Nuclear Medicine and Biology 59 (2018) 16–21

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Nuclear Medicine and Biology
journal homepage: www.elsevier.com/locate/nucmedbio

Development of radioiodine labeled acetaminophen for speciﬁc,high-contrast imaging of malignant melanoma☆
Wen Jing Zhu a, Masato Kobayashi b,⁎, Kohei Yamada c, Kodai Nishi d, Kotaro Takahashi a, Asuka Mizutani e,f,
Ryuichi Nishii g, Leo G. Flores II h, Naoto Shikano i, Munetaka Kunishima c, Keiichi Kawai a,ja

Department of Health Sciences, College of Medical, Pharmaceutical and Health Sciences, Kanazawa University, Kanazawa, Japan
Wellness Promotion Science Center, College of Medical, Pharmaceutical and Health Sciences, Kanazawa University, Kanazawa, Japanc
Department of Pharmaceutical Sciences, College of Medical, Pharmaceutical and Health Sciences, Kanazawa University, Kanazawa, Japand
Department of Radioisotope Medicine, Atomic Bomb Disease Institute, Nagasaki University, Nagasaki, Japane
Graduate School of Medicine, Division of Health Science, Osaka University, Osaka, Japanf
Department of Radiology, Kanazawa University Hospital, Kanazawa, Japang
Molecular Imaging Center, National Institute of Radiological Sciences, Chiba, Japanh
Department of Cancer Systems Imaging, University of Texas M.D. Anderson Cancer Center, Houston, TX, USAi
Department of Radiological Sciences, Ibaraki Prefectural Sciences of Health Sciences, Ibaraki, Japanj
Biomedical Imaging Research Center, University of Fukui, Fukui, Japanba r t i c l e

i n f oArticle history:
Received 4 July 2017
Received in revised form 21 December 2017
Accepted 26 December 2017
Keywords:
Malignant melanoma
Acetaminophen
Radioiodine
SPECT imaging
Tyrosinasea b s t r a c t
Introduction: Due to its poor prognosis, speciﬁc imaging for early detection of malignant melanoma is strongly
desired. Although radioiodine labeled 4-hydroxyphenylcysteamine, which we previously developed, has good
afﬁnity for tyrosinase, an enzyme in the melanin metabolic pathway, image contrast of the melanoma:organ ratios is not sufﬁciently high for detection of primary melanoma and metastases at early injection times. In thisstudy, we developed radioiodine labeled acetaminophen (I-AP) for speciﬁc, high-contrast imaging of malignant
melanoma.
Methods: Radioiodine-125-labeled AP (125I-AP) was prepared using the chloramine-T method under no carrieradded conditions. Accumulation of radioactivity and the mechanism were evaluated in vitro using B16 melanoma cells incubated with 125I-AP or 14C(U)-labeled AP (14C-AP) with and without L-tyrosine as a substrate of tyrosinase, phenylthiourea as an inhibitor of tyrosinase, and thymidine as an inhibitor of DNA polymerase. The
biological distribution of radioactivity in B16 melanoma-bearing mice was evaluated to determine the accumulation of 125I-AP. The stability of 125I-AP over time was evaluated in mice.
Results: The labeling efﬁciency and radiochemical purity of 125I-AP were N80% and 95%, respectively. Accumulation of 125I-AP was higher than that of 14C-AP at 60 min of incubation in vitro. The afﬁnity of 14C-AP for tyrosinase
and DNA polymerase was higher than that of 125I-AP, whereas the Vmax of 125I-AP was higher than that of 14C-AP.
125
I-AP showed the highest accumulation in the gall bladder, and clearance from the blood and kidney was rapid.
Melanoma:muscle and melanoma:normal skin ratios of 125I-AP for imaging contrast were the highest at 15 minafter injection, whereas the melanoma:blood and melanoma:bone ratios gradually increased over time. 125I-AP
remained stable for 60 min after injection in mice.
Conclusions: 125I-AP has afﬁnity for tyrosinase and high image contrast at early time points after injection. Therefore, 123I-AP imaging has great potential for speciﬁc, high-contrast detection of malignant melanoma.
Advances in knowledge: 123I-AP will provide speciﬁc, high-contrast imaging for malignant melanoma at early injection times.
Implications for patient care: 123I-AP has good potential for the diagnosis of malignant melanoma compared with
123
I-labeled 4-hydroxyphenylcysteamine, which we previously developed.
© 2017 Elsevier Inc. All rights reserved.

