Targeting the Neuropeptide Y1 Receptor for Cancer Imaging by

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Targeting the neuropeptide Y1 receptor for cancer imaging by positron emission tomography using novel truncated peptides Chengcheng Zhang, Jinhe Pan, Kuo-Shyan Lin, Iulia Dude, Joseph Lau, Jutta Zeisler, Helen Merkens, Silvia Jenni, Brigitte Guérin, and Francois Benard Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.6b00464 • Publication Date (Web): 26 Sep 2016 Downloaded from http://pubs.acs.org on September 27, 2016

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Molecular Pharmaceutics

Targeting the neuropeptide Y1 receptor for cancer imaging by positron emission tomography using novel truncated peptides

Chengcheng Zhang1, Jinhe Pan1, Kuo-Shyan Lin1, 2, Iulia Dude1, Joseph Lau1, Jutta Zeisler1, Helen Merkens1, Silvia Jenni1, Brigitte Guérin3, François Bénard*1, 2

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Department of Molecular Oncology, BC Cancer Agency, Vancouver, BC, Canada

Department of Radiology, University of British Columbia, Vancouver, BC, Canada

Département de médecine nucléaire et radiobiologie, Université de Sherbrooke, Sherbrooke, Québec, Canada

AUTHOR INFORMATION *Corresponding Author Dr. François Bénard. Address: Department of Molecular Oncology, BC Cancer Agency, 675 West 10th Avenue, Rm 4-113, Vancouver, BC V5Z 1L3, Canada. Phone: 604-675-8206. Fax: 604-675-8218. E-mail: [email protected]. Notes The authors declare no competing financial interest.

ACS Paragon Plus Environment

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Table of Contents Graphic


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ABSTRACT The neuropeptide Y1 receptor (Y1R) is overexpressed in many human cancers, particularly breast cancer. Due to stability issues, limited success has been achieved for Y1R imaging agents, including full length and truncated neuropeptide Y (NPY) analogues. The goal of this study was to evaluate the possibility of using radiolabeled truncated NPY analogues to visualize Y1R expression in a preclinical model of Y1R-positive tumor. Four truncated NPY analogues were synthesized based on the sequence of [Pro30, Tyr32, Leu34]NPY(28-36), also known as BVD15. We substituted Tyr5 and Arg6 with unnatural amino acids aiming to enhance plasma stability while maintain good receptor binding affinity to Y1R. In addition, we substituted Leu4 to Lys4 in order to conjugate via an optional linker the DOTA chelator for 68Ga labeling. Receptor binding affinity and plasma stability of these compounds were evaluated. Positron emission tomography/computed tomography (PET/CT) imaging and biodistribution studies were performed using immune-compromised mice bearing HEK293T::WT and HEK293T::hY1R tumors. [Lys(Ga-DOTA)4, Bip5]BVD15 (CCZ01035), [Lys(Ahx-Ga-DOTA)4, Bip5]BVD15 (CCZ01053), and [Lys(Pip-Ga-DOTA)4, Bip5]BVD15 (CCZ01055) demonstrated good binding affinity to Y1R (Ki = 23.4 – 32.3 nM), while [Lys(Ga-DOTA)4, Har6]BVD15 (P05067) showed poor binding affinity (Ki > 1,000 nM). In addition, CCZ01055 exhibited low binding affinity (Ki > 1,000 nM) to Y2R and Y4R, demonstrating its selectivity to Y1R. The former three peptides showed improved in vitro plasma stability of 7-16% remaining intact after 1 h incubation. PET/CT imaging and biodistribution studies for 68Ga-labeled CCZ01053, CCZ01035 and CCZ01055 showed that radioactivity was mainly cleared by the renal pathway, and HEK293T::hY1R tumors were clearly visualized with minimal background activity with the latter two. Of these two tracers, [68Ga]CCZ01055 provided lower kidney accumulation and


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higher contrast, i.e. average uptake ratios of Y1R tumor to wild type tumor, blood and muscle are 3.87 ± 0.83, 4.12 ± 1.14 and 17.6 ± 4.64, respectively. Furthermore, Y1R tumor uptake with [68Ga]CCZ01055 was significantly reduced with co-injection of 100 µg peptide YY, confirming the specificity of tumor accumulation was receptor mediated. We successfully developed the first Y1R-targeting truncated NPY analogues for PET imaging in a preclinical model, and [68Ga]CCZ01055 is a critical template for designing improved imaging agents to detect Y1R expressing cancers.

Keywords: Neuropeptide Y1R; PET; Cancer imaging; Breast cancer; Ga-68.


