Monomeric and Dimeric 68Ga-Labeled Bombesin Analogues for

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Monomeric and dimeric Ga-labeled bombesin analogues for positron-emission tomography imaging (PET) of tumors expressing gastrin-releasing peptide receptors (GRPrs) Christos Liolios, Benjamin Buchmuler, Ulrike Bauder-Wüst, Martin Schaefer, Karin Leotta, Uwe Haberkorn, Matthias Eder, and Klaus Kopka J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b01856 • Publication Date (Web): 12 Feb 2018 Downloaded from http://pubs.acs.org on February 13, 2018

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Journal of Medicinal Chemistry

Title page

Monomeric and dimeric

68

Ga-labeled bombesin analogues for positron-emission

tomography imaging (PET) of tumors expressing gastrin-releasing peptide receptors (GRPrs) Christos Liolios1*, Benjamin Buchmuller1, Ulrike Bauder-Wüst1, Martin Schäfer1, Karin Leotta2, Uwe Haberkorn2,3,4, Matthias Eder1,3,5, Klaus Kopka1,3. 1

2

Division of Radiopharmaceutical Chemistry, Clinical Cooperation Unit Nuclear Medicine, 3German Cancer

Consortium (DKTK), German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69120 Heidelberg, Germany 4

Department of Nuclear Medicine, University of Heidelberg, Im Neuenheimer Feld 400, 69120 Heidelberg,

Germany. 5

Division of Radiopharmaceutical Development, German Cancer Consortium (DKTK) Freiburg, Department of

Nuclear Medicine, Faculty of Medicine, Medical Center - University of Freiburg , Hugstetter Straße 55, 79106 Freiburg, Germany.

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-CBN(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-carboxymethyl-piperidine) 3 and HBED-CC-{PEG2-Y-[D-Phe6,Sta13,Leu14]-BN(6– 1 ACS Paragon Plus Environment

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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, [68Ga]2 were mainly internalized, while antagonist [68Ga]3, [68Ga]4 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.

Keywords: Bombesin, GRPr ligands, 68Ga, PET, prostate cancer, breast cancer, HBED-CC.

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Introduction

The gastrin-releasing peptide receptor (GRPr), also called bombesin receptor 2 (BB2), is a promising target for non-invasive 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 breast1,2. Especially for prostate cancer, GRPr is overexpressed in comparison to sparse expression in normal prostate tissue3. 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 score1,4. In addition GRPr expression has been found in high density in 72-96 % of ductal breast cancer specimens5–7. 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 cancer1,7–9. Bombesin (BBN), a 14-mer GRPr peptide agonist was initially found in the skin of the fire-bellied toad Bombina bombina10 and since then several peptide GRPr agonists and antagonists have been synthesized, radiolabeled and tested pre-clinically and clinically1,11. BBN agonists internalize after binding to GRPr which is considered advantageous for ligands carrying therapeutic nuclides, but they have proliferating effect on cancer cells. On the other side BBN antagonists are mainly surface bound and they don’t have a proliferation effect on cancer cells, they also have less acute adverse effects (e.g. gastrointestinal)1,12. Over the last decade the positron emitter applications, due to its availability by

68

68

Ga has attracted a lot of interest for PET imaging

Ge/68Ga radionuclide generators and lately due to the idea of

labeling using kit-based preparations of the PET tracer comparable with those in 99

68

Ga-

99m

Tc radiopharmacies using

Mo/99mTc generators12. Radiometals with a short half-life such as 68Ga (T1/2 = 68 min) are ideally combined

with chelators that have fast kinetics and can form stable complexes at ambient temperature13. Such an agent is HBED-CC, N,N′-bis[2-hydroxy-5-(carboxyethyl)benzyl]ethylenediamine-N,N′-diacetic acid, which can be conveniently labeled with

68

Ga (25 oC, 10–20 min, pH 4–4.5) and has two additional carboxylic groups



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(propionic acid moieties), that can be used for the conjugations to a pharmacophore / binding vector14. 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 interactions17. Several binding modes have been proposed as an explanation for this effect i.e. the ligand simultaneous 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 increased17. So far only a few studies on BBN-based homodimers have been reported with varying results18–22. In the present study we investigated the effects of linking the HBED-CC chelator to different BBN pharmacophores. HBED-CC was linked to two agonists, the naturally occurring peptide H2N-BN(2-14) and the modified peptide sequence H2N-PEG2-[D-Tyr6,β-Ala11,Thi13,Nle14]BN(6–14), as well as to the antagonist, H2NY-[D-Phe6,Sta13,Leu14]-BN(6–14), (Y = 4-amino-1-carboxymethyl-piperidine) resulting in ligands 1, 2 and 3, respectively. The latter two BBN pharmacophores connected to a DOTA chelator have been used in clinical studies, i.e. BZH3 and RM223–25. 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, internalisation 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-231, low expression). Internal controls for the in vitro studies included BBN, BZH3 and RM2. [68Ga]i (i = 1-4) were also tested in vivo in mice bearing PC-3 tumors for their tumor-targeting ability and biodistribution behaviour 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 (2,3,5,6-tetrafluorophenol) 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 15-20 %, 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 (1M HCl) and elution with AcCN/H2O (8:2, v/v), to afford iron-free products for labeling with 68Ga and section). The analyses results of the pure products and their

nat

Ga (see next

nat

Ga complexes with RP-HPLC and MALDI-MS

are summarized in Table 1.

