pharmaceutics
ArticleIndirect SPECT Imaging Evaluation for Possible Nose-to-Brain
Drug Delivery Using a Compound with Poor Blood–Brain
Barrier Permeability in Mice
Asuka Mizutani 1,† , Masato Kobayashi 1,† , Makoto Ohuchi 1 , Keita Sasaki 2 , Yuka Muranaka 1 , Yusuke Torikai 2 ,Shota Fukakusa 2,3 , Chie Suzuki 3 , Ryuichi Nishii 4 , Shunji Haruta 2 , Yasuhiro Magata 3 and Keiichi Kawai 1,5, *
1

2

3

4

5

Citation: Mizutani, A.; Kobayashi,M.; Ohuchi, M.; Sasaki, K.; Muranaka,Y.; Torikai, Y.; Fukakusa, S.; Suzuki,*†

Faculty of Health Sciences, Institute of Medical, Pharmaceutical and Health Sciences, Kanazawa University,
5-11-80 Kodatsuno, Kanazawa 920-1192, Japan; mizutani.a@staff.kanazawa-u.ac.jp (A.M.);
kobayasi@mhs.mp.kanazawa-u.ac.jp (M.K.); makohhm359@gmail.com (M.O.);
yukarisa93@stu.kanazawa-u.ac.jp (Y.M.)
R&D Department, TR Company, Shin Nippon Biomedical Laboratories, Ltd., 2438 Miyanoura,
Kagoshima 891-1394, Japan; sasaki-keita@snbl.co.jp (K.S.); torikai-yusuke@snbl.co.jp (Y.T.);
fukakusa-shota@snbl.co.jp (S.F.); haruta-shunji@snbl.co.jp (S.H.)
Department of Molecular Imaging, Institute for Medical Photonics Research,
Preeminent Medical Photonics Education & Research Center, Hamamatsu University School of Medicine,
1-20-1 Handayama, Higashi-ku, Hamamatsu 431-3192, Japan; csuzuki@hama-med.ac.jp (C.S.);
ymagata@hama-med.ac.jp (Y.M.)
Department of Molecular Imaging and Theranostics, Institute for Quantum Medical Science,
Quantum Life and Medical Science Directorate, National Institutes for Quantum Science and Technology,
4-9-1 Anagawa, Inage, Chiba 263-8555, Japan; nishii.ryuichi@qst.go.jp
Biomedical Imaging Research Center, University of Fukui, 23-3 Matsuokashimoaizuki, Eiheiji, Yoshida-gun,
Fukui 910-1193, Japan
Correspondence: kei@mhs.mp.kanazawa-u.ac.jp; Tel.: +81-76-265-2527; Fax: +81-76-234-4366
These authors contributed equally to this work.

C.; Nishii, R.; Haruta, S.; et al.
Indirect SPECT Imaging Evaluation
for Possible Nose-to-Brain Drug
Delivery Using a Compound with
Poor Blood–Brain Barrier
Permeability in Mice. Pharmaceutics
2022, 14, 1026. https://doi.org/
10.3390/pharmaceutics14051026
Academic Editors: Frank Wuest andLeonard I. Wiebe
Received: 30 March 2022
Accepted: 7 May 2022
Published: 10 May 2022
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims inpublished maps and institutional affil-

Abstract: Single-photon emission computed tomography (SPECT) imaging using intravenous radioactive ligand administration to indirectly evaluate the time-dependent effect of intranasal drugs
with poor blood-brain barrier permeability on brain drug distributions in mice was evaluated. The
biodistribution was examined using domperidone, a dopamine D2 receptor ligand, as the model drug,with intranasal administration at 0, 15, or 30 min before intravenous [123 I]IBZM administration. In
the striatum, [123 I]IBZM accumulation was significantly lower after intranasal (IN) domperidone administration than in controls 15 min after intravenous [125 I]IBZM administration. [123 I]IBZM SPECT
was acquired with intravenous (IV) or IN domperidone administration 15 min before [123 I]IBZM, andtime–activity curves were obtained. In the striatum, [123 I]IBZM accumulation was clearly lower in theIN group than in the control and IV groups. Time–activity curves showed no significant difference
between the control and IV groups in the striatum, and values were significantly lowest during
the first 10 min in the IN group. In the IN group, binding potential and % of receptor occupancy
were significantly lower and higher, respectively, compared to the control and IV groups. Thus,
brain-migrated domperidone inhibited D2R binding of [123 I]IBZM. SPECT imaging is suitable forresearch to indirectly explore nose-to-brain drug delivery and locus-specific biological distribution.

iations.

