pharmaceutics
ArticleBiological Distribution of Orally Administered [123I]MIBG for
Estimating Gastrointestinal Tract Absorption
Masato Kobayashi 1, * , Asuka Mizutani 1 , Yuka Muranaka 1 , Kodai Nishi 2 , Hisakazu Komori 3 ,Ryuichi Nishii 4 , Naoto Shikano 5 , Takeo Nakanishi 6 , Ikumi Tamai 3 and Keiichi Kawai 1,7
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5

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Citation: Kobayashi, M.; Mizutani,

7*

Faculty of Health Sciences, Institute of Medical, Pharmaceutical and Health Sciences, Kanazawa University,
5-11-80 Kodatsuno, Kanazawa 920-0942, Japan; mizutani.a@staff.kanazawa-u.ac.jp (A.M.);
yukarisa93@stu.kanazawa-u.ac.jp (Y.M.); kei@mhs.mp.kanazawa-u.ac.jp (K.K.)
Department of Radioisotope Medicine, Atomic Bomb Disease Institute, Nagasaki University, 1-12-4 Sakamoto,Nagasaki 852-8523, Japan; koudai@nagasaki-u.ac.jp
Faculty of Pharmaceutical Sciences, Institute of Medical, Pharmaceutical and Health Sciences,
Kanazawa University, Kakuma, Kanazawa 920-1192, Japan; hkomori@p.kanazawa-u.ac.jp (H.K.);
tamai@p.kanazawa-u.ac.jp (I.T.)
Department of Molecular Imaging and Theranostics, Institute for Quantum Medical Science Quantum Life
and Medical Science Directorate, National Institutes for Quantum Science and Technology (QST),
4-9-1 Anagawa, Inage, Chiba 263-8555, Japan; nishii.ryuichi@qst.go.jp
Department of Radiological Sciences, Ibaraki Prefectural University of Health Sciences, 4669-2 Ami, Inashiki,
Ibaraki 300-0394, Japan; sikano@ipu.ac.jp
Faculty of Pharmacy, Takasaki University of Health and Welfare, 60 Nakaorui-machi,
Takasaki 370-0033, Japan; nakanishi@takasaki-u.ac.jp
Biomedical Imaging Research Center, University of Fukui, 23-3 Matsuokashimoaizuki, Eiheiji,
Fukui 910-1193, Japan
Correspondence: kobayasi@mhs.mp.kanazawa-u.ac.jp; Tel.: +81-76-265-2524; Fax: +81-76-234-4366

A.; Muranaka, Y.; Nishi, K.; Komori,
H.; Nishii, R.; Shikano, N.; Nakanishi,T.; Tamai, I.; Kawai, K. BiologicalDistribution of Orally Administered[123 I]MIBG for Estimating
Gastrointestinal Tract Absorption.
Pharmaceutics 2022, 14, 61.
https://doi.org/10.3390/
pharmaceutics14010061
Academic Editor: Emanuela
Fabiola Craparo
Received: 30 November 2021
Accepted: 24 December 2021
Published: 28 December 2021
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims inpublished maps and institutional affiliations.

Abstract: Gastrointestinal tract absorption of cationic anticancer drugs and medicines was estimatedusing whole-body imaging following oral [123 I]MIBG administration. [123 I]MIBG was added to
cultures of human embryonic kidney (HEK)293 cells expressing human organic anion transporting
polypeptide (OATP)2B1, carnitine/organic cation transporter (OCTN)1 and OCTN2, and organic
cation transporter (OCT)1, OCT2, and OCT3 with and without cimetidine (an OCTN and OCT
inhibitor) and L-carnitine (an OCTN inhibitor). Biodistribution analyses and single-photon emission
computed tomography (SPECT) imaging in normal and dextran sodium sulfate (DSS)-induced
experimental colitis mice were conducted using [123 I]MIBG with and without cimetidine. [123 I]MIBG
uptake was significantly higher in HEK293/OCTN1, 2, and OCT1-3 cells than in mock cells. Uptake
via OCTN was inhibited by L-carnitine, whereas OCT-mediated uptake was inhibited by cimetidine.
Biodistribution analyses and SPECT imaging studies showed significantly lower accumulation of[123 I]MIBG in the blood, heart, liver, and bladder in DSS-induced experimental colitis mice and micewith cimetidine loading compared with normal mice, whereas significantly higher accumulationin the stomach and kidney was observed after [123 I]MIBG injection. [123 I]MIBG imaging after oral
administration can be used to estimate gastrointestinal absorption in the small intestine via OCTN
and/or OCT by measuring radioactivity in the heart, liver, and bladder.
Keywords: [123 I]MIBG; SPECT; imaging; drug transporters; gastrointestinal tract absorption

Copyright: © 2021 by the authors.
Licensee MDPI, Basel, Switzerland.

