i

uitr

3, GR 265 04, Rion, Greece, Technoucts, N.C

d Charleston Radiation Therapy Consultants, Charleston, W

a r t i c l e i n f o

Article history:Received 18 September 2012Received in revised form 23 October 2012

3

Nuclear Medicine and Biology 40 (2013) 262272

Contents lists available at SciVerse ScienceDirect

Nuclear Medicin

j ourna l homepage: www.e lsevthe preparation of multifunctional SPECT/MRI contrast agents (RGD-conjugated nanoparticles) for dualmodality imaging of integrin expressing tumors should be further investigated.

2013 Elsevier Inc. All rights reserved.

1. Introduction

Angiogenesis is the development of new vessels in both normalsituations (embryo development, wound healing, mammal menstru-ation) and cancer (tumor growth andmetastasis) [1]. Tumors producemany angiogenic factors, which are able to activate endothelial cells inestablished blood vessels and induce endothelial proliferation,migration, and new vessel formation through a series of sequential,

brane glycoproteins 3-integrin play an important role duringtumor angiogenesis and has stimulated extensive investigation of3-mediated antiangiogenic strategies for cancer treatment as wellas prevention of cancer recurrence or metastasis [4].

Noninvasive imaging of integrin 3 expression by molecularimaging techniques can also be a signicant contribution to the earlydetection of malignancy, the evaluation of tumor progress, and themonitoring of treatment efcacy of antiangiogenic drugs [5,6]. Morebut partially overlapping steps [2,3]. The he

Corresponding author. Department of Medical PhysUniversity of Patras, P.O.BOX: 132 73, Rion, GR 265 04, Gfax: +30 2610 969166.

E-mail addresses: gkagad@gmail.com, george.kagadi(G.C. Kagadis).

0969-8051/$ see front matter 2013 Elsevier Inc. Alhttp://dx.doi.org/10.1016/j.nucmedbio.2012.10.015Blocking experiments indicated target specicity for integrin receptors in U87MG glioblastoma cells.Conclusion: Due to its relatively high tumor uptake, renal elimination and negligible abdominal localization,the new 99mTc-RGD peptide is considered promising in the eld of imaging -positive tumors. However,Accepted 24 October 2012

Keywords:RGDIntegrin 3TechnetiumRadiolabelingChelatorOrnithine

derivative, c(RGDfK)-(Orn)3-CGG. This new peptide availing the polar linker (Orn)3 and the 99mTc-chelatingmoiety CGG (Cys-Gly-Gly) is appropriately designed for 99mTc-labeling, as well as consequent conjugationonto nanoparticles.Methods: A tumor imaging agent, c(RGDfK)-(Orn)3-[CGG-99mTc], is evaluated with regard to itsradiochemical, radiobiological and imaging characteristics.Results: The complex c(RGDfK)-(Orn)3-[CGG-99mTc] was obtained in high radiochemical yield (N98%) andwas stable in vitro and ex vivo. It presented identical to the respective, fully analytically characterized 185/187Recomplex retention time in RP-HPLC. In contrary to other RGD derivatives, we showed that the newradiopeptide exhibits kidney uptake and urine excretion due to the ornithine linker. High tumor uptake(3.870.48% ID/g at 60 min p.i.) was observed and was maintained relatively high even at 24 h p.i. (1.830.05 % ID/g), thus providing well-dened scintigraphic imaging. Accumulation in other organs was negligible..S.R. Demokritos, GR 153 10 Aghia Paraskevi, GreeceV 253 04, USA

a b s t r a c t

Introduction: Radiolabeled RGD peptides that specically target integrin 3 have great potential in earlytumor detection through noninvasive monitoring of tumor angiogenesis. Based on previous ndings of ourgroup on radiopeptides containing positively charged aminoacids, we developed a new cyclic cRGDfKb Department of Medical Instruments Technologyc Institute of Radioisotopes-Radiodiagnostic Prodlogical Educational Institute, GR 122 10 Athens, Greecea Department of Medical Physics, School of Medicine, University of Patras, P.O. BOX: 132 7Biological evaluation of an ornithine-modangiogenesis imaging agent

Irene Tsiapa a, George Loudos b, Alexandra VarvarigoTheodoros Tsotakos b,c, Stavros Xanthopoulos c, DimGeorge C. Nikiforidis a, George C. Kagadis a,terodimeric transmem-

ics, School of Medicine,reece. Tel.: +30 2610 969146;

s@med.upatras.gr

l rights reserved.ed 99mTc-labeled RGD peptide as an

c, Eirini Fragogeorgi b,c, Dimitrios Psimadas b,c,is Mihailidis d, Penelope Bouziotis c,

e and Biology

i e r .com/ locate /nucmedb iospecically, the cyclic Arg-Gly-Asp (RGD) peptide moiety presentshigh afnity for the 3 integrin [712]. Thus, 3-integrin canpotentially be a suitable target for radioligands such as radiolabeledpeptides containing the RGDmotif [8,10]. Up to now, a large variety ofradiolabeled analogs have been developed as potential agents fornuclear medicine imaging and therapy [7,11]. Many RGD peptidesserve as targeting biomolecules carrying radionuclides to the 3-integrin over-expressed in tumor cells and endothelial cells of tumor

263I. Tsiapa et al. / Nuclear Medicine and Biology 40 (2013) 262272neovasculature [12,13]. Many research groups have been using cyclicRGD peptides in the development of integrin-3-targeted radio-tracers in order to image rapidly growing and metastatic tumors inseveral tumor-bearing animal models [1115]. In particular, it hasbeen shown that cyclic (Arg-Gly-Asp-D-Phe-Lys) cRGDfK presentshigh afnity to the 3 receptor and can specically accumulate intumors including osteosarcomas, glioblastomas, melanomas, lungcarcinomas, and breast cancer [1115].