Keywords: nose-to-brain drug delivery; poor BBB-permeable drug; brain drug distribution; dopamine
D2 receptor antagonist; SPECT; [123 I]IBZM
Copyright: © 2022 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms andconditions of the Creative Commons
Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

1. Introduction
Almost all potential therapies are greatly restricted from entering the brain because
the blood-brain barrier (BBB) is highly successful at protecting the brain from the entryof drugs [1,2]. P-glycoprotein (P-gp) is considered one of the most important efflux transporters located at the BBB, and it protects the brain from chemicals and toxins. For thisPharmaceutics 2022, 14, 1026. https://doi.org/10.3390/pharmaceutics14051026

https://www.mdpi.com/journal/pharmaceutics

Pharmaceutics 2022, 14, 1026

2 of 10reason, many drugs have an affinity for P-gp and drugs administered intravenously cannot
accumulate in the brain due to the BBB. Nose-to-brain (N2B) drug delivery is an attractive route for direct drug delivery from the nose to the brain that bypasses the BBB [3].
Neotrofin, a P-gp substrate, administered intracerebroventricularly in the lateral cerebral
ventricle is inhibited from being excreted from the brain when combined with verapamil, a
P-gp inhibitor [4]. Thus, P-gp substrate drugs may temporarily accumulate in the brain
via N2B drug delivery because they are excreted from the brain when they are recognizedby P-gp in the brain. However, it remains unclear whether P-gp-mediated efflux has aneffect on time-dependent drug accumulation in the brain. Therefore, it is useful to establish
a method for accurate evaluation of the effect of intranasal P-gp substrate drugs.
Radionuclide imaging using positron emission tomography (PET), or single-photon
emission computed tomography (SPECT), enables direct, non-invasive measurement offunctional proteins (e.g., receptors, transporters) in a living organism. A previous PET
study evaluated brain uptake of 11 C-labeled CH3 -Orexin A after intranasal delivery inrodents and nonhuman primates. However, these imaging methods have issues that
include the technical difficulty of labeling an evaluation compound with a radioisotope,a high likelihood of changes in properties of an evaluation compound by labeling, as
well as uncertainties in detecting labeled metabolites rather than a labeled intact (nonlabeled) compound. In addition to these issues, if the above method is applied to N2B drugdelivery, there is great difficulty in achieving rigorous detection of very slight and sensitive
brain uptake of a radiolabeled evaluation compound because of extreme differences inradioactivity between the nasal cavity and the brain [5].
Instead of the above method of direct evaluation of a radiolabeled compound in thebrain by PET, indirect evaluation using brain PET imaging of specific receptor occupancy
has been performed to examine the time-dependent effect of naloxone administered intranasally on mu opioid receptor occupancy in nonhuman primates and human subjects,
with intravenous injection of [11 C]carfentanil as a PET tracer ligand into the receptor [6,7].
Since the naloxone used in these studies is highly nasally absorbable, BBB-permeable, anddoes not activate efflux transporter P-gp [8,9], it is possible that the results of receptor
occupancy with nasal naloxone in these studies are based on brain naloxone distributions via the BBB route rather than the N2B route. Although brain PET imaging witha 11 C-labeled compound of receptor occupancy enables the use of an intact evaluation
compound and includes evaluation of its pharmacodynamics in the brain, the volumeof 11 C-labeled compounds injected into the subject is usually high because 11 C-labeled
compounds in new radiopharmaceuticals have a very short radioactive half-life and low radioactive concentration. In this study, we demonstrated a SPECT imaging method that uses
intravenous radioactive ligand administration to indirectly evaluate the time-dependent
effect of intranasal drugs with poor BBB permeability on brain drug distribution in mice.
To evaluate brain drug distribution in mice via only the N2B route, the present mousestudy used domperidone, a dopamine D2 receptor (D2R) antagonist, which has been reported to have only peripheral effects due to its poor BBB permeability caused by active
efflux transport of P-gp [10,11], as a model drug for N2B delivery. In addition, (S)-(-)-3iodo-2-hydroxy-6-methoxy-N-[(1-ethyl-2-pyrrolidinyl)-methyl]benzamide ([123 I]IBZM), a
D2R ligand with known high BBB permeability, was selected as the radiopharmaceuticalto indirectly evaluate brain domperidone distribution by competing D2R binding using
SPECT imaging.
2. Materials and Methods
2.1. Materials(S)-(-)-3-trimetylstannyl-2-hydroxy-6-methoxy-N-[(1-ethyl-2-pyrrolidinyl)methyl]-benzamide(TBZM; >95% purity) and S-(-)-IBZM ([127I]IBZM, >95% purity) were purchased from the NARD Institute, Ltd. (Hyogo, Japan) and ABX (Radeberg, Germany), respectively. 125I-NaI (3.3–4.1 GBq/mL)
and 123I-NaI (8.5–15.0 GBq/mL) were purchased from PerkinElmer, Inc. (Waltham, MA, USA) andFUJIFILM Toyama Chemical Co., Ltd. (Tokyo, Japan), respectively. Chloramine-T (>95% purity),Pharmaceutics 2022, 14, x FOR PEER REVIEW

