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A Preclinical Study on GRPR-Targeted 68GaProbes for PET Imaging of Prostate Cancer Yao Sun, Xiaowei Ma, Zhe Zhang, Ziyan Sun, Mathias Loft, Bingbing Ding, Changhao Liu, Liying Xu, Meng Yang, Yuxin Jiang, Jianfeng Liu, Yuling Xiao, Zhen Cheng, and Xuechuan Hong Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.6b00279 • Publication Date (Web): 11 Jul 2016 Downloaded from http://pubs.acs.org on July 12, 2016
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Bioconjugate Chemistry
A Preclinical Study on GRPR-Targeted
68
Ga-Probes for PET Imaging of Prostate
Cancer Yao Sun1,
2†
, Xiaowei Ma2†, Zhe Zhang2, Ziyan Sun2, Mathias Loft2, Bingbing Ding1,
Changhao Liu2, Liying Xu2, Meng Yang3, Yuxin Jiang3, Jianfeng, Liu4, Yuling Xiao1, Zhen Cheng2*, Xuechuan Hong1* 1. Department State Key Laboratory of Virology, Key Laboratory of Combinatorial Biosynthesis and Drug Discovery (MOE) and Hubei Province Engineering and Technology Research
Center
for
Fluorinated
Pharmaceuticals,
Wuhan
University
School
of
Pharmaceutical Sciences, Wuhan 430071, China;
2. Molecular Imaging Program at Stanford (MIPS), Department of Radiology and Bio-X Program, Canary Center at Stanford for Cancer Early Detection, Stanford University, Stanford, CA94305, USA. 3. Chinese Academy of Medical Science, Peking Union Medical College Hospital, Department of Ultrasound, Beijing, 100730, China 4. Chinese Academy of Medical Science, Institute of Radiation Medicine, Department of Molecular Nuclear Medicine, Tianjin, 300192, China ‡
These two authors contributed equally to this work
Corresponding Authors: Phone: 027-68759734, Email:
[email protected] Phone: 650-210-6001, Email:
[email protected] 1
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Abstract Gastrin-releasing peptide receptors (GRPR) targeted positron emission tomography (PET) is a highly promising approach for imaging of prostate cancer (PCa) in small animal models and patients. Developing a GRPR-targeted PET probe with excellent in vivo performance such as high tumor uptake, high contrast and optimal pharmacokinetics is still very challenging. Herein, a novel bombesin (BBN) analog (named as SCH1) based on JMV594 peptide modified with an 8-amino octanoic acid spacer (AOC) was thus designed and conjugated with the metal chelator 1,4,7-triazacyclononane,1-glutaric acid-4,7-acetic acid (NODAGA). The resulting NODAGA-SCH1 was then radiolabeled with 68Ga and evaluated for PET imaging of PCa. Compared with
68
Ga-NODAGA-JMV594 probe,
68
Ga-NODAGA-SCH1 exhibited
excellent PET/CT imaging properties on PC-3 tumor-bearing nude mice, such as high tumor uptake (5.80 ± 0.42 vs 3.78 ± 0.28 %ID/g, 2h) and high tumor/muscle contrast (16.6 ± 1.50 vs 8.42 ± 0.61 %ID/g, 2h). Importantly, biodistribution data indicated a relatively similar accumulation of
68
Ga-NODAGA-SCH1 was observed in the liver (4.21 ± 0.42 %ID/g) and
kidney (3.41 ± 0.46 %ID/g) suggesting that the clearance is both through the kidney and the liver. Overall, 68Ga-NODAGA-SCH1 showed promising in vivo properties and is a promising candidate for translation into clinical PET-imaging of PCa patients.
