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`2012; 2(5):481-501. doi: 10.7150/thno.4024
`Review
`Radiolabeled Peptides: Valuable Tools for the Detection and Treatment of
`Cancer
`M. Fani1, H. R. Maecke1, S. M. Okarvi2 
`1. Department of Nuclear Medicine, University Hospital Freiburg, Freiburg, Germany
`2. Cyclotron and Radiopharmaceuticals Department, King Faisal Specialist Hospital and Research Centre, Riyadh 11211,
`Saudi Arabia
` Corresponding author: S. M. Okarvi , Cyclotron and Radiopharmaceuticals Department. King Faisal Specialist Hospital
`and Research Centre. MBC -03, PO Box 3354, Riyadh 11211, Saudi Arabia . Tel: +9661-442-4812, Fax: +9661-442-4743. E-mail:
`sokarvi@kfshrc.edu.sa
`© Ivyspring International Publisher. This is an open-access article distributed under the terms of the Creative Commons License (http://creativecommons.org/
`licenses/by-nc-nd/3.0/). Reproduction is permitted for personal, noncommercial use, provided that the article is in whole, unmodified, and properly cited.
`Received: 2011.12.29; Accepted: 2012.03.31; Published: 2012.05.16
`Abstract
`Human cancer cells overexpress many peptide receptors as molecular targets. Radiolabeled
`peptides that bind with high affinity and specificity to the receptors on tumor cells hold great
`potential for both diagnostic imaging and targeted radionuclide therapy. The advantage of
`solid-phase peptide synthesis, the availability of different chelating agents and prosthetic
`groups and bioconjugation techniques permit the facile preparation of a wide variety of
`peptide-based targeting molecules with diverse biological and tumor targeting properties.
`Some of these peptides, including somatostatin, bombesin, vasoactive intestinal peptide,
`gastrin, neurotensin, exendin and RGD are currently under investigation. It is anticipated that
`in the near future many of these peptides may find applications in nuclear oncology. This ar-
`ticle presents recent developments in the field of small peptides, and their applications in the
`diagnosis and treatment of cancer.
`Key words: Radiolabeled peptides, tumor imaging, radionuclide therapy, radionuclides
`Introduction
`Molecular imaging techniques are increasingly
`being used in the localization of disease, the staging of
`disease and for therapy control. The most sensitive
`imaging methods are those using nuclear probes for
`single photon emission computed tomography
`(SPECT) and positron emission tomography (PET). A
`variety of imaging probes have been developed for
`different molecular targets. Radiolabeled peptides are
`valuable biological tools for tumor receptor imaging
`and targeted radionuclide therapy. This attributed to
`the favorable pharmacokinetics and specific tumor
`targeting characteristics, together with the overe x-
`pression of their receptors on the tumor cells, making
`these peptides attractive agents for imaging and
`therapy [1 -4]. Peptides primarily synthesized in th e
`brain, especially in neurons, are called “neurope p-
`tides” [2, 3]. However, since most of these peptides
`are also found in the gut, lymphatic tissue, endocrine
`system, etc., the terminology “regulatory peptide” is
`often used [3]. Some of the most important regulatory
`peptides and their receptors overexpressed on tumors
`are listed in Table 1. The action of neuropeptides is
`mediated by their binding to specific, me m-
`brane-associated receptors. The majority of these r e-
`ceptors belong to the family of G -protein-coupled
`receptors [2]. These receptors are consisting of a single
`polypeptide chain, with seven transmembrane d o-
`mains, an extracellular domain with the ligand bin d-
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`ing site, and an intracellular domain linked to
`G-proteins and arrestin for the activation of second
`messengers and internalization [5]. These receptors
`represent useful molecular targets for the detection
`and treatment of cancer because they are located on
`the plasma membrane and upon binding of the rad i-
`oligand, the receptor-ligand complex is internalized,
`allowing long retention of radioactivity in tumor cells
`[6].
`
`
`Table 1. Peptide receptor expression patterns. The main
`receptor types overexpressed in human tumor cells are in
`bold.