Labeling with 68Ga and natGa In all cases, radiolabeling with

68

Ga 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

68

Ga-labeled

compounds (obtained by radio-HPLC analysis) are summarized in Table 1 (comparative HPLC results for pure [natGa]i can be found in supplementary material, Figure S2). In general, sequence dependent differences proved more important than size for hydrophilicity. More specifically, [68Ga]1 (Molecular weight, MW = 2091.0), 5 ACS Paragon Plus Environment


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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 post 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 125

I[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 BZH323–25 and natural BBN. The results of the IC50 values (nM) for both cell lines are summarized in Table 2 (Displacement curves of

125

I[Tyr4]-BBN for ligands 1-4 and reference compounds

natural BBN, RM2, BZH3 are available in Figure S1 supplementary data). All ligands presented high binding affinities in the nM range close to the values of the reference compound BBN (0.26 nM, T47D and 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. 6 ACS Paragon Plus Environment

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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 show increased internalized radioactivity (76 % and 72 %), while antagonists [68Ga]3, [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 x 105 cells) were investigated in GRPr positive cell lines PC-3 (prostate cancer) and T47D (breast cancer), in negative cell lines LNCaP (prostate cancer) and MDA-MB-231 (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 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 cell-bound activity after approx. 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 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 0.8-1.6 % of the given radioactivity (Figure 4a). The amount of cell binding for [68Ga]i (i = 1-4) after blocking PC-3 cells with an 1000x excess of the corresponding pharmacophore ranged between 0.5-0.7 % of the given radioactivity.



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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) from the biodistribution experiments (30 and 60 min p.i.) are summarized in Figure 5a (for the organ distribution values see also Table S1, supplementary data). 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, supplementary data). 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 3-4 % 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 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 8 ACS Paragon Plus Environment

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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. supplementary material). 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 non-target 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 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 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 the supplementary data. 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, (Figure 6-7) 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 9 ACS Paragon Plus Environment


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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 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 non-target organs (liver, 19.2) and blood (heart, 25.7) than [68Ga]4 (liver, 65.2; heart, 24.7).

Discussion

The positron emitter

68

Ga 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]GaDOTA-TOC, [68Ga]Ga-DOTA-TATE or [68Ga]Ga-PSMA-11 has been established in daily clinical PET imaging during the last decade for highly sensitive and selective noninvasive imaging of distinct tumor entities26,27. 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 cells28. 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 interest14,15. Thus, HBED-CC is well suited for the synthesis of dimeric compounds, either homo- or heterodimeric15,16. 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 non-invasive PET-imaging1,2. In previous studies of our group15,16 the HBEDCC chelator has been successfully combined with the BBN pharmacophore, [68Ga]2, for the investigation of 10 ACS Paragon Plus Environment

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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 results18,20,21,29, 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[D-Tyr6,β-Ala11,Thi13,Nle14]BN(6–14), [68Ga]2, and a BBN antagonist, H2N-Y-[D-Phe6,Sta13,Leu14]-BN(6–14), (Y = 4-amino-1-carboxymethyl-piperidine) [68Ga]3. The latter was also used for the synthesis of the homodimer, [68Ga]4. This choice was made because BBN antagonists are expected to have less side effects and non-mitogenic properties29,30. All compounds tested in vitro in GRPr expressing cell lines, PC-3 and T47D (Table 2) presented IC50 values in the low nM 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, possible due to the interference of HBED-CC with the GRPrbinding. 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 (X-PEG2-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 and X= DOTAGA, 10.0 nM and X = AlnatF-NOTA, 4.4 nM, respectively31,32. 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



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influenced by the statistical effect17. This means if monovalent binding is assumed the homodimer has a higher probability of rebinding after dissociation from the receptor17,19. 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 agonists7,36 i.e. when 111In-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 bound radio-ligand non-significant 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) in 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 significantly 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 4a and 4b). Blocking experiments of [68Ga]i (i = 1-4) showed a minimal cell uptake, providing thus further proof for their specific binding to GRPr. 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 activity after 60 min, which is in accordance with previous studies with GRPr ligands15,25. 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


