* Corresponding author: Amir R. Jalilian, Radiation Application Research School, Nuclear Science and Technology Research Institute (NSTRI), North Kargar Street, Tehran, Iran. Tel: 098 21 88221103; Fax: 098 21 88221105; Email: ajalili@aeoi.org.ir © 2014 mums.ac.ir All rights reserved. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.  

   

Developmentof111In‐labeledporphyrinsforSPECTimagingShaghayegh Sadeghi, Mohammad Mirzaei, Mohammad Rahimi, Amir R.Jalilian*RadiationApplicationResearchSchool,NuclearScienceandTechnologyResearchInstitute(NSTRI),IranARTICLEINFO ABSTRACT

Articletype:OriginalArticle

Objective(s): The aim of this research was the development of 111In‐labeledporphyrinsaspossibleradiopharmaceuticalsfortheimagingoftumors.Methods: Ligands, 5, 10, 15, 20‐tetrakis (3, 5‐dihydroxyphenyl) porphyrin)(TDHPP),5,10,15,20‐tetrakis(4‐hydroxyphenyl)porphyrin(THPP)and5,10,15,20‐tetrakis(3,4‐dimethoxyphenyl)porphyrin)(TDMPP)werelabeledwith111InCl3(producedfromprotonbombardmentofnatCdtarget)in60minat80ºC.Qualitycontrol of labeled compounds was performed via RTLC and HPLC followed bystabilitystudiesinfinalformulationandpresenceofhumanserumat37ºCfor48h as well as partition coefficient determination. The biodistribution studiesperformedusingtissuedissectionandSPECTimagingupto24h.Results:Thecomplexeswerepreparedwithmorethan99%radiochemicalpurity(HPLCandRTLC)andhighstabilityto48h.Partitioncoefficients(calculatedaslogP) for 111In‐TDHPP, 111In‐THPP and 111In‐TDMPP were 0.88, 0.8 and 1.63respectively.Conclusion: Due to urinary excretion with fast clearance for 111In‐TDMPP, thiscomplex is probably a suitable candidate for considering as a possible tumorimagingagent.

Articlehistory:Received:20Feb2014Revised:27Mar2014Accepted:1Apr2014

Keywords:Hydroxyphenylporphyrins111InBiodistributionRadiolabelingSPECT

►Pleasecitethispaperas:Sadeghi Sh, Mirzaei M, Rahimi M, Jalilian AR. Development of 111In‐labeled porphyrins for SPECT imaging. AsiaOceaniaJNuclMedBiol.2014;2(2):95‐103.

IntroductionPorphyrins can be appropriate ligands for

designing metal complex including radio‐pharmaceuticals because porphyrins and theirrelated compounds are used as tumor seekingdrugs, especially as photosensitizers in thephotodynamic therapy (PDT) of cancer (1, 2)where the combination of light andphotosensitizer,generatesactiveoxygenspeciesnear the tumor, to damage the malfunctionedtissues. Varieties of porphyrins includinganionic porphyrins (3), hematoporphyrins (4),cationic porphyrins (5) and phethalocyanines(6) have been successfully used for tumortreatment.

Porphyrins taken into thebloodcirculationas tumor localizing agents are transported totarget tissue by human serum albumin (HSA)andotherplasmaproteinssuchaslowandhigh‐

density lipoproteins (7). Other investigatorshave suggested that porphyrin accumulation inthe tumor is related to low‐density lipoprotein(LDL) uptake that occurs through receptor‐mediatedendocytosis(8).

Mesotetrakis(4‐hydroxyphenyl)porphyrin(THPP)hasbeenusedasapotentialmoleculeinPDT with high photosensitivity for cancertreatment, leading to the destruction ofintrahepatic tumors with better efficacy andfewersideeffects(9).

Several investigators have reported thesynthesisandradiolabelingofwidevarietiesofporphyrin derivatives with various types ofperipheral moieties with several importantmedical radionuclides for developing an idealtumor localizing agent (10‐15). However, noneof these radiolabeled porphyrins have

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Figure1.Chemicalstructuresforligandsusedinthisworksucceededasapopularregularproduct. In‐111is a cyclotron produced radionuclide, decayingby electron capture (EC) with subsequentemissionofgammaphotonsof173and247keV(89%and94%intensity,respectively),iswidelyused ingammascintigraphy. It is reported that111In‐labeled porphyrins exhibit specificaccumulationintumortissues(16,17).

