[DTPA°)CCK@;1111n;tumor scintigraphy; radionuclide therapy;medullarythyroidcancers

J NuclMed 1999;40:2081—2087

diolabeled tumor receptor-binding peptides can beused for in vivo scintigraphic imaging. The peptides mostwidely used now are stable somatostatin analogs that bind totheir receptors on tumors of neuroendocrine origin (1). Anexample is the octapeptide [@In-DTPA°]octreotide, consisting of octreotide and the chelator diethylenetriamine pentaacetic acid (DTPA), enabling instant radiolabeling with aradiometal such as WIn. We have described its use forscintigraphic imaging of somatostatin receptor-positive lesions, such as gastroenteropancreatic neuroendocrine tumors, neuroblastoma, pheochromocytoma, breast cancer,Hodgkin's lymphoma and small cell lung cancer (2,3).However, unlike in other neuroendocrine tumors, somatostatin receptor expression is rather low in medullary thyroidcancer (MTC) and is completely absent in clinically aggressive forms of the disease (4,5). Recently, the presence ofcholecystokinin (CCK)-B (gastrin) receptors was shown inmore than 90% of MTCs, and the presence of these receptorswas shown in a high percentage of small cell lung cancers,stromal ovarium cancers, astrocytomas and several othertumor types (6). On the basis ofthese findings, Behr et al. (7)evaluated the suitability of radioiodinated gastrin, a specificligand for the CCK-B receptor, for targeting CCK-B receptorexpressing tumors in vivo. Their data suggest that gastrinand its analogs may represent a useful new class ofreceptor-binding peptides for diagnosis and therapy of avariety of tumor types, including MTC. Furthermore, Reubiet at. (8) developed DTPA-conjugated CCK-B receptorbinding CCK analogs, evaluated their receptor-bindingcharacteristics and obtained initial preclinical biodistribution data in nontumor-bearing rats. For the tetraazacyclododecanetetraacetic acid (DOTA) counterpart of the most

The presence ofcholecystokinin (CCK)-B(gastrin) receptors hasbeen shown in more than 90% of medullary thyroid cancers(MTC5) and in a high percentage of small cell lung cancers,stromal ovanum cancers and several other tumor types. Methods: The aim of this study was to evaluate in vitro and in vivowhether 111ln-labeled CCK-B receptor-specific CCK@analog[D-Asp@,Nle28'31]CCK@.@(D-Asp-Tyr-NIe-Gly-Trp-Nle-Asp-PheNH2) is suitable for CCK-B receptor scintigraphy based on thefinding that unlabeled nonsulfated diethylenetriamine pentaacidic acid [DTPA°]CCK@and tetraazacyclododecanetetraacetic acid [DOTA°]CCK@analogs show high and specific bindingfor CCK-B receptors in human tumors. Fifty percent inhibitoryconcentrationswerein the lownanomolarrange.Results:Invitro, [111ln-DOTA°]CCK@showed specific internalization in CCK-Breceptor-positiverat pancreatictumorcellsAR42J. Intemalization of the analog appeared to be time and temperature dependent and receptorspecific.Fromthe data obtainedwith[‘111n-DOTA°]CCK@and12511-gastnn,thelatterbeingaspecificligandforthe CCK-B receptor, the rat pancreatic cell line CA20948 alsoappearedtobeCCK-Breceptorpositive.Thisprovidesaninvitroand in vivo rat tumor model because this cell line can be grown tosolidtumorsinLewisrats.InvivobiodistributionexperimentsinCA20948 tumor-bearing Lewis rats showed rapid clearance of

[111ln-DOTA°]CCK@,andspecificuptakewasfoundintheCCK-Breceptor-expressingstomachandtumor.Furthermore,comparing [111In-DOTA°]CCK@with the radioiodinated nonsulfated CCK10analog(D-Tyr-GIy-Asp-Tyr-Nle-Gly-Trp-Nle-Asp-Phe-NH2),bothligandshavinghighaffinityforthe CCK-Breceptor,tumor-toblood ratioswere significantlyhigher for [111In-DOTA°]CCK@thanfor 1@I-CCK10,analogousto the findings with radioiodinatedand111In-labeledoctreotide. The study in humans with [111InDTPA°]CCK@showed receptor-specific uptake in the CCK-Breceptor-positivestomachandin metastasesinthe neckregionup to 48 h after injection. Conclusion: [111ln-DOTA°]CCK@ismostpromisingfor scintigraphyand,aftercouplingto therapeuticradionuclides,for radionuclidetherapyof humanCCK-Breceptorpositive tumors such as MTC and small cell lung cancer.

