Imaging Bradykinin B1 Receptor with 68Ga-Labeled [des-Arg10

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Imaging bradykinin B1 receptor with Ga-labeled [des-Arg ]kallidin derivatives: effect of the linker on biodistribution and tumor uptake Guillaume Amouroux, Jinhe Pan, Silvia Jenni, Chengcheng Zhang, Zhengxing Zhang, Navjit Hundal-Jabal, Nadine Colpo, Zhibo Liu, Francois Benard, and Kuo-Shyan Lin Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.5b00070 • Publication Date (Web): 23 Jun 2015 Downloaded from http://pubs.acs.org on June 25, 2015

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Molecular Pharmaceutics

Imaging bradykinin B1 receptor with 68Ga-labeled [desArg10]kallidin derivatives: effect of the linker on biodistribution and tumor uptake Guillaume Amouroux†,§, Jinhe Pan†,§, Silvia Jenni§, Chengcheng Zhang§, Zhengxing Zhang§, Navjit Hundal-Jabal§, Nadine Colpo§, Zhibo Liu‡, 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: 604-675-8218. 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: 604-675-8218. E-mail: [email protected]. Author Contributions †

Guillaume Amouroux and Jinhe Pan 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 overexpressed in cancers but minimally expressed in normal healthy tissues represents an attractive biomarker for the development of cancer imaging agents. The goal of this study was to evaluate the effect of different linkers on the pharmacokinetics and tumor uptake of a B1R-targeting radio-peptide sequence: 68Ga-DOTALinker-Lys-Arg-Pro-Hyp-Gly-Cha-Ser-Pro-Leu. Four peptides, SH01078, P03034, P04115 and P04168, with 6-aminohexanoic acid, 9-amino-4,7-dioxanonanoic acid, Gly-Gly, and 4amino-(1-carboxymethyl)piperidine, respectively as the linker were synthesized and evaluated. In vitro competition binding assays showed that the Ki values of SH01078, P03034, P04115 and P04168 were 27.8 ± 4.9, 16.0 ± 1.9, 11.4 ± 2.5 and 3.6 ± 0.2 nM, respectively. Imaging and biodistribution studies were performed in mice bearing both B1Rpositive HEK293T::hB1R and B1R-negative HEK293T tumors. All tracers showed mainly renal excretion with excellent tumor visualization and minimal background activity except for kidneys and bladder. The average uptake of 68Ga-labeled SH01078, P03034 and P04115 in HEK293T::hB1R tumor was similar (1.96 – 2.17 %ID/g) at 1 h post-injection. 68Ga-P04168 generated higher HEK293T::hB1R tumor uptake (4.15 ± 1.13 %ID/g) and lower background activity, leading to > 2-fold improvement in HEK293T::hB1R tumor-to-background (HEK293T tumor, blood, muscle, and liver) contrasts over 68Ga-labeled SH01078, P03034 and P04115. Our results indicate that the choice of linker affects binding affinity, pharmacokinetics, and tumor targeting. The use of the cationic 4-amino-(1carboxymethyl)piperidine linker improved tumor visualization, and the resulting 68Ga-P04168 might be promising for clinical application for imaging B1R-expressing tumors with positron emission tomography.

KEYWORDS: Bradykinin B1 receptor; Gallium-68; Antagonist; Kallidin; Linker; Positron emission tomography



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INTRODUCTION Bradykinin B1 and B2 receptors (B1R and B2R) are G-protein-coupled receptors and have long been known to function in pain and inflammatory pathways.1-2 The endogenous ligands of B2R are bradykinin and its close analog kallidin (Table 1), whereas their des-Arg analogs ([des-Arg9]bradykinin and [des-Arg10]kallidin) are the agonists of B1R.3 While B2R is constitutively and ubiquitously expressed, B1R is minimally expressed in healthy normal tissues but has been shown to be up-regulated in a variety of cancers.4-13 The activation of B1R was reported to induce angiogenesis and enhance survival, proliferation and migration/metastasis of cancer cells.14-22 Consequently, B1R has been proposed as a promising therapeutic target of cancer.23 The use of non-invasive modalities such as positron emission tomography (PET) for imaging B1R expression could potentially facilitate the development of B1R-targeted therapies. Suitable B1R-targeting PET tracers can be used to select potential responders for treatment, optimize treatment dosage, and monitor treatment response. Using the [Leu9,des-Arg10]kallidin sequence24-25 as the targeting vector, we previously reported the synthesis and evaluation of 68Ga-P03083 (Table 1) for imaging B1R expression with PET.26 Despite good B1R binding affinity (Ki = 2.6 ± 0.7 nM) 68Ga-P03083 was ineffective at visualizing B1R-positive (B1R+) HEK293T::hB1R tumors due to fast in vivo degradation of the native [des-Arg10]kallidin sequence.24,26 Subsequent substitution of Pro4 and Phe6 of P03083 with unnatural amino acids hydroxyproline (Hyp) and cyclohexylalanine (Cha), respectively to improve in vivo stability led to the discovery of 68Ga-SH01078 (Table 1).26 Although it had lower B1R binding affinity (Ki = 27.8 ± 4.9 nM), 68Ga-SH01078 greatly enhanced visualization of B1R+ tumors. Replacing the linear-carbon 6-aminohexanoic acid (Ahx) linker of 68Ga-SH01078 with a polyethylene glycol linker, 9-amino-4,7-dioxanonanoic acid (dPEG2), resulted in 68Ga-P03034 (Table 1) with enhanced B1R binding affinity (Ki = 16.0 ± 1.9 nM).26 In addition to B1R binding affinity, the choice of linker could potentially affect their pharmacokinetics, uptake in target of interest, and overall target-to-background contrast. Therefore, besides the previously reported 68Ga-SH01078 and 68Ga-P03034, we synthesized 68 Ga-P04115 and 68Ga-P04168 (Table 1) with additional two different types of linkers (GlyGly and 4-amino-(1-carboxymethyl)piperidine (Pip), respectively) for comparison. Herein, we present the synthesis of 68Ga-P04115 and 68Ga-P04168, and comparative studies of these four 68 Ga-labeled B1R-targeting tracers based on the same general construct: 68Ga-DOTA-LinkerLys-Arg-Pro-Hyp-Gly-Cha-Ser-Pro-Leu.