3 of 10

Pharmaceutics 2022, 14, 1026

3 of 10PerkinElmer, Inc. (Waltham, MA, USA) and FUJIFILM Toyama Chemical Co., Ltd. (Tokyo, Japan), respectively. Chloramine-T (>95% purity), sodium metabisulfite (>95% purity), andammoniumacetate(>97%
purity) werepurchasedfrom Nacalai
Tesque,fromInc.
sodiummetabisulfite(>95%
purity),and ammonium
acetate(>97% purity)
were purchased(Kyoto,Japan);
domperidone(>90%
purity),ethanol(>99.5%purity),andhydrogencarNacalai Tesque, Inc. (Kyoto, Japan); domperidone (>90% purity), ethanol (>99.5% purity), andbonate (>95%
purity)were
providedby FUJIFILM
Wako
Pure
Chemicalhydrogencarbonate(>95%
purity)were provided
by FUJIFILM
Wako
Pure
ChemicalCorporation
Corporation(Osaka,Japan).
(Osaka, Japan).
125/123I]IBZM
2.2.LabelingLabelingofof[[125/123
2.2.
I]IBZM
125/123I]IBZM following the method of Kung [12] and the schematic di125/123
Tosynthesize[
To synthesize [I]IBZM following the method of Kung [12] and the schematic
agram shown
diluted byby addingadding 0.5
0.5 M
M ammonium
ammonium
diagramshownininFigure
Figure1,1,455
455μMµM TBZM
TBZM (20(20 μL)µL) diluted
125125123123
acetate(100(100µL)μL)andandethanol
ethanol
were
mixed
with
I-NaI
I-NaIandand0.10.1µM
μM
chloramineacetate
were
mixed
with
I-NaI
oror
I-NaI
chloramine-T
T (25
μL).
reaction
mixtureincubated
at room
temperature
5 min.
oxida(25µL).
TheThereaction
mixturewaswasincubated
at room
temperaturefor 5formin.
TheTheoxidation
tion
reactionwasstoppedbytheadditionof
1/10
saturated
sodiummetabisulfite.
reaction was stopped by the addition of 1/10 saturated sodium metabisulfite. Then, pHThen,was
pH was toadjusted
tousing
about700
7.0µL
using
700saturated
μL of 1/20.12
saturated
0.12 M
hydrogencarbonate.
adjustedabout 7.0
of 1/2
M hydrogencarbonate.
Thelabeling
125/123I]IBZM was >80% using a thin layer chromatography, and the
Theoflabelingrate of [wasrate
[125/123 I]IBZM>80% using a thin layer chromatography, and the radiochemical
radiochemical
purity>95% using high-performanceliquid chromatography(HPLC),
puritywas >95%
usingwashigh-performanceliquid chromatography(HPLC), comprisingacomprising
pump (model
HitachiHigh-Tech
Global,a UV
Ibaraki,Japan),a UV depump(modelaL-7100;
Hitachi L-7100;
High-Tech
Global,Ibaraki, Japan),detector(ChromastertectorHitachi(Chromaster
5410,Global),
Hitachiand
High-Tech
Global), (modeland a γ-ray
detector(modelRLC5410,High-Techa γ-ray detector
RLC-701,
Hitachi,
Ibaraki,
701,
Hitachi,
Ibaraki,Japan)
equipped
with
a
5C
18
AR-II-column
(4.6
×
250
mm;
5
μm,
Japan) equipped with a 5C18 AR-II-column (4.6 × 250 mm; 5 µm, Nacalai Tesque). The
Nacalaiphase
Tesque).
The mobile
phase
consistedof 10%phosphate
4 mM ammonium
phosphate
mobileconsistedof 10%
4 mM
ammoniumbuffer (pH7.0) andbuffer
90%
acetonitrile
at90%
a flowrate of 1.0
radioactive
concentrationsof [125 I]IBZM(pH 7.0) and
acetonitrile
at mL/min.
a flow rateTheof 1.0mL/min. The
radioactive concentrations
and[123
I]IBZM
wereapproximately
3.7 MBq/mL3.7
and
130 MBq/mL,The
of [125I]IBZM
and[123I]IBZM
were approximately
MBq/mLand 130respectively.
MBq/mL, respec125/123
spectrumof spectrum[I]IBZM
wasI]IBZM
identified
comparison
with the spectrum
ofspectrum[127 I]IBZM
tively. Theof [125/123
wasin
identified
in comparison
with the
of
127
in
the
HPLC
system(Supplemental
Materials).
[ I]IBZM in the HPLC system (Supplemental Materials).

125/123

SnBu3
OH

N
O

OH

125/123I-NaI

H
N
OMe

I

Et

H
N

Chloramine-T
RT, 5min

N
OMe

O

Et

Figure 1. Schematic of 125/123 I labelling of TBZM. TBZM is labelled with 125/123 I-NaI by oxidation

Figure 1.
Schematicof 125/123forI labelling
TBZM.
TBZM is labelled with 125/123I-NaI by oxidation reacreaction
withchloramine-T
5 min atofroom
temperature.
tion with chloramine-T for 5 min at room temperature.

