Dalton Transactions Dynamic Article Links Cite this: DOI: 10.1039/c0dt01599g www.rsc.org/dalton PERSPECTIVE Radiometallated peptides for molecular imaging and targeted therapy João D. G. Correia, António Paulo, Paula D. Raposinho and Isabel Santos* Received 17th November 2010, Accepted 11th January 2011 DOI: 10.1039/c0dt01599g In developed countries, cancer is the second leading cause of death, being only surpassed by cardiovascular diseases. To develop tumor-targeted tools to localize and treat cancer at an early stage is a multidisciplinary area fuelled by the convergence of biology, medicine, chemistry, physics and engineering. Chemists, in particular, play a critical role in this effort, as they are continuously challenged to use innovative chemical strategies to develop ‘smart drugs’. The in vitro observation that peptide receptors are overexpressed in certain tumors, as compared to endogenous expression levels, has prompted the use of such receptors as targets and the design of radiolabelled peptide-based tools for targeted nuclear molecular imaging and therapy. Such approach has gained increased interest over the last two decades, driven in particular by the success of OctreoScan R© and by the increasing knowledge concerning overexpression of regulatory peptide receptors in tumor tissues. Selected peptides that target a variety of disease related receptors are in place and have been labeled with different radiometals, using mainly the bifunctional approach. This review begins by summarizing some relevant aspects of the coordination chemistry of the metals studied for labeling peptides. Then, we provide an overview of the chemical strategies explored to improve the biological performance of different families of radiometallated peptides for nuclear molecular imaging and/or targeted radionuclide tumor therapy. Introduction Despite the advances in medical sciences, cancer is still a lead- ing cause of death worldwide. The World Health Organization reported that, in developed countries, cancer is the second leading cause of death, being only surpassed by cardiovascular diseases.1 Nevertheless, during recent decades, remarkable insights into the cell and molecular biology of malignancies has been acquired, and a myriad of differences in the biological make-up of cancers compared with their healthy-tissue counterparts have been catalogued.2–4 The increasing knowledge generated by such achievements has led to the identification of several biomarkers, and some of them have been considered as potential targets for in vivo molecular imaging and/or therapeutic purposes.2,3,5–7 Among others, antigens, membrane receptors and enzymes have been considered as interesting biomarkers, since they play important roles in pathological processes, being in most cases overexpressed or upregulated compared to endogenous expression levels. The validation and potential interest of those targets have been intensively studied, and the identification and design of high- affinity binders for such targets has been – and remains – an area of intense research. The optimization of endogenous ligands has been the most widely used strategy to generate high-affinity ligands.2,3,5–7 Unidade de Ciências Quı́micas e Radiofarmacêuticas, Instituto Tecnológico e Nuclear, Estrada Nacional 10, Sacavém, 2686-953, Portugal. E-mail: isantos@itn.pt; Fax: +351 21-9946185; Tel: +351 21-9946201 Nuclear medicine uses radiolabelled compounds for in vivo imaging and therapeutic purposes. Such compounds are named radiopharmaceuticals and are used in such low concentrations that they have no pharmacological effect. When specific, ra- diopharmaceuticals consist of a target-specific moiety, such as an antibody or antibody fragment, peptides or low molecular weight ligands, linked to an appropriate radionuclide. Depending on the intrinsic physical characteristics of the radionuclide, the radiopharmaceuticals are used for in vivo imaging or targeted- radionuclide therapy (TRT). Single-photon emission computed tomography (SPECT) and positron emission tomography (PET) are the two imaging modalities used in nuclear medicine. These modalities are able to determine the concentration of specific molecules in the human body in a non-invasive way, and are sensitive enough to visualize interactions between physiological targets and ligands.3,5–7 TRT involves specific localization of a radionuclide emitting ionizing radiation to deliver a cytotoxic radiation dose to cancerous tissues, while sparing the surrounding healthy ones.6,8,9 Table 1 summarizes the most relevant radionuclides with medical interest in nuclear medicine, for both diagnostic (g or b+ emitters) and therapeutic applications (b-, a or Auger electron emitters). In terms of target-specific moieties, monoclonal antibodies have long been considered interesting biomolecules for cancer diagnosis and therapy, and represent the start of a new era in cancer management.8–10 Owing to some drawbacks, namely poor pharmacokinetics, some improvements through protein This journal is © The Royal Society of Chemistry 2011 Dalton Trans. D ow nl oa de d by I ns ti tu to T ec no lo gi co e N uc le ar ( IT N ) on 2 5 F eb ru ar y 20 11 P ub li sh ed o n 25 F eb ru ar y 20 11 o n ht tp :/ /p ub s. rs c. or g | d oi :1 0. 10 39 /C 0D T 01 59 9G View Online http://dx.doi.org/10.1039/c0dt01599g http://dx.doi.org/10.1039/c0dt01599g http://dx.doi.org/10.1039/C0DT01599G Table 1 Relevant radionuclides for medical applications Nuclide Physical half-life Mode of decay (%) Application 99m Tc 6.0 h IT (100) SPECT 186 Re 89.2 h b- (92) Therapy EC (8) 188 Re 17 h b- (100) Therapy 123 I 13.2 h EC (100) SPECT 131 I 8.02 d b- (100) Therapy 18 F 109.8 min b+ (97) PET EC (3) 11 C 20.3 min b+ (100) PET 86 Y 14.7 h b+ (33) PET EC (66) 90 Y 64.1 h b- (100) Therapy 111 In 2.80 d EC (100) SPECT 67 Ga 3.26 d EC (100) SPECT 68 Ga 67.8 min b+ (99) PET EC (10) 60 Cu 0.4 h b+ (93) PET EC (7) 61 Cu 3.3 h b+ (62) PET EC (38) 62 Cu 0.16 h b+ (98) PET EC (2) 64 Cu 12.7 h b- (40), b+ (19) PET/Therapy EC (41) 67 Cu 61.8 h b- (100) Therapy 89 Zr 78.5 h b+ (22.7) PET EC (77) 153 Sm 46.3 d b- (100) Therapy 166 Ho 26.8 d b- (100) Therapy 177 Lu 6.73 d b- (100) Therapy engineering have been made in the last few years, and the dream of targeted radionuclide therapy was partially fulfilled with the introduction of radiolabelled antibodies for clinical use, such as Zevalin (murine antibody-90Y) and Bexxar (murine antibody- 131I). Despite these successful examples, there is still room for improvement, and attempts to find targeted radionuclide therapy for solid tumors8–10 makes this a very active research area. Following the finding that small regulatory peptide receptors are often overexpressed in certain human cancers and that derivatives of their natural ligands can be used for tumor targeting, the use of peptides has appeared as another approach for delivering ra- dioactivity to tumors. This approach has gained increased interest over the last two decades, driven in particular by the success of OctreoScan R© (111In-labelled somatostatin analog) in the late 1980s and by the increasing knowledge concerning overexpression of regulatory peptide receptors in tumor tissues.2,3,5–7,11 The avail- ability of different techniques to generate potential high-affinity peptides for a selected target is also responsible for the large pool of bioactive synthetic peptides. Indeed, target-specific delivery of radioactive peptides, both for molecular imaging and therapy, is increasingly considered a promising strategy. Well-established solid-phase peptide synthesis allows reproducible preparation of a variety of peptides with accurate chemical structures, which can be modulated to optimize affinity and specificity for the target, metabolic stability and pharmacokinetics. Most of the naturally occurring peptides have a short biological half-life due to rapid degradation by various peptidases and proteases found in plasma. Once the biological portion of the peptides has been identified, they can be engineered to prolong their biological half-lives in vivo. Such improvement can be done by the introduction of D-amino acids, incorporation of amino alcohol, use of unusual amino acids or side-chains and amidation and/or acetylation of peptide C- and N-termini. The pharmacokinetics of peptides can also be tuned by altering the hydrophilic and hydrophobic balance of the peptide structure, through the introduction of charged amino acids (e.g. glutamic acid), carbohydrates or poly(ethylene)glycol (PEG) chains in the peptide backbone. Another advantage of peptides is their tolerance towards the modifications necessary for their labeling with different radionu- clides. For radiometallation, for example, the most explored ap- proach makes use of a bifuncional chelator (BFC) that coordinates the metal and presents an adequate functionality for the coupling of the targeting peptide. Additionally, an appropriate linker that separates the chelating moiety and the bioactive fragment can also be used. The nature of such linkers is variable, and generally they are also used as pharmacokinetic modifiers. However, it must be kept in mind that the design of a peptide- based radiopharmaceutical is a non-trivial task, due to the relatively small size of the targeting peptide. All the structural modifications have to be done with retention of its affinity and selectivity to the putative receptors. Moreover, the radiolabeled peptide must be obtained with high specific activity, show a high stability under physiological conditions, and present high selectivity and target-specific uptake, with low accumulation in non-target tissues. Herein, we will present an overview of the chemical efforts made to find metallated peptides for nuclear molecular imaging and TRT. In the first section, we will present relevant aspects of the coordination chemistry of metals with medical interest in nuclear medicine, for both diagnostic and therapeutic appli- cations. The second section will present a broad view of the chemical strategies explored to synthesize different families of radiometallated peptides, as well as the chemical efforts made to improve their biological performance. This contribution intends to update previous reviews, but will not cover work on radiolabeled somatostatin analogs for imaging or therapy of tumors, since these radiopeptides have been the focus of various comprehen- sive reviews recently published.12–17 To provide some context to the current manuscript, some overlap with earlier reviews is unavoidable.18,19 Relevant coordination chemistry Acyclic and cyclic polyaminopolycarboxylic ligands (Fig. 1), such as diethylenetriaminepentacetic acid (DTPA), 1,4,7,10- tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), 1,4,8,11-tetraazacyclododecane-1,4,8,11-tetraacetic acid (TETA), 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) and cross- bridged (CB) tetraazamocycle derivatives, have been the most extensively evaluated BFCs for the labeling of peptides with trivalent and bivalent radiometals like Ga3+, In3+, Y3+ and Ln3+ or Cu2+.20–23 One of the carboxylic arms of polyaminopolycarboxylic ligands can be used for the coupling of the peptide, typically via formation of amide bonds with primary amines from lysine residues or the N-terminus of peptides, without compromising the stability of the respective metal complexes. Alternatively, the functional group used to couple the peptide can be introduced in the methylenic backbone of the chelator, Dalton Trans. This journal is © The Royal Society of Chemistry 2011 D ow nl oa de d by I ns ti tu to T ec no lo gi co e N uc le ar ( IT N ) on 2 5 F eb ru ar y 20 11 P ub li sh ed o n 25 F eb ru ar y 20 11 o n ht tp :/ /p ub s. rs c. or g | d oi :1 0. 