in vivo 19: 9-30 (2005)

Review Radiometallation of Receptor-specific Peptides for Diagnosis and Treatment of Human Cancer

MICHAEL F. GIBLIN1,2, BHADRASETTY VEERENDRA2 and CHARLES J. SMITH1,2,3

1The Harry S. Truman Memorial Veterans’ Hospital, Columbia, MO 65201; 2Department of Radiology, University of Missouri-Columbia School of Medicine, Columbia, MO 65211; 3University of Missouri-Columbia Research Reactor Center, Columbia, MO 65211, U.S.A.

Abstract. Radiolabeled, receptor-specific peptides are becoming peptides with various therapeutic or diagnostic nuclides, increasingly popular as targeting vectors for the design and including 90Y, 188Re, 64Cu, 111In, 105Rh and 99mTc (15-20). development of new diagnostic and therapeutic radio- Lower molecular weight, receptor-specific peptides are pharmaceuticals. The over-expression of functioning receptors on more suitable for delivery of diagnostic or therapeutic a variety of human cancers makes this method of drug radionuclides to specific binding sites when compared to development a viable tool for tumor targeting in vivo. This review monoclonal antibodies. For example, peptides exhibit describes some of the more recent efforts that are currently distinct advantages over their higher molecular weight underway towards development of new receptor-specific ‘cousins’ including rapid clearance from blood serum, ease radiopharmaceuticals. Diagnostic/therapeutic radionuclides, of penetration into tumor vascular endothelium, and specific metal co-ordinating ligands/chelating systems, spacer increased diffusion rates into human tissue (21-25). As a technology, radiolabeling protocols, and specific peptides/peptide result of their small size, receptor-avid peptides possess conjugates will be discussed in detail. relatively low immunogenicity (21-25). Furthermore, high affinity receptors, expressed on a variety of neoplastic cells, Clinical applications of radiolabeled monoclonal antibodies in have been identified for a number of small, receptor-avid recent years (1-4) have demonstrated the potential of site- peptides (21-26). Therefore, certain radiolabeled, receptor- directed, biologically-active compounds for development of new specific peptides are ideal candidates as new diagnostic and successful diagnostic and therapeutic radiopharmaceuticals and/or therapeutic radiopharmaceuticals. for human cancers. However, the pitfalls of slow clearance from blood serum/non-target tissue, and the less-than-desirable Diagnostic Radionuclides tumor uptake have limited the clinical applications of these conjugates (1-5). Therefore, radiolabeled, biologically-active, Metallic radionuclides can be utilized in gamma scintigraphy receptor-specific peptides continue to hold some promise for and positron emission tomography (PET) to diagnose diagnosis or treatment of certain human cancers (6-14). This is, neurological disorders, heart perfusion, renal disorders and in part, due to the recent successes of Octreoscan® ([111In- specific cancers. The requirements for SPECT (Single Photon DTPA-octreotide]), a peptide-based radiopharmaceutical that Emission Computed Tomography) imaging radioisotopes are, is currently being used to image neuroendocrine tumors (15). ideally, ready availability, a short physical half-life, and For example, the clinical utility of Octreoscan® has catalyzed emission of a single gamma (Á) photon in the 100-250keV research efforts in radiolabeling other biologically-active range (27). The physical half-life of diagnostic radiometals is of utmost importance in radiopharmaceutical development. If the radiometal is not readily available (i.e., available from a radionuclide generator), the half-life of the radionuclide must Correspondence to: Dr. Charles J. Smith, Radiopharmaceutical be sufficiently long to accommodate travel/delivery, Sciences Institute and the Department of Radiology. University of radiopharmaceutical preparation and possible purification, and Missouri-Columbia School of Medicine, 143 Major Hall, dose delivery to the patient. However, while the half-life should Columbia, MO 65211, U.S.A. e-mail: [email protected] be long enough to overcome these particular concerns, it Key Words: Peptides, therapy, diagnosis, radionuclide, somatostatin, should also be as short as possible in order to deliver a minimal bombesin, review. dose to non-target tissue within the patient (27). PET imaging

0258-851X/2005 $2.00+.40 9 in vivo 19: 9-30 (2005)

Table I. Selected, metal-based radionuclides (Á and ‚+) which are Indium-111 is a cyclotron-produced radionuclide with a suitable for diagnostic imaging. sufficiently long half-life (67.9h) to be considered readily available for site-directed radiopharmaceutical preparation. Radionuclide Production/ Decay EÁ/E‚+(keV) Reference In-111 is produced by irradiation of Cd-111 via the Availability 111Cd(p,n)111In reaction. In-111 emits two imagable Tc-99m 99Mo/99mTc IT Á, 142.7 28-31 photons (171keV, 89% and 245keV, 95%) and decays by Generator electron capture (27). In3+ is considered to be a hard metal center, preferentially complexing to hard donor atoms such In-111 Cyclotron EC Á, 171.3 and 15,27,31 as oxygen and nitrogen (27). An example of a FDA- 245.4 approved, peptide-based, site-directed radiopharmaceutical ® Ga-67 Cyclotron EC Á, 93.3, 184.6, 27,31 used routinely for clinical application is Octreoscan , an and 300.2 111In-containing diagnostic agent for identification of neuroendocrine tumors (15). Cu-64 Cyclotron EC, ‚-, Á, 1345.8 19,27,31 + - Gallium radioisotopes continue to receive considerable and ‚ ‚ , 578, 67 ‚+, 651 interest as SPECT or PET imaging radionuclides. Ga is a cyclotron-produced radionuclide having suitable physical Ga-68 Cyclotron EC Á, 1077.3 27,31 characteristics for diagnostic imaging. For example, 67Ga and ‚+ ‚+, 1899 has a half-life of 3.261 days, and emits a host of gamma photons ranging in energy from 91 to 388keV (27,31). Like Ga-66 Cyclotron EC Á, 1039.3 and 27,34,35 3+ 3+ and ‚+ 2752.2 In , Ga is also considered to be a hard metal center. ‚+, 4153 However, in terms of the hard/soft acid base theory, gallium is considered to be a harder center than indium, Y-86 Cyclotron EC Á, 1076.7, 627.8, 27,36 forming complexes with harder oxygen/nitrogen atom + and ‚ and 1153.1 donors (i.e., EDTA or DTPA). Indium, on the other hand, ‚+, 1248 also forms stable complexes with sulfur atom-donating ligands (27,32,33). PET imaging radionuclides. 64Cu is a readily available, cyclotron-produced radionuclide ((64Zn(n,p)64Cu) or radiometals are positron-emitting (‚+) radionuclides. Positron (64Ni(p,n)64Cu)) with attractive physical characteristics for emission results in the production of two 511 keV gamma PET imaging and radionuclide therapy. For example, 64Cu photons emitted at an angle of 180Æ. Therefore, PET requires has a half-life of 12.7h, emits a 0.651MeV ‚+, and decays by coincidence counting over many angles around the body axis ‚- (0.578MeV) emission (19). There are other ‚+-emitting of the patient. PET radionuclides often have relatively short isotopes of copper that are also suitable for PET imaging. half-lives and are produced by either an accelerator or However, they suffer from very short half-lives, requiring the cyclotron, limiting their ready availability (27). Table I lists a presence of an in-house cyclotron for availability. variety of selected, metallic radionuclides that have suitable As previously mentioned, gallium isotopes continue to physical characteristics for SPECT or PET imaging. hold some promise as radiolabels to produce site-directed, diagnostic radiopharmaceuticals. 68Ga is a readily available, SPECT imaging radionuclides. 99mTc continues to be the most positron-emitting (1.899MeV) radionuclide that is produced versatile radioisotope in nuclear medicine applications today. via the 68Ge/68Ga generator system. 68Ga has a very short This is due primarily to ready availability, ideal nuclear half-life (1.13h), which could limit its usefulness for decay/imaging characteristics (t1/2 = 6.04h, EÁ = 143keV production of site-directed bioconjugates. However, a shelf- 68 68 (89%)), and well-established radiolabeling chemistries (28-29). life of ~2 years for the Ge/ Ga generator system (t1/2 of In fact, 99mTc accounts for more than 85% of all diagnostic 68Ge=270.8d) provides for an ample supply of radionuclide applications performed in medical facilities each year. 99mTc is for PET imaging without the need for an in-house cyclotron 99m - 66 available at a relatively low cost as Na TcO4 radionuclide facility (27). Ga is another gallium radionuclide with ideal and is produced via the 99Mo/99mTc generator system. 99Mo physical characteristics for PET imaging (34,35). However, a has a half-life of 2.78 days and decays by ‚- emission to 9.5h half-life and the need for an on-site cyclotron facility 99m 99 99m - Tc/ Tc. Na TcO4 is eluted from the generator using limits its usefulness in production of site-directed isotonic saline solution. While the 99mTc solution is not radiopharmaceuticals. entirely carrier-free, the specific activity of the eluted Yttrium-86 continues to receive considerable attention as precursor is considered to still be very high and is ideal for a useful radionuclide for PET imaging. 86Y has ideal radiometallation of biologically-active molecules (30). physical characteristics (see Table I) including a 14.7h half-

10 Giblin et al: Radiolabeling of Receptor-specific Peptides life, making it a readily available cyclotron-produced Table II. Selected, metal-based radionuclides (· and ‚-) which are (86Sr(p,n)86Y) radionuclide. Another unique feature of 86Y potentially useful for radiotherapy. is that it is a true "matched pair" of 90Y, a pure beta- Radionuclide Production/ Decay E‚-/E·(MeV) Reference emitting radionuclide that has been investigated extensively Availability for use in radiotherapeutic applications (36,37). Re-188 188W/188Re ‚- and Á ‚-, 2.118, 31,38,46 Therapeutic Radionuclides Generator 1.962 Á, 0.155

Therapeutic radiopharmaceuticals utilize metallic Re-186 Reactor ‚- and Á ‚-, 1.071, 31,38 radionuclides and specific "transport vehicles" (i.e., peptides, 0.933 Mabs, specific ligating frameworks) to deliver ionizing Á, 0.1372 radiation to diseased tissue. Particle-emitting radionuclides - - tend to be very effective at delivering cytotoxic ionizing Rh-105 Reactor ‚ and Á ‚ , 0.566, 47-49 0.248 radiation to a localized site (38-40). However, as with those Á, 0.3192 diagnostic radionuclides previously mentioned (vide supra), there are inherent requirements that must be considered Cu-67 Accelerator ‚- and Á ‚-, 0.577, 27 when selecting a therapeutic radiometal for treatment of 0.484, 0.395 specific disease. For example, physical half-life, ready Á, 0.185 availability, emission characteristics, nuclear decay properties, Y-90 90Sr/90Y ‚- ‚-, 2.27 27,31,38 specific activity, and linear energy transfer (LET) of the Generator radionuclide must be considered (38-40). Ideally, the physical half-life of the radionuclide should be Lu-177 Reactor ‚- and Á ‚-, 0.497 31,38,43 well matched to the biological half-life of the delivery vehicle. In Á, 0.2084, 0.1129 other words, the physical half-life should be very similar to the in vivo localization and clearance properties of the biomolecular Pm-149 Reactor ‚- and Á ‚-, 1.072 31,38,43 targeting vector (40-42). Furthermore, the physical half-life of Á, 0.286 the radionuclide must once again be sufficiently long to - - accommodate travel or delivery to the radiopharmacy, Sm-153 Reactor ‚ and Á ‚ , 0.69, 31,41,43 0.64 radiopharmaceutical preparation and possible purification, and Á, 0.1032, dose delivery/administration to the patient. For shorter half- 0.0697 lived radionuclides, ready availability and cost effectiveness is very critical for widespread clinical efficacy (40-42). Ho-166 Reactor ‚- and Á ‚-, 1.855, 31,41,43 Decay properties and emission characteristics are also to 1.773 Á, 0.0806, be considered during the design and development of site- 1.3794 directed, therapeutic radiopharmaceuticals. Beta (‚-) particle emitters, Alpha (·) particle emitters, and Auger At-211 Accelerator EC, ·, Á, 0.687, 38,53 electron emitters (not to be discussed in this brief review) and Á 0.6696 offer a diverse spectrum of effective range in tissue and ·, 5.868 LET (38). The choice of radioisotope can depend entirely Bi-212 224Ra/212Bi ‚-, Á, ‚-, 2.251 39.53 on the application for which it is to be used. For example, Generator and · Á, 0.7273 tumor size and homogeneity might influence whether or not ·, 6.051 a low-energy, low-penetrating or high-energy, high- 225 213 - - penetrating radionuclide is used. Luckily, a diverse array of Bi-213 Ac/ Bi ‚ , Á, ‚ , 1.42, 1.02 38,55 Generator and · Á, 0.4405, metallic radionuclides is available for therapeutic 0.3237 applications (vide infra) (38,43). ·, 5.87, 5.55 Lastly, the specific activity of the radionuclide should be considered during the design and development of radiometallated, site-directed radiopharmaceuticals for patient use. Ideally, the isotope should be available as a carrier-free radionuclide, free of any non-radioactive target material. The conjugate will be inseparable from the radiometallated presence of a non-radioactive metal precursor can and will conjugate using traditional chemical methods of separation compete for complexation to ligand binding sites on the (i.e., RP or size exclusion HPLC) and will compete for low biologically-active molecule. The non-radioactive, metallated capacity binding sites on cancerous tissue (39,43,44).

