preko receptorja EP4. Vrednosti EC50 L-902,688 in PgE1-OH so bile statistično značilno manjše na celicah KLL v primerjavi z limfoblastoidnimi celičnimi linijami (LCL) in perifernimi mononuklearnimi krvnimi celicami (PBMC), s čimer smo potrdili selektivnost delovanja na maligne limfocite B. V nadaljevanju smo ovrednotili delovanje najpogosteje uporabljanega zdravila pri KLL fludarabina in novih tarčnih zdravil ibrutiniba in idelalisiba. Po 24 urah je imel fludarabin statistično značilno večje vrednosti EC50 v primerjavi z L-902,688 in PgE1-OH, idelalisib pa le v primerjavi z L-902,688. Po 48 urah so bile vrednosti EC50 (IC50) fludarabina, ibrutiniba in idelalisiba 4,65 μM, 3,15 μM in 8,84 μM primerljive z vrednostmi EC50 za L-902,688 in PgE1-OH. Ker je uspešnost zdravljenja KLL večja pri kombinacijah učinkovin z različnimi mehanizmi delovanja, smo ovrednotili potencialni sinergizem L-902,688 in PgE1-OH s fludarabinom, ibrutinibom ter idelalisibom. Pri kombinaciji agonistov receptorja EP4 s fludarabinom in ibrutinibom smo zaznali sinergizem pri manjših koncentracijah spojin, medtem ko je bil z idelalisibom, ki ima med testiranimi spojinami najmanj citotoksično delovanje (IC50 po 24 urah je bila 20,64 μM, po 48 urah pa 8,84 μM), sinergizem prisoten pri vseh koncentracijah spojin. Z aneksinom V smo dokazali, da L-902,688 in PgE1-OH inducirata apoptozo v malignih limfocitih B, s testom CFSE pa smo pokazali, da delujeta tudi citostatično. Po 48 urah sta obe spojini ustavili delitev celic, kar pomeni, da bi lahko zavirali razmnoževanje celic KLL tudi in vivo. Pokazali smo tudi, da spojini ne vplivata na ekspresijo receptorja EP4, kar potrjuje pomen receptorja EP4 kot potencialne terapevtske tarče. Selektivni agonisti receptorja EP4 imajo citotoksično in citostatično delovanje ter izkazujejo sinergistične učinke z novejšimi terapevtiki in vitro, kar bi lahko vodilo v nove možnosti zdravljenja KLL.

• Jezik: Slovenski jezik
• Ključne besede: kronična limfocitna levkemija, ibrutinib, fludarabin, sinergizem, idelalisib, L-902, 688, PgE1-OH, prostaglandinski receptor tipa 4 (Ep4)
• Vrsta gradiva: Magistrsko delo/naloga
• Tipologija: 2.09 - Magistrsko delo
• Organizacija: FFA - Fakulteta za farmacijo
• Kraj izida: Ljubljana
• Založnik: [D. Avsec]
• Leto izida: 2018
• Št. strani: XII, 66 f., I-X f. pril.
• PID: 20.500.12556/RUL-121656
• UDK: 616.155.392+616-076.3(043.3)
• COBISS.SI-ID: 4534385
• Datum objave v RUL: 21.10.2020

Sekundarni jezik

• Jezik: Angleški jezik
• Naslov: Synergistic effects of ibrutinib and idelalisib with EP4 receptor agonists on chronic lymphocytic leukemia cells

Izvleček

Chronic 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.

• Ključne besede: chronic lymphocytic leukemia (CLL), prostaglandin receptor type 4 (EP4), L-902, 688, PgE1-OH, fludarabine, ibrutinib, idelalisib, synergy