GRPR-selective PET imaging of prostate cancer using [18F]-lanthionine-bombesin analogs

GRPR-selective PET imaging of prostate cancer using [18F]-lanthionine-bombesin analogs

Peptides 67 (2015) 45–54 Contents lists available at ScienceDirect Peptides journal homepage: www.elsevier.com/locate/peptides GRPR-selective PET i...

2MB Sizes 0 Downloads 8 Views

Peptides 67 (2015) 45–54

Contents lists available at ScienceDirect

Peptides journal homepage: www.elsevier.com/locate/peptides

GRPR-selective PET imaging of prostate cancer using [18 F]-lanthionine-bombesin analogs G. Carlucci a,b , A. Kuipers c , H.J.K. Ananias a , D. de Paula Faria b , R.A.J.O. Dierckx b , W. Helfrich d , R. Rink c , G.N. Moll c,e , I.J. de Jong a , P.H. Elsinga b,∗ a

Department of Urology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands Department of Nuclear Medicine and Molecular Imaging, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands c Lanthio Pharma, Groningen, The Netherlands d Surgical Research Laboratory, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands e Department of Molecular Genetics, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Groningen, The Netherlands b

a r t i c l e

i n f o

Article history: Received 10 November 2014 Received in revised form 2 March 2015 Accepted 9 March 2015 Available online 20 March 2015 Keywords: GRPR Bombesin Al18 F PC-3 PET Stability

a b s t r a c t The gastrin-releasing peptide receptor (GRPR) is overexpressed in a variety of human malignancies, including prostate cancer. Bombesin (BBN) is a 14 amino acids peptide that selectively binds to GRPR. In this study, we developed two novel Al18 F-labeled lanthionine-stabilized BBN analogs, designated Al18 FNOTA-4,7-lanthionine-BBN and Al18 F-NOTA-2,6-lanthionine-BBN, for positron emission tomography (PET) imaging of GRPR expression using xenograft prostate cancer models. (Methyl)lanthioninestabilized 4,7-lanthionine-BBN and 2,6-lanthionine-BBN analogs were conjugated with a NOTA chelator and radiolabeled with Al18 F using the aluminum fluoride strategy. Al18 F-NOTA-4,7-lanthionine-BBN and Al18 F-NOTA-2,6-lanthionine-BBN was labeled with Al18 F with good radiochemical yield and specific activity > 30 GBq/␮mol for both radiotracers. The log D values measured for Al18 F-NOTA-4,7-lanthionineBBN and Al18 F-NOTA-2,6-lanthionine-BBN were −2.14 ± 0.14 and −2.34 ± 0.15, respectively. In athymic nude PC-3 xenografts, at 120 min post injection (p.i.), the uptake of Al18 F-NOTA-4,7-lanthionine-BBN and Al18 F-NOTA-2,6-lanthionine-BBN in prostate cancer (PC-3) mouse models was 0.82 ± 0.23% ID/g and 1.40 ± 0.81% ID/g, respectively. An excess of unlabeled ␧-aminocaproic acid-BBN(7–14) (300-fold) was co-injected to assess GRPR binding specificity. Tumor uptake of Al18 F-NOTA-4,7-lanthionine-BBN and Al18 F-NOTA-2,6-lanthionine-BBN in PC-3 tumors was evaluated by microPET (␮PET) imaging at 30, 60 and 120 min p.i. Blocking studies showed decreased uptake in PC-3 bearing mice. Stabilized 4,7-lanthionineBBN and 2,6-lanthionine-BBN peptides were rapidly and successfully labeled with 18 F. Both tracers may have potential for GRPR-positive tumor imaging. © 2015 Elsevier Inc. All rights reserved.

Introduction Prostate cancer (PCa) is the third-leading cause of cancer related deaths and the most frequently diagnosed cancer among men in the Western World [1]. Early detection of prostate cancer may lead to an improved cure rate. Although transrectal ultrasoundguided biopsies is the gold standard procedure for histological diagnosis, chances of under- or overstaging due to sampling errors in multifocal disease are still common [2]. Furthermore, transrectal ultrasound-guided biopsies have a suboptimal sensitivity,

∗ Corresponding author at: Department of Nuclear Medicine and Molecular Imaging, University Medical Center Groningen, Hanzeplein 1, 9713 EZ Groningen, The Netherlands. Tel.: +31 50 361 3247; fax: +31 50 3611697. E-mail address: [email protected] (P.H. Elsinga). http://dx.doi.org/10.1016/j.peptides.2015.03.004 0196-9781/© 2015 Elsevier Inc. All rights reserved.

as they can miss up to 35% of cancers [3,4]. Therefore, a sensitive, specific imaging procedure to detect prostate cancer is needed. Such an imaging technique might also be of use for detection and local staging of prostate cancer, guidance for prostate biopsies, application of intensity-modulated radiotherapy on hotspots, detection of distant metastases or local recurrence and therapy response monitoring. Nuclear imaging techniques such as positron emission tomography (PET) and single photon emission computed tomography (SPECT) are effective diagnostic tools in oncology and can fully fulfill these purposes [5]. Some radiopharmaceuticals such as 11 C-choline or 18 F-fluoroacetate have already been employed for prostate cancer detection [6]. However, they have limitations due to the relatively low uptake of choline at low PSA levels, the unselective uptake of choline and acetate in both normal and inflamed prostate and the low sensitivity for small-sized metastases. Therefore it is highly important

46

G. Carlucci et al. / Peptides 67 (2015) 45–54

to develop accurate new radiopharmaceuticals that specifically target prostate cancer-associated, overexpressed antigens. Gastrinreleasing peptide receptor (GRPR) has emerged, over the years, as a tumor-associated antigen of particular interest. Different reports have demonstrated that GRPR is overexpressed in a variety of cancers such as lung, colon, gastric, pancreatic, breast and prostate, and that the expression levels of GRPR has prognostic value [7–9]. Bombesin (BBN) is a 14-amino acid peptide which shares sequence homology with GRPR [9]. Several BBN sequences have been developed for GRPR-positive tumor-targeted imaging with PET and SPECT [10–14]. Mainly two types of bombesin sequences have been reported, full-length BBN and truncated sequences. Full-length BBN sequences can be easily modified and truncated. Alternatively, synthetic sequences with one or more substituted and/or deleted amino acids can be easily obtained. The major limitation of full-length BBN is its poor in vivo stability. The aim of this study is to evaluate two full-length lanthionine-stabilized BBN analogs (named 4,7-lanthionine-BBN and 2,6-lanthionineBBN) with respect to their ability to target GRPR and to their increased stability. Lanthionines are thioether crosslinked amino acids that can confer resistance to peptidases even in peptidaserich homogenates of kidney cortex, liver and pancreas [15–17]. The thioether bridges in lanthionines are much more stable than disulphide bridges and even more stable than peptide bonds [18]. The selected sequences were radiolabeled by 18 F via the aluminium 18 F-fluoride (Al18 F) one-pot method pioneered by McBride and co-workers [19–21]. NOTA was used as chelator because of its properties to stabilize the +2 charge of the Al18 F2+ complex. Here, we describe the radiosynthesis and subsequently the in vitro and in vivo targeting characteristics of the two different lanthioninestabilized BBN peptides labeled by 18 F. Methods Reagents and materials All reagents were purchased from Sigma-Aldrich Chemical (St. Louis, Missouri, USA), and were used as received without further purification. NOTA-NHS was purchased from CheMatech (Dijon, France) and used as received without further purification. Aqueous 18 F-fluoride was produced by irradiation of 18 O-water with a Scanditronix MC-17 cyclotron via the 18 O(p,n)18 F nuclear reaction. The 18 F-fluoride solution was passed through a SepPak® Light Accell plus QMA anion exchange cartridge (Waters, Milford, MA, USA) to recover the 18 O-enriched water. C18 cartridges with 55–105 ␮m particle size were purchased from Waters Corporation (Milford, MA, USA) and were pre-treated with water and acetonitrile before use. 125 I-Tyr4 -BBN was purchased from PerkinElmer (Boston, MA). 4-d-Cysteine, 7-l-cysteine BBN and 2d-cysteine,6-l-cysteine-BBN peptides were purchased from JPT Peptide Technologies (Berlin, Germany) and peptide purity was over 95%. Radioactivity was detected by a Bicron Frisk-Tech area monitor (Umass Lowell, MA). The GRPR-positive human prostate cancer cell line PC-3 (ATCC, Manassas, VA, USA) was cultured in RPMI 1640 medium (Lonza, Verviers, France) supplemented with 10% fetal calf serum (Thermo Fisher Scientific Inc., Logan, UT) at 37 ◦ C in a humidified 5% CO2 atmosphere. Male athymic nude mice (6 weeks of age, 18–20 g) were purchased from Harlan (Zeist, The Netherlands). Reversed phase high-performance liquid chromatography (RP-HPLC) was performed on a HITACHI L-2130 HPLC system (Hitachi High Technologies America Inc., Pleasanton, CA) equipped with a Bicron Frisk-Tech area monitor (Umass Lowell, MA). Purification of radiolabeled peptides was performed using a reversed phase Alltima Alltech RP-C18 column (10 mm × 250 mm, 5 ␮m) (Delta Technical Products, Des Plaines, IL). The flow was set at 2.5 mL/min using a gradient system starting from 90% solvent

