Toward the Optimization of Bombesin-Based Radiotracers for Tumor

Subscriber access provided by UNIV OF GEORGIA

Article

Towards the Optimization of BombesinBased Radiotracers for Tumor Targeting Ibai E. Valverde, Sandra Vomstein, and Thomas L. Mindt J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b00025 • Publication Date (Web): 07 Apr 2016 Downloaded from http://pubs.acs.org on April 8, 2016

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Journal of Medicinal Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 39

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

Journal of Medicinal Chemistry

Towards the Optimization of Bombesin-Based Radiotracers for Tumor Targeting Ibai E. Valverde*1, Sandra Vomstein1, Thomas L. Mindt*12† 1

Division of Radiopharmaceutical Chemistry, University of Basel Hospital, Petersgraben 4, 4031

Basel, Switzerland.2 Ludwig Boltzmann Institute for Applied Diagnostics, General Hospital of Vienna, Währinger Gürtel 18-20, A-1090 Vienna, Austria. † Current address: Institute of Pharmaceutical Sciences, ETH Zurich, Vladimir-Prelog-Weg 4, 8093 Zurich, Switzerland.

ABSTRACT

The peptide bombesin (BBN) is a peptide with high affinity for the Gastrin-Releasing Peptide Receptor (GRPr), a receptor that is overexpressed by, e.g., breast and prostate cancers. Thus, GRPr agonists can be used as cancer-targeting vectors to shuttle diagnostic and therapeutic agents into tumor cells. With the aim of optimizing the tumor targeting properties of a radiolabeled [Nle14]BBN(7–14) moiety, novel BBN(7–14)- and BBN(6–14)-based radioconjugates were synthesized, labeled with Lu-177, and fully evaluated in vitro and in vivo. The effect of residue and backbone modification on several parameters such as the internalization of the radiolabeled peptides into PC3 and AR42J tumor cells, their affinity towards the human GRPr, metabolic stability in blood plasma, and biodistribution in mice bearing GRPr-expressing PC3 xenografts was studied. As a result of our investigations, a novel radiolabeled GRPr agonist with a high tumor uptake and a high tumor-to-kidney ratio was identified. 1

ACS Paragon Plus Environment


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 2 of 39

TEXT Introduction The amphibian peptide bombesin (BBN) and its mammalian counterpart, the gastrin-releasing peptide, are receiving increasing attention due to their ability to bind to the gastrin-releasing peptide receptor (GRPr) with high specificity and affinity.1 The GRPr is a G-protein coupled receptor that is overexpressed in high incidence by several types of tumors such as prostate, breast, gastrointestinal stromal, and small cell lung cancer.2-4 Therefore, the employment of bombesin derivatives as tumor-targeting vectors is of particular interest for the specific delivery of, e.g., nanoparticles,5 quantum dots,6 cytotoxic moieties,7,

8

and photosensitizers to GRPr-

expressing tumors.9 One of the currently most promising applications of the tumor targeting ability of BBN-based conjugates is found in nuclear medicine, where different radiolabeled bombesin analogs have been successfully used as theranostic probes for the diagnosis (imaging) and endoradiotherapy of GRPr-expressing tumors in humans.10-16 A crucial criteria in the development of peptide-based radiopharmaceuticals for application in nuclear oncology is a high tumor uptake relative to a low or negligible uptake in non-targeted tissues including excretion organs, e.g., the kidneys, which are sensitive to ionizing radiation. However, the fast metabolic degradation of BBN-based radiotracers impacts their tumor targeting capabilities and thus represents a limitation for medical applications.17 As a consequence, substantial research efforts have been made towards the stabilization of the peptide moiety in order to increase the tumor uptake of BBN-based radiotracers.18-20 Most studies have focused on the modification of amino acid side chains,18,

20, 21

and, more recently, backbone modification strategies have been

explored.22, 23 We have recently reported a novel strategy for the improvement of the biological 2


Page 3 of 39

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


properties of different radiotracers based on short, tumor targeting peptides.22-25 In these studies, stabilization of the BBN derivative [Nle14]BBN(7–14) was successfully achieved by replacing amide bonds with metabolically stable 1,4-disubstituted, 1,2,3 triazoles. The resulting radiolabeled peptidotriazoles displayed an improved stability in vitro and an up to two-fold increased tumor uptake in vivo.22,

23

Despite promising preliminary results, our radiolabeled

peptidomimetics did not reach the level of tumor uptakes reported for other radiometalated GRPr agonists, such as the benchmark peptide conjugate 3 (DOTA–PESIN, Table 1) or AMBA (see abbreviations).26-29 Intrigued by these observations and with the aim to optimize the peptide vector, we first set out to investigate the effect of C-terminal modifications on the properties of [177Lu]1 (Tables 1-2). In a second step, we studied the influence of an additional amino acid at the N-terminus of the most promising compound identified. Finally, we combined the results of the amino acid substitution study with our amide-to-triazole substitution approach for the design of a novel radiopeptidomimetic. In an effort to identify peptide-based GRPr-specific agonistic radiotracers with improved tumor targeting properties, we herein report the synthesis of a series of radiolabeled BBN(7–14) and BBN(6–14) derivatives and a systematic comparison of their pharmacological properties in vitro and in vivo (including cell internalization, receptor affinity, metabolic stability, and biodistributions).

Results Synthesis. The different peptide 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 (see also the Supporting Information).22, 23 For the synthesis of 7, the peptidomimetic sequence was elongated by standard Fmoc-SPPS up to 3



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 4 of 39

the histidyl residue at position 12. The free N-terminal α-amino function was then converted to an azide by a diazotransfer using imidazolylsulfonyl azide as the reagent.30, 31 The copper-catalyzed cycloaddition was then performed using (9H-fluoren-9-yl)methyl prop-2-yn-1-ylcarbamate,22 [(CH3CN)4Cu][PF6] as a copper (I) source, tris-(benzyltriazolylmethyl)amine (TBTA) as a stabilizing ligand for the copper catalyst, and diisopropylethylamine to yield the amide bond mimic between Gly11-His12.22 The conjugate sequence was then completed following standard SPPS protocols. All non-radioactive peptide conjugates were obtained in satisfying yields and characterized by RP-HPLC and high resolution mass spectrometry (Table 1). Radiolabeling. DOTA-substituted peptides were radiolabeled with Lu-177 at specific activities ranging from 2.9 to 18.5 GBq/µmol (not optimized) depending on the experiments planned. Radiometalations were conducted by incubation of the DOTA-substituted peptide conjugates with [177Lu]LuCl3 in a NH4OAc buffer (pH 5.0) at 95 °C for 30 min. After radiolabeling, the remaining non-metalated conjugates were complexed with natLuCl3.22, 23, 32 The Lu-177 complexes were obtained in radiochemical yields and purities exceeding 95% (Supporting Information). Addition of an excess methionine prevented oxidation of the methionine-containing [177Lu]3 during radiolabeling, and kept the radiochemical purity >95%.26 Radiochemical yields and purities of all radiolabeled compounds were determined by γ-HPLC. Cell binding and internalization studies. Several studies have shown that bombesin-like peptides may have different structure–activity relationships depending on the species from which the GRPr are derived from (e.g. human versus rat/mouse GPRr).23, 33, 34 Thus, in vitro evaluation of the radioconjugates in both murine and human cell lines is essential in order to understand potential discrepancies in tissue uptakes, e.g., in a xenogeneic mouse model (e.g., high radiotracer uptake in a xenografted tumor expressing the human receptor and a low uptake in tissues expressing the murine receptor; see the discussion of in vivo experiments below).35 Thus, 4


Page 5 of 39

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


the kinetics of cell internalization properties of all radiolabeled compounds were evaluated with two different cell lines that overexpress either the murine (AR42J) or the human GRP receptor (PC3).33 Receptor specific cell internalization of radiolabeled compounds is expressed as the percent of added radioactivity per million cells. Receptor specificity of the cell binding and internalization of the radiolabeled conjugates was confirmed for all compounds by addition of a 1000-fold excess of bombesin (referred to as blocking). The receptor blocking experiments decreased the internalization of radiolabeled compounds to less than 0.5 % in all cases hence demonstrating their receptor specificity (see supporting information). Specific internalization kinetics and rates after 4 hours of incubation with both cell lines are given in Table 2 and Figure 2. With the PC3 cell line, the

177

Lu-labeled compounds internalized quickly up to 21-33% of administered dose

after 4 h of incubation. With the exception of [177Lu]4, all compounds internalized at a similar rate and reached a maximum after 1-2 h of incubation. A more pronounced difference in the internalization kinetics of the radiotracers was observed when using AR42J cells with which the internalized fraction ranged from 5-25% of the added radioactivity after 4 h of incubation. Methionine-containing compound [177Lu]3 showed the quickest and highest internalization in rat cells after 4 hours of incubation (25%) whereas [DAla6, Hms14]BBN(6–14)-based compound [177Lu]4 displayed the lowest one (5%) (Table 2). Receptor binding affinity. Since we are ultimately interested in the use of the radioconjugates in humans, the binding affinities (KD) of the radiolabeled conjugates were determined by receptor saturation binding assays only with PC3 cells that overexpress the human GRPr. Dissociation constants (KD) and maximal receptor occupancies (Bmax) were evaluated using increasing concentrations of

