[⁶⁴Cu]Cu-diacetyl-bis(N⁴-methylthiosemicarbazone) ([⁶⁴Cu]Cu-ATSM) is a radioactive hypoxia-targeting therapeutic agent being investigated in clinical trials for malignant brain tumors. For the quality management of [⁶⁴Cu]Cu-ATSM, understanding trace metal impurities’ effects on the chelate formation of ⁶⁴Cu and ATSM is important. In this study, we conducted coordination chemistry studies on metal–ATSM complexes. First, the effects of nonradioactive metal ions (Cu²⁺, Ni²⁺, Zn²⁺, and Fe²⁺) on the formation of [⁶⁴Cu]Cu-ATSM were evaluated. When the amount of Cu²⁺ or Ni²⁺ added was 1.2 mol or 288 mol, equivalent to ATSM, the labeling yield of [⁶⁴Cu]Cu-ATSM fell below 90%. Little effect was observed even when excess amounts of Zn²⁺ or Fe²⁺ were added to the ATSM. Second, these metals were reacted with ATSM, and chelate formation was measured using ultraviolet–visible (UV-Vis) absorption spectra. UV-Vis spectra showed a rapid formation of Cu²⁺ and the ATSM complex upon mixing. The rate of chelate formation by Ni²⁺ and ATSM was lower than that by Cu-ATSM. Zn²⁺ and Fe²⁺ showed much slower reactions with the ATSM than Ni²⁺. Trace amounts of Ni²⁺, Zn²⁺, and Fe²⁺ showed little effect on [⁶⁴Cu]Cu-ATSM’ quality, while the concentration of impurity Cu²⁺ must be controlled. These results can provide process management tools for radiopharmaceuticals.

Managing metastasis at the early stage and detecting and treating submillimeter tumors at early metastasis are crucial for improving cancer prognosis. Angiogenesis is a critical target for developing drugs to detect and inhibit submillimeter tumor growth; however, drug development remains challenging because there are no suitable models for observing the submillimeter tumor mass and the surrounding blood vessels in vivo. We have established a xenograft subcutaneous submillimeter tumor mouse model with HT-29-RFP by transplanting a single spheroid grown on radiation-crosslinked gelatin hydrogel microwells. Here, we developed an in vivo dual-observation method to observe the submillimeter tumor mass and tumor-surface blood vessels using this model. RFP was detected to observe the tumor mass, and a fluorescent angiography agent FITC-dextran was administered to observe blood vessels via stereoscopic fluorescence microscopy. The anti-angiogenesis agent regorafenib was used to confirm the usefulness of this method. This method effectively detected the submillimeter tumor mass and tumor-surface blood vessels in vivo. Regorafenib treatment revealed tumor growth inhibition and angiogenesis downregulation with reduced vascular extremities, segments, and meshes. Further, we confirmed that tumor-surface blood vessel areas monitored using in vivo dual-observation correlated with intratumoral blood vessel areas observed via fluorescence microscopy with frozen sections. In conclusion, this method would be useful in developing anti-angiogenesis agents against submillimeter tumors.

Copper (Cu) is a critical element for cancer cell proliferation and considerably accumulates in the nucleus. ⁶⁴Cu²⁺ is an anticancer radiopharmaceutical that targets the copper requirement of cancer cells. However, intravenously injected ⁶⁴Cu²⁺ ions primarily accumulate in the liver. Ligand complexation of ⁶⁴Cu²⁺ may be a promising method for increasing tumor delivery by reducing liver uptake. In this study, we used three tripodal amine ligands [tris(2-aminoethyl)amine (Tren), diethylenetriamine (Dien), and tris(2-pyridylmethyl)amine (TPMA)] to enclose ⁶⁴Cu²⁺ ions and compared their in vivo tumor and liver uptakes using a tumor-bearing xenograft mouse model of the extrahepatic bile duct carcinoma cell line TFK-1. We examined intracellular Cu distribution using microparticle-induced X-ray emission (micro-PIXE) analysis of these compounds. ⁶⁴Cu²⁺-Tren and ⁶⁴Cu²⁺-Dien showed higher tumor uptake than ⁶⁴Cu²⁺-TPMA and ⁶⁴Cu²⁺ ions in TFK-1 tumors. Among the three ⁶⁴Cu²⁺ complexes and ⁶⁴Cu²⁺ ions, liver uptake was inversely correlated with tumor uptake. Micro-PIXE analysis showed that in vitro cellular uptake was similar to in vivo tumor uptake, and nuclear delivery was the highest for ⁶⁴Cu²⁺-Tren. Conclusively, an inverse correlation between tumor and liver uptake was observed using three ⁶⁴Cu²⁺ complexes of tripodal amine ligands and ⁶⁴Cu²⁺ ions. These results provide useful information for the future development of anticancer ⁶⁴Cu radiopharmaceuticals.

Copper (Cu) is a critical element for cancer cell proliferation and considerably accumulates in the nucleus. 64Cu2+ is an anticancer radiopharmaceuticals targeting the copper requirement of cancer cells. However, intravenously injected 64Cu2+ ions primarily accumulate in the liver. Ligand complexation of 64Cu2+ would be a promising method for increasing tumor delivery by reducing liver uptake. Herein, we used three tripodal amine ligands [tris(2-aminoethyl)amine (Tren), diethylenetriamine (Dien), and tris(2-pyridylmethyl)amine (TPMA)] to enclose 64Cu2+ ions and compared their in vivo tumor and liver uptake using a tumor-bearing xenograft mouse model of the extrahepatic bile duct carcinoma cell line TFK-1. We examined intracellular Cu distribution using microparticle-induced X-ray emission (micro-PIXE) analysis for these compounds. 64Cu2+-Tren and 64Cu2+-Dien showed higher tumor uptake than 64Cu2+-TPMA and 64Cu2+ ions in TFK-1 tumors. Among the three 64Cu2+ complexes and 64Cu2+ ions, liver uptake was inversely correlated with tumor uptake. Micro-PIXE analysis showed that in vitro cellular uptake was similar to in vivo tumor uptake and nuclear delivery was the highest for 64Cu2+-Tren. Conclusively, an inverse correlation between tumor and liver uptake was observed using three 64Cu2+ complexes of tripodal amine ligands and 64Cu2+ ions. These results provide useful information for the future development of anticancer 64Cu radiopharmaceuticals.