New Gastrin Releasing Peptide Receptor-Directed [99mTc

Article Cite This: J. Med. Chem. 2018, 61, 3138−3150

pubs.acs.org/jmc

New Gastrin Releasing Peptide Receptor-Directed [99mTc]Demobesin 1 Mimics: Synthesis and Comparative Evaluation Berthold A. Nock,† David Charalambidis,† Werner Sallegger,‡ Beatrice Waser,§ Rosalba Mansi,∥ Guillaume P. Nicolas,⊥ Eleni Ketani,† Anastasia Nikolopoulou,† Melpomeni Fani,∥ Jean-Claude Reubi,§ and Theodosia Maina*,† †

Molecular Radiopharmacy, INRASTES, National Center for Scientific Research “Demokritos”, GR-153 10 Athens, Greece piCHEM, A-8045 Graz, Austria § Cell Biology and Experimental Cancer Research, Institute of Pathology, University of Berne, CH-3010 Berne, Switzerland Divisions of ∥Radiopharmaceutical Chemistry and ⊥Nuclear Medicine, University Hospital Basel, CH-4031 Basel, Switzerland ‡

S Supporting Information *

ABSTRACT: We have previously reported on the gastrin releasing peptide receptor (GRPR) antagonist [99mTc]1, ([99mTc]demobesin 1, 99mTc-[N4′-diglycolate-DPhe6,Leu-NHEt13]BBN(6−13)). [99mTc]1 has shown superior biological profile compared to analogous agonist-based 99mTc-radioligands. We herein present a small library of [99mTc]1 mimics generated after structural modifications in (a) the linker ([99mTc]2, [99mTc]3, [99mTc]4), (b) the peptide chain ([99mTc]5, [99mTc]6), and (c) the C-terminus ([99mTc]7 or [99mTc]8). The effects of above modifications on the biological properties of analogs were studied in PC-3 cells and tumor-bearing SCID mice. All analogs showed subnanomolar affinity for the human GRPR, while most receptoraffine 4 and 8 behaved as potent GRPR antagonists in a functional internalization assay. In mice bearing PC-3 tumors, [99mTc]1− [99mTc]6 exhibited GRPR-specific tumor uptake, rapidly clearing from normal tissues. [99mTc]4 displayed the highest tumor uptake (28.8 ± 4.1%ID/g at 1 h pi), which remained high even after 24 h pi (16.3 ± 1.8%ID/g), well surpassing that of [99mTc]1 (5.4 ± 0.7%ID/g at 24 h pi).

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INTRODUCTION

radionuclide therapy whereby higher peptide doses are typically administered.18 Our search toward GRPR-targeting radiopeptides has involved GRPR antagonist-based radioligands already in the early days of our work.19 In a more recent study, we have directly compared the biological profile of the antagonist-based [99mTc]1 ([99mTc]demobesin 1 or [99mTc]DB1, 99mTc-[N4′-diglycolate-DPhe6,LeuNHEt13]BBN(6−13),20,21 N4′ = 6-{p-[(carboxymethoxy)acetyl]-aminobenzyl}-1,4,8,11-tetraazaundecane), and the agonist-based [99mTc]demobesin 422,23 ([99mTc]DB4, 99mTc[N4-Pro1,Tyr4,Nle14]BBN, N4 = 6-(carboxy)-1,4,8,11-tetraazaundecane) in several cell and animal models.24 [99mTc]1 showed a superior pharmacokinetic profile in mice bearing human PC-3 xenografts. Specifically, radioactivity cleared more rapidly from the mouse GRPR-rich pancreas than from the implanted

1

The gastrin releasing peptide receptor (GRPR) has emerged as an attractive molecular target in nuclear oncology owing to its high-density expression in a wide spectrum of human cancers, including the frequently occurring prostate and breast cancers.2−11 For this purpose, peptide radionuclide carriers have been developed based on analogs either of the frog tetradecapeptide bombesin (BBN) or of the native human GRP and neuromedin C (NMC, GRP(18−27)) sequences.12−14 These frog BBN- or human GRP/NMC-derived radiopeptides bind with high affinity to the human GRPR and internalize rapidly within target cells, displaying typical receptor-agonist profiles. Until recently, internalization of agonist radioligands was considered essential for a strong diagnostic signal or for optimum delivery of cytotoxic doses to cancer lesions. However, after intravenous injection in patients, GRPR agonists may elicit adverse effects following receptor binding and activation.15−17 Such undesirable effects are expected to intensify during © 2018 American Chemical Society

Received: February 3, 2018 Published: March 8, 2018 3138

DOI: 10.1021/acs.jmedchem.8b00177 J. Med. Chem. 2018, 61, 3138−3150

Journal of Medicinal Chemistry

Article

Chart 1. Chemical structures of [99mTc]1−[99mTc]8, Including the Bifunctional Chelator N4′ (Blue) or N4 (Green) Binding 99mTc, the Linker (Magenta), and the Peptide Chain with Modifications at Positions 7, 11, and the C-Terminal Highlighted (Red)a

a

No spacer is used in [99mTc]2. *In the case of the two hydrazide analogs, labeling with

tumor as compared to [99mTc]DB4. Subsequent studies have corroborated these findings in animal models and in patients leading to a shift of paradigm from GRP/BBN-based radioagonists to GRPR radioantagonists.25−30 The molecular basis for this unexpected behavior is currently under investigation and may provide opportunities for safer and more efficacious diagnosis and therapy of GRPR-expressing tumors in man. Following this line of research, we now introduce a series of new [99mTc]1 mimics, generated after structural modifications in (a) the linker (N4 coupled without a linker, [99mTc]2; via the PEG2 linker, [99mTc]3; or via the AMA-DGA (aminomethylaniline diglycolate) linker, [99mTc]4), (b) the peptide chain (N4-AMA-DGA to mono-DAla11- ([99mTc]5) or bis-DGln7,DAla11substituted peptide ([99mTc]6)) aiming at higher metabolic stability, and (c) the C-terminus ((N4′-DGA, [99mTc]7; or N4AMA-DGA, [99mTc]8; both C-terminal hydrazides31) (Chart 1). The biological profile of new analogs was directly compared in several in vitro and in vivo GRPR-positive models using [99mTc]1 as reference. A major goal of this search was to obtain GRPRdirected 99mTc radiotracers with longer tumor retention compared to our “gold standard” [99mTc]1, which undesirably washed out of the tumor lesions at time points beyond 4 h pi.19 Yet for successful clinical translation in patients with GRPR-positive tumors, longer retention in pathological lesions is highly desirable, not only because it would provide flexibility of time for acquiring scans in a routine clinical setting. Moreover, prolonged tumor retention, in combination with the faster background clearance typical for receptor antagonists, would offer the opportunity of advantageous later-time imaging, anticipated to result in higher-contrast images and enhanced diagnostic accuracy. It should be added that

99m

Tc led to a mixture of unidentified species.

prolonged tumor retention may pave the way for radionuclide therapy with the analogous [186Re/188Re]1 mimics owing to the similarities of Tc and Re chemistries.