A (0.01 M phosphate buffer, pH = 6.0) and 10% solvent B (acetonitrile) (5 min), followed by a linear gradient mobile phase going to 35% solvent A and 65% solvent B at 35 min and then back to 90% solvent A and 10% solvent B at 40 min (Method A). Quality control was performed using a reversed phase Grace Smart RP-C18 column (Grace, Lokeren, Belgium) (4.6 mm × 250 mm, 5 ␮m). The flow was set at 1 mL/min using a gradient system starting from 90% solvent A (0.01 M phosphate buffer, pH = 6.0) and 10% solvent B (acetonitrile) (2 min) to 35% solvent A and 65% solvent B at 32 min (Method B). Lyophilization and sample concentration was done in a Speedvac concentrator (Thermo Fisher Scientific Inc., Logan, UT). Mass spectra were recorded with a Matrix-assisted laser desorption/ionization (MALDI) equipped with Time-of-Flight (ToF) (Bruker Daltonics Inc., Billerica, MA). All radioactive counting measurements were obtained on a Compugamma CS1282 (LKB-Wallac, Turku, Finland). All PET imaging experiments were conducted on a microPET INVEON camera equipped with a CT scanner (Siemens, Knoxville, TN) and images reconstructed using INVEON Acquisition Workplace software (Siemens Inveon Software, Erlangen, Germany). Homogenization of organs for stability studies were conducted using an IKA Ultra-Turrax T8 (IKA-Werke GmbH & Co. KG, Staufen, Germany). Synthesis of stabilized BBN analogs A small library of lanthionine bombesin variants with lanthionine rings of three or four amino acids considering a lanthionine (Ala-S-Ala) as one amino acid (thioether crosslinks of positions i, i+3 or i, i+4), throughout the bombesin peptide was designed followed by enzymatic and chemical synthesis as described below. Enzymatic synthesis depended on the substrate specificity of the lanthionine-installing enzymes [22,23]. Lanthionine-stabilized peptides with the lanthionine in the C-terminal half could be produced stereospecifically by a three-step method. Lanthionine was firstly introduced via a Lactococcus lactis production system [24,25] followed by pGlu formation and finally amidation [26]. Nonspecific base-assisted sulfur extrusion was used for obtaining lanthionine-stabilized peptides with the lanthionines in the N-terminal half [22–24]. Briefly, d-Cys, l-Cys bombesin mutants were dissolved in water [2 mg/mL]. Ammonia was added to a 0.3% final concentration and the reaction mixture incubated at 37 ◦ C overnight. After that, ammonia was removed, the sample was concentrated and lanthionine-bombesin was purified by HPLC. The final isomeric mixtures of the lanthionine bombesin analogs were analyzed by mass spectrometry. Introduction of a lanthionine by desulphurization, causes a loss in mass of 34 Da. We hypothesized that limited reduction in receptor affinity could be more than compensated by increased stability. Two variants were eventually selected on the basis of successful synthesis and receptor affinity. These selected peptides, 4,7lanthionine-bombesin and 2,6-lanthionine-bombesin, are shown in Fig. 1. Synthesis of NOTA-4,7-lanthionine-BBN and NOTA-2,6-lanthionine-BBN NOTA-NHS (50 ␮mol) and 4,7-lanthionine-BBN (2.6 ␮mol) or 2,6-lanthionine-BBN (2.6 ␮mol) were dissolved in 1.5 mL of dimethylformamide (DMF). After addition of excess triethylamine (3 equivalents, 7.8 ␮mol), the reaction mixture was stirred overnight at room temperature to make sure the reaction was complete. The product was purified by HPLC (HPLC method 1) and collected at 26 min. Lyophilization of the collected fraction gave the final product (∼24%) with >95% purity by HPLC. For NOTA-4,7-lanthionine-BBN

G. Carlucci et al. / Peptides 67 (2015) 45–54

47

Fig. 1. Structure of Al18 F-NOTA-4,7-lanthionine-BBN (A) and Al18 F-NOTA-2,6-lanthionine-BBN (B).

(C94 H126 N24 O24 S2 , calculated molecular weight 1930.86) a m/z of 1931.65 [M+H]+ was observed. For NOTA-2,6-lanthionine-BBN (C94 H131 N23 O23 S2 ) m/z calculated was 1929.86, observed 1930.49 [M+H]+ . In vitro competitive binding assay The in vitro competitive binding assay was performed as reported previously [7]. The 50% inhibitory concentration IC50 values were calculated over n = 4 by fitting the data with nonlinear regression using GraphPad Prism 5.0 (GraphPad Software, San Diego, CA, USA) and expressed as an average plus the standard deviation.

In vitro stability The stability of the radiolabeled BBN analogs was investigated in human plasma at various incubation times (0–240 min) at 37 ◦ C, as described previously [9]. PBS was used as a control. Samples were collected at different time points (0, 5, 15, 30, 60, 120, 180, 240 min) after incubation. 250 ␮L sample of the human serum mixture was precipitated with 750 ␮L of ethanol/acetonitrile solution (Vethanol :Vacetonitrile = 1:1). After centrifugation, the supernatants were collected, filtered by 0.22 ␮m filter and finally analysed by RPHPLC analysis. The PBS control was diluted with 0.5 mL acetonitrile and analysed by analytical HPLC.

