Bioconjugate Chem. 2003, 14, 3-17
`
`3
`
`REVIEWS
`
`Synthesis of Target-Specific Radiolabeled Peptides for Diagnostic
`Imaging
`
`Jakub Fichna and Anna Janecka*
`
`Department of Medicinal Chemistry, Medical University of Lodz, Lindleya 6, 90-131 Lodz, Poland.
`Received April 23, 2002
`
`INTRODUCTION
`Radiopharmaceuticals are drugs containing atoms of
`some radioactive elements. They are designed for diag-
`nostic or therapeutic purposes, to deliver small doses of
`ionizing radiation to the disease sites in the body.
`Radiopharmaceuticals, unlike classical chemotherapeu-
`tics, act against malignant cells with high specificity.
`Radiopharmaceuticals are mostly small organic mol-
`ecules, such as peptides or peptidomimetics, but they can
`also be macromolecules, for example antibodies.
`Biodistribution of radiopharmaceuticals can be deter-
`mined either by their chemical and physical properties
`or by their biological interactions. Radiopharmaceuticals,
`which act through receptor binding, are called target-
`specific. Ideally, these radiopharmaceuticals are designed
`to locate with high specificity at cancerous tumors, even
`if their location in the body is unknown, while producing
`minimal radiation damage to normal tissues (1-6). In
`the past decade significant progress has been made in
`the development of peptide-based target-specific radiop-
`harmaceuticals, which have become an important class
`of imaging agents for the detection of various diseases,
`such as tumors, thrombosis, and inflammation.
`Many excellent reviews have been published discussing
`different aspects of radionuclide chemistry and therapy
`(7-9) and the use of radiopharmaceuticals for diagnosis
`and treatment of different pathological conditions (10-
`19). Most popular technetium radiopharmaceuticals (20-
`28), as well as those incorporating other radionuclides
`(29, 30), have been reviewed. Antibodies (24, 25), peptides
`(23, 30-35), and steroids (36) as targeting molecules have
`been described.
`In this review, which is limited to the use of small
`peptides as targeting molecules, we attempt to sum-
`marize, from the chemical point of view, the development
`of labeling methods, in particular the application of
`different bifunctional chelating agents. We also give a
`short description of radionuclides used with these agents.
`Radiopharmaceuticals based on small peptides, which are
`in clinical use or under investigation in preclinical and
`clinical trials, are also mentioned.
`
`LABELING METHODS
`Direct Labeling. Direct labeling methods (Table 1)
`are mostly based on the binding of a radionuclide to thiol
`groups in the targeting molecule, that seems relatively
`
`* Address for correspondence: Dr. Anna Janecka, Depart-
`ment of Medicinal Chemistry, Medical University of Lodz,
`Lindleya 6, 90-131 Lodz, Poland. Tel/fax:
`(4842) 6784277.
`E-mail: ajanecka@csk.am.lodz.pl.
`
`easy to perform (37, 38). However, such a labeling process
`is difficult to control, for its detailed chemistry is un-
`known and may lead to unplanned changes in the
`structure, stability, and pharmacokinetic properties of
`the labeled molecule. Furthermore, very little is known
`about the number of donor atoms in the labeled molecule
`and the geometry of radionuclide coordination. The
`stability in vivo of a synthesized complex also remains
`uncertain.
`The direct labeling approach is rather unsuitable for
`small peptides, which either do not possess disulfide
`linkage or are unable to maintain their activity after
`reduction. For example Thakur (39) has reported the
`alteration of the receptor binding properties of the
`radiolabeled somatostatin analogues, when the disulfide
`bridge was reduced to free thiol groups and subsequently
`radiolabeled with 99mTc. However, direct labeling has
`been successfully applied for labeling of the platelet
`receptor-binding peptide with 99mTc (40, 41) and for high
`molecular weight proteins such as antibodies and their
`fragments (42, 43).
`Chelate Methods. In chelate methods (Table 1) a
`radionuclide is bound to the targeting molecule in-
`directly, through a bifunctional chelating agent (BFCA)
`(Figure 1).
`In general, a radiopharmaceutical containing a BFCA
`consists of the following parts: a targeting molecule,
`BFCA, radionuclide, and a linker (22). The targeting
`molecule is a carrier of a radionuclide to the receptor site
`in vivo. A radionuclide serves as a radiation source. A
`BFCA, covalently attached to the targeting molecule,
`functions as the coordinator of the radionuclide. A linker,
`not always necessary, is a spacer residue, which sepa-
`rates a targeting molecule from a chelating agent.
