Radiochemistry: [18F]Altanserin, [11C]Cimbi-36, [11C]Pimavanserin, [18F]MH.MZ, [18F]Fallypride, [11C]Cimbi-701, [11C]Cimbi-717 and [11C]BA-10 were synthesized as previously reported [12–19]. Chemical structures of all compounds are displayed in the Electronic Supplementary Material (Supplementary Fig. S2).
Animals: 200-300 g (8-10 weeks) Long-Evans WT (or Sprague-Dawley in the case of Altanserin) female rats (Charles River, Calco, Italy) were housed in groups of 2-3 animals per cage in a climate-controlled rodent facility with a 12h light/dark cycle. The animals had free access to water and were fed ad libitum. All procedures were conducted in accordance with the European Commission’s Directive 2010/63/EU, FELASA and ARRIVE guidelines for animal research and, with approval from The Danish Council for Animal Ethics (license numbers: 2017-15-0201-01283, 2012-15-2934-00156, 2007-561-1320) as well as the Department of Experimental Medicine, University of Copenhagen.
Pig PET experiments: Description of PET experiments in pigs and analysis of obtained data can be found in the Electronic Supplementary Material.
Rat PET scanning protocol: On the day of scanning, rats were transported to the scanner at least one hour prior to the experiment. Isoflurane, 3-3.5 % in 0.6% oxygen, was used to induce anaesthesia, while anaesthesia was maintained with 2.0-2.5 % isoflurane during the scans. The PET tracers were administered as intravenous (i.v.) bolus injections via tail vein catheters (BD Neoflon 25G, Stockholm, Sweden) at the beginning of the scan with doses being between 5-20 MBq. The rats were subsequently scanned in the high-resolution research tomography (HRRT) scanner (Siemens AG, Munich, Germany) using a custom-made 2×2 rat holder, which enabled simultaneous scanning of four rats (Fig. 1) [11]. The animals were scanned for either 60 min (11C) or 90 min (18F), followed by a transmission scan at speed 10 (acquisition time approximately 6 min). The animals were scanned at baseline (no pre-treatment before tracer injection), and after receiving P-gp inhibition with or without target block. Details of inhibition and blocking regimens are described in Table 1. For combined inhibition and target block scans, rats were pre-treated with the P-gp inhibitor elacridar (5 mg/kg, Carbosynth, Compton, United Kingdom) and the 5-HT7 antagonist SB-269970 (3 mg/kg, Tocris Bioscience, Abingdon, United Kingdom), the sigma and dopamine D2/3 antagonist haloperidol (1 mg/kg, Janssen-Cilag, Birkerød, Denmark) or the 5-HT2A antagonist ketanserin (3 mg/kg, Sigma-Aldrich, Saint Louis, Missouri, USA). Elacridar was given 30 min prior to tracer injection through the intravenous catheter, and receptor blocking drugs were given 15 min before tracer injection. Chosen dosages and pre-treatment intervals were based on literature data [20–23].
Studies with [18F]Altanserin were performed in a MicroPET Focus 120 scanner (Siemens Medical Solutions, Malvern, PA, USA). Hyponorm/midazolam (VetaPharma Ltd., Leeds, United Kingdom/Hameln Pharmaceuticals, Hameln, Germany) was used as anaesthesia. The rats received 11±2 MBq of the tracer as a bolus injection. Three of the rats were administered with 22.5 mg/kg bolus of Cyclosporin A (Sandimmun, Novartis, Basel, Switzerland) followed by a constant infusion of 7.5 mg/kg/h, starting 20-25 min before radiotracer administration, as previously described [10].
Rat PET image reconstruction: For HRRT scans (all tracers except [18F]Altanserin), 60-minute list-mode PET data was transformed into 33 dynamic frames (6 × 10, 6 × 20, 6 × 60, 8 × 120, and 7 × 300 seconds), while 90-minute list-mode PET data was transformed into 35 dynamic frames (6 × 10, 8 × 30, 5 × 60, and 16 × 300 seconds). Attenuation maps were reconstructed from transmission scans using maximum a posteriori algorithm for transmission data (MAP-TR) with human head (HH) segmentation/thresholding scans [11]. All images were reconstructed using ordinary Poisson 3D ordered subset expectation maximization (OP-OSEM3D) algorithm with point spread function modelling. PET image frames consisted of 207 planes of 256 × 256 voxels of 1.22 × 1.22 × 1.22 mm. [18F]Altanserin scans were performed on the Focus 120 camera. 90-minute list-mode PET data was transformed into 25 dynamic frames (10 × 30, 5 × 120, 5 × 30, and 5 × 600 seconds) and reconstructed using the filtered backprojection method. Reconstructed PET image frames consisted of 95 planes of 128 × 128 voxels of 0.87 × 0.87 × 0.80 mm.