`Peptide
`
`Receptor
`types/subtypes
`
`Tumor expression
`Somatostatin sst1, sst2, sst3, sst4,
`sst5
`Neuroendocrine tumors (gas-
`troenteropancreatic tumors),
`lymphoma, paraganglioma,
`carcinoids, breast, brain, renal,
`small cell lung cancer, me-
`dullary thyroid cancer
`Bombesin/GRP BB1 (NMB-R), BB2
`(GRP-R),
`BB3, BB4
`Prostate, breast, pancreas,
`gastric, colorectal, small cell
`lung cancer
`VIP VPAC1, VPAC2 Adenocarcinomas of breast,
`prostate, stomach and liver;
`neuroendocrine tumors
`α-M2 α-M2-R Breast cancer
`α-MSH MC1-5R Melanomas
`CCK/gastrin CCK1, CCK2 Medullary thyroid cancer,
`small cell lung cancer, gas-
`trointestinal stromal tumor,
`stromal ovarian cancer, as-
`trocytomas
`Neurotensin NTR1, NTR2, NTR3 Small cell lung cancer, colon,
`exocrine ductal pancreatic
`cancer, Ewing sarcoma, men-
`ingioma, astrocytoma, breast,
`prostate cancer
`LHRH LHRH-R Prostate, breast cancer
`Substance P
`
`NK1, NK2, NK3 Glial tumors (glioblastoma,
`medullary thyroid cancer),
`pancreas, breast, small cell
`lung cancer
`Exendin
`
`GLP-1 Insulinomas, gastrinomas,
`pheochromocytomas, para-
`gangliomas and medullary
`thyroid carcinomas
`RGD αvβ3-integrin Glioma, breast, prostate can-
`cer
`GRP, gastrin-releasing peptide; VIP, vasoactive intestinal peptide;
`α-MSH, α-melanocyte-stimulating hormone; CCK, cholecystokinin;
`LHRH, luteinizing hormone-releasing hormone; GLP, gluca-
`gon-like peptide; RGD, Arg-Gly-Asp.
`
`Peptide-based radiopharmaceuticals were i n-
`troduced into the clinic more than two decades ago [7,
`8]. The first and most successful registered imaging
`agent to date is the somatostatin (SST) analog,
`111In-DTPA0-octreotide ( 111In-OctreoScan, 111In-pente-
`treotide) (Figure 1) [9, 10]. The Food and Drug A d-
`ministration (FDA) approved 111In-DTPA0-octreotide
`is proven to be effective for imaging SST rece p-
`tor-positive lesions, such as neuroendocrine tumors
`(NETs), mammary cancer and small cell lung cancer
`[9-11]. Another important application of radiolabeled
`octreotide and other SST peptide analogs is peptide
`receptor-mediated radionuclide therapy (PRRT)
`[11-14]. The thriving advent of 111In-DTPA0-octreotide
`raised interest in the development of radiolabeled
`peptides to target other tumor-related peptide rece p-
`tor systems [1, 4]. This interest resulted in the disco v-
`ery of bombesin peptide analogs to target
`bombesin/gastrin-releasing peptide receptors, which
`are overexpressed in many common human cancers
`[2, 15-18]. Other radiolabeled peptides, such as the
`analogs of vasoactive intestinal peptide [19-21], ne u-
`rotensin [22-25], cholecystokinin/gastrin [26-29], e x-
`endin [30-33] and RGD (Arg-Gly-Asp) [34-37], have
`been developed and are currently under preclinical or
`clinical evaluation to establish their applicability for
`the diagnosis or treatment of cancers [2, 4].
`Because of rapid progress in the field of radi o-
`labeled peptides it is difficult to cover all aspects pe r-
`taining to the subject. The focus of this article remains
`the chief applications of radiolabeled peptides in
`cancer research and development, with major e m-
`phasis on recent development of small peptides as
`targeting agents in nuclear oncology.
`Characteristics and limitations of small ra-
`diopeptides
`The distinctive advantages of small radiope p-
`tides over other biologically active molecules, such as
`proteins and antibodies, are summarized in Table 2.
`Receptors for peptides are often found in higher de n-
`sity on tumor cells than in normal tissues; hence sp e-
`cifically designed receptor-binding radiolabeled pe p-
`tides could enable efficient visualization of tumors.
`Because of their small size, peptides usually exhibit
`rapid pharmacokinetics, and good tumor targeting
`characteristics, with the ability to penetrate into t u-
`mors efficiently [1-3]. Peptides can easily be synth e-
`sized using conventional peptide synthesizers and the
`desired pharmacokinetic characteristics can be m o-
`lecularly engineered (by making appropriate changes
`in the peptide sequence) during synthesis and/or by
`adding a biomodifying molecule [38, 39]. Automated
`peptide synthesizers are available for parallel synth e-
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`sis procedures allowing the synthesis of peptide l i-
`braries in short time.
