Neurokinin-3 receptor binding in Guinea pig, monkey, and human brain: In vitro and in vivo imaging using the novel radioligand, [18F]Lu AF10628

Article abstract of DOI:10.1093/ijnp/pyw023

Background: Previous autoradiography studies have suggested a marked interspecies variation in the neuroanatomical localization and expression levels of the neurokinin 3 receptor, with high density in the brain of rat, gerbil, and Guinea pig, but at the t

Full text of DOI:10.1093/ijnp/pyw023

International Journal of Neuropsychopharmacology Advance Access published April 7, 2016
International Journal of Neuropsychopharmacology, 2016, 1–10
doi:10.1093/ijnp/pyw023
Advance Access Publication: March 18, 2016
Research Article
research article
Neurokinin-3 Receptor Binding in Guinea Pig,
Monkey, and Human Brain: In Vitro and in Vivo
Imaging Using the Novel Radioligand,
[18F]Lu AF10628
Katarina Varnäs, PhD; Sjoerd J. Finnema, PhD; Vladimir Stepanov, PhD;
Akihiro Takano, MD, PhD; Miklós Tóth, PhD; Marie Svedberg, PhD;
Søren Møller Nielsen, PhD; Nikolay A. Khanzhin, PhD; Karsten Juhl, PhD;
Benny Bang-Andersen, PhD; Christer Halldin, PhD; Lars Farde, MD, PhD
Karolinska Institutet, Department of Clinical Neuroscience, Centre for Psychiatry Research, Stockholm,
Sweden (Drs Varnäs, Finnema, Stepanov, Takano, Tóth, Svedberg, Halldin, and Farde); Lundbeck Research, H.
Lundbeck A/S, 9 Ottiliavej, DK-2500 Copenhagen-Valby, Denmark (Drs Møller Nielsen, Khanzhin, Juhl, and
Bang-Andersen); AstraZeneca Translational Science Centre at Karolinska Institutet, PET CoE, Stockholm,
Sweden (Dr Farde).
Present address: Glycom A/S, Diplomvej 373, 1, DK-2800 Kgs. Lyngby, Denmark (N.A.K.).
Correspondence: Katarina Varnäs, PhD, Karolinska Institutet, Department of Clinical Neuroscience, Centre for Psychiatry Research, R5:02 Karolinska
University Hospital, SE-17176 Stockholm, Sweden (katarina.varnas@ki.se).
Abstract
Background: Previous autoradiography studies have suggested a marked interspecies variation in the neuroanatomical
localization and expression levels of the neurokinin 3 receptor, with high density in the brain of rat, gerbil, and guinea pig,
but at the time offered no conclusive evidence for its presence in the human brain. Hitherto available radioligands have
displayed low affinity for the human neurokinin 3 receptor relative to the rodent homologue and may thus not be optimal for
cross-species analyses of the expression of this protein.
Methods: A novel neurokinin 3 receptor radioligand, [18F]Lu AF10628 ((S)-N-(cyclobutyl(3-fluorophenyl)methyl)-8-fluoro-
2-((3-[18F]-fluoropropyl)amino)-3-methyl-1-oxo-1,2-dihydroisoquinoline-4-carboxamide), was synthesized and used
for autoradiography studies in cryosections from guinea pig, monkey, and human brain as well as for positron emission
tomography studies in guinea pig and monkey.
Results: The results confirmed previous observations of interspecies variation in the neurokinin 3 receptor brain localization
with more extensive distribution in guinea pig than in primate brain. In the human brain, specific binding to the neurokinin
3 receptor was highest in the amygdala and in the hypothalamus and very low in other regions examined. Positron emission
tomography imaging showed a pattern consistent with that observed using autoradiography.The radioactivity was, however,
found to accumulate in skull bone, which limits the use of this radioligand for in vivo quantification of neurokinin 3 receptor
binding.
Received: September 15, 2015; Revised: February 6, 2016; Accepted: March 10, 2016
© The Author 2016. Published by Oxford University Press on behalf of CINP.
.This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://
creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium,
1
provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

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| International Journal of Neuropsychopharmacology, 2016
Conclusion: Species differences in the brain distribution of neurokinin 3 receptors should be considered when using animal
models for predicting human neurokinin 3 receptor pharmacology. For positron emission tomography imaging of brain
neurokinin 3 receptors, additional work is required to develop a radioligand with more favorable in vivo properties.
Keywords: autoradiography, positron emission tomography, tachykinin receptor, NK3 receptor
Introduction
The tachykinins, substance P, neurokinin A, and neurokinin B
(NKB) constitute a group of peptide neurotransmitters that are
known to bind to 3 G protein-coupled receptor subtypes, the
NK1, NK2, and NK3 receptors (Gerard et al., 1993). These neuro-
peptides and receptors have long been thought to be implicated
in the pathophysiology and putative treatment of a range of CNS
disorders (Hökfelt et al., 1980; Pantaleo et al., 2010).
