Eur J Nucl Med Mol Imaging (2009) 36:446–453
DOI 10.1007/s00259-008-0968-x
ORIGINAL ARTICLE
Effect of cyclosporin A administration on the biodistribution
and multipinhole μSPECT imaging of [123I]R91150
in rodent brain
P. Blanckaert & I. Burvenich & S. Staelens &
S. De Bruyne & L. Moerman & L. Wyffels & F. De Vos
Received: 3 March 2008 / Accepted: 21 September 2008 / Published online: 5 November 2008
# Springer-Verlag 2008
Abstract
Purpose P-glycoprotein (Pgp) is an efflux protein found
amongst other locations in the blood–brain barrier. It is
important to investigate the effect of Pgp modulation on
clinically used brain tracers, because brain uptake of the tracer
can be altered by blocking of the Pgp efflux transporter. The
function of Pgp can be blocked with cyclosporin A.
Methods We investigated the effect of cyclosporin A admin-
istration on the biodistribution of [123I]R91150 in rodents,
and the effect of Pgp blocking on the quality of multipinhole
μSPECT imaging with [123I]R91150. The influence of
increasing doses of cyclosporin A on the brain uptake of
[123I]R91150 was investigated in NMRI mice. A biodistri-
bution study with [123I]R91150 was performed in male
Sprague-Dawley rats pretreated with cyclosporin A and not
pretreated. Brain uptake of [123I]R91150 after cyclosporin A
injection was compared to the brain uptake in untreated
animals, and a displacement study with ketanserin was
performed in both groups. A multipinhole μSPECT brain
imaging study was also performed using a Milabs U-SPECT-II
camera in male Sprague-Dawley rats. To exclude the effect of
possible metabolites, a metabolite study was also performed.
Results At the highest cyclosporin A dose (50 mg/kg), a
sevenfold increase in brain radioactivity concentration was
P. Blanckaert (*) : I. Burvenich : S. De Bruyne : L. Moerman :
L. Wyffels : F. De Vos
Laboratory for Radiopharmacy, Faculty of Pharmaceutical Sciences,
Ghent University,
Harelbekestraat 72,
9000 Gent, Belgium
e-mail: peter.blanckaert@hotmail.com
S. Staelens
MEDISIP, Faculty of Engineering, Ghent University - IBBT,
De Pintelaan 185 (blok B),
9000 Gent, Belgium
observed in NMRI mice. Also, a dose-response relationship
was established between the dose of cyclosporin A and the
brain uptake of [123I]R91150 in mice. Compared to the con-
trol group, a five-fold increase in [123I]R91150 radioactivity
concentration was observed in the brain of Sprague-Dawley
rats after cyclosporin A treatment (50 mg/kg). Radioactivity
concentration in the frontal cortex increased from 0.24±
0.0092 to 1.58±0.097% injected dose per gram of tissue after
treatment with cyclosporin A (at the 1-h time-point). Blood
radioactivity concentrations did not increase to the same
extent. The cortical activity was displaced by administration
of ketanserin. A metabolite study confirmed that there was no
increased metabolism of [123I]R91150 due to cyclosporin A.
The visual quality of multipinhole μSPECT images with
[123I]R91150 in Sprague-Dawley rats improved markedly
after cyclosporin A pretreatment.
Conclusion From the results obtained in the biodistribution
studies, it can be concluded that [123I]R91150 is a substrate
for Pgp in rodents. A relationship between the administered
dose of cyclosporin A and the increase in [123I]R91150
brain radioactivity concentration was established. The
overall quality of our multipinhole μSPECT images with
[123I]R91150 in rats improved markedly after pretreatment
of the animals with cyclosporin A.
Keywords Cyclosporin A . P-glycoprotein .