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Table 1 Amino acid sequences and B1R binding affinities of bradykinin and related peptides Peptide

Radiolabel complex

Linker

Bradykinin Kallidin [des-Arg10]Kallidin [Leu9,des-Arg10]Kallidin P03083 SH01078 P03034 P04115 P04168 a pEC50. bpIC50.

Ga-DOTA Ga-DOTA Ga-DOTA Ga-DOTA Ga-DOTA

Ahx Ahx dPEG2 Gly-Gly Pip

B1R binding domain Lys 1 Lys Lys Lys Lys Lys Lys Lys

Arg Arg 2 Arg Arg Arg Arg Arg Arg Arg

Pro Pro 3 Pro Pro Pro Pro Pro Pro Pro

Pro Pro 4 Pro Pro Pro Hyp Hyp Hyp Hyp

Gly Gly 5 Gly Gly Gly Gly Gly Gly Gly

Phe Phe 6 Phe Phe Phe Cha Cha Cha Cha


Ser Ser 7 Ser Ser Ser Ser Ser Ser Ser

Pro Pro 8 Pro Pro Pro Pro Pro Pro Pro

Phe Phe 9 Phe Leu Leu Leu Leu Leu Leu

Arg Arg 10

Ki (nM) (mean ± SD) 5.7a 7.4a 9.7a 8.9b 2.6 ± 0.7 27.8 ± 4.9 16.0 ± 1.9 11.4 ± 2.5 3.6 ± 0.2

Ref 25 25 25 25 26 26 26 -


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Figure 1. Chemical structures of (A) SH01078, (B) P03034, (C) P04115, and (D) P04168. Linkers (A: Ahx; B: dPEG2; C: Gly-Gly; D: Pip) and unnatural amino acids (Hyp and Cha) in the receptor-binding domain are shown in blue and red, respectively.


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MATERIALS AND METHODS General P03034, SH01078, and their 68Ga analogs were prepared according to previously reported procedures26. All other chemicals and solvents were obtained from commercial sources, and used without further purification. Balb/c mouse plasma for stability studies was obtained from Innovative Research (Novi, MI). 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 68Ga-labeled 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 radiodetector was connected to a Bioscan B-FC-1000 Flow-count System, and the output from the Bioscan Flow-count system was fed into an Agilent 35900E Interface which converted the analog signal to digital signal. The operation of the Agilent HPLC system was controlled using the Agilent ChemStation software. The HPLC columns used were a semi-preparative column (Phenomenex C18, 5 µ, 250 × 10 mm) and an analytical column (Eclipse XOB-C18, 5 µ, 150 × 4 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 a Bruker (Billerica, MA) Autoflex MALDI-TOF system and a Bruker Esquire-LC/MS system with ESI ion source. 68Ga was obtained from an Eckert & Ziegler (Berlin, Germany) IGG100 68Ga generator, and was purified according to the previously published procedures using DGA resin column.26 Radioactivity of 68Ga-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 Packard (Meriden, CT) Cobra II 5000 Series auto-gamma counter. Synthesis of DOTA-Gly-Gly-[Hyp4,Cha6,Leu9,des-Arg10]kallidin This peptide was synthesized via the Nα-Fmoc solid-phase peptide synthesis strategy starting from Fmoc-Leu-Wang resin. The resin was treated with 20% piperidine in DMF to remove the Nα-Fmoc protecting group. The following Fmoc-protected amino acids (3 equivalents) including Fmoc-Pro-OH, Fmoc-Ser(tBu)-OH, Fmoc-Cha-OH, Fmoc-Gly-OH, FmocHyp(tBu)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Lys(Boc)-OH, and the chelator DOTA tri-t-butyl ester were subsequently coupled according to sequence. The coupling was carried out in NMP with standard in situ activating reagent HBTU (3 equivalents) in the presence of DIEA (6 equivalents). At the end of elongation, the peptide was de-protected and simultaneously cleaved from the resin by treating with 95/2.5/2.5 TFA/H2O/TIS (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 precipitated crude peptides were collected by centrifugation, and purified by HPLC using the semi-preparative column eluted with 80/20 A/B at a flow rate of 4.5 mL/min. The retention time of DOTA-Gly-Gly-[Hyp4,Cha6,Leu9,desArg10]kallidin was 16.7 min, and the yield of the peptide was 26 %. MALDI-MS: calculated [M+H]+ for DOTAGly-Gly-[Hyp4,Cha6,Leu9,desArg10]kallidin C67H114N19O21 1520.8, found 1520.9 (see Supplemental Figure 1); ESI-MS: found [M+H]+ 1521.0. Synthesis of DOTA-Pip-[Hyp4,Cha6,Leu9,des-Arg10]kallidin