☆ Financial support: This study was partly funded by Grants-in-Aid for Scientiﬁc Research from the Japan Society for the Promotion of Science (Nos. 25293260, 15K09949,
15K15452, and 16KK0200) and the Program of the Network-Type Joint Usage/ResearchCenter for Radiation Disaster Medical Science of Hiroshima University, Nagasaki University, and Fukushima Medical University (105-N).
⁎ Corresponding author at: Wellness Promotion Science Center, College of Medical,
Pharmaceutical and Health Science, Kanazawa University, 5-11-80 Kodatsuno, Kanazawa
920-0942, Japan.
E-mail address: kobayasi@mhs.mp.kanazawa-u.ac.jp (M. Kobayashi).
https://doi.org/10.1016/j.nucmedbio.2017.12.008
0969-8051/© 2017 Elsevier Inc. All rights reserved.

1. Introduction
The 5-year survival rate with stage IV metastatic melanoma is currently b20% because no effective treatments have been established [1,2]. Because survival is associated with an early stage at detection, specific detection of the primary and metastatic malignant melanoma tumors

W.J. Zhu et al. / Nuclear Medicine and Biology 59 (2018) 16–21

is strongly desired. Melanomas develop due to an increase in activity ofthe melanin biosynthetic pathway in melanin-producing cells (melanocytes) [3]. Melanin biosynthesis involves conversion of tyrosine to dopa,
and then subsequent oxidation of dopa to dopaquinone by the tyrosinase enzyme. Therefore, radiopharmaceuticals with afﬁnity for tyrosinase are potential speciﬁc imaging agents.
Single photon emission computed tomography (SPECT) [4–14] andpositron emission tomography (PET) imaging [14–16] have been used
to detect the primary melanoma and metastases. With SPECT imaging,some radiopharmaceuticals (melanocortin-1 receptor-targeting peptide [11] and iodobenzamide [7, 12, 13]) can image melanomas bytargeting tumor receptors. Melanocortin-1 receptor-targeting peptide
may not be useful for clinical imaging of melanoma because of highrenal accumulation, although methods to reduce kidney uptake havebeen suggested [11]. 123I-N-(2-diethylaminoethyl)-2-iodobenzamide,
an iodobenzamide derivative, showed low uptake for detection of melanoma metastases in a clinical study [13]. Therefore, imaging with thisderivative would require a very high radiation dose in clinical studies.
Thus, these radiopharmaceuticals that allow speciﬁc imaging of melanoma have not been fully developed. On the other hand, 18Fﬂuorodeoxyglucose (FDG)-PET imaging was useful for detection of the
primary melanoma and metastases, but FDG-PET does not have highenough speciﬁcity to detect melanoma [14, 15], and the availability of
PET is still lower than that of SPECT because many facilities do nothave a cyclotron for synthesis of PET tracers.
We previously developed 125I-labeled 4-hydroxyphenyl-L-cysteine[4] and radioiodine labeled 4-hydroxyphenylcysteamine (I-PCA) [5]that have good afﬁnity for tyrosinase. However, the image contrast of
the melanoma:organ ratios might not be sufﬁciently high for detectionof primary and metastatic melanoma at early injection times. Acetaminophen (AP) for chemotherapy is selectively bioactivated by tyrosinase
to form active quinone metabolites that are toxic to melanoma cells.
The selective toxicity and efﬁcacy of AP for melanoma has been previously reported in B16 melanoma tumor-bearing C57BL/6 mice [17, 18]. However, the effect of chemotherapeutic drugs such as AP cannot be
visualized. We hypothesize that AP may be a good molecule for detection of primary and metastatic melanoma. In this study, we developed

