OPEN ACCESS
`
`
`
`molecules
`
`Molecules 2013, 18, 3379-3409; doi:10.3390/molecules18033379
`
`ISSN 1420-3049
`www.mdpi.com/journal/molecules
`
`Review
`Synthesis of Peptide Radiopharmaceuticals for the Therapy and
`Diagnosis of Tumor Diseases
`
`Mazen Jamous, Uwe Haberkorn and Walter Mier *
`
`Department of Nuclear Medicine, University Hospital Heidelberg, Im Neuenheimer Feld 400,
`D-69120 Heidelberg, Germany
`
`* Author to whom correspondence should be addressed; E-Mail: walter.mier@med.uni-heidelberg.de;
`Tel.: +49-6221-56-7720; Fax: +49-6221-65-33629.
`
`Received: 29 December 2012; in revised form: 25 February 2013 / Accepted: 7 March 2013 /
`Published: 14 March 2013
`
`
`Abstract: Despite the advances in molecular biology and biochemistry, the prognosis of
`patients suffering from tumor diseases remains poor. The limited therapeutic success can
`be explained by the insufficient performance of the common chemotherapeutic drugs that
`lack the ability to specifically target tumor tissues. Recently peptide radiopharmaceuticals
`have been developed that enable the concurrent imaging and therapy of tumors expressing
`a specific target. Here, with a special emphasis on the synthesis of the building blocks
`required for the complexation of metallic radioisotopes, the requirements to the design and
`synthesis of radiolabeled peptides for clinical applications are described.
`
`Keywords: radionuclides; chelator; prosthetic groups; carrier molecules; peptides;
`medicinal application; radiopharmaceutical; diagnostic imaging; radiotherapeutics
`
`
`
`1. Introduction
`
`The incidence of human malignant tumor diseases is still increasing worldwide. Generally, cancer
`treatment can be performed using one or a combination of the following methods: surgery,
`chemotherapy and radiation therapy. Their side effects limit the efficiency of chemo- and
`radiotherapeutic agents, but can be avoided and a much more effective therapy is possible if the drugs
`used have tumor selectivity. This involves the determination of biochemical processes that distinguish
`tumor tissue samples from healthy tissue (Table 1). As a result, tumor-specific biomarkers are used in
`oncology. Several types of agents have been developed for specific accumulation in the malignant cells
`
`Petitioner GE Healthcare – Ex. 1009, p. 3379
`
`

`

`3380
`
`Molecules 2013, 18
`
`to reduce the cytotoxic effect on the normal cells. These agents can be labeled with radionuclides that
`accumulate in the tissue of interest. Depending on the purpose, gamma or positron emitters are used
`for diagnosis and beta, alpha or Auger electron emitters are used for therapeutic applications in cancer
`treatment. The higher the specific activity of a drug, the better the imaging and the lower the cytotoxic
`side-effects in therapeutic applications [1].
`Modern imaging methods include computer tomography (CT), magnetic resonance tomography
`(MRI), ultrasound, single-photon emission computed tomography (SPECT) and positron emission
`tomography (PET). They provide information about the phenotypic functional changes associated with
`the development of the disease. New treatment modalities based on the biological properties of tissues
`have been developed, where important progress has been achieved using antibodies and peptides [2].
`When labeled with therapeutic radioisotopes, these agents are suitable for endoradiotherapy and
`exploit their high specificity. This has been realized for antibodies against the tumor associated epitope
`CD20 [3] or peptides binding to the somatostatin receptors [4].
`
`Table 1. Biomarkers used in clinical routine for tumor-diagnosis [5].
`
`Perfusion
`Glucose metabolism
`Bone metabolism
`Choline metabolism
`DNA synthesis
`Amino acid transport and protein synthesis
`Receptor binding
`Antigen binding
`PSMA
`Angiogenesis
`Lipid synthesis
`Hypoxia
`Apoptosis
`Gene expression
`
`[15O]H2O
`[18F]FDG
`[18F]Fluoride
`[18F]Choline
`[18F]FLT
`[18F]FET, [11C]MET, [18F]FDOPA
`[68Ga]-DOTA-TOC
`[111In]-anti-CD20 mAb
`[68Ga]-PSMA
`[18F]Galacto-RGD
`[11C]AcOH
`[18F]FAZA, [18F]MISO
`[124I]Annexin V
`[18F]FHBG
`
`Many specific radiopharmaceuticals have been developed in various preclinical and clinical stages for
`imaging and therapy of tumor diseases and some of them are currently in routine clinical use. They can be
`classified into three major categories according to the molecular weight of the carrier: (a) radiolabeled
`monoclonal antibodies (b) receptor specific small proteins and peptides and (c) small molecules.
`
`2. Carrier Molecules
`
`Many tumors overexpress specific targets on the surface of their cells. The target ligands are used
`with radiolabels in cancer diagnosis and therapy in accordance with the key-lock principle (Figure 1).
`As the number of receptors on the surface of tumor cells compared with that in normal tissues often is
`higher, the effect on the tumor cells is stronger than that on the normal cells resulting in a wide
`therapeutic window [6].
`
`
`
`Petitioner GE Healthcare – Ex. 1009, p. 3380
`
`