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This peptide was synthesized following the procedures described above for the synthesis of DOTA-Gly-Gly-[Hyp4,Cha6,Leu9,desArg10]kallidin by replacing the linker Gly-Gly with Pip. The crude peptide was purified by HPLC using the semi-preparative column eluted with 81/19 A/B at a flow rate of 4.5 mL/min. The retention time of DOTA-Pip[Hyp4,Cha6,Leu9,desArg10]kallidin was 13.0 min. The yield of the peptide was 26 %. MALDI-MS: calculated [M+H]+ for DOTA-Pip-[Hyp4,Cha6,Leu9,desArg10]kallidin C70H120N19O20 1546.9, found 1546.9 (see Supplemental Figure 2); ESI-MS: found [M+H]+ 1546.4. Synthesis of Ga-DOTA-Gly-Gly-[Hyp4,Cha6,Leu9,des-Arg10]kallidin (P04115) A solution of DOTA-Gly-Gly-[Hyp4,Cha6,Leu9,des-Arg10]kallidin (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 a semi-preparative column eluted with 80/20 A/B at a flow rate of 4.5 mL/min. The retention time of P04115 was 16.7 min, and the yield was 88%. MALDI-MS: calculated [M+H]+ for P04115 C67H111GaN19O21 1586.7, found 1586.8 (see Supplemental Figure 3); ESI-MS: found [M+H]+ 1586.8. Synthesis of Ga-DOTA-Pip-[Hyp4,Cha6,Leu9,desArg10]kallidin (P04168) P01468 was synthesized according to the procedures described above for the synthesis of P04115. The reaction mixture was purified by HPLC using the semi-preparative column eluted with 80/20 A/B at a flow rate of 4.5 mL/min. The retention time of P04168 was 15.3 min, and the yield was 89%. MALDI-MS: calculated [M+H]+ for P04168 C70H117GaN19O20 1612.8, found 1612.8 (See Supplemental Figure 4); ESI-MS: found [M+H]+ 1613.1. Synthesis of 68Ga-DOTA-Gly-Gly-[Hyp4,Cha6,Leu9,des-Arg10]kallidin (68Ga-P04115) The radiolabeling experiment was performed following previously published procedures.26 68 GaCl3 solution (0.5 mL in water) was added to a 4-mL glass vial preloaded with 0.7 mL of HEPES buffer (2 M, pH 5.0) and DOTA-Gly-Gly-[Hyp4,Cha6,Leu9,des-Arg10]kallidin (100 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 18/82 CH3CN/PBS (pH 7.1) at a flow rate of 4.5 mL/min. The retention time of 68Ga-DOTA-GlyGly-[Hyp4,Cha6,Leu9,desArg10]kallidin was 15.7 min. Synthesis of 68Ga-DOTA-Pip-[Hyp4,Cha6,Leu9,des-Arg10]kallidin (68Ga-P04168) 68

Ga-P04168 was prepared according to the procedures described above for the synthesis of Ga-P04115 by using 70 µg of DOTA-Pip-[Hyp4,Cha6,Leu9,desArg10]kallidin as the radiolabeling precursor. The reaction mixture was purified by HPLC using the semipreparative column eluted with 18/82 CH3CN/PBS (pH 7.1) at a flow rate of 4.5 mL/min. The retention time of 68Ga-DOTA-Pip-[Hyp4,Cha6,Leu9,desArg10]kallidin was 16.7 min. 68

In vitro competition binding assays The binding affinity to B1R was measured using competition binding assays on B1Rexpressing CHO-K1 cell membranes as reported previously.26 Fluorometric measurement of calcium release


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Intracellular calcium release in cells upon binding with B1R-targeting [des-Arg10]kallidin analogs was investigated using a FLIPR Calcium 6 Assay Kit from Molecular Devices (Sunnyvale, CA) following manufacturer’s experimental protocols. Briefly, 50,000 HEK293T::B1R cells were seeded overnight in 96-well clear bottom black plates (product #3603) from Corning Inc. (Corning, NY) to reach 90% confluency at the time of the assay. Loading buffer (calcium-sensitive dye in HBSS, 20 mM HEPES, pH 7.4, 100 µl) was added to each 100 µl of cells, and incubated with cells for 30 min at 37°C under 5% CO2. After the incubation, the cells were placed in a Molecular Devices FlexStation 3 microplate reader. The baseline of the fluorescent signal was established for 20 sec, and the tested peptides (or PBS) were added to the cells to reach a final concentration of 5 and 50 nM. [des-Arg10]kallidin and [Leu9,desArg10]kallidin were used as the agonist and antagonist control, respectively. The fluorescent signal was then monitored for 100 sec. Relative fluorescence unit (RFU = Max Min) was used to evaluate the extent of calcium release induced by each peptide. LogD7.4 measurements LogD7.4 values of 68Ga-labeled peptides were measured using the shake flask method as previously reported.27 Stability in Mouse Plasma Radiotracer stability was assessed according to previously published procedures in balb/c mouse plasma for 5, 15, 30, and 60 min at 37°C.26 In vivo plasma stability, biodistribution and PET/CT imaging studies Biodistribution and PET/CT imaging studies were performed as previously reported.26 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 in vivo plasma stability study, mice were sedated with 2% isoflurane, and injected with ~ 15 MBq of 68Ga-labeled peptides via the tail vein. At 5 min post-injection (p.i.), the mice were euthanized by CO2 asphyxiation. The blood was promptly drawn via cardiac puncture, and mixed with acetonitrile to reach a final concentration of acetonitrile at ~ 10 – 15%. The mixture was centrifuged for 15 min, and the supernatant was collected and analyzed by an Agilent radio-HPLC system consisting of a model 1260 Infinity pump, a model 1260 Infinity UV absorbance detector (set at 220 nm), a Bioscan NaI scintillation detector, and a semipreparative column (Phenomenex C18, 5 µ, 250 × 10 mm) eluted with 18/82 MeCN/PBS (pH 7.1) at a flow rate of 4.5 mL/min. 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. Each mouse had both a B1R+ and B1R- tumor. After 2 weeks of growth, palpable tumors measuring approximately 7 mm in diameter were obtained. Mice were injected with ~ 3.7 MBq of 68Galabeled 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 of interest were harvested, rinsed with normal saline, blotted dry,



