A new 18F-labeled BBN-RGD peptide heterodimer with a symmetric linker for prostate cancer imaging

Article abstract of DOI:10.1007/s00726-010-0762-5

A peptide heterodimer comprises two different receptor-targeting peptide ligands. Molecular imaging probes based on dual-receptor targeting peptide heterodimers exhibit improved tumor targeting efficacy for multi-receptor expressing tumors compared with their parent single-receptor targeting peptide monomers. Previously we have developed bombesin (BBN)-RGD (Arg-Gly-Asp) peptide heterodimers, in which BBN and RGD are covalently connected with an asymmetric glutamate linker (J Med Chem 52:425-432, 2009). Although ¹⁸F-labeled heterodimers showed significantly better microPET imaging quality than ¹⁸F-labeled RGD and BBN monomers in a PC-3 xenograft model which co-expresses gastrin-releasing peptide receptor (GRPR) and integrin αvβ3, tedious heterodimer synthesis due to the asymmetric nature of glutamate linker restricts their clinical applications. In this study, we report the use of a symmetric linker AEADP [AEADP = 3,3′-(2-aminoethylazanediyl) dipropanoic acid] for the synthesis of BBN-RGD peptide heterodimer. The ¹⁸F-labeled heterodimer (¹⁸F-FB-AEADP-BBN-RGD) showed comparable microPET imaging results with glutamate linked BBN-RGD heterodimers, indicating that the replacement of glutamate linker with AEADP linker did not affect the biological activities of BBN-RGD heterodimer. The heterodimer synthesis is rather easy and straightforward. Because tumors often co-express multiple receptors, the use of a symmetric linker provides a general method of fast assembly of various peptide heterodimers for imaging multi-receptor expressing tumors.

Full text of DOI:10.1007/s00726-010-0762-5

Amino Acids (2011) 41:439–447
DOI 10.1007/s00726-010-0762-5
ORIGINAL ARTICLE
18
A new F-labeled BBN-RGD peptide heterodimer
with a symmetric linker for prostate cancer imaging
•
Yongjun Yan Kai Chen Min Yang
•
•
•
Xilin Sun Shuanglong Liu Xiaoyuan Chen
•
Received: 27 August 2010 / Accepted: 23 September 2010 / Published online: 10 October 2010
Ó Springer-Verlag (outside the USA) 2010
Abstract A peptide heterodimer comprises two different
receptor-targeting peptide ligands. Molecular imaging
probes based on dual-receptor targeting peptide heterodi-
mers exhibit improved tumor targeting efficacy for multi-
receptor expressing tumors compared with their parent
single-receptor targeting peptide monomers. Previously we
have developed bombesin (BBN)-RGD (Arg-Gly-Asp)
peptide heterodimers, in which BBN and RGD are cova-
lently connected with an asymmetric glutamate linker
(J Med Chem 52:425–432, 2009). Although 18F-labeled
heterodimers showed significantly better microPET imag-
ing quality than 18F-labeled RGD and BBN monomers in a
PC-3 xenograft model which co-expresses gastrin-releasing
peptide receptor (GRPR) and integrin avb3, tedious het-
erodimer synthesis due to the asymmetric nature of gluta-
mate linker restricts their clinical applications. In this
study, we report the use of a symmetric linker AEADP
[AEADP = 3,30-(2-aminoethylazanediyl)dipropanoic acid]
for the synthesis of BBN-RGD peptide heterodimer.
The 18F-labeled heterodimer (18F-FB-AEADP-BBN-RGD)
showed comparable microPET imaging results with glu-
tamate linked BBN-RGD heterodimers, indicating that the
replacement of glutamate linker with AEADP linker did
not affect the biological activities of BBN-RGD hetero-
dimer. The heterodimer synthesis is rather easy and
straightforward. Because tumors often co-express multiple
receptors, the use of a symmetric linker provides a general
method of fast assembly of various peptide heterodimers
for imaging multi-receptor expressing tumors.
Y. Yan and K. Chen have contributed equally in this work.
Y. Yan ꢀ M. Yang ꢀ X. Sun ꢀ X. Chen (&)
Laboratory of Molecular Imaging and Nanomedicine (LOMIN),
National Institute of Biomedical Imaging and Bioengineering
(NIBIB), National Institutes of Health (NIH),
31 Center Drive, Suite 1C14, Bethesda,
MD 20892-2281, USA
Keywords Integrin avb3 ꢀ Gastrin-releasing peptide
receptor ꢀ BBN-RGD heterodimer ꢀ PET ꢀ 18F
e-mail: shawn.chen@nih.gov
Introduction
Y. Yan ꢀ K. Chen ꢀ S. Liu ꢀ X. Chen
Molecular Imaging Program at Stanford,
Department of Radiology and Bio-X Program,
School of Medicine, Stanford University,
1201 Welch Road, Stanford, CA 94305-5484, USA
Molecular imaging probes based on receptor-targeting
peptides have attracted intense attention in the last decade
due to their fast clearance, excellent tissue penetration and
low immunogenicity (Benedetti et al. 2004; Lee et al.