2
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Bioconjugate Chemistry
INTRODUCTION Prostate cancer (PCa) remains one of the most frequently diagnosed cancers and leading cause of cancer-related death among men in the United States and Europe.1 Failure of current therapies to prolong patient survival provides some impetus to develop new and innovative diagnostic and treatment strategies for patients with prostate cancer. Because of their intrinsic limitations for the detection and staging of PCa, conventional diagnostic techniques such as ultrasound (US) and computed tomography (CT) play a relatively minor role in the management of PCa.2-3 Molecular imaging techniques, and in particular positron emission tomography (PET), are expected to play a pivotal role in the detection and staging of localized and locally recurrent PCa.4-6 For example, PET-imaging probe in cancer detection and staging, 2-deoxy-2-18F-fluoro-D-glucose (18F-FDG), has been used to evaluate late-stage, recurrent or metastatic PCa.4,7-8 However,
18
F-FDG has shown lower tumor uptake in well-differentiated
PCa than in other tumor types.9 The dominated urinary excretion of 18F-FDG, which leads to high bladder activity, also poses a potential problem in PCa detection as the background activity can mask the signal from PCa tumors. Therefore, the development of novel PET probes targeting specific biomarkers of PCa with high tumor uptake and optimal pharmacokinetics remains crucial for optimization of detection and staging of PCa. Such agents are expected to dramatically facilitate the diagnosis and prognosis of patients and could potentially better stratify PCa patients for effective therapeutic regimens.10-12 Recently, the presence of the gastrin-releasing peptide receptor (GRPR) has been documented in many human cancers including prostate cancer, breast cancer and lung cancer.13-15 High GRPR expression was identified in tissue biopsy samples of human PCa.16 3
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Reubi has demonstrated that GRPR expression in primary prostatic invasive carcinoma was present in 100% of the tissues tested, and in 83% of these cases GRPR expression was determined to be very high (1000 dpm/mg tissue).16 Thus, GRPR can serve as a promising target for PCa diagnosis and therapy.17-19 Taking advantages of highly sensitive and quantitative molecular imaging methods such as PET, GRPR targeting PET probes can be used for detection and monitoring of PCa in a noninvasive and specific manner. Bombesin (BBN) peptide, an amphibious analog of mammalian gastrin-releasing peptide (GRP), exhibits nanomolar affinity and high specificity towards GRPR.20-21 Since natural BBN exhibits a limited in vivo metabolic stability,22 several agonistic and antagonistic BBN analogs have been developed and radiolabeled with a variety of radionuclides for GRPR targeted imaging and treatment of PCa in small animal models and patients over the last several years.23-27 Among these BBN analogs, D-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH2 (JMV594) has shown great potential for further development of GRPR targeted agents.28 Herein, we report a novel GRPR targeting JMV594-based PET probe with high clinical translational ability. To optimize the in vivo properties of the JMV594, we introduced an 8-amino octanoic acid (AOC) spacer to the N-terminal part of JMV594 (named as SCH1). Then SCH1 conjugated with the metal chelator NODAGA-NHS ester. The resulting compound, NODAGA-SCH1, was radiolabeled with
68
Ga (Figure1) and evaluated in small
animal models for PET imaging of PCa. For comparison, 68Ga-NODAGA-JMV594, without the AOC spacer, was also prepared and evaluated. Our results demonstrated excellent in vivo performance of the
68
Ga-NODAGA-SCH1 probe which is thus a promising candidate for
clinical translation into PET imaging of PCa patients. 4
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Bioconjugate Chemistry
Figure 1. The schematic structure of 68Ga-NODAGA-SCH1 and 68Ga-NODAGA-JMV594 RESULTS Chemistry, Radiochemistry and Serum Stability The Aoc-D-Phe-Gln-Trp-Ala-Val-Gly-His-Sat-Leu-NH2 (abbreviated as SCH1) and D-Phe-Gln-Trp-Ala-Val-Gly-His-Sat-Leu-NH2 (abbreviated as JMV594) peptides were synthesized on Tentagel S RAM resin using traditional Fmoc solid-phase peptide chemistry (SPPS) and subsequently purified by reversed-phase high-performance liquid chromatography (RP-HPLC) and the desired products, SCH1 and JMV594, were characterized by ESI-MS (Figure S1 and Figure S2). The retention time (Rt) of the purified SCH1 and JMV594 was 19.7 min and 17.2 min, respectively. These results suggested that the incorporation of the AOC motif increased the hydrophobicity of the original JMV594 peptide. The purified peptides were then conjugated with commercially available chelating agent NODAGA-NHS and purified by RP-HPLC to obtain NODAGA-SCH1 (2h, yield 80%, purity 95%, MS Calcd. for C78H119N18O19+ ([M+H]+): 1611.9, found: MALDI-TOF-MS: m/z 1611.9, Figure S3 and S5) and NODAGA-JMV594 (4h, yield 55%, purity 98%, MS Calcd. for C70H103N17O18+ 5
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([M+H]+): 1470.8, found: MALDI-TOF-MS: m/z 1470.8, Figure S4 and S5) respectively. The higher conjugation yield for NODAGA-SCH1 is likely attributed to the reduced steric hindrance due to the insertion of the long hydrocarbon amino group of the AOC spacer.