The NK3 receptor has attracted particular attention as a
potential target for the treatment of psychiatric disorders such
as schizophrenia (Spooren et al., 2005; Dawson and Porter, 2013).
In preclinical studies, NK3 receptor agonists have been shown to
have a stimulatory effect on dopaminergic neurotransmission
(Keegan et al., 1992; Seabrook et al., 1995; Alonso et al., 1996;
Nalivaiko et al., 1997; Marco et al., 1998), whereas NK3 recep-
tor antagonists inhibit drug-induced dopaminergic cell firing
(Gueudet et al., 1999) and dopamine release (Dawson et al.,
2008), supporting potential utility of these agents in the treat-
ment of psychosis.
The notion that NK3 receptor antagonists may have anti-
psychotic properties has, however, not been supported by clini-
cal trials. Though initial evidence for antipsychotic efficacy was
reported for the NK3 receptor antagonist osanetant (Meltzer
et al., 2004), subsequent studies with osanetant and 2 additional
NK3 receptor antagonists, talnetant and AZD2624, have failed to
demonstrate clinical efficacy in the treatment of schizophrenia
(Griebel and Beeske, 2012; Dawson and Porter, 2013). However,
available data regarding the therapeutic potential of NK3 recep-
tor antagonists for the treatment of psychiatric disorders remain
inconclusive. First, given the lack of a suitable positron emission
tomography (PET) radioligand for NK3 receptors, it is not known
whether sufficient receptor occupancy had been achieved in
the clinical trials. Second, information regarding the anatomi-
cal localization of NK3 receptors in the human brain is limited,
and it is therefore not known whether initial findings in animal
models can be extrapolated to human conditions.
hypothalamus, cortex, and hippocampus (Mileusnic et al., 1999;
Koutcherov et al., 2000; Tooney et al., 2000).
The lack of binding in early autoradiography studies using
human brain tissue could possibly be explained by limitations
of 125I-labeled eledoisin as a radioligand, having suboptimal
affinity for human NK3 receptors. Thus, the affinity of eledoi-
sin for human NK3 receptors (Ki >300nM; Buell et al., 1992) has
been reported to be more than 10-fold lower than for rat (19nM;
Bergström et al., 1987) and guinea pig (35nM; Guard et al., 1990).
In addition to the low affinity for human NK3 receptors, suscep-
tibility of the radioligand to degradation by peptidases in brain
tissue could contribute to the lack of a detectable signal (Dietl
and Palacios, 1991; Rigby et al., 2005).
In the present study, [18F]Lu AF10628 ((S)-N-(cyclobutyl(3-
fluorophenyl)methyl)-8-fluoro-2-((3-[18F]-fluoropropyl)amino)-
3-methyl-1-oxo-1,2-dihydroisoquinoline-4-carboxamide),
a selective nonpeptide NK3 receptor antagonist radioligand
with high affinity across species, was used to examine the
anatomical distribution of NK3 receptors. The positron emit-
ting isotope 18F offers the advantage of high image resolution,
combined with a relatively long half-life (approximately 110
minutes) and thereby allows for in vitro and in vivo imaging
with autoradiography and PET using the same radioligand.
Autoradiography studies in guinea pig, monkey, and human
brain tissue was used for the interspecies comparison, as this
radioligand has been found to have similar affinity for guinea
pig and primate NK3 receptors (Ki values of 0.21 and 0.24nM for
guinea pig and human receptors, respectively; for details, see
supplementary Material). In addition, the in vivo properties of
[18F]Lu AF10628 as a PET radioligand for brain NK3 receptors
were characterized in guinea pig and monkey.
