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

Abstract

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 [(11)C]Me@HAPTHI by describing its affinity, selectivity, metabolic stability, plasma free fraction, blood-brain barrier (BBB) penetration and binding behaviour in in vitro autoradiography.
[(11)C]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 [(11)C]Me@HAPTHI is a feasible and improved NET radioligand and might prospectively facilitate clinical NET imaging.

Full text

O R I G I N A L R E S E A R C H Open Access
Radiosynthesis and first preclinical
evaluation of the novel norepinephrine
transporter pet-ligand [
11
C]ME@HAPTHI
Christina Rami-Mark
1,2
, Neydher Berroterán-Infante
1,2
, Cecile Philippe
1,3
, Stefanie Foltin
1
, Chrysoula Vraka
1
,
Alexander Hoepping
4
, Rupert Lanzenberger
5
, Marcus Hacker
1
, Markus Mitterhauser
1,3*†
and Wolfgang Wadsak
1,2†
Abstract
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
[
11
C]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: [
11
C]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 [
11
C]Me@HAPTHI is a feasible and improved NET radioligand and might prospectively facilitate
clinical NET imaging.
Keywords: NET; PET; Autoradiography; Radiosynthesis; HAPTHI
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 hyper-
activity 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 pri-
marily 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 [
11
C]MeNER ([
11
C]MRB, ((S,S)-2-(α-(2-
[
11
C]methoxyphenoxy)benzyl)morpholine), [
11
C]MeNET
and [
18
F]FMeNER-D
2
((S,S)-2-(α-(2-[
18
F]fluoro[
2
H
2
]
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 radiosynth-
esis [10, 15–18]. 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
* Correspondence: markus.mitterhauser@meduniwien.ac.at
†
Equal contributors
1
Department of Biomedical Imaging and Image-guided Therapy, Division of
Nuclear Medicine, Medical University of Vienna, Vienna, Austria
3
Faculty of Life Sciences, Department of Technology and Biopharmaceutics,
University of Vienna, Vienna, Austria
Full list of author information is available at the end of the article
© 2015 Rami-Mark et al. This is an Open Access article distributed under the terms of the Creative Commons Attribution
License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any
medium, provided the original work is properly credited.
Rami-Mark et al. EJNMMI Research (2015) 5:34
DOI 10.1186/s13550-015-0113-3

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-reboxetine-
based 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 de-
signed benzothidiazole dioxides exhibits excellent affin-
ity and selectivity as well as slightly reduced flexibility
compared to other previously published benzoimidazo-
lones [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;
Theup-scalingandset-upofafullyautomated
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.
Methods
Materials
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 de-
scribed 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), tetrabu-
tylammonium hydroxide 30-hydrate (TBAH), ammonium
formate, ammonium acetate, sodium hydroxide, triethyla-
mine 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 pre-
pared by reaction of 1 g of silver trifluoromethanesulfo-
nate (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 re-
duced pressure.
For formulation of the product, 0.9 % saline solution
from B. Braun (Melsungen, Germany), 3 % saline solution
(Landesapotheke Salzburg, Austria) and sodium dihydro
genphosphate-monohydrate and disodiumhydrogenphos
phate-dihydrate (both from Merck, Darmstadt, Germany)
Fig. 1 Chemical structures of reboxetine and
11
C-labelled NET PET tracers [
11
C]Me@APPI and [
11
C]MeNER, [
11
C]MeNET and our novel NET PET
ligand [
11
C]ME@HAPTHI. The red coloured atom indicates the position of the radioisotope introduced by radiolabeling
Rami-Mark et al. EJNMMI Research (2015) 5:34 Page 2 of 12

were used. Sterile water was purchased from Meditrade
Medicare Medizinprodukte (Kufstein, Austria). Phosphate
buffer (125 mM) was prepared by dissolving 0.224 g so-
dium dihydrogenphosphate-monohydrate and 1.935 g
disodiumhydrogenphosphate-dihydrate in 100 mL ster-
ile water. For solid phase extraction, C18 plus SepPak®
cartridges were purchased from Waters (Waters® Asso-
ciates, 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 prepara-
tions were obtained from Perkin Elmer (MA, USA). An
ODP-50column(20×4.0mm,5μm) 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 (7–9hpostmortem
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 neurodegen-
erative 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 re-
moved and quick-frozen in i-pentan. Research using ani-
mal 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 de-
tection 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 Autoradiog-
raphy Cassette FBCS 1417) were purchased from Fisher
Scientific (PA, USA).
TheNET-antibody(SLC6A2AntibodyH-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). Phos-
phate 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.
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 semi-
preparative reversed phase HPLC using the built-in
semi-preparative HPLC system equipped with a radio-
activity 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,
5μm, 250 × 10 mm (Supelco®, Bellefonte, PA, USA) col-
umn was used with a mobile phase of ACN/0.1 M am-
monium 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 mea-
sured with a Wescor osmometer Vapro® 5600 (Sanova
Medical Systems, Vienna, Austria).
Rami-Mark et al. EJNMMI Research (2015) 5:34 Page 3 of 12

