Nuclear Medicine and Biology xxx (2014) xxx–xxx

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Nuclear Medicine and Biology journal homepage: www.elsevier.com/locate/nucmedbio

In vivo evaluation of the brain

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F-labeled TCO for pre-targeted PET imaging in

Leonie wyffels a, b, David Thomae a, c, Ann-Marie Waldron a, d, Jens Fissers a, c, Stefanie Dedeurwaerdere d, Pieter Van der Veken c, Jurgen Joossens c, Sigrid Stroobants a, b, Koen Augustyns c, Steven Staelens a,⁎ a

Molecular Imaging Center Antwerp, University of Antwerp, Antwerp, Belgium University Hospital Antwerp, Department of Nuclear Medicine, Edegem, Belgium Laboratory of Medicinal Chemistry, University of Antwerp, Antwerp, Belgium d Department of Translational Neurosciences, University of Antwerp, Belgium b c

a r t i c l e

i n f o

Article history: Received 27 January 2014 Received in revised form 18 March 2014 Accepted 27 March 2014 Available online xxxx Keywords: 18 F-trans-cyclooctene Pre-targeted imaging PET

a b s t r a c t Introduction: The tetrazine-trans-cylooctene cycloaddition using radiolabeled tetrazine or radiolabeled transcyclooctene (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 pretargeted 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. © 2014 Elsevier Inc. All rights reserved.

1. 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 ⁎ Corresponding author at: Antwerp University, Faculty of Medicine and Health Sciences, Molecular Imaging (MICA), Campus Drie Eiken – Building Uc, Universiteitsplein 1, B-2610 Antwerpen (Wilrijk). Tel.: +32 3 265 28 20. E-mail address: [email protected] (S. Staelens).

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 electrondeficient 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

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Please cite this article as: wyffels L., et al, In vivo evaluation of (2014), http://dx.doi.org/10.1016/j.nucmedbio.2014.03.023

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F-labeled TCO for pre-targeted PET imaging in the brain, Nucl Med Biol

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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 halflives 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 pretargeted imaging has so far mainly been described with longer living isotopes like 111In, 64Cu and 177Lu [5–8]. 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-tetrazinetargeting molecules in which [ 18F]TCO was evaluated as a new 18Flabeling strategy rather than a bioorthogonal chemical reaction [11– 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. 2. Materials and methods 2.1. 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 ® ISOLERA One flash system equipped with an internal variable dual-wavelength diode array detector (200–400 nm). No carrier-added [ 18F]F − was produced in a Siemens Eclipse HP cyclotron by bombardment of [ 18O]H2O (Rotem Industries, Israel) by the 18O(p,n) 18F reaction. Radiosynthesis of [ 18F]TCO was carried out on an automated synthesis module (Fluorsynthon III, Veenstra Instruments, The Netherlands) specifically adapted to this radiosynthesis. Radiochemical yields were calculated from the theoretical initial amount of [ 18F]F − and decay corrected to end of bombardment (EOB). Purification of [ 18F]TCO following radiosynthesis was done by semi-preparative HPLC using a Knauer HPLC pump and a Smartline UV detector (λ = 254 nm) in line with a Hi-Rad 1000-CD-X CdWO4 scintillation detector (Scionix, The Netherlands). Radiochemical and chemical purity was determined by analytical reverse phase HPLC using Please cite this article as: wyffels L., et al, In vivo evaluation of (2014), http://dx.doi.org/10.1016/j.nucmedbio.2014.03.023

