Radiolabeled B9958 Derivatives for Imaging Bradykinin B1 Receptor

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Radiolabeled B9958 derivatives for imaging bradykinin B1 receptor expression with positron emission tomography: effect of the radiolabel-chelator complex on biodistribution and tumor uptake Zhengxing Zhang, Guillaume Amouroux, Jinhe Pan, Silvia Jenni, Jutta Zeisler, Chengcheng Zhang, Zhibo Liu, David M. Perrin, Francois Benard, and Kuo-Shyan Lin Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.6b00428 • Publication Date (Web): 27 Jun 2016 Downloaded from http://pubs.acs.org on June 29, 2016

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Molecular Pharmaceutics

Radiolabeled B9958 derivatives for imaging bradykinin B1 receptor expression with positron emission tomography: effect of the radiolabel-chelator complex on biodistribution and tumor uptake Zhengxing Zhang†,§, Guillaume Amouroux†,§, Jinhe Pan§, Silvia Jenni§, Jutta Zeisler§, Chengcheng Zhang§, Zhibo Liu‡, David M. Perrin‡, François Bénard*,§,‖ , Kuo-Shyan Lin*,§,‖

§

Department of Molecular Oncology, BC Cancer Agency, Vancouver, BC V5Z 1L3, Canada

‡

Chemistry Department, University of British Columbia, Vancouver, BC V6T 1Z1, Canada

‖Department

of Radiology, University of British Columbia, Vancouver, BC V5Z 4E3, Canada

AUTHOR INFORMATION Corresponding Author *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: 604-6758218. E-mail: [email protected]. *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: 604-6758218. E-mail: [email protected]. Author Contributions †

Zhengxing Zhang and Guillaume Amouroux contributed equally to this work.

Notes The authors declare no competing financial interest.

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ABSTRACT GRAPHIC


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ABSTRACT Bradykinin B1 receptor (B1R) that is up-regulated in a variety of malignancies is an attractive cancer imaging biomarker. In this study we optimized the selection of radiolabel-chelator complex to improve tumor uptake and tumor-to-background contrast of radiolabeled analogs of B9958 (Lys-Lys-Arg-Pro-Hyp-Gly-Cpg-Ser-D-Tic-Cpg), a potent B1R antagonist. Peptide sequences were assembled on solid-phase. Cold standards were prepared by incubating DOTA/NODA-conjugated peptides with GaCl3, and by incubating AlOH-NODA-conjugated peptide with NaF. Binding affinities were measured via in vitro competition binding assays. 68Ga and 18F labeling experiments were performed in acidic buffer and purified by HPLC. Imaging/biodistribution studies were performed in mice bearing both B1R-positive (B1R+) HEK293T::hB1R and B1R-negative (B1R-) HEK293T tumors. Z02176 (Ga-DOTA-Pip-B9958; Pip: 4-amino-(1-carboxymethyl)piperidine), Z02137 (Ga-NODA-Mpaa-Pip-B9958; Mpaa: 4methylphenylacetic acid) and Z04139 (AlF-NODA-Mpaa-Pip-B9958) bound hB1R with high affinity (Ki = 1.4 – 2.5 nM). 68Ga-/18F-labeled peptides were obtained on average in ≥ 32% decay-corrected radiochemical yield with > 99% radiochemical purity and 100 - 261 GBq/µmol specific activity. Biodistribution/imaging studies at 1 h post-injection showed that all tracers cleared rapidly from background tissues (except kidneys), and were excreted predominantly via the renal pathway. Only kidneys, bladders and B1R+ tumors were clearly visualized in PET images. Uptake in B1R+ tumor was higher by using 68Ga-Z02176 (28.9 ± 6.21 %ID/g) and 18FZ04139 (22.6 ± 3.41 %ID/g) than 68Ga-Z02137 (14.0 ± 4.86 %ID/g). The B1R+ tumor-to-blood and B1R+ tumor-to-muscle contrast ratios were also higher for 68Ga-Z02176 (56.1 ± 17.3 and 167 ± 57.6) and 18F-Z04139 (58.0 ± 20.9 and 173 ± 42.9) than 68Ga-Z02137 (34.3 ± 15.2 and 103 ± 30.2). With improved target-to-background contrast 68Ga-Z02176 and 18F-Z04139 are promising for imaging B1R expression in cancers with PET.

KEY WORDS Bradykinin B1 receptor; B9958; Radiolabel-chelator complex; Fluorine-18; Gallium-68; Positron emission tomography



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INTRODUCTION Bradykinin (Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg) and its close analog kallidin (Lysbradykinin, Table 1) are endogenous peptides that are known to function in pain and inflammatory pathways.1-2 Bradykinin and kallidin elicit their biological effects through the activation of two G-protein-coupled receptors, bradykinin B1 and B2 receptors (B1R and B2R).3 B2R is constitutively and ubiquitously expressed in the body, whereas B1R has minimal expression in healthy tissues but has been shown up-regulated in a variety of cancers.3-4 Due to its low expression in normal tissues, B1R is a promising imaging biomarker for the detection of cancers. We initiated the development of B1R imaging agents labeled with positron emitters as positron emission tomography (PET) is considered one of the most powerful molecular imaging modalities because of its quantification capability and high sensitivity. Our first candidate 68GaP03083 (68Ga-DOTA-Ahx-[Leu9,des-Arg10]kallidin, Table 1) showed minimal uptake in B1Rexpressing tumors because of its fast in vivo degradation by peptidases.5 Tumor uptake was greatly improved with co-injection of peptidase inhibitor (phosphoramidon or enalaprilat), or using a more metabolically stable derivative 68Ga-SH01078 (68Ga-DOTA-Ahx[Hyp4,Cha6,Leu9,des-Arg10]kallidin, Table 1).5 Using [Hyp4,Cha6,Leu9,des-Arg10]kallidin as the B1R-targeting sequence, we further optimized the selection of linker to improve binding affinity and tumor uptake (Table 1).6 68Ga-P04168 (68Ga-DOTA-Pip-[Hyp4,Cha6,Leu9,desArg10]kallidin) that contains a cationic linker Pip (4-amino-(1-carboxymethyl)piperidine) showed improved binding affinity, tumor uptake and tumor-to-nontarget contrast ratios.6 By conjugating Ga-DOTA-dPEG2 (dPEG2: 9-amino-4,7-dioxanonanoic acid) at the N-terminus, we recently reported that B9958 (Lys-[Hyp4,Cpg6,D-Tic8,Cpg9,des-Arg10]kallidin) was superior to B9858 (Lys-[Hyp4,Igl6,D-Igl8,Oic9,des-Arg10]kallidin) and [Hyp4,Cha6,Leu9,des10 Arg ]kallidin as the targeting vector for the design of B1R imaging agents (68Ga-Z02090 vs. 68 Ga-P04158 and 68Ga-P03034, Table 1).7 In a separate study by conjugating the AmBF3-Mta (4(N-trifluoroborylmethyl-N,N-dimethylammonio)methyl-1,2,3-triazole-1-acetic acid) moiety at the N-terminus, we further confirmed that the B9958 derivative 18F-L08060 (previously called 18 F-AmBF3-B9958, Table 1) was preferable to the B9858 derivative 18F-L08064 (previously called 18F-AmBF3-B9858, Table 1) for imaging B1R expression in xenografted tumors.8 The aim of this study was to investigate if the tumor uptake and tumor-to-background contrast ratios could be further improved via the selection of radiolabel-chelator complexes (68Ga-DOTA, 68 Ga-NODA and 18F-AlF-NODA) coupled to the preferred B9958 sequence and the cationic linker Pip. Herein, we present the synthesis and comparative evaluation of three new tracers (Fig. 1): 68Ga-DOTA-Pip-B9958 (68Ga-Z02176), 68Ga-NODA-Mpaa-Pip-B9958 (Z02137; Mpaa: 4methylphenylacetic acid) and 18F-AlF-NODA-Mpaa-Pip-B9958 (Z04139). The results were compared with those previously obtained using 18F-L08060 for imaging B1R expression with PET.