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INTRODUCTION Neuropeptide Y (NPY) is a 36 amino acid neurotransmitter that is involved in various human physiological functions, e.g. vasoconstriction, food uptake, anxiety and memory retention.1, 2 These processes are regulated by G-protein coupled receptors NPY Y1, Y2, Y4 and Y5 in humans, and additional NPY receptors in other species, Y6 (in mice and rabbit) and Y7 (in fish and chicken).3-6 NPY Y1 receptor (Y1R) has been shown to be overexpressed in 85% primary breast carcinomas and 100% lymph node metastases.7 Although it is also expressed in various other cancer types, such as ovarian adenocarcinoma, nephroblastoma and renal cell carcinoma, Y1R has the highest density in breast cancer.8 Furthermore, a dose-dependent inhibitory effect of NPY on the growth of Y1R expressing breast cancer cells was observed, suggesting a functional role of Y1R in breast cancer.7 The high incidence and density of Y1R in breast cancer has led to several attempts in developing radiopharmaceuticals targeting Y1R. The Beck-Sickinger group synthesized [Lys(DOTA)4, Phe7, Pro34]NPY and radiolabeled with 111In, in vivo biodistribution studies showed moderate tumor uptake, providing proof of principle for designing Y1R-binding radioligands.9 Recently, a similar NPY analogue [Pra4, Phe7, Pro34]NPY was designed and radiolabeled with 18F via click chemistry-based fluoroglycosylation.10 Although tumor uptake was observed by positron emission tomography (PET) images and biodistribution studies, it was not blocked by excess amount of the non-radioactive counterpart or BIBP3226, a potent Y1R ligand. Meanwhile, truncated NPY analogues as potent Y1R ligands have attracted increasing attention due to simpler chemical synthesis and ease of introducing modifications to improve the metabolic stability of the peptide. NPY(27-36) with substitutions at Tyr32 and Leu34 was first described by the Daniels group, which possessed antagonistic activity at both Y1R and Y2R.11


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The same study also proposed [Pro30, Tyr32, Leu34]NPY(28-36), also known as BVD15 or BW1911U90, and showed high binding affinity to Y1R.11 Later studies revealed that BVD15 also binds to Y4R with high affinity.12, 13 More recently, our group showed that amino acid substitution at position 4 of BVD15 was tolerable, and [Lys4]BVD15 showed good binding affinity at 7 ± 3 nM.14 The lysine substitution allowed the conjugation of a DOTA chelator, the resulting [Lys(DOTA)4]BVD15 retained moderate binding affinity to Y1R at 63 ± 25 nM, and the binding at Y2R and Y4R were abolished.14 Our group also attempted to label an azidecontaining analogue [Lys(CO-CH2-N3)4]BVD15 with an alkyne-bearing prosthetic group [18F]FPy5yne. Although good binding affinity was obtained, minimal tumor uptake was observed by in vivo biodistribution studies.15 This might be caused by stability issues as [Lys4]BVD15 was shown to have a very short half-life (< 0.5 min) in human plasma.16 In this study, we aimed to design novel truncated NPY analogues based on the [Lys4]BVD15 sequence to improve both stability and binding affinity to Y1R. We substituted Arg6 with an unnatural amino acid homoarginine (Har), and conjugated DOTA to Lys4 to obtain [Lys(GaDOTA)4, Har6]BVD15 (P05067). Similarly, we substituted Tyr5 with an unnatural amino acid 4,4'-biphenylalanine (Bip) to obtain [Lys(Ga-DOTA)4, Bip5]BVD15 (CCZ01035). Furthermore, we synthesized [Lys(Ahx-Ga-DOTA)4, Bip5]BVD15 (CCZ01053) and [Lys(Pip-Ga-DOTA)4, Bip5]BVD15 (CCZ01055). We radiolabeled the BVD15 analogues that showed high binding affinity with 68Ga and evaluated their potential for imaging Y1R with PET.


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EXPERIMENTAL SECTION Peptide synthesis Peptide synthesis was performed using the solid phase approach on an Endeavor 90 peptide synthesizer (Aapptec). P05067 and CCZ01035 were synthesized using standard Fmoc chemistry. Starting from Fmoc-Tyr(tBu)-Rink-Amide-MBHA resin, the Nα-Fmoc protecting group was removed by treating the resin with 20% piperidine in DMF. Subsequently, Fmoc-protected amino acids (3 equivalents), Fmoc-Arg(Pbf)-OH, Fmoc-Leu-OH, Fmoc-Arg(Pbf)-OH (CCZ01035) or Fmoc-Har(Pbf)-OH (P05067), Fmoc-Bip-OH (CCZ01035) or Fmoc-Tyr(tBu)OH (P05067), Fmoc-Lys(Mtt)-OH, Fmoc-Pro-OH, Fmoc-Asn(Trt)-OH and Boc-Ile-OH were coupled to the sequence in presence of standard in situ activating reagent HBTU (3 equivalents) and HOBt (3 equivalents) with DIEA (6 equivalents) in DMF. The Mtt protection group was selectively removed by incubating with 1.8% trifluoroacetic acid (TFA), and the chelator DOTA tri-t-butyl ester was coupled to the amino side chain of Lys4 in presence of Nhydroxysuccinimide (NHS, 3.6 equivalents) and N,N'-dicyclohexylcarbodiimide (DCC, 3 equivalents). For CCZ01053 and CCZ01055, before DOTA conjugation, Fmoc-Ahx-OH and Fmoc-Pip-OH, was coupled to the amino side chain of Lys4, respectively. The peptides were deprotected and simultaneously cleaved from the resin by incubating with 92.5/2.5/2.5/2.5 TFA/Phenol/H2O/TIS for 4 h at room temperature. The solution was filtered and the peptide was precipitated in 10-time volume of diethyl ether, and purified by HPLC (Agilent) on a semipreparative column (Phenomenex C18, 5 µ, 250 × 10 mm) using 23% acetonitrile containing 0.1% TFA at a flow rate of 4.5 mL/min. The HPLC eluates containing the desired peptide were collected and lyophilized. Mass analyses of peptides were performed on a 5600 mass spectrometer (AB/Sciex).