10 39 /C 0D T 01 59 9G View Online http://dx.doi.org/10.1039/C0DT01599G Fig. 1 Acyclic and cyclic polyaminocarboxylic ligands. leaving all of the carboxylic pendant arms available for coordi- nation to the metal. A variety of possibilities can be explored to couple the peptide to the chelator. If the coupling involves a carboxylic group from the chelator, it is possible to perform the ac- tivation of the carboxyl group in situ using the common activation strategies, like those based on the formation of tetrafluorophenyl or N-hydroxysuccinimide activated esters. Another alternative is the introduction of maleimide or isocyanate functions in the chelator framework, which promptly react with thiol or amino groups of the peptide with formation of thioether or thiourea bonds.24,25 Macrocyclic chelators provide metal complexes that are ther- modynamically more stable and kinetically more inert than the complexes with their acyclic counterparts, as a consequence of the ability of the free macrocycles to adopt preorganized conformations.26 Table 2 summarizes the stability constants (K ML) for complexes of some of the metals reviewed herein with the most common acyclic or macrocyclic polyaminopolycarboxylic ligands (Fig. 1). By themselves, these K ML values can indicate the relative affinity of the different chelators for a given metal. However, one has to consider that it can be difficult to compare stability constants for ligands of different basicity. To overcome such difficulty, the respective pM values must be considered. Among the several polyaminopolycarboxylic ligands, DOTA- like chelators do not always provide for the most stable complexes (Table 2). Nevertheless, so far, DOTA-like chelators have been extensively used for the radiometallation of peptides, most proba- Table 2 Stability constants (logK ML a ) for complexes of polyaminocar- boxylates with divalent and trivalent metal ions Metal Chelator Cu(II) Ga(III) In(III) Y(III) Lu(III) DTPA 21.5b 25.5b 29.5h 22.5j — DOTA 22.3c 21.3g 23.9f 24.3j 25.5l ,m TETA 21.7d 19.7f 21.8f 14.8k 15.3k NOTA 21.63e 31.0i 26.2i — — a K ML = [ML]/[M][L]. b Ref. 27 c Ref. 28. d Ref. 29. e Ref. 30. f Ref. 31. g Ref. 32. h Ref. 33. i Ref. 34. j Ref. 35. k Ref. 36. l Ref. 37. m Ref. 38. bly due to the commercial availability of several activated DOTA derivatives ready for conjugation. Gallium and indium The group 13 elements gallium (Ga) and indium (In) are post- transition metals presenting radionuclides suitable for SPECT (67Ga, 111In) and PET (68Ga) imaging, or for Auger-therapy (111In) (Table 1). 67Ga and 111In are cyclotron-produced gamma emitters obtained at reasonable cost and are deliverable to different users over relatively large distances. 68Ga is a positron emitter readily accessible from the 68Ge/68Ga generator, offering the possibility to obtain on site a PET radionuclide without needing the presence of a nearby cyclotron.23,39 This journal is © The Royal Society of Chemistry 2011 Dalton Trans. D ow nl oa de d by I ns ti tu to T ec no lo gi co e N uc le ar ( IT N ) on 2 5 F eb ru ar y 20 11 P ub li sh ed o n 25 F eb ru ar y 20 11 o n ht tp :/ /p ub s. rs c. or g | d oi :1 0. 10 39 /C 0D T 01 59 9G View Online http://dx.doi.org/10.1039/C0DT01599G The chemistry of gallium and indium in aqueous media is exclusively limited to the oxidation state III. In aqueous solu- tion, the M3+ (M = Ga, In) ions have a marked tendency to undergo hydrolysis, which is even more pronounced for Ga(III). At physiological pH, gallium forms essentially the soluble gallate anion [Ga(OH)4]-, while indium precipitates as the tris(hydroxide) [In(OH)3]. When designing radiopharmaceuticals, namely ra- diometallated peptides, it is of particular importance to obtain Ga and In complexes resistant to hydrolysis. These complexes must also have resistance towards transchelation reactions with transferrin, which is a protein present in the plasma and involved in the receptor-mediated transport of iron into cells. This is particularly relevant for Ga(III), that presents the highest affinity to transferrin due to the similarity of the coordination chemistry of trivalent gallium and iron.40,41 Both Ga(III) and In(III) are rather hard Lewis acids and, for this reason, the formation of stable complexes with these metal ions usually requires the use of polydentate chelators presenting anionic oxygen donor groups, such as acyclic or macrocyclic polyaminocarboxylic ligands (Fig. 1). The difference on the ionic radius of Ga3+ (47–62 pm, CN = 4–6) and In3+ (62–92 pm, CN = 4–8) is another important aspect to take into consideration when selecting a proper ligand for labeling a biomolecule with their radioisotope. The maximum coordination number (CN) attained by Ga(III) complexes is 6 while In(III), being larger, forms complexes with CN = 7 and even with CN = 8. These differences are well documented by several X-ray structures of Ga(III) and In(III) complexes with polyaminocarboxylic ligands, as exemplified in Fig. 2 for a DOTA derivative containing a pendant arm functionalized with a triphenylphosphonium (TPP) group. The Ga(III) complex is hexacoordinated with a distorted octahedral geometry, and the In(III) complex is heptacoordinated with a monocapped trigonal prismatic geometry.42 Fig. 2 Molecular structures of Ga-DOTA-TPP and In-DOTA-TPP.42 The labeling of peptides with 67Ga/68Ga has been performed using mainly DOTA or NOTA derivatives as bifunctional chela- tors, while DTPA and DOTA derivatives have been used for 111In- labeling of peptides. DTPA is potentially octadentate and forms complexes of higher stability with In(III) compared to Ga(III) (Table 2). The Ga(III)-NOTA complex has an exquisite stability among gallium complexes, presenting a thermodynamic stability (logK = 31.0, pM = 26.4) approximately 10 orders of magnitude higher than the one of Ga(III)-DOTA (logK = 21.3, pM = 15.2). Moreover, the kinetics of complexation of Ga(III) is faster for NOTA than for DOTA, necessitating longer reaction times and higher tempera- tures to label peptides with 67/68Ga using DOTA-like chelators. For this reason, NOTA-like chelators are very favorable for67/68Ga labeling of peptides. The high stability constant of Ga(III)-NOTA complexes and their kinetics certainly reflect a better fitting of the NOTA cavity size with the size of the Ga3+ ion and the involvement of all pendant arms in the coordination to the metal. To keep the possibility of a N3O3-hexadentate coordination after linkage of the biomolecule, NOTA-like chelators containing a diacid pendant arm, such as NODASA (1,4,7-triazacyclononane-N-succinic acid- N¢,N¢¢-diacetic acid) and NODAGA ((1,4,7-triazacyclononane- N-glutamic acid-N¢,N¢¢-diacetic acid), have been designed and synthesized (Fig. 1).43,44 Unlike Ga(III), the coordination require- ments of In(III) are not fulfilled by NOTA-like chelators which, for this reason, are not the best suited bifunctional chelators for the labeling of peptides with 111In. Yttrium and the lanthanides Yttrium (Y) and the lanthanides (Ln) are trivalent metals that offer differing b-emitting radioisotopes relevant for therapeutic applications. Among these radioisotopes, 90Y and the radiolan- thanide 177Lu have been the most extensively explored to obtain radiometallated peptides for peptide receptor radionuclide therapy (PRRT).20,45,46 The aqueous coordination chemistry of yttrium and lanthanides shows a great similitude due to their common tricationic charge and similar ionic radii. The Y3+ and Ln3+ metal ions show a hard acidic character and tend to form complexes with hard donor atom ligands, displaying high coordination numbers, usually 8 or 9. Therefore, the labeling of peptides with these radiometals has been performed using mainly polyaminocarboxylic ligands. Acyclic DTPA derivatives form by far more stable complexes with In(III) than with Y(III) or Ln(III) (Table 1). The latter metal ions are coordinated more avidly by DOTA derivatives, due to the higher thermodynamic stability and enhanced kinetic inertness of the corresponding complexes. These features explain why DOTA- like compounds can be considered as the best choice for labeling peptides with 90Y or 177Lu, although DTPA derivatives have been used in several instances for that purpose. The high stability of Y-DOTA and Lu-DOTA complexes can be accounted for by the good match of the DOTA cavity size to the ionic radii of these trivalent metal ions. A poor match between Y3+ and Lu3+ ions and the cavity size of TETA derivatives justifies the much lower stability of M-TETA complexes (M = Y, Lu). For this reason, TETA chelators are not a good option for 90Y- or 177Lu-labeling of peptides. Even after functionalization of one pendant arm with a targeting biomolecule, it is considered that DOTA-like chelators act as N4O4-octadentate donor ligands towards Ln3+ and Y3+ ions, Dalton Trans. This journal is © The Royal Society of Chemistry 2011 D ow nl oa de d by I ns ti tu to T ec no lo gi co e N uc le ar ( IT N ) on 2 5 F eb ru ar y 20 11 P ub li sh ed o n 25 F eb ru ar y 20 11 o n ht tp :/ /p ub s. rs c. or g | d oi :1 0. 10 39 /C 0D T 01 59 9G View Online http://dx.doi.org/10.1039/C0DT01599G as the amide oxygen from the conjugating arm also coordinates to the metal. This coordination mode has been confirmed by X-ray structural analysis of the model compound Y-DOTA-DPheNH2 that contains a DOTA derivative with a carboxymethyl arm func- tionalized with phenylalanine (Fig. 3).47 NMR studies of a related yttrium complex bearing a p-aminoanilide (AA) functionalized pendant arm, Y-DOTA-AA, have shown the retention of the octadentate coordination of the DOTA derivative in solution.42 This macrocyclic ligand in the congener In-DOTA-AA is also octa- coordinated but the In(III) complex is fluxional in solution at room temperature, most probably due to de-coordination/coordination of the amide oxygen from the functionalized pendant arm.48 Such differences can affect the in vivo behavior of congener In(III) and Y(III) complexes and, eventually, may explain the discrepancies observed for the biological performance of similar 90Y or 111In-DOTA-AA complexes. Despite such differences, in radiopharmaceutical chemistry 111In complexes are often used as surrogates to estimate the biodistribution and radiation dosimetry of congener 90Y complexes. However, such studies need to take into consideration the differences in solution of Y(III) and In(III) complexes. Fig. 3 Molecular structure of Y-DOTA-DPheNH2 .47 Yttrium(III) and lanthanide(III) complexes of DOTA derivatives can exist in two interconverting diastereoisomers of square antiprism (SAP) and twisted square antiprism (TSAP) geometries. The ratio SAP/TSAP depends very much on the size of the trivalent ion being preferred the SAP geometry for the largest ions, like Y3+ or Lu3+. At macroscopic level, the formation and intercon- version of these coordination isomers has been intensively studied by different research groups using NMR techniques.49,50 Studies on the formation of different coordination isomers of DOTA- like chelators using trivalent radiometals are scarce. Recently, it has been shown that a DOTA derivative ((S)-p-NH2-Bn-DOTA) bearing a benzyl amine substituent at the methylenic backbone of the macrocycle forms a mixture of two isomers when labeled with 86Y (b+ emitter; t1/2 = 14.7 h).51 As expected, the SAP isomer is predominant, being observed a SAP/TSAP ratio of 3 : 1. After separation by HPLC, the biodistribution profile of each isomer of [86Y((S)-p-NH2-Bn-DOTA)] was assessed using Wistar rats. Only minor differences were observed in their biological behavior, which may indicate that the isomerism of Y(III) complexes with DOTA- like chelators does not strongly influence their in vivo behavior. Copper Copper has a unique combination of diagnostic (60Cu, 61Cu, 62Cu and 64Cu) and therapeutic radionuclides (64/67Cu) (Table 1). Moreover, due to its nuclear properties, 64Cu is suitable for PET imaging and for TRT.21,23 From the three accessible oxidation states (I–III) of copper under aqueous solution, Cu(II) has been the most widely used to obtain 64Cu complexes potentially useful as radiopharmaceuticals. This reflects the fact that Cu(III) is relatively rare and difficult to stabilize in aqueous solution, while