Synthesis and evaluation of a radioiodinated bladder cancer specific peptide

Article abstract of DOI:10.1016/j.bmc.2012.05.049

Bladder cancer is the second most common cancer of the urinary tract, however the invasive cystoscopy is still the standard technique for diagnosis and surveillance of bladder cancer. Herein, we radiolabel bladder cancer specific peptide with radioactive iodine (^131/124I) and evaluate its potential as a new radiopharmaceutical for the non-invasive diagnosis of bladder cancer. A 9-mer bladder cancer specific peptide (BP) was conjugated with tyrosine and cyclized by disulfide bond formation to give Y-BP, which was further radioiodinated to give [^131/124I]Y-BP in good radiochemical yield. The biodistribution data showed the high selectivity of [ ¹²⁴I]Y-BP in HT1376 human bladder cancer xenograft models with a tumor-to-muscle ratio of 6.2. This tumor targeting was not observed in control B16F10 melanoma tumor models. In microPET studies, while the control scrambled peptide, [¹²⁴I]Y-sBP, did not accumulate in either the bladder cancer or melanoma, [¹²⁴I]Y-BP showed high tumor uptake only in animals with HT1376 bladder cancer cells. Furthermore, [¹²⁴I]Y-BP showed superior bladder cancer uptake even compared to most commonly used cancer imaging tracer, [¹⁸F]FDG. The experimental results suggest the potential of [¹²⁴I]Y-BP as a new radiopharmaceutical for the non-invasive diagnosis of bladder cancer with high binding affinity and selectivity.

Full text of DOI:10.1016/j.bmc.2012.05.049

Bioorganic & Medicinal Chemistry 20 (2012) 4330–4335
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and evaluation of a radioiodinated bladder cancer specific peptide
Yeong Su Ha a, , Hwa Young Lee a, , Gwang Il An b, Jonghee Kim a, Wonjung Kwak a, Eun-Ju Lee c,
Seung-Min Lee c, Byung-Heon Lee c, In-San Kim c, Takele Belay a, Woonghee Lee a, Byeong-Cheol Ahn d,
Jaetae Lee d, Jeongsoo Yoo a,
⇑
a Department of Molecular Medicine, Kyungpook National University, School of Medicine, Daegu 700-422, South Korea
b Molecular Imaging Research Center, Korea Institute of Radiological and Medical Sciences, Seoul, South Korea
c Department of Biochemistry and Cell Biology and Cell & Matrix Research Institute, Kyungpook National University, School of Medicine, Daegu, South Korea
d Department of Nuclear medicine, Kyungpook National University, School of Medicine, Daegu, South Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Bladder cancer is the second most common cancer of the urinary tract, however the invasive cystoscopy
is still the standard technique for diagnosis and surveillance of bladder cancer. Herein, we radiolabel
bladder cancer specific peptide with radioactive iodine (131/124I) and evaluate its potential as a new radio-
pharmaceutical for the non-invasive diagnosis of bladder cancer. A 9-mer bladder cancer specific peptide
(BP) was conjugated with tyrosine and cyclized by disulfide bond formation to give Y-BP, which was fur-
ther radioiodinated to give [131/124I]Y-BP in good radiochemical yield. The biodistribution data showed
the high selectivity of [124I]Y-BP in HT1376 human bladder cancer xenograft models with a tumor-to-
muscle ratio of 6.2. This tumor targeting was not observed in control B16F10 melanoma tumor models.
In microPET studies, while the control scrambled peptide, [124I]Y-sBP, did not accumulate in either the
bladder cancer or melanoma, [124I]Y-BP showed high tumor uptake only in animals with HT1376 bladder
cancer cells. Furthermore, [124I]Y-BP showed superior bladder cancer uptake even compared to most
commonly used cancer imaging tracer, [18F]FDG. The experimental results suggest the potential of
Received 12 April 2012
Revised 19 May 2012
Accepted 21 May 2012
Available online 29 May 2012
Keywords:
Bladder cancer
Disease-specific peptide
Radiopharmaceuticals
Radioiodination
PET
[
124I]Y-BP as a new radiopharmaceutical for the non-invasive diagnosis of bladder cancer with high bind-
ing affinity and selectivity.