Methods
Radiochemistry
Translational research on the NK3 receptor has indeed been
hampered by species differences in pharmacology (Maggi, 1995)
and receptor distribution patterns (Dietl and Palacios, 1991;
Langlois et al., 2001; Rigby et al., 2005).Whereas previous autora-
diography studies using 125I-labeled eledoisin (a peptide analogue
of NKB derived from mollusks) as a radioligand have demon-
strated high density of NK3 receptors in the brain of rat, gerbil,
and guinea pig, no evidence has been obtained for binding of
radiolabeled eledoisin analogues to NK3 receptors in the human
brain (Dietl and Palacios, 1991; Rigby et al., 2005). An autoradio-
graphic study using the radioligand [3H]senktide has, however,
provided support for binding to human brain NK3 receptors,
although only data for frontal and sensorimotor cortices were
reported (Mileusnic et al., 1999). The presence of NK3 receptors
in the primate brain has also been supported by evidence of NK3
mRNA expression, as detected using the polymerase chain reac-
tion, in several regions of the monkey (Nagano et al., 2006) and
human brain (Buell et al., 1992) and by immunohistochemistry
studies, suggesting expression of NK3 receptors in the human
Lu AF10628 and (S)-N-(cyclopropyl(4-fluorophenyl)methyl)
-2-(ethylamino)-8-fluoro-3-methyl-1-oxo-1,2-dihydroiso-
quinoline-4-carboxamide as well as the precursor, (S)-3-
((tert-butoxycarbonyl)(4-((cyclobutyl(3-fluorophenyl)methyl)
carbamoyl)-8-fluoro-3-methyl-1-oxoisoquinolin-2(1H)-yl)
amino)propyl 4-methylbenzenesulfonate, for synthesis of [18F]
Lu AF10628 were synthesized at H. Lundbeck A/S, Copenhagen-
Valby, Denmark. Other compounds and chemicals were obtained
from commercially available sources and were of analytical
grade wherever possible. [18F]Lu AF10628 (Figure 1) was synthe-
sized and purified using a semiautomatic synthesis module
(DM Automation). [18F]Fluoride was produced via the 18O(p,n)18F
nuclear reaction using a GE Medical Systems PETtrace cyclo-
tron with a silver liquid water target. The radionuclide was then
trapped on a QMA Light Sep-Pak cartridge (bicarbonate form),
and [18F]fluoride was eluted into the reaction vessel using 2mL
of acetonitrile/water (96/4vol/vol) containing 9.8mg of Kryptofix
2.2.2 and 1.8mg of potassium carbonate. The solvent was then

Varnäs et al.
| 3
Figure 1. Radiosynthesis of [18F]Lu AF10628.
evaporated by heating at 140°C under a stream of nitrogen
(100mL/min). To the dried [18F]fluoride/Kryptofix complex, 0.5
to 0.7mg of (S)-3-((tert-butoxycarbonyl)(4-((cyclobutyl(3-fluoro-
phenyl)methyl)carbamoyl)-8-fluoro-3-methyl-1-oxoisoquinolin-
2(1H)-yl)amino)propyl 4-methylbenzenesulfonate dissolved in
500–600µL of N,N-dimethylformamide was added. The reaction
mixture was heated for 20minutes at 120°C in a glass reactor
without stirring. After 20 minutes, the reactor was cooled to
room temperature and 200 to 300 µL of 6M aq. hydrochloric acid
was added and reaction mixture heated for 120°C for another 10
minutes. After deprotection, the reaction mixture was diluted
with water and the crude product was injected directly onto
semipreparative HPLC column, Waters μBondapak C18 column
(10µm, 125A, 7.8×300mm), and purified using HPLS system
consisting of the following components: Smartline Pump 100
(Knauer), automatic sample injector (Rheodyne-type) with a
2-mL loop; Smartline UV Detector 2500 (Knauer) and a gamma-
radioactivity PIN diode detector (Caroll & Ramsey Associates)
using acetonitrile-water 450:550 mixture as the mobile phase
with flow of 6mL/min, with UV detector set to 254nm. The
desired fraction was collected into a vial containing 50mL of
water. The resulting solution was pushed through a Sep-Pak
tC18 Plus Short cartridge; after trapping of the product the car-
tridge was rinsed with 10mL of distilled water, and the product
([18F]Lu AF10628) was then eluted with approximately 1mL of
ethanol and collected in a sterile receiving vial prefilled with 9
to 10mL of sterile phosphate buffered saline. Finally, the product
was passed through a sterile filter (0.22-µm pore size, Millipore)
in a clean-room environment.
Semmelweis University Human Ethical Committee (registration
no. 113/1995, 180/2001).
Brain tissue from 6 guinea pigs and 6 cynomolgus monkeys
(Macaca fascicularis) was used in the study. The brains were fro-
zen in dry ice-cooled isopentane (-40°C) and stored at -25°C until
use. For detailed anatomical analyses, brains were cut at 10 μm
(guinea pig brain tissue; n=2) and 20 μm (monkey brain tissue;
n=3) coronal and sagittal sections on a freezing microtome
(Leica, Heidelberg, Germany) and thaw mounted onto gelatin-
coated slides. In addition, for quantitative analyses comparing
radioligand binding density across species, brain tissue from
4 guinea pigs and 3 monkeys were sectioned, using a freezing
microtome (Leica, Heidelberg, Germany), into 100–μm-thick
sections.
Human brains were obtained at clinical autopsy at the
National Institute of Forensic Medicine, Karolinska Institutet
(Stockholm, Sweden) and the Department of Forensic Medicine,
Semmelweis Medical University, Budapest, Hungary. Whole
hemispheres were removed, frozen, and cryosectioned in
accordance with previously described procedures (Hall et al.,
1998), using a heavy-duty cryomicrotome (Leica cryomacrocut
CM3600, Leica, Nussloch, Germany). Tissue was obtained from
2 male and 1 female donor (ages 32, 58, and 59 years) with no
known neurological or psychiatric diagnosis at the time of
death. The postmortem times ranged between 11 and 15 hours.
