Radiosynthesis and first preclinical evaluation of the novel norepinephrine transporter pet-ligand [11C]ME@HAPTHI

Background The norepinephrine transporter (NET) has been demonstrated to be relevant to a multitude of neurological, psychiatric and cardiovascular pathologies. Due to the wide range of possible applications for PET imaging of the NET together with the limitations of currently available radioligands, novel PET tracers for imaging of the cerebral NET with improved pharmacological and pharmacodynamic properties are needed. Methods The present study addresses the radiosynthesis and first preclinical evaluation of the novel NET PET tracer [11C]Me@HAPTHI by describing its affinity, selectivity, metabolic stability, plasma free fraction, blood–brain barrier (BBB) penetration and binding behaviour in in vitro autoradiography. Results [11C]Me@HAPTHI was prepared and displayed outstanding affinity and selectivity as well as excellent in vitro metabolic stability, and it is likely to penetrate the BBB. Moreover, selective NET binding in in vitro autoradiography was observed in human brain and rat heart tissue samples. Conclusions All preclinical results and radiosynthetic key-parameters indicate that the novel benzothiadiazole dioxide-based PET tracer [11C]Me@HAPTHI is a feasible and improved NET radioligand and might prospectively facilitate clinical NET imaging. Electronic supplementary material The online version of this article (doi:10.1186/s13550-015-0113-3) contains supplementary material, which is available to authorized users.


Background
The noradrenergic system-and specifically the presynaptic norepinephrine transporter (NET)-is proposed to be altered in a variety of neurological, neuropsychiatric and cardiovascular diseases. For example, alterations have been shown in Alzheimer's disease, Morbus Parkinson, major depressive disorder and attention deficit hyperactivity disorder [1][2][3][4][5][6][7][8][9]. Therefore, a reliable non-invasive molecular imaging technique-such as positron emission tomography (PET)-would be of great benefit for early stage in vivo diagnostics, visualization of treatment response and further elucidation of underlying pathophysiological mechanisms.
Great efforts have been made to develop PET tracers for the NET over the last two decades. Focus was primarily placed on reboxetine-derived ligands [10][11][12][13][14]. However, previous studies have shown that the in vivo and in vitro behaviour of these reboxetine analogues, more specifically [ 11  ] methoxyphenoxy)benzyl) morpholine), is not favourable for viable imaging of the NET by PET. Limitations include their metabolic stability, late reaching of equilibrium, unexplainable striatal uptake and complexity of radiosynthesis [10,[15][16][17][18]. Recently, we aimed at the preparation of a benzo[d]imidazolone derivative-[ 11 C]Me@APPI as new NET PET tracer [19]. Despite its favourable properties and straightforward production, its affinity was not sufficient and its lipophilicity high. Hence, there is ample demand for a novel, improved radioligand for in vivo NET imaging. Therefore, this study highlights a novel, non-reboxetinebased NET PET tracer based on a benzothiadiazole scaffold: [ 11 C]Me@HAPTHI ((S)-1-(3-hydroxy-4-([ 11 C] methylamino)butyl)-3-phenyl-1,3-dihydrobenzo[c] [1,2,5]thiadiazole 2,2-dioxide) (Fig. 1). In general, the designed benzothidiazole dioxides exhibits excellent affinity and selectivity as well as slightly reduced flexibility compared to other previously published benzoimidazolones [20,21]. Hence, these substances offer an ideal basis for the further development of novel NET ligands for PET imaging.
The objectives of this investigation were as follows: The set-up of a small-scale radiosynthetic procedure for the preparation of the carbon-11 labelled [ 11 C]Me@HAPTHI and its optimization; The up-scaling and set-up of a fully automated preparation of [ 11 C]Me@HAPTHI, including purification and formulation; The in vitro evaluation of Me@HAPTHI and its precursor HAPTHI. Evaluation includes binding studies for the determination of affinity and selectivity of both Me@HAPTHI and its precursor HAPTHI towards NET using NET, serotonin transporter (SERT) and dopamine transporter (DAT) expressing membranes, metabolic stability testing in vitro against Cytochrom P 450 enzymes, logP analysis and immobilized artificial membrane (IAM) chromatography for indirect measurement of the blood-brain barrier (BBB) penetration and determination of plasma free fraction. Comparative in vitro autoradiography on human and rodent tissue slices.
Microsomal preparations (human/rat liver microsomes) for stability testing were obtained from BD Bioscience (NJ, USA). Pooled human and rat plasma was obtained from Innovative Research (MI, USA).
The human postmortem tissue (7-9 h postmortem time, no history of neurological diseases) was obtained from the Neurobiobank of the Medical University of Vienna and approved by the local ethics committee ("Molecular neuropathologic investigation of neurodegenerative diseases" Nr.396/2011) following the principles of the Helsinki Declaration. Wild-type male rats were deeply anesthesized by isoflurane and sacrificed by decapitation. The organs of interest (i.e. brain, heart and testis) were removed and quick-frozen in i-pentan. Research using animal tissue was carried out under institutional approval in accordance with the Austrian Animal Care Law. Tissues were cut at −20°C in a micro-cryotome (Microm HM 560, Thermo Scientific, Austria). Frozen slices were thaw-mounted onto superfrost slides (Menzel-Gläser SUPERFROST plus microscopy slides, Thermo Scientific, Germany). A barrier pen (Mini PAP Pen, Invitrogen, USA) was used for immunohistochemistry only. For detection of autoradiography, a Cyclone Phospho-Imager (Cyclone Plus Storage Phosphor System, Perkin Elmer, Germany) and Phosphor Imager plates (Multisensitive Phosphor Screens Long Type MS, PPN 7001724, Perkin Elmer, Germany) were used. The lead shielded and light-protected cassettes (Fisher Biotech Autoradiography Cassette FBCS 1417) were purchased from Fisher Scientific (PA, USA).