1. Introduction

This article is an open access article

Iodine-123 labeled m-iodobenzylguanidine ([123 I]MIBG), an analog of the adrenergic
neurotransmitter norepinephrine, has been used as a cardiac [1] and adrenomedullary
scintigraphic agent [2] since the early 1980 s. [123 I]MIBG is primarily transported into
cardiac and neuroblastoma cells via organic cation transporter (OCT)1-3, including the
norepinephrine transporter [3,4].

distributed under the terms andconditions of the Creative Commons
Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

Pharmaceutics 2022, 14, 61. https://doi.org/10.3390/pharmaceutics14010061

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

Pharmaceutics 2022, 14, 61

2 of 10

Many anticancer drugs (e.g., imatinib) and medicines (e.g., metformin, cimetidine,
procainamide) are incorporated into the blood from the small intestinal epithelial cell
membrane via solute carrier (SLC) transporters. At the small intestinal epithelial cell
membrane in humans, organic anion transporting polypeptide (OATP)2B1 (SLCO2B1),
organic cation/carnitine transporter (OCTN)1 (SLC22A4), OCTN2 (SLC22A5), organic
cation transporter (OCT)1-3, and peptide transporter 1 (SLC15A1) isoforms are expressedprimarily as SLC transporters [5–9]. Although the enterocyte membrane localization of thetransporters, especially OATP2B1 and OCT1, changes along the gastrointestinal tract [10],the SLC transporters OATP2B1, OCTN1, 2, and peptide transporter 1 are highly expressedon the basolateral side in the small intestine, whereas OCT1-3 expression occurs primarilyon the apical side.
Gastrointestinal tract absorption of anticancer drugs and medicines is typically measured using liquid chromatography/mass spectrometry (LC/MS) [11]. Although LC/MS
requires liquid samples, such as blood or urine to measure gastrointestinally absorbed
anticancer drugs and medicines, such liquid samples are affected by not only gastrointestinal tract absorption but also distribution throughout the body. Therefore, the purpose ofthis study was to estimate gastrointestinal tract absorption of cationic anticancer drugs
and medicines via whole-body imaging following oral [123 I]MIBG administration. After
gastrointestinal tract absorption, cationic anticancer drugs and medicines primarily accumulate in the liver for metabolism and/or excretion via the urine and/or feces. It may
be possible to estimate gastrointestinal tract absorption by focusing on drug-associatedradioactivity in the liver and/or bladder.
2. Materials and Methods
2.1. Materials[123 I]MIBG (>95% of the radiochemical purity) was purchased from FUJIFILM Toyama
Chemical Co., Ltd. (Tokyo, Japan). [3 H]Methyl-4-phenylpyridinium (2.96 TBq/mmol) andp-[14 C]aminohippuric acid (2.04 GBq/mmol) were purchased from American Radiolabeled
Chemicals Inc (St. Louis, MO, USA) and PerkinElmer Inc. (Waltham, MA, USA). For in vivo
study, cimetidine was purchased from Nacalai-Tesque (Kyoto, Japan). Dextran sodium
sulfate (DSS, MW 36,000–50,000 Da) was purchased from Tokyo Chemical Industry Co., Ltd.
(Tokyo, Japan). OCTN1/2 antibody (H-9) was purchased from Santa Cruz (Dallas, TX, USA).
2.2. Human Embryonic Kidney (HEK)293 Cells for SLC Transporters
SLC transporters were investigated using HEK293 cells expressing OATP2B1 (SLCO2B1,
NM_007256), OCTN1 (SLC22A4, NM_003059), OCTN2 (SLC22A5, NM_003060), OCT1
(SLC22A1, NM_003057), OCT2 (SLC22A2, NM_003058), and OCT3 (SLC22A3, NM_021977).
Briefly, HEK293 cells were transfected with plasmids encoding the respective transporters
and then selected using appropriate antibiotics to establish HEK293/OATP2B1,
HEK293/OCTN1, HEK293/OCTN2, HEK293/OCT1, HEK293/OCT2, HEK293/OCT3,
and mock control cell lines. All cell lines were cultured at 37 ◦ C and 5% CO2 in Dulbecco’s
modified Eagle’s medium (Wako Pure Chemical Industries Ltd., Osaka, Japan) including 10% (v/v) fetal bovine serum (Life Technologies, Carlsbad, CA, USA), 100 µg/mL
streptomycin, and 100 U/mL penicillin.
2.3. Analysis of [123 I]MIBG Uptake by HEK293 Cells
Expression of OATP2B1 was confirmed in HEK293 cells using p-[14 C]aminohippuric acid,and expression of OCTN and OCT was confirmed using [3 H]methyl-4-phenylpyridinium [3,12].
One day before the uptake experiments, HEK293 cells expressing the respective SLC transporter were seeded at 4 × 105 cells/well in 12-well plastic plates. Cells of each type werepre-incubated for 10 min in modified Hank’s balanced salt solution (MHBS) and thenincubated with [123 I]MIBG (37 kBq) for 5 min (n = 4), after which the cells were detachedfrom the tissue culture plate using 0.25% trypsin-EDTA solution (Sigma-Aldrich, St. Luis,
MO, USA). The radioactivity associated with the cells was measured using a gamma