Our attempt was to prepare a series ofwater soluble suitable radiolabeled porphyrinsfortumordiagnosis,duetotheavailabilityofIn‐111.StructuresofligandsusedinthisstudyareshowninFigure1.

In this research, synthesis, radiolabeling,partition coefficient, quality control and bio‐distribution studies using SPECT andscarification of 111In‐TDHPP, 111In‐THPP and111In‐TDMPPinwild‐typeratsarereported.

Methods

In‐111 was produced at the Agricultural,Medical and Industrial Research School(AMIRS), 30 MeV cyclotron (Cyclone‐30, IBA)using natCd(p,x)111In reaction. Natural cadmiumsulfate with a purity of more than 99% wasobtainedfromMerckCo.Germany.Allchemicalswere purchased from Sigma‐Aldrich ChemicalCo. U.K. Radio‐chromatographywas performedby Whatman paper using a thin layerchromatography scanner, Bioscan AR2000,Paris,France.AnalyticalHPLCtodeterminethespecific activity was performed by a ShimadzuLC‐10AT, armed with two detector systems,flowscintillationanalyzer(Packard‐150TR)andUV‐visible (Shimadzu) using WhatmanPartisphere C‐18 column 250 × 4.6 mm(WhatmanCo.NJ,USA).Calculationswerebasedonthe172keVpeakforIn‐111.Allvalueswere

expressedasmean standarddeviation (Mean SD) and the data were compared usingstudentT‐test. Animalstudieswereperformedin accordance with the United KingdomBiological Council's Guidelines on the Use ofLiving Animals in Scientific Investigations,secondedition.

ElectroplatingofthenaturalCdtargets:

In order to prepare Cd targets for theproduction, cadmium electroplating wasperformedoveracoppersurfacewasperformedaccording to the previously reported method(18). Cadmium was electroplated over thecopperbackingaccordingtothemethodgiveninthe literature (19).AmixtureofCdSO4.8/3H2O,KCN, BrijTM detergent solution and traces ofhydrazinehydratewithafinalvolumeof450mldouble‐distilled water (DDH2O) at pH=13 wasused as the electroplating bath (constantcurrent:320mA,stirringrate780rpm,time0.5h). After the deposition of an about 500 mgcadmium layer, the targets were wrapped inParafilmcoatingstoavoidatmosphericoxygenexposure. Finally, the target was sent forirradiation.Production and quality control of 111InCl3solution

Indium‐111 chloride was prepared by 22MeV proton bombardment of the cadmiumtarget at a 30MeV cyclotron,with a current of100μAfor48min(80μAh).Afterdissolutionofthe irradiated target by conc.HBr, the solutionwas passed through a cation exchange dowex508 resin, pre‐conditioned by 25 ml of conc.HBr. The resin was then washed by HBr conc.solution (50 ml). In order to remove theundesiredimpuritiesofCdandCu,theresinwastotally washed with DDH2O. Indium‐111 waseluted with 1 N HCl (25 ml) as 111InCl3 forlabelinguse.

Qualitycontroloftheproduct

Control of Radionuclide purity: Gammaspectroscopyofthefinalsamplewascarriedoutcounting in an HPGe detector coupled to aCanberra multi‐channel analyzer for 1000seconds.

Chemical purity control: This step wascarried out to ensure that the amounts ofcadmium and copper ions resulting from thetargetmaterialandbackinginthefinalproductare acceptable regarding internationallyacceptedlimits.Chemicalpuritywascheckedbydifferential‐pulsed anodic stripping polaro‐

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graphy. The detection limit of our system was0.1ppmforbothcadmiumandcopperions.