`Tumor receptor imaging creates distinct cha l-
`lenges for the design of peptide-based radiopharm a-
`ceuticals and imaging approaches [40]. Most receptors
`have high affinities for their ligands and are active at
`nanomolar concentrations of the ligands. Therefore,
`radiopharmaceutical preparations with high specific
`radioactivity are crucial. Even small molar quantities
`of imaging agents may saturate a receptor, limiting
`the ability to visualize receptor expression and i n-
`creasing the background of nonspecific binding. For
`this reason, molecular imaging of tumor receptors has
`been mainly confined to radionuclide imaging
`(SPECT and PET), with which it is possible to generate
`medical images with micromolar to picomolar co n-
`centrations of imaging probes [40]. It is important to
`use a minimum possible amount of a peptide in h u-
`mans to reduce any adverse pharmacologic effects.
`For instance, it has been shown that VIP is pharm a-
`cologically very potent peptide molecule and doses
`even in the submicrogram range will produce toxic
`effects, including hypertension, bronchospasm and
`diarrhea [4], requiring efficient purification step b e-
`fore administration in order to reduce the admini s-
`tered dose to subpharmacologic levels.
`
`
`Figure 1. Structures of somatostatin-14 and -28 and [DTPA0-D-Phe1]octreotide (OctreoScan).
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`I 2 3 4 5 6
`® -®-~-8-G-8-
`7
`s
`I s
`@-e-®-e-
`14 13 12 11
`Somatostatin-14
`16
`5
`6
`7
`8 Cys ---0---®
`28 27 26
`S omatostatin-28
`8
`9
`20
`21
`22
`23
`24
`25
`I 2
`HOOC~ ~~~ CO-N H-S-
`N N N
`HOO C __/ l ~ COOH COOH
`DTPA 0-D-Pbe1-octreotide (OctreoS can) s
`8 7 6
`
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`Table 2. Requirements of small peptides.
` Small in size
` Easy to synthesize
` Easy to radiolabel
` feasibility of kit formulation
` Amenable to chemical/molecular modifications
` Ability to attach a chelating agent at the C- or N-terminus of the
`peptide
` High receptor binding affinity
` High tumor penetration
` Favorable pharmacokinetics
` Attain a high concentration in the target tissues
` Rapid clearance from the blood and non-target tissues
` Rate and route of excretion can be modified
` Few side effects
` Not immunogenic
` Many biologically important targets
`
`
`For smaller biomolecules, such as peptides the
`radioisotope label may significantly affect binding to
`the receptor and in vivo metabolism. In this situation,
`the choices of radionuclide, labeling position or loc a-
`tion of the radiolabel can be critical [40]. Radiolabeling
`at a specific-site (chelation-site) remote from the
`binding region is important to prevent the loss of
`binding affinity and biological activity of the radi o-
`labeled peptides [1, 3].
`The biologic role of most receptor systems i m-
`portant in cancer is derived from their role in the ti s-
`sue of cancer origin. In general, tumor receptors are
`expressed in the parent cell lineage and have an e s-
`tablished physiologic function. For example, estrogen
`receptor expression is vital to the function of normal
`mammary gland epithelial cells. Many tumor rece p-
`tors also play an important role in promoting carci n-
`ogenesis or tumor growth, as is the case for steroid
`receptors in breast and prostate cancer. The depen d-
`ence on the receptor pathway for tumor growth
`makes the receptor a suitable target for therapy, b e-
`cause interruption of the receptor-initiated signal will
`result in a cessation of tumor growth and often tumor
`cell death. Thus, knowledge of the levels of receptor
`expression, which may vary significantly in different
`types of tumors and even in different sites in the same
`tumor, is required to infer the possibility that rece p-
`tor-directed targeting will be effective [40].
`One major problem associated with unmodified
`linear peptides is their often short biological half-life
`due to rapid proteolysis in plasma. A short half-life in
`blood is a major obstacle for the successful in vivo
`application as radiopharmaceuticals since they may
`be degraded before reaching the intended target.
