Preparation and preliminary biological evaluation of 177Lu- labeled GluDTPA-cyclo(RGDfK) for integrin αvβ3 receptor-positive tumor targeting

Article abstract of DOI:10.1002/jlcr.1944

Integrin α_vβ₃ is a receptor and is highly expressed on activated and proliferating endothelial cells during the growth and metastasis of solid tumors but not on resting endothelial cells and normal organs. Because RGD peptide binds to integrin α_vβ ₃ receptor, a variety of radiolabeled RGD peptides have been evaluated for non-invasive imaging of integrin α_vβ ₃-positive tumors. In an attempt to develop RGD-based radiopharmaceuticals, a novel GluDTPA-cyclo arginine-glycine-aspartic acid-d-phenylalanine-lysine (GluDTPA-cycloRGDfK) was simply synthesized and radiolabeled with ¹⁷⁷Lu. Also, tumor targeting and retention of the radiolabeled complex were evaluated in U87MG glioma-bearing mice. The ¹⁷⁷Lu-labeled GluDTPA-cyclo(RGDfK) was formulated with a high radiolabeling yield (>98%) under mild condition, and the radiochemical purity was sustained in both saline and serum for over 4days at 37°C. The radiolabeled compounds were rapidly cleared from the blood pool and non-target tissue. Tumor-to-blood ratio was 12.09 at 2h post injection and increased to 134.67 at 24h, while tumor to liver ratio was 2.01 at 24h similar to that of 2h. Though it is inappropriate for targeted therapy due to its low uptake in tumor (～ 1 %ID/g), the acceptable results on radiochemistry and biodistribution propose to take a further assessment for non-invasive imaging and detection of integrin α_vβ₃-positive tumors by applying diagnostic radionuclides.

Full text of DOI:10.1002/jlcr.1944

Research Article
Received 27 July 2011,
Revised 10 September 2011,
Accepted 4 October 2011
Published online 25 November 2011 in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/jlcr.1944
Preparation and preliminary biological
177
evaluation of Lu-labeled GluDTPA-cyclo
(RGDfK) for integrin anb3 receptor-positive
tumor targeting
Jin-Hwan Kim,a Jae-Cheong Lim,b Ki-Cheol Yun,b Sun-Ju Choi,b
and Young-Don Honga,b
*
Integrin anb3 is a receptor and is highly expressed on activated and proliferating endothelial cells during the growth
and metastasis of solid tumors but not on resting endothelial cells and normal organs. Because RGD peptide binds to
integrin anb3 receptor, a variety of radiolabeled RGD peptides have been evaluated for non-invasive imaging of integrin
anb3-positive tumors.
In an attempt to develop RGD-based radiopharmaceuticals, a novel GluDTPA-cyclo arginine-glycine-aspartic acid-D-
phenylalanine-lysine (GluDTPA-cycloRGDfK) was simply synthesized and radiolabeled with 177Lu. Also, tumor targeting
and retention of the radiolabeled complex were evaluated in U87MG glioma-bearing mice.
The 177Lu-labeled GluDTPA-cyclo(RGDfK) was formulated with a high radiolabeling yield (>98%) under mild condition,
and the radiochemical purity was sustained in both saline and serum for over 4 days at 37ꢀC. The radiolabeled compounds
were rapidly cleared from the blood pool and non-target tissue. Tumor-to-blood ratio was 12.09 at 2 h post injection and
increased to 134.67 at 24 h, while tumor to liver ratio was 2.01 at 24 h similar to that of 2 h.
Though it is inappropriate for targeted therapy due to its low uptake in tumor (~ 1 %ID/g), the acceptable results on
radiochemistry and biodistribution propose to take a further assessment for non-invasive imaging and detection of integrin
anb3-positive tumors by applying diagnostic radionuclides.
