Pharm Res (2019) 36: 18
https://doi.org/10.1007/s11095-018-2548-5

RESEARCH PAPER

Different Efflux Transporter Aff inity and Metabolismof 99m Tc-2-Methoxyisobutylisonitrile and 99m Tc-Tetrofosminfor Multidrug Resistance Monitoring in Cancer
Masato Kobayashi 1 & Takafumi Tsujiuchi 2 & Yuya Okui 2 & Asuka Mizutani 2 & Kodai Nishi 3 &Takeo Nakanishi 4 & Ryuichi Nishii 5 & Kazuki Fukuchi 6 & Ikumi Tamai 4 & Keiichi Kawai 2,7Received: 2 September 2018 / Accepted: 21 November 2018 / Published online: 29 November 2018
# Springer Science+Business Media, LLC, part of Springer Nature 2018

ABSTRACT
Background Little is known about the affinity and stability of99m
Tc-labeled 2-methoxyisobutylisonitrile (99mTc-MIBI) andtetrofosmin (99mTc-TF) for imaging of multiple drug resistancetransporters in cancer. We examined the affinity of 99mTc-labeled compounds for these transporters and their stability.
Methods 99mTc-MIBI and 99mTc-TF were incubated in vesicles expressing P-glycoprotein (MDR1), multidrug resistanceassociated protein (MRP)1–4, or breast cancer resistance protein with and without verapamil (MDR1 inhibitor) or MK571 (MRP inhibitor). Time activity curves of 99mTc-labeled
compounds were established using SK-N-SH neuroblastoma,
SK-MEL-28 melanoma, and PC-3 prostate adenocarcinoma* Masato Kobayashi
kobayasi@mhs.mp.kanazawa-u.ac.jp

1

Wellness Promotion Science Center, Institute of Medical, Pharmaceuticaland Health Sciences Kanazawa University
5-11-80 Kodatsuno, Kanazawa 920-0942, Japan

2

School of Health Sciences, Institute of Medical, Pharmaceutical and
Health Sciences Kanazawa University
Kanazawa, Japan3

Department of Radioisotope Medicine
Atomic Bomb Disease Institute Nagasaki University
Nagasaki, Japan

4

School of Pharmaceutical Sciences, Institute of Medical, Pharmaceuticaland Health Sciences Kanazawa University
Kanazawa, Japan

5

Department of Molecular Imaging and Theranostics
National Institute of Radiological Sciences
Chiba, Japan

6

Graduate School of Medicine, Division of Health Sciences
Osaka University
Osaka, Japan

7

Biomedical Imaging Research Center University of Fukui
Fukui, Japan

cell lines, and transporter expression of multiple drug resistance was measured in these cells. The stability was evaluated.
Results In vesicles, 99mTc-labeled compounds had affinity for
MDR1 and MRP1. 99mTc-TF had additional affinity for
MRP2 and MRP3. In SK-N-SH cells expressing MDR1 and
MRP1, MK-571 produced the highest uptake of both 99mTclabeled compounds. 99mTc-MIBI uptake with inhibitors washigher than 99mTc-TF uptake with inhibitors. 99mTc-TF was
taken up more in SK-MEL-28 cells expressing MRP1 and
MRP2 than PC-3 cells expressing MRP1 and MRP3. 99mTcMIBI was metabolized, whereas 99mTc-TF had high stability.
Conclusion 99mTc-MIBI is exported via MDR1 and MRP1
(MRP1 > MDR1) at greater levels and more quickly comparedto 99mTc-TF, which is exported via MDR1 and MRP1–3(MRP1 > MDR1; MRP1, 2 > MRP3). Because 99mTc-MIBI
is metabolized, clinical imaging for monitoring MDR andshorter examination times may be possible with an earlier scanning time on late phase imaging. 99mTc-TF has high stability
and accurately reflects the function of MDR1 and MRP1–3.

KEY WORDS 99mTc-MIBI . 99mTc-tetrofosmin . adenosine
triphosphate-binding cassette transporters . multidrug resistance .
multidrug resistance-associated protein

INTRODUCTION
Multiple drug resistance (MDR) in cancer is often associatedwith an adenosine triphosphate (ATP)-dependent decrease incellular drug accumulation and is attributed to the overexpression of certain ATP-binding cassette (ABC) transporter
proteins. ABC transporters belong to the largest transporter
gene family and generally use energy derived from ATP hydrolysis for translocation of different substrates across biological membranes. ABC transporters are classified into sevensubfamilies based on phylogenetic analysis and are designated
ABCA to ABCG (1). In tumor cell lines, ABC proteins mainlyPharm Res (2019) 36: 18

