SNAPshots of the MCHR1: a Comparison Between the PET-Tracers [18F]FE@SNAP and [11C]SNAP-7941

Purpose The melanin-concentrating hormone receptor 1 (MCHR1) has become an important pharmacological target, since it may be involved in various diseases, such as diabetes, insulin resistance, and obesity. Hence, a suitable positron emission tomography radiotracer for the in vivo assessment of the MCHR1 pharmacology is imperative. The current paper contrasts the extensive in vitro, in vivo, and ex vivo assessments of the radiotracers [18F]FE@SNAP and [11C]SNAP-7941 and provides comprehensive information about their biological and physicochemical properties. Furthermore, it examines their suitability for first-in-man imaging studies. Procedures Kinetic real-time cell-binding studies with [18F]FE@SNAP and [11C]SNAP-7941 were conducted on adherent Chines hamster ovary (CHO-K1) cells stably expressing the human MCHR1 and MCHR2. Small animal imaging studies on mice and rats were performed under displacement and baseline conditions, as well as after pretreatment with the P-glycoprotein/breast cancer resistant protein inhibitor tariquidar. After the imaging studies, detailed analyses of the ex vivo biodistribution were performed. Ex vivo metabolism was determined in rat blood and brain and analyzed at various time points using a quantitative radio-HPLC assay. Results [11C]SNAP-7941 demonstrates high uptake on CHO-K1-hMCHR1 cells, whereas no uptake was detected for the CHO-K1-hMCHR2 cells. In contrast, [18F]FE@SNAP evinced binding to CHO-K1-hMCHR1 and CHO-K1-hMCHR2 cells. Imaging studies with [18F]FE@SNAP and [11C]SNAP-7941 showed an increased brain uptake after tariquidar pretreatment in mice, as well as in rats, and exhibited a significant difference between the time-activity curves of the baseline and blocking groups. Biodistribution of both tracers demonstrated a decreased uptake after displacement. [11C]SNAP-7941 revealed a high metabolic stability in rats, whereas [18F]FE@SNAP was rapidly metabolized. Conclusions Both radiotracers demonstrate appropriate imaging properties for the MCHR1. However, the pronounced metabolic stability as well as superior selectivity and affinity of [11C]SNAP-7941 underlines the decisive superiority over [18F]FE@SNAP.

The current paper contrasts novel and previously attained in vitro, in vivo, and ex vivo assessments of [ 11 C]SNAP-7941 and [ 18 F]FE@SNAP to determine the superior radioligand with respect to its biological and physicochemical properties. A comprehensive illustration of the applied in vitro, in vivo, and ex vivo experiments is shown in Fig. 1c.

Tracer Preparation
The radiosynthesis of [ 11 C]SNAP-7941, the radiolabeled analog of (±)-SNAP-7941, was performed in a fully automated synthesizer (TRACERlab™ FX C Pro, GE Healthcare, Germany) as previously reported [30,33]. Radiosynthesis of [ 18 F]FE@SNAP was performed in a microfluidic device (Advion NanoTek®, Ithaca, NY, USA) as described elsewhere [37], followed by purification in a conventional synthesizer unit (Nuclear Interface®, GE Medical Systems, Uppsala, Sweden) [36,38]. Radiochemical purity and molar activity of [ 11 [37]. The cells were cultured in Ham's F-12 medium (Gibco®, Life Technologies) with additives (1 % penicillin-streptomycin-glutamine (PSG), 10 % fetal bovine serum (FBS), and 300-μg/m Geneticin (G-418)) and incubated in a humidified 5 % CO 2 atmosphere at 37°C. For the preparation of the binding experiment, approximately 10 6 cells were seeded as a monolayer on the bottom of a tilted cell culture dish (100 mm × 20 mm, CELLSTAR®, Greiner Bio-One) and incubated with 2-ml medium for 24 h to avoid a spreading of the cells over the whole Petri dish. In the next step, the medium was discarded and the Petri dish was placed in a horizontal position with 10-ml medium for additional 24 h. Afterwards, the binding experiment was performed at ambient temperature with LigandTracer® Yellow (Ridgeview Instruments AB, Uppsala, Sweden) using 3-ml fresh medium (Ham's F-12, serum-free). Unspecific radiotracer uptake was determined with native CHO-K1 cells. The experiments were initiated with baseline measurements for 10-15 min followed by radioligand incubation of [ 11 C]SNAP-7941 and [ 18 F]FE@SNAP. Binding to the seeded cells was ensured by adding different concentrations of the radiotracer (0.05-1000 nM). Association-time curves were monitored in real-time until the binding equilibrium was achieved. The observed rate constant of the association reaction (k obs ) was determined using non-linear regression curve fitting algorithms implemented in GraphPad Prism 6.0 (GraphPad Software, Inc., San Diego, CA, USA), as previously reported [39,40]. Cell survival was continuously