Keywords: Lutetium-177 (177Lu); DTPA; RGD; integrin; tumor targeting; angiogenesis
In designing radiolabeled peptides for labeling with diag-
Introduction
nostic radioisotopes such as 99mTc, 111In, 68Ga, and 64Cu for
imaging of tumor sites and with particle emitters such as 177Lu,
Integrins that are composed of non-covalently associated a and
90Y, 188Re, 153Sm, and 213Bi for targeted radionuclide therapy,
b subunits are involved in a wide range of interaction between
cell–cell as well as cell–extracellular matrixes.1,2 Among the
bifunctional chelating agents (BFCAs) have received increas-
integrin family members, it is well-known that the integrin anb3
is highly expressed on activated endothelial cells but not on
ing interest because of their important role in developing
radiolabeled bioactive molecules.17,18 Among many BFCA
resting vessels during angiogenesis. Additionally, it plays an
series such as ethylenediamine tetraacetic acid (EDTA); 1,4,7,
important role in the regulation for tumor growth, local invasive-
10-tetraazacyclododecome-1,4,7,10-tetraacetic acid (DOTA); and
ness, and metastatic potential.2 Therefore, intensive investiga-
diethylenetriamine pentaacetic acid (DTPA), DTPA is good for
tion of integrin anb3 receptor targeted anti-angiogenic strategies
its higher affinity and reasonable stability to almost all kinds of
radionuclides used in clinical applications.19 However, when
for cancer treatment, and the prevention of cancer recurrence
or metastasis has been underway. Also, non-invasive imaging
DTPA is directly used for the preparation of a DTPA conjugate,
of integrin anb3 expression by molecular imaging techniques
would be of great help for early detection of malignancy,
evaluation of tumor progress, and monitoring treatment efficacy
aDepartment of Applied Chemical Engineering, Chonnam National University,
Gwangju 500-757, Korea
of anti-angiogenic drugs.3 Actually, in recent years, RGD (Arg-Gly-
Asp) peptide-based multimodality probes were developed
for non-invasive imaging of integrin anb3 expression.4–10 Several
positron emission tomography probes using 18F have been used in
clinical investigations.11–16 In particular, 18F-labeled galacto-RGD
derivatives showed rapid and predominant excretion, resulting
in a low concentration in most of the non-target organs and
visualization of integrin anb3 expressing tumor.5,11
bRadioisotope Research Division, Research Reactor Utilization and Development,
Korea Atomic Energy Research Institute (KAERI), Daejeon 305-353, Korea
*Corresponndence to: Young-Don Hong, Ph.D., Radioisotope Research Division,
Research Reactor Utilization and Development, Korea Atomic Energy Research
Institute (KAERI), Daejeon 305–353, Korea.
E-mail: ydhong@kaeri.re.kr
J. Label Compd. Radiopharm 2012, 55 10–17
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J.-H. Kim et al.
the compound possesses only seven coordination sites that Preparation of chelator conjugated peptide; GluDTPA-cyclo
results in reduced kinetic stability in vivo. The typical example (RGDfK)
is the 111In-DTPA-DPhe-octreotide for detecting of tumors express-
Synthesis of glutamic acid DTPA (tBu)5
ing somatostatin receptors. When the DTPA-DPhe-octreotide was
labeled with 90Y, yttrium was detached from its DTPA complex
Synthetic glutamic acid DTPA penta-tert-butyl-ester was prepared
and presented in serum because of its deficiency of the coordina-
as described in Figure 1 according to the previous reported
tion demand.20
procedure.23
In particular, the DTPA-conjugated biomolecule should
have eight coordination sites to form a stable complex with
therapeutic radionuclide, such as 90Y, 177Lu, 153Sm, and 188Re.
To synthesize DTPA-conjugated peptide for using radiotracer
and radionuclide therapy, a DTPA chelator containing six
carboxylates is needed. Because peptides can be easily produced
by using solid phase peptide synthesis method, the DTPA
chelator system should possess monocarboxylic acid and
penta-protected carboxylate in the structure directly applicable
for the peptide synthesis method, which forms up to eight
bonds with radionuclides even though it was conjugated
to peptides.
