high contrast pet imaging of grpr expression in prostate...

Research ArticleHigh Contrast PET Imaging of GRPR Expression in ProstateCancer Using Cobalt-Labeled Bombesin Antagonist RM26

Bogdan Mitran1 Helge Thisgaard23 Ulrika Rosenstroumlm4 Johan Hygum Dam2

Mats Larhed5 Vladimir Tolmachev6 and Anna Orlova1

1Division of Molecular Imaging Department of Medicinal Chemistry Uppsala University Uppsala Sweden2PET amp Cyclotron Unit Department of Nuclear Medicine Odense University Hospital Odense Denmark3Department of Clinical Research University of Southern Denmark Odense Denmark4Division of Organic Pharmaceutical Chemistry Department of Medicinal Chemistry Uppsala University Uppsala Sweden5Science for Life Laboratory Department of Medicinal Chemistry Uppsala University Uppsala Sweden6Department of Immunology Genetics and Pathology Uppsala University Uppsala Sweden

Correspondence should be addressed to Anna Orlova annaorlovapetmedchemuuse

Received 22 April 2017 Accepted 22 June 2017 Published 10 August 2017

Academic Editor Ralf Schirrmacher

Copyright copy 2017 Bogdan Mitran et alThis is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

High gastrin releasing peptide receptor (GRPR) expression is associated with numerous cancers including prostate and breastcancer The aim of the current study was to develop a 55Co-labeled PET agent based on GRPR antagonist RM26 for visualizationof GRPR-expressing tumors Labeling with 57Co and 55Co stability binding specificity and in vitro and in vivo characteristics of57Co-NOTA-PEG2-RM26were studied NOTA-PEG2-RM26was successfully radiolabeled with 57Co and 55Cowith high yields anddemonstrated high stabilityThe radiopeptide showed retained binding specificity to GRPR in vitro and in vivo 57Co-NOTA-PEG2-RM26 biodistribution in mice was characterized by rapid clearance of radioactivity from blood and normal non-GRPR-expressingorgans and low hepatic uptake The clearance was predominantly renal with a low degree of radioactivity reabsorption Tumor-to-blood ratios were approximately 200 (3 h pi) and 1000 (24 h pi)The favorable biodistribution of cobalt-labeled NOTA-PEG2-RM26translated into high contrast preclinical PETCT (using 55Co) and SPECTCT (using 57Co) images of PC-3 xenografts The initialbiological results suggest that 55Co-NOTA-PEG2-RM26 is a promising tracer for PET visualization of GRPR-expressing tumors

1 Introduction

Receptors for small regulatory peptides are often overex-pressed in human malignant tumors This observation hasled to a number of studies exploring the opportunity forclinical application of peptide analogs in tumor diagnosisand therapy [1] One promising approach is to target thegastrin releasing peptide receptor (GRPR) using bombesinanalogs HighGRPR expressionwas documented in the earlyandrogen-dependent stages of prostate cancer (PCa) but notin the hyperplastic prostate Therefore GRPR has emerged asan attractive target in PCa a notoriously difficult cancer todiagnose and stage using conventional imaging techniques

Bombesin (BN) is a 14-amino acid peptide that bindsto GRPR with high affinity Over the past decade severalderivatives of bombesin have been investigated in preclinical

and clinical studies Initially the consensus was that agonistswere superior to antagonists due to the rapid radioligandinternalization The high internalization was considered tobe essential for optimal imaging because it provides efficientintracellular trapping of radiometal labels [2] However thestrong physiological action and mitogenicity associated withBN-based GRPR-agonists [3] and the superior tumor uptakeseen for somatostatin antagonists [4] led to a paradigmshift towards the use of potent GRPR antagonists in thedevelopment of BN analogs during recent years [5]

Previously our group reported a high affinity GRPRantagonist based on RM26 (DPhe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH2) [6] conjugated to a 147-triazacyclononane-NN1015840N10158401015840-triacetic acid (NOTA) chelator via a diethyleneglycol (PEG2) spacer (NOTA-PEG2-RM26) This constructwas initially radiolabeled with 68Ga and 18F [7 8] for positron

HindawiContrast Media amp Molecular ImagingVolume 2017 Article ID 6873684 10 pageshttpsdoiorg10115520176873684

2 Contrast Media amp Molecular Imaging

emission tomography (PET) imaging and 111In [7] for singlephoton emission computer tomography (SPECT) imagingThe 68Ga and 111In-labeled constructs showed favorablepharmacokinetic properties and high affinity to GRPR Inan attempt to further improve the biodistribution profile forpotential clinical use we have also explored the use of dif-ferent radionuclide-chelator complexes It is known that thecombination of radionuclide and chelator could have a stronginfluence on the targeting properties of radiopeptides [910] For example the somatostatin analog [57Co-(dotatoc)]had the highest reported affinity for somatostatin receptorsubtype 2 (SSTR2) [11] Our studies on PEG2-RM26 labeledwith 68Ga and 111In using different macrocyclic chelatorsdemonstrated that charge and structure of the radionuclide-chelator complex have a strong influence on binding affinityand liver uptake of the radioconjugates [12 13]

One interesting finding from our studies was that thetumor-to-background ratios increased significantly over timeup to 24 h after injection (pi) [7 13] due to the high uptake andlong retention in tumors in combination with the fast clear-ance from blood and GRPR-expressing organs underscoringthe importance of radionuclide half-life in high sensitivitymolecular imagingThese data are in agreement with findingsfrom the head-to-head evaluation of 55Co- and 68Ga-labeledGRPR agonist NOTA-AMBA In this study 55Co-NOTA-AMBA provided superior imaging contrast compared tothe 68Ga-labeled counterpart because of the possibility ofperforming imaging at 24 h pi However both 68Ga- and5557Co-labeled NOTA-AMBA had a prolonged retention inGRPR-expressing tissues typical for agonists [14] This issuemight be solved by the use of a high affinity GRPR antagonist

Thus the use of a long lived radionuclide for nextday imaging may enable the detection of low abdominallymph node involvement which requires the highest possiblesensitivity and is an ultimate goal in prostate cancer imaging[15 16] Labeling of RM26with a long lived positron-emittingradionuclide could further increase diagnostic accuracy dueto the inherent high sensitivity and imaging quantitationcapability of PET compared to SPECT For next day imaginga positron-emitting radiometal with a half-life of 10ndash20 hwould be optimal Possible PET radionuclides for suchpurposes include 64Cu (11987912 = 127 h) 86Y (11987912 = 147 h)and 55Co (11987912 = 175 h) Among them 55Co (76 120573+) hasa higher positron abundance compared to 64Cu and a betterratio between annihilation photons and coemitted gammascompared to 86Y providing better image quality [17] The175 h half-life of 55Co not only allows for delayed imaginginvestigations but also enables shipment to stand-alone PETscanners

The goal of this study was to evaluate the tumor targetingproperties of GRPR antagonist NOTA-PEG2-RM26 labeledwith 55Co

2 Materials and Methods

21 Radiochemistry NOTA-PEG2-RM26 (RM26 = [D-Phe6Sta13 Leu14] bombesin [6-14]) was synthesized by standard

manual solid-phase peptide synthesis based on previouslypublished procedures [7]57Co cobalt chloride was purchased from PerkinElmer

