Gastrin releasing peptide receptor-directed radioligands based on a bombesin antagonist: Synthesis, 111in-labeling, and preclinical profile

Article abstract of DOI:10.1021/jm301692p

Novel bombesin (BBN) antagonists were synthesized by coupling the chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) to H-d-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH₂ (JMV594) through linkers of increasing number of (βAla)_x residues (x = 1-3). Labeling with ¹¹¹In afforded the respective radiotracers in high purity and high specific activity. Bioconjugate affinity for the gastrin releasing peptide receptor (GRPR) as determined against [¹²⁵I-Tyr⁴]BBN was high (IC₅₀ values in the lower nanomolar range). Radioligands poorly internalized in PC-3 cells at 37 C. Radiopeptides remained >60% intact 5 min after entering the bloodstream of healthy mice. After injection in SCID mice bearing human PC-3 xenografts all analogues showed high tumor uptake and rapid background clearance via the kidneys into urine. Interestingly, pancreatic uptake, albeit GRPR-specific, declined rapidly with time. ¹¹¹In-DOTA- (βAla)₂-JMV594 achieved the highest tumor values among the group (17.0 ± 2.8%ID/g vs. 8-10%ID/g, respectively, at 4 h pi) indicating that the (βAla)₂-linker favors in vivo interaction of radiopeptides with the GRPR.

Full text of DOI:10.1021/jm301692p

Article
pubs.acs.org/jmc
Gastrin Releasing Peptide Receptor-Directed Radioligands Based on
111
a Bombesin Antagonist: Synthesis, In-Labeling, and Preclinical
Profile
Panteleimon J. Marsouvanidis,†,‡ Berthold A. Nock,*,† Bouchra Hajjaj,§ Jean-Alain Fehrentz,§ Luc Brunel,§
Celine M’Kadmi,§ Linda van der Graaf,‡ Eric P. Krenning,‡ Theodosia Maina,† Jean Martinez,§
́
and Marion de Jong‡
†Molecular Radiopharmacy, INRASTES, National Center for Scientific Research “Demokritos”, Ag. Paraskevi Attikis, GR-153 10
Athens, Greece
§IBMM, UMR 5247, Universites
́ ́
Montpellier 1 & 2, CNRS, Faculte de Pharmacie, 34093 Montpellier Cedex 5, France
‡Department of Nuclear Medicine, Erasmus MC, 3015 GD, Rotterdam, The Netherlands
ABSTRACT: Novel bombesin (BBN) antagonists were synthesized
by coupling the chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-
tetraacetic acid (DOTA) to H-D-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-
Leu-NH2 (JMV594) through linkers of increasing number of (βAla)x
residues (x = 1−3). Labeling with 111In afforded the respective
radiotracers in high purity and high specific activity. Bioconjugate
affinity for the gastrin releasing peptide receptor (GRPR) as
determined against [125I-Tyr4]BBN was high (IC50 values in the
lower nanomolar range). Radioligands poorly internalized in PC-3
cells at 37 °C. Radiopeptides remained >60% intact 5 min after
entering the bloodstream of healthy mice. After injection in SCID
mice bearing human PC-3 xenografts all analogues showed high
tumor uptake and rapid background clearance via the kidneys into
urine. Interestingly, pancreatic uptake, albeit GRPR-specific, declined rapidly with time. 111In-DOTA-(βAla)2-JMV594 achieved
the highest tumor values among the group (17.0 2.8%ID/g vs. 8−10%ID/g, respectively, at 4 h pi) indicating that the (βAla)2-
linker favors in vivo interaction of radiopeptides with the GRPR.
administration of potent BBN agonist-based (radio)peptides to
patients, even at low doses. Most frequently observed adverse
reactions were associated with abdominal smooth muscle
contraction, stimulation of gastrointestinal hormone secretion,
and thermoregulation, elicited by activation of bombesin
receptors, which are physiologically expressed in peripheral
tissues and especially in the gut.3,9,11−13 These findings had a
negative impact on the clinical application of a few promising
BBN agonist-based radioligands as well as on their commerci-
alization prospects. Receptor-targeted radiotherapy, in partic-
ular, is often associated with more severe side effects, because a
higher peptide amount needs to be injected into patients to
reach an effective tumoricidal dose of the therapeutic
radionuclide.14,15
Biosafety problems may be averted with the advent of
radioligands based on GRPR-antagonists instead of GRPR-
agonists. In addition, the proliferative action of BBN
propagated via the GRPR on cancer cells16−19 strengthens
the rationale to use GRPR-antagonists as anticancer drugs. For
this purpose, several BBN-receptor antagonists successfully
INTRODUCTION
■
Among the three bombesin receptors identified in mammals,1
the gastrin releasing peptide receptor (GRPR) is nowadays
regarded as a major molecular target for radiolabeled bombesin
(BBN) analogues owing to its abundant expression in many
frequently occurring human cancers, including prostate cancer,
mammary carcinoma, or lung cancer.2−7 As a result, several
radiolabeled analogues of the amphibian tetradecapeptide BBN
and its C-terminal octapeptide fragment BBN(7−14) have
been developed by coupling suitable chelators to their N-
terminus to allow for labeling with radiometals used in SPECT/
PET imaging or in radionuclide therapy. The resulting
radiopeptides rapidly internalize into target cells after binding
to the GRPR and have shown high accumulation in human
GRPR-expressing xenografts in immunosuppressed mice.8−10
Hence, until recently the consensus has been to develop
agonist-based radioligands, which would effectively accumulate
into malignant lesions by virtue of their fast in vivo
internalization into cancer cells, a prerequisite widely
considered essential for optimal diagnostic imaging and
radionuclide therapy of tumors.
Translation of preclinical results into the clinic, however,
revealed biosafety hazards linked to the intravenous (iv)
Received: November 18, 2012
Published: February 22, 2013
© 2013 American Chemical Society
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Figure 1. Synthesis of DOTA-(βAla)x-JMV594 conjugates, 1 (JMV4705, x = 1); 2 (JMV4168, x = 2); and 3 (JMV4706, x = 3). Fragments Ax′ and B
were synthesized separately and then condensed in solution; after coupling of the chelator DOTA and removal of side chain protecting groups
peptide analogues were isolated by chromatographic methods and lyophilized.
inhibiting the growth of human xenografts in nude mice have
been developed.20−23 The advantages of exploiting such
receptor antagonists as molecular vehicles of radionuclides to
GRPR-expressing cancer were first evident with our analogue
99mTc-Demobesin 1.24 This radiotracer was generated by
coupling an acyclic tetraamine chelator to D-Phe6 of the potent
GRPR-antagonist [H-D-Phe6,LeuNHEt13,desMet14]BBN(6−14)
followed by 99mTc-labeling. Despite its poor ability to
internalize 99mTc-Demobesin 1 displayed unprecedentedly
high and GRPR-specific accumulation in human prostate
cancer PC-3 xenografts in mice and a rapid background
clearance, even from the strongly GRPR-positive mouse
pancreas, via the kidneys into urine. This pharmacokinetic
profile in mice was found to be superior to that of agonist-based
99mTc-Demobesin 4 ([99mTc−N4 ,Pro1,Tyr4,Nle14]BBN) in a
0
direct comparison study.25 Most importantly, the favorable
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Table 1. Analytical Data for 1, 2, 3
HPLC tR (min), UV trace
b
c
compound
sequence
DOTA-(βAla)-JMV594
MW calculated/found (m/z)
system 1
system 2
a
1
2
3
1570.83/[M + 2H+]/2 = 786.18
1641.90/[M + 2H+]/2 = 821.5
1712.98/[M + 2H+]/2 = 857.2
1.35
1.37
1.37
16.3
17.2
17.3
DOTA-(βAla)2-JMV594
DOTA-(βAla)3-JMV594
a
b
JMV594: D-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH2. System 1: RP-HPLC with UV detection; Column, Chromatolith Speed ROD (50 mm ×
4.6 mm); solvents, A = 0.1% TFA(aq) (v/v) and B = 0.1% TFA (v/v) in MeCN; linear gradient, 0% B to 100% B in 4 min; flow rate, 5 mL/min.
c
System 2: RP-HPLC with UV detection; Column, C18 XSelect CSH (5 μm, 4.6 mm × 150 mm); solvents, A = 0.1% TFA in H2O (v/v) and B =
MeCN; linear gradient, 20% B to 25% B in 10 min followed by isocratic elution; flow rate, 1 mL/min.
clearance and excellent tolerability of 99mTc-Demobesin 1 were
reproduced in a small number of prostate cancer patients.26
It is interesting to note that radiolabeled somatostatin
receptor antagonists have likewise shown high lesion uptake
and fast background clearance, even from tissues physiologically
expressing somatostatin receptors.27 Furthermore, results
originally acquired with 99mTc-Demobesin 1 were soon
corroborated by data obtained by a broader spectrum of
BBN antagonist-based radioligands modified with similar or
different chelators and labeled with a wide range of radio-
metals.28,29 Nevertheless, the exact molecular mechanism(s)
shaping the superior in vivo profile of both bombesin and
somatostatin receptor antagonists as compared to agonists have
not been completely understood yet, although binding of
antagonists to a higher number of receptors expressed on target
cells has been suspected.
