Monomeric and Dimeric 68Ga-Labeled Bombesin Analogues for

Article Cite This: J. Med. Chem. 2018, 61, 2062−2074

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Monomeric and Dimeric 68Ga-Labeled Bombesin Analogues for Positron Emission Tomography (PET) Imaging of Tumors Expressing Gastrin-Releasing Peptide Receptors (GRPrs) Christos Liolios,*,† Benjamin Buchmuller,† Ulrike Bauder-Wüst,† Martin Schaf̈ er,† Karin Leotta,‡ Uwe Haberkorn,‡,§,∥ Matthias Eder,†,§,⊥ and Klaus Kopka†,§ †

Division of Radiopharmaceutical Chemistry, ‡Clinical Cooperation Unit Nuclear Medicine, and §German Cancer Consortium (DKTK), German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69120 Heidelberg, Germany ∥ Department of Nuclear Medicine, University of Heidelberg, Im Neuenheimer Feld 400, 69120 Heidelberg, Germany ⊥ Division of Radiopharmaceutical Development, German Cancer Consortium (DKTK) Freiburg, and Department of Nuclear Medicine, Faculty of Medicine, Medical Center, University of Freiburg, Hugstetter Straße 55, 79106 Freiburg, Germany S Supporting Information *

ABSTRACT: The GRPr, highly expressed in prostate PCa and breast cancer BCa, is a promising target for the development of new PET radiotracers. The chelator HBED-CC (N,N′-bis[2-hydroxy-5-(carboxyethyl)benzyl]ethylenediamine-N,N′-diacetic acid) was coupled to the bombesin peptides: HBED-C-BN(2−14) 1, HBED-CC-PEG2-[D-Tyr6,β-Ala11,Thi13,Nle14]-BN(6−14) 2, HBED-CC-Y-[D-Phe6,Sta13,Leu14]-BN(6−14) (Y = 4-amino-1-carboxymethylpiperidine) 3, and HBED-CC-{PEG2-Y-[DPhe6,Sta13,Leu14]-BN(6−14)}2 4 (homodimer). Compounds 1−4 presented high binding affinities for GRPr (T47D, 0.56−3.51 nM; PC-3, 2.12−4.68 nM). In PC-3 and T47D cells, agonists [68Ga]1 and [68Ga]2 were mainly internalized while antagonists [68Ga]3 and [68Ga]4 were surface bound. Cell-related radioactivity reached a maximum after 45 min, while tracer levels followed GRPr expression (PC-3 > T47D > LNCaP > MDA-MB-231). [68Ga]4 showed the highest cell-bound radioactivity (PC-3 and T47D). In vivo, tumor (PC-3) targeting for [68Ga]3 and [68Ga]4 increased over time, with dynamic μPET showing clearer tumors images at later time points. [68Ga]3 and [68Ga]4 can be considered suitable PET tracers for imaging PCa and BCa expressing GRPr.

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bombina,10 and since then several peptide GRPr agonists and antagonists have been synthesized, radiolabeled, and tested preclinically and clinically.1,11 BBN agonists internalize after binding to GRPr which is considered advantageous for ligands carrying therapeutic nuclides, but they have a proliferating effect on cancer cells. On the other hand BBN antagonists do not have a proliferation effect on cancer cells, they are mainly surface bound and they have shown less acute adverse effects (e.g. gastrointestinal).1,12 Over the past decade the positron emitter 68Ga has attracted a lot of interest for PET imaging applications, due to its availability from 68Ge/68Ga generators and due to the idea of 68 Ga-labeling using kit-based preparations comparable with those used in 99mTc radiopharmacies with 99Mo/99mTc generators.12 Radiometals with a short half-life such as 68Ga (T1/2 = 68 min) are ideally combined with chelators that have

INTRODUCTION The gastrin-releasing peptide receptor (GRPr), also called bombesin receptor 2 (BB2), is a promising target for noninvasive PET imaging of various types of cancer. GRPr is a glycosylated seven-transmembrane G-protein-coupled receptor, which is expressed in numerous cancers, such as lung, colon, prostate, and breast.1,2 Especially for prostate cancer, GRPr is overexpressed in comparison to sparse expression in normal prostate tissue.3 GRP binding to this receptor stimulates the growth of prostate cancer cells both in vitro and in vivo. A significant inverse correlation was found between GRPr expression and increased Gleason score.1,4 In addition GRPr expression has been found in high density in 72−96% of ductal breast cancer specimens.5−7 There are multiple preclinical and a few clinical studies published evaluating GRPr as imaging target/imaging biomarker mainly for prostate cancer and secondary for breast cancer.1,7−9 Bombesin (BBN), a 14-mer GRPr peptide agonist, was initially found in the skin of the fire-bellied toad Bombina © 2018 American Chemical Society

Received: December 15, 2017 Published: February 12, 2018 2062

DOI: 10.1021/acs.jmedchem.7b01856 J. Med. Chem. 2018, 61, 2062−2074

Journal of Medicinal Chemistry

Article

Scheme 1. Synthesis of GRPr Agonists and Antagonists Coupled with the Chelator HBED-CCa

a

(a) FeCl3, 1.2 equiv, 5% DIPEA, MeOH/H2O 1/1, v/v; (b) TFP (2,3,5,6-tetrafluorophenol), 10 equiv, 0.19 mmol, 311 mg, DIC, 4 equiv, 0.08 mmol, 118 μL, in DMF (1.0 mL), rt, 1 day; (c) pharmacophore (i−iii) 1.2 equiv 6.6 μmol, (i) 10.7 mg, (ii) 8.5 mg, and (iii) 8.3 mg; (d) pharmacophore (iv), 4 equiv, 13.0 mg, 8.5 μmol, excess DIPEA, in DMF (1.0 mL), rt, 1 day; where (i) H2N-BN(2−14); (ii) H2N-PEG2-[D-Tyr6,βAla11,Thi13,Nle14]BN(6−14); (iii) H2N−Y-[D-Phe6,Sta13,Leu14]-BN(6−14); (iv) H2N-PEG2-Y-[D-Phe6,Sta13,Leu14]-BN(6−14) and Y = 4-amino-1carboxymethylpiperidine; (e) [68Ga]GaCl3, 40 μL (80−100 MBq), 1−4 (0.3−1.0 nmol) in 0.1 M HEPES buffer (pH = 7.5, 100 μL), 10 μL of HEPES solution (2.1 M). pH 4.2, 98 °C, 10 min.

