Animals

Experiments with animals ([¹⁸F]4, n = 17; [¹⁸F]FMPEP-d₂, n = 19) were conducted under the guidelines of the International Council of Laboratory Animal Science (ICLAS). This study was approved by the Finnish National Animal Experiment Board (ESAVI/16273/2019 and ESAVI/4660/04.10.07/2016). Experimental animals were housed at the Central Animal Laboratory of the University of Turku under standardized conditions (temperature 21 °C, humidity 55% ± 5%, lights on from 7:00 a.m. to 7:00 p.m.), with free access to certified standard laboratory soy-free chow (RM3 soya-free, 801710, Special Diets Service) and tap water. Studies with [¹⁸F]4 were conducted with healthy wild-type 2 (n = 11, males, weight 25.5 ± 1.3 g) and 4 to 5 month old (n = 6, 2 females, weight 32.1 ± 4.2 g) C57BL/6NRj mice (Charles River Laboratories, Research Models and Services, Germany). The ex vivo biodistribution study with [¹⁸F]FMPEP-d₂ was conducted with healthy wild-type 3–4 month old (n = 7, males, weight 32.8 ± 4.6 g) and 1.5–6 month old (n = 12, 5 females, weight 26.5 ± 6.6 g) C57BL/6N mice. The injected volume of the tracer was 155 ± 42 μL.

Dynamic PET scans were performed for 5 month old mice with an Inveon Multimodality PET/CT scanner (Siemens Medical Solutions USA, Knoxville, Tennessee). Mice were anesthetized with 2.5% isoflurane/oxygen gas, and body temperature was maintained with a heating pad and a bubble-wrap cover. CT transmission scans were performed to correct for attenuation. PET scans were initiated in parallel with an intravenous (iv) bolus injection of [¹⁸F]4 to the mouse tail vein (4–5 month old, n = 6, 2 females; injected radioactivity 4.0 ± 0.4 MBq; injected mass 56 ± 25 ng, 1.7 ± 0.7 μg/kg).

Dynamic emission scans were acquired for 120 min in 3D list mode, with an energy window of 350–650 keV. Scanning times were 120 min (54 frames 30 × 10 s, 15 × 60 s, 4 × 300 s, 2 × 600 s, 3 × 1200 s). The list mode data were stored in 3D sinograms. Data were reconstructed with OSEM3D/MAP software (2 OSEM3D iterations and 18 MAP iterations). The PET/CT images were preprocessed in Matlab R2017a (The MathWorks, Natick, Massachusetts) with an in-house semiautomated pipeline for preclinical images that uses SPM12 (Wellcome Department of Cognitive Neurology, London, UK) preprocessing functionalities and analysis routines. Images were first cropped to a bounding box containing the heads, and individual PET images were coregistered through a rigid-body transformation to their corresponding CT scan. Subjects were spatially normalized through a two-step registration (a rigid followed by an affine transformation) of each subject’s CT to a template CT that was previously constructed as an average of several subjects and was aligned with an atlas T2-weighted MRI template. The combination of transformations was then applied to the PET images, which were also resampled to a voxel size of 0.2 × 0.2 × 0.2 mm³ (trilinear interpolation), matching the anatomical atlas dimensions.

Volume of interest (VOI) analysis of the whole brain, neocortex, and hippocampus was performed on each subject by averaging the signal inside a slightly modified version of the Ma et al.⁴⁰ atlas delineated VOIs, and data were obtained as standardized uptake values (SUVs). SUVs were calculated as follows: SUV = tissue activity concentration (Bq/mL)/(injected dose (Bq)/body weight (g)); it was assumed that 1 mL of tissue equals 1 g. Tissue activity and dose were decay corrected to the same time point.

All in vivo animals (n = 6, 2 females) and additional 2 month old mice (n = 11, males, injected radioactivity 7.0 ± 3.0 MBq; injected mass 160 ± 60 ng, 6.2 ± 2.6 μg/kg) were used for ex vivo studies to examine the biodistribution and metabolism of [¹⁸F]4. Thus, the injected dose and injected mass to all the animals used (n = 17) were 5.9 ± 2.8 MBq and 120 ± 70 ng, 4.6 ± 3.0 μg/kg, respectively. After iv injection of [¹⁸F]4, mice were sacrificed in deep anesthesia of 4.0% isoflurane with cardiac puncture at 30 (n = 4), 60 (n = 2), and 120 min (in vivo animals, n = 6 and 2 month old mice n = 5). Organs were immediately dissected, weighed, and measured for ¹⁸F-radioactivity in a 2480 Wizard Gamma Counter (Wallac PerkinElmer, Turku, Finland).

