Preliminary in vivo evaluation of a 11C-labeled tetrazine for bioorthogonal reaction within CNS

Objectives Due to its fast reaction kinetics and orthogonality towards biological functionalities, the inverse-electron-demand Diels-Alder cycloaddition between an electron deficient 1,2,4,5-tetrazine and a trans-cyclooctene has proven to be a useful application for in vivo chemistry in the field of molecular imaging. We have previously reported a 11C-labeled tetrazine, which rapidly reacts with trans-cyclooctene. Here, we describe its preliminary evaluation in respect to in vitro and in vivo stability, its capability to cross the blood-brain-barrier and its conjugation to a trans-cyclooctene functionalized polyglumatic acid (PGA), which was used as a model to test reaction kinetics and feasibility of the inverse-electron-demand Diels-Alder cycloaddition of this particular reaction.

Methods A 4-(hydroxy)benzamide substituted bispyridyl-tetrazine was radiolabeled with 11C using [11C]CH3I. The [11C]tetrazine was conjugated to a polyglutamic acid functionalized with trans-cyclooctene (11.4%). Analysis was performed by radio-HPLC using size exclusion chromatography and by radio-TLC. In vitro metabolism studies were conducted in human plasma and analyzed by radio-HPLC. The in vivo stability assessment and PET imaging were performed in Danish landrace pigs.

Results The [11C]tetrazine was formed in a radiochemical yield of 33% and >98% radiochemical purity. The subsequent inverse-electron-demand Diels-Alder cycloaddition with trans-cyclooctene tagged PGA was performed in water at 40 °C and completed within 1 min. The in vitro metabolic stability studies of the free [11C]tetrazine in human plasma showed 82% intact tracer after 30 min, whereas the in vivo experiments showed 26% intact tracer after 10 min. Furthermore, a summed PET image of the pig brain 6-20 min after injection of the free [11C]tetrazine demonstrated brain uptake.

Conclusions The 11C-labeled tetrazine is to our knowledge the first tetrazine described to cross the blood brain barrier. In addition, it reacts rapidly and efficiently with a trans-cyclooctene tagged polymer. We aim to use these results as a starting point for future pretargeted neuroimaging using click chemistry.

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