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
risk populations. However, clinical experience has shown limita-
tions of urine cytology in low grade cancers, in which the sensitiv-
ity is low.7,8 Several urine markers have shown higher sensitivity
than urine cytology, however most of these markers tend to be less
specific for bladder cancer than cytology.6 These characteristics of
current diagnosis techniques have prompted the search for more
reliable non-invasive imaging methods for bladder cancer.
Bladder cancer is the second most common cancer of the uri-
nary tract after prostate cancer.1 About 70% of all bladder cancer
patients have superficial cancers. Although superficial cancers
can be easily removed by trans-urethral resection, they are associ-
ated with a high recurrence rate and 10–20% will progress to mus-
cle invasion.2 Furthermore, following radical cystectomy, up to 50%
of patients will develop metastases.3 Because of its high recurrence
rate, which is the highest of all cancers, surveillance of bladder can-
cer patients needs to be very rigorous.
Cystoscopy and urine cytology are the traditional techniques for
diagnosis and surveillance of bladder cancer. Cystoscopy is inva-
sive and lacks specificity for flat malignancies4–6, though it can
visualize the urothelium of the bladder wall directly. And, the
use of cystoscopy is restricted to primary cancer inside bladder,
but the metastasized tumor to other organs cannot be diagnosed
by this technique. Cytology is a convenient non-invasive test for
patients with history of bladder cancer and for screening of high-
Positron emission tomography (PET) using fluorine-18 fluoro-2-
deoxy-D
-glucose ([18F]FDG) has been shown to be an accurate tech-
nique for cancer detection and staging and for the monitoring of
therapy in patients with various malignant cancers.9,10 Because
[
18F]FDG is an analog of glucose, it is taken up by the cells via
the glucose transporter and are then phosphorylated by the hexo-
kinase, but the product, fluoro-2-deoxyglucose-6-phosphate is not
further processed and trapped in the cells. Hence, the accumula-
tion of [18F]FDG is an indicator of high glucose uptake rate and
hexokinase activity in the tissue. However, clinical experience with
FDG–PET in bladder cancer has revealed a major disadvantage of
FDG: the excretion of radioactivity into urine, causing accumula-
tion of activity in the bladder.11,12 Furthermore, because of rela-
tively short half-life of F-18 (110 min), the late imaging, in which
most activity is cleared out from non-specific organs, is not
feasible. Another disadvantage of FDG–PET imaging is an inherent
⇑
Corresponding author. Tel.: +82 53 420 4947; fax: +82 53 426 4944.
E-mail address: yooj@knu.ac.kr (J. Yoo).
These authors contributed equally.
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.05.049

Y. S. Ha et al. / Bioorg. Med. Chem. 20 (2012) 4330–4335
4331
background signal.13 Basically, all cells metabolize glucose and
some glucose-avid organs such as brain, heart and brown fat al-
ways show intense FDG background signal.
method was changed to Iodogen (Pierce) from Iodo-beads, the
radiolabeling yield with 131I and 124I dramatically increased to
>90% and >60%, respectively. The radiolabeling yield was always
higher when using I-131 compared to I-124, presumably due to
higher specific activity of I-131 over I-124. The radiolabeling yields
of Y-sBP was comparable with those of Y-BP. The labeled peptides
were purified by reverse-phase HPLC to give [131/124I]Y-BP and
In the past decade, new radiopharmaceuticals based on small
synthetic peptides have been developed for tumor imaging and tar-
geted radiotherapy.14–20 Radiopharmaceuticals based on peptides
have a number of distinct advantages over those based on proteins
or antibodies, including their small size, easy preparation, site-spe-
cific radiolabeling, toleration of harsh conditions of conjugation or
radiolabeling, rapid clearance from blood and non-target tissues,
high penetration into tumor tissue, and low immunogenicity.14,16
Recently, Lee et al. identified a small peptide which shows high
binding affinity for bladder cancer using phage-displayed peptide
libraries.21 The fluorescein-conjugated CSNRDARRC peptide was
found to bind selectively to bladder cancer tissue and it also selec-
tively labeled cancer cells in urine of patients with bladder cancer.