`Functional groups, naturally present in the peptide or
`introduced synthetically, are responsible for covalent
`attachment of BFCA. Naturally occurring functional
`groups include terminal, as well as side-chain amino and
`carboxy groups, thiol groups from cysteine, and p-
`hydroxyphenyl from tyrosine. BFCA must contain a
`conjugation group, which is used for attachment of the
`peptide.
`Several types of conjugation groups, active esters,
`isothiocyanates, maleimides, hydrazides, and R-haloam-
`ides are used to form BFCA-peptide linkages (Figure 2).
`Active esters can be used to form an amide bond
`between a carboxy group of a BFCA and an amino group
`in a peptide ligand. Since they are at a low level of
`activation, side-reactions during coupling are generally
`less of a problem than with most amide bond forming
`procedures. Commonly used are p-nitrophenyl, pen-
`
`© 2003 American Chemical Society
`10.1021/bc025542f CCC: $25.00
`Published on Web 12/19/2002
`
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`Petitioner GE Healthcare – Ex. 1018, p. 3
`
`

`

`4 Bioconjugate Chem., Vol. 14, No. 1, 2003
`
`Fichna and Janecka
`
`Table 1. General Overview of Applied Labeling Methods
`labeling method
`main principle
`targeting molecule
`high molecular
`direct labeling
`radionuclide
`weight molecules
`binds directly to
`active groups
`present in the
`targeting molecule
`
`advantages
`easy to perform
`
`disadvantages
`unknown chemistry
`
`ref
`(32, 35,
`37, 38)
`
`unknown geometry of
`a radionuclide-targeting
`molecule complex
`possible damage to
`targeting molecule
`during labeling process
`
`chelate methods
`prelabeling
`
`postlabeling
`
`labeling of BFCA
`followed by
`conjugation with
`the targeting
`molecule
`
`conjugation of
`BFCA to targeting
`molecule, followed
`by labeling
`of conjugate
`
`small peptides
`
`relatively easy to control,
`well-defined chemistry
`
`time-consuming
`
`(26, 32)
`
`targeting molecule functional
`groups remain unlabeled
`
`small peptides
`
`most popular method
`
`complicated purification
`of obtained
`radiopharmaceutical
`possible damage to targeting
`molecule during labeling
`process
`
`(35, 44)
`
`well-defined chemistry
`possible use of classical
`solid-phase or solution
`methods of the peptide
`synthesis
`
`Figure 1. Schematic structure of a radiopharmaceutical. BFCA
`can complex a metal and also contains a functional group which
`forms a covalent linkage to a biological molecule, such as
`peptide.
`
`tafluorophenyl, N-hydroxysuccinimide, and sulfo-N-hy-
`droxysuccinimide active esters. The N-hydroxysuccinim-
`ide esters are very reactive with high selectivity toward
`aliphatic amines (22, 45). The choice of an active ester
`to be used for BFCA-peptide bond formation is partly
`dictated by sheer reactivity, but the ease of coproduct
`removal is also an important consideration. Thus, for
`water-insoluble BFCA-peptide conjugates, a succinimide
`ester coupling is especially convenient because both
`N-hydroxysuccinimide and sulfo-N-hydroxysuccinimide
`are very water-soluble (46, 47) and easy to remove. But
`for water-soluble BFCA-peptide conjugates p-nitrophe-
`nyl or pentafluorophenyl esters, which are ether-soluble,
`may be a better choice (48).
`Isothiocyanates, like active esters, react with amino
`groups of a peptide forming thiourea bonds. Since they
`react best in the higher pH, they cannot be used with
`peptides susceptible to alkaline conditions.
`The third class are maleimides, which react with thiols
`and form thioether bonds. The optimum pH for the
`reaction is near 7. At higher pH maleimides may hydro-
`lyze to form nonreactive maleanic acids (49, 50).
`Hydrazides are conjugation groups which react with
`the aldehyde group of a peptide to form a hydrazone-
`
`Figure 2. Reaction schemes for BFCA-peptide conjugation.
`peptide conjugate. They are suitable for peptides contain-
`ing 2-amino alcohol structure like in serine, threonine,
`and hydroxylysine, which can be very rapidly oxidized
`
`Petitioner GE Healthcare – Ex. 1018, p. 4
`
`

`

`Reviews
`by periodate at pH ) 7 to generate an aldehyde. The use
`of a low molar ratio of periodate to peptide minimizes
`the potential for side-reactions during the oxidation. The
`formed hydrazones are stable at pH 6-8 for at least 12
`h at 22 °C (51).
`R-Haloamides are suitable for conjugation with pep-
`tides containing a free SH group. Chloroacetyl- or bro-
`moacetylamides are used (52, 53).