Preparation and quality control ofradiolabeledporphyrins

Complexionof In‐111withporphyrinswascarried out by using acidic solution of 111InCl3,acetate buffer and porphyrins in absoluteethanol.Thereactionwasperformedbyaddingacidic solution (100 µl) of 111InCl3 (185MBq, 5mCi) to a 5 mL‐borosilicate vial. The solutionwas heated under a flowof nitrogen till itwasdried at 50ºC. A volume (100 µl) of porphyrindissolved in absolute ethanol (20 mg/ml, 290‐300 nmol)was transferred to the vial. The pHwas adjusted to 5.5‐7 by adding 2000 µl ofacetatebuffer.Resultingsolutionwasstirredat25 ⁰C for 30–60 min at pH=5. Radiochemicalpurity of the solution was measured by RTLCandHPLC.Radiothinlayerchromatographywasdone with 10% ammonium acetate: methanol(1:1) mixture as mobile phase andchromatography Whatman No. 2 paper asstationary phase. For high performance liquidchromatography, a mixture of acetonitrile:water (40:60), used as elution and reversedphase columnWhatman Partisphere C18 4.6 ×250 mm, used as stationary phase. HPLC wasperformed with a flow rate of 1 ml/min,pressure: 130 kgF/cm2 for 20 min. Thereafter,the solution was filtered through a 0.22 µmfilter.

Determinationofpartitioncoefficient

For calculation of partition coefficient (logp), P (ratio of specific activities of organic andaqueousphases)wasdetermined.37MBqofthe

radiolabeled indium complex was added to amixtureof1mlof1‐octanoland1mlofisotonicacetate buffered saline (pH=7) and then thesolutionwasstirredat37ºCandkeptfor5min.Afterthatthesolutionwascentrifugedat1200gfor5min.Finally500µloftheoctanolphaseandaqueous phaseswere sampled and the specificactivityinbothphaseswascalculated(20).

Stabilitytests

Forevaluationofstabilitytest,ITLCmethodwas done. ITLC was carried out for 2 samplesand then compared to each otherwhich one is37MBq of 111In‐porphyrin complexes thatwaskept at room temperature for 2 days andanother isamixtureof500µl freshlycollectedhumanserumand36.1MBq(976µCi)of 111In‐porphyrin complexes thatwas incubated at 37⁰Cfor48h.

Biodistributionof labeled compound inwild‐typerats

Biodistribution of 111In‐porphyrincomplexesamongtissuesofwild‐typeratsweredetermined. Animals were sacrificed by CO2asphyxiationafterinjection(2,4and24h).Dosecalibratorwithfixedgeometrycountedthetotalamountofradioactivitywhichinjectedintoeachrats. The final activity of theradiopharmaceutical for injection was 50 µCi.After that the tissues (blood, heart, lung, brain,intestine, faces, skin, stomach, kidneys, spleen,bone, liver and muscle) were weighed andwashed with normal saline. For calculation ofpercentageofinjecteddosepergramoftissues,HPGe detector armed with a sample holderdevice used to determine specific activity of

Figure2. ITLCchromatogramsof 111InCl3 solution inDTPAsolution(pH=5) (left)and10%ammoniumacetate :methanol (1:1)solution(right)usingWhatmanno.2paper

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Figure3.RTLCchromatogramsof111In‐porphyrinsusing10% ammonium acetate:methanol (1:1) solution (right) onWhatmanno.2

percentageofinjecteddosepergramoftissues,HPGe detector armed with a sample holderdevice used to determine specific activity oftissues.Imagingof111In‐porphyrincomplexesinwild‐typerats

Imageswere taken2, 4 and24hours afteradministrationof the radiopharmaceutical by adual‐head SPECT system. The mouse‐to‐highenergy septadistancewas12 cm. Imagesweretaken from both normal and tumor bearingmice. The useful field of view (UFOV)was 540mm×400mm.ResultsandDiscussionRadionuclideproduction

Indium‐111, in formof InCl3,waspreparedby22MeVprotonbombardmentoftheenrichedCd‐112 target atCyclone‐30ona regularbasis.Radionuclidic control showed the presence of172 and 245 keV gamma energies, alloriginating from 111In and showed aradionuclidic purity higher than 99% (E.O.S.).The concentrations of cadmium (from targetmaterial) and copper (from target support)were determined using polarography andshowntobebelowtheinternationallyacceptedlevels,i.e.0.1ppmforCdandCu(21,22).

