Aloj, Luigi

Boerman, Otto C

European Journal of Nuclear Medicine

English

PubMed

Otto C. Boerman

Luigi Aloj

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Evaluation of CCK2 receptor binding ligands: the inheritance of Thomas Behr

Apollo (Cambridge)

EDITORIAL COMMENTARYEvaluation of CCK2 receptor binding ligands:the inheritance of Thomas BehrOtto C. Boerman & Luigi AlojPublished online: 7 June 2011# The Author(s) 2011. This article is published with open access at Springerlink.comThis issue of the European Journal of Nuclear Medicineand Molecular Imaging contains a series of studiescomparing the in vitro and in vivo characteristics of aseries of 12 cholecystokinin (CCK) receptor (CCKR)binding ligands [1–3]. Nine European research groupsfrom Athens, Basel, Freiburg, Innsbruck, Ljubljana, Lon-don, Naples, Nijmegen and Rotterdam supported by aCOST Action (BM0607) of the European Union havecooperated to carry out these studies. The project aimed toevaluate the in vitro and in vivo properties of 12 DOTA-conjugated CCK2R ligands. The ligands were synthesizedby the groups in Athens, Freiburg, Innsbruck, Nijmegen,London and Ljubljana and the following characteristics ofthe ligands were studied in standardized model systems:– The affinity (IC50) of the ligands for the CCK2R wasdetermined in Berne– The internalization kinetics of the ligands weredetermined in Naples and in Rotterdam– The stability of the ligands was evaluated in Innsbruck– The in vivo targeting properties of CCK2R-expressingtumours were evaluated in Nijmegen and in LondonCCK/gastrin analogues were first proposed for radionu-clide imaging and therapy of tumours approximately15 years ago. The name of the late Thomas Behr isinextricably connected to the in vivo use of CCK/gastrinanalogues for in vivo targeting of CCK2R-expressingtumours (Fig. 1). Thomas Behr recognized the fact thatexcellent sensitivity of the pentagastrin test for detectingmalignant C cells in patients with medullary thyroidcarcinoma (MTC) suggested a high incidence of gastrinreceptors in human MTC [4]. Indeed, at that time theautoradiography studies of Reubi et al. confirmed theexpression of CCK/gastrin receptor in more than 90% ofMTCs and in several other tumour types, such as small celllung cancer, stromal ovarian cancers and astrocytomas [5].At that time Thomas Behr worked at the Department ofNuclear Medicine at the Georg-August University inGöttingen headed by the late Prof. Wolfgang Becker. Therehe showed that a 131I-labelled truncated gastrin analogueaccumulated specifically in CCK2R-expressing tumours inmice and men [4]. In a subsequent study Thomas Behrscreened a series of 20(!) CCK/gastrin analogues [6]. Allpeptides were radioiodinated and the affinity for the CCK2Rwas determined and a few of these peptides were tested innude mice with CCK2R-expressing xenografts. From thesestudies Thomas Behr selected minigastrin, a 12-amino acidpeptide containing the pentaglutamic acid sequence as theoptimal peptide for targeting CCK2R-expressing tumours.Fig. 1 Thomas Behr: *21 May1966–†20 August 2010O. C. Boerman (*)Department of Nuclear Medicine, Radboud University NijmegenMedical Centre,PO Box 9101, 6500 HB Nijmegen, The Netherlandse-mail: O.Boerman@nucmed.umcn.nlL. AlojDepartment of Nuclear Medicine, Istituto Nazionale Tumouri,Fondazione “G.Pascale”,Naples, ItalyEur J Nucl Med Mol Imaging (2011) 38:1407–1409DOI 10.1007/s00259-011-1848-3Thomas realized that for in vivo application these internal-izing peptides should be labelled with a residualizingradiolabel and therefore, minigastrin was conjugated withDTPA and labelled with 111In and tested in xenografted nudemice and in a patient with MTC.Ever since Thomas Behr carried out this pioneeringwork numerous research groups have synthesized newCCK/gastrin analogues in an attempt to further improve thetumour targeting properties. As part of the COST ActionBM0607 on targeted radionuclide therapy we designed a setof in vitro