177/nat

Lu-labeled conjugates. The results of the receptor

saturation experiments are summarized in Table 2. Receptor affinities of compounds [177Lu]2-7 5



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 6 of 39

were in the single digit nanomolar range (KD=2.0-4.7 nM) thus illustrating the high affinity of the compounds towards the human GRP receptor. Among the radiolabeled conjugates evaluated, [177Lu]1 exhibited the highest affinity towards the human GRPr (KD = 2.0 ± 0.6 nM). Compounds bearing an electronegative atom in the side chain of the amino acid residue in position 14 ([177Lu]2 and [177Lu]3) displayed an increased Bmax (approx. 2.3 nM) compared to [177Lu]1 (1.3 ± 0.1 nM), whereas [177Lu]3 exhibited the highest value in the series (2.5 ± 0.1 nM). Stability studies. The stability of the different conjugates is expressed as their respective halflifes in hours (t½). The results are summarized in Table 2. The half-lives of the radioconjugates were determined by incubation of the radiolabelled peptides in human blood plasma for different time periods followed by analysis by γ-HPLC after precipitation of proteins (Table 2). The halflives of compounds [177Lu]1-6 were found to be between 5 and 17 h. The insertion of a triazole in the sequence as an amide bond mimic in position Gly11-His12 led to a substantially increased metabolic stability of radiolabeled conjugate [177Lu]7 (t1/2= 75 h).22 Biodistribution studies. Uptakes of radioactivity in tissue and organs are expressed in % injected dose per gram of tissue (% ID/g). Experiments were performed in groups of 3-4 female nude mice xenografted on the right shoulder with PC3 cells. Results of the biodistribution studies in athymic Foxn1nu nude mice bearing PC3 xenografts are reported in detail in Tables 3-4, and summarized in Figure 3 (see also the Discussions and the Supporting Information). The conjugates showed characteristics common to short radiolabeled peptides, i.e. fast blood clearance and renal excretion. Additionally, no uptake was observed in GRPr negative organs (e.g., heart, lungs, or liver) at 4 hours post-injection of the radiotracer via tail vein injection. Blocking experiments performed demonstrated the specificity of the uptake in GRPr-expressing tissues and tumors. In all cases, co-administration of the conjugates with a 2000-fold excess of BBN led to a significant reduction of the uptake in GRPr-expressing organs such as the 6


Page 7 of 39

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


intestines, stomach, colon, adrenals, pancreas, and the PC3 tumor xenografts. Tumor uptake of the derivatives [177Lu]2-7 was significantly higher in comparison to the one of the parent compound [177Lu]1. Among the compounds tested, conjugates [177Lu]3-5 displayed the highest tumor uptake (up to 3.90 % ID/g). The replacement of Met14 ([177Lu]3 ) with Nle14 ([177Lu]1) or Hms14 ([177Lu]2) caused the uptake of radioactivity in the kidneys to drop from 2.5 ± 0.4 % ID/g to approx. 1.5 ± 0.3 % ID/g (at 4h p.i.)Figure 4). This decrease was statistically significant and resulted in a favorable tumor-to-kidney ratio for compounds [177Lu]4-7 (Tables 3-4, Figures 3-4). Of the compounds investigated, [177Lu]5 displayed the lowest kidney uptake (0.9 ± 0.2 % ID/g) whereas [177Lu]3 the lowest tumor-to-kidney ratio (Figure 4).

Discussion The overexpression of the GRPr by a variety of clinically relevant cancer (e.g., breast, prostate and small cell lung cancer) has provided the molecular basis for the targeting of these tumors with ligands of the receptor. In this context, the minimal binding sequence (BBN(7–14); HQWAVGHLM-NH2) of the tetradecapeptide bombesin BBN has been successfully used for the specific delivery of diagnostic probes and therapeutic agents to GRPr-positive tumors.26, 27, 29, 36 We are interested in the development of novel radiolabeled, BBN-based radiotracers that display improved tumor targeting properties. One the most important requirement for a tumor-targeting vector is a high uptake in tumors and a low or negligible accumulation in non-targeted tissue. Tumor uptake of a receptor-specific radiotracer can be increased by improving the interactions of the peptide moiety with the receptor (high receptor occupancy),37 and/or by improving the stability of the peptidic vector.22, 25, 38 As a matter of fact, BBN and BBN(7–14) derivatives are quickly degraded in vivo by regulatory proteases, which results in the delivery of only a fraction of the theoretically possible amount of radioactivity to tumors and metastases. Thus, increasing 7



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 8 of 39

the stability of the peptide moiety of BBN-based radiopharmaceuticals can potentially lead to an increased tumor uptake.22, 25, 38 We have recently published two reports that illustrate the use of backbone modification as a means to improve the metabolic stability of [Nle14]BBN(7–14)-based radiotracer in vitro and, as a result, their uptake in GRPr expressing tumors in vivo. As part of our ongoing efforts to optimize the tumor targeting properties of bombesin-based radiotracers, the present study aims at improving the pharmacological properties of this class of compounds by both amino acid substitutions and backbone modifications. Modifications of the C-terminus of the peptide Based on literature data, we hypothesized that the use of amino acids other that methionine in position 14 of BBN(7–14) could influence important biological parameters such as its metabolic stability and/or tumor uptake.18,

21, 39

In addition, the use of methionine in peptide-based

radiotracers is usually avoided because the thioether functional group is readily oxidized to the corresponding sulfoxide during radiolabeling reactions.18, 27, 40 The formation of oxidation side products has to be minimized since they cannot only lead to a decrease of the radiochemical purity of the product but also in a loss of affinity of the peptide towards its corresponding receptor (as in the case of bombesin).41 The use of Nle14 as a suitable replacement for Met14 in the bombesin sequence was first suggested by Jensen and co-workers. Their work illustrated that the replacement of different amino acids in the BBN(6–14) sequence did not result in a significant loss of its affinity for the GRPr.42,

43

As a consequence, a number of [Nle14]BBN(7–14)

derivatives have since been proposed as suitable radiotracers for the detection of GRPrexpressing tumors.18-21, 39 However, the exchange of a Met for a Nle at position 14 has also been shown to decrease the uptake of BBN(7–14)-based radiotracers in tumors in vivo despite a retained affinity towards the GRPr in vitro.21, 39

8


Page 9 of 39

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


With the aim of identifying a suitable substitute of Met14 that would fully preserve the biological properties of the sequence BBN(7–14) while being resistant to oxidation, we first set out to compare systematically the pharmacological properties of three BBN(7–14) derivatives. We used [177Lu]DOTA-PEG4-[Nle14]BBN(7–14), ([177Lu]1), as a reference compound for studying the effect of the amino acid substitutions at the C-terminus of the peptide sequence. In addition to the well-studied Met14-Nle14 exchange, we also investigated the utility of the amino acid Hms (homoserine-methylether; methoxinine) as a potential new substitute for Met. To the best of our knowledge, the present work represents the first investigation of Hms as a structurally similar but electronically more closely related amino acid substitute for Met than the currently used Nle. The properties of [177Lu]1 were compared with those of [177Lu]DOTA-PEG4-[Hms14]BBN(7–14), ([177Lu]2),

and

the

benchmark

compound

[177Lu]DOTA-PEG4-BBN(7–14),

([177Lu]3;

[177Lu]DOTA-PESIN), a reported radiotracer for GRPr targeting.26-28 In vitro experiments with PC3 cells showed that compounds [177Lu]2-3 exhibited superior cell internalization properties in comparison to [177Lu]1, particularly at 2 h and 4 h of incubation (Table 2, Figure 2). With AR42J cells, compound [177Lu]3 displayed the fastest and highest and compound [177Lu]1 the slowest cell internalization properties. Radiolabeled peptides [177Lu]1-3 all displayed comparable affinity towards the human GRPr with single-digit nanomolar KD values (approx. 2-4 nmol). However, compounds [177Lu]2-3 showed a two-fold increased maximal receptor occupancy (BMax) in comparison to [177Lu]1 (2.5 nM versus 1.3 nM, respectively). Regarding their stability in human blood plasma, compound [177Lu]3 had an almost twofold increased half-life in comparison to [177Lu]1 and 2 (Table 2). This result is consistent with the report of Zhang et al. who also observed an increased stability by switching a Nle14 to a Met14 in a BBN(6–14) analog.39

