Expression of the gastrin-releasing peptide receptor (GRPR) in prostate cancer suggests that this receptor can be used as a potential molecular target to visualize and treat these tumors. We have previously investigated an antagonist analog of bombesin (D-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH2, RM26) conjugated to 1,4,7-triazacyclononane-N,N',N''-triacetic acid (NOTA) via a diethylene glycol (PEG2) spacer (NOTA-P2-RM26) labeled with 68Ga and 111In. We found that this conjugate has favorable properties for in vivo imaging of GRPR-expression. The focus of this study was to develop a 18F-labelled PET agent to visualize GRPR. NOTA-P2-RM26 was labeled with 18F using aluminum-fluoride chelation. Stability, in vitro binding specificity and cellular processing tests were performed. The inhibition efficiency (IC50) of the [natF]AlF-NOTA-P2-RM26 was compared to that of the natGa-loaded peptide using 125I-Tyr4-BBN as the displacement radioligand. The pharmacokinetics and in vivo binding specificity of the compound were studied. NOTA-P2-RM26 was labeled with 18F within 1 h (60-65% decay corrected radiochemical yield, 55 GBq/µmol). The radiopeptide was stable in murine serum and showed high specific binding to PC-3 cells. [natF]AlF-NOTA-P2-RM26 showed a low nanomolar inhibition efficiency (IC50=4.4±0.8 nM). The internalization rate of the tracer was low. Less than 14% of the cell-bound radioactivity was internalized after 4 h. The biodistribution of [18F]AlF-NOTA-P2-RM26 demonstrated rapid blood clearance, low liver uptake and low kidney retention. The tumor uptake at 3 h p.i. was 5.5±0.7 %ID/g, and the tumor-to-blood, -muscle and -bone ratios were 87±42, 159±47, 38±16, respectively. The uptake in tumors, pancreas and other GRPR-expressing organs was significantly reduced when excess amount of non-labeled peptide was co-injected. The low uptake in bone suggests a high in vivo stability of the Al-F bond. High contrast PET image was obtained 3 h p.i. The initial biological results suggest that [18F]AlF-NOTA-P2-RM26 is a promising candidate for PET imaging of GRPR in vivo.
Citation: Varasteh Z, Åberg O, Velikyan I, Lindeberg G, Sörensen J, Larhed M, et al. (2013) In Vitro and In Vivo Evaluation of a 18F-Labeled High Affinity NOTA Conjugated Bombesin Antagonist as a PET Ligand for GRPR-Targeted Tumor Imaging. PLoS ONE 8(12): e81932. https://doi.org/10.1371/journal.pone.0081932
Editor: Juri G. Gelovani, Wayne State University, United States of America
Received: July 17, 2013; Accepted: October 17, 2013; Published: December 3, 2013
Copyright: © 2013 Varasteh et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This research was financially supported by the Swedish Cancer Society (Cancerfonden) and the Swedish Research Council (Vetenskapsradet). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Prostate cancer (PC) is the most frequently diagnosed non-cutaneous cancer and is the second cause of cancer-related mortality after lung and bronchus cancers in men . An optimal PC treatment is guided by clinically staging the cancer, determining the extent of bone and soft tissue involvement, and selecting treatment options based on the stage. Different treatment modalities are used for organ-confined disease or PC beyond the confines of the prostate gland . Although methods are available to visualize bone metastases , imaging of soft tissue metastases remains a significant problem for disease staging. Sensitive staging agents are urgently needed, especially to monitor soft tissue involvement.
Cell membrane antigen targeting using radiolabeled peptides is a promising approach that can provide adequate staging of PC. Although prostate stem cell antigen (PSCA) and prostate-specific membrane antigen (PSMA) are expressed in primary prostate tumors and the vast majority of metastases, the natural ligands for the aforementioned antigens are unknown. The gastrin-releasing peptide receptor (GRPR) is an interesting alternative molecular target for visualization of PC that can be easily targeted by its natural ligand GRP or bombesin-like small peptides . High GRPR-density expression in prostate carcinomas and prostatic intraepithelial neoplasms and the absence of receptor expression in normal prostate tissue and benign hyperplastic prostate tissue has previously been reported . The data concerning the high levels of receptor expression in prostate tissues, which are in the earliest phase of the malignant transformation process, suggest that GRPR expression can be a marker of choice for the early detection of prostate carcinoma and local metastases .
Bombesin (BN) is a tetradecapeptide that binds to GRPR with high affinity. BN-agonists elicit a strong physiological response due to the activation of BN-receptors in the nervous system and the gut . For instance, a DOTA-conjugated BN[7-14] agonist labeled with 177Lu (177Lu-AMBA) showed some side effects in a phase I dose escalation study . The observed side effects for BN agonists have been hypothesized to be absent for antagonistic analogs [9,10,11]. BN-based antagonists were shown to have superior in vivo biodistribution and targeting properties to agonists .
Very recently, we reported data supporting the potential utility of a new radiolabeled BN-antagonist conjugate, NOTA-P2-RM26 (Figure 1), to image GRPR-expressing tumors in vivo . In this conjugate, the chelator NOTA (1,4,7-triazacyclononane-N,N',N''-triacetic acid) was coupled to D-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH2 (RM26) [11,13] via diethylene glycol (PEG2) and labeled with radiometals: 111In was used for single photon emission computer tomography (SPECT), and 68Ga was used for positron emission tomography (PET) imaging. Fast clearance from the blood and receptor-positive organs together with high uptake and long retention in tumors led to increasing tumor-to-background ratios over time for this conjugate.