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RESULTS Ligands and Radioligands. Synthesis of 1−8. The ethylamide analogs 1−6 were assembled on the solid support following typical Fmoc-protection methodology, and either the Boc-protected bifunctional chelator N4′ (1) or the Boc-protected bifunctional chelator N4 (2−6) was attached to their N-terminus directly (2) or via a linker (1, 3−6). In the cases of 1−6 an ethylindole AM resin, typically used for the preparation of NH-ethylamide peptides, led to poor yields. Therefore, synthesis of analogs 1−6 was performed using another strategy involving several consecutive steps. In the first step, the analog sequences comprising the chelator precursors were assembled on a trityl resin. Cleavage with 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) delivered the protected analog fractions, which could be coupled with H-Leu-NHEt to generate the protected C-ethylamide derivatives. In a final step, all protecting groups were removed by treatment with TFA. Synthesis of C-terminal hydrazides 7 and 8 was performed with assembling peptide sequences on a 4-hydroxymethylbenzoic acid AM resin (HMBA), following the same methodology as in the case of C-terminal ethylamides 1 and 4, respectively. Release from the resin was accomplished with hydrazine hydrate solution. Lateral protecting groups could be subsequently removed by treatment with TFA. Purification by preparative HPLC and lyophilization afforded analogs 1−8 as white solids in good yield and high purity (≥95%), as verified by 3139



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analytical HPLC in two different systems; MALDI-TOF spectra of the products were consistent with the expected formulas (Chart 1, Table 1). Table 1. Analytical Data for 1−8 HPLC tR (min), UV trace compd % puritya MWb calcd MWb found, m/z 1 2 3 4 5 6 7 8

100 98.5 98.5 99.6 97.4 97.1 97.9 97.6

1347.7 1169.7 1316.1 1390.7 1404.7 1404.7 1334.6 1377.6

system 1c

system 2d

13.0 6.5 15.4 18.0 18.4 18.4 18.9

6.8 6.5 6.5 6.8 6.8 6.8 6.4 6.6

+

1348.2 [M + H] 1171.1 [M + H]+ 1316.4 [M + H]+ 1391.2 [M + H]+ 1406.5 [M + H]+ 1406.7 [M + H]+ 1336.0 [M + H]+ 1378.3 [M + H]+

a

Purity was determined by HPLC systems 1 and 2. bAverage mass. System 1: RP-HPLC with UV detection at 220 nm. An Xterra RP18 column (5 μm, 4.6 mm × 150 mm) was eluted at a flow rate of 1 mL/min with linear gradient 15% B to 30% B in 30 min, with A = 0.1% TFA in H2O (v/v) and B = MeCN. dSystem 2: A Nucleosil-100 C18 column (5 μm, 150 mm × 4 mm) was eluted at a flow rate of 1 mL/min with a linear gradient 0.1% TFA in MeCN (from 10% to 90% in 15 min) and 0.1% aqueous TFA as complementary phase. Runs were monitored by UV detection at 215 nm. c

Radiolabeling and Quality Control of [99mTc]1 Mimics. After labeling with 99mTc, [99mTc]1−[99mTc]6 formed in >95% yield and >98% radiochemical purity, as verified by RP-HPLC. However, the presence of the hydrazide function at the C-terminus of 7 and 8 interfered with 99mTc-labeling, and as a result a mixture of unidentified species formed.32 In all other cases, 99mTcradiotracers were obtained as single radioactive species at typical specific activities of 18−37 MBq/nmol peptide in ≥98% radiochemical purity, as verified by RP-HPLC and TLC; therefore, the resulting [99mTc]1−[99mTc]6 were used without purification in all subsequent experiments. In Vitro Studies. Binding Affinity of 1−8 for the Human GRPR. All eight 1−8 analogs displaced [125I-Tyr4]BBN from GRPR binding sites in PC-3 cell membranes in a monophasic and dose-dependent manner exhibiting subnanomolar affinities for the human GRPR (Figure 1), revealing a positive influence of the pendent cationic N4-framework at the N-terminus of 1−8 on receptor affinity.33 The two hydrazides and especially 8 (IC50 = 0.17 ± 0.01 nM) as well as 4 (IC50 = 0.26 ± 0.03 nM) showed the highest affinity for the GRPR,31 while 3 containing the PEG2 linker displayed the lowest affinity for the GRPR (IC50 = 0.93 ± 0.01 nM) in this series of analogs. It should be noted that the affinity of [Tyr4]BBN for the GRPR under the applied experimental conditions was slightly lower (IC50 = 1.22 ± 0.18 nM) compared to 1−8. Antagonistic Properties of 4 and 8. The antagonistic properties of the two most GRPR-affine analogs 4 and 8 were confirmed by immunofluorescence-based internalization assays with HEK293-GRPR cells. As illustrated in Figure 2, 10 nM BBN could trigger a massive GRPR internalization in HEK293-GRPR cells compared with the condition without peptide. Conversely, 4 or 8 alone could not trigger the internalization of the GRPR at 1 μM. Furthermore, when given at a concentration of 1 μM together with 10 nM BBN, 4 or 8 was able to completely prevent the BBN-induced GRPR internalization. Cell-Association/Internalization of [99mTc]1−[99mTc]6 in PC-3 Cells. The internalization properties of radioligands [99mTc]1−[99mTc]6 were studied in PC-3 cells by 1 h incubation

Figure 1. (A) Displacement of [125I-Tyr4]BBN from GRPR binding sites in PC-3 cell membranes by increasing concentrations of 1 (∗, IC50 = 0.70 ± 0.08 nM), 2 (▼ in magenta, IC50 = 0.72 ± 0.06 nM), 3 (● in blue, IC50 = 0.93 ± 0.01 nM), and 4 (◆ in red, IC50 = 0.26 ± 0.03 nM) and (B) of 5 (∗ in green, IC50 = 0.56 ± 0.09 nM), 6 (▼ in violet, IC50 = 0.71 ± 0.08 nM), 7 (● in wine-red, IC50 = 0.30 ± 0.03 nM), and 8 (◆ in greenishblue, IC50 = 0.17 ± 0.01 nM).

Figure 2. The GRPR resides on the cell membrane of in HEK293-GRPR cells (A) in the absence of peptide in the medium, but (B) massive receptor-internalization is observed in the presence of 10 nM BBN. Analogs 4 (C) and 8 (E) were unable to trigger the internalization of GRPR in HEK293-GRPR cells at 1 μM but were effective (D and F, respectively) in preventing the stimulation of GRPR internalization by 10 nM BBN, as consistent with a receptor-antagonist profile.

at 37 °C in the absence or presence of excess [Tyr4]BBN to define nonspecific involvement. As shown in the comparative 3140



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additionally acquired from saturation binding assays in PC-3 cell membranes. Due to the high specific activity of 99mTc (19.24 TBq/μmol) and radiation safety limitations, the required concentration series (0.01−10 nM) for a single radioligand chemical entity could be achieved using the pseudo-stable β emitter 99gTc. As depicted in Figure 3B, [99mTc/99gTc]4 demonstrated a strong and dose-dependent interaction to a single class of high affinity binding sites in the PC-3 cell membranes. The Kd value of 0.24 ± 0.03 nM illustrates the high affinity of [99mTc/99gTc]4 to the human GRPR. This finding showed that incorporation of the radiometal by the N4-framework and the presence of the positively charged [Tc(O)2(N4)]+ chelate at the peptide N-terminus were well-tolerated by the receptor, as previously reported.33 Metabolic Studies in Mice. Analysis of mouse blood collected 5 min after administration of [99mTc]1−[99mTc]6 in the animals by HPLC revealed a different extent of radiotracer degradation in mouse peripheral blood, as well as a diverse pattern of radiometabolites (Figure 4). Apparently, not only the type of linker but also key amino acid replacements in the peptide chain affected the in vivo stability of these analogs. Specifically, the mono-DAla11-substituted [99mTc]5 and the PEG2-containing [99mTc]3 analogs represented the most in vivo robust members in the series (75% and 70% intact at 5 min postinjection, pi). Interestingly, doubly DGln7,DAla11-substituted [99mTc]6 was less stable in vivo displaying comparable stability with the reference [99mTc]1 and its mimic [99mTc]4 (60% intact at 5 min pi). The least in vivo stable radiotracer turned out to be [99mTc]2 (45% intact at 5 min pi), altogether lacking a linker. Biodistribution of [99mTc]1−[99mTc]6 in PC-3 XenograftBearing SCID Mice. Biodistribution results of [99mTc]1− [99mTc]6 in SCID mice bearing GRPR-positive PC-3 xenografts for 1, 4, and 24 h pi are summarized in Tables 2, 3, and 4. Results are presented as percent injected dose per gram (%ID/g) tissue and correspond to mean values ± sd, n = 4. In addition, tumor-tophysiological tissue ratios can be found in Figure S3 (Supporting Information). All radiotracers cleared rapidly from blood and the background mainly via the kidneys and the urinary system. The analogs [99mTc]1, [99mTc]4, [99mTc]5, and [99mTc]6, containing an aromatic residue between the linear tetraamine framework and the peptide chain, exhibited some transient bowel activity. In the case of [99mTc]4 intestinal accumulation could be partially blocked by co-injection of excess [Tyr4]BBN, implying specific interaction with bombesin binding sites in the intestinal tract.34 It is interesting to also observe the lower liver uptake for [99mTc]4 (3.19 ± 0.20%ID/g at 1 h pi) compared to [99mTc]1 (9.62 ± 0.97%ID/g at 1 h pi). All radiotracers specifically localized in the human PC-3 xenografts as well as in mouse pancreata, via a GRPR-mediated process, as demonstrated by the significantly lower values in these tissues during in vivo GRPR blockade with excess [Tyr4]BBN. Tumor uptake for all analogs was initially high at 1 h pi, resulting in the following radiotracer rank: [99mTc]4 (28.80 ± 4.13%ID/g) > [99mTc]1 (24.61 ± 1.98%ID/g) ≈ [99mTc]5 (24.39 ± 2.45%ID/g) > [99mTc]3 (15.48 ± 2.59%ID/g) > [99mTc]6 (12.08 ± 3.35% ID/g) > [99mTc]2 (9.88 ± 3.25%ID/g). Surprisingly, [99mTc]4 surpassed the tumor uptake of our “gold standard” [99mTc]1, despite the high structural similarity of the chain connecting the tetraamine backbone to the N-terminus of the peptide moiety, in both cases including an aromatic residue. Pancreatic values at 1 h pi were also initially high for most radiotracers, with the exception of [99mTc]2 and [99mTc]3, but rapidly declined for all analogs at later time points. Washout of radioactivity from the implanted tumors was observed as well but proceeded in an

