18F-labeled bombesin analogs for targeting GRP receptor-expressing prostate cancer

Article (PDF Available)inJournal of Nuclear Medicine 47(3):492-501 · March 2006with70 Reads
Source: PubMed
Abstract
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.
18
F-Labeled Bombesin Analogs for Targeting
GRP Receptor-Expressing Prostate Cancer
Xianzhong Zhang, PhD
1
; Weibo Cai, PhD
1
; Feng Cao, MD, PhD
1
; Eduard Schreibmann, PhD
2
;YunWu,PhD
1
;
Joseph C. Wu, MD, PhD
1,3
; Lei Xing, PhD
2
; and Xiaoyuan Chen, PhD
1
1
Molecular Imaging Program at Stanford (MIPS), Department of Radiology and Bio-X Program, Stanford University School of Medicine,
Stanford, California;
2
Department of Radiation Oncology, Stanford University School of Medicine, Stanford, California; and
3
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
18
F-labeled bombesin analogs for PET
of GRPR expression in prostate cancer xenograft models.
Methods: [Lys
3
]Bombesin ([Lys
3
]BBN) and aminocaproic acid-
bombesin(7–14) (Aca-BBN(7–14)) were labeled with
18
F by cou-
pling the Lys
3
amino group and Aca amino group, respectively,
with N-succinimidyl-4-
18
F-fluorobenzoate (
18
F-SFB) under slightly
basic condition (pH 8.5). Receptor-binding affinity of FB-
[Lys
3
]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.
18
F-FB-
[Lys
3
]BBN was also tested for orthotopic PC-3 tumor delineation.
Metabolic stability of
18
F-FB-[Lys
3
]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
18
F
2
.
18
F-FB-[Lys
3
]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.
18
F-FB-[Lys
3
]BBN had predominant renal
excretion.
18
F-FB-Aca-BBN(7–14) exhibited both hepatobiliary
and renal clearance. Dynamic microPET imaging studies revealed
that the PC-3 tumor uptake of
18
F-FB-[Lys
3
]BBN in PC-3 tumor
was much higher than that of
18
F-FB-Aca-BBN(7–14) at all time
points examined (P , 0.01). The receptor specificity of
18
F-FB-
[Lys
3
]BBN in vivo was demonstrated by effective blocking of
tumor uptake in the presence of [Tyr
4
]BBN. No obvious blockade
was found in PC-3 tumor when
18
F-FB-Aca-BBN(7–14) was used
as radiotracer under the same condition.
18
F-FB-[Lys
3
]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. Conclusion: This study demonstrates that
18
F-FB-
[Lys
3
]BBN and PET are suitable for detecting GRPR-positive pros-
tate cancer in vivo.
Key Words: prostate cancer; GRP receptor;
18
F-bombesin;
microPET; microCT
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
delivery.
Being the most widely applied radionuclide for diagnos-
tic purposes, a great deal of research has been done to de-
velop
99m
Tc- and
111
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,
90
Y,
188
Re, and
177
Lu have been used to radiolabel BBN analogs
for potential peptide receptor radiotherapy applications
(15,16).
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.
E-mail: shawchen@stanford.edu
492 THE JOURNAL OF NUCLEAR MEDICINE Vol. 47 No. 3 March 2006
64
Cu (half-life [t
1/2
] 5 12.7 h; b
1
, 17.4%)-labeled BBN
analog,
64
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
64
Cu-
labeled BBN analog,
64
Cu-DOTA-[Lys
3
]BBN, for targeting
GRPR expression in both PC-3 and 22RV1 tumor models
(19). Very recently, another BBN analog, [
D-Tyr
6
,b-Ala
11
,
Thi
13
,Nle
14
]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
Ga (t
1/2
5
68 min; b
1
, 88%), obtained from a
68
Ge/
68
Ga generator for
imaging AR42J rat pancreatic cancer -bearing nude mice (20).
18
F(t
1/2
5 109.7 min; b
1
, 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).