Sweden (Upplands Vasby Sweden) 55Co was produced in-house as previously described [18] with an extra purificationstep added [19] Buffers including 02M ammonium acetatepH 55 and 02M citric acid were produced in-housefrom chemicals supplied by Merck (Darmstadt Germany)Phosphate buffered saline (PBS) pH 75 01Mwas purchasedfrom the National Veterinary Institute SVA Sweden Buffersfor labeling were purified from free metals contamination byChelex 100 resin (Bio-Rad Laboratories Hercules CA)

Radiolabeling with 57Co was performed using38ndash10 nmol (aq 1 nmol120583L) of NOTA-PEG2-RM26 bufferedwith 80 120583L ammonium acetate (02M pH 55) The mixturewas incubated with 57Co cobalt chloride (10ndash50MBq) for30min at 60∘C For 55Co labeling 13 nmol of NOTA-PEG2-RM26bufferedwith 80 120583L ammoniumacetate (02M pH55)was heated by microwave irradiation for 1min at 850W in asealed vial using a conventional microwave oven (SamsungMW82N-B) To evaluate the labeling stability 57Co-NOTA-PEG2-RM26 was incubated in the presence of 1000-foldmolar excess of EDTA disodium salt (Sigma) for 1 h at RTLabeling yield radiochemical purity and in vitro stabilitywere analyzed using instant thin-layer chromatography(ITLC) strips (Agilent Technologies) eluted with citric acid(02M pH 20) In this system free 5755Comigrates with theeluent front (119877119891 = 10) and the radiolabeled construct stayson the application point (119877119891 = 00) The system was previ-ously validated by sodium dodecyl sulfate polyacrylamidegel electrophoresis (SDS-PAGE) and radio-HPLC [7 8]

22 In Vitro Studies PC-3 human prostate cancer cellsexpressing GRPR were purchased form ATCC via LGCPromochem The cells were cultured in RPMI-1640 mediasupplemented with 10 fetal calf serum PEST (penicillin100 IUmL streptomycin 100120583gmL) and 2mML-glutamine(all from Biochrom AG Berlin Germany) This mediumis referred to as complete medium in the text Trypsin-EDTA (005 trypsin 002 EDTA in buffer BiochromAG)was used for cell detachment All in vitro experiments wereperformed in triplicate

The binding specificity of 57Co-NOTA-PEG2-RM26 wasevaluated using PC-3 cells incubated with 1 nM 57Co-NOTA-PEG2-RM26 solution for 1 h at 37∘C In this experiment oneset of dishes was preincubated for 10min at RT with anexcess (100-fold) of nonlabeled peptide before applying theradioactivity After incubation the cells were washed withserum-free media and detached with 05mL trypsin-EDTAsolution Radioactivity of cells wasmeasured in an automatedgamma-counter (3-inch NaI(Tl) detector 2480 WIZARD2PerkinElmer) and presented as cell-associated percentagefrom added radioactivity

Cellular processing of the conjugate was evaluated usingPC-3 cells incubated with 1 nM of 57Co-NOTA-PEG2-RM26at 37∘C 5 CO2 At predetermined time points (1 2 4 8and 24 h after the start of incubation) the medium from 3dishes was discarded and the cells were washed with ice-cold

Contrast Media amp Molecular Imaging 3

serum-free mediumThemembrane-bound and internalizedradioactivity were collected using the acid wash methoddescribed earlier [7]

The half maximal inhibitory concentration (IC50) wasdetermined for natCo-NOTA-PEG2-RM26 using the uni-versal BN radioligand 125I-Tyr4-BBN (Perkin Elmer) Forcomparison an additional experiment was performed in par-allel for the previously evaluated natIn-NOTA- PEG2-RM26under the same experimental conditions [7] Briefly cellmonolayerswere incubatedwith 125I-Tyr4-BBN (01 pmol) for5 h at 4∘C in the presence of increasing concentrations ofnatCo-loaded and natIn-loaded conjugates (0ndash1000 nM) Afterincubation the cells were collected and the cell-associatedradioactivity wasmeasured in an automated gamma-counterThe IC50 values were calculated by nonlinear regression usingGraphPad Prism software (GraphPad Software Inc)

The binding kinetics were measured in real-time usingLigandTracer Yellow Instruments (Ridgeview InstrumentsAB) at room temperature as described earlier [20] Theuptake curves were recorded at 033 and 6 nM After mea-suring the uptake curves the 57Co-NOTA-PEG2-RM26-containing medium was replaced by fresh medium andthe dissociation curve was monitored overnight Datawere collected and evaluated using TracerDrawer software(Ridgeview Instruments AB)

23 In Vivo Studies The animal experiments were plannedand performed in accordance with the national legislation onthe protection of laboratory animals and the study planswereapproved by the local committee for animal research ethicsGroups of 3-4 mice were used per data point Biodistributionstudies were performed on female immunocompetent NMRImice (weight 30 plusmn 2 g) female Balbc nunu (inoculated10 million PC-3 cellsmouse 21 days before the experimentweight 17 plusmn 2 g) and male inbred immunodeficient NOD-SCID mice (5 million PC-3 cellsmouse 35 days before theexperiment 31 plusmn 2 g)

24 Biodistribution and In Vivo Binding Specificity in NMRIMice FemaleNMRImicewere injectedwith 45 pmol (100120583Lin PBS) 57Co-NOTA-PEG2-RM26 into the tail vein Theinjected radioactivity was adjusted to 30 kBq per animalTo test the in vivo binding specificity of 57Co-NOTA-PEG2-RM26 to murine GRPRs one group of animals wasintravenously coinjected with 20 nmol of unlabeled peptideThe mice were euthanized at 30min pi by intraperitonealinjection of Ketalar-Rompun solution (10mgmLKetalar and1mgmLRompun 20120583Lg bodyweight) Blood samples werecollected by cardiac puncture Lung liver spleen pancreasstomach small intestines kidneys muscle bone and the restof intestines with their content were collected The organswere weighed and their radioactivity content was measuredin a gamma-counterThe data were corrected for backgroundradiation The organ uptake values were expressed as apercentage of injected dose per gram of tissue ( IDg)except for the gastrointestinal tract and the remaining carcasswhich were calculated as ID per whole sample

25 Biodistribution and In Vivo Binding Specificity in PC-3 Xenografted Mice Tumor targeting specificity and biodis-tribution over time were studied on PC-3 xenograftedfemale Balbc nunu and male NOD-SCID mice respec-tively For the in vivo binding specificity experiment theanimals were injected with 45 pmol (20 kBq 100 120583L in 01BSA in PBS) of 57Co-NOTA-PEG2-RM26 An additionalgroup of animals was coinjected with an excess of non-labeled peptide (20 nmol) The mice were sacrificed at 3 hpi by intraperitoneal injection of Ketalar-Rompun solution(10mgmL Ketalar and 1mgmL Rompun 20120583Lg bodyweight) Other groups of mice were intravenously injectedinto the tail vein with 168 plusmn 36 pmol of 57Co-NOTA-PEG2-RM26 (55 plusmn12 kBq 100 120583L in 01 BSA in PBS) The micewere euthanized at 3 and 24 h pi by cervical dislocationTumors blood and other organs of interest were collectedweighed andmeasured on radioactivity content in a gamma-counter The data were corrected for background radiationThe organ uptake values were expressed as a percentage ofinjected dose per gram of tissue ( IDg) except for thegastrointestinal tract and the remaining carcass which werecalculated as ID per whole sample