Pharmaceutics 2022, 14, 61

3 of 10counter. Cellular protein content was measured using a bicinchoninic acid protein assay kit(Thermo Fisher Scientific Inc., Waltham, MA, USA) with bovine serum albumin as the standard. Uptake of [123 I]MIBG by HEK293/OATP2B1, HEK293/OCTN1, HEK293/OCTN2,HEK293/OCT1, HEK293/OCT2, and HEK293/OCT3 cells was compared with uptake bymock control HEK293 cells. The effects of the OCTN and OCT inhibitor cimetidine [13,14]
and OCTN inhibitor L-carnitine [15] were assessed by incubating HEK293 cells in a medium
containing [123 I]MIBG with and without 1 mM cimetidine and L-carnitine in MHBS. Uptakeof [123 I]MIBG by HEK293/OCTN1, HEK293/OCTN2, HEK293/OCT1, HEK293/OCT2,
and HEK293/OCT3 cells was examined using this assay method, with [123 I]MIBG uptake
shown as % injected dose (%ID)/g protein.
2.4. DSS-Induced Experimental Colitis Mice
Animal experiments were approved by the Kanazawa University Committee for theCare and Use of Laboratory Animals (AP-194046) and were carried out in accordancewith the guidelines for the Care and Use of Laboratory Animals. Male mice (4 weeks old,
18.9 ± 1.3 g, SLC Inc., Hamamatsu, Japan) were permitted free access to drinking watercontaining 2% (w/v) DSS from days 1 to 8 and days 22 to 29 and free access to DSS-freewater from days 8 to 22 and days 29 to 31 [16]. Change in body weight was assessed
every two days. Hematoxylin and eosin (HE) staining along with anti-OCTN1/2 (H9)immunohistochemical analysis were performed on specimens of tissue from the small andlarge intestines, liver, and kidney.
2.5. Biological Distribution of [123 I]MIBG in DSS-Induced Experimental Colitis Mice
A total of 12 normal ddY mice (male, 9 weeks old, 33.7 ± 2.0 g) and 12 DSS-induced
experimental colitis mice (male, 9 weeks old, 31.9 ± 2.4 g) were fasted overnight before
experiments, with water supplied ad libitum. Each mouse with a clip on the penis to prevent
urination was then intragastrically injected with approximately 185 kBq of [123 I]MIBG
via oral administration using feeding needles (Fuchigami). At 5, 10, 30, and 60 min after
injection, the mice were euthanized under isoflurane (n = 3 per time point) after blood
was sampled via cardiocentesis, and the following tissues were collected: thyroid, heart,
stomach, liver, kidney, and bladder. The tissues were then weighed, and the radioactivity inthe weighed tissue samples was measured using a gamma counter (AccuFLEX γARC-7010,
Hitachi, Ibaraki, Japan). Data are expressed as %ID per g wet tissue (%ID/g tissue).
2.6. Single-Photon Emission Computed Tomography (SPECT) Imaging of [123 I]MIBG in Normal
and DSS-Induced Experimental Colitis Mice
Normal ddY mice (male, 9 weeks old, 37.4 ± 1.5 g, n = 5) and DSS-induced experimental colitis mice (male, 9 weeks old, 33.2 ± 2.9 g, n = 5) were intragastrically injectedwith [123 I]MIBG (23.5 ± 2.0 MBq) under isoflurane gas anesthesia. SPECT acquisition wasstarted 5 min after intragastric injection using a VECTor+ system (MILabs, Utrecht, The
Netherlands). The acquisition data were reconstructed using the ordered subset expectation
maximization method with 16 subsets and 6 iterations including resolution, attenuationand scatter correction. In the other reconstruction parameters, the voxel size was selected
to 0.8 × 0.8 × 0.8 mm3 and a 1.0-mm Gaussian filter was applied as post-reconstructionsmoothing filtering. For imaging analysis, the medical image data analysis software
programs Pmod (ver. 3.7, PMOD Technologies LLC, Zurich, Switzerland) and Amide(exe. 1.0.4-1) were used [17]. SPECT images were obtained at 5–10 min, 30–35 min, and
60–65 min after oral administration of [123 I]MIBG. Time-activity curves for the thyroid,
heart, stomach, liver, kidney, and bladder were generated from the SPECT images, andcoronal images are displayed as similar section images.
2.7. Biological Distribution of [123 I]MIBG in the Presence/Absence of Cimetidine
One day before the experiments, a total of 24 ddY mice (male, 9 weeks old,
31.7 ± 1.8 g) were fasted with no food overnight, but water supplied ad libitum. The