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Beta-emitting radionuclides. An exciting approach for the be produced in high specific activity in a nuclear reactor development of new diagnostic and therapeutic facility by the neutron irradiation of an enriched 104Ru radiopharmaceuticals is that of the "matched pair" concept. target. Chemical separation of parent 105Ru from daughter "Matched pairs" (i.e., 99mTc/188Re) provide the opportunity 105Rh has been reported (47-49). 105Rh has a half-life of to use information derived from routine patient diagnostic 36h, and emits two ‚- particles of medium (0.56MeV) and Single Photon Emission Computed Tomography (SPECT) low (0.25MeV) energy. Emission of two imagable gamma studies (99mTc) to determine the receptor availability on photons (306 and 319keV) would allow for ex vivo primary and metastatic tissues prior to administration of the evaluation of the injected therapeutic dose (47-49). 188 corresponding therapeutic analog ( Re). In this way, Copper-67 (t1/2=62h) is an accelerator-produced treatment would only be administered to patients previously radionuclide (67Zn(n,p)67Cu) that emits a host of ‚- demonstrating expression of the target receptor. particles that are suitable for development of site-directed Furthermore, the diagnostic radiopharmaceutical would be therapeutic conjugates. For example, Cu-67 emits three ‚- invaluable in pre-screening receptor-positive patients for particles (0.577, 0.484 and 0.395 Mev) and an imagable therapy with respect to drug pharmacokinetics, receptor gamma photon of 0.185 Mev (27). density and patient dosimetry, potentially reducing or Yttrium-90 exists as a 3+ cation and exhibits labeling eliminating unsuccessful radiotherapeutic regimens (45). chemistries very similar to 111In3+. In fact, the two species Rhenium-188 continues to hold potential as an isotope for are often mistaken for "matched pair" radionuclides. 90Y is therapeutic nuclear medicinal applications because of its a pure beta-emitting radionuclide (E‚max=2.27MeV) and is widespread availability (188W/188Re Generator) and attractive available in carrier-free form from a 90Sr/90Y generator - 90 physical characteristics (t1/2=16.94h, ‚ max=2.12MeV, and system. Y has a half-life of 2.7days, making radionuclide 186 EÁ=155keV). Re is another rhenium isotope that is shipment and subsequent preparation/purification of available for radiotherapy (38,46). 186Re (Table II) is radiolabeled conjugates quite feasible. Like 111In3+, 90Y is prepared by direct neutron irradiation of enriched 185Re in a considered to be a hard metal center, preferentially nuclear reactor facility. 186Re has a 3.7 day half-life, decays complexing to donor ligands containing the elements - by ‚ emission (E‚max=1.07MeV), and emits an imagable nitrogen or oxygen (27,38). gamma photon of 137keV. Although its physical The rare-earth radionuclides, much like In3+, Ga3+ and characteristics make it an attractive isotope for use in Y3+, exist primarily in the 3+ oxidation state and are therapeutic application, its low specific activity limits its considered to be hard metal centers, requiring multidentate, usefulness (31,38). hard donor ligands for in vivo kinetic inertness (27,43). 188/186Re-labeled radiopharmaceuticals often parallel the Furthermore, the radiolanthanides possess very similar chemistries used to develop technetium-based labeling chemistries while offering a diverse spectrum of radiopharmaceuticals. However, 188/186Re is used to a lesser nuclear decay properties (See Table II). Therefore, it is extent than other conventional therapeutic isotopes (i.e., possible to match a desired set of nuclear properties, (i.e., rare-earth elements) due to the complex radiolabeling t1/2, E‚max, etc.) to a particular clinical application (43). The chemistries and purification protocols required for radiolanthanides are produced, primarily, via direct or formation of an in vivo stable radiopharmaceutical. The indirect neutron capture of an irradiated target in a nuclear formulation of rhenium-based radiopharmaceuticals, like reactor facility (43,50). For example, most lanthanide their technetium surrogates, most often involves complexes elements have sufficiently large cross sections to capture a in the +5 oxidation state. However, the reduction potential neutron and are, therefore, considered to be suitable targets of rhenium is ~200mV higher than that of technetium and, for production of radiolanthanide elements. The therefore, the reaction conditions necessary for formation radiolanthanides are considered to be readily available. of a *Re-conjugate often necessitate excess reducing agent However, for those rare-earth radionuclides produced by (i.e., Sn2+) and/or more harsh reaction conditions (i.e., direct neutron capture, only moderate specific activities can heating, pH, etc.). Furthermore, oxidative instability in vivo be obtained. However, production of no-carrier-added can be an issue for rhenium complexes/conjugates of this radiolanthide elements (i.e., 177Lu and 149Pm) has recently type. As the radiometal *Re is more difficult to reduce, it is been reported, and these radionuclides do hold significant also more easily oxidized, as compared to technetium (46). promise for future therapeutic applications (43). Rhodium-105 has, in recent years, received considerable interest as a therapeutic radionuclide that would be suitable Alpha-emitting radionuclides. While ‚- particles tend to for the formulation of a site-directed radiopharmaceutical deposit their decay energy over a range of 5-150 cell primarily due to the formation of low-spin, d6 Rh(III) diameters (depending upon the isotope), · particles (high- complexes that would be stable to in vivo transligation energy helium nuclei) tend to have ranges across only a few reactions to proteins such as transferrin (47-49). 105Rh can cells (38,51,52). Therefore, they are ideally suited for

12 Giblin et al: Radiolabeling of Receptor-specific Peptides

Figure 1. Specific ligand frameworks capable of stabilizing the Tc(V) metal center.

smaller diameter tumors that are more homogeneous in Radiolabeling Strategies nature (38). Alpha-emitting radionuclides are considered to be high LET radiation, producing a very high density of Methods of radiolabeling proteins or peptides generally follow ionization along their linear tracks (52-54). Alpha-emitters one of two approaches: 1) the direct labeling method, and 2) are therefore considered to be ideal candidates for site- the indirect (bifunctional chelate) labeling approach (56). To directed radiotherapeutic applications as collateral damage be successful, both radiolabeling strategies require that high to surrounding normal tissue is often minimal (52-54). specific activity products are obtained (7). A high specific Although there are many ·-emitting radionuclides, only activity radiolabeled bioconjugate is one in which the maximum a handful have been considered for therapeutic number of molecules possible possess a radionuclide. radiopharmaceutical application (38,51). Astatine-211 is an accelerator-produced (209Bi(·,2n)211At) radionuclide The direct radiolabeling strategy. This method of radiolabeling having ideal physical characteristics for radionuclide therapy peptides is predominantly used for 99mTc/186/188Re (53). For example, 211At has a half-life of 7.21h and decays radiopharmaceutical production. The direct labeling strategy by ·-emission (E·=5.8 and 5.2MeV). Bismuth-212 is offers a relatively short, easy radiosynthetic approach for readily available from a 224Ra/212Bi generator system. 212Bi producing radiolabeled peptides or proteins. In this method of has a half-life of 1.009h and decays by emission of a 6.1MeV peptide/protein labeling, naturally occurring functional groups ·-particle (53). Bismuth-213(E·=5.8 and 5.5MeV) has also (i.e., -SH, -NH2, -OH, etc.) within the peptide/protein sequence been considered to be potentially useful for are used to complex to the radioisotope (2,57-60). While this radiotherapeutic applications in nuclear medicine (55). With method offers a simplistic approach to peptide/protein labeling, a half-life of 45.6 min and ready availability from an on-site there are distinct disadvantages that must also be considered. 225Ac/213Bi generator system, this nuclide does hold some For example, this radiolabeling strategy often suffers from lack promise for site-directed radiopharmaceutical development of specificity. For example, radiometals can complex within or in the near future (55). near the binding region of the biologically-active compound,

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Figure 2. Specific ligand frameworks capable of stabilizing the Tc(I)(CO)3 metal fragment. thereby inhibiting receptor-binding affinity. Secondly, the Metal-specific Bifunctional Chelating/Co- reduction of disulfide bonds via external reducing agents ordinating Ligand Frameworks promotes protein degradation and, ultimately, lack of receptor binding. Lastly, this method provides for effective radiolabeling Entire symposia and several reviews have been devoted to of large proteins/monoclonal antibodies, as smaller peptides the discussions of design and development of specific and peptide receptor fragments often lack reducible disulfide ligating frameworks necessary to stabilize a radioactive bonds necessary for metal chelation. If reducible disulfide element for in vivo applications (27-30, 38). We intend to bonds do exist in these smaller systems, they are often a only discuss the general frameworks of specific ligand necessary component for effective biological activity (58,61). entities for this review, but will refer the reader elsewhere for more detailed information. The indirect labeling strategy. The indirect labeling method overcomes the problems of the direct labeling strategy by Technetium/Rhenium-based. Technetium/Rhenium(V) introduction of a bifunctional chelating moiety onto the conjugates have been extensively investigated for use as site- biologically-active compound at some distance away from the directed tumor-targeting vectors for diagnosis and therapy region necessary for receptor binding. The bifunctional of human cancers. For conjugates of this type, the metal chelating ligand serves to form a stable, high-yield complex center most often exists as the [MO]3+ core. For example, with the metallic nuclide, while at the same time covalently square pyramidal complexes of the [MO]3+ core with linking the radioligand to the biologically-active region of the tetradentate ligand frameworks such as PNAO (propylene protein or peptide (62). The indirect labeling strategy can be amine oxime), N2S2 constructs (N2S2 = diaminedithiols, categorized into two groups: 1) the preformed chelate diamidedithiols, or monoamidemonoaminedithiols), and approach, and 2) the post-conjugate method of radiolabeling N3S triamidethiols have been reported (Figure 1) (64-69). (58,63). The preformed chelate approach is most often used Radiolabeling conditions are relatively straight-forward for when the isotope-BFCA complex can only be generated under conjugates of this type. For example, radiolabeling is often harsh reaction conditions, such as extreme heating or pH, that performed at either ambient temperature or with minor could otherwise destroy the biospecificity of the targeting heating and at normal or basic pH in the presence of a vector. The post-conjugate method of radiolabeling reducing agent, typically Sn2+. For the most part, 99mTc- biologically-active molecules is most often the method of conjugates of this type tend to be relatively stable in choice, as it offers a one-step synthetic approach to effective aqueous environments (64-69). labeling. This method requires the covalent attachment of the PNAO conjugates of specific biomolecules have been BFCA at some point in the sequence away from the binding reported (64). PNAO complexes of technetium are square site of the peptide/protein, typically on the N-terminus or on pyramidal, with deprotonation of the secondary amine an Â-amine of a lysine residue (62,63). atoms upon co-ordination to the metal center. The terminal

14 Giblin et al: Radiolabeling of Receptor-specific Peptides

Figure 3. Poly(aminocarboxylate) bifunctional chelating ligand frameworks for lanthanide and lanthanide-like elements.

hydroxides of the ligand share a single proton upon metal conventional labeling of biologically-active compounds with co-ordination, giving the complex an overall neutral charge 99mTc(V) or 188Re(V), via N3S donor ligands (Figure 1) is (70,71). Conjugates of this type are ideal from the an attractive radiolabeling alternative (72,73). Triamidethiol standpoint of preparation. For example, the conjugate can complexes of technetium have an overall zero charge. While be prepared at ambient temperature in the presence of conjugates of this type tend to show more favorable in vivo reducing agent, typically Sn2+. However, while the pharmacokinetics of the radiopharmaceutical when advantages of conjugates of this type seem to be ideal from radiolabeled with 99mTc, development of a therapeutic a clinical standpoint, PNAO derivatives often suffer from surrogate (186Re/188Re) often proves futile due to instability in aqueous solution and high lipophilicity (30). insufficient stability of the radiopharmaceutical in vivo. N2S2 ligand frameworks have become the cornerstone of Hence, there is a significant trade-off between ligands of 99m production for anionic, neutral, and cationic Tc- either N2S2 or N3S origin. bioconjugates (30,65-67). Diamidedithols, for example, An alternative to traditional 99mTcO3+-radiolabeling of deprotonate on the thiolate sulfurs and the amide nitrogens peptides/proteins is the use of 2-hydrazinonicotinamide, to produce anionic technetium conjugates (Figure 1). On HYNIC (Figure 1), as a bifunctional complexing ligand. The the other hand, diaminedithiols can deprotonate on the use of the 99mTc-HYNIC core was first reported by Abrams thiolate sulfurs and on one or both nitrogen atoms to form (74), for the labeling of polyclonal IgG. Since then, HYNIC neutral or cationic species. Monoamidemonoaminedithiols has been conjugated to various biomolecules including contain an amide nitrogen, an amine nitrogen, and two antibodies (75), chemotactic peptides (76), somatostatin thiolate sulfurs. Co-ordination of the donor atoms is analogs (77), antisense-oligonucleotides (78), interleukin- 99m tribasic, forming an overall neutral Tc-complex. N2S2 879 and many others (80,81). Technetium-99m binds to the conjugates tend to be very stable in vivo (30,65-67). hydrazino-moiety forming a 99mTc-nitrogen bond (81,82). 99m 188 However, Tc/ Re-N2S2 conjugates often suffer from As HYNIC alone cannot satisfy the coordination significant in vivo hydrophobicity, hence slower clearance requirements of Tc(V) (HYNIC can only occupy one or two from blood serum and non-target tissue. Therefore, coordination sites on the radionuclide), coligands are

