Evaluation of Three Different Families of Bombesin Receptor

Article pubs.acs.org/jmc

Evaluation of Three Different Families of Bombesin Receptor Radioantagonists for Targeted Imaging and Therapy of Gastrin Releasing Peptide Receptor (GRP-R) Positive Tumors Rosalba Mansi,†,‡ Keelara Abiraj,†,⊥ Xuejuan Wang,† Maria Luisa Tamma,†,∇ Eleni Gourni,‡ Renzo Cescato,§,# Sandra Berndt,∥ Jean Claude Reubi,§ and Helmut R. Maecke*,†,‡ †

Division of Radiological Chemistry, University Hospital Basel, Petersgraben 4, CH-4031 Basel, Switzerland Department of Nuclear Medicine, University Hospital Freiburg, Hugstetter Strasse 55, 79106 Freiburg, Germany § Division of Cell Biology and Experimental Cancer Research, Institute of Pathology, University of Berne, Murtenstrasse 31, CH-3010 Berne, Switzerland ∥ Global Drug Discovery, Bayer Healthcare, Berlin, Muellerstrasse 178, 13342 Berlin, Germany ‡

S Supporting Information *

ABSTRACT: Two new classes of radiolabeled GRP receptor antagonists are studied and compared with the well-established statine-based receptor antagonist DOTA-4-amino-1-carboxymethylpiperidine-D-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH2 (RM2, 1; DOTA:1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid; Sta:(3S,4S)-4-amino-3-hydroxy-6-methylheptanoic acid). The bombesin-based pseudopeptide DOTA-4-amino-1-carboxymethylpiperidine-D-Phe-Gln-Trp-Ala-Val-Gly-His-Leuψ(CHOH-CH2)-(CH2)2-CH3 (RM7, 2), and the methyl ester DOTA-4-amino-1-carboxymethylpiperidine-D-Phe-Gln-Trp-AlaVal-Gly-His-Leu-OCH3 (ARBA05, 3) analogues are labeled with 111In and evaluated in vitro in PC-3 cell line and in vivo in PC-3 tumor-bearing nude mice. Antagonist potency was assessed by immunofluorescence-based receptor internalization and Ca2+ mobilization assays. The conjugates showed good binding affinity, the IC50 value of 2 (3.2 ± 1.8 nM) being 2 and 10 times lower than 1 and 3. Compared to 111In-1, 111In-2 showed higher uptake in target tissues such as pancreas (1.5 ± 0.5%IA/g and 39.8 ± 9.3%IA/g at 4 h, respectively), whereas the compounds had similar tumor uptake (11.5 ± 2.4%IA/g and 11.8 ± 3.9%IA/g at 4h, respectively). The displacement of the radioligand in vivo was different in different receptor positive organs and depended on the displacing peptide.

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The recent finding of the superior properties of radiolabeled somatostatin receptor antagonists15 prompted numerous research groups to investigate the potential use of radiolabeled GRP-R antagonists. Several analogues have been explored, which showed promising results encouraging development of bombesin antagonist based radiopharmaceuticals for the diagnosis and treatment of prostate cancer.16−21 For instance, Abd-Elgaliel et al.,19 developed a propyl amide bombesin derivative with the spacer aminohexanoyl and the chelator DOTA at the N-terminus (111In-DOTA-aminohexanoyl-[D-Phe,6Leu-NHCH2CH2CH3,13des-Met14]-bombesin(6−14)) supporting the use of radiolabeled bombesin antagonists as potential candidates for specific in vivo imaging of GRP-R positive tumors.

INTRODUCTION

A technique emerging in cancer management is the targeting of specific peptide receptors overexpressed on cancer cells.1,2 The three mammalian bombesin receptors are overexpressed on human tumors; in particular the gastrin releasing peptide receptor (GRP-R) has high expression on prostate cancer, breast cancer, small cell lung cancer, ovarian cancer, endometrial cancer, and gastrointestinal stromal tumors.3−6 Radiolabeled GRP-R peptides7−10 have been studied for imaging of prostate cancer, which is now considered the leading cause of cancer related death in men in the USA and Europe.11 In the early 80s, on the basis of the observation of growth stimulatory effects of bombesin agonists in several solid malignancies in preclinical models,12,13 a series of bombesin antagonists have been synthesized and evaluated in experimental tumor systems.14 © 2014 American Chemical Society

Received: August 6, 2014 Published: December 4, 2014 682

dx.doi.org/10.1021/jm5012066 | J. Med. Chem. 2015, 58, 682−691

Journal of Medicinal Chemistry

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Figure 1. Structural formulas of 1, 2, and 3.

labeled bombesin-based peptide agonists may be the probes of choice for prostate cancer imaging due to their high tumor uptake and retention and hypothesized that this may be a property of the radiometal labeled bombesin antagonists solely. In addition, the Missouri group (Hoffman T. J., Smith C. J., et al.)27 compared NODAGA conjugated and 64Cu labeled agonist vs antagonist with similar GRP-R affinity and concluded as well that based on microPET imaging the agonist is a superior molecular imaging agent for targeting the GRP-R. On the basis of the good in vitro and in vivo properties of 5 and clear superiority over the bombesin agonist, 4, we continued to study new statine-based bombesin antagonists having different spacers and chelators aiming to improve pharmacokinetics and binding affinity for use in imaging and targeted radionuclide therapy.17,21 Our promising preclinical results with radiolabeled receptor antagonists led to early clinical translation. 64Cu-CB-TE2A-AR0618 was studied in an early human-use approach in primary prostate cancer patients showing its high potential in the visualization of these tumors and 68Ga-1 (68Ga-RM221) completed dosimetry and safety studies in human volunteers28 and a phase 1 study in a limited number of prostate cancer patients.29,30 For therapeutic application, high tumor accumulation and long retention are needed. With this in mind and the growing debate regarding the superiority of one or the other (agonists