Pharmaceutics 2022, 14, 61

4 of 10mice were then intragastrically injected with approximately 185 kBq of [123 I]MIBG withor without cimetidine via oral administration using feeding needles (Fuchigami). Eachmouse with a clip on the penis to prevent urination was then intragastrically injectedwith approximately 185 kBq of [123 I]MIBG via oral administration using feeding needles(Fuchigami). At 5, 10, 30, and 60 min after injection, the mice were euthanized under isoflurane (n = 3 per time point) after blood was sampled via cardiocentesis, and the following
tissues were collected: thyroid, heart, stomach, liver, kidney, and bladder. The tissues werethen weighed, and the radioactivity in the weighed tissue samples was measured usinga gamma counter (AccuFLEX γARC-7010, Hitachi, Ibaraki, Japan). Data are expressed
as %ID per g wet tissue (%ID/g tissue). For studies with cimetidine loading (an OCT
inhibitor with a high safety level in humans), ddY mice were orally administrated witha mixture of [123 I]MIBG and cimetidine (approximately 7.5 µg/g mouse body weight in
100 µL of saline [18]), and the biological distribution analyses were performed using thesame protocol used for control mice.
2.8. Statistical Analysis
P-values were analyzed using the two-tailed paired Student’s t-test for comparisons between two groups and analysis of variance and the Tukey test for comparisonsamong three groups using GraphPad Prism 8 statistical software (GraphPad Software, Inc.,
La Jolla, CA, USA) after normality testing using the Kolmogorov-Smirnov test. A p < 0.01
or 0.05 was considered as a significant difference.
3. Results
Figure 1 shows the uptake of [123 I]MIBG by HEK293 cells expressing different SLC
transporters and the inhibition of uptake by cimetidine and L-carnitine. The function of the
SLC transporters in HEK293 cells was confirmed using p-[14 C]aminohippuric for OATP
and [3 H]methyl-4-phenylpyridinium for OCTN and OCT. [123 I]MIBG uptake was significantly higher in HEK293/OCTN1, HEK293/OCTN2, HEK293/OCT1, HEK293/OCT2,
and HEK293/OCT3 cells than in mock cells. Uptake of [131 I]MIBG by HEK293/OCT1,
HEK293/OCT2, and HEK293/OCT3 cells was significantly inhibited only by cimetidine,
whereas L-carnitine significantly inhibited [131 I]MIBG uptake by HEK293/OCTN1 andHEK293/OCTN2 cells.
The biodistribution in normal mice, DSS-induced experimental colitis mice, andmice with oral loading of cimetidine with [123 I]MIBG is shown in Table 1. In all mice,[123 I]MIBG accumulated maximally in the stomach and kidney at 5 min and was thenwashed out. The accumulation of [123 I]MIBG in the liver and bladder increased gradually
over time. There was no diiodine because of little thyroid accumulation. Compared withnormal mice, both DSS-induced experimental colitis mice and mice with cimetidine loading
showed significantly lower accumulation in the blood at 5, 10, and 30 min, in the heart
at 5, 10, and 30 min for DSS-induced experimental colitis mice and 5 min for mice withcimetidine loading, in the liver at all time points for DSS-induced experimental colitis
mice and at 5 and 10 min for mice with cimetidine loading, and in the bladder at alltime points for DSS-induced experimental colitis mice and at 5 and 10 min for mice withcimetidine loading.
SPECT images of [123 I]MIBG in normal and DSS-induced experimental colitis miceat 10, 30, and 60 min after oral administration of [123 I]MIBG are shown in Figure 2. A
visually notable difference was observed in the bladder between normal and DSS-induced
experimental colitis mice. Accumulation of [123 I]MIBG increased in the small intestine at
5–10 min and 30–35 min after oral administration.