BBN-based homodimers have been reported with varying results.18−22 In the present study we investigated the effects of linking the HBED-CC chelator to different BBN pharmacophores. HBEDCC was linked to two agonists, the naturally occurring peptide H2N-BN(2−14) and the modified peptide sequence H2NPEG2-[D-Tyr6,β-Ala11,Thi13,Nle14]BN(6−14), as well as to the antagonist, H2N−Y-[D-Phe6,Sta13,Leu14]-BN(6−14), (Y = 4amino-1-carboxymethylpiperidine) resulting in ligands 1, 2, and 3, respectively. The last two BBN pharmacophores connected to a DOTA chelator have been used in clinical studies, i.e., BZH3 and RM2.23−25 In addition, by taking advantage of dual conjugation possibilities of HBED-CC, a homodimer was synthesized, HBED-CC-{PEG2-Y-[D-Phe6,Sta13,Leu14]-BN(6− 14)}2 4 based on the antagonist’s sequence (Scheme 1). The new peptides were evaluated in vitro for their GRPr affinity, internalization, and time kinetic cell binding in prostate cancer (PC-3, high GRPr expression, LNCaP, low expression) and breast cancer cells (T47D, high GRPr expression, MDA-MB-

fast kinetics and can form stable complexes at ambient temperature.13 Such an agent is HBED-CC, N,N′-bis[2hydroxy-5-(carboxyethyl)benzyl]ethylenediamine-N,N′-diacetic acid, which can be conveniently labeled with 68Ga (25 °C, 10− 20 min, pH 4−4.5) and has two additional carboxylic groups (propionic acid moieties) that can be used for the conjugations to a pharmacophore/binding vector.14 This functionality is also ideal for the synthesis of dimeric compounds, resulting in radiolabeled homodimers (same pharmacophore), heterodimers (different pharmacophores),15,16 or dual-labeled tracers, e.g., a radiolabeled pharmacophore and a dye for a combination of PET with optical imaging. The multivalency approach (e.g., homodimers) is considered to increase ligand affinity through multiple binding interactions.17 Several binding modes have been proposed as an explanation for this effect; i.e., the ligand simultaneously binds to two receptors on the cell surface or the improved statistical effect, where the ligand binds to one receptor, but its apparent local concentration is increased.17 So far only a few studies on 2063



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Table 1. Calculated Mass Results with MALDI-MS and Retention Times after Radio-RP-HPLC Analysis of the Ligands (L) and Their [natGa]L and [68Ga]L Complexes [M + H]+ or [M]+

calculated mass

a

nat

a

compd

chelator

linker−pharmacophore

L

[ Ga]L

1 2 3 4

HBED-CC

BN(2−14) PEG2-[D-Tyr6,β-Ala11,Thi13,Nle14]-BN(6−14) Y-[D-Phe6,Sta13,Leu14]-BN(6−14) (PEG2-Y-[D-Phe6,Sta13,Leu14]-BN(6−14))2

2023.3 1799.0 1768.1 3293.9

2091.0 1866.7 1834.8 3359.6a

Theoretical calculations for [

nat

L

[natGa]Lb

tR (min) [68Ga]L

2024.8 1800.8 1769.5 3294.2

2091.0 1867.5 1836.3 3359.7

2.15 2.42 2.25 2.39

GaH2L] complex, where L equals ligand. bExperimental values, Y = 4-amino-1-carboxymethylpiperidine.

Figure 1. The γ-trace of the comparative RP-radio-HPLC analysis after labeling with 68Ga is presented with the tR (min) noted at the top of each peak. Radiochemical purity was above 98% in all cases. RP-HPLC analysis was performed on a Chromolith RP-18e (100 mm × 4.6 mm; Merck, Darmstadt, Germany), using a linear A−B gradient (0% B to 100% B in 5 min), flow of 4 mL/min, A consisting of 0.1% aqueous TFA, and B consisting of 0.1% TFA in CH3CN.

analyses results of the pure products and their natGa complexes with RP-HPLC and MALDI-MS are summarized in Table 1. Labeling with 68Ga and natGa. In all cases, radiolabeling with 68Ga resulted in one single species as determined by analytical RP-HPLC, while the radiochemical yield was >98% (Figure 1). The retention times (tR) of the 68Ga-labeled compounds (obtained by radio-HPLC analysis) are summarized in Table 1 (comparative HPLC results for pure [natGa]i can be found in Supporting Information, Figure S2). In general, sequence dependent differences proved more important than size for hydrophilicity. More specifically, [68Ga]1 (molecular weight, MW = 2091.0), containing the native BBN sequence, was the most hydrophilic (tR = 2.15 min), followed by the antagonist [68Ga]3 (tR = 2.25 min, MW = 1868) and the homodimer [68Ga]4 (tR = 2.39 min, MW = 3359.7) and finally [68Ga]2 (tR = 2.42 min, MW = 1836.3). All compounds were proven stable at room temperature in saline even after 2 h after labeling. After complexation with natGa compounds were purified with semipreparative RP HPLC and analyzed with MALDI-MS (Table 1). [natGa]i (i = 1−4) complexes showed the same RP-HPLC retention times as the [68Ga]i. Determination of Binding Affinity for PSMA and GRPr. An in vitro competitive cell binding assay for the compounds under study was performed against 125I[Tyr4]-BN in order to determine the binding potency for GRPr, with PC-3 (human prostate cancer) and T47D cells (human breast cancer). As internal references, two peptides known from the literature were also included in the assay, i.e., RM2 and BZH3,23−25 and natural BBN. The results of the IC50 values (nM) for both cell lines are summarized in Table 2.

231, low expression). Internal controls for the in vitro studies included BBN, BZH3, and RM2. [68Ga]i (i = 1−4) compounds were also tested in vivo in mice bearing PC-3 tumors for their tumor-targeting ability and biodistribution behavior with [68Ga]RM2 as internal control.

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RESULTS Chemistry. The peptidic parts of the tracers were synthesized on a Rink amide resin according to standard Fmoc peptide synthesis protocols. For the reference compounds RM2 and BZH3 the DOTA chelator was coupled as an amino acid and then the final product was cleaved from the resin and purified with RP-HPLC. For the HBED-CC conjugates 1−4 initially the phenolic and carboxylate groups of HBED-CC were selectively protected by complexation with Fe3+ to form [Fe(HBED-CC)]− and then the two remaining carboxylate groups were selectively activated with TFP (2,3,5,6tetrafluorophenol) resulting in the mono- and bis-TFP esters of [Fe(HBED-CC]−. At the final step, the mono-TFP ester of [Fe(HBED-CC]− was reacted with the corresponding peptidic parts resulting into monomers 1−3, while the bis TFP ester of [Fe(HBED-CC]− resulted in the formation of dimer 4. The synthesis of ligands 1−4 is summarized in Scheme 1. The final products were of purity greater than 98%. Overall yield for 1−3 ranged between 15% and 20%, while for the homodimer 4 it was 10%. At the final step, iron was removed from the ligands via SEP-PAK cartridge after acidic treatment (1 M HCl) and elution with AcCN/H2O (8:2, v/v) to afford iron-free products for labeling with 68Ga and natGa (see next section). The 2064