Cell uptake and efflux studies 18 F

Radiolabeling

NOTA-4,7-lanthionine-BBN and NOTA-2,6-lanthionine-BBN were labeled with [F-18] in a one-pot strategy as described by McBride and co-workers [19] producing the Al18 F-NOTA4,7-lanthionine-BBN and Al18 F-NOTA-2,6-lanthionine-BBN PET imaging agents. In brief, 200 ␮L acetonitrile and 50 ␮L aqueous [18 F]fluoride (0.74–1.11 GBq) was added to a 1-mL plastic tube. To this, 5 ␮L of metal-free glacial acetic acid was added to adjust the pH to ∼4.1 followed by 5 ␮L of 2 mM AlCl3 in 0.1 M sodium acetate buffer (pH ∼4.1). Finally, 75 ␮L of this solution was added to 400 ␮L of ACN and 25 ␮g of 4,7-lanthionine-BBN in 0.5 M sodium acetate buffer [1 mg/mL] (pH ∼4.1). The reaction mixture was heated for 20 min at 95 ◦ C. After cooling the reaction mixture, the radiolabeled peptides were isolated and purified by HPLC. The collected product was directly loaded onto a C18 cartridge and was eluted with 0.4 mL 60% ethanol. The radiochemical purity was checked by analytical HPLC and the final product was buffered in PBS. Al18 F-NOTA-2,6-lanthionine-BBN was synthesized by a similar procedure. Octanol/water partition coefficient Al18 F-NOTA-4,7-lanthionine-BBN or Al18 F-NOTA-2,6lanthionine-BBN (37 kBq) were dissolved in a mixture of 0.5 mL n-octanol and 0.5 mL of 25 mM PBS (pH 7.4) and well mixed for 5 min at room temperature. After that, the mixture was centrifuged at 3000 rpm for 5 min, 100 ␮L samples were obtained from n-octanol and aqueous layers and ␥-counted. The log D value is reported as an average of three different measurements (mean ± SD).

PC-3 cells were placed in 6-well plates (0.5 million cells/well) one day prior to the assay and incubated overnight. The day after, the cells were washed twice with PBS and incubated with Al18 F-NOTA-4,7-lanthionine-BBN or Al18 F-NOTA-2,6-lanthionineBBN (0.04 MBq/well) to allow for cellular uptake at 37 ◦ C up to 180 min (n = 3). ␧-Aminocaproic acid-BBN(7–14) unlabeled peptide (20 ␮g) was co-incubated with Al18 F-NOTA-4,7-lanthionine-BBN or Al18 F-NOTA-2,6-lanthionine-BBN (0.04 MBq/well) in the blocking study. After incubating at 37 ◦ C for 15, 30, 60, and 120 min, cells were rinsed three times with PBS, were incubated for ∼3 min with 1 mL glycine acid solution (50 mM glycine–HCl/100 mM NaCl, pH 2.8) and washed again with ice-cold PBS. Radioactivity of collected glycine acid and PBS solutions was measured in ␥-counter as membrane receptor bound radioactivity. Then, the 6-well plates were washed with 1 M NaOH and put at 37 ◦ C to allow cell lysis. The cell lysate was collected in measurement tubes, ␥-counted and reported as internalized activity. Total cellular uptake of Al18 FNOTA-4,7-lanthionine-BBN or Al18 F-NOTA-2,6-lanthionine-BBN was plotted as sum of internalized and membrane-receptor-bound radioactivity (Fig. 2A). Results were expressed as mean ± SD (n = 3). For cell efflux experiments, PC-3 cells were seeded on 6-well plates at a density of 5 × 105 cells/well 24 h before the assay and incubated overnight. The subsequent day, cells were rinsed three times with PBS and incubated with Al18 F-NOTA-4,7-lanthionine-BBN or Al18 FNOTA-2,6-lanthionine-BBN (0.04 MBq/well) at 37 ◦ C for 60 min for maximal internalization. After incubation, the cells were washed with PBS, and then reincubated with serum-free medium. At six different time points (0, 15, 30, 60, 120, and 180 min), the cells were washed with ice-cold PBS and then washed twice for 3 min with acid (50 mM glycine–HCl/100 mM NaCl, pH 2.8) to remove cell-surface bound radiotracer. Finally, the cells were incubated

48

G. Carlucci et al. / Peptides 67 (2015) 45–54

Fig. 2. In vitro stability of Al18 F-NOTA-4,7-lanthionine-BBN and Al18 F-NOTA-2,6-lanthionine-BBN in saline and human serum solution.

with 1 M NaOH at 37 ◦ C to induce cell lysis. Subsequently, the cell lysate was collected in measurement tubes for ␥-counting. Results are calculated as percentage of maximum intracellular radioactivity (remaining activity at specific time-point/activity at time-point 0) (mean ± SD).

(10 mg/kg body weight) were sacrificed 2 h p.i. to determine nonspecific tissue uptake. Biodistribution data are reported as mean ± SD (n = 4). Metabolism of Al18 F-NOTA-4,7-lanthionine-BBN or Al18 F-NOTA-2,6-lanthionine-BBN

Mouse models 6 weeks old athymic nude mice were injected subcutaneously with 1 × 106 PC-3 cells (in 0.1 mL of sterile saline) in the left shoulder. Animals were anesthetized with gas (3.5% isoflurane in an air/oxygen mixture) during inoculation. Four weeks after inoculation, mice were subjected to microPET scan and biodistribution studies. All animal experiments were performed in accordance with the regulations of Dutch law on animal welfare and according to the protocol which was approved by the institutional ethics committee for animal procedures. MicroPET/CT imaging and biodistribution The PC-3 xenografted athymic mice were used for animal experiments when the tumor volume reached 250–300 mm3 (4–5 weeks after inoculation). PET scans and image analysis were performed using an INVEON microPET. Mice with subcutaneous PC-3 were injected intravenously with ∼7.4 MBq (200 ␮Ci) of Al18 F-NOTA-4,7-lanthionine-BBN or Al18 F-NOTA-2,6-lanthionineBBN. Representative coronal images were obtained at 30, 60, and 120 min after injection of the radiolabeled BBN analogs (Fig. 4). Scans were reconstructed with INVEON software using a threedimensional posteriori algorithm with the following parameters: matrix, 128/128/159; pixel size, 0.86/0.86/0.8 mm; and b-value, 1.5, with uniform resolution. For the blocking experiment, PC-3 tumor-bearing mice were injected 30 min before with blocking dose (10 mg/kg bodyweight) of ␧-aminocaproic acid-BBN(7–14) unlabeled peptide followed by and ∼7.4 MBq (200 ␮Ci) of Al18 FNOTA-4,7-lanthionine-BBN or Al18 F-NOTA-2,6-lanthionine-BBN, respectively. Fifteen-minute static PET scans were acquired at 30 min, 60 min and 120 min post injection (n = 6 per group). Biodistribution studies Mice were sacrificed at 2 h after injection of the tracer. Blood, tumor, major organs and tissue samples were collected, wetweighed and measured by ␥-counter for each sample. For each mouse, the amount of radioactivity was determined as percentage of injected dose per gram of tissue (% ID/g). Mice that received a coinjection of unlabeled ␧-aminocaproic acid-BBN(7–14) peptide

Stability studies were conducted as previously published by Zhang and co-workers [27]. Male nude athymic mice bearing PC3 tumors were injected intravenously with approximately 10 MBq (∼270 ␮Ci) of the 18 F-BBN derivative. Animals were sacrificed and dissected at 60 min after injection. Plasma, tumor, kidneys, liver and urine were collected. Plasma and urine were mixed with acetonitrile and centrifuged for 10 min at 13,200 × g for protein precipitation. Tumor, kidneys and liver were separately collected in different tubes, suspended in 1 mL of water and homogenized. Each homogenate was further passed through a 0.22-␮m Millipore filter and injected onto an analytic HPLC column the gradient described earlier (Method B). Radioactivity was monitored using a Bicron Frisk-Tech area monitor detector and fractions were collected every 0.5 min. Finally the activity of each collected fraction was measured using HPLC chromatograms, and obtained by plotting the data obtained from the ␥-counter fraction measurements in GraphPad Prism 5.0 (n = 4). Mass spectrometry analysis was also performed to confirm the identity of the recovered HPLC fractions. Statistical analysis All data are expressed as mean ± SD. Differences between mouse cohorts were analyzed with the 2-sided unpaired Student test and were considered statistically significant when P < 0.05. Results Radiolabeling, partition coefficient, in vitro stability The overall synthesis time for Al18 F-NOTA-4,7-lanthionineBBN or Al18 F-NOTA-2,6-lanthionine-BBN was around 60 min (including formulation) with a decay-corrected yield ranging from 50 to 60% and a radiochemical purity of more than >95%, as determined by HPLC. Al18 F-NOTA-4,7-lanthionine-BBN or Al18 F-NOTA-2,6-lanthionine-BBN were clearly separated from cold precursor (∼1 min shift in RT) and were collected at a retention time of ∼26 min. The calculated specific activity was generally ≥63 GBq/␮mol for Al18 F-NOTA-4,7-lanthionine-BBN and ≥88 GBq/␮mol for Al18 F-NOTA-2,6-lanthionine-BBN based on the

G. Carlucci et al. / Peptides 67 (2015) 45–54

49

Fig. 3. (A) Cellular uptake of Al18 F-NOTA-4,7-lanthionine-BBN and Al18 F-NOTA-2,6-lanthionine-BBN in the PC-3 prostate cancer cell line. (B) Efflux of Al18 F-NOTA-4,7lanthionine-BBN and Al18 F-NOTA-2,6-lanthionine-BBN in the PC-3 prostate cancer cell line.

