Copper-64 Labeled Macrobicyclic Sarcophagine Coupled to a GRP Receptor Antagonist Shows Great Promise for PET Imaging of Prostate Cancer.

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Article

Copper-64 labeled Macrobicyclic Sarcophagine Coupled to a GRP Receptor Antagonist Shows Great Promise for PET Imaging of Prostate Cancer. Eleni Gourni, Luigi Del Pozzo, Emilie Kheirallah, Christiane Smerling, Beatrice Waser, JeanClaude Reubi, Brett M Paterson, Paul S. Donnelly, Philipp T Meyer, and Helmut R. Maecke Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/mp500671j • Publication Date (Web): 01 Jul 2015 Downloaded from http://pubs.acs.org on July 7, 2015

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Molecular Pharmaceutics

Copper-64 labeled Macrobicyclic Sarcophagine Coupled to a GRP Receptor Antagonist Shows Great Promise for PET Imaging of Prostate Cancer.

Eleni Gourni,*,†,‡,§ Luigi Del Pozzo‡, Emilie Kheirallah‡, Christiane Smerling¦, Beatrice Waserǁ, JeanClaude Reubiǁ, Brett M. Paterson#, Paul S. Donnelly#, Philipp T. Meyer‡, Helmut R. Maecke‡ †

German Cancer Consortium (DKTK), Heidelberg, Germany, ‡Department of Nuclear Medicine,

University Hospital Freiburg, Freiburg, Germany, §German Cancer Research Center (DKFZ), Heidelberg, Germany, ¦3B Pharmaceuticals, Berlin, Germany, ǁDepartment of Pathology, University Hospital Bern, Bern, Switzerland,

#

School of Chemistry and Bio21 Molecular Science and

Biotechnology Institute, The University of Melbourne, Australia.

*Corresponding author: Eleni Gourni, PhD, University Hospital Freiburg, Department of Nuclear Medicine, Hugstetter Str. 55, 79106 Freiburg, Germany. E-mail: [email protected] Tel: +49(0)76127039860. Fax: +49(0)76127039980.

1 ACS Paragon Plus Environment


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Table of Contents


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Abstract The gastrin-releasing peptide receptor (GRPr) is an important molecular target for the visualization and therapy of tumors and can be targeted with radiolabeled bombesin derivatives. The present study aims to develop statine-based bombesin receptor antagonists suitable for labeling with

64

Cu for

imaging by positron emission tomography (PET). The potent GRPr antagonist D-Phe-Gln-Trp-AlaVal-Gly-His-Sta-Leu-NH2 was conjugated to the sarcophagine (3,6,10,13,16,19-hexaazabicyclo[6.6.6] icosane=Sar)

derivative

5-(8-methyl-3,6,10,13,16,19-hexaaza-bicyclo[6.6.6]icosan-1-ylamino)-5-

oxopentanoic acid (MeCOSar) via PEG4 (LE1) and PEG2 (LE2) spacers and radiolabeled with 64Cu2+ with >95% yield and specific activities of about 100 MBq/nmol. Both Cu(II) conjugates have high affinity for GRPr (IC50:

nat

Cu-LE1; 1.4±0.1 nM and

nat

Cu-LE2; 3.8±0.6 nM). The antagonistic

2+

properties of both conjugates were confirmed by Ca -flux measurements. Biodistribution studies of Cu-64-LE1 exhibited specific targeting of the tumor (19.6 ± 4.7 % IA/g at 1 h p.i.) and GRPr-positive organs. Biodistribution and PET images at 4 and 24 h post injection showed increasing tumor-tobackground ratios with time. This was illustrated by the acquisition of PET images showing high tumor-to-normal tissue contrast. This study demonstrates the high affinity of the MeCOSar-PEGxbombesin conjugates to GRPr. The stablity of 64Cu complexes of MeCOSar, the long half-life of 64Cu and the suitable biodistribution profile of the

64

Cu-labeled peptides lead to PET images of high

contrast suitable for potential translation into the clinic.

Keywords: gastrin–releasing peptide receptor antagonists, sarcophagine MeCOSar chelator,

64

Cu,

PET-imaging.

Abbreviations GRPr, the gastrin-releasing peptide receptor; HATU, 1-[Bis(dimethylamino)methylene]-1H-1,2,3triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate; MRI,

magnetic resonance imaging; SPECT,

single positron emission computed tomography; PET, positron emission tomography; MIP, Maximum intensity projection; 18

64

Cu, copper-64;

99m

Tc, technecium-99m;

111

In, indium-111;

68

Ga, gallium-68;

F, fluorine-18; IC50, half maximum inhibitory concentration; RGD, Arginine-Glycine-Aspartate;

NODAGA,

1,4,7-triazacyclononane,

1-glutaric

acid-4,7

acetic

tetraazacyclododecane-1,4,7,10-tetraacetic acid.


acid;

DOTA,

1,4,7,10-


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INTRODUCTION The bombesin receptor family, in particular the gastrin-releasing peptide receptor (GRPr), is a very attractive target in the field of nuclear oncology due to the high density of these receptors on several human tumors.1,2 The overexpression of the GRPr on the membrane of most prostate, breast and small cell lung cancers, gastrointestinal stromal tumors and in tumoral vessels of urinary cancers has led to the development of GRPr-based radiopharmaceuticals.1 A variety of radiolabeled GRPr-based agonists, mainly derived from the N-terminal truncated octapeptide bombesin(7-14), have been studied in vitro and in vivo and have shown promising results.3 Although some of them were studied in patients with prostate and breast cancer either for diagnostic or therapeutic purposes,4-6 rapid translation to the clinic was hampered due, in part, to side effects after their intravenous administration.7 GRPr antagonists have been investigated by several research groups, including ours, with a view to generating new GRPr radiolabeled analogues which can be safely administrated to patients. The shift to antagonists was partially stimulated by the discovery that radiolabeled somatostatin antagonists recognize more binding sites and found to have superior pharmacokinetic properties when compared to agonists.8 Radionuclides of copper offer considerable potential in the development of new radiopharmaceuticals because of their decay characteristics which combine PET imaging or/and targeted radiotherapy capabilities.9 Several