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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 the radiotracer (~ 3.7 MBq) with or without the presence of 100 µg of excess unlabeled peptide, then allowed to recover and roam freely in their cages for 55 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 harvested for biodistribution. Statistical analysis Statistical analyses were performed by Student’s t-test using the Microsoft Excel software. 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, muscle, liver and kidney) ratios between the control and blocked mice, whereas a paired one-tailed test was used to compare the uptake in B1R+ and B1R- tumors in the same mice. The difference was considered statistically significant when P value was < 0.05.


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RESULTS Chemistry and radiochemistry DOTA-Gly-Gly-[Hyp4,Cha6,Leu9,des-Arg10]kallidin and DOTA-pip-[Hyp4,Cha6,Leu9,desArg10]kallidin were both obtained in 26% yield. After complexation with non-radioactive Ga, P04115 and P04168 were obtained in 88 and 89% yields, respectively. The identities of these peptides were confirmed by MALDI-MS (see Supplemental Figures 1-4) and ESI-MS analyses. The radiochemical data are summarized in Table 2. These four 68Ga-labeled B1R-targeting [des-Arg10]kallidin derivatives were obtained in average decay-corrected radiochemical yields ranging from 55 – 69% with > 99% radiochemical purity after HPLC purification (see Supplemental Figures 5-9). Their average specific activities were ≥ 111 GBq/µmol at the end of synthesis. Table 2 Radiolabeling, LogD7.4 and in vitro/in vivo plasma stability data of 68Ga-labeled [des-Arg10]kallidin derivativesa Plasma stability (% intact) Radiochem. Specific In vitro In vivo yield (%, Radiochem. activity LogD7.4 Tracers decaypurity (%) (GBq/µmol, (n = 3) 5 15 30 60 5 corrected) n ≥ 3) min min min min min 68 Ga-SH01078b 56 ± 20 (n=4) > 99 189 ± 59 -2.69 ± 0.25 99 99 99 99 9 ± 2 (n=3) 68 Ga-P03034b 69 ± 8 (n=7) > 99 222 ± 37 -2.76 ± 0.11 99 97 94 91 8 ± 2 (n=3) 68 Ga-P04115 55 ± 12 (n=9) > 99 130 ± 67 -2.99 ± 0.22 92 89 87 84 11 ± 3 (n=3) 68 Ga-P04168 58 ± 14 (n=9) > 99 111 ± 59 -2.82 ± 0.08 98 97 94 88 17 ± 4 (n=3) a Data are presented as mean ± SD. bThe data for 68Ga-SH01078 and 68Ga-P03034 were reported previously.26

Lipophilicity and in vitro/vivo plasma stability The lipophilicity of these 68Ga-labeled peptides was measured via traditional shake flask method and the results are shown in Table 2. These peptides are highly hydrophilic with average LogD7.4 values in the range of -2.69 to -2.99. The stability of these 68Ga-labeled peptides was assessed both in vitro and in vivo (Table 2). All tracers showed relatively good in vitro stability in mouse plasma with ≥ 84% remaining intact after 1-h incubation at 37 °C. 68 Ga-SH01078, in particular, was the most stable one with negligible degradation over the course of 1 h. However, the in vivo stability study showed that these 68Ga-labeled [desArg10]kallidin derivatives were metabolized quickly in mice. At 5 min p.i., only 9 ± 2, 8 ± 2, 11 ± 3 and 17 ± 4 of 68Ga-labeled SH01078, P03034, P04115 and P04168, respectively, remained intact (see Supplemental Figures 10-13).



