Neurotensin (8-13) and Neuromedin N Neuropeptides Radiolabelling with Copper-64 Produced on Solid or Liquid Targets.

On the verge of a theranostic approach to personalised medicine, copper-64 is one of the emerging radioisotopes in nuclear medicine due to its exploitable nuclear and biochemical characteristics. The increased demand for copper-64 for preclinical and clinical studies has prompted the development of production routes. This research aims to compare the (p,n) reaction on nickel-64 solid versus liquid targets and evaluate the effectiveness of [64Cu]CuCl2 solutions prepared by the two routes. As new treatments for neurotensin receptor-overexpressing tumours have developed, copper-64 was used to radiolabel Neurotensin (8-13) and Neuromedin N. High-quality [64Cu]CuCl2 solutions were prepared using ACSI TR-19 and IBA Cyclone Kiube cyclotrons. The radiochemical purity after post-irradiation processing reached 99% (LT) and 99.99% (ST), respectively. The irradiation of a solid target with 11.8 MeV protons and 150 µAh led to 704 ± 84 MBq/µA (17.6 ± 2.1 GBq/batch at EOB). At the end of the purification process (1 h, 90.90% activity yield), the solution for peptide radiolabelling had a radioactive concentration of 1340.4 ± 70.1 MBq/mL (n.d.c.). The irradiation of a liquid target with 16.9 MeV protons and 230 µAh resulted in 3.7 ± 0.2 GBq/batch at EOB, which corresponds to an experimental production yield of 6.89 GBq.cm3/(g.µA)sat. Benefiting from a shorter purification process (40 min), the activity yielded 90.87%, while the radioactive concentration of the radiolabelling solution was lower (492 MBq/mL, n.d.c.). The [64Cu]CuCl2 solutions were successfully used for the radiolabelling of DOTA-NT(8-13) and DOTA-NN neuropeptides, resulting in a high RCP (>99%) and high molar activity (27.2 and 26.4 GBq/µmol for LT route compared to 45 and 52 GBq/µmol for ST route, respectively). The strong interaction between the [64Cu]Cu-DOTA-NT(8-13) and the colon cancerous cell lines HT29 and HCT116 proved that the specificity for NTR had not been altered, as shown by the uptake and retention data.

Fully Automated Production of [68Ga]GaFAPI-46 with Gallium-68 from Cyclotron Using Liquid Targets.

68Ga-based radiopharmaceuticals are routinely used for PET imaging of multiple types of tumors. Gallium-68 is commonly obtained from 68Ge/68Ga generators, which are limited in the quantity of activity produced. Alternatively, gallium-68 can easily be produced on a cyclotron using liquid targets. In this study, we optimized the GMP production of [68Ga]GaFAPI-46 using gallium-68 produced via a standard medical cyclotron using liquid targets. Starting from the published synthesis and quality control procedures described for other 68Ga-based radiopharmaceuticals, we have validated the synthesis process and the analytical methods to test the quality parameters of the final product to be used for routine clinical studies. [68Ga]GaFAPI-46 was successfully produced with high radiochemical purity and yield using an IBA Synthera® Extension module. Gallium chloride was produced on a medical cyclotron using a liquid target with activity of 4.31 ± 0.36 GBq at the end of purification (EOP). Analytical methods were established and validated, meeting Ph. Eur. standards. Full GMP production was also validated in three consecutive batches, producing 2.50 ± 0.46 GBq of [68Ga]GaFAPI-46 at the end of synthesis (EOS), with 98.94 ± 0.72% radiochemical purity measured via radio-HPLC. Quality was maintained for up to 3 h after the EOS. Production of [68Ga]GaFAPI-46 was performed and validated using a standard medical cyclotron with liquid targets. The quality control parameters (e.g., sterility, purity, and residual solvents) conformed to Ph. Eur. and a shelf life of 3 h was established. The activity of [68Ga]GaFAPI-46 produced was substantially higher than the one obtained with generators, enabling a better response to the clinical need for this radiopharmaceutical.

Antibody and Nanobody Radiolabeling with Copper-64: Solid vs. Liquid Target Approach.