18
F-Labeled pros-
thetic groups such as N-succinimidyl-4-
18
F-fluorobenzoate
(
18
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
3
]bombesin ([Lys
3
]BBN)
and aminocaproic acid-bombesin(7–14) (Aca-BBN(7–14))
with
18
F for GRPR imaging of subcutaneous and orthotopic
PC-3 tumor models with PET.
MATERIALS AND METHODS
Materials
All chemicals obtained commercially were used without further
purification. [Lys
3
]BBN and Aca-BBN(7–14) were synthesized
using solid-phase Fmoc chemistry by American Peptide, Inc. No-
carrier-added
18
F
2
was obtained from PETNET Inc. The received
18
F
2
was trapped on an anion-exchange resin and eluted with 0.5 mL
K
2
CO
3
(2 mg/mL in H
2
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
3
]BBN and FB-
Aca-BBN(7–14)) were synthesized as reference standards. In brief,
an equimolar amount of SFB (in acetonitrile) and [Lys
3
]BBN or
Aca-BBN(7–14) (in H
2
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
3
]BBN and 19.1 min for FB-Aca-BBN(7–14),
respectively.
4-
18
F-Fluorobenzoyl-[Lys
3
]bombesin (
18
F-FB-[Lys
3
]BBN) and
4-
18
F-fluorobenzoyl-Aca-bombesin(7–14) (
18
F-FB-Aca-BBN(7–
14)) were synthesized by coupling the corresponding BBN peptide
with
18
F-SFB (25–27).
18
F-SFB was purified by semipreparative
HPLC, concentrated to about 200 mL, and added to [Lys
3
]BBN or
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
2
. In vitro binding affinity and spec-
ificity of FB-BBN analogs for GRPR were evaluated using com-
petitive receptor-binding assay.
125
I-[Tyr
4
]BBN (Perkin-Elmer
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
(IC
50
) value for the displacement binding of
125
I-[Tyr
4
]BBN by
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
18
F-FB-[Lys
3
]BBN and
18
F-FB-
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.
Animal Models
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
6
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
5
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
running fashion.
Biodistribution
The subcutaneous tumor-bearing mice were used for biodis-
tribution when the tumor volume reached 300–400 mm
3
(3–4 wk
after inoculation). Three mice were each injected intravenously
with about 370 kBq (10 mCi)
18
F-FB-[Lys
3
]BBN or
18
F-FB-Aca-
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
4
]BBN as a blocking agent
18
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
radiotracer (
18
F-FB-[Lys
3
]BBN or
18
F-FB-Aca-BBN(7–14)) under
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 5.2.4.0) 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
ROI–derived %ID/g.
Metabolic Stability
Male mice bearing PC-3 tumors were injected intravenously
with 3.7 MBq of
18
F-FB-[Lys
3
]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
18
cartridges. The urine sample was
directly diluted with 1 mL of PBS and passed through Sep-Pak
C
18
cartridge. The cartridges were washed with 2 mL of H
2
O
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
18
F-FB-[Lys
3
]BBN
directly to the same tissue samples.
microCT Imaging
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.
Histology
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
1.0.0.4 (Carl Zeiss vision GmbH) software.
Statistical Analysis
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.
RESULTS
Radiosynthesis
18
F-Fluorination of bombesin analogs ([Lys
3
]BBN and
Aca-BBN(7–14)) were achieved via
18
F-SFB (Fig. 1).
Starting with
18
F-F
2
in Kryptofix 2.2.2./K
2
CO
3
solution,
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
18
F-SFB
was estimated by radio-HPLC to be 2002250 TBq/mmol.
18
F-FB-[Lys
3
]BBN and
18
F-FB-Aca-BBN(7–14) were well
separated from [Lys
3
]BBN and Aca-BBN(7–14), respec-
tively, rendering the specific activity of these 2 PET tracers
virtually the same as that of
18
F-SFB.
