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Radiolabeled R954 derivatives for imaging bradykinin B1 receptor expression with positron emission tomography Hsiou-Ting Kuo, Jinhe Pan, Joseph Lau, Chengcheng Zhang, Jutta Zeisler, Nadine Colpo, Francois Benard, and Kuo-Shyan Lin Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.6b01055 • Publication Date (Web): 17 Jan 2017 Downloaded from http://pubs.acs.org on January 20, 2017

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Molecular Pharmaceutics

Radiolabeled R954 derivatives for imaging bradykinin B1 receptor expression with positron emission tomography Hsiou-Ting Kuo†, Jinhe Pan†, Joseph Lau†, Chengcheng Zhang†, Jutta Zeisler†, Nadine Colpo†, François Bénard*,†,‡,§, Kuo-Shyan Lin*,†,‡,§

†

Department of Molecular Oncology, BC Cancer Agency, Vancouver, BC V5Z 1L3, Canada Department of Functional Imaging, BC Cancer Agency, Vancouver, BC, V5Z 4E6, Canada § Department of Radiology, University of British Columbia, Vancouver, BC V5Z 4E3, Canada ‡

AUTHOR INFORMATION Corresponding Authors *Dr. François Bénard. Address: Department of Molecular Oncology, BC Cancer Agency, 675 West 10th Avenue, Rm 14-111, Vancouver, BC V5Z 1L3, Canada. Phone: 604-675-8206. Fax: 604-675-8218. E-mail: [email protected] *Dr. Kuo-Shyan Lin. Address: Department of Molecular Oncology, BC Cancer Agency, 675 West 10th Avenue, Rm 4-123, Vancouver, BC V5Z 1L3, Canada. Phone: 604-675-8208. Fax: 604-675-8218. E-mail: [email protected]

Notes The authors declare no competing financial interest.

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ABSTRACT GRAPHIC


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ABSTRACT Peptide receptors have emerged as promising targets for diagnosis and therapy. The aberrant overexpression of these receptors in different cancer subtypes allows for the adoption of new treatment strategies that complement conventional chemotherapies. Bradykinin B1 receptor (B1R) is a G protein-coupled receptor that is overexpressed in many cancers, with limited expression in healthy tissues. Previously, we developed 68Ga- and 18F-labeled derivatives of B1R antagonist peptides B9858 and B9958, and successfully targeted B1R-expressing tumor xenografts in vivo. R954 (Ac-Orn-Arg-Oic-Pro-Gly-αMePhe-Ser-D-2-Nal-Ile), a potent B1R antagonist, is reportedly more stable than B9858 against peptidase degradation. We evaluated two radiolabeled derivatives of R954 (68Ga-HTK01083 and 18F-HTK01146) for B1R PET imaging. Peptides were assembled on were synthesized via solid phase strategy. Non-radioactive standards were obtain by reacting GaCl3 with DOTA-dPEG2-R954, and by clicking Npropargyl-N,N-dimethylammoniomethyl-trifluoroborate with azidoacetyl-dPEG2-R954. Binding affinity for B1R was determined by an in vitro competition binding assay. 68Ga-HTK01083 was obtained by incubating DOTA-dPEG2-R954 with 68GaCl3 under acidic conditions, while 18FHTK01146 was prepared via an 18F-19F isotope exchange reaction. Biodistribution and imaging studies were conducted at 1 h post-injection (p.i.) in mice inoculated with B1R-expressing (B1R+) and B1R-nonexpressing (B1R-) cells. HTK01083 and HTK01146 bound B1R with good affinity (Ki = 30.5 and 24.8 nM, respectively). 68Ga/18F-labeled R954 were obtained on average in ≥ 10% decay-corrected radiochemical yield with > 99% radiochemical purity and ≥ 52 GBq/µmol specific activity. For both tracers, clearance was predominantly renal with minimal involvement of the hepatobiliary system. For PET images, B1R+ tumors, kidneys and bladder were visible. At 1 h p.i., uptake in B1R+ tumor was comparable between 68Ga-HTK01083 (8.46 ± 1.44 %ID/g) and 18F-HTK01146 (9.25 ± 0.69 %ID/g). B1R+ tumor-to-blood and B1R+ tumorto-muscle ratios were 6.32 ± 1.44 and 20.7 ± 3.58 for 68Ga-HTK01083, and 7.24 ± 2.56 and 19.5 ± 4.29 for 18F-HTK01146. Our results indicate R954 is a good lead sequence for optimization of B1R tracers for cancer imaging.

KEY WORDS Bradykinin B1 receptor; R954; Fluorine-18; Gallium-68; Positron emission tomography