Several classes of potent bombesin receptor antagonists have
been developed by structural interventions in the C-terminal
Leu13-Met14NH2 dipeptide fragment of frog BBN,20 as for
example development of desmethionine Leu13-alkylamide
analogues (as in 99mTc-Demobesin 1),30 by replacement of
the peptide bond between the two last amino acid residues by a
pseudopeptide bond,31 or by replacement of Leu13 by a γ amino
acid statyl residue (Sta = statine, (3S,4S)4-amino-3-hydroxy-6-
methylheptanoic acid).32 Several of these peptides were able to
recognize BBN binding sites on rat pancreatic acini in vitro and
to antagonize BBN stimulated amylase secretion in the
nanomolar range, while in vivo they were shown to inhibit
the BBN-induced pancreatic enzyme secretion in rodents.
Among these analogues the linear nonapeptide JMV594 (H-D-
Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH2) has attracted our
attention. JMV594 was previously developed by us from the
frog BBN(6−14) sequence after replacement of the C-terminal
dipeptide fragment by Sta13-Leu14NH2 and substitution of Asn6
by D-Phe6 and has shown high binding affinity and potent
antagonistic properties at the GRPR.32
In this study we have coupled the universal chelator DOTA
(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) to the
N-terminus of JMV594 via a series of linkers containing an
increasing number of βAla-residues, thereby generating the
following DOTA-(βAla)x-JMV594 peptide-conjugates:
JMV4705, x = 1; JMV4168, x = 2; and JMV4706, x = 3
(Figure 1). Coupling of DOTA will allow labeling with a wide
range of diagnostic (PET: 68Ga; and SPECT: 111In, 67Ga) and
therapeutic (177Lu, 90Y, 213Bi) radiometals. On the other hand,
linker length variations will demonstrate the metal-chelate
position leading to radiopeptides of improved biological
features, especially of higher tumor uptake.33 After labeling
with 111In BBN analogues are directly compared in GRPR-
positive in vitro models and in SCID mice bearing human
prostate cancer PC-3 xenografts as potential candidates for
application in the diagnostic imaging of GRPR-expressing
tumors in man. Furthermore, 111In-radiotracers may serve as
surrogates during early analogue-screening approaches for the
development of the respective 177Lu/90Y/213Bi-radioligands
intended for therapeutic applications.
RESULTS
■
Synthesis of DOTA-(βAla)x-JMV594 Conjugates. We
chose a fragment condensation approach for the synthesis of
the conjugates and combined solid phase peptide synthesis
(SPPS) and synthesis in solution. In fact, solid phase synthesis
of peptides containing a statine residue is challenging, as
acylation of the hydroxyl side chain of statine can occur during
elongation of the peptide with large excess of activated Nα-
protected amino acids. Furthermore, epimerization during
fragment condensation cannot occur in this case, because
fragment A is terminated with a glycine residue. First, fragments
Ax′ (Fmoc-(βAla)x-D-Phe-(Trt)Gln-(Boc)Trp-Ala-Val-Gly-
OH; x = 1−3) were synthesized on the solid support and
fragment B′ (Fmoc-His(Trt)-Sta-Leu-NH2) in solution, as
shown in Figure 1. Fragment Ax′ (x = 1−3) was released from
the 2-chlorotrityl chloride resin and the Fmoc-group was
removed from the N-terminus of fragment B′ with diethyl-
amine in DMF. The two fragments were condensed in DMF
using BOP in the presence of DIEA as coupling agent to
produce the respective constructs Ax′-B′ (Fmoc-(βAla)x-D-Phe-
(Trt)Gln-(Boc)Trp-Ala-Val-Gly-His(Trt)-Sta-Leu-NH2; x =
1−3); the Fmoc groups were removed from the Nα-amino
acids by diethylamine in DMF. At the final stage, coupling of
the DOTA-tris(tBut)ester chelator to the Nα-amino acid of
individual Ax′-B construct was achieved in high yields using
HATU as a coupling agent. After treatment with TFA to
remove side chain protecting groups, the conjugates DOTA-
(βAla)x-JMV594: 1 (JMV4705, x = 1); 2 (JMV4168, x = 2);
and 3 (JMV4706, x = 3) were isolated by preparative HPLC
and lyophilized (Figure 1). BBN analogues were obtained with
an average yield of ∼40% and a >97% purity as confirmed by
RP-HPLC; ES/MS spectra were in accordance with the
expected formulas (Table 1).
Labeling with 111In. Incorporation of 111In by the DOTA
framework was achieved by heating a mixture of each peptide-
conjugate and 111InCl3 in acidic medium for 20 min at 90 °C
(>95% yield).34 As demonstrated by RP-HPLC analysis of the
labeling reaction mixtures, all three 111In-radiotracers eluted as a
single radiochemical species (>95% purity), and typical specific
activities of ≈4−7 MBq/nmol. Elution times (tR) determined
by HPLC (system 3, column III) for the three radiopeptides
were [111In]1 ([111In]JMV4705), 18.2 min; [111In]2 ([111In]-
JMV4168), 19.6 min; [111In]3 ([111In]JMV4706), 20.0 min. A
representative radiochromatogram for [111In]2 is shown in
Figure 2.
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Figure 4. Intracellular Ca2+ mobilization induced in GRPR-positive
PC-3 cells by 1 h incubation at 37 °C with increasing concentrations of
Figure 2. RP-HPLC analysis of labeling reaction mixture of [111In]2
with chemical structure included (system 3, column III).
BBN (Δ) BBN (EC50 = 0.73
0.14 nM); Ca2+ release inhibition
■
curves by increasing concentrations of ( ) 1 (EC50 = 11.5 1.6 nM),
◆
▼
) 2 (EC50 = 130 34 nM), and ( ) 3 (EC50 = 137 16 nM) in
(
Affinity of Peptide Conjugates to Human GRPR.
Binding affinities of peptide conjugates for the human GRPR
were determined by competition binding assays at 4 °C in
prostate adenocarcinoma PC-3 cells35 against [125I-Tyr4]BBN.
As shown in Figure 3, all analogues displaced the radioligand
the presence of 1 nM BBN; effects induced by BBN at 10 μM were set
as maximum.
Binding of 111In-Radiopeptides to PC-3 Cells. During 1
h incubation at 37 °C, all three 111In-radiopeptides were bound
to PC-3 cells adhered as confluent monolayers in six-well plates
(36% of total added activity was associated to PC-3 cells for
[
111In]2 and ≈27% for [111In]1 and [111In]3; ≈1.5 × 106 cells/
well were measured). Only 6% ([111In]2) and 4.5% ([111In]1
and [111In]3) of total-added activity internalized into the cells
and the rest of radioactivity was bound to the cell-membrane, as
observed also for other BBN-receptor radioantagonists.25,28
Total association to cells dropped to base levels (<0.7% of total
added activity) in the presence of excess [Tyr4]BBN (1 μM)
implying that activity bound and/or internalized in PC-3 cells
via the GRPR.