2.3. Animals
2.3. Animals
All applicable institutional guidelines of Kanazawa University for the care and useof animals
were followed.
All procedures
were
performedUniversity
in accordance
withcaretheandethicalAll applicableinstitutionalguidelinesof Kanazawa
for theuse
standards
ofKanazawa
University(AnimalCare
CommitteeofKanazawa
University,
APof animals were followed. All procedures were performed in accordance with the ethical173851)
andofwithinternational
standards
for animal
welfare andofinstitutional
standards
Kanazawa
University(AnimalCare Committee
Kanazawa guidelines.
University,
The
mice used
the international
study (ddY, males,
28.5for± animal
0.9 g, 29
mice in
total)
were obtained
AP-173851)
andinwithstandards
welfareandinstitutionalguidefrom
SLC,usedTokyo,
Japan.
cannulation
methodof Hirailines.JapanThe micein thestudyTracheal(ddY, males,
28.5 ± 0.9accordingg, 29 micetointhe
total)
were obtainedetal. [13]was
performed
in all mice
to increase
N2B drugdeliveryduringthe intranasal
from
Japan
SLC,
Tokyo, Japan.
Trachealcannulationaccordingto themethodof Hirai etadministrationof
domperidone.
The
drug
solutionwasadministered
into
the
nasal
cavityal. [13] was performed in all mice to increase N2B drug delivery during the intranasalvia
thetracheal
catheter.
The
sectionoftrachea
to
the
lung
was
also
cannulatedtomaintainadministration of domperidone. The drug solution was administered into the nasal cavity
spontaneous
breathing.
via the tracheal
catheter. The section of trachea to the lung was also cannulated to maintain spontaneous breathing.
2.4. Biological Distribution Study after an Intravenous Dose of [125 I]IBZM with an Intranasal
Domperidone or Saline Dose
2.4. Biological Distribution Study after an Intravenous Dose of [125I]IBZM with an Intranasal
All proceduresin Dosemice were conducted under isoflurane inhalation anesthesia. DomDomperidone
or Saline
peridone diluted with saline was intranasally administered at a dose of 2 µg/g body weight
All procedures in mice were conducted under isoflurane inhalation anesthesia.
and at a volume of 20 µL, using a micro-syringe via the tracheal catheter. Saline was
Domperidone diluted with saline was intranasally administered at a dose of 2 μg/g body
intranasally administered as the control in the same manner. Approximately 200 kBq
weight and at a volume of 20 μL, using a micro-syringe via the tracheal catheter. Saline[125 I]IBZM were injected into the tail vein of mice at 0, 15, and 30 min after intranasal
was intranasally administered as the control in the same manner. Approximately 200 kBq
domperidone administration, or at 0 min after intranasal saline administration (n = 4
at each time point, 16 mice in total) (Figure 2). At 30 min after the intravenous dose ofPharmaceutics 2022, 14, x FOR PEER REVIEW

Pharmaceutics 2022, 14, 1026

4 of 10[125I]IBZM were injected into the tail vein of mice at 0, 15, and 30 min after intranasal
4 of 10domperidone administration, or at 0 min after intranasal saline administration (n = 4 ateach time point, 16 mice in total) (Figure 2). At 30 min after the intravenous dose of[125
I]IBZM, cardiac blood was collected under isoflurane inhalation anesthesia, and the[125 I]IBZM, cardiac blood was collected under isoflurane inhalation anesthesia, and theanimals
animalswere
wereeuthanizedeuthanizedandandquickly
quicklyfractionatedfractionatedinto
intothetheolfactory
olfactorybulb,
bulb,cerebral
cerebralcortex,
cortex,striatum,hippocampus,
andcerebellum
under
ice-cold
conditions.
The
wetstriatum, hippocampus, and cerebellum under ice-cold conditions. The wettissues
tissueswere
were
then
thenweighed,weighed,and
andthetheradioactivity
radioactivityin
inthe
theweighedweighedtissue
tissuesamples
sampleswas
wasmeasured
measuredusing
usingaagamma(AccuFLEXγARC-7010;
γARC-7010;Hitachi).
Hitachi).
Dataexpressed
as %injected
the %injectedgamma countercounter (AccuFLEX
Data
areareexpressed
as the
dose
dose
per
g
wet
tissue(%ID/gtissue).
per g wet tissue (%ID/g tissue).

Figure 2.
2. Procedure
Procedureof
ofthe
thebiologicalbiologicaldistribution
distributionstudy
studyand
and(a)(a)SPECT
SPECTimaging
imagingunder
underisoflurane
isoflurane
Figure123
123
inhalation
anesthesia
(b)
using
intravenous[I]IBZMadministration
withintravenousor
inhalation anesthesia (b) using intravenous [ I]IBZM administration with intravenous orintranasal
intranadomperidone
administration.
intranasally administeredadministeredsaldomperidone
administration.Normal
NormalddY
ddYmicemiceareare intravenously
intravenously or intranasally
22μg/gweight/20
μL µL
domperidone(control,n = 5;
group,
n = 3 (onedied);µg/gbody
bodyweight/20
domperidone(control,n intravenous= 5; intravenous
group,
n = 3mouse(one mouse
intranasal
group, group,n = 4; foradministration).
After mice
aremicesacrificed,
brain anatomy
is imme-isdied); intranasal
n =each4; foreach administration).
After
are sacrificed,
brain anatomy
diatelyexamined,and thenis measured.
immediately
examined,
andradioactivity
then radioactivity
is measured.