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Table 1 Binding affinity, overall charge, selected tissue uptake and B1R+ tumor-to-background contrast ratios of previously reported B1R-targeting peptides.

Peptide

Sequence

Ki (nM)

Bradykinin Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg Kallidin Lys-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg Lys-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Leu [Leu9,desArg10]kallidin 68 68 Ga-P03083 Ga-DOTA-Ahx-[Leu9,des-Arg10]kallidin 68 68 Ga-SH01078 Ga-DOTA-Ahx-[Hyp4,Cha6,Leu9,des-Arg10]kallidin 68 68 Ga-P04115 Ga-DOTA-Gly-Gly-[Hyp4,Cha6,Leu9,des-Arg10]kallidin 68 68 Ga-P03034 Ga-DOTA-dPEG2-[Hyp4,Cha6,Leu9,des-Arg10]kallidin 68 68 Ga-P04168 Ga-DOTA-Pip-[Hyp4,Cha6,Leu9,des-Arg10]kallidin B9858 Lys-Lys-Arg-Pro-Hyp-Gly-Igl-Ser-D-Igl-Oic B9958 Lys-Lys-Arg-Pro-Hyp-Gly-Cpg-Ser-D-Tic-Cpg 68 68 Ga-P04158 Ga-DOTA-dPEG2-B9858 68 68 Ga-Z02090 Ga-DOTA-dPEG2-B9958 18 18 F-L08064 F-AmBF3-Mta-Pip-B9858 18 18 F-L08060 F-AmBF3-Mta-Pip-B9958 a pEC50. bpIC50.

5.7a 7.4a 8.9b 2.6 ± 0.7 28 ± 4.9 11 ± 2.5 16 ± 1.9 3.6 ± 0.2 10.1b 0.089 1.5 ± 1.9 1.1 ± 0.8 0.1 ± 0.1 0.5 ± 0.3

Overall charge +1 +2 +1 +1 +1 +1 +1 +2 +2 +2 +2 +2 +3 +3


Tissue uptake (1-h p.i., %ID/g) B1R+ tumor 0.79 ± 0.22 2.06 ± 0.52 1.96 ± 0.83 2.17 ± 0.49 4.15 ± 1.13 19.6 ± 4.50 14.1 ± 1.63 3.94 ± 1.24 4.20 ± 0.98

B1R+ tumor-tobackground ratio (1-h p.i.)

Kidney

To blood

To muscle

4.95 ± 0.86 3.14 ± 0.62 4.02 ± 2.40 4.50 ± 2.17 4.02 ± 1.22 69.2 ± 7.39 50.1 ± 9.68 36.2 ± 5.78 30.9 ± 6.74

10.4 ± 3.78 7.78 ± 2.20 6.37 ± 3.82 5.72 ± 2.20 15.9 ± 6.84 19.2 ± 8.21 29.9 ± 5.58 6.69 ± 3.60 14.7 ± 3.56

8.18 ± 1.69 30.2 ± 7.42 26.1 ± 8.91 25.5 ± 13.1 78.1 ± 28.5 66.1 ± 17.0 124 ± 28.1 21.3 ± 4.33 48.6 ± 10.7

Ref.

25 25 25 5 5,6 6 5-7 6 24 21 7 7 8 8


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Figure 1. Chemical structures of radiolabeled B9958 derivatives.


