Radiosynthesis and first preclinical evaluation of the novel norepinephrine transporter pet-ligand [11C]ME@HAPTHI
- Christina Rami-Mark1, 2,
- Neydher Berroterán-Infante1, 2,
- Cecile Philippe1, 3,
- Stefanie Foltin1,
- Chrysoula Vraka1,
- Alexander Hoepping4,
- Rupert Lanzenberger5,
- Marcus Hacker1,
- Markus Mitterhauser†1, 3Email author and
- Wolfgang Wadsak†1, 2
© Rami-Mark et al. 2015
Received: 10 April 2015
Accepted: 22 May 2015
Published: 10 June 2015
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.
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.
[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.
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.
KeywordsNET PET Autoradiography Radiosynthesis HAPTHI
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 [1–9]. 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 [10–14]. However, previous studies have shown that the in vivo and in vitro behaviour of these reboxetine analogues, more specifically [11C]MeNER ([11C]MRB, ((S,S)-2-(α-(2-[11C]methoxyphenoxy)benzyl)morpholine), [11C]MeNET and [18 F]FMeNER-D2 ((S,S)-2-(α-(2-[18 F]fluoro[2H2] 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, 15–18]. Recently, we aimed at the preparation of a benzo[d]imidazolone derivative—[11C]Me@APPI as new NET PET tracer . 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.
The set-up of a small-scale radiosynthetic procedure for the preparation of the carbon-11 labelled [11C]Me@HAPTHI and its optimization;
The up-scaling and set-up of a fully automated preparation of [11C]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.
Precursor, HAPTHI ((S)-1-(4-amino-3-hydroxybutyl)-3-phenyl-1,3-dihydrobenzo[c][1, 2, 5]thiadiazole 2,2-dioxide, and cold reference compound Me@HAPTHI ((S)-1-(3-hydroxy-4-(methylamino)butyl)-3-phenyl-1,3-dihydrobenzo[c][1, 2, 5]thiadiazole 2,2-dioxide) were custom-synthesized by ABX Advanced Biochemical Compounds (Radeberg, Germany). Briefly, synthesis of (2S)-4-(2,2-dioxido-3-phenyl-2,1,3-benzothiadiazol-1(3H)-yl)-1-(methylamino)butan-2-ol followed the route described by Neill et al. [20, 21]. For more details, see Additional file 1.
2-Butanone (MEK, <99.0 % ACS reagent), acetonitrile (ACN, HPLC grade), dimethylsulfoxide (DMSO), tetrabutylammonium hydroxide 30-hydrate (TBAH), ammonium formate, ammonium acetate, sodium hydroxide, triethylamine and ethanol (absolute) were purchased from Sigma-Aldrich (Vienna, Austria) in the highest available grades. In addition, iodine (sublimated grade for analysis; ACS, Pharm. Eur.) was obtained from Merck (Darmstadt, Germany). Silver triflate impregnated carbon was prepared by reaction of 1 g of silver trifluoromethanesulfonate (Sigma Aldrich, Vienna, Austria) in 20 mL ACN with 3 g of Graphpac-GC (80/100 mesh, Alltech, Deerfield, USA). The suspension was stirred under protection from light and in an argon atmosphere for 30 min. After removal of the solvent, the resulting powder was dried under protection from light for further 2 h under reduced pressure.
For formulation of the product, 0.9 % saline solution from B. Braun (Melsungen, Germany), 3 % saline solution (Landesapotheke Salzburg, Austria) and sodium dihydrogenphosphate-monohydrate and disodiumhydrogenphosphate-dihydrate (both from Merck, Darmstadt, Germany) were used. Sterile water was purchased from Meditrade Medicare Medizinprodukte (Kufstein, Austria). Phosphate buffer (125 mM) was prepared by dissolving 0.224 g sodium dihydrogenphosphate-monohydrate and 1.935 g disodiumhydrogenphosphate-dihydrate in 100 mL sterile water. For solid phase extraction, C18 plus SepPak® cartridges were purchased from Waters (Waters® Associates, Milford, USA). Low-protein binding Millex® GS 0.22 μm sterile filters were obtained from Millipore (Bedford, USA).
All other chemicals and solvents for the radiosyntheses were obtained from Merck (Darmstadt, Germany) and Sigma-Aldrich (Vienna, Austria) with at least analytical grade and used without further purification.
