ORIGINAL ARTICLE

MicroPET/CT imaging ofαvβ3 integrin via a novel68Ga-NOTA-RGD

peptidomimetic conjugate in rat myocardial infarction

Luca Menichetti & Claudia Kusmic & Daniele Panetta & Daniela Arosio &

Debora Petroni & Marco Matteucci & Piero A. Salvadori & Cesare Casagrande &

Antonio L’Abbate & Leonardo Manzoni

Received: 14 January 2013 /Accepted: 15 April 2013# Springer-Verlag Berlin Heidelberg 2013

AbstractPurpose The αvβ3 integrin is expressed in angiogenic vesselsand is a potential target for molecular imaging of evolvingpathological processes. Its expression is upregulated in cancerlesions and metastases as well as in acute myocardial infarction(MI) as part of the infarct healing process. The purpose of ourstudywas to determine the feasibility of a new imaging approachwith a novel 68Ga-2,2′,2″-(1,4,7-triazonane-1,4,7-triyl)triaceticacid (NOTA)-arginine-glycine-aspartic acid (RGD) construct toassess integrin expression in the evolving MI.Methods A straightforward labelling chemistry to attach theradionuclide 68Ga to a NOTA-based chelating agent conjugat-ed with a cyclic RGD peptidomimetic is described. Affinityfor αvβ3 integrin was assessed by in vitro receptor bindingassay. The proof-of-concept in vivo studies combined the

68Ga-NOTA-RGD with the flow tracer 13N-NH3 imaging inorder to obtain positron emission tomography (PET)/CT im-aging of both integrin expression and perfusion defect at4 weeks after infarction. Hearts were then processed for im-munostaining of integrin β3.Results NOTA-RGD conjugate displayed a binding affinityfor αvβ3 integrin of 27.9±6.8 nM. 68Ga-NOTA-RGD showedstability without detectable degradation or formation of by-products in urine up to 2 h following injection in the rat. MIhearts exhibited 68Ga-NOTA-RGD uptake in correspondenceto infarcted and border zone regions. The tracer signal drew aparallel with vascular remodelling due to ischaemia-inducedangiogenesis as assessed by immunohistochemistry.Conclusion As compared to similar imaging approachesusing the 18F-galacto-derivative, we documented for the firsttime with microPET/CT imaging the 68Ga-NOTA-RGD de-rivative that appears eligible for PET imaging in animalmodels of vascular remodelling during evolving MI. Thesimple chemistry employed to synthesize the 68Ga-basedradiotracer may greatly facilitate its translation to a clinicalsetting.

Keywords MicroPET/CT imaging . Angiogenesis .

Myocardial infarction . Integrin . RGD peptidomimetic .68Ga-NOTA-RGD

Introduction

Myocardial infarction (MI) is a dynamic process initiatingwith coronary occlusion and rapidly progressing towardsirreversible contractile tissue damage. Considerable researchhas been devoted in the last few decades to studying theearly revascularization of the ischaemic territory in order to

Electronic supplementary material The online version of this article(doi:10.1007/s00259-013-2432-9) contains supplementary material,which is available to authorized users.

L. Menichetti (*) :C. Kusmic :D. Panetta :D. Petroni :P. A. Salvadori :A. L’AbbateCNR-Institute of Clinical Physiology (IFC), Via Moruzzi 1,56124 Pisa, Italye-mail: luca.menichetti@ifc.cnr.it

D. Arosio : L. Manzoni (*)CNR-Institute of Molecular Science and Technologies (ISTM),Via C. Golgi 19,20133 Milan, Italye-mail: leonardo.manzoni@istm.cnr.it

M. Matteucci :A. L’AbbateScuola Superiore Sant’Anna, Piazza Martiri della Libertà 33,56127 Pisa, Italy

C. CasagrandeDepartment of Chemistry, University of Milan, Via C. Golgi 19,20133 Milan, Italy

Eur J Nucl Med Mol ImagingDOI 10.1007/s00259-013-2432-9

reduce infarct size and improve outcome. However, beyondearly reperfusion, infarct outcome is actually determined bythe efficacy of a long series of biological modifications lead-ing to tissue repair, ventricular remodelling and conservationof contractile function. Among biological factors interveningafter the ischaemic event, angiogenesis has a prominent role intissue repair and regeneration and in ventricular remodelling[1, 2], a process involving residual viable myocardium whichplays a crucial role in the progression toward chronic heartfailure. The clinical evaluation of angiogenesis, i.e. its pres-ence, extent and time course, might significantly improvepost-infarction risk stratification and possibly guide noveltherapeutic interventions.

Integrins and vascular endothelial growth factor (VEGF)have been associated with post-infarction angiogenesis [3, 4].All integrins are composed of two subunits, pertaining to theαand β sets, and each subunit consists of a large extracellulardomain, a single transmembrane domain and a short cytoplas-mic tail. The extracellular domain is responsible for the adhe-sion to proteins of the extracellular matrix (ECM) or specificproteins expressed on other cells. On ligation, two-way sig-nalling is initiated with the intracellular domain, which in-duces cell spreading, migration, survival, proliferation anddifferentiation, playing a crucial role in tissue regenerationand ventricular remodelling.

In tissue ischaemia, the two main integrins of the endo-thelial cells, i.e. αvβ3 (with a minor participation of therelated αvβ5) and α5β1, become overexpressed favouringthe cell adhesion to ECM, whereby cell-to-cell interactioninitiates the construction of new microvessels. Recognitionand adhesion to specific ECM proteins are driven toward theArg-Gly-Asp (RGD) sequences [5, 6]. Investigators havedevoted considerable study to ligands of the integrin fortherapeutic and diagnostic purposes [7] and specifically totheir use in the construction of probes for non-invasive invivo molecular imaging of tumours, by single photon emis-sion computed tomography (SPECT), positron emissiontomography (PET) and other techniques [8–11].

