The bradykinin receptor B1R is overexpressed in many human cancers where it might be used as a general target for cancer imaging. In this study, we evaluated the feasibility of using radiolabeled kallidin derivatives to visualize B1R expression in a preclinical model of B1R-positive tumors. Three synthetic derivatives were evaluated in vitro and in vivo for receptor binding and their ability to visualize tumors by PET. Enalaprilat and phosphoramidon were used to evaluate the impact of peptidases on tumor visualization. While we found that radiolabeled peptides based on the native kallidin sequence were ineffective at visualizing B1R-positive tumors, peptidase inhibition with phosphoramidon greatly enhanced B1R visualization in vivo. Two stabilized derivatives incorporating unnatural amino acids (68Ga-SH01078 and 68Ga-P03034) maintained receptor-binding affinities that were effective, allowing excellent tumor visualization, minimal accumulation in normal tissues, and rapid renal clearance. Tumor uptake was blocked in the presence of excess competitor, confirming that the specificity of tumor accumulation was receptor mediated. Our results offer a preclinical proof of concept for noninvasive B1R detection by PET imaging as a general tool to visualize many human cancers. Cancer Res; 75(2); 387–93. ©2014 AACR.

The bradykinin receptor family is constituted of two subtypes, B1 and B2, which are involved in the response to tissue damage and are important mediators of pain and inflammation. These G-protein–coupled receptors bind bradykinin and kallidin, which are produced by enzymatic cleavage of kininogens (1). The overexpression of the B1 and B2 receptors (B1R and B2R) has been documented in many cancers (2). While B2R is ubiquitously expressed, B1R has low endogenous expression in normal tissues. Molina and colleagues showed that B1R was expressed in a high proportion of ductal breast carcinomas in situ as well as invasive ductal breast carcinomas (3). Increased B1R expression was also noted in prostate carcinomas (4). Activation of B1R and/or B2R has been reported to promote the phosphorylation of extracellular regulated kinases (3), upregulate matrix metalloproteinases (5), and stimulate cell invasion and metastasis (6, 7). Crosslinked dimers of bradykinin analogs have also been proposed as therapeutic agents for prostate cancer and small cell lung cancers (8, 9).

Because of low expression in normal tissues, B1R is an attractive target for the development of imaging probes to visualize B1R-positive (B1R+) tumors. Furthermore, because bradykinin receptors are important mediators of pain and inflammation, radiopharmaceuticals targeting these receptors might be useful to understand the role of the kinin-kallikrein system in human tumors in vivo and explore the relationship between these receptors, the immune system, and cancer progression. Although fluorescent derivatives have been reported for microscopy and flow cytometry (10, 11), there have been so far few successful attempts to visualize B1R-expressing tissues noninvasively, in vivo. Stahl and colleagues described a 99mTc-labeled peptide (HOE 140) to visualize B2R expression, and evaluated the biodistribution of this compound in healthy mice (12). Fuchs and colleagues reported the visualization of chronic cutaneous delayed type hypersensitivity reaction in mice using a 11C-labeled sulfonamide targeting B1R, but high, nondisplaceable background activity was observed with the radiotracer (13). The purpose of this study was to develop and evaluate radiolabeled peptides to noninvasively visualize B1R expression in vivo.

All chemicals and solvents were obtained from commercial sources, and used without further purification.

Peptide synthesis

B1R-targeting peptides were synthesized on solid phase and the methods are further described in the Supplementary Data section. A semipreparative (Phenomenex C18, 5μ, 250 × 10 mm) and an analytical column (Eclipse XOB-C18, 5μ, 150 × 4 mm) were used for high-performance liquid chromatography (HPLC) purification and analysis. To obtain gallium conjugates, a solution of the DOTA-conjugated peptide (4 μmol) and GaCl3 (20 μmol) in 500 μL sodium acetate buffer (0.1 mol/L, pH 4.0) was incubated at 80°C for 15 minutes. The methods for conjugation with nonradioactive gallium are described in the Supplementary Data section. We prepared three conjugated peptides: Ga-DOTA-Ahx-[Leu9, desArg10]kallidin (hereafter referred as P03083), Ga-DOTA-Ahx-[Hyp4, Cha6, Leu9, desArg10]kallidin (SH01078), and Ga-DOTA-PEG2-[Hyp4, Cha6, Leu9, desArg10]kallidin (P03034). The chemical structures of the peptides are shown in the Supplementary Data.