17

radioiodine labeled AP (I-AP) (Fig. 1), which is a speciﬁc imaging radiotracer of malignant melanoma.
2. Materials and methods
2.1. Preparation of 125I-AP and 127I-AP
Radioiodine-125-labeled AP (Nakalai Tesque, Kyoto, Japan) ( 125IAP) was synthesized with the chloramine-T (Nakalai Tesque) method
under no carrier-added conditions [4, 5]. In brief, 4 mM chloramine-T
was added to a mixture of 10 mM AP in ethanol (≥99.5%, SigmaAldrich, St. Louis, MO) and 3.7–37 MBq Na 125I (American Radiolabeled
Chemicals, Inc., St. Louis, MO) in the absence of carriers. Thirty minutes
later, 280 mM sodium sulﬁte (Sigma-Aldrich) in 0.05 M phosphate buffer (pH 6.2) was added to stop the reaction. The labeling efﬁciency of125
I-AP was determined with thin-layer chromatography (TLC, Silica
gel 60 F254; Merck Millipore, Darmstadt, Germany) with the solvent system of hexane:ethylacetate:diethylether at 1:4:1 (Rf value: 125I-AP;
0.15–0.25, 125I −; 0.0) and/or methanol:acetic acid at 100:1 (Rf value:125
I-AP; 0.70–0.80, 125I −; 0.90–0.95). The Rf value for TLC and thepeak position for high performance liquid chromatography (HPLC) of
125
I-AP were conﬁrmed using Na 127I-labeled AP (127I-AP). To synthesize
127
I-AP, AP in a mixture of 0.1 M CHCl3 and 0.8 M K2CO3 was reactedwith 0.8 M Na 127I in 0.1 M ethanol at room temperature. About 10
min later, the solution was cooled to about 0 °C, and then 0.1 M CHCl3
was added to stop the reaction. After washing with 1 M HCl and brine,
the ﬁnal solution was dried by Na2SO4. 127I-AP was obtained by the preparative TLC method. The structure of 127I-AP was conﬁrmed by gas
chromatography-mass spectrometry (MS, JMS-700 electron impact;
Japan Electron Optics Laboratory, Tokyo, Japan).
The radiochemical purity of 125I-AP was examined with HPLC
(Hitachi, Ibaraki, Japan) using a 5C18-MS-II column (Cosmosil, Nakalai
Tesque), a combination gamma counter, and 225 nm ultraviolet light.
The initial solvent was methanol:50 mM KH2PO4 (pH 4.7) at 20:80 for
the ﬁrst 10 min after injection, and then the same solvent at a 50:50
ratio for the next 10 min as the eluent at a ﬂow rate of 1.0 mL/min.
2.2. B16 melanoma cells
Mouse skin melanoma B16 cells were obtained from RIKEN Cell
Bank in Japan and cultured in Dulbecco's Modiﬁed Eagle's Medium(Sigma-Aldrich) supplemented with 10% fetal bovine serum (Dainippon
Sumitomo, Osaka, Japan) at 37 °C in a 5% CO2 incubator.
2.3. In vitro assays with B16 melanoma cells without and with a substrate
and inhibitors

Fig. 1. Structures of 14C-AP (A) and 125I-AP (B). On the 14C-AP structure (A), * means 14Clabeling.