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INTRODUCTION Bradykinin B1 and B2 receptors (B1R and B2R) belong to the G protein-coupled receptor (GPCR) family, and mediate different biological processes including cardiovascular permeability, inflammatory response, and nociception1-3. Unlike B2R which is found throughout the body, B1R expression is limited and induced by pathophysiological conditions3. In the last decade, there is growing evidence implicating the B1R system in cancer progression. Activation of B1R can lead to cell proliferation, migration, and angiogenesis – selected hallmarks of malignant behavior3. The pro-inflammatory effect of B1R is also suggested to help establish metastatic microenvironments for aggressive tumors4, 5. Notably, the overexpression of B1R has been observed in prostate, breast, renal, esophageal, cervical, gastric, and lung cancers6-12. As B1R is pathologically expressed by cancers and readily accessible, it has been proposed as a potential theranostic target3. Since 2012, our research group has been developing radiopharmaceuticals targeting B1R for positron emission tomography (PET) imaging13-18. PET imaging is routinely used in the clinic for diagnosis and staging, treatment planning and monitoring. Novel B1R-targeted PET tracers can be used for patient stratification and to facilitate development of B1R-targeted therapies. As a means for quantifying B1R expression, PET can be used to identify patients that are likely to respond to treatment. Promising agents can potentially be repurposed for therapy by substituting imaging isotopes with therapeutic isotopes. Initial imaging effort targeting B1R with [Leu9,desArg10]kallidin, a close analog of the endogenous peptide [des-Arg10]kallidin, was unsuccessful due to the metabolic lability of [des-Arg10]kallidin13. Subsequently, we selected two antagonist peptide sequences, B9858 (Lys-Lys-Arg-Pro-Hyp-Gly-Igl-Ser-D-Igl-Oic) and B9958 (Lys-LysArg-Pro-Hyp-Gly-Cpg-Ser-D-Tic-Cpg), each containing four unnatural amino acids as targeting vectors15-17. 68Ga and 18F-labeled derivatives of B9858 and B9958 successfully targeted B1Rexpressing tumor xenografts to generate high contrast images in preclinical studies15-17. Neugebauer et al. reported the synthesis of R954 (Ac-Orn-Arg-Oic-Pro-Gly-αMePhe-Ser-D-2Nal-Ile)19, a potent B1R antagonist with four unnatural amino acids as well. Reportedly, R954 is more stable against peptidase degradation than B985819. Herein, we report the synthesis and evaluation of two novel R954 derivatives for B1R-targeted PET imaging (Figure 1).


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Figure 1. Chemical structures of R954 and its derivatives. The radiolabeled complexes (GaDOTA and AmBF3-Mta) in blue were coupled to N-terminus of the peptide via a dPEG2 linker. The unnatural amino acids within the sequences are highlighted in red. AmBF3-Mta: 4-(Ntrifluoroborylmethyl-N,N-dimethylammonio)methyl-1,2,3-triazole-1-acetic acid; dPEG2: 9amino-4,7-dioxanonanoic acid.



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MATERIALS AND METHODS General Methods All chemicals and solvents were obtained from commercial sources, and used without further purification. [3H][Leu9,des-Arg10]kallidin was purchased from PerkinElmer (Waltham, MA). B1R-targeting peptides were synthesized using solid phase approach on an AAPPTec (Louisville, KY) Endeavor 90 peptide synthesizer. Purification and quality control of cold and radiolabeled peptides were performed on Agilent HPLC systems equipped with a model 1200 quaternary pump, a model 1200 UV absorbance detector (set at 220 nm), and a Bioscan (Washington, DC) NaI scintillation detector. The operation of Agilent HPLC systems was controlled using the Agilent ChemStation software. The HPLC columns used were a semipreparative column (C18, 5 µ, 250 × 10 mm) and an analytical column (C18, 5 µ, 250 × 4.6 mm) purchased from Phenomenex (Torrance, CA). The collected HPLC eluates containing the desired peptide were lyophilized using a Labconco (Kansas City, MO) FreeZone 4.5 Plus freeze-drier. Mass analyses were performed using an AB SCIEX (Framingham, MA) 4000 QTRAP mass spectrometer system with an ESI ion source. 18F-Fluoride Trap & Release columns were purchased from ORTG Inc. (Orkdale, TN), and C18 Sep-Pak cartridges (1 cm3, 50 mg) were obtained from Waters (Milford, MA). 68Ga was eluted from an iThemba Labs (Somerset West, South Africa) generator, and was purified according to the previously published procedures using a DGA resin column from Eichrom Technologies LLC (Lisle, IL)13. 18F-Fluoride was produced via the 18O(p,n)18F reaction using an Advanced Cyclotron Systems Inc. (Richmond, Canada) TR19 cyclotron. Radioactivity of 68Ga and 18F-labeled peptides was measured using a Capintec (Ramsey, NJ) CRC®-25R/W dose calibrator, and the radioactivity of mouse tissues collected from biodistribution studies were counted using a Perkin Elmer (Waltham, MA) Wizard2 2480 automatic gamma counter. Synthesis of DOTA-dPEG2-R954 The assembly of dPEG2-R954 sequence (dPEG2-Orn(Boc)-Arg(Pbf)-Oic-Pro-Gly-αMePheSer(tBu)-D-2-NaI-Ile) was conducted on solid phase starting from Fmoc-Ile-Wang resin. The resin was treated with 20% piperidine in N,N-dimethylformamide (1 × 5 min and 1 × 10 min) to remove the Nα-Fmoc protecting group. Side-chain protected amino acids including FmocOrn(Boc)-OH, Fmoc-Arg(Pbf)-OH, and Fmoc-Ser(tBu)-OH were used for the synthesis. The coupling was carried out in N-methyl-2-pyrrolidone using Fmoc-protected amino acid (3 equivalents), N,N,N′,N′-tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate (3 equivalents) and N,N-diisopropylethylamine (6 equivalents). The chelator DOTA tri-t-butyl ester (3 equivalents) was coupled to the N-terminus of dPEG2-R954 bound resin with N,N’diisopropylcarbodiimide (3 equivalents) and N-hydroxysuccinimide (3.6 equivalents) in the presence of N,N-diisopropylethylamine (10 equivalents). At the end, the peptide was deprotected and simultaneously cleaved from the resin by treating with 95/5 trifluoroacetic acid (TFA)/ triisopropylsilane (TIS) for 2 h at room temperature. After filtration, the peptide was precipitated by the addition of cold diethyl ether to the TFA solution. The crude peptide was purified by HPLC using the semi-preparative column eluted with 30 - 60% acetonitrile in water in 25 min with 0.1% TFA at a flow rate of 4.5 mL/min. The retention time was 10.6 min, and the yield of the peptide was 7%. ESI-MS: calculated [M+2H]2+ for DOTA-dPEG2-R954 C82H125N18O21 849.5; found [M+2H]2+ 849.6.


