Translational assessment of a DATA-functionalized FAP inhibitor with facile 68Ga-labeling at room temperature.

PURPOSE The present study aims at evaluating the preclinical and the clinical performance of [68Ga]Ga-DATA5m.SA.FAPi, which has the advantage to be labeled with gallium-68 at room temperature. METHODS [68Ga]Ga-DATA5m.SA.FAPi was assessed in vitro on FAP-expressing stromal cells, followed by biodistribution and in vivo imaging on prostate and glioblastoma xenografts. Moreover, the clinical assessment of [68Ga]Ga-DATA5m.SA.FAPi was conducted on six patients with prostate cancer, aiming on investigating, biodistribution, biokinetics, and determining tumor uptake. RESULTS [68Ga]Ga-DATA5m.SA.FAPi is quantitatively prepared in an instant kit-type version at room temperature. It demonstrated high stability in human serum, affinity for FAP in the low nanomolar range, and high internalization rate when associated with CAFs. Biodistribution and PET studies in prostate and glioblastoma xenografts revealed high and specific tumor uptake. Elimination of the radiotracer mainly occurred through the urinary tract. The clinical data are in accordance with the preclinical data concerning the organ receiving the highest absorbed dose (urinary bladder wall, heart wall, spleen, and kidneys). Different to the small-animal data, uptake of [68Ga]Ga-DATA5m.SA.FAPi in tumor lesions is rapid and stable and tumor-to-organ and tumor-to-blood uptake ratios are high. CONCLUSION The radiochemical, preclinical, and clinical data obtained in this study strongly support further development of [68Ga]Ga-DATA5m.SA.FAPi as a diagnostic tool for FAP imaging