In vitro assays were performed using the methods of Kobayashi
et al., with some modiﬁcations [5, 19]. 14C(U)-labeled AP ( 14C-AP)
was purchased from American Radiolabeled Chemicals Inc., and 125IAP was synthesized by the method described above. Brieﬂy, B16 melanoma cells were seeded into 24-well cell culture multiwell plates at adensity of 5 × 105 cells/well. Assays were conducted 24 h after seeding.
After the culture medium was removed, each well was incubated with
0.5 M HEPES buffer (pH 7.4) for 10 min at 37 °C. Then, 18.5 kBq/well
125
I-AP or 14C-AP was added and incubated for 5, 15, 30, 60, and 120
min at 37 °C as the control condition. Michaelis-Menten analyses werecarried out using AP as a parent compound, L-tyrosine (Sigma-Aldrich)as a tyrosinase substrate, N-phenylthiourea (PTU; Sigma-Aldrich) as
an inhibitor of tyrosinase, and thymidine (Nakalai Tesque) as an inhibitor of DNA polymerase at concentrations of 0, 0.01, 0.1, 0.5, 1, 1.5, 2.0,
3.0, 4.0, and 5.0 mM [20, 21]. B16 melanoma cells were incubated for
30 min with 125I-AP or 14C-AP plus one of these inhibitors at 37 °C. At
the end of the incubation, each well was rapidly washed twice with
500 μL of 0.5 M HEPES buffer. Cells were then solubilized in 500 μL of
0.1 M NaOH. The 125I-AP radioactivity that accumulated in B16

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W.J. Zhu et al. / Nuclear Medicine and Biology 59 (2018) 16–21

for 5 min. A total of 30 μL perchloric acid was added to the supernatant,which was centrifuged again, after which the ﬁnal supernatant was
spotted onto the TLC plate as plasma. Krebs-Ringer phosphate buffer(pH 7.4) was added to the liver samples, followed by homogenization
with an ultrasonic homogenizer (SONIFIER250, Branson, MO, USA).
Then, ethanol was added to the homogenate to remove proteins, andthe sample was centrifuged at 18,000 ×g at 4 °C for 5 min. The ﬁnal supernatant was spotted onto the TLC plate, and the TLC spots were developed using the above-mentioned solvents. After development andcomplete drying, the TLC plates were cut into 21 fractions, and the radioactivity associated with each fraction was quantiﬁed using a γ-ray
counter. The fraction ratios of 125I− and 125I-AP were calculated by dividing the radioactive counts for each fraction by the total radioactivity
count.

Fig. 2. In vitro assays of 14C-AP and 125I-AP in B16 melanoma cells. 18.5 kBq/well 125I-AP(mass; 278.1) or 18.5 kBq/well 14C-AP (mass; 151.2) was incubated for 5, 15, 30, 60, and
120 min. 14C-AP was taken up earlier into the tumor cells and reached a steady state
after about 30 min of incubation. In contrast, 125I-AP was taken up gradually, and theaccumulation of 125I-AP exceeded that of 14C-AP after about 60 min of incubation. Data
are the mean ± standard deviation. **P b .01 and *P b .05 between 125I-AP and 14C-AP.

2.6. Statistical analysis

melanoma cells was measured with a gamma counter (ARC-380;
Hitachi-Aloka Medical, Tokyo, Japan), and that of 14C-AP was mixed
with scintillation ﬂuid (Ultima Gold, PerkinElmer, Inc., Waltham, MA)
and measured with a liquid scintillation counter (LSC-5100; HitachiAloka Medical). B16 melanoma cells were detached with trypsin, andthe protein in the cells was measured using a protein assay. All experimental conditions were examined with quadruplicate assays.

3. Results

2.4. Biological distribution of 125I-AP in B16 melanoma-bearing mice

Data are presented as mean and standard deviation (SD). P values
were calculated using a two-tailed Student's t-test. Results were considered to be signiﬁcant at P b 0.05.

3.1. Preparation of 125I-AP
MS data revealed an MH(+) of 278, which matched the calculated
MH(+) of 278 for C8H8INO2. With TLC analysis, using the results of
the Rf values for 127I-AP, the Rf values of 125I-AP and 125I − were 0.150.25 and 0.00, respectively. 125I-AP was labeled with an efﬁciency of
N80%. With HPLC analysis, the retention times were about 3 min for