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Figure 4. Tetrathiamacrocycles capable of forming stable complexes with Rh(III).

necessary to complete the coordination sphere of the suited for preparing 99mTc-labeled bioconjugates containing technetium(V) core (76). Coligands that have been used to disulfide bonds since excess reducing agent (sodium improve the 99mTc-radiolabeling of HYNIC-biomolecules borohydride or borane-ammonia complex) is destroyed prior include glucoheptonate (83), tricine and ethylenediamine to the radiolabeling procedure (87-89). Furthermore, the new 99m + diacetic acid (EDDA) (75,77,81). Liu and co-workers have [ Tc(H2O)3(CO)3] aqua ion is stable over a wide range of described the use of ternary ligand systems to complete the pH values, presumably due to the low-spin, d6 electronic coordination environment of the Tc(V)-HYNIC metal configuration of Tc/Re(I). Lastly, the lability of the three water fragment. For example, they have used a water soluble molecules coordinated to the fac-M(CO)3 moiety account for phosphine or an imine-N-containing heterocycle as an the excellent labeling efficiencies with a number of donor additional coligand to form a ternary ligand framework ligands including amines, thioethers, phosphines, and thiols 99m + (84,85). Other ternary ligand systems that have been (Figure 2) (87-94). [ Tc(H2O)3(CO)3] is now available as reported include tricine/pyridine (77,84), tricine/nicotinic Isolink® from Mallinckrodt Inc., St. Louis, MO, USA. acid (77,79,81) and tricine/trisodium triphenylphosphine- 3,3’,3"-trisulfonate (TPPTS) (80). Lanthanide and lanthanide-like based. Bifunctional chelating Current techniques for radiolabeling peptides containing ligands necessary to stabilize the lanthanide (i.e., 153Sm, disulfide bonds with Tc-99m continue to be a problem. For 149Pm, 177Lu) and lanthanide-like elements (i.e., 90Y and example, production of a high specific activity, well-defined 111In) for in vivo therapeutic applications are centrally product via direct or indirect radiolabeling methods is focused around the poly(aminocarboxylates) (15,16,27,43). sometimes difficult, as disulfide bond reduction can occur. This Central to the development of kinetically inert *M3+- is by virtue of excess reducing agent (Sn2+) in the labeling conjugates for the diagnosis or therapy of human cancers cocktail (86). Clearly, this is a problem that plagues formation are the polydentate-ligating amino(carboxylates) including of 99mTc(V)- and 188Re(V)-bioconjugates appended with DTPA (diethylenetriaminepentaacetic acid), DOTA N2S2/N3S/HYNIC chelators. More recently, an (1,4,7,10-tetraazacyclododecane-N,N’,N",N’"-tetraacetic "organometallic" radiolabeling strategy has been identified. acid), and TETA (1,4,8,11-tetraazacyclotetradecane- Recent investigations by Alberto and co-workers have led to N,N’,N",N"’-tetraacetic acid) (Figure 3). the development of some remarkable Tc(I) and Re(I) Acyclic poly(aminocarboxylic acids) such as DTPA have chemistry (87-91). With the development of the new been extensively investigated as stabilizing ligand frameworks 99m + organometallic triaqua ion [ Tc(H2O)3(CO)3] , a new for the radiolanthanide/lanthanide-like elements due to the avenue for the successful radiolabeling of bioactive molecules successes of Octreoscan® (111In-DTPA-octreotide) for with low-valent 99mTc/188Re has been achieved (90,91). diagnosis of somatostatin, receptor-positive tumors (15). The + 99m 188 [*M(H2O)3(CO)3] (*M= Tc or Re) eliminates the in vivo stability of ligands of this type is presumably due to problem of labeling S-S-containing biomolecules and is well the chelating capacity of the multidentate ligand network

16 Giblin et al: Radiolabeling of Receptor-specific Peptides

Figure 5. Cyclam-based ligand framworks capable of forming stable complexes with Cu(II).

about the metal center. Expanded co-ordination spheres for as DOTA and TETA tend to form complexes with these elements necessitate multidentate ligands for in vivo lanthanide and lanthanide-like radionuclides that exhibit a kinetic inertness in order to reduce radiation toxicity to higher degree of in vivo kinetic inertness than acyclic ligands normal tissues such as the bone marrow (43). The chelating such as DTPA, presumably due to the inherent macrocyclic capacity of DTPA can be diminished if one of the pendant effect of such ligand-metal complexes (15,16,27,43). Ligands arms necessary for complexation to the metal center is used of this type, unlike DTPA and other acyclic ligand for covalent linkage to the biomolecular targeting vector. In frameworks, provide rigidity to the metallic complexes and vivo decomplexation of the metal center from the ligating result in in vivo, kinetically-inert, site-directed framework can result in isotopic uptake by serum proteins bioconjugates. Complexes of DOTA and TETA with and other non-target tissues such as the bone marrow. The numerous cationic radionuclides have been extensively consequences of demetallation are undesirable and can be investigated (15,16,27,43). Kinetically inert complexes/ lethal to the patient (43). In order to overcome the inherent conjugates under the most stringent of conditions have been difficulties of such, many researchers have developed reported (15,16,27,43). functionalized derivatives of DTPA so that octadenticity Radiolabeling strategies employed for complexing M3+ toward the metal center can be maintained (95,96). radionuclides to targeting vectors containing DOTA continues to be the most widely-used ligand poly(aminocarboxylate) ligand frameworks tend to follow a framework for complexation of 3+ metallic radionuclides universal approach (50,96-101). The radionuclide is (i.e., the rare-earth radionuclides) to biologically-active generally shipped in a 0.05M HCl solution as *MCl3. molecules. Macrocyclic, polyaza amino(carboxylates) such Complexation of the radionuclide solution to the targeting

17 in vivo 19: 9-30 (2005)

Figure 6. Structures of somatostatin and somatostatin-like receptor-specific peptides.

vector is often performed in ammonium acetate or production of site-directed, diagnostic/therapeutic tetraethylammonium acetate solution, pH = 5.5 to 6.0. radiopharmaceuticals. Venkatesh, Goswami and Li have Reaction times of 30min to 1h (80ÆC) often provide for found that tetrathiamacrocycles (Figure 4) are able to radiolabeling yields of ≥90% (50,100,101). stabilize the Rh3+ metal center to form kinetically inert, in vivo stable conjugates (47-49). They have suggested that Specific ligand frameworks for copper and rhodium a combination of the macrocyclic effect and -acidity of radionuclides. 64Cu2+ and 105Rh3+ are two aforementioned the sulfur donor atoms is responsible for stability of these radionuclides that have also been investigated for Rh3+ complexes (47-49, 102-104).

18 Giblin et al: Radiolabeling of Receptor-specific Peptides

Figure 7. Structures of VIP, BBN and ·-MSH peptides.

Anderson and co-workers have investigated the relationship have reported that an increase in the hydrophilic nature of between kinetic inertness and in vivo stability for peptide- the conjugate can be accomplished by introduction of based conjugates of 64Cu2+ (19,27). They and others have "innocent" peptide sequences such as polylysine, -glycine, or found that derivatives of 1,4,8,11-tetraazacyclotetradecane -aspartic acid residues into the peptide sequence (30). (CYCLAM, Figure 5) produce conjugates with the desirable Decristoforo has recently reported that more hydrophobic pharmacokinetics, tumor uptake and in vivo kinetic inertness 99mTc-labeled peptides are accumulated in liver tissue and to be used as site-directed tumor-targeting agents (19). For excreted predominantly by the hepatobiliary pathway (105). example, *Cu2+-conjugates of CPTA (4-[(1,4,8,11- Therefore, hydrophilic site-directed radioconjugates can tetraazacyclotetradec-1-yl)-methyl]benzoic acid), BAT ([6-(p- provide diagnostically useful abdominal images if needed. bromoacetamido)benzyl]-1,4,8,11-tetraazacyclotetradecane- N,N’,N",N"’-tetraacetic acid), and TETA (1,4,8,11- Receptor-specific Peptides as Targeting Vectors tetraazacyclotetradecane-N,N’,N",N"’-tetraacetic acid) have shown considerable promise as Mab/peptide-based tumor- There is a tremendous body of research relating to the targeting vectors (Figures 3 and 5) (27). topics of peptide-receptor scintigraphy and peptide-receptor radiotherapy. This review necessarily touches on only a Spacer Technology small subset of recent results. The five classes of peptides reviewed here have all received considerable attention in Spacing moieties (i.e., amino acids or aliphatic spacers) are radiopharmaceutical development efforts. However, often a critical element in site-directed radiopharmaceutical numerous other peptides have also been studied, including preparation. These linkers are placed at a point between the neurotensin, cholecystokinin, E. coli heat-stable enterotoxin, bifunctional chelating ligand and the receptor-binding substance P, and endothelin, among others. For discussion region of the molecule in order to preserve biological of these peptide-targeting vectors, as well as more detailed integrity or receptor specificity. Furthermore, specific amino information on the peptides reviewed here, readers are acid or aliphatic spacers can also be used to tune the degree referred to several excellent reviews (102, 106-109). of hydrophilicity/hydrophobicity of radiometallated conjugates. An increase in the hydrophilic nature of a Somatostatin. Analogs of somatostatin (SST) are the most radiolabeled peptide will serve to increase renal clearance successful examples of peptide-receptor imaging agents to and decrease residence time in blood of the new date (26). SST is a peptide hormone that is expressed in radiopharmaceuticals, imparting ideal pharmacokinetic both the central and peripheral nervous systems. It exists criteria on the radiolabeled conjugates. Liu and Edwards naturally in two forms. SST-14 is a 14 amino acid peptide,

