Pharmaceutical development of the ISOL technique for the production of radionuclides and their applications in targeted radionuclide therapy

Radiopharmaceuticals are the fundamental tool for nuclear medicine procedures, enabling imaging and therapy of cancer after administration of a radioligand. Imaging or therapy are performed with radionuclides that are bound to a ligand, which selectively accumulates into cancer tissues. The discovery of new radionuclides on one side and the optimization of ligands on the other are fundamental parts of the research in the radiopharmaceutical field. In the first part of this thesis work we aimed at the development of a new technique for the production of pure radionuclides. The study was born from the collaboration between the Department of Pharmaceutical and Pharmacological Sciences (DSF) of the University of Padua (UniPD) and the Legnaro National Laboratories (LNL) of the National Institute for Nuclear Physics (INFN), where the Selective Production of Exotic Species (SPES) project is under development. This project aims at the production of Radioactive Ion Beams (RIBs) by means of the Isotope Separation On-Line technique (ISOL), which can be exploited for radionuclides production. Thanks to mass separation, a feature of the ISOL technique, radionuclides can be produced at high purity because isotopic contaminants can be cleared away easily. Suitable secondary targets (chapter 2) were developed to allow ion beams collection, depending on the accelerated ions. To verify the suitability of the method for radionuclides production, tests with accelerated stable ion beams were carried out and are described in chapter 3. The SPES test bench was used for the elements ionization and acceleration. The beams were collected on the produced secondary targets and quantified. The tests were carried out for yttrium, iodine and copper, elements interesting for the production of 90Y, 125/127/131I and 64/67Cu. The ISOL method guarantees the purification of the radionuclides from the isotopic contaminants, but isobaric and pseudo-isobaric contaminants can be present in the beam and thus in the final solution. For this reason chemical purification methods have to be developed for radionuclides purification. In chapter 4 the synthesis of an inorganic material (sodium nonatitanate) and its use for strontium and yttrium purification is described. The second part of this thesis, chapters 5 and 6, was developed in collaboration with the group of PD Dr. Cristina Müller at the Center for Radiopharmaceutical Sciences (CRS) of Paul Scherrer Institute (PSI). Targeted radionuclide therapy (TRT) represents one of the most promising therapeutic strategies for cancer therapy ; it is based on the use of energetic particles (alpha, beta- or Auger electrons) selectively delivered in proximity of tumors thanks to specific biologic features. Small molecules are an attractive strategy for active targeted delivery because they are amenable for chemical synthesis and have some advantages if compared with high molecular weight vehicles, such as antibodies. A potential challenge of small molecular radiotherapeutics is their fast elimination from the body preventing sufficient accumulation of radioactivity in the malignancies due to glomerular filtration and fast renal clearance; high accumulation of radioactivity in the kidneys increases at the same time the risk of nephrotoxicity. For this reason, new strategies to allow a higher translation from preclinical to clinical phase of small molecule-based radiopharmaceuticals should be developed. In this thesis project two strategies to improve TRT with small molecules were developed. The first relies on the development of radioligands with a serum protein-binding domain. This strategy is based on the consideration that serum proteins have high molecular weight, and for this reason do not undergo glomerular filtration (filtration cut-off 30-50 kDa). In particular, three serum proteins-binding compounds were radiolabeled and characterized in vitro and in vivo (chapter 5). Another strategy to improve TRT efficacy while decreasing side effects is the use of combined-modality regimens. This approach is very popular in cancer therapy since targeting the cancer cell on diverse molecular path mechanisms makes the cell less prone to developing resistance to the therapy. At the same time, the use of multimodality therapies allows to decrease the dosage of single therapies, thus reducing side-effects. In the last chapter of this thesis (chapter 6) a preliminary study combining radionuclide therapy (177Lu-cm10) with two tyrosine kinases inhibitors is described, based on the assumptions of a possible synergistic effect of the two agents

Preclinical investigations using [177Lu]Lu-Ibu-DAB-PSMA toward its clinical translation for radioligand therapy of prostate cancer