Figure 2. HPLC Radio-chromatogram for a)
purified
68
GaNODAGA-SCH1
in
68
GaNODAGA-JMV594; d)
after 68
2
h
incubation
68
mouse
GaNODAGA-SCH1; b) serum;
c)
purified
GaNODAGA-JMV594 after 2 h incubation in mouse serum.
QC analysis of the radiolabeled peptides was carried out on the radio-HPLC (Dionex P680) and C18 Column in the radio-chemistry lab. NODAGA-SCH1 and NODAGA-JMV594 were then efficiently labeled with
68
Ga [2
mCi] under mild conditions (NaOAc buffer, pH=4.5) within 15 min and were purified by radio-HPLC resulting in greater than 90% purity (Figure 2a and Figure 2c). The specific 6
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activities of
68
Ga-NODAGA-SCH1 and
68
Ga-NODAGA-JMV594 were determined to be
~30GBq/µmol. The in vitro stability of the PET probes was evaluated by incubation with mouse serum (1 mL) at 37 oC. Both PET probes displayed high in vitro stability and showed no degradation products or release of
68
Ga after 2 h of incubation in mouse serum at 37°C
(Figure 2b and 2d). Overall, two PET probes were easily and reliably produced and their high in vitro stability warrants further investigation as novel PET probes for imaging of PCa. Octanol/Water Partition Coefficient To establish lipophilicity of the
68
Ga-NODAGA-SCH1 and
68
Ga-NODAGA-JMV594, the
octanol/water partition coefficients were determined. The log Poctanol/water values for the 68
Ga-NODAGA-SCH1 and 68Ga-NODAGA-JMV594 were -1.24 ± 0.04 and -2.26 ± 0.03,
respectively. Cell-Binding Affinity Assays The
receptor-binding
assay
results
for
SCH1,
NODAGA-SCH1,
JMV594
and
NODAGA-JMV594 were shown in Figure 3a. All of these peptides inhibited the GRPR-binding of
125
I-[Tyr4]BBN on PC-3 cells in a concentration-dependent manner. The
IC50 values for SCH1, NODAGA-SCH1, JMV594 and NODAGA-JMV594 were 0.12 ± 0.03, 1.30 ± 0.35, 0.32 ± 0.08 and 3.50 ± 0.85 nM (n = 4) respectively. The affinity binding results suggested that the incorporation of the metal chelator NODAGA directly into JMV594 can dramatically reduce the binding affinity of peptides (3.50 vs. 0.32 nM), whereas adding the AOC spacer can improve the GRPR-binding affinity of SCH1 over JMV594 (0.12 vs. 0.32 nM). Further modification of SCH1 with NODAGA still maintains high binding affinity (1.30 nM) which is higher than that of NODAGA-JMV594 (3.50 nM). 7
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Figure 3. a) IC50 values of SCH1, JMV594, NODAGA-SCH1 and NODAGA-JMV594 in human 68
prostate
PC-3
Cells;
b)
Uptake
of
68
Ga-NODAGA-SCH1
and
Ga-NODAGA-JMV594 with or without blocking agent (JMV594, 1µM) in PC-3 cells for
0.5 h, 1h and 2h incubation. All results, expressed as percentage of applied radioactivity, are mean of triplicate measurements ± SD Cell Uptake For cell uptake studies,
68
Ga-NODAGA-SCH1 exhibited greater (p