Radiochemistry

An Eckert & Ziegler IGG100 68Ga generator was used to obtain 68Ga. Radioactivity of 68Ga-labeled peptides was measured using a Capintec CRC-25R/W dose calibrator. The 68Ga generator was eluted with a total of 4 mL of 0.1 mol/L HCl. The elution that contained the activity was mixed with 2 mL concentrated HCl. The mixture was passed through a DGA resin column and the column was washed by 3 mL 5 mol/L HCl. After the column was dried by passage of air, 68Ga was eluted off with 0.5 mL water. The purified 68Ga solution was added to a 4-mL glass vial preloaded with 0.7 mL of HEPES buffer (2 mol/L, pH 5.0) and the DOTA-conjugated precursor. All labeling procedures were performed in a conventional microwave oven, using a 1-minute reaction time. Microwave labeling was used because of previous experience achieving high yields in a short time using similar peptides—it is likely that similar results can be obtained with conventional heating. This step was followed by HPLC purification to ensure high-specific activity for preclinical imaging. For 68Ga-P03083, 50 μg of peptide was used. The reaction mixture was purified by HPLC using the semipreparative column eluted with 17/83 CH3CN/PBS (pH 7.1) at a flow rate of 4.5 mL/minute. For 68Ga-P03034, 50 μg of precursor was used and 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/minute. 68Ga-SH01078 was synthesized using 100 μg of the radiolabeling precursor and purified using the same conditions as 68Ga-P03034. The quality control was performed by HPLC on the analytical column eluted with 16/84, 18/82, and 19/81 CH3CN/PBS at a flow rate 2 mL/minute, for 68Ga-P03083, 68Ga-P03034, and 68Ga-SH01078, respectively. The retention times of 68Ga-P03083, 68Ga-P03034, and 68Ga-SH01078 were 6.8, 7.7, and 6.1 minutes, respectively. The specific activity of the 68Ga-labeled peptides was measured using the analytical HPLC system. It was calculated by dividing the injected radioactivity (∼37 MBq) of final products by the mass of the peptides in the injected solution. The mass of 68Ga-labeled peptides was estimated by comparing the UV absorbance obtained from the injection with a previously prepared standard curve.

Stability in mouse plasma

Balb/c mouse plasma for stability studies was obtained from Innovative Research. Aliquots (100 μL) of the 68Ga-labeled peptide (P03083, P03034, and SH01078) were incubated with 400 μL of balb/c mouse plasma for 5, 15, 30, and 60 minutes at 37°C. At the end of each incubation period, samples were quenched with 500 μL 70% CH3CN and centrifuged for 20 minutes. The metabolites were measured using a semipreparative HPLC system (Agilent). The suspension was loaded onto the HPLC and eluted with 17/83 CH3CN/PBS (pH 7.1), 23% MeCN (0.1% TFA), and 23% MeCN (0.1% TFA) at a flow rate of 4.5 mL/minute, for 68Ga-P03083, 68Ga-P03034, and 68Ga-SH01078, respectively. The retention times of 68Ga-P03083, 68Ga-P03034, and 68Ga-SH01078 were 16.6, 12.1, and 12.5 minutes, respectively.

LogD7.4 measurements

Aliquots (2 μL) of the 68Ga-labeled peptides were added to a vial containing 3 mL of octanol and 3 mL of 0.1 mol/L phosphate buffer (pH 7.4). The mixture was vortexed for 1 minute and then centrifuged at 5,000 rpm for 10 minutes. Samples of the octanol (1 mL) and buffer (1 mL) layers were taken and counted in a well counter. LogD7.4 was calculated using the following equation: LogD7.4 = log10[(counts in octanol phase)/(counts in buffer phase)].