Cu(II) complexes display an increased kinetic inertness compared with Cu(I) complexes. To find labeling methodologies to prepare 64Cu(II) complexes stable in vivo, it is necessary to take into consideration basic aspects of the aqueous coordination chemistry of Cu(II), as well as the behavior of copper as an essential trace metal in human biochemistry. The 64Cu(II) complexes must be resistant towards transchelation to proteins involved in the transport and storage of copper, and must not undergo reduction to Cu(I), as it will increase the probability of releasing the radiometal in vivo. DOTA and TETA have been largely used as bifunctional chelators for 64Cu-labeling of peptides, although they are not ideal chelators for Cu(II), as well documented by the in vivo instability of their complexes. In vivo experiments in rat models have shown that both 64Cu-DOTA and 64Cu-TETA undergo transchelation of 64Cu(II) to liver and blood proteins, with this behavior being more pronounced in the case of 64Cu-DOTA.52 These macrocyclic complexes present a high thermodynamic stability with almost coincident K ML values (Table 2), indicating that their kinetic inertness has a more crucial influence on their in vivo instability. The 9-membered triazamacrocycle NOTA has also a good affinity for divalent copper, and the corresponding Cu(II)-NOTA complex presents a stability constant similar to those with DOTA and TETA (Table 2).53–56 NOTA-based bifunctional chelators allowed the 64Cu-labeling of different bioactive peptides in very high specific activity and under mild reaction conditions. As reviewed below, the resulting metallopeptides have shown a better biodistribution profile than those labeled with 64Cu using DOTA or TETA derivatives as BFCs, pointing out the best properties of NOTA-derivatives to stabilize the radiometal in vivo.57 Different investigators have synthesized cross-bridged (CB) tetraazamocycles, aiming to introduce novel classes of bifunctional chelators suited for the in vitro and in vivo stabilization of Cu(II) complexes.20,23 The cyclen-based 4,10-bis(carboxymethyl)- 1,4,7,10-tetraazabicyclo-[5.5.2]tetradecane (CB-DO2A) and the cyclam-based 4,11-bis(carboxymethyl)-1,4,8,11-tetraazabicyclo- [6.6.2]tetradecane (CB-TE2A) were used to prepare the complexes 64Cu-CB-DO2A and 64Cu-CB-TE2A. Metabolic studies in rat models showed that 64Cu-CB-DO2A and 64Cu-CB-TE2A pre- sented an increased in vivo stability compared with 64Cu-DOTA and 64Cu-TETA complexes, confirming that the introduction of the ethylenic bridge enhances the stability of these macrocyclic Cu(II) complexes.52,58 In particular, the combination of the cyclam backbone with the cross-bridge significantly enhanced the in vivo stability of 64Cu-CB-TE2A. However, the kinetics of Cu(II) complexation by CB-TE2A is rather slow, and the formation of 64Cu-CB-TE2A requires relatively harsh radiolabeling conditions that may induce damage to some biomolecules. Hence, there is still room for finding This journal is © The Royal Society of Chemistry 2011 Dalton Trans. D ow nl oa de d by I ns ti tu to T ec no lo gi co e N uc le ar ( IT N ) on 2 5 F eb ru ar y 20 11 P ub li sh ed o n 25 F eb ru ar y 20 11 o n ht tp :/ /p ub s. rs c. or g | d oi :1 0. 10 39 /C 0D T 01 59 9G View Online http://dx.doi.org/10.1039/C0DT01599G bifunctional chelators that efficiently bind to Cu(II) under mild reaction conditions. Looking to achieve such goals, cryptand macrocyclic ligands of the hexaaminemacrobicyclic type (Fig. 1), known as sarcophagines (Sar’s), have been used as BFCs to label a few antibodies and peptides with 64Cu.59–65 The Sar ligands encapsulate the Cu(II) ion, forming hexacoordinated and octahedral Cu(II) complexes with thermodynamic stability constants as high as the ones found with DOTA and TETA derivatives. At room temperature, the Sar ligands bind to 64Cu(II) with fast complexation kinetics, at remarkably low concentrations over a pH range of 4–9. The resulting complexes show a high kinetic inertness, as shown by negligible in vitro transchelation. Potentially hexadentate acyclic ligands of the bispidine type (Fig. 1) have been also envisaged as promising bifunctional chela- tors for 64Cu-labeling of biomolecules, offering different positions of the ligand framework to couple the targeting molecule. These extremely rigid N-donor ligands efficiently encapsulate Cu(II) leading to octahedral complexes that present stability constants in the same range as those of macrocyclic Cu(II) complexes. Also, these acyclic ligands still keep relatively fast complexation kinetics like other open-chain amine-pyridine based ligands. These favorable features prompted the synthesis of the model complex 64Cu-bispidine and its in vitro evaluation. No transchelation or demetalation was found in the presence of superoxide dismutase (SOD) or in rat plasma.66 Square planar bis(thiosemicarbazone) Cu(II) complexes were explored for the development of 64Cu radiopharmaceuticals several years ago (Fig. 4). Specifically, 64Cu-ATSM (ATSM: diacetyl-bis(N 4-methylthiosemicarbazone) has been considered a promising hypoxia-specific PET tracer.67 Recently, a new ATSM derivative bearing a pendant hexanoic acid arm (ATSM-Ahx) was synthesized (Fig. 4), conjugated to a bombesin analog and labeled with 64Cu.68 In vitro studies have shown that the resulting radiometallated peptide resisted to histidine and cysteine challenge. It is of crucial importance to evaluate the in vivo stability of such Cu complexes. Technetium and rhenium 99mTc is among the most widely used SPECT radionuclides for labeling bioactive peptides, due to its ideal nuclear properties, low-cost and availability from commercial 99Mo/99mTc generators. The radiometallation of peptides with 99mTc is done in aqueous solution, starting from the Tc(VII) permetallate anion (99mTcO4 -), which needs to be reduced prior to its complexation by an adequate BFC carrying the biomolecule. The diverse and rich chemistry of this radiometal allows the use of different strategies for labeling peptides with 99mTc, in terms of metal cores and/or oxidation states and selection of BFCs (Fig. 5).69–74 One of the approaches used for labeling peptides with 99mTc relies on the use of square-pyramidal Tc(V) oxocomplexes of the type [TcO(NxS4-x)] containing the [TcO]3+ core and tetradentate NxS4-x bifunctional chelators, namely the tripeptide mercap- toacetiltriglycine (MAG-3) that acts as a N3S-donor ligand and presents a pendant carboxylic arm for biomolecule coupling (Fig. 5).75 This class of complexes can give syn and anti isomers that may present different biological properties. In addition, the functionalization of the tetradentate chelator with the biologi- cally active molecule can be quite demanding, requiring tedious protection/deprotection strategies. To overcome some of the drawbacks associated with the use of Tc(V) monoxocomplexes, other approaches based on the trans-[TcO2]+ and the [Tc-HYNIC] (HYNIC = 6-hydrazinonicotinic acid) cores (Fig. 5) have been exploited, and these approaches led, in several instances, to radiometallated peptides with promising biological profiles. The trans-[TcO2]+ core has been used in combination with acyclic tetraamine ligands, which form well-defined octahedral Tc(V) dioxocomplexes, and can be C-functionalized with pendant arms suitable for the coupling of peptides, as shown in Fig. 5.76 The [Tc- HYNIC] core offers the advantage of a straightforward functional- ization with the biomolecule, avoiding the use of tedious and time- consuming protection strategies. HYNIC can coordinate as a uni- or bidentate ligand and, therefore, does not fulfill the coordination requirements of the metal, making necessary the use of chelating coligands. Hydrophilic N- and O-donors, like ethylenediamine diacetic acid (EDDA), gluconate or tricine, are among the most explored coligands.74,77–79 The use of such coligands offers the advantage of an easy adjustment of the physico-chemical proper- ties (e.g. charge, hydrophilicity) of the final complexes, which can strongly influence the pharmacokinetics and excretory pathways of 99mTc-labeled peptides. However, the resulting binary mixed- ligand complexes show a relatively low stability. The improvement of the stability of Tc-HYNIC complexes has been achieved by the introduction of a ternary ligand, such as a water-soluble phosphine, or by exploring phosphine- and nicotinyl-containing HYNIC chelators.79–82 The nature of the Tc–N bonds involved in the coordination of HYNIC is still unknown, which can be a serious drawback since the full characterization and chemical identification of a potential radiopharmaceutical is mandatory to get a marketing authorization. The so-called tricarbonyl approach has gained considerable attention in the last few years, following the introduction by Alberto and co-workers of a convenient and fully aqueous-based kit preparation of the organometallic precursor fac-[99mTc(OH2)3(CO)3]+ directly from [TcO4]-.83,84 The chemical robustness of the fac-[Tc(CO)3]+ core and the lability of the three water molecules offer the possibility of exploring a Fig. 4 Bis(thiosemicarbazone) Cu(II) complexes. Dalton Trans. This journal is © The Royal Society of Chemistry 2011 D ow nl oa de d by I ns ti tu to T ec no lo gi co e N uc le ar ( IT N ) on 2 5 F eb ru ar y 20 11 P ub li sh ed o n 25 F eb ru ar y 20 11 o n ht tp :/ /p ub s. rs c. or g | d oi :1 0. 10 39 /C 0D T 01 59 9G View Online http://dx.doi.org/10.1039/C0DT01599G Fig. 5 Selected 99m Tc-complexes with different cores, oxidation states and BFCs. well-defined chemistry that is easily amenable to bioconjugation. Labeling of peptides based on this organometallic approach has been reported by several research groups, using bidentate or tridentate BFCs.69–74 In general, complexes anchored by tridentate chelators are more stable in vivo compared with those involving bidentate BFCs (Fig. 5). Rhenium has two b-emitting isotopes, 186Re and 188Re (Table 1), with nuclear properties suitable for the development of therapeutic radiopharmaceuticals, namely for TRT. The chemistry of rhenium is quite similar to that of the 7th group congener technetium, in terms of the large variety of oxidation states, metallic cores, and bifunctional chelators adequate for the design of radiopharmaceuticals.71,85 In fact, for a given class of ligands and metal oxidation state, Re and Tc complexes are usually isostructural. However, there are important differences in the kinetics of ligand exchange reactions and redox chemistry of Re and Tc complexes, which are key issues in the radiopharmaceutical chemistry of these metals. Rhenium compounds are more difficult to reduce than the Tc congeners. Moreover, ligand exchange reactions are faster for Tc complexes. The labeling of peptides with 186Re/188Re can be attempted using the strategies mentioned above for 99mTc, starting from aqueous perrhenate. However, the achievement of 186Re and 188Re-labeled peptides with high in vivo stability can be quite challenging due to more pronounced tendency of Re complexes to undergo in vivo oxidation reactions. Target-specific radiometallated peptides Radiopeptides targeting the avb3 integrin receptor Integrins are a family of heterodimeric receptors that play a pivotal role in many cell–cell and cell–extracellular matrix interactions.86,87 They consist on transmembrane glycoproteins that contain two non-covalently bound a and b subunits. In mammals, 18 a and 8 b subunits have been characterized, which selectively combine to afford at least 24 different integrin receptors.88 The integrin receptor avb3, also known as the vitronectin receptor, is expressed on endothelial cells and modulates cell migration and survival during angiogenesis. Being overexpressed in a variety of tumor cell types, such as gliobastoma, melanoma, ovarian, breast and prostate cancer, it potentiates tumor invasion and metastasis.89–92 Thus, the avb3 receptor has become a target of choice for the diagnosis and therapy of rapidly growing and metastatic tumors.93 Additionally, the non-invasive assessment of avb3 expression in vivo can be helpful to select patients likely to respond to treatment with antiangiogenic drugs, as well as allowing treatment follow-up. The avb3-integrin recognizes selectively extracellular matrix proteins, such as vitronectin or fibronectin, which contain the exposed Arg-Gly-Asp (RGD) sequence.94 The discovery of the canonical RGD sequence motivated an intense research work on small peptide-based molecules aimed at finding avb3-integrin antagonists suitable for antiangiogenic therapy.95,96 Moreover, a plethora of mono- and multivalent RGD-containing peptides have been labeled with a variety of radionuclides.97–102 So far, the most promising results have been obtained with [18F]galacto-RGD,103 which has been evaluated in patients with melanoma, sarcoma and breast cancer. However, this tracer has a relatively low tumor uptake, high cost and is obtained by a tedious and relatively low-yield radiosynthesis. Looking for a better alternative, intense research efforts have been devoted to radiometallated RDG- containing peptides for PET or SPECT imaging of avb3-integrin receptors. Incorporation of the RGD sequence into a cyclic pentapeptide structure provides avb3-antagonists with enhanced affinity and selectivity, as in the case of cyclo(Arg-Gly-Asp-DTyr-Val). Re- placement of the Val5 in c(RGDfV) by Lys led to c(RGDfK) (Fig. 6) without altering the integrin avb3 binding-affinity.104,105 This journal is © The Royal Society of Chemistry 2011 Dalton Trans. D ow nl oa de d by I ns ti tu to T ec no lo gi co e N uc le ar ( IT N ) on 2 5 F eb ru ar y 20 11 P ub li sh ed o n 25 F eb ru ar y 20 11 o n ht tp :/ /p ub s. rs c. or g | d oi :1 0. 