18 Page 2 of 10

include P-glycoprotein (MDR1) (gene symbol ABCB1), themultidrug resistance protein 1 (MRP1, gene symbol ABCC1),
MRP2 (gene symbol ABCC2), MRP3 (gene symbol ABCC3),
MRP4 (gene symbol ABCC4), and breast cancer resistance
protein (BCRP, gene symbol ABCG2).
99m
Tc-labeled 2-methoxyisobutylisonitrile (99mTc-MIBI)
99m
Tc-labeled tetrofosmin (99mTc-TF) are lipophilicandmonocationic radiotracers that are widely used for myocardial
perfusion imaging (2) and tumor imaging (3) in single photon
emission computed tomography (SPECT) studies. In tumor
imaging, MDR to anticancer drugs has been estimated using
both 99mTc-labeled compounds in clinical studies (4,5). ABC
transporters, which are associated with MDR, are often highly
expressed in cancer cells. 99mTc-labeled compounds are mainly exported via MDR1 (6–8) and MRP1 (7). Many ABC transporters, not only MDR1 and MRP1 but also MRP2–4 (9–11)
and BCRP (12), are expressed in cancer cells. However,whether MRP2–4 and BCRP are associated with export of99m
Tc-labeled compounds has not been clearly determined.
If 99mTc-labeled compounds are metabolized completely
and immediately after injection in vivo, monitoring of MDR
in cancers may be affected in clinical SPECT imaging because
metabolites of 99mTc-labeled compounds may not be taken up
in cancers and undergo efflux through MDR when clinicalimaging is performed in the early phase (10–30 min) and thelate phase (2–3 h) after injection (4,5). However, the stability of99m
Tc-labeled compounds has not been sufficiently evaluatedin vivo. We examined whether 99mTc-labeled compounds were
exported via MRP2–4 and BCRP and whether these compounds had affinity for these transporters including MDR1
and MRP1 in cancer cells. Furthermore, stability of the
99m
Tc-labeled compounds over time was investigated in ahuman-derived cancer cell line, mouse liver, and human liver,which is an important organ for metabolism.

MATERIALS AND METHODS

Vesicles
We used ABC transporter vesicles (GenoMembrane Inc.,
Kanagawa, Japan) transfected with human MDR1, MRP1–
4, and BCRP. Experimental kits were also purchased from
GenoMembrane Inc. and were used for experiments witheach ABC transporter.

Vesicle Assay
After pre-incubation of vesicles for 10 min, 37 kBq 99mTclabeled compound was incubated for 5 min with eachvesicle solution and ATP, which supplies energy for
ABC transporters, or adenosine monophosphate (AMP),
which does not provide energy and was used for comparison to ATP, on nitrocellulose filters, and the radioactivity
was measured using a γ-ray counter (AccuFLEXγ7000,
Aloka, Tokyo, Japan) (13). Uptake of 99m Tc-MIBI or
99m
Tc-TF in ATP solution was compared with that in
AMP solution. When uptake of 99mTc-MIBI or 99mTcTF in ATP solution was higher than that in AMP solution, this indicated an effect on ABC transporters for99m
Tc-MIBI or 99mTc-TF. In assays with inhibitors, uptake of 99mTc-MIBI or 99mTc-TF was examined in ATP
solution with verapamil, a MDR1 inhibitor, or MK-571,
a MRP inhibitor.

Cancer Cells
The following cultured human cancer cell lines were purchased from American Type Culture Collection (Manassas,
VA, USA): SK-N-SH neuroblastoma, SK-MEL-28 melanoma, and PC-3 prostate adenocarcinoma. Cancer cells wereincubated in αMEM (Wako, Osaka, Japan; SK-N-SH cells)
or Dulbecco’s Modified Eagle’s Medium (Wako; SK-MEL-28
cells) or RPMI-1640 Medium (Sigma; PC-3 cells) with 10%
fetal bovine serum.

Materials
99m

Tc-MIBI (300 MBq/mL) and 99mTc-TF (592 MBq/mL)
injection kits were purchased from FUJIFILM RI Pharma Co.,
Ltd. (Chiba, Japan) and Nihon Medi-physics Co., Ltd. (Chiba,
Japan), respectively. 99mTcO4− was eluted from a 99Mo/99mTccolumn generators (Nihon Medi-physics Co., Ltd., Chiba,
Japan). β-nicotinamide-adenine dinucleotide phosphate (βNADP+) and glucose-6-phosphate dehydrogenase were purchased from Oriental Yeast (Osaka, Japan). Verapamil hydrateand MK-571 sodium salt were purchased from Sigma-Aldrich(St. Louis, MO, USA) and Cayman Chemical (Ann Arbor, MI,
USA), respectively. Human liver S9 was purchased from
Corning Gentest (New York, NY, USA).

