Improvement Strategies for Peptide ReceptorScintigraphy and Radionuclide Therapy
Monique de Visser,* Suzanne M. Verwijnen,* and Marion de JongDepartment of Nuclear Medicine, Erasmus MC, Rotterdam, The Netherlands
SUMMATION
Somatostatin receptor-targeting peptides are widely used for the imaging and therapy of neuroendocrinetumors. Peptide-receptor radionuclide therapy (PRRT) in neuroendocrine tumor patients with radiola-beled somatostatin analogs has resulted in symptomatic improvement, prolonged survival, and enhancedquality of life. The side-effects of PRRT are few and mostly mild, certainly when using kidney protectiveagents. If a more widespread use of PRRT is possible, such therapy might become the therapy of firstchoice in patients with metastasized or inoperable neuroendocrine gastroenteropancreatic tumors. Yet,much profit can be gained from improving the receptor-targeting strategies available and developing newstrategies. This review presents an overview of several options to optimize receptor-targeted imaging andradionuclide therapy. These include the optimization of peptide analogs, increasing the number of re-ceptors on the tumor site, and combining PRRT with other treatment strategies. The development of newpeptide analogs with increased receptor-binding affinity and improved stability might lead to a higher ac-cumulation of radioactivity inside tumor cells. Analogs of somatostatin have been widely studied. How-ever, much profit can be gained in improving peptide analogs targeting other tumor-related receptors,including gastrin-releasing peptide (GRP) receptors, neurotensin (NT) receptors, cholecystokinin (CCK)receptors, and glucagon-like peptide-1 (GLP-1) receptors. Several peptide analogs targeting these re-ceptors are well on their way to clinical utilization. The literature shows that it is possible to increase thereceptor density on tumor cells by using different methods, which results in higher binding and internal-ization rates and thus a higher contrast during peptide-receptor scintigraphy. In PRRT treatment, thiswould enable the administration of higher therapeutic doses to tumors, which might lead to a higher curerate in patients. Combinations of radionuclide therapy with other treatment modalities, such as chemo-therapy or pretreatment with radiosensitizers, might increase the impact of the treatment. Further, the ad-ministration of higher dosages of radioactivity to the patient, enabled by combinations of PRRT withstrategies reducing the radiation dose to healthy organs, will improve the outcome of tumor treatment.Also, targeting one or several tumor-specific receptors by using combinations of therapeutic agents, aswell as by reducing nontarget uptake of radioactivity, will enlarge the therapeutic window of PRRT. Clin-ical studies will provide more insight in the effects of combining treatment strategies in cancer patients.
Address reprint requests to: Marion de Jong; Department of Nuclear Medicine, Erasmus MC; Room V-218, ‘s Gravendijk-wal 230, 3015 CE Rotterdam, The Netherlands; Tel.: �31-10-7035781; Fax: �31-10-7035997E-mail: [email protected]*Both authors contributed equally to this manuscript
INTRODUCTION
Radiolabeled receptor-binding peptides are pow-erful tools for the imaging and therapy of tumorsexpressing peptide-binding receptors. Especially,analogs of somatostatin are suitable for receptor-targeted localization, staging, and treatment ofsomatostatin (sst)-receptor-expressing neuroen-docrine tumors.1 The somatostatin receptor fam-ily consists of five receptor subtypes: sst1–sst5.The majority of neuroendocrine tumors feature astrong overexpression of sst, mainly subtype 2(sst2).
The introduction of radiolabeled somatostatinanalogs started with the development of the sst-targeting somatostatin analog, [111In-DTPA0]octreotide (Octreoscan®, Covidien, Hazelwod,MO). This analog is being used to visualize sst-receptor-positive tumors and their metastases.2,3
The therapeutic efficacy of this analog was foundto be promising, although no effects were foundin patients with larger tumors and advanced dis-ease.4 The next generation of modified somato-statin analogs, including [DOTA0,Tyr3]octreotide,is being used for sst-receptor-targeted radionuclidetherapy as well. This analog has a higher affinityfor sst2 and has 1,4,7,10-tertraazacyclododecane-N�,N�,N�,N��-tetraacetic acid (DOTA) instead ofdiethylenetriaminepentaacetic acid (DTPA) as thechelator, allowing the stable radiolabeling withbeta-emitting radionuclides, such as 90Y and177Lu. Several phase-1 and -2 peptide-receptorradionuclide therapy (PRRT) trials were per-formed using [90Y-DOTA0-Tyr3]octreotide (90Y-DOTATOC; OctreoTher®),5–9 objective re-sponses in patients with gastroenteropancreatic(GEP) tumors ranged from 9% to 33%.10 Theseresults were better than those obtained with[111In-DTPA0]octreotide, despite differences inthe [90Y-DOTA0,Tyr3]octreotide protocols ap-plied. [177Lu-DOTA0,Tyr3] octreotate is a third-generation sst analog for PRRT and has been usedin our medical center since 2000. [DOTA0,Tyr3]-octreotate differs from [DOTA0,Tyr3]octreotide,in that the C-terminal threoninol has been replacedwith threonine. Compared with [DOTA0,Tyr3]oc-treotide, it shows considerable improvement inbinding to sst2-positive tissues in vitro and invivo.11,12 [177Lu-DOTA0,Tyr3]octreotate repre-sents an important improvement because of thehigher absorbed radiation doses that can beachieved to most tumors with about equal radia-tion doses to dose-limiting organs.13,14 90Y- and177Lu-labeled peptides have greater therapeutic
potential, compared to 111In-labeled peptides, fortheir emitted �-particle range exceeds the cell di-ameter, enabling the irradiation of neighboringtumor cells, which is favorable in the case of het-erogeneous receptor expression. 177Lu, as com-pared to 90Y, has a lower tissue penetration range,which is favorable for the treatment of small tu-mors, whereas 90Y might be more effective in tu-mors with a larger diameter.15,16 In contrast to90Y, 177Lu also emits low-energy �-rays, whichdirectly allows the imaging and dosimetry fol-lowing [177Lu-DOTA0,Tyr3]octreotate therapy.Treatment with [177Lu-DOTA0,Tyr3]octreotate inpatients with GEP tumors resulted in complete orpartial remission in 28% of patients.17 Median timeto progression was more than 36 months in patientswho had either stable disease or tumor regressionafter treatment. In addition, patients treated with[177Lu-DOTA0,Tyr3]octreotate indicated a signifi-cant improvement of their quality of life.18
In summary, PRRT with radiolabeled sstanalogs is a promising treatment option for pa-tients with inoperable or metastasized neuroen-docrine tumors. The side-effects of PRRT are fewand mostly mild.
This review discusses several options to opti-mize receptor-targeted imaging and radionuclidetherapy, outlining the efforts to develop opti-mized radiopharmaceuticals, to increase the tar-get density and combine treatment modalities(see also Fig. 1):
1. Developing new peptide analogs with in-creased receptor-binding affinity and im-proved stability might lead to a higher accu-mulation of radioactivity inside tumor cells.Many new analogs of sst have been developedand widely studied; much profit can also begained by improving peptide analogs target-ing other tumor-related receptors, includinggastrin-releasing peptide (GRP) receptors,neurotensin (NT) receptors, cholecystokinin(CCK) receptors, and glucagon-like peptide-1(GLP-1) receptors.
2. Increasing the number of receptors that can be targeted on the tumor cells will result in ahigher contrast during peptide-receptor scinti-graphy (PRS) and a higher radiation dose tothe tumor during PRRT.
3. Combinations of radionuclide therapy withother treatment modalities, such as chemo-therapy or radiosensitizer pretreatment, mightincrease the treatment impact. Also, the ad-ministration of higher radioactivity doses, en-
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abled by combinations of PRRT with strate-gies reducing the radiation dose to normal or-gans, will improve the outcome of tumor treat-ment.
Development of New Peptide Analogs
The natural structure of peptides makes them sen-sitive to peptidases. They are rapidly brokendown in blood and other tissues, thereby re-stricting their potential use as radiopharmaceuti-cals. Metabolically stable analogs are, therefore,preferable for clinical application. Strategies tostabilize peptides include the introduction of non-biodegradable peptide bonds, stabilized aminoacid derivatives replacing the natural aminoacids, and cyclization.
High in vivo stability is advantageous, but notsufficient, for good target-to-nontarget ratios. Im-portant factors are also long retention time of ra-dioactivity at the tumor site and rapid clearanceof radioactivity from nontarget tissues and blood.Internalization of radiolabeled peptides by tumorcells may lead to a longer retention of radioac-tivity.19 Peptide agonists often undergo receptor-mediated endocytosis, enabling the internaliza-tion of the radionuclide into tumor cells, whereasantagonists do, most often, not internalize.20 The
majority of research efforts to design peptide-based radiopharmaceuticals have focused on re-ceptor agonists. Recently, somatostatin antago-nists were shown to be most suitable for ssttargeting as well.21
Subtle changes in peptide structures, as de-scribed above, can have dramatic effects on thereceptor-binding capacity and biodistribution ofthe compound. Attempts to improve the stabilityof the radiolabeled peptide can, at the same time,be fatal for its targeting abilities owing to the lossof receptor-binding affinity. As described below,we reviewed the most recent efforts to developnew radiolabeled peptides for the imaging andtherapy of receptor-expressing tumors.
SST-receptor-targeting peptides99mTc-labeled somatostatin analogs, including hy-drazinonicotinamide (Hynic)-derivatized 99mTc-[Hynic-Tyr3]octreotide, 99mTc-[Hynic-Tyr3]octreo-tate,22–26 and tetra-amine-functionalized derivative99mTc-[N4
0,Tyr3]octreotate (Demotate 1)27–29 canbe regarded as promising new radiopharmaceuticalsfor sst scintigraphy. Both Hynic- and N4-derivatizedanalogs were capable of detecting sst-expressing le-sions in patients.
Compared to single-photon emission computed
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Figure 1. Improvement strategies for peptide receptor radionuclide therapy.
tomography (SPECT) imaging, clinical positronemission tomography (PET) imaging provides ahigher spatial resolution and the possibility tomore accurately quantitate tumor and normal or-gan uptake. For PET imaging, peptides can be la-beled with positron-emitting radionuclides, suchas 68Ga, 18F, 64Cu, 86Y, 89Zr, and 124I. Several18F- and 64Cu-labeled sst analogs have been re-ported, including Gluc-Lys([18F]FP)-TOCA30
and 64Cu-TETA-octreotide31 which both ap-peared promising for the imaging of patientsbearing neuroendocrine tumors. In contrast toPET radionuclides that require a cyclotron forproduction, 68Ga can be produced in-house by us-ing a 68Ge/68Ga generator.32 PET imaging, using68Ga-[DOTA0-Tyr3]octreotide, has been shownpreferable to SPECT imaging with Octreoscan,especially in small lesions and tumors with lowsst density.33,34
The radiolabeled analogs of octreotide and oc-treotate, including the analogs described above,have high binding affinity for sst2,11 the most fre-quently expressed subtype in neuroendocrinecancers. In some cancers, however, sst2 is not oris only low density expressed, whereas other subtype receptors can be present.35,36 The het-erogeneous and concomitant sst-receptor subtype expression strongly pleads for tracers, or combi-nations of tracers, that can target more than onesst. Ginj et al. evaluated 24 DOTA-somatostatinanalogs, all based on octreotide, using a system-atic modification at amino acid position 3.37 Twoanalogs, namely [DOTA0-Nal3]octreotide and[DOTA0-BzThi3]octreotide, showed high bind-ing affinity for sst2, sst3, and sst5. 68Ga-labeled[DOTA0-Nal3]octreotide has been shown to be agood tracer for primary diagnostic and follow-upstudies in patients suspected from, or with,proven sst-expressing tumors.38
As mentioned above, peptide agonists inter-nalize into the cell after receptor binding, whichis thought to be essential for the good retentionof radionuclides in target cells. Ginj et al., how-ever, recently reported promising results in a pre-clinical study comparing targeting characteristicsof sst2- or sst3-binding agonists versus antago-nists.21 They found that these antagonists, eventhough they did not internalize, showed a higheraccumulation in tumor cells, compared to ago-nists, whereas the receptor affinity of agonistsand antagonists was in the same range. In addi-tion, accumulation in nontumor tissues, exceptfor that in the kidneys, was less for antagoniststhan for agonists up to 24 hours after injection.