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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 an Agilent HPLC system 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 the Agilent HPLC system was controlled using the Agilent ChemStation software. The HPLC columns used were a semipreparative column (Phenomenex C18, 5 µ, 250 × 10 mm) and an analytical column (Phenomenex C18, 5 µ, 250 × 4.6 mm). The HPLC solvents were A: H2O containing 0.1% TFA, and B: CH3CN containing 0.1% TFA. 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 either a Bruker (Billerica, MA) Esquire-LC/MS system with ESI ion source or a Bruker Autoflex MALDI-TOF spectrometer. 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 generator (Somerset West, South Africa), and was purified according to the previously published procedures using a DGA resin column.5 18F-Fluoride was produced by 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-Pip-B9958 The assembling of Fmoc-protected Pip-B9958 sequence (Fmoc-Pip-Lys(Boc)-Lys(Boc)Arg(Pbf)-Pro-Hyp(tBu)-Gly-Cpg-Ser(tBu)-D-Tic-Cpg) on 2-chlorotrityl chloride resin was performed according the previously published procedures.7 The resin was treated with 20% piperidine in N,N-dimethylformamide to remove the Nα-Fmoc protecting group. The chelator DOTA tri-t-butyl ester (3 equivalents) was coupled to the N-terminus in N-methyl-2-pyrrolidone with in situ activating reagent N,N’-diisopropylcarbodiimide and N-hydroxysuccinimide (3 equivalents) in the presence of N,N-diisopropylethylamine (20 equivalents). At the end, the peptide was deprotected and simultaneously cleaved from the resin by treating with 95/2.5/2.5 TFA/H2O/TIPS (triisopropylsilane) for 4 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 gradient 20/80 A/B to 30/70 A/B in 30 min at a flow rate of 4.5 mL/min. The retention time of DOTA-Pip-B9958 was 20.4 min, and the yield of the peptide was 18 %. ESI-MS: calculated [M+2H]2+ for DOTA-Pip-B9958 C80H129N21O21 860.98; found [M+2H]2+ 861.67. Synthesis of NODA-Mpaa-Pip-B9958 The assembling of Fmoc-protected Pip-B9958 sequence (Fmoc-Pip-Lys(Boc)-Lys(Boc)Arg(Pbf)-Pro-Hyp(tBu)-Gly-Cpg-Ser(tBu)-D-Tic-Cpg) on 2-chlorotrityl chloride resin was



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performed as described above. The resin was treated with 20% piperidine in N,Ndimethylformamide to remove the Nα-Fmoc protecting group. 4-(Bromomethyl)phenylacetic acid (3 equivalents) was activated with N,N’-diisopropylcarbodiimide (3 equivalents) in 5 mL Nmethyl-2-pyrrolidone and then coupled to the N-terminus. Subsequently, 1,4,7triazacyclononane-1,4-bis(t-butyl acetate) (3 equivalents) was coupled to the peptide sequence by secondary amine alkylation in 5 mL N-methyl-2-pyrrolidone. The peptide was deprotected and cleaved from the resin as described above for the synthesis of DOTA-Pip-B9958. The crude peptide was purified by HPLC using the semi-preparative column eluted with 24/76 A/B at a flow rate of 4.5 mL/min. The retention time of NODA-Mpaa-Pip-B9958 was 18.7 min, and the yield of the peptide was 10 %. MALDI-MS: calculated [M+2H]2+ for NODA-Mpaa-Pip-B9958 C83H128N20O19 855.48; found [M+2H]2+ 855.70. Synthesis of Ga-DOTA-Pip-B9958 (Z02176) A solution of DOTA-PiP-B9958 (4 µmol) and GaCl3 (20 µmol) in 500 µL sodium acetate buffer (0.1 M, pH 4.0) was incubated at 80 °C for 15 min. The reaction mixture was purified by HPLC using the semi-preparative column eluted with gradient 20/80 A/B to 30/70 A/B in 30 min at a flow rate of 4.5 mL/min. The retention time of Z02176 was 20.6 min, and Z02176 was obtained quantitatively. ESI-MS: calculated [M+2H]2+ for Z02176 C80H126GaN21O21 893.93; found [M+2H]2+ 894.13. Synthesis of Ga-NODA-Mpaa-Pip-B9958 (Z02137) Z02137 was synthesized according to the procedures described above for the synthesis of Z02176 by replacing DOTA-Pip-B9958 with NODA-Mpaa-Pip-B9958. The reaction mixture was purified by HPLC using the semi-preparative column eluted with 24/76 A/B at a flow rate of 4.5 mL/min. The retention time of Z02137 was 15.0 min, and the yield was 43%. ESI-MS: calculated [M+2H]2+ for Z02137 C83H126GaN20O19 888.94; found [M+2H]2+ 889.20. Synthesis of AlOH-NODA-Mpaa-Pip-B9958 To a solution of NODA-Mpaa-Pip-B9958 (6.4 mg, 3.7 µmol) in 0.3 mL sodium acetate buffer solution (2 mM, pH 7.0) was added AlCl3 (1.5 mg, 11.2 µmol), and the mixture was stirred at 110 °C for 30 min. The reaction mixture was purified by HPLC using the semi-preparative column eluted with gradient 75/25 A/B to 72/28 A/B in 20 min at a flow rate of 4.5 mL/min. The retention time of AlOH-NODA-Mpaa-Pip-B9958 was 10.0 min, and the yield was 61%. ESIMS: calculated [M+2H]2+ for AlOH-NODA-Mpaa-Pip-B9958 C83H127AlN20O20 876.47; found [M+2H]2+ 876.69. Synthesis of AlF-NODA-Mpaa-Pip-B9958 (Z04139) A solution of AlOH-NODA-Mpaa-Pip-B9958 (2 mg, 1.1 µmol) and NaF (48 µg, 1.1 µmol) in 0.2 mL sodium acetate buffer solution (2 mM, pH 7.0) and 0.2 mL ethanol was stirred at 105 °C for 15 min. The reaction mixture was purified by HPLC using the semi-preparative column eluted with 77/23 A/B at a flow rate of 4.5 mL/min. The retention time of AlF-NODA-Mpaa-PipB9958 was 19.5 min, and the yield was 65%. ESI-MS: calculated [M+2H]2+ for AlF-NODAMpaa-Pip-B9958 C83H126AlFN20O19 877.47; found [M+2H]2+ 877.67. In vitro competition binding assays


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The binding affinity of B9958 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 radioligand.5 Synthesis of 68Ga-DOTA-Pip-B9958 (68Ga-Z02176) The radiolabeling experiment was performed following previously published procedures.5 The 68 Ga generator was eluted with a total of 4 mL of 0.6 M HCl. The elution which contained the activity was mixed with 2 mL concentrated HCl. The mixture was passed through a 35-mg DGA resin (Eichrom Technologies LLC, Lisle, IL) column and the column was washed by 3 mL 5 M HCl. After the column was dried by passage of air, 68Ga was eluted off with 0.5 mL water. 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-Pip-B9958 (20 µg). The radiolabeling reaction was carried out under microwave heating for 1 min. The reaction mixture was purified by HPLC using the semi-preparative column eluted with 20/80 CH3CN/PBS (0.1 M, pH 7.4) at a flow rate of 4.5 mL/min. The retention time of 68Ga-Z02176 was 19.6 min. The eluate fraction containing 68 Ga-Z02176 was collected, diluted with water (50 mL), and passed through a C18 Sep-Pak cartridge. The 68Ga-labeled Z02176 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 same semi-preparative column eluted with the same solvent conditions. Synthesis of 68Ga-NODA-Mpaa-Pip-B9958 (68Ga-Z02137) 68

