Preparation and First Preclinical Evaluation of [18F]FE@SNAP: A Potential PET Tracer for the Melanin-Concentrating Hormone Receptor-1 (MCHR1)

The melanin-concentrating hormone (MCH) system is a new target for the treatment of human disorders. Since the knowledge of the MCH system’s involvement in a variety of pathologies (obesity, diabetes, and deregulation of metabolic feedback mechanism) is based on in vitro or preclinical studies, a suitable positron emission tomography (PET) tracer needs to be developed. We herein present the preparation and first preclinical evaluation of [18F]FE@SNAP – a new PET tracer for MCH receptor-1 (MCHR1). The synthesis was performed using a microfluidic device. Preclinical evaluation included binding affinity, plasma stability, plasma free fraction, stability against the cytochrome P-450 (CYP450) system using liver microsomes, stability against carboxyl-esterase, and methods to assess the penetration of the blood-brain barrier (BBB) such as logD analysis and immobilized artificial membrane (IAM) chromatography. Levels at 374 ± 202 MBq [18F]FE@SNAP were obtained after purification. The obtained Kd value of [18F]FE@SNAP was 2.9 nM. [18F]FE@SNAP evinced high stability against carboxylesterase, CYP450 enzymes, and in human plasma. LogD (3.83) and IAM chromatography results (Pm=0.51) were in the same range as for known BBB-penetrating compounds. The synthesis of [18F]FE@SNAP was reliable and successful. Due to high binding affinity and stability, [18F]FE@SNAP is a promising tracer for MCHR1.


Physicochemical Parameters
The logD value of FE@SNAP was 3.83 ± 0.1 (n=3) and the value of the permeability through the membrane (P m ) was 0.51 ± 0.1 (n=3). Preliminary experiments on hMCHR2 showed poor binding of FE@SNAP (K i > 1000 nM).

Discussion
Due to the low density of the MCH receptors in the human brain (B max =5.8 ± 0.3 fmol/mg, [22]), a high binding affinity in a low nanomolar range of [ 18  1. Due to incomplete priming of the solution into the loop to guarantee bubble-free filling (which is a systematic problem in the microfluidic system that was used), 16.1 ± 0.3% of the activity remained in the concentrator vial of the microfluidic system after azeotropic drying and was not accessible for the synthesis [23].
2. Furthermore, 8.0 ± 3.5% remained in the lines and was thereby not accessible for the reaction [23].
3. The syntheses were performed in the discovery mode of the microfluidic system. There, approximately half of the amount of activity from the loop was used for the synthesis. The residual activity was kept as a reserve in the loop to have the option for a second consecutive synthesis.
However, 374 ± 202 MBq [ 18 F]FE@SNAP were sufficient for any subsequent preclinical evaluation studies. Higher radiochemical yields can be achieved using the sequence mode of the microfluidic system.
The tested quality control parameters of the physiologically formulated [ 18 F]FE@SNAP solution were in accordance with the standards for human application. Specific activity was relatively low (24.8 ± 12 GBq/µmol). Higher specific activities are expected for future syntheses with higher yields. We note that no conversion of [ 18 F]FE@SNAP could be achieved using conventional synthesizing modules [21].
We evaluated the stability of [ 18 F]FE@SNAP not only in human, but also in rat tissues (plasma, liver microsomes) to be prepared for species differences in future small animal PET experiments. [ 18 F]FE@SNAP was highly stable against human and rat liver microsomes (consisting of the multienzyme complex cytochrome P-450: 5.39 ± 1.6% (human) and 2.59 ± 1.8% (rat) decomposition after 60 min) and in human plasma (only 3.87 ± 3.9% metabolism after 120 min). In contrast, [ 18 F]FE@SNAP was completely metabolized in rat plasma within 120 min.
Porcine carboxylesterase was used to assess MMK, due to its wide use as a biochemical model for in vitro studies [25]. With a K m of 347.3 µM, FE@SNAP again showed very high stability.
For the prediction of blood-brain barrier (BBB) penetration, the lipophilicity expressed as logD was measured in the first step. Since the logP/logD values were shown to be poor predictors for BBB penetration [26], immobilized artificial membrane (IAM) chromatography was additionally performed. Under the modified conditions from Tavares et al. [27], FE@SNAP (P m =0.51) is situated well in between β-CIT (P m =0.31) and DASB (P m =1.23)two known BBB penetrating compounds. Therefore, considering only passive diffusion, a penetration through the BBB seems possible.  a high binding affinity to MCHR1 in a low nanomolar range,  high metabolic stability to assure enough intact tracer for the visualization of MCHR1-specific tissues and  reasonable lipophilicity to expect BBB penetration.
FE@SNAP binds to hMCHR1 in a nanomolar range (K d =2.9 nM) and is highly selective to this receptor subtype. It showed very high stability against porcine carboxylesterase, the cytochrome P-450 fraction of human and rat liver microsomes and in human plasma. Furthermore, the human plasma free fraction (f 1 =12.6 ± 0.2%) is high enough for potential brain imaging. The fact that decomposition in rat plasma is complete within 120 min has to be considered for further preclinical studies in rats. As IAM chromatography experiments showed comparable behavior to known BBB-penetrating compounds, passive BBB penetration is possible. Collectively, [ 18 F]FE@SNAP is a promising tracer for MCHR1, and further preclinical evaluation steps (e.g. autoradiography and small-animal PET) will thus elucidate its potential.