In the last years, several groups have compared the in vivo tumor targeting performance of different bombesin-based radioconjugates. Still there is no consensus in regard to the superiority of bombesin-based radioantagonists over agonists. Three manuscripts clearly seem to support the superiority of the antagonists over the agonists by a side-by-side comparison.16,20,22 The ethyl amide derivative Demobesin 1,23 a 99mTc-N4-[DPhe,6Leu-NHEt,13des-Met14]bombesin(6−14) was chosen by Cescato et al.20 to demonstrate the superiority of the antagonist as tumor targeting agent with respect to the potent radioagonist 99m Tc-Demobesin 4 (N4-[Pro,1Tyr,4Nle14]Bombesin).24 Mansi et al.16 compared the potent radiolabeled agonist 111 In-DO3A-CH2 CO-G-aminobenzoyl-Q-W-A-V-G-H-L-MNH2 (111In-AMBA,25 111In-4) with the antagonist 111In-DO3ACH2CO-G-aminobenzoyl-D-Phe-Q-W-A-V-G-H-Sta-Leu-NH2 (111In-RM1,16111In-5) and found a distinct superiority of the 111 In-5, showing approximately 3.5-fold higher tumor uptake and superior tumor-to-major normal tissue ratios. Recently Liu et al.22 studied NODAGA-conjugated 4 and 5, labeled with 64Cu and 18F-AlF. They concluded that the two antagonists show better tumor uptake and pharmacokinetics and favor 18F-AlF-NODAGA-5 as a highly promising probe for clinical PET imaging. On the contrary, Chen et al.,26 in the direct comparison among bombesin agonist and antagonist, concluded that 18F683



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Table 1. Chemical and Physicochemical Properties of the Three Studied Conjugates CODE 1 2 3

amino acid sequence

purity (%)

DOTA-Pip*-D-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH2a DOTA- Pip*-D-Phe-Gln-Trp-Ala-Val-Gly-His-Leuψ(CHOH-CH2)-(CH2)2-CH3a DOTA- Pip*-D-Phe-Gln-Trp-Ala-Val-Gly-His-Leu-OCH3a

97.4 98.1 97.9 98.2 98.5

nat

In-1 In-2

nat

a

m/z observed 1678.1 1564.3 1497.7 1755.7 1640.3

[M [M [M [M [M

+ + + + +

K+] K+] H+] H+] K+]

IC50 (nmol/L) 7.7 3.2 34 9.3 2.5

± ± ± ± ±

3.3 1.8 9.5 3.3 0.4

Pip*: 4-amino-1-carboxymethylpiperidine.

Figure 2. Internalization of 111In-2 was found higher compared to the other two radioantagonists (A). 111In-1 and 111In-2 showed similar receptor surface-bound activity at each time points, higher in comparison to 111In-3 (B). Values and standard deviations are the result of two independent experiments with triplicates in each experiment.

Figure 3. Fate of the GRP-R bound 111In-1, 111In-2, and 111In-3. The amount of radioactivity present as surface-bound (●), dissociated (■), and internalized (▲) radioligand was measured with respect to the total receptor bound radioligand in 2 h at 4 °C (100%). Each time point is the average of triplicate wells corrected for nonspecific binding.

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or antagonists), we decided to explore two classes of bombesin receptor antagonists, the methyl ester, as a representative of the “ester” class, and the pseudopeptide analogues, both reported to show strong antagonist potency.31,32 The choice of the 4amino-1-carboxymethylpiperidine as spacer and DOTA as chelator would allow us the direct comparison with the highly potent statine-based analogue 1. In this paper, we report the synthesis, characterization, radiolabeling, in vitro, and in vivo properties of the two novel radioantagonists, the pseudopeptide analogue, DOTA-4-amino-1-carboxymethylpiperidine-D-PheGln-Trp-Ala-Val-Gly-His-Leuψ(CHOH-CH 2 )-(CH 2 ) 2 -CH 3 (RM7, 2), and the methyl ester analogue, DOTA-4-amino-1carboxymethylpiperidine-D-Phe-Gln-Trp-Ala-Val-Gly-His-LeuOCH3 (ARBA05, 3). This is a part of a program studying structure−activity relationship of bombesin-based radioantagonists to better understand their pharmacological properties and support the statement that antagonists outperform agonists.

RESULTS

Chemistry; Radiochemistry. The peptide-chelator conjugates 1, 2, and 3 (Figure 1) were obtained with an overall yield of about 30%. The pure products (purity ≥97%) after lyophilization, were analyzed by analytical RP-HPLC and characterized using ESI-MS (Table 1). 111In-labeled conjugates were obtained by incubation at elevated temperature (95 °C, 30 min) with labeling yields of ≥95% at a maximum specific activity of 30 GBq/μmol. 68Ga-2 was obtained with labeling yields of ≥95% at a specific activity of 25 GBq/μmol. Binding Affinity. GRP-R binding affinities of the metalated and unmetalated conjugates were determined by a competitive binding assay using [125I-Tyr4]BN as radioligand by in vitro GRP-R autoradiography on cryostat sections of well characterized prostate carcinomas (Table 1). The IC50 value of 2 (3.2 ± 1.8 nM) is 2 and 10 times lower than 1 (7.7 ± 3.3 nM) and 3 (34 ± 9.5 nM), respectively. The binding affinity of 2 is retained when metalated with natIn. (IC50 of natIn-2 = 2.5 ± 0.4 nM). Cellular Uptake Studies. The cell uptake and the internalization kinetics of the three conjugates were studied 684