From examination at autopsy and during sectioning, none of the
brains exhibited damage, abnormalities, or neurologic features.
The whole hemispheres were oriented so that a line connect-
ing the anterior and posterior commissures was parallel to the
surface of the cryostat specimen holder. Subsequently, whole
hemispheres were cryosectioned into 100-µm-thick coronal or
horizontal cryosections, transferred to gelatinized glass plates
(10–22cm), dried at room temperature, and then stored with
dehydrating agents (-25°C) until use.
The radiochemical purity of [18F]Lu AF10628 was determined
using analytical radio-HPLC under the following conditions:
C-18 μBondapak analytical column (3.9×300mm, 5µm, acetoni-
trile:water 60:40, flow 2mL/min, UV 254nm). The identity of [18F]
Lu AF10628 was confirmed by co-injection with an authentic
nonradioactive standard. [18F]Lu AF10628 was produced with
sufficient radiochemical yield (10–30%, decay corrected), high
specific radioactivity (>2000 Ci/mmol), and high radiochemical
purity (>95.0%). The total amount of unknown chemical impuri-
ties present in the formulation was <2 μg/injection.
In addition to the above-mentioned whole hemisphere
brain cryosections, the other human brain specimens had
been obtained at autopsy. The tissue was immediately cut into
coronal slabs, frozen in dry-ice-cooled isopentane, and stored
at -70oC. The slabs were subsequently cut into coronal blocks
of tissue containing the brainstem. Twenty-micron-thick cryo-
sections were taken from these blocks using a Leica cryostat
(Heidelberg, Germany), dried onto glass slides, and stored frozen
at -70oC until later use.
In Vitro Autoradiography Studies
Brain Tissue
Studies using experimental animals were approved by the
Animal Ethics Committee of the Swedish Animal Welfare
Agency (registration no. 4820/06-600 and 399/08). Studies includ-
ing human brain tissue were approved by the Ethics Committee
at Karolinska Institutet (registration no. 03-767) and the
Autoradiography
Autoradiography was carried out essentially as described ear-
lier (Hall et al., 1998). The sections were preincubated at room
temperature for 5 minutes in Tris-HCl buffer (pH 7.4, 50mM).

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| International Journal of Neuropsychopharmacology, 2016
Subsequently, the sections were incubated for 90 minutes at
room temperature with [18F]Lu AF10628 (0.02 MBq/mL) in Tris-
HCl buffer (pH 7.4, 50mM) containing 3mM MnCl2. After incuba-
tion, sections were washed with cold Tris buffer (pH 7.4, 50mM;
3 x 30 minutes for whole hemisphere sections and 3 x 5 minutes
for sections from smaller tissue blocks), briefly dipped into dis-
tilled water, and dried on a warm plate.
Nonspecific binding was defined in adjacent sections co-
incubated with 10 µM osanetant. Specificity and selectivity of
the binding were further assessed in a series of consecutive sec-
tions incubated in the absence and presence of the selective NK3
receptor antagonists SB222200 (100nM; Sarau et al., 2000) or (S)-
N-(cyclopropyl(4-fluorophenyl)methyl)-2-(ethylamino)-8-fluoro-
3-methyl-1-oxo-1,2-dihydroisoquinoline-4-carboxamide
(100nM), respectively, or the NK2 receptor selective antagonist
saredutant (100nM; Emonds-Alt et al., 1992).
Radioactivity was detected and quantified with a phosphor
imager (Fuji BAS-5000 image reader). Brain regions of interest
(ROI) were defined in autoradiography images with reference
to brain anatomical atlases (Rapisarda and Bacchelli, 1977;
Mikula et al., 2007; Mai et al., 2008). Nuclei of amygdala were
defined according to the description by Yilmazer-Hanke (2012).
The measured photostimulated luminescence/mm2 values were
subsequently transformed into radioactivity units (kBq/mm3)
based on intensity values obtained using radioactivity stand-
ards from serial dilutions of the incubation solution assuming
that the volume of the region of interest was the product of the
area and section thickness (100 µm). Finally, specific binding
was calculated by subtracting nonspecific binding, defined in
the presence of 10 µM osanetant, from the total [18F]Lu AF10628
binding.
Two cynomolgus monkeys weighing 4.0 and 6.3kg, respec-
tively, were supplied by Astrid Fagraeus Laboratory, Karolinska
Institutet, Solna, Sweden. MRIs of the monkey brains were
obtained using a 1.5 T General Electrics Signa (GE, Milwakee,
WI) system. A spoiled gradient recalled sequence was acquired
in the coronal plane with the following parameters: TR=21ms;
TE=4ms; flip angle=35º; slice thickness=1.0mm; FOV=12.8cm;
NEX=2; voxel size = 0.5 x 0.5 x 1mm3.
