In vitro and in vivo characterization of novel 18F-labeled bombesin analogues for targeting GRPR-positive tumors

Article abstract of DOI:10.1021/bc100222u

The gastrin-releasing peptide receptor (GRPR) is overexpressed on a number of human tumors and has been targeted with radiolabeled bombesin analogues for the diagnosis and therapy of these cancers. Seven bombesin analogues containing various linkers and peptide sequences were designed, synthesized, radiolabeled with ¹⁸F, and characterized in vitro and in vivo as potential PET imaging agents. Binding studies displayed nanomolar binding affinities toward human GRPR for all synthesized bombesin analogues. Two high-affinity peptide candidates 6b (K_i = 0.7 nM) and 7b (K_i = 0.1 nM) were chosen for further in vivo evaluation. Both tracers revealed specific uptake in GRPR-expressing PC-3 tumors and the pancreas. Compared to [¹⁸F]6b, compound [¹⁸F]7b was characterized by superior tumor uptake, higher specificity of tracer uptake, and more favorable tumor-to-nontarget ratios. In vivo PET imaging allowed for the visualization of PC-3 tumor in nude mice suggesting that [¹⁸F]7b is a promising PET tracer candidate for the diagnosis of GRPR-positive tumors in humans.

Full text of DOI:10.1021/bc100222u

1864
Bioconjugate Chem. 2010, 21, 1864–1871
18
In Vitro and in ViWo Characterization of Novel F-Labeled Bombesin
Analogues for Targeting GRPR-Positive Tumors
Linjing Mu,† Michael Honer,† Jessica Becaud,† Miljen Martic,† Pius A. Schubiger,† Simon M. Ametamey,*,†
Timo Stellfeld,‡ Keith Graham,‡ Sandra Borkowski,‡ Lutz Lehmann,‡ Ludger Dinkelborg,‡ and Ananth Srinivasan‡
Center for Radiopharmaceutical Sciences of ETH, PSI and USZ, CH-8093 Zurich, Switzerland, and Bayer Schering Pharma AG,
Global Drug Discovery, D-13342 Berlin, Germany. Received May 6, 2010; Revised Manuscript Received July 19, 2010
The gastrin-releasing peptide receptor (GRPR) is overexpressed on a number of human tumors and has been
targeted with radiolabeled bombesin analogues for the diagnosis and therapy of these cancers. Seven bombesin
analogues containing various linkers and peptide sequences were designed, synthesized, radiolabeled with 18F,
and characterized in Vitro and in ViVo as potential PET imaging agents. Binding studies displayed nanomolar
binding affinities toward human GRPR for all synthesized bombesin analogues. Two high-affinity peptide candidates
6b (Ki ) 0.7 nM) and 7b (Ki ) 0.1 nM) were chosen for further in ViVo evaluation. Both tracers revealed specific
uptake in GRPR-expressing PC-3 tumors and the pancreas. Compared to [18F]6b, compound [18F]7b was
characterized by superior tumor uptake, higher specificity of tracer uptake, and more favorable tumor-to-nontarget
ratios. In ViVo PET imaging allowed for the visualization of PC-3 tumor in nude mice suggesting that [18F]7b is
a promising PET tracer candidate for the diagnosis of GRPR-positive tumors in humans.
research groups focus on new synthetic strategies that avoid
the use of prosthetic groups. Recent studies have demonstrated
the feasibility of labeling organoboron and organosilicon
bioconjugates with 18F in a single step (32-35). In our
laboratories, we have successfully labeled bombesin peptides
with 18F in one step and applying silicon-fluorine chemistry
(36). However, the 18F-labeled silicon-based peptides showed
relatively low tumor accumulation and retention as well as
unfavorable hepatobiliary excretion in nude mice with PC-3
xenografts. This might be due to the introduction of the highly
lipophilic di-tert-butyl silyl labeling moiety into the peptide. In
order to improve the tumor targeting and imaging potential of
18F-labeled bombesin peptides, another approach via 18F-for-
+N(CH3)3 substitution using a less lipophilic benzonitrile
labeling moiety has been exploited in our laboratories (37). The
in Vitro binding affinity of these fluorinated bombesin peptides
toward GRPR were assessed by a competitive displacement
assay using [125I]-Tyr4-bombesin as the radioligand. More than
11-fold higher binding affinity with benzonitrile as the labeling
moiety was obtained compared to using di-tert-butyl silyl as
the labeling moiety with the same peptide sequence (36).
Therefore, in this study, several new BBS peptides were
synthesized and 18F-labeled via the aforementioned 18F-for-
+N(CH3)3 substitution approach. Our efforts have been focused
on improving the pharmacokinetics of BBS derivatives by using
different linkers, peptide sequences, and non-natural amino
acids. Two bombesin analogues, one being positively and
another negatively charged, were chosen for further in ViVo
evaluation in nude mice with PC-3 xenografts.
INTRODUCTION
Bombesin (BBS) is a 14-amino acid neuropeptide that was
originally isolated from the skin of the Bombina frog and binds
with high affinity to gastrin-releasing peptide (GRP) receptors
(1-4). Four subtypes of BBS/GRP receptors have been identi-
fied. The bombesin receptor subtype 2 has been shown to be
overexpressed on a variety of human tumors, including small-
cell lung cancer (SCLC), prostate, breast, gastric, colon,
pancreatic, and gastrointestinal cancer (5-9). Thus, radiolabeled
BBS analogues having high affinity for this receptor subtype
can be used as radiopharmaceuticals either for the detection or
treatment of these cancers (10-12).