The radioisotope was dissolved in acidicmedia as a starting sample and was furtherdilutedandevaporatedforobtainingthedesiredpHandvolumefollowedbysterilefiltering. TheradiochemicalpurityoftheIn‐111solutionwascheckedintwosolventsystems,in1mMDTPA,free In3+ cation is converted tomore lipophilicIn‐DTPA form and migrates to higher Rf=0.8while any small radioactive fraction remainingat the origin could be related to other Indium‐111ionicspecies,notformingIn‐DTPAcomplex,such as InCl4‐, etc. and/or colloids (notobserved).

On the other hand, 10 % ammoniumacetate:methanol mixture was also used todetermine the radiochemical purity. The fasteluting species was possibly the ionic In‐111cations other than In3+ (not observed) and theremaining fraction at Rf=0 was a possiblemixtureofIn3+and/orcolloids(Figure2).

The radiolabeling process was checked byITLC using 10 % ammonium acetate:methanol(1:1)solutionasshowninFigure3. Inallcasesradiolabeled porphyrins migrate to higher Rfdue to higher lipophilicity compared to freeindium cation. Among the radiolabeledporphyrins In‐TDMPP demonstrated higher Rf

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Figure4.HPLCchromatogramsof 111In‐THPP(sectionA), 111In‐TDHPP(sectionB), 111In‐TDMPP(sectionC)ona reversedphasecolumnusingacetonitrile:water40:60,(inallsections;upper:UVchromatogram,scintillationchromatogram;below)

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Figure 5. Biodistribution of 111InCl3 (1.85 MBq, 50 Ci) in wild‐type rats 2, 4 and 24 h after injection via tail vein (ID/g%:percentageofinjecteddosepergramoftissue)(n=5)

Figure6.Biodistributionof111In‐THPP(50µCi)inwildtyperats2,4and24hafterivinjectionviatailvein(ID/g%:percentageofinjecteddosepergramoftissuecalculatedbasedontheareaundercurveof245keVpeakingammaspectrum)(n=3)duetomorelipophilicitycomparedtotwootherphenolicderivatives.Dihydroxycomplex (111In‐TDHPP) showedmorehydrophilicity comparedto the mono hydroxyl compound. ITLC studiesapprovedtheproductionofasingleradiolabeledcompoundineachcase.

HPLC studies also demonstrated theexistenceofonlyoneradiolabeledspeciesusingbothUVandscintillationdetectors. Inallcasesamore fast‐eluting compound observed withscintillation detector was almost time‐coincident with a related peak of UV detector,demonstrating a more lipophilic compoundcomparedto111Incation(Figure4).

Stabilitystudies

Studies on 111In‐porphyrins showed thatstability of the labeled compounds was highenough to carry out further studies. ITLCshowed no loss of In‐111 from labeled

compound after incubation of 111In‐porphyrinsin freshlypreparedhumanserum for2daysat37 ⁰C. At physiologic conditions theradiochemical purity of labeled compoundremainedmorethan99%for2days.

Biodistributionstudies

Freeindiumcation:Forbettercomparisonbiodistribution study was performed for freeIn3+.The%ID/gdataaresummarized inFigure5.

Indium cation similarity with ferric ion isimportant in the development of indiumradiopharmaceuticals, since iron is an essentialelement in the human body and a number ofiron binding proteins, such as transferrin (inblood),which used in transporting and storingironinvivo(23).

AsshowninFigure5, indiumcationalmostmimicstheferriccationbehaviorandisrapidly

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Figure7.Biodistributionof111In‐TDMPP(50µCi)inwildtyperats2,4and24hafterivinjectionviatailvein(ID/g%:percentageofinjecteddosepergramoftissuecalculatedbasedontheareaundercurveof245keVpeakingammaspectrum)(n=3)

Figure8.Biodistributionof111In‐TDHPP(50µCi)inwildtyperats2,4and24hafterivinjectionviatailvein(ID/g%:percentageofinjecteddosepergramoftissuecalculatedbasedontheareaundercurveof245keVpeakingammaspectrum)(n=3)removed from the circulation and isaccumulatedintheliver,alsoamajorfractionisexcreted rapidly through the kidney leading tohigh accumulation in urine in 2‐24 h postinjectionasawatersolublecation.

Radiolabeledporphyrins

111In‐THPP: Biodistribution data of 111In‐THPP(Figure6),2hafter injection thehighestactivitywas seen in and blood pool, while thisamountisreducedduetoothertissue’suptake.Due to the presence of polar groups in thestructure of the compound, a major portion ofthe radioactive compoundwas concentrated inkidneys,2‐24hafterinjection.Althoughliverisanother accumulation site due to lipoproteintransport into this organ leading to significantfecesactivitycontent.