Key Words: cholecystokenin receptors; [DOTA°]CCK@

ReceivedDec.14,1998;revisionacceptedApr.9, 1999.Forcorrespondenceorreprintscontact:MariondeJong,PhD,Departmentof

NuclearMedicine,V220,UniversityHospitalRotterdam,3015GDRotterdam,The Netherlands.

EVALUATIONOF RADIOLABELEDCCK Ar@ioos •de Jong et al. 2081

Preclinical and Initial Clinical Evaluation of1111n-.LabeledNonsulfated CCK8 Analog:A Peptide for CCK-B Receptor-TargetedScintigraphy and Radionuclide TherapyMarion de Jong, Willem H. Bakker, Bert F. Bernard, Roelf Valkema, Dik J. Kwekkeboom, Jean-Claude Reubi,Ananth Srinivasan, Michelle Schmidt and Eric P. Krenning

Department ofNuclear Medicine, University Hospital Dijkzigt, Rotterdam, The Netherlands; Department of Pathology,University ofBerne, Berne, Switzerland; Mallinckrodt, Inc., St. Louis, Missouri

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promising analog, a high CCK-B receptor affinity wasfound. They concluded that CCK analogs are promising forhuman CCK-B receptor scintigraphy as well (8).

A new application is the use of radiolabeled peptides forradionuclide therapy. Promising results in tumor growthinhibition using [@In-DTPA°]octreotide have been reportedin humans (9). Besides Auger electron emitters, such aslllIn, 13- particle emitters, such as 90Y, may appear suitable

for this purpose. The 90Y-DTPA complex is unstable, resulting in hematopoietic toxicity in vivo. Therefore, the DOTAchelator, which forms stable complexes with 90Y and 111In,was coupled to CCK8, enabling future radionucide therapyof MTC.

The success of the therapeutic strategy relies on theamount of radioligand that can be concentrated within tumorcells and thus the rates of internalization, degradation andrecycling of both ligand and receptor. Binding of severalpeptide hormones to specific surface receptors is generallyfollowed by internalization of the ligand-receptor complex.Williams et al. (10) and Svoboda et al. (11) have reportedinternalization of unchelated CCK. The internalization process of this peptide appears to be CCK receptor specific andtemperature dependent. One aim of this study was toevaluate internalization of [I @In-DOTA°]CCK@in rat pancreatic tumor cells in vitro.

Biodistribution and tumor visualization also were investi

gated in vivo in tumor-bearing animals. Biodistribution of[WIn@DOTA0JCCKg was compared with that of ‘251-CCK10analog (8), analogous to our studies using radioiodinatedand WIn-labeled octreotide, to compare the differences inbiodistribution and cellular retention of the peptides radiolabeled with either a residualizing radiolabel like @Inor witha nonresidualizing radiolabel like 125!.We performed toxicity studies with unlabeled [DOTA°]CCK8and [DTPA°]CCK8in rats and mice, and, thereafter, an initial evaluation of[@In-DTPA°]CCK8 in humans was performed.

MATERIALS AND METHODS

Compounds‘251-gastrin(74 X 1012Bq/mmol) was obtained from Amersham

Pharmacia Biotech (Buckinghamshire, UK). Synthetic gastrmnwasfrom Bachem (Bubendorf, Switzerland). This is a heptadecapeptide, specific for the CCK-B/gastrin receptor with an affinityconstant in the subnanomolar range, whereas its affinity for theCCK-A receptoris lower by more than four ordersof magnitude.Mallinckrodt, Inc. (Petten, The Netherlands) provided ‘@1nCl3.DOTAand DTPAanalogs of CCK8(D-Asp-T@rr-Nle-Gly-Trp-NleAsp-Phe-NH2) were synthesized according to previously describedmethods (8). @I-CCK10(D-T@r-Gly-Mp-1'@,r-Nle-Gly-Trp-Nle-AspPhe-NH2) was synthesized by Chiron (8). “In-labelingof theDTPA and DOTA analogs was as described for [DTPA°]octreotide(2) and [DOTA°,1@yr@1octreotide(12), respectively. ‘@I-labelingof

@I-CCK10was performedas describedfor [Tyr@]octreotide(13).

