Ethylene cross-bridged cyclam with two acetate pendant arms, ECB-TE2A, is known to form the most kinetically stable (64)Cu complexes. However, its usefulness as a bifunctional chelator is limited because of its harsh radiolabeling conditions. Herein, we report new cross-bridged cyclam chelator for the development of ultrastable (64)Cu-radiolabeled bioconjugates. Propylene cross-bridged TE2A (PCB-TE2A) was successfully synthesized in an efficient way. The Cu(II) complex of PCB-TE2A exhibited much higher kinetic stability than ECB-TE2A in acid decomplexation studies, and also showed high resistance to reduction-mediated demetalation. Furthermore, the quantitative radiolabeling of PCB-TE2A with (64)Cu was achieved under milder conditions compared to ECB-TE2A. Biodistribution studies strongly indicate that the (64)Cu complexes of PCB-TE2A cleared out rapidly from the body with minimum decomplexation.

A facile method of preparing triple‐modality, optical–nuclear–magnetic imaging probes using radiolabeled superparamagnetic nanoparticles is developed. Cerenkov luminescence imaging based on radionuclides showed great potential as a new optical imaging modality. The complementary nature of the optical/PET/MR hybrid nanoprobes facilitated non‐invasive differentiation between tumor‐metastasized sentinel lymph nodes (SLNs) and tumor‐free SLNs.

The purpose of this study was to develop Cu-labeled trastuzumab with improved pharmacokinetics for human epidermal growth factor receptor 2. Trastuzumab was conjugated with SCN-Bn-NOTA and radiolabeled with Cu. Serum stability and immunoreactivity ofCu-NOTA-trastuzumab were tested. Small animal PET imaging and biodistribution study were performed in HER2-positive breast cancer xenograft model (BT-474). Internal dosimetry of experimental animals was performed using the image-based approach with the Monte Carlo N-Particle Code. Cu-NOTA-trastuzumab was prepared with high radiolabel yield and radiochemical purity (>98%) and showed high stability in serum and good immunoreactivity. Uptake of Cu-NOTA-trastuzumab was highest at 48 h after injection determined by PET imaging and biodistribution results in BT-474 tumors. The blood radioactivity concentrations ofCu-NOTA-trastuzumab decreased bi-exponentially with time in both mice with and without BT-474 tumor xenografts. The calculated absorbed dose of Cu-NOTA-trastuzumab was 0.048 mGy/MBq for the heart, 0.079 for the liver and 0.047 for the spleen.Cu-NOTA-trastuzumab was effectively targeted to the HER2-expressing tumor and, and it exhibited relatively low absorbed dose due to short residence time. Therefore, Cu-NOTA-trastuzumab could be applied to select the right patients/right timing for HER2 therapy, to monitor the treatment response after HER2-targeted therapy, and to detect distal or metastatic spread.

Immuno-PET provides valuable information about tumor location, phenotype, susceptibility to therapy, and treatment response, especially to targeted radioimmunotherapy. In this study, we prepared antiepidermal growth factor receptor (EGFR) antibody via identical chelator, 3,6,9,15-tetraazabicyclo[9.3.1]-pentadeca-1(15),11,13-trience-3,6,9,-triacetic acid (PCTA), labeled with 64 Cu or 177 Lu to evaluate the EGFR expression levels using immuno-PET and the feasibility of radioimmunotherapy in an esophageal squamous cell carcinoma (ESCC) model. Methods: Cetuximab was conjugated with p-SCN-Bn-PCTA and radiolabeled with 64 Cu or 177 Lu. In vitro EGFR expression levels were determined and compared using flow cytometry and cell binding assay. In vivo EGFR expression levels were evaluated via immuno-PET imaging of 64 Cu-cetuximab and biodistribution analysis. Micro-SPECT/CT imaging, biodistribution, and radioimmunotherapy studies of 177 Lu-cetuximab were performed in the ESCC model. Therapeutic responses were monitored using 18 F-FDG PET and immunohistochemical staining. Results: 64 Cu-or 177 Lu-labeled antibodies showed high radiolabeling yield (.98%), stability (.90%), and favorable immunoreactivity. In vitro EGFR status measured by cell binding assay was correlated with the flow cytometry data. Immuno-PET, micro-SPECT/CT, and biodistribution demonstrated specific uptake in ESCC tumors depending on the EGFR expression levels. Tumor accumulation of 64 Cu-and 177 Lu-cetuximab was peaked at 48 and 120 h, respectively. Radioimmunotherapy with 177 Lu-cetuximab showed significant inhibition of tumor growth (P , 0.01) and marked reduction of 18 F-FDG SUV compared with that of control (P , 0.05). Terminal deoxynucleotidyl transferase dUTP nick-end labeling positivity and Ki-67 staining indices increased and decreased, respectively, in the radioimmunotherapy group compared with other groups (P , 0.01). Conclusion: 64 Cu-cetuximab immuno-PET represented EGFR expression levels in ESCC tumors, and 177 Lu-cetuximab radioimmunotherapy effectively inhibited the tumor growth. The diagnostic and therapeutic convergence radiopharmaceutical 64 Cu-/ 177 Lu-PCTA-cetuximab may be useful as a diagnostic tool in patient selection and a potent radioimmunotherapy agent in EGFR-positive ESCC tumors.