[$^{177}$Lu]Lu-Ibu-DAB-PSMA was previously characterized with moderate albumin-binding properties enabling high tumor accumulation but reasonably low retention in the blood. The aim of this study was to investigate [$^{177}$Lu]Lu-Ibu-DAB-PSMA in preclinical in vivo experiments and compare its therapeutic efficacy and potential undesired side effects with those of [$^{177}$Lu]Lu-PSMA-617 and the previously developed [$^{177}$Lu]Lu-PSMA-ALB-56. BALB/c nude mice without tumors were investigated on Day 10 and 28 after injection of 10 MBq radioligand. It was revealed that most plasma parameters were in the same range for all groups of mice and histopathological examinations of healthy tissue did not show any alternations in treated mice as compared to untreated controls. Based on these results, a therapy study over twelve weeks was conducted with PC-3 PIP tumor-bearing mice for comparison of the radioligands’s therapeutic efficacy up to an activity of 10 MBq (1 nmol) per mouse. In agreement with the increased mean absorbed tumor dose, [$^{177}$Lu]Lu-Ibu-DAB-PSMA (~ 6.6 Gy/MBq) was more effective to inhibit tumor growth than [$^{177}$Lu]Lu-PSMA-617 (~ 4.5 Gy/MBq) and only moderately less potent than [$^{177}$Lu]Lu-PSMA-ALB-56 (~ 8.1 Gy/MBq). As a result, the survival of mice treated with 2 MBq of an albumin-binding radioligand was significantly increased (p < 0.05) compared to that of mice injected with [$^{177}$Lu]Lu-PSMA-617 or untreated controls. The majority of mice treated with 5 MBq or 10 MBq [$^{177}$Lu]Lu-Ibu-DAB-PSMA or [$^{177}$Lu]Lu-PSMA-ALB-56 were still alive at study end. Hemograms of immunocompetent mice injected with 30 MBq [$^{177}$Lu]Lu-Ibu-DAB-PSMA or 30 MBq [$^{177}$Lu]Lu-PSMA-617 showed values in the same range as untreated controls. This was, however, not the case for mice treated with [$^{177}$Lu]Lu-PSMA-ALB-56 which revealed a drop in lymphocytes and hemoglobin at Day 10 and Day 28 after injection. The data of this study demonstrated a significant therapeutic advantage of [$^{177}$Lu]Lu-Ibu-DAB-PSMA over [$^{177}$Lu]Lu-PSMA-617 and a more favorable safety profile as compared to that of [$^{177}$Lu]Lu-PSMA-ALB-56. Based on these results, [$^{177}$Lu]Lu-Ibu-DAB-PSMA may has the potential for a clinical translation

Early Evaluation of Copper Radioisotope Production at ISOLPHARM

The ISOLPHARM (ISOL technique for radioPHARMaceuticals) project is dedicated to the development of high purity radiopharmaceuticals exploiting the radionuclides producible with the future Selective Production of Exotic Species (SPES) Isotope Separation On-Line (ISOL) facility at the Legnaro National Laboratories of the Italian National Institute for Nuclear Physics (INFN-LNL). At SPES, a proton beam (up to 70 MeV) extracted from a cyclotron will directly impinge a primary target, where the produced isotopes are released thanks to the high working temperatures (2000 \ub0C), ionized, extracted and accelerated, and finally, after mass separation, only the desired nuclei are collected on a secondary target, free from isotopic contaminants that decrease their specific activity. A case study for such project is the evaluation of the feasibility of the ISOL production of 64Cu and 67Cu using a zirconium germanide target, currently under development. The producible activities of 64Cu and 67Cu were calculated by means of the Monte Carlo code FLUKA, whereas dedicated off-line tests with stable beams were performed at LNL to evaluate the capability to ionize and recover isotopically pure copper

Combination of terbium-161 with somatostatin receptor antagonists—a potential paradigm shift for the treatment of neuroendocrine neoplasms