9



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 10 of 39

The different radiotracers behaved similar in vivo in our mouse model. Compounds [177Lu]1-3 showed favorable fast renal excretion as indicated by the absence of radioactivity in the blood and the liver at 4 h p.i. (Figure 3 and Table 3); unspecific uptake of radiotracer in the kidneys is the result of renal elimination as typically observed for radiotracers based on small peptides. Receptor specific accumulation was observed in the gastrointestinal tract due to the expression of the GRPr in the intestines, stomach, colon, adrenals, and pancreas. An increased tumor uptake was observed for compounds [177Lu]2 and 3 in comparison to [177Lu]1 (3.4 and 3.9 % ID/g vs. 1.5 % ID/g, respectively) (Figure 4). The increased tumor uptake of [177Lu]2 and 3 may reflect the higher maximal receptor occupancy (BMax) of the compounds as observed in vitro (Table 2). To conclude, of the C-terminally modified derivatives investigated, [177Lu]3 showed a favorable high uptake in receptor positive tissue and tumor xenografts. However, the new derivative based on the [Hms14]BBN(7–14) peptide moiety, [177Lu]2, not only exhibited a tumor uptake comparable to [177Lu]3 but also the highest tumor-to-kidney ratio among the three compounds tested (Table 3 and Figure 4). Thus, we selected compound [177Lu]2 for further optimizations. Modification of the N-terminus of the peptide In the next step, we investigated the influence of an additional amino acid attached to the Nterminus of the [Hms14]BBN(7–14) peptide moiety of [177Lu]2. Surprisingly, the potential advantage of the use of extended BBN(6–14) derivatives in comparison to the shorter BBN(7–14) analogues has yet not been thoroughly studied and discussed in the literature. However, the addition of D-Phenylalanine and D-Tyrosine in position 6 of the minimal binding sequence of BBN(7–14) can be found frequently in reported non-radioactive,44-47 as well as radiolabeled bombesin derivatives.19, 20, 37 We thus decided to investigate the effect of an additional D-amino acid in position 6. In addition to the reported D-Phenylalanine and D-Tyrosine residues, we included D-Alanine in this series to study the influence of an non-aromatic residue at this 10


Page 11 of 39

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


position. Thus, compounds included in this part of the work were [177Lu]DOTA-PEG4[DAla6,Hms14]BBN(6–14),

([177Lu]4),

[177Lu]DOTA-PEG4-[DPhe6,Hms14]BBN(6–14),

([177Lu]5), and [177Lu]DOTA-PEG4-[DTyr6, Hms14]BBN(6–14), ([177Lu]6). Radiolabeled peptides [177Lu]5 and [177Lu]6 modified with an aromatic D-amino acid residue at the N-terminus displayed similar cell internalization properties with PC3 cells in comparison to [177Lu]2, which served as a truncated reference compound for this part of our study (Table 2 and Figure 2A). In comparison, compound [177Lu]4 bearing the aliphatic D-amino acid D-Ala at the N-terminus showed the slowest and lowest cell internalization kinetics of the BBN(6–14) derivatives tested in this series. In the rat tumor cell line AR42J, compounds [177Lu]5 and [177Lu]6 displayed similar cell internalization kinetics to the one of the [177Lu]2, whereas [177Lu]4 performed significantly poorer under the same experimental conditions (Figure 2B). This marked difference in the cell internalization kinetics is likely the result of a decreased affinity of [177Lu]4 towards the rat GRPr caused by the multiple structural modifications (see also discussion of in vivo data below). Receptor saturation experiments revealed that compounds [177Lu]4-6 had a retained high affinity towards the human GRPr with single digit nanomolar dissociation constants (KD=2.4-4.7 nM). The BBN(6–14) derivatives [177Lu]4-6 also preserved the high receptor occupancy (Bmax) observed for analogues with the shorter BBN(7–14) moieties ([177Lu]2 and [177Lu]3 respectively ; approx. 2.0 nM). The addition of an additional D-amino acid residue in position 6 of the peptide moiety resulted in a significant increased stability of the radioactive conjugates in human blood plasma. In comparison to the shorter peptide [177Lu]2 (t1/2= 5 h), the half-lifes of conjugates [177Lu]5-6 were increased by a two-fold in human blood serum whereas the one of [177Lu]4 was improved by a three-fold (Table 2). In

general, the DOTA-PEG4-BBN(6–14)

derivatives

[177Lu]4-6

displayed

a similar

pharmacokinetic profile in vivo as discussed above for the truncated versions [177Lu]1-3. This 11



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 12 of 39

included fast blood clearance of the radiotracers and renal excretion, low accumulation in nontargeted tissues (see above), and GRPr-specific uptake in receptor positive organs and in the tumor xenografts (Tables 3-4 and Figure 3). Investigation of this series of compounds, [177Lu]4-6, aimed at the clarification whether or not the addition of a (aromatic or aliphatic) D-amino acid placed in position 6 of the [Hms14]BBN(6–14) sequence would be beneficial in terms of the tumor uptake and/or tumor-to-kidney ratios of the radiotracer in vivo. In general, tumor uptakes of conjugates [177Lu]4 and 5 were found similar to the one observed for reference compound [177Lu]2, whereas the one of compound [177Lu]6 was decreased. The latter observation is consistent with results from Zhang et al. who reported that the removal of the DTyr residue in position 6 of radiolabeled [DTyr6]BBN(6–14)-based peptides led to an increase of the tumor uptake of the corresponding shorter BBN(7–14) analogue.39 While the kidney uptakes of [177Lu]4 and 6 were comparable to the one of the parent compound [177Lu]2 (Figure 3), [177Lu]5 showed a slight but statistically significant decreased kidney uptake (Tables 3-4, and Figures 3-4). Interestingly, [177Lu]4 displayed very low uptake in murine receptor positive organs (e.g., pancreas, colon, intestines) but an unaltered accumulation in the tumor xenografts which express the human GRPr. This result is consistent with the results of our in vitro experiments using AR42J cells (see above), in which [177Lu]4 displayed the lowest cell internalization rate in cells expressing the murine GRPr (Table 2). We therefore conclude that the low in vivo uptake of [177Lu]4 in organs expressing the mouse GRPr is the result of a partial loss of affinity towards the murine GRPr for compound [177Lu]4.23,

33

Such interspecies differences need to be taken into

consideration during the preclinical development of radiopharmaceuticals. Of the N-terminally modified BBN(6–14) derivatives investigated in this series, compound [177Lu]5 based on a [DPhe6, Hms14]BBN(6–14) peptide moiety, displayed a favorable tumor uptake and, to the best of our knowledge, the highest tumor-to-kidney ratio reported to date for 12


Page 13 of 39

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


radiolabeled agonist of the GRPr (Figure 3 and 4).26 Thus compound [177Lu]5 was carried forward as a lead compound for further investigations. Modification of the peptide backbone We have recently reported a novel methodology for the stabilization of peptide-based radiotracers based on the isosteric replacement of amide bonds by nonhydrolyzable 1,2,3-triazoles.22,

25

In

previous studies, it was shown that the substitution of the amide bond between Gly11 and His12 of BBN(7–14) by a 1,2,3 triazole in compound [177Lu]1 resulted in a radiopeptidomimetic with an improved stability in vitro and a two-fold increased tumor uptake in vivo.22, 23 Thus, we applied this backbone modification to the lead structure [177Lu]5 identified during the above presented amino acid substitution study. [177Lu]DOTA-PEG4-[DPhe6, Gly11ψ[Tz]His12, Hms14]BBN(6–14), ([177Lu]7, Table 2), was synthesized on solid support by a combination of standard Fmoc/tBu solid phase peptide synthesis, a diazo transfer reaction and the Cu(I)-catalyzed, azide-alkyne cycloaddition (CuAAC) by methods previously described (see experimental part)22,

23

and its

pharmacological properties were compared to the ones of (all amide bond) reference compound [177Lu]5. In vitro, [177Lu]7 showed a slower cell internalization kinetics than those of the parent compound [177Lu]5 in both cell lines tested (Table 2 and Figure 2). Saturation binding assays revealed that peptidomimetic [177Lu]7 displayed a retained high affinity towards the GRP receptor but a decreased receptor occupancy (Bmax) in comparison to conjugate [177Lu]5 (Table 2 and Supporting Information). Remarkably, the introduction of a 1,2,3-triazole in the backbone of the BBN(6–14) motif resulted in a more than 7-fold increased stability of compound [177Lu]7 in human blood serum (Table 2). Thus, backbone modified peptide [177Lu]7 represents another successful example which illustrates the general utility of 1,2,3-triazoles as amide bond mimics for the stabilization of bioactive peptides.22-25, 48-53

13



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 14 of 39

Biodistribution experiments revealed that radiolabeled peptidomimetic [177Lu]7 preserved most of the main in vivo characteristics of the parent compound [177Lu]5 (Table 4, Figures 3-4; for a discussion of the general pharmacological features see discussion above). Despite its remarkably improved stability, tumor uptake of [177Lu]7 was found statistically significant lower than that of [177Lu]5 (2.71 % ID/g and 3.84 % ID/g, respectively; Table 4, Figure 4). These unexpected results in terms of tumor uptake of compound [177Lu]7 in comparison to [177Lu]5 may reflect the observed differences of their respective receptor occupancy values (Bmax) as determined in vitro (Table 2). It is likely that this is the result of the subtle structural changes induced by the insertion of the 1,2,3-triazole heterocycle into the tumor targeting peptide sequence BBN(7–14) which in turn might have caused alterations of its binding conformation.