Fluorine-18 is the most commonly used radioisotope for PET. The nuclear properties of 18F make it attractive as a label for peptide-based imaging agents. Its half-life (109.7 min) matches with the rapid pharmacokinetics of short peptides. Its low positron energy (Eβ+,max = 0.64 MeV) results in a short positron range in tissues (theoretically calculated path length in water = 2.39 mm), making it well suited for high resolution PET images .
Until recently, the most common approach to the fluorination of peptides was a multistep synthesis of 18F-labeled precursors containing thiol-reactive malemides or primary amine-reactive succinimides and their coupling to peptides . The conjugation was often non-regiospecific and generally resulted in low radiochemical yields . Over the past several years, another convenient alternative for 18F-labeling, the silicon-fluoride acceptor (SiFA) approach, was developed. While the radiochemical yields are generally high, the increased overall lipophilicity of the peptides that were 18F-labeled via SiFA-radiochemistry resulted in an unfavorable biodistribution with high liver uptake and reduced bioavailability of tracers .
Recently, a new simple, one-step labeling method for the radiofluorination of peptides was reported by McBride et al. . The method utilized the strength of the Al-F bond and the ability of NOTA to chelate aluminum. In many aspects this method resembles the labeling procedures for radiometals, such as 68Ga and 111In. The advantages of this approach are the relatively high yield, simplicity and robustness as well as the hydrophilic character of the label.
The aim of this study was to evaluate a 18F-labeled competitive antagonistic analog of BN for PET imaging of GRPR expression in PC. To this end, NOTA-P2-RM26 was labeled with 18F via NOTA-AlF chelation chemistry. The labeling stability, in vitro binding specificity, inhibition efficiency and cellular processing of [18F]AlF-NOTA-P2-RM26 were investigated. Finally, the in vivo specificity and pharmacokinetics of [18F]AlF-NOTA-P2-RM26 were studied in NMRI and Balb/c nu/nu PC-3 tumor xenografted mice.
Materials and Methods
NOTA-P2-RM26) with a molecular mass of 1543.8 Da has been previously reported . Fluorine-18 was produced via the 18O(p,n)18F nuclear reaction using a Scanditronic MC-17 cyclotron (Uppsala, Sweden). A silver body target filled with 25% 18O-enriched water (Rotem) was used. High-performance liquid chromatography (HPLC) analysis was conducted on an Elite LaChrom system (Hitachi, VWR) consisting of an L-2130 pump, a UV detector (L-2400) and a radiation flow detector (Bioscan) coupled in series. Data acquisition and handling were performed using the EZChrom Elite Software Package. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and instant thin-layer chromatography (ITLC) were used for analysis. The distribution of radioactivity along the ITLC strips and SDS-PAGE gels was measured on a Cyclone™ Storage Phosphor System (PerkinElmer). The radioactivity was measured in an automated ɤ-counter with a 3-inch NaI(Tl) detector (1480 WIZARD, WallacOy). The data were analyzed by an unpaired, two-tailed t-test using GraphPad Prism (version 4.00 for Windows GraphPad Software, San Diego, California, USA) to differentiate significant statistical differences (p<0.05).
Radiolabeling and in vitro stability test
[18F]Fluoride (2-4 GBq) was trapped using a Chromafix 30-PS-HCO3 cartridge (Macherey-Nagel GMBH & Co.KG, Geramny). The cartridge was washed with 3 ml deionized water, and the 18F was eluted into siliconized Eppendorf tubes using a mixture of 0.462 M aq. NaCl:EtOH (25:75). Two hundred microliter fractions were collected. The second fraction, containing approximately 80% of the radioactivity (1-2 GBq), was buffered to pH 4.0 with 15 µl of 0.5 M sodium acetate (pH 4.0), followed by the addition of an aqueous solution of NOTA-P2-RM26 (25 µl, 25 nmol, Milli-Q water), 1.5 µl (15 nmol) AlCl3 in 0.1 M sodium acetate buffer (pH 4.0) and 100 µl EtOH. The reaction mixture was heated to 100°C for 20 min. The reaction mixture was then diluted with 3 ml of deionized water and passed through a 1 ml Oasis HLB cartridge (Waters). The cartridge was then washed with 5 ml of deionized water to remove any unreacted 18F. The radiolabeled product was eluted with 5×200 µl fractions of 1:1 EtOH/water. The chemical and radiochemical purity of [18F]AlF-NOTA-P2-RM26 was checked by ITLC (Biodex Medical Systems) using 0.2 M citric acid (pH 2.0) as the running buffer (Rf=0.0 for the peptide and Rf=1.0 for the free fluoride) and HPLC. The conditions were as follows: A=10 mM TFA; B=70% acetonitrile (MeCN), 30% H2O, and 10 mM TFA with UV-detection at 220 nm; gradient elution: 0-2 min at 35% B, 2-9 min at 35 to 100% B, 9-12 min at 100% B; and flow rate was 2.0 mL/min. The analytes were separated using an analytical column with a stationary phase consisting of covalently bonded pentylsilane (Discovery BIO Wide Pore C5; 5cm x 4.6 mm). The analyte was spiked with the original conjugate in order to confirm the identity.
