18F- and 68Ga-Labeled Neurotensin Peptides for PET Imaging of

Subscriber access provided by Caltech Library Services

Article 18

68

F- and Ga-labeled Neurotensin Peptides for PET Imaging of Neurotensin Receptor 1 Simone Maschauer, Juergen Einsiedel, Harald Hübner, Peter Gmeiner, and Olaf Prante J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b00675 • Publication Date (Web): 23 Jun 2016 Downloaded from http://pubs.acs.org on June 24, 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 41

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

18

F- and 68Ga-labeled Neurotensin Peptides for PET Imaging of Neurotensin Receptor 1

Simone Maschauer,‡ Jürgen Einsiedel,⊥ Harald Hübner,⊥ Peter Gmeiner,⊥ Olaf Prante‡,* ‡

Department of Nuclear Medicine, Molecular Imaging and Radiochemistry, Friedrich-

Alexander University (FAU), Schwabachanlage 6, 91054 Erlangen, Germany, ⊥

Department of Chemistry and Pharmacy, Medicinal Chemistry, Emil Fischer Center,

Friedrich-Alexander University (FAU), Schuhstraße 19, 91052 Erlangen, Germany.

Title running head: 18F- and 68Ga-labeled Neurotensin-Peptides

*

Corresponding author: Prof. Olaf Prante, Molecular Imaging and Radiochemistry, Nuclear

Medicine Clinic, Friedrich-Alexander University (FAU), Schwabachanlage 6, D-91054 Erlangen,

Germany.

Tel:

+49-9131-8544440;

Fax:

+49-9131-8539288,

[email protected].

ACS Paragon Plus Environment

E-mail:


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

ABSTRACT The neurotensin (NT) receptor-1 (NTS1) is overexpressed in a variety of carcinomas and is therefore an interesting target for imaging with positron emission tomography (PET). The aim of this study was the development of new NT derivatives based on the metabolically stable peptide sequence NLys-Lys-Pro-Tyr-Tle-Leu suitable for PET imaging. The NT peptides were synthesized by SPPS and elongated with respective chelators (NODA-GA, DOTA) for 68

Ga-labeling or propargylglycine for

18

F-labeling via copper-catalyzed azide-alkyne

cycloaddition (CuAAC). Receptor affinities of the peptides for NTS1 were in the range of 19110 nM. Biodistribution studies using HT29 tumor-bearing mice showed highest tumor uptake for [68Ga]6 and [68Ga]8 and specific binding in small animal PET studies. The tumor uptake of 68Ga-labeled peptides in vivo significantly correlated with the in vitro Ki values for NTS1. [68Ga]8 displayed an excellent tumor-to-background ratio and could therefore be considered as an appropriate molecular probe for NTS1 imaging by PET.

Keywords: fluorine-18, gallium-68, neurotensin, neurotensin receptor, positron emission tomography.


Page 2 of 41

Page 3 of 41

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


Introduction The G-protein-coupled neurotensin (NT) receptor consists of seven-transmembrane domains in two subtypes: the high-affinity neurotensin receptor 1 (NTS1) and the low affinity neurotensin receptor 2 (NTS2). The NTS1 has been described to be highly expressed in 75% of primary pancreatic ductal adenocarcinomas as demonstrated by the pioneering work of Reubi et al. using autoradiography.1 Moreover, NTS1 overexpression has been shown in various carcinomas, e.g. prostate, mamma, lung and colon carcinoma,1-5 whereas they show negligible expression in healthy tissues where these tumors arise from.1, 6, 7 In addition, the proliferative effect of neurotensin in such tumors has been shown to be mediated by NTS1.8, 9 The NTS2 has been described to be expressed in prostate carcinomas cells,4 however, until now, literature data on the expression of NTS2 in tumors is scarce. Therefore, especially the NTS1 represents an interesting target for the design of radiolabeled ligands for imaging of NTS1-positive tumors by positron emission tomography (PET) or for NTS1-mediated endoradiotherapy. The endogenous ligand for NT receptors is the 13 amino acids containing peptide NT, which is widely distributed in the brain and in the gut acting as modulator for actions on the nervous, endocrine, reticuloendothelial and gastrointestinal systems.10 The biologically active Cterminal part NT(8-13) with the sequence Arg8-Arg9-Pro10-Tyr11-Ile12-Leu13 is rapidly degraded by endogenous peptidases in vivo, attacking the bond between the amino acids Arg8Arg9, Pro10-Tyr11 and Tyr11-Ile12.11 In order to enhance metabolic stability while preserving affinity of the molecule for NT receptors, a large variety of studies aimed at the modification of the amino acid sequence of NT(8–13), e.g. by the introduction of non-natural amino acids or by variations of the amino bonds.12-23 In principle, PET allows non-invasive in vivo imaging of receptors expressed on tumors with excellent sensitivity and precise quantification of receptor densities and therefore PET is a highly powerful imaging modality in nuclear medicine, provided that suitable radioligands for 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 4 of 41

in vivo use are available.24 Most of the previously developed radiolabeled NT derivatives were labeled with nuclides suitable for single photon emission tomography (SPECT) or radiotherapy,25-27 however, only a limited number of NT derivatives were radiolabeled with an isotope suitable for PET imaging studies, such as fluorine-18 or gallium-68.20, 28-32 The first NT derivative which was labeled with fluorine-18 and tested in vivo was published by Bergmann et al 2002.28 In that study 4-([18F]fluoro)benzoyl-neurotensin(8–13) (18FB-Arg8Arg9-Pro10-Tyr11- Ile12-Leu13-OH) and two stabilized analogs (18FB-Arg8ψ(CH2NH)Arg9Pro10-Tyr11- Ile12-Leu13-OH and 18FB-Arg8ψ(CH2NH)Arg9-Pro10-Tyr11-Tle12-Leu13-OH) were synthesized and characterized in vitro and in vivo. Despite modification of the amino acid sequence, these radioligands showed only low uptake in HT29 tumors, which was not sufficient for tumor imaging, presumably due to rapid degradation and fast elimination by the kidneys. In another study, NT peptides with C- and/or N-terminal β-amino acid residues for metabolic stabilization were synthesized and labeled with gallium-68.20 Again, fast degradation occurred in vivo and only low specific uptake in HT29 tumors was observed, making these radioligands unsuitable for PET imaging. The group of Shively et al. developed a NT derivative (18F-DEG-VS-NT) which was labeled with

18

F-DEG-VS (18F-(2-(2-(2-

fluoroethoxy)ethoxy)ethylsulfonyl)ethane) as prosthetic group.31 This prosthetic group was prepared in 35% isolated radiochemical yield (decay-corrected) and after coupling to the Cysbearing NT peptide the end product was isolated in 31% yield.33 Their NT derivative allowed successful PET imaging of HT29 tumor bearing mice, however, it showed several metabolites in vivo.31 So far, only one imaging study is dealing with the development of an NTS2 selective radioligand for PET imaging.32 The shift from NTS1 to preferred NTS2 affinity was achieved by exchange of Tyr11 to N-homo-Tyr11 in the amino acid sequence, a strategy which was successfully demonstrated previously for a series of NTS2 subtype-selective peptides.34 The 18

F-fluoroglycoslated NT analog displayed high affinity towards NTS2 (7 nM, ACS Paragon Plus Environment

Page 5 of 41

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


NTS1/NTS2=260), however, it was fastly degraded in the blood and therefore displayed relatively low specific tumor uptake in vivo.32 Taken together, all these published radiopeptide ligands for the neurotensin receptor do not demonstrate sufficient properties for in-vivo use, due to their fast metabolization or low tumor uptake. In our previous work,29,

30

on the one hand, we developed the first

68

Ga-labeled NT

([68Ga]2)30 derivative by attaching the DOTA chelator directly to the N-terminus of the amino acid sequence NLys8-Lys9-Pro10-Tyr11-Tle12-Leu13 that has a proven metabolic stability as previously demonstrated.29,

30

Unfortunately, this modification led to a significant loss of

affinity to NTS1 (Ki = 180 nM). On the other hand, we developed a 18F-glycosyl labeled NT peptoid, that showed promising tumor uptake in HT29 tumor-bearing nude mice. However, the 2-deoxy-2-[18F]fluoroglucosyl moiety of this candidate led to a very high kidney uptake that needs to be lowered to reduce difficulties in PET imaging studies of peripheral tumors in the abdominal regions. Consequently, it is our intention here to design and develop a new series of NT derivatives based on the metabolically stable peptide sequence NLys8-Lys9-Pro10-Tyr11-Tle12-Leu13. First, we studied the effect of the chelator distance from the binding sequence on the binding affinity by the use of different linkers in our search for a

68

Ga-labeled NT analog with

improved binding affinity. Second, starting from the previously published alkyne 13 (NT4),29 we aimed at the syntheses of the 6-deoxy-6-[18F]fluoroglucosyl, the 18F-maltosyl and one less hydrophilic NT analog to study their biodistribution, assuming that these candidates could demonstrate lower kidney uptake or improved kidney clearance together with sufficient tumor uptake. We evaluated the NTS1 and NTS2 binding affinities of the new series of gallium and fluoro substituted peptides and studied their radiopharmaceutical properties in vitro and in vivo using HT29 tumor-bearing nude mice and small animal PET. The most suitable radiopeptide from 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 6 of 41

this series could be a candidate PET ligand for imaging of NTS1-positive tumors and for planning and monitoring of NTS1-targeted 177Lu-radiotherapy.