2.5.SPECTSPECTImaging
Imagingafter
afterananIntravenous
IntravenousDose
Doseofof[123[123
I]IBZMwith
withananIntranasal
IntranasalororIntravenous
Intravenous
2.5.
I]IBZM
Doseof
Domperidone
Dose of Domperidone
All procedures
procedures in
under
isoflurane
inhalation
anesthesia(Figure2).
Allinmicemicewere
wereconductedconducted
underisoflurane
inhalation
anesthesia(FigDomperidone
dilutedwithsalinewasadministeredintravenously
into
the
tail
vein
(n
=
3;
one
ure 2). Domperidone diluted with saline was administered intravenously into the tail vein
mouse
died)
or intranasally(n = 4) using(na micro-syringe
via the tracheal
at a doseof(n= 3; onemousedied) or intranasally= 4) using a micro-syringe
viacatheter
the tracheal
cath2
µg/g
body
weightandvolume
of
20
µL.
As
a
control,salinewasintranasallyadministeredeter at a dose of 2 μg/g body weight and volume of 20 μL. As a control, saline
was intranato theadministeredmice (n = 5) toin thethe micesame(nmanner.
14.0 ±
1.6 MBq [12314.0
I]IBZMsally= 5) in Furthermore,
the same manner.
Furthermore,± 1.6diluted
MBqwithsalinewasintravenously
administered
into
the
tail
vein
15
min
after
the
intranasal
123[ I]IBZM diluted with saline was intravenously administered into the tail vein 15 dosemin
(IN groups)
and intravenous(IV groups)of domperidoneor(IVsaline,and SPECT
acquisition
after
the intranasal
dose (INdosegroups)andintravenous
dose
groups)ofdomperidone
started immediately after the dose of [123 I]IBZM, at 5 min/frame for 90 min using a VECTor+or saline, and SPECT acquisition started immediately after the dose of [123I]IBZM, at 5
system (MILabs, Utrecht, The Netherlands). SPECT images were obtained and reconstructedmin/frame for 90 min using a VECTor+ system (MILabs, Utrecht, The Netherlands). SPECT
using the ordered subset expectation maximization method with 16 subsets and 6 iterations,images were obtained and reconstructed using the ordered subset expectation maximiza-3
including scatter and attenuation correction. The voxel size was set to 0.8 × 0.8 × 0.8 mm .
tion method with 16 subsets and 6 iterations, including scatter and attenuation correction.
Post-reconstruction smoothing filtering was applied using a 1.0-mm Gaussian filter. Image
The voxel size was set to 0.8 × 0.8 × 0.8 mm3. Post-reconstruction smoothing filtering was
displays were obtained using the medical image data analysis software programs Pmod (ver.
applied using a 1.0-mm Gaussian filter. Image displays were obtained using the medical
3.7, PMOD Technologies LLC, Zurich, Switzerland) and Amide (exe. 1.0.4-1). Time–activity
image data analysis software programs Pmod (ver. 3.7, PMOD Technologies LLC, Zurich,curves (TACs) for the striatum and cerebellum were generated from the SPECT images, andSwitzerland) and Amide (exe. 1.0.4-1). Time–activity curves (TACs) for the striatum andcoronal images were displayed as similar section images. Specific binding was calculated by
cerebellum were generated from the SPECT images, and coronal images were displayed
subtracting SUVmean in the cerebellum from that in the striatum. Binding potential (BP) and %assimilar section images. Specific binding was calculated by subtracting SUVmean in theof receptor occupancy (%RO) using SUVmean in the equilibrium state were calculated in the
cerebellum
fromthat in the
striatum andcerebellum[14].striatum. Binding potential (BP) and % of receptor occupancy

Pharmaceutics 2022, 14, 1026

5 of 102.6. Statistical Analysis
Data are presented as means and standard deviation. After normality testing using
the Kolmogorov–Smirnov test for statistical analysis of TACs and specific binding, and theShapiro–Wilk test for analysis of BP and %RO, p values were calculated using Welch’s t-test
for comparisons between two groups and analysis of variance. Tukey’s test was used forcomparisons among the three groups. All analyses were conducted using GraphPad Prism
8 statistical software (GraphPad Software, Inc., La Jolla, CA, USA). A P value of less than
0.05 was considered indicative of a significant difference.
3. Results
The appropriate intranasal dose schedule of domperidone was assessed prior to the
SPECT study. In the biological distribution study, we decided to sacrifice the mice 30 min
after [125 I]IBZM injection because the ratio of striatum to cerebellum was highest in eachbrain tissue 30 min after an intravenous dose of [125 I]IBZM.
Table 1 shows the biological distributions (%ID/g) for each brain tissue and organ.
Compared with the controls that received saline instead of domperidone, a significant
decrease in accumulation of [125 I]IBZM was observed only for the striatum, which expresses
high levels of D2R [15], in the group that was dosed with intranasal domperidone at 15 min
before the [125 I]IBZM dose. This result suggested that the dose schedule of intranasal
domperidone at 15 min before the [125 I]IBZM dose was the most suitable for use in the
SPECT study to evaluate whether intranasal domperidone has a competitive inhibition
effect on striatal D2R binding by [125 I]IBZM.
Table 1. Biological distribution of [125 I]IBZM in control mice and following intranasal domperidone administration.
Accumulation After [125 I]IBZM Administration (%ID/g)
Organ

Control GroupBlood
Olfactory bulb
Cerebral cortex
Striatum
Hippocampus
Cerebellum

2.03 ± 0.28
5.02 ± 1.00
5.07 ± 1.10
11.06 ± 3.33
4.37 ± 1.12
2.97 ± 0.19

IN Groups
0 min

15 min30 min1.22 ± 0.16
3.56 ± 0.56
4.72 ± 0.57
9.27 ± 1.26
4.04 ± 0.54
3.00 ± 0.14

1.69 ± 0.31
3.87 ± 0.70
4.54 ± 0.35
6.87 ± 1.95 *3.47 ± 0.36
3.52 ± 0.42

1.67 ± 0.21
4.63 ± 1.19
4.59 ± 1.02
9.92 ± 2.01
3.83 ± 0.94
2.90 ± 0.86

%ID/g, percent injected dose per gram of tissue. Values are the mean ± standard deviation obtained from four
mice in each group. * p < 0.05 compared with control mice.