HPLC analysis of purified product. Log D values of Al18 F-NOTA4,7-lanthionine-BBN or Al18 F-NOTA-2,6-lanthionine-BBN were −2.14 ± 0.1 and −2.34 ± 0.1, respectively. The in vitro stability of both 18 F-tracers was evaluated in saline and human serum as shown in Fig. 2. The results are plotted as the percentage of parent Al18 F labeled BBN analogs at different time points. For both tracers, more than 90% of the radioactivity kept its initial form after 4 h incubation in saline. In human plasma (at 37 ◦ C) more than 75% of radioactivity detected was still identified as parent compound after 4 h (Fig. 2). In vitro competitive receptor binding assay The affinity of Al19 F-NOTA-4,7-lanthionine-BBN and Al19 FNOTA-4,7-lanthionine-BBN for GRPR was determined by performing competitive binding assay with 125 I-[Tyr4 ]BBN as the radioligand. Binding of 125 I-[Tyr4 ]BBN peptide to GRPR was displaced by BBN analogs in a concentration-dependent manner. The IC50 for 4,7-lanthionine-BBN was 251 ± 8 nM and for Al19 FNOTA-4,7-lanthionine-BBN was 114 ± 3 nM. The IC50 observed for 2,6-lanthionine-BBN was 23 ± 4 nM and for Al19 F-NOTA-2,6lanthionine-BBN 15 ± 2 nM. Cell uptake, internalization and efflux kinetics Uptake of Al18 F-NOTA-4,7-lanthionine-BBN and Al18 F-NOTA2,6-lanthionine-BBN in PC-3 cells gradually increased during the first 60 min of incubation and reached a plateau after this time point. The measured total cellular activity was

∼30% for Al18 F-NOTA-4,7-lanthionine-BBN and ∼41% for Al18 FNOTA-2,6-lanthionine-BBN. For Al18 F-NOTA-4,7-lanthionine-BBN the internalized activity was ∼23% and for Al18 F-NOTA-2,6lanthionine-BBN ∼32% (Fig. 3A). The internalization was already observed within 15 min after incubation with both the analogs. The maximum cellular uptake observed with Al18 F-NOTA4,7-lanthionine-BBN was lower than that of Al18 F-NOTA-2,6lanthionine-BBN. The cellular uptake was negligible in case of co-injection of both tracers with blocking agent. The efflux kinetics of Al18 F-NOTA-4,7-lanthionine-BBN and 18 Al F-NOTA-2,6-lanthionine-BBN were also compared in the PC-3 prostate cancer cell line (Fig. 3B). Both tracers showed similar efflux characters in the PC-3 cell line. For Al18 F-NOTA-4,7-lanthionineBBN and Al18 F-NOTA-2,6-lanthionine-BBN, 40 ± 5% and 48 ± 9% of internalized radioactivity remained in PC-3 cells after 3 h incubation respectively. Biodistribution experiments The biodistribution studies of Al18 F-NOTA-4,7-lanthionine-BBN and Al18 F-NOTA-2,6-lanthionine-BBN were performed at 0.5, 1, and 2 h after injection in nude mice bearing PC-3 tumors (n = 6 per group). The results are summarized in Table 1 and Figs. 4B and 5B. Both tracers showed significantly decrease in uptake values from 0.5 h to 2 h after injection in the PC-3 tumors and in all examined organs. The specificity of both tracers was assessed by preinjection of an excess of unlabeled ␧-aminocaproic acid-BBN(7–14) (10 mg/kg). The tumor uptake of Al18 F-NOTA-4,7-lanthionine-BBN was 1.3 ± 0.2% ID/g at 1 h p.i. and 0.82 ± 0.23% ID/g at 120 min

50

G. Carlucci et al. / Peptides 67 (2015) 45–54

Table 1 Biodistribution data of Al18 F-NOTA-4,7-lanthionine-BBN and Al18 F-NOTA-2,6-lanthionine-BBN in PC3 tumor-bearing mice at 2 h p.i. Data are expressed as normalized accumulation of activity in % ID/g ± SD (n = 6). Al18 F-NOTA-4,7lanthioBBN Tumor Heart Lung Blood Liver Spleen Pancreas Kidney Small Intestine Large Intestine Stomach Muscle Bone

0.82 0.32 1.21 1.15 0.62 0.14 6.84 8.42 4.04 1.52 1.23 0.09 0.08

± ± ± ± ± ± ± ± ± ± ± ± ±

0.23 0.13 0.61 0.85 0.42 0.07 0.56 4.8 1.82 0.91 0.09 0.04 0.06

Al18 F-NOTA-4,7-lanthioBBN blocking (2 h p.i.) 0.19 0.31 0.72 1.51 0.40 0.23 0.52 7.45 2.73 2.23 0.71 0.07 0.07

± ± ± ± ± ± ± ± ± ± ± ± ±

0.09 0.21 0.34 1.32 0.12 0.17 0.21 3.76 1.43 1.32 0.42 0.02 0.05

p.i. The tumor uptake of Al18 F-NOTA-2,6-lanthionine-BBN was 2.7 ± 0.2% ID/g at 1 h p.i. and 1.4 ± 0.2% ID/g at 120 min p.i. In a blocking study, the tumor uptakes were significantly reduced at 30 min, 60 min and 120 min p.i. in both the cases with Al18 F-NOTA4,7-lanthionine-BBN (from 0.83 ± 0.7 at 0.5 h p.i. to 0.19 ± 0.09% ID/g at 2 h p.i.) (Fig. 4, panel C) and Al18 F-NOTA-2,6-lanthionineBBN (from 1.2 ± 0.6% ID/g at 0.5 h p.i. to 0.4 ± 0.35% ID/g at 2 h p.i.) (Fig. 5, panel C). The blocking agent decreased also the uptake of pancreas, and intestines whereas the uptake in the blood, kidneys, and liver was increased (Table 1). The kidney uptake decreased from 15.2 ± 7.2% ID/g (30 min p.i.) to 7.4 ± 3.9% ID/g (120 min p.i.) for Al18 F-NOTA-4,7-lanthionine-BBN and from 13.2 ± 6.2% ID/g (30 min p.i.) to 6.74 ± 2.72% ID/g (120 min p.i.) for Al18 F-NOTA-2,6-lanthionine-BBN. Relatively high uptakes of Al18 FNOTA-4,7-lanthionine-BBN and Al18 F-NOTA-2,6-lanthionine-BBN at 2 h time point were observed in the small intestines (4.04 ± 1.82 and 5.23 ± 1.43% ID/g, respectively) and pancreas (6.84 ± 0.56 and 7.47 ± 0.75% ID/g, respectively). The other non-targeting organs and not GRPR expressing tissue showed lower % ID/g (Table 1) uptake