Expression of ABC Transporters in Human Cancer Cells
Expression of ABC transporters in human cancer cells was evaluated as described (14). The following genes were analyzed
using real-time polymerase chain reaction with a Mx3005P
thermocycler (Agilent Technologies, Santa Clara, CA, USA):
MDR1 (ABCB1) and MRP1, 2, 3 (ABCC1, 2, and 3) as summarized in Table I. Three different housekeeping genes,
glyceraldehyde-3-phosphate dehydrogenase (GAPDH), beta actin (ACTB), and hypoxanthine phosphoribosyltransferase-1(HPRT1), were amplified to control for the differences between
the initial RNA and cDNA amounts.

Pharm Res (2019) 36: 18

Page 3 of 10 18

Table I Expression of Genes Encoding the ABC Transporters in Human
Cancer Cells
Transporter

Gene symbol

SK-N-SH

SK-MEL-28

PC-3

MDR1

ABCB1

25.83

0.02

0.00

MRP1

ABCC1

23.41

7.81

20.60

MRP2
MRP3

ABCC2
ABCC3

0.16
0.75

11.61
0.00

0.26
21.39

Data are expressed as copy number per 1000 copies of housekeeping genes

Assay with Human Cancer Cells
Transport assays were performed as described (13). One day
after seeding of cancer cells, each well was pre-incubated with
1 mL incubation medium for 10 min. The cells were thenincubated with 37 kBq 99mTc-MIBI or 99mTc-TF for 5, 10,30, or 60 min at 37°C.
For the competitive inhibition assay, the cells were incubated for 5 min with 99mTc-MIBI or 99mTc-TF in the presence of
inhibitor: final concentration 50 μM verapamil hydrate for
MDR1 (15) or 50 μM MK-571 sodium salt for MRP1–4(16). 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 was measured with a γ-ray counter.
SPECT Imaging of 99mTc-Labeled Compounds
in SK-N-SH-Bearing Mice
All animal studies were conducted following approval by the
Animal Care Committee of Kanazawa University (AP122339). Five Scid mice (female, 5 weeks old) weretransplanted with SK-N-SH cells (5 × 105 cells/100 μL) andMatrigel (#354230, Corning, NY, USA) into the lower abdomen of the mice. The mice were housed for about 5–7 weeks
under a 12-h light/12-h dark cycle with free access to food andwater. Each 99mTc-labeled compound (20–30 MBq) was
injected into the tail vein of cancer-bearing mice, and
SPECT acquisition was started 5 min after injection and continued for 90 min every 5 min using a U-SPECT-II/CT system (MILabs, Utrecht, The Netherlands). The data were reconstructed using the ordered subset expectation maximization method with 16 subsets and six iterations including no
scatter and attenuation correction. The voxel size was set to
0.8 × 0.8 × 0.8 mm. Post-reconstruction smoothing filtering
was applied using a 1.0-mm Gaussian filter. Image displays
were obtained using medical image data analysis software,
AMIDE (ver. 1.0.2). Coronal images were displayed as maximum intensity projections. In these images, three to five regions of interest were placed over the parathyroid, heart, liver,gallbladder, and kidney, and the time activity curve of each
was obtained.

Time Stability of 99mTc-Labeled Compounds
in SK-N-SH-Bearing Mice and Human Liver S9Three mice each (total 15 mice) were killed at 5, 10, 20, 30,
and 60 min after injection of 185 MBq 99mTc-labeled compounds. After blood sampling via cardiocentesis, the SK-NSH cells and mouse livers were removed. Metabolites in SKN-SH cells, mouse liver, and plasma were analyzed by thinlayer chromatography (TLC). Human liver S9 was also used
for metabolic 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 were developed using acetonitrile:methanol:0.5 mol/L ammonium
acetate:tetrahydrofuran at a ratio of 4:3:2:1 for 99mTc-MIBI(17) and dichloromethane:acetone at a ratio of 13:7 for 99mTcTF (17–19). The rates of flow (Rf) for 99mTc-MIBI and 99mTcTF were in the ranges of 0.15–0.25 and 0.20–0.30, respectively. After development and complete drying, the TLC plates
were cut into 20 fractions, and the radioactivity associatedwith each fraction was measured using a γ-ray counter. The
fractional ratios of metabolites were calculated by dividing theradioactive counts for each fraction by the total radioactivity
count.
Statistical Analysis
Data are presented as the means and standard deviation (SD).
P values were calculated using the two-tailed paired Student’s
t test for comparison between two groups. A P value less than
0.05 was considered significant.

