Radiooznačeni peptidi v nuklearni medicini

Nuklearna medicina pri obravnavi onkoloških bolnikov omogoča slikovni prikaz oziroma lokalizacijo tumorjev, ciljano zdravljenje ter spremljanje uspešnosti zdravljenja. Visoko specifične radiooznačene peptidne učinkovine lahko izkoristimo za ciljanje tumorskih celic, ki imajo na svoji površini prekomerno izražene receptorje za te učinkovine. Enako peptidno učinkovino lahko radiooznačimo tako z diagnostičnimi kot terapevtskimi radionuklidi, kar nam omogoča personaliziran pristop pri obravnavi bolnikov. V preglednem članku opisujemo dva sistema teranostičnih parov, ki jih v nuklearni medicini uporabljamo za diagnosticiranje ter zdravljenje tumorjev. Klasičen primer takšnih teranostičnih parov so radiooznačeni analogi somatostatina, ki se rutinsko uporabljajo v diagnostiki ter zdravljenju nevroendokrinih tumorjev (NET) s prekomerno izraženimi receptorji za somatostatin. Po drugi strani pa so radiooznačeni analogi minigastrina, ki se vežejo na receptorje za holecistokinin-2, primerni za diagnostiko medularnega raka ščitnice (MTC), za njihovo varno zdravljenje pa še potekajo klinična preskušanja

COST Action CA19114, Network for Optimized Astatine labelled Radiopharmaceuticals

Cancer is a major health concerns for European citizens. Thus, the main research aim of this Network for Optimized Astatine labeled Radiopharmaceuticals (NOAR) COST Action is to successfully demonstrate that one of the most promising radionuclides for Targeted Alpha Therapy (TAT), namely astatine-211, can become the European standard for treatment of certain cancerous pathologies. To this end, an efficient networking is essential among all European stakeholders interested in promoting astatine-211 for medical applications. NOAR COST Action brings together European and international excellence labs, astatine-211 production centers, hospitals, industry and patient associations from more than 20 countries, thus covering the whole value chain of innovation: production, chemistry, radiochemistry, biology, preclinical and clinical research and delivery of radiopharmaceuticals to patients. A European web portal will be created containing information for patients, practitioners, researchers, Industry and as a contact point for National and European patient associations. The idea is to gather forces at the European level in order to implement actions to leverage hurdles to the development of this powerful radionuclide and to identify pathologies in which it will be particularly relevant. A special emphasis will be given to train a new generation of young researchers and PhD students, promoting interdisciplinary competencies through international and inter-sectoral mobility. The long-term goal of this project is to make Astatine-211 technology available to all European citizen

Design, synthesis and evaluation of radiolabeled ligands for targeting of somatostatin and cholecistokinin-2 receptor

Medularni rak ščitnice (ang. Medullary Thyroid Cancer, MTC) predstavlja okoli 3,5 % vseh rakov ščitnice in se uvršča med redke bolezni. MTC je nevroendokrini tumor, ki v nasprotju z ostalimi vrstami raka ščitnice ne izhaja iz folikularnih celic ščitnice, njegove metastaze pa ne kopičijo radioaktivnega jodida. Zato je obravnava bolnikov z medularnim rakom ščitnice bolj zahtevna. Ob postavitvi diagnoze je pri večini bolnikov bolezen že napredovala, saj MTC ponavadi metastazira že pred pojavom simptomov. Trenutni pristopi zdravljenja (agresivno kirurško zdravljenje, kemoterapija, novejši zaviralci tirozin kinaz) so le deloma učinkoviti in so povezani s pojavom neželenih učinkov. Iskanje učinkovitega zdravljenja bolnikov z napredovalim MTC ostaja danes še vedno izziv za mnoge raziskovalne skupine. Nuklearna medicina ima v obravnavi onkoloških bolnikov pomembno vlogo tako pri diagnostiki kot tudi pri zdravljenju. Radiooznačeni peptidi predstavljajo pomemben razred radiofarmakov za diagnostiko in zdravljenje tumorjev, ki imajo na celični površini prekomerno izražene receptorje za regulatorne peptide. Ti peptidi imajo visoko afiniteto in specifičnost do receptorjev. Odkritje, da