10 39 /C 0D T 01 59 9G View Online http://dx.doi.org/10.1039/C0DT01599G The c(RGDfK) motif has been the most extensively explored for development of radiometallated peptides, profiting from the presence of the e-NH2 group of Lys5 to conjugate to a BFC and/or pharmacokinetic modifiers (Fig. 6). For evaluation of compounds with maximized binding affinity via the bivalency/multivalency approach, the c(RGDfK) motif has also been used to syn- thesize congener multimeric molecules (e.g. E[c(RGDfK)]2 or E[E[c(RGDfK)]2]2) via either a glutamic acid tree, by assembling to the Regioselectivity Adressable Functionalized Template (RAFT), or by click chemistry (Fig. 6).106–117 The linear peptides Gly1-Arg2-Gly3-Asp4-Ser5-Pro6-Cys7 and Arg1-Gly2-Asp3-Ser4-Cys5-Arg6-Gly7-Asp8-Ser9-Tyr10 were the first 99mTc-labeled RGD-containing compounds.118,119 The result- ing radiometallated peptides correspond most likely to Tc(V) oxocomplexes stabilized by the cysteine side chain. The radio- labeled decapeptide was able to localise metastatic melanoma lesions in several patients but with low tumor-to-background ratios.119 A doubly cyclized RGD-contaning peptide (NC100692), bearing a PEGylated C-terminus and a diamine-dioxime chela- tor for complexation of Tc(V), was used to prepare 99mTc- NC100692 (Fig. 7).120–122 The ability of 99mTc-NC100692 to de- tect metastatic lesions in 15 patients with lung cancer and 10 patients with breast cancer was investigated in a multicenter phase 2a clinical trial. It has been concluded that the sensitivity of 99mTc-NC100692 to detect liver metastases was poor and the detection of bone metastases equivocal. However, lung and brain metastases from both breast and lung cancer could be detected.122 Different monomeric or multimeric cyclic RGD-containing peptides have been labeled with 99mTc using the HYNIC approach.97,99 The 99mTc complex 99mTc-HYNIC-E[c(RGDfK)2] has shown a tenfold higher affinity for avb3-integrin com- pared to the monomeric congener 99mTc-HYNIC-E-c(RGDfK). In agreement, the dimeric compound has shown an increased tumor uptake and retention in a OVCAR-3 ovarian carcinoma xenograft. However, kidney retention of the dimeric peptide was higher than that of the corresponding monomer.107,123 To improve the biodistribution profile of radiolabeled dimeric peptides of the 99mTc-HYNIC-E[c(RGDfK)2] type, different strategies were explored, namely the use of different co- ligands such as trisodium triphenylphosphine-3,3¢,3¢¢-trisulfonate (TPPTS), isonicotinic acid (ISONIC) or 2,5-pyridinedicarboxylic acid (PDA). The resulting ternary complexes, [99mTc-HYNIC- E[c(RGDfK)]2(tricine)(L)] (L = TPPTS, ISONIC, PDA) showed a high tumor uptake and improved tumor to kidney and tumor to liver ratios.124 Fig. 6 Monomeric, dimeric and tetrameric cyclic RGD peptides. Dalton Trans. This journal is © The Royal Society of Chemistry 2011 D ow nl oa de d by I ns ti tu to T ec no lo gi co e N uc le ar ( IT N ) on 2 5 F eb ru ar y 20 11 P ub li sh ed o n 25 F eb ru ar y 20 11 o n ht tp :/ /p ub s. rs c. or g | d oi :1 0. 10 39 /C 0D T 01 59 9G View Online http://dx.doi.org/10.1039/C0DT01599G Fig. 7 Structure of NC100692. A series of cyclic dimeric RGD peptides containing triglycine (G3) and PEG4 linkers between the E[c(RGDfK)2] binding mo- tifs were recently introduced and labeled with 99mTc using the HYNIC approach and TPPTS as the co-ligand. The complexes [99mTc(HYNIC-3G3-dimer)(tricine)(TPPTS)] and [99mTc(HYNIC- 3PEG4-dimer)(tricine)(TPPTS)] have shown a higher avb3- integrin binding affinity and much higher tumor uptake in MDA- MB-435 breast cancer xenograft than [99mTc(HYNIC-PEG4- dimer)(tricine)(TPPTS)].125,126 These differences can be accounted for by the longest distances between the two cyclic RGD motifs providing for the best-performing complex. The related radiopep- tide [99mTc(HYNIC-PEG4-tetramer)(tricine)(TPPTS)], bearing a tetrameric RGD derivative, also presented a high tumor uptake, but has shown more pronounced kidney and liver retention compared to the dimeric congeners. The tricarbonyl approach has been also used in several instances for labeling linear or cyclic RGDf/yK peptides, using histidine, N,N-picolylamine diacetic acid (PADA), iminodiacetic acid (IDA) or pyrazolyl-diamine (pzNN) as BFCs.127–129 The radiometallation of RGD-containing peptides did not compromise their affinity for avb3-integrin receptors, although all the compounds presented a relatively low tumor accumulation. A variety of monomeric or multimeric RGD-containing peptides have been labeled with 111In, 68Ga and 64Cu, using polyaminocarboxylic ligands as BFCs. The initial studies were done with DTPA- and DOTA-c(RGDf/yK) derivatives, which were further optimized using pharmacokinetic modifiers such as additional charged amino acids (e.g. glutamic acid) or PEGylated linkers.130,131 In the case of [111In-DTPA-E-E[c(RGDfK)]2] and [64Cu-DOTA-PEG(3400)-c(RGDfK)] the presence of glutamic acid and PEG (3400 Da) spacers led to enhanced tumor to kidney, and tumor to liver ratios without compromising tumor uptake.113,132 Some dimeric RGD derivatives (Fig.6) were coupled to NOTA and DOTA chelators, using triglycine (G3) or PEG4 linkers, and labeled with 68Ga, 64Cu or 111In. The radioconjugates [68Ga-NOTA-X-dimer] (X = 2G3, 2PEG4), [M-DOTA-3PEG4- dimer] and [M-DOTA-3G3-dimer] (M = 64Cu, 111In) have shown high tumor uptake and prolonged tumor retention with favorable tumor to background ratios.133–136 Interestingly, [64Cu-DOTA- 3PEG4-dimer] and [111In-DOTA-3PEG4-dimer], sharing the same BFC, have shown almost superimposed tumor uptake and tumor to background values in the same animal model, suggesting a min- imal impact of the radiometal on the biodistribution profile.133,135 [111In-DTPA-3PEG4-dimer] has also shown a high initial tumor uptake with excellent tumor-to-liver and tumor-to-kidney ratios. However, the DTPA-conjugate showed a much faster tumor washout and poorer tumor-to-background ratios compared to [111In-DOTA-3PEG4-dimer].135 Altogether, these findings seem to indicate that 3PEG4-dimer and 3G3-dimer are among the most suitable RGD-containing compounds to design radiometallated peptides for SPECT and PET imaging of avb3-integrin expression, as well as for PRRT (Peptide Receptor Radionuclide Therapy) of avb3-positive tumors. Radiopeptides targeting the cholecystokinin 2 (CCK2)/gastrin receptor Cholecystokinin (CCK) is an endogenous regulatory peptide that displays a wide variety of physiological functions both in the gastrointestinal tract and central nervous system. All the biologically active forms of the peptide (e.g. CCK33, CCK8 and CCK4) are derived from a 115-amino acid peptide precursor, with CCK8 (Asp-Tyr-Met-Gly-Trp-Met-Asp-Phe-NH2) being the most abundant form in the brain.137–139 So far, three distinct subtypes of CCK receptors have been identified: CCK1 or CCK- A, CCK2/gastrin receptor or CCK–B, and CCK2idsv.137–140 A high incidence of CCK2 receptor protein was found in medullary thyroid carcinomas (MTC) (92%), small cell lung cancer (SCLC) This journal is © The Royal Society of Chemistry 2011 Dalton Trans. D ow nl oa de d by I ns ti tu to T ec no lo gi co e N uc le ar ( IT N ) on 2 5 F eb ru ar y 20 11 P ub li sh ed o n 25 F eb ru ar y 20 11 o n ht tp :/ /p ub s. rs c. or g | d oi :1 0. 10 39 /C 0D T 01 59 9G View Online http://dx.doi.org/10.1039/C0DT01599G (89%), stromal ovarian cancers (100%), astrocytomas (65%), some of the neuroendocrine gastroenteropancreatic tumors, and in several soft tissue tumors, in particular in leiomyosarcomas.2,141,142 Thorough research efforts have been directed toward the development of radioactive peptides for targeting CCK2 receptor in vivo, aiming at the visualization/detection or treatment of CCK2 receptor-expressing tumors such as MTC or SCLC.143–145 As can be seen in Tables 3 and 4, the peptides studied include gastrin- or CCK-related analogs, which share the C-terminal CCK receptor-binding tetrapeptide sequence Trp-Met-Asp-Phe-NH2. In some of these analogs, the methionine amino acid may be replaced by leucine or norleucine.145 The initial promising results obtained with 131I-labeled gastrin I at the preclinical and preliminary clinical level prompted several Table 3 Gastrin and gastrin analogsa Amino acid sequence: pGlu-Gly-Pro-Trp-Leu-(Glu)5 -Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH2 (Human Gastrin I) Leu-(Glu)5 -Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH2 (Minigastrin (MG)) DGlu-(Glu)5 -Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH2 (MG0) DGlu-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH2 (MG11) His-His-Glu-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH2 (H2Met) His-His-Glu-Ala-Tyr-Gly-Trp-Nle-Asp-Phe-NH2 (H2Nle) (His)6 -Glu-Ala-Tyr-Gly-Trp-Nle-Asp-Phe-NH2 (H6Nle) Gly-Ser-Cys(succinimidopropionyl-Glu-Ala-Tyr-Gly-Trp-Nle-Asp-Phe- NH2 )-Glu-Ala-Tyr-Gly-Trp-Nle-Asp-Phe-NH2 (divalent peptide MGD5) Gly-DGlu-(Glu)5 -Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH2 ([Gly0 -DGlu1 ]MG) Glu-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH2 (Peptide1) Gly-His-Glu-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH2 (Peptide2) Gly-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH2 (Peptide3) a Amino acid residues in bold type are important for the biological activity of the peptide. Table 4 Cholecystokinin (CCK) analogsa b Amino acid sequence: DAsp-Tyr-Met-Gly-Trp-Met-Asp-Phe-NH2 (CCK8) DAsp-Tyr(OSO3 H)-Met-Gly-Trp-Met-Asp-Phe-NH2 (sCCK8) DAsp-Tyr-Nle-Gly-Trp-Nle-Asp-Phe-NH2 ([Nle3,6 ]CCK8; CCK8(Nle)) DAsp-Phe(p-CH2 SO3 H)-Nle-Gly-Trp-Nle-Asp-Phe-NH2 (sCCK8[Phe2 (p-CH2 SO3 H), Nle3,6 ]) DAsp-Phe(p-CH2 SO3 H)-HPG-Gly-Trp-HPG-Asp-Phe-NH2 (sCCK8[Phe2 (p-CH2 SO3 H), HPG3,6 ]) Trp-Nle-Asp-Phe-NH2 (CCK4) Ahx-Ahx-Trp-Nle-Asp-Phe-NH2 ((Ahx)2 -CCK4) DAsp-Tyr-Met-Gly-Trp-Nle-Asp-Phe-NH2 ([Nle6 ]CCK8) a HPG = homopropargylglycine; Ahx = 6-aminohexanoic acid. b Amino acid residues in bold type are important for the biological activity of the peptide. research groups to label gastrin and CCK analogs with metals such as 111In or 99mTc using adequate BFCs.146,147 Beher and Béhé demonstrated that [111In-DTPA-DGlu1]MG (111In-DTPA-MG0) showed improved in vitro and in vivo stability over [111In-DTPA-]MG.147 In tumor-bearing nude mice, fast and specific uptake in