Methods
Radiochemistry
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 reduc-
tion 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
Iwastrapped
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
Ithroughapre-heated
(200 °C) column containing 300 mg silver triflate impreg-
nated graphitized carbon at a flow rate of 40 mL/min [24].
Small-scale reactions
For optimization of reaction conditions, small-scale reac-
tions using [
11
C]CH
3
Ior[
11
C]CH
3
OTf were performed.
Either [
11
C]CH
3
Ior[
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 v-
vials. 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 equi-
volume 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 precur-
sor 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, con-
taining 80 mL of distilled water. The now predominantly
Fig. 2 Radiosynthesis of [
11
C]Me@HAPTHI starting from the precursor molecule HAPTHI
Rami-Mark et al. EJNMMI Research (2015) 5:34 Page 4 of 12

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 re-
moval 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
Fig. 3 Flow scheme of the fully automated radiosynthesis of [
11
C]Me@HAPTHI
Fig. 4 aSemi-preparative and banalytical HPLC chromatogram
Rami-Mark et al. EJNMMI Research (2015) 5:34 Page 5 of 12

under aseptic conditions (laminar air flow hot cell, class A)
to avoid microbial contamination.
Quality control
Chemical and radiochemical impurities were assessed
using analytical radio- and UV-HPLC according to the
monograph in the European Pharmacopoeia [25]. Radio-
chemical identity and purity were measured via analyt-
ical 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 sol-
vents were determined by standard procedures routinely
performed at the PET Centre of the Vienna General
Hospital/Medical University of Vienna and follow the re-
spective 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 two-
tailed ttest (α= 0.95) was performed using Microsoft®
Excel. Pvalues of <0.05 were considered to be signifi-
cant. Unless otherwise stated, error bars in figures repre-
sent 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 subse-
quently IC
50
and K
i
values were calculated using GraphPad
Prism® software (San Diego, USA).
Assays similar to those described for NET were per-
formed 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%methanolwithin9.4minataflow-rateof
1.5 mL/min was applied. Internal standards were tri-
phenylene and toluene; detection was performed at 260
and 285 nm.
As lipophilicity alone was shown to be a tenuous pre-
dictor for blood–brain barrier penetration, other in vitro
methods have been described, such as immobilized artifi-
cial membrane (IAM) chromatography and further calcu-
lation 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) ac-
cording 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 stand-
ard. 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 in-
vestigate the metabolization of [
11
C]Me@HAPTHI. As
the results, both the percentage of test compound me-
tabolized 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 previ-
ously published methods was used [35–38]. Briefly,
aliquots of pooled human plasma were spiked with
[
11
C]Me@HAPTHI and centrifuged using ultrafiltra-
tion 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-
Rami-Mark et al. EJNMMI Research (2015) 5:34 Page 6 of 12

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 concen-
trations. 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 map-
ping 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 per-
formed 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].
Results
Radiochemistry
The optimum parameters were examined in small-scale
reactions. Thus, the influence of different
11
C-methyla-
tion 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 iod-
ide. 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 con-
centration and even above 50 % RCY were obtained
with 2 mg/mL precursor concentration. A further in-
crease in basicity—facilitated by 0.5 μL5MNaOH
(aq.) instead of 1 M NaOH (aq.)—did not lead to im-
proved results (in a total reaction volume of 100 μL);
only <0.5 % RCY were obtained.
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 % radio-
chemical yield was achieved.
Therefore, these optimum reaction parameters were
transferred to the fully automated radiosynthesis within
the Tracerlab FxC Pro synthesizer. In Table 1, an over-
view on the automated syntheses, their conversion and
yield is given. The crude reaction mixture was purified
via semi-preparative radio-HPLC using isocratic condi-
tions (0.1 M ammonium acetate and acetonitrile (60/40;
v/v)) at a flow rate of 5 mL/min. An exemplary semi-
preparative HPLC chromatogram is outlined in Fig. 4a.
The precursor HAPTHI was found to be eluted at a re-
tention 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 per-
formed, yielding 2.2 ± 2.0 GBq (18.9 ± 13.3 %, corrected
Fig. 5 Dependence of the radiochemical yield of [
11
C]Me@HAPTHI (n≥3) on the
11
C-methylation agent a[
11
C]methyliodide or b
[
11
C]methyltriflate) in DMSO and 2-butanone using different bases (NaOH, triethylamine or TBAH) at 2-min reaction time
Rami-Mark et al. EJNMMI Research (2015) 5:34 Page 7 of 12