It has been shown that the C-terminal amino acid sequences,
Trp8-Ala9-Val10-Gly11-His12-Leu13-Met14-NH2, are neces-
sary for retaining receptor binding affinity and preserving the
biological activity of BBS-like peptides; hence, the N-terminal
region of the peptide can be used for radiolabeling. A number
of potent BBS analogues have been labeled with various
radionuclides, such as mTc, 111In, 90Y, 64Cu, 177Lu, 68Ga, or
99
18F, for targeting GRPR-expressing cancer cells in both animal
models and human subjects (13-25). Among the available PET
nuclides, 18F has ideal characteristics for peptide receptor
imaging studies in terms of its half-life (109.7 min) and low
ꢀ+ energy (0.64 MeV) (26). Current known methods of
radiolabeling peptides with 18F rely on using prosthetic groups
such as the [18F]succinimidyl 4-fluorobenzoate ([18F]SFB) (27),
[18F]4-fluorobenzaldehyde (28, 29), and thiol-selective 18F-
labeling reagents (30, 31). Although great improvements have
been achieved, the established methods have disadvantages in
that the synthetic methods comprise multistep radiosyntheses
and time-consuming workup procedures. Therefore, several
EXPERIMENTAL PROCEDURES
General. All chemicals unless otherwise stated were pur-
chased from Sigma-Aldrich or Merck and used without further
purification. Fmoc-aminoacids were purchased from IRIS Bio-
tech except Fmoc-Statine, FA01010, and Fmoc-Ala(SO3Na)-
OH), which were purchased from NeoMPS (now Polypeptide).
The solvents were of HPLC quality. Peptide syntheses were
carried out using Rink-Amide resin (0.68 mmol/g) following
the standard Fmoc strategy (29). All amino acid residues were,
* Corresponding author. Center for Radiopharmaceutical Sciences
of ETH, PSI and USZ, CH-8093 Zurich, ETH Ho¨nggerberg D-CHAB
IPW HCI H427, Wolfgang-Pauli-Strasse 10, Switzerland. Tel: +41 44
633 74 63. Fax: +41 44 633 13 67. E-mail: simon.ametamey@pharma.
ethz.ch.
† Center for Radiopharmaceutical Sciences of ETH, PSI and USZ.
‡ Bayer Schering Pharma AG.
10.1021/bc100222u  2010 American Chemical Society
Published on Web 09/21/2010

Novel 18F-Labeled Bombesin Analogues
Bioconjugate Chem., Vol. 21, No. 10, 2010 1865
if not further specified, L-amino acid residues. FA01010 denotes
(4R,5S)-4-amino-5-methylheptanoic acid, statine (Sta) denotes
(3S,4S)-4-amino-3-hydroxy-6-methylheptanoic acid, Ala(SO3H)
denotes cysteine sulfonic acid, and Ava denotes 5-aminopen-
tanoic acid. High-performance liquid chromatography (HPLC)
analyses were performed using an ACE C18 column (50 × 4.6
mm, 3 µm) under the indicated conditions. Analytical HPLC
chromatograms were obtained using an Agilent 1100 system
with Gina software, equipped with UV multiwavelength and
Raytest Gabi Star detectors. Semipreparative HPLC purifications
were carried out using a semipreparative ACE C18 column (250
× 10 mm, 5 µm) under the indicated conditions. The semi-
preparative HPLC system used was a Merck-Hitachi L6200A
system equipped with a Knauer variable wavelength detector
and an Eberline radiation detector.
equiv), and DIPEA (N,N′-di-iso-propylethylamine, 70 µL, 4
equiv) in DMF (2 mL) was added to the resin-bound side chain
protected peptide (0.1 mmol, based on the initial resin loading)
which was prepared by following the general procedures
described above. The reaction mixture was shaken intensively
for 4 h. The resin was then filtered and washed with DMF (3
× 4 mL) and CH2Cl2 (3 × 4 mL). Thereafter, the resin was
treated with a mixture of trifluoroacetic acid, distilled water,
phenol, and triisopropylsilane (85/5/5/5, 1.5 mL) for 3 h. The
mixture was added to ice cold methyl tert-butylether, and the
precipitate was separated by centrifugation. Water was added
to the pellet, and the supernatant was lyophilized. The residue
was purified by preparative RP-18 HPLC-MS with a gradient
of 5-50% acetonitrile in 20 min and 0.1% trifluoroacetic acid
as cosolvent. The desired fraction was collected and lyophilized,
and the product was analyzed by HPLC-MS.
Solid-Phase Peptide Synthesis. Peptide synthesis was carried
out using Fmoc-Rink Amide-linker functionalized polystyrene
resin (0.68 mmol/g). The peptide chain was elongated in cycles
of Fmoc deprotection, followed by coupling of the subsequent
Fmoc amino acid. Acid-labile side chain protecting groups were
used for the following amino acids: Fmoc-His(Trt)-OH, Fmoc-
Trp(Boc)-OH, and Fmoc-Gln(Trt)-OH. Fmoc deprotection
was achieved by shaking a suspension of the resin in 20%
piperidine in DMF for 5 min and repeatedly doing so for another
20 min. The resin was subsequently washed with DMF (2×),
CH2Cl2 (2×), and DMF (2×). A solution of Fmoc-Xaa-OH
(Xaa ) amino acid, 4 equiv), HBTU (O-(benzotriazol-1-yl)-
N,N,N′,N′-tetramethyluronium hexafluorophosphate, 4 equiv),
HOBT (1-hydroxybenzotriazole, 4 equiv), DIPEA (N,N′-di-iso-
propylethylamine, 8 equiv) in DMF was added to the resin-
bound free amine peptide and shaken for 90 min at room
temperature. This step was repeated with a reaction time of 60
min, and the resin was washed with DMF (2×), CH2Cl2 (2×),
and DMF (2×). The two steps of Fmoc-deprotection and amino-
acid addition were then repeated until the final elongation of
the desired peptide was achieved. The peptides were typically
prepared starting with 147 mg (0.1 mmol) of the resin. The
amounts of reagents and building blocks in all subsequent
reactions were calculated on the basis of this amount.