`

`Molecules 2013, 18
`
`
`3381
`
`Figure 1. Binding of ligand to target like a peptide-receptor has been visualized by a “lock
`and key” arrangement, where the peptide fits into a binding pocket of the receptor on the
`surface of tumor cells in a similar manner to a key fitting into a lock.
`
`
`
`reporting unitreporting unit
`
`
`
`spacerspacer
`
`
`
`carrier moleculecarrier molecule
`
`
`
`2.1. Small Molecules
`
`A variety of molecular and functional alterations has been shown to change the morphology and
`functional status of tumor tissue. Molecular imaging has been established as a tool to measure
`biomarkers or indicators of disease or therapeutic effects [7]. There are numerous different carriers that
`have been designed and developed for the targeting of tumors. Several radiolabeled small molecules
`have been applied in vivo for PET imaging [5]. PET radiopharmaceuticals have a significant potential
`for routine clinical imaging studies. The efficiency of these radiotracers is based on their ability to
`accumulate in the tumor cells (Table 1).
`
`2.2. Antibodies
`
`Antibodies with a very high specificity for their target antigen overexpressed in tumors can display
`a direct therapeutic effect and must therefore not necessarily be combined with a drug for application
`as anticancer drugs. However, as many antibodies are not sufficiently cytotoxic, radionuclides have
`been shown to significantly enhance the therapeutic effects of monoclonal antibodies (mAb).
`Radiolabeled antibodies exert a certain cytotoxic effect on surrounding cells, depending on the emitted
`energy of radionuclide radiation over its reach in the tissue decides. In contrast, the unlabeled
`antibodies interaction is limited on the targeted cells [8]. Zevalin®, a 90Y-anti-CD20 mAb and
`Bexxar®, a 131I-anti-CD20 mAb have been shown to ideally fulfill this task by selectively transporting
`radionuclides to tumors [9,10].
`
`
`
`Petitioner GE Healthcare – Ex. 1009, p. 3381
`
`