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0.56 nM, PC-3). The IC50 values observed for most cases were lower for T47D than for PC-3. Ligand 1 (0.56 nM, T47D and 2.12 nM, PC-3) presented the lowest IC50 (highest affinities for GRPr), followed by the antagonist 3 (1.07 nM, T47D; 2.32 nM, PC-3) and its dimer 4 (3.03 nM, T47D; 2.45 nM, PC-3). The modified agonist H2N-PEG2-[D-Tyr6, β-Ala11,Thi13, Nle14]BN(6−14) presented the higher IC50 values (lowest affinity for GRPr) (3.51 nM, T47D; 4.68 nM, PC-3). Internalization Experiments in PC-3 and T47D Cell Lines. The HBED-CC BBN analogues [68Ga]i (i = 1, 2, and 3) were tested in vitro in PC-3 and T47D cells (as internal reference [68Ga]RM2 was also included in the assay). The results are summarized in Figures 2 and 3. For the two agonists, [68Ga]1 and [68Ga]2, the main fraction of cell-bound (surface plus internalized) radioactivity was internalized in PC-3 cells (66% and 54% of total), while the antagonists [68Ga]3 and [68Ga]4 were mainly surface bound (84% and 73% of total). Analogously, in T47D cells the two agonists [68Ga]1 and [68Ga]2 showed increased internalized radioactivity (76% and

Table 2. IC50 Values of the Compounds 1−4 Determined against 125I-BBN (30 nM) in T47D and PC-3 Cellsa compd

T47D (nM)

1 2 3 4 BBN RM2 BZH3

0.56 3.51 1.07 3.03 0.26 0.63 1.85

log IC50 (St.Er.) −0.25 0.54 0.03 0.51 −0.59 −0.20 0.28

(0.04) (0.05) (0.07) (0.06) (0.02) (0.04) (0.04)

PC-3 (nM) 2.12 4.68 2.12 2.45 0.65 1.33 2.08

log IC50 (St.Er.) 0.33 0.67 0.33 0.39 −0.19 0.12 0.32

(0.03) (0.07) (0.12) (0.06) (0.03) (0.05) (0.03)

a

As internal references (italics), three known compounds from the literature, i.e., BBN, the antagonist RM2, and the agonist and BZH3, were also used.

(Displacement curves of 125I[Tyr4]-BBN for ligands 1−4 and reference compounds natural BBN, RM2, BZH3 are available in Figure S1 in Supporting Information.) All ligands presented high binding affinities in the nanomolar range close to the values of the reference compound BBN (0.26 nM, T47D and

Figure 2. Surface, internalized, and total cell bound ligands [68Ga]1, [68Ga]2, [68Ga]3, and [68Ga]4 expressed as cpm at 37 °C (45 min incubation time) in PC-3 (a) and T47D (b) cells. The values at the top of the bars represent the percentage of the total cell related activity that was surface bound or internalized. Statistical results are expressed as asterisks at the top of the bars (one way nonparametric ANOVA, P < 0.05). 2065



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Figure 3. Total cell related [68Ga]i agonists (i = 1, 2) and antagonists and (i = 3, 4) in PC-3 (a) and T47D (b) cells. The results are presented as specifically bound radioactivity (cpm) of 68Ga-labeled compounds (30 nM in 2 × 105 PC-3 or T47D cells/well seeded 24 h before the experiment). As reference [68Ga]RM2 was included in the assay. Asterisks at the top of the bars represent the statistically significant differences against [68Ga]RM2 (ANOVA, one way nonparametric, P < 0.05).

Figure 4. Total cell-bound radioactivity over time of (a) [68Ga]4 (antagonist) in PC-3 and PC-3 blocked, [68Ga]3 (antagonist) in PC-3 and PC-3 blocked, T47D, LNCaP, and MDA-MB-231 cells and (b) [68Ga]1 in PC-3, PC-3 blocked and [68Ga]2 (agonists) in PC-3, PC-3 blocked and LNCaP cells. Results are expressed as % of the total activity added in 105 cells. The GRPr positive cell lines (PC-3, T47D) are presented with solid lines, while the GRPr negative cell lines (LNCaP and MBA-MB-231) and blocking experiments are presented with dashed lines. Blocking experiments were conducted with an addition of 1000-fold excess of the corresponding bombesin pharmacophore.

72%), while antagonists [68Ga]3 and [68Ga]4 increased surface bound (89% and 95%) (Figure 2). In both PC-3 and T47D cells the total cell-related radioactivity for the dimer [68Ga]4 was higher than the monomers and at the same levels as the DOTA analogue [68Ga]RM2 (Figure 3).

In Vitro Time Kinetic Binding Studies. Time kinetic data (0−90 min) for 68Ga-labeled agonists and antagonists (30 nM in 1.4 × 105 cells) were investigated in GRPr positive cell lines PC-3 (prostate cancer) and T47D (breast cancer) and in negative cell lines LNCaP (prostate cancer) and MDA-MB-231 2066



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Figure 5. (a) Comparative biodistribution studies of [68Ga]i (i =1−4) and [68Ga]RM2 (control) at 30 min (top) and 60 min (bottom) p.i. in Balb nu/nu male mice bearing PC-3 tumors. The results are expressed as percentage of the injected dose per g for each organ or tissue (mean %ID/g ± SD, n = 3−4). Asterisk represent statistically significant differences against [68Ga]RM2 (ANOVA, one way nonparametric, P < 0.05). (b) Tumor to normal tissue ratios (mean value ± SD) for the two antagonists under study, [68Ga]3 and [68Ga]4, and the reference compound [68Ga]RM2 at 30 min (top) and 60 min p.i.(bottom).

from the biodistribution experiments (30 and 60 min p.i.) are summarized in Figure 5a (for the organ distribution values see also Table S1, Supporting Information). Tumor-to-background ratios for the antagonists [68Ga]i (i = 3−4) and reference antagonistic compound [68Ga]RM2 have been calculated for 30 and 60 min p.i. and are presented as a bar graph in Figure 5b (see also Table S2, Supporting Information). For the two antagonists [68Ga]3 and [68Ga]4 dynamic μPET imaging studies were also conducted which are being presented in Figure 6. The biodistribution experiments showed a similar tumor uptake for all compounds tested at 30 min p.i. ranging between 3 and 4 %ID/g, i.e., 3.06% ± 0.28 %ID/g for [68Ga]2 (agonist) and 4.36% ± 0.90 %ID/g for [68Ga]1 (agonist containing the native BBN sequence). At 60 min p.i. tumor-localized tracer activity was increased for both antagonists. More specifically for [68Ga]3 it increased from 4.32 ± 0.25 %ID/g (30 min) to 4.74 ± 1.43 %ID/g (60 min) and for [68Ga]4 from 3.67 ± 0.27 % ID/g (30 min) to 5.07 ± 1.06 %ID/g (60 min), while for both agonists tumor radioactivity was decreased ([68Ga]1 was 1.84 ± 0.93 %ID/g and [68Ga]2 1.75 ± 0.30 %ID/g, 60 min p.i.). The reference antagonistic compound [68Ga]RM2 showed a significantly higher tumor uptake (6.40 ± 1.05 %ID/g) than [68Ga]2 and [68Ga]4 at 30 min p.i., but compared to [68Ga]1 and [68Ga]3 differences were not significant. At 60 min p.i. tumor uptake for [68Ga]RM2 (5.74 ± 1.22 %ID/g) was identical to the other antagonists [68Ga]3 and [68Ga]4 and