Pharmaceutics 2022,
2022, 14,
14, 61x FOR PEER REVIEW
Pharmaceutics

55 of
of 11
10

*

*
350

Uptake (%ID/mg protein)Control for OATP
300
250

*

*

Control for OCTN
and OCT**

Cimetidine
200

*

*

*
*

L-Carnitine

150
100
50

0
Mock

OATP2B1

OCTN1

OCTN2

OCT1

OCT2

OCT3

Uptakeofof[123[123
I]MIBG
humanembryonic
kidney(HEK)293
expressing
a solute
Figure 1. Uptake
I]MIBG
byby
human
embryonic
kidney(HEK)293
cellscells
expressing
a solute
car123 I]MIBG,
riertransporter(n =(n4).
Beforethetheexperimentswith[123[I]MIBG,
thethefunctionof SLCtransportersin
carriertransporter= 4).
Beforeexperimentswithfunctionof SLCtransporters
3H-3 or 14C-substrates.
3 H-labeled
HEK293cells
wasconfirmed
using
Uptakeof theH-labeledsubstrates
into
in HEK293
cells
wasconfirmedusing
H- or 14 C-substrates.
Uptakeof 3thesubstrates
HEK293
mockmockcells cells
as control
cells cells
is 107.1± 18.5%ID/g
proteinprotein
for HEK-Mockcells fororganic
anion
into HEK293
as control
is 107.1± 18.5%ID/g
for HEK-Mockcells
for organic
transporting
polypeptide(OATP),
84.9 ±84.9
22.7%ID/g
proteinfor HEK-Mockcells cells
for organic
catanion transporting
polypeptide(OATP),± 22.7%ID/g
proteinfor HEK-Mockfor organicion/carnitine transporter (OCTN) and organic cation transporter (OCT). On the other hand, uptake
cation/carnitine transporter (OCTN) and organic cation transporter (OCT). On the other hand,of the 3H- or 14C-labeled
substrates into HEK293 cells expressing a SLC transporter is as follows: 363
uptake of the 3 H- or 14 C-labeled substrates into HEK293 cells expressing a SLC transporter is as± 38.6%ID/g protein for HEK293-OATP2B1, 387 ± 44.1%ID/g protein for HEK293-OCTN1, 379 ±follows: 363± 38.6%ID/g
protein for HEK293-OATP2B1,
387 for± 44.1%ID/g
protein
HEK29341.6%ID/g
proteinfor HEK293-OCTN2,
403 ± 48.1%ID/g protein
HEK293-OCT1,
429for
± 58.5%ID/g
123
OCTN1,
379±
41.6%ID/g
proteinfor
HEK293-OCTN2,
403±
48.1%ID/g
proteinfor
HEK293-OCT1,
protein for HEK293-OCT2, and 346 ± 37.1%ID/g protein for HEK293-OCT3. [ I]MIBG uptake is429 ± 58.5%ID/g
for HEK293-OCT2,
and 346 ± 37.1%ID/g
proteinHEK293/OCT2,
for HEK293-OCT3.
significantly
higherprotein
in HEK293/OCTN1,
HEK293/OCTN2,HEK293/OCT1,
and123
HEK293/OCT3
cellsisthan
in mockhigher
cells. Cimetidine
inhibits HEK293/OCT1,
HEK293/OCT2,
and[ I]MIBG uptake
significantly
in HEK293/OCTN1,
HEK293/OCTN2,HEK293/OCT1,
HEK293/OCT3,
inhibits
HEK293/OCTN1
and HEK293/OCTN2.
* pHEK293/OCT1,< 0.01 vs. mock
HEK293/OCT2,and
andL-carnitine
HEK293/OCT3
cells
than in mock cells.
Cimetidine inhibits
cells
and
HEK293cells
loadedwithbothinhibitors.
HEK293/OCT2, and HEK293/OCT3, and L-carnitine inhibits HEK293/OCTN1 and HEK293/OCTN2.
* p < 0.01 vs. mock cells and HEK293 cells loaded with both inhibitors.

Mice Types

Normal mice

DSS-induced
experimental
colitis mice

The biodistribution in normal mice, DSS-induced experimental colitis mice, and micewith oralloading of cimetidinewith [123I]MIBGradioactivity
is shown in following
Table 1. Inoral
all mice,[123I]MIBG
Table
1. Biodistributionof [123 I]MIBG-associatedadministrationinaccumulated
maximally experimental
in the stomachandkidneyat 5withminoralandcimetidine
was thenloading.
washed out.
normal mice, DSS-induced
colitismice,
and mice
The accumulation of [123I]MIBG in the liver and bladder increased gradually over time.
Timebecause
after Oralof[123I]MIBG
Administration
There was no diiodine
littlethyroid
accumulation. Compared with normal
mice,
both
DSS-induced
experimental
colitismiceand30mice
Organ (%ID/g)
5 min
10 minmin with cimetidine
60 minloading
showed significantly
lower accumulationin the blood
at 5, 10, and 30 min,in the heart at
Blood
2.40 ± 0.41
2.11 ± 0.45
1.92 ± 0.52
1.61 ± 0.43
5,
10,
and
30
min
for
DSS-induced
experimental
colitis
mice
and
5
min
for
Thyroid0.05 ± 0.01
0.08 ± 0.03
0.08 ± 0.04
0.10 ±mice
0.04 with cimetidine loading,in the liver at1.97
all time
experimental
colitis miceHeart
2.02 ± 0.36
± 0.52points for DSS-induced
1.65 ± 0.48
1.31 ± 0.43
Stomach and at 5 and45.8
3.22
41.9cimetidine± 3.8438.9and± 3.11
36.1
10 ±
min
for mice withloading,in the bladderat ±all2.63
time points
Liver
1.74 ± experimental
0.59
2.74
± 0.620.93
3.92± 0.81for DSS-induced
colitismice and at 53.21
and±10min for micewithcimetidine
Kidney1.14 ± 0.31
1.01 ± 0.38
0.72 ± 0.42
0.51 ± 0.39
loading.
Bladder

10.1 ± 2.31

16.3 ± 3.05

21.2 ± 3.94

38.2 ± 4.31

Blood
ThyroidHeart
StomachLiver
KidneyBladder

1.39 ± 0.19 *0.06 ± 0.03
0.91 ± 0.11 *85.1 ± 9.22 *0.81 ± 0.15 *1.59 ± 0.47 †2.41 ± 0.36 *1.73 ± 0.48 †0.09 ± 0.04
1.35 ± 0.22 *82.9 ± 10.84 *1.25 ± 0.27 *1.28 ± 0.41
4.39 ± 0.85 *1.58 ± 0.39 *0.11 ± 0.03
1.21 ± 0.18 *78.5 ± 9.43 *1.68 ± 0.31 *0.88 ± 0.54
13.8 ± 2.53 *1.42 ± 0.43
0.10 ± 0.03
0.81 ± 0.11
77.4 ± 9.89 *2.12 ± 0.53 *0.60 ± 0.41
21.8 ± 3.89 *Pharmaceutics 2022, 14, 61