2010a, b). However, one major drawback of peptide probes
is their relatively low tumor affinity and short retention
time. To improve the binding affinity of peptide based
probes, a variety of methods have been developed. Among
them, a new approach using peptide heterodimers has
recently shown its great potential in developing high
affinity molecular imaging probes (Yan and Chen 2010).
M. Yang
Key Laboratory of Nuclear Medicine,
Jiangsu Institute of Nuclear Medicine,
Wuxi 214063, China
X. Sun
Department of Medical Imaging and Nuclear Medicine,
The Fourth Affiliated Hospital, Harbin Medical University,
Harbin 150001, China
123

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Y. Yan et al.
18F
18F
A peptide heterodimer is comprised of two different
A
receptor-targeting peptide ligands which are covalently
linked. Molecular imaging probes based on peptide hetero-
dimers exhibit improved tumor targeting efficacy compared
with their parent single receptor targeting peptide monomers
(Yan and Chen 2010; Li et al. 2008; Liu et al. 2009b, c, d, e;
Reubi and Waser 2003; Vagner et al. 2008; Yang et al. 2009).
The improvement is believed to be a result of the following:
increased receptor number for targeting, increased local
ligand concentration and increased binding kinetics.
+
HN
O
HN
O
O
O
O
O
BBN
RGD
RGD
BBN
18F-FB-Glu-BBN-RGD
O
B
C
O
O
N
H
O
O
18F
HN
O
O
Androgen independent prostate cancer tissues have been
discovered to co-express both gastrin-releasing peptide
receptor (GRPR) (di Sant’Agnese 1998; Chung et al. 1992;
Glover et al. 2003; Vashchenko and Abrahamsson 2005)
and integrin avb3 (Friedlander et al. 1995; Horton 1997;
Bello et al. 2001). Both bombesin (BBN) peptides that
target GRPR (Chen et al. 2004b; Zhang et al. 2006a; Yang
et al. 2006; Shi et al. 2008; Ananias et al. 2008) and RGD
peptides that target avb3 (Cai et al. 2005, 2008; Chen
2006; Liu 2009) have been used in prostate cancer imag-
ing. However, their imaging results are not so satisfactory
in term of binding affinity and retention time.
18F-FB-PEG-Glu-BBN-RGD
RGD
BBN
18F
OH
NH
O
O
HN
O
NH
HN
HN
O
NH
N
H
HOOC
NH
BBN
RGD
N
HN
O
O
NH2
O
O
18F-FB -A EA DP-BB N- RG D
RGD
Previously we have developed two 18F-labeled BBN-
RGD heterodimers, which showed significantly better
microPET imaging quality than 18F-labeled RGD and
BBN(7–14) monomers in a PC-3 xenograft model (Li et al.
2008; Liu et al. 2009d). However, there are alsodrawbacksof
these BBN-RGD heterodimers. Our first 18F-labeled BBN-
RGD heterodimer (18F-FB-Glu-BBN-RGD), is not a pure
compound, instead it is a mixture of two isomers as shown in
Fig. 1a. These two regional isomers have similar physical
properties and cannot be separated by HPLC. Two isomeric
products were formed due to the non-regioselective synthetic
strategy we initially used for the preparation of peptide
heterodimer. Because the two isomers are inseparable, exact
determination of the proportion in which component isomers
are present in the mixture is impossible. Because of his, the
application of our first generation 18F-FB-Glu-BBN-RGD
heterodimer in clinical study is restricted. In order to make a
clinically translatable imaging tracer, we further developed a
strategy to synthesize a PEGylated BBN-RGD heterodi-
mer with definite structure through solid phase synthesis
using orthogonal Alloc protected glutamate. Figure 1b
shows the structure of our second 18F-labeled heterodimer
(18F-FB-PEG-Glu-BBN-RGD). Although the new peptide
heterodimer is a pure compound and has the desired definite
structure, the synthesis of this peptide is rather lengthy and
cannot use standard Fmoc chemistry. This drawback resulted
in high cost for the peptide production and may hamper its
further translational application in the clinic even though this
peptide heterodimer has also been labeled with other iso-
topes and fluorophores for prostate cancer and breast cancer
imaging (Liu et al. 2009b, c, e).
NH
O
O
NH2
O
O
O
O
H
H
N
H
N
H
N
N
N
N
H
N
H
N
NH 2
H
H
O
O
O
O
HN
S
BBN
Fig. 1 Structures of 18F-labeled BBN-RGD peptide heterodimers
As part of our efforts to develop prostate cancer imaging
probes suitable for clinical applications, we designed a new
BBN-RGD heterodimer with definite structure by using
a symmetric linker AEADP [AEADP = 3,30-(2-amino-
ethylazanediyl)dipropanoic acid]. The peptide heterodimer
can be easily prepared in solution and the 18F-labeled het-
erodimer (18F-FB-AEADP-BBN-RGD, as shown in Fig. 1c)
showed comparable imaging results with BBN-RGD het-
erodimers linked with a glutamate linker. Herein, we would
like to report its preparation and applications in microPET
imaging of PC-3 tumor mice.