Figure 3. (Α) Specific binding of [99mTc]1−[99mTc]6 to PC-3 cells after 1 h incubation at 37 °C: percentage of specific cell-bound activity of total added = percentage of specific membrane bound fraction (solid colored bars) + percentage of specific internalized fraction (checkered color bars): gray, [99mTc]1 (12.87 ± 1.05); magenta, [99mTc]2 (2.19 ± 0.05); blue, [99mTc]3 (3.84 ± 0.39); red, [99mTc]4 (36.05 ± 0.43); green, [99mTc]5 (2.2 ± 0.1%); violet, [99mTc]6 (3.8 ± 0.4). (Β) Specific saturation binding curve of [99mTc/99gTc]4 in PC-3 cell membranes (Kd = 0.24 ± 0.03 nM; Bmax = 380 ± 25 fmol/mio cells).

data in Figure 3A, the major portion of cell-associated radioactivity remained bound on the cell membrane of PC-3 cells with only a minor fraction of radioactivity detected inside the cells, as expected for receptor radioantagonists.19 In all cases, cell-associated activity was significantly reduced in the presence of 1 μM [Tyr4]BBN, suggesting a GRPR-mediated process. Interestingly, differences could be observed in the amount of cell-associated activity across this series of [99mTc]1 mimics, depending both upon the linker and on modifications in the peptide chain. Thus, [99mTc]1 and [99mTc]4, combining an aromatic ring-containing bifunctional anchor or linker, respectively, with the unmodified peptide chain, displayed the highest overall cell association (12.9 ± 1.1% and 36.1 ± 0.4%, respectively). Conversely, the absence of linker ([99mTc]2, 2.2 ± 0.1%) or the introduction of a shorter and hydrophilic PEG2 linker ([99mTc]3, 3.8 ± 0.4%) resulted in the poorest cell association in the series. Mono amino acid substitution in [99mTc]5 only slightly deteriorated cell-association levels (10.6 ± 0.4%) vs the [99mTc]1 reference, whereas double amino acid substitution in [99mTc]6 further reduced the uptake in PC-3 cells (6.0 ± 0.5%). In short, [99mTc]4 showed the highest binding to PC-3 cells, well surpassing that of the lead compound [99mTc]1. Saturation Binding of [99mTc/99gTc]4. Binding data for the highest cell-binding metalated analog, [99mTc/99gTc]4, was 3141



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Figure 4. Radiochromatograms (HPLC system 4) of mouse blood samples 5 min after injection of (A) [99mTc]1 (gray line, 60% intact radioligand), (B) [99mTc] 2 (magenta line, 45% intact radioligand), (C) [99mTc]3 (blue line, 70% intact radioligand), (D) [99mTc]4 (red line, 60% intact radioligand), (E) [99mTc] 5 (green line, 75% intact radioligand), and (F) [99mTc]6 (violet line, 60% intact radioligand). The tR of the parent radioligand is indicated by the arrows.

exceptionally slower pace for [99mTc]4. The latter showed significant retention in the tumor even at 24 h pi (16.32 ± 1.82% ID/g), notably surpassing the retention of reference [99mTc]1 (5.38 ± 0.72%ID/g) and of all other [99mTc]1 mimics. SPECT/CT Imaging of [99mTc]1 and [99mTc]4 in PC-3 Xenograft-Bearing Mice. Static SPECT/CT images of PC-3 tumor-bearing mice injected with [99mTc]1 or [99mTc]4 are shown in Figure 5. Both radiotracers clearly delineated the GRPR-expressing tumor revealing comparable abdominal radioactivity levels. Despite the fact that the overall radioactivity distribution patterns were not strikingly different for the two tracers in the 15 h pi images (Figure 5A and Figure 5B), still higher tumor uptake is evident for [99mTc]4 compared to [99mTc] 1. The observed difference in the tumor uptake is expected to become more pronounced at later time points, according to the quantitative biodistribution data retrieved at 24 h pi (Tables 1 and 2; Figure S3). However, SPECT/CT images could not be acquired at this later time point due to technical limitations, such as the rather short half-life of 99mTc, the amount of peptide that can be injected at a practically accessible specific activity, and the sensitivity threshold of the preclinical camera. The GRPR specificity of uptake in the PC-3 tumor could be well-visualized at

1 h pi by comparing the transaxial tumor images of [99mTc]4 injected alone (20 pmol/2.4 MBq) or with co-injection of excess [Tyr4]BBN (20 nmol) for in vivo GRPR blockade (Figure 5C and Figure 5D, respectively).

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DISCUSSION Molecular imaging with the aid of systemically administered radiopeptide probes, such as radiolabeled BBN-like peptides, which are directed at specific tumor biomarkers, like GRPR expressed in human cancer, is a rapidly expanding field in oncology.35−37 Besides allowing for noninvasive evaluation of key disease parameters, such as localization and spread, it is gradually being established as a reliable tool for patient stratification for subsequent targeted therapies, monitoring of therapeutic responses, and overall disease follow-up. Radiolabeled BBN-like agonists at the GRPR were originally preferred on the basis of their ability to internalize in cancer cells for enhancing diagnostic sensitivity.24 However, GRPR agonists inadvertently activate the receptor eliciting acute pharmacological effects after intravenous injection in human,15−17 as those observed during the clinical evaluation of 177Lu-AMBA38 for systemic therapy of prostate cancer.18 3142



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Table 2. Biodistribution of [99mTc]1 and [99mTc]2 in PC-3 Xenograft-Bearing SCID Micea %ID/g tissue ± SD, n = 4 [99mTc]1 organ

1h

blood

1.36 ± 0.09

liver

9.62 ± 0.97

heart

0.76 ± 0.08

kidneys

8.08 ± 0.65

stomach

2.10 ± 0.30

intestines

10.27 ± 0.55

spleen

1.66 ± 0.25

muscle

0.24 ± 0.02

lung

1.37 ± 0.12

pancreas

104.39 ± 3.59

tumor

24.61 ± 1.98

[99mTc]2

4h

24 h

1h

4h

24 h

0.24 ± 0.06 0.14 ± 0.01 6.62 ± 0.66 14.48 ± 0.96 0.19 ± 0.03 0.25 ± 0.03 4.33 ± 0.44 9.01 ± 0.30 2.12 ± 0.48 0.57 ± 0.04 10.36 ± 0.46 10.72 ± 0.85 1.68 ± 0.94 0.78 ± 0.27 0.06 ± 0.01 0.07 ± 0.01 0.38 ± 0.05 1.35 ± 0.49 49.82 ± 6.69 6.48 ± 0.20b 22.66 ± 2.20 5.19 ± 1.35b

