18F-labeled bombesin analogs for targeting GRP receptor-expressing prostate cancer
The gastrin-releasing peptide receptor (GRPR) is found to be overexpressed in a variety of human tumors. The aim of this study was to develop 18F-labeled bombesin analogs for PET of GRPR expression in prostate cancer xenograft models. [Lys3]Bombesin ([Lys3]BBN) and aminocaproic acid-bombesin(7-14) (Aca-BBN(7-14)) were labeled with 18F by coupling the Lys3 amino group and Aca amino group, respectively, with N-succinimidyl-4-18F-fluorobenzoate (18F-SFB) under slightly basic condition (pH 8.5). Receptor-binding affinity of FB-[Lys3]BBN and FB-Aca-BBN(7-14) was tested in PC-3 human prostate carcinoma cells. Internalization and efflux of both radiotracers were also evaluated. Tumor-targeting efficacy and in vivo kinetics of both radiotracers were examined in male athymic nude mice bearing subcutaneous PC-3 tumors by means of biodistribution and dynamic microPET imaging studies. 18F-FB-[Lys3]BBN was also tested for orthotopic PC-3 tumor delineation. Metabolic stability of 18F-FB-[Lys3]BBN was determined in mouse blood, urine, liver, kidney, and tumor homogenates at 1 h after injection. The typical decay-corrected radiochemical yield was about 30%-40% for both tracers, with a total reaction time of 150 +/- 20 min starting from 18F-. 18F-FB-[Lys3]BBN had moderate stability in the blood and PC-3 tumor, whereas it was degraded rapidly in the liver, kidneys, and urine. Both radiotracers exhibited rapid blood clearance. 18F-FB-[Lys3]BBN had predominant renal excretion. 18F-FB-Aca-BBN(7-14) exhibited both hepatobiliary and renal clearance. Dynamic microPET imaging studies revealed that the PC-3 tumor uptake of 18F-FB-[Lys3]BBN in PC-3 tumor was much higher than that of 18F-FB-Aca-BBN(7-14) at all time points examined (P < 0.01). The receptor specificity of 18F-FB-[Lys3]BBN in vivo was demonstrated by effective blocking of tumor uptake in the presence of [Tyr4]BBN. No obvious blockade was found in PC-3 tumor when 18F-FB-Aca-BBN(7-14) was used as radiotracer under the same condition. 18F-FB-[Lys3]BBN was also able to visualize orthotopic PC-3 tumor at early time points after tracer administration, at which time minimal urinary bladder activity was present to interfere with the receptor-mediated tumor uptake. This study demonstrates that 18F-FB-[Lys3]BBN and PET are suitable for detecting GRPR-positive prostate cancer in vivo.
F-Labeled Bombesin Analogs for Targeting
GRP Receptor-Expressing Prostate Cancer
Xianzhong Zhang, PhD
; Weibo Cai, PhD
; Feng Cao, MD, PhD
; Eduard Schreibmann, PhD
Joseph C. Wu, MD, PhD
; Lei Xing, PhD
; and Xiaoyuan Chen, PhD
Molecular Imaging Program at Stanford (MIPS), Department of Radiology and Bio-X Program, Stanford University School of Medicine,
Department of Radiation Oncology, Stanford University School of Medicine, Stanford, California; and
Division of Cardiology, Department of Medicine, Stanford University School of Medicine, Stanford, California
The gastrin-releasing peptide receptor (GRPR) is found to be
overexpressed in a variety of human tumors. The aim of this
study was to develop
F-labeled bombesin analogs for PET
of GRPR expression in prostate cancer xenograft models.
]BBN) and aminocaproic acid-
bombesin(7–14) (Aca-BBN(7–14)) were labeled with
F by cou-
pling the Lys
amino group and Aca amino group, respectively,
F-SFB) under slightly
basic condition (pH 8.5). Receptor-binding afﬁnity of FB-
]BBN and FB-Aca-BBN(7–14) was tested in PC-3 human
prostate carcinoma cells. Internalization and efﬂux of both radio-
tracers were also evaluated. Tumor-targeting efﬁcacy and in vivo
kinetics of both radiotracers were examined in male athymic
nude mice bearing subcutaneous PC-3 tumors by means of
biodistribution and dynamic microPET imaging studies.
]BBN was also tested for orthotopic PC-3 tumor delineation.
Metabolic stability of
]BBN was determined in mouse
blood, urine, liver, kidney, and tumor homogenates at 1 h after in-
jection. Results: The typical decay-corrected radiochemical yield
was about 30%–40% for both tracers, with a total reaction time of
150 6 20 min starting from
]BBN had moderate
stability in the blood and PC-3 tumor, whereas it was degraded
rapidly in the liver, kidneys, and urine. Both radiotracers exhibited
rapid blood clearance.