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a Shimadzu LC-20AT HPLC pump equipped with a SPD-20A UV/VIS detector (λ = 220 nm, Shimadzu, Japan) in series with a NaIscintillation detector for radiation detection (Raytest, Germany). The recorded data were processed by the GINA-Star 5 software (Raytest, Germany). Radioactivity in samples from animal studies was measured in a Wizard 2 2480 automatic gamma counter (Perkin Elmer, Finland). Full body PET/CT acquisitions were acquired on an Inveon microPET/CT scanner (Siemens Preclinical Solutions Inc., USA). 2.2. Chemistry 2.2.1. Synthesis of 5-(tert-butyldimethylsiloxy)-1,2-E-cyclooctene (5 and 6) To sodium hydride (NaH; 0.753 g of a 60% dispersion in mineral oil, 18.84 mmol) in 25 mL dry dimethylformamide (DMF), was slowly added to the mixture of 4a and 4b (4.32 g, 9.42 mmol) in 25 mL dry DMF at 0 °C. After 30 min reaction a precipitate was observed. Reaction was stirred for 5 h at room temperature (RT). Saturated ammonium chloride solution was then added (150 mL) and the reaction mixture was extracted with ethylacetate (EtOAc; 3 × 50 mL). The organic layers were dried with sodium sulfate (Na2SO4), filtered and concentrated under vacuum. The residue was purified with flash column chromatography (SNAP cartridge 100 g) eluting first with Hexanes (100%, 50 mL/min) to isolate the first diastereoisomer 5 as a colorless oil (770 mg, 34%). 1H-NMR (CDCl3, 400 MHz): δ 5.64 (ddd, J = 16 Hz, J = 12 Hz, J = 8 Hz, 1H), 5.45 (ddd, J = 16 Hz, J = 12 Hz, J = 8 Hz, 1H), 4.00 (dd, J = 12 Hz, J = 8 Hz, 1H), 2.41 (m, 1H), 2.22 (m, 1H), 1.50–2.07 (m, 7H), 1.20 (m, 2H), 0.94 (m, 9H), 0.03 (s, 3H), 0.01 (m, 3H). 13C-NMR (CDCl3, 100 MHz): δ 136.1, 131.7, 67.8, 44.2, 35.4, 34.0, 29.9, 27.9, 26.4, 18.6, − 4.5, − 4.6. Then the column was eluted with EtOAc (100%, 50 mL/min) to isolate the second diastereoisomer 6 as a colorless oil (410 mg, 18%). 1H-NMR (CDCl3, 400 MHz): δ 5.55 (ddd, J = 16 Hz, J = 12 Hz, J = 8 Hz, 1H), 5.36 (ddd, J =16 Hz, J = 12 Hz, J = 8 Hz, 1H), 3.40 (m, 1H), 2.17–2.36 (m, 3H), 1.87–2.02 (m, 1H), 1.46–1.76 (m, 7H), 0.87 (m, 9H), 0.03 (s, 6H). 13C-NMR (CDCl3, 100 MHz): δ 135.7, 132.7, 79.8, 45.1, 42.2, 34.8, 33.2, 31.5, 26.3, 18.5, −4.2, −4.3. 2.2.2. Synthesis of rel-(1R, 4E, pS)-cyclooct-4-enol (7) 1 M solution of tetrabutylammoniumfluoride (TBAF; 23.7 mL, 23.70 mmol) in tetrahydrofuran (THF) was added to compound 5 (570 mg, 2.37 mmol). The reaction was refluxed for 35 min and the solvent was evaporated. The residue was quenched with 20 mL of water and extracted with EtOAc (3 × 20 mL) and water was added. The organic layer was washed two more times with water, before drying with Na2SO4, filtered and concentrated under vacuum. The residue was purified with flash column chromatography (SNAP 25g) eluting with a gradient elution (Hex/EtOAc 5–40% in 14 min, 25 mL/min) to obtain the alcohol 7 as a colorless oil (160 mg, 40%). 1H-NMR (CDCl3, 400 MHz): δ 5.51–5.62 (m, 2H), 4.04 (m, 1H), 2.32–2.42 (m, 1H), 2.07–2.27 (m, 4H), 1.74–1.91 (m, 3H), 1.61–1.70 (m, 1H), 1.19–1.30 (m, 2H). 2.2.3. Synthesis of rel-(1R, 4E, pR)-cyclooct-4-enol (8) 1 M solution of TBAF (8.32 mL, 8.32 mmol) in THF was added to compound 6 (200 mg, 0.83 mmol). The reaction was refluxed for 35 min and the solvent was evaporated. The residue was quenched with 20 mL of water and extracted with EtOAc (3 × 20 mL) and water was added. The organic layer was washed two more times with water, before drying with Na2SO4, filtered and concentrated under vacuum. The residue was purified with flash column chromatography (SNAP 10 g) eluting with a gradient elution (Hex/EtOAc 12–100% in 8 min, 12 mL/min) to obtain the alcohol 7 as a colorless oil. 1H-NMR (CDCl3, 400 MHz): δ 5.57 (ddd, J = 16 Hz, J = 12 Hz, J = 8 Hz, 1H), 5.38 (ddd, J = 16 Hz, J = 12 Hz, J = 8 Hz, 1H), 3.46 (m, 1H), 2.26–2.36 (m, 3H), 1.92–1.99 (m, 4H), 1.60–1.71 (m, 3H).

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2.2.4. 2-[rel-(1R, 4E, pR)-cyclooct-4-en-1yloxy] ethanol (9) via alkylation and reduction NaH (101 mg of a 60% dispersion in mineral oil, 2.54 mmol) was added to an oven dried round bottom flask under nitrogen atmosphere. The NaH was rinsed twice with 5 mL of hexanes. Dry THF was added (2 mL) followed by a solution of 8 (80 mg, 0.634 mmol) in THF (1 mL). The reaction mixture was refluxed for 1 h. α-Iodo acetic acid (118 mg, 0.634 mmol) dissolved in THF (2.5 mL) was added dropwise to the solution and reflux was continued for 1 h. The mixture was then allowed to cool to room temperature (RT), and the mixture was concentrated under reduced pressure. The residue was basified with sodium hydroxide (NaOH 10%; 5 mL) and extracted with diethyl ether (Et2O; 2 × 5 mL). Then the aqueous phase was cooled to 0 °C and acidified with hydrochloric acid (HCl 37%) and then extracted with dichloromethane (CH2Cl2; 3 × 10 mL). The organic phase was dried over Na2SO4, filtered and evaporated under vacuum. The resulting residue was used without further purification. A solution of rel-(1R, 4E,pS)-(cyclooct-4-enyloxy)-acetic acid (0.1 g, 0.543 mmol) Et2O (1 mL) was added at 0 °C to a suspension of lithium aluminum hydride (LiAlH4; 0.019 g, 0.500 mmol) in Et2O (5 mL). The reaction was stirred overnight at room temperature and quenched with HCl 2 N until all salts were dissolved. The solution was extracted with Et2O (3 × 10 mL), the organic phase was dried over Na2SO4, filtered and evaporated under vacuum. The residue was dissolved in CH2Cl2 (5 mL) and washed with NaOH 10% (10 mL) to remove traces of starting material. Then the organic phase was dried over Na2SO4, filtered and evaporated under vacuum to obtain a colorless oil (52 mg, 56%). The NMR data were in agreement with previously reported data [16]. 2.2.5. 2-[rel-(1R, 4E, pR)-cyclooct-4-en-1yloxy] ethanol (9) and 2-[rel-(1R, 4E, pS)-cyclooct-4-en-1yloxy] ethanol (12) via UV irradiation (Z)-2-(cyclooct-4-en-1-yloxy)ethanol 11 (1.302 g, 7.65 mmol) and methyl benzoate (1.068 g, 7.84 mmol) were dissolved in 125 mL of a solution of hexane/Et2O (1/9, V/V). The beaker was placed in an UVP-CL1000 crosslinker reactor (UVP, UK) and irradiated for 8 h under icecooling. In intervals of 30 min the reaction mixture was passed through a column packed with 13 g of silver nitrate (AgNO3, 10%) impregnated silica (commercially available from Aldrich). The mixture when passed through the column was placed back into the UV crosslinker for further irradiation. After 8 h the irradiation was stopped and the silica was added to an ammonium hydroxide (NH4OH) solution (90 mL, 28%). The suspension was stirred for 5 min, 90 mL of Et2O were added and stirring continued for 5 min. The aqueous layer was extracted two more times with Et2O (2 × 90 mL) and the combined organic phases were washed with water, dried over magnesium sulfate (MgSO4) and concentrated under vacuum. The residue was purified by column chromatography (SNAP 10 g) eluting with pentane/Et2O (2/1, V/V) at 12 mL/min to obtain both diastereisomers 9 and 12: Rf = 0.4 (pentane/Et2O (2/1, V/V)). Yield: 10%, 0.13 g. 1H-NMR (CDCl3, 400 MHz): δ 5.42–5.59 (m, 2H), 3.70–3.73 (m, 2H), 3.50–3.55 (m, 1H), 3.39–3.45 (m, 1H), 2.12–2.53 (m, 5H), 3.70–3.73 (m, 2H), 1.98– 2.03 (m, 1H), 1.70–1.85 (m, 3H), 1.44–1.53 (m, 1H), 1.12–1.23 (m, 2H). Compound 9 has the same Rf as the starting material 11 and it was obtained as a mixture (1:1) of 9 and 11 (0.39 g). The mixture was purified passing through a column packed with 4 g of AgNO3 (10%) impregnated silica and eluted with 50 mL of EtOAc to remove the starting material 11. The silica was then added to an NH4OH solution (28 mL, 28%). The suspension was stirred for 5 min, 28 mL of Et2O was added and stirring continued for 5 min. The aqueous layer was extracted 2 more times with Et2O (2 × 28 mL) and the combined organic phases were washed with water, dried over MgSO4 and concentrated under vacuum to obtain 9 as a colorless oil. Rf = 0.2 (pentane/Et2O (2/1, V/V)). Yield: 9%, 0.12 g. 1H-NMR (CDCl3, 400 MHz): δ 5.54–5.64 (m, 1H), 5.34–5.41 (m, 1H), 3.64–3.70 (m, 2H), 3.46–3.54 (m, 1H), 3.37–3.42 (m, 1H), 3.01–3.05 (m, 1H), 2.34–2.40 (m, 2H), 2.07–2.29 (m, 2H), 1.90–2.03 (m, 2H), 1.74–1.86 (m, 3H), 1.44– 1.56 (m, 2H). Please cite this article as: wyffels L., et al, In vivo evaluation of (2014), http://dx.doi.org/10.1016/j.nucmedbio.2014.03.023