RESULTSIn the vesicle assay (Fig. 1), uptake of neither 99mTc-MIBI nor
99m
Tc-TF in ATP solution was significantly different fromuptake in AMP solution in control vesicles. The uptake of99m
Tc-MIBI was significantly different in ATP solution compared to AMP solution in MDR1 and MRP1 vesicles, whereas
99m
Tc-TF showed significantly higher uptake in not only vesicles expressing MDR1 and MRP1 but also vesicles expressing
MRP2 and MRP3. Loading with verapamil for MDR1 or
MK-571 for MRP restored the uptake to levels similar to thatin AMP solution for both 99m Tc-labeled compounds.
Regarding the expression of ABC transporters in cancer cells(Table I), all cancer cells showed high MRP1 expression. In
addition, we observed high expression of MDR1 in SK-N-SH
cells, MRP2 in SK-MEL-28 cells, and MRP3 in PC-3 cells.

Pharm Res (2019) 36: 18

18 Page 4 of 10

a
350
*

300

99mTc-TF uptake (nmol/mg protein)

99mTc-MIBI uptake (nmol/mg protein)

b
*

250
200
150
100
50
0

Control

MDR1

MRP1

MRP2

MRP3

MRP4

300

*

*

*

MRP2

MRP3

250
200
150
100

BCRP

ATP
99m

*

350

50
0

AMP

Control

MDR1

MRP1

MRP4

BCRP

Inhibitor

99m

Fig. 1 Uptake of Tc-MIBI (a) and Tc-TF (b) after 5 min of incubation in vesicles that highly express each ABC transporter. For 99mTc-MIBI, uptake via MDR1
and MRP1 in ATP solution was significantly higher than uptake in AMP solution, and each inhibitor in ATP solution decreased the uptake to the level in AMP
solution. For 99mTc-TF, uptake via MDR1, MRP1, MRP2, and MRP3 in ATP solution was significantly higher than uptake in AMP solution, and uptake was blockedby each inhibitor. *P < 0.01 between uptake in ATP and AMP solution (n = 5; paired t test).

In the time activity curves for 99mTc-MIBI and 99mTc-TF
in SK-N-SH cells (Fig. 2), verapamil or MK-571 loading produced significantly higher uptake than in the control at all
incubation times. At 5 min of incubation, uptake of 99mTcMIBI and verapamil loading was 2.1-fold higher than in thecontrol, and that with MK-571 loading was 2.8-fold higher(Fig. 2a). Uptake of 99mTc-TF and verapamil loading was 1.8-

afold higher than the control condition, and that with MK-571
loading was 2.3-fold higher (Fig. 2b).
99m
Tc-MIBI with MK-571 loading produced higher accumulation than in the control condition in both SK-MEL-28
and PC-3 cells at all incubation times (Fig. 3a). Time activity
curves of 99mTc-MIBI with MK-571 loading showed very
little difference in accumulation in both cancer cell lines.

b

Fig. 2 Time activity curves for 99mTc-MIBI (a) and 99mTc-TF (b) in SK-N-SH cells, which highly expressed MDR1 and MRP1. For both 99mTc-labeled compounds,
loading with MK-571, a MRP inhibitor, produced the highest uptake despite similar expression levels of MDR1 and MRP1. Time activity curves of 99mTc-MIBI witheach inhibitor showed higher uptake than that of 99mTc-TF with each inhibitor. *P < 0.01 between uptake of 99mTc-labeled compounds and control (n = 4; pairedt test).

Pharm Res (2019) 36: 18

Page 5 of 10 18abFig. 3 Time activity curves of 99mTc-MIBI (a) and 99mTc-TF (b) in SK-MEL-28 and PC-3 cells. 99mTc-MIBI with MK-571 loading (●; SK-MEL-28 and ▲; MK-571)
produced higher accumulation than the control condition (○; SK-MEL-28 and △; MK-571) in both cell lines at all incubation times. Time activity curves of 99mTcMIBI with MK-571 loading showed very little difference in accumulation in both cancer cell lines. Time activity curves of 99mTc-TF with MK-571 loading in SK-MEL28 cells, which expressed MRP1 and MRP2, showed higher uptake than that in the control condition and PC-3 cells, which expressed MRP1 and MRP3.
*P < 0.01 between the uptake of 99mTc-labeled compounds in SK-MEL-28 and PC-3 cells compared to the control condition (n = 4; paired t test).