Purpose: The β¯-emitting terbium-161 also emits conversion and Auger electrons, which are believed to be effective in killing single cancer cells. Terbium-161 was applied with somatostatin receptor (SSTR) agonists that localize in the cytoplasm (DOTATOC) and cellular nucleus (DOTATOC-NLS) or with a SSTR antagonist that localizes at the cell membrane (DOTA-LM3). The aim was to identify the most favorable peptide/terbium-161 combination for the treatment of neuroendocrine neoplasms (NENs). Methods: The capability of the 161Tb- and 177Lu-labeled somatostatin (SST) analogues to reduce viability and survival of SSTR-positive AR42J tumor cells was investigated in vitro. The radiopeptides' tissue distribution profiles were assessed in tumor-bearing mice. The efficacy of terbium-161 compared to lutetium-177 was investigated in therapy studies in mice using DOTATOC or DOTA-LM3, respectively. Results: In vitro, [161Tb]Tb-DOTA-LM3 was 102-fold more potent than [177Lu]Lu-DOTA-LM3; however, 161Tb-labeled DOTATOC and DOTATOC-NLS were only 4- to fivefold more effective inhibiting tumor cell viability than their 177Lu-labeled counterparts. This result was confirmed in vivo and demonstrated that [161Tb]Tb-DOTA-LM3 was significantly more effective in delaying tumor growth than [177Lu]Lu-DOTA-LM3, thereby, prolonging survival of the mice. A therapeutic advantage of terbium-161 over lutetium-177 was also manifest when applied with DOTATOC. Since the nuclear localizing sequence (NLS) compromised the in vivo tissue distribution of DOTATOC-NLS, it was not used for therapy. Conclusion: The use of membrane-localizing DOTA-LM3 was beneficial and profited from the short-ranged electrons emitted by terbium-161. Based on these preclinical data, [161Tb]Tb-DOTA-LM3 may outperform the clinically employed [177Lu]Lu-DOTATOC for the treatment of patients with NENs

Pharmaceutical development of the ISOL technique for the production of radionuclides and their applications in targeted radionuclide therapy