so pri medularnem raku ščitnice (ter še nekaterih ostalih rakih) prekomerno izraženi receptorji za holecistokininski receptor 2 (CCK2R), je omogočilo razvoj radiooznačenih analogov minigastrina za slikovno diagnostiko ter ciljano radionuklidno terapijo. Hkratna ekspresija somatostatinskih receptorjev in CCK2R pri MTC in drugih tipih rakov pa omogoča uporabo radiooznačenih analogov somatostatina pri obravnavi bolnikov z MTC ter tudi načrtovanje in razvoj t.i. hibridnih molekul za ciljanje multireceptorskih tarč. Glavni namen doktorske disertacije je bil načrtovanje, sinteza, radiooznačevanje in ovrednotenje novih analogov minigastrina in somatostatina z namenom optimizacije radiofarmacevtskih ter farmakokinetičnih lastnosti. Uspešno radiooznačevanje načrtovanih peptidov z različnimi radionuklidi (indij-111, galij-68, lutecij-177, itrij-90, bizmut-213) ter njihovo ustrezno ovrednotenje omogoča tudi translacijo (prenos) novih dognanj v klinično prakso. Raziskave smo tako zasnovali v več sklopih in jih načrtovali tako, da bomo lahko izsledke doktorske disertacije tudi implementirali v klinično prakso pri rutinski pripravi radiofarmakov in obravnavi bolnikov. Osnova za načrtovanje ter razvoj novih radiooznačenih analogov minigastrina v doktorskem delu so bili tudi izsledki raziskav, opisani v poglavju 1. Pri ciljani radionuklidni terapiji s peptidnimi radiofarmaki je največja težava nefrotoksičnost, saj se peptidne učinkovine večinoma izločajo skozi ledvice, kjer se tudi deloma ponovno privzamejo. Visoko zadrževanje radiooznačenih analogov minigastrina v ledvicah lahko zmanjšamo s sočasnim injiciranjem poliglutaminskih kislin, gelofuzina, albumina ali albuminskih fragmentov. Glavni mehanizem zadrževanja minigastrinskih analogov v ledvicah so povezovali z N-terminalnimi glutaminskimi kislinami (torej celokupnim nabojem molekule). S primerjavo dveh analogov, ki se razlikujeta le v stereokemiji aminokislin v distančniku (L- in D-Glu), ne pa tudi v kemijski sestavi in naboju, smo ugotovili velike razlike v metabolični stabilnosti ter v privzemu in zadrževanju v ledvicah. Pokazali smo tudi pomembno vlogo sekundarne strukture peptidnih molekul na farmakokinetične lastnosti. V prvem sklopu doktorskega dela smo sintetizirali ter ovrednotili več novih, z indijem-111 (111In) radiooznačenih analogov minigastrina. Z uporabo analogov z različnimi distančniki med bifunkcionalnem kelatorjem ter C-terminalnim delom minigastrina, ki je odgovoren za vezavo na receptor, smo sintetizirali novo serijo analogov minigastrina. Analogi s t.i. mešanim distančnikom (izmenjajoče nenaravne aminokisline s kislimi ali bazičnimi funkcionalnimi skupinami v stranski verigi ali brez njih(D-Gln-D-Glu)3 in (D-Gln-D-Asp)3) v primerjavi z analogom, ki imajo v distančniku samo neionske (6 D-Gln) aminokisline, izkazujejo nižjo metabolično stabilnost, v primerjavi z analogom z ionskim distančnikom (6 D-Glu) pa podobno metabolično stabilnost. Vsi novi analogi imajo podobno vezavno afiniteto do CCK2R v nM območju. Metionin v C-terminalnem delu minigastrinskih analogov se pri radiooznačevanju pri povišani temperaturi oksidira in s tem izgubi vezavno afiniteto do CCK2R, kar smo pokazali tudi s študijo na živalskem modelu in vivo. Z izosterno zamenjavo z norlevcinom se tej težavi enostavno izognemo. Ker literaturni podatki kažejo nasprotujoče si rezultate pri študijah analogov z norlevcinom in vitro, smo s primerjavo rezultatov raziskav in vitro ter in vivo pokazali, da menjava bistveno ne vpliva na vezavno afiniteto do CCK2R, metabolično stabilnost ter biodistribucijo na živalskih modelih. Pokazali smo, da se ti analogi hitro nakopičijo v tumorju ter se tam zadržijo tudi 4h po injiciranju. Dolgo zadrževanje radiooznačenih peptidov je predpogoj za njihovo uporabo v ciljani radionuklidni terapiji s peptidnimi radiofarmaki (ang. Peptide Receptor Radionuclide Therapy, PRRT). Glavne prednosti peptidnih analogov z norlevcinom so lažja formulacija peptida za radiooznačevanje, izboljšana radiokemijska čistota ter odsotnost stranskih produktov pri radiooznačevanju, kar v našem primeru pomeni varnejša in učinkovitejša zdravila - radiofarmake. Uporaba peptidnih analogov, radiooznačenih z različnimi radionuklidi (diagnostičnimi, kot sta 111In in 68Ga, in terapevtskimi, kot so 177Lu, 90Y, 213Bi), omogoča t.i. teranostični pristop k obravnavi bolnikov. 