Step (1) 5-benzyl 1-tert-butyl glutamic acid
g-benzyl glutamate-tert-butyl-ester (14.82 g, 62.4 mmol) was
dissolved at tert-butylacetate (219 ml, 26 eq, 1.624 mol) in a
500-ml round-bottom flask with a magnetic bar. 70% HClO4
(6.13 ml, 1.62 eq, 101 mmol) was added slowly and reacted for
24 h at room temperature. 10% Na2CO3 (188 ml, 2.83 eq) was
poured to neutralize the solution and filtrated. The pH of filtered
solution was adjusted to 10 by adding 10 M NaOH and dried
with MgSO4. The solvent was concentrated to afford a residue
as yellowish oil. Purification was performed with flash column
chromatography to yield an oily compound (1) (ethylacetate :
hexane = 0:10–3:7).
Because 177Lu decays by a half-life of 6.71 days with emission
of beta particles and lower gamma photons, it came into
spotlight for imaging and therapy of tumor among many radio-
nuclides. So, 177Lu can monitor in vivo localization of the injected
therapeutic radiopharmaceutical during treatment performing
dosimetric evaluation.21
Infrared (neat) 3371 (w), 2977 (w), 1728 (s), 1455 (w), 1367 (w),
1152 (m), 1024 (w); 1H NMR (CDCl3, 400 MHz) d 1.45 (s, 9H),
1.79–1.86 (q, 1H), 2.01–2.10 (q, 1H), 2.51 (t, 2H), 3.33 (t, 1H),
5.10 (s, 2H), 7.28–7.38 (m, 5H).
In this study, a glutamic acid-based DTPA was prepared by
applying the synthetic method for lysine-DTPA previously
reported.22 The synthesized GluDTPA was easily incorporated
into cyclo arginine-glycine-aspartic acid-D-phenylalanine-lysine
(cycloRGDfK) for targeting the anb3 integrin receptor using a
conventional peptide synthesis strategy. The prepared
GluDTPA-cyclo(RGDfK) has eight coordination sites to form a
stable complex with a variety of radionuclides. Sequentially,
GluDTPA-cyclo(RGDfK) was labeled with 177Lu and was evalu-
ated for its radiochemical stability. The biological behavior of
177Lu-labeled GluDTPA-cyclo(RGDfK) in anb3 integrin receptor-
positive U87MG xenografted mice was performed not only to
determine the aspect of tumor uptake, retention, and excretion
but also to investigate its correlation between tumor uptake
and tumor size.
Step (2) 5-benzyl-1-tert-2-(bis(2-(bis(2-tert-butoxy-2-oxoethyl)
amino)ethyl)amino)pentanedioate
To solution of g-benzyl-glutamate-tert butyl-ester (2.36 g,
8.03 mmol) in 100 ml of acetonitrile, tert-butyl 2,2′-(2-bromoethy-
lazanediyl) diacetate (6.50 g, 2.3 eq, 18.46 mmol) and 100 ml of
2 M phosphate buffer (pH = 8) were added, and the mixture was
reacted for 48 h at room temperature. Then, acetonitrile layer
was concentrated and extracted with diethylether–distilled
water. The organic layer was separated, dried with MgSO4, and
evaporated in vacuo. An oily crude was purified using flash
column chromatography (ethylacetate : hexane = 1:9 ~ 1:4) to
give a clear oily compound (2).
Infrared (neat) 2978 (w), 1731 (s), 1367 (m), 1218 (m), 1144 (s),
1
846 (w); H NMR (CDCl3, 400 MHz) d 1.44 (s, 45H), 1.80–1.89 (m,
1H), 1.95–2.01 (m, 1H), 2.44 (m, 2H), 2.67–2.78 (m, 8H), 3.33 (m,
1H), 3.40 (s, 8H), 5.10 (s, 2H), 7.30–7.36 (m, 5H).
