Theranostics 2011, 1
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`58
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`
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`TThheerraannoossttiiccss
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`2011; 1:58-82
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`Review
`
`Radiolabeled Cyclic RGD Peptides as Radiotracers for Imaging Tumors and
`Thrombosis by SPECT
`
`Yang Zhou, Sudipta Chakraborty and Shuang Liu 
`
`School of Health Sciences, Purdue University, West Lafayette, IN 47907, USA
`
` Corresponding author: Dr. Shuang Liu, School of Health Sciences, Purdue University, 550 Stadium Mall Drive, West
`Lafayette, IN 47907, USA; Tel: 765-494-0236; E-mail: liu100@purdue.edu
`
`© 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.
`
`Published: 2011.01.18
`
`Abstract
`
`The integrin family is a group of transmembrane glycoprotein comprised of 19 - and 8 -subunits
`that are expressed in 25 different / heterodimeric combinations on the cell surface. Integrins
`play critical roles in many physiological processes, including cell attachment, proliferation, bone
`remodeling, and wound healing. Integrins also contribute to pathological events such as throm-
`bosis, atherosclerosis, tumor invasion, angiogenesis and metastasis, infection by pathogenic mi-
`croorganisms, and immune dysfunction. Among 25 members of the integrin family, the v3 is
`studied most extensively for its role of tumor growth, progression and angiogenesis. In contrast,
`the IIb3 is expressed exclusively on platelets, facilitates the intercellular bidirectional signaling
`(“inside-out” and “outside-in”) and allows the aggregation of platelets during vascular injury. The
`IIb3 plays an important role in thrombosis by its activation and binding to fibrinogen especially in
`arterial thrombosis due to the high blood flow rate. In the resting state, the IIb3 on platelets does
`not bind to fibrinogen; on activation, the conformation of platelet is altered and the binding sites of
`IIb3 are exposed for fibrinogen to crosslink platelets. Over the last two decades, integrins have
`been proposed as the molecular targets for diagnosis and therapy of cancer, thrombosis and other
`diseases. Several excellent review articles have appeared recently to cover a broad range of topics
`related to the integrin-targeted radiotracers and their nuclear medicine applications in tumor
`imaging by single photon emission computed tomography (SPECT) or a positron-emitting radi-
`onuclide for positron emission tomography (PET). This review will focus on recent developments
`of v3-targeted radiotracers for imaging tumors and the use of IIb3-targeted radiotracers for
`thrombosis imaging, and discuss different approaches to maximize the targeting capability of cyclic
`RGD peptides and improve the radiotracer excretion kinetics from non-cancerous organs. Im-
`provement of target uptake and target-to-background ratios is critically important for tar-
`get-specific radiotracers.
`
`Key words: Integrin αvβ3; Integrin αIIbβ3; cyclic RGD peptides; tumor; thrombosis; SPECT.
`
`1. INTRODUCTION
`
`Radiopharmaceuticals, which are also called ra-
`diotracers, are drugs containing a radionuclide. Ra-
`diotracers are used routinely in nuclear medicine for
`diagnosis or therapy of diseases, such as cancer, in-
`flammation and myocardial infarction [1-6]. Radio-
`
`tracers can be classified according to the biodistribu-
`tion characteristics: those whose biodistribution is
`determined exclusively by their chemical and physi-
`cal properties; and those whose biological properties
`are determined by the receptor binding capability of
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`Theranostics 2011, 1
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`radiolabeled biomolecules. The latter class is often
`called target-specific radiotracers [3, 4]. Diagnostic
`radiotracers are molecules labeled with either a
`-emitting isotope for single photon emission com-
`puted tomography (SPECT) or a positron-emitting
`radionuclide
`for positron emission
`tomography
`(PET), and provide a method of assessing the disease
`or disease states by SPECT or PET. They are also
`useful for monitoring the treatment efficacy of a spe-
`cific therapeutic regimen in a noninvasive fashion.
`
`
`
`
`
`
`Figure 1. Schematic presentation of the target-specific
`radiotracer. Radionuclide is the radiation source. BM is the
`targeting biomolecule for receptor binding. A multidentate
`bifunctional chelator is used for chelation of metallic radi-
`onuclides. A spacer is used to bridge the radiometal chelate
`and targeting biomolecule.
`
`
`
`Fig. 1 shows the schematic illustration of the
`target-specific radiotracers, which are often radio-
`metal complexes of a chelator-biomolecule conjugate.