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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%). The amount of maximum cellbound [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 mitogenesis20. An enhancement of potency for the BBN dimers has also been reported on melanophores transfected with plasmid encoding GRPr18. 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-NOTAdimer 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 cells21. Other studies on BBN dimers, [99mTc]Tc-HYNIC(Tricine/TPPTS)-Glu[Aca-BN(7–14)]2, have reported Ki values being five 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 monomers19. According to the authors, this effect was either due to bivalent binding or if monovalent binding was assumed to an increased probability of rebinding after dissociation. Either way, eventually resulting in higher internalization rates19. 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 was 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 didn’t show significant differences regarding tumor uptake (30 and 60 min p.i.), while for the dimer 13 ACS Paragon Plus Environment


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[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 both at 30 and 60 min p.i. The exchange of the DOTA chelator for HBED-CC didn’t affect the pharmacokinetics of the tracer as much as the dimerization of the pharmacophore on the HBED-CC 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 x) and at 60 min (1/3 x) p.i. Additionally, the HBED-CC exchange for DOTA resulted in a slightly slower elimination of [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. This was possible 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 possibly 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 6-7). 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 non-target 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]CuNOTA-monomer and [64Cu]Cu-NOTA-dimer 2 (mice bearing PC-3 tumors) reported initially a higher tumor


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uptake for the monomer (until 30 min p.i) which was then reversed in favor of the dimer21. The inclusion of charged groups in [68Ga]4 could possible improve its pharmacokinetic properties towards a faster clearance from the blood pool and non-target organs i.e. liver and GRPr expressing pancreas.

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, [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 (nM range) and high and fast binding in PC-3 and T47D cells, indicating 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 demonstrates 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 (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 multi-wavelength-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 gamma detector (Bioscan; Washington, USA). Both RP-HPLC systems were equipped with a Chromolith RP18e (4.6 × 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 µΜ 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: 4 mL/min and for purifications: 10-90 % B in 20 min flow: 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) gamma counter (Packard Cobra II, GMI, Minnesota, USA) was used for the measurement of radioactive probes.


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Chemistry All peptides were synthesized on a rink amide resin (4-methyl-benzhydrylamine 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 RM225. 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(HBEDCC)]- according to previously published methods14,37. 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 three days in DMF (0.5 mL, RT). The mono and bis-TFP ester of [Fe(HBED-CC)]¯ were isolated with semipreparative HPLC (overall yield: 30 %, tR: 2.9 min; 25 % tR: 4.8 min, respectively) and identified with mass spectrometry: C32H28F4FeN2O10(-); MW: 732,1; m/z 733.2 [M+H]+; C38H28F8FeN2O10(-); MW: 880.5; m/z 881.0 [M+H]+. The mono TFP 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 H2N-Y-[D-Phe6,Sta13,Leu14]-BN(6–14) (Y = 4-amino-1-carboxymethyl-piperidine) (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-[D-Phe6,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 immoblilisation of the corresponding chelates on a C-18-SEP-PAK and by flushing with acid treatment (1.0 M HCl) according to previously published methods15. For calculations of the exact masses ChemBioDraw ultra 13.0 was used.



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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 support38. Typically, 1 GBq of 68Ga was eluted as tetrachlorogallate using 5.5 M HCl. [68Ga]GaCl4− was trapped on an anion-exchange resin cartridge (AG1X8, Biorad, Richmond, CA, USA).

68

Ga 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 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 to 100 MBq). The pH of the labeling solution was adjusted to 4.2 using 30% NaOH. The reaction mixture was incubated at 98 ᴏC for 10 min. Labeling efficiency was determined via analytical RPHPLC, solvent gradient A-B: 0-100% B in 6 min, flow: 4 mL/min, A: of 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.

nat

Ga 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 2.1 M HEPES solution and 2 µL 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 CRL-1435) and T47D (human breast cancer, ATCC® HTB-133™). Cells were cultured in DMEM medium supplemented with 10 % fetal calf serum and 2 mM of 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 18 ACS Paragon Plus Environment

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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 protocols15. 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 non labeled competitor ranging from 0−5000 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 2x100 µL and 1x200 µL ice-cold binding buffer. Cell-bound radioactivity on the filters was measured using a gamma 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 described16. 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°C 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-carboxymethyl-piperidine) (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 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



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radioactivity). The surface-bound and the internalized fraction were measured in a gamma 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 protocol39. Briefly, solutions of the