NET, DAT and SERT expressing membrane preparations were obtained from Perkin Elmer (MA, USA). An ODP-50 column (20 × 4.0 mm, 5 μm) was purchased from Shodex® (Showa Denko Europe GmbH, Munich, Germany). For prediction of BBB penetration, a Redistech IAM.PC.DD2 column (Regis Technologies Inc., Morton Grove, USA) was used.
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).
The NET-antibody (SLC6A2 Antibody H-67, sc-67216) was purchased from Santa Cruz Biotechnology (TX, USA). An endogenous Avidin-Biotin blocking kit (ab64212) as well as the DAB (=3,3′-diaminobenzidine) substrate kit (94665) was obtained from abcam (Cambridge, UK). A rabbit primary antibody isotype control was purchased from Invitrogen (CA, USA). A peroxidase-based Vectastain ABC kit (Rabbit IgG, PK-4001) was obtained from Vector Laboratories (CA, USA). Phosphate buffered saline (PBS pH 7.4, tenfold concentrate, 11237) was obtained from Morphisto Evolutionsforschung und Anwendung GmbH (Germany). Mayer’s Hemalaun solution was purchased from Merck Millipore (Germany). Histofluid (Marienfeld Superior, Germany) was used as a mounting medium. Coverslips from Menzel Gläser (24 × 60 mm, Thermo Fisher Scientific, Germany) were used for conservation of mounted slides. All other chemicals were obtained from Sigma-Aldrich.
[11C]CO2 was produced within a GE PETtrace cyclotron (General Electric Medical System, Uppsala, Sweden) by a 14 N(p,α)11C nuclear reaction under irradiation of a gas target filled with N2 (+1 % O2) (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, [11C]Me@HAPTHI-synthesis was transferred to the TRACERlab™ FX C Pro synthesizer and a fully automated synthesis was established.
Crude [11C]Me@HAPTHI was purified by semi-preparative 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 SupelcosilTM 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 analytical HPLC was performed on a Merck-Hitachi LaChrom HPLC system (L-7100 pump; LaChrom L-7400 UV detector) using a NaI radio-detector (Bertholdt Technologies, Bad Wildbach, Germany) and a GinaStar® processing software (Raytest, Straubenhardt, Germany). A Phenomenex® Prodigy, Phenyl-3(PH-3), 5 μm, 250 × 4.6 mm (Phenomenex®, Aschaffenburg, Germany) column with a mobile phase consisting of ACN/0.1 M ammonium formate 50/50 v/v% at a flow rate of 2 mL/min was used while detection of the cold compounds was performed at 280 nm.
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 [11C]CO2 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, [11C]CO2 was converted into [11C]CH4 by a Ni-catalysed reduction with H2 at 400 °C. [11C]CH3I was produced within the same synthesizer using the dry method (gas phase conversion) described by Larsen et al.  with adopted modifications described by Kniess et al. . Briefly, the resulting [11C]CH4 was reacted with sublimated iodine at 738 °C in a recirculating process for 4 min to give [11C]CH3I. The produced [11C]CH3I was trapped on-line on a Porapak® N column and finally released by heating the trap to 190 °C. [11C]CH3OTf was prepared on-line at the passage of [11C]CH3I through a pre-heated (200 °C) column containing 300 mg silver triflate impregnated graphitized carbon at a flow rate of 40 mL/min .
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
Full automation of radiosyntheses
Chemical and radiochemical impurities were assessed using analytical radio- and UV-HPLC according to the monograph in the European Pharmacopoeia . 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 .
All quantitative data described in the text and figures are specified as arithmetic mean ± standard deviation. For the determination of significance, a Student’s two-tailed 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
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 IC50 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. IC50 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 . 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 [29–31]. Therefore, IAM chromatography was performed using a Redistech IAM.PC.DD2 column (15 cm × 4.6 mm) according to previously published methods [19, 32–35]. 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 [11C]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 [35–38]. Briefly, aliquots of pooled human plasma were spiked with [11C]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 [11C]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].
Hence, the best results were obtained with NaOH-catalysis in 2-butanone for 2 min at 75 °C using 2 mg/mL precursor HAPTHI. Thereby, 54.0 ± 8.3 % radiochemical yield was achieved.