Materials and methods
All chemicals purchased were of analytical grade and were
used as received without further purification. No-carrier-
added 18F-F- was obtained from an in-house GE Health-
care PETtrace cyclotron. Reversed-phase extraction C18
Sep-Pak cartridges were purchased from Waters and were
pretreated with ethanol (5 mL) and water (10 mL) before
123

A new 18F-labeled BBN-RGD peptide heterodimer
441
use. The syringe filter and polyethersulfone membranes
(pore size, 0.22 mm; diameter, 13 mm) were purchased
from Nalge Nunc International. 125I-Echistatin, labeled
by the lactoperoxidase method to a specific activity
of 74 TBq/mmol (2,000 Ci/mmol) and 125I-[Tyr4]BBN
[74 TBq/mmol (2,000 Ci/mmol)] were purchased from
PerkinElmer The peptides Aca-BBN(7–14) and c(RGDyK)
were purchased from Peptides International. Analytical and
semi-preparative reversed-phase high-performance liquid
chromatography (RP-HPLC) was performed on a Dionex
680 chromatography system with a UVD 170U absorbance
detector and model 105S single-channel radiation detector
(Carroll & Ramsey Associates). The recorded data were
processed using Chromeleon version 6.50 software. A C18
Vydac protein and peptide column (218TP510; 5 lm,
250 9 10 mm) was used for purification. The flow was set
at 5 mL/min using a gradient system starting from 95%
solvent A (0.1% trifluoroacetic acid [TFA] in water) and
5% solvent B (0.1% TFA in acetonitrile [ACN]) (0–2 min)
and ramped to 35% solvent A and 65% solvent B at
32 min. The flow rate of the analytic HPLC was set at
1 mL/min using the same gradient system, but with a C18
Vydac column (218TP54, 5 lm, 250 9 4.6 mm). The
ultraviolet (UV) absorbance was monitored at 218 nm and
the identification of the peptides was confirmed based on
the UV spectrum acquired using a photodiode array
detector and matrix-assisted laser desorption/ionization
time-of-flight mass spectrometry (MALDI-TOF-MS).
(5 mL) and the solvent was evaporated to yield 3,30-(2-
(tert-butoxycarbonylamino)ethylazanediyl)dipropanoic acid
1
as a white solid (0.7 g, 74%). H NMR (DMSO): d 2.93
(m, 2 H), 2.65 (t, J = 7.1 Hz, 4 H), 2.41 (t, J = 7.1 Hz,
2 H), 2.29 (t, J = 9.93 Hz, 4 H), 1.38 (s, 9 H).
Synthesis of AEADP-BBN-RGD hetero dimer
To a solution of 3,30-(2-(tert-butoxycarbonylamino)ethyl-
azanediyl)dipropanoic acid (3.04 mg, 10 lmol) in DMF
(300 lL) was added a solution of O-(N-succinimidyl)-
1,1,3,3-tetramethyluronium tetrafluoroborate (TSTU)
(6.02 mg, 20 lmol) in DMF (600 lL) followed by N,N-
diisopropylethylamine (DIPEA) (20 lL). The reaction
mixture was stirred for 30 min at room temperature. Aca-
BBN(7–14) (abbreviated as BBN) (10.5 mg, 10 lmol) in
DMF (1.0 mL) was added and the reaction mixture was
stirred for 20 min at room temperature. After adding
c(RGDyK) (abbreviated as RGD) (12.4 mg, 20 lmol) in
DMF (1.2 mL), the reaction mixture was heated at 60°C
for 1 h. The reaction progress was monitored by analytical
HPLC. Once the reaction reached completion, HOAc
solution (2 mL, 5%) was added to quench the reaction. The
Boc protected crude product was purified by preparative
HPLC and lyophilized to yield a white powder. The Boc
protection group was removed by dissolving the product in
3 mL TFA and stirred for 10 min at room temperature.
After removing excess TFA under reduced pressure, the
final product was purified by preparative HPLC and
lyophilized to afford AEADP-BBN-RGD heterodimer as a
white powder (5.9 mg, purity [ 95%, 32% overall yield,
two steps). Analytical HPLC (Rt = 18.1 min) and MS
(MALDI-TOF: m/z 1,841.76 for [MH]? (C84H129N25O20S,
calculated [MW] 1,840.96)) analyses confirmed the product
identification.
Synthesis of benzyl 3,30-(2-(tert-butoxycarbonylamino)
ethylazanediyl)dipropanoate
Tert-butyl 2-aminoethylcarbamate (1.6 g, 10 mmol) and
benzyl acrylate (16.2 g, 100 mmol) were heated at 70°C
under N2 for 7 days. The excess of benzyl acrylate was
distilled off at 60°C and the residue was purified by column
chromatography to yield 3,30-(2-(tert-butoxycarbonylami-
no)ethylazanediyl)dipropanoate as colorless oil (4.3 g,
89%). 1H NMR (CDCl3): d 7.26–7.36 (m, 10 H), 5.11 (br s,
1 H), 5.04 (bs, 4 H), 3.14–3.15 (m, 2 H), 2.76
(t, J = 6.93 Hz, 4 H), 2.51 (t, J = 5.73 Hz, 2 H), 2.44
(t, J = 6.93 Hz, 4 H), 1.40 (s, 9H).
