Probing the Backbone Function of Tumor Targeting Peptides by an

Article pubs.acs.org/jmc

Probing the Backbone Function of Tumor Targeting Peptides by an Amide-to-Triazole Substitution Strategy Ibai E. Valverde, Sandra Vomstein, Christiane A. Fischer, Alba Mascarin, and Thomas L. Mindt* Division of Radiopharmaceutical Chemistry, University of Basel Hospital, Petersgraben 4, 4031 Basel, Switzerland

Downloaded by UNIV OF SHEFFIELD on September 10, 2015 | http://pubs.acs.org Publication Date (Web): September 2, 2015 | doi: 10.1021/acs.jmedchem.5b00994

S Supporting Information *

ABSTRACT: Novel backbone-modified radiolabeled analogs based on the tumor targeting peptide bombesin were synthesized and fully evaluated in vitro and in vivo. We have recently introduced the use of 1,4-disubstituted 1,2,3-triazoles as metabolically stable trans-amide bond surrogates in radiolabeled peptides in order to improve their tumor targeting. As an extension of our approach, we now report several backbone-modified analogs of the studied bombesin peptide bearing multiple triazole substitutions. We investigated the effect of the modifications on several biological parameters including the internalization of the radiopeptidomimetics into tumor cells, their affinity toward the gastrin releasing peptide receptor (GRPr), metabolic stability in blood plasma, and biodistribution in mice bearing GRPr-expressing xenografts. The backbone-modified radiotracers exhibited a significantly increased resistance to proteolytic degradation. In addition, some of the radiopeptidomimetics retained a nanomolar affinity toward GRPr, resulting in an up to 2-fold increased tumor uptake in vivo in comparison to a (all amide bond) reference compound.

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INTRODUCTION The gastrin releasing peptide receptor (GRPr) has attracted considerable attention as a molecular target in oncology over the past years because of its overexpression in high incidence by a variety of clinically relevant tumors such as prostate, breast, gastrointestinal stromal, and small cell lung cancer.1,2 Bombesin (BBN) is an amphibious analog of the gastrin releasing peptide (GRP), the natural agonistic ligand of GRPr. BBN and derivatives thereof exhibit a nanomolar affinity and high specificity toward the GRPr, features that make them ideal vector molecules for the delivery of diagnostic probes or therapeutic agents to tumors and metastases.3,4 For example, several radiolabeled BBN conjugates are currently under investigation for diagnostic (imaging) and therapeutic applications in nuclear medicine for the management of cancer.5−10 However, the low metabolic stability of BBNbased radiotracers impacts their tumor targeting capabilities and thus represents a limitation for medical applications. Consequently, substantial research efforts have been made to optimize and stabilize the peptide vector with the aim of increasing the uptake of the radiotracer in tumors.3 In a first step, the original 14 amino acid sequence was shortened to the last eight residues, resulting in the minimum binding sequence BBN(7−14) (QWAVGHLM-NH2).11−14 Efforts to increase the metabolic stability of radiolabeled BBN © XXXX American Chemical Society

derivatives have focused on modifications of amino acid side chains, including the replacement of Met14, Leu13, and Gly11 by other amino acids.14−16 In the course of the development of bombesin-based radiotracers, surprisingly little attention has been paid to backbone modification as an alternative strategy for peptide stabilization even though the potential of this approach has been reported for other peptide-based radiotracers.17 With respect to BBN(7−14), Jensen et al. investigated the biological activity of several carbonyl-reduced nonradioactive analogs.18 In addition, covalent cyclization, Nmethylation of amide bonds, and an alanine scan of the peptide sequence have been reported.19,20 These investigations provided insights with regard to which amino acid residues and amide bonds are crucial in the BBN(7−14) sequence for its biological function; however, the metabolic stability of such peptidomimetics and their application as radiotracers have not yet been investigated. We are interested in the development of novel radiolabeled, BBN-based radiotracers that display improved tumor targeting properties. Specifically, we are investigating synthetic approaches for the development of radiopeptidomimetics of BBN with a high affinity toward the GRPr and an improved Received: June 25, 2015

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DOI: 10.1021/acs.jmedchem.5b00994 J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

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Figure 1. Structure of reference compound 1 with numbering of amino acid residues.

1,4-disubstituted 1,2,3-triazole was inserted in the amino acid sequence of the peptide by the CuAAC using the corresponding α-aminoalkyne, [(CH3CN)4Cu][PF6] as a copper(I) source, and diisopropylethylamine as a base. The remaining amino acids to complete the peptide were coupled following standard Fmoc/tBu protocols as was the N-terminal conjugation of the universal macrocyclic chelator 1,4,7,10tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) via a hydrophilic tetraethylene glycol spacer (PEG4). All nonradioactive peptide conjugates were obtained in satisfying yields and characterized by RP-HPLC and high resolution mass spectrometry (Table 1). Radiolabeling. DOTA-conjugated peptides were radiolabeled at specific activities ranging from 2.9 to 17.5 GBq/μmol depending on the planned experiments (see Experimental Section). Radiometalations were achieved by incubation of the DOTA-functionalized peptides with [177Lu]LuCl 3 in a NH4OAc buffer (400 mM, pH 5.0) at 95 °C for 30 min. The respective radiometal complexes were obtained in radiochemical yields, and purities exceeded 95% as determined by γ-HPLC. Cell Binding and Internalization Studies. The kinetics of internalization, expressed as percent of added activity per 1 million cells, was evaluated for all compounds with PC3 cells overexpressing the human GRPr (n ≥ 2 in triplicates; Table 2). Receptor specificity of the cell internalization of the radiopeptides was confirmed for all compounds by blocking experiments. Addition of a 1000-fold excess of unlabeled bombesin (1−14) decreased the internalization rates of the radiolabeled peptidomimetics to less than 0.5% in all cases. Internalization rates after 4 h of incubation with PC3 cells are given in Table 2. Compounds [177Lu]1−6 internalized into the cells up to 20−25% after 4 h of incubation with the exception of [177Lu]4 (approximately 7.6% ± 0.4 uptake after 4 h).21 Compounds [177Lu]7−9 showed very low cell internalization (0.1−3.5% uptake after 4 h of incubation) under the same conditions, and thus, they were not further evaluated. Low accumulation of [177Lu]4 in receptor positive organs observed in vivo (see below) prompted us to evaluate the compound with AR42J cells, which express the rat GRPr. In identical experimental conditions as described above for PC3 cells, reference compound [177Lu]1 internalized up to 10.1% ± 0.5 whereas [177Lu]4 showed only 0.4% ± 0.1 of specific internalization (see Discussion and Supporting Information). Receptor Binding Affinity. Binding affinities of the radiolabeled peptidomimetics toward the GRPr were determined by receptor saturation binding assays (Table 2). The dissociation constants (KD) were determined with PC3 cells using increasing concentrations of 177Lu-labeled conjugates. Dissociation constants of compounds [177Lu]2−5 with a single