`In some cases, they can be biomolecules attached with
`a non-metallic radionuclide, such as 18F and 123I. A
`target-specific radiotracer is based on the receptor
`binding of the radiolabeled receptor ligand in the
`diseased tissue [7-20]. The metal-containing tar-
`get-specific radiotracer can be divided into four parts:
`targeting biomolecule (BM), spacer, bifunctional che-
`lating agent (BFC), and radionuclide. The targeting
`biomolecule serves as a ―carrier‖ for target-specific
`delivery of radionuclide to the diseased tissue with
`many targeted receptors. The radiolabeled receptor
`ligand binds to these receptors with high affinity and
`specificity, resulting in selective uptake of the radio-
`tracer. The choice of a radionuclide depends on the
`clinical utility of the radiotracer. Table 1 lists several
`selected radionuclides useful for planar imaging and
`SPECT, along with their nuclear characteristics. For
`SPECT, more than 80% of radiotracers used in nuclear
`medicine departments are 99mTc compounds mainly
`due to the optimal nuclear properties of 99mTc and its
`easy availability at low cost [1-5]. The 6 h half-life is
`long enough to allow radiopharmacists to carry out
`radiosynthesis and for physicians to collect clinically
`
`useful images. It is also short enough to permit ad-
`ministration of 20 – 30 mCi of 99mTc radiotracer
`without imposing a significant radiation dose to the
`patient. 111In is also widely used in gamma scintigra-
`phy (only second to 99mTc in clinical applications). It
`decays by electron capture and emits two -photons of
`173 and 247 keV (90% and 94% abundance, respec-
`tively). 111In radiotracers are often used as the imaging
`surrogates for biodistribution and dosimetry deter-
`mination of their corresponding therapeutic 90Y ana-
`logs, which might be useful for treatment of cancer.
`67Ga is a cyclotron-produced radionuclide, and has a
`half-life of 78 h. 67Ga has little use in the development
`of target-specific radiotracers since 68Ga radiotracers
`offer significant advantages because of the high spa-
`tial resolution of PET as compared to that of SPECT.
`Due to the low solution stability of 201Tl(I) complexes,
`201Tl is used exclusively as its chloride salt for myo-
`cardial perfusion imaging in the patients with cardi-
`ovascular diseases.
`
`
`
`Table 1. Selected radionuclides for SPECT.
`
`Radionuclide Half-life Mode of
`decay
`
`99mTc
`
`6.01 h
`
`
`
`Principal  emis-
`sion in keV (%
`abundance)
`140.5 (87.2)
`
`123I
`
`131I
`
`67Ga
`
`111In
`
`201Tl
`
`13.27 h
`
`EC
`
`159.0 (83.3)
`
`8.02 d
`
`- & 
`
`364.5 (81.2)
`
`3.261 d
`
`2.805 d
`
`3.038 d
`
`EC
`
`EC
`
`EC
`
`93.3 (37.0), 184.6
`(20.4)
`171.3 (90.2), 245.4
`(94.0)
`167.4 (9.4)
`
`
`
`Nuclear imaging techniques are widely used for
`clinical applications because of their high sensitivity.
`Nuclear imaging modalities (PET and SPECT) are able
`to determine concentrations of specific molecules in
`the human body in the picomolar range and provide
`enough sensitivity needed to visualize most interac-
`tions between physiological targets and receptor lig-
`ands. Many biomolecules (monoclonal antibodies,
`peptides, or non-peptide receptor ligands) have been
`successfully used for target-specific delivery of radi-
`onuclides. Among them, small peptides with less than
`30 amino acids or molecular weight less than 3500
`Daltons are of particular interest. Compared to mon-
`oclonal antibodies and antibody fragments, small
`peptides offer several advantages. Peptides are nec-
`essary elements in more fundamental biological pro-
`cesses than any other class of molecule. They can also
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`Theranostics 2011, 1
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`tolerate harsher conditions for chemical modification
`or radiolabeling. Small peptides are easy to synthesize
`and modify, less likely to be immunogenic, and can
`have rapid blood clearance. The faster blood clearance
`results in adequate T/B ratios earlier so that it is
`practical to use 99mTc, which is the preferred radionu-
`clide for diagnostic nuclear medicine. In most cases,
`the primary sites of interactions of peptides are re-
`ceptors on the outer surface of cell membranes (ex-
`tracellular). All these factors make small bioactive
`peptides excellent candidates for development of
`target-specific radiotracers. The peptide-based radio-
`tracers have been reviewed extensively [7-20].
`The integrin family is comprised of 25 identified
`members, which are heterodimers of 19 α- and 8
`β-subunits imbedded non-covalently into the cell
`membrane [21]. The member of this family is still ex-
`panding as observed from human genome studies
`[22]. The cell-cell and cell-matrix adhesion processes
`through binding of integrins to their ligands play
`critical roles in physiological processes, including cell
`attachment, proliferation [23-25], bone remodeling
`[26], and wound healing [27]. Besides, integrins also
`contribute to pathological events such as thrombosis,
`atherosclerosis [28, 29], tumor invasion, angiogenesis
`and metastasis [30-33], infection by pathogenic mi-
`croorganisms [34, 35], and immune dysfunction [36].
