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PET Imaging of Prostate Cancer with Ga-68 Labeled GRPR Agonist BBN7-14 and Antagonist RM26 Siyuan Cheng, Lixin Lang, Zhantong Wang, Orit Jacobson, Bryant C. Yung, Guizhi Zhu, Dongyu Gu, Ying Ma, Xiaohua Zhu, Gang Niu, and Xiaoyuan Chen Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.7b00726 • Publication Date (Web): 18 Dec 2017 Downloaded from http://pubs.acs.org on December 20, 2017
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Bioconjugate Chemistry
PET Imaging of Prostate Cancer with Ga-68 Labeled GRPR Agonist BBN7-14 and Antagonist RM26 Siyuan Cheng†, ‡, Lixin Lang‡, Zhantong Wang ‡, Orit Jacobson‡, Bryant Yung‡, Guizhi Zhu‡, Dongyu Gu‡, Ying Ma‡, Xiaohua Zhu†*, Gang Niu‡*, and Xiaoyuan Chen‡*
†
Department of Nuclear Medicine and PET, Tongji Hospital, Tongji Medical College, Huazhong
University of Science and Technology, Wuhan 430000, P. R. China
‡
Laboratory of Molecular Imaging and Nanomedicine, National Institute of Biomedical Imaging and
Bioengineering (NIBIB), National Institutes of Health (NIH), Bethesda, Maryland (MD) 20892, United States of America (USA)
*
For correspondence or reprint contact either of the following:
Dr. Xiaohua Zhu (
[email protected]); Dr. Gang Niu (
[email protected]); Dr. Xiaoyuan Chen (
[email protected])
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ABSTRACT
2 3
Radiolabeled bombesin (BBN) analogs have long been used for developing
4
gastrin-releasing peptide receptor (GRPR) targeted imaging probes, and tracers with
5
excellent in vivo performance including high tumor uptake, high contrast, and
6
favorable pharmacokinetics are highly desired. In this study, we compared
7
68
8
antagonist (D-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH2, RM26) for PET imaging
9
of prostate cancer. The in vitro stabilities, receptor binding, cell uptake, internalization,
Ga-labeled GRPR agonist (Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH2, BBN7-14) and
10
and
11
68
12
abilities and kinetics were investigated using PC-3 tumor xenografted mice. BBN7-14,
13
P3-RM26, NOTA-Aca-BBN7-14, and NOTA-PEG3-RM26 showed similar binding
14
affinity
15
68
efflux
properties
of
the
probes
68
Ga-NOTA-Aca-BBN7-14
and
Ga-NOTA-PEG3-RM26 were studied in PC-3 cells, and the in vivo GRPR targeting
to
GRPR.
In
PC-3
tumor-bearing
mice,
the
tumor
uptake
of
Ga-NOTA-PEG3-RM26 remained at around 3.00 %ID/g within 1 h after injection,
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Bioconjugate Chemistry
1
in contrast with 68Ga-NOTA-Aca-BBN7-14, which demonstrated rapid elimination and
2
high background signal. Additionally, the majority of
3
remained intact in mouse serum at 5 min after injection, while almost all of
4
68
5
more favorable in vivo pharmacokinetic properties and metabolic stabilities of the
6
antagonist probe relative to its agonist counterpart. Overall, the antagonistic GRPR
7
targeted probe
8
agonist 68Ga-NOTA-Aca-BBN7-14 for PET imaging of prostate cancer patients.