Results and Discussion Chemistry The synthesis of NT derivatives 3, 5 and 7 as well as their Ga-bearing analogs 4, 6 and 8 is depicted in Scheme 1. Solid phase supported peptide synthesis (SPPS) was performed starting from Fmoc-leucinyl loaded Wang resin. Microwave irradiation turned out advantageous for the acceleration of both the Fmoc deprotection and the amino acid coupling procedures. The amino

acids

were

routinely

activated

in

presence

of

(benzotriazol-1-

yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyBOP) and 1-hydroxybenzotriazole (HOBt). For several couplings to sterically demanding amino acids or for activating costly building blocks, we relied on the more powerful reagent 2-(1H-7-azabenzotriazol-1-yl)1,1,3,3-tetramethyl uronium hexafluorophosphate (HATU) to ensure complete acylation. In detail, the common precursor H-NLys(Boc)-Lys(Boc)-Pro-Tyr(tBu)-Tle-Leu-Wang resin was synthesized by sequentially coupling the amino acids Fmoc-tert-leucine, Fmoc-Tyr(tBu)-OH, Fmoc-Pro-OH, Fmoc-Lys(Boc)-OH and N-Fmoc-N-(4-Boc-aminobutyl)-Gly-OH (FmocNLys(Boc)-OH). For the synthesis of peptide 3, we attached the polyethylene glycol (PEG)spacer Fmoc-21-amino-4,7,10,13,16,19-hexaoxaheneicosanoic acid and the gallium chelator building block NODA-GA-tri-t-Bu-ester. Prior to peptide cleavage with TFA, we treated the resin with 10% sulfuric acid in dioxane at 8 °C in order to preclude tert-butylation of the peptide, which can occur in the course of Boc and tert-butyl deprotection.30 Peptide 5 was synthesized by coupling Fmoc-Lys(Alloc)-OH, followed by the cleavage of the Alloc-group with tetrakis(triphenylphosphine)palladium(0) and coupling of DOTA-tri-tBu-ester. The cleavage from the resin was done as described for peptide 3. Elongation with Boc-Lys(Fmoc)OH, the above mentioned PEG spacer and NODA-GA-tri-tBu-ester, followed by the same ACS Paragon Plus Environment

Page 7 of 41

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


two step cleavage protocol, led to the formation of peptide 7. The

nat

GaIII complexes of

compounds 3, 5 and 7 were prepared by incubation of 3, 5 and 7 with aq. Ga(NO3)3 in sodium acetate buffer at pH 4.5 at room temperature for 45 min (for 4 and 8) or 90°C for 15 min (for 6). After preparative HPLC the peptides were obtained in yield of 54-86% in sufficient purity. The glycosylated peptides 10 and 11 were readily obtained by a copper-catalyzed azidealkyne cycloaddition (CuAAC)35, 36 with 6-deoxy-6-fluoro-β-glucosyl azide37 or 6’-deoxy-6’fluoro-β-maltosyl azide,37 respectively. Furthermore, the cycloaddition of 13 with 3-(4azidophenoxy)propyl fluoride38 resulted in the formation of peptide 12. The CuSO4 / sodium ascorbate promoting system35 proved advantageous for fast reactions and good yields.

In vitro studies Ki values for NTS1 and NTS2 were determined for all synthesized NT-peptides and are listed in Table 1. Interestingly, the NODA-GA- and DOTA-peptides with free chelator (compounds 3, 5 and 7) revealed significantly lower affinities for NTS1 than their Ga-bearing analogs by a factor of 2.5 – 17. This fact was also observed in our previous study for peptide 1,30 and may be explained by 1) the different chemical structures, 2) the different hydrophilicities, and 3) the different overall charges and charge distribution induced by the complexation of gallium. A similar effect was reported for a CXCR4 radioligand, which showed an increase in CXCR4 affinity by a factor of 35 for the gallium compound compared to the uncomplexed form,39 and also for a series of DOTA-octreotides/-octreotates.40 All peptides, except 5 and 6, showed a considerably higher affinity for NTS1 than for NTS2 by a factor of 2-35. Obviously, the free N-terminus in 6 and 8 lead to significantly higher affinities for NTS1 (19-20 nM) and NTS2 (22-87 nM) compared to 2 and 4 (110-3800 nM), which were derivatized at the N-terminus, whereas the PEG-linker and the type of the chelator (DOTA or NODA-GA) did not significantly affect the affinity to NTS1. The fluoro substituted NT-peptides 9, 10, 11 and 12 also showed good to moderate NTS1 affinity (22-71 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 8 of 41

nM) and selectivity (NTS2/NTS1 = 1.4 – 19) with slightly higher affinities for the fluoroglycosylated derivatives 9, 10, 11 compared to 12.

Radiosyntheses The radiosyntheses of the 68Ga-labeled peptides [68Ga]4, [68Ga]6 and [68Ga]8 was performed as depicted in Scheme 1. They were successfully achieved using standard labeling procedures, i.e. NODA-GA linked peptides 3 and 7 were 68Ga-labeled with [68Ga]GaCl3 in sodium acetate buffer at room temperature and pH 4-4.5 and the DOTA-peptide 5 was HEPES buffer at 98°C and pH 3.5-4.0. The RCY for all

68

68

Ga-labeled in

Ga-labeled NT-peptides were

>95%, so that these peptides were used for further evaluation without further purification. The

18

F-fluoroglycosylated NT-peptides [18F]10 and [18F]11 were synthesized by following

our previously described methodology29, 37 in good overall yields (referred to [18F]fluoride, nondecay-corrected) of 16-21% after a total synthesis time of 80-85 min (Scheme 3, Table 2). The copper(I)-catalyzed alkyne-azide cycloaddition (CuAAC) of deacetylated

18

F-glycosyl

azides [18F]14 and [18F]15 proceeded in high RCY of 70-80% during 10 min, applying a mixture of Cu(OAc)2 and sodium ascorbate in aqueous NaCl and a minimum amount of the precursor peptide 13 (100 µg, 0.3 mM). In contrast to the previously published procedure for the synthesis of [18F]9,29 the HPLC separation of the 18F-fluoroglycosylated products [18F]10 and [18F]11 from their respective precursor 13 was not possible. To overcome this obstacle, an additional reaction step with per-acetylated glucopyranosyl azide was performed as described recently for the radiosynthesis of an 18F-labeled NTS-2-radioligand,32 to completely scavenge the alkyne precursor 13. Following this strategy, the amount of 13 as an impurity in the fraction of the final radiopeptide product was reduced to 5-10 nmol, leading to an acceptable specific activity of the final radiopeptide of 2-21 GBq/µmol (Table 2). After HPLC separation [18F]10 and [18F]11 were obtained in high radiochemical purities of >99%. For the radiosynthesis of [18F]12 3-(4-azidophenoxy)propyl methansulfonate41 was applied as ACS Paragon Plus Environment

Page 9 of 41

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


labeling precursor (Scheme 1). The intermediate product 3-(4-azidophenoxy)propyl [18F]fluoride ([18F]16) was obtained in high RCY of 80±3 % and was isolated by radio-HPLC (Scheme 2). The CuAAC of [18F]16 and 13 (100 µg, 0.55 mM) proceeded in a mixture of PBS/ethanol, CuSO4 and sodium ascorbate at 60 °C, providing [18F]12 in excellent RCY of >90 %. [18F]12 was isolated by semipreparative HPLC and obtained in high radiochemical purity of >99% after a total synthesis time 70-75 min with specific activities of 10-44 GBq/µmol (Table 2).

Stability, logD7.4 and cell uptake studies After the successful radiosyntheses, we studied the stability of the 18F- and 68Ga-labeled NTpeptides in human serum in vitro at 37 °C. The HPLC data indicated high stability of at least 93% after an incubation time of 60 min for all tested compounds (Table 2), which is in accordance with our previously published peptides [18F]9 and [68Ga]2 bearing the same peptide sequence.29, 30 The highest stability demonstrated the 18F-fluoroglycosylated peptides [18F]10 and [18F]11 as well as the NODA-GA-peptide [68Ga]8 that showed no degradation products at all after 60 min. Since previously published peptides [18F]9 and [68Ga]2 showed high stability not only in vitro but also in vivo, it is likely that the peptides in this study are equally stable in vivo. The lipophilicity properties were determined by the n-octanol/PBS distribution method; the logD7.4 values measured by this way were -4.0 to -4.2 for the 68Ga-labeled peptides and -2.1 for [18F]12 (Table 2). Internalization studies were performed in vitro using human NTS1 expressing HT29 cells, with a special focus on studying the effect of the linker on the internalization rate of the 68Ga-labeled peptides. The internalization rate of the peptide/NTS1 complex into HT29 cells was lowest for [68Ga]4 with an internalization of 36% after 30 min and much higher for [68Ga]6, [68Ga]8 and [18F]12 with values of 63-78% (Table 2, Figure 1A). The lower internalization rate of [68Ga]4 may be explained by low affinity of only 110 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 10 of 41

nM for NTS1 of this peptide. Efflux rates were determined for [68Ga]6 and [68Ga]8 using HT29 cells after 30 min of internalization, revealing slow efflux for both peptides with 65 – 80% still internalized [68Ga]6 and [68Ga]8 after 60 min (Figure 1B).