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For gallium complexation, a solution of DOTA-conjugated peptides and GaCl3 (5 equivalents) in sodium acetate buffer (0.1 M, pH 4.0) was incubated at 80 °C for 15 min. The mixture was purified by HPLC on a semi-preparative column using 23% acetonitrile containing 0.1% TFA at a flow rate of 4.5 mL/min.

Radiochemistry 68

Ga was obtained from a

68

Ge/68Ga generator (iThemba Labs), and was purified according to

previously published procedures using DGA resin column.17 The purified

68

Ga solution was

added to a 4-mL glass vial preloaded with HEPES buffer (2 M, pH 5.0) and 50 µg peptides. The radiolabeling reaction was carried out under microwave heating for 1 min. The reaction mixture was purified by HPLC using a semi-preparative column eluted with 24% acetonitrile containing 0.1% TFA at a flow rate of 4.5 mL/min. Radiochemical purity of > 99% was achieved for the labeled peptides as determined by radio HPLC. The specific activity was measured using an analytical HPLC system (Phenomenex C18, 5 µ, 250 × 4.6 mm). It was calculated by dividing the injected radioactivity (~1 mCi) in the final product solution by the mass in the injected solution. The mass of injected product was estimated by comparing the UV absorbance obtained from the injection with a previously prepared standard curve.

In vitro plasma stability In vitro radiotracer stability was assessed according to previously published procedures in balb/c mouse plasma (Innovative Research) for 5, 15, 30, and 60 min at 37°C.18

LogD7.4 measurements


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LogD7.4 values of

68

Ga-labeled peptides were measured using the shake flask method as

previously reported.18

Creation of stable Y1R and Y4R expression cell lines The pCEP-EGFP-sp-Y1 cloning vector, which was generously provided by Dr. Bernard Bucher,19 was linearized with AvrII , treated with Calf intestinal alkaline phosphatase (CIP) to prevent re-circularization and transfected into human embryonic kidney cells HEK293T (Clontech Laboratories) using the TransIT 2020 Transfection Reagent (Mirus Bio). The HEK293T cell line was validated by CellCheck9 test (IDEXX BioResearch). The gene for GFPY1R fusion protein was constitutively expressed under an hCMV promoter. An antibiotic hygromycin marker was present in the expression vector to allow for selection of transfected cells. The presence of GFP and Y1R expression in vitro was confirmed by flow cytometry using the 488 nm channel and an APC-conjugated Y1R antibody (R&D Systems), respectively. The presence of Y1R expression in vivo was confirmed by autoradiography. The Y4R gene was sub-cloned into the pLenti-CMV-GFP-2A-Puro vector (ABM). A GFP marker and a puromycin resistance marker were expressed independently, and the Y4R gene was expressed under an hCMV promoter. The vector was linearized with AclI, CIP-treated and transfected into HEK293T cells using the TransIT-293 Transfection Reagent (Mirus Bio). Selection for transfected cells was performed through puromycin resistance. The presence of GFP and Y4R expression in vitro was confirmed by flow cytometry using the 488 nm channel and a Y4R primary antibody (abcam) with an APC-conjugated secondary antibody (Life technologies), respectively.


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Receptor binding assays In vitro competitive binding assays were performed using HEK293T::hY1R, SK-N-BE2 (for hY2R), and HEK293T::hY4R cells. 100,000 cells were seeded onto a 24 well poly-D-lysine coated plate (Corning) overnight. Growth media was removed, and reaction buffer containing 4.8 mg/mL HEPES, 1,000 µg/mL penicillin/streptavidin and 2 mg/mL BSA was added, and allowed to incubate with cells at 37°C for at least an hour. 125I-human Peptide YY (125I-hPYY, Perkin Elmer) for hY1R and hY2R binding assays or 125I-human Pancreatic Polypeptide (125I-hPP, Perkin Elmer) for hY4R binding assays were added to each well with non-radioactive Gacoupled peptides. The final concentration of 125I-hPYY and 125I-hPP in each sample was 17.3 and 50 pM, respectively. The binding of 125I-hPYY or 125I-hPP was competed by ligands of interest at increasing concentrations from 5 pM to 50 µM in three replicates. The reaction mixture was incubated at 37°C with moderate shaking for 45 min. After the incubation, the reaction mixture was removed, and cells were washed with ice-cold PBS three times. 0.25% Trypsin solution was used to harvest the cells, which were counted for radioactivity using a Wallac WIZARD2 gamma counter (Perkin Elmer). The binding assays were performed in three replicates for each compound.