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2.3. Radiochemistry [ 18F]TCO was synthesized by modification of the method described by Li et al [10] on a Veenstra FluorSynthon III synthesis module. Cyclotron produced [ 18F]F − solution was transferred to the hotcell and passed over a preconditioned QMA Sep-Pak Light cartridge (Waters) to recover the [ 18O]H2O. The fixed [ 18F]F − was then eluted from the QMA cartridge with a solution of Kryptofix 222 (4 mg) and K2CO3 (26.7 mg) in 1 mL acetonitrile (CH3CN)/H2O (95/5, V/V) and the eluate was collected in a reaction vial. The solvents in the reaction vial were removed under a stream of helium and vacuum at 130 °C for 7 min. The remaining traces of water were removed by azeotropic distillation with CH3CN (2 × 1 mL) and heating at 140 °C under gentle vacuum and a stream of helium. The reaction vial was left to cool to 90 °C after which the precursor solution (2 mg of 14 in 800 μL anhydric CH3CN) was added and the mixture was left to react at 90 °C for 10 min. After cooling the reaction to 75 °C, 1 mL of sodium acetate (NaOAc 0.05 M) was added and the suspension was loaded onto a HPLC loop through a preconditioned Alumina N Light cartridge (Waters). [ 18F]TCO was purified using a Phenomenex Luna C18(2), 250 × 10 mm, 10 μm HPLC column and a mixture of NaOAc 0.05 M pH 5.5/ethanol (60/40, V/V) as mobile phase at a flow rate of 4 mL/min. The fraction containing [18F]TCO (Rt = 10–11 min) was collected and transferred to a shielded LAF cabinet where it was diluted with 0.9% NaCl to reduce the ethanol concentration to b 10% and sterile filtered (Pall Acrodisc syringe filter, 13 mm, 0,2 μm Supor membrane). To evaluate the chemical and radiochemical purity reverse phase analytical HPLC analysis (Phenomenex Luna C18, 150 × 4.6 mm, 10 μm) was performed using an isocratic elution of 0.1% trifluoroacetic acid (TFA) in water/0.1% TFA in CH3CN (45/55, V/V) with sodium fluoride (NaF 0.01 M) at a flow rate of 1 mL/min. Co-injection with FTCO (15, 1 μg/mL) was used to confirm the identity of [ 18F]TCO. 2.4. Measurement of partition coefficient The partition coefficient (logDn-octanol/PBS pH 7.4) was determined according to the shake-flask method. For this, 2 mL of n-octanol (d = 0.827 g/mL) and 2 mL of phosphate buffered saline (PBS), pH 7.4 were mixed and left to equilibrate for 30 min. Next, 5 μL of purified [ 18F]TCO was added and the mixture was vortexed at room temperature for 2 min. The tube was centrifuged for 10 min at 1500 × g (Hermle Z326K, Hermle Labortechnik, Germany). Aliquots of 500 μL n-octanol and 500 μL PBS were pipetted into tared test tubes and the samples were weighed and counted for radioactivity in the automatic gamma counter. Corrections were made for the mass difference and density between the two phases. The partition coefficient D was calculated as: [radioactivity (cpm/mL) in n-octanol/radioactivity (cpm/mL) in PBS]. The logD values are reported as an average of three different measurements. 2.5. Animals Male C57BL/6J mice weighing 20–24 g (Charles River Laboratories, Belgium) were used. The animals were kept under environmentally controlled conditions (12h light/dark cycle, 20–24°C and 40–70% relative humidity) in IVC cages with food and water ad libitum. The study protocol was approved by the local Animal Experimental Ethical Committee of the University of Antwerp, Belgium (2012-25). All animal studies were ethically reviewed and carried out in accordance with European Directive 86/609/EEC Welfare and Treatment of Animals. 2.6. Ex-vivo biodistribution Male C57BL/6J mice (n = 18) received 18.5 MBq of [ 18F]TCO via the lateral tail vein and were sacrificed by cervical dislocation under isoflurane anesthesia at 5, 30, 60, 90, 120 or 240 min pi with n = 3 per