Metabolism of 111In-Radiopeptides in Mice. The in vivo
metabolic stability of all three 111In-radiopeptides was studied
in healthy Swiss albino mice. Blood samples were collected 5
min post injection (pi) and analyzed by RP-HPLC coupled to a
γ-detector. As shown in the representative radiochromatograms
of Figure 5, more than 60% of intact radiopeptides were still
detected. All three analogs displayed similar degradation
patterns, characterized by formation of two major hydrophilic
radiometabolites (A − tR = 6.1−9.2 min; B − tR = 17.9−19.8
min, respectively).
Figure 3. Displacement of [125I-Tyr4]BBN from GRPR-positive PC-3
cells by increasing concentrations of the following peptide analogues,
(Δ) [Tyr4]BBN (IC50 = 1.8 0.2 nM); ( ) 1 (IC50 = 7.2 0.6 nM);
■
◆
(
▼
) 2 (IC50 = 8.2 0.7 nM); and ( ) 3 (IC50 = 5.4 0.7 nM).
from GRPR-binding sites in a monophasic and dose-dependent
way. The IC50 values were found in the lower nanomolar range
with minor differences across analogs: 3 (IC50 = 5.4 0.7 nM)
> 1 (IC50 = 7.2 0.6 nM) ≈ 2 (IC50 = 8.2 0.7 nM) but
higher to the IC50 value determined for frog [Tyr4]BBN (IC50
= 1.8 0.2 nM).
Intracellular Calcium Release. The antagonistic proper-
ties of 1, 2, and 3 were evaluated by an intracellular Ca2+-release
experiment in GRPR-positive PC-3 cells, and representative
curves are shown in Figure 4. Native BBN strongly triggered
the mobilization of intracellular Ca2+ during 1 h incubation at
37 °C showing a typical agonist profile at the GRPR in this
assay (EC50 = 0.73 0.14 nM).36 In contrast, 1, 2, and 3 were
unable to induce Ca2+ mobilization even at a concentration of
10 μM. Instead, they inhibited the effect of 1 nM BBN on
intracellular Ca2+ release in a monophasic and dose-dependent
manner, albeit with different potencies (EC50 = 11.5 1.6 nM,
EC50 = 130 34 nM, and EC50 = 137 16 nM, respectively),
all displaying typical antagonistic behavior at the GRPR.
Biodistribution of 111In-Radiopeptides in PC-3 Xeno-
graft-Bearing SCID Mice. Biodistribution data for 111In-
radiolabeled analogs in female SCID mice bearing human PC-3
xenografts is summarized in Table 2 and in Figure 6 for the 4 h,
and 24 h pi time points; in a separate 4 h-pi group mice
received excess [Tyr4]BBN (40 nmol) together with the
radioligand (10 pmol) to determine changes of uptake in the
implanted tumor and in physiological tissues of interest during
in vivo GRPR-blockade.24,25 Results are expressed as average %
ID/g values SD, n ≥ 4.
All three 111In-radiopeptides showed high uptake in the
human GRPR-expressing PC-3 xenografts at 4 h pi with the
(βAla)2-containing radiotracer, [111In]2, displaying almost twice
as high tumor values as compared to the other two analogues
(8.0 2.0%ID/g, 17.0 2.9%ID/g and 9.8 1.8%ID/g for
[
111In]1, [111In]2 and [111In]3, respectively). In all cases, tumor
uptake was significantly reduced (P < 0.001) during in vivo
GRPR-blockade (<1.4%ID/g) demonstrating GRPR-specificity.
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Figure 5. Radiochromatograms by RP-HPLC analysis of mouse blood
collected 5 min pi of (A) [111In]1, 60% intact; (B) [111In]2, 64%
intact; and (C) [111In]3, 66% intact; the arrow indicates the elution of
parent radiopeptide (tR in min) determined by coinjection with the
respective radioligands (system 4, column IV).
Tumor values slowly declined with time, but at 24 h pi [111In]2
still displayed the highest tumor uptake compared to the other
two radioligands (Table 2). Moderate, but receptor-specific
uptake was also observed in the strongly GRPR-positive mouse
pancreas with [111In]2 again displaying the highest values (0.6
0.2%ID/g, 3.0 1.3%ID/g and 0.8 0.3%ID/g for [111In]1,
[
111In]2, and [111In]3, respectively, at 4 h pi). Interestingly,
radioactivity in the pancreas rapidly declined to very low levels
(≤0.8%ID/g) at 24 h pi, in agreement with the rapid pancreatic
washout of previously reported radiolabeled BBN-receptor
antagonists.25,28,29
Blood clearance of 111In-radiopeptides was very rapid. The
radioactivity washed out very fast from the body of mice via the
kidneys into the urine, whereas the hepatobiliary pathway
played a minor role in excretion. No kidney retention was
observed; the low renal values at 4 h pi (<3.5%ID/g) further
declined at 24 h pi (<2.0%ID/g). The high and GRPR-specific
tumor uptake attained by 111In-radiolabeled analogues, and
especially by [111In]2, in combination with their rapid body
clearance led to excellent overall pharmacokinetic profiles.
DISCUSSION
■
The search for radiolabeled peptides, which are able to interact
with target receptors on cancer cells and specifically localize on
tumor lesions in vivo, has been quite intensive in the past two
decades and has made available new powerful tools for clinical
oncology.2,37,38 Much interest has been focused on GRPRs in
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99mTc-radioligand based on a desmethionine Leu13-ethylamide
BBN receptor antagonist30 showed superior pharmacokinetic
profile in a mouse model when directly compared to the
agonist 99mTc-Demobesin 4. Specifically, 99mTc-Demobesin 1
displayed higher uptake in human PC-3 xenografts in mice
combined with a faster body clearance via the kidneys into
urine, and a rapid radioactivity washout from the GRPR-rich
pancreas. These findings were soon corroborated by reports on
other radioantagonists21,29 and are of great clinical significance,
especially when considered in combination with tolerability
issues.15 It should be noted that biosafety breaches have been
reported during injection of radiolabeled BBN-agonists to
patients due to inadvertent activation of bombesin receptors
physiologically expressed in the body.11−13,15 Apparently,
switching paradigm from GRPR-agonists to antagonists for
tumor visualization and therapy with radionuclides not only
improves the in vivo targeting of GRPR-positive lesions and
background clearance but, most importantly, minimizes
biosafety risks.
Despite recent progress, however, little is known about the
structural parameters affecting the clinical efficacy of radio-
metal-labeled BBN receptor antagonists. In addition to the
influence of peptide chain per se, the impact of the metal-chelate
type and position warrants further investigation. Metal-chelate
position is determined by the length of the linker introduced
between the reporting unit and the pharmacophore site. In this
work, we have coupled the universal chelator DOTA33 to the
N-terminus of linkers of increasing length coupled to the
potent GRPR-antagonist JMV594.32 Thus, by introducing
growing number of βAla residues in the linker, the position
of the metal-chelator could be adjusted to maximize tumor
uptake in vivo. Herein we report the preclinical evaluation of
DOTA-peptide conjugates labeled with 111In. Results will assist
in the selection of the best candidate for labeling with a wider
range of diagnostic and therapeutic radiometals and for
studying the suitability of resulting radioligands for the clinic.