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Synthesis of Azidoacetyl-dPEG2-R954 Wang resin containing dPEG2-R954 sequence was prepared as described above. 2-Azidoacetic acid (5 equivalents) was coupled to the N-terminus of dPEG2-R954 sequence with N,N’diisopropylcarbodiimide (5 equivalents) and N-hydroxysuccinimide (6 equivalents). At the end, the peptide was deprotected and simultaneously cleaved from the resin by treating with 95/5 TFA/TIS for 2 h at room temperature. After filtration, the peptide was precipitated by the addition of cold diethyl ether to the TFA solution. The crude peptide was purified by HPLC using the semi-preparative column eluted with 36% acetonitrile in water with 0.1% TFA at a flow rate of 4.5 mL/min. The retention time was 16.3 min, and the yield of the peptide was 36%. ESI-MS: calculated [M+2H]2+ for azidoacetyl-dPEG2-R954 C68H99N17O15 697.9; found [M+2H]2+ 697.9. Synthesis of Ga-DOTA-dPEG2-R954 (HTK01083) A solution of DOTA-dPEG2-R954 (4.0 mg, 2.4 µmol) and GaCl3 (2.1 mg, 11.8 µmol) in 500 µL sodium acetate buffer (0.1 M, pH 4.2) was incubated at 80 °C for 15 min. The reaction mixture was purified by HPLC using the semi-preparative column eluted with 30 - 60% acetonitrile in water with 0.1% TFA in 25 min at a flow rate of 4.5 mL/min. The retention time was 10.9 min, and the yield of the peptide was 71%. ESI-MS: calculated [M+2H]2+ for HTK01083 C82H121N18O2169Ga 882.4; found [M+2H]2+ 882.3. Synthesis of AmBF3-Mta-dPEG2-R954 (HTK01146) A solution of azidoacetyl-dPEG2-R954 (10.5 mg, 7.5 µmol), N-propargyl-N,N-dimethylammoniomethyl-trifluoroborate (8.0 mg, 48.6 µmol), 1 M CuSO4 (30 µL), and 1 M sodium ascorbate (72 µL) in acetonitrile (100 µL) and 5% NH4OH (100 µL) was incubated at 45 °C oil bath for 2 h. The reaction mixture was purified by HPLC using the semi-preparative column eluted with 36% acetonitrile and 64% ammonia formate buffer (40 mM, pH 6.0) at a flow rate of 4.5 mL/min. The retention time was 12.6 min, and the yield of the peptide was 42%. ESI-MS: calculated [M+2H]2+ for HTK01146 C74H110BF3N18O15 780.4; found [M+2H]2+ 780.4. In Vitro Competition Binding Assays The binding affinity of R954 derivatives to B1R was measured as reported previously via competition binding assays using B1R-expressing CHO-K1 cell membranes and [3H][Leu9,desArg10]kallidin as the radioligand13. Cell Lines HEK293T and HEK293T::hB1R cell lines were cultured in Dulbecco’s Modified Eagle Medium, high glucose, GlutaMAXTM supplement with 10% fetal bovine serum, and 100 U/mL penicillinstreptomycin. Cells were incubated at 37 °C in an atmosphere containing 5% CO2 and used for in vitro or in vivo experiments when 80-90% confluence was reached. The HEK293T::h1BR cell line was previously established in our lab with lentiviral transduction to overexpress human B1R13. Characterization of hB1R expression was conducted with fluorescence microscopy13.