Study and biological evaluation of radiolabeled bombesin like peptides

Bombesin (BN) is a 14-amino acid neuropeptide with high affinity and specificity for the gastrin-releasing-peptide (GRP) receptors. Specific receptors for BN are expressed on a variety of human tumors including prostate, gastric, colon, breast, lung and pancreatic cancers. Aim of the present study is the evaluation of the 99mTc-complexes of the two following new Bombesin (BN)-like peptides: Gly-Gly-Cys-Aca-BN[2-14], (BN1.1), and Gly-Gly-Cys-Aca-BN[7-14] (BN1.1p), where Aca: 6-amino-hexanoic acid. Pyroglutamic acid in the bombesin molecule has been replaced by the chemical moiety Gly-Gly-Cys-Aca, which bears an amino-acid combination capable of complexing radiometals. The new BN derivatives were synthesized according to the SPPS method, purified and characterized by analytical methods. The complexes 185/187Re-BN-1.1 and 185/187Re-BN-1.1p were formed via the precursor rhenium gluconate and were identified by analytical methods. 99mTc-labeling was performed via the precursor 99mTc-gluconate. Chromatographic analysis showed for both derivatives that 99mTc-labeling led to the formation of a single radioactive species in high yield (>98%). The stability of the radiolabeled derivatives obtained was examined with time and in human plasma. Metabolic studies were performed in kidney and liver homogenates. Both 99mTc-BN1.1 and 99mTc-BN1.1p were practically stable with time and in human plasma, whereas they degraded rapidly in kidney and liver homogenates, as indicated by metabolic studies. IC50 values were determined, comparatively, for the BN-derivatives, the respective185/187Re complexes and Tyr4-BN. Both unlabeled and labeled peptides demonstrated high binding affinity for the human prostate adenocarcinoma PC-3 cell line, which over-expresses bombesin receptors. Internalization was investigated in PC-3 cells at 37 0C. The degree of the residualization of 99mTc into the cells was examined with time. The rate of internalization of the labeled peptides was found to be time-dependent. Despite this high internalization rate, there was no longer-term retention of radioactivity in the cells. Tissue distribution of the radiopeptides was evaluated in normal mice and in prostate cancer experimental models. Significant uptake of radioactivity was observed in the pancreas of normal mice. Receptor-blocking studies with native BN confirmed the specificity of 99mTc-BN1.1 and 99mTc-BN1.1p toward the GRP receptors. Excretion took place mainly via the urinary system for 99mTc-BN1.1 and through the hepatobiliary tract in the case of 99mTc-BN1.1p. Satisfactory tumor images were obtained with both radiolabeled peptides. The above preliminary results indicate that these new Bombesin derivatives are promising for human cancer detection.Η μπομπεσίνη (BN) είναι ένα δεκατετραπεπτίδιο που παρουσιάζει μεγάλη συγγένεια και εξειδίκευση για τους υποδοχείς του πεπτιδίου απελευθέρωσης της γαστρίνης (GRP). Υποδοχείς ειδικοί για την μπομπεσίνη εκφράζονται σε πολλούς ανθρώπινους καρκινικούς όγκους, όπως για παράδειγμα σε καρκίνο του προστάτη, του παχέος εντέρου, του μαστού, των πνευμόνων, καθώς και σε καρκίνο του παγκρέατος. Σκοπός της παρούσας εργασίας είναι η μελέτη δυο νέων πεπτιδικών παραγώγων της μπομπεσίνης επισημασμένων με 99mTc. Η αμινοξική αλληλουχία των νέων αυτών πεπτιδικών παραγώγων είναι: Gly-Gly-Cys-Aca-BN[2-14], (BN1.1) και Gly-Gly-Cys-Aca-BN[7-14] (BN1.1p), όπου Aca: το 6-άμινο εξανονοϊκο οξύ. Το πυρογλουταμικό οξύ στο μόριο της μπομπεσίνης έχει αντικατασταθεί και στις δυο περιπτώσεις από την αλληλουχία αμινοξέων Gly-Gly-Cys-Aca, η οποία διαθέτει κατάλληλα άτομα δότες για την σύμπλεξη με ραδιομέταλλα. Η σύνθεση των νέων πεπτιδικών παραγώγων πραγματοποιήθηκε σε στερεά φάση (SPPS) και ο καθαρισμός-χαρακτηρισμός τους έγινε με αναλυτικές μεθόδους. Ο σχηματισμός των συμπλόκων 185/187Re-BN-1.1 και 185/187Re-BN-1.1p έγινε μέσω του προδρόμου συμπλόκου γλυκονικό-ρήνιο και ο χαρακτηρισμός του πραγματοποιήθηκε με αναλυτικές μεθόδους. Η επισήμανση τους με 99mTc έγινε μέσω του προδρόμου συμπλόκου γλυκονικό-99mTc και οδήγησε στο σχηματισμό ενός μόνο επισημασμένου παραγώγου, σε κάθε περίπτωση, με ποσοστό επισήμανσης >98%. Η σταθερότητα των επισημασμένων προϊόντων εξετάσθηκε με την πάροδο του χρόνου, καθώς και σε παρουσία ανθρώπινου πλάσματος. Μεταβολικές μελέτες πραγματοποιήθηκαν σε ομογενοποίημα νεφρών και ήπατος. Τα 99mTc-BN1.1 και 99mTc-BN1.1p είναι πρακτικά σταθερά με τον χρόνο, καθώς και σε παρουσία ανθρώπινου πλάσματος, ενώ αποικοδομούνται ταχύτατα σε ομογενοποίημα νεφρών και ήπατος. Υπολογίστηκαν οι τιμές IC50 των νέων πεπτιδίων, καθώς και των συμπλόκων τους με 185/187Re, συγκριτικά με την Tyr4-BN. Τόσο τα πεπτίδια όσο και τα σύμπλοκά τους με 185/187Re παρουσιάζουν μεγάλη συγγένεια για τους υποδοχείς της μπομπεσίνης. Πραγματοποιήθηκαν μελέτες εσωτερικοποίησης και ελευθέρωσης του 99mTc, χρησιμοποιώντας την καρκινική σειρά PC-3, στους 37 0C. Ο βαθμός εσωτερικοποίησης των επισημασμένων πεπτιδίων στα καρκινικά κύτταρα εξαρτάται από τον χρόνο. Παρόλο που τα επισημασμένα πεπτίδια εσωτερικοποιούνται σε μεγάλο ποσοστό δεν παρατηρείται διατήρηση της ραδιενέργειας μέσα στα καρκινικά κύτταρα για μεγάλο χρονικό διάστημα. Μελέτες βιοκατανομής των επισημασμένων πεπτιδίων πραγματοποιήθηκαν σε φυσιολογικά πειραματόζωα και παθολογικά πρότυπα καρκίνου του προστάτη. Σημαντικό ποσοστό πρόσληψης της ραδιενέργειας παρατηρείται στο πάγκρεας των φυσιολογικών πειραματόζωων. Πειράματα ταυτόχρονης χορήγησης φυσικής μπομπεσίνης και 99mTc-BN1.1 και 99mTc-BN1.1p επιβεβαίωσαν την εξειδίκευση των ραδιοπεπτιδίων για τους υποδοχείς του GRP. Η αποβολή έγινε κυρίως από το ουροποιητικό σύστημα για το 99mTc-BN1.1, ενώ από το ηπατοχολοκό στην περίπτωση του 99mTc-BN1.1p. Ικανοποιητικές απεικονίσεις των όγκων λήφθηκαν και στις δυο περιπτώσεις των επισημασμένων πεπτιδίων. Τα παραπάνω αρχικά αποτελέσματα δείχνουν ότι τα δυο αυτά νέα παράγωγα της μπομπεσίνης είναι πολλά υποσχόμενα ως διαγνωστικά ραδιοφάρμακα