Instrumentation
[ 11 C]CO 2 was produced within a GE PETtrace cyclotron (General Electric Medical System, Uppsala, Sweden) by a 14 N(p,α) 11 C nuclear reaction under irradiation of a gas target filled with N 2 (+1 % O 2 ) (Air Liquide Austria GmbH, Schwechat, Austria).
The evaluation of the reaction conditions was performed manually with starting activities <2 GBq. After optimization of the reaction parameters, [ 11 C]Me@HAPTHI-synthesis was transferred to the TRACERlab™ FX C Pro synthesizer and a fully automated synthesis was established.
Crude [ 11 C]Me@HAPTHI was purified by semipreparative reversed phase HPLC using the built-in semi-preparative HPLC system equipped with a radioactivity and a UV detector (Linear Instruments Model 200 Detector UV/VIS) and a LaPrep HPLC pump (VWR International, Radnor, USA). A Supelcosil TM LC-ABZb, 5 μm, 250 × 10 mm (Supelco®, Bellefonte, PA, USA) column was used with a mobile phase of ACN/0.1 M ammonium acetate 40/60 v/v% at a flow rate of 6 mL/min.
The osmolality of the final sterile product was measured with a Wescor osmometer Vapro® 5600 (Sanova Medical Systems, Vienna, Austria).

