www.nature.com/scientificreports

Corrected: Author Correction

OPEN

Received: 8 March 2019
Accepted: 3 September 2019
Published online: 16 September 2019

Imaging of hepatic drugtransporters with [131I]6-βiodomethyl-19-norcholesterol
Masato Kobayashi1, Kodai Nishi2, Asuka Mizutani1, Tsuzumi Hokama1, Miki Matsue1,
Tetsuya Tsujikawa3, Takeo Nakanishi4,5, Ryuichi Nishii6, Ikumi Tamai4 & Keiichi Kawai1,3
We examined whether [131I]6-β-iodomethyl-19-norcholesterol (NP-59), a cholesterol analog, can beused to measure function of hepatic drug transporters. Hepatic uptake of NP-59 with and without
rifampicin was evaluated using HEK293 cells expressing solute carrier transporters. The stability of
NP-59 was evaluated using mouse blood, bile, and liver, and human liver S9. Adenosine triphosphatebinding cassette (ABC) transporters for bile excretion were examined using hepatic ABC transporter
vesicles expressing multidrug resistance protein 1, multidrug resistance-associated protein (MRP)1-4,
breast cancer resistance protein (BCRP), or bile salt export pump with and without MK-571 and Ko143.
Single photon emission computed tomography (SPECT) was performed in normal mice injected with
NP-59 in the presence or absence of Ko143. Uptake of NP-59 into HEK293 cells expressing organic
anion transporting polypeptide (OATP)1B1 and OATP1B3 was significantly higher than that into mockcells and was inhibited by rifampicin. NP-59 was minimally metabolized in mouse blood, bile, andliver, and human liver S9 after 120 min of incubation. In vesicles, NP-59 was transported by MRP1 and
BCRP. Excretion of NP-59 into bile via BCRP was observed in normal mice with and without Ko143 in
the biological distribution and SPECT imaging. NP-59 can be used to visualize and measure the hepaticfunction of OATP1B1, OATP1B3, and BCRP.
Nuclear medicine imaging may potentially be used to directly detect and quantify the activity of drug transporters
involved in drug clearance. In addition, imaging holds great promise in personalized medicine because it allows
for characterization of individual patients before treatment initiation for a certain molecular target1,2. In clinicalstudies, [99mTc]2-methoxyisobutylisonitrile and [99mTc]tetrofosmin have been used for imaging of multiple drug
resistance transporters in cancer before chemotherapy treatment3. For improved personalized medicine, accurate
measurement of not only the drug resistance transporters but also the function of hepatic transporters is important because of inter-individual variability in plasma pharmacokinetics that controls drug clearance.
In the human liver, organic anion transporting polypeptide (OATP)1B1 (SLCO1B1), OATP1B3 (SLCO1B3),
OATP2B1 (SLCO2B1), organic anion transporter (OAT)2 (SLC22A7), organic cation transporter (OCT)1(SLC22A1), and Na+-taurocholate cotransporting polypeptide (NTCP, SLC10A1) isoforms are mainly expressedat the sinusoidal membrane as solute carrier (SLC) transporters4,5. On the other hand, adenosine triphosphate(ATP)-binding cassette (ABC) transporters are mainly located on the bile canalicular or basolateral membrane in
the liver. ABC transporters include multidrug resistance protein 1 (MDR1, P-glycoprotein, ABCB1 gene), multidrug resistance-associated protein 2 (MRP2, ABCC2), breast cancer resistance protein (BCRP, ABCG2), and bile
salt export pump (BSEP, ABCB11) on the bile canalicular membrane and MRP1 (ABCC1), MRP3 (ABCC3), and
MRP4 (ABCC4) on the basolateral hepatocyte membrane4,5.
During hepatobiliary scintigraphy with single photon emission computed tomography (SPECT), [99mTc]
diisopropyl iminodiacetic acid ([99mTc]disofenin) undergoes high hepatic extraction via human OATPs6,7 and1

School of Health Sciences, Institute of Medical, Pharmaceutical and Health Sciences, Kanazawa University,
Ishikawa, Japan. 2Department of Radioisotope Medicine, Atomic Bomb Disease Institute, Nagasaki University,
Nagasaki, Japan. 3Biomedical Imaging Research Center, University of Fukui, Fukui, Japan. 4School of Pharmaceutical
Sciences, Institute of Medical, Pharmaceutical and Health Sciences, Kanazawa University, Ishikawa, Japan. 5Facultyof Pharmacy, Takasaki University of Health and Welfare, Gunma, Japan. 6Department of Molecular Imaging andTheranostics, National Institute of Radiological Sciences, Chiba, Japan. Ryuichi Nishii and Masato Kobayashi
contributed equally. Correspondence and requests for materials should be addressed to M.K. (email: kobayasi@mhs.
mp.kanazawa-u.ac.jp)
Scientific Reports | (2019) 9:13413 | https://doi.org/10.1038/s41598-019-50049-8