19 in vivo 19: 9-30 (2005) while SST-28 contains those same 14 amino acids, plus a 14 resulting, for example, in molecules modified in positions 3 amino acid N-terminal extension. Each peptide possesses a and 8 of octreotide to yield tracers with high affinities for cyclic domain by virtue of a single disulfide bond (Figure 6). SSTR subtypes 2, 3 and 5 (122). These modified analogs may There are 5 known SST receptor subtypes, SSTR1-5, which prove to be superior vectors for the imaging and treatment mediate the diverse biological functions of SST. These of tumors expressing these SSTR subtypes. receptors belong to the family of G protein-coupled receptors, are highly expressed on tumors of Bombesin. Bombesin (BBN) is a tetradecapeptide neuroendocrine origin, and bind both SST-14 and SST-28 originally isolated from the skin of the frog Bombina with nanomolar affinity. bombina (123). In its native form, it is C-terminally Native SST has a very limited half-life in vivo (1-3 min). amidated and contains an N-terminal pyroglutamate As a result, much work has been devoted to the development residue (Figure 7). Gastrin-releasing peptide (GRP) is a of SST analogs with increased stability to proteolytic mammalian analog of the amphibian BBN peptide, as is degradation, which could be used pharmaceutically for the neuromedin B. Bombesin shares its 8-14 C-terminal treatment of diseases including cancer (110). Three sequence with GRP, and this sequence (-Trp-Ala-Val-Gly- octapeptide analogs, octreotide, vapreotide and lanreotide, His-Leu-Met-NH2) has been shown to be sufficient for are currently available in the clinic (111). Each of these is a binding to the bombesin receptor (124). truncated analog of SST-14, with a disulfide bond directly Four GRP receptor subtypes are known – BB1 adjacent to the sequence analogous to the four most critical (neuromedin B receptor subtype) (125), BB2 (GRP binding residues in SST-14, Phe-Trp-Lys-Thr. Each contains receptor subtype) (126), BB3 ("orphan receptor" subtype) a D-Trp substitution for the L isomer, which increases (127) and BB4 (128). GRP receptors are G protein-coupled, peptide resistance to proteolytic degradation and also 7 transmembrane receptors which are efficiently increases binding affinity. The analogs differ in N- and C- endocytosed upon agonist binding (129). The ability of GRP terminal residues that lie outside of the constrained disulfide agonists to be internalized is one factor that has led to their loop, and in substitutions at positions 3 and 6 of the use as vehicles for radionuclide imaging and therapy, truncated molecule (Figure 6). despite their proliferative effect on cells expressing GRP In addition to their clinical utility as antiproliferative receptors (130). GRP receptors are overexpressed on agents that modulate hormonal secretion, SST analogs such various tumor types, including prostate (131, 132), breast as octreotide have had extensive application as radiolabeled (133, 134), and small cell lung cancer (135). This agents for both imaging and treatment of cancer. Since the overexpression in various neoplasias relative to normal first 123I-labeled octreotide analog was developed (112), a tissues is another factor that has driven BBN-based host of different labeled SST analogs have been reported. radiopharmaceutical development. OctreoScan® is a commercially available SST analog (111In- The development of GRP receptor-specific radio- DTPA-D-Phe1-Octreotide, 111In-pentetreotide) that is pharmaceuticals has recently been reviewed (45). Several routinely used for imaging of neuroendocrine tumors (15). 99mTc- and 188Re-bombesin conjugates have been 99mTc depreotide (NeoSpect®, NeoTect®, 99mTc-p829) and synthesized using a wide range of chelators, including 99m 3 99m Tc-tricine-HYNIC-Tyr -octreotide are two Tc-labeled DADT (136), N3S (137), P2S2 (138) and carbonyl (46) 99m SST analogs that have been tested in the clinic (113). moieties. The clinical utility of RP-527 ([ Tc(V)-N3S-5- Use of the DOTA chelator in SST analogs has widened Ava-BBN[7-14]NH2]) as a cancer specific imaging agent the range of radionuclides available for imaging and was recently demonstrated in human patients with either treatment with radiolabeled SST analogs (114). Both 90Y- prostate or breast cancer. These studies showed that 90 1 3 99m DOTA-lanreotide and Y-DOTA-DPhe -Tyr -octreotide [ Tc-N3S-5-Ava-BBN[7-14]NH2] localizes in tumors with (90Y-DOTATOC) have been extensively studied as internal high specificity, producing good tumor-to-normal tissue radiotherapeutic agents (115, 116). In order to predict the uptake ratios and high quality SPECT images (72). internal dosimetry of such analogs, surrogate molecules have Extensive work has been done with DOTA-labeled BBN been synthesized that employ radionuclides such as 86Y117. anaolgs, both for imaging with 111In (100) and for A series of PET agents has also been synthesized using treatment with various radiolanthanides (101). Most DOTA- and TETA-SST analogs, including tracers based on recently, PET imaging agents have been developed using 68Ga (118), 86Y (119) and 64Cu (19,120). Such tracers will radionuclides such as 64Cu (139,140). allow more accurate determination of individual internal The majority of research into BBN-based dosimetry, thereby perhaps reducing the occurrence of side- radiopharmaceuticals has been carried out using GRP effects such as nephrotoxicity, which can currently be dose- agonists (129, 140). Again, one reason for this has been that limiting for peptide receptor radiotherapy (121). Further GRP agonists are internalized upon receptor binding, refinement of SST analogs is currently being studied, thereby residualizing the attached radiometal within the

20 Giblin et al: Radiolabeling of Receptor-specific Peptides targeted cell. One recent study utilizing a 99mTc-labeled RGD-dependent tumor uptake of this agent was not GRP antagonist adds complexity to this picture (141). Since observed in a C26/BalbC in vivo model, probably due to GRP antagonists are presumably not internalized to the both the relatively low affinity of the linear molecule for the degree that GRP agonists are, agonists have been expected integrin receptor (IC50 = 20 ÌM) and to the rapid rate of to more effectively residualize label within tumor cells. proteolytic breakdown of the construct. However, in a study using 99mTc-labeled Demobesin-1, Cyclization of RGD-containing peptides has been shown tumor localization was 3-8 times higher than for various to increase both the specificity and affinity of peptides for GRP agonists at 1 h pi (45). Furthermore, at 24 h pi, 99mTc- defined integrin subtypes (148,149). In one instance, Demobesin-1 was well retained at the tumor site (5.24 + screening of a phage display library yielded an RGD peptide 0.67 %ID/g), at 3- to 5-fold higher levels than previously cyclized via two disulfide bonds, termed RGD-4C (150). An reported for GRP agonists (141). This study suggests that abbreviated version of this cyclic RGD peptide has been the concept of GRP agonists being preferable for used as substrate for 99mTc labeling (151). The affinity of the 99m radiopharmaceutical development, while intuitively Tc-labeled peptide for the ·v‚3 integrin was estimated to compelling, may bear reexamination. be approximately 70-fold lower than that of RGD-4C, suggesting that some aspect of the tracer synthesis could be RGD-containing peptides. Peptides containing the amino acid responsible for a lack of specific tumor uptake in vivo. sequence Arg-Gly-Asp (RGD) have been used extensively to A large number of studies have been carried out utilizing target integrin receptors up-regulated on tumor cells and head-to-tail cyclized RGD peptides, and this represents the neovasculature. The RGD consensus sequence appears in most promising class of RGD-based imaging agents (152). several proteins of the extracellular matrix, including Early investigations into the effect of head-to-tail cyclization vitronectin, fibronectin, fibrinogen, von Willebrand factor, on RGD affinity for the ·v‚3 integrin led to the thrombospondin and osteopontin (142). Integrin recognition identification of cyclo(RGDfV), an ·v‚3 antagonist with a of the canonical RGD sequence plays a prominent role in low nanomolar IC50. Further characterization led to the many cell-cell and cell-ECM interactions. Integrins are cell observation that a bulky hydrophobic residue was required surface transmembrane glycoproteins that exist as ·‚ in position 4 for maximum affinity, while position 5 was heterodimers. At least 24 different combinations of ·‚ tolerant of a range of substitutions (153). heterodimers are known (143). The integrins of most interest One result of these observations was that D- or L-tyrosine in cancer imaging and therapy contain the ·v subunit, was inserted into either position 4 or 5 to yield RGD peptide 125 particularly the ·v‚3 and ·v‚5 subtypes. The structure of the substrates for iodination reactions. The resulting I-labeled extracellular domain of the ·v‚3 integrin was determined peptides have been tested in vitro and in vivo to examine the crystallographically to 3.1 Å resolution in 2001 (144). feasibility of using cyclic RGD peptides as in vivo imaging The ·v‚3 integrin is known to be overexpressed in many agents (153). These peptides displayed rapid blood clearance tumor types, and expressed at lower levels in normal tissues through hepatobiliary excretion, and tumor uptake at 1 h pi (145). Both ·v‚3 and ·v‚5 subtypes are expressed in of 1.30±0.13 %ID/g in M21 melanoma human tumor neovasculature during angiogenesis (145). These expression xenografts. In an attempt to improve the pharmacokinetics patterns form the basis of attempts to image angiogenesis of these compounds, a glycosylated analog was synthesized and tumor formation in vivo using RGD-based peptide- and tested (154). Substitution of a lysine residue permitted targeting vectors. RGD peptides used to target integrin the attachment of a carbohydrate moiety to the  amino receptors generally fall into one of three categories: linear, group of lys5. The resulting molecule showed decreased disulfide-cyclized, and head-to-tail cyclized. uptake in liver and intestine relative to an analog lacking At least two examples of integrin targeting using linear carbohydrate, with 1.22±0.32 %ID/g vs. 11.23±1.95 %ID/g RGD-containing peptides have been described. In one in the liver at 1 h pi. Additionally, the glycosylated species instance, two RGDS peptide sequences from human showed increased tumor uptake and residualization, with fibronectin were linked in series and labeled with 99mTc 2.05±0.55 %ID/g in M21 melanoma human tumor (146). A single cysteine residue intervening between the two xenografts. Similar biodistribution results were obtained 99m 18 RGDS sequences presumably served to localize Tc using an F-labeled galacto-RGD analog (155). ·v‚3- binding at that position within the molecule. This conjugate targeted cyclic pentapeptides commonly include a lysine was studied in fourteen melanoma patients. In this group, residue in position 5 for the purpose of appending a diverse eleven metastatic lesions were visualized, with high array of labeling moieties. PET tracers have been background activity in the lung and abdomen (146). A synthesized by appending a PEG- [18F]fluorobenzoate second study described the N-terminal labeling of an RGD- domain (156). Metal chelators such as DTPA and DOTA containing peptide (KPQVTRGDVFTEG-NH2) with an have also been used to coordinate a range of radionuclides [18F]fluorobenzoyl moiety by solid phase synthesis (147). useful for imaging and therapy (157, 158).

21 in vivo 19: 9-30 (2005)

Increasing attention is currently focused on the development Much current effort is being expended on the of dimeric or multimeric cyclic RGD peptide constructs. development of VIP analogs with specificity for VPAC1 and Multimerization is expected to increase the apparent affinity VPAC2 subtypes (162, 171), and with increased stability of targeting vectors for their cognate receptors due to avidity toward in vivo proteolytic degradation (172). For example, a effects. In one instance, a dimeric cyclo(RGDfK) construct was modified VIP analog was synthesized with 9 alanine synthesized by bridging two lysine  amino groups with a substitutions at positions 2, 8, 9, 11, 19, 24, 25, 27 and 28 DOTA-glutamic acid moiety (159). This peptide, when labeled (171). This analog was equipotent to the wild-type peptide 111 with In, showed high receptor-mediated tumor uptake of at VPAC1 receptors (IC50=1.6±0.1 nM), yet showed 7.5%ID/g at 2 h pi in an OVCAR-3/nude mouse in vivo model. significantly increased stability to proteolytic degradation. Receptor-mediated uptake in nontarget tissues such as spleen, Incorporation of an arginine residue in the nonessential liver, and lung was also surprisingly high in this study. In a position 8 of VIP resulted in a fluorescent dye-labeled second study, cyclo(RGDfE) multimers were formed using analog with increased tumor-targeting capacity over dye- variable-length PEG spacers linked to a diaminopropionic labeled wild-type VIP (172). Continuing structure/function acid/lysine backbone (160). Monomeric, dimeric, and studies will provide new lead compounds for development tetrameric forms were synthesized and labeled with 18F. of VIP-based imaging agents. Biodistribution of the dimer compared favorably with that of an 18F-labeled galacto-RGD analog. ·-MSH. ·-Melanocyte stimulating hormone (·-MSH) is an N-terminally acetylated, C-terminally amidated 1 13 Vasoactive intestinal peptide. Vasoactive intestinal peptide tridecapeptide [Ac-S YSMEHFRWGKPV -NH2] that (VIP) is a 28 amino acid peptide neurotransmitter with a regulates skin pigmentation in most vertebrates (173). The wide range of biological activities in mammals (161). VIP is a biological activity of this peptide in vivo is mediated by member of a larger family of peptide hormones that includes interactions with the melanocortin 1 receptor (MC1R), one secretin, glucagon, peptide histidine methionine amide, of five known subtypes of G-protein-coupled melanocortin growth hormone-releasing factor (GRF), and pituitary receptors. ·-MSH receptors are expressed on melanoma adenylate cyclase-activating peptide (PACAP) (162). VIP cell lines and on human melanoma tissue samples (174, receptors are highly expressed in various tumor types (163), 175). This, coupled with the low nanomolar affinity of ·- including intestinal adenocarcinomas (164) and breast MSH for its receptor and the ability of the peptide-receptor cancers (165). Recognition of this fact has led to the use of complex to rapidly internalize, has catalyzed investigation labeled VIP analogs for peptide receptor scintigraphy (166). into the potential of ·-MSH-based imaging and therapeutic VIP action is mediated through two known receptor radiopharmaceuticals. The ·-MSH-based peptide subtypes, VPAC1 and VPAC2. VPAC1 and VPAC2 are radiopharmaceuticals studied to date can be broadly members of the G protein-coupled receptor (GPCR) classified as either linear or cyclic structures. superfamily. These receptors can be distinguished The class of linear ·-MSH peptide radiopharmaceuticals pharmacologically through the use of subtype-specific VIP is based upon a superpotent analog (NDP-MSH) developed analogs. Such experiments have demonstrated the over 20 years ago (176). This is a highly potent, protease- predominance of VPAC1 expression in the majority of resistant analog identical to wild-type ·-MSH, but for the human tumors (163). substitution of D-Phe at position 7 and norleucine at The use of radiolabeled VIP analogs for in vivo imaging position 4. The first radiopharmaceutical in this class of cancer has been documented in numerous studies. VIP consisted of two NDP-MSH molecules bridged by a single analogs have been labeled with 123I (164, 167), 99mTc (168), 111In-DPTA moiety (177). Although this construct did 64Cu (169), and 18F (170). 123I-labeling occurs at tyr10 demonstrate specific tumor uptake, high nonspecific kidney and/or tyr22 of the native molecule (Figure 7), while and liver uptake limited its clinical utility (178). incorporation of 99mTc and 64Cu occurred via addition of C- Dehalogenation of 125I-NDP-MSH in vivo (179) has been terminal chelating moieties (169). 123I-VIP was studied in overcome through the use of N-Succinimidyl 3-125I- over 200 patients, demonstrating the ability to specifically Iodobenzoate (180). Use of this reagent results in addition localize intestinal adenocarcinomas and carcinoid tumors in of 125I-IBA to the  amino group of Lys11, and lends the vivo. The 123I-VIP preparations used were purified by RP- resulting labeled peptide increased inertness to HPLC prior to injection, and even so caused a small, dehalogenation. The NDP-MSH molecule has also been transient blood pressure drop due to the vasodilatory effect conjugated to the DOTA moiety to provide a targeting of VIP (164). High receptor-mediated uptake was also vector capable of coordinating a wide range of observed in the lungs in these studies due to high expression radioisotopes. The DOTA group in one study was attached of VIP receptors in lung acini, which reduces the ability to to the N-terminus of both NDP-MSH and an altered observe pulmonary lesions. octapeptide fragment of NDP-MSH and used for