Production of [ 11 C]CH 3 I and [ 11 C]CH 3 OTf
The cyclotron production of [ 11 C]CO 2 was terminated at desired target activities between 40 and 50 GBq at currents between 48 and 53 μA (20-25 min) and trapped upon delivery on a molecular sieve (4 Å) within the Tracerlab FxC Pro synthesizer. Subsequently, [ 11 C]CO 2 was converted into [ 11 C]CH 4 by a Ni-catalysed reduction with H 2 at 400°C. [ 11 C]CH 3 I was produced within the same synthesizer using the dry method (gas phase conversion) described by Larsen et al. [22] with adopted modifications described by Kniess et al. [23]. Briefly, the resulting [ 11 C]CH 4 was reacted with sublimated iodine at 738°C in a recirculating process for 4 min to give [ 11 C]CH 3 I. The produced [ 11 C]CH 3 I was trapped on-line on a Porapak® N column and finally released by heating the trap to 190°C. [ 11 C]CH 3 OTf was prepared on-line at the passage of [ 11 C]CH 3 I through a pre-heated (200°C) column containing 300 mg silver triflate impregnated graphitized carbon at a flow rate of 40 mL/min [24].

Small-scale reactions
For optimization of reaction conditions, small-scale reactions using [ 11 C]CH 3 I or [ 11 C]CH 3 OTf were performed. Either [ 11 C]CH 3 I or [ 11 C]CH 3 OTf was trapped in 500 μL of the solvent of choice at room temperature (RT) and aportioned for further experiments in 1 mL Wheaton vvials. All evaluation reactions were performed manually (shielded hood; starting activity <2 GBq). The influence of various reaction conditions was investigated: -Reaction temperature: 25°C, 75°C -Base as catalyst: NaOH, triethylamine (TEA) and TBAH -Precursor concentration: 1 or 2 mg/mL -Solvent: MEK or DMSO Finale reaction volumes of small-scale reactions were 10-200 μL. The reactions were quenched with an equivolume solution of ammonium acetate (aq., pH 3.5), and the radiochemical yield (RCY) was determined using analytical radio-HPLC. In Fig. 2, the reaction scheme is presented.

Full automation of radiosyntheses
The automation of the N-11 C-methylation reaction was done on the TRACERlab TM FX C Pro (GE Healthcare). A schematic flowchart of the synthesis is depicted in Fig. 3.
After conversion of cyclotron-produced [ 11 C]CO 2 to [ 11 C]methane, [ 11 C]methyl iodide and [ 11 C]CH 3 OTf, it was trapped at RT in a glass reactor containing precursor HAPTHI (1 mg, 3 μmol) and 0.5 μL of an aqueous NaOH-solution (5 M) in 500 μL MEK. After heating of the sealed reaction vessel to 75°C for 2 min, the crude reaction mixture was cooled to 25°and quenched by addition of 1 mL HPLC eluent. The entire volume was then transferred to the 5 mL injection loop. The crude mixture was (fluid detector controlled) injected into the semi-preparative HPLC column (Fig. 4). The pure [ 11 C]Me@HAPTHI peak was cut into a round bulb, containing 80 mL of distilled water. The now predominantly aqueous product solution was subjected to solid phase extraction by transferring over a preconditioned (10 mL EtOH, air, 20 mL water) C18 SPE cartridge. After rinsing of the C18 SepPak® with water (V6) for complete removal of residual HPLC solvents, the pure product was eluted with 1.5 mL EtOH (V5) into a two-neck vial and the cartridge and transfer lines rinsed with further 5 mL 0.9 % saline into the same vial. After formulation with 9 mL 0.9 % saline, 1 mL 3 % saline and 1 mL 125 mM phosphate buffer, sterile filtration (0.22 μm) was performed

Quality control
Chemical and radiochemical impurities were assessed using analytical radio-and UV-HPLC according to the monograph in the European Pharmacopoeia [25]. Radiochemical identity and purity were measured via analytical radio-HPLC by comparison of retention times with authentic samples. Specific radioactivity was determined by quantification of the non-radioactive product (HPLC UV channel at 280 nm) and inclusion of the overall radiochemical yield (GBq at end of synthesis). Sterility, absence of endotoxins, pH, osmolality and residual solvents were determined by standard procedures routinely performed at the PET Centre of the Vienna General Hospital/Medical University of Vienna and follow the respective monograph in the European Pharmacopoeia [25].