]BBN had predominant renal
F-FB-Aca-BBN(7–14) exhibited both hepatobiliary
and renal clearance. Dynamic microPET imaging studies revealed
that the PC-3 tumor uptake of
]BBN in PC-3 tumor
was much higher than that of
F-FB-Aca-BBN(7–14) at all time
points examined (P , 0.01). The receptor speciﬁcity of
]BBN in vivo was demonstrated by effective blocking of
tumor uptake in the presence of [Tyr
]BBN. No obvious blockade
was found in PC-3 tumor when
F-FB-Aca-BBN(7–14) was used
as radiotracer under the same condition.
also able to visualize orthotopic PC-3 tumor at early time points
after tracer administration, at which time minimal urinary bladder
activity was present to interfere with the receptor-mediated tumor
uptake. Conclusion: This study demonstrates that
]BBN and PET are suitable for detecting GRPR-positive pros-
tate cancer in vivo.
Key Words: prostate cancer; GRP receptor;
J Nucl Med 2006; 47:492–501
Neuroendocrine (NE) cells are believed to play a para-
crine regulatory role in the prostate (1). Prostatic NE cells
contain abundant secretory granules ﬁlled with numerous
bioactive compounds collectively called NE products (NEP)
(2). In particular, members of the gastrin-rele asing peptide
(GRP) family and its analog bombesin (BBN) have been
implicated in the biology of several human malignancies,
including lung, colon, breast, and prostate cancers (1–4). To
date, 3 mammalian GRP/BBN receptor subtypes have been
cloned and characterized: the GRP receptor (GRPR), the
BBN-receptor subtype 3 (BRS-3), and the neuromedin-B
receptor (NMBR) (5). Only GRPR was found in prostate
carcinoma (6), although NMBR and BSR-3 have been found
in other cancer types (7,8). Antagonists of GRPR are de-
signed to bind to human GRPR with high afﬁnity and block
the receptor-activ ated signal transduction pathways and,
thus, inhibit the growth of prostate cancer both in vitro and in
vivo (9). GRP/BBN analogs have also been used as carriers
to deliver drugs, radionuclides, and toxins to target prostate
carcinoma and other cancer types that are GRPR positive
(10,11). Therefore, the ability to document GRPR density in
vivo is crucial for the application of GRPR-targeted drug
Being the most widely applied radionuclide for diagnos-
tic purposes, a great deal of research has been done to de-
In-labeled BBN-like peptides involving
a wide range of chelators, peptide sequences, and bifunc-
tional linkers (12). To date, 2 of the de novo radiolabeled
GRP-like peptides, RP527 (13) and the BN1 (14), are under
clinical evaluation with satisfactory results. In addition,
Lu have been used to radiolabel BBN analogs
for potential peptide receptor radiotherapy applications
PET for cancer imaging of GRPR status in vivo has been
less studied. Rogers et al. developed a truncated form of a
Received Sep. 28, 2005; revision accepted Nov. 15, 2005.
For correspondence or reprints contact: Xiaoyuan Chen, PhD, Molecular
Imaging Program at Stanford (MIPS), Department of Radiology and Bio-X
Program, Stanford University School of Medicine, 1201 Welch Rd., P095,
Stanford, CA 94305-5484.
492 THE JOURNAL OF NUCLEAR MEDICINE • Vol. 47 • No. 3 • March 2006
Cu (half-life [t
] 5 12.7 h; b
, 17.4%)-labeled BBN
Cu-DO TA-Aoc-BBN(7–14) (Aoc is 8-aminooctanoic
acid), for microPET imaging of an androgen-independent PC-3
tumor xenograft model (17). Incorporation of a poly(ethylene
glycol) (PEG) linker (molecular weight 3,400) resulted in
signiﬁcantly reduced receptor avidity and lower receptor spe-
ciﬁc activity accumulation in vi vo (18). We recently reported
the synthesis and pharmacologic evaluation of another
labeled BBN analog,
]BBN, for targeting
GRPR expression in both PC-3 and 22RV1 tumor models
(19). Very recently, another BBN analog, [
]BBN(6–14) amide (BZH3), was conjugated with
1,4,7,10-tetraazac yclododecane-N,N9,N99,N999-tetraacetic acid
(DOTA) through a PEG2 linker and labeled with
68 min; b
, 88%), obtained from a
Ga generator for
imaging AR42J rat pancreatic cancer -bearing nude mice (20).
5 109.7 min; b
, 99%) is an idea l short-lived
PET isotope for labeling small molecular recognition units
such as antigen- binding domain of antibody fragments and
small biologically active peptides (21).
thetic groups such as N-succinimidyl-4-
F-SFB) have been developed, which can be attached to
either N-terminal or lysine e-amino groups with little or no
loss of bioactivity of the peptide ligand (22,23). In the
present study, we labeled both [Lys
and aminocaproic acid-bombesin(7–14) (Aca-BBN(7–14))
F for GRPR imaging of subcutaneous and orthotopic
PC-3 tumor models with PET.
MATERIALS AND METHODS
All chemicals obtained commercially were used without further
]BBN and Aca-BBN(7–14) were synthesized
using solid-phase Fmoc chemistry by American Peptide, Inc. No-
was obtained from PETNET Inc. The received
was trapped on an anion-exchange resin and eluted with 0.5 mL
(2 mg/mL in H
O) combined with 1 mL Kryptoﬁx 2.2.2.