1

www.nature.com/scientificreports/

www.nature.com/scientificreports

rapid bile canalicular excretion via human MRP1 and MRP28. [99mTc]3-bromo-2, 4, 6-trimethyl iminodiacetic
acid ([99mTc]mebrofenin) is taken up into the liver by human OATP1B1 and OATP1B39,10, and is then excretedby MRP211. Among the [99mTc]pyridoxylaminates, [99mTc]N-pyridoxyl-5-methyl-tryptophan is transported byhuman OATP1B1 and OATP1B3 and excreted by MDR1 and MRP212. Clinical hepatobiliary SPECT scintigraphyrarely involves BCRP and/or BSEP. On the other hand, some positron emission tomography (PET) agents with
11
C and 18F labeling are also delivered via drug transporters13,14. However, 11C-labeled PET agents cannot be usedin general imaging centers because a cyclotron is required for synthesis of the PET agents.
Some glucocorticoid drugs and sulfate conjugates of estradiol and estrone, which are biosynthesizedfrom cholesterol, are transported via BCRP15. In clinical nuclear medicine imaging, [131I]6-β-iodomethyl19-norcholesterol ([131I]adsterol, [131I]NCL-6, NP-59), which is a cholesterol analog, is used to localize adrenal
cortical lesions16,17. NP-59 is most useful for evaluation of incidentally detected adrenal adenomas and hyperaldosteronism. When considering the whole-body distribution, NP-59 prominently accumulates in the liver, and isthen excreted into bile18. In this study, we examined if NP-59 was useful for measurement of the function of thehepatic BCRP transporter.

Materials and Methods

Materials. NP-59 was purchased from FUJIFILM Toyama Chemical Co., Ltd. (Tokyo, Japan). For evaluationof SLC transporters, rifampicin was purchased from Nacalai Tesque (Kyoto, Japan). For evaluation of ABC transporters, MK-571 sodium salt and Ko143 were purchased from Cayman Chemical (Ann Arbor, MI, USA) andSigma-Aldrich (St. Louis, MO, USA), respectively. Human liver S9 was purchased from Corning Gentest (NewYork, NY, USA).
Cells and vesicles.

To investigate SLC transporters, human embryonic kidney (HEK)293 cells expressing human OATP1B1, OATP2B1, OATP1B3, OAT2, OCT1, or NTCP plasmid vector alone were prepared as
described previously19. Briefly, HEK293 cells were transfected with the respective plasmid DNA, and were thenselected with the appropriate antibiotics; cells were designated HEK293/OATP1B1, HEK293/OATP2B1, HEK293/
OATP1B3, HEK293/OAT2, HEK293/OCT1, and HEK293/NTCP, respectively. We also tested mock-transfected
cells. All cell lines were grown in Dulbecco’s modified Eagle medium (Wako Pure Chemical Industries Ltd.,
Osaka, Japan) supplemented with 10% (v/v) fetal bovine serum (Life Technologies, Carlsbad, CA, USA), 100 U/ml
penicillin, and 100 μg/ml streptomycin at 37 °C in a 5% CO2 incubator.
To investigate ABC transporters, we used vesicles (GenoMembrane Inc., Kanagawa, Japan) without (control)
and with high expression of human MDR1, MRP1, MRP2, MRP3, MRP4, BCRP, or BSEP. Experimental kits werealso purchased from GenoMembrane Inc. and were used for experiments testing each ABC transporter.

Partition coefficients.

All our methods were performed in accordance with the relevant guidelines andregulations. Partition coefficients of NP-59 were measured using 2.0 ml n-octanol as the organic phase and 2.0 ml
0.1 M phosphate buffer (pH 7.4 for plasma) as the aqueous phase. n-Octanol and buffer were pre-mixed twice
using a mechanical mixer for 1 min at room temperature. Then, 20 μl NP-59 in saline was added and mixed twicefor 1 min at room temperature. Samples were centrifuged, and then the radioactivity of 200 μl of each phase wasmeasured using a gamma counter. The partition coefficient was calculated by log(counts per min in n-octanol
phase/counts per min in phosphate buffer). The partition coefficient of N-isopropyl-p-[123I]iodoamphetamine(123I-IMP) was used as a reference because 123I-IMP has high lipophilicity20.

Transport assays with HEK293 cells and vesicles.