26 SPECTCT Imaging Using 57Co-NOTA-PEG2-RM26Mice bearing PC-3 xenografts were intravenously injectedwith 100 pmol of 57Co-NOTA-PEG2-RM26 (600 kBq) Theanimals were euthanized by CO2 asphyxiation 3 h and 24 hpi immediately before being placed in the camera Wholebody SPECTCT scans were performed using the TriumphTrimodality System (TriFoil Imaging Inc Northridge CAUSA) at the following parameters CT 80mm field of view(FOV) 148 magnification 1 projection and 512 framesSPECT 80mm FOV and 75A10 collimators (5 pinholes) CTraw files were reconstructed by Filter Back Projection (FBP)SPECT raw data were reconstructed using an ordered SubsetExpectation Maximization (OSEM) iterative reconstructionalgorithm SPECT and CT data were analyzed using PMODv3508 (PMOD Technologies Ltd Zurich Switzerland) andwere presented as maximum intensity projections (MIPs)

27 PETCT Imaging Using 55Co-NOTA-PEG2-RM26 MaleNOD-SCID mice bearing PC-3 xenografts were intra-venously injected with 018 nmol (approx 3MBq) of 55Co-NOTA-PEG2-RM26 in the tail vein At 3 h pi the animals (119899 =3) were anesthetized using isoflurane and PETCT scannedusing the Siemens Inveon Multimodality preclinical scanner(Siemens Healthcare Knoxville USA) At 24 h pi the animalswere euthanized by intraperitoneal injection of pentobarbitaland PETCT scanned again CT parameters were 2 bedpositions 360 projections in 360 degreesrsquo rotation and bin4 The PET acquisition times of the static scans were 900 sand 1800 s at 3 h and 24 pi respectively The PET data wereattenuation corrected using the coregistered CT scan andthe sinograms were reconstructed using the OSEM3D-MAPreconstruction algorithm (4 OSEM3D iterations 16 MAPsubsets and 18 MAP iterations target resolution 08mm)PET and CT data were analyzed using Inveon ResearchWorkplace (Siemens Healthcare) and presented as MIPs


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Figure 1 Schematic overview of the structure of 5557Co-NOTA-PEG2-RM26

adjusted to display a color scale from 0 to the maximumtumor uptake value

3 Results and Discussion

Prostate cancer is a complex disease with a heterogeneousgrowth pattern where some patients may not require activetreatment while others need drastic intervention In viewof this biologic and clinical heterogeneity it is imperative todistinguish between rapidly and slowly progressing diseaseand to accurately determine the stage of PCa in order to pre-scribe appropriate treatments Molecular imaging of GRPRexpression can contribute substantially in the initial diagnosisand staging of PCa bynoninvasively assessing the lymphnodeinvolvement and other metastases and distinguishing low-from high-risk disease However imaging of low abdominallymph node involvement in PCa is challenging and requiresthe highest possible sensitivity

In a recent study we have evaluated the effect ofmacrocyclic chelators on the biodistribution and target-ing properties of 111In-labeled bombesin antagonist RM26(111In-X-PEG2-RM26 (X = NOTA NODAGA DOTA andDOTAGA)) [13] In addition to the main conclusions relatedto aspects that underlie the influence of radionuclide-chelatorcomplexes the study also highlighted the crucial role ofradionuclide half-life in high sensitivity molecular imagingand pointed that next day imaging may be preferable dueto higher contrast In this regard all conjugates evaluatedin that study demonstrated significantly higher tumor-to-organ ratios at 24 h pi compared to 4 h pi Therefore we haveinvestigated in this project a new probe 55Co-NOTA-PEG2-RM26 for next day PET imaging of GRPR expression in PCathat would combine the advantages of later imaging with thehigh sensitivity and quantitative accuracy of PET

Labeling of NOTA-PEG2-RM26 with radiocobalt wasperformed in acidic conditions that should prevent oxida-tion of Co(II) by atmospheric oxygen to Co(III) that iscommon in alkaline solutions [21] Labeling was successfulwith yields exceeding 99 for 57Co and 98 for 55Co57Co-NOTA-PEG2-RM26 (Figure 1) was characterized byexcellent stability (Table 1) and retained the capacity to bindspecifically to GRPR-expressing PC-3 cells (Figure 2(a))Binding of 57Co-NOTA-PEG2-RM26 to GRPR-expressingPC-3 cells was significantly (119901 lt 11 times 10minus8) reduced when

Table 1 Labeling and stability of 5557Co-NOTA-PEG2-RM265557Co-NOTA-PEG2-RM26

Labeling yield for 57Co 998 plusmn 02Labeling yield for 55Co 986Release in the presence of excess EDTAlowast 07 plusmn 02Release in PBSlowast 007 plusmn 011lowastStability of 57Co-NOTA-PEG2-RM26 was checked after 1 h incubationat room temperature in the presence of 1000x excess of EDTA Data arepresented as average plusmn standard deviation

presaturating the receptorswith a largemolar excess of nonla-beled peptide indicating that binding of 57Co-NOTA-PEG2-RM26 is receptor-dependent The binding pattern of 57Co-NOTA-PEG2-RM26 was characterized by a rapid increasein total cell-associated radioactivity within the first hour ofincubation followed by a slower increase until the 24 h timepoint (Figure 2(b)) An interesting find was the very lowinternalized fraction that reached only 12 of cell-associatedradioactivity after 24 h incubation (Figure 2(b)) Although alow internalized fraction is expected from antagonists theinternalized fraction for 57Co-labeled NOTA-PEG2-RM26was threefold lower compared to the same radioconjugatelabeled with 111In [7 13] This might reflect lower residualiz-ing properties of cobalt-NOTA containing radiocatabolites

The binding properties of the peptide loaded with cobaltand for comparison indium were evaluated in a competitivebinding assay using 125I-Tyr4-BBN as displacement radioli-gand (Figure 3) While both IC50 values were in the lownanomolar range the cobalt-loaded analog natCo-NOTA-PEG2-RM26 had two fold higher IC50 values (55 plusmn 04 nM)compared to the natIn-loaded counterpart (25 plusmn 01 nM)These findings are in good agreement with previous resultsobtained for bombesin and somatostatin analogs confirmingthat the net charge of the N-terminus of the conjugate hasa strong impact on the binding affinity [12 13 22 23] Inthis case the positive N-terminus charge of indium-NOTAcomplex resulted in higher affinity while a neutral charge(cobalt-NOTA complex) decreased the binding affinity Real-time measurements of 57Co-NOTA-PEG2-RM26 bindingaffinity to GRPR-expressing PC-3 cells revealed119870119863 values inthe low picomolar range (22 plusmn 10 pM) with a rapid binding(association rate of 83 times 104 plusmn 87 times 103 1M times sec) and slow