Instrumentation
The radiosynthesis of [ 18 F]FE@SNAP was carried out within an Advion NanoTek® unit (Ithaca, NY, USA) comprising a concentrator unit (CE) and a liquid flow reaction unit (LF) with dedicated control software (Advion, version 1.4). Microreactors were made of fused silica tubing (ID, 0.1 µm; length 2.0 m), wound up and held in a brass ring, and filled with a thermoresistant polymer to hold the tubing in place. The purification of the resulting crude product solution and the final formulation of [ 18 F]FE@SNAP was carried out within a Nuclear Interface® PET synthesizer (GE Medical Systems, Uppsala, Sweden) remote controlled via GINAstar software (Raytest Isotopenmessgeräte GmbH, Straubenhardt, Germany) installed on a standard PC. Analytical HPLC was performed using an Agilent system (Boeblingen, Germany) consisting of an autosampler 1100, a quartenary pump 1200, a diode array detector 1200 (operated at 254 nm), and a lead-shielded BGOradiodetector. The osmolality was measured using a Wescor osmometer Vapro® 5600 (Sanova Medical Systems, Vienna, Austria), pH was measured using a WTW inoLab 740 pH meter (WTW, Weilheim, Germany). Gas chromatography was performed using a 430-GC system (Burker Daltonik GmbH, Bremen, Germany). For the binding experiments, a Sorvall Ultracentrifuge Combi OTD (Thermo Fisher Scientific Inc, Waltham, MA, USA) and a 2480 WIZARD 2 Automatic Gamma Counter (PerkinElmer, Waltham, MA, USA) were used. For the stability experiments, sample incubation was conducted within a Thermomixer compact from Eppendorf® (Vienna, Austria) and sample centrifugation with a Universal 30 RF centrifuge (Hettich, Tuttlingen, Germany). The same centrifuge was used for the determination of the plasma free fraction.

Binding Affinity
The method used was conducted according to Mashiko

Plasma Stability
The stability of [ 18 F]FE@SNAP in human and rat plasma was determined according to Nics et al. [31]. 1800 μL of lithium-heparinized plasma (rat and human, respectively) were pre-incubated under physiological conditions (PBS, pH 7.4, 37°C) in a shaking incubator for 5 minutes. 36 µL [ 18 F]FE@SNAP (corresponding to 2% ethanol v/v in the total volume) were added and the plasma vial was vortexed for at least 10 seconds. After defined points in time (0 and 120 min), 500 µL of the incubation-mixture were added to a preconditioned (with 5 mL methanol followed by 5 mL water) SPE-cartridge (Oasis). The cartridge was then eluted into a collection tube, washed with 5 mL of 5% methanol in water (v/v) into a second tube, and eluted with 3 mL of THF into a third tube. 20 µL of the eluate-solution of tube two and three were injected into the analytical HPLC (mobile phase: (water/ acetic acid 97.5/2.5 v/v; 2.5 g/L ammonium acetate; pH 3.5)/acetonitrile 70/30 v/v; flow: 2mL/min).

Plasma Free Fraction
The method used was modified from Parsey et al. [24]. 1 mL of heparinized plasma (rat and human, respectively) were mixed with 10-50 µL [ 18 F]FE@SNAP. 200 µL aliquots were pipetted into the centrifugal filter units and the total radioactivity was measured in a Gamma Counter. After the centrifugation step (2.000 × g, 50 min), 50 µL of the obtained filtrate was back-measured for radioactivity. For the determination of the plasma free fraction (f 1 ), the ratio of filtrate to total activity concentration was calculated.

Stability Against Liver Microsomes (CYP450)
The method used was described by Nics et al. [31]. Briefly, liver microsomes (pooled from human or rat origin) were pre-incubated under physiological conditions (PBS, pH 7.4, 37°C) with a NADPH-generating system (solution-A: NADP+, glucose-6-phosphate and magnesium-chloride in H 2 O and solution-B: glucose-6-phosphate dehydrogenase in sodium citrate) for 5 min. 6 µL of [ 18 F]FE@SNAP, which correspond to 2% ethanol (v/v) in the total volume, were added. Enzymatic reactions were stopped after defined points in time (0, 2, 5, 10, 20, 40, and 60 min) by adding the same amount of ice-cold acetonitrile/methanol (10:1). The mixtures were vortexed, followed by a centrifugation step (23.000 × g, 5 min). Aliquots of the obtained supernatant were analyzed by an analytical HPLC (for conditions see Plasma Stability).

Stability Against Carboxylesterase
The method used was slightly modified from Nics et al. [32].

LogD Analysis
LogD values were determined using an HPLC-based assay according to Donovan and Pescatore [33]. A cocktail of two internal standards (toluene and triphenylene) with known logD and k' values and FE@SNAP in methanol were injected into a short polymeric ODP-50 column. A linear gradient from 10% methanol/90% phosphate buffer (pH 7.4) to 100% methanol within 9.4 min at a flow rate of 1.5 mL/min was applied. Detection was performed at 260 nm and 285 nm.

IAM Chromatography
IAM chromatography was modified from Tavares et al. [29]. A 0.01 M phosphate buffer (pH 7.0) and acetonitrile (ranging from 50% to 35%, v/v) were used as the mobile phase at a flow rate of 1 mL/min. FE@SNAP was injected onto the IAM column. As a result, the permeability through the membrane (P m ) was calculated and compared with the P m of the known BBB-penetrating compounds (DASB, β-CIT) as external standards.