Experiments were conducted on four young adult male cynomolgus monkeys (Macaca fascicularis, 5.2 ± 1.1 kg, 4.4 ± 0.7 years). Animal use procedures were in accordance with the recommendations of the European regulations (EU Directive 2010/63) and approved by the local ethical committee (CETEA n°44), and the French Ministry of Education and Research (NEUROMODEL: APAFIS#389-20150327162135690v02). The experimental data reported in this study are in compliance with the ARRIVE (Animal Research: Reporting in Vivo Experiments) guidelines (26).
Radiosynthesis of the enantiomeric ligand [18F]UCB-H was realized through a one-step radiolabelling of a pyridyliodonium precursor as previously described (27). [18F]UCB-H was formulated in 0.9% aqueous saline with 3% ethanol (v/v). The radiochemical purity of [18F]UCB-H was > 98% and the molar activity at the time of injection was 54±32 GBq/μmol.
Experimental Design - All four non-human primates (NHP) underwent a 2-hour (2hr) test and retest PET scan; two NHP underwent an additional 4hr retest PET scan. Three NHP underwent one 4-hour (4hr) displacement PET scan. Arterial blood sampling was performed during test and retest scans with PET imaging spaced by at least 3 weeks between each scan. To this end, the femoral artery opposite to the saphenous vein used for radioligand injection was cannulated. For the 2hr scans, we collected in total 28 blood samples of 1 mL: 16 samples during the first 5 minutes followed by 3 samples every 5 minutes and 11 samples every 10 minutes. For the 4hr scans, we collected an additional 6 samples every 20 minutes during the last two hours (34 samples in total). Larger samples (2 – 3 mL) were collected at 5, 15, 30, 60, 90, 120 minutes (2hr) and at 180, 240 minutes (4hr) for metabolite analysis. Displacement of [18F]UCB-H was done by intravenous (IV) administration 90 minutes after [18F]UCB-H injection of a bolus of 30 mg/kg LEV, or of 80 µg/kg (≈250 nmol/kg) or 5 mg/kg (≈15 µmol/kg) of cold [19F]-UCB-H.
Drug Formulation - Solvents and LEV (C8H14N2O2; MM 170.21 g/mol) were obtained at Sigma-Aldrich® (France), and [19F]-UCB-H (C16H12F4N2O; MM 324.28 g/mol) was synthesised as previously described (27). Injectable solutions of [19F]-UCB-H for displacement studies were prepared with a mixture of Tetrahydrofuran (THF), Médialipide® and glucose at 2.5% as previously described (28). LEV was dissolved in a glucose solution of 2.5% to reach a concentration of 90 mg/mL.
PET imaging – PET imaging was performed on the microPET FOCUS220 (Siemens) under standard anesthesia and monitoring procedures (29). Data acquisition started with the IV bolus injection of [18F]UCB-H (32.9±1.0 MBq/kg, 0.35±0.07 µg/kg). Dynamic PET images were reconstructed using standard OSEM-2D algorithms while correcting for radioactive decay, scatter, attenuation and detectors inhomogeneity, which were measured prior to PET scanning using respectively 57Co and 68Ge external sources.
Blood Measurement & Analysis - Plasma was separated from whole-blood by centrifugation (5 minutes, 2054xg, 4°C) and 50 µL of plasma and whole-blood were counted using a PET cross-calibrated gamma well counter (WIZARD2, PerkinElmer, France) to obtain the whole-blood and plasma activity curves. All data were corrected for radioactive decay from the injection time. For the larger blood samples, 500 µL plasma was deproteinized with acetonitrile. The supernatant was injected in high-performance liquid chromatography, equipped with an Atlantis® T3 5μm 4.6x150mm column (Waters) and an Atlantis® T3 5μm 3.9x5mm pre-column (Waters), with an LB-513 radioactivity flow detector (Berthold, La Garenne Colombes, France, MX Z100 cell). The eluant was collected in interval of 15 seconds (fraction collector III, Waters, France) and counted in the gamma well counter (WIZARD2, PerkinElmer, France) to measure total activity. The unmetabolized parent [18F]UCB-H was calculated as a percentage of the total radioactivity (metabolites and parent).
For each animal, a 2-exponential decay function was fitted to the parent fraction of [18F]UCB-H , which was time multiplied with the plasma activity curve to obtain the metabolite-corrected arterial plasma input function (mcAIF) used for the kinetic modeling.
The fraction of [18F]UCB-H in NHP plasma samples not bound to plasma protein was measured before PET injection using a previously described ultrafiltration method (30). In brief, standard amounts of [18F]UCB-H (≈15 kBq) were added to 200 μL plasma that was applied to Microcon® filtration devices containing an YM-10 membrane (Millipore, France). The devices were centrifuged for 10 min at 10000 g (Sigma 2-16KL, France). [18F]UCB-H activity concentration in the resulting ultrafiltrate (≈70 μL, CFP) and a sample of plasma (CP) were counted. The free fraction (fp) was calculated as: fp=CFP/CP and measured in triplicates.
PET data analysis – PET image analysis was performed using PMOD software version 3.8 (PMOD Technologies Ltd., Zurich, Suisse). After individual co-registration of PET-MR images, a cynomolgus atlas published by Ballanger and coworkers (31) was normalized to PET images to extract time activity curves in different brain regions. Volumes of interest (VOI) were cerebral white matter, striatum, thalamus, cerebellum, frontal -, parietal -, and temporal cortex, and whole brain as a composite region of all regions in the atlas. [18F]UCB-H pharmacokinetics were evaluated by analyzing the time activity curves of the test- and retest scans using 1- and 2-tissue compartment models (unconstrained and constrained with global K1/k2 coupled fit across all regions) (32), and Logan graphical analysis with a fixed t* at 60 minutes (33) using the arterial plasma input function corrected for radio-metabolites to derive the volume of distribution in each region. Percentage standard error (%SE) was estimated from the theoretical parameter covariance matrix. Only VT and k3/k4 values with reliably estimates, i.e. %SE less than 25%, were included in the current analyses. For the three drug studies, percent displacement of the total activity in the whole brain was estimated relative to an average baseline constructed from the two 4hr baseline studies. The curves of the average baseline and the three displacement studies were normalized by the activity immediately before the administration of the drugs. The percent displacement of total activity was then calculated as (baseline-displacement)/baseline.
Statistical Analysis – Statistical analysis was performed using R software (version 3.3.1.). Plasma, intact parent fraction and time activity curves were statistically compared between subjects using a one-factor variance analysis. Absolute Test-Retest variability (aTRV) of PET quantification parameters was calculated as ABS(test - retest) / AVERAGE(test , retest). All values are expressed as average ± standard error of the mean (SEM; significance level was fixed at p < 0.05).