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Fluorometric Calcium Release Assays To determine agonistic/antagonistic properties of the modified peptides, calcium release assays were performed according to previously published procedures14. Synthesis of 68Ga-DOTA-dPEG2-R954 (68Ga-HTK01083) The radiolabeling experiments were performed following previously published procedures13. The aqueous 68GaCl3 solution (751 – 980 MBq) was added to a 4-mL glass vial preloaded with 0.7 mL of HEPES buffer (2 M, pH 5.0) and DOTA-dPEG2-R954 (50 µg). The reaction was heated using a Danby DMW7700WDB microwave (Findlay, OH) at power setting 2 for 1 min. The reaction mixture was purified by HPLC using the semi-preparative column eluted with 35% acetonitrile in water with 0.1% TFA at a flow rate of 4.5 mL/min. The retention time of 68GaHTK01083 was 15.7 min. The eluate fraction containing 68Ga-HTK01083 was collected, diluted with water (50 mL), and passed through a C18 Sep-Pak cartridge. The 68Ga-HTK01083 trapped on the cartridge was then eluted off with ethanol (0.4 mL), and diluted with saline for imaging, biodistribution and in vivo stability studies. Quality control was performed using the analytical column eluted with 35% acetonitrile in water with 0.1% TFA at a flow rate of 2 mL/min. The retention time of 68Ga-HTK01083 was 7.7 min. Synthesis of 18F-AmBF3-Mta-dPEG2-R954 (18F-HTK01146) The radiolabeling experiment was optimized from previously published procedures16, 20. 19FAmBF3-Mta-dPEG2-R954 (19F-HTK01146, 100 nmol) was dissolved in a mixture of aqueous pyridazine-HCl buffer (15 µL, 1 M, pH 2.0) and N,N-dimethylformamide (15 µL) in a 4.5 mL Falcon tube. H218O containing 18F-fluoride (29.9 – 38.4 GBq) was passed through a short anion exchange Trap & Release column (pre-activated with 3 mL brine), and the column was washed with de-ionized water (3 mL × 2). The 18F-fluoride was eluted off the column with 70 µL saline into the Falcon tube containing 19F-HTK01146. The tube was heated at 85 °C for 20 min. The reaction mixture was subsequently quenched with PBS (1 mL), and purified by HPLC using the semi-preparative column eluted with 38% acetonitrile in water with 0.1% TFA at a flow rate of 4.5 mL/min. The retention time of 18F-HTK01146 was 13.3 min. 18F-HTK01146 was further purified by C18 Sep-Pak cartridge to remove TFA and acetonitrile, and formulated with saline as described above for the synthesis of 68Ga-HTK01083. Quality control was performed on the analytical column eluted with 37% acetonitrile in water with 0.1% TFA at a flow rate of 2 mL/min. The retention time of 18F-HTK01146 was 9.0 min. LogD7.4 Measurements LogD7.4 values of radiolabeled peptides were measured using the shake flask method as previously reported13. Biodistribution and PET Imaging Biodistribution and PET/CT imaging studies were performed as previously reported13-18. Male immunodeficient NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice were obtained from a breeding colony at the Animal Resource Centre of the BC Cancer Research Centre. All experiments were


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conducted in accordance with the guidelines established by the Canadian Council on Animal Care and approved by the Animal Ethics Committee of the University of British Columbia. For biodistribution study, HEK293T and HEK293T::hB1R tumors were inoculated by subcutaneous injection of 1 × 106 cells on each dorsal flank of the mice. Each mouse had a B1R+ tumor and a B1R- tumor. After 2 weeks of growth, palpable tumors measuring approximately 7 9 mm in diameter were obtained. Mice were injected with ~ 1 - 3 MBq of radiolabeled peptides. For blocking experiments, the radioactive compound was co-injected with 100 µg of the unmodified R954 peptide. After a 1 h uptake period, the mice were anesthetized by isoflurane inhalation, followed by CO2 asphyxiation. Blood was promptly withdrawn, and the organs/tissues of interest were harvested, rinsed with normal saline, blotted dry, and weighed. The radioactivity of the collected mouse tissues was counted and expressed as the percentage of the injected dose per gram of tissue (%ID/g). PET/CT imaging experiments were conducted using a Siemens (Knoxville, TN) Inveon microPET/CT scanner. Mice bearing two tumors derived from HEK293T and HEK293T::hB1R cells as described above were used. For dynamic imaging study, the mice were sedated with 2% isoflurane inhalation and positioned in the scanner. A baseline CT scan was obtained for localization and attenuation correction before radiotracer injection, using 60 kV X-rays at 500 mA, three sequential bed position with 33% overlap, and 220 degree continuous rotation. The mice were kept warm by a heating pad during acquisition. The dynamic acquisition of 60 minutes was started at the time of intravenous injection with 6 - 8 MBq of the radiotracer. The list mode data were rebinned into time intervals (12 × 10, 6 × 30, 5 × 60, 6 × 300, and 2 × 600 seconds) to obtain tissue time-activity curves. The mice were euthanized at the end of imaging section, and the tissues were collected, counted and analyzed as described above. For static imaging study, the mice were briefly sedated for intravenous injection of the radiotracer (6 – 8 MBq), and then allowed to recover and roam freely in their cages for 45 minutes. At that point, the mice were sedated with 2% isoflurane inhalation, placed in the scanner, and an attenuation correction CT scan was obtained as described above. A single static emission scan was acquired for 10 minutes. The mice were euthanized and the organs harvested for biodistribution as described above. In Vivo Stability Radiolabeled R954 derivatives (3 - 5 MBq) were intravenously injected in male NSG mice. After a 5 min uptake period, mice were euthanized and blood was collected. The plasma fraction was isolated and subsequently analyzed with radio-HPLC following published procedures14. For 68 Ga-HTK01083, sample was analyzed on the semi-preparative column with 38% acetonitrile in water with 0.1% TFA at a flow rate of 4.5 mL/min. The retention time was 12.3 min. For 18FHTK01146, sample was analyzed on the semi-preparative column with 40% acetonitrile in water with 0.1% TFA at a flow rate of 4.5 mL/min. The retention time was 14.1 min. Statistical Analysis Statistical analyses were performed by GraphPad Prism 7 or Microsoft Excel software. The differences in binding affinity between R954 and HTK01083, and R954 and HTK01146 were compared by unpaired two-tailed t-test. For calcium release assays (Figure 2), differences



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between treatment and control group were analyzed by one way ANOVA. In Table 1, comparison of uptake in B1R- tumors and other tissues between control and blocked groups was performed using unpaired, two-tailed t-test. The unpaired one-tailed t-test was used to compare B1R+ tumor uptake and B1R+ tumor-to-background (B1R- tumor, blood and muscle) ratios between the control and blocked groups. The difference was considered statistically significant when p value was < 0.05.