Experimental
Step (3) Glutamic acid-DTPA penta-tert-butyl-ester; [GluDTPA-(tBu)5]
All chemicals were analytical grade purchased from chemical
company and used without further purification. The preparation
of peptide was accomplished by the use of an automated Multiple
Biomolecular Synthesizer (Peptron, Daejeon, Korea). Analytical
and preparative reverse-phase high performance liquid chromatog-
raphy (RP-HPLC) for the prepared peptide was performed on a
Shimadzu prominence HPLC (Kyoto, Japan) by using Shiseido
capcell pak C-18 column. The wavelength used for UV detection
was 220 nm for analytical RP-HPLC. The liquid chromatography-
mass spectrometer was performed by using HP 1100 series. 177Lu
was produced at the Hanaro research reactor (30 MW) installed in
Korea Atomic Energy Research Institute (KAERI). The radioactivity
was measured by using an ionizing chamber (Atomlab 200,
Biodex, Shirley, NY, USA). The radiolabeling yield and the radio-
chemical purity were determined by using Cyclone Storage Phos-
phor System (Perkin Elmer, Wellesley, MA, USA). Radioactivity for
To a solution of compound 2 (4.18 g, 5.00 mmol) in methanol
(100 ml), 10% Pd/C powder (0.42 g, 10 wt%) was added por-
tionwise followed by the addition of 3–4 drops of distilled
water. After filling with H2 gas, it was reacted for 3 h at room
temperature. After the solid residue was filtered off, the filtrated
solution was concentrated and purified with flash column chro-
matography (ethylacetate : hexane = 1:4–1:2) to give a colorless
oily compound (3).
Infrared (neat) 2976 (w), 1725 (s), 1367 (m), 1219 (m), 1147 (s),
1
848 (w); H NMR (CDCl3, 400 MHz) d 1.45 (s, 45H), 1.88–2.02 (m,
2H), 2.65 (m, 2H), 2.80–2.94 (m, 8H), 3.42 (s, 8H), 3.88 (dd, 1H),
LC/MS: calculated 745.7; found 746.4 (M + 1).
Conjugation of glutamic acid DTPA with cyclo(RGDfK): GluDTPA-
cyclo(RGDfK)
biodistribution experiments was determined with a Wallac 1470 GluDTPA-cyclo(RGDfK) was synthesized by applying standard
automated gamma counter (Perkin Elmer).
Fmoc strategy as detailed in Figure 2. Briefly, Wang resin
J. Label Compd. Radiopharm 2012, 55 10–17
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J.-H. Kim et al.
Figure 1. Reaction route for glutamic acid-based DTPA-penta-tert-butyl ester, GluDTPA-(tBu)5.
Figure 2. Solid phase synthesis of GluDTPA-cyclo(RGDfK).
conjugated Fmoc–Aspartic acid allylester was used as a solid sup- (RGDfK) product was purified by Shimadzu HPLC equipped with
port the reaction. After the Fmoc protecting group was removed capcell pak C18 column, using mobile phase with 0.1% TFA/water
from resin bounded Fmoc–Aspartic acid allylester under standard (A) and 0.1% TFA/acetonitrile (B) and under gradient conditions of
condition (20% piperidine in N,N-dimethylformamide), the linear 0–10% B in 2 min, 10–40% B in 10 min, 40–70% B in 2 min at a flow
sequence peptide was prepared by the sequential coupling of rate of 1 ml/min. The purified GluDTPA-cyclo(RGDfK) was analyzed
Fmoc-Gly-OH, Fmoc-Arg (Pbf)-OH, Fmoc-Lys (Mtt)-OH, Fmoc-D- by LC/MS (HP1100).
Phe-OH in which Fmoc group was deprotected after every
coupling step. O-benzotriazole-N,N,N′N′-tetramethyl-uronium-
Radiochemistry of 177Lu-labeled GluDTPA-cyclo(RGDfK)
hexafluoro-phosphate and N-hydroxybenzotriazole were applied
Preparation of 177Lu-labeled GluDTPA-cyclo(RGDfK)
as an activating reagent to ensure efficient coupling. Cyclization
of RGDfK was accomplished by the reaction between an amine Radiolabeling of the GluDTPA-cyclo(RGDfK) was accomplished
of phenylalanine and a carboxylic acid of aspartic acid under a by simply mixing with 177Lu solution at room temperature as
conventional amide coupling procedure after deprotection of an previously described.21
allyl group of aspartic acid using tetrakis (triphenylphosphine) (0)
GluDTPA-cyclo(RGDfK) was dissolved in distilled water to give
palladium and 1,3-dimethyl-barbituric acid in dichloromethane. a concentration of 10À6 mole/ml and 177Lu was diluted in 50 mM
To introduce the DTPA chelators, resin bounded cyclo(RGDfK) sodium acetate buffer (pH = 5.5) to give a concentration of
compounds were elongated by the coupling of GluDTPA-(tBu)5. 37 MBq/ml. 177Lu solution was injected to GluDTPA-cyclo
After the last assembly step, the resulting peptide of GluDTPA- (RGDfK) solution vial and stood for 15 min at room temperature.