`Therefore, the integrins have been proposed as the
`molecular targets for the treatment of cancer [37-42],
`thrombosis [43, 44] and other diseases [45, 46] in the
`last two decades. The role of integrins has been re-
`viewed extensively [21, 47-50].
`Many integrin family members are crucial to the
`initiation, progression and metastasis of solid tumors.
`Epithelial-derived tumor cells generally retain integ-
`rins expressed by epithelial cells including α6β4, α6β1,
`αvβ5, α2β1 and α3β1, and mediate the adhesion, migra-
`tion, proliferation and survival of tumor cells. Differ-
`ent integrins can promote or suppress the tumor de-
`velopment.
`For
`example,
`integrin α2β1
`is
`down-regulated in tumor cells, the phenomenon as-
`sociated with increased tumor cell dissemination [51].
`This suggests that α2β1 could function as a tumor
`suppressor [52]. On the other hand, the expression of
`αvβ3, αvβ5, α5β1, α6β4, α4β1 and αvβ6 on tumor cells is
`correlated with disease progression in various tumor
`types [53-58]. More importantly, the expression of
`integrins αvβ3, α5β1 and αvβ6 are usually at low or
`undetectable levels in most adult epithelia. Among 25
`members of the integrin family, integrin αvβ3 is stud-
`ied most extensively for its role in the tumor growth
`and angiogenesis. While the αvβ3 plays pivotal role in
`the tumor growth and progression, the αIIBβ3 is critical
`for platelet aggregation during thrombosis. It is be-
`
`lieved that the interaction between the tumor αvβ3 and
`platelet αIIbβ3 is also related to the increased tumor
`metastasis via a bridge such as fibrinogen, von Wil-
`lebrand factor or thrombospondin [59]. This interac-
`tion is believed to facilitate the tumor cell adhesion to
`the vasculature, and often leads to metastasis to var-
`ious secondary sites, including bone marrow [60].
`Integrin αIIBβ3 is exclusively expressed on plate-
`lets, although αvβ3, α2β1, α5β1 and α6β1 can also medi-
`ate platelet adhesion functions [61]. On the surface of
`platelet, there are 70~90 thousand copies of αIIBβ3,
`which facilitate the intercellular bidirectional signal-
`ing (―inside-out‖ and ―outside-in‖) and allow the ag-
`gregation of platelets during the vascular injury. The
`αIIBβ3 plays an important role in thrombosis formation
`by its activation and binding to fibrinogen especially
`in arterial thrombi due to the high blood flow rate. In
`the resting state, the αIIBβ3 on platelets does not bind
`to fibrinogen. On activation, the conformation of
`platelet is altered and the binding sites of αIIBβ3 are
`exposed for fibrinogen to crosslink with the activated
`platelets. Integrin αIIBβ3 antagonists have been widely
`used in the antithrombotic therapy in the patients
`with percutaneous coronary interventions and unsta-
`ble angina [47, 48, 62-65].
`The αvβ3 and αIIBβ3 receptor ligands share a
`common RGD tripeptide binding sequence. General-
`ly,
`linear RGD peptides,
`such
`as GRGDS
`(Gly-Arg-Gly-Asp-Ser), often have low affinity (IC50 >
`100 nM) and selectivity for αvβ3 and αIIBβ3 [66], and
`undergo rapid degradation in serum by a variety of
`proteases [67, 68]. It has been shown that cyclization
`of RGD peptides via linkers, such as S-S disulfide,
`thioether and rigid aromatic rings, often leads to the
`increased receptor binding affinity and selectivity
`[67-77]. It has been reported that the αIIBβ3 is less sen-
`sitive to variations in the RGD backbone structure and
`can accommodate a larger distance or spacer than
`αvβ3 and v5 [66]. On the basis of extensive struc-
`ture-activity-relationship studies, it was found that
`incorporation of the RGD unit into a cyclic the pen-
`tapeptide framework (Fig. 2: top) increases binding
`affinity and selectivity for v3 over αIIBβ3 [66, 68-77],
`while addition of a rigid aromatic ring (Fig. 2:
`DMP728 and DMP757) into the cyclic hexapeptide
`structure enhance the receptor binding affinity and
`selectivity for αIIBβ3 over both v3 and v5 [66, 79,
`80]. It was also found that the valine residue in
`c(RGDfV) could be readily replaced by lysine (K) or
`glutamic acid (E) to afford c(RGDfK) or c(RGDfE),
`without significantly changing the v3 binding affin-
`ity [69-71]. Similar behavior was also seen for
`αIIBβ3-selective hexapeptides [66].
