Nuclear Medicine Communications, 2003, 24, 575±582

A novel radiopharmaceutical for detection ofmalignant melanoma, based on melanin formation:
3-iodo-4-hydroxyphenyl-L-cysteine
R. NISHII,1,* K. KAWAI,2 L. GARCIA FLORES II,2 H. KATAOKA,3 S. JINNOUCHI,1
S. NAGAMACHI,1 Y. ARANO4 and S. TAMURA1
1

Department of Radiology, 2Central Research Laboratories, and 3Department of 2nd Pathology, Miyazaki Medical
College, 5200 Kihara, Kiyotake-cho, Miyazaki-gun, Miyazaki 889-1692, Japan; and 4Faculty of Pharmaceutical
Sciences, Chiba University, Yayoi-cho, Chiba 263-8522, Japan
Received 2 May 2002, in revised form 7 October 2002 and accepted 21 October 2002

Summary
The aim of this study was to develop a new artificial amino acid radiopharmaceutical labelled withradioiodine for detection of malignant melanoma, based on melanin formation. By considering the affinity
for tyrosinase, a starting enzyme on the branching point to melanin biosynthesis, 3-[125I]iodo-4hydroxyphenyl-L-cysteine (125I-L-PC) was synthesized and evaluated biologically. Labelling of 125I-L-PC
using the chloramine-T method was carried out conveniently and efficiently in a short period of time, withhigh specific activity. In a biodistribution study, 125I-L-PC showed a low accumulation in normal tissue andrelative retention in B16 melanoma. A high contrast image of peripheral tumour was obtained during
autoradiography. During an in vitro accumulation study, inhibition of 125I-L-PC with a tyrosinase inhibitor
suggested interaction of this tracer with tyrosinase. It indicates that the uptake mechanism of 125I-L-PC tomelanoma tissue was dependent on high tyrosinase activity in melanoma cells. Thus, 125I-L-PC appears to
be a promising radioiodinated amino acid radiopharmaceutical for imaging malignant melanoma in
relation to melanin formation, namely specific metabolism with high tyrosinase activity. (# 2003
Lippincott Williams & Wilkins)
Keywords: 3-[125I]iodo-4-hydroxyphenyl-L-cysteine, malignant melanoma, tyrosinase, amino acid radiopharmaceutical.

Introduction
Melanin biosynthesis of melanocytes is an essential
metabolic pathway for melanomas. It consists of conversion of tyrosine to dopa and then to dopaquinone bytyrosinase oxidation (Fig. 1). In malignant melanoma,
melanin formation is associated with high tyrosinase
activity and has been the target of antimelanoma
chemotherapeutic agents [1]. Graham et al. [2] and Wick[3] reported a possible mechanism of selective toxicity ofcatechols to melanoma cells. A similar attempt to use a*Address all correspondence to Dr Ryuichi Nishii, Department ofRadiology, Miyazaki Medical College, 5200 Kihara, Kiyotake-cho,Miyazaki-gun, Miyazaki 889-1692, Japan.
e-mail: rnisii@post1.miyazaki-med.ac.jp0143±3636/03 # 2003 Lippincott Williams & Wilkins

melanin precursor as an antitumour agent has also beenreported [4].
As antimelanoma chemotherapeutic agents that are
sulfur homologues of tyrosinase substrates, 4-hydroxyphenyl-L-cysteine, 4-hydroxyphenyl-L-cysteamine, and its
N-acetyl and N-propionyl derivatives were synthesizedby Ito et al. [5] and Miura et al. [6]. These compounds havealso been reported to have selective cytotoxicity to humanneoplastic cells, especially to tyrosinase positive melanoma cells. However, there is no irreversible damage toamelanotic, tyrosinase negative cells [5±7].
In recent years, the contribution of nuclear medicine tooncology has expanded, particularly in the field of
tumour imaging. Where radiological techniques aim atanatomical delineation of tumour, nuclear medicine
specializes on its physiology. Thereby the focus ofinterest is shifting from tumour detection to tumour
DOI: 10.1097/01.mnh.0000071254.54690.86

576

Nishii et al.