The new conjugates retained a high affinity for the human
GRPR (IC50= 5.4−8.2 nM) as determined by competition
binding assays against [125I-Tyr4]BBN in PC-3 cells. This
affinity, although inferior to Demobesin 1 (0.7 0.1 nM),25 is
in the same range with values reported for similarly conjugated
analogues.28 In a Ca2+-release assay in PC-3 cells,36 1, 2, and 3
inhibited the BBN-induced calcium mobilization behaving as
true antagonists at the GRPR with potencies in the nanomolar
range. In accordance to this finding, radiolabeled analogues
tested, poorly internalized in PC-3 cells at 37 °C. Instead, they
were retained on the cell-membrane of PC-3 cells by a GRPR-
mediated process, as reported for other radiolabeled GRPR-
antagonists.25,28,29 It is interesting to note that the (βAla)2-
linker containing radioligand [111In]2 showed significantly
higher association to PC-3 cells (both in the membrane-
bound and internalized fraction) than the other two radio-
peptides (P < 0.001). Therefore, in this series of analogues
111
□
■
Figure 6. . Comparative biodistribution data of ( ) [ In]1, ( )
[
111In]2 and (checkered squares) [111In]3 in PC-3 tumor-bearing
SCID mice shown for (A) tumor, (B) pancreas, and (C) kidneys
expressed as %ID/g for 4 h, and 24 h pi; for in vivo GRPR-blockade at
4 h pi animals received excess [Tyr4]BBN along with the radioligand.
view of their high density expression in frequently occurring
human cancers, including breast and prostate carcinoma, or
lung cancer.3−7 These receptors represent an efficient
molecular means to visualize and combat GRPR-expressing
cancer using radiolabeled BBN analogues following the
paradigm of somatostatin receptors expressed in neuro-
endocrine tumors recognized by radiolabeled somatostatin
analogues. Until recently the prevailing theory held agonist-
based radioligands as the secure choice for successful in vivo
tumor targeting due to their rapid internalization in cancer cells,
which in turn would lead to high tumor-to-background ratios in
patients.27
[
111In]2 displayed the most favorable position of the radio-
metal-chelate for GRPR-specific cell-association with potential
implications for favorable in vivo uptake in PC-3 tumors (vide
infra).
The in vivo stability of the three 111In-radiopeptides was
comparable, with more than 60% intact peptides detected by
RP-HPLC in blood samples collected 5 min pi in mice.
Metabolic stabilities of radiopeptide ligands are usually
monitored by in vitro incubation in blood serum of
mice.24,39,40 This method, however, often leads to over-
However, important advantages became unexpectedly
evident by the introduction of radioantagonists in the imaging
of GRPR-expressing cancer.25 First 99mTc-Demobesin 1, a
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estimation of radiotracer stability because enzymes anchored in
the vasculature walls or situated in major organs in the body are
not present in sufficient amounts in the serum thereby having
no opportunity to act. Therefore, in vitro studies do not
accurately reflect the actual situation in the living organism, as
reported in detail for other radiopeptides, such as for
177LuAMBA ([177Lu-DOTA-CH2CO-Gly-p-aminobenzoyl]-
BBN(7−14)).40 Metabolic patterns were very much alike for
the three radiolabeled analogues revealing two major hydro-
philic radiometabolites, concordant with results reported for
both agonist and antagonist BBN-based radioligands.24,39,40
The three radiopeptides displayed high and specific localization
in human PC-3 xenografts in mice demonstrating that in vivo
degradation had not compromised their efficient delivery at the
target. It is interesting to note that in this group of compounds
human origin tissues and from complementary animal models,
will be unequivocally settled by studies in human.
The overall pharmacokinetic profile of the three radio-
peptides in PC-3 tumor-bearing mice was consistent with their
hydrophilic nature with the kidneys and the urinary tract
representing the preferred excretion pathway. However, renal
accumulation declined with time and the bulk of radioactivity in
the kidneys was gradually transferred into urine with no
evidence of prolonged retention, which is often observed for
111In-labeled peptide analogues.27,34,37,38 Again, this feature
renders the new radioantagonists, and especially [111In]2, very
attractive candidates for clinical application. In addition,
hepatobiliary excretion was less preferred and radioactivity
levels in the gut were lower favoring high contrast imaging in
the abdomen, especially for [111In]2. Translation of this
excellent preclinical pharmacokinetic profile in GRPR-positive
cancer patients, in particular in the case of [111In]2, warrants
further study to reveal potential benefits associated with the use
of radio-antagonists in addition to higher biosafety. Moreover,
these positive results have set the basis for the development and
preclinical evaluation of the respective therapeutic 177Lu- or
90Y-based radioligands.
[
111In]2, consistent with its higher binding in PC-3 cells in vitro,
displayed significantly higher tumor uptake in mice both at 4
and 24 h pi. This finding demonstrates that subtle changes in
the linker may have a remarkable impact on in vivo targeting
capacities, as well. Nevertheless, tumor values significantly
declined for all analogues between 4 and 24 h pi most probably
due to lack of internalization that could slow down radioactivity
washout from the tumor. This pattern was also exhibited by
radioligands based on JMV594 and on other BBN-antagonist
sequences.24,28,29 Thus, in the same model the tumor uptake of
99mTc-Demobesin 1 declined from 22.7%ID/g at 4 h pi to 5.4%
ID/g at 24 h pi.25 Likewise, for the BBN-receptor antagonist
CONCLUSIONS
■
Three new analogues of the GRPR-antagonist JMV594 carrying
the universal chelator DOTA via linkers of increasing number
of βAla-residues have been synthesized and labeled with 111In.
The peptide conjugates displayed high affinity for the human
GRPR expressed in PC-3 cells during competition binding
assays against [125I-Tyr4]BBN. The antagonistic properties at
the GRPR were confirmed for 1, 2, and for 3 during an
intracellular Ca2+ release inhibition assay in PC-3 cells.
Consistent with their antagonistic character the respective
111In-radioligands showed lack of internalization in the same
cell line. Most importantly, they showed a fast, high, and
GRPR-mediated localization in human PC-3 xenografts in mice
combined with rapid clearance from nontarget tissues via the
kidneys into urine, with [111In]2 demonstrating the highest
tumor uptake. Ongoing studies will reveal the applicability of
new analogs for radionuclide therapy of GRPR-positive cancer
and will show how well these promising qualities translate in
man.
[
111In]-RM1 ([111In-DOTA-CH2CO-Gly-p-aminobenzoyl]-
JMV594) uptake dropped from 13.5%ID/g at 4 h pi to 6.5%
ID/g at 24 h pi in PC-3 xenografts grown in female nude
mice.28 This quality may represent an inherent disadvantage of
radiolabeled BBN-receptor antagonists, narrowing their appli-
cation perspectives in GRPR-targeted radionuclide therapy.
Therefore, the therapeutic efficacy of noninternalizing radio-
antagonists warrants further investigation both by the develop-
ment of elegantly designed analogues with prolonged tumor
retention and by competent translational studies in patients.
Given that shorter range particle emitters will not be able to
access the cell nucleus while longer half-life radionuclides will
run the risk of premature washout from tumor sites at later time
points when radioantagonists are applied, care should be taken
that radionuclide selection and therapy dose-planning is
accordingly adopted.
EXPERIMENTAL SECTION
Another interesting feature of the new GRPR-radio-
antagonists is their accumulation and washout from mouse
GRPR-positive pancreas. Pancreatic uptake was specific but not
high at 4 h pi following the same trend across analogs as tumor
uptake, with [111In]2 again showing the highest pancreatic
uptake at all time intervals (Table 2). However, radioactivity
rapidly washed out from pancreas following a consistent pattern
of BBN receptor antagonist radioligands in mice, such as for
99mTc-Demobesin 1 (50%ID/g at 4 h pi to 1.3%ID/g at 24 h
pi).24,28,29 The significance of this observation for the human
pancreatic uptake needs further investigation in view of
interspecies differences in the affinity and density of bombesin
receptor subtype expression in several mouse and human
tissues.41,42 The low pancreatic uptake of these analogues
rapidly declining with time, as opposed to their much slower
washout from the tumor, is a very attractive feature for
application in GRPR-expressing cancer patients, especially in
the case of radionuclide therapy. This issue, currently addressed
by ongoing studies comparing results from mouse versus
■
Materials and Instrumentation. Chemicals. Unless otherwise
stated all chemicals were reagent grade and were used without further
purification. The amino acid precursors (Fmoc-βAla, Fmoc-D-Phe,
Fmoc-Gln(Trt), Fmoc-Trp(Boc), Fmoc-Ala, Fmoc-Val and Fmoc-
Gly) used in SPPS and those (Fmoc-His(Trt), Boc-Sta and Boc-Leu)
used in solution synthesis were purchased from Senn Chemicals
(Dielsdorf, Switzerland) or Iris Biotech GmbH (Marktredwitz,
Germany). The 2-chlorotrityl chloride resin (Iris Biotech GmbH,
Mesh size 100−200, loading 1.55 mmol/g) was used as solid support.