494 THE JOURNAL OF NUCLEAR MEDICINE Vol. 47 No. 3 March 2006
In Vitro Receptor-Binding Assay
The binding affinities of [Lys
3
]BBN, Aca-BBN(7–14),
FB-[Lys
3
]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
125
I-[Tyr
4
]BBN from PC-3 cells as a function of increasing
concentration of bombesin analogs. The IC
50
values were
determined to be 3.3 6 0.4 nmol/L for [Lys
3
]BBN, 20.8 6
0.3 nmol/L for Aca-BBN(7–14), 5.3 6 0.6 nmol/L for FB-
[Lys
3
]BBN, and 48.7 6 0.1 nmol/L for FB-Aca-BBN(7–14)
on 1 · 10
5
PC-3 cells. [Lys
3
]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,
18
F-
FB-[Lys
3
]BBN and
18
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
18
F-FB-[Lys
3
]BBN and 58% for
18
F-FB-Aca-BBN(7–14),
respectively. After approximately 15 min of incubation, in-
ternalization of both tracers reached a maximum (85% for
18
F-FB-[Lys
3
]BBN and 60% for
18
F-FB-Aca-BBN(7–14))
and then decreased slowly through 120 min of incubation
(61% for
18
F-FB-[Lys
3
]BBN and 50% for
18
F-FB-Aca-
BBN(7–14) at 120 min). When blocked with 200 mmol/L
of [Tyr
4
]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).
18
F-FB-[Lys
3
]
BBN and
18
F-FB-Aca-BBN(7–14) tracers exhibited similar
efflux curves. After 30 min of incubation, approximately
54% of
18
F-FB-[Lys
3
]BBN had effluxed out of the cells. At
the end of the 3-h incubation period, approximately 77% of
the radiotracer had effluxed. For
18
F-FB-Aca-BBN(7–14)
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
18
F-FB-Aca-BBN(7–14) is
faster than that of
18
F-FB-[Lys
3
]BBN, which might be
due to the lower affinity of
18
F-FB-Aca-BBN(7–14) for the
GRPR than
18
F-FB-[Lys
3
]BBN, as determined from the in
vitro cell-binding assay.
Biodistribution
Biodistribution of
18
F-FB-[Lys
3
]BBN and
18
F-FB-Aca-
BBN(7–14) was evaluated in athymic nude mice bearing
subcutaneous PC-3 tumors. The results were shown in
Figure 3. For
18
F-FB-[Lys
3
]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
4
]BBN (10 mg/kg mice body weight).
[Tyr
4
]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-
served. For
18
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
18
F-FB-Aca-BBN(7–14) and
18
F-FB-
[Lys
3
]BBN.
18
F-BOMBESIN PET FOR GRPR Zhang et al. 495
18
F-FB-[Lys
3
]BBN. A blocking dose of [Tyr
4
]BBN de-
creased the uptake of
18
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
of
18
F-FB-[Lys
3
]BBN were significantly higher than those of
18
F-FB-Aca-BBN(7–14) for all organs and tissues examined
(P , 0.001) (Fig. 3C).
Dynamic microPET Imaging of Subcutaneous PC-3
Tumor Model
The dynamic microPET scans were performed on the
subcutaneous PC-3 tumor model with
18
F-FB-[Lys
3
]BBN
and
18
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
18
F-FB-[Lys
3
]BBN
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
18
F-FB-[Lys
3
]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
18
F-FB-
[Lys
3
]BBN,
18
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
(B) of
18
F-FB-[Lys
3
]BBN and
18
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
18
F-FB-[Lys
3
]BBN (A) and
18
F-
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
of [Tyr
4
]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
uptake of
18
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
18
F-FB-
[Lys
3
]BBN, with and without coinjection of 10 mg/kg
[Tyr
4
]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.
Metabolism of
18
F-FB-[Lys
3
]BBN
The metabolic stability of
18
F-FB-[Lys
3
]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
18
F-FB-[Lys
3
]BBN directly
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
defluoridation of
18
F-FB-[Lys
3
]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
18
F-FB-[Lys
3
]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
18
F-FB-[Lys
3
]BBN (A) or
18
F-FB-
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
18
F-FB-[Lys
3
]BBN in absence (Control)
and presence (Block) of coinjected blocking dose of [Try
4
]BBN
(10 mg/kg mice body weight). Tumors are indicated by white
arrows in all cases.
