⁶⁴Cu is a cyclotron-produced radionuclide suitable for positron emission tomography (PET) and internal radiotherapy. We have been focused on ⁶⁴Cu radioisotope due to its favorable nuclear characteristics (β⁺ emission, 18% and β⁻ emission, 39%). Moreover, the electron capture (43%) implies Auger electron emission with high linear energy transfer in the targeted cells. Also, the 12.7 hours half-life allows a good uptake and distribution of the radiopharmaceuticals to obtain qualitative images with high contrast. The production of no-carrier-added ⁶⁴Cu via the ⁶⁴Ni(p,n)⁶⁴Cu nuclear reaction using a variable energy cyclotron (TR-19, ACSI, Canada) was studied in our work involving the implementation of a fully automated solid target irradiation and processing system. In this paper we report the validation of the process for the automated production and purification of ⁶⁴Cu produced in a variable energy (14-19 MeV) cyclotron, using a commercially available automated solid target system (ALCEO, Comecer S.p.A, Italy), comprising modules for electrodeposition, pneumatic transfer, irradiation unit, dissolving and purification. The obtained ⁶⁴CuCl₂ solution complies with the requirements of a radiopharmaceutical for medical applications. The process is to be translated for production of other copper radioisotopes of medical interest.

The use of radioisotopes in nuclear medicine applications became essential for the diagnosis and follow-up imaging of many oncological, cardiovascular, and neurodegenerative diseases, in a safe and non-invasive way. Their use in the personalized treatment of tumors is on the verge to change the oncological patient management. Optimization of the radioisotopes production aims to maximize the production efficiency while minimizing side reactions and costs. A practical approach to balance these non-converging ways is to employ the simulation tools in the process design phase and experimental setup. In this way, the production yield can be estimated and the radionuclide impurities content that appears during the bombardment of the target of interest can be optimally set below acceptable limits. Copper-64 is an emerging radionuclide in nuclear medicine theragnostic applications due to three decay modes, namely electron capture, electron (β⁻) and positron (β⁺) emissions, and a 12.7 h half-life, favorable for visualization of fast biological processes. Optimization of ⁶⁴Cu production by irradiation of enriched ⁶⁴Ni targets with protons in a particular geometry on a TR-19 cyclotron is discussed in this work. The simulated activity produced on different levels of enrichment of the ⁶⁴Ni targets was calculated using the Monte Carlo simulation in the Geant4 platform, where a customized solid target irradiation system set-up was replicated; the obtained parameters were implemented in the experiments and the results were compared, aiming to validate the simulation parameters through experimental data.As the simulated and experimental results regarding the production of ⁶⁴Cu via⁶⁴Ni(p,n)⁶⁴Cu reaction are in good compliance, the tool can be further applied for the optimization of the production of other radionuclides on the same set-up.

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.

Due to combined therapeutical emissions, a high linear energy transfer Auger-electrons with the longer ranged β⁻ particles, ⁶⁴Cu-based radiopharmaceuticals raise particular theragnostic interest in cancer, by joined therapeutic and real-time PET imaging properties. The in vitro study aimed to investigate the biological and molecular background of ⁶⁴CuCl₂ therapy by analyzing the damages and stress responses inflicted in various human normal and tumor cell lines. Colon (HT29 and HCT116) and prostate carcinoma (DU145) cell lines, as well as human normal BJ fibroblasts, were treated up to 72 h with 2–40 MBq/mL ⁶⁴CuCl₂. Radioisotope uptake and retention were assessed, and cell viability/death, DNA damage, oxidative stress, and the expression of 84 stress genes were investigated at various time points after [⁶⁴Cu]CuCl₂ addition. All the investigated cells incorporated ⁶⁴Cu ions similarly, independent of their tumoral or normal status, but their fate after exposure to [⁶⁴Cu]CuCl₂ was cell-dependent. The most striking cytotoxic effects of the radioisotope were registered in colon carcinoma HCT116 cells, for which a substantial decrease in the number of metabolically active cells, and an increased DNA damage and oxidative stress were registered. The stress gene expression study highlighted the activation of both death and repair mechanisms in these cells, related to extrinsic apoptosis, necrosis/necroptosis or autophagy, and cell cycle arrest, nucleotide excision repair, antioxidant, and hypoxic responses, respectively. The in vitro study indicated that 40 MBq/mL [⁶⁴Cu]CuCl₂ delivers a therapeutic effect in human colon carcinoma, but its use is limited by harmful, yet lower effects on normal fibroblasts. The exposure of tumor cells to 20 MBq/mL [⁶⁴Cu]CuCl₂, might be used for a softer approach aiming for a lower radiotoxicity in normal fibroblasts as compared to tumor cells. This radioactive concentration was able to induce a persistent decrease in the number of metabolically active cells, accompanied by DNA damage and oxidative stress, associated with significant changes in stress gene expression in HCT116 colon cancer cells.

Zirconium-89 (⁸⁹Zr) is a highly valued diagnostic radionuclide for positron emission tomography (PET) due to its long physical half-life of 78.4 h and decay characteristics, being preferred for the radiolabelling of nanoparticles and slow kinetics macromolecules, such as antibodies. ⁸⁹Zr-based high-resolution PET images can be employed to scan tumours and localize the tracer on a longer timeframe, which allows for real-time therapy monitoring. The goal of this study was to maximize the ⁸⁹Zr production yield by fine-tunning the irradiation parameters of a solid target, in two different experimental set-ups, using a variable energy 14-19 MeV TR-19 cyclotron. Monte Carlo programs simulated the irradiation geometry and estimated the activity and irradiation yields produced by the ⁸⁹Y(p, n)⁸⁹Zr reaction, at the process optimal parameters. The resulted data were compared with the experimental data collected in our particular irradiation setups. ⁸⁹Zr was obtained from ^natY foil target using: (A) the solid target holder placed on the extraction port, and (B) the automated solid target irradiation station, installed on a sloped-down extension of the proton beamline. The two irradiation geometries are differentiated by the distances from the respective extraction ports, beam-geometry and shape, cooling capacity, and degrader's thickness. Based on the specific geometries, A and B, the Monte Carlo simulations output determined the optimal experimental irradiation parameters (extracted energy, degrader thickness, proton current intensity), as well as the target thickness. The 250 μm ^natY foils were irradiated with 14 MeV protons and an integrated current of 32 µA·h, on the solid target configuration A, and with 15.2 MeV protons, 100 µA·h on the solid target configuration B. After the dissolution and purification of the targets, [⁸⁹Zr]Zr-oxalate solutions of 1.28 ± 0.18 GBq, and 2.95 ± 0.31 GBq respectively, were evaluated, to determine the radionuclidic purity and contaminant levels of ⁸⁹Zr solutions across different incident proton beam energies. The pharmaceutical specifications require the solutions radionuclidic purity to be above 99.9% of the total radioactivity, as criteria of their suitability for use as radiopharmaceutical precursors for antibodies radiolabelling. Simulations were providing optimized input parameters to maximize the production yield of ⁸⁹Zr and subsequently, to achieve the highest possible activity with no detriment to radionuclide purity, as per the [⁸⁹Zr]Zr-oxalate solution pharmaceutical specification. The parameters were then implemented in the experiments, and the production processes were tested on two particular irradiation configurations. The yields and activities produced through ⁸⁹Y(p, n)⁸⁹Zr reaction, at the TR-19 cyclotron were in good agreement with the simulations, within 18.4-21.3%, which include activity losses during irradiation and post-processing and uncertainties resulted from activity measurements and cross-section values.

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.