Theranostics 2012, 2(5)
`
` e
`
`Ivyspring
`
`International Publisher
`
`481
`
`TThheerraannoossttiiccss
`
`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 overex-
`pression of their receptors on the tumor cells, making
`these peptides attractive agents for imaging and
`
`therapy [1-4]. Peptides primarily synthesized in the
`brain, especially in neurons, are called “neuropep-
`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, mem-
`brane-associated receptors. The majority of these re-
`ceptors belong to the family of G-protein-coupled
`receptors [2]. These receptors are consisting of a single
`polypeptide chain, with seven transmembrane do-
`mains, an extracellular domain with the ligand bind-
`
`http://www.thno.org
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`Theranostics 2012, 2(5)
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`482
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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 radi-
`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
`
`
`Somatostatin
`
`Receptor
`types/subtypes
`
`
`sst1, sst2, sst3, sst4,
`sst5
`
`Bombesin/GRP BB1 (NMB-R), BB2
`(GRP-R),
`BB3, BB4
`
`Tumor expression
`
`Neuroendocrine tumors (gas-
`troenteropancreatic tumors),
`lymphoma, paraganglioma,
`carcinoids, breast, brain, renal,
`small cell lung cancer, me-
`dullary thyroid cancer
`Prostate, breast, pancreas,
`gastric, colorectal, small cell
`lung cancer
`
`VIP
`
`VPAC1, VPAC2
`
`α-M2
`
`α-MSH
`
`α-M2-R
`
`MC1-5R
`
`CCK/gastrin CCK1, CCK2
`
`Adenocarcinomas of breast,
`prostate, stomach and liver;
`neuroendocrine tumors
`Breast cancer
`Melanomas
`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
`Prostate, breast cancer
`Glial tumors (glioblastoma,
`medullary thyroid cancer),
`pancreas, breast, small cell
`lung cancer
`Insulinomas, gastrinomas,
`pheochromocytomas, para-
`gangliomas and medullary
`thyroid carcinomas
`
`LHRH
`
`LHRH-R
`
`Substance P
`
`
`NK1, NK2, NK3
`
`Exendin
`
`
`GLP-1
`
`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 in-
`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 Ad-
`ministration (FDA) approved 111In-DTPA0-octreotide
`is proven to be effective for imaging SST recep-
`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 recep-
`tor systems [1, 4]. This interest resulted in the discov-
`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], neu-
`rotensin [22-25], cholecystokinin/gastrin [26-29], ex-
`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 radio-
`labeled peptides it is difficult to cover all aspects per-
`taining to the subject. The focus of this article remains
`the chief applications of radiolabeled peptides in
`cancer research and development, with major em-
`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 radiopep-
`tides over other biologically active molecules, such as
`proteins and antibodies, are summarized in Table 2.
`Receptors for peptides are often found in higher den-
`sity on tumor cells than in normal tissues; hence spe-
`cifically designed receptor-binding radiolabeled pep-
`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 tu-
`mors efficiently [1-3]. Peptides can easily be synthe-
`sized using conventional peptide synthesizers and the
`desired pharmacokinetic characteristics can be mo-
`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 synthe-
`
`
`http://www.thno.org
`
`Petitioner GE Healthcare – Ex. 1032, p. 482
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`Theranostics 2012, 2(5)
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`483
`
`sis procedures allowing the synthesis of peptide li-
`braries in short time.
`Tumor receptor imaging creates distinct chal-
`lenges for the design of peptide-based radiopharma-
`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 in-
`creasing the background of nonspecific binding. For
`this reason, molecular imaging of tumor receptors has
`
`
`imaging
`to radionuclide
`been mainly confined
`(SPECT and PET), with which it is possible to generate
`medical images with micromolar to picomolar con-
`centrations of imaging probes [40]. It is important to
`use a minimum possible amount of a peptide in hu-
`mans to reduce any adverse pharmacologic effects.
`For instance, it has been shown that VIP is pharma-
`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 be-
`fore administration in order to reduce the adminis-
`tered dose to subpharmacologic levels.