Plasma Protein Binding Using Bioaffinity Chromatography
The binding of (±)-SNAP-7941 and FE@SNAP to human serum albumin (HSA) was examined by bioaffinity chromatography according to a previously published manuscript [40]. In short, the analytes were diluted in 2-propanol and ammonium acetate buffer (0.5 mg/ml) and injected on the CHIRALPAK®HSA stationary phase (50 × 3 mm, 5 μm pore size, column-batch: H13L-2405, Daicel Chemical Industries, West Chester, PA, USA). Prior the experiments, the column was calibrated with reference standards, and the resulting regression equation was used to convert the logarithmic capacity factors (log(k′)) to the percent of plasma protein binding (%PPB). The calibration curves, as well as the experiments, were performed by triplicate injections and at least three times.

Animals
Ten-week-old male rats (412 ± 58 g, Sprague-Dawley, HIM:OFA, n = 30) and 12-week-old male mice (24 ± 6 g, BALB/cAnNRj, n = 7) were purchased from the Division of Laboratory Animal Science and Genetics, Himberg, Austria. Animals were kept under conventional housing conditions (22 ± 1°C; 40-70 % humidity) with food and water supply ad libitum and 12-h day/night cycle. All animals were treated according to the European Union rules on animal care and respective animal experiments were approved by the Austrian Ministry of Sciences, Research and Economy (BMWFW-66.009/0029-WF/V/3b/20159). For in vivo imaging, animals were anesthetized using 1.5-2 % isoflurane mixed with oxygen (1.5-2 l/min) to avoid movement during the examination. Anesthesia as well as vital parameters were monitored during the time interval of PET acquisition. Radioligands and non-labeled compounds were administered intravenously via the lateral tail vein.

Small Animal Imaging
Anesthetized rats and mice were immobilized in a multimodal animal carrier unit (MACU; medres®-medical research GmbH, Cologne, Germany). The body temperature was preserved at 37°C throughout the whole experiment. Rats received either [ 11 C]SNAP-7941 or [ 18 F]FE@SNAP, followed by an injection of (±)-SNAP-7941 (15-mg/kg body weight; displacement study; n = 4 for each radiotracer) or the respective solvent serving as the vehicle (baseline condition; n = 4 for each radiotracer). The MCHR1 antagonist, (±)-SNAP-7941, and the vehicle were administered either 15 ([ 11 C]SNAP-7941) or 20 min ([ 18 F]FE@SNAP) after the radiotracer application via the lateral tail vein. To investigate a potential binding to the P-glycoprotein (P-gp) and/or breast cancer resistance protein (BCRP) brain efflux transporter system, rats as well as mice were pretreated either with the P-gp/BCRP inhibitor TQD (15 mg/kg body weight; P-gp/ BCRP inhibition group; rats: n = 4 for each radiotracer; mice: n = 4 for [ 18 F]FE@SNAP) or the respective solvent (baseline condition; rats: n = 3 for each radiotracer; mice: n = 3 for [ 18 F]FE@SNAP)). Mice received the P-gp/BCRP inhibitor 30 min and rats 60 min before the radiotracer application. Experiments were initiated with a 7-min cone beam attenuation CT (CBCT) of the brain (

Image Reconstruction and Data Post-Processing
Image reconstruction of the CT raw data was performed with a Feldkamp algorithm using a ramp filter followed by standard rat beam-hardening correction and noise reduction (matrix size 1024 × 1024; effective pixel size: 97.56 μm). All CT image data was calibrated to Hounsfield units (HU After the imaging studies, the animals were sacrificed by decapitation; blood and tissues were removed and collected in tubes, weighed, and subjected to radioactivity measurements in a Gamma Counter (2480 WIZARD 2 , PerkinElmer, Waltham, MA, USA). Values were normalized to the applied dose, the organ, and body weight and expressed as the standardized uptake value (SUV [g/ml]).

Statistical Analysis
Experimental data are expressed as mean ± SEM of independent experiments (n ≥ 3) with different lots of radiolabeled and non-labeled compounds. Statistical testing was performed using GraphPad Prism 7.0 (GraphPad Software, Inc., San Diego, CA). Differences among groups and conditions were determined using either a two-tailed, unpaired Student's t test with Welch's correction or a two-tailed parametric paired t test. Post hoc testing for multiple comparisons was performed using either ordinary one-way ANOVA with Tukey's correction or ordinary two-way ANOVA with Sidak's correction. Values of P G 0.05 were considered as statistically significant.