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time point. Blood was collected via cardiac puncture and thereafter the brain and main organs to be examined were rapidly removed, rinsed in PBS and blotted dry. Radioactivity in the samples was measured using an automatic gamma-counter. The uptake of radioactivity in blood and organs was expressed as percentage of the injected dose per gram of tissue plus or minus the standard deviation (%ID/g ± SD). 2.7. μPET Imaging Prior to ex-vivo biodistribution a subset of mice first underwent in vivo μPET imaging (n = 6). In this instance tracer injection was performed on the scanner under isoflurane anesthesia (5% for induction, 2% for maintenance) via a lateral tail vein catheterization. Accordingly a dynamic scan of 120 min was acquired and rebinned in static frames centered at 5, 30, 60, 90 and 120 min with a duration of 164 s, 272 s, 319 s, 385 s, 466 s and 563 s respectively. Thereafter the mouse was removed from the scanner and allowed to regain consciousness. At the time corresponding to 240 min pi the mouse was repositioned on the scanner and a 20 min static scan was acquired to which the durations of the earlier frames were matched for equal Poisson noise. At each of the aforementioned μPET scanning time points one animal was sacrificed for the ex-vivo biodistribution as described in 2.6. For quantitative analysis PET data were reconstructed with 2dimensional ordered subset estimation maximisation (OSEM2D) using 4 iterations and 16 subsets [17] following Fourier rebinning (FORE) [18]. Additionally for visual inspection the PET data were also reconstructed with OSEM3D/MAP using 2 OSEM3D iterations and 18 MAP iterations [19]. The PET images were reconstructed on a 128 × 128 × 159 grid with a pixel size of 0.776 × 0.776 × 0.776 mm. Normalization, dead time, random, CT-based attenuation and single scatter stimulation (SSS) [20] scatter corrections were applied. A 5 min CT was acquired subsequent to all PET scans, performed using a 220 degree rotation with 12-rotation steps, voltage and amperage were set to 80 keV and 500 uA respectively. For the visualization of cerebral uptake each individual PET image was transformed into the same stereotaxic coordinates of a standard mouse brain template [21] using brain normalization in PMOD v3.3 (PMOD Technologies, Switzerland) transforming the hardwarematched individual animal-specific CT to the template CT. Average images of cerebral uptake were then generated from the total number of mice at each time point and overlaid on a predefined T2-weighted MRI template which was a priori registered with the CT template for anatomical reference. For an absolute measure of tracer uptake, normalized images were scaled according to the percent injected dose (tissue uptake[kBq/cc]/injected dose[kBq] * 100).

To evaluate the in vivo plasma and brain stability of [ 18F]TCO, a metabolite study was performed. C57BL/6J mice (n = 7) were injected via the lateral tail vein with purified [ 18F]TCO (18.5– 37 MBq in max 200 μL). Five min (n = 3), 30 min (n = 3) or 120 min (n = 1) pi the animals were anesthetized using isoflurane and blood was withdrawn by cardiac puncture. Immediately thereafter the mice were sacrificed by cervical dislocation and the brain was rapidly removed. The blood was centrifuged at 4 °C for 7 min at 4500 × g to collect the plasma. An aliquot of the plasma (200–400 μL) was mixed with an equal amount of ice cold CH3CN for deproteination and spiked with 10 μL of 15 (1 mg/mL). The sample was counted for radioactivity in the automatic gamma counter, vortexed for 30 s and then centrifuged for 4 min at 4000 × g. The supernatant was separated from the pellet and both fractions were counted in a gamma counter to calculate the extraction efficiency (percent recovery of radioactivity in the CH3CN). Hundred microliters of the supernatant was analyzed by reverse phase HPLC using a Phenomenex Luna C18, 150 × 4.6 mm, 10 μm HPLC column coupled to a Phenomenex security guard pre-column. A mobile phase of 0.1% TFA in water/0.1% TFA in CH3CN (45/55, V/V) with 0.01 M NaF was used at a flow rate of 1 mL/min. The eluate was collected in fractions of 0.5 min and counted for radioactivity in the gamma counter. The brain was transferred to a tube and homogenized with 1 mL of ice cold CH3CN. Ten microliters of 15 (1 mg/mL) was added, the sample was counted for radioactivity, vortexed for 30 s and then centrifuged for 4 min at 4000 × g. The supernatant was separated from the pellet and both fractions were counted for radioactivity in the gamma counter. Hundred microliters of the supernatant was analyzed by reverse phase HPLC as described above. The radioactivity due to unchanged [ 18F]TCO was expressed as a percentage of the total peak areas. 2.10. In vitro metabolite analysis The metabolite profile of [ 18F]TCO was further evaluated by incubation of the tracer with human liver microsomes (BD Gentest). For this, 400 μL [ 18F]TCO (about 9 MBq dissolved in 0.1 M NaKHPO4 buffer, pH 7.4) was pre-incubated with 500 μL liver microsomes (2 mg/mL in 0.1 M NaKHPO4 buffer, pH 7.4) for 5 min at 37 °C with gentle shaking. Next, 100 μL NADPH (10 mM in 0.1 M NaKHPO4 buffer pH 7.4) was added and the mixture was left to incubate at 37 °C for 0 min (n = 3), 2 min (n = 3), 5 min (n = 3) or 30 min (n = 2) while gently shaking. The incubation was followed by addition of 2 mL of CH3CN after which the sample was mixed on a vortex and spun at 4 °C for 5 min at 4000 × g. The supernatant was removed and 100 μL was analyzed by HPLC as described above. The amount of unchanged [ 18F]TCO was expressed as a percentage of the total peak areas. 3. Results and discussion