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`Theranostics 2011, 1
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`Figure 2. Examples of monomeric cyclic RGD peptides. Incorporation of the RGD sequence into a cyclic pentapeptide
`framework increases the binding affinity and selectivity for αvβ3 over v5 and αIIBβ3, while the addition of one or two rigid
`aromatic rings into cyclic hexapeptide structure enhance the binding affinity and selectivity for the αIIBβ3 over αvβ3 and v5.
`
`
`
`
`
`Several excellent review articles have appeared
`recently to cover a broad range of topics related to
`integrin-targeted radiotracers and their nuclear med-
`icine applications in tumor imaging by SPECT and
`PET [81-97]. This review is not intended to be an ex-
`haustive review on all radiolabeled cyclic RGD pep-
`tides. Instead, it will focus on recent development of
`v3-targeted SPECT radiotracers for imaging tumor
`angiogenesis and the use of the IIb3-targeted radio-
`tracers for thrombosis imaging by SPECT. Because of
`the limited space, authors would apologize to those
`whose work has not been presented in detail, and for
`the omission of 123I-labeled cyclic RGD peptides as
`radiotracers in this review.
`
`2. v3–TARGETED RADIOTRACERS FOR
`TUMOR IMAGING
`
`Integrin v3 and tumor angiogenesis. Tumor cells
`produce many angiogenic factors, which are able to
`activate endothelial cells on the established blood
`vessels and induce endothelial proliferation, migra-
`
`(angiogenesis)
`formation
`tion, and new vessel
`through a series of sequential but partially overlap-
`ping steps [98-103]. Angiogenesis is a key requirement
`for both the tumor growth and metastasis. Without
`the formation of the new blood vessels which provide
`oxygen and nutrients, tumors cannot grow beyond 1 –
`2 mm in size [98, 103]. Angiogenesis is regulated by
`many proteins, such as vascular endothelial growth
`factor (VEGF), vascular endothelial growth factor re-
`ceptors (VEGFR), G-protein coupled receptors for
`angiogenesis modulating proteins, endogenous an-
`giogenesis inhibitors and integrins [102-105]. Among
`the angiogenesis factors, integrins are responsible for
`the cellular adhesion to extracellular matrix proteins
`in the intercellular spaces and basement membranes
`and subsequent migration of cells, and regulate cel-
`lular entry and withdraw from cell cycle [100,
`107-110]. Among the integrins identified so far, the
`v3 is studied most extensively since serves as a re-
`ceptor for a variety of extracellular matrix proteins
`with the exposed RGD tripeptide sequence. These
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`Theranostics 2011, 1
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`include vitronectin, fibronectin, fibrinogen, laminin,
`collagen, von Willebrand factor, and osteopontin
`[111-119]. The v3 is usually expressed in relatively
`low levels on epithelial cells and mature endothelial
`cells, but is highly expressed in the tumors including
`osteosarcomas, neuroblastomas, glioblastomas, mel-
`anomas, breast,
`lung and prostate carcinomas
`[112-120]. Recently, it has been reported that the v3
`is overexpressed on not only tumor cells but also en-
`dothelial cells of the tumor neovasculature [121]. The
`v3 expressed on the activated endothelial cells can
`modulate cell adhesion and migration during tumor
`angiogenesis, and its expression on carcinoma cells
`potentiates metastasis by facilitating invasion and
`movement of tumor cells across blood vessels [121]. It
`has also been demonstrated that the v3 expression
`level correlates well with the potential for metastasis
`and the aggressiveness of many tumors including
`glioblastomas, melanoma, ovarian, breast and lung
`cancers [113, 119-121]. Therefore, the v3 has been
`identified as an interesting molecular target for the
`early diagnosis of rapidly growing and metastatic
`tumors [81-97].
`Integrin v3-targeted radiotracers under clinical in-
`vestigation. Many radiolabeled cyclic RGD peptides
`have been evaluated as the v3-targeted radiotracers
`[122-158]. Significant progress has been made on their
`use in tumor imaging by either SPECT or PET. Among
`the radiotracers evaluated in many preclinical tu-
`mor-bearing animal models, [18F]Galacto-RGD (Fig. 3:
`top) and [18F]AH111585 (Fig. 3: middle) are currently
`under clinical investigation for non-invasive imaging
`of the v3 expression in cancer patients [159-164].