(Sigma-Aldrich) (10 mg/mL in acetonitrile). The semipreparati ve
rev ersed-phase high-performance liquid chromatography (HPLC)
system has been described elsewhere (24).
Chemistry and Radiochemistry
4-Fluorobenzoyl-bombesin analogs (FB-[Lys
]BBN and FB-
Aca-BBN(7–14)) were synthesized as reference standards. In brief,
an equimolar amount of SFB (in acetonitrile) and [Lys
Aca-BBN(7–14) (in H
O) was mixed and the pH was adjusted
to 8.3 by addition of 0.1N sodium borate buffer. The reaction
mixture was incubated at 40C for 80 min and then quenched by
triﬂuoroacetic acid (TFA). Semipreparative HPLC puriﬁcation
gave the desired products. The HPLC retention times were around
20.8 min for FB-[Lys
]BBN and 19.1 min for FB-Aca-BBN(7–14),
14)) were synthesized by coupling the corresponding BBN peptide
F-SFB was puriﬁed by semipreparative
HPLC, concentrated to about 200 mL, and added to [Lys
Aca-BBN(7–14) peptide (200 mg) in 800 mL of sodium borate
buffer (50 mmol/L, pH 8.5). The reaction mixture was gently
mixed at 40C for 30 min. Final puriﬁcation was accomplished
by semipreparative HPLC and the tracers were reconstituted in
phosphate-buffered saline (PBS, pH 7.4) and passed through a
0.22-mm Millipore ﬁlter (Millipore Corp.) for in vivo applications.
In Vitro Cell-Binding Assay
The PC-3 human prostate carcinoma cell line was purchased
from American Type Culture Collection. PC-3 cells were grown in
F-12K nutrient mixture (Kaighn’s modiﬁcation) (Invitrogen Corp.)
supplemented with 10% (v/v) fetal bovine serum (FBS) (Invitrogen
Corp.) at 37Cwith5%CO
. In vitro binding afﬁnity and spec-
iﬁcity of FB-BBN analogs for GRPR were evaluated using com-
petitive receptor-binding assay.
Life Science Products, Inc.; speciﬁc activity, 74 TBq/mmol) was
used as the GRPR-speciﬁc radioligand. Experiments were performed
as described previously (19). The 50% inhibitory concentration
) value for the displacement binding of
those ligands was calculated by nonlinear regression analysis using
GraphPad Prism software (Graph-Pad Software Inc.). All experi-
ments were performed twice with triplicate samples.
Internalization and Efﬂux Studies
Internalization and efﬂux of
Aca-BBN(7–14) into PC-3 cells were examined following a pro-
tocol reported earlier (19). The data was normalized as percentage
of the total amount of radioactivity added per cell.
All animal experiments were performed under a protocol ap-
proved by the Stanford University Administrative Panel on Lab-
oratory Animal Care (A-PLAC). Both subcutaneous and orthotopic
tumor model were established in 4- to 6-wk-old male athymic
nu/nu mice (Harlan). For the subcutaneous prostate cancer model,
5 · 10
PC-3 cells suspended in 50 mL serum-free F-12K medium
and 50 mL Matrigel (BD Biosciences) were injected into the right
shoulder of the mice. For the orthotopic PC-3 tumor model, 5 · 10
cells in 20 mL PBS were injected into the prostate gland of male
nude mice. The prostate of anesthetized mice was exposed through
a midline laparotomy incision and by retraction of the bladder and
male sex accessory glands anteriorly. Injection of cells was per-
formed with a 27-gauge needle inserted into the prostatic lobe
located at the base of the seminal vesicles as described (28). The
abdominal wound was sutured using a 4.0 chromic gut suture in a
The subcutaneous tumor-bearing mice were used for biodis-
tribution when the tumor volume reached 300–400 mm
after inoculation). Three mice were each injected intravenously
with about 370 kBq (10 mCi)
BBN(7–14). The mice were sacriﬁced at 60 min after injection
and the body weight was recorded. Blood, tumor, major organs,
and tissues were collected, wet weighed, and counted by
g-counter. The percentage of injected dose per gram (%ID/g) was
determined for each sample. For each mouse, radioactivity of the
tissue samples was calibrated against a known aliquot of radio-
tracer. Values are expressed as mean 6 SD. To test the speciﬁc
binding of the radiotracers to PC-3 tumor, GRPR-blocking studies
were performed by examining the biodistribution of each radio-
labeled tracer in the presence of [Try
]BBN as a blocking agent
F-BOMBESIN PET FOR GRPR • Zhang et al. 493
(10 mg/kg mice body weight). Mice were also sacriﬁced at 60 min
after injection (n 5 3).
microPET Imaging and Image Analysis
microPET scans were performed on a microPET R4 rodent
model scanner (Concorde Microsystems Inc.). The scanner has a
computer-controlled bed and 10.8-cm transaxial and 8-cm axial
ﬁelds of view (FOVs). It has no septa and operates exclusively in
the 3-dimensional (3D) list mode. Animals were placed near the
center of the FOV of the scanner, where the highest image reso-
lution and sensitivity are available. The microPET studies were
performed by tail-vein injection of 3.7 MBq (100 mCi) of
isoﬂurane anesthesia. The 60-min dynamic (5 · 1 min, 5 · 2 min,
5 · 3 min, 6 · 5 min) microPET data acquisition (total of 21
frames) was started 4 min after injection. Static images at 2.5-, 3-,
and 4-h time points were also acquired as 10-min static images.