metabolic stability. To achieve this goal, we are employing a novel approach that uses 1,4-disubstituted 1,2,3-triazoles as metabolically stable trans-amide bond bioisosters.21−25 The introduction of 1,2,3-triazoles into the backbone of linear, high affinity peptides is conveniently achievable at any position of the amino acid sequence by a solid phase synthesis approach that combines standard Fmoc chemistry, diazo transfer reaction, and the Cu(I)-catalyzed azide−alkyne cycloaddition (CuAAC; click chemistry).26 We have previously reported that the systematic replacement of single amide bonds in the backbone of BBN(7−14) by a 1,2,3-triazole, a strategy we termed a “triazole scan”, provided several peptidomimetics with improved stability and retained biological function.21 Preliminary evaluation of a 177Lu-labeled, triazole-containing peptide based on a BBN(7−14) analog in mice bearing GRProverexpressing tumor xenografts demonstrated the superior tumor targeting properties of the peptidomimetic in comparison to an analogous BBN(7−14) (all amide bond) reference compound. After the identification of positions in [Nle14]BBN(7−14) that allow for an amide-to-triazole switch, we herein report an extension of our methodology that employs multiple 1,2,3-triazoles in the backbone of the BBN analog. In addition we report full details of the in vitro and in vivo evaluation of 177Lu-labeled, mono- and multiple-triazole containing BBN(7−14) analogs in a side-by-side comparison with a reference compound based on the [Nle14]BBN(7−14) peptide moiety (Figure 1).

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RESULTS Synthesis. Conjugates were synthesized using standard Fmoc/tBu solid phase peptide synthesis (SPPS) and solid phase copper-catalyzed azide−alkyne cycloaddition (CuAAC) according to methods previously described.21 Alkyne derivatives of α-amino acids were obtained by a Seyferth−Gilbert homologation of the corresponding Weinreb amides using the Bestmann−Ohira reagent.27 The enantiomeric purity of alkyne derivatives of amino acids was verified in each case by formation of dipeptides and analysis of their diastereoisomeric purity by HPLC and NMR.21 The strategy of solid phase diazo transfer followed by CuAAC is analogous to the procedures described by Angelo and Arora for the synthesis of triazolecontaining oligomers.28 The synthesis of peptidomimetics bearing multiple contiguous triazoles is illustrated as an example by the synthesis of compound 6 shown in Scheme 1. Briefly, the peptidomimetic sequence was elongated by standard Fmoc-SPPS up to the amide bond to be substituted with a 1,2,3-triazole. After removal of the N-terminal Fmoc protective group, the free α-amino function of the peptide was converted to an azide by a diazo transfer using the reagent imidazolyl-1-sulfonyl azide.29,30 The amide bond mimicking B



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Scheme 1. Solid Supported Synthesis of Multitriazole Compound 6 as an Illustrative Example

agreement with the internalization experiments with PC3 cells described above. Stability Studies. The biological half-lives (t1/2) of the radioconjugates were determined by incubation of the radiometalated complexes in human blood plasma (Table 2). The half-lives of compounds [177Lu]2−5 ranged between 6 and 25 h and have been published previously.21 The values for novel multitriazole containing peptidomimetics ranged from 27 h ([177Lu]6) to 66 h ([177Lu]8). All triazole-containing radio-

triazole in the peptide moiety were in the single digit nanomolar range (3.0−5.9 nM) with the exception of [177Lu] 4 (48.6 ± 11.5 nM).21 Of the novel multitriazole containing peptidomimetics described herein, [177Lu]6 exhibited a promising affinity toward the human GRPr (KD = 25.6 ± 6.9 nM). Receptor saturation with compounds [177Lu]7−9 could not be achieved with the experimental setup applied because of their low affinity toward the GRPr. These results are in C



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Table 1. Summary of Purity, Yields, and MS Analysis of Peptide Conjugates 1−9 compd

structure

yield [%]

m/z found [Da]

1 2 3 4 5 6 7 8 9

DOTA-PEG4-Gln-Trp-Ala-Val-Gly-His-Leu-Nle-NH2 DOTA-PEG4-Gln-Trp-Ala-Val-Gly-His-Leu- Nleψ[Tz]-H DOTA-PEG4-Gln-Trp-Ala-Val- Glyψ[Tz]His-Leu-Nle-NH2 DOTA-PEG4-Gln-Trp-Ala- Valψ[Tz]Gly-His-Leu-Nle-NH2 DOTA-PEG4-Gln-Trp- Alaψ[Tz]Val-Gly-His-Leu-Nle-NH2 DOTA-PEG4-Gln-Trp-Alaψ[Tz]Val-Glyψ[Tz]His-Leu-Nle-NH2 DOTA-PEG4-Gln-Trp-Ala-Valψ[Tz]Glyψ[Tz]His-Leu-Nle-NH2 DOTA-PEG4-Gln-Trp-Alaψ[Tz]Valψ[Tz]Gly-His-Leu-Nle-NH2 DOTA-PEG4-Gln-Trp-Alaψ[Tz]Valψ[Tz]Glyψ[Tz]His-Leu-Nle-NH2

35 32 35 24 54 37 13 23 20

1555.894a 1579.889a 1579.890a 1579.880a 1579.867a 1603.870b 1603.870b 1603.870b 1627.882b

a

Molecular masses of peptides were measured as [M + H]+ by MALDI-MS. Synthesis and characterization of compounds 1−5 have been previously reported.21 bMolecular masses of peptides were measured as [M + H]+ by ESI-MS.

Table 3. Biodistribution (% ID/g) of Compounds [177Lu]2 and [177Lu]4 in Athymic Nude Mice 4 h after Intravenous Injectiona


Table 2. Gastrin Releasing Peptide Receptor Binding Affinities, Internalization Rates in PC3 Cells, and Plasma Stabilities of 177Lu-Labeled Conjugates 1−9a compd 177

[ Lu]1 [177Lu]2 [177Lu]3 [177Lu]4 [177Lu]5 [177Lu]6 [177Lu]7 [177Lu]8 [177Lu]9

KD (nM)