68

Ga-labeled compounds (30 nM, 30 MBq/nmol), were added to 1.4 × 106 cells (PC-3)

suspended in 0.1 mL 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 x 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 12000 rpm for 2 min. After freezing 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 gamma counter. Non-specific binding was determined by competitive blocking with a 1000-fold excess of native BBN or H2NPEG2-Y-[D-Phe6,Sta13,Leu14]-BN(6–14) (Y = 4-amino-1-carboxymethyl-piperidine), 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 x 106) were suspended in Opti-MEM 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 20 ± 2.0 g, Jackson Laboratory, Maine, USA). The tumors were allowed to grow for 3 to 4 weeks until approximately 1 cm3 in size (0.100 - 0.250 g). The 68Ga-labeled compounds were injected into a tail vein (100µL, in saline pH 7, 1-2 MBq/mouse; 80 pmol). At predetermined time points after


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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 gamma 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 (approx. 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 co-administering an 1000-fold excess of non-radioactive substance, H2N-PEG2-Y-[D-Phe6,Sta13,Leu14]-BN(6–14) (Y = 4-amino-1carboxymethyl-piperidine) along with the tracer. The anaesthetized animals (1 vol % Sevofluran , AbbVie, Ludwigshafen, Germany) were placed in prone position into a small animal PET scanner (Inveon Micro PET Scanner, Siemens, Knoxville TN, USA) to perform a 60 min-dynamic microPET scan in list mode, starting 3 sec before injection (emission 0-1h, in list mode), for attenuation correction transmission scan 10 min (2 rotating 57

Co 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 2x15s; 8x30s; 5x60s; 5x120s; 8x300s and 3 frames 0-20 min; 20-40 min; 40-60 min. Reconstruction settings for 3D-OSEM MAP, 16 subsets, 2 iteration, 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 mean ± SD. If applicable, means were compared using Student’s t-test or ordinary one way ANOVA (P values < 0.05). For all statistical calculations GraphPad Prism 6 was used. 21 ACS Paragon Plus Environment


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Associated content Supporting information Table of contents of supporting information Figure S1 Displacement curves of 125I[Tyr4]-BN for ligands 1-4 and reference compounds BBN, RM2,

Page

BZH3

S2

Figure S2 RP-HPLC results for the [natGa]i (i = 1-4) complexes

S4 68

Table S1 Biodistribution data after the iv administration of the tracers [ Ga]i (i = 1-4)

S5 68

Table S2 Contrast ratios tumor to normal tissue 30 and 60 min p.i. after the administration of [ Ga]i (i =

S6

1-4) and reference compound [68Ga]RM2

Author information *Corresponding Author: Christos Liolios, phone (DKFZ): +49 6221 42 2431, e-mail: [email protected] Current address: Division of Radiopharmaceutical Chemistry, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280 , 69120 Heidelberg, Germany Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes The authors declare no competing financial interest

Abbreviations: ACMP: 4-amino-1-carboxymethyl-piperidine, AUC: area under the curve, BBN: bombesin, BSA: bovine serum albumin, DIPC: N,N′-Diisopropylcarbodiimide, DIPEA: N,N-Diisopropylethylamine, HBED-CC: N,N'bis[2-hydroxy 5-(ethylen β carboxy) benzyl] ethylenediamine N,N' diacetic acid, GRPR:


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gastrin releasing peptide receptor, p.i.: post-injection, MW: Molecular weight, SUV: standardized uptake value, VOI: volume of interest.

References

(1) Sancho, V.; Di Florio, A.; Moody, T. W.; Jensen, R. T. Bombesin Receptor-Mediated Imaging and Cytotoxicity: Review and Current Status. Curr. Drug Deliv. 2011, 8 (1), 79–134. (2) Parry, J. J.; Andrews, R.; Rogers, B. E. MicroPET Imaging of Breast Cancer Using Radiolabeled Bombesin Analogs Targeting the Gastrin-Releasing Peptide Receptor. Breast Cancer Res. Treat. 2007, 101 (2), 175–183. (3) Rybalov, M.; Ananias, H. J. K.; Hoving, H. D.; van der Poel, H. G.; Rosati, S.; de Jong, I. J. PSMA, EpCAM, VEGF and GRPR as Imaging Targets in Locally Recurrent Prostate Cancer after Radiotherapy. Int. J. Mol. Sci. 2014, 15 (4), 6046–6061. (4) Wieser, G.; Mansi, R.; Grosu, A. L.; Schultze-Seemann, W.; Dumont-Walter, R. A.; Meyer, P. T.; Maecke, H. R.; Reubi, J. C.; Weber, W. A. Positron Emission Tomography (PET) Imaging of Prostate Cancer with a Gastrin Releasing Peptide Receptor Antagonist - from Mice to Men. Theranostics 2014, 4 (4), 412–419. (5) Reubi, J. C.; Fleischmann, A.; Waser, B.; Rehmann, R. Concomitant Vascular GRP-Receptor and VEGF-Receptor Expression in Human Tumors: Molecular Basis for Dual Targeting of Tumoral Vasculature. Peptides 2011, 32 (7), 1457–1462. (6) Reubi, J. C.; Maecke, H. R. Peptide-Based Probes for Cancer Imaging. J. Nucl. Med. 2008, 49 (11), 1735–1738. (7) Dalm, S. U.; Martens, J. W. M.; Sieuwerts, A. M.; van Deurzen, C. H. M.; Koelewijn, S. J.; de Blois, E.; Maina, T.; Nock, B. A.; Brunel, L.; Fehrentz, J.-A.; Martinez, J.; de Jong, M.; Melis, M. In Vitro and In Vivo Application of Radiolabeled Gastrin-Releasing Peptide Receptor Ligands in Breast Cancer. J. Nucl. Med. 2015, 56 (5), 752–757. (8) Prasanphanich, A. F.; Retzloff, L.; Lane, S. R.; Nanda, P. K.; Sieckman, G. L.; Rold, T. L.; Ma, L.; Figueroa, S. D.; Sublett, S. V; Hoffman, T. J.; Smith, C. J. In Vitro and in Vivo Analysis of [64Cu-NO2A-8-Aoc-BBN(7-14)NH2]: A Site-Directed Radiopharmaceutical for Positron-Emission Tomography Imaging of T47D Human Breast Cancer Tumors. Nucl. Med. Biol. 2009, 36 (2), 171–181.