Statistical analysis
All quantitative data described in the text and figures are specified as arithmetic mean ± standard deviation. For the determination of significance, a Student's twotailed t test (α = 0.95) was performed using Microsoft® Excel. P values of <0.05 were considered to be significant. Unless otherwise stated, error bars in figures represent the standard deviation; if not visual, they are within the icon margin.

NET-expressing membrane binding studies
The affinity of new radiolabelled ligand was determined in a NET-expressing membrane binding protocol [26,27]. For details, see Additional file 1.
Data from the competition plots (as arithmetic means of values derived from three different assays, each in triplicate for each compound) were analyzed and subsequently IC 50 and K i values were calculated using GraphPad Prism® software (San Diego, USA). Assays similar to those described for NET were performed in order to determine the selectivity of the tested compounds towards NET in comparison to DAT and SERT. IC 50 and K i values were obtained in analogy to NET experiments. Ratios DAT/NET and SERT/NET were determined.

LogD analysis, IAM chromatography and blood-brain barrier penetration
LogD values were assessed using a HPLC-based protocol according to Donovan and Pescatore [28]. All compounds (as cold reference standards) were injected together with two known compounds-with known logD and k′ values-according to a standard protocol. A polymeric ODP-50 column was used; a linear gradient from 10 % MeOH 90 % 25 mM phosphate buffer (pH 7.4) to 100 % methanol within 9.4 min at a flow-rate of 1.5 mL/min was applied. Internal standards were triphenylene and toluene; detection was performed at 260 and 285 nm.
As lipophilicity alone was shown to be a tenuous predictor for blood-brain barrier penetration, other in vitro methods have been described, such as immobilized artificial membrane (IAM) chromatography and further calculation of total polar surface area (tPSA) values [29][30][31]. Therefore, IAM chromatography was performed using a Redistech IAM.PC.DD2 column (15 cm × 4.6 mm) according to previously published methods [19,[32][33][34][35]. For analysis, 0.01 M phosphate buffer (pH 7.4) and ACN (in different ratios) were used isocratically as mobile phase at a flow rate of 1 mL/min. Resulting K m (membrane partition coefficient) and P m (permeability) values were obtained after data analysis using Microsoft Excel. The resulting data were compared with those derived from compounds known to penetrate BBB as external standard. Additionally, tPSA values were determined in silico using Chem Bio Draw Ultra (Cambridge Software, Perkin Elmer, USA).

Metabolic stability testing
Pooled human and rat liver microsomes are subcellular fractions that are rich in endoplasmatic reticuli, which contain many drug-metabolizing enzymes, e.g. cytochrome P450s, flavin monooxygenases and epoxide hydrolase. Microsomal incubations were performed in order to investigate the metabolization of [ 11 C]Me@HAPTHI. As the results, both the percentage of test compound metabolized after a certain time and the biological half-life were determined.

Plasma protein binding
For the determination of free fraction in human pooled plasma, an ultrafiltration protocol according to previously published methods was used [35][36][37][38]. Briefly, aliquots of pooled human plasma were spiked with [ 11 C]Me@HAPTHI and centrifuged using ultrafiltration vials (Amicon Centrifree; Millipore, Bedford, USA). The plasma free fraction was calculated, and the percentage of unspecific binding of [ 11 C]Me@HAPTHI to the filter matrix evaluated. For a detailed method, see Additional file 1.