InternalizationAR42J cells were grown in RPMI-1640 medium (Gibco, Grand

Island, NY), CA20948 cells were grown in Dulbecco's modified

Eagle's medium (DMEM) (Gibco) and ARO cells were grown inDMEM/Fl2 medium (Gibco). For all cell lines, medium wassupplemented with 2 mmollL glutamine and 10% fetal calf serum(Gibco). Before the experiment, subconfluent cell cultures weretransferred to six-well plates.

The binding of the radiolabeled peptides to tumor cells andsubsequent internalization were studied as described (14). Cellswere washed and incubation was started by addition of 1 mLinternalization medium per well (culture medium without fetal calfserum but with 1% bovine serum albumin [Sigma, St. Louis, MO])with about 80 kBq of radiotracer. Peptide concentration range inthe different experiments was 0.1—1pmol/L. Cells were incubatedat 37°Cfor indicated periods of time. To determine nonspecificinternalization, cells were incubated with an excess unlabeledpeptide (0.1 pmolIL). Cellular uptake was stopped by removingmedium from the cells and washing with 2 mL ice-cold phosphatebuffered saline. To discriminate between internalized and noninternalized (surface-bound) radiopharmaceutical, intact cells wereincubated with 1 mL 20 mmol/L sodium acetate. The internalizedand noninternalized fractions were determined by measuringradioactivity. The internalized fraction was expressed as percentageof the applied dose per milligram cellular protein. The latter wasdetermined using a commercially available kit (Bio-Rad,Veenendaal, The Netherlands).

Data are expressed as mean ±SD for incubations assayed intriplicate, with each experiment performed at least three times.

In Vivo Tissue DistributionAnimal experiments were performed in compliance with the

regulations of this institution and with generally accepted guidelines governing such work. Male Lewis rats (200—250g), bearingthe CA20948 pancreatic tumor, were used in the experiments. Ratswere injected under ether anesthesia with 3 MBq (0.5 pig)“In-labeledpeptide in 200 pL saline into the dorsal vein of thepenis. To determine nonspecific binding of the radiopharmaceutical, a separate group of rats was coinjected intravenously with 0.1mg [DOTA°]CCK8.At the indicated time points, rats were killedunder ether anesthesia. Organs and blood were collected, and theradioactivity in these samples was determined. Results are cxpressed as mean ±SD of at least six rats per group.

Toxicity Study of Unlabeled [DOTA°]CCK@,and [DTPA°JCCK@,

On experimental day 0, eight treatment groups of five Wistar ratsand five BALB/c mice were injected intravenously with saline (0times), 10 times, 100 times or 1000 times the human concentrationof 15 j.tg/75kg of [DOTA°]CCK8or [DTPA°]CCKS,respectively.All test solutions were injected through the penis vein at a rate notexceeding 1 mL/min. Until 24 h after injection the animals weremonitored for changes in behavior (eating, sleeping, motion,posture) and signs of toxicosis. Any reaction to the treatment wasrecorded. All rats were killed by ether narcosis 24 h after injectionand subjected to detailed macroscopic postmortem examination.After examination of the external surfaces, the chest and abdomenwere opened by midline incision. Thoracic and abdominal viscerawere examined for abnormalities. Aberrations were recorded.Organs were investigated macroscopically (e.g., for bleedings).Liver, kidneys, stomach, spleen, lungs and intestines were isolatedand fixed in 4% buffered formalin. The organs were sectioned,stained and evaluated by light microscopy.