Transport assays were performed according to ourpreviously described method12. Before the transport assays with NP-59, we confirmed the transporter activity in
HEK293 cells expressing each SLC transporter using [6,7-3H(N)]estrone-3-sulfate (PerkinElmer Inc., Waltham,
MA, USA, 2.04 GBq/mmol, 2.22 TBq/mmol) for OATP1B1, OATP2B1, and OATP1B3; p-[14C]aminohippuric
acid (PerkinElmer Inc., 2.04 GBq/mmol) for OAT2; [3H]methyl-4-phenylpyridinium (American Radiolabeled
Chemicals Inc., St. Louis, MO, USA, 2.96 TBq/mmol) for OCT1; and [3H(G)]taurocholic acid (PerkinElmer Inc.,
37 GBq/mmol) for NTCP. One day before the uptake experiments, HEK293 cells expressing a SLC transporter
were plated at 4 × 105 cells/well in 12-well plastic dishes. Cells were pre-incubated for 10 min in modified Hank’sbalanced salt solution. Then, 37 kBq NP-59 with or without the OATP inhibitor, 1.0 mM rifampicin, was added toeach well. At the end of the incubation, each well was rapidly washed twice with 1 ml ice-cold incubation medium.
The cells were then solubilized in 0.5 ml 0.1 N NaOH, and radioactivity in an aliquot of the solution was measured
with a gamma counter (AccuFLEXγ7000, Aloka, Tokyo, Japan). Subsequently, the protein concentration in therest of the solution was measured using a BCA Protein Assay Kit (Thermo Fisher Scientific, Kanagawa, Japan).
The accumulation rates of NP-59 in HEK293 cells were calculated as % injected dose (%ID) /g protein. Uptake of
NP-59 into HEK293 cells expressing SLC transporters was compared with that in mock-transfected HEK293 cells.
Assays were performed in octuplicate (n = 8 per HEK293 cells expressing each SLC transporter).
Excretion into bile was represented using ABC transporter vesicles transfected with MDR1, MRP2, BCRP, or
BSEP. Excretion from liver into the blood was represented using ABC transporter vesicles transfected with MRP1,
MRP3, or MRP4. Vesicles were pre-incubated for 10 min in reaction buffer. Each vesicle solution was incubatedwith NP-59 (37 kBq) for 5 min with adenosine triphosphate (ATP), which supplies energy for ABC transporters,or adenosine monophosphate (AMP), which does not provide energy and was used for comparison to ATP. At theend of the incubation, the vesicle solution was filtered through nitrocellulose filters, and the filters were rapidly
washed twice with 1 ml ice-cold incubation medium. The vesicles were then solubilized in 0.5 ml 0.1 N NaOH,
and the radioactivity was measured using a γ-ray counter (AccuFLEXγ7000, Aloka, Tokyo, Japan). Export of
NP-59 from the vesicles into the ATP solution was compared with that in AMP solution. For the competitive
inhibition assay, the vesicles were incubated with NP-59 for 5 min in the presence of inhibitor: final concentration
Scientific Reports | (2019) 9:13413 | https://doi.org/10.1038/s41598-019-50049-82

www.nature.com/scientificreports/

www.nature.com/scientificreportsof 50 µM MK-571 sodium salt for MRP and 50 µM Ko143 for BCRP21–23. At the end of the incubation, the vesicle
solution was filtered through nitrocellulose filters, and the filters were rapidly washed twice with 1 ml ice-cold
incubation medium. The vesicles were then solubilized in 0.5 ml 0.1 N NaOH, and radioactivity and protein concentration were measured as above. Assays were performed in octuplicate (n = 8 per vesicles expressing each
ABC transporter). The accumulation rates of the vesicles of NP-59 were calculated as nmol/mg protein using thespecific radioactivity of NP-59.

Stability of NP-59 in mouse liver, mouse blood, and human liver S9.

All animal studies were conducted following approval by the Animal Care Committee of Kanazawa University (AP-122339). Three ddy normal mice each (total of nine mice) were killed at 10 min, 30 min, and 60 min after injection of 370 kBq NP-59.
After blood sampling via cardiocentesis, mouse livers and gall bladders were removed. Metabolites in mouseplasma, bile, and liver were analyzed with thin layer chromatography (TLC).
Human liver S9 was also used for metabolite analysis. Briefly, Krebs-Ringer phosphate buffer (pH 7.4) was
added to the samples, followed by homogenization with an ultrasonic homogenizer (SONIFIER250, Branson,
MO, USA). Then, ethanol was added to the homogenate to remove proteins, and the sample including blood wascentrifuged for 5 min at 18,000 × g. The final supernatant was spotted onto the TLC plate, and the TLC spots weredeveloped using hexane:ether:ethyl acetate at a ratio of 1:1:424. After development and complete drying, the TLC
plates were cut into 20 fractions, and the radioactivity associated with each fraction was measured using a γ-ray
counter. The fractional ratios of metabolites were calculated by dividing the radioactive counts per minute (cpm)
for each fraction by the total cpm.

Biological distribution and SPECT imaging of NP-59 with and without Ko143 loading in normal
mice. For the distribution experiments, normal ddy normal mice (male, 6 weeks old) were housed with a