0.07 ± 0.01

1.19 ± 0.16

0.05 ± 0.01

2.61 ± 0.16

2.61 ± 0.11

0.14 ± 0.03

1.13 ± 0.58

1.02 ± 0.18

13.64 ± 4.15

1.37 ± 0.98

0.61 ± 0.15

1.35 ± 0.51

2.40 ± 0.45

0.90 ± 0.17

1.16 ± 0.77

0.04 ± 0.01

0.54 ± 0.22

0.15 ± 0.02

1.99 ± 1.00

1.29 ± 0.46

2.97 ± 1.52

5.38 ± 0.72

9.88 ± 3.25

0.07 ± 0.06 0.29 ± 0.21 1.72 ± 0.48 3.12 ± 0.31 0.33 ± 0.47 0.01 ± 0.00 4.10 ± 0.87 0.70 ± 0.11 0.33 ± 0.14 0.11 ± 0.01 2.30 ± 0.70 5.81 ± 0.40 0.50 ± 0.29 0.03 ± 0.01 0.03 ± 0.01 0.42 ± 0.05 0.23 ± 0.18 0.04 ± 0.01 0.35 ± 0.08 0.02 ± 0.01 4.71 ± 1.00 0.84 ± 0.15b

0.39 ± 0.08 0.06 ± 0.01 0.87 ± 0.22 0.43 ± 0.11 0.59 ± 0.35 0.42 ± 0.16 0.04 ± 0.01 0.10 ± 0.03 0.12 ± 0.02 1.33 ± 0.20

a Values in italics at 4 h pi represent animals co-injected with 20 nmol of [Tyr4]BBN for in vivo GRPR blockade. bHighly significant (P < 0.005) difference between blocked and unblocked animals (unpaired two-tailed Student’s t test).

Table 3. Biodistribution of [99mTc]3 and [99mTc]4 in PC-3 Xenograft-Bearing SCID Micea %ID/g tissue ± SD, n = 4 [99mTc]3 organ

[99mTc]4

1h

4h

24 h

blood

1.15 ± 0.37

0.02 ± 0.01

1.36 ± 0.47

liver

1.67 ± 0.34

0.36 ± 0.05

3.19 ± 0.20

heart

0.45 ± 0.14

0.06 ± 0.02

0.99 ± 0.25

kidneys

5.71 ± 1.23

0.58 ± 0.23

11.75 ± 0.42

stomach

0.58 ± 0.12

0.59 ± 0.35

3.23 ± 0.62

intestines

1.42 ± 0.27

0.28 ± 0.23

12.01 ± 0.32

spleen

0.71 ± 0.10

0.29 ± 0.08

3.20 ± 1.61

muscle

0.22 ± 0.07

0.04 ± 0.01

0.32 ± 0.07

lung

1.02 ± 0.32

0.08 ± 0.02

3.19 ± 2.53

pancreas

5.71 ± 0.41

0.19 ± 0.02

161.00 ± 10.37

tumor

15.48 ± 2.59

0.30 ± 0.03 0.08 ± 0.03 0.89 ± 0.14 2.11 ± 0.43 0.04 ± 0.01 0.09 ± 0.03 1.48 ± 0.03 2.38 ± 0.56 0.32 ± 0.2 0.45 ± 0.34 1.27 ± 0.27 2.85 ± 0.39 0.27 ± 0.06 1.33 ± 0.37 0.15 ± 0.01 0.02 ± 0.01 0.14 ± 0.05 0.55 ± 0.26 0.68 ± 0.06 0.37 ± 0.09 4.78 ± 1.47 0.53 ± 0.20b

1h

1.02 ± 0.25

28.80 ± 4.13

4h

24 h

0.50 ± 0.40 0.43 ± 0.31 2.78 ± 1.67 4.26 ± 0.79 0.33 ± 0.28 0.27 ± 0.10 4.87 ± 2.46 6.22 ± 2.92 3.81 ± 1.26 0.95 ± 0.62 11.40 ± 2.33 5.04 ± 0.82 2.76 ± 1.64 2.98 ± 1.88 0.15 ± 0.15 0.10 ± 0.06 1.95 ± 0.40 0.93 ± 0.12 119.04 ± 44.87 9.88 ± 1.08b 21.49 ± 3.18 5.66 ± 1.92b

0.13 ± 0.03 1.05 ± 0.19 0.11 ± 0.02 1.99 ± 0.65 2.20 ± 0.58 3.85 ± 1.06 0.81 ± 0.26 0.05 ± 0.02 0.86 ± 0.81 30.26 ± 14.65 16.32 ± 1.82

a

Values in italics at 4 h pi represent animals co-injected with 20 nmol [Tyr4]BBN for in vivo GRPR blockade. bHighly significant (P < 0.005) difference between blocked and unblocked animals (unpaired two-tailed Student’s t test).

Conversely, GRPR antagonists are not expected to trigger acute adverse responses after systemic administration and are consequently characterized by higher inherent biosafety. In the

preceding decades, a great number of GRPR antagonists have been developed for the study of the bombesin receptor system.39 They have also been evaluated as antiproliferative agents in 3143



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Table 4. Biodistribution of [99mTc]5 and [99mTc]6 in PC-3 Xenograft-Bearing SCID Micea %ID/g tissue ± SD, n = 4 [99mTc]6b

[99mTc]5 organ

1h

blood

1.98 ± 0.37

liver

3.14 ± 0.46

heart

0.88 ± 0.05

kidneys

11.29 ± 1.53

stomach

3.15 ± 0.37

intestines

10.77 ± 0.52

spleen

2.77 ± 2.56

muscle

0.25 ± 0.02

lung

1.76 ± 0.18

pancreas

181.05 ± 15.78

tumor

24.39 ± 2.45

4h

24 h

0.62 ± 0.11 0.32 ± 0.01 2.59 ± 0.80 4.13 ± 0.62 0.32 ± 0.07 0.29 ± 0.03 6.49 ± 1.97 5.57 ± 1.15 3.06 ± 1.05 0.57 ± 0.34 7.58 ± 1.26 6.14 ± 3.32 1.16 ± 0.34 1.15 ± 0.37 0.10 ± 0.03 0.07 ± 0.01 0.71 ± 0.15 0.76 ± 0.28 88.39 ± 6.23 19.43 ± 6.01 18.91 ± 4.98 4.82 ± 1.55c

0.07 ± 0.01

1.69 ± 0.35

1h

0.61 ± 0.17

4.16 ± 0.19

0.14 ± 0.02

0.98 ± 0.15

1.10 ± 0.38

12.59 ± 1.03

0.60 ± 0.32

1.75 ± 0.51

1.22 ± 0.06

6.14 ± 0.96

0.48 ± 0.06

1.33 ± 0.15

0.05 ± 0.02

0.31 ± 0.03

0.17 ± 0.02

2.69 ± 1.24

4.43 ± 1.92

60.98 ± 11.67

4.54 ± 1.30

12.08 ± 3.35

4h 0.17 ± 0.02 0.16 ± 0.04 2.83 ± 0.64 4.73 ± 0.11 0.16 ± 0.04 0.20 ± 0.01 4.96 ± 0.88 7.68 ± 1.65 1.75 ± 0.77 0.91 ± 0.93 3.11 ± 0.98 2.67 ± 0.06 0.58 ± 0.15 1.17 ± 0.19 0.05 ± 0.01 0.05 ± 0.01 0.28 ± 0.05 0.60 ± 0.16 6.83 ± 2.55 0.92 ± 0.11c 3.53 ± 1.49 0.74 ± 0.07c

a Values in italics at 4 h pi represent animals co-injected with 20 nmol [Tyr4]BBN for in vivo GRPR blockade. bFor [99mTc]6 the 24 h pi results have not been included due to the very low and declining tumor uptake already in the earlier time points. cHighly significant (P < 0.005) difference between blocked and unblocked animals (unpaired two tailed Student’s t test).