The labeling stability was evaluated using SDS-PAGE analysis (200 V constant) on NuPAGE 4–12% bis-Tris gels (Invitrogen) with the aims to check the release of fluorine-18, transchelation of [18F]AlF to serum proteins, and cleavage of labeled protein by peptidases. Briefly, 5 nmol of [18F]AlF-NOTA-P2-RM26 was incubated in 0.5 ml of murine serum for 30 min at 37°C. After incubation, the samples were treated with NuPAGE® LDS Sample Buffer according to the manufacturer’s instructions and finally 0.05 nmol of sample loaded on the gel. Na18F-sodium fluoride was loaded in a separate lane as a low molecular weight internal reference.
To determine the IC50, NOTA-P2-RM26 was loaded with stable gallium and aluminum fluoride. The cold gallium loading procedure has been previously reported . For fluorine loading, AlCl3 and NaF (both Sigma-Aldrich) were dissolved in Milli-Q water and mixed in equimolar amounts (30 nmol). The solution was buffered to pH 4.0 with 10 µl 0.5 M sodium acetate (pH 4.0) followed by the addition of NOTA-P2-RM26 (25 nmol, 25 µl in Milli-Q water) and 100 µl EtOH. The reaction mixture was heated to 100°C for 20 min. Both natGa- and AlnatF-NOTA-P2-RM26 were analyzed by HPLC as described above.
In vitro studies
PC-3 human prostate cancer cells (ATCC, LGC Standards AB) expressing GRPR were cultured in RPMI media supplemented with 10% (v/v) FCS, 2 mM L-glutamine, and PEST (100 U/mL penicillin and 100 µg/mL streptomycin) (all from Biochrom AG). All in vitro experiments were performed in triplicate with 1×106 cells/dish seeded one day before the experiment.
In vitro binding specificity assay.
Two groups of cultured cell dishes were incubated with a [18F]AlF-NOTA-P2-RM26 solution (1 nM) for 1 h at 37 °C. One set of dishes was pre-saturated with a 1000-fold excess of unlabeled peptide that was added 5 minutes before the addition of [18F]AlF-NOTA-P2-RM26. After the incubation, the cells were washed and treated with 1 ml trypsin-ethylenediaminetetraacetic acid (EDTA) solution (0.25% trypsin, 0.02% EDTA in buffer; Biochrom AG). The detached cells were collected, and the cell-associated radioactivity was measured. The cell-bound radioactivity was expressed as a percentage of added radioactivity.
In vitro competitive binding assay: IC50 determination.
An in vitro competition experiment was performed using 125I-Tyr4-BBN (Perkin Elmer). Cell monolayers were incubated with natGa- or AlnatF-NOTA-P2-RM26 (0 - 460 nM) in the presence of 0.1 pmol (100,000 cpm) 125I-Tyr4-BBN for 3 h at 4 °C. After the incubation, the cells were collected, and the cell-associated radioactivity was determined as described above. The IC50 values were determined using GraphPad software.
Cellular uptake and internalization assay.
The cells were incubated with 2 nM of [18F]AlF-NOTA-P2-RM26 medium at 37 °C. At predetermined time points (1, 2, 3 and 4 h), the incubation medium was discarded, the cells were washed once and the membrane-bound and internalized radioactivity were collected using the method described earlier .
In vivo studies
All animal studies were planned and performed in accordance with the national legislation on the protection of laboratory animals and the Ethic Committee for Animal Research of the Uppsala University approved the study plans. Randomly divided groups of 4 mice per data point were used in all experiments. The biodistribution of [18F]AlF-NOTA-P2-RM26 was evaluated in male NMRI mice (weight 37±3 g). BALB/c nu/nu male mice with PC-3 xenografts (weight 20±1 g at the time of the experiment) were used for tumor targeting and imaging studies. The tumors were grafted by subcutaneous injection of PC-3 cells (107 cells/mouse) into the right hind legs 3 weeks before the experiment. The average tumor size was 0.42±0.05 g at the time of the experiment.
Biodistribution and in vivo binding specificity study in NMRI mice.
NMRI male mice were intravenously injected with 45 pmol of [18F]AlF-NOTA-P2-RM26 (60 kBq in 100 µL of phosphate buffered saline (PBS)). One group of animals was co-injected with 20 nmol of unlabeled peptide to test the in vivo binding specificity of [18F]AlF-NOTA-P2-RM26 to murine GRPRs. The mice were euthanized 1 h post-injection (p.i.) by intraperitoneal administration of Ketalar-Rompun solution (10 mg/mL Ketalar and 1 mg/mL Rompun; 20 µL of solution per gram of body weight). Blood samples were collected by cardiac puncture. The organs of interest were collected, weighed and their radioactivity was measured. The organ uptake values were expressed as a percentage of the injected dose per gram of tissue (%ID/g) except for the gastrointestinal tract (GI) and carcass, which were expressed as %ID per whole sample.
Biodistribution and in vivo binding specificity studies in PC-3 tumor-bearing BALB/c nu/nu mice.
PC-3 xenografted male BALB/c nu/nu mice were intravenously injected with 45 pmol of [18F]AlF-NOTA-P2-RM26 (100 µL in PBS). The mice were sacrificed at 1, 3 and 6 h p.i. to determine the uptake in tumors and other receptor-positive organs. A group of animals was co-injected with an excess of non-labeled peptide (20 nmol) and sacrificed 1 h p.i. The blood and other organs of interest were collected and treated as described above.