64

Cu-labeled GRPr-targeting conjugates have been reported.10-18 Although the

majority of them successfully targeted the GRPr positive tumors, they exhibited high background ratios, with the liver being the organ with the highest accumulated activity, as it is the major site of copper metabolism, leading to very low tumor-to-background ratios over time. The goal of this study was to develop GRPr antagonists which can be stably labeled with 64Cu and at the same time provide an optimum overall pharmacokinetic performance. Our previous experience using the statine-based GRPr-antagonistic peptide H-D-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH2 functionalized with a variety of chelators via different spacers showed that this peptide motif can be considered as one of the most promising candidates for the targeting of GRPr positive tumors which allows for efficient targeting of the GRPr exhibiting excellent in vivo behavior.17,19-23 Cage amine sarcophagine derived chelators form stable complexes with 64Cu in vitro and in vivo.24-30 In order to develop 64Cu-labeled GRPr antagonists with improved tumor-to-background ratios we proceeded with the functionalization of the statine-based GRPr antagonist with the sarcophagine (3,6,10,13,16,19hexaazabicyclo[6.6.6]

icosane=Sar)

derived

chelator,

5-(8-methyl-3,6,10,13,16,19-hexaaza-

bicyclo[6.6.6]icosan-1-ylamino)-5-oxopentanoic acid (MeCOSar), via the spacers PEG4 and PEG2 to obtain LE1 and LE2, respectively, and radiolabel them with

64

Cu. The MeCOSar chelator has

excellent pharmacokinetics when coupled to the potent somatostatin receptor targeting peptide Tyr3octreotate and labeled with 64Cu.30 We also describe the in vitro and in vivo evaluation of 64Cu-labeled conjugates with a particular focus on the most promising candidate, 64Cu-LE1.


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MATERIALS AND METHODS

Reagents and Instrumentation All reagents and solvents were obtained from commercial sources and used without further purification. The precursor MeCOSar(Boc)4 was synthesized as described.30 All culture reagents were from Gibco BRL, Life Technologies (Grand Island, NY). 64CuCl2 was obtained from the University of Tübingen (Germany) and from ACOM (Montecorsaro Scalo,Italy). The purification of the peptides was performed by semipreparative RP-HPLC on a 120-5 C18 Nucleosil column (250 x 21 mm) applying a linear gradient of 15-90% solvent B in 25 min at a flow rate of 12 mL/min (solvent A, 0.1% TFA/H2O; solvent B, 0.1% TFA/Acetonitrile). Ultraviolet detection was performed using a Knauer detector at 280 nm. The quality control of the peptides as well as the radiolabeled compounds was performed by analytical RP-HPLC on an analytical 120-5 C18 Nucleosil column (250 x 4.5 mm) applying the same gradient as described before at a flow rate of 1 mL/min. For radioactivity measurement, a Na(Tl) well-type scintillation Gina star was used. Ultraviolet detection was performed using a Knauer detector at λ = 280 and λ = 220 nm. The radiotracer solutions were prepared by dilution with 0.9% NaCl. ESI-MS mass spectra were acquired on a Bruker Daltonics Esquire 3000 plus device. Quantitative γ-counting was performed with a COBRA 5003 γ-system well counter from Packard Instrument (USA). For PET studies a dedicated small-animal PET scanner (Focus 120 microPET scanner; Concorde Microsystems Inc.) was used. All experiments were carried out twice in triplicates.

Synthesis of Chelator-Peptide Conjugates The peptide-chelator conjugates were synthesized manually using standard Fmoc chemistry and Rink amide 4-methylbenzhydrylamine resin. The spacers (PEG4 and PEG2) and the protected chelator (MeCOSar(Boc)4) were consecutively coupled to the peptide using HATU as an activating agent. The cleavage of the peptides and the simultaneous deprotection of the side chain-protecting groups were perfomed using trifluoroacetic acid/triisopropylsilane/H2O (95/2.5/2.5). The crude conjugates were further purified by semipreparative RP-HPLC. Preparation of the natCu Metallopeptides The natCu metallopeptides were prepared using a 1.2-fold excess of

nat

CuCl2 x 2H2O 0.3 M. Briefly, a

mixture of LE1 (1.5 mg, 0.847 µmol) and LE2 (1.5 mg, 0.898 µmol,) in ammonium acetate buffer pH 5.4 (0.5 mol/L, 300 µL) and an aliquot (3.4 µL, ~1.01 µmol) and (3.6 µL, ~1.07 µmol), respectively, of an aqueous solution of

nat

CuCl2 x 2H2O 0.3 M, were reacted at room temperature for 30 min. The

complexation was confirmed by analytical RP-HPLC. Excess Cu2+ was removed by SepPak C-18



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purification (Waters). The metallopeptides were eluted with methanol, followed by evaporation to dryness, dilution in water, and lyophilization. Quality control of the

nat

Cu-containing complexes was

carried out by analytical HPLC following the conditions described above where only one single peak was detected. The natCu-complexes were also characterised by ESI-MS.

Radiochemistry 64

Cu-LE1 and 64Cu-LE2, were prepared by dissolving 6 nmol of each peptide in ammonium acetate

buffer (300 µL, 0.5 mol/L, pH 5.4) followed by incubation with

64

CuCl2 (600 MBq) for 30 min at

room temperature or 10 min at 95 ºC. To minimize radiolysis ethanol (40 µL) was added. The 64Culabeled radiotracers were used for in vitro and in vivo studies without further purification.

