doktorska disertacija
Doroteja Novak (Author), Marko Anderluh (Mentor), Petra Kolenc-Peitl (Co-mentor)

Abstract

Zaradi prekomernega izražanja holecistokininskega-2/gastrinskega receptorja (CCK2R) na površini celic nekaterih vrst tumorjev predstavlja CCK2R privlačno tarčo za razvoj radiooznačenih ligandov. Razvoj teh je v zadnjih letih še vedno v največjem obsegu omejen na razvoj radiooznačenih agonistov na osnovi minigastrina. Analoge minigastrina pesti nizek privzem v tumor in/ali visoko zadrževanja v ledvicah, pa tudi možnih potencialnih stranskih učinkov. Slednjim bi se lahko izognili z uporabo radiooznačenih antagonistov, zato je bil glavni cilj doktorske disertacije načrtovati, sintetizirati in biološko ovrednotiti nove radiooznačene antagoniste CCK2R. S primerjavo radiooznačenih antagonistov in agonistov CCK2R smo skušali odgovoriti na vprašanje, ali so antagonistično delujoči radiooznačeni ligandi CCK2R primernejši za nadaljnji razvoj od radiooznačenega agonista PP11, ki se trenutno nahaja v kliničnem preskušanju. Nove radiooznačene antagoniste CCK2R smo tako načrtovali na osnovi nizkomolekularnega antagonista CCK2R - nastorazepida/Z360, ki smo ga funkcionalizirali z ustreznim distančnikom, katerega dolžino smo določili na podlagi homolognega modeliranja in molekulskega sidranja in silico, ter bifunkcionalnim kelatorjem DOTA, ki bi že v osnovi omogočal teranostični pristop. Literaturne izhodne spojine smo v nadaljevanju poskusili optimizirati s podaljšanjem distančnika s polietilenglikolnim distančnikom različnih dolžin, identifikacijo ključnih aminokislinskih preostankov v distančniku in zamenjavo bifunkcionalnega kelatorja. Vsem sintetiziranim in analitsko ovrednotenim spojinam (RP-HPLC, ESI-MS, HRMS) smo v celičnem funkcijskem testu na celični liniji A431-CCK2R+ potrdili antagonistične lastnosti z določanjem vrednosti IC50 ter jih v nadaljevanju uspešno radioaktivno označili z različnimi radionuklidi - z galijem-68 (68Ga), z indijem-111 (111In) in lutecijem-177 (177Lu). Radioaktivno označenim spojinam smo določili vrednosti logD7,4, stabilnost v humanem serumu in obseg vezave na beljakovine seruma, pri čemer so pripravljene spojine izkazovale izboljšano hidrofilnost v primerjavi z izhodno spojino, visoko metabolno stabilnost in ugoden obseg vezave na beljakovine seruma. Na celični liniji A431-CCK2R+ so izbrani radiooznačeni antagonisti pri primerljivih vrednostih konstante disociacije izkazovali višji ali primerljiv obseg privzema in zadrževanja v celice z izraženim CCK2R kot radiooznačen agonist PP11. Za radiooznačene antagoniste so vrednosti maksimalnega obsega vezave (Bmax) signifikantno višje od Bmax za radiooznačen agonist PP11. Omenjene razlike v ovrednotenju in vitro med radiooznačenimi antagonisti in agonistom so se izrazile tudi pri slikovnem prikazu PET/CT za z galijem-68 (68Ga) radiooznačena antagonista in biodistribuciji in vivo, kjer sta izbrana z lutecijem-177 (177Lu) radiooznačena antagonista izkazovala znatno višji privzem v tumor kot [177Lu]Lu-PP11. Na podlagi rezultatov ovrednotenja in vitro in in vivo lahko zaključimo, da pripravljeni antagonisti CCK2R predstavljajo ustrezno podlago za nadaljnji razvoj radiooznačenih ligandov CCK2R ter da agonistično delovanje ni predpogoj za zagotavljanje visokega privzema v tumorske celice in tumorje z izraženim CCK2R.

Keywords

holecistokininski-2/gastrinski receptor;holecistokininski-1 receptor;radionuklidi;analog minigastrina;antagonisti CCK2R;sinteza na trdnem polimernem nosilcu;

Data

Language: Slovenian
Year of publishing:
Typology: 2.08 - Doctoral Dissertation
Organization: UL FFA - Faculty of Pharmacy
Publisher: [D. Novak]
UDC: 615.4:54:616-006(043.3)
COBISS: 80452867 Link will open in a new window
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Other data

Secondary language: English
Secondary title: Novel radiolabelled cholecystokinin-2/gastrin receptor antagonists
Secondary abstract: The overexpression of cholecystokinin-2/gastrin receptor (CCK2R) on the cell surface of certain tumour types makes CCK2R a promising target for the development of radiolabelled ligands, which is in the last years still mainly focused on the optimization and development of radiolabelled agonists based on minigastrin (MG). The use of MG analogues is limited by the low tumour uptake and/or high kidney retention, and also the possible adverse side effects caused by the agonistic properties. The latter could be bypassed by the use of radiolaelled CCK2R antagonists; therefore, the main aim of the doctoral thesis was to design, synthesiszed and evaluate novel radiolabelled CCK2R antagonists. Additionally, by comparising such compounds with the radiolabelled agonists currently under clinical investigation (PP11), we sought to answer whether radiolabelled CCK2R antagonists represent suitable option for further development of radiolabelled CCK2R ligands. Novel radiolabelled CCK2R antagonists were designed based on the small-molecule CCK2R antagonist % nastorazepide/Z360, which was further functionalized with spacer and bifunctional chelator DOTA to ensure the theranostic approach. After the determination of the minimal length of the spacer determined by using in silico homology modelling and docking the literature reference compound was chosen. To optimize the reference compounds three approaches were undertaken: a) the extension of spacer by the introduction of polyethylene glycol chains of variable length, b) the identification of crucial aminoacid residues in the spacer region, and c) replacement of bifunctional chelator. For all synthesized and analytically evaluated (by RP-HPLC, ESI-MS, HRMS) compounds the antagonistic properties were determined in the functional cell-based test on the A431-CCK2R+ cell line. Afterwards, the compounds were successfully radiolabelled with different radionuclides e.g. gallium-68, indium-111 and lutetium-177. For radiolabelled compounds high stability in human serum and reasonably low serum protein binding were achieved, whereas lower logD7,4 values compared to reference compound were determined. Compared to the cell uptake of radiolabelled PP11 determined on the A431-CCK2R+ cell line, the selected radiolabelled CCK2R antagonists exhibited higher or comparable cell uptake and retention (at comparable values of the constant of dissociation (Kd)). For radiolabelled antagonists significantly higher Bmax values were achived compared to the radiolabelled agonists PP11. The differences observed between the radiolabelled antagonists and agonist in in vitro setting translated also to the differences in in vivo PET/CT imaging performed for gallium-68 radiolabelled compounds and biodistribution. The two selected antagonists radiolabelled with lutetium-177 had considerably higher tumour uptake compared to the [177Lu]Lu-PP11 uptake. Based on the results of the in vitro and in vivo testing, we can conclude that the radiolabelled CCK2R antagonists should be considered as suitable for further development of CCK2R radiolabelled compounds, as the agonistic behaviour does not seem to be prerequisite to achive high uptake in CCK2R expressing tumours.
Secondary keywords: Farmacevtska kemija;Disertacije;Tumorji;
Type (COBISS): Dissertation
Thesis comment: Univ. v Ljubljani, Fak. za farmacijo
Pages: 173 f.
ID: 15504110