`
`I
`
`2
`
`3
`
`4
`
`5
`
`6
`
`® -®-~-8-G-8- 7
`
`s
`I
`s
`
`@-e-®-e-
`
`14
`
`13
`
`12
`
`11
`
`Somatostati n-14
`16
`
`8
`
`9
`
`20
`
`21
`
`22
`
`23
`
`24
`
`25
`
`Cys ---0---®
`
`27
`
`26
`
`28
`S omatostatin-28
`
`5
`
`6
`
`7
`
`8
`
`I
`
`N
`
`N
`
`N
`
`HOOC~ ~~~ CO-NH -S -
`l ~ COOH
`DTPA0-D-Pbe1-octreotide (OctreoS can) s 8
`
`HOOC __ /
`
`COOH
`
`Figure 1. Structures of somatostatin-14 and -28 and [DTPA0-D-Phe1]octreotide (OctreoScan).
`
`2
`
`7
`
`6
`
`
`
`
`http://www.thno.org
`
`Petitioner GE Healthcare – Ex. 1032, p. 483
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`Theranostics 2012, 2(5)
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`484
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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 loca-
`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 radio-
`labeled peptides [1, 3].
`The biologic role of most receptor systems im-
`portant in cancer is derived from their role in the tis-
`sue of cancer origin. In general, tumor receptors are
`expressed in the parent cell lineage and have an es-
`tablished physiologic function. For example, estrogen
`receptor expression is vital to the function of normal
`mammary gland epithelial cells. Many tumor recep-
`tors also play an important role in promoting carcin-
`ogenesis or tumor growth, as is the case for steroid
`receptors in breast and prostate cancer. The depend-
`ence on the receptor pathway for tumor growth
`makes the receptor a suitable target for therapy, be-
`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 recep-
`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 syn-
`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 un-
`natural amino acids or amino acid residues with
`modified side-chains without compromising the re-
`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 half-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 ne-
`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 radio-
`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
`
`
`http://www.thno.org
`
`Petitioner GE Healthcare – Ex. 1032, p. 484
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`Theranostics 2012, 2(5)
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`485
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`to the peptide either directly or through a link-
`er/spacer group, (iv) radiolabeling that allow high
`labeling efficiency and high specific activity radio-
`labeled peptide preparation, (v) in vitro characteriza-
`tion, such as the binding of a radiopeptide with tumor
`cells, determination of receptor binding affinity, in-
`ternalization into the tumor cells and dissociation
`from the tumor cells, (vi) in vivo evaluation to assess
`the biological behavior, biokinetics and tumor tar-
`geting capacity of the radiolabeled peptide in animal
`models. Many aspects should be considered for fur-
`ther development, such as the accumulation of radio-
`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 radi-
`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 develop-
`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 pre-
`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.
`
`- Target identification and validat ion
`- 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
`
`- In itial 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)
`
`
`
`Figure S1. (Scheme 1) Different phases of drug (peptide radiopharmaceutical) development process.
`
`
`http://www.thno.org
`
`Petitioner GE Healthcare – Ex. 1032, p. 485
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`Theranostics 2012, 2(5)
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`486
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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 radio-