HO

CH2

CH COOH
MelaninNH2
L-Tyrosine

O
Tyrosinehydroxylase

Tyrosinase
O

CH2

CH

COOH

NH2
HO
DopaquinoneHO

CH2 CH

COOH

NH2
HO
Dopa
HO

CH2

CH2

NH2

Dopa Decarboxylase
Dopamine

Fig. 1. Metabolic pathway of melanin formation.

characterization [8]. A number of radiopharmaceuticals
with affinity to melanin have been synthesized, but few
have achieved clinical application for early detection ofmetastasis, staging, follow-up and radionuclide therapy
of melanoma [8].
The authors have developed and investigated radioiodinated tyrosine derivatives such as 3-[123I]iodo-amethyl-L-tyrosine (123I-L-AMT) [9, 10]. It has been provento be a metabolically stable radiopharmaceutical and has
high tissue affinity to the amino acid active transport.
123
I- L-AMT also has been introduced for imaging
tumours [11]. It has been reported to be taken up in
brain tumour, reflecting amino acid transport and issuitable for single photon emission computed tomography (SPECT) studies [12].
For melanoma specific detection based on its melaninbiosynthesis, development of a new radiopharmaceuticalthat could interact with elevated tyrosinase activity isessential. 4-Hydroxyphenyl-L-cysteine (4-L-PC), a tyrosine derivative with a phenol moiety that provides a site
for easy iodination, was selected as a tyrosinase substrateanalogue. Introduction of iodine and sulfur to the Ltyrosine molecule would give metabolic stability andrapid renal excretion [9, 13]. This would lead to low
background and high contrast in tumour imaging. 3Iodo-4-hydroxyphenyl-L-cysteine (Fig. 2), a new artificial
amino acid radiopharmaceutical labelled with radioiodine, which can be widely available for SPECT studies,
was prepared and evaluated.

Nuclear Medicine Communications (2003) 24

Materials and methods
Synthesis of 4-hydroxyphenyl-L-cysteine
4-hydroxyphenyl-L-cysteine (4-L-PC) was synthesizedaccording to the modified method described by Miuraet al. [6]. All chemicals used were of reagent grade. A
mixture of 28.2 g (300 mmol) of phenol and 18.0 g(75 mmol) of L-cystine was added in 500 ml of 47%
HBr and refluxed for 2 h (Fig. 2). The solution was
evaporated with 15 ml of thioglycolic acid. The residue
was dissolved in water and was adjusted to approximately pH 5 with sodium acetate. After a week ofrefrigeration, colourless plates were obtained. Isomeric
purity was confirmed by high-performance liquid chromatography (HPLC) using the modified method of
Somayaji et al. [14] which was performed on a Nova
Pak C18 (Waters) column. The 4-L-PC was separated fromthe 2-hydroxy isomer using water as the eluent.
The structure of 4-L-PC was confirmed by 1H NMR,
mass spectrometry and elemental analysis. For 1H NMR:
d 3.33 (2H, d, J = 5.5 Hz, CH2), 4.28 (1H, t, J = 5.5 Hz, CH),
6.95 (2H, d, J = 8.5 Hz, Ar-H2 and H6), 7.47 (2H, d,
J = 8.5 Hz, Ar-H3 and H5). For mass spectrometry: MH(+) 214. For elemental analysis, calculated for
C9H11NO3S: H 5.20%, C 50.69%, N 6.57%, O 22.52%, S
15.01%. Found: H 5.09%, C 49.53%, N 6.36%.
It was clear that only the L-configuration of the aminoacid was obtained, since a single peak was found with theA new radiopharmaceutical for detection of melanoma
S

577

CH

CH2

COOH

NH2

HO
S

CH COOH

CH2

NH2
L-Cystine

Phenol
HBr

HO

S

CH2

Reflux, 2 h

CH

COOH

NH2
4-Hydroxyphenyl-L-cysteine(4-L-PC)
I
I–/Chloramine-T
HO

S

CH2

CH

COOH

NH2

RT, 5 min

3-Iodo-4-hydroxyphenyl-L-cysteine(I-L-PC)

Fig. 2. Chemical reaction and radioiodination of 3-iodo-4-hydroxyphenyl-L-cysteine. RT, room temperature.

chiral HPLC, performed on Crownpak CR (7) (Daicel)
column. The retention time of 4-L-PC at a flow rate of
0.5 ml×min71 was 16 min.
Preparation of 3-[125I]iodo-4-hydroxyphenyl-L-cysteine
125