DOTA-tris(tBut)ester was purchased from TCI Europe N.V.
(Zwijndrecht, Belgium). The coupling reagent 2-(1H-7-azabenzotria-
zol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU)
was purchased from Molekula GmbH (Munchen, Germany) and
̈
benzotriazole-1-yl-oxy-tris-(dimethylamino)-phosphonium hexafluoro-
phosphate (BOP) was purchased from Iris Biotech GmbH
(Marktredwitz, Germany). 111In was purchased as a solution of
111InCl3 in 0.05 M HCl (0.5 mL, 370−185 MBq) from Covidien
(Petten, The Netherlands). Pyr-Gln-Arg-Tyr-Gly-Asn-Gln-Trp-Ala-
Val-Gly-His-Leu-Met-NH2 ([Tyr4]BBN) was purchased from Bachem
(Bubendorf, Switzerland).
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Analysis − Radiochemistry. The formation and purity of synthetic
intermediates and final products were monitored by RP-HPLC
(Beckman, LC-126) connected with a photodiode array UV detector
(Beckman, 168 Detector) on a reverse phase-18 (RP-18) Chromato-
lith Speed ROD column from Merck (50 mm × 4.6 mm, column I).
The crude product purification was accomplished by a preparative
HPLC (Waters Delta Preparative, Waters 4000 system controller)
connected with a column (C18 40 mm × 100 mm, Waters Delta Pack,
column II) and a UV detector set at 254 nm (Waters 486 tunable
absorbance detector). Further verification of intermediate formation
was achieved by ES/MS (Waters micromass ZQ, Waters 2695
Separation Module). The lyophilizer (Cryonext, Saint Gely du Fesc,
France) was used with a freezing trap at −90 °C. Products were
obtained with an average yield of ∼40% and a purity >97% as
confirmed by RP-HPLC; ES/MS spectra were in accordance with the
expected formulas (Table 1).
Radiochemical HPLC analyses were performed on a Waters
Chromatograph (Waters, Vienna, Austria) with a 600 solvent delivery
system coupled to both a 996 photodiode array UV detector (Waters,
Vienna, Austria) and a Gabi gamma detector (Raytest RSM
Analytische Instrumente GmbH, Germany), with the Millennium
Software (Waters, Vienna, Austria) applied to control the HPLC
system and process the data. Analyses were conducted on a C18
XSelect CSH (5 μm, 4.6 mm × 150 mm, Waters, Vienna, Austria;
column III) cartridge column applying the elution systems described
in the text. Metabolic studies were conducted using a Chromatograph
(600 solvent delivery system, coupled to both a 2998 photodiode array
UV detector; Waters, Vienna, Austria) and a Gabi gamma detector
(Raytest RSM Analytische Instrumente GmbH, Germany) with the
Empower Software (Waters, Vienna, Austria) used to control the
HPLC system and process the data; a RP18 Symmetry Shield column
was employed (5 μm, 3.9 mm × 20 mm, Waters, Vienna, Austria;
column IV). For radioactivity measurements an automatic well-type
gamma counter calibrated for 111In was used [NaI(Tl) 3″-crystal,
Canberra Packard Auto-Gamma 5000 series instrument].
mmol, 1 g) following a two-step reaction. After deprotection the
dipeptide (H-Sta-Leu-NH2) was coupled with 1 equiv of Fmoc-
His(Trt)−OH (2.94 mmol, 1.82 g) to afford Fmoc-His(Trt)-Sta-Leu-
NH2 (fragment B′).
3rd Step − Condensation of Fragments Ax′and B, Coupling of
DOTA and Product Isolation. The Nα-Fmoc protecting group was
removed from fragment B′ by diethylamine in DMF to afford fragment
B (H-His(Trt)-Sta-Leu-NH2). Fragments Ax′ (x = 1−3) (74.5 nmol)
were then reacted with 1 equiv of fragment B using BOP as coupling
agent in the presence of N,N-diisopropylethylamine (DIEA) in DMF.
After removal of the Fmoc-group by addition of diethylamine in DMF,
DOTA-tris(tBut)ester (2 equiv, 100 mg) was coupled to the free Nα-
amino acid of Ax′-B constructs using HATU (2 equiv, 60 mg) as
coupling reagent in the presence of DIEA. Side chain groups were
removed by treatment with TFA in the presence of either 1,2-
ethanediol or triisopropylsilane (Tis) as free radical scavengers and
diethylether was added directly to the solution. The mixture was
filtered under vacuum and the precipitate (free peptide conjugates)
was dissolved in a mixture of H2O/MeCN (50/50 v/v) and freeze-
dried. Crude products were purified by preparative HPLC (column II)
applying a linear gradient A = 75% - B = 25% to A = 50% - B = 50% in
25 min at a 50 mL/min flow rate, where A = 0.1% TFA in H2O and B
= 0.1% TFA in MeCN (system 2). Analytical data for the isolated
bioconjugates 1, 2, and 3 is presented in Table 1.
Radiolabeling. Lyophilized peptide conjugates were dissolved in
HPLC-grade water at a final 1 mM concentration. The bulk solution
was distributed in 50 μL aliquots in Eppendorf vials that were stored at
−20 °C. To an Eppendorf vial containing 1 M sodium acetate buffer
pH 4.6 (3 μL), freshly prepared 0.1 M sodium ascorbate solution was
added (5 μL) followed by 111InCl3 solution (30 μL/12−22 MBq) and
finally the peptide analogue stock solution (3 μL, 3 nmol). The
reaction mixture was allowed to react in a boiling water bath for 20
min, and samples thereof were prepared for HPLC analysis by mixing:
(i) H2O (24 μL), (ii) 1 M sodium acetate buffer pH 4.6 (2 μL), (iii)
0.1 M Na2EDTA (2 μL), and (iv) test labeling solution for analysis (2
μL). After 10 min incubation at room temperature aliquots were
analyzed by HPLC applying the following gradient system at a 1 mL/
min flow rate: from 20% B to 25% B in 10 min followed by isocratic
elution; solvent A = 0.1% TFA in H2O and solvent B = MeCN
(system 3, column III). Prior to further use, the labeling solution was
neutralized with 0.5 M phosphate buffer.
Synthesis of DOTA-(βAla)x-JMV594. Synthesis of DOTA-
(βAla)x-JMV594 (x = 1, 2, 3) was conducted in three consecutive
steps, first step: synthesis of fragments Ax′ on the solid support;
second step: synthesis of fragment B′ in solution; and third step:
condensation of fragments Ax′ and B′, DOTA-coupling, isolation of
peptide conjugates.
Radioiodination of [Tyr4]BBN (Bachem, Bubendorf, Switzerland)
was performed as previously described.39 Methionine was added to the
purified radioligand solution to prevent oxidation of Met14 to the
corresponding sulfoxide; a stock solution thereof in 0.1% BSA-PBS
was kept at −20 °C, and aliquots were withdrawn for competition
binding assays (specific activity of 2 Ci/μmol).
ATTENTION!!! All manipulations with radioactive solutions were
performed by authorized and trained personnel behind sufficient lead
shielding in supervised and licensed laboratories following national and
international rules.
Cell Culture. Human androgen-independent prostate adenocarci-
noma PC-3 cells (LGC Promochem, Teddington, UK) endogenously
expressing the human GRPR35 were cultured in Dulbecco’s modified
Eagle medium with GlutaMAX-I (DMEM) supplemented with 10%
(v/v) heat-inactivated fetal bovine serum (FBS; Gibco BRL, Life
Technologies, Grand Island, NY/USA), 100 U/mL penicillin and 100
μg/mL streptomycin (Biochrom KG Seromed, Berlin, Germany).
Cells were kept in a controlled humidified atmosphere containing 5%
CO2 at 37 °C. Cells were inspected for confluency and 1:3 or 1:4
passages were performed (usually every 4−5 days) using a trypsin/
EDTA (0.05%/0.02% w/v) solution.