Biodistribution studies Since [68Ga]4 and [18F]12 demonstrated the lowest NTS1 affinities, these radiopeptides were not further evaluated in biodistribution studies. The biodistribution studies of [68Ga]6, [68Ga]8, [18F]10 and [18F]11 were evaluated in HT29 tumor-bearing nude mice at 10 min, 30 min and 60 min p.i (Table 3 and 4, Figure 2). The 68Ga-labeled peptides [68Ga]6 and [68Ga]8 showed higher tumor uptake at 60 min p.i. (1.40 and 1.55 %ID/g, respectively) than

18

F-

fluoroglycosylated peptides [18F]10 and [18F]11 (0.75 and 1.02 %ID/g, respectively; Figure 2B), whereas the highest tumor retention from 30 min to 60 min was observed for [68Ga]8 (84%) and [18F]10 (83%). Besides the uptake in tumor, all peptides displayed considerably high uptake in the kidneys with values of 35-45 %ID/g for [68Ga]6 and [68Ga]8 and with values of 17-19 %ID/g about half as high uptake for [18F]10 and [18F]11, indicating renal excretion of the tracers. In comparison with [18F]9, which displayed high kidney uptake values of >50 %ID/g after 65 min p.i.,29 the positive influence of the 6-fluoroglucosyl ([18F]10) and the 6’-fluoromaltosyl residue ([18F]11) on the kidney clearance rate has been verified once more; a characteristic which we already observed before in the case of glycosylated cyclic RGD peptides.37 Uptake values in all other organs investigated were similar for all labeled peptides used in this study (Figure 2A). Clearance from blood was fast for all tested peptides leading to tumor-to-blood ratios of 6 and 4 for [18F]10 and [18F]11, respectively, and, noteworthy, 9 and 31 for [68Ga]6 and [68Ga]8 at 60 min p.i. (Figure 2C). The tumor-to-blood values were qualitatively confirmed by the tumor-to-muscle ratios (Table 3 and Table 4). To confirm the specificity of the uptake of the radiotracers in the HT29 tumor in vivo, we performed blocking studies by coninjection of an excess of a nonradioactive NTS1 ACS Paragon Plus Environment

Page 11 of 41

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


ligand to saturate NTS1 binding sites. Therefore, the high-affinity NTS1 ligand 13 (2.5 mg/kg) was coinjected together with the radiotracer under study, leading to significantly diminished tumor uptake (0.21-0.45 %ID/g) at 60 min p.i. when compared to animals injected with the radiotracer alone (Figure 2B, Table 3 and 4).

Small animal PET studies Small animal PET studies using HT29 tumor-bearing nude mice were performed with all 68

Ga-labeled NT-peptides and confirmed the specific uptake of [68Ga]4, [68Ga]6 and [68Ga]8

in HT29 tumors in vivo (Figure 3). The standard uptake values (SUV) in the tumor were only in the range of 0.2-0.5, but due to favorable tumor-to-background ratios the HT29 tumors were clearly visualized even by the low-affine NT-peptide [68Ga]4. As shown by the biodistribution studies, nonspecific uptake of [68Ga]6 in the tumor, as determined by the blocking study with 13, was slightly higher compared to [68Ga]4 or [68Ga]8. Including the values for the previously published [68Ga]2, there was a significant correlation of the tumor uptake expressed as SUV for the 68Ga-labeled peptides as measured by PET and the Ki values determined in vitro with a regression factor of r = -0.965 (Spearman correlation, P96%, with the exception of compound 3, which could only be synthesized with a purity of 94.1%) and identites were assessed by analytical HPLC (Agilent 1100 analytical series, equipped with a quarternary pump and variable wavelength detector; column Zorbax Eclipse XDB-C8 analytical column, 4.6 mm × 150 mm, 5 µm, flow rate 0.5 mL/min using solvent systems as specified below) coupled to a ACS Paragon Plus Environment

Page 15 of 41

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


Bruker Esquire 2000 mass detector equipped with an ESI-trap, if not otherwise stated. ESITOF high mass accuracy and resolution experiments were performed on a BRUKER maXis MS (Bruker Daltonics, Bremen) in the laboratories of the Chair of Organic Chemistry (Prof. Dr. Rik Tykwinski), Department of Pharmacy and Chemistry, Friedrich-Alexander University of Erlangen Nuremberg. The purity of key compounds 4, 6, 8, 10, 11, 12 determined by RPHPLC, was >96% throughout. The labeling precursors 2,3,4-tri-O-acetyl-6-O-toluenesulfonyl-β-D-glucopyranosyl azide and 2’,3’,4’,2,3,6-hexa-O-acetyl-6’-O-p-tolylsulfonyl-β-D-maltosyl azide and the glycosyl azides 6-deoxy-6-fluoro-D-glucopyranosyl azide and 6’-deoxy-6’-fluoro-β-maltosyl azide were prepared as published previously.37 The alkyne-bearing NT-peptide 13 and 2-deoxy-2fluoro-glucosylated peptide 9 were prepared as described before.29 Radio-HPLC was performed on an Agilent 1100 system with a quarternary pump and variable wavelength detector and radio-HPLC detector D505TR (Canberra Packard). Computer analysis of the HPLC data was performed using FLO-One software (Canberra Packard). Synthesis of peptide 3. Remark: Since only racemic NODA-GA-tri-tBu-ester is commercially available, peptide 3 was obtained as mixture of the corresponding (S)-and (R)NODA-GA epimers which were not separable by RP-HPLC. The amino acids were incorporated in the following order: Fmoc-Tle-OH (a, acylation completed by a second coupling with aa (amino acid) / HATU / DIPEA 5 eq / 5 eq / 5 eq), Fmoc-Tyr(tBu)-OH (b), Fmoc-Pro-OH (c), Fmoc-Lys(Boc)-OH (d), N-Fmoc-N-(4-Boc-aminobutyl)-Gly-OH (e), Fmoc-21-amino-4,7,10,13,16,19-hexaoxaheneicosanoic acid (f) and NODA-GA-tri-tBu-ester (g). Coupling conditions: aa / PyBOP / DIPEA / HOBt (5 eq / 5 eq / 5 eq / 7.5 eq for a,b,c,d and 4 eq/ 4 eq / 4 eq / 6 eq for f,g); aa / HATU / DIPEA (4 eq / 4 eq / 4 eq for e). Further treatment of the resin was done as described in “General”. Preparative RP-HPLC gradient: CH3OH in H2O (0.1% HCO2H) 10-50% in 16 min (tR: 14.1 min). Analytical HPLC: linear



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

gradient 10–55% CH3OH in H2O (0.1% HCO2H) in 18 min, purity: >99% (tR: 16.2 min). [M+H]+; calcd for C68H117N12O22: 1453.8, found: 1454.2. HR-ESI-TOF: [M+H]+ calcd for C68H117N12O22: 1453.8405, found: 1453.8390. Synthesis of peptide 4. Remark: peptide 4 is obtained as a mixture of the corresponding (S)-and (R)-NODA-GA epimers. To a solution of peptide 3 (4.0 mg, 2.4 µmol) in 0.1 M sodium acetate buffer pH 4.5 (2 mL) a solution of Ga(NO3)3 × H2O (1.9 mg, 7.3 µmol) in H2O (0.1 mL, prepared as a stock solution) was added. After stirring at rt for 45 min, the solution was immediately subjected to preparative HPLC to afford 1.7 mg (54%) 4. Preparative RP-HPLC gradient: CH3OH in H2O (0.1% HCO2H) 10-55% in 18 min (tR: 14.7 min). Analytical HPLC: linear gradient 10–55% CH3OH in H2O (0.1% HCO2H) in 18 min, purity: >99% (tR: 16.6 min). [M+H]+; calcd for C68H114N12O22Ga 1519.7, found: 1520.1. Synthesis of peptide 5. The basic sequence Fmoc-NLys(Boc)-Lys(Boc)-Pro-Tyr(tBu)-TleLeu-Wang resin (0.9 mmol) was synthesized as described for peptide 3. After removing the Fmoc-group, Fmoc-Lys(Alloc)-OH was coupled by using aa / HATU / DIPEA (5 eq / 5 eq / 5 eq) in DMF and the Alloc group was removed by agitating the resin in a solution of tetrakis(triphenylphosphine)palladium(0) (293.2 mg, 0.25 mmol) in CHCl3 / morpholine / CH3CO2H 37:2:1 (7.5 mL) for 2 h at rt. After removing of the solvent, washings with 0.5% DIPEA in DMF (5×), 0.5% sodium diethyldithiocarbamate (5×) and DMF (5×) were performed. DOTA-tri-tBu-ester was coupled employing aa / HATU / DIPEA (5 eq / 5 eq/ 5 eq) in DMF and the Fmoc group was removed. Further treatment of the resin was done as described in “General”. Preparative RP-HPLC gradient: CH3OH in H2O (0.1% HCO2H) 1028% in 22 min (tR: 16.3 min). Analytical HPLC: linear gradient 10–40% CH3OH in H2O (0.1% HCO2H) in 18 min, purity: 94.1% (tR: 14.2 min). [M+H]+; calcd for C60H103N14O16: 1275.8, found: 1275.8. HR-ESI-TOF: [M+Na]+ calcd for C60H102N14O16Na 1297.7496, found: 1297.7442.


Page 16 of 41

Page 17 of 41

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


Synthesis of peptide 6. To a solution of peptide 5 (3.0 mg, 2.1 µmol) in 0.1 M sodium acetate buffer pH 4.5 (1.5 mL) a solution of Ga(NO3)3 × H2O (0.68 mg, 2.5 µmol) in H2O (0.1 mL, prepared as a stock solution) was added. After stirring at 90 °C for 15 min, the solution was immediately subjected to preparative HPLC to afford 3.6 mg (86%) 6. Preparative RPHPLC gradient: CH3OH in H2O (0.1% HCO2H) 10-55% in 18 min (tR: 14.8 min). Analytical HPLC: linear gradient 10–40% CH3OH in H2O (0.1% HCO2H) in 18 min, purity: 96.8% (tR: 12.3 min). [M]+; calcd for C60H100N14O16Ga: 1341.7, found: 1341.7. Synthesis of peptide 7. Remark: since only racemic NODA-GA-tri-tBu-ester is commercially available, peptide 7 was obtained as a mixture of the corresponding (S)-and (R)NODA-GA epimers. The basic sequence Fmoc-NLys(Boc)-Lys(Boc)-Pro-Tyr(tBu)-Tle-LeuWang resin (0.9 mmol) was synthesized as described for peptide 3. After removing the Fmocgroup, a double coupling of Boc-Lys(Fmoc)-OH was performed using aa / HATU / DIPEA (5 eq / 5 eq / 5 eq) in DMF, followed by N-ε-Fmoc-deprotection. Fmoc-21-amino4,7,10,13,16,19-hexaoxaheneicosanoic acid and, after a further the Fmoc-deprotection, NODA-GA-tri-tBu-ester was coupled by using aa / PyBOP / DIPEA / HOBt (4 eq / 4 eq / 4 eq / 6 eq) in DMF, respectively. Further treatment of the resin was done as described in “General”. Preparative RP-HPLC gradient: CH3OH in H2O (0.1% HCO2H) 10-40% in 18 min (tR: 16.5 min). Analytical HPLC: linear gradient 10–40% CH3OH in H2O (0.1% HCO2H) in 18 min, then 40%-40% for 5 min, purity: 97.8% (tR: 19.1 min). [M+H]+; calcd for C74H129N14O23: 1581.9, found: 1581.8. Synthesis of peptide 8. Remark: peptide 8 is obtained as a mixture of the corresponding (S)-and (R)-NODA-GA epimer. To a solution of peptide 7 (4.1 mg, 2.3 µmol) in 0.1 M sodium acetate buffer pH 4.5 (2 mL) a solution of Ga(NO3)3 × H2O (1.9 mg, 7.0 mmol) in H2O (0.1 mL, prepared as a stock solution) was added. After stirring at rt for 45 min, the solution was immediately subjected to preparative HPLC to afford 3.4 mg (79%) of peptide 8.