68
Ga-NOTA-PEG3-RM26
Ga-NOTA-Aca-BBN7-14 was degraded under the same conditions, demonstrating
68
Ga-NOTA-PEG3-RM26 is a more promising candidate than the
9
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INTRODUCTION
2
Prostate cancer (PCa) accounts for almost 20% of the newly diagnosed
3
cancers among men in the United States in 2017 and remains the third leading cause
4
of cancer related male death.1 Typical diagnosis of PCa relies on histopathological
5
examination of suspected prostate biopsy tissues or specimens from benign prostatic
6
enlargement surgeries or transurethral resection of the prostate following detection of
7
elevated prostate-specific antigen (PSA) levels, and/or abnormal digital rectal
8
examination (DRE), and bone scanning. X-ray computed tomography and magnetic
9
resonance imaging (MRI) are currently the major imaging techniques for further
10
identification of PCa.2 However, the capacity of conventional diagnostic techniques
11
for primary lesion detection, staging, or relapse monitoring of PCa is limited.3 For
12
example, the PSA test can be interfered by noncancerous factors such as prostate
13
enlargement, old age, and prostatitis, and low levels of PSA do not necessarily rule
14
out the incidence of PCa.4 The sensitivity and specificity of either ultrasound or MRI
15
is also limited by abnormal signals confounded by prostatitis or benign prostatic
16
hyperplasia (BPH).5,6 The notable multiparametric MRI (MP-MRI) remains imperfect
17
as well with a pooled sensitivity up to 89% and a specificity up to 73%.7
18
Interest in applying molecular imaging to PET has grown and a plethora of
19
radiotracers have been developed and investigated actively for PCa. The classical
20
2-deoxy-2-18F-fluoro-D-glucose (18F-FDG) has been used for evaluating late-stage or
21
recurrent PCa, but is not particularly avid.8,9 Other promising agents targeting
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metabolites like fatty acids and amino acids (e.g.
2
18
3
antigens such as prostate-specific membrane antigen (PSMA).11,12 These tracers are
4
proven beneficial for recurrent PC diagnosis and staging. The PSMA targeted tracers
5
have also been applied specially for predicting the optimal timing of PSMA-based
6
therapies.13 However, almost all these tracers show limited diagnostic accuracy for
7
primary lesions,3,10,14 and few of those tracers have been sufficiently investigated and
8
clinically validated to date.
11
C/18F-choline,
11
C-acetate,
F-FACBC) have been further introduced,3,10 as well as agents targeting specific PCa
9
The gastrin-releasing peptide receptor (GRPR) is a G protein-coupled receptor
10
expressed in various organs of mammals, especially in the gastrointestinal tract and
11
the pancreas. Upon binding with the ligand gastrin-releasing peptide (GRP), GRPR
12
can be activated and elicit certain exocrine or endocrine secretions to regulate
13
multiple physiological processes.15 Notably, GRPR overexpression is presented in
14
several types of tumors such as prostate, urinary tract, gastrointestinal stromal, breast,
15
and lung, and related to proliferation and growth of these malignancies.16,17 Especially,
16
GRPR is almost 100% expressed in clinical PCa samples investigated by PCR,
17
immunohistochemistry, or radionuclide binding assay,16 which makes GRPR an
18
attractive target for PCa imaging and therapy.
19
As an amphibian homolog of GRP, bombesin (BBN) was found to bind to
20
GRPR with a high affinity. For decades, the BBN motifs have been used extensively
21
in radioactive imaging or in radionuclide therapy for GRPR overexpressing
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1
cancers.18,19 For example, the GRPR agonist BBN7-14, a truncated form of BBN with
2
the sequence of Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH2, has been studied as PET or
3
single photon emission computed tomography (SPECT) tracers in both preclinical and
4
clinical research.20-23 In the meantime, numerous clinical trials have been performed
5
using
6
68
7
Recently,
8
D-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH2 (RM26) has been developed as an
9
antagonist against GRPR and has been applied actively in preclinical studies.29-32
10
Based on these observations, both BBN7-14 and RM26 are considered as high-quality
11
candidates for further clinical translation.
antagonistic
Ga-RM2,24,25 a
68
GRPR
Ga-SB3,26
targeting 68
PET
radiopharmaceuticals
Ga-BAY86-7458,27 and
nine-amino-acid
analog
of
64
including
Cu-CB-TE2A-AR06.28
nonapeptide
BBN6-14,
12
Despite the outstanding tumor targeting potential, BBN related research is
13
accompanied by a debate on the superiority of GRPR antagonist- versus agonist-based
14
tracers.33-36 It is generally claimed that even though antagonists are not internalized,
15
radiolabeled antagonists may depict clearer images and pharmacokinetic profiles than
16
agonists. More data are expected to emerge for direct comparison of specific
17
radiolabeled agonist and antagonist tracers to address this controversy, especially
18
among tracers that are promising for clinical translation.