Synthesis of FB-AEADP-BBN-RGD
N-Succinimidyl-4-fluorobenzoate (SFB) (2 mg, 8.4 lmol)
in DMF (200 lL), AEADP-BBN-RGD (1.8 mg, 1.0 lmol)
in DMF (200 lL) and DIPEA (20 lL) were mixed. The
reaction mixture was heated at 60°C for 30 min. The reaction
mixture was quenched with 2 mL 5% HOAc. The crude
product FB-AEADP-BBN-RGD was purified by preparative
HPLC and lyophilized to give a white powder in 85% yield.
Analytical HPLC (Rt = 20.3 min) and MS (MALDI-TOF:
m/z 1,961.95 for [MH]? (C91H132FN25O21S, calculated [MW]
1,961.98)) analyses confirmed the product identification.
Synthesis of 3,30-(2-(tert-butoxycarbonylamino)
ethylazanediyl)dipropanoic acid
To a solution of benzyl 3,30-(2-(tert-butoxycarbonylami-
no)ethylazanediyl)dipropanoate (1.5 g, 3.1 mmol) in EtOH
(100 ml) was added Pd/C (50 mg, 10%). The reaction
mixture was bubbled with H2 overnight. The reaction
mixture was filtered through a plug of Celite, washed with
EtOH (5 mL) and evaporated to dryness to give the crude
product as colorless oil. The residue was added CH2Cl2
Radiochemistry
To a mixture of AEADP-BBN-RGD (200 lg) in DMSO
(20 lL) and DIPEA (20 lL) was added 18F-SFB which
123

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Y. Yan et al.
was prepared according to previously reported procedures
(Wu et al. 2007; Chen et al. 2004a; Zhang et al. 2006b; Liu
et al. 2009a). The reaction mixture was heated for 15 min
at 90°C. The reaction was quenched with 800 lL of 5%
HOAc. The 18F labeled peptide was purified by semi-
preparative HPLC. The desired fractions were combined
and the solvent was removed under reduced pressure. The
18F-labeled peptide was then formulated in normal saline
and passed through an 0.22-lm Millipore filter into a sterile
multi-dose vial for in vitro and in vivo experiments. Yield:
42%.
plates at a density of 5 9 105 cells/well and incubated
overnight. PC-3 cells were washed thrice with PBS
(phosphate buffered saline) and then 18F-FB-AEADP-
BBN-RGD was added in triplicate (*0.3 lCi/well). PC-3
cells were then re-incubated at 37°C. At five different time
points (5, 15, 30, 60, and 120 min), cells were rinsed thrice
with PBS and lysed with NaOH-SDS (sodium dodecyl
sulfate) (0.2 M NaOH, 1% SDS). The cell lysate was
collected and measured in a c counter (Packard, Meriden,
CT, USA). In the cell efflux experiment, PC-3 cells were
seeded into 12-well plate at a density of 5 9 105 cells/well
and incubated overnight. Cells were rinsed thrice with PBS
and then 18F-FB-AEADP-BBN-RGD was added in tripli-
cate (*0.3 lCi/well). After incubation at 37°C for 2 h, the
cells were washed with PBS, and then re-incubated with
serum-free medium. At six different time points (0, 15, 30,
60, 120, 180 min), the cells were washed with PBS and
lysed with NaOH-SDS (0.2 M NaOH, 1% SDS). The cell
lysate was collected and measured with a c counter
(Packard, Meriden, CT, USA). The cell uptake and efflux
values were normalized in terms of added radioactivity and
expressed as the percent added radioactivity. All experi-
ments were performed twice with triplicate wells.
Cell lines and animal models
The PC-3 human prostate carcinoma cell line was purchased
from American Type Culture Collection (ATCC). PC-3 cells
were grown in F-12K nutrient mixture (Kaighn’s modifi-
cation) (Invitrogen) supplemented with 10% (v/v) fetal
bovine serum (Invitrogen) at 37°C with 5% CO2. Animal
procedures were performed according to a protocol
approved by the Stanford University Institutional Animal
Care and Use Committee. The PC-3 tumor model was
generated by subcutaneous injection of 5 9 106 tumor cells
into the front flank of male athymic nude mice (Harlan).
Small animal PET studies were performed on the mice
3–4 weeks after inoculation when the tumor volume reached
100–300 mm3.