Autoradiography, Nissl staining and immunohistochemistry
Human brain tissue (cortex, thalamus, hippocampus, cerebellum and hypothalamus) was obtained deeply frozen from the Neurobiobank of the Medical University Vienna and was stored at −80°C. Before cutting, tissue blocks were thawed slowly within 12 h to −20°C. The organs were cut at −20°C in a micro-cryotome into 10-μm-thick slices and thaw mounted onto object slides. Slices were again stored at −80°C until the beginning of the experiment.
In vitro autoradiography was performed with slight modifications according to previously published protocols [13,39,40]. Non-specific binding was determined by co-incubation with excess Nisoxetine (10 μM). For competition experiments, non-radioactive FMeNER-D2, an established NET PET tracer, and Me@HAPTHI were added to the incubation solution in different concentrations. After 1 h at room temperature, incubation was stopped and slices were processed on phosphor imaging films.
All data was exported to Microsoft Excel for statistical analysis, and the percentage of total specific binding was calculated.
Post-autoradiographic processing of the slices was done by Nissl staining in order to facilitate morphological mapping of hot areas in the autoradiography. The same tissue slices were stained after autoradiography with cresyl violet [28,41,42] to demonstrate the Nissl substance in the neurons and cell nuclei. For a detailed procedure, see Additional file 1.
Immunohistochemical staining experiments were performed on rat and human tissue cryo-slices, vicinal to the slices used for autoradiographic experiments. The staining procedure was a modification of a general protocol as published previously in detail [28,43].

Radiochemistry
The optimum parameters were examined in small-scale reactions. Thus, the influence of different 11 C-methylation agents, solvent, precursor concentration, reaction temperature and base were investigated (Fig. 5a-d). Radiochemical yields (RCY) of [ 11 C]Me@HATPHI were below 6 % for all examined conditions using [ 11 C]CH 3 I as methylation agent. Hereby, DMSO proved to be the best solvent for the SN 2 reaction using [ 11 C]methyl iodide. In contrast, very promising results were obtained using [ 11 C]CH 3 OTf as radio-methylation agent (Fig. 5c-d).
Interestingly, the use of DMSO as solvent did not result in high yields, less than 1 % RCY was observed using [ 11 C]CH 3 OTf. Applying 2-butanone resulted in high radiochemical yields. Furthermore, the influence of basic catalysis was examined: TBAH catalysis could not shift the reaction kinetics to favourable outcomes, as it did not result in any methylation of HAPTHI. Up to 12.8 ± 4.7 % RCY was observed when using 0.5 μL triethylamine instead. Conducting the experiments with 0.5 μL of 1 M NaOH (aq.), however, yielded 42.9 ± 5.2 % radiochemical yield with 1 mg/mL precursor concentration and even above 50 % RCY were obtained with 2 mg/mL precursor concentration. A further increase in basicity-facilitated by 0.5 μL 5 M NaOH (aq.) instead of 1 M NaOH (aq.)-did not lead to improved results (in a total reaction volume of 100 μL); only <0.5 % RCY were obtained.
Therefore, these optimum reaction parameters were transferred to the fully automated radiosynthesis within the Tracerlab FxC Pro synthesizer. In Table 1, an overview on the automated syntheses, their conversion and yield is given. The crude reaction mixture was purified via semi-preparative radio-HPLC using isocratic conditions (0.1 M ammonium acetate and acetonitrile (60/40; v/v)) at a flow rate of 5 mL/min. An exemplary semipreparative HPLC chromatogram is outlined in Fig. 4a. The precursor HAPTHI was found to be eluted at a retention time of 4.5 min (k′ = 0.55) and the product [ 11 C]Me@HAPTHI at 7.6 min (k′ = 1.62), respectively.
Overall, seven large-scale radiosyntheses were performed, yielding 2.2 ± 2.0 GBq (18.9 ± 13.3 %, corrected  Fig. 4b. The retention times in the analytical HPLC assay were 3.37 min (k′ = 2.17) for precursor HAPTHI, 1.8 min (k′ = 0.7) for [ 11 C]MeOH, 2.7 min (k′ = 1.55) for [ 11 C]CH 3 OTf and 3.1 min (k′ = 1.9) for [ 11 C]CH 3 I, respectively. The product [ 11 C]Me@HAPTHI was eluted at a retention time of 4.38 min (k′ = 3.08). Radiochemical purity always exceeded 98 %. Osmolality and pH values were at all times found to be in a physiological range. Residual solvent analysis using GC revealed MEK <5 ppm and ACN <20 ppm, besides 8.5 % ethanol present in the product formulation (total product volume 17.5 mL). Moreover, sterility and absence of endotoxins was approved for all produced batches of [ 11 C]Me@HAPTHI upon complete decay of radioactivity as in-process control.