Al18 F-NOTA-2,6lanthioBBN 1.40 0.53 0.61 1.13 0.50 0.53 7.47 5.40 5.23 1.92 1.96 0.21 0.09

± ± ± ± ± ± ± ± ± ± ± ± ±

0.81 0.32 0.32 0.51 0.31 0.38 0.75 2.9 1.43 1.14 1.04 0.12 0.08

Al18 F-NOTA-2,6-lanthioBBN blocking (2 h p.i.) 0.4 0.41 0.54 2.57 0.4 0.45 1.37 6.74 1.70 0.57 0.97 0.11 0.03

± ± ± ± ± ± ± ± ± ± ± ± ±

0.32 0.21 0.48 1.36 0.35 0.29 0.83 2.72 1.52 0.54 0.27 0.05 0.02

than the tumor and due to the clearance, the T/NT ratios increased with time for both tracers (Figs. 4 and 5, panels D). MicroPET/CT imaging Representative coronal microPET images of PC-3 tumor-bearing mice (n = 6 per group) intravenously injected with approximately 7.8 MBq (200 ␮Ci) of Al18 F-NOTA-4,7-lanthionine-BBN and Al18 F-NOTA-2,6-lanthionine-BBN are shown in Figs. 4 and 5 (panels A). The standardized uptake value (SUV) was 1.21 ± 0.5, 1 ± 0.32 at 60 min p.i., and 0.73 ± 0.12 at 120 min p.i. for Al18 FNOTA-4,7-lanthionine-BBN. Calculated SUVs for Al18 F-NOTA-2,6lanthionine-BBN were, 1.98 ± 0.61 at 60 min p.i. and 1.03 ± 0.45 at 120 min p.i. The tumor uptake value of the blocking groups, at 2 h p.i., were 0.3 ± 0.2 for Al18 F-NOTA-4,7-lanthionine-BBN and 0.5 ± 0.3 for Al18 F-NOTA-2,6-lanthionine-BBN, respectively. Quantitative analysis also showed that the average tumor-to-muscle (T/M) ratios at 30, 60, and 120 min p.i. were 6.2 ± 0.3, 9.4 ± 0.6, and 9.1 ± 1.2 for Al18 F-NOTA-4,7-lanthionine-BBN and 5.2 ± 0.7,

Fig. 4. (A) Coronal microPET imaging reconstruction of Al18 F-NOTA-4,7-lanthionine-BBN at 0.5 h, 1 h and 2 h p.i. (n = 6). (B) Biodistribution observed in control mice at 0.5 h, 1 h and 2 h p.i. (n = 6). (C) Biodistribution observed in blocked mice at 0.5 h, 1 h and 2 h p.i. (n = 6). (D) Tumor-to-non-target tissue ratio (non-blocked vs. blocked PC-3 bearing mice) at 2 h after injection (n = 6).

G. Carlucci et al. / Peptides 67 (2015) 45–54

51

Fig. 5. (A) Coronal microPET imaging reconstruction of Al18 F-NOTA-2,6-lanthionine-BBN at 0.5 h, 1 h and 2 h p.i. (n = 6). (B) Biodistribution observed in control mice at 0.5 h, 1 h and 2 h p.i. (n = 6). (C) Biodistribution observed in blocked mice at 0.5 h, 1 h and 2 h p.i. (n = 6). (D) Tumor-to-non-target tissue ratio (non-blocked vs. blocked PC-3 bearing mice) at 2 h after injection (n = 6).

10.6 ± 0.6, and 14.6 ± 1.7 for Al18 F-NOTA-2,6-lanthionine-BBN. In addition prominent uptake of both the tracers was revealed in kidneys and bladder confirming that these imaging agents do mainly excrete via renal–urinary route (not considerable liver uptake was observed). In vivo stability of Al18 F-NOTA-4,7-lanthionine-BBN and Al18 F-NOTA-2,6-lanthionine-BBN The in vivo stability of Al18 F-NOTA-4,7-lanthionine-BBN and Al18 F-NOTA-2,6-lanthionine-BBN was evaluated in PC-3 tumor bearing athymic nude mice at 1 h after injection. HPLC analysis results of the acetonitrile-eluted fractions are shown in Fig. 6A and B. The eluted fractions were plotted in a chromatogram and the in vivo stability was measured in different compartments of interest as percentage of parent compound. The average fraction of intact tracer in the tumor area was 88% for Al18 F-NOTA-4,7lanthionine-BBN and 87% for Al18 F-NOTA-2,6-lanthionine-BBN, indicating a similar stability of both the compounds in vivo. Although we did not identify the composition of the metabolites, our analysis showed that all metabolites eluted from the HPLC column earlier than the parent compound. Any relevant bone uptake was neither observed in microPET scans nor in any of biodistribution studies. These findings, combined to our metabolism studies, are indicative of a high in vivo stability of the described imaging probes. Discussion Prostate cancer is a heterogeneous disease that ranges from indolent forms to high-risk tumors [5]. Despite a variety of treatments, prostate cancer has a high rate of morbidity [1,28]. Therefore early detection of the tumor and an accurate follow up are urgently needed [29]. Gastrin-releasing peptide receptor (GRPR) is overexpressed in prostate, breast cancer and other malignancies [7,8]. This receptor has been widely studied and reported as a promising molecular target for use in molecular diagnostic imaging, and therapeutic follow-up [7,11,27,30–32]. Because of their high affinity to GPRP, bombesin analogs are widely used to image GRPR positive cancers. Bombesin (BBN) is a GRP homologue that

Fig. 6. HPLC profiles of soluble fractions of tumor homogenates (blue), blood (green) and urine (orange). Panel (A) is of Al18 F-NOTA-4,7-lanthionine-BBN and (B) Al18 FNOTA-2,6-lanthionine-BBN. Quality control traces are shown in red for both of them. Samples were collected at 1 h p.i. from male athymic PC-3 tumor-bearing nude mouse (n = 4). As a comparison, the quality control HPLC profile of intact tracer is shown as red trace. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

shares a highly conserved carboxyl-terminal sequence of seven amino acids. The continuous interest and design of novel BBN agents is due to the discovery that primary prostatic carcinomas often express GRPR at much higher levels than non-neoplastic prostate glands. However, due to unfavorable in vivo stability, only a few BBN analogs have entered the clinical phase [33–39].

52

G. Carlucci et al. / Peptides 67 (2015) 45–54

Table 2 Overview of BBN analogs which have been studied in the last years. Analog

Amino acid sequence

Radioisotope

Chelator

Linker

Refs.