2082 THE Joum@ OF NUCLEARMEDICINE•Vol. 40 •No. 12 •December 1999

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0.1 1 10 100 1000

, ARO

fl!

0.1

10

8

8

4

2

Initial Human Evaluation of [111In-DTPA°]CCK@A 45-y-old woman with local recurrenceof MTC was injected

with 222 MBq tHIn@labeled [DTPA°JCCK8(10 @igpeptide). Scanswere acquired 4, 24 and 48 h after injection as described (2,3).Urine was collected during the first 24 h after injection.

RESULTS

Radlolabeling11‘In-labeling efficiency of the different peptides and

radioiodination efficiency of ‘25I-CCK,0ranged from 95% to100%. Radiochemical purity always exceeded 90%.

In Vitro Internalization StudiesFigure 1A shows the time- and temperature-dependent

internalization ‘‘In-DOTA°]CCK@,in the CCK-B receptorpositive AR42J rat pancreatic tumor cells and in the CCK-Breceptor-negative ARO human anaplastic thyroid tumor cellline. Internalization was dose dependent and was reduced inthe presence of increasing concentrations of unlabeledpeptide, indicating that this process is receptor specific.Furthermore, it was reduced at 6°Cversus 37°C,indicatingtemperature dependence, and increased over time. Theacid-removable (“surface-bound―)uptake was about 5%—10% of the internalized fraction (not shown). The ARO cellswere used as negative controls. In these cells, internalizationof [“1In-DOTA°]CCK8was indeed low and showed nospecific temperature-dependent accumulation. Figure lBshows the inhibitory effect in AR42J cells of excess mediumgastrin concentrations on internalization of 1251-gastrin, aspecific ligand with high affinity for CCK-B receptors,indicating specific internalization in these AR42J cells.

Figure 2A shows the time- and temperature-dependentinternalization of [I I‘In-DOTA°]CCK8in the CA20948 rat

pancreatic tumor cells. Internalization was reduced in thepresence of increasing concentrations of unlabeled peptide.Furthermore, it was reduced at 6°C,indicating temperaturedependence, and increased over time. The acid-removable(surface-bound) uptake was about 5%—10%of the internalized fraction (not shown). Figure 2B shows internalization

of ‘251-gastrinin the presence or absence of excess mediumgastrin concentrations. In the presence of unlabeled gastrin,internalization was significantly reduced, indicating thepresence ofCCK-B receptors on the CA20948 rat pancreatictumor cells.

Tissue Distribution In RatsTable 1 compares the radioactivity in several organs after

injection of [“In-DOTA°}CCK8and [‘@In-DTPA°]CCK8.Arapid clearance from the blood through renal excretion wasfound for both radiolabeled compounds, with a pronounceduptake in the kidneys. Receptor-negative organ radioactivityparalleledblood clearance, resulting in a very low background radioactivity 24 h after injection. Comparison ofbiodistribution data and tumor uptake of[@In-DOTA°]CCK8and [lUIn@DTPAO]CCKgshowed no major differences.

Figure 3 shows that uptake of [‘‘‘In-DOTA°]CCK8instomach and tumor, 4 h after injection, was at least partiallyreceptor specific because uptake was significantly reducedafter coinjection with 0.1 mg unlabeled peptide. Uptake inthe other organs and radioactivity in blood did not changesignificantly after coinjection of unlabeled peptide (notshown).

Figure 4 shows a comparison of tissue radioactivityafter injection of [@In-DOTA°]CCK8 and ‘25I-CCK,0,1 and4 h after injection,in severalrat organs.Clearancefromthe blood was slower for ‘251-CCK10than for [l 1IInDOTA°]CCK8,and uptake in receptor-negative organs, ofwhich the liver is shown as an example, was higher for‘25I-CCK,0than for [@In-DOTA°]CCK8.A higher uptake of1251-CCK,0than of [‘@In-DOTA°]CCK8was also found inthe receptor-positive stomach, which seemed to be favorablefor ‘25I-CCK,0.However, the stomach-to-blood ratio, whichrepresents the target-to-background ratio, was significantlylower for ‘251-CCK10than for [@In-DOTA°]CCK8 at 4 hafter injection.