Overview on the fully automated, large-scale radiosyntheses of [11C]Me@HAPTHI
[11C]Me@HAPTHI (n = 7)
Starting activity [11C]CO2
Trapped [11C]CH3OTf in reactor
Loss during injection in loop waste
Product [11C]Me@HAPTHI (EOS)
Yield (decay corr. to EOB)
Specific activity [GBq/μmol] (EOS)
Overall, seven large-scale radiosyntheses were performed, yielding 2.2 ± 2.0 GBq (18.9 ± 13.3 %, corrected for decay to EOB) of sterile, formulated [11C]Me@HAPTHI within 41 min including 5 min of radiopharmaceutical quality control. A mean specific activity of 46.8 ± 28.5 GBq/μmol was found in the large-scale syntheses (calculated using an HPLC-based method). A representative analytical HPLC chromatogram of the purified, sterile [11C]Me@HAPTHI is shown in 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 [11C]MeOH, 2.7 min (k′ = 1.55) for [11C]CH3OTf and 3.1 min (k′ = 1.9) for [11C]CH3I, respectively. The product [11C]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 [11C]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 [11C]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 .
Metabolic stability testing
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 [11C]Me@HAPTHI was in the same range as that of [11C]ADAM .
In vitro autoradiography, immunohistochemistry and Nissl staining
Overview of specific NET binding of the radioligand [11C]Me@HAPTHI vs. Me@HAPTHI and FMeNER-D2 on rat and human tissue origin
n ≥ 3
FMeNER 1 μM
88.8 ± 11.2
99.00 ± 0.07
Me@HAPTHI 1 μM
92.5 ± 7.5*
104.5 ± 4.5
FMeNER 1 μM
71.9 ± 7.9*
86.3 ± 11.2*
Me@HAPTHI 1 μM
66.3 ± 5.9*
82.1 ± 13.9*
FMeNER 1 μM
68.36 ± 2.11
77.6 ± 9.8
Me@HAPTHI 1 μM
85.9 ± 18.5
92.5 ± 17.3
FMeNER 1 μM
77.4 ± 14.5
FMeNER 100 nM
97.8 ± 14.6
Me@HAPTHI 1 μM
62.0 ± 3.6*
Me@HAPTHI 100 nM
83.7 ± 1.7*
FMeNER 1 μM
67.3 ± 8.2
FMeNER 100 nM
97.1 ± 10.3
Me@HAPTHI 1 μM
68.3 ± 5.3
Me@HAPTHI 100 nM
84.1 ± 9.3
Human nucleus caudatus
FMeNER 1 μM
107.6 ± 17.7
102.6 ± 14.5
Me@HAPTHI 1 μM
110.0 ± 21.0
93.5 ± 12.5
FMeNER 1 μM
108.2 ± 17.3
103.9 ± 12.2
Me@HAPTHI 1 μM
107.2 ± 20.8
124.7 ± 10.8
Immunohistochemical staining was used to allocate areas with high uptake in autoradiography with regions known high NET abundance. Hence, the NET antibody-dye 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.
[11C]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 [11C]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 [11C]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 11C-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 [11C]Me@HAPTHI against human liver microsomes, containing all human liver cytochrome P450 enzymes, is excellent (99.6 ± 0.3 % intact tracer after 60 min). In contrast, other existing PET tracers show significant metabolic degradation within this timeframe (e.g. [11C]MeNER, [11C]DASB or [11C]WAY-100635 [15, 44, 45]). Also in the rodent setting, where highly increased turnover rates of the enzymes are present, a sufficient metabolic stability of [11C]Me@HAPTHI was observed (29.26 ± 1.95 % intact, 60 min).
Furthermore, a plasma free fraction of 8.4 % was determined in ultrafiltration experiments, which was in a similar range with other clinically successful PET-tracers (e.g. [11C]ADAM).
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 [11C]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 [11C]Me@HAPTHI could be confirmed on a cellular and on a human and rat tissue level.
Thus, [11C]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 [11C]Me@HAPTHI.
A number of key properties have been discussed in the presented study, indicating that the benzothiadiazole dioxide [11C]Me@HAPTHI presents a viable and improved NET PET tracer.
We demonstrated its outstanding affinity and selectivity, its great stability in human liver microsomes, as well as promising results from in vitro autoradiography. Therefore, these data encourage us for an in vivo application of this compound in small animal PET experiments in the future. On these grounds, [11C]Me@HAPTHI might improve clinical NET imaging.
The authors would like to thank Vanessa Fröhlich and Thomas Zenz for their practical and technical support. The authors are grateful to Marie Spies, MD, for native English editing.