Over the last decade, a variety of radiolabeled cyclic RGD peptideshave been evaluated as radiotracers for imaging tumors by singlephoton emission computed tomography (SPECT) or positron emis-sion tomography (PET) techniques and some of them are already inclinical studies [12,1619]. Especially, SPECT imaging is widelyavailable and has signicant imaging potential using tumor-specicradiopharmaceuticals. It should be noted that as far as diagnosticimaging is concerned, 99mTc is considered to be an ideal radionuclideand 99mTc-radiopharmaceuticals are in high demand in nuclearmedicine due to their low cost, widespread availability and favorablephysical and imaging characteristics ( ray=142 keV, half-life=6.02 h) [20,21].

A traditional approach for radiolabeling RGD derivatives with99mTc is via stabilization of the [Tc(V)O]3+ metal core with ligandsof the so-called N3S type [2226]. Smith et al. have studied 99mTc-complexes containing a N3S chelating group [27]. Our group hasalso focused on the design of Bombesin (BN) derivatives in whichthe chelating moiety is comprised of an N3S-combination of aminoacids, presenting the sequence CGG (Cys-Gly-Gly) [22,23,25]. Wedesigned the new RGD derivative, namely c(RGDfK)-(Orn)3-CGG(Fig. 1), based on previous ndings of our group on radiopeptidesthat contain positively charged aminoacids, such as ornithine andarginine in order to improve excretion kinetics and biodistributionprole [22,25]. The aim was to investigate the characteristics andthe biological and imaging properties of this radiolabeled RGDcompound, which contains in its moiety such a polar linker and a99mTc-chelator.

Increased interest for RGD conjugated nanoparticles [2830] hasprompted researchers to design RGD derivatives as successfulreceptor integrin 3 targeting moieties in SPECT/MRI dual-modality tumor imaging [31]. Nanoparticles produce multivalenteffects due to multiple simultaneous interactions between thebiomolecules conjugated on the nanoparticle surface and the speciccell surface receptors. Taking into consideration the advantagesoffered by a combined SPECT/MRI system, it is critical that multi-functional probe development is improved for future use of thisbifunctional imaging modality and accessed for further investigationof c(RGDfK)-(Orn)3-CGG, after conjugation with magnetic nano-particles. The new RGD derivative, containing the radionuclidechelating moiety CGG, is considered to be appropriately designedfor both 99mTc-labeling, and conjugation with nanoparticles, via theCys moiety.

Within the framework of the present study, the new RGDderivative, c(RGDfK)-(Orn)3-CGG, was complexed with both 99mTcand the respective 185/187Re(V) gluconate precursors. The non-radioactive rhenium complex was characterized by liquid chroma-tography-electrospray ionization mass spectrometry (ESI-MS).Radiochemical identication of the 99mTc-RGD derivative, metabolicstability studies, in vitro assays, in vivo evaluation in normal Swissmice and tumor-bearing SCID mice are reported. The nal goalwas to evaluate the potential use of the new RGD derivative,c(RGDfK)-(Orn)3-CGG, as a tumor imaging agent in nuclear medicine,in order to head for the design and synthesis of a multifunctionalSPECT/MRI contrast agent, RGD conjugated nanoparticles, for imagingof integrin expressing tumors [15,2831]. The multimodal imagingapproach could facilitate verication of the accuracy in tumordetection and provide additional information regarding the pathologyof the tumor.2. Materials and methods

2.1. General

The RGD derivative, c(RGDfK)-(Orn)3-CGG, was designed by ourgroup and consequently obtained from Caslo Laboratory at theTechnical University of Denmark. All other chemicals were of reagentgrade and used without further purication. Ionization massspectroscopy (ESI-MS) analysis was performed on a Finnigan AQANavigator, using a Harvant syringe pump. The 185/187Re-complex waspuried by semi-preparative reverse phase high performance liquidchromatography (RP-HPLC) on a Waters system (pump 600E,detector UV-484) equipped with a 10 Nucleosil 7 C18 column[25012.7 mm ID; Macherey-Nagel]. Technetium-99 m, in the formof 99mNaTcO4 in saline, was eluted from a commercial 99Mo-99mTcgenerator (Drytec, GE Healthcare). The 99mTc-complexes wereidentied by comparative RP-HPLC analysis with their respectiverhenium complexes, on a Waters 600 chromatography systemcoupled to both a Waters 2487 Dual absorbance detector and aGabi gamma detector from Raytest. Radiochromatographic analysiswas performed on a C-18 RP (25.4 cm2.5 cm, 5 m porosity)column eluted with a binary gradient system at a 1 mL/min ow rate.Instant thin layer chromatography (ITLC) measurements wereperformed by electronic autoradiography (Instant Imager Packard-Canberra). Scintigraphic images were acquired on a small mouse-sized camera manufactured by our team [32]. The detection device isbased on two Position Sensitive Photomultiplier Tubes (H85000,Hamamatsu, Japan), a parallel hole collimator and a NaI(Tl) pixilatedscintillator. The spatial resolution of the systems is ~1.5 mm at 0 mmdistance from the collimator's surface. The high resolution gammacamera is in ideal position for planar studies, minimizing the distancebetween the animal and the collimator, so that maximum resolutionand sensitivity are achieved.

2.2. Conjugation and analysis of the c(RGDfK)-(Orn)3-[CGG-185/187Re(V)]

derivative (Nonradioactive Complex)

The Rhenium(V) complex of the RGD derivative was prepared by aligand exchange reaction, between the Re(V)O gluconate precursorand the RGD derivative according to a slightly modied literaturemethod [33]. Briey, an aqueous solution of crude c(RGDfK)-(Orn)3-CGG and the 185/187Re(V)O gluconate precursor was allowed to reactovernight at 37 C. The crude c(RGDfK)-(Orn)3-[CGG-185/187Re(V)]complexwas puried by semi-preparative RP-HPLC and characterizedby ESI-MS and RP-HPLC analysis.

2.3. ESI-MS mass spectral analysis

The ESI-MS mass spectral analysis of the c(RGDfK)-(Orn)3-CGGand 185/187Re complexes with the peptide was performed at the MassSpectrometry and Dioxin Analysis Lab, NCSR Demokritos. A testsolution in 50% aqueous ACNwas infused at a ow rate of 0.1 mL/min,under a stream of hot nitrogen gas (Dominic-Hunter UHPLCMS-10)for desolvation at 170 C. Negative or positive ion ESI-MS spectrawere acquired by adjusting the needle and cone voltages.