These results suggest that antagonists may be bet-ter candidates to target tumors than agonists. Theresearchers attribute the superior antagonist ac-cumulation to the binding of antagonists to alarger variety of receptor configurations. If thepresent observation can be translated to other re-ceptors as well, the use of radiolabeled antago-nists may considerably improve tumor imagingand PRRT efficacy.
GRP receptor-targeting peptides
Overexpression of GRP receptors has been dem-onstrated in a large number of human tumors, in-cluding prostate and breast tumors,39 which areamong the major causes of cancer death world-wide.40 Bombesin (BN) is a 14-amino-acid pep-tide with high affinity for the GRP receptor; radi-olabeled analogs of BN might, therefore, be usefulfor GRP-receptor-targeted imaging and therapy.First attempts to develop radiolabeled BN analogsfor diagnostic imaging were aimed at radioiodi-nated peptides. These compounds were found tobe very unstable, and iodine was rapidly clearedfrom the tumor cells.41 Now, more than 10 yearslater, several 111In- and 99mTc-labeled BN analogshave been developed with favorable in vivo char-acteristics for SPECT imaging of GRP-receptor-expressing tumors.42–47
99mTc-labeled BN analogs have a tendency to ac-cumulate in the liver and intestines as a result oftheir high lipophilicity. This high unspecific accu-mulation of radioactivity interferes with the de-tection of GRP-receptor-positive lesions in the abdominal area. Much effort has been put into reducing the lipophilicity of the 99mTc-labeled BNanalogs. Ferro-Flores et al. conjugated the bifunc-tional chelator, hydrazino nicotinate (HYNIC) andthe coligand, EDDA (ethylenediamine-N,N�-di-acetic acid), to BN for the preparation of 99mTc-EDDA/HYNIC-[Lys3]-BN. This conjugation re-sulted in less lipophilic properties of the peptideand, consequently, lower hepatobiliary and, pre-dominantly, renal excretion.48 Further, GarciaGarayoa et al. recently showed that the introduc-tion of a hydrophilic spacer between the peptide se-quence and the 99mTc-binding complex can reducethe high lipophilicity and impove tumor-to-nontu-mor ratios.49
Next to tumor diagnosis, staging, and localiza-tion, 111In-labeled peptide analogs are often usedas surrogates to determine the biodistribution anddosimetry of therapeutic radiopharmaceuticals la-beled with radiometals, such as 90Y. DTPA and
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DOTA are being used as chelating systems cou-pled to the BN analogs for this purpose.50
111In-DTPA-BN analogs (e.g., [111In-DTPA-Pro1,Tyr4]BN20,47) were reported to have goodtumor uptake and rapid clearance from nontargettissues and blood. Substitution of the DTPAchelator system in [DTPA-Pro1,Tyr4]BN byDOTA was previously found to have favorableeffects on the receptor-binding characteristics ofthis radioligand.47 We recently synthesized a new DTPA-coupled BN analog, [111In-DTPA-ACMpip5,Tha6,�Ala11,Tha13,Nle14]BN(5-14)(Cmp 3) with a significantly higher GRP-recep-tor-mediated tumor uptake in vivo in animal stud-ies.51 As 111In-Cmp 3 seems promising for theSPECT imaging of GRP-receptor-expressing tu-mors, replacing the DTPA chelator by DOTAwould enable the therapeutic use of the com-pound and diagnostic PET imaging.
Most of the recent studies on newly developedBN peptide analogs focus on the DOTA-chelatingsystem for its multipurpose utilization options:SPECT, PET, and PRRT.19,52–58 For example,DOTA-PESIN (DOTA-PEG4-BN(7-14)) showedto be a very promising new compound. Althoughit has only a moderate affinity for the GRP recep-tor, it showed good in vivo tumor uptake in animalstudies.52 Clearance of the compound proceededthrough the kidneys and urinary tract with a fastwashout from GRP-receptor-negative tissues but arather high accumulation in the kidneys. The highkidney retention could not be reduced by lysine.
Another very promising DOTA-BN analog is177Lu-AMBA.58 This compound consists ofDOTA attached to BN(7-14) by a short linker.177Lu-AMBA, like DOTA-PESIN, showed in an-imals high GRP-receptor-mediated tumor uptakeand favorable tumor-to-background ratios. Invivo tumor treatment with 177Lu-AMBA resultedin a significantly prolonged survival of tumor-bearing mice and a decreased tumor growth rateover that of the controls. Like DOTA-PESIN,177Lu-AMBA is excreted through the kidneys,and the relatively high kidney retention cannot bereduced by a coinjection of lysine, which is prob-ably owing to the lack of lysine residues in thesepeptide sequences. However, the accumulation ofradioactivity in the kidneys is still 50% lower forDTPA- and DOTA-derivatized BN analogs, com-pared to that of somatostatin analogs.
PRRT, using BN analogs, as described above,may be promising. Clinical scintigraphy with99mTc- and 68Ga-labeled BN analogs clearly de-lineated tumor lesions, including lymph nodes, and
metastases.44,59,60 However, also relatively highuptake in nontarget, GRP-receptor-positive tissues,such as the pancreas and intestines, was found,which is unfavorable for PRRT. In a preclinicalstudy using 111In-Cmp 3, we found that increasingamounts of injected peptide mass in tumor-bear-ing rats decreased uptake in receptor-positive nor-mal tissues more than that in the tumor. Also, thepreinjection of excess unlabeled peptide before ad-ministration of radiolabeled compound showed tobe profitable for tumor uptake, compared to that inreceptor-expressing normal tissues (Fig. 2).61
These effects were also found with 177Lu-AMBAin tumor-bearing mice.58 Thus, the injection ofhigher peptide mass and/or the preinjection of ex-cess BN may increase tumor-to-nontumor ratios.Taking into account the biologic activity of BN ag-onists in patients, the preinjection of GRP-recep-tor antagonists might be preferable.
Radiolabeled BN analogs are of particular in-terest for PRRT of advanced prostate cancer pa-tients who do not respond to hormone therapy.So far, the best treatment strategies available forthis group of patients are only marginally effec-tive.62,63 However, in a study evaluating GRP-re-ceptor expression in human prostate cancerxenograft models representing the differentstages of prostate tumor development, includingthe shift from androgen-dependent toward inde-pendent tumor growth, we found high GRP-re-ceptor density only in androgen-dependentprostate cancer xenografts. These results suggesthigh GRP-receptor expression in the early, an-drogen-dependent stages of prostate tumor de-velopment and not in later stages. In addition, thesimulation of androgen ablation treatment in theanimal model (i.e., castration) strongly reducedGRP-receptor expression in androgen-dependenttumors, suggesting that GRP-receptor expressionin human prostate cancer is androgen regulated.64
Studies evaluating GRP-receptor expression onclinical prostate cancer tissue samples are under-way to determine whether these results are clin-ically relevant.
The application of BN peptides in cancer pa-tients is still in its infancy.44,59,60 However, re-cent developments in the synthesis of new,promising BN analogs are encouraging for fur-ther utilization in clinical studies.
NT-receptor-targeting peptides
Neuroendocrine pancreatic tumors can be suc-cessfully localized and treated by using radiola-
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beled somatostatin analogs.65–67 Exocrine pan-creatic cancer, however, does not express a suf-ficient level of sst for scintigraphic imaging.Reubi et al. reported that 75% of ductal pancre-atic carcinomas overexpressed NT receptors,whereas normal pancreatic tissue, pancreatitis,and endocrine pancreatic cancers were NT-re-ceptor-negative.68 NT is a 13-amino-acid peptidelocalized in both the central nervous system andin peripheral tissues, mainly the gastrointestinaltract.69,70 The instability of native NT prompted
several groups71–76 to synthesize NT analogs thatwere less susceptible to degradation while main-taining the binding affinity to NT receptors. Preclinical studies using 111In-labeled DTPA(MP2530) and DOTA (MP2656)-linked NTanalogs demonstrated that subtle changes as theintroduction of non-natural amino acids on spe-cific positions can be made in the C-terminal partof the peptide, the crucial part for binding and bi-ologic activity, without markedly affecting thebinding properties.77 These NT analogs showed
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Figure 2. Gastrin-releasing peptide (GRP) receptor saturation: uptake of 111In-Cmp 3 in GRP-receptor-expressing pancreas (A)and CA20948 tumor (B) in rats (4 hours postinjection, lowest peptide mass is set at 100%). Receptor availability: uptake of 0.1�g 111In-Cmp 3 in rat pancreas (C) and tumor (D) without (control) or with 100 �g of unlabeled bombesin (BN) either coin-jected (block) or preinjected 0.5 (0.5-hour preblock) or 4 hours (4-hour preblock) prior to the administration of the radiolabeledcompound (4 hours postinjection, control is set at 100%). Injection of a higher peptide mass and/or preinjection of excess BNaffected the uptake in nontarget GRP-receptor-expressing tissues more than that in tumor.
good receptor-mediated uptake in NT-receptor-expressing HT29 xenografts and were thuspromising tools for the imaging of exocrine pan-creatic tumors. PRRT, using these analogs,might, however, be hampered by the relativelyhigh kidney uptake of 111In-NT analogs. Re-cently, Maes et al.73 reported a triply stabilized99mTc-labeled NT (NT-XIX) analog with high tu-mor uptake and reduced kidney uptake, which ledto a superior tumor-to-kidney ratio, compared to111In-labeled analogs. Also, 99mTc-demotensin 4,a doubly-stabilized NT analog,72 showed a fa-vorable tumor-to-kidney ratiol. Still, the tumor-to-intestine and tumor-to-liver ratios were con-siderably higher for 111In-labeled analogs, whichis favorable for the visualization of pancreatictumors in patients.78
Only one clinical evaluation study using a ra-diolabeled NT analog has been reported.79 Thisstudy included four exocrine pancreatic cancerpatients, who were injected with the NT analog,99mTc-NT-XI. Scintigraphic imaging showedmoderate tumor uptake in 1 patient, whereas theother 3 patients showed no tumor uptake. Two(2) of these 3 patients were found to have a NT-receptor-negative tumor.
CCK2-receptor-targeting peptides
Unlike other neuroendocrine tumors, sst-receptorexpression is rather low in medullary thyroid can-cer (MTC) and is completely absent in clinicallyaggressive forms of the disease.80,81 The presenceof cholecystokinin-2 (CCK2) receptors wasshown in more than 90% of MTCs and in a highpercentage of small-cell lung cancers, stromalovarian cancers, astrocytomas and several othertumor types.82 On the basis of these findings,Behr et al.83 evaluated the suitability of radioio-dinated gastrin, a specific high-affinity ligand forthe CCK2 receptor, for targeting CCK2-receptor-expressing tumors in vivo. Their data suggestedthat gastrin analogs may represent a useful newclass of receptor-binding peptides for the diag-nosis and therapy of a variety of tumor types, in-cluding MTC. Reubi et al.84 developed DTPA-CCK2-receptor binding CCK analogs, evaluatedreceptor-binding characteristics, and obtainedinitial preclinical biodistribution data in nontu-mor-bearing rats. For the DOTA counterpart ofthe most promising analog [111In-DOTA0]CCK8,a high CCK2-receptor affinity was found. The latter analog could visualize CCK2-receptor-expressing tumors in vivo in rats,85 and also in
patients with advanced metastatic MTC [111In-DTPA0] CCK8 was able to visualize the tumorlesions.86
Besides CCK analogs, radiolabeled analogs ofminigastrin have also shown to be suitable forCCK2-receptor targeting. For example, a clinicalstudy in MTC patients showed that most tumorsites could be visualized with 111In-DTPA-mini-gastrin.83,87 Nock et al. synthesized 99mTc-la-beled N4-derivatized analogs of minigastrin.88
Preclinical evaluation studies resulted in the se-lection of [N4
0–1,Gly0,(D)Glu1]minigastrin (De-mogastrin 2) as the most promising CCK2-tar-geting analog for tumor imaging. The qualities of[99mTc]Demogastrin 2 could be confirmed in apatient with metastatic MTC; tumor depositswere clearly delineated (see also Fig. 3).