Conclusions We have recently initiated a research program which aims at the improvement of the tumor targeting properties of agonistic, bombesin-based radiolabeled peptides for applications in nuclear oncology as diagnostic imaging probes or endoradiotherapeutics. In a previous study, we successfully improved the metabolic stability and tumor uptake of GRPr-specific, radioactive labeled [Nle14]BBN(7–14) derivatives by means of backbone modifications of the peptide moiety.22 In order to optimize further the tumor targeting properties of BBN-derived radiolabeled peptide conjugates, we report in the present study several amino acid substitutions at different positions of the peptidic vector that were selected based on literature data.18-20, 26, 27, 39, 46, 47, 54 Finally, we describe for the first time the combination of our amide-to-triazole replacement strategy with the more traditional amino acid residue substitution approach in an attempt to identify novel derivatives with improved biological properties.

14


Page 15 of 39

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


C-terminal variations of the parent compound

[177Lu]1, revealed that Hms (homoserin-

methylether, methoxinine) is a suitable substitute for methionine in radiolabeled peptide conjugates based on the BBN(7–14) moiety. In comparison to the aliphatic norleucine (Nle) currently used as a stable analog of Met in pre- and clinical studies, Hms includes an oxygen heteroatom in its side chain which is not only resistant to oxidative side reactions during radiolabeling experiments like Nle but also resembles more closely the electronic properties of Met. To the best of our knowledge, this is the first report that describes an alternative amino acid substitute of Met other than Nle for the development of radiolabeled peptide conjugates. In comparison to parent compound [177Lu]1, Hms14-containing [177Lu]2 displayed improved cell internalization kinetics and receptor occupancy (Bmax) in vitro as well as a favorable tumor uptake and tumor-to-kidney ratio in vivo. Modifications of the N-terminus of lead compound [177Lu]2, revealed that an additional (aliphatic or aromatic) amino acid in position 6 of the minimum binding sequence of BBN yielded elongated BBN(6–14) analogues with enhanced blood plasma stability. While tumor uptake was not influenced to a great extent by the additional amino acid, tumor-to-kidney ratios were found improved because of decreased kidney uptake. As a result of these investigations, compound [177Lu]5 was identified as a novel radiolabeled agonistic GRPrspecific radiopeptide which exhibits, to the best of our knowledge, an unprecedented high tumorto-kidney ratio in vivo.26 Insertion of a 1,4-disubstituted 1,2,3-triazoles as a stable amide bond mimic at position Gly11-His12 of compound [177Lu]5 yielded radiolabeled peptidomimetic [177Lu]7 which displayed one of the highest stabilities in blood plasma reported in the literature for a radiolabeled peptide-based GRPr agonist;26 however, the backbone modification did not result in an further improved tumor uptake in vivo. In summary, we report here the application of different strategies for the optimization of the tumor-targeting properties of radiolabeled bombesin derivatives by structural modifications of the 15



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 16 of 39

peptide vector. Both, amino acid substitutions and backbone modifications were applied to the parent compound [177Lu]1. The study yielded several novel agonistic BBN(7–14) and BBN(6– 14) analogues with promising pharmacological properties which may be suitable for clinical applications in nuclear medicine for the detection and treatment of GRPr-positive tumors.

Experimental section Materials and methods. Unless stated otherwise, all reagents and solvents were purchased from Acros Organics (Geel, Belgium), Merck (Darmstadt, Germany), and Sigma Aldrich (Buchs, Switzerland) and used without further purification. Fmoc-amino acids, Rink Amide MBHA LL resin (100-200 mesh), and HATU were purchased from Merck Biosciences (Nottingham, UK). Fmoc-15-amino-4,7,10,13-tetraoxapentadecanoic acid (Fmoc-PEG4-OH) was purchased from Polypeptide Group (Strasbourg, France). N-(Fmoc)-O-methyl-L-homoserine (Fmoc-Hms-OH) was purchased from Iris Biotech and DOTA(tBu)3 and DOTA-NHS ester from CheMatech (Dijon, France). Bombesin trifluoroacetate was purchased from Bachem (Bubendorf, 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 in 0.05 M HCl was provided by ITM (Munich, Germany). HPLC analysis and purification were performed on Bischoff HPLC systems. The analytical HPLC 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. HPLC preparative purification was carried out on a Bischoff HPLC system composed of a Bischoff LCCaDi 22-14 interface, a UV-vis Lambda 1010 detector and two HPLC compact pumps 2250. A Phenomenex Jupiter 4 µm Proteo 90 Å 250 × 4.6 mm was used for analytical separations. A Nucleodur C18 ISIS, 5 µm, 250 × 16 mm column (Macherey Nagel) was used for preparative 16


Page 17 of 39

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


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 correspond to the monoisotopic ions. Quantitative γ-counting was performed on a COBRA 5003 γ-system well counter from Packard Instruments (Meriden, CT, USA). 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. Peptide 1 has been previously described.22 Syntheses of compounds 2 to 6 were performed following the general procedure described below. Synthesis of the backbone-modified peptidomimetic conjugate 7 was performed manually following previously published procedures.22, 23 General procedure 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 mmol or 0.03 mmol, swollen in peptide-synthesis grade DMF) were successively added the Fmoc-protected amino acid (2 equiv., 0.2 mmol 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% 17



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 18 of 39

Piperidine in DMF to the peptide-resin. The suspension was then allowed to react for three minutes. The treatment was repeated three times. The solvent and excess reagents were then removed by filtration and the resin was washed thoroughly with DMF and CH2Cl2. With the exception of 3 (see below), the peptide resin was cleaved 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 (gradient B/A 20% to 40% in 15 min at 8 mL/min) and the peptide conjugates were isolated as white powders after lyophilization. DOTA-PEG4-Gln-Trp-Ala-Val-His-Leu-Hms-NH2 (2). Peptide 2 was prepared by manual synthesis, using commercial DOTA-(tris-tBu), Fmoc-15-amino-4,7,10,13-tetraoxapentadecanoic acid, Fmoc-Gln(Trt)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Val-OH, Fmoc-Gly-OH, Fmoc-His(Trt)OH, Fmoc-Leu-OH, Fmoc-Hms-OH. After synthesis and purification, conjugate 2 was obtained in 36% yield. ESI-MS observed [M+2H]2+ m/z = 779.4184 (calcd. for [C70H114N18O22]2+ : 779.4179). DOTA-PEG4-Gln-Trp-Ala-Val-His-Leu-Met-NH2 (3). Peptide 3 was prepared by manual synthesis, using commercial DOTA-NHS ester, Fmoc-15-amino-4,7,10,13-tetraoxapentadecanoic acid, Fmoc-Gln(Trt)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Val-OH, Fmoc-Gly-OH, Fmoc-His(Trt)OH, Fmoc-Leu-OH, Fmoc-Met-OH. DOTA-NHS ester was coupled by the following procedure. A solution of DOTA-NHS ester (5 equiv.) and DIPEA (10 equiv.) in DMF was added to the peptide resin and the suspension was allowed to stir overnight and RT. After synthesis of the DOTA functionalized conjugate on solid support, the peptide resin was then cleaved and deprotected by a 2 h treatment with a mixture of TFA/H2O/i-Pr3SiH/thioanisole/ethanedithiol (90:2.5:2.5:2.5:2.5), and the peptide was precipitated with ice-cold tert-butyl methyl ether, recovered by centrifugation and washed twice with cold tert-butyl methyl ether. The precipitate 18