The images were reconstructed by a 2-dimensional ordered-
subsets expectation maximum (OSEM) algorithm and no correc-
tion was necessary for attenuation or scatter (29).
For each microPET scan, regions of interest (ROIs) were drawn
over the tumor, normal tissue, and major organs by using vendor
software (ASI Pro 220.127.116.11) on decay-corrected whole-body coro-
nal images. The maximum radioactivity concentration (accumu-
lation) within a tumor or an organ was obtained from mean pixel
values within the multiple ROI volume, which were converted to
counts/mL/min by using a conversion factor. Assuming a tissue
density of 1 g/mL, the ROIs were converted to counts/g/min and
then divided by the administered activity to obtain an imaging
Male mice bearing PC-3 tumors were injected intravenously
with 3.7 MBq of
]BBN. The animals were sacriﬁced
and dissected at 60 min after injection Blood, urine, liver, kidneys,
and tumor were collected. Blood was immediately centrifuged for
5 min at 13,200 rpm. Organs were homogenized using an IKA
Ultra-Turrax T8 (IKA Works Inc.), suspended in 1 mL of PBS, and
centrifuged for 5 min at 13,200 rpm. After removal of the super-
natants, the pellets were washed with 500 mL of PBS. For each
sample, supernatants of both centrifugation steps were combined
and passed through Sep-Pak C
cartridges. The urine sample was
directly diluted with 1 mL of PBS and passed through Sep-Pak
cartridge. The cartridges were washed with 2 mL of H
and eluted with 2 mL of acetonitrile containing 0.1% TFA. The
combined aqueous and organic solutions were concentrated to about
1 mL by rotary evaporation, passed through a 0.22-mm Millipore
ﬁlter, and injected onto an analytic HPLC column at a ﬂow rate of
1 mL/min using the gradient described earlier. Radioactivity was
monitored using a solid-state radiation detector. At the same time,
the eluent was also collected by a fraction collector (0.5 min/
fraction), and the activity of each fraction was measured by the
g-counter. The HPLC analysis was performed in duplicate and the
extraction efﬁciency was determined in triplicate. Data obtained
from the g-counter were plotted to reconstruct the HPLC chro-
matograms. Control experiments were also performed to test the
extraction and elution efﬁciency by adding
directly to the same tissue samples.
To perform a microCT scan, an anesthetized male nude mouse
bearing an orthotopic PC-3 tumor (4–6 wk after inoculation) was
mounted on a turntable bed that could be moved automatically in
the axial direction. The high-resolution 3D images were obtained
by a commercial microCAT II system (ImTek Inc.). This scanner
uses a SourceRay SB-80-50 x-ray tube with about 40-mm focal
spot providing 30-mm resolution in high-resolution conﬁguration.
A total of 220 rotation steps was taken over 220 with one axial
bed position. A standard convolution-backprojection procedure
with a Shepp–Logan ﬁlter was used to reconstruct the CT images
in 512 · 512 pixel matrices.
microPET and microCT Image Fusion
For the microPET and microCT coregistration, we used a
narrow-band approach, which is a hybrid method combining the
advantages of pixel-based and distance-based registration tech-
niques (30). In essence, this technique is a 2-step image registra-
tion in which the tumor to be registered is ﬁrst represented by a
data structure containing the signed distance values from its
boundaries, followed by an image registration using a pixel-based
metric. The optimization aligns the zero set of the narrow band
obtained from the CT images with the tumor gradients in the PET
dataset, eliminating the assumption of uniform pixel intensities
within one organ used in the mutual information (MI) approach. In
our setup, the normalized correlation was used as the metric and a
gradient-based algorithm was used to ﬁnd the optimal match.
After imaging, both subcutaneous and orthotopic tumors were
dissected for histology to verify tumor pathology. Tumor tissues
were frozen at 280C in optimal cutting temperature (OCT)
medium. Frozen sections (5 mm; Leica Microsystems, Inc.) were
ﬁxed in acetone at 220C for 15 min and air-dried overnight
(4C). They were then stained with hematoxylin–eosin (BD Bio-
sciences). Slides were examined under a ZEISS AxioVert 25 re-
search microscope (Carl Zeiss) equipped with a ZEISS digital
camera (model AxioCam MRc5) and captured with MRGrab
18.104.22.168 (Carl Zeiss vision GmbH) software.
Quantitative data are expressed as mean 6 SD. Means were
compared using 1-way ANOVA and the Student t test. P values ,
0.05 were considered signiﬁcant.
F-Fluorination of bombesin analogs ([Lys
Aca-BBN(7–14)) were achieved via
F-SFB (Fig. 1).
in Kryptoﬁx 2.2.2./K
the total reaction time, including ﬁnal HPLC puriﬁ cation,
was about 150 6 20 min. The overall radiochemical yield
with decay correction was 31.4% 6 4.6% (n 5 12). The
radiochemical purity of the labeled peptides was .98% ac-
cording to analytic HPLC. The speciﬁc activity of
was estimated by radio-HPLC to be 2002250 TBq/mmol.