FIGURE 5. Time–activity curves of
18
F-FB-[Lys
3
]BBN (A) and
18
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
kidney.
18
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.
DISCUSSION
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
64
Cu,
86
Y, and
68
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
injection of
18
F-FB-[Lys
3
]BBN to a male athymic PC-3 tumor-
bearing nude mouse. As a comparison, the HPLC profile of
intact tracer (Standard) is also shown.
TABLE 1
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
Unsoluble fraction
y
38.6 (2.0) ND 5.0 (32.3) 8.9 (6.8) 2.2 (0.3)
Soluble fraction
z
61.4 (98.0) ND 95.0 (67.7) 91.1 (93.2) 97.8 (99.7)
Elution efficiency (%)
Nonretained fraction
§
52.5 (7.9) ND 55.3 (2.4) 57.1 (1.4) 2.9 (1.7)
Wash water
k
3.2 (1.8) ND 4.9 (1.8) 1.4 (1.3) 1.7 (0.6)
Acetonitrile eluent
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
18
F-FB-[Lys
3
]BBN with tissue homogenates.
y
Amount of activity retained in pellets.
z
Amount of activity that was extracted to PBS solution.
§
Amount of activity that could not be trapped onto C
18
cartridge.
k
Amount of activity that was eluted from C
18
cartridge using 2 mL of water.
Amount of activity that was eluted from C
18
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 (
64
Cu and
68
Ga) and SPECT (
99m
Tc and
111
In)
imaging applications (14,15,17,18,20,37).
18
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
with
18
F through an amine-reactive prosthetic labeling
group such as
18
F-SFB (22). Thus, we decided to label
both peptides ([Lys
3
]BBN and Aca-BBN(7–14)) with
18
F
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
3
]BBN.
18
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
18
F-FB-[Lys
3
]
BBN and
18
F-FB-Aca-BBN(7–14) are of note.
18
F-FB-
[Lys
3
]BBN with higher receptor affinity than
18
F-FB-Aca-
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
125
I-[Tyr
4
]BBN but very different
from radiometal-labeled BBNs. The prolonged retention of
99m
Tc-,
111
In-, or
64
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
18
F-labeled bombesin
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
18
F-FB-[Lys
3
]BBN
and
18
F-FB-Aca-BBN(7–14), with the less-potent
18
F-FB-Aca-
BBN(7–14) effluxed even more rapidly than
18
F-FB-
[Lys
3
]BBN (Fig. 2B). Such in vitro characters of
18
F-labeled
bombesin analogs tally with the relatively short half-life of
18
F.
18
F-FB-[Lys
3
]BBN with higher receptor affinity and pro-
longed cell retention than
18
F-FB-Aca-BBN(7–14) also exhib-
ited superior tumor-targeting efficacy and pharmacokine tics
in vivo. Although
18
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
character of
18
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
observed for
18
F-FB-[Lys
3
]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
64
Cu-DOTA-[Lys
3
]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
18
F-FB-
[Lys
3
]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
18
F-FB-[Lys
3
]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.
18
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
18
F-FB-
[Lys
3
]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
18
F-
FB-[Lys
3
]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.
CONCLUSION
This study demonstrated the successful coupling of
[Lys
3
]BBN and Aca-BBN(7–14) with positron-emitting
radionuclide
18
F through the prosthetic labeling group
18
F-SFB. The bombesin analog with the full tetradecapep-
tide sequence (
18
F-FB-[Lys
3
]BBN) is superior to that with a
truncated C-terminal sequence (
18
F-FB-Aca-BBN(7–14))
in terms of GRPR avidity, receptor-mediated internalization
rate, intracellular retention, tumor-targeting efficacy, and
in vivo pharmacokinetics. Although
18
F-FB-[Lys
3
]BBN is
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.