The contribution of our group to the design of integrinligands started from the discovery of RGD mimics in whichthe peptide sequence is conformationally constrained by a 1-aza-2-oxobicyclo[X.3.0]alkane scaffold, resulting in potentand selective αvβ3 ligands [12, 13]. Thereafter, we providednovel related ligands with functionalized side chains, suit-able for conjugation with imaging probes [14–16] and withtherapeutic agents [17], targeting endothelial and tumourcells in vitro [14] and tumour xenografts in vivo [15–17].

With the present study we extend our research interest tothe cardiovascular field, and specifically to MI. We focusedon the potential of PET imaging of αvβ3 integrin with68Ga3+ chelates, which appears attractive because of stimu-lating in vivo results in animal models of cancer [9, 18]. Atpresent, 68Ga-labelled tracers have been used for PET

imaging of atherosclerosis [19, 20] or myocardial perfusion[21]. However, 68Ga imaging of MI has not yet beenreported.

We decided to test our tracer on a model of MI bycoronary ligation in rats. Thus, we chose one of ourfunctionalized ligands, namely compound DB58, for theconstruction of a novel 68Ga complex (compound 1, Fig. 1)including the suitable chelating moiety 2,2′,2″-(1,4,7-triazonane-1,4,7-triyl)triacetic acid (NOTA), with the goal ofexploring by microPET/CT the extent of αvβ3 integrinexpression in MI.

This direct approach allows testing one-pot synthesiswith a different strategy from that for other PET labellingagents, e.g. the 18F labelling of glycosylated RGD moieties.Indeed, PET imaging not only can provide a map of theintegrin, but also a direct comparison with perfusionmeasures, an important factor in the repair of the post-infarction myocardium.

Materials and methods

Chemistry

All chemicals and solvents were of reagent grade and wereused without further purification. NODA-GA(tBu)3 waspurchased from CheMatech, and all the other reagents werepurchased from Sigma-Aldrich. 1H and 13C NMR spectrawere recorded at 300 K on a Bruker AVANCE 400 and600 MHz. Chemical shifts δ are expressed in ppm relativeto internal Me4Si as standard. Mass spectra were obtainedwith an ESI apparatus Bruker Esquire 3000 plus. Columnchromatography was carried out with the Biotage SP1 sys-tem using Biotage cartridges. Semi-preparative HPLC was

Fig. 1 Peptidomimetic integrin ligand DB58 and the 68Ga-NOTA-RGD conjugate 1

Eur J Nucl Med Mol Imaging

carried out with an Atlantis Prep T3 OBD, 5 μm, 19×150 mm Waters column using a gradient mixture ofCH3CN/H2O+0.1 % trifluoroacetic acid (TFA). The syntheticscheme for the preparation of the conjugated compound isreported in the “Electronic supplementary material”.

Synthesis of NODA-GA(tBu)3-RGD conjugate Protectedc-RGD-NH2 (48.5 mg, 0.06 mmol), HATU (27.0 mg,0.072 mmol), HOAt (10.0 mg, 0.072 mmol) and NODA-GA(tBu)3 (41.0 mg, 0.075 mmol) were dissolved, under N2

and at room temperature, in dry DMF and finally DIPEA(50 μl, 0.3 mmol) was added. The reaction mixture wasstirred overnight and after reaction completion the solventwas evaporated under reduced pressure. The pure productwas obtained after reverse-phase column chromatographyon a Biotage SP1 system using C18 25+M cartridge elutingwith water/(MeOH+ iPrOH 4:1) from 95:5 to 10:90 Y: 30 %(white solid). The 1H NMR, 13C NMR and mass character-izations of the conjugated compound have been reported inthe “Electronic supplementary material”.

Synthesis of NOTA-RGD conjugate A solution of protectedcompound (21.0 mg, 0.016 mmol) in TFA/thioanisole/1,2-ethanedithiol/anisole 90:5:3:2 (2 ml) was stirred at room tem-perature for 6 h. After reaction completion, the solvent wasevaporated under reduced pressure; next, the crude wasdissolved in H2O and washed with iPr2O. The aqueous phasewas evaporated under reduced pressure and the crude purifiedby HPLC (Atlantis Prep T3 OBD column 19×150 mm, 5 μm;gradient from 0–100 % MeCN/H2O+0.1 % TFA to 30–70 %MeCN/H2O+0.1 % TFA over 15 min, then washed with 30–70 % MeCN/H2O+0.1 % TFA to 100-0 % MeCN/H2O+0.1 % TFA over 1 min and 100-0 % MeCN/H2O+0.1 %TFA for 4 min, flow rate 15 ml/min, λ=216 nm). TheNOTA-RGD conjugate peak was collected at a retention timeof 10.6 min Y: 66 %. The 1H NMR, 13C NMR and masscharacterizations of compound 5 have been reported in the“Electronic supplementary material”.

Synthesis of cold Ga-NOTA-RGD 6 Gallium(III) chloride(1.0 mg, 0.0056 mmol) in water (0.5 ml) was added to asolution of NOTA-RGD conjugate (1.0 mg, 0.001 mmol)dissolved in water (5.0 ml). The mixture was stirred at roomtemperature for 15 min. After reaction completion, waterwas removed and the crude was purified by HPLC (AtlantisPrep T3 OBD column 19×150 mm, 5 μm; gradient from 0–100 % MeCN/H2O+0.1 % TFA to 30–70 % MeCN/H2O+0.1 % TFA over 15 min, then washed with 30–70 %MeCN/H2O+0.1 % TFA to 100-0 % MeCN/H2O+0.1 %TFA over 1 min and 100-0 % MeCN/H2O+0.1 % TFA for4 min, flow rate15 ml/min, λ=216 nm). The peak of thecomplex was collected at a retention time of 9.3 min Y:>99 %.