xenografts in mice clearing faster from physiological tissues than from tumor sites, resulting in attractive in vivo profiles.24,25 Yet sufficient retention at the tumor site(s) for longer periods of time remains a significant challenge for a wider use of GRPR radioantagonists in tumor diagnosis in a clinical setting and particularly in radionuclide therapy. In our search for anti-GRPR radiotracers of clinical interest, we have previously presented [99mTc]1,19 based on the potent GRPR antagonist [DPhe6,Leu13-NHEt]BBN(6−13)20,21 and exhibiting excellent GRPR-specific accumulation in human PC-3 xenografts in nude mice and much faster washout from physiological tissues, including the GRPR-rich mouse pancreas. However, at 24 h pi the tumor uptake declined to less than 25% of its value at 4 h pi, leaving space for improvements.24 On the other hand, longer tumor-residing times of the radiotracer, especially when combined with the faster background clearance of a receptorantagonist, may lead to favorably enhanced-contrast imaging and hence to improved diagnostic accuracy. Such prolonged tumor retention of a diagnostic 99mTc-radiotracer may provide opportunities for radionuclide therapy with the analogous 186 Re/188Re-therapeutic agent, based on the similarities of Tc and Re chemistries. Aiming toward prolonged tumor retention, we hereby introduce a series of [99mTc]1 mimics generated by diverse structural modifications on the [99mTc]1 motif (Chart 1; Introduction). The impact of these changes on the GRPR affinity of 1−8 was minor, with receptor affinities of all analogs remaining in the subnanomolar range (Figure 1). This finding is in agreement with previous reports on the favorable influence of the pendant cationic N-terminal tetraamine framework on the binding affinity of BBN-like analogs to the GRPR.33 It is interesting to note that by replacing the tetraamine chelator by negatively charged

Figure 5. Maximum intensity projection (MIP) SPECT/CT static images of mice bearing subcutaneous PC-3 xenografts on their right shoulders euthanized 15 h after injection of (A) [99mTc]1 or (B) [99mTc]4; in both cases animals received 20 MBq radiotracer corresponding to 200 pmol of peptide conjugate. The GRPR specificity of tumor uptake could be demonstrated for [99mTc]4 by comparing the transaxial images of PC-3 tumors in mice euthanized 1 h after receiving 20 pmol/2.4 MBq [99mTc]4 (C) alone or (D) after coinjection of 20 nmol of [Tyr4]BBN.

several cell lines and, most importantly, in animal models bearing GRPR-positive xenografts.39−42 The above efforts have generated a broad collection of GRPR antagonists potentially serving as motifs for the development of the respective radiolabeled GRPR antagonists for diagnostic imaging of human cancer. Despite their inability to internalize, several GRPR radioantagonists were able to successfully visualize GRPR-expressing 3144



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led to a drop in stability ([99mTc]6, 60% intact). Currently ongoing studies are expected to reveal the role of neutral endopeptidase or other peptidase(s) operating alone or together in the in vivo catabolism of these analogs,44,47,49,50 as suggested by the discrepancies observed in radiometabolite patterns. The much faster in vivo degradation [99mTc]1 compared to previously reported results from in vitro mouse plasma incubates (>85% intact at 1 h)19 is in agreement with recent reports on the limited relevance of such studies to reliably evaluate radiopeptide stability. Similar observations may be deduced by comparing the metabolic stability of [99mTc]4 determined in vivo in mice (60% intact at 5 min pi; Figure 4D) and in vitro in mouse plasma incubates included in this work for further comparison purposes (>67% intact at 30 min; Figure S1C). Both [99mTc]1 and [99mTc] 4 were excreted predominantly in the form of radiometabolites in the urine of mice at 30 min pi (Figure S2C). However, the observed complete degradation of [99mTc]4 in the urine (and also [99mTc]1)19 should be rather assigned to the action of enzymes encountered after glomerular filtration of the tracer in the kidneys and should be not erroneously linked to the actual metabolic stability of circulating [99mTc]4 at later time points. This assumption is strongly supported by the fast degradation of [99mTc]4 in mouse kidney homogenates observed in vitro (Figure S2B). In mice bearing human PC-3 xenografts, all analogs showed GRPR-specific tumor uptake and rapid clearance from background tissues. However, tumor uptake significantly differed at 1 h pi across analogs as a combined result of individual cell binding capacity assessed in vitro and bioavailability. Notably, the highest cell-associating member with an acceptable in vivo metabolic stability [99mTc]4 exceeded the lead compound [99mTc]1 in tumor uptake, whereas the least cell-binding and least in vivo stable member [99mTc]2 displayed the lowest tumor uptake. Remarkably, [99mTc]4 was the only member in this series displaying not only the highest tumor uptake at 1 h pi (28.80 ± 4.13%ID/g) but also the longest retention at 24 h pi (16.32 ± 1.82%ID/g). Similar tumor retention values were reported in the same animal model for another 99mTc labeled tetraaminefunctionalized tracer based on a C-terminal Sta13-Leu14-NH2 GRPR antagonist.43 By achieving significantly improved tumor values at 24 h pi compared to [99mTc]1 (5.38 ± 0.72%ID/g), [99mTc]4 represents a promising candidate for further validation in man. In comparison to the [99mTc]1 reference, longer retention of [99mTc]4 is observed in mouse pancreas even at 24 h pi, but at this stage it is difficult to predict if and to what extent this attribute will be translated in human. Furthermore, the radioactivity patterns of [99mTc]4 and [99mTc]1 at 15 h pi in a preclinical SPECT/CT camera appeared to be comparable. Interestingly, the uptake in the PC-3 tumor was superior for [99mTc]4 despite the technical limitations not allowing a later-point imaging and/or a reliable quantitative comparison. In this regard, it is interesting to compare the respective quantitative tumor-tobackground ratios of [99mTc]1 mimics over time (Figure S3). In most cases, [99mTc]4 resulted not only in superior absolute tumor-uptake but in higher tumor-to-background ratio values too, especially at later time intervals. This observation is very promising for high quality imaging in patients. This option becomes attractive when taking into account contemporary exciting technological advances in clinical SPECT instrumentation, including the development of state-of-the-art hybrid SPECT/CT and SPECT/MRI systems for hospitals.51 The observed “renaissance” of SPECT is also reflected in latest efforts to enhance the production capacity of 99 Mo, and thus, its 99m Tc-daughter adopting new processes to ensure unperturbed