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in PC-3 cells. 111In-1 and 111In-2 showed similar receptor surface-bound activity (15.9 ± 0.9% and 17.7 ± 0.3%, respectively) and higher receptor bound activity and internalization rate in comparison to 111In-3 (9.9 ± 0.1%). At 4 h, the internalized activity of 111In-2 was 8.1 ± 0.1%, 2.2 times higher than the one of 111In-1 (3.7 ± 0.4%) and 111In-3 (3.0 ± 0.1%) (Figure 2). The Fate of GRP-R Bound Radiopeptides in Vitro. This experiment, performed on PC-3 cells, was designed to comparatively study the fate of the radiopeptides when bound to the receptor at 4 °C, followed by a temperature shift to 37 °C. The fraction of radiopeptide bound to the receptor, internalized or dissociated, was determined and is shown as a function of time in Figure 3. At 4 h, 70% of 111In-2 is still bound to the receptor and 16% is internalized, while for 111 In-3 the amount of ligand dissociated from the cells was 55% of the total ligand prebound. 111In-1 showed 64% of the prebound ligand still bound to the receptor at 4 h. Immunofluorescence Microscopy. The antagonist properties of the bombesin analogues were confirmed by the immunofluorescence-based internalization assay using HEKGRP-R cells. Figure 4A illustrates that 10 nmol/L bombesin can trigger internalization of the receptors. The conjugates were not able to stimulate GRP-R internalization. However, when given at a concentration of 1000 nmol/L together with 10

nmol/L bombesin, the peptides were able to prevent bombesininduced receptor internalization. Calcium Release. The antagonist potency of the three peptides was evaluated by using a calcium-mobilization assay in PC-3 cells. Figure 4B illustrates the efficacy of the antagonists to inhibit the intracellular calcium mobilization stimulated by the agonist Tyr4−bombesin. Addition of the agonist (100 nmol/L) induces maximum calcium mobilization, which decreases with an increase in the concentration of the antagonists. At a concentration of >100 mmol/L, all the antagonists completely inhibited the Tyr4−bombesin-induced calcium mobilization. Biodistribution Studies. Biodistribution studies of 111In-2 (Table 2) were done in athymic nude mice bearing subcutaneously implanted PC-3 tumor at 1, 4, 24, 48 and 72 h, while 111In-3 (Supporting Information, Table 1) was studied only at 4 h. This antagonist showed low tumor and receptor positive organs uptake probably due to the metabolically unstable methyl ester group. Biodistribution data on 111In-1 were previously reported.17 A direct comparison of the accumulation in the main tissues is shown in Figure 5. 111 In-1 and 111In-2 are characterized by fast blood and kidney clearance. However, the pharmacokinetics of the two compounds is different. 111In-1 accumulates fast in the tumor (15.2 ± 4.8% IA/g at 1 h pi) and in GRP-R positive organs such as pancreas (22.6 ± 4.7% IA/g at 1 h). At 4 h, the tumor uptake of 111In-1 was still high (11.7 ± 2.4%IA/g) but it was washed out from the other organs (1.5 ± 0.5%IA/g for the pancreas). Accumulation of 111In-2 in the tumor is high at 1 h and stays high even at 4 h (11.80 ± 3.99%IA/g). The uptake in GRP-R positive organs such as pancreas is high at 1 h (65.69 ± 8.14%IA/g), but it is washed out reaching the value of 39.4 ± 11.4%IA/g at 4 h. For both compounds, the washout from the tumor is slower than the washout from the pancreas. The tumor uptake stays reasonably high also at 24 h for both radiopeptides. Blocking with the cold ligand reduced the uptake by >97%. In Vivo Radioligand Displacement Using Excess of Agonist and Antagonist. We studied if the receptor bound radioligands can be displaced by an excess of 2 (20 nmol) or of Tyr4-bombesin (20 nmol) at 1, 4, and 24 h pi and if there is a difference when displaced with agonist or antagonist. The data of selected GRP-R positive organs and the tumor are shown in Table 3. The displacement study in vivo showed that there is a difference in receptor positive tissues depending if agonist or antagonist is the displacing agent. If the competing agent is the agonist, the tumor uptake is not displaced while the uptake in the receptor positive organs is displaced by about 50% at each time point. When the displacement is performed by adding an excess of the antagonist 42% of the radioligand can be displaced from the tumor at 1 h, while 92% can be displaced in the pancreas. The displacement in the tumor seems not to be related to time, while in the pancreas the percentage of displacement decreases with time, indicating a faster washout of the radioligand in this organ in respect to the tumor. Small-Animal PET Studies. Representative PET images obtained upon injection of 68Ga-2 on PC-3 xenografts at 1 h and 3 h pi (Figure 6) very well visualize the tumor and represent the results of the biodistribution studies. The radiotracer was taken up by the tumor and the GRP-R positive organs, such as pancreas, at early time points (1 h pi). The accumulated activity in the pancreas declined with time contrary to the tumor, where uptake remained still high after