Anesthesia was induced by i.m. injection of ketamine hydro-
chloride (approximately 10mg/kg) and maintained after endotra-
cheal intubation by administration of a mixture of sevoflurane,
oxygen, and medical air.The monkey was observed continuously
during the PET experimental day. Body temperature was main-
tained by Bair Hugger Model 505 (Arizant Healthcare Inc.) and
monitored by an esophageal thermometer. Electrocardiogram,
heart rate, respiratory rate, oxygen saturation, and arterial blood
pressure were continuously monitored throughout the experi-
ment. No anesthesia- or drug-related effects on vital parameters
were noted. A head fixation system was used to secure a fixed
position of the monkey’s head throughout the PET measure-
ment (Karlsson et al., 1993).
A sterile physiological phosphate buffer (pH=7.4) solution of
[18F]Lu AF10628 (153 or 157 MBq) was injected as a bolus into
a sural vein during 5 seconds with simultaneous start of PET-
data acquisition. The PET measurement was conducted using
the High Resolution Research Tomograph (Siemens Molecular
Imaging, Knoxville, TN). List mode data were reconstructed
using the ordinary Poisson-3D-ordered subset expectation max-
imization algorithm, with 10 iterations and 160 subsets includ-
ing modeling of the point spread function. The corresponding
in-plane resolution with ordinary Poisson-3D-ordered subset
expectation maximization point spread function was 1.5mm in
the center of the field of view and 2.4mm at 10-cm off-center
directions (Varrone et al., 2009). Attenuation correction was
acquired with a 6-minute transmission measurement using a
single 137Cs source. List-mode data were acquired continuously
for 180 minutes starting at the time of radioligand injection.
Positron emission tomography images were then reconstructed
with a series of 28 frames.
Venous blood samples (2mL) were obtained manually at
5, 30, 60, 90, 120, and 150min after injection of [18F]Lu AF10628
in the monkeys. After centrifugation, 0.5 to 0.8mL plasma was
pipetted and plasma radioactivity was measured in a well coun-
ter (Farde et al., 1989).The fraction of plasma radioactivity corre-
sponding to unchanged radioligand in plasma was determined
as previously described for other PET radioligands (Halldin et al.,
2001). Briefly, venous plasma samples were deproteinized with
acetonitrile and analyzed by gradient HPLC with radiodetection.
Coregistrations and ROI delineations were performed using
PMOD v. 3 (Pixel-wise modeling software, PMOD Group, Zurich,
Switzerland). For guinea pigs, ROIs were delineated for the cor-
tex, amygdala, cerebellum, caudate-putamen, thalamus, the
whole brain, and skull on a guinea pig brain T2-weighted MRI,
which was used as a template for manual coregistration of PET
images.The monkey brain MRI was manually coregistered to the
average PET image and ROIs for skull, temporal cortex, amyg-
dala, cerebellum, putamen, thalamus and the whole brain con-
tour were delineated with reference to the coregistered MRI.
The ROIs were displayed on the corresponding PET images
and pair-wise pooled for each anatomical region. Regional
radioactivity was calculated for the sequence of time frames,
corrected for radioactivity decay, and plotted vs time. For
each region a time-radioactivity curve was generated and the
radioactivity concentration expressed as kBq/cm3. Regional
PET Studies in Guinea Pig and Monkey
Studies in guinea pigs were conducted in accordance with
the guidelines of the Swedish National Board of Laboratory
Animals and Karolinska Institutet’s guidelines for planning,
conducting, and documenting experimental research (registra-
tion no. 4820/06-600) under protocols approved by the Animal
Ethics Review Board of Northern Stockholm, Sweden (N557/11).
Two Dunkin Hartley guinea pigs, weighing 0.84 and 0.85kg,
respectively, obtained from Harlan Laboratories were housed at
the animal department of Karolinska University Hospital in a
temperature- (approx. 21ºC) and humidity- (approximately 40%)
controlled environment on a 12-h-light/-dark cycle (lights on
7:00 am) with access to food and water ad libitum. Animals were
allowed at least 1 week to habituate to the animal department
before start of the imaging sessions.
All experiments were conducted during the light phase of
the cycle. The imaging experiments were performed under iso-
flurane anesthesia (induction: 4%-5%, maintenance: 1.5%-2% in
50/50 air/oxygen). [18F]Lu AF10628 (16.8 and 16.9 MBq, respec-
tively) was injected i.v. into the 2 guinea pigs. Radioactivity was
measured for 123 minutes according to a preprogrammed series
of 35 frames, using the small-animal nanoScan PET/CT and PET/
MRI systems (Mediso Ltd.; Szanda et al., 2011; Nagy et al., 2013)
at Karolinska Experimental Research Imaging Center.