Radiochemistry and Preclinical PET Imaging of 68Ga-Desferrioxamine Radiotracers Targeting Prostate-Specific Membrane Antigen

Radiotracers incorporating the urea-based Glu-NH-C(O)-NH-Lys group have gained prominence due to their role in targeting prostate-specific membrane antigen (PSMA)—a clinical biomarker of prostate cancer. Here, the synthesis, radiolabeling, and in vitro and in vivo characterization of two 68Ga-radiolabeled Glu-NH-C(O)-NH-Lys radiotracers conjugated to the desferrioxamine B (DFO) chelate were evaluated. Two linker groups based on amide bond and thiourea coupling chemistries were employed to develop 68Ga-DFO-Nsucc-PSMA (68Ga-4) and 68Ga-DFO-pNCS-Bn-PSMA (68Ga-7), respectively. Radiosynthesis proceeded quantitatively at room temperature with high radiochemical yields, chemical/radiochemical purities, and specific activities. Pharmacokinetic profiles of 68Ga-4 and 68Ga-7 were assessed using positron-emission tomography (PET) in mice bearing subcutaneous LNCaP tumors. Data were compared to the current clinical benchmark radiotracer 68Ga-HBED-CC-PSMA (68Ga-1) (HBED ¼ N,N0-Bis(2-hydroxy-5-(ethylene-beta-carboxy)benzyl)ethylenediamine N,N0-diacetic acid). Results indicated that the target binding affinity, protein association, blood pool and background organ clearance properties, and uptake in PSMApositive lesions are strongly dependent on the nature of the chelate, the linker, and the spacer groups. Protein dissociation constants (Kd values) were found to be predictive of pharmacokinetics in vivo. Compared to 68Ga-1, 68Ga-4 and 68Ga-7 resulted in decreased tumor uptake but enhanced blood pool clearance and reduced residence time in the kidney. The study highlights the importance of maximizing protein binding affinity during radiotracer optimization

Metal-Based PSMA Radioligands

Prostate cancer is one of the most common malignancies for which great progress has been made in identifying appropriate molecular targets that would enable efficient in vivo targeting for imaging and therapy. The type II integral membrane protein, prostate specific membrane antigen (PSMA) is overexpressed on prostate cancer cells in proportion to the stage and grade of the tumor progression, especially in androgen-independent, advanced and metastatic disease, rendering it a promising diagnostic and/or therapeutic target. From the perspective of nuclear medicine, PSMA-based radioligands may significantly impact the management of patients who suffer from prostate cancer. For that purpose, chelating-based PSMA-specific ligands have been labeled with various diagnostic and/or therapeutic radiometals for single-photon-emission tomography (SPECT), positron-emission-tomography (PET), radionuclide targeted therapy as well as intraoperative applications. This review focuses on the development and further applications of metal-based PSMA radioligands