Creation of a stable B1R expression cell line

We used human embryonic kidney cells HEK293T transduced with both GFP and B1R for this study, using separate vectors. HEK293T cells were obtained from Clontech Laboratories. For GFP transduction, we used the Lenti-XTM Expression System (Clontech Laboratories), using the pGIPz(TurboGFP) cloning vector. For B1R overexpression, GFP-positive HEK293T cells were transduced using premade inducible lentiviral particles at 1 × 107 IFU/mL, obtained from GenTarget (cat. no. LVP291). The B1R open reading frame was constitutively expressed under a suCMV promoter. An antibiotic blasticidin—RFP (red fluorescence protein) fusion marker under Rous sarcoma virus promoter was present in the expression vector to allow for selection and verification of transduced cells. The presence of both GFP and RFP expression was confirmed by fluorescence microscopy using a Nikon Eclipse TE2000 E confocal microscope.

Receptor-binding assays

The affinity of the peptides for B1R was measured using competitive binding assays. Chinese hamster ovary cell membranes overexpressing the recombinant human B1R receptor (PerkinElmer) were used for those assays. Briefly, 96-well MultiScreen plates with glass fiber filter and polyvinylidene difluoride (PVDF) support (Millipore) were presoaked with 0.5% of cold polyethyleneimine (Sigma-Aldrich) for 30 minutes. Afterward, wells were washed once with 50 mmol/L of Tris-HCl, pH 7.4. The wells were loaded with the assay buffer containing 50 mmol/L of Tris-HCl, pH 7.4, and 5 mmol/L of MgCl2. Varying concentration of nonradioactive control Lys-(Des-Arg9)-Bradykinin (Bachem) or the peptides of interest were added in the presence of 4.8 nmol/L of [3H]-(des-Arg10, Leu9)-Kallidin (PerkinElmer). B1R membranes were added to each well to a final protein concentration of 50 μg/well. The MultiScreen plate was incubated at 27°C for 15 minutes with gentle agitation at 300 rpm. The assay was stopped by aspirating the reaction solution through the PVDF membrane filter, followed by washing with ice-cold 50 mmol/L Tris-HCl, pH 7.4. The filter membranes were dried before adding scintillation liquid, and the activity in the plates was measured using a 1450 MicroBeta Counter (PerkinElmer). Data analysis was performed with GraphPad Prism 5, using a one-site competitive binding model. The Ki was calculated from the IC50 using the Cheng–Prusoff equation.

The successful transduction of B1R was also measured by a saturation assay on B1R+ HEK293T cell membranes. Briefly, the cells were disrupted using a Dounce homogenizer, and cell membranes were isolated by sequential centrifugation. Protein concentration was determined using a Bradford assay. Of note, 50 μg of membrane protein per well was used for the saturation assay. The assay conditions were similar to the competitive binding assays with the exception that progressively higher concentrations of radioactive [3H]-(des-Arg10, Leu9)-Kallidin were used (range 0.05–20 nmol/L), with and without the presence of excess competitor [30 μmol/L Lys-(Des-Arg9)-Bradykinin, Bachem]. GraphPad Prism 5 was used to calculate the binding affinity (Kd) and receptor concentration (Bmax), normalized in fmol/mg of protein.

Biodistribution experiments

Animal experiments were approved by the University of British Columbia Animal Care Committee (Vancouver, BC, Canada). Male immunodeficient NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice were obtained from an in-house breeding colony at the Animal Resource Centre of the BC Cancer Agency Research Centre. B1R+ and negative (B1R) HEK293T tumors were inoculated by subcutaneous injection of 1 × 106 cells on each dorsal flank of the mice. Thus, each mouse had a positive and negative control. After growth period ranging from 10 days to 14 days, palpable tumors measuring approximately 7 mm in diameter were obtained. Mice (n = 4–7 per group) were injected with either 0.37 MBq or 3.7 MBq of 68Ga-labeled peptides, depending on whether the mice were used solely for biodistribution or for imaging followed by biodistribution. For blocking experiments, the radioactive compound was coinjected with 100 μg of the same nonradioactive (e.g., natGa) compound. After 60 minutes of uptake, the mice were anesthetized by isoflurane inhalation, and then euthanized by CO2 inhalation. Blood was promptly withdrawn, and the organs of interest were harvested, rinsed with normal saline, blotted dry, and inserted into preweighted tubes. The tubes were weighed again to obtain the exact organ weight. The radioactivity of the collected mouse tissues was counted using a Cobra II gamma counter (Packard), normalized to the injected dose using a standard curve and expressed as the percentage of the injected dose per gram of tissue (%ID/g).