Radiolabeling. No-carrier added [18F] fluoride was produced
via the 18O (p, n) 18F nuclear reaction by irradiation of enriched
[18O] water. [18F]Fluoride was immobilized on an anion-
exchange cartridge (QMA light, Waters, AG) and eluted with
a solution of Kryptofix K2.2.2 (5 mg), Cs2CO3 (2.3 mg), or K2CO3
(1 mg) in acetonitrile (1.5 mL) and water (0.5 mL). The fluoride
was dried by azeotropic distillation of acetonitrile at 110 °C
under vacuum with a stream of nitrogen. The azeotropic drying
was repeated three times with acetonitrile (3 × 1 mL). To the
dried [18F] fluoride complex, the TMA-based bombesin peptide
precursor was added (2 mg, in 150 µL of DMSO). The reaction
solution was heated at the indicated reaction temperatures. An
aliquot was taken from the reaction mixture and analyzed by
analytical HPLC. The identity of the tracer was confirmed by
coinjection of the authentic standard on the same analytical
HPLC system ACE C18 column (50 × 4.6 mm, 3 µm). Eluting
conditions: 10 mM K2HPO4 in water (solvent A), 10 mM
K2HPO4 in water/acetonitrile 3/7 (solvent B); 0-7.0 min,
5-95% B; 7.0-7.1 min, 95-100% B; 7.1-8.8 min, 100% B;
8.8-9.0 min, 100-5% B. The flow rate was 2 or 1.8 mL/min.
Semipreparative HPLC purifications were carried out using a
semipreparative ACE C18 column (250 × 10 mm, 5 µm).
Elution system: 0.1% TFA in water (solvent A) and 0.1% TFA
in acetonitrile/water 9/1 (solvent B).
Procedure for the Synthesis of (4-Trimethylammonium-
3-cyano-benzoyl)-Functionalized Peptide Trifluoroacetate
Salts (Precursor Compounds 1a-7a). Representative procedure:
N-methylmorpholine (NMM, 22 µL, 0.2 mmol) was added to a
solution of 2-cyano-4-carboxyl-phenyl)-trimethylammonium tri-
flate salt (71 mg, 0.2 mmol) and 4-(4,6-dimethoxy-1,3,5-triazin-
2-yl)-4-methylmorpholinium tetrafluoroborate (66 mg, 0.2 mmol)
in DMF (2 mL). The mixture was added to the resin-bound,
side chain protected, Fmoc-deprotected peptide (0.1 mmol, based
on the initial resin loading) which was prepared by following
the general procedures described above. The reaction mixture
was shaken intensively for 4 h. The resin was then filtered and
washed with DMF (3 × 4 mL) and CH2Cl2 (3 × 4 mL). The
coupling step was repeated. Thereafter, the resin was treated
with a mixture of trifluoroacetic acid, distilled water, phenol,
and triisopropylsilane (85/5/5/5, 1.5 mL) for 3 h. The mixture
was added to ice cold methyl tert-butylether, and the precipitate
was separated by centrifugation. Water was added to the pellet,
and the supernatant was lyophilized. The residue was purified
by preparative RP-18 HPLC-MS with a gradient of 5-30%
acetonitrile in 20 min and 0.1% trifluoroacetic acid as cosolvent.
The desired fraction was collected and lyophilized, and the
product was analyzed by HPLC-MS.
3-Cyano-4-[18F]fluoro-benzoyl-AVa-Gln-Trp-Ala-Val-Gly-
His(3Me)-Sta-Leu-NH2 ([18F]1b). A solution of compound 1a (2
mg) in anhydrous DMSO (150 µL) was added to a reaction
vial containing the dry K[18F]F/K2.2.2 complex. After heating at
50 °C for 15 min, the crude reaction mixture was analyzed using
an ACE C18 column. The 18F-incorporation determined by
HPLC was 77% (tR ) 4.92 min).
3-Cyano-4-[18F]fluoro-benzoyl-Arg-AVa-Gln-Trp-Ala-Val-Gly-
His(3Me)-Sta-Leu-NH2 ([18F]2b). A solution of compound 2a (2
mg) in anhydrous DMSO (150 µL) was added to a reaction
vial containing the dry Cs[18F]F/K2.2.2 complex (3.4 GBq). After
heating at 70 °C for 15 min, the crude reaction mixture was
analyzed using ACE C18 column. The 18F-incorporation deter-
mined by HPLC was 67% (tR ) 4.87 min). The reaction mixture
was diluted with water (4 mL) and injected onto an ACE
semipreparative HPLC column (0.1% TFA in water (solvent
A); B, 0.1% TFA in acetonitrile/water 9/1 (solvent B), 0-2.0
min, 20% B; 2.0-22.0 min, 20-60% B; 22.0-23.0 min,
60-100% B; 23.0-28.0 min, 100% B; flow rate, 3 mL/min),
and the product was collected (1.1 GBq). The decay corrected
radiochemical yield of the isolated product was around 47%,
and radiochemical purity was greater than 99%.
Syntheses of Fluorinated Peptides (Reference Compounds
1b-7b). Representative procedure: A solution of 3-cyano-4-
fluorobenzoic acid (66 mg, 4 equiv) (37), HBTU (O-(benzot-
riazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate,
151 mg, 4 equiv), HOBT (1-hydroxybenzotriazole, 61 mg, 4
3-Cyano-4-[18F]fluoro-benzoyl-Arg-ꢀAla-Gln-Trp-Ala-Val-Gly-
His(3Me)-Sta-Leu-NH2 ([18F]3b). A solution of compound 3a (2
mg) in anhydrous DMSO (150 µL) was added to a reaction
vial containing dry K[18F]F/K2.2.2 complex (1.49 GBq). After
heating at 50 °C for 15 min, the crude reaction mixture was

1866 Bioconjugate Chem., Vol. 21, No. 10, 2010
Mu et al.
analyzed using an ACE C18 column. The 18F-incorporation
determined by HPLC was 56% (tR ) 4.78 min). The reaction
mixture was diluted with water (4 mL) and injected onto an
ACE semipreparative HPLC (0-5 min, 29% B; 5-25 min,
29-34% B; flow rate 3 mL/min). The desired 18F-labeled
product was collected (150 MBq, 21.1% d.c. RCY) with a
specific activity of 72.5 GBq/µmol.
and centrifuged for 10 min at 520g at room temperature. Signals
were measured in Top Count (Perkin-Elmer) for 1 min integra-
tion time per well. Nonspecific binding determined by an excess
of bombesin was subtracted from total binding to yield the
specific binding at each concentration. The IC50 and Ki values
were calculated by nonlinear regression using GraFit 5 data
analysis software (Erithacus Software Ltd.).