MP2346

Pro1 -Gln2 -Arg3 -Tyr4 -Gly5 -Asn6 -Gln7 -Trp8 -Ala9 -Val10 -Gly11 -His12 Leu13 -Met14 -NH2 Gln7 -Trp8 -Ala9 -Val10 -Gly11 -His12 -Leu13 -Met14 -NH2 Gln7 -Trp8 -Ala9 -Val10 -Gly11 -His12 -Cha13 -Nle14 -NH2 Asn6 -Gln7 -Trp8 -Ala9 -Val10 -Gly11 -His12 -Leu-NHEt13 Pro1 -Gln2 -Arg3 -Tyr4 -Gly5 -Asn6 -Gln7 -Trp8 -Ala9 -Val10 -Gly11 -His12 Leu13 -Met14 -NH2 Gln7 -Trp8 -Ala9 -Val10 -Gly11 -His12 -Cha13 -Nle14 -NH2 ACMPip5 -Tha6 -Gln7 -Trp8 -Ala9 -Val10 -␤Ala11 -His12 -Tha13 -Nle14 NH2 DTyr6 -Gln7 -Trp8 -Ala9 -Val10 -␤Ala11 -His12 -Thi13 -Nle14 -NH2 Gln7 -Trp8 -Ala9 -Val10 -Gly11 -His12 -Leu13 -Met14 -NH2 Gln7 -Trp8 -Ala9 -Val10 -Gly11 -His12 -Leu13 -Met14 -NH2 Gln7 -Trp8 -Ala9 -Val10 -Gly11 -His12 -Leu13 -Met14 -NH2 pGlu1 -Gln2 -Lys3 -Leu4 -Gly5 -Asn6 -Gln7 -Trp8 -Ala9 -Val10 -Gly11 His12 -Leu13 -Met14 -NH2 Pro1 -Gln2 -Arg3 -Tyr4 -Gly5 -Asn6 -Gln7 -Trp8 -Ala9 -Val10 -Gly11 -His12 Leu13 -Met14 -NH2 pGlu1 -Gln2 -Lys3 -Tyr4 -Gly5 -Asn6 -Gln7 -Trp8 -Ala9 -Val10 -Gly11 His12 -Leu13 -Met14 -NH2 Cys1 -Gln2 -Arg3 -Leu4 -Gly5 -Asn6 -Gln7 -Trp8 -Ala9 -Val10 -Gly11 His12 -Leu13 -Met14 -NH2 Gln7 -Trp8 -Ala9 -Val10 -Gly11 -His12 -Cha13 -Nle14 -NH2 Gln7 -Trp8 -Ala9 -Val10 -Gly11 -His12 -StaLeu13 -NH2 Gln7 -Trp8 -Ala9 -Val10 -NMeGly11 -His12 -StaLeu13 -NH2

111

DOTA



[44]

Tc Tc(CO)3 99m Tc 99m Tc

N3S (N␣His)Ac N4 N4

Gly-5-Ava Lys(Sha)-␤Ala-␤Ala Bzdig Bzdig

[37,38] [48] [49,50] [51]

68

DOTA DTPA

– –

[52] [53]

In Ga, 111 In 177 Lu, 111 In 64 Cu 18 F

DOTA DOTA DOTA NO2A SFB

␥-Aminobutyric acid PEG4 Gly-4-aminobenzyl 8-Aoc –

[54] [55] [56] [57] [27]

64

DTPA



[47]

RP527 [Cha13 Nle14 ]BN(7–14) DEMOBESIN-1 DEMOBESIN-4 DOTABOM MP2653 BZH2 PESIN AMBA BN(7–14) [Lys3]BN MP2248 [DTPA,Lys3 (Pm-DADT),Tyr4 ]BN [Leu13 ]BN BN(7–14) RM1 BAY-86-4367

To overcome the lack of in vivo stability of radiolabeled BBNs, other moieties such as chelators, linkers, unnatural amino acids and, more generally, prosthetic groups have been attached to BBNs. In this paper, we describe lanthionine-stabilized bombesin analogs with improved resistance to breakdown by peptidases and thereby improved in vivo stability. Selection of BBN candidates for in vivo evaluation has been made on the basis of ease of synthesis and receptor affinity. Following the desulphurization method described by Galande [26], two lanthionine-BBN analogs presented in this paper were radiolabeled using Al18 F methodology (McBride and co-workers) [19–21]. Al18 F-NOTA-4,7lanthionine-BBN and Al18 F-NOTA-2,6-lanthionine-BBN are both highly hydrophilic, as indicated by their log D values (−2.14 ± 0.14 for and −2.34 ± 0.15). The overall synthesis time, including RPHPLC purification and formulation, took approximately 60 min. One important difference between the two analogs concerns their respective IC50 values. In a competitive binding assay, the calculated IC50 value of Al19 F-NOTA-2,6-lanthionine-BBN was 15 ± 2 nM whereas the IC50 value of Al19 F-NOTA-4,7-lanthionine-BBN was 114 ± 3 nM. The calculated IC50 values were, in both cases, lower than the NOTA unconjugated companion peptides. This indicated an improvement in the affinity of the conjugate upon loading with metal cations. Similar findings were also observed by Varasteh and co-workers [40]. The difference in IC50 played a major role in the cellular uptake studies. As expected, Al19 F-NOTA-2,6-lanthionineBBN showed the highest cellular uptake in vitro consistent with its low nanomolar affinity. The tumor uptake values of Al18 F-NOTA2,6-lanthionine-BBN (2.1 ± 0.3% ID/g) at 1 h p.i. were similar to the tumor uptake observed with 18 F-FB-[Lys3 ]BBN (±2.61% ID/g) [27] and with 18 F-AlF-NODAGA-AMBA (2.4 ± 0.24% ID/g at 1 h p.i.) [12]. The in vitro and the ␮PET results suggested that Al18 F-NOTA-2,6lanthionine-BBN might be a valuable imaging agent for human clinical translation since it binds specifically to GRPR, is mostly stable in vivo and rapidly washed-out by renal clearance. In agreement with the results obtained in this paper and the encouraging enzymatic resistance previously described for lanthionine-stabilised agents elsewhere [5,22,23], we decided to validate the in vivo metabolic stability after intravenous administration of the probes. Our HPLC tissue analysis confirmed that the thioether bridge is a

In, 68 Ga

99m

99m

Ga In

111

111

67/68

Cu

99m

Tc

Pm-DADT



[58]

99m

Tc

-

Aca

[34,35]

DOTA DOTA +N(CH3)3

Aoc Gly-4-aminobenzyl Ala(SO3)-Ala(SO3)-Ava

[59] [60] [61]

64

Cu Lu, 111 In 18 F 177

valuable stabilization approach for peptides applied in imaging, and also for radiotherapeutic peptides. We developed our in vivo stability assay based on a previously published method [27]. Minor degradation products were observed in the tumor and, importantly, some of the intact radiotracer was seen in kidneys, plasma and urine. This demonstrated the high stabilization potential of this method in comparison to other BBN sequences that were radiolabeled by different approaches which have been reported to be mostly unstable in vivo [27,32,39]. Rapid and highly selective proteolytic cleavage of bioactive peptides is a common aspect of the autonomic regulation of the biologic effects of peptides. In this respect, ecto-enzymes located on the cell surface that are shed in blood and are highly expressed in the liver and kidneys could be major players in this process [41]. Earlier studies reported that BBN-like peptides can be easily metabolized by the neutral endopeptidase E.C.3.4.24.11 and the angiotensin-converting enzyme [42–44]. To more accurately determine radiochemical stability, Ocak et al. suggested to use liver and kidney homogenates [45]. By contrast, in another study by the same author, it was shown that rat liver and kidney homogenates were no good predictors for in vivo stability [46]. It might be important to identify the cleavage sites in order to design and characterize peptides of enhanced metabolic stability. For years, the use of truncated sequences such as Aca-BN(7–14) were described as useful tool to increase the metabolic stability of BBN peptides. A list of BBN analogs considered “promising” has been enclosed in Table 2. At the moment, an accurate standardized comparison of available analogs for PC detection is not yet available because of differences in the preclinical studies. As a consequence, it is also difficult to unequivocally establish which analog displays the overall best characteristics for clinical use. The published clinical data are those obtained from the clinical studies on 99m Tc-RP527 [37,38], 99m Tc-[Leu13 ]BN [34], 177 Lu-AMBA [53] and 99m Tc-HABBN [39]. Al18 F-NOTA-2,6-lanthionine-BBN has similar tumor-to-muscle ratio and tumor uptake to other 18 F labeled BBN agonists but is more stable than many other BBN agents that have been evaluated in the past (Table 2). Hence, the lanthionine stabilization method described in this paper is a promising platform for the development of more effective peptides which are less sensitive to in vivo enzymatic degradation.