Biodistribution studies Animal experiments were approved by the University of British Columbia Animal Care Committee (Vancouver, BC, Canada). Male immunodeficient NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice were obtained from an in-house breeding colony at the Animal Resource Centre of the BC Cancer Agency Research Centre. HEK293T::WT (wild type) and HEK293T::hY1R cells (2 million) were subcutaneously inoculated on left and right dorsal flank of the mice,


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respectively. After growth period ranging from 14 to 18 days, palpable tumors measuring approximately 7-10 mm in diameter were obtained. Mice were anesthetized by 2% isoflurane inhalation, and injected with 1-2 MBq of 68Ga-labeled peptides. For blocking studies, 100 µg of human PYY (Polypeptide) was co-injected with the radioactive compound. After the injection, mice were allowed to roam freely in cages for 60 min, anesthetized by 2% isoflurane inhalation, and then euthanized by CO2 inhalation. Blood was promptly withdrawn, the organs of interest were harvested, rinsed with PBS, blotted dry, and the weights were measured. The radioactivity of the collected mouse tissues was counted using a Wallac WIZARD2 gamma counter (Perkin Elmer), normalized to the injected dose using a standard curve and expressed as the percentage of the injected dose per gram of tissue (%ID/g).

Preclinical PET/CT imaging PET/CT imaging experiments were conducted using an Inveon microPET/CT scanner (Siemens) following the same procedures as previously reported.17 Mice bearing HEK293T::WT and HEK293T::hY1R tumors, as described above, were used for the experiments. Dynamic PET scans were performed to determine the time activity curve of the radiopharmaceuticals in organs of interest. A baseline CT scan was obtained for localization and attenuation correction, and then a 60-min PET dynamic acquisition was started at the time of intravenous injection with 4-6 MBq of 68Ga-labeled peptide. For static PET imaging, 10-min PET scans were acquired following baseline CT scans. For blocking studies, 100 µg of PYY was co-injected with the radioactive compound. The mice were euthanized after static PET imaging and the organs harvested for biodistribution.


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Statistical analysis Data analysis was performed using GraphPad Prism (6.0h). Two-way ANOVA analysis was performed for all organs in biodistribution studies and the uptake ratios, and multiple comparisons were corrected using the Bonferroni method. The difference was considered statistically significant when P value was < 0.05.


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RESULTS Chemical properties and in vitro binding affinity All four BVD15 analogues, P05067, CCZ01035, CCZ01053 and CCZ01055, were prepared with high purity (> 98%), and their molecular weights were verified by mass spectrometry (Table 1). The chemical structures of the tracers are shown in Figure 1. The inhibition constant (Ki) of these BVD15 analogues to Y1R was measured using competitive binding assays with 125I-hPYY using HEK293T::hY1R cells (Table 1, Fig. 2A). Binding affinity of P05067 was very low at micromolar level, therefore this compound was not further characterized in this study. [Lys4, Bip5]BVD15 showed high affinity to Y1R at 3.4 ± 1.5 nM. The Ga-DOTA-conjugated peptide, CCZ01035, showed good affinity at 25.0 ± 5.3 nM. Also, adding an Ahx (CCZ01053) or a Pip (CCZ01055) linker between the Ga-DOTA and Lys4 did not affect binding affinity. The binding affinity of CCZ01055 to Y2R and Y4R was also characterized with 125I-hPYY using SK-N-BE2 cells and with 125I-hPP using HEK293T::hY4R cells, respectively (Fig. 2B-C). Micromolar binding affinities (Ki > 1,000 nM) were observed for both NPY receptor subtypes. In addition, no specific binding was observed for HEK293T::WT cells using either 125I-hPYY or 125I-hPP.

Radiochemical characteristics, plasma stability and internalization The radiochemical data are summarized in Table 2. The 68Ga-labeled CCZ01035, CCZ01053 and CCZ01055 were prepared with > 99% radiochemical purity in average decay-corrected radiochemical yields ranging from 42 – 69%. Their average specific activity were > 92.5 GBq / µmol (n = 3) at the end of synthesis. In addition, all three 68Ga-labeled tracers were fairly hydrophilic with low LogD7.4 values ranging from -1.62 to -2.29. After 1 h incubation in mouse plasma in vitro, 7%, 16% and 14% of [68Ga]CCZ01035, [68Ga]CCZ01053 and [68Ga]CCZ01055


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remained intact, respectively (Table 2). Also, internalization study showed that [Ga68]CCZ01055 remained mostly membrane-bound on HEK293T::hY1R cells after 90 min incubation with only a small fraction of the peptide being internalized (Supplementary Fig. 1).