Lipophilicity: log D7.4 Determination. The distribution
coefficient D (log D7.4) was determined by the “shake flask
method” (38): the peptide was dissolved in a mixture of
phosphate buffer (600 µL, pH 7.4) and n-octanol (600 µL) at
20 °C. The sample was equilibrated by shaking for 4 min
followed by centrifugation. The concentrations of the peptide
in each phase were measured by HPLC-UV absorption. The
log D value was calculated as the log ratio of UV peak area at
254 nm in the organic and aqueous phases of the tested
compound.
3-Cyano-4-[18F]fluoro-benzoyl-Arg-Ser-Gln-Trp-Ala-Val-Gly-
His(3Me)-Sta-Leu-NH2 ([18F]4b). A solution of compound 4a (2
mg) in anhydrous DMSO (150 µL) was added to a reaction
vial containing dry K[18F]F/K2.2.2 complex (2.9 GBq). After
heating at 50 °C for 15 min, the crude reaction mixture was
analyzed using ACE C18 column. The 18F-incorporation deter-
mined by HPLC was 35% (tR ) 4.94 min). The reaction mixture
was diluted with water (4 mL) and injected onto an ACE
semipreparative HPLC (0-2 min, 35% B; 2-22 min, 35-55%
B; flow rate 3 mL/min). The desired 18F-labeled product was
collected (28 MBq, 4% d.c. RCY).
Biodistribution Studies. All animal experiments were per-
formed in compliance with the current version of the German
and Swiss law on the Protection of Animals. The biodistribution
experiments were performed using nude mice bearing human
prostate tumors (PC-3). For the induction of tumor xenografts,
PC-3 cells (2 × 106 cells/mouse) were injected subcutaneously
and allowed to grow for four to five weeks. Animals were
injected intravenously with approximately 177 kBq of [18F]6b
and 230 kBq of [18F]7b (100 µL). The animals were sacrificed
at different postinjection time points (from 0.5 to 4 h, n ) 3
for each time point). Organs and tissues of interest were
collected and weighed. The amount of radioactivity was
determined in the γ-counter to calculate the uptake (% injected
dose per g tissue). In addition, three mice received 100 µg of
bombesin coinjected with the radiolabeled compound and were
sacrificed at 1 h postinjection to determine nonspecific uptake.
PET Study. Four nude mice were inoculated subcutaneously
with PC-3 tumor cells (see above) next to the right shoulder.
Animals were injected intravenously with 10-15 MBq of
[18F]7b and scanned in a whole body configuration from 60 to
105 min p.i. in the dedicated small-animal PET system quad-
HIDAC. Two animals were coinjected with 50 µg of bombesin
(1 mg/mL), while two animals received a corresponding volume
of vehicle. PET data acquisition and monitoring of anesthesia
were performed as described elsewhere (39). After the termina-
tion of the PET scan, animals were sacrificed at 107 min p.i.
by decapitation for tissue sampling. PET data were reconstructed
in a single 45 min time frame and normalized to the injected
dose per body weight.
3-Cyano-4-[18F]fluoro-benzoyl-Arg-AVa-Gln-Trp-Ala-Val-Gly-
His-FA01010-Leu-NH2 ([18F]5b). A solution of compound 5a (2
mg) in anhydrous DMSO (150 µL) was added to a reaction
vial containing dry K[18F]F/K2.2.2 complex (11 GBq). After
heating at 70 °C for 15 min, the crude reaction mixture was
analyzed using ACE C18 column. The 18F-incorporation deter-
mined by HPLC was 63% (tR ) 5.51 min).
3-Cyano-4-[18F]fluoro-benzoyl-Arg-AVa--Gln-Trp-Ala-Val-NMeG-
ly-His-Sta-Leu-NH2 ([18F]6b). Radiolabeling based on the pub-
lished procedure (37).
3-Cyano-4-[18F]fluoro-benzoyl-Ala(SO3H)-AVa-Gln-Trp-Ala-
Val-NMeGly-His-Sta-Leu-NH2 ([18F]7b). A solution of compound
7a (2 mg) in anhydrous DMSO (150 µL) was added to a reaction
vial containing dry Cs[18F]F/K2.2.2 complex (20 GBq). After
heating at 90 °C for 15 min, the crude reaction mixture was
analyzed using ACE C18 column. The 18F-incorporation deter-
mined by HPLC was 76% (tR ) 4.30 min). The reaction mixture
was diluted with water (3 mL) and injected onto an ACE
semipreparative HPLC column (0.1% TFA in water (solvent
A), B, 0.1% TFA in acetonitrile/water 9/1 (solvent B), 0-2.0
min, 20% B; 2.0-22.0 min, 20-60% B; 22.0-23.0 min,
60-100% B; flow rate, 3 mL/min), and the product was
collected (2.1 GBq). The decay corrected radiochemical yield
of the isolated product was around 14%, and radiochemical
purity was greater than 99%. Specific activity was 77 GBq/
µmol at the end of the synthesis.