Figure 4. (A) Immunofluorescence microscopy-based internalization assay with HEK-GRP-R cells, 2 (c,d), 1 (e,f), 3 (g,h). Control experiment shows membrane-bound GRP-R in absence of peptide (a); bombesin agonist (10 nmol/L) triggers massive GRP-R internalization (b). Antagonist conjugates failed to induce GRP-R internalization, even at concentration of 1 mmol/L (c,e,g); antagonist conjugates at a concentration of 1 mmol/L efficiently blocked bombesin agonistmediated GRP-R internalization (d,f,h). (B) Representative inhibition curves of bombesin antagonists obtained by the calcium release assay in PC-3 cells (RFU: relative fluorescence unit). Cells were loaded with Ca-dye and different concentrations of antagonists ranging from 0.1 nM to 100 μM. The curves show the potential of 1 (■), 2 (●), and 3 (▲) to inhibit the intracellular calcium mobilization stimulated by the addition of the agonist Tyr4-bombesin (100 nM). 685



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Table 2. Biodistribution Studies in PC-3 Tumor-Bearing Mice (n = 3−4 Mice per Time Point) after Injection of Tumor to Tissue Radioactivity Ratiosa

a

organ

1h

4h

blood heart liver spleen lung kidney stomach intestine adrenal pancreas muscle bone tumor tumor/blood tumor/kidney tumor/liver tumor/muscle

0.43 ± 0.10 0.17 ± 0.03 0.73 ± 0.12 1.13 ± 0.41 0.45 ± 0.05 5.23 ± 3.01 4.43 ± 2.48 3.61 ± 0.61 10.66 ± 2.64 65.69 ± 8.14 0.22 ± 0.06 1.31 ± 1.29 9.18 ± 1.16 21.1 1.8 12.7 42.6

0.12 ± 0.03 0.09 ± 0.02 0.49 ± 0.14 0.57 ± 0.24 0.21 ± 0.06 2.43 ± 0.47 5.72 ± 4.04 3.31 ± 1.61 5.16 ± 1.55 39.80 ± 9.25 0.14 ± 0.04 0.71 ± 0.25 11.80 ± 3.99 109.7 5.4 26.7 94.7

111

In-2 and

4 h blockingb

24 h

48 h

72 h

± ± ± ± ± ± ± ± ± ± ± ± ±

0.01 ± 0.003 0.03 ± 0.01 0.20 ± 0.02 0.21 ± 0.06 0.08 ± 0.04 1.41 ± 0.22 1.08 ± 0.15 0.35 ± 0.05 2.31 ± 0.96 4.52 ± 0.53 0.05 ± 0.01 0.29 ± 0.14 8.39 ± 0.88 631.4 6.0 42.4 169.2

0.01 ± 0.002 0.02 ± 0.00 0.11 ± 0.01 0.15 ± 0.04 0.05 ± 0.02 0.97 ± 0.06 0.43 ± 0.10 0.16 ± 0.07 2.24 ± 1.43 2.30 ± 0.19 0.03 ± 0.01 0.23 ± 0.13 5.89 ± 0.35 803.8 6.1 53.0 191.9

0.01 ± 0.001 0.08 ± 0.03 0.09 ± 0.02 0.15 ± 0.04 0.18 ± 0.07 0.55 ± 0.15 0.28 ± 0.09 0.10 ± 0.01 1.84 ± 0.36 1.06 ± 0.19 0.06 ± 0.02

0.04 0.15 0.40 0.23 0.38 2.88 0.16 0.14 1.36 0.18 0.13 1.61 0.38

0.001 0.05 0.02 0.03 0.06 1.52 0.03 0.02 0.56 0.04 0.07 0.52 0.08

4.22 ± 0.34 842.7 7.7 48.4 69.3

Data are expressed as %IA/g ± SD. bBlocked with 20 nmol of cold peptide 2

biological processes and to explore its possible role in the growth of human neoplasms. In particular, the groups of Jensen et al. and Coy et al.14 advanced the development of bombesin receptor family antagonists. They performed thorough structure−activity relationship studies. In their study they found four major families of peptides with antagonistic properties. The first class is represented by various D-amino acid substituted substance P analogues that are reported to have a broad inhibitory activity against bombesin action.33,34 The substitution of the His residue in position 12 with a DPhe residue (class 2) conferred antagonistic properties to [Leu14]Bn in pancreatic acini as described by Heinz-Erian.35 On the basis of the successful strategy to obtain antagonists by the modification of the peptide backbone, substituting the amide bond (CONH) with the pseudopeptide bond (CH2NH), Coy and co-workers36 developed a new series of bombesin antagonists (class 3). [Leu13-ψ-CH2NH-Leu14] bombesin was shown to be a strong antagonist, inhibiting bombesin-stimulated growth of Swiss 3T3 cells and of various small cell lung cancer cell lines. [desMet14]BN belongs to the fourth class of bombesin receptor antagonists. Among these peptides, the [DPhe6]-

Figure 5. Comparison of the accumulation in the main tissues of 111In1 and 111In-2 at 4 h and 48 h.

3 h pi. Blocking experiment, using a high excess of the peptide (20 nmol), demonstrated that the accumulation of 68Ga-2 in the tumor and in the positive organs is specific.