`The bifunctional approach is often associated with the
`term ‘pharmacokinetic modifier’ (PKM). Liu and Edwards
`(22) define PKM as a linker between a targeting molecule
`and a BFCA. PKM is a spacer residue, separating the
`radionuclide from the binding site of the molecule to
`minimize the risk of its undesired modification. The use
`of a linker helps to choose a BFCA and a radionuclide
`more independently. The most popular linkers are long
`poly(ethylene glycol) (PEG) or hydrocarbon chains to
`increase the lipophilicity and polyamino acid sequences,
`such as polyglycine, to increase the hydrophilicity, as well
`as esters and disulfides capable of rapid metabolism.
`The distinction between chelate methods can be made
`depending on the sequence of the steps used for the
`synthesis of a radiolabeled peptide.
`Prelabeling Method. The pre-labeling method (pre-
`formed chelate approach) is based on the labeling of a
`BFCA with its subsequent activation and conjugation,
`through covalent bonds, to a peptide. In this approach
`the chemistry of a process is well defined and easily
`controlled. As the labeling and conjugation steps are
`separated, it can be ensured that the radionuclide is
`attached directly to a chelate moiety and the peptide
`amino groups remain unlabeled (22). However, the prela-
`beling of a BFCA may impair the conjugation process and
`complicate purification of a radiopharmaceutical. The
`method is time-consuming, so not appropriate for use
`with short-lived isotopes, generally associated with imag-
`ing. As such the prelabeling method is rarely used for
`synthesis of radiolabeled peptides.
`Postlabeling Method. The postlabeling method (indirect
`labeling approach), the most popular approach in the
`synthesis of radiopharmaceuticals, requires the synthesis
`of a BFCA-peptide conjugate and is followed by its
`labeling. In this method BFCA may be attached to N- or
`C-terminus, as well as to a side chain of a peptide or it
`can be even incorporated into a peptide backbone. The
`postlabeling method is characterized by a well-defined
`chemistry and relative simplicity. For convenient, high
`yield synthesis of radiopharmaceuticals, applied BFCA
`should be compatible with the solid-phase or solution
`methods of peptide synthesis. However, harsh conditions
`required for effective labeling of conjugates may some-
`times cause changes in the amino acid sequence or
`peptide backbone conformation and even begin the
`decomposition of the whole radiopharmaceutical (35).
`
`BIFUNCTIONAL CHELATING AGENTS
`BFCAs are used to connect a radionuclide and a
`targeting molecule to form a radiopharmaceutical. An
`ideal BFCA should coordinate the radionuclide with a
`high yield, to form a relatively stable complex. The agent
`must comply with the nature and oxidation state of a
`radionuclide and should prevent any accidental changes
`in its redox potential.
`It is important to carefully choose a proper BFCA, as
`the conjugation with targeting molecule requires specific
`conditions: pH, temperature, reaction time. The stere-
`ochemistry of a BFCA is important when synthesizing
`radiopharmaceuticals targeting specific receptors.
`
`Bioconjugate Chem., Vol. 14, No. 1, 2003 5
`
`Figure 3. Structure of DTPA and its analogues:
`(b) cDTPA; (c) mDTPA.
`
`(a) DTPA;
`
`DTPA. DTPA (NR-diethylenetriaminopentaacetic acid)
`belongs to the group of polyaminocarboxy chelates (Fig-
`ure 3a). It is a strong chelating group, mostly linked with
`111In, a trivalent radionuclide. It can be attached to larger
`proteins, e.g., albumins and antibodies (49, 50, 54), as
`well as to small peptides, like somatostatin analogues
`(55, 56). The conjugation of DTPA with macromolecules
`has been successfully performed by the use of isobutyl
`chloroformate as a coupling reagent (57). For small
`peptides, however, DTPA derivatives such as DTPA
`bicyclic anhydride (cDTPA) and monoreactive DTPA
`derivative, 3,6-bis(carboxymethyl)-9-(((2-maleimidoethyl)-
`carbamoyl)methyl)-3,6,9-triazaundecanedioic acid (mDT-
`PA), have been applied (Figure 3b and 3c).
`Hnatowich et al. (54) have developed a simple method
`of covalent coupling of cDTPA to peptides at their amino
`groups. The efficiency of this method is relatively high
`and it has several advantages. The coupling reaction runs
`in an aqueous solution and is a simple, one-step process.