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Binding affinity and agonist/antagonist characteristics In vitro competition binding assays were performed to measure their binding affinity to hB1R. As shown in Fig. 2, these four Ga-labeled peptides effectively inhibited the binding of [3H][Leu9,des-Arg10]kallidin to the hB1R expressed on CHO-K1 cell membranes in a dosedependent manner. The calculated Ki values of SH01078, P03034, P04115 and P04168 were 27.8 ± 4.9, 16.0 ± 1.9, 11.4 ± 2.5, and 3.6 ± 0.2 nM, respectively. The profiles of calcium release in HEK293T::hB1R cells induced by B1R-targeting peptides are shown in Fig. 3. [Des-Arg10]kallidin (Fig. 3A), an endogenous B1R agonist, caused the release of calcium in a dose-dependent manner: 145 ± 20 and 178 ± 25 RFU at 5 and 50 nM, respectively. The synthetic B1R antagonist [Leu9,des-Arg10]kallidin (Fig. 3B) caused moderate calcium release only at high concentration (64 ± 34 RFU at 50 nM). SH01078 (Fig. 3C) and P04115 (Fig. 3E) did not induce significant amount of calcium release even at 50 nM. Similar to the antagonist control ([Leu9,des-Arg10]kallidin) P03034 (Fig. 4D) and P04168 (Fig. 4F) caused moderate calcium release (44 ± 8 and 24 ± 16 RFU, respectively) only at 50 nM.

Figure 2. Representative displacement curves of [3H]-[Leu9,des-Arg10]kallidin by SH01078, P03034, P04115 and P04168.


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Figure 3. Calcium release in HEK293T::hB1R cells induced by B1R-targeting peptides: (A) [des-Arg10]kallidin, (B) [Leu9,des-Arg10]kallidin, (C) SH01078, (D) P03034, (E) P04115, and (F) P04168. Data are presented as mean ± SD (n = 3).



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Biodistribution and PET/CT imaging The biodistribution data of these four 68Ga-labeled [des-Arg10]kallidin derivatives in tumorbearing mice are summarized in Table 3. At 1 h p.i., most organs/tissues except right B1R+ tumors and kidneys had uptake level < 1 %ID/g for all the tracers tested. The uptake values of kidney and B1R+ tumor were 3.14 ± 0.62 and 2.06 ± 0.52 %ID/g for 68Ga-SH01078; 4.50 ± 2.17 and 2.17 ± 0.49 % ID/g for 68Ga-P03034; 4.02 ± 2.40 and 1.96 ± 0.83 %ID/g for 68GaP04115; and 4.02 ± 1.22 and 4.15 ± 1.13 %ID/g for 68Ga-P04168, respectively. Co-injection with 100 µg of their non-radioactive standard significantly reduced the uptake of 68GaSH01078, 68Ga-P03034, 68Ga-P04115 and 68Ga-P04168 in B1R+ tumors to 0.40 ± 0.03, 0.41 ± 0.08, 0.48 ± 0.07 and 0.57 ± 0.10 %ID/g, respectively. Representative PET/CT images obtained at 1 h p.i. are shown in Fig. 4. Consistent with their biodistribution data, very low background radioactivity level was observed for all four 68Galabeled [des-Arg10]kallidin derivatives. Only B1R+ tumors, kidneys and bladders were clearly visualized in the PET images. Co-injection of their non-radioactive standard reduced the uptake of these tracers in B1R+ tumors to near background level.


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Table 3 Biodistribution and uptake ratios of 68Ga-labeled [des-Arg10]kallidin derivatives in tumor-bearing micea 68

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

Ga-SH01078 Control Blocked (n = 8) (n = 5) 0.28 ± 0.08 0.24 ± 0.07 0.07 ± 0.04 0.07 ± 0.04 0.09 ± 0.02 0.13 ± 0.06 0.11 ± 0.04 0.12 ± 0.03 0.22 ± 0.13 0.13 ± 0.07 0.13 ± 0.03 0.11 ± 0.03 0.14 ± 0.02 0.18 ± 0.10 0.08 ± 0.04 0.07 ± 0.05 0.10 ± 0.03 0.10 ± 0.04 3.14 ± 0.62 3.40 ± 0.67 0.27 ± 0.06 0.21 ± 0.06 0.12 ± 0.04 0.09 ± 0.03 0.34 ± 0.18 0.32 ± 0.27 2.06 ± 0.52 0.40 ± 0.03*** 0.07 ± 0.02 0.06 ± 0.02 0.08 ± 0.01 0.07 ± 0.01 0.02 ± 0.00 0.01 ± 0.00

68

Ga-P03034 Control Blocked (n = 5) (n = 4) 0.46 ± 0.34 0.23 ± 0.06 0.11 ± 0.10 0.08 ± 0.08 0.16 ± 0.10 0.12 ± 0.11 0.19 ± 0.12 0.14 ± 0.02 0.22 ± 0.26 0.13 ± 0.07 0.23 ± 0.17 0.11 ± 0.03 0.18 ± 0.10 0.14 ± 0.05 0.07 ± 0.08 0.06 ± 0.01 0.15 ± 0.16 0.15 ± 0.17 4.50 ± 2.17 3.19 ± 0.72 0.53 ± 0.38 0.20 ± 0.05 0.15 ± 0.07 0.09 ± 0.02 0.36 ± 0.09 0.25 ± 0.09 2.17 ± 0.49 0.41 ± 0.08*** 0.13 ± 0.13 0.06 ± 0.01 0.10 ± 0.10 0.09 ± 0.03 0.01 ± 0.01 0.02 ± 0.02