Page 19 of 39

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


was purified to obtain conjugate 3 in 17% yield. ESI-MS observed [M+H]+ m/z = 1573.8043 (calcd. for [C70H113N18O21S]+ : 1573.8048). DOTA-PEG4-DAla-Gln-Trp-Ala-Val-Gly-His-Leu-Hms-NH2 (4). Peptide 4 was prepared by manual

synthesis,

using

commercial

DOTA-(tris-tBu),

Fmoc-15-amino-4,7,10,13-

tetraoxapentadecanoic acid, Fmoc-DAla-OH, Fmoc-Gln(Trt)-OH, Fmoc-Trp(Boc)-OH, FmocVal-OH, Fmoc-Gly-OH, Fmoc-His(Trt)-OH, Fmoc-Leu-OH, Fmoc-Hms-OH. After synthesis and purification, conjugate 4 was obtained in 33% yield. ESI-MS observed [M+2H]2+ m/z = 814.9366 (calcd. for [C73H119N19O23]2+ : 814.9363). DOTA-PEG4-DPhe-Gln-Trp-Ala-Val-Gly-His-Leu-Hms-NH2 (5). Peptide 5 was prepared by manual

synthesis,

using

commercial

DOTA-(tris-tBu),

Fmoc-15-amino-4,7,10,13-

tetraoxapentadecanoic acid, Fmoc-DPhe-OH, Fmoc-Gln(Trt)-OH, Fmoc-Trp(Boc)-OH, FmocVal-OH, Fmoc-Gly-OH, Fmoc-His(Trt)-OH, Fmoc-Leu-OH, Fmoc-Hms-OH. After synthesis and purification, conjugate 5 was obtained in 32% yield. ESI-MS observed [M+2H]2+ m/z = 852.9521 (calcd. for [C79H123N19O23]2+: 852.9520). DOTA-PEG4-DTyr-Gln-Trp-Ala-Val-Gly-His-Leu-Hms-NH2 (6). Peptide 6 was prepared by manual

synthesis,

using

commercial

DOTA-(tris-tBu),

Fmoc-15-amino-4,7,10,13-

tetraoxapentadecanoic acid, Fmoc-DTyr-OH, Fmoc-Gln(Trt)-OH, Fmoc-Trp(Boc)-OH, FmocVal-OH, Fmoc-Gly-OH, Fmoc-His(Trt)-OH, Fmoc-Leu-OH, Fmoc-Hms-OH. After synthesis and purification, conjugate 6 was obtained in 32% yield. ESI-MS observed [M+2H]2+ m/z = 860.9501 (calcd. for [C79H123N19O24]2+ : 860.9494). DOTA-PEG4-DPhe-Gln-Trp-Ala-Val-Glyψ[Tz]His-Leu-Hms-NH2 (7). Peptide 7 was prepared by

manual

synthesis,

using

commercial

DOTA-(tris-tBu),

Fmoc-15-amino-4,7,10,13-

tetraoxapentadecanoic acid, Fmoc-D-Phe-OH, Fmoc-Gln(Trt)-OH, Fmoc-Trp(Boc)-OH, Fmoc19



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

Val-OH,

Fmoc-His(Trt)-OH,

Fmoc-Leu-OH,

Fmoc-Hms-OH

Page 20 of 39

(N-(Fmoc)-O-methyl-L-

homoserine). The peptidomimetic moiety was introduced as previously described, after deprotection of the Histidyl residue, a diazotransfer reaction was performed in order to introduce an azide functional group.31 Diazotransfer reaction was performed by addition of a solution of imidazolylsulfonyl azide hydrochloride (2 equiv., 0.06 mmol) and DIPEA (5 equiv., 0.15 mmol) to the resin (0.03 mmol) and subsequent shaking of the resin for one hour. The completion of the reaction was checked by the Kaiser test. Coupling of (9H-fluoren-9-yl)methyl prop-2-yn-1ylcarbamate was performed as previously described.22 Briefly, to the resin (0.03 mmol) was added a solution of the Fmoc-protected amino alkyne (2 equiv., 0.06 mmol) and i-Pr2NEt (1 equiv., 0.03 mmol) in degassed DMF. To the suspension was added a solution of tetrakis(acetonitrile)copper(I) hexafluorophosphate (0.5 equiv., 0.015 mmol) and tris[(1-benzyl1H-1,2,3-triazol-4-yl)methyl]amine (TBTA; 0.5 equiv., 0.015 mmol) and was vigorously shaken at RT overnight. The resin was then successively and extensively washed with DMF, a solution of 0.5% of diethyldithiocarbamate in DMF (3x), CH2Cl2 and DMF. After synthesis of the DOTA functionalized conjugate on solid support, cleavage from the resin, and deprotection, the precipitate was purified to obtain conjugate 7 in 23% yield. ESI-MS observed [M+2H]2+ m/z = 864.9578 (calcd. for [C80H123N21O22]2+: 864.9576). Radiolabeling. DOTA-conjugated peptides were radiolabeled at different specific activities depending on the experiments planned. For in vitro cell studies, the specific activity was approx. 2.9 GBq/µmol. For stability experiments in human blood plasma and for in vivo studies the peptides were labeled at 18.5 GBq/µmol (not optimized). To 10 µg (6.14 - 6.30 nmol in 10 µL water) of DOTA-conjugates were added approx. 300 µL of 0.4 M NH4OAc buffer (pH 5.0) in a pre-lubricated Eppendorf tube. To this solution of the peptide were added 37-111 MBq of a commercial solution

177

LuCl3 in 0.05 M HCl and the mixture was heated at 95°C for 30 min. 20


Page 21 of 39

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


After quality control by γ-RP-HPLC, 5 nmol (approx. 80% of the amount of peptide) of a 1 mM nat

LuCl3 solution was added and the mixture was heated for additional 30 min at 95°C.32

Peptide 3 was labeled in the presence of 40 µL of a fresh 20 mg/mL solution of Methionine (approx. 1000 equiv.) to prevent oxidation of the thioether function of the methionine to a sulfoxide during incubation with Lu-177 at 95°C. The addition of Methionine during radiolabeling maintained the amount of sulfoxide below 5%.27,

40

Radiolabeling yields were

determined by γ-HPLC. All radiometallated peptides were obtained in radiochemical yields and purities of > 95%. 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. 2.5 pmol of [177/natLu]-labeled peptides were added and the cells were incubated at 37 °C (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 (see Supporting Information). 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 gamma counter. The percentage of added activity (% of applied dose) was calculated for each time point. Non-specific binding and internalization was determined by incubation of the cells with the radiopeptides and a large excess of bombesin trifluoroacetate (2.5 nmol per well). Experiments were performed two to three times in triplicates. Receptor binding affinity. KD determination was performed as previously described.22 Briefly, PC-3 cells at confluence were placed in 6-well plates (0.8-1×106 cells/well). The cells were 21



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

incubated with increasing concentrations of

Page 22 of 39

177/nat

Lu-radiolabeled peptides (0.1, 0.5, 1, 5, 10, 25,

50, 75, 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 1M NaOH aqueous solution for 10 min at 37°C, the supernatant was collected and the radioactivity was measured in a gamma counter. Non-specific binding was determined by incubation of the cells with the radiopeptides and a large excess of bombesin (10 µM per well). Experiments were performed two to three times in triplicates. The receptor saturation curves were fitted by non-linear regression using GraphPad Prism 5.0. Stability studies. 90 pmol (1.6 MBq) of radioactive labeled peptides (specific activity: 18.5 Bq/nmol) were added to 1 mL of fresh human blood plasma and incubated (37°C, 5% CO2). Samples of 100 µL were collected at pre-selected time points (1, 2, 4, 6, 24 h), and the plasma proteins precipitated by addition of 200 µL ethanol. After centrifugation at 5200 rpm for 5 min, the supernatant was then transferred into an Eppendorf tube and the remaining plasma proteins were precipitated again 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(-λ·t) using GraphPad Prism 5.0. Plasma stability of the radiopeptides is expressed as the half-life (t½, in hours). The protein pellet contained < 5% of the added radioactivity in all cases. This observation is consistent with literature data.19, 39 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 to 8 weeks old female athymic nude Foxn1nu mice (2022


Page 23 of 39

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


25 g), purchased from Charles River or Harlan Laboratories. The mice were xenografted with 510·106 PC-3 cells and the grafts were allowed to grow until the tumors reached an approximate diameter of 0.5 cm (approx. 2-3 weeks). On the day of the experiment, the mice received an intravenous injection of 100 µL of

177/nat

Lu-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). Non-specific uptake was determined by co-injection of an excess of bombesin using the same formulation (20 nmol, n = 3/group). The animals were sacrificed 4 hours post-injection of the radiotracer by CO2 asphyxiation. Tumors, 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 (% I.D./g). Statistical analysis was conducted using unpaired two-tailed Student’s t-test using GraphPad Prism 5.0.

FIGURES

23



Figure 1. Structure of compounds 1 to 7 with numbering of amino acid residues.

Specific internalization (%)

A

40

[177Lu]1

30

[177Lu]2 [177Lu]3

20

[177Lu]4 [177Lu]5

10

[177Lu]6 [177Lu]7

0 0

B

Specific internalization (%)

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 24 of 39

50

100

150

200

250

Time (min) 30

[177Lu]1 [177Lu]2

20

[177Lu]3 [177Lu]4 [177Lu]5

10

[177Lu]6 [177Lu]7

0 0

50

100

150

200

250

Time (min)

Figure 2. Time-dependent, receptor specific internalization in % of administered dose of compounds [177/natLu]1−7 in PC3 cells (A) and AR42J cells (B) at 37°C (n=2 in triplicates). nonspecific binding (< 0.5%) was omitted for clarity (see supporting information). Data of previously reported compound [177Lu]1 is included here for comparison.22

24


Page 25 of 39

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


Figure 3. Comparison of tracer uptakes in selected organs of PC3 cell-xenografted cell xenografted athymic nude mice at 4 h p.i. (n = 4). (r) indicates receptor-positive positive organs. Biodistribution data of previously reported compound [177Lu]1 is included here for comparison.22

25



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 26 of 39

Figure 4. Comparison of tumor uptakes of [177Lu]1−7 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 compound [177Lu]1 is reproduced here for comparison.22

TABLES

Cpd.