`

`Molecules 2013, 18
`
`2.3. Peptides
`
`3382
`
`Several receptors with small regulatory peptide ligands are overexpressed in certain human cancers,
`offering the possibility to target these tumors with radiopeptides. The somatostatin analogs DOTA-TOC
`and DOTA-TATE (1) can be labeled with 111In or 68Ga for imaging, or with 90Y, 177Lu for radiotherapy
`of somatostatin receptor (SSTR)-positive tumors (Figure 2). The excellent results obtained
`led to the development of analogs of other peptide families, such as bombesin, neurotensin,
`cholecystokinin/gastrin, exendin, RGD (Arg-Gly-Asp) and substance P. Numerous radiolabeled
`peptides are currently under preclinical research or clinical evaluation for both diagnostic imaging of
`peptide receptor expression [11,12] and peptide receptor mediated therapy (PRRT) [13–15].
`
`Figure 2. Chemical structures of DOTA-TATE and [18F]Galacto-RGD, two typical
`radiolabeled peptide tracers.
`
`HN
`
`NH2
`NH
`
`
`
`O
`
`O
`
`O
`
`HN
`
`NH
`
`NH
`
`NH
`
`HN
`
`O
`HO
`
`O
`
`O
`
`2
`
`HO
`HO
`
`18F
`
`HN
`
`O
`
`OH
`H
`O
`
`O
`
`HN
`
`NH
`
`NH2
`
`OH
`
`HN
`
`O
`
`HN
`
`O
`
`HN
`
`O
`
`O
`
`NH
`
`OS
`
`NH
`
`HO
`
`HN
`
`S
`
`O
`
`O
`
`HN
`
`1
`
`OO
`
`H
`
`O
`
`NH
`
`O
`
`HO
`
`HO
`
`HO
`
`O
`
`O
`
`HO
`
`N
`
`N
`
`N
`
`N
`
`2.3.1. Peptides and Radiopeptides as Targeting Agents
`
`The overexpression of peptide receptors in human tumors led to the development of peptide
`radio-pharmaceuticals for specific diagnostic imaging and/or therapy of cancers. Table 2 summarizes
`the receptors-binding peptides and their specificity of overexpression in tumors. Neuroendrocrine
`tumors (NETs), including primaries and metastases, overexpress somatostatin receptor types
`(sst1-sst5) [6], particularly sst2 [16]. These receptors present the molecular basis for peptide-based
`probes for cancer imaging and therapy. The somatostatin analogs DOTA-TOC and DOTA-TATE (1)
`can be labeled with 111In, 64Cu and 67/68Ga for in vivo imaging of SST receptor-expressing tumors [17]
`or with β-emitters (90Y or 177Lu) or -emitters (213Bi or 225Ac), these labeled analogs can be utilized for
`peptide receptor mediated therapy (PRRT) [14]. For bombesin receptors family, four subtypes are
`known (BB1-BB4). Gastrin-releasing peptide receptor (GRPR/BB2) has been found to be
`overexpressed in a variety of tumors, including prostate, breast, pancreas, gastrointestinal and small
`cell lung cancer [6]. Several radiolabeled bombesin-like peptides, which bind to BN/GRP receptors
`with high affinity, have been developed in order to be used for diagnostic and/or therapeutic purposes.
`Bracco has developed the first radiolabeled BN analog [177Lu]-AMBA for imaging and PRRT [18,19].
`Bombesin antagonists with favorable tumor-to-normal tissue ratios have been by developed
`Manci et al. [20–22]. The preliminary clinical study shows that [64Cu]-CB-TE2A-AR-06 is a
`
`
`
`Petitioner GE Healthcare – Ex. 1009, p. 3382
`
`

`

`3383
`
`Molecules 2013, 18
`
`promising ligand for imaging GRP-Receptor-positive tumors in humans [23]. An other application of
`peptide-ligands as attractive agents is radiolabeled peptides based on the lead structure cyclo(Arg-Gly-
`Asp-D-Phe-Val) as the integrin αvβ3-targeted radiotracers. Many radiolabeled cyclic RGD peptide
`antagonists have been evaluated for imaging integrin αvβ3-positive tumors by SPECT or PET [24,25].
`Among the radiotracers evaluated in preclinical tumor-bearing models, [18F]Galacto-RGD (2) is
`currently under clinical studies in patients suffering from malignant melanomas, sarcomas, head and
`neck cancer, glioblastomas, and breast cancer [5]. Cholecystokinin (CCK) receptors have been
`identified in numerous human cancers, like medullary thyroid carcinomas, small cell lung cancers,
`stromal ovarian cancers and astrocytomas [6]. Radiolabelled CCK/gastrin analogues have been
`synthesized and characterized for imaging using positron emission tomography and single photon
`emission computed tomography imaging. All peptides are mostly based on the C-terminal CCK
`receptor-binding tetrapeptide sequence Trp-Met-Asp-Phe-NH2. 99mTc-demogastrin 2 has been
`evaluated and compared with [111In]-DOTA-CCK8 and [111In]-DOTA-MG11 in patients with
`medullary thyroid cancers (MTC) [26]. The results obtained show that [99mTc]-demogastrin 2 showed
`the best visualization, which may be due to better imaging properties of 99mTc as compared to 111In.
`The glucagon-like peptide-1 receptor (GLP-1R) is one of the most frequently studied peptide
`receptors. The high density of glucagon-like peptide-1 receptors (GLP-1R) in human insulinomas
`provides an attractive target for molecular imaging and internal radiotherapy [6]. For this purpose
`DTPA- and DOTA-conjugate of exendin-4 were synthesized. The peptide [Lys40(Ahx-DOTA)-NH2]-
`Exendin-4 radiolabeled with 111In shows success in the detection of tumors in patients with
`insulinomas [27–29]. Using the Auger electrons of 111In, [Lys40(Ahx-DOTA)-NH2]-Exendin-4 was
`evaluated as a radiotherapeutic for glucagon-like peptide-1 receptor-targeted therapy for insulinoma [30].
`The peptide receptors, melanocortin receptors exist in five subtypes. The melanocortin 1 receptor
`(MC1R) is overexpressed in most murine and human melanoma metastases [6], and hence is an
`attractive target for the detection and treatment of these cancers. Radiolabeled -MSH analogs, contain
`the sequence His-Phe-Arg-Trp. They have been developed for MC1R targeting. Recently data
`radiolabeled α-MSH analogs DOTA-Nle-CycMSHhex and DOTA-Re-
`demonstrates
`that
`CCMSH(Arg11) are potential candidates for diagnostic imaging or radiotherapy of melanoma tumors
`[31,32]. The overexpression of neurotensin receptor NTR1 has been found in several human cancers
`including Ewing sarcomas, meningiomas, astrocytomas, medulloblastomas and pancreatic carcinomas
`[6], and several NT analogs have been synthesized and conjugated with a chelator, like DTPA or
`DOTA. Among all the radiopeptides, DOTA-NT-20.3 is a promising candidate for 68Ga-PET imaging
`of neurotensin receptor-positive tumors [33]. Human adenocarcinomas of the gastroenteropancreatic
`system overexpress vasoactive intestinal peptide (VIP) receptors [6] and therefore represent logical
`diagnostic targets for receptor scintigraphy. 99mTc labeled VIP analog (TP3654) is a promising agent
`for imaging colorectal cancer [34]. Neurokinin type 1 (NK-1) receptors are overexpressed in malignant
`gliomas. The radiopeptide [111In]/[90Y]-DOTAGA-substance P binds to these receptors and can be
`used for treatment of brain tumors [35]. Neuropeptide Y receptors involve Y1R and/or Y2R have been
`found to be expressed in neuroblastoma, breast carcinomas, ovarian cancers [6]. The chemokine receptors
`CXCR4 are highly expressed in breast and prostate cancer. These receptors (NPY1R and CXXR4) are
`promising additional candidates in the oncology field and their advanced status is under preclinical studies.
`
`
`
`Petitioner GE Healthcare – Ex. 1009, p. 3383
`
`