68

Ga-P04115 Control Blocked (n = 8) (n = 4) 0.39 ± 0.17 0.35 ± 0.11 0.07 ± 0.03 0.08 ± 0.02 0.14 ± 0.06 0.21 ± 0.20 0.19 ± 0.15 0.15 ± 0.09 0.26 ± 0.21 0.17 ± 0.08 0.24 ± 0.13 0.20 ± 0.06 0.14 ± 0.02 0.15 ± 0.05 0.09 ± 0.01 0.08 ± 0.02 0.23 ± 0.20 0.18 ± 0.10 4.02 ± 2.40 3.06 ± 0.92 0.30 ± 0.08 0.27 ± 0.09 0.15 ± 0.06 0.14 ± 0.04 0.28 ± 0.10 0.46 ± 0.02** 1.96 ± 0.83 0.48 ± 0.07** 0.09 ± 0.01 0.14 ± 0.12 0.12 ± 0.05 0.20 ± 0.12 0.02 ± 0.01 0.02 ± 0.01

68

Ga-P04168 Control Blocked (n = 8) (n = 4) 0.28 ± 0.08 0.23 ± 0.05 0.06 ± 0.03 0.04 ± 0.01 0.09 ± 0.02 0.07 ± 0.02 0.10 ± 0.03 0.08 ± 0.02 0.20 ± 0.13 0.15 ± 0.09 0.14 ± 0.06 0.18 ± 0.09 0.11 ± 0.03 0.12 ± 0.01 0.07 ± 0.02 0.05 ± 0.02 0.06 ± 0.05 0.07 ± 0.02 4.02 ± 1.22 3.53 ± 0.29 0.28 ± 0.05 0.25 ± 0.03 0.11 ± 0.03 0.10 ± 0.03 0.30 ± 0.16 0.16 ± 0.03 4.15 ± 1.13 0.57 ± 0.10*** 0.06 ± 0.01 0.05 ± 0.01 0.09 ± 0.02 0.08 ± 0.04 0.01 ± 0.00 0.01 ± 0.00

B1R+T:B1R-T 7.24 ± 2.74 2.01 ± 0.96** 6.23 ± 1.69 1.85 ± 0.88** 7.58 ± 3.86 1.05 ± 0.15** 17.8 ± 9.50 3.65 ± 0.37** B1R+T:Blood 7.78 ± 2.20 1.74 ± 0.38*** 5.72 ± 2.20 1.92 ± 0.58** 6.37 ± 3.82 1.47 ± 0.43* 15.9 ± 6.84 2.49 ± 0.22** B1R+T:Muscle 30.2 ± 7.42 7.83 ± 1.94*** 25.5 ± 13.1 6.61 ± 1.54* 26.1 ± 8.91 5.33 ± 3.60** 78.1 ± 28.5 11.6 ± 1.46*** B1R+T:Liver 15.9 ± 5.12 2.86 ± 1.14*** 14.6 ± 6.16 3.10 ± 1.05** 14.9 ± 5.15 3.64 ± 1.35** 42.9 ± 14.2 4.97 ± 1.19*** B1R+T:Kidney 0.67 ± 0.17 0.12 ± 0.02*** 0.54 ± 0.18 0.13 ± 0.04** 0.66 ± 0.38 0.17 ± 0.06* 1.13 ± 0.53 0.16 ± 0.02** a Studies were performed at 1 h p.i. with (blocked) or without (control) co-injection of non-radioactive standard (100 µg). Data are displayed as mean ± SD, and significance of differences between control and blocked groups: *p < 0.05; **p < 0.01; ***p < 0.001.



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Figure 4. Maximum intensity projection (MIP) PET/CT images of 68Ga-labeled [desArg10]kallidin derivatives at 1-h p.i. in mice bearing both B1R+ (red arrows) and B1R(yellow arrows) tumors without (top row) or with (bottom row) co-injection of the nonradioactive standard (100 µg).