6 of 10

Table 1. Cont.
Time after Oral [123 I]MIBG Administration

Mice Types
Organ (%ID/g)

Cimetidine
loading mice

Blood
ThyroidHeart
StomachLiver
KidneyBladder

5 min

10 min30 min2.14 ± 0.30 †1.89 ± 0.23 †1.64 ± 0.34 †0.03 ± 0.01
1.75 ± 0.34 †58.9 ± 4.44 †1.39 ± 0.48 †1.51 ± 0.35 †7.90 ± 1.53 †0.05 ± 0.02
1.91 ± 0.45
49.4 ± 4.93 †2.46 ± 0.59 †1.10 ± 0.45
10.5 ± 2.95 †0.08 ± 0.03
1.68 ± 0.50
41.8 ± 4.12
3.18 ± 0.87
0.79 ± 0.46
18.5 ± 4.51

60 min1.49 ± 0.41
0.10 ± 0.03
1.40 ± 0.52
38.8 ± 3.91
3.88 ± 0.97
0.58 ± 0.37
32.9 ± 5.09

Pharmaceutics 2022, 14, x FOR PEER REVIEW
7 of 11%ID/g indicates the percent injected dose per gram of tissue. Values are the mean ± standard deviation obtained
from three mice. * p < 0.01 and † p < 0.05 compared with normal mice.

Esophagus
StomachNormalBladder

DSS

5-10 min30-35 min

55-60 minFigure 2. Representative [123I]MIBG single-photon emission computed tomography (SPECT) imaging
Figure2. Representative[ I]MIBG
single-photon
emissioncomputed
tomography(SPECT)imagin normal
and dextran sodium
sulfate(DSS)−induced
experimental
colitismice at 5–10
min, 30–35
ing
innormalanddextransodiumsulfate(DSS)−
inducedexperimental
colitis
mice
at
5–10
min,
123
min, and 60–65 min after oral administration of approximately 20 MBq of [ I]MIBG. DSS-induced
123 I]MIBG. DSS123
30–35
min,
and
60–65
min
after
oral
administrationofapproximately
20
MBq
of
[
experimental colitis mice show decreased accumulation of [ I]MIBG in the bladder at 30–35 min
inducedmice administration
show decreased
accumulationof [123 I]MIBG
in thebladderatand 60–65experimental
min after oralcolitis[123I]MIBG
andincreased accumulationin thesmall
intestine
123I]MIBG administration.
123
at
5–10
min
and
30–35
min
after
oral
[
30–35 min and 60–65 min after oral [administration and increased accumulation in thesmall intestine at 5–10 min and 30–35 min after oral [123 I]MIBG administration.
123

Time-activity curves generated from the SPECT images of control and DSS-induced
experimental colitis mice orally injected with [123I]MIBG is shown in Figure 3. In DSS-induced experimental colitis mice, accumulation of [123I]MIBG was significantly lower in theheart, liver, and bladder compared with normal mice, whereas [123I]MIBG accumulation
was higher in the stomach at the early time points after oral administration.

Pharmaceutics 2022, 14, 61

7 of 10Time-activity curves generated from the SPECT images of control and DSS-induced
experimental colitis mice orally injected with [123 I]MIBG is shown in Figure 3. In DSSinduced experimental colitis mice, accumulation of [123 I]MIBG was significantly lower in
Pharmaceutics 2022, 14, x FOR PEER REVIEW
8 of 11the heart, liver, and bladder compared with normal mice, whereas [123 I]MIBG accumulation
was higher in the stomach at the early time points after oral administration.

Figure 3. Time activity curves for the heart (a), stomach (b), liver (c), and bladder (d) in SPECT
Figure 3. Time activity curves for the heart (a), stomach (b), liver (c), and bladder(d) in SPECT
images of control and DSS-induced experimental colitis mice orally injected with [123I]MIBG.
In DSS123 I]MIBG. In
imagesofcontroland
DSS-induced
experimental
colitismiceorallyinjectedwith[123
induced experimental colitis mice, accumulation of [ I]MIBG is significantly lower in the heart,
DSS-induced
experimental
colitis mice,of [123 I]MIBG
is significantly
lower inliver, and bladderthan in normal
mice,accumulation
whereas accumulation
is higher
in the stomach
inthetheheart,
early
123
†
liver,
andbladderthaninnormalmice,
whereasaccumulationishigherinthestomach
in
the
early
time points after oral [ I]MIBG administration. * p < 0.01 and p < 0.05 between normal and DSS modeltime
mice.points after oral [123 I]MIBG administration. * p < 0.01 and † p < 0.05 between normal and DSS
model mice.

HE staining in conjunction with anti-OCTN1/2 immunohistochemical analysis was
HE staining
in conjunction
withintestine,
anti-OCTN1/2
immunohistochemical
analysiswasperformed
on tissue
from the smalllarge intestine,
liver, and kidney(Figure4).
performed
ontissue
from
the
small
intestine,
large
intestine,
liver,
andkidney(Figure4).
The DSS-induced experimental colitis mice exhibited lower OCTN1/2 expression (purple
The
DSS-induced
colitismice
exhibitedlower
expression(purpledots)
than controlexperimental
mice in the small
and
large
intestines.
DSSOCTN1/2
had no notableeffects
on thedots)
thancontrol
mice
in
the
small
and
large
intestines.
DSS
had
no
notableeffectsontheliver and kidney.
liver and kidney.