a
c

b
d

e

f

g

h

Fig. 4 Whole-body images of SK-N-SH-bearing mice under 2.0% isoflurane anesthesia injected with 20–30 MBq 99mTc-MIBI (A) or 99mTc-TF (B) at 5–10 min(c, f), 30–35 min (d, g), and 55–60 min (e, h). The arrows show solid cancers of SK-N-SH cells. The accumulation between brain and lung was in brown
adipocytes around the neck. Although accumulation in SK-N-SH cells was lower than in abdominal organs at all acquisition times, similar accumulation levels wereseen between 99mTc-MIBI (c) and 99mTc-TF (f) at 5–10 min after injection. At 30–35 min, we observed that the accumulation of 99mTc-MIBI was lower than that of99m
Tc-TF. Background levels of 99mTc-TF were higher than those of 99mTc-MIBI. Time activity curves were obtained for the whole body (Fig. 5) and SK-N-SH cells(Fig. 6).

Pharm Res (2019) 36: 18

18 Page 6 of 10bRadioactivity (kBq/g body weight)

Radioactivity (kBq/g body weight)

a
700
600
500
400
300
200
100
0
0

10

20

30

40

50

60

70

80

90

Time (min)

700
600
500
400
300
200
100
0
0

10

20

30

40

50

60

70

80

90

Time (min)Fig. 5 Time activity curves of each organ obtained from 5-min acquisition images in SK-N-SH-bearing mice injected with 20–30 MBq 99mTc-MIBI (a) or 99mTc-TF(B). In the gallbladder (●), accumulation was higher than in other organs, whereas faster excretion was found in kidney (■). In liver (○), 99mTc-TF showed higher
accumulation than 99mTc-MIBI at 10 min after injection. In the parathyroid (▲) and heart (□), accumulation did not change much over time, but 99mTc-MIBI
showed higher accumulation than 99mTc-TF. The error bars show standard deviation of data from five miceTime activity curves of 99mTc-TF with MK-571 loading in
SK-MEL-28 cells showed the highest uptake (Fig. 3b).
Whole-body images of SK-N-SH-bearing mice were obtained for 99mTc-MIBI (Fig. 4a) and 99mTc-TF (Fig. 4b) at 5–
10 min (c, f), 30–35 min (d, g), and 55–60 min (e, h). The
accumulation between brain and lung was in brown adipocytes around the neck. Accumulation in SK-N-SH cells was
lower than in abdominal organs at all acquisition times.
Although we observed similar accumulation levels in SK-NSH cells between 99mTc-MIBI (Fig. 4Ac) and 99mTc-TF (Fig.
4Bf) at 5–10 min after injection, accumulation of 99mTc-MIBI
in liver was lower than that of 99mTc-TF. At 30–35 min, we
observed that the accumulation of 99mTc-MIBI in SK-N-SH
cells was lower than that of 99mTc-TF. Background levels of
99m
Tc-MIBI were also lower than those of 99mTc-TF.
The whole-body distributions of 99mTc-MIBI (Fig. 5a) and99m
Tc-TF (Fig. 5b) were obtained from whole-body images.
Accumulation in the gallbladder was higher than that in otherorgans, and excretion via the kidneys was fast. In liver, 99mTcTF had higher accumulation than 99mTc-MIBI at 10 min
after injection as shown in Fig. 4. In the parathyroid and heart,these accumulations did not change much over time, but
99m
Tc-MIBI accumulated more than 99mTc-TF.
Time activity curves of SK-N-SH cells (Fig. 6) from wholebody images in Fig. 4 showed that more 99m Tc-MIBI
underwent significant efflux from the tumors than 99mTc-TF
during the first 50 min after injection. 99mTc-MIBI underwent
rapid efflux from the tumors until about 30 min after injection, and then showed moderate efflux, whereas 99mTc-TF
underwent gradual efflux from the tumors over time (Fig.
6a). Regarding accumulation in SK-N-SH cells of 99mTcMIBI (Fig. 6b) and 99mTc-TF (Fig. 6c) with verapamil or