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(9) Stoykow, C.; Erbes, T.; Maecke, H. R.; Bulla, S.; Bartholomä, M.; Mayer, S.; Drendel, V.; Bronsert, P.; Werner, M.; Gitsch, G.; Weber, W. A.; Stickeler, E.; Meyer, P. T. Gastrin-Releasing Peptide Receptor Imaging in Breast Cancer Using the Receptor Antagonist 68Ga-RM2 And PET. Theranostics 2016, 6 (10), 1641–1650. (10) Anastasi, A.; Erspamer, V.; Bucci, M. Isolation and Amino Acid Sequences of Alytesin and Bombesin, Two Analogous Active Tetradecapeptides from the Skin of European Discoglossid Frogs. Arch. Biochem. Biophys. 1972, 148 (2), 443–446. (11) Cornelio, D. B.; Roesler, R.; Schwartsmann, G. Gastrin-Releasing Peptide Receptor as a Molecular Target in Experimental Anticancer Therapy. Ann. Oncol. 2007, 18 (9), 1457–1466. (12) Fani, M.; Maecke, H. R.; Okarvi, S. M. Radiolabeled Peptides: Valuable Tools for the Detection and Treatment of Cancer. Theranostics 2012, 2 (5), 481–501. (13) Price, E. W.; Orvig, C. Matching Chelators to Radiometals for Radiopharmaceuticals. Chem. Soc. Rev. 2014, 43 (1), 260–290. (14) Eder, M.; Wängler, B.; Knackmuss, S.; LeGall, F.; Little, M.; Haberkorn, U.; Mier, W.; Eisenhut, M. Tetrafluorophenolate of HBED-CC: A Versatile Conjugation Agent for 68Ga-Labeled Small Recombinant Antibodies. Eur. J. Nucl. Med. Mol. Imaging 2008, 35 (10), 1878–1886. (15) Liolios, C.; Schäfer, M.; Haberkorn, U.; Eder, M.; Kopka, K. Novel Bispecific PSMA/GRPr Targeting Radioligands with Optimized Pharmacokinetics for Improved PET Imaging of Prostate Cancer. Bioconjug. Chem. 2016, 27 (3), 737–751. (16) Eder, M.; Schäfer, M.; Bauder-Wüst, U.; Haberkorn, U.; Eisenhut, M.; Kopka, K. Preclinical Evaluation of a Bispecific Low-Molecular Heterodimer Targeting Both PSMA and GRPR for Improved PET Imaging and Therapy of Prostate Cancer. Prostate 2014, 74 (6), 659–668. (17) Handl, H. L.; Vagner, J.; Han, H.; Mash, E.; Hruby, V. J.; Gillies, R. J. Hitting Multiple Targets with Multimeric Ligands. Expert Opin. Ther. Targets 2004, 8 (6), 565–586. (18) Carrithers, M. D.; Lerner, M. R. Synthesis and Characterization of Bivalent Peptide Ligands Targeted to G-ProteinCoupled Receptors. Chem. Biol. 1996, 3 (7), 537–542. (19) Abiraj, K.; Jaccard, H.; Kretzschmar, M.; Helm, L.; Maecke, H. R. Novel DOTA-Based Prochelator for Divalent Peptide Vectorization: Synthesis of Dimeric Bombesin Analogues for Multimodality Tumor Imaging and Therapy. Chem. Commun. (Camb). 2008, 28, 3248–3250.