F-FB-Aca-BBN(7–14) were well
separated from [Lys
]BBN and Aca-BBN(7–14), respec-
tively, rendering the speciﬁc activity of these 2 PET tracers
virtually the same as that of
494 THE JOURNAL OF NUCLEAR MEDICINE • Vol. 47 • No. 3 • March 2006
In Vitro Receptor-Binding Assay
The binding afﬁnities of [Lys
]BBN, and FB-Aca-BBN(7–14) for GRPR were
evaluated for PC-3 cells. Results of the cell-binding assay
were plotted in sigmoid curves for the displacement of
]BBN from PC-3 cells as a function of increasing
concentration of bombesin analogs. The IC
determined to be 3.3 6 0.4 nmol/L for [Lys
]BBN, 20.8 6
0.3 nmol/L for Aca-BBN(7–14), 5.3 6 0.6 nmol/L for FB-
]BBN, and 48.7 6 0.1 nmol/L for FB-Aca-BBN(7–14)
on 1 · 10
PC-3 cells. [Lys
]BBN with the full sequence
of the bombesin peptide is substantially more potent than
Aca-BBN(7–14) with the truncated sequence. Coupling of
the ﬂuorobenzoyl group had a minimal effect on the binding
afﬁnity for both compounds.
Internalization and Efﬂux Studies
The results for the internalization of both tracers,
F-FB-Aca-BBN(7–14), are shown in
Figure 2A. For both tracers, internalization occurred during
5 min of incubation after the preincubation step: 51% for
]BBN and 58% for
respectively. After approximately 15 min of incubation, in-
ternalization of both tracers reached a maximum (85% for
]BBN and 60% for
and then decreased slowly through 120 min of incubation
]BBN and 50% for
BBN(7–14) at 120 min). When blocked with 200 mmol/L
]BBN, the nonspeciﬁc uptake for both tracers was
,10% over the incubation period (data not shown).
Efﬂux studies were performed for up to 3 h of incubation
to further characterize both tracers (Fig. 2B).
F-FB-Aca-BBN(7–14) tracers exhibited similar
efﬂux curves. After 30 min of incubation, approximately
]BBN had efﬂuxed out of the cells. At
the end of the 3-h incubation period, approximately 77% of
the radiotracer had efﬂuxed. For
tracer, after 30 min of incubation, appro ximately 39% of
the radioactivity efﬂuxed out of the PC-3 cells and, after 3 h
of incubation, approximately 83% of the radioactivity had
efﬂuxed. The efﬂux rate of
faster than that of
]BBN, which might be
due to the lower afﬁnity of
F-FB-Aca-BBN(7–14) for the
]BBN, as determined from the in
vitro cell-binding assay.
BBN(7–14) was evaluated in athymic nude mice bearing
subcutaneous PC-3 tumors. The results were shown in
Figure 3. For
]BBN (Fig. 3A), the tumor up-
take was 5.94 6 0.78 %ID/g at 60 min after injection,
which decreased to 0.50 6 0.11 %ID/g in the presence of a
blocking dose of [Tyr
]BBN (10 mg/kg mice body weight).
]BBN was also able to substantially reduce the activity
accumulation in the pancreas, intestines, and kidneys,
demonstrating that these organs are also GRPR positive.
Increased uptake in the lung, liver, and spleen was ob-
F-FB-Aca-BBN(7–14) (Fig. 3B), the tumor
uptake (0.43 6 0.18 %ID/g at 60 min after injection) was
more than one order of magnitude lower than that for
FIGURE 1. Schematic structures of
F-BOMBESIN PET FOR GRPR • Zhang et al. 495
]BBN. A blocking dose of [Tyr
creased the uptake of
F-FB-Aca-BBN(7–14) in the tumor,
pancreas, and intestines, whereas the uptake in the liver,
kidneys, and lung was increased. Tumor-to-nontarget ratios
]BBN were signiﬁcantly higher than those of
F-FB-Aca-BBN(7–14) for all organs and tissues examined
(P , 0.001) (Fig. 3C).
Dynamic microPET Imaging of Subcutaneous PC-3
The dynamic microPET scans were performed on the
subcutaneous PC-3 tumor model with
F-FB-Aca-BBN(7–14). Selected coronal images at
different time points after administration of the appropriate
tracers are shown in Figure 4 for comparison. Tumor con-
trast was observed as early as 10 min after injection for
both radiotracers. The tumor uptake of
was 3.50, 3.68, and 2.61 %ID/g at 10, 30, and 60 min after
injection, respectively. The tumor-to-contralateral back-
ground (muscle) ratio was 3.95 at 60 min after injection
Time–activity curves derived from the 60-min dynamic
microPET scan showed that
]BBN was ex-
creted predominantly through the renal route (Fig. 5A).