% internalization rate after 4 h

2.0 ± 0.6 3.0 ± 0.5 3.1 ± 1.0 48.6 ± 11.5 5.9 ± 1.8 25.6 ± 6.9 >1000 >1000 >1000

24.9 ± 1.1 26.4 ± 0.9 26.0 ± 1.1 7.6 ± 0.4 22.4 ± 2.3 21.7 ± 0.2 3.5 ± 0.6 0.1 ± 0.1 0.3 ± 0.1

half-life (h) organ

5 6 17 25 16 27 40 66 61

blood muscle liver spleen lung kidneys stomach intestine colon pancreas PC3 tumor

a

Characterization data of compounds [177Lu]1−5 reproduced here for comparison.21

[177Lu]2 0.01 0.01 0.08 0.60 0.02 1.47 0.42 0.55 2.28 6.65 1.92

± ± ± ± ± ± ± ± ± ± ±

0.00 0.02 0.03 0.25 0.03 0.18 0.21 0.21 1.48 2.49 0.73

[177Lu]2 blocking 0.01 0.04 0.15 0.05 0.06 1.59 0.05 0.06 0.07 0.10 0.17

± ± ± ± ± ± ± ± ± ± ±

0.00 0.03 0.03 0.00 0.02 0.25 0.02 0.02 0.03 0.03 0.01

[177Lu]4 0.01 0.01 0.08 0.04 0.02 1.87 0.04 0.05 0.16 0.23 2.41

± ± ± ± ± ± ± ± ± ± ±

0.00 0.01 0.01 0.01 0.01 0.30 0.01 0.02 0.05 0.03 0.35

[177Lu]4 blocking 0.01 0.02 0.12 0.07 0.05 2.11 0.07 0.11 0.06 0.08 0.27

± ± ± ± ± ± ± ± ± ± ±

0.01 0.01 0.02 0.01 0.01 0.44 0.03 0.10 0.01 0.01 0.34

a

Blocking was performed by co-injection of 2000-fold excess bombesin. Data are expressed as a percentage of the injected dose per gram of tissue and are mean ± SD (n = 4 for nonblocked experiments, n = 3 for blocking experiments).

peptidomimetics showed an increased half-life in plasma in comparison to reference [177Lu]1. Biodistribution Studies. Results of the biodistribution studies of investigated radiopeptides and -mimetics in athymic Fox-n1 nude mice bearing human GRPr-expressing PC-3 cell xenografts are summarized in Figure 2, Table 3, and Table 4 (for details on compounds [177Lu]1 and [177Lu]3 see ref 21). Uptake of radioactivity in tissue and organs is expressed as % of injected dose per gram (% ID/g). Experiments were performed in groups of four mice per time point (three mice for blocking experiments). The compounds showed pharmacokinetic characteristics common to radiolabeled peptides, i.e., fast blood clearance and renal excretion (Figure 3). Also, no uptake

of radioactivity was observed in receptor negative organs (e.g., heart, lungs, or liver) at 4 h postinjection (p.i.) of the radiolabeled peptides. Blocking experiments demonstrated the specificity of the uptake in GRPr-expressing tissues. In all cases, coadministration of the conjugates with a 2000-fold excess of BBN led to a significant reduction of the uptake in GRPr expressing tissues such as the intestines, stomach, colon, adrenals, pancreas, and the tumor xenografts (see Supporting

Figure 2. Comparison of tracer uptakes in selected organs of PC3 cell-xenografted athymic nude mice at 4 h p.i. (n = 4). (r) indicates receptorpositive tissue. Biodistribution data of compounds [177Lu]1 and -3 are reproduced here for comparison.21 D



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Table 4. Biodistribution (% ID/g) of Compounds [177Lu]5 and [177Lu]6 in Athymic Nude Mice 4 h after Intravenous Injectiona organ blood muscle liver spleen lung kidneys stomach intestine colon pancreas PC3 tumor

[177Lu]5 0.01 0.03 0.07 0.36 0.04 1.63 1.04 0.64 4.72 7.01 2.05

± ± ± ± ± ± ± ± ± ± ±

0.00 0.04 0.01 0.22 0.01 0.23 1.06 0.17 1.02 1.02 0.35

[177Lu]5 blocking 0.00 0.01 0.08 0.05 0.03 1.47 0.04 0.05 0.09 0.12 0.17

± ± ± ± ± ± ± ± ± ± ±

0.00 0.03 0.02 0.01 0.01 0.33 0.02 0.04 0.05 0.04 0.06

[177Lu]6 0.01 0.04 0.06 0.18 0.05 1.63 0.88 0.83 1.43 6.03 2.50

± ± ± ± ± ± ± ± ± ± ±

0.00 0.02 0.02 0.08 0.02 0.34 0.27 0.15 0.57 0.26 0.63

DISCUSSION GRPr is a G-protein-coupled receptor that is overexpressed in a variety of clinically relevant cancers, e.g., prostate and breast carcinoma. The expression of the GRPr in high density and incidence on the surface of tumor cells has provided the molecular basis for the use of its ligands as vectors to target specifically tumor cells for diagnostic and therapeutic applications.31 BBN is an amphibian tetradecapeptide sharing high homology with the human GRP. As a result, a significant number of radiolabeled, BBN, and BBN(7−14) analogs have been investigated for their ability to target GRPr-expressing tumors.3,5,14,16 However, BBN and its radiolabeled analogs are quickly degraded in vivo by proteases, which results in the delivery of only a fraction of the theoretically possible amount of radioactivity to tumors and metastases and thus potentially limits their use as tumor-targeting radiopharmaceuticals.10,15,21,32 Reported attempts to stabilize the peptide moiety via modifications of amino acid residues had only moderate success.15,33 With the aim of developing novel radiolabeled BBN-based conjugates that display both a high affinity to the GRPr and an improved metabolic stability, we set out to investigate a novel method for the stabilization of the BBN(7− 14) moiety based on peptide backbone modifications. We chose 1,4-disubstituted 1,2,3-triazoles as metabolically stable heterocycles to mimic trans-amide bonds in the [Nle14]BBN(7−14) sequence, an analog of BBN(7−14) in which Met14 is replaced by Nle14 resulting in a derivative resistant to oxidative side reactions during radiolabeling reactions.14 For our studies, the peptidomimetics were functionalized N-terminally with a hydrophilic tetraethylene glycol (PEG4) spacer, which has been reported to have a positive effect on the clearance of radioactivity from radiation sensitive kidneys.9,34,35 The conjugates were coupled via the PEG4 spacer to the universal macrocyclic chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10tetraacetic acid (DOTA) because of its ability to complex stably a wide range of metallic radionuclides suitable for both imaging (e.g., 111In, 68Ga) and targeted radionuclide therapy (e.g., 90Y, 177Lu). In this study, lutetium-177 (t1/2 = 6.7 d) was used because of its concomitant γ- and β−-emission, which makes it an attractive radionuclide for theranostic applications.36 We have previously reported that the systematic replacement of single amide bonds by 1,2,3-triazoles in the peptide backbone of DOTA-PEG4-[Nle14]BBN(7−14) led to a series of novel peptidomimetics.21 To our delight, almost half of the peptidomimetics obtained by the triazole scan had a preserved high affinity toward the GRPr in vitro. Amide bonds between Ala9-Val10, Val10-Gly11, Gly11-His12, and the C-terminally amidated bond of Nle14 could be successfully replaced by 1,2,3-triazoles without significantly affecting the biological activity of the peptide vector. These findings demonstrate the potential of the use of 1,4-disubstituted, 1,2,3-triazoles as transamide bond surrogates, especially in comparison with other reported backbone modification strategies techniques. For example, studies on the N-methylation and carbonyl reduction of amide bonds of BBN(7−14) revealed that only the amide bond between Gly11-His12 and Val10-Gly11, respectively, could be modified without resulting in a significant loss of receptor affinity.18,20 The influence of the amide-to-triazole substitution on the proteolytic stability of these conjugates was variable (t1/2 = 6−25 h). Triazole substitutions in the peptide vector ([177Lu] 3−5) afforded radiotracers with half-lives increased by up to 5-

[177Lu]6 blocking 0.01 0.01 0.08 0.05 0.04 1.40 0.07 0.06 0.20 0.37 0.72

± ± ± ± ± ± ± ± ± ± ±

0.00 0.01 0.02 0.02 0.01 0.23 0.03 0.03 0.10 0.20 0.53

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Blocking was performed by co-injection of 2000-fold excess bombesin. Data are expressed as a percentage of the injected dose per gram of tissue and are mean ± SD (n = 4 for non-blocked experiments, n = 3 for blocking experiments).