All animal studies were conducted following approval by the Animal
Care Committee of Kanazawa University. C57BL6 male mice (5 weeksold) were transplanted with B16 melanoma cells (5 × 10 5 cells/100μL) into the thigh of the mice [6]. The tumors appeared and increased
gradually after about 10 days. The mice were housed for 2 weeks
under a 12-h light/12-h dark cycle with free access to food and water.
Before the bio-distribution experiment, B16 melanoma-bearing mice
were fasted overnight with only water available. 125I-AP (37 kBq) was
injected into the tail vein of B16 melanoma-bearing mice, and ﬁve
mice each were killed at 5, 10, 15, 30, and 60 min after injection. Afterblood sampling via cardiocentesis, the brain, pancreas, spleen, stomach,small intestine, kidney, liver, heart, lung, thyroid, gall bladder, skin,muscle, bone, and melanoma were removed. Radioactivity in weighed
tissue samples was measured using a gamma counter (ARC-380;
Aloka, Tokyo, Japan). Data were expressed in terms of the injected
dose (%ID/organ) for stomach, small intestine, and thyroid and injected
dose per weight of tissues (%ID/g tissue) for the other organs.

%ID=organ ¼ ðradioactivity of tissue ½cpm=radioactivity of injection ½cpmÞ  100%ID=g ¼ ð%ID=weight of tissues ½gÞ  100

2.5. Stability analysis in mice125

I-AP was prepared to a speciﬁc activity of 37 MBq/mL by additionof saline. Fasted C57BL6 male mice (6–8 weeks old) were administered
125
I-AP via the tail vein (3.7 MBq/mouse). At 5, 10, 15, 30, and 60 min
after injection, mice were euthanized with isoﬂurane (n = 3), andblood and liver were collected and analyzed with TLC. Brieﬂy, heparin
was added to 300 μL blood, which was centrifuged at 18,000 ×g at 4 °C

Fig. 3. Michaelis-Menten analyses using 125I-AP (A) or 14C-AP (B) with cold AP, L-tyrosine,
PTU, and thymidine in B16 melanoma cells. Km values of 125I-AP with AP, L-tyrosine, PTU,
and thymidine were 2.56 ± 0.31, 2.73 ± 0.39, 1.93 ± 0.29, and 2.10 ± 0.41 mM,
respectively, whereas those of 14C-AP were 0.71 ± 0.08, 2.47 ± 0.67, 1.23 ± 0.30, and1.26 ± 0.18 mM, respectively. Vmax values of 125I-AP with AP, L-tyrosine, PTU, andthymidine were 490.3 ± 27.3, 473.5 ± 32.8, 433.1 ± 27.0, and 413.9 ± 35.2 pmol/min/
mg protein, respectively, whereas those of 14C-AP were 242.3 ± 7.6, 349.5 ± 44.2, 284.7± 23.7, and 296.2 ± 15.1 pmol/min/mg protein, respectively.

W.J. Zhu et al. / Nuclear Medicine and Biology 59 (2018) 16–21

19

Table 1
Biological distribution of 125I-AP in B16 melanoma-bearing C57BL6 mice.
Organ or tumor

Blood
Brain
Pancreas
SpleenStomacha
Small intestinea
Kidney
Liver
Heart
Lung
Thyroida
Gall bladderSkin
MuscleBoneMelanoma

Time (minutes)
5

10

15

30

60

13.51 ± 1.0
0.64 ± 0.1
3.50 ± 0.5
2.95 ± 0.3
0.78 ± 0.0
6.89 ± 0.3
17.32 ± 2.1
5.20 ± 0.6
4.34 ± 0.5
13.09 ± 0.1
0.08 ± 0.0
22.28 ± 8.4
4.21 ± 0.8
1.71 ± 0.5
2.01 ± 0.7
2.43 ± 0.1

8.92 ± 1.4
0.26 ± 0.1
4.25 ± 0.2
2.97 ± 0.2
0.69 ± 0.2
6.08 ± 0.1
12.19 ± 2.3
4.51 ± 0.9
3.28 ± 0.6
8.24 ± 1.5
0.08 ± 0.0
27.77 ± 2.0
4.24 ± 0.7
1.24 ± 0.8
2.07 ± 0.3
3.61 ± 0.2