DOTA in SB3, the GRPR affinity was found >19-fold lower (IC50 = 5.0 ± 0.04 nM)26 compared to 4 (IC50 = 0.26 ± 0.03 nM), in support of this observation. It should be noted that an analogous switch in GRPR affinity (from 3.7 ± 1.3 nM to 35 ± 0.5 nM) was observed when substituting the N4-to-DOTA in another set of GRPR antagonists, coupled to the Gly-4-aminobenzoyl-DPheGln-Trp-Ala-Val-Gly-His-Sta-Leu-NH2 sequence.43 As exemplified by the binding affinity of [99mTc/99gTc]4 for the GRPR (Kd = 0.24 ± 0.03 nM), incorporation of the radiometal by the tetraamine framework under formation of a monocationic metal chelate did not negatively affect interaction with the receptor, in agreement with previous observations on [99mTc/99gTc]1 and other tetraamine-derivatized peptide receptor-ligands.19,33 The antagonistic properties at the GRPR were preserved after N-terminal coupling of the chelator as verified by an immunofluorescence microscopy-based receptor internalization assay for the two representative analogs with the highest GRPR affinity, 4 and 8, as previously reported also for reference 1.24 Access to the corresponding [99mTc]1 mimics was straightforward, as reported for several other acyclic tetraamine-functionalized peptides, with the exception of the two hydrazides, 7 and 8. In the latter case a mixture of unidentified radiochemical species formed, most probably due to interaction of the hydrazide function with the metal center.32 The rest of the radioligands [99mTc]1−[99mTc]6 showed a consistent GRPR radioantagonist behavior during a cell internalization assay, with the bulk of radioactivity localizing on the cell membrane. The highest cellbinding member in the series was [99mTc]4, which well-surpassed the cell-binding of [99mTc]1. This finding showed that minor linker “architecture” differences were sufficient to provoke such pronounced response differences in PC-3 cells. The exceptionally high PC-3 cell association of [99mTc]4 may be indicative of higher uptake and longer retention in PC-3 tumor lesions in mice (vide infra). A significant prerequisite for effective in vivo tumor targeting of radiopeptides is bioavailability. Radiopeptides due to their small size exhibit very rapid in vivo kinetics and localize very fast in tumor sites.44 Therefore, metabolic integrity in the first critical minutes when they need to reach their target is decisive for optimum supply and uptake to tumor sites. Several recent studies have shown that in vitro serum stability results may not be representative of the real situation encountered by the circulating radiotracer in the body. Metabolism often occurs much faster in vivo simply because of the bigger pool of proteases present in vivo, leading to a higher number of potential degradation pathways. Accordingly, the action of highly abundant cell-surface proteases (often not or only in traces present in serum) anchored on blood cells, endothelial cells of blood vasculature, and major organs is totally disregarded during in vitro serum incubation assays.44−47 Furthermore, in vitro stability studies using incubates of tissue homogenates are also problematic, because cell compartmentalization is disrupted and radioligands are exposed to enzymes which they do not normally encounter in systemic circulation.19,44 In view of the above, the metabolic stability of [99mTc]1 mimics was tested in peripheral blood 5 min after intravenous administration of the radiotracers. The percentage of intact radioligand detected was in the range of 45−75%. The Gly11-to-DAla11 replacement improved metabolic stability ([99mTc]5, 75% intact), as well as the use of the PEG2 linker ([99mTc]3, 70% intact), while the absence of linker deteriorated stability ([99mTc]2, 45% intact). Additional Gln7-to11 DGln replacement undertaken based on previous studies on the high potency of similarly bis-substituted BBN(7−14) analogs48 3145



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global supply of the preeminent SPECT radiometal, 99mTc.52 It is imperative that exciting evolutions in clinical imaging instrumentation are matched by new generation 99mTc radiotracers for high quality imaging of disease, as represented by [99mTc]4. Furthermore, Tc and its congener Re are governed by similar chemistries and indeed acyclic tetraamines form isostructural Tc/Re chelates.53 Thus, the option of making available [99mTc] 4/[186Re/188Re]4 radiopeptide pairs for theranostic applications is particularly compelling and warrants further investigation.

excess of the respective Fmoc amino acid derivatives, N,N′-diisopropylcarbodiimide (DIC), 1-hydroxybenzatriazole (HOBt), and DIPEA in a 90:10 v/v mixture of N,N-dimethylformamide (DMF) and dichlormethane (DCM) as solvent. All used amino acids were protected with Nα Fmoc, and His and Gln were protected with Trt in the side chain. Finally the tetra-Boc protected tetraamine chelator N4′ (N,N′,Ν″,Ν′′′tetra-(tert-butoxycarbonyl)-6-{p-[(carboxymethoxy)acetyl]aminobenzyl}1,4,8,11-tetraazaundecane) was coupled (in 3 equiv excess) to the assembled amino acid sequence on the resin in the presence of (benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyBOP) and DIPEA in DMF. Cleavage of 1 from the solid support was achieved by treatment with TFA. The crude product was precipitated with cold diethyl ether, collected, and dissolved in a MeCN/H2O mixture for lyophilization. Synthesis of Ethylamides 2−6. Synthesis of 2−6 involved several steps. First the sequences, beginning with the C-terminal His and ending with N-terminal DPhe, were built on a Fmoc-His(Trt)-trityl resin (substitution >0.15 mmol/g) following solid phase peptide synthesis (SPPS) methodologies. After Fmoc deprotection, anchoring of each amino acid was achieved with a 10-fold excess of the Fmoc amino acid derivative in the presence of DIC, HOBt, and DIPEA in a 90:10 v/v mixture of DMF and DCM. Coupling of Fmoc-O2Oc-OH 8-(9fluorenylmethyloxycarbonylamino)-3,6-dioxaoctanoic acid (for 3) and Fmoc-p-aminomethylaniline diglycolate (for 4−6) to the N-terminal amine was completed in the presence of PyPOB and DIPEA in DMF, and Fmoc-deprotection was carried out with 30% piperidine in DMF. The tetra-Boc protected tetraamine chelator N4 (N,N′,Ν″,Ν′′′tetrakis(tert-butoxycarbonyl)-6-(carboxy)-1,4,8,11-tetraazaundecane) was then coupled to the assembled sequences with PyBOP and DIPEA in DMF, and the resin was subsequently treated for 15 min at ambient temperature with a mixture of 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP), triethylsilane (TES), and DCM in a ratio 20/5/75 v/v/v. The solvent was removed by rotary evaporation in vacuum to afford the protected crude “His conjugates”. The analogs were purified by RP-HPLC on an AKZO Nobel Kromasil Semi/Prep C18 column (250 mm × 20 mm). Fractions containing the desired analog were collected, and the solvent was removed by lyophilization. The protected conjugates were subsequently coupled with H-Leu-NHEt hydrochloride by addition of PyPOB and DIPEA in DMF. The reaction mixture was diluted with H2O and freeze-dried. A mixture of trifluoroacetic acid (TFA), 1,2-ethandithiole (EDT), thioanisole, and water in a ratio 90:4:4:2 v/v/v/v was added to the solid and incubated at ambient temperature for 1 h to remove all protecting groups. Transfer of the cocktail into ice-cooled diethyl ether led to precipitation of the crude ethylamides 2−6, which were collected, dissolved in MeCN/H2O 40/60 v/v, and lyophilized. Synthesis of Hydrazides 7 and 8. Synthesis of the C-terminal hydrazide analogs 7 and 8 was performed on a 4-hydroxymethylbenzoic acid AM resin (HMBA) with a capacity of 0.7 mmol/g. Coupling of Fmoc-Leu-OH to the resin was achieved by preswelling for 30 min at ambient temperature and subsequent addition of a cocktail of 10 equiv of Fmoc-Leu-OH and 5 equiv of DIC with respect to the loading in DMF, which was stirred 20 min at 0 °C in advance. After incubation of the reaction mixture at ambient temperature for 1 h the resin was filtered off and washed with DMF several times. The assembling of the peptide sequences and the coupling of diglycolate and the tetra-Boc protected tetraamine chelator N4′ (for 7) or of Fmoc-p-aminomethylaniline diglycolate and the chelator N4 (for 8) were completed, as described above for 1 and 4, respectively. A solution of 5% hydrazine hydrate in DMF was added to the resin, and the suspension was left to stand at ambient temperature for over 1 h. The resin was filtered off and washed with DMF. The liquid layer was lyophilized to remove the DMF, and the residue was treated with a mixture of TFA, EDT, thioanisole, and water in a ratio 90:4:4:2 v/v/v/v to remove all protective groups. Finally, the crude hydrazine derivatives were precipitated with cold diethyl ether, collected, dissolved in MeCN/water 50/50 v/v, and lyophilized. Crude products for all above synthesized analogs 1−8 were purified by RP-HPLC on an AKZO Nobel Kromasil Semi/Prep C18 column (250 mm × 20 mm). Fractions containing the desired peptide were collected, and the solvent was removed by lyophilization. The compound

■

CONCLUSIONS This structure−activity-relationships study has revealed the profound impact of minor linker variations on several biological parameters of this small library of [99mTc]1 mimics, which synergistically affect end-pharmacokinetics and in particular tumor uptake and retention. It has further revealed [99mTc]4 as the best candidate for clinical translation owing to its advantageously high uptake and prolonged retention in GRPRexpressing tumors, which significantly surpassed our “gold standard” [99mTc]1, as well as to excellent tumor-to-background ratios that increased with time. These qualities, besides offering practical advantages in terms of time flexibility for conveniently completing diagnostic imaging in a clinical setting, provide further opportunities for later-time imaging, anticipated to result in advantageous higher-contrast images and enhanced diagnostic accuracy. They may also pave the way for theranostic patient management with the application of twin diagnostic [99mTc]4 and therapeutic [186Re/188Re]4 pairs.