In Vitro Binding Affinity Studies. The receptor binding
affinities of bombesin (BBS) analogues 1b-7b were determined
in quadruplicate in a scintillation proximity assay (SPA) using
cellular membranes transfected with human bombesin 2 recep-
tors (GRPR) from PerkinElmer (RBHBS2M). The membranes
and agglutinin-coupled SPA beads type A (PVT PEI Treated
Wheatgerm, Amersham Bioscience) were mixed in assay buffer
(50 mM Tris/HCl at pH 7.2; 5 mM MgCl2; 1 mM EGTA;
protease inhibitor (Roche Diagnostics GmbH; 1 tablet/50 mL);
and 0.3% polyethylenemine) to give final concentrations of
approximately 20 µg/mL protein and 8 mg/mL PVT-SPA beads.
The ligand [125I]-Tyr4-bombesin (PerkinElmer; specific activity,
81.4 TBq/mmol) was diluted to 0.2 nM in assay buffer. The
test compounds were dissolved in DMSO to give 1 mM stock
solutions. They were further diluted in assay buffer to 2
pM-300 nM. Nonspecific binding was determined by an excess
of 10 µM Tyr4-bombesin (Sigma).
RESULTS AND DISCUSSION
Peptide Synthesis. On the basis of the amino acid sequence
7-14 of natural bombesin, we have designed and synthesized
a series of new BBS analogues. To overcome the rapid
degradation of natural BBS, some modifications were introduced
at positions 13 and 14. Met14 was replaced by Leu for
stabilization against aminopeptidase 3.4.11.1, and Leu13 was
replaced by a non-natural amino acid (FA01010 or Sta) to
prevent cleavage by neutral endopeptidase 3.4.24.11. Further
modifications were the introduction of the methylated versions
of His12 or Gly11. Additionally, a polar spacer, such as -Ava-,
-Ava-Arg-, -Arg-ꢀAla-, -Arg-Ser-, or -Ala(SO3H)-Ava- was
inserted between the labeling moiety and the binding sequence
in order to further improve the pharmacokinetics and to avoid
interference of the radiolabel with the binding region.
The assay was then performed as follows: First, 10 µL of
compound solution to be tested for binding was placed in white
384 well plates (Lumitrac 200, Greiner). Next, 20 µL of the
GRPR/PVT-SPA bead mixture and 20 µL of the ligand solution
were added. After 120 min of incubation at room temperature,
another 50 µL of assay buffer was added; the plate was sealed
Nonradioactive peptides were synthesized by solid phase
peptide synthesis (SPPS) following standard Fmoc strategies.
Coupling of the resin-bound peptide with 3-cyano-4-fluoro-
benzoic acid or 3-cyano-4-trimethylamino-benzoic acid afforded
the corresponding nonradioactive (19F) peptide standards and

Novel 18F-Labeled Bombesin Analogues
Bioconjugate Chem., Vol. 21, No. 10, 2010 1867
Figure 1. Synthesized bombesin analogues and structures of non-natural amino acids.
Table 1. Mass Spectrometric Analysis of Synthesized Bombesin
Peptide Analogues and Their Yieldsa
in Figure 2 using peptide 7a as an example. Under the indicated
radiolabeling conditions (Table 2), most peptides showed high
18F-labeling efficiency. For peptide 4a, which contains an Arg-
Ser linker, a 29% 18F-incorporation was obtained under mild
radiolabeling conditions (50 °C, 15 min). For peptide 7a
containing the Ala(SO3H)-Ava linker, a similar 18F-labeling
efficiency (26%, Table 2, entry 7) was achieved. These results
successfully demonstrated that aromatic nucleophilic substitu-
tions of 18F-for-+N(CH3)3 are feasible not only in the presence
of histidine, tryptophan, and arginine amino acids but also in
the presence of the unprotected acid and hydroxyl functionalities
(serine amino acid). In addition, it was found that the 18F-
labeling efficiency could be dramatically improved from 26%
to 75% by increasing the reaction temperature from 70 to 90
°C (Table 2, entries 7 and 8).
yield
(%)
peptides
molecular formula
calculated
found
M+: 1265.5
1a
1b
2a
2b
3a
3b
4a
4b
5a
5b
6a
6b
7a
7b
C63H93N16O12 (M+)
C60H84FN15O12
1265.7
1225.6
1421.8
1381.7
1393.8
1353.7
1409.8
1369.7
1391.8
1351.7
1421.8
1381.7
1416.7
1376.6
11
10
13
3
22
5
20
12
8
12
11
8
[M + H]+: 1226.6
[(M + H)/2]+: 711.8
[M + H]+: 1383.1
M+: 1393.8
C69H105N20O13 (M+)
C66H96FN19O13
C67H101N20O13 (M+)
C64H92FN19O13
[M + H]+: 1354.8
[(M + H)/2]+: 705.7
[(M + 2H)/2]+: 686.6
M+: 1391.9
C67H101N20O14 (M+)
C64H92FN19O14
C68H103N20O12 (M+)
C65H94FN19O12
[M + H]+: 1352.9
[(M + H)/2]+: 711.9
[(M + 2H)/2]+: 692.4
M+: 1417.4
C69H105N20O13 (M+)
C66H96FN19O13
C66H98N17O16S (M+)
C63H89FN16O16S
17
34
[M + H]+: 1378.0
a Isolated yields (not optimized; single syntheses) based on the
amount and loading of commercial Fmoc-Rink-amide resin used for the
synthesis.
Concerning the stereochemistry of the peptides, similar
peptides have been labeled under basic conditions (pH 8-10)
and at elevated temperatures (75-100 °C); no racemization of
the peptides was observed (40-42). Because most of our
radiolabeling conditions (pH < 8, temperature 50-70 °C) are
even milder than those previously reported, peptide racemization
is not expected to occur under the above-mentioned radiola-
beling conditions (Table 2). In addition, binding analysis of a
18F-labeled peptide containing the same peptide sequence as 7b
revealed Kd and IC50 values of 1.43 nM and 0.94 nM,
respectively, showing that the 18F-labeled peptide still retains
high binding affinity toward GRPR after heating at 90 °C for
10 min.
precursors for 18F-labeling, respectively. The nonradioactive
BBS analogues served as a surrogate for 18F-labeled compounds
in the determination of binding affinity and as a standard
reference for radio HPLC analysis. Figure 1 shows the sequences
and the structures of the non-natural amino acid derivatives.