(breast cancer), while for the highly GRPr expressing PC-3 cell lines blocking experiments were also conducted. Results for antagonists [68Ga]3 and (PC-3 and PC-3 blocked, T47D, MDA-MB-231, LNCaP) and [68Ga]4 (PC-3 and PC-3 blocked) are presented in Figure 4a, while results for agonists [68Ga]2 (PC-3 and PC-3 blocked, LNCaP) and [68Ga]1 (PC-3 and PC-3 blocked) are shown in Figure 4b. All compounds tested presented similar time kinetics reaching maximal cellbound activity after approximately 45 min. Antagonists [68Ga]3 and [68Ga]4 reached the plateau slightly faster than agonists [68Ga]1 and [68Ga]2, while for all compounds the cell related activity declined after 60 min. In PC-3 cells the homodimeric antagonist [68Ga]4 presented higher binding than the others, and agonist [68Ga]2 presented the lowest. For the antagonist [68Ga]3 the amount of cell bound activity for both negative cell lines LNCaP (prostate cancer) and MDA-MB-231 (breast cancer) was similar and ranged between 0.8% and 1.6% of the given radioactivity (Figure 4a). The amount of cell binding for [68Ga]i (i = 1−4) after blocking PC-3 cells with a 1000× excess of the corresponding pharmacophore ranged between 0.5% and 0.7% of the given radioactivity. Biodistribution and Imaging μPET Results. The in vivo biodistribution behavior of the radiolabeled analogues [68Ga]i (i = 1−4) was examined with organ distribution (30 and 60 min p.i.) and imaging experiments in PC-3 tumor bearing mice. For the two antagonists a comparative organ distribution study (30 and 60 min p.i.) was also conducted against reference antagonistic compound [68Ga]RM2. Results (%ID/g ± SD) 2067



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Figure 6. Whole-body μPET images of athymic male nu/nu mice bearing PC-3 tumor xenografts (from left to right, MIP 20−40 min, MIP 120−140 min, and MIP 120−140 min blocked) after iv administration of (a) [68Ga]3 (100 pmol, 35−45 MBq) and (b) [68Ga]4 (100 pmol, 35−45 MBq), where T = tumor; K = kidneys, B = bladder, as indicated with arrows.

and higher uptake in the kidneys (11.31 ± 4.24 %ID/g, 30 min, 17.67 ± 2.00 %ID/g, 60 min) and liver (6.71 ± 0.22 %ID/g, 30 min and 4.56 ± 0.69 %ID/g, 60 min, respectively). Off-target tissues such as blood, muscle, and the rest of the organs sampled showed minimal uptake and thus low background noise for tumor detection (Figure 5a). More specifically based on the organ distribution results, the contrast ratios of tumor to normal tissue were calculated for [68Ga]i (i = 1−4) and [68Ga]RM2, respectively and the results are summarized in Figure 5b and Table S2 in Supporting Information. As indicated by the bar graph, the monomeric antagonist [68Ga]3 and the reference [68Ga]RM2 (Figure 5b) were superior to the other compounds, presenting the highest values for most cases at both 30 and 60 min p.i. For the two antagonists [68Ga]3 and [68Ga]4, dynamic μPET imaging studies (Figure 6) were additionally conducted in order to examine their pharmacokinetic profile at later time points (120 min p.i.). The dynamic μPET imaging and blocking studies of [68Ga]3 and [68Ga]4, (Figures 6 and 7) showed that both tracers were able to efficiently and specifically visualize the PC-3 tumor. After 1 h p.i., both tracers were washed out from the body though the kidneys and the urinary bladder while remaining in the tumor. Consequently, later time points provided clearer images of PC-3 tumors as shown in the MIPs at 120−140 min p.i. Time−activity curves were generated for selected organs and the tumor (VOIs) (Figure 7), and the area under the curve (AUC) was calculated for the first hour. The results of this analysis matched the findings of the biodistribution experiments. Briefly, both tracers presented

significantly higher than [68Ga]1 and [68Ga]2, both having shown a fast clearance from the tumor. The uptake in pancreas was high for all compounds under study due to the expression of GRPr receptors on pancreatic cells. Pancreatic uptake for compounds [68Ga]1, [68Ga]2, and [68Ga]3 was decreased from 30 to 60 min with the exception of [68Ga]4, showing a slight increase in the pancreatic radioactivity accumulation (23.83 ± 1.71 %ID/g, 30 min and 35.24 ± 3.29 %ID/g, 60 min). Blocking studies at 30 min p.i. for the antagonistic dimer [68Ga]4 showed a decrease in both sites of GRPr expression pancreas (4.68 ± 1.67 %ID/g) and tumor (1.17 ± 0.08 %ID/g), providing further proof of its specific binding to GRPr (Table S1, Supporting Information). The reference compound [68Ga]RM2 also showed high pancreatic uptake at 30 min (19.59 ± 3.15 %ID/g) and 60 min (15.23 ± 2.77 %ID/g) p.i., which was less than the dimer [68Ga]4 but more than the monomer [68Ga]3. All compounds were mainly excreted via the kidneys into the urinary bladder. The agonists [68Ga]1 and [68Ga]2 presented a high kidney uptake at 30 min p.i. (18.75 ± 0.82 and 5.40 ± 1.41 %ID/g, respectively), which was later significantly decreased (4.10 ± 0.55 and 2.02 ± 0.33 %ID/g, 60 min p.i.). The antagonist [68Ga]3 showed minimal kidney uptake both at 30 min p.i. (3.77 ± 0.67 %ID/g) and at 60 min p.i. (4.23 ± 0.22 % ID/g), likewise [68Ga]RM2 (4.00 ± 1.82 %ID/g, 30 min p.i. and 2.09 ± 0.25 %ID/g, 60 min p.i.). Regarding uptake in nontarget organs both [68Ga]3 and [68Ga]RM2 showed a similar pharmacokinetic profile. On the other hand the dimer [68Ga]4 showed slower elimination rates from the circulation 2068



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Figure 7. Time−activity curves expressed as SUVs around the tumor (■), muscle (●), heart (▲), liver (▼) for (a) [68Ga]3 (100 pmol, 35−45 MBq) and (b) [68Ga]4 (100 pmol, 35−45 MBq) at 0−60 min p.i. (c) Area under the curve (AUC0−57min) for [68Ga]3 (left) and [68Ga]4 (right) for selected organs.

nearly the same values for tumor AUC (Figure 7) with [68Ga]4 being slightly better (18.5) than [68Ga]3 (16.0). Nevertheless, [68Ga]3 presented a better pharmacokinetic profile by showing lower AUC values for nontarget organs (liver, 19.2) and blood (heart, 25.7) than [68Ga]4 (liver, 65.2; heart, 24.7).