2.4. Formation of the c(RGDfK)-(Orn)3-[CGG-99mTc(V)] complex

Radiolabeling of the new RGD derivative, c(RGDfK)-(Orn)3-[CGG-99mTc] (Fig. 1), was performed according to an alreadypublished method via the 99mTc-gluconate precursor (sodiumgluconate being used as an intermediate exchange ligand for 99mTcwith stannous chloride as the reducing agent) [22]. Briey, a solidmixture containing 1.0 g sodium gluconate (C6H11NaO7), 2.0 gsodium bicarbonate (NaHCO3) and 15 mg stannous chloride (SnCl2)was homogenized and kept dry. Then, 3.0 mg of the above mixture

Fig. 1. Radiochemical structure of c(RGDfK)-(Orn)3-[CGG-99mTc].

264 I. Tsiapa et al. / Nuclear Medicine and Biology 40 (2013) 262272

was dissolved in 1.0 ml of a sodium pertechnetate solution

analyzed by gradient RP-HPLC in order to determine potentiatranschelation effects.

2.8. Metabolic studies

2.8.1. In plasmaThe metabolic stability of the radiolabeled RGD derivative was

studied in human plasma ex vivo. Human blood (approximately 3 mL)was collected in heparinized polypropylene tubes and centrifuged at

Table 1Analytical HPLC retention times (tR) for c(RGDfK)-(Orn)3-CGG and its complex.

HPLC tR (min)

Compd UV detector Radioactivity detector

c(RGDfK)-(Orn)3-CGG 16.6c(RGDfK)-(Orn)3-[CGG-99mTc] 17.8c(RGDfK)-(Orn)3-[CGG-185/187Re] 17.7

265I. Tsiapa et al. / Nuclear Medicine and Biology 40 (2013) 262272(Na99mTcO4), containing 370555 MBq of 99mTc and was left toreact for 10 min at room temperature. An amount of the lyophilizedRGD derivative was then dissolved in an aliquot of 500 l of theabove solution, the mixture (pH 78) was homogenized byvortexing, allowed to react at 37 C for 30 min and subsequently,the radiolabeled formed peptide was puried by RP-HPLC. Theeffect of peptide concentration on the labeling yield was studied byusing peptide solutions of different concentrations varying from8.6105 M to 8.3107 M.

2.5. Radiochemical analysis

The labeling efciency of the puried derivativewas assessed up to24 h post-labeling and determined by analytical RP-HPLC. Elution wasperformed with a solvent system consisting of 0.1% TFA in water(solvent A) and 0.1% TFA in 90%CH3CN (solvent B) at a ow rate of1 mL/ min. The elution gradient was 95% A, followed by a lineargradient to 75% A for 20 min; this composition was maintained foranother 5 min. After a column wash with 95% B for 5 min, the columnwas re-equilibrated by applying the initial conditions that is 95% A for15 min prior to the next injection. The same system was used for the185/187Re complex analysis. The radioactive RGD derivative was alsoanalyzed by ITLC on Silica Gel (ITLC-SG) strips using saline (0.9%NaCl), acetone and pyridine/CH3COOH/H2O as the mobile phases.

2.6. Lipophilicity: logP values

To a mixture of 300 l n-octanol and 300 l phosphate-bufferedsaline (PBS, pH 7.4), 30 l of puried radiolabeled RGD derivativewere added and vortexed. The nal mixture was centrifuged at6000 rpm for 10 min at 4 C. An aliquot of both saline and octanollayers was collected and the activity of each sample was measuredin a -counter. The log P value was calculated as the log ratio ofthe radioactivity in the organic and aqueous phases (mean ofthree samples).

2.7. Cysteine challenge tests

In order to examine the in vitro stability of the new radiolabeled RGDderivative, the radiolabeled solution was challenged with cysteine.Aliquots of 100 l of the puried radiolabeled peptide derivative wereadded to 900 l of cysteine solutions in saline (103 M and 104 M).The samples were incubated at 37 C for 2 h. At two time points(60 and 120 min), 100 l of each sample were removed andTable 2Data for c(RGDfK)-(Orn)3-CGG peptide and its 185/187Re complex by MS Analysis.

Compd CalculatedMW

Chargedion (m/z)

Theoreticallyexpected m/z

Found m/z

c(RGDfK)-(Orn)3-CGG 1163.4 [M+H]+ 1164.4 1164.0[M+2 H]2+ 582.7 582.6

c(RGDfK)-(Orn)3-[CGG-185/187Re]

1362.6 [M+H]+ 1363.6 1363.4

[M+2 H]2+ 682.3 682.45000 rpm at 4 C for 5 min. The supernatant layer (450 l plasma)was collected and incubated with 50 l (1.853.70 MBq) of thepuried radiolabeled RGD derivative at 37 C. Fractions werewithdrawn at 15, 30, 60 and 120 min, treated with ethanol (2:1EtOH/aliquot, v/v) and centrifuged at 14000 rpm for 30 min.Supernatants were ltered through Millex GP lters (0.22 m) andanalyzed by RP-HPLC using the conditions described for radio-chemical analysis.

2.8.2. In urineTo detect metabolites excreted in urine, 100 l (22.2 MBq) of

c(RGDfK)-(Orn)3-[CGG-99mTc(V)] were injected via the tail vein infemale Swiss Albino mice. Animals were kept at metabolic cages for30 min and thenwere sacriced by cardiac puncture undermild etheranesthesia. Urinewas immediately collected from their bladder with asyringe and analyzed by ITLC and HPLC for detection of metabolitesand free pertechnetate.

2.9. Cell culture

Human glioblastoma cancer cells (U87MG) were maintained inDulbecco's DMEM-high glucose supplemented with 10% FBS, 1% L-Glutamine, 1% penicillin/streptomycin, 1% GlutaMAX. The cells wereincubated in a controlled humidied atmosphere containing 5% CO2 at37 C and were subcultured weekly. They were detached from theask surface by using a trypsin/EDTA solution (0.25%).