For autoradiography, brains were further frozen and cut into 20 μm coronal cryosections for digital autoradiography, and the slices were exposed on an imaging plate (BAS-TR2025, Fuji Photo Film Co.) for approximately 2 half-lives and scanned with a Fuji BAS5000 phosphorimager (FUJIFILM Life Science, Stamford, Connecticut). Digital autoradiographic images were analyzed for count densities with the Aida 4 program (Raytest Isotopenmessgeräte GmbH, Straubenhardt, Germany). For brain slices, regions of interest (ROIs) were drawn over the parietotemporal cortex, striatum, frontal cortex, hippocampus, cerebellar gray matter, and thalamus. The autoradiography was quantified as ratios of the different ROIs relative to the thalamus, which was used as the reference region.

After iv injection of [¹⁸F]FMPEP-d₂ to the mouse tail vein, the mice were sacrificed at the following time points: 30 min (3–4 month old, n = 3, males; injected radioactivity 1.3 ± 0.3 MBq; injected mass 1.5 ± 0.5 ng), 60 min (3–4 months old, n = 4, males; injected radioactivity 1.2 ± 0.8 MBq; injected mass 1.3 ± 1.0 ng), and 120 min (1.5–6 month old, n = 12, 5 females; injected radioactivity 2.6 ± 0.8 MBq; injected mass 3.1 ± 1.1 ng). Mice were sacrificed in deep anesthesia of 4.0% isoflurane with cardiac puncture. Organs were operated in the similar manner as in the biodistribution study with [¹⁸F]4.

The specificity of [¹⁸F]4 on mouse cerebral CB1Rs was examined in a 120 min in vivo study, and then, the same mice were used for ex vivo biodistribution and brain autoradiography studies. An inverse CB1R agonist rimonabant (90 μL, 2 mg/kg in 10% EtOH in Kleptose β-cyclodextrin; rimonabant, Merck; Kleptose β-cyclodextrin, APL pharma specials, Stockholm, Sweden) was administered iv into the mouse tail vein (n = 3, 1 female) 10 min prior to injection of [¹⁸F]4. Control mice (n = 3, 1 female) were the given vehicle (90 μL, 10% EtOH in Kleptose β-cyclodextrin). The binding specificity of [¹⁸F]4 was estimated by comparing the binding of [¹⁸F]4 in the presence and absence of rimonabant. Mice were sacrificed 120 min after iv injection of [¹⁸F]4, and the ¹⁸F-radioactivity distribution differences between pretreated and control mice were examined with in vivo PET imaging, ex vivo autoradiography, and 2480 Wizard Gamma Counter, and the data were analyzed as described in the previous sections.

The 2 month old animals (n = 11, used only for ex vivo studies) were used for metabolite analysis. Plasma and brain samples were collected at 30 (n = 4), 60 (n = 2), and 120 min (n = 5) p.i. Plasma samples were treated with MeCN and centrifuged to remove the proteins from the solution. Brain tissue samples were treated with the 40/60 (v/v) 1% TFA (aq)/MeCN solution and then centrifuged. The supernatants were applied to HPTLC silica gel 60 RP-18 plates (Art. 1.05914.0001, Merck, Darmstadt, Germany), and the plates were developed in 40/60 (v/v) 1% TFA (aq)/ MeCN. After drying, the TLC plates were exposed to an imaging plate for approximately 4 h and the imaging plate was scanned with a Fuji Analyzer BAS5000. The amount of [¹⁸F]4 and its radioactive metabolites were analyzed with the Aida 4 program, and the curves were fitted with GraphPad Prism 6.0 (GraphPad Software, San Diego, California) using a double exponential decay equation.

Values are expressed as mean ± standard deviation (SD). For ex vivo brain autoradiography and biodistribution, statistical analyses were performed with the Mann–Whitney U test (GraphPad Prism 6.0).