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Bombesin receptors and especially subtype bombesin receptor 2 (BB2), also called gastrin-releasing peptide receptor (GRPr), are expressed in various types of cancer (i.e., lung, colon, prostate, breast) and thus considered as promising target for noninvasive PET imaging.1,2 In previous studies of our group15,16 the HBED-CC chelator has been successfully combined with the BBN pharmacophore, [68Ga]2, for the investigation of monomers and heterodimers (combining PSMA and BBN), as potential radiotracers for PET imaging of prostate cancer. In this study we further investigated the effects of combing the HBED-CC chelator with different BBN pharmacophores (agonists, antagonists) in an effort to further improve GRPr targeting and biodistribution properties in view of future clinical translation. Since only a few studies regarding BBN homodimers have been reported so far, with varying results,18,20,21,29 the potential of using the complexing agent HBED-CC for the construction of homodimers was also investigated. As pharmacophores, we tested two BBN agonists, the naturally occurring peptide H2N-BN(2−14) (reference), [68Ga]1, and the modified peptide sequence H2N-PEG2-[DTyr6,β-Ala11,Thi13,Nle14]BN(6−14), [68Ga]2, and a BBN antagonist, H2N−Y-[D-Phe6,Sta13,Leu14]-BN(6−14), (Y = 4amino-1-carboxymethylpiperidine) [68Ga]3. The last was also used for the synthesis of the homodimer, [68Ga]4. This choice

DISCUSSION

The positron emitter 68Ga through its availability by elution of 68 Ge/68Ga generators12 and usage as radionuclide of choice for a number of clinically relevant radiopharmaceutical precursors such as [68Ga]Ga-DOTA-TOC, [68Ga]Ga-DOTA-TATE, or [68Ga]Ga-PSMA-11 has been established in daily clinical PET imaging during the past decade for highly sensitive and selective noninvasive imaging of distinct tumor entities.26,27 68Ga has a short half-life (T1/2 = 68 min); thus it is ideally combined with tracers which can be rapidly radiolabeled at ambient temperature with high yields13 and which target highly expressed receptors on the surface of cancer cells.28 HBED-CC represents a versatile chelator as it can be easily labeled with 68Ga at room temperature, while its two additional carboxylic groups not participating at the complexation of the radiometal can be linked to a corresponding pharmacophore of interest.14,15 Thus, HBED-CC is well suited for the synthesis of dimeric compounds, either homo- or heterodimeric.15,16 2069



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activity after 60 min, which is in accordance with previous studies on GRPr ligands.15,25 The homodimeric antagonist [68Ga]4 showed higher binding than the others. The amount of cell related activity for the negative cell lines LNCaP (prostate cancer) and MDA-MB-231 (breast cancer) was similar and ranged between 0.8% and 1.6% of the given radioactivity, which was close to cell binding values for highly expressing GRPr cells, PC-3, during the blocking experiments (0.5−0.7%). This provides further proof for their specific binding on GRPr. The amount of maximum cell-bound [68Ga]3 followed in order previously reported GRPr expression levels for each cell line PC-3 (2.5 × 105 GRPr/cell), LNCaP (5.9 × 103 GRPr/cell),33 T47D (3.6 × 104 GRPr/cell), MDA-MB-231 (2.27 × 103 GRPr/cell).34,35 Early studies on BBN dimers on Swiss 3T3 cells suggested that dimeric forms of Lys3-BN have antagonistic properties for GRPr and they were 100- to 1000-fold more potent than the monomers for the inhibition of mitogenesis.20 An enhancement of potency for the BBN dimers has also been reported on melanophores transfected with plasmid encoding GRPr.18 Regarding radiolabeled compounds previous studies on two [64Cu]Cu-NOTA-Y-BBN(6−14) (Y = -Suc-PEG-Lys/-Gly or -PEG-Gly) dimers have reported nonsignificant differences between the Ki values of monomers (2.51 ± 1.54 nM) and dimers ([64Cu]Cu-NOTA-dimer 1, 1.76 ± 1.30; and dimer 2, 2.00 ± 1.59 nM) on PC-3 cells, while the expected higher cellular uptake after dimerization was not observed. On the contrary, the dimers showed lower uptake than the monomer but slower efflux from the cells.21 Other studies on BBN dimers, [99mTc]Tc-HYNIC(tricine/TPPTS)-Glu[Aca-BN(7− 14)2, have reported Ki values being 5 times higher than the monomer (63.40 ± 11.70 nM, PC-3 cells) and a slower but eventually higher cellular accumulation for the dimer [99mTc]Tc-HABN2 (after 40 min).22 A higher cell uptake was also observed in the case of [177Lu]Lu-DOTA-BBN(1−14) and -Ahx-BBN(4−14) dimers in comparison with the monomers.19 According to the authors, this effect was due to bivalent binding or, if monovalent binding was assumed, to an increased probability of rebinding after dissociation. Either way dimerization eventually resulted in higher internalization rates.19 The investigation of the pharmacokinetics of the 68Ga labeled tracers [68Ga]i (i = 1−4) showed minimal radioactivity in blood and muscle and the rest of organs sampled, resulting in low background noise for tumor detection (Figure 5a). At 30 min p.i. tumor uptake for agonist [68Ga]1, antagonist [68Ga]3 and reference [68Ga]RM2 were significantly higher than that of [68Ga]2 and [68Ga]4, while at 60 min p.i. all antagonists including the reference showed significantly higher tumor uptake than the agonists. The reference [68Ga]RM2 and [68Ga] 3 did not show significant differences regarding tumor uptake (30 and 60 min p.i.), while for the dimer [68Ga]4 this was significantly less at 30 min p.i. and the same at 60 min p.i. Among the HBED-CC analogues studied the antagonist [68Ga] 3 has shown the best tumor to normal tissue ratios at both 30 and 60 min p.i. The exchange of the DOTA chelator for HBED-CC did not affect the pharmacokinetics of the tracer as much as the dimerization of the pharmacophore on the HBEDCC chelator. Especially regarding off-target accumulation, the biggest difference between [68Ga]RM2 and [68Ga]3 was in the pancreas, where [68Ga]3 showed less uptake both at 30 min (1/2×) and at 60 min (1/3×) p.i. Additionally, the HBED-CC exchange for DOTA resulted in a slightly slower elimination of