Ga-Z02137 was prepared according to the procedures described above for the synthesis of Ga-Z02176 by using 610 - 1106 MBq aqueous 68GaCl3 solution and 25 µg of NODA-MpaaPip-B9958 as the radiolabeling precursor. The reaction mixture was purified by HPLC using the semi-preparative column eluted with 76/24 A/B at a flow rate of 4.5 mL/min. The retention time of 68Ga-Z02137 was 15.0 min. The collected 68Ga-Z02137 in HPLC solvents was further purified by C18 Sep-Pak cartridge to remove TFA and CH3CN, and formulated with saline as described for the synthesis of 68Ga-Z02176. Quality control was performed using an analytical column eluted with 24/76 A/B at a flow rate of 2 mL/min. The retention time of 68Ga-Z02137 was 7.5 min. 68

Synthesis of 18F-AlF-NODA-Mpaa-Pip-B9958 (18F-Z04139) H218O containing 18F-fluoride was passed through a short anion exchange Trap & Release column (pre-activated with 5 mL brine), and the column was washed with de-ionized water (1 mL × 2). The 18F-fluoride was eluted off with saline (1 mL), and an aliquot (100 µL, 2.85 – 3.68 GBq) was added into a vial pre-loaded with ethanol (110 µL) and AlOH-NODA-Mpaa-PipB9958 (1 mM, 10 µL) solution in sodium acetate buffer (2 mM, pH 4.4). The reaction mixture was incubated at 105 °C for 15 min and then purified by HPLC using the semi-preparative column eluted with 76/24 A/B at a flow rate of 4.5 mL/min. The retention time of 18F-Z04139 was 15.5 min. The collected 18F-Z04139 in HPLC solvents was further purified by C18 Sep-Pak cartridge to remove TFA and CH3CN, and formulated with saline as described for the synthesis of 68Ga-Z02176. Quality control was performed on an analytical column eluted with 24/76 A/B at a flow rate of 2 mL/min. The retention time of 18F-Z04139 was 10.0 min. LogD7.4 measurements



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LogD7.4 values of radiolabeled peptides were measured using the shake flask method as previously reported.5 Imaging and biodistribution study in tumor-bearing mice Biodistribution and PET/CT imaging studies were performed as previously reported.5-8 Male immunodeficient NODSCID IL2RKO mice were obtained from a breeding colony at the Animal Resource Centre of the BC Cancer Research Centre. All experiments were 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, wild-type HEK293T and HEK293T::hB1R tumors were inoculated by subcutaneous injection of 1 × 106 cells on each dorsal flank of the mice. This way, 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 - 2 MBq (0.007 – 0.048 µg) of radiolabeled peptides. For blocking experiments, the radioactive compound was co-injected with 100 µg of the same non-radioactive 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 (Erlangen, Germany) Inveon microPET/CT scanner. Mice bearing two tumors derived from HEK293T and HEK293T::hB1R cells as described above were used. Mice were sedated with 2% isoflurane inhalation for i.v. injection of 6 – 8 MBq (0.044 -0.272 µg) of the radiotracer with or without the presence of 100 µg of excess unlabeled peptide, then allowed to recover and roam freely in their cages for 45 min. At that point, the mice were sedated again and placed onto the scanner. A baseline CT scan was obtained for localization and attenuation correction using 60 kV x-rays at 500 µA, using 3 sequential bed positions with 33% overlap and 220° continuous rotation. Body temperature was maintained by a heating pad during acquisition. A single static emission scan was acquired for 10 min. PET data were acquired in list mode acquisition, reconstructed using the 3d-OSEM-MAP algorithm with CT-based attenuation correction, and co-registered for alignment. The mice were euthanized afterwards and the organs/tissues were harvested for biodistribution as described above. In vivo stability In vivo stability of radiolabeled B9958 derivatives in male NODSCID IL2RKO mice was conducted following previously published procedures, and monitored by radio-HPLC.6 Statistical analysis Statistical analyses were performed by Student’s t-test using the Microsoft Excel software. In Table 3, comparison of uptake in B1R- tumors and other tissues between control and blocked mice was performed using unpaired, two-tailed test. The unpaired one-tailed test was used to compare B1R+ tumor uptake and B1R+ tumor-to-background (B1R- tumor, blood and muscle) ratios between the control and blocked mice. In Table 4, the unpaired two-tailed test was used to


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compare the tissue uptake and contrast ratios between imaged and biodistribution mice. The difference was considered statistically significant when p value was < 0.05.



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RESULTS Peptide synthesis and radiolabeling DOTA-Pip-B9958 and NODA-Mpaa-Pip-B9958 were assembled on solid phase using N-Fmoc protected amino acids, and obtained in 18 and 10% yield, respectively, after purification by HPLC. The HPLC spectra of purified DOTA-Pip-B9958 and NODA-Mpaa-Pip-B9958 are provided as Supplemental Figures 1 and 2, respectively. AlOH-NODA-Mpaa-Pip-B9958 was prepared by incubating NODA-Mpaa-Pip-B9958 with AlCl3 in acetate buffer (2 mM, pH 7.0), and was obtained in 61% yield. The cold standards of Z02176 and Z02137 (Fig. 1) were obtained in 100 and 43% yield, respectively, by reacting their respective precursor with GaCl3 in acetate buffer (0.1 M, pH 4.0). Cold Z04139 (Fig. 1) was prepared by incubating AlOH-NODA-MpaaPip-B9958 with NaF in 1:1 ethanol/acetate buffer (2 mM, pH 7.0), and was obtained in 65% yield. The radiolabeling data are summarized in Table 2. The synthesis times for 68Ga-Z02176, 68 Ga-Z02137 and 18F-Z04139 were 33, 37 and 79 min, respectively. 18F-Z04139 and 68Galabeled Z02176 and Z02137 were prepared in average decay-corrected radiochemical yields of 32 - 47% and with > 99% radiochemical purity. Their average specific activities ranged 100 261 GBq/µmol at the end of synthesis. These tracers were used for imaging and biodistribution studies within 2 h from the end of synthesis, providing the average specific activity of > 50 GBq/µmol at the time of injection.