Kinetic Real-Time Cell-Binding Studies
Kinetic real-time cell-binding studies were performed in a reliable manner with high temporal resolution as shown in Fig. 2 (Fig. 2b).

Small Animal Imaging
[ 18 F]FE@SNAP imaging experiments in mice showed a 1.93-fold increased brain uptake in the TQD blocking group. As illustrated in Fig. 3a, the differences between the TACs of the vehicle and blocking groups were statistically significant (P G 0.0001). Furthermore, both radiotracers showed an increased brain uptake for the TQD blocking group in rats. Brain uptake was increased 2.45-times for [ 18 F]FE@SNAP (Fig. 3b) and 3.04-times for [ 11 C]SNAP-7941 (Fig. 3c), respectively. Both radiotracers exhibited a significant difference between the TACs of the vehicle and blocking groups (P G 0.0001). TACs of selected regions were analyzed before and after displacement with 15 mg/kg (±)-SNAP-7941. A clear difference in the binding pattern was observed for both tracers, [ 18 F]FE@SNAP and [ 11 C]SNAP-7941, for the brown adipose tissue (BAT), brain, and lung. Corresponding TACs are depicted in Fig. 4b-d. The associated blood input curve for both radiotracers is presented in Fig. 4a, showing no significant difference in the uptake behavior (P = 0.3117). Differences in the binding profiles before and after displacement with 15

Ex Vivo Metabolites
Ten minutes after the radiotracer application, 38.40 ± 2.3 % of intact [ 18 F]FE@SNAP was present in rat whole blood; at 30 min after administration, 31.59 ± 4.0 % was left and 14.42 ± 3.3 % at 60 min. Moreover, the formation of a radioactive hydrophilic metabolite was observed. On the contrary, [ 11 C]SNAP-7941 evinced a high metabolic stability in rat whole blood, resulting in 93.52 ± 0.1 % of intact tracer at 10 min and 93.74 ± 6.2 % at 45 min (Fig. 6). The investigation of brain metabolites evinced a strong degradation of the parent compound at 45 min (22.37 ± 5.8 % of intact tracer) for [ 18 F]FE@SNAP.