`metal be stably attached to the peptide using a bi-
`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 radio-
`labeling of peptides with different radionuclides.
`Several clinically relevant radionuclides have been
`used for labeling bioactive peptides either for diag-
`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
`
`diated upon binding with high affinity receptors. The
`high overexpression of these receptors on various
`tumor cells as compared to their low density in nor-
`mal tissues has provided the molecular basis for the
`clinical use of radiolabeled peptides as tumor receptor
`imaging and therapeutic agents [40]. After intrave-
`nous injection, the radiolabeled peptide will extra-
`vascate and bind to sites with high receptor density,
`e.g. tumor. Imaging and/or therapy follows depend-
`ing on the radionuclide used. In recent years, many
`radiolabeled peptide analogs, such as somatostatin,
`bombesin, vasoactive intestinal peptide, cholecysto-
`kinin/gastrin, neurotensin, exendin and RGD deriva-
`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 affin-
`ity to five different receptor subtypes (sst15), but have
`a short plasma half-life (3 min), owing to rapid en-
`zymatic degradation by endogenous peptidases,
`preventing their in vivo use [9, 49-51]. Several syn-
`thetic SST peptide analogs that are more resistant to
`enzymatic degradation have been prepared by mo-
`lecular modifications preserving most of the biologi-
`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 in-
`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 dif-
`ferent binding profiles for SST receptor subtypes, but
`all show high affinity for sst2, 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 fol-
`low-up response to therapy. Also, SST receptor bind-
`ing peptides allows the detection of small metastatic
`
`
`http://www.thno.org
`
`Fluorine-18 (18F)
`
`1.83 h
`
` Technetium-99m
`(99mTc)
`
`6.02 h
`
` MAG3, DADT,
`HYNIC
`SFB
`SIB, SIPC
`13.2 h
`Iodine-123 (123I)
`Gallium-68 (68Ga) 1.13 h NOTA, DOTA
`Copper-64 (64Cu) 12.7 h
`TETA, DOTA,
`NOTA
`Indium-111 (111In) 67.2 h DTPA, DOTA
`160.8 h DOTA
`Lutetium-177
`(177Lu)
`
`diagnosis
`
`diagnosis
`
`diagnosis
`
`diagnosis
`
`diagnosis/therapy
`
`diagnosis/therapy
`
`therapy
`
`therapy
`
`therapy
`
`Yttrium-90 (90Y)
`
`64.1 h DOTA
`
`Bismuth-213
`(213Bi)
`
`45.6 min DOTA
`
`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 challeng-
`es for the design and development of a peptide-based
`agent. The biological actions of the peptides are me-
`
`Petitioner GE Healthcare – Ex. 1032, p. 486
`
`

`

`Theranostics 2012, 2(5)
`
`487
`
`sites, which underline the importance and strength of
`molecular imaging in nuclear oncology [55]. In addi-
`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 anti-
`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.
`I
`
`HOOe~ ~ ~ eO-NH - s-
`
`[DOT AO, D-Phe1 Joctreotide CN
`
`(DOTA-OC)
`
`N=
`
`N ~
`N
`HOOe _ / \__J "'------ eOOH ~
`8
`7
`2
`
`I
`
`HOOC~ ~ ~ eO-NH - s -
`
`[DOTA0,D-Phe1,Tyr3Joctreotide [N NJ
`
`(DOTA-TOC)
`
`N ~
`N
`Hooe_/ \__J "'------ eOO H ~
`8
`7
`1
`2
`HOOe~ ~ ~ eO-NH-9-
`
`[DOTA0,D-J3Nal1]1anreotide [N
`
`(DOT A-IAN)
`
`NJ
`
`6
`
`3
`
`6
`
`N ~
`N
`Hooe_ / \__J "'------ eOOH ~
`8
`7
`
`HOOe~ ~ ~ eO-NH - s -
`
`l
`
`6
`
`2
`
`[DOTA0,D-Phe1,Tyr3Jvapreotide [N
`
`N I
`
`(DOTA-VAP) (RC-160)
`)
`N
`N
`H OOe _ / \__J "'------ eOOH
`
`8
`
`HOOe~ ~ ~ CO-NH- s -
`
`I
`
`[DOTA0,D-Phe1,Tyr3)octreotate [N NJ
`
`(DOTA-TATE)
`
`6
`
`7
`
`2
`
`N ~
`N
`Hooe _ / \__J "'------ eOOH ~
`8
`7
`
`6
`
`
`
`Figure 2. Structures of DOTA-coupled somatostatin analogs.
`
`
`http://www.thno.org
`
`Petitioner GE Healthcare – Ex. 1032, p. 487
`
`

`

`Theranostics 2012, 2(5)
`
`Table 4. Radiolabeled somatostatin analogs under preclinical/clinical evaluation.