[123I]R91150 . 5-HT2A receptors
Introduction
The serotonin 2A receptor (5-HT2AR) is a well-known G-
protein-coupled receptor that plays a role in the regulation
of a number of central nervous system processes and
dysfunctions [1] such as mood, appetite, anxiety and
Eur J Nucl Med Mol Imaging (2009) 36:446–453
anorexia. High levels of 5-HT2AR are found in the brain,
primarily in cortical areas (neocortex, entorhinal and pyri-
form cortex, claustrum), caudate nucleus, nucleus accum-
bens, olfactory tubercle and hippocampus [2]. Low levels
are found in the cerebellum, among other locations. A
number of tracers has been used for PET and SPECT imag-
ing of the 5-HT2AR, for example [11C]MDL100907 [3–5],
[123I]R91150 (radioiodinated 4-amino-N-1-[3-(4-fluoro-
phenoxy)propyl]-4-methyl-4-piperidinyl 5-iodo-2-methoxy-
benzamide) [6, 7] and [18F]altanserin [3, 8–11].
Several papers have been published recently concerning
the importance of P-glycoprotein (Pgp) function/modula-
tion in PET or SPECT studies [12–15]. Pgp and multidrug
resistance-associated protein (MRP) are drug efflux pumps
that play a role in so-called multidrug resistance (MDR),
together with decreased cellular topoisomerase II and
increased cellular glutathione [16]. Several drugs are
substrates for Pgp and MRP, such as different chemother-
apeutic drugs (vincristine, paclitaxel), cardiovascular drugs
(verapamil, digoxin), immunosuppressive drugs (cyclo-
sporin A) [17] and antiepileptic drugs [18]. The function
of Pgp can be blocked by MDR modulators such as calcium
channel blockers [19] and cyclosporin A. Pgp is expressed
in different tissues such as the blood capillaries of the brain
and the adrenal cortex, and in different tumour types such
as colon cancer and renal cell carcinoma [20]. Due to the
function of Pgp in the blood–brain barrier as a drug efflux
pump, the impact of possible Pgp modulation on PET and
SPECT brain scans may be significant: if a tracer is a Pgp
substrate, increased or decreased Pgp expression will have
an impact on tracer brain uptake [21]. Genetic variations in
Pgp function can lead to successful scans in one subject
and failed scans in another. This effect also needs to be
taken into account when performing PET or SPECT
brain scans in patients treated with possible modulators
of Pgp.
Several tracers have already been evaluated for Pgp
modulation, including amongst others [11C]verapamil [22–
24], [99mTc]sestamibi [17, 25], [131I]MIBG [26] and [11C]-
(R)-(−)-RWAY [21]; all tracers except [131I]MIBG are Pgp
substrates. [123I]R91150 is an antagonist with very high
affinity for the 5-HT2AR (Kd 0.11±0.01 nM). Selectivity for
other neurotransmitter receptor binding sites (5-HT1A, 5-
HT1B, 5-HT1D, 5-HT2C, 5-HT3, α1 and α2 adrenoreceptors,
histamine H1 and dopamine D2 receptors) amounts to at
least a factor of 50 [27]. [123I]R91150 has been shown to
be suitable for imaging of the 5-HT2AR in human brain [6,
7, 27].
In this study we investigated the influence of Pgp
blocking with cyclosporin A on the biodistribution and
brain penetration of [123I]R91150. First a dose-response
study was performed to assess the impact of increasing
cyclosporin A dose on the brain uptake of [123I]R91150 in
447
NMRI mice, and to determine whether the increased brain
uptake of the tracer was due to a decreased brain efflux. A
regional brain biodistribution study in Sprague-Dawley rats
was performed to assess the impact of Pgp modulation on
the distribution and brain penetration of the tracer. Also, a
displacement study (with and without cyclosporin A
administration) in Sprague-Dawley rats was performed to
evaluate the specificity of tracer binding. A metabolite
assay was conducted to exclude possible increased tracer
metabolism under the influence of cyclosporin A. Finally, a
multipinhole μSPECT imaging study with [123I]R91150
was performed using a Milabs U-SPECT-II camera in
Sprague-Dawley rats pretreated with cyclosporin A, to
asses the influence of Pgp modulation on the quality of the
obtained SPECT images.