I-L-PC was prepared by the chloramine-T method
under a no-carrier added condition [9] (Fig. 2). Chloramine-T (Aldrich) at a concentration of 1.061078 mol in
10 ml of 0.05 M phosphate buffer (pH 8.5) was added to a
mixture of 4-L-PC (1.061077 mol) in 10 ml of phosphoricacid and no-carrier added Na125I (Amersham) in 80 ml of
0.4 M phosphate buffer (pH 8.5). Five minutes later,
1.061078 mol of sodium metabisulfite in 10 ml of 0.05 M
phosphate buffer (pH 8.5) was added to stop the reaction.
125
I-L-PC was purified by HPLC, performed on Nova
Pak C18 (Waters) column using 15% v/v acetic acid aseluent at a flow rate of 0.5 ml×min71 (retention times: 125I125
L-PC, 38 min;I, 6 min; unlabelled 4-L-PC, 28 min).
The labelling efficiency and radiochemical purity were
examined by thin-layer chromatography using silica gel(Merck; Art. 5553) and three solvent systems: methanol/
acetic acid, 100 : 1 (RF values: 125I-L-PC, 0.45±0.55; 125I,
0.70±0.80); methanol/10% ammonium acetate, 10 : 1 (RFvalues: 125I-L-PC, 0.50±0.60; 125I, 0.75±0.85); and ethanol/ammonia solution (28%), (Nacalai Tesque), 3 : 1 (RFvalues: 125I-L-PC, 0.40±0.50; 125I, 0.65±0.75).
Tumour cells
B16 melanoma cells obtained from the Riken Cell Bank
were cultured in Dulbecco's Modified Eagle's Medium(DMEM) (Sigma) medium containing 10% of fetal bovine
serum (Gibco). B16 melanoma was maintained in ourlaboratory by serial transplantation in C57BL6 mice for 2
week intervals. The tumour was dissociated in saline
solution. Then the suspension was centrifuged at 5006g
for 10 min, and the sediment was suspended in saline.
Melanoma cells (16106) in 0.3 ml saline were injectedsubcutaneously on the right thigh of a 6-week-old
C57BL6 male mouse. Ten days later, the tumour became
palpable and was examined. The experimental procedures were in accordance with the guidelines on the useof living animals for scientific investigation which were
established in our institution.
Biodistribution in normal mice
Biodistribution of 125I-L-PC was determined using ddY
male mice (6 weeks old) as amelanotic ones. 125I-L-PC

Nuclear Medicine Communications (2003) 24

578(37 kBq) in 100 ml of saline was injected via the tail vein.
The mice were killed by ether anaesthesia at various timeintervals, and the organs of interest were rapidly
dissected. The radioactivity in weighed tissue samples
was measured using a gamma counter (Aloka; ARC1000M). Data are expressed in mean+SD per centinjected dose per g wet tissue.
Biodistribution in B16 melanoma-bearing mice
Biodistribution of 125I-L-PC in B16 melanoma-bearing
C57BL6 mice was performed using the same method
mentioned above. Moreover, 3-[125I]iodo-a-methyl-L-tyrosine (125I-L-AMT) was compared with 125I-L-PC at
15 min intervals. 125I-L-AMT was prepared from amethyl-L-tyrosine (Aldrich) using our previously described method [9]. The radioactivity in tissue samples
was measured using a gamma counter (Aloka; ARC-360).
Autoradiography
A tumour-bearing C57BL6 mouse was injected with
100 ml of 125I-L-PC (1.85 MBq) intravenously and was
killed by ether anaesthesia 60 min after the tracer
injection. For whole body autoradiography, the mouse
was frozen in an embedding medium at 7158C for 24 hand then was sliced using an Autocryotome (Nakagawa;NA-200F). After drying overnight, freeze-dried slices
were placed in contact with the imaging plate (Fuji Photo
Film; BAS-TR2040) for 10 days. Images were processedwith a Bio-Imaging Analyzer (Fuji Photo Film; BAS2000).
In vitro accumulation study in B16 melanoma cells
Melanoma cells (56105) in 0.9 ml of HEPES buffer(pH 7.4) were preincubated for 10 min for temperatureequilibration. The 0.1 ml of the buffer containing radioactive tracers was added and incubated at 378C for
30 min. Inhibition studies of the membrane active
transport and tyrosinase, the starting enzyme of melaninformation, were carried out using 561074 M ouabain(Sigma), 161073 M L-tyrosine (Sigma) and 161074 M
phenylthiourea (PTU) (Aldrich), respectively, added
before preincubation.
At the end of incubation period, the cells were trappedon a glass filter (Advantec; GC-50) and were washedtwice with the ice-cold buffer under air vacuum. The
radioactivities of the cells were measured and theaccumulation percentage of the injected dose per eachsample was then calculated.
To further investigate the localization in melanosome,a cell fraction study was performed. After the incubation,
each sample was centrifuged at 800 rpm (1306g) for