Figure 5 shows a gamma camera scan, made 20 mm afterinjection, of two tumor-bearing rats and a control rat,

FIGURE 1. (A) Internalizationof [111lnDOTA°]CCK@in CCK-Breceptor-positiveAR42J rat pancreatic tumor cells and inCCK-Breceptor-negativeAROhumananaplastic thyroid tumor cell line. Data areexpressedas percentageadded dose/mgprotein (mean ±SD). (B) InternalizatIon,after 60-mm incubation, of 125I-gastrin,specific ligand with high affinity for CCK-Breceptor, in AR42J cells. Data are expressed as percentage added dose/mg protein(mean±SD).

A 37C 6C B1o@ 10

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AR42JAR42J

0.1 1 10 100 1000 0.1

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EVALUATIONOF RADIOLABELEDCCK A@ioos •de Jong et al. 2083

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15tumour*

DOTA*timeDTPA*@administration

Tsssue lh 4h 24administrationh24h

*flasueradioactivityisexpressedas percentageinjecteddose/gtissue(mean±SD).Foreachgroup,n 4.

DOTA = tetraazacyclododecanetetraaceticacid CCK@analog;DTPA= [111lnjdiethylenetriaminepentaaceticacid CCK@analog.

A 37C 6C B4r.@ 10 C

0) CA209A0 •@53.@ 8 “° .@@ CA20948

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CA20948

•6ominLll2Omin

0.1 1 10 100 1000

FIGURE 2. (A) Internalizationof [111In-DOTA°]CCK@in CCK-B receptor-positiveCA20948 rat pancreatic tumor cells. Data areexpressedas percentageaddeddose/mgprotein(mean±SD). (B) Internalization,after60-mmincubation,of 125l-gastrin,specificligandwithhighaffinityforCCK-Breceptor,inCA20948cells.Dataareexpressedaspercentageaddeddose/mgprotein(mean±SD).

injected with [@In-DOTA°]CCK8 without or with excessunlabeled peptide or with ‘251-CCK10.The radioiodinatedanalog had a different distribution pattern with much higherliver uptakethan did [@‘In-DOTA°]CCK8,whichis clearedthrough the kidneys. Furthermore, the CCK-B receptorpositive tumor was clearly visualized in the left rat but wasnot detected after coinjection with unlabeled peptide, asshown in the middle rat.

Toxicity StudyNo abnormalities were found in animal behavior during

the 24 h after injection. No macroscopic pathology was

TABLE IRadioactivity in Organs and Tumor of CA20948

Tumor-BearingRats 1, 4 and 24 HoursAfterAdministrationof [111ln-DOTA°]CCK@or 24 HoursAfterAdministration of

observed. Microscopic examination of representative tissuesections from rats treated with [DOTA°]CCK8revealed noabnormalities that could be attributed to the treatment.

Initial Human Evaluation of [111ln-DTPA°]CCK@Intravenous injection of 10 pg peptide was well tolerated

by the patient, with no adverse reactions. Figure 6 shows a

0.05

0.

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stomach

*

@ control blocked

Blood0.099 (0.010)0.018(0.002)0.005(0.001)0.005(0.001)Spleen0.041(0.004)0.034(0.004)0.033(0.003)0.023(0.003)Pancreas0.037(0.002)0.013 (0.001)0.011(0.000)0.012(0.000)Adrenals0.086(0.016)0.017 (0.002)0.019(0.000)0.018(0.000)Kidneys0.706(0.11)0.439 (0.061)0.379(0.050)0.322(0.03)Liver0.040(0.011)0.027 (0.004)0.026(0.005)0.045(0.007)Stomach0.08(0.02)0.044 (0.004)0.028(0.006)0.035(0.007)Colon0.041(0.013)0.013 (0.003)0.037 (0.008)0.01(0.001)Muscle0.012(0.001)0.003 (0.000)0.003(0.000)0.003(0.000)Femur0.035(0.002)0.014 (0.001)0.012 (0.001)0.011(0.001)Pituftary0.012(0.002)0.004(0.001)0.003(0.000)Tumor0.160(0.021)0.130 (0.020)0.082 (0.011)0.094(0.02)

control blocked

FIGURE 3. Uptakein rat stomachand CA20948 tumor,4 hafter injectionof [111In-DOTA°JCCK@,withor withoutcoinjectionwith 100 pg unlabeled peptide. %ID/g = percentage injecteddose/gtissue(mean±SD).