The experiment was performed to obtain a visual confirmation of the ex vivo biodistribution experiments. A PC-3 tumor-bearing mouse was sacrificed 3 h after injection of 45 pmol of [18F]AlF-NOTA-P2-RM26 (~ 2 MBq); the urinary bladder was excised; and the image was acquired using a clinical PET-CT scanner (GE Discovery VCT). The PET emission events were collected over 5 min. The image was acquired in 3-D mode and reconstructed in a 128×128 matrix with a 50-cm field of view (FOV).
Radiolabeling and in vitro stability test
NOTA-P2-RM26 was successfully labeled with 18F. Within 1 h, a labeling yield of 60-65% (decay corrected) was achieved starting from 2.8 GBq Na18F (at end of bombardment). An effective specific activity of 55 GBq·μmol-1 was obtained.
The radiochemical purity exceeded 98% according to the HPLC analysis (Figure 2A). SDS-PAGE analysis of the sample incubated in serum for 1 h at 37 °C showed a single radioactivity peak (Figure 2B). No peak corresponding to free 18F was detected.
A. UV-radio-HPLC analyses of [18F]AlF-NOTA-P2-RM26. Upper panel: UV profile with signals corresponding to the original conjugate, NOTA-P2-RM26 (6.5 min), and the product, [18F]AlF-NOTA-P2-RM26 (6.8 min). The recording wavelength was 220 nm. The analyte was spiked with the original conjugate in order to confirm the identity. Lower panel: Radioactivity profile with the principle signal (>98%) corresponding to the product, [18F]AlF-NOTA-P2-RM26. B. SDS-PAGE analysis of [18F]AlF-NOTA-P2-RM26 after a 1 h incubation in murine serum at 37°C (1). Radiolabeled sample (2). Na18F-sodium fluoride was used as low molecular weight radioactivity marker on the same gel.
In vitro studies
In vitro binding specificity assay.
To ensure that the GRPR-binding capacity of NOTA-P2-RM26 was preserved after labeling, the PC-3 cells were incubated with a [18F]AlF-labeled peptide. Binding of [18F]AlF-NOTA-P2-RM26 to the cells was specific as demonstrated by displacement by a non-radioactive peptide. A more than 57-fold decrease in radioactivity was observed for the cells pretreated with a 1000-fold excess of non-labeled peptide (Figure 3A).
A. In vitro binding specificity of [18F]AlF-NOTA-P2-RM26 to GRPRs was tested on PC-3 cells. Blocked dishes were pretreated with a 1000-fold excess of non-labeled peptide 5 min prior to the addition of 1 nM labeled compound. The cell-associated radioactivity is presented as a percentage of the total added radioactivity. B. Inhibition of 125I-Tyr4-BBN binding to PC3 cells with natGa-NOTA-P2-RM26 or AlnatF-NOTA-P2-RM26. C. Cell-associated (internalized, membrane and total) radioactivity as a function of time after continuous incubation of PC-3 cells with [18F]AlF-NOTA-P2-RM26. Data are mean values ± SD of 3 culture dishes. Not all error bars are visible due to the small standard deviations.
In vitro competitive binding assay: IC50 determination.
The binding properties of AlnatF-NOTA-P2-RM26 were compared to those of the natGa-loaded sample in a competitive binding assay using 125I-Tyr4-BBN as the displacement radioligand (Figure 3B). The IC50 values were in the same low nanomolar range with no significant difference (4.4±0.8 nM for [natF]-AlF-NOTA-P2-RM26 and 3.5±0.5 nM for natGa-NOTA-P2-RM26).
Cellular uptake and internalization assay.
The binding kinetics of [18F]AlF-NOTA-P2-RM26 to the GRPR were tested on PC-3 cells (Figure 3C). The cellular uptake of [18F]AlF-NOTA-P2-RM26 reached a plateau within 2 h, when almost 22% of the added radioactivity was associated with the cells. The internalized radioactivity constituted less than 14% (13.9% ± 1.3%) of the cell-associated radioactivity after 4 h of continuous incubation of the cells at 37°C.
In vivo studies
Biodistribution study in NMRI mice.
Data concerning the biodistribution of [18F]AlF-NOTA-P2-RM26 in NMRI mice at 1 h p.i. are presented in Figure 4 and Table S1. The obtained data agreed well with a previous study that investigated the biodistribution of 111In and 68Ga-labeled NOTA-P2-RM26 . Receptor-positive abdominal organs (pancreas, small intestine and stomach) showed high and specific uptake. The saturation of GRPR using a co-injection of non-labeled peptide significantly decreased the uptake of [18F]AlF-NOTA-P2-RM26 in these organs. [18F]AlF-NOTA-P2-RM26 also showed fast blood clearance via kidney excretion together with low kidney retention and liver uptake.
Biodistribution of [18F]AlF-NOTA-P2-RM26 in male NMRI mice 1 h p.i. The total injected mass of radiolabeled conjugate was 45 pmol, and all animals in the blocked group were co-injected with 20 nmol of the non-labeled peptide. The data are presented as the mean percentage of the injected dose per gram of tissue (%ID/g ± SD, n=4). The red asterisks denote significant differences between the groups injected with 45 pmol (non-blocked) and 20 nmol (blocked) (p<0.05).