`Therefore, most peptides have to be modified sy n-
`thetically to minimize rapid enzymatic degradation
`[1, 41]. Great efforts have been focused on developing
`metabolically-stable peptides suitable for clinical use
`by carrying appropriate molecular modifications,
`such as the use of more stable D-amino acids for
`L-amino acids, the use of pseudo-peptide bonds, the
`inclusion of amino alcohols and the insertion of u n-
`natural amino acids or amino acid residues with
`modified side-chains without compromising the r e-
`ceptor binding affinity and biological activity of the
`peptide [1]. It is the specific amino acid sequence of a
`peptide and usually the nature and type of particular
`amino acid side-chains that determine resistance to
`enzymatic degradation. For example, native SST, has
`a plasma half-life of approximately 2-3 min, but its
`molecularly modified synthetic peptide derivative,
`octreotide has a ha lf-life of 1.5-2 h, making it suitable
`for clinical application [40].
`Another issue related to radiolabeled peptides is
`often their high uptake and retention by the kidneys,
`which is of a concern, particularly for radionuclide
`therapy because of the potential nephrotoxicity [14,
`42-44]. Though procedures have been developed and
`applied successfully in the clinics to manage n e-
`phrotoxicity that includes the infusion of a mixture of
`amino acids, such as lysine and arginine [45]; there is
`a need to reduce kidney uptake and/or enhanced
`renal clearance of the radiopeptide. Recently, it has
`been shown that the use of the cytoprotective drug
`amifostine [46], and low doses of the plasma expander
`succinylated gelatin and gelatin-based gelofusine can
`inhibit the renal uptake of radiolabeled octreotide
`analogs [47, 48].
`Development of a peptide-based radio-
`pharmaceutical
`The main steps involved in developing a radi o-
`labeled peptide for clinical application are as follows:
`(i) identification of the molecular target (receptor)
`with relevance to human disease and search for a lead
`peptide, which may be a natural or synthetic peptide,
`(ii) solid-phase peptide synthesis of a peptide or its
`analogs. In general, the design of a peptide is based on
`the structural composition of the endogenous ligand
`(natural peptide ligand), which exhibits high affinity
`for the corresponding receptor system. The natural
`peptide molecule is often structurally modified to
`produce a metabolically stabilized analog, which
`preserves most of the biological activity and receptor
`affinity of the original peptide molecule, (iii) covalent
`attachment of a chelating agent or a prosthetic group
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`to the peptide either directly or through a lin k-
`er/spacer group, (iv) radiolabeling that allow high
`labeling efficiency and high specific activity radi o-
`labeled peptide preparation, (v) in vitro characteriz a-
`tion, such as the binding of a radiopeptide with tumor
`cells, determination of receptor binding affinity, i n-
`ternalization into the tumor cells and dissociation
`from the tumor cells, (vi) in vivo evaluation to assess
`the biological behavior, biokinetics and tumor ta r-
`geting capacity of the radiolabeled peptide in animal
`models. Many aspects should be considered for fu r-
`ther development, such as the accumulation of radi o-
`labeled peptide in target and non-target tissues, the
`rate and extent of the clearance of radioactivity from
`the body, the excretory pathway and in vivo stability
`of the radiolabeled peptide. (vii) The radiolabeled
`peptides, which successfully passed all the preclinical
`tests, after toxicological studies and established rad i-
`opharmaceutical preparation, may enter clinical
`studies in humans [10].
`From the design of a new peptide molecule until
`the use in the clinical settings is a long way and from a
`huge number of developed radiolabeled peptides
`only few meet the criteria of a radiopharmaceutical
`for clinical application. Thus, in the drug develo p-
`ment process, considerable preclinical work and the
`need for validation at each step of development is
`require in order to transfer a drug from the laboratory
`bench to the clinical bedside and ultimately obtaining
`the regulatory approval (see Figure S1/Scheme 1). As
`mentioned above, several characteristics are desirable
`when developing a molecularly engineered peptide to
`be used as a tumor imaging agent: it must bind with
`high affinity to the target receptor that is found pr e-
`dominantly on cancer cells and not on normal tissues,
`be specific for its target and not bind to the nontarget
`tissues, be sufficiently stable to reach the tumor le-
`sions in an intact state, and be cleared rapidly from
`the blood and nontarget tissues in order to minimize
`the background radioactivity.