In vitro and in vivo cellular expression of Y1R Successful in vitro Y1R transfection was confirmed using flow cytometry, where HEK293T::hY1R showed high expression level of GFP and Y1R (Supplementary Fig. 2). To further verify Y1R expression in vivo, autoradiography was performed on HEK293T::hY1R tumor sections with 125I-hPYY (Supplementary Fig. 3). Strong intensity was observed, and the signal decreased to baseline level by increasing concentration of a Y1R selective ligand, BIBO 3304. This confirms that the binding observed by autoradiography represented Y1R expression.

PET/CT imaging and biodistribution For all three tracers at 1 h p.i., radioactivity was cleared primarily through the renal pathway, with accumulation in the bladder and kidneys (Fig. 3). Moderate activity was observed in the liver, and minimal background activity for other normal tissues. The 1-h biodistribution data for these tracers are summarized in Supplementary Table 1. For CCZ01053, limited Y1R tumor uptake was observed. The biodistribution results showed that CCZ01055 generated similar Y1R tumor uptake at 0.98 ± 0.21%ID/g, compared to CCZ01035 at 1.14 ± 0.26 %ID/g. For both CCZ01035 and CCZ01055, Y1R tumors were clearly visualized on the PET images (Fig. 4). The average uptake ratios of Y1R tumor to WT tumor, blood and muscle for CCZ01035 at 1 h p.i. were 2.67 ± 0.85, 2.46 ± 0.85 and 9.75 ± 5.96, respectively. For CCZ01055 at 1 h p.i., these ratios were 3.87 ± 0.83, 4.12 ± 1.14 and 17.6 ± 4.64, respectively. Despite similar tumor uptakes,


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CCZ01055 provided higher contrast than CCZ01035. Moreover, much lower kidney uptakes were obtained for CCZ01055 at 2.53 ± 0.36 %ID/g, compared to 45.5 ± 10.4 %ID/g for CCZ01035. In addition, time activity curves for all three radiotracers were generated from dynamic PET images, and showed progressive clearance from blood and muscle, and Y1R tumor uptake also reduced over the 1 h period (Fig. 5). Blocking experiments were performed for CCZ01055 at 1 h p.i. with 100 µg PYY co-injection, and tumor uptake was significantly reduced (Fig. 4, Supplementary Table 1, p < 0.01).


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DISCUSSION In this study, we developed and evaluated successful truncated NPY analogues for Y1R-targeted cancer imaging with PET. Due to the reported high incidence of expression and high receptor density in breast cancer cells, and low expression level in normal tissue, Y1R is an attractive target for breast cancer detection. A small molecule-based agent, [18F]Y1-973, was developed targeting Y1R in monkey brain.20 The ability of this lipophilic compound to target Y1R expression in tumors and peripheral tissues is unknown. Peptides are generally hydrophilic, and it is often easier to modify their pharmacokinetic properties to optimize tumor visualization. Full length NPY analogue labeled with 99mTc targeting Y1R was applied to breast cancer patients using Single-Photon Emission Computed Tomography (SPECT).21 Although SPECT is more widely available, PET offers superior sensitivity and specificity, particularly when coupled with CT,22 which might translate into more accurate diagnosis. Furthermore, full-length NPY analogues are more challenging to produce, and offer a larger number of potential peptidase degradation sites. Despite high binding affinity, the tumor-to-blood ratios and imaging contrast obtained with a glycosylated 18F-labeled full-length NPY analogue were suboptimal.10 Truncated NPY analogues suffer from low plasma stability, and are rapidly cleared from circulation before reaching targeted tumor sites. The Beck-Sickinger group reported high human plasma stability for BVD15 analogues with unnatural amino acid substitutions at position 4 (Nle) and/or 5 (Bpa or Nal), and identified that enzymatic cleavage of these analogues occurred at Nterminus of positions 6 and 8.23 The same group also reported that substitution at position 5 with Bip showed similar response, and improved photo-instability of the Bpa residue.24 Therefore, we designed our tracers with Bip5 for increased stability. We have observed much improved in vitro mouse plasma stability of 7-16% remaining intact after 1 h incubation, compared to the short