22 Giblin et al: Radiolabeling of Receptor-specific Peptides coordination of 111In (181). The DOTA moiety has also 5 Jain RK: Delivery of novel therapeutic agents in tumors: been linked to the  amino group of Lys11 in a similar physiological barriers and strategies. J Natl Cancer Inst 81: octapeptide fragment of NDP-MSH and used to coordinate 570-576, 1989. 6 Bakker WH, Albert R, Bruns C, Breeman WA, Hofland LJ, 67Ga/68Ga (182). In a B16F1/B6D2F1 in vivo murine Marbach P, Pless J, Pralet D, Stolz B and Koper JW: [111In- melanoma model, tumor uptake of these peptides at 24h pi DTPA-D-Phe1]-octreotide, a potential radiopharmaceutical was between 1.17±0.13 and 3.10±0.36%ID/g, while kidney for imaging of somatostatin receptor-positive tumors: uptake at that time point ranged between 2.04±0.17 and synthesis, radiolabeling and in vitro validation. Life Sci 49: 6.56±0.77%ID/g. 1583-1591, 1991. The cyclic ·-MSH radiopharmaceuticals under current 7 Eckelman, WC and Gibson RE: The design of site-directed study are based upon a superpotent ·-MSH analog, Cys4,10, radiopharmaceuticals for use in drug discovery. In: Nuclear Imaging in Drug Discovery, Development, and Approval D-Phe7-·-MSH4-13, first described in 1985 (183). Using a (Burns HD, Gibson RE, Dannals R and Siegl P, eds), Boston, rational design approach, the structure of this peptide was Birkhauser, 1993, pp 113-134. modified such that incorporation of technetium or rhenium 8 Fischman AJ, Babich JW and Strauss HW: A ticket to ride: led to ring closure and cyclization of the peptide via a peptide radiopharmaceuticals. J Nucl Med 34: 2253-2263, 1993. Tc(V)O3+ or Re(V)O3+ core (17). Subsequent studies have 9 Nagy A, Szoke B and Schally AV: Selective coupling of extended this approach, for example, by altering the Lys11 methotrexate to peptide hormone carriers through a gamma- residue in order to reduce nonspecific kidney uptake (184, carboxamide linkage of its glutamic acid moiety: benzotriazol- 1-yloxytris(dimethylamino)phosphonium hexafluorophosphate 185). Further work has resulted in peptides that retain a 3+ activation in salt coupling. Proc Natl Acad Sci USA 90: 6373- nonradioactive Re(V)O core to preserve peptide stability 6376, 1993. and affinity, while also including an N-terminal DOTA 10 Reubi JC: Neuropeptide receptors in health and disease: the moiety for the coordination of a more diverse array of molecular basis for in vivo imaging. J Nucl Med 36: 1825- radionuclides (186). 1835, 1995. 11 Larson S: Receptors on tumors studied with radionuclide Conclusion scintigraphy. J Nucl Med 32: 1189-1191, 1991. 12 Pinski J, Reile H, Halmos G, Groot K and Schally AV: This review is but a brief introduction into the design and Inhibitory effects of analogs of luteinizing hormone-releasing development of site-directed, diagnostic/therapeutic, hormone on the growth of the androgen-independent Dunning peptide-based radiopharmaceuticals. In brief, we have R-3327-AT-1 rat prostate cancer. Int J Cancer 59: 51-55, 1994. 13 Reubi JC: In vitro identification of vasoactive intestinal described many of the important aspects necessary to peptide receptors in human tumors: implications for tumor develop conjugates of this type, keeping in mind the imaging. J Nucl Med 36: 1846-1853, 1995. capacity of these conjugates to target specific human tissues. 14 Stokkel MP and Pauwels EK: Octreotide scintigraphy for small In order to continue the development of, or improve, cell lung carcinoma: past, present or future? Eur J Nucl Med existing radiopharmaceuticals of this type, a collaborative 21: 1276-1278, 1994. research effort among scientists and physicians in inorganic 15 Krenning EP, Kweekkeboom DJ, Bakker WH, Breeman WA, chemistry, organic/medicinal chemistry, analytical chemistry, Kooij PP, Oei HY, van Hagen M, Postema PT, de Jong M and Reubi JC: Somatostatin receptor scintigraphy with [111In- biochemistry, molecular biology, radiology and nuclear DTPA-D-Phe1]- and [123I-Tyr3]-octreotide: the Rotterdam medicine is paramount. Great strides will continue to be experience with more than 1000 patients. Eur J Nucl Med 20: made in the field of radiopharmaceutical chemistry, but only 716-731, 1993. through interdisciplinary research efforts. 16 Liu S and Edwards DS: Bifunctional chelators for therapeutic lanthanide radiopharmaceuticals. Bioconjugate Chem 12: 7- References 34, 2001. 17 Giblin MF, Wang N, Hoffman TJ, Jurisson SS and Quinn TP: 1 Eary JF, Schroff RW, Abrams PG, Fritzberg AR, Morgan AC, Design and characterization of alpha-melanotropin peptide Kasina S, Reno JM, Srinivasan A, Woodhouse CS and Wilbur analogs cyclized through rhenium and technetium metal DS: Successful imaging of malignant melanoma with coordination. Proc Natl Acad Sci USA 95: 12814-12818, 1998. technetium-99m-labeled monoclonal antibodies. J Nucl Med 18 Ning L, Ochrymowcycz LA, Higginbotham C, Struttmann M, 30: 25-32, 1989. Abrams MJ, Vollano JF, Skerlj RT, Ketring AR and Volkert 2 Fritzberg AR and Wilbur DS: Radiolabeling of antibodies for WA: Pharmacokinetic studies of 105Rh(III) complexes with targeted diagnostics. In: Targeted Delivery of Imaging Agents. macrocycles. J Labelled Compd Radiopharm 37: 426-428, Boca Raton, FL, CRC Press, Inc., 1995, pp 83-101. 1995. 3 Goldenberg DM and Larson SM: Radioimmunodetection in 19 Lewis JS, Lewis MR, Srinivasan A, Schmidt MA, Wang J and cancer identification. J Nucl Med 33: 803-814, 1992. Anderson CJ: Comparison of four 64Cu-labeled somatostatin 4 Press OW, Appelbaum FR, Early JF and Bernstein ID: analogues in vitro and in a tumor-bearing rat model: evaluation Radiolabeled antibody therapy of lymphomas. Biolog Ther of new derivatives for positron emission tomography imaging Cancer Update 4: 1-13, 1994. and targeted radiotherapy. J Med Chem 42: 1341-1347, 1999.

23 in vivo 19: 9-30 (2005)

20 Stolz B, Smith-Jones PM, Weckbecker G, Albert R, Knecht H, 40 Schubiger PA, Alberto R and Smith A: Vehicles, chelators, Haller R, Tolcsvai L, Hofman G, Pollehn K and Bruns C: and radionuclides: choosing the "building blocks" of an Radiotherapy with yttrium-90 labeled DOTA-Tyr3-octreotide effective therapeutic radioimmunoconjugate. Bioconjugate in tumor bearing rodents. J Nucl Med 38: 18, 1997. Chem 7: 165-179, 1996. 21 Blok D, Feitsma RI, Vermeij P and Pauwels EJ: Peptide 41 Volkert WA, Goeckeler WF, Ehrhardt GJ and Ketring AR: radiopharmaceuticals in nuclear medicine. Eur J Nucl Med 26: Therapeutic radionuclides: production and decay property 1511-1519, 1999. considerations. J Nucl Med 32: 174-185, 1991. 22 Boerman OC, Oyen WJG and Corstens FHM: Radio-labeled 42 Ehrhardt GJ, Ketring AR and Ayers LM: Reactor-produced receptor-binding peptides: a new class of radiopharmaceuticals. radionuclides at the University of Missouri Research Reactor. Sem Nucl Med 30: 195-208, 2000. Appl Radiat Isot 49: 295-297, 1998. 23 Okarvi SV: Recent developments in 99mTc-labelled peptide- 43 Cutler CS, Smith CJ, Ehrhardt GJ, Tyler TT, Jurisson SS and based radiopharmaceuticals: an overview. Nuclear Med Deutsch E: Current and potential therapeutic uses of Commun 20: 1093-1112, 1999. Lanthanide radioisotopes. Cancer Biotherap Radiopharmaceut 24 Schally AV, Comaru-Schally AM, Plonowski A, Nagy A, 15(6): 531-545, 2000. Halmos G and Rekasi Z: Peptide analogs in the therapy of 44 Eckelman WC: Radiolabeling with technetium-99m to study prostate cancer. Prostate 45: 158-166, 2000. high-capacity and low-capacity biochemical systems. Eur J 25 Trejtnar F, Laznicek M, Laznickova A and Mather SJ: Nucl Med 22: 249, 1995. Pharmacokinetics and renal handling of 99mTc-labeled 45 Smith CJ, Volkert WA and Hoffman TJ: Gastrin releasing peptides. J Nucl Med 41: 177-182, 2000. peptide (GRP) receptor targeted radiopharmaceuticals: a 26 Kwekkeboom D, Krenning EP and de Jong M: Peptide concise update. Nucl Med Biol 30: 861-868, 2002. receptor imaging and therapy. J Nucl Med 41: 1704-1713, 46 Smith CJ, Sieckman GL, Owen NK, Hayes DL, Mazuru DG, 2000. Volkert WA and Hoffman TJ: Radiochemical investigations of 188 27 Anderson CJ and Welch MJ: Radiometal-labeled agents [ Re(H2O)(CO)3-Diaminopropionic acid-SSS-bombesin(7- (non-technetium) for diagnostic imaging. Chem Rev 99: 14)NH2]: syntheses, radiolabeling and in vitro/in vivo GRP 2219-2234, 1999. receptor targeting studies. Anticancer Res 23: 63-70, 2003. 28 Jurisson S, Berning D, Jia W and Ma D: Coordination 47 Pillai MRA, Lo JM and Troutner DE: Labeling of compounds in nuclear medicine. Chem Rev 93: 1137-1156, 1993. hematoporphyrin with Rhodium-105 and binding studies 29 Jurisson S and Lydon JD: Potential technetium small molecule with human gamma globulin. Appl Radiat Isot 41: 69-73, radiopharmaceuticals. Chem Rev 99: 2205-2218, 1999. 1990. 30 Liu S and Edwards DS: 99mTc-labeled small peptides as 48 Venkatesh M, Goswami N, Volkert WA, Schlemper EO, diagnostic radiopharmaceuticals. Chem Rev 99: 2235-2268, 1999. Ketring AR, Barnes CL and Jurisson S: An 105Rh complex of 31 Chart of the Nuclides, Fifteenth Edition. Copyright©1996, tetrathiacyclohexadecane diol with potential for formulating General Electric Co. and KAPL Inc.. bifunctional chelates. Nucl Med Biol 23: 33, 1996. 32 Pearson RG: Hard and soft acids and bases. J Am Chem Soc 49 Goswami M, Alberto R, Barnes CL and Jurisson S: Rhodium 22: 3533-3539, 1963. (III) complexes with acyclic tetrathioether ligands. Effects of 33 Martell AE and Hancock RD: Metal Complexes in Aqueous backbone chain length on the conjugation of the Rh Solutions. New York Plenum, 1996. (III)complex. Inorg Chem 35: 7546-755, 1996. 34 Goethals P, Coene M, Slegers G, Agon P, Deman J and 50 Smith CJ, Hoffman TJ, Hayes DL, Owen NK, Sieckman GL Schelstraete K: Cyclotron production of carrier-free 66Ga as a and Volkert WA: Radiochemical investigations of 177Lu- positron emitting label of albumin colloids for clinical use. Eur DOTA-8-Aoc-BBN[7-14]NH2: a new gastrin releasing peptide J Nucl Med 14: 152, 1988. receptor (GRPr) targeting radiopharmaceutical. J Labelled 35 Daube-Witherspoon, ME, Green SL, Plascjak P, Wu C, Compd Radiopharm 44: 706-708, 2001. Carson RE, Brechbiel M and Eckelman WC: Pet imaging 51 Howell RW, Azure MT, Narra VR and Rao DV: Relative characteristics of 66Ga. J Nucl Med 38: 200, 1997. biological effectiveness of alpha-particle emitters in vivo at low 36 Herzog H, Rosch F, Stocklin G, Lueders C, Qaim SM and doses. Radiat Res 137: 352-360, 1994. Feinendegen LE: Measurement of pharmacokinetics of 52 Kozak RW, Atcher RW, Gansow OA, Friedman AM, Hines yttrium-86 radiopharmaceuticals with PET and radiation dose JJ and Waldman TA: Bismuth-212-labeled anti-TAC calculation of analogous yttrium-90 radiotherapeutics. J Nucl monoclonal antibody: alpha-particle-emitting radionuclides as Med 34: 2222, 1993. modalities for radioimmunotherapy. Proc Natl Acad Sci USA 37 Herzog H, Rosch F, Brockmann J, Muhlensiepen H, Kohle 83 (2): 474-478, 1986. M, Stolz B, Marbach P and Muller-Gartner HW: Quantitative 53 Zalutsky MR and Bigner DD: Radioimmunotherapy with evaluation of 90Y-DOTA-Tyr3-OCTREOTIDE as measured alpha-particle emitting radioimmunoconjugates. Acta Oncol in baboons by means of Yttrium-86 and PET. J Nucl Med 38: 35: 373-379, 1996. 60, 1997. 54 Kassis AI, Harris CR, Adelstein SJ, Ruth TJ, Lambrecht R 38 Volkert WA and Hoffman TJ: Therapeutic radiopharmaceuticals. and Wolf AP: The in vitro radiobiology of astatine-211 decay. Chem Rev 99: 2269-2292, 1999. Radiat Res 105(1): 27-36, 1986. 39 Fritzberg AR, Gustavson LM, Hylarides MD and Reno JM: 55 Kennel SJ, Stabin M, Yoriyaz H, Brechbiel MW and Mirzadeh Chemical and structural approaches to rational drug design. S: Treatment of lung tumor colonies with 90Y targeted to In: Weiner DB, Williams MV, eds. Boca Raton, FL, CRC blood vessels: comparison with the alpha-particle emitter 213Bi. Press, p 125, 1995. Nucl Med Biol 26: 149-157, 1999.