Here, we radiolabel this bladder cancer specific peptide with
PET radionuclide, 124I, and evaluate its potential as a new radio-
pharmaceutical for the diagnosis of bladder cancer in in vivo tumor
models.
[
124I]Y-sBP in >98% radiochemical purities (Fig. 3). The purified
radioiodinated peptides were used for following in vitro and
in vivo studies.
2.2. Serum stability test
To ensure that the radiolabeled peptide remains intact in vivo
long enough to target the bladder cancer specific receptors, we
studied the stability of radioiodinated bladder cancer specific pep-
tide in serum. The HPLC purified [131I]Y-BP was incubated with FBS
and PBS at 37 °C for up to 24 h. The relative amount of intact parent
radiolabled peptide was determined using radio-TLC over the
study period. The results of radio-TLC analyses indicated that more
than 60% of intact peptide remained after a 5 h incubation in FBS,
which could be fairly enough for microPET imaging (Fig. 4). After
24 h in FBS, only 37% of intact peptide remained, which is far lower
than 88% in PBS.
2. Results and discussion
2.1. Peptide preparation and radiochemistry
2.3. MicroPET imaging
The bladder cancer specific peptide, which was found by phage
display screening,21,22 was first conjugated to tyrosine residue for
radioiodination and cyclized using the two cysteine residues at
the terminal positions to inhibit the in vivo proteolysis of the pep-
tides.16 Both bladder cancer specific peptide, Y-BP, and its scram-
bled version, Y-sBP were prepared in >98% chemical purities,
purified by HPLC, and characterized by mass analysis (Fig. 1).
Experimental MS (ESI) data were well matched with the expected
values (calculated for Y-BP: 1240.52, found: 1241; calculated for Y-
sBP: 1240.52, found: 1240).
The specific binding of the radiolabeled [124I]Y-BP to the bladder
cancer was evaluated by microPET imaging studies using xenograft
tumor models.
The typical images of the comparative microPET imaging stud-
ies of [124I]Y-BP and [124I]Y-sBP in the nude mice bearing HT1376
bladder cancer and B16F10 melanoma cells on each flank side
are shown in Figure 6. While the bladder cancer HT1376 was
clearly visualized, the uptake level of [124I]Y-BP in melanoma
B16F10 is as low as the background at 5.5 h post-injection. The up-
take activity in HT1376 bladder cancer model (1.2%ID/g in tumor)
was 4.6 fold higher than that of B16F10 melanoma tumor model.
At earlier time points (30 min, 2 h), the tumor-to-background ratio
was insufficiently high (1.7 and 2.0, respectively) for the tumor re-
These two peptides were then radiolabeled with 131I or 124I
using tyrosine group on N-terminal (Fig. 2). Among the various
radiolabeling methods, radioiodination was chosen, because vari-
ous iodine radionuclides such as 123I, 124I, 125I, 131I are available
and radiolabeling can be achieved easily by direct iodination via
the phenolic ring of the tyrosine residue in the presence of an oxi-
dizing agent such as Iodo-beads and Iodogen.15,23 We used a posi-
tron-emitting 124I (23% of disintegration, t1/2 4.2 d) for microPET
gion to be clearly visualized. Thanks to enough long half-life of 124
I
(4.2 d), up to one day image could be measured without difficulty
even though tumor-to-background ratio was slowly decreased
after 5.5 h (5.4 and 1.9 at 5.5 and 15 h, respectively). In addition,
the control peptide [124I]Y-sBP did not show any noticeable tumor
uptake both in HT1376 bladder tumor (0.4%ID/g) and B16F10 mel-
anoma xenografts. This microPET data is well consistent with the
previous in vivo targeting studies of the fluorescein-conjugated
CSNRDARRC peptide.23 In both microPET studies, the thyroid and
stomach showed very intense activity uptake due to the free 124I
ions released from the injected peptides under physiological condi-
tions. This is not unexpected, as de-iodination in vivo of directly
radioiodinated peptide has already been reported as an inevitable
phenomenon.25 This de-iodination in physiological condition was
also expected from the in vitro serum stability test of [131I]Y-BP.