`Imaging studies clearly showed that the accumulation
`of 18F-labeled RGD peptide radiotracers correlated
`well with the tumor v3 expression levels in cancer
`patients [159-164]. However, their relatively low tu-
`mor uptake, high cost and lack of preparative mod-
`ules for routine radiosynthesis will limit their con-
`tinued clinical utilities. In addition, several steps of
`manual radiosynthesis and post-labeling purification
`can cause significant radiation exposure to radio-
`pharmacists in the clinics. 99mTc-NC100692 (Fig. 3:
`bottom) is a 99mTc-labeled cyclic RGD peptide mono-
`mer reportedly to have high integrin v3 binding
`affinity [165]. In breast cancer patients, 19 of 22 ma-
`lignant lesions (86%) were detected by SPECT [165].
`However, its intensive liver uptake and hepatobiliary
`excretion due to its lipophilic Tc-chelate (Fig. 3) will
`limit its continued clinical applications. Thus, there is
`a continuing need for more efficient v3-specific
`99mTc radiotracers that can be readily prepared from a
`kit formulation at low cost.
`
`Multimer concept. Since interactions between the
`v3 and RGD-containing proteins (e.g. vitronectin,
`fibronectin and fibrinogen) may involve multiple
`binding sites, the idea to use multimeric cyclic RGD
`peptides might provide more effective v3 antago-
`nists with tumor targeting capability and hence high-
`er cellular uptake for their corresponding radiotracers
`[166]. Multivalent interactions are used in such a way
`that weak ligand-receptor interactions may become
`biologically relevant. The multimer concept has been
`used for enhancing the radiotracer tumor-targeting
`capability. For example, biodistribution studies
`showed that the divalent 99mTc-[sc(Fv)2]2 had ap-
`proximately 3-fold higher
`tumor uptake
`than
`99mTc-sv(Fv)2 [167]. The increased binding affinity and
`tumor targeting capability were also reported for the
`125I-labeled divalent recombinant antibody fragment
`[168].
`Multimeric cyclic RGD peptides. To improve v3
`binding affinity, dimeric RGD peptides, such as
`E[c(RGDfK)]2 (Fig. 4: RGD2), have been used to de-
`velop the v3-targeted radiotracers. Rajopadhye et al
`were the first to use E[c(RGDfK)]2 to develop diag-
`nostic (99mTc and 64Cu) and therapeutic (90Y and 177Lu)
`radiotracers [146-157, 169, 170]. Dijkgraff et al found
`that the tumor uptake of 111In-labeled E[c(RGDfK)]2
`was >2x of that for its corresponding monomeric an-
`alog in athymic mice with xenografted SK-RC-52 tu-
`mors [154]. The same group also reported the
`DOTA-conjugated cyclic RGD dimers and tetramers
`[154, 155], but no in vivo data was presented. Recent-
`ly, Chen and coworkers reported 64Cu and 18F-labeled
`E[c(RGDyK)]2 as PET radiotracers [140, 141]. Poethko
`et
`al
`also
`found
`that
`the RGDfE dimer
`[c(RGDfE)-HEG]2-K (Fig. 4) had much better targeting
`capability
`than
`the monomer
`c(RGDfE)-HEG
`[128-130]. The multimer concept was also used to
`prepare cyclic RGD tetramers [142, 144, 153, 155,
`171-173] and octamers [173]. For example, Boturyn et
`al reported a series of cyclic RGDfK tetramers [172],
`and found that increasing the peptide multiplicity
`significantly enhanced the v3 binding affinity and
`internalization. Kessler et al reported a cyclic RGDfE
`tetramer (Fig. 5) that had better v3 binding affinity
`than its corresponding dimer counterpart [128-130].
`Liu et al used E[E[c(RGDfK)]2]2 (Fig. 5: RGD4) for the
`development of v3-targeted diagnostic (99mTc and
`64Cu) radiotracers [142, 153]. Chen et al also reported
`the use of 64Cu and 18F-labeled cyclic RGD peptide
`tetramer
`E[E[c(RGDyK)]2]2
`and
`octamer
`E[E[E[c(RGDyK)]2]2]2 for tumor imaging by PET [173].
`Both the in vitro assays and the ex vivo biodistribu-
`tion studies showed that the radiolabeled multimeric
`cyclic RGD peptides had better tumor uptake with
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`Theranostics 2011, 1
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`longer tumor retention time than their dimeric ana-
`logs. However, their T/B ratios were not substantially
`better due to their high uptake in the normal organs
`[173]. It remains unclear if the multimeric cyclic RGD
`peptides, such as E[E[E[c(RGDyK)]2]2]2, are really
`multivalent. Moreover, the cost for synthesis of the
`RGD octamer E[E[E[c(RGDyK)]2]2]2 is prohibitively
`
`high for future development of the v3-targeted di-
`agnostic radiotracers. Thus, an alternate approach is
`needed to improve the v3-targeting capability of the
`radiotracer and minimize its accumulation in normal
`organs.