G. Carlucci et al. / Peptides 67 (2015) 45–54

Conclusions To overcome the stability issues of radiolabeled peptides for receptor-mediated cancer imaging, we synthesized a series of lanthionine-stabilized full-length BBN-like peptides and two promising candidates were successfully labeled with 18 F. The effect of stabilization was evaluated in vitro and in vivo. PET studies demonstrated the possibility of lanthionine-stabilized BBN tracers to target PC-3 tumor in xenograft models showing GRPR binding potential. Author contributions The manuscript was written by contributions of all authors. All authors have given approval to the final version of the manuscript. Conflict of interest A. Kuipers, R. Rink and G.N. Moll declare no competing financial interests in the findings of this study with Lanthio Pharma, Rozenburglaan 13 B, 9727 DL Groningen, The Netherlands. Acknowledgements This work was made possible by a financial contribution from CTMM, project PCMM, project number 03O-203. We thank V.R. Wiersma and L.D. Kluskens for initial studies on synthesis of lanthionine-stabilized BBN analogs, D.F. Samplonius for technical assistance on cell culturing and J. A. Sijbesma for assisting animal experiments. References [1] Jemal A, Bray F, Ferlay J. Global cancer statistics. CA Cancer J Clin 2011;61:69–90. [2] Arora R, Koch MO, Eble JN, Ulbright TM, Li L, Cheng L. Heterogeneity of Gleason grade in multifocal adenocarcinoma of the prostate. Cancer 2004;11:2362–6. [3] Presti Jr JC, Chang JJ, Bhargava V, Shinohara K. The optimal systematic prostate biopsy scheme should include 8 rather 6 biopsies: results of a prospective clinical trial. J Urol 2000;163:163–7. [4] Chang JJ, Shinohara K, Bhargava, Presti Jr JC. Prospective evaluation of lateral biopsies of the peripheral zone for prostate cancer detection. J Urol 1998;6:2111–4. [5] Zanzonico P. Principles of nuclear medicine imaging: planar, SPECT, PET, multimodality, and autoradiography systems. Radiat Res 2012;177:349–64. [6] Jadvar H. Prostate cancer: PET with 18 F-FDG, 18 F- or 11 C-acetate, and 18 F- or 11 C-choline. J Nucl Med 2011;52:81–9. [7] Ananias HJK, van den Heuvel MC, Helfrich W, de Jong IJ. 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 2009;69:1101–8. [8] Mansi R, Fleischmann A, Macke HR, Reubi JC. Targeting GRPR in urological cancers from basic research to clinical application. Nat Rev Urol 2013;10:235–44. [9] Markwalder R, Reubi JC. Gastrin-releasing peptide receptors in the human prostate: relation to neoplastic transformation gastrin-releasing peptide receptors in the human prostate: relation to neoplastic transformation. Cancer Res 1999:1152–9. [10] Moody TW, Crawley JN, Jensen RT. Pharmacology and neurochemistry of bombesin-like peptides. Peptides 1982;3:559–63. [11] Zhang H, Abiraj K, Thorek DLJ, Waser B, Smith-Jones PM, Honer M, et al. Evolution of bombesin conjugates for targeted PET imaging of tumors. PLOS ONE 2012;7, e44046-e. [12] Liu Y, Hu X, Liu H, Bu L, Ma X, Cheng K, et al. A comparative study of radiolabeled bombesin analogs for the PET imaging of prostate cancer. J Nucl Med 2013;54:2132–8. [13] Dijkgraaf I, Franssen GM, McBride WJ, D’Souza CA, Laverman P, Smith CJ, et al. PET of tumors expressing gastrin-releasing peptide receptor with an 18 F-labeled bombesin analog. J Nucl Med 2012;53:947–52. [14] Zhang X, Cai W, Cao F, Schreibmann E, Wu Y. Bombesin analogs for targeting GRP receptor-expressing prostate cancer. J Nucl Med 2014:492–501. [15] Rink R, Arkema-Meter A, Baudoin I, Post E, Kuipers A, Nelemans SA, et al. To protect peptide pharmaceuticals against peptidases. J Pharmacol Toxicol Methods 2010;61:210–8. [16] de Vries L, Reitzema-Klein CE, Meter-Arkema A, van Dam A, Rink R, Moll GN, et al. Oral and pulmonary delivery of thioether-bridged angiotensin-(1–7). Peptides 2010;31:893–8.

53

[17] Kluskens LD, Nelemans SA, Rink R, de Vries L, Meter-Arkema A, Wang Y, et al. Angiotensin-(1–7) with thioether bridge: an angiotensin-converting enzyme-resistant, potent angiotensin-(1–7) analog. J Pharmacol Exp Ther 2009;328:849–54. [18] Tugyi R, Mezö G, Fellinger E, Andreu D, Hudecz F. The effect of cyclization on the enzymatic degradation of herpes simplex virus glycoprotein D derived epitope peptide. J Pept Sci 2005;11:642–9. [19] McBride WJ, Sharkey RM, Goldenberg DM. Radiofluorination using aluminumfluoride (Al18 F). EJNMMI Res 2013;1:3–36. [20] Goldenberg DM, Sharkey RM, McBride WJ, Boerman OC. Al18 F: a new standard for radiofluorination. J Nucl Med 2013;54:1170. [21] D’Souza CA, McBride WJ, Sharkey RM, Todaro LJ, Goldenberg DM. Highyielding aqueous 18 F-labeling of peptides via Al18 F chelation. Bioconjug Chem 2011;22:1793–803. [22] Rink R, Kuipers A, de Boef E, Leenhouts KJ, Driessen AJM, Moll GN, et al. Lantibiotic structures as guidelines for the design of peptides that can be modified by lantibiotic enzymes. Biochemistry 2005;44:8873–82. [23] Rink R, Wierenga J, Kuipers A, Kluskens LD, Driessen AJM, Kuipers OP, et al. Production of dehydroamino acid-containing peptides by Lactococcus lactis. Appl Environ Microbiol 2007;73:1792–6. [24] Kluskens LD, Kuipers A, Rink R, de Boef E, Fekken S, Driessen AJM, et al. Posttranslational modification of therapeutic peptides by NisB, the dehydratase of the lantibiotic nisin. Biochemistry 2005;44:12827–34. [25] Rink R, Kluskens LD, Kuipers A, Driessen AJM, Kuipers OP, Moll GN. NisC, the cyclase of the lantibiotic nisin, can catalyze cyclization of designed nonlantibiotic peptides. Biochemistry 2007;46:13179–89. [26] Galande AK, Trent JO, Spatola AF. Understanding base-assisted desulfurization using a variety of disulfide-bridged peptides. Pept Sci 2003;71:534–51. [27] Zhang X, Cai W, Cao F, Schreibmann E, Wu Y, Wu JC, et al. 18 F-labeled bombesin analogs for targeting GRP receptor-expressing prostate cancer. J Nucl Med 2006;47:492–501. [28] Conteduca V, Aieta M, Amadori D, De Giorgi U. Neuroendocrine differentiation in prostate cancer: current and emerging therapy strategies. Crit Rev Oncol Hematol 2014;92:11–24. [29] Pinto F, Totaro A, Calarco A, Sacco E, Volpe A, Racioppi M, et al. Imaging in prostate cancer diagnosis: present role and future perspectives. Urol Int 2011;86:373–82. [30] Körner M, Waser B, Rehmann R, Reubi JC. Early over-expression of GRP receptors in prostatic carcinogenesis. Prostate 2014;74:217–24. [31] Pan D, Xu YP, Yang RH, Wang L, Chen F, Luo S, et al. A new (68)Ga-labeled BBN peptide with a hydrophilic linker for GRPR-targeted tumor imaging. Amino Acids 2014;46:1481–9. [32] Nanda PK, Pandey U, Bottenus BN, Rold TL, Sieckman GL, Szczodroski AF, et al. Bombesin analogues for gastrin-releasing peptide receptor imaging. Nucl Med Biol 2012;39:461–71. [33] Schroeder RPJ, Weerden WMv, Bangma C, Krenning EP, Jong Md. Peptide receptor imaging of prostate cancer with radiolabelled bombesin analogues. Methods 2009;48:200–4. [34] De Vincentis G, Scopinaro F, Varvarigou A, Ussof W, Schillaci, ArchimandritisS, et al. Phase I trial of technetium [Leu13] bombesin as cancer seeking agent: possible scintigraphic guide for surgery? Tumori 2002;3:S28–30. [35] De Vincentis G, Remediani S, Varvarigou AD, Di Santo G, Iori F, Laurenti C, et al. Role of 99m Tc-bombesin scan in diagnosis and staging of prostate cancer. Cancer Biother Radiopharm 2004;19:81–4. [36] Scopinaro F, De Vincentis G, Varvarigou A, Laurenti C, Iori F, Remediani S, et al. 99m Tc-bombesin detects prostate cancer and invasion of pelvic lymph nodes. Eur J Nucl Med Mol Imaging 2003;30:1378–82. [37] Van de Wiele C, Dumont F, Dierckx RA, Peers SH, Thornback JR, Slegers G, et al. Biodistribution and dosimetry of 99m Tc-RP527, a gastrin-releasing peptide (GRP) agonist for the visualization of GRP receptor-expressing malignancies. J Nucl Med 2001;42:1722–7. [38] Van de Wiele C, Phonteyne P, Pauwels P, Goethals I, Van den Broecke R, Cocquyt V, et al. Gastrin-releasing peptide receptor imaging in human breast carcinoma versus immunohistochemistry. J Nucl Med 2008;49:260–4. [39] Ananias HJK, Yu Z, Hoving HD, Rosati S, Dierckx RA, Wang F, et al. Application of 99m Technetium-HYNIC(tricine/TPPTS)-Aca-Bombesin(7–14) SPECT/CT in prostate cancer patients: a first-in-man study. Nucl Med Biol 2013;40: 933–8. [40] Varasteh Z, Velikyan I, Lindeberg G, Sörensen J, Larhed M, Sandström M, et al. Synthesis and characterization of a high-affinity NOTA-conjugated bombesin antagonist for GRPR-targeted tumor imaging. Bioconjug Chem 2013;24:1144–53. [41] Konkoy CS, Davis TP. Ectoenzymes as sites of peptide regulation. Trends Pharmacol Sci 1996;17:288–94. [42] Grady EF, Slice LW, Brant WO, Walsh JH, Payan DG, Bunnett NW. Direct observation of endocytosis of gastrin releasing peptide and its receptor. J Biol Chem 1995;270:4603–11. [43] Slice LW, Yee Jr HF, Walsh JH. Visualization of internalization and recycling of the gastrin releasing peptide receptor-green fluorescent protein chimera expressed in epithelial cells. Receptors Channels 1998;6:201–12. [44] Coy DH, Mungan Z, Rossowski WJ, Cheng BL, Lin JT, Mrozinski Jr JE, et al. Development of a potent bombesin receptor antagonist with prolonged in vivo inhibitory activity on bombesin-stimulated amylase and protein release in the rat. Peptides 1992;13:775–81. [45] Ocak M, Helbok A, von Guggenberg E, Ozsoy Y, Kabasakal L, Kremser L, et al. Influence of biological assay conditions on stability assessment of