Lipophilicity The lipophilicity (LogD, pH 7.4) was estimated by the “shake-flask” method: The labeled conjugates (10 µL of 100 nM, 6.3 MBq.nmol-1) were added to a solution of 1-octanol (500 µL) and of PBS (500 µL, pH 7.4). The mixture was vortexed for 1 h to reach the equilibrium and then centrifuged (3000 rpm) for 10 min. From each phase, an aliquot (100 µL) was pipetted out and measured in a γ-counter. Each measurement was repeated five times. Care was taken to avoid cross-contamination between the phases. The partition coefficient was calculated as the average log ratio of the radioactivity in the organic fraction and the PBS fraction.

Determination of Binding Affinity The binding affinity profiles (IC50 values) of natCu-LE1, natCu-LE2 and native BN were determined by in vitro GRPr autoradiography on cryostat sections of well-characterized prostate carcinomas as described previously.30 The radioligand used was

125

I-Tyr4-bombesin (2000 Ci/mmol), known to

preferentially label GRPr. Calcium flux was determined using a Flexstation II 384 from Molecular Devices. PC3 cells were seeded at a density of 25000 cells per well in 96-well plates and incubated with 100 µL Ca5 dye R8186 solution including probenecid (5 mmol/L) diluted with washing buffer. To determine antagonistic properties, cells were pre-incubated with serial dilutions of the antagonists followed by the determination of the Tyr4-BN concentration which gives the 80% of the maximal Ca2+ response (EC80-concentration). The change of the fluorescent signal was recorded continually for 90 sec at 538 nm.

Cell Line – Animal Models The PC3 human prostate cancer cell line which is known to overexpress GRPr was cultured at 37 °C and 5% CO2 in DMEM containing 10% FBS, 100 U/mL penicillin and 100 µg/mL streptomycin.


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Female athymic nude mice (age: 4-6 weeks, weight: 17-20 g) were purchased from Janvier, France. For implantation, the tumor cells were harvested by trypsinization and 5 x 106 cells in PBS (100 µL) were inoculated subcutaneously into the right shoulder of the mice. After an average of three weeks, tumor size reached 200 to 300 mg and the animals were used for biodistribution and PET imaging studies. All animal experiments were approved by local authorities and are in compliance with the institutional guidelines (Registration number: G-13/30).

Human Serum Stability To human serum (500 µL) at 37 ºC was added either a solution of 64Cu-LE1 or

64

Cu-LE2 in saline

(100 µL, 1 nmole, ~ 40MBq) and the mixtures were incubated at 37 ºC for 30 min. Samples (300 mL) of serum were transferred to an ultrafiltration device (Vivacon 500; 30,000 molecular weight cutoff [Sartorius Stedium Biotech GmbH]), followed by centrifugation (10 min, 9,660g, 4 ºC) for the separation of proteins. Samples from the ultrafiltrate and

64

Cu-LE1 and

64

Cu-LE2 solutions were

analyzed by RP-HPLC following the conditions described above for the quality control of the radiolabeled peptides.

Internalization / The Fate of GRPr-bound Radiopeptide in vitro For internalization experiments, PC3 cells were seeded at a density of 0.8-1 million cells per well in 6well plates and incubated overnight with medium (DMEM containing 1% FBS, 100 U/mL penicillin and 100 µg/mL streptomycin). Approximately 0.25 pmol / 16 KBq of the respective radiopeptide were added to the medium and the cells were incubated (in triplicates) for 0.5, 1, 2, 4 and 6 h at 37 °C, 5% CO2. To determine nonspecific membrane binding and internalization, excess of Tyr4-BN (final concentration 1 µΜ) was added to selected wells. At each time point, the internalization was stopped by removing the medium and washing the cells twice with ice-cold PBS. To remove the receptorbound radioligand, an acid wash was carried out twice with a 0.1 M glycine buffer pH 2.8 for 5 min on ice. Finally, cells were solubilized with 1 N NaOH. The radioactivity of the culture medium, the receptor-bound, and the internalized fractions were measured in a γ-counter. To determine the fate of GRPr-bound radiopeptide, PC3 cells were seeded into 6-well plates and treated as described above. The plates were placed on ice for 30 min and 0.25 pmol of the respective radiotracers were added in presence or absence of Tyr4-BN (final concentration 1 µΜ) for the determination of nonspecific binding. After the addition of the radioligand, the cells were incubated for 120 min at 4 °C. After the end of the incubation, the cells were washed twice with ice-cold PBS and 1 mL of fresh pre-warmed (37 °C) culture medium was added to each well followed by incubation for 10, 20, 30 min and 1, 2 and 4 h (37 °C, 5% CO2). At each time point the plates were treated as described in the internalization studies.

Biodistribution Studies in PC3 Xenografts and Blood Clearance



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100 pmol / 3 - 5 MBq of 64Cu-LE1 in 100 µL NaCl solution (0.9 %, NaCl) were injected intravenously into the tail vein of PC3 tumor bearing mice. Animals were sacrificed by isoflurane anesthesia at 1, 4 and 24 h after injection of the 64Cu-labeled vector. The organs of interest were dissected and weighted, and the radioactivity in tissue samples was counted in a γ-counter. Biodistribution data are given as percent of injected activity per gram of tissue (% IA/g) and are means ± SD (n = 4). To demonstrate the specificity of binding, PC3 mice were preinjected (3-5 min) with non-radioactive peptide (20 nmol). Animals were sacrificed at 1 h after injection by isoflurane anesthesia. A separate group of mice (n=2) was intravenously injected into the tail vein with 100 pmol / 3 - 5 MBq of 64Cu-LE1 and blood samples were obtained from the facial vein at 1, 3, 5, 7, 10, 15, 20, 30, 45, 60, 240 min after the injection of the radiotracer. The radioactivity in blood samples was counted in a γ-counter data are given as percent of injected activity per gram of blood (% IA/g) and are means ± SD (n = 2).