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RESULTS Peptide Synthesis, Radiolabeling, and Hydrophilicity The radiolabeling precursor DOTA-dPEG2-R954 was obtained in 7% yield. Complexation with cold gallium to produce the standard HTK01083 led to 71% yield. Azidoacetyl-dPEG2-R954 was obtained in 36% yield. HTK01146, the radiolabeling precursor and standard, was obtained in 42% yield following click chemistry. For radiolabeling, 68Ga-HTK01083 was obtained in 58 ± 6% (n = 3) decay-corrected radiochemical yields, with an average specific activity of 100 ± 43 GBq/µmol, and in > 99% radiochemical purity. 18F-HTK01146 was obtained in 10 ± 8% (n = 3) decay-corrected radiochemical yields, with an average specific activity of 52 ± 15 GBq/µmol, and in > 99% radiochemical purity. To quantify the hydrophilic properties of the modified peptides, the LogD7.4 (D7.4: distribution coefficient in octanol/water at pH 7.4) values were determined. 68Ga-HTK01083 was slightly more hydrophilic (-1.64 ± 0.33) compared to 18F-HTK01146 (-1.36 ± 0.07). The experiment was performed in triplicate.



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Binding Affinity and Agonist/Antagonist Characterization Per Figure 2, R954 and its derivatives all successfully inhibited B1R binding by [3H][Leu9,desArg10]kallidin in a dose dependant manner. The Ki values for R954, HTK01083, and HTK01146 were 10.0 ± 3.12, 30.5 ± 7.57, and 24.8 ± 2.76 nM, respectively. In both cases, the reduction in binding affinity was statistically significant (p < 0.05). The levels of calcium release in HEK293T::hB1R cells induced by R954 and its derivatives are shown in Figure 3. [des-Arg10]Kallidin, a positive control, induced calcium release of 148 ± 19.6 and 145 ± 28.6 relative fluorescence unit (RFU) at 5 and 50 nM. On the other hand, R954, HTK01083, and HTK01146 did not induce any statistically significant amount of calcium release compared to PBS control at 5 and 50 nM.

Figure 2. Representative displacement curves of [3H][Leu9,des-Arg10]kallidin by R954, HTK01083, and HTK01146. Experiment was performed in triplicate.


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Figure 3. Calcium release assays for R954, HTK01083 and HTK01146. Data are displayed as means ± SD, and ** indicates the difference between the control (PBS) and treatment groups is significant (p < 0.01). [Des-Arg10]Kallidin, a natural agonist of the B1R system, was included as a positive control.



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Biodistribution, PET/CT imaging, and In Vivo Stability The biodistribution data for 68Ga-HTK01083 and 18F-HTK01146 at 1 h p.i. are summarized in Table 1. 68Ga-HTK01083 and 18F-HTK01146 were able to differentiate between B1R+ tumor and B1R- tumor, derived from HEK293T::hB1R and HEK293T cells respectively, as uptake in B1R+ tumor was approximately 10-fold higher. For both tracers, the kidneys had the highest uptake (66.1 - 77.0 %ID/g), immediately followed by B1R+ tumor (8.46 - 9.25 %ID/g). Tracers were cleared mainly by the kidneys with minimal involvement of the hepatobiliary system (≤ 1.23 %ID/g in liver and intestines). Blocking with R954 significantly reduced the uptake of both tracers in B1R+ tumor by > 70% (p < 0.001), but increased uptake in most of non-target tissues/organs. Representative maximum intensity projection PET images are shown in Figure 4. 68GaHTK01083 and 18F-HTK01146 generated high contrast images at 1 h p.i. B1R+ tumor was clearly visualized, with uptake only lower than kidneys and bladder. With blocking, uptake in B1R+ tumor was reduced to background intensity, and the background activity level was elevated especially in the mouse injected with 18F-HTK01146. Time activity curves for 68GaHTK01083 and 18F-HTK01146 based on regions-of-interest (ROIs) are shown in Figure 5. For both tracers, activity accumulated progressively in B1R+ tumors, and cleared quickly from the rest of the body except kidneys. Stability of 68Ga-HTK01083 and 18F-HTK01146 was assessed in NSG mice. Based on HPLCanalysis (Figure 6), 68Ga-HTK01083 and 18F-HTK01146 were relatively stable in vivo with 96 ± 6 and 97 ± 4% (n = 3), respectively, of the tracers remaining intact at 5 min p.i.