CCK–B-receptor-positive tissues and a fast renal clearance pattern was found for both peptides. However, [111In- DTPA-]MG showed higher background activity in the whole body. In humans, fast tumor and stomach uptake was observed for both 111In-labeled compounds, but 111In-DTPA-MG0 lacked the liver, spleen and bone marrow uptake observed with its Leu1 analog.148 Following a preliminary pilot clinical study with 111In-DTPA- MG0 in four MTC patients, where CCK2 receptor expression was identified both in physiologically CCK2 receptor-expressing tissues and in metastatic lesions, a larger clinical study in 75 patients was performed.147,149 These clinical studies allowed the visualization of all tumors detected by other imaging modalities and, interestingly, in 29 out of 32 MTC patients with occult disease, at least one lesion was detected. In the same study, the therapeutic effect of 90Y-DTPA-MG0 was studied in 8 MTC patients and, despite severe nephrotoxicity in two of them, four patients experienced stabilization of the disease, which lasted for up to 36 months. The utility of 111In-DTPA-MG0 for visualization of CCK2 receptor-expressing tumors was confirmed by an independent clinical study conducted by Gottardt et al.150 Aiming to reduce the high renal retention associated with MG0, the MG11, minigastrin analogs, missing five glutamic acid residues in positions 2–6, have been developed. The biological properties of111In-DTPA-MG0 were also compared with those of the ra- diopeptide 111In-DOTA-MG11 in AR4-2J-tumor bearing Lewis rats. The reduction of the number of glutamates increased tumor- to-kidney ratio but, additionally, resulted also in a considerably lower metabolic stability.151,152 A new family of 111In- DOTA-minigastrin analogs, containing a variable number of His residues at the N-terminal (111In-DOTA- H2Met, 111In-DOTA-H2Nle, 111In-DOTA-H6Nle) was assessed in pancreatic xenografted models (Table 3).153 Among these peptides, 111In-DOTA-H2Met has shown the most interesting properties in terms of tumor-to-kidney ratios, with saturable uptake in target organs and low uptake by nontarget tissues other than the kidney. However, a high level of oxidation of the methionine residues was observed during the labeling procedure. Replacement of Met by Nle, a non-oxidizable amino acid, led to a significant reduction of receptor affinity and in vivo tumor uptake, contrary to what has been described for other analogs.154,155 To improve the in vivo performance of these monomeric CCK2R-binding minigastrin analogs, Sosabowski et al. la- beled the divalent gastrin peptide conjugate DOTA-Gly-Ser- Cys(succinimidopropionyl-Glu-Ala-Tyr-Gly-Trp-Nle-Asp-Phe- NH2)-Glu-Ala-Tyr-Gly-Trp-Nle-Asp-Phe-NH2 (DOTA-MGD5) with 111In, and compared the tumor-targeting properties of the resulting radiocomplex with those of 111In-DOTA-H2Met. Biological studies have shown that dimerization of the receptor binding site resulted in an increase in tumor uptake. However, such effect must still be confirmed in humans.156,157 One of the most successful approaches to target CCK2 receptor- expressing tumors in vivo with radiometal-based probes has been developed by Nock et al., who synthesized a minigastrin analog labeled with the trans-[99mTcO2]+ core stabilized by a tetraamine Dalton Trans. This journal is © The Royal Society of Chemistry 2011 D ow nl oa de d by I ns ti tu to T ec no lo gi co e N uc le ar ( IT N ) on 2 5 F eb ru ar y 20 11 P ub li sh ed o n 25 F eb ru ar y 20 11 o n ht tp :/ /p ub s. rs c. or g | d oi :1 0. 10 39 /C 0D T 01 59 9G View Online http://dx.doi.org/10.1039/C0DT01599G Fig. 8 Metallated CCK4 derivatives of the type [M(O)(SN2 O-PhSCN-X n -CCK4)] (M = 99m Tc, Re; X = b-alanine, Ahx; n = 0, 2 and 4). ligand. Different spacers between the chelator and the peptide have been explored. Among all of them, [99mTc(O2)(N4 0–1)Gly0- DGlu1]MG (Demogastrin 2) was the most promising. The biolog- ical behavior of Demogastrin 2 has been compared with that of 111In-DOTA-MG11 and 111In-DOTA-CCK8 both at the preclinical and clinical level. Demogastrin 2 was the best diagnostic tool in MTC patients, not only because of its superior in vivo stability, but also due its high sensitivity and better quality of the scintigraphic images. Renal uptake was similar to all radiopeptides studied, but could be reduced by co-injection of polyglutamic acid.158–160 The analogs MG0 and MG11 were also labeled with 99mTc, using the HYNIC approach, and the resulting complexes, 99mTc-EDDA- HYNIC-MG0 and 99mTc-EDDA-HYNIC-MG11, were evaluated in AR4-2J rat pancreatic tumor cells and in AR4-2J tumor-bearing nude mice. The 99mTc-EDDA-HYNIC-MG11 derivative showed advantages over 99mTc-EDDA-HYNIC-MG0, in terms of lower kidney retention with unchanged uptake in tumors and CCK-2 receptor-positive tissue. However, the lower metabolic stability and impurities formed in the labeling process still leave room for further improvement.161 Attempting to improve stability, cyclic variants of MG11 have been proposed, and the resulting peptides were labeled with the 99mTc-HYNIC moiety, yielding the radiometalated peptides 99mTc-EDDA-HYNIC-cycloMG1 and 99mTc-EDDA- HYNIC-cycloMG2. Both radiopeptides showed rapid internal- ization in receptor expressing cells (AR42J cells) and high tumor uptake in AR42J tumor xenografts. However, the cyclization of MG had only a limited effect on the overall stability, and the biodistribution profile of 99mTc-EDDA-HYNIC-cyclo-MG1 was similar to the linear analog 99mTc- EDDA-HYNIC-MG11.162 King et al. reported the synthesis of three peptide-HYNIC conjugates containing the -Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH2 C-terminal sequence and combinations of histidine, glutamic acid, and glycine. The peptide conjugates were labeled with 99mTc using either tricine or EDDA as a coligand, and their biological properties evaluated in AR42J cells and AR42J tumor xenograft. It was found that the insertion of histidine into the sequence of peptide-HYNIC conjugates resulted in more stable, more homogeneous 99mTc complexes (99mTc-Tricine-HYNIC-Lys- Peptide2) (Table 3), with improved tumor-targeting performance both in vitro and in vivo.163 In addition to the previously mentioned radioiodination studies of gastrin analogs for targeting CCK receptors in vivo, Beher et al. also investigated the utility of CCK derivatives, and concluded that sulfated (s) CCK analogs and some non-sulfated (ns) gastrin analogs displayed the highest binding affinities (Tables 3 and 4). Desulfation or the complete removal of the N-terminal Tyr led to a loss of affinity.147 Reubi et al. has introduced a family of highly potent and selec- tive DTPA- and DOTA-CCK (non-sulfated) analog conjugates: DTPA-CCK8(Nle) and DTPA-CCK8. The corresponding 111In complexes were prepared, and their biological properties indicated that these compounds have substantial promise for the in vivo visualization of CCK–B receptor-expressing tumors.154 Only 111In- DOTA-CCK8(Nle) was evaluated in humans and it was shown that this complex holds great potential for both scintigraphy and radionuclide therapy of human CCK2 receptor positive tumors such as MTC and SCLC.164 More recently, Aloj et al. studied the in vitro and in vivo properties of 111In-DTPAGlu-Gly-CCK8, a complex containing the chelating moiety DTPAGlu bound through a glycine linker at the N-terminal end of the bioactive peptide CCK8. It was found that this highly stable radiopeptide presents a high- binding affinity to the receptor and presented avid uptake in CCK2R overexpressed xenographs, with rapid clearance of unbound radioactivity through the kidneys.146,165 The radiopeptide 111In-BPCA-(Ahx)2-CCK4, which contains two 6-aminohexanoic acid (Ahx) moieties between the BFC (BPCA) and the CCK4 derivative, presented a high and spe- cific tumor uptake and a low renal accumulation in mice bearing E151A-CCK2R tumors compared with the internal control, 111In-trans-cyclohexyldiethylenetriaminepentaacetic acid cholecystokinin octapeptide (111In-SCN-CHX-A¢¢-DTPA-[Nle]6- CCK8).166 The same research team has also proposed a set of 99mTc(V)-radiolabelled short peptide conjugates of the type indicated in Fig. 8.167 Laverman et al. have shown that sulfated and non-sulfated CCK8 peptides labeled with the 99mTc-HYNIC moiety using tricine/nicotinic acid as coligands bind with high affinity to the CCK2 receptor. 99mTc-HYNIC-sCCK8 also showed high affinity toward the CCK1 receptor. Studies in athymic mice bearing subcutaneous tumors expressing either CCK1 or CCK2 receptors revealed that uptake of 99mTc-HYNIC-sCCK8 in CCK1 or CCK2 receptor-positive tumors was fifteen-fold higher than that of 99mTc- HYNIC-nsCCK8.168 Owing to the fact that sCCK8 contains an easily hy- drolyzable sulfated tyrosine residue and two methionine residues prone to oxidation, Roosenburg et al. replaced the Tyr(OSO3H) moiety in sCCK8 by a robust isos- teric sulfonate, Phe(p-CH2SO3H), and replaced the methio- nine by norleucine (Nle) or homopropargylglycine (HPG). The peptides sCCK8[Phe2(p-CH2SO3H),Met3,6], sCCK8[Phe2(p- CH2SO3H),Nle3,6], and sCCK8[Phe2(p-CH2SO3H),HPG3,6] were N-terminally conjugated to DOTA and labeled with 111In. Biodistribution studies in mice with AR42J tumors showed This journal is © The Royal Society of Chemistry 2011 Dalton Trans. D ow nl oa de d by I ns ti tu to T ec no lo gi co e N uc le ar ( IT N ) on 2 5 F eb ru ar y 20 11 P ub li sh ed o n 25 F eb ru ar y 20 11 o n ht tp :/ /p ub s. rs c. or g | d oi :1 0. 10 39 /C 0D T 01 59 9G View Online http://dx.doi.org/10.1039/C0DT01599G [111In-DOTA-sCCK8[Phe2(p-CH2SO3H),Nle3,6] to have the highest tumor uptake.155 CCK8 has been derivatized with a Cys-Gly unit and labeled with the metal fragment 99m[Tc(N)(PNP3)]2+ (PNP3 = N,N- bis(dimethoxypropylphosphinoethyl)methoxyethylamine), giving the complex [99mTc(N)(NS-Cys-Gly-CCK8)(PNP3)]+. Biodistri- bution studies in nude mice bearing CCK2-R positive A431 xenografts showed rapid and specific targeting to CCK2-R, a four- fold higher accumulation compared to nonreceptor-expressing tumors.169 The CCK8 peptide was modified at its N-terminus by addition of two Lys-His units and histidine was coupled to the side chain of the lysine ((Lys-His)2-CCK8). The conjugate was labeled with fac- [99mTc(CO)3] and biodistribution experiments showed negligible tumor accumulation in A431-CCK2R xenografts.170 Radiopeptides targeting the vasoactive intestinal peptide receptor (VPAC-1) VIP, an endogenous growth hormone, is a 28 amino acid peptide with a wide range of biological activities such as vasodilatation, secretion of different hormones, immunomodulation and prolif- eration of normal and malignant cells. These actions are mediated trough the cell surface receptors VPAC1 and VPAC2, which are expressed in various tissues in different densities.171–174 These receptors, predominantly the VPAC1 subtype, are over- expressed in the great majority of the most frequently occurring human tumors, including breast (100%), prostate (100%), pancreas (65%), lung (58%), colon (96%), stomach (54%), liver (49%), and urinary bladder (100%) carcinomas as well as lymphomas (58%) and meningiomas (100%).175 VIP or VIP derivatives labeled mainly with 99mTc, or more recently 64Cu, have been explored extensively toward the in vivo detection/visualization of VPACR-expressing tumors (Table 5). Aiming to label the VIP peptide with 99mTc and to assess its properties for imaging colorectal cancer, the peptide was modified at the C-terminal by conjugation to a 4-aminobutyric acid (Aba) spacer, followed by 4 terminal amino acids (Gly-Gly-DAla-Gly), which provide a N4 donor atom set for metal stabilization. The pharmacokinetic profile of the resulting labeled peptide (99mTc-TP3654) (Table 5) was more favorable than that of 111In- DTPA-Octreotide or 99mTc-anti-CEA in the same tumor model. Preliminary clinical studies revealed that within 20 min all of the tumors could be delineated.176–178 Aimed at targeting VIP/PACAP receptors in breast tumors, a new VIP analog (TP3982) derivatized