Small-Animal PET Studies PET images were obtained upon injection of 100 pmol of the radioligands (64Cu-LE1 or 64Cu-LE2: 3 5 MBq / 100 µL) on PC3 tumor bearing mice. Imaging was acquired for a time period of 30 min at 1, 4 and 24 h post injection. To visualize the extent of GRP-specific tumour uptake of the 64Cu-labeled LE1 and LE2 radioconjugates, blocking studies were performed as described above and static scans were obtained as previously described. PET-images were corrected for 64Cu decay and reconstructed with filtered backprojection. No correction was applied for attenuation. Images were generated using AMIDE software. The color scale was set from 0 to 20 % IA/g to allow for qualitative comparison among the images.

Statistical Analysis All data are expressed as the mean of values ± standard deviation (mean ± SD). Prism 5 software (GraphPad Software) was used to determine statistical significance at the 95% confidence level, with a P value of less than 0.05 being considered significantly different.


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RESULTS

Synthesis and Radiochemistry The synthesis yields of the peptide-chelator conjugates (Scheme 1) ranged from 30 to 40%. The purity of the peptides was >95% and the identity was determined by HPLC and mass spectrometry (Table 1). LE1 and LE2 were efficiently labeled with 64Cu at room temperature, with labeling yields >98% and specific activities of up to 100 GBq/µmol. Additionally, 64Cu-LE1 is more hydrophilic than 64Cu-LE2 (logDoctanol/PBS are given in Table 1).

Binding Affinities / Calcium Flux Assay The binding of [125I-Tyr4]-BN to the GRPr measured on human tumors was inhibited by various concentrations of

nat

Cu-LE1 and

nat

Cu-LE2, respectively and the IC50 values were found to be in the

low nanomolar range (Table 1). Calcium flux studies showed that natCu-LE1 is an antagonist as it does not induce any calcium flux in PC3 cells in culture.

Human Serum Stability As demonstrated by RP-HPLC metabolite analysis of human serum samples after 30 min of incubation either with 64Cu-LE1 or 64Cu-LE2, the amount of the intact radiotracer in both cases was found to be between 85 and 90% and three possible metabolites were detected. Loss of 64Cu form the sarcophagine ligand was not observed as 'free' 64CuII was not detected (Fig 1).

Internalization / The Fate of GRPr-bound Radiopeptide in vitro 64

Cu-LE1 and

64

Cu-LE2 were found to be well associated with the PC3 cells within 6 h-incubation

(Fig 2). Continued exposure of the cells to the radioactive ligands resulted in a gradual increase of the total cell associated uptake from 30 min to 6 h, which was higher for

64

Cu-LE1 (41.7 ± 6.1%)

64

compared to Cu-LE2 (23.3 ± 1.1%), at 6 h (P=0.0035). At 6 h the amount of specifically internalized activity was 12.5 ± 2.3% for 64Cu-LE1 and 7.2 ± 0.65% for 64Cu-LE2 (P=0.0054). The fate of the receptor-bound radiopeptide 64Cu-LE1 was studied by a temperature shift experiment (Fig 3). More than 60% of the labeled peptide was still bound to the receptor after 4 h at 37 ºC. The internalized fraction after 4 h at 37 ºC was less than 20%.

Biodistribution Studies in PC3 Xenografts and Blood Clearance The biodistribution data and tumor-to-tissue ratios of 64Cu-LE1 are summarized in Table 2. 64Cu-LE1 showed fast blood clearance (only 0.06 ± 0.02 % IA/g left in blood at 4 h p.i.). Tumor was found to be the tissue with the highest accumulated activity with a maximum of 19.6 ± 4.7 % IA/g at 1 h after injection. Even after 24 h p.i., the tumor uptake remained high with a value of 7.9 ± 1.4 % IA/g. Tumor uptake was reduced by 95 % after the in vivo saturation of the receptors with 20 nmol of LE1



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demonstrating the high specificity of 64Cu-LE1 towards GRPr. The pancreas, a GRPr positive organ, accumulated high activity at 1 h p.i. (14.7 ± 2.0 % IA/g) but fast washout (1.7 ± 0.6 % IA/g at 4 h p.i. and 0.07 ± 0.00 % IA/g at 24 h p.i.) leads to increased tumor to pancreas ratios over time. The kidneys showed a remarkably low uptake which was reduced to 0.5 ± 0.1 %IA/g 24 h after injection from 3.1 ± 0.7 % IA/g at 1 h p.i. and 1.7 ± 0.1 % IA/g at 4 h p.i. The blood kinetics were determined for 64Cu-LE1 (Fig 4). Assessment of the in vivo LE1 stability of 64

Cu-LE1 in circulation in mice revealed an initial half-life of 1.6 min and a terminal half-life of 18.5

min.

Small-Animal PET Studies The small-animal maximum intensity projection (MIPs) PET images and quantification analysis of 64

Cu-LE1 and

64

Cu-LE2 are reported in Figures 5 and 6, respectively. Both radiotracers selectively

accumulate in GRPr positive organs, such as the tumor and the pancreas at early time points. The 64

Cu-PET studies of both GRPr antagonists at 4 and 24 h p.i. showed excellent pharmacokinetics, with

only marginal uptake in non-tumor tissue. Tumor uptake of 64Cu-LE1 is higher compared to 64Cu-LE2 and it also has longer retention. Administration of both 64Cu-labeled conjugates resulted in remarkably low liver uptake.