2.10. Animal models

All animal experiments were performed in compliance with theEuropean legislation for animal welfare. Animal protocols have beenapproved by the Greek Authorities. Normal Swiss and SCID mice(average weight of 20 g) were obtained from the breeding facilities ofthe Institute of Biology NCSR Demokritos and were used forbiodistribution and dynamic imaging studies. SCID mice wereinoculated subcutaneously with 100 l of cell suspension (approxi-mately 107 U87MG cells/animal) just above the left anterior leg, understerile conditions. Tumors were allowed to grow for up to 4 weeks. Allexperiments were carried out in compliance with the relevantnational laws relating to the conduct of animal experimentation.

2.11. Biodistribution evaluation in normal Swiss mice

ormal Swiss mice were injected via the tail vein with 100 l(~3.70 MBq per animal) of the puried radiolabeled RGD derivative,diluted in saline (pH 7.0). Different groups of animals were sacriced5, 30, 60 min p.i. (post injection) by cardiac puncture under mild

Table 3Stability data obtained by the cysteine challenge test. The % intact c(RGDfK)-(Orn)3-[CGG-99mTc] in the presence of different concentrations of cysteine (103 M) and (104

M), incubated at 37 C for 60 min and 120 min, respectively.

% Intact c(RGDfK)-(Orn)3-[CGG-99mTc]

Time (min) Cys(104 M) Cys(103 M)

60 93.3 80.8120 92.7 62.1l

weight. The animals were placed on the camera at approximately5 min after tracer injection. Data were acquired continuously andsequential images of 10 and 3.5 min frames were stored, respec-tively, for normal and tumor bearing mice. The images were storedin raw format and processed with ImageJ software (NIH, USA,version 1.47c).

3. Results

3.1. Rhenium(V) complex analysis

Chemical purity of the peptide, c(RGDfK)-(Orn)3-CGG, waschecked by HPLC and was found higher than 95%. The nonradioactivecomplex, c(RGDfK)-(Orn)3-[CGG-185/187Re], of the crude RGD deriv-ative was obtained via the 185/187Re-gluconate precursor. The

Fig. 2. RP-HPLC radioactivity detector analysis of mouse urine, collected 30 min postinjection of c(RGDfK)-(Orn)3-[CGG-99mTc].

266 I. Tsiapa et al. / Nuclear Medicine and Biology 40 (2013) 262272ether anesthesia. The main organs were subsequently removed and,together with samples of muscles and urine, were weighed andcounted in a NaI well counter. Stomach and intestines were notemptied before the measurements. Uptake of the radiotracer in eachtissue was calculated and expressed as the percentage injected doseper gram of the tissue (%ID/g). The %ID in whole blood wasestimated assuming a whole-blood volume of 6.5% of the total bodyweight. Biodistribution data are reported as %ID/g and are meansSD (n=35).

2.12. Biodistribution study in U87MG tumor bearing SCID mice

SCID mice bearing U87MG tumors were injected via the tail veinwith 100 l (3.70 MBq) of the puried radiolabeled RGD derivative,diluted in saline, pH 7.0 and were sacriced at 5, 30, 60, 120 min and24 h p.i.. Receptor blocking studies were also carried out, as follows: Agroup of mice was co-injected additionally with an excess amount(100 l, 1 mg/mL) of unlabeled RGD derivative for receptor blocking.The main organs and tissues were subsequently removed, weighed,and counted as previously described.

2.13. Dynamic imaging studies in normal and tumor bearing mice

Evaluation of the new radiolabeled RGD derivative, c(RGDfK)-(Orn)3-[CGG-99mTc(V)] was performed by dynamic imaging at a highresolution small animal -camera. Localization over time of the99mTc-labeled RGD derivative in normal mice and mice (n=4)bearing U87MG tumors was determined by -camera imaging, atdifferent time intervals from 5 minutes until 24 hours afteradministering 100 l (~3.70 MBq) of puried radiolabeled peptideintravenously into the tail vein of the animals. The mice weresubsequently anesthetized by the subcutaneous injection of aproper anesthetizing solution (1.0 g of 2,2,2-tribromomethanol in1.0 mL of 2-methyl-2-butanol) at a dose of 300 l per 30 g bodyFig. 3. Biodistribution results of c(RGDfK)-(Orn)3-[CGG-99mTc] in normal mice atpurication and characterization of the peptide was performedby semi-preparative RP-HPLC, and analytical RP-HPLC and ESI-MS respectively (Tables 1 and 2). The ESI-MS results forc(RGDfK)-(Orn)3-CGG and c(RGDfK)-(Orn)3-[CGG-185/187Re] alongwith the theoretically expected charged ion values are summarized inTable 2. The retention time of the 185/187Re(V)O-N3S-RGD derivative,compared to the respective radioactive complex, was similarafter their co-injection into the RP-HPLC column, conrming thatboth 185/187Re and 99mTc form complexes of similar structure (Table 1).

3.2. Radiolabeling and quality control

The new RGD derivate was efciently labeled with 99mTc with ahigh specic activity of 5.55 GBq/mol. HPLC analysis indicatedthat the puried product remained stable at a yield higher than98% for at least 24 h post labeling when stored at roomtemperature. The retention time of the puried 99mTc complex issimilar to that of the analogous 185/187Re complex (Table 1). Thepeptide could be efciently radiolabeled at a yield higher than 95%even at low concentrations down to 8.3107 M. The lipophilicityof the radiolabeled RGD derivative was evaluated by determiningthe log P partition coefcient values between n-octanol and PBS atpH 7.4. Partition coefcient for the radiolabeled compound waslogP(O/PBS):2.450.01, a value compatible with a hydrophilicmolecule [34].

3.3. In vitro stability studies

In vitro stability was examined by the cysteine challenge test, usingtwo different concentrations of cysteine (103 M and 104 M). Thepuried c(RGDfK)-(Orn)3-[CGG-99mTc] remained stable, at a yieldhigher than 90 % up to 120 min in the presence of excess cysteine(104 M) and N60 % until 120 min in the presence of an even higherconcentration of cysteine (103 M) (Table 3).