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halflife (< 0.5 min) of [Lys4]BVD15 in human plasma. We also attempted replacing Arg6 with an unnatural amino acid Har, to decrease N-terminal cleavage of this position. However, the binding affinity of the resulting [Lys(Ga-DOTA)4, Har6]BVD15 (P05067) to Y1R was poor, suggesting substitution at position 6 of BVD15 was not well tolerated. BVD15 with Ga-DOTA-conjugated Lys4 and Bip5 (CCZ01035) showed good binding affinity to Y1R at 25.0 ± 5.3 nM, which was an improvement compared to the [Lys(DOTA)4]BVD15 at 63 ± 25 nM. We aimed to further increase the binding affinity by introducing a linker between Lys4 and Ga-DOTA, since we observed 4-fold increase of binding affinity when adding an Ahx linker on [Lys(NOTA)4]BVD15.25 Similarly, we also observed around 8-fold increase in binding affinity when replacing an Ahx to a Pip linker in bradykinin receptor-targeting tracers.26 However, adding an Ahx or a Pip linker between the lysine and Ga-DOTA in CCZ01035 did not improve the binding affinity. The binding affinity of the best candidate, CCZ01055, to Y2R and Y4R was further characterized, which demonstrated the selective binding of CCZ01055 to Y1R. This is consistent with our previous observation that [Lys(DOTA)4]BVD15 exhibited selective binding to Y1R.14 Also, the Bip5 substitution did not disrupt the selectivity of the peptide. Furthermore, BVD15 has been shown to be a Y1R antagonist.12 The internalization study revealed that [68Ga]CCZ01055 remained an antagonist, since it did not internalize into HEK293T::hY1R cells. Due to low binding affinities, internalization assays were not performed for CCZ01055 using the Y2R and the Y4R-expressing cell lines. High contrast PET/CT images and excellent tumor visualization were obtained for both CCZ01035 and CCZ01055, despite moderate absolute tumor uptake values. Higher values were achieved with similar small peptide based radiotracers;17, 27 therefore, it might be possible to improve tumor visualization by further optimizing binding affinity, stability, hydrophobicity


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and/or charge state of these peptides. Between these two radiotracers, although the absolute Y1R tumor uptake values were similar, PET images with [68Ga]CCZ01055 showed higher tumor to blood, tumor to muscle ratios and lower kidney activity retention. The extra positive charge the Pip linker provided might contribute to faster clearance of the radiolabeled peptide from circulation. The mechanism of renal retention is still unclear, but the number of charges on peptides plays an important role.28 Furthermore, PET images showed that co-injection of a known Y1R ligand, PYY, largely reduced the tumor uptake, suggesting the [68Ga]CCZ01035 and [68Ga]CCZ01055 selectively bound Y1R in vivo. This was further confirmed by biodistribution studies for [68Ga]CCZ01055, in that the Y1R-positive tumors showed statistically significant reduction, approximately 30%, in uptake values when PYY was co-injected. The moderate reduction might be due to the low plasma stability of PYY.29 Overall, the normal tissue uptake was low, and only the liver and spleen showed a statistically significant reduction with PYY coinjections. This could be due to the higher total injected mass of peptides, which could saturate peptidases or peptide transporters and reduce nonspecific accumulation of [68Ga]CCZ01055 or degradation fragments from the liver and spleen. As there is no data to suggest expression of Y1R in mouse liver and spleen, further studies will be needed to explore the significance of this observation. We used a transfected tumor model to evaluate biochemical characteristics of the radioligand in vitro and in vivo. Overexpression of Y1R on HEK293T::hY1R cells were confirmed using flow cytometry as well as autoradiography. This model allowed a direct comparison between a tumor overexpressing the target receptor and the same tumor that does not express the receptor endogenously in the same animal. Such models have been used widely in preclinical settings for radioligands, and we have also successfully employed this system to investigate bradykinin


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receptor imaging with PET.17 However, since the transfected tumor model might not reflect the actual receptor density in the clinical setting, further verifications using established Y1R expressing cell lines would be needed. In conclusion, we demonstrated that Y1R imaging with the truncated NPY analogues was possible. Also, [68Ga]CCZ01055 selectively bound Y1R in vivo and produced high contrast PET images of HEK293T::hY1R tumor xenografts in mice. Our results suggest that [68Ga]CCZ01055 can be used as a template for designing NPY analogues with higher stability and affinity for PET imaging of Y1R overexpressed tumors.


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ACKNOWLEDGEMENT This work was supported by the Canadian Institutes of Health Research grant MOP-119361 and the BC Leading Edge Endowment Fund. We would like to thank Navjit Hundal-Jabal and Nadine Colpo for technical support.


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Supporting Information. Methods and figures for flow cytometry, autoradiography and internalization assays, as well as a table for biodistribution results are provided in Supporting Information. This information is available free of charge via the Internet at http://pubs.acs.org/.