was made because BBN antagonists are expected to have less side effects and nonmitogenic properties.29,30 All compounds tested in vitro in GRPr expressing cell lines PC-3 and T47D (Table 2) presented IC50 values in the low nanomolar range, comparable with those of the three reference compounds (BBN, RM2, BZH3). The combination of the HBED-CC with H2N-[D-Tyr6,Bal11,Thi13,Nle14]BN(6−14) 2 resulted in slightly higher IC50 values than the reference compound BZH3, possibly due to the interference of HBEDCC with the GRPr binding. The monomeric antagonist 3 and the agonist containing the native BBN sequence 1 presented the lowest IC50 values in both T47D and PC-3. Previously reported studies with similar antagonistic BBN peptides (XPEG2-RM26) conjugated with different chelators have reported IC50 values (PC-3 cells) in the same range of concentrations, i.e., natGa complexes of X = NOTA, 2.3 nM; X = NODAGA, 3.0 nM; X = DOTA, 2.9 nM; X= DOTAGA, 10.0 nM; and X = AlnatF-NOTA, 4.4 nM.31,32 Dimer 4 presented slightly higher IC50 values than the monomer 3 regarding T47D but no significant difference in PC-3. This observation could be related to the differences between the two cell lines regarding the expression levels and proximity of receptors on the cell surface (PC-3, 2.5 × 105 GRPr/cell; T47D, 36.0 × 103/cell).33−35 A sparse receptor expression on the cell surface would make multiple binding less possible, in which case the ligand’s binding affinity would be more influenced by the statistical effect.17 This means if monovalent binding is assumed, the homodimer has a higher probability of rebinding after dissociation from the receptor.17,19 Further in vitro testing in PC-3 and T47D cells of antagonists [68Ga]3 and [68Ga]4 showed that cell bound activity was mainly bound on the surface, while for agonists [68Ga]1 and [68Ga]2 it was internalized (Figure 2). These results are in agreement with previous studies on similar BBN antagonists, i.e., RM2631,32 and BBN agonists;7,36 i.e., when 111 In-AMBA was tested in T47D cells, it showed higher amounts for the internalized cell fraction (∼60−55%) than the membrane fraction (surface bound, ∼40−45%) for the same range of ligand concentrations (10−1 nM).7 Regarding the amount of cell related radioligand nonsignificant differences between the monomeric HBED-CC analogues [68Ga]1, [68Ga] 2, [68Ga]3 have been observed in both PC-3 and T47D. In contrast, the dimer [68Ga]4 presented significantly higher total cell binding than the monomers, indicating bivalence had indeed an impact on the binding capacity. However, the overall affinity was slightly reduced which might be attributed to the chemical characteristic and increased size of the dimeric molecule. The tracers [68Ga]1, [68Ga]2, [68Ga]3, and [68Ga]4 were also compared with the antagonist [68Ga]RM2 (reference) with respect to their total cell binding behavior. Statistical analysis of the results in both cell lines, PC-3 and T47D (Figure 3), showed that the dimer [68Ga]4 was not different from the reference compound, while all other compounds [68Ga]1, [68Ga]2, and [68Ga]3 showed less total cell binding. Radiolabeled agonists and antagonists [68Ga]i (i = 1−4) were investigated for their cell binding kinetics in GRPr positive cell lines, PC-3 (prostate cancer) and T47D (breast cancer), and GRPr negative cell lines, LNCaP (prostate cancer) and MDA-MB-231 (breast cancer), while for the high GRPr expressing PC-3 cells blocking experiments were also conducted (Figure 4). The antagonists [68Ga]3 and [68Ga]4 presented slightly faster binding kinetics than the agonists, while for all compounds there was a decline in the cell related 2070



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[68Ga]3 from the blood pool; thus reference [68Ga]RM2 showed higher tumor/blood values, although at 60 min p.i. [68Ga]3 has shown higher values for tumor/muscle, tumor/ spleen, tumor/brain than [68Ga]RM2 (Figure 5b). Tracer [68Ga]4 on the other hand despite showing a higher in vitro uptake had inferior pharmacokinetic properties than the monomer [68Ga]3. Possibly because its higher lipophilicity resulted in slower elimination from the circulation and prolonged retention in off-target organs and tissues (i.e., kidneys, liver, and intestines). Previous biodistribution studies on BBN dimers have reported PC-3 tumor uptake of 1.58 ± 0.18 %ID/g (60 min p.i.) for [99mTc]Tc-HABN2 and for [64Cu]Cu-NOTA-dimer 2 (3.95 ± 0.26 %ID/g, 30 min p.i. and 6.28 ± 2.87 %ID/g, 120 min p.i.). It should also be commented that the differences between the literature values for the tumor uptake at 60 min p.i. for reference [68Ga]RM2 (14.11 ± 1.88 % ID/g)25 and reported values (5.74 ± 1.22 %ID/g) are most likely due to differences in the labeling procedure (i.e., specific activity, use of cartridge) and the animal models (i.e., tumor size, age of the animals). The two HBED-CC antagonists [68Ga]3 and [68Ga]4 were further evaluated at later time points with dynamic imaging μPET studies, in mice bearing PC-3 tumors (Figures 6 and 7). After 1 h, both tracers were washed out from the body though the kidneys and the urinary bladder, while radioactivity accumulated in the tumor. Thus, later time points provided clearer images of PC-3 tumors. The AUCtumor calculated for the first hour after administration showed [68Ga]4 being slightly better (18.5) than [68Ga]3 (16.0). Nevertheless, [68Ga]3 presented a better pharmacokinetic profile by showing lower AUC values for nontarget organs (liver, 19.2) and blood (heart, 25.7) than [68Ga]4 (liver, 65.2; heart, 24.7). Previous dynamic imaging μPET studies for [64Cu]Cu-NOTA-monomer and [64Cu]Cu-NOTA-dimer 2 (mice bearing PC-3 tumors) reported initially a higher tumor uptake for the monomer (until 30 min p.i.) which was then reversed in favor of the dimer.21 The inclusion of charged groups in [68Ga]4 could possible improve its pharmacokinetic properties toward a faster clearance from the blood pool and nontarget organs, i.e., liver and GRPr expressing pancreas.

