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Pretargeting is a powerful nuclear imaging strategy to achieve enhanced imaging contrast for nanomedicines. It reduces the radiation burden to healthy tissue. Pretargeting is based on bioorthogonal chemistry. The most attractive reaction for this purpose is currently the tetrazine ligation, which occurs between trans-cyclooctene (TCO) tags and tetrazines (Tzs). Pretargeted imaging beyond the blood-brain-barrier (BBB) has not been reported thus far. In this study, we developed Tz imaging agents that are capable to ligate in vivo to targets beyond the BBB. We chose to develop 18F-labeled Tzs as they can be applied to positron emission tomography (PET) - the most powerful molecular imaging technology. Fluorine-18 is an ideal radionuclide for PET due to its almost ideal decay properties. Fluorine-18 also allows - as a non-metal radionuclide - to develop Tzs with physicochemical properties enabling passive brain diffusion. In order to develop these imaging agents, we applied a rational drug design approach. This approach was based on estimated and experimental determined parameters such as the BBB score, pretargeted autoradiography contrast, in vivo input and washout curves as well as on metabolism studies. From initially 18 developed structures, five Tzs were selected to be tested on their in vivo click performance. Whereas all selected structures clicked in vivo into the brain, [18F]18 displayed the most favorable characteristics with respect to brain pretargeting. [18F]18 is our lead compound for future pretargeted imaging studies based on BBB-penetrant monoclonal antibodies. Pretargeting beyond the BBB will allow us to image targets beyond the BBB that are currently not imageable. For example, soluble protein isoforms could be imaged. These proteins are valuable drug targets for several neurodegenerative diseases and can currently not be imaged. Imaging would allow for diagnosis of these diseases, identifying responders from non-responders or to monitor treatment. Consequently, imaging will provide valuable information to accelerate drug development and greatly benefit patient care.

The glymphatic system is a brain-wide waste drainage system that promotes cerebrospinal fluid circulation through the brain to remove waste metabolites. Currently, the most common methods for assessing glymphatic function are ex vivo fluorescence microscopy of brain slices, macroscopic cortical imaging, and MRI. While all these methods have been crucial for expanding our understanding of the glymphatic system, new techniques are required to overcome their specific drawbacks. Here, we evaluate SPECT/CT imaging as a tool to assess glymphatic function in different anesthesia-induced brain states using two radiolabeled tracers, [111In]-DTPA and [99mTc]-NanoScan. Using SPECT, we confirmed the existence of brain state-dependent differences in glymphatic flow and we show brain state-dependent differences of CSF flow kinetics and CSF egress to the lymph nodes. We compare SPECT and MRI for imaging glymphatic flow and find that the two imaging modalities show the same overall pattern of CSF flow, but that SPECT was specific across a greater range of tracer concentrations than MRI. Overall, we find that SPECT imaging is a promising tool for imaging the glymphatic system, and that qualities such as high sensitivity and the variety of available tracers make SPECT imaging a good alternative for glymphatic research.

Pretargeting imaging of nanomedicines have attracted considerable interest in nuclear medicine since it has the potential to increase imaging contrast while simultaneously reducing radiation burden to healthy tissue. Currently, the tetrazine ligation is the fastest bioorthogonal reaction available for this strategy and consequently, the state-of-art choice for in vivochemistry. We have recently identified key properties for tetrazines to be applied in pretargeting. We have also developed a method to 18F-label highly reactive tetrazines using an aliphatic nucleophilic substitution strategy. In this study, we combined this knowledge and developed an 18F-labeled tetrazine for pretargeted imaging. In order to develop this ligand, a small structure-property study was carried out. The most promising compound - with respect to reactivity, hydrophilicity and ex vivo blocking effect - was selected for labeling and subsequent PET in vivo imaging. Radiolabeling was achieved in satisfying radiochemical yields, molar activities as well as in high radiochemical purities. The tracer displayed favorable pharmacokinetics and remarkable target-to-background ratios in pretargeted experiments - already one hour post injection. We believe that the developed pretargeting imaging agent is a promising candidate for translation into clinical studies.