54

[46]

[47] [48]

[49]

[50]

[51]

[52]

[53]

[54]

G. Carlucci et al. / Peptides 67 (2015) 45–54 radiometal-labelled peptides exemplified using a 177Lu-DOTA-minigastrin derivative. Nucl Med Biol 2011;38:171–9. Ocak M, Helbok A, Rangger C, Peitl PK, Nock BA, Morelli G, et al. Comparison of biological stability and metabolism of CCK2 receptor targeting peptides, a collaborative project under COST BM0607. Eur J Nucl Med Mol Imaging 2011;8:1426–35. Froberg AC, Visser M, Maina T, Erion J, de Swart J, de Jong M, et al. Are GRPreceptors present in the human pancreas? J Nucl Med 2006:429. Garcia Garayoa E, Ruegg D, Blauenstein P, Zwimpfer M, Khan IU, Maes V, et al. Chemical and biological characterization of new Re(CO)3 /[99m Tc](CO)3 bombesin analogues. Nucl Med Biol 2007;1:17–28. Nock B, Nikolopoulou A, Chiotellis E, Loudos G, Maintas D, Reubi JC, et al. [99m Tc]Demobesin 1, a novel potent bombesin analogue for GRP receptortargeted tumour imaging. Eur J Nucl Med Mol Imaging 2003;2:247–58. Nock BA, Nikolopoulou A, Galanis A, Cordopatis P, Waser B, Reubi JC, et al. Potent bombesin-like peptides for GRP-receptor targeting of tumors with 99m Tc: a preclinical study. J Med Chem 2005;1:100–10. Mather SJ, Nock BA, Maina T, Gibson V, Ellison D, Murray I, et al. GRP receptor imaging of prostate cancer using [99m Tc]demobesin 4: a first-in-man study. Mol Imaging Biol 2014;6:888–95. Hofmann M, Machtens S, Stief C, et al. Feasibility of Ga-68-DOTABOM PET in prostate carcinoma patients. Eur J Nucl Med Mol Imaging 2004;31:S253 [abstr 207]. Schroeder RP, Müller C, Reneman S, Melis ML, Breeman WA, de Blois E, et al. A standardised study to compare prostate cancer targeting efficacy of five radiolabelled bombesin analogues. Eur J Nucl Med Mol Imaging 2010;7:1386–96. Zhang H, Chen J, Waldherr C, Hinni K, Waser B, Reubi JC, et al. Synthesis and evaluation of bombesin derivatives on the basis of pan-bombesin peptides

[55]

[56]

[57]

[58]

[59]

[60]

[61]

labeled with indium-111, lutetium-177, and yttrium-90 for targeting bombesin receptor-expressing tumors. Cancer Res 2004;18:6707–15. Zhang H, Schuhmacher J, Waser B, Wild D, Eisenhut M, Reubi JC, et al. DOTAPESIN, a DOTA-conjugated bombesin derivative designed for the imaging and targeted radionuclide treatment of bombesin receptor-positive tumours. Eur J Nucl Med Mol Imaging 2007;8:1198–208. Lantry LE, Cappelletti E, Maddalena ME, Fox JS, Feng W, Chen J, et al. 177Lu-AMBA: synthesis and characterization of a selective 177Lu-labeled GRP-R agonist for systemic radiotherapy of prostate cancer. J Nucl Med 2006;7:1144–52. Rogers BE, Bigott HM, McCarthy DW, Della Manna D, Kim J, Sharp TL, et al. MicroPET imaging of a gastrin-releasing peptide receptor-positive tumor in a mouse model of human prostate cancer using a 64 Cu-labeled bombesin analogue. Bioconjug Chem 2003;4:756–63. Lin KS, Luu A, Baidoo KE, Hashemzadeh-Gargari H, Chen MK, Brenneman K, et al. A new high affinity technetium-99m-bombesin analogue with low abdominal accumulation. Bioconjug Chem 2005;1:43–50. Parry JJ, Kelly TS, Andrews R, Rogers BE. In vitro and in vivo evaluation of 64 Culabeled DOTA-linker-bombesin(7–14) analogues containing different amino acid linker moieties. Bioconjug Chem 2007;4:1110–7. Mansi R, Wang X, Forrer F, Kneifel S, Tamma ML, Waser B, et al. 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 2009;16:5240–9. Honer M, Mu L, Stellfeld T, Graham K, Martic M, Fischer CR, et al. 18 F-labeled bombesin analog for specific and effective targeting of prostate tumors expressing gastrin-releasing peptide receptors. J Nucl Med 2011;2:270–8.