12-hour light/12-hour dark cycle with free access to only water. Mice that had fasted all night were administered
NP-59 via the tail vein (18.5 kBq/mouse). At 5, 10, 20, 30, 60, and 120 min, normal mice were euthanized underisoflurane (n = 5) anesthesia, and the following tissues were collected: blood, brain, thyroid, lung, heart, stomach, liver, gall bladder, small intestine, large intestine, kidney, adrenal gland, and bladder. Tissues were weighed,and radioactivity was quantified using a γ-ray counter to calculate the percent injected dose (%ID, radioactive
cpm in tissue sample divided by cpm of injected radioactivity × 100) or percent injected dose per gram of tissue(%ID/g). Liver-to-blood, gall bladder-to-blood, and kidney-to-blood ratios were calculated by dividing %ID/g
of liver, gall bladder, and kidney, respectively, by %ID/g of blood. For the inhibition study, mice were injectedwith about 1.0 mM (2.0 μg/g weight/100 μl) Ko143 immediately before injection of NP-5922. At 5, 10, and 30 min
after injection, mice were euthanized under isoflurane (n = 5) anesthesia, and the same tissues were collected
and quantified. Liver-to-blood, gall bladder-to-blood, and kidney-to-blood ratios were also calculated as above.
SPECT acquisition with a single pinhole collimator was started at 5 min after an intravenous bolus administration of NP-59 (about 5.0 MBq) using a micro-injection pump CFX1010 (ISIS, Osaka, Japan) for 15–20 sec
into the tail vein of normal mice (n = 5) under 1.2–1.5% isoflurane anesthesia (Abbot Laboratories, Green Oaks,
IL, USA) and continued for about 20 min with the step-and-shoot mode (20 sec/step) using a small animal
SPECT/PET/computed tomography (CT) imaging scanner (TriFoil Imaging, Inc., Northridge, CA, USA). After
SPECT imaging, CT scans (75 kV and 130 μA) were performed. The data were reconstructed using the maximum
likelihood-expectation maximization with six iterations including attenuation and no scatter correction. The
voxel size was set to 0.53 × 0.53 × 0.37 mm. Post-reconstruction smoothing filtering was applied using a 1.0-mm
Gaussian filter. Image displays were obtained using medical image data analysis software called Amide’s a Medical
Image Data Examiner, an open-source software tool (ver. 1.04). Transaxial and coronal images were displayed as
maximum intensity projections. In these images, three to five regions of interest were placed over the liver and gall
bladder with reference to CT images. Other normal mice (n = 5) were used for experiments using administrationof about 1.0 mM Ko143, a BCRP inhibitor, immediately before injection of about 5.0 MBq NP-59.

Statistical analysis. Data are presented as the means and standard deviation (SD). P values were calculatedusing the two-tailed unpaired Student’s t test for in vitro experiments after the F test, and the Wilcoxon rank
sum test for in vivo experiments for comparison between two groups. A P value less than 0.05 was consideredsignificant.

Results

The partition coefficient of NP-59 was 1.62 ± 0.10, which was higher than that of 123I-IMP (0.94 ± 0.01) as a reference of lipophilicity.
Uptake of NP-59 into HEK293 cells expressing SLC transporters is shown in Fig. 1. Uptake of NP-59 into
HEK293/OATP1B1 and HEK293/OATP1B3 cells was significantly higher than that into HEK293/mock cells(control) and that with rifampicin (P < 0.01, respectively).
The stability of NP-59 in mouse blood, mouse liver, and human liver S9 is shown in Table 1. The rates offlow (Rf values) for NP-59 and [125I]NaI were in the ranges of 0.60–0.70 and 0.00–0.05, respectively, in hexane:ether:ethyl acetate. Although deiodination of NP-59 was identified, NP-59 had high stability in mouseplasma, bile, and liver, and human liver S9. Figure 2 shows the uptake of NP-59 in the presence of ATP or AMP
solution into vesicles expressing hepatic ABC transporters (MDR1, MRP1–4, BCRP, or BSEP) after 5 min of incubation. We compared the uptake of NP-59 in ATP and AMP solution. Uptake into vesicles with high expression of
MRP1 and BCRP in ATP solution was significantly higher than that in AMP solution and each inhibitor.
Regarding the biological distribution of NP-59 in normal mice (Table 2), radioactivity in the blood, heart,
liver, and kidney rapidly increased immediately after injection and then gradually decreased. Radioactivity inthe lung and gall bladder also rapidly increased immediately after injection, continued to gradually increase by
Scientific Reports | (2019) 9:13413 | https://doi.org/10.1038/s41598-019-50049-8

3

www.nature.com/scientificreports

www.nature.com/scientificreports/

Figure 1. Uptake of NP-59 into HEK293 cells expressing SLC transporters. Uptake into HEK293/OATP1B1
and HEK/OATP1B3 cells was significantly higher than that in HEK293 mock cells. Uptake of NP-59 was also
inhibited by rifampicin, an OATP inhibitor (gray bars). *P < 0.01 vs. HEK293 mock cells and the inhibitor(n = 8; unpaired Student’s t test).

Figure 2. Uptake of NP-59 after 5 min of incubation in vesicles that highly express each ABC transporter.
Uptake via MRP1 and BCRP in ATP solution (white bars) was significantly higher than uptake in AMP solution(black bars), and the uptake was blocked by MK-571 or Ko143 loading (gray bars). *P < 0.01 and **P < 0.05
between uptake in ATP and AMP solution and each inhibitor (n = 8; unpaired Student’s t test).

Time
(min)

Mouse
plasmaMouse bile

Mouse liver

Human liverS9

10

95.4 ± 2.5

95.0 ± 3.8

95.1 ± 3.6

95.0 ± 4.3

3094.8 ± 3.2

94.2 ± 4.2

93.5 ± 4.5

94.4 ± 2.1

6093.9 ± 3.6

93.3 ± 3.5

92.8 ± 3.3

93.3 ± 3.2

Table 1. Stability of NP-59 in mouse plasma, mouse bile, mouse liver, and human liver S9 fractions. The
fractional ratios of NP-59 (non-metabolite) were calculated by dividing the radioactive counts for each fraction
by the total radioactivity count.