`
`
`
`
`
`Figure 3. Examples of radiolabeled cyclic RGD peptide monomers as radiotracers ([18F]Galacto-RGD, [18F]AH111585 and
`99mTc-NC100692) for imaging tumor angiogenesis. They are currently under clinical investigation for noninvasive visualiza-
`tion of the αvβ3 expression in cancer patients.
`
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`
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`Theranostics 2011, 1
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`Figure 4. Examples of RGD dimers (E[c(RGDfK)]2, E[c(RGDyK)]2 and [c(RGDfE)HEG]2-K) for αvβ3-targeting.
`
`
`
`Fiure 5. Structure of a cyclic RGD tetramer [[c(RGDfE)HEG]2K]2-K.
`
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`Theranostics 2011, 1
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`their DOTA-conjugates against 125I-c(RGDyK) bound
`to U87MG glioma cells [176]: DOTA-RGD4 (IC50 = 1.3
`± 0.3 nM) ~ DOTA-3P-RGD2 (IC50 = 1.3 ± 0.3 nM) ~
`DOTA-3G-RGD2 (IC50 = 1.1 ± 0.2 nM) > DOTA-RGD2
`(IC50 = 8.0 ± 2.8 nM) >> DOTA-P-RGD (IC50 = 42.1 ±
`3.5 nM) ~ c(RGDfK) (IC50 = 38.5 ± 4.5 nM) >>
`DOTA-3P-RGK2 (IC50 = 452 ± 11 nM). These data
`suggest that the G3 and PEG4 linkers between two
`RGD motifs are responsible for the improved αvβ3
`binding
`affinity
`of HYNIC-3P-RGD2
`and
`HYNIC-3G-RGD2 as compared to HYNIC-P-RGD2
`[174, 175]. The higher αvβ3 binding affinity of
`HYNIC-RGD4 is likely due to the presence of its two
`extra RGD motifs in RGD4 as compared to those in
`HYNIC-3P-RGD2 and HYNIC-3G-RGD2 [174].
`It is important to note that the IC50 values of cy-
`clic RGD peptides are largely dependent on the type
`of assay (the immobilized v3-binding assay vs
`whole-cell v3 competition assay), the radioligand
`(125I-c(RGDyK) vs 125I-echistatin) and tumor cell lines
`(U87MG vs MDA-MB-435). Caution should be taken
`when comparing their IC50 values. Whenever possi-
`ble, a ―control compound‖, such as c(RGDfK) and
`c(RGDyK), should be used in each experiment. In
`addition, the IC50 values obtained from the in vitro
`assays cannot be used as the ―absolute proof‖ to
`support the concept of bivalency. They must be used
`in combination with the biodistribution data of their
`corresponding radiotracers.
`To prove the bivalency of cyclic RGD dimers
`(Fig. 7: 3P-RGD2 and 3G-RGD2),
`complexes
`99mTc-3P-RGD2 and 99mTc-3G-RGD2 (Fig. 8) were
`evaluated in the athymic nude mice bearing U87MG
`human glioma and MDA-MB-435 human breast tu-
`mor xenografts [174, 175]. For comparison purposes,
`99mTc-P-RGD2 and 99mTc-RGD4 (Fig. 8) were also
`evaluated using the same tumor-bearing animal
`models [174, 175]. As expected, the breast tumor up-
`take of 99mTc-3P-RGD2 and 99mTc-3G-RGD2 was com-
`parable to that of 99mTc-RGD4 (Fig. 8), and was >2x
`higher than that of 99mTc-P-RGD2 [174]. These data
`strongly suggest that RGD4, 3P-RGD2 and 3G-RGD2
`are bivalent and P-RGD2 is only monodentate in
`binding to the integrin αvβ3 even though it has two
`RGD motifs. Similar conclusion was also made for
`3P-RGD2 in 64Cu(DOTA-3P-RGD2) [176], 3G-RGD2 in
`64Cu(DOTA-3G-RGD2)
`[176], G3-2P4-RGD2
`in
`99mTc-G3-2P4-RGD2 [177], and 2P-RGD2 in their 68Ga
`and 18F radiotracers [178, 179]. If P-RGD2 were biva-
`lent, HYNIC-P-RGD2 would have had the same αvβ3
`binding
`affinity
`as HYNIC-3P-RGD2
`and
`HYNIC-3G-RGD2 while 99mTc-P-RGD2 would have
`shared the same tumor uptake with 99mTc-3P-RGD2
`and 99mTc-3G-RGD2.