and in vivo studies to select the peptides withoptimal characteristics for in vivo targeting (Table 1). Thegroup of Prof. Reubi in Berne, Switzerland determined theaffinity of each of the 12 peptide analogues for the CCK2Rusing in vitro competitive autoradiography. This studyshowed that the IC50 of all ligands was in the nanomolarrange (0.2–3.4 × 10−9 M). The internalization kinetics ofthe 12 peptides as determined in Rotterdam and in Napleswere also similar with most of the radiolabelled peptidesinternalized within 2 h [2].A successful ligand for targeted radionuclide therapyshould be stable in vivo at least during hours. Therefore thebiological stability of the 12 analogues was studied in detailby the group in Innsbruck [3]. Variable stability in humanserum was found for the different peptides with calculatedhalf-lives between 4.5±0.1 and 198±0.1 h. In urine ofnormal mice only metabolized peptide fragments weredetected even at short times after injection for all peptides.Matrix-assisted laser desorption/ionization time-of-flightmass spectrometry (MALDI-TOF MS) revealed a majorcleavage site of all minigastrin derivatives between Asp andPhe-NH2 at the C-terminal end of the peptides.The in vivo properties of the 12 radiolabelled peptideswere studied in Nijmegen and in London. The Nijmegengroup determined the biodistribution of the 111In-labelledpeptides in mice with subcutaneous CCK2/gastrin receptor-expressing tumours in the flank and with receptor-negativetumours contralaterally [1]. The two CCK analogues bothdisplayed relatively low tumour uptake as compared to theten minigastrin analogues. Two linear minigastrin peptidesdisplayed good accumulation in the CCK2R+ tumour, butwith very high kidney uptake. The linear analogue lackingthe penta-Glu sequence (MG11) showed lower tumouruptake and also low kidney uptake. Varying the N-terminalGlu residues in the minigastrin analogues led toimproved tumour targeting properties, with the analoguein which D-amino acids were used in the pentaglutamicacid sequence (PF11), displaying high tumour uptake incombination with low retention in the kidneys. Further-more these studies showed that the cyclized peptide andthe divalent peptide also showed high tumour to kidneyratios in this animal model. In addition, the group inLondon studied the in vivo tumour targeting properties ofthe 12 peptides by microSPECT/CT imaging. SCID micewith A431-CCK2R-positive and A431-CCK2R-negativetumours on the left and right shoulders, respectively, wereinjected with the 111In-labelled minigastrin/CCK peptideanalogues radiolabelled with 111In to a specific activity of30 MBq/nmol. Whole-body SPECT/CT images after 1 and4 h were obtained with a four-head microSPECT scannerequipped with 2-mm pinhole collimators. All imagesobtained showed preferential uptake in the CCK2R-expressing tumours. Peptides were ranked based on thequantitation of tumour, kidney uptake and retention in thetumour during 4 h. Four compounds appeared to have optimalproperties for therapeutic application: the cyclic gastrinanalogue, two minigastrin analogues (PP-F11 and PP-F16)and the dimeric compound MGD5.In summary, in the studies described in this issue of theEuropean Journal of Nuclear Medicine and MolecularTable 1 Nomenclature, sequence and molecular weight of the 12 CCK2R binding ligands that were evaluated in the projectName Sequence MW (g/mol)G-CCK8 DOTA-Gly-Asp-Tyr-Met-Gly-Trp-Met-Asp-Phe-NH2 1,507.0SA106 DOTA-DAsp-Phe(p-CH2SO3H)-HPG-Gly-Trp-HPG-Asp-Phe-NH2 1,482.6MG0 DOTA-DGlu-Glu-Glu-Glu-Glu-Glu-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH2 2,037.0Sargastrin DOTA-Gln-Gly-Pro-Trp-Leu-Glu-Glu-Glu-Glu-Glu-Ala-Tyr-Gly-Trp-Nle-Asp-Phe-NH2 2,483.6MG11 DOTA-DGlu-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH2 1,531.0APH070 DOTA-His-His-Glu-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH2 1,677.8PP-F10 DOTA-DGln-DGln-DGln-DGln-DGln-DGln-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH2 