In vivo evaluation of 18F-labeled TCO for pre-targeted PET imaging in the brain

https://doi.org/10.1016/j.nucmedbio.2014.03.023Get rights and content

Abstract

Introduction

The tetrazine-trans-cylooctene cycloaddition using radiolabeled tetrazine or radiolabeled trans-cyclooctene (TCO) has been reported to be a very fast, selective and bioorthogonal reaction that could be useful for in vivo radiolabeling of molecules. We wanted to evaluate the in vivo biodistribution profile and brain uptake of 18F-labeled TCO ([18F]TCO) to assess its potential for pre-targeted imaging in the brain.

Methods

We evaluated the in vivo behavior of [18F]TCO via an ex vivo biodistribution study complemented by in vivo μPET imaging at 5, 30, 60, 90, 120 and 240 min post tracer injection. An in vivo metabolite study was performed at 5 min, 30 min and 120 min post [18F]TCO injection by RP-HPLC analysis of plasma and brain extracts. Incubation with human liver microsomes was performed to further evaluate the metabolite profile of the tracer.

Results

μPET imaging and ex-vivo biodistribution revealed an high initial brain uptake of [18F]TCO (3.8%ID/g at 5 min pi) followed by a washout to 3.0%ID/g at 30 min pi. Subsequently the brain uptake increased again to 3.7%ID/g at 120 min pi followed by a slow washout until 240 min pi (2.9%ID/g). Autoradiography confirmed homogenous brain uptake. On the μPET images bone uptake became gradually visible after 120 min pi and was clearly visible at 240 min pi. The metabolite study revealed a fast metabolization of [18F]TCO in plasma and brain into three main polar radiometabolites.

Conclusions

Although [18F]TCO has previously been described to be a useful tracer for radiolabeling of tetrazine modified targeting molecules, our study indicates that its utility for in vivo chemistry and pre-targeted imaging will be limited. Although [18F]TCO clearly enters the brain, it is quickly metabolized with a non-specific accumulation of radioactivity in the brain and bone.

Introduction

PET and SPECT imaging of radiolabeled peptides, proteins, antibodies and antibody fragments is a powerful in vivo research tool. However, compounds with a molecular weight over the glomerular filtration threshold of ~ 60–70 kDa can take several days to even weeks to clear from the body. This long retention time is associated with a high radiation dose and possible low target to non target ratios. Radiolabeling with shorter half-life isotopes like fluorine-18 (110 min) also represents a considerable challenge as the short half-life does not match with the long circulation time of these high molecular weight compounds and additionally 18F-labeling conditions are often harsh and thus not compatible with biological molecules like proteins. A solution for the above mentioned obstacles was offered by the bioorthogonal inverse-electron demand Diels-Alder reaction between strained trans-cyclooctene (TCO) and electron-deficient tetrazines [1], [2]. The tetrazine-TCO cycloaddition proved to be a very fast reaction (k2 = 2000 M 1s 1 in MeOH/water (9/1)), that proceeds in high yields in organic solvents, water, buffer, cell media, or lysate [1]. The reaction is extremely selective and will thus tolerate a broad range of functional groups commonly present in peptides and proteins [3], [4]. The method also avoids the use of cytotoxic Cu-catalysts as is required for azide-alkyne cycloaddition reactions, allowing in vivo use of this chemical reaction.

A promising biological application of the tetrazine-TCO cycloaddition is pre-targeted imaging. In this two-step targeting approach, a non-radiolabeled high molecular weight molecule modified with a tag (tetrazine or TCO) is injected into a living subject and is allowed to reach maximum uptake at its target site and sufficient clearance from non-target sites. Then a relatively small, radiolabeled molecule (TCO or tetrazine respectively) is administered which will selectively and covalently bind to the pre-targeted molecule via the tag while the non-bound radiolabeled molecule is rapidly cleared from the body. This allows in vivo imaging of the target with superior image contrast and reduced radiation doses compared to directly labeled peptides, proteins, antibodies or antibody fragments. This two step in vivo labeling approach facilitates the use of radioisotopes with short half-lives that would otherwise not be compatible with the long circulation times of high molecular weight molecules.