2.8. Autoradiography

3.1. Chemistry

For the ex-vivo visualization of cerebral tracer distribution at 5, 30, 60 and 90 min pi (n = 1 per time point), the brain was hemisected along the sagittal midline and processed for autoradiography. The right hemisphere was snap-frozen and subsequently sectioned on a cryostat (Leica CM1950). Sections (20 μm) were dried at 37 °C for 30 min and exposed to Fujifilm BAS IP MS 2025 plates overnight. The plates were imaged using a Fuji Phosphoimager system (FLA7000).

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-1yloxy)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 (2:1) of diastereoisomers 4a and 4b. Treatment of the phosphine oxides 4a and 4b with NaH allows the formation of the double bond and gave access to substituted TCO 5 and 6. The two diastereoisomers were separated by column chromatography and were also obtained in a 2:1 ratio (5, 34% and 6, 18%). Deprotection of the t-butyltrimethylsilyl (TBDMS) group was performed using a solution of TBAF 1 M in THF. Monitoring the reaction is crucial to avoid isomerization of the double bond.

2.9. In vivo metabolite analysis To determine the recovery of [ 18F]TCO, as well as the stability of the tracer during the workup, control experiments (n = 3) were done using blood and brain spiked in vitro with 185 kBq of [ 18F]TCO. Sample workup was identical as described hereafter for the main metabolite experiment. Please cite this article as: wyffels L., et al, In vivo evaluation of (2014), http://dx.doi.org/10.1016/j.nucmedbio.2014.03.023

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Scheme 1. Synthesis of functionalized trans cyclooctenes. Reagents and Conditions: a) LiAlH4, THF, reflux 4 h, 93%; b) TBDMS-Cl; Et3N, Imidazole, CH2Cl2; c) MCPBA, CH2Cl2, RT 5 h, 42% over two steps; d) lithium diphenyl phosphate, RT, overnight; acetic acid and hydrogen peroxide, RT 1 h, 63%; e) NaH, RT 3 h, 18–34%; f) TBAF, THF, reflux, 40%; g) TBAF, THF, reflux, 76%; h) NaH, THF, BrCH2COOH, reflux, 1 h; i) LiAlH4, THF, 0 °C to RT, overnight, 47% over two steps.

After 35–45 min of reflux, the reaction was completed and the NMR of the crude showed no presence of the cis-cyclooctene 2. Alkylation of 8 with 2-iodoacetic acid followed by reduction of the carboxylic acid using LiAlH4 afforded TCO 9 in a 55% yield (Scheme 1). In most of the current literature on TCO, photoisomerization of the double bond of the (Z)-2-(cyclooct-4-ene-1-yloxy)ethanol 11 is performed using a cycle/trap system [3,12,16]. More recently, an alternative to this system has been published by Schoch et al. using a UVP-CL-1000-ultraviolet crosslinker as UV lamp which significantly reduces the cost of the synthesis [24]. We successfully used this UV method to do the photoisomerization of 11. The mixture of 9 and 12 was purified by column chromatography and the minor diastereoisomer 12 was isolated in 10% yield. The major isomer 9 was obtained in a 15% yield but it appeared to have the same Rf as the starting material and was thus contaminated with cis-isomer 11. To remove 11 an extra purification on silver nitrate impregnated silica was necessary but resulted in lowering the yield of the major isomer 9 to 9%. This method is preferable to the phosphine oxide elimination (Scheme 1) because it is four steps shorter and results in a better overall yield (6.18% instead of 1.58% for phosphine oxide elimination). Compared to the method described by Fox et al. [16], we had a lower yield but the compounds are still obtained in a 100 mg scale

which is acceptable for preparation of labeling precursor and cold reference standard. For the preparation of the precursor for radiolabeling, we initially tried to do the nosylation on compound 9 but we observed formation of 30% of the cis cyclooctene 13 (Scheme 3) [16]. In the literature, nosylate 12 is purified from this cis cyclooctene 13 by preparative HPLC [16]. Tosylation of the alcohol 9 afforded the expected product 14 (55%, Scheme 3) without traces of the isomerization to the cis form. We therefore chose to proceed with the tosylate precursor for radiosynthesis. Fluorination with TBAF afforded the cold reference standard 15 in 55% yield (Scheme 3). 3.2. Radiosynthesis of [ 18F]TCO and partition coefficient The radiosynthesis of [ 18F]TCO was performed in a Fluorsynthon III automated synthesis module by modification of the procedure described by Li et al. [10]. Tosylate precursor 14 in dry CH3CN was reacted with dried [ 18F]KF-K2.2.2 solution for 10 min at 90 °C and diluted with NaOAc buffer following reaction. By passing the crude reaction mixture through an Alumina N cartridge, most of the unreacted [ 18F]F − was removed before injection onto the HPLC. The reaction mixture was purified by reverse phase semi-preparative

Scheme 2. Synthesis of functionalized trans cyclooctenes via UV irradiation. Reagents and conditions: a) NaH, THF, BrCH2COOH, reflux, 1 h, 85%; b) LiAlH4, THF, 0 °C to RT, overnight, 87%; c) 254 nm, methyl benzoate, Et2O, Hexanes, 0 °C, 8 h, 9–10%.