Yield

m/z found

[%]

[Da] a

Structure

1

DOTA-PEG4-Gln-Trp-Ala-Val-Gly-His-Leu-Nle-NH2

35b

1555.894b

2

DOTA-PEG4-Gln-Trp-Ala-Val-Gly-His-Leu-Hms-NH2

36

779.4184

3

DOTA-PEG4-Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH2

17

1573.8043

4

DOTA-PEG4-DAla-Gln-Trp-Ala-Val-Gly-His-Leu-Hms-NH2

33

814.9366

5

DOTA-PEG4-DPhe-Gln-Trp-Ala-Val-Gly-His-Leu-Hms-NH2

32

852.9521

6

DOTA-PEG4-DTyr-Gln-Trp-Ala-Val-Gly-His-Leu-Hms-NH2

32

860.9501

7

DOTA-PEG4-DPhe-Gln-Trp-Ala-Val-Glyψ[Tz]His-Leu-Hms-NH2

23

864.9578

a

With the exception of 3, all the conjugates masses were observed [M+2H]2+ by ESI-HRMS, b synthesis and characterization of compound 1 has been previously reported.22 Table 1. Summary of structure, yield and MS analysis of peptide 1 to 7

% internalization

% internalization

Cpd. 177

[

177

[

177

[

177

[

KD (nM)

BMax (nM)

Half-life (hrs)

after 4h in PC3 cells

after 4h in AR42J cells

Lu]1a

24.9 ± 1.1

10.1 ± 0.5

2.0 ± 0.6

1.3 ± 0.1

5

Lu]2

32.9 ± 1.3

15.7 ± 0.4

4.1 ± 0.8

2.1 ± 0.1

5

Lu]3

30.6 ± 1.8

24.7 ± 0.4

4.5 ± 0.9

2.5 ± 0.1

10

Lu]4

21.2 ± 1.3

5.4. ± 0.2

4.7 ± 0.9

1.7 ± 0.1

17

26


Page 27 of 39

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


177

[

177

[

177

[

a

Lu]5

30.8 ± 3.8

19.0 ± 0.1

2.4 ± 0.5

2.1 ± 0.1

11

Lu]6

30.2 ± 0.8

21.7 ± 0.6

3.4 ± 0.6

1.9 ± 0.1

14

Lu]7

26.9 ± 2.0

10.7± 0.1

4.1 ± 1.2

1.5 ± 0.1

75

Data of compound [177Lu]1 reproduced here for comparison.22

Table 2. Internalization rates determined in PC3 and AR42J cells, GRPr binding affinities (KD) and maximal receptor occupancies (BMax) determined in PC3 cells, and human plasma stabilities of [177Lu]1-7 (n=2-3 in triplicates).

177

[ Lu]2 blocking

[

0.01±0.00

muscle

177

177

Lu]3

[ Lu]3 blocking

[

0.01±0.00

0.01±0.00

0.01±0.01

0.00±0.00

0.00±0.00

0.02±0.02

0.03±0.02

0.02±0.01

0.04±0.02

0.02±0.04

0.02±0.03

liver

0.07 ±0.01

0.10±0.01

0.10±0.01

0.16±0.07

0.07±0.02

0.12±0.01

spleen

0.26±0.02

0.07±0.01

1.24±0.23

0.07±0.01

0.13±0.04

0.05±0.00

lung

0.04±0.01

0.05±0.02

0.06±0.01

0.08±0.03

0.01±0.02

0.07±0.01

kidneys

1.45±0.32

1.48±0.18

2.53±0.39

1.97±0.30

1.34±0.17

2.05±0.49

stomach

0.59±0.06

0.05±0.02

1.25±0.30

0.07±0.01

0.35±0.25

0.04±0.01

intestine

1.32±0.59

0.08±0.02

1.24±0.47

0.09±0.02

0.25±0.07

0.08±0.02

colon

2.76±1.06

0.13±0.05

7.37±1.36

0.11±0.04

1.21±0.66

0.09±0.03

pancreas

4.68±1.37

0.17±0.08

18.03±2.44 0.27±0.03

0.65±0.47

0.06±0.03

PC3 tumor

3.39±0.78

0.29±0.13

3.94±0.88

0.27±0.02

3.86±0.68

0.19±0.03

-

1.57±0.36

-

2.89±0.47

-

177

Organ

[

blood

Lu]2

Tumor/kidney 2.45±0.94

177

177

Lu]4

[ Lu]4 blocking

a

Blocking experiments were performed by co-injection of a 2000 fold excess of bombesin. Data is expressed as the percentage of the injected dose per gram of tissue, represented as mean values ± SD (n = 4 for non-blocked experiments, n=3 for blocking experiments). Table 3. Biodistribution (% I.D./g) of compounds [177Lu]2-4 in PC3 xenografted athymic nude mice 4 h post i.v. injection.a 27



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

177

177

Page 28 of 39

177

Organ

[

Lu]5

[ Lu]5 blocking

[

Lu]6

[ Lu]6 blocking

[

blood

0.02±0.01

0.01±0.00

0.02±0.00

0.00±0.01

0.02±0.00

0.01±0.00

muscle

0.01±0.04

0.01±0.00

0.01±0.03

0.00±0.03

0.01±0.02

0.01±0.01

liver

0.05±0.01

0.05±0.02

0.04±0.01

0.05±0.02

0.04±0.01

0.06±0.01

spleen

0.40±0.33

0.04±0.01

0.32±0.08

0.04±0.00

0.19±0.03

0.03±0.00

lung

0.02±0.02

0.04±0.00

0.04±0.01

0.03±0.01

0.04±0.01

0.07±0.01

kidneys

0.89±0.21

0.72±0.24

1.32±0.15

0.99±0.21

0.97±0.25

0.90±0.15

stomach

0.61±0.09

0.02±0.01

0.58±0.11

0.03±0.02

0.50±0.21

0.03±0.01

intestine

1.08±0.34

0.03±0.01

1.64±0.26

0.03±0.02

0.93±0.41

0.02±0.02

colon

3.02±0.64

0.14±0.14

2.96±0.44

0.11±0.05

3.16±1.09

0.07±0.03

pancreas

3.76±1.27

0.07±0.04

3.09±0.99

0.23±0.15

4.34±0.52

0.17±0.03

PC3 tumor

3.84±0.38

0.13±0.02

2.64±0.24

0.20±0.06

2.71±0.60

0.24±0.03

-

2.05±0.09

-

2.81±0.34

-

177

Tumor/kidney 4.44±0.27

177

177

Lu]7

[ Lu]7 blocking

a

Blocking experiments were performed by co-injection of 2000 fold excess bombesin. Data is expressed as the percentage of the injected dose per gram of tissue, represented as mean values ± SD (n = 4 for non-blocked experiments, n=3 for blocking experiments). Table 4. Biodistribution (% I.D./g) of compounds [177Lu]5-7 in PC3 xenografted athymic nude mice 4 h post i.v. injection.a ASSOCIATED CONTENT Supporting Information. Full characterization of peptides 2-7 and [177Lu]2-7 (UV- and γHPLC, mass spectrometric data) and detailed results of in vitro (cell internalization and receptor saturation experiments, stability studies in blood plasma) and in vivo experiments can be found in the Supporting Information. This material is available free of charge via the Internet at http://pubs.acs.org.

28


Page 29 of 39

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


AUTHOR INFORMATION Corresponding Authors I. E. Valverde, phone: +41 61 265 46 74, email: [email protected] T. L. Mindt, phone: +41 44 633 76 48, e-mail: [email protected] Present Addresses † Institute of Pharmaceutical Sciences, ETH Zurich, Vladimir-Prelog-Weg 4, 8093 Zürich, Switzerland Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Funding Sources This work was supported in part by the Swiss National Science Foundation (Grants 205321_132280 and 200020_146385 to T.L.M.). Notes The authors declare no competing financial interest ACKNOWLEDGMENT The authors thank Alba Mascarin for helpful scientific discussions, Rudolf Von Wartburg (University of Basel Hospital, Basel, Switzerland) for technical support, and ITM (Munich, Germany) for the supply of non-carrier-added Lu-177.. ABBREVIATIONS 29



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 30 of 39

AMBA, DOTA-Gly-4-aminobenzoyl- Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH2 ; BBN, bombesin (pGlu-Gln-Arg-Leu-Gly-Asn-Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH2); modified

Eagle’s

medium

;

DMF,

N,N-dimethylformamide;

DMEM,

Dulbecco

DOTA,

1,4,7,11-

tetraazacyclododecane-1,4,7,11 tetraacetic acid; FBS, fetal bovine serum; GRP, gastrin releasing peptide; GRPr, gastrin-releasing peptide receptor; HSA, human serum albumin; Hms, O-methylhomoserine, methoxinine ; PBS, phosphate buffered saline; PEG4, 1-amino-3,6,9,12tetraoxapentadecan-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. REFERENCES (1) Jensen, R. T.; Battey, J. F.; Spindel, E. R.; Benya, R. V. International Union of Pharmacology. LXVIII. Mammalian Bombesin Receptors: Nomenclature, Distribution, Pharmacology, Signaling, and Functions in Normal and Disease States. Pharmacol Rev 2008, 60, 1-42. (2) Markwalder, R.; Reubi, J. C. Gastrin-Releasing Peptide Receptors in the Human Prostate: Relation to Neoplastic Transformation. Cancer Res. 1999, 59, 1152-1159. (3) Gugger, M.; Reubi, J. C. Gastrin-Releasing Peptide Receptors in Non-Neoplastic and Neoplastic Human Breast. Am J Pathol 1999, 155, 2067-2076. (4) Reubi, J. C.; Wenger, S.; Schmuckli-Maurer, J.; Schaer, J.-C.; Gugger, M. Bombesin Receptor Subtypes in Human Cancers Detection with the Universal Radioligand

125

I-[D-Tyr6, β-

Ala11, Phe13, Nle14] Bombesin(6–14). Clin Cancer Res 2002, 8, 1139-1146.