`

`Molecules 2013, 18
`
`
`3384
`
`Somatostatin
`
`sst2
`
`Bombesin
`
`GRPR
`
`Table 2. Peptide receptors, disease indications and peptide probe in clinical use.
`Peptide
`Receptor
`Tumor Type
`Peptide probe
`Gastroenteropancreatic
`DTPA-octreotide/
`neuro-endocrine tumors
`DOTA-TOC/DOTA-TATE
`Breast, prostate and
`AMBA/CB-TE2A-AR-06
`gastro-intestinal stromal cancer
`BZH3
`[18F]Galacto-RGD
`Melanomas
`[99mTc]-demogastrin 2
`Medullary thyroid carcinomas
`[Lys40(Ahx-DOTA)-NH2]-
`Exendin-4
`DOTA-Nle-CycMSHhex
`DOTA-Re-CCMSH(Arg11)
`TP3654
`DOTAGA-substance P
`
`RGD
`CCK/gastrin
`GLP-1/
`exendin
`
`-MSH
`
`VIP
`substance P
`
`αvβ3
`CCK2R
`
`GLP-1R
`
`MC1R
`
`VIPR
`NK-1R
`
`Insulinomas
`
`Melanomas
`
`Colorectal cancer
`Glioblastoma
`
`The fact that various receptor subtypes can be expressed simultaneously on tumors provides the
`possibility to improve the efficiency of peptide tracers in vivo multireceptor targeting. As neuroendrocrine
`tumors (NETs) usually overexpress somatostatin receptors, enables the use of radiolabeled
`somatostatin analogues. As other peptide receptors have been found to be overexpessed on certain
`NETs, they can be targeted for radionuclide therapy and imaging of NETs. Examples are radiolabelled
`gastrin analogues for MTCs and radiolabelled exendin analogues for insulinomas [17,36–38].
`In the case of tumors simultaneously expressing several types of receptors, targeting of
`multireceptor overexpressed tumors can be performed by the use of heterodimeric peptides as
`molecular imaging agents. Better tumor affinity and pharmacokinetics can be achieved through these
`multivalent interactions. The development of a heterodimeric RGD-bombesin derivatives such as
`X-RGD-Glu-6-Ahx-BBN(7-14)-NH2 [X = [18F]SFB (11), DOTA (28) and NOTA (29)] demonstrated
`favorable pharmacokinetic properties, resulting in a more specific targeting and higher imaging quality
`of gastrin-releasing peptide receptor (GRPR) [39–42]. Josan et al. have prepared a peptide heterodimer
`MSH(7)-CCK-6 that binds to two G protein-coupled receptors: melanocortin-4 (MC4R) and
`cholecystokinin-2 Receptors (CCK2R) [43]. By using solid-phase synthetic strategy, heterobivalent
`ligands targeted to melanocortin-4 (MC4R) and -opioid (-OR) receptors were prepared [44]. The
`heterodimeric peptides are provided to illustrate the relative enhancement in binding affinity to
`receptors overexpressed tumor cells.
`
`2.3.2. Characteristics and Challenges of the Synthesis of Peptide-based Radiopharmaceuticals
`
`Peptides are important regulators of growth and cellular functions in normal tissue and tumors. In
`oncology, major progress has been made with radiolabeled peptide analogs for in vivo localization and
`therapy of tumors. With the advances in organic, bioconjugate and coordination chemistry, solid phase
`peptide synthesis and phage display techniques radiolabeled peptides with high receptor binding
`affinity for a selected target have been developed [45]. Generally, peptides offer distinctive advantages
`over other carriers like small molecules, proteins and antibodies. Peptides, cover many biologically
`important targets, have high receptor binding affinity, are of relatively low molecular weight, easy to
`synthesize, accessible to modification like conjugation with chelators for radiolabeling which allows
`
`
`Petitioner GE Healthcare – Ex. 1009, p. 3384
`
`

