Kit Method for ⁶⁴Cu-BaBaSar-RGD₂ One-Step Production

Objectives: Peptides containing Arg-Glu-Asp (RGD) amino acid sequence have a high binding affinity and selectivity for integrin α_vβ₃. Herein, we report a one-step production of ⁶⁴Cu-radiopharmaceutical, ⁶⁴Cu-BaBaSar-RGD₂ with a kit preloaded with all the precursors.

Methods: The 18.2 MΩ·cm water from in-house GenPure^TM station was treated with chelex 100 resin 48 hours before use. All the solution hereafter was prepared with this treated water. The lyophilized BaBaSar-RGD₂ (1.0 mg) was dissolved in 1.0 mL sodium acetate buffer (NaOAc, 0.1 M, pH 5.5). The pH of BaBaSar-RGD₂ solution was adjusted to pH 5.5 using 0.1 M sodium hydroxide (NaOH). Then, the BaBaSar-RGD₂ solution was equally aliquoted to 20 Eppendorf vials. The filled vials were frozen using dry ice and then transferred to the bottles of the Labconco Freeze Dry System (pressure < 100 mTorr). After the solvent was removed, the vials containing BaBaSar-RGD₂ powder were then sealed and stored at -18°C. ⁶⁴CuCl₂ (5-30 mCi) purchased from Washington University at St. Louis was reconstituted using 200-300 µL NaOAc buffer (0.1 M, pH 5.5) and added to a vial prepared in above section. The vial was gently shaking at room temperature for 5 min. After the reaction was quenched with 5.0 mL saline, the activity passed through a 0.22 µm sterile filter (Pall Corp.) into a 10 mL Allergy vial for quality control test and animal injection. Results A cold kit contained 50 µg BaBaSar-RGD₂ ligand with buffer salts for the suitable labelling conditions. The kits were prepared in a lyophilized form and had a long shelf life over 3 months at room temperature. When the cold kits were stored in a refrigerator at -18°C, the shelf life was over a year. The ⁶⁴Cu-labeling yield for ⁶⁴Cu-BaBaSar-RGD₂ was > 99% based on HPLC analysis. However, after passing through 0.22 µm Pall filter to remove pyrogen, approximately 15-20% ⁶⁴Cu-BaBaSar-RGD₂ was trapped onto the filter and the overall recovered yield for ⁶⁴Cu-BaBaSar-RGD₂ was about 80% calculated from the loaded ⁶⁴Cu. The radiochemical purity of ⁶⁴Cu-BaBaSar-RGD₂ was > 99% based on radiotrace analytical HPLC. The retention times for free ⁶⁴CuCl₂ and ⁶⁴Cu-BaBaSar-RGD₂ on HPLC were 2.5 and 13.9 min, respectively. The reaction crude without purification did not show free ⁶⁴Cu in HPLC chromatograms. Therefore, no further purification is needed for the final product. Conclusions Integrin αvβ3-targeted radiopharmaceutical ⁶⁴Cu-BaBaSar-RGD₂ have been successfully synthesized with the one-step kit

Methods: The straightforward method will greatly simplify the production process and benefit the clinical application of ⁶⁴Cu-BaBaSar-RGD₂. We also estimated human radiation dosimetry of ⁶⁴Cu-BaBaSar-RGD₂ after intravenous administration in macaque monkey, by PET imaging and OLINDA/EXM calculations. The findings from this investigation show great promise of ⁶⁴Cu-BaBaSar-RGD₂ as an integrin marker, with a desirable biodistribution and safety characteristics in monkey. Therefore, ⁶⁴Cu-BaBaSar-RGD₂ can safely be used in human scan for further evaluation of its performance as an integrin-targeting probe.

• Download figure
• Open in new tab
• Download powerpoint