All of the newly synthesized bombesin precursors and standard
references were purified by reverse-phase HPLC and character-
ized by mass spectrometry (Table 1).
Radiolabeling. The direct 18F-fluorination protocol developed
previously in our laboratories was applied to these new
bombesin peptide analogues. The synthetic scheme is depicted

1868 Bioconjugate Chem., Vol. 21, No. 10, 2010
Mu et al.
Figure 2. Synthetic scheme for the generation of [18F]7b.
Table 2. 18F-Radiolabeling of TMA-Based Bombesin Peptidesa
yielded picomolar affinity (Ki ) 0.1 nM) of this peptide
candidate toward the GRPR.
The log D7.4 values of the peptides are in the similar range
(-0.8 to -1.4) with respect to the different polar linkers of
Arg-Ava, Arg-ꢀAla, or Ala(SO3H)-Ava and peptide sequences.
Compounds 6b and 7b gave the same log D7.4 value of -1.1
(Table 3).
temp
(°C)
reaction
time
18F-incorporation
(% HPLC)
d. c.
RCY (%)
entry
compd.
1
2
3
4
5
6
7
8
1a
2a
3a
4a
5a
6a
7a
7a
50
70
70
50
70
70
70
90
15 min
15 min
15 min
15 min
15 min
15 min
15 min
15 min
69 ( 8 (n ) 2)
69 ( 7 (n ) 3)
56 ( 1 (n ) 2)
29 ( 6 (n ) 2)
63 (n ) 1)
51 ( 5 (n ) 2)
26 ( 2 (n ) 2)
74 ( 3 (n ) 2)
n.d.
18%
21%
4%
n.d.
20%
n.d.
14%
Biodistribution Studies. The biodistributions of [18F]6b and
[18F]7b were carried out using nude mice bearing subcutaneous
human prostate tumors (PC-3). The results are shown in Table
4. For [18F]6b, the tumor uptake was 2.36 ( 0.47% ID/g at 30
min after injection, which slightly decreased to 1.80 ( 1.56%
ID/g at 60 min postinjection and 1.61 ( 0.23% ID/g at 4 h
postinjection. The binding of [18F]6b toward GRPR could be
blocked by around 40% in the tumor and 60% in the pancreas
at 60 min postinjection, indicating that a large part of the tracer
uptake in the tumor and pancreas can be attributed to specific
GRPR binding. Besides the tumor and pancreas, the highest
accumulation of the radiotracer [18F]6b was found in excretory
organs such as the gallbladder, intestine, kidneys, and liver.
Activity uptake in the gall bladder and intestine was much higher
than that in the kidneys, suggesting a preponderance of
hepatobiliary elimination versus renal excretion. In the intestine
there was, however, no differentiation between GRPR binding
naturally expressed in intestinal smooth muscle cells and
intestinal content. Furthermore, the increasing uptake in bone
over time indicated that 18F-fluoride represents a major radio
metabolite. In comparison to the bombesin analogue [18F]6b,
the radiotracer [18F]7b was characterized by clearly superior
tumor targeting. The absolute tumor accumulation was more
than double reaching about 5% ID/g at 30 min p.i., and this
uptake was persistent over four hours. Tracer uptake in the
GRPR-rich pancreas amounted to almost 50% ID/g at 1 h
postinjection. Uptake of compound [18F]7b in the tumor and
pancreas was highly specific since accumulation could be
effectively blocked by coadministration of unlabeled bombesin
(83% and 97% specificity, respectively). The biodistribution of
[18F]7b was also characterized by a fast blood clearance leading
to favorable tumor-to-blood ratios of >30 at 60 min postinjection
and later. In line with compound [18F]6b, the bombesin analogue
[18F]7b was mainly excreted via hepatobiliary pathways result-
ing in bile-to-tumor ratios of >20. It is clear that at physiological
pH, Arg and Ala(SO3H) become positively and negatively
a
18F-labeling was carried out in DMSO (150 µL) with a 2 mg
amount of precursor using Cs2CO3 as the base. Conversion was
determined from
a
radio-HPLC chromatogram representing the
percentage of radioactivity area of the product related to the total
radioactivity area. n.d.: not determined.
In Vitro Receptor Binding Affinity and Distribution
Coefficient log D7.4. The binding affinities of the novel
bombesin analogues toward the human GRPR were determined
by a scintillation proximity assay using human GRPR-trans-
fected cell membranes. All synthesized bombesin analogues
showed nanomolar binding affinities toward human GRPR
(Table 3). For peptide 1b with Ava as a linker, a binding affinity
of 28 nM was obtained. However, the attachment of one more
hydrophilic amino acid Arg to the linker resulted in a more
than 100 times higher binding affinity (Table 3; 2b, Ki ) 0.2
nM). Encouraged by these results, Arg-ꢀAla and Arg-Ser were
tested with the same peptide sequence. In both cases, binding
affinities in the high nanomolar range were obtained (Table 3.
