New Macrobicyclic Chelator for the Development of Ultrastable ⁶⁴Cu-Radiolabeled Bioconjugate

Ethylene cross-bridged cyclam with two acetate pendant arms, ECB-TE2A, is known to form the most kinetically stable ⁶⁴Cu 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 ⁶⁴Cu-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 ⁶⁴Cu was achieved under milder conditions compared to ECB-TE2A. Biodistribution studies strongly indicate that the ⁶⁴Cu complexes of PCB-TE2A cleared out rapidly from the body with minimum decomplexation

Visualization and Quantification of Radiochemical Purity by Cerenkov Luminescence Imaging

Determination of radiochemical purity is essential for characterization of all radioactive compounds, including clinical radiopharmaceuticals. Radio-thin layer chromatography (radio-TLC) has been used as the gold standard for measurement of radiochemical purity; however, this method has several limitations in terms of sensitivity, spatial resolution, two-dimensional scanning, and quantification accuracy. Here, we report a new analytical technique for determination of radiochemical purity based on Cerenkov luminescence imaging (CLI), whereby entire TLC plates are visualized by detection of Cerenkov radiation. Sixteen routinely used TLC plates were tested in combination with three different radioisotopes (¹³¹I, ¹²⁴I, and ³²P). All TLC plates doped with a fluorescent indicator showed excellent detection sensitivity with scanning times of less than 1 min. The new CLI method was superior to the traditional radio-TLC scanning method in terms of sensitivity, scanning time, spatial resolution, and two-dimensional scanning. The CLI method also showed better quantification features across a wider range of radioactivity values compared with radio-TLC and classical zonal analysis, especially for β^–-emitters such as ¹³¹I and ³²P

New Bifunctional Chelator for ⁶⁴Cu-Immuno-Positron Emission Tomography

A new tetraazamacrocyclic bifunctional chelator, TE2A-Bn-NCS, was synthesized in high overall yield from cyclam. An extra functional group (NCS) was introduced to the <i>N</i>-atom of TE2A for specific conjugation with antibody. The Cu complex of TE2A-Bn-NCS showed high kinetic stability in acidic decomplexation and cyclic voltammetry studies. X-ray structure determination of the Cu-TE2A-Bn-NH₂ complex confirmed octahedral geometry, in which copper atom is strongly coordinated by four macrocyclic nitrogens in equatorial positions and two carboxylate oxygen atoms occupy the elongated axial positions. Trastuzumab was conjugated with TE2A-Bn-NCS and then radiolabeled with ⁶⁴Cu quantitatively at room temperature within 10 min. Biodistribution studies showed that the ⁶⁴Cu-labeled TE2A-Bn-NCS-trastuzumab conjugates maintain high stability in physiological conditions, and NIH3T6.7 tumors were clearly visualized up to 3 days by ⁶⁴Cu-immuno-positron emission tomography imaging in animal models

Vivid Tumor Imaging Utilizing Liposome-Carried Bimodal Radiotracer

By developing a new bimodal radioactive tracer that emits both luminescence and nuclear signals, a trimodal liposome for optical, nuclear, and magnetic resonance imaging is efficiently prepared. Fast clearance of the radiotracer from reticuloendothelial systems enables vivid tumor imaging with minimum background

Phosphonate Pendant Armed Propylene Cross-Bridged Cyclam: Synthesis and Evaluation as a Chelator for Cu-64

A propylene cross-bridged macrocyclic chelator with two phosphonate pendant arms (PCB-TE2P) was synthesized from cyclam. Various properties of the synthesized chelator, including Cu-complexation, Cu-complex stability, ⁶⁴Cu-radiolabeling, and in vivo behavior, were studied and compared with those of a previously reported propylene cross-bridged chelator (PCB-TE2A)

High in Vivo Stability of ⁶⁴Cu-Labeled Cross-Bridged Chelators Is a Crucial Factor in Improved Tumor Imaging of RGD Peptide Conjugates

Although the importance of bifunctional chelators (BFCs) is well recognized, the chemophysical parameters of chelators that govern the biological behavior of the corresponding bioconjugates have not been clearly elucidated. Here, five BFCs closely related in structure were conjugated with a cyclic RGD peptide and radiolabeled with Cu-64 ions. Various biophysical and chemical properties of the Cu­(II) complexes were analyzed with the aim of identifying correlations between individual factors and the biological behavior of the conjugates. Tumor uptake and body clearance of the ⁶⁴Cu-labeled bioconjugates were directly compared by animal PET imaging in animal models, which was further supported by biodistribution studies. Conjugates containing propylene cross-bridged chelators showed higher tumor uptake, while a closely related ethylene cross-bridged analogue exhibited rapid body clearance. High in vivo stability of the copper–chelator complex was strongly correlated with high tumor uptake, while the overall lipophilicity of the bioconjugate affected both tumor uptake and body clearance

Non-Cross-Bridged Tetraazamacrocyclic Chelator for Stable ⁶⁴Cu-Based Radiopharmaceuticals

<i>N</i>-mono/dimethylated TE2A tetraazamacrocycles (MM-TE2A and DM-TE2A) were synthesized in high yields. Both Cu-MM/DM-TE2A complexes showed increased kinetic stability compared to that of Cu-TE2A, whereas Cu-DM-TE2A showed even higher in vitro stability than that of Cu-ECB-TE2A. MM-TE2A and DM-TE2A were quantitatively radiolabeled with ⁶⁴Cu ions and showed rapid clearance from the body to emerge as a potential efficient bifunctional chelator

High in Vivo Stability of ⁶⁴Cu-Labeled Cross-Bridged Chelators Is a Crucial Factor in Improved Tumor Imaging of RGD Peptide Conjugates

Although the importance of bifunctional chelators (BFCs) is well recognized, the chemophysical parameters of chelators that govern the biological behavior of the corresponding bioconjugates have not been clearly elucidated. Here, five BFCs closely related in structure were conjugated with a cyclic RGD peptide and radiolabeled with Cu-64 ions. Various biophysical and chemical properties of the Cu­(II) complexes were analyzed with the aim of identifying correlations between individual factors and the biological behavior of the conjugates. Tumor uptake and body clearance of the ⁶⁴Cu-labeled bioconjugates were directly compared by animal PET imaging in animal models, which was further supported by biodistribution studies. Conjugates containing propylene cross-bridged chelators showed higher tumor uptake, while a closely related ethylene cross-bridged analogue exhibited rapid body clearance. High in vivo stability of the copper–chelator complex was strongly correlated with high tumor uptake, while the overall lipophilicity of the bioconjugate affected both tumor uptake and body clearance

Synthesis and Evaluation of New Generation Cross-Bridged Bifunctional Chelator for ⁶⁴Cu Radiotracers

Bifunctional chelators have been successfully used to construct ⁶⁴Cu-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 ⁶⁴Cu ions in an acetate buffer at 60 °C within 60 min. The electrophoresis study revealed that the ⁶⁴Cu-PCB-TE1A1P complex has net negative charge in aqueous solution. The biodistribution and in vivo stability study profiles of ⁶⁴Cu-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 ⁶⁴Cu-based radiopharmaceuticals, especially those involving peptides