at the C-terminal with a N2S2 chelating unit (-(Dap-(BMA)2)) has been synthesized and fully characterized. Smooth-muscle relaxivity assays demon- strated functional integrity of the peptide conjugate TP3982, when compared with VIP. The conjugate was labeled with 64Cu and 99mTc in high yields, giving the stable metal-complexes 64Cu-TP3982 and 99mTc-TP3982, respectively. Imaging and tissue distribution studies after injection of 64Cu-TP3982, 99mTc-TP3982 or 99mTc-TP3654 in nude mice bearing human T47D breast tumor xenografts, revealed a significantly greater (21.2–74-fold) receptor-specific tumor uptake for 64Cu-TP3982.179 Following these promising results, the same team has synthe- sized and characterized three new VIP analogs (P3939, TP4200 and TP3805) containing also a N2S2 chelating unit for metal coordination. The peptide conjugates TP3939, TP4200, TP3805 and TP3982 retained the biological activity as demonstrated by smooth muscle relaxivity assays and cell binding assays (T47T human breast cancer line). The labeling yields of all analogs with 64Cu were higher than 92%. In vitro receptor autoradiography studies showed 2.17 to 10.93 times greater quantity of 64Cu-peptide analogs (including also TP3982) bound to breast cancer tissue (13 human breast cancer tissue) than to the normal breast tissue. These data indicated that a greater number of VAPAC1 receptors were expressed on malignant cells than on the normal. This finding was corroborated by RT-PCR studies using the same samples.180 64Cu-TP3939 has been investigated as a PET imaging probe to detect prostate cancer, its metastatic or recurrent lesions and to determine the effectiveness of its treatment. Biodistribution studies in PC3 tumor-bearing nude mice demonstrated rapid blood clearance, high stability and receptor-specific tumor uptake. The PET images delineated the xenografted PC in nude mice, as well Table 5 Vasoactive intestinal peptide (VIP) and analogsa b Amino acid sequence: His-Ser-Asp-Ala-Val-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-Met-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser-Ile-Leu-Asn-NH2 (VIP) His-Ser-Asp-Ala-VaI-Phe-Thr-Asp-Mn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-Met-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser-lle-Leu-Asn-Aba-Gly-Gly-DAla-Gly (TP3654) His-Ser-Asp-Ala-Val-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-Met-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser-lle-Leu-Asn-Aba-Lys-(Dap-(BMA)2) (TP3982) His-Ser-Asp-Ala-Val-Phe-Thr-Asp-Asn-Tyr-Thr-Lys-Leu-Arg-Lys-Gln-Nle-Ala-Val-Lys-Lys-(3-OCH3,4-OH)Phe-Leu-Asn-Ser-Val-Leu-Thr-Aba- Lys-(Dap-(BMA)2) (TP3939) Ac-His-Ala-Asp-Ala-Val-Phe-Thr-Glu-Asn-Tyr-Thr-Lys-Leu-Arg-Lys-Gln-Nle-Ala-Ala-Lys-c(-Lys-Tyr-Leu-Asn-Asp-)-Leu-Lys-Lys-Ala-Ala-Ala- Aba-Lys(Dap-BMA)2) (TP4200) His-Ser-Asp-Gly-lle-Phe-Thr-Asp-Ser-Tyr-Ser-Arg-Tyr-Arg-Lys-Gln-Met-Ala-Val-Lys-Lys-Tyr-Leu-Ala-Ala-Val-Leu-Aba-Lys-(Dap-(BMA)2) (TP3805) His-Ser-Asp-Deg-Val-4-Cl-DPhe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-Leu-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser-Ile-Leu-Asn-NH2 (VP05) His-DPhe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-Leu-Aib-Val-Lys-Lys-Tyr-Leu-NH2 (VD4) His-ACP-DPhe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-Leu-Aib-Val-Lys-Lys-Tyr-Leu-NH2 (VD5) a Aba = 4-aminobutyric acid; Dap = diaminopropionic acid. b Amino acid residues in bold type are important for the biological activity of the peptide. Some chelating units are also displayed in bold type. Dalton Trans. This journal is © The Royal Society of Chemistry 2011 D ow nl oa de d by I ns ti tu to T ec no lo gi co e N uc le ar ( IT N ) on 2 5 F eb ru ar y 20 11 P ub li sh ed o n 25 F eb ru ar y 20 11 o n ht tp :/ /p ub s. rs c. or g | d oi :1 0. 10 39 /C 0D T 01 59 9G View Online http://dx.doi.org/10.1039/C0DT01599G as spontaneous occult PC in TRAMP II mice, which was not delineated by 18F-FDG in the same animal model. As expected, 64Cu-TP3939 did not detect prostate with hyperplasia in TRAMP I, confirming the specific nature of the probe. Brought together, the results confirm the potential of 64Cu-TP3939 for PET imaging of prostate cancer and its metastatic or recurrent lesions.181 Thakur et al. also studied the ability of 94Cu-TP3805 to detect breast cancer (BC) in MMTVneu mice using 18F-FDG as a gold standard. PET imaging studies in mice have shown that 94Cu- TP3805 could identify all malignant lesions that overexpressed VPAC1 receptors. Interestingly, benign tumors that did not express the receptor could only be imaged by 18F-FDG and not by 94Cu- TP3805.182 A set of three other VIP analogs (VP05, VD4 and VD5) have been directly labeled with the moiety fac-[99mTc(CO)3]+, and the tumor-targeting properties of the resulting radioactive species evaluated in human colon carcinoma cells (PTC cells) and in a animal tumor model. Despite the specific in vitro cell uptake of 99mTc(CO)3-labeled VP05 analog, its tumor uptake was modest.183 Radiopeptides targeting the glucagon-like peptide-1 receptor (GLP-1) Glucagon-like peptide-1 is a intestinal hormone that plays an important role in glucose metabolism and homeostasis. GLP-1 stimulates postprandial insulin secretion from pancreatic b-cells in a manner dependent on blood-glucose levels. This receptor was shown to be overexpressed in various neuroendocrine tumors, particularly in human insulinomas, as well as in brain tumors and embryonic tumors but not in carcinomas or lymphomas. Additionally, GLP-1R could not be identified in specific tissue compartments of several organs (e.g. pancreas, intestine, and lung). Such findings have made this receptor a promising molecular target for in vivo imaging or therapeutic proposals.184,185 The proof-of-principle for in vivo GLP-1 receptor targeting was provided in a pioneer study by Gottarhardt et al., who detected insulinomas in NEDH rats and RINm5F cells, using radioiodinated GLP-1(7–36)amide and exendin-3 ([123I]GLP-1(7– 36)amide and [123I]exendin-3, Table 6).186 These promising results prompted further studies with radiometallated (111In, 68Ga and 99mTc) GLP-1 analogs. DTPA conjugates of exendin-4 were synthesized and labeled with 111In. Among others, the stable radiometallated compound Table 6 Glucagon-like peptide 1 (GLP-1) analogsa Amino acid sequence: His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly- Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-NH2 (GLP-1(7–36)amide) His-Ser-Asp-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu3-Ala- Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly2-Pro-Ser2 -Gly-Ala- Pro3- Ser-(Lys40 )-NH2 (Lys40 -Exendin-3) His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu3-Ala- Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly2-Pro-Ser2 -Gly-Ala- Pro3- Ser-(Lys40 )-NH2 (Lys40 -Exendin-4) a Amino acid residues in bold type are important for the biological activity of the peptide. [Lys40(Ahx-DTPA-111In)NH2]exendin-4 accumulates significantly and specifically in the tumor of Rip1Tag2 mice, a transgenic mouse model of pancreatic b-cell carcinogenesis, which exhibits a GLP- 1R expression comparable with human insulinoma. The high tumor uptake resulted in excellent tumor visualization by pinhole SPECT/MRI and SPECT/CT.187,188 The therapeutic potential of [Lys40(Ahx-DTPA-111In)NH2]exendin-4 has been evaluated also in the same transgenic mouse model (Rip1Tag2 mice). A single injection of the radiopeptide resulted in a reduction of the tumor volume by up to 94% in a dose-dependent manner without significant acute organ toxicity. The authors claim that these results prove that the Auger-emitting compound is able to produce relevant therapeutic effects.189 The same radiopeptide successfully detected tumors in patients with insulinomas that were not detected by other imaging modalities.190 Following the successful use of [Lys40(Ahx-DTPA- 111In)NH2]exendin-4 for the detection of insulinomas in rodents and humans, the radiopeptide [Lys40(Ahx-DOTA- 111In)NH2]exendin-4 has been prepared and tested in six patients.191 GLP-1R scans detected the insulinomas in all six cases. By using a g-probe intra-operatively, the radiopeptide allowed successful surgical removal of all insulinomas, presenting a high density of GLP-1R as confirmed by autoradiography. To overcome some of the drawbacks associated with the use of 111In for imaging, the new radiopeptides [Lys40(Ahx-DTPA-68Ga)NH2]exendin-4 and [Lys40(Ahx-HYNIC- 99mTc/EDDA)NH2]exendin-4 were prepared. Biodistribution studies in Rip1Tag2 mice have shown a high tumor uptake for [Lys40(Ahx-DTPA-68Ga)NH2]exendin-4, comparable to that of [Lys40(Ahx-DTPA-111In)NH2]exendin-4 and significantly higher than that of [Lys40(Ahx-HYNIC-99mTc/EDDA)NH2]exendin-4. However, the lower tumor uptake obtained with the 99mTc complex did not result in reduced image quality as all the radiopetides showed high tumor-to-background ratios. Such results make 99mTc- and 68Ga-labeled exendin-4 suitable candidates for clinical GLP-1R imaging studies.192 The biodistribution profile of the new radiopeptide [Lys40(DOTA-68Ga)NH2]exendin-3 was evaluated in BALB/c nude mice with subcutaneous INS-1 tumors and compared with that of [Lys40(DOTA-111In)NH2]exendin-3 and [Lys40(DTPA- 111In)NH2]exendin-3 in the same animal model. The chelator used did not affect the biodistribution profile of [Lys40]exendin-3 as evidenced by the almost identical concentrations of [Lys40(DOTA- 111In)NH2]exendin-3 and [Lys40(DTPA-111In)NH2]exendin-3 in all tissues examined. The biodistribution of the latter was also iden- tical to the biodistribution of [Lys40(DTPA-111In)NH2]exendin- 4. Tumor uptake of 68Ga-labelled [Lys40(DOTA)]exendin-3 was lower than tumor uptake of 111In-labelled [Lys40(DTPA)]exendin- 3. Despite this difference in insulinoma uptake, the authors claim that clinical studies should be conducted to elucidate the potential of [Lys40(68Ga-DOTA)]exendin-3 for insulinoma PET imaging in Humans.193 Peptides targeting chemokine receptor CXCR4 Chemokines are structurally related small glycoproteins (8–14 kDa) that chemoattract leukocytes by binding to cell surface receptors.194 CXCR4 is highly expressed in breast and prostate cancer, and plays a crucial role in tumor metastasis.5,195,196 This journal is © The Royal Society of Chemistry 2011 Dalton Trans. D ow nl oa de d by I ns ti tu to T ec no lo gi co e N uc le ar ( IT N ) on 2 5 F eb ru ar y 20 11 P ub li sh ed o n 25 F eb ru ar y 20 11 o n ht tp :/ /p ub s. rs c. or g | d oi :1 0. 10 39 /C 0D T 01 59 9G View Online http://dx.doi.org/10.1039/C0DT01599G Fig. 9 DTPA–AcTZ14011 peptide conjugate. Fig. 10 Labeling of SDF-1a with 99m Tc. Additionaly, chemokines and their receptors are also associated with cardiac disfunction.197–199 Aiming to prepare novel radiolabeled probes for the in vivo imag- ing of CXCR4 expression on tumors, Koglin et al. radiolabeled CPCR4, a cyclic peptide, and tested its biological properties.200 The authors claim that the tracer binds with high affinity and specificity in an antagonistic manner to its binding site and allowed a clear delineation of CXCR4 positive tumors in vivo. However, further optimization of the in vivo behavior of the tracer needs to be done. Hanaoka et al. designed a cyclic 14-residue peptidic CXCR4 inhibitor, AcTZ14011 (Fig. 9), attached it to DTPA through the side chain of DLys8, and labeled the resulting DTPA–AcTZ14011 conjugate with 111In.201 Biodistribution studies in nude mice bearing pancreatic carci- noma AsPC-1 have shown that the receptor-specific accumulation of [111In-DTPA–AcTZ14011] in the CXCR4-expressing tumor was greater than that in the blood or muscle. The authors claimed that this radiopeptide was a potential radiopharmaceu- tical for the imaging of CXCR4 expression in metastatic tumors in vivo. Aimed at the non-invasive quantification of CXCR4 expression in vivo, for the understanding of its importance in diverse processes including cardiac response to injury, recombinant SDF-1a was derivatized with a tetradentate N3S chelator (S- acetylmercaptoacetyltriserine: MAS3), and labeled with 99mTc, yielding the highly stable complex [99mTc-MAS3]-SDF-1a (Fig. 10).202 Biodistribution studies in a rat model of ischemia reperfusion have shown that after induction of myocardial infarction, CXCR4 expression levels in the myocardium increased more than 5-fold, as quantified using [99mTc-MAS3]-SDF-1a and confirmed using confocal immunofluorescence. The main conclusion drawn by the authors is that CXCR4 levels can be quantifiable in vivo in a variety of animal models, using appropriate radioactive probes such as [99mTc-MAS3]-SDF-1a. Peptides targeting neuropeptide Y receptors Neuropeptide Y (NPY), a member of the pancreatic polypeptide family, consists of 36 amino acids residues and binds to the five Y receptor subtypes (Y1, Y2, Y4, Y5 and y6) with nanomolar Dalton Trans. This journal is © The Royal Society of Chemistry 2011 D ow nl oa de d by I ns ti tu to T ec no lo gi co e N uc le ar ( IT N ) on 2 5 F eb ru ar y 20 11 P ub li sh ed o n 25 F eb ru ar y 20 11 o n ht tp :/ /p ub s. rs c. or g | d oi :1 0. 