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Figure 2 (a) In vitro specificity test of 57Co-NOTA-PEG2-RM26 binding to GRPR on PC-3 (human prostate cancer) cells Presaturation ofreceptors with unlabeled NOTA-PEG2-RM26 caused significant (119901 lt 005) reduction of cell-bound 57Co-NOTA-PEG2-RM26 radioactivity(b) Binding and internalization of 57Co-NOTA-PEG2-RM26 at 37∘C by PC-3 cells Data are normalized to a maximum cell-boundradioactivity and presented as average value from 3 cell dishes plusmn SD Not all error bars are visible due to the small standard deviations

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(83 plusmn 09) times 104

(18 plusmn 08) times 10minus6

22 plusmn 10

(b)

Figure 3 (a) Inhibition of 125I-Tyr4-BBN binding to PC-3 cells with natCo-NOTA-PEG2-RM26 and natIn-NOTA-PEG2-RM26 Data arepresented asmean values of three dishesplusmn SD (b) LigandTracer sensorgram and fitted binding curve of 57Co-NOTA-PEG2-RM26 interactionwith GRPR-expressing PC-3 cells at room temperature Uptake curves were recorded at 033 and 6 nM The data were fitted to a one-to-oneinteraction model

dissociation (dissociation rate of 18times 10minus6 plusmn 80times 10minus7 1sec)One important advantage of using real-timemeasurements isthe ability to detect the time to equilibrium which eliminatesthe risk of premature interruption of incubation in otherassays In such a way the intrinsic limitation of the end-point in vitro assays to study high affinity conjugates (119870119863determination in saturation assay IC50 determination ininhibition assay) is avoided In the end-point assays the

values of high affinity conjugates (in low picomolar range) areunderestimated because of the extremely long time requiredto reach equilibrium [24] It should be noted that highaffinity binding is amajor precondition when aiming for highcontrast next day imaging

The biodistribution of 57Co-NOTA-PEG2-RM26 infemale NMRI mice was characterized by rapid clearancefrom blood and normal tissues already at 30min pi


lowast lowastlowast

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Figure 4 In vivo binding specificity of 57Co-NOTA-PEG2-RM26 in (a) NMRImice at 30min pi and (b) PC-3 xenografted Balbc nunumiceat 3 h piThe total injected mass of radioconjugate was 45 pmol All animals in the blocked group were coinjected with 20 nmol of nonlabeledpeptide Data are presented as an average IDg and standard deviation for four mice lowast stands for significant difference (119901 lt 005) in uptakeof radioactivity in the respective organs between the blocked and nonblocked groups of mice

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Figure 5 (a) Biodistribution of 57Co-NOTA- PEG2-RM26 in male NOD-SCID mice bearing PC-3 xenografts at 3 and 24 h pi (total injectedmass of 168 plusmn 36 pmol) (b) Tumor-to-organ ratios of 57Co-NOTA- PEG2-RM26 in NOD-SCID mice with subcutaneous PC-3 xenograftsData are presented as an average value and standard deviation for three mice

(Figure 4(a)) The clearance was predominantly renal witha low kidney retention (6 plusmn 2 IDg) and low liver uptake(065 plusmn 005 IDg) Receptor-positive abdominal organs(pancreas small intestine and stomach) showed highuptake In these organs the accumulated radioactivity wassignificantly lower in the group of mice coinjected withexcess amount of unlabeled peptide indicating specificuptake of 57Co-NOTA-PEG2-RM26 These results werefurther confirmed in female Balbc nunu mice bearingsubcutaneous PC-3 tumors at 3 h pi where the tumor uptakeand the uptake in GRPR-expressing organs were inhibitedby coinjection of an excess amount of unlabeled peptide

(Figure 4(b)) The accumulation of radioactivity in normalorgans and excretory organs was not significantly differentupon blocking

Data concerning the biodistribution of 57Co-NOTA-PEG2-RM26 over time in PC-3 xenografted mice are pre-sented in Figure 5 The overall biodistribution pattern of thecobalt-labeled peptide and the tumor uptake were in goodagreement with the data published for this peptide labeledwith indium-111 gallium-68 and fluorine-18 via aluminumfluoride [7 8 12 13] A surprising finding of this studywas the significantly faster whole body and blood clearanceof 57Co-NOTA-PEG2-RM26 compared to the previously


Table 2 Long-lived positron-emitting radiometals Data are taken from [10]

Nuclide (half-life) Mode of decay 119864max (keV) Principal photon emissions keV (abundance in )55Co (175 h) 120573+ 76

EC 24 1498 477 (202) 931 (75) 1317 (71) 1408 (169)

64Cu (127 h)120573+ 18120573minus 37EC 24

653 1346 (05)

86Y (147 h) 120573+ 33EC 67 3141 443 (169) 628 (326) 646 (92) 703 (154) 778 (224) 1077 (825) 1153

(305) 1854 (172) 1920 (208)

(a) (b)

Figure 6 Coronal MIP preclinical PETCT images showing tracer distribution in PC-3 xenografted NOD-SCID mice The animals wereinjected with 018 nmol of 55Co-NOTA-PEG2-RM26 (approx 3MBq) and scanned at (a) 3 h and (b) 24 h piThe tumor is shown by the arrow

evaluated 68Ga and 111In-labeled NOTA-PEG2-RM26 [7]In this regard at the 3 h time point the blood levels ofradioactivity for 57Co-labeled NOTA-PEG2-RM26 were 2-fold lower than 111In- and 6-fold lower compared to 68Ga-labeled analogs Different clearance rates from circulationfor peptides and proteins labeled with different radiometalsusing the same chelator are a known phenomenon [25ndash27] This could be due to different coordination geometriesthat could affect the binding to blood proteins thereforemodulating the excretion rate The excretion of radioactivitywas predominantly through the kidneys as the radioactivityuptake in both the liver and gastrointestinal tract was low atall time points

The rapid clearance of radioactivity from blood (005 plusmn002 IDg) and normal organs resulted in high tumor-to-organ ratios already at 3 h pi (Figure 5(b)) Remarkably thetumor uptake exceeded the uptake in all other organs andwas7-fold higher than pancreas the organ with the second high-est uptake at 3 h pi This discrepancy in retention of radioac-tivity between xenografts and receptor-positive organs waspreviously reported for 68Ga and 111In-labeled NOTA-PEG2-RM26 [7] as well as other bombesin analogs [28ndash30] andcould be attributed to interspecies differences betweenmouseand human GRPR or to receptor density in tissues The rapidradioactivity washout from normal and receptor-positiveorgans together with a higher retention of the radioconjugate

in tumors resulted in a further increase of tumor-to-organratios over time Therefore exceptionally high tumor-to-blood tumor-to-pancreas tumor-to-stomach and tumor-to-kidney ratios were achieved at 24 h pi For comparison forradiocobalt-labeled bombesin agonist AMBA the tumor-to-blood ratio at this time point was 7-fold lower tumor-to-kidney 9-fold lower tumor-to-liver 15-fold lower tumor-to-colon 80-fold lower and tumor-to pancreas 300-fold lower[14]

The high potential of radiocobalt-labeled NOTA-PEG2-RM26 for imaging of GRPR-expressing tumors was con-firmed by 120583PETCT images using 55Co-NOTA-PEG2-RM26(Figure 6) Similar to the biodistribution results the predom-inant radioactivity uptake was seen in tumors The kidneyand urinary bladder uptake could also be visualized at both3 h and 24 h pi due to the predominant renal excretion ofthe radiotracer The SPECTCT and 120583PETCT MIP imagestogether with the biodistribution results suggest 24 h pi as theoptimal time point for image acquisition