`
`
`http://www.thno.org
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`
`
`Figure 6. Schematic illustration of interactions between a
`cyclic RGD tetramer and the integrin v3 receptor.
`
`Improve αvβ3 binding affinity via bivalency. Fig. 6
`illustrates the interaction between v3 and a cyclic
`RGD tetramer. The targeting moiety is c(RGDfK). The
`spacer is glutamic acid (E) or its derivatives. Two
`factors contribute to the high v3 binding affinity of
`multimeric cyclic RGD peptides: bivalency and the
`enhanced local RGD concentration. The key for biva-
`lency is the distance between two adjacent cyclic RGD
`motifs. If this distance is long enough for simultane-
`ous v3 binding, the cyclic RGD multimer will bind
`to v3 in a bivalent fashion. If this distance is too
`short, the local cyclic RGD peptide concentration is
`still ―enriched‖ in the vicinity of neighboring v3
`sites once the first RGD motif is bound. The combina-
`tion of simultaneous v3 binding (bivalency factor)
`and the locally enriched RGD concentration (concen-
`tration factor) will result in higher v3 binding affin-
`ity for cyclic RGD multimers and better tumor uptake
`with longer tumor retention for their corresponding
`radiotracers.
`To demonstrate the proof-of-principle for the
`bivalency concept, Shi et al recently reported a series
`of cyclic RGD dimers (Fig. 7) with G3 (Gly-Gly-Gly)
`and PEG4 (15-amino-4,7,10,13-tetraoxapentadecanoic
`acid) linkers [174-181]. The G3 and PEG4 linkers were
`used to increase the distance between two RGD motifs
`from 6 bonds in RGD2 to 24 bonds in 3G-RGD2 and 38
`bonds in 3P-RGD2 [174, 175]. The αvβ3 binding affini-
`ties (Table 2) against 125I-echistatin bound to U87MG
`human glioma cells follow the order of HYNIC-RGD4
`(IC50 = 7 ± 2 nM) > HYNIC-2P-RGD2 (IC50 = 52 ± 7 nM)
`~ HYNIC-3P-RGD2 (IC50 = 60 ± 4 nM) ~ HYNIC-3G-
`RGD2 (IC50 = 61 ± 2 nM) > HYNIC-P-RGD2 (IC50 = 84 ±
`7 nM) ~ HYNIC-RGD2 (IC50 = 112 ± 21 nM) >>
`HYNIC-G-RGD (IC50 = 358 ± 8 nM) > HYNIC-P-RGD
`(IC50 = 452 ± 11 nM). A similar trend was observed for
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`Theranostics 2011, 1
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`66
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`Figure 7. Examples of cyclic RGD dimers with PEG4 and G3 linkers, which are used to increase the distance between two
`RGD motifs and to improve radiotracer excretion kinetics from normal organs.
`
`
`
`
`
`
`http://www.thno.org
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`Petitioner GE Healthcare – Ex. 1052, p. 66
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`Theranostics 2011, 1
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`67
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`Figure 8. Comparison of the tumor uptake for 99mTc-P-RGD2, 99mTc-3G-RGD2, 99mTc-3P-RGD2 and 99mTc-RGD4 in the
`athymic nude mice bearing MDA-MB-435 breast cancer xenografts.
`
`
`
`
`
`Table 2. Integrin αvβ3 binding data for cyclic RGD peptides and their corresponding HYNIC and DOTA conjugates against
`125I-echistatin bound to the αvβ3–positive U87MG human glioma cells.
