Development of 18F-Labeled Bispyridyl Tetrazines for In Vivo Pretargeted PET Imaging

García-Vázquez, Rocio; Jørgensen, Jesper Tranekjær; Bratteby, Klas; Shalgunov, Vladimir; Hvass, Lars; Herth, Matthias M.; Kjær, Andreas; Battisti, Umberto Maria

doi:10.3390/ph15020245

Cited by 21 publications

(30 citation statements)

References 44 publications

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“…Suitable Tzs should be stable, highly reactive, clear rapidly from the blood stream, and easy to radiolabel. We have recently developed robust methods to radiolabel highly reactive Tzs with fluorine-18 as it is the most relevant radionuclide for clinical PET studies [ 48 , 49 , 50 , 51 ]. For brain pretargeted imaging, the Tz must also enter the brain.…”

Section: Introductionsupporting

confidence: 84%

“…In the second step, the brain sections were incubated with 111 In-Tz for one hour. This timeframe was sufficient to achieve complete ligation of the available TCOs as 111 In-Tz is a bispyridyl tetrazine with an expected reaction timeframe within minutes [ 49 ]. Afterward, the excess 111 In-Tz was washed away and the sections were quantified and analyzed using the uptake values in the cortex and in cerebellum.…”

Section: Resultsmentioning

confidence: 99%

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Pretargeted Imaging beyond the Blood–Brain Barrier—Utopia or Feasible?

et al. 2022

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Pretargeting is a promising nuclear imaging technique that allows for the usage of antibodies (Abs) with enhanced imaging contrast and reduced patient radiation burden. It is based on bioorthogonal chemistry with the tetrazine ligation—a reaction between trans-cyclooctenes (TCOs) and tetrazines (Tzs)—currently being the most popular reaction due to its high selectivity and reactivity. As Abs can be designed to bind specifically to currently ‘undruggable’ targets such as protein isoforms or oligomers, which play a crucial role in neurodegenerative diseases, pretargeted imaging beyond the BBB is highly sought after, but has not been achieved yet. A challenge in this respect is that large molecules such as Abs show poor brain uptake. Uptake can be increased by receptor mediated transcytosis; however, it is largely unknown if the achieved brain concentrations are sufficient for pretargeted imaging. In this study, we investigated whether the required concentrations are feasible to reach. As a model Ab, we used the bispecific anti-amyloid beta (Aβ) anti-transferrin receptor (TfR) Ab 3D6scFv8D3 and conjugated it to a different amount of TCOs per Ab and tested different concentrations in vitro. With this model in hand, we estimated the minimum required TCO concentration to achieve a suitable contrast between the high and low binding regions. The estimation was carried out using pretargeted autoradiography on brain sections of an Alzheimer’s disease mouse model. Biodistribution studies in wild-type (WT) mice were used to correlate how different TCO/Ab ratios alter the brain uptake. Pretargeted autoradiography showed that increasing the number of TCOs as well as increasing the TCO-Ab concentration increased the imaging contrast. A minimum brain concentration of TCOs for pretargeting purposes was determined to be 10.7 pmol/g in vitro. Biodistribution studies in WT mice showed a brain uptake of 1.1% ID/g using TCO-3D6scFv8D3 with 6.8 TCO/Ab. According to our estimations using the optimal parameters, pretargeted imaging beyond the BBB is not a utopia. Necessary brain TCO concentrations can be reached and are in the same order of magnitude as required to achieve sufficient contrast. This work gives a first estimate that pretargeted imaging is indeed possible with antibodies. This could allow the imaging of currently ‘undruggable’ targets and therefore be crucial to monitor (e.g., therapies for intractable neurodegenerative diseases).

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Section: Introductionsupporting

confidence: 84%

Section: Resultsmentioning

confidence: 99%

Pretargeted Imaging beyond the Blood–Brain Barrier—Utopia or Feasible?

et al. 2022

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“…[39][40][41] They can be labeled with fluorine-18 40,41 and modified with a second handle, i.e., their physicochemical properties can also be manipulated. 41,42 Consequently, these scaffolds are ideal to develop a BBB penetrant Tz. Since previous studies have shown that addition of a single methyl group can dramatically affect the pharmacokinetics and distribution of CNS tracers, a homologation approach was employed stepwise increasing the length/bulk of the chosen side chain…”

Section: Tetrazine In Silico Parametersmentioning

confidence: 99%

“…39 We have recently developed methods to prepare highly reactive Tzs labeled with fluorine-18. [40][41][42][43] These methods were used to label all Tzs in this study. The respective development strategy is displayed in Figure 1.…”

Section: Introductionmentioning

confidence: 99%

Pretargeted Imaging Beyond the Blood-Brain-Barrier

Shalgunov

Broek

Andersen

et al. 2022

Preprint

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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.

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“…This method allowed for the use of non-nucleophilic bases for the elution of fluoride-18 from anion exchange cartridges (fluoride-18 is standardly trapped on these cartridges during its work-up), and reduced the basicity of the reaction [18]. Our group has advanced this methodology for the 18 F-labeling of highly reactive Tzs [17,21,[23][24][25]. In particular, we reported the first direct aromatic fluorination of Tzs from stannane precursors [23].…”

Section: Introductionmentioning

confidence: 99%

Optimization of Direct Aromatic 18F-Labeling of Tetrazines

et al. 2022

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Radiolabeling of tetrazines has gained increasing attention due to their important role in pretargeted imaging or therapy. The most commonly used radionuclide in PET imaging is fluorine-18. For this reason, we have recently developed a method which enables the direct aromatic 18F-fluorination of tetrazines using stannane precursors through copper-mediated fluorinations. Herein, we further optimized this labeling procedure. 3-(3-fluorophenyl)-1,2,4,5-tetrazine was chosen for this purpose because of its high reactivity and respective limited stability during the labeling process. By optimizing parameters such as elution conditions, precursor amount, catalyst, time or temperature, the radiochemical yield (RCY) could be increased by approximately 30%. These conditions were then applied to optimize the RCY of a recently successfully developed and promising pretargeting imaging agent. This agent could be isolated in a decay corrected RCY of 14 ± 3% and Am of 201 ± 30 GBq/µmol in a synthesis time of 70 min. Consequently, the RCY increased by 27%.

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Development of 18F-Labeled Bispyridyl Tetrazines for In Vivo Pretargeted PET Imaging

Cited by 21 publications

References 44 publications

Pretargeted Imaging beyond the Blood–Brain Barrier—Utopia or Feasible?

Pretargeted Imaging beyond the Blood–Brain Barrier—Utopia or Feasible?

Pretargeted Imaging Beyond the Blood-Brain-Barrier

Optimization of Direct Aromatic 18F-Labeling of Tetrazines

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