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DISCUSSION Bombesin receptor antagonists have been developed in the last 30 years to better understand the role of bombesin in various

Table 3. Radioligand Displacement of 111In-2 in Selected GRP-R Positive Tissues and the PC-3 Tumor at Different Time Points (1, 4, 24 h) (A) displacement performed with 2000-fold excess of Tyr4-Bombesin organ tumor pancreas stomach intestine organ tumor pancreas stomach intestine

1h 9.18 65.69 4.43 3.61

± ± ± ±

1 h displ 1.16 8.14 2.48 0.61

1h 9.18 65.69 4.43 3.61

± ± ± ±

9.73 32.74 2.47 2.25 (B)

1 h displ 1.16 8.14 2.48 0.61

5.32 5.23 0.60 0.47

4h

4 h displ

24 h

± 1.36 11.80 ± 3.99 8.85 ± 2.12 ± 7.20 39.80 ± 9.25 19.70 ± 3.63 ± 0.42 5.72 ± 4.04 1.97 ± 0.08 ± 0.54 3.31 ± 1.61 1.11 ± 0.17 displacement performed with 2000-fold excess of cold 2 ± ± ± ±

0.60 0.54 0.09 0.05

4h 11.80 39.80 5.72 3.31

± ± ± ±

4 h displ 3.99 9.25 4.04 1.61

686

7.20 7.59 0.91 0.63

± ± ± ±

8.39 4.52 1.08 0.35

± ± ± ±

0.88 0.53 0.15 0.05

24 h

0.88 2.02 0.11 0.17

8.39 4.52 1.08 0.35

± ± ± ±

0.88 0.53 0.15 0.05

24 h displ 6.13 3.58 0.61 0.27

± ± ± ±

0.61 0.34 0.11 0.12

24 h displ 5.17 3.31 0.32 0.18

± ± ± ±

0.54 0.39 0.10 0.04



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Figure 6. PET imaging of PC-3 tumor bearing mice upon injection of 68Ga-2 at 1 h and 3 h pi along with blocking studies.

high instability with the presence of a second peak on the HPLC chromatogram already at early time points that was identified as the carboxylic acid free analogue by coinjection of the two peptides (data not shown). The main difference in the pharmacokinetic of 2 when compared to 1 is the high pancreas uptake at early time points that can be correlated to its higher binding affinity. High pancreas uptake is reported as well for other bombesin receptor antagonists at early time point (1 h), such as 99mTc-Demobesin 1 (104 ± 3.59%IA/g,), 99mTc-N4-AR17 (66.01 ± 4.89%IA/g) and 64Cu-CB-TE2A-AR17 (63.31 ± 2.69%IA/g) having binding affinities comparable to 2. 111 In-2 and 111In-1 have similar biodistribution profiles except for some GRP-R expressing normal tissues such as pancreas at early time point. The pancreas uptake of 111In-2 decreased rapidly with time, while the washout from the tumor is much slower; this characteristic is very attractive for a potential clinical translation. No significant uptake is observed in the nontarget tissues, and the washout is fast while the tumor accumulation is maintained high even at later time points, resulting in a very high tumor to background ratio (tumor to kidney ratio is 5.4 at 4 h and 7.7 at 72 h). In our attempt to understand the strong receptor−peptide interaction (slow washout from the tumor), we performed an in vivo displacement study (Table 3). At different time points after the injection of 111In-2, an excess of the peptide 2 or of the agonist Tyr4-bombesin was injected and the animals sacrificed 1 h later. When the displacement is done with an excess of 2, we observed a partial displacement in the tumor (42% at 1 h) that is constant over time (38% at 24 h), while in the GRP-R positive organs such as pancreas the displacement is around 92% at 1 h and decreases with time (27% at 24 h). We had previously performed this experiment for the statine-based analogue 5,16 having a lower binding affinity. 111In-2 showed a completely different behavior compared to 111In-5 that was displaced by 89% at 1 h and 57% at 24 h using an excess of 5. We explained these results by hypothesizing a slow in vivo internalization or a slow migration of the radioantagonist to an inaccessible binding site. The low displacement rate of 2 can be explained with the high binding affinity of this analogue and the presence of the “extra” binding site that can render the radioantagonist even more inaccessible. The complete absence of displacement when this is done with the agonist Tyr4−

BN6−13 methyl ester and its analogues exhibit high affinity and selectivity for the GRP receptor over the neuromedin B receptor in rat and human and strong antagonistic properties.37 Because of controversies concerning agonists vs antagonists and our interest in structure−activity relationship studies, we decided to introduce two new families (pseudopeptide and methyl ester) of antagonistic peptides into the nuclear medicine field. The pseudopeptide (D-Phe-Gln-Trp-Ala-Val-Gly-His-Leuψ(CHOH-CH2)-(CH2)2-CH3) and the methyl ester derivative ((D-Phe6)Bombesin(6−13)-OMe) were coupled to the 4amino-1-carboxymethylpiperidine and to DOTA to allow the direct comparison with the statine-based 1 that, labeled with 68 Ga, is already in clinical trials for the detection of primary prostate cancer and recurrent disease. In-2, showed a 3.7-fold improvement in IC50 value compared to In-1. These data reflect somehow the difference in binding affinity between the two bombesin antagonists, the statine derivative H- D -Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH 2 (JMV594,38 6) and the pseudopeptide H-D-Phe-Gln-Trp-AlaVal-Gly-His-Leuψ(CHOH-CH2)-(CH2)2-CH3 (JMV641,31 7). 7 (IC50 = 0.72 ± 0.29 nM) showed 7.7 times higher binding affinity than 6 (IC50= 5.6 ± 1.8 nM). Tokita et al.39 investigated the molecular basis for the GRP-R selectivity of these two classes of bombesin antagonists. The authors stated that the fourth extracellular domain of the GRP-R is the principal receptor region responsible for the high affinity and selectively of both peptides for the GRP-R over the NMB-R. Moreover, site-directed mutagenesis studies suggested an important role of the second and third extracellular domains for the GRP-R selectively of 7. This “extra” binding site can, most probably, explain the higher binding affinity and stability of the receptor− 2 complex. Studying the fate of the peptides in a temperature shift experiment, the dissociation rate of 2 was 1.6 times lower than the one of the other two analogues studied. 111 In-3 showed inferior in vitro properties with high dissociation rate: this is in accordance with the higher IC50 value observed for 3. On the basis of this data, biodistribution experiments were performed for this compound only at 4 h. We explained the absence of uptake in tumor and in GRP-R positive organs considering the low metabolic stability of the methyl ester group, probably due to the action of esterases. Serum stability studies performed on this conjugate showed 687