`
`Compound
`c(RGDyK)
`HYNIC-G-RGD
`HYNIC-P-RGD
`HYNIC-RGD2
`HYNIC-P-RGD2
`HYNIC-2G-RGD2
`HYNIC-2P-RGD2
`HYNIC-3G-RGD2
`HYNIC-3P-RGD2
`HYNIC-RGD4
`DOTA-RGD2
`DOTA-3G3-RGD2
`DOTA-3PEG4-RGD2
`DOTA-RGD4
`NOTA-RGD2
`NOTA-2G3-RGD2
`NOTA-2PEG4-RGD2
`
`IC50 (nM)
`458 ± 45
`358 ± 8
`452 ± 11
`112 ± 21
`84 ± 7
`60 ± 4
`52 ± 7
`61 ± 2
`62 ± 5
`7 ± 2
`102 ± 5
`74 ± 3
`62 ± 6
`10 ± 2
`100 ± 3
`66 ± 4
`54 ± 2
`
`Radiotracer
`
`[99mTc(HYNIC-G-RGD)(tricine)(TPPTS)]
`[99mTc(HYNIC-P-RGD)(tricine)(TPPTS)]
`[99mTc(HYNIC-RGD2)(tricine)(TPPTS)]
`[99mTc(HYNIC-P-RGD2)(tricine)(TPPTS)]
`[99mTc(HYNIC-2G-RGD2)(tricine)(TPPTS)]
`[99mTc(HYNIC-2P-RGD2)(tricine)(TPPTS)]
`[99mTc(HYNIC-3G-RGD2)(tricine)(TPPTS)]
`[99mTc(HYNIC-3P-RGD2)(tricine)(TPPTS)]
`[99mTc(HYNIC-RGD4)(tricine)(TPPTS)]
`64Cu(DOTA-RGD2)/111In(DOTA-RGD2)
`64Cu(DOTA-3G-RGD2)/111In(DOTA-3G-RGD2)
`64Cu(DOTA-3P-RGD2)/111In(DOTA-3P-RGD2)
`64Cu(DOTA-RGD4)/111In(DOTA-RGD4)
`68Ga(NOTA-RGD2)
`68Ga(NOTA-2G-RGD2)
`68Ga(NOTA-2P-RGD2)
`
`
`http://www.thno.org
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`Petitioner GE Healthcare – Ex. 1052, p. 67
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`Theranostics 2011, 1
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`Impact of radiometal chelate on tumor uptake and
`pharmacokinetics. Shi et al [180, 181] also prepared the
`cyclic RGD
`conjugates: MAG2-3P-RGD2
`and
`MAG2-3G-RGD2.
`It
`was
`found
`that
`99mTcO(MAG2-3P-RGD2) had better tumor uptake
`than 99mTc-3P-RGD2 [180], while their liver and kidney
`uptake was almost identical at >60 min p.i. On the
`other hand, 99mTcO(MAG2-3G-RGD2) had the same
`tumor uptake as 99mTc-3G-RGD2 at <60 min p.i., but its
`liver and kidney uptake was much lower than that of
`99mTc-3G-RGD2 [181]. Among 99mTc-labeled cyclic
`RGD dimers evaluated in the U87MG glioma-bearing
`model, 99mTcO(MAG2-3P-RGD2) has the highest gli-
`oma uptake (~15 %ID/g over 2 h study period) while
`99mTcO(MAG2-3G-RGD2) has the best tumor/kidney
`(2.49 ± 0.25) and tumor/liver (8.29 ± 1.50) ratios at 120
`min
`p.i.
`Obviously,
`replacing
`[99mTc(HYNIC)(tricine)(TPPTS)] (M.W. = ~1000 Dal-
`tons) with 99mTcO(MAG2) (M.W. = ~350 Daltons) had
`a significant impact on both tumor uptake and phar-
`macokinetics of 99mTc radiotracers. In contrast, sub-
`stituting the bulky [99mTc(HYNIC)(tricine)(TPPTS)]
`with a much smaller and more hydrophilic
`111In(DOTA) chelate had little impact on the radio-
`tracer tumor uptake [182, 183]. However, the liver and
`kidney uptake of 111In(DOTA-3P-RGD2) is signifi-
`cantly lower than that of 99mTc-3P-RGD2, probably due
`to higher hydrophilicity of 111In(DOTA) [82]. Similar
`conclusion could be made by directly comparing
`111In(DOTA-3G-RGD2) and 99mTc-3G-RGD2 [181, 183].
`111In(DOTA-3P-RGD2)
`and
`64Cu(DOTA-3P-
`RGD2) share the same DOTA-conjugate. The tumor
`uptake of 111In(DOTA-3P-RGD2) was very close to that
`of 64Cu(DOTA-3P-RGD2) [176, 182]. They also have a
`similar uptake in normal organs. For example, the
`kidney uptake of 111In(DOTA-3P-RGD2) was com-
`pared well with that of 64Cu(DOTA-3P-RGD2) within
`the experimental errors. The
`liver uptake of
`111In(DOTA-3P-RGD2) was 2.52 ± 0.57 %ID/g at 30
`min and 1.61 ± 0.06 %ID/g at 240 min p.i., while
`64Cu(DOTA-3P-RGD2) had the liver uptake of 2.80 ±
`0.35 %ID/g at 30 min p.i. and 1.87 ± 0.51 %ID/g at 240
`min p.i. These data suggest that the radiometal (64Cu
`vs. 111In) has little impact on the radiotracer tumor
`uptake and excretion kinetics, probably due to the
`overwhelmingly large size of the dimeric RGD pep-
`tides as compared to that of the radiometal chelate.
`The same conclusion was also made by directly com-
`paring the uptake in tumor and normal