for decay to EOB) of sterile, formulated [
11
C]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 represen-
tative analytical HPLC chromatogram of the purified,
sterile [
11
C]Me@HAPTHI is shown in Fig. 4b. The reten-
tion 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 re-
vealed >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, se-
lectivity 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 pre-
cursor 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)re-
vealed 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 micro-
somes revealed a higher metabolic degradation. The per-
centage of intact tracer over time is presented in Fig. 6.
Overall, 29.3 ± 1.9 % tracer was still intact after 1-h incu-
bation 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 reboxetine-
derived PET tracer [
18
F]FMeNER-D2.
Plasma protein binding
The mean percentage of plasma free fraction (ff ) and per-
centage 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 re-
gions identified with immunohistochemistry (Fig. 7).
Blocking was performed with non-radioactive NET li-
gands FMeNER-D2 and Me@HAPTHI in two different
concentrations each (100 nM, 1 μM). A concentration-
dependent binding displacement was observed using
human tissue samples for both cold competitors. In
Table 2, an overview on the percentage of specific dis-
placeable binding of [
11
C]Me@HAPTHI and fmol/mm
2
values of relative transporter protein density on the dif-
ferent tissue sections is given. All values are given in %
as mean n≥3 triplicates. Autoradiography of human
Table 1 Overview on the fully automated, large-scale
radiosyntheses of [
11
C]Me@HAPTHI
[
11
C]Me@HAPTHI (n= 7) Mean SD Median
Starting activity [
11
C]CO2 53.4 2.4 53.9
Trapped [
11
C]CH4 34.6 4.6 32
Trapped [
11
C]CH3I 29.6 2.4 29
Trapped [
11
C]CH3OTf in reactor 16.6 5.5 17.2
After quenching 8.8 3.6 8.9
Loss during injection in loop waste 1.0 0.5 0.8
Product [
11
C]Me@HAPTHI (EOS) 2.2 2.0 1.9
Yield (decay corr. to EOB) 13.7 13.5 15.9
Specific activity [GBq/μmol] (EOS) 43.4 29.7 59.2
Reaction conditions: [
11
C]MeOTf, NaOH, MEK, 2 mg/mL
precursor concentration
EOS end of synthesis, EOB end of bombardment
Rami-Mark et al. EJNMMI Research (2015) 5:34 Page 8 of 12

cerebellum revealed NET specific uptake in NET-rich
regions identified with IHC, though blocking experi-
ments were not possible due to the vast inhomogeneity
of the tissue samples. In human nucleus caudatus, a re-
gion 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 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 anti-
body 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).
Fig. 6 Metabolic stability of [
11
C]Me@HAPTHI against human and rat liver microsomes
Fig. 7 aNET-autoradiography and bimmunohistochemistry of [
11
C]Me@HAPTHI on 10 μm slices of human cortex, thalamus, hypothalamus,
cerebellum and nucleus caudatus as well as rat heart tissue and blocking with 100 nM FMeNER-D2, 1 μM FMeNER-D2, 100 nM Me@HAPTHI
and 1 μM Me@HAPTHI. The scale shows the radioactivity from high (red ) to low levels of radiotracer present on the Phosphor imager film
Rami-Mark et al. EJNMMI Research (2015) 5:34 Page 9 of 12