Nevertheless, the use of the tetrazine-TCO cycloaddition for pre-targeted imaging has so far mainly been described with longer living isotopes like 111In, 64Cu and 177Lu [5], [6], [7], [8]. In all of these examples monoclonal antibodies (mAb) directed against tumor antigens are modified with TCO while a tetrazine radiolabeled with 111In [5], [6], [8] or with 64Cu [7] is used for reaction with the TCO (attached to the mAb) thus enabling SPECT or PET imaging of the tumor. To date, there is only one example of tetrazine radiolabeled with a shorter living isotope [9]. In this paper, Weissleder and colleagues described an 18F labeled polymer-tetrazine adduct for in vivo labeling of a tumor targeting mAb modified with TCO [9]. These studies demonstrated efficient labeling of the mAb and obtained high tumor-to-background ratios, especially when used in combination with a clearing agent [6]. However due to the polarity of tetrazines, the bioorthogonal tetrazine-TCO cycloaddition using radiolabeled tetrazines is only applicable for imaging of non-internalizing and peripheral targets. For pre-targeted imaging of internalizing targets or the brain for instance, the inverse strategy using tetrazine modified targeting molecules and radiolabeled TCO may be recommended. Fox and co-workers were the first to describe 18F-labeled TCO ([18F]TCO) for radiolabeling of tetrazine-modified targeting molecules [10]. So far, applications have been limited to studies with pre-assembled 18F-TCO-tetrazine-targeting molecules in which [18F]TCO was evaluated as a new 18F-labeling strategy rather than a bioorthogonal chemical reaction [11], [12], [13], [14], [15]. To our knowledge, no biological evaluation of [18F]TCO has been described so far. The aim of the present study was therefore to evaluate the potential of [18F]TCO for in vivo pre-targeted imaging of tetrazine modified targeting molecules, exemplified here for the brain. With this purpose we evaluated the biodistribution profile of [18F]-TCO via ex-vivo biodistribution as well as μPET imaging and we have additionally characterized the in vitro and in vivo stability.

Section snippets

General procedures and materials

Unless stated otherwise, all chemical reagents were obtained from commercial sources and used without further purification. Characterization of all compounds was done with 1H NMR, 13C NMR and mass spectrometry. 1H NMR and 13C NMR spectra were recorded on a 400 MHz BrukerAvance III nanobay spectrometer, where necessary flash purification was performed by silica gel column chromatography using Biotage ® SNAP cartridges (silica gel cartridge size: 10–100 g; flow rate 10–50 mL/min) on a Biotage ®

Chemistry

In the literature, trans-cyclooctene (TCO) 9 can be prepared by elimination of phosphine oxide [22], [23] (Scheme 1) and as more recently described, by UV irradiation of (Z)-2-(cyclooct-4-ene-1-yloxy)ethanol 11 using a cycle/trap system (Scheme 2) [10], [12]. First, we repeated the synthesis of protected hydroxy trans-cyclooctenes 5 and 6 as depicted in Scheme 1 [22], [23]. Opening of the epoxycyclooctane 3 with lithium diphenylphosphamide and oxidation with hydrogen peroxide lead to a mixture

Conclusion

The rapid, selective, bioorthogonal Diels-Alder cycloaddition between tetrazine and trans-cyclooctene has become of great interest for pre-targeted imaging to reduce radiation dose and increase image contrast. The aim of the current study was to evaluate the use of [18F]TCO as a tracer for pre-targeted imaging of tetrazine modified targeting probes in the brain. The study revealed that although [18F]TCO is able to enter the brain, it is quickly metabolized in vivo in mouse plasma and brain into

Acknowledgments

The authors are thankful to Katie De Wagter (In vitro PK, Janssen Pharmaceutica) for help in the in vitro metabolite study and Philippe Joye (Molecular Imaging Center Antwerp) for support with in vivo experiments. We also want to thank Sophie Lyssens (UAMC) for excellent technical support in chemistry. Steven Deleye and Jeroen Verhaeghe (Molecular Image Center Antwerp) are also gratefully acknowledged for help in the image processing. This work supported in part by IWT grant 42/FA020000/5970.

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