Liver had low initial uptake (5.15 %ID/g at 5 min after
injection) and the radioactivity was also washed out rapidly
(1.75 %ID/g at 1 h after injection). The activity accumu-
lation in the kidneys was moderately low at early time
points (4.85 %ID/g at 5 min after injection) but rapidly
increased to 47.00 %ID/g at 50 min after injection followed
by a steep decline afterward (28.49 %ID/g at 60 min and
1.01 %ID/g at 2 h after injection). Compared with
F-FB-Aca-BBN(7–14) had a signiﬁcantly
lower tumor uptake, which corroborates the biodistribution
results obtained from direct tissue sampling. The tumor
FIGURE 2. Comparison of internalization (A) and efﬂux rate
F-FB-Aca-BBN(7–14) using PC-3
cells. Data are from 2 experiments with triplicate samples and
are expressed as mean 6 SD.
FIGURE 3. Biodistribution of
]BBN (A) and
FB-Aca-BBN(7–14) (B) in male athymic nude mice bearing
subcutaneous PC-3 tumors. Mice were injected intravenously
with 370 kBq of radioligand with or without the presence
]BBN at 10 mg/kg mice body weight and euthanized at
60 min after injection. (C) Tumor-to-nontarget ratios of 2 radio-
tracers resulting from the biodistribution are also shown. Data
are presented as mean %ID/g 6 SD (n 5 3).
496 THE JOURNAL OF NUCLEAR MEDICINE • Vol. 47 • No. 3 • March 2006
F-FB-Aca-BBN(7–14) was 0.92, 0.71, and 0.78
%ID/g at 10, 30, and 60 min after injection, respectively.
Liver had low uptake at all time points (1.35, 3.29, and 1.75
%ID/g at 5, 25, and 60 min after injection, respectively).
The activity accumulation in the kidneys was also low at
early time points (4.77 %ID/g at 5 min after injection) but
increased to 11.19 %ID/g at 45 min after injection and
remained steady over the remaining dynamic scan period.
Figure 4C shows the transaxial microPET images of PC-3
tumor-bearing mice at 1 h after administration of
]BBN, with and without coinjection of 10 mg/kg
]BBN. The blocking reduced the tumor uptake to
0.58 %ID/g at 1 h after injection, 4- to 5-fold lower than
that of the control animals.
The metabolic stability of
]BBN was deter-
mined in mouse blood, urine, liver, kidney, and tumor homog-
enates at 6 0 min after injection The extraction efﬁciencies
were 61.4% for the blood, 95.0% for the liver, 91.1% for
the kidneys, and 97.8% for the PC-3 tumor, respectively.
The elution efﬁciencies of the soluble fractions were 44.4%
for the blood, 39.8% for the liver, 41.5% for the kidneys,
and 95.5% for the PC-3 tumor. HPLC analysis results of
the acetonitrile-eluted fractions are shown in Figure 6. The
average fraction of intact tracer was between 0.7% and
47.2% (Table 1). Incubation of
with tissue and organ homogenates revealed that the extrac-
tion efﬁciency was .90% in all cases, except for the liver,
for which the extraction efﬁciency was only 67.7%. The
elution efﬁciency was also .9 0% for all samples tested.
Although we did not identify the composition of the metab-
olites, we found that all metabolites came off the HPLC
column earlier than those for the parent compound. No
]BBN was observed as no visi-
ble bone uptake was observed in any of the microPET scans.
PET and CT Imaging of Orthotopic PC-3 Tumor Model
We also evaluated
]BBN in orthotopic PC-3
tumor-bearing mice. The representative microPET images
shown in Figure 7A were at 17 min after injection The
orthotopic tumor uptake was calculated to be 2.07 %ID/g
from microPET imaging, which is somewhat lower than
that of subcutaneous PC-3 tumor (3.74 %ID/g at 17 min
after injection). Dynamic scans indicated that the tumor
was clearly visualized between 10 and 30 min, after which
a signiﬁcant amount of urinary bladder activity interferes
FIGURE 4. microPET images of athymic nude mice bearing
PC-3 tumor on the right shoulder. Coronal images (decay
corrected to time of tracer injection) were collected at multiple
time points after injection of
]BBN (A) or
Aca-BBN(7–14) (B) (370 kBq/mouse). (C) Transaxial microPET
images of PC-3 tumor-bearing mice at 1 h after tail vein
injection of 3.7 MBq of
]BBN in absence (Control)
and presence (Block) of coinjected blocking dose of [Try
(10 mg/kg mice body weight). Tumors are indicated by white
arrows in all cases.
FIGURE 5. Time–activity curves of
]BBN (A) and
F-FB-Aca-BBN(7–14) (B) derived from 60-min dynamic micro-
PET scans. ROIs are shown for PC-3 tumor, liver, muscle, and
F-BOMBESIN PET FOR GRPR • Zhang et al. 497
with the tumor delineation. The presence of the well-
established tumor grown in the prostate gland was con-
ﬁrmed by microCT without a contrast agent (Fig. 7A). Good
visual agreement after registration was obtained in all
sagittal, coronal, and transaxial images (Fig. 7A). The reg-
istration is focused on the tumor region and did not use
markers that can be shifted or displaced. The whole reg-
istration procedure took ,15 min on a standard personal
computer, as the narrow-band approach used is a compact
representation of a structure where only pixels close to the
structure boundaries are considered (30). Both subcutane-
ous and orthotopic PC-3 tumor tissues were also resected
for histology to verify the characterization of tumors ex
vivo. The hematoxylin–eosin staining results (Fig. 7B) of
both PC-3 tumors showed simi lar morphology characteris-
tic of cancer cells.