GRPR-targeting radiotheranostics for breast cancer management

Breast Cancer (BC) is the most common cancer worldwide and, despite the advancements made toward early diagnosis and novel treatments, there is an urgent need to reduce its mortality. The Gastrin-Releasing Peptide Receptor (GRPR) is a promising target for the development of theranostic radioligands for luminal BC with positive estrogen receptor (ER) expression, because GRPR is expressed not only in primary lesions but also in lymph nodes and distant metastasis. In the last decades, several GRPR-targeting molecules have been evaluated both at preclinical and clinical level, however, most of the studies have been focused on prostate cancer (PC). Nonetheless, given the relevance of non-invasive diagnosis and potential treatment of BC through Peptide Receptor Radioligand Therapy (PRRT), this review aims at collecting the available preclinical and clinical data on GRPR-targeting radiopeptides for the imaging and therapy of BC, to better understand the current state-of-the-art and identify future perspectives and possible limitations to their clinical translation. In fact, since luminal-like tumors account for approximately 80% of all BC, many BC patients are likely to benefit from the development of GRPR-radiotheranostics

Effect of the versatile bifunctional chelator AAZTA5 on the radiometal labelling properties and the in vitro performance of a gastrin releasing peptide receptor antagonist.

BACKGROUND Gastrin Releasing Peptide receptor (GRPr)-based radioligands have shown great promise for diagnostic imaging of GRPr-positive cancers, such as prostate and breast. The present study aims at developing and evaluating a versatile GRPr-based probe for both PET/SPECT imaging as well as intraoperative and therapeutic applications. The influence of the versatile chelator AAZTA5 on the radiometal labelling properties and the in vitro performance of the generated radiotracers were thoroughly investigated. The GRPr-based antagonist D-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH2 was functionalized with the chelator 6-[Bis (carboxymethyl)amino]-1,4-bis (carboyxmethyl)-6-methyl-1,4-diazepane (AAZTA5) through the spacer 4-amino-1-carboxymethyl-piperidine (Pip) to obtain AAZTA5-Pip-D-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH2 (LF1). LF1 was radiolabelled with gallium-68 (PET), indium-111 (SPECT, intraoperative applications) and lutetium-177 (therapy, SPECT). In vitro evaluation included stability studies, determination of lipophilicity, protein-binding studies, determination of Kd and Bmax as well as internalization studies using the epithelial human prostate cancer cell line PC3. In vitro monotherapy as well as combination therapy studies were further performed to assess its applicability as a theranostic compound. RESULTS LF1 was labelled with gallium-68, indium-111 and lutetium-177 within 5 min at room temperature (RT). The apparent molar activities (Am) were ranging between 50 and 60 GBq/μmol for the 68Ga-labelled LF1, 10-20 GBq/μmol for the 111In- and 177Lu-labelled LF1. The radiotracers were stable for a period of 4 h post labeling exhibiting a hydrophilic profile with an average of a LogDoctanol/PBS of - 3, while the bound activity to the human serum protein was approximately 10%. 68/natGa-LF1, 177/natLu-LF1 and 111/natIn-LF1 exhibited high affinity for the PC3 cells, with Kd values of 16.3 ± 2.4 nM, 10.3 ± 2.73 nM and 5.2 ± 1.9 nM, respectively, and the required concentration of the radiotracers to saturate the receptors (Bmax) was between 0.5 and 0.8 nM which corresponds to approximately 4 × 105 receptors per cell. Low specific internalization rate was found in cell culture, while the total specific cell surface bound uptake always exceeded the internalized activity. In vitro therapy studies showed that inhibition of PC3 cells growth is somewhat more efficient when combination of 177Lu-labelled LF1 with rapamycin is applied compared to 177Lu-laballed LF1 alone. CONCLUSION Encouraged by these promising in vitro data, preclinical evaluation of the LF1 precursor are planned in tumour models in vivo

The Glucose-Dependent Insulinotropic Polypeptide Receptor: A Novel Target for Neuroendocrine Tumor Imaging-First Preclinical Studies