`

`3385
`
`Molecules 2013, 18
`
`straightforward kit-preparation of peptide radiopharmaceuticals, provide favorable pharmacokinetics
`resulting in a rapid whole body clearance, good tumor penetration and reach it in high concentration
`and For therapeutic purposes, they are applied at doses lower than conventional drugs, and therefore
`cause few side effects and in addition lack immunogenicity [46].
` A variety of strategies have been applied to enhance the bioavailability of radiolabeled peptides.
`The introduction of unnatural or D-amino acids and shortening of the sequence of natural molecules to
`the biological active sequence are strategies to prolong the biological half-life. A typical example is the
`optimization of radiolabeled RGD peptide including multimererization for improvement of the binding
`affinity for the v3 receptor [47]. Pharmacokinetic modifications such as introduction of charged
`amino acids, glycosylation [48] and PEGylation have also been applied [49].
`The radioactive halogens or metals are the most frequently used elements to prepare peptide based
`radiopharmaceuticals. The radiolabeled peptides provide a class of targeting molecules appropriate for
`both molecular imaging and radiotherapy. The fact that many metallic radionuclides form stable
`complexes with similar chelators allows for the labeling of the same peptide or peptide conjugate with
`various radionuclides for different purposes. The labeling protocols include covalent labeling, either
`direct or indirect using prosthetic groups, or labeling strategies using bifunctional chelating agents
`(BFCAs). Ligands that contain two different moieties, a chelating unit to complex the radiometal and a
`functional group for the covalent attachment of the peptide, are known as bifunctional chelating agents
`(BFCAs). Prosthetic groups are bifunctional agents that consist of a suitable site for radioiodination or
`fluorination and functional groups to allow covalent attachment of the peptide. Several of them have
`been designed and evaluated to engineer high thermodynamically and kinetically stable radiolabeled
`peptides to prevent the release of the radionuclide. In the following sections, the methods for
`radiolabeling with clinically relevant radionuclides and the developments of BFCAs, based on
`polyaminopolycaboxylate, including acyclic and macrocyclic chelator are discussed.
`
`3. Label Types
`
`3.1. Iodine-Labeled Peptide Radiopharmaceuticals
`
`Generally, radioiodination of peptides can be performed using one of following methods:
`radioiodination by electrophilic substitution (direct) or radioiodination via conjugation (indirect). The
`tyrosine or histidine side chains in peptides offer the possibility of electrophilic aromatic substitution
`by electrophilic radioiodine (*I+) with high efficiency under mild conditions. Several oxidizing agents
`can be used for the generation of electrophilic iodine (*I+) such as chloramine T (sodium
`tosylchloroamide) (3) or Iodogen® (1,3,4,6-tetrachloro-3,6-diphenylglycoluril) (4) (Figure 3).
`Polymer-bound chloramine T (IodoBeads®) (5) or vials coated with Iodogen® have the advantage that
`no reducing agent is needed to quench the labeling reaction, since they are insoluble and can be easily
`separated from the reaction mixture.
`
`
`
`Petitioner GE Healthcare – Ex. 1009, p. 3385
`
`