1st Step − SPPS of Fragments Ax′. Synthesis followed standard 9-
fluorenylmethyloxycarbonyl (Fmoc)/tert-butyl (tBu) methodology on
solid support. The protected peptide sequences Fmoc-(βAla)x-D-Phe-
(Trt)Gln-(Boc)Trp-Ala-Val-Gly (x = 1 fragment A1′, x = 2 fragment
A2′, and x = 3, fragment A3′) were assembled on the 2-chlorotrityl
chloride resin. For couplings a 10-fold excess of Fmoc-amino acid was
added using BOP as coupling reagent in N,N-dimethylformamide
(DMF) or dichloromethane (DCM) or mixtures thereof. Reaction
progress was monitored after 1−2 h by the Kaiser test and in case of
incomplete coupling, the procedure was repeated. Nα-Fmoc protecting
group removal was achieved by addition of 20% piperidine in DMF for
5 min, followed by a 10 min treatment. Resin samples were treated
with a mixture of trifluoroethanol (TFE)/acetic acid (AcOH)/DCM
(2/1/7 v/v/v) for 1.5 h to release intermediate peptides from the
resin. These fragments were checked for purity and complete
deprotection by HPLC. In a typical HPLC experiment an aliquot
(10 μL) of the solution was injected on the column (column I).
Elution at 5 mL/min flow rate using a gradient pattern with 0−100%
solvent B in 4 min (solvent A = 0.1% trifluoroacetic acid (TFA) in
H2O (v/v) and solvent B = 0.1% TFA in acetonitrile (MeCN) (system
1)).
2nd Step − Synthesis of Fragment B′. Solution synthesis was
performed following standard procedure of Nα-Boc deprotection with
TFA and coupling of amino-acids in the presence of BOP in
consecutive steps. In a preliminary stage, Boc-Leu-OH.H2O (8.66
mmol, 2.16 g) was activated by isobutyl-chloroformate (IBCF) and
treated with 2.5 equiv of NH4OH (21.5 mmol, 3 mL) in 1,2-
dimethoxyethane (DME) to afford Boc-Leu-NH2. After removal of the
Boc group, Leu-NH2 was coupled with 1 equiv of Boc-Sta-OH (3.63
Competition Binding of Peptide-Conjugates in PC-3 Cells.
Confluent PC-3 cells were seeded in 12-well plates (1.0 × 106 cells per
well) and were incubated for 24 h. On the day of the experiment, cells
were rinsed twice with ice-cold washing buffer containing DMEM
GlutaMAX-I supplemented with 1% (v/v) FBS. Competition was
conducted by incubation of [125I-Tyr4]BBN (18 fmol, ∼40 000 cpm)
in the presence of increasing concentrations in triplicate of test peptide
in binding buffer (total volume per well 600 μL, 50 mM HEPES, 5.5
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mM MgCl2, 0.1 mg/mL bacitracin, 1% BSA) for 90 min at 4 °C.
Competition was interrupted by discarding supernatants and washing
the cells with ice-cold PBS. Cells were then lysed and detached from
the plates by 1 N NaOH treatment (2 × 600 μL). Lysates were
collected and counted for their radioactivity content in a γ-counter.
IC50 values were calculated by nonlinear regression according to a one-
site model applying the PRISM 2 program (GraphPad Software, San
Diego, CA) and are the average of three experiments. Nonspecific
binding was defined as the amount of activity bound in the presence of
1 μM [Tyr4]BBN.
blood (0.5−1 mL) was collected from the heart of animals while under
ether-anesthesia and immediately transferred in prechilled polypropy-
lene tubes containing EDTA and placed on ice. Blood samples were
centrifuged (10 min, 2000g/4 °C, in a Hettich, Universal 320R,
centrifuge, Tuttlingen, Germany), the plasma was collected, mixed
with MeCN in a 1/1 v/v ratio and centrifuged again (10 min, 15000g/
4 °C). Supernatants were concentrated to a small volume under a
gentle N2-flux at 40 °C, diluted with physiological saline (≈ 400 μL)
and filtered through a Millex GV filter (0.22 μm). Aliquots thereof
were analyzed by RP-HPLC at a 1 mL/min flow rate applying the
following gradient: 100% A − 0% B to 50% A − 50% B in 50 min; A =
0.1% TFA in H2O and B = MeCN (system 4, column IV). The elution
times (tR) of intact radiotracers were determined by coinjection of the
respective 111In-radiopeptide.
Calcium Release Assay. Intracellular calcium release was
measured using the benchtop scanning fluorometer FlexStation II
machine (Pharmacologie & Screening Plateform of the Institut
́ ́
Federatif de Recherche 3, Montpellier, France). In brief, PC-3 cells
Biodistribution of 111In-Radiopeptides in PC-3 Xenograft-
Bearing SCID Mice. Female SCID mice of 6 weeks of age on the day
of arrival (NCSR “Demokritos” Animal House) were inoculated in the
right flank with a ≈150 μL bolus containing a suspension of (1.5−2) ×
107 freshly harvested human PC-3 cells in sterile physiological saline.
The animals were kept under aseptic conditions and ≈3 weeks later
developed well-palpable tumors at the inoculation site. For
biodistribution, a bolus of test 111In-radiopeptide solution (100 μL,
37−74 kBq, 10 pmol total peptide) in physiological saline:EtOH (9:1,
v/v) was injected in the tail vein of mice. Animals were sacrificed in
groups of 4 by cardiac puncture under a mild ether anesthesia at 4 and
24 h pi; in an additional 4-h group animals iv received a high excess
[Tyr4]BBN (100 μL, ≈40 nmol) in normal saline/EtOH 9/1 v/v
along with the radioligand. Samples of blood and urine as well as
organs of interest were collected, weighed and measured for
radioactivity in a gamma counter. Intestines and stomach were not
emptied of their contents. Data were calculated as percent injected
dose per gram tissue (%ID/g) with the aid of standard solutions and
applying the Excel Microsoft. Results represent mean values
standard deviation (SD) as calculated by the PRISM 2.01 (GraphPad,
San Diego, CA) software program.
(80 000 cells per well) were seeded in 96-well Costar black clear
bottom plates (Corning) and cultured in Ham’s F-12K medium
supplemented with 7% fetal bovine serum and 2 mM glutamine at 37
°C in a humidified atmosphere containing 5% CO2. After 24 h cells
were washed with 150 μL of buffer A (Hanks’ balanced salt solution,
0.5% BSA, 20 mM CaCl2, 2.5 mM probenecid, pH 7.4) and then
loaded with 1 μM of the fluorescent calcium indicator Fluo-4AM
prepared in buffer A, containing 0.06% pluronic acid (a mild-ionic
detergent which facilitates Fluo-4AM ester loading). The cells were
incubated for 1 h in the dark at 37 °C. Then, excess Fluo-4AM was
removed by washing the cells 2 × 100 μL of buffer A; 50 μL/well of
the same buffer was added. The cells were left at room temperature for
30 min to allow complete de-esterification of intracellular Fluo-4AM
esters. The black-bottom plate containing the cells as well as the plate
containing the test-compounds were then placed into the temperature-
regulated FlexStation machine. The machine records the fluorescence
output over a period of 60 s, with the compounds (0−10−5 M) being
automatically distributed into the wells containing the cells after 15 s.
The excitation and emission wavelengths were 485 and 525 nm,
respectively. The ratio between the fluorescence peak upon maximal
response and the basal fluorescence intensity of dye-loaded cells was
about 7. To assess the ability to induce calcium mobilization,
compounds were tested at a concentration of 10 μM in triplicate, in
at least two independent experiments. In each case, the change in
fluorescence upon addition of the compound was compared with the
basal fluorescence output measured with the control (addition of
buffer A only). The maximum fluorescent output was equivalent to
that achieved during cell stimulation by 10 μM BBN.36 In the case of
high affinity antagonists, the EC50 were determined using antagonist
inhibition curves in the presence of 1 nM BBN. The EC50 was
calculated as the molar concentration of receptor antagonist that
reduced the maximal response of BBN by 50%.
All animal experiments were carried out in compliance with
European and national regulations and after approval of protocols by
national Authorities.