(Marktredwitz, Germany). For all reaction products the chemical purity was greater than 95% as determined by HPLC. The following RP-HPLC systems were used for analysis and purification: Agilent 1100 series, equipped with a multiwavelength detector (MWD) and Latek P402 (Latek Labortechnik-Geraete, Eppelheim, Germany) equipped with a HITACHI variable UV detector (absorbance was measured at 214 and 254 nm) and a γ detector (Bioscan; Washington, USA). Both RP-HPLC systems were equipped with a Chromolith RP-18e (4.6 mm × 100 mm; Merck, Darmstadt, Germany) analytical column. For purifications the following system was used: VWR International, La Prep UV/vis detector P314, pumps P110, equipped with a Nucleodur Sphinx RP, 5 μm VP 250/21 (MACHEREY-NAGEL GmbH & Co. KG, Düren, Germany). Unless stated otherwise the following gradient (A−B) was used for analysis: 0−100% B in 6 min; (A) 0.1% TFA in H2O and (B) 0.1% TFA in CH3CN; flow of 4 mL/min. For purifications, the following gradient was used: 10−90% B in 20 min, flow of 20 mL/min. Mass spectrometry was performed with a MALDI-MS Daltonics Microflex system (Bruker Daltonics, Bremen, Germany). Full-scan single mass spectra were obtained by scanning m/z = 200−4000 (2,5dihydroxybenzoic acid in H2O/AcCN 1:1 was used as matrix). For all in vitro and in vivo experiments a NaI (TI) γ counter (Packard Cobra II, GMI, Minnesota, USA) was used for the measurement of radioactive probes. Chemistry. All peptides were synthesized on a Rink amide resin (4-methylbenzhydrylamine resin, 200−400 mesh). Amino acid (a.a.) coupling was according to standard Fmoc peptide synthesis protocols (a.a./HBTU/DIPEA, 4.0/3.9/4.0 equiv, 30 min, rt). The same protocol was used for coupling of DOTA-tris(tBu)ester (CheMatech, Dijon, France) for the reference compound RM2.25 At the final step the peptides were cleaved from the resin with the following mixture TFA/TIPS/H2O (95/2.5/2.5, v/v/v), precipitated in ice-cold (0 °C) diethyl ether, and purified with semipreparative HPLC. For the HBED-CC compounds 1−4 the phenolic and carboxylate groups of HBED-CC (synthesized in house) were selectively protected by complexation with Fe3+ to form [Fe(HBED-CC)]− according to previously published methods.14,37 The two propionic acid functions remaining in [Fe(HBED-CC]− (0.01 M solution in DMF, 700 μL) reacted with an excess of TFP (2,3,5,6-tetrafluorophenol) (10 equiv, 0.19 mmol, 311 mg) and DIPC (4 equiv, 0.08 mmol, 118 μL) for 3 days in DMF (0.5 mL, rt). The mono- and bis-TFP ester of [Fe(HBED-CC]− compounds were isolated with semipreparative HPLC (overall yield 30%, tR = 2.9 min; 25%, tR = 4.8 min, respectively) a nd identified with mass spectrometry: C 32 H 28 F 4 FeN 2 O 10 (−) ; MW, 732,1; m/z 733.2 [M + H] + ; C38H28F8FeN2O10(−); MW, 880.5; m/z 881.0 [M + H]+. The monoTFP ester (4.0 mg, 5.5 μmol) then reacted with the purified peptides H2N-BN(2−14); H2N-PEG2-[D-Tyr6,β-Ala11,Thi13,Nle14]BN(6−14); and H 2N−Y-[D -Phe 6 ,Sta 13 ,Leu 14]-BN(6−14) (Y = 4-amino-1carboxymethylpiperidine) (1.2 equiv, 6.6 μmol, 10.7 mg; 8.5 mg and 8.3 mg) resulting in the monomers 1−3, respectively (rt, 1 day). For the synthesis of the dimer, 4, the peptide H2N-PEG2-Y-[DPhe6,Sta13,Leu14]-BN(6−14) (4 equiv 13.0 mg, 8.5 μmol) reacted with the bis-TFP ester of [Fe(HBED-CC)]− (1.9 mg, 2.1 μmol) and an excess of DIPEA in DMF (1.0 mL) (rt, 1 day) (Scheme 1). The above crude products were purified with preparative HPLC, and their masses were analyzed with MALDI-MS. In the final step iron was removed from the HBED-CC chelators after immobilization of the corresponding chelates on a C-18-SEP-PAK and by flushing with acid treatment (1.0 M HCl) according to previously published methods.15 For calculations of the exact masses ChemBioDraw ultra 13.0 was used. Labeling with 68Ga and natGa. For radiolabeling, 68Ga (t1/2 = 68 min, β+ 88%, Eβ+ max 1.9 MeV) eluate was obtained from a 68Ge/68Ga generator based on pyrogallol resin support.38 Typically, 1 GBq of 68 Ga was eluted as tetrachlorogallate using 5.5 M HCl. [68Ga]GaCl4− was trapped on an anion-exchange resin cartridge (AG1X8, Biorad, Richmond, CA, USA). 68Ga was subsequently eluted from the cartridge in a final volume of 300 μL of 0.1 M HCl (Merck, Darmstadt, Germany) as [68Ga]GaCl3. A solution of each radiolabeling

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CONCLUSION In the present study we synthesized four HBED-CC conjugates of GRPr ligands. The HBED-CC chelator was combined with bombesin analogues for the synthesis of two agonists [68Ga]1 and [68Ga]2 (monomers) and two antagonists [68Ga]3 (monomer) and [68Ga]4 (homodimer). Overall [68Ga]3 and its dimer [68Ga]4 showed the best in vitro and in vivo results. The two antagonists presented low IC50 values (nanomolar range) and high and fast binding in PC-3 and T47D cells, indicating that they can be considered as good candidates for imaging prostate and breast cancer tumors. In addition the high and specific PC-3 tumor uptake and the good tumor to background ratio at each time point demonstrate their potential as candidates for further PET/CT human studies in the future.

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EXPERIMENTAL SECTION

General Methods. All commercially available chemicals were of analytical grade and were used without further purification. The chemical suppliers were from Sigma-Aldrich (Taufkirchen, Germany) and Merck (Darmstadt, Germany), unless otherwise indicated. Protected amino acids (a.a.) and resins were supplied from Novabiochem (Merck, Darmstadt, Germany) and IRIS Biotech 2071