Materials and methods
Synthesis and analysis of [123I]R91150
[123I]R91150 was synthesized according to the original
method [28] with modification. Radioiodination was per-
formed by direct electrophilic substitution on the precursor
(4-amino-N-[1-[3-(4-fluorophenoxy)-propyl]-4-methyl-4-
piperidinyl]-2-methoxybenzamide). The 5-position of the
methoxybenzamide group was iodinated. The precursor
was kindly donated by Janssen Pharmaceutics (J&J, Beerse,
Belgium).
Precursor (0.5–1 mg) was dissolved in 350 μl glacial
acetic acid in a V-vial, and carrier solution with no added
[123I] (NaI in 0.05 M NaOH, Amersham) was added. Cold
sodium iodide was added as carrier (0.1 nmol cold NaI per
37 MBq of 123I). At 0, 10, 20 and 25 min, 35 μl of a 30%
solution of H2O2 (Sigma-Aldrich Belgium) was added to
the vial. At 30 min the reaction was quenched by adding
1 ml of sodium sulphite solution (126 mg/ml in 3 M NaOH
solution). The reaction mixture was transferred to a 2-ml
HPLC loop and passed through a short column (filled with
Lichroprep RP-8; Merck, Germany) with 1 mM NaOH. The
column was rinsed with 1 mM NaOH and the tracer was
purified by injecting onto a HPLC column (Lichrospher RP
Select B, 250×4 mm; Merck, Germany). A sterile sodium
acetate/ethanol solution (2.28 g NaOAc·3H2O in 60 ml
H2O + 40 ml EtOH) was used as eluent. The fraction
corresponding to [123I]R91150 (TR 18–20 min, 3.2 ml) was
collected in a sterile vial after filtration through a 0.22-μm
filter (FP 13/0.2 RC-S; Schleicher&Schuell, pretreated with
HPLC eluent) and diluted with water for injection (9.6 ml).
Quality control of the tracer was performed by injecting an
aliquot onto an analytic HPLC column (Waters Lichrospher
RP-8, 125×4 mm, 5 μm) using methanol/acetonitrile/water
(190:260:550) as eluent.
448
Cyclosporin A dose-response study in NMRI mice
Cyclosporin A formulated in Cremophor EL (polyoxyethy-
lated castor oil) was obtained from Sandoz (Sandimmune
Injection, cyclosporin A 50 mg/ml, Cremophor EL 650 mg/ml;
Basel, Switzerland) and was diluted with sterile saline
solution. Adult male NMRI mice (three per dosage group,
weight 20–25 g) were injected in the tail vein with various
amounts of cyclosporin A (the doses used were 10, 20, 35
and 50 mg/kg). For the control group, only the vehicle was
injected. [123I]R91150 (300–500 KBq) was administered
via the tail vein 60 min after administration of cyclosporin
A, and 1 h after tracer injection the animals were killed by
decapitation under isoflurane anaesthesia. Blood was
collected and the brain was rapidly removed. Blood and
brain were weighed and counted for radioactivity in an
automated gamma counter (Cobra, Packard Canberra).
Three aliquots of the injected tracer solution were weighed
and counted for radioactivity to determine the injected
radioactivity dose received by the animals. The results were
corrected for decay, and tissue radioactivity concentrations
are expressed as a percentage of the injected dose per gram
of tissue (% ID/g tissue, mean±standard deviation values,
n=3).
Regional brain biodistribution in Sprague-Dawley rats
The dose of cyclosporin A used was 50 mg/kg [12]. Adult
male Sprague-Dawley rats (weight 200–250 g) were
divided into two groups: one group received an injection
of cyclosporin A (50 mg/kg) into the penile vein 1 h before
injection of [123I]R91150. The second group received no
pretreatment. The animals (three per time point) were killed
by decapitation under isoflurane anaesthesia at selected
time-points (10 min, 30 min, 60 min, 2 h and 4 h) after
injection of [123I]R91150 (5–10 MBq per animal). Blood
and brain were removed, the brain was rapidly dissected
and the brain parts were weighed. The radioactivity of the
samples was measured with an automated gamma counter
(Cobra, Packard Canberra).