Radiolabelling with 68Ga

Labelling was performed at room temperature with a one-potsynthesis approach with solid phase extraction (SPE) purifi-cation (fully automated direct labelling applying fractionatedelution; pre-concentration of the eluate on SPE column).68Ge/68Ga Generators were obtained from Radius with theactivity of 74 MBq (20 mCi). Acetone, HEPES (biochemicalgrade, Fluka, Germany) and ultrapure water (Milli-Q,Millipore). A PC-controlled radiopharmaceutical synthesisdevice based on a modular system (Eckert & ZieglerEurotope, Berlin, Germany) was used for all steps in thesynthesis. 68Ge/68Ga Ionic generators (Eckert & ZieglerObninsk, 20 mCi capacity) were eluted with 0.1 M HCl(biochemical grade, Fluka, Germany) using a peristalticpump driven by the synthesizer through the SPE cationexchange cartridge (Strata-X-C 30 mg, Phenomenex, Tor-rance, CA, USA) [22]. To extract 68Ga3+, the column waseluted by 0.7 ml 98 % acetone/0.02 N HCl solution [22].NOTA moiety was labelled at room temperature using≈90 nmol of peptide precursor and one-pot/one-step synthesis.The 68Ga3+ eluate was directly transferred into 0.9 ml 0.5 MHEPES buffer (pH 3.9) and 0.9 ml ultrapure water (Milli-Q,Millipore) containing the NOTA-RGD chelating moiety.After 3 min, the residual acetone in the mixture was removedby distillation: the reactor was flushed with N2, gentlyheating the bulk of the solution at 55 °C for 5 min. After thisstep, the solution was passed through a preconditioned C18reversed-phase cartridge (Sep-Pak Plus) to ensure a finalpurification from any 68Ga-hydrolysed species present [22],and finally it was recovered for delivery. Osmolarity wasadjusted by adding≈500 μl of 2 % NaCl in the receiver vial.Radiochemical synthesis was set up and checked by radio-thin-layer chromatography (TLC) (with BGO detector, Gina-Star TLC, Raytest Isotopenmeßgeräte GmbH, Straubenhardt,Germany) as reported by another group [19]. PhotodiodeArray (PDA)-Radio-HPLC (HPLC Waters Controller/Delta600, PAD Waters 996; NAI Radiodetector Gabi, Raytest)water, column Atlantis (C18, 150×4.6 mm, 5 μm), eluent A:TFA 0.1 %, B: CH3CN/0.1 % TFA, 10 min gradient, A: 95–30 %, B: 5–70 %, was used (Rt

68Ga-NOTA-RGD≈7±0.2 min). Prior to each in vivo study we tested the qualitycontrol of the labelled compound 68Ga-NOTA-RGD bymeans of PAD-radio-HPLC using the NOTA-RGD derivativeas reference compound (Rt NOTA-RGD≈8.1±0.2 min). Theshelf life of the radiopharmaceutical and the in vivo stability ina urine sample of 68Ga-NOTA-RGD were monitored usingthe same analytical conditions. Urine was obtained directlyfrom the urinary bladder and was filtered through 0.22 μm(Millipore) filters and then centrifuged at 9,500 g for 10 minfor the separation of proteins. Filtrates were analysed by radio-HPLC as described above. Residual solvent was analysed byGC-FID (Clarus 600, PerkinElmer, Elite 624 column).

Eur J Nucl Med Mol Imaging

Biology

Solid phase receptor binding assay Purified αvβ3 receptor(Chemicon International, Inc., Temecula, CA, USA) wasdiluted to 0.5 μg/ml in coating buffer containing 20 mmol/LTris–HCl (pH 7.4), 150 mmol/L NaCl, 1 mmol/L MnCl2,2 mmol/L CaCl2 and 1 mmol/L MgCl2. An aliquot ofdiluted receptor (100 μl/well) was added to 96-well micro-titer plates (NUNC MW 96F MEDISORP STRAIGHT)and incubated overnight at 4 °C. The plates were thenincubated with blocking solution [coating buffer plus 1 %bovine serum albumin (Sigma)] for an additional 2 h atroom temperature to block non-specific binding followedby 3 h incubation at room temperature with variousconcentrations (10−12–10−5 M) of test compounds in thepresence of 1 μg/ml vitronectin-biotinylated using theEZ-Link Sulfo-NHS-Biotinylation kit (Pierce, Rockford,IL, USA). After washing, the plates were incubated for1 h at room temperature with streptavidin-biotinylatedperoxidase complex (Amersham Biosciences, Uppsala,Sweden) followed by 30 min incubation with 100 μlsubstrate reagent solution (R&D Systems, Minneapolis,MN, USA) before stopping the reaction by addition of50 μl of 2 N H2SO4. Absorbance at 415 nm was read ina Synergy™ HT Multi-Detection Microplate Reader(BioTek Instruments, Inc.). Each data point is the resultof the average of triplicate wells and was analysed bynon-linear regression analysis with the Prism GraphPadprogram.

In vivo studies

Rat model of myocardial infarction The experimental pro-tocol, conforming to the Guide for the Care and Use ofLaboratory Animals published by the US National Institutesof Health (NIH Publication No. 85–23, revised 1996), wasapproved by the Animal Care Committee of the ItalianMinistry of Health (Endorsement n.135/2008-B).

Male Wistar rats 10–12 weeks old and weighing 310±3 gwere used in the study. Rats were anaesthetized usingZoletil®+xylazine (50 and 3 mg/kg, respectively). A standardlimb D1-D3 electrocardiogram (ECG) was monitored withsubcutaneous stainless steel electrodes. The rats (n=8) wereconnected to a respirator through an oropharyngeal cannula andthe heart was rapidly exposed through a left lateral thoracotomyand pericardial incision. The coronary artery was permanentlyligated about 2–3mm from its origin. Ischaemia was confirmedby ST segment elevation in the ECG and visually assessed byregional cardiac cyanosis. Then the heart was returned to itsnormal position and the thorax closed. An additional four ratsunderwent all surgical procedures except the final ligation ofthe thread around the left anterior descending (LAD) artery(sham-operated).

Post-operatively, all rats were hydrated with physiologicalsaline and given buprenorphine 0.05 mg/kg s.c. (Temgesic®,Schering-Plough, Brussels, Belgium) twice a day for 3 daysfor analgesia.

