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 [ 64 Cu][Cu-diacetyl-bis(N(4)methylthiosemicarbazone)] ([ 64 Cu][Cu(ATSM)]), as demonstrated in both preclinical and clinical studies. In addition, emerging studies suggest using [ 64 Cu][Cu(ATSM)] for molecular radiotherapy, mainly due to the release of therapeutic Auger electrons from copper-64, making [ 64 Cu][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 [ 60/61/62/64 Cu][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 [ 64 Cu][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 [ 64 Cu][Cu(ATSM)]. Comparisons between [ 64 Cu][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.

The widest scan that had been demonstrated previously for rapid scan EPR was a 155 G sinusoidal scan. As the scan width increases, the voltage requirement across the resonating capacitor and scan coils increases dramatically and the background signal induced by the rapidly changing field increases. An alternate approach is needed to achieve wider scans. A field-stepped direct detection EPR method that is based on rapid-scan technology is now reported, and scan widths up to 6200 G have been demonstrated. A linear scan frequency of 5.12 kHz was generated with the scan driver described previously. The field was stepped at intervals of 0.01 to 1 G, depending on the linewidths in the spectra. At each field data for triangular scans with widths up to 11.5 G were acquired. Data from the triangular scans were combined by matching DC offsets for overlapping regions of successive scans. This approach has the following advantages relative to CW, several of which are similar to the advantages of rapid scan. (i) In CW if the modulation amplitude is too large, the signal is broadened. In direct detection field modulation is not used. (ii) In CW the small modulation amplitude detects only a small fraction of the signal amplitude. In direct detection each scan detects a larger fraction of the signal, which improves the signal-to-noise ratio. (iii) If the scan rate is fast enough to cause rapid scan oscillations, the slow scan spectrum can be recovered by deconvolution after the combination of segments. (iv) The data are acquired with quadrature detection, which permits phase correction in the post processing. (v) In the direct detection method the signal typically is oversampled in the field direction. The number of points to be averaged, thereby improving the signal-to-noise ratio, is determined in post processing based on the desired field resolution. A degased lithium phthalocyanine sample was used to demonstrate that the linear deconvolution procedure can be employed with field-stepped direct detection EPR signals. Field-stepped direct detection EPR spectra were obtained for Cu2+ doped in Ni(diethyldithiocarbamate)2, Cu2+ doped in Zn tetratolylporphyrin, perdeuterated tempone in sucrose octaacetate, vanadyl ion doped in a parasubstituted Zn tetratolylporphyrin, Mn2+ impurity in CaO, and an oriented crystal of Mn2+ doped in Mg(acetylacetonate)2(H2O)2.

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.