68Ga je eden redkih PET (ang. Positron Emission Tomography) radionuklidov, ki za pridobivanje ne potrebuje ciklotrona. Rokovanje z visokoenergijskimi PET ali β- sevalci je kritično z vidika varstva pred ionizirajočem sevanjem, zato je v vsakdanji klinični in tudi raziskovalni praksi zelo pomembna avtomatizacija procesov. V drugem sklopu raziskav smo z uporabo modularnega sistema za popolnoma avtomatizirano pripravo radiofarmakov razvili metode za radiooznačevanje klinično pomembnih analogov somatostatina za diagnostiko (68Ga-DOTATATE) ter PRRT (90Y-, 177Lu-DOTATATE). Uspešno smo razvili metodo za avtomatizirano radiooznačevanje dveh minigastrinskih analogov z 68Ga ter izračunali zaščitne faktorje pred radioaktivnim sevanjem. Izbrane minigastrinske analoge smo uspešno radiooznačili z 213Bi ter primerjali lastnosti in vitro z 111In radiooznačenimi analogi. Primerjava je pokazala, da menjava kovinskega radionuklida ne vpliva na lastnosti in vitro in bi te analoge lahko uporabili v ciljani terapiji z delci alfa. Ker MTC sočasno izraža več različnih receptorjev smo z vidika multireceptorskega pristopa k obravnavi MTC optimizirali tako analoge minigastrina kot somatostatina. Razvili smo nove analoge minigastrina z izboljšanimi lastnostmi ter razvili metode radiooznačevanja za nove analoge minigastrina ter nekatere že klinično uveljavljene analoge somatostatina. Na ta način smo postavili osnovo za uporabo teh radiooznačenih peptidov v multireceptorskem pristopu k prikazovanju ali ciljani terapiji MTC. Načrtovali smo tudi t.i. »hibridno« molekulo, ki bi združevala minigastrinski ter somatostatinski del in bi omogočala tudi radiooznačevanje. Uspešno smo sintetizirali združen minigastrinski ter somatostatinski del, neuspešni pa smo bili pri konjugaciji te molekule z bifunkcionalnim kelatorjem, ki bi omogočil tudi radiooznačevanje ter nadaljnjo ovrednotenje s študijami in vitro ter in vivo. V zadnjem delu opisujemo pomembnost merjenja radioaktivnosti z radionuklidnim kalibratorjem pred aplikacijo pripravljenih odmerkov preiskovancem. Posledica napačnih meritev in posledično odmerjanja radiofarmakov je lahko prevelika izpostavljenost preiskovanca ionizirajočem sevanju, v najslabšem primeru tudi toksičnost, po drugi strani pa bi prenizka radioaktivnost za preiskovanca pomenila neustrezno kakovost slikovne diagnostike ali neučinkovito zdravljenje. Na meritev radioaktivnosti vplivajo vrsta radionuklida, vrsta vsebnika (geometrijsko telo), v katerem se radionuklid nahaja, in volumen raztopine v samem vsebniku. Z meritvami 90Y in 111In v različnih radionuklidnih kalibratorjih smo ugotovili, da se lahko izmerjene meritve z uporabo tovarniško določenih kalibracijskih faktorjev od dejanskih razlikujejo tudi za do 25 %. Odstopanja so še posebej kritična pri meritvah radioaktivnosti terapevtskih sevalcev β-, saj je za učinkovito in varno PRRT izjemnega pomena natančno odmerjanje terapevtskih radiofarmakov.Medullary thyroid cancer (MTC) is a rare disease and represents 3.5 % of all thyroid cancers. MTC is a neuroendocrine tumour which in comparison to other thyroid cancers does not derive from thyroid follicular cells, while its metastases does not take up radioiodine. Thus, the management of patients with MTC is more demanding. The majority of MTC patients presents with the advanced disease at the time of diagnosis, since it is metastasizing before the onset of the symptoms. The currently available therapeutic options (surgery, conventional chemotherapy, latest tyrosine kinase inhibitors) are only partially effective and connected to side effects. The search for effective treatment is still a challenge for many research groups. Nuclear medicine plays an important part in the field of oncology for imaging of tumour cells and/or targeted radionuclide therapy. Radiolabelled peptides are an important class of radiopharmaceuticals in imaging