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

Preparative RP-HPLC gradient: CH3CN in H2O (0.1% HCO2H) 3-30% in 26 min (tR: 15.1 min). Analytical HPLC: linear gradient 10–40% CH3OH in H2O (0.1% HCO2H) in 18 min: purity: 97.2% (tR: 18.5 min). [M+H]+; calcd for C74H126N14O23Ga: 1647.8, found: 1647.8. Synthesis of peptide 10. To a solution of 6-deoxy-6-fluoro-β-glucosyl azide37 (4.5 mg, 21.5 µmol) in iPrOH (0.6 mL) subsequently 1329 (9.2 mg, 9.3 µmol) in H2O (0.2 mL) copper(II)sulfate pentahydrate (5.4 mg, 21.5 µmol) in H2O (0.2 mL) and sodium ascorbate (12.8 mg, 64.5 µmol) in H2O (0.2 mL) were added. The mixture was placed in an ultrasonic bath for 1 h. The mixture was diluted with 5% CH3OH in water (0.1% HCO2H) and then preparative RP-HPLC was performed directly to yield 3.5 mg (31%): Preparative RP-HPLC gradient: CH3OH in H2O (0.1% HCO2H) 10-28% in 18 min (tR: 14.1 min). Analytical HPLC: linear gradient 10–40% CH3OH in H2O (0.1% HCO2H) in 18 min: 96.6% (tR: 14.5 min). [M+H]+; calcd for C49H80FN12O13: 1063.6, found: 1063.7. HR-ESI-TOF: [M+H]+ calcd for C49H80FN12O13 1063.5952, found: 1063.5957. Synthesis of peptide 11. To a solution of 6’-deoxy-6’-fluoro-β-maltosyl azide37 (2 mg, 5 µmol) and 1329 (2.5 mg, 2.6 µmol) in EtOH / H2O 1:5 (0.5 mL) a solution of copper(II)acetate (0.1 M, 30 µL) and sodium ascorbate (0.1 M, 90 µL) was added. The mixture was stirred at room temperature for 30 min, then diluted with H2O / CH3CN (9:1, 0.5 mL) and subjected to semipreparative RP-HPLC for purification of the desired product. After lyophilization of the product fraction, the glycopeptide was obtained as white solid in a yield of 75% (1.2 mg). Semipreparative RP-HPLC gradient: Kromasil C8, 125 × 8 mm, 4 mL/min, linear gradient 10-35% CH3CN in H2O (0.1% TFA) in 20 min (tR: 11.5 min). Analytical HPLC: Kromasil C8, 250 × 4.6 mm, 1.5 mL/min, linear gradient 10-50% CH3CN in H2O (0.1% TFA) in 30 min, purity: 96.3% (tR: 13.7 min). [M+H]+; calcd for C55H89FN12O18 1225.7, found: 613.6 ([M+2H]2+). Synthesis of peptide 12. To a solution of 3-(4-azidophenoxy)propyl fluoride38 (5.3 mg, 27.1 µmol) in tBuOH (1.15 mL) and 1329 (12.0 mg, 12.1 µmol) in H2O (0.75 mL) was added ACS Paragon Plus Environment

Page 18 of 41

Page 19 of 41

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


copper(II)sulfate pentahydrate (8.0 mg, 32.0 µmol) and sodium ascorbate (6.4 mg, 32.0 µmol). The mixture was placed in an ultrasonic bath for 5 min and then allowed to stand for 50 min at RT. The mixture was diluted with 5% CH3OH in water (0.1% HCO2H) and then pre-purified by submitting to a C18-cartridge and eluting with 5% CH3OH in water (0.1% HCO2H) (10 mL). After removing the solvent in vacuo, purification was done using preparative RP-HPLC to yield 3.0 mg (21%) of 12: Preparative RP-HPLC gradient: CH3OH in H2O (0.1% HCO2H) 10-65% in 18 min (tR: 12.7 min). Analytical HPLC: linear gradient 10–50% CH3OH in H2O (0.1% HCO2H) in 18 min: 96.9% (tR: 18.3 min). [M+H]+; calcd for C52H80FN12O10: 1051.6, found: 1051.7. Receptor Binding Assays. Receptor binding data were determined according to protocols as described previously.42 In detail, NTS1 binding was measured using homogenates of membranes from CHO cells stably expressing human NTS1 at a final concentration of 2-4 µg/well and the radioligand [3H]neurotensin (specific activity 101 Ci/mmol; PerkinElmer, Rodgau, Germany) at a concentration of 0.30-0.50 nM. Specific binding of the radioligand was determined at KD values of 0.23-0.54 nM and a Bmax of 1200-7000 fmol/mg protein. Nonspecific binding was determined in the presence of 10 µM neurotensin. NTS2 binding was done using homogenates of membranes from HEK 293 cells, which were transiently transfected with the pcDNA3.1 vector containing the human NTS2 gene (Missouri S&T cDNA Resource Center (UMR), Rolla, MO) by the calcium phosphate method.43 Membranes were incubated at a final concentration of 4-6 µg/well together with 0.50 nM of [3H]NT(8-13) (specific activity 136 Ci/mmol; custom synthesis of [leucine-3H]NT(8-13) by GE Healthcare, Freiburg, Germany) at KD values in the range from 0.78-1.4 nM and a Bmax of 700-2400 fmol/mg protein. Nonspecific binding was determined in the presence of 10 µM NT(8-13) and the protein concentration was generally determined by the method of Lowry using bovine serum albumin as standard.44



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 20 of 41

Data analysis of the competition curves from the radioligand binding experiments was accomplished by non-linear regression analysis using the algorithms in PRISM6.0 (GraphPad Software, San Diego, CA). EC50 values derived from the resulting dose response curves were transformed into the corresponding Ki values utilizing the equation of Cheng and Prusoff.45 Radiosynthesis of [68Ga]4 and [68Ga]8. To 7 nmol peptide 3 or 7 in sodium acetate (2.5 M, 170 µl) [68Ga]GaCl3 (in 0.6 M HCl, 400 µl, 200 MBq), freshly eluted from a

68

Ge/68Ga

generator (IDB Holland BV (The Netherlands)/ iThemba LABS (South Africa)) was added, resulting in a final pH of 4-4.5. The solution was incubated at room temperature for 5 min. The radiochemical yield (RCY) was >99% as determined by radio-HPLC (Chromolith RP18e, 10 × 4.6 mm, 10-50% CH3CN in water (0.1% TFA) in a linear gradient over 5 min, 4 mL/min, tR ([68Ga]4) = 2.30 min. Chromolith C8, 10 × 4.6 mm, 10-50% CH3CN in H2O (0.1% TFA) in a linear gradient over 5 min, 4 mL/min, tR ([68Ga]8) = 2.13 min). The resulting solution was neutralized with aqueous NaHCO3 (1 M) and used for all in vitro and in vivo studies without further purification. Radiosynthesis of [68Ga]6. To 20 nmol DOTA-peptide 5 in HEPES buffer (2.5 M, 180 µl) [68Ga]GaCl3 (in 0.6 M HCl, 400 µl, 200 MBq), freshly eluted from a

68

Ge/68Ga generator

(IDB Holland BV (The Netherlands)/ iThemba LABS (South Africa)) was added, resulting in a final pH of 3.5-4. The solution was incubated at 98 °C for 10 min. The RCY was >95% as determined by radio-HPLC (Chromolith RP-18e, 10 × 4.6 mm, 10-50% CH3CN in H2O (0.1% TFA) in a linear gradient over 5 min, 4 mL/min, tR (68Ga-3) = 1.93 min). The resulting solution was neutralized with aqueous NaHCO3 (1 M) and used for all in vitro and in vivo studies without further purification. Production of [18F]fluoride. No-carrier-added (n.c.a.) [18F]fluoride was produced by the 18