Furthermore,
18
F-FB-[Lys
3
]BBN may also be used for local-
ization of other tumors that are GRPR positive.
ACKNOWLEDGMENTS
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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F-BOMBESIN PET FOR GRPR Zhang et al. 501
    • "The conjugation yield was comparable or higher than for other reported proteins35363738. Higher conjugation yields can be achieved using organic solvents (acetonitrile, ethanol) and/or higher amounts of peptide3940414243 . However, the use of high amounts of organic solvents is not applicable to nbs because it causes precipitation and denaturation of the protein. "
    [Show abstract] [Hide abstract] ABSTRACT: Introduction: Radiolabeled nanobodies are exciting new probes for molecular imaging due to high affinity, high specificity and fast washout from the blood. Here we present the labeling of an anti-HER2 nanobody with 18F and its validation for in vivo assessment of HER2 overexpression. Methods: The GMP grade anti-HER2 nanobody was labeled with the prosthetic group, N-succinimidyl-4-[18F]fluorobenzoate ([18F]-SFB), and its biodistribution, tumor targeting and specificity were evaluated in mouse and rat tumor models. Results: [18F]FB-anti-HER2 nanobody was prepared with a 5-15% global yield (decay corrected) and a specific activity of 24.7 ± 8.2 MBq/nmol. In vivo studies demonstrated a high specific uptake for HER2 positive xenografts (5.94 ± 1.17 and 3.74 ± 0.52%IA/g, 1 and 3 h p.i.) with high tumor-to-blood and tumor-to-muscle ratios generating high contrast PET imaging. The probe presented fast clearance through the kidneys (4%IA/g at 3 h p.i.). [18F]FB-anti-HER2 nanobody is able to image HER2 expressing tumors when co-administered with the anti-HER2 therapeutic antibody trastuzumab (Herceptin), indicating the possibility of using the tracer in patients undergoing Herceptin therapy. Conclusions: The GMP grade anti-HER2 nanobody was labeled with 18F. This new PET probe for imaging HER2 overexpression in tumors has ample potential for clinical translation.
    Full-text · Article · Jan 2016 · Nuclear Medicine and Biology
    • "Target Organ Dosimetric data(×10 The emergence of various probes has become an indispensable factor for SMM. In previous studies, several one-target based radiotracers have been developed, such as a series of arginineglycine-aspartic acid (RGD) containing peptides and radiolabeled BBN analogue which can specifically bind to integrin α v β 3 and GRPR respectively141516171819. Some have been successfully applied to human clinical trials [20]. "
    [Show abstract] [Hide abstract] ABSTRACT: Purpose: This study aimed to explore the diagnostic performance of single photon emission computed tomography / computerized tomography (SPECT/CT) using a new radiotracer 99mTc-RGD-BBN for breast malignant tumor compared with 99mTc-3P4-RGD2. Methods: 6 female patients with breast malignant tumors diagnosed by fine needle aspiration cytology biopsy (FNAB) who were scheduled to undergo surgery were included in the study. 99mTc-3P4-RGD2 and 99mTc-RGD-BBN were performed with single photon emission computed tomography (SPECT) at 1 hour after intravenous injection of 299 ± 30 MBq and 293 ± 32 MBq of radiotracers respectively at separate day. The results were evaluated by the Tumor to non-Tumor ratios (T/NT). 99mTc-RGD-BBN and 99mTc-3P4-RGD2 SPECT/CT images were interpreted independently by 3 experienced nuclear medicine physicians using a 3-point scale system. All of the samples were analyzed immunohistochemically to evaluate the integrin αvβ3 and gastrin-releasing peptide receptor (GRPR) expression. The safety, biodistribution and radiation dosimetry of 99mTc-RGD-BBN were also evaluated in the healthy volunteers. Results: No serious adverse events were reported in any of the patients during the study. The effective radiation dose entirely conformed to the relevant standards. A total of 6 palpable malignant lesions were detected using 99mTc-RGD-BBN SPECT/CT with clear uptake. All malignant lesions were also detected using 99mTc-3P4-RGD2 SPECT/CT. The results showed that five malignant lesions were with clear uptake and the other one with barely an uptake. 4 malignant cases were found with both αvβ3 and GRPR expression, 1 case with only GRPR positive expression (integrin αvβ3 negative) and 1 case with only integrin αvβ3 positive expression (GRPR negative). Conclusion: 99mTc-RGD-BBN is a safe agent for detecting breast cancer. 99mTc-RGD-BBN may have the potential to make up for the deficiency of 99mTc-3P4-RGD2 in the detection of breast cancer with only GRPR positive expression (integrin αvβ3 negative). The preliminary application of 99mTc-RGD-BBN has demonstrated its powerful potential in breast cancer diagnosis and therapy.