There has been an exponential growth in the development
of radiolabeled peptides for diagnostic and therapeutic
applications in the last decade. Peptidic radiopharmaceuti-
cals have many favorable properties, including fast clear-
ance, rapid tissue penetration, and low antigenecity, and can
be produced easily and inexpensively (31). However, there
may be problems with the in vivo catabolism, unwanted
physiologic effects, and chelate attachment. Most studies
have been focused on radiometal labeling of peptides for
SPECT imaging of receptors that are overexpressed on the
diseased cells (32–34). More recently, peptides have been
conjugated to macrocyclic chelators for labeling of
Ga for PET applications (17,20,35,36). Becaus e
of the overexpression of GRPR in a variety of cancers,
bombesin analogs—derived either from the full tetradeca-
peptide sequence or from a truncated C-terminal portion of
the peptide—have been labeled with various radiometals
FIGURE 6. HPLC proﬁles of soluble fractions of blood, urine,
liver, kidney, and tumor homogenates collected at 1 h after
]BBN to a male athymic PC-3 tumor-
bearing nude mouse. As a comparison, the HPLC proﬁle of
intact tracer (Standard) is also shown.
Extraction Efﬁciency and Elution Efﬁciency Data and HPLC Analysis of Soluble Fraction of Tissue Samples
at 60 Minutes After Injection
Extraction efﬁciency* (%)
Fraction Blood Urine Liver Kidney PC-3
38.6 (2.0) ND 5.0 (32.3) 8.9 (6.8) 2.2 (0.3)
61.4 (98.0) ND 95.0 (67.7) 91.1 (93.2) 97.8 (99.7)
Elution efﬁciency (%)
52.5 (7.9) ND 55.3 (2.4) 57.1 (1.4) 2.9 (1.7)
3.2 (1.8) ND 4.9 (1.8) 1.4 (1.3) 1.7 (0.6)
44.4 (90.3) ND 39.8 (95.8) 41.5 (97.3) 95.5 (97.7)
HPLC analysis (%)
Intact tracer 19.7 0.7 8.4 3.2 47.2
*Results in parentheses are from direct mixing of
]BBN with tissue homogenates.
Amount of activity retained in pellets.
Amount of activity that was extracted to PBS solution.
Amount of activity that could not be trapped onto C
Amount of activity that was eluted from C
cartridge using 2 mL of water.
Amount of activity that was eluted from C
cartridge using 2 mL of acetonitrile with 0.1% TFA.
ND 5 not determined.
498 THE JOURNAL OF NUCLEAR MEDICINE • Vol. 47 • No. 3 • March 2006
for both PET (
Ga) and SPECT (
imaging applications (14,15,17,18,20,37).
F is an ideal
short-lived PET isotope for labeling small molecular
recognition units, such as biologically active peptides,
and it is easily produced in the small biomedical cyclotrons.
Most peptides have the N-term inal primary amine group
and one or more lysine e-amino residues that can be labeled
F through an amine-reactive prosthetic labeling
group such as
F-SFB (22). Thus, we decided to label
both peptides ([Lys
]BBN and Aca-BBN(7–14)) with
for in vitro and in vivo characterizations.
Our cell-binding assay experiment demonstrated that the
truncated peptide sequence Aca-BBN(7–14) had signiﬁcantly
lower receptor-binding afﬁnity than that of [Lys
F-labeled Aca-BBN(7–14) was also less potent than the
corresponding bombesin peptide analogs. Both tracers are
metabolically unstable after intravenous administration.
Multiple metabolites were found but not characterized here.
Identiﬁcation of the composition of the degradation products
may be important to identify the cleavage sites to design and
characterize peptides of enhanced metabolic stability.
The internalization and efﬂux patterns of
F-FB-Aca-BBN(7–14) are of note.
]BBN with higher receptor afﬁnity than
BBN(7–14) showed signiﬁcantly higher cellular uptake.
Both tracers, however, had a rapid washout after reaching a
maximum at 15 min of incubation with PC-3 cells (Fig.