5, 30, 60 min (% dose/g). Each value is the average of three to ve animals.

t 5, 30, 60, 120 min and 24 h p.i. (%ID/g). Each value is the average of three to ve animals

120 min 24 h 60 min/blocked

.07 0.790.13 0.210.11 0.240.07

.22 1.621.06 1.090.38 3.530.22

.06 0.750.22 0.440.05 1.460.061.31 59.3911.65 59.168.80 38.8910.79.15 0.590.12 0.210.03 1.090.15.30 0.990.44 0.910.21 2.810.30.55 0.820.35 1.240.01 3.640.55.02 0.340.15 0.310.06 0.800.02.16 1.290.48 0.620.27 4.180.16.12 0.480.16 0.330.09 3.100.12.48 3.391.05 1.830.05 1.230.48

267I. Tsiapa et al. / Nuclear Medicine and Biology 40 (2013) 2622723.4. Metabolic stability

The human plasma stability study, performed as described above,showed that c(RGDfK)-(Orn)3-[CGG-99mTc] remained stable inplasma (N90%) after a 120 min incubation at 37 C. Specically,almost the 30% value of radioactivity remained in the supernatantafter treatment with ethanol and centrifugation. Moreover, HPLCanalysis of a urine sample collected 30 min after injection of theradiopeptide in mice, indicated metabolism in the form of hydrophilicproducts. Injected activity was 22.2 MBq/ 100 l and proved to besufcient to show the metabolic prole of the radiotracer in urine byHPLC. A representative radiochromatogram of c(RGDfK)-(Orn)3-[CGG-99mTc] and its metabolites in mouse urine is presented(Fig. 2), showing that a signicant portion of intact radiopeptide isstill detected in the urine (tR=17.8 min), in addition to catabolicproducts, which have tR=2.08.5 min.

3.5. Biodistribution evaluation in normal Swiss mice

An in vivo study in physiological experimental mice was carriedout and the biodistribution results are presented in Fig. 3. Rapid bloodclearancewas observedwith blood levels of about 0.280.07% ID/g at60 min p.i.. Low values were observed in the stomach, indicating thatthere is minimal, if any, reoxidation to free 99mTcO4. In addition, liveruptake was low even at 60 min p.i. (0.770.02% ID/g) indicating nohepatobiliary excretion of the radiopeptide. It is worth noting thateven at 60 min p.i., kidney uptake was high (77.541.68% ID/g).

3.6. Biodistribution analysis in U87MG tumor-bearing SCID mice

The results of the in vivo analysis in tumor-bearing SCID mice arepresented in Table 4. The biodistribution study was performed inanimals bearing experimentally-induced U87MG tumors that over-express the a receptors. The new radiopeptide, c(RGDfK)-(Orn) -

Table 4Biodistribution of c(RGDfK)-(Orn)3-[CGG-99mTc], in U87MG tumor bearing SCID mice aValues are corrected for 99mTc decay.

Organ 5 min 30 min 60 min

Blood 7.210.11 0.910.11 0.880Liver 3.090.38 1.530.38 1.600Heart 4.120.05 0.870.05 0.970Kidneys 36.436.80 58.448.80 72.171Stomach 3.500.03 1.210.03 1.190Intestines 2.230.21 0.940.21 1.100Spleen 2.260.01 1.570.01 1.980Muscles 2.120.06 0.380.06 0.400Lungs 7.600.27 1.990.27 2.270Pancreas 2.090.09 0.550.09 0.440Tumor 4.000.05 3.320.09 3.870 3 3

[CGG-99mTc], was capable of targeting the 3-positive U87MGtumor at 30 min p.i. (3.320.09% ID/g) and remained at high valuesat 120 min p.i. (3.391.05% ID/g). It is worth noting that even 24 hp.i. tumor uptake was signicant (1.830.05 % ID/g). Regarding thetumor-bearing SCID mice, the predominant excretion route was viathe urinary system, which is in agreement with the biodistributionresults in normal mice. High kidney values (59.168.80% ID/g) wereobserved even at 24 h p.i.. Insignicant uptake was observed in allother organs such as liver, intestines and muscles.

The tumor/blood, tumor/muscle, tumor/intestines and tumor/liverratios at 5, 30, 60 and 120 min p.i. for the radiolabeled derivativec(RGDfK)-(Orn)3-[CGG-99mTc] are presented in Fig. 4. The tumor tobackground ratio was signicantly high, due to high tumor uptake andlow uptake in the rest of the organs. As can be observed, the tumor/blood and tumor/muscle contrast ratios are high with values of 4.39and 10.08 at 60 min and 120 min p.i., respectively, indicating hightumor uptake and retention of the new radiopeptide.

In vivo blocking experiments, performed by co-administration ofan excess amount of native RGD, resulted in a signicantly reduceduptake of c(RGDfK)-(Orn)3-[CGG-99mTc] in tumor 60 min p.i.. Tumoruptake was decreased from 3.870.48 %ID/g to 1.230.48 %ID/g,advocating for specicity of the radiopeptide under study for theabove-referred experimental tumor (Fig. 5).