Nuclear Medicine Communications (2003) 24

Nishii et al.
5 min. The supernatant was removed and the tumour
cells were suspended in 0.25 M sucrose. The suspensionof melanoma cells was homogenized using a Potter±
Elvehjem homogenizer (Iuchi) and was centrifugedaccording to the method of Menon and Haberman [15]using ultracentrifugation (Tomy, SRX-200). The radioactivity of the precipitate was measured as the melanin
granule fraction.
Measurement of partition coefficients
The partition coefficients of 125I-L-PC was measured
using 2.0 ml of n-octanol as the organic phase and2.0 ml of 0.1 M phosphate buffer (pH 7.0 for tumour
tissue and pH 7.4 for plasma) as the aqueous phase. The
n-octanol and the buffer were pre-mixed twice using amechanical mixer for 1 min at room temperature. Then,
20 ml of 125I-L-PC in saline was added and mixed twicefor 1 min at room temperature. The radioactivity of
200 ml of each phase was measured after centrifugation[13].
Results
Radiosynthesis
Chemical reaction and iodination of 3-iodo-4-hydroxyphenyl-L-cysteine are shown in Fig. 2. A labelling
efficiency of more than 65% resulted in the labelling of
4-L-PC to 125I-L-PC. After the purification, no-carrier
added 125I-L-PC with a radiochemical purity greater than
95% was obtained and separation from unlabelled 4-LPC, which had biological activity, was confirmed.
Biodistribution in normal mice
Table 1 summarizes the distribution data of 125I-L-PC in
normal mice. Blood clearance was faster than othermetabolically stable radioiodinated amino acids such as
125
I-L-AMT [10]. The accumulation of 125I-L-PC in thekidney and the liver reached the highest value within
3 min post-injection of tracer and the radioactivity rapidly
decreased without re-uptake of this tracer. Meanwhile,less accumulation was observed in the pancreas andbrain, which have been known for their high utilization ofamino acids. Furthermore, the accumulation in thestomach was not elevated, indicating metabolic stability
against de-iodination in the in vivo study.
Biodistribution in B16 melanoma-bearing mice
The biodistribution study of 125I- L-PC in the B16
melanoma is shown in Table 2. The tumour accumula-

A new radiopharmaceutical for detection of melanoma
tions at 5 min and 60 min were 1.71+0.27% and0.35+0.21% injected dose per g (ID/g) tissue. Higher
accumulation was observed in the tumour than in theskin and muscle at 60 min. Rapid blood clearance was579
also noted. The comparison of the tumour-to-blood ratio
and the tumour-to-muscle ratio between 125I-L-PC and125
I-L-AMT is summarized in Table 3. The tumour-toblood ratio of 125I-L-PC reached approximately 1 at

Table 1. Biodistribution of 125I-L-PC in normal ddY mice.
Time (min)
Organ
Blood
Brain
Pancreas
SpleenStomachIntestine
Adrenal gland
Kidney
Liver
Heart
Lung
Eyeball

1

2

3

5

10

15

30

60

6.23+0.67
0.30+0.05
3.35+0.63
1.55+0.16
0.96+0.14
1.10+0.20
1.19+0.40
18.71+5.51
4.73+0.60
4.01+0.82
4.04+0.70
0.75+0.27

4.31+0.49
0.18+0.02
1.93+0.33
1.46+0.39
0.62+0.11
0.89+0.13
1.22+0.18
30.04+3.14
4.79+0.30
2.35+0.33
3.05+0.31
0.85+0.60

3.29+0.95
0.26+0.27
1.27+0.40
1.08+0.66
0.95+0.43
0.83+0.21
0.74+0.56
31.46+9.68
3.63+1.07
1.47+0.38
2.31+0.55
0.77+0.64