More recent clinical studies by Gotthardt etal.,89,90 in patients with metastatic/recurrentMTC, compared the results of CCK2 (gastrin) receptor scintigraphy (GRS), using [111In-(D)Glu1]minigastrin, with somatostatin-receptorscintigraphy (SRS), CT, and 18F-flourodeoxyglu-cose (FDG) PET. They found that GRS had ahigher tumor detection rate than SRS and 18F-FDG PET. GRS in combination with CT wasmost effective in the detection of metastaticMTC. Further, GRS in patients bearing neuroen-docrine tumors other than MTC detected addi-tional tumor sites that were missed in SRS in 20%of patients. The researchers concluded that GRSmay become the scintigraphic imaging modalityof choice in MTC patients. In conclusion,(pre)clinical studies have shown the suitability ofradiolabeled CCK and gastrin analogs for thescintigraphy of tumors, such as MTC. PRRT, us-ing these radioligands, is still preliminary, but itsfuture is promising.
GLP-1-receptor-targeting peptides
A new promising candidate for in vivo tumor tar-geting is the GLP-1 receptor, a member of theglucagon-receptor family.91 The GLP-1 receptorwas recently shown to be highly overexpressed inhuman endocrine tumors, in particular, insulino-mas, gastrinomas,92 and pheochromocytomas.93
Similar to other naturally occurring receptor-binding ligands, native GLP-1-receptor agonistsare rapidly degraded in the blood.94,95 Therefore,Gotthardt et al. evaluated the more stable GLP-1selective analog, exendin, which showed to havethe potential for the scintigraphic imaging ofGLP-1-receptor-expressing tumors.96 Recently,
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the exendin analog has been further optimized,which has led to two new 111In-DTPA-conju-gated, exendin-4 analogs: 111In-DTPA-Lys40-ex-endin-497 and [Lys40(Ahx-DTPA-111In)NH2]ex-endin-4.98 Both analogs showed encouragingpreclinical characteristics with high GLP-1-re-ceptor-mediated uptake in target tissues and goodtarget-to-background ratios in vivo in animalmodels. In addition, Wicki et al. showed that[Lys40(Ahx-DTPA-111In)NH2]exendin-4 efficiently
repressed insulinoma growth in mice.99 Kidneytoxicity was found to be the limiting factor in thistreatment strategy.
No clinical study using GLP-1-receptor-target-ing analogs has been reported thus far. For therapeutic purposes, high kidney retention of exendin-4 analogs could be problematic. Never-theless, when this high accumulation in the kid-neys can be overcome, high GLP-1-receptor expression on tumors, in combination with the
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Figure 3. 99mTc-demogastrin 2 scintigraphy (3 hours postinjection) in a 44-year-old female medullary thyroid cancer patientwith elevated serum calcitonin. No tumor localization with ultrasound, somatostatin-receptor scintigraphy, computed tomogra-phy, magnetic resonance imaging, and 18fluorodeoxyglucose positron emission tomography. Anterior (Ant) and posterior (Post)images of the thoracic (upper images) and pelvic (lower images) regions showing metastatic lesions (some marked with arrows).
favorable in vivo characteristics of the recent ex-endin-4 analogs, gives GLP-1-receptor-targetedPRRT serious potential.
�v�3 integrin-targeting peptides
Cell-matrix interactions are of fundamental im-portance for tumor invasion and formation ofmetastases as well as tumor-induced angiogenesis.
The �v�3 integrin is a transmembrane proteinthat is preferentially expressed on proliferatingendothelial cells,100 whereas it is absent on qui-escent endothelial cells. For growth beyond thesize of 1–2 mm in diameter, tumors require theformation of new blood vessels. The �v�3 re-ceptors are overexpressed on these newly formedblood vessels of actively growing tumors, andare, therefore, potential targets for receptor-me-diated tumor imaging and therapy and for plan-ning and monitoring of �v�3 targeting treatmentstrategies.
It was found that the essential amino-acid se-quence for the binding of extracellular matrixproteins to �v�3 receptors is arginine-glycine-as-partic acid (RGD).101 Several studies have beenaimed to develope optimized �v�3 targeting com-pounds. In summary, it was found that cyclicanalogs of RGD containing five amino acids(RGD sequence � hydrophobic amino acid inposition 4 � additional amino acid in position 5)have the highest �v�3 binding affinities.102,103
Radiolabeled analogs containing this five-amino-acid sequence have been synthesized and evalu-ated for their �v�3 targeting characteristics.Among them are DTPA- and DOTA-conjugatedanalogs for radiolabeling with 111In, 90Y, 177Lu,68Ga, and 64Cu, enabling SPECT and PET imag-ing and PRRT.104,105 Also 18F-labeled cyclicRGD analogs for PET imaging have been char-acterized.105–107 In patients, Beer et al. showedthat PET imaging using the RGD analog, 18F-galacto-RGD, can effectively show the level of�v�3 expression in man.108–110
Dijkgraaf et al.111 developed multivalent RGDpeptides in an attempt to increase receptor-bind-ing affinity. They synthesized and compared thein vitro and in vivo �v�3 targeting characteristicsof DOTA-linked monomeric, dimeric, andtetrameric RGD peptides radiolabeled with 111In.They found enhanced receptor affinity in vitroand better tumor uptake in vivo for the tetramericcompound, compared to its monomeric anddimeric analogs. Alternatively, they synthesizedmultimeric RGD peptides as dendrimers: macro-
molecules consisting of multiple perfectlybranched monomers. Consistent with their pre-vious results, the tetrameric RGD dendrimershowed enhanced affinity and significantlyhigher tumor uptake, compared to its monomericand dimeric analogs.112 The researchers ascribedthe improved targeting characteristics of the mul-timer to the enhanced local concentration of RGDunits in the vicinity of the receptor (statistical re-binding) and not to the binding of the compoundto multiple �v�3 receptors. Unfortunately, thekidney retention of the mulitimeric peptides wasalso increased, resulting in unfavorable tumor-to-kidney ratios. Introduction of a linker in betweenthe peptide moiety and the DOTA chelator, in anattempt to improve the target-to-background ra-tios of the peptide, led to a marginal enhancementof the tumor-to-kidney ratio only.113 In a studyevaluating the targeting potential of a cyclic RGDanalog in an intraperitoneally (i.p.) growing tu-mor model, Dijkgraaf et al. found that i.p. versusintravenous (i.v) injection of the radiolabeledRGD peptide resulted in markedly higher tumoruptake after i.p. administration, whereas uptakein the other organs, such as the kidneys, were un-affected by the route of administration. PRRT ex-periments in this model indicated that i.p. grow-ing tumors can be inhibited significantly by thei.p. injection of a therapeutic dose of 177Lu-la-beled RGD analog.114 Multimeric RGD peptidesare promising tools for in vivo imaging of tumorangiogenesis in cancer patients. �v�3 targetedPRRT with these compounds might particularlybe used for i.p. growing tumors. Currently, 18F-galacto-RGD is the only �v�3-targeting peptideshown effective for tumor imaging in patients.110
Improving the Therapeutic Effect:Increasing Receptor Density on Target Cells
By increasing the receptor density on tumor cellsin patients to be treated with radiolabeled pep-tides, and thereby increasing radioactivity uptakein the tumor, the therapeutic window can be en-larged.
Upregulation of receptors
During the last three decades, several reportshave been published concerning hormones andgrowth factors inducing a higher number of re-ceptors on tumor cells.115–123 For an overview ofreferences and findings, see Table 1. In this re-view, the focus is on radiation-induced receptorupregulation.
145
Upregulation of peptide receptors on tumorcells after irradiation was first reported by Béhéet al.,124,125 who showed that a total dose of 4–16Gy of external beam irradiation led to a time-de-pendent upregulation of both sst2 and gastrin re-ceptors on AR42J cells, in vitro as well as in vivo.This phenomenon was also investigated in vitroin NCI-H69 small-cell lung cancer cells.126
which were irradiated with a total dose of 4 Gy,and the subsequent internalization of [177Lu-DTPA0,Tyr3]octreotate was 1.5 to three times in-creased, compared to that in the control cells.
Not only the use of external beam radiation,but also low therapeutic doses of radiolabeledpeptides were found to induce sst2 upregulation.CA20948 rat pancreatic tumor-bearing rats127,128
were treated with a relatively low, noncurativedose of either [111In-DTPA0]octreotide127 or[177Lu-DOTA0,Tyr3]octreotate,128 and sst2-re-ceptor expression in different phases of tumor re-sponse was determined versus baseline (control).Both studies revealed an increased sst2 density on
tumors regrowing after initial therapy-induced re-gression, compared to control: Treatment with[111In-DTPA0]octreotide resulted in a 2-fold increase, while [177Lu-DOTA0,Tyr3]octreotatetreatment showed a more pronounced effect (2–5-fold increase). This radiation-induced upregula-tion of receptor expression might be importantfor improving the response rate in clinical PRRT,although the clinical value has to be determined.
Gene therapy
In general, gene-transfer methods can be ap-plied to induce the expression of a desired genein a cell. This concept has been used mostly forthe treatment of cancer.129 By using a vector, ei-ther viral or nonviral, a peptide-receptor-encod-ing gene (or several genes) can be transferred intoa tumor cell, with the aim to enhance the uptakeof radiolabeled peptide analogs. Gene therapy ap-proaches in combination with PRRT might havesome advantages: First, transduction of receptors
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Table 1. An Overview of References Concerning Receptor Up- and Downregulation
Reference(first author) Cell type Origin of cell Modulator In vitro/in vivo Effect on expr.
115Kimura, 1986 Anterior pit. cells Rat Estradiol In vitro Up (2-fold)116Presky, 1988 GH4C1 Rat pit. tumor cells SRIF (chronic) In vitro Up117Kimura, 1989 Anterior pit. cells Rat Estradiol In vivo Up118Slama, 1992 Arcuate nucleus Female rat brain Estradiol In vivo Up119Vidal, 1994 AR42J Rat panc. tumor cells EGF/Gastrin In vitro Up120Visser-Wisselaar, 7315b Pit. tumor cells Estradiol In vitro � in vivo Up (� up of sst3)
1997121Froidevaux, 1999 AR42J Rat panc. tumor cells Octreotide In vivo Up
(continuous)
122Viguerie, 1987 AR42J Rat panc. tumor cells Dexamethasone In vitro Down121Froidevaux, 1999 AR42J Rat panc. tumor cells Octreotide In vitro Down
Octreotide In vivo Down(single injection)
Octreotide In vivo Down(discontinuous)
123Gunn, 2006 IMR-32 Human neuroblastoma Octreotide In vitro Down
124Behe, 2003 AR42J Rat panc. tumor cells External beam In vitro � in vivo Up (also gastrin)irradiation
125Behe, 2004 AR42J Rat panc. tumor cells External beam In vivo Up (also gastrin)irradiation
127Capello, 2005 CA20948 Rat panc. tumor cells 111ln-octreotate In vivo Up (2-fold)(low dose)
126Oddstig, 2006 NCI-H69 Small-cell lung cancer X-ray (100 keV) In vitro Up (1.5–3-time)128Melis, 2007 CA20948 Rat panc. tumor cells 177Lu-octreotate In vivo Up (2–5-time)
is locally achieved (only in the tumor), leadingto a higher tumor-to-background ratio. Second,constitutive receptor expression in the tumor isnot required; therefore, also receptor-negative tu-mors could theoretically be treated. And third, thetherapeutic effect might be enormously increasedby performing a dual gene transfer. For example,a “suicide” gene can be cotransferred with the re-ceptor gene into the tumor cell, which can be si-multaneously or subsequently used for treatment.On the other hand, patients with metastatic dis-ease are probably difficult to treat with gene ther-apy, since this requires a systemic administrationof gene therapy vectors, with all related risks.Therefore, patients with circumscribed tumor le-sions would probably benefit from gene-therapystrategies, which is the case in ovarian andglioblastoma cancer patients.