cyclo(RGDfK) was cleaved from the polymeric support by treat- Saline was added to the solution to give a radiotracer concen-
ment with the mixture solvent of 90% trifluoroacetic acid (TFA) tration of 370 KBq/10À9 mol/200 ml (177Lu/ligand/volume). The
containing 2.5% triisopropylsilane (TIS), 2.5% ethanedithiol (EDT), radiolabeling yield and radiochemical purity of radiolabeled
2.5% thioanisole, and 2.5% deionized water. (TFA: TIS : EDT : compound was analyzed using silica TLC plate with methanol/
thioanisole: H2O = 90:2.5:2.5:2.5:2.5). The crude GluDTPA-cyclo water (3:1, v/v).
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Copyright © 2011 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2012, 55 10–17

J.-H. Kim et al.
Serum stability assay
experiment, four animals were pre-administered 2 Â 10À7 mole
of cyclo(RGDyK) in 0.2 ml of saline via tail vein 30 min before
the injection of 177Lu-labeled GluDTPA-cyclo(RGDfK), and after
2 h p.i., biodistribution experiment was performed.
177Lu-labeled GluDTPA-cyclo(RGDfK) was added to 200 ml of 25%
human serum in phosphate buffered saline (PBS) and incubated
at 37ꢀC for 4 days. After incubation, serum samples were
analyzed by using silica TLC at given time intervals. The stability
of radiolabeled compound was determined by Cyclone Storage
Phosphor System.
Statistical analysis
The results are expressed as the mean Æ standard deviation.
All the experiments were tested with an analysis of variance.
In some experiments, means were compared with the control
using the Bonferroni–Dunn test. A p value < 0.05 was considered
significant.
Determination of log p value
37 KBq of 177Lu-labeled GluDTPA-cyclo(RGDfK) was dissolved in
an equal volume mixture of 1-octanol and PBS buffer (1 ml : 1 ml).
After stirring vigorously for 20 min, the mixture was centrifuged
at a speed of 8000 rpm for 5 min. One hundred microliters of
samples from both 1-octanol and PBS layers were transferred
and measured the radioactivity using a Wallac 1470 Wizard
automated gamma counter. Partition coefficients were mea-
sured three different times. The log p values were reported as
the average of three independent measurements.
Results and discussion
The glutamic acid DTPA penta-tert-butyl-ester and the novel
glutamic acid DTPA-conjugated RGD peptide via the solid phase
synthetic method were easily prepared as described in Figures 1
and 2, and their final structures were shown in Figure 3. The
major peak counterpart for GluDTPA-cyclo(RGDfK) was purified
using HPLC. As shown in Figure 4, the retention time on analyti-
cal HPLC for GluDTPA-cyclo(RGDfK) was found at 7.35 min, and
the MS data were consistent with their proposed formula. The
measured molecular weight was 1051.5 for GluDTPA-cyclo
(RGDfK) (calculated molecular weight = 1051.06).
Biological evaluation of 177Lu-labeled GluDTPA-cyclo(RGDfK)
Cell culture
U87MG cells were obtained from the American Type Culture
Collection (Manassas, VA, USA) and grown in 100-mm culture
dishes (Corning, Lowell, MA, USA). The cells were cultured in
Eagle’s minimum essential medium (Lonza, Walkersville, MD,
USA), supplemented with 10% fetal bovine serum, 100units/ml
penicillin, and 100g/ml streptomycin in an atmosphere of 5%
CO2 in air at 37ꢀC up to approximately 90% confluence.