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several times with DCM, and dried. The chelator-spacer-peptide, in DMF, was activated for 20 min using HATU (1.2 equiv) and HLeuψ(CHOH)-(CH2)3-CH3 was added. The pH was adjusted to 8 with DIEA, and the reaction mixture was stirred for 4 h at RT. The mixture was concentrated, and the fully protected peptide was precipitated with H2O on ice, centrifuged, and separated from the solvent by decantation. To get the peptide fully deprotected, it was treated for 4 h in a mixture of DCM/TFA/TIS/H2O (10/85/2.5/2.5) and then it was precipitated using a mixture of 50% diethyl ether and 50% diisopropylether on ice. The synthesis, purification, and characterization of 1 are described.21 3 was synthesized manually according to standard Fmoc chemistry40 using Rink acid resin as described above. The cleavage of the fully protected peptide from the resin was accomplished by incubating it for 1 h in DCM:AcOH:H2O (90:9:1). The resin was then filtered, washed with the above mixture, added excess of toluene, and evaporated. The residue was coevaporated with toluene several times to obtain solid conjugate, which was further dried in a desiccator overnight. For the methylation, 20 μmol of the peptide conjugate were dissolved in THF (2−3 mL) and the same amount of dry MeOH was added. The solution was stirred, and 100 μL of (trimethylsilyl) diazomethane solution (2.0 M in diethyl ether) was added. Stirring was continued for 3 h, and the solvent evaporated followed by trituration with diethyl ether yielded the crude DOTA−peptide conjugate. The final deprotection was performed using trifluoroacetic acid/triisopropylsilane/H2O (95/2/2/1). The conjugates were further purified by semipreparative highperformance liquid chromatography (HPLC) and characterized by ESI-MS. The conjugates were complexed with natInCl3 using the procedure described previously.7 After lyophilization, the pure product (yields ranging from 70%−80%) was analyzed by analytical RP-HPLC and characterized using ESI-MS. Radiolabeling. 111In-labeled conjugates were prepared by dissolving 10 μg of peptide in 250 μL of sodium acetate buffer (0.4 mol/L, pH 5.0) and by incubating with 111InCl3 (110−220 MBq) for 30 min at 95 °C. To obtain structurally characterized homogeneous ligands, 1 equiv of natInCl3·5H2O was added and the final solution incubated again at 95 °C for 30 min. For biodistribution studies, the labeling was performed accordingly without the addition of In3+-salt. 68 Ga-2 was prepared in sodium acetate buffer (0.2 mol/L, pH 4.0) at 95 °C for 8 min as described elsewhere.17 Receptor Binding Affinity. IC50 values were determined by in vitro GRP-R autoradiography on cryostat sections of well characterized prostate carcinomas, as described previously.3,41 The radioligand used was [125I-Tyr4]-bombesin, known to preferentially bind to the GRPR.42 Internalization. Confluent PC-3 cells were seeded in six-well plates (∼1.0 × 106 cells) 24 h before starting the experiments. The medium was removed and the cells washed and incubated for 1 h at 37 °C with fresh medium (DMEM with 1% FBS). For internalization experiments, approximately 3 kBq of 111/natIn-labeled peptides (0.25 pmol) were added to the medium and the cells were incubated (in triplicates) for 0.5, 1, 2, and 4 h at 37 °C, 5% CO2. Blocking experiments were performed using the antagonist 8 (2 μmol/L, 100 μL) at 37 or 4 °C. At appropriate time points, the internalization was stopped by removal of the medium followed by washing the cells with ice-cold PBS (pH 7.2). Cells were then treated 5 min (twice) with glycine buffer (0.05 mol/L glycine solution, pH adjusted to 2.8 with 1 mol/L HCl) to distinguish between cell surface-bound (acid releasable) and internalized (acid resistant) radioligand. Finally, cells were detached from the plates by incubation with 1 mol/L NaOH for 10 min at 37 °C, and the radioactivity was measured in a γ-counter. The Fate of GRP-R Bound Radiopeptides in Vitro. PC-3 cells were seeded into 6-well plates and treated as described above. The plates were placed on ice for 30 min; an excess of blocking agent was added to selected wells to determine nonspecific binding. The radioligands (0.25 pmol, 3 kBq) were added to the medium and allowed to bind to the cells for 2 h at 4 °C. After the incubation, the

bombesin can be explained by the fact that the excess of the agonist may induce internalization of the antagonist bound due to clustering. The PET images of a PC-3 tumor bearing mouse injected with 68Ga-2 in Figure 6 show the specificity of tracer uptake in the tumor and the abdominal receptor-positive organs. The 1 h PET images reflect the 111In-biodistribution profile showing high and selective accumulation in tumor and positive target tissues at early time point. The late PET image (3 h pi) confirms the pharmacokinetics of 111In-2 with a slower washout from the tumor in respect to the receptor positive target tissues. The good biodistribution profile of 68Ga-2 indicates this analogue as a good candidate for clinical translation. The high accumulation in the tumor, and its strong peptide− receptor complex, may play an important role in the choice of 2, labeled with therapeutic radionuclides such as Lu-177 or Y90, as potential candidates for therapeutic applications.