Figure 3. Comparison of tumor uptakes of [177Lu]1−6 in PC3 cells xenografted athymic nude mice at 4 h p.i. (n = 4). Unpaired two-tailed Student’s t test: (∗∗∗) P < 0.001, (∗∗) P < 0.005, (∗) P < 0.05. Tumor uptake of compounds [177Lu]1 and -3 are reproduced here for comparison.21

Information). In general, triazole-modified peptides exhibited an increased tumor uptake in comparison to reference conjugate [177Lu]1 (1.5% ID/g; Figure 3). [177Lu]3 showed the highest (2.8% ± 0.2) and [177Lu]2 the lowest (1.9% ± 0.4) tumor uptake in the series (Figure 3). Compound [177Lu]6 with two amide bonds substituted by 1,2,3-triazoles did not accumulate in the tumors more than compounds having only a single backbone modification. Interestingly, [177Lu]4 had the second highest tumor uptake despite the slow cell internalization rate observed in vitro (∼8% of administered dose at 4 h of incubation; Table 2). Additionally, [177Lu]4 showed low accumulation in GRPr-expressing organs which is likely the result of its low affinity for the rat GRP receptor (see Discussion and Supporting Information). Finally, all compounds displayed low accumulation and retention in the kidneys (less than 2% at 4 h p.i.). E




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fold in comparison to that of reference [177Lu]1 (t1/2 = 5 h). In vivo evaluation of one selected radiolabeled compound, [177Lu] 3, illustrated that the stabilization of the peptide led to a 2-fold increase of tumor uptake in comparison to reference [177Lu]1.21 Encouraged by these first results, we set out to explore the possibility of using multiple triazole substitutions in the peptide backbone in an effort to improve further the stability and therefore potentially the tumor uptake of BBN-based radiotracers. The only region of [Nle14]BBN(7−14) that tolerated the introduction of triazoles without decreasing the affinity toward the GRPr was in the middle portion of the peptide ([177Lu]3, [177Lu]4, and [177Lu]5) and the C-terminal amide ([177Lu]2) (Table 2). On the other hand, the C-terminal modification had only little effect on the stability of [177Lu]2.21 As a consequence, the substitution of the C-terminal acetamide of the peptide with a triazole was not further investigated. The present report describes the synthesis and biological evaluation of a series of novel bombesin-based peptidomimetics that feature several amide-to-triazole replacements in the same molecule. To explore the limits of the amide-to-triazole substitution methodology, we designed compounds 6−9 which include all possible combinations of triazole modifications at positions previously identified during a “triazole scan” of [Nle14]BBN(7−14). The synthesis of the conjugates was conveniently achieved on solid phase by a combination of Fmoc-tBu SPPS, diazo transfer reaction, and CuAAC (Scheme 1). Required chiral α-aminoalkyne building blocks were synthesized as described previously by us and others.21,27,37,38 In brief, the corresponding amino acids were converted to Weinreb amides, reduced with DIBAL, and the resulting αamino aldehydes were subjected in situ to a Seyferth−Gilbert homologation using the Bestmann−Ohira reagent. Azides were obtained from the corresponding N-terminal amines of the peptides directly on solid support using imidazolyl-1-sulfonyl azide as a diazo transfer reagent (Scheme 1).30,39 In comparison to our previous work,21 the time-consuming synthesis of chiral α-azido acid building blocks in solution could thus be avoided. With the exception of the preparation of alkyne derivatives, the otherwise solid phase synthesis approach efficiently yielded peptidomimetics with up to three contiguous triazoles in good yields. This illustrates the full compatibility of the amide-totriazole substitution strategy with standard solid phase peptide chemistry.24,40 While derivatives [177Lu]7−9 displayed very low internalization rates into PC3 cells and nearly abolished affinities to GRPr, compound [177Lu]6 bearing two noncontiguous triazole substitutions exhibited internalization kinetics comparable to that of reference [177Lu]1 and a receptor affinity of 25.6 ± 7.5 nM (Table 2). These results suggest that the high conformational constraint induced by two or more contiguous triazole modifications ([177Lu]7−9) can result in a significantly decreased GRPr binding. These results are consistent with observations of Coy et al., who showed that conformationally restrained cyclic analogs of bombesin had affinities 1000 times lower than their linear counterparts.19 Incubation of the radiolabeled, multiple-triazole containing compounds in blood plasma showed that the stability of the peptidomimetics investigated increased with the number of amide-to-triazole substitutions. The half-lives of multiple triazole containing conjugates reached more than 60 h, representing an up to 12-fold increase in comparison to that of reference [177Lu]1. Unfortunately, compounds [177Lu]7−9 did not retain their biological function in terms of affinities