19
Herein we would like to establish the distinction of GRPR targeted agonist
20
and antagonist with similar sequences by applying
21
for side-by-side comparative studies, including in vitro receptor binding, cell uptake,
68
Ga-labeled BBN7-14 and RM26
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1
internalization, and efflux studies on PC-3 cells, and in vivo microPET imaging study
2
of PC-3 tumor-bearing mice. The in vitro and in vivo stabilities of both radio
3
conjugates were presented and compared as well.
4
RESULTS
5
Synthesis and Radiolabeling
6
With excess amounts of NOTA-NHS, the NOTA-Aca-BBN7-14 and
7
NOTA-PEG3-RM26 conjugate were produced in > 95% yield. A m/z of 1338 for
8
[M+H+] was identified for NOTA-Aca-BBN7-14 using matrix-assisted laser desorption
9
ionization–time of flight mass spectrometry (MALDI-TOF MS). NOTA-PEG3-RM26
10
was synthesized and characterized by the same method (m/z = 1601 for [M+H+]).
11
Both conjugates were labeled with 68Ga within 20 min, with the specific activities of
12
21.6 ~ 40.01 MBq/nmol and 26.7 ~ 53.33 MBq/nmol respectively for
13
68
14
yield was > 90-95 %, radiochemical purity was > 98 %. The chemical structures of
15
68
Ga-NOTA-Aca-BBN7-14 and
68
Ga-NOTA-PEG3-RM26, and both radiochemical
Ga-NOTA-Aca-BBN7-14 and 68Ga-NOTA-PEG3-RM26 were presented in Figure 1.
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1 2
Figure 1. Schematic structures of GRPR agonist
3
antagonist 68Ga-NOTA-PEG3-RM26 (B).
4
In vitro Stability
5
68
In vitro stabilities of
68
Ga-NOTA-Aca-BBN7-14 (A) and
Ga-NOTA-Aca-BBN7-14 and
68
Ga-NOTA-PEG3-RM26
6
in saline and non-heat-inactivated fetal bovine serum (FBS) (Gibco) were determined
7
according to peak integration of analytical high-performance liquid chromatography
8
(HPLC).
9
68
At
0
min
of
Ga-NOTA-Aca-BBN7-14 and
the 68
incubation,
the
radiochemical
purities
of
Ga-NOTA-PEG3-RM26 were all > 95 % in both
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1
saline and FBS (Figure 2). After 2 h incubation, the parent compound of
2
68
3
peak of 11.58%, while this metabolism for
4
FBS was not as obvious. Metabolites represented by radio peaks of slightly higher
5
lipophilicity than the parent compounds were observed for both after 2 h incubation in
6
FBS, accompanied by the percentages of the parent compounds dropping to 89.24%
7
and
8
68
Ga-NOTA-Aca-BBN7-14 in saline dropped to 88.42% along with a more hydrophilic
80.58%,
respectively,
68
Ga-NOTA-Aca-BBN7-14 incubated in
for
68
stabilities
of
Ga-NOTA-Aca-BBN7-14
and
Ga-NOTA-PEG3-RM26.
9 10
Figure
11
68
12
after incubation. (A) In vitro radioactive stabilities of 68Ga-NOTA-Aca-BBN7-14 in saline
13
at 0 and 120 min after incubation. (B) In vitro radioactive stabilities of
2.
In
vitro
radioactive
68
Ga-NOTA-Aca-BBN7-14
and
Ga-NOTA-PEG3-RM26 in saline and Fetal Bovine Serum (FBS) for 0 and 120 min
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1
68
2
radioactive stabilities of
3
incubation.
4
and 120 min after incubation.