Figure 3 shows SPECT images of the brain obtained approximately 30 min after a doseof intravenous [123 I]IBZM with a dose of intranasal saline (control), in the IV group, and in
the IN group. One mouse in the IV group died during SPECT imaging. Compared withthe strong accumulation of [123 I]IBZM seen in the striatum in the control image, there wasless accumulation in the IV group, and very little accumulation in the IN group. This result
corresponds closely to the result of the [125 I]IBZM biological distribution study.
Figure 4 shows mean TACs of [123 I]IBZM for the striatum and cerebellum in the threegroups. For the cerebellum, there was no significant difference in SUVmean values at any
time point among the groups. For the striatum, there was no significant difference in
SUVmean values at any time point between the control and IV groups, whereas SUVmean
values in approximately the first 10 min were significantly lower in the IN group than in
the other two groups.

Pharmaceutics 2022, 14, x FOR PEER REVIEW
Pharmaceutics 2022, 14, 1026

6 of 10
6 of 10Figure 3. Representative [123I]IBZM SPECT images of the control group, (a) intravenous domperidone administration (IV group), (b) intranasal domperidone administration (IN group), and (c) at
25–30 min after intravenous administration of approximately 14 MBq [123I]IBZM. There is avid accumulation in the striatum in the control. Accumulation in the striatum is not much lower in the IV
group with domperidone compared to the control, but clearly lower in the IN group with domperidone. There is little difference in accumulation in the cerebellum among the control, IV, and IN
groups.
Figure 3. Representative [123I]IBZM SPECT images of the control group, (a) intravenous domperiFigure 3. Representative [123 I]IBZM SPECT
images of the control group, (a) intravenous domperidone
4 shows(IV
mean
TACs
[123I]IBZM
for the striatum
and cerebellumin thedoneFigure
administrationgroup),(b)ofintranasal
domperidone
administration(IN group),
andthree(c) atadministration (IV group), (b) intranasal domperidone administration (IN 123
group), and (c) at 25–30 mingroups.
Fortheintravenous
cerebellum,
there was nodifferencein
SUV
meanThere
valuesat any25–30 minafter
administrationof significant
approximately
14MBq[I]IBZM.
is
avid
ac123
after
intravenous
approximately
14 MBqin[ theI]IBZM.
There
is muchavid accumulationincumulationin theadministration
striatumin theofFor
control.
is notlower inintheIV
timepoint among
the groups.
the Accumulation
striatum,
there wasstriatum
no significant
difference
SUthe
striatumin the control.compared
Accumulationinthe striatum
is notmuch
the IV
group with
groupwith domperidone
to the thecontrol,but andclearly
in lower
the
IN in
group
with
V
mean values
at any time point
betweencontrol
IVlowergroups,whereasSUV
meandompervalues
domperidone
comparedto the control,
but clearly in
lowerin
the IN groupwiththedomperidone.
isidone.
Thereislittledifferenceinaccumulation
thecerebellum
amongcontrol,andIN
in approximately the first 10 min were significantly lower in the IN group than inIV,
theThereotherlittledifference in accumulation in the cerebellum among the control, IV, and IN groups.
groups.

two groups.

Barin(SUV
accumulation(SUVmean)Barin accumulation
mean)

Figure 4 shows mean TACs of [123I]IBZM for the striatum and cerebellum in the three
10.0
Control
Striatum
Cerebellum
groups. For the cerebellum,
there wasno significant Control
differencein SUVmean values at any
time point
For the striatum, there was no significant difference in SU9.0 among the groups.
IV group Striatum
IV group Cerebellum
Vmean values at any time point between the control and IV groups, whereas SUVmean values
8.0
in approximately
the first 10INmin
wereStriatum
significantly lower
in the IN
group than in the othergroup
IN group
Cerebellum
two groups.

7.0

10.0
6.0

Control Striatum

Control Cerebellum

9.0
5.0

IV group Striatum

IV group Cerebellum

IN group Striatum

IN group Cerebellum

8.0
4.0

*

*

7.0
3.0
6.0
2.0
5.0
1.0
4.0
0.0
3.0

0

*

*
10

20

30

40

50

60

70

80

90

Time (min)2.0

123 I]IBZM SPECT images of the
Figure 4.
4. Time–activity
Time–activity curves
curves forfor theFigurethe striatum
striatum andand cerebellum
cerebellum inin [[123I]IBZM
SPECT images of thecontrol,IV, andandIN
INgroups.
groups.InIn
thestriatum,there
is significant
no significant
difference
betweenthe control
1.0
control,IV,thestriatum,
there
is no
difference
betweenthe control
andandgroups
IV groups
domperidone,
but values
are significantly
lower
in theIN group
duringIV
withwith
domperidone,
but values
are significantly
lower
in theIN group
duringthe thefirstfirst10
0.0
10 mincomparedwith
controlandgroups.InInthethecerebellum,
cerebellum,there
thereisisno
nosignificant
significantdifference
differencemincomparedwiththethecontrolandIVIVgroups.
0102030control
40and
60or
80
90
among
** pp <<0.05
between
IN50
group
or
IV IV
and70
IN IN
group.
among thethe groups.
groups.
0.05
betweencontrol
and
IN
group
and
group.