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Scheme 3. Synthesis the nosylate (A) and tosylate (B) precursor for radiosynthesis and the cold reference standard (B). Reagents and conditions: a) pNs-Cl, Et2O, Et3N, b) pTs-Cl, DCM, Et3N, DMAP, RT overnight, 56%; c) TBAF, reflux, 3 h, 55%.

HPLC using a mixture of NaOAc 0.05 M pH 5.5 and ethanol as mobile phase. By using this bio-compatible mobile phase for purification of the tracer, the need for a post HPLC SPE purification could be eliminated. By simply diluting the collected fraction with sterile 0.9% NaCl to reduce the ethanol concentration to b 10% followed by a sterile filtration, an injectable tracer solution could be obtained. Using this procedure, [ 18F]TCO could be obtained in a total synthesis time of

55 min from EOB, including formulation. [ 18F]TCO could be obtained in a decay corrected radiochemical yield of 14 ± 5% and with a radiochemical purity of N 99%. By co-elution of [ 18F]TCO and 15, the identity of the tracer could be confirmed. The tracer was stable for at least 4 h post radiosynthesis as determined by HPLC analysis. Using the shake-flask method, a log D value of 1.68 ± 0.02 was obtained. A logD value between 1 and 4 is considered to be the

Fig. 1. Average brain images from C57/BL6J mice injected with 18.5 MBq of [18F]TCO and scanned under isoflurane anesthesia. PET images, expressed in %ID/g, are presented in the coronal, transverse and sagittal orientation and are co-registered to a T2-weighted template for anatomical reference. Cerebral uptake of [18F]TCO and its metabolites follows a distinctive pattern with a high initial uptake (1 min) and rapid washout (5–60 min). At later time points a second influx of radioactivity, corresponding to non-specific uptake, is evident (90–120 min) and gradually clears from the brain (240 min).

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Fig. 2. In vivo biodistribution of radioactivity in a male C57/BL6J mouse scanned under isoflurane anesthesia up to 240 min post injection of 18.5 MBq [18F]TCO. All images are expressed as %ID/g, and are presented in the transverse and sagittal orientation. These images indicate clearance of [18F]TCO by predominantly renal and hepatobiliary excretion and demonstrate high bone uptake at 240 min post injection; note: images were smoothed with a three dimensional isotropic Gaussian filter of 1.4 mm for visualization purposes.

optimum for blood brain barrier penetration. Therefore, [ 18F]TCO is expected to be able to enter the brain.

3.3. μPET imaging and biodistribution studies To evaluate the brain penetration and pharmacokinetic profile of [ 18F]TCO, ex-vivo biodistribution in parallel with in vivo μPET was performed. Adult C57BL/6J mice were injected intravenously (iv) with radiotracer and imaged up to 240 min pi. [ 18F]TCO clearly shows a high initial brain uptake followed by a washout (Fig. 1). At 60 min pi, a gradual increase in uptake of radioactivity in the brain is visible with a peak uptake at 120 min pi. Radioactivity uptake in the brain then starts decreasing again until 240 min pi. Representative transverse whole body images at 1 min, 5 min, 30 min, 60 min, 90 min, 120 min and 240 min pi are shown in Fig. 2. A combination of hepatobiliary and renal clearance of the radioactivity could be detected with bladder activity dominating at later time points. At 120 min post tracer skeletal uptake of radioactivity becomes gradually visible. At 240 min

pi high bone uptake is clearly visible especially in the spine, tail bone and joints. Quantitatively, the distribution of 18F radioactivity in various tissues as a function of time following iv administration of [ 18F]TCO is presented in Fig. 3. The biodistribution study showed an initial high accumulation of radioactivity in the kidneys (7.00 ± 0.97%ID/g at 5 min pi) followed by a decrease until 240 min pi (2.46 ± 0.63%ID/g) and an increase in uptake of radioactivity until 90 min pi in the small (4.50 ± 1.04%ID/g at 5 min and 5.56 ± 2.04%ID/g at 90 min pi) and large intestine (3.15 ± 0.45%ID/g at 5 min and 7.10 ± 3.08%ID/g at 90 min pi) indicating a mixed renal and hepatobiliary clearance of [ 18F]TCO as was also evidenced by the PET images. The tracer showed an initial slow blood clearance (3.71 ± 0.40%ID/g at 5 min and 3.45 ± 0.45%ID/g at 60 min pi) which was followed by a gradual increase in blood pool activity to 5.44 ± 1.37%ID/g at 120 min pi. At 240 min pi the blood pool activity finally decreased to 2.83 ± 0.94%ID/g. No significant accumulation of activity over time could be detected in heart, lungs, stomach, spleen, pancreas, liver and muscle. Only in fat tissue an increasing uptake of radioactivity could be detected until

Fig. 3. Tissue distribution (%ID/g ± SD) at different time points post injection of [18F]TCO in C57BL/6J mice.

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60 min pi (7.50 ± 4.08%ID/g) followed by a gradual decrease until the end of the study (0.90 ± 0.36%ID/g). In line with the μPET data, the ex-vivo biodistribution study demonstrated an initial high brain uptake of [ 18F]TCO (3.74 ± 0.68%ID/g at 5 min pi) followed by a decline until 30 min pi (3.03 ± 0.52%ID/g). Similarly, at later time points the uptake of activity in the brain started rising again (3.26 ± 0.52%ID/g at 60 min pi) with a plateau occurring between 90 and 120 min (3.66 ± 0.84%ID/g and 3.66 ± 1.0%ID/g, respectively). At

240 min pi, radioactivity uptake in the brain declined again to 2.83 ± 0.94%ID/g. The initial brain uptake followed by a wash-out clearly indicates that the tracer is able to pass the blood brain barrier. However, the increased uptake of radioactivity in the brain at later time points is indicative of an unfavorable metabolite profile with possible penetration of the blood brain barrier and accumulation of metabolites in the brain. Highest uptake could be detected in bone with an increase from 2.04 ± 0.70%ID/g at 5 min pi to 15.2 ±

Fig. 4. Representative radiochromatograms of plasma and brain samples at 5 min (A), 30 min (B) and 120 min (C) post iv injection of [18F]TCO (18.5–37 MBq).