- Zhou J. Norepinephrine transporter inhibitors and their therapeutic potential. Drugs Future. 2004;29:1235–44.PubMed CentralPubMedView ArticleGoogle Scholar
- Zhou J, Zhang A, Klaess T, Johnson KM, Wang CZ, Ye YP, et al. Biaryl analogues of conformationally constrained tricyclic tropanes as potent and selective norepinephrine reuptake inhibitors: synthesis and evaluation of their uptake inhibition at monoamine transporter sites. J Med Chem. 2003;46:1997–2007.PubMedView ArticleGoogle Scholar
- Curatolo P, D’Agati E, Moavero R. The neurobiological basis of ADHD. Ital J Pediatr. 2010;36:79.PubMed CentralPubMedView ArticleGoogle Scholar
- Mash DC, Ouyang Q, Qin Y, Pablo J. Norepinephrine transporter immunoblotting and radioligand binding in cocaine abusers. J Neurosci Methods. 2005;143:79–85.PubMedView ArticleGoogle Scholar
- Schlessinger A, Geier E, Fan H, Irwin JJ, Shoichet BK, Giacomini KM, et al. Structure-based discovery of prescription drugs that interact with the norepinephrine transporter, NET. Proc Natl Acad Sci U S A. 2011;108:15810–5.PubMed CentralPubMedView ArticleGoogle Scholar
- Vazey EM, Aston-Jones G. The emerging role of norepinephrine in cognitive dysfunctions of Parkinson’s disease. Front Behav Neurosci. 2012;6:48.PubMed CentralPubMedView ArticleGoogle Scholar
- Moldovanova I, Schroeder C, Jacob G, Hiemke C, Diedrich A, Luft FC, et al. Hormonal influences on cardiovascular norepinephrine transporter responses in healthy women. Hypertension. 2008;51:1203–9.PubMedView ArticleGoogle Scholar
- Harik S, Duckrow R, LaManna J, Rosenthal M, Sharma V, Banerjee S. Cerebral compensation for chronic noradrenergic denervation induced by locus ceruleus lesion: recovery of receptor binding, isoproterenol- induced adenylate cyclase activity, and oxidative metabolism. J Neurosci. 1981;1:641–9.PubMedGoogle Scholar
- Rommelfanger KS, Edwards GL, Freeman KG, Liles LC, Miller GW, Weinshenker D. Norepinephrine loss produces more profound motor deficits than MPTP treatment in mice. Proc Natl Acad Sci U S A. 2007;104:13804–9.PubMed CentralPubMedView ArticleGoogle Scholar
- Wilson AA, Patrick Johnson D, Mozley D, Hussey D, Ginovart N, Nobrega J, et al. Synthesis and in vivo evaluation of novel radiotracers for the in vivo imaging of the norepinephrine transporter. Nucl Med Biol. 2003;30:85–92.PubMedView ArticleGoogle Scholar
- Takano A, Gulyás B, Varrone A, Halldin C. Comparative evaluations of norepinephrine transporter radioligands with reference tissue models in rhesus monkeys: (S, S)-[18F]FMeNER-D2 and (S, S)-[11C]MeNER. Eur J Nucl Med Mol Imaging. 2009;36:1885–91.PubMedView ArticleGoogle Scholar
- Schou M, Halldin C, Sóvágó J, Pike VW, Hall H, Gulyás B, et al. PET evaluation of novel radiofluorinated reboxetine analogs as norepinephrine transporter probes in the monkey brain. Synapse. 2004;53:57–67.PubMedView ArticleGoogle Scholar
- Gulyás B, Brockschnieder D, Nag S, Pavlova E, Kása P, Beliczai Z, et al. The norepinephrine transporter (NET) radioligand (S, S)-[18F]FMeNER-D2 shows significant decreases in NET density in the human brain in Alzheimer’s disease: a post-mortem autoradiographic study. Neurochem Int. 2010;56:789–98.PubMedView ArticleGoogle Scholar
- Rami-Mark C, Zhang MR, Mitterhauser M, Lanzenberger R, Hacker M, Wadsak W. [18F]FMeNER-D2: reliable fully-automated synthesis for visualization of the norepinephrine transporter. Nucl Med Biol. 2013;40:1049–54.PubMed CentralPubMedView ArticleGoogle Scholar
- Schou M, Zoghbi S, Shetty H, Shchukin E, Liow J-S, Hong J, et al. Investigation of the metabolites of (S, S)-[11C]MeNER in humans, monkeys and rats. Mol Imaging Biol. 2009;11:23–30.PubMed CentralPubMedView ArticleGoogle Scholar