The potential of [124I]Y-BP to be used as a radiotracer for the
diagnosis of bladder cancer was further examined by direct micro-
PET comparison studies with the most widely employed tumor
imaging tracer, [18F]FDG in the same tumor model.13 In this exper-
iment, the free radioactive iodine uptake in thyroid and stomach
was minimized by pre-injection of sodium perchlorate (NaClO4)
before [124I]Y-BP injection.26
imaging,24 but in pre-imaging studies, -ray emitting 131I was em-
c
ployed as radioactive iodine source because high purity 131I is eas-
ily available while 124I is produced in limited quantity only for
research purpose yet. The another beauty of 131I is its long half-life
(8.0 d) and the following experiments can be performed without
restriction from decay loss.
The radiolabeling yield of Y-BP with 131I and 124I was >30% and
>23%, respectively, using Iodo-beads. However, when the labeling
Peptide
Y-BP
Sequence
Tyr-Cys-Ser-Asn-Arg-Asp-Ala-Arg-Arg-Cys
S
S
Y-sBP
Tyr-Cys-Asp-Ala-Ser-Arg-Arg-Asn-Arg-Cys
S
S
The tumor lesion in HT1376 bladder cancer bearing xenografts
was recognized using both [124I]Y-BP and [18F]FDG as a PET radio-
tracers at 5.5 h and 40 min post-injection, respectively (Fig. 7).
However, while HT1376 cancer was barely recognized by
Figure 1. Peptide sequences of bladder cancer specific peptide (Y-BP) and control
peptide having scrambled sequence (Y-sBP).

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Y. S. Ha et al. / Bioorg. Med. Chem. 20 (2012) 4330–4335
NH
NH
H2N
H2N
HN
NH
HN
NH
HN
NH2
NH2
HN
O
O
O
O
O
O
N
N
[
131/124I]NaI
Iodogen
HN
HN
H
H
HOOC
HOOC
NH
NH
O
O
O
O
HN
HN
HN
HN
HN
HN
COOH
COOH
S
S
S
S
O
O
N
H
N
H
H
N
H
N
HO
NH
HN
HO
NH
HN
NH
NH
NH2
NH2
NH2
NH2
O
O
O
O
O
O
131/124I
O
O
OH
OH
NH2
NH2
[
131/124I]Y-BP
NH
NH
H2N
H2N
NH2
NH2
HN
HN
HN
O
HN
O
NH2
NH2
O
O
NH
O
NH
O
O
O
N
N
H
HN
HN
H
HOOC
HOOC
NH
NH
131/124I]NaI
Iodogen
NH2
NH
H
N
[
O
O
H
N
O
O
HN
HN
HN
NH2
NH
HN
HN
HN
S
S
S
O
S
O
N
H
OH
N
H
OH
H
N
H
N
HO
131/124I
HO
NH
NH2
NH
NH2
O
O
O
O
O
O
COOH
COOH
[
131/124I]Y-sBP
Figure 2. Chemical structures of radiolabeled [131/124I]Y-BP and [131/124I]Y-sBP.
1.0
0.8
0.6
0.4
0.2
0.0
0.2
100
80
60
PBS
40
FBS
20
0
5
10
15
20
25
0
0h
1h
2h
3h
4h
5h
6h
24h
20
16
12
8
Figure 4. Serum stability of [131I]Y-BP in PBS and FBS.
[
124I]Y-BP and [18F]FDG was calculated to be 5.4 and 2.5, respec-
tively, which clearly says that [124I]Y-BP is superior to [18F]FDG
in visualizing bladder cancer. Thanks to the blocking of free 124I
in thyroid and stomach using sodium perchlorate, the background
level in [124I]Y-BP image was lower compared to the previous
images in Fig. 6. However, the clearance organ, bladder showed
much higher radioactivity. In [18F]FDG image, high glucose-con-
suming lesions such as brain and brown fat showed higher uptake
of [18F]FDG than tumor.27
4
0
4
0
5
10
15
20
25
Time (min)
Figure 3. UV chromatogram of non-radiolabeled Y-BP at 220 nm wavelength (top)
and radio tracer of [124I]Y-BP after HPLC purification (bottom).
