⁶⁴Cu production via the ⁶⁸Zn(p,nα)⁶⁴Cu nuclear reaction: An untapped, cost-effective and high energy production route

Introduction: Copper-64 (⁶⁴Cu, t_1/2 = 12.7 h) is a positron emitter well suited for theranostic applications with beta-emitting ⁶⁷Cu for targeted molecular imaging and radionuclide therapy. The present work aims to evaluate the radionuclidic purity and radiochemistry of ⁶⁴Cu produced via the ⁶⁸Zn(p,nα)⁶⁴Cu nuclear reaction. Macrocyclic chelators DOTA, NOTA, TETA, and prostate-specific membrane antigen ligand PSMA I&T were radiolabeled with purified ⁶⁴Cu and tested for in vitro stability. [⁶⁴Cu]Cu-PSMA I&T was used to demonstrate in vivo PET imaging using ⁶⁴Cu synthesized via the ⁶⁸Zn(p,nα)⁶⁴Cu production route and its suitability as a theranostic imaging partner alongside ⁶⁷Cu therapy.

Methods: ⁶⁴Cu was produced on a 24 MeV TR-24 cyclotron at a beam energy of 23.5 MeV and currents up to 70 μA using 200 mg ⁶⁸Zn encapsulated within an aluminum‑indium-graphite sealed solid target assembly. ⁶⁴Cu semi-automated purification was performed using a NEPTIS Mosaic-LC synthesis unit employing CU, TBP, and TK201 (TrisKem) resins. Radionuclidic purity was measured by HPGe gamma spectroscopy, while ICP-OES assessed elemental purity. Radiolabeling was performed with NOTA at room temperature and DOTA, TETA, and PSMA I&T at 95 °C. ⁶⁴Cu incorporation was studied by radio-TLC. ⁶⁴Cu in vitro stability of [⁶⁴Cu]Cu-NOTA, [⁶⁴Cu]Cu-DOTA, [⁶⁴Cu]Cu-TETA, and [⁶⁴Cu]Cu-PSMA I&T was assessed at 37 °C from 0 to 72 h in human blood serum. Preclinical PET imaging was performed at 1, 24, and 48 h post-injection with [⁶⁴Cu]Cu-PSMA I&T in LNCaP tumor-bearing mice and compared with [⁶⁸Ga]Ga-PSMA I&T.

Results: Maximum purified activity of 4.9 GBq [⁶⁴Cu]CuCl₂ was obtained in 5 mL of pH 2-3 solution, with 2.9 GBq ⁶⁴Cu concentrated in 0.5 mL. HPGe gamma spectroscopy of purified ⁶⁴Cu detected <0.3 % co-produced ⁶⁷Cu at EOB with no other radionuclidic impurities. ICP-OES elemental analysis determined <1 ppm Al, Zn, In, Fe, and Cu in the [⁶⁴Cu]CuCl₂ product. NOTA, DOTA, TETA, and PSMA I&T were radiolabeled with ⁶⁴Cu, resulting in maximum molar activities of 164 ± 6 GBq/μmol, 155 ± 31 GBq/μmol, 266 ± 34 GBq/μmol, and 117 ± 2 GBq/μmol, respectively. PET imaging in PSMA-expressing LNCaP xenografts resulted in high tumor uptake (SUV_mean = 1.65 ± 0.1) using [⁶⁴Cu]Cu-PSMA I&T, while [⁶⁸Ga]Ga-PSMA I&T yielded an SUV_mean of 0.76 ± 0.14 after 60 min post-injection.

Conclusions: ⁶⁴Cu was purified in a small volume amenable for radiolabeling, with yields suitable for preclinical and clinical application. The ⁶⁴Cu production and purification process and the favourable PET imaging properties confirm the ⁶⁸Zn(p,nα)⁶⁴Cu nuclear reaction as a viable ⁶⁴Cu production route for facilities with access to a higher energy proton cyclotron, compared to using expensive ⁶⁴Ni target material and the ⁶⁴Ni(p,n)⁶⁴Cu nuclear reaction.

Advances in knowledge and implications for patient care: Our ⁶⁴Cu production technique provides an alternative production route with the potential to improve ⁶⁴Cu availability for preclinical and clinical studies alongside ⁶⁷Cu therapy.