`

`Molecules 2013, 18
`
`
`Figure 3. Chemical structure of N-chloroamide oxidizing agents and prosthetic groups for
`radioiodination of peptides.
`
`3386
`
`
`
`Indirect labeling is another strategy for iodination of peptides, when direct labeling is not possible.
`The incorporation of radioiodine can be performed by the utilization of radioiodinated prosthetic
`groups, which can be used for conjugation with specific functionalities introduced previously into the
`biomolecule or peptide precursors such as amine, aminooxy or thiol groups. Due to the disadvantage of
`the Bolton-Hunter reagent (N-succinimidyl-3-(4-hydroxy,5-[*I]iodophenyl)-propionate) (6) of low in vivo
`stability, other active esters have been developed (Figure 3). Among these, SIB (N-succinimidyl-3-
`[*I]iodobenzoate) (7) and SIPC (N-succinimidyl-5-[*I]iodo-3-pyridine carboxylate) (8) are very stable
`against in vivo deiodonation and allow high-yield conjugation with amino groups of peptides [50,51].
`Aldehydes, such as 4-[*I]iodobenzaldehyde (9), have been used for the coupling of peptides to form
`stable radiolabeled oximes. This methodology has been proposed for radioiodination of multimeric
`cyclic RGD peptides [52]. Maleimides allow the chemoselective conjugation to thiols in peptides. A
`radiolabeled maleimide derivative of 10 has been used for a radioiododestannylation approach
`followed by conjugation with a Cys-peptide under very mild conditions in one step in high yield [53].
`
`3.2. Fluorine-Labeled Peptide Radiopharmaceuticals
`
`18F-labeling of peptides by direct labeling is not possible via nucleophilic substitution under mild
`conditions. Mild conditions are required as the elevated temperatures and strong bases that are used for
`radiofluorination destroy the peptidic biomolecules. Therefore, 18F-labeled prosthetic groups have been
`developed. For this purpose specific functionalities, such as amine, aminooxy, hydrazine, alkyne or
`azide groups have to be introduced into the peptide precursor. Amino reactive prosthetic groups
`(Figure 4) are widely used for [18F]fluorination of peptides [24,54,55], since [18F]SFB
`(N-succinimidyl-4-[18F]fluorobenzoate) (11) and [18F]NPFP (4-nitrophenyl-2-[18F]fluoropropionate) (12)
`allow conjugation in good yield and poses high metabolic stability [56,57]. Numerous [18F]fluorinated
`prosthetic groups based on thiol-maleimide coupling chemistry or thiol-selective alkylation reactions,
`N-(4-[18F]fluorobenzyl)-2-bromoacetamide
`1-[3-(2-[18F]fluoropyridin-3-yloxy)propyl]
`(13),
`e.g.,
`pyrrole-2,5-dione ([18F]FPyMe, 14) and N-2-(4-[18F]-fluorobenzamido) ethylmaleimide ([18F]FBEM, 15),
`have been developed for the conjugating to peptides [58,59]. As the synthesis of these prosthetic
`groups include multistep procedures, there is still the need for 18F-labeling methods suitable for faster
`peptide labeling. Chemoselective conjugation methods using aldehydes, alkyne or azide derivatives
`labeled with 18F seem to be more efficient for clinical application, such as [18F]FBA (4-[18F]
`fluorobenzaldehyde, 16), [18F]SiFA-A (p-(di-t-butyl[18F]fluorosilyl)benzaldehyde) (17), [18F]fluoro-
`
`
`Petitioner GE Healthcare – Ex. 1009, p. 3386
`
`