MK-571 loading, MK-571 loading yielded higher accumulation with the two 99mTc-labeled compounds because MRP in
SK-N-SH cells was inhibited by MK-571. In the time activity
curves of 99mTc-MIBI (Fig. 6b), both inhibitors provided significantly higher accumulation than the control condition atabout 30 min after injection. In the time activity curves of99m
Tc-TF (Fig. 6c), verapamil loading led to an increase inaccumulation at about 10 min, whereas MK-571 loading led
to an increase in accumulation at about 50 min.
Regarding the stability over time for both 99mTc-labeled
compounds in SK-N-SH cells, mouse liver, plasma, and human liver S9 fractions (Table II), the Rf values of 99mTc-MIBI,
99m
Tc-TF, and 99mTcO4− were in the ranges of 0.15–0.25,
0.20–0.30, and 0.85–0.95 in our study, respectively. Althoughboth 99mTc-labeled compounds were slightly metabolized up
to 20 min after injection in all tissues, the radiochemical fraction of 99mTc-MIBI shifted from 97.5 ± 3.2% to 32.4 ±
17.5%, 26.3 ± 14.2%, 79.9 ± 18.5%, and 62.9 ± 12.1% in
SK-N-SH cells, mouse liver, human liver S9 fractions, andFig. 6 Time activity curves of SK-N-SH cells obtained from 5-min acquisition
images in SK-N-SH-bearing mice injected with 20–30 MBq 99mTc-MIBI or
99m
Tc-TF. In the control condition (a), more 99mTc-MIBI (〇) underwent efflux
from the tumors than 99mTc-TF (□) during the first 50 min after injection.
99m
Tc-MIBI underwent rapid efflux from the tumors until about 30 min after
injection and then underwent moderate efflux, whereas 99mTc-TF underwent
gradual efflux from the tumors over time. For both 99mTc-labeled compoundswith inhibitors, MK-571 loading (● or ■ solid line) for MRP inhibition yielded
the highest accumulation. 99mTc-MIBI (b) with verapamil loading (● and dotted line) and MK-571 loading (● and solid line) accumulated at significantly
higher levels than in the control condition during the first ~30 min. 99mTc-TF(c) with verapamil loading (■ and dotted line) showed increased accumulation
during the first 10 min, whereas MK-571 loading (■ and solid line) increased
the accumulation during the first 50 min

Pharm Res (2019) 36: 18

Page 7 of 10 18ab

plasma, respectively, at 30 min after injection, and from 97.1
± 4.0% to 35.7 ± 16.8%, 35.9 ± 16.8%, 85.1 ± 20.3%, and72.4 ± 21.2% in SK-N-SH cells, mouse liver, human liverc

S9 fractions, and plasma, respectively, at 60 min after injection. On the other hand, 99mTc-TF was stable at about morethan 95% at all injection times in all tissues.

Pharm Res (2019) 36: 18

18 Page 8 of 10
Table IITime (min)Metabolism Ratios of 99mTc-MIBI and 99mTc-TF in SK-N-SH Cells, Mouse Liver, Human Liver S9 Fractions, and Plasma
99m

99m

Tc-MIBI (%)

Tc-TF (%)

SK-N-SH

Mouse liver

Human liver S9

PlasmaSK-N-SH

Mouse liver

Human liver S9

Plasma30

32.4 ± 17.5

26.3 ± 14.2

79.9 ± 18.5

62.9 ± 12.1

1.1 ± 0.2

1.3 ± 0.3

3.5 ± 0.7

1.8 ± 0.2

60

35.7 ± 16.8

35.9 ± 16.8

85.1 ± 20.3

72.4 ± 21.2

3.5 ± 1.3

4.0 ± 0.8

4.6 ± 0.9

4.3 ± 0.2

The fractional ratios of metabolites were calculated by dividing the radioactive counts for each fraction by the total radioactivity count

DISCUSSION
Although 99mTc-MIBI and 99mTc-TF are known to be exported
via efflux transporters (6–12), several different kinds of efflux
transporters have not been evaluated yet. In the vesicle assay,
99m
Tc-MIBI had affinity for MDR1 and MRP1, whereas
99m
Tc-TF showed affinity for not only MDR1 and MRP1 but
also MRP2 and MRP3, as indicated by the significant difference
in uptake between ATP and AMP loading, which shows theeffect of each efflux transporter in the vesicle study (Fig. 1). In
addition, uptake of the 99mTc-labeled compounds in vesicles
was inhibited by verapamil for MDR1 and MK-571 for
MRP. The data confirmed that 99mTc-MIBI and 99mTc-TF
are exported via MDR1 (6–8) and MRP1 (7), and our data
show that 99mTc-TF is also transported by MRP2 and MRP3.
To confirm the mechanism and the affinity of efflux of99m
Tc-labeled compounds via the ABC transporters in human cancer cells, we selected SK-N-SH cells, which express
MDR1 and MRP1, as model cancer cells that highly express
ABC transporters. In the assay (Fig. 2), because uptake of99m
Tc-labeled compounds was significantly increased by verapamil and MK-571 inhibitors, the efflux mechanism of
99m
Tc-labeled compounds could be identified as involving
MDR1 and MRP1. The time activity curves of both 99mTclabeled compounds and MK-571 loading showed significantly
higher uptake than in the control condition or with verapamilloading, although MDR1 and MRP1 are expressed at similarly high levels in SK-N-SH cells. Thus, when MDR1 and
MRP1 are highly expressed in cancers, 99mTc-labeled compounds will be exported via MDR1 and MRP1, but MRP1 ismore sensitive than MDR1. Time activity curves of 99mTcMIBI with each inhibitor showed higher uptake than that of99m
Tc-TF with each inhibitor. Therefore, 99mTc-MIBI isexported via MDR1 and MRP1 (MDR1 < MRP1) at greaterlevels and more quickly than 99mTc-TF, and this is importantfor MDR monitoring. Although Gomes et al. have also reported that 99mTc-MIBI is more sensitive to MRP1 than 99mTcTF in vitro (8), they evaluated the relationship between 99mTclabeled compounds and efflux transporters using a small-celllung cancer cell line with single overexpression of MDR1 or
MRP1. Because cancer cells usually overexpress multiple
transporters, e.g., MDR1 and/or MRP1–3, our results
strongly certified that 99mTc-MIBI is more sensitive to