Several groups have explored the possibility toincrease sst expression on tumors by using genetransfer modalities. One of the first studies usingthe adenoviral vector AdCMVhSSTr2, encodingthe human sst2, was performed in i.p. growingSKOV3.ip1 human ovarian cancer tumor.130
Biodistribution and gamma-camera imagingshowed a higher uptake of various radiolabeled sstanalogs in infected tumors than in control tumors.
Zinn et al. and Hemminki et al. introduced theconcept of dual gene transfer by using a replication-incompetent adenoviral vector encoding sst2 and aso-called “suicide gene”: the herpes simplex virustype 1 thymidine kinase (HSV1-tk).131,132 Thisgene encodes the thymidine kinase (tk) enzyme,that unlike mammalian tk, preferentially phos-phorylates acycloguanosines, such as acyclovir(ACV) and ganciclovir (GCV), into monophos-phate compounds, which are then converted intodi- and triphosphates by cellular enzymes. Thetriphosphates are subsequently trapped inside thecell. Acycloguanosines are so-called “prodrugs,”since only the phosphorylated forms are incor-porated as chain-terminating derivatives into theDNA and/or inhibit DNA polymerase activity,eventually leading to cell kill. Moreover, thymi-dine analogs (e.g., FIAU, FIRU) do not showtoxic effects following phosphorylation and can,therefore, be used for the imaging of HSV1-tkexpression. Zinn et al. showed that expression ofboth sst2 and HSV1-tk following AdTKSSTR in-fection could be measured with 99mTc-P2045 andradioiodinated FIAU, respectively, in mice bear-ing an A-427 tumor.133
In 2002, Hemminki et al. reported the effectsof gene transfer and subsequent treatment of sub-
cutaneous (s.c.) and i.p. SKOV3.ip1 tumors invivo: Mice were infected intratumorally (i.t.) witheither AdTKSSTR, the infectivity-enhancedcounterpart, RGDTKSSTR, or control virus,134
followed by GCV treatment. The use of the in-fectivity-enhanced virus RGDTKSSTR resultedin an improved therapeutic effect, compared tocontrols. Expression of sst2 could be detectedwith 99mTc-P2045 imaging for 15 days after vi-ral infection, although the uptake of the tracer de-creased over time.
Using the same viral vector, our group showeda nonhomogeneous uptake of specific sst2 andHSV1-tk tracers in U87MG human glioma-bear-ing nude mice i.t. infected with Ad5.tk.sst2, us-ing small-animal SPECT/CT imaging (manu-script in progress). Herewith, a major hurdle ofgene therapy was visualized: Poor viral spread isnot favorable for the therapeutic outcome.
Rogers et al. transfected A-427 tumors in vivowith an adenovirus expressing sst2, AdSSTr2.They performed therapy studies in animals, re-ceiving AdSSTr2 and 400–500 �Ci [90Y]SMT-487.135 Animals that received viral infection plusradiolabeled peptide treatment showed a signifi-cantly reduced tumor quadrupling time, com-pared to the control animals.
Dual-gene transfer modalities offer the use oftwo therapeutic pathways: (1) sst2-targeted ther-apy with peptides radiolabeled with, for example,a �-particle emitting radionuclide, such as 117Luor 90Y, or (2) suicide therapy with a prodrug.Both possibilities can have a widespread effect:The first can lead to a high tumoricidal effect,since the crossfire effect of 177Lu and 90Y cancause double stranded, unrepairable DNA breaks,which leads to cell-cycle arrest and eventuallyapoptosis.136 In addition, noninfected cells arealso treated owing to the long particle range of177Lu and 90Y. In the second pathway, GCV-triphosphate can migrate through gap junctionsto the surrounding cells that might not have beentransfected and apoptosis is induced: the so-called“bystander effect.”137 These two effects are impor-tant, since the homogeneous transduction of a solidtumor has been rarely achieved.138,139
The use of molecular imaging in gene therapyexperiments offers the opportunity to provide in-formation about, for example, the location of vec-tor delivery and the extent and magnitude of genetransfer and gene expression. Integrating imag-ing techniques, such as SPECT and PET, intothese gene therapy protocols will make it possi-ble to determine optimal treatment time points
147
following vector administration. Further, imag-ing will help to optimize treatment protocols forgene therapy modalities.
Combination Treatment: RadiolabeledPeptides Plus Other Therapeutic Agents
Chemotherapeutics and radiosensitizers
Recently, investigations have been started tocombine PRRT with either chemotherapy orother radiosensitizing agents to increase thera-peutic effects in patients with neuroendocrine tu-mors. Gotthardt et al. performed mono- and com-bination treatment in nude mice bearing AR42Jtumors.140 They examined [177Lu-DOTA0,Tyr3]octreotide (177Lu-DOTATOC) either alone or incombination with doxorubicin (DX) or cisplat-inum (CS) during a 4-week period. They foundthat the combination of 177Lu-DOTATOC plusDX was 14% and that of 177Lu-DOTATOC plusCS was 23% more effective than 177Lu-DOTA-TOC treatment alone, making the combination“PRRT plus chemotherapy” an effective ap-proach to increase therapeutic efficacy in sst-ex-pressing tumors.
In patients, the radiosensitizing agent, 5-fluo-rouracil (5-FU), was investigated in combinationwith high-dose 111In-labeled octreotide.141 In 21patients with neuroendocrine tumors, the efficacyand toxicity of this combination treatment wasevaluated. The investigators found that the com-bination of high-dose [111In-DTPA0]octreotideand 5-FU was safe, and that symptomatic re-sponse rates were at least comparable to those re-ported for [111In-DTPA0]octreotide treatmentalone. Stable disease or improvements in hor-monal and functional scan abnormalities in pa-tients with previous progression were achievedwith the combination treatment. Our group re-cently started a pilot trial, using the oral prodrugof 5-FU, capecitabine, in combination with[177Lu-DOTA0,Tyr3]octreotate in patients withGEP tumors to investigate the feasibility of com-bination treatment in these patients.
Johnson et al. recently investigated combina-tion treatment of radiolabeled BN analogs withchemotherapy in a preclinical setting.56 They ex-amined the chemotherapeutic agents docetaxel(DC) and estramustine (EMP) in combinationwith 177Lu-labeled DOTA-8-AOC-BBN(7-14)NH2 (177Lu-BBN) in a PC-3 flank xenograftmodel. These chemotherapeutics were chosensince they are currently evaluated in clinical tri-
als for the treatment of androgen-independentprostate cancer. They work synergistically as mi-crotubule inhibitors and offer an increased cyto-toxic effect; they also exhibit radiosensitizationproperties. The results showed that mice treatedwith 177Lu-BBN combined with either DC aloneor DC � EMP showed a statistically significantlonger survival, 107 and 109 days, respectively,than the control animals (50 days). Further, com-bination therapy demonstrated a significant sur-vival advantage, compared to the 177Lu-BBNtherapy alone. Blood was analyzed during the ex-periment until 2 weeks after the final therapy ad-ministration, and no differences in blood-cellcounts were found.
Unfortunately, kidney damage was not evalu-ated in these studies. It is of interest to investi-gate the effect of chemotherapeutics combinedwith PRRT on radiation uptake in the kidneys andon the long-term renal damage. Wild et al. re-ported therapy studies investigating the combi-nation of the GLP-receptor-binding analog,[111In-DTPA0]exendin-4, and the angiogenesisinhibitor, PTK, in Rip1Tag2 mice. They foundthat combination therapy resulted in a signifi-cantly lower median tumor volume, compared tomonotherapy. In addition, this study did not re-veal renal toxicity in the group that was treatedwith the combination.142
An issue that also needs to be addressed is theeffect that chemotherapeutic agents might haveon receptor expression on the tumor. Fueger etal. examined the possible influence of cytotoxicor cytostatic agents on binding characteristics ofan sst ligand in vitro,143 and they found a reducedexpression of high-affinity DOTA-LAN bindingsites in response to the incubation with gemc-itabine, camptotecin, mitomycin C, and doxoru-bicin. In the case of gemcitabine, sst was againoverexpressed after a 4-day recovery period, in-dicating that the downregulation of receptor ex-pression can be reversed. However, in vivo stud-ies need to be performed to investigate the effectof chemotherapeutic agents on receptor expres-sion, especially when combination treatment isgiven.
Combining different radionuclides
In preclinical studies, we found that the anti-tumor effect of radiolabeled sst analogs is de-pendent on tumor size.65,66 In a study comparingtwo radionuclides coupled to sst analogs, we
148
demonstrated that [177Lu-DOTA0-Tyr3]octreo-tate has a very good tumor cure rate in small tu-mors of approximately 0.5 cm2, while larger tu-mors of about 7–9 cm2 were better treated with[90Y-DOTA-Tyr3]octreotide.16 These resultsagreed with the mathematical model proposed byO’Donoghue et al.15 For different radionuclideenergies, the model predicts the chance of cura-tion for different tumor diameters: According tothis model, radionuclides with lower energies(e.g., 177Lu) are optimal for small tumors and ra-dionuclides with higher energies (e.g., 90Y) areoptimal for larger tumors. This indicates thatPRRT in patients with sst2-positive tumors of dif-ferent sizes might have better potential with acombination of radionuclides with higher andlower energy �-particles. However, the feasibil-ity of this combination treatment should be fur-ther evaluated in patients, preferably in a ran-domized, clinical trial.