toward GRP-r and were thus not further evaluated. Despite the observed sensitivity of the peptide moiety to multiple backbone modifications, we identified [177Lu]6 with a double substitution in the peptide backbone that still displays nanomolar affinity toward the GRPr. This result is somewhat surprising with regard to the decreased affinity of compounds [177Lu]7−9 but consistent with literature reports that point out the importance of the amide bond between the Val10-Gly11 residues for the binding of BBN(7−14). On the basis of the results of an Ala scan and studies of carbonyl reduction and N-methylation of different BBN sequences, Horwell et al., proposed a binding conformation of Ac-BBN(7−14) that presents a sharp bend involving residues Val10 and Gly11.18,20 The presence of a bent structure between residues Val10-Gly11 is further supported by results by Cristau et al., who demonstrated that Val10 can be substituted by a constrained turn mimicking seven-membered lactam diazepin-2-one without a significant loss of affinity toward the GRPr.41 Thus, it could be possible that the presence of 1,2,3-triazoles between Ala9-Val10 and Gly11-His12 (compound [177Lu]6) might not alter the conformation of the peptide required to bind to the GRPr, whereas this appears to be the case for compounds with two to three triazoles between contiguous residues (such as [177Lu]7−9). Biodistribution studies of compounds [177Lu]2−6 and reference [177Lu]1 were performed in nude mice bearing PC3 xenografts at 4 h p.i. for a side-by-side comparison of the radiolabeled conjugates (Figure 2). In analogy to our preliminary in vivo study with compound [177Lu]3, no accumulation of radioactivity in receptor negative tissue and organs (e.g., bones, heart, lungs, or liver) was observed for compounds [177Lu]2−6 resulting in a favorable low background signal. The low levels of radioactivity in the blood and the liver indicate fast blood clearance and the absence of hepatobiliary excretion of the radiotracer and/or its metabolites. Unspecific uptake of radioactivity in the kidneys at 4 h p.i. is the result of renal excretion, a typical characteristic for radiolabeled peptides. Tumor uptake of metabolically more stable radiolabeled peptidomimetics was higher than that of reference [177Lu]1 in all cases (Figure 3). The uptake of radioactivity in the xenografted tumors was almost doubled for compounds with a biological half-life of >15 h (Figure 3). As expected, compound [177Lu]2 without an improved stability showed similar tumor uptake as reference compound [177Lu]1 (Figure 3). These results illustrate the general relationship between tumor uptake and plasma stability of a radiolabeled peptide.21,42,43 However, even though the stability of a peptidebased radiotracer can be used as general predictor for tumor uptake, this relationship did not correlate well in all examples studied. For example, compounds [177Lu]3, -4, and -6 showed similar tumor uptakes (approximately 2.5% ± 0.4, Figure 3) despite varying plasma stabilities (17−27 h), which might reflect in part differences in their receptor affinities. It is worth noticing that [177Lu]4 exhibited almost no accumulation in the receptor-positive organs expressing the rat GRPr (Figure 2) despite a high uptake in PC3 tumor xenografts expressing the human GRPr (2.41% ID/g, Table 3). Internalization and saturation binding assays performed with AR42J cells expressing the rat GRPr showed that the uptake of [177Lu]4 was negligible in comparison to [177Lu]1 (Table 5 and Supporting Information). These results demonstrate that [177Lu]4 has a low affinity toward the rat GRPr but retained high affinity toward the human GRPr. The fact that the rat, mice, and human GRPr can have substantial F



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Table 5. Internalization of Compounds [177Lu]1 and [177Lu] 4 in Human and Rat Cell Lines at 4 h after Administration (n = 3)

14) trifluoroacetate was purchased from Bachem (Bubendorf, Switzerland). Solvents were purchased from Acros Organics (Geel, Belgium), Merck (Darmstadt, Germany), and Sigma-Aldrich (Buchs, Switzerland). All other chemicals were from Sigma-Aldrich (Buchs, Switzerland). Polypropylene syringes fitted with polypropylene frits and a polypropylene plunger were obtained from MultiSyntech (Witten, Germany) and Teflon taps from Biotage (Uppsala, Sweden). [177Lu]LuCl3 was provided by ITM (Munich, Germany). HPLC analysis was performed on Bischoff HPLC systems. The injections were carried out in two ways, manual or fully automated. The HPLC system with a manual injection system was composed of a Bischoff LC-CaDi 22-14 interface, a UV−vis Lambda 1010 detector, Berthold LB509 radioflow detector, and two HPLC compact pumps 2250. The HPLC system with a fully automated injection system was composed of a Bischoff LC-CaDi 22-14 interface, a DAD100-EU detector, an autosampler AL 3110, and two HPLC compact pumps 2250. HPLC preparative purification was carried out on a Bischoff HPLC system composed of a Bischoff LC-CaDi 22-14 interface, a UV−vis Lambda 1010 detector, and two HPLC compact pumps 2250. A Phenomenex Jupiter 4 μm Proteo 90 Å, or a 4 μm, 250 mm × 4.6 mm was used for analytical separations. A Nucleodur C18 ISIS, 5 μm, 250 mm × 16 mm column (Macherey Nagel) was used for preparative purification. HPLC solvents A and B were a 0.1% solution of TFA in H2O and 0.1% solution of TFA in MeCN, respectively. All compounds used for in vitro and in vivo experiments were analyzed by γ-HPLC, confirming ≥95% purity. High resolution mass spectrometry measurements were performed by ESI-MS on a maXis 4G (Bruker). The observed m/z values correspond to the monoisotopic ions. Quantitative γ-counting was performed on a COBRA 5003 γ-system well counter from Packard Instruments (Meriden, CT, USA). Unless stated otherwise, all CuAAC were carried out under an argon atmosphere in oxygen-free solvents. Cell Culture. PC-3 and AR42J cells were cultured at 37 °C and 5% CO2 in DMEM (high glucose) and RPMI 1640, respectively. The cell mediums contained 10% (v/v) heat-inactivated fetal bovine plasma (FBS superior, OXOID, Pratteln, Switzerland), L-glutamine (200 mM), 100 IU/mL penicillin, and 100 μg/mL streptomycin. All culture reagents, except FBS, were purchased from BioConcept (Allschwil, Switzerland). Synthesis. Optically pure protected α-aminoalkynes were synthesized following previously published methods.21 Synthesis of the backbone-modified peptidomimetic conjugates was performed manually following previously published procedures.21 Briefly, Fmocamino acids were coupled on solid support following procedure A described below on a Rink amide MBHA LL resin (100−200 mesh) (0.02−0.03 mmol). Diazo transfer reactions were performed according to general procedure B in order to obtain the azido precursor of the heterocycle.30 The corresponding α-aminoalkyne was then coupled on the resin via CuAAC using procedure C. The synthesis of the peptide was finished by coupling the remaining amino acids and DOTA(tBu)3 using procedure A. The conjugate was then cleaved from the resin and deprotected by a standard 5 h treatment with a mixture of TFA/H2O/ i-Pr3SiH/PhOH (87.5:5:2.5:5). The conjugate was then precipitated with ice-cold diethyl ether, recovered by centrifugation, and washed twice with cold diethyl ether. The precipitate was purified by preparative HPLC. General Procedure A for Manual Solid Phase Peptide Synthesis. Fmoc-protected amino acids were manually coupled onto the resin in a syringe fitted with a polypropylene frit and a Teflon tap. Briefly, to the resin (0.1 or 0.03 mmol, swollen in peptide-synthesis-grade DMF) were successively added the Fmoc-protected amino acid (2 equiv, 0.2 or 0.06 mmol), HATU (2 equiv), and i-Pr2NEt (5 equiv), and the suspension was shaken at rt for 1 h. The solvent and excess reagents were then removed by filtration, and the resin was thoroughly washed by DMF and CH2Cl2. The completion of the reaction was checked by the Kaiser test and repeated if necessary. Fmoc-protected amino acids were deprotected by addition of a solution of 20% piperidine in DMF to the peptide resin. The suspension was then allowed to react for 3 min. The treatment was repeated three times. The solvent and excess

% internalization after 4 h compd

PC3 cells

AR42J cells

[177Lu]1 [177Lu]4

24.9 ± 1.1 7.6 ± 0.4

10.1 ± 0.5 0.4 ± 0.1

differences in their affinity toward the same ligand has been reported in the literature, and thus, such interspecies receptor selectivity should be considered in the process of preclinical radiotracer development.44−46