MRP1 than 99mTc-TF using cancer cells that overexpress
both MDR1 and MRP1.
Time activity curves of 99mTc-MIBI with MK-571 loading
showed very little difference in accumulation in SK-MEL-28
and PC-3 cells (Fig. 3a). Regarding MDR expression in PC-3
and SK-MEL-28 cells (Table I), although little MDR1 expression was seen in either cell line, MRP1 expression in PC-3 cells
was higher than that in SK-MEL-28 cells. When expression of
MRP1 is somewhat high in cancer cells, time activity curves of99m
Tc-MIBI, which is exported via MDR1 and MRP1, may besimilar. In the time activity curves of 99mTc-TF (Fig. 3b), 99mTcTF with MK-571 loading in SK-MEL-28 cells, which express
MRP1 and MRP2, showed higher accumulation than that in
PC-3 cells, which express MRP1 and MRP3. Although 99mTcTF was exported by not only MDR1 and MRP1 but also
MRP2 and MRP3, MRP3 may have a smaller effect than
MRP1 and MRP2 in export of 99mTc-TF.
In whole-body imaging of a SK-N-SH-bearing mouse
injected with 99mTc-MIBI (Fig. 4a) or 99mTc-TF (Fig. 4b) at 5–
10 min (c, f), 30–35 min (d, g), and 55–60 min (e, h), accumulation in SK-N-SH cells was lower than in abdominal organs at all
acquisition times. Although we observed similar accumulationlevels between 99mTc-MIBI (Fig. 4Ac) and 99mTc-TF (Fig. 4Bf)
at 5–10 min after injection in SK-N-SH cells, accumulation of
99m
Tc-MIBI in liver was lower than that of 99mTc-TF. Livermainly expresses MDR1, MRP2, BCRP, and the bile salt export
pump, which are ABC transporters that are the same types of
MDR efflux transporters expressed in tumor cells. We suggestthat 99mTc-MIBI is transferred quickly from liver to gallbladder
after injection. At 30–35 min after injection, the accumulation of
99m
Tc-MIBI (Fig. 4Ad) was likely lower than that of 99mTc-TF(Fig. 4Bg) in SK-N-SH cells, and background levels of 99mTcMIBI were also lower than those of 99mTc-TF because excretionof 99mTc-MIBI was faster than that of 99mTc-TF (Fig. 5).
The whole-body distribution of 99mTc-MIBI (Fig. 5a) and99m
Tc-TF (Fig. 5b) was obtained from whole-body images.
Because liver and kidney express MDR1 but not MRP1 for
efflux and excretion of both 99mTc-labeled compounds (20),99m
Tc-MIBI showed faster transport to the gallbladder andquicker excretion via MDR1 than 99mTc-TF. Therefore, we
found that 99mTc-MIBI is sensitive to the ABC transporter,
MDR1, in whole-body imaging. In the parathyroid and heart,accumulation did not change much over time, but 99mTc-MIBI