The 177Lu radiolabeling was accomplished by simple mixing
of DTPA-conjugated RGD peptide with 177Lu solution. Because
the separation of radiolabeled peptide using HPLC with various
solvent systems was not achieved successfully, the instant TLC
impregnated with silica gel using a solvent mixture of methanol/
water (3:1, v/v) was applied to discriminate the radiolabeled
peptide from un-bounded 177Lu and 177Lu-colloids. 177Lu-labeled
GluDTPA-cyclo(RGDfK) was migrated to the solvent front,
whereas unbounded 177Lu solution and colloids were retained
at the starting point (Figure 5). A high labeling yield (>98%)
was achieved and used directly without any further purification.
177Lu-labeled GluDTPA-cyclo(RGDfK) showed excellent in vitro
stability in saline and serum at 37ꢀC for 4 days (>98%) (Figure 6).
Decomposition was not observed more than 4 days of incuba-
tion, and only about 10% of the radiopeptide was decomposed
after 7 days. The log p value of 177Lu-labeled GluDTPA-cyclo
(RGDfK) was À4.8, which indicates a relative high hydrophilicity
of the radiolabeled compound.
Animal models
Female BALB/c nu/nu mice were purchased at 6 weeks of age
from Oriental, Inc. (Seoul, Korea) and were allowed 1 week
for quarantine and acclimatization. For the induction of tumor
xenografts, U87MG cells were subcutaneously injected in the
right upper flank at a concentration of 1 Â 106 cells/mouse with
200 ml of 1:1 mixture of RPMI1640 culture medium and Matrigel.
The animals were housed in a room that was maintained at
23 Æ 2ꢀC and 50 Æ 5% relative humidity, with artificial lighting
from 08:00 h to 20:00 h and 13–18 air changes per hour. They
housed four animals per cage and given tap water and commer-
cial rodent chow (Samyang Feed, Seoul, Korea) ad libitum. The
Institutional Animal Care and Use Committee at KAERI approved
the protocols used in this study, and the animals were cared for
in accordance with the Guidelines for Animal Experiments.
In an attempt to evaluate the correlation between tumor mass
and 177Lu-labeled GluDTPA-cyclo(RGDfK) uptake, the results of
two different U87MG biodistribution experiments were analyzed.
The %ID of 177Lu-labeled GluDTPA-cyclo(RGDfK) within each
tumor was plotted against the respective tumor mass. As shown
in Figure 7, the %ID of 177Lu-labeled GluDTPA-cyclo(RGDfK) in
tumors was correlated (R2 = 0.909) with tumor weight. Although
Biodistribution of 177Lu-labeled GluDTPA-cyclo(RGDfK) in U87MG
xenograft-bearing mice
Biodistribution studies were carried out using the athymic nude further study is needed, it was assumed from this analysis that
mice bearing U87MG human glioma xenograft. U87MG tumor- as the tumor size increases, tumor-associated 177Lu-labeled
bearing mice with 200 ~ 400 mg tumors were randomly divided GluDTPA-cyclo(RGDfK) uptake remains readily accessible to
into three groups (n = 4). The 177Lu-labeled GluDTPA-cyclo blood-borne RGD conjugates.
(RGDfK) (370 KBq) in 0.2 ml of saline was administered via tail
The results of biodistribution studies were summarized in
vein. After 2 and 24 h post injection (p.i.), all animals were Figure 6, when U87MG xenografted mice were injected
sacrificed by using 60 ~ 70% CO2 chamber. Blood samples were 177Lu-labeled GluDTPA-cyclo(RGDfK) with or without RGD
withdrawn from the heart. The tumor and normal organs (liver, blocking. By blocking studies, it was confirmed that 177Lu-
kidney, spleen, heart, intestine, lung, stomach) were excised, labeled GluDTPA-cyclo(RGDfK) uptake in U87MG tumors was
washed with saline, dried with absorbent tissue, weighed, and specifically mediated by integrin anb3 receptor. As shown in
counted on a Wallac 1470 Wizard automated gamma counter. Figure 8, the uptake of the radiolabeled compounds was the
The organ uptake was calculated as a percentage of the injected highest in tumor and kidney, in turn. In other organs, such
dose per gram of tissue (%ID/g). In parallel, for the blocking as blood, heart, lung, and stomach, the accumulation kept in
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J.-H. Kim et al.