A new family of peptide receptors, the incretin receptor family, overexpressed on many neuroendocrine tumors (NETs) is of great importance because it may enable the in vivo peptide-based receptor targeting of a category of NETs that does not express the somatostatin receptor. Impressive in vivo diagnostic data were published for glucagonlike peptide 1 receptor-targeting radiopeptides. Recently, promising in vitro data have appeared for the second member of the incretin family, the glucose-dependent insulinotropic polypeptide (GIP) receptor. This prompted us to develop and evaluate a new class of radioligands with the potential to be used for the in vivo targeting of GIP receptor-positive tumors. METHODS GIP(1-42) was modified C-terminally, and the truncated peptides [Lys(30)(aminohexanoic acid [Ahx]-DOTA)]GIP(1-30)NH2 (EG1), [Lys(16)(Ahx-DOTA)]GIP(1-30)NH2 (EG2), and [Nle(14), Lys(30)(Ahx-DOTA)]GIP(1-30)NH2 (EG4) were conjugated with Ahx-DOTA via the Lys(16) and Lys(30) side chains. Their inhibitory concentration of 50% (IC50) was determined using [(125)I-Tyr(10)]GIP(1-30) as radioligand and GIP(1-30) as control peptide. The DOTA conjugates were labeled with (111)In and (68)Ga. In vitro evaluation included saturation and internalization studies using the pancreatic endocrine cell line INR1G9 transfected with the human GIP receptor (INR1G9-hGIPr). The in vivo evaluation consisted of biodistribution and PET imaging studies on nude mice bearing INR1G9-hGIPr tumors. RESULTS Binding studies (IC50 and saturation studies) showed high affinity toward GIP receptor for the GIP conjugates. Specific in vitro internalization was found, and almost the entire cell-associated activity was internalized (>90% of the cell-bound activity), supporting the agonist potency of the (111)In-vectors. (111)In-EG4 and (68)Ga-EG4 were shown to specifically target INR1G9-hGIPr xenografts, with tumor uptake of 10.4% ± 2.2% and 17.0% ± 4.4% injected activity/g, 1 h after injection, respectively. Kidneys showed the highest uptake, which could be reduced by approximately 40%-50% with a modified-fluid-gelatin plasma substitute or an inhibitor of the serine protease dipeptidyl peptidase 4. The PET images clearly visualized the tumor. CONCLUSION The evaluation of EG4 as a proof-of-principle radioligand indicated the feasibility of imaging GIP receptor-positive tumors. These results prompt us to continue the development of this family of radioligands for imaging of a broad spectrum of NETs

AMD3100: A Versatile Platform for CXCR4 Targeting 68 Ga-Based Radiopharmaceuticals

International audienceCXCR4 is a G protein-coupled receptor (GPCR), which is overexpressed in numerous diseases, particularly in multiple cancers. Therefore, this receptor represents a valuable target for imaging and therapeutic purposes. Among the different approaches, which were developed for CXCR4 imaging, a CXCR4 antagonist biscyclam system (AMD3100, also called Mozobil), currently used in the clinic for the mobilization of hematopoietic stem cells, was radiolabeled with different radiometals such as 62Zn, 64Cu, 67Ga, or 99mTc. However, cyclam is not an ideal chelator for most of these radiometals, and could lead to the release of the radionuclide in vivo. In the current study, a new family of CXCR4 imaging agents is presented, in which AMD3100 is used as a carrier for specific delivery of an imaging reporter, i.e., a 68Ga complex for PET imaging. AMD3100 was functionalized on the phenyl moiety with different linkers, either ethylenediamine or diamino-polyethylene glycol 3 (PEG3). The resulting AMD3100 analogues were further coupled with two different chelators, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) or 1,4,7-triazacyclononane-1-glutaric acid-4,7-acetic acid (NODAGA). Five potential CXCR4 targeting agents were obtained. The derived AMD3100-based ligands were labeled with 68Ga, highlighting the influence of the spacer nature on the 68Ga-labeling yield. The lipophilic character of the different systems was also investigated, as well as their affinity for the CXCR4 receptor. The most promising compound was further evaluated in vivo in H69 tumor xenografts by biodistribution and PET imaging studies, validating the proof of principle of our concept