`

`Molecules 2013, 18
`
`ethylazide (18), [18F]fluoroalkynes 19 and [18F]-glycosyl azide (20) [60–64]. The derivatives are
`synthesized in one step and used to form oximes, hydrazones or 1,2,3-triazoles of unprotected peptides.
`
`3387
`
`Figure 4. Chemical structure of prosthetic groups for the fluorination of peptides.
`
`
`
`The high lipophilicity of the resulting peptide radiopharmaceuticals derived from the fluorination
`strategy discussed above leads to a high unspecific liver and low tumour uptake. Glycosylation or
`polyethylene glycol (PEG) conjugation yields peptides showing lower lipophilicity thus more
`significantly favorable radiolabeled peptide pharmacokinetics. For example glycosyl-Lys([18F]FP)-
`TOCA and [18F]galactosyl-RGD, glycosylated analogs, have been developed [24,65] and evaluated in
`patients [25,66].
`
`3.3. 99mTc-Labeled Peptide Radiopharmaceuticals
`
`99mTc is still the most frequently used radionuclide in diagnostic applications of nuclear medicine,
`due to its ideal nuclear physical properties, availability through a commercial 99Mo-99mTc generator,
`the low production cost and easy and rich labeling chemistry. Most radiopharmaceuticals have
`99mTc-complexes in the oxidation state of +V. 99mTc is eluted from the generator in physiological
`saline in its chemically inert oxidation state of +VII as the complex ion 99mTcO4−. 99mTc(+VII) must be
`reduced to a lower oxidation. For this purpose different reducing agents, such as Na2S2O4, SnCl2,
`phosphines or zinc, can be used in the presence of suitable ligands. The labeling of peptide based
`radiopharmaceuticals usually follows the postconjugation labeling strategy. A bifunctional chelator is
`first covalently bound to the peptide. Subsequently, 99mTcO4¯ is reduced with Sn(II) and complexed by
`the chelator. For this strategy a variety of bifunctional chelators have been designed and tested (Figure 5).
`The structure of the resulting complexes and the oxidation state of technetium depend on the reducing
`agent, the ligand as well as coligands. The tetradentate bifunctional chelators based on N3S [67,68],
`N2S2 [69], such as MAG3 (mercaptoacetyltriglycine) (21), form square pyramidal complexes
`containing the [Tc=O]3+ core. The trans-[O=Tc=O]+ core, which forms octahedral complexes can be
`prepared with tetraamine ligands [70–76]. The use of N4 cores (22) offers the advantages of
`hydrophilic Tc-complex without isomeric structural influence. HYNIC (hydrazinonicotinic acid, 23) is
`widely used for the coupling of technetium to peptides [77–79]. It acts as a mono or bidentate ligand [80].
`In both cases coligands, such as EDDA, tricine or nicotinic acid are required to complete the
`coordination of the [Tc]-HYNIC core. The use of coligands can have a positive side effect, since they
`
`
`
`Petitioner GE Healthcare – Ex. 1009, p. 3387
`
`

`

`Molecules 2013, 18
`
`can influence the lipophilicity of the radiopharmaceutical and the in vivo stability of the
`99mTc-complexes, which in turn affects the radiopeptide pharmacokinetics [81].
`
`3388
`
`Figure 5. Chemical structure of chelators for the labeling of peptides with 99mTc.
`
`
`Another strategy is the labeling of biomolecules with the organometallic [99mTc(CO)3]+ core. The
`99mTc tricarbonyl approach has been used for the development of new radiopharmaceuticals with the
`organometallic precursors fac-[99mTc(CO)3(H2O)3]+. 99mTc-tricarbonyl complexes which are formed
`with a tridentate BFC conjugated peptide, such as (N-His)Ac (24) or picolylamine diacetic acide
`(PADA) (25) show better stability in vivo, compared to mono and bidentate ligands such as histidine
`(26) [82]. Finally, the application of HYNIC and the N4-approach for peptide conjugation results in
`products with highly favorable pharmacokinetics in animal models and patients [12].
`
`3.4. 111In/67/68Ga/86/90Y/177Lu/64/67Cu-Labeled Peptide Radiopharmaceuticals
`
`Both Ga3+ and In3+ are hard Lewis acids, because of their high charge density and small ionic
`radius. For this reason, hard ligands form thermodynamically stable complexes with these ions (HSAB
`concept). These ligands contain nitrogen and oxygen donor atoms. In3+ is softer and larger than Ga3+.
`This difference often leads to a different coordination chemistry. The first clinically established
`peptide radiopharmaceutical for the visualization of neuroendocrine tumors was DTPA-octreotide
`labeled with 111In (under the name of Octreoscan®) [83]. DTPA (diethylenetriaminepentaacetic acid,
`27, Figure 6) is still one of the choices of BFCs for the labeling of peptides with 111In. It forms stable
`111In-complexes with fast labeling kinetic. However, it does not suit well for the labeling with many of
`lanthanides [84]. The macrocyclic chelator DOTA
`the clinically used -emitters such as
`(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, 28, Figure 6) has been evaluated for the
`labeling of peptides with divalent and trivalent radiometals, such as Ga3+, In3+, Y3+, Lu3+ and Cu2+. It
`forms thermodynamically and kinetically stable complexes. Several DOTA-peptide conjugates labeled
`with gallium and indium have been used in clinical routine like the somatostatin conjugates
`DOTA-TOC and DOTA-TATE, bombesin analogs, RGD analogs, minigastrin analogs etc. The
`macrocyclic chelator NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid, 29, Figure 6) is most
`favorable for the 67/68Ga-labeling of peptides [85]. The difference in cavity size of NOTA and DOTA
`is another important aspect to consider when selecting a proper chelator for the labeling with
`radiometalls. The thermodynamic stability constant of Ga-NOTA complex is approximately 10 orders
`higher than that of Ga-DOTA [86]. NOTA derivatives containing an additional coupling moiety that
`was introduced into the macrocycle such as benzyl-isothiocyanate (NOTA-Bz-NCS) or at the
`-position of one carboxylate arm such as aspartic acid (NODASA), glutamic acid (NODAGA) and
`
`
`
`Petitioner GE Healthcare – Ex. 1009, p. 3388
`
`