Figure 3. Structure of glutamic acid-based DTPA derivative, GluDTPA-(tBu)5 (A) and GluDTPA-Cyclo(RGDfK) (B).
Figure 4. HPLC analysis (A) and LC/MS profile (B) of GluDTPA-cyclo(RGDfK).
Figure 5. Typical instant thin layer chromatography profiles of 177Lu solution (A) and 177Lu-labeled GluDTPA-cyclo(RGDfK) (B) determined by Cyclone storage phosphor
system using instant thin layer chromatography impregnated with silica gel with methanol/water (3:1, v/v) as a mobile phase.
a low level. 177Lu-labeled GluDTPA-cyclo(RGDfK) had the tumor data revealed that the tumor uptake of 177Lu-labeled
uptake of 1.05 Æ 0.03 %ID/g at 2 h and 0.70 Æ 0.13 %ID/g at GluDTPA-cyclo(RGDfK) was the highest compared with the
24 h p.i. with very fast blood clearance (0.088 Æ 0.017 %ID/g other tissues.
at 2 h and 0.009 Æ 0.007 %ID/g at 24 h). A relatively high radio-
Radiolabeled cyclic RGD (Arg-Gly-Asp) peptides represent a
activity in the kidney was found (0.95 Æ 0.04 %ID/g at 2 h and new class of radiotracers that target the integrin anb3 receptor
0.69 Æ 0.03 %ID/g at 24 h). The tumor-to-blood ratios increased overexpressed on both tumor cells and activated endothelial
from 12.09 at 2 h to 134.67 at 24 h, whereas the tumor-to-liver cells of the neovasculature during tumor growth, invasion, and
(T/L) and the tumor-to-kidney (T/K) ratios were kept at certain metastasis.24 In addition to specific expression on the activated
proportion. The T/L ratios were 2.39 at 2 h and 2.01 at 24 h, tumor vasculature, integrin anb3 receptor also overexpressed
and the T/K ratios were 1.11 at 2 h and 1.08 at 24 h. Those on some tumor cells, including late-stage glioblastoma, breast
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J.-H. Kim et al.
Figure 6. Stability of 177Lu-labeled GluDTPA-cyclo(RGDfK) for 4 days in saline (A) and human serum (B).
1.2
1
y = 1.0828x - 0.0804
R² = 0.9092
0.8
0.6
0.4
0.2
0
0
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9
1
Tumor weight (g)
Figure 8. Biodistribution of 177Lu-labeled GluDTPA-cyclo(RGDfK) in BALB/c nude
mice bearing U87MG tumor.
Figure 7. 177Lu-labeled GluDTPA-cyclo(RGDfK) uptake in U87MG tumors varying
on tumor weight.
demands on incubation an elevated temperature to form a
and prostate cancers, malignant melanoma, and ovarian metal complex, DTPA can complex with cationic metal ions at
carcinoma.25,26 It was previously reported that U87MG cells room temperature.21,27–38
are one of the tumor cells that express a very high level of
Accordingly, a glutamic acid-based DTPA derivative can
human integrin anb3 receptor.3 In this study, we synthesized bind with various radionuclides, such as 213Bi, 64Cu, 67Cu, 67 Ga,
a GluDTPA-conjugated RGD peptide, labeled with 177Lu and 68 Ga, 64Gd, 166Ho, 111In, 177Lu, 188Re, 153Sm, and 99mTc. In par-
examined its affinity for U87MG xenografted mice.
ticular, the prepared GluDTPA-penta-tert-butyl-ester can be
A variety of BFCAs have been prepared and conjugated to applicable to various fields including targeting diagnosis and
tumor-specific biomolecules such as peptides and antibodies therapy using metal ions for computed tomography/magnetic
for the development of radiotracers and magnetic resonance resonance imaging contrast-enhanced imaging and radionuclide
imaging contrast-enhancing agents. Among many BFCAs, DTPA imaging/therapy because of its easiness for the conjugation
is reasonable for their high complexity with metal cation in with biomolecules.