`The side product of the reaction, a double substituted
`DTPA derivative, and unreacted material can be both
`easily separated from the main product by gel chroma-
`tography. The conjugated peptide maintains its affinity
`toward specific receptors. The sample, purified before the
`addition of a radionuclide, can be stored and labeled only
`when required. For peptides containing lysine the con-
`jugation occurs especially at its (cid:15)-amino group, as more
`basic than N-terminal amino group. The method is
`therefore inappropriate for somatostatin analogues, as
`the lysine residue is situated within the active site of the
`molecule and the conjugation may result in the loss of
`receptor binding activity. For that reason a modified
`method, proposed by Bakker et al. (55) for DTPA-
`octreotide, is used to conjugate the somatostatin analogue
`with cDTPA. In this approach lysine residue within the
`active site of the peptide is protected with tert-butyloxy-
`carbonyl group (Boc), before the reaction with cDTPA and
`deprotected after the conjugation (Figure 4). This method
`enables a selective reaction of the N-terminal amino
`group with BFCA, whereas the lysine residue within the
`bioactive site remains unsubstituted.
`A monoreactive DTPA derivative, mDTPA, with four
`carboxy groups protected as tert-butyl esters, was intro-
`duced by Arano et al. (57) (Figure 5). Since mDTPA
`possesses only one free carboxy group, the formation of
`undesired intermolecular linkages with peptides is pre-
`vented. High solubility of mDTPA in various solvents
`makes this BFCA appropriate for both liquid- and solid-
`phase peptide synthesis.
`A great obstacle in the efficient radiolabeling of DTPA
`conjugates is the presence of trace metals in the prepara-
`
`Petitioner GE Healthcare – Ex. 1018, p. 5
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`

`

`6 Bioconjugate Chem., Vol. 14, No. 1, 2003
`
`Fichna and Janecka
`
`very stable complexes with a variety of trivalent radio-
`nuclides, such as 66,67,68Ga,86,90Y, 111In, 149Pm, 177Lu (68-
`73) and divalent radionuclides, 27Mg,47Ca,64Cu (74).
`Two different approaches for DOTA conjugation with
`peptides have been developed. In the first approach one
`of the four carboxy groups in DOTA is activated to
`facilitate the reaction with primary amines in the peptide
`and form a stable amide bond linkage. In the second
`approach DOTA derivatives with additional side chains
`are used. A peptide ligand is attached to the side chain
`of DOTA derivative. Several DOTA derivatives have been
`synthesized so far, like PA-DOTA (R-[2-(4-aminophenyl)-
`ethyl]-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraace-
`tic acid) and p-NCS-Bz-DOTA ((2-(4-isothiocyanatoben-
`zyl)-1,4,7,10-tetraazacyclododecane-N,N¢ ,N¢¢
`,N¢¢¢
`-
`tetraacetic acid)
`(Figure 8b and 8c)
`(69). Recently
`Eisenwiener et al. (44) have introduced two new DOTA
`derivatives, DOTASA(t-Bu)4, (1-(1-carboxy-2-carbo-tert-
`butoxyethyl)-4,7,10-(carbo-tert-butoxymethyl)-1,4,7,10-
`tetraazacyclododecane) and DOTAGA(t-Bu)4, (1-(1-carboxy-
`3-carbo-tert-butoxypropyl)-4,7,10-(carbo-tert-butoxymethyl)-
`1,4,7,10-tetraazacyclododecane) (Figure 8d), which con-
`venient synthesis is outlined in Figure 9. The conjugation
`of all DOTA derivatives to a peptide is performed through
`an amino group of a peptide.
`Fully eight coordinate structure has been reported for
`all DOTA complexes (60) using four amino and four
`carboxy groups. In the case when one carboxy group is
`used for conjugation, the amide carbonyl oxygen occupies
`the eighth position around the radionuclide.
`DOTA and derivatives were successfully conjugated to
`a number of somatostatin analogues, and obtained ra-
`diopharmaceuticals had good pharmacological param-
`eters (72-78). DOTA conjugates are especially suitable
`for radionuclide therapy, as they can be radiolabeled with
`67Ga (75), 90Y (71, 76), and 111In (73). De Jong et al. (68)
`have demonstrated that 90Y-DOTA conjugates have very
`good pharmacokinetic properties in vivo. However, in
`these conjugates the chelate is situated closer to the
`peptide, the labeled conjugate is more rigid and less
`flexible, which makes binding with the receptor more
`difficult. Reubi et al. (79) reported the best pharmacologi-
`cal properties for 67Ga-DOTA complexes. The radionu-
`clide coordination geometry, including the number of
`uncomplexed carboxy and amino groups, increases the
`flexibility of a ligand and allows its better adjustment to
`the receptor binding site.
`TETA. TETA (1,4,8,11-tetraazacyclotetradecane-1,4,8,-
`11-tetraacetic acid) (Figure 10) is one of the most studied
`chelating agents for copper in peptide targeted radio-
`
`Figure 4. Synthesis of DTPA-octreotide, starting with cDTPA.