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L. wyffels et al. / Nuclear Medicine and Biology xxx (2014) xxx–xxx Table 1 Metabolite profile in C57BL/6J mice at 5 min (n = 3), 30 min (n = 3) and 120 min (n = 1) after iv injection of [18F]TCO. Tissue

Time pi (min)

5 min⁎ 30 min⁎ 120 min plasma 5 min⁎ 30 min⁎ 120 min brain

Retention time on RP-HPLC (min) 2.4 M3

3.2 M4

Table 2 Metabolite profile of [18F]TCO following incubation with human liver microsomes for 0 min (n = 3), 2 min (n = 3), 5 min (n =3) or 30 min (n = 2). Incubation time (min)

1.5 M1

1.8 M2

8.7 [18F]TCO

6.7 ± 0.9 20.8 ± 3.4 87.8 23.2 ± 2.0 59.3 ± 5.8 88.6

28.5 ± 4.8 32.6 ± 5.5 32.2 ± 5.8 70.0 ± 10.1 7.3 ± 4.2 1.9 ± 1.7 12.2 27.3 ± 5.8 10.6 ± 0.3 2.3 ± 4.5 10.7 ± 5.6 38.7 ± 5.2 2.0 ± 1.8 11.9

9

0 min 2 min 5 min 30 min

Retention time on RP-HPLC (min) 1.6 M1

1.8 M2

5.9 ± 0.5 5.2 ± 0.2

0.9 3.0 4.1 24.5

± ± ± ±

0.5 1.4 0.7 0.2

2.6 M3

3.2 M4

3.1 ± 1.7 6.4 ± 1.1 9.5 ± 0.3

2.1 23.6 70.4 59.8

8.5 [18F]TCO ± ± ± ±

0.1 14.9 0.8 0.5

97.3 70.4 13.2 1.0

± ± ± ±

0.7 17.8 1.2 0.2

Values are expressed % of total radioactivity, mean ± S.D.

⁎ Values are expressed % of total radioactivity, mean ± S.D. (n = 3).

2.27%ID/g at 240 min pi. This accumulation of radioactivity in the bone is also an indication of tracer instability. The high uptake in the bone might be the result of gradual defluorination of the tracer or a metabolite. [ 18F]F - is known to have a high affinity for bone and the resulting skull uptake will render quantification of brain uptake of radiotracers inaccurate due to ‘spill-over’ of radioactivity from the skull to the nearby brain tissue through the partial volume effect. Since 18F bound to an aliphatic carbon atom is often prone to defluorination, we envisioned that this might also be problematic for [ 18F]TCO. Although tracer defluorination is known to result in high skull and other bone uptake [25,26], it is not until 240 min pi that the bone uptake becomes prominently visible in the PET images. This makes us believe that defluorination of [ 18F]TCO or its metabolites only becomes problematic after 120 min pi. 3.4. Autoradiography analysis To further explore brain distribution of 18F radioactivity, half of the brain of 4 animals were cut in 20 μm-thick sections for autoradiography analysis (data not shown). From this analysis, no regional differences in brain uptake of radioactivity could be detected. A uniform brain distribution was visible at all the time points (5, 30, 60, 90 min) investigated indicating no region specific binding of the tracer or its metabolites. 3.5. In vivo metabolite assay Since the μPET imaging and the biodistribution study indicated a possible problematic metabolite profile for [ 18F]TCO, a metabolite assay was performed. For this, adult C57BL/6J mice were iv injected with radiotracer and at different time points pi plasma and brain were analyzed by HPLC for presence of radiometabolites. In the validation study extraction yields of 83.2 ± 1.60% for brain and 81.3 ± 9.98% for plasma were determined and no degradation of the tracer occurred during work-up. The radio-HPLC metabolite analysis revealed a fast degradation of [ 18F]TCO in plasma as well as in brain tissue following iv injection of the tracer. In plasma at 5 min pi, the concentration of unmetabolized

[ 18F]TCO (Rt = 8.7 min) represented 10.7 ± 5.6% of the total radioactivity (Fig. 4, Table 1). Four main polar radiometabolite peaks could be detected in the plasma eluting with retention times of 1.5 min (M1; void volume), 1.8 min (M2), 2.4 min (M3) and 3.2 min (M4) respectively (Fig. 3). At 30 min pi no intact tracer could be detected in plasma and metabolites M3 (not present) and M4 (2.0 ± 1.8%) were significantly reduced while the most polar metabolites M1 and M2 increased to 59.3 ± 5.8% and 38.7 ± 5.2% of total radioactivity, respectively. At 120 min pi, M1 accounted for most of the plasma radioactivity. Isomerization of trans-cyclooctene to ciscyclooctene, as has been described for a TCO analogue by Rossin et al. following incubation in mouse serum [8], was not observed. In the brain on the other hand, more intact [ 18F]TCO could be detected at 5 min pi (32.2 ± 5.8% of the total radioactivity), in the presence of polar metabolites M1, M2 and M4. These polar metabolites might thus also be generated in the brain or be taken up from the plasma into the brain. The polar metabolite M3 was not detectable at any time point investigated. At 30 min pi only a very small amount of intact tracer (b2%) was detected in the brain and the ratio of the three polar metabolites changed with a negligible presence of M4 and a higher amount of M2 in comparison to M1. Thus while in the plasma at 30 min pi, M1 is the dominant metabolite, in the brain M2 is the more dominant metabolite. At 120 min pi, the metabolite profile of the brain resembled that of the plasma with no intact [ 18F]TCO present and M1 representing the main metabolite. While the extraction yields from plasma (88.0 ± 0.29%) remained relatively constant, the extraction of radioactivity from the brain decreased as a function of time (81.7%, 65.8% and 59.3% for 5 min, 30 min and 60 min respectively). This is presumably because of the higher proportion of radioactivity in the brain represented by poorly extractable 18F-labeled metabolite(s). Biodistribution studies revealed a substantial accumulation of radioactivity in bone at the later time points of 120 and 240 min pi. We hypothesized the source of this activity to be [ 18F]F −, a common product of defluorination which binds avidly to bone. To discern whether one of the radiometabolites of [ 18F]-TCO was indeed [ 18F]F − we spiked 1 mL of blood with cyclotron-produced [ 18F]F − and processed the sample as described in 2.9. Fig. 5 shows the obtained radiochromatogram after injection of the supernatant onto HPLC. The polar radioactive peak representing [ 18F]F − was broad and eluted at a retention time of 2.2 min and did not resemble the sharper peaks of