In vivomicroPET/CTimaging and PETimage analysis Twenty-eight days following surgery, the MI rats were re-anaesthetized as described above and imaged for αvβ3

integrin expression. Animals were excluded from the studyif lesions to the ribs due to the surgical procedure weredetected by microCT imaging. In fact, integrin expressionin osteoclasts and inflammatory cells involved in boneremodelling [23] of ribs could mask the actual angiogenesisassociated with myocardial wound healing. MicroPET im-ages were acquired using the YAP-(S)PET scanner [24];microCT images were acquired with the Xalt-HR scanner[25]. Coregistration between PET and CT images wasperformed by rigid geometric transformations. Regions ofinterest (ROI) for focal tracer uptake were defined on a mid-myocardial section in both infarcted and remote regions ofinfarcted rats (Fig. S8 in the “Electronic supplementarymaterial”). The ROI definition was the same in both sham-operated and infarcted animals.

As a reference for the localization of infarcted myocardi-um, a perfusion PET study with 13N-NH3 as flow tracer(activity≈0.12 MBq/g body weight) was carried out. Thetracer was injected in the jugular vein, and data acquisitionof 45 min (3 frames×15 min/frame) was started just afterinjection. This acquisition sequence allowed evaluation ofthe infarct location and size, by selecting a time of observa-tion at which the tracer accumulation into the myocardiumhas reached a plateau (typically at 15 min post-injection).Once the 13N-NH3 imaging was completed, activity of0.1 MBq/g body weight of 68Ga-NOTA-RGD was adminis-tered, and 60 min acquisition (2×30 min) was started 45minafter the tracer injection (see Fig. S9 in the “Electronicsupplementary material”). A microCT scan (7 min acquisitiontime, 50 kVp, 300 mAs) for anatomical reference of PETimages was performed after the PET scans. Immediately afterthe imaging session, all rats were euthanized by injecting alethal dose of anaesthetics and hearts were excised for afurther imaging session of ex vivo detection of 68Ga-NOTA-RGD uptake in isolated heart. Briefly, for localizationpurposes, the left ventricular (LV) cavity was filled withdiluted iodinated contrast medium (Iomeron, Bracco ImagingSpA, Milan, Italy), 50 % in volume. The coregistrationbetween PET and CT images was facilitated by a compatiblemechanical interface, which allowed imaging of each animalon the same bed in the two modalities. This procedure wasvalidated via specially designed phantoms with dual-modalityfiducial markers.

All data were acquired in three-dimensional (3-D) modeand reconstructed with a 3-D model-based maximum

Eur J Nucl Med Mol Imaging

likelihood expectation maximization algorithm [26] forPET and with a modified Feldkamp-type filteredbackprojection (FBP) for CT; the isotropic voxel sizewas 0.753 mm3 for PET and 803 μm3 for CT. PET datawere normalized and corrected for random, dead timeand radioactive decay. AMIDE software was used forPET image analysis [27]. Spherical ROIs of 4.5 mmdiameter were drawn around hypoperfused regions, asdetected in the 13N-NH3 PET images, and in the con-tralateral region (Fig. S8 in the “Electronic supplemen-tary material”). The same ROIs were used for theanalysis of the image with both tracers. The signalwithin the ROI was expressed as kilobecquerels permillilitre after well counter calibration of the PETscanner.

Histology and immunohistochemistry Hearts from sham-operated and infarcted rats were arrested in diastole, ventri-cles were weighed and fixed in 10 % buffered formalin for24–48 h, dehydrated through alcohol series, cleared in xy-lene and embedded in paraffin. Serial 5- to 7-μm transversesections starting from the mid-ventricular (1 mm distal tothe ligature) level were obtained and stained withhaematoxylin and eosin. Immunoperoxidase staining wasused to identify endothelial cells, by a mouse monoclonalanti-CD31 primary antibody (DakoCytomation, Glostrup,Denmark) and by a rabbit monoclonal anti-integrin β3

(CD61) subunit primary antibody (Abcam, Cambridge,UK). Tissue slides were pretreated with target retrievalsolution (sodium citrate solution 10 mM, pH 6.0) at 95 °Cfor 15 min before successive procedures. Endogenous per-oxidase was blocked in a solution of 3 % H2O2 for 15 min atroom temperature. After incubation with 5 % goat serum for30 min, sections were incubated with primary antibodies at37 °C for 1 h followed by three washes with phosphate-buffered saline (PBS). Chromogenic detection of antigenswas done by the VECTASTAIN ABC system (Vector Labora-tories, Burlingame, CA, USA) based on avidin-biotinimmunoperoxidase. Sections were counterstained withhaematoxylin. Slides were examined using light microscopy(Leitz Orthoplan and Olympus BX43) at ×10 to ×40 originalmagnification and digitized by a video system (Olympus DP20camera) interfaced with a computer with dedicated software(cellSens Dimension, Olympus) for image acquisition.

Statistical analysis

Statistical analysis was performed with MedCalc 12.2. Datawere expressed as mean ± standard deviation (SD). For eachcondition the comparison of the two data sets wasperformed using a two-tailed paired or unpaired t test whenappropriate. A p value less than 0.05 was considered statis-tically significant.

Results

Chemistry

Among a small collection of functionalized cyclic RGD, wechose compound DB58 (Fig. 1), which showed the highestaffinity for αvβ3 integrin in an in vitro binding assay [28] asa good candidate for labelling with 68Ga. In our study, aNOTA-based chelating agent was selected for conjugationwith the RGD ligand on account of its ability to form ahighly stable neutral complex with gallium.

The RGD amine derivative employed in the conjugationstep was easily obtained from DB58 following a known pro-cedure [14, 28]. Briefly, the hydroxy group was transformedinto the azido derivative via mesylate displacement, and thenthe azido compound was subjected to standard hydrogenationon Pd/C affording the corresponding amine. The couplingreaction between the RGD amine derivative and the fullyprotected chelating agent NODA-GA(tBu)3 was performedunder standard conditions (HATU, HOAt, DIPEA) (SchemeS1). Finally, the cleavage of the side chain protecting groups(Mtr and tBu) by TFA in the presence of scavengers, followedby HPLC purification, afforded the NOTA-RGD conjugate.