Hybrid molecules: Apoptosis-inducing peptides
The receptor-targeted delivery of cytotoxicagents was first proposed to reduce the toxicityof chemotherapeutic drugs in patients.144 In or-der to achieve this, chemotherapeutic agents werelinked to peptide analogs, resulting in the inter-nalization of the complete molecule into the tu-mor cell. It is conceivable that these hybrid pep-tides can be used to improve PRRT, for example,in tumors with a low receptor expression or in nonresponding receptor-expressing tumortypes.145 Nagy et al. and Hofland et al. have de-scribed the development and antitumor action ofdifferent cytotoxic sst analogs.145,146 Recently,new publications showed that the targeted cyto-toxic analog, AN-238, a conjugate based on thesst analog, RC-121, coupled to a derivative ofdoxorubicin, could offer a more effective therapythan RC-121 treatment alone in mice bearing hu-man melanoma tumors147 or endometrial tu-mors.148 In addition, the combination of targetedcytotoxic conjugates of luteinizing hormone-re-leasing hormone (LHRH) (AN-207), sst (AN-238), and BN (AN-215) were tested in mice bear-ing ovarian tumors.149 Results showed thatAN-238 and AN-215 significantly inhibited tu-mor growth, the combination being equally ef-fective. The researchers concluded that combina-tion treatment is feasible and effective with a lowtoxicity risk.149 Other studies showed that micebearing human glioblastomas, U118MG and
U87MG, could also be effectively treated withthese agents. Both AN-215 and AN-238 stronglyreduced tumor growth in glioblastoma-bearingmice.150–152 These studies show that a wide va-riety of receptor-expressing tumors can be treatedwith receptor-targeted chemotherapeutic agents,although tumor cure was not achieved yet in theseanimal studies. It would be of great interest to in-vestigate the effects on tumor growth with theseagents radiolabeled with therapeutic radionu-clides or combined with PRRT strategies. Mean-while, clinical trials using these (unlabeled) targetedchemotherapeutic agents are ongoing.145,148
Other examples of hybrid peptides are camp-tothecin-conjugated analogs of sst153,154 orBN.155,156 Several in vitro studies have shown in-creased efficacy of treatment with camptothecin-sst and camptothecin-BN conjugates, comparedto camptothecin alone.153–156 This concept wasfurther investigated in mice bearing NCI-H1299human non-small-cell lung tumors, which weretreated with the camptothecin-BN conjugate anda camptothecin-BN analog that does not specifi-cally bind the receptor. Tumor growth was sig-nificantly reduced after the incubation with thecamptothecin-BN conjugate, demonstrating theimportance of receptor-specific binding and in-ternalization of the conjugate to the tumor cellfor therapeutic purposes.155
Recently, we investigated the hybrid peptide[RGD-DTPA0]octreotate radiolabeled with111In.146,157–159 Arg-Gly-Asp (RGD) binds theintegrin receptor �v�3 and is known as an apoptosis-inducing agent by the direct activationof caspase-3.160 We found that [RGD-111In-DTPA0]octreotate predominantly internalizes bythe sst2, probably owing to the higher affinity ofoctreotate for the sst2 than that of RGD for the�v�3.157 Further, when [RGD-111In-DTPA0]oc-treotate was compared with either [111In-DTPA0]RGD or [111In-DTPA0]octreotate in aclonogenic survival assay, using sst2/�v�3 ex-pressing tumor cells, [RGD-111In-DTPA0]octreo-tate showed the highest tumoricidal effects.158
Caspase-3 activity assays confirmed that [RGD-111In-DTPA0]octreotate had the most pronouncedactivation of this executioner protease in theapoptosis pathway. Unfortunately, in vivo stud-ies showed that renal uptake of [RGD-111In-DTPA0]octreotate was high, a disadvantage forPRRT.159 However, caspase-3 activity after in-cubation with the unlabeled hybrid peptide washigher than after RGD or DTPA-octreotide alone,
149
making unlabeled [RGD-DTPA0]octreotate dur-ing or after PRRT interesting as well.159
Combining different peptides: Multireceptor targeting
Many cancer types simultaneous overexpressseveral peptide receptors.92 There are a numberof possible advantages in utilizing multiple radi-olabeled ligands for the therapeutic application ofneuroendocrine tumors: (1) in vivo application ofmultireceptor targeting selectively increases theradioactivity accumulation in tumors, (2) some ofthe receptors are not homogeneously expressed,and by multireceptor targeting it is possible toachieve a higher tumoricidal effect, and (3) thereis a reduced risk of the loss of some peptide re-ceptors during therapy owing to tumor dediffer-entiation and the subsequent loss of some pep-tide receptors.16
Reubi and Waser performed in vitro autoradi-ography on neuroendocrine tumors, includingileal carcinoids, bronchial carcinoids, insulino-mas, gastrinomas, glucagonomas, and vipomas.92
They found that all neuroendocrine tumors theyexamined expressed two or more receptors; sev-eral combinations of peptides are of interest foroptimal targeting of neuroendocrine tumors invivo: (1) combination of ligands for the GLP-1and CCK2 receptors for insulinomas and (2) amixture of sst2, GLP-1, and GRP-radiolabeled li-gands for gastrinomas.
Radiation protection in normal organs
Increasing the therapeutic window can also beachieved by reducing the radiation toxicity tonormal organs. In peptide(sst)-based therapy, thekidney is one of the dose-limiting organs, andsome clinical studies showed renal toxicity fol-lowing PRRT.161–163 It is, therefore, favorable toreduce renal radiation, making it feasible to in-crease the total amount of injected radioactivity.
It has been found that radiolabeled sst analogsare filtered and reabsorbed in the proximaltubules of rat kidneys.164 Also, in the human kid-ney, radioactivity was mostly concentrated in thecortex and the megalin/cubulin system was foundto play an essential role in the reabsorption of oc-treotide.165,166 In addition, it was shown that 18%of the renal uptake of sst2-targeting peptides canbe dedicated to sst-mediated uptake.167
Standard procedure to reduce renal uptake dur-ing PRRT in our institution is a 4-hour infusionof a mixture of lysine and arginine.17 We inves-
tigated whether an oral administration of lysinecould also reduce renal uptake.168 In rats, oral ad-ministration of lysine reduced renal uptake with40%, comparable to the reduction found with i.v.administration of lysine.169
Moreover, other agents, such as gelofu-sine,170,171 colchicine,172 and the radioprotectivedrug amifostine,173 could improve kidney-pro-tection strategies currently used in the clinic.
CONCLUSIONS
Many tumors overexpress one or more receptors,which can be targeted using receptor-specific ra-diolabeled peptides. So far, sst-targeting peptidesare widely used for the imaging and therapy ofcancer patients. PRRT with 177Lu-labeled sstanalogs has resulted in symptomatic improve-ment, prolonged survival, and enhanced qualityof life of neuroendocrine tumor patients. PRS andPRRT targeting other tumor-specific receptors,such as GRP and CCK receptors, are well on theirway to clinical utilization as well.
The literature shows that it is possible to in-crease the receptor density on tumor cells by us-ing different methods. In PRRT treatment, thiswould enable the administration of higher thera-peutic doses to tumors, which might lead to ahigher cure rate in patients.
Targeting one or several tumor-specific recep-tors by combinations of therapeutic agents, aswell as by reducing nontarget uptake of radioac-tivity, will enlarge the therapeutic window ofPRRT. Clinical studies will provide more insightin the effects of combination treatment strategiesin cancer patients.
REFERENCES
1. Krenning EP, Teunissen JJ, Valkema R, et al. Molec-ular radiotherapy with somatostatin analogs for (neuro)endocrine tumors. J Endocrinol Invest 2005;28:146.
2. Krenning EP, Kwekkeboom DJ, Bakker WH, et al. So-matostatin receptor scintigraphy with [111In-DTPA-D-Phe1]- and [123I-Tyr3]-octreotide: The Rotterdam ex-perience with more than 1000 patients. Eur J Nucl Med1993;20:716.
3. Kwekkeboom D, Krenning EP, de Jong M. Peptide re-ceptor imaging and therapy. J Nucl Med 2000;41:1704.
4. Valkema R, De Jong M, Bakker WH, et al. Phase Istudy of peptide receptor radionuclide therapy with[In-DTPA]octreotide: The Rotterdam experience.Semin Nucl Med 2002;32:110.
150
5. Bodei L, Cremonesi M, Zoboli S, et al. Receptor-me-diated radionuclide therapy with 90Y-DOTATOC inassociation with amino acid infusion: A phase I study.Eur J Nucl Med Mol Imaging 2003;30:207.
6. Valkema R, Pauwels S, Kvols LK, et al. Survival andresponse after peptide receptor radionuclide therapywith [90Y-DOTA0,Tyr3]octreotide in patients with ad-vanced gastroenteropancreatic neuroendocrine tumors.Semin Nucl Med 2006;36:147.
7. Otte A, Herrmann R, Heppeler A, et al. Yttrium-90DOTATOC: First clinical results. Eur J Nucl Med1999;26:1439.
8. Waldherr C, Pless M, Maecke HR, et al. Tumor re-sponse and clinical benefit in neuroendocrine tumorsafter 7.4 GBq (90)Y-DOTATOC. J Nucl Med2002;43:610.
9. Chinol M, Bodei L, Cremonesi M, et al. Receptor-me-diated radiotherapy with Y-DOTA-D-Phe-Tyr-oc-treotide: The experience of the European Institute ofOncology Group. Semin Nucl Med 2002;32:141.
10. van Essen M, Krenning EP, de Jong M, et al. Peptidereceptor radionuclide therapy with radiolabelled so-matostatin analogues in patients with somatostatin re-ceptor positive tumours. Acta Oncologica 2007;46:723.
11. Reubi JC, Schar JC, Waser B, et al. Affinity profilesfor human somatostatin receptor subtypes SST1–SST5of somatostatin radiotracers selected for scintigraphicand radiotherapeutic use. Eur J Nucl Med 2000;27:273.
12. de Jong M, Breeman WA, Bakker WH, et al. Com-parison of (111)In-labeled somatostatin analogues fortumor scintigraphy and radionuclide therapy. CancerRes 1998;58:437.
13. Esser JP, Krenning EP, Teunissen JJ, et al. Compari-son of [(177)Lu-DOTA(0),Tyr(3)]octreotate and[(177)Lu-DOTA(0),Tyr(3)]octreotide: Which peptideis preferable for PRRT? Eur J Nucl Med Mol Imaging2006;33:1346.
14. Kwekkeboom DJ, Bakker WH, Kooij PP, et al. [177Lu-DOTA0Tyr3]octreotate: Comparison with [111In-DT-PAo]octreotide in patients. Eur J Nucl Med 2001;28:1319.
15. O’Donoghue JA, Bardies M, Wheldon TE. Relation-ships between tumor size and curability for uniformlytargeted therapy with beta-emitting radionuclides. JNucl Med 1995;36:1902.
16. de Jong M, Breeman WA, Valkema R, et al. Combi-nation radionuclide therapy using 177Lu- and 90Y-labeled somatostatin analogs. J Nucl Med 2005;46(Suppl. 1):13S.
17. Kwekkeboom DJ, Teunissen JJ, Bakker WH, et al. Radiolabeled somatostatin analog [177Lu-DOTA0,Tyr3]octreotate in patients with endocrine gastroenteropan-creatic tumors. J Clin Oncol 2005;23:2754.
18. Teunissen JJ, Kwekkeboom DJ, Krenning EP. Qual-ity of life in patients with gastroenteropancreatic tu-mors treated with [177Lu-DOTA0,Tyr3]octreotate. JClin Oncol 2004;22:2724.
19. Zhang H, Chen J, Waldherr C, et al. Synthesis andevaluation of bombesin derivatives on the basis of pan-
bombesin peptides labeled with indium-111, lutetium-177, and yttrium-90 for targeting bombesin receptor-expressing tumors. Cancer Res 2004;64:6707.
20. Breeman WA, Hofland LJ, de Jong M, et al. Evalua-tion of radiolabelled bombesin analogues for receptor-targeted scintigraphy and radiotherapy. Int J Cancer1999;81:658.
21. Ginj M, Zhang H, Waser B, et al. Radiolabeled so-matostatin receptor antagonists are preferable to ago-nists for in vivo peptide receptor targeting of tumors.Proc Natl Acad Sci USA 2006;103:16436.
22. Gabriel M, Decristoforo C, Donnemiller E, et al. Anintrapatient comparison of 99mTc-EDDA/HYNIC-TOC with 111In-DTPA-octreotide for diagnosis of so-matostatin receptor-expressing tumors. J Nucl Med2003;44:708.
23. Bangard M, Behe M, Guhlke S, et al. Detection of so-matostatin receptor-positive tumours using the new99mTc-tricine-HYNIC-D-Phe1-Tyr3-octreotide: Firstresults in patients and comparison with 111In-DTPA-D-Phe1-octreotide. Eur J Nucl Med 2000;27:628.
24. Hubalewska-Dydejczyk A, Fross-Baron K, GolkowskiF, et al. 99mTc-EDDA/HYNIC-octreotate in detectionof atypical bronchial carcinoid. Exp Clin EndocrinolDiabetes 2007;115:47.
25. Hubalewska-Dydejczyk A, Fross-Baron K, Mikola-jczak R, et al. 99mTc-EDDA/HYNIC-octreotate scinti-graphy, an efficient method for the detection and stag-ing of carcinoid tumours: Results of 3 years’experience. Eur J Nucl Med Mol Imaging 2006;33:1123.