3.7. Dynamic imaging studies in normal and in tumor-bearing mice

Dynamic imaging of anesthetized normal mice was performed at ahigh resolution, dedicated small animal -camera, up to 65 min p.i..Data were continuously recorded and images were stored every10 min, providing fast and low-cost dynamic planar view of thebiodistribution of the radiopeptide. It has been possible to record datain list mode format and construct images at shorter intervals. Therespective images are shown in Fig. 6. The localization ofc(RGDfK)-(Orn)3-[CGG-99mTc] in a U87MG tumor-bearing mouse indynamic imaging is presented in Fig. 7. The experimental tumor at theleft anterior leg was delineated with high accumulation. Regions ofInterest (ROI) analysis was performed in the collected images toobtain semi-quantitative information at 3.5 min intervals from5 min up to 82 min p.i., as shown in Fig. 8. Due to the high valuesobserved in kidneys and in order to provide better visualization, thekidneys values are reported to the secondary y-axis on the right,while the tumor, muscle and bladder values to the primary y-axis onthe left. Comparative imaging of the new radiolabeled derivative,c(RGDfK)-(Orn)3-[CGG-99mTc], in normal mice and in U87MG tumor-bearing models, was assessed in representative planar -cameraimages in Fig. 9. The same tumor was not visualized in another SCIDmouse, which had received a high dose of unlabeled RGD along withthe radiolabeled RGD derivative, for receptor blocking (Fig. 9). It isworth noting that imaging data were recorded for 24 h p.i. indicatingFig. 4. Graphical presentation of the tumor/blood, tumor/muscle, tumor/intestines andtumor/liver ratios, at 5, 30, 60 and 120 min p.i. of the radiolabeled compound (n=3)..

signicant retention into the experimental U87MG tumor over-expressing the 3 integrin receptors (Fig. 10). Planar imagingstudy is in agreement with biodistribution results.

4. Discussion

Integrin 3 is overexpressed on the surface of activatedendothelial cells as well as cancer cells during neovascularizationinduced by carcinogenesis. Radiolabeled peptides based on the Arg-Gly-Asp (RGD) sequence have been reported as radiopharmaceuticalswith high afnity and selectivity for the 3 integrin and are

(diagnostic or therapeutic). For therapeutic radiopharmaceuticals,high uptake in kidneys and prolonged kidney retention of radiotracersimposes a serious challenge for radiolabeled cyclic RGD peptides[39,42]. In the last several years, a variety of pharmacokineticmodifying linkers have been used to improve excretion kinetics ofradiolabeled RGD peptides. It has been reported that introduction ofthe polyethylene glycol (PEG) linker can improve not only excretionkinetics but also tumor uptake of 125I- and 18 F-labeled c(RGDyK) and64Cu-labeled E[c(RGDyK)]2 [36,43,44]. We designed a new RGDderivative, as a hydrophilic molecule, namely c(RGDfK)-(Orn)3-CGG(Fig. 1), based on previous ndings of our group on radiopeptides

Fig. 5. The specicity of the novel radiopeptide, c(RGDfK)-(Orn)3-[CGG-99mTc], derivative was demonstrated by receptor blocking, after co-administration of an excess amount(100 l, 1 mg/mL) of the non-radiolabeled RGD derivative at 60 min p.i. (n=3).

268 I. Tsiapa et al. / Nuclear Medicine and Biology 40 (2013) 262272therefore useful in the noninvasive monitoring of tumor angiogenesisby molecular imaging techniques. For the past decade, many RGDpeptides and non-peptide RGD mimetics have been labeled withdifferent radioisotopes to develop angiogenesis-targeting radiocom-pounds for both diagnosis (125I, 99mTc, 111In, 18 F, and 64Cu) andtumor-specic radiotherapy (90Y and 177Lu), some of them beingalready introduced in clinical trials [12,1619,3441]. In general, theradiolabeled RGD peptide radiotracer of clinical importance shouldhave high afnity and high specicity for integrin 3, a goodelimination prole and no toxicity. The pharmacokinetic requirementfor an optimal radiotracer depends largely on its medical applicationsFig. 6. Gamma-ray dynamic images of normal mouse administered with 3.70 MBq of c(RGDMouse was anesthetized by the subcutaneous injection of a proper anesthetizing solution acontaining non-natural aminoacids, such as ornithine and arginine[22,25]. To the best of our knowledge, there are no other studies thathave evaluated the radiolabeled cyclic RGDfK sequence conjugated toan N3S chelator via a spacer containing the triaminoacid sequence ofornithine. The intention was to obtain a radiolabeled RGD derivativewith main excretion route, the renal system, and consequentlyreducing the upper abdominal area radioactivity [17,19,4547]. Inthe present study, the new radiopeptide, c(RGDfK)-(Orn)3-[CGG-99mTc] showed high and stable 99mTc-labeling, high tumoruptake, and rapid blood clearance, while the main route of excretionwas found to be the renal system. Moreover, the new RGD derivative,fK)-(Orn)3-[CGG-99mTc]. Images were stored 10 min per frame 5 min up to 65 min p.i..t a dose of 300 l per 30 g body weight.

.70thet

269I. Tsiapa et al. / Nuclear Medicine and Biology 40 (2013) 262272containing the radionuclide chelating moiety CGG, is considered tobe appropriately designed for both 99mTc-labeling and conjugation

Fig. 7. Gamma-ray dynamic images of U87MG tumor-bearing mice, administered with 3to 82 min p.i.. Mouse was anesthetized by the subcutaneous injection of a proper aneswith nanoparticles.In the present study, the 99mTc-labeled peptide was HPLC-puried

in order to be sufcient pure for further in vitro and in vivoinvestigation. HPLC analysis indicated that the puried productremained stable at a yield higher than 98% for at least 24 h afterlabeling when stored at room temperature. The above results wereconsistent with previous studies, which demonstrated that N3S-typeligand systems such as CGG form stable 99mTc-peptide conjugates

Fig. 8. Time activity curves showing the percentage of radioactivity in major organs, using 3.5in Figure 7. The kidney values correspond to the secondary y-axis on the right, while the tum~4.5 times higher than concentration in normal tissue.[22,23,25,48]. According to the literature, the addition of a positivelycharged spacer chain did not seem to affect the labeling efciency of

MBq of c(RGDfK)-(Orn)3-[CGG-99mTc]. Images were stored 3.5 min per frame 5 min upizing solution at a dose of 300 l per 30 g body weight.the CGG chelator [25]. The claim that c(RGDfK)-(Orn)3-[CGG-185/187Re] and c(RGDfK)-(Orn)3-[CGG-99mTc] derivatives have the samestructure was also veried by their similar Rt after co-injection on aRP-HPLC column (Table 1).