Preclinical imaging

PET imaging experiments were conducted using a Siemens Inveon microPET/CT scanner. Mice bearing B1R+ and B1R tumors, as described above, were used for the experiments. In some mice, dynamic scanning was performed to determine the time-activity course of the radiopharmaceuticals in organs of interest. 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 μA, 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 3.7 MBq of 68Ga-labeled peptide, with or without the presence of 100 μg of excess unlabeled natGa-peptide. The list mode data were rebinned into time intervals (12 × 10, 6 × 30, 5 × 60, 6 × 300, 2 × 600 seconds) to obtain tissue time-activity curves. As we noticed higher renal accumulation and lower urinary excretion in these mice due to the effects of prolonged isoflurane sedation, these animals were not used for biodistribution experiments or for the final analysis of tumor uptake by imaging at 60 minutes. Static imaging was done in the other mice. The mice were briefly sedated for intravenous injection of the radiotracer, and then allowed to recover and roam freely in their cages for 55 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.

Peptidase inhibition with phosphoramidon or enalaprilat

To determine the effects of peptidase inhibition on radiotracer uptake in target tissues, groups of mice injected with 68Ga-P03083 were coinjected with either 0.3 mg of phosphoramidon or 0.25 mg of enalaprilat in normal saline. Imaging and/or biodistribution was performed 55 to 60 minutes later, as described above.

Statistical analysis

Statistical analysis was performed using the Prism 6 software (GraphPad). Biodistribution data were analyzed by two-way ANOVA, with the organs of interest as a factor, and the radiotracers with or without peptidase inhibition as a second factor. Tukey multiple comparison test was used to compare the uptake in the tumors and organs between the following groups: P03083 injected without peptidase inhibition, P03083 injected with enalaprilat, and P03083 injected with phosphoramidon, 68Ga-SH01078 and 68Ga-P03034. An adjusted P value of less than 0.05 was considered significant.

Radiochemistry and plasma stability

The radiochemistry and plasma stability data are reported in Table 1. Because of the use of HPLC, all radiopeptides were obtained in good yield, at high-specific activities, suitable for use in mice for receptor imaging. The stability tests performed in vitro using mouse plasma showed relatively good stability for all peptides in mouse plasma, with the best results obtained with 68Ga-SH01078.

Table 1.

Radiochemistry and plasma stability data for 68Ga peptides

Plasma stability (%)
PeptideRadiochemical yieldSpecific activity (GBq/μmol)LogD7.45 min15 min30 min60 min
68Ga-P03083 70% ± 16% 185 ± 33 −2.83 ± 0.13 96 94 92 87 
68Ga-P03034 69% ± 8% 222 ± 37 −2.76 ± 0.11 99 97 94 91 
68Ga-SH01078 56% ± 20% 189 ± 59 −2.69 ± 0.25 99 99 99 99 
Plasma stability (%)
PeptideRadiochemical yieldSpecific activity (GBq/μmol)LogD7.45 min15 min30 min60 min
68Ga-P03083 70% ± 16% 185 ± 33 −2.83 ± 0.13 96 94 92 87 
68Ga-P03034 69% ± 8% 222 ± 37 −2.76 ± 0.11 99 97 94 91 
68Ga-SH01078 56% ± 20% 189 ± 59 −2.69 ± 0.25 99 99 99 99 

Cellular expression of B1R and binding affinity

Following transduction with the hB1R, successful gene transfer from the lentiviral construct was confirmed by the expression of RFP (Fig. 1). No red fluorescence was observed in the control HEK293T cell line. Saturation assays performed with [3H]-(des-Arg10, Leu9)-kallidin showed a maximal binding capacity (Bmax) of 451 ± 88 fmol/μg of protein for the B1R+ HEK293T cell membranes and 56 ± 48 fmol/μg of protein for the control HEK293T cells. The binding affinities (Kd) of [3H]-(des-Arg10, Leu9)-kallidin for those cell membranes were 1.9 ± 0.5 and 1.6 ± 0.2 nmol/L, respectively. By competitive binding assays, the inhibition constant (Ki) of P03083 was 2.6 ± 0.7 nmol/L. The Ki of SH01078 and P03034 were 27.8 ± 4.9 nmol/L and 16.0 ± 1.9 nmol/L, respectively.