Antibody and nanobody-based copper-64 radiopharmaceuticals are increasingly being proposed as theranostic tools in multiple human diseases. While the production of copper-64 using solid targets has been established for many years, its use is limited due to the complexity of solid target systems, which are available in only a few cyclotrons worldwide. In contrast, liquid targets, available in virtually in all cyclotrons, constitute a practical and reliable alternative. In this study, we discuss the production, purification, and radiolabeling of antibodies and nanobodies using copper-64 obtained from both solid and liquid targets. Copper-64 production from solid targets was performed on a TR-19 cyclotron with an energy of 11.7 MeV, while liquid target production was obtained by bombarding a nickel-64 solution using an IBA Cyclone Kiube cyclotron with 16.9 MeV on target. Copper-64 was purified from both solid and liquid targets and used to radiolabel NODAGA-Nb, NOTA-Nb, and DOTA-Trastuzumab conjugates. Stability studies were conducted on all radioimmunoconjugates in mouse serum, PBS, and DTPA. Irradiation of the solid target yielded 13.5 ± 0.5 GBq with a beam current of 25 ± 1.2 µA and an irradiation time of 6 h. On the other hand, irradiation of the liquid target resulted in 2.8 ± 1.3 GBq at the end of bombardment (EOB) with a beam current of 54.5 ± 7.8 µA and an irradiation time of 4.1 ± 1.3 h. Successful radiolabeling of NODAGA-Nb, NOTA-Nb, and DOTA-Trastuzumab with copper-64 from both solid and liquid targets was achieved. Specific activities (SA) obtained with the solid target were 0.11, 0.19, and 0.33 MBq/µg for NODAGA-Nb, NOTA-Nb, and DOTA-trastuzumab, respectively. For the liquid target, the corresponding SA values were 0.15, 0.12, and 0.30 MBq/µg. Furthermore, all three radiopharmaceuticals demonstrated stability under the testing conditions. While solid targets have the potential to produce significantly higher activity in a single run, the liquid process offers advantages such as speed, ease of automation, and the feasibility of back-to-back production using a medical cyclotron. In this study, successful radiolabeling of antibodies and nanobodies was achieved using both solid and liquid targets approaches. The radiolabeled compounds exhibited high radiochemical purity and specific activity, rendering them suitable for subsequent in vivo pre-clinical imaging studies.

Production of GMP-Compliant Clinical Amounts of Copper-61 Radiopharmaceuticals from Liquid Targets.

PET imaging has gained significant momentum in the last few years, especially in the area of oncology, with an increasing focus on metal radioisotopes owing to their versatile chemistry and favourable physical properties. Copper-61 (t1/2 = 3.33 h, 61% ß+, Emax = 1.216 MeV) provides unique advantages versus the current clinical standard (i.e., gallium-68) even though, until now, no clinical amounts of 61Cu-based radiopharmaceuticals, other than thiosemicarbazone-based molecules, have been produced. This study aimed to establish a routine production, using a standard medical cyclotron, for a series of widely used somatostatin analogues, currently labelled with gallium-68, that could benefit from the improved characteristics of copper-61. We describe two possible routes to produce the radiopharmaceutical precursor, either from natural zinc or enriched zinc-64 liquid targets and further synthesis of [61Cu]Cu-DOTA-NOC, [61Cu]Cu-DOTA-TOC and [61Cu]Cu-DOTA-TATE with a fully automated GMP-compliant process. The production from enriched targets leads to twice the amount of activity (3.28 ± 0.41 GBq vs. 1.84 ± 0.24 GBq at EOB) and higher radionuclidic purity (99.97% vs. 98.49% at EOB). Our results demonstrate, for the first time, that clinical doses of 61Cu-based radiopharmaceuticals can easily be obtained in centres with a typical biomedical cyclotron optimised to produce 18F-based radiopharmaceuticals.

Improved Characteristics of RANKL Immuno-PET Imaging Using Radiolabeled Antibody Fab Fragments.

PURPOSE: RANKL expression in the tumor microenvironment has been identified as a biomarker of immune suppression, negating the effect of some cancer immunotherapies. Previously we had developed a radiotracer based on the FDA-approved RANKL-specific antibody denosumab, [89Zr]Zr-DFO-denosumab, enabling successful immuno-PET imaging. Radiolabeled denosumab, however, showed long blood circulation and delayed tumor uptake, potentially limiting its applications. Here we aimed to develop a smaller radiolabeled denosumab fragment, [64Cu]Cu-NOTA-denos-Fab, that would ideally show faster tumor accumulation and better diffusion into the tumor for the visualization of RANKL. EXPERIMENTAL DESIGN: Fab fragments were prepared from denosumab using papain and conjugated to a NOTA chelator for radiolabeling with 64Cu. The bioconjugates were characterized in vitro using SDS-PAGE analysis, and the binding affinity was assessed using a radiotracer cell binding assay. Small animal PET imaging evaluated tumor targeting and biodistribution in transduced RANKL-ME-180 xenografts. RESULTS: The radiolabeling yield of [64Cu]Cu-NOTA-denos-Fab was 58 ± 9.2%, with a specific activity of 0.79 ± 0.11 MBq/µg (n = 3). A radiotracer binding assay proved specific targeting of RANKL in vitro. PET imaging showed fast blood clearance and high tumor accumulation as early as 1 h p.i. (2.14 ± 0.21% ID/mL), which peaked at 5 h p.i. (2.72 ± 0.61% ID/mL). In contrast, [64Cu]Cu-NOTA-denosumab reached its highest tumor uptake at 24 h p.i. (6.88 ± 1.12% ID/mL). [64Cu]Cu-NOTA-denos-Fab specifically targeted human RANKL in transduced ME-180 xenografts compared with the blocking group and negative ME-180 xenograft model. Histological analysis confirmed RANKL expression in RANKL-ME-180 xenografts. CONCLUSIONS: Here, we report on a novel RANKL PET imaging agent, [64Cu]Cu-NOTA-denos-Fab, that allows for fast tumor imaging with improved imaging contrast when compared with its antibody counterpart, showing promise as a potential PET RANKL imaging tool for future clinical applications.