Biodistribution and in vivo binding specificity studies in PC-3 tumor-baring BALB/c nu/nu mice.
The results of the biodistribution experiment in PC-3 xenografted mice are presented in Figure 5 and 6 and in Table S2. Xenografts and receptor-positive normal organs showed GRPR-specific uptake, which agreed with the data obtained from the NMRI mice. Pre-saturating the receptors with co-injection of non-labeled peptide decreased the tumor uptake more than 8-fold, from 6.3±0.9 %ID/g to 0.8±0.2 %ID/g (Figure 5). Rapidly decreasing radioactive concentrations were observed in all receptor-expressing organs (e.g., pancreas uptake dropped from 13±2 %ID/g 1 h p.i. to 0.15±0.05 %ID/g 6 h p.i.) and the kidneys (from 3.6±0.3 %ID/g 1 h p.i. to 0.27±0.06 %ID/g 6 h p.i.) (Figure 6A). The radioactive concentration in the tumors exceeded the radioactivity concentration in all organs and tissues 3 h p.i. (e.g., the tumor-to-blood ratio was 87±42 and the tumor-to-pancreas ratio was 2.2±0.6 3 h p.i.) (Figure 6B). However, the radioactivity concentration in the tumors was stable between 1 and 3 h p.i. and then decreased more than 2-fold between 3 and 6 h p.i. High tumor-to-normal organ ratios were observed 3 h p.i.
Biodistribution of [18F]AlF-NOTA-P2-RM26 in male Balb/c nu/nu mice bearing PC-3 xenografts 1 h p.i. The total injected mass of radiolabeled conjugate was 45 pmol, and the animals in the blocked group were co-injected with 20 nmol of the non-labeled peptide. The data are presented as the mean percentage of the injected dose per gram of tissue (%ID/g ± SD, n=4). The red asterisks denote significant differences between the groups injected with 45 pmol (non-blocked) and 20 nmol (blocked) (p<0.05).
A. Biodistribution of [18F]AlF-NOTA-P2-RM26 in male BALB/c nu/nu mice bearing PC-3 xenografts at different time points (total injected mass of 45 pmol). B. Tumor to normal organ ratios after injection of 45 pmol of [18F]AlF-NOTA-P2-RM26 in male BALB/c nu/nu mice bearing PC-3 xenografts. The data are presented as the mean percentage of the injected dose per gram of tissue (%ID/g ± SD, n=4).
A mouse with PC-3 xenograft injected with fluorine-labeled peptide was imaged 3 h p.i. (Figure 7). The tumor was clearly visualized, a finding that agreed well with the biodistribution data. Accumulation of radioactivity was also detected in the kidneys and abdominal area.
Imaging of GRPR expression in PC-3 xenografts in a BALB/c nu/nu male mouse. The animals was injected with 45 pmol of [18F]AlF-NOTA-P2-RM26 (~2 MBq). After 3 h, the animal was euthanized, the urinary bladder was dissected and the PET/CT images were acquired.
Detection of soft tissue metastases in PC is a challenge. Conventional anatomical imaging modalities, such as transrectal ultrasound (TRUS), computed tomography (CT) and magnetic resonance imaging (MRI), have demonstrated limited accuracy for the diagnosis of primary prostate tumors . These methods have poor sensitivity, especially for soft tissue metastases, resulting in understaging and overtreatment of patients with extraprostatic disease . Moreover, a variety of imaging agents in nuclear medicine which are used to visualize cellular metabolic activity ([18F]FDG, [11C]acetate, [11C]methionine) failed to detect PC soft tissue metastases due to low cancer cell metabolism [21,22,23]. [11C]/[18F]Choline uptake by tumors has also been studied. However, due to its moderate sensitivity and specificity, choline derivatives cannot be recommended as first-line imaging agents .
To be optimally useful in the current clinical context, a PET scan must be able to detect very small deposits of PC in the lymph nodes to guide therapy.
A receptor-mediated tumor-targeting concept might show a higher specificity for the site of action. [18F]fluorodihydrotestosterone (FDHT)-based imaging was evaluated in prostatic carcinoma with increased androgen receptor (AR) expression. It is used to assess the receptor status and quantify changes in the receptors. However, the oncogenic changes in the AR in PC patients over time may affect the specificity of the ligand . Until recently, only 111In-labeled full-length monoclonal antibody (mAb) capromab pendetide (ProstaScint) was available to image prostate-specific membrane antigen (PSMA). This antibody is directed against an epitope on the cytoplasmic domain of PSMA. However, the sensitivity and specificity of this imaging agent is far from optimal . The labeling of two anti-PSMA mAbs (7E11 and J591, targeting the intra- and extra- cellular domains of PSMA, respectively) with 89Zr [25,26] has recently been reported. In a murine xenograft model, both mAbs demonstrated high and specific tumor uptake (up to 40%ID/g at 4 d p.i.) as well as high liver, spleen and bone uptake. The slow blood clearance of antibodies is also a limitation of this class of proteins as imaging probes. PSMA inhibitors based on glutamate-urea-lysine analogues have recently been developed . Two derivatives, 123I-MIP-1072 and 123I-MIP-1095, were tested in a Phase 1 study of men and were capable of detecting lesions in soft tissue, bone, and the prostate gland as early as 1-4 h after injection . Other radiolabeled small molecule PSMA inhibitors are under evaluation for PET and SPECT imaging of PC .