Figure 1.

Fluorescent images of successful B1R transduction. The bright-field images are shown on the left panel. The cells were previously transduced by GFP (middle). RFP was transduced in the same lentiviral vector as B1R (right).

Figure 1.

Fluorescent images of successful B1R transduction. The bright-field images are shown on the left panel. The cells were previously transduced by GFP (middle). RFP was transduced in the same lentiviral vector as B1R (right).

Close modal

Biodistribution and imaging

The results of the biodistribution experiments are presented in Fig. 2, and the entire data can be found in Supplementary Data. The peptides cleared rapidly by urinary excretion, with accumulation in the bladder and kidneys. No significant uptake was seen in other normal organs, with minimal background activity. When 68Ga-P03083 was injected without peptidase inhibitors, tumor uptake was 4-fold higher in the transduced B1R+ compared with the control B1R tumor. This did not improve significantly when 68Ga-P03083 was coinjected with enalaprilat. Enalaprilat increased renal uptake of 68Ga-P03083, but did not improve tumor accumulation. However, when coinjected with phosphoramidon, a significant increase (P < 0.0001) in radiotracer uptake was noted in the B1R+ tumors, with 9-fold higher uptake compared with the B1R tumors (Fig. 3). Similarly, the metabolically stable peptides (68Ga-SH01078 and 68Ga-P03034) led to significantly higher accumulation in B1R+ compared with B1R tumors (Fig. 4). With 68Ga-P03083 coinjected with phosphoramidon, the average ratios of B1R+ tumor to plasma, B1R+ tumor to muscle, and B1R+ to B1R tumors were 9.2 ± 3.7, 35 ± 3, and 9.2 ± 3.7, respectively. With 68Ga-SH01078, the average ratios were 5.2 ± 2.3, 28 ± 5, and 4.4 ± 1.0, respectively. Similar results were obtained using a PEG linker, as evidenced by the high tumor-to-background ratios obtained with 68Ga-P03034. Thus, although the absolute tumor uptake remained moderate due to rapid renal clearance of the radiolabeled peptides, excellent contrast was achieved using either 68Ga-P03083 coinjected with phosphoramidon or with the stable derivatives 68Ga-SH01078 and 68Ga-P03034. Blocking experiments were performed for 68Ga-P03034. The uptake of 68Ga-P03034 in the B1R+ tumor was completely blocked by the coinjection with 100 μg of P03034 (Fig. 5). Time-activity curves of the radiolabeled peptides showed progressive clearance from the blood and nontarget tissues, with retention in B1R+ tumors and high activity in the kidneys (Fig. 6).

Figure 2.

Biodistribution data showing organ or tumor uptake (expressed as %ID/g) for selected organs and tumors with or without B1R expression.

Figure 2.

Biodistribution data showing organ or tumor uptake (expressed as %ID/g) for selected organs and tumors with or without B1R expression.

Close modal
Figure 3.

Maximum intensity projection PET images obtained with 68Ga-P03083 without (left), with enalaprilat (middle), and with coinjection of phosphoramidon (right). The B1R+ tumor (red arrow) was located on the right shoulder (the animal is viewed on a coronal projection, ventral viewpoint). The B1R tumor (blue arrow) had no significant uptake. The gray scale bar to the right of each image is set in units of %ID/g.

Figure 3.

Maximum intensity projection PET images obtained with 68Ga-P03083 without (left), with enalaprilat (middle), and with coinjection of phosphoramidon (right). The B1R+ tumor (red arrow) was located on the right shoulder (the animal is viewed on a coronal projection, ventral viewpoint). The B1R tumor (blue arrow) had no significant uptake. The gray scale bar to the right of each image is set in units of %ID/g.

Close modal
Figure 4.

Maximum intensity projection (left) and fused PET/CT images (right) of 68Ga-SH01078 showing high tumor accumulation in the B1R+ tumor (red arrow) and negligible accumulation in the negative control (B1R, blue arrow) tumor. The gray scale bar is set in units of %ID/g.