Time (min)Figure 55 shows
shows thethe specificspecific binding
binding value–timevalue–time curves
curves forfor thethe striatum
striatum inin thethe three
three
Figure123
groups,the
SUVvaluesforthestriatumandcerebellum.
groups,which
werecalculated
using
mean
valuesforthestriatumandcerebellum.
mean
Figure 4. Time–activity curves for the striatum and cerebellum in [ I]IBZM SPECT images of theSpecificbinding
atIn
approximately
theisfirst
20 min was
significantly
lower
in theandINcontrol, IV,
and INvalue
groups.
the striatum, there
no significant
difference
betweenthe control
group
comparedwith the othertwo groups.
The equilibriumstate
the during
specificthe
binding
IV groups
with domperidone,
but values
are significantly
lower in the
IN of
group
first 10min comparedwithforthethecontroland IV
groups.
In theno significant
difference
value–time
curvestriatumwasachieved
atcerebellum,
25–35 min there
in all isthreegroups. The
mean
among
the
groups.
*p<
0.05
betweencontrol
and
IN
group
or
IV
and
IN
group.
BP values in the equilibrium state were 1.27, 1.08, and 0.67 for the control group, IV group,and IN group, respectively; the BP value was significantly lower in the IN group than in
Figure5 shows
specificbinding
for the striatum
in inthethethree
the othertwogroupsthe(Table2). Themeanvalue–time%RO valuecurves
was significantly
higherIN
groups,
which
were
using
group
than
in theIV calculatedgroup (Table2). the SUVmean values for the striatum and cerebellum.

Pharmaceutics 2022, 14, 1026

Specific binding value at approximately the first 20 min was significantly lower in the IN
group compared with the other two groups. The equilibrium state of the specific binding
value–time curve for the striatum was achieved at 25–35 min in all three groups. The mean
BP values in the equilibrium state were 1.27, 1.08, and 0.67 for the control group, IV group,and IN group, respectively; the BP value was significantly lower in the IN group than in
7 of IN10
the other two groups (Table 2). The mean %RO value was significantly higher in thegroup than in the IV group (Table 2).

Figure 5. Specific binding of the striatum in the control, IV, and IN groups. Values are significantly
Figurein5.theSpecificbindingof the20
striatumin intravenous
the control, IV,
IN groups.
Values are
significantly
lower
IN group
for about
min after[123andI]IBZMadministrationcompared
withlower in the IN group for about 20 min after intravenous [123I]IBZM administration compared withthe control and IV groups.* p < 0.05 between control and IN groups.
the control and IV groups.* p < 0.05 between control and IN groups.
Table
Mean BPBP andand %RO%RO values
valuesininthe
theequilibrium
equilibriumstate
stateinincontrol
control
mice
andTable2.2. Mean
mice
and
in in
thetheIV IVandandIN
IN
groups.
groups.

Control
Controlgroup
IVIVgroup
IN group
IN group

BPBP± 0.16
1.271.27± 0.16± 0.111.081.08± 0.110.67 ± 0.08 *0.67 ± 0.08 ** p < 0.05 compared with control mice or IV group.

%RO
%RO

−4.38± ±
10.95
−4.38
10.95
35.63 ± 7.35 *35.63 ± 7.35 ** p < 0.05 compared with control mice or IV group.

4. Discussion
4. Discussion
In the present study, a SPECT imaging method was demonstrated in mice to indirectly
In the present study, a SPECT imaging method was demonstrated in mice to indievaluate whether temporal accumulation of drugs with an affinity for P-gp occurred in the
rectly evaluate whether temporal accumulation of drugs with an affinity for P-gp occurred
brain via N2B drug delivery. Domperidone, a D2R ligand that is well-known to be poorly
in the brain via N2B drug delivery. Domperidone, a D2R ligand that is well-known to be
BBB permeable, was used as the model drug for N2B delivery. The mouse animal model was
poorly BBB permeable, was used as the model drug for N2B delivery. The mouse animal
appropriate for N2B delivery because the mouse nose has a high ratio of olfactory region tomodel was appropriate for N2B delivery because the mouse nose has a high ratio of olfacnasal mucosa, and a surgical procedure was performed to place the tip of a catheter in the
tory region to nasal mucosa, and a surgical procedure was performed to place the tip of anasal cavity for intranasal drug administration. A rodent study is appropriate for new drug
catheter in the nasal cavity for intranasal drug administration. A rodent study is approscreening tests by N2B delivery for the reasons of cost-effectiveness and convenience of use
priate for new drug screening tests by N2B delivery for the reasons of cost-effectivenessin comparison with nonhuman primates and other animals. It is important to synthesize
and convenienceuse highin comparison
nonhumantoprimatesotheranimals.
It isradioactive
ligandsofwith
radioactivewith
concentration
ensure a andsmall
injection
volume.
importanttosynthesize
radioactive
ligands
withhighradioactive
concentration
toensure125/123
The radioactive concentration of [I]IBZM used in this study was very high. Based
125/123I]IBZM used in this study
a smallinjection volume.
The data
radioactive
concentrationof [theon
the biologicaldistributionof [125 I]IBZM(Table 1),schedule for the dosing of
intranasal domperidone for the SPECT study was set at 15 min before the intravenous[123 I]IBZM. The significantly lower accumulation of [125 I]IBZM in the striatum alone in the
pretreatment group with intranasal domperidone 15 min before the intravenous [125 I]IBZM
compared with the control indicates the binding of domperidone to D2R in the striatum.
This result corresponds closely to the result of the SPECT image analysis for the intranasal
domperidone group, in which the accumulation of [123 I]IBZM in the striatum was lower
than that in the intranasal saline group. Conversely, the lack of a significant decreasein [125 I]IBZM accumulation in the striatum for the pretreatment group of 30 min withintranasal domperidone compared with the control may mean that the distribution ofPharmaceutics 2022, 14, 1026