The PET study in nonhuman primates was approved by
the Animal Ethics Committee of the Swedish Animal Welfare
Agency (registration no. 399/08) and was performed according
to the “Guidelines for planning, conducting and document-
ing experimental research” (registration no. 4820/06-600) of
Karolinska Institutet as well as the “Guide for the Care and Use
of Laboratory Animals” (Clark et al., 1997).

Varnäs et al.
| 5
radioactivity was normalized to injected radioactivity and body
weight and expressed as standard uptake value ([SUV] fraction
of injected radioactivity/cm3 brain x body weight [g]).
differences observed between the forebrain regions examined,
although the cortical binding displayed a laminar pattern with
more intense binding in internal layers (Figure 2C). In the human
brain, specific binding was very low in most of the regions ana-
lyzed, with exception for the amygdala, which showed intense
binding of [18F]Lu AF10628. For all of the species investigated,
a very low level of specific binding was obtained for the hip-
pocampus, striatum, globus pallidus, and cerebellum.
Results
In Vitro Autoradiography Studies
Autoradiographic images of [18F]Lu AF10628 binding in brain tis-
sue of guinea pig, monkey, and human are shown in Figure 2.
In tissue sections incubated with [18F]Lu AF10628, a markedly
higher image intensity was observed for the cerebral cortex of
guinea pig than for primates (Figure 2A,C,E). Nonspecific bind-
ing, as determined in adjacent sections in the presence of the
NK3 receptor antagonist osanetant (10 μM), was low and homo-
geneously distributed (Figure 2B,D,F).
The density of regional specific binding for the 3 species is
presented inTable 1. In the guinea pig brain, high [18F]Lu AF10628
binding was observed in isocortical regions and the amygdala,
with low binding in other regions. In monkey brain tissue,
the binding was homogeneously distributed with no evident
Binding was unevenly distributed among subregions of the
human amygdala and was highest in the ventromedial part of
the basolateral nucleus and paralaminar nucleus, extending to
the parahippocampal-amygdaloid transition area and entorhi-
nal cortex (Figure 3; Table 1). Binding was lower in the medial
nucleus and the dorsal part of the basolateral amygdaloid
nucleus (Figure 3).
Specific binding, defined as the difference between total [18F]
Lu AF10628 binding and the binding remaining in the presence
of 10 µM osanetant, was moderate (approximately 0.4 kBq/mm3)
in the human hypothalamus (paraventricular and supraoptic
nuclei; Figures 2E and 3) and low (0.15–0.16 kBq/mm3) in the bed
nucleus of stria terminalis and the basal nucleus of Meynert
Figure 2. Autoradiograms showing binding of [18F]Lu AF10628 to neurokinin 3 (NK3) receptors in coronal sections of the guinea pig (A-B), monkey (C-D), and human
brain (E-F). Total binding (A, C, E) and nonspecific binding in the presence of the NK3 receptor compound osanetant (10 µM; B, D, F). Amg, amygdaloid nuclei; BL, baso-
lateral amygdaloid nucleus; Ca, caudate nucleus; FC, frontal cortex; GP, globus pallidus; Hy, hypothalamus; La, lateral amygdaloid nucleus; Pa, paraventricular nucleus
of the hypothalamus; Pu, putamen; SO, supraoptic nucleus of the hypothalamus; TC, temporal cortex; Th, thalamus. Scale bar = 1cm.

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| International Journal of Neuropsychopharmacology, 2016
Table 1. Binding of [18F]Lu AF10628 to NK3 Receptors In Autoradiography Studies of the Guinea Pig, Monkey, and Human Brain
Guinea Pig
Monkey
Human
n
Region
n
4
Mean
Range
1.0–1.1
n
3
Mean
0.10
Range
Mean
Range
Amygdala
1.1
0.08–0.12
Basolateral nucleus,
ventromedial part
Lateral nucleus
Cortex
Frontal
Temporal
3
3
1.1
0.09
0.93–1.2
0.04–0.15
4
2
1.4
1.2–1.6
1
3
3
3
0.13
0.12
0.17
0.08
3
3
1
0.05
0.09
0.05
0.03–0.09
0.02–0.16
0.12–0.13
0.13–0.21
0.07–0.09
Occipital
Striatum
0.11
0.05–0.17
Caudate nucleus
Putamen
Thalamus
Hippocampus
Substantia nigra
Cerebellum
3
3
3
3
1
1
0.06
0.05
0.04
0.06
0.10
0.05
0.05–0.08
0.03–0.09
0.01–0.09
0.01–0.09
4
2
2
4
0.21
0.02
0.20
0.09
0.18–0.24
0.00–0.04
0.17–0.24
0.03–0.12
1
3
3
3
0.14
0.07
0.10
0.08
0.05–0.08
0.09–0.11
0.08-0.08
Radioactivity concentration expressed in kBq/mm3 tissue.