Because GRPR is expressed in 63-100% of primary prostate tumors and over 50% of metastases, it presents an attractive target for both therapeutic and diagnostic applications . The GRPR expression level in human prostate tumors differs over time. Its expression is markedly upregulated in early-stage tumors, while lower expression has been reported in progressive prostate tumors . This expression pattern makes GRPR useful as a prognostic biomarker. Prostate cancers with a higher GRPR density showed a better prognosis than ones with low or negative expression . Development of PET tracers for GRPR expression could potentially add prognostic value by receptor quantification.
A recent paradigm shift toward G protein-coupled receptor antagonists after sst2 and sst3-targeting with somatostatin analogs has been observed . Bombesin antagonists also showed superior targeting properties compared to GRPR agonists. High and sustained tumor uptake of Demobesin 1 increased the appreciation of the radiolabeled bombesin antagonists used as an imaging agent . The potential capacity of the bombesin antagonist NOTA-P2-RM26 as an imaging agent for GRPR-expressing tumors has been verified in our previous work . In the present work, the use of 18F-labeled NOTA-P2-RM26 to image GRPR with PET was investigated. NOTA-P2-RM26 was stably radiolabeled with 18F using a chelator-based labeling method, AlF-chemistry , with reasonably high yield within 1 h and a high radiochemical purity after a simple cartridge purification. The obtained specific radioactivity for [18F]AlF-NOTA-P2-RM26 was in the range of reported for other BN-antagonists [9,33] which corresponds to approximately 5.8 GBq injected radioactivity into human if protein dose per body weight would be the same as in this study. SDS-PAGE analysis suggests that fluorine-18 is not released, the radiopeptide is not cleaved by peptidases and [18F]AlF does not undergo transchelation to serum proteins.
The in vitro characteristics of [18F]AlF-NOTA-P2-RM26 were investigated and it was found that the radiolabeled conjugate bound specifically to human GRPRs.
The binding properties of [natF]AlF-NOTA-P2-RM26 did not differ from those of the natGa-loaded peptide. Both [natF]AlF- and natGa-NOTA-P2-RM26 showed similar low nanomolar affinity in a competitive binding assay. In accordance with the data for 111In- and 68Ga-labeled peptides from our previous work , the PC-3 cells slowly internalized [18F]AlF-NOTA-P2-RM26 in the cellular processing experiment. This finding indicates that the antagonistic function of RM26 is preserved after modification by radiolabeling.
Overall, the biodistribution of [18F]AlF-NOTA-P2-RM26 was similar to that of 111In- and 68Ga-labeled NOTA-P2-RM26 . The biodistribution of [18F]AlF-NOTA-P2-RM26 in normal mice demonstrated specific uptake in receptor-positive organs (e.g., pancreas, stomach and small intestine) that was significantly decreased by the co-injection of an excess amount of non-labeled peptide. This finding suggests that the peptide cross-reacts with murine GRPRs. The tracer showed also specific tumor uptake in PC-3 xenografts. The peptide was mainly excreted via the kidneys, and low liver and gastrointestinal tract (with content) uptake rates were observed. A fast excretion and low degree of renal re-absorption was found. The rapid blood clearance observed for this conjugate agreed well with the 111In- and 68Ga-NOTA-P2-RM26 data . Low uptake by the bones indicates a high in vivo stability of Al-F bond in [18F]AlF-NOTA-P2-RM26. The same pharmacokinetic behavior of rapid radioactivity release from normal GRPR-positive organs that was observed for 111In/68Ga-NOTA-P2-RM26  was also found for [18F]AlF-NOTA-P2-RM26. In contrast to previous data, the washout of [18F]AlF-NOTA-P2-RM26 from PC-3 tumors was faster than that of 111In-NOTA-P2-RM26 . The tumor uptake for [18F]AlF-NOTA-P2-RM26 decreased by more than 2.4-fold between 3 and 6 h p.i., while a less than 1.3-fold drop was observed previously for 111In-NOTA-P2-RM26 uptake in PC-3 xenografts between 3 and 24 h p.i. . The data suggest that the optimal imaging time point for this tracer would be 3 h p.i., at which time the tumor-to-normal organ ratios are maximized. The potential of 18F-labeled conjugate for visualization of GRPR-positive tumors was confirmed by an additional imaging study. Images obtained with high contrast 3 h after the injection of [18F]AlF-NOTA-P2-RM26 clearly visualized GRPR-expressing PC-3 xenografts. However, the accumulation of radioactivity in the kidneys and abdominal organs was appreciably lower for [68Ga]-NOTA-P2-RM26, which resulted in higher image contrast (Table 1) . Still, because of the wide availability and the superior imaging characteristics of 18F, [18F]AlF-NOTA-P2-RM26 could be a suitable alternative to [68Ga]-NOTA-P2-RM26 for clinical PET imaging.
[18F]AlF-NOTA-P2-RM26 exhibited a high affinity for GRPRs, demonstrated specific targeting to GRPRs both in vitro and in vivo and showed favorable in vivo pharmacokinetics in normal and tumor-xenografted mice. An 18F-labeled tracer could be a suitable alternative for high-resolution PET imaging to stage PC and quantify GRPRs.
Biodistribution of [18F]AlF-NOTA-P2-RM26 in male NMRI mice 1 h p.i.