Pharmaceutics 2022, 14, x FOR PEER REVIEW
Pharmaceutics 2022, 14, 61

9 of 118 of 10Figure 4. Anti-OCTN1/2 immunohistochemical analysis in the small intestine, large intestine, liver,
Figure4. Anti-OCTN1/2
immunohistochemical
analysisThein thesmall intestine,large intestine,
liver,
andkidney.
Purple indicates
expression of OCTN1/2.
DSS-induced
experimental
colitis miceandkidney.
Purpleindicates
expressionof
OCTN1/2.
The
DSS-induced
experimental
colitisshow relatively lower expression of OCTN1/2 than control mice in the small and large intestine. mice
DSS
show relatively lower expression of OCTN1/2 than control mice in the small and large intestine. DSS
has no effect on the liver and kidney.
has no effect on the liver and kidney.

4. Discussion
4. Discussion
In this study, [123 I]MIBG was administered orally to estimate the gastrointestinal
this study,I]MIBGanticancer
was administeredorallyto estimate
thesmallgastrointestinal
tract
tract In
absorptionof[123
cationic
drugs andmedicines
in theintestine. Thisabsorptionofcationic
anticancer
drugs
and
medicines
in
the
small
intestine.
Thisisimis important because individual patients can exhibit differences in gastrointestinal tract
portant
becauseindividual
patients
canexhibitdifferencesingastrointestinal
tract
absorpabsorption, which can affect the activity of cationic anticancer drugs and medicines. Antion, which
can affectactivityof cationicdrugs tract
and medicines.
imaging
methodthat theenablesestimationof anticancer
gastrointestinal
absorptionAnof imaging
cationic
method thatenablesof thegastrointestinal
tract
absorptionof usefulcationic
anticancer
anticancer
drugs
and estimation
medicines insmall intestine
would
be a veryclinical
tool.
drugs
and
medicines
in the small intestine
would be a very useful[123I]MIBG
is a water-soluble,
cationic radiopharmaceutical
thatclinicalis takentool.
up by various[123viaI]MIBG
is a water-soluble,
cationic
radiopharmaceuticalis taken
up byatvaritissues
OCT [3,4].
We hypothesizedthat OCTN1/2
and OCT1-3 thatfunctionprimarilythesmall
intestinal
epithelial
in theof cationicdrugs function
and medicines[9].
ous tissues
via OCT[3,4]. cell
We membrane
hypothesized
thatuptake
OCTN1/2
and OCT1-3
primarily
123 I]MIBG exhibited a high affinity for OCTN1/2 and OCT1-3 (Figure 1). We,
Indeed,[at the small intestinal epithelial cell membrane in the uptake of cationic drugs and meditherefore,
expect thatoral administrationof affinity[123 I]MIBG
would enable
the estimationofcines [9]. Indeed,[123I]MIBG
exhibited a highfor OCTN1/2
and OCT1-3(Figure 1).
gastrointestinal
tract absorptionof cationic drugsofand
medicines
in theenable
small theintestine.
DSSWe, therefore, expectthat oral administration[123I]MIBG
wouldestimationinducedexperimental
mice exhibit
variationsin theexpressionof OCTN,
mainlyof gastrointestinal
tractcolitis
absorptionof cationicdrugs andmedicines
in thesmall intestine.
OCTN1,
at theexperimental
basolateral sideon theof small intestinal
epithelialofcells[16].
DSS-induced
colitismicemembrane
exhibit variationsin the expression
OCTN,
Changesintheexpression
levelsof
OCTN1/2
inducedbyDSS
wereevaluatedthroughmainly OCTN1, at the basolateral side on the membrane of small intestinal epithelial cells
anti-OCTN1/2
analysis(Figure 4)induced
because by
OCTN
the basolateral[16]. Changes immunohistochemicalin the expression levelsof OCTN1/2
DSSonwere
evaluated
side
is
more
important
than
OCT
on
the
apical
side
in
the
small
intestine
to estimate
through anti-OCTN1/2 immunohistochemical analysis (Figure 4) because OCTN
on the123 I]MIBG. There was
gastrointestinal
absorption
followingoralonadministrationbasolateral side tract
is moreimportant
than OCT
the apical sideofin[ thesmall intestine toanincreasein the number tractof purple
dots indicatingof OCTN1/2
the
small
estimate
gastrointestinal
absorption
followingexpression
oral administration
ofin[123I]MIBG.
andlarge
intestinesof
DSS-induced
experimental
colitismicecomparedwithnormalmice.
There was an increase in the number of purple dots indicating expression of OCTN1/2
In
addition,thecollapseofthemucosalstructureandinfiltrationof
inflammatory
in the small and large intestines of DSS-induced experimental colitis mice compared cells
withwasconfirmed
both thethe
small
and large
Thus, DSS-induced
experimental
normalmice. Ininaddition,
collapseof theintestines.
mucosal structure
and infiltrationof inflamcolitis
relativelyinincreased
OCTN1
and OCTN2
tomatorymice
cellsshowed
was confirmed
both theexpression
small and oflarge
intestines.
Thus, compared
DSS-induced
control
mice.
experimental colitis mice 123
showed relatively increased expression of OCTN1 and OCTN2Visualinspectionof[compared to control mice. I]MIBG SPECT images (Figure 2) indicated reduced accumulation in
the bladderof DSS-induced
experimental
colitismice2)comparednormalmice.
Visualinspectionof [123I]MIBG
SPECT images(Figureindicatedwith
reducedaccumu123 I]MIBG administration (Figure 3) indicated significantly
Time-activity
curvesoforal[lation in the bladder of DSS-induced experimental colitis mice compared with normal
lower accumulation of [123 I]MIBG in123the heart, liver, and bladder of DSS-induced experimice. Time-activity curves of oral [ I]MIBG administration (Figure 3) indicated signifimental colitis mice compared withnormal mice. In contrast, accumulation was higher incantly lower accumulation of [123I]MIBG in the heart, liver, and bladder of DSS-induced
the stomach, because DSS reduces gastrointestinal tract absorption via OCTN1/2 at theexperimental colitis mice compared with normal mice. In contrast, accumulation wasbasolateral side on the small intestinal epithelial cell membrane.