O(p,n)18F reaction on

18

O-enriched H2O (>98%, Rotem Ind. LTD) using a proton beam of

11 MeV generated by a RDS 111 cyclotron (Siemens / CTI).


Page 21 of 41

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


Radiosynthesis of [18F]10 and [18F]11. A QMA-cartridge (Sep-Pak® Accell Plus QMA Plus Light Cartridge, 130 mg, Waters) with [18F]fluoride was eluted with a solution of 10 mg Kryptofix® 2.2.2, 0.1 M K2CO3 (17.5 µL) and 0.1 M KH2PO4 (17.5 µL) in 1 mL CH3CN / H2O (8:2). The solution was evaporated using a stream of nitrogen at 85 °C and co-evaporated to dryness with CH3CN (3 × 500 µL). The respective labeling precursor (2,3,4-tri-O-acetyl-6O-toluenesulfonyl-β-D-glucopyranosyl

azide37

or

2’,3’,4’,2,3,6-hexa-O-acetyl-6’-O-p-

tolylsulfonyl-β-D-maltosyl azide,37 15 µmol) in anhydrous CH3CN (450 µL) was added and the solution was stirred for 10 min at 85 °C. The labeling products 2,3,4-tri-O-acetyl-6-deoxy6-[18F]fluoroglucosyl

azide

([18F]14)

or

2’,3’,4’,2,3,6-hexa-O-acetyl-6’-deoxy-6’-

[18F]fluoromaltosyl azide ([18F]15), respectively, were isolated by evaporating the solvent in a stream of nitrogen, dissolving the residue with CH3CN / H2O (30:70, 500 µL) and separation by semipreparative HPLC (Kromasil C8, 125 × 8 mm, 4 mL/min, 30-70% CH3CN in H2O (0.1% TFA) in a linear gradient over 30 min). The product fraction was diluted with H2O (1:10) and transferred to a C18-cartridge (LiChrolut®, Merck, 200 mg), dried in a stream of nitrogen and eluted with ethanol (1 mL). The solvent was evaporated at 60 °C in a stream of nitrogen and subsequently NaOH (250 µL, 60 mM, 10% ethanol) was added. Deacetylation was complete after stirring for 5 min at 60 °C, and the crude product 6-deoxy-6[18F]fluoroglucosyl azide or 6’-deoxy-6’-[18F]fluoromaltosyl azide, respectively, was used for the click chemistry reaction with alkyne 1329 in a one-pot-procedure. This was accomplished by neutralizing the solution with HCl (1 M, 13.5 µL), followed by addition of a mixture of 13 (100 µg in 20 µL H2O), Cu(OAc)2 (0.1 M, 30 µL) and sodium ascorbate (0.1 M, 60 µL). The reaction mixture was stirred for 15 min at 60 °C, 2,3,4,6-tetra-O-acetylglucopyranosyl azide (300 µg in 20 µl DMSO) was added and the mixture was stirred for 10 min at 60 °C. The RCY was 70-80% for [18F]10 and [18F]11 as determined by analytical HPLC from a sample withdrawn from the reaction mixture. tR ([18F]10) = 2.18 min, tR ([18F]11) = 2.07 min (radio-



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 22 of 41

HPLC: Chromolith C8, 10 × 4.6 mm, 10-50% CH3CN in H2O (0.1% TFA) in a linear gradient over 5 min, 4 mL/min). 18F-glycopeptides [18F]10 or [18F]11 were isolated by semipreparative HPLC (Kromasil C8, 125 × 8 mm, 4 mL/min. tR ([18F]10) = 11.0 min, tR ([18F]11) = 10.5 min (10-35% CH3CN in H2O (0.1% TFA) in a linear gradient over 20 min). The product fraction was diluted in H2O, passed through a RP-18 cartridge (LiChrolut®, Merck, 100 mg) and the product was eluted with a solution of ethanol / 0.9% saline (1:1, 1 mL). For in vitro and in vivo experiments the solvent was evaporated in vacuo and the

18

F-glycopeptides were

formulated with 0.9% saline. Starting from [18F]fluoride (500 MBq), this procedure yielded 80-105 MBq (16-21% nondecay-corrected yield) of [18F]10 or [18F]11 in a total synthesis time of 80-85 min. Radiosynthesis of [18F]12. A QMA-cartridge with [18F]fluoride was eluted with a solution of 10 mg Kryptofix® 2.2.2, 0.1 M K2CO3 (17.5 µL) and 0.1 M KH2PO4 (17.5 µL) in 1 mL acetonitrile / H2O (8:2). The solution was evaporated using a stream of nitrogen at 85 °C and co-evaporated to dryness with CH3CN (3 × 500 µL). The labeling precursor (3-(4azidophenoxy)propyl methansulfonate,41 18 µmol) in anhydrous CH3CN (450 µL) was added and the solution was stirred for 5 min at 85 °C. The RCY was determined by radio-TLC from a sample withdrawn from the reaction mixture and was 80±3 %. The labeling product 3-(4azidophenoxy)propyl [18F]fluoride ([18F]16) was isolated by evaporating the solvent in a stream of nitrogen, dissolving the residue with CH3CN / H2O (1:1, 500 µL) and separation by semipreparative HPLC (Kromasil C8, 125 × 8 mm, 4 mL/min, 30-70% CH3CN / H2O (0.1% TFA) 50:50, tR = 9 min). The product fraction was diluted with H2O (1:10) and transferred to a C18-cartridge (LiChrolut®, Merck, 100 mg), dried in a stream of nitrogen and eluted with ethanol (1 mL). The solvent was evaporated at 60 °C in a stream of nitrogen, subsequently a mixture of 1329 (100 µg in 80 µL phosphate buffered saline (PBS, pH 5-6) and 80 µL ethanol), CuSO4 (0.2 M, 10 µL) and sodium ascorbate (0.6 M, 10 µL) was added and the reaction mixture was stirred for 20 min at 60 °C. The RCY of [18F]12 was >90 % as ACS Paragon Plus Environment

Page 23 of 41

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


determined by analytical HPLC from a sample withdrawn from the reaction mixture. tR ([18F]12) = 1.95 ± 0 03 min (radio-HPLC: Chromolith C18, 10 × 4.6 mm, 10-50% CH3CN in H2O (0.1% TFA) in a linear gradient over 5 min, 4 mL/min). [18F]12 was isolated by semipreparative HPLC (Kromasil C8, 125 × 8 mm, 4 mL/min, 20-70% acetonitrile in H2O (0.1% TFA) in a linear gradient over 30 min, tR ([18F]12) = 11 min). The product fraction was diluted in H2O, passed through a RP-18 cartridge (LiChrolut®, Merck, 100 mg) and the product was eluted with a solution of ethanol / 0.9% saline (1:1, 1 mL). For in vitro and in vivo experiments the solvent was evaporated in vacuo and [18F]12 was formulated with 0.9% saline. Starting from [18F]fluoride (500 MBq), this procedure yielded 95-110 MBq (19-22% nondecay-corrected yield) of [18F]12 in a total synthesis time of 70-75 min. Stability in Serum in Vitro. An aliquot of the

68

Ga- and

18

F-labeled peptides in saline

(0.9%) (20 µL) was added to human serum (200 µL) and incubated at 37 °C. Aliquots (25 µL) were taken at various time intervals (5-90 min) and quenched in CH3OH / H2O (1:1, 100 µL). The samples were centrifuged, and the supernatants were analyzed by radio-HPLC. Determination of the Partition Coefficients (log D7.4). The lipophilicity of the radioligands was assessed by determination of the water-octanol partition coefficient as described before.37 Cell Culture. The human NTS1 and NTS2 expressing colon cancer cell line HT29 (ECACC Nº 91072201) was grown in culture medium (McCoy's 5a medium containing glutamine (2 mM) supplemented with fetal bovine serum (FBS, 10%)) at 37 °C in a humidified atmosphere of 5% CO2. Cells were routinely subcultured every 3-4 d. Routine test of the HT29 cells for contamination with mycoplasma were always negative. Internalization and Efflux. Internalization and efflux experiments were conducted using HT29 cells in 24-multiwell plates and 0.5 MBq of each radiotracer as described before.21 The experiments were performed in quadruplicate and were repeated at least twice.



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

Animal Model. All animal experiments were performed in compliance with the protocols approved by the local Animal Protection Authorities (Regierung Mittelfranken, Germany, No. 54-2532.1-15/08). Female athymic NMRI nude mice (nu/nu) were obtained from Harlan Winkelmann GmbH (Borchen, Germany) at 4 weeks of age and were kept under standard conditions (12 h light/dark) with food and water available ad libitum for at least 5 weeks. HT29 cells were harvested and suspended in sterile PBS at a concentration of 1×107 cells/mL. Viable cells (1×106) in PBS (100 µL) were injected subcutaneously in the back. One to two weeks after inoculation the mice, now weighing about 35 g and bearing tumors of 150–500 mg, were used for biodistribution and small-animal PET studies. Biodistribution Studies. HT29 xenografted nude mice were injected with [18F]10, [18F]11, [68Ga]6 or [68Ga]8 into a tail vein (2-8 MBq/mouse). The animals were killed by cervical dislocation 10, 30 or 60 min post-injection (p.i.). The tumors, other tissues (lung, liver, kidneys, heart, spleen, brain, muscle, femur and intestine) and blood were removed and weighed. Radioactivity of the samples was measured using a γ-counter, and expressed as percentage of injected dose per gram of tissue (%ID/g), from which tumor-to-blood ratios were calculated. Blocking experiments were carried out by co-injecting randomly chosen mice with 100 µg 13 (2.5 mg/kg body weight) together with the radiotracer. These mice were killed by cervical dislocation at 60 min p.i. and organs and tissue were removed, weighed and counted as described above. Small-animal PET Imaging. PET scans and image analysis were performed using a smallanimal PET scanner (Inveon, Siemens Medical Solutions). About 3-8 MBq of [68Ga]4, [68Ga]6 and [68Ga]8 were intravenously injected into each mouse (n = 3-4) under isoflurane anesthesia (4%). Animals were subjected to a 15 min static scan starting 45 min p.i. After iterative maximum a posteriori (MAP) image reconstruction of the decay and attenuation corrected images, regions of interest (ROIs) were drawn over the tumor. The radioactivity concentration within the tumor region was obtained from the mean value within the multiple ACS Paragon Plus Environment

Page 24 of 41

Page 25 of 41

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


ROIs and then converted to standard uptake values (SUV) relative to the injected dose and the individual body weight. For receptor-blocking experiments HT29 tumor-bearing mice were scanned for the period 45-60 min p.i. after coinjection with radiotracer containing 13 (2.5 mg/kg).