Figure 3. Details of human brain whole hemisphere autoradiograms showing binding of the neurokinin 3 (NK3) receptor antagonist [18F]Lu AF10628 in the amygdala
and hypothalamus. Cresyl violet stained section (left), total binding (middle), and nonspecific binding in the presence of the NK3 receptor compound osanetant (10 µM;
right). BL, basolateral amygdaloid nucleus; BL pv, basolateral amygdaloid nucleus, parvicellular part; La, lateral amygdaloid nucleus; LH, lateral hypothalamic area; Me,
medial amygdaloid nucleus; PHA, parahippocampal-amygdaloid transition area; PL, paralaminar amygdaloid nucleus; SO, supraoptic nucleus of the hypothalamus.
Scale bar = 1cm.
(results not shown). Very low specific binding was observed
in cortical regions (frontal, temporal, insular, cingulate, and
occipital cortex; Table 1) and in the brainstem substantia nigra
(supplementary Figure 1A-B) and raphe nuclei (supplementary
Figure 1C-F).
The selectivity of the binding to human NK3 receptors
was examined in adjacent sections co-incubated with the 2
chemically distinct NK3 receptor antagonists, SB222200 and
(S)-N-(cyclopropyl(4-fluorophenyl)methyl)-2-(ethylamino)-
8-fluoro-3-methyl-1-oxo-1,2-dihydroisoquinoline-4-carboxam-
ide, or with the NK2 receptor selective antagonist saredutant.The
binding was inhibited by 100-nM concentrations of SB222200
and (S)-N-(cyclopropyl(4-fluorophenyl)methyl)-2-(ethylamino)-
8-fluoro-3-methyl-1-oxo-1,2-dihydroisoquinoline-4-carboxam-
ide, but remained at the same level as the total binding in tissue
cryosections co-incubated with 100nM saredutant (Figure 4A-D).
Images of the averaged radioactivity for the initial 30 minutes
after [18F]Lu AF10628 injection showed a pattern of preferential
binding in cortical regions of the guinea pig brain, whereas the
binding in monkey was homogeneously distributed throughout
the brain regions studied (Figure 5). For both species, brain radio-
activity was low (<0.7 SUV) at late time (after 30 minutes) of PET
data acquisition when a much larger proportion of radioactivity
was detected outside the brain within the skull. The pattern of
the observed regional radioactivity distribution could be con-
firmed by time curves for radioactivity in the skull and different
brain regions (Figure 6A-B). The ratios of AUC calculated based
on 5 to 30 minutes of data acquisition for cortex to the cerebel-
lum were 2.2 (mean; n=2) and 1.2 (mean; n=2) for guinea pig
and monkey, respectively.
The percentage of parent [18F]Lu AF10628 in monkey plasma
was 39% to 45 % at 30 minutes postinjection and 18% to 27 % at
90 minutes postinjection. At 30 minutes after radioligand injec-
tion, the average ratio of regional brain radioactivity concentra-
tion to that for metabolite corrected plasma was 2.3 for cortex
and 1.6 for the cerebellum.
PET Studies in Guinea Pig And Monkey
After i.v. administration of [18F]Lu AF10628 in guinea pig, brain
radioactivity reached peak concentration within 1 minute (0.90–
0.95 SUV) after which it rapidly decreased. A similar time course
was seen in monkey, where the maximum brain radioactivity
concentration (1.0–1.2 SUV) was reached at 1.5 minutes after
radioligand administration.
Discussion
The aim of the present study was to examine and compare the
regional localization of NK3 receptors in the brain of 3 species.

Varnäs et al.
| 7
Figure 4. Selectivity of [18F]Lu AF10628 binding to the human brain neurokinin 3 (NK3) receptor. (A) Total binding. Binding in the presence of saturating concentra-
tion (100nM) of the NK3 receptor-selective compounds SB222200 (B) and (S)-N-(cyclopropyl(4-fluorophenyl)methyl)-2-(ethylamino)-8-fluoro-3-methyl-1-oxo-1,2-dihy-
droisoquinoline-4-carboxamide (C), respectively, and the NK2 receptor selective compound saredutant (100nM; D). Scale bar = 1cm.
Figure 5. Positron emission tomography images of radioactivity in the guinea pig brain (top) and monkey brain (bottom) after i.v. injection of [18F]Lu AF10628. The left
panel shows the guinea pig MRI template and the individual monkey MR images, respectively, used for anatomical definition of regions of interest.The middle and right
panels show average radioactivity from 0 to 30 minutes and 30 to 123 minutes, respectively, after [18F]Lu AF10628 injection. Image intensity is presented in SUV units.