Pharm Res (2019) 36: 18

showed higher accumulation than 99mTc-TF because few ABC
transporters are expressed in normal conditions in the parathyroid and heart. Vrachimis et al. showed similar results as ours inliver and heart (21). Additionally, initial uptake of 99mTc-MIBI
may be increased compared with 99mTc-TF.
Time activity curves of SK-N-SH cells in SK-N-SH-bearing
mice from whole-body images in Fig. 4 showed that more
99m
Tc-MIBI underwent efflux from the tumors than 99mTcTF during the first 50 min after injection (Fig. 6A). Although
Gomes et al. also showed that export of 99mTc-MIBI is fasterthan that of 99mTc-TF in an in vitro study (8), we confirmed thisobservation in an in vivo study. The time activity curve of 99mTcMIBI shifted from fast export to slightly moderate export at
about 30 min after injection because we estimate that about halfof 99mTc-MIBI was metabolized in cancer cells and liver about
35 min after injection, and the metabolites of 99mTc-MIBI will
undergo minimal efflux from the tumors via MDR1 and
MRP1. MK-517 loading for MRP inhibition in SK-N-SH cells
provided the highest significant accumulation at about 35 min,whereas verapamil loading also yielded significantly higher accumulation than the control condition at about 30 min (Fig. 6B).
The results of our in vivo study emphasized that export of 99mTcMIBI is more sensitive to MRP1 than MDR1, and the metabolites of 99mTc-MIBI will be minimally exported via MDR1 and
MRP1. On the other hand, 99mTc-TF underwent gradual efflux
from the tumors via MDR1 and MRP1 in SK-N-SH cells overtime (Fig. 4A) because we estimate that 99mTc-TF was much
more stable at all scanning times. In the time activity curves of99m
Tc-TF (Fig. 6C), verapamil and MK-571 loading increased the accumulation compared to the control condition at 10 min and 50 min, respectively. This resultshows that export of 99mTc-TF is also more sensitive to
MRP1 than MDR1. However, the effect of both inhibitors was smaller than the effect on 99mTc-MIBI because
involvement of 99mTc-TF with MDR1 and MRP1–3
may be smaller than that of 99mTc-MIBI.
In clinical settings, MDR in solid cancers has been evaluated by comparing washout rates between the early timephase (10–30 min) and the late time phase (2–3 h) after injection of 99mTc-labeled compounds (4,5). If 99mTc-labeled compounds are metabolized in cancers and the liver completely
and immediately after injection, monitoring of MDR in cancers may be affected because metabolites of 99mTc-labeled
compounds may not be taken up in cancers and undergoefflux through MDR1 and MRP. In our study, 99mTc-MIBI
was metabolized about 32%, 26%, and 80% in SK-N-SH
cells, mouse liver, and human liver S9 at 30 min and about
36%, 36%, and 85% at 60 min after injection (Table II),
respectively. Perek and Le Jeune evaluated monoglutathionyl
conjugation in glioma cells with high levels of glutathione fordrug metabolism using both 99mTc compounds (22,23). They
showed that 99mTc-MIBI was metabolized to about 90% at
30 min, whereas 99mTc-TF was metabolized to about 20% at

Page 9 of 10 18

30 min. The amounts of metabolites of 99mTc-MIBI weresimilar to those of human liver S9, but higher than in SK-NSH cells because SK-N-SH cells may have less drug metabolizing enzyme including glutathione S-transferase than glioma
cells (23). On the other hand, little metabolism of 99mTc-TF
occurs in all tissues. These results were different from Jeune’s
results because 99mTc-TF may not be susceptible to metabolism by glutathione S-transferase in mice, human liver, and
SK-N-SH cells. Additionally, mouse and human liver
may have lower expression levels of glutathione Stransferase than glioma cells. Therefore, metabolites of99m
Tc-TF may not have been present in our study.
However, the influence of these metabolites will besmall because 99mTc-MIBI shows MDR in the earlytime phase before metabolism of 99mTc-MIBI occurs(Fig. 4a). On the other hand, 99mTc-TF imaging showed
accurate MDR using two scans in the early time phase andlate time phase because 99mTc-TF is stable (Table II).

CONCLUSION
The efflux mechanism and metabolism of 99mTc-labeled compounds differed in human cancer cells and SK-N-SH-bearing
mice. When cancers express MDR1 and MRP1, 99mTc-MIBI isexported via MDR1 and MRP1 (MRP1 > MDR1) at greaterlevels and more quickly compared to 99mTc-TF. 99mTc-TF isexported by not only MDR1 and MRP1 but also MRP2 and
MRP3, although MRP3 has a smaller influence than MRP1 and
MRP2 (MRP1 > MDR1; MRP1, 2 > MRP3). Because
99m
Tc-MIBI is metabolized in human-derived cancers
and human liver S9 fractions, clinical imaging to monitor MDR1 and MRP1 will be possible, and shorter
SPECT examination times may be possible by switching
to an earlier scan time (0.5–1 h etc.) for late phaseimaging at 2–3 h after injection. 99mTc-TF has highstability and reflects the function of MDR1 and MRP1–3.

ACKNOWLEDGMENTS AND DISCLOSURESThe 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 Researchfrom the Japan Society for the Promotion of Science (Nos.
25,293,260, 15 K09949, 15 K15452, and 16KK0020).

Publisher’s NoteSpringer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Pharm Res (2019) 36: 18

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