`
`
`Figure S1. (Scheme 1) Different phases of drug (peptide radiopharmaceutical) development process.
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`-Target identification and validation
`- Selection of targeting molecule ( drug)
`-Chemical synthesis of a drug
`- Radio labeling and characterization
`-Drug efficacy testing
`-In vitro chemical and metabolic stability
`-In vitro tumor cell binding and cellular
`internalization
`-In vivo animal biodistribution and tumor
`targeting characteristics
`- Initial safety, efficacy and phannacokinetic
`studies in volunteers and patients
`- Dose determination
`- Drug efficacy evaluation
`- Drug safety and side effects determination
`-Diagnosis and disease staging of patients
`Preclinical research
`and development phase
`Clinical development phase
`Phase I
`Phase II
`PhaseUl
`Regulatory approval
`(market)
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`Radiolabeling of peptides
`Peptide-based targeted agents either design for
`diagnostic imaging or radionuclide therapy involve
`the use of a radiometal. This requires that the radi o-
`metal be stably attached to the peptide using a b i-
`functional chelating agent (BFCA). An important
`property of BFCA is that it chelates radiometals with
`high in vivo stability, resulting in minimal deposition
`of free radiometal in normal tissues. A wide variety of
`BFCAs and prosthetic groups have been developed in
`recent years, allowing rapid and convenient radi o-
`labeling of peptides with different radionuclides.
`Several clinically relevant radionuclides have been
`used for labeling bioactive peptides either for dia g-
`nostic imaging ( 99mTc, 111In, 68/66Ga, 18F, 123I, 64Cu), or
`for therapy (111In, 64/67Cu, 90Y, 177Lu, 213Bi) are listed in
`Table 3.
`
`
`Table 3. Methods for labeling peptides with different di-
`agnostic and therapeutic radionuclides.
`Radionuclide Half-life BFCA/prosthetic
`group
`Application
` Technetium-99m
`(99mTc)
`6.02 h MAG3, DADT,
`HYNIC
`diagnosis
`Fluorine-18 (18F) 1.83 h SFB diagnosis
`Iodine-123 (123I) 13.2 h SIB, SIPC diagnosis
`Gallium-68 (68Ga) 1.13 h NOTA, DOTA diagnosis
`Copper-64 (64Cu) 12.7 h TETA, DOTA,
`NOTA
`diagnosis/therapy
`Indium-111 (111In) 67.2 h DTPA, DOTA diagnosis/therapy
`Lutetium-177
`(177Lu)
`160.8 h DOTA therapy
`Yttrium-90 (90Y) 64.1 h DOTA therapy
`Bismuth-213
`(213Bi)
`45.6 min DOTA therapy
`BFCA, bifunctional chelating agent; MAG3, mercaptoacetyltri-
`glycine; DADT, diaminedithiol; HYNIC, 2-hydrazinonicotinic acid;
`SFB, N-succinimidyl-4-[18F]fluorobenzoate; TETA,
`1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid; SIB,
`N-succinimidyl-3-iodobenzoate; SIPC,
`N-succinimidyl-5-iodo-3-pyridinecarboxylate; DTPA, diethylene-
`triaminepentaacetic acid; DOTA,
`1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid; NOTA,
`1,4,7-triazacyclononane-1,4,7-triacetic acid.
`
`
`Peptide radiopharmaceuticals for tumor
`imaging
`Tumor receptor imaging poses unique challen g-
`es for the design and development of a peptide-based
`agent. The biological actions of the peptides are m e-
`diated upon binding with high affinity receptors. The
`high overexpression of these receptors on various
`tumor cells as compared to their low density in no r-
`mal tissues has provided the molecular basis for the
`clinical use of radiolabeled peptides as tumor receptor
`imaging and therapeutic agents [40]. After intrav e-
`nous injection, the radiolabeled peptide will extr a-
`vascate and bind to sites with high receptor density,
`e.g. tumor. Imaging and/or therapy follows depen d-
`ing on the radionuclide used. In recent years, many
`radiolabeled peptide analogs, such as somatostatin,
`bombesin, vasoactive intestinal peptide, cholecyst o-
`kinin/gastrin, neurotensin, exendin and RGD deriv a-
`tives, have been developed for scintigraphic detection
`of different tumor types [1-4]. These are described
`below.