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DISCUSSION Prostate cancer is the most common malignancy found in men and the second leading cause of cancer death in the US. Even though prostate cancer is one of the few slowly-growing cancers, it becomes potentially lethal if it metastasizes. Consequently, early detection is particularly important for effective treatment. Functional imaging methods to detect prostate cancer include MRI, SPECT and PET. MRI has shown high sensitivity in prostate cancer localization, although performance varies with the patient population studied. It is also not sensitive in detecting cancer in regions other than the peripheral zone of the prostate.32 The over-expression of peptide receptors in various tumors has generated interest in the development of radiolabeled peptides for the targeting of those tumors either for radiodiagnostic or targeted radiotherapeutic applications. To date, a variety of radiolabeled GRPr-peptidic analogues have been developed and labeled with different SPECT (99mTc,

111

In) and PET (68Ga,

64

Cu,

18

F)

radionuclides, using different chelators, with GRPr-radioantagonists having shown improved pharmacokinetics compared to GRPr-radioagonists.32,33 Amongst the PET-radioisotopes

64

Cu has gained particular attention because of its decay

characteristics (t1/2=12.7 h; β+, Emax=0.653 MeV [17.8%]; β-, Emax=0.579 MeV [38.4%]) and the wellestablished coordination chemistry with a variety of chelators. The longer half-life of 64Cu compared to 68Ga (t1/2=67.8 min) and 18F (T1/2=109.8 min), is particularly attractive as it allows PET-images at later time points with improved tumor-to-background ratios.9

64

Additionally,

Cu-labeled

radiopharmaceuticals can be produced at a central facility and distributed to remote hospitals. The successful application of 64Cu as a diagnostic agent may also lead to potential targeted radionuclide therapy with 67Cu (t1/2=61.9 h; β-, Emax=0.141 MeV [100%]) providing a promising theranostic pair. Within the frame of this project we continued our previous studies using the statine-based GRPr antagonist D-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH234 which was functionalized with a variety of chelators via different spacers and showed excellent pharmacokinetic performance in GRPr positive tumor models and in humans.17,19-21,23 The GRPr-antagonist was N-terminally modified by conjugation to the sarcophagine derivative, MeCOSar, via the PEG4 and PEG2 spacers to obtain LE1 and LE2, respectively, and radiolabeled with

64

Cu. MeCOSar was coupled to PEG4 and PEG2 via an amide

bond. To our knowledge, this is the first time that a GRPr antagonist was functionalized with a Sarderived chelator. Sar derivatives form stable dicationic complexes with Cu2+.24-28 The in vivo stability of

64

64

Cu-complexes is important, as dissociated

Cu2+ accumulates after binding to albumin in the

nuclei or in the mitochondria of non-targeted organs such as the liver.35,36 Sar-based chelators coordinate

64

Cu2+ rapidly at conditions compatible for most targeting molecules (small molecules,

antibodies, peptides)37: pH between 3.7-7, at room temperature, and an incubation time between 10 min-60 min. The non-radioactive analogues,

nat

Cu-LE1 and

nat

Cu-LE2, have high affinity towards GRPr compared

to native bombesin, with IC50 values in the low nanomolar range. The importance of the PEG4 spacer is highlighted by the 2.7-fold higher affinity of

nat

Cu-LE1 when compared to


nat

Cu-LE2. The


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Page 12 of 37

superiority of PEG4 compared to PEG2 has also been reported by us when the same GRPr-antagonist was coupled to several DOTA-PEGx moieties (x= 2 to 12) and PEG4 showed the highest binding affinity and the optimum pharmacokinetic profile.21 Both LE1 and LE2 could be radiolabelled with

64

Cu at room temperature, with a maximal specific

activity of 100 MBq/nmol at 98 % radiochemical purity. Reported specific activities for GRPr-analogs coupled with various Sar derived chelators were about 12 MBq/nmol.15,37 When the same GRPr antagonists was coupled with 4,11-bis(carboxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane (CB-TE2A) via PEG4, the labeling with 64Cu was performed at elevated temperatures (95 ºC, 30 min) and led to the formation of a single radioactive species with a specific activity of 25 MBq/nmol.17 In vitro studies demonstrated high and selective binding of overexpressing cells, with

64

64

Cu-LE1 and

64

Cu-LE2 to GRPr

Cu-LE1 exhibiting a superior profile in terms of total cell associated

uptake and % of specific internalized fraction, which might be due to the higher affinity of compared to

nat

Cu-LE1

nat

Cu-LE2 towards GRPr. In both cases the amount of surface associated activity

exceeded the amount of internalized activity at all-time points. These results in combination with the Ca2+-flux assay, which showed no Ca2+-release when PC3 cells in culture were incubated with

nat

Cu-

LE1, safely led us to conclude that the N-terminal modifications did not change the antagonist features of the statine-based antagonist and LE1 retains its GRPr-antagonistic profile. Both 64Cu-LE1 and 64Cu-LE2 accumulate in GRPr positive organs, in PC3 xenografted mice, at early time points by a receptor mediated process (as blocking studies with non-radioactive peptide lead to reduced uptake). The tumor was the tissue with the highest accumulated activity followed by the pancreas. Whilst the radioactivity in the pancreas washed out quickly the tumor showed longer retention for both radiotracers, a finding which is in accordance with previous results for other GRPr antagonists labeled with different radionuclides.17,19-20,23,39-40 The low liver uptake of

64

Cu-LE1 and

64

Cu-LE2 needs to be highlighted since this is a strong indication that MeCOSar stably encapsulates

64

Cu, resulting in high in vivo stability. The resistance to loss of 64Cu2+ from sarcophagine ligands in

vivo has been reported by several groups. Smith et al, compared the biodistribution profiles of 64CuSar (1,8-diamine-3,6,10,13,16,19-hexaazabicyclo[6.6.6]icosane),

64

Cu-SarAr (1-N-(4-aminobenzyl)-

3,6,10,13,16,19-hexaazabicyclo[6.6.6]eicosane-1,8-diamine) and free 64Cu, where 64Cu-Sar as well as 64

Cu-SarAr showed significantly lower liver uptake compared to free 64Cu at 30 min p.i..24 Cai and co-

workers performed a comparative study between

64

Cu-DOTA and

64

Cu-AmBaSar (4-((8-amino-

3,6,10,13,16,19-hexaazabicyclo[6.6.6.]icosane-1-ylamino)methyl)benzoic acid) with the later having shown less uptake in the liver as well as in the abdomen.25 AmBaSar was also coupled by the same group to a cyclic RGD peptide motif, and 64Cu-AmBaSar-RGD was directly compared to 64Cu-DOTARGD in U87MG glioma xenografts. While they both showed similar tumor uptake after 20 h p.i. 64CuAmBaSar-RGD exhibited lower liver uptake resulting in 3-fold increase of tumor-to-liver ratio.41 Another point worth noting is the long blood circulation of 64Cu-DOTA-RGD (about 0.3% IA/g even after 20 h p.i.) indicative of transmetallation to albumin. On the other hand 64Cu-LE1 exhibits very fast