Tissue radioactivity concentrations are expressed as a
percentage of the injected dose per gram of tissue (% ID/g).
The results after cyclosporin A treatment were compared to
the values obtained without pretreatment. The effect of the
isoflurane anaesthesia on the brain uptake of [123I]R91150
was investigated earlier, and no effect was observed (data
not shown).
Displacement study in Sprague-Dawley rats
Ketanserin tartrate (1 mg/kg, dissolved in water/ethanol 95/
5; Tocris, Bristol, UK) was used for displacement studies.
The amount of radioactivity injected was approximately 5–
Eur J Nucl Med Mol Imaging (2009) 36:446–453
10 MBq of [123I]R91150. Male Sprague-Dawley rats
(weight 200–250 g, three per group) were divided into
two groups: one group was treated with cyclosporin A
(50 mg/kg) 1 h before the injection of [123I]R91150; the
second group received no pretreatment. Ketanserin tartrate
(1 mg/kg) was injected into the penile vein 30 min after
injection of [123I]R91150. The animals were killed by
decapitation under isoflurane anaesthesia 1 h after tracer
injection. Blood was collected and the brain was rapidly
removed. The brain was dissected into different regions:
cortex (frontal, temporal, parietal and occipital cortex),
cerebellum and subcortical region. Blood and brain parts
were weighed and counted for radioactivity with an
automated gamma counter (Cobra, Packard Canberra).
The results were corrected for decay and tissue radioactivity
concentrations are expressed as a percentage of the injected
dose per gram of tissue (% ID/g). To exclude possible
interference of Cremophor EL, three animals were injected
with only the vehicle (Cremophor EL, saline and 10%
ethanol) 1 h before [123I]R91150 injection.
Metabolite determination
For determination of possible lipophilic metabolites of
[123I]R91150, three male Sprague-Dawley rats (200–250 g)
were injected with [123I]R91150 (20 MBq) 1 h after
administration of cyclosporin A (50 mg/kg). The animals
were killed by decapitation 1 h after tracer injection, blood
was collected into heparinized tubes, and the brain was
quickly removed by dissection. The blood was centrifuged
for 3 min at 3,000×g and the plasma was separated from the
pellet. Plasma (500 μl) was mixed with acetonitrile (1 ml).
The mixture was vortexed on ice for 10 s and centrifuged
for 3 min at 3,000×g. Brain tissue was homogenized in
acetonitrile (2 ml) for 30 s and the mixture was centrifuged
for 3 min at 3,000×g. For both brain and plasma, an aliquot
of the acetonitrile fraction was spotted onto a silica thin-
layer chromatography (TLC) plate (Polygram TLC Sil G/
UV254 20×20 cm, 200 μm layer thickness; Machery-Nagel,
Germany). An aliquot of authentic [123I]R91150 solution
was also spotted as a reference. The TLC plate was
developed in a dichloromethane/methanol mixture (88/12
with 5% triethylamine). The TLC plate was dried at room
temperature, exposed to a phosphor imaging screen (plate
type SR, 12.5×25.2 cm; Perkin Elmer Life Sciences, USA)
for 30 min and analysed using a Cyclone phosphor imager
(Perkin Elmer Life Sciences).
Multipinhole μSPECT imaging in Sprague-Dawley rats
μSPECT images were acquired on a U-SPECT-II camera
(Milabs, Utrecht, The Netherlands) equipped with a
dedicated 75 pinhole high-resolution rat collimator. Male
Eur J Nucl Med Mol Imaging (2009) 36:446–453
Sprague-Dawley rats (250–266 g) were anaesthetized with
isoflurane and injected with cyclosporin A (50 mg/kg).