and therapy of tumours, overexpressing receptors for regulatory peptides. These molecules have high affinity and specificity for receptors and enable visualisation and/or targeted radionuclide therapy. Overexpression of CCK2R on MTC cells is the molecular basis for the development of radiolabelled CCK/gastrin analogues. Thus, nuclear medicine can play a major role in both diagnosis (scintigraphy, PET/CT) and therapy of advanced MTC. Besides CCK2R, MTC cells also overexpress somatostatin receptors. Receptor co-expression enables the use of multiple radiolabelled ligands in the management of MTC and development of multireceptor (hybride) targeting molecules. The main goal of dissertation was design, synthesis, radiolabelling and characterisation of minigastrin and somatostatin analogues. The radiolabelling of these molecules with different radionuclides (indium-111, gallium-68, lutetium-177, yttrium-90, bismuth-213) and their characterisation enables translation of these molecules into clinical practice. We have designed the research work in such a way that we also can influence the daily clinical practice regarding preparation of radiopharmaceuticals and patient management. The results from the study in chapter one are the basis for the development of novel radiolabelled minigastrin analogues. Nephrotoxicity is the main side effect in targeted radionuclide therapy due to renal reuptake and accumulation of radiolabelled peptides. The main mechanism of high kidney retention of radiolabelled minigastrin analogues has been related to N-terminal glutamic acids (the overall charge of molecule) and can be reduced by coinjection of polyglutamic acids or gelofusine. The comparison of two radiolabelled minigastrin analogues with the same chemical composition and charge, differing only in the stereochemistry of the amino acids that form the spacer, showed the difference in enzymatic stability and large difference in their kidney uptake and retention. We also showed the influence of secondary structure on the pharmacokinetics of the radiopeptides. In the first part of the research, we synthesised and characterised several novel, indium-111 radiolabelled minigastrin analogues. We developed a series of minigastrin analogues with mixed (non-ionic/ionic) amino acids that form the spacer. The introduction of mixed ionic/non-ionic spacers lead to a lower enzymatic stability in vitro in comparison to non-ionic spacer (6 D-Gln) analogue, whereas, relative to the charged analogue with 6 D-Glu as a spacer, there was no significant change in the stability. All radiopeptides tested show similar internalisation rate in cell lines and have similar binding affinity for CCK2R in nM range. Methionine in the C-terminal part of the peptide is oxidation-prone, especially during the radiolabelling step at high temperature. The binding affinity for the CCK2R is lost, if the methionine residue is oxidized (sulphones, sulphoxides), which was also demonstrated in the experiment in vivo. To overcome the problem of oxidation, we introduced isosteric replacement (norleucine) of the oxidation-prone methionine in the C-terminus of the analogues. In contrast to literature data, direct comparison between methionine and norleucine congeners in vitro and in vivo showed no significant difference in the behaviour of these analogues. We also showed that these analogues are quickly accumulated in the tumour and retained there for up to 4h p.i. Good tumour retention is a prerequisite for the use of these analogues in PRRT. The main advantages of norleucine congeners are more effective formulation for radiolabelling, better radiolabelling results and fewer side products which, in our case, can lead to safer and more effective radiopharmaceuticals. Radiolabelling of the same molecule with different radionuclides (diagnostic – 111In and 68Ga and therapeutic - 177Lu, 90Y, 213Bi) enables so called theranostic approach to patient management. 