`Somatostatin peptide analogs
`Somatostatin (SST) is a naturally occurring cyclic
`disulphide-containing peptide with either 14 or 28
`amino acids (Figure 1), which binds to SST receptors.
`Both natural SST-14 and SST-28 bind with high affi n-
`ity to five different receptor subtypes (sst15), but have
`a short plasma half-life ( 3 min), owing to rapid e n-
`zymatic degradation by endogenous peptidases,
`preventing their in vivo use [9, 49-51]. Several sy n-
`thetic SST peptide analogs that are more resistant to
`enzymatic degradation have been prepared by m o-
`lecular modifications preserving most of the biolog i-
`cal activity of the original SST peptide [9, 42, 49, 50].
`Introduction of the D-amino acids, and decreasing the
`ring size to the bioactive core sequence, resulted in an
`8-amino acid- containing SST analog, “octreotide”
`(Figures 1 and 2), that preserves the 4-amino acid
`motif (Phe- D-Trp-Lys-Thr) of native SST-14 involved
`in receptor binding and has a significantly longer
`plasma half-life as compared to endogenous SST.
`Other SST receptor-targeting analogs developed i n-
`clude lanreotide and vapreotide (RC-160) (Figure 2),
`all with enhanced metabolic stability [9, 50]. These
`analogs preserved the cyclic form via a disulfide
`bond. These stabilized SST peptide analogs have di f-
`ferent binding profiles for SST receptor subtypes, but
`all show high affinity for sst 2, which is the most
`prominent SST receptor on human tumors [2, 52, 53].
`Somatostatin receptors have been identified on
`the surface of many NETs and represent a valid target
`for in vivo tumor imaging [2, 55]. SPECT and
`PET-based agents have been applied successfully to
`image and quantify the uptake of receptor-specific
`radiopeptides in SST receptor-positive tumors for
`cancer staging, treatment planning as well as fo l-
`low-up response to therapy. Also, SST receptor bin d-
`ing peptides allows the detection of small metastatic
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`sites, which underline the importance and strength of
`molecular imaging in nuclear oncology [55]. In add i-
`tion to its usefulness as a diagnostic imaging agent,
`non-radioactive SST peptide analogs have been used
`for symptomatic treatment of hormone-secreting
`NETs [2, 11, 42]. It has been shown that SST peptide
`analogs reduce the symptoms associated with excess
`hormone secretion and may also have direct ant i-
`tumor effects [11, 40]. Several SST-derived peptides
`labeled with radiohalogens, such as 18F via prosthetic
`groups, or linked to the chelating agents to facilitate
`labeling with radiometals, such as 99mTc, 111In, 64Cu
`and 67/68Ga have been used for in vivo imaging of SST
`receptor-expressing tumors. When labeled with
`β-emitters ( 90Y or 177Lu), these SST peptide analogs
`can be utilized for receptor-mediated radionuclide
`therapy [1, 11-14]. A few of SST peptide analogs are
`summarized in Table 4.
`
`Figure 2. Structures of DOTA-coupled somatostatin analogs.
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`I
`HOOe~ ~ ~ eO -NH-s -
`[DOT AO, D-Phe1 Joctreotide C
`N N=
`(DOTA-OC)
`N N ~
`HOOe _/ \__J "'------eOOH ~
`8 7
`I 2
`HOOC~ ~ ~ eO -NH-s-
`[DOTA 0,D-Phe1,Tyr3Joctreotide [N NJ
`(DOTA-TOC)
`N N ~
`Hooe_/ \__J "'------eOO H ~
`8 7
`1 2
`HOOe~ ~ ~ eO-NH-9-
`[DOTA 0,D-J3Nal1]1anreotide [N NJ
`(DOT A-IAN)
`N N ~
`Hoo e_/ \__J "'------eOOH ~
`8 7
`l 2
`HOOe~ ~ ~ eO -NH-s-
`[DOTA 0,D-Phe1,Tyr3Jvapreotide [N NI
`(DOTA-VAP) (RC-160) )
`N N
`H OOe _/ \__J "'------eOOH
`8 7
`I 2
`HOOe~ ~ ~ CO -NH -s-
`[DOTA 0,D-Phe1,Tyr3)octreotate [N NJ
`(DOTA-TATE)
`N N ~
`Hooe _/ \__J "'------eOO H ~
`8 7
`6
`3
`6
`6
`6
`6
`
`
`
`
`
`
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`Table 4. Radiolabeled somatostatin analogs under preclinical/clinical evaluation.