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(20) Gawlak, S. L.; Kiener, P. A.; Braslawsky, G. R.; Greenfield, R. S. Homodimeric Forms of Bombesin Act as Potent Antagonists of Bombesin on Swiss 3T3 Cells. Growth Factors 1991, 5 (2), 159–170. (21) Fournier, P.; Dumulon-Perreault, V.; Ait-Mohand, S.; Langlois, R.; Bénard, F.; Lecomte, R.; Guérin, B. Comparative Study of 64Cu/NOTA-[D-Tyr6,βAla11,Thi13,Nle14]BBN(6-14) Monomer and Dimers for Prostate Cancer PET Imaging. EJNMMI Res. 2012, 2 (1), 8. (22) Yu, Z.; Carlucci, G.; Ananias, H. J. K.; Dierckx, R. A. J. O.; Liu, S.; Helfrich, W.; Wang, F.; de Jong, I. J.; Elsinga, P. H. Evaluation of a Technetium-99m Labeled Bombesin Homodimer for GRPR Imaging in Prostate Cancer. Amino Acids 2013, 44 (2), 543–553. (23) Strauss, L. G.; Koczan, D.; Seiz, M.; Tuettenberg, J.; Schmieder, K.; Pan, L.; Cheng, C.; Dimitrakopoulou-Strauss, A. Correlation of the Ga-68-Bombesin Analog Ga-68-BZH3 with Receptors Expression in Gliomas as Measured by Quantitative Dynamic Positron Emission Tomography (dPET) and Gene Arrays. Mol. Imaging Biol. 2012, 14 (3), 376–383. (24) Minamimoto, R.; Hancock, S.; Schneider, B.; Chin, F. T.; Jamali, M.; Loening, A.; Vasanawala, S.; Gambhir, S. S.; Iagaru, A. Pilot Comparison of

68

Ga-RM2 PET and

68

Ga-PSMA-11 PET in Patients with Biochemically Recurrent

Prostate Cancer. J. Nucl. Med. 2016, 57 (4), 557–562. (25) Mansi, R.; Wang, X.; Forrer, F.; Waser, B.; Cescato, R.; Graham, K.; Borkowski, S.; Reubi, J. C.; Maecke, H. R. Development of a Potent DOTA-Conjugated Bombesin Antagonist for Targeting GRPr-Positive Tumours. Eur. J. Nucl. Med. Mol. Imaging 2011, 38 (1), 97–107. (26) Zaknun, J. J.; Bodei, L.; Mueller-Brand, J.; Pavel, M. E.; Baum, R. P.; Hörsch, D.; O’Dorisio, M. S.; O’Dorisiol, T. M.; Howe, J. R.; Cremonesi, M.; Kwekkeboom, D. J. The Joint IAEA, EANM, and SNMMI Practical Guidance on Peptide Receptor Radionuclide Therapy (PRRNT) in Neuroendocrine Tumours. Eur. J. Nucl. Med. Mol. Imaging 2013, 40 (5), 800–816. (27) Eder, M.; Eisenhut, M.; Babich, J.; Haberkorn, U. PSMA as a Target for Radiolabelled Small Molecules. Eur. J. Nucl. Med. Mol. Imaging 2013, 40 (6), 819–823. (28) Al-Nahhas, A.; Win, Z.; Szyszko, T.; Singh, A.; Khan, S.; Rubello, D. What Can Gallium-68 PET Add to Receptor and Molecular Imaging? Eur. J. Nucl. Med. Mol. Imaging 2007, 34 (12), 1897–1901. (29) Abiraj, K.; Mansi, R.; Tamma, M.-L.; Fani, M.; Forrer, F.; Nicolas, G.; Cescato, R.; Reubi, J. C.; Maecke, H. R. Bombesin Antagonist-Based Radioligands for Translational Nuclear Imaging of Gastrin-Releasing Peptide Receptor-



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Positive Tumors. J. Nucl. Med. 2011, 52 (12), 1970–1978. (30) Halmos, G.; Schally, A. V. Reduction in Receptors for Bombesin and Epidermal Growth Factor in Xenografts of Human Small-Cell Lung Cancer after Treatment with Bombesin Antagonist RC-3095. Proc. Natl. Acad. Sci. U. S. A. 1997, 94 (3), 956–960. (31) Varasteh, Z.; Mitran, B.; Rosenström, U.; Velikyan, I.; Rosestedt, M.; Lindeberg, G.; Sörensen, J.; Larhed, M.; Tolmachev, V.; Orlova, A. The Effect of Macrocyclic Chelators on the Targeting Properties of the

68

Ga-Labeled

Gastrin Releasing Peptide Receptor Antagonist PEG2-RM26. Nucl. Med. Biol. 2015, 42 (5), 446–454. (32) Varasteh, Z.; Aberg, O.; Velikyan, I.; Lindeberg, G.; Sörensen, J.; Larhed, M.; Antoni, G.; Sandström, M.; Tolmachev, V.; Orlova, A. In Vitro and in Vivo Evaluation of a

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F-Labeled High Affinity NOTA Conjugated

Bombesin Antagonist as a PET Ligand for GRPR-Targeted Tumor Imaging. PLoS One 2013, 8 (12), e81932. (33) Maddalena, M. E.; Fox, J.; Chen, J.; Feng, W.; Cagnolini, A.; Linder, K. E.; Tweedle, M. F.; Nunn, A. D.; Lantry, L. E.