30


Page 31 of 39

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


(5) Kulhari, H.; Pooja, D.; Shrivastava, S.; V, G. M. N.; Sistla, R. Peptide Conjugated Polymeric Nanoparticles as a Carrier for Targeted Delivery of Docetaxel. Colloids Surf. B 2014, 117, 166-173. (6) Hu, K.; Wang, H.; Tang, G.; Huang, T.; Tang, X.; Liang, X.; Yao, S.; Nie, D. In Vivo Cancer Dual-Targeting and Dual-Modality Imaging with Functionalized Quantum Dots. J Nucl Med 2015, 56, 1278-1284. (7) Nagy, A.; Armatis, P.; Cai, R. Z.; Szepeshazi, K.; Halmos, G.; Schally, A. V. Design, Synthesis, and in Vitro Evaluation of Cytotoxic Analogs of Bombesin-Like Peptides Containing Doxorubicin or Its Intensely Potent Derivative, 2-Pyrrolinodoxorubicin. Proc. Natl. Acad. Sci. U. S. A. 1997, 94, 652-656. (8) Moody, T. W.; Mantey, S. A.; Pradhan, T. K.; Schumann, M.; Nakagawa, T.; Martinez, A.; Fuselier, J.; Coy, D. H.; Jensen, R. T. Development of High Affinity Camptothecin-Bombesin Conjugates That Have Targeted Cytotoxicity for Bombesin Receptor-Containing Tumor Cells. J. Biol. Chem. 2004, 279, 23580-23589. (9) Ranyuk, E.; Cauchon, N.; Klarskov, K.; Guérin, B.; van Lier, J. E. Phthalocyanine–Peptide Conjugates: Receptor-Targeting Bifunctional Agents for Imaging and Photodynamic Therapy. J. Med. Chem. 2013, 56, 1520-1534. (10) Shariati, F.; Aryana, K.; Fattahi, A.; Forghani, M. N.; Azarian, A.; Zakavi, S. R.; Sadeghi, R.; Ayati, N.; Sadri, K. Diagnostic Value of

99m

Tc-Bombesin Scintigraphy for Differentiation of

Malignant from Benign Breast Lesions. Nucl Med Commun 2014, 35, 620-625.

31



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 39

(11) Van de Wiele, C.; Phonteyne, P.; Pauwels, P.; Goethals, I.; Van den Broecke, R.; Cocquyt, V.; Dierckx, R. A. Gastrin-Releasing Peptide Receptor Imaging in Human Breast Carcinoma Versus Immunohistochemistry. J Nucl Med 2008, 49, 260-264. (12) 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, 1-8. (13) Roivainen, A.; Kähkönen, E.; Luoto, P.; Borkowski, S.; Hofmann, B.; Jambor, I.; Lehtiö, K.; Rantala, T.; Rottmann, A.; Sipilä, H.; Sparks, R.; Suilamo, S.; Tolvanen, T.; Valencia, R.; Minn, H. Plasma Pharmacokinetics, Whole-Body Distribution, Metabolism, and Radiation Dosimetry of

68

Ga Bombesin Antagonist BAY 86-7548 in Healthy Men. J Nucl Med 2013, 54,

867-872. (14) 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. Nuc. Med. Mol. Imaging 2000, 27, 1694-1699. (15) Scopinaro, F.; De Vincentis, G.; Varvarigou, A. D.; Laurenti, C.; Iori, F.; Remediani, S.; Chiarini, S.; Stella, S.

99m

Tc-Bombesin Detects Prostate Cancer and Invasion of Pelvic Lymph

Nodes. Eur. J. Nuc. Med. Mol. Imaging 2003, 30, 1378-1382. (16) Bodei, L.; Ferrari, M.; Nunn, A.; Llull, J.; Cremonesi, M.; Martano, L.; Laurora, G.; Scardino, E.; Tiberini, S.; Bufi, G.; Eaton, S.; de Cobelli, O.; Paganelli, G. Lu-177-AMBA Bombesin Analogue in Hormone Refractory Prostate Cancer Patients: A Phase I Escalation Study with Single-Cycle Administrations. Eur. J. Nuc. Med. Mol. Imaging 2007, 34, S221-S221. 32


Page 33 of 39

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


(17) Linder, K. E.; Metcalfe, E.; Arunachalam, T.; Chen, J. Q.; Eaton, S. M.; Feng, W. W.; Fan, H.; Raju, N.; Cagnolini, A.; Lantry, L. E.; Nunn, A. D.; Swenson, R. E. In Vitro and in Vivo Metabolism of Lu-AMBA, a GRP-Receptor Binding Compound, and the Synthesis and Characterization of Its Metabolites. Bioconjugate Chem. 2009, 20, 1171-1178. (18) 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. Nuc. Med. Biol. 2007, 34, 17-28. (19) Zhang, H.; Chen, J.; 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 Indium-111, Lutetium-177, and Yttrium-90 for Targeting Bombesin ReceptorExpressing Tumors. Cancer Res. 2004, 64, 6707-6715. (20) Visser, M.; Bernard, H. F.; Erion, J. L.; Schmidt, M. A.; Srinivasan, A.; Waser, B.; Reubi, J. C.; Krenning, E. P.; Jong, M. Novel

111

In-Labelled Bombesin Analogues for Molecular

Imaging of Prostate Tumours. Eur. J. Nuc. Med. Mol. Imaging 2007, 34, 1228-1238. (21) 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

99m

Tc: A

Preclinical Study. J. Med. Chem. 2005, 48, 100-110. (22) Valverde, I. E.; Bauman, A.; Kluba, C. A.; Vomstein, S.; Walter, M. A.; Mindt, T. L. 1,2,3-Triazoles

as

Amide

Bond

Mimics:

Triazole

Scan

Yields

Protease-Resistant

Peptidomimetics for Tumor Targeting. Angew. Chem. Int. Ed. 2013, 52, 8957-8960.

33



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 39

(23) Valverde, I. E.; Vomstein, S.; Fischer, C. A.; Mascarin, A.; Mindt, T. L. Probing the Backbone Function of Tumor Targeting Peptides by an Amide-to-Triazole Substitution Strategy. J. Med. Chem. 2015, 58, 7475-7484. (24) Valverde, I. E.; Huxol, E.; Mindt, T. L. Radiolabeled Antagonistic Bombesin Peptidomimetics for Tumor Targeting. J. Label Compd. Radiopharm 2014, 57, 275-278. (25) Mascarin, A.; Valverde, I. E.; Vomstein, S.; Mindt, T. L. 1,2,3-Triazole Stabilized Neurotensin-Based Radiopeptidomimetics for Improved Tumor Targeting. Bioconjugate Chem. 2015, 26, 2143-2152. (26) Schroeder, R. P. J.; Müller, C.; Reneman, S.; Melis, M. L.; Breeman, W. A. P.; Blois, E.; Bangma, C. H.; Krenning, E. P.; Weerden, W. M.; Jong, M. A Standardised Study to Compare Prostate Cancer Targeting Efficacy of Five Radiolabelled Bombesin Analogues. Eur. J. Nuc. Med. Mol. Imaging 2010, 37, 1386-1396. (27) 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. Nuc. Med. Mol. Imaging 2007, 34, 1198-1208. (28) Wild, D.; Frischknecht, M.; Zhang, H.; Morgenstern, A.; Bruchertseifer, F.; Boisclair, J.; Provencher-Bolliger, A.; Reubi, J.-C.; Maecke, H. R. Alpha- Versus Beta-Particle Radiopeptide Therapy in a Human Prostate Cancer Model (213Bi-DOTA-PESIN and 177

213

Bi-AMBA Versus

Lu-DOTA-PESIN). Cancer Res. 2011, 71, 1009-1018.