`Peptide Major application Reference
`Gluc/Cel-S- Dpr([18F]FBOA)TOCA  Targeting of sstr-positive AR42J tumor in mice [114]
`64Cu-CB-TE2A-Y3-TATE  microPET imaging of sstr-positive AR42J tumor in rats [115]
`(DOTA0),1-Nal3,Thr8]-octreotide
`(111In-DOTA-NOC-ATE)/
`[DOTA0,BzThi3,Thr8]-octreotide
`(111In-DOTA-BOC-ATE)
` Targeting of sstr-expressing tumors in AR4-2J tumor- bearing rats [116]
`99mTc-EDDA/HYNIC-TOC
`(99mTc-TOC)
` Diagnostic imaging of sstr-positive tumors in patients [117]
`99mTc-EDDA/HYNIC-octreotate - Imaging of sstr-expressing carcinoid tumors in patients [118]
`Nα-(1-deoxy-D-fructosyl)-N-(2-[18F]fluoropropionyl)-Lys0-
`Tyr3-octreotate
`(Glu-Lys([18F] FP)-TOCA)
` PET imaging of sstr-expressing tumors in patients [119]
`[18F]FP-Glu-TOCA  PET imaging of sstr-positive tumors in patient with
` metastatic carcinoid in the liver
`[120]
`68Ga-DOTA-D-Phe1-Tyr3-octreotide
` (68Ga-DOTA-TOC)
` PET imaging of sstr-positive meningioma tumors in
` Patients
`[121]
`[DOTA]-1-Nal3-octreotide
`(68Ga-DOTA-NOC)
` PET imaging of sstr (subtypes 2 and 5) expressing tumors in pa-
`tients
`[122]
`111In-DOTA-TATE  Imaging of sstr-positive tumors in patients [123]
`[111In-DOTA]-lanreotide
`(111In-DOTA-LAN)
` Detection of sstr-expressing tumors in patients [124]
`Maltotriose-[123I] Tyr3-octreotate ([123I]Mtr-TOCA)  Scintigraphic detection of sstr-positive tumors in
` patients
`[125]
`sstr, somatostatin receptor; Glu, glucose; Cel, cellobiose; CB-TE2A, cross-bridged-tetraazamacrocycle
`4,11-bis(carboxymethyl)-1,4,8,11-tetraazabicyclo [6.6.2]hexadecane; BzThi, benzothienyl-Ala. 99mTc-TOC,
`99mTc-hydrazinonicotinyl-Tyr3-octreotide; EDDA, ethylenediamine-N,N'-diacetic acid; [18F]FP-Gluc-TOCA,
`N-(1-deoxy-D-fructosyl)-Nε-(2-[18F]fluoropropionyl)-Lys0-Tyr3-octreotate.
`
`There are a number of factors that usually d e-
`termine the tumor uptake capacity of radiolabeled
`SST peptide analogs. These include: (i) high specific
`radioactivity preparation of the radioligand, (ii) the in
`vivo stability of the radioligand, (iii) the density of SST
`receptor-expression in the tumor, (iv) the
`type/subtype of SST receptors expression by the t u-
`mor, (v) the receptor binding affinity of the radi o-
`labeled SST analog for the particular SST receptor
`type, (vi) the efficiency of SST receptor-mediated i n-
`ternalization and recycling of radiopeptide, (vii) the
`final trapping of the radiolabeled SST peptide analog
`within tumor cells, and (viii) the amount of the pe p-
`tide administered [5, 11, 54].