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16. Liu, M.; Mountford, S. J.; Zhang, L.; Lee, I.-C.; Herzog, H.; Thompson, P. E. Synthesis of BVD15 peptide analogues as models for radioligands in tumour imaging. Int. J. Pept. Res. Ther. 2013, 19, (1), 33-41. 17. Lin, K.-S.; Pan, J.; Amouroux, G.; Turashvili, G.; Mesak, F.; Hundal-Jabal, N.; Pourghiasian, M.; Lau, J.; Jenni, S.; Aparicio, S. In vivo radioimaging of bradykinin receptor B1, a widely overexpressed molecule in human cancer. Cancer Res. 2015, 75, (2), 387-393. 18. Lin, K.-S.; Amouroux, G.; Pan, J.; Zhang, Z.; Jenni, S.; Lau, J.; Liu, Z.; Hundal-Jabal, N.; Colpo, N.; Bénard, F. Comparative Studies of Three 68Ga-Labeled [Des-Arg10] Kallidin Derivatives for Imaging Bradykinin B1 Receptor Expression with PET. J. Nucl. Med. 2015, 56, (4), 622-627. 19. Gicquiaux, H.; Lecat, S.; Gaire, M.; Dieterlen, A.; Mély, Y.; Takeda, K.; Bucher, B.; Galzi, J.-L. Rapid internalization and recycling of the human neuropeptide Y Y(1) receptor. J. Biol. Chem. 2002, 277, (8), 6645-6655. 20. Hostetler, E. D.; Sanabria-Bohórquez, S.; Fan, H.; Zeng, Z.; Gantert, L.; Williams, M.; Miller, P.; O'Malley, S.; Kameda, M.; Ando, M. Synthesis, characterization, and monkey positron emission tomography (PET) studies of [18 F] Y1-973, a PET tracer for the neuropeptide Y Y1 receptor. Neuroimage 2011, 54, (4), 2635-2642. 21. Khan, I. U.; Zwanziger, D.; Böhme, I.; Javed, M.; Naseer, H.; Hyder, S. W.; Beck‐Sickinger, A. G. Breast-cancer diagnosis by neuropeptide Y analogues: from synthesis to clinical application. Angew. Chem. Int. Ed. Engl. 2010, 49, (6), 1155-1158. 22. Even-Sapir, E.; Metser, U.; Mishani, E.; Lievshitz, G.; Lerman, H.; Leibovitch, I. The detection of bone metastases in patients with high-risk prostate cancer: 99mTc-MDP Planar bone scintigraphy, single- and multi-field-of-view SPECT, 18F-fluoride PET, and 18F-fluoride PET/CT. J. Nucl. Med. 2006, 47, (2), 287-297. 23. Zwanziger, D.; Böhme, I.; Lindner, D.; Beck‐Sickinger, A. G. First selective agonist of the neuropeptide Y1-receptor with reduced size. J. Pept. Sci. 2009, 15, (12), 856-866. 24. Hofmann, S.; Frank, R.; Hey-Hawkins, E.; Beck-Sickinger, A.; Schmidt, P. Manipulating Y receptor subtype activation of short neuropeptide Y analogs by introducing carbaboranes. Neuropeptides 2013, 47, (2), 59-66. 25. Guérin, B.; Ait-Mohand, S.; Tremblay, M.-C.; Dumulon-Perreault, V.; Fournier, P.; Bénard, F. Total solid-phase synthesis of NOTA-functionalized peptides for PET imaging. Organic letters 2009, 12, (2), 280-283. 26. Amouroux, G.; Pan, J.; Jenni, S.; Zhang, C.; Zhang, Z.; Hundal-Jabal, N.; Colpo, N.; Liu, Z.; Bénard, F.; Lin, K.-S. Imaging Bradykinin B1 Receptor with 68Ga-Labeled [des-Arg10] Kallidin Derivatives: Effect of the Linker on Biodistribution and Tumor Uptake. Molecular pharmaceutics 2015, 12, (8), 2879-2888. 27. Lin, M.; Welch, M. J.; Lapi, S. E. Effects of chelator modifications on 68Ga-labeled [Tyr3] octreotide conjugates. Mol. Imaging Biol. 2013, 15, (5), 606-613. 28. Gotthardt, M.; van Eerd-Vismale, J.; Oyen, W. J.; de Jong, M.; Zhang, H.; Rolleman, E.; Maecke, H. R.; Béhé, M.; Boerman, O. Indication for different mechanisms of kidney uptake of radiolabeled peptides. Journal of Nuclear Medicine 2007, 48, (4), 596-601. 29. Addison, M. L.; Minnion, J. S.; Shillito, J. C.; Suzuki, K.; Tan, T. M.; Field, B. C.; Germain-Zito, N.; Becker-Pauly, C.; Ghatei, M. A.; Bloom, S. R. A Role for Metalloendopeptidases in the Breakdown of the Gut Hormone, PYY3–36. Endocrinology 2011, 152, (12), 4630-4640.