10 39 /C 0D T 01 59 9G View Online http://dx.doi.org/10.1039/C0DT01599G Table 7 Neuropeptide Y (NPY) and analogsa Amino acid sequence: Tyr-Pro-Ser-Lys-Pro-Asp-Asn-Pro-Gly-Glu-Asp-Ala-Pro-Ala-Glu-Asp- Met-Ala-Arg-Tyr-Tyr-Ser-Ala-Leu-Arg-His-Tyr-Ile-Asn-Leu-Ile-Thr- Arg-Gln-Arg-Tyr-NH2 (human NPY) Tyr-Pro-Ser-Lys-Pro-Asp-Phe-Pro-Gly-Glu-Asp-Ala-Pro-Ala-Glu-Asp- Met-Ala-Arg-Tyr-Tyr-Ser-Ala-Leu-Arg-His-Tyr-Ile-Asn-Leu-Ile-Thr- Arg-Pro-Arg-Tyr-NH2 ([Phe7 ,Pro34 ]-NPY) Ile-Asn-Pro-Ile-Tyr-Arg-Leu-Arg-Try-NH2 (BVD15) Ile-Asn-Pro-Lys-Ile-Tyr-Arg-Leu-Arg-Tyr-NH2 ([Lys4 ]-BVD15) Ile-Asn-Pro-Nle-Bpa-Arg-Leu-Arg-Tyr-NH2 (NPY1) a Amino acid residues in bold type are important for Y1R-binding. affinity (Table 7).203,204 NPY receptors are promising candidates in the oncology field since Y1R and/or Y2R have been found to be expressed in neuroblastoma, breast carcinomas, ovarian cancers, the Ewing sarcoma family of tumors, and high-grade glioblastomas among others.205–212 Beck–Sickinger et al. labelled a Y2 receptor-preferring NPY analog (Ac-[Ahx 5-24,Lys4,Ala26]- NPY) with the organometallic fac-[99mTc(CO)3]+ moiety using PADA (2-picolylamine-N,N-diacetic acid) as a chelator. A stable radiopeptide with selective Y2 binding in neuroblastoma cells was obtained.213 With regard to breast cancer, Reubi et al. have shown that Y1R is expressed in 85% of all tumors in large quantities and in 100% of the examined metastases.211,212 Interestingly, a shift of the receptor subtype from Y2 in healthy tissue to Y1 during neoplasm was found. Thus, Y1R selective peptides have been considered promising for imaging and therapy of breast cancer.205,214 A Y1R-specific NPY analog was labelled with 111In, using DOTA, and the resulting complex [111In-DOTA- Lys4,Phe7,Pro34]-NPY showed a high kidney and low tumor uptake in MCF-7 breast cancer xenografts.215 The same NPY analog was labeled with 99mTc using a N a-histidinyl acetyl (N aHis-ac) chelator and evaluated in breast cancer patients.216 A clear tumor uptake of [99mTc–N aHis-ac-Lys4,Phe7,Pro34]-NPY was observed, whereas normal tissues and organs only showed background radiation.216 The short NPY analog [Pro30, Nle31, Bpa32, Leu34]-NPY(28– 36) (NPY1, Table 7),217 with high affinity and selectivity for Y1 receptor, was labelled with 99mTc and 67Ga using pzNN and DOTA chelators, respectively.218 The biological interest of such radiopeptides has still to be demonstrated. Another short NPY analog, [Pro30, Tyr32, Leu34]-NPY(28–36) (BVD15), conjugated to DOTA at different positions was also described and the affinity to Y1R evaluated.219 It has been shown that the introduction of the BFC at the N-terminus of the peptide led to poor affinity, but the conjugate [Lys4(DOTA)]-BVD15 (Table 7) and the respective Cu-complex presented good affinity in the MCF-7 breast cancer cell line.219 Peptides targeting the melanocortin 1 (MC1) receptor a-Melanocyte-stimulating hormone (a-MSH), a linear tride- capeptide (Table 8), binds to five subtypes of melanocortin receptors (MC1–MC5).220 Among these, the melanocortin type 1 receptor (MC1R) is overexpressed in both melanotic and amelanotic murine and human melanoma cells.221–223 Furthermore, Table 8 a-MSH and analogsa Amino acid sequence: Ac-Ser-Tyr-Ser-Met4 -Glu5 -His-Phe7 -Arg-Trp-Gly-Lys-Pro-Val-NH2 (a-MSH) Ac-Ser-Tyr-Ser-Nle4 -Glu5 -His-DPhe7 -Arg-Trp-Gly-Lys-Pro-Val-NH2 (NDP-MSH) Ac-Nle4 -Asp5 -His-DPhe7 -Arg-Trp-Gly-Lys-NH2 (NAPamide) Ac-Cys3 -Cys4 -Glu5 -His-DPhe7 -Arg-Trp-Cys10 -Lys-Pro-Val-NH2 (CCMSH) Ac-Cys3 -Cys4 -Glu5 -His-DPhe7 -Arg-Trp-Cys10 -Arg11 -Pro-Val-NH2 (CCMSH(Arg11 )) Ac-cyclo[Cys4 -Glu5 -His-DPhe7 -Arg-Trp-Cys10 ]-Lys-Pro-Val-NH2 (CMSH) Ac- Nle4 -cyclo[Asp5 -His-DPhe7 -Arg-Trp-Lys10 ]-NH2 (Melanotan-II: MTII) bAla3 -Nle4 -cyclo[Asp5 -His-DPhe7 -Arg-Trp-Lys10 ]-NH2 (bAlaMT-II) bAla3 -Nle4 -Asp5 -His-DPhe7 -Arg-Trp-Lys10 -NH2 (MSHoct) a Amino acid residues in bold type are important for the biological activity of the peptide. more than 80% of human metastatic melanoma samples have also been identified to display MC1R receptors. Thus, MC1R became a potential target for the diagnosis and therapy of melanoma and metastases, and several linear and cyclic radiolabeled a-MSH analogs have been studied as candidates for MC1R targeting (Table 8).5,18,224,225 Structure–bioactivity studies have shown that the minimal sequence of a-MSH required for biological activity is His-Phe-Arg-Trp, and that the replacement in a-MSH of Met4 and Phe7 by Nle and DPhe, respectively, leads to the potent [Nle4,DPhe7]-aMSH analog (NDP-MSH, IC50 = 0.21 nM), which is enzymatically stable and has a long half-life.220 The peptide NDP-MSH was radiolabeled with 99mTc and 188Re using as BFCs mercapto-acetylglycylglycine (MAG2) or the tetrapeptide Ac-Cys-Gly-Cys-Gly (CGCG).226 The short linear peptide [bAla3,Nle4,Asp5,DPhe7,Lys10]-aMSH(3–10) (MSHoct) was conjugated to DOTA and to pzNN through the N-terminus and labeled with 111In and 99mTc, respectively.227,228 Despite the im- proved potency of NDP-MSH and MSHoct analogs, the resulting radiolabeled peptides have shown poor in vivo behavior. Another analog, [Ac-Nle4,Asp5,DPhe7]-aMSH(4–11) (NAPamide), with high affinity for MC1R, was also conjugated to DOTA and to pzNN through the lateral chain of Lys11. The DOTA derivative was labeled with 111In, 68Ga and 64Cu, while the pzNN-conjugate was labeled with 99mTc.229–231 In vivo evaluation has shown that the 64Cu-labeled peptide was unstable, with high liver and kidney uptake. The other labeled peptides were stable in vivo but have shown only a moderate tumor uptake. To reduce kidney reabsorption, several glycosylated derivatives of NAPamide were conjugated to DOTA and labeled with 111In. In vivo studies (B16F1-melanoma mice) confirmed the improvement of pharmacokinetics but the tumor uptake and retention was relatively low.232 Several homobivalent NAPamide derivatives containing DOTA or pzNN were labeled with 111In and with the tricarbonyl core, respectively.233,234 In the case of 111In, the dimeric peptides displayed excellent receptor affinity and internalization, but the tumor uptake was low and the kidney accumulation high, compared to the monomeric 111In-DOTA-NAPamide.234 Cyclized a-MSH analogs have also been used to improve binding affinity, in vivo stability, and receptor selectivity. Among This journal is © The Royal Society of Chemistry 2011 Dalton Trans. D ow nl oa de d by I ns ti tu to T ec no lo gi co e N uc le ar ( IT N ) on 2 5 F eb ru ar y 20 11 P ub li sh ed o n 25 F eb ru ar y 20 11 o n ht tp :/ /p ub s. rs c. or g | d oi :1 0. 10 39 /C 0D T 01 59 9G View Online http://dx.doi.org/10.1039/C0DT01599G Fig. 11 Structures of Ac-ReCCMSH, 99m Tc-CCMSH(Arg11 ), and 111 In-DOTA-Re-CCMSH(Arg11 ). cyclization strategies, the disulfide-, lactam- and metal-based cyclizations have been the most explored.5,18,224 The peptide [Cys4,10,DPhe7]a-MSH (CMSH), cyclized via a disulfide bond, was conjugated to DOTA and labeled with 111In. In vivo evaluation of the resulting radiopeptide 111In-DOTA-CMSH has shown moderate tumor uptake and high kidney accumulation.235 Another approach consisted of the cyclization of an a-MSH analog through site-specific rhenium (Re) and technetium (Tc) metal coordination. Such cyclic analogs were structurally characterized and their ability to bind melanoma cells evaluated.236 It was found that the metal promotes cyclization by coordination to Cys3,4,10 sulfhydryls and to Cys4 amide nitrogen (Fig. 11). The resulting Re-peptide complex Re-[Cys3,4,10,DPhe7]-a-MSH(3–13) (ReCCMSH), displayed a high receptor-binding affinity. The cor- responding 99mTc complex 99mTc-CCMSH, although having high kidney uptake, successfully targeted B16F1-melanoma becoming the proof-of-principle for the potential use of metal-cyclized radiolabeled compounds for melanoma imaging or therapy.236 Another interesting approach was the conjugation of the metal- cyclized complex Re-CCMSH to DOTA, followed by labelling with 111In.235 Compared to the linear analog 111In-DOTA-CCMSH, the Re-cyclized radiopeptide 111In-DOTA-Re-CCMSH presented increased tumor-targeting capacity, higher tumor retention and enhanced renal clearance in murine melanoma-bearing mice.235 By comparing the two metal-based cyclized CCMSH analogs, 111In-DOTA-Re-CCMSH versus99mTc-CCMSH, similar tumor up- take was found, but the Re-mediated cyclized radiopeptide had an enhanced whole-body clearance and a higher tumor-to- blood ratio.237 Despite the favorable features of 111In-DOTA-Re- CCMSH, a relatively high level of radioactivity still remained in the kidneys. Aiming to reduce kidney retention, four new 111In- DOTA-derivatized Re-CCMSH analogs were designed, and the analog 111In-DOTA-Re-CCMSH(Arg11) (Lys11 replaced by Arg, Fig. 11) showed the highest tumor uptake and the lowest kidney radioactivity accumulation in a murine melanoma model.238 The analog CCMSH(Arg11) was also cyclized through labelling with 99mTc (Fig. 11).239 Also in this case, the replacement of Lys11 by Arg improved tumor uptake and reduced kidney accumulation. Com- pared to the Re-cyclized analog 111In-DOTA-Re-CCMSH(Arg11), 99mTc-CCMSH(Arg11) exhibited favorable and comparable tumor- targeting properties, and both allowed clear micro-SPECT/CT images of flank melanoma tumors as well as of B16F10 pulmonary melanoma metastases, with 99mTc-CCMSH(Arg11) providing im- ages with greater resolution of metastatic lesions.239 These promising properties prompted the direct labeling of the linear peptide CCMSH(Arg11) with 188Re, yielding the complex 188Re-CCMSH(Arg11).240,241 Its therapeutic efficacy was assessed in C57BL6 mice bearing B16/F1 murine melanoma tumors and in TXM-13 human melanoma xenografted SCID mice.242 In both, the therapeutic effect of 188Re-CCMSH(Arg11) on tumor growth was dose-dependent. For PET imaging of MC1R, the DOTA-Re-CCMSH(Arg11) conjugate was labeled with 64Cu and 86Y.243 Complex 64Cu- DOTA-Re-CCMSH(Arg11) had high radioactivity accumulation in non-target organs, due most likely to the in vivo instability of the complex and consequent release of 64Cu. To avoid such behaviour, the DOTA chelator was replaced by CBTE2A.244 The in vitro MC1R-binding properties of CBTE2A-Re-CCMSH(Arg11) were unchanged relative to DOTA-Re-CCMSH(Arg11).244 Fur- thermore, the complex 64Cu-CBTE2A-Re-CCMSH(Arg11) pre- sented a B16F1-melanoma uptake comparable to 64Cu-DOTA-Re- CCMSH(Arg11) but a significantly higher ratios of tumor to non- target tissues. In vivo studies have also shown that 64Cu-CBTE2A- Re-CCMSH(Arg11) provided better PET images than 86Y-DOTA- Re-CCMSH(Arg11), due to increased tumor retention and kidney clearance. Still for PET, 68Ga-DOTA-Re-CCMSH(Arg11) of low and high specific activity was prepared and evaluated.245,246 Despite some differences in the biological profile, in both cases the tumor uptake was low compared to the linear a-MSH analog 68Ga- DOTA-NAPamide in the same melanoma animal model. Such results indicated that the Re-mediated cyclization did not bring significant advantages when the radiometal is 68Ga. To evaluate the therapeutic potential of these cyclic peptides, 177Lu-DOTA- Re-CCMSH(Arg11) was prepared. Its in vivo evaluation showed a high and prolonged tumor uptake, but also high non-specific kidney accumulation.247,248 The tumor growth rate of treated mice was substantially reduced. The authors have also studied the same peptide conjugate labeled with 212Pb. They have found dramatic dose-dependent reductions in tumor growth rates for 212Pb-DOTA-Re(Arg11)CCMSH, and postmortem histopatholog- ical examination of the tumor and other major organs showed no sign of primary or metastatic melanoma.249 All treated groups displayed a significant improvement in mean survival time with minor kidney damage.249 A heterobivalent complex, 99mTc-RGD-Lys-CCMSH(Arg11), was synthesized for dual imaging of integrin and MC1 receptor- expressing tumors. Biodistribution studies in B16F1 melanoma- bearing C57BL6 mice have shown a tumor accumulation and retention higher than those found for 99mTc-CCMSH(Arg11), but an extremely high kidney uptake was observed.250 As an alternative to metal-cyclized a-MSH analogs, a promis- ing cyclization approach based on a side chain lactam-bridge was recently introduced.224 Using the potent lactam-cyclized Dalton Trans. This journal is © The Royal Society of Chemistry 2011 D ow nl oa de d by I ns ti tu to T ec no lo gi co e N uc le ar ( IT N ) on 2 5 F eb ru ar y 20 11 P ub li sh ed o n 25 F eb ru ar y 20 11 o n ht tp :/ /p ub s. rs c. or g | d oi :1 0. 