Cobalt-55 was used in ionic form in several clinicalstudies as tracer that may reflect calcium (Ca) influx [31ndash33]This PET radioisotope has favorable properties for noninva-sive imaging relatively long half-life (175 h) production atlow-energy cyclotrons available in most PET facilities andthe highest positron abundancy among positron-emittingradiometals with comparable half-life (Table 2) It can be


(a) (b)

Figure 7 Coronal MIP preclinical SPECTCT images showing tracer distribution in PC-3 xenografted BALBC nunu mice The animalsused for SPECT camera imaging were injected with 45 pmol of 57Co-NOTA- PEG2-RM26 (300 kBq) and euthanized at (a) 3 h and (b) 24 hpi

produced by low-energy medical cyclotrons available inmost PET facilities and due to the long half-life it canbe distributed to distant sites The production costs of abatch of cobalt-55 should be in the same range as a batchof copper-64 However until recently radiocobalt was notused for labeling of peptides and proteins for molecularimaging due to undeveloped labeling chemistry Within thelast years several groups reported labeling with radiocobaltisotopes of scaffold proteins affibody molecules DOTA-ZHER2 [27] andDOTA-ZHER1 [34] GRPR-agonistic BNanalogNOTA-AMBA [14] and somatostatin analogs DOTATATEDOTATOC and DOTANOC [18 19] Both tetra- and tri-aza-chelators demonstrated stable coordination of divalentcobalt For preclinical development cobalt-55 is rather expen-sive Cobalt-57 has half-life of 2716 days emits photonswith convenient energy (122 keV (86) 136 keV (10)) andcan be used for preclinical experiments in the same way asiodine-125 is used for development of radiotracers insteadof iodine-124 (PET isotope) iodine-123 (SPECT isotope)and iodine-131 (therapeutic isotope) To prove this we per-formed 120583SPECTCT imaging of GRPR-expressing tumorsusing 57Co-NOTA-PEG2-RM26 at 3 h and 24 h pi (Figure 7)Similar to the 120583PETCT images and the biodistributionresults the predominant radioactivity uptake was seen intumors Traces of radioactivity could also be observed in thekidneys at 3 h pi At the 24 h pi due to the fast clearance ofradioactivity from normal organs only the tumor could bevisualized

4 Conclusions

We have developed a peptide-based targeting agent labeledwith 55Co for next day high contrast PET imaging of GRPR-expressing tumorsWe have also demonstrated that long lived57Co can be used as a surrogate for 55Co in preclinical studies

Disclosure

Helge Thisgaard is co-first author

Conflicts of Interest

The authors declare that they have no conflicts of interest

Authorsrsquo Contributions

Bogdan Mitran and Helge Thisgaard made equal contribu-tion

Acknowledgments

The molecular imaging work in this publication has beensupported by the Wallenberg infrastructure for PET-MRI(WIPPET) at SciLifeLab Pilot Facility for Preclinical PET-MRI a Swedish nationally available imaging platform atUppsala University Sweden financed by Knut and AliceWallenberg Foundation (120583SPECTCT) This work was sup-ported by the Swedish Cancer Society (Grants CAN2014474(Anna Orlova) and CAN2015350 (Vladimir Tolmachev))and the Swedish ResearchCouncil (Grants 2015-02509 (AnnaOrlova) and 2015-02353 (Vladimir Tolmachev)) which areacknowledged for financial support The authors acknowl-edge the Danish Molecular Biomedical Imaging Center(DaMBIC University of Southern Denmark) for the useof the bioimaging facilities and they thank Christina BaunDepartment of Nuclear Medicine Odense University Hospi-tal for her technical assistance

References

[1] J C Reubi ldquoOld and new peptide receptor targets in cancerFuture directionsrdquo Recent Results in Cancer Research vol 194pp 567ndash576 2013


[2] J C Reubi ldquoPeptide receptors as molecular targets for cancerdiagnosis and therapyrdquo Endocrine Reviews vol 24 no 4 pp389ndash427 2003

[3] L Bodei M Ferrari A Nunn et al ldquo177Lu-AMBA bombesinanalogue in hormone refractory prostate cancer patients aphase I escalation study with single-cycle administrationsrdquoEuropean Journal of Nuclear Medicine and Molecular Imagingvol 34 supplement 2 p S221 2007

[4] M Ginj H Zhang B Waser et al ldquoRadiolabeled somatostatinreceptor antagonists are preferable to agonists for in vivo pep-tide receptor targeting of tumorsrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 103 no44 pp 16436ndash16441 2006

[5] R Cescato T Maina B Nock et al ldquoBombesin receptorantagonists may be preferable to agonists for tumor targetingrdquoJournal of Nuclear Medicine vol 49 no 2 pp 318ndash326 2008

[6] M Llinares C Devin O Chaloin et al ldquoSyntheses andbiological activities of potent bombesin receptor antagonistsrdquoJournal of Peptide Research vol 53 no 3 pp 275ndash283 1999

[7] Z Varasteh I Velikyan G Lindeberg et al ldquoSynthesis andcharacterization of a high-affinity NOTA-conjugated bombesinantagonist for GRPR-targeted tumor imagingrdquo BioconjugateChemistry vol 24 no 7 pp 1144ndash1153 2013

[8] Z Varasteh O Aberg I Velikyan et al ldquoIn vitro and invivo evaluation of a18F-labeled high affinity NOTA conjugatedbombesin antagonist as a PET ligand for GRPR-targeted tumorimagingrdquo PLoS ONE vol 8 no 12 Article ID e81932 2013

[9] V Tolmachev and A Orlova ldquoInfluence of labelling methodson biodistribution and imaging properties of radiolabelledpeptides for visualisation of molecular therapeutic targetsrdquoCurrent Medicinal Chemistry vol 17 no 24 pp 2636ndash26552010

[10] V Tolmachev and S Stone-Elander ldquoRadiolabelled proteinsfor positron emission tomography Pros and cons of labellingmethodsrdquo Biochimica et Biophysica Acta - General Subjects vol1800 no 5 pp 487ndash510 2010

[11] A Heppeler J P Andre I Buschmann et al ldquoMetal-ion-dependent biological properties of a chelator-derived somato-statin analogue for tumour targetingrdquo Chemistry - A EuropeanJournal vol 14 no 10 pp 3026ndash3034 2008

[12] Z Varasteh B Mitran U Rosenstrom et al ldquoThe effect ofmacrocyclic chelators on the targeting properties of the 68Ga-labeled gastrin releasing peptide receptor antagonist PEG2-RM26rdquo Nuclear Medicine and Biology vol 42 no 5 pp 446ndash454 2015

[13] B Mitran Z Varasteh R K Selvaraju et al ldquoSelection ofoptimal chelator improves the contrast of GRPR imaging usingbombesin analogue RM26rdquo International Journal of Oncologyvol 48 no 5 pp 2124ndash2134 2016

[14] J H Dam B B Olsen C Baun P-F Hoslashilund-Carlsen andH Thisgaard ldquoIn Vivo Evaluation of a Bombesin AnalogueLabeled with Ga-68 and Co-5557rdquo Molecular Imaging andBiology vol 18 no 3 pp 368ndash376 2016