Table 1: Target blocking drugs, P-gp inhibitors and PET image summation timespan for rat PET experiments.
Tracer
|
Target blocking drug
|
Dose
|
P-gp Inhibitor
|
Dose
|
Time span for image summation
|
[18F]MH.MZ
|
ketanserin
|
3 mg/kg
|
elacridar
|
5 mg/kg
|
5-90 min
|
[18F]Altanserin
|
ketanserin
|
3 mg/kg
|
Cyclosporin A
|
22.5 mg/kg*
|
5-90 min
|
[11C]Pimavanserin
|
ketanserin
|
3 mg/kg
|
elacridar
|
5 mg/kg
|
5-60 min
|
[11C]Cimbi-36
|
ketanserin
|
3 mg/kg
|
elacridar
|
5 mg/kg
|
5-60 min
|
[11C]Cimbi-717
|
SB269970
|
3 mg/kg
|
elacridar
|
5 mg/kg
|
2-60 min
|
[11C]Cimbi-701
|
SB269970
|
3 mg/kg
|
elacridar
|
5 mg/kg
|
2-60 min
|
[11C]BA-10
|
SB269970
|
3 mg/kg
|
elacridar
|
5 mg/kg
|
30-60 min
|
[18F]Fallypride
|
haloperidol
|
1 mg/kg
|
elacridar
|
5 mg/kg
|
5-90 min
|
*After bolus injection of 22.5 mg/kg Cyclosporin A, rats received constant infusion of 7.5 mg/kg/h.
Quantification of rat PET data: For data analysis, the software PMOD 3.7 (PMOD Technologies, Zürich, Switzerland) was used. Summed PET images were generated based on all counts recorded in the time intervals specified in Table 1. Images were then aligned to a standardized MRI-based atlas of the rat brain [24] from where pre-defined regions of interest (ROIs) were extracted. Regions known to possess high densities of relevant receptors were selected as target ROIs: medial prefrontal cortex (mPFC) and frontal cortex (FC) for 5-HT2A receptor PET tracers [25], thalamus (Tha) for 5-HT7 receptor PET tracers [16] and striatum (Str) for dopamine D2/3 receptor tracer [26]. Cerebellum region (Cb), having low densities of receptors targeted by all investigated tracers, was chosen as a reference region (Supplementary Fig. S3). In addition, whole brain (Wb) ROI was chosen to monitor overall tracer penetration into the brain. The time-activity curves (TACs) for target ROIs were extracted from the PET images, and the activity was converted into standardized uptake values (SUV). SUV, expressed in g/mL, is equal to the concentration of radioactivity measured in the ROI divided by the injected radioactivity dose per body weight. Area under the curve (AUC) values were calculated from the TACs using GraphPad Prism 7 (GraphPad Software, California, USA) and expressed in min×g/mL. As scanning durations were different for 11C-labeled and 18F-labeled tracers (60 min and 90 min, respectively), AUC values of 18F-labeled tracers were calculated for both the full duration and the first 60 min of the scan.
Rat data analysis: Apparent target-specific binding of the tracers in the target regions was expressed as specific binding ratios (SBR), which were calculated from mean full scan length AUC values applying Equation 1; cerebellum was used as a reference region for all tracers. Changes in apparent specific binding of the tracers in response to target receptor blockade were calculated from SBR values using Equation 2; SBR changes were calculated for “baseline – target block” and “P-gp inhibition alone – combined P-gp inhibition and target block” condition pairs. Changes in tracer uptake in the target-rich region in response to P-gp inhibition were calculated from mean AUC values for the first 60 min of the scan as shown in Equation 3.
Evaluation of a tracer was considered successful if (whether with or without P-gp inhibition) the tracer showed an SBR value of at least 0.15 (15% higher uptake in the target region relative to reference region), and this SBR value decreased by at least 30% under target block condition.