Fig. 5. Representative radiochromatogram of extracted plasma sample obtained after spiking of whole mouse blood with [18F]F−.

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Fig. 6. Possible metabolic pathway for [18F]TCO.

M1 (1.5 min), M2 (1.8 min) or M3 (2.4 min). Therefore none of the radiometabolites observed at 5, 30 or 120 min in the brain or plasma can be unambiguously identified as [ 18F]F −. 3.6. In vitro metabolite assay To further evaluate the metabolite profile, an in vitro metabolite assay was performed by incubating [ 18F]TCO with human liver microsomes. For the in vitro assay, the same radiometabolites as those detected in plasma were observed but the relative amount of individual metabolites differed between the in vitro (Table 2) and in vivo assay (Table 1). Where M1 and M2 were the more dominant polar metabolites in vivo for the plasma, M4 is the dominant metabolite in the in vitro study. At 2 min post incubation, 70.4 ± 17.8% of intact tracer was still remaining with the main metabolite arising at a retention time of 3.2 min (M4). At 5 min post incubation, only 13.2 ± 1.2% of intact tracer remained in the presence of M4 and three minor more polar metabolites M1, M2 and M3. At 30 min post incubation almost no intact [ 18F]TCO could be detected (1.0 ± 0.2%) while M4 remained the most dominant metabolite (59.8 ± 0.5%) in the presence of M1 (5.2 ± 0.2%), M2 (24,5 ± 0.2%) and M3 (9.5 ± 0.3%). It must be noted that human liver microsomes were used for this in vitro assay what might explain the difference in relative amounts of polar metabolites in the in vitro assay compared to the in vivo assay which was performed in mice. Liver microsomes contain many metabolizing enzymes of which the super family of cytochrome P450s (CYPS) is the most prominent one. It therefore is likely that CYPs are involved in the formation of the polar metabolites. Comparison of the in vitro and in vivo radiochromatograms indicates that [ 18F]TCO is metabolized to a more polar radiometabolite M4 which might in turn undergo oxidative metabolism to M2 and further to M1. The polar metabolites probably cross the blood-brain-barrier. Since M1 is the predominant metabolite at later time points, the non-specific accumulation of radioactivity in brain tissue and bone might be related to this metabolite. This non-specific uptake of radiometabolites in the brain will attenuate the signal coming from the intact tracer in PET imaging and will seriously exacerbate quantification. Based on the obtained data, a possible metabolic pathway was proposed for [ 18F]TCO in Fig. 6. CYP mediated O-deethylation has been described before for other tracer containing a [ 18F]fluoroethyl group like [ 18F]DPA-714 and [ 18F]PBR102 [27]. This dealkylation will result in the formation of radiometabolites [ 18F]fluoroethanol, [ 18F]fluoroacetaldehyde and [ 18F]fluoroacetic acid which are expected to be metabolically interchangeable in vivo and to be able to cross the blood-brain-barrier [28]. In rodents, in vivo defluorination of [ 18F] Please cite this article as: wyffels L., et al, In vivo evaluation of (2014), http://dx.doi.org/10.1016/j.nucmedbio.2014.03.023

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fluoroacetic acid has been described, resulting in accumulation of radioactivity in skeletal structures [29]. The latter can explain the increasing bone uptake that was seen in the biodistribution study and μPET imaging. To fully characterize the metabolite profile an LC-MS analysis should be carried out. 4. 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 three main radiometabolites, all of them more polar than the intact tracer. At later time points bone uptake becomes clearly visible indicating possible further defluorination of the metabolites. The fast metabolism and presence of radiometabolites in the brain will seriously hamper the usefulness of [ 18F]TCO for in vivo pre-targeted imaging in the brain using the Diels-Alder cycloaddition reaction. New, metabolically more stable [ 18F]TCO analogues need to be developed that are able to penetrate the blood-brain-barrier while preserving selectivity and a fast rate of reactivity towards tetrazines. 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. References [1] Blackman ML, Royzen M, Fox JM. Tetrazine Ligation: Fast Bioconjugation Based on Inverse-Electron-Demand Diels-Alder Reactivity. J Am Chem Soc 2008;130:13518–9. [2] Devaraj NK, Weissleder R, Hilderbrand SA. Tetrazine-Based Cycloadditions: Application to Pretargeted Live Cell Imaging. Bioconjug Chem 2008;19:2297–9. [3] Royzen M, Yap G, Fox J. A photochemical synthesis of functionalized transcyclooctenes driven by metal complexation. J Am Chem Soc 2008;130:3760–1.

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F-labeled TCO for pre-targeted PET imaging in the brain, Nucl Med Biol