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EXPERIMENTAL SECTION

Materials and Methods. All chemicals were obtained from commercial sources and used without additional purification. Rink amide 4-methyl-benzhydrylalanine (MBHA) resin and all the Fmocprotected amino acids are commercially available from NovaBiochem (Laeufelfingen, Switzerland), DOTA(tBu)3 from Chematec (Dijon, France), Fmoc-4-amino-1-carboxymethylpiperidine from NeoMPS (Strasbourg, France), and 111InCl3 from Covidien Medical (Petten, Netherlands). 68Ge/68Ga-generator was available from Eckert & Ziegler (Berlin, Germany). [ D -F 5 Phe, 6 D -Ala 11 ]BN(6−13)OMe (BIM26226,37 8) was provided by Ipsen Biotech (Paris, France). Electrospray ionization mass spectroscopy was carried out with a Finnigan SSQ-7000 spectrometer (Bremen, Germany). Analytical high-performance liquid chromatography (HPLC) was performed on a Hewlett-Packard 1050 HPLC system with a multiwavelength detector and a flow-through Berthold LB-506-Cl γ-detector using a MachereyNagel Nucleosil 120 C18-column (Oensingen, Switzerland) (eluents, A = 0.1% TFA in water and B = acetonitrile; gradient, 0−30 min, 95%−30% A; flow, 0.750 mL/min). Preparative HPLC was performed on a Metrohm HPLC-system LC-CaDI 22−14 (Herisau, Switzerland) with a Macherey-Nagel VP 250/21 Nucleosil 100−5 C18-column (gradient, 0−20 min, 90%−50% A; flow, 10 mL/min). Quantitative γcounting was performed on a COBRA 5003 γ-system well counter from Packard Instruments (Packard, Meridan, CT, USA). Cell Lines. Human embryonic kidney 293 (HEK293) cells, stably expressing the HA-epitope-tagged human GRP-R (HEK-GRP-R), were generated as previously described20 and cultured at 37 °C and 5% CO2 in Dulbecco’s Modified Eagle Medium (DMEM) with GlutaMAX-I containing 10% (v/v) fetal bovine serum (FBS), 100 U/mL penicillin, 100 μg/mL streptomycin, and 750 μg/mL G418. Human prostate cancer cells (PC-3) were obtained from ATCC (Virginia, USA), cultured in DMEM or in Ham’s F-12K medium, supplemented with vitamins, amino acids, penicillin/streptomycin, and 10% FBS in a humidified 5% CO2 atmosphere at 37 °C. All culture reagents were from Invitrogen (Basel, Switzerland) or from BioConcept (Allschwil, Switzerland). Synthesis of Peptide Conjugate and Metalation. The peptide conjugate 2 was synthesized in solution by condensation of the peptidic moiety DOTA(tBu)3-4-amino-1-carboxymethylpiperidineDPhe-Gln(Trt)-Trp(Boc)-Ala-Val-Gly-His(Trt)-OH with the modified moiety H-Leuψ(CHOH)-(CH2)3-CH3. The peptide was synthesized manually on 2-chlorotrityl chloride resin using Fmoc strategy.40 The spacer and the prochelator were coupled using HATU as activating agent. The fully protected peptide was cleaved from the solid support by suspending the resin for 1 h in a mixture of TFA/TIS/DCM (1/5/94), and it was characterized by RPHPLC and ESI-MS. The synthesis of Boc-Leuψ(CHOH)-(CH2)3-CH3 involves three steps starting from Boc-Leu-OH as described earlier.38 Boc-Leuψ(CHOH)-(CH2)3-CH3 was deprotected using a solution of 80% TFA in DCM. After 1 h, the solution was concentrated, washed 688



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5 min) with 20 nmol of cold peptide and afterward with 200 pmol of 68 Ga-2, and static scans were obtained at 1 h post injection, as described above. PET-images were corrected for 68Ga decay and reconstructed with filtered back projection. No correction was applied for attenuation. Images were generated using AMIDE software.43