MicroPET studies
MicroPET scans and image data analysis were performed
using a microPET R4 rodent model scanner (Siemens
Medical Solutions) (Li et al. 2008; Liu et al. 2009d). Mice
bearing PC-3 tumor were each tail-vein injected with
*3.7 MBq (100 lCi) of 18F-FB-AEADP-BBN-RGD
under isoflurane anesthesia. Five-minute static PET images
were then acquired at three time points post injection
(30 min, 1 h, and 2 h). The image reconstruction was done
by two-dimensional ordered subsets expectation maximum
(OSEM) algorithm. No attenuation or scatter correction
was used. For the integrin and GRPR receptor-blocking
experiment, c(RGDyK) (10 mg/kg), Aca-BBN(7–14)
(15 mg/kg), or mixture of both RGD and BBN (10 mg/kg
RGD and 15 mg/kg BBN) were co-injected with 3.7 MBq
of 18F-FB-AEADP-BBN-RGD into PC-3 tumor mice. At
1 h after injection, the 5-min static microPET scans were
acquired. For each microPET scan, regions of interest
(ROIs) were drawn over the tumor, normal tissue, and
major organs on decay-corrected whole-body coronal
images using vendor software (ASI Pro 5.2.4.0). The
maximum radioactivity concentration (accumulation)
within a tumor or an organ was obtained from mean pixel
values within the multiple ROI volume, which were con-
verted to MBq/mL/min by using a conversion factor.
Assuming a tissue density of 1 g/mL, the ROIs were con-
verted to MBq/g/min and then divided by the administered
activity to obtain an imaging ROI-derived %ID/g.
In vitro cell-binding assay
In vitro integrin avb3-binding affinity of FB-AEADP-
BBN-RGD was assessed via displacement cell-binding
assays using 125I-echistatin as the radioligand. Experiments
were performed on U87MG human glioblastoma cells by a
previously described method (Wu et al. 2005). In vitro
GRPR binding affinity of FB-AEADP-BBN-RGD was
assessed via displacement cell-binding assays using
125I-[Tyr4]BBN as the radioligand. Experiments were
performed on PC-3 human prostate carcinoma cells by a
previously described method (Chen et al. 2004b). IC50 (the
best-fit 50% inhibitory concentration) values were calcu-
lated by fitting the data with nonlinear regression using
Graph-Pad Prism (GraphPad Software, Inc.). The binding
of 125I-echistatin on PC-3 and 125I-[Tyr4]BBN on U87MG
were performed as negative control. Experiments were
performed with triplicate samples.
Cell uptake and efflux studies
Cell uptake and efflux studies of 18F-FB-AEADP-BBN-
RGD were performed as follows with PC-3 tumor cells (Li
et al. 2008; Yang et al. 2006; Chen et al. 2004b). In the cell
uptake experiment, PC-3 cells were seeded into 12-well
123

A new 18F-labeled BBN-RGD peptide heterodimer
Statistical analysis
443
the unlabeled peptide could be efficiently separated from the
product, the effective specific activity was estimated to be
100 TBq/mmol on the basis of the labeling agent 18F-SFB.
Quantitative data are expressed as mean SD. Means
were compared using 1-way ANOVA and the Student’s
t test. P values of \0.05 were considered statistically
significant.
Cell binding assay
125I-c(RGDyK) was used as the radioligand for competitive
binding assay to determine the integrin avb3 receptor-
binding affinity of FB-AEADP-BBN-RGD. The heterodi-
meric peptide inhibited the binding of 125I-c(RGDyK) to
integrin expressing U87MG cells in a concentration-
dependent manner (Fig. 3a). The IC50 value for FB-AE-
ADP-BBN-RGD was determined to be 553 1.08 nM.
The binding affinities of FB-AEADP-BBN-RGD for GRPR
was evaluated using 125I-[Tyr4]BBN as the radioligand in
GRPR positive PC-3 cells. Results of the cell-binding assay
were plotted in sigmoid curves for the displacement of
125I-[Tyr4]BBN from PC-3 cells as a function of increasing
concentration of BBN analogues. The IC50 values were
determined to be 167 1.41 nM on PC-3 cells (Fig. 3b).
Similar as the previously reported FB-Glu-BBN-RGD
heterodimer (Liu et al. 2009d), FB-AEADP-BBN-RGD
showed good binding affinities for integrin avb3 and
GRPR receptors.
Results
Chemistry and radiochemistry
The synthesis of AEADP-BBN-RGD peptide heterodimer
was depicted in Fig. 2. The AEADP linker was synthesized
in two steps: (1) Michael addition of the mono-Boc-pro-
tected diamine with excess benzyl acrylate at 70°C for
7 days afforded the dipropanoate in 89% yield. (2)
Deprotection of benzyl group by Pd/C catalyzed hydroge-
nation produced the bisacid in 74% yield. Assembly of
RGD and BBN was carried out stepwise and stoichiomet-
rically, which represents the most critical operation of the
whole synthesis. The bisacid was first activated with
2 equiv. of TSTU at room temperature for 30 min, then
1 equiv. of BBN was added. After conjugation for 20 min
at room temperature, 1 equiv. of RGD was added and the
reaction mixture was heated up to 60°C for 1 h to ensure
high conversion. The resulting Boc-protected AEADP-
BBN-RGD heterodimer was further treated with anhydrous
TFA to remove Boc-protection group to afford AEADP-
BBN-RGD heterodimer bearing the free amine function-
ality. The overall yield was 32% for the assembly and
de-protection steps. The crude product mixture contained
RGD-BBN, RGD-RGD, and BBN-BBN dimers. Only the
desired RGD-BBN heterodimer was isolated. The purity
and identity of final product was confirmed by HPLC and
MALDI-TOF mass spectrometry.