(29) Lantry, L. E.; Cappelletti, E.; Maddalena, M. E.; Fox, J. S.; Feng, W.; Chen, J.; Thomas, R.; Eaton, S. M.; Bogdan, N. J.; Arunachalam, T.; Reubi, J. C.; Raju, N.; Metcalfe, E. C.; 34


Page 35 of 39

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


Lattuada, L.; Linder, K. E.; Swenson, R. E.; Tweedle, M. F.; Nunn, A. D. Synthesis and Characterization of a Selective

177

177

Lu-AMBA:

Lu-Labeled GRP-R Agonist for Systemic

Radiotherapy of Prostate Cancer. J Nucl Med 2006, 47, 1144-1152. (30) Goddard-Borger, E. D.; Stick, R. V. An Efficient, Inexpensive, and Shelf-Stable Diazotransfer Reagent: Imidazole-1-Sulfonyl Azide Hydrochloride. Org. Lett. 2007, 9, 37973800. (31) Hansen, M. B.; van Gurp, T. H. M.; van Hest, J. C. M.; Löwik, D. W. P. M. Simple and Efficient Solid-Phase Preparation of Azido-Peptides. Org. Lett. 2012, 14, 2330-2333. (32) Wang, X.; Fani, M.; Schulz, S.; Rivier, J.; Reubi, J. C.; Maecke, H. R. Comprehensive Evaluation of a Somatostatin-Based Radiolabelled Antagonist for Diagnostic Imaging and Radionuclide Therapy. Eur. J. Nuc. Med. Mol. Imaging 2012, 39, 1876-1885. (33) Maina, T.; Nock, B. A.; Zhang, H. W.; Nikolopoulou, A.; Waser, B.; Reubi, J. C.; Maecke, H. R. Species Differences of Bombesin Analog Interactions with GRP-R Define the Choice of Animal Models in the Development of GRP-R-Targeting Drugs. J Nucl Med 2005, 46, 823-830. (34) Uehara, H.; González, N.; Sancho, V.; Mantey, S. A.; Nuche-Berenguer, B.; Pradhan, T.; Coy, D. H.; Jensen, R. T. Pharmacology and Selectivity of Various Natural and Synthetic Bombesin Related Peptide Agonists for Human and Rat Bombesin Receptors Differs. Peptides 2011, 32, 1685-1699. (35) de Jong, M.; Maina, T. Of Mice and Humans: Are They the Same?--Implications in Cancer Translational Research. J Nucl Med 2010, 51, 501-504.

35



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 36 of 39

(36) 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, 79-134. (37) Mansi, R.; Wang, X.; Forrer, F.; Kneifel, S.; Tamma, M.-L.; Waser, B.; Cescato, R.; Reubi, J. C.; Maecke, H. R. Evaluation of a 1,4,7,10-Tetraazacyclododecane-1,4,7,10-Tetraacetic Acid-Conjugated Bombesin-Based Radioantagonist for the Labeling with Single-Photon Emission

Computed

Tomography,

Positron

Emission

Tomography,

and

Therapeutic

Radionuclides. Clin Cancer Res 2009, 15, 5240-5249. (38) Good, S.; Walter, M. A.; Waser, B.; Wang, X. J.; Muller-Brand, J.; Behe, M. P.; Reubi, J. C.; Maecke, H. R. Macrocyclic Chelator-Coupled Gastrin-Based Radiopharmaceuticals for Targeting of Gastrin Receptor-Expressing Tumours. Eur. J. Nuc. Med. Mol. Imaging 2008, 35, 1868-1877. (39) Zhang, H.; Abiraj, K.; Thorek, D. L.; Waser, B.; Smith-Jones, P. M.; Honer, M.; Reubi, J. C.; Maecke, H. R. Evolution of Bombesin Conjugates for Targeted PET Imaging of Tumors. PloS one 2012, 7, e44046. (40) Chen, J.; Linder, K. E.; Cagnolini, A.; Metcalfe, E.; Raju, N.; Tweedle, M. F.; Swenson, R. E. Synthesis, Stabilization and Formulation of [177Lu]Lu-AMBA, a Systemic Radiotherapeutic Agent for Gastrin Releasing Peptide Receptor Positive Tumors. Appl Radiat Isot 2008, 66, 497505. (41) Vigna, S. R.; Giraud, A. S.; Reeve, J. R., Jr.; Walsh, J. H. Biological Activity of Oxidized and Reduced Iodinated Bombesins. Peptides 1988, 9, 923-926. (42) Mantey, S. A.; Weber, H. C.; Sainz, E.; Akeson, M.; Ryan, R. R.; Pradhan, T. K.; Searles, R. P.; Spindel, E. R.; Battey, J. F.; Coy, D. H.; Jensen, R. T. Discovery of a High Affinity 36


Page 37 of 39

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


Radioligand for the Human Orphan Receptor, Bombesin Receptor Subtype 3, Which Demonstrates That It Has a Unique Pharmacology Compared with Other Mammalian Bombesin Receptors. J. Biol. Chem. 1997, 272, 26062-26071. (43) Coy, D. H.; Heinz-Erian, P.; Jiang, N. Y.; Sasaki, Y.; Taylor, J.; Moreau, J. P.; Wolfrey, W. T.; Gardner, J. D.; Jensen, R. T. Probing Peptide Backbone Function in Bombesin. A Reduced Peptide Bond Analogue with Potent and Specific Receptor Antagonist Activity. J. Biol. Chem. 1988, 263, 5056-5060. (44) Coy, D. H.; Jiang, N. Y.; Kim, S. H.; Moreau, J. P.; Lin, J. T.; Frucht, H.; Qian, J. M.; Wang, L. W.; Jensen, R. T. Covalently Cyclized Agonist and Antagonist Analogues of Bombesin and Related Peptides. J. Biol. Chem. 1991, 266, 16441-16447. (45) 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. (46) Mantey, S. A.; Coy, D. H.; Pradhan, T. K.; Igarashi, H.; Rizo, I. M.; Shen, L.; Hou, W.; Hocart, S. J.; Jensen, R. T. Rational Design of a Peptide Agonist That Interacts Selectively with the Orphan Receptor, Bombesin Receptor Subtype 3. J. Biol. Chem. 2001, 276, 9219-9229. (47) Mantey, S. A.; Gonzalez, N.; Schumann, M.; Pradhan, T. K.; Shen, L.; Coy, D. H.; Jensen, R. T. Identification of Bombesin Receptor Subtype-Specific Ligands: Effect of N-Methyl Scanning, Truncation, Substitution, and Evaluation of Putative Reported Selective Ligands. J. Pharmacol. Exp. Ther. 2006, 319, 980-989. (48) Valverde, I. E.; Mindt, T. L. 1,2,3-Triazoles as Amide-Bond Surrogates in Peptidomimetics. Chimia 2013, 67, 262-266. 37



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 38 of 39

(49) Valverde, I. E.; Lecaille, F.; Lalmanach, G.; Aucagne, V.; Delmas, A. F. Synthesis of a Biologically Active Triazole-Containing Analogue of Cystatin a through Successive Peptidomimetic Alkyne–Azide Ligations. Angew. Chem. Int. Ed. 2012, 51, 718-722. (50) Aucagne, V.; Valverde, I. E.; Marceau, P.; Galibert, M.; Dendane, N.; Delmas, A. F. Towards the Simplification of Protein Synthesis: Iterative Solid-Supported Ligations with Concomitant Purifications. Angew. Chem. Int. Ed. 2012, 51, 11320-11324. (51) Beltramo, M.; Robert, V.; Galibert, M.; Madinier, J. B.; Marceau, P.; Dardente, H.; Decourt, C.; De Roux, N.; Lomet, D.; Delmas, A. F.; Caraty, A.; Aucagne, V. Rational Design of Triazololipopeptides Analogs of Kisspeptin Inducing a Long-Lasting Increase of Gonadotropins. J Med Chem 2015, 58, 3459-3470. (52) Tischler, M.; Nasu, D.; Empting, M.; Schmelz, S.; Heinz, D. W.; Rottmann, P.; Kolmar, H.; Buntkowsky, G.; Tietze, D.; Avrutina, O. Braces for the Peptide Backbone: Insights into Structure–Activity Relationships

of

Protease

Inhibitor

Mimics

with

Locked

Amide

Conformations. Angew. Chem. Int. Ed. 2012, 51, 3708-3712. (53) Pedersen, D. S.; Abell, A. 1,2,3‐Triazoles in Peptidomimetic Chemistry. Eur. J. Org. Chem. 2011, 2011, 2399-2411. (54) Darker, J. G.; Brough, S. J.; Heath, J.; Smart, D. Discovery of Potent and Selective Peptide Agonists at the GRP-Preferring Bombesin Receptor (BB2). J. Pept. Sci. 2001, 7, 598-605.

38


Page 39 of 39

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


Table of Contents Graphic

39


Toward the Optimization of Bombesin-Based Radiotracers for Tumor

Recommend Documents