Conceived and designed the experiments: ZV JS ML GA VT AO. Performed the experiments: ZV OÅ IV MS. Analyzed the data: ZV IV MS VT AO. Contributed reagents/materials/analysis tools: GL. Wrote the manuscript: ZV OÅ VT AO.
- 1. Siegel R, Ward E, Brawley O, Jemal A (2011) Cancer Statistics, The Impact of Eliminating Socioeconomic and Racial Disparities on Premature Cancer Deaths. CA: a Cancer Journal for Clinicians 61: 212-236. doi:https://doi.org/10.3322/caac.20121.
- 2. Kelloff GJ, Choyke P, Coffey DS et al. (2009) Challenges in Clinical Prostate Cancer: Role of Imaging. AJR Am J Roentgenol 192: 1455-1470. doi:https://doi.org/10.2214/AJR.09.2579. PubMed: 19457806.
- 3. Even-Sapir E (2005) Imaging of Malignant Bone Involvement by Morphologic, Scintigraphic, and Hybrid Modalities. J Nucl Med 46: 1356-1367. PubMed: 16085595.
- 4. Ananias HJ, van den Heuvel MC, Helfrich W, de Jong IJ (2009) Expression of the gastrin-releasing peptide receptor, the prostate stem cell antigen and the prostate-specific membrane antigen in lymph node and bone metastases of prostate cancer. Prostate 69: 1101-1108. doi:https://doi.org/10.1002/pros.20957. PubMed: 19343734.
- 5. Markwalder R, Reubi JC (1999) Gastrin-releasing peptide receptors in the human prostate: relation to neoplastic transformation. Cancer Res 59: 1152-1159. PubMed: 10070977.
- 6. de Visser M, van Weerden WM, de Ridder CM, Reneman S, Melis M et al. (2007) Androgen-dependent expression of the gastrin-releasing peptide receptor in human prostate tumor xenografts. J Nucl Med 48: 88-93. PubMed: 17204703.
- 7. Jian Xiaoying, Sainz Eduardo, Clark William A, Jensen Robert T, Battey. et al James F. (1999) The bombesin receptor subtypes have distinct G protein specificities. J Biol Chem 274: 11573-11581. doi:https://doi.org/10.1074/jbc.274.17.11573. PubMed: 10206964.
- 8. Bodei LFM, Nunn J, Llull J, Cremonesi M, Martano L et al. (2007) 177Lu-AMBA Bombesin analogue in hormone refractory prostate cancer patients: a phase I escalation study with single-cycle administrations. European Association of Nuclear; Medicine (2007) abstract 463.
- 9. Abiraj K, Mansi R, Tamma ML, Fani M, Forrer F et al. (2011) Bombesin antagonist-based radioligands for translational nuclear imaging of gastrin-releasing peptide receptor-positive tumors. J Nucl Med 52: 1970-1978. doi:https://doi.org/10.2967/jnumed.111.094375. PubMed: 22080443.
- 10. Cescato R, Maina T, Nock B, Nikolopoulou A, Charalambidis D et al. (2008) Bombesin Receptor Antagonists May Be Preferable to Agonists for Tumor Targeting. J Nucl Med 49: 318-326. doi:https://doi.org/10.2967/jnumed.107.045054. PubMed: 18199616.
- 11. Mansi R, Wang X, Forrer F, Kneifel S, Tamma ML et al. (2009) Evaluation of a 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-conjugated bombesin-based radioantagonist for the labeling with single-photon emission computed tomography, positron emission tomography, and therapeutic radionuclides. Clin Cancer Res 15: 5240-5249. doi:https://doi.org/10.1158/1078-0432.CCR-08-3145. PubMed: 19671861.
- 12. Varasteh Z, Velikyan I, Lindeberg G, Sörensen J, Larhed M et al. (2013) Synthesis and characterization of a high-affinity NOTA-conjugated bombesin antagonist for GRPR-targeted tumor imaging. Bioconjug Chem, 24: 1144–53. doi:https://doi.org/10.1021/bc300659k. PubMed: 23763444.
- 13. Mansi R, Wang X, Forrer F, Waser B, Cescato R et al. (2011) Development of a potent DOTA-conjugated bombesin antagonist for targeting GRPr-positive tumours. Eur J Nucl Med Mol Imaging 38: 97-107. doi:https://doi.org/10.1007/s00259-010-1596-9. PubMed: 20717822.
- 14. Cho ZH, Chan JK, Ericksson L, Singh M, Graham S et al. (1975) Positron ranges obtained from biomedically important positron-emitting radionuclides. J Nucl Med 16: 1174-1176. PubMed: 1194970.
- 15. Tolmachev V, Stone-Elander S (2010) Radiolabelled proteins for positron emission tomography: Pros and cons of labelling methods. Biochim Biophys Acta. 1800: 487–510. doi:https://doi.org/10.1016/j.bbagen.2010.02.002. PubMed: 20153401.
- 16. Schirrmacher R, Bradtmöller G, Schirrmacher E, Thews O, Tillmanns J et al. (2006) 18F-labeling of peptides by means of an organosilicon-based fluoride acceptor. Angew Chem Int Ed Engl 45: 6047-6050. doi:https://doi.org/10.1002/anie.200600795. PubMed: 16888726.