[123I]R91150 (29.6 MBq) was injected into the tail vein
30 min later. After a further 40 min, a U-SPECT-II scan was
performed using the rat collimator (75 pinholes of 1.0 mm)
for a continuous 60 min (12 frames of 5 min) with list-
mode output enabled. As a reference, animals were also
scanned in a similar protocol but without administration of
cyclosporin A. A 20% photopeak window was selected
around 159 keV, while background correction was per-
formed using two additional 10% windows around 133 and
188 keV. The time-frames from 45 min to 75 min after
injection were selected and the data were reconstructed on
0.75-mm voxels by three iterations of 16 OSEM subsets.
The images were 3-D postfiltered by a 15-pixel Gaussian
kernel with a width of 1.5 mm. Two regions of interest
(ROIs) were drawn around the frontal cortex and the
cerebellum using Amide (OpenSource software package)
with 3-D freehand on the coronal slices. The mean value
and standard deviation over all voxels in the respective
ROIs were calculated for post-analysis.
All experiments were conducted following the principles
of laboratory animal care and the Belgian law on the pro-
tection of animals. Our research protocol was approved by the
Ghent University Hospital ethics committee (ECP 06-18).
Statistical analysis
Differences between [123I]R91150 accumulation in the
normal and the cyclosporin A groups were analysed using
nonparametric analysis (Mann-Whitney U-test). Only p
values <0.05 were considered statistically significant.
Results
[123I]R91150 synthesis
[123I]R91150 was prepared from the precursor (4-amino-N-
[1-[3-(4-fluorophenoxy)-propyl]-4-methyl-4-piperidinyl]-2-
methoxybenzamide) with an overall radiochemical yield of
75.6±8.6% (n=12; mean±standard deviation). Radiochemi-
cal purity at the time of injection was always >95%. Specific
activity of the tracer was approximately 350 TBq/mmol.
Cyclosporin A dose-response study in NMRI mice
The effects of administering increasing doses of cyclo-
sporin A on the blood concentration and brain uptake of
[123I]R91150 were investigated in male NMRI mice. The
results are shown in Fig. 1.
Administration of cyclosporin A had a dose-dependent
effect on the brain uptake of [123I]R91150 in NMRI mice.
449
Fig. 1 Effects of cyclosporin A dose on the blood concentration and
brain uptake of [123I]R91150 in NMRI mice. The results are expressed
as means±standard deviation (n=3 per dosage group)
The greatest effect was observed at the highest dose of
cyclosporin A used (50 mg/kg). After administration of cyclo-
sporin A at this dose, a brain tracer concentration of 7.16±
0.25% ID/g brain tissue was reached 1 h after tracer injection.
For comparison, brain uptake of [123I]R91150 without cyclo-
sporin A pretreatment averaged at 1.0±0.08% ID/g tissue.
Lower doses of cyclosporin A were associated with lower
radioactivity concentrations in the brain. A slight increase in
blood radioactivity concentration was observed after admin-
istration of increasing doses of cyclosporin A. At the highest
cyclosporin A dose used, a radioactivity concentration of
2.68±0.82% ID/g tissue was observed in the blood. Blood
radioactivity concentration without cyclosporin A pretreat-
ment averaged at 0.70±0.18% ID/g tissue.
Regional brain biodistribution in Sprague-Dawley rats
The effect of cyclosporin A administration on the biodis-
tribution of [123I]R91150 in male Sprague-Dawley rats was
investigated. Radioactivity concentrations in the different
brain regions of the cyclosporin A-treated rats are shown in
Fig. 2, and of the rats with no pretreatment are shown in
Fig. 3.