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DISCUSSION Although significant effort have been made toward the development of B1R-targeted therapies,23,27-29 only few attempts were reported on developing non-invasive in vivo B1R imaging probes. In 2011, Fuchs et al. presented a meeting abstract on the evaluation of a 11Clabeled sulfonamide for imaging B1R expression in chronic inflammation in a mouse model.30 Despite uptake in inflamed tissues, high non-displaceable binding was observed. Recently, using peptides as targeting vectors we reported the preparation and evaluation of several 68Ga-labeled [des-Arg10]kallidin derivatives for imaging B1R expression with PET.26 Instead of small molecule inhibitors, peptides were chosen for our work due to the flexibility of optimizing their binding affinity and pharmacokinetics through the selection of receptorbinding domains, linkers, and/or radiolabel-chelator complexes. Two of our promising candidates, 68Ga-SH01078 and 68Ga-P03034 (Table 1 and Fig. 1), were constructed using the same B1R-targeting sequence [Hyp4,Cha6,Leu9,des-Arg10]kallidin with two unnatural amino acid substitutions (Hyp4 and Cha6) to improve their in vivo stability. Both peptides were radiolabeled with 68Ga via the DOTA (1,4,7,10-tetraazacyclododecanetetraacetic acid) chelator which was separated from the receptor-binding domain via a liner carbon chain (Ahx in 68Ga-SH01078) or a polyethylene glycol linker (dPEG2 in 68GaP03034). It is well documented that the choice of linker could have a profound effect on binding affinity and pharmacokinetics via the introduced charge and/or enhanced hydrophilicity/lipophilicity.31-32 Besides the previously tested linear carbon chain and polyethylene glycol, there are two other types of linkers, polyamino acid and the cationic 4amino-(1-carboxymethyl)piperidine (Pip), that are also commonly used for the design of radio-peptides.33-38 For comparison and in hopes to optimize the tumor-to-background contrast through the selection of linker, we synthesized two more analogs, 68Ga-P04115 and 68 Ga-P04168 (Table 1 and Fig. 1), containing Gly-Gly and Pip, respectively as the linker. The Gly-Gly was selected as a representative polyamino acid linker due to its structural simplicity and a similar linker length comparable to that of Ahx and Pip. As shown in Table 2, all tracers (68Ga-SH01078, 68Ga-P03034, 68Ga-P04115 and 68GaP04168) had similar average radiochemical yields (55 – 69%, decay-corrected). The relatively higher average specific activity of 68Ga-SH01078 and 68Ga-P03034 (189 – 222 GBq/nmol) than 68Ga-P04115 and 68Ga-P04168 (130 – 111 GBq/nmol) reflected the radioactivity decay of the 68Ge-68Ga generator used for this study. Most of the work of 68Ga-SH01078 and 68GaP03034 was performed shortly after receiving the 68Ge-68Ga generator, whereas 68Ga-P04115 and 68Ga-P04168 were prepared using 68Ga eluted from the same generator at a much later time point, resulting in a relatively lower specific activity. The order of their LogD7.4 values was determined by the hydrophilicity of the linker as these tracers shared the same Ga-DOTA complex and the receptor-binding domain [Hyp4,Cha6,Leu9,des-Arg10]kallidin. 68Ga-SH01078 equipped with the linear carbon chain linker Ahx had the lowest hydrophilicity (LogD7.4 = 2.69 ± 0.25), whereas with the polyamino acid linker Gly-Gly 68Ga-P04115 displayed the highest hydrophilicity (LogD7.4 = -2.99 ± 0.22). This order is consistent with the order of predicted LogD7.0 values of these linkers obtained from ACS SciFinder: Ahx, -2.47; Pip, 2.57; dPEG2, -3.66; Gly-Gly, -4.97). In vitro stability study showed that 68Ga-SH01078 had the highest stability with no detectable degradation after being incubated in mouse plasma for 1 h at 37 °C. The other three tracers also displayed relatively high stability with ≥ 84% of the tracers remaining intact after 1 h incubation. The reduced stability of 68Ga-P04115 could be due to the introduction of an



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additional amide bond from the Gly-Gly linker, which could be susceptible to peptidase cleavage. For comparison, we also performed in vivo metabolic stability studies. In contrast to their high in vitro stability, these four 68Ga-labeled [des-Arg10]kallidin derivatives decomposed quickly in vivo with on average 8 - 17% remaining intact at 5 min p.i.. Our results indicate that in vitro plasma study is not suitable for evaluating the stability of radiolabeled kallidin derivatives that were reported to be metabolized mainly by angiotensinconverting enzyme,24 a tissue peptidase, rather than soluble circulating peptidases. In vitro competition assays (Fig. 2 and Table 1) showed that these four Ga-labeled [desArg10]kallidin derivatives retained high binding affinity (Ki = 3.6 – 27.8 nM) to B1R after the addition of a linker and the Ga-DOTA complex. These data are in agreement with previous reports that modification at the N-terminus of B1R-targeting peptides was tolerable, and the resulting derivatives could retain good binding affinity to B1R.39-42 Of the four [desArg10]kallidin derivatives, P04168 displayed the highest B1R binding affinity. At physiological pH, the Pip linker can be protonated to confer an additional +1 charge at the Nterminus. Mechanistically, this electrostatic potential could draw the charged peptide closer to cellular surface and facilitate binding. Other potent B1R-targeting antagonists such as B9858 (Lys-[Hyp4,Igl6,D-Igl8,Oic9,des-Arg10]kallidin)24 and B9958 ((Lys-[Hyp4,Cpg6,D8 9 10 39 Tic ,Cpg ,des-Arg ]kallidin) both had increased binding affinity when an cationic Lys was added to the N-terminus of the substituted [des-Arg10]kallidin sequence. The same phenomenon has also been observed for another G-protein-coupled receptor system. Zhang et al. reported that N-terminally positively charged peptide ligands had significantly higher affinity to human gastrin releasing peptide receptor (GRPR) than negatively charged or uncharged ligands.43 This could be the main reason that the potent GRPR-targeting 68GaBAY86-7548 currently evaluated in clinical trial for prostate cancer imaging has the cationic Pip as the linker to separate the 68Ga-DOTA complex and the receptor-binding domain.44 While this study further supports that binding affinity of B1R-targeting peptides are robust against modifications at the N-terminus, it is unclear if agonist/antagonist properties of these peptides are affected. Reubi et al. reported that a somatostatin antagonist was converted into an agonist after a DOTA chelator was added to its sequence.45 Therefore, we performed fluorometric measurement of calcium release induced by these four Ga-labeled [desArg10]kallidin derivatives, and compared the results with those obtained using the endogenous agonist [des-Arg10]kallidin and the reported synthetic antagonist [Leu9,des-Arg10]kallidin. As shown in Fig. 3 the antagonist characteristics of these four Ga-labeled [des-Arg10]kallidin derivatives were confirmed by their inability to induce calcium release in HEK293::hB1R cells at a lower concentration (5 nM). At a higher concentration (50 nM) P03034 and P04158 along with [Leu9,des-Arg10]kallidin induced ~ 15 - 36% of the level of calcium release induced by using the same concentration of [des-Arg10]kallidin. Therefore, P03034, P04158, and [Leu9,des-Arg10]kallidin could potentially behave as a partial agonist at such concentration. The creation of the HEK293T::hB1R cell model and the confirmation of B1R expression in these cells were reported previously.26 In this study, we further confirmed that the expressed B1Rs in these cells are functional, and could induce intracellular calcium mobilization upon being activated by a B1R agonist. As shown in the biodistribution data (Table 3) and PET/CT images (Fig. 4) these four 68Galabeled [des-Arg10]kallidin derivatives had very fast renal excretion and minimal background uptake including in the hepatobiliary excretion tract (liver and intestines). This is a desirable feature for cancer imaging probes with the ability to detect primary and/or metastatic lesions within the abdomen. The average uptake values (1.96 – 2.17 %ID/g) in B1R+ tumors were