24 Giblin et al: Radiolabeling of Receptor-specific Peptides

56 Okarvi SV: Recent developments in 99mTc-labelled peptide-based 73 Smith CJ, Gali H, Sieckman GL, Higginbotham C, Volkert radiopharmaceuticals. Nucl Med Commun 20: 1093-1112, 1999. WA and Hoffman TJ: Radiochemical investigations of 99mTc- 99m 57 Rhodes BA: Direct labeling of proteins with Tc. Nucl Med N3S-X-BBN[7-14]NH2: an in vitro/in vivo structure-activity Biol 18: 667-676, 1991. relationship study where X = 0-, 3-, 5-, 8-, and 11-carbon 58 Rhodes BA, Lambert CR, Marek MJ, Knapp Jr FF and Harvey tethering moieties. Bioconj Chem 14: 93-102, 2003. EB: 188Re labeled antibodies. Appl Radiat Isot 47: 7-14, 1996. 74 Abrams MJ, Juweid M, tenKate CI, Schwartz DA, Hauser 59 John E, Thakur ML, DeFulvio J, McDevitt MR and Damjanov MM, Gaul FE, Fuccello AJ, Rubin RH, Strauss W and I: Rhenium-186-labeled monoclonal antibodies for radio- Fischman AJ: Technetium-99m-human polyclonal IgG immunotherapy: preparation and evaluation. J Nucl Med 34: radiolabeled via the hydrazino nicotinamide derivative for 260-267, 1993. imaging focal sites of infection in rats. J Nucl Med 31: 2022- 60 Wong DW, Mishkin F and Lee T: A rapid chemical method 2028, 1990. of labeling human plasma proteins with 99mTc-pertechnetate 75 Verbeke K, Kieffer D, Vanderheyden JL, Reutelingsperger C, at pH 7.4. Int J Appl Radiat Isot 29: 251-253, 1978. Steinmetz N, Green A and Verbruggen A: Optimization of the 61 Hnatowich DJ: Antibody radiolabeling, problems and preparation of 99mTc-labeled HYNIC-derivatized annexin V promises. Nucl Med Biol 17: 49-55, 1990. for human use. Nucl Med Biol 30: 771-778, 2003. 62 Sundberg MW, Meares CF, Goodwin DA and Diamanti CI: 76 Babich JW and Fischman AJ: Effect of "co-ligand" on the Selective binding of metal ions to macromolecules using biodistribution of 99mTc-labeled hydrazino nicotinic acid bifunctional analogs of EDTA. J Med Chem 17: 1304, 1974. derivatized chemotactic peptides. Nucl Med Biol 22: 25-30, 63 Paik CH, Phan LNB and Hong JJ: The labeling of high affinity 1995. sites of antibodies with 99mTc. Int J Nucl Med Biol 12: 3, 1985. 77 Decristoforo C and Mather SJ: Technetium-99m somatostatin 64 Maina T, Stolz B, Albert R, Bruns C, Koch P and Macke H: analogues: effect of labelling methods and peptide sequence. Synthesis, radiochemistry and biological evaluation of a new Eur J Nucl Med 26: 869-876, 1999. somatostatin analogue(SDZ 219-387) labeled with technetium- 78 Hnatowich DJ, Winnard Jr P, Virzi F, Fogarsi M, Sano T, 99m. Eur J Nucl Med 21: 437, 1994. Smith CL, Cantor CL and Rusckowski M: Technetium-99m 65 Liu S, Edwards DS, Looby RJ, Poirier MJ, Rajopadhye M, labeling of DNA. J Nucl Med 36: 2306-2314, 1995. Bourque JP and Carroll TR: Labeling cyclic glycoprotein 79 Rennen HJJM, van Eerd JE, Oyen WJG, Corstens FHM, IIb/IIIa receptor antagonists with 99mTc by the preformed Edwards DS and Boerman OC: Effects of coligand variation chelate approach: effects of chelators on properties of [99mTc] on the in vivo characteristics of 99mTc-labeled interleukin-8 in chelator-peptide conjugates. Bioconj Chem 7: 196, 1996. detection of infection. Bioconj Chem 13(2): 370-377, 2002. 66 Rao TN, Adhikesavalu D, Camerman A and Fritzberg AR: 80 Guo W, Hinkle GH and Lee RJ: 99mTc-HYNIC-folate: A Technetium (V) and rhenium (V) complexes of 2,3-bis novel receptor-based targeted radiopharmaceutical for tumor (mercaptoacetamido)propanoate. Chelate ring stereochemistry imaging. J Nucl Med 40: 1563-1569, 1999. and influence on chemical and biological properties. J Am Chem 81 Decristoforo C and Mather S: 99mTc-technetium-labelled Soc 112: 5798, 1990. peptide-HYNIC conjugates: effects of lipophilicity and 67 Eshima D, Taylor A, Fritzberg AR, Kasina S, Hansen L and stability on biodistribution. Nucl Med Biol 26: 389-396, 1999. Sorenson JF: Animal evaluation of technetium-99m triamide 82 Fichna J and Janecka A: Synthesis of target-specific radiolabeled mercaptide complexes as potential renal imaging agents. J peptides for diagnostic imaging. Bioconj Chem 14: 3-17, 2003. Nucl Med 28: 1180, 1987. 83 Liu S, Edwards DS, Looby RJ, Harris AR, Poirier MJ, Barrett JA, 68 Subhani M, Cleynhens B, Bormans G, Hoogmartens M, Heminway SJ and Carrol TR: Labeling a hydrazino nicotinamide- DeRoo M and Verbruggen AM: Technetium, Rhenium and modified cyclic IIb/IIIa receptor antagonist with 99mTc using Other Metals in Chemistry and Nuclear Medicine (Nicolini M, aminocarboxylates as coligands. Bioconj Chem 7: 63-71, 1996. Banoli G and Ulderico M, eds.). Verona, Cortina 84 Liu S, Edwards DS and Harris AR: A novel ternary ligand International, 1989, Vol. 3, pp 453-461. system for 99mTc-labeling of hydrazino nicotinamide-modified 69 Vanbilloen HP, Bormans GM, DeRoo MJ and Verbruggen biologically active molecules using imine-N-containing AM: Complexes of technetium-99m with tetrapeptides, a new heterocycles as coligands. Bioconj Chem 9: 583-595, 1998. class of 99mTc-labelled agents. Nucl Med Biol 22: 325 1996. 85 Liu S, Edwards DS and Barrett JA: 99mTc-labeling of highly 70 Jurisson S, Schlemper EO, Troutner DE, Canning LR, Nowotnik potent small peptides. Bioconj Chem 8: 621-636, 1997. DP and Neirinckx RD: Synthesis, characterization, and x-ray 86 Zalutsky MR and Narula AS: Radiohalogenation of a monoclonal structural determinations of technetium(V)-oxo-tetradentate antibody using an N-succinimidyl 3-(tri-n-butylstannyl) benzoate amine oxime complexes. Inorg Chem 25(4): 543-549, 1986. intermediate. Cancer Res 48: 1446-1450, 1988. 71 Jurisson S, Aston K, Fair KF, Schlemper EO, Sharp PR and 87 Alberto R, Egli A, Schibli R, Waibel R, Abram U, Kaden TA, Troutner DE: Effect of ring size on properties of technetium Schaffland A, Schwarzbach R and Schubiger PA: From - amine oxime complexes. X-ray structures of TcO2Pent(AO)2, [TcO4] to an organometallic aqua-ion: synthesis and chemistry 99m + which contains an unusual eight-membered chelate ring, and of [ Tc(OH2)3(CO)3] . In: Technetium, Rhenium and of TcOEn(AO)2. Inorg Chem 26: 3576, 1987. Other Metals in Chemistry and Nuclear Medicine (Nicolini M, 72 Van de Wiele C, Dumont F, Broecke RV, Oosterlinck W, and Ulderico M, eds.), SGE, Italy, 1999, pp27-34. Cocquyt V, Serreyn R, Peers S, Thornback J, Slegers G and 88 Alberto R, Schibli R, Waibel R, Abram U and Schubiger PA: + Dierckx RA: Technetium-99m RP527, a GRP analogue for Basic aqueous chemistry of [M(OH2)3(CO)3] (M = Re, Tc) visualization of GRP receptor-expressing malignancies: a directed towards radiopharmaceutical application. Coord feasible study. Eur J Nucl Med 27: 1694-1699, 2000. Chem Rev 190-192, 901-919, 1999.

25 in vivo 19: 9-30 (2005)