2,042.9PP-F6 DOTA-DGln-DGln-DGln-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH2 1,658.7PP-F16 DOTA-DGln-DGlu-DGln-DGlu-DGln-DGlu-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH2 2,045.9PP-F11 DOTA-DGlu-DGlu-DGlu-DGlu-DGlu-DGlu-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH2 2,048.8Cyclo-MG2 DOTA-DGlu-(Ala-Tyr)-DLys-Trp-Nle-Asp-Phe-NH2 (cyclo DGlu-DLys) 1,455.7MGD5 DOTA-Gly-Ser-Cys-(Glu-Ala-Tyr-Gly-Trp-Nle-Asp-Phe-NH2)2 2,782.9HPG homopropargyl glycine1408 Eur J Nucl Med Mol Imaging (2011) 38:1407–1409Imaging the in vitro [2, 3] and in vivo [1] properties of aseries of 12 CCK2R binding ligands were determinedextensively. The studies indicated that three of theseanalogues have optimal characteristics for application intargeted radionuclide therapy of CCK2R-expressing can-cers. In future studies the groups will further investigate thetherapeutic potential of the selected compounds in mousetumour models. Based on these studies a set of new ligandswill be designed and synthesized. In addition, a workinggroup aims to transfer one of these compounds for clinicalstudies in cancer patients. In conclusion, 15 years after thepioneering work of Thomas Behr, his gastrin analogues willbe applied clinically for radionuclide imaging and possiblyfor radionuclide therapy.Open Access This article is distributed under the terms of theCreative Commons Attribution Noncommercial License whichpermits any noncommercial use, distribution, and reproduction inany medium, provided the original author(s) and source arecredited.References1. Laverman P, Joosten L, Eek A, Roosenburg S, Peitl PK, Maina T,et al. Comparative biodistribution of 12 (111)In-labelled gastrin/CCK2 receptor-targeting peptides. Eur J Nucl Med Mol Imaging,doi:10.1007/s00259-011-1806-0.2. Aloj L, AurilioM, Rinaldi V, D’ambrosio L, Tesauro D, Peitl PK, et al.Comparison of the binding and internalization properties of 12 DOTA-coupled and (111)In-labelled CCK2/gastrin receptor binding peptides:a collaborative project under COSTAction BM0607. Eur J Nucl MedMol Imaging, doi:10.1007/s00259-011-1816-y.3. Ocak M, Helbok A, Rangger C, Peitl PK, Nock BA, Morelli G, et al.Comparison of biological stability and metabolism of CCK2 receptortargeting peptides, a collaborative project under COSTBM0607. Eur JNucl Med Mol Imaging, doi:10.1007/s00259-011-1818-9.4. Behr TM, Jenner N, Radetzky S, Béhe M, Gratz S, Yücekent S, et al.Targeting of cholecystokinin-B/gastrin receptors in vivo: preclinicaland initial clinical evaluation of the diagnostic and therapeuticpotential of radiolabelled gastrin. Eur J Nucl Med 1998;25(4):424–30.5. Reubi JC, Schaer JC,Waser B. Cholecystokinin(CCK)-A and CCK-B/gastrin receptors in human tumors. Cancer Res 1997;57(7):1377–86.6. Behr TM, Jenner N, Béhé M, Angerstein C, Gratz S, Raue F, et al.Radiolabeled peptides for targeting cholecystokinin-B/gastrinreceptor-expressing tumors. J Nucl Med 1999;40(6):1029–44.Eur J Nucl Med Mol Imaging (2011) 38:1407–1409 1409

Evaluation of CCK2 receptor binding ligands: the inheritance of Thomas Behr.

Boerman, O.C.

Aloj, L.

Radboud Repository

NARCIS 

Comparative biodistribution of 12 (111)In-labelled gastrin/ CCK2 receptor-targeting peptides.

Comparisonofthebindingandinternalizationpropertiesof12DOTAcoupled and (111)In-labelled CCK2/gastrin receptor binding peptides: a collaborative project under COSTAction BM0607.

Radiolabeled peptides for targeting cholecystokinin-B/gastrin receptor-expressing tumors.

ReubiJC,SchaerJC,WaserB.Cholecystokinin(CCK)-AandCCK-B/ gastrin receptors in human tumors.

Targeting of cholecystokinin-B/gastrin receptors in vivo: preclinical and initial clinical evaluation of the diagnostic and therapeutic potentialofradiolabelledgastrin.EurJNuclMed1998;25(4):424–30.

Evaluation of CCK2 receptor binding ligands: the inheritance of Thomas Behr.

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Apollo (Cambridge)

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