Page 13 of 37

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blood clearance with values which drop to almost zero already at 4 h p.i. indicating no transfer to albumin. MeCOSar was previously synthesized and coupled to Tyr3-octreotate by Paterson et al to obtain

64

Cu-MeCOSar-TATE. In a comparative study between

DOTA-TATE using A427-7 tumor bearing mice,

64

64

Cu-MeCOSar-TATE and

64

Cu-

Cu-MeCOSar-TATE exhibited superior

pharmacokinetic performance in terms of tumor uptake and retention and liver uptake with the only drawback of higher, by a factor of more than 3, kidney uptake compared to

64

Cu-DOTA-TATE a

30

finding which can be attributed to the different net charge of the radiotracers. In contrary, when Wei et al coupled DiamSar (1,8-diamine-3,6,1013,16,19-hexaazabicyclo[6.6.6]icosane) and CB-TE2A to a cyclic RGD peptide, they reported that although the accumulated activity in liver at 1 h p.i. was lower for

64

64

Cu-DiamSar-c(RGDfD) compared to

surprisingly, the wash out from the liver of

Cu-CBTE2A-c(RGDyK) by a factor of about 0.6,

64

Cu-DiamSar-c(RGDfD) compared to

64

Cu-CBTE2A-

42

c(RGDyK) was much slower.

The tumor-to-liver and tumor-to-kidney ratios of 64Cu-LE1 ranged from 6 to 17 and from 1 to 24 h p.i. and were somewhat improved compared to what we previously reported for

64

Cu-CB-TE2A-AR

where the same ratios ranged between 5-13 the same time points.17 Although the tumor uptake for 64

Cu-LE1 was lower compared to

64

Cu-CB-TE2A-AR the considerably decreased kidney and liver

uptakes of 64Cu-LE1, led to higher ratios over time. The reported tumor-to-liver and tumor-to-kidney ratios for the

64

Cu-labeled GRPr agonists

Bombesin(7-14) and respectively. 64

10,13,16

64

64

Cu-CB-TE2A-Aoc-bombesin(7-14)

64

Cu-NOTA-Aoc-

Cu-NOTA-Bn-SCN-Aoc-BN(7-14) were 0.5-2 from 1 to 24 h p.i.,

The truncated GRPr agonist BN(7-14) has also been functionalized with SarAr and

Cu-SarAr-SA-Aoc-bombesin(7-14) and

64

Cu-SarAr-SA-Aoc-GSG-bombesin(7-14) (SA: succinic

acid, Aoc: 8-aminooctanoic acid, GSG: Gly-Ser-Gly), were evaluated in vitro and in vivo.15 Although the tumor uptake for both radiotraces appeared to be relatively high (13 % IA/g for 64Cu-SarAr-SAAoc-bombesin(7-14) and 8.5 IA/g for 64Cu-SarAr-SA-Aoc-GSG-bombesin(7-14) at 1 h p.i.), the high and sustained liver and kidney uptake resulted in tumor-to-liver and tumor-to-kidney ratios varying from 1.3 to 1.7. Liu et al have coupled NODAGA to RM1 (NODAGA-Gly-4-aminobenzoyl-D-PheGln-Trp-Ala-Val-Gly-His-Sta-Leu-NH2; antagonist) and to AMBA (NODAGA-Gly-4-aminobenzoylGln-Trp-Ala-Val-Gly-His-Leu-Met-NH2; agonist) and labeled the two GRPr-targeting conjugates with 64

Cu. They found low but specific tumor uptake for both with the radioantagonist showing

significantly lower abdominal uptake. Additionally the clearance from the tumor was more rapid for 64

Cu-NODAGA-AMBA.11

The PET images reflect the results from the biodistribution studies. Both radiotracers are specifically taken up by the GRPr positive organs at early time points while at 4 and 24 h p.i. only marginal uptake in non-tumor tissue was observed. Both 64Cu-LE1 and 64Cu-LE2 show excellent pharmacokinetics, as demonstrated by biodistribution as well as PET imaging. The microPET images of clearly superior to other

64

10-16,18

Cu-labeled GRPr peptidic analogues.

64

Cu-LE1 are

Translation into the clinic is

undoubtedly an arduous process but the data presented here are sufficiently promising to further



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

clinical studies. Despite the numerous challenges of introducing peptide-based PET imaging agents into the clinic three of our bombesin based GRPr antagonists are currently in clinical studies in different hospitals and we conclude that the experimental data of 64Cu-LE1 warrant clinical translation as well.


Page 14 of 37

Page 15 of 37

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CONCLUSIONS The data presented here demonstrate that the coupling of the MeCOSar chelator via the spacers PEG2 and PEG4 to a statine-based GRPr antagonist and the subsequent complexation with Cu(II) improve the affinity of

nat

Cu-LE1 and

naz

Cu-LE2 towards GRPr compared to the native bombesin. The ease of

LE1 and LE2 synthesis, the efficient radiolabeling with high specific activities, the stable encapsulation of 64Cu by MeCOSar and the suitable biodistribution profile of the 64Cu-labeled peptides lead to PET images of high contrast. On the basis of these results, 64Cu-LE1 and 64Cu-LE2 appear to have considerable potential to be translated into the clinic for the targeting of GRPr positive tumors.

ACKNOWLEDGMENT Financial support: this work was supported by the German Consortium for Translational Cancer Research (DKTK). We thank Yvonne Kiefer and Roswitha Toennesmann for their technical assistance.

CONFLICT OF INTEREST The authors declare that they have no conflict of interest.