89 Schibli R, Schwarzbach R, Alberto R, Ortner K, Schmalle H, 101 Smith CJ, Gali H, Sieckman GL, Hayes DL, Owen NK, Dumas C, Egli A and Schubiger PA: Steps toward high specific Mazuru DG, Volkert WA and Hoffman TJ: Radiochemical 177 activity labeling of biomolecules for therapeutic application: investigations of Lu-DOTA-8-BBN[7-14]NH2: an in vitro/in 188 + preparation of precursor [ Re(OH2)3(CO)3] and synthesis vivo assessment of the targeting ability of this new of tailor-made bifunctional ligand systems. Bioconj Chem 13: radiopharmaceutical for PC-3 human prostate cancer cells. 750-756, 2002. Nucl Med Biol 30: 101-109, 2002. 90 Alberto R, Schibli R, Egli A and Schubiger PA: A novel 102 Hoffman TJ, Quinn TP and Volkert WA: Radiometallated organometallic aqua complex of technetium for the labeling of receptor-avid peptide conjugates for specific in vivo targeting 99m + biomolecules: synthesis of [ Tc(OH2)3(CO)3] from of cancer cells. Nucl Med Biol 28: 527-539, 2001. 99m - [ TcO4] in aqueous solution and its reaction with a 103 Ning L, Ochrymowcycz LA, Higginbotham C, Struttmann M, bifunctional ligand. J Am Chem Soc 120: 7987-7988, 1998. Abrams MJ, Vollano JF, Skerlj RT, Ketring AR and Volkert 91 Egli A, Alberto R, Tannahill L, Schibli R, Abram U, Schaffland WA: Pharmacokinetic studies of 105Rh(III) complexes with A, Waibel R, Tourwe D, Jeannin L, Iterbeke K and Schubiger macrocycles. J Labelled Compd Radiopharm 37: 426-428, 1995. PA: Organometallic 99mTc-aquaion labels peptide to an 104 Hoffman TJ, Li N, Volkert WA, Sieckman GL, Higginbotham unprecedented high specific activity. J Nucl Med 40(11): 1913- C and Ochrymowcycz LA: Synthesis and characterization of 1917, 1999. 105Rh labeled bombesin analogues: enhancement of GRP 92 Abram U, Abram S, Alberto R and Schibli R: Ligand receptor binding affinity utilizing aliphatic carbon chain exchange reactions starting from [Re(CO)3Br3]2-. Synthesis, linkers. J Labelled Compd Radiopharm 40: 490-493, 1997. characterization, and structures of rhenium(I) tricarbonyl 105 Decristoforo C and Mather SJ: The influence of chelator on complexes with thiourea and thiourea derivatives. Inorg Chem the pharmacokinetics of 99mTc-labeled peptides. Q J Nucl Med Acta 248: 193-202, 1996. 46: 195-205, 2002. 93 Schibli R, Katti KV, Higginbotham C, Volkert WA and 106 Reubi JC: Peptide receptors as molecular targets for cancer Alberto R: In vitro and in vivo evaluation of bidentate, water- diagnosis and therapy. Endocrine Rev 24: 389-427, 2003. soluble phosphine ligands as anchor groups for the 107 Gotthardt M, Boermann OC, Behr TM, Behe MP and Oyen 99m + organometallic fac-[ Tc(CO)3] -Core. Nucl Med Biol 26: WJG: Development and clinical application of peptide-based 711-716, 1999. radiopharmaceuticals. Curr Pharm Des 10: 2951-2963, 2004. 94 Egli A, Alberto R, Tannahill L, Schibli R, Abram U, Schaffland 108 Britz-Cunningham SH and Adelstein SJ: Molecular targeting + A, Tourwe D and Schubiger PA: [99mTc(OH2)3(CO)3] labels with radionuclides: state of the science. J Nucl Med 44: 1945- peptide to an unprecedented high specific activity. A labeling 1961, 2003. study with amino acids and neurotensin. In: Technetium, 109 Krenning EP, Kwekkeboom DJ, Valkema R, Pauwels S, Kvols Rhenium and Other Metals in Chemistry and Nuclear LK and de Jong M: Peptide receptor radionuclide therapy. Medicine (Nicolini M and Ulderico M, eds.), SGE, Italy, 1999, Ann NY Acad Sci 1014: 234-245, 2004. pp507-512. 110 Weckbecker G, Lewis I, Albert R, Schmid HA, Hoyer D and 95 Roselli M, Schlom J, Gansow OA, Brechbiel MW, Mirzadeh Bruns C: Opportunities in somatostatin research: biological, S, Pipppin CG, Milenic EE and Colcher D: Comparative chemical and therapeutic aspects. Nature Rev Drug Disc 2: biodistribution studies of DTPA-derivative bifunctional 999-1017, 2003. chelates for radiometal labeled monoclonal antibodies. Int J 111 Froidevaux S and Eberle AN: Somatostatin analogs and Rad Appl Instrum B 18: 389-394, 1991. radiopeptides in cancer therapy. Biopolymers 66: 161-183, 2002. 96 Cummins CH, Rutter EW Jr and Fordyce WA: A convenient 112 Krenning EP, Bakker WH, Breeman WAP, Koper JW, Kooij synthesis of bifunctional chelating agents based on PPM, Ausema L, Lameris JS, Reubi JC and Lamberts SWJ: diethylenetriaminepentaacetic acid and their coordination Localisation of endocrine-related tumours with radioiodinated chemistry with yttrium (III). Bioconj Chem 2: 18-186, 1991. analogue of somatostatin. Lancet 1: 242-244, 1989. 97 Kwekkeboom DJ, Bakker WH, Kooij PPM, Konijnenberg 113 Lebtahi R, Le Cloirec J, Houzard C, Daou D, Sobhani I, MW, Srinivasan A, Erion LJ, Schmidt MA, Bugaj LJ, de Jong Sassolas G, Mignon M, Bourguet P and Le Guludec D: M and Krenning EP: [177Lu-DOTA0,Tyr3]octreotate: Detection of neuroendocrine tumors: 99mTc-P829 scintigraphy comparison with [111In-DTPA0]octreotide in patients. Eur J compared with 111In-pentetreotide scintigraphy. J Nucl Med Nucl Med 28: 1319-1325, 2001. 43: 889-895, 2002. 98 Lewis JS, Wang W, Laforest R, Wang F, Erion LJ, Bugaj JE, 114 Virgolini I, Traub T, Novotny C, Leimer M, Fuger B, Li SR, Srinivasan A and Anderson CJ: Toxicity and dosimetry of Patri P, Panger T, Angelberger P, Raderer M, Burggasser G, 177Lu-DOTA-Y3-Octreotate in a rat model. Int J Cancer 94: Andreae F, Kurtaran A and Dudczak R: Experience with 873-877, 2001. indium-111 and yttrium-90-labeled somatostatin analogs. Curr 99 Paganelli G, Zoboli S, Cremonesi M, Maecke HR and Chinol Pharm Des 8: 1781-1807, 2002. M: Receptor-mediated radiolanthanide therapy with 90Y- 115 Virgolini I, Szilvasi I, Kurtaran A, Angelberger P, Raderer M, DOTA-D-Phe1-Tyr3-octreotide: preliminary report in cancer Havlik E, Vorbeck F, Bischof C, Leimer M, Dorner G, Kletter patients. Cancer Biother Radiopharm 14: 477-483, 1999. K, Niederle B, Scheithauer W and Smith-Jones P: Indium-111- 100 Gali H, Hoffman TJ, Owen NK, Sieckman GL and Volkert DOTA-lanreotide: biodistribution, safety and radiation absorbed WA: In vitro and in vivo evaluation of 111In-labeled DOTA-8- dose in tumor patients. J Nucl Med 39: 1928-1936, 1998. Aoc-BBN[7-14]NH2 conjugate for specific targeting of tumors 116 Otte A, Harrmann R, Heppeler A, Behe M, Jermann E, expressing gastrin releasing peptide receptors (GRP-R). J Powell P, Maecke HR and Muller J: Yttrium-90 DOTATOC: Nucl Med (Supplement) 41(5): 119P, 2000. first clinical results. Eur J Nucl Med 26: 1439-1447, 1999.

26 Giblin et al: Radiolabeling of Receptor-specific Peptides

117 Rosch F, Herzog H, Stolz B, Brockmann J, Kohle M, 130 Fischer JB and Schonbrunn A: The bombesin receptor is Muhlensiepen H, Marbach P and Muller-Gartner H: Uptake coupled to a guanine nucleotide-binding protein which is kinetics of the somatostatin receptor ligand [86Y]DOTA- insensitive to pertussis and cholera toxins. J Biol Chem 263: DPhe1-Tyr3-octreotide ([86Y]SMT487) using positron emission 2808-2816, 1988. tomography in non-human primates and calculation of 131 Markwalder R and Reubi JC: Gastrin releasing peptide radiation doses of the 90Y-labelled analogue. Eur J Nucl Med receptors in the human prostate: relation to neoplastic 26: 358-366, 1999. transformation. Cancer Res 59: 1152-1159, 1999. 118 Hofmann M, Maecke H, Borner AR, Weckesser E, Schoffski 132 Sun B, Halmos G, Schally AV, Wang X and Martinez M: P, Oei ML, Schumacher J, Henze M, Heppeler A, Meyer GJ Presence of receptors for bombesin/gastrin-releasing peptide and Knapp WH: Biokinetics and imaging with the and mRNA for three receptor subtypes in human prostate somatostatin receptor PET radioligand 68Ga-DOTATOC: cancers. Prostate 42: 295-303, 2000. preliminary data. Eur J Nucl Med 28: 1751-1757, 2001. 133 Halmos G, Wittliff JL and Schally AV: Characterization of 119 Forster GJ, Engelbach M, Brockmann J, Reber H, Buchholz bombesin/gastrin-releasing peptide receptors in human breast H, Maecke HR, Rosch F, Herzog H and Bartenstein P: cancer and their relationship to steroid receptor expression. Preliminary data on biodistribution and dosimetry for therapy Cancer Res 55: 280-287, 1995. planning of somatostatin receptor positive tumors: comparison 134 Gugger M and Reubi JC: Gastrin-releasing peptide receptors of 86Y-DOTATOC and 111In-DTPA-octreotide. Eur J Nucl in non-neoplastic and neoplastic human breast. Am J Pathol Med 28: 1743-1750, 2001. 155: 2067-2076, 1999. 120 Anderson CJ, Dehdashti F, Cutler PD, Schwarz SW, Laforest 135 Toi-Scott M, Jones CL and Kane MA: Clinical correlates of R, Bass LA, Lewis JS and McCarthy DW: 64Cu-TETA- bombesin-like peptide receptor subtype expression in human octreotide as a PET imaging agent for patients with lung cancer cells. Lung Cancer 15: 341-354, 1996. neuroendocrine tumors. J Nucl Med 42: 213-221, 2001. 136 Baidoo KE, Lin K-S, Zhan Y, Finley P, Scheffel U and 121 Boerman OC, Oyen WJG and Corstens FHM: Between the Scylla Wagner HN: Design, synthesis, and initial evaluation of high- and Charybdis of peptide radionuclide therapy: hitting the tumor affinity technetium bombesin analogues. Bioconj Chem 9: 218- and saving the kidney. Eur J Nucl Med 28: 1447-1449, 2001. 225, 1998. 122 Wild D, Schmitt JS, Ginj M, Maecke HR, Bernard BF, 137 Smith CJ, Gali H, Sieckman GL, Higginbothman C, Volkert Krenning E, de Jong M, Wenger S and Reubi JC: DOTA- WA and Hoffman TJ: Radiochemical investigations of 99mTc- NOC, a high-affinity ligand of somatostatin receptor subtypes N3S-X-BBN(7-14)NH2: an in vitro/in vivo structure-activity 2, 3 and 5 for labelling with various radiometals. Eur J Nucl relationship study where X = 0, 3, 5, 8, and 11-carbon Med Mol Imaging 30: 1338-1347, 2003. tethering moieties. Bioconj Chem 14: 93-102, 2003. 123 Anastasi A, Erspamer V and Nucci M: Isolation and amino 138 Gali H, Hoffman TJ, Sieckman GL, Owen NK, Katti KV and acid sequences of alytesin and bombesin, two analogous active Volkert WA: Synthesis, characterization, and labeling with tetradecapeptides from the skin of european discoglossid 99mTc/188Re of peptide conjugates containing a dithia- frogs. Arch Biochem Biophys 148: 443-446, 1972. bisphosphine chelating agent. Bioconj Chem 12: 354-363, 2001. 124 Girard F, Bachelard H, St-Pierre S and Rioux F: The 139 Rogers BE, Bigott HM, McCarthy DW, Della Manna D, Kim contractile effect of bombesin, gastrin releasing peptide and J, Sharp TL and Welch MJ: MicroPET imaging of a gastrin- various fragments in the rat stomach strip. Eur J Pharmacol releasing peptide receptor-positive tumor in a mouse model of 102: 489-497, 1984. human prostate cancer using a 64Cu-labeled bombesin 125 Von Schrenck T, Heinz-Erian P, Moran T, Mantey SA, analogue. Bioconj Chem 14: 756-763, 2003. Gardner J and Jensen R: Neuromedin B receptor in 140 Chen X, Park R, Hou Y, Tohme M, Shahinian AH, Bading JR esophagus: evidence for subtypes of bombesin receptors. Am J and Conti PS: MicroPET and autoradiographic imaging of Physiol 256: G747-G758, 1989. GRP receptor expression with 64Cu-DOTA-[Lys3]bombesin in 126 Spindel ER, Giladi E, Brehm P, Goodman RH and Segerson human prostate adenocarcinoma xenografts. J Nucl Med 45: TP: Cloning and functional characterization of a 1390-1397, 2004. complementary DNA encoding the murine fibroblast 141 Nock B, Nikolopoulou A, Chiotellis E, Loudos G, Maintas D, bombesin/gastrin releasing peptide receptor. Mol Endocrinol Reubi JC and Maina T: [99mTc]demobesin 1, a novel potent 4: 1956-1963, 1990. bombesin analogue for GRP receptor-targeted tumour 127 Fathi Z, Corjay MH, Shapira H, Wada E, Benya R, Jensen R, imaging. Eur J Nucl Med Mol Imag 30: 247-258, 2003. Viallet J, Sausville EA and Battey JF: BRS-3: a novel 142 Healy JM, Murayama O, Maeda T, Yoshino K, Sekiguchi K and bombesin receptor subtype selectively expressed in testis and Kikuchi MM: Peptide ligands for integrin ·V‚3 selected from lung carcinoma cells. J Biol Chem 268: 5979-5984, 1993. random phage display libraries. Biochemistry 34: 3948-3955, 1995. 128 Nagalla SR, Barry BJ, Creswick KC, Eden P, Taylor JT and 143 Reinmuth N, Liu W, Ahmad SA, Fan F, Stoeltzing O, Parikh Spindel ER: Cloning of a receptor for amphibian AA, Bucana CD, Gallick GE, Nickols MA, Westlin WF and 13 [Phe ]bombesin distinct from the receptor for gastrin Ellis LM: ·V‚3 integrin antagonist S247 decreases colon releasing peptide: identification of a fourth bombesin receptor cancer metastasis and angiogenesis and improves survival in subtype (BB4). Proc Natl Acad Sci USA 92: 6205-6209, 1995. mice. Cancer Res 63: 2079-2087, 2003. 129 Van de Wiele C, Dumont F, Van Belle S, Slegers G, Peers SH 144 Xiong J, Stehle T, Diefenbach B, Zhang R, Dunker R, Scott and Dierckx RA: Is there a role for agonist gastrin-releasing DL, Joachimiak A, Goodman SL and Arnaout MA: Crystal peptide receptor radioligands in tumor imaging? Nucl Med structure of the extracellular segment of integrin ·V‚3. Science Comm 22: 5-15, 2001. 294: 339-345, 2001.