Statistics. Statistical analysis using the unpaired two-tailed
Student’s t test was performed to compare values between control
and the in vivo GRPR-blockade animal groups at 4 h pi; values P < 0.05
were considered statistically very significant.
AUTHOR INFORMATION
■
Corresponding Author
*Phone: +30-210-6503908; e-mail: nock_berthold.a@hotmail.
com.
Internalization of 111In-Radiopeptides in PC-3 Cells. Con-
fluent PC-3 cells were seeded in six-well plates ((1.0−1.5) × 106 cells
per well) and were incubated for 24 h. Cells were rinsed twice with ice-
cold internalization buffer containing DMEM GlutaMAX-I supple-
mented with 1% (v/v) FBS and then fresh internalization medium (37
°C) was added (1.2 mL). Test 111In-radiopeptide (50000−100000
cpm, 250 fmol of total peptide in 150 μL of 0.5%BSA PBS − I-PBS)
was added followed by I-PBS alone (150 μL, triplicates of total series)
or by [Tyr4]BBN solution in I-PBS (150 μL, triplicates of nonspecific
series at final concentration 1 μM). Cells were incubated at 37 °C for
60 min; incubation was interrupted by placing the plates on ice,
removing the supernatants and rapid rinsing with ice-cold I-PBS. Cells
were then treated 2 × 5 min with acid wash buffer (2 × 0.6 mL, 50
mM glycine buffer pH 2.8, 0.1 M NaCl) at room temperature and
supernatants were collected (membrane-bound radioligand fraction).
After being rinsed with chilled I-PBS, cells were lysed by treatment
with 1 N NaOH (2 × 0.6 mL) and lysates were collected (internalized
radioligand fraction). Sample radioactivity was measured in a gamma
counter and total cell-associated (internalized + membrane bound)
radioactivity was determined with the Microsoft Excel program.
Metabolism of 111In-Radiopeptides in Mice. A bolus of test
111In-radiopeptide solution (150 μL, 11−22 MBq, 3 nmol of total
peptide in physiological saline) was injected in the tail vein of healthy
Swiss albino mice (NCSR “Demokritos” Animal House). At 5 min pi
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
Networking was facilitated by the COST Action BM0607:
“Targeted Radionuclide Therapy”.
ABBREVIATIONS USED
■
SPECT, single photon emission computed tomography; PET,
positron emission tomography; RP-HPLC, reverse phase high
performance liquid chromatography; tR, retention or elution
time; ES-MS, electrospray mass spectrometry; PC-3 cells,
androgen insensitive human prostate adenocarcinoma cells
REFERENCES
■
(1) Kroog, G. S.; Jensen, R. T.; Battey, J. F. Mammalian bombesin
receptors. Med. Res. Rev. 1995, 15 (5), 389−417.
(2) Reubi, J. C. Peptide receptors as molecular targets for cancer
diagnosis and therapy. Endocr. Rev. 2003, 24 (4), 389−427.
2382
dx.doi.org/10.1021/jm301692p | J. Med. Chem. 2013, 56, 2374−2384

Journal of Medicinal Chemistry
Article
(3) Sancho, V.; Di Florio, A.; Moody, T. W.; Jensen, R. T. Bombesin
receptor-mediated imaging and cytotoxicity: review and current status.
Curr. Drug Delivery 2011, 8 (1), 79−134.
antagonist RC-3095 on growth of PC-3 human prostate-cancer
xenografts in nude mice. Int. J. Cancer 1993, 55 (6), 963−967.
(22) Szepeshazi, K.; Schally, A. V.; Halmos, G.; Groot, K.; Radulovic,
S. Growth inhibition of estrogen-dependent and estrogen-independent
MXT mammary cancers in mice by the bombesin and gastrin-releasing
peptide antagonist RC-3095. J. Natl. Cancer Inst. 1992, 84 (24), 1915−
1922.
(23) Thomas, F.; Arvelo, F.; Antoine, E.; Jacrot, M.; Poupon, M. F.
Antitumoral activity of bombesin analogues on small cell lung cancer
xenografts: relationship with bombesin receptor expression. Cancer
Res. 1992, 52 (18), 4872−4877.
(24) Nock, B.; Nikolopoulou, A.; Chiotellis, E.; Loudos, G.; Maintas,
D.; Reubi, J. C.; Maina, T. [99mTc]Demobesin 1, a novel potent
bombesin analogue for GRP receptor-targeted tumour imaging. Eur. J.
Nucl. Med. Mol. Imaging 2003, 30 (2), 247−258.
(25) Cescato, R.; Maina, T.; Nock, B.; Nikolopoulou, A.;
Charalambidis, D.; Piccand, V.; Reubi, J. C. Bombesin receptor
antagonists may be preferable to agonists for tumor targeting. J. Nucl.
Med. 2008, 49 (2), 318−326.
(26) Froeberg, A. V., M.; Maina, T.; Erion, J.; de Swart, J.; de Jong,
M.; Krenning, E. P. GRP-receptors are likely present in human
pancreas. J. Nucl. Med. 2006, 47 (Suppl. 1), 429P.
(4) Markwalder, R.; Reubi, J. C. Gastrin-releasing peptide receptors
in the human prostate: relation to neoplastic transformation. Cancer
Res. 1999, 59 (5), 1152−1159.
(5) Gugger, M.; Reubi, J. C. Gastrin-releasing peptide receptors in
non-neoplastic and neoplastic human breast. Am. J. Pathol. 1999, 155
(6), 2067−2076.
(6) Guinee, D. G., Jr.; Fishback, N. F.; Koss, M. N.; Abbondanzo, S.
L.; Travis, W. D. The spectrum of immunohistochemical staining of
small-cell lung carcinoma in specimens from transbronchial and open-
lung biopsies. Am. J. Clin. Pathol. 1994, 102 (4), 406−414.
(7) Reubi, J. C.; Korner, M.; Waser, B.; Mazzucchelli, L.; Guillou, L.
High expression of peptide receptors as a novel target in gastro-
intestinal stromal tumours. Eur. J. Nucl. Med. Mol. Imaging 2004, 31
(6), 803−810.
(8) Zhang, H.; Abiraj, K.; Thorek, D. L.; Waser, B.; Smith-Jones, P.
M.; Honer, M.; Reubi, J. C.; Maecke, H. R. Evolution of bombesin
conjugates for targeted PET imaging of tumors. PLoS One 2012, 7 (9),
e44046.
(9) Maina, T.; Nock, B.; Mather, S. Targeting prostate cancer with
radiolabelled bombesins. Cancer Imaging 2006, 6, 153−157.
(10) Nanda, P. K.; Pandey, U.; Bottenus, B. N.; Rold, T. L.;
Sieckman, G. L.; Szczodroski, A. F.; Hoffman, T. J.; Smith, C. J.
Bombesin analogues for gastrin-releasing peptide receptor imaging.
Nucl. Med. Biol. 2012, 39 (4), 461−471.
(27) Ginj, M.; Zhang, H.; Waser, B.; Cescato, R.; Wild, D.; Wang, X.;
Erchegyi, J.; Rivier, J.; Macke, H. R.; Reubi, J. C. Radiolabeled
somatostatin receptor antagonists are preferable to agonists for in vivo
peptide receptor targeting of tumors. Proc. Natl. Acad. Sci. U. S. A.
2006, 103 (44), 16436−16441.
(28) Mansi, R.; Wang, X.; Forrer, F.; Kneifel, S.; Tamma, M. L.;
Waser, B.; Cescato, R.; Reubi, J. C.; Maecke, H. R. Evaluation of a
1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-conjugated
bombesin-based radioantagonist for the labeling with single-photon
emission computed tomography, positron emission tomography, and
therapeutic radionuclides. Clin. Cancer Res. 2009, 15 (16), 5240−5249.
(29) Abiraj, K.; Mansi, R.; Tamma, M. L.; Fani, M.; Forrer, F.;
Nicolas, G.; Cescato, R.; Reubi, J. C.; Maecke, H. R. Bombesin
antagonist-based radioligands for translational nuclear imaging of
gastrin-releasing peptide receptor-positive tumors. J. Nucl. Med. 2011,
52 (12), 1970−1978.