6.42 ± 1.1
0.21 ± 0.0
2.96 ± 0.3
2.12 ± 0.4
1.00 ± 0.2
6.53 ± 0.6
9.94 ± 2.4
3.50 ± 0.6
2.08 ± 0.3
6.26 ± 0.7
0.07 ± 0.0
18.94 ± 6.4
3.05 ± 0.3
0.79 ± 0.1
1.35 ± 0.5
4.65 ± 1.6

2.82 ± 0.0
0.09 ± 0.0
1.03 ± 0.3
0.73 ± 0.2
1.21 ± 0.1
3.31 ± 0.3
3.39 ± 0.7
1.88 ± 1.6
0.87 ± 0.2
3.42 ± 0.7
0.05 ± 0.0
72.06 ± 20.1
2.38 ± 0.2
0.56 ± 0.8
0.48 ± 0.4
2.30 ± 0.2

1.10 ± 0.3
0.05 ± 0.0
1.92 ± 1.7
0.63 ± 0.1
0.76 ± 0.1
8.93 ± 2.2
1.30 ± 0.2
1.26 ± 0.5
0.31 ± 0.0
1.68 ± 0.0
0.01 ± 0.0
47.34 ± 17.2
0.96 ± 0.0
0.28 ± 0.3
0.18 ± 0.0
1.06 ± 0.1

Percent injected dose per g tissue (%ID/g), mean ± standard deviation of data from ﬁve mice.
a%ID/organ was calculated by dividing the radioactivity in the tissue by the radioactivity that was injected.
125 −I and 16–17 min for 127I-AP and 125I-AP. Non-carrier-added 125I-AP
was obtained with a radiochemical purity of N95% after puriﬁcation.

3.2. In vitro assays with B16 melanoma cells without and with a substrate
and inhibitors
The comparison of the accumulation of 125I-AP and 14C-AP in B16
melanoma cells is shown in Fig. 2. 125I-AP was gradually taken up by
the cells, and accumulation of 125I-AP exceeded that of 14C-AP after
about 60 min of incubation. In contrast, 14C-AP was taken up into thetumor cells early and reached a steady-state after about 30 min of
incubation.
Fig. 3 shows the Michaelis-Menten analyses using 125I-AP (A) or 14CAP (B) with cold AP, L-tyrosine, PTU, and thymidine in B16 melanoma
cells. Km values of 125I-AP with AP, L-tyrosine, PTU, and thymidine
were 2.56 ± 0.31, 2.73 ± 0.39, 1.93 ± 0.29, and 2.10 ± 0.41 mM, respectively, whereas those of 14C-AP were 0.71 ± 0.08, 2.47 ± 0.67,
1.23 ± 0.30, and 1.26 ± 0.18 mM, respectively. Vmax values of 125I-AP
with AP, L-tyrosine, PTU, and thymidine were 490.3 ± 27.3, 473.5 ±
32.8, 433.1 ± 27.0, and 413.9 ± 35.2 pmol/min/mg protein, respectively, whereas those of 14C-AP were 242.3 ± 7.6, 349.5 ± 44.2, 284.7 ±
23.7, and 296.2 ± 15.1 pmol/min/mg protein, respectively.