    Full-text · Article · Apr 2015
    • "We investigated the metabolic stability of [ 18 F] BBN-2 in murine blood in vivo and showed that 65% of the radiopeptide was intact after 60 min p.i. This is a significant increase in metabolic stability compared to the full-length tetradecapeptide 18 F- [Lys 3 ]BBN, which only gave 20% of intact peptide after 60 min p.i. in murine blood [23]. Metabolism of [ 18 F]BBN-2 in urine and the liver resulted in several more hydrophilic radiometabolites after 60 min p.i. "
    [Show abstract] [Hide abstract] ABSTRACT: Introduction: Bombesin (BBN) and BBN analogues have attracted much attention as high-affinity ligands for selective targeting of the gastrin-releasing peptide (GRP) receptor. GRP receptors are overexpressed in a variety of human cancers including prostate cancer. Radiolabeled BBN derivatives are promising diagnostic probes for molecular imaging of GRP receptor-expressing prostate cancer. This study describes the synthesis and radiopharmacological evaluation of various metabolically stabilized fluorobenzoylated bombesin analogues (BBN-1, BBN-2, BBN-3). Methods: Three fluorobenzoylated BBN analogues containing an aminovaleric (BBN-1, BBN-2), or an aminooctanoic acid linker (BBN-3) were tested in a competitive binding assay against (125)I-[Tyr(4)]-BBN for their binding potency to the GRP receptor. Intracellular calcium release in human prostate cancer cells (PC3) was measured to determine agonistic or antagonistic profiles of fluorobenzoylated BBN derivatives. Bombesin derivative BBN-2 displayed the highest inhibitory potency toward GRP receptor (IC50 = 8.7 ± 2.2 nM) and was subsequently selected for radiolabeling with fluorine-18 ((18)F) through acylation with N-succinimidyl-4-[(18)F]fluorobenzoate ([(18)F]SFB). The radiopharmacological profile of (18)F-labeled bombesin [(18)F]BBN-2 was evaluated in PC3 tumor-bearing NMRI nude mice involving metabolic stability studies, biodistribution experiments and dynamic small-animal PET studies. Results: All fluorobenzoylated BBN derivatives displayed high inhibitory potency toward the GRP receptor (IC50=8.7-16.7 nM), and all compounds exhibited antagonistic profiles as determined in an intracellular calcium release assay. The (18)F-labeled BBN analogue [(18)F]BBN-2 was obtained in 30% decay-corrected radiochemical yield with high radiochemical purity >95% after semi-preparative HPLC purification. [(18)F]BBN-2 showed high metabolic stability in vivo with 65% of the radiolabeled peptide remaining intact after 60 min p.i. in mouse plasma. Biodistribution experiments and dynamic small-animal PET studies demonstrated high tumor uptake of [(18)F]BBN-2 in PC3 xenografts (2.75 ± 1.82 %ID/g after 5 min and 2.45 ± 1.25 %ID/g after 60 min p.i.). Specificity of radiotracer uptake in PC3 tumors was confirmed by blocking experiments. Conclusion: The present study demonstrates that (18)F-labeled BBN analogue [(18)F]BBN-2 is a suitable PET radiotracer with favorable metabolic stability in vivo for molecular imaging of GRP receptor-positive prostate cancer.
    Full-text · Article · Aug 2013
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