2.58+0.08
0.16+0.09
0.99+0.07
0.79+0.16
0.77+0.15
1.03+0.36
2.95+4.34
24.12+3.04
3.79+1.41
1.16+0.11
2.00+0.25
0.74+0.63

1.52+0.12
0.07+0.01
0.71+0.25
0.36+0.02
1.95+0.71
1.95+1.18
0.48+0.19
15.79+2.39
2.64+0.56
0.60+0.05
1.29+0.14
0.26+0.09

0.99+0.13
0.04+0.01
0.47+0.10
0.24+0.03
2.41+0.71
2.24+1.76
50.01
9.05+1.62
1.39+0.62
0.38+0.06
0.94+0.15
0.14+0.09

0.41+0.09
0.01+0.09
0.16+0.03
0.07+0.04
0.62+0.11
1.41+0.20
50.01
5.87+4.91
0.46+0.13
0.35+0.30
0.41+0.04
50.01

0.14+0.04
50.01
0.07+0.02
0.02+0.01
1.23+0.45
2.52+1.64
50.01
1.93+2.11
0.33+0.12
0.02+0.01
0.15+0.03
50.01%ID/g tissue, mean+(SD) for 3±4 mice.

Table 2. Biodistribution of 125I-L-PC in B16 melanoma-bearing C57BL6 mice.
Time (min)Organ or tumour
Blood
Brain
Pancreas
SpleenStomachIntestine
Kidney
Liver
Heart
Lung
Tumour
MuscleSkin
Eyeball

5

15

30

60

120

4.82+0.44
0.18+0.01
1.92+0.21
1.12+0.03
1.36+0.33
2.70+0.11
53.87+6.32
5.76+0.45
1.89+0.26
3.18+0.11
1.71+0.27
1.01+0.01
2.72+0.15
0.71+0.16

2.07+0.07
0.07+0.01
0.84+0.03
0.52+0.08
0.02+0.38
2.28+1.27
20.48+1.58
2.17+0.20
0.71+0.01
1.65+0.10
0.86+0.17
0.26+0.14
1.44+0.21
0.32+0.14

0.56+0.12
0.02+0.01
0.24+0.05
0.18+0.03
3.59+2.88
3.39+0.25
4.09+1.98
1.02+0.30
0.18+0.07
0.41+0.05
0.43+0.03
0.09+0.03
0.48+0.17
0.11+0.05

0.41+0.04
0.01+0.00
0.41+0.24
0.01+0.02
5.55+4.25
5.79+2.12
1.50+0.59
1.44+0.32
0.18+0.07
0.32+0.03
0.35+0.21
0.05+0.03
0.24+0.03
0.05+0.01

0.30+0.07
0.01+0.00
0.07+0.02
0.13+0.03
1.53+0.17
3.37+0.46
0.49+0.05
0.56+0.16
0.07+0.02
0.29+0.07
0.12+0.04
0.02+0.01
0.11+0.02
0.04+0.01%ID/g tissue, mean+(SD) for three mice.

Table 3. Tumour-to-blood ratios and tumour-to-muscle ratios after intravenous injection of tumour-seeking tracers in B16
melanoma-bearing C57BL6 mice.
Tumour-to-blood ratio
Amino acid
125
125

I-L-PC
I-L-AMT

Tumour-to-muscle ratio

5 min

15 min60 min

5 min

15 min60 min0.38+0.07
1.13+0.11

0.50+0.16
2.22+0.69

0.97+0.48
1.87+0.38

1.84+0.03
4.86+0.57

3.55+0.84
4.26+0.36

6.47+0.76
3.62+0.59

All values were for three mice.

Nuclear Medicine Communications (2003) 24

580

Nishii et al.

60 min after injection. Tumour-to-muscle ratios increased
up to 6.67 at 60 min after 125I-L-PC injection and werehigher than 125I-L-AMT. The results showed relative
retention in melanoma tissue of 125I-L-PC.
Autoradiography
Fig. 3 shows autoradiograms obtained at 60 min after
intravenous injection of 125I-L-PC to a melanoma-bearing
mouse. The tumour image was clearly visualized withhigh contrast to soft tissue. Low accumulation in thebrain and the lung supported the results of thebiodistribution study. No uptake into areas of necrosis
was noted on this image. The specific localization within
the tumour itself is of particular interest for clinicalimaging.
In vitro accumulation study in B16 melanoma cells
The accumulation of 125I-L-PC (3.03+0.10% ID per 56105
melanoma cells) was similar to 125I-L-AMT. Table 4shows the percentage effect of control accumulation with
inhibitor loading. Significant inhibition of 125I-L-AMT
was noted with ouabain (P50.05) and L-tyrosine

Brain

Lung
Liver
StomachKidneyTumourFig. 3. Autoradiography of a mouse bearing B16 melanoma
injected with 125I-L-PC.