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Page 16 of 37

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(22) Wieser, G.; Mansi, R.; Grosu, A.L.; Schultze-Seemann, W.; Dumont-Walter, R.A.; Meyer, P.T.; Maecke, H.R.; Reubi, J.C.; Weber, W.A. Positron emission tomography (PET) imaging of prostate cancer with a gastrin releasing peptide receptor antagonist - from mice to men. Theranostics 2014, 4, 412-419. (23) Gourni, E.; Mansi, R.; Jamous, M.; Waser, B.; Smerling, C.; Burian, A.; Buchegger, F.; Reubi, , J.C.;

Mäcke,

H.R.

N-Terminal

modifications

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receptor

affinity

and

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Cu radiopharmaceutcals.

Nucl. Med. Biol. 2010, 37, 57-65. (26) Cai, H.; Fissekis, J.; Conti, P.S. Synthesis of a novel bifunctional chelator AmBaSar based sarcophasine for peptide conjugation and 64Cu radiolabeling. J. Chem. Soc. Dalton Trans. 2009, 27, 5395-5400. (27) Ma, M.T.; Karas, J.A.; White, J.M.; Scanlon, D.; Donnelly, P.S. A new bifunctional chelator for copper radiopharmaceuticals: a cage amine ligand with a carboxylate functional group for conjugation to peptides. Chem. Commun. 2009, 3237-3239. (28) Di Bartolo, N.M.; Sargeson, A.M.; Donlevy, T.M.; Smith, S.V. Synthesis of a new cage ligand, SarAr, and its coplexation with selected transition metal ions for potential use in radioimaging. J. Chem. Soc. Dalton Trans. 2001, 15, 2303–2309. (29) Voss, S.T.; Smith, S.V.; DiBartolo, N.; McIntosh, L.J.; Cyr, E.M.; Bonab, A.A.; Dearling, J.L.J.; Carter, E.A.; Fishman, A.J.; Treves, S.T.; Gillies, S.D.; Sargeson, A.M.; Huston, J.S.; Packard, A.B. Positron emission tomography (PET) imaging of neuroblastoma and melanoma with 64Cu-SarAr immunoconjugates. Proc. Natl. Acad. Sci. USA 2007, 104, 17489-17493. (30) Paterson, B.M.; Roselt, P.; Denoyer, D.; Cullinane, C.; Binns, D.; Noonan, W.; Jeffery, C.M.; Price, R.I.; White, J.M.; Hicks, R.J.; Donnelly, P.S. PET imaging of tumors with a 64Cu labeled macrobicyclic cage amine ligand tethered to Tyr3-octreotate. Dalton Trans. 2014, 43, 13861396. (31) Markwalder, R.; Reubi, J.C. Gastrin-releasing peptide receptors in the human prostate: relation to neoplastic transformation. Cancer Res. 1999, 59, 1152-1159. (32) Hricak, H.; Choyke P.L.; Eberhardt, S.C.; Leibel, S.A.; Scardino, P.T. Imaging prostate cancer: A multidisciplinary perspective. Radiology 2007, 243, 28-53.


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Page 20 of 37

Table 1: Analytical and affinity data for natCu-LE1 and natCu-LE2. MS (ESI):

Theoretical m/z

m/z ([M+H]2+)

([M+H]2+)

nat

916.0

916.8

nat

866.4

865.8

Compound

logDoctanol/PBS

Rt

IC50

(min)

(nmol/L) 3.4 ± 0.7

-2.6 ± 0.1

23.20

1.4 ± 0.1

-2.1 ± 0.1

20.19

3.8 ± 0.6

native BN Cu-LE1

Cu-LE2

Analytical RP-HPLC: 15-90% solvent B in 25 min, flow rate:1 mL/min, solvents: A: 0.1% TFA/H2O, and B: 0.1% TFA/Acetonitrile.


Page 21 of 37

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Table 2: Biodistribution data of 64Cu-LE1 in PC3 xenografts, 1, 2 and 24 h after injection.a Organ

1h

1 h blocked

4h

24 h

Blood

0.5 ± 0.1

0.3 ± 0.05

0.06 ± 0.02

0.03 ± 0.00

Heart

0.2 ± 0.05

0.1 ± 0.01

0.08 ± 0.01

0.07 ± 0.01

Liver

2.9 ± 0.4

2.3 ± 0.6

2.7 ± 0.3

1.5 ± 0.1

Spleen

0.7 ± 0.1

0.4 ± 0.2

0.4 ± 0.2

0.1 ± 0.01

Lung

0.5 ± 0.1

0.9 ± 0.6

0.2 ± 0.08

0.1 ± 0.01

Kidney

3.1 ± 0.7

2.2 ± 0.3

1.7 ± 0.1

0.5 ± 0.09

Stomach

3.0 ± 0.5

0.8 ± 0.7

1.5 ± 0.2

0.2 ± 0.02

Intestine

2.7 ± 1.2

1.5 ± 1.2

0.7 ± 0.2

0.1 ± 0.02

Adrenal

2.1 ± 0.4

0.4 ± 0.05

0.9 ± 0.3

0.4 ± 0.2

Pancreas

14.7 ± 2.0

0.3 ± 0.01

1.7 ± 0.6

0.07 ± 0.00

Muscle

0.2 ± 0.08

0.09 ± 0.04

0.04 ± 0.02

0.02 ± 0.00

Bone

0.5 ± 0.2

0.2 ± 0.0

0.2 ± 0.08

0.1 ± 0.03

PC3-tumor

19.6 ± 4.7

0.9 ± 0.1

17.7 ± 1.0

7.9 ± 1.4

Tumor/Blood

42 ± 3.7

345 ± 109

255 ± 35

Tumor/Liver

6.1 ± 1.4

7.2 ± 0.8

5.4 ± 1.1

Tumor/Kidney

5.9 ± 0.8

10.4 ± 0.4

17 ± 4.2

Tumor/Pancreas

1.2 ± 0.3

11.5 ± 4.4

117 ± 19

Tumor/Muscle

97 ± 40

534 ± 282

482 ± 151

a

Data are expressed in percentage of injected activity per gram of tissue (%IA/g) and are presents as

the mean ± SD (n = 3-4).