The results confirmed previous observations of a marked cross-
species variation, with higher density and a more widespread
anatomical distribution in guinea pig than in primate brain.
However, contrary to previous evidence, specific radioligand
binding to the NK3 receptor was obtained also for some human
brain regions, notably in specific nuclei of amygdala.
of the monkey (Figure 2C; supplementary Figure 2). Conversely, a
layer-specific cortical binding pattern, consistent with that pre-
viously reported using autoradiography in nonhuman primate
brain (Rigby et al., 2005), was found for monkey but was not
identified in human tissue sections. This discrepancy is unex-
pected given the close agreement in binding patterns commonly
reported for other neurotransmitter receptors in the human and
nonhuman primate brain. Given that no information is available
Despite the conspicuous binding in the human amygdala, a
correspondingly high binding was not evident in the amygdala

8
| International Journal of Neuropsychopharmacology, 2016
Figure 6. Time curves for radioactivity in brain regions and skull after administration of [18F]Lu AF10628 in guinea pig (A) and monkey (B).
regarding the affinity of Lu AF10628 for monkey NK3 receptors,
it is possible that the discrepant binding patterns observed
may reflect species differences in affinity between monkey and
human receptors. However, this interpretation seems unlikely,
based on similar amino acid sequence (Nagano et al., 2006),
and affinity of established NK3 receptor compounds (Nagano
et al., 2006; Malherbe et al. 2011), for nonhuman primate and
human NK3 receptors. Nevertheless, the unique binding pattern
observed in the cerebral cortex supports that [18F]Lu AF10628
binds specifically to monkey NK3 receptors under the present
experimental conditions.
To analyze the binding to brain NK3 receptors, a novel radioli-
gand, [18F]Lu AF10628, was used as the imaging tool. Lu AF10628
was selected from a chemical series where in vitro affinities
for the NK1 receptor in general were in the micromolar range
and where high selectivity vs other targets was observed. The
in vitro Ki value at the human NK2 receptor was 1 µM, but this
specific compound has not been tested for NK1 receptor affinity.
In support of selective labeling of NK3 receptors in guinea pig
brain, the binding pattern showed a good correspondence with
that found using other NK3 receptor radioligands in this species
(Langlois et al., 2001; Rigby et al., 2005). Further, selectivity of the
signal for human NK3 vs NK2 receptors was confirmed by the
observation that the binding could be inhibited by 2 chemically
distinct NK3 receptor ligands, but remained at the same level in
the presence of a saturating concentration of an NK2 receptor
selective compound. The findings support that the binding pat-
tern observed with [18F]Lu AF10628 corresponds to the regional
distribution of NK3 receptors in brain.
To our knowledge, this study provides the first detailed
autoradiographic mapping of the presence of NK3 receptors in
human brain. The findings corroborate previous results from
immunohistochemistry studies indicating the presence of
NK3 receptors in several regions of the human brain, including
hypothalamus, hippocampus, and cortex (Mileusnic et al., 1999;
Koutcherov et al., 2000; Tooney et al., 2000). In general, immu-
nohistochemistry studies may be limited by lack of specificity
of antibodies for the target protein. For instance, the NK3 recep-
tor antibody may show potential cross-reactivity with a kappa-
like opioid receptor given sequence similarity with this receptor
(Mileusnic et al., 1999). Confirmation of the findings with auto-
radiography studies using a selective NK3 receptor radioligand
now provides conclusive support for the presence of NK3 recep-
tors in human brain.

Varnäs et al.
| 9
However, previous studies using 125I-labeled eledoisin as a
radioligand have reported no evidence for binding to NK3 recep-
tors in human brain cryosections (Dietl and Palacios, 1991; Rigby
et al., 2005). A likely explanation for the different findings could
be the superior properties of [18F]Lu AF10628 as a radioligand in
terms of affinity for human NK3 receptors (Ki = 0.24nM for Lu
AF10628 vs >300nM for eledoisin; Buell et al., 1992).A direct com-
parison of our findings to those in previous studies is not pos-
sible, as it is unknown whether the regions showing dense [18F]
Lu AF10628 binding (amygdala, hypothalamus) were included in
the previous studies.
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Acknowledgments
The authors thank Siv Eriksson, Åsa Södergren, and all members
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The work was supported by the Innovative Medicines
Initiative Joint Undertaking under grant agreement no 115008
of which resources are composed of EFPIA in-kind contribution
and financial contribution from the European Union’s Seventh
Framework Programme (FP7/2007–2013).
Statement of Interest
Søren Møller Nielsen, Nikolay A. Khanzhin, Karsten Juhl, and
BennyBang-AndersenarecurrentorpastemployeesofLundbeck.
Lars Farde is employed by AstraZeneca Pharmaceuticals. The
other authors declare no conflicts of interest.
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