Affinity and selectivity testings
Affinity of reference compounds (Me@HAPTHI and its radiolabeling progenitor HAPTHI) was determined using human NET membranes as K d = 0.21 ± 0.07 nM for Me@HAPTHI and 24.2 ± 10.9 nM for HAPTHI, respectively (n ≥ 9 triplicates). For determination of selectivity, the affinity of both reference substances was assessed on human DAT and SERT membranes and revealed >10 μM for both compounds for DAT and 409 ± 43 nM (Me@HAPTHI) and 10,274 ± 1207 nM (HAPTHI) towards SERT, respectively, (n ≥ 5 triplicates). Hence, selectivity of Me@HAPTHI towards NET was determined as DAT/NET >1947.6 and SERT/NET = 9757. Both values clearly elucidate the ideal binding properties of our novel NET PET ligand [ 11 C]Me@HAPTHI.

LogD analysis, IAM chromatography and blood-brain barrier penetration
The lipophilicity of the novel NET PET radioligand Me@HAPTHI was found to be in a decent range (logD = 2.27 ± 0.01) for a potential penetration of the BBB. The precursor HAPTHI showed a logD value of 2.30 ± 0.01. Additionally, BBB penetration experiments using IAM chromatography revealed a permeability of P m = 1.15 ± 0.25 for Me@HAPTHI and P m = 1.14 ± 0.27 for the precursor HAPTHI, respectively. Both values were within the identical, ideal range (P m = 0.01-4.21) from other PET tracers, known to easily penetrate the BBB [34].

Metabolic stability testing
Stability testing using human liver microsomes (n = 4) revealed no significant metabolism of [ 11 C]Me@HAPTHI within the tested timeframe. After 60 min, 99.6 ± 0.3 % of the tracer was found to be still intact. Incubation of [ 11 C]Me@HAPTHI with pooled male rat liver microsomes revealed a higher metabolic degradation. The percentage of intact tracer over time is presented in Fig. 6. Overall, 29.3 ± 1.9 % tracer was still intact after 1-h incubation time. Thus, the stability of the novel NET PET tracer [ 11 C]Me@HAPTHI is encouraging in a human and rodent setting and superior to the established reboxetinederived PET tracer [ 18 F]FMeNER-D2.

Plasma protein binding
The mean percentage of plasma free fraction (ff) and percentage of unspecific binding to the filter matrix of the Centrifuge vials was determined. A plasma free fraction of ff = 8.2 ± 0.3 % (n = 7 triplicates) as well as an unspecific filter retention of 51.26 ± 0.78 % was found. Overall, the ff of our novel NET PET tracer [ 11 C]Me@HAPTHI was in the same range as that of [ 11 C]ADAM [35].