Table 4. Effects of inhibition on tracer accumulation in B16
melanoma cells.
Amino acid125

I-L-PC
125
I-L-AMT

Ouabain

L-Tyrosine

Phenylthiourea

113.66+15.82
72.64+0.87*

77.59+14.94
58.32+2.48*

55.24+12.32*
84.82+5.37

Per cent of control accumulation, mean+(SD); *P50.05.

Nuclear Medicine Communications (2003) 24(P50.05), while there was no significant inhibition in
I-L-PC. With PTU, a specific tyrosinase inhibitor, astronger inhibitory effect on the accumulation of 125I-L-PC(P50.05) was noted in contrast to 125I-L-AMT.
In a cell fraction study, high localization of 125I-L-PC(37.3% of the total cell uptake) in melanin granule fraction
was observed different from 125I-L-AMT (4%). The
radioactivity of 125I-L-PC, especially in the melanosome
fraction, was also reduced after pretreatment with PTU.

125

Partition coefficients
The role of partition coefficients in 125I-L-PC was also
evaluated (Table 5). The partition coefficients of 123I-LAMT and 14C-L-tyrosine are also shown as references,which were previously reported [9, 13]. The lipophilicity
of 125I-L-PC was higher than that of 123I-L-AMT and 14C-Ltyrosine.
Discussion
In malignant melanoma, the synthesis of melanin ishighly elevated because of a marked increase oftyrosinase activity [16]. Many attempts have been madeto develop rational chemotherapy for this disease bytaking advantage of this unique physiological process ofmelanin synthesis. Ito and Miura have synthesizedvarious phenolic compounds as substrates for tyrosinase[5, 6], and they found that 4-L-PC was a good substrate of
mammalian tyrosinase for forming melanin-like pigments [17].
Affinity to melanoma tissue, with elevated tyrosinase
activity, is of paramount importance in detecting melanoma. In the present study, which aimed to develop a
new radiopharmaceutical for detecting malignant melanoma based on its melanin biosynthesis, 4-L-PC was
selected as a mother compound for iodination. It is atyrosine derivative of tyrosinase substrates with a phenol
moiety which provides a site for easy iodination. In
addition, radioiodination and sulfurization of the tyrosine molecule were expected to result in metabolicstability and rapid excretion [9, 13].

Table 5. Partition coefficients of 125I-L-PC, 123I-L-AMT and
14
C-tyrosine at pH 7.0 and 7.4.
Amino acid125

I-L-PC
I-L-AMT
14
C-tyrosine
123

pH 7.0pH 7.470.28+0.04
70.88+0.00
71.98+0.02

70.37+0.04
70.91+0.00
72.00+0.05

log (n-octanol/0.1 M phosphate buffer).