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CONCLUSION We have recently introduced the concept of using 1,4disubstituted 1,2,3-triazoles as proteolytically resistant bioisosteres of trans-amide bonds for the stabilization of radiotracers based on high affinity, linear peptides in an effort to improve their tumor targeting properties.21,24,47 In the current study, we present an extension of our amide-to-triazole substitution strategy that aims at the development of peptidomimetics bearing multiple triazole heterocycles in their backbone. As previously reported, the systematic replacement of single amide bonds within the modified binding sequence of BBN, [Nle14]BBN(7−14), by 1,2,3-triazoles (a methodology we termed a “triazole scan”) provided a series of stabilized peptidomimetics with retained biological activity at a higher success rate in comparison to other peptide backbone modification strategies (e.g., N-methylation or reduction of amide bonds).18,20 On the other hand, the introduction of multiple triazoles in the backbone of the peptide at position identified to be tolerable for such a modification proved to be less straightforward. In particular the insertion of multiple, contiguous triazoles resulted in a diminished receptor affinity of the peptide vector. However, we were able to identify bistriazolyl peptidomimetic [177Lu]6, which represents to the best of our knowledge one of the very few examples of a biologically active peptidomimetic reported having several amide bonds replaced by triazoles.24,48,49 In summary, an amide-to-triazole substitution can be achieved at any position of a given peptide of interest by convenient solid phase chemistry employing methods that are fully compatible with standard SPPS. We have demonstrated the high potential of this approach for the development of metabolically stabilized peptide-based radiopharmaceuticals which display improved in vivo properties for applications in tumor targeting. Investigations toward the combination of triazoles with other peptide stabilization strategies as well as further optimizations of our BBN-based lead compounds by modification of the amino acid sequence are currently ongoing and will be reported in due time.

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EXPERIMENTAL SECTION

Materials and Methods. Unless stated otherwise, all reagents and anhydrous solvents were purchased from Sigma-Aldrich Chemicals and used without further purification. Fmoc-amino acids, Rink amide MBHA LL resin (100−200 mesh), HATU, TBTU, and BOP were purchased from Merck Biosciences (Nottingham, U.K.). Fmoc-15amino-4,7,10,13-tetraoxapentadecanoic acid (Fmoc-PEG4-OH) was purchased from Polypeptide Group (Strasbourg, France). DOTA(tBu)3 was purchased from Chematech (Dijon,France). Bombesin (1− G




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reagents were then removed by filtration, and the resin was thoroughly washed by DMF and CH2Cl2. General Procedure B for Solid Phase Diazo Transfer. The procedure was analogous to the one described by Hansen et al.30 In a syringe fitted with a frit and a tap, the resin (0.03 mol) was swollen with DMF. The solvent was drained off, and a solution of imidazolyl-1sulfonyl hydrochloride (2 equiv, 0.06 mmol) and i-Pr2NEt (2.5 equiv, 0.075 mmol) in DMF (approximately 1−2 mL) was added to the resin (0.03 mmol). The suspension was shaken for 1 h. The resin was then successively and repetitively washed with DMF, CH2Cl2, and DMF. The completion of the reaction was checked by the Kaiser test and repeated if necessary until completion. General Procedure C for Solid Phase Copper(I) Catalyzed Cycloaddition. In a syringe fitted with a frit and a tap, the resin was swollen in degassed DMF. The solvent was drained off thoroughly, and to the resin (0.03 mmol) was added a solution of the Fmocprotected amino alkyne (2 equiv, 0.06 mmol), tetrakis(acetonitrile)copper(I) hexafluorophosphate (0.5 equiv, 0.015 mmol), tris[(1benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA; 0.5 equiv, 0.015 mmol), and i-Pr2NEt (1 equiv, 0.03 mmol) in degassed DMF. The suspension was then vigorously shaken at rt overnight. The resin was then successively and repetitively washed with DMF, a solution of 0.5% of diethyl dithiocarbamate in DMF (3 times), CH2Cl2, and DMF. The yield of the CuAAC was determined by UV titration of the fluorenylmethylpiperidine adduct after treatment of the resin with a 20% piperidine/DMF solution (3 × 3 min). DOTA-PEG4-Gln-Trp-Alaψ[Tz]Val-Glyψ[Tz]His-Leu-Nle-NH2 (6). Synthesis was performed as described above. Compound 6 was prepared using commercial DOTA-(tris-tBu), Fmoc-15-amino4,7,10,13-tetraoxapentadecanoic acid, Fmoc-Gln(Trt)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Val-OH, Fmoc-His(Trt)-OH, Fmoc-Leu-OH, Fmoc-Nle-OH, (9H-fluoren-9-yl)methyl (S)-but-3-yn-2-ylcarbamate, and (9H-fluoren-9-yl)methyl prop-2-yn-1-ylcarbamate. After synthesis of the DOTA functionalized conjugate on solid support, cleavage from the resin, and deprotection, the precipitate was purified by preparative HPLC (gradient B/A 24−33% in 15 min at 8 mL/min) to obtain peptide 6 in 37% yield. ESI-MS [M + H]+ m/z = 1603.870 (calcd for C73H115N22O19: 1603.871). DOTA-PEG4-Gln-Trp-Ala-Valψ[Tz]Glyψ[Tz]His-Leu-Nle-NH2 (7). Peptide 7 was prepared using commercial DOTA-(tris-tBu), Fmoc-15-amino-4,7,10,13-tetraoxapentadecanoic acid, Fmoc-Gln(Trt)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Ala-OH, Fmoc-His(Trt)-OH, Fmoc-Leu-OH, Fmoc-Nle-OH, (9H-fluoren-9-yl)methyl (S)-(4-methylpent-1-yn-3-yl)carbamate, and (9H-fluoren-9-yl)methyl prop-2-yn-1ylcarbamate. After synthesis of the DOTA functionalized conjugate on solid support, cleavage from the resin, and deprotection, the precipitate was purified by preparative HPLC (gradient B/A 26% to 29% in 15 min at 8 mL/min) to obtain peptide 7 in 13% yield. ESI-MS [M + H]+ m/z = 1603.870 (calcd for C73H115N22O19: 1603.871). DOTA-PEG4-Gln-Trp-Alaψ[Tz]Valψ[Tz]Gly-His-Leu-Nle-NH2 (8). Peptide 8 was prepared using commercial DOTA-(tris-tBu), Fmoc-15-amino-4,7,10,13-tetraoxapentadecanoic acid, Fmoc-Gln(Trt)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Gly-OH, Fmoc-His(Trt)-OH, Fmoc-Leu-OH, Fmoc-Nle-OH, (9H-fluoren-9-yl)methyl (S)-but-3-yn2-ylcarbamate, and (9H-fluoren-9-yl)methyl (S)-(4-methylpent-1-yn3-yl)carbamate. After synthesis of the DOTA functionalized conjugate on solid support, cleavage from the resin, and deprotection, the precipitate was purified by preparative HPLC (gradient B/A 26−29% in 15 min at 8 mL/min) to obtain peptide 8 in 23% yield. ESI-MS [M + H]+ m/z = 1603.870 (calcd for C73H115N22O19: 1603.871). DOTA-PEG4-Gln-Trp-Alaψ[Tz]Valψ[Tz]Glyψ[Tz]His-Leu-NleNH2 (9). Peptide 9 was prepared using commercial DOTA-(tris-tBu), Fmoc-15-amino-4,7,10,13-tetraoxapentadecanoic acid, Fmoc-Gln(Trt)-OH, Fmoc-Trp(Boc)-OH, Fmoc-His(Trt)-OH, Fmoc-LeuOH, Fmoc-Nle-OH, (9H-fluoren-9-yl)methyl (S)-but-3-yn-2-ylcarbamate, (9H-fluoren-9-yl)methyl (S)-(4-methylpent-1-yn-3-yl)carbamate, and (9H-fluoren-9-yl)methyl prop-2-yn-1-ylcarbamate. After synthesis of the DOTA functionalized conjugate on solid support, cleavage from the resin, and deprotection, the precipitate was purified by preparative HPLC (gradient B/A 25−33% in 15 min at 8