The influence of cyclosporin A administration on the
regional brain biodistribution of [123I]R91150 in rats can be
clearly seen by comparing Figs. 2 and 3. On average, there
was a five- to sixfold increase in [123I]R91150 concentra-
tion in the brain after cyclosporin A treatment. For
example, at 1 h after injection, the radioactivity concentra-
tion in the frontal cortex increased from 0.24±0.0092 to
1.58±0.097% ID/g tissue after treatment with cyclosporin
A. In the reference tissue, the cerebellum, radioactivity
concentration increased from 0.038±0.0023% ID/g tissue at
1 h after injection of [123I]R91150 to 0.45±0.017% ID/g
450
Fig. 2 Regional brain biodistribution of [123I]R91150 in male Sprague-
Dawley rats after cyclosporin A treatment (50 mg/kg). The results are
expressed as means±standard deviation (n=3 per time-point)
tissue after treatment of the animals with cyclosporin A.
The other brain regions (temporal, parietal and occipital
cortex) showed similar increases in [123I]R91150 radioac-
tivity concentrations after treatment of the animals with
cyclosporin A. On the other hand, the large increases in
brain radioactivity concentrations after cyclosporin A
treatment were not found in the blood, indicating that the
increased brain uptake was not the result of an increased
supply of tracer to the brain.
The increase in [123I]R91150 concentrations in the brain
of male Sprague-Dawley rats is as expected after cyclo-
sporin A treatment because of the effect of Pgp in
decreasing efflux of tracer out of the brain (since its
function is blocked by cyclosporin A). Theoretically, an
Fig. 3 Regional brain biodistribution of [123I]R91150 in male
Sprague-Dawley rats with no pretreatment. The results are expressed
as means±standard deviation (n=3 per time-point)
Eur J Nucl Med Mol Imaging (2009) 36:446–453
increased brain uptake could be the result of a higher blood
radioactivity concentration, resulting in an increased supply
of tracer to the brain. Because the increase in blood
radioactivity concentration was not as pronounced after
cyclosporin A treatment, this theory could be ruled out.
To exclude possible effects of Cremophor EL, a blank
test was performed (injecting only the vehicle, diluted ten
times). No effects of Cremophor EL on [123I]R91150
accumulation in the brain of Sprague-Dawley rats were
found (data not shown). These results are in accordance
with the findings of Hendrikse et al. [23].
Displacement study in Sprague-Dawley rats
The effect of displacement of [123I]R91150 with ketanserin
on the biodistribution of [123I]R91150 in the brain of male
Sprague-Dawley rats was investigated. Radioactivity con-
centrations of different brain regions in the different
treatment groups are shown in Fig. 4.
[123I]R91150 was displaced by ketanserin at the 1-h time-
point in target tissues (cortex tissues, especially the frontal
cortex) but not in the cerebellum or blood, which is in
accordance with the use of the cerebellum as a reference
region (i.e. reference tissue model).
The radioactivity concentration in the frontal cortex of the
animals with no cyclosporin A pretreatment decreased from
0.24±0.0092% ID/g tissue in those without ketanserin dis-
placement to 0.18±0.005% ID/g tissue in those with ketan-
serin displacement (i.e. a decrease of 25% after displacement
with ketanserin). The radioactivity concentration in the frontal
cortex of the animals pretreated with cyclosporin A decreased
from 1.58±0.097% ID/g tissue in those without ketanserin
displacement to 1.14±0.15% ID/g tissue in those with
ketanserin displacement (i.e. a decrease also of 25% after
displacement with ketanserin). As mentioned above, in the
cerebellum there was no significant difference between the
groups with and without ketanserin, in both the animals with
no pretreatment (0.038±0.002 and 0.0379±0.004% ID/g
Fig. 4 Effect of displacement with ketanserin on the biodistribution of
[123I]R91150 in Sprague-Dawley rats. Animals were pretreated with
cyclosporin A (50 mg/kg) 30 min before tracer injection. Ketanserin
tartrate was administered 30 min after [123I]R91150. The results are
expressed as means±standard deviation (n=3 per group). *p<0.05
Eur J Nucl Med Mol Imaging (2009) 36:446–453
tissue, respectively) and the animals pretreated with cyclo-
sporin A (0.456±0.017 and 0.450±0.038% ID/g tissue,
respectively). Radioactivity concentration in the blood did
not change significantly after ketanserin displacement.