5
Competitive Binding Assay
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Ga-NOTA-PEG3-RM26 in saline at 0 and 120 min after incubation. (C) In vitro
6
68
Ga-NOTA-Aca-BBN7-14 in FBS at 0 and 120 min after
(D) In vitro radioactive stabilities of
68
Ga-NOTA-PEG3-RM26 in FBS at 0
The GRPR-binding affinities of BBN7-14, P3-RM26, NOTA-Aca-BBN7-14 and
7
NOTA-PEG3-RM26
8
125
9
Binding of 125I-[Tyr4]BBN to GRPR was displaced by the cold analogs in a
10
concentration-dependent manner. The half maximal inhibitory concentration
11
(IC50) values of BBN7-14, P3-RM26, NOTA-Aca-BBN7-14 and NOTA-PEG3-RM26
12
were 0.32 ± 0.10, 0. 41 ± 0.13, 1.80 ± 0.67 and 2.05 ± 0.50 nM, respectively. The
13
results indicated that the intermolecular targeting abilities of BBN7-14 and P3-RM26
14
for GRPR were comparable. After the NOTA conjugation, the affinities of both
15
compounds decreased to some extent. However, there were no distinct disparities
16
discovered between NOTA-Aca-BBN7-14 and NOTA-PEG3-RM26 either.
were
assessed
by
competitive
binding
assay
using
I-[Tyr4]BBN as the radioligand. The results of these assays were shown in Figure 3.
17
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Bioconjugate Chemistry
1 2
Figure 3. Inhibition of
3
P3-RM26, NOTA-Aca-BBN7-14, and NOTA-PEG3-RM26 (n = 3/group, mean ± SD).
125
I-[Tyr4]BBN binding to GRPR on PC-3 cells by BBN7-14,
4 5
Cell Uptake, Internalization and Efflux
6
Time dependent cellular uptake pattern in GRPR positive PC-3 cells was
7
observed for both
8
uptake of
9
incubation, and that of 68Ga-NOTA-PEG3-RM26 was slightly lower (Figure 4A). The
68
68
Ga-NOTA-Aca-BBN7-14 and
68
Ga-NOTA-PEG3-RM26. The
Ga-NOTA-Aca-BBN7-14 increased rapidly to nearly 27% within 1 h of
10
agonist
11
around 74 % of the radioactivity uptake was internalized within 1 h of incubation. By
12
contrast,
13
uptake) (Figure 4A). After washing and medium replacement, both the tracers showed
14
efflux with a similar pattern (Figure 4B). At 60 min, 50% of radioactivity uptake was
15
still retained with the cells.
68
Ga-NOTA-Aca-BBN7-14 showed distinctively high internalization and
68
Ga-NOTA-PEG3-RM26 showed very low internalization (< 15 % of total
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1 2
Figure
3
68
4
and internalization assay of 68Ga-NOTA-Aca-BBN7-14 and 68Ga-NOTA-PEG3-RM26 on
5
PC-3 tumor cells (n = 3, mean ± SD). (B) Cell efflux assay of 68Ga-NOTA-Aca-BBN7-14
6
and 68Ga-NOTA-PEG3-RM26 on PC-3 tumor cells (n = 3, mean ± SD).
7
In vivo PET Imaging
4.
In
vitro
cell
Ga-NOTA-Aca-BBN7-14 and
uptake, 68
internalization,
and
efflux
studies
of
Ga-NOTA-PEG3-RM26 on PC-3 cells. (A) Cell uptake
8
Representative coronal PET images of PC-3 tumor-bearing mice at different
9
time points are shown in Figure 5. The tumors were clearly visualized with high
10
contrast at all the time points for 68Ga-NOTA-PEG3-RM26 (n = 3), as well at 15 min
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Bioconjugate Chemistry
1
and 30 min for 68Ga-NOTA-Aca-BBN7-14 (n = 4). However, at 60 min post injection,
2
the
3
with 68Ga-NOTA-Aca-BBN7-14 (Figure 5A). Meanwhile, both the tracers showed
4
considerable accumulation and retention in the abdominal regions including pancreas
5
and intestines, though less was observed for
6
68
7
radioactivity was observed while the radioactivity in the bladder was constantly high
8
for these two probes, suggesting the tracers were excreted mainly by the renal system.
9
Activity accumulation in the tumor was quantified by measuring the regions of
10
interest (ROIs) on the coronal images (Figure 5B, 5C). The mean tumor uptake was
11
determined to be 4.40 ± 0.29, 3.28 ± 0.47, and 2.04 ± 0.34 %ID/g (percentage of
12
injected dose per gram of tissue) for 68Ga-NOTA-Aca-BBN7-14, and 2.99 ± 0.44, 2.96
13
± 0.45, and 3.01 ± 0.45 %ID/g for
14
with the corresponding P value of