A new radiopharmaceutical for detection of melanoma
An amino acid in the L-configuration is required foraffinity of biological mechanism. After one step ofchemical reaction and separation of the 2-hydroxyisomer, only the L-configuration of 4-PC was obtained.
The labelling efficiency of 125I-L-PC was from 65% to 82%,
and the radiochemical purity was greater than 95%. It
was shown that our labelling procedure conveniently
gave high labelling efficiency and high specific activity ina short time. Because radioiodine has the advantage ofavailability, low cost, adequate half-life and radiationenergy, radiopharmaceuticals labelled with radioiodine
are widely used for SPECT imaging. Use of 131I could beavailable for radiotherapy.
In the biodistribution study of 125I-L-PC in normal ddY
mice, blood clearance and renal excretion were fasterthan those of metabolically stable radioiodinated aminoacids such as 125I-L-AMT [10]. Meanwhile, less accumulation was observed in pancreas and brain in which 125I-LAMT showed high accumulation (Table 1). Because of
non-affinity to normal organs, 125I-L-PC showed uniquecharacteristics as an L-amino acid. The rapid clearance of
this tracer from normal organs and relative retention in
the tumour was also noted in the biodistribution study in
B16 melanoma-bearing mice (Table 2). When comparedwith 125I-L-AMT, which are available for clinical use,tumour-to-muscle ratios increased up to 6.67 at 60 min
after 125I-L-PC injection and were higher than 125I-L-AMT.
In order to visualize malignant melanoma andmetastases, a potential radiopharmaceutical must meet
important criteria: high tumour-to-tissue (blood, muscle,
brain, liver or lung) ratios. In spite of non-affinity of 125IL-PC to normal tissue, tumour-to-blood ratio reachedapproximately 1 at 60 min after injection. Tumour-tomuscle ratio increased up to 6.67 at 60 min after injectionof this tracer (Table 3). This result indicates relative
retention of the tracer in the melanoma. Therefore, 125I-LPC has the potential of good contrast in a tumour-seeking
agent.
Indeed, autoradiography in B16 melanoma-bearing
mouse showed tumour localization of radioactivity after
injection of 125I-L-PC (Fig. 3). It provided a clear
delineation of the tumour. We noted that, on autoradiographic images, there was less uptake into the necrotic
region. The specific localization within the tumour itself
is of particular interest for clinical imaging. It isconsidered that 125I-L-PC could be a suitable radiopharmaceutical for imaging melanoma.
Furthermore, the accumulation of 125I-L-PC in melanotic tissues, the skin and the uvea, in C57BL6 mouse
was higher than that in amelanotic ddY mouse. Thissuggested that the localization of 125I-L-PC could be
related to the biosynthesis of melanin.
In in vitro accumulation study with B16 melanoma
cells, ouabain which is an efficient Na+±K+ ATPase581
inhibitor has been used to suppress the Na+ dependent
concentrative uptake of amino acids [18]. L-tyrosine has
been used as a competitive inhibitor of the neutralamino acid transport [9, 19]. No inhibitory effect ofouabain and L-tyrosine on the uptake of 125I-L-PC
suggested less involvement of amino acid active
transport for 125I-L-PC accumulation into the melanoma
cells. This lower participation of amino acid transport
accounts for the lower accumulation in the brain andthe pancreas in contrast with high cerebral andpancreatic accumulation of 125I-L-AMT, a marker ofamino acid transport.
Although the transport system of 125I-L-PC is lessrelated by amino acid active transport, it is consideredthat the relative increase of lipophilicity of this tracer
could explain the permeability into cells.
In inhibition studies, PTU is frequently used as atyrosinase inhibitor in melanin biosynthesis [20]. PTU
had an inhibitory effect on only the accumulation of 125I125
L-PC (Table 4). The decrease in the uptake of
I-L-PC
125
I-L-PC interacted with
with PTU indicated thattyrosinase and that is similar to 4-L-PC. Furthermore,
the specific localization of 125I-L-PC in melanin granulefractions supports these results. In melanoma cells, itinteracts with tyrosinase confirmed by PTU inhibition
and then localized in melanin granules fraction. Theinteraction with tyrosinase and the localization inmelanin granules might lead to the retention of thiscompound in melanoma cells.
In summary, the preparation of 125I-L-PC was carriedout conveniently and efficiently in a short period of time,with high specific activity. The biodistribution of 125I-LPC showed low accumulation in normal tissue andrelative retention in B16 melanoma. The high imaging
contrast of peripheral tumour obtained with this tracer isideal for clinical application. In vitro accumulation study
revealed an interaction of 125I-L-PC with tyrosinase. It
indicates that the accumulation and retention of 125I-L-PC
to melanoma tissue is dependent on high tyrosinase
activity in melanoma. With affinity to tyrosinase activity,
125
I-L-PC could image melanoma tissue with specificity
and might be able to determine the viability of tumour
cells. Moreover, because I-L-PC accumulated low innormal tissue, it might be applied as a therapeutic
radiopharmaceutical when labelled with 131I.
Conclusion
I-L-PC, a new artificial amino acid radiopharmaceutical,
has a high potential for diagnosis and treatment ofmalignant melanoma. Its accumulation in melanoma wasdependent on high tyrosinase activity in melanin formation.