60 min and 30 min, and then decreased. In the thyroid, stomach, small intestine, adrenal gland, and bladder, radioactivity gradually increased after injection. Small amounts of radioactivity were present in the brain and large
intestine. In normal mice given NP-59 and Ko143 loading (Table 3), radioactivity in the blood, brain, liver, andkidney significantly increased compared to normal mice without Ko143 loading, whereas radioactivity in the gall
bladder significantly decreased.
Figure 3 shows SPECT images of a mouse at 5–25 min after injection of NP-59 without (Fig. 3A) and with
(Fig. 3B) Ko143 loading. NP-59 primarily accumulated in the lung, heart, liver, and gall bladder in normal mice.
In mice with Ko143 loading, accumulation of NP-59 in the liver increased from 14.0 ± 4.5 (normal mice) to
16.7 ± 3.8 counts (mice with Ko143 loading), and accumulation in the gall bladder slightly decreased from33.9 ± 8.4 (normal mice) to 26.5 ± 10.9 counts (mice with Ko143 loading).

Scientific Reports | (2019) 9:13413 | https://doi.org/10.1038/s41598-019-50049-8

4

www.nature.com/scientificreports

www.nature.com/scientificreports/
Organ (%ID/g) or %ID/
organ)

5 min

10 min20 min30 min60 min

120 minBlood

141.89 ± 9.24

100.36 ± 9.15

88.65 ± 11.71

56.98 ± 9.09

54.30 ± 5.12

57.17 ± 6.97

Brain

0.27 ± 0.04

0.20 ± 0.04

0.18 ± 0.05

0.16 ± 0.00

0.16 ± 0.02

0.16 ± 0.03

Thyroid*0.05 ± 0.05

0.10 ± 0.03

0.14 ± 0.02

0.16 ± 0.03

0.17 ± 0.10

0.20 ± 0.09
12.93 ± 5.62

Lung

7.55 ± 2.17

10.21 ± 2.53

10.03 ± 0.68

10.67 ± 2.54

15.96 ± 4.89

Heart

2.28 ± 0.42

1.98 ± 0.29

2.00 ± 0.33

1.97 ± 0.25

1.93 ± 0.31

1.69 ± 0.36

Stomach*1.04 ± 0.23

0.80 ± 0.11

0.95 ± 0.17

1.05 ± 0.26

1.10 ± 0.21

1.13 ± 0.23

Liver

14.39 ± 0.89

15.93 ± 1.77

15.09 ± 1.87

13.75 ± 1.63

13.30 ± 1.35

11.53 ± 1.80

Gall bladder12.98 ± 0.94

13.05 ± 1.08

23.20 ± 2.76

23.61 ± 2.87

14.80 ± 1.65

10.79 ± 1.25

Small intestine*

0.88 ± 0.07

0.99 ± 0.31

1.35 ± 0.24

1.74 ± 0.43

2.32 ± 0.52

3.11 ± 0.75

Large intestine*

0.27 ± 0.02

0.26 ± 0.08

0.26 ± 0.04

0.26 ± 0.05

0.25 ± 0.06

0.22 ± 0.08

Kidney2.07 ± 0.37

1.88 ± 0.31

1.91 ± 0.28

1.88 ± 0.28

2.15 ± 0.05

2.26 ± 0.38

Adrenal gland

0.51 ± 0.12

0.80 ± 0.20

1.02 ± 0.28

1.15 ± 0.47

2.09 ± 0.32

3.05 ± 1.02

Bladder

0.53 ± 0.45

0.67 ± 0.61

1.33 ± 0.54

1.43 ± 0.91

1.50 ± 0.80

2.11 ± 0.88

Table 2. Biological distribution of NP-59 in normal mice. %ID/g indicates percent injected dose per gram oftissue. *%ID/organ was calculated from %ID per organ. Values are the mean ± standard deviation obtained
from five mice.
Organ (%ID/g or%ID/organ)

5 min

10 min30 min60 minBlood

165.51† ± 18.31

124.42† ± 10.82

70.36† ± 10.68

60.96† ± 10.06

Brain

0.34 ± 0.00

0.28 ± 0.02

0.24 ± 0.01

0.20 ± 0.02

Thyroid*0.06 ± 0.03

0.08 ± 0.03

0.11 ± 0.02

0.16 ± 0.08
11.89 ± 5.12

††

††

††

Lung

6.73 ± 1.11

16.47 ± 8.05

12.25 ± 7.39

Heart

3.13† ± 1.12

2.76 ± 0.60

1.96 ± 0.21

1.92 ± 0.28

Stomach*0.72 ± 0.31

1.08† ± 0.22

1.56† ± 0.28

1.21 ± 0.32

Liver

16.44† ± 1.99

20.52† ± 2.35

18.87†† ± 0.57

14.21 ± 2.12

Gall bladder10.25 ± 0.85

10.88 ± 1.04

18.65†† ± 2.08

14.91 ± 1.27

Small intestine*

1.05 ± 0.23

1.15 ± 0.76

1.42 ± 0.64

2.28 ± 0.55

Large intestine*

0.37 ± 0.15

0.33 ± 0.12

0.36 ± 0.13

0.27 ± 0.12

Kidney2.71† ± 0.59

2.59† ± 0.31

2.61† ± 0.24

2.59† ± 0.15††††

Adrenal gland

0.53 ± 0.10

0.84 ± 0.17

1.19 ± 0.21

2.55† ± 0.23

Bladder

0.62† ± 0.29

0.62 ± 0.56

1.57 ± 0.97

1.71 ± 1.02

Table 3. Biological distribution of NP-59 with Ko143 loading. %ID/g indicates percent injected dose per gramof tissue. *%ID/organ was calculated from %ID per organ. Values are the mean ± standard deviation obtained
from five mice. †P < 0.05 and ††P < 0.01 compared with normal mice.

