Pretargeted imaging beyond the blood-brain barrier

Abstract

Pretargeting is a powerful nuclear imaging strategy to achieve enhanced imaging contrast for nanomedicines and reduce 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) is challenging and has not been reported thus far. In this study, we developed Tz imaging agents that are capable of ligating 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. As a non-metal radionuclide, fluorine-18 also allows for development of Tzs with physicochemical properties enabling passive brain diffusion. To develop these imaging agents, we applied a rational drug design approach. This approach was based on estimated and experimentally determined parameters such as the BBB score, pretargeted autoradiography contrast, in vivo brain influx and washout as well as on peripheral metabolism profiles. From 18 initially developed structures, five Tzs were selected to be tested for their in vivo click performance. Whereas all selected structures clicked in vivo to TCO-polymer deposited into the brain, [¹⁸F]18 displayed the most favorable characteristics with respect to brain pretargeting. [¹⁸F]18 is our lead compound for future pretargeted neuroimaging studies based on BBB-penetrant monoclonal antibodies. Pretargeting beyond the BBB will allow us to image targets in the brain that are currently not imageable, such as soluble oligomers of neurodegeneration biomarker proteins. Imaging of such currently non-imageable targets will allow early diagnosis and personalized treatment monitoring. This in turn will accelerate drug development and greatly benefit patient care.

Fig. 1. A) The concept of pretargeting beyond the BBB. In the first step, a BBB-penetrating TCO-tagged mAb is injected. The mAb is actively transported over the BBB and binds to its target. In the second step, a ¹⁸F-radiolabeled Tz is injected. The Tz clicks to the TCO-tagged mAbs, thus enabling the imaging of the selected target. B) Comparison between pretargeted and conventional imaging C) strategy and workflow of this study to develop BBB permeable Tz imaging agent that is able to click in vivo to targets within the brain. The starting point was a Tz probe developed for cancer pretargeting. A library was designed based on this scaffold to have optimal parameters to cross the BBB and click in vivo. All the designed molecules were synthesized to evaluate labeling feasibility and in vitro stability. The ¹⁸F-Tzs were then tested for imaging contrast with in vitro autoradiography. Finally, compounds were injected into rats to evaluate the brain uptake. Metabolism was then evaluated for the best compounds.

Fig. 2. A) Designed Tz structures. B) Radiolabeling of Tzs [¹⁸F]1–18 and HPLC traces of [¹⁸F]18. C) In silico properties of Tzs along with key screening results.

Fig. 3. A) Workflow of pretargeted autoradiography. B) Pretargeted autoradiography images for selected Tzs. C) Brain time-activity curves for selected Tzs. D) Structures of Tzs selected for further evaluation. E) Ranking of evaluated Tzs by in vitro contrast and in vivo brain uptake kinetics. Tzs selected for further evaluation are marked with filled symbols, relative rankings (in vitro/in vivo) are shown in parentheses. In vitro contrast for [¹⁸F]18 was estimated by regression (see ESI Section S7).

Fig. 4. A) Intracerebral TCO-polymer injection model B) confirmation of TCO-polymer retention by SPECT/CT C) averaged TACs and representative PET images showing in vivo uptake of ¹⁸F-Tzs in the left (TCO-injected) and right (TCO-free) striata. D) Absolute and relative contrast between left and right striatum for selected Tzs. PET images and contrast measurements are based on average ¹⁸F-activity uptake at 60–90 min post-injection.

Fig. 5. A) In vivo metabolic stability of selected Tzs in rat plasma B) metabolite-corrected arterial plasma input curves for selected Tzs. C and D) Correlation of relative imaging contrast with clog P and in vitro contrast, respectively. Dash-dot lines represent putative non-monotonous relationships for highly lipophilic Tzs, E) correlation of absolute imaging contrast with in vivo brain AUC, F) correlation of absolute imaging contrast with in vivo metabolic stability of ¹⁸F-Tzs. Dashed line represents putative trend without 12. G) Summary rankings of Tzs displaying the most promising characteristics for pretargeting beyond the BBB. Rankings are relative to other Tzs. [¹⁸F]18 was identified to possess the best parameters.