26. Hubalewska-Dydejczyk A, Szybinski P, Fross-BaronK, et al. (99m)Tc-EDDA/HYNIC-octreotate—a new ra-diotracer for detection and staging of NET: A case ofmetastatic duodenal carcinoid. Nucl Med Rev CentEast Eur 2005;8:155.
27. Gabriel M, Decristoforo C, Maina T, et al. 99mTc-N4-[Tyr3]octreotate versus 99mTc-EDDA/HYNIC-[Tyr3]octreotide: An intrapatient comparison of two noveltechnetium-99m-labeled tracers for somatostatin re-ceptor scintigraphy. Cancer Biother Radiopharm2004;19:73.
28. Nikolopoulou A, Maina T, Sotiriou P, et al.Tetraamine-modified octreotide and octreotate: Label-ing with 99mTc and preclinical comparison in AR4-2Jcells and AR4-2J tumor-bearing mice. J Pept Sci 2006;12:124.
29. Decristoforo C, Maina T, Nock B, et al. 99mTc-De-motate 1: First data in tumour patients—results of apilot/phase I study. Eur J Nucl Med Mol Imaging 2003;30:1211.
30. Meisetschlager G, Poethko T, Stahl A, et al. Gluc-Lys([18F]FP)-TOCA PET in patients with SSTR-pos-itive tumors: Biodistribution and diagnostic evaluationcompared with [111In]DTPA-octreotide. J Nucl Med2006;47:566.
31. Anderson CJ, Dehdashti F, Cutler PD, et al. 64Cu-TETA-octreotide as a PET imaging agent for patientswith neuroendocrine tumors. J Nucl Med 2001;42:213.
151
32. Breeman WA, de Jong M, de Blois E, et al. Radiola-belling DOTA-peptides with 68Ga. Eur J Nucl MedMol Imaging 2005;32:478.
33. Kowalski J, Henze M, Schuhmacher J, et al. Evalua-tion of positron emission tomography imaging using[68Ga]-DOTA-D-Phe(1)-Tyr(3)-Octreotide in compar-ison to [111In]-DTPAOC SPECT. First results in pa-tients with neuroendocrine tumors. Mol Imaging Biol2003;5:42.
34. Gabriel M, Decristoforo C, Kendler D, et al. 68Ga-DOTA-Tyr3-octreotide PET in neuroendocrine tu-mors: Comparison with somatostatin receptor scintig-raphy and CT. J Nucl Med 2007;48:508.
35. Kulaksiz H, Eissele R, Rossler D, et al. Identificationof somatostatin receptor subtypes 1, 2A, 3, and 5 inneuroendocrine tumours with subtype specific anti-bodies. Gut 2002;50:52.
36. Reubi JC, Waser B, Schaer JC, et al. Somatostatin re-ceptor sst1–sst5 expression in normal and neoplastichuman tissues using receptor autoradiography with sub-type-selective ligands. Eur J Nucl Med 2001;28:836.
37. Ginj M, Schmitt JS, Chen J, et al. Design, synthesis,and biological evaluation of somatostatin-based ra-diopeptides. Chem Biol 2006;13:1081.
38. Wild D, Macke HR, Waser B, et al. 68Ga-DOTANOC:A first compound for PET imaging with high affinityfor somatostatin receptor subtypes 2 and 5. Eur J NuclMed Mol Imaging 2005;32:724.
39. Reubi JC, Wenger S, Schmuckli-Maurer J, et al.Bombesin receptor subtypes in human cancers: De-tection with the universal radioligand (125)I-[D-TYR(6), beta-ALA(11), PHE(13), NLE(14)]bombesin(6-14). Clin Cancer Res 2002;8:1139.
40. Jemal A, Siegel R, Ward E, et al. Cancer statistics,2007. CA Cancer J Clin 2007;57:43.
41. Breeman WA, de Jong M, Bernard B, et al. Tissue dis-tribution and metabolism of radioiodinated DTPA0, D-Tyr1, and Tyr3 derivatives of octreotide in rats. Anticancer Res 1998;18:83.
42. Nock B, Nikolopoulou A, Chiotellis E, et al.[(99m)Tc]Demobesin 1, a novel potent bombesin ana-logue for GRP-receptor-targeted tumour imaging. EurJ Nucl Med Mol Imaging 2003;30:247.
43. Nock BA, Nikolopoulou A, Galanis A, et al. Potentbombesin-like peptides for GRP-receptor targeting oftumors with 99mTc: A preclinical study. J Med Chem2005;48:100.
44. Van de Wiele C, Dumont F, Vanden Broecke R, et al.Technetium-99m RP527, a GRP analogue for visuali-sation of GRP-receptor-expressing malignancies: Afeasibility study. Eur J Nucl Med 2000;27:1694.
45. van Bokhoven A, Varella-Garcia M, Korch C, et al.Molecular characterization of human prostate carci-noma cell lines. Prostate 2003;57:205.
46. Hoffman TJ, Gali H, Smith CJ, et al. Novel series of111In-labeled bombesin analogs as potential radio-pharmaceuticals for specific targeting of gastrin-re-leasing peptide receptors expressed on human prostatecancer cells. J Nucl Med 2003;44:823.
47. Breeman WA, de Jong M, Erion JL, et al. Preclinicalcomparison of (111)In-labeled DTPA- or DOTA-bombesin analogs for receptor-targeted scintigraphyand radionuclide therapy. J Nucl Med 2002;43:1650.
48. Ferro-Flores G, Arteaga de Murphy C, Rodriguez-Cortes J, et al. Preparation and evaluation of 99mTc-EDDA/HYNIC-[Lys 3]-bombesin for imaging gastrin-releasing peptide receptor-positive tumours. Nucl MedCommun 2006;27:371.
49. Garcia Garayoa E, Ruegg D, Blauenstein P, et al.Chemical and biological characterization of newRe(CO)(3)/[(99m)Tc](CO)(3) bombesin analogues.Nucl Med Biol 2007;34:17.
50. Reubi JC, Macke HR, Krenning EP. Candidates forpeptide receptor radiotherapy today and in the future.J Nucl Med 2005;46(Suppl.1):67S.
51. de Visser M, Bernard HF, Erion JL, et al. Novel(111)In-labelled bombesin analogues for molecularimaging of prostate tumours. Eur J Nucl Med MolImaging 2007;34:1228.
52. Zhang H, Schuhmacher J, Waser B, et al. DOTA-PESIN, a DOTA-conjugated bombesin derivative de-signed for the imaging and targeted radionuclide treat-ment of bombesin receptor-positive tumours. Eur JNucl Med Mol Imaging 2007;34:1198.
53. Yang YS, Zhang X, Xiong Z, et al. Comparative invitro and in vivo evaluation of two 64Cu-labeledbombesin analogs in a mouse model of human prostateadenocarcinoma. Nucl Med Biol 2006;33:371.
54. Smith CJ, Gali H, Sieckman GL, et al. Radiochemicalinvestigations of (177)Lu-DOTA-8-Aoc-BBN[7-14]NH(2): An in vitro/in vivo assessment of the target-ing ability of this new radiopharmaceutical for PC-3 hu-man prostate cancer cells. Nucl Med Biol 2003;30:101.
55. Rogers BE, Bigott HM, McCarthy DW, et al. Micro-PET imaging of a gastrin-releasing peptide-receptor-positive tumor in a mouse model of human prostatecancer using a 64Cu-labeled bombesin analogue. Bio-conjug Chem 2003;14:756.
56. Johnson CV, Shelton T, Smith CJ, et al. Evaluation ofcombined (177)Lu-DOTA-8-AOC-BBN (7-14)NH(2)GRP-receptor-targeted radiotherapy and chemother-apy in PC-3 human prostate tumor cell xenograftedSCID mice. Cancer Biother Radiopharm 2006;21:155.
57. Biddlecombe GB, Rogers BE, Visser MD, et al. Mo-lecular imaging of gastrin-releasing peptide-receptor-positive tumors in mice using (64)Cu- and (86)Y-DOTA-(Pro(1),Tyr(4))-Bombesin(1-14). BioconjugChem 2007.
58. Lantry LE, Cappelletti E, Maddalena ME, et al. 177Lu-AMBA: Synthesis and characterization of a selective177Lu-labeled GRP-R agonist for systemic radiother-apy of prostate cancer. J Nucl Med 2006;47:1144.
59. Van de Wiele C, Dumont F, van Belle S, et al. Is therea role for agonist gastrin-releasing peptide receptor ra-dioligands in tumour imaging? Nucl Med Commun2001;22:5.
60. Baum R, Prasad V, Mutloka N, et al. Molecular imag-ing of bombesin receptors in various tumors by Ga-68
152
AMBA PET/CT: First results. J Nucl Med 2007;48:79P.
61. de Visser M, van Weerden WM, Melis M, et al. Ra-diolabeled bombesin analogs in preclinical studies. JNucl Med 2007;48 (Suppl. 2):24P.
62. Mancuso A, Oudard S, Sternberg CN. Effective che-motherapy for hormone-refractory prostate cancer(HRPC): Present status and perspectives with taxane-based treatments. Crit Rev Oncol Hematol 2007;61:176.
63. Oudard S, Banu E, Beuzeboc P, et al. Multicenter ran-domized phase II study of two schedules of docetaxel,estramustine, and prednisone versus mitoxantrone plusprednisone in patients with metastatic hormone-re-fractory prostate cancer. J Clin Oncol 2005;23:3343.
64. de Visser M, van Weerden WM, de Ridder CM, et al.Androgen-dependent expression of the gastrin-releas-ing peptide receptor in human prostate tumor xeno-grafts. J Nucl Med 2007;48:88.
65. de Jong M, Breeman WA, Bernard BF, et al. Tumorresponse after [(90)Y-DOTA(0),Tyr(3)]octreotide ra-dionuclide therapy in a transplantable rat tumor modelis dependent on tumor size. J Nucl Med 2001;42:1841.
66. de Jong M, Breeman WA, Bernard BF, et al. [177Lu-DOTA(0),Tyr3] octreotate for somatostatin-receptor-targeted radionuclide therapy. Int J Cancer 2001;92:628.
67. De Jong M, Valkema R, Jamar F, et al. Somatostatin-receptor-targeted radionuclide therapy of tumors: Pre-clinical and clinical findings. Semin Nucl Med 2002;32:133.
68. Reubi JC, Waser B, Friess H, et al. Neurotensin re-ceptors: A new marker for human ductal pancreaticadenocarcinoma. Gut 1998;42:546.
69. Kuhar MJ. Imaging receptors for drugs in neural tis-sue. Neuropharmacology 1987;26:911.
70. Ehlers RA, Kim S, Zhang Y, et al. Gut-peptide-re-ceptor expression in human pancreatic cancers. AnnSurg 2000;231:838.
71. Zhang K, An R, Gao Z, et al. Radionuclide imagingof small-cell lung cancer (SCLC) using 99mTc-labeledneurotensin peptide 8-13. Nucl Med Biol 2006;33:505.
72. Nock BA, Nikolopoulou A, Reubi JC, et al. Towardstable N4-modified neurotensins for NTS1-receptor-targeted tumor imaging with 99mTc. J Med Chem2006;49:4767.
73. Maes V, Garcia-Garayoa E, Blauenstein P, et al. Novel99mTc-labeled neurotensin analogues with optimizedbiodistribution properties. J Med Chem 2006;49:1833.
74. Garcia-Garayoa E, Maes V, Blauenstein P, et al. Dou-ble-stabilized neurotensin analogues as potential ra-diopharmaceuticals for NTR-positive tumors. NuclMed Biol 2006;33:495.
75. Garcia-Garayoa E, Allemann-Tannahill L, BlauensteinP, et al. In vitro and in vivo evaluation of new radio-labeled neurotensin(8-13) analogues with high affinityfor NT1 receptors. Nucl Med Biol 2001;28:75.