`
`tion, as they compete with radionuclides in the process
`of labeling. For that reason a significant, 40- to 70-fold
`molar excess of peptide conjugate and ultrapure radio-
`nuclide derivative of the highest possible specific activity
`are required (55).
`DTPA conjugates have been shown to form 111In-
`chelate structures (Figure 6), which are eight coordinate,
`using all three amino and four carboxy groups, while the
`eighth position around the radionuclide is occupied by
`the amide carbonyl oxygen (58). 111In-DTPA conjugates
`possess excellent in vivo stability (59-61).
`Many research groups put much effort in the synthesis
`of kinetically stable DTPA-peptide conjugates that form
`complexes with 90Y (62). Substitutions, particularly in the
`carbon atoms of the DTPA backbone, sterically hinder
`the opening of the chelate ring that must occur during
`radionuclide complex dissociation and increase the in vivo
`stability of the radiopharmacutical. The first class of
`modified DTPA conjugates was constructed by attaching
`p-isothiocyanatobenzyl moiety to one DTPA backbone
`ethylene group and appending methyl to another ethyl-
`ene group in the same backbone (Figure 7a,b). The second
`class of modified DTPA conjugates was developed by
`replacing one of the ethylene groups by a cyclohexyl
`moiety (Figure 7c). Such modifications increase the
`rigidity in the DTPA backbone and the in vivo stability
`of obtained radiopharmaceuticals (62-64). Synthesis of
`new derivatives constructed on DTPA core has been
`recently reported (65, 66).
`99mTc is less suitable for the labeling of DTPA-peptide
`conjugates, as this radionuclide, even at high concentra-
`tions, has low affinity and poor selectivity to the binding
`sites of this BFCA (67).
`DOTA.
`DOTA
`(1,4,7,10-tetraazacyclododecane-
`N,N¢,N¢¢,N¢¢¢-tetraacetic acid) (Figure 8a) and its deriva-
`tives proved to be a good alternative for DTPA. They play
`an important role in clinical applications, as they form
`
`Figure 5. Reaction scheme for synthesis of mDTPA.
`
`Petitioner GE Healthcare – Ex. 1018, p. 6
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`

`

`Reviews
`
`Bioconjugate Chem., Vol. 14, No. 1, 2003 7
`
`Figure 6. Possible structure of In3+-DTPA-peptide.
`
`Figure 7. Structures of DTPA derivatives: (a) 2-(p-isothiocy-
`anatobenzyl) diethylenetriaminopentaacetic acid; (b) 2-(p-iso-
`thiocyanatobenzyl)-6-methyldiethylene-triaminopentaacetic acid
`(1B4H-DTPA); (c) 2-(p-isothiocyanatobenzyl) cyclohexyldieth-
`ylene-triaminopentaacetic acid (CHX-DTPA).
`
`therapy. TETA has been successfully used as a BFCA
`with somatostatin analogues (30).
`NOTA. NOTA (1,4,7-triazacyclononane-1,4,7-triacetic
`acid) (Figure 11a), its phosphonate analogue NOTP
`(1,4,7-triazacyclononane-N,N¢ ,N¢¢ -tris(methylenephospho-
`nic) acid) (Figure 11b) and the monoethyl ester of NOTP,
`NOTPME (1,4,7-triazacyclononane-N,N¢ ,N¢¢ -tris(methyl-
`enephosphonate-monoethyl ester)) (Figure 11c) were
`studied for possible use in radiopharmaceuticals. Com-
`plexes with 67Ga (80) and 111In (81) were reported.
`A monoreactive NOTA derivative, NODAGA(tBu)3 (1-
`(1-carboxy-3-carbo-tert-butoxypropyl)-4,7-(carbo-tert-bu-
`toxymethyl)-1,4,7-triazacyclononane) (Figure 11d) was
`synthesized by Eisenwiener et al. (82). The synthesis is
`outlined in Figure 12. This BFCA is useful for the
`coupling to the N-terminus of peptides on solid-phase and
`in solution. The NODAGA-peptide conjugates were
`labeled with 67Ga and 111In in high yields and good
`specific activities. NODAGA-based derivatives carry a
`spacer function between the BFCA and the peptide which
`improves the receptor binding affinity.
`HYNIC. HYNIC (2-hydrazinonicotinic acid) (Figure
`13), first described by Abrams et al. (83) has been used
`as a BFCA for radiolabeling of different groups of
`molecules, such as (cid:231)-globulins (83, 84), chemotactic
`peptides (85, 86), and somatostatin analogues (87-90).