(30) Wang, L. H.; Coy, D. H.; Taylor, J. E.; Jiang, N. Y.; Moreau, J.
P.; Huang, S. C.; Frucht, H.; Haffar, B. M.; Jensen, R. T. des-Met
carboxyl-terminally modified analogues of bombesin function as
potent bombesin receptor antagonists, partial agonists, or agonists. J.
Biol. Chem. 1990, 265 (26), 15695−15703.
(31) Severi, C.; Coy, D. H.; Jensen, R. T.; Boschero, L.; Anania, M.
C.; Delle Fave, G. Pharmacological characterization of [Leu13-psi-
CH2NH-Leu14]-bombesin as a specific bombesin receptor antagonist
on isolated smooth muscle cells. J. Pharmacol. Exp. Ther. 1989, 251
(2), 713−717.
(32) Azay, J.; Nagain, C.; Llinares, M.; Devin, C.; Fehrentz, J. A.;
Bernad, N.; Roze, C.; Martinez, J. Comparative study of in vitro and in
vivo activities of bombesin pseudopeptide analogs modified on the C-
terminal dipeptide fragment. Peptides 1998, 19 (1), 57−63.
(33) Wadas, T. J.; Wong, E. H.; Weisman, G. R.; Anderson, C. J.
Coordinating radiometals of copper, gallium, indium, yttrium, and
zirconium for PET and SPECT imaging of disease. Chem. Rev. 2010,
110 (5), 2858−2902.
(34) Breeman, W. A.; De Jong, M.; Visser, T. J.; Erion, J. L.;
Krenning, E. P. Optimising conditions for radiolabelling of DOTA-
peptides with 90Y, 111In and 177Lu at high specific activities. Eur. J. Nucl.
Med. Mol. Imaging 2003, 30 (6), 917−920.
(35) Reile, H.; Armatis, P. E.; Schally, A. V. Characterization of high-
affinity receptors for bombesin/gastrin releasing peptide on the human
prostate cancer cell lines PC-3 and DU-145: internalization of receptor
bound 125I-(Tyr4) bombesin by tumor cells. Prostate 1994, 25 (1), 29−
38.
(36) Aprikian, A. G.; Han, K.; Chevalier, S.; Bazinet, M.; Viallet, J.
Bombesin specifically induces intracellular calcium mobilization via
gastrin-releasing peptide receptors in human prostate cancer cells. J.
Mol. Endocrinol. 1996, 16 (3), 297−306.
(11) Bruzzone, R.; Tamburrano, G.; Lala, A.; Mauceri, M.; Annibale,
B.; Severi, C.; de Magistris, L.; Leonetti, F.; Delle Fave, G. Effect of
bombesin on plasma insulin, pancreatic glucagon, and gut glucagon in
man. J. Clin. Endocrinol. Metab. 1983, 56 (4), 643−647.
(12) Delle Fave, G.; Kohn, A.; De Magistris, L.; Annibale, B.;
Bruzzone, R.; Sparvoli, C.; Severi, C.; Torsoli, A. Effects of bombesin
on gastrin and gastric acid secretion in patients with duodenal ulcer.
Gut 1983, 24 (3), 231−235.
(13) Delle Fave, G.; Annibale, B.; de Magistris, L.; Severi, C.;
Bruzzone, R.; Puoti, M.; Melchiorri, P.; Torsoli, A.; Erspamer, V.
Bombesin effects on human GI functions. Peptides 1985, 6 (Suppl. 3),
113−116.
(14) Maddalena, M. E.; Fox, J.; Chen, J.; Feng, W.; Cagnolini, A.;
Linder, K. E.; Tweedle, M. F.; Nunn, A. D.; Lantry, L. E. 177Lu-AMBA
biodistribution, radiotherapeutic efficacy, imaging, and autoradiog-
raphy in prostate cancer models with low GRP-R expression. J. Nucl.
Med. 2009, 50 (12), 2017−2024.
(15) Bodei, L. F., M.; Nunn, A.; Llull, J.; Cremonesi, M.; Martano, L.;
Laurora, G.; Scardino, E.; Tiberini, S.; Bufi, G.; Eaton, S.; de Cobelli,
O.; Paganelli, G. 177Lu-AMBA Bombesin analogue in hormone
refractory prostate cancer patients: A phase I escalation study with
single-cycle administrations. Eur. J. Nucl. Med. Mol. Imaging 2007, 34,
S221.
(16) Rozengurt, E. Bombesin stimulation of mitogenesis. Specific
receptors, signal transduction, and early events. Am. Rev. Respir. Dis.
1990, 142 (6 Pt 2), S11−S15.
(17) Cuttitta, F.; Carney, D. N.; Mulshine, J.; Moody, T. W.;
Fedorko, J.; Fischler, A.; Minna, J. D. Bombesin-like peptides can
function as autocrine growth factors in human small-cell lung cancer.
Nature 1985, 316 (6031), 823−826.
(18) Aprikian, A. G.; Tremblay, L.; Han, K.; Chevalier, S. Bombesin
stimulates the motility of human prostate-carcinoma cells through
tyrosine phosphorylation of focal adhesion kinase and of integrin-
associated proteins. Int. J. Cancer 1997, 72 (3), 498−504.
(19) Nelson, J.; Donnelly, M.; Walker, B.; Gray, J.; Shaw, C.;
Murphy, R. F. Bombesin stimulates proliferation of human breast
cancer cells in culture. Br. J. Cancer 1991, 63 (6), 933−936.
(20) de Castiglione, R.; Gozzini, L. Bombesin receptor antagonists.
Crit. Rev. Oncol. Hematol. 1996, 24 (2), 117−151.
(21) Pinski, J.; Schally, A. V.; Halmos, G.; Szepeshazi, K. Effect of
somatostatin analog RC-160 and bombesin/gastrin releasing peptide
2383
dx.doi.org/10.1021/jm301692p | J. Med. Chem. 2013, 56, 2374−2384

Journal of Medicinal Chemistry
Article
(37) Kwekkeboom, D.; Krenning, E. P.; de Jong, M. Peptide receptor
imaging and therapy. J. Nucl. Med. 2000, 41 (10), 1704−1713.
(38) Reubi, J. C.; Macke, H. R.; Krenning, E. P. Candidates for
̈
peptide receptor radiotherapy today and in the future. J. Nucl. Med.
2005, 46 (Suppl. 1), 67S−75S.
(39) Nock, B. A.; Nikolopoulou, A.; Galanis, A.; Cordopatis, P.;
Waser, B.; Reubi, J. C.; Maina, T. Potent bombesin-like peptides for
GRP-receptor targeting of tumors with 99mTc: a preclinical study. J.
Med. Chem. 2005, 48 (1), 100−110.
(40) Linder, K. E.; Metcalfe, E.; Arunachalam, T.; Chen, J.; Eaton, S.
M.; Feng, W.; Fan, H.; Raju, N.; Cagnolini, A.; Lantry, L. E.; Nunn, A.
D.; Swenson, R. E. In vitro and in vivo metabolism of Lu-AMBA, a
GRP-receptor binding compound, and the synthesis and character-
ization of its metabolites. Bioconjug. Chem. 2009, 20 (6), 1171−1178.
(41) Uehara, H.; Gonzalez, N.; Sancho, V.; Mantey, S. A.; Nuche-
Berenguer, B.; Pradhan, T.; Coy, D. H.; Jensen, R. T. Pharmacology
and selectivity of various natural and synthetic bombesin related
peptide agonists for human and rat bombesin receptors differs. Peptides
2011, 32 (8), 1685−1699.
(42) Maina, T.; Nock, B. A.; Zhang, H.; Nikolopoulou, A.; Waser, B.;
Reubi, J. C.; Maecke, H. R. Species differences of bombesin analog
interactions with GRP-R define the choice of animal models in the
development of GRP-R-targeting drugs. J. Nucl. Med. 2005, 46 (5),
823−830.
2384
dx.doi.org/10.1021/jm301692p | J. Med. Chem. 2013, 56, 2374−2384