`

`Molecules 2013, 18
`
`benzyl-isothiocyanate (NODAPA-NCS) have been developed. The advantage of this additional
`coupling moity is that all of the carboxylic arms are available to saturate the hexadentate coordination.
`
`3389
`
`Figure 6. Chemical structures of chelators that are suited for the labeling of peptides with
`radiometals such as 111In/67/68Ga/86/90Y/177Lu/64/67Cu.
`
`
`
`Yttrium and lanthanide ions with an oxidation state of 3+ are hard Lewis acids as well, and tend to
`form very stable complexes with hard ligands. Because of their large size, their complexes have the
`high coordination numbers of 8 and even 9. The labeling of peptides with these radionuclides has been
`performed using mainly macrocyclic polyaminopolycarboxlic bifunctional chelating agents [87]. Due
`to the favorable pharmacokinetic profiles, DOTA derivatives have been used for the 86/90Y and 177Lu
`labeling of various peptides. DOTA provides eight donor atoms and the appropriate cavity size to form
`more stable complexes with these radionuclides than acyclic chelating agents and TETA (1,4,8,11-
`tetraazacyclododecane-1,4,8,11-tetraacetic acid, 30, Figure 6) derivatives. Thermodynamic stability
`and kinetic inertness are the most important factors for in vivo applications. DOTA derivatives were
`efficiently coupled to peptides as DOTA monoamides, where the kinetic inertness in vivo is not
`changed in comparison to DOTA. DOTA and TETA have been used for the production of 67/64Cu-
`labeled peptides. They show moderate kinetic stability under in vivo conditions. Cu-complexes with
`cross-bridged cyclam such as CB-TE2A (4,11-bis(carboxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2]-
`hexadecane, 31, Figure 6) improve the kinetic inertness and thus the pharmacokinetics of the
`radiolabeled peptide.
`
`4. Chelator Types
`
`4.1. Acyclic Chelators
`
`[111In]-DTPA-octreotide (OctreoScan®) is an octreotide derived somatostatin analog known to act
`as a selective molecular targeting agent for the imaging of neuroendocrine tumors. It has been widely
`used as imaging agent in single photon emission computer tomography (SPECT). DTPA
`(diethylenetriaminopentaacetic acid, 27) was first synthesized by Frost [88]. DTPA and its derivatives
`(Figure 7) can be used for the complexation of radiometals like 111In, 213Bi, 86/90Y, 177Lu, 99mTc, 67/68Ga
`for nuclear medicine applications and for Gd for MRT applications. The conjugation of DATP using
`
`
`Petitioner GE Healthcare – Ex. 1009, p. 3389
`
`

`

`3390
`
`Molecules 2013, 18
`
`its reactive mixed anhydride [89] or cyclic bisanhydride 32 [90] can lead to undesired conjugates, in
`particular double substituted DTPA side products [91]. To avoid this drawback, DTPA-tetra(t-Bu ester)
`(33) or DTPA-tetra(All ester) (34) have been synthesized starting from diethylenetriamine [92,93]. The
`activation of the free carboxylic group with activation agents like DIC or HBTU allows the d