No abstract

Copper is an essential cofactor for a variety of biochemical processes including oxidative phosphorylation, cellular antioxidant activity, and elimination of free radicals. The copper transporter 1 is known to be involved in cellular uptake of copper ions. In this study, we evaluated the utility of human copper transporter 1 (hCTR1) gene as a new reporter gene for 64 Cu PET imaging. Methods: Human breast cancer cells (MDA-MB-231) were infected with a lentiviral vector constitutively expressing the hCTR1 gene under super cytomegalovirus promoter, and positive clones (MDA-MB-231-hCTR1) were selected. The expression of hCTR1 gene in MDA-MB-231-hCTR1 cells was measured by reverse transcription polymerase chain reaction, Western blot, and 64 Cu uptake assay. To evaluate the cytotoxic effects induced by hCTR1 expression, the dose-dependent cell survival rate after treatment with cisplatin (Cis-diaminedichloroplatinum (II) [CDDP]) was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and trypan blue dye exclusion. Small-animal PET images were acquired in tumorbearing mice from 2 to 48 h after an intravenous injection of 64 Cu.

Bifunctional chelators have been successfully used to construct (64)Cu-labeled radiopharmaceuticals. Previously reported chelators with cross-bridged cyclam backbones have various essential features such as high stability of the copper(II) complex, high efficiency of radiolabeling at room temperature, and good biological inertness of the radiolabeled complex, along with rapid body clearance. Here, we report a new generation propylene-cross-bridged chelator with hybrid acetate/phosphonate pendant groups (PCB-TE1A1P) developed with the aim of combining these key properties in a single chelator. The PCB-TE1A1P was synthesized from cyclam with good overall yield. The Cu(II) complex of our chelator showed good robustness in kinetic stability evaluation experiments, such as acidic decomplexation and cyclic voltammetry studies. The Cu(II) complex of PCB-TE1A1P remained intact under highly acidic conditions (12 M HCl, 90 °C) for 8 d and showed quasi-reversible reduction/oxidation peaks at -0.77 V in electrochemical studies. PCB-TE1A1P was successfully radiolabeled with (64)Cu ions in an acetate buffer at 60 °C within 60 min. The electrophoresis study revealed that the (64)Cu-PCB-TE1A1P complex has net negative charge in aqueous solution. The biodistribution and in vivo stability study profiles of (64)Cu-PCB-TE1A1P indicated that the radioactive complex was stable under physiological conditions and cleared rapidly from the body. A whole body positron emission tomography (PET) imaging study further confirmed high in vivo stability and fast clearance of the complex in mouse models. In conclusion, PCB-TE1A1P has good potential as a bifunctional chelator for (64)Cu-based radiopharmaceuticals, especially those involving peptides.

The first macrocyclic bifunctional chelator incorporating propylene cross-bridge was efficiently synthesized from cyclam in seven steps. After the introduction of an extra functional group for facile conjugation onto the propylene cross-bridge, the two carboxylic acid pendants could contribute to strong coordination of Cu(II) ions, leading to a robust Cu complex. The cyclic RGD peptide conjugate of PCB-TE2A-NCS was prepared and successfully radiolabeled with (64)Cu ion. The radiolabeled peptide conjugate was evaluated in vivo through a biodistribution study and animal PET imaging to demonstrate high tumor uptake with low background.