2A), which is similar to
]BBN but very different
from radiometal-labeled BBNs. The prolonged retention of
Cu-labeled BBNs is most likely due to
the lack of cell permeability of the hydrophilic macrocyclic
conjugate (14,15,17). In the case of
analogs, after GRPR-mediated internalization, both the intact
tracer and the metabolized peptide fractions that are radio-
active remain to be lipophilic and, thus, more amenable to
penetration in and out of the cells. It is, thus, not surprising
to obser ve rapid externalization of both
F-FB-Aca-BBN(7–14), with the less-potent
BBN(7–14) efﬂuxed even more rapidly than
]BBN (Fig. 2B). Such in vitro characters of
bombesin analogs tally with the relatively short half-life of
]BBN with higher receptor afﬁnity and pro-
longed cell retention than
F-FB-Aca-BBN(7–14) also exhib-
ited superior tumor-targeting efﬁcacy and pharmacokine tics
in vivo. Although
F-FB-Aca-BBN(7–14) showed some
contrast at early time points, the activity accumulation in
the tumor was quickly washed out. Because of the lipophilic
F-FB-Aca-BBN(7–14), it exhibited both
hepatobiliary and renal clearance routes as evidenced by
very strong signal in the liver, gallbladder, and lower abdo-
men, eliminating the potential of this compound for detecting
the orthotopic prostate cancer that is located very close to the
urinary bladder. A strong tumor-to-background contrast was
]BBN in PC-3 tumor, although the
magnitude of tracer uptake is signiﬁcantly lower than that
obtained from biodistribution studies. A similar phenomenon
has been observed for
]BBN (19). We rea-
son that the amount of tracer adm inistered for PET is about an
order of magnitude higher than that for biodistribution, which
may have caused partial self-inhibition of receptor-speciﬁc
uptake in PC-3 tumor. We also noticed that nonradioactive
BBN is able to effectively inhibit the tumor uptake of
]BBN despite of the relatively low metabolic stability of
FIGURE 7. (A) microPET and microCT visualization of ortho-
topic PC-3 tumor. Representative transverse, coronal, and
sagittal images that contain the tumor at 17 min after injection
of 3.7 MBq of
]BBN are shown. The tumor grown in
mouse prostate gland is conﬁrmed by microCT scan without
contrast agent. Coregistration of microPET (slice thickness, 1.2
mm) and microCT (slice thickness, 80 mm) is accomplished by
using a narrow-band approach without the need for ﬁducial
markers. (B) Hematoxylin–eosin staining (·400) of subcutane-
ous (left) and orthotopic (right) PC-3 tumor tissues.
F-BOMBESIN PET FOR GRPR • Zhang et al. 499
the tracer. The substantial blockade of tumor uptake by
unlabeled BBN suggests that some of the degraded radioac-
tive components accumulated in the tumor may also have
afﬁnity for GRP receptor, which can be replaced by BBN.
microPET/microCT coregistration using
]BBN is a powerful tool for orthotopic prostate cancer
imaging. The high-resolution microCT scan provides good
contrast for PC-3 tumor without the need of contrast-
enhancing media, whereas microPET imaging with
]BBN offers the GRPR expression level of the
tumor. In general, image registration can be formulated as
an optimization problem where the variables are a group of
transformation parameters that lead to the best match
between the input images. The match is quantiﬁed in
mathematic terms by the use of a metric, which ranks a
potential matching based on the image histograms, resolu-
tion, or pixel values of the involved organs. Usage of MI
has been widely adopted when dealing with multimodality
image registration (38). However, MI cannot be applied
directly to PET/CT registration for soft tissue because
the wide pixel intensities within an organ as imaged in
the PET images produce multiple correspondences with the
CT images that act as noise to the registration algorithm,
hindering its convergence ( 39). Therefore, only marker-
based techniques have been reported for PET/CT registra-
tion of mice studies (40). The narrow-band approach used
in this study was originally devised for magnetic resonance
spectroscopic imaging (MRSI)/CT registration, where a
similar noncorrelation of pixel intensities was observed
(30). Previous studies have suggested that this 2-step image
registration technique improves the convergence behavior
of the calculation and reduces the computational efforts
because sophisticated statistical considerations can be
replaced with simpler pixel-based metrics computed only
in the regions of clinical interest.
This study demonstrated the successful coupling of
]BBN and Aca-BBN(7–14) with positron-emitting
F through the prosthetic labeling group
F-SFB. The bombesin analog with the full tetradecapep-
tide sequence (
]BBN) is superior to that with a
truncated C-terminal sequence (
in terms of GRPR avidity, receptor-mediated internalization
rate, intracellular retention, tumor-targeting efﬁcacy, and
in vivo pharmacokinetics. Although
relatively metabolically unstable, dynamic PET scans dem-
onstrated the ability of this tracer to visualize both subcutane-
ous and orthotopic PC-3 tumor in murine xenograft models.
]BBN may also be used for local-
ization of other tumors that are GRPR positive.
This work was supported, in part, by Department of
Defense (DOD) Prostate Cancer Research Program (PCRP)
New Investigator Award (NIA) DAMD1717-03-1-0143,
National Cancer Institute (NCI) grant R21 CA102123,
National Institute of Biomedical Imaging and Bioengineer-
ing (NIBIB) grant R21 EB001785, DOD Breast Cancer
Research Program (BCRP) Concept Award DAMD17-03-
1-0752, DOD BCRP IDEA Award W81XWH-04-1-0697,
American Lung Association California, Society of Nuclear
Medicine Education and Research Foundation, NCI Small
Animal Imaging Resource Program (SAIRP) grant R24
CA93862, and NCI In Vivo Ce llular Molecular Imaging
Center (ICMIC) grant P50 CA114747. Dr. Zhengming
Xiong is acknowledged for cell culture and the authors
thank Pauline Chu for histology.
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