Propylene Cross-Bridged Macrocyclic Bifunctional Chelator: A New Design for Facile Bioconjugation and Robust Cu-64 Complex Stability

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 64Cu 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. © 2014 American Chemical Society.

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

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)

Propylene Cross-Bridged Macrocyclic Bifunctional Chelator: A New Design for Facile Bioconjugation and Robust ⁶⁴Cu Complex Stability

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 ⁶⁴Cu 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

Synthesis and Evaluation of New Generation Cross-Bridged Bifunctional Chelator for Cu-64 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. (Chemical Equation Presented). © 2015 American Chemical Society.

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

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