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Table 1. Biodistribution and uptake ratios of radiolabeled R954 derivatives in tumor-bearing micea. 68

18

Blood Fat Testes Intestine Spleen Liver Pancreas Adrenal glands Kidneys Lungs Heart B1R+tumor B1R- tumor Muscle Bone Brain

Ga-HTK01083 Baseline Blocked (n = 6) (n = 5) 1.14 ± 0.42 3.53 ± 0.45*** 0.22 ± 0.05 0.47 ± 0.12** 0.36 ± 0.11 0.80 ± 0.08*** 0.45 ± 0.06 0.98 ± 0.23*** 0.52 ± 0.15 1.25 ± 0.17*** 1.08 ± 0.11 1.80 ± 0.16*** 0.36 ± 0.08 0.58 ± 0.05*** 0.75 ± 0.29 1.94 ± 0.83** 66.1 ± 9.70 43.1 ± 6.55** 1.41 ± 0.78 2.35 ± 0.21* 0.53 ± 0.12 1.17 ± 0.15*** 8.46 ± 1.44 1.51 ± 0.34*** 0.94 ± 0.22 1.29 ± 0.20* 0.42 ± 0.10 0.97 ± 0.34** 0.30 ± 0.17 0.71 ± 0.14** 0.05 ± 0.02 0.11 ± 0.02**

F-HTK01146 Baseline Blocked (n = 4) (n = 4) 1.53 ± 0.93 4.89 ± 1.37** 0.29 ± 0.12 0.46 ± 0.18 0.45 ± 0.21 0.79 ± 0.18 0.69 ± 0.21 1.21 ± 0.31* 0.68 ± 0.07 1.73 ± 0.50** 1.23 ± 0.19 2.40 ± 0.53** 0.40 ± 0.08 0.87 ± 0.33* 0.68 ± 0.17 1.41 ± 0.33* 77.0 ± 19.5 81.1 ± 12.2 1.69 ± 0.80 3.92 ± 1.20* 0.65 ± 0.23 1.49 ± 0.57* 9.25 ± 0.69 2.64 ± 0.78*** 0.93 ± 0.35 1.99 ± 0.79* 0.49 ± 0.13 0.98 ± 0.35* 1.70 ± 0.64 1.44 ± 0.39 0.07 ± 0.01 0.09 ± 0.03

B1R+T:B1R-T B1R+T:Blood B1R+T:Muscle

9.43 ± 2.32 6.32 ± 1.44 20.7 ± 3.58

10.8 ± 3.16 7.24 ± 2.56 19.5 ± 4.29

Tissue (%ID/g)

1.18 ± 0.29*** 0.43 ± 0.11*** 1.69 ± 0.59***

a

1.60 ± 1.05*** 0.54 ± 0.08*** 2.92 ± 1.20***

Studies were conducted with (blocked) or without (baseline) co-injection of nonradioactive R954 (100 µg), and mice were euthanized at 1 h p.i. Tissue uptake values are presented as means ± SD. The significance of differences between groups (baseline and blocked) was: *p < 0.05; **p < 0.01; ***p < 0.001.



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Figure 4. Maximal intensity projection images of PET/CT and PET with 68Ga-HTK01083 and F-HTK01146 in mice bearing HEK293T and HEK293T::hB1R tumors at 1 h post-injection. Blocking studies were performed with co-injection of 100 µg of R954. t+: B1R+ tumor; t-: B1Rtumor; k: kidney; bl: bladder.

18


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Figure 5. Time activity curves for 68Ga-HTK01083 and 18F-HTK01146 using ROIs drawn around tumors, kidneys, heart, muscle, and bone. Both 68Ga-HTK01083 and 18F-HTK01146 show progressive increase in B1R+ tumor with clearance from non-target tissues except kidneys.

Figure 6. Representative radio-HPLC chromatograms of 68Ga-HTK01083 and 18F-HTK01146 from QC (top) and mouse plasma samples (bottom) taken at 5 min post-injection.