Table 2 Overall charge, radiolabeling and LogD7.4 data of radiolabeled B1R-targeting tracers. Tracers

Overall charge

Ga-Z02176 Ga-Z02137 18 F-Z04139

+3 +4 +3

68 68

Radiochem. yield (%, decaycorrected) 46 ± 2 (n = 4) 47 ± 11 (n = 3) 32 ± 11 (n = 3)

Radiochem. purity (%)

Specific activity (GBq/µmol, n ≥ 3)

LogD7.4 (n = 3)

> 99 > 99 > 99

210 ± 72 261 ± 74 100 ± 8

-4.15 ± 0.04 -3.35 ± 0.03 -3.90 ± 0.21


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Affinity and lipophilicity measurements Binding affinity to B1R was measured via in vitro competition binding assay using B1Roverxepressing CHO cell membrane and [3H]-[Leu9,des-Arg10]kallidin as the radioligand. Z02176, Z02137 and Z04139 inhibited the binding of [3H]-[Leu9,des-Arg10]kallidin to hB1R on CHO cell membrane in a dose-dependent manner (Fig. 2), and their corresponding Ki values were 2.5 ± 0.8, 2.6 ± 0.7 and 1.4 ± 0.7 nM, respectively. The lipophilicity of radiolabeled Z02176, Z02137 and Z04139 was measured using the traditional shake flask method. These radiolabeled B1R-targeting peptides were highly hydrophilic with average LogD7.4 (D: distribution coefficient) in the range of -4.15 – -3.35 (Table 2).

Figure 2. Representative displacement curves of [3H]-[Leu9,des-Arg10]kallidin by Z02176, Z02137 and Z04139.



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PET/CT imaging The 1-h post-injection (p.i.) static PET/CT images of 68Ga-Z02176, 68Ga-Z02137 and 18FZ04139 in tumor-bearing mice are shown in Fig. 3. All tracers cleared rapidly from background tissues/organs (except kidneys), and were excreted mainly through the renal pathway. Only tumors, kidneys and bladders were clearly visualized in the images. The uptake in tumor was higher by using 68Ga-Z02176 and 18F-Z04139 than 68Ga-Z02137. Co-injection with their respective cold standard (100 µg) effectively blocked uptake of all tracers into B1R+ tumors.

Figure 3. Representative 1 h post-injection MIP (maximum intensity projection) and axial PET/CT images of radiolabeled Z02176, Z02137 and Z04139 in mice bearing both B1R+ (pointed by yellow arrows) and B1R- (pointed by red arrows) tumors (A) without and (B) with co-injection of their cold standard (100 µg).


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Biodistribution The biodistribution data of these three B1R-targeting tracers in tumor-bearing mice are summarized in Table 3, and they are consistent with the observations from PET/CT images. No brain uptake indicates that these tracers did not cross blood-brain barrier. The high kidney uptake (on average 85.2 – 101 %ID/g) and minimal uptake (< 1 %ID/g) in liver and intestines also confirms that these tracers were excreted predominantly via the renal pathway. The uptake in B1R+ tumor was higher by using 68Ga-Z02176 (28.9 ± 6.21 %ID/g) and 18F-Z04139 (22.6 ± 3.41 %ID/g) than 68Ga-Z02137 (14.0 ± 4.86 %ID/g). The uptake in B1R- wild-type HEK293T tumors was low and less than that in blood for all tracers, confirming the high uptake in B1R+ HEK293T::hB1R tumors was mediated by B1R. Blocking with cold standard reduced the uptake of all three tracers in B1R+ tumors by > 90%.

Table 3 Biodistribution data of radiolabeled B1R-targeting tracers at 1 h post-injection in tumorbearing mice. 68

Tissue (%ID/g) Blood Fat Testes Large intestine Small intestine Spleen Liver Pancreas Adrenal glands Kidney Lungs Heart B1R- tumor B1R+ tumor Muscle Bone Brain

Ga-Z02176 Control Blocked (n = 9) (n = 5) 0.55 ± 0.15 0.84 ± 0.11** 0.10 ± 0.04 0.17 ± 0.04** 0.19 ± 0.07 0.30 ± 0.11* 0.23 ± 0.09 0.30 ± 0.12 0.33 ± 0.17 0.36 ± 0.09 0.41 ± 0.25 0.32 ± 0.01 0.43 ± 0.18 0.49 ± 0.23 0.14 ± 0.05 0.18 ± 0.04 0.11 ± 0.07 0.24 ± 0.11** 90.9 ± 22.8 51.2 ± 2.70** 0.69 ± 0.25 0.83 ± 0.23 0.25 ± 0.08 0.31 ± 0.07 0.38 ± 0.17 0.35 ± 0.06 28.9 ± 6.21 0.89 ± 0.13*** 0.19 ± 0.07 0.18 ± 0.04 0.30 ± 0.11 0.34 ± 0.07 0.03 ± 0.01 0.02 ± 0.01

68

Ga-Z02137 Control Blocked (n = 9) (n = 4) 0.46 ± 0.16 0.51 ± 0.21 0.10 ± 0.04 0.09 ± 0.03 0.16 ± 0.05 0.20 ± 0.07 0.22 ± 0.10 0.20 ± 0.07 0.25 ± 0.08 0.29 ± 0.05 0.42 ± 0.28 0.54 ± 0.26 0.48 ± 0.12 0.50 ± 0.13 0.14 ± 0.06 0.13 ± 0.04 0.18 ± 0.11 0.18 ± 0.02 85.2 ± 12.1 70.2 ± 13.5 0.79 ± 0.30 0.65 ± 0.20 0.28 ± 0.16 0.30 ± 0.08 0.31 ± 0.06 0.38 ± 0.21 14.0 ± 4.86 1.06 ± 0.47*** 0.14 ± 0.04 0.22 ± 0.05* 0.30 ± 0.09 0.36 ± 0.07 0.03 ± 0.02 0.02 ± 0.01

18

Control (n = 9) 0.43 ± 0.15 0.09 ± 0.03 0.16 ± 0.04 0.25 ± 0.10 0.36 ± 0.13 0.29 ± 0.07 0.47 ± 0.10 0.12 ± 0.04 0.18 ± 0.05 101 ± 14.4 0.52 ± 0.12 0.23 ± 0.07 0.31 ± 0.02 22.6 ± 3.41 0.14 ± 0.04 0.21 ± 0.05 0.03 ± 0.01