`
`488
`
`Reference
`
`[114]
`
`Peptide
`Gluc/Cel-S- Dpr([18F]FBOA)TOCA
`
`64Cu-CB-TE2A-Y3-TATE
`(DOTA0),1-Nal3,Thr8]-octreotide
`(111In-DOTA-NOC-ATE)/
`[DOTA0,BzThi3,Thr8]-octreotide
`(111In-DOTA-BOC-ATE)
`
`99mTc-EDDA/HYNIC-TOC
`(99mTc-TOC)
`
`99mTc-EDDA/HYNIC-octreotate
`
`Nα-(1-deoxy-D-fructosyl)-N-(2-[18F]fluoropropionyl)-Lys0-
`Tyr3-octreotate
`(Glu-Lys([18F] FP)-TOCA)
`[18F]FP-Glu-TOCA
`
`68Ga-DOTA-D-Phe1-Tyr3-octreotide
` (68Ga-DOTA-TOC)
`
`[DOTA]-1-Nal3-octreotide
`(68Ga-DOTA-NOC)
`111In-DOTA-TATE
`
`[111In-DOTA]-lanreotide
`(111In-DOTA-LAN)
`Maltotriose-[123I] Tyr3-octreotate ([123I]Mtr-TOCA)
`
`Major application
`
` Targeting of sstr-positive AR42J tumor in mice
`
` microPET imaging of sstr-positive AR42J tumor in rats
`[115]
` Targeting of sstr-expressing tumors in AR4-2J tumor- bearing rats [116]
`
` Diagnostic imaging of sstr-positive tumors in patients
`
`- Imaging of sstr-expressing carcinoid tumors in patients
` PET imaging of sstr-expressing tumors in patients
`
` PET imaging of sstr-positive tumors in patient with
` metastatic carcinoid in the liver
` PET imaging of sstr-positive meningioma tumors in
` Patients
`
` PET imaging of sstr (subtypes 2 and 5) expressing tumors in pa-
`tients
`
` Imaging of sstr-positive tumors in patients
` Detection of sstr-expressing tumors in patients
`
` Scintigraphic detection of sstr-positive tumors in
` patients
`
`[117]
`
`[118]
`
`[119]
`
`[120]
`
`[121]
`
`[122]
`
`[123]
`
`[124]
`
`[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 de-
`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 tu-
`mor, (v) the receptor binding affinity of the radio-
`labeled SST analog for the particular SST receptor
`type, (vi) the efficiency of SST receptor-mediated in-
`ternalization and recycling of radiopeptide, (vii) the
`final trapping of the radiolabeled SST peptide analog
`within tumor cells, and (viii) the amount of the pep-
`tide administered [5, 11, 54].
`It is worth mentioning here that common to all
`receptors is the interaction of a ligand and the recep-
`tor, in which specific binding of the ligand to the re-
`ceptor results in downstream biochemical or physio-
`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 “an-
`tagonists” [40]. It is generally believed that receptor
`
`agonist radioligands are more suited for tumor tar-
`geting as they exhibit good receptor-mediated inter-
`nalization into the tumor cell upon binding to the
`respective cell-surface receptors, thus promoting ac-
`tive accumulation in the target results in optimal vis-
`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 tu-
`mor cells, can also be effective in terms of in vivo tu-
`mor uptake characteristics in animal models as com-
`pared to the corresponding agonists, which highly
`internalized into tumor cells. This observation which
`was made both for sst2 and sst3-selective SST peptide
`analogs, demonstrates that the SST receptor antago-
`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 eval-
`uated for in vivo PET imaging of sst2-positive tumors
`and compared to 64Cu-CB-TE2A-TATE [57]. The
`pharmacokinetic characteristics indicated the slight
`superiority of the radioantagonists over receptor ag-
`onists.
`The most recent study on the newly developed
`
`
`http://www.thno.org
`
`Petitioner GE Healthcare – Ex. 1032, p. 488
`
`

`

`Theranostics 2012, 2(5)
`
`489
`
`sst2-antagonist, namely LM3, demonstrated high and
`persistent tumor uptake of radiolabeled SST antago-
`nists. Also, profound influence of the chelator and the
`radiometal was observed on the receptor binding af-
`finity of the radiolabeled conjugates [58]. The first
`clinical study of SST antagonists confirmed the pre-
`clinical data as it showed higher tumor uptake of the
`antagonist 111In-DOTA-sst2-ANT compared to the
`agonist 111In-DTPA0-octreotide and improved tu-
`mor-to-background
`contrast,
`in particular
`tu-
`mor-to-kidney [59].
`
`Bombesin peptide analogs
`
`ceptors, BN and GRP possess a variety of physiologi-
`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 re-
`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-PEG2-[D-Tyr6, β-Ala11,
`Thi13, Nle14] BN(6-14)amide (68Ga-BZH3), was per-
`formed on patients with gastrointestinal stromal tu-
`mors in order to determine the impact of peptide re-
`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 recep-
`tor-expressing tumors.
`
`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 re-
`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, gastro-
`intestinal and small cell lung cancer [2, 15, 16]. The BN
`receptor
`family
`comprises
`four
`subtypes of
`G-pro