Ki ) 6.2 nM and 6.4 nM for 3b and 4b, respectively). With
Arg-Ava as the linker, further investigations on the peptide
sequence by exchanging Sta with FA01010 demonstrated that
Sta was superior to FA01010 in terms of binding affinity. While
the difference between the methylated His or the methylated
Gly was not so obvious in this study (Table 3, Ki ) 0.2 nM
and 0.7 nM for 2b and 6b, respectively), the methylated Gly
BBS analogue showed a 10-fold higher binding affinity than
its corresponding methylated His analogue in our previous
studies (36). Thus, the most promising peptide sequence toward
human GRPR in this study was Gln-Trp-Ala-Val-NMeGly-His-
Sta-Leu-NH2. Further derivatization of the optimal peptide
sequence by exchanging the linker Arg-Ava to Ala(SO3H)-Ava

Novel 18F-Labeled Bombesin Analogues
Bioconjugate Chem., Vol. 21, No. 10, 2010 1869
Table 3. Sequence of the Tested Bombesin Analoguess and Their Binding Affinity (Ki) Towards Human GRPR and Lipophilicities (log D7.4
)
peptide
linker
peptide sequence
IC50 (nM)
Ki (nM)
log D7.4
1b
2b
3b
4b
5b
6b
7b
Ava
Arg-Ava
Arg-ꢀAla
Arg-Ser
Arg-Ava
Arg-Ava
Ala(SO3H)-Ava
Gln-Trp-Ala-Val-Gly-His(3Me)-Sta-Leu-NH2
Gln-Trp-Ala-Val-Gly-His(3Me)-Sta-Leu-NH2
Gln-Trp-Ala-Val-Gly-His(3Me)-Sta-Leu-NH2
Gln-Trp-Ala-Val-Gly-His(3Me)-Sta-Leu-NH2
Gln-Trp-Ala-Val-Gly-His-FA01010-Leu-NH2
Gln-Trp-Ala-Val-NMeGly-His-Sta-Leu-NH2
Gln-Trp-Ala-Val-NMeGly-His-Sta-Leu-NH2
97
0.8
25
28.0
0.2
6.2
6.4
1.5
0.7
0.1
n.d.a
-1.4
-1.0
n.d.a
-0.8
-1.1
-1.1
69
6.0
2.7
0.4
a n.d.: not determined.
Table 4. Biodistribution of [18F]6b and [18F]7b in Nude Mice Bearing PC-3 Xenografts at Different Times p.i.a
tissue 0.5 h 1.0 h 2.0 h
1.0 hb
3-CN-4-[18F]Fluoro-Bz-Arg-Ava--Gln-Trp-Ala-Val-NMeGly-His-Sta-Leu-NH2 ([18F]6b)
4.0 h
blood
kidneys
liver
spleen
lung
muscle
gallbladder
bone
intestine
pancreas
adrenals
tumor
0.93 ( 0.28
2.92 ( 0.44
3.26 ( 0.60
0.56 ( 0.10
1.13 ( 0.27
0.29 ( 0.03
82.06 ( 19.10
4.36 ( 0.34
12.90 ( 1.53
2.57 ( 0.03
0.86 ( 0.11
2.36 ( 0.47
2.59 ( 0.24
0.27 ( 0.04
1.35 ( 0.11
1.23 ( 0.17
0.28 ( 0.06
0.48 ( 0.16
0.15 ( 0.05
81.38 ( 52.78
4.64 ( 0.42
6.76 ( 5.84
1.30 ( 0.59
0.65 ( 0.40
1.80 ( 1.56
6.82 ( 6.19
0.32 ( 0.02
1.46 ( 0.13
1.35 ( 0.24
0.21 ( 0.04
0.69 ( 0.19
0.20 ( 0.08
132.49 ( 73.06
4.04 ( 0.35
9.66 ( 2.06
0.51 ( 0.10
0.70 ( 0.39
1.11 ( 0.30
3.46 ( 0.97
0.15 ( 0.03
0.87 ( 0.18
0.61 ( 0.06
0.17 ( 0.03
0.28 ( 0.04
0.07 ( 0.02
64.13 ( 34.51
7.80 ( 0.89
8.92 ( 1.83
1.26 ( 0.09
0.26 ( 0.07
1.28 ( 0.27
8.55 ( 2.29
0.21 ( 0.16
1.10 ( 0.74
0.72 ( 0.39
0.19 ( 0.09
0.29 ( 0.14
0.07 ( 0.02
58.81 ( 20.33
7.98 ( 1.58
11.04 ( 0.10
1.54 ( 0.47
0.44 ( 0.04
1.61 ( 0.23
11.37 ( 7.23
T/blood
3-CN-4-[18F]Fluoro-Bz-Ala(SO3H)-Ava--Gln-Trp-Ala-Val-NMeGly-His-Sta-Leu-NH2 ([18F]7b)
blood
kidneys
liver
spleen
lung
muscle
gallbladder
bone
intestine
pancreas
adrenals
tumor
0.52 ( 0.13
3.00 ( 2.20
1.37 ( 0.18
0.63 ( 0.23
0.51 ( 0.18
0.10 ( 0.01
230.12 ( 88.04
0.84 ( 0.08
11.54 ( 2.91
41.95 ( 5.37
4.49 ( 1.27
4.67 ( 0.04
9.50 ( 2.81
0.16 ( 0.01
0.66 ( 0.05
0.50 ( 0.13
0.30 ( 0.02
0.28 ( 0.03
0.05 ( 0.00
102.63 ( 74.25
1.52 ( 0.22
11.75 ( 2.86
49.01 ( 8.07
3.45 ( 0.24
4.88 ( 0.36
30.59 ( 1.05
0.48 ( 0.21
1.16 ( 0.48
1.76 ( 0.56
1.32 ( 0.49
23.04 ( 6.99
0.14 ( 0.05
137.40 ( 106.91
1.61 ( 0.35
11.81 ( 5.52
1.63 ( 0.65
0.97 ( 0.10
0.84 ( 0.26
1.80 ( 0.29
0.10 ( 0.01
0.42 ( 0.04
0.37 ( 0.08
0.31 ( 0.12
0.20 ( 0.03
0.06 ( 0.02
66.80 ( 54.48
1.60 ( 0.12
9.88 ( 2.76
34.49 ( 4.96
2.31 ( 0.73
5.40 ( 0.58
51.77 ( 2.52
0.09 ( 0.02
0.36 ( 0.12
0.34 ( 0.12
0.28 ( 0.35
0.11 ( 0.02
0.04 ( 0.02
137.22 ( 109.07
1.75 ( 0.23
12.14 ( 2.38
30.70 ( 5.14
1.17 ( 0.19
5.16 ( 1.32
55.03 ( 0.56
T/blood
a Results are expressed as % ID/g ( SD (n ) 3). b Blockade study: animals received 100 µg of bombesin coinjected with the radiolabeled compound.
charged, respectively. Although bearing the same binding
peptide sequence and having the same lipophilicity, the charge
of the linker obviously plays a major role in in ViVo pharma-
cokinetics. Similar results have been reported for different
bombesin analogues labeled with 99 mTc, 64Cu, and 68Ga (43-46)
suggesting that a negatively charged linker has a profound effect
on the biodistribution profile of bombesin analogues. Further-
more, significant in ViVo defluorination of compound [18F]7b
was not evident from the % ID/g values in the bone.