111In‐TDMPP: despite the presence of twoCH3O‐ groups in TMPP complex and lowwatersolubilityleadingtohigherlogP,kidneysarethemajoraccumulationsitesofexcretionwhileliverstayedaminorexcretionroute.Lungandspleenalsoshowsignificantactivity(Figure7).

111In‐TDHPP: Similar to 111In‐THPP, di‐hydroxycompoundisalsoaccumulatedmajorlyin the liver and kidneys which are typicalaccumulation sites for porphyrins. Due tooxidative/reductiveenzymespresent in lungsasignificantuptakeisalsoobserved(Figure8).

Due to the importance of low liveraccumulation compared to the kidneysexcretion, a kidney: liver uptake ratio can besuggested as a suitable criterion for rapidclearance of the radiolabeled complexes astumor imaging agents, while imposing less

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Table1. The kidney: liver uptake ratio of the radiolabeledcomplexesin2,4and24hpostinjection(n=3)Time/complex 111In‐TDHPP 111In‐THPP 111In‐TDMPP

2h 1.46 1.27 1.744h 2.08 1.37 3.0824h 1.65 2.47 4.13

irradiationdoseasshowninTable1.As shown in Table 1, TDMPP complex

demonstratesthebestkidney:liveruptakeratioamongthethreecomplexesatalltimeintervalsdespitethehighlipophilicbehaviorobservedinpartition coefficient and chromatographicstudies.

Itcanbeproposedthatametabolicpathwayleading to the formationofmorewater solublemetabolitesexistsfordi‐methoxycomplex.Thus111In‐TDMPP complex is probably a suitablecandidate for considering as a possible tumorimagingagent.Imagingofwild‐typerats

TheIn‐111labeledporphyrinimaginginthewild‐typeratsshowedadistinctaccumulationofthe radiotracer in the chest region all the timeafterinjection.Mostoftheactivityiswashedoutfrom the body after 24 h and the picturecontrastweakened. Figure 9, demonstrates theliver and spleen uptake of 111In‐THPP complexamong the rat tissues at all time intervals,however a minor kidney uptake is alsoobserved. These findings are in full agreementwiththedissectionstudiesshowninFigure6.

Figure9.SPECTimagesof111In‐THPP(70Ci)inwild‐typerats2,4and24hpostinjection

Figure10.SPECT imagesof 111In‐TDMPP (70Ci) inwild‐typerats2,4and24hpostinjection

Figure11. SPECT images of 111In‐TDHPP (70Ci) inwild‐typerats2,4and24hpostinjection

In case of 111In‐TDMPP, kidneys are thesignificantexcretingorgansalthough lung, liverand spleen uptakes are observed in a singlesignal (Figure 10). These findings are in fullagreementwiththedissectionstudiesshowninFigure7.

Figure 11, also demonstrates the liver andspleen uptake of 111In‐TDHPP complex amongtherattissuesatalltimeintervals.Asignificantkidney uptake is also observed. These findingsare in agreement with the dissection studiesshowninFigure8.

All porphyrins are transferred in the bodyasbondedtothelipoproteins,thusanypossibletargetsoflipoproteinsaretheaccumulationsite,and among many other porphyrins this is themajordrawback.Conclusion

111In‐TDHPP, 111In‐THPP and 111In‐TDMPPwere prepared using 111InCl3 (produced fromprotonbombardmentof natCdtarget) in60minat 80 ºC. The complexes were prepared withmorethan99%radiochemicalpurity(HPLCandRTLC) and high stability to 48h. Partitioncoefficients (calculated as log P) for 111In‐TDHPP,111In‐THPPand111In‐TDMPPwere0.88,0.8 and 1.63 respectively. Biodistributionstudies using SPECT imaging and tissuedissection demonstrated significant urinaryexcretion with fast clearance for 111In‐TDMPP,while the two other complexes demonstratedliver uptake and longer retention in animaltissues.Thus111In‐TDMPPcomplexisprobablyasuitablecandidate forconsideringasapossibletumorimagingagent.

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