`Structural organization of HYNIC determines its ap-
`plication, as it can only occupy one or two coordination
`sites of the radionuclide. That is why a coligand such as
`tricine or EDDA (ethylenediaminodiacetic acid) should
`be also coordinated to complete the coordination sphere
`
`Figure 8. Structures of DOTA and its derivatives: (a) DOTA;
`(b) PA-DOTA; (c) p-NCS-Bz-DOTA; (d) DOTASA(t-Bu)4, n )
`1 and DOTAGA(t-Bu)4, n ) 2.
`
`of a radionuclide (91, 92) (Figure 14). The conjugation of
`coligands helps in modifying the properties of obtained
`radiopharmaceutical, such as hydrophilicity or pharma-
`cokinetics. However, the requirement for the use of
`coligands makes the chemistry of the synthesis more
`complicated, and multiple possible products and side-
`products can be obtained.
`HYNIC-coligand conjugates were reported to have low
`stability (22). The search for stable HYNIC-coligand
`complexes is now carried on and phosphines seem to be
`the most promising coligands so far (92, 93). HYNIC
`derivatives, together with phosphines and tricine, form
`ternary complexes [99mTc(HYNIC-TM)(tricine)(phosphine)]
`(TM-targeting molecule). Such complexes are stable in
`solution, and their hydrophilicity can be modified by
`changing functional groups attached to phosphine back-
`bone or by substitution of tricine by other glycine deriva-
`tives.
`HYNIC is often used as a BFCA for somatostatin
`analogues. The desired amide bond formation should
`occur between the carboxy group of HYNIC and the
`N-terminal amino group of a peptide. However, in soma-
`tostatin analogues the presence of lysine makes it dif-
`ficult to obtain a monosubstituted product. Krois et al.
`(87) have compared available methods of HYNIC-oct-
`reotide conjugation, and none of them seemed efficient
`enough. The method of protecting the lysine amino group
`with (Boc)2O reported by Bakker et al. (55) proved to be
`unsatisfactory, as the final product was contaminated
`with Boc-disubstituted derivative, difficult to separate.
`The activation of HYNIC to N-hydroxysuccinimide ester
`to facilitate the conjugation step, a method suggested by
`Abrams et al. (83), also produced poor results. An
`improved method of HYNIC-octreotide conjugation,
`reported by Krois et al. (87), is based on the incorporation
`of the chelator at an early stage of ab ovo peptide
`synthesis performed by solution method. That approach
`produced high yield of conjugate, practically free of any
`contamination.
`
`Petitioner GE Healthcare – Ex. 1018, p. 7
`
`

`

`8 Bioconjugate Chem., Vol. 14, No. 1, 2003
`
`Fichna and Janecka
`
`Figure 9. Synthesis of DOTASA(t-Bu)4 and DOTAGA(t-Bu)4.
`
`Figure 10. Structure of TETA.
`
`Figure 11. Structures of NOTA and its derivatives: (a) NOTA,
`(b) NOTP, (c) NOTPME, (d) NODAGA(t-Bu)3.
`
`Multidentate Chelators. The class of tetradentate
`chelators includes N3S triamidothiols, N2S2 diamidodithi-
`ols (94), and N2S4 diaminotetrathiols (26) (Figure 15) and
`a number of their derivatives, containing sulfur and
`nitrogen atoms, incorporated into chelating backbones.
`N2S4 chelators contain two amino and four thiol donors
`and they have been used for 99mTc- and 186Re-labeling of
`antibodies (95, 96). N2S2 containing two amido and two
`thiol donors have been used for 99mTc- and 186Re-labeling
`of proteins, peptides, and oligonucleotides. Since most of
`the N2S2 derivatives form highly lipophilic rhenium and
`technetium complexes, they are used whenever hydro-
`phobic targeting systems are applied (97). Both N3S- and
`
`N2S2-derived BFCAs form high specific activity com-
`plexes, and for that reason they can be used in the
`preformed chelate labeling method. In case of small
`molecules (steroids, low molecular weight peptides) these
`BFCAs complex 186/188Re in the postconjugation labeling
`process (28). BFCAs based on N3S backbone are used for
`the synthesis of 168/188Re radiopharmaceuticals (98).
`The most frequently applied BFCAs from that group
`are MAG3 (mercaptoacetyl-glycylglycylglycine) and MAG2-
`GABA (mercaptoacetylglycylglycyl-(cid:231)-butyric acid), con-
`taining the spacer groups glycine and GABA, respectively
`(Figure 16). GABA spacer group is two carbon units
`longer than glycine; therefore, it may exhibit more useful
`properties, such as higher yield in radionuclide labeling
`or better receptor binding due to the changes in intramo-
`lecular organization (99-101).