10 39 /C 0D T 01 59 9G View Online http://dx.doi.org/10.1039/C0DT01599G Fig. 12 Structures of 99m Tc-pzNN-bAlaMT-II and 11 In-DOTA-Nle-CycMSHhex . agonist Melanotan II (MT-II) as model, the cyclic peptide bAla-Nle-c[Asp-His-DPhe-Arg-Trp-Lys]-NH2 (bAlaMT-II) was synthesized, conjugated to the pzNN chelator, and labeled with fac-[99mTc(CO)3]+ (Fig. 12).228,251 The radiometallated peptide 99mTc-(CO)3-pzNN-bAlaMT-II had high binding affinity and a remarkable internalization in B16F1 cells when compared with its linear analog and with the metal-cyclized 99mTc-CCMSH.228,252 In B16F1 melanoma-bearing mice, 99mTc-(CO)3-pzNN-bAlaMT- II has also showed a significant MC1R-mediated tumor uptake comparable to that obtained for metal-based cyclic peptides 99mTc- CCMSH and99mTc-CCMSH(Arg11).236,238 Longer lactam-based cyclic a-MSH analogs, CycMSH and GlyGlu-CycMSH (12-amino acid ring), were conjugated to DOTA, through the peptide N-terminal, and labeled with 111In.253 Both radiopeptides exhibited high receptor-mediated tumor up- take in B16F1 melanoma-bearing mice. These values were com- parable to those found for the lactam-based cyclic 99mTc-pzNN- bAlaMT-II and for the metal-cyclized 111In-DOTA-Re-CCMSH, but lower than those found for 111In-DOTA-Re-CCMSH(Arg11). The introduction of GlyGlu in the CycMSH sequence has reduced kidney accumulation. These results have prompted the synthesis of 67Ga-DOTA- GlyGlu-CycMSH and its biological assessment in B16/F1 melanoma-bearing mice. Kidney accumulation higher than tumor uptake was observed, but both flank primary B16/F1 melanoma and B16/F10 pulmonary melanoma metastases could be clearly visualized by SPECT/CT imaging.254 To evaluate the effect of DOTA position in the peptide backbone, GlyGlu-CycMSH was acetylated in the N-terminus, conjugated to DOTA through the Lys in the cyclic ring, and labeled with 111In. The overall pharmacokinetic profile of the resulting radiopeptide did not improve.255 The same authors also evaluated the effect of the ring size on biodistribution.256 Thus, DOTA was conjugated to the N-terminus of MT-II (6- amino acid peptide ring) and labeled with 111In (Fig. 12). The resulting radiopeptide, 111In-DOTA-Nle-CycMSHhex presented a rapid and high tumor uptake, a prolonged tumor retention and a moderate kidney accumulation. When compared to 111In-DOTA- GlyGlu-CycMSH with a 12-amino acid ring, the reduction of the peptide ring size dramatically increased the melanoma uptake and decreased the renal retention.253,256 The tumor-targeting properties and pharmacokinetics of 111In-DOTA-Nle-CycMSHhex were more favorable than those presented by the first lactam-cyclized 6-amino Table 9 Neurotensin and analogsa Amino acid sequence: pGlu-Leu-Tyr-Glu-Asn-Lys-Pro-Arg-Arg-Pro-Tyr-Ile-Leu-OH (NT) H-Arg-Arg-Pro-Tyr-Ile-Leu-OH (NT(8-13)) Lys-w(CH2 -NH)-Arg-Pro-Tyr-Ile-Leu-OH (NT-VI) Lys-w(CH2 -NH)-Arg-Pro-Tyr-Tle-Leu-OH (NT-XI) Ahx-Arg-Me-Arg-Pro-Tyr-Tle-Leu-OH (NT-XII) Arg-Me-Arg-Pro-Dmt-Tle-Leu-OH (NT-XIX) Ac-Lys-Pro-Me-Arg-Arg-Pro-Tyr-Tle-Leu-OH (NT-20.3) a Amino acid residues in bold type are important for the biological activity of the peptide. acid ring radiopeptide 99mTc-pzNN-bAlaMT-II.228,256 Finally, the lactam-cyclized 111In-DOTA-Nle-CycMSHhex displayed a tumor- to-kidney ratio comparable to the best metal-cyclized radiopeptide 111In-DOTA-Re-CCMSH(Arg11), suggesting a high potential of lactam-based cyclic radiolabeled a-MSH analogs for MC1R- melanoma targeting. Peptides targeting the neurotensin (NT) receptor Neurotensin (NT) is a 13 amino acid peptide (Table 9) that binds to three NT receptor subtypes (NTS1-NTS3). Most NT biological effects are mediated by NTS1, and NT(8–13) is the minimal sequence that mimics the effects of full-lengh NT.257 Overexpression of NT receptors has been found in many tumors, namely Ewing’s sarcoma, meningiomas, SCLC, MTC, ductal pancreatic adenocarcinomas (>70% overexpression) and invasive ductal breast cancers.258–261 Numerous NT(8–13) analogues (Table 9) containing the (NaHis)Ac chelator and labeled with 99mTc or 188Re have been synthesized.262–268 Among all the radiometallated neurotensin analogs, [99mTc(CO)3-(NaHis)Ac-NT-XIX] displayed the highest tumor uptake with low accumulation in non-target organs, particularly in kidneys.265,266 Encouraging therapeutic effects were also obtained in nude mice injected with the 188Re- analog.265 NTS1-binding NT analogs conjugated to DTPA or DOTA chelators have been synthesized and labeled with 111In.269 These radiopeptides had unfavorable ratios of tumor to non-target organs. In order to increase tumor uptake and reduce kidney accumulation, two novel families of DTPA-NT analogs have been proposed. The first family comprises a series of DTPA-NT(8–13) analogs (DTPA-NT-VI, DTPA-NT-XI, DTPA-Ahx-NT-XII and This journal is © The Royal Society of Chemistry 2011 Dalton Trans. D ow nl oa de d by I ns ti tu to T ec no lo gi co e N uc le ar ( IT N ) on 2 5 F eb ru ar y 20 11 P ub li sh ed o n 25 F eb ru ar y 20 11 o n ht tp :/ /p ub s. rs c. or g | d oi :1 0. 10 39 /C 0D T 01 59 9G View Online http://dx.doi.org/10.1039/C0DT01599G Fig. 13 Structures of 99m TcO-RP-527 and 99m TcO2 -demobesin 1. DTPA-Ahx-NT-XIX), which share the same peptide sequence with the analogs bearing the (Na-His)Ac moiety described previously. The second series of DTPA-peptides are analogs of NT(6–13) peptide (Table 9), in which DTPA was coupled to the e-NH2 of Lys6.270 Structure activity studies demonstrated that the attachment of DTPA induces an important loss of affinity, unless the distance between the BFCA and the biologically active sequence (NT(8–13)) is increased. Among all the radiopeptides, 111In-DTPA-NT-20.3 was the most promising, with high tumor uptake but still with a high kidney accumulation. In spite of the kidney retention, the tumor-to-intestine ratio was higher than that found for [99mTc(CO)3-(NaHis)Ac-NT-XIX]. Radiopeptides targeting the gastrin-releasing peptide (GRP) receptor Bombesin (BBN, pGlut1-Gln2-Arg3-Leu4-Gly5-Asn6-Gln7-Trp8- Ala9-Val10-Gly11-Hist12-Leu13-Met14-NH2) is an amphibian homo- logue of mammalian gastrin-releasing peptide (GRP) that has very high affinity and specificity for the GRP receptor (GRPr).271 To date, four different GRPr subtypes have been characterized, and overexpression of GRPrs has been observed on a variety of tumors including breast, prostate, pancreatic, and small-cell lung cancer (SCLC).2 The C-terminal 7–14 amino acid sequence of BBN is essential for high-affinity binding to GRPr. Therefore, various BBN analogs based on the BBN[7–14]NH2 agonist have been used by many research groups to design radiometallated peptides suitable for diagnostic and therapeutic of GRPr-positive tumors. Profiting from the diversified chemistry of technetium, different strategies have been used for labeling BBN derivatives with this ra- diometal. Such strategies involved the use of Tc-HYNIC, [TcO]3+, trans-[TcO2]+, fac-[Tc(CO)3]+ and the Tc(III) ‘4 + 1’ approach, in combination with a variety of bifunctional chelators.272–290 The pre-clinical evaluation of these 99mTc-labelled BBN derivatives led to some encouraging results, but only a few have been tested in the clinic. The radiopeptide 99mTc-RP-527 (Fig. 13), containing a N3S chelator coupled to BBN[7–14] via a Gly-5-aminovaleric acid, was able to identify primary breast cancer and prostate cancer and their metastatases.272 A more recent achievement has been the introduction of 99mTc-Demobesin 1 ([99mTc–N4-DPhe6,Leu- NHEt3,des-Met14]-BBN6-14), which contains a TcO2 + core and a linear tetraamine as BFC (Fig. 13). 99mTc-Demobesin 1 exhibited the highest absolute tumor uptake described in the literature for a PC3 xenograft model, while showing a high stability in vivo and a favourable pharmacokinetic profile.275,287 This radiopeptide has an antagonist character and does not internalize significantly into PC-3 cells, which suggests a change of paradigm on the diagnostic and PRRT of GRPr-positive tumors. The BBN[7–14]NH2 analog has been labeled with 64Cu using DOTA, CB-TE2A and NOTA-derivatives and different linkers to modulate pharmacokinetics.57,291–300 The resulting radioconju- gates behave as agonists and were able to target GRPr-positive xenografted human tumors in a specific way. Due to a prolonged retention of radioactivity in kidneys and gastrointestinal tract, the 64Cu-BBN-DOTA derivatives have showed less favourable target– non-target ratios compared to the radioconjugates-CB-TE2A and NOTA.57 These differences have been considered to reflect the Dalton Trans. This journal is © The Royal Society of Chemistry 2011 D ow nl oa de d by I ns ti tu to T ec no lo gi co e N uc le ar ( IT N ) on 2 5 F eb ru ar y 20 11 P ub li sh ed o n 25 F eb ru ar y 20 11 o n ht tp :/ /p ub s. rs c. or g | d oi :1 0. 10 39 /C 0D T 01 59 9G View Online http://dx.doi.org/10.1039/C0DT01599G Fig. 14 Chemical structure of [64 Cu-NO2A-(X)-BBN[7–14]]. highest in vivo stability of Cu(II) complexes with CB-TE2A and NOTA chelators. The most promising results were reported by Smith and co-workers for the radiopeptide [64Cu-NO2A-(X)- BBN[7–14]], where NO2A (1,4,7-triazacyclononane-1,4-diacetic acid) is a NOTA derivative and X are pharmacokinetic modifiers (Fig. 14). The radiopeptide [64Cu-NO2A-(AMBA)-BBN[7–14]], containing the shorter and more hydrophilic linker, exhibited the highest tumor accumulation and the fastest clearance from non- target tissues, emerging as a good candidate for further evaluation in humans.300 Several BBN derivatives were also labeled with the trivalent radiometals 67/68Ga, 111In and 177Lu, using DOTA-like chelators and different linkers to improve the pharmokinetics.301–314 Two of these derivatives, 68Ga-DOTABOM and 177Lu-AMBA, underwent clinical trials for PET detection or PRRT of prostate cancer (PC), respectively. 68Ga-DOTABOM allowed the detection of malignant PC lesions in 13 out of 15 patients.301 Within a phase I study and aiming at PC therapy, 177Lu-AMBA (177Lu- DOTA-G-4-aminobenzyl-BBN7-14) detected lesions in 5 out of 7 patients.311 Concluding remarks and perspectives Peptide-based nuclear tools for molecular imaging and therapy have now become an established approach, mainly due to the success achieved with somatostatin analogs, increasing knowledge into the cell and molecular biology of malignancies, advances in the coordination chemistry of radiometals, and bioconjugation. 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