2084 THEJoui@.r@@iOFNUCLEARMEDICINE•Vol. 40 •No. 12 •December 1999

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lh 4h lh 4h

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0 FIGURE 4. Radioactivityin several ratorgans, 1 and 4 h after injectionof 125ICCK10 and [111In-DOTA°]CCK@.%ID/g =percentageinjecteddose/gtissue(mean±SD).

scan of the upper abdomen and neck, taken 48 h afterinjection. As in the preclinical studies in rats, receptorspecific uptake was seen in the CCK-B receptor-expressingfundus of the stomach and in metastases in the neck region.Cumulative excretion of radioactivity in the urine was morethan 90% of the dose at 24 h after injection.

DISCUSSION

Autoradiographic studies by Reubi et al. (6) revealed thepresence of CCK-B receptors in more than 90% of MTCsand in a high percentage of other tumors. Therefore, CCK-Breceptors appeared to represent a new and promising targetfor radiolabeled peptides for tumor scintigraphy and radionuclide therapy. Behr et al. (7) evaluated the suitability ofradioiodinated gastrin, a specific ligand for the CCK-Breceptor, for targeting CCK-B receptor-expressing tumors invivo. Reubi et al. (8) also developed DOTA- and DTPAconjugated CCK-B receptor-binding CCK analogs, evaluated their receptor binding characteristics and obtainedinitial preclinical biodistribution data using [DTPA°]CCK8in nontumor-bearing control rats. They concluded that thepeptide analogs used were promising for human CCK-Breceptor scintigraphy and radionuclide therapy.

For these studies, we chose nonsulfated octapeptideCCK8, derivatized with either DOTA or DTPA, because this

peptide has shown high affinity for CCK-B receptors but lowaffinity for CCK-A receptors (8), the latter in contrast tosulfated analogs that bear a sulfate ester auached to the Tyrmoiety. CCK-A receptors are expressed at a much higherdensity in normal tissues, especially in the abdomen. Theselective CCK-B receptor affinity of the chosen nonsulfatedanalog will result therefore in a favorable low backgroundradioactivity during scanning.

For the success of radionuclide therapy, it is importantthat the tumor cells internalize the radiopharmaceutical afterbinding to the receptor. We demonstrated receptor-specific,time- and temperature-dependent internalization of [11‘InDOTA°]CCK8in AR42J cells, indicating that the DOTAgroup does not prevent the CCK analog from internalization.In this study we also demonstrated receptor-specific internalization of ‘251-gastrinand [‘‘‘In-DOTA°]CCK8in CA20948cells in culture, consistent with the presence of CCK-Breceptors in these tumor cells. This provides a tumor modelfor research on CCK analogs in vivo because these cells canalso be grown to solid tumors in vivo in Lewis rats.

For [‘‘‘In-DTPA°]octreotide,the internalization pathwayproceeds through endosomes fusing with lysosomes wheredegradation of the radiolabeled peptide occurs. After dissociation of the ligand, the receptor protein may return to theplasma membrane, whereas ‘‘‘In-DTPA-containing degrada

EVALUATION OF RADIOLABELED CCK ANALOGS •de Jong et al. 2085

::5JI:@1r=1

stomach/bloodratio

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4h

h post injection

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4

FIGURE5. Scan,made20 mmafterinjection, of two tumor-bearing rats (left andmiddle)andcontrolrat(right),injectedwith[111ln-DOTA°]CCK@(left),[111In-DOTA°]CCK@inthepresenceofexcessunlabeledpeptide(middle)or1@I-CCK10(right).Leftandmiddlearrows indicate tumor uptake; right arrowindicatesliveruptake.

tion products are retained in the lysosomes, causing the longretention time of radioactivity in receptor-positive cells (15).The assumption that this intracellular route holds also forCCK-B receptor-specific peptides is in accordance with thelong retention time of the radiolabel shown in the humanstudy, where CCK-B receptors in stomach and metastaseswere visualized up to 48 h after injection.