cells were quickly washed twice with ice-cold PBS and 1 mL of fresh prewarmed (37 °C) culture medium was added to each well followed by incubation for 10, 20, and 30 min and 1, 2, and 4 h (37 °C, 5% CO2). At each time point, the plates were treated as described recently. Immunofluorescence Microscopy. Immunofluorescence microscopy-based internalization assays with HEK-GRP-R cells were performed as previously described.20 HEK-GRP-R cells were treated either with 10 nmol/L bombesin or with 1 μmol/L of all conjugates, or to evaluate potential antagonism, with 10 nmol/L bombesin in the presence of a 100-fold excess of all conjugates for 30 min at 37 °C, 5% CO2 in growth medium and then processed for immunofluorescence microscopy using the mouse monoclonal HA-epitope antibody at a dilution of 1:1000 as first antibody (Covance, Berkeley, CA) and Alexa Fluor 488 goat antimouse IgG (H+L) at a dilution of 1:600 as secondary antibody (Molecular Probes, Eugene, OR). The cells were imaged using a Leica DM RB immunofluorescence microscope and an Olympus DP10 camera. Ca2+-Mobilization Assay. Intracellular calcium mobilization was measured in PC-3 cells using the Calcium 3 Assay kit (Molecular Probes, Inc.) following the protocol described earlier with slight modifications.20 In brief, PC-3 cells (25000 cells per well) were seeded in 96-well plates and cultured for 1 d at 37 °C and 5% CO2. On the day of the experiment, the medium was removed and the cells were then loaded with 100 μL per well of Ca-dye in assay buffer (Hank’s Balanced Salt Solution and 20 mM N-(2-hydroxyethyl)piperazine-N9(2-ethanesulfonic acid), HEPES) containing 2.5 mM probenecid for 1 h at 37 °C. The agonist Tyr4-bombesin was diluted in a dilution buffer (HBSS with 20 mM HEPES, 0.1% BSA, 0.05% pluronic acid) to a concentration of 100 nM and dispensed into a reagent source 96-well plate. Varying concentrations of antagonists (InIII-1, InIII-2, and InIII-3) ranging from 0.1 nM to 100 μM were prepared in the dilution buffer and loaded in the same 96-well plate. Cell- and peptide-containing plates were loaded into a FLEX station 3 microplate reader (Molecular Devices). Intracellular calcium mobilization was recorded at room temperature for 4 min in a kinetic monitoring fluorescence emission at 525 nm (with λex = 485 nm), and the data were analyzed by SoftMax Pro software (Molecular Devices). The instrument was programmed such that the antagonist (20 μL at varying concentrations) was added to cell plates and preincubated for 1 min prior to the addition of the agonist (20 μL). Biodistribution Experiments. All animal experiments were performed in compliance with the Swiss (no. 798) and German regulations for animal treatment. The pharmacokinetics of 111In-labeled peptides were performed using female athymic nude mice (3 weeks old), implanted subcutaneously with 10 million PC-3 tumor cells, freshly expanded on sterilized solution of phosphate-buffered saline (pH 7.4). Eleven days after inoculation, 10 pmol of radiolabeled peptides (about 0.18 MBq, 100 μL) were injected into the tail vein of the mice (20−22 g). For the determination of nonspecific uptake in tumor or receptor positive organs, a group of four animals was preinjected (5 min) with 0.02 μmol of the corresponding unlabeled peptide. The biodistribution of 111In-3 was performed at 4 h, while 111In-2 was studied at 1, 4, 24, 48, and 72 h. At each time point, the mice (in groups of 4−11) were sacrificed and organs of interest were collected, rinsed of blood, blotted, weighed, and counted in a γ-counter. The percentage of injected activity per gram (%IA/g) was calculated for each tissue. In Vivo Radioligand Displacement Using Excess of Cold Peptide. Mice were injected with 10 pmol of 111In-2 (0.18 MBq, 100 μL), as described above, to study if the radioligand can be displaced in vivo by excess of agonist or antagonist. Then 20 nmol of the cold peptides 2 or Tyr4-bombesin (100 μL saline) were injected at 1 h, 4 h, and 24 h and biodistribution was performed 1 h pi as described above. Small-Animal PET Studies. MicroPET scans were performed using a dedicated small-animal PET scanner (Focus 120 microPET scanner; Concorde Microsystems Inc.). PET images were obtained upon injection of 200 pmol of 68Ga-2 (approximately 5 MBq/100 μL) on PC-3 tumor bearing mice. Static imaging was acquired for a time period of 30 min at 1 and 3 h post injection. To visualize the extent of GRP-specific tumor uptake of 68Ga-2, PC-3 mice were preinjected (3−

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ASSOCIATED CONTENT

S Supporting Information *

Biodistribution data on PC-3 tumor bearing nude mice of 111In3 at 4 h. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: ++49 761 270 74220. Fax: ++49 761 270 39300 Email: [email protected]. Present Addresses ⊥

For K.A.: Roche Pharmaceutical Research and Early Development, Roche Innovation Center Basel, F. Hoffmann-La Roche Ltd., Grenzacherstrasse 124, 4070 Basel, Switzerland. # For R.C.: Universitaet Bern, Department Klinische Forschung, Inselspital, 3010 Bern, Switzerland. ∇ For M.L.T.: Centro di Biotecnologie ‘A.O.R.N.’, A. Cardarelli, 80138 Naples, Italy. Author Contributions

The manuscript was written through contributions of all authors. R.M. has performed synthesis, labeling and cell studies, analysis of the data and wrote part of the manuscript; A.K. has performed synthesis of the methyl ester analogue, Ca-flux studies; X.W. and M.L.T. have done the biodistribution studies with In-111; E.G. has performed the animal PET studies; R.C. did immunofluorescence and determined IC(50) values; S.B. designed experiments; J.C.R. designed research; H.M. is the PI, designed research, and edited the manuscript. Notes

The authors declare the following competing financial interest(s): R. Mansi, H. R. Maecke and J. C. Reubi declare associations with the following company: Bayer. S. Berndt is an employee of Bayer. The other authors declare no competing interests. Abbreviations of the common amino acids are in accordance with the recommendations of IUPAC-IUB (http://www.chem. qmul.ac.uk/iupac/AminoAcid).

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ACKNOWLEDGMENTS We thank Novartis Pharma for analytic assistance, Sibylle Tschumi, Yvonne Kiefer, and Roswitha Toennesmann for their expert technical assistance, Bayer Schering Pharma for financial support, and the COST action BM0607.

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ABBREVIATIONS USED GRP, gastrin releasing peptide; PET, positron emission tomography; RP-HPLC, reverse phase high performance liquid chromatography; ESI-MS, electrospray mass spectrometry; PC3 cells, androgen insensitive human prostate adenocarcinoma cells; IC50, half-maximum inhibitory concentration; DOTA, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid; NODAGA, 1,4,7-triazacyclononane, 1-glutaric acid-4,7 acetic acid; N4, 6-carboxy-1,4,7,11-tetraazaundecane; CB-TE2A, 4,11-bis(carboxymethyl)-1,4,8,11-tetraazabicyclo-[6.6.2]hexadecane 689



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