F-Z04139 Blocked (n = 5) 0.65 ± 0.14 0.13 ± 0.03** 0.24 ± 0.03 0.22 ± 0.04 0.54 ± 0.37 0.35 ± 0.06 0.64 ± 0.22 0.15 ± 0.03 0.18 ± 0.08 106 ± 18.4 0.68 ± 0.13 0.30 ± 0.05 0.44 ± 0.24 2.05 ± 0.81*** 0.21 ± 0.07* 0.24 ± 0.06 0.02 ± 0.01

B1R+T:B1R-T 85.4 ± 30.1 2.63 ± 0.70*** 47.5 ± 19.6 1.89 ± 1.35** 74.5 ± 15.1 *** B1R+T:Blood 56.1 ± 17.3 1.08 ± 0.20 34.3 ± 15.2 1.31 ± 0.95** 58.0 ± 20.9 B1R+T:Muscle 167 ± 57.6 5.00 ± 1.23*** 103 ± 30.2 5.23 ± 2.76*** 173 ± 42.9 Significance of differences between control and blocked groups: *p < 0.05; **p < 0.01; ***p < 0.001.


5.16 ± 2.40*** 3.31 ± 1.50*** 10.8 ± 5.10***


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In vivo stability The in vivo stability of 68Ga-Z02176, 68Ga-Z02137, and 18F-Z04139 were evaluated in mice (n = 3), and the blood samples were taken at 5 min p.i. The plasma was isolated, and injected into HPLC to check the percentage of intake tracers. Representative radio-HPLC chromatograms are shown in Fig. 4. Both 68Ga-Z02176 and 18F-Z04139 were fairly stable in vivo in mice with 93.7 ± 0.6 and 95.8 ± 1.0 %, respectively, remaining intact at 5 min p.i. On the other hand, 68GaZ02137 showed faster degradation with only 67.6 ± 4.0 % remaining intact at the same time point.

Figure 4. Representative radio-HPLC chromatograms of (A) 68Ga-Z02176, (B) 68Ga-Z02137, and (C) 18F-Z04139 from QC (left chromatograms) and mouse plasma samples (right chromatograms) taken at 5 min post-injection.


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DISCUSSION Our previous work on the development of B1R-targeting PET tracers led to the identification of B9958 and Pip as the preferred targeting vector and linker, respectively.6-8 However, the only reported B1R-trageting tracer composed of B9958 and Pip was 18F-L08060 (Fig. 1).8 L08060 was designed for radiolabeling with 18F via an 18F-19F isotope exchange reaction on the ammoniomethyl-trifluoroborate (AmBF3) moiety.9-10 Such reaction could be performed directly in aqueous solution, and therefore the tedious drying step needed for the traditional 18F-labeling reaction via a nucleophilic substitution reaction could be eliminated. In addition, direct 18F-19F isotope exchange reaction on AmBF3-conjugated molecules has been shown to generate 18Flabeled analogs with good radiochemical yield (~ 10 - 20%) and specific activity (~ 74 GBq/µmol), and has been successfully applied for the design and preparation of a variety of PET tracers.8,11-15 Despite excellent binding affinity (Ki = 0.5 nM), the uptake in B1R+ tumor and tumor-tobackground contrast ratios of 18F-L08060 were inferior to those of 68Ga-P04158 and 68GaZ02090 (Table 1). 68Ga-P04158 and 68Ga-Z02090 were derived from different targeting sequences (B9858 and B9958, respectively), but shared the same radiolabel-chelator complex (68Ga-DOTA) and linker (dPEG2).7 This observation prompted us to think that maybe 18FAmBF3 motif is not the optimal radiolabel-chelator complex in conjunction with B9958 and Pip for the design of B1R-targeting tracers. To optimize selection of the radiolabel-chelator complex, we subsequently prepared and evaluated 68Ga-DOTA-, 68Ga-NODA-Mpaa- and 18F-AlF-NODAMpaa-conjugated Pip-B9958 derivatives (68Ga-Z02176, 68Ga-Z02137 and 18F-Z04139, respectively; Fig. 1), and compared the results with those previously obtained using 18F-L08060. We focused on the radioisotopes 68Ga and 18F as they are the most widely used positron emitters for labeling peptides for imaging. 68Ga could be readily available from the 68Ge-68Ga generator, and be used at imaging facilities that do not have a cyclotron. On the other hand, 18F could be easily produced in a multi-Curie level by a medical cyclotron, and 18F-labeled tracers could be prepared at centralized pharmacies and distributed for use to imaging facilities up to few hours away. For 18F-labeling, we compared two strategies: formation of aluminum 18F-fluoride (18FAlF)16 and the 18F-19F isotope exchange reaction on AmBF3 in L08060. Both strategies could be performed directly in aqueous solution, and have been successfully applied for the design of 18Flabeled peptide-based PET tracers.8,14-20 For 68Ga-labeling, we compared the use of NODA and a well-established chelator DOTA. Z02176, Z02137 and Z04139 all retained high affinity binding to B1R, and their Ki values (1.4 – 2.6 nM) were comparable to those of previously obtained B9958 derivatives, L08060 and Z02090 (0.5 and 1.1 nM, respectively; Table 1). This is consistent with previous observations that modification at the N-terminus of B9958 was tolerable and the modified derivatives would still retain high binding affinity to B1R.7-8,21 As shown in Table 2, the radiochemical yield (32 ± 11%, decay-corrected) and specific activity (100 ± 8 GBq/µmol) of 18F-Z04139 were similar to those of previously reported 18F-L08060 (24 ± 1.8%, non-decay-corrected; up to 87 GBq/µmol).8 These data demonstrated that both 18Flabeling methods (via the formation of 18F-AlF-NODA in 18F-Z04139 and via 18F-19F isotope exchange on the AmBF3 motif of L08060) were effective and comparable strategies for the preparation of 18F-labeled peptides. The radiochemistry data of 68Ga-Z02137 and 68Ga-Z02176