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similar for 68Ga-SH01078, 68Ga-P03034 and 68Ga-P04115, whereas 68Ga-P04168 displayed ~2-fold higher uptake (4.15 ± 1.13 %ID/g) in B1R+ tumors at 1-h p.i. The higher uptake of 68 Ga-P04168 in B1R+ tumor could be attributed to the improved binding affinity resulted from incorporation of the cationic Pip liner. Higher uptake of 68Ga-P04168 along with relatively lower background activity led to > 2-fold improved B1R+ tumor-to-background (HEK293T tumor, blood, muscle, and liver) contrasts compared to those of 68Ga-labeled SH01078, P03034 and P04115. To verify the specific targeting of these 68Ga-labeled [des-Arg10]kallidin derivatives to B1R, biodistribution and PET/CT imaging studies were perform in mice bearing both B1R+ HEK293T::hB1R tumor and B1R- tumor derived from parent HEK293T cells. In the baseline biodistribution/imaging studies (Table 3, Figs. 4-5), significantly higher uptake in B1R+ than B1R- tumors was observed for all four tracers (2.06 ± 0.52 vs. 0.34 ± 0.18 %ID/g for 68GaSH01078; 2.17 ± 0.49 vs. 0.36 ± 0.09 %ID/g for 68Ga-P03034; 1.96 ± 0.83 vs. 0.28 ± 0.10 %ID/g for 68Ga-P04115, and 4.15 ± 1.13 vs. 0.30 ± 0.16 %ID/g for 68Ga-P04168). In addition, co-injection of their non-radioactive standard (100 µg) reduced ~ 80% uptake of 68GaSH01078, 68Ga-P03034 and 68Ga-P04115, and ~ 90% of 68Ga-P04168 in B1R+ tumors. This further confirmed the uptake of these tracers in B1R+ tumors was specific. Another interesting finding was that significantly higher uptake in B1R+ than B1R- tumors was observed not only in the control cohort, but also in the blocked mice when 68Ga-P03034 and 68 Ga-P04168 were used (Fig. 5). Presumably, co-injection of 100 µg standard could lead to blockade of most B1R in HEK293T::hB1R tumors, and only a small fraction of B1R remained unblocked and was available for targeting. The capability of 68Ga-P03034 and 68GaP04168 to distinguish B1R+ over B1R- tumors even in the blocked mice demonstrated their high sensitivity for in vivo imaging of B1R expression. 68Ga-P04168, in particular, showed the highest B1R tumor-to-background (B1R- tumor, blood, muscle and liver) contrasts in not only the control cohort but also the blocked mice warrants further evaluation for its potential clinical applications. The potential applications include cancer imaging as well as imaging other pathophysiological conditions such as infection,46-47 atherosclerosis,48-49 myocardial infarction,50 and diabetes,51-52 that have been reported to induce upregulation of B1R expression in animal models. In summary, we demonstrated that linker selection is critical for binding affinity, pharmacokinetics and tumor targeting for B1R imaging. Among the four types of linker compared in this study, the application of the cationic Pip linker led to 68Ga-P04168 with superior binding affinity and tumor-to-background contrasts. While it appears attractive to apply this strategy with other ligands and receptor systems, caution should be exercised as this phenomenon is unlikely to be broadly applicable. A thorough and systematic investigation remains critical for linker selection or optimizing other parameters for any ligand/receptor systems.



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Figure 5. Comparison of uptake of 68Ga-labeled [deg-Arg10]kallidin derivatives in B1R+ and B1R- tumors in mice in the (A) control and (B) blocked groups. NS, * and *** indicate the P value is > 0.05, < 0.05, or < 0.001, respectively.


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ASSOCIATED CONTENT Supporting Information MALDI-MS spectra of DOTA-Gly-Gly-[Hyp4,Cha6,Leu9,des-Arg10]kallidin, DOTA-Pip[Hyp4,Cha6,Leu9,des-Arg10]kallidin, P04115 and P04168 as well as HPLC chromatograms of 68 GaCl3, reaction mixture and purified of radiolabeled/cold [desArg10]kallidin derivatives, and their metabolite profiles in plasma are provided in Supporting Information. This information is available free of charge via the Internet at http://pubs.acs.org/.

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: 604-675-8218. 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: 604-675-8218. E-mail: [email protected]. Author Contributions †

Guillaume Amouroux and Jinhe Pan 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) and the BC Leading Edge Endowment Fund.



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