Metabolite study
Metabolites in blood and brain of Sprague-Dawley rats
were determined to exclude possible interference of cyclo-
sporin A with in vivo metabolism of [123I]R91150. No
lipophilic metabolites were detected in the blood or brain.
The only radiolabelled product found in brain samples was
unmetabolized [123I]R91150, by comparing the retention
times with a [123I]R91150 reference standard. Besides
unmetabolized [123I]R91150, a small amount of free radio-
iodine was present in the blood samples, probably due to in
vivo deiodination of the tracer.
Multipinhole μSPECT imaging study
The μSPECT images obtained are shown in Fig. 5. Based
on anatomical data, two ROIs were drawn on the coronal
view around the frontal cortex and the cerebellum (Fig. 5).
Fig. 5 Multipinhole μSPECT
images with [123I]R91150 in
Sprague-Dawley rats. Animals
were scanned around 1 h after
injection of [123I]R91150
(29.6 MBq): A without pretreat-
ment; B after pretreatment with
cyclosporin A (50 mg/kg).
ROI’s were drawn around front-
al cortex and cerebellum. The
lower part shows high resolu-
tion [123I]R91150 μSPECT
images after cyclosporin A
treatment (transversal, coronal
and sagittal views)
451
In general, a low brain uptake was observed after
injection of [123I]R91150 in the animals without pretreat-
ment. As evident from Fig. 5 part A, hardly any activity
was visible on the μSPECT scans. The count rate obtained
was too low to perform reliable statistical analysis on the
data from the animals without pretreatment. Radioactivity
uptake in the brain was considerably higher after treatment
of the animals with cyclosporin A. Highest uptake of [123I]
R91150 was observed in the frontal cortex, and the lowest
concentration of radioactivity was found in cerebellum. As
can be seen in Fig. 5 part B, radioactivity concentrations
after cyclosporin A treatment were higher than the values
obtained with no cyclosporin A pretreatment.
Tracer uptake in the cerebellum was used as a reference
for nonspecific binding, based on the reference tissue
model. For semiquantification, the binding index was
estimated as (counts/voxel in frontal cortex ROI)/(counts/
voxel in cerebellum ROI). A binding index value of 3.16
was obtained from the μSPECT scans in the animals
pretreated with cyclosporin A. This result is in accordance
to the binding index data calculated from the biodistribution
study with [123I]R91150 in Sprague-Dawley rats (a binding
index of around 4 was obtained).
452
The lower part of Fig. 5 again shows the results of a
[123I]R91150 scan after administration of cyclosporin A,
but now optimized for optimal spatial resolution (the data
were reconstructed on 0.375 mm voxels by three iterations
of 16 OSEM subsets). The images were 3-D postfiltered by
a 15 pixel Gaussian kernel with a width of 1.125 mm.
Discussion
The human mdr1 gene (that encodes Pgp) has several single
nucleotide polymorphisms that can change the pharmaco-
kinetics of drugs that are substrates for this transporter [21].
This can lead to successful scans in one patient, and failed
scans in another, and is very important in patients treated
with Pgp modulators such as cyclosporin A. Therefore it is
necessary in the development of new tracers to determine if
they are modulated by Pgp or not. It is also of interest to
know whether the tracers presently used clinically are
modulated by Pgp. In this regard, several clinically used
tracers have already been examined as Pgp substrates [13,
20, 21, 25, 26, 29].
The Sprague-Dawley rat biodistribution studies with
[123I]R91150 and the rat μSPECT imaging study revealed a
four- to sixfold increase in brain [123I]R91150 radioactivity
concentration after blocking of Pgp with cyclosporin A.