3.3. Biological distribution of 125I-AP in B16 melanoma-bearing mice
Table 1 summarizes the biological distribution of 125I-AP in B16
melanoma-bearing mice. Accumulation of 125I-AP in the melanoma
was the highest at 15 min after injection. 125I-AP accumulation was
highest in the gall bladder, and clearance of 125I-AP from the bloodand kidney was rapid.
Fig. 4 shows the melanoma:blood ratios, melanoma:normal skin ratios,melanoma:muscle ratios, and melanoma:bone ratios in B16 melanomabearing mice at each time after injection. The melanoma:muscle ratios
and melanoma:normal skin ratios were the highest at 15 min after injection,
whereas the melanoma:blood ratios and melanoma:bone ratios gradually
increased over time.
3.4. Stability analysis in mice
Table 2 shows the stability over time of 125I-AP in blood and liver ofmice. In the blood, 125I-AP showed high stability over time, but in theliver, 125I-AP was slightly metabolized at 5 min after injection. Then,
125
I-AP remained stable during 60 min after injection.
4. Discussion
Speciﬁc imaging of primary and metastatic melanoma is importantfor patient survival because detection and treatment at earlier stages
are more effective. As general clinical examination methods for detection of melanoma, visual inspection [22], dermoscopy [23], and ultrasonography [23, 24] are useful for detection of mainly primary melanoma,
whereas computed tomography [25, 26] and magnetic resonance imaging [25] are effective for detection of primary and metastatic melanoma
in the whole body. Although these methods are very useful for ﬁnding
structural changes in melanoma, detection of primary and metastaticmelanoma at earlier stages may be insufﬁcient. Nuclear medicine imaging, which we focused on in this study, can detect biological functionalchanges in melanoma before structural changes in the melanoma
occur. We developed SPECT imaging radiotracers with high availability
and speciﬁcity for detection of the primary melanoma and metastases.
Table 2
Stability analysis of 125I-AP in blood and liver of mice.

Fig. 4. Ratios of melanoma:blood, melanoma:skin, melanoma:muscle, andmelanoma:bone in B16 melanoma-bearing C57BL6 mice at 5, 10, 15, 30, and 60 min
after 37 kBq 125I-AP injection. The melanoma:muscle and melanoma:normal skin ratios
were the highest at 15 min after injection, whereas the melanoma:blood andmelanoma:bone ratios increased over time. All values were obtained using ﬁve miceeach. Data are the mean ± standard deviation.

Time (minutes)

5

10

15

30

60

Blood (%)
Liver (%)

94.8 ± 7.2
90.9 ± 6.5

94.4 ± 5.4
89.3 ± 5.1

94.2 ± 8.2
90.6 ± 6.8

93.8 ± 1.4
89.9 ± 4.7

94.1 ± 5.1
88.2 ± 5.5

All data are the mean ± standard deviation from triplicate measurements.

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W.J. Zhu et al. / Nuclear Medicine and Biology 59 (2018) 16–21

