A .sup.99mTc-Labelled Tetrazine for Bioorthogonal Chemistry. Synthesis and Biodistribution Studies with Small Molecule trans-Cyclooctene Derivatives

A convenient strategy to radiolabel a hydrazinonicotonic acid (HYNIC)-derived tetrazine with .sup.99m Tc was developed, and its utility for creating probes to image bone metabolism and bacterial infection using both active and pretargeting strategies was demonstrated. The .sup.99m Tc-labelled HYNIC-tetrazine was synthesized in 75% yield and exhibited high stability in vitro and in vivo. A trans-cyclooctene (TCO)-labelled bisphosphonate (TCO-BP) that binds to regions of active calcium metabolism was used to evaluate the utility of the labelled tetrazine for bioorthogonal chemistry. The pretargeting approach, with .sup.99m Tc-HYNIC-tetrazine administered to mice one hour after TCO-BP, showed significant uptake of radioactivity in regions of active bone metabolism (knees and shoulders) at 6 hours post-injection. For comparison, TCO-BP was reacted with .sup.99m Tc-HYNIC-tetrazine before injection and this active targeting also showed high specific uptake in the knees and shoulders, whereas control .sup.99m Tc-HYNIC-tetrazine alone did not. A TCO-vancomycin derivative was similarly employed for targeting Staphylococcus aureus infection in vitro and in vivo. Pretargeting and active targeting strategies showed 2.5- and 3-fold uptake, respectively, at the sites of a calf-muscle infection in a murine model, compared to the contralateral control muscle. These results demonstrate the utility of the .sup.99m Tc-HYNIC-tetrazine for preparing new technetium radiopharmaceuticals, including those based on small molecule targeting constructs containing TCO, using either active or pretargeting strategies.