76. Lugrin D, Vecchini F, Doulut S, et al. Reduced pep-tide bond pseudopeptide analogues of neurotensin:
Binding and biological activities and in vitro metabolicstability. Eur J Pharmacol 1991;205:191.
77. de Visser M, Janssen PJ, Srinivasan A, et al. Stabilised111In-labelled DTPA- and DOTA-conjugated neurotensinanalogues for imaging and therapy of exocrine pancre-atic cancer. Eur J Nucl Med Mol Imaging 2003;30:1134.
78. Emami B, Lyman J, Brown A, et al. Tolerance of nor-mal tissue to therapeutic irradiation. Int J Radiat On-col Biol Phys 1991;21:109.
82. Reubi JC, Schaer JC, Waser B. Cholecystokinin(CCK)-A and CCK-B/gastrin receptors in human tumors. Can-cer Res 1997;57:1377.
83. Behr TM, Jenner N, Radetzky S, et al. Targeting ofcholecystokinin-B/gastrin receptors in vivo: Preclini-cal and initial clinical evaluation of the diagnostic andtherapeutic potential of radiolabelled gastrin. Eur JNucl Med 1998;25:424.
84. Reubi JC, Waser B, Schaer JC, et al. UnsulfatedDTPA- and DOTA-CCK analogs as specific high-affinity ligands for CCK-B receptor-expressing humanand rat tissues in vitro and in vivo. Eur J Nucl Med1998;25:481.
85. de Jong M, Bakker WH, Bernard BF, et al. Preclini-cal and initial clinical evaluation of 111In-labeled non-sulfated CCK8 analog: A peptide for CCK-B-recep-tor-targeted scintigraphy and radionuclide therapy. JNucl Med 1999;40:2081.
86. Kwekkeboom DJ, Bakker WH, Kooij PP, et al. Chole-cystokinin receptor imaging using an octapeptideDTPA-CCK analogue in patients with medullary thy-roid carcinoma. Eur J Nucl Med 2000;27:1312.
87. Behr TM, Jenner N, Behe M, et al. Radiolabeled pep-tides for targeting cholecystokinin-B/gastrin receptor-expressing tumors. J Nucl Med 1999;40:1029.
88. Nock BA, Maina T, Behe M, et al. CCK-2/gastrin re-ceptor-targeted tumor imaging with (99m)Tc-labeledminigastrin analogs. J Nucl Med 2005;46:1727.
89. Gotthardt M, Behe MP, Beuter D, et al. Improved tu-mour detection by gastrin receptor scintigraphy in pa-tients with metastasised medullary thyroid carcinoma.Eur J Nucl Med Mol Imaging 2006;33:1273.
90. Gotthardt M, Behe MP, Grass J, et al. Added value ofgastrin receptor scintigraphy in comparison to so-matostatin receptor scintigraphy in patients with car-cinoids and other neuroendocrine tumours. Endocr Re-lat Cancer 2006;13:1203.
91. Mayo KE, Miller LJ, Bataille D, et al. InternationalUnion of Pharmacology. XXXV. The glucagon re-ceptor family. Pharmacol Rev 2003;55:167.
153
92. Reubi JC, Waser B. Concomitant expression of sev-eral peptide receptors in neuroendocrine tumours: Mo-lecular basis for in vivo multireceptor tumour target-ing. Eur J Nucl Med Mol Imaging 2003;30:781.
93. Korner M, Stockli M, Waser B, et al. GLP-1 receptorexpression in human tumors and human normal tis-sues: Potential for in vivo targeting. J Nucl Med2007;48:736.
94. Meier JJ, Nauck MA. Glucagon-like peptide 1 (GLP-1) in biology and pathology. Diabetes Metab Res Rev2005;21:91.
95. Hassan M, Eskilsson A, Nilsson C, et al. In vivo dy-namic distribution of 131I-glucagon-like peptide-1 (7-36) amide in the rat studied by gamma camera. NuclMed Biol 1999;26:413.
96. Gotthardt M, Fischer M, Naeher I, et al. Use of the in-cretin hormone glucagon-like peptide-1 (GLP-1) forthe detection of insulinomas: Initial experimental re-sults. Eur J Nucl Med Mol Imaging 2002;29:597.
97. Gotthardt M, Lalyko G, van Eerd-Vismale J, et al. Anew technique for in vivo imaging of specific GLP-1binding sites: First results in small rodents. Regul Pept2006;137:162.
98. Wild D, Behe M, Wicki A, et al. [Lys40(Ahx-DTPA-111In)NH2]exendin-4, a very promising ligand forglucagon-like peptide-1 (GLP-1) receptor targeting. JNucl Med 2006;47:2025.
99. Wicki A, Wild D, Storch D, et al. [Lys40(Ahx-DTPA-111In)NH2]-exendin-4 is a highly efficient radiothera-peutic for glucagon-like peptide-1 receptor-targetedtherapy for insulinoma. Clin Cancer Res 2007;13:3696.
100. Brooks PC. Role of integrins in angiogenesis. Eur JCancer 1996;32A:2423.
101. Plow EF, Haas TA, Zhang L, et al. Ligand binding tointegrins. J Biol Chem 2000;275:21785.
102. Gurrath M, Muller G, Kessler H, et al. Conforma-tion/activity studies of rationally designed potent anti-adhesive RGD peptides. Eur J Biochem 1992;210:911.
103. Aumailley M, Gurrath M, Muller G, et al. Arg-Gly-Asp constrained within cyclic pentapeptides. Strongand selective inhibitors of cell adhesion to vitronectinand laminin fragment P1. FEBS Lett 1991;291:50.
104. van Hagen PM, Breeman WA, Bernard HF, et al. Eval-uation of a radiolabelled cyclic DTPA-RGD analoguefor tumour imaging and radionuclide therapy. Int JCancer 2000;90:186.
105. Chen X, Park R, Tohme M, et al. Micro-PET and au-toradiographic imaging of breast cancer alpha v-inte-grin expression using 18F- and 64Cu-labeled RGD pep-tide. Bioconjug Chem 2004;15:41.
106. Cai W, Zhang X, Wu Y, et al. A thiol-reactive 18F-labeling agent, N-[2-(4-18F-fluorobenzamido)ethyl]male-imide, and synthesis of RGD-peptide-based tracer forPET imaging of alpha v-beta-3 integrin expression. J Nucl Med 2006;47:1172.
108. Beer AJ, Haubner R, Sarbia M, et al. Positron emis-sion tomography using [18F]galacto-RGD identifiesthe level of integrin alpha(v)beta-3 expression in man.Clin Cancer Res 2006;12:3942.
109. Beer AJ, Haubner R, Wolf I, et al. PET-based humandosimetry of 18F-galacto-RGD, a new radiotracer forimaging alpha v-beta3-expression. J Nucl Med2006;47:763.
110. Beer AJ, Haubner R, Goebel M, et al. Biodistributionand pharmacokinetics of the alpha-v-beta-3-selectivetracer 18F-galacto-RGD in cancer patients. J Nucl Med2005;46:1333.
111. Dijkgraaf I, Kruijtzer JA, Liu S, et al. Improved tar-geting of the alpha(v)beta (3) integrin by multimeri-sation of RGD peptides. Eur J Nucl Med Mol Imag-ing 2007;34:267.
112. Dijkgraaf I, Rijnders AY, Soede A, et al. Synthesis ofDOTA-conjugated multivalent cyclic-RGD peptidedendrimers via 1,3-dipolar cycloaddition and their bi-ological evaluation: Implications for tumor targetingand tumor imaging purposes. Org Biomol Chem2007;5:935.
113. Dijkgraaf I, Liu S, Kruijtzer JA, et al. Effects of linkervariation on the in vitro and in vivo characteristics ofan 111In-labeled RGD peptide. Nucl Med Biol 2007;34:29.
114. Dijkgraaf I, Kruijtzer JA, Frielink C, et al. Alpha-v-beta-3 integrin-targeting of intraperitoneally growingtumors with a radiolabeled RGD peptide. Int J Can-cer 2007;120:605.
115. Kimura N, Hayafuji C, Konagaya H, et al. 17-beta-estradiol induces somatostatin (SRIF) inhibition ofprolactin release and regulates SRIF receptors in ratanterior pituitary cells. Endocrinology 1986;119:1028.
116. Presky DH, Schonbrunn A. Somatostatin pretreatmentincreases the number of somatostatin receptors inGH4C1 pituitary cells and does not reduce cellular re-sponsiveness to somatostatin. J Biol Chem 1988;263:714.
117. Kimura N, Hayafuji C, Kimura N. Characterization of17-beta-estradiol-dependent and -independent somato-statin receptor subtypes in rat anterior pituitary. J BiolChem 1989;264:7033.
118. Slama A, Videau C, Kordon C, et al. Estradiol regu-lation of somatostatin receptors in the arcuate nucleusof the female rat. Neuroendocrinology 1992;56:240.
119. Vidal C, Rauly I, Zeggari M, et al. Upregulation of so-matostatin receptors by epidermal growth factor andgastrin in pancreatic cancer cells. Mol Pharmacol1994;46:97.
120. Visser-Wisselaar HA, Van Uffelen CJ, Van KoetsveldPM, et al. 17-beta-estradiol-dependent regulation ofsomatostatin receptor subtype expression in the 7315bprolactin secreting rat pituitary tumor in vitro and invivo. Endocrinology 1997;138:1180.
121. Froidevaux S, Hintermann E, Torok M, et al. Differ-ential regulation of somatostatin receptor type 2 (sst2) expression in AR4-2J tumor cells implanted intomice during octreotide treatment. Cancer Res1999;59:3652.
154
122. Viguerie N, Esteve JP, Susini C, et al. Dexamethasoneeffects on somatostatin receptors in pancreatic acinarAR4-2J cells. Biochem Biophys Res Commun1987;147:942.
123. Gunn SH, Schwimer JE, Cox M, et al. In vitro mod-eling of the clinical interactions between octreotide and111In-pentetreotide: Is there evidence of somatostatinreceptor downregulation? J Nucl Med 2006;47:354.
124. Béhé M, Püsken M, Henzel M, et al. Upregulation ofgastrin and somatostatin receptor after irradiation. EurJ Nucl Med Mol Imaging 2003;30:S218.
125. Béhé M, Koller S, Püsken M, et al. Irradiation-inducedupregulation of somatostatin and gastrin receptors invitro and in vivo. Eur J Nucl Med Mol Imaging 2004;31:S237.
126. Oddstig J, Bernhardt P, Nilsson O, et al. Radiation-in-duced upregulation of somatostatin receptor expres-sion in small-cell lung cancer in vitro. Nucl Med Biol2006;33:841.
127. Capello A, Krenning E, Bernard B, et al. 111In-labelledsomatostatin analogues in a rat tumour model: So-matostatin receptor status and effects of peptide re-ceptor radionuclide therapy. Eur J Nucl Med MolImaging 2005;32:1288.
128. Melis M, Forrer F, Capello A, et al. Upregulation ofsomatostatin receptor density on rat CA20948 tumoursescaped from low dose [177Lu-DOTA0,Tyr3]octreotatetherapy. Q J Nucl Med 2007.
129. Seth P. Vector-mediated cancer gene therapy: Anoverview. Cancer Biol Ther 2005;4:512.
130. Rogers BE, McLean SF, Kirkman RL, et al. In vivolocalization of [(111)In]-DTPA-D-Phe1-octreotide tohuman ovarian tumor xenografts induced to expressthe somatostatin receptor subtype 2 using an adenovi-ral vector. Clin Cancer Res 1999;5:383.
131. Zinn KR, Chaudhuri TR, Buchsbaum DJ, et al. Simul-taneous evaluation of dual gene transfer to adherent cellsby gamma-ray imaging. Nucl Med Biol 2001;28:135.