68Ga is a generator-based PET radionuclide. The full automation of the processes in everyday clinical and research practice is required when handling the high-energy PET or β- emitters. In the second part of the research, we used a novel, self-shielded radiosynthesis box for fully automated preparation of radiopharmaceuticals and determined the shielding properties of the modular synthesis system. We developed radiolabelling methods of novel minigastrin analogues and optimized methods for radiolabelling of clinical relevant somatostatin analogues with 68Ga, 90Y and 177Lu. We radiolabelled minigastrin analogues with 213Bi and compared in vitro characteristics with 111In labelled ones. There was no significant difference between two analogues in vitro. We showed the feasibility of 213Bi radiolabelling and thus the potential use of these analogues in alpha radionuclide therapy. Co-expression of both cholecystokinin and somatostatin receptors on the surface of MTC cells is a basis for multireceptor targeting of these tumour cells. We developed minigastrin analogues with improved characteristics and developed radiolabelling methods for novel minigastrin analogues and optimized radiolabelling of clinical relevant somatostatin analogues. The optimisation of both radioligands is a prerequisite for a possible multireceptor targeting of MTC. We also designed a hybride molecule with both minigastrin and somatostatin moiety. We successfully synthesised hybride molecule employing principles of “click” chemistry. However, the conjugation of bifunctional chelator to the molecule, which enables radiolabelling and further characterization of the molecule, was not successful. The importance of correct measurements of radioactivity in radionuclide calibrator is described in the last chapter. Imprecise measurement leads to administration of either excessive or too low activity to the patient. Excessive activity administered is not in agreement with patient radioprotection and in the worst case can lead to toxicity. On the other hand, too low activity administered can result in insufficient quality of diagnostic scans or inadequate response in case of targeted radionuclide therapy. Type of radiation, geometry and volume of radiation influence the measurement of radioactivity. Measurements of 90Y and 111In in different radionuclide calibrators showed significant difference between measured and true activity, which can be as much as 25%, by either overestimation or underestimation of true activity. Measuring true activity is especially critical for efficient and safe targeted PRRT with β- emitters

Development of the 99mTc-labelled SST2 antagonist TECANT-1 for a first-in-man multicentre clinical study

Broad availability and cost-effectiveness of 99Mo/99mTc generators worldwide support the use, and thus the development, of novel 99mTc-labelled radiopharmaceuticals. In recent years, preclinical and clinical developments for neuroendocrine neoplasms patient management focused on somatostatin receptor subtype 2 (SST2) antagonists, mainly due to their superiority in SST2-tumour targeting and improved diagnostic sensitivity over agonists. The goal of this work was to provide a reliable method for facile preparation of a 99mTc-labelled SST2 antagonist, [99mTc]Tc-TECANT-1, in a hospital radiopharmacy setting, suitable for a multi-centre clinical trial. To ensure successful and reproducible on-site preparation of the radiopharmaceutical for human use shortly before administration, a freeze-dried three-vial kit was developed. The final composition of the kit was established based on the radiolabelling results obtained during the optimisation process, in which variables such as precursor content, pH and buffer, as well as kit formulations, were tested. Finally, the prepared GMP-grade batches met all predefined specification parameters together with long-term kit stability and stability of the product [99mTc]Tc-TECANT-1. Furthermore, the selected precursor content complies with micro-dosing, based on an extended single-dose toxicity study, where histopathology NOEL was established at 0.5 mg/kg BW, being more than 1000 times higher than the planned human dose of 20 µg. In conclusion, [99mTc]Tc-TECANT-1 is suitable to be advanced into a first-in-human clinical trial