Challenge stability studies showed that the radiopeptide remainedstable when incubated with excess of a strong competitor for the99mTc-core such as Cys. A worth noticing degree of transmetalationoccurred only when the radiopeptide was incubated with a high Cys

min frames, from 5 to 82 min post injection in the U87MG tumor-bearing mice shownor, muscle and bladder values to the primary axis on the left. Concentration in tumor is

a) a, 1 mre an

270 I. Tsiapa et al. / Nuclear Medicine and Biology 40 (2013) 262272concentration (103 M) for 2 hours. Metabolic stability studies,which ran in parallel, interestingly showed that metabolites detectedin human plasma and urine did not include free 99mTcO4 (tR=3.5 min) expected to be released in the case of metal chelatedegradation, which is consistent with previous studies reporting thehigh in vivo stability of CGG chelates [22,23,25]. This nding wasadditionally veried by ITLC tests, running in parallel.

It is worthwhile noting that predominant excretion route ofradioactivity for c(RGDfK)-(Orn)3-[CGG-99mTc] took place via thekidneys to the urine as indicated by the obtained kidney values(58.448.80% ID/g) at 30 min p.i.. High kidney values may beattributed to the high overall positive charge of the peptide. The

Fig. 9. Representative planar images of 3.70 MBq of c(RGDfK)-(Orn)3-[CGG-99mTc]):(U87MG onto the left front ank) after co-administration of an excess amount (100 lpresence of the tumor (or blocked tumor), kidneys, bladder and injection point. Mice we300 l per 30 g body weight.overall charge of the radiometalchelatorspacer conjugate affectsthe hydrophilicity of the molecule, especially when we refer topeptides with similar spacer moieties [22,25]. Lipophilicity of theradiolabeled RGD derivative was evaluated by determining the log Ppartition coefcient values between n-octanol to PBS, with partitioncoefcient logP(O/PBS):2.450.01, a value compatible with ahydrophilic molecule. Other RGD derivatives with similar lipophili-city, such as 99mTc-PGC-c(RGDyK) showed hepatobiliary excretionand high intestinal uptake (22.165.66% ID/g) at 120 min p.i., whilethe new RGD derivative, c(RGDfK)-(Orn)3-[CGG-99mTc], exhibitedurine excretion and high kidney uptake (59.3911.65% ID/g) at120 min p.i. [34]. This is due to the ornithine linker and is inagreement with previous work from our group with BN peptidesbearing (Orn)3 linker [22,23,25].

Several groups have developed radiolabeled RGD compounds thathave shown promise in terms of tumor uptake and tumor-to-background ratios [3440]. Tumor uptake of c(RGDfK)-(Orn)3-[CGG-99mTc] was relatively high at 30 min p.i. (3.320.09% ID/g)and remained at high values at 120 min p.i. (3.391.05% ID/g). Eventhough a direct comparison among radiolabeled RGD derivatives wasnot appropriate since a wide variety of linkers and chelating groupsfor 99mTc were used, we can note that tumor uptake ofc(RGDfK)-(Orn)3-[CGG-99mTc] is relative high in comparison toother RGD monomers with tumor uptake (2.81.5% ID/g for 99mTc-His-cRGDfK and 1.380.30% ID/g for 99mTc-PGC-c(RGDyK) at 30 minp.i. and 120 min p.i. respectively) [15,34]. It is worth noting thattumor accumulation of c(RGDfK)-(Orn)3-[CGG-99mTc] was signi-cantly high (1.830.05 % ID/g) at 24 h p.i. compared to other 99mTc-labeled RGD peptides [34]. The tumor to background ratio issignicantly high, due to high tumor uptake and also to low uptakein the rest of the organs allowing clear nuclear imaging (Fig. 4). As canbe observed, the tumor/blood and tumor/muscle ratios are high withvalues 4.39 and 10.08 at 60 min and 120 min p.i., respectively,indicating high tumor uptake and retention of c(RGDfK)-(Orn)3-[CGG-99mTc]. The addition of the charged spacer in the structure wasadvantageous for biodistribution and tumor targeting ability, becauseit reduced the upper abdominal radioactivity levels observedelsewhere [17,19,34] and increased tumor/muscle tissue contrastratio. High kidney uptake observed at 30 min p.i. remained stable

normal mouse, b) a U87MG tumor-bearing mouse c) a blocked tumor bearing mouseg/mL) of the non-radiolabeled RGD derivative at 60 min p.i. The arrows indicate theesthetized by the subcutaneous injection of a proper anesthetizing solution at a dose of(59.168.80% ID/g) even at 24 h p.i.. This can be attributed to kidneytubular reabsorption, which is a process that can inuence theresidence time of radiolabeled peptides into the kidneys [48,49] andcan be manipulated by making use of the electrostatic interactionbetween the negatively charged surface of the proximal tubular cellsand the cationic portion of peptides. Accumulation in other organs

Fig. 10. Representative planar -image of a U87MG tumor-bearing mouse, 24 h afterintravenous injection of 3.70 MBq of c(RGDfK)-(Orn)3-[CGG-99mTc]. Tumor, kidneysand bladder are properly indicated. Mouse was anesthetized by the subcutaneousinjection of a proper anesthetizing solution at a dose of 300 l per 30 g body weight.

271I. Tsiapa et al. / Nuclear Medicine and Biology 40 (2013) 262272such as muscle and spleen was insignicant. It is worth examining thepharmacokinetic inuence of a hydrophilic RGD derivative,c(RGDfK)-(Orn)3-CGG, after conjugation with nanoparticles. Due tothe colloidal nature of all nanoparticles, the high liver and spleenuptake is difcult to overcome and can often be higher than tumoruptake [50,51]. The multimodal imaging approach can facilitateverication of the accuracy in tumor detection and provide additionalinformation regarding the pathology of the tumor.