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 ro-
dent 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 evalu-
ation of this tracer. Moreover, a lower non-specific bind-
ing can be expected, as the described radioligand is less
lipophilic than previous NET PET tracers based on rebox-
etine (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 mem-
brane 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 reac-
tion 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
C]Me@HAPTHI against
human liver microsomes, containing all human liver cyto-
chrome 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 time-
frame (e.g. [
11
C]MeNER, [
11
C]DASB or [
11
C]WAY-100635
[15, 44, 45]). Also in the rodent setting, where highly
increased turnover rates of the enzymes are present, a
Table 2 Overview of specific NET binding of the radioligand
[
11
C]Me@HAPTHI vs. Me@HAPTHI and FMeNER-D2 on rat and
human tissue origin
n≥3[
11
C]Me@HAPTHI
% BL-competitor fmol
Rat heart
FMeNER 1 μM 88.8 ± 11.2 <0.01
FMeNER 100nM 99.00 ± 0.07 <0.01
Me@HAPTHI 1 μM 92.5 ± 7.5* <0.01
Me@HAPTHI 100nM 104.5 ± 4.5 <0.01
Human cortex
FMeNER 1 μM 71.9 ± 7.9* 0.86
FMeNER 100nM 86.3 ± 11.2* <0.01
Me@HAPTHI 1 μM 66.3 ± 5.9* 1.32
Me@HAPTHI 100nM 82.1 ± 13.9* 0.02
Human thalamus
FMeNER 1 μM 68.36 ± 2.11 0.71
FMeNER 100nM 77.6 ± 9.8 0.47
Me@HAPTHI 1 μM 85.9 ± 18.5 0.09
Me@HAPTHI 100nM 92.5 ± 17.3 0.26
Human hypothalamus
FMeNER 1 μM 77.4 ± 14.5 0.02
FMeNER 100 nM 97.8 ± 14.6 0.11
Me@HAPTHI 1 μM 62.0 ± 3.6* 0.04
Me@HAPTHI 100 nM 83.7 ± 1.7* 0.05
Human hippocampus
FMeNER 1 μM 67.3 ± 8.2 <0.01
FMeNER 100 nM 97.1 ± 10.3 <0.01
Me@HAPTHI 1 μM 68.3 ± 5.3 <0.01
Me@HAPTHI 100 nM 84.1 ± 9.3 <0.01
Human nucleus caudatus
FMeNER 1 μM 107.6 ± 17.7 n.d.
FMeNER 100nM 102.6 ± 14.5 n.d.
Me@HAPTHI 1 μM 110.0 ± 21.0 n.d.
Me@HAPTHI 100nM 93.5 ± 12.5 nd
Human cerebellum
FMeNER 1 μM 108.2 ± 17.3 n.d.
FMeNER 100nM 103.9 ± 12.2 n.d.
Me@HAPTHI 1 μM 107.2 ± 20.8 n.d.
Me@HAPTHI 100nM 124.7 ± 10.8 n.d.
fmol values reflect calculated relative concentration (fmol/mm
2
) of transporter
protein). Limit of detection = 0.01 fmol; BL=baseline
n.d. not determined
*p< 0.05
Fig. 8 NET-binding of [
11
C]Me@HAPTHI on human NET expressing
cell membranes using a harvesting protocol. Competition was done
using different concentrations of Me@HAPTHI and FMeNER-D2 (1, 3,
10, 30, 100 and 1000 nM)
Rami-Mark et al. EJNMMI Research (2015) 5:34 Page 10 of 12

sufficient metabolic stability of [
11
C]Me@HAPTHI was ob-
served (29.26 ± 1.95 % intact, 60 min).
Furthermore, a plasma free fraction of 8.4 % was de-
termined in ultrafiltration experiments, which was in a
similar range with other clinically successful PET-tracers
(e.g. [
11
C]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 competi-
tion 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 promis-
ing 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 eluci-
date the in vivo behaviour of [
11
C]Me@HAPTHI.
Conclusions
A number of key properties have been discussed in the
presented study, indicating that the benzothiadiazole diox-
ide [
11
C]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. There-
fore, these data encourage us for an in vivo application of
this compound in small animal PET experiments in the
future. On these grounds, [
11
C]Me@HAPTHI might im-
prove clinical NET imaging.
Additional file
Additional file 1: Supplementary data on affinity testing, metabolic
stability assessments and autoradiography. Detailed methods for
synthesis of precursor and reference compounds, the affinity testing of
the new radiolabelled ligand via NET-expressing membrane binding
protocol, as well as detailed procedures to autoradiography,
immunohistochemistry and metabolic stability testings.
Competing interests
Dr. Alexander Hoepping is a full employee at the ABX Advanced Biochemical
Compounds. All other authors declare that they have no competing interests.
Authors’contributions
CRM performed all radiosyntheses and preclinical in vitro experiments,
autoradiography, immunohistochemistry and writing of the paper. NB
contributed to all radiosyntheses and metabolite studies. CP contributed to
in vitro autoradiography and immunohistochemistry. SF contributed to the
affinity and selectivity testing procedures. CV contributed to IAM
chromatography experiments and plasma free fraction. AH performed
the synthesis of the cold reference standard Me@HAPTHI and the
precursor HAPTHI. RL participated in the design of the study and
proofread the manuscript. MH designed parts of the research and
proofread the manuscript. MM conceived and supervised the preclinical
experiments and proofread the manuscript. WW conceived and
supervised the radiosyntheses and proofread the manuscript. All authors
read and approved the manuscript.
Acknowledgements
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.
Author details
1
Department of Biomedical Imaging and Image-guided Therapy, Division of
Nuclear Medicine, Medical University of Vienna, Vienna, Austria.
2
Department
of Inorganic Chemistry, University of Vienna, Vienna, Austria.
3
Faculty of Life
Sciences, Department of Technology and Biopharmaceutics, University of
Vienna, Vienna, Austria.
4
ABX Advanced Biochemical Compounds, Radeberg,
Germany.
5
Department of Psychiatry and Psychotherapy, Division of
Biological Psychiatry, Medical University of Vienna, Vienna, Austria.
Received: 10 April 2015 Accepted: 22 May 2015
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