`BFCAs listed above have been widely used as metal
`chelating agents in both diagnostic and therapeutic
`procedures. Attempts to design new derivatives with
`improved properties are still under way (66, 102).
`
`RADIONUCLIDES
`Each radionuclide is characterized by a specific coor-
`dination chemistry and therefore must be conjugated
`with a specific BFCA with specific donor atoms and
`ligand frameworks to retain its own radioactivity.
`The selection of an appropriate radionuclide is an
`inherent determinant in developing any therapeutic
`radiopharmaceutical. Important factors to consider in-
`clude half-life of the radioactive nuclide, its mode of
`decay, and its cost and availability (Table 2). The half-
`life is a critical factor. For diagnostic imaging the half-
`life of a radionuclide must be long enough to enable the
`synthesis of the labeled compound and to facilitate the
`accumulation in the target tissue, while allowing clear-
`
`Figure 12. Synthesis of NODAGA(t-Bu)3.
`
`Petitioner GE Healthcare – Ex. 1018, p. 8
`
`

`

`Reviews
`
`Bioconjugate Chem., Vol. 14, No. 1, 2003 9
`
`Table 2. (cid:231)-, (cid:226)--, and (cid:226)+-Emitting Radionuclidesa Used for Radiolabeling of Radiopharmaceuticals (103)
`(cid:226)--energy (keV)
`(cid:226)+-energy (keV)
`radionuclide
`(cid:231)-energy (keV)
`t1/2 (h)
`64Cu
`12.7
`579
`
`653
`
`-
`
`66Ga
`
`67Ga
`68Ga
`
`86Y
`
`90Y
`99mTc
`111In
`149Pm
`177Lu
`
`9.5
`
`78.3
`1.1
`
`14.7
`
`64.1
`6.0
`67.9
`53.0
`160.8
`
`-
`
`91, 93, 185, 296, 388
`-
`
`-
`
`-
`141
`245, 172
`286
`-
`
`-
`
`-
`-
`
`-
`
`2288
`-
`-
`1070
`236
`
`4150, 935
`
`-
`1880, 770
`
`2335, 2019, 1603, 1248, 1043
`
`-
`-
`-
`-
`-
`
`decay mode
`(cid:226)+ (17.4%)
`(cid:226)- (39%)
`EC
`(cid:226)+ (56%)
`EC (44%)
`EC (100%)
`(cid:226)+ (90%)
`EC (10%)
`(cid:226)+ (33%)
`EC (66%)
`(cid:226)- (72%)
`IT (100%)
`EC (100%)
`(cid:226)- (95.9%)
`(cid:226)- (100%)
`
`Figure 13. Structure of HYNIC.
`
`Figure 14. Structures of HYNIC-technetium complexes: (a)
`[Tc(HYNIC)(tricine)2]; (b) [Tc(HYNIC)(EDDA)Cl]; (c) [Tc(HYNIC)-
`(tricine)(L)].
`
`Figure 15. Structures of multidentate chelators: (a) N3S tri-
`amidethiol; (b) N2S4 diaminotetrathiol; (c) N2S2 diamidodithiol.
`
`Figure 16. Structures of: (a) MAG3; (b) MAG2-GABA.
`
`ance through the nontarget organs. Ideally, the half-life
`should be as short as possible to reach these two goals.
`Radionuclides most commonly used in radiopharmaceu-
`ticals range in half-lives from minutes (62Cu) to days (177-
`Lu).
`
`Copper (64Cu). There are two radionuclides of copper
`used in the radiopharmaceutical labeling. 64Cu (t0.5)12,7
`h) is a (cid:226)+-, 0.653 MeV (17.4% abundance), and (cid:226)--emitter,
`0.579 MeV (39% abundance), and it decays by electron
`capture. A good review on the biological chemistry of
`copper is a book by Linder (104).
`Gallium (66Ga, 67Ga, 68Ga). Gallium radionuclides can
`be either used for (cid:231)-scintigraphy or positron emission
`tomography (PET) imaging. 66Ga (t0.5 ) 9.5 h) is a
`medium half-life (cid:226)+-emitting radionuclide obtained from
`66Zn(p,n)66Ga cyclotron, applied in the limited number
`of cases. 67Ga (t0.5 ) 78.3 h) is produced in 68Zn(p,2n)67Ga
`cyclotron. 68Ga (t0.5 ) 1.1 h) is obtained from the 68Ge/68-
`Ga generator and it has been used in a limited number
`of clinical studies (105, 106).