The co-administration of an excess amount of unlabeled RGDderivative signicantly decreased the uptake of c(RGDfK)-(Orn)3-[CGG-99mTc] in the tumor, while tumor uptake was reduced to 1.230.48 %ID/g in the blockage group, compared to 3.870.48 %ID/g in thenon-blocked group at 60 min p.i. (Fig. 5). Therefore, blocking experi-ments showed that target specicity could be obtained for integrinreceptors in U87MG glioblastoma cells. It is worth noting that highkidney uptake (72.1711.31% ID/g) was decreased (38.8910.79% ID/g) after receptor blocking. According to the literature, thereceptor mediated endocytosis and the peptide reabsorption could beprevented by blocking the receptor by the administration of positivelycharged amino acids [52,53]. High kidney accumulation of theradiolabeled peptide, c(RGDfK)-(Orn)3-[CGG-99mTc], is under investiga-tion and may be overcome by co-administration of gelofusine or lysine.

The biodistribution and the dynamic -camera imaging studies innormal and U87MG tumor-bearing SCID mice have shown signicantearly tumor uptake, combined with fast blood clearance and renalelimination. The predominant excretion route for the tumor-bearingSCIDmice was also via the kidneys to the urine, in agreement with thebiodistribution results in normal mice. Insignicant uptake wasobserved in all other organs such as liver, intestines and muscles.The images also showed high background activity in the kidneys andbladder, verifying that the predominant excretion route of radioac-tivity is via the urinary tract, in full accordance to the biodistributiondata. Dynamic imaging study in U87MG tumor-bearing mouseshowed accumulation to the tumor 5 min p.i., in clear contrast tothe adjacent background. The tumor remained clearly observable upto 82 min p.i. (Fig. 7). Since in planar imaging organs overlap, ROIswere drawn only on the tumor, kidneys, bladder andmuscle (left backfoot) (Fig. 8). These data provide radioactivity concentration perorgan and are complementary to the biodistribution results presentedabove. Concentration in kidneys is slowly increased from 31.5% to 39%,while an almost stable concentration in bladder (~4.5%) is observed.Tumor concentration presented a slow decrease from ~7% to 5.9%,while muscle values ranges from ~1.7% to ~1.3%. The tumor to muscleratio has an average value of ~4.5, which is in good agreement withthe value of 4.39 reported in biodistribution studies 1 h p.i.. Finally,ROI analysis also shows a slow increase in tumor to muscle ratio.Comparative imaging of c(RGDfK)-(Orn)3-[CGG-99mTc] in normalmice and U87MG tumor-bearing models was assessed in represen-tative planar -camera images (Fig. 9). It is worth noting that theU87MG tumor is delineated with clear contrast, while the other organradioactivity uptake is the same in both normal mice and experi-mental tumor models. In contrast, the same tumor was not visualizedin another SCID mouse, which had received a high dose of unlabeledRGD along with the radiolabeled RGD derivative, for receptorblocking. Moreover, it is worth noting that imaging data recordedfor 24 h p.i., indicating signicant uptake into the experimentalU87MG tumor over-expressing the 3 integrin receptors (Fig. 10).Planar imaging study is in agreement with biodistribution results.

5. Conclusion

We designed a new RGD derivative, c(RGDfK)-(Orn)3-CGG, forintegrin 3 targeting. We chose the triaminoacid sequence ofornithines as a spacer with the intention to investigate the inuenceof the ornithine linker on the in vitro and in vivo properties ofc(RGDfK)-(Orn) -[CGG-99mTc]. Labeling with 99mTc led to a radio-3active compound which remained stable up to 24 h post-labeling.Administration of the 99mTc-RGD derivative in tumor-bearing miceresulted in scintigraphic detection of experimentally induced tumors.The primary route of clearance was the renal system. Tumor uptakeremained high and it is worth noting that even 24 h p.i., tumor uptakevalues were signicant. Low tumor uptake was obtained in thereceptor blocking study, indicating high tumor specicity of theradiopeptide. The nal aim was to evaluate the potential use ofthe new derivative as a tumor imaging agent in nuclear medicine, aswell as to apply it further in nanoparticle 99mTc-labeling. Taking intoconsideration the advantages offered by a combined SPECT/MRIsystem, it is critical that multifunctional probe development isimproved for the future use of this bifunctional imaging modalityand accessed for further investigation of the RGD derivative,c(RGDfK)-(Orn)3-CGG, after conjugation with magnetic nanoparti-cles. Investigation of the functionalized magnetic nanoparticles withc(RGDfK)-(Orn)3-CGG peptide is currently in progress.

Acknowledgments

This research has been co-nanced by the European Union(European Social Fund ESF) and Greek national funds through theOperational Program "Education and Lifelong Learning" of theNational Strategic Reference Framework (NSRF) Research FundingProgram: Heracleitus II. Investing in knowledge society through theEuropean Social Fund. The authors would like to thank Dr. LeondiosLeondiadis for ESI-MS analysis performed at the Mass Spectrometryand Dioxin Analysis Lab and Dr. Maria Paravatou-Petsotas for cellculture at Radiobiology and Biodosimetry Lab of NCSR Demokritos.

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Biological evaluation of an ornithine-modified 99mTc-labeled RGD peptide as an angiogenesis imaging agent1. Introduction2. Materials and methods2.1. General2.2. Conjugation and analysis of the c(RGDfK)-(Orn)3-[CGG-185/187Re(V)] derivative (Nonradioactive Complex)2.3. ESI-MS mass spectral analysis2.4. Formation of the c(RGDfK)-(Orn)3-[CGG-99mTc(V)] complex2.5. Radiochemical analysis2.6. Lipophilicity: logP values2.7. Cysteine challenge tests2.8. Metabolic studies2.8.1. In plasma2.8.2. In urine

2.9. Cell culture2.10. Animal models2.11. Biodistribution evaluation in normal Swiss mice2.12. Biodistribution study in U87MG tumor bearing SCID mice2.13. Dynamic imaging studies in normal and tumor bearing mice

3. Results3.1. Rhenium(V) complex analysis3.2. Radiolabeling and quality control3.3. In vitro stability studies3.4. Metabolic stability3.5. Biodistribution evaluation in normal Swiss mice3.6. Biodistribution analysis in U87MG tumor-bearing SCID mice3.7. Dynamic imaging studies in normal and in tumor-bearing mice

4. Discussion5. ConclusionAcknowledgmentsReferences