Actinium-225 (²²⁵Ac) is a promising radionuclide used in targeted alpha therapy (TAT). Although ²²⁵Ac labeling of bifunctional chelating ligands is effective, previous in vivo studies reported that free ²²⁵Ac can be released from the drugs and that such free ²²⁵Ac is predominantly accumulated in the liver and could cause unexpected toxicity. To accelerate the clinical development of ²²⁵Ac TAT with a variety of drugs, preparing methods to deal with any unexpected toxicity would be valuable. The aim of this study was to evaluate the feasibility of various chelators for reducing and excreting free ²²⁵Ac and compare their chemical structures. Nine candidate chelators (D-penicillamine, dimercaprol, Ca-DTPA, Ca-EDTA, CyDTA, GEDTA TTHA, Ca-TTHA, and DO3A) were evaluated in vitro and in vivo. The biodistribution and dosimetry of free ²²⁵Ac were examined in mice before an in vivo chelating study. The liver exhibited pronounced ²²⁵Ac uptake, with an estimated human absorbed dose of 4.76 Sv_RBE5/MBq. Aminopolycarboxylate chelators with five and six carboxylic groups, Ca-DTPA and Ca-TTHA, significantly reduced ²²⁵Ac retention in the liver (22% and 30%, respectively). Significant ²²⁵Ac reductions were observed in the heart and remainder of the body with both Ca-DTPA and Ca-TTHA, and in the lung, kidney, and spleen with Ca-TTHA. In vitro interaction analysis supported the in vivo reduction ability of Ca-DTPA and Ca-TTHA. In conclusion, aminopolycarboxylate chelators with five and six carboxylic groups, Ca-DTPA and Ca-TTHA, were effective for whole-body clearance of free ²²⁵Ac. This feasibility study provides useful information for reducing undesirable radiation exposure from free ²²⁵Ac.

Detecting tumor lesions <1 cm in size using current imaging methods remains a clinical challenge, especially in pancreatic cancer. Previously, we developed a method to identify pancreatic tumor lesions ≥3 mm using positron emission tomography (PET) with an intraperitoneally administered ⁶⁴Cu-labeled anti-epidermal growth factor receptor (EGFR) antibody (⁶⁴Cu-NCAB001 ipPET). Here, we conducted an extended single-dose toxicity study of ⁶⁴Cu-NCAB001 ipPET in mice based on approach 1 of the current ICH M3 [R2] guideline, as our new drug formulation contains 45 μg of the antibody. We used NCAB001 labeled with stable copper isotope instead of ⁶⁴Cu. The total content of size variants was approximately 6.0% throughout the study. The relative binding potency of Cu-NCAB001 to recombinant human EGFR was comparable to that of cetuximab. The general and neurological toxicities of Cu-NCAB001 ipPET at 62.5 or 625 μg/kg were assessed in mice. The no-observed-adverse-effect level of Cu-NCAB001 was 625 μg/kg, a dose approximately 1000-fold higher at the μg/kg level than the dose of ⁶⁴Cu-NCAB001 in our formulation (45 µg). The size variants did not affect the safety of the formulation. Therefore, clinical studies on the efficacy of ⁶⁴Cu-NCAB001 ipPET for early detection of pancreatic cancer using PET imaging can be safely conducted.

[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.

Peritoneal dissemination of pancreatic cancer has a poor prognosis. We have reported that intraperitoneal radioimmunotherapy using a ⁶⁴Cu-labeled antibody (⁶⁴Cu-ipRIT) is a promising adjuvant therapy option to prevent this complication. To achieve personalized ⁶⁴Cu-ipRIT, we developed a new in vitro tumor cell-binding assay (⁶⁴Cu-TuBA) system with a panel containing nine candidate ⁶⁴Cu-labeled antibodies targeting seven antigens (EGFR, HER2, HER3, TfR, EpCAM, LAT1, and CD98), which are reportedly overexpressed in patients with pancreatic cancer. We investigated the feasibility of ⁶⁴Cu-TuBA to select the highest-binding antibody for individual cancer cell lines and predict the treatment response in vivo for ⁶⁴Cu-ipRIT. ⁶⁴Cu-TuBA was performed using six human pancreatic cancer cell lines. For three cell lines, an in vivo treatment study was performed with ⁶⁴Cu-ipRIT using high-, middle-, or low-binding antibodies in each peritoneal dissemination mouse model. The high-binding antibodies significantly prolonged survival in each mouse model, while low-and middle-binding antibodies were ineffective. There was a correlation between in vitro cell binding and in vivo therapeutic efficacy. Our findings suggest that ⁶⁴Cu-TuBA can be used for patient selection to enable personalized ⁶⁴Cu-ipRIT. Tumor cells isolated from surgically resected tumor tissues would be suitable for analysis with the ⁶⁴Cu-TuBA system in future clinical studies.

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.