Nanobody-Based Theranostic Agents for HER2-Positive Breast Cancer: Radiolabeling Strategies.

The overexpression of human epidermal growth factor 2 (HER2) in breast cancer (BC) has been associated with a more aggressive tumor subtype, poorer prognosis and shorter overall survival. In this context, the development of HER2-targeted radiotracers is crucial to provide a non-invasive assessment of HER2 expression to select patients for HER2-targeted therapies, monitor response and identify those who become resistant. Antibodies represent ideal candidates for this purpose, as they provide high contrast images for diagnosis and low toxicity in the therapeutic setting. Of those, nanobodies (Nb) are of particular interest considering their favorable kinetics, crossing of relevant biological membranes and intratumoral distribution. The purpose of this review is to highlight the unique characteristics and advantages of Nb-based radiotracers in BC imaging and therapy. Additionally, radiolabeling methods for Nb including direct labeling, indirect labeling via prosthetic group and indirect labeling via complexation will be discussed, reporting advantages and drawbacks. Furthermore, the preclinical to clinical translation of radiolabeled Nbs as promising theranostic agents will be reported.

Advances in the automated synthesis of 6-[18F]Fluoro-L-DOPA.

The neurotracer 6-[18F] FDOPA has been, for many years, a powerful tool in PET imaging of neuropsychiatric diseases, movement disorders and brain malignancies. More recently, it also demonstrated good results in the diagnosis of other malignancies such as neuroendocrine tumours, pheochromocytoma or pancreatic adenocarcinoma.The multiple clinical applications of this tracer fostered a very strong interest in the development of new and improved methods for its radiosynthesis. The no-carrier-added nucleophilic 18F-fluorination process has gained increasing attention, in recent years, due to the high molar activities obtained, when compared with the other methods although the radiochemical yield remains low (17-30%). This led to the development of several nucleophilic synthetic processes in order to obtain the product with molar activity, radiochemical yield and enantiomeric purity suitable for human PET studies.Automation of the synthetic processes is crucial for routine clinical use and compliance with GMP requirements. Nevertheless, the complexity of the synthesis makes the production challenging, increasing the chance of failure in routine production. Thus, for large-scale clinical application and wider use of this radiopharmaceutical, progress in the automation of this complex radiosynthesis is of critical importance.This review summarizes the most recent developments of 6-[18F]FDOPA radiosynthesis and discusses the key issues regarding its automation for routine clinical use.

The importance of drug metabolites synthesis: the case-study of cardiotoxic anticancer drugs.

Anticancer drugs are presently guarantying more survivors as a result of more powerful drugs or combinations of drugs used in therapy. Thus, it has become more crucial to study and overcome the side effects of these therapies. Cardiotoxicity is one of the most relevant side effects on the long-term cancer survivors, because of its high social and economic impact. Drug metabolism can result in active metabolites or toxic metabolites that can lead to important side effects. The metabolites of anticancer drugs are possible culprits of cardiotoxicity; however, the cardiotoxicity of many of the metabolites in several drug classes was not yet suitably studied so far. On the other hand, the use of prodrugs that are bioactivated through metabolism can be a good alternative to obtain more cardio safe drugs. In this review, the methods to obtain and study metabolites are summarized and their application to the study of a group of anticancer drugs with acknowledged cardiotoxicity is highlighted. In this group of drugs, doxorubicin (DOX, 1), mitoxantrone (MTX, 2), cyclophosphamide (CTX, 3) and 5-fluorouracil (5-FU, 4) are included, as well as the tyrosine kinase inhibitors, such as imatinib (5), sunitinib (6) and sorafenib (7). Only with the synthesis and purification of considerable amounts of the metabolites can reliable studies be performed, either in vitro or in vivo that allow accurate conclusions regarding the cardiotoxicity of anticancer drug metabolites and then pharmacological prevention or treatment of the cardiac side effects can be done.