Uptake of the “In-labeled peptide in CCK-B receptorexpressing tissues (stomach and CA20948 tumor) in vivo inrats was also found to be specific because uptake wasreduced significantly in the presence of excess unlabeledhormone. Furthermore, in agreement with the assumptionthat unsulfated CCK analogs will result in low backgroundradioactivity, uptake in receptor-negative organs was indeedlow, which is favorable during scintigraphy and radionuclidetherapy. These findings also showed that the tumor-tobackground ratio for [“In-DOTA°]CCK8already was significantly higher than that for ‘251-CCK,0at 4 h after injection,analogous to the findings with octreotide (16). Because weknow that the intracellular retention of radioactivity is muchlonger for the residualizing “In-chelator peptides than for

radioiodinated peptides (16), this tumor-to-background ratiowill be increasingly favorable for the “In-labeled peptide.Furthermore, the high liver uptake found with the radioiodinated compound is very unfavorable during scintigraphy oftumors in the upper abdomen. Behr et al. (7), using‘311-gastrin,found that a tumor-to-blood ratio of about 5could be reached in nude mice bearing the human MTCtumor. In this study, a tumor-to-blood ratio of 16 wasreached 24 h after injection, showing that residualizingradiolabels, such as the radiometal “In,have advantagesover ‘@‘I.This was also shown in human studies. Afterinjection of ‘31I-gastrin,good tumor-to-nontumor ratioswere maintained for about 3 h (7), whereas in this study,using the “In-labeled peptide, the receptor-positive stomach (17) and tumor metastases were clearly visualized even48 h afterinjection.

Despite the fact that the excretion of [“In-DOTA°]CCK8was through renal excretion, the uptake of radioactivity inthe kidneys was about one order of magnitude lower thanthat of [“In-DTPA]octreotide, a DTPA-coupled peptidealso excreted in the urine. [“In-DOTA°]CCK8contains two

A .:.,..,.@ BFIGURE

6 VisualizationofCCK B receptors in 45-y-old woman with MTC after intravenous administrationof 222 MBq [111InDTPA°]CCK@(10pgpeptide)Scansat48hafter injection show uptake in lymph nodemetastasesinneckregion(A,arrow)andinreceptor-positivestomach(B,arrow).:@

,@

@ @‘

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@ i@'

2086 THEJOURNALOFNUCLEARMEDICINE•Vol. 40 •No. 12 •December 1999

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4. Reubi JC. Chayvialle JA. Franc B, Cohen R, Calmettes C, Modigliani E.Somatostatin receptors and somatostatin content in meduilary thyroid carcinomas.Lab Invest. 1991;64:567—573.

5. Kwekkeboom Di, Reubi JC, Lamberts 5WJ, et ai. In vivo somatostatin receptorimaging in medullary thyroid carcinoma. J Clin EndocrinolMetab. 1993;76: 1413—1417.

6. Reubi JC, Schaer JC, Waser B. Cholecystokinin (CCK)-A and CCK-B/gastrinreceptors in human tumors. Cancer Res. 1997;57: 1377—1386.

7. Behr TM, Jenner N. Radetzky S. et al. Targeting of choiecystokinin-B/gastrinreceptors in vivo: preclinical and initial clinical evaluation of the diagnostic andtherapeutic potential of radiolabelled gastrin. Eur I Nucl Med. 1998:25:424—430.

8. Reubi JC, Waser B, Schaer JC. et al. Unsuifated DTPA- and DOTA-CCK analogsasspecifichighaffinityugandaforCCK-Breceptor-expressinghumanandrattissues in vitro and in vivo. EurJ NuciMed. 1998;25:481—490.

9. Krenning EP, Kooij PPM, Pauweis 5, et al. Somatostatin receptor scintigraphy andradionuclide therapy. Digestion. 1996;57:57—61.