Radiopharmaceuticals are the fundamental tool for nuclear medicine procedures, enabling imaging and therapy of cancer after administration of a radioligand. Imaging or therapy are performed with radionuclides that are bound to a ligand, which selectively accumulates into cancer tissues. The discovery of new radionuclides on one side and the optimization of ligands on the other are fundamental parts of the research in the radiopharmaceutical field. In the first part of this thesis work we aimed at the development of a new technique for the production of pure radionuclides. The study was born from the collaboration between the Department of Pharmaceutical and Pharmacological Sciences (DSF) of the University of Padua (UniPD) and the Legnaro National Laboratories (LNL) of the National Institute for Nuclear Physics (INFN), where the Selective Production of Exotic Species (SPES) project is under development. This project aims at the production of Radioactive Ion Beams (RIBs) by means of the Isotope Separation On-Line technique (ISOL), which can be exploited for radionuclides production. Thanks to mass separation, a feature of the ISOL technique, radionuclides can be produced at high purity because isotopic contaminants can be cleared away easily. Suitable secondary targets (chapter 2) were developed to allow ion beams collection, depending on the accelerated ions. To verify the suitability of the method for radionuclides production, tests with accelerated stable ion beams were carried out and are described in chapter 3. The SPES test bench was used for the elements ionization and acceleration. The beams were collected on the produced secondary targets and quantified. The tests were carried out for yttrium, iodine and copper, elements interesting for the production of 90Y, 125/127/131I and 64/67Cu. The ISOL method guarantees the purification of the radionuclides from the isotopic contaminants, but isobaric and pseudo-isobaric contaminants can be present in the beam and thus in the final solution. For this reason chemical purification methods have to be developed for radionuclides purification. In chapter 4 the synthesis of an inorganic material (sodium nonatitanate) and its use for strontium and yttrium purification is described. The second part of this thesis, chapters 5 and 6, was developed in collaboration with the group of PD Dr. Cristina Müller at the Center for Radiopharmaceutical Sciences (CRS) of Paul Scherrer Institute (PSI). Targeted radionuclide therapy (TRT) represents one of the most promising therapeutic strategies for cancer therapy ; it is based on the use of energetic particles (alpha, beta- or Auger electrons) selectively delivered in proximity of tumors thanks to specific biologic features. Small molecules are an attractive strategy for active targeted delivery because they are amenable for chemical synthesis and have some advantages if compared with high molecular weight vehicles, such as antibodies. A potential challenge of small molecular radiotherapeutics is their fast elimination from the body preventing sufficient accumulation of radioactivity in the malignancies due to glomerular filtration and fast renal clearance; high accumulation of radioactivity in the kidneys increases at the same time the risk of nephrotoxicity. For this reason, new strategies to allow a higher translation from preclinical to clinical phase of small molecule-based radiopharmaceuticals should be developed. In this thesis project two strategies to improve TRT with small molecules were developed. The first relies on the development of radioligands with a serum protein-binding domain. This strategy is based on the consideration that serum proteins have high molecular weight, and for this reason do not undergo glomerular filtration (filtration cut-off 30-50 kDa). In particular, three serum proteins-binding compounds were radiolabeled and characterized in vitro and in vivo (chapter 5). Another strategy to improve TRT efficacy while decreasing side effects is the use of combined-modality regimens. This approach is very popular in cancer therapy since targeting the cancer cell on diverse molecular path mechanisms makes the cell less prone to developing resistance to the therapy. At the same time, the use of multimodality therapies allows to decrease the dosage of single therapies, thus reducing side-effects. In the last chapter of this thesis (chapter 6) a preliminary study combining radionuclide therapy (177Lu-cm10) with two tyrosine kinases inhibitors is described, based on the assumptions of a possible synergistic effect of the two agents.I radiofarmaci rappresentano uno strumento fondamentale per la medicina nucleare, rendendo possibile, grazie alla somministrazione di un radioligando, procedure diagnostiche e terapeutiche in ambito oncologico. I radiofarmaci sono costituiti da un radionuclide e da un ligando; quest’ultimo permette la localizzazione della radioattività solamente nei tessuti bersaglio. La ricerca in ambito radiofarmaceutico è dedicata sia alla scoperta di nuovi radionuclidi, sia all’ottimizzazione dei ligandi. Scopo della prima parte di questo lavoro di tesi è stato lo studio e lo sviluppo di una tecnica innovativa per la produzione di radionuclidi ad alta purezza per la medicina nucleare. Lo studio è nato dalla collaborazione presente tra il Dipartimento di Scienze del Farmaco (DSF) dell’Università di Padova (UniPD) e i Laboratori Nazionali di Legnaro (LNL) dell’Istituto Nazionale di Fisica Nucleare (INFN). Presso questi ultimi, infatti, è in fase di costruzione il progetto Selective Production of Exotic Species (SPES), che prevede la produzione di fasci di ioni radioattivi (RIBs), prodotti tramite la tecnologia Isotope Separation On Line (ISOL). La separazione in massa prevista dalla tecnica ISOL, la rende un’ottima via di produzione di radionuclidi, perché permette di eliminare tutti i contaminanti isotopici del radionuclide d’interesse. Per la deposizione dei fasci sono stati sviluppati e costruiti dei bersagli secondari; questi oggetti, posizionati alla fine della linea di fascio, sono utilizzati per depositare e recuperare gli ioni accelerati. La progettazione e realizzazione di tali bersagli è descritta nel capitolo 2. In seguito, con lo scopo di verificare la possibilità di utilizzare l’apparato sperimentale di SPES per la produzione di radionuclidi per la medicina nucleare, sono stati condotti dei test utilizzando fasci di ioni stabili degli elementi di interesse, in particolare sono stati studiati fasci di ittrio, iodio e rame, ai fini della produzione futura di 90Y, 125/127/131I e 64/67Cu. Il metodo ISOL consente grazie alla separazione in massa di purificare i radionuclidi dai contaminanti isotopici, ma contaminanti isobarici e pseudo-isobarici devono essere rimossi tramite altri metodi, in particolare tramite metodi chimici. Per questo motivo nel capitolo 4 vengono descritti la sintesi e l’uso di una resina inorganica a scambio ionico per la purificazione di ittrio e stronzio. La seconda parte di questa tesi è stata sviluppata in collaborazione con il Center for Radiopharmaceutcal Sciences (CRS) presso il Paul Scherrer Institute (PSI), Villigen (CH), in particolare nel gruppo della Dott.ssa PD Cristina Müller. La targeted radionuclide therapy (TRT) è un strategia terapeutica molto promettente in campo oncologico e si basa sull’uso radionuclidi che emottono particelle ad energia medio-alta (alfa, beta- o elettroni di Auger) che vengono selettivamente direzionati nel tessuto canceroso in seguito al riconoscimento di una particolare caratteristica della cellula tumorale. L’utilizzo di piccole molecole per il direzionamento dei radionuclidi rappresenta una strategia vantaggiosa rispetto all’uso di veicoli ad alto peso molecolare (come gli anticorpi monoclonali) ed inoltre possono essere prodotte tramite sintesi chimica. Tuttavia, nonostante il successo di alcune small molecules in clinica, molte volte questa tipologia di radiofarmaci va incontro ad una veloce eliminazione ematica a causa della loro filtrazione glomerulare. In questo modo l’acccumulo della radioattivià nel tumore è diminuito, mentre può esserci un accumulo indesiderato nei reni con aumentato rischio di nefrotossicità. In questa tesi vengono descritte due strategie per il miglioramento della TRT basata sull’uso di piccole molecole. La prima prevede la modifica della struttura chimica dei ligandi con un dominio di legame alle proteine plasmatiche. Questa strategia permette, infatti, di evitare la filtrazione glomerulare del radioligando. Questo, se dopo la somministrazione si lega in modo reversibile ad una proteina plasmatica, viene ad assumere un peso molecolare >30 kDa, valore del cut-off della filtrazione nel glomerulo ed aumenta per questo motivo la sua emivita plasmatica. Nel capitolo 5 vengono descritte la radiomarcatura e la caratterizzazione in vitro ed in vivo di tre radioligandi con affinità per le proteine plasmatiche. La seconda strategia è quella dello sviluppo di terapie combinate con altri agenti chemioterapeutici. Questo approccio è molto popolare in quanto permette da una parte di ridurre le dosi dei singoli agenti e ridurre così gli effetti collaterali, dall’altra di ridurre la resistenza alla terapia. Nel capitolo 6 della tesi, è descritto lo studio preliminare dell’associazione di un radioligando per la terapia (177Lu-cm10) con due inibitori tirosin chinasici