Nuclear Medicine Communications (2003) 24

582
References
1. Jimbow K, Miura T, Ito S, Ishikawa K. Phenolic melaninprecursors provide a rational approach to design ofantitumor agents for melanoma. Pig Cell Res 1989; 2: 34±39.
2. Graham GD, Tiffany SM, Vogel FS. The toxicity of melaninprecursors. J Invest Dermatol 1978; 70: 113±116.
3. Wick MM. Levodopa and dopamine analogs as DNA
polymerase inhibitors and antitumor agents in humanmelanoma. Cancer Res 1980; 40: 1414±1418.
4. Yamada K, Jimbow K, Engelhardt R, Ito S. Selective
cytotoxicity of phenolic melanin precursor, 4-S-cysteamynylphenol, on in vitro melanoma cell. Biochem Pharmacol
1989; 38: 2217±2221.
5. Ito S, Inoue S, Yamamoto Y, Fujita K. Synthesis andantitumor activity of cysteinyl-3,4-dihydroxyphenylalanines and related compounds. J Med Chem 1981; 24: 673±
677.
6. Miura S, Ueda T, Jimbow K, Ito S, Fujita K. Synthesis ofcysteinylphenol, cysteaminylphenol, and related compounds, and in vivo evaluation of antimelanoma effect.
Arch Dermatol Res 1987; 279: 219±225.
7. Ito S, Kato T, Ishikawa K, Kasuga T, Jimbow K. Mechanismof selective toxicity of 4-S-cysteinylphenol and 4-S-cysteaminylphenol to melanocytes. Biochem Pharmacol 1987; 36:
2007±2011.
8. Hoefnagel CA. Role of nuclear medicine in melanoma. Eur J
Nucl Med 1998; 25: 1567±1574.
9. Kawai K, Fujibayashi Y, Saji H, et al. A strategy for the studyof cerebral amino acid transport using iodine-123-labeledamino acid radiopharmaceutical: 3-iodo-alpha-methyl-Ltyrosine. J Nucl Med 1991; 32: 819±824.
10. Kawai K, Fujibayashi Y, Yonekura Y, et al. An artificial
amino acid radiopharmaceutical for single photon emission
computed tomographic study of pancreatic amino acidtransports: 123I-3-iodo-alpha-methyl-L-tyrosine. Anal Nucl
Med 1992; 6: 169±175.

Nuclear Medicine Communications (2003) 24

Nishii et al.
11. Jager PL, Franssen EJF, Kool W, et al. Feasibility of tumor
imaging using L-3-[iodine-123]-iodo-a-methyl-tyrosine inexperimental tumors. J Nucl Med 1998; 39: 1736±1743.
12. Langen KJ, Roosen N, Coenen HH. Brain and brain tumor
uptake of L-3-[123I]iodo-alpha-methyl-tyrosine: competition
with natural L-amino acids. J Nucl Med 1991; 32: 1225±1228.
13. Kawai K, Flores II LG, Nakagawa M, et al. Brain uptake ofiodinated L-meta-tyrosine, a metabolically stable amino acidderivative. Nucl Med Commun 1999; 20: 153±157.
14. Somayaji V, Ito S, Jimbow K, Wiebe LI. The synthesis of 4-Scysteinyl-[U-14C]phenol, an experimental antimelanoma
agent. Appl Radiat Isot 1989; 40: 539±540.
15. Menon IA, Haberman HF. Isolation of melanin granules.
Methods Enzymol 1974; 31: 389±394.
16. Pawelek JM. Factors regulating growth and pigmentation ofmelanoma cells. J Invest Dermatol 1976; 66: 201±209.
17. Jimbow K, Ito S, Maeda K, et al. Mechanism regulating
selective growth inhibition disintegration of melanoma cellsby 4-S-cysteinyl phenol and related compounds. J Invest
Dermatol 1985; 84: 355±360.
18. Hughes CCW, Lantos PL. Uptake of leucine and alanine bycultured cerebral capillary endothelial cells. Brain Res 1989;
480: 126±132.
19. Oxender DL, Christensen HN. Distinct mediating systems
for the transport of neutral amino acids by the Ehrlich cell. J
Biol Chem 1963; 238: 3686±3699.
20. Prezioso JA, Epperly MN, Wang N, Bloomer WD. Effects oftyrosinase activity on the cytotoxicity of 4-S-cysteaminylphenol and N-acetyl-4-S-cysteaminylphenol in melanoma
cells. Cancer Lett 1992; 63: 73±79.