In order to set up the labelling conditions, the chelationreaction was first attempted with cold gallium. Labellingwas performed at room temperature using GaCl3 andproceeded over 10 min, as monitored by HPLC-mass spec-trometry (MS). The complex formation was confirmed byMS. Ga-NOTA-RGD showed an earlier retention time(3.5 min method A, 5.9 min method B) in comparison withunlabelled compound (4.0 min method A, 6.8 min methodB) (see “Electronic supplementary material”). 1H NMRspectroscopy showed a change in the chemical shift of theprotons of the NOTA after complexation, while chemicalshift of the protons of the RGD ligand remained unaffected(see “Electronic supplementary material”).

Receptor binding assay

The NOTA-RGD conjugate was examined in vitro for itsability to inhibit the binding of biotinylated vitronectin tothe isolated, immobilized αvβ3 receptor. The functionalizedcyclic pentapeptide NOTA-RGD displayed a binding affin-ity toward αvβ3 integrin (27.9±6.8 nM) comparable to thatof the reference ligand DB58 (20.2±1.9 nM) indicating thatthe conjugation of NOTA to the RGD did not substantiallychange the affinity for the integrin αvβ3 (see Fig. S10 in the“Electronic supplementary material”)

Labelling reaction

The 68Ge/68Ga generator was eluted and washed with 5 mlof ultrapure 0.1 M HCl solution through a cation exchange

Eur J Nucl Med Mol Imaging

resin (Strata-X-C). Removal of 68Ge by >90 % andrecovery of 68Ga >85 % was reported to be achievedwith Strata-X-C cation exchanger with 0.8 ml 98 %acetone/0.02 M HCl [22]. Labelling conditions wereset up using radio-TLC, while the radiochemical puritiesof 68Ga were assessed by radio-HPLC. The optimallabelling condition was obtained with 1:1 v:v pre-solubilized peptide and HEPES buffer (900 μl, pH3.9). The labelling reaction took place at room temper-ature, while a slight amount of the reactor temperaturewent up to 55 °C and a mild vacuum was required toremove the solvent from the bulk of the solution: bygas chromatography/flame ionization detector (GC/FID)the amount of acetone was checked and was typicallyless than 5 mg/ml in the final solution. Considering anoverall synthesis time of less than 20 min, this corre-sponds to up to 90 % decay-corrected overall yield ofthe available activity that could be achieved in the fullyautomated system. Main losses resulted from residues ofactivity on the cation exchanger. The labelled com-pound, 68Ga-NOTA-RGD, was detected using theradio-HPLC system (Rt≈7.0 min) with a 97 % radio-chemical purity. In vivo stability of the compound waschecked by radio-HPLC in a urine sample and demon-strated the stability of 68Ga-NOTA coordination with nodetectable degradation of the complex and/or formationof downstream byproducts until 2 h after injection (seeFig. S11 in the “Electronic supplementary material” forradiochromatogram of a urine sample taken from ananimal bladder). Shelf life of the complexed NOTA-RGD compound was monitored until 2 h after the endof the synthesis without detectable degradation of theNOTA complex.

In vivo detection of 68Ga-NOTA-RGD uptake usingmicroPET/CT imaging

LV myocardium was visualized by 13N-NH3 imaging in allanimals, and the images were used as a reference for localiza-tion of 68Ga-NOTA-RGD uptake. Homogeneous 13N-NH3

distribution throughout the LV myocardium was observed insham-operated rats (Fig. 2a). However, 13N-NH3 imagesshowed focal decrease of tracer accumulation, visually assessedin the LV anterolateral wall of the LAD ligated animals, indi-cating underperfused infarcted myocardium (Fig. 2b).

No focal 68Ga-NOTA-RGD uptake was found in themyocardium of sham-operated animals. On the contrary,increased 68Ga-NOTA-RGD uptake was recorded in themid-apical portion of the anterolateral LV wall of infarctedrats. By superimposing images relative to the two PETtracers on the CT heart image, a perfusion defect becameapparent in the same area of integrin expression in infarctedrats.

The best trade-off between tracer uptake and countingstatistics for imaging was obtained at 45–60 min after in-jection. For this reason, the tracer accumulation at the focalmyocardial uptake area was measured 45 min after injectionand expressed as the ratio between ROI 1 and ROI 2(Fig. 3a). The 13N-NH3 uptake in the anterior wall was 0.4±0.1 of that in the contralateral ROI and about half of the valuemeasured in the sham group (p<0.001), thus confirming thepresence of underperfused myocardium in the LAD territory.In contrast, the uptake of 68Ga-NOTA-RGD was 1.7±0.2times greater in the anterior wall than in the contralateralregion, differently from sham animals (p<0.001) where up-take was nearly the same in the two ROIs. Standardizeduptake values (SUVmean) of

68Ga-NOTA-RGD in each ROI

Fig. 2 Uptake differences of 13N-NH3 (blue) and 68Ga-NOTA-RGD(red) tracers in sham-operated and infarcted rats. a Representative 13N-NH3 images of a sham-operated animal showing homogeneous myo-cardial perfusion (13N-NH3) in the absence of significant integrin

overexpression (68Ga-NOTA-RGD). b Representative images of in-farcted rat showing a perfusion defect (absence of blue) in the samearea of 68Ga-NOTA-RGD uptake (red spot)

Eur J Nucl Med Mol Imaging

were also calculated. SUVmean values measured in the LVposterior wall were comparable between sham and MI groups(0.41±0.1 and 0.44±0.05, respectively; n.s.). In contrast,SUVmean of 68Ga-NOTA-RGD in the LV anterior wall wassignificantly higher in the MI group than in sham-operatedanimals (0.7±0.06 and 0.40±0.1, respectively, p=0.028).

Ex vivo detection of 68Ga-NOTA-RGD uptake in isolatedhearts using microPET/CT imaging

Ex vivo PET/CT imaging of isolated hearts confirmed thetracer uptake in the infarcted region of the myocardium(Fig. 4) revealing a good match between in vivo and exvivo analysis.