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were also very similar, indicating that NODA can chelate 68Ga as efficiently as DOTA does. Giving that several NODA-conjugates have already been developed and successfully radiolabeled with 18F-AlF for imaging,20,22-23 these conjugates could potentially be used directly as precursors to generate 68Ga-labeled tracers as well. The three novel B1R-targeting tracers reported here all generated high-contrast PET images and the B1R+ tumors were clearly visualized (Fig. 3A). Co-injection with their corresponding cold standard (100 µg) reduced the uptake in B1R+ tumors by > 90%, demonstrating the uptake of these tracers was specific (Fig. 3B). An interesting finding is that in blocked mice the uptake in B1R+ tumor and the B1R+ tumor-to-background contrast ratios of 18F-Z04139 were ~ 2-fold of those obtained using 68Ga-Z02176 and 68Ga-Z02137 (uptake in B1R+ tumor: 2.05 vs. 0.89 and 1.06 %ID/g; B1R+ T/B ratio: 3.31 vs. 1.08 and 1.31; B1R+ T/M ratio: 10.8 vs. 5.00 and 5.23; Table 3). Co-injection with the cold standard significantly reduced the specific activity (> 500fold) of the radiotracers, and presumably blocked most of the B1R, and left only a small fraction of B1R available for binding to the injected radiotracers. The fact that the good uptake in B1R+ tumor and tumor-to-background contrast ratios in the blocked group of 18F-Z04139 suggests that 18 F-Z04139 might be more sensitive for imaging low expression level of B1R even with reduced specific activity. To verify this, we reanalyzed the biodistribution data of 68Ga-Z02176 and 18F-Z04139, and divided the data of the control group in Table 3 into two sub-groups (imaged and biodistribution mice) as shown in Table 4. The imaged mice were the mice that received a higher dose of tracer, and were imaged at ~ 50 – 60 min p.i., and euthanized afterwards for tissue collection. The biodistribution mice were the mice that received a lower dose of the tracer, and euthanized at 1-h p.i. for tissue collection. For 68Ga-Z02176, the imaged and biodistribution mice received 5.86 – 8.40 and 0.58 – 1.48 MBq of the tracer, respectively. For 18F-Z04139, the imaged and biodistribution mice received 8.06 – 8.16 and 1.32 – 2.30 MBq of the tracer, respectively. Despite a limited number (n = 3 for the imaged mice) the reanalyzed biodistribution data showed that uptake of 68Ga-Z02176 in B1R+ tumor was significantly lower (p < 0.001) in imaged mice (21.3 ± 1.66 %ID/g) than that in biodistribution mice (32.8 ± 2.76 %ID/g). However, no difference in B1R+ tumor uptake was observed between imaged and biodistribution mice receiving 18F-Z04139 (Table 4). Since all mice were euthanized at ~ 1 h p.i., timing should not be the cause for the uptake difference. Compared with biodistribution mice that were anesthetized again at 1 h p.i. for euthanasia, imaged mice were anesthetized at ~ 45 min p.i., and remained anesthetized during a 10-min imaging session before being euthanized at ~ 1 h p.i. Because radiolabeled peptides typically have a very fast pharmacokinetic profile, we believe the additional anesthesia time at a later time point should not be the reason causing the reduced uptake of 68Ga-Z02176 in B1R+ tumor in imaged mice. Most likely, the main cause was the higher (4- to 5-fold) injected dose (mass) received by the imaged mice compared with the biodistribution mice. However, to confirm this hypothesis, a more comprehensive imaging/biodistribution study in mice with various amount of injected does (mass) of 68Ga-Z02176 is needed. Nevertheless, our preliminary data indicate that for 68Ga-Z02176 the quality of its PET images could be very susceptible to the amount of the injected mass. Once translated to the clinic, 68Ga-Z02176 should be used with the highest possible specific activity to ensure the good quality of PET images. On the contrary, no uptake difference was observed between imaged and biodistribution mice receiving 18F-Z04139


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suggesting the image quality of this tracer is tolerable within a certain mass (specific activity) range. Therefore, 18F-Z04139 could be potentially prepared at a centralized radiopharmacy, and used for imaging at remote hospitals few hours later without losing the quality of PET images due to reduced specific activity of the tracer at the time of injection.

Table 4 Biodistribution data (1 h p.i.) of 68Ga-Z02176 and 18F-Z04139 in tumor-bearing mice used for imaging or biodistribution study. 68

Tissue (%ID/g) Blood Fat Testes Large intestine Small intestine Spleen Liver Pancreas Adrenal glands Kidney Lungs Heart B1R- tumor B1R+ tumor Muscle Bone Brain

Ga-Z02176 Imaged mice Biodistribution (n = 3) mice (n = 6) 0.54 ± 0.23 0.55 ± 0.12 0.09 ± 0.05 0.11 ± 0.03 0.17 ± 0.06 0.20 ± 0.08 0.24 ± 0.08 0.22 ± 0.10 0.23 ± 0.10 0.38 ± 0.18 0.31 ± 0.15 0.46 ± 0.28 0.43 ± 0.20 0.43 ± 0.19 0.15 ± 0.09 0.14 ± 0.03 0.09 ± 0.04 0.12 ± 0.03 65.3 ± 8.51 104 ± 14.6** 0.78 ± 0.36 0.65 ± 0.21 0.26 ± 0.13 0.25 ± 0.06 0.30 ± 0.14 0.42 ± 0.19 21.3 ± 1.66 32.8 ± 2.76*** 0.18 ± 0.09 0.19 ± 0.06 0.26 ± 0.13 0.31 ± 0.10 0.02 ± 0.01 0.03 ± 0.01

18

F-Z04139 Imaged mice Biodistribution (n = 3) mice (n = 6) 0.52 ± 0.19 0.39 ± 0.13 0.09 ± 0.02 0.12 ± 0.08 0.17 ± 0.04 0.15 ± 0.04 0.33 ± 0.03 0.21 ± 0.11 0.27 ± 0.03 0.40 ± 0.13 0.29 ± 0.08 0.29 ± 0.07 0.50 ± 0.07 0.46 ± 0.11 0.13 ± 0.04 0.12 ± 0.04 0.20 ± 0.04 0.17 ± 0.05 88.4 ± 7.36 108 ± 12.7 0.54 ± 0.12 0.51 ± 0.13 0.25 ± 0.06 0.21 ± 0.07 0.31 ± 0.03 0.31 ± 0.02 22.2 ± 3.49 22.7 ± 3.69 0.16 ± 0.03 0.13 ± 0.04 0.23 ± 0.05 0.21 ± 0.05 0.03 ± 0.00 0.02 ± 0.01

B1R+T:B1R-T 76.9 ± 23.5 89.7 ± 34.1 72.4 ± 17.3 76.1 ± 15.8 B1R+T:Blood 43.0 ± 14.3 62.6 ± 15.6 50.6 ± 31.3 61.7 ± 16.0 B1R+T:Muscle 132 ± 49.3 184 ± 57.1 150 ± 54.8 185 ± 35.2 Significance of differences between groups of imaging and biodistribution mice: **p < 0.01; ***p < 0.001.