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DISCUSSION Peptide receptors such as somatostatin, gastrin-releasing peptide, glucagon-like peptide, integrin ανβ3, and chemokine receptor 4 have become molecular targets for cancer diagnosis and therapy21. The aberrant overexpression of these receptors in different cancer subtypes provides an opportunity to use radiolabeled peptides to visualize disease spread and also to predict response to treatments like peptide receptor radionuclide therapy21, 22. Peptides are excellent imaging vectors, as their small size and high affinity to receptors facilitate efficient tumor penetrance and binding, and rapid clearance from non-target tissues22. Overexpressed in cancers, B1R is a cell-surface receptor that can be readily targeted by a peptide-based approach. One of the challenges associated with using peptides as targeting vectors is their metabolic lability. Due to their functions in normal physiology, certain signaling peptides have very short biological half-lives23. With the potential of inducing hyperalgesia, the endogenous ligands for B1R, [des-Arg9]bradykinin and [des-Arg10]kallidin, are regulated and cleaved by peptidases: angiotensin-converting enzyme, aminopeptidase and carboxypeptidase3. In previous studies, we demonstrated that tracer stability is crucial for B1R imaging. For tracers derived from [Leu9,des-Arg10]kallidin, co-administration of peptidase inhibitors such as phosphoramidon greatly improved tumor targeting and contrast. In parallel, we investigated the use of peptide sequences that incorporated unnatural amino acids as B1R targeting agents (ex. B9858 and B9958), which generated higher contrast PET images for B1R expressing xenografts15-17. As shown in Figure 1, R954 is a nonapeptide with four unnatural amino acids including ornithine (Orn), octahydroindole-2-carboxylic acid (Oic), N-methylphenylalanine (αMePhe) and 3-(β-naphthyl)-D-alanine (D-2-Nal). Each amino acid substitution was specifically integrated to confer resistance to the aforementioned peptidases19. R954 was first identified in an effort to develop potent B1R antagonists with prolonged duration of action. Since then, B1R antagonism by R954 has been evaluated in many preclinical disease models. R954 was tested as a therapeutic to relieve symptoms associated with diabetes (neuropathic pain, inflammatory edema, and hyperglycemia), renal ischemia-reperfusion injury, and osteoarthritis24-29. Most recently, Shen et al. reported that subcutaneous injections of R954 were able to retard the growth rate of 4T1 murine breast cancer xenografts30. Gobeil Jr. et al. reported that R954 has at least 100-fold specificity for B1R over 133 other evaluated human targets (GPCRs, ion channels and transporters)31. The same group also reported pharmacology, stability, pharmacokinetics, and toxicology data for R954 in rats31. Based on favourable pharmacokinetics, stability, and safety profile, R954 was approved for a Phase I clinical study31. Collectively, these observations provide support and rationale for using R954 as a lead compound for B1R probe development. To synthesize the two radiotracers evaluated in this study, R954 was first assembled on standard Fmoc solid phase support. At the N-terminus, a hydrophilic dPEG2 linker was inserted as a spacer to separate the targeting sequence from the ensuing radioprosthetic group. To prepare the gallium derivative (HTK01083) a DOTA chelator was directly added after the linker, while the fluorine derivative (HTK01146) was synthesized via Cu2+ catalyzed click reaction between 2-azidoacetic acid and an ammoniomethyl-trifluroborate (AmBF3)-conjugated alkyne. Once nonradioactive standards were prepared, cell competition and calcium release assays were conducted to determine binding affinity and agonist/antagonist properties. While N-terminal modifications of previous B1R peptides were well-tolerated, this was not the case with R954 (Figure 2).


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HTK01146 exhibited better binding affinity (Ki: 24.8 ± 2.76 nM) than HTK01083 (Ki: 30.5 ± 7.57 nM); however, both compounds had reduced binding affinity to B1R compared to unmodified R954 (Ki: 10.0 ± 3.12 nM). HTK01083 and HTK01146 were characterized as antagonists as neither compound induced calcium release in B1R expressing cells (Figure 3)32. Although they forego internalization, antagonists can bind multiple conformational states of the receptor increasing overall uptake. For certain GPCRs, antagonists have shown superiority over agonists (ex: somatostatin analogs) for molecular imaging33, 34. 68

Ga-gallium and 18F-fluorine were selected as PET imaging isotopes for this study, as they have short radioactive half-lives (67.7 and 109.8 min) that makes them ideal for radiolabeling molecules with fast pharmacokinetics21. 68Ga decays 89% via positron emission (Eβ+: 1880 keV) while 18F decays 97% via positron emission (Eβ+: 634 keV)21. Based on their decay and positron emission energy, 18F provides better spatial resolution. In terms of convenience, they can be considered as complementary. 68Ga can be eluted from 68Ge/68Ga generators for on-demand synthesis and 18F can be produced by cyclotrons. Using DOTAdPEG2-R954 as the precursor for 68Ga-labeling, 68Ga-HTK01083 was obtained in good radiochemical yields (58 ± 6%) and specific activities (100 ± 43 GBq/µmol). For 18F-labeling, we used a one-step 18F-19F isotope exchange reaction on the trifluoroborate motif of 19FHTK01146. Although 18F-HTK01146 was obtained in moderate radiochemical yields (10 ± 8%), the amount of 18F-HTK01146 and its specific activity (52 ± 15 GBq/µmol) were sufficient for imaging studies. The biodistribution of 68Ga-HTK01083 was similar to that of 18F-HTK01146, including renal clearance (66.1 ± 9.70 vs. 77.0 ± 19.5 %ID/g), high B1R+ tumor uptake (8.46 ± 1.44 vs. 9.25 ± 0.69 %ID/g), and low uptake in non-target tissues. At 1 h p.i., the contrast ratios for B1R+ tumor-to-B1R-tumor, -to-blood, and -to-muscle were 9.43 ± 2.32 vs. 10.8 ± 3.16, 6.32 ± 1.44 vs. 7.24 ± 2.56, and 20.7 ± 3.58 vs. 19.5 ± 4.29 respectively. Neither tracer was capable of penetrating the blood-brain barrier as uptake in brain was lowest of the collected tissues (≤ 0.07 ± 0.01 %ID/g). The primary difference between the two tracers was higher bone uptake (1.70 ± 0.64 %ID/g) for 18F-HTK01146, which can be attributed to minor defluorination in vivo. The defluorination observed for 18F-HTK01146 is higher compared to reported radiofluorinated B1Rtargeting peptides16, 17. When co-injected with 100 µg of R954, uptake in B1R+ tumor was reduced by approximately 82% for 68Ga-HTK01083 and 71% for 18F-HTK01164, indicating that uptake was specific and receptor-mediated. Of note, the decrease was observed with concomitant increase uptake in non-target tissues. Based on the residual activity in blood, it is believed that the dosage of blocking agent administered may have saturated clearance pathways. PET images show excellent agreement with biodistribution data. With the exception of excretory organs (bladder and kidneys), highest uptake was observed in B1R+ tumor (Figure 4). The tracers were able to delineate B1R+ tumor from non-target tissues, with higher uptake observed for 18F-HTK01164. With R954 blockade, uptake in B1R+ tumor was significantly reduced to near background levels. Analyzing the time activity curves for each tracer (Figure 5), two similar pharmacokinetic profiles were observed. 18F-HTK01164 had higher residual activity in blood at 1 h p.i., but both tracers were quickly excreted through the kidneys. Given that B1R+