PET Study. The excellent tumor targeting of compound
[18F]7b encouraged us to test the capability of [18F]7b to
visualize PC3 tumors in ViVo by PET imaging. Two nude mice
bearing subcutaneous PC-3 tumor xenografts next to the right
shoulder were scanned from 60 to 105 min p.i. in a dedicated
small-animal PET system both under baseline and blockade
conditions (Figure 3). The tumor was clearly imaged under
baseline conditions, whereas no tumor visualization was possible
under blocking conditions. As expected from the ex ViVo
Figure 3. Series of horizontal whole body slices (ventral to dorsal) of two nude mice bearing subcutaneous PC-3 tumor xenografts next to the right
shoulder (see arrows), which were injected with [18F]7b and scanned from 60 to 105 min p.i., both under baseline (left) and blockade conditions
(right). Image data were normalized to the injected dose per body weight.

1870 Bioconjugate Chem., Vol. 21, No. 10, 2010
Mu et al.
(7) Zhou, J. H., Chen, J., Mokotoff, M., and Ball, E. D. (2004)
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and Gugger, M. (2002) Bombesin receptor subtypes in human
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biodistribution study, highest activity concentrations in the in
ViVo PET data were identified in the gall bladder and the
intestine. The pancreas could not be visualized by PET due to
the proximity to the bowel. Postmortem tissue sampling of these
mice after PET scanning (107 min p.i.) confirmed the data which
were obtained in the biodistribution study (Table 4). The
absolute radioactivity uptake values in the GRPR-rich tissues
were 5% ID/g for the tumor and 30% ID/g for the pancreas.
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In-111-labeled bombesin analogs as potential radiopharmaceu-
ticals for specific targeting of gastrin-releasing peptide receptors
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Volkert, W. A., and Hoffman, T. J. (2003) Radiochemical
investigations of Tc-99m-N3S-X-BBN[7-14]NH2: An in vitro/
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14, 93–102.
CONCLUSIONS
The direct 18F-fluorination protocol developed previously in
our lab was successfully applied to new bombesin peptide
analogues. Amino acids such as histidine, tryptophan, and
arginine, and non-natural amino acids such as statine and
cysteine sulfonic acid in the peptide sequence did not require
any protection group during radiosynthesis. In Vitro binding
studies of the synthesized bombesin analogues revealed nano-
molar binding affinities toward the human GRPR. The most
promising peptide sequence in this study was Gln-Trp-Ala-Val-
NMeGly-His-Sta-Leu-NH2. Two bombesin analogues containing
this optimal peptide sequence, one positively (Arg-Ava) and
another one negatively (Ala(SO3H)-Ava) charged, were radio-
labeled with 18F and evaluated in biodistribution studies in nude
mice bearing PC-3 xenografts. The comparative in ViVo analysis
of [18F]6b and [18F]7b showed superior characteristics for
[18F]7b with respect to (1) the total tracer accumulation in
tumors and the GRPR-rich pancreas, (2) the specificity of uptake
in target tissues, and (3) the target to nontarget ratios. The
positive in ViVo results of [18F]7b suggest that this compound
represents a promising PET tracer candidate for the visualization
of GRPR-positive tumors in humans.
(15) Smith, C. J., Gali, H., Sieckman, G. L., Hayes, D. L., Owen,
N. K., Mazuru, D. G., Volkert, W. A., and Hoffman, T. J. (2003)
Radiochemical investigations of Lu-177-DOTA-8-Aoc-BBN[7-
14]NH2: An in vitro/in vivo assessment of the targeting ability
of this new radiopharmaceutical for PC-3 human prostate cancer
cells. Nucl. Med. Biol. 30, 101–109.
ACKNOWLEDGMENT
(16) Rogers, B. E., Bigott, H. M., McCarthy, D. W., Della Manna,
D., Kim, J., Sharp, T. L., and Welch, M. J. (2003) MicroPET
imaging of a gastrin-releasing peptide receptor-positive tumor
in a mouse model of human prostate cancer using a Cu-64-
labeled bombesin analogue. Bioconjugate Chem. 14, 756–763.
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Maintas, D., Reubi, J. C., and Maina, T. (2003) [Tc-99m]De-
mobesin 1, a novel potent bombesin analogue for GRP receptor-
targeted tumour imaging. Eur. J. Nucl. Med. Mol. Imaging 30,
247–258.
(18) Lin, K. S., Luu, A., Baidoo, K. E., Hashemzadeh-Gargari,
H., Chen, M. K., Pili, R., Pomper, M., Carducci, M., and Wagner,
H. N. (2004) A new high affinity technetium analogue of
bombesin containing DTPA as a pharmacokinetic modifier.
Bioconjugate Chem. 15, 1416–1423.
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A. H., Bading, J. R., and Conti, P. S. (2004) MicroPET and
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64-DOTA-[Lys(3)]bombesin in human prostate adenocarcinoma
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stabilization and formulation of [Lu-177]Lu-AMBA, a systemic
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We thank Claudia Keller, Cindy Fischer, Selahattin, Ede,
Marion Zerna, Ingo Horn, Jo¨rg Pioch, Rene´ Zernicke, Volker
Stickel, Ingolf Weber, Melanie Appel, Peter Friese, and Katrin
Dinse for their excellent technical assistance.
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