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The average uptake of these B9958 derivatives (68Ga-Z02176, 68Ga-Z02137 and 18F-Z04139) in B1R+ tumor was ≥ 14.0 %ID/g (Table 3) that was much higher than those previously obtained using 68Ga-DOTA derivatives of [Hyp4,Cha6,Leu9,des-Arg10]kallidin (1.96 – 4.15 %ID/g, Table 1).6 The increase in B1R+ tumor uptake could be partly due to improved binding affinity of these B9958 derivatives (Ki = 1.4 - 2.6 nM) over previously reported 68Ga-labeled [Hyp4,Cha6,Leu9,des-Arg10]kallidin derivatives (Ki = 3.6 – 28 nM, Table 1). Another possible factor for the increased uptake in B1R+ tumor is the enhanced in vivo stability of radiolabeled B9958 derivatives. B9958 (Lys[Hyp4,Cpg6,D-Tic8,Cpg9,des-Arg10]kallidin) contains four unnatural amino acids, whereas the previously tested [Hyp4,Cha6,Leu9,des-Arg10]kallidin contains only two unnatural amino acids. With more unnatural amino acid substitutions in the targeting sequence, B9958 derivatives are expected to have a higher in vivo stability against degradation by peptidases. To verify this, in vivo stability study was conducted. Indeed, these B9958 derivatives were fairly stable in vivo with on average > 67% of the traces remaining intact at 5 min p.i. (Fig. 4). In comparison, only 8 – 17% of 68Ga-[Hyp4,Cha6,Leu9,des-Arg10]kallidin remained intact under the same assay conditions.6 The four radiolabeled Pip-B9958 derivatives (Fig. 1) showed comparable high binding affinity to B1R (Ki = 0.5 – 2.6 nM). However, 18F-L08061 showed the lowest uptake in B1R+ tumor (4.20 %ID/g) and B1R+ tumor-to-background contrast ratios (T/B: 14.7; T/M: 48.6), while 68GaZ02176 and 18F-Z04139 showed the highest uptake in B1R+ tumor (≥ 22 %ID/g) and B1R+ tumor-to-background contrast ratios (T/B: > 56; T/M: > 160). These data clearly demonstrated that binding affinity alone is not very useful for use to predict which tracers would be more suitable for imaging. Other factors such as pharmacokinetics and in vivo stability all need to be taken into account. Therefore, unless better prediction could be successfully achieved using other strategies such as molecular docking, systematic selection and evaluation of binding sequence, linker and radiolabel-chelator complex are still needed to optimize the design of peptide-based radiotracers. It should be noted that we did not conduct a head-to-head comparison between these radiolabeled Pip-B9958 derivatives (Fig. 1) as 18F-L08061 has been previously reported.8 Ideally they should be compared in biodistribution and imaging studies in the same group of mice bearing tumors derived from the same batches of cells. This is to eliminate the biological variation between different groups of mice, and to eliminate the variation of B1R expression level between different batches of cells as B1R expression level could be changed after several passages. Another noticeable features for these three tracers reported here are their extremely high kidney uptake (on average > 85 %ID/g, Table 3) and overall charge (+3 - +4). The kidney uptake and overall charge of previously reported B1R-targeting tracers are provided in Table 1. By using the sequences of [Leu9,des-Arg10]kallidin and [Hyp4,Cha6,Leu9,des-Arg10]kallidin, the overall charge of these tracers was +1 - +2, and their average kidney uptake ranged 3 – 5 %ID/g.5-7 When combining the sequence of B9858/B9958 with 68Ga-DOTA-dPEG2 or 18F-AmBF3-Mta, the overall charge of these tracers increased to +2 - +3, and their average uptake in kidneys increased to 30 – 70 %ID/g.7-8 The increased kidney uptake among these tracers could be potentially caused by the introduction of additional positive charge(s) by using B9858/B9958 sequence instead of [Hyp4,Cha6,Leu9,des-Arg10]kallidin, and by using the cationic Pip instead of the neutral dPEG2 linker. Therefore, investigation on the use of more negatively charged chelator (such as DOTAGA instead of DOTA) to balance the overall charge and minimize the kidney uptake is currently underway.


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In summary, we have synthesized and evaluated three novel B1R-targeting PET tracers (68GaZ02176, 68Ga-Z02137 and 18F-Z04139) bearing the same B9958 sequence and Pip linker but a different radiolabel-chelator complex. 68Ga-Z02176 and 18F-Z04139 outperformed 68Ga-Z02137, and previously reported 68Ga- and 18F-labeled B1R-targeting tracers. With superior tumor uptake and target-to-background contrast ratios, 68Ga-Z02176 and 18F-Z04139 are promising B1Rtrageting PET tracers, and warrant further investigation for cancer imaging.

AUTHOR INFORMATION Corresponding Author *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: 604-6758218. E-mail: [email protected]. *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: 604-6758218. E-mail: [email protected]. Author Contributions †

Zhengxing Zhang and Guillaume Amouroux contributed equally to this work.

Notes The authors declare no competing financial interest.

ACKNOWLEDGEMENTS This work was supported by the Canadian Institutes of Health Research (MOP-126121). We thank Navjit Hundal-Jabal, Nadine Colpo, Wade English, Julius Klug, and Milan Vuckovic for their technical assistance.

ASSOCIATED CONTENT Supporting Information HPLC spectra of purified DOTA-Pip-B9958 and NODA-Mpaa-Pip-B9958 are provided in Supporting Information. This information is available free of charge via the Internet at http://pubs.acs.org/.



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