8 of 10domperidone in the brain via the N2B route is transient due to complex factors, including
the effects of P-gp expressed in endothelial cells lining the brain capillaries, as well as
the olfactory mucosa [16,17]. In support of this hypothesis, a recent similar study was
conducted to assess the N2B delivery of quinidine as a poor BBB-permeable drug in rats.
This experiment demonstrated the possibility that quinidine, a P-gp substrate, could betransported into the striatum by intranasal administration in a pretreatment with the P-gp
specific inhibitor [18].
The transport pathways in N2B drug delivery include the intracellular route (axonaltransport), the paracellular route, the transcellular route through olfactory epithelium, andthe trigeminal nerve route through respiratory epithelium [19,20]. Although we could notclarify the detailed mechanism of N2B delivery of domperidone in this study, we consider
that the main pathway for nasal transport of domperidone is the paracellular route throughthe lamina propria of olfactory epithelium. In fact, fluorophore-conjugated dextran, anon-directional tracer, has been shown to be easily and quickly transported into the lamina
propria of olfactory epithelium [21,22]. However, further experiments are necessary to
clarify in more detail the mechanism of transport of domperidone into the brain.
In the present study, TACs (Figure 4) and specific binding value–time curves (Figure 5)of [123 I]IBZM for the striatum were significantly lower in the intranasal domperidone groupcompared with the intranasal saline and intravenous domperidone groups. In addition, BP
and %RO values were significantly lower in the intranasal domperidone group comparedwith the intravenous domperidone group. From the perspective of ligand-receptor binding
competition, these results also indicate that the intact (non-labeled) intranasal domperidone
could be transported to the brain via the N2B pathway and compete with binding of[123 I]IBZM in the striatum.
As a limitation, the striatum, which has the highest expression of D2R and the highest
accumulation of [123 I]IBZM SPECT imaging in the mouse brain, was analyzed. Because thebrain size is small, and the resolution of SPECT images is low, it was difficult to set volumesof interest for each brain region. Imaging studies using rats and monkeys, which havelarger brain areas than mice, are needed. In addition, it is necessary to confirm the dosedependent increase in %RO of domperidone by IN administration and to confirm detailed
brain region-selective %RO changes in rats and primates, which have larger brain sizes thanmice, in the future. In addition, whether P-gp substrates other than domperidone, such as
amisulpride and sulpiride, can also be evaluated for brain migration needs to be examined.
5. Conclusions
Indirect SPECT imaging evaluation using intravenous [123 I]IBZM administration could
evaluate the time-dependent effect of intranasal domperidone in a mouse study and may
be a suitable method for use in N2B drug delivery research to explore time-dependent
brain uptakes and locus-specific biological distribution of such drugs as the P-gp substrate
domperidone and other poorly BBB-permeable agents.
Supplementary Materials: The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/pharmaceutics14051026/s1, Figure S1: In the UV and radioisotope(RI) chromatograph, the peak waveform in UV of [127 I]IBZM (a) and RI of [125 I]IBZM (b) is confirmed
at the same retention time of about 8.5 min.
Author Contributions: Conceptualization, S.H. and K.K.; methodology, A.M., M.O. and S.F.; software,
M.K. and K.S.; validation, C.S., R.N. and Y.M. (Yasuhiro Magata); formal analysis, M.K. and K.S.;
investigation, A.M., M.O., C.S. and Y.M. (Yuka Muranaka); resources, K.S., Y.T., S.F. and S.H.; data
curation, A.M., M.K. and M.O.; writing—original draft preparation, A.M. and M.K.; writing—review& editing, K.S., Y.T. and S.H.; visualization, A.M. and M.O.; supervision, R.N.; project administration,
S.H. and K.K.; funding acquisition, M.K. and K.K. All authors have read and agreed to the publishedversion of the manuscript.

Pharmaceutics 2022, 14, 1026

9 of 10

Funding: The present study was funded in part by a Grant-in-Aid for Scientific Research fromthe Japan Society for the Promotion of Science (Nos. 18K19508 and 21H02865) and Shin Nippon
Biomedical Laboratories, Ltd.
Institutional Review Board Statement: The study was conducted in accordance with the ethicalstandards of Kanazawa University (Animal Care Committee of Kanazawa University, AP-173851)
and with international standards for animal welfare and institutional guidelines.
Informed Consent Statement: Not applicable.
Data Availability Statement: The authors confirm that the data supporting the findings of this study
are available within the article.
Acknowledgments: The authors would like to thank research members of the Faculty of Health
Sciences, Kanazawa University and Shin Nippon Biomedical Laboratories, Ltd.
Conflicts of Interest: This study was funded by Shin Nippon Biomedical Laboratories, Ltd. (SNBL).
K.S., Y.T., S.F., and S.H. are employees of SNBL. The company had no role in the design of the study;in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in thedecision to publish the results.

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