Supporting Information •

Molecular formula strings (CSV)

Corresponding Author Information Corresponding author: Prof. Olaf Prante, Molecular Imaging and Radiochemistry, Nuclear

Medicine Clinic, Friedrich-Alexander University (FAU), Schwabachanlage 6, D-91054 Erlangen,

Germany.

Tel:

+49-9131-8544440;

Fax:

+49-9131-8539288,

E-mail:

[email protected].

Competing interests The authors declare no competing financial interest.

Authors’ contributions SM designed and coordinated the study, carried out radiosyntheses, in vitro and in vivo experiments, data analyses and interpretations and drafted the manuscript. JE designed and synthesized the NT peptides and contributed to writing of the manuscript. HH supervised the neurotensin receptor binding assays, analyzed the data, and contributed to writing of the manuscript. PG contributed to writing of the manuscript and critically reviewed the manuscript. OP designed and supervised all aspects of the current study and contributed to writing of the manuscript. All authors approved the final manuscript.



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 41

Acknowledgment The authors thank Manuel Geisthoff, Bianca Weigel, Hanna Hübner, Simon Kurzhals, Iris Torres, Dr. Philipp Tripal and Dr. Carsten Hocke for expert technical support. Anthony Budach, Philipp Trautner and Viola Hamann are acknowledged for their skilful work in our laboratories. Furthermore, we thank Prof. Rik Tykwinski, Dr. Frank Hampel and Margarete Dzialach (Department of Chemistry and Pharmacy, Chair of Organic Chemistry, Friedrich Alexander University Erlangen-Nuremberg) for measuring high resolution ESI-TOF mass spectra. This work was supported by the Deutsche Forschungsgemeinschaft (DFG, grants MA 4295/1-2).

Abbreviations Used Alloc, allyloxycarbonyl; CuAAC, copper-catalyzed azide-alkyne cycloaddition; DIPEA, diisopropylethylamine; DOTA, 1,4,7,10-tetraazacyclododecane-N,N′,N′′,N′′′- tetraacetic acid; FBS, fetal bovine serum; HATU, 2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uronium hexafluorophosphate; NODA-GA,

HEPES,

2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic

1-(1,3-carboxypropyl)-4,7-carboxymethyl-1,4,7-triazacyclononane;

acid; NT,

neurotensin; NTS1, neurotensin receptor 1; NTS2, neurotensin receptor 2; p.i., post injection; pipAmGly,

4-piperidinyl(N-amidino)-S-glycine;

PyBOP,

(benzotriazol-1-

yloxy)tripyrrolidinophosphonium hexafluorophosphate; RCY, radiochemical yield; ROI, region of interest; SUV, standard uptake value; SUVmax, maximum standard uptake value; Tle, tert-leucine.

References 1. Reubi, J. C.; Waser, B.; Friess, H.; Buchler, M.; Laissue, J. Neurotensin receptors: a new marker for human ductal pancreatic adenocarcinoma. Gut 1998, 42, 546-550.


Page 27 of 41

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


2. Dupouy, S.; Mourra, N.; Doan, V. K.; Gompel, A.; Alifano, M.; Forgez, P. The potential use of the neurotensin high affinity receptor 1 as a biomarker for cancer progression and as a component of personalized medicine in selective cancers. Biochimie 2011, 93, 1369-1378. 3. Wu, Z.; Martinez-Fong, D.; Tredaniel, J.; Forgez, P. Neurotensin and its high affinity receptor 1 as a potential pharmacological target in cancer therapy. Front. Endocrinol. 2012, 3, 184. 4. Swift, S. L.; Burns, J. E.; Maitland, N. J. Altered expression of neurotensin receptors is associated with the differentiation state of prostate cancer. Cancer Res. 2010, 70, 347-356. 5. Carraway, R. E.; Plona, A. M. Involvement of neurotensin in cancer growth: evidence, mechanisms and development of diagnostic tools. Peptides 2006, 27, 2445-2460. 6. Gromova, P.; Rubin, B. P.; Thys, A.; Erneux, C.; Vanderwinden, J. M. Neurotensin receptor 1 is expressed in gastrointestinal stromal tumors but not in interstitial cells of Cajal. PLoS One 2011, 6, e14710. 7. Maoret, J. J.; Pospai, D.; Rouyer-Fessard, C.; Couvineau, A.; Laboisse, C.; Voisin, T.; Laburthe, M. Neurotensin receptor and its mRNA are expressed in many human colon cancer cell lines but not in normal colonic epithelium: binding studies and RT-PCR experiments. Biochem. Biophys. Res. Commun. 1994, 203, 465-471. 8. Moody, T. W.; Chan, D. C.; Mantey, S. A.; Moreno, P.; Jensen, R. T. SR48692 inhibits nonsmall cell lung cancer proliferation in an EGF receptor-dependent manner. Life Sci. 2014, 100, 25-34. 9. Wang, J. G.; Li, N. N.; Li, H. N.; Cui, L.; Wang, P. Pancreatic cancer bears overexpression of neurotensin and neurotensin receptor subtype-1 and SR 48692 counteracts neurotensin induced cell proliferation in human pancreatic ductal carcinoma cell line PANC-1. Neuropeptides 2011, 45, 151156. 10. Carraway, R.; Reinecke, M. Neurotensin and related peptides. In The Comparative Physiology of Regulatory Peptides, Holmgren, S., Ed. Springer Netherlands: 1989; pp 87-111. 11. Kitabgi, P.; De Nadai, F.; Rovère, C.; Bidard, J.-N. Biosynthesis, maturation, release, and degradation of neurotensin and neuromedin N. Ann. N.Y. Acad. Sci. 1992, 668, 30-42. 12. Buchegger, F.; Bonvin, F.; Kosinski, M.; Schaffland, A. O.; Prior, J.; Reubi, J. C.; Blauenstein, P.; Tourwé, D.; Garayoa, E. G.; Delaloye, A. B. Radiolabeled neurotensin analog, Tc99m-NT-XI, evaluated in ductal pancreatic adenocarcinoma patients. J. Nucl. Med. 2003, 44, 16491654. 13. Garcia-Garayoa, E.; Maes, V.; Blauenstein, P.; Blanc, A.; Hohn, A.; Tourwé, D.; Schubiger, P. A. Double-stabilized neurotensin analogues as potential radiopharmaceuticals for NTR-positive tumors. Nucl. Med. Biol. 2006, 33, 495-503. 14. Maina, T.; Nikolopoulou, A.; Stathopoulou, E.; Galanis, A. S.; Cordopatis, P.; Nock, B. A. [99mTc]Demotensin 5 and 6 in the NTS1-R-targeted imaging of tumours: synthesis and preclinical results. Eur. J. Nucl. Med. Mol. Imaging 2007, 34, 1804-1814. 15. Myers, R. M.; Shearman, J. W.; Kitching, M. O.; Ramos-Montoya, A.; Neal, D. E.; Ley, S. V. Cancer, chemistry, and the cell: molecules that interact with the neurotensin receptors. ACS Chem. Biol. 2009, 4, 503-525. 16. Nock, B. A.; Nikolopoulou, A.; Reubi, J. C.; Maes, V.; Conrath, P.; Tourwe, D.; Maina, T. Toward stable N-4-modified neurotensins for NTS1-receptor-targeted tumor imaging with Tc-99m. J. Med. Chem. 2006, 49, 4767-4776.



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. Orwig, K. S.; Lassetter, M. R.; Hadden, M. K.; Dix, T. A. Comparison of N-terminal modifications on neurotensin(8-13) analogues correlates peptide stability but not binding affinity with in vivo efficacy. J. Med. Chem. 2009, 52, 1803-1813. 18. Zhang, K. J.; An, R.; Gao, Z. R.; Zhang, Y. X.; Aruva, M. R. Radionuclide imaging of smallcell lung cancer (SCLC) using Tc-99m-labeled neurotensin peptide 8-13. Nucl. Med. Biol. 2006, 33, 505-512. 19. Held, C.; Plomer, M.; Hübner, H.; Meltretter, J.; Pischetsrieder, M.; Gmeiner, P. Development of a metabolically stable neurotensin receptor 2 (NTS2) ligand. ChemMedChem 2013, 8, 75-81. 20. Sparr, C.; Purkayastha, N.; Yoshinari, T.; Seebach, D.; Maschauer, S.; Prante, O.; Hübner, H.; Gmeiner, P.; Kolesinska, B.; Cescato, R.; Waser, B.; Reubi, J. C. Syntheses, receptor bindings, in vitro and in vivo stabilities and biodistributions of DOTA-neurotensin(8-13) derivatives containing betaamino acid residues - a lesson about the importance of animal experiments. Chem. Biodivers. 2013, 10, 2101-2121. 21. Maschauer, S.; Ruckdeschel, T.; Tripal, P.; Haubner, R.; Einsiedel, J.; Hubner, H.; Gmeiner, P.; Kuwert, T.; Prante, O. In vivo monitoring of the antiangiogenic effect of neurotensin receptormediated radiotherapy by small-animal positron emission tomography: a pilot study. Pharmaceuticals 2014, 7, 464-481. 22. Mascarin, A.; Valverde, I. E.; Vomstein, S.; Mindt, T. L. 1,2,3-Triazole stabilized neurotensinbased radiopeptidomimetics for improved tumor targeting. Bioconjugate Chem. 2015, 26, 2143-2152. 23. Mascarin, A.; Valverde, I. E.; Mindt, T. L. Structure–activity relationship studies of amino acid substitutions in radiolabeled neurotensin conjugates. ChemMedChem 2016, 11, 102-107. 24. Li, Z.; Conti, P. S. Radiopharmaceutical chemistry for positron emission tomography. Adv. Drug Deliv. Rev. 2010, 62, 1031-1051. 25. Schottelius, M.; Wester, H.-J. Molecular imaging targeting peptide receptors. Methods 2009, 48, 161-177. 26. Morgat, C.; Mishra, A. K.; Varshney, R.; Allard, M.; Fernandez, P.; Hindié, E. Targeting neuropeptide receptors for cancer imaging and therapy: perspectives with bombesin, neurotensin, and neuropeptide-Y receptors. J. Nucl. Med. 2014, 55, 1650-1657. 27. Jia, Y.; Shi, W.; Zhou, Z.; Wagh, N. K.; Fan, W.; Brusnahan, S. K.; Garrison, J. C. Evaluation of DOTA-chelated neurotensin analogs with spacer-enhanced biological performance for neurotensinreceptor-1-positive tumor targeting. Nucl. Med. Biol. 2015, 42, 816-823. 28. Bergmann, R.; Scheunemann, M.; Heichert, C.; Mäding, P.; Wittrisch, H.; Kretzschmar, M.; Rodig, H.; Tourwé, D.; Iterbeke, K.; Chavatte, K.; Zips, D.; Reubi, J. C.; Johannsen, B. Biodistribution and catabolism of 18F-labeled neurotensin(8–13) analogs. Nucl. Med. Biol. 2002, 29, 61-72. 29. Maschauer, S.; Einsiedel, J.; Haubner, R.; Hocke, C.; Ocker, M.; Hübner, H.; Kuwert, T.; Gmeiner, P.; Prante, O. Labeling and glycosylation of peptides using click chemistry: a general approach to 18F-glycopeptides as effective imaging probes for positron emission tomography. Angew. Chem. Int. Ed. 2010, 49, 976-979. 30. Maschauer, S.; Einsiedel, J.; Hocke, C.; Hübner, H.; Kuwert, T.; Gmeiner, P.; Prante, O. Synthesis of a 68Ga-labeled peptoid-peptide hybrid for imaging of neurotensin receptor expression in vivo. ACS Med. Chem. Lett. 2010, 1, 224-228.