mL/min) to obtain peptide 9 in 20% yield. ESI-MS [M + H]+ m/z = 1627.882 (calcd for C74H115N24O18: 1627.882) Radiolabeling. DOTA-conjugated peptides were radiolabeled at different specific activities depending on the application planned. For in vitro studies, the specific activity was 2.9 GBq/μmol. For plasma stability experiments and in vivo studies the peptides were labeled at 17.5 GBq/μmol (not optimized). An amount of 10 μg (6.14−6.30 nmol in 10 μL of water) of DOTA conjugates was added to approximately 300 μL of 0.4 M NH4OAc buffer (pH 5.0) in a prelubricated Eppendorf tube. To this solution of peptide was added 37−111 MBq of a commercial solution 177LuCl3, and the mixture was heated at 95 °C for 30 min. Radiolabeling yields were determined via γ-HPLC. In the case of in vitro studies, after quality control by γ-RPHPLC, 5 nmol (approximately 80% of the amount of peptide) of a 1 mM natLuCl3 solution was added, and the mixture was heated for an additional 30 min at 95 °C.50 Cell Binding and Internalization Studies. On the day prior to the experiment, PC3 or AR42J cells (106 cells per well) were placed in six-well plates with cell culture medium (1% FBS) and incubated at 37 °C, 5% CO2 overnight. On the day of the experiment, the medium was removed, and cells were incubated with fresh medium (1.3 mL) for 1 h. An amount of 2.5 pmol of [177Lu]-labeled peptide was added to the incubation medium, and the cells were incubated at 37° (5% CO2). At 30, 60, 120, and 240 min of incubation, the medium was removed and the cells were washed with an ice-cold 0.01 M PBS buffer, pH 7.4, to determine the amount of tracer not bound to the cells. Cells were incubated with saline glycine buffer (0.05 M, pH 2.8) twice for 5 min at 4 °C to determine the cell surface bound fraction. Finally, cells were detached and lysed from the plates by incubation with 1 M NaOH aqueous solution for 10 min at 37 °C, and the radioactivity was measured in a γ counter. The percentage of added activity (% of applied dose) was calculated for each time point. Nonspecific binding and internalization were determined by incubation of the cells with the radiopeptides and a large excess of bombesin (1−14) (2.5 nmol per well). Receptor Binding Affinity. KD determination was performed as previously described.21 Briefly, PC-3 cells at confluence were placed in six-well plates ((0.8−1) × 106 cells/well). To the cells were added increasing concentrations of the 177Lu-radiolabeled peptides (1, 5, 10, 50, 100, 250, 500, 750, 1000 nM) corresponding to a final concentration of peptide per well of 0.1 to 100 nM. The different plates were then incubated for 2 h at 4 °C (final volume, 1 mL/well). After incubation, the supernatant was collected and the cells were washed twice with 1 mL of ice-cold 0.01 M PBS (pH 7.4). The cells were then incubated with 1 M NaOH aqueous solution for 10 min at 37 °C, the supernatant was collected, and the radioactivity was measured in a γ counter. Nonspecific binding was determined by incubation of the cells with the radiopeptides and a large excess of bombesin 1−14 (10 μM per well). Stability Studies. An amount of 90 pmol (1.6 MBq) of radioactive labeled peptides (specific activity, 0.47 mCi/nmol) was added to 1 mL of fresh human blood plasma and incubated (37 °C, 5% CO2). Samples of 100 μL were collected at preselected time points (1, 2, 4, 6, 24 h), and the plasma proteins precipitated by addition of 200 μL of ethanol. After centrifugation at 5200 rpm for 5 min, the supernatant was transferred into a tube and the remaining plasma proteins were precipitated by addition of another 200 μL of ethanol. The supernatant was then removed by centrifugation as described above and analyzed by γ-HPLC. The kinetics of the metabolic degradation of the peptides was fitted with the equation A(t) = A0 exp(−λt) using GraphPad Prism 5.0. The plasma stability of the radiopeptides is expressed as the halflife (t1/2, in hours). Biodistribution Experiments. All animal experiments were conducted in compliance with the Swiss animal protection laws and with the ethical principles and guidelines for scientific animal trials established by the Swiss Academy of Medical Sciences and the Swiss Academy of Natural Sciences. Studies were performed with 6−8 weeks old female athymic nude Foxn1nu mice (20−25 g), purchased from Charles River or Harlan Laboratories. The mice were xenografted with (5−10) × 106 PC-3 cells, and the grafts were allowed to grow until the H



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tumors reached an approximate diameter of 0.5 cm (approximately 2− 3 weeks). On the day of the experiment, the mice received an intravenous injection of 100 μL of 177Lu-radiolabeled peptides (10 pmol, 185 kBq, diluted in 0.9% NaCl, containing 0.1% HSA, pH 7.4) via the tail vein, n = 4/group. Nonspecific uptake was determined by co-injection of an excess of bombesin (1−14) (20 nmol), n = 3/group. The animals were sacrificed 4 h postinjection by CO2 asphyxiation. Tumor, selected organs, and aliquots of blood were collected, weighed, and the amount of radioactivity was measured in a γ-counter. Results are expressed as a percentage of the injected dose per gram of tissue (% ID/g). Statistical analysis was conducted using unpaired two-tailed Student’s t-test.