2.4. Biodistribution studies
[
[
[
18F]FDG, the same tumor was unambiguously visualized using
124I]Y-BP. There is little background in microPET image of
124I]Y-BP except hot bladder. The tumor-to-background ratio of
The in vivo characteristics and cancer targeting of [131I]Y-BP
was investigated in BALB/c nude mice (n = 5) with subcutaneous
HT1376 bladder cancers. B16F10 xenograft tumor models (n = 5)

Y. S. Ha et al. / Bioorg. Med. Chem. 20 (2012) 4330–4335
4333
served, was chosen as dissection time point based on a series of
microPET images at different time points.
Radioactivity uptake for most non-specific organs was compa-
rable or lower in HT1376 cancer bearing mice than in B16F10 tu-
mor bearing mice. However, tumor uptake of
[
131I]Y-BP in
animals with HT1376 cancer cell almost six times higher in com-
parison to B16F10 tumor bearing mice. The tumor/blood, tumor/
muscle, tumor/kidney and tumor/liver uptake ratios in HT1376 tu-
mor bearing mice were found to be 2.7, 6.2, 2.7 and 5.9, respec-
tively. This tumor targeting of the radiotracer was not observed
in B16F10 tumor model at all, which clearly indicates that tumor
accumulation of [131I]Y-BP is a specific cell line targeting process.
It was also observed that [131I]Y-BP was cleared from the blood
mainly via the renal pathway and to a lesser extent via the liver
to the intestines.
Figure 5. Biodistribution data of [131I]Y-BP in mice bearing HT1376 human bladder
cancer and B16F10 melanoma cells (n = 5). The mice were sacrificed at 5.5 h post-
injection of [131I]Y-BP.
The direct imaging of bladder cancer using [124I]Y-BP seems to
be hampered by high urine activity in bladder at early time point.
However, the long half-life of 124I (4.2 d) enables late imaging such
as at 1 d post-injection. At 1 d post-injection of [124I]Y-BP, the urine
activity will be dropped dramatically and can be even lowered by
using the diuretic agent. However, the better application of
were also used as a negative control. The organ distribution of
[
131I]Y-BP at 5.5 h post-injection both in HT1376 and B16F10 xeno-
graft tumor models was summarized in Figure 5. The 5.5 h post-
injection, at which the highest tumor-to-background ratio was ob-
Coronal
Transverse
6 %ID/g
B16F10
HT1376
St
[
124I]Y-BP
0
5 %ID/g
Th
St
B16F10
HT1376
[
124I]Y-sBP
0
Figure 6. Typical microPET images of BALB/c nu/nu female mice bearing HT1376 bladder cancer and B16F10 melanoma on opposite flanks. Coronal (middle) and transverse
images (right) at 5.5 h post-injection of [124I]Y-BP and [124I]Y-sBP are shown. The arrows indicate the HT1376 tumors. Th = thyroid, St = stomach.
Coronal
Sagittal
17 %ID/g
Br
[18F]FDG
0
0.5 %ID/g
[
124I]Y-BP
Bl
0
Figure 7. Comparative microPET images of [18F]FDG and [124I]Y-BP in the same xenograft mouse bearing HT1376 bladder cancer on the right flank. The transverse (right) and
coronal (middle) images are shown. The microPET images were obtained at 40 min and 5.5 h after injection of 20 MBq of [18F]FDG and 7.4 MBq of [124I]Y-BP, respectively. The
arrows indicate the HT1376 tumors. Br = brain, Bl = bladder. The white dotted circle shows the region selected for tumor-to-muscle ratio calculation.

4334
Y. S. Ha et al. / Bioorg. Med. Chem. 20 (2012) 4330–4335
[
124I]Y-BP will be found in the metastasized bladder cancer in other
of 1 mL/min, linear gradient from 97% solvent A (water with 0.1%
organs rather than primary bladder cancer.
trifluoroacetic acid) and 3% solvent B (acetonitrile with 0.1% triflu-
oroacetic acid) to 20% solvent A and 80% solvent B over a period of
30 min. The UV chromatogram was monitored using 220 nm wave-
length. The collected fraction was concentrated under reduced
pressure using rotary evaporator at maximum 40 °C bath setting,
then diluted with saline for use in following in vitro and in vivo
animal experiments.