`Yttrium (86Y, 90Y). There are two radionuclides of
`yttrium used in the radiopharmaceutical labeling. 86Y (t0.5
`) 14.7 h) is a (cid:226)+-emitting radionuclide, often used as an
`equivalent for 90Y in the PET imaging. 90Y (t0.5 ) 64 h) is
`a (cid:226)--emitter, which is probably the most frequently used
`radionuclide for targeted radiotherapy in human studies.
`It has a relatively high energy transfer and therefore can
`be used for imaging of bulky disease sites of solid tumors.
`90Y can be relatively easily obtained in a high-specific
`activity from 90Sr (107-109). 90Y is also available as a
`GMP commercial product.
`Technetium (99mTc). 99mTc is used in about 85% of
`all diagnostic applications. It has ideal properties for
`diagnostic imaging. The half-life of 6 h is long enough to
`synthesize the 99mTc-labeled radiopharmaceuticals and
`perform imaging studies, yet short enough to minimize
`the radiation dose to a patient. 99mTc emits a 140 keV
`(cid:231)-ray with 89% abundance which is close to optimum for
`imaging with the present (cid:231) cameras. This energy is
`sufficient for emerging from inside the body and for
`imaging internal organs. 99mTc is readily available at low
`costs from its parent nuclide 99Mo (t0.5 ) 66 h) from a
`99Mo/99mTc generator. At the concentration levels used for
`imaging (<10-6 M) neither its (cid:231)-radiation nor the soft
`(cid:226)-decay is hazardous (110).
`Indium (111In). 111In has the half-life of 67 h which
`makes this isotope ideal for labeling immunoglobulins,
`where imaging is performed over intervals of several
`days. This nuclide decays by electron capture with
`emission of (cid:231)-photons of 173 and 247 keV (89% and 95%
`abundance, respectively), which allows its use in (cid:231)-scin-
`tigraphy.
`The first peptide radiopharmaceutical approved for
`clinical use was the 111In labeled somatostatin analogue
`OctreoScan. Nevertheless, because of its less favorable
`half-life, 111In is inferior to 99mTc for diagnosis. 111In is
`often used as an equivalent for 90Y in scintigraphic
`
`Petitioner GE Healthcare – Ex. 1018, p. 9
`
`

`

`10 Bioconjugate Chem., Vol. 14, No. 1, 2003
`
`Table 3. Regulatory Peptides
`
`regulatory peptide
`SST (somatostatin)
`
`BN/GRP (bombesin/gastrin
`releasing peptide)
`VIP (vasoactive
`intestinal peptide)
`RGD-containing
`peptides/RGD-peptidomimetics
`R-MSH (R-melanocyte
`stimulating hormone)
`NT (neurotensin)
`
`SP (substance P)
`
`no. of amino acid
`residues
`14
`
`14
`
`28
`
`-
`
`13
`
`13
`
`11
`
`Fichna and Janecka
`
`receptor type (subtypes)
`SST receptors (sst1/sst2/sst3/sst4/sst5)
`
`BN/GRP receptors (GRP, NMB, BRS-3)
`
`VIP receptors (-)
`
`GPIIb/IIIa/platelet and
`vitronectin/integrin receptors
`R-MSH receptors (-)
`
`NT receptors (NT1, NT2, NT3)
`
`SP receptors (NK1)
`
`in vivo activity
`inhibition of hormone and
`exocrine secretion
`gut hormone release,
`regulation of exocrine secretion
`vasodilation, water, and
`electrolyte secretion in the gut
`inhibition of adhesive and
`aggregatory functions of platelets
`melanogenesis
`
`vasoconstriction, regulation of cardiac
`activity raise in vascular permeability
`hypotension, salivary gland secretion,
`transmission of pain
`
`BFCA
`DTPA
`TETA
`HYNIC
`TETA
`HYNIC
`S-S (direct)
`HYNIC
`N2S2
`HYNIC
`MAG3
`HYNIC
`
`targeted disease
`tumor (neuroendocrine)
`
`tumor (neuroendocrine)
`
`tumor (neuroendocrine)
`
`tumor
`tumor
`tumor
`thrombosis
`
`ref
`(3, 113)
`(152, 153)
`(61, 118)
`(152, 153)
`(114, 115)
`(116)
`(117)
`(94)
`(119)
`(120-122)
`(40, 41, 123, 124)
`
`VIP analogues
`RGD-containing peptides/
`RGD-peptidomimetics
`S-S (direct)
`R-MSH analogues
`(125, 126)
`tumor (breast, prostate)
`CCMSH
`(127, 128)
`tumor
`DTPA
`SP
`SP analogues
`(129, 130)
`in