Cell uptake studies
The cell uptake of 18F-FB-AEADP-BBN-RGD was eval-
uated in PC-3 tumor cells that coexpress GRPR and inte-
grin avb3. The results are shown in Fig. 3c. During the first
15 min of incubation, 18F-FB-AEADP-BBN-RGD was
taken up by tumor cells rapidly. After 15 min, the uptake
was slow and finally reached a plateau. The PC-3 cell
uptake of 18F-FB-AEADP-BBN-RGD was 2.64 0.22,
3.90 0.16, 4.38 0.17, 4.68 0.26, and 4.72 0.36%
at 5, 15, 30, 60, and 120 min, respectively. The cell uptake
values of 18F-FB-AEADP-BBN-RGD are comparable to
those of our previously reported 18F-FB-Glu-BBN-RGD
tracer in which RGD and BBN were connected by a glu-
tamate linker (Liu et al. 2009d; Li et al. 2008). Efflux of
The 18F-radiolabeling of AEADP-BBN-RGD heterodi-
mer with 18F-SFB was performed in DMSO at 90°C for
15 min. The radiolabeling yield was around 42% from
18F-SFB with high radiochemical purity ([95%). Because
Fig. 2 Synthesis of AEADP-
BBN-RGD heterodimer
O
O
N
NH2
+
O
BocHN
BocHN
O
7d
O
O
O
O
OH
OH
BBN
RGD
1. 2 eq. TSTU, DIPEA
2. 1 eq. BBN
3. 1 eq. RGD
4. TFA
N
N
Pd/C
H2
BocHN
H2N
O
O
123

444
Y. Yan et al.
Fig. 3 a Inhibition of
B
125I-[Tyr3]-BBN binding to
GRPR on PC-3 cells by
FB-AEADP-BBN-RGD
A
125
100
75
50
25
0
125
100
75
50
25
0
(n = 3). b Inhibition of
125I-echistatin binding to
integrin avb3 on U87MG cells
by FB-AEADP-BBN-RGD
(n = 3). c Cell uptake assay of
18F-FB-AEADP-BBN-RGD on
PC-3 tumor cells (n = 3). d Cell
efflux assay of 18F-FB-AEADP-
BBN-RGD on PC-3 tumor
cells (n = 3)
-10
-9
-8
-7
-6
-5
-4
-11
-10
-9
-8
-7
-6
-5
Log(mol/L)
Log(mol/L)
C
6
5
4
3
2
1
0
60
50
40
30
20
10
0
D
0
25
50
75
100
125
150
0
25
50
75
100
125
150
Incubation time (min)
Incubation time (min)
radioactivity from PC-3 tumor cell was almost linear dur-
ing the 2 h incubation time (Fig. 3d). After 2 h incubation,
about 50% of the radioactivity was released from the tumor
cells, which was slightly less than what we observed with
18F-FB-Glu-BBN-RGD.
of 18F-AEADP- BBN-RGD are comparable to previously
reported 18F-FB-Glu-BBN-RGD (Liu et al. 2009d; Li et al.
2008) indicating that the structure of AEADP linker did not
have negative effects on tumor targeting in vivo.
In vivo blocking study also demonstrated that dual-
receptor binding ability of 18F-FB-AEADP-BBN-RGD for
integrin avb3 and GRPR was not changed when symmetric
AEADP linker was used. Illustrated in Fig. 5a are the rep-
resentative coronal images of PC-3 tumor mice at 1 h
postinjection of 18F-FB-AEADP-BBN-RGD when co-
injected with RGD (10 mg/kg of mouse body weight), BBN
(15 mg/kg of mouse body weight), or mixture of both RGD
and BBN (10 mg/kg for RGD and 15 mg/kg for BBN).
The tumor uptake of 18F-FB-AEADP-BBN-RGD (Fig. 5b)
decreased from 4.56 0.87 %ID/g to 1.76 0.17 and
1.40 0.14 %ID/g in the presence of only RGD or BBN,
respectively, indicating partial inhibition. The biggest
decrease in tumor uptake was observed when the tracer was
co-injected with mixture of both RGD and BBN, the tracer
uptake values decreased to 1.06 0.20 %ID/g.
MicroPET imaging
Around 3.7 MBq (100 lCi) of 18F-FB-AEADP-BBN-RGD
was intravenous injected into PC-3 tumor-bearing mice
(n = 3/group). Figure 4a shows representative coronal
microPET images at different time points post-injection.
Tumor can be clearly seen at all time points (30, 60 and
120 min). The tumor to contralateral background contrast
is also very good. The regions of interest (ROIs) that
encompass the entire organ on the microPET coronal
images were measured to determine the tumor and major
organ activity accumulation. The tumor uptake of 18F-FB-
AEADP- BBN-RGD was calculated to be 5.20 1.04,
4.56 0.87 and 4.00 0.59 %ID/g at 30, 60, and
120 min, respectively. The kidneys showed high uptake,
indicating that the tracer is mainly excreted via renal route.