Histology and immunohistochemistry

At 28 days after surgery, sham animals showed an insignif-icant area of LV damage (<2 %), mainly due to tissue traumacaused by the small curved needle and suture passedthrough the myocardium beneath the proximal portion ofthe LAD. In the animals with LAD ligation infarct size was33.9±1.5 % of LV (p<0.001 vs sham-operated) (Table 1 andFig. 5). Thickness of the core infarcted wall was about 43 %of the corresponding anterior wall in the sham group (p<0.001), whereas opposite wall thickness was about 118 %, avalue significantly higher than in sham animals (p<0.05),indicating already established remodelling of residual viablemyocardium.

As compared with sham-operated rats, in LAD ligatedrats immunohistochemistry showed an increased number ofCD31-positive vessels mainly localized in the peri-infarctregion, and to a lesser extent within the infarcted area. Thesevessels also displayed stronger β3 integrin staining relativeto viable myocardium or vessels in remote regions.

Discussion

In the present study, we report the synthesis and biologicalevaluation of a new 68Ga-NOTA-RGD (compound 1) con-jugate characterized by structural novelty due to theazabicycloalkane-RGD ligand. Compound 1 is a novel de-rivative of DB58, chosen on account of its affinity andselectivity for αvβ3 integrin; moreover, the peptidomimeticnature envisages in vivo enhanced stability of the finalconstruct. This kind of RGD peptide binds to αvβ3 integrinwith high selectivity, as shown in our previous studies [14, 28].There are several advantages to using 68Ga for labelling. Thepositron-emitting 68Ga isotope has become commerciallyavailable through a 68Ge/68Ga generator. The parent nuclide,68Ge, possesses a half-life of 271 days, and so 68Ge/68Gagenerators can produce sufficient quantities of 68Ga for up to1 year. This generator system provides a relatively inexpensiveand reliable source of a positron-emitting radionuclide withoutthe need for an on-site cyclotron. The favourable decay profileof 68Ga (positron emission, Emax 1.9 keV, 90 %), coupled to ahalf-life of 68 min, has already prompted clinical imaging

Fig. 3 Histograms representing13N-NH3 uptake (a) and

68Ga-NOTA-RGD (b) in sham-operated (n=4) and infarctedrats (n=8). Data are expressedas the mean ± SD of the ratiobetween ROI 1 (anterior wall)and ROI 2 (posterior wall).*p<0.001 vs sham-operatedgroup

Fig. 4 Short axis (left) andlongitudinal long axis (right) exvivo microPET/CT images ofthe isolated heart, at the end ofthe in vivo imaging study. Thefocal uptake of 68Ga-NOTA-RGD is compatible with theinfarcted and peri-infarct zones,as shown by the thinner scarredLV wall segment on CT images

Eur J Nucl Med Mol Imaging

applications based on peptide receptor-targeted constructs [29,30]. Finally, the labelling of the NOTA-RGD conjugate pro-ceeds smoothly at room temperature with a one-potsynthesis approach leading to high yield (Y≈90 % de-cay corrected) and radiochemical purity≈98 % checkedby radio-HPLC.

We chose to test 68Ga-NOTA-RGD in a rat model ofchronic MI by LAD ligation, and in order to evaluate theperfusion and the cardiac remodelling we set up a dual-isotope imaging of perfusion with 13N-NH3 and αvβ3

integrin expression with the labelled RGD peptide.In vivo focal 68Ga-NOTA-RGD uptake was observed at

28 days after LAD coronary ligation in correspondence toinfarcted and peri-infarct myocardial regions. The data wereconfirmed by the subsequent ex vivo imaging of the isolatedheart. This result is in line with findings from other studiesperformed by using 111In-RP748 as a SPECT radiotracerlinked to a non-peptide αvβ3 integrin ligand [31–33] or an

18F-labelled glycosylated RGD αvβ3 integrin ligand [4, 34] or18F-AlF-NOTA-PPRGD2 [35] in rats subjected to myocardialischaemia and reperfusion. In addition, the [18F]galacto-RGDtracer has been previously used in man to image integrinexpression in acute MI [36].

As far as the tracer specificity is concerned, the in vitro testshowed that the displacement of biotinylated vitronectin byour conjugated RGD compound was comparable to that of itsunconjugated counterpart. Although the possible artefact offree 68Ga contribution to the 68Ga myocardial imaging cannotbe quantified in the present study, we are confident regardingits scarce relevance to our conclusions since we ascertained,on the one hand, the very high radiochemical purity (97 %)and a low fraction of free 68Ga(III) species in the injected dose(≤3 %), and on the other, the integrity of the 68Ga-NOTA-RGD in the urine up to 2 h post-injection, documenting thestability of the construct during the study period. This issuemerits further studies devoted to in vivo competitive receptorblocking experiments.

The in vivo and ex vivo uptake, detected 4 weeks aftersurgery, correlated well with immunohistochemical stainingby CD31 and CD61 (the β3 subunit of integrin) of newlyformed vessels in the infarcted and, mostly, in the peri-infarct area. In addition, vessel β3 integrin staining in thesespecific areas was stronger relative to viable myocardium orvessels in remote regions, the latter also characterized byweak 68Ga-NOTA-RGD uptake.

The results of our study document the feasibility ofin vivo monitoring of angiogenesis following MI by68Ga-NOTA-RGD. The animals were imaged at 4 weeksafter infarction, a time consistent with the healing and

Table 1 Macroscopic morphometric parameters at 28 days aftersurgery

Sham group n=4 MI group n=8

Infarct size (% of LV) <2 33.9±1.5**

WT infarcted (mm) 2.66±0.05 1.13±0.06**

WT opposite (mm) 2.60±0.05 3.07±0.13*

Values are mean ± SD

n number of animals tested, WT wall thickness

*p<0.02

**p<0.001 vs sham-operated

Fig. 5 Macro- andmicrosections of rat hearts. aCoronal section of sham heartstained with haematoxylin andeosin. b Coronal section ofinfarcted heart stained withhaematoxylin and eosin; curvedarrow marks the infarcted area.c Freshly cut transversal sectionof the heart at mid-level (4–5mmbelow the LAD ligation)showing absence ofcardiomyocytes in the scar (paletissue). The white dotted boxoutlines corresponding region inimmunostained sections. d and eRepresentative immunostainingfor CD61 (d) and CD31 (e) fromthe border region of infarctedheart at low magnification (×10,calibration bar 50 μm)

Eur J Nucl Med Mol Imaging

regenerative processes that follow the acute phase ofMI, among which angiogenesis is thought to play acrucial role in the maintenance of ventricular functionand in the prevention of heart failure.