Theoretically, an increased brain uptake may be the result
of an increased tracer influx to the brain, or a decreased
tracer efflux out of the brain. Since the drug efflux protein
function of Pgp was blocked by the administration of
cyclosporin A, the increase in [123I]R91150 brain uptake
should have been the result of decreased tracer efflux out of
the brain. This is in accordance with the localization and
function of Pgp as an efflux protein at the blood–brain
barrier [21]. Moreover, a dose-response relationship was
found between the doses of cyclosporin A that were
administered and the tracer concentration in the brain of
NMRI mice. Although blood radioactivity concentrations
did increase slightly after treatment of the mice with
cyclosporin A, this increase was not as pronounced as the
resulting increased tracer concentration in the brain, and
could have been the result of an influence of cyclosporin A
on the pharmacokinetics of [123I]R91150 [30].
The increased [123I]R91150 radioactivity concentration
in rodent brain after cyclosporin A treatment could not be
fully displaced by ketanserin (Fig. 4), indicating that at least
part of the increase in [123I]R91150 uptake is due to
increased aspecific binding in brain. This is also evident
from the increased radioactivity concentration after cyclo-
sporin A treatment in the reference area, the cerebellum.
Our results are in accordance with those of other
previously published studies investigating the effect of
cyclosporin A administration on the brain uptake of
Eur J Nucl Med Mol Imaging (2009) 36:446–453
different radiotracers [22]. Similar to the results obtained
by Liow et al. [21] with the 5-HT1A tracer [11C]RWAY
(who observed a five-fold increase in brain tracer concen-
tration after cyclosporin A treatment), brain uptake of [123I]
R91150 increased four- to sixfold. Our results are also in
agreement with the findings of Hendrikse et al. [23], where
a dose-dependent relationship was observed between the
brain uptake in mice of [11C]verapamil and the dose of
cyclosporin A; a tenfold increase in brain radioactivity
concentration was found after treatment of the animals with
cyclosporin A (50 mg/kg).
In the multipinhole μSPECT imaging study, the effect of
cyclosporin A administration could be seen as an increase
in brain uptake of [123I]R91150 (Fig. 5, part B), while the
brain was hardly visible when only the tracer [123I]R91150
was administered (Fig. 5, part A), in contrast with, for
example, human trials where the tracer does show ade-
quate brain uptake [6, 7]. After pretreatment with cyclo-
sporin A, cortical tissues could be clearly distinguished
(Fig. 5, part B).
Due to possible peripheral effects of cyclosporin A (for
example CYP3A4 inhibition), we found it necessary to
perform a metabolite analysis after cyclosporin A treatment.
Since no radiolabelled metabolites were found, the in-
creased radioactivity uptake in the brain can only be
attributed to an increased concentration of authentic [123I]
R91150.
Future work will investigate possible changes in [123I]
R91150 free plasma fraction under the influence of cyclo-
sporin A, to exclude possible displacement of the tracer
from plasma proteins, also resulting in an increased tracer
influx to the brain.
Conclusion
We evaluated the effect of cyclosporin A pretreatment on
the [123I]R91150 radioactivity concentrations in the brain of
NMRI mice and Sprague-Dawley rats. A relationship
between the administered dose of cyclosporin A and the
radioactivity concentration of [123I]R91150 in rodent brain
was established. The effect of cyclosporin A administration
on the quality of multipinhole μSPECT images with [123I]
R91150 was also evaluated in Sprague-Dawley rats. From
the results obtained it can be concluded that [123I]R91150 is
an in vivo substrate for Pgp in rodents. Pretreatment of the
animals with cyclosporin A resulted in an increased brain
uptake of [123I]R91150 and improved the quality of the
multipinhole μSPECT images obtained.
Therefore, we suggest further exploration of the use of
cyclosporin A (or other less toxic Pgp blocking agents) for
improving the otherwise limited brain uptake of some
radioligands for brain imaging.
Eur J Nucl Med Mol Imaging (2009) 36:446–453
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