- Zeng F, Jarkas N, Stehouwer JS, Voll RJ, Owens MJ, Kilts CD, et al. Synthesis, in vitro characterization, and radiolabeling of reboxetine analogs as potential PET radioligands for imaging the norepinephrine transporter. Bioorg Med Chem. 2008;16:783–93.PubMedView ArticleGoogle Scholar
- Zeng F, Mun J, Jarkas N, Stehouwer JS, Voll RJ, Tamagnan GD, et al. Synthesis, radiosynthesis, and biological evaluation of carbon-11 and fluorine-18 labeled reboxetine analogues: potential positron emission tomography radioligands for in vivo imaging of the norepinephrine transporter. J Med Chem. 2008;52:62–73.View ArticleGoogle Scholar
- Zeng F, Stehouwer JS, Jarkas N, Voll RJ, Williams L, Camp VM, et al. Synthesis and biological evaluation of 2β,3α-(substituted phenyl)nortropanes as potential norepinephrine transporter imaging agents. Bioorg Med Chem Lett. 2007;17:3044–7.PubMedView ArticleGoogle Scholar
- Mark C, Bornatowicz B, Mitterhauser M, Hendl M, Nics L, Haeusler D, et al. Development and automation of a novel NET-PET tracer: [11C]Me@APPI. Nucl Med Biol. 2013;40:295–303. doi:10.1016/j.nucmedbio.2012.11.009.PubMedView ArticleGoogle Scholar
- O’Neill DJ, Adedoyin A, Bray JA, Deecher DC, Fensome A, Goldberg JA, et al. Discovery of novel selective norepinephrine inhibitors: 1-(2-morpholin-2-ylethyl)-3-aryl-1,3-dihydro-2,1,3-benzothiadiazole 2,2-dioxides (WYE-114152). J Med Chem. 2011;54:6824–31. doi:10.1021/jm200733r.PubMedView ArticleGoogle Scholar
- O'Neill DJ, Adedoyin A, Alfinito PD, Bray JA, Cosmi S, Deecher DC, et al. Discovery of novel selective norepinephrine reuptake inhibitors: 4-[3-aryl-2,2-dioxido-2,1,3-benzothiadiazol-1(3H)-yl]-1-(methylamino)butan-2-ols (WYE-103231). J Med Chem. 2010;53:4511–21.PubMedView ArticleGoogle Scholar
- Larsen P, Ulin J, Dahlstrøm K, Jensen M. Synthesis of [11C]iodomethane by iodination of [11C]methane. Appl Radiat Isot. 1997;48:153–7.View ArticleGoogle Scholar
- Kniess T, Rode K, Wuest F. Practical experiences with the synthesis of [11C]CH3I through gas phase iodination reaction using a TRACERlabFXC synthesis module. Appl Radiat Isot. 2008;66:482–8.PubMedView ArticleGoogle Scholar
- Jewett DM. A simple synthesis of [11C]methyl triflate. Int J Radiat Appl Instrum Part A. 1992;43:1383–5.View ArticleGoogle Scholar
- Council of Europe. Radioactive pharmaceuticals (European Pharmacopoeia (Europäisches Arzneibuch). 6th ed. Vienna: Verlag Österreich GmbH; 2008.Google Scholar
- Zeng F, Jarkas N, Owens MJ, Kilts CD, Nemeroff CB, Goodman MM. Synthesis and monoamine transporter affinity of front bridged tricyclic 3β-(4′-halo or 4′-methyl)phenyltropanes bearing methylene or carbomethoxymethylene on the bridge to the 2β-position. Bioorg Med Chem Lett. 2006;16:4661–3.PubMedView ArticleGoogle Scholar
- Tejani-Butt SM. [3H]nisoxetine: a radioligand for quantitation of norepinephrine uptake sites by autoradiography or by homogenate binding. J Pharmacol Exp Ther. 1992;260:427–36.PubMedGoogle Scholar
- Donovan SF, Pescatore MC. Method for measuring the logarithm of the octanol-water partition coefficient by using short octadecyl-poly(vinyl alcohol) high-performance liquid chromatography columns. J Chromatogr A. 2002;952:47–61.PubMedView ArticleGoogle Scholar
- Naik P, Cucullo L. In vitro blood–brain barrier models: current and perspective technologies. J Pharm Sci. 2012;101:1337–54.PubMed CentralPubMedView ArticleGoogle Scholar
- Neuhaus W, Freidl M, Szkokan P, Berger M, Wirth M, Winkler J, et al. Effects of NMDA receptor modulators on a blood–brain barrier in vitro model. Brain Res. 2011;1394:49–61.PubMedView ArticleGoogle Scholar