Research and technological development of beta-emitting radiopharmaceuticals produced with radioactive ion beams

An innovative method is proposed for the production of carrier-free \uf062- radiopharmaceuticals, based on the Isotope Separation On-Line (ISOL) technique in the framework of the SPES (Selective Production of the Exotic Species) project. SPES is a nuclear physics project under advanced construction at Legnaro National Laboratories of INFN (Istituto Nazionale di Fisica Nucleare). The innovation of the method relies not only on the new physical technique but mainly on the final product. The class of radiopharmaceutical obtainable, indeed, can be considered completely new, since they have a high purity in terms of specific activity (5 orders of magnitude bigger than traditional ones); specific activity is the ratio between radioactivity and mass of the element

Impact of the mouse model and molar amount of injected ligand on the tissue distribution profile of PSMA radioligands

Purpose Various preclinical study designs are described in the literature for the evaluation of PSMA radioligands. In this study, [177Lu]Lu-Ibu-DAB-PSMA, an albumin-binding radioligand, and [177Lu]Lu-PSMA-617 were investigated and compared under variable experimental conditions. Methods In vitro cell uptake studies were performed with PC-3 PIP and LNCaP tumor cells using a range of molar concentrations (0.75–500 nM) of both radioligands. Biodistribution and SPECT/CT imaging studies were carried out with the respective tumor mouse models using 0.05 nmol and 1.0 nmol injected ligand per mouse. Results In both tumor cell lines, the uptake of the radioligands was increased when using low molar concentrations of the respective ligand. The observed saturation effect at high ligand concentrations was more pronounced for LNCaP cells that express PSMA at lower levels than for PC-3 PIP cells. At all investigated timepoints, the in vivo uptake of both radioligands was higher in PC-3 PIP tumors than in LNCaP tumors. A low molar amount of injected ligand increased the PC-3 PIP tumor uptake mainly for [177Lu]Lu-Ibu-DAB-PSMA; however, the molar amount of ligand was relevant for both radioligands when using LNCaP tumors. Renal retention of both radioligands was, however, up to fourfold higher during the first hours after application of a low ligand amount compared to the high ligand amount. Conclusion The results of this preclinical study underline the relevance of the tumor model and applied ligand amount for the characterization of PSMA radioligands. The application of equal preclinical study designs is crucial to allow the comparison of novel radioligands with existing ones and, thus, predict potential advantages of new radioligands in view of a clinical application.ISSN:1619-7070ISSN:1619-708