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Table 1. Analytical data for BVD15 analogues and affinities for NPY1R. Non-radioactive peptides were used for determination of Y1R binding affinity. Peptide P05067 [Lys ,Bip5]BVD15 CCZ01035 CCZ01053 CCZ01055 4

Mass calculated 1687.82 1280.75 1733.84 1846.92 1873.94

Mass found 1688.58 (M+2H) 1282.82 (M+2H) 1735.20 (M+2H) 1848.15 (M+2H) 1874.78 (M+1H)

Purity

Ki (nmol/L)

>99% >99% >98% >99% >99%

> 1000 3.4 ± 1.5 25.0 ± 5.3 32.3 ± 9.7 23.4 ± 9.0


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Table 2. Radiochemistry and plasma stability data for 68Ga-labeled CCZ01035, CCZ01053 and CCZ01055. Peptide

Radiochemical yield (decaycorrected)

Specific activity (GBq/µmol)

LogD7.4

[68Ga]CCZ01035 [68Ga]CCZ01053 [68Ga]CCZ01055

42 ± 21% (n=7) 69 ± 17% (n=3) 65 ± 8% (n=7)

159.1 ± 11.1 (n=3) 92.5 ± 51.8 (n=3) 159.1 ± 11.1 (n=3)

-1.62 ± 0.17 -1.93 ± 0.03 -2.29 ± 0.28


Plasma stability (%) 5 15 30 60 min min min min 73 53 27 7 89 66 40 16 72 59 39 14

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Figure 1. Chemical structures of A. P05067, B. CCZ01035, C. CCZ01053 and D. CCZ01055.


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Figure 2. Representative displacement curves of A. 125I-hPYY by [Lys4,Bip5]BVD15, CCZ01035, CCZ01053, CCZ01055 and P05067 using HEK293T::hY1R cells, B. 125I-hPYY by CCZ01055 using SKN-BE2 cells expressing hY2R, and C. 125I-hPP by CCZ01055 using HEK293T::hY4R cells. Nonradioactive peptides were used for determination of binding affinity. The binding assays were performed in three replicates for each compound.


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Figure 3. PET images of A. [68Ga]CCZ01035, B. [68Ga]CCZ01035 with co-injection of 100 µg PYY, C. [68Ga]CCZ01053, D. [68Ga]CCZ01055 and E. [68Ga]CCZ01055 with co-injection of 100 µg PYY at 1 h p.i. in mice bearing HEK293::WT (black arrow) and HEK293::hY1R tumors (grey arrow). Uptake in the Y1R tumors was significantly reduced in the presence of PYY, confirming receptor-mediated uptake. Scale bar unit is %ID/g.


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Figure 4. Biodistribution data of [68Ga]CCZ01055 showing organ or tumor uptake expressed as %ID/g at 1 h p.i. with (n=10) or without (n=8) 100 µg PYY co-injection (Two-way ANOVA analysis was performed for all organs, and multiple comparisons were corrected using the Bonferroni method; *p < 0.05; **p < 0.01; ***p < 0.001).


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Figure 5. Time activity curves of A. [68Ga]CCZ01035, B. [68Ga]CCZ01053, C. [68Ga]CCZ01055 and D. [68Ga]CCZ01055 with co-injection of 100 µg PYY, showing the average activity in regions-of-interest located around the Y1R and wild type tumors, blood (left ventricle), kidney and muscle. %ID/g is in log scale.


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Figure 1. Chemical structures of A. P05067, B. CCZ01035, C. CCZ01053 and D. CCZ01055. 109x75mm (300 x 300 DPI)



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Figure 2. Representative displacement curves of A. 125I-hPYY by [Lys4,Bip5]BVD15, CCZ01035, CCZ01053, CCZ01055 and P05067 using HEK293T::hY1R cells, B. 125I-hPYY by CCZ01055 using SK-N-BE2 cells expressing hY2R, and C. 125I-hPP by CCZ01055 using HEK293T::hY4R cells. Non-radioactive peptides were used for determination of binding affinity. The binding assays were performed in three replicates for each compound. 131x210mm (300 x 300 DPI)


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Figure 3. PET images of A. [68Ga]CCZ01035, B. [68Ga]CCZ01035 with co-injection of 100 µg PYY, C. [68Ga]CCZ01053, D. [68Ga]CCZ01055 and E. [68Ga]CCZ01055 with co-injection of 100 µg PYY at 1 h p.i. in mice bearing HEK293::WT (black arrow) and HEK293::hY1R tumors (grey arrow). Uptake in the Y1R tumors was significantly reduced in the presence of PYY, confirming receptor-mediated uptake. Scale bar unit is %ID/g. 82x114mm (300 x 300 DPI)



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Figure 4. Biodistribution data of [68Ga]CCZ01055 showing organ or tumor uptake expressed as %ID/g at 1 h p.i. with (n=10) or without (n=8) 100 µg PYY co-injection (Two-way ANOVA analysis was performed for all organs, and multiple comparisons were corrected using the Bonferroni method; *p < 0.05; **p < 0.01; ***p < 0.001). 109x68mm (300 x 300 DPI)


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Figure 5. Time activity curves of A. [68Ga]CCZ01035, B. [68Ga]CCZ01053, C. [68Ga]CCZ01055 and D. [68Ga]CCZ01055 with co-injection of 100 µg PYY, showing the average activity in regions-of-interest located around the Y1R and wild type tumors, blood (left ventricle), kidney and muscle. %ID/g is in log scale. 112x72mm (300 x 300 DPI)



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Table of contents graphic 35x15mm (300 x 300 DPI)


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Targeting the Neuropeptide Y1 Receptor for Cancer Imaging by

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