[15] G J Kelloff P Choyke and D S Coffey ldquoChallenges inclinical prostate cancer role of imagingrdquo American Journal ofRoentgenology vol 192 no 6 pp 1455ndash1470 2009

[16] W C Eckelman M R Kilbourn and C A Mathis ldquoSpecific tononspecific binding in radiopharmaceutical studies itrsquos not sosimple as it seemsrdquo Nuclear Medicine and Biology vol 36 no3 pp 235ndash237 2009

[17] P E N Braad S B Hansen H Thisgaard and P F Hoslashilund-Carlsen ldquoPET imaging with the non-pure positron emitters

55Co 86Y and 124Irdquo Physics in Medicine and Biology vol 60 no9 pp 3479ndash3497 2015

[18] H Thisgaard M L Olesen and J H Dam ldquoRadiosynthesisof 55Co- and 58119898Co-labelled DOTATOC for positron emissiontomography imaging and targeted radionuclide therapyrdquo Jour-nal of Labelled Compounds and Radiopharmaceuticals vol 54no 12 pp 758ndash762 2011

[19] H Thisgaard B B Olsen J H Dam P Bollen J Mollen-hauer and P F Hoslashilund-Carlsen ldquoEvaluation of cobalt-labeledoctreotide analogs for molecular imaging and Auger electron-based radionuclide therapyrdquo Journal of Nuclear Medicine vol55 no 8 pp 1311ndash1316 2014

[20] H Bjorkelund L Gedda and K Andersson ldquoComparing theepidermal growth factor interaction with four different celllines Intriguing effects imply strong dependency of cellularcontextrdquo PLoS ONE vol 6 no 1 Article ID e16536 2011

[21] W Maret and B L Vallee ldquoCobalt as probe and label ofproteinsrdquoMethods in Enzymology vol 226 pp 52ndash70 1993

[22] K Abiraj R Mansi M-L Tamma et al ldquoBombesin antagonist-based radioligands for translational nuclear imaging of gastrin-releasing peptide receptor-positive tumorsrdquo Journal of NuclearMedicine vol 52 no 12 pp 1970ndash1978 2011

[23] E Gourni R Mansi M Jamous et al ldquoN-terminal modifi-cations improve the receptor affinity and pharmacokinetics ofradiolabeled peptidic gastrin-releasing peptide receptor antag-onists examples of 68Ga- and 64Cu-labeled peptides for PETimagingrdquo Journal of Nuclear Medicine vol 55 no 10 pp 1719ndash1725 2014

[24] Z Varasteh and A Orlova ldquoComparing the measured affinityof 111In-labeled ligands for cellular receptors by monitoringgamma beta or X-ray radiation with three different Lig-andTracer devicesrdquo Journal of Radioanalytical and NuclearChemistry vol 304 no 2 pp 823ndash828 2015

[25] H Zhang J Schuhmacher B Waser et al ldquoDOTA-PESIN aDOTA-conjugated bombesin derivative designed for the imag-ing and targeted radionuclide treatment of bombesin receptor-positive tumoursrdquo European Journal of Nuclear Medicine andMolecular Imaging vol 34 no 8 pp 1198ndash1208 2007

[26] E Koumarianou RMikołajczakD Pawlak et al ldquoComparativestudy on DOTA-derivatized bombesin analog labeled with 90Yand 177Lu in vitro and in vivo evaluationrdquo Nuclear Medicineand Biology vol 36 no 6 pp 591ndash603 2009

[27] H Wallberg S Ahlgren C Widstrom and A OrlovaldquoEvaluation of the radiocobalt-labeled [MMA-DOTA-Cys61]-Z HER22395-Cys affibody molecule for targeting of HER2-expressing tumorsrdquoMolecular Imaging and Biology vol 12 no1 pp 54ndash62 2010

[28] T Maina B A Nock H Zhang et al ldquoSpecies differences ofbombesin analog interactions with GRP-R define the choice ofanimal models in the development of GRP-R-targeting drugsrdquoJournal of Nuclear Medicine vol 46 no 5 pp 823ndash830 2005

[29] P Fournier V Dumulon-Perreault S Ait-Mohand et alldquoNovel radiolabeled peptides for breast and prostate tumorPET imaging 64Cuand 68GaNOTA-PEG-[D-Tyr6120573Ala11Thi13Nle14]BBN(6-14)rdquo Bioconjugate Chemistry vol 23 no8 pp 1687ndash1693 2012

[30] S R Lane P Nanda T L Rold et al ldquoOptimization biolog-ical evaluation and microPET imaging of copper-64-labeledbombesin agonists [64Cu-NO2A-(X)-BBN(7-14)NH2] in aprostate tumor xenografted mouse modelrdquo Nuclear Medicineand Biology vol 37 no 7 pp 751ndash761 2010


[31] P Goethals A Volkaert C Vandewielle R Dierckx and NLameire ldquo55Co-EDTA for renal imaging using positron emis-sion tomography (PET) A feasibility studyrdquo Nuclear Medicineand Biology vol 27 no 1 pp 77ndash81 2000

[32] H M L Jansen J Pruim A M V D Vliet et al ldquoVisualizationof damaged brain tissue after ischemic stroke with cobalt-55positron emission tomographyrdquo Journal of Nuclear Medicinevol 35 no 3 pp 456ndash460 1994

[33] H M L Jansen A T M Willemsen L G F Sinnige etal ldquoCobalt-55 positron emission tomography in relapsing-progressive multiple sclerosisrdquo Journal of the Neurological Sci-ences vol 132 no 2 pp 139ndash145 1995

[34] J Garousi K G Andersson J H Dam et al ldquoThe use ofradiocobalt as a label improves imaging of EGFR using DOTA-conjugated Affibody moleculerdquo Scientific Reports vol 7 no 1Article ID 5961 2017

Submit your manuscripts athttpswwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


emission tomography (PET) imaging and 111In [7] for singlephoton emission computer tomography (SPECT) imagingThe 68Ga and 111In-labeled constructs showed favorablepharmacokinetic properties and high affinity to GRPR Inan attempt to further improve the biodistribution profile forpotential clinical use we have also explored the use of dif-ferent radionuclide-chelator complexes It is known that thecombination of radionuclide and chelator could have a stronginfluence on the targeting properties of radiopeptides [910] For example the somatostatin analog [57Co-(dotatoc)]had the highest reported affinity for somatostatin receptorsubtype 2 (SSTR2) [11] Our studies on PEG2-RM26 labeledwith 68Ga and 111In using different macrocyclic chelatorsdemonstrated that charge and structure of the radionuclide-chelator complex have a strong influence on binding affinityand liver uptake of the radioconjugates [12 13]

One interesting finding from our studies was that thetumor-to-background ratios increased significantly over timeup to 24 h after injection (pi) [7 13] due to the high uptake andlong retention in tumors in combination with the fast clear-ance from blood and GRPR-expressing organs underscoringthe importance of radionuclide half-life in high sensitivitymolecular imagingThese data are in agreement with findingsfrom the head-to-head evaluation of 55Co- and 68Ga-labeledGRPR agonist NOTA-AMBA In this study 55Co-NOTA-AMBA provided superior imaging contrast compared tothe 68Ga-labeled counterpart because of the possibility ofperforming imaging at 24 h pi However both 68Ga- and5557Co-labeled NOTA-AMBA had a prolonged retention inGRPR-expressing tissues typical for agonists [14] This issuemight be solved by the use of a high affinity GRPR antagonist