`It is worth mentioning here that common to all
`receptors is the interaction of a ligand and the rece p-
`tor, in which specific binding of the ligand to the r e-
`ceptor results in downstream biochemical or physi o-
`logic changes. Ligands that cause physiological
`changes with receptor binding, typically the naturally
`occurring ligands, are called “agonists”. Ligands that
`bind to the receptor and block the binding of agonists
`but that do not activate changes are known as “a n-
`tagonists” [40]. It is generally believed that receptor
`agonist radioligands are more suited for tumor ta r-
`geting as they exhibit good receptor-mediated inte r-
`nalization into the tumor cell upon binding to the
`respective cell-surface receptors, thus promoting a c-
`tive accumulation in the target results in optimal vi s-
`ualization [3]. Interestingly, however, a recent study
`performed with two potent SST receptor-selective
`antagonists demonstrated that the high-affinity SST
`receptor antagonists that poorly internalized into t u-
`mor cells, can also be effective in terms of in vivo tu-
`mor uptake characteristics in animal models as co m-
`pared to the corresponding agonists, which highly
`internalized into tumor cells. This observation which
`was made both for sst 2 and sst 3-selective SST peptide
`analogs, demonstrates that the SST receptor antag o-
`nists are preferable to SST receptor agonists for in vivo
`tumor targeting [56]. In another recent study,
`64Cu-CB-TE2A-sst2-ANT, a SST antagonist was eva l-
`uated for in vivo PET imaging of sst 2-positive tumors
`and compared to 64Cu-CB-TE2A-TATE [57]. The
`pharmacokinetic characteristics indicated the slight
`superiority of the radioantagonists over receptor a g-
`onists.
`The most recent study on the newly developed
`Petitioner GE Healthcare – Ex. 1032, p. 488
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`Theranostics 2012, 2(5)
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`h
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`489
`sst2-antagonist, namely LM3, demonstrated high and
`persistent tumor uptake of radiolabeled SST antag o-
`nists. Also, profound influence of the chelator and the
`radiometal was observed on the receptor binding a f-
`finity of the radiolabeled conjugates [58]. The first
`clinical study of SST antagonists confirmed the pr e-
`clinical data as it showed higher tumor uptake of the
`antagonist 111In-DOTA-sst2-ANT compared to the
`agonist 111In-DTPA0-octreotide and improved t u-
`mor-to-background contrast, in particular t u-
`mor-to-kidney [59].
`Bombesin peptide analogs
`Bombesin (BN), an amphibian 14-amino acid
`peptide (Figure 3), is a homolog of the 27-amino acid
`mammalian gastrin-releasing peptide (GRP) (Figure
`3). Bombesin and GRP share a highly conserved
`7-amino acid C-terminal sequence
`(Trp-Ala-Val-Gly-His-Leu-Met-NH2), which is r e-
`quired for immunogenicity and for high-affinity
`binding to the BN/GRP-preferring receptor [1, 2, 60].
`Both BN and GRP show high affinity binding to the
`human GRP receptor, which is overexpressed by a
`variety of cancers, including prostate, breast, gastr o-
`intestinal and small cell lung cancer [2, 15, 16]. The BN
`receptor family comprises four subtypes of
`G-protein-coupled receptors, including the neurom e-
`din B (NMB) receptor (BB1), the GRP receptor (BB2),
`the orphan receptor (BB3), and the amphibian rece p-
`tor (BB4) [2, 15, 16]. Following activation of these r e-
`ceptors, BN and GRP possess a variety of physiolog i-
`cal and pharmacological functions and also play an
`important role in stimulating the growth of different
`types of cancers [2]. Of the four receptors, three r e-
`ceptor subtypes (BB1, BB2, and BB3) have shown to be
`expressed in variable degrees on various cancers. Of
`particular interest is the GRP receptor (BB2) that has
`been found to be overexpressed in a variety of tumors,
`including lung, breast, GIST, brain and prostate [2, 15,
`16], and hence is an attractive target for the detection
`and treatment of these cancers. Radiolabeled BN-like
`peptides, which bind to BN/GRP receptors with high
`affinity and specificity, have potential to be used for
`site-directed diagnostic and/or therapeutic purposes
`[1, 3, 4]. A recent study with 68Ga-labeled
`Pan-bombesin analog, DOTA-PEG 2-[D-Tyr6, β -Ala11,
`Thi13, Nle 14] BN(6-14)amide ( 68Ga-BZH3), was pe r-
`formed on patients with gastrointestinal stromal t u-
`mors in order to determine the impact of peptide r e-
`ceptor scintigraphy on the diagnosis and the potential
`therapy [17]. Also in vivo kinetics of the BN peptide
`was compared with 18F-fluorodeoxyglucose (18F-FDG)
`on the same patients. The study demonstrates that
`68Ga-BZH3 showed poor tumor targeting potential as
`compared to 18F-FDG. Further studies on larger
`number of patients are required to determine the real
`potential of 68Ga-BZH3 for targeting BN rece p-
`tor-expressi