which DTPA forms potentially an octadentate ligand. So, the
To prepare the RGD derivative containing DTPA chelator,
formation constant of metal-DTPA complex is about 100 times an cyclo(RGDfK) peptide was conjugated with GluDTPA using a
greater than that of EDTA.27 Also, contrary to DOTA, which solid phase synthesis where the DTPA served as a metal chelator
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J.-H. Kim et al.
to be radiolabeled with 177Lu that forms an eight-coordinate time (T/B ratios = 12.09 at 2 h, 134.67 at 24 h p.i., respectively).
complex. Because the DTPA was attached apart from the Moreover, T/L ratios (2.39 at 2 h, 2.01 at 24 h p.i., respectively)
targeting site of the peptide, the binding affinity may not be and T/K ratios (1.11 at 2 h, 1.08 at 24 h p.i., respectively) were
affected by the radiolabeling process. After the preparation of desirable.
177Lu-labeled GluDTPA-cyclo(RGDfK) at ambient temperature
The results of blocking experiments demonstrated that
by simple mixing of reactants, HPLC analysis was performed the tumor uptake was derived by receptor-mediated specific
but failed its discrimination from unbounded 177Lu. This phe- binding. The pre-injection of excess cyclo(RGDyK) decreased
nomenon has shown most of the radiolabeled DTPA-containing considerably the tumor uptake of 177Lu-labeled GluDTPA-cyclo
compounds. The aspect that radiolabeled DTPA-conjugated (RGDfK), in which tumor uptake was reduced to 0.60 Æ 0.10 %
compounds were not separated from impurities using HPLC ID/g with blockage, compared with 1.05 Æ 0.03 %ID/g without
might be caused by its anionic charge and its higher hydrophi- blockage (p < 0.05).
licity. When the reaction solution was differentiated with TLC,
the labeling yield of 177Lu-labeled GluDTPA-cyclo(RGDfK) was
Conclusions
greater than 98% and its radiochemical stability was maintained
over 4 days even in human serum at 37ꢀC without any degrada-
tion, which is sufficient for further investigation.
In this study, a glutamic acid-based DTPA (GluDTPA) was
prepared and conjugated with cyclo(RGDfK) by applying a
conventional solid phase synthesis method. The 177Lu-labeled
GluDTPA-cyclo(RGDfK) was formulated by simple mixing of the
peptide with 177Lu at mild condition and confirmed its biological
affinity as a targeting agent for integrin anb3-positive tumor. The
results for 177Lu-labeled GluDTPA-cyclo(RGDfK) suggest that
further assessments of the peptide of GluDTPA-cyclo(RGDfK)
with a different radionuclide such as 68 Ga, 111In, and 99mTc for
non-invasive imaging of integrin anb3-positive tumor.
The therapeutic efficacy of drugs can be decreased by a large
tumor volume via various mechanisms. Actually, a solid tumor is
likely to form vasculatures poorly with intermittent blood flow
and large distances between functional blood vessels. Addition-
ally, increased interstitial pressure in the center of large tumors
causes radial fluid flow from the center to the periphery. The
drugs must penetrate through multiple layers of solid tissue to
reach all of the tumor cells, and this requirement is a formidable
barrier to many therapeutic drugs. For 177Lu-labeled GluDTPA-
cyclo(RGDfK), it was found that its tumor uptake was not signifi-
cantly decreased by an increase of tumor volume. There was a
consistent increase in the relative amount of 177Lu-labeled
GluDTPA-cyclo(RGDfK) in the tumor as a function of tumor
weight over the range of 100–1000 mg in U87MG xenografted
mice. These results correspond with the report by Chakraborty
et al. that there are linear relationships between the tumor
volume and %ID of 111In-labeled DOTA-3 G-RGD2 uptake.24
Taken together, we suggest that the RGD conjugate can readily
penetrate deeply into solid RGD-positive tumors, and the net
Acknowledgement
This study was supported by KAERI Major Project, Development
of radioisotopes production and application technology based
on research reactor and Chonnam National University.
Conflict of interest
The authors did not report any conflict of interest.
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