The liver uptake was relatively low, with the uptake values
calculated to be 2.58 0.53, 1.93 0.21 and 1.21
0.10 %ID/g at 30, 60 and 120 min, respectively. As the
tracer is cleared rapidly from normal nontargeted organs,
the tumor/nontumor (T/NT) ratio increased with time. At
120 min postinjection, the T/NT ratios reached highest
points (1.18 0.19, 3.31 0.36 and 5.98 0.39 for
kidneys, liver and muscle, respectively). The T/NT ratios
Discussion
Androgen-independent prostate cancer cells coexpress both
gastrin-releasing peptide receptor (GRPR) and integrin
avb3. Previously, we developed two 18F-labeled BBN-
RGD peptide heterodimers, which target both GRPR and
integrin and showed significant improvement over BBN or
RGD monopeptides. However, their application in the
123

A new 18F-labeled BBN-RGD peptide heterodimer
445
5% ID/g
5% ID/g
A
A
0% ID/g
0% ID/g
0.5 h
1 h
2 h
RGD+ BBN
Block
RGD Block
BBN Block
B
10
8
6
5
4
3
2
1
0
B
0.5 h
1 h
2 h
6
4
2
0
Control
RGD Block
BBN Block RGD + BBN Block
Tumor
0.5 h
Kidney
Liver
Muscle
C
7
6
5
4
3
2
1
0
Fig. 5 a Decay-corrected whole-body coronal microPET images of
PC-3 tumor-bearing mice at 1 h after injection of 18F-FB-BBN-RGD
and a blocking dose of c(RGDyk) (10 mg/kg of mouse body weight),
BBN (15 mg/kg mouse body weight), or mixture of RGD
(10 mg/kg) ? BBN (15 mg/kg). b Comparison between the uptake
of 18F-FB-AEADP-BBN-RGD in PC-3 tumor with or without
co-injection of blocking dose of c(RGDyk) (10 mg/kg of mouse
body weight), BBN (15 mg/kg mouse body weight), or mixture of
RGD (10 mg/kg) ? BBN (15 mg/kg)
1 h
2 h
Kidney
Liver
Muscle
Glu-BBN-RGD heterodimer, the free amino group was
attached directly to a secondary carbon atom of the glu-
tamate linker. While in AEADP-BBN-RGD, the protruding
free amino group which attached to the AEADP backbone
through two methylene groups encounters less steric
hindrance.
Fig. 4 Representative coronal microPET images (a) and the radio-
activity accumulation quantification (b) in selected organs of the PC-3
tumor-bearing mice at 30, 60 and 120 min after injection of 3.7 MBq
of 18F-FB-AEADP-BBN-RGD (n = 3/group). All microPET images
were decay-corrected to the injection time. c Calculated tumor/
nontumor (T/NT) ratios from (b)
Our receptor-binding assay results demonstrated that,
similar to our previous developed Glu-BBN-RGD hetero-
dimer, the new AEADP-BBN-RGD heterodimer also pos-
sesses dual receptor binding ability. The replacement of
glutamate linker with AEADP linker did not affect the
biological activities of BBN-RGD heterodimer.
Comparison of the PET imaging results of 18F-FB-AE-
ADP-BBN-RGD and 18F-FB-Glu-BBN-RGD revealed that
they have comparable tumor uptake and nonspecific tissue
uptake. The in vivo blocking experiment demonstrated that
co-injection of either RGD or BBN could only partially
inhibit the PC-3 tumor uptake of 18F-FB-AEADP-BBN-
RGD. When one type of receptors was blocked in the
presence of blocking amount of non-radiolabeled peptide
monomer, there were still unblocked receptors of other
type available for tumor targeting. Co-injection of RGD
and BBN mixture could further decrease the tumor uptake
because both GRPR and integrin avb3 receptors were
blocked and no receptors were available for targeting. This
clinic is restricted due to either the mixture nature or
inefficient synthesis of the tracers.
A PET tracer of potential large scale clinical applica-
tions should be capable of providing high quality images
and should be easy to prepare. With this goal in mind,
we designed a new BBN-RGD heterodimer based on an
AEADP linker. The linker is comprised of one amino and
two carboxylic acid functional groups. The two carboxylic
acid groups were used to link RGD and BBN peptides by
forming amide bonds with the free amino groups of the
peptides, the free amino group in AEADP linker was used
for 18F labeling through SN2 nucleophilic reaction with
18F-SFB. Activation of both carboxylic acid functional
groups of the linker followed by sequential addition of
BBN and RGD peptides produced the desired heterodimer.
Due to the symmetric nature of the linker, only one het-
erodimer product with definite structure was formed. In
123

446
Y. Yan et al.
observation confirmed that the ability of 18F-FB-AEADP-
BBN-RGD to bind both GRPR and integrin avb3 was
retained in vivo.