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Models with Low GRP-R Expression. J. Nucl. Med. 2009, 50 (12), 2017–2024. (34) Retzloff, L. B.; Heinzke, L.; Figureoa, S. D.; Sublett, S. V; Ma, L.; Sieckman, G. L.; Rold, T. L.; Santos, I.; Hoffman, T. J.; Smith, C. J. Evaluation of [99mTc-(CO)3-X-Y-bombesin(7-14)NH2] Conjugates for Targeting Gastrin-Releasing Peptide Receptors Overexpressed on Breast Carcinoma. Anticancer Res. 2010, 30 (1), 19–30. (35) Giacchetti, S.; Gauvillé, C.; de Crémoux, P.; Bertin, L.; Berthon, P.; Abita, J. P.; Cuttitta, F.; Calvo, F. Characterization, in Some Human Breast Cancer Cell Lines, of Gastrin-Releasing Peptide-like Receptors Which Are Absent in Normal Breast Epithelial Cells. Int. J. cancer 1990, 46 (2), 293–298. (36) Liolios, C. C.; Fragogeorgi, E. A.; Zikos, C.; Loudos, G.; Xanthopoulos, S.; Bouziotis, P.; Paravatou-Petsotas, M.; Livaniou, E.; Varvarigou, A. D.; Sivolapenko, G. B. Structural Modifications of

99m

Tc-Labelled Bombesin-like

Peptides for Optimizing Pharmacokinetics in Prostate Tumor Targeting. Int. J. Pharm. 2012, 430 (1–2). (37) Zoller, M.; Schuhmacher, J.; Reed, J.; Maier-Borst, W.; Matzku, S.; Zöller, M.; Schuhmacher, J.; Reed, J.; MaierBorst, W.; Matzku, S. Establishment and Characterization of Monoclonal Antibodies against an Octahedral Gallium Chelate Suitable for Immunoscintigraphy with PET. J. Nucl. Med. 1992, 33 (7), 1366–1372. (38) Schuhmacher, J.; Maier-Borst, W. A New 68Ge/68Ga Radioisotope Generator System for Production of 68Ga in Dilute HCl. Int. J. Appl. Radiat. Isot. 1981, 32 (1), 31–36. (39) Eder, M.; Schäfer, M.; Bauder-Wüst, U.; Hull, W.-E.; Wängler, C.; Mier, W.; Haberkorn, U.; Eisenhut, M.

68

Ga-


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Complex Lipophilicity and the Targeting Property of a Urea-Based PSMA Inhibitor for PET Imaging. Bioconjug. Chem. 2012, 23 (4), 688–697.



Page 28 of 40

Schemes

Scheme 1 Synthesis of GRPr agonists and antagonists coupled with the chelator HBED-CC. (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,β-Ala11,Thi13,Nle14]BN(6–14); (iii) H2N-Y-[DPhe6,Sta13,Leu14]-BN(6–14);

(iv)

H2N-PEG2-Y-[D-Phe6,Sta13,Leu14]-BN(6–14)

and

Y

=

4-amino-1-

carboxymethyl-piperidine, (e) [68Ga]GaCl3, 40 µL (80 to 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 ᴏC, 10 min. 28 ACS Paragon Plus Environment

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Figures

Figure 1 The gamma-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 × 4.6 mm; Merck, Darmstadt, Germany), using a linear A−B gradient (0 % B to 100 % B in 5 min), flow: 4 mL/min, A: 0.1 % aqueous TFA, and B: 0.1 % TFA in CH3CN.



Page 30 of 40

** * *

(2a)


Page 31 of 40

*** **** ****

te d

ed

la

is

to ta l

ce

ll

re

rn al in te

su rf

ac e

bo un d

cpm

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(2b) Figure 2 Surface, internalized and total cell bound ligands [68Ga]1, [68Ga]2, [68Ga]3 and [68Ga]4 expressed as cpm at 37 o

C (45 min incubation time) in PC-3 (2a) and T47D (2b) cells. The values on the top of bars represent the

percentage of the total cell related activity that was surface bound or internalised. Statistical results are expressed as stars on the top of the bars (one way non parametric ANOVA, P < 0.05)



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 x 105 PC-3 or T47D cells/well seeded 24 h before the experiment). As reference [68Ga]RM2 was included in the assay. Stars on the top of the bars represent the statistical difference against [68Ga]RM2 (ANOVA, one way non parametric, P < 0.05).