Pharmaceutics 2022, 14, 61

9 of 10In the biodistribution study of oral [123 I]MIBG administration in normal mice, DSSinduced experimental colitis mice, and cimetidine loading mice (Table 1), a significant
decrease in radioactivity was observed in the blood, heart, liver, and bladder of DSSinduced experimental colitis mice and cimetidine loading mice compared with normal
mice 5 min after oral [123 I]MIBG administration, whereas the radioactivity increased in
the stomach and kidney. Because the kidney expresses high levels of OCTN and OCT [5],the accumulation of radioactivity in the kidney should be increased in DSS-induced experimental colitis mice and mice with cimetidine loading. Thus, we considered that the
cationic drug uptake activity of OCTN and OCT in the small intestine was inhibited by theeffects of DSS and cimetidine, which inhibit OCT. As mentioned above, the accumulationof [123 I]MIBG-associated radioactivity was also significantly lower in the heart, liver, andbladder in the DSS-induced experimental colitis mice compared with normal mice, as determined from time-activity curves generated based on [123 I]MIBG SPECT images (Figure 2).
A marked difference in uptake in the bladder between normal mice and DSS-induced
experimental colitis mice was particularly notable.
Although intravenous administration of [131 I]MIBG for radionuclide therapy has noserious hepatic or renal toxicities, side effects include mild nausea and transient elevationof arterial blood pressure [19]. Because [123 I]MIBG has the same biological distribution
as [131 I]MIBG, oral administration of [123 I]MIBG will not have serious side effects. As alimitation, the tight junction may have an effect on gastrointestinal tract absorption oforally administered [123 I]MIBG [20]. However, OCTNs and/or OCTs were involved in
gastrointestinal tract absorption of [123 I]MIBG in a mouse study, because the effect of DSS
to change to lower OCTN expression and that of cimetidine loading to inhibit OCT wereconfirmed (Table 1). Therefore, gastrointestinal tract absorption via OCTN and/or OCT in
the small intestine can be estimated by measuring time-activity curves for the heart, liver,and bladder after oral [123 I]MIBG administration.
5. Conclusions[123 I]MIBG imaging after oral administration can be used to estimate gastrointestinal
tract absorption via OCTN and/or OCT in the small intestine based on analysis of timeactivity curves for the heart, liver, and bladder.
Author Contributions: Conceptualization, M.K. and K.K.; methodology, M.K.; software, A.M.;
validation, Y.M., K.N. and H.K.; formal analysis, M.K.; investigation, R.N.; resources, H.K., T.N. and
I.T.; data curation, N.S.; writing—original draft preparation, M.K.; writing—review and editing,
M.K.; visualization, M.K., A.M. and Y.M.; supervision, K.K.; project administration, R.N.; funding
acquisition, M.K., R.N. and K.K. All authors have read and agreed to the published version of
the manuscript.
Funding: This study was funded in part by Grants-in-Aid for Scientific Research from the Japan
Society for the Promotion of Science (nos. 16KK0200, 16H05397 and 18K07747) and the Network-type
Joint Usage/Research Center for Radiation Disaster Medical Science and The Mitani Foundation for
Research and Development.
Institutional Review Board Statement: The animal study protocol was approved by the Institutional
Review Board of Kanazawa University (protocol code AP-194046 and date of approval 17 March 2021).
Informed Consent Statement: Informed consent was not required because this was not a clinical study.
Acknowledgments: The authors would like to thank Mikie Ohtake and other staff of the Faculty of
Health Sciences, Kanazawa University.
Conflicts of Interest: There are no conflict of interest to disclose.

Pharmaceutics 2022, 14, 61

10 of 10

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