In vitro autoradiography, immunohistochemistry and Nissl staining
In the autoradiographic experiments, the highest uptake of [ 11 C]Me@HAPTHI was observed in NET-rich regions identified with immunohistochemistry (Fig. 7). Blocking was performed with non-radioactive NET ligands FMeNER-D2 and Me@HAPTHI in two different concentrations each (100 nM, 1 μM). A concentrationdependent binding displacement was observed using human tissue samples for both cold competitors. In Table 2, an overview on the percentage of specific displaceable binding of [ 11 C]Me@HAPTHI and fmol/mm 2 values of relative transporter protein density on the different tissue sections is given. All values are given in % as mean n ≥ 3 triplicates. Autoradiography of human cerebellum revealed NET specific uptake in NET-rich regions identified with IHC, though blocking experiments were not possible due to the vast inhomogeneity of the tissue samples. In human nucleus caudatus, a region known to be low in NET density, only unspecific binding was observed. Immunohistochemical staining was used to allocate areas with high uptake in autoradiography with regions known high NET abundance. Hence, the NET antibodydye complexes were found highly abundant in the heart fibres, hippocampus, thalamus and hypothalamus and to a minor extent in all other brain regions (Fig. 7). NET specificity of staining was validated using a rabbit antibody isotype control.
Moreover, harvesting experiments with [ 11 C]Me@HAP THI using hNET expressing membranes were performed according to the affinity testing protocol. Thereby, a concentration-dependent displacement of [ 11 C]Me@HA PTHI was observed for all tested competitor substances (cold FMeNER-D2 or Me@HAPTHI), and the counts were corrected for decay (Fig. 8). Using Graph Pad Prism, data correlation revealed akin-binding displacement behaviour for both cold Me@HAPTHI as well as the established NET ligand FMeNER-D2 (n ≥ 3 triplicates).  Discussion [ 11 C]Me@HAPTHI presents a large stride towards an improved, novel, conveniently producible PET tracer for NET imaging. This study comprises the first radiochemical preparation, quality control and in vitro evaluation of this novel candidate PET-tracer. We describe its affinity, selectivity, lipophilicity and its potential to penetrate the BBB as well as metabolic stability. Moreover, the in vitro binding behaviour of [ 11 C]Me@HAPTHI to human NET cell membranes as well as human and rodent tissue slices was examined.
The excellent affinity of Me@HAPTHI (K d hNET = 0.21 ± 0.07 nM) and exceptional selectivity of our candidate NET PET ligand present the ideal ground for a further evaluation of this tracer. Moreover, a lower non-specific binding can be expected, as the described radioligand is less lipophilic than previous NET PET tracers based on reboxetine (logD Me@HAPTHI = 2.21, logD FMeNER-D2 = 2.73). Based on the in vitro data acquired, successful BBB penetration by [ 11 C]Me@HAPTHI may be expected. This assumption is supported by immobilized artificial membrane chromatography results showing Me@HAPTHI to be within the discussed range of permeability P m values.
Additionally, the high radiochemical yields and feasible radiosynthetic availability favour our newly developed NET radioligand. The employed 11 C-methylation reaction can be implemented at any PET facility with a cyclotron. Hence, this study presents a large stride towards a highly affine, selective and routinely available radiotracer. Moreover, in vitro stability of [ 11 [15,44,45]). Also in the rodent setting, where highly increased turnover rates of the enzymes are present, a In vitro binding studies revealed specific displaceable binding in human brain regions and rat heart, indicating towards a promising further use of this tracer in in vivo studies. Binding displacement was observed in competition experiments with different NET ligands FMeNER-D2 and Me@HAPTHI in a concentration-dependent manner. The high radiotracer uptake areas matched with the high NET-density regions identified by immunohistochemistry. Therefore, specific NET uptake of [ 11 C]Me@HAPTHI can be affirmed. While this specific NET binding may be valid on ex vivo tissue, the question of binding behaviour on a cellular level was raised. Therefore, in vitro binding studies on human NET membranes were performed in a cell harvesting protocol. In these cell-based experiments, which used the same parameters as autoradiography studies (i.e. incubation time and buffer), a comparable concentration-dependent binding displacement was found for both competitors FMeNER-D2 and Me@HAPTHI. Therefore, selective NET-uptake for our novel PET ligand [ 11 C]Me@HAPTHI could be confirmed on a cellular and on a human and rat tissue level.
Thus, [ 11 C]Me@HAPTHI was showing highly promising results in vitro so far and might therefore become an improved, routine NET PET tracer. As a next step, small animal experiments will be performed to further elucidate the in vivo behaviour of [ 11 C]Me@HAPTHI.