3. Conclusion
In this study, the bladder cancer specific peptide, Y-BP with its
scrambled version, Y-sBP, were radiolabeled with [131I]NaI or
[
124I]NaI and evaluated in xenograft bladder cancer models as a po-
tential bladder cancer imaging agent. In consistent with the biodis-
tribution result, [124I]Y-BP showed highly selective uptake in
HT1376 human bladder cancer models in microPET imaging stud-
ies. However, tumor targeting of [124I]Y-BP was not observed when
tested using B16F10 melanoma tumor model, indicating that tu-
mor accumulation was a receptor-mediated specific process. Fur-
thermore, [124I]Y-BP showed superior tumor-to-background ratio
in bladder cancer model compared to [18F]FDG. All these experi-
mental data suggest that [124I]Y-BP can be considered as a poten-
tial radiopharmaceutical for the non-invasive diagnosis of
bladder cancer, especially metastasized bladder cancer using PET.
4.3. Serum stability test
Stability of the radiolabeled peptide, [131I]Y-BP in either Phos-
phate Buffered Saline (PBS) or Fetal Bovine Serum (FBS) was eval-
uated by incubation at 37 °C for 24 h. Briefly, radiolabeled [131I]Y-
BP (50
either PBS or FBS (450
l
L, 100
l
Ci) was incubated at 37 °C with gently shaking in
L). The reaction aliquot were removed at
l
0, 1, 2, 3, 4, 5, 6 and 24 h post-incubation and the relative amount
of intact parent radiolabled peptide was determined using radio-
TLC over the study period. The same radio-TLC condition was used
as above.
4. Materials and methods
4.4. Animal models
4.1. Peptides
All animal experiments were conducted in compliance with the
Animal Care and Use Committee requirements of Kyungpook Na-
tional University. Xenograft tumor models of human bladder can-
cer (HT1376) and melanoma (B16F10) cell lines for comparison
study were prepared using 6 week-old BALB/c nu/nu female nude
mice.21 5 Â 106 HT1376 cells were inoculated subcutaneously into
one side of flank of mice with or without B16F10 cells co-inocu-
lated into the other flank of the mice. Tumors of appropriate size
usually grew within 30 d (HT1376) and 12 d (B16F10) after the
implantation of the respective tumor cells.
Bladder cancer-specific peptides having the sequence, Cys-Ser-
Asn-Arg-Asp-Ala-Arg-Arg-Cys (CSNRDARRC, all in L-isomer form),
were synthesized by standard Fmoc methods and tyrosine residue
was added to the N-terminal position before their cleavage from
the resin. Then, the cleaved peptides were cyclized by disulfide
bond formation between the two cysteine residues at the terminal
positions (Y-BP). As a control, the seven amino acid sequences be-
tween the two cysteines were randomized to give scrambled pep-
tides of the sequence, Cys-Asp-Ala-Ser-Arg-Arg-Asn-Arg-Cys which
latter were also conjugated with tyrosine in the same way as above
(Y-sBP). Both peptides were then purified and characterized by
HPLC and LC-MS, respectively.
4.5. Biodistribution Studies
Biodistribution of [131I]Y-BP was studied in BALB/c nu/nu fe-
male mice bearing HT1376 or B16F10 tumor. Mice were anesthe-
tized with 1–2% isoflurane in O2, and a volume of 0.1 mL of the
purified [131I]Y-BP solution (0.74 MBq) was then injected via a tail
vein. Animals were sacrificed under anesthesia (isoflurane) at 5.5 h
post-injection (n = 5). Blood, tumor, and tissues of interest were
4.2. Radiolabeling study
[
131I]NaI in 0.1 M NaOH was purchased from Korea Atomic En-
ergy Research Institute (Korea) and [124I]NaI was kindly provided
from the Korea Institute of Radiology and Science (KIRAMS, Seoul,
Korea). The two peptides, Y-BP and Y-sBP, were labeled with
dissected, weighed, and counted in a
c-counter (Wallach, Turku,
Finland). The percentage injected dose per gram (%ID/g) for each
tissue was calculated.