25

% cell bound radioactivity

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 32 of 40

[68Ga]4 (PC-3)

20

[68Ga]4 (PC-3) blocked [68Ga]3 (PC-3) [68Ga]3 (PC-3) blocked

15

[68Ga]3 (T47D) [68Ga]3 (MDA-MB-231) [68Ga]3 (LNCaP)

10

5

0 0

20

40 60 Time (min)

80

100 (4a) 32

ACS Paragon Plus Environment

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(4b) 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; (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 with dashed lines. Blocking experiments conducted with an addition of 1000-fold excess of the corresponding bombesin pharmacophore.



B ra Tu in m or (P C -3 )

M us cl e In te st in es Pa nc re as

r K id ne ys

Li ve

Sp le en

Lu ng s

rt H ea

B lo od

** *

or (

PC -3 )

Br ai n m Tu

M us cl e In te st in es Pa nc re as

ey s Ki dn

Li ve r

Sp le en

gs Lu n

ea rt H

lo od

**

B

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 34 of 40

(5a)


Page 35 of 40 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(5b) Figure 5 (5a) Comparative biodistribution studies at 30 min (top) and 60 min (bottom) p.i. of [68Ga]1, [68Ga]2, [68Ga]3,



Page 36 of 40

[68Ga]4 and control [68Ga]RM2 in Balb nu/nu male mice bearing PC-3 tumors and A solution in saline (pH 7) of each compound was injected into the tail vein of the animals (100 µL, 1-2 MBq/mouse; 0.1-0.2 nmol). The results are expressed as percentage of the injected dose per g (% ID/g ±SD) for each organ or tissue (mean ± SD, n = 3-4). (5b) Tumor to normal tissue ratios (Mean value ± SD) for the two antagonists under study [68Ga]3, [68Ga]4 and the reference compound [68Ga]RM2 at 30 min (top) and 60 min p.i.(bottom).


Page 37 of 40 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(6a)

(6b) 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 i.v. administration of (6a) [68Ga]3 (100 pmol, 35-45 MBq) and (6b) [68Ga]4 (100 pmol, 35-45 MBq). Where T = Tumor; K= kidneys, B = bladder, as indicated with arrows.



Page 38 of 40

Figure 7 Time−activity curves expressed as SUVs around the tumor ( ); muscle (); heart (); liver (); for [68Ga]3 (100 pmol, 35-45 MBq) (a) and [68Ga]4 (100 pmol, 35-45 MBq), (b) 0-60 min p.i. Area Under the Curve (AUC0-57 min) for [68Ga]3 (left) and [68Ga]4 (right) for selected organs.


Page 39 of 40

Table 1 Calculated mass results with MALDI-MS and retention times after radio RP-HPLC analysis of the ligands (L)

Chelator

and their [natGa]L and [68Ga]L complexes respectively

1

HBED-CC

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


2 3

Calculated mass linker

RT (min)

L

[natGa]L*

L

[natGa]L§

[68Ga]L

-

H2N-BN(2–14)

2023.3

2091.0

2024.8

2091.0

2.15

-PEG2

[D-Tyr6,β-Ala11,Thi13,Nle14]-BN(6–14)

1799.0

1866.7

1800.8

1867.5

2.42

1768.1

1834.8

1769.5

1836.3

2.25

3293.9

3359.6*

3294.2

3359.7

2.39

-Y [D-Phe6,Sta13,Leu14]-BN(6–14) -PEG2-Y

4

[M + H]+or [M]+

pharmacophore

*Theoretical calculations for [ natGaH2L] complex, where L equals ligand, § experimental values, Y = 4-amino-1-carboxymethylpiperidine

Table 2 The IC50 values of the compounds 1-4 determined against

125

I-BBN (30 nM) in T47D and PC-3 cells. As

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

T47D (nM)

Log IC50 (St.Er.)

PC-3 (nM)

Log IC50 (St.Er.)

1

0.56

-0.25 (0.04)

2.12

0.33 (0.03)

2

3.51

0.54 (0.05)

4.68

0.67 (0.07)

3

1.07

0.03 (0.07)

2.12

0.33 (0.12)

4

3.03

0.51 (0.06)

2.45

0.39 (0.06)

BBN

0.26

-0.59 (0.02)

0.65

-0.19 (0.03)

RM2

0.63

-0.20 (0.04)

1.33

0.12 (0.05)

BZH3

1.85

0.28 (0.04)

2.08

0.32 (0.03)



Page 40 of 40

Table of Contents graphic


Monomeric and Dimeric 68Ga-Labeled Bombesin Analogues for

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