27 in vivo 19: 9-30 (2005)

145 Kerr JS, Slee AM and Mousa SA: Small molecule ·V integrin 160 Poethko T, Schottelius M, Thumshirn G, Hersel U, Herz M, antagonists: novel anticancer agents. Exp Opin Invest Drugs Henriksen G, Kessler H, Schwaiger M and Wester HJ: Two- 9: 1271-1279, 2000. step methodology for high-yield routine radiohalogenation of 146 Sivolapenko GB, Skarlos D, Pectasides D, Stathopoulou E, peptides: 18F-labeled RGD and octreotide analogs. J Nucl Milonakis A, Sirmalis G, Stuttle A, Courtenay-Luck NS, Med 45: 892-902, 2004. Konstantinides K and Epenetos AA: Imaging of metastatic 161 Dockray GJ: Vasoactive intestinal polypeptide and related melanoma utilizing a technetium-99m labelled RGD-containing peptides. In: Gut Peptides (Walsh JH and Dockray GJ, eds.) synthetic peptide. Eur J Nucl Med 25: 1383-1389, 1998. New York, Raven Press, 1994, pp 447-472. 147 Sutcliffe-Goulden JL, O’Doherty MJ, Marsden PK, Hart IR, 162 Nicole P, Lins L, Rouyer-Fessard C, Drouot C, Fulcrand P, Marshall JF and Bansal SS: Rapid solid phase synthesis and Thomas A, Couvineau A, Martinez J, Brasseur R and biodistribution of 18F-labelled linear peptides. Eur J Nucl Med Laburthe M: Identification of key residues for interaction of 29: 754-759, 2002. vasoactive intestinal peptide with human VPAC1 and VPAC2 148 Pierschbacher MD and Ruoslahti E: Influence of receptors and development of a highly selective VPAC1 stereochemistry of the sequence Arg-Gly-Asp-Xaa on binding receptor agonist. J Biol Chem 275: 24003-24012, 2000. specificity in cell adhesion. J Biol Chem 262: 17294-17298, 1987. 163 Reubi JC, Laderach U, Waser B, Gebbers J, Robberecht P 149 Gottschalk K and Kessler H: The structures of integrins and and Laissue JA: Vasoactive intestinal peptide/pituitary integrin-ligand complexes: implications for drug design and adenylate cyclase-activating peptide receptor subtypes in signal transduction. Angew Chem Int Ed 41: 3767-3774, 2002. human tumors and their tissues of origin. Cancer Res 60: 150 Assa-Munt N, Jia X, Laakkonen P and Ruoslahti E: Solution 3105-3112, 2000. structures and integrin binding activities of an RGD peptide 164 Virgolini I, Raderer M, Kurtaran A, Angelberger P, Banyai S, with two isomers. Biochemistry 40: 2373-2378, 2001. Yang Q, Li S, Banyai M, Pidlich J, Niederle B, Scheithauer W 151 Su Z, Liu G, Gupta S,Zhu Z, Rusckowski M and Hnatowich and Valent P: Vasoactive intestinal peptide-receptor imaging DJ: In vitro and in vivo evaluation of a technetium-99m-labeled for the localization of intestinal adenocarcinomas and cyclic RGD peptide as a specific marker of ·V‚3 integrin for endocrine tumors. N Engl J Med 33: 1116-1121, 1994. tumor imaging. Bioconj Chem 13: 561-570, 2002. 165 Moody TW, Leyton J, Gozes I, Lang L and Eckelman WC: 152 Haubner R and Wester HJ: Radiolabeled tracers for imaging VIP and breast cancer. Ann NY Acad Sci 865: 290-296, 1998. of tumor angiogenesis and evaluation of anti-angiogenic 166 Virgolini I: Receptor nuclear medicine: vasointestinal peptide therapies. Curr Pharm Des 10: 1439-1455, 2004. and somatostatin receptor scintigraphy for diagnosis and 153 Haubner R, Wester HJ, Reuning U, Senekowitsch-Schmidtke treatment of tumor patients. Eur J Clin Invest 27: 793-800, 1997. R, Diefenbach B, Kessler H, Stocklin G and Schwaiger M: 167 Raderer M, Kurtaran A, Leimer M, Angelberger P, Niederle B, Radiolabeled ·V‚3 integrin antagonists: a new class of tracers Vierhapper H, Vorbeck F, Hejna MHL, Scheithauer W, Pidlich for tumor targeting. J Nucl Med 40: 1061-1071, 1999. J and Virgolini I: Value of peptide receptor scintigraphy using 154 Haubner R, Wester HJ, Burkhart F, Senekowitsch-Schmidtke 123I-vasoactive intestinal peptide and 111In-DTPA-D-Phe1- R, Weber W, Goodman SL, Kessler H and Schwaiger M: octreotide in 194 carcinoid patients: Vienna University Glycosylated RGD-containing peptides: tracer for tumor experience, 1993 to 1998. J Clin Oncol 18: 1331-1336, 2000. targeting and angiogenesis imaging with improved biokinetics. 168 Thakur ML, Marcus CS, Saeed S, Pallela V, Minami C, Diggles J Nucl Med 42: 326-336, 2001. L, Le Pham H, Ahdoot R and Kalinowski EA: 99mTc-labeled 155 Haubner R, Wester HJ, Weber W, Mang C, Ziegler SI, vasoactive intestinal peptide analog for rapid localization of Goodman SL, Senekowitsch-Schmidtke R, Kessler H and tumors in humans. J Nucl Med 41: 107-110, 2000. Schwaiger M: Noninvasive imaging of ·V‚3 integrin expression 169 Thakur ML, Aruva MR, Gariepy, J, Acton P, Rattan S, Prasad using 18F-labeled RGD-containing glycopeptide and positron S, Wickstrom E and Alavi A: PET imaging of oncogene emission tomography. Cancer Res 61: 1781-1785, 2001. overexpression using 64Cu vasoactive intestinal peptide (VIP) 156 Chen X, Park R, Hou Y, Khankaldyyan V, Gonzales-Gomez I, analog: comparison with 99mTc-VIP analog. J Nucl Med 45: Tohme M, Bading JR, Laug WE and Conti PS: MicroPET 1381-1389, 2004. imaging of brain tumor angiogenesis with 18F-labeled PEGylated 170 Jagoda EM, Aloj L, Seidel J, Lang L, Moody TW, Green S, RGD peptide. Eur J Nucl Med Mol Imag 31: 1081-1089, 2004. Caraco C, Daube-Witherspoon M, Green MV and Eckelman 157 van Hagen PM, Breeman WAP, Bernard HF, Schaar M, Mooij WC: Comparison of an 18F labeled derivative of vasoactive CM, Srinivasan A, Schmidt, MA, Krenning EP and de Jong M: intestinal peptide and 2-deoxy-2-[18F]fluoro-D-glucose in nude Evaluation of a radiolabelled cyclic DTPA-RGD analogue for mice bearing breast cancer xenografts. Mol Imaging Biol 4: tumor imaging and radionuclide therapy. Int J Cancer 90: 186- 369-379, 2002. 198, 2000. 171 Igarishi H, Ito T, Hou W, Mantey SA, Pradhan TK, Ulrich 158 Chen X, Park R, Tohme M, Shahinian AH, Bading JR and CD, Hocart SJ, Coy DH and Jensen RT: Elucidation of Conti PS: MicroPET and autoradiographic imaging of breast vasoactive intestinal peptide pharmacophore for VPAC1 cancer alpha v-integrin expression using 18F- and 64Cu-labeled receptors in human, rat, and guinea pig. J Pharm Exp Ther RGD peptide. Bioconj Chem 15: 41-49, 2004. 301: 37-50, 2002. 159 Janssen ML, Oyen WJ, Dijkgraaf I, Massuger LF, Frielink C, 172 Bhargava S, Licha K, Knaute T, Ebert B, Becker A, Edwards DS, Rajopadhye M, Boonstra H, Corstens FH and Grotzinger C, Hessenius C, Wiedenmann B, Schneider- Boerman OC: Tumor targeting with radiolabeled ·V‚3 Mergener J and Volkmer-Engert R: A complete substitutional integrin binding peptides in a nude mouse model. Cancer Res analysis of VIP for better tumor imaging properties. J Mol 62: 6146-6151, 2002. Recog 15: 145-153, 2002.

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173 Hruby VJ, Sharma SD, Toth K, Jaw JY, al-Obeidi F, Sawyer 181 Froidevaux S, Calame-Christe M, Tanner H, Sumanovski L TK and Hadley ME: Design, synthesis, and conformation of and Eberle AN: A novel DOTA-·-melanocyte-stimulating superpotent and prolonged acting melanotropins. Ann NY hormone analog for metastatic melanoma diagnosis. J Nucl Acad Sci 680: 51-63, 1993. Med 43: 1699-1706, 2002. 174 Siegrist W, Solca F, Stutz S, Giuffre L, Carrel S, Girard J and 182 Froidevaux S, Calame-Christe M, Schuhmacher J, Tanner H, Eberle AN: Characterization of receptors for alpha- Saffrich R, Henze M and Eberle AN: A gallium-labeled melanocyte-stimulating hormone on human melanoma cells. DOTA-·-melanocyte-stimulating hormone peptide analog for Cancer Res 49: 6352-6358, 1989. PET imaging of melanoma metastases. J Nucl Med 45: 116- 175 Tatro JB, Atkins M, Mier JW, Hardarson S, Wolfe H, Smith 123, 2004. T, Entwistle ML and Reichlin S: Melanotropin receptors 183 Cody WL, Mahoney M, Knittel JJ, Hruby VJ, Castrucci AM demonstrated in situ in human melanoma. J Clin Invest 85: and Hadley ME: Cyclic melanotropins. 9. 7-D-Phenylalanine 1825-1832, 1990. analogues of the active-site sequence. J Med Chem 28: 583- 176 Sawyer TK, Sanfilippo PJ, Hruby VJ, Engel MH, Heward CB, 588, 1985. Burnett JB and Hadley ME: 4-Norleucine, 7-D-phenylalanine- 184 Chen J, Cheng Z, Hoffman TJ, Jurisson SS and Quinn TP: alpha-melanocyte-stimulating hormone: a highly potent alpha- Melanoma-targeting properties of 99mtechnetium-labeled melanotropin with ultralong biological activity. Proc Natl Acad cyclic ·-melanocyte-stimulating hormone peptide analogues. Sci USA 77: 5754-5758, 1980. Cancer Res 60: 5649-5658, 2000. 177 Bard DR, Knight CG and Page-Thomas DP: A chelating 185 Miao Y, Whitener D, Feng W, Owen NK, Chen J and Quinn derivative of alpha-melanocyte stimulating hormone as a TP: Evaluation of the human melanoma targeting properties potential imaging agent for malignant melanoma. Br J Cancer of radiolabeled ·-stimulating hormone peptide analogues. 62: 919-922, 1990. Bioconj Chem 14: 1177-1184, 2003. 178 Wraight EP, Bard DR, Maughan TS, Knight CG and Page- 186 Cheng Z, Chen J, Miao Y, Owen NK, Quinn TP and Jurisson Thomas DP: The use of a chelating derivative of alpha SS: Modification of the structure of a metallopeptide: melanocyte stimulating hormone for the clinical imaging of Synthesis and biological evaluation of 111In-labeled DOTA- malignant melanoma. Br J Radiol 65: 112-118, 1992. conjugated rhenium-cyclized ·-MSH analogues. J Med Chem 179 Tatro JB and Reichlin S: Specific receptors for ·-melanocyte- 45: 3048-3056, 2002. stimulating hormone are widely distributed in tissues of rodents. Endocrinol 121: 1900-1907, 1987. 180 Garg PK, Alston KL, Welsh PC and Zalutsky MR: Enhanced binding and inertness to dehalogenation of ·-melanotropic peptides labeled using N-Succinimidyl 3-iodobenzoate. Bioconj Received November 2, 2004 Chem 7: 233-239, 1996. Accepted December 17, 2004

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