Page 28 of 41

Page 29 of 41

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


31. Wu, Z.; Li, L.; Liu, S.; Yakushijin, F.; Yakushijin, K.; Horne, D.; Conti, P. S.; Li, Z.; Kandeel, F.; Shively, J. E. Facile preparation of a thiol-reactive 18F-labeling agent and synthesis of 18F-DEGVS-NT for PET imaging of a neurotensin receptor–positive tumor. J. Nucl. Med. 2014, 55, 1178-1184. 32. Maschauer, S.; Greff, C.; Einsiedel, J.; Ott, J.; Tripal, P.; Hübner, H.; Gmeiner, P.; Prante, O. Improved radiosynthesis and preliminary in vivo evaluation of a 18F-labeled glycopeptide-peptoid hybrid for PET imaging of neurotensin receptor 2. Bioorg. Med. Chem. 2015, 23, 4026-4033. 33. Liu, S.; Wu, Z.; Yap, L.-P.; Kandeel, F.; Shively, J.; Conti, P.; Li, Z. Evaluation of 18F-DEGVS-NT for NTR1 targeted imaging in prostate cancer. J. Nucl. Med. 2014, 55, 1041. 34. Einsiedel, J.; Held, C.; Hervet, M.; Plomer, M.; Tschammer, N.; Hübner, H.; Gmeiner, P. Discovery of highly potent and neurotensin receptor 2 selective neurotensin mimetics. J. Med. Chem. 2011, 54, 2915-2923. 35. Rostovtsev, V. V.; Green, L. G.; Fokin, V. V.; Sharpless, K. B. A stepwise huisgen cycloaddition process: copper(I)-catalyzed regioselective "ligation" of azides and terminal alkynes. Angew. Chem. Int. Ed. 2002, 41, 2596-2599. 36. Tornoe, C. W.; Christensen, C.; Meldal, M. Peptidotriazoles on solid phase: [1,2,3]-triazoles by regiospecific copper(I)-catalyzed 1,3-dipolar cycloadditions of terminal alkynes to azides. J. Org. Chem. 2002, 67, 3057-3064. 37. Maschauer, S.; Haubner, R.; Kuwert, T.; Prante, O. 18F-Glyco-RGD peptides for PET imaging of integrin expression: efficient radiosynthesis by click chemistry and modulation of biodistribution by glycosylation. Mol. Pharm. 2014, 11, 505-515. 38. Mercier, F.; Paris, J.; Kaisin, G.; Thonon, D.; Flagothier, J.; Teller, N.; Lemaire, C.; Luxen, A. General method for labeling siRNA by click chemistry with fluorine-18 for the purpose of PET imaging. Bioconjugate Chem. 2011, 22, 108-114. 39. Gourni, E.; Demmer, O.; Schottelius, M.; D'Alessandria, C.; Schulz, S.; Dijkgraaf, I.; Schumacher, U.; Schwaiger, M.; Kessler, H.; Wester, H.-J. PET of CXCR4 expression by a 68Galabeled highly specific targeted contrast agent. J. Nucl. Med. 2011, 52, 1803-1810. 40. Reubi, C. J.; Schär, J.-C.; Waser, B.; Wenger, S.; Heppeler, A.; Schmitt, S. J.; Mäcke, R. H. Affinity profiles for human somatostatin receptor subtypes SST1–SST5 of somatostatin radiotracers selected for scintigraphic and radiotherapeutic use. Eur. J. Nucl. Med. 2000, 27, 273-282. 41. Sirion, U.; Kim, H. J.; Lee, J. H.; Seo, J. W.; Lee, B. S.; Lee, S. J.; Oh, S. J.; Chi, D. Y. An efficient F-18 labeling method for PET study: Huisgen 1,3-dipolar cycloaddition of bioactive substances and F-18-labeled compounds. Tetrahedron Lett. 2007, 48, 3953-3957. 42. Lang, C.; Maschauer, S.; Hübner, H.; Gmeiner, P.; Prante, O. Synthesis and evaluation of a 18 F-labeled diarylpyrazole glycoconjugate for the imaging of NTS1-positive tumors. J. Med. Chem. 2013, 56, 9361-9365. 43. Jordan, M.; Schallhorn, A.; Wurm, F. M. Transfecting mammalian cells: optimization of critical parameters affecting calcium-phosphate precipitate formation. Nucleic Acids Res. 1996, 24, 596-601. 44. Lowry, O. H.; Rosebrough, N. J.; Farr, A. L.; Randall, R. J. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 1951, 193, 265-275.



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

45. Cheng, Y.; Prusoff, W. H. Relationship between the inhibition constant (K1) and the concentration of inhibitor which causes 50 per cent inhibition (I50) of an enzymatic reaction. Biochem. Pharmacol. 1973, 22, 3099-3108.


Page 30 of 41

Page 31 of 41

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 1. In vitro binding affinities of a series of neurotensin peptides containing the metabolically stable sequence NLys8-Lys9-Pro10-Tyr11-Tle12-Leu13-OH to the human NTS1 and NTS2.a Peptide

Structure (R= -Lys-Pro-Tyr-Tle-Leu-OH)

Ki (NTS1, nM)

Ki (NTS2, nM)

NT(8-13)

Arg8-Arg9-Pro10-Tyr11-Ile12-Leu13

0.29±0.03

1.4±0.11b

130

2300±1400c,d

1800±140c,d

0.8

230

180±47c

350±270c,d

1.9

3

280±14d

2800±640d

10

4

110±22

3800±1300

35

5

320±71d

260±57d

0.8

6

19±6.4d

22±5.7d

1.1

7

160±10

640±44

4

8

20±1.2

87±23

4.4

929

22±2.2

66±13

3


NTS2 / NTS1


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

a

Page 32 of 41

10

26±5.9

86±20

3.3

11

33±14d

620±7.1d

19

12

71±5.0d

100±69d

1.4

Data are expressed as mean values ± SEM (standard error of the mean) from 3-7 independent experiments each

determined in triplicate. b KD value ± SEM determined with the radioligand [3H]NT(8-13) in saturation binding experiments each performed in quadruplicates.

c

Values from ref. 30 for comparison.

d

Data are expressed as

mean values ± SD (standard deviation) from two independent experiments each done in triplicate.

Table 2. Radiochemical yields (RCY), specific activities (AS), logD7.4 values, stabilities in human serum and internalization rates of the radiolabeled NT peptides.

a

Peptide

RCY

AS (GBq/µmol)a

logD7.4

Stability in vitrob

Internalizationc

[68Ga]4

100 %d

30 (n=3)

-4.2

95 %

36 %

[68Ga]6

>95 %d

10 (n=3)

-4.0

93 %

63 %

[68Ga]8

100 %d

30 (n=3)

-4.1

100 %

70 %

[18F]10

16-21 %e

2-9 (n=6)

n.d.

100 %

n.d.

[18F]11

16-21 %e

2-21 (n=2)

n.d.

100 %

n.d.