Cai W, Niu G, Chen X (2008) Imaging of integrins as biomarkers for
tumor angiogenesis. Curr Pharm Des 14(28):2943–2973
Chen X (2006) Multimodality imaging of tumor integrin avb3
expression. Mini Rev Med Chem 6(2):227–234
By using the new AEADP linker, we have demonstrated
that a BBN-RGD peptide heterodimer with definite struc-
ture and good imaging quality can be easily prepared
without tedious synthesis. Such strategy can be generally
applied to other peptide heterodimer preparations. Many
tumors co-express multiple receptors, and many targeting
peptide ligands are now commercially available. With our
strategy of using a symmetric linker, assembly of peptide
heterodimers from peptide monomers would be very
straightforward (one pot synthesis within 2 h).
Chen X, Park R, Hou Y, Khankaldyyan V, Gonzales-Gomez I, Tohme
M, Bading JR, Laug WE, Conti PS (2004a) Micropet imaging of
brain tumor angiogenesis with 18F-labeled pegylated RGD
peptide. Eur J Nucl Med Mol Imaging 31(8):1081–1089
Chen X, Park R, Hou Y, Tohme M, Shahinian AH, Bading JR, Conti
PS (2004b) Micropet and autoradiographic imaging of grp
receptor expression with 64Cu-DOTA-[Lys3]bombesin in
human prostate adenocarcinoma xenografts.
45(8):1390–1397
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Chung DH, Evers BM, Beauchamp RD, Upp JR Jr, Rajaraman S,
Townsend CM Jr, Thompson JC (1992) Bombesin stimulates
growth of human gastrinoma. Surgery 112(6):1059–1065
di Sant’Agnese PA (1998) Neuroendocrine cells of the prostate and
neuroendocrine differentiation in prostatic carcinoma: a review
of morphologic aspects. Urology 51(5A Suppl):121–124
Friedlander M, Brooks PC, Shaffer RW, Kincaid CM, Varner JA,
Cheresh DA (1995) Definition of two angiogenic pathways by
distinct av integrins. Science 270(5241):1500–1502
Glover SC, Tretiakova MS, Carroll RE, Benya RV (2003) Increased
frequency of gastrin-releasing peptide receptor gene mutations
during colon-adenocarcinoma progression. Mol Carcinog 37(1):
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It also should be noted that although using a peptide
heterodimer is beneficial for tumor detection, there are
potential limitations of this approach. For instance, the lack
of target specificity, i.e., it cannot determine which specific
target is imaged. In addition, binding in normal tissue
might be increased since multiple targets are imaged.
Horton MA (1997) The alpha v beta 3 integrin ‘‘Vitronectin
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Conclusion
Lee S, Xie J, Chen X (2010a) Peptide-based probes for targeted
molecular imaging. Biochemistry 49(7):1364–1376
Lee S, Xie J, Chen X (2010b) Peptides and peptide hormones for
molecular imaging and disease diagnosis. Chem Rev 110(5):
3087–3111
Li ZB, Wu Z, Chen K, Ryu EK, Chen X (2008) 18f-labeled bbn-rgd
heterodimer for prostate cancer imaging. J Nucl Med 49(3):
453–461
Liu S (2009) Radiolabeled cyclic rgd peptides as integrin avb3-
targeted radiotracers: Maximizing binding affinity via bivalency.
Bioconjug Chem 20(12):2199–2213
In this study, we successfully designed and synthesized a
dual GRPR and integrin receptor-targeting heterodimeric
peptide, AEADP-BBN-RGD, using a symmetric linker.
18F-labeled AEADP-BBN-RGD showed high tumor uptake
and good tumor-to-background contrast in a PC-3 tumor
model. The peptide heterodimer was easy to prepare,
especially from commercially available monomeric pep-
tides. Because tumors often co-express multiple receptors,
this strategy thus provides a general method of fast
assembly of various peptide heterodimers for imaging dual
receptor expressing tumors.
Liu S, Liu Z, Chen K, Yan Y, Watzlowik P, Wester HJ, Chin FT,
Chen X (2009a) 18F-labeled galacto and pegylated rgd dimers for
EPT imaging of avb3 integrin expression. Mol Imaging Biol.
doi:10.1007/s11307-009-0284-2
Liu Z, Li ZB, Cao Q, Liu S, Wang F, Chen X (2009b) Small-animal
pet of tumors with 64Cu-labeled rgd-bombesin heterodimer.
J Nucl Med 50(7):1168–1177
Acknowledgments This work was supported in part by the National
Cancer Institute (NCI) grants R01 CA119053, P50 CA114747, and
U54 CA119367) and the Intramural Research Program of the National
Institute of Biomedical Imaging and Bioengineering (NIBIB),
National Institutes of Health (NIH).
Liu Z, Niu G, Wang F, Chen X (2009c) 68Ga-labeled NOTA-RGD-
BBN peptide for dual integrin and GRPR-targeted tumor
imaging. Eur J Nucl Med Mol Imaging 36(9):1483–1494
Liu Z, Yan Y, Chin FT, Wang F, Chen X (2009d) Dual integrin and
gastrin-releasing peptide receptor targeted tumor imaging using
18F-labeled pegylated rgd-bombesin heterodimer 18F-FB-PEG3-
Glu-RGD-BBN. J Med Chem 52(2):425–432
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