Figure 4.

Maximum intensity projection (left) and fused PET/CT images (right) of 68Ga-SH01078 showing high tumor accumulation in the B1R+ tumor (red arrow) and negligible accumulation in the negative control (B1R, blue arrow) tumor. The gray scale bar is set in units of %ID/g.

Close modal
Figure 5.

Maximum intensity projection images of 68Ga-P03034 images with (right) and without (left) injection of competitor. Uptake in the B1R+ tumor is blocked in the presence of excess unlabeled P03034, confirming receptor-mediated uptake. The gray scale bar is set in units of %ID/g.

Figure 5.

Maximum intensity projection images of 68Ga-P03034 images with (right) and without (left) injection of competitor. Uptake in the B1R+ tumor is blocked in the presence of excess unlabeled P03034, confirming receptor-mediated uptake. The gray scale bar is set in units of %ID/g.

Close modal
Figure 6.

Time activity curve of 68Ga-P03034 using regions-of-interest located around the tumors, heart, and kidney (A, linear scale; B, log scale). There was rapid clearance from the blood, fast renal excretion, and sustained tumor accumulation.

Figure 6.

Time activity curve of 68Ga-P03034 using regions-of-interest located around the tumors, heart, and kidney (A, linear scale; B, log scale). There was rapid clearance from the blood, fast renal excretion, and sustained tumor accumulation.

Close modal

Bradykinin receptors are overexpressed in several human cancers (2). Although B2R is expressed in normal tissues, normal physiologic expression of B1R is low (1). Thus, B1R is a potentially attractive target for breast and prostate cancer detection. B1R expression is stimulated by the presence of bradykinin, TNFα, and IL1β (14). The soluble receptor of the globular heads of C1q (sgC1qR) has also been shown to induce B1R expression in endothelial cells (15). The cause of B1R overexpression in cancers is not known. The presence of tissue kallikreins (notably kallikrein-2 and kallikrein-3, also known as the prostate-specific antigen) might play a role in the overexpression of B1R (4). In particular, kallikrein-2 might release bradykinin locally from low- and high-molecular weight kininogens, which could chronically stimulate the overexpression of B1R in cancer cells. Alternatively, the immune system might contribute to enhance B1R expression due to leukocyte infiltration associated with secretion of cytokines.

In this study, we observed that despite the addition of a chelator and radiometal at the N-terminus of [Leu9, desArg10]kallidin, receptor-binding affinity was maintained using a short spacer. We also showed that replacing proline and phenylalanine by hydroxyproline and cyclohexylalanine at positions 4 and 6 deteriorated binding affinity to B1R but improved metabolic stability in vitro. More importantly, we demonstrated that it is possible to visualize noninvasively B1R in vivo, by PET.

We found that using the native antagonist sequence did not lead to good tumor visualization, and confirmed that this was due to peptidase activity. In vitro plasma stability assays did not predict such low in vivo stability for 68Ga-P03083, suggesting that the peptidase inactivating this peptide was not circulating in plasma.

Peptidase inhibition with phosphoramidon, but not enalaprilat, led to improved tumor uptake in vivo. The beneficial effects of phosphoramidon parallel the recent finding of Nock and colleagues, who observed higher levels of circulating intact radiopeptides and improved tumor visualization following phosphoramidon coadministration (16). Enalaprilat is an inhibitor of the angiotensin conversion enzyme (ACE), whereas phosphoramidon inhibits endopeptidase 24.11 in addition to being a potent inhibitor of ACE and other peptidases (15). ACE, carboxypeptidase N, and endopeptidase 24.11 have been implicated in the degradation of bradykinin (7, 17). We did not see a statistically significant effect of enalaprilat on tumor accumulation of the antagonist radiopeptides in this study. However, enalaprilat administration led to slightly higher renal accumulation of 68Ga-P03083. Whether this was caused by the effects of enalaprilat on kidney glomerular filtration rate, or due to a change in the nature of peptide fragments from 68Ga-P03083 degradation remains uncertain.