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(2) Reubi, J. C.; Wenger, S.; Schmuckli-Maurer, J.; Schaer, J.-C.; Gugger, M. Bombesin receptor subtypes in human cancers detection with the universal radioligand 125I-[D-Tyr6, β-Ala11, Phe13, Nle14] Bombesin(6−14). Clin. Cancer Res. 2002, 8, 1139−1146. (3) Sancho, V.; Di Florio, A.; Moody, T. W.; Jensen, R. T. Bombesin receptor-mediated imaging and cytotoxicity: review and current status. Curr. Drug Delivery 2011, 8, 79−134. (4) Majumdar, S.; Siahaan, T. J. Peptide-mediated targeted drug delivery. Med. Res. Rev. 2012, 32, 637−658. (5) Van de Wiele, C.; Dumont, F.; Vanden Broecke, R.; Oosterlinck, W.; Cocquyt, V.; Serreyn, R.; Peers, S.; Thornback, J.; Slegers, G.; Dierckx, R. A. Technetium-99m RP527, a GRP analogue for visualisation of GRP receptor-expressing malignancies: a feasibility study. Eur. J. Nucl. Med. Mol. Imaging 2000, 27, 1694−1699. (6) Dimitrakopoulou-Strauss, A.; Hohenberger, P.; Haberkorn, U.; Mäcke, H. R.; Eisenhut, M.; Strauss, L. G. 68Ga-Labeled bombesin studies in patients with gastrointestinal stromal tumors: Comparison with 18F-FDG. J. Nucl. Med. 2007, 48, 1245−1250. (7) Chatalic, K. L. S.; Franssen, G. M.; van Weerden, W. M.; McBride, W. J.; Laverman, P.; de Blois, E.; Hajjaj, B.; Brunel, L.; Goldenberg, D. M.; Fehrentz, J.-A.; Martinez, J.; Boerman, O. C.; de Jong, M. Preclinical comparison of Al18F- and 68Ga-labeled gastrinreleasing peptide receptor antagonists for PET imaging of prostate cancer. J. Nucl. Med. 2014, 55, 2050−2056. (8) Mather, S. J.; Nock, B. A.; Maina, T.; Gibson, V.; Ellison, D.; Murray, I.; Sobnack, R.; Colebrook, S.; Wan, S.; Halberrt, G.; Szysko, T.; Powles, T.; Avril, N. GRP Receptor imaging of prostate cancer using [99mTc]demobesin 4: a first-in-man study. Mol. Imaging Biol. 2014, 16, 888−895. (9) Zhang, H.; Schuhmacher, J.; Waser, B.; Wild, D.; Eisenhut, M.; Reubi, J.; Maecke, H. DOTA-PESIN, a DOTA-conjugated bombesin derivative designed for the imaging and targeted radionuclide treatment of bombesin receptor-positive tumours. Eur. J. Nucl. Med. Mol. Imaging 2007, 34, 1198−1208. (10) Zhang, H. W.; Chen, J. H.; Waldherr, C.; Hinni, K.; Waser, B.; Reubi, J. C.; Maecke, H. R. Synthesis and evaluation of bombesin derivatives on the basis of pan-bombesin peptides labeled with indium111, lutetium-177, and yttrium-90 for targeting bombesin receptorexpressing tumors. Cancer Res. 2004, 64, 6707−6715. (11) Broccardo, M.; Falconieri Erspamer, G.; Melchiorri, P.; Negri, L.; de Castiglione, R. Relative potency of bombesin-like peptides. Br. J. Pharmacol. 1975, 55, 221−227. (12) Lin, J.-T.; Coy, D. H.; Mantey, S. A.; Jensen, R. T. Comparison of the peptide structural requirements for high affinity interaction with bombesin receptors. Eur. J. Pharmacol. 1995, 294, 55−69. (13) La Bella, R.; García-Garayoa, E.; Bähler; Bläuenstein, P.; Schibli, R.; Conrath, P.; Tourwé, D.; Schubiger, P. A. A 99mTc(I)-Postlabeled high affinity bombesin analogue as a potential tumor imaging agent. Bioconjugate Chem. 2002, 13, 599−604. (14) Nock, B. A.; Nikolopoulou, A.; Galanis, A.; Cordopatis, P.; Waser, B.; Reubi, J.-C.; Maina, T. Potent bombesin-like peptides for GRP-receptor targeting of tumors with 99mTc: A Preclinical Study. J. Med. Chem. 2005, 48, 100−110. (15) García Garayoa, E.; Rüegg, D.; Bläuenstein, P.; Zwimpfer, M.; Khan, I. U.; Maes, V.; Blanc, A.; Beck-Sickinger, A. G.; Tourwé, D. A.; Schubiger, P. A. Chemical and biological characterization of new Re(CO)3/[99mTc](CO)3 bombesin analogues. Nucl. Med. Biol. 2007, 34, 17−28. (16) de Visser, M.; Bernard, H. F.; Erion, J. L.; Schmidt, M. A.; Srinivasan, A.; Waser, B.; Reubi, J. C.; Krenning, E. P.; de Jong, M. Novel 111In-labelled bombesin analogues for molecular imaging of prostate tumours. Eur. J. Nucl. Med. Mol. Imaging 2007, 34, 1228− 1238. (17) Alshoukr, F.; Prignon, A. l.; Brans, L.; Jallane, A.; Mendes, S.; Talbot, J.-N. l.; Tourwé, D.; Barbet, J.; Gruaz-Guyon, A. Novel DOTAneurotensin analogues for 111In scintigraphy and 68Ga PET imaging of neurotensin receptor-positive tumors. Bioconjugate Chem. 2011, 22, 1374−1385.

ASSOCIATED CONTENT


S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.5b00994. Full characterization of peptides 6−9 and [177Lu]6−9 (UV and γ-HPLC, mass spectrometric data) and detailed results of in vitro (cell internalization and receptor saturation experiments, stability studies in human blood plasma) and in vivo experiments (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Phone: +41 (0)78 707 3616. E-mail: [email protected]. 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.

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ACKNOWLEDGMENTS This work was supported by the Swiss National Science Foundation (Grants 205321_132280 and 200020_146385), the Nora van Meeuwen-Haeflinger Foundation (Switzerland), and the Cancer League Basel (Switzerland). The authors thank Prof. Dr. H.-J. Wester (Technical University Munich, Munich, Germany) for scientific discussions, Karolin Roemhild (University of Basel Hospital, Basel, Switzerland) for technical support, and ITM (Munich, Germany) for the supply of noncarrier-added Lu-177.

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ABBREVIATIONS USED BBN, bombesin (pGlu-Gln-Arg-Leu-Gly-Asn-Gln-Trp-Ala-ValGly-His-Leu-Met-NH2); DMEM, Dulbecco modified Eagle medium; DMF, N,N-dimethylformamide; DOTA, 1,4,7,11tetraazacyclododecane-1,4,7,11-tetraacetic acid; FBS, fetal bovine serum; GRP, gastrin releasing peptide; GRPr, gastrinreleasing peptide receptor; HSA, human serum albumin; PBS, phosphate buffered saline; PEG4, 1-amino-3,6,9,12-tetraoxapentadecan-15-oic acid; RP-HPLC, reverse-phase high performance liquid chromatography; TFA, trifluoroacetic acid; ψ[Tz], designates the use of a 1,4-disubstituted, 1,2.3-triazole pseudopeptide bond between two amino acid residues

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REFERENCES

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