It is also established that the co-presence of conditions suchas diabetes, metabolic syndrome, hypercholesterolaemia andothers, all leading to endothelial dysfunction, may challengeangiogenic response to ischaemic insult and negatively affectinfarct outcome. Thus, the non-invasive, high-resolution,high-sensitivity molecular imaging described in this studycan specifically provide direct information regarding myocar-dial angiogenesis in patients with healing MI. It can alsocontribute to better defining risk stratification and hopefullyencourage new therapeutic strategies based on the control ofthe molecular processes underlying post-infarction structuraland functional changes in the LV.

From the perspective of its clinical application, 68Ga-NOTA-RGD presents some technical advantages: it is adiffusible compound targeting intra- and extravascularmolecules, thus increasing imaging sensitivity; as a PETtracer, unlike 18F, it does not require a cyclotron andcomplex radiochemistry; compared to SPECT tracers itcan provide semi/quantitative results with higher spatialresolution. Finally, from a clinical perspective, its usecombined with a flow tracer might easily provide information,as in the present study, on both protein expression andperfusion defect.

Conclusion

In this study we documented myocardial uptake of the68Ga-NOTA-RGD conjugate (compound 1) in corre-spondence to 4-week-old MI in a rat model of LADcoronary ligation.

In this model of ischaemia-induced angiogenesis, weconfirmed the increased expression of the αvβ3 integrin byimmunohistochemistry and demonstrated a stronger β3

integrin staining relative to viable myocardium or vesselsin the remote region.

Then we verified the ability of 68Ga-NOTA-RGDmicroPET/CT imaging in in vivo evaluation of ischaemia-induced angiogenesis. The imaging findings were congruentwith increased density of neovascularization in infarcted andperi-infarct myocardium at histological examination.

In conclusion, non-invasive PET imaging with this new68Ga-NOTA-RGD tracer appears to be a promising clinicaltool for improving risk stratification and monitoring thetemporal evolution of the cardiac healing and the effects ofpro-angiogenic therapies in ischaemic heart disease.As compared to similar imaging approaches using the18F-galacto-derivative, the very simple chemistry of ourapproach might greatly facilitate its translation to clinicalapplications.

Acknowledgments This work was supported by the ConsiglioNazionale delle Ricerche, Italy (grant CNR-DG.RSTL.035.007-035),Scuola Superiore Sant’Anna, Italy (grant PNAZ.M6010AL).

Conflicts of interest None.

References

1. Banai S, Jaklitsch MT, Shou M, Lazarous DF, Scheinowitz M,Biro S, et al. Angiogenic-induced enhancement of collateral bloodflow to ischemic myocardium by vascular endothelial growthfactor in dogs. Circulation 1994;89:2183–9.

2. Ren G, Dewald O, Frangogiannis NG. Inflammatory mechanismsin myocardial infarction. Curr Drug Targets Inflamm Allergy2003;2(3):242–56.

3. Wu JC, Chen IY, Wang Y, Tseng JR, Chhabra A, Salek M, et al.Molecular imaging of the kinetics of vascular endothelial growthfactor gene expression in ischemic myocardium. Circulation2004;110:685–91.

4. Higuchi T, Bengel FM, Seidl S, Watzlowik P, Kessler H, HegenlohR, et al. Assessment of αvβ3 integrin expression after myocardialinfarction by positron emission tomography. Cardiovasc Res2008;78:395–403.

5. Horton MA. The alpha ν beta 3 integrin “vitronectin receptor”. IntJ Biochem Cell Biol 1997;29:721–5.

6. Arnaout MA, Goodman SL, Xiong JP. Coming to grips withintegrin binding to ligands. Curr Opin Cell Biol 2002;14:641–51.

7. Auzzas L, Zanardi F, Battistini L, Burreddu P, Carta P, Rassu G, etal. Targeting alphavbeta3 integrin: design and applications ofmono- and multifunctional RGD-based peptides and semipeptides.Curr Med Chem 2010;17(13):1255–99.

8. Schottelius M, Laufer B, Kessler H, Wester H-J. Ligands formapping αvβ3-integrin expression in vivo. Acc Chem Res2009;42(7):969–80.

9. Liu Z, Niu G, Shi J, Liu S, Wang F, Liu S, et al. (68)Ga-labeledcyclic RGD dimers with Gly3 and PEG4 linkers: promising agentsfor tumor integrin alphavbeta3 PET imaging. Eur J Nucl Med MolImaging 2009;36:947–57.

10. Beer AJ, Kessler H, Wester H-J, Schwaiger M. PET imaging ofintegrin alphaνbeta3 expression. Theranostics 2011;1:48–57.

11. Gaertner FC, Kessler H, Wester H-J, Schwaiger M, Beer AJ.Radiolabelled RGD peptides for imaging and therapy. Eur J NuclMed Mol Imaging 2012;39 Suppl 1:S126–38.

12. Belvisi L, Bernardi A, Checchia A, Manzoni L, Potenza D,Scolastico C, et al. Potent integrin antagonist from a small libraryof RGD-including cyclic pseudopeptides. Org Lett 2001;3:1001–4.

13. Belvisi L, Riccioni T, Marcellini M, Vesci L, Chiarucci I, Efrati D,et al. Biological and molecular properties of a new alpha(v)beta3/alpha(v)beta5 integrin antagonist. Mol Cancer Ther 2005;4:1670–80.

14. Arosio D, Manzoni L, Araldi EMV, Caprini A, Monferini E,Scolastico C. Functionalized cyclic RGD peptidomimetics:conjugable ligands for ανβ3 receptor imaging. Bioconjug Chem2009;20:1611–7.