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precursor (0.3−1.0 nmol) in 0.1 M HEPES buffer, (pH = 7.5, 100 μL) was added to a mixture of 10 μL of HEPES solution (2.1 M) and 40 μL of [68Ga]Ga3+ eluate (80−100 MBq). The pH of the labeling solution was adjusted to 4.2 using 30% NaOH. The reaction mixture was incubated at 98 °C for 10 min. Labeling efficiency was determined via analytical RP-HPLC, solvent gradient A−B: 0−100% B in 6 min, flow of 4 mL/min, (A) 0.1% TFA in H2O and (B) 0.1% TFA in CH3CN. Radiolabeled ligands were obtained with radiochemical purity (as determined by radio-HPLC) above 98% and used without further purification for in vitro and in vivo experiments. natGa complexes were formed after the reaction of 10× molar excess of Ga(III) nitrate (Sigma-Aldrich, Germany) in 0.1 N HCl (10 μL) with the compounds under study (1 mM in 0.1 M HEPES buffer, pH 7.5, 40 μL) in a mixture of 10 μL of 2.1 M HEPES solution and 2 μL of 1 N HCl for 2 min at 80 °C.39 Cell Culture. Binding studies and in vivo experiments were performed with the following GRPr positive cell lines, PC-3 cells (bone metastasis of a grade IV prostatic adenocarcinoma, ATCC CRL1435) and T47D (human breast cancer, ATCC HTB-133). Cells were cultured in DMEM medium supplemented with 10% fetal calf serum and 2 mM L-glutamine (Invitrogen, Carlsbad, CA, USA). Cells were incubated in a controlled humidified atmosphere containing 5% CO2 at 37 °C and were subcultured weekly after being detached from the flask surface by trypsin/ethylenediaminetetraacetic acid (EDTA) solution (0.25% trypsin/0.02% EDTA, Invitrogen, Carlsbad, CA, USA). For the cellular assays the cells were harvested using trypsin/ EDTA solution and washed with PBS, counted, and seeded on 24- and 96-well plates (internalization and IC50 studies) or low protein binding Eppendorf tubes (kinetic studies) containing Opti-MEM medium (Gibco, Auckland, New Zealand). Determination of Binding Affinity for GRPr in PC-3 and T47D Cells. Binding affinities (IC50) were determined by a competitive cell binding assay with the GRPr positive PC-3 and T47D cells, according to known protocols.15 On a 96-well plate (MultiScreen HTS-DV 0.65 μm) the cells (105 per well) were incubated with 0.05 nM [125I]Tyr4-BN (82.8 μCi/mL, 2200 Ci/mmol, PerkinElmer, USA) in the presence of 12 different concentrations of each nonlabeled competitor ranging from 0 to 5000 nM (100 μL/ well). After incubation at rt for 45 min with gentle agitation, the binding buffer was removed using a multiscreen vacuum manifold (Millipore, Billerica, MA). Cells were washed with 2 × 100 μL and 1 × 200 μL of ice-cold binding buffer. Cell-bound radioactivity on the filters was measured using a γ counter (Packard Cobra II, GMI, Ramsey, MN, USA). The 50% inhibitory concentrations (IC50) and values were calculated by fitting the data using a nonlinear regression algorithm (GraphPad Software, La Jolla, CA, USA). Experiments were performed in triplicate. Internalization Experiments in PC-3 Cells. Internalization experiments were performed as previously described.16 Briefly, 105 PC-3 or T47D cells were seeded in 24-well cell culture plates 24 h before the day of the experiment. The cells were incubated with the radiolabeled compounds (30 nM, in reduced serum, Opti-MEM, Gibco) for 45 min at 37 and 4 °C, respectively. To determine specific cellular uptake, cells were blocked by competition with 1000-fold excess of native BBN or H2N-PEG2-Y-[D-Phe6,Sta13,Leu14]-BN(6−14) (Y = 4-amino-1-carboxymethylpiperidine) (30 μM). After incubation the supernatant was removed and the cells were washed with ice-cold PBS. To remove surface-bound radioactivity, cells were incubated twice with 0.5 mL of glycine-HCl in PBS (50 mM, pH 2.8) for 5 min. Then cells were washed with 1.0 mL of ice-cold PBS and lysed using 0.5 mL of 0.3 N NaOH (internalized radioactivity). The surface-bound and the internalized fraction were measured in a γ counter (Packard Cobra II, GMI, Ramsey, MN, USA). Time Kinetic Binding Studies on Cancer Cell Lines. Specificity of binding over time was analyzed using a modified previously published protocol.39 Briefly, solutions of the 68Ga-labeled compounds (30 nM, 30 MBq/nmol) were added to 1.4 × 106 cells (PC-3) suspended in 0.1 mL of Opti-MEM (1% BSA) medium (Gibco, Auckland, New Zealand) and incubated at 37 °C. Samples were briefly vortexed and a 10 μL aliquot (1−1.4 × 105 cells) was taken at

predetermined time points (15, 30, 45, 60, 90 min). The aliquot was then transferred to a 400 μL microcentrifuge tube (Roth, Germany) containing 350 μL of a 75:25 mixture of silicon oil, density 1.05 (Sigma-Aldrich, Germany), and mineral oil, density 0.872 (Acros, Nidderau, Germany). Separation of cells from the medium was performed by centrifugation at 12 000 rpm for 2 min. After freezing of the tubes using liquid nitrogen, the bottom tips containing the cell pellet were cut off. The cell pellets and the supernatants were separately counted in a γ counter. Nonspecific binding was determined by competitive blocking with a 1000-fold excess of native BBN or H 2N-PEG 2 -Y-[ D-Phe 6,Sta 13,Leu 14]-BN(6−14) (Y = 4-amino-1carboxymethylpiperidine), 100 mM solution in DMSO, respectively. Cell binding (cell counts) was determined as the percentage of the total counts added to the cell suspension (mean of four replicas ± SD). Biodistribution and Imaging μPET Studies. For the experimental tumor models PC-3 cells (5 × 106) were suspended in OptiMEM medium (Gibco, Auckland, New Zealand) and subcutaneously inoculated (100 μL) into the right trunk of male 7−8 week-old BALB/ c nu/nu mice (average weight of 20 ± 2.0 g, Jackson Laboratory, Maine, USA). The tumors were allowed to grow for 3−4 weeks until approximately 1 cm3 in size (0.100−0.250 g). The 68Ga-labeled compounds were injected into a tail vein (100 μL, in saline pH 7, 1−2 MBq/mouse; 80 pmol). At predetermined time points after injection (30 and 60 min p.i.), the animals were sacrificed and the organs of interest were dissected, blotted dry, and weighed. The radioactivity was measured using a γ counter and calculated as percentage injected dose per gram (%ID/g). Results were expressed as mean ± SD (3 animals per time point). The μPET studies were conducted with 5 MBq (approximately 100 pmol) of [68Ga]3 and [68Ga]4 injected via a lateral tail vein into PC-3 tumor-bearing mice. Blocking experiments were conducted by coadministering an 1000-fold excess of nonradioactive substance, H 2N-PEG 2 -Y-[ D-Phe 6,Sta 13,Leu 14]-BN(6−14) (Y = 4-amino-1carboxymethylpiperidine) along with the tracer. The anesthetized animals (1 vol % sevofluran, AbbVie, Ludwigshafen, Germany) were placed in prone position into a small animal PET scanner (Inveon microPET scanner, Siemens, Knoxville TN, USA) to perform a 60 min dynamic microPET scan in list mode, starting 3 s before injection (emission 0−1 h, in list mode), for attenuation correction transmission scan 10 min (two rotating 57Co sources). After 2 h the animals were measured again (20 min; attenuation correction, 10 min transmission scan). Scans reconstruction software Inveon Acquisition Workplace (IAW) Software, Siemens; protocols, 28 frames 2 × 15 s; 8 × 30 s; 5 × 60 s; 5 × 120 s; 8 × 300 s, and 3 frames 0−20 min; 20−40 min; 40− 60 min. Reconstruction settings for 3D-OSEM MAP, 16 subsets, 2 iterations, output interval: 10, MAP iterations:18, output interval: 20, image x−y size 256; image z size 161, size of voxel in mm, y = 0.388; z = 0.796. VOIs around the heart, liver, kidneys, urinary bladder, muscle, and tumor were manually defined and time−activity curves were generated to calculate SUVs (0−1 h, SUV body weight−time graphs). The area under the curve (AUC) was calculated from the SUV−time curves using GraphPad Prism 6. All animal experiments have been conducted according to the German animal welfare law (TierSchG, Approval Number 35-9185.81/G187/15), which implements European Guideline 2010/63/EU, for animal experimentation. Statistical Aspects. All experiments were performed at least in triplicate. Quantitative data were expressed as the mean ± SD. If applicable, mean values were compared using Student’s t test or ordinary one-way ANOVA (P values of

Monomeric and Dimeric 68Ga-Labeled Bombesin Analogues for

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