# Development of a 99mTc-labeled tetrazine for pretargeted SPECT imaging using an alendronic acid-based bone targeting model

## TL;DR

This study develops a new 99mTc-labeled tetrazine for improved bone imaging in nuclear medicine using a pretargeting approach.

## Contribution

The study introduces a novel 99mTc-labeled tetrazine scaffold with improved bone imaging performance in a pretargeted model.

## Key findings

- 3,6-bis(2-pyridyl)-1,2,4,5-Tz showed higher bone uptake and lower background activity compared to other tetrazine variants.
- Improved bone/blood ratios were observed in pretargeted animals compared to directly labeled models.
- The scaffold 3,6-bis(2-pyridyl)-1,2,4,5-Tz is identified as a promising candidate for 99mTc-labeled tetrazines.

## Abstract

Pretargeting, which is the separation of target accumulation and the administration of a secondary imaging agent into two sequential steps, offers the potential to improve image contrast and reduce radiation burden for nuclear imaging. In recent years, the tetrazine ligation has emerged as a promising approach to facilitate covalent pretargeted imaging due to its unprecedented kinetics and bioorthogonality. Pretargeted bone imaging with TCO-modified alendronic acid (Aln-TCO) is an attractive model that allows the evaluation of tetrazines in healthy animals without the need for complex disease models or targeting regimens. Recent structure-activity relationship studies of tetrazines evaluated important parameters for the design of potent tetrazine-radiotracers for pretargeted imaging. However, limited information is available for 99mTc-labeled tetrazines. In this study, four tetrazines intended for labeling with fac-[99mTc(OH2)3 (CO)3]+ were synthesized and evaluated using an Aln-TCO mouse model. 3,6-bis(2-pyridyl)-1,2,4,5-Tz without additional linker showed higher pretargeted bone uptake and less background activity compared to the same scaffold with a PEG8 linker or 3-phenyl-1,2,4,5-Tz-based compounds. Additionally, improved bone/blood ratios were observed in pretargeted animals compared to animals receiving directly labeled Aln-TCO. The results of this study implicate 3,6-bis(2-pyridyl)-1,2,4,5-Tz as a promising scaffold for potential 99mTc-labeled tetrazines.