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tumor uptake was sustained and increasing progressively, the imaging window can potentially be extended beyond the 1 h time point evaluated in this study. We compared in vivo data obtained for 68Ga-HTK01083 and 18F-HTK01164 against previously reported 9859/B9958 derivatives (Table 2)15-17, performed with the identical animal xenograft model. In general, 68Ga-HTK01083 and 18F-HTK01164 exhibited lower tumor uptake and contrast ratios than previously synthesized tracers. 18F-HTK01164 (18F-AmBF3-Mta-dPEG2R954) had higher uptake in B1R+ tumors than 18F-L08064 (18F-AmBF3-Mta-Pip-B9858) or 18FL08060 (18F-AmBF3-Mta-Pip-B9958). However, contrast ratios were lower than those of 18FL08060. We hypothesize that these observations can be attributed to differences in binding affinity and hydrophilicity. The former modulates tumor uptake, while the latter guides the clearance kinetics from non-target tissues. Focusing on the best 68Ga and 18F-labeled peptides in the list, 68Ga-Z02176 (68Ga-DOTA-Pip-B9958) and 18F-Z04139 (Al18F-NODA-Mpaa-PipB9958), we see that both tracers had approximately 10-fold better affinity and were 100-fold more hydrophilic than 68Ga-HTK01083 and 18F-HTK01164. 68Ga-Z02176 and 18F-Z04139 were only identified after methodical testing of linkers and radiolabel complexes14,17. Given the promising preliminary results obtained by 68Ga-HTK01083 and 18F-HTK01164, we believe R954 warrants further investigation as a lead sequence for the design of B1R tracers.


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Table 2. Comparison of binding affinity, hydrophilicity, tracer uptake, and uptake ratios of selected B1R-targeting peptidesa. n/a: not available; Pip: 4-amino-(1-carboxymethyl)piperidine; Mpaa: 4-methylphenylacetic acid.

Tracer name

B9858 derivatives Ga-P04158 68 Ga-P05022 18 F-L08064 68

B9958 derivatives Ga-Z02090 68 Ga-Z02176 68 Ga-Z02137 18 F-L08060 18 F-Z04139 68

R954 derivatives Ga-HTK01083 18 F-HTK01146 68

a

Peptide sequence

68

Ki (nM)

LogD7.4

Average tissue uptake (1 h p.i., %ID/g) B1R+ tumor

Kidneys

Ave. B1R+ tumor-tobackground contrast ratio (1 h p.i.) To To blood muscle

Ref

Ga-DOTA-dPEG2-B9858 Ga-DOTA-Pip-B9858 18 F-AmBF3-Mta-Pip-B9858

1.5 5.5 0.1

-2.50 -2.65 n/a

19.6 11.6 3.94

69.2 93.7 36.2

19.2 11.2 6.69

66.1 40.2 21.3

15 Unpublished 16

68

1.1 2.5 2.6 0.5 1.4

-2.71 -4.15 -3.35 n/a -3.90

14.4 28.9 14.0 4.20 22.6

50.1 90.9 85.2 30.9 101

29.9 56.1 34.3 14.7 58.0

124 167 103 48.6 173

15 17 17 16 17

30.5 24.8

-1.64 -1.36

8.46 9.25

66.1 75.9

6.32 7.24

20.7 19.5

-

68

Ga-DOTA-dPEG2-B9958 Ga-DOTA-Pip-B9958 68 Ga-NODA-Mpaa-Pip-B9958 18 F-AmBF3-Mta-Pip-B9958 Al18F-NODA-Mpaa-Pip-B9958 68

68 18

Ga-DOTA-dPEG2-R954 F-AmBF3-Mta-dPEG2-R954

Studies were performed with mice bearing the same B1R- HEK293T and B1R+ HEK293T::hB1R tumors.



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ASSOCIATED CONTENT Supporting Information HPLC and MS spectra of purified HTK01083 and HTK01146 are provided in Supporting Information. This information is available free of charge via the Internet at http://pubs.acs.org/. AUTHOR INFORMATION Corresponding Authors *Dr. François Bénard. Address: Department of Molecular Oncology, BC Cancer Agency, 675 West 10th Avenue, Rm 14-111, Vancouver, BC V5Z 1L3, Canada. Phone: 604-675-8206. Fax: 604-675-8218. E-mail: [email protected] *Dr. Kuo-Shyan Lin. Address: Department of Molecular Oncology, BC Cancer Agency, 675 West 10th Avenue, Rm 4-123, Vancouver, BC V5Z 1L3, Canada. Phone: 604-675-8208. Fax: 604-675-8218. E-mail: [email protected]

Notes The authors declare no competing financial interest. ACKNOWLEDGEMENTS This work was supported by the Canadian Institutes of Health Research (MOP-126121) and the BC Leading Edge Endowment Fund. We thank Wade English, Baljit Singh, and Milan Vuckovic for their technical assistance.


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