- 17. Wängler C, Niedermoser S, Chin J, Orchowski K, Schirrmacher E et al. (2012) One-step (18)F-labeling of peptides for positron emission tomography imaging using the SiFA methodology. Nat Protoc 7: 1946-1955. doi:https://doi.org/10.1038/nprot.2012.109. PubMed: 23037309.
- 18. McBride WJ, Sharkey RM, Karacay H, D'Souza CA, Rossi EA et al. (2009) A Novel Method of 18F Radiolabeling for PET. J Nucl Med 50: 991-998. doi:https://doi.org/10.2967/jnumed.108.060418. PubMed: 19443594.
- 19. Schwarzenböck S, Souvatzoglou M, Krause BJ (2012) Choline PET and PET/CT in Primary Diagnosis and Staging of Prostate Cancer. Theranostics 2: 318-330. doi:https://doi.org/10.7150/thno.4008. PubMed: 22448198.
- 20. Pinto F, Totaro A, Palermo G, Calarco A, Sacco E et al. (2012) Imaging in prostate cancer staging: present role and future perspectives. Urol Int 88: 125-136. doi:https://doi.org/10.1159/000335205. PubMed: 22286304.
- 21. Jadvar H (2013) Imaging evaluation of prostate cancer with (18)F-fluorodeoxyglucose PET/CT: utility and limitations. Eur J Nucl Med Mol Imaging 40: 5-10. doi:https://doi.org/10.1007/s00259-013-2361-7.
- 22. Dimitrakopoulou-Strauss A, Strauss LG (2003) PET Imaging of Prostate Cancer with 11 C-Acetate. J Nucl Med 44: 556-558. PubMed: 12679399.
- 23. Shiiba M, Ishihara K, Kimura G, Kuwako T, Yoshihara H et al. (2012) Evaluation of primary prostate cancer using 11C-methionine-PET/CT and 18F-FDG-PET/CT. Ann Nucl Med 26: 138-145. doi:https://doi.org/10.1007/s12149-011-0551-6. PubMed: 22069194.
- 24. Dehdashti F, Picus J, Michalski JM, Dence CS, Siegel BA et al. (2005) Positron tomographic assessment of androgen receptors in prostatic carcinoma. Eur J Nucl Med Mol Imaging 32: 344-350. doi:https://doi.org/10.1007/s00259-005-1764-5. PubMed: 15726353.
- 25. Ruggiero A, Holland JP, Hudolin T, Shenker L, Koulova A et al. (2011) Targeting the internal epitope of prostate-specific membrane antigen with 89Zr-7E11 immuno-PET. J Nucl Med 52: 1608-1615. doi:https://doi.org/10.2967/jnumed.111.092098. PubMed: 21908391.
- 26. Holland JP, Divilov V, Bander NH, Smith-Jones PM, Larson SM et al. (2010) 89Zr-DFO-J591 for immunoPET of prostate-specific membrane antigen expression in vivo. J Nucl Med 51: 1293-1300. doi:https://doi.org/10.2967/jnumed.110.076174. PubMed: 20660376.
- 27. Hillier SM, Maresca KP, Lu G, Merkin RD, Marquis JC et al. (2013) 99mTc-Labeled Small-Molecule Inhibitors of Prostate-Specific Membrane Antigen for Molecular Imaging of Prostate. Cancer. J - Nucl Med. doi:https://doi.org/10.2967/jnumed.112.116624.
- 28. Barrett JA, Coleman RE, Goldsmith SJ, Vallabhajosula S, Petry NA et al. (2013) First-in-man evaluation of 2 high-affinity PSMA-avid small molecules for imaging prostate cancer. J Nucl Med 54: 380-387. doi:https://doi.org/10.2967/jnumed.112.111203. PubMed: 23303962.
- 29. Eder M, Eisenhut M, Babich J, Haberkorn U (2013) PSMA as a target for radiolabelled small molecules. Eur J Nucl Med Mol Imaging 40: 819-823. doi:https://doi.org/10.1007/s00259-013-2374-2. PubMed: 23463331.
- 30. Ananias HJ, van den Heuvel MC, Helfrich W, de Jong IJ (2009) Expression of the gastrin-releasing peptide receptor, the prostate stem cell antigen and the prostate-specific membrane antigen in lymph node and bone metastases of prostate cancer. Prostate 69: 1101-1108. doi:https://doi.org/10.1002/pros.20957. PubMed: 19343734.
- 31. Beer M, Montani M, Gerhardt J, Wild PJ, Hany TF et al. (2012) Profiling gastrin-releasing peptide receptor in prostate tissues: Clinical implications and molecular correlates. Prostate 72: 318-325. doi:https://doi.org/10.1002/pros.21434. PubMed: 21739464.
- 32. Ginj M, Zhang H, Waser B, Cescato R, Wild D et al. (2006) Radiolabeled somatostatin receptor antagonists are preferable to agonists for in vivo peptide receptor targeting of tumors. Proc Natl Acad Sci U S A 103: 16436-16441. doi:https://doi.org/10.1073/pnas.0607761103. PubMed: 17056720.
- 33. Nock B, Nikolopoulou A, Chiotellis E, Loudos G, Maintas D et al. (2003) [99mTc]Demobesin 1, a novel potent bombesin analogue for GRP receptor-targeted tumour imaging. Eur J Nucl Med Mol Imaging 30: 247–258. doi:https://doi.org/10.1007/s00259-002-1040-x. PubMed: 12552343.