■

EXPERIMENTAL SECTION

Materials and Instrumentation. Compounds and Radionuclides. All chemicals used were reagent grade. Fmoc-O2-Oc-OH 8-(9-fluorenylmethyloxycarbonylamino)-3,6-dioxaoctanoic acid was purchased from Iris Biotech (Germany) and Fmoc-ethylindole AM resin as well as 4-hydroxymethyl benzoic acid AM resin from Novabiochem. BBN (Pyr-Gln-Arg-Phe-Gly-Asn-Gln-Trp-Ala-Val-Gly-HisLeu-Met-NH2 ) and [Tyr 4 ]BBN were obtained from Bachem (Bubendorf, Switzerland). For radiolabeling, 99mTcO4− was eluted in normal saline from a commercial 99Mo/99mTc generator (Ultratechnekow, Tyco Healthcare, Petten, The Netherlands), while Na125I was purchased from MDS Nordion, SA (Canada) in a 10−5 M NaOH solution (10 μL). Technetium-99g was purchased from Oak Ridge National Laboratories, USA, as NH499gTcO4. The impure black solid was purified prior to use by overnight treatment with H2O2 and NH4OH in MeOH. Evaporation of the solvent afforded NH499gTcO4 as a white powder. Analysis, Radiochemistry. MALDI-TOF mass spectrometry data were acquired from a MALDI-TOF spectrometer Kompact Kratos Axima Analytical, Shimadzu; Manchester, U.K. HPLC analyses and separations were conducted on a Waters RP-18 XTerra column (5 μm, 3.9 mm × 20 mm) using a Waters chromatograph with a 600 solvent delivery system coupled to a 996 photodiode array UV detector and a Gabi γ detector (Raytest RSM Analytische Instrumente GmbH, Germany). The Millennium Software was used for data processing and for controlling the HPLC system. An automated well-type γ counter [NaI(Tl) 3 in. crystal, Canberra Packard Auto-Gamma 5000 series instrument] calibrated for 99mTc or 125I was used for radioactivity measurements. Synthesis of 1−8 on the Solid Support. Synthesis of 1. Synthesis of 1 was carried out on an ethylindole AM resin (capacity 0.83 mmol/g) as solid support. First the Fmoc group was removed by incubation of the resin with a solution of piperidine in DMF in a ratio 30/70 v/v. Fmoc-Leu-OH was coupled on the resin by use of a 3-fold excess of the amino acid, O-(7-azabenzotriazolyl-1,1,3,3-tetramethylammonium hexafluorophosphate (HATU) and N-ethyldiisopropylamine (DIPEA) in DMF. After confirmation of the effective amino acid loading via UV absorption measurement of the Fmoc group, elongation of the sequence was performed on an automated synthesizer using a 10-fold 3146



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100 U/mL penicillin, 100 μg/mL streptomycin, and 750 μg/mL G418. Cells were kept in a controlled humidified air containing 5% CO2 at 37 °C. Splitting of cells with a ratio of 1:3 to 1:5 was performed when approaching confluency using a trypsin/EDTA solution (0.05%/0.02% w/v). In Vitro Assays. Competition Binding Assays with 1−8 in PC-3 Cell Membranes. PC-3 cell membrane homogenates were prepared as previously described.57 The [125I-Tyr4]BBN radioligand (∼40 000 cpm per assay tube, at a 50 pM concentration) was incubated at 22 °C for 45 min in triplicates in PC-3 cell membrane homogenates and increasing concentrations of test peptide, either each of unlabeled analogs 1−8 or [Tyr4]BBN as reference, in a total volume of 300 μL binding buffer (BB, 50 mM HEPES, pH 7.4, 1% BSA, 5.5 mM MgCl2, 35 μM bacitracin) in an incubator−orbital shaker (MPM Instr. SrI, Italy). Binding was interrupted by ice-cold washing buffer (WB, 10 mM HEPES, pH 7.4, 150 mM NaCl) and rapid filtration (Whatman GF/B filters presoaked in BB) on a Brandel cell harvester (Adi Hassel Ing. Büro, Munich, Germany). Filters were washed with ice-cold WB and counted in the γ counter. The half maximal inhibitory concentration (IC50) values were calculated using nonlinear regression according to a one-site model applying the PRISM 2 program (Graph Pad Software, San Diego, CA). Immunofluorescence Microscopy-Based Internalization Assay with 4 and 8. Immunofluorescence microscopy-based internalization assays with HEK293-GRPR cells were performed as previously described.24 In brief, HEK293-GRPR cells were grown on poly-D-lysine (20 μg/mL) (Sigma-Aldrich, St. Louis, MO) coated 35 mm four-well plates (Cellstar, Greiner Bio-One GmbH, Frickenhausen, Germany). For the experiment, cells were treated either with vehicle alone (no peptide) or with 10 nM BBN or 1 μM unlabeled analog 4 or 8 alone, or (to evaluate potential antagonistic properties) with 10 nM BBN in the presence of 1 μM 4 or 8 for 30 min at 37 °C and 5% CO2 in growth medium, and then they were processed for immunofluorescence microscopy using a mouse monoclonal HA-epitope antibody (Covance; Berkeley, CA) at a dilution of 1:1000 as first antibody and Alexa Fluor 488 goat anti-mouse IgG (H+L) (Molecular Probes, Inc.; Eugene, OR) at a dilution of 1:600 as secondary antibody. The cells were imaged using a Leica DM RB immunofluorescence microscope and an Olympus DP10 camera. Cell-Association/Internalization of [99mTc]1−[99mTc]6 in PC-3 Cells. Confluent PC-3 cells were seeded in six-well plates ((1.0−1.5) × 106 cells per well) and were incubated for 24 h. Cells were rinsed twice with ice-cold internalization medium containing DMEM GlutaMAX-I supplemented with 1% (v/v) FBS, and then fresh internalization medium (37 °C) was added (1.2 mL). Approximately 300 000 cpm of test radioligand (∼200 fmol total peptide in 150 μL of 0.5% BSA PBS) was added per well, and the experiment was performed after 1 h incubation at 37 °C, as previously described. Nonspecific cell-binding was determined by a parallel triplicate series containing 1 μM [Tyr4]BBN. Surface bound radioligand was separated from the internalized radioactivity by treatment of the cells with acidic buffer (50 mM glycine buffer, pH 2.8, 0.1 M NaCl). Samples were measured for their radioactivity content, and the percent total cell bound, surface-bound, and internalized radioactivity could be calculated. Saturation Binding of [99mTc/99gTc]4 in PC-3 Cell Membranes. For saturation binding experiments two sets of [99mTc/99gTc]4 triplicates of different radioligand concentrations were prepared for total and nonspecific binding. Each assay tube contained 50 μL of binding buffer (50 mM HEPES, pH 7.6 containing 0.3% BSA, 10 mM MgCl2, 14 mg/L bacitracin), 50 μL of [99mTc/99gTc]4 solution of the corresponding concentration (final concentration series in the range of 0.01−10 nM), and 200 μL of PC-3 cell membrane homogenates containing 40 μg of protein. For the nonspecific series, instead of 50 μL of binding buffer 20 μL of binding buffer plus 30 μL of 10 μM [Tyr4]BBN solution was used. Tubes were incubated at ambient temperature for 1 h and binding was interrupted by addition of ice-cold buffer (10 mM HEPES, 150 mM NaCl, pH 7.6) and rapid filtration through glass fiber filters (Whatman GF/B, presoaked in 0.3% BSA) on the Brandel cell harvester. Filter activity was measured on the γ counter. Nonspecific binding was defined as the amount of activity binding in the presence of 1 μM [Tyr4]BBN, and the equilibrium dissociation constant (Kd) was calculated after fitting the data to a one-site model.