Figure 3. Whole-body SPECT images of normal mice without (A) and with Ko143 loading (B) under 2.0%
isoflurane anesthesia injected with 5.0 MBq NP-59 at 5–25 min. The arrows show the gall bladder, and dashedlines show the level of the transaxial images. NP-59 accumulated in the lung, liver, and gall bladder in normal
mice. In mice with Ko143 loading, accumulation of NP-59 in the liver was increased, whereas accumulation in
the gall bladder was slightly decreased compared with normal mice.

Scientific Reports | (2019) 9:13413 | https://doi.org/10.1038/s41598-019-50049-8

5

www.nature.com/scientificreports

www.nature.com/scientificreports/
Time (min)

5

10

30

60

Liver-to-blood
NP-59 without Ko143

0.10 ± 0.02

0.10 ± 0.02

0.24 ± 0.03

0.25 ± 0.05

NP-59 with Ko143

0.10 ± 0.01

0.14 ± 0.02* 0.27 ± 0.03*

0.23 ± 0.05

Gall bladder-to-blood
NP-59 without Ko143

0.09 ± 0.01

0.13 ± 0.02

NP-59 with Ko143

0.06 ± 0.01

0.09 ± 0.02* 0.27 ± 0.05**

0.41 ± 0.05

0.27 ± 0.05
0.24 ± 0.04*

Kidney-to-blood
NP-59 without Ko143

0.02 ± 0.00

0.02 ± 0.00

0.03 ± 0.00

0.04 ± 0.00

NP-59 with Ko143

0.02 ± 0.00

0.02 ± 0.00

0.04 ± 0.00

0.04 ± 0.00

Table 4. Organ-to-blood ratios at 5, 10, 30, and 60 min after NP-59 injection with and without Ko143 loading.
All data are the mean ± standard deviation measured in five mice. **P < 0.01 and *P < 0.05 between NP-59
without Ko143 and with Ko143 loading.

Discussion

Measurement of hepatic transporter function is important for prediction of the effect of chemotherapeutic agents.
In this study, we aimed to develop imaging of hepatic function using NP-59, a cholesterol analog. NP-59 was
confirmed to be a lipophilic agent because it showed a higher partition coefficient than 123I-IMP, which is generally known as a lipophilic agent in nuclear medicine20. Due to its high lipophilicity, the radioactivity of NP-59 in
the blood was higher than that in other tissues (Table 2) because about 74% of NP-59 binds to serum proteins inblood as shown with ultrafiltration in our previous study (unpublished observations). In addition, some NP-59
was non-specifically bound and/or adsorbed to the HEK293 cell surface membrane, vesicles, and the experimental apparatus (Figs 1 and 2) although the cells and vesicles were washed twice with 1 ml ice-cold incubation
medium.
Regarding SLC transporters for liver uptake of NP-59, we observed involvement of OATP1B1 and OATP1B3(Fig. 1). Although other SPECT and PET imaging agents used for hepatobiliary scintigraphy are also taken up
via OATP1B1 and OATP1B36,7,9,10,12–14, the liver uptake of NP-59 was slower than that of other agents because weestimate that NP-59 has too high lipophilicity for uptake into the liver via these transporters. Although humanliver S9 does not contain all hepatic enzymes, NP-59 had high stability at 120 min after injection in mouse blood,
mouse bile, mouse liver, and human liver (Table 1). Kojima et al. reported that NP-59 has higher stability thanother cholesterol analogs16. NP-59 underwent minimal deiodination because radioactivity in the thyroid andstomach, in which accumulation indicates deiodination, was low, as seen in the biological distribution studyof normal mice (Table 2). Therefore, we concluded that NP-59 is stable in the body and can be used to measure
hepatic ABC transporter function.
NP-59 was excreted via MRP1 and BCRP (Fig. 2). MRP1 is expressed on the basolateral side of hepatic epithelial cells for export from liver to blood, whereas BCRP is expressed on the apical side of hepatic epithelial cells for
excretion into bile. We observed a significant difference between BCRP and MRP1 (BCRP was lower than MRP1)
for NP-59 transport (Fig. 2). Thus, efflux of NP-59 may be higher via BCRP than MRP1.
In our study investigating the biological distribution of NP-59 in normal mice (Table 2), higher radioactivity was mainly found in the blood, lung, liver, and gall bladder. Uptake of NP-59 into the liver may depend on
OATP1B1 and OATP1B3, and bile secretion of NP-59 into the gall bladder may be due to BCRP. Although NP-59
accumulated in adrenal glands more than 1 day after injection16, the uptake of NP-59 in the liver and the gall bladder peaked by 10 min and 30 min, respectively, and then decreased (Table 2) because NP-59 was taken up into theliver, transferred to the gall bladder, and then transferred to the small intestine and bladder. Therefore, uptake intoadrenal glands has little influence on measurement of hepatic functional imaging with NP-59.
Because Allen reported that mouse Bcrp cDNA encodes a 657-amino acid protein with 81% identity and86% similarity to human BCRP25, we used mice loaded with Ko143, which is a BCRP inhibitor to confirm thebile secretion of NP-59 into the gall bladder via BCRP. Ko143 loading led to higher radioactivity of NP-59 in
the blood, brain, liver, and kidney (Table 3). BCRP is expressed not only in the liver but also in the kidney on
the apical side26. Liver-to-blood, gall bladder-to-blood, and kidney-to-blood ratios were calculated to decrease
the influence of radioactivity in blood (Table 4). Ko143 loading increased NP-59 accumulation in the liver, anddecreased the accumulation in the gall bladder due to inhibition of BCRP. No influence was seen on the accumulation in kidney.
Although planar imaging with NP-59 can usually be applied for detection of adrenal adenomas and hyperaldosteronism in clinical adrenal gland scintigraphy, we estimate that SPECT/CT imaging can be useful to separate
the gall bladder and right adrenal gland because the radioactivity of NP-59 in the gall bladder may be confusedfor that in the right adrenal gland. In SPECT imaging (Fig. 3), NP-59 accumulated in the lung, liver, and gall
bladder in normal mice. In mice with Ko143 loading, accumulation of NP-59 in the liver was increased, whereas
accumulation in the gall bladder was slightly decreased compared with normal mice. The ratios of radioactivity inthe gall bladder to the liver were significantly different between normal mice (2.45 ± 0.45) and mice with Ko143
loading (1.60 ± 0.74). If the function of BCRP is low in clinical studies due to certain diseases, the ratios will besignificantly decreased compared to healthy subjects with normal function of hepatic BCRP, as suggested by the
results of Ko143 loading.
NP-59 is useful for measuring human hepatic function of OATP1B1, OATP1B3, and BCRP. If 131I labeling of
NP-59 is changed to 123I, the imaging quality will be improved. However, NP-59 may not be the best agent for
Scientific Reports | (2019) 9:13413 | https://doi.org/10.1038/s41598-019-50049-8