[
131I]NaI or [124I]NaI using Iodo-beads or Iodogen (Pierce Biochem-
ical Co., Illinois, USA) to give the radioiodinated peptides,
[
131/124I]Y-BP and [124I]Y-sBP, respectively.28 Briefly, in case of
4.6. MicroPET imaging
Iodo-beads method, a bead was washed in phosphate buffered sal-
ine (PBS, pH 7.2), dried on filter paper at room temperature and
MicroPET images were acquired on a Concorde MicroPET R4 Ro-
dent Model Scanner (Concorde Microsystems Inc., Knoxville, USA).
Animals were anesthetized with isoflurane (1–2% in oxygen) and
fixed in prone position on the bed.
then was added to a solution of [131/124I]NaI in 100
l
L PBS. Follow-
ing 5 min of shaking, peptide (20 lg in 20 lL of distilled water)
was added to the solution and incubated at room temperature
for 15 min under gentle shaking. The iodination was stopped by
removing bead from the reaction tube. When Iodogen tube was
MicroPET images of animals bearing HT1376 tumor and B16F10
on both flanks (n = 3) were acquired at 5.5 h post-injection of
either radioiodinated
0.15 mL saline) via tail vein.
[ [
124I]Y-BP or 124I]Y-sBP (7.4 MBq in
used, 80
amount of [131/124I]NaI was added. Then, the same amount of pep-
tide (20 g in 20 L of distilled water) was added to the Iodogen
lL PBS was added to Iodogen tube and the necessary
For comparison study, tumor uptake of our radiotracer ([124I]Y-
BP) was compared with that of [18F]FDG, the most widely used PET
tracer for tumor detection. The microPET images of animals bear-
ing HT1376 cancer cells (n = 3) were acquired for 20 min at
40 min post-injection of [18F]FDG via tail vein (20 MBq). After
1 day, the same mice were injected again with 7.4 MBq of
l
l
tube and the mixture was allowed to stand for 15 min at room
temperature after gentle shaking. The mixture was filtered through
0.22 lm syringe filter before HPLC injection.
The labeling yield was monitored by a radio-TLC imaging scan-
ner (Bioscan, Inc., Washington, DC, USA) on a silica plate using 10%
ammonium acetate:methanol (3:7) as the developing solvent. The
labeled peptides were purified using an HPLC system (Waters 600,
Milford, MA, USA), incorporating a reverse-phase GraceSmart RP
[
124I]Y-BP and scanned for 60 min at 5.5 h post-injection. Each
200 L solution of NaClO4 (20 mg/mL concentration) was injected
orally and intraperitoneally 30 min earlier before [124I]Y-BP injec-
tion to block free iodine uptake.29 Another 200
L solutions of Na-
l
C18 column (5
l
m, 4.6 Â 250 mm; Deerfield, IL, USA) at a flow rate
l

Y. S. Ha et al. / Bioorg. Med. Chem. 20 (2012) 4330–4335
9. Reske, S. N.; Kotzerke, J. Eur. J. Nucl. Med. 2001, 28, 1707.
124I]Y-BP and microPET image was obtained at 5.5 h.
4335
ClO4 solution was injected in the same way at 3 h post-injection of
[
10. Younes-Mhenni, S.; Janier, M. F.; Cinotti, L.; Antoine, J. C.; Tronc, F.; Cottin, V.;
Ternamian, P. J.; Trouillas, P.; Honnorat, J. Brain 2004, 127, 2331.
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All microPET images were reconstructed by a 2-dimensional or-
dered-subsets expectation maximum (OSEM) algorithm and focal
accumulations on microPET images were quantified by region of
interest (ROI) analysis. Tumor-to-background ratios (T/B) were cal-
culated using average counts per voxel on coronal images.
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Acknowledgments
17. Schottelius, M.; Wester, H.-J. Methods 2009, 48, 161.
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This work was supported by the Nuclear R&D (Grant code:
20090081817) and BAERI (Grant code: 20090078235) Programs
of NRF, funded by MEST, and by the Korea Healthcare technology
R&D Project, Ministry for Health, Welfare
(A102132). We thank Dr. Kyeong Min Kim for helpful discussion.
& Family Affairs
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