purity was tested by analytical HPLC in two different systems: system 1, RP-HPLC with UV detection at 220 nm, and a Waters Xterra RP18 column (5 μm, 4.6 mm × 150 mm) eluted at a flow rate of 1 mL/min with linear gradient 15% B to 30% B in 30 min, with A = 0.1% TFA in H2O (v/v) and B = MeCN; system 2, an Agilent system with UV detection at 215 nm and with a Nucleosil-100 C18 column (5 μm, 150 mm × 4 mm) eluted at a flow rate of 1 mL/min with a linear gradient 0.1% TFA in MeCN (from 10% to 90% in 15 min) and 0.1% aqueous TFA as complementary phase. Analytical RP-HPLC data acquired from two separate HPLC systems (system 1 and system 2) confirmed the ≥95% purity of all analogs and are included in Table 1, along with MALDITOF MS results, consistent with the expected formulas. Radiolabeling and Quality Control. The lyophilized peptide analogs were dissolved in 50 mM acetic acid/EtOH 8/2 v/v to a final 1 mM concentration and stored at −20 °C in 50 μL aliquots. For 99mTclabeling the following solutions were added into an Eppendorf tube containing 0.5 M phosphate buffer, pH 11.5 (25 μL): 0.1 M sodium citrate (3 μL), [99mTc]NaTcO4 (210 μL, 140−280 MBq) generator eluate, 1−8 stock solution (7.5 μL, 7.5 nmol), and finally fresh SnCl2 solution in EtOH (5 μg, 5 μL). After 30 min incubation at ambient temperature the reaction mixture was neutralized by addition of 1 M HCl (4 μL), and EtOH was added (25 μL). Quality control comprised radioanalytical HPLC and instant thin-layer chromatography (ITLC). HPLC analyses were performed on a Waters chromatograph coupled to a 996 photodiode array UV detector (Waters, Vienna, Austria) and a Gabi γ detector (Raytest RSM Analytische Instrumente GmbH, Germany). For analysis, a Waters RP-8 XTerra cartridge column was eluted at a 1.0 mL/min flow rate with the following gradient: 0% B to 40% B in 20 min, where A = 0.1% aq TFA, B = MeCN (system 3). Under these conditions 99mTcO4− eluted at 1.8 min and [99mTc]1 mimics with a tR > 13 min. For the detection of reduced hydrolyzed technetium (99mTcO2·xH2O) ITLC was conducted on ITLC-SG strips (Pall Corporation, NY/USA), as previously described.19 [99gTc]4 was prepared by following a similar protocol as its [99mTc]4 counterpart at tracer level. Due to the higher mass of [99gTc]NH4TcO4 utilized (0.9 μg, 5 nmol), an increased amount of SnCl2 (20 μg, 0.1 μmol) was required for complete reduction of Tc(VII) to Tc(V). The peptide was purified by RP-HPLC adopting binary γ and photometric detection modes. A Waters Symmetry Shield RP-18 cartridge column (5 μm, 3.9 mm × 20 mm) was eluted at 1 mL/min flow rate with the following gradient system: 0−1 min 0% to 15% B, 1−31 min 15% to 30% B, with A = 0.1% TFA and B = MeCN. Under these conditions, [99mTc/99gTc]4 (tR = 17.6 min; both γ and UV trace) was easily separated as a single species from nonmetalated 4 (tR = 13.6; min UV trace). Triplicates in the 10−12−10−6 M concentration range were prepared and used for saturation binding experiments, as previously described.19 Radioiodination of [Tyr4]BBN was accomplished following the chloramine-T method. The forming sulfoxide (Met14O) was reduced by dithiothreitol, and [125I-Tyr4]BBN was isolated in a highly pure form by HPLC.22,54 Methionine was added to the purified radioligand solution to prevent oxidation of Met14 to the corresponding sulfoxide, and the resulting stock solution in 0.1% BSA-PBS was kept at −20 °C; aliquots thereof were used in competition binding assays (74 GBq /μmol). All manipulations with β (e.g., the pseudostable long-lived 99gTc, β− 294 keV, t1/2 = 2.1 × 105 years) and γ emitting (e.g., 99mTc, t1/2 = 6 h, monoenergetic γ photons 140 keV) radionuclides and their solutions were performed behind suitable shielding in dedicated laboratories in compliance with national and international radiation safety guidelines and supervised by the Greek Atomic Energy Commission. Cell Culture. Human androgen-independent prostate adenocarcinoma PC-3 cells endogenously expressing the GRPR (by LGC Promochem, Teddington, U.K.)55 and HEK293 cells transfected to stably express the HA epitope-tagged human GRPR24,56 were used in this study. All culture media were purchased from Gibco BRL, Life Technologies (Grand Island, NY, USA), and supplements were supplied by Biochrom KG Seromed (Berlin, Germany). PC-3 cells were cultured in Dulbecco’s modified Eagle medium (DMEM) with GlutaMAX-I supplemented with 10% (v/v) fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin. HEK293-GRPR cells were grown in DMEM with GlutaMAX-I supplemented with 10% (v/v) FBS, 3147



Article

Metabolic Stability. In Vivo Stability of [99mTc]1−[99mTc]6 in Healthy Mice. A bolus of [99mTc]1−[99mTc]6 (100 μL, 50−60 MBq, 3 nmol total peptide; in saline/EtOH 9/1 v/v) was injected in the tail vein of male Swiss albino mice (30 ± 5 g, NCSR “Demokritos” Animal House Facility). Blood withdrawn 5 min pi was directly placed in prechilled polypropylene tubes containing EDTA on ice. Samples were centrifuged (10 min, 2000g/4 °C, in a Hettich, Universal 320R centrifuge, Tüttlingen, Germany), the plasma was collected, mixed with chilled MeCN in a 1/1 v/v ratio, and centrifuged again (10 min, 5000g/4 °C). Supernatants were concentrated to a small volume under a gentle N2 flux at 40 °C, diluted with physiological saline (400 μL), and filtered through a Millex GV filter (0.22 μm). Aliquots thereof were analyzed by HPLC (system 4) on a Waters RP-18 Symmetry Shield column (5 μm, 3.9 mm × 150 mm) adopting the following gradient: 100% A/0% B to 40% A/60% B within 60 min (A = 0.1% aqueous TFA and B = MeCN) at a flow rate of 1 mL/min; the tR of intact [99mTc]1−[99mTc]6 was determined by co-injection of a parent radioligand sample. ITLC-SG was performed in parallel using acetone as the eluent to detect traces of 99m TcO4− release (99mTcO4− Rf = 0.9). Biodistribution in SCID Mice Bearing Human PC-3 Xenografts. Suspensions of freshly harvested PC-3 cells (∼150 μL, 1.4 × 107) in saline were subcutaneously injected in the flanks of female SCID mice (15 ± 3 g, 6 weeks of age animals at the day of arrival were employed, NCSR “Demokritos” Animal House Facility). The animals were kept under aseptic conditions and 3 weeks later developed well-palpable tumors at the inoculation site (80−150 mg). On the day of the experiment, each of [99mTc]1−[99mTc]6 was injected as a bolus (100 μL, 185 kBq, 10 pmol total peptide; in saline/EtOH 9/1 v/v) in the tail vein of mice; for in vivo GRPR blockade separate 4 h animal groups were co-injected with excess [Tyr4]BBN (20 nmol). Animals were euthanized in groups of four at 1, 4, and 24 h pi; samples of blood were immediately collected, and tissues of interest and tumors were excised, weighed, and counted for radioactivity in the γ counter. Data were calculated as percent injected dose per gram tissue (%ID/g) with the aid of suitable standards of the injected dose and are presented as mean ± sd. The unpaired two tailed Student’s t test of GraphPad Prism Software (San Diego, CA) was applied to evaluate statistically significant differences. P values of

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