Discussion
To quantify the biomolecular mechanisms of the MCHR1 in vivo, a selective PET radioligand is indispensable. Hence, a specific MCHR1 PET-tracer would provide deeper insights on the receptor's involvement in lifestyle diseases, such as obesity and diabetes, and promote drug development for related pathologies. Nevertheless, only three PET radioligands for the visualization of the MCHR1 have been developed [32,36,41,42]. This paper focuses on the first MCHR1 PET ligands, [ 18 F]FE@SNAP and [ 11 C]SNAP-7941, and contrasts their advantages and disadvantages. Table 1 gives an overview of the results, which are discussed in this section.
Since MCHR2 is not expressed in rodents, additional kinetic real-time cell-binding studies were performed to substantiate the target selectivity. In this context, both radiotracers demonstrated a specific accumulation profile on the CHO-K1-hMCHR1 cells and negligible accumulation on the native CHO-K1 cells (Fig. 2a). While [ 11 C]SNAP-7941 evinced selective binding to the CHO-K1-hMCHR1 cells, which is in good agreement with previously published data [32], [ 18 F]FE@SNAP additionally exhibited accumulation to the CHO-K1-hMCHR2 cells (Fig. 2b). This phenomenon contradicts previously elaborated findings on CHO-K1-hMCHR2 membranes [34] and might be explained by the difference in the biochemical approach (competition experiments with the unlabeled ligand vs. direct binding with the radiolabeled ligand) and the experimental setup (membranes vs. living cells). In this context, it has to be highlighted that experiments on living cells, as performed in the present study, enhance the understanding of the complex interplay between the radiotracer and the dedicated biological target [39]. Moreover, previous experiments revealed higher binding affinity for (±)-SNAP-7941 (K i = 3.91 ± 0.74 nM) compared to FE@SNAP (K i = 9.98 ±  1.12 nM) [25]. Based on current and previous results, [ 11 C]SNAP-7941 exhibits an improved target affinity and superior selectivity. Considering the in vivo pharmacology, it has been demonstrated in preceding [32,43] and recent experiments that [ 11 C]SNAP-7941 is a P-gp/BCRP substrate, as confirmed in small animal imaging studies of rat brains (3.04times higher uptake after TQD pretreatment, Fig. 3c). A similar behavior was observed for [ 18 F]FE@SNAP (2.45times higher uptake after TQD pretreatment, Fig. 3b). Additionally, no species differences in P-gp/BCRP inhibition between mouse and rat were found as shown in small animal imaging studies of mouse brains with [ 18 F]FE@SNAP, revealing also an increased brain uptake after TQD pretreatment (Fig. 3a). Detailed quantitative assessment of the whole brain uptake evinced a more distinct difference between the vehicle and TQD-treated groups for [ 11  7941, these results were in accordance with the bioaffinity chromatography outcome, indicating that (±)-SNAP-7941 utterly binds to serum albumin. Whereas, the results for FE@SNAP diverged around 10 % when applying the chromatographic method. One reason might be that FE@SNAP also binds to other plasma components, such as alpha1-acid glycoprotein or diverse lipoproteins. Furthermore, the higher brain uptake of [ 18 F]FE@SNAP compared to [ 11 C]SNAP-7941 results from the formation of radiometabolites (e.g., [ 18 F]fluoroethanol) supported by the findings from the ex vivo metabolism studies in rat whole blood (Fig. 6), which underlines the superior imaging contrast of [ 11 C]SNAP-7941 in the brain.
Considering the vehicle and displacement groups, the analysis of the whole brain resulted in a clear displacement for [ 11 C]SNAP-7941, whereas for [ 18 F]FE@SNAP a displacement could not be detected (Fig. 4c). This finding is supported by previously conducted experiments [25] and is likely a result of the high unspecific binding and metabolic degradation of [ 18 F]FE@SNAP. Furthermore, a drop in the TAC of both radiotracers after displacement with 15 mg/kg (±)-SNAP-7941 could be detected for the lung (Fig. 4d). The detailed analysis of the TACs of other target regions, such as tongue, pancreas, and colon, was not possible due to the limited field of view of the imaging modality and spillover effects of the surrounding tissue. Interestingly, the TAC of the BAT depicts an increased uptake for both tracers after the administration of (±)-SNAP-7941 (Fig. 4b), which indicates a potential involvement of the MCHergic system and a further interaction of important regulatory pathways. The representative TAC of the blood pool confirms the proper administration and further bioavailability of both radiotracers (Fig. 4a). . Differences among groups were tested using a twotailed parametric paired t test (ns = P 9 0.05; * = P G 0.05; ** = P G 0.01; **** = P G 0.0001). If not visible, error bars are within the margin of the symbols.  Global analysis of the ex vivo biodistribution depicts reduced tracer uptake after displacement with (±)-SNAP-7941 for both radiotracers (Fig. 5a). This finding is highlighted by previously conducted studies [38] and confirmed by tissueto-blood analyses [32]. The reduced uptake in BAT stands in contrast to the enhanced uptake shown in the TAC (Fig. 4b).
This phenomenon may be explained by the differences in the experimental setup due to the high biodiversity when using different animals as used in biodistribution studies. In contrast, the in vivo displacement analysis of the TACs was performed within the same animal. The involvement of the MCHR1 in BAT is part of ongoing investigations. Regional analysis of selected target regions (colon, pancreas, eye) contrasts the higher suitability of [ 11 C]SNAP-7941. Even if both radiotracers demonstrate a significant decrease in uptake in the pancreas and eye after displacement with (±)-SNAP-7941, [ 11 C]SNAP-7941 revealed a more pronounced difference. However, a significant displacement in the colon was only observed for [ 11 C]SNAP-7941 ( Fig. 5b-g) (Fig. 6), which was validated by previous in vitro as well as in vivo studies. A detailed analysis of the brain metabolism demonstrated an extensive degradation of [ 18 F]FE@SNAP, while [ 11 C]SNAP-7941 remained metabolically stable [32,36,38].
Even though both tracers exhibit suitable properties for the imaging of the MCHR1, [ 11 C]SNAP-7941 clearly demonstrated superior imaging properties due to its higher selectivity, affinity, and metabolic stability. Based on the combined data, we recommend [ 11 C]SNAP-7941 as the tracer of choice for the imaging of MCHR1.

Conclusions
The MCHR1 has become an interesting pharmacological target for clinical medicine, as well as for biomedical research, since it may be involved in a plethora of lifestyle diseases. In this context, the availability of a suitable PET radiotracer is a crucial step for the quantitative in vivo assessment of MCHR1 pharmacology. Extensive in vitro, in vivo, and ex vivo assessments of [ 18 F]FE@SNAP and [ 11 C]SNAP-7941 demonstrate appropriate imaging properties for the MCHR1. Yet, some physiological processes influenced by the MCH system, as for instance its contribution to BAT stimulation, remain unclear and demand further elucidation.
However, the pronounced metabolic stability as well as superior affinity and selectivity of [ 11 C]SNAP-7941 reveal the decisive superiority over [ 18 F]FE@SNAP. Since humans express both the MCHR1 and MCHR2, tracer selectivity is essential for prospective first-in-man imaging studies. Therefore, [ 11 C]SNAP-7941 is the ideal candidate for initial clinical trials, addressing the imaging of endocrinological and psychiatric disorders.