Poor tumor uptake of the 68Ga-P03083, a peptide derived from the native sequence of bradykinin, led us to design radiopeptides, 68Ga-SH01078 and 68Ga-P03034, with unnatural amino acids to improve stability. Despite lower affinity to B1R than P03083, images obtained using these modified peptides had significantly improved tumor uptake and tumor:background ratios when used in the absence of peptidase inhibitors. All three peptides evaluated in this study presented significant renal accumulation. It is known that small peptides are filtered by glomerular filtration, but can be reabsorbed by endocytic receptors in proximal tubular cells, notably megalin. The exact mechanisms by which the B1R peptides presented in this study accumulate in the kidneys remain unknown, but likely follow similar mechanisms (18).

We obtained high-contrast images with minimal background activity and excellent tumor visualization, despite moderate absolute tumor uptake values. It is worth emphasizing that the absolute tumor uptake is in part dependent on the clearance rate of a radiopharmaceutical—typically much higher uptake is observed with radiotracers that remain in circulation for many days, such as antibodies, compared with lower molecular weight radiotracers. Because higher tumor uptake values have been achieved with other radiopeptides with similar molecular weight (19), it is likely feasible to improve tumor visualization by further improvements in metabolic stability, receptor-binding affinity, or by imaging at later time points to optimize the tumor-to-background ratio.

Although the overexpression of B1R has been well documented in studies conducted on resected human tumor specimen, there are no established models of a B1R-expressing tumor for preclinical studies. We thus used a transfected tumor model to avoid the confounding factor of potentially limited target expression on various commonly used in vitro cancer cell lines. This model allowed us to directly compare, in the same animal, a tumor overexpressing the target with the same tumor that expresses low endogenous levels of B1R. Such models are commonly used in preclinical studies for some radiotracers such as somatostatin receptor ligands (20), but may not reflect the actual expression levels that may be encountered in the clinical setting. Further studies will be required to identify a suitable model with endogenous overexpression of B1R to more closely reflect the clinical situation.

In conclusion, we demonstrated in this study that bradykinin receptor imaging was possible using metabolically stable derivatives of [Leu9, desArg10]kallidin, a bradykinin receptor antagonist sequence. Good receptor-binding affinities to B1R were observed after the addition of a radiometal chelator at the N-terminus of these peptides. Protection from peptidase activity was critical to achieve good tumor visualization in vivo for noninvasive imaging of B1R expression. These radiolabeled peptides can readily be translated for noninvasive imaging in human subjects. 68Ga, a radioisotope that is readily available from a long-lived 68Ge generator, does not require an on-site cyclotron and provides excellent image quality with low radiation exposure.

The causes and significance of B1R expression in human cancers will require further investigations. B1R imaging could be a potential surrogate biomarker of the activity of some tissue kallikreins (notably kallikrein 2), reflect an immune response to cancer cells due to the known relationship between B1R expression and TNFα and IL1β, or be intrinsically driven by other cellular processes. Potential clinical applications of B1R imaging peptides in oncology include the early detection of breast and prostate cancers, and the noninvasive assessment of B1R expression to predict potential benefits from drugs targeting this receptor. Provided that renal accumulation can be mitigated or reduced, other potential applications include the possibility of using this receptor for the delivery of peptide–drug conjugates, or for radionuclide therapy, using peptides labeled with therapeutic α or β emitters.

No potential conflicts of interest were disclosed.

Conception and design: K.-S. Lin, J. Pan, F. Bénard

Development of methodology: K.-S. Lin, J. Pan, G. Amouroux, F. Mesak, F. Bénard

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): K.-S. Lin, J. Pan, G. Amouroux, G. Turashvili, F. Mesak, N. Hundal-Jabal, M. Pourghiasian, J. Lau, S. Jenni, S. Aparicio, F. Bénard

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): J. Pan, G. Amouroux, G. Turashvili, F. Mesak, S. Jenni, F. Bénard

Writing, review, and/or revision of the manuscript: K.-S. Lin, J. Pan, G. Amouroux, G. Turashvili, F. Bénard

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): K.-S. Lin, J. Pan, F. Mesak, N. Hundal-Jabal, S. Jenni, F. Bénard

Study supervision: K.-S. Lin, S. Aparicio, F. Bénard

This work was supported by the Canadian Institutes of Health Research and the BC Leading Edge Endowment Fund.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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