6

www.nature.com/scientificreports/

www.nature.com/scientificreports

hepatobiliary scintigraphy because uptake of NP-59 into the liver and gall bladder is slower than that of otherimaging agents used for hepatobiliary scintigraphy due to the possibility of a somewhat low affinity of the agent
for OATP and BCRP as shown by the results of other studies10–12. Therefore, chemical analogs of NP-59 may havehigher affinity for these transporters and thus be more suitable for use in hepatobiliary scintigraphy.

Conclusion

NP-59, which is used clinically for adrenal gland scintigraphy, can be used to visualize and measure the functionof human hepatic OATP1B1, OATP1B3, and BCRP.

References

1. Smith, S. V. Challenges and opportunities for positron-emission tomography in personalized medicine. IDrugs. 8, 827–833 (2002).
2. Kjaer, A. Molecular imaging of cancer using PET and SPECT. Adv Exp Med Biol. 587, 277–284 (2006).
3. Kostakoglu, L. et al. Association of tumor washout rates and accumulation of technetium-99 m-MIBI with expression of
P-glycoprotein in lung cancer. J Nucl Med. 39, 228–234 (1998).
4. Shitara, Y. et al. Clinical significance of organic anion transporting polypeptides (OATPs) in drug disposition: their roles in hepatic
clearance and intestinal absorption. Biopharm Drug Dispos. 34, 45–78 (2013).
5. Shitara, Y., Sato, H. & Sugiyama, Y. Evaluation of drug-drug interaction in the hepatobiliary and renal transport of drugs. Annu Rev
Pharmacol Toxicol. 45, 689–723 (2005).
6. Krishnamurthy, G. T. & Turner, F. E. Pharmacokinetics and clinical application of technetium 99m-labeled hepatobiliary agents.
Semin Nucl Med. 20, 130–149 (1990).
7. Michael, M. et al. Relationship of hepatic functional imaging to irinotecan pharmacokinetics and genetic parameters of drug
elimination. J Clin Oncol. 24, 4228–4235 (2006).
8. Hendrikse, N. H. et al. In vivo imaging of hepatobiliary transport function mediated by multidrug resistance associated protein andP-glycoprotein. Cancer Chemother Pharmacol. 54, 131–138 (2004).
9. Ghibellini, G., Leslie, E. M., Pollack, G. M. & Brouwer, K. L. Use of Tc-99m mebrofenin as a clinical probe to assess alteredhepatobiliary transport: integration of in vitro, pharmacokinetic modeling, and simulation studies. Pharm Res. 25, 1851–1860(2008).
10. de Graaf, W. et al. Transporters involved in the hepatic uptake of 99mTc-mebrofenin and indocyanine green. J Hepatol. 54, 738–745(2011).
11. Swift, B., Yue, W. & Brouwer, K. L. Evaluation of 99mTechnetium-mebrofenin and 99mTechnetium-sestamibi as specific probes forhepatic transport protein function in rat and human hepatocytes. Pharm Res. 27, 1987–1998 (2010).
12. Kobayashi, M. et al. Transport mechanisms of hepatic uptake and bile excretion in clinical hepatobiliary scintigraphy with 99mTc-Npyridoxyl-5-methyltryptophan. Nucl Med Biol. 41, 338–342 (2014).
13. Mairinger, S., Erker, T., Muller, M. & Langer, O. PET and SPECT radiotracers to assess function and expression of ABC transporters
in vivo. Curr Drug Metab. 12, 774–792 (2011).
14. Langer, O. Use of PET Imaging to Evaluate Transporter-Mediated Drug-Drug Interactions. J Clin Pharmacol. 56, S143–156 (2016).
15. Pavek, P. et al. Human breast cancer resistance protein: interactions with steroid drugs, hormones, the dietary carcinogen 2-amino1-methyl-6-phenylimidazo(4,5-b)pyridine, and transport of cimetidine. J Pharmacol Exp Ther. 312, 144–152 (2005).
16. Kojima, M., Maeda, M., Ogawa, H., Nitta, K. & Ito, T. New adrenal-scanning agent. J Nucl Med. 16, 666–668 (1975).
17. Shapiro B., Sandler M.D. Adrenocortical Scintigraphy. (Chapter 64) In: Murray P.S., Ell ICP, editors. Nuclear Medicine in clinicaldiagnosis and treatment (Volume 2). Edinburgh UK: Churchill Livingston. p. 737 (1994).