[18F]12

20-21 %e

10-44 (n=4)

-2.1

96 %

78 %

calculated at end of synthesis

b

intact tracer (%) as determined by analytical radio-HPLC after incubation in human serum in vitro for 60 min c

internalization (%) of tracer after 30 min using HT29 cells

d

RCY determined by radio-HPLC from a sample withdrawn from the reaction mixture after the reaction time of 10 min e

nondecay-corrected yield after two-step radiosynthesis related to [18F]fluoride


Page 33 of 41

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 3. Biodistribution data expressed as %ID/g tissue for 18F-labeled NT-peptides [18F]10 and [18F]11 measured in HT29 tumor bearing nude mice.a [18F]10 (n=2-7)

[18F]11 (n=2)

10 min

30 min

60 min

60 min b blocking

30 min

60 min

60 min b blocking

Blood

1.99 ± 0.27

0.52 ± 0.15

0.12 ± 0.07

0.46 ± 0.32

0.58 ± 0.07

0.26 ± 0.17

0.58 ± 0.30

Lung

4.41 ± 1.10

0.50 ± 0.11

0.23± 0.07

0.64 ± 0.32

0.78 ± 0.16

0.32 ± 0.05

0.66 ± 0.33

Liver

2.04 ± 1.49

0.19 ± 0.04

0.13 ± 0.04

0.29 ± 0.16

0.34 ± 0.12

0.19 ± 0.03

0.59 ± 0.21

Kidney

31.06 ± 6.05

20.21 ± 3.95

18.81 ± 2.46

14.38 ± 2.23

19.74 ± 1.70

17.40 ± 1.11

8.00 ± 3.65

Heart

0.98 ± 0.18

0.19 ± 0.07

0.08 ± 0.04

0.63 ± 0.89

0.29 ± 0.05

0.20 ± 0.08

0.64 ± 0.67

Spleen

1.70 ± 1.19

0.20 ± 0.03

0.14 ± 0.03

0.30 ± 0.17

0.38 ± 0.13

0.20 ± 0.04

0.32 ± 0.21

Brain

0.25 ± 0.08

0.04 ± 0.01

0.03 ± 0.01

0.04 ± 0.01

0.07 ± 0.02

0.08 ± 0.01

0.08 ± 0.03

Muscle

1.77 ± 1.46

0.35 ± 0.36

0.27 ± 0.33

0.27 ± 0.16

0.28 ± 0.12

0.11 ± 0.04

1.67 ± 1.83

Femur

0.97 ± 0.61

0.21 ± 0.12

0.20 ± 0.32

0.37 ± 0.25

0.50 ± 0.54

0.18 ± 0.08

0.39 ± 0.29

Tumor

1.69 ± 0.20

0.90 ± 0.04

0.75 ± 0.25

0.32 ± 0.10

1.33 ± 0.47

1.02 ± 0.17

0.35 ± 0.04

Intestine

1.70 ± 0.96

0.24 ± 0.05

0.20 ± 0.11

0.44 ± 0.18

0.48 ± 0.26

0.25 ± 0.08

0.50 ± 0.36

0.9

1.7

6.3

2.3

3.9

T/Kd

0.05

0.04

0.04

0.07

0.06

T/Me

0.95

2.6

2.8

4.8

9.3

T/B

c

a

Data represent mean values ± standard deviation

b

Coinjection of 13 (2.5 mg/kg)

c

Tumor-to-blood ratio

d

Tumor-to-kidney ratio

e

Tumor-to-muscle ratio



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 41

Table 4. Biodistribution data expressed as %ID/g tissue for 68Ga-labeled NT-peptides [68Ga]6 and [68Ga]8 measured in HT29 tumor bearing nude micea [68Ga]6 (n=3)

[68Ga]8 (n=2-4)

10 min

30 min

60 min

60 min blockingb

10 min

30 min

60 min

60 min blockingb

Blood

2.67 ± 1.09

0.81 ± 0.59

0.15 ± 0.06

0.38 ± 0.26

1.87 ± 0.52

0.52 ± 0.69

0.05 ± 0.02

0.08 ± 0.04

Lung

2.07 ± 0.42

0.85 ± 0.38

0.36 ± 0.28

0.51 ± 0.38

1.52 ± 0.35

0.51 ± 0.43

0.20 ± 0.01

0.42 ± 0.15

Liver

1.16 ± 0.10

0.80 ± 0.19

0.45 ± 0.13

0.62 ± 0.17

0.77 ± 0.13

0.32 ± 0.13

0.30 ± 0.10

0.28 ± 0.03

Kidney

30.64 ± 7.26

31.37 ± 6.26

34.87 ± 2.48

26.71 ± 4.95

48.74 ± 2.99

40.14 ± 3.83

44.59 ± 6.46

37.13 ± 5.66

Heart

0.86 ± 0.21

0.31 ± 0.21

0.10 ± 0.05

0.14 ± 0.10

0.64 ± 0.23

0.22 ± 0.27

0.05 ± 0.02

0.07 ± 0.04

Spleen

0.83 ± 0.12

0.43 ± 0.08

0.25 ± 0.14

0.28 ± 0.15

0.67 ± 0.15

0.21 ± 0.16

0.14 ± 0.05

0.12 ± 0.01

Brain

0.15 ± 0.03

0.07 ± 0.04

0.04 ± 0.03

0.03 ± 0.02

0.09 ± 0.03

0.03 ± 0.02

0.02 ± 0.00

0.02 ± 0.01

Muscle

0.92 ± 0.76

0.22 ± 0.12

0.16 ± 0.09

0.12 ± 0.09

0.44 ± 0.20

0.10 ± 0.11

0.48 ± 0.54

0.20 ± 0.14

Femur

1.00 ± 0.23

0.35 ± 0.19

0.11 ± 0.05

0.21 ± 0.15

0.71 ± 0.16

0.29 ± 0.28

0.06 ± 0.01

0.08 ± 0.03

Tumor

4.12 ± 2.51

2.22 ± 0.42

1.40 ± 0.13

0.45 ± 0.19

2.70 ± 0.47

1.84 ± 0.31

1.55 ± 0.35

0.21 ± 0.06

Intestine

0.86 ± 0.35

0.45 ± 0.22

0.69 ± 0.44

0.17 ± 0.07

0.71 ± 0.18

0.29 ± 0.18

0.17 ± 0.04

0.08 ± 0.02

T/Bc

1.5

2.7

9.3

1.4

3.5

31

T/Kd

0.13

0.07

0.04

0.06

0.05

0.03

4.5

10

8.8

6.1

18

3.2

e

T/M

a

Data represent mean values ± standard deviation

b

Coinjection of 13 (2.5 mg/kg)

c

Tumor-to-blood ratio

d

Tumor-to-kidney ratio

e

Tumor-to-muscle ratio


Page 35 of 41

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


Fmoc-Tle-OH Fmoc-Tyr(tBu)-OH Fmoc-Pro-OH Fmoc-Lys(Boc)-OH Fmoc-NLys(Boc)-OH

Fmoc LeuO

Leu-Tle-(tBu)Tyr-Pro-(Boc)Lys-(Boc)LysN-H O

= wang resin

-wave assisted SPPS: Fmoc-cleavage: piperidine/DMF 25:75, 100 W, 7x 5 s AA-coupling: AA, PyBOP, HOBt, DIPEA or AA, HATU, DIPEA both with 50 W, 15x 10 s

f or 3: - coupling of the Fmoc-PEG-Spacer and NODA-GA tri-t-Bu ester: PyBOP, HOBt, DIPEA f or 5: - coupling of Fmoc-Lys(Alloc)-OH: HATU, DIPEA: - Alloc cleavage: Pd(PPh3)4, CHCl3, morpholine, HOAc - coupling of DOTA-tri-t-Bu ester: HATU, DIPEA f or 7: - coupling of Fmoc-Lys(Boc)-OH: HATU, DIPEA - further coupling steps: see peptide 3 cleavage f rom the resin: 1. H2SO4/dioxane (1:9), 8 °C 2. TFA, phenol, H2O, TIS

R

NLys-Lys-Pro-Tyr-Tle-Leu-OH O O

O N( H

HO2C 3: R = N CO2H

N

O)

O

N( H

NH

6

HO2C 5: R =

N CO2H

OH O

HO

N

N

N

N

O

O

H2N

N

7: R = N OH

CO2H

O)

6

O HN

N CO2H H2N

Ga-complex f ormation: for 4 and 8: Ga(NO3)3, sodium acetate buffer pH 4.5, rt for 6: Ga(NO3)3, sodium acetate buffer pH 4.5, 90°C for [68Ga]4 and [68Ga]8: [68Ga]GaCl3, sodium acetate buffer pH 4-4.5, rt for [68Ga]6: [68Ga]GaCl3, HEPES buffer, pH 3.5-4, 98°C 4, 6, 8, [68Ga]4, [68Ga]6, [68Ga]8 (see Table 1)

Scheme 1: Synthesis of the peptide precursors 3, 5, 7 and the peptide-gallium-complexes 4, 6, 8, and [68Ga]4, [68Ga]6 and [68Ga]8.



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

Scheme 2. Synthesis of the reference compounds 10, 11 and 12.


Page 36 of 41

Page 37 of 41

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


Scheme 3. Radiosynthesis of 18F-labeled NT-peptides [18F]10, [18F]11 and [18F]12.



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 1. A. Internalization rates of [18F]12, [68Ga]4, [68Ga]6 and [68Ga]8 in human HT29 cells. B. Efflux rates of [68Ga]6 and [68Ga]8 on human HT29 cells. Each data point represents the mean ± standard error of the mean of two experiments performed in quadruplicate.


Page 38 of 41

Page 39 of 41

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 2. A. Biodistribution data of [18F]10, [18F]11, [68Ga]6 and [68Ga]8 using HT29 tumor bearing nude mice (n=2-7) at 60 min p.i. B. HT29 tumor uptake of the radiotracers in comparison to animals injected with radiotracer plus blocking compound (13, 2.5 mg/kg) at 60 min p.i. C. Tumor-to-blood ratios at 60 min p.i.



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. Representative coronal small animal PET images of HT29 tumor bearing nude mice injected with the radiolabeled NT-peptides [68Ga]4, [68Ga]6 and [68Ga]8 at 45-60 min p.i. Left animal: control, right animal: blocking, i.e. coinjection of radiotracer and 13 (2.5 mg/kg). Red arrows indicate the HT29 tumors.

Figure 4. Standard uptake values (SUVmax) of HT29 tumors in correlation to Ki values (nM) of the 68Ga-labeled peptides. Single data points from four animals per peptide are depicted. r = -0.965 (Spearman correlation, P

18F- and 68Ga-Labeled Neurotensin Peptides for PET Imaging of

Recommend Documents