Tyrosine analogues, which are substrates of the tyrosinase enzyme,
are good candidates for drugs for melanoma-targeting chemotherapies[27]. In addition, the afﬁnity of tyrosine analogues for detection of elevated tyrosinase enzyme activity in melanin biosynthesis indicates thepossibility of application for speciﬁc detection of melanoma. Although
we previously developed two radiotracers, which are good substrates
for tyrosinase and are retained longer in malignant melanoma cells [4,5], improved image contrast due to the tumor:organ ratios is requiredfor clinical imaging of melanoma for high-throughput examination.
Vad et al. reported that AP has afﬁnity for tyrosinase in melanoma
cells [17, 18]. We hypothesized that 125I-AP would be a useful SPECT imaging agent for speciﬁc detection of melanoma. In our previous study,
iodination of AP was estimated at the ortho position to the 4-hydroxyl
group [4, 5, 28]. In our in vitro study, 125I-AP accumulation was signiﬁcantly higher than that of 14C-AP at 60 min of incubation, although accumulation of 14C-AP was higher than that of 125I-AP at early incubation
times (Fig. 2). As the in vitro study was conducted using equal amountsof radioactivity with about 300-fold different speciﬁc activities (14C-AP:
1.85–2.22 GBq/mmol and 125I-AP: about 600 GBq/mmol), the relative
uptake velocities into melanoma cells may be much different. In ourprevious study, although 125I-L-tyrosine had higher afﬁnity for system
L amino acid transporters than 14C-L-tyrosine due to the effect ofradioiodine labeling, accumulation of 14C-L-tyrosine was higher thanthat of 125I-L-tyrosine at early incubation times [28]. In this study, we estimate that accumulation of 14C-AP was higher than that of 125I-AP due
to the effect of the accumulation mechanism.
In Fig. 3, Michaelis-Menten analyses were performed in B16 melanoma cells using 125I-AP (A) or 14C-AP (B) with cold AP as the parent compound, L-tyrosine and PTU as a substrate and inhibitor of tyrosinase,respectively, and thymidine as an inhibitor of DNA polymerase. Km
values of 125I-AP with AP, L-tyrosine, PTU, and thymidine were 2.56 ±
0.31, 2.73 ± 0.39, 1.93 ± 0.29, and 2.10 ± 0.41 mM, respectively,
whereas those of 14C-AP were 0.71 ± 0.08, 2.47 ± 0.67, 1.23 ± 0.30,
and 1.26 ± 0.18 mM, respectively. Thus, the afﬁnity of 14C-AP for tyrosinase and DNA polymerase was higher than that of 125I-AP in B16 melanoma cells. From these results, we estimated that accumulation of 14CAP was higher than that of 125I-AP at early incubation times (Fig. 2).
Vmax values of 125I-AP with AP, L-tyrosine, PTU, and thymidine were490.3 ± 27.3, 473.5 ± 32.8, 433.1 ± 27.0, and 413.9 ± 35.2 pmol/min/
mg protein, respectively, whereas those of 14C-AP were 242.3 ± 7.6,
349.5 ± 44.2, 284.7 ± 23.7, and 296.2 ± 15.1 pmol/min/mg protein, respectively. Thus, the Vmax of 125I-AP for tyrosinase and DNA polymerase
was higher than that of 14C-AP in B16 melanoma cells. From these results, we also estimated that 125I-AP accumulation was signiﬁcantly
higher than that of 14C-AP at 60 min of incubation (Fig. 2).
In the biological distribution experiment with B16 melanomabearing mice (Table 1), accumulation of 125I-AP in the melanoma was
the highest at 15 min after injection, whereas 125I-PCA showed thehighest accumulation at 30 min after injection [5]. Therefore, 125I-AP
may allow earlier acquisition than 125I-PCA in whole body imaging. In
addition, 125I-AP was washed out faster from blood and was excreted
as quickly by the kidney as 125I-PCA [5]. The washout of 125I-AP was
also faster than that of other radioligands for melanoma imaging [7,10–12]. Melanoma:muscle and melanoma:normal skin ratios for imaging contrast were the highest at 15 min after injection (Fig. 4). The average melanoma:muscle ratio (5.9) for primary melanoma andmelanoma:bone ratio (3.5) for melanoma metastases of 125I-AP weresigniﬁcantly greater than those of 125I-PCA (2.1, 1.5, respectively),
whereas the average melanoma:blood (0.7) and melanoma:normalskin ratios (1.5) for 125I-AP were similar to those of 125I-PCA (0.8 and
1.8, respectively) [5]. From results of these melanoma:organ ratios,
125
I-AP is likely to be an effective radiotracer for speciﬁc, high-contrast
imaging of malignant melanoma. However, radioiodide-131-labeled
AP may not be applicable in radionuclide therapy because themelanoma:muscle and melanoma:normal skin ratios gradually decreased 15 min after injection.

Regarding the stability over time, 125I-AP was quite stable with
about 95% in mouse blood and 90% in mouse liver remaining 60 min
after injection. 125I-AP is minimally deiodinated because it does not accumulate in the thyroid, and thus, metabolites of 125I-AP may have little
inﬂuence on accumulation of 125I-AP in melanoma.
5. Conclusion
125

I-AP has afﬁnity for tyrosinase and high imaging contrast at earlytimes after injection. Therefore, 123I-AP imaging has great potential for
speciﬁc, high-contrast imaging of malignant melanoma.
Conﬂict of interest statement
There is no conﬂict of interest.
Acknowledgments
The authors would like to thank Mikie Ohtaki and the other laboratory staff of Kanazawa University. This study was partly funded by
Grants-in-Aid for Scientiﬁc Research from the Japan Society for the Promotion of Science (Nos. 25293260, 15K09949, and 15K15452) and theProgram of the Network-Type Joint Usage/Research Center for Radiation
Disaster Medical Science of Hiroshima University, Nagasaki University,and Fukushima Medical University.
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