18. Gross, M.D., Shapiro, B., Shreve, P., Shulkin, B.I. Scintigraphy of Adrenal Disorders. In: De Herder W.W. (ed.). Functional andMorphological Imaging of the Endocrine System. Endocrine Updates. vol. 7. Boston: Kluwer Academic Publishers. pp. 163–189(2000).
19. Tamai, I. et al. Functional characterization of human organic anion transporting polypeptide B (OATP-B) in comparison with liverspecific OATP-C. Pharm Res. 18, 1262–1269 (2001).
20. Winchell, H. S., Baldwin, R. M. & Lin, T. H. Development of I-123-labeled amines for brain studies: localization of I-123
iodophenylalkyl amines in rat brain. J Nucl Med. 21, 940–946 (1980).
21. Kobayashi, M. et al. Different efflux transporter affinity and metabolism of 99mTc-2-Methoxyisobutylisonitrile and 99mTc-Tetrofosmin
for multidrug resistance monitoring in cancer. Pharm Res. 36, 18 (2018).
22. Weidner, L. D. et al. The inhibitor Ko143 is not specific for ABCG2. J Pharmacol Exp Ther. 354, 384–393 (2015).
23. Sakamoto, S., Suzuki, H., Kusuhara, H. & Sugiyama, Y. Efflux mechanism of taurocholate across the rat intestinal basolateral
membrane. Mol Pharm. 3, 275–281 (2006).
24. Louis-Ferdinand, R. T., Therriault, D. G., Blatt, W. F. & Mager, M. Application of thin-layer chromatography to the quantitation ofplasma neutral lipids and free fatty acids. Clin Chem. 13, 773–787 (1967).
25. Allen, J. D., Brinkhuis, R. F., Wijnholds, J. & Schinkel, A. H. The mouse Bcrp1/Mxr/Abcp gene: amplification and overexpression incell lines selected for resistance to topotecan, mitoxantrone, or doxorubicin. Cancer Res. 59, 4237–4241 (1999).
26. Nakanishi, T. & Ross, D. D. Breast cancer resistance protein (BCRP/ABCG2): its role in multidrug resistance and regulation of its
gene expression. Chin J Cancer. 31, 73–99 (2012).

Acknowledgements

The authors thank the staff of the School of Health Sciences, Kanazawa University for their technical support.
This study was partly funded by Grants-in-Aid for Scientific Research from the Japan Society for the Promotionof Science (Nos. 15K09949, 15K15452, 16KK0200, and 18K07747).

Author Contributions

Masato Kobayashi was the research coordinator for the study and wrote the main manuscript text. Asuka
Mizutani performed the biological distribution experiments shown in Tables 2 and 3 and SPECT imaging shownin Fig. 3. Tsuzumi Hokama, Takeo Nakanishi, and Ikumi Tamai were responsible for the in vitro study with HEK
cells and vesicles shown in Figs 1 and 2. Miki Matsue evaluated the time stability of [131I]6-β-iodomethyl-19norcholesterol in mouse blood, liver, and human liver S9 in Table 1. Tetsuya Tsujikawa and Ryuichi Nishii are
medical doctors and served as research advisers. Kodai Nishi and Keiichi Kawai were advisors for handling the[131I]6-β-iodomethyl-19-norcholesterol.

Additional Information

Competing Interests: The authors declare no competing interests.
Scientific Reports | (2019) 9:13413 | https://doi.org/10.1038/s41598-019-50049-8

7

www.nature.com/scientificreports/

www.nature.com/scientificreports

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps andinstitutional affiliations.
Open Access This article is licensed under a Creative Commons Attribution 4.0 International
License, which permits use, sharing, adaptation, distribution and reproduction in any medium or
format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in thisarticle are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to thematerial. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the
copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
© The Author(s) 2019

Scientific Reports | (2019) 9:13413 | https://doi.org/10.1038/s41598-019-50049-88