Thus the use of a long lived radionuclide for nextday imaging may enable the detection of low abdominallymph node involvement which requires the highest possiblesensitivity and is an ultimate goal in prostate cancer imaging[15 16] Labeling of RM26with a long lived positron-emittingradionuclide could further increase diagnostic accuracy dueto the inherent high sensitivity and imaging quantitationcapability of PET compared to SPECT For next day imaginga positron-emitting radiometal with a half-life of 10ndash20 hwould be optimal Possible PET radionuclides for suchpurposes include 64Cu (11987912 = 127 h) 86Y (11987912 = 147 h)and 55Co (11987912 = 175 h) Among them 55Co (76 120573+) hasa higher positron abundance compared to 64Cu and a betterratio between annihilation photons and coemitted gammascompared to 86Y providing better image quality [17] The175 h half-life of 55Co not only allows for delayed imaginginvestigations but also enables shipment to stand-alone PETscanners

The goal of this study was to evaluate the tumor targetingproperties of GRPR antagonist NOTA-PEG2-RM26 labeledwith 55Co

2 Materials and Methods

21 Radiochemistry NOTA-PEG2-RM26 (RM26 = [D-Phe6Sta13 Leu14] bombesin [6-14]) was synthesized by standard

manual solid-phase peptide synthesis based on previouslypublished procedures [7]57Co cobalt chloride was purchased from PerkinElmer

Sweden (Upplands Vasby Sweden) 55Co was produced in-house as previously described [18] with an extra purificationstep added [19] Buffers including 02M ammonium acetatepH 55 and 02M citric acid were produced in-housefrom chemicals supplied by Merck (Darmstadt Germany)Phosphate buffered saline (PBS) pH 75 01Mwas purchasedfrom the National Veterinary Institute SVA Sweden Buffersfor labeling were purified from free metals contamination byChelex 100 resin (Bio-Rad Laboratories Hercules CA)

Radiolabeling with 57Co was performed using38ndash10 nmol (aq 1 nmol120583L) of NOTA-PEG2-RM26 bufferedwith 80 120583L ammonium acetate (02M pH 55) The mixturewas incubated with 57Co cobalt chloride (10ndash50MBq) for30min at 60∘C For 55Co labeling 13 nmol of NOTA-PEG2-RM26bufferedwith 80 120583L ammoniumacetate (02M pH55)was heated by microwave irradiation for 1min at 850W in asealed vial using a conventional microwave oven (SamsungMW82N-B) To evaluate the labeling stability 57Co-NOTA-PEG2-RM26 was incubated in the presence of 1000-foldmolar excess of EDTA disodium salt (Sigma) for 1 h at RTLabeling yield radiochemical purity and in vitro stabilitywere analyzed using instant thin-layer chromatography(ITLC) strips (Agilent Technologies) eluted with citric acid(02M pH 20) In this system free 5755Comigrates with theeluent front (119877119891 = 10) and the radiolabeled construct stayson the application point (119877119891 = 00) The system was previ-ously validated by sodium dodecyl sulfate polyacrylamidegel electrophoresis (SDS-PAGE) and radio-HPLC [7 8]

22 In Vitro Studies PC-3 human prostate cancer cellsexpressing GRPR were purchased form ATCC via LGCPromochem The cells were cultured in RPMI-1640 mediasupplemented with 10 fetal calf serum PEST (penicillin100 IUmL streptomycin 100120583gmL) and 2mML-glutamine(all from Biochrom AG Berlin Germany) This mediumis referred to as complete medium in the text Trypsin-EDTA (005 trypsin 002 EDTA in buffer BiochromAG)was used for cell detachment All in vitro experiments wereperformed in triplicate

The binding specificity of 57Co-NOTA-PEG2-RM26 wasevaluated using PC-3 cells incubated with 1 nM 57Co-NOTA-PEG2-RM26 solution for 1 h at 37∘C In this experiment oneset of dishes was preincubated for 10min at RT with anexcess (100-fold) of nonlabeled peptide before applying theradioactivity After incubation the cells were washed withserum-free media and detached with 05mL trypsin-EDTAsolution Radioactivity of cells wasmeasured in an automatedgamma-counter (3-inch NaI(Tl) detector 2480 WIZARD2PerkinElmer) and presented as cell-associated percentagefrom added radioactivity

Cellular processing of the conjugate was evaluated usingPC-3 cells incubated with 1 nM of 57Co-NOTA-PEG2-RM26at 37∘C 5 CO2 At predetermined time points (1 2 4 8and 24 h after the start of incubation) the medium from 3dishes was discarded and the cells were washed with ice-cold











NHNH

OH(S)

(S)(R)

O

O OO

O

O

O

O

NN

N

NH

NH

O

O

NH

NH

NH

(S) NH (S)

(S)

O

O O

N

N

HNH

O

NH

(S)(S) (S)

O

ONH

RM26

PEG2NOTA

5557Co

minus

minus

（2

（2












NonblockedBlocked

BlockedNonblocked0

20

40

60

80

o

f add

ed ra

dioa

ctiv

ity

(a)

0

20

40

60

80

100

Cell

asso

ciat

ed ra

dioa

ctiv

ity (

)

4 8 12 16 20 240Time (h)

Total


(b)


Variant

0

50

100

Nor

mal

ised

o

f add

ed ra

dioa

ctiv

ity

ＨＮInＨＮCo



247 plusmn 01

554 plusmn 04

(a)

25

20

15

10

5

0 200 400 600 800 1000Time (minutes)

Sign

al (C

PS)

0






22 plusmn 10

(b)






lowast lowastlowast

Panc

reas

Stom

ach

Sple

en

Lung

s

Kidn

eys

Mus

cle

Smal

l int

Bone

Bloo

d

Live

r

0

2

4

6

8

10

12U

ptak

e (

IAg

)


(a)

lowast lowastlowast

Panc

reas

Stom

ach

Sple

en

Lung

s

Kidn

eys

Mus

cle

Smal

l int

Bone

Bloo

d

Live

r

Tum

or

0

1

2

3

4

Upt

ake (

IA

g)


(b)


3 Ｂ

24 Ｂ

Panc

reas

Stom

ach

Adre

nals

Sple

en

Smal

l int

Kidn

eys

Lung

s

Bloo

d

Tum

or

Col

on

Live

r

Hea

rt

0

1

2

Upt

ake (

IA

g) 3

6

9

(a)

3 Ｂ

24 Ｂ

Panc

reas

Stom

ach

Sple

en

Adre

nals

Lung

s

Kidn

eys

Smal

l int

Bloo

d

Live

r

Col

on

Hea

r t

0

100

200

300

TO

1000200030004000

(b)








EC 24 1498 477 (202) 931 (75) 1317 (71) 1408 (169)

64Cu (127 h)120573+ 18120573minus 37EC 24

653 1346 (05)