- Yoon CH, Kim SJ, Shin BS, Lee KC, Yoo SD. Rapid screening of blood–brain barrier penetration of drugs using the immobilized artificial membrane phosphatidylcholine column chromatography. J Biomol Screen. 2006;11:13–20.PubMedView ArticleGoogle Scholar
- Tavares AA, Lewsey J, Dewar D, Pimlott SL. Radiotracer properties determined by high performance liquid chromatography: a potential tool for brain radiotracer discovery. Nucl Med Biol. 2012;39:127–35.PubMedView ArticleGoogle Scholar
- Rami-Mark C, Bornatowicz B, Fink C, Otter P, Ungersboeck J, Vraka C, et al. Synthesis, radiosynthesis and first in vitro evaluation of novel PET-tracers for the dopamine transporter: [(11)C]IPCIT and [(18)F]FE@IPCIT. Bioorg Med Chem Lett. 2013;21:7562–9.View ArticleGoogle Scholar
- Vraka C, Nics L, Weiss V, Wagner K-H, Hacker M, Wadsak W, et al. Combination of high throughput HPLC methods for rapid prediction of blood brain barrier penetration of newly developed radiotracers—EANM 14. Eur J Nucl Med Mol Imaging. 2014;41:442.Google Scholar
- Huang Y, Hwang D-R, Narendran R, Sudo Y, Chatterjee R, Bae S-A, et al. Comparative evaluation in nonhuman primates of five PET radiotracers for imaging the serotonin transporters[11C]McN 5652, [11C]ADAM, [11C]DASB, [11C]DAPA, and [11C]AFM. J Cereb Blood Flow Metab. 2002;22:1377–98.PubMedView ArticleGoogle Scholar
- Gandelman MS, Baldwin RM, Zoghbi SS, Zea-Ponce Y, Innis RB. Evaluation of ultrafiltration for the free-fraction determination of single photon emission computed tomography (SPECT) radiotracers: beta-CIT, IBF, and iomazenil. Thai J Pharm Sci. 1994;83:1014–9.View ArticleGoogle Scholar
- Price JC, Mayberg HS, Dannals RF, Wilson AA, Ravert HT, Sadzot B. Measurement of benzodiazepine receptor number and affinity in humans using tracer kinetic modeling, positron emission tomography, and [11C]flumazenil. Journal of cerebral blood flow and Metabolism. 1993;13:656–67.PubMedView ArticleGoogle Scholar
- Sadzot B, Price JC, Mayberg HS, Douglass KH, Dannals RF, Lever JR, et al. Quantification of human opiate receptor concentration and affinity using high and Low specific activity [11C]diprenorphine and positron emission tomography. J Cereb Blood Flow Metab. 1991;11:204–19.PubMedView ArticleGoogle Scholar
- Schou M, Halldin C, Pike VW, Mozley PD, Dobson D, Innis RB, et al. Post-mortem human brain autoradiography of the norepinephrine transporter using (S, S)-[18F]FMeNER-D2. Eur Neuropsychopharmacol. 2005;15:517–20.PubMedView ArticleGoogle Scholar
- Philippe C, Haeusler D, Fuchshuber F, Spreitzer H, Viernstein H, Hacker M, et al. Comparative autoradiographic in vitro investigation of melanin concentrating hormone receptor 1 ligands in the central nervous system. Eur J Pharmacol. 2014;735:177–83.PubMedView ArticleGoogle Scholar
- Kádár A, Wittmann G, Liposits Z, Fekete C. Improved method for combination of immunocytochemistry and Nissl staining. J Neurosci Methods. 2009;184:115–8.PubMed CentralPubMedView ArticleGoogle Scholar
- Fukuda T, Koelle GB. The cytological localization of intracellular neuronal acetylcholinesterase. J Biophys Biochem Cytol. 1959;5:433–40.PubMed CentralPubMedView ArticleGoogle Scholar
- Immunohistochemistry HF. Current protocols in immunology. 2001.Google Scholar
- Någren K, Halldin C, Pike VW, Allonen T, Hietala J, Swahn CG, et al. Radioactive metabolites of the 5-HT1A receptor pet radioligand, [carbonyl-11C]way-100635, measured in human plasma samples. J Label Compd Radiopharm. 2001;44:S472–4.View ArticleGoogle Scholar
- Parsey RV, Ojha A, Ogden RT, Erlandsson K, Kumar D, Landgrebe M, et al. Metabolite considerations in the in vivo quantification of serotonin transporters using 11C-DASB and PET in humans. J Nucl Med. 2006;47:1796–802.PubMedGoogle Scholar
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