Background: Tumor hypoxia (low tissue oxygenation) is an adverse condition of the solid tumor environment, associated with malignant progression, radiotherapy resistance, and poor prognosis. One method to detect tumor hypoxia is by positron emission tomography (PET) with the tracer [⁶⁴Cu][Cu-diacetyl-bis(N(4)-methylthiosemicarbazone)] ([⁶⁴Cu][Cu(ATSM)]), as demonstrated in both preclinical and clinical studies. In addition, emerging studies suggest using [⁶⁴Cu][Cu(ATSM)] for molecular radiotherapy, mainly due to the release of therapeutic Auger electrons from copper-64, making [⁶⁴Cu][Cu(ATSM)] a “theranostic” agent. However, the radiocopper retention based on a metal-ligand dissociation mechanism under hypoxia has long been controversial. Recent studies using ionic Cu(II) salts as tracers have raised further questions on the original mechanism and proposed a potential role of copper itself in the tracer uptake. We have reviewed the evidence of using the copper radiopharmaceuticals [^60/61/62/64Cu][Cu(ATSM)]/ionic copper salts for PET imaging of tumor hypoxia, their possible therapeutic applications, issues related to the metal-ligand dissociation mechanism, and possible explanations of copper trapping based on studies of the copper metabolism under hypoxia. Results: We found that hypoxia selectivity of [⁶⁴Cu][Cu(ATSM)] has been clearly demonstrated in both preclinical and clinical studies. Preclinical therapeutic studies in mice have also demonstrated promising results, recently reporting significant tumor volume reductions and improved survival in a dose-dependent manner. Cu(II)-[Cu(ATSM)] appears to be accumulated in regions with substantially higher CD133⁺ expression, a marker for cancer stem cells. This, combined with the reported requirement of copper for activation of the hypoxia inducible factor 1 (HIF-1), provides a possible explanation for the therapeutic effects of [⁶⁴Cu][Cu(ATSM)]. Comparisons between [⁶⁴Cu][Cu(ATSM)] and ionic Cu(II) salts have showed similar results in both imaging and therapeutic studies, supporting the argument for the central role of copper itself in the retention mechanism. Conclusions: We found promising evidence of using copper-64 radiopharmaceuticals for both PET imaging and treatment of hypoxic tumors. The Cu(II)-[Cu(ATSM)] retention mechanism remains controversial and future mechanistic studies should be focused on understanding the role of copper itself in the hypoxic tumor metabolism.

Cyclotron‐produced copper‐64 radioisotope tracers offer the possibility to perform both diagnostic investigation by positron emission tomography (PET) and radiotherapy by a theranostic approach with bifunctional chelators. The versatile chemical properties of copper add to the importance of this isotope in medicinal investigation. [⁶⁴Cu][Cu (ATSM)] has shown to be a viable candidate for imaging of tumor hypoxia; a critical tumor microenvironment characteristic that typically signifies tumor progression and resistance to chemo‐radiotherapy. Various production and radiosynthesis methods of [⁶⁴Cu][Cu (ATSM)] exist in labs, but usually involved non‐standardized equipment with varying production qualities and may not be easily implemented in wider hospital settings. [⁶⁴Cu][Cu (ATSM)] was synthesized on a modified GE TRACERlab FXN automated synthesis module. End‐of‐synthesis (EOS) molar activity of [⁶⁴Cu][Cu (ATSM)] was 2.2–5.5 Ci/μmol (HPLC), 2.2–2.6 Ci/μmol (ATSM‐titration), and 3.0–4.4 Ci/μmol (ICP‐MS). Radiochemical purity was determined to be >99% based on radio‐HPLC. The final product maintained radiochemical purity after 20 h. We demonstrated a simple and feasible process development and quality control protocols for automated cyclotron production and synthesis of [⁶⁴Cu][Cu (ATSM)] based on commercially distributed standardized synthesis modules suitable for PET imaging and theranostic studies.

Purpose: Hypoxic tumors are associated with therapy resistance and poor cancer prognosis, but methods to detect and counter tumor hypoxia remain insufficient. Our purpose was to investigate ⁶⁴Cu(II)-elesclomol ([⁶⁴Cu][Cu(ES)]) as a novel theranostic agent for hypoxic tumors, by implementing an improved production method and assessing its therapeutic and diagnostic potential compared to the established Cu-64 radiopharmaceuticals [⁶⁴Cu]CuCl₂ and [diacetyl-bis(N4-methylthiosemicarbazone) [⁶⁴Cu][Cu(ATSM)]. Methods: Cu-64 was produced using a biomedical cyclotron at 12 MeV with the reaction ⁶⁴Ni(p,n)⁶⁴Cu, followed by synthesis of [⁶⁴Cu]CuCl₂, [⁶⁴Cu][Cu(ATSM)], and [⁶⁴Cu][Cu(ES)]. In vitro therapeutic effects were assessed in both normoxic and hypoxic cells (22Rv1 and PC3 prostate cancer cells, and U-87MG glioblastoma cells) using the clonogenic assay and analyzing cellular uptake and internalization. In vivo therapeutic effects were assessed in 22Rv1 xenografts in BALB/cAnN-Foxn1nu/nu/Rj mice receiving a single or multiple doses of radiopharmaceutical, before their feasibility to detect tumor hypoxia was assessed by positron emission tomography (PET) in 22Rv1 and U-87MG xenografts. Results: In vitro and in vivo studies demonstrated that [⁶⁴Cu][Cu(ES)] reduced cell survival and inhibited tumor growth more effectively than [⁶⁴Cu][Cu(ATSM)] and [⁶⁴Cu]CuCl₂. Hypoxia increased the cellular uptake and internalization of [⁶⁴Cu][Cu(ES)] and [⁶⁴Cu][Cu(ATSM)]. [⁶⁴Cu][Cu(ES)]-PET tumor hypoxia detection was feasible and also revealed an unexpected finding of uptake in the brain. Conclusion: To the best of our knowledge, this is the first time that ES is radiolabeled with [⁶⁴Cu]CuCl₂ to [⁶⁴Cu][Cu(ES)]. We demonstrated superior therapeutic effects of [⁶⁴Cu][Cu(ES)] compared to [⁶⁴Cu][Cu(ATSM)] and [⁶⁴Cu]CuCl₂ and that [⁶⁴Cu][Cu(ES)]-PET is feasible. [⁶⁴Cu][Cu(ES)] is a promising theranostic agent for hypoxic solid tumors.