132. Hemminki A, Belousova N, Zinn KR, et al. An ade-novirus with enhanced infectivity mediates molecularchemotherapy of ovarian cancer cells and allows imag-ing of gene expression. Mol Ther 2001;4:223.
133. Zinn KR, Chaudhuri TR, Krasnykh VN, et al. Gammacamera dual imaging with a somatostatin receptor andthymidine kinase after gene transfer with a bicistronicadenovirus in mice. Radiology 2002;223:417.
134. Hemminki A, Zinn KR, Liu B, et al. In vivo molecu-lar chemotherapy and noninvasive imaging with an in-fectivity-enhanced adenovirus. J Natl Cancer Inst2002;94:741.
135. Rogers BE, Zinn KR, Lin CY, et al. Targeted radio-therapy with [(90)Y]-SMT 487 in mice bearing humannon-small-cell lung tumor xenografts induced to ex-press human somatostatin receptor subtype 2 with anadenoviral vector. Cancer 2002;94:1298.
136. Payne CM, Bjore CG, Jr, Schultz DA. Change in thefrequency of apoptosis after low- and high-dose X-ir-radiation of human lymphocytes. J Leukoc Biol1992;52:433.
137. Freeman SM, Abboud CN, Whartenby KA, et al. The“bystander effect”: Tumor regression when a fractionof the tumor mass is genetically modified. Cancer Res1993;53:5274.
138. Buchsbaum DJ, Chaudhuri TR, Yamamoto M, et al.Gene expression imaging with radiolabeled peptides.Ann Nucl Med 2004;18:275.
139. ter Horst M, Verwijnen SM, Brouwer E, et al. Lo-coregional delivery of adenoviral vectors. J Nucl Med2006;47:1483.
140. Gotthardt M, Librizzi D, Wolf D, et al. Increased ther-apeutic efficacy through combination of Lu-177-DOTATOC and chemotheray in neuroendocrine tumoursin vivo. Eur J Nucl Med Mol Imaging 2006;33:S115.
141. Kong G, Lau E, Ramdave S, et al. High-dose In-111
octreotide therapy in combination with radiosensitiz-ing 5-FU chemotherapy for treatment of SSR-express-ing neuroendocrine tumors. J Nucl Med 2005;46:151P.
142. Wild D, Wicki A, Christofori G. Combination therapywith [(Lys40(Ahx-[111In-DTPA])]-exendin-4 andVEGF-receptor tyrosine kinase inhibitor PTK in aglucagon-like-peptide-1 receptor-positive transgenicmouse tumor model. J Nucl Med 2007;48(Suppl.2):83P.
143. Fueger BJ, Hamilton G, Raderer M, et al. Effects ofchemotherapeutic agents on expression of somato-statin receptors in pancreatic tumor cells. J Nucl Med2001;42:1856.
144. Schally AV, Nagy A. Cancer chemotherapy based ontargeting of cytotoxic peptide conjugates to their re-ceptors on tumors. Eur J Endocrinol 1999;141:1.
145. Nagy A, Schally AV. Targeting cytotoxic conjugatesof somatostatin, luteinizing hormone-releasing hor-mone, and bombesin to cancers expressing their re-ceptors: A “smarter” chemotherapy. Curr Pharm Des2005;11:1167.
146. Hofland LJ, Capello A, Krenning EP, et al. Inductionof apoptosis with hybrids of Arg-Gly-Asp moleculesand peptides and antimitotic effects of hybrids of cytostatic drugs and peptides. J Nucl Med 2005;46(Suppl.1):191S.
147. Keller G, Schally AV, Nagy A, et al. Effective ther-apy of experimental human malignant melanomas witha targeted cytotoxic somatostatin analogue without in-duction of multidrug resistance proteins. Int J Oncol2006;28:1507.
148. Engel JB, Schally AV, Halmos G, et al. Targeted ther-apy with a cytotoxic somatostatin analog, AN-238, in-hibits growth of human experimental endometrial carcinomas expressing multidrug resistance proteinMDR-1. Cancer 2005;104:1312.
149. Buchholz S, Keller G, Schally AV, et al. Therapy ofovarian cancers with targeted cytotoxic analogs ofbombesin, somatostatin, and luteinizing hormone-re-leasing hormone and their combinations. Proc NatlAcad Sci USA 2006;103:10403.
150. Kanashiro CA, Schally AV, Nagy A, et al. Inhibitionof experimental U-118MG glioblastoma by targetedcytotoxic analogs of bombesin and somatostatin is
155
associated with a suppression of angiogenic and anti-apoptotic mechanisms. Int J Oncol 2005;27:169.
151. Kiaris H, Schally AV, Nagy A, et al. Regression of U-87 MG human glioblastomas in nude mice aftertreatment with a cytotoxic somatostatin analog AN-238. Clin Cancer Res 2000;6:709.
152. Szereday Z, Schally AV, Nagy A, et al. Effective treat-ment of experimental U-87MG human glioblastoma innude mice with a targeted cytotoxic bombesin ana-logue, AN-215. Br J Cancer 2002;86:1322.
153. Moody TW, Fuselier J, Coy DH, et al. Camptothecin-somatostatin conjugates inhibit the growth of small-cell lung cancer cells. Peptides 2005;26:1560.
154. Sun LC, Luo J, Mackey LV, et al. A conjugate ofcamptothecin and a somatostatin analog againstprostate cancer cell invasion via a possible signalingpathway involving PI3K/Akt, alphavbeta3/alphav-beta5, and MMP-2/-9. Cancer Lett 2007;246:157.
155. Moody TW, Sun LC, Mantey SA, et al. In vitro andin vivo antitumor effects of cytotoxic camptothecin-bombesin conjugates are mediated by specific inter-action with cellular bombesin receptors. J PharmacolExp Ther 2006;318:1265.
156. Sun LC, Luo J, Mackey VL, et al. Effects of camp-tothecin on tumor cell proliferation and angiogenesiswhen coupled to a bombesin analog used as a targeteddelivery vector. Anticancer Drugs 2007;18:341.
157. Bernard B, Capello A, van Hagen M, et al. Radiola-beled RGD-DTPA-Tyr3-octreotate for receptor-tar-geted radionuclide therapy. Cancer Biother Radio-pharm 2004;19:173.
158. Capello A, Krenning EP, Bernard BF, et al. Increasedcell death after therapy with an Arg-Gly-Asp-linkedsomatostatin analog. J Nucl Med 2004;45:1716.
159. Capello A, Krenning EP, Bernard BF, et al. Anticanceractivity of targeted proapoptotic peptides. J Nucl Med2006;47:122.
160. Buckley CD, Pilling D, Henriquez NV, et al. RGDpeptides induce apoptosis by direct caspase-3 activa-tion. Nature 1999;397:534.
161. Lambert B, Cybulla M, Weiner SM, et al. Renal tox-icity after radionuclide therapy. Radiat Res 2004;161:607.
162. Kwekkeboom DJ, Mueller-Brand J, Paganelli G, et al.Overview of results of peptide receptor radionuclide
therapy with three radiolabeled somatostatin analogs.J Nucl Med 2005;46(Suppl. 1):62S.
163. Valkema R, Pauwels SA, Kvols LK, et al. Long-termfollow-up of renal function after peptide receptor ra-diation therapy with (90)Y-DOTA(0),Tyr(3)-oc-treotide and (177)Lu-DOTA(0), Tyr(3)-octreotate. JNucl Med 2005;46(Suppl. 1):83S.
164. Melis M, Krenning EP, Bernard BF, et al. Localisa-tion and mechanism of renal retention of radiolabelledsomatostatin analogues. Eur J Nucl Med Mol Imaging2005;32:1136.
165. De Jong M, Valkema R, Van Gameren A, et al. Inho-mogeneous localization of radioactivity in the humankidney after injection of [(111)In-DTPA]octreotide. JNucl Med 2004;45:1168.
166. de Jong M, Barone R, Krenning E, et al. Megalin isessential for renal proximal tubule reabsorption of(111)In-DTPA-octreotide. J Nucl Med 2005;46:1696.
167. Rolleman EJ, Kooij PP, de Herder WW, et al. So-matostatin-receptor-subtype-2-mediated uptake of ra-diolabelled somatostatin analogues in the human kid-ney. Eur J Nucl Med Mol Imaging 2007;34:1854.
168. Verwijnen SM, Krenning EP, Valkema R, et al. Oralversus intravenous administration of lysine: Equal ef-fectiveness in reduction of renal uptake of [111In-DTPA]octreotide. J Nucl Med 2005;46:2057.
169. Bernard BF, Krenning EP, Breeman WA, et al. D-ly-sine reduction of indium-111 octreotide and yttrium-90octreotide renal uptake. J Nucl Med 1997;38:1929.
170. van Eerd JE, Vegt E, Wetzels JF, et al. Gelatin-basedplasma expander effectively reduces renal uptake of111In-octreotide in mice and rats. J Nucl Med 2006;47:528.
171. Vegt E, Wetzels JF, Russel FG, et al. Renal uptake ofradiolabeled octreotide in human subjects is efficientlyinhibited by succinylated gelatin. J Nucl Med 2006;47:432.
172. Rolleman EJ, Krenning EP, Van Gameren A, et al. Up-take of [111In-DTPA0]octreotide in the rat kidney is in-hibited by colchicine and not by fructose. J Nucl Med2004;45:709.
173. Rolleman EJ, Forrer F, Bernard B, et al. Amifostineprotects rat kidneys during peptide receptor radionu-clide therapy with [(177)Lu-DOTA (0),Tyr (3)]oc-treotate. Eur J Nucl Med Mol Imaging 2007;34:763.
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About the Authors
Dr. Monique de Visser re-ceived her B.Sc. degree fromBrabant College in theNetherlands. In December2007, she received her Ph.D.degree from the ErasmusUniversity in Rotterdam. Dr.de Visser joined the group of Professor Dr. Marion deJong at the department ofNuclear Medicine, Erasmus
MC in Rotterdam in 2000. The research of Pro-fessor de Jong mainly focuses on preclinical mo-lecular imaging and radionuclide therapy, usingpeptide-based radiopharmaceuticals. Dr. de Visseris specialized in the targeting of gastrin-releas-ing peptide receptors for prostate cancer imag-ing and treatment, using radiolabeled analogs ofbombesin. She is currently working on the re-duction of renal radiation damage during pep-tide-receptor radionuclide therapy (PRRT).
Dr. Suzanne Verwijnen re-ceived her B.Sc. degreefrom Utrecht College in theNetherlands. In December2007, she received her Ph.D.degree from the ErasmusUniversity in Rotterdam. Dr.Verwijnen joined the groupof Professor Dr. Marion de Jong at the ErasmusMC (Department of Nuclear
Medicine) in 2001, where she is now a postdoc-toral fellow. One area of interest is in improvingthe treatment of malignant brain cancer by gene-transfer strategies or PRRT. She is currentlyworking on the use of dual-isotope imaging insmall animal models and on multimodality imag-ing using single photon emission computed to-mography and positron emission tomographywith computed tomography and magnetic reso-nance imaging.
Professor Dr. Marion deJong received her M.Sc. de-gree in biology and biochem-istry from the AgriculturalUniversity in Wageningen,the Netherlands. She ob-tained her Ph.D. degreefrom the Erasmus Univer-
sity in Rotterdam in 1993, where her thesis dealtwith the significance of thyroid hormone trans-port systems (Department of Internal Medicine).She joined the research group of Professor Dr.Eric Krenning at the department of Nuclear Med-icine of the Erasmus MC in 1994. Her researchmainly focuses on the use of radiolabeled pep-tides and other radiopharmaceuticals for multi-modal molecular imaging and radionuclide ther-apy. In 2006, she was appointed Professor ofNuclear Biology. Marion is a member of the Mo-lecular Imaging Task Group of the EANM, andshe serves as a member of the editorial board andas reviewer for several international journals inthe field of nuclear medicine and molecularimaging.