86Y (147 h) 120573+ 33EC 67 3141 443 (169) 628 (326) 646 (92) 703 (154) 778 (224) 1077 (825) 1153

(305) 1854 (172) 1920 (208)

(a) (b)








(a) (b)



4 Conclusions


Disclosure






Acknowledgments


References











































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


























NHNH

OH(S)

(S)(R)

O

O OO

O

O

O

O

NN

N

NH

NH

O

O

NH

NH

NH

(S) NH (S)

(S)

O

O O

N

N

HNH

O

NH

(S)(S) (S)

O

ONH

RM26

PEG2NOTA

5557Co

minus

minus

（2

（2












NonblockedBlocked

BlockedNonblocked0

20

40

60

80

o

f add

ed ra

dioa

ctiv

ity

(a)

0

20

40

60

80

100

Cell

asso

ciat

ed ra

dioa

ctiv

ity (

)

4 8 12 16 20 240Time (h)

Total


(b)


Variant

0

50

100

Nor

mal

ised

o

f add

ed ra

dioa

ctiv

ity

ＨＮInＨＮCo



247 plusmn 01

554 plusmn 04

(a)

25

20

15

10

5

0 200 400 600 800 1000Time (minutes)

Sign

al (C

PS)

0






22 plusmn 10

(b)






lowast lowastlowast

Panc

reas

Stom

ach

Sple

en

Lung

s

Kidn

eys

Mus

cle

Smal

l int

Bone

Bloo

d

Live

r

0

2

4

6

8

10

12U

ptak

e (

IAg

)


(a)

lowast lowastlowast

Panc

reas

Stom

ach

Sple

en

Lung

s

Kidn

eys

Mus

cle

Smal

l int

Bone

Bloo

d

Live

r

Tum

or

0

1

2

3

4

Upt

ake (

IA

g)


(b)


3 Ｂ

24 Ｂ

Panc

reas

Stom

ach

Adre

nals

Sple

en

Smal

l int

Kidn

eys

Lung

s

Bloo

d

Tum

or

Col

on

Live

r

Hea

rt

0

1

2

Upt

ake (

IA

g) 3

6

9

(a)

3 Ｂ

24 Ｂ

Panc

reas

Stom

ach

Sple

en

Adre

nals

Lung

s

Kidn

eys

Smal

l int

Bloo

d

Live

r

Col

on

Hea

r t

0

100

200

300

TO

1000200030004000

(b)








EC 24 1498 477 (202) 931 (75) 1317 (71) 1408 (169)

64Cu (127 h)120573+ 18120573minus 37EC 24

653 1346 (05)

86Y (147 h) 120573+ 33EC 67 3141 443 (169) 628 (326) 646 (92) 703 (154) 778 (224) 1077 (825) 1153

(305) 1854 (172) 1920 (208)

(a) (b)








(a) (b)



4 Conclusions


Disclosure






Acknowledgments


References











































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















NonblockedBlocked

BlockedNonblocked0

20

40

60

80

o

f add

ed ra

dioa

ctiv

ity

(a)

0

20

40

60

80

100

Cell

asso

ciat

ed ra

dioa

ctiv

ity (

)

4 8 12 16 20 240Time (h)

Total


(b)


Variant

0

50

100

Nor

mal

ised

o

f add

ed ra

dioa

ctiv

ity

ＨＮInＨＮCo



247 plusmn 01

554 plusmn 04

(a)

25

20

15

10

5

0 200 400 600 800 1000Time (minutes)

Sign

al (C

PS)

0






22 plusmn 10

(b)






lowast lowastlowast

Panc

reas

Stom

ach

Sple

en

Lung

s

Kidn

eys

Mus

cle

Smal

l int

Bone

Bloo

d

Live

r

0

2

4

6

8

10

12U

ptak

e (

IAg

)


(a)

lowast lowastlowast

Panc

reas

Stom

ach

Sple

en

Lung

s

Kidn

eys

Mus

cle

Smal

l int

Bone

Bloo

d

Live

r

Tum

or

0

1

2

3

4

Upt

ake (

IA

g)


(b)


3 Ｂ

24 Ｂ

Panc

reas

Stom

ach

Adre

nals

Sple

en

Smal

l int

Kidn

eys

Lung

s

Bloo

d

Tum

or

Col

on

Live

r

Hea

rt

0

1

2

Upt

ake (

IA

g) 3

6

9

(a)

3 Ｂ

24 Ｂ

Panc

reas

Stom

ach

Sple

en

Adre

nals

Lung

s

Kidn

eys

Smal

l int

Bloo

d

Live

r

Col

on

Hea

r t

0

100

200

300

TO

1000200030004000

(b)








EC 24 1498 477 (202) 931 (75) 1317 (71) 1408 (169)

64Cu (127 h)120573+ 18120573minus 37EC 24

653 1346 (05)

86Y (147 h) 120573+ 33EC 67 3141 443 (169) 628 (326) 646 (92) 703 (154) 778 (224) 1077 (825) 1153

(305) 1854 (172) 1920 (208)

(a) (b)








(a) (b)



4 Conclusions


Disclosure






Acknowledgments


References











































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















lowast lowastlowast

Panc

reas

Stom

ach

Sple

en

Lung

s

Kidn

eys

Mus

cle

Smal

l int

Bone

Bloo

d

Live

r

0

2

4

6

8

10

12U

ptak

e (

IAg

)


(a)

lowast lowastlowast

Panc

reas

Stom

ach

Sple

en

Lung

s

Kidn

eys

Mus

cle

Smal

l int

Bone

Bloo

d

Live

r

Tum

or

0

1

2

3

4

Upt

ake (

IA

g)


(b)


3 Ｂ

24 Ｂ

Panc

reas

Stom

ach

Adre

nals

Sple

en

Smal

l int

Kidn

eys

Lung

s

Bloo

d

Tum

or

Col

on

Live

r

Hea

rt

0

1

2

Upt

ake (

IA

g) 3

6

9

(a)

3 Ｂ

24 Ｂ

Panc

reas

Stom

ach

Sple

en

Adre

nals

Lung

s

Kidn

eys

Smal

l int

Bloo

d

Live

r

Col

on

Hea

r t

0

100

200

300

TO

1000200030004000

(b)








EC 24 1498 477 (202) 931 (75) 1317 (71) 1408 (169)

64Cu (127 h)120573+ 18120573minus 37EC 24

653 1346 (05)

86Y (147 h) 120573+ 33EC 67 3141 443 (169) 628 (326) 646 (92) 703 (154) 778 (224) 1077 (825) 1153

(305) 1854 (172) 1920 (208)

(a) (b)








(a) (b)



4 Conclusions


Disclosure






Acknowledgments


References











































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















EC 24 1498 477 (202) 931 (75) 1317 (71) 1408 (169)

64Cu (127 h)120573+ 18120573minus 37EC 24

653 1346 (05)

86Y (147 h) 120573+ 33EC 67 3141 443 (169) 628 (326) 646 (92) 703 (154) 778 (224) 1077 (825) 1153

(305) 1854 (172) 1920 (208)

(a) (b)








(a) (b)



4 Conclusions


Disclosure






Acknowledgments


References











































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(a) (b)



4 Conclusions


Disclosure






Acknowledgments


References











































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















high contrast pet imaging of grpr expression in prostate...

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