15. Manzoni L, Belvisi L, Arosio D, Bartolomeo MP, Bianchi A,Brioschi C, et al. Synthesis of Gd and (68)Ga complexes inconjugation with a conformationally optimized RGD sequence aspotential MRI and PET tumor-imaging probes. ChemMedChem2012;7(6):1084–93.

16. Lanzardo S, Conti L, Brioschi C, Bartolomeo MP, Arosio D,Belvisi L, et al. A new optical imaging probe targeting ανβ3integrin in glioblastoma xenografts. Contrast Media Mol Imaging2011;6:449–58.

Eur J Nucl Med Mol Imaging

17. Pilkington-Miksa M, Arosio D, Battistini L, Belvisi L, De MatteoM, Vasile F, et al. Design, synthesis, and biological evaluation ofnovel cRGD-paclitaxel conjugates for integrin-assisted drug deliv-ery. Bioconjug Chem 2012;23(8):1610–22.

18. Jeong JM, Hong MK, Chang YS, Lee YS, Kim YJ, Cheon GJ, et al.Preparation of a promising angiogenesis PET imaging agent: 68Ga-labeled c(RGDyK)-isothiocyanatobenzyl-1,4,7-triazacyclononane-1,4,7-triacetic acid and feasibility studies in mice. J Nucl Med2008;49(5):830–6.

19. Haukkala J, Laitinen I, Luoto P, Iveson P, Wilson I, Karlsen H, etal. 68Ga-DOTA-RGD peptide: biodistribution and binding intoatherosclerotic plaques in mice. Eur J Nucl Med Mol Imaging2009;36(12):2058–67.

20. Li X, Samnick S, Lapa C, Israel I, Buck AK, Kreissl MC, et al.68Ga-DOTATATE PET/CT for detection of inflammation of largearteries: correlation with 18F-FDG, calcium burden and riskfactors. EJNMMI Res 2012;2:52.

21. Tarkia M, Saraste A, Saanijoki T, Oikonen V, Vähäsilta T,Strandberg M, et al. Evaluation of 68Ga-labeled tracers for PETimaging of myocardial perfusion in pigs. Nucl Med Biol2012;39:715–23.

22. Ocak M, Antretter M, Knopp R, Kunkel F, Petrik M, Bergisadi N,et al. Full automation of (68)Ga labelling of DOTA-peptides in-cluding cation exchange prepurification. Appl Radiat Isot2010;68(2):297–302.

23. Teitelbaum SL. Osteoclasts, integrins, and osteoporosis. J BoneMiner Metab 2000;18:344–9.

24. Del Guerra A, Bartoli A, Belcari N, Herbert D, Motta A, Vaiano A,et al. Performance evaluation of the fully engineered YAP-(S)PETscanner for small animal imaging. IEEE Trans Nucl Sci2006;53(3):1078–83.

25. Panetta D, Belcari N, Del Guerra A, Bartolomei A, Salvadori PA.Analysis of image sharpness reproducibility on a novel engineeredmicro-CT with variable geometry and embedded recalibrationsoftware. Phys Med 2012;28:166–73.

26. Moehrs S, Defrise M, Belcari N, Del Guerra A, Bartoli A, FabbriS, et al. Multi-ray-based system matrix generation for 3D PETreconstruction. Phys Med Biol 2008;53:6925–45.

27. Loening AM, Gambhir SS. AMIDE: a free software tool formultimodality medical image analysis.Mol Imaging 2003;2(3):131–7.

28. Manzoni L, Belvisi L, Arosio D, Civera M, Pilkington-Miksa M,Potenza D, et al. Cyclic RGD-including functionalizedazabicycloalkane amino acids as potent integrin antagonists fortumor targeting. ChemMedChem 2009;4:615–32.

29. Al-Nahhas A, Win Z, Szyszko T, Singh A, Nanni C, Fanti S, et al.Gallium-68 PET: a new frontier in receptor cancer imaging.Anticancer Res 2007;27:4087–94.

30. Breeman WA, de Blois E, Sze Chan H, Konijnenberg M,Kwekkeboom DJ, Krenning EP. (68)Ga-labeled DOTA-peptidesand (68)Ga-labeled radiopharmaceuticals for positron emissiontomography: current status of research, clinical applications, andfuture perspectives. Semin Nucl Med 2011;41:314–21.

31. Meoli DF, Sadeghi MM, Krassilnikova S, Bourke BN, GiordanoFJ, DioneDP, et al. Noninvasive imaging ofmyocardial angiogenesisfollowing experimental myocardial infarction. J Clin Invest2004;113:1684–91.

32. Kalinowski L, Dobrucki LW, Meoli DF, Dione DP, Sadeghi MM,Madri JA, et al. Targeted imaging of hypoxia-induced integrinactivation in myocardium early after infarction. J Appl Physiol2008;104:1504–12.

33. Dobrucki LW, Meoli DF, Hu J, Sadeghi MM, Sinusas AJ. Regionalhypoxia correlates with the uptake of a radiolabeled targeted markerof angiogenesis in rat model of myocardial hypertrophy and ischemicinjury. J Physiol Pharmacol 2009;60 Suppl 4:117–23.

34. Sherif HM, Saraste A, Nekolla SG, Weidl E, Reder S, Tapfer A, etal. Molecular imaging of early αvβ3 integrin expression predictslong-term left-ventricle remodeling after myocardial infarction inrats. J Nucl Med 2012;53(2):318–23.

35. Gao H, Lang L, Guo N, Cao F, Quan Q, Hu S, et al. PET imagingof angiogenesis after myocardial infarction/reperfusion using aone-step labeled integrin-targeted tracer 18F-AIF-NOTA-PRGDD2. Eur J Nucl Med Mol Imaging 2012;39:683–92.

36. Makowski MR, Ebersberger U, Nekolla S, Schwaiger M. In vivomolecular imaging of angiogenesis, targeting αvβ3 integrinexpression, in a patient after acute myocardial infarction. EurHeart J 2008;29(18):2201.

Eur J Nucl Med Mol Imaging