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Page 22 of 37

SCHEME AND FIGURE CAPTIONS

Scheme 1: Schematic representation of the chemical structures of LE1 and LE2. Figure 1: HPLC profiles of: 64Cu-LE1 (A), 64Cu-LE2 (C) and human plasma sample after 30 min of incubation with 64Cu-LE1 (B) or 64Cu-LE2 (D). Figure 2: Specific cell uptake and internalization rate after the incubation of PC3 cells with 64Cu-LE1 and

64

Cu-LE1 within 6 h at 37 °C. A. Cell uptake calculated as cell surface-bound and internalized

fraction. B. Receptor-specific internalization expressed as percentage of the applied radioactivity. Nonspecific binding was determined in the presence of 1 µM Tyr4-BN. Figure 3: The fate of the GRPr-bound 64Cu-LE1 as measured with PC3 cells. The radioactive ligand was measured with respect to the total receptor bound radioactive ligand in 2 h at 4 °C (100%). Figure 4: Radiocativity concentration in the blood over time (mean and standard deviation) and biexponential fit of the data. The initial half-life of 64Cu-LE1 is 1.6 min and the terminal half-life is 18.5 min. Figure 5: Maximum intensity projections (MIPs) of

64

Cu-LE1 (A) and

64

Cu-LE2 (B) upon their

injection on PC3 tumor bearing mice at 1 h, 4 h and 24 h p.i along with blocking studies at 1h p.i.. Figure 6: Quantitative analysis of the PET images; A:

64

Cu-LE1 and

background ratios.


64

Cu-LE2, B: Tumor-to-

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Scheme 1



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Figure 1


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Figure 2



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Figure 3


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Figure 4



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Figure 5


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Figure 6



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44x36mm (300 x 300 DPI)

ACS Paragon Plus Environment

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Schematic representation of the chemical structures of LE1 and LE2. 74x33mm (600 x 600 DPI)



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HPLC profiles of:

64

Cu-LE1 (A),

64

Cu-LE2 (C) and human plasma sample after 30 min of incubation with LE1 (B) or 64Cu-LE2 (D). 68x40mm (600 x 600 DPI)


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64

Cu-

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Specific cell uptake and internalization rate after the incubation of PC3 cells with 64Cu-LE1 and 64Cu-LE1 within 6 h at 37 °C. A. Cell uptake calculated as cell surface-bound and internalized fraction. B. Receptorspecific internalization expressed as percentage of the applied radioactivity. Nonspecific binding was determined in the presence of 1 µM Tyr4-BN. 116x168mm (600 x 600 DPI)



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The fate of the GRPr-bound 64Cu-LE1 as measured with PC3 cells. The radioactive ligand was measured with respect to the total receptor bound radioactive ligand in 2 h at 4 °C (100%). 67x53mm (600 x 600 DPI)


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Radiocativity concentration in the blood over time (mean and standard deviation) and bi-exponential fit of the data. The initial half-life of 64Cu-LE1 is 1.6 min and the terminal half-life is 18.5 min. 57x40mm (300 x 300 DPI)



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Maximum intensity projections (MIPs) of 64Cu-LE1 (A) and 64Cu-LE2 (B) upon their injection on PC3 tumor bearing mice at 1 h, 4 h and 24 h p.i along with blocking studies at 1h p.i.. 95x79mm (300 x 300 DPI)


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Quantitative analysis of the PET images; A: 64Cu-LE1 and 64Cu-LE2, B: Tumor-to-background ratios. 122x101mm (600 x 600 DPI)


PET imaging of tumours with a 64Cu labeled macrobicyclic cage amine ligand tethered to Tyr3-octreotate.

(64)Cu labeled sarcophagine exendin-4 for microPET imaging of glucagon like peptide-1 receptor expression.

GRP receptor imaging of prostate cancer using [(99m)Tc]Demobesin 4: a first-in-man study.

Positron emission tomography (PET) imaging of prostate cancer with a gastrin releasing peptide receptor antagonist--from mice to men.

Dual-Modality Imaging of Prostate Cancer with a Fluorescent and Radiogallium-Labeled Gastrin-Releasing Peptide Receptor Antagonist.

Breast cancer vaccine shows promise.

GRP-R signaling contributes to castration-resistant prostate cancer progression.

Synthesis of a new fluorine-18-labeled bexarotene analogue for PET imaging of retinoid X receptor.

[Elastography shows promise in testicular cancer detection].

Pembrolizumab Shows Promise for NSCLC.

Improving PET spatial resolution and detectability for prostate cancer imaging.

CT imaging of gastric cancer.

Hepatitis A vaccine shows promise.

CT: a novel radiotracer for imaging of prostate carcinoma.

In Vivo Stabilization of a Gastrin-Releasing Peptide Receptor Antagonist Enhances PET Imaging and Radionuclide Therapy of Prostate Cancer in Preclinical Studies.

Prostate-specific G-protein-coupled receptor collaborates with loss of PTEN to promote prostate cancer progression.

A Meta-Analysis and Indirect Comparison of Endothelin A Receptor Antagonist for Castration-Resistant Prostate Cancer.

CT imaging.

PET imaging detection of macrophages with a formyl peptide receptor antagonist.

Gastrin-releasing Peptide Receptor Imaging in Breast Cancer Using the Receptor Antagonist (68)Ga-RM2 And PET.

CT imaging in prostate cancer.

Discovery of BMS-641988, a Novel Androgen Receptor Antagonist for the Treatment of Prostate Cancer.

Temporal Heterogeneity of Estrogen Receptor Expression in Bone-Dominant Breast Cancer: 18F-Fluoroestradiol PET Imaging Shows Return of ER Expression.

Erratum to: A new F-18 labeled PET tracer for fatty acid imaging.