10. WilliamsJA, BaileyAC, RoachE.Temperaturedependenceof high-affinityCCKreceptor binding and CCK internalization in rat pancreatic acini. Am J Physiol.1988;254(suppi):G513-G521.

I1. SvobodaM. DupucheMH, LambertM, Bui D, ChristopheJ. Internalizationsequestration and degradation of cholecystokinin (CCK) in tumoral rat pancreaticAR4—2Jcells. Biochim BiophysActa. 1990:1055:207—216.

12. de Jong M, Krenning EP, Bakker WH, et al. @°Yand ‘@ ‘Inlabelling, receptorbinding and biodistribution of [DOTA°,D-Phe',Tyr3loctreotide,a promisingsomatostatin analogue for radionuclide therapy. Eur J Nuci Med. 1997:24:368—371.

13. Bakker WE, Krenning EP, Breeman WAP. et al. Receptor scintigraphy with a

radioiodinated somatostatin analogue: radiolabeling, purification, biologic activity and in vivo application in animals. J Nucl Med. 1990:31:1501—1509.

14. de Jong M. Bernard HF, De Bruin E, et al. Internalization of radiolabeled[DTPA°]octreotideand [DOTA°,'tyr3loctreotide: peptides for somatostatin receptortargeted scintigraphy and radionuclide therapy. NuclMed Commun. 1998:19:283—288.

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17. Reubi JC, Waser B, Laderach U, et al. Localization of cholecystokinin A and

cholecystokinin G-gastrin receptors in the human stomach. Gastroenterologv.1997:112:1197—1205.

18. MogensenCE, Soiling K. Studies on renal tubularproteinreabsorption:partialand near complete inhibition by certain amino acids. Scan J Clin Lab Invest.1977:37:477—486.

EVALUATIONOF RADIOLABELEDCCK ANALOGS •de Jong et al. 2087

Asp moieties, giving an anionic charge to the molecule,whereas octreotide contains the positively charged Lysmoiety. Negatively charged peptides apparently have alower renal uptake in the environment of negatively chargedmembranes of tubular cells than neutral or cationic ones,consistent with the finding that positively charged aminoacids can block peptide reabsorption by binding to thenegatively charged membranes of the tubular cells (18). Thislow kidney uptake is very favorable during scintigraphy inthe perirenal region; during radionuclide therapy studies, itwill prevent renal radiotoxicity.

CONCLUSION

‘‘In-DOTA°]CCK8 is most promising for scintigraphy

and, after coupling to therapeutic radionuclides, for radionuclide therapy of CCK-B receptor-positive tumors, such asMTC and small cell lung cancer.

ACKNOWLEDGMENTS

The authors thank Prof. W.J. Mooi for sharing hispathology expertise in the evaluation of tissue slices fromthe toxicology studies and Marcel van der Pluijm, Arthurvan Gameren, Michael Schaar and Elisa de Bruin for theirexcellent help during the experiments.

REFERENCES

I. Lamberts swi, Bakker WH, Reubi JC, Krenning EP. Somatostatin receptorimaging in the localization ofendocrine wmors. NewEngliMed. 1990;323:1246—1249.

2. Krenning EP. Kwekkeboom DJ. Bakker WH, et al. Somatostatin receptorscintigraphy with [“In-DTPA-D-Phe']- and [‘231-TS'r31-octreotide:the Rotterdamexperience with more than 1000 patients. Eur J Nuci Med. 1993;20:716—773.

3. Krenning EP. Kwekkeboom DJ, Pauweis5, Kvols LK, Reubi JC. Somatostatinreceptor scintigraphy. In: Freeman, LH, ed. Nuclear Medicine Annual. New York,NY: Raven Press; 1995:1—50.

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1999;40:2081-2087.J Nucl Med.   Srinivasan, Michelle Schmidt and Eric P. KrenningMarion de Jong, Willem H. Bakker, Bert F. Bernard, Roelf Valkema, Dik J. Kwekkeboom, Jean-Claude Reubi, Ananth  Peptide for